Basic Medical Biochemistry - Marks 4th ed

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Marks’

Basic Medical Biochemistry

A Clinical Approach Fourth Edition

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Marks’

Basic Medical Biochemistry

A Clinical Approach Fourth Edition

Michael Lieberman, PhD Distinguished Teaching Professor Department of Molecular Genetics, Biochemistry and Microbiology University of Cincinnati College of Medicine Cincinnati, Ohio

Allan Marks, MD Associate Professor of Internal Medicine (Emeritus) Department of Internal Medicine Section of Endocrinology and Metabolism Temple University School of Medicine Philadelphia, Pennsylvania

Alisa Peet, MD Associate Professor Director, Internal Medicine Clerkship Department of Internal Medicine Section of General Internal Medicine Temple University School of Medicine Philadelphia, Pennsylvania

Illustrations by Matthew Chansky

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Acquisitions Editor: Susan Rhyner Product Manager: Stacey Sebring Marketing Manager: Joy Fisher-Williams Vendor Manager: Bridgett Dougherty Designer: Doug Smock Compositor: Absolute Service, Inc. Fourth Edition Copyright © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business. 351 West Camden Street Baltimore, MD 21201

530 Walnut Street Philadelphia, PA 19106

Printed in China All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Lippincott Williams & Wilkins at 530 Walnut Street, Philadelphia, PA 19106, via email at [email protected], or via website at lww.com (products and services). 9

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Library of Congress Cataloging-in-Publication Data Lieberman, Michael, 1950Marks’ basic medical biochemistry : a clinical approach / Michael Lieberman, Allan Marks, Alisa Peet ; illustrations by Matthew Chansky. — 4th ed. p. ; cm. Basic medical biochemistry Includes bibliographical references and index. ISBN 978-1-60831-572-7 (pbk.) I. Marks, Allan D. II. Peet, Alisa. III. Title. IV. Title: Basic medical biochemistry. [DNLM: 1. Biochemistry. 2. Clinical Medicine. QU 4] 612.1'111—dc23 2011053244 DISCLAIMER Care has been taken to confirm the accuracy of the information present and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this information in a particular situation remains the professional responsibility of the practitioner; the clinical treatments described and recommended may not be considered absolute and universal recommendations. The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with the current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in this publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in their clinical practice. To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. International customers should call (301) 223-2300. Visit Lippincott Williams & Wilkins on the Internet: http://www.lww.com. Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6:00 pm, EST.

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Preface to the Fourth Edition It has been 4 years since the third edition was completed. The third edition retained the major pedagogic tools of the first and second editions, whereas the text had been completely revised, and certain features updated from the first edition. The fourth edition has, based on extensive reviews by both faculty and students, enhanced the features of the third edition in the following ways: 1. A major effort for this edition has been to update the patient histories to better reflect changes in treatment options for the disorders presented in this text. Every patient history has been reviewed and updated, if needed, to reflect the standard of care in 2011. 2. The “methods” notes of the third edition have been expanded such that there is at least one such note per chapter. These notes allow students to understand how biochemistry is used as a foundation for many of the laboratory tests ordered during the diagnosis of a patient. 3. Biochemical comments in several chapters have been updated to reflect recent research, particularly as it applies to signaling, type 2 diabetes, and metabolic syndrome. 4. Every chapter now contains a Table, at the end of the chapter, summarizing the diseases discussed in the chapter. 5. All chapters were reviewed and updated, where appropriate. Errors found, or pointed out by observant readers, were corrected. 6. The online question bank has been expanded by 5 questions per chapter, bringing the total number of questions (and explanations) in that bank to 468. The text itself contains 245 questions and explanations for student review. 7. Chapter references are now only available as an online supplement to the text. The references, where appropriate, are linked to the original research article in PubMed. In revising a text geared primarily toward medical students, the authors always struggle with new advances in biochemistry and whether such advances should be included in the text. In this text, the authors include only advances that will enable the student to better relate biochemistry to medicine, and future diagnostic tools. Although providing incomplete, but exciting, advances to graduate students is best for their education, medical students require a more directed approach, one that emphasizes how biochemistry is useful for the practice of medicine. This is a major goal of this book. Any errors are the responsibility of the authors, and we would like to be notified when such errors are found. We have retained the interesting names of the patients in this edition. The use of such names is meant as an educational tool and is not meant to be disrespectful to the patients or their conditions. The accompanying website for this edition of Marks’ Basic Medical Biochemistry: A Clinical Approach contains the aforementioned additional questions for review, patient summaries (complete stories of all the patients discussed in the text), and the references for each chapter. v

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PREFACE TO THE FOURTH EDITION

HOW TO USE THIS BOOK Icons identify the various components of the book: the patients who are presented at the start of each chapter; the clinical notes, methods notes, questions, and answers that appear in the margins; and the key concepts, clinical comments, and biochemical comments that are found at the end of each chapter. Each chapter starts with an abstract that summarizes the information so that students can recognize the key words and concepts they are expected to learn. The next component of each chapter is “The Waiting Room,” describing patients with complaints and detailing the events that led them to seek medical help. indicates a female patient indicates a male patient indicates a patient who is an infant or young child As each chapter unfolds, icons appear in the margin, identifying information related to the material presented in the text: indicates a clinical note, usually related to the patients in “The Waiting Room” for that chapter. These notes explain signs or symptoms of a patient or give some other clinical information relevant to the text. indicates a methods note, which elaborates on how biochemistry is required to perform, and interpret, common laboratory tests. Questions and answers also appear in the margin and should help to keep students thinking as they read the text: indicates a question indicates the answer to the question. The answer to a question is always located on the next page. If two questions appear on one page, the answers are given in order on the next page. Each chapter ends with “Key Concepts,” “Clinical Comments,” and “Biochemical Comments”: The “Key Concepts” summarize the important take-home messages from the chapter. The “Clinical Comments” give additional clinical information, often describing the treatment plan and the outcome. The “Biochemical Comments” add biochemical information that is not covered in the text or explore some facet of biochemistry in more detail or from another angle. Finally, “Review Questions” are presented. These questions are written in a USMLE-like format and many of them have a clinical slant. Answers to the review questions, along with detailed explanations, are provided in the Appendix.

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PREFACE TO THE FOURTH EDITION

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ACKNOWLEDGMENTS The authors would like to thank Drs. Jerry Lingrel, Paul Rosevear, and Peter Stambrook for reviewing chapters as they were being revised. Their suggested revisions were thoughtful and improved the chapters they were reviewing. Matt Chansky, the illustrator, deserves a tremendous amount of credit for creating the new figures and revising existing figures. Stacey Sebring, our developmental editor, was very patient with the authors and was always there when needed. Her expertise and patience was greatly appreciated by the authors we worked through this revision. Susan Rhyner, our acquisitions editor, was also extremely supportive as the work was in progress. We would also like to acknowledge the initial contributions of Dawn Marks, whose vision of a textbook geared toward medical students led to the first edition of this book. Her vision is still applicable today.

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Contents SECTION ONE. Fuel Metabolism / 1 1 Metabolic Fuels and Dietary Components / 3 2 The Fed or Absorptive State / 21 3 Fasting / 30 SECTION TWO. Chemical and Biologic Foundations of Biochemistry / 39 4 Water, Acids, Bases, and Buffers / 41 5 Structures of the Major Compounds of the Body / 54 6 Amino Acids in Proteins / 70 7 Structure–Function Relationships in Proteins / 88 8 Enzymes as Catalysts / 112 9 Regulation of Enzymes / 135 10 Relationship between Cell Biology and Biochemistry / 152 11 Cell Signaling by Chemical Messengers / 171 SECTION THREE. Gene Expression and the Synthesis of Proteins / 191 12 Structure of the Nucleic Acids / 193 13 Synthesis of DNA / 209 14 Transcription: Synthesis of RNA / 227 15 Translation: Synthesis of Proteins / 248 16 Regulation of Gene Expression / 265 17 Use of Recombinant DNA Techniques in Medicine / 288 18 The Molecular Biology of Cancer / 310 SECTION FOUR. Fuel Oxidation and the Generation of Adenosine Triphosphate / 333 19 Cellular Bioenergetics: ATP and O2 / 336 20 Tricarboxylic Acid Cycle / 355 21 Oxidative Phosphorylation and Mitochondrial Function / 377 22 Generation of Adenosine Triphosphate from Glucose: Glycosis / 396 23 Oxidation of Fatty Acids and Ketone Bodies / 414 24 Oxygen Toxicity and Free Radical Injury / 437 25 Metabolism of Ethanol / 457

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CONTENTS

SECTION FIVE. Carbohydrate Metabolism / 473 26 Basic Concepts in the Regulation of Fuel Metabolism by Insulin, Glucagon, and Other Hormones / 477 27 Digestion, Absorption, and Transport of Carbohydrates / 493 28 Formation and Degradation of Glycogen / 512 29 Pathways of Sugar Metabolism: Pentose Phosphate Pathway, Fructose, and Galactose Metabolism / 529 30 Synthesis of Glycosides, Lactose, Glycoproteins, and Glycolipids / 544 31 Gluconeogenesis and Maintenance of Blood Glucose Levels / 559 SECTION SIX. Lipid Metabolism / 583 32 Digestion and Transport of Dietary Lipids / 585 33 Synthesis of Fatty Acids, Triacylglycerols, and the Major Membrane Lipids / 597 34 Cholesterol Absorption, Synthesis, Metabolism, and Fate / 626 35 Metabolism of the Eicosanoids / 663 36 Integration of Carbohydrate and Lipid Metabolism / 678 SECTION SEVEN. Nitrogen Metabolism / 693 37 Protein Digestion and Amino Acid Absorption / 696 38 Fate of Amino Acid Nitrogen: Urea Cycle / 707 39 Synthesis and Degradation of Amino Acids / 723 40 Tetrahydrofolate, Vitamin B12, and S-Adenosylmethionine / 744 41 Purine and Pyrimidine Metabolism / 759 42 Intertissue Relationships in the Metabolism of Amino Acids / 774 SECTION EIGHT. Tissue Metabolism / 795 43 Actions of Hormones That Regulate Fuel Metabolism / 797 44 The Biochemistry of Erythrocytes and Other Blood Cells / 823 45 Blood Plasma Proteins, Coagulation, and Fibrinolysis / 847 46 Liver Metabolism / 863 47 Metabolism of Muscle at Rest and during Exercise / 884 48 Metabolism of the Nervous System / 903 49 The Extracellular Matrix and Connective Tissue / 927 Appendix: Answers to Review Questions / 944 Index / 969

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Dietary components Fuels: Carbohydrate Fat Protein

Vitamins Minerals H2O

Xenobiotics Digestion absorption, transport Compounds in cells Fuel storage pathways

Biosynthetic pathways Body components

Fuel stores

Detoxification and waste disposal pathways

Waste products

CO2 H2O

O2 Fuel oxidative pathways

For example, a message that we have just had a meal, carried by the hormone insulin, signals adipose tissue to store fat. In the following section, we will provide an overview of various types of dietary components and examples of the pathways involved in using these components. We will describe the fuels in our diet, the compounds produced by their digestion, and the basic patterns of fuel metabolism in the tissues of our bodies. We will describe how these patterns change when we eat, when we fast for a short time, and when we starve for prolonged periods. Patients with medical problems that involve an inability to deal normally with fuels will be introduced. These patients will appear repeatedly throughout the book and will be joined by other patients as we delve deeper into biochemistry. In order for students to be able to remember the patients as they progress through the book, the patients have been given names that reflect their condition. This is not meant to demean the patient or the condition, but rather, to allow the student to better follow the progress of the patient throughout the book. It is important to note that this section of the book contains an overview of basic metabolism, which allows patients to be presented at an elementary level and to whet the student’s appetite for the biochemistry to come. Its goal is to enable the student to taste and preview what biochemistry is all about. It is not designed to be all-inclusive because all of these topics will be discussed in greater detail in Sections IV through VIII of the text. The next section of the text (Section II) begins with the basics of biochemistry and the relationship of basic chemistry to processes that occur in all living cells.

Energy

FIG. I.1. An overview of the general metabolic routes for dietary components in the body. The types of pathways are named in red.

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Metabolic Fuels and Dietary Components

Fuel Metabolism. We obtain our fuel primarily from carbohydrates, fats, and proteins in our diet. As we eat, our foodstuffs are digested and absorbed. The products of digestion circulate in the blood, enter various tissues, and are eventually taken up by cells and oxidized to produce energy. To completely convert our fuels to carbon dioxide (CO2) and water (H2O), molecular oxygen (O2) is required. We breathe to obtain this oxygen and to eliminate the carbon dioxide that is produced by the oxidation of our foodstuffs. Fuel Stores. Any dietary fuel that exceeds the body’s immediate energy needs is stored, mainly as triacylglycerol (fat) in adipose tissue; as glycogen (carbohydrate) in muscle, liver, and other cells; and, to some extent, as protein in muscle. When we are fasting, between meals and overnight while we sleep, fuel is drawn from these stores and is oxidized to provide energy (Fig. 1.1). Fuel Requirements. We require enough energy each day to drive the basic functions of our bodies and to support our physical activity. If we do not consume enough food each day to supply that much energy, the body’s fuel stores supply the remainder, and we lose weight. Conversely, if we consume more food than required for the energy we expend, our body’s fuel stores enlarge, and we gain weight. Other Dietary Requirements. In addition to providing energy, the diet provides precursors for the biosynthesis of compounds necessary for cellular and tissue structure, function, and survival. Among these precursors are the essential fatty acids and essential amino acids (those that the body needs but cannot synthesize). The diet must also supply vitamins, minerals, and water. Waste Disposal. Dietary components that we can use are referred to as nutrients. However, both the diet and the air we breathe contain xenobiotic compounds, compounds that have no use or value in the human body and may be toxic. These compounds are excreted in the urine and feces together with metabolic waste products.

Excess dietary fuel Fed Fuel stores: Fat Glycogen Protein Fasting Oxidation

Energy

FIG. 1.1. Fate of excess dietary fuel in fed and fasting states.

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SECTION ONE

Fuel Metabolism

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n order to survive, humans must meet two basic metabolic requirements: We must be able to synthesize everything our cells need that is not supplied by our diet, and we must be able to protect our internal environment from toxins and changing conditions in our external environment. In order to meet these requirements, we metabolize our dietary components through four basic types of pathways: fuel oxidative pathways, fuel storage and mobilization pathways, biosynthetic pathways, and detoxification or waste disposal pathways. Cooperation between tissues and responses to changes in our external environment are communicated through transport pathways and intercellular signaling pathways (Fig. I.1). The foods in our diet are the fuels that supply us with energy in the form of calories. This energy is used for carrying out diverse functions such as moving, thinking, and reproducing. Thus, several of our metabolic pathways are fuel oxidative pathways that convert fuels into energy that can be used for biosynthetic and mechanical work. But what is the source of energy when we are not eating, such as between meals and while we sleep? How does a person on a hunger strike that you read about in the morning headlines survive so long? We have other metabolic pathways that are fuel storage pathways. The fuels that we store can be mobilized during periods when we are not eating or when we need increased energy for exercise. Our diet also must contain the compounds we cannot synthesize, as well as all the basic building blocks for compounds we do synthesize in our biosynthetic pathways. For example, we have dietary requirements for some amino acids, but we can synthesize other amino acids from our fuels and a dietary nitrogen precursor. The compounds required in our diet for biosynthetic pathways include certain amino acids, vitamins, and essential fatty acids. Detoxification pathways and waste disposal pathways are metabolic pathways devoted to removing toxins that can be present in our diets or in the air we breathe, introduced into our bodies as drugs, or generated internally from the metabolism of dietary components. Dietary components that have no value to the body and must be disposed of are called xenobiotics. In general, biosynthetic pathways (including fuel storage) are referred to as anabolic pathways, that is, pathways that synthesize larger molecules from smaller components. The synthesis of proteins from amino acids is an example of an anabolic pathway. Catabolic pathways are those pathways that break down larger molecules into smaller components. Fuel oxidative pathways are examples of catabolic pathways. In the human, the need for different cells to carry out different functions has resulted in cell and tissue specialization in metabolism. For example, our adipose tissue is a specialized site for the storage of fat and contains the metabolic pathways that allow it to carry out this function. However, adipose tissue is lacking many of the pathways that synthesize required compounds from dietary precursors. To enable our cells to cooperate in meeting our metabolic needs during changing conditions of diet, sleep, activity, and health, we need transport pathways into the blood and between tissues and intercellular signaling pathways. One means of communication is for hormones to carry signals to tissues about our dietary state.

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SECTION I ■ FUEL METABOLISM

THE WAITING ROOM Percy Veere is a 59-year-old school teacher who was in good health until his wife died suddenly. Since that time, he has experienced an increasing degree of fatigue and has lost interest in many of the activities he previously enjoyed. Shortly after his wife’s death, one of his married children moved far from home. Since then, Mr. Veere has had little appetite for food. When a neighbor found Mr. Veere sleeping in his clothes, unkempt, and somewhat confused, she called an ambulance. Mr. Veere was admitted to the hospital psychiatric unit with a diagnosis of mental depression associated with dehydration and malnutrition.

Heat

ATP

CO 2 Energy production via oxidation of Carbohydrate Lipid Protein

O2

Otto Shape is a 25-year-old medical student who was very athletic during high school and college but is now “out of shape.” Since he started medical school, he has been gaining weight (at 5 ft 10 in tall, he currently weighs 187 lb). He has decided to consult a physician at the student health service before the problem becomes worse. Energy utilization Biosynthesis Detoxification Muscle contraction Active ion transport Thermogenesis

ADP + Pi

FIG. 1.2. The ATP–ADP cycle. The energygenerating pathways are shown in red; the energy-utilizing pathways in blue.

Fatty acids Glucose

Amino acids e–

e–

e– Acetyl CoA TCA cycle CO2 CO2 e– Electron-

ATP

transport chain H2O

O2

FIG. 1.3. Generation of adenosine ATP from fuel components during respiration. Glucose, fatty acids, and amino acids are oxidized to acetyl-CoA, a substrate for the tricarboxylic acid (TCA) cycle. In the TCA cycle, they are completely oxidized to CO2. As fuels are oxidized, electrons (e⫺) are transferred to O2 by the electron-transport chain, and the energy is used to generate ATP.

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Ivan Applebod is a 56-year-old accountant who has been morbidly obese for several years. He exhibits a pattern of central obesity, referred to as an “apple shape,” which is caused by excess adipose tissue being disproportionally deposited in the abdominal area. His major recreational activities are watching TV while drinking scotch and soda and doing occasional gardening. At a company picnic, he became very “winded” while playing baseball and decided it was time for a general physical examination. At the examination, he weighed 264 lb at 5 ft 10 in tall. His blood pressure was elevated, 155 mm Hg systolic (normal ⫽ 120 mm Hg or less, with prehypertensive defined as between 121 and 139), and 95 mm Hg diastolic (normal ⫽ 80 mm Hg or less, with prehypertensive defined as between 81 and 89). Ann O’Rexia is a 23-year-old buyer for a woman’s clothing store. Despite the fact that she is 5 ft 7 in tall and weighs 99 lb, she is convinced she is overweight. Two months ago, she started a daily exercise program that consists of 1 hour of jogging every morning and 1 hour of walking every evening. She also decided to consult a physician about a weight reduction diet.

I.

DIETARY FUELS

The major fuels we obtain from our diet are carbohydrates, proteins, and fats. When these fuels are oxidized to CO2 and H2O in our cells, energy is released by the transfer of electrons to O2. The energy from this oxidation process generates heat and adenosine triphosphate (ATP) (Fig. 1.2). Carbon dioxide travels in the blood to the lungs, where it is expired, and water is excreted in urine, sweat, and other secretions. Although the heat that is generated by fuel oxidation is used to maintain body temperature, the main purpose of fuel oxidation is to generate ATP. ATP provides the energy that drives most of the energy-consuming processes in the cell, including biosynthetic reactions, muscle contraction, and active transport across membranes. As these processes use energy, ATP is converted back to adenosine diphosphate (ADP) and inorganic phosphate (Pi). The generation and utilization of ATP is referred to as the ATP–ADP cycle. The oxidation of fuels to generate ATP is called respiration (Fig. 1.3). Before oxidation, carbohydrates are converted principally to glucose, fat to fatty acids, and protein to amino acids. The pathways for oxidizing glucose, fatty acids, and

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CHAPTER 1 ■ METABOLIC FUELS AND DIETARY COMPONENTS

CH2OH O

CH2OH O O

OH

OH

HO CH2OH O

HO CH2OH O

O

OH

O

CH2 O O

OH

HO

HO Starch (diet)

or

O

OH HO

Glycogen (body stores)

CH2OH O C H H H C C H OH HO OH C C H OH Glucose

FIG. 1.4. Structure of starch and glycogen. Starch, our major dietary carbohydrate, and glycogen, the body’s storage form of glucose, have similar structures. They are polysaccharides (many sugar units) composed of glucose, which is a monosaccharide (one sugar unit). Dietary disaccharides are composed of two sugar units.

amino acids have many features in common. They first oxidize the fuels to acetyl coenzyme A (acetyl-CoA), a precursor of the tricarboxylic acid (TCA) cycle. The TCA cycle is a series of reactions that completes the oxidation of fuels to CO2 (see Chapter 20). Electrons lost from the fuels during oxidative reactions are transferred to O2 by a series of proteins in the electron-transport chain (see Chapter 21). The energy of electron transfer is used to convert ADP and Pi to ATP by a process known as oxidative phosphorylation. Oxidative pathways are catabolic; that is, these pathways break molecules down. In contrast, anabolic pathways build molecules up from component pieces. In discussions of metabolism and nutrition, energy is often expressed in units of calories. “Calorie” in this context really means kilocalorie (kcal). Calorie was originally spelled with a capital C, but the capitalization was dropped as the term became popular. Thus, a 1-cal soft drink actually has 1 cal (1 kcal) of energy. Energy is also expressed in joules. One kilocalorie equals 4.18 kilojoules (kJ). Physicians tend to use units of calories, in part, because that is what their patients use and understand. One kilocalorie of energy is the amount of energy required to raise the temperature of 1 L of water by 1°C.

A. Carbohydrates The major carbohydrates in the human diet are starch, sucrose, lactose, fructose, and glucose. The polysaccharide starch is the storage form of carbohydrates in plants. Sucrose (table sugar) and lactose (milk sugar) are disaccharides, and fructose and glucose are monosaccharides. Digestion converts the larger carbohydrates to monosaccharides, which can be absorbed into the bloodstream. Glucose, a monosaccharide, is the predominant sugar in human blood (Fig. 1.4). Oxidation of carbohydrates to CO2 and H2O in the body produces approximately 4 kcal/g (Table 1.1). In other words, every gram of carbohydrate we eat yields approximately 4 kcal of energy. Note that carbohydrate molecules contain a significant amount of oxygen and are already partially oxidized before they enter our bodies (see Fig. 1.4).

B. Proteins Proteins are composed of amino acids that are joined to form linear chains (Fig. 1.5). In addition to carbon, hydrogen, and oxygen, proteins contain approximately 16%

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Table 1.1

Caloric Content of Fuels kcal/g

Carbohydrate Fat Protein Alcohol

4 9 4 7

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SECTION I ■ FUEL METABOLISM

Peptide bonds R1 O

N C

C

H H

H N

C

O

R

R3 O +

H3N

C N C C

H R 2

C COO– H

H H

Protein

Amino acid

FIG. 1.5. General structure of proteins and amino acids. Each amino acid in this figure is indicated by a different color. R ⫽ side chain. Different amino acids have different side chains. For example, R1 might be –CH3; R2, –CH2OH; R3, –CH2–COO⫺. In a protein, the amino acids are linked by peptide bonds.

nitrogen by weight. The digestive process breaks down proteins to their constituent amino acids, which enter the blood. The complete oxidation of proteins to CO2, H2O, and ammonium (NH4⫹) in the body yields approximately 4 kcal/g.

C. Fats

An analysis of Ann O’Rexia’s diet showed she ate 100 g of carbohydrate, 20 g of protein, and 15 g of fat each day. Approximately how many calories did she consume per day?

Fats are lipids composed of triacylglycerols (also called triglycerides). A triacylglycerol molecule contains three fatty acids esterified to one glycerol moiety (Fig. 1.6). Fats contain much less oxygen than is contained in carbohydrates or proteins. Therefore, fats are more reduced and yield more energy when oxidized. The complete oxidation of triacylglycerols to CO2 and H2O in the body releases approximately 9 kcal/g, more than twice the energy yield from an equivalent amount of carbohydrate or protein.

O O CH3

(CH2)7

CH

CH

(CH2)7

C

CH2

O

CH2

C

(CH2)14 CH3

O

CH

O

O

C

(CH2)16 CH3

Triacylglycerol

CH2 OH HO

C H

O CH3

CH2OH

(CH2)14 C

O–

Palmitate

Glycerol O CH3 (CH2)7

CH

CH

(CH2)7

C

O–

Oleate O CH3

(CH2)16

C

O–

Stearate

FIG. 1.6. Structure of a triacylglycerol. Palmitate and stearate are saturated fatty acids (i.e., they have no double bonds). Oleate is monounsaturated (one double bond). Polyunsaturated fatty acids have more than one double bond.

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CHAPTER 1 ■ METABOLIC FUELS AND DIETARY COMPONENTS

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D. Alcohol

Miss O’Rexia consumed

Many people used to believe that alcohol (ethanol, in the context of the diet) has no caloric content. In fact, ethanol (CH3–CH2–OH) is oxidized to CO2 and H2O in the body and yields approximately 7 kcal/g, which is more than carbohydrate or protein but less than fat.

100 ⫻ 4 ⫽ 400 kcal as carbohydrate 20 ⫻ 4 ⫽ 80 kcal as protein 15 ⫻ 9 ⫽ 135 kcal as fat for a total of 615 kcal/day.

II. BODY FUEL STORES Although some of us may try, it is virtually impossible to eat constantly. Fortunately, we carry supplies of fuel within our bodies (Table 1.2), which are similar to the fuel supplies in the plants and animals we eat. These fuel stores are light in weight, large in quantity, and readily converted into oxidizable substances. Most of us are familiar with fat, our major fuel store, which is located in adipose tissue. Although fat is distributed throughout our bodies, it tends to increase in quantity in our hips and thighs and in our abdomens as we advance into middle age. In addition to our fat stores, we also have important, although much smaller, stores of carbohydrate in the form of glycogen located primarily in our liver and muscles. Glycogen consists of glucose residues joined together to form a large, branched polysaccharide (see Fig. 1.4). Body protein, particularly the protein of our large muscle masses, also serves to some extent as a fuel store, and we draw on it for energy when we fast.

Ivan Applebod ate 585 g of carbohydrate, 150 g of protein, and 95 g of fat each day. In addition, he drank 45 g of alcohol daily. How many calories did he consume per day?

A. Fat Our major fuel store is adipose triacylglycerol (triglyceride), a lipid more commonly known as fat. The average 70-kg man has approximately 15 kg of stored triacylglycerol, which accounts for approximately 85% of his total stored calories (see Table 1.2). Two characteristics make adipose triacylglycerol a very efficient fuel store: the fact that triacylglycerol contains more calories per gram than carbohydrate or protein (9 kcal/g vs. 4 kcal/g) and the fact that adipose tissue does not contain much water. Adipose tissue contains only about 15% water, compared to tissues such as muscle that contain about 80%. Thus, the 70-kg man with 15 kg of stored triacylglycerol has only about 18 kg of adipose tissue.

B. Glycogen Our stores of glycogen in liver, muscle, and other cells are relatively small in quantity but perform important functions. Liver glycogen is used to maintain blood glucose levels between meals, which is necessary for optimal functioning of the nervous system. Thus, the size of this glycogen store fluctuates during the day; an average 70-kg man might have 200 g or more of liver glycogen after a meal but only 80 g after an overnight fast. Muscle glycogen supplies energy for muscle contraction during exercise. At rest, the 70-kg man has approximately 150 g of muscle glycogen. Almost all cells, including neurons, maintain a small emergency supply of glucose as glycogen. It is not practical to store all of the energy in triacylglycerol as glycogen. Consider what would happen to a 70-kg man if the 135,000 kcal stored as triacylglycerols in his 18 kg of adipose tissue was stored instead as skeletal muscle glycogen. It would take approximately 34 kg of glycogen to store as many calories. Glycogen, because Table 1.2 Fuel Composition of the Average 70-kg Mana after an Overnight Fast Fuel Glycogen Muscle Liver Protein Triglyceride

Amount (kg)

Percentage of Total Stored Calories

0.15 0.08 6.0 15.0

0.4 0.2 14.4 85.0

a

In biochemistry and nutrition, the standard reference is often the 70-kg (154-lb) man. This standard was probably chosen because in the first half of the 20th century, when many nutritional studies were performed, young healthy medical and graduate students (who were mostly men) volunteered to serve as subjects for these experiments.

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Mr. Applebod consumed 585 ⫻ 4 ⫽ 2,340 kcal as carbohydrate 150 ⫻ 4 ⫽ 600 kcal as protein 95 ⫻ 9 ⫽ 855 kcal as fat 45 ⫻ 7 ⫽ 315 kcal as alcohol for a total of 4,110 kcal/day.

it is a polar molecule with hydroxyl groups, binds approximately four times its weight as water, or 136 kg. Thus, his fuel stores would weigh 170 kg, as opposed to 18 kg when stored as triacylglycerol.

C. Protein Protein serves many important roles in the body; unlike fat and glycogen, it is not solely a fuel store. Muscle protein is essential for body movement. Other proteins serve as enzymes (catalysts of biochemical reactions) or as structural components of cells and tissues. Only a limited amount of body protein can be degraded, approximately 6 kg in the average 70-kg man, before our body functions are compromised.

III. DAILY ENERGY EXPENDITURE If we want to stay in energy balance, neither gaining nor losing weight, we must, on average, consume an amount of food equal to our daily energy expenditure (DEE). The DEE includes the energy to support our basal metabolism (basal metabolic rate [BMR] or resting metabolic rate [RMR]) and our physical activity, plus the energy required to process the food we eat (diet-induced thermogenesis [DIT]). Thus, the DEE, in kilocalories per day ⫽ RMR ⫹ the energy needed for physical activity ⫹ DIT.

A. Resting Metabolic Rate

What are Ivan Applebod’s and Ann O’Rexia’s RMR? (Compare the method for a rough estimate to values obtained with equations in Table 1.4.)

Table 1.3 Factors Affecting BMR Expressed per Kilogram (kg) of Body Weight Gender (males higher than females) Body temperature (increased with fever) Environmental temperature (increased in cold) Thyroid status (increased in hyperthyroidism) Pregnancy and lactation (increased) Age (decreases with age)

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The resting metabolic rate is a measure of the energy required to maintain life: the functioning of the lungs, kidneys, and brain; the pumping of the heart; the maintenance of ionic gradients across membranes; the reactions of biochemical pathways; and so forth. Another term used to describe basal metabolism is the basal metabolic rate. The BMR was originally defined as the energy expenditure of a person mentally and bodily at rest in a thermoneutral environment 12 to 18 hours after a meal. However, when a person is awakened and his or her heat production or oxygen consumption is measured, he or she is no longer sleeping or totally at mental rest, and his or her metabolic rate is called the resting metabolic rate. It is also sometimes called the resting energy expenditure (REE). The RMR and BMR differ very little in value. The BMR, which is usually expressed in kilocalories per day, is affected by body size, age, sex, and other factors (Table 1.3). It is proportional to the amount of metabolically active tissue (including the major organs) and to the lean (or fat-free) body mass. Obviously, the amount of energy required for basal functions in a large person is greater than the amount required in a small person. However, the BMR is usually lower for women than for men of the same weight because women usually have more metabolically inactive adipose tissue. Body temperature also affects the BMR, which increases by 12% with each degree centigrade increase in body temperature (i.e., “feed a fever; starve a cold”). The ambient temperature affects the BMR, which increases slightly in colder climates as thermogenesis is activated. Excessive secretion of thyroid hormone (hyperthyroidism) causes the BMR to increase, whereas diminished secretion (hypothyroidism) causes it to decrease. The BMR increases during pregnancy and lactation. Growing children have a higher BMR per kilogram body weight than adults because a greater proportion of their bodies is composed of brain, muscle, and other more metabolically active tissues. The BMR declines in aging individuals because their metabolically active tissue is shrinking and body fat is increasing. In addition, large variations exist in BMR from one adult to another, determined by genetic factors. A rough estimate of the BMR may be obtained by assuming it is 24 kcal/day/kg body weight and multiplying by the body weight. An easy way to remember this is 1 kcal/kg/hr. This estimate works best for young individuals who are near their ideal weight. More accurate methods for calculating the BMR use empirically derived equations for different gender and age groups (Table 1.4). Even these calculations do not take into account variation among individuals.

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Table 1.4 Equation for Predicting BMR from Body Weight (W) in Kilograms (kg) Males

Females

Age Range (years)

BMR (kcal/day)

Age Range (years)

BMR (kcal/day)

0–3 3–10 10–18 18–30 30–60 ⬎60

60.9 W ⫺ 54 22.7 W ⫹ 495 17.5 W ⫹ 651 15.3 W ⫹ 679 11.6 W ⫹ 879 13.5 W ⫹ 487

0–3 3–10 10–18 18–30 30–60 ⬎60

61.0 W ⫺ 51 22.5 W ⫹ 499 12.2 W ⫹ 746 14.7 W ⫹ 496 8.7 W ⫹ 829 10.5 W ⫹ 596

From World Health Organization. Energy and Protein Requirements: Report of a Joint FAO/WHO/UNU Expert Consultation. Geneva, Switzerland: World Health Organization; 1987:71. Technical report series no. 724. See also Schofield WN. Predicting basal metabolic rate, new standards and review of previous work. Hum Nutr Clin Nutr. 1985;39(suppl 1):5–41.

9

Mr. Applebod weighs 264 lb or 120 kg (264 lb divided by 2.2 lb/kg). His estimated RMR ⫽ 24 kcal/kg/day ⫻ 120 kg ⫽ 2,880 kcal/day. His RMR calculated from Table 1.4 is only 2,271 kcal (11.6 W ⫹ 879 ⫽ (11.6 ⫻ 120) ⫹ 879). Miss O’Rexia weighs 99 lb or 45 kg (99 lb divided by 2.2 lb/kg). Her estimated RMR ⫽ 24 kcal/kg/day ⫻ 45 kg ⫽ 1,080 kcal/day. Her RMR from Table 1.4 is very close to this value (14.7 W ⫹ 496 ⫽ 1,157 kcal/ day). Thus, the rough estimate does not work well for obese patients because a disproportionately larger proportion of their body weight is relatively inactive metabolically.

B. Physical Activity In addition to the RMR, the energy required for physical activity contributes to the DEE. The difference in physical activity between a student and a lumberjack is enormous, and a student who is relatively sedentary during the week may be much more active during the weekend. Table 1.5 gives factors for calculating the approximate energy expenditures associated with typical activities. A rough estimate of the energy required per day for physical activity can be made by using a value of 30% of the RMR (per day) for a very sedentary person (such as a medical student who does little but study) and a value of 60% to 70% of the RMR (per day) for a person who engages in about 2 hours of moderate exercise per day (see Table 1.5). A value of 100% or more of the RMR is used for a person who does several hours of heavy exercise per day.

C. Diet-Induced Thermogenesis Our DEE includes a component related to the intake of food known as diet-induced thermogenesis or the thermic effect of food (TEF). DIT was formerly called the specific dynamic action (SDA). After the ingestion of food, our metabolic rate increases because energy is required to digest, absorb, distribute, and store nutrients. The energy required to process the types and quantities of food in the typical American diet is probably equal to approximately 10% of the kilocalories ingested. This amount is roughly equivalent to the error involved in rounding off the caloric content of carbohydrate, fat, and protein to 4, 9, and 4, respectively. Therefore,

Table 1.5

Typical Activities with Corresponding Hourly Activity Factorsa

Activity Category Resting: sleeping, reclining Very light: seated and standing activities, driving, laboratory work, typing, sewing, ironing, cooking, playing cards, playing a musical instrument Light: walking on a level surface at 2.5–3 mph, garage work, electrical trades, carpentry, restaurant trades, house cleaning, golf, sailing, table tennis Moderate: walking 3.5–4 mph, weeding and hoeing, carrying loads, cycling, skiing, tennis, dancing Heavy: walking uphill with a load, tree felling, heavy manual digging, mountain climbing, basketball, football, soccer a

Hourly Activity Factor (for Time in Activity) 1.0 1.5 2.5

5.0 7.0

The hourly activity factor is multiplied by the BMR (RMR) per hour times the number of hours engaged in the activity to give the caloric expenditure for that activity. If this is done for all of the hours in a day, the sum over 24 hours will approximately equal the daily energy expenditure. Reprinted with permission from National Research Council. Recommended Dietary Allowances. 10th ed. Washington, DC: National Academy Press; 1989.

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Registered dieticians use extensive tables for calculating energy requirements, based on height, weight, age, and activity level. A more accurate calculation is based on the fat-free mass (FFM), which is equal to the total body mass minus the mass of the person’s adipose tissue. With FFM, the BMR is calculated using the equation BMR ⫽ 186 ⫹ FFM ⫻ 23.6 kcal/kg/day. This formula eliminates differences between sexes and between aged versus young individuals that are attributable to differences in relative adiposity. However, determining FFM is relatively cumbersome— one technique requires weighing the patient under water and measuring the residual lung volume. Indirect calorimetry, a technique that measures O2 consumption and CO2 production, can be used when more accurate determinations are required for hospitalized patients. A portable indirect calorimeter is used to measure oxygen consumption and the respiratory quotient (RQ), which is the ratio of O2 consumed to CO2 produced. The RQ is 1.00 for individuals oxidizing carbohydrates, 0.83 for protein, and 0.71 for fat. From these values, the DEE can be determined. A simplified method to measure the DEE also uses indirect calorimetry, but only measures oxygen production. Because the oxidation of nutrients requires molecular oxygen, through the measurement of the volume of total inspired and expired air, and the amount of oxygen in that air, a very good estimate of the BMR can be determined. The device required for this method is less cumbersome than the device that measures both oxygen and carbon dioxide consumption, and it is easier to use.

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Based on the activities listed in Table 1.5, the average US citizen is rather sedentary. Sedentary habits correlate strongly with risk for cardiovascular disease, so it is not surprising that cardiovascular disease is the major cause of death in this country. What are reasonable estimates for Ivan Applebod’s and Ann O’Rexia’s DEE?

DIT is often ignored and calculations are based simply on the RMR and the energy required for physical activity.

D. Calculations of Daily Energy Expenditure The total DEE is usually calculated as the sum of the RMR (in kilocalories per day) plus the energy required for the amount of time spent in each of the various types of physical activity (see Table 1.5). An approximate value for the DEE can be determined from the RMR and the appropriate percentage of the RMR required for physical activity (given previously). For example, a very sedentary medical student would have a DEE equal to the RMR plus 30% of the RMR (or 1.3 ⫻ RMR), and an active person’s daily expenditure could be two times the RMR.

E. Healthy Body Weight Ideally, we should strive to maintain a weight consistent with good health. Overweight people are frequently defined as more than 20% above their ideal weight. But what is the ideal weight? The body mass index (BMI), calculated as weight per height2 (kg/m2), is currently the preferred method for determining whether a person’s weight is in the healthy range. This formula, in the English system, is (weight [in pounds] ⫻ 704)/height2 (with height in inches). In general, adults with BMI values below 18.5 are considered underweight. Those with BMIs between 18.5 and 24.9 are considered to be in the healthy weight range, between 25 and 29.9 are in the overweight or preobese range, and above 30 are in the obese range. Morbid obesity is defined as a BMI of 40 or greater.

F. Weight Gain and Loss

Are Ivan Applebod and Ann O’Rexia in a healthy weight range?

To maintain our body weight, we must stay in caloric balance. We are in caloric balance if the kilocalories in the food we eat equal our DEE. If we eat less food than we require for our DEE, our body fuel stores supply the additional calories, and we lose weight. Conversely, if we eat more food than we require for our energy needs, the excess fuel is stored (mainly in our adipose tissue), and we gain weight. When we draw on our adipose tissue to meet our energy needs, we lose approximately 1 lb whenever we expend approximately 3,500 kcal more than we consume. In other words, if we eat 1,000 kcal less than we expend per day, we will lose about 2 lb/week. Because the average individual’s food intake is only about 2,000 to 3,000 kcal/day, eating one-third to one-half the normal amount will cause a person to lose weight rather slowly. Fad diets that promise a loss of weight much more rapid than this have no scientific merit. In fact, the rapid initial weight loss the fad dieter typically experiences is attributable largely to loss of body water. This loss of water occurs in part because muscle tissue protein and liver glycogen are degraded rapidly to supply energy during the early phase of the diet. When muscle tissue (which is approximately 80% water) and glycogen (approximately 70% water) are broken down, this water is excreted from the body.

IV. DIETARY REQUIREMENTS In addition to supplying us with fuel and with general-purpose building blocks for biosynthesis, our diet also provides us with specific nutrients that we need to remain healthy. We must have a regular supply of vitamins and minerals and of the essential fatty acids and essential amino acids. “Essential” means that they are essential in the diet; the body cannot synthesize these compounds from other molecules and therefore must obtain them from the diet. Nutrients that the body requires in the diet only under certain conditions are called conditionally essential. The Recommended Dietary Allowance (RDA) and the Adequate Intake (AI) provide quantitative estimates of nutrient requirements. The RDA for a nutrient is the average daily dietary intake level necessary to meet the requirement of nearly all (97% to 98%) healthy individuals in a particular gender and life stage group.

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Life stage group is a certain age range or physiologic status (i.e., pregnancy or lactation). The RDA is intended to serve as a goal for intake by individuals. The AI is a recommended intake value that is used when not enough data are available to establish an RDA.

A. Carbohydrates No specific carbohydrates have been identified as dietary requirements. Carbohydrates can be synthesized from amino acids, and we can convert one type of carbohydrate to another. However, health problems are associated with the complete elimination of carbohydrate from the diet, partly because a lowcarbohydrate diet must contain higher amounts of fat to provide us with the energy we need. High-fat diets are associated with obesity, atherosclerosis, and other health problems.

11

Mr. Applebod’s BMR is 2,271 kcal/ day. He is sedentary, so he only requires approximately 30% more calories for his physical activity. Therefore, his daily expenditure is approximately 2,271 ⫹ (0.3 ⫻ 2,271) or 1.3 ⫻ 2,271 or 2,952 kcal/day. Miss O’Rexia’s BMR is 1,157 kcal/day. She performs 2 hours of moderate exercise per day (jogging and walking), so she requires approximately 65% more calories for her physical activity. Therefore, her daily expenditure is approximately 1,157 ⫹ (0.65 ⫻ 1,157) or 1.65 ⫻ 1,157 or 1,909 kcal/day.

B. Essential Fatty Acids Although most lipids required for cell structure, fuel storage, or hormone synthesis can be synthesized from carbohydrates or proteins, we need a minimal level of certain dietary lipids for optimal health. These lipids, known as essential fatty acids, are required in our diet because we cannot synthesize fatty acids with these particular arrangements of double bonds. The essential fatty acids, ␣-linoleic and ␣-linolenic acid, are supplied by dietary plant oils, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are supplied in fish oils. They are the precursors of the eicosanoids (a set of hormone-like molecules that are secreted by cells in small quantities and have numerous important effects on neighboring cells). The eicosanoids include the prostaglandins, thromboxanes, leukotrienes, and other related compounds.

Ivan Applebod’s weight is classified as obese. His BMI is (264 lb ⫻ 704)/(70 in)2 ⫽ 37.9. Ann O’Rexia is underweight. Her BMI is (99 lb ⫻ 704)/(67 in)2 ⫽ 15.5.

C. Protein The RDA for protein is approximately 0.8 g high-quality protein per kilogram of ideal body weight, or approximately 60 g/day for men and 50 g/day for women. “High-quality” protein contains all of the essential amino acids in adequate amounts. Proteins of animal origin (milk, egg, and meat proteins) are high quality. The proteins in plant foods are generally of lower quality, which means they are low in one or more of the essential amino acids. Vegetarians may obtain adequate amounts of the essential amino acids by eating mixtures of vegetables that complement each other in terms of their amino acid composition. 1.

ESSENTIAL AMINO ACIDS

Different amino acids are used in the body as precursors for the synthesis of proteins and other nitrogen-containing compounds. Of the 20 amino acids commonly required in the body for synthesis of protein and other compounds, 9 amino acids are essential in the diet of an adult human because they cannot be synthesized in the body. These are lysine, isoleucine, leucine, threonine, valine, tryptophan, phenylalanine, methionine, and histidine. Certain amino acids are conditionally essential, that is, required in the diet only under certain conditions. Children and pregnant women have a high rate of protein synthesis to support growth and require some arginine in the diet, although it can be synthesized in the body. Histidine is essential in the diet of the adult in very small quantities because adults efficiently recycle histidine. The increased requirement of children and pregnant women for histidine is therefore much larger than their increased requirement of other essential amino acids. Tyrosine and cysteine are considered conditionally essential. Tyrosine is synthesized from phenylalanine, and it is required in the diet if phenylalanine intake is inadequate, or if an individual is congenitally deficient in an enzyme required to convert phenylalanine to tyrosine (the congenital disease phenylketonuria). Cysteine is synthesized by using sulfur from methionine, and it also may be required in the diet under certain conditions.

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To evaluate a patient’s weight, physicians need standards of obesity applicable in a genetically heterogeneous population. Life insurance industry statistics have been used to develop tables giving the weight ranges, based on gender, height, and body frame size, that are associated with the greatest longevity, such as the Metropolitan Height and Weight Tables. However, these tables are considered inadequate for several reasons (e.g., they reflect data from upper-middle-class white groups). The BMI is the classification that is currently used clinically. It is based on two simple measurements, height without shoes and weight with minimal clothing. Height* 6'6" 6'5" 6'4" 6'3" 6'2" 6'1" 6'0" 5'11" 5'10" 5'9" 5'8" 5'7" 5'6" 5'5" 5'4" 5'3" 5'2" 5'1" 5'0" 4'11" 4'10" 50

BMI (body mass index) 25

18.5

75

100

125

150 Pounds

175

200

225

* Without shoes

250

30

2.

NITROGEN BALANCE

The proteins in the body undergo constant turnover; that is, they are constantly being degraded to amino acids and resynthesized. When a protein is degraded, its amino acids are released into the pool of free amino acids in the body. The amino acids from dietary proteins also enter this pool. Free amino acids can have one of three fates: They are used to make proteins; they serve as precursors for the synthesis of essential nitrogen-containing compounds (e.g., heme, DNA, RNA); or they are oxidized as fuel to yield energy. When amino acids are oxidized, their nitrogen atoms are excreted in the urine principally in the form of urea. The urine also contains smaller amounts of other nitrogenous excretory products (uric acid, creatinine, and ammonium ion) derived from the degradation of amino acids and compounds synthesized from amino acids. Some nitrogen is also lost in sweat, feces, and cells that slough off. Nitrogen balance is the difference between the amount of nitrogen taken into the body each day (mainly in the form of dietary protein) and the amount of nitrogen in compounds lost (Table 1.6). If more nitrogen is ingested than excreted, a person is said to be in positive nitrogen balance. Positive nitrogen balance occurs in growing individuals (e.g., children, adolescents, pregnant women) who are synthesizing more protein than they are breaking down. Conversely, if less nitrogen is ingested than excreted, a person is said to be in negative nitrogen balance. A negative nitrogen balance develops in a person who is eating either too little protein or protein that is deficient in one or more of the essential amino acids. Amino acids are continuously being mobilized from body proteins. If the diet is lacking an essential amino acid or if the intake of protein is too low, new protein cannot be synthesized, and the unused amino acids will be degraded, with the nitrogen appearing in the urine. If a negative nitrogen balance persists for too long, bodily function will be impaired by the net loss of critical proteins. In contrast, healthy adults are in nitrogen balance (neither positive nor negative), and the amount of nitrogen consumed in the diet equals its loss in urine, sweat, feces, and other excretions.

275

Without clothes

Patients can be shown their BMI in a nomogram and need not use calculations. The healthy weight range coincides with the mortality data derived from life insurance tables. The BMI also shows a good correlation with independent measures of body fat. The major weakness of the use of the BMI is that some very muscular individuals may be classified as obese when they are not. Other measurements to estimate body fat and other body compartments, such as weighing individuals under water, are more difficult, expensive, and time consuming and have generally been confined to research purposes. If patients are above or below ideal weight (such as Ivan Applebod or Ann O’Rexia), the physician, often in consultation with a registered dietician, prescribes a diet designed to bring the weight into the ideal range.

D. Vitamins Vitamins (Latin vita, life) are a diverse group of organic molecules required in very small quantities in the diet for health, growth, and survival. The absence of a vitamin from the diet or an inadequate intake results in characteristic deficiency signs and, ultimately, death. Table 1.7 lists the signs and symptoms of deficiency for each vitamin, its RDA or AI for young adults, and common food sources. The amount of each vitamin required in the diet is small (in the microgram or milligram range) compared with essential amino acid requirements (in the gram range). The vitamins are often divided into two classes: water-soluble vitamins and fat-soluble vitamins. This classification has little relationship to their function but is related to the absorption and transport of fat-soluble vitamins with lipids. Most vitamins are used for the synthesis of coenzymes, complex organic molecules that assist enzymes in catalyzing biochemical reactions, and the deficiency symptoms reflect an inability of cells to carry out certain reactions. However, some vitamins also act as hormones. We will consider the roles played by individual vitamins as we progress through the subsequent chapters of this text. Although the RDA or AI for each vitamin varies with age and sex, the difference is usually not very large once adolescence is reached. For example, the RDA for riboflavin is 0.9 mg/day for males between 9 and 13 years of age, 1.3 mg/day Table 1.6

Nitrogen Balancea

Positive nitrogen balance Nitrogen balance Negative nitrogen balance

Growth (e.g., childhood, pregnancy) Normal healthy adult Dietary deficiency of total protein or amino acids: catabolic stress

Dietary N ⬎ Excreted N Dietary N ⫽ Excreted N Dietary N ⬍ Excreted N

a

N refers to nitrogen.

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Table 1.7

Vitamin

Water-soluble vitamins RDA Vitamin C F: 75 mg M: 90 mg UL: 2 g

Thiamin

RDA F: 1.1 mg M: 1.2 mg

Riboflavin

RDA F: 1.1 mg M: 1.3 mg

Niacina

RDA F: 14 mg NEQ M: 16 mg NEQ UL: 35 mg RDA F: 1.3 mg M: 1.3 mg UL: 100 mg

Vitamin B6 (pyridoxine)

Folate

RDA F: 400 ␮g M: 400 ␮g UL: 1,000 ␮g

Vitamin B12

RDA F: 2.4 ␮g M: 2.4 ␮g AI F: 30 ␮g M: 30 ␮g

Biotin

Pantothenic acid

AI F: 5 mg M: 5 mg

Choline

AI F: 425 mg M: 550 mg UL: 3.5 g

Fat-soluble vitamins RDA Vitamin A F: 700 ␮g M: 900 ␮g UL: 3,000 ␮g Vitamin K

Lieberman_CH01.indd 13

Are Ivan Applebod and Ann O’Rexia gaining or losing weight?

Vitamins Dietary Reference Intakes (DRI) Females (F) Males (M) Some Common (18–30 yrs old) Food Sources

RDA F: 90 ␮g M: 120 ␮g

Citrus fruits; potatoes; peppers, broccoli, spinach; strawberries Enriched cereals and breads; unrefined grains; pork; legumes, seeds, nuts Dairy products; fortified cereals; meats, poultry, fish; legumes

Meat: chicken, beef, fish; enriched cereals or whole grains; most foods

Consequences of Deficiency (Names of Deficiency Diseases Are in Bold) Scurvy: defective collagen formation leading to subcutaneous hemorrhage, aching bones, joints, and muscle in adults, rigid position and pain in infants Beriberi: (wet) edema; anorexia, weight loss; apathy, decrease in short-term memory, confusion; irritability; muscle weakness; an enlarged heart Ariboflavinosis: Sore throat, hyperemia, edema of oral mucosal membranes; cheilosis, angular stomatitis; glossitis, magenta tongue; seborrheic dermatitis; normochromic normocytic anemia Pellagra: Pigmented rash in areas exposed to sunlight; vomiting; constipation or diarrhea; bright red tongue; neurologic symptoms Seborrheic dermatitis; microcytic anemia; epileptiform convulsions; depression and confusion

Chicken, fish, pork; eggs; fortified cereals, unmilled rice, oats; starchy vegetables; noncitrus fruits; peanuts, walnuts Citrus fruits; dark Impaired cell division and green vegetables; growth; megaloblastic anefortified ceremia; neural tube defects als and breads; legumes Megaloblastic anemia; neuroAnimal productsb logic symptoms Liver Egg yolk Wide distribution in foods, especially animal tissues; whole grain cereals; legumes Milk; liver; eggs; peanuts

13

Conjunctivitis; central nervous system abnormalities; glossitis; alopecia; dry, scaly dermatitis Irritability and restlessness; fatigue, apathy, malaise; gastrointestinal symptoms; neurologic symptoms

Malnutrition, the absence of an adequate intake of nutrients, occurs in the United States principally among children of families with incomes below the poverty level, the elderly, individuals whose diet is influenced by alcohol and drug usage, and those who make poor food choices. More than 13 million children in the United States live in families with incomes below the poverty level. Of these, approximately 10% have clinical malnutrition, most often anemia resulting from inadequate iron intake. A larger percentage have mild protein and energy malnutrition and exhibit growth retardation, sometimes as a result of parental neglect. Childhood malnutrition may also lead to learning failure and chronic illness later in life. A weight-for-age measurement is one of the best indicators of childhood malnourishment because it is easy to measure, and weight is one of the first parameters to change during malnutrition. The term kwashiorkor refers to a disease originally seen in African children suffering from a protein deficiency (although overall caloric intake may be normal). It is characterized by marked hypoalbuminemia (low levels of albumin in the blood), anemia, edema (buildup of fluids in the interstitial spaces), pot belly, loss of hair, and other signs of tissue injury. The term marasmus is used for prolonged protein and calorie malnutrition, particularly in young children. Children with marasmus usually do not develop edema. The term proteincalorie malnutrition can be used to describe both disorders.

Liver damage

Carrots; dark Night blindness; xerophgreen and leafy thalmia; keratinization of vegetables; sweet epithelium in GI, respiratory potatoes and and genitourinary tract, skin squash; broccoli becomes dry and scaly Green leafy vegDefective blood coagulation; etables; cabbage hemorrhagic anemia of the family (brassica); newborn bacterial flora of intestine (continued)

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Mr. Applebod expends about 2,952 kcal/day and consumes 4,110 kcal. By this calculation, he consumes 1,158 more kcal than he expends each day and is gaining weight. Miss O’Rexia expends 1,909 kcal/day while she consumes only 615 kcal. Therefore, she expends 1,294 more kcal/day than she consumes, so she is losing weight.

Table 1.7

Vitamin Vitamin D

Vitamin E

Multiple vitamin deficiencies accompanying malnutrition are far more common in the United States than the characteristic deficiency diseases associated with diets lacking just one vitamin, because we generally eat a variety of foods. The characteristic deficiency diseases arising from single vitamin deficiencies were often identified and described in humans through observations of populations consuming a restricted diet because that was all that was available. For example, thiamine deficiency was discovered by a physician in Java, who related the symptoms of beriberi to diets composed principally of polished rice. Today, single vitamin deficiencies usually occur as a result of conditions that interfere with the uptake or utilization of a vitamin or as a result of poor food choices or a lack of variety in the diet. For example, peripheral neuropathy associated with vitamin E deficiency can occur in children with fat malabsorption, and alcohol consumption can result in beriberi. Vegans, individuals who consume diets lacking all animal products, can develop deficiencies in vitamin B12.

Vitamins (continued) Dietary Reference Intakes (DRI) Females (F) Males (M) Some Common (18–30 yrs old) Food Sources AIc F: 5 ␮g M: 5 ␮g UL: 50 ␮g RDA F: 15 mg M: 15 mg UL: 1 g

Fortified milk; exposure of skin to sunlight

Consequences of Deficiency (Names of Deficiency Diseases Are in Bold) Rickets (in children); inadequate bone mineralization (osteomalacia)

Vegetable oils, Muscular dystrophy, neurologic margarine; wheat abnormalities germ; nuts; green leafy vegetables

RDA, Recommended Dietary Allowance; AI, Adequate Intake; UL, Tolerable Upper Intake Level. a NEQ ⫽ niacin equivalents. Niacin can be synthesized in the human from tryptophan, and this term takes into account a conversion factor for dietary tryptophan. b Vitamin B12 is found only in animal products. c Dietary requirement assumes the absence of sunlight. Information for this table is from Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001). Washington, DC: National Academy Press. This information can also be obtained via the Web, at http://fnic.nal.usda.gov/, and click on Dietary Guidance, then Dietary Reference Intakes, and then DRI tables.

for males 19 to 30 years of age, still 1.3 mg/day for males older than 70 years, and 1.1 mg/day for females aged 19 to 30 years. The largest requirements occur during lactation (1.6 mg/day). Vitamins, by definition, cannot be synthesized in the body, or are synthesized from a very specific dietary precursor in insufficient amounts. For example, we can synthesize the vitamin niacin from the essential amino acid tryptophan, but not in sufficient quantities to meet our needs. Niacin is therefore still classified as a vitamin. Excessive intake of many vitamins, both fat-soluble and water-soluble, may cause deleterious effects. For example, high doses of vitamin A, a fat-soluble vitamin, can cause desquamation of the skin and birth defects. High doses of vitamin C cause diarrhea and gastrointestinal disturbances. One of the Dietary Reference Intakes is the Tolerable Upper Intake Level (UL), which is the highest level of daily nutrient intake that is likely to pose no risk of adverse effects to almost all individuals in the general population. As intake increases above the UL, the risk of adverse effects increases. Table 1.7 includes the ULs for vitamins known to pose a risk at high levels. Intake above the UL occurs most often with dietary or pharmacologic supplements of single vitamins and not from foods.

E. Minerals Many minerals are required in the diet. They are generally divided into the classifications of electrolytes (inorganic ions that are dissolved in the fluid compartments of the body), minerals (required in relatively large quantities), trace minerals (required in smaller quantities), and ultratrace minerals (Table 1.8). Sodium (Na⫹), potassium (K⫹), and chloride (Cl⫺) are the major electrolytes (ions) in the body. They establish ion gradients across membranes, maintain water balance, and neutralize positive and negative charges on proteins and other molecules. Calcium and phosphorus serve as structural components of bones and teeth and are thus required in relatively large quantities. Calcium (Ca2⫹) plays many other roles in the body; for example, it is involved in hormone action and blood clotting. Phosphorus is required for the formation of ATP and of phosphorylated intermediates in metabolism. Magnesium activates many enzymes and also forms a complex

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Table 1.8

Minerals Required in the Diet

Electrolytes

Minerals

Trace Minerals

Ultratrace or Trace Mineralsa

Sodium Potassium Chloride

Calcium Phosphorus Magnesium Iron Sulfur

Iodine Selenium Copper Zinc

Manganese Fluoride Chromium Molybdenum

a

These minerals can be classified as trace or as ultratrace.

with ATP. Iron is a particularly important mineral because it functions as a component of hemoglobin (the oxygen-carrying protein in the blood) and is part of many enzymes. Other minerals, such as zinc or molybdenum, are required in very small quantities (trace or ultratrace amounts). Sulfur is ingested principally in the amino acids cysteine and methionine. It is found in connective tissue, particularly in cartilage and skin. It has important functions in metabolism, which we will describe when we consider the action of coenzyme A (CoA), a compound used to activate carboxylic acids. Sulfur is excreted in the urine as sulfate. Minerals, like vitamins, have adverse effects if ingested in excessive amounts. Problems associated with dietary excesses or deficiencies of minerals are described in subsequent chapters in conjunction with their normal metabolic functions.

15

In the hospital, it was learned that Mr. Percy Veere had lost 32 lb in the 8 months since his last visit to his family physician. On admission, his hemoglobin (the iron-containing compound in the blood, which carries O2 from the lungs to the tissues) was 10.7 g/dL (reference range, males ⫽ 12 to 15.5 g/dL), his serum ferritin was 4 ng/mL (reference range, males ⫽ 40 to 200 ng/mL), and other hematologic indices that reflect nutritional status were also abnormal. These values are indicative of an iron deficiency anemia. His serum folic acid level was 0.9 ng/ mL (reference range ⫽ 3 to 20 ng/dL), indicating a low intake of this vitamin. His vitamin B12 level was 190 pg/mL (reference range ⫽ 180 to 914 pg/mL). A low blood vitamin B12 level can be caused by decreased intake, absorption, or transport, but it takes a long time to develop. His serum albumin was 3.2 g/dL (reference range ⫽ 3.5 to 5.0 g/dL), which is an indicator of protein malnutrition or liver disease.

F. Water Water constitutes one-half to four-fifths of the weight of the human body. The intake of water required per day depends on the balance between the amount produced by body metabolism and the amount lost through the skin, through expired air, and in the urine and feces.

V. DIETARY GUIDELINES Dietary guidelines or goals are recommendations for food choices that can reduce the risk of developing chronic or degenerative diseases while maintaining an adequate intake of nutrients. Many studies have shown an association between diet and exercise and decreased risk of certain diseases, including hypertension, atherosclerosis, stroke, diabetes, certain types of cancer, and osteoarthritis. Thus, the American Heart Institute and the American Cancer Institute, as well as several other groups, have developed dietary and exercise recommendations to decrease the risk of these diseases. The Dietary Guidelines for Americans (2010), prepared under the authority of the U.S. Department of Agriculture (USDA) Center for Nutrition Policy and Promotion, merges many of these recommendations, which are revised every 5 years (you can view these at the Web site listed in the references). Recommended servings of different food groups can be customized for individuals by going to the USDA MyPlate Web site (see references). Issues of special concern for physicians who advise patients include the following:

A dietary deficiency of calcium can lead to osteoporosis and osteomalacia, a disorder in which bones are insufficiently mineralized and consequently are fragile and easily fractured. Osteoporosis is a particularly common problem among elderly women. Deficiency of phosphorus results in bone loss along with weakness, anorexia, malaise, and pain. Iron deficiencies lead to anemia, a decrease in the concentration of hemoglobin in the blood.

A. General Recommendations • Aim for a healthy weight and be physically active each day. For maintenance of a healthy weight, caloric intake should balance caloric expenditure. Accumulate at least 30 minutes of moderate physical activity (such as walking 2 miles) daily. A regular exercise program helps in achieving and maintaining ideal weight, cardiovascular fitness, and strength. • Choose foods in the proportions recommended by your personalized Plan from MyPlate, including a variety of grains and a variety of fruits and vegetables daily. • Keep food safe to eat. For example, refrigerate leftovers promptly.

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Which foods would provide Percy Veere with good sources of folate and vitamin B12?

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Folate is found in fruits and vegetables: citrus fruits (e.g., oranges), green leafy vegetables (e.g., spinach, broccoli), fortified cereals, and legumes (e.g., peas) (see Table 1.7). Conversely, vitamin B12 is found only in foods of animal origin, including meats, eggs, and milk.

B. Vegetables, Fruits, and Grains • Diets rich in vegetables, fruits, and grain products should be chosen. Four and one-half cups of vegetables and fruits should be eaten each day, particularly green and yellow vegetables and citrus fruits. Three or more ounce-equivalents of whole grain products should be eaten per day (starches and other complex carbohydrates, in the form of breads, fortified cereals, rice, and pasta). In addition to energy, vegetables, fruits, and grains supply vitamins, minerals, protective substances (such as carotenoids), and fiber. Fiber, the indigestible part of plant food, has various beneficial effects, including relief of constipation. • The consumption of refined sugar in foods and beverages should be reduced to below the American norm. Refined sugar has no nutritional value other than its caloric content, and it promotes tooth decay.

C. Fats • Fat intake should be reduced. Fat should account for no more than 35% of total dietary calories (but ⬎20%), and saturated fatty acids should account for 10% or less. Fats derived from fish, nuts, and vegetables, which are primarily polyunsaturated and monosaturated fatty acids, are preferred. Foods high in saturated fat, which should be limited, include cheese, whole milk, butter, regular ice cream, and many cuts of beef. Trans-fatty acids, such as the partially hydrogenated vegetable oils used in margarine, should also be avoided. • Cholesterol intake should be ⬍300 mg/day in subjects without atherosclerotic disease and ⬍200 mg/day in those with established atherosclerosis. Cholesterol is obtained from the diet and synthesized in most cells of the body. It is a component of cell membranes and the precursor of steroid hormones and of the bile salts used for fat absorption. High concentrations of cholesterol in the blood, particularly the cholesterol in lipoprotein particles called low-density lipoproteins (LDL), contribute to the formation of atherosclerotic plaques inside the lumen of arterial vessels, particularly in the heart and brain. These plaques (fatty deposits on arterial walls) can obstruct blood flow to these vital organs causing heart attacks and strokes. A high content of saturated fat in the diet tends to increase circulatory levels of LDL cholesterol and contributes to the development of atherosclerosis.

D. Proteins • Based on the year 2000 report, and still present in the 2010 report, protein intake for adults should be approximately 0.8 g/kg ideal body weight per day. The protein should be of high quality and should be obtained from sources low in saturated fat (e.g., fish, lean poultry, dry beans). Vegetarians should eat a mixture of vegetable proteins that ensures the intake of adequate amounts of the essential amino acids.

E. Alcohol • Alcohol consumption should not exceed moderate drinking. Moderation is defined as no more than one drink per day for women and no more than two drinks per day for men. A drink is defined as 1 regular beer, 5 oz of wine (a little over 0.5 cup), or 1.5 oz of an 80-proof liquor, such as whiskey. Pregnant women should drink no alcohol. The ingestion of alcohol by pregnant women can result in fetal alcohol syndrome (FAS), which is marked by prenatal and postnatal growth deficiency; developmental delay; and craniofacial, limb, and cardiovascular defects.

F. Vitamins and Minerals

The high intake of sodium and chloride (in table salt) of the average American diet appears to be related to the development of hypertension (high blood pressure) in individuals who are genetically predisposed to this disorder.

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• Sodium intake should be decreased in most individuals. Sodium is usually consumed as salt, NaCl. Less than 2.3 g of sodium should be consumed daily, which is about 4.5 g, or 1 tbsp, of salt. • Many of the required vitamins and minerals can be obtained from eating a variety of fruits, vegetables, and grains (particularly whole grains). However, calcium and iron are required in relatively high amounts. Low-fat or nonfat dairy products and dark green leafy vegetables provide good sources of calcium. Lean meats, shellfish, poultry, dark meat, cooked dry beans, and some green leafy vegetables provide good sources of iron. Vitamin B12 is found only in animal sources.

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CHAPTER 1 ■ METABOLIC FUELS AND DIETARY COMPONENTS

• Dietary supplementation in excess of the recommended amounts (e.g., megavitamin regimens) should be avoided. • Fluoride should be present in the diet, at least during the years of tooth formation, as a protection against dental caries.

VI. XENOBIOTICS In addition to nutrients, our diet also contains a large number of chemicals called xenobiotics, which have no nutritional value, are of no use in the body, and can be harmful if consumed in excessive amounts. These compounds occur naturally in foods, can enter the food chain as contaminants, or can be deliberately introduced as food additives. Dietary guidelines of the American Cancer Society and the American Institute for Cancer Research make recommendations relevant to the ingestion of xenobiotic compounds, particularly carcinogens. The dietary advice that we eat a variety of food helps to protect us against the ingestion of a toxic level of any one xenobiotic compound. It is also suggested that we reduce consumption of salt-cured, smoked, and charred foods, which contain chemicals that can contribute to the development of cancer. Other guidelines encourage the ingestion of fruits and vegetables that contain protective chemicals called antioxidants.

17

The prevalence of obesity in the US population is increasing. In 1962, 12.8% of the population had a BMI ⱖ30 and therefore were clinically obese. That number increased to 14.5% by 1980 and to 22.5% by 1998. An additional 30% were preobese in 1998 (BMI ⫽ 25.0 to 29.9). In 2009, 26.7% of adults had a BMI ⱖ30. It is apparent, therefore, that more than 50% of the population is currently overweight, that is, obese or preobese. Increased weight increases cardiovascular risk factors, including hypertension, diabetes mellitus, and alterations in blood lipid levels. It also increases the risk of respiratory problems, gallbladder disease, and certain types of cancer.

CLINICAL COMMENTS Otto Shape. Otto Shape sought help in reducing his weight of 187 lb (BMI of 27) to his previous level of 154 lb (BMI of 22, in the middle of the healthy range). Otto Shape was 5 ft 10 in tall, and he calculated that his maximum healthy weight was 173 lb. He planned on becoming a family physician, and he knew that he would be better able to counsel patients in healthy behaviors involving diet and exercise if he practiced them himself. With this information and assurances from the physician that he was otherwise in good health, Otto embarked on a weight loss program. One of his strategies involved recording all the food he ate and the portions. To analyze his diet for calories, saturated fat, and nutrients, he used the personalized MyPlate Plan (see references), available online from the USDA Food and Nutrition Information Center. Ivan Applebod. Ivan Applebod weighed 264 lb and was 5 ft 10 in tall with a heavy skeletal frame. For a male of these proportions, a BMI of 18.5 to 24.9 would correspond to a weight between 129 lb and 173 lb. He is currently almost 100 lb overweight, and his BMI of 37.9 is in the obese range. Mr. Applebod’s physician cautioned him that exogenous obesity (caused by overeating) represents a risk factor for atherosclerotic vascular disease, particularly when the distribution of fat is primarily “central” or in the abdominal region (apple shape, in contrast to the pear shape, which results from adipose tissue deposited in the buttocks and hips). In addition, obesity may lead to other cardiovascular risk factors such as hypertension (high blood pressure), hyperlipidemia (high blood lipid levels), and type 2 diabetes mellitus (characterized by hyperglycemia). He already has elevated blood pressure. Furthermore, his total serum cholesterol level was 296 mg/dL, well above the desired normal value (200 mg/dL). Mr. Applebod was referred to the hospital’s weight reduction center, where a team of physicians, dieticians, and psychologists could assist him in reaching his ideal weight range. Ann O’Rexia. Because of her history and physical examination, Ann O’Rexia was diagnosed as having early anorexia nervosa, a behavioral disorder that involves both emotional and nutritional disturbances. Miss

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O’Rexia was referred to a psychiatrist with special interest in anorexia nervosa, and a program of psychotherapy and behavior modification was initiated. Percy Veere. Percy Veere weighed 125 lb and was 5 ft 11 in tall (without shoes) with a medium frame. His BMI was 17.5, which is significantly underweight. At the time his wife died, he weighed 147 lb. For his height, a BMI in the healthy weight range corresponds to weights between 132 lb and 178 lb. Mr. Veere’s malnourished state was reflected in his admission laboratory profile. The results of hematologic studies were consistent with an iron deficiency anemia complicated by low levels of folic acid and vitamin B12, two vitamins that can affect the development of normal red blood cells. His low serum albumin level was caused by insufficient protein intake and a shortage of essential amino acids, which result in a reduced ability to synthesize body proteins. The psychiatrist requested a consultation with a hospital dietician to evaluate the extent of Mr. Veere’s marasmus (malnutrition caused by a deficiency of both protein and total calories) as well as his vitamin and mineral deficiencies. BIOCHEMICAL COMMENTS Dietary Reference Intakes. DRIs are quantitative estimates of nutrient intakes that can be used in evaluating and planning diets for healthy people. They are prepared by the Standing Committee on the Scientific Evaluation of DRIs of the Food and Nutrition Board, Institute of Medicine, and the National Academy of Science, with active input of Health Canada. The four reference intake values are the Recommended Dietary Allowance (RDA), the Estimated Average Requirement (EAR), the Adequate Intake (AI), and the Tolerable Upper Intake Level (UL). For each vitamin, the committee has reviewed available literature on studies with humans and established criteria for AI, such as prevention of certain deficiency symptoms, prevention of developmental abnormalities, or decreased risk of chronic degenerative disease. The criteria are not always the same for each life stage group. A requirement is defined as the lowest continuing intake level of a nutrient able to satisfy these criteria. The EAR is the daily intake value that is estimated to meet the requirement in half of the apparently healthy individuals in a life stage or gender group. The RDA is the EAR plus two standard deviations of the mean, which is the amount that should satisfy the requirement in 97% to 98% of the population. The AI level instead of an RDA is set for nutrients when there is not enough data to determine the EAR. The UL refers to the highest level of daily nutrient intake consumed over time that is likely to pose no risks of adverse effects for almost all healthy individuals in the general population. Adverse effects are defined as any significant alteration in the structure or function of the human organism. The UL does not mean that most individuals who consume more than the UL will suffer adverse health effects, but that the risk of adverse effects increases as intake increases above the UL. An example of the difference between the AI and the EAR is provided by riboflavin. Very few data exist on the nutrient requirements of very young infants. However, human milk is the sole recommended food for the first 4 to 6 months, so the AI of the vitamin riboflavin for this life stage group is based on the amount in breast milk consumed by healthy full-term infants. Conversely, the riboflavin EAR for adults is based on several studies in humans relating dietary intake of riboflavin to biochemical markers of riboflavin status and development of clinical deficiency symptoms.

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CHAPTER 1 ■ METABOLIC FUELS AND DIETARY COMPONENTS

Table 1.9

19

Diseases Discussed in Chapter 1

Disorder or Condition

Genetic or Environmental

Depression

Both

Obesity

Both

Anorexia

Environmental

Kwashiorkor

Environmental

Marasmus Osteoporosis/ osteomalacia

Environmental Environmental

Comments Diagnosed by behavioral changes, can be treated with a variety of pharmacologic agents and counseling therapy Long-term effects of obesity affect cardiovascular system and may lead to metabolic syndrome Self-induced reduction of food intake, distorted body image, considered at least in part a psychiatric disorder Protein and mineral deficiency yet normal amount of calories in the diet. Leads to marked hypoalbuminemia, anemia, edema, pot belly, loss of hair, and other indications of tissue injury Prolonged calorie and protein malnutrition Calcium-deficient diet leading to insufficient mineralization of the bones, which produces fragile and easily broken bones.

Diseases that may have a genetic component are indicated as genetic; disorders caused by environmental factors (with or without genetic influences) are indicated as environmental.

Key Concepts • • • •

•

• • • • • •

Fuel is provided in the form of carbohydrates, fats, and proteins in our diet. Energy is obtained from the fuel by oxidizing it to carbon dioxide and water. Unused fuel can be stored as triacylglycerol (fat) or glycogen (carbohydrate) within the body. Weight gain or loss is a balance between the energy consumed in our diet and the energy required each day to drive the basic functions of our body and our physical activity. The daily energy expenditure (DEE) is the amount of fuel consumed in a 24-hour period. The resting metabolic rate (RMR) is a measure of the energy required to maintain nonexercise bodily functions such as respiration, contraction of the heart muscle, biosynthetic processes, and establishment of ion gradients across neuronal membranes. The DEE is determined by the RMR and the individual’s activity level while awake. The body mass index (BMI) is a rough measure of determining an ideal weight for an individual, and whether a person is underweight or overweight. In addition to nutrients, the diet provides vitamins, minerals, essential fatty acids, and amino acids. The Recommended Dietary Allowance (RDA) and the Adequate Intake (AI) provide quantitative estimates of nutrient requirements. The Tolerable Upper Intake Level (UL) indicates the highest level of daily nutrient uptake that is likely to pose no risk of adverse effects. A summary of the diseases/disorders discussed in this chapter are presented in Table 1.9.

REVIEW QUESTIONS—CHAPTER 1 Directions: For each question that follows, select the single best answer. 1.

In the process of respiration, fuels most often undergo which one of the following fates? A. They are stored as triacylglycerols. B. They are oxidized to generate ATP. C. They release energy principally as heat. D. They combine with CO2 and H2O and are stored. E. They combine with other dietary components in anabolic pathways.

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2.

Mrs. Jones is a sedentary 83-year-old woman who is 5 ft 4 in tall and weighs 125 lb. She has been at this weight for about a year. She says that a typical diet for her includes a breakfast of toast (white bread, no butter), a boiled egg, and coffee with cream. For lunch, she often has a cheese sandwich (white bread) and a glass of whole milk. For supper, she prefers cream of chicken soup and a slice of frosted cake. Mrs. Jones’ diet is most likely to be inadequate in which one of the following? A. Vitamin C B. Protein C. Calcium

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C. The minimum amount of a nutrient ingested daily that prevents deficiency symptoms. D. A reasonable dietary goal for the intake of a nutrient by a healthy individual. E. It is based principally on data obtained with laboratory animals.

D. Vitamin B12 E. Calories 3.

4.

The resting metabolic rate is best explained by which one of the following statements? A. It is equivalent to the caloric requirement of our major organs and resting muscle. B. It is generally higher per kilogram body weight in women than in men. C. It is generally lower per kilogram body weight in children than adults. D. It is decreased in a cold environment. E. It is approximately equivalent to the daily energy expenditure. The RDA is best described by which one of the following? A. The average amount of a nutrient required each day to maintain normal function in 50% of the US population. B. The average amount of a nutrient ingested daily by 50% of the US population.

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5.

A 35-year-old sedentary male patient weighing 120 kg was experiencing angina (chest pain) and other signs of coronary artery disease. His physician, in consultation with a registered dietician, conducted a 3-day dietary recall. The patient consumed an average of 585 g of carbohydrate, 150 g of protein, and 95 g of fat each day. In addition, he drank 45 g of alcohol. The patient’s diet is best described by which one of the following? A. He consumed between 2,500 and 3,000 kcal/day. B. He had a fat intake within the range recommended in current dietary guidelines (i.e., year 2010). C. He consumed 50% of his calories as alcohol. D. He was deficient in protein intake. E. He was in negative caloric balance.

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2

The Fed or Absorptive State

The Fed State. During a meal, we ingest carbohydrates, lipids, and proteins, which are subsequently digested and absorbed. Some of this food is oxidized to meet the immediate energy needs of the body. The amount consumed in excess of the body’s energy needs is transported to the fuel depots, where it is stored. During the period from the start of absorption until absorption is completed, we are in the fed or absorptive state. Whether a fuel is oxidized or stored in the fed state is determined principally by the concentration of two endocrine hormones in the blood: insulin and glucagon. Fate of Carbohydrates. Dietary carbohydrates are digested to monosaccharides, which are absorbed into the blood. The major monosaccharide in the blood is glucose (Fig. 2.1). After a meal, glucose is oxidized by various tissues for energy, enters biosynthetic pathways, and is stored as glycogen, mainly in the liver and muscles. Glucose is the major biosynthetic precursor in the body, and the carbon skeletons of most of the compounds we synthesize can be synthesized from glucose. Glucose is also converted to triacylglycerols. The liver packages triacylglycerols, made from glucose or from fatty acids obtained from the blood, into very low-density lipoproteins (VLDL) and releases them into the blood. The fatty acids of the VLDL are stored mainly as triacylglycerols in adipose tissue, but some may be used to meet the energy needs of cells. Fate of Proteins. Dietary proteins are digested to amino acids, which are absorbed into the blood. In cells, the amino acids are converted to proteins or used to make various nitrogen-containing compounds such as neurotransmitters and heme. The carbon skeleton may also be oxidized for energy directly, or be converted to glucose. Fate of Fats. Triacylglycerols are the major lipids in the diet. They are digested to fatty acids and 2-monoacylglycerols, which are resynthesized into triacylglycerols in intestinal epithelial cells, packaged in chylomicrons and secreted by way of the lymph into the blood. The fatty acids of the chylomicron triacylglycerols are stored mainly as triacylglycerols in adipose cells. They are subsequently oxidized for energy or used in biosynthetic pathways, such as synthesis of membrane lipids.

Glucose Oxidation Energy

Storage Glycogen TG

Synthesis Many compounds

Amino acids Protein synthesis

Synthesis of nitrogen-containing compounds

Oxidation Energy

Fats Storage TG

Oxidation Energy

Synthesis Membrane lipids

FIG. 2.1. Major fate of fuels in the fed state. TG, triacylglycerol.

21

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THE WAITING ROOM The body can make fatty acids from a caloric excess of carbohydrate and protein. These fatty acids, together with the fatty acids of chylomicrons (derived from dietary fat), are deposited in adipose tissue as triacylglycerols. Thus, Ivan Applebod’s increased adipose tissue is coming from his intake of all fuels in excess of his caloric need.

Ivan Applebod returned to his doctor for a second visit. His initial efforts to lose weight had failed dismally. In fact, he now weighed 270 lb, an increase of 6 lb since his first visit 2 months ago (see Chapter 1). He reported that the recent death of his 45-year-old brother from a heart attack had made him realize that he must pay more attention to his health. Because Mr. Applebod’s brother had a history of hypercholesterolemia and because his serum total cholesterol had been significantly elevated (296 mg/dL) at his first visit, his blood lipid profile was determined, his blood glucose level was measured, and several other blood tests were ordered. (The blood lipid profile is a test that measures the content of the various triacylglycerol- and cholesterol-containing particles in the blood.) His blood pressure was 162 mm Hg systolic and 98 mm Hg diastolic or 162/98 mm Hg (normal ⫽ ⬍120/80 mm Hg, with prehypertension 120 to 139/80 to 89 mm Hg and hypertension defined as ⬎140/90 mm Hg). His waist circumference was 48 in (healthy values for men, ⬍40 in; for women, ⬍35 in).

I. DIGESTION AND ABSORPTION A. Carbohydrates Dietary carbohydrates are converted to monosaccharides. Starch, a polymer of glucose, is the major carbohydrate of the diet. It is digested by the enzyme salivary ␣-amylase and then by pancreatic ␣-amylase, which acts in the small intestine. Enzymes are proteins that catalyze biochemical reactions (usually, they increase the speed at which the reactions occur). Disaccharides, trisaccharides, and oligosaccharides (disaccharide refers to two linked sugars; a trisaccharide to three linked sugars, and an oligosaccharide to N-linked sugars) produced by these ␣-amylases are cleaved to glucose by digestive enzymes located on the surface of the brush border of the intestinal epithelial cells. Dietary disaccharides also are cleaved by enzymes in this brush border. Sucrase converts the disaccharide sucrose (table sugar) to glucose and fructose, and lactase converts the disaccharide lactose (milk sugar) to glucose and galactose. Monosaccharides produced by digestion and dietary monosaccharides are absorbed by the intestinal epithelial cells and released into the hepatic portal vein, which carries them to the liver.

B. Proteins Proteins contain amino acids that are linked through peptide bonds (see Chapter 1). Dipeptides contain two amino acids, tripeptides contain three amino acids, and so on. Dietary proteins are cleaved to amino acids by enzymes known as proteases (Fig. 2.2, circle 3), which cleave the peptide bond between amino acids (see Fig. 1.5). Pepsin acts in the stomach, and the proteolytic enzymes produced by the pancreas (trypsin, chymotrypsin, elastase, and the carboxypeptidases) act in the lumen of the small intestine. Aminopeptidases, dipeptidases, and tripeptidases associated with the intestinal epithelial cells complete the conversion of dietary proteins to amino acids, which are absorbed into the intestinal epithelial cells and released into the hepatic portal vein.

C. Fats The digestion of fats is more complex than that of carbohydrates or proteins because fats are not very soluble in water. The triacylglycerols of the diet are emulsified in the intestine by bile salts, which are synthesized in the liver and stored in the gallbladder. The enzyme pancreatic lipase converts the triacylglycerols in the lumen of

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CHAPTER 2 ■ THE FED OR ABSORPTIVE STATE

23

Glucose

Blood 4 Intestine

Glucose

Glucagon

1

CHO

Liver

Insulin

Acetyl CoA

Acetyl CoA +

2

Fat (TG)

8

Glycogen

5 I

Glucose

I 6

+

+

7 TG

TCA

Chylomicrons [ATP]

Brain

I

TCA CO2

[ATP]

CO2

3 Protein

AA VLDL

RBC 12

Pyruvate

FA + Glycerol

Lactate

9

14 10 Tissues AA

Glucose +

Protein Important compounds

TCA [ATP] CO2

Muscle

I +

+

I

Acetyl CoA

I

11

13 TG

+

I

CO2

TCA [ATP]

Adipose Glycogen

FIG. 2.2. The fed state. The circled numbers indicate the approximate order in which the processes occur. TG, triacylglycerols; FA, fatty acid; AA, amino acid; RBC, red blood cell; VLDL, very low-density lipoprotein; I, insulin; 䊝, stimulated by.

the intestine to fatty acids and 2-monoacylglycerols (glycerol with a fatty acid esterified at carbon 2), which interact with bile salts to form tiny microdroplets called micelles. The fatty acids and 2-monoacylglycerols are absorbed from these micelles into the intestinal epithelial cells, where they are resynthesized into triacylglycerols. The triacylglycerols are packaged with proteins, phospholipids, cholesterol, and other compounds into the lipoprotein complexes known as chylomicrons, which are secreted into the lymph and ultimately enter the bloodstream (see Fig. 2.2, circle 2). Fats must be transported in the blood bound to protein or in lipoprotein complexes because they are insoluble in water. Thus, both triacylglycerols and cholesterol are found in lipoprotein complexes.

II. CHANGES IN HORMONE LEVELS AFTER A MEAL After a typical high-carbohydrate meal, the pancreas is stimulated to release the hormone insulin, and release of the hormone glucagon is inhibited (see Fig. 2.2, circle 4). Endocrine hormones are released from endocrine glands, such as the pancreas, in response to a specific stimulus. They travel in the blood, carrying messages between tissues concerning the overall physiologic state of the body. At their target tissues, they adjust the rate of various metabolic pathways to meet the changing conditions. The endocrine hormone insulin, which is secreted from

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The laboratory studies ordered at the time of his second office visit show that Ivan Applebod has hyperglycemia, an elevation of blood glucose greater than normal values. At the time of this visit, his blood glucose, determined after an overnight fast, was 162 mg/dL (normal is 80 to 100 mg/dL). Because this blood glucose measurement was significantly above normal, the fasting blood glucose levels were tested the next day, with a result of 170 mg/dL. This led to a diagnosis of type 2 diabetes mellitus, formerly known as non–insulin-dependent diabetes mellitus (NIDDM). In this disease, liver, muscle, and adipose tissue are relatively resistant to the action of insulin in promoting glucose uptake into cells and storage as glycogen and triacylglycerols. Therefore, more glucose remains in his blood.

the ␤-cells of the pancreas in response to a high-carbohydrate meal, carries the message that dietary glucose is available and can be used and stored. The release of another hormone, glucagon, from the ␣-cells of the pancreas is suppressed by glucose and insulin. Glucagon carries the message that glucose must be generated from endogenous fuel stores. The subsequent changes in circulating hormone levels cause changes in the body’s metabolic patterns, involving several different tissues and metabolic pathways.

III. FATE OF GLUCOSE AFTER A MEAL A. Conversion to Glycogen, Triacylglycerols, and Carbon Dioxide in the Liver Because glucose leaves the intestine via the hepatic portal vein, the liver is the first organ it passes through. The liver extracts a portion of this glucose from the blood. Some of the glucose that enters hepatocytes (liver cells) is oxidized in adenosine triphosphate (ATP)-generating pathways to meet the immediate energy needs of these cells, and the remainder is converted to glycogen and triacylglycerols or used for biosynthetic reactions. In the liver, insulin promotes the uptake of glucose by increasing its use as a fuel and its storage as glycogen and triacylglycerols (see Fig. 2.2, circles 5, 6, and 7). As glucose is being oxidized to CO2, it is first oxidized to pyruvate in the pathway of glycolysis. Pyruvate is then oxidized to acetyl coenzyme A (acetyl-CoA). The acetyl group enters the tricarboxylic acid (TCA) cycle, where it is completely oxidized to CO2. Energy from the oxidative reactions is used to generate ATP. The ATP that is generated is used for anabolic and other energy-requiring processes in the cell. Coenzyme A (CoA), which makes the acetyl group more reactive, is a cofactor derived from the vitamin pantothenate. Liver glycogen stores reach a maximum of approximately 200 to 300 g after a high-carbohydrate meal, whereas the body’s fat stores are relatively limitless. As the glycogen stores begin to fill, the liver also begins converting some of the excess glucose it receives to triacylglycerols. Both the glycerol and the fatty acid moieties of the triacylglycerols can be synthesized from glucose. The fatty acids are also obtained preformed from the blood. The liver does not store triacylglycerols, however, but packages them along with proteins, phospholipids, and cholesterol into the lipoprotein complexes known as very low-density lipoproteins (VLDL), which are secreted into the bloodstream. Some of the fatty acids from the VLDL are taken up by tissues for their immediate energy needs, but most are stored in adipose tissue as triacylglycerols.

B. Glucose Metabolism in Other Tissues The glucose from the intestine that is not metabolized by the liver travels in the blood to the peripheral tissues (most other tissues), where it can be oxidized for energy. Glucose is the one fuel that can be used by all tissues. Many tissues store small amounts of glucose as glycogen. Muscle has relatively large glycogen stores. Insulin greatly stimulates the transport of glucose into the two tissues that have the largest mass in the body: muscle and adipose tissue. It has much smaller effects on the transport of glucose into other tissues. Fuel metabolism is often discussed as though the body consisted only of brain, skeletal and cardiac muscle, liver, adipose tissue, red blood cells, kidney, and intestinal epithelial cells (“the gut”). These are the dominant tissues in terms of overall fuel economy, and they are the tissues we describe most often. Of course, all tissues require fuels for energy, and many have a very specific fuel requirement.

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CHAPTER 2 ■ THE FED OR ABSORPTIVE STATE

1.

25

BRAIN AND OTHER NEURAL TISSUES

The brain and other neural tissues are very dependent on glucose for their energy needs. They generally oxidize glucose, via glycolysis and the TCA cycle, completely to CO2 and H2O, generating ATP (see Fig. 2.2, circle 8). Except under conditions of starvation, glucose is their only major fuel. Glucose is also a major precursor of neurotransmitters, the chemicals that convey electrical impulses (as ion gradients) between neurons. If our blood glucose drops much lower than normal levels, we become dizzy and light-headed. If blood glucose continues to drop, we become comatose and, ultimately, die. Under normal, nonstarving conditions, the brain and the rest of the nervous system require roughly 150 g of glucose each day. 2.

RED BLOOD CELLS

Glucose is the only fuel used by red blood cells because they lack mitochondria. Fatty acid oxidation, amino acid oxidation, TCA cycle, electron-transport chain, and oxidative phosphorylation (ATP generation that is dependent on oxygen and the electron-transport chain) occur principally in the mitochondria. Glucose, in contrast, generates ATP from anaerobic glycolysis in the cytosol and, thus, red blood cells obtain all their energy by this process. In anaerobic glycolysis, the pyruvate formed from glucose is converted to lactate and then released into the blood (see Fig. 2.2, circle 9). Without glucose, red blood cells could not survive. Red blood cells carry O2 from the lungs to the tissues. Without red blood cells, most of the tissues of the body would suffer from a lack of energy because they require O2 to completely convert their fuels to CO2 and H2O. 3.

MUSCLE

Exercising skeletal muscles can use glucose from the blood or from their own glycogen stores, converting glucose to lactate through glycolysis or oxidizing it completely to CO2 and H2O. Muscle also uses other fuels from the blood such as fatty acids (Fig. 2.3). After a meal, glucose is used by muscle to replenish the glycogen

Glycogen [ATP] Glucose Fatty acids (from blood)

Lactate Acetyl CoA TCA [ATP]

(to liver via blood)

CO2

FIG. 2.3. Oxidation of fuels in exercising skeletal muscle. Exercising muscle uses more energy than resting muscle, and, therefore, fuel utilization is increased to supply more ATP.

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SECTION I ■ FUEL METABOLISM

stores that were depleted during exercise. Glucose is transported into muscle cells and converted to glycogen by processes that are stimulated by insulin. 4. ADIPOSE TISSUE

Insulin stimulates the transport of glucose into adipose cells as well as into muscle cells. Adipocytes oxidize glucose for energy, and they also use glucose as the source of the glycerol moiety of the triacylglycerols they store (see Fig. 2.2, circle 10).

IV. FATE OF LIPOPROTEINS IN THE FED STATE

Ivan Applebod’s total cholesterol level is now 315 mg/dL, slightly higher than his previous level of 296 mg/ dL. (The currently recommended level for total serum cholesterol is 200 mg/dL or less.) His triacylglycerol level is 250 mg/dL (normal is between 60 and 150 mg/dL). These lipid levels clearly indicate that Mr. Applebod has a hyperlipidemia (a high level of lipoproteins in the blood) and therefore is at risk for the future development of atherosclerosis and its consequences, such as heart attacks and strokes.

Two types of lipoproteins, chylomicrons and VLDL, are produced in the fed state. The major function of these lipoproteins is to provide a blood transport system for triacylglycerols, which are very insoluble in water. However, these lipoproteins also contain the lipid cholesterol, which is also somewhat insoluble in water. The triacylglycerols of chylomicrons are formed in intestinal epithelial cells from the products of digestion of dietary triacylglycerols. The triacylglycerols of VLDL are synthesized in the liver. When these lipoproteins pass through blood vessels in adipose tissue, their triacylglycerols are degraded to fatty acids and glycerol (see Fig. 2.2, circle 12). The fatty acids enter the adipose cells and combine with a glycerol moiety that is produced from blood glucose. The resulting triacylglycerols are stored as large fat droplets in the adipose cells. The remnants of the chylomicrons are cleared from the blood by the liver. The remnants of the VLDL can be cleared by the liver, or they can form a low-density lipoprotein (LDL), which is cleared by the liver or by peripheral cells. Most of us have not even begun to reach the limits of our capacity to store triacylglycerols in adipose tissue. The ability of humans to store fat appears to be limited only by the amount of tissue we can carry without overloading the heart.

V. FATE OF AMINO ACIDS IN THE FED STATE The amino acids derived from dietary proteins travel from the intestine to the liver in the hepatic portal vein (see Fig. 2.2, circle 3). The liver uses amino acids for the synthesis of serum proteins as well as its own proteins, and for the biosynthesis of nitrogen-containing compounds that need amino acid precursors, such as the nonessential amino acids, heme, hormones, neurotransmitters, and purine and pyrimidine bases (e.g., adenine and cytosine in DNA). The liver also may oxidize the amino acids or convert them to glucose or ketone bodies and dispose of the nitrogen as the nontoxic compound urea. Many of the amino acids will go into the peripheral circulation, where they can be used by other tissues for protein synthesis and various biosynthetic pathways, or can be oxidized for energy (see Fig. 2.2, circle 14). Proteins undergo turnover; they are constantly being synthesized and degraded. The amino acids released by protein breakdown enter the same pool of free amino acids in the blood as the amino acids from the diet. This free amino acid pool in the blood can be used by all cells to provide the right ratio of amino acids for protein synthesis or for biosynthesis of other compounds. In general, each individual biosynthetic pathway using an amino acid precursor is found in only a few tissues in the body.

VI. SUMMARY OF THE FED (ABSORPTIVE) STATE After a meal, the fuels that we eat are oxidized to meet our immediate energy needs. Glucose is the major fuel for most tissues. Excess glucose and other fuels are stored as glycogen mainly in muscle and liver, and as triacylglycerols in adipose tissue. Amino acids from dietary proteins are converted to body proteins or oxidized as fuels.

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CHAPTER 2 ■ THE FED OR ABSORPTIVE STATE

CLINICAL COMMENTS Ivan Applebod. Mr. Applebod was advised that his obesity represents a risk factor for future heart attacks and strokes. He was told that his body has to maintain a larger volume of circulating blood to service his extra fat tissue. This expanded blood volume not only contributes to his elevated blood pressure (itself a risk factor for vascular disease), but also puts an increased workload on his heart. This increased load will cause his heart muscle to thicken and eventually to fail. Mr. Applebod’s increasing adipose mass has also contributed to his development of type 2 diabetes mellitus, which is characterized by hyperglycemia (high blood glucose levels). The mechanism behind this breakdown in his ability to maintain normal levels of blood glucose is, at least in part, a resistance by his triacylglycerolrich adipose cells to the action of insulin. In addition to diabetes mellitus, Mr. Applebod has a hyperlipidemia (high blood lipid level—elevated cholesterol and triacylglycerols), another risk factor for cardiovascular disease. A genetic basis for Mr. Applebod’s disorder is inferred from a positive family history of hypercholesterolemia and premature coronary artery disease in his brother. At this point, the first therapeutic step should be nonpharmacologic. Mr. Applebod’s obesity should be treated with caloric restriction and a carefully monitored program of exercise. A reduction of dietary fat and sodium would be advised in an effort to correct his hyperlipidemia and his hypertension, respectively. He should also monitor his carbohydrate intake because of his type 2 diabetes. BIOCHEMICAL COMMENTS Anthropometric Measurements. Anthropometry uses measurements of body parameters to monitor normal growth and nutritional health in well-nourished individuals, and to detect nutritional inadequacies or excesses. In adults, the measurements most commonly used are height, weight, triceps skinfold thickness (SFT), arm muscle circumference (AMC), and waist circumference. In infants and young children, length and head circumference are also measured. Weight and Height. Weight should be measured by using a calibrated beam or lever balance-type scale, and the patient should be in a gown or in underwear. Height for adults should be measured while the patient stands against a straight surface, without shoes, with the heels together, and with the head erect and level. The weight and height are used in calculation of the body mass index (BMI). Skinfold Thickness. More than half of the fat in the body is deposited in subcutaneous tissue under the skin, and the percentage increases with increasing weight. To provide an estimate of the amount of body fat, a standardized caliper is used to pinch a fold of the skin, usually in more than one site (e.g., the biceps, triceps, subscapular and suprailiac areas). Obesity by this physical anthropometric technique is defined as a fatfold thickness greater than the 85th percentile for young adults, that is, 18.6 mm for males and 25.1 mm for females. Midarm Anthropometry. The AMC, also called the mid-upper arm muscle circumference (MUAMC), reflects both caloric adequacy and muscle mass and can serve as a general index of marasmic-type malnutrition. The arm circumference is measured at the midpoint of the left upper arm by a flexible fiberglass-type tape. The AMC can be calculated from a formula that subtracts a factor related to the SFT from the arm circumference: MUAMC (cm) ⫽ arm circumference (cm) ⫺ (3.14 ⫻ SFT [mm])/10

where MUAMC is the mid-upper arm muscle circumference in centimeters, and SFT is the skinfold thickness, expressed in millimeters.

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Ivan Applebod’s waist circumference indicates that he has the android pattern of obesity (apple shape). Fat stores are distributed in the body in two different patterns: android and gynecoid. After puberty, men tend to store fat in and on their abdomens and upper body (an android pattern), whereas women tend to store fat around their breasts, hips, and thighs (a gynecoid pattern). Thus, the typical overweight male tends to have more of an apple shape than the typical overweight woman, who is more pear-shaped. Abdominal fat carries a greater risk of hypertension, cardiovascular disease, hyperinsulinemia, diabetes mellitus, gallbladder disease, stroke, and cancer of the breast and endometrium. It also carries a greater risk of overall mortality. Because more men than women have the android distribution, they are more at risk for most of these conditions. However, women who deposit their excess fat in a more android manner have a greater risk than women whose fat distribution is more gynecoid. Upper-body fat deposition tends to occur more by hypertrophy of the existing cells, whereas lower-body fat deposition is by differentiation of new fat cells (hyperplasia). This may partly explain why many women with lowerbody obesity have difficulty losing weight. The constellation of symptoms exhibited by Mr. Applebod—abdominal obesity, hyperglycemia, hyperlipidemia, and high blood pressure— can all be related to the disorder known as metabolic syndrome or “syndrome X.” We will discuss more aspects of metabolic syndrome as Mr. Applebod’s case progresses.

To obtain reliable measures of SFT, procedures are carefully defined. For example, in the triceps measurement, a fold of skin in the posterior aspect of the nondominant arm midway between shoulder and elbow is grasped gently and pulled away from the underlying muscle. The SFT reading is taken at a precise time, 2 to 3 seconds after applying the caliper, because the caliper compresses the skin. Even when these procedures are performed by trained dieticians, reliable measurements are difficult to obtain.

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SECTION I ■ FUEL METABOLISM

The waist-to-hip ratio has been used instead of the waist circumference as a measure of abdominal obesity in an attempt to correct for differences between individuals with respect to body type or bone structure. In this measurement, the waist circumference is divided by the hip circumference (measured at the iliac crest). The average waist-to-hip ratio for men is 0.93 (with a range of 0.75 to 1.10), and the average for women is 0.83 (with a range of 0.70 to 1.00). However, the waist circumference may actually correlate better with intra-abdominal fat and the associated risk factors than the waist-to-hip ratio.

Table 2.1

Diseases Discussed in Chapter 2

Disorder or Condition

Genetic or Environmental

Hypercholesterolemia

Both

Hyperglycemia

Both

Hyperlipidemia

Both

Comments Elevated cholesterol caused by mutation within a specific protein, or excessive cholesterol intake High blood glucose levels caused by either mutations in specific proteins, or tissue resistance to insulin High levels of blood lipids, may be caused by mutations in specific proteins, or ingestion of high-fat diets

MUAMC values can be compared with reference graphs available for both sexes and all ages. Protein-calorie malnutrition and negative nitrogen balance induce muscle wasting and decrease muscle circumference. Waist Circumference. The waist circumference is another anthropometric measurement that serves as an indicator of body composition but is used as a measure of obesity and body fat distribution (the “apple shape”), not malnutrition. It is the distance around the natural waist of a standing individual (at the umbilicus). A high-risk waistline is larger than 35 in (88 cm) for women and larger than 40 in (102 cm) for men. Key Concepts • • • • • •

• • •

During a meal, we ingest carbohydrate, lipids, and proteins. Two endocrine hormones—insulin and glucagon—primarily regulate fuel storage and retrieval. The predominant carbohydrate in the blood is glucose. Blood glucose levels regulate the release of insulin and glucagon from the pancreas. Under the influence of insulin (fed state), glucose can be used as a fuel and also as a precursor for storage via conversion to glycogen or triacylglycerol. Insulin stimulates the uptake of glucose into adipose and muscle cells. Triacylglycerol obtained from the diet is released into circulation in the form of chylomicrons. Triacylglycerol synthesized from glucose in the liver is released as VLDL. Adipose tissue is the storage site for triacylglycerol. Brain and red blood cells use glucose as their primary energy source under normal conditions. Amino acids obtained from the diet are used for the biosynthesis of proteins and nitrogen-containing molecules and as an energy source. Diseases discussed in this chapter are summarized in Table 2.1.

REVIEW QUESTIONS—CHAPTER 2 1.

During digestion of a mixed meal, which one of the following is most likely to occur? A. Starch and other polysaccharides are transported to the liver. B. Proteins are converted to dipeptides, which enter the blood. C. Dietary triacylglycerols are transported in the portal vein to the liver. D. Monosaccharides are transported to adipose tissue via the lymphatic system. E. Glucose levels increase in the blood.

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2.

After digestion of a high-carbohydrate meal, which one of the following is most likely to occur? A. Glucagon is released from the pancreas. B. Insulin stimulates the transport of glucose into the brain. C. Liver and skeletal muscle use glucose as their major fuel. D. Skeletal muscles convert glucose to fatty acids. E. Red blood cells oxidize glucose to CO2.

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CHAPTER 2 ■ THE FED OR ABSORPTIVE STATE

3.

A B C D E F

4.

Insulin release in the fed state will lead to which of the following metabolic changes? Increased Glucose Transport by Muscle

VLDL Synthesis by the Liver

Fatty Acid Synthesis in Fat Cells

Glycogen Synthesis in the Liver

Yes Yes Yes No No No

Yes No Yes No Yes Yes

Yes Yes No Yes Yes No

No No Yes Yes Yes Yes

Elevated levels of chylomicrons were measured in the blood of a patient. A dietary therapy, which decreased which one of the following answer choices would be most helpful in lowering chylomicron levels? A. Overall calories B. Fat

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C. Cholesterol D. Starch E. Sugar 5.

A male patient exhibited a BMI of 33 kg/m2 and a waist circumference of 47 in. What dietary therapy would you consider most helpful? A. Decreased intake of total calories, because all fuels can be converted to adipose tissue triacylglycerols. B. The same amount of total calories, but substitution of carbohydrate calories for fat calories. C. The same amount of total calories, but substitution of protein calories for fat calories. D. A pure-fat diet, because only fatty acids synthesized by the liver can be deposited as adipose triacylglycerols. E. A limited food diet, such as the ice cream and sherry diet.

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3

Fasting The Fasting State. Fasting begins approximately 2 to 4 hours after a meal, when blood glucose levels return to basal levels, and continues until blood glucose levels begin to rise after the start of the next meal. Within about 1 hour after a meal, blood glucose levels begin to fall. Consequently, insulin levels decline and glucagon levels rise. These changes in hormone levels trigger the release of fuels from the body stores. Liver glycogen is degraded by the process of glycogenolysis, which supplies glucose to the blood. Adipose triacylglycerols are mobilized by the process of lipolysis, which releases fatty acids and glycerol into the blood. Use of fatty acids as a fuel increases with the length of the fast; they are the major fuel oxidized during overnight fasting. Fuel Oxidation. During fasting, glucose continues to be oxidized by glucose-dependent tissues such as the brain and red blood cells, and fatty acids are oxidized by tissues such as muscle and liver. Muscle and most other tissues oxidize fatty acids completely to CO2 and H2O. However, the liver partially oxidizes fatty acids to smaller molecules called ketone bodies, which are released into the blood. Muscle, kidney, and certain other tissues derive energy from completely oxidizing ketone bodies in the tricarboxylic acid (TCA) cycle. Maintenance of Blood Glucose. As fasting progresses, the liver produces glucose not only by glycogenolysis (the release of glucose from glycogen), but also by a second process called gluconeogenesis (the synthesis of glucose from noncarbohydrate compounds). The major sources of carbon for gluconeogenesis are lactate, glycerol, and amino acids. When the carbons of the amino acids are converted to glucose by the liver, their nitrogen is converted to urea. Starvation. When we fast for 3 or more days, we are in the starved state. Muscle continues to burn fatty acids but decreases its use of ketone bodies. As a result, the concentration of ketone bodies rises in the blood to a level at which the brain begins to oxidize them for energy. The brain then needs less glucose, so the liver decreases its rate of gluconeogenesis. Consequently, less protein in muscle and other tissues is degraded to supply amino acids for gluconeogenesis. Protein sparing preserves vital functions for as long as possible. Because of these changes in the fuel use patterns of various tissues, humans can survive for extended periods without ingesting food.

30

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31

THE WAITING ROOM Percy Veere had been admitted to the hospital with a diagnosis of mental depression associated with malnutrition (see Chapter 1). At the time of admission, his body weight of 125 lb gave him a body mass index (BMI) of 17.5 (healthy range, 18.5 to 24.9). His serum albumin was 10% below the low end of the normal range, and he exhibited signs of iron and vitamin deficiencies. Additional tests were made to help evaluate Mr. Veere’s degree of malnutrition and his progress toward recovery. His arm circumference and triceps skinfold were measured, and his mid-upper arm muscle circumference (MUAMC) was calculated (see Chapter 2, Anthropometric Measurements). His serum prealbumin as well as his serum albumin were measured. Fasting blood glucose and serum ketone body concentration were determined on blood samples drawn the next day before breakfast. A 24-hour urine specimen was collected to determine ketone body excretion and creatinine excretion for calculation of the creatinine–height index (CHI), a measure of protein depletion from skeletal muscle. Ann O’Rexia was receiving psychological counseling for anorexia nervosa, but with little success (see Chapter 1). She saw her gynecologist because she had not had a menstrual period for 5 months. She also complained of becoming easily fatigued. The physician recognized that Ann’s body weight of 85 lb was now ⬍65% of her ideal weight, and he calculated that her BMI was now 13.3. The physician recommended immediate hospitalization. The admission diagnosis was severe malnutrition (grade III protein-calorie malnutrition) secondary to anorexia nervosa. Clinical findings included decreased body core temperature, blood pressure, and pulse (adaptive responses to malnutrition). Her physician ordered measurements of blood glucose and ketone body levels and made a spot check for ketone bodies in the urine as well as ordering tests to assess the functioning of her heart and kidneys.

I.

THE FASTING STATE

Percy Veere has grade I proteincalorie malnutrition. At his height of 71 in, his body weight would have to be ⬎132 lb to achieve a BMI of ⬎18.5. Ann O’Rexia has grade III malnutrition. At 67 in, she needs a body weight that is ⬎118 lb to achieve a BMI of 18.5. Degrees of protein-calorie malnutrition (marasmus) are classified as types I, II, and III according to BMI. Type I refers to a BMI in the 17.0 to 18.4 range, type II to a BMI in the 16.0 to 16.9 range, and type III is designated for a BMI of ⬍16.0.

Creatinine is usually released from the muscles at a constant rate, and it is proportional to muscle mass. The creatinine is removed from the circulation by the kidneys and appears in the urine. Thus, elevated creatinine in the blood relates to impaired renal function. To measure creatinine in biologic specimens, the Jaffé reaction is used. Creatinine is reacted with picric acid in an alkaline solution to form a red-orange product, which can be quantitated via spectrophotometry. To increase specificity, a kinetic Jaffé reaction is run, and the rate of formation of the product is determined. Creatinine can be measured in plasma, serum, and urine.

Blood glucose levels peak approximately 1 hour after eating and then decrease as tissues oxidize glucose or convert it to storage forms of fuel. By 2 hours after a meal, the level returns to the fasting range (between 80 and 100 mg/dL). This decrease in blood glucose causes the pancreas to decrease its secretion of insulin, and the serum insulin level decreases. The liver responds to this hormonal signal by starting to degrade its glycogen stores and release glucose into the blood. If we eat another meal within a few hours, we return to the fed state. However, if we continue to fast for a 12-hour period, we enter the basal state (also known as the postabsorptive state). A person is generally considered to be in the basal state after an overnight fast, when no food has been eaten since dinner the previous evening. By this time, the serum insulin level is low and glucagon is rising. Figure 3.1 illustrates the main features of the basal state.

A. Blood Glucose and the Role of the Liver during Fasting The liver maintains blood glucose levels during fasting, and its role is thus critical. Glucose is the major fuel for tissues such as the brain and neural tissue and the sole fuel for red blood cells. Most neurons lack enzymes required for oxidation of fatty acids, but they can use ketone bodies to a limited extent. Red blood cells lack mitochondria, which contain the enzymes of fatty acid and ketone body oxidation,

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SECTION I ■ FUEL METABOLISM

Blood

Glycogen

Glucose

1

Liver

Acetyl CoA

3

2 Glucose

Insulin

Brain

Glucose

Glucagon

12

CO2

[ATP] FA

Acetyl CoA

11

7

Glycerol

TCA

KB Lactate

[ATP]

4 RBC Lactate

Urea

10 Adipose

9

KB

5 TG

Kidney

AA FA

8 6

AA Acetyl CoA

Protein Urine

TCA

Muscle

CO2

[ATP]

FIG. 3.1. Basal state. This state occurs after an overnight (12-hour) fast. The circled numbers serve as a guide indicating the approximate order in which the processes begin to occur. KB, ketone bodies; other abbreviations are defined in Figure 2.2.

O NH2

C

NH2

Urea

FIG. 3.2. The structure of urea, the highly soluble nitrogen disposal molecule.

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and can use only glucose as a fuel. Therefore, it is imperative that blood glucose not decrease too rapidly nor fall too low. Initially, liver glycogen stores are degraded to supply glucose to the blood, but these stores are limited. This pathway is known as glycogenolysis (the lysis, or splitting of glycogen to form glucose subunits). Although liver glycogen levels may increase to 200 to 300 g after a meal, only approximately 80 g remain after an overnight fast. Fortunately, the liver has another mechanism for producing blood glucose, known as gluconeogenesis. Gluconeogenesis means formation (genesis) of new (neo) glucose, and, by definition, converts new (noncarbohydrate) precursors to glucose. In gluconeogenesis, lactate, glycerol, and amino acids are used as carbon sources to synthesize glucose. As fasting continues, gluconeogenesis progressively adds to the glucose produced by glycogenolysis in the liver. Lactate is a product of glycolysis in red blood cells and exercising muscle; glycerol is obtained from lipolysis of adipose triacylglycerols; and amino acids are generated by the breakdown of protein. Because our muscle mass is so large, most of the amino acid is supplied from degradation of muscle protein. These compounds travel in the blood to the liver, where they are converted to glucose by gluconeogenesis. Because the nitrogen of the amino acids can form ammonia, which is toxic to the body, the liver converts this nitrogen to urea. Urea has two amino groups for just one carbon (Fig. 3.2). It is a very soluble, nontoxic compound that can be readily excreted by the kidneys and thus is an efficient means for disposing of excess ammonia.

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CHAPTER 3 ■ FASTING

As fasting progresses, gluconeogenesis becomes increasingly more important as a source of blood glucose. After a day or so of fasting, liver glycogen stores are depleted, and gluconeogenesis is the only source of blood glucose.

B. Role of Adipose Tissue during Fasting Adipose triacylglycerols are the major source of energy during fasting. They supply fatty acids, which are quantitatively the major fuel for the human body. Fatty acids are oxidized not only directly by various tissues of the body; they are also partially oxidized in the liver to four-carbon products called ketone bodies. Ketone bodies are subsequently oxidized as a fuel by other tissues. As blood insulin levels decrease and blood glucagon levels rise, adipose triacylglycerols are mobilized by a process known as lipolysis (lysis of triacylglycerol). They are converted to fatty acids and glycerol, which enter the blood. It is important to realize that most fatty acids cannot provide carbon for gluconeogenesis. Thus, of the vast store of food energy in adipose tissue triacylglycerols, only the small glycerol portion travels to the liver to enter the gluconeogenic pathway. Fatty acids serve as a fuel for muscle, kidney, and most other tissues. They are oxidized to acetyl coenzyme A (acetyl-CoA), and subsequently to CO2 and H2O in the tricarboxylic acid (TCA) cycle, producing energy in the form of adenosine triphosphate (ATP). In addition to the ATP required to maintain cellular integrity, muscle uses ATP for contraction, and the kidney uses it for urinary transport processes. Most of the fatty acids that enter the liver are converted to ketone bodies rather than being completely oxidized to CO2. The process of conversion of fatty acids to acetyl-CoA produces a considerable amount of energy (ATP), which drives the reactions of the liver under these conditions. The acetyl-CoA is converted to the ketone bodies acetoacetate and ␤-hydroxybutyrate, which are released into the blood (Fig. 3.3). The liver lacks an enzyme required for ketone body oxidation. Nevertheless, ketone bodies can be further oxidized by most other cells with mitochondria, such as muscle and kidney. In these tissues, acetoacetate and ␤-hydroxybutyrate are converted to acetyl-CoA and then oxidized in the TCA cycle, with subsequent generation of ATP.

C. Summary of the Metabolic Changes during a Brief Fast In the initial stages of fasting, stored fuels are used for energy (see Fig. 3.1). The liver plays a key role by maintaining blood glucose levels in the range of 80 to 100 mg/dL, first by glycogenolysis (Fig. 3.1, circle 2) and subsequently by gluconeogenesis (Fig. 3.1, circles 9, 11, and 12). Lactate, glycerol, and amino acids serve as carbon sources for gluconeogenesis. Amino acids are supplied by muscle (via proteolysis, lysis of proteins to individual amino acids). Their nitrogen is converted in the liver to urea (Fig. 3.1, circle 10), which is excreted by the kidneys. Fatty acids, which are released from adipose tissue by the process of lipolysis (Fig. 3.1, circle 5), serve as the body’s major fuel during fasting. The liver oxidizes most of its fatty acids only partially, converting them to ketone bodies (Fig. 3.1, circle 7), which are released into the blood. Thus, during the initial stages of fasting, blood levels of fatty acids and ketone bodies begin to increase. Muscle uses fatty acids, ketone bodies (Fig. 3.1, circles 6 and 8), and (when exercising and while supplies last) glucose from muscle glycogen. Many other tissues use either fatty acids or ketone bodies. However, red blood cells, the brain, and other neural tissues use mainly glucose (Fig. 3.1, circles 3 and 4). The metabolic capacities of different tissues with respect to pathways of fuel metabolism are summarized in Table 3.1.

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33

Percy Veere had not eaten much on his first day of hospitalization. His fasting blood glucose determined on the morning of his second day of hospitalization was 72 mg/dL (normal, overnight fasting ⫽ 80 to 100 mg/dL). Thus, in spite of his malnutrition and his overnight fast, his blood glucose was being maintained at nearly normal levels through gluconeogenesis using amino acid precursors. If his blood glucose had decreased to ⬍50 to 60 mg/dL during fasting, his brain would have been unable to absorb glucose fast enough to obtain the glucose needed for energy and neurotransmitter synthesis, resulting in coma and eventual death. Although many other tissues, such as red blood cells, are also totally or partially dependent on glucose for energy, they are able to function at lower concentrations of blood glucose than the brain. On his second day of hospitalization, Percy Veere’s serum ketone body level was 110 ␮M. (Normal value after a 12-hour fast is approximately 70 ␮M.) No ketone bodies were detectable in his urine. At this stage of protein-calorie malnutrition, Mr. Veere still has remaining fat stores. After 12 hours of fasting, most of his tissues are using fatty acids as a major fuel, and the liver is beginning to produce ketone bodies from fatty acids. As these ketone bodies increase in the blood, their use as a fuel will increase.

OH CH3

CH

CH2

–

COO

β-Hydroxybutyrate

O CH3

C

CH2

COO–

Acetoacetate O CH3

C

CH3

Acetone

FIG. 3.3. The ketone bodies ␤-hydroxybutyrate, acetoacetate, and acetone. ␤-hydroxybutyrate and acetoacetate are formed in the liver. Acetone is produced by nonenzymatic decarboxylation of acetoacetate. However, acetone is expired in the breath and is not metabolized to a significant extent in the body, whereas ␤-hydroxybutyrate and acetoacetate are used by muscle and the nervous system as an energy source.

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SECTION I ■ FUEL METABOLISM

Table 3.1

Metabolic Capacities of Various Tissues

Process

Liver

Adipose Tissue

Kidney Cortex

Muscle

Brain

Red Blood Cells

TCA cycle (acetyl-CoA → CO2 ⫹ H2O) ␤-Oxidation of fatty acids Ketone body formation Ketone body utilization

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺⫺

⫹⫹ ⫺⫺ ⫺⫺ ⫹

⫹⫹⫹ ⫹⫹ ⫹ ⫹

⫹⫹⫹ ⫹⫹⫹ ⫺⫺ ⫹⫹⫹

⫺⫺ ⫺⫺ ⫺⫺ ⫺⫺

Glycolysis (glucose → CO2 ⫹ H2O) Lactate production (glucose → lactate)

⫹⫹⫹ ⫹

⫹⫹ ⫹

⫹⫹ ⫺⫺

Glycogen metabolism (synthesis and degradation) Gluconeogenesis (lactate, amino acids, glycerol → glucose) Urea cycle (ammonia → urea) Lipogenesis (glucose → fatty acids)

⫹⫹⫹ ⫹⫹⫹

⫹ ⫺⫺

⫹ ⫹

⫹⫹⫹ ⫹⫹⫹ (Exercise) ⫹⫹⫹ ⫺⫺

⫹⫹⫹ ⫺⫺ ⫺⫺ ⫹⫹⫹ (Prolonged starvation) ⫹⫹⫹ ⫹ ⫹ ⫺⫺

⫺⫺ ⫺⫺

⫹⫹⫹ ⫹⫹⫹

⫺⫺ ⫹

⫺⫺ ⫺⫺

⫺⫺ ⫺⫺

⫺⫺ ⫺⫺

⫺⫺ ⫺⫺

⫺⫺ ⫹⫹⫹

TCA, tricarboxylic acid; acetyl-CoA, acetyl coenzyme A. ⫹⫹ indicates use of the fuel; ⫹⫹⫹ is maximal use, whereas ⫹ is minimal use. ⫺ ⫺ indicates no use of the fuel.

The liver synthesizes several serum proteins and releases them into the blood. These proteins decrease in the blood during protein malnutrition. Two of these serum proteins, albumin and prealbumin (a liver-derived protein that transports thyroid hormone), are often measured to assess the state of protein malnutrition. Serum albumin is the traditional standard of protein malnutrition. Neither measurement is specific for protein malnutrition. Serum albumin and prealbumin levels decrease with hepatic disease (although prealbumin levels are less affected by liver disease than albumin levels), certain renal diseases, surgery, and several other conditions in addition to protein malnutrition. Percy Veere’s values were below the normal range for both of these proteins, indicating that his muscle mass was unable to supply sufficient amino acids to sustain both gluconeogenesis and the synthesis of serum proteins by the liver.

Ann O’Rexia’s admission laboratory studies showed a blood glucose level of 65 mg/dL (normal fasting blood glucose ⫽ 80 to 100 mg/dL). Her serum ketone body concentration was 4,200 ␮M (normal ⫽ ⬃70 ␮M). The Ketostix (Bayer Diagnostics, Mishawaka, Indiana) urine test was moderately positive, indicating that ketone bodies were present in the urine. In her starved state, ketone body use by her brain was helping to conserve protein in her muscles and vital organs.

Lieberman_CH03.indd 34

II. METABOLIC CHANGES DURING PROLONGED FASTING If the pattern of fuel utilization that occurs during a brief fast were to persist for an extended period, the body’s protein would be quite rapidly consumed to the point at which critical functions would be compromised. Fortunately, metabolic changes occur during prolonged fasting that conserve (spare) muscle protein by causing muscle protein turnover to decrease. Figure 3.4 shows the main features of metabolism during prolonged fasting (starvation).

A. Role of Liver during Prolonged Fasting After 3 to 5 days of fasting, when the body enters the starved state, muscle decreases its use of ketone bodies and depends mainly on fatty acids for its fuel. The liver, however, continues to convert fatty acids to ketone bodies. The result is that the concentration of ketone bodies rises in the blood (Fig. 3.5). The brain begins to take up these ketone bodies from the blood and oxidizes them for energy. Therefore, the brain needs less glucose than it did after an overnight fast (Table 3.2). Glucose is still required, however, as an energy source for red blood cells, and the brain continues to use a limited amount of glucose, which it oxidizes for energy and uses as a source of carbon for the synthesis of neurotransmitters. Overall, however, glucose is “spared” (conserved). Less glucose is used by the body and, therefore, the liver needs to produce less glucose per hour during prolonged fasting than during shorter periods of fasting. Because the stores of glycogen in the liver are depleted by approximately 30 hours of fasting, gluconeogenesis is the only process by which the liver can supply glucose to the blood if fasting continues. The amino acid pool, produced by the breakdown of protein, continues to serve as a major source of carbon for gluconeogenesis. A fraction of this amino acid pool is also used for biosynthetic functions (e.g., synthesis of heme and neurotransmitters) and new protein synthesis, processes that must continue during fasting. However, as a result of the decreased rate of gluconeogenesis during prolonged fasting, protein is “spared”; less protein is degraded to supply amino acids for gluconeogenesis. While converting amino acid carbon to glucose in gluconeogenesis, the liver also converts the nitrogen of these amino acids to urea. Consequently, because glucose production decreases during prolonged fasting compared with early fasting, urea production also decreases (Fig. 3.6).

B. Role of Adipose Tissue during Prolonged Fasting During prolonged fasting, adipose tissue continues to break down its triacylglycerol stores, providing fatty acids and glycerol to the blood. These fatty acids serve as the major source of fuel for the body. The glycerol is converted to glucose, whereas the fatty

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CHAPTER 3 ■ FASTING

Blood Glucose

Glycogen (depleted)

Liver

Acetyl CoA

Brain

Glucose

Insulin

TCA

Glucose

Glucagon

CO2

[ATP] FA

[ATP]

Acetyl CoA

Glycerol

KB

RBC

Lactate

Lactate Urea

Adipose

KB AA

TG

Kidney

FA

AA Acetyl CoA

Protein Urine

TCA

Muscle

CO2

[ATP]

FIG. 3.4. Starved state. Abbreviations are defined in Figures 2.2 and 3.1. Dashed lines indicate processes that have decreased (the use of amino acids as a gluconeogenic precursor), and the red solid line indicates a process that has increased relative to the fasting state (the use of ketone bodies by the brain as a major energy source).

Muscle use of ketone bodies Brain use of ketone bodies Brain use of glucose Liver gluconeogenesis Muscle protein degradation Liver production of urea

Lieberman_CH03.indd 35

Decreases Increases Decreases Decreases Decreases Decreases

Plasma level (mM)

Table 3.2 Metabolic Changes during Prolonged Fasting Compared with Fasting for 24 Hours

Death by starvation occurs with loss of roughly 40% of body weight, when approximately 30% to 50% of body protein has been lost, or 70% to 95% of body fat stores. Generally, this occurs at body mass indices (BMIs) of approximately 13 for men and 11 for women.

6 5 4 3

90 Glucose 70 50 Ketone bodies

5 4 3 2 1 0

Fatty acids 2

4

6

Plasma level (mg/dL)

acids are oxidized to CO2 and H2O by tissues such as muscle. In the liver, fatty acids are converted to ketone bodies that are oxidized by many tissues including the brain. Several factors determine how long we can fast and still survive. The amount of adipose tissue is one factor, because adipose tissue supplies the body with its major source of fuel. However, body protein levels can also determine the length of time we can fast. Glucose is still used during prolonged fasting (starvation), but in significantly reduced amounts. Although we degrade protein to supply amino acids for gluconeogenesis at a slower rate during starvation than during the first days of a fast, we are still losing protein that serves vital functions for our tissues. Protein can become so depleted that the heart, kidney, and other vital tissues stop functioning, or we can develop an infection and not have adequate reserves to mount an immune response. In addition to fuel problems, we are also deprived of the vitamin and mineral precursors of coenzymes and other compounds necessary for tissue function. Because of either a lack of ATP or a decreased intake of electrolytes, the electrolyte composition of the blood or cells could become incompatible with life. Ultimately, we die of starvation.

8

Days of starvation

FIG. 3.5. Changes in the concentration of fuels in the blood during prolonged fasting.

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SECTION I ■ FUEL METABOLISM

CLINICAL COMMENTS

Glucose 700 g/d Fasting 12 hours Starvation 3 days Starvation 5–6 weeks 5 10 15 Urea excreted (g/d)

FIG. 3.6. Changes in urea excretion during fasting. Urea production is very low in a person who is consuming only glucose. It increases during fasting as muscle protein is broken down to supply amino acids for gluconeogenesis. However, as fasting progresses, urea synthesis decreases. Because the brain meets some of its energy needs by oxidizing ketone bodies after 3 to 5 days of fasting, gluconeogenesis decreases, sparing protein in muscle and other tissues.

Creatinine–height index. The most widely used biochemical marker for estimating body muscle mass is the 24-hour urinary creatinine excretion. Creatinine is a degradation product formed in active muscle at a constant rate, in proportion to the amount of muscle tissue present in a patient. In a protein-malnourished individual, urinary creatinine will decrease in proportion to the decrease in muscle mass. To assess depletion of muscle mass, creatinine excreted is expressed relative to the height, the CHI. The amount of creatinine (in milligrams) excreted by the subject in 24 hours is divided by the amount of creatinine excreted by a normal, healthy subject of the same height and sex. The resulting ratio is multiplied by 100 to express it as a percentage. Percy Veere’s CHI was 85% (80% to 90% of normal indicates a mild deficit; 60% to 80% of normal indicates a moderate deficit; and 1 mM Ca2+

Cell 2

Cell membrane

B HH

p120

␤ ␣

Catenins

Release of inhibition

Activate GL1 complex

Actin filament

GL1 Nucleus Activate target gene transcription

B. ␤-Catenin and APC in gene transcription

FIG. 18.12. The patched/smoothened signaling system. A. In the absence of a hedgehog (HH) signal, smoothened is inactive because of inhibition by the HH receptor patched, and the GL1 transcription factor complex acts as a repressor of transcription. B. When a ligand binds to the patched receptor, the inhibition of smoothened is repressed, leading to an activation of the GL1 complex, and active transcription of the target genes.

Degradation

␤-Catenin

APC

Inactivation of APC Activation of gene transcription DNA myc

FIG. 18.13. A. Catenins and cadherins. E-cadherin molecules form intercellular, calcium-dependent homodimers with cadherins from another cell, resulting in cell– cell adhesion. The cytoplasmic portion of E-cadherin is complexed to various catenins, which anchor the cadherin to the actin cytoskeleton. B. ␤-Catenin and adenomatous polyposis coli (APC) in transcription. The APC complex activates ␤-catenin for proteolytic degradation. If APC is inactivated, ␤-catenin levels increase. It acts as a transcription factor that increases synthesis of myc and other genes that regulate cell cycle progression.

C. Tumor Suppressor Genes that Affect Cell Adhesion The cadherin family of glycoproteins mediates calcium-dependent cell–cell adhesion. Cadherins form intercellular complexes that bind cells together (Fig. 18.13A). They are anchored intracellularly by catenins, which bind to actin filaments. Loss of E-cadherin expression may contribute to the ability of cancer cells to detach and migrate in metastasis. Individuals who inherit a mutation in E-cadherin (this mutation is designated CDH1) are sharply predisposed to developing diffuse-type gastric cancer. The catenin proteins have two functions: In addition to anchoring cadherins to the cytoskeleton, they act as transcription factors (see Fig. 18.13B). ␤-Catenin also binds to a complex that contains the regulatory protein APC (adenomatous polyposis coli), which activates it for degradation. When the appropriate signal inactivates APC, ␤-catenin levels increase, and it travels to the nucleus where it activates myc and cyclin D1 transcription, leading to cell proliferation. APC is a tumor suppressor gene. If it is inactivated, it cannot bind ␤-catenin and inhibit cell proliferation. Mutations in APC or proteins that interact with it are found in most sporadic human colon cancers. Inherited mutations in APC lead to the most common form of hereditary colon cancer, familial adenomatous polyposis (FAP).

V. CANCER AND APOPTOSIS In the body, superfluous or unwanted cells are destroyed by a pathway called apoptosis, or programmed cell death. Apoptosis is a regulated energy-dependent sequence of events by which a cell self-destructs. In this suicidal process, the cell shrinks, the chromatin condenses, and the nucleus fragments. The cell membrane

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forms blebs (outpouches), and the cell breaks up into membrane-enclosed apoptotic vesicles (apoptotic bodies) containing varying amounts of cytoplasm, organelles, and DNA fragments. Phosphatidylserine, a lipid on the inner leaflet of the cell membrane, is exposed on the external surface of these apoptotic vesicles. It is one of the phagocytic markers recognized by macrophages and other nearby phagocytic cells that engulf the apoptotic bodies. Apoptosis is a normal part of multiple processes in complex organisms: embryogenesis, the maintenance of proper cell number in tissues, the removal of infected or otherwise injured cells, the maintenance of the immune system, and aging. It can be initiated by injury, radiation, free radicals, or other toxins; withdrawal of growth factors or hormones; binding of proapoptotic cytokines; or interactions with cytotoxic T cells in the immune system. Apoptosis can protect organisms from the negative effects of mutations by destroying cells with irreparably damaged DNA before they proliferate. Just as an excess of a growth signal can produce an excess of unwanted cells, the failure of apoptosis to remove excess or damaged cells can contribute to the development of cancer.

323

Death receptor

Mitochondrion

cytC Active initiator caspases Execution procaspases Proteolysis

A. Normal Pathways to Apoptosis Apoptosis can be divided into three general phases: an initiation phase, a signal integration phase, and an execution phase. Apoptosis can be initiated by external signals that work through death receptors, such as tumor necrosis factor (TNF), or deprivation of growth hormones (Fig. 18.14). It can also be initiated by intracellular events that affect mitochondrial integrity (e.g., oxygen deprivation, radiation), and irreparably damaged DNA. In the signal integration phase, these proapoptotic signals are balanced against antiapoptotic cell survival signals by several pathways, including members of the Bcl-2 family of proteins. The execution phase is carried out by proteolytic enzymes called caspases. 1.

CASPASES

Caspases are cysteine proteases that cleave peptide bonds next to an aspartate residue. They are present in the cell as procaspases, zymogen-type enzyme precursors that are activated by proteolytic cleavage of the inhibitory portion of their polypeptide chain. The different caspases are generally divided into two groups according to their function: initiator caspases, which specifically cleave other procaspases; and execution caspases, which cleave other cellular proteins involved in maintaining cellular integrity (see Fig. 18.14). The initiator caspases are activated through two major signaling pathways: the death receptor pathway and the mitochondrial integrity pathway. They activate the execution caspases, which cleave protein kinases involved in cell adhesion, lamins that form the inner lining of the nuclear envelope, actin and other proteins required for cell structure, and DNA repair enzymes. They also cleave an inhibitor protein of the endonuclease CAD (caspase-activated DNase), thereby activating CAD to initiate the degradation of cellular DNA. With destruction of the nuclear envelope, additional endonucleases (Ca2⫹- and Mg2⫹-dependent) also become activated. 2.

Active execution caspases Cellular proteins Apoptotic fragments

FIG. 18.14. Major components in apoptosis. The release of cytochrome c from mitochondria or activation of death receptors can both lead to the initiation of apoptosis.

THE DEATH RECEPTOR PATHWAY TO APOPTOSIS

The death receptors are a subset of TNF-1 receptors, which includes Fas/CD95, TNF-receptor 1 (TNF-R1), and death receptor 3 (DR3). These receptors form a trimer that binds TNF-1 or another death ligand on its external domain and binds adaptor proteins to its intracellular domain (Fig. 18.15). The activated TNF– receptor complex forms the scaffold for binding two molecules of procaspase 8 (or procaspase 10), which autocatalytically cleave each other to form active caspase 8 (or caspase 10). Caspases 8 and 10 are initiator caspases that activate execution caspases 3, 6, and 7. Caspase 3 also cleaves a Bcl-2 protein, Bid, to a form that activates the mitochondrial integrity pathway to apoptosis.

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SECTION III ■ GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS

Cytotoxic T-cell

Ligand

Death receptor

FADD

Cell membrane Adaptor protein Initiator caspases Bcl-2protein tBid

+

Procaspase 8 (procaspase 10) Autocatalysis

+

Caspase 8 (caspase 10) +

Inactive execution caspases Procaspase 3 Procaspase 6 Procaspase 7

Mitochondrial permeability increase

Bid Execution caspases 3 (Active) 6 7

Chromatin condensation DNA fragmentation Surface alterations

FIG. 18.15. The death receptor pathway to apoptosis. The ligand (either a free ligand or a cell surface–associated protein from another cell) binds to the death receptor, which makes a scaffold for autocatalytic activation of caspases 8 (and sometimes 10). Active caspases 8 (and sometimes 10) cleave apoptotic execution caspases directly. However, the pathway also activates Bid, which acts on mitochondrial membrane integrity.

3.

Death signals

Mitochondrion

Apaf-1

Cytochrome c ATP Apoptosome

4.

Procaspase 9

Active caspase 9

Execution procaspases

Active

FIG. 18.16. The mitochondrial integrity pathway releases cytochrome c, which binds to Apaf and forms a multimeric complex called the apoptosome. The apoptosome converts procaspase 9 to active caspase 9, an initiator caspase, which is released by the apoptosome into the cytosol.

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THE MITOCHONDRIAL INTEGRITY PATHWAY TO APOPTOSIS

Apoptosis is also induced by intracellular signals indicating that cell death should occur. Examples of these signals include growth factor withdrawal, cell injury, the release of certain steroids, and an inability to maintain low levels of intracellular calcium. All of these treatments or changes lead to release of cytochrome c from the mitochondria (Fig. 18.16). Cytochrome c is a necessary protein component of the mitochondrial electron-transport chain that is loosely bound to the outside of the inner mitochondrial membrane. Its release initiates apoptosis. In the cytosol, cytochrome c binds Apaf (proapoptotic protease-activating factor). The Apaf/cytochrome c complex binds caspase 9, an initiator caspase, to form an active complex called the apoptosome. The apoptosome, in turn, activates execution caspases (3, 6, and 7) by zymogen cleavage. INTEGRATION OF PROAPOPTOTIC AND ANTIAPOPTOTIC SIGNALS BY THE BCL-2 FAMILY OF PROTEINS

The Bcl-2 family members are decision makers that integrate prodeath and antideath signals to determine whether the cell should commit suicide. Both proapoptotic and antiapoptotic members of the Bcl-2 family exist (Table 18.3). Bcl-2 family members contain regions of homology, known as Bcl-2 homology (BH) domains. There are four such domains. The antiapoptotic factors contain all four domains (BH1–BH4). The channel-forming proapoptotic factors contain just three domains (BH1–BH3), whereas the proapoptotic BH3-only family members contain just one BH domain (BH3). The antiapoptotic Bcl-2–type proteins (including Bcl-2, Bcl-L, and Bcl-w) have at least two ways of antagonizing death signals. They insert into the outer mitochondrial membrane to antagonize channel-forming proapoptotic factors, thereby decreasing cytochrome c release. They may also bind cytoplasmic Apaf so that it cannot form the apoptosome complex (Fig. 18.17). These antiapoptotic Bcl-2 proteins are opposed by proapoptotic family members that fall into two categories: ion channel–forming members and BH3-only members. The prodeath ion channel–forming members, such as Bax, are very similar

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CHAPTER 18 ■ THE MOLECULAR BIOLOGY OF CANCER

to the antiapoptotic family members, except that they do not contain the binding domain for Apaf. They have the other structural domains, however, and when they dimerize with proapoptotic BH3-only members in the outer mitochondrial membrane, they form an ion channel that promotes cytochrome c release rather than inhibiting it (see Fig. 18.17). The prodeath BH3-only proteins (e.g., Bim and Bid) contain only the structural domain that allows them to bind to other Bcl-2 family members (the BH3 domain) and not the domains for binding to the membrane, forming ion channels, or binding to Apaf. Their binding activates the prodeath family members and inactivates the antiapoptotic members. When the cell receives a signal from a prodeath agonist, a BH3 protein like Bid is activated (see Fig. 18.17). The BH3 protein activates Bax (an ion channel–forming proapoptotic channel member), which stimulates release of cytochrome c. Normally, Bcl-2 acts as a death antagonist by binding Apaf and keeping it in an inactive state. However, at the same time that Bid is activating Bax, Bid also binds to Bcl-2, thereby disrupting the Bcl-2/Apaf complex and freeing Apaf to bind to released cytochrome c to form the apoptosome.

B. Cancer Cells Bypass Apoptosis Apoptosis should be triggered by several stimuli, such as withdrawal of growth factors, elevation of p53 in response to DNA damage, monitoring of DNA damage by repair enzymes, or release of TNF or other immune factors. However, mutations in oncogenes can create apoptosis-resistant cells. One of the ways this occurs is through activation of growth factor–dependent signaling pathways that inhibit apoptosis, such as the PDGF/Akt/BAD pathway. Nonphosphorylated BAD acts like Bid in promoting apoptosis (see Fig. 18.17). Binding of the platelet-derived growth factor to its receptor activates PI-3 kinase, which phosphorylates and activates the serine–threonine kinase Akt (protein kinase B, see Chapter 11, Section III.B.3). Activation of Akt results in the phosphorylation of the proapoptotic BH3-only protein BAD, which inactivates it. The PDGF/ Akt/BAD pathway illustrates the requirement of normal cells for growth factor stimulation to prevent cell death. One of the features of neoplastic transformation is the loss of growth factor dependence for survival. The MAP kinase pathway is also involved in regulating apoptosis and sends cell survival signals. MAP kinase kinase phosphorylates and activates another protein kinase known as RSK. Like Akt, RSK phosphorylates BAD and inhibits its activity. Thus, BAD acts as a site

Stimulus Growth-factor deprivation Steroids Irradiation Chemotherapeutic drugs

Anti-apoptosis Bcl-2

BH3-only (Bid)

Mitochondrion

Bax

325

Table 18.3 Examples of Bcl-2 Family Members Antiapoptotic Bcl-2 Bcl-x Bcl-w Proapoptotic Channel forming Bax Bak Bok Proapoptotic BH3 only Bad Bid Bim Roughly 30 Bcl-2 family members are currently known. These proteins play tissue-specific as well as signal pathway–specific roles in regulating apoptosis. The tissue specificity is overlapping. For example, Bcl-2 is expressed in hair follicles, kidney, small intestines, neurons, and the lymphoid system, whereas Bcl-x is expressed in the nervous system and hematopoietic cells.

When Bcl-2 is mutated, and oncogenic, it is usually overexpressed, for example, in follicular lymphoma and CML. Overexpression of Bcl-2 disrupts the normal regulation of proapoptotic and antiapoptotic factors and tips the balance to an antiapoptotic stand. This leads to an inability to destroy cells with damaged DNA, such that mutations can accumulate within the cell. Bcl-2 is also a multidrug-resistant transport protein and if it is overexpressed, it will block the induction of apoptosis by antitumor agents by rapidly removing them from the cell. Thus, strategies are being developed to reduce Bcl-2 levels in tumors that overexpress it before initiating drug or radiation treatment.

Apaf/Bcl-X (inactive) Bcl-X Apaf

Caspase 9 Apoptosome

cyclochrome C

Pro-apoptosis

FIG. 18.17. Roles of the Bcl-2 family members in regulating apoptosis. Bcl-2, which is antiapoptotic, binds Bid (or tBid) and blocks formation of channels that allow cytochrome c release from the mitochondria. Death signals result in activation of a BH3-only protein such as Bid, which can lead to mitochondrial pore formation, swelling, and release of cytochrome c. Bid binds to and activates the membrane ion channel proapoptotic protein Bax, activating cytochrome c release, which binds to Apaf and leads to formation of the apoptosome.

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SECTION III ■ GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS

of convergence for the PI-3 kinase/Akt and MAP kinase pathways in signaling cell survival. Gain-of-function mutations in the genes that control these pathways, such as ras, create apoptosis-resistant cells.

C. MicroRNAs and Apoptosis Recent work has identified several miRNAs that regulate apoptotic factors. Bcl-2, for example, is regulated by at least 2 miRNAs, designated as miR-15 and miR-16. Expression of these miRNAs will control Bcl-2 (an antiapoptotic factor) levels in the cell. If, for any reason, the expression of these miRNAs is altered, Bcl-2 levels will also be altered, promoting either apoptosis (if Bcl-2 levels decrease) or cell proliferation (if Bcl-2 levels increase). Loss of both of these miRNAs is found in 68% of chronic lymphocytic leukemia (CLL) cells, most often caused by a deletion on chromosome 13q14. Loss of miR-15 and miR-16 expression would lead to an increase in Bcl-2 levels, favoring increased cell proliferation. Other miRNA species have been identified, which regulate factors involved in apoptosis. miR-21 regulates the expression of the programmed cell death 4 gene (PDCD4). PDCD4 is upregulated during apoptosis and functions to block translation. Loss of miR-21 activity would lead to cell death, as PDCD4 would be overexpressed. However, overexpression of miR-21 would be antiapoptotic, as PDCD4 expression would be ablated. The miR-17 cluster regulates the protein kinase B/akt pathway by regulating the levels of PTEN (the enzyme that converts PIP3 to PIP2), as well as the levels of the E2F family of transcription factors. An upregulation of miR-17, acting as an oncogene, would decrease PTEN levels such that cellular proliferation is favored over apoptosis because of the constant activation of the akt pathway.

Cell type Gene alteration Normal epithelium Loss of APC Hyperproliferative epithelium

Early adenoma Activation of Ras Intermediate adenoma Loss of a tumorsuppressor gene Late adenoma Loss of p53 activity Carcinoma Other alterations Metastasis

FIG. 18.18. Possible steps in the development of colon cancer. The changes do not always occur in this order, but the most benign tumors have the lowest frequency of mutations, and the most malignant have the highest frequency.

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VI. CANCER REQUIRES MULTIPLE MUTATIONS Cancer takes a long time to develop in humans because multiple genetic alterations are required to transform normal cells into malignant cells (see Fig. 18.1). A single change in one oncogene or tumor suppressor gene in an individual cell is not adequate for transformation. For example, if cells derived from biopsy specimens of normal cells are not already “immortalized,” that is, able to grow in culture indefinitely, addition of the ras oncogene to the cells is not sufficient for transformation. However, additional mutations in a combination of oncogenes, for example, ras and myc, can result in transformation (Fig. 18.18). Epidemiologists have estimated that four to seven mutations are required for normal cells to be transformed. Cells accumulate multiple mutations through clonal expansion. When DNA damage occurs in a normally proliferative cell, a population of cells with that mutation is produced. Expansion of the mutated population enormously increases the probability of a second mutation in a cell containing the first mutation. After one or more mutations in proto-oncogenes or tumor suppressor genes, a cell may proliferate more rapidly in the presence of growth stimuli and with further mutations grow autonomously, that is, independent of normal growth controls. Enhanced growth increases the probability of further mutations. Some families have a strong predisposition to cancer. Individuals in these families have inherited a mutation or deletion of one allele of a tumor suppressor gene, and as progeny of that cell proliferate, mutations can occur in the second allele, leading to a loss of control of cellular proliferation. These familial cancers include familial retinoblastoma, familial adenomatous polyps of the colon, and multiple endocrine neoplasia (MEN), one form of which involves tumors of the thyroid, parathyroid, and adrenal medulla (MEN type II). Studies of benign and malignant polyps of the colon show that these tumors have several different genetic abnormalities. The incidence of these mutations increases with the level of malignancy. In the early stages, normal cells of the intestinal epithelium proliferate, develop mutations in the APC gene, and develop polyps

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VII. AT THE MOLECULAR LEVEL, CANCER IS MANY DIFFERENT DISEASES

2009 Estimated cancer deaths, United States percent distribution of sites by sex 2% Brain Esophagus 4% Lung 30% Liver 4% Pancreas 6% Kidney 3% Colon & rectum 9% Prostate 9% Urinary 4%

15% Breast 26% Lung 6% Pancreas 9% Colon & rectum 5% Ovary 4% Uterus

Leukemia & Lymphomas

8%

All other 23%

7% Leukemia & Lymphomas 26% All other

FIG. 18.19. Estimated cancer deaths by site and sex. Data from the American Cancer Society, Inc., Cancer Facts and Figures, 2006.

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5,000

200

4,000 Smoking

150

Lung cancer

3,000 Smoking

2,000

100

Men Women

1,000

50

Annual deaths from lung cancer (per 100,000 population)

More than 20% of the deaths in the United States each year are caused by cancer, with tumors of the lung, the large intestine, and the breast being the most common (Fig. 18.19). Different cell types typically use different mechanisms through which they lose the ability to control their own growth. An examination of the genes involved in the development of cancer shows that a particular type of cancer can arise in multiple ways. For example, patched and smoothened are the receptor and coreceptor for the signaling peptide, sonic hedgehog. Either mutation of smoothened, an oncogene, or inactivation of patched, a tumor suppressor gene, can give rise to basal cell carcinoma. Similarly, transforming growth factor ␤ and its signal transduction proteins Smad4/DPC are part of the same growth-inhibiting pathway, and either may be absent in colon cancer. Thus, treatments that are successful for one patient with colon cancer may not be successful in a second patient with colon cancer because of the differences in the molecular basis of each individual’s disease (this now appears to be the case with breast cancer as well). Medical practice in the future will require identifying the molecular lesions involved in a particular disease and developing appropriate treatments accordingly. The use of proteomics and gene chip technology (see Chapter 17) to genotype tumor tissues, and to understand which proteins they express, will aid greatly in allowing patient-specific treatments to be developed.

Nick O’Tyne had been smoking for 40 years before he developed lung cancer. The fact that cancer takes so long to develop has made it difficult to prove that the carcinogens in cigarette smoke cause lung cancer. Studies in England and Wales show that cigarette consumption by men began to increase in the early 1900s. Followed by a 20-year lag, the incidence in lung cancer in men also began to rise. Women began smoking later, in the 1920s. Again, the incidence of lung cancer began to increase after a 20-year lag.

Annual per-capita consumption of cigarettes

(see Fig. 18.18). This change is associated with a mutation in the ras proto-oncogene that converts it to an active oncogene. Progression to the next stage is associated with a deletion or alteration of a tumor suppressor gene on chromosome 5. Subsequently, mutations occur in chromosome 18, inactivating a gene that may be involved in cell adhesion, and in chromosome 17, inactivating the p53 tumor suppressor gene. The cells become malignant, and further mutations result in growth that is more aggressive and metastatic. This sequence of mutations is not always followed precisely, but an accumulation of mutations in these genes is found in a large percentage of colon carcinomas.

Lung cancer

0 1900

0 1920

1940

1960

1980

A treatment for CML based on rational drug design has been developed. The fusion protein Bcr-Abl is found only in transformed cells that express the Philadelphia chromosome and not in normal cells. Once the structure of Bcr-Abl was determined, the drug Gleevec was designed to specifically bind to and inhibit only the active site of the fusion protein and not the normal protein. Gleevec was successful in blocking Bcr-Abl function, thereby stopping cell proliferation, and in some cells inducing apoptosis, so the cells would die. Because normal cells do not express the hybrid protein, they were not affected by the drug. The problem with this treatment is that some patients suffered relapses, and when their Bcr-Abl proteins were studied, it was found that in some patients, the fusion protein had a single amino acid substitution near the active site that prevented Gleevec from binding to the protein. Other patients had an amplification of the Bcr-Abl gene product. Other tyrosine kinase inhibitors (such as dasatinib and nilotinib) can also be used in treating CML if a resistance to Gleevec is encountered.

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VIII. VIRUSES AND HUMAN CANCER Three RNA retroviruses are associated with the development of cancer in humans: HTLV-1, HIV, and hepatitis C. There are also DNA viruses associated with cancer, such as hepatitis B, Epstein-Barr virus (EBV), human papillomavirus (HPV), and herpesvirus (HHV-8). HTLV-1 causes adult T-cell leukemia. The HTLV-1 genome encodes a protein Tax, which is a transcriptional coactivator. The cellular proto-oncogenes c-sis and c-fos are activated by Tax, thereby altering the normal controls on cellular proliferation and leading to malignancy. Thus, tax is a viral oncogene without a counterpart in the host cell genome. Infection with HIV, the virus that causes AIDS, leads to the development of neoplastic disease through several mechanisms. HIV infection leads to immunosuppression and, consequently, loss of immune-mediated tumor surveillance. HIV-infected individuals are predisposed to non-Hodgkin lymphoma, which results from an overproduction of T-cell lymphocytes. The HIV genome encodes a protein, Tat, a transcription factor that activates transcription of the interleukin-6 (IL-6) and interleukin-10 (IL-10) genes in infected T cells. IL-6 and IL-10 are growth factors that promote proliferation of T cells, and thus their increased production may contribute to the development of non-Hodgkin lymphoma. Tat can also be released from infected cells and act as an angiogenic (blood vessel–forming) growth factor. This property is thought to contribute to the development of Kaposi sarcoma. DNA viruses also cause human cancer but by different mechanisms. Chronic hepatitis B infections will lead to hepatocellular carcinoma. A vaccine currently is available to prevent hepatitis B infections. EBV is associated with B- and T-cell lymphomas, Hodgkin disease, and other tumors. The EBV encodes a Bcl-2 protein that restricts apoptosis of the infected cell. HHV-8 has been associated with Kaposi sarcoma. Certain strains of papillomavirus have been shown to be a major cause of cervical cancer, and a vaccine has been developed against the specific papillomavirus strains that often lead to cancer development.

CLINICAL COMMENTS Mannie Weitzels. The treatment of a symptomatic patient with chronic myelogenous leukemia (CML) whose white blood cell count is in excess of 50,000 cells/mL is usually initiated with a tyrosine kinase inhibitor. If the patient is intolerant to the tyrosine kinase inhibitor, then busulfan, a DNA-alkylating agent, may be used. Other alkylating agents, such as cyclophosphamide, have also been used alone or in combination with busulfan. Purine and pyrimidine antagonists and hydroxyurea (an inhibitor of the enzyme ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides for DNA synthesis) are sometimes effective in CML as well. In addition, past experience with both ␥- and ␤-interferon has shown promise in increasing survival in these patients if they are intolerant to the tyrosine kinase inhibitors. Interestingly, the interferons have been associated with the disappearance of the Philadelphia chromosome in dividing marrow cells of some patients treated in this way. Nick O’Tyne. Surgical resection of the primary lung cancer with an attempt at cure was justified in Nick O’Tyne who had a good prognosis with a T1N0M0 staging classification preoperatively. Without some evidence of spread to the central nervous system at that time, a preoperative CT scan of the brain would not have been justified. This conservative approach would require scanning of all of the potential sites for metastatic disease from a non–small-cell cancer of the lung in all patients who present in this way. In an era of runaway costs of health care delivery, such an approach could not be considered cost-effective.

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Unfortunately, Mr. O’Tyne developed a metastatic lesion in the right temporal cortex of his brain. Because metastases were almost certainly present in other organs, Mr. O’Tyne’s brain tumor was not treated surgically. In spite of palliative radiation therapy to the brain, Mr. O’Tyne succumbed to his disease just 9 months after its discovery, an unusually virulent course for this malignancy. On postmortem examination, it was found that his body was riddled with metastatic disease. Colin Tuma. Colin Tuma requires regular colonoscopies to check for new polyps in his intestinal tract. Because the development of a metastatic adenoma requires several years (because of the large numbers of mutations that must occur), frequent checks will enable new polyps to be identified and removed before malignant tumors develop. Mel Anoma. The biopsy of Mel Anoma’s excised mole showed that it was not malignant. The most important clinical sign of a malignant melanoma is a change in color in a pigmented lesion. Unlike benign (nondysplastic) nevi, melanomas exhibit striking variations in pigmentation, appearing in shades of black, brown, red, dark blue, and gray. Additional clinical warning signs of a melanoma are enlargement of a preexisting mole, itching or pain in a preexisting mole, development of a new pigmented lesion during adult life, and irregularity of the borders of a pigmented lesion. Mel Anoma was advised to conduct a monthly self-examination, to have a clinical skin examination once or twice yearly, to avoid sunlight, and to use appropriate sunscreens.

329

The TNM system standardizes the classification of tumors. The T stands for the stage of tumor (the higher the number, the worse the prognosis), the N stands for the number of lymph nodes that are affected by the tumor (again, the higher the number, the worse the prognosis), and M stands for the presence of metastasis (0 for none, 1 for the presence of metastatic cells).

Mutations associated with malignant melanomas include ras (gain of function in growth signal transduction oncogene), p53 (loss of function of tumor suppressor gene), p16 (loss of function in Cdk inhibitor tumor suppressor gene), Cdk4 (gain of function in a cell cycle progression oncogene), and cadherin/␤-catenin regulation (loss of regulation that requires attachment).

BIOCHEMICAL COMMENTS Hereditary nonpolyposis colorectal cancer (HNPCC) and hereditary breast cancer both result from inherited mutations in genes involved in DNA repair. HNPCC is estimated to account for between 5% and 6% of all colon cancer cases. These syndromes are heterogeneous, caused, most likely, by the finding that mutations in any of five genes could lead to the finding of right-sided colon cancer. The disease genes include hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6. These genes all play a role in DNA mismatch repair, and all act as tumor suppressors (a loss of function is required for the tumor to develop). It is important to understand that the lack of a DNA mismatch repair enzyme actually does not directly lead to cancer (such as an activating mutation in myc would do, for example). However, the lack of a functional mismatch repair system increases the frequency at which new mutations are introduced into somatic cells (particularly rapidly proliferating cells, such as the colonic epithelium), such that eventually a mutation will result in a gene necessary for proper growth control. Once that mutation occurs, tumors can begin to develop. Familial breast cancer results in only 10% of all breast cancer cases and has been traced to inherited mutations in either one of two genes, BRCA1 and BRCA2. BRCA1 maps to chromosome 17 and acts as a tumor suppressor. The biochemical function of BRCA1 is to participate in the response to DNA damage. BRCA1 is phosphorylated by various kinases, each of which is activated by a different form of DNA damage. BRCA1 is primarily involved in repairing double-strand breaks in DNA and in transcription-coupled repair. Once BRCA1 is phosphorylated, it will signal for cell cycle arrest to allow the DNA damage to be repaired. Women who carry a BRCA1 mutation have an 80% risk of developing breast cancer, and a 40% risk of ovarian cancer, by the age of 70 years. Men who carry BRCA1 mutations do not develop breast cancer, and there is mixed data in the

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SECTION III ■ GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS

literature whether these men have an increased risk of prostate cancer. BRCA1 mutations are correlated with 40% to 50% of all hereditary breast cancer cases. The other gene involved in hereditary breast cancer is BRCA2 located on chromosome 13. BRCA2 is required for DNA double-strand break repair, which is usually caused by ionizing radiation. As such, loss of BRCA2 activity is required for cancer to develop, classifying BRCA2 as a tumor suppressor. BRCA2 is also required for homologous recombination between sister chromatids during meiosis and mitosis. BRCA2 mutations have been liked to increased incidence of breast and ovarian cancer in women and breast cancer in men. An understanding of the roles of BRCA1 and BRCA2 in repairing double-strand breaks in DNA has led to the development of poly-ADP ribose polymerase (PARP-1) inhibitors for the treatment of BRCA1- or BRCA2-induced breast cancers. Doublestrand break repair occurs either by homologous recombination (requiring the activities of BRCA1 and BRCA2 proteins) or through nonhomologous end-joining (NHEJ), which is an error-prone process, caused by trimming of the DNA ends before ligation. Single-strand breaks in DNA are more common than double-strand breaks. The cellular mechanism for repairing single-strand breaks is dependent on PARP-1. PARP-1 produces large branched chains of poly (ADP-ribose) (derived from NAD⫹) at the site of damage, which acts as a docking station for proteins involved in repairing the single-strand break. Inhibiting PARP-1 would lead to an accumulation of single-strand breaks in the DNA. PARP-1 inhibitors are effective in killing BRCA1 or BRCA2 mutated cells in that when single-strand breaks are not repaired, they often are converted to doublestrand breaks when the replisome tries to replicate through the break. In a cell lacking BRCA1 or BRCA2 activity, the only way the DNA can be repaired is by NHEJ, which is an error-prone process. This leads to the cells accumulating a large number of mutations, eventually leading to cell death. Cells with functional BRCA1 or BRCA2 activity will not undergo that fate. Drugs that inhibit PARP-1 activity in cell culture are now in clinical trials with very promising results. Key Concepts • • •

•

•

•

• •

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Cancer is the term applied to a group of diseases in which cells no longer respond to normal constraints on growth. Cancer arises because of mutations in the genome (either inherited or formed in somatic cells). The mutations that lead to cancer occur in certain classes of genes, including Those that regulate cellular proliferation and differentiation Those that suppress growth Those that target cells for apoptosis Those that repair damaged DNA Mutations that lead to cancer can be either gain-of-function mutations or loss of activity of a protein. Gain-of-function mutations occur in proto-oncogenes, resulting in oncogenes. Loss-of-function mutations occur in tumor suppressor genes. Examples of proto-oncogenes are those involved in signal transduction and cell cycle progression. Growth factors and growth factor receptors Ras (a GTP-binding protein) Transcription factors Cyclins and proteins that regulate them MicroRNAs, which regulate growth-inhibitory proteins Examples of tumor suppressor genes include Retinoblastoma gene product (Rb), which regulates the G1 to S phase of the cell cycle p53, which monitors DNA damage and arrests cell cycle progression until the damage has been repaired Regulators of ras MicroRNAs, which regulate growth-promoting signals Apoptosis, programmed cell death, leads to the destruction of damaged cells that cannot be repaired. Apoptosis consists of three phases: Initiation phase (external signals or mitochondrial release of cytochrome c) Signal integration phase Execution phase

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CHAPTER 18 ■ THE MOLECULAR BIOLOGY OF CANCER

Table 18.4

331

Diseases Discussed in Chapter 18 Environmental or Genetic

Comments

Chronic myelogenous leukemia

Disease or Disorder

Environmental

Lung adenocarcinoma

Environmental

Intestinal adenocarcinoma

Both

Melanoma

Environmental

Burkitt lymphoma

Environmental

Li-Fraumeni syndrome

Genetic

Neurofibromatosis (NF-1)

Genetic

Chromosomal translocation leading to the novel Bcr-Abl protein being produced, leading to uncontrolled cell growth. Rational drug design has led to Bcr-Abl targeted agents such as Gleevec, which have a high rate of initial success in controlling tumor cell proliferation. Lung tumor caused by inhalation of mutagenic compounds over several years. Longitudinal data indicates a 20-year lag from the initiation of smoking and a rise in cancer incidence in such individuals. Colon tumors may result from environmental insult, leading to mutations, or an inherited mutation in a tumor suppressor gene, such as APC. HNPCC is caused by inherited mutations in proteins involved in DNA mismatch repair. Tumor of the melanocyte, leading to uncontrolled cell growth. Mutations associated with malignant melanomas include Ras, p53, p16 (a regulator of cdk4), cdk4, and cadherin/␤-catenin regulation. Disorder caused by a chromosomal translocation, in this case chromosomes 8 and 14, leading to the transcription factor myc being moved from chromosome 8 to 14. This leads to inappropriate and overexpression of c-myc, leading to uncontrolled cell proliferation. An inherited mutation in the protein p53, which is responsible for protecting the genome against environmental damage. Loss of p53 activity will lead to an increased mutation rate, eventually leading to a mutation in a gene which regulates cell proliferation. A mutation in a protein (neurofibromin-1) that regulates the GTPase activity of ras, which leads to numerous, benign tumors of the nervous system.

APC, adenomatous polyposis coli; GTPase, guanosine triphosphatase; HNPCC, hereditary nonpolyposis colorectal cancer.

• • • • • •

Apoptosis is regulated by a group of proteins of the Bcl-2 family, which consists of both proapoptotic and antiapoptotic factors. Cancer cells have developed mechanisms to avoid apoptosis. Multiple mutations are required for a tumor to develop in a patient, acquired over several years. Both RNA and DNA viruses can cause a normal cell to become transformed. Exploitation of DNA repair mechanisms may provide a novel means for regulating tumor cell growth. The diseases discussed in this chapter are summarized in Table 18.4.

REVIEW QUESTIONS—CHAPTER 18

1.

The ras oncogene in Colin Tuma’s malignant polyp differs from the c-ras proto-oncogene only in the region that encodes the N-terminus of the protein. This portion of the normal and mutant sequences is

2.

The mechanism through which Ras becomes an oncogenic protein is which of the following? A. Ras remains bound to GAP. B. Ras can no longer bind cAMP. C. Ras has lost its GTPase activity. D. Ras can no longer bind GTP. E. Ras can no longer be phosphorylated by MAP kinase.

3.

Which of the following statements best describes a characteristic of oncogenes? A. All retroviruses contain at least one oncogene. B. Retroviral oncogenes were originally obtained from a cellular host chromosome. C. Proto-oncogenes are genes, found in retroviruses, which have the potential to transform normal cells when expressed inappropriately. D. The oncogenes that lead to human disease are different from those that lead to tumors in animals. E. Oncogenes are mutated versions of normal viral gene products.

10 20 30 Normal A T G A C G G A A T A T A A G C T G G T G G T G G T G G G C G C C G G C G G T Mutant A T G A C G G A A T A T A A G C T G G T G G T G G T G G G C G C C G T C G G T

This mutation is similar to the mutation found in the ras oncogene in various tumors. What type of mutation converts the ras proto-oncogene to an oncogene? A. An insertion that disrupts the reading frame of the protein B. A deletion that disrupts the reading frame of the protein C. A missense mutation that changes one amino acid within the protein D. A silent mutation that produces no change in amino acid sequence of the protein E. An early termination that creates a stop codon in the reading frame of the protein

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4.

SECTION III ■ GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS

When p53 increases in response to DNA damage, which of the following events occurs? A. p53 induces transcription of cdk4. B. p53 induces transcription of cyclin D. C. p53 binds E2F to activate transcription. D. p53 induces transcription of p21. E. p53 directly phosphorylates the transcription factor jun.

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5.

A tumor suppressor gene is best described by which of the following? A. A gain-of-function mutation leads to uncontrolled proliferation. B. A loss-of-function mutation leads to uncontrolled proliferation. C. When it is expressed, the gene suppresses viral genes from being expressed. D. When it is expressed, the gene specifically blocks the G1/S checkpoint. E. When it is expressed, the gene induces tumor formation.

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SECTION FOUR

Fuel Oxidation and the Generation of Adenosine Triphosphate

A

ll physiologic processes in living cells require energy transformation. Cells convert the chemical bond energy in foods into other forms, such as an electrochemical gradient across the plasma membrane, the movement of muscle fibers in an arm, or the assembly of complex molecules such as DNA (Fig. IV.1). These energy transformations can be divided into three principal phases: (1) oxidation of fuels (fat, carbohydrate, and protein), (2) conversion of energy from fuel oxidation into the high-energy phosphate bonds of adenosine triphosphate (ATP), and (3) use of ATP phosphate bond energy to drive energy-requiring processes. The first two phases of energy transformation are part of cellular respiration, the overall process of using O2 and energy derived from oxidizing fuels to generate ATP. We need to breathe principally because our cells require O2 to generate adequate amounts of ATP from the oxidation of fuels to CO2. Cellular respiration uses ⬎90% of the O2 we inhale. In phase 1 of respiration, energy is conserved from fuel oxidation by enzymes that transfer electrons from the fuels to the electron-accepting coenzymes nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), which are reduced to NADH and FAD(2H), respectively (Fig. IV.2). The pathways for the oxidation of most fuels (glucose, fatty acids, ketone bodies, and many amino acids) converge in the generation of the activated 2-carbon acetyl group in acetyl coenzyme A (acetyl-CoA). The complete oxidation of the acetyl group to CO2 occurs in the tricarboxylic acid (TCA) cycle, which collects the energy mostly as NADH and FAD(2H). In phase 2 of cellular respiration, the energy derived from fuel oxidation is converted to the high-energy phosphate bonds of ATP by the process of oxidative phosphorylation (see Fig. IV.2). Electrons are transferred from NADH and FAD(2H) to O2 by the electron-transport chain, a series of electron-transfer proteins that are located in the inner mitochondrial membrane. Oxidation of NADH and FAD(2H) by O2 generates an electrochemical potential across the inner mitochondrial membrane in the form of a transmembrane proton gradient (⌬p). This electrochemical potential drives the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) by a transmembrane enzyme called ATP synthase (or F0F1ATPase). In phase 3 of cellular respiration, the high-energy phosphate bonds of ATP are used for processes such as muscle contraction (mechanical work), maintaining low intracellular Na⫹ concentrations (transport work), synthesis of larger molecules such as DNA in anabolic pathways (biosynthetic work), or detoxification (biochemical work). As a consequence of these processes, ATP is either directly or indirectly hydrolyzed to ADP and Pi or to adenosine monophosphate (AMP) and pyrophosphate (PPi). Cellular respiration occurs in mitochondria (Fig. IV.3). The mitochondrial matrix, which is the compartment enclosed by the inner mitochondrial membrane, contains almost all of the enzymes for the TCA cycle and oxidation of fatty acids,

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O2 Fuels

ATP Pi

O2 CO2 Cellular response

ADP

FIG. IV.1. Energy transformations in fuel metabolism. When ATP energy is transformed into cellular responses, such as muscle contraction, ATP is cleaved to ADP and Pi. In cellular respiration, O2 is used for regenerating ATP from oxidation of fuels to CO2.

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Fatty acids Glucose

Amino acids ATP

NADH

NADH Pyruvate

FAD(2H)

NADH

Nitrogen Urea

Acetyl CoA Ketone bodies

Phase 1 of respiration The oxidation of fuels

TCA cycle CO2 CO2 FAD(2H) NADH O2 H2O

ATP ADP + Pi

Electrontransport chain

Phase 2 of respiration ATP generation from oxidative phosphorylation

+ + H+ + Δp +

FIG. IV.2. Cellular respiration. ⌬p, proton gradient.

TCA cycle enzymes

Outer mitochondrial membrane

␤-Oxidation of fatty acids ATP

ATP synthase

Intermembrane space Inner mitochondrial membrane

Electrontransport chain

Matrix

Mitochondrial DNA

Permeable membrane

FIG. IV.3. Oxidative metabolism in mitochondria. The inner mitochondrial membrane forms infoldings, called cristae, which enclose the mitochondrial matrix. Most of the enzymes for the TCA cycle, the ␤-oxidation of fatty acids, and for mitochondrial DNA synthesis are found in the matrix. ATP synthase and the protein complexes of the electron-transport chain are embedded in the inner mitochondrial membrane. The outer mitochondrial membrane is permeable to small ions, but the inner mitochondrial membrane is impermeable. 334

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ketone bodies, and most amino acids. The inner mitochondrial membrane contains the protein complexes of the electron-transport chain and ATP synthase, the enzyme complex that generates ATP from ADP and Pi. Some of the subunits of these complexes are encoded by mitochondrial DNA, which resides in the matrix. ATP is generated in the matrix, but most of the energy-using processes in the cell occur outside of the mitochondrion. As a consequence, newly generated ATP must be continuously transported to the cytosol by protein transporters in the impermeable inner mitochondrial membrane and by diffusion through pores in the more permeable outer mitochondrial membrane. The rates of fuel oxidation and ATP use are tightly coordinated through feedback regulation of the electron-transport chain and the pathways of fuel oxidation. Thus, if less energy is required for work, more fuel is stored as glycogen or fat in adipose tissue. The basal metabolic rate (BMR), caloric balance, and ⌬G (the change in Gibbs free energy, which is the amount of energy available to do useful work) are quantitative ways of describing energy requirements and the energy that can be derived from fuel oxidation. The various types of enzyme regulation described in Chapter 9 are all used to regulate the rate of oxidation of different fuels to meet energy requirements. Fatty acids are a major fuel in the body. After eating, we store excess fatty acids and carbohydrates that are not oxidized as fat (triacylglycerols) in adipose tissue. Between meals, these fatty acids are released and circulate in blood bound to albumin. In muscle, liver, and other tissues, fatty acids are oxidized to acetyl-CoA in the pathway of ␤-oxidation. NADH and FAD(2H) generated from ␤-oxidation are reoxidized by O2 in the electron-transport chain, thereby generating ATP (see Fig. IV.2). Small amounts of certain fatty acids are oxidized through other pathways that convert them to either oxidizable fuels or urinary excretion products (e.g., peroxisomal ␤-oxidation). Not all acetyl-CoA generated from ␤-oxidation enters the TCA cycle. In the liver, acetyl-CoA generated from ␤-oxidation of fatty acids can also be converted to

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the ketone bodies acetoacetate and ␤-hydroxybutyrate. Ketone bodies are taken up by muscle and other tissues, which convert them back to acetyl-CoA for oxidation in the TCA cycle. They become a major fuel for the brain during prolonged fasting. Amino acids derived from dietary or body proteins are also potential fuels that can be oxidized to acetyl-CoA or converted to glucose and then oxidized (see Fig. IV.2). These oxidation pathways, like those of fatty acids, generate NADH or FAD(2H). Ammonia, which can be formed during amino acid oxidation, is toxic. It is therefore converted to urea in the liver and excreted in the urine. There are more than 20 different amino acids, each with a somewhat different pathway for oxidation of the carbon skeleton and conversion of its nitrogen to urea. Because of the complexity of amino acid metabolism, use of amino acids as fuels is considered separately in Section VII. Glucose is a universal fuel used to generate ATP in every cell type in the body (Fig. IV.4). In glycolysis, 1 mol of glucose is converted to 2 mol of pyruvate and 2 mol of NADH by cytosolic enzymes. Small amounts of ATP are generated when high-energy pathway intermediates transfer phosphate to ADP in a process termed substrate-level phosphorylation. In aerobic glycolysis, the NADH produced from glycolysis is reoxidized by O2 via the electron-transport chain, and pyruvate enters the TCA cycle. In anaerobic glycolysis, the NADH is reoxidized by conversion of pyruvate to lactate, which enters the blood. Although anaerobic glycolysis has a low ATP yield, it is important for tissues with a low oxygen supply and few mitochondria (e.g., the kidney medulla) or tissues that are experiencing diminished blood flow (ischemia). All cells continuously use ATP and require a constant supply of fuels to provide energy for the generation of ATP. Chapters 1 through 3 of this text outlined the basic patterns of fuel use in the human and provided information about dietary components. The pathologic consequences of metabolic problems in fuel oxidation can be grouped into one of two categories: (1) lack of a required product or (2) excess of a substrate or pathway intermediate. The product of fuel oxidation is ATP, and an inadequate rate of ATP production occurs under a wide variety of medical conditions. Extreme conditions that interfere with ATP generation from oxidative phosphorylation, such as complete oxygen deprivation (anoxia) or cyanide poisoning, are fatal. A myocardial infarction is caused by a lack of adequate blood flow to regions of the heart (ischemia), thereby depriving cardiomyocytes of oxygen and fuel. Hyperthyroidism is associated with excessive heat generation from fuel oxidation, and in hypothyroidism, ATP generation can decrease to a fatal level. Conditions such as malnutrition, anorexia nervosa, or excessive alcohol consumption may decrease availability of thiamine, Fe2⫹, and other vitamins and minerals required by the enzymes of fuel oxidation. Mutations in mitochondrial DNA or nuclear DNA result in deficient ATP generation from oxidative metabolism. In contrast, problems arising from an excess of substrate or fuel are seen in diabetes mellitus, which may result in a potentially fatal ketoacidosis. Lactic acidosis occurs with a reduction in oxidative metabolism.

Glucose ATP NADH Pyruvate NADH Lactate Anaerobic glycolysis

Acetyl CoA TCA cycle

FIG. IV.4. Glycolysis. In glycolysis, glucose is converted to pyruvate. If the pyruvate is reduced to lactate, the pathway does not require O2 and is called anaerobic glycolysis (in red). If this pyruvate is converted instead to acetyl-CoA and oxidized in the TCA cycle, glycolysis requires O2 and is aerobic (in black).

Definitions of prefixes and suffixes used in describing clinical conditions: an-emia hyperhypo-osis -uria

without blood excessive, above normal deficient, below normal abnormal or diseased state urine

335

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19

Cellular Bioenergetics: ATP and O2

Heat

ATP

CO2 Energy production Carbohydrate Lipid Protein

O2

FIG. 19.1.

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Energy utilization Muscle contraction Active ion transport Biosynthesis Detoxification Thermogenesis

ADP + Pi The ATP–ADP cycle.

Bioenergetics refers to cellular energy transformations. The ATP–ADP Cycle. In cells, the chemical bond energy of fuels is transformed into the physiologic responses that are necessary for life. The central role of the high-energy phosphate bonds of adenosine triphosphate (ATP) in these processes is summarized in the ATP–ADP (adenosine diphosphate) cycle (Fig. 19.1). To generate ATP through cellular respiration, fuels are degraded by oxidative reactions that transfer most of their chemical bond energy to NAD⫹ and FAD to generate the reduced form of these coenzymes: NADH and FAD(2H). When NADH and FAD(2H) are oxidized by O2 in the electron-transport chain, the energy is used to regenerate ATP in the process of oxidative phosphorylation. Energy available from cleavage of the highenergy phosphate bonds of ATP can be used directly for mechanical work (e.g., muscle contraction) or for transport work (e.g., a Na⫹ gradient generated by Na⫹,K⫹-ATPase). It can also be used for biochemical work (energyrequiring chemical reactions), such as anabolic pathways (biosynthesis of large molecules such as proteins) or detoxification reactions. Phosphoryl transfer reactions, protein conformational changes, and the formation of activated intermediates containing high-energy bonds (e.g., nucleotide-sugars) facilitate these energy transformations. Energy released from foods that is not used for work against the environment is transformed into heat. ATP Homeostasis. Fuel oxidation is regulated to maintain ATP homeostasis (“homeo,” same; “stasis,” state). Regardless of whether the level of cellular fuel utilization is high (with increased ATP consumption) or low (with decreased ATP consumption), the available ATP within the cell is maintained at a constant level by appropriate increases or decreases in the rate of fuel oxidation. Problems in ATP homeostasis and energy balance occur in obesity, hyperthyroidism, and myocardial infarction. Energy from Fuel Oxidation. Fuel oxidation is exergonic: It releases energy. The maximum quantity of energy released that is available for useful work (e.g., ATP synthesis) is called ⌬G0⬘, the change in Gibbs free energy at pH 7.0 under standard conditions. Fuel oxidation has a negative ⌬G0⬘; that is, the products have a lower chemical bond energy than the reactants and their formation is energetically favored. ATP synthesis from ADP and inorganic phosphate is endergonic: It requires energy and has a positive ⌬G0⬘. To proceed in our cells, all pathways must have a negative ⌬G0⬘. How is this accomplished for anabolic pathways such as glycogen synthesis? These metabolic pathways incorporate reactions that expend high-energy bonds to compensate for the energy-requiring steps. Because the ⌬G0⬘ values for a sequence of reactions are additive, the overall pathway becomes energetically favorable. Fuels are oxidized principally by donating electrons to NAD⫹ and FAD, which then donate electrons to O2 in the electron-transport chain. The caloric value of a fuel is related to its ⌬G0⬘ for transfer of electrons to O2 and its reduction potential, E0⬘ (a measure of its willingness to donate or accept electrons). Because fatty acids are more reduced than carbohydrates, they have a higher caloric value. The high affinity of oxygen for electrons

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CHAPTER 19 ■ CELLULAR BIOENERGETICS: ATP AND O2

337

(a high positive reduction potential) drives fuel oxidation forward, with release of energy that can be used for ATP synthesis in oxidative phosphorylation. However, smaller amounts of ATP can be generated without the use of O2 in anaerobic glycolysis. Fuel oxidation can also generate NADPH, which usually donates electrons to biosynthetic pathways and detoxification reactions. For example, in some reactions catalyzed by oxygenases, NADPH is the electron donor and O2 is the electron acceptor.

THE WAITING ROOM Otto Shape is a 26-year-old medical student who has completed his first year of medical school. He is 70 in tall and began medical school weighing 154 lb—within his ideal weight range (see Chapter 1). By the time he finished his last examination in his first year, he weighed 187 lb. He had calculated his basal metabolic rate (BMR) at approximately 1,680 kcal and his energy expenditure for physical exercise equal to 30% of his BMR. He planned on returning to his premedical school weight in 6 weeks over the summer by eating 576 kcal less each day and playing 7 hours of tennis every day. However, he did a summer internship instead of playing tennis. When Otto started his second year of medical school, he weighed 210 lb. X. S. Teefore is a 26-year-old man who noted heat intolerance, with heavy sweating, heart palpitations, and tremulousness. Over the past 4 months, he has lost weight in spite of a good appetite. He is sleeping poorly and describes himself as feeling “jittery inside.” On physical examination, his heart rate is rapid (116 beats/minute) and he appears restless and fidgety. His skin feels warm, and he is perspiring profusely. A fine hand tremor is observed as he extends his arms in front of his chest. His thyroid gland appears to be diffusely enlarged and, on palpation, is approximately three times normal size. Thyroid function tests confirm that Mr. Teefore’s thyroid gland is secreting excessive amounts of the thyroid hormones T4 (tetraiodothyronine) and T3 (triiodothyronine), the major thyroid hormones present in the blood. Cora Nari is a 64-year-old woman who had a myocardial infarction 8 months ago. Although she has managed to lose 6 lb since that time, she remains overweight and has not reduced the fat content of her diet adequately. The graded aerobic exercise program she started 5 weeks after her infarction is now followed irregularly, falling far short of the cardiac conditioning intensity prescribed by her cardiologist. She is readmitted to the hospital cardiac care unit (CCU) after experiencing a severe “viselike pressure” in the midchest area while cleaning ice from the windshield of her car. The electrocardiogram (ECG) shows evidence of a new posterior wall myocardial infarction. Signs and symptoms of left ventricular failure are present.

I.

ENERGY AVAILABLE TO DO WORK

The basic principle of the ATP–ADP cycle is that fuel oxidation generates adenosine triphosphate (ATP), and hydrolysis of ATP to adenosine diphosphate (ADP) provides the energy to perform most of the work required in the cell. ATP has,

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To assess for thyroid function, one must understand how the hormones T3 and T4 are released from the thyroid (see Chapter 43). The hypothalamus and the pituitary gland both monitor the level of free T3 in the blood bathing them. When the concentration of free T3 in the blood drops, the pituitary releases thyroid-stimulating hormone (TSH), which stimulates the thyroid to release T3 and T4. The pituitary is under the control of the hypothalamus, which releases TSH-releasing hormone (TSHRH) under the appropriate conditions. Thus, if one notices low serum T3 or T4 levels, it may represent a thyroid or pituitary problem. Understanding the physiology enables the appropriate tests to be run to determine where the defect lies. T3 and T4 are measured using sensitive techniques that involve antibody recognition (radioimmunoassay; see Chapter 43). TSH levels can be determined in a similar fashion, using a sandwich technique (which requires the use of two distinct antibodies that recognize TSH). Through the appropriate interpretation of these tests, one can determine if thyroid or pituitary function is impaired, and design treatment accordingly.

Cora Nari suffered a heart attack 8 months ago and had a significant loss of functional heart muscle. The pain she is experiencing is called angina pectoris and is a crushing or constricting pain located in the center of the chest, often radiating to the neck or arms (see Ann Jeina, Chapters 6 and 7). The most common cause of angina pectoris is partial blockage of coronary arteries from atherosclerosis. The heart muscle cells beyond the block receive an inadequate blood flow and oxygen, and they die when ATP production falls too low.

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therefore, been called the energy currency of the cells. Like the $1 bill, it has a defined value, is required to obtain goods and services, and disappears before we know it. To keep up with the demand, we must constantly replenish our ATP supply through the use of O2 for fuel oxidation. The amount of energy from ATP cleavage available to do useful work is related to the difference in energy levels between the products and substrates of the reaction and is called the change in Gibbs free energy, ⌬G (⌬, difference; G, Gibbs free energy). In cells, the ⌬G for energy production from fuel oxidation must be greater than the ⌬G of energy-requiring processes, such as protein synthesis and muscle contraction, for life to continue.

The heart is a specialist in the transformation of ATP chemical bond energy into mechanical work. Each single heartbeat uses approximately 2% of the ATP in the heart. If the heart were not able to regenerate ATP, all its ATP would be hydrolyzed in less than 1 minute. Because the amount of ATP required by the heart is so high, it must rely on the pathway of oxidative phosphorylation for generation of this ATP. In Cora Nari’s heart, hypoxia is affecting her ability to generate ATP.

A. The High-Energy Phosphate Bonds of ATP The amount of energy released or required by bond cleavage or formation is determined by the chemical properties of the substrates and products. The bonds between the phosphate groups in ATP are called phosphoanhydride bonds (Fig. 19.2). When these bonds are hydrolyzed, energy is released because the products of the reaction (ADP and phosphate) are more stable, with lower bond energies, than the reactants (ATP and water [H2O]). The instability of the phosphoanhydride bonds arises from their negatively charged phosphate groups, which repel each other and strain the bonds between them. It takes energy to make the phosphate groups stay together. In contrast, there are fewer negative charges in ADP to repel each other. The phosphate group, as a free anion, is more stable than it is in ATP because of an increase in resonance structures (i.e., the electrons of the oxygen double bond are shared by all the oxygen atoms). As a consequence, ATP hydrolysis is energetically favorable and proceeds with release of energy as heat. In the cell, ATP is not hydrolyzed directly. Energy released as heat from ATP hydrolysis cannot be transferred efficiently into energy-requiring processes such as biosynthetic reactions or maintaining an ion gradient. Instead, cellular enzymes transfer the phosphate group directly to a metabolic intermediate or protein that is part of the energy-requiring process (a phosphoryl transfer reaction).

B. Change in Free Energy (⌬G) during a Reaction How much energy can be obtained from ATP hydrolysis to do the work required in the cell? The maximum amount of useful energy that can be obtained from a reaction

NH2 C

High-energy phosphate bonds O

O –

O

P –

O

P

HC

C N

C

C

N

OCH2

P –

O

O

O

␥

␤

␣

H H OH

N

Adenine CH

HC

N

H2O

O O

–

N

NH2

O

O

Hydrolysis H

H

O

O –

P

OH

–

O

+

–

O

N

C

N CH N

O

P

O –

O

D-Ribose

+ H+

OH

C

P

OCH2 –

O

O

H H OH

H

H

OH

Adenosine 5'-triphosphate

Phosphate

Adenosine 5'-diphosphate

ATP

Pi

ADP

FIG. 19.2. Hydrolysis of ATP to ADP and inorganic phosphate (Pi). Cleavage of the phosphoanhydride bonds between either the ␤- and ␥-phosphates or between the ␣- and ␤-phosphates releases the same amount of energy, approximately 7.3 kcal/mol. However, hydrolysis of the phosphate–adenosine bond (a phosphoester bond) releases less energy (⬇3.4 kcal/mol), and consequently, this bond is not considered a highenergy phosphate bond. During ATP hydrolysis, the change in disorder during the reaction is small and so ⌬G values at physiologic temperature (37°C) are similar to those at standard temperature (25°C). ⌬G is affected by pH, which alters the ionization state of the phosphate groups of ATP and by the intracellular concentration of Mg2⫹ ions, which bind to the ␤- and ␥-phosphate groups of ATP.

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Table 19.1

339

Thermodynamic Expressions, Laws, and Constants

Definitions ⌬G ⌬G0 ⌬G0⬘ ⌬H ⌬S K⬘eq ⌬E0⬘ P

Change in free energy, or Gibbs free energy Standard free-energy change, ⌬G starting with 1 M concentrations of substrates and products Standard free-energy change at 25°C, pH 7.0 Change in enthalpy, or heat content Change in entropy, or increase in disorder Equilibrium constant at 25°C, pH 7.0, incorporating [H2O] ⫽ 55.5 M and [H⫹] ⫽ 10⫺7 M in the constant Change in reduction potential Biochemical symbol for a high-energy phosphate bond (i.e., a bond that is hydrolyzed with the release of more than about 7 kcal/mol of heat)

Laws of Thermodynamics First law of thermodynamics, the conservation of energy: In any physical or chemical change, the total energy of a system, including its surroundings, remains constant. Second law of thermodynamics: The universe tends toward disorder. In all natural processes, the total entropy of a system always increases. Constants Units of ⌬G and ⌬H ⫽ cal/mol or J/mol: 1 cal ⫽ 4.18 J T, absolute temperature: K, kelvin ⫽ 273 ⫹ °C (25°C ⫽ 298 K) R, universal gas constant: 1.99 cal/mol-K or 8.31 J/mol-K F, Faraday constant: F ⫽ 23 kcal/mol-V or 96,500 J/V-mol Units of E0⬘, V Formulas ⌬G ⫽ ⌬H ⫺ T ⌬S ⌬G0⬘ ⫽ ⫺RT ln Keq⬘ ⌬G0⬘ ⫽ ⫺n F ⌬E0⬘ ln ⫽ 2.303 log10

is called ⌬G—the change in Gibbs free energy. The value of ⌬G for a reaction can be influenced by the initial concentration of substrates and products, temperature, pH, and pressure. The ⌬G0 for a reaction refers to the energy change for a reaction starting at 1 M substrate and product concentrations and proceeding to equilibrium (equilibrium, by definition, occurs when there is no change in substrate and product concentrations with time). ⌬G0⬘ is the value for ⌬G0 under standard conditions (pH ⫽ 7.0, [H2O] ⫽ 55 M, and 25°C), as well as standard concentrations (Table 19.1). ⌬G0⬘ is equivalent to the chemical bond energy of the products minus that of the reactants, corrected for energy that has gone into entropy (an increase in amount of molecular disorder). This correction for change in entropy is very small for most reactions that occur in cells and, thus, the ⌬G0⬘ for hydrolysis of various chemical bonds reflects the amount of energy available from that bond. The value ⫺7.3 kcal/mol (⫺30.5 kJ/mol) that is generally used for the ⌬G0⬘ of ATP hydrolysis is thus the amount of energy available from hydrolysis of ATP under standard conditions that can be spent on energy-requiring processes; it defines the “monetary value” of our “ATP currency.” Although the difference between cellular conditions (pH 7.3, 37°C) and standard conditions is very small, the difference between cellular concentrations of ATP, ADP, and inorganic phosphate (Pi) and the standard 1 M concentrations is huge and greatly affects the availability of energy in the cell.

C. Exothermic and Endothermic Reactions The value of ⌬G0⬘ tells you whether the reaction requires or releases energy, the amount of energy involved, and the ratio of products to substrates at equilibrium. The negative value for the ⌬G0⬘ of ATP hydrolysis indicates that, if you begin with equimolar (1 M) concentrations of substrates and products, the reaction proceeds in the forward direction with the release of energy. From initial concentrations of 1 M, the ATP concentration will decrease, and ADP and Pi will increase until equilibrium is reached.

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The reaction catalyzed by phosphoglucomutase (PGM) is reversible and functions in the synthesis of glycogen from glucose as well as the degradation of glycogen back to glucose. If the ⌬G0⬘ for conversion of glucose 6-phosphate to glucose 1-phosphate is ⫹1.65 kcal/mol, what is the ⌬G0⬘ of the reverse reaction?

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The ⌬G0⬘ for the reverse reaction is ⫺1.65 kcal. The change in free energy is the same for the forward and reverse directions but has opposite sign. Because negative ⌬G0⬘ values indicate favorable reactions, this reaction under standard conditions favors the conversion of glucose 1-P to glucose 6-P.

O –

O

O

P

CH2 O

–

O

H H HO OH H

H H OH OH

Glucose 6-phosphate (G6P) PGM HOCH2 O H H

H

HO OH

H O P

H

OH

O

O

O– –

Glucose 1-phosphate (G1P)

[G1P] [G6P]

FIG. 19.3. The phosphoglucomutase reaction. The forward direction (formation of glucose 1-phosphate) is involved in converting glucose to glycogen, and the reverse direction in converting glycogen to glucose 6-phosphate. PGM, phosphoglucomutase.

For a reaction in which a substrate S is converted to a product P, the ratio of the product concentration to the substrate concentration at equilibrium is given by Equation 19.1: ⌬G0⬘ ⫽ ⫺RT ln[P] / [S]

(see Table 19.2 for a more general form of this equation; R is the gas constant [1.99 cal/mol oK], and T is the temperature in degrees Kelvin). Thus, the difference in chemical bond energies of the substrate and product (⌬G0⬘) determines the concentration of each at equilibrium. Reactions such as ATP hydrolysis are exergonic (release energy) or exothermic (release heat). They have a negative ⌬G0⬘ and release energy while proceeding in the forward direction to equilibrium. Endergonic or endothermic reactions have a positive ⌬G0⬘ for the forward direction (the direction shown), and the backward direction is favored. For example, in the pathway of glycogen synthesis, phosphoglucomutase converts glucose 6-phosphate (glucose 6-P) to glucose 1-phosphate (glucose 1-P). Glucose 1-P has a higher phosphate bond energy than glucose 6-P because the phosphate is on the aldehyde carbon (Fig. 19.3). The ⌬G0⬘ for the forward direction (glucose 1-P → glucose 6-P) is therefore positive. Beginning at equimolar concentrations of both compounds, there is a net conversion of glucose 1-P back to glucose 6-P and, at equilibrium, the concentration of glucose 6-P is higher than glucose 1-P. The exact ratio is determined by the ⌬G0⬘ for the reaction. It is often said that a reaction with a negative ⌬G0⬘ proceeds spontaneously in the forward direction, meaning that products accumulate at the expense of reactants. However, ⌬G0⬘ is not an indicator of the velocity of the reaction or of the rate at which equilibrium can be reached. In the cell, the velocity of the reaction depends on the efficiency and amount of enzyme available to catalyze the reaction (see Chapter 9), so a reaction proceeding “spontaneously” in this context can be misleading. The equations for calculating ⌬G are based on the first law of thermodynamics (see Table 19.1). The change in chemical bond energy that occurs during a reaction is ⌬H—the change in enthalpy of the reaction. At constant temperature and pressure, ⌬H is equivalent to the chemical bond energy of the products minus that of the reactants. ⌬G—the maximum amount of useful work available from a reaction—is equal to ⌬H ⫺ T ⌬S. T ⌬S is a correction for the amount of energy that has gone into an increase in the entropy (disorder in arrangement of molecules) of the system. Thus, ⌬G ⫽ ⌬H ⫺ T ⌬S, where ⌬H is the change in enthalpy, T is the temperature of the system in kelvin, and ⌬S is the change in entropy, or increased disorder of the system. ⌬S is often negligible in reactions such as ATP hydrolysis, in which the numbers of substrates (H2O, ATP) and products (ADP, Pi) are equal and no gas is formed. Under these conditions, the values for ⌬G at physiologic temperature (37°C) are similar to those at standard temperature (25°C).

Table 19.2

A General Expression for ⌬G

To generalize the expression for ⌬G, consider a reaction in which aA ⫹ bB ← → cC ⫹ dD The lowercase letters denote that a moles of A will combine with b moles of B to produce c moles of C and d moles of D.

The ⌬G0⬘ for the conversion of glucose 6-P to glucose 1-P is ⫹1.65 kcal/mol. What is the ratio of [glucose 1-P] to [glucose 6-P] at equilibrium?

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[C]ceq[D]deq ⌬G0⬘ ⫽ ⫺RT ln Keq ⫽ ⫺RT ln _________ [A]aeq[B]beq And [C]c[D]d ⌬G ⫽ ⌬G0⬘ ⫹ RT ln _______ [A]a[B]b

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As shown in Table 19.2, ⌬G0⬘ ⫽ ⫺RT ln Keq. For this reaction, Keq ⫽ [glucose 1-phosphate] / [glucose 6phosphate]. The constant R is 1.99 ⫻ 10⫺3 kcal/ mol-K, and T is (273 ⫹ 25) K, so RT ⫽ ⫺0.593 kcal/mol. Substituting in the previous equation then gives 1.65 ⫽ ⫺0.593 ln[glucose 1-P] / [glucose 6-P]. Thus, ln[glucose 1-P] / [glucose 6-P] ⫽ ⫺2.78, and [glucose 1-P] / [glucose 6-P] ⫽ e⫺2.78, or 0.062. So the ratio of [glucose 1-P] to [glucose 6-P] at equilibrium is 0.062.

II. ENERGY TRANSFORMATIONS TO DO MECHANICAL AND TRANSPORT WORK To do work in the cell, a mechanism must be available for converting the chemical bond energy of ATP into another form, such as a Na⫹ gradient across a membrane. These energy transformations usually involve intermediate steps in which ATP is bound to a protein, and cleavage of the bound ATP results in a conformational change of the protein.

A. Mechanical Work In mechanical work, the high-energy phosphate bond of ATP is converted into movement by changing the conformation of a protein (Fig. 19.4). For example, in contracting muscle fibers, the hydrolysis of ATP while it is bound to myosin ATPase changes the conformation of myosin so that it is in a “cocked” position— ready to associate with the sliding actin filament. Thus, exercising muscle fibers have almost a 100-fold higher rate of ATP utilization and caloric requirements than resting muscle fibers. Motor proteins, such as kinesins that transport chemicals along fibers, provide another example of mechanical work in a cell.

B. Transport Work In transport work, called active transport, the high-energy phosphate bond of ATP is used to transport compounds against a concentration gradient (see Chapter 10, Fig. 10.10). In plasma membrane ATPases (P-ATPases) and vesicular ATPases (V-ATPases), the chemical bond energy of ATP is used to reversibly phosphorylate the transport protein and change its conformation. For example, as Na⫹,K⫹-ATPase binds and cleaves ATP, it becomes phosphorylated and changes its conformation to release 3 Na⫹ ions to the outside of the cell, thereby building up a higher extracellular than intracellular concentration of Na⫹. Na⫹ reenters the cell on cotransport proteins that drive the uptake of amino acids and many other compounds into the

1

2

1

Actin filament Myosin head Myosin thick filament

2

ATP

ATP Dissociation of actin–myosin

Hydrolysis of ATP by myosin head

1

2

1

3

2

1

ADP

2

ADP + Pi

Actin filament slides to new position ADP

H2O

Pi

FIG. 19.4. A simplified diagram of myosin ATPase. Muscle fiber is made of thick filaments composed of bundles of the protein myosin, and thin filaments composed of the protein actin (which is activated by Ca2⫹ binding). At many positions along the actin filament, a terminal domain of a myosin molecule, referred to as the “head,” binds to a specific site on the actin. The myosin head has an ATP-binding site and is an ATPase; it can hydrolyze ATP to ADP and Pi. (1) As ATP binds to myosin, the conformation of myosin changes, and it dissociates from the actin. (2) Myosin hydrolyzes the ATP, again changing conformation. (3) When Pi dissociates, the myosin head reassociates with activated actin at a new position (position 2 in the figure). (4) As ADP dissociates, the myosin again changes conformation, or tightens. This change of conformation at multiple association points between actin and myosin slides the actin filament forward.

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Otto Shape has not followed his proposed diet and exercise regimen and has been gaining weight. He has a positive caloric balance, because his daily energy expenditure is less than his daily energy intake (see Chapter 2). Although the energy expenditure for physical exercise is only approximately 30% of the BMR in a sedentary individual, it can be 100% or more of the BMR in a person who exercises strenuously for several hours or more each day. The large increase in ATP utilization for muscle contraction during exercise accounts for its contribution to the daily energy expenditure. In the thermodynamic perspective of energy expenditure, when energy intake to the body exceeds energy expended, the difference is effectively stored as fat.

Total energy expenditure = heat produced + work on environment

Energy intake (food)

Metabolism

Energy storage (fat)

Physical activity variable Adaptive thermogenesis Obligatory energy expenditure Cellular and organ functions

The portion of food that is metabolized is regulated to match the total energy expenditure of the body. A certain amount of the energy is obligatory (the amount of energy expended to do the work of the cells; the BMR). Some energy is also expended for adaptive thermogenesis, which is heat generated in response to cold or diet. An additional amount of energy is used for physical exercise (work against the environment). To voluntarily store less energy as fat, we can vary our caloric intake through dietary changes or our energy expenditures through changes in our physical exercise.

Lieberman_Ch19.indd 342

cell. Thus, Na⫹ must be continuously transported back out. The expenditure of ATP for Na⫹ transport occurs even while we sleep and is estimated to account for 10% to 30% of our basal metabolic rate (BMR). A large number of other active transporters also convert ATP chemical bond energy into an ion gradient (membrane potential). V-ATPases pump protons into lysosomes. Ca2⫹-ATPases in the plasma membrane move Ca2⫹ out of the cell against a concentration gradient. Similar Ca2⫹-ATPases pump Ca2⫹ into the lumen of the endoplasmic reticulum and the sarcoplasmic reticulum (in muscle). Thus, a considerable amount of energy is expended in maintaining a low cytoplasmic Ca2⫹ level.

III. BIOCHEMICAL WORK The high-energy phosphate bonds of ATP are also used for biochemical work. Biochemical work occurs in anabolic pathways, which are pathways that synthesize large molecules (e.g., DNA, glycogen, triacylglycerols, and proteins) from smaller compounds. Biochemical work also occurs when toxic compounds are converted to nontoxic compounds that can be excreted (e.g., the liver converts NH4⫹ ions to urea in the urea cycle). In general, formation of chemical bonds between two organic molecules (e.g., C–C bonds in fatty acid synthesis or C–N bonds in protein synthesis) requires energy and is, therefore, biochemical work. How do our cells get these necessary energy-requiring reactions to occur? To answer this question, the next sections consider how energy is used to synthesize glycogen from glucose (Fig. 19.5). Glycogen is a storage polysaccharide consisting of glucosyl units linked together through glycosidic bonds. If an anabolic pathway, such as glycogen synthesis, were to have an overall positive ⌬G0⬘, the cell would be full of glucose and intermediates of the pathway, but very little glycogen would be formed. To avoid this, cells do biochemical work and spend enough of their ATP currency to give anabolic pathways an overall negative ⌬G0⬘.

A. ⌬G0 Values Are Additive Reactions in which chemical bonds are formed between two organic molecules are usually catalyzed by enzymes that transfer energy from cleavage of ATP in a phosphoryl transfer reaction or by enzymes that cleave a high-energy bond in an activated intermediate of the pathway. Because the ⌬G0⬘ values in a reaction sequence are additive, the pathway acquires an overall negative ⌬G0⬘, and the reactions in the pathway will occur to move toward an equilibrium state in which the concentration of final products is greater than that of the initial reactants. 1.

PHOSPHORYL TRANSFER REACTIONS

One of the characteristics of Gibbs free energy is that ⌬G0 values for consecutive steps or reactions in a sequence can be added together to obtain a single value for the overall process. Thus, the high-energy phosphate bonds of ATP can be used to drive a reaction forward that would otherwise be highly unfavorable energetically. Consider, for example, synthesis of glucose 6-P from glucose, the first step in glycolysis and glycogen synthesis (see Fig. 19.5, circle 2). If the reaction were to proceed by addition of inorganic phosphate to glucose, glucose 6-P synthesis would have a positive ⌬G0⬘ value of 3.3 kcal/mol (Table 19.3). However, when this reaction is coupled to cleavage of the high-energy ATP bond through a phosphoryl transfer reaction, the ⌬G0⬘ for glucose 6-P synthesis acquires a net negative value of ⫺4.0 kcal/mol, which can be calculated from the sum of the two reactions. Glucose 6-P cannot be transported back out of the cell and, therefore, the net negative ⌬G0⬘ for glucose 6-P synthesis helps the cell to trap glucose for its own metabolic needs. The net value for synthesis of glucose 6-P from glucose and ATP would be the same whether the two reactions were catalyzed by the same enzyme, were catalyzed by two separate enzymes, or were not catalyzed by an enzyme at all because it is dictated by the amount of energy in the chemical bonds being broken and formed.

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Table 19.3 ⌬G0⬘ for the Transfer of a Phosphate from Adenosine Triphosphate to Glucose Glucose ⫹ Pi → glucose 6-P ⫹ H2O ATP ⫹ H2O → ADP ⫹ Pi Sum: glucose ⫹ ATP → glucose 6-P ⫹ ADP

343

Glucose

⌬G0⬘ ⫽ ⫹3.3 kcal/mol ⌬G0⬘ ⫽ ⫺7.3 kcal/mol ⌬G0⬘ ⫽ ⫺4.0 kcal/mol

Glucose transport

1

Glucose

2.

ATP

ACTIVATED INTERMEDIATES IN GLYCOGEN SYNTHESIS

To synthesize glycogen from glucose, energy is provided by the cleavage of three high-energy phosphate bonds in ATP, uridine triphosphate (UTP), and pyrophosphate (PPi) (see Fig. 19.5, Steps 2, 5, and 6). Energy transfer is facilitated by phosphoryl group transfer and by the formation of an activated intermediate (UDPglucose). Step 4—the conversion of glucose 6-P to glucose 1-P—has a positive ⌬G0⬘. This step is pulled and pushed in the desired direction by the accumulation of substrate and removal of product in reactions that have a negative ⌬G0⬘ from cleavage of high-energy bonds. In Step 5, the UTP high-energy phosphate bond is cleaved to form the activated sugar, UDP-glucose (Fig. 19.6). This reaction is further facilitated by cleavage of the high-energy bond in the PPi (Step 6) that is released in Step 5 (approximately ⫺7.7 kcal). In Step 7, cleavage of the bond between UDP and glucose in the activated intermediate provides the energy for attaching the glucose moiety to the end of the glycogen molecule (approximately ⫺3.3 kcal). In general, the amount of ATP phosphate bond energy used in an anabolic pathway, or detoxification pathway, must provide the pathway with an overall negative ⌬G0⬘, so that the concentration of products is favored over that of reactants.

B. ⌬G Depends on Substrate and Product Concentrations

⌬G0⬘ reflects the energy difference between reactants and products at specific concentrations (each at 1 M) and standard conditions (pH 7.0, 25°C). However, these are not the conditions that prevail in cells, in which variations from “standard conditions” are relevant to determining actual free-energy changes and, hence, the direction in which reactions are likely to occur. One aspect of freeenergy changes contributing to the forward direction of anabolic pathways is the dependence of ⌬G, the free-energy change of a reaction, on the initial substrate and product concentrations. Reactions in the cell with a positive ⌬G0⬘ can proceed in the forward direction if the concentration of substrate is raised to a high enough level, or if the concentration of product is decreased to a very low level. Product concentrations can be very low if, for example, the product is rapidly used in a subsequent energetically favorable reaction, or if the product diffuses or is transported away.

O HOCH2

C

O

HN

H H

H

HO OH

H O P

H

OH

O

O

O O

–

P O

O C O CH2

–

H H HO

CH N

CH

O H

2

ADP

Glucose 6- P

3

4

Glycolysis H2O 2Pi

6

Glucose 1- P UTP

5 PPi UDP-glucose

7

Glycogenn Glycogenn +1

UDP

FIG. 19.5. Energetics of glycogen synthesis. Compounds containing high-energy bonds are shown in red. (1) Glucose is transported into the cell. (2) Glucose phosphorylation uses the high-energy phosphate bond (⬃P) of ATP in a phosphoryl transfer step. (4) Conversion of glucose 6-phosphate to glucose 1-phosphate by phosphoglucomutase. (5) UDP-glucose pyrophosphorylase cleaves a ⬃P bond in UTP, releasing pyrophosphate (PPi) and forming UDP-glucose, an activated intermediate. (6) The PPi is hydrolyzed, releasing additional energy. (7) The phosphoester bond of UDPglucose is cleaved during the addition of a glucosyl unit to the end of a glycogen polysaccharide chain. The UDP acts as the leaving group in this reaction. Glucose 6-phosphate also can be metabolized via glycolysis (3) when energy is required.

Given a ⌬G0⬘ of ⫹1.65 kcal/mol for the conversion of glucose 6-P to glucose 1-P and a ⌬G0⬘ of ⫺4.0 kcal/ mol for the conversion of glucose ⫹ ATP to glucose 6-P ⫹ ADP, what is the value of ⌬G0⬘ for the conversion of glucose to glucose 1-P?

H

OH

Uridine diphosphate glucose (UDP-glucose)

FIG. 19.6. UDP-glucose contains a high-energy pyrophosphate (PPi) bond, shown in the green box.

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X. S. Teefore has increased blood levels of thyroid hormones, which accelerate basal metabolic processes that use ATP in our organs (e.g., Na⫹,K⫹-ATPase), thereby increasing the BMR. An increased BMR was used for a presumptive diagnosis of hyperthyroidism before development of the tests to measure T3 and T4. Because X. S. Teefore did not fully compensate for his increased ATP requirements with an increased caloric intake, he was in negative caloric balance and lost weight.

Approximately 70% of our resting daily energy requirement arises from work carried out by our largest organs: the heart, brain, kidneys, and liver. Using their rate of oxygen consumption and a P/O ratio of 2.5 (see Chapter 21), it can be estimated that each of these organs is using and producing several times its own weight in ATP each day. Estimated Daily Use of ATP (g ATP/g Tissue) Heart Brain Kidneys Liver Skeletal muscle (rest) Skeletal muscle (running)

16 6 24 6 0.3 23.6

The heart, which contracts rhythmically, is using this ATP for mechanical work. In contrast, skeletal muscles in a resting individual use far less ATP per gram of tissue. The kidney has an ATP consumption per gram of tissue similar to that of the heart and uses this ATP largely for transport work to recover usable nutrients and to maintain pH and electrolyte balance. The brain, likewise, uses most of its ATP for transport work, maintaining the ion gradients necessary for conduction of nerve impulses. The liver, in contrast, has a high rate of ATP consumption and utilization to carry out metabolic work (biosynthesis and detoxification). Otto Shape realizes that his resting daily energy requirement will remain constant, and for him to lose weight, he will have to eat less, exercise more, or both.

1.

THE DIFFERENCE BETWEEN ⌬G AND ⌬G0ⴕ

The driving force toward equilibrium starting at any concentration of substrate and product is expressed by ⌬G, and not by ⌬G0⬘, which is the free-energy change to reach equilibrium starting with 1 M concentration of substrate and product. For a reaction in which the substrate S is converted to the product P, Equation 19.2: ⌬G ⫽ ⌬G0⬘ ⫹ RT ln[P] / [S]

(see Table 19.2, for the general form of this equation). The expression for ⌬G has two terms: ⌬G0⬘, the energy change to reach equilibrium starting at equal and 1 M concentrations of substrates and products; and the second term, the energy change to reach equal concentrations of substrate and product starting from any initial concentration. (When [P] ⫽ [S] and [P] / [S] ⫽ 1, ln [P] / [S] ⫽ 0, and ⌬G ⫽ ⌬G0⬘.) The second term will be negative for all concentrations of substrate greater than the product, and the greater the substrate concentration, the more negative this term will be. Thus, if the substrate concentration is suddenly raised high enough or the product concentration is decreased low enough, ⌬G (the sum of the first and second terms) will also be negative, and conversion of substrate to product becomes thermodynamically favorable. 2.

THE REVERSIBILITY OF THE PHOSPHOGLUCOMUTASE REACTION IN THE CELL

The effect of substrate and product concentration on ⌬G and the direction of a reaction in the cell can be illustrated with conversion of glucose 6-P to glucose 1-P, the reaction catalyzed by phosphoglucomutase in the pathway of glycogen synthesis (see Fig. 19.3). The reaction has a small positive ⌬G0⬘ for glucose 1-P synthesis (⫹1.65. kcal/mol), and at equilibrium, the ratio [glucose 1-P] / [glucose 6-P] is approximately 6/94 (which was determined in Question 2). However, if another reaction uses glucose 1-P such that this ratio suddenly becomes 3/94, there is now a driving force for converting more glucose 6-P to glucose 1-P and restoring the equilibrium ratio. Substitution in Equation 19.2 gives ⌬G, the driving force to equilibrium, as ⫹1.65 ⫹ RT ln[glucose 1-P] / [glucose 6-P] ⫽ 1.65 ⫹ (⫺2.06) ⫽ ⫺0.41, which is a negative value. Thus, a decrease in the ratio of product to substrate has converted the synthesis of glucose 1-P from a thermodynamically unfavorable to a thermodynamically favorable reaction that will proceed in the forward direction until equilibrium is reached.

C. Activated Intermediates with High-Energy Bonds Many biochemical pathways form activated intermediates containing high-energy bonds to facilitate biochemical work. The term high-energy bond is a biologic term defined by the ⌬G0⬘ for ATP hydrolysis; any bond that can be hydrolyzed with the release of approximately as much—or more—energy than ATP is called a high-energy bond. The high-energy bonds in activated intermediates, such as UDPglucose in glycogen synthesis, facilitate energy transfer. 1.

ATP, UTP, GTP, AND CTP

Cells use guanosine triphosphate (GTP) and cytidine triphosphate (CTP), as well as UTP and ATP, to form activated intermediates. Different anabolic pathways generally use different nucleotides as their direct source of high phosphate-bond energy: UTP is used for combining sugars, CTP for lipid synthesis, and GTP for protein synthesis. The high-energy phosphate bonds of UTP, GTP, and CTP are energetically equivalent to ATP and are synthesized from ATP by nucleoside diphosphokinases and nucleoside monophosphokinases. For example, UTP is formed from UDP by a nucleoside diphosphokinase in the reaction ATP ⫹ UDP ↔ UTP ⫹ ADP

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ADP is converted back to ATP by the process of oxidative phosphorylation, using energy supplied by fuel oxidation. Energy-requiring reactions often generate the nucleoside diphosphate ADP. Adenylate kinase, an important enzyme in cellular energy balance, is a nucleoside monophosphate kinase that transfers a phosphate from one ADP to another ADP to form ATP and adenosine monophosphate (AMP): ADP ⫹ ADP ↔ AMP ⫹ ATP

This enzyme can thus regenerate ATP under conditions in which ATP use is required. 2.

OTHER COMPOUNDS WITH HIGH-ENERGY BONDS

In addition to the nucleoside triphosphates, other compounds containing highenergy bonds are formed to facilitate energy transfer in anabolic and catabolic pathways (e.g., 1,3-bisphosphoglycerate in glycolysis and acetyl coenzyme A in the tricarboxylic acid cycle) (Fig. 19.7). Creatine phosphate contains a high-energy phosphate bond that allows it to serve as an energy reservoir for ATP synthesis and transport in muscle cells, neurons, and spermatozoa. All of these high-energy bonds are “unstable,” and their hydrolysis yields substantial free energy because the products are much more stable, because of electron resonance within their structures.

345

⌬G0⬘ for the overall reaction is the sum of the individual reactions, or ⫺2.35 kcal. The individual reactions are Glucose ⫹ ATP → glucose 6-P ⫹ ADP ⌬G0⬘ ⫽ ⫺4.0 kcal/mol Glucose 6-P → glucose 1-P ⌬G0⬘ ⫽ ⫹1.65 kcal/mol Therefore, Glucose ⫹ ATP → glucose 1-P ⫹ ADP ⌬G0⬘ ⫽ ⫺2.35 kcal/mol Thus, the cleavage of ATP has made the synthesis of glucose 1-P from glucose energetically favorable.

O 2–

C ~ OPO3

IV. THERMOGENESIS According to the first law of thermodynamics, energy cannot be destroyed. Thus, energy from oxidation of a fuel (its caloric content) must be equal to the amount of heat released, the work performed against the environment, and the increase in order of molecules in our bodies. Some of the energy from fuel oxidation is converted into heat as the fuel is oxidized and some heat is generated as ATP is used to do work. If we become less efficient in converting energy from fuel oxidation into ATP, or if we use an additional amount of ATP for muscular contraction, we will oxidize an additional amount of fuel to maintain ATP homeostasis (constant cellular ATP levels). With the oxidation of additional fuel, we release additional heat. Thus, heat production is a natural consequence of “burning fuel.” The term thermogenesis refers to energy expended for the purpose of generating heat in addition to that expended for ATP production. To maintain the body at 37°C despite changes in environmental temperature, it is necessary to regulate fuel oxidation and its efficiency (as well as heat dissipation). In shivering thermogenesis, we respond to sudden cold with asynchronous muscle contractions (shivers) that increase ATP utilization and, therefore, fuel oxidation and the release of energy as heat. In nonshivering thermogenesis (adaptive thermogenesis), the efficiency of converting energy from fuel oxidation into ATP is decreased. More fuel needs to be oxidized to maintain constant ATP levels and, thus, more heat is generated.

H C

2–

1,3-Bisphosphoglycerate

O C

A. Energy Transfer from Fuels through Oxidative Phosphorylation Fuel oxidation is our major source of ATP and our major means of transferring energy from the chemical bonds of the fuels to cellular energy-requiring

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O– 2–

C ~ OPO3 CH2

Phosphoenolpyruvate

H +

H2N

C

O N ~ P O– O– N CH3 CH2 COO–

Creatine phosphate

V. ENERGY FROM FUEL OXIDATION Fuel oxidation provides energy for bodily processes principally through generation of the reduced coenzymes NADH and FAD(2H). They are used principally to generate ATP in oxidative phosphorylation. However, fuel oxidation also generates NADPH, which is most often used directly in energy-requiring processes. Carbohydrates also may be used to generate ATP through a nonoxidative pathway, called anaerobic glycolysis.

OH

CH2OPO3

O CH3

C ~ SCoA

Acetyl CoA

FIG. 19.7. Some compounds with highenergy bonds. 1,3-bisphosphoglycerate and phosphoenolpyruvate are intermediates of glycolysis. Creatine phosphate is a high-energy phosphate reservoir and shuttle in brain, muscle, and spermatozoa. Acetyl coenzyme A (acetyl-CoA) is a precursor of the TCA cycle. The high-energy bonds are shown in red.

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X. S. Teefore has increased thyroid hormone levels that increase his rate of ATP utilization and fuel oxidation. An excess of thyroid hormones may also affect the efficiency of ATP production, resulting in fewer ATP produced for a given level of O2 consumption. The increased rate of ATP utilization and diminished efficiency stimulate oxidative metabolism, resulting in a much greater rate of heat production. The hyperthyroid patient, therefore, complains of constantly feeling hot (heat intolerance) and sweaty. (Perspiration allows dissipation of excess heat through evaporation from the skin surface.)

Inner mitochondrial membrane Pyruvate

Pyruvate

1

NADH

O2 H2O H+ H+ + + +

2 ⌬pH ⌬␷

Δp – – –

Acetyl CoA

FAD (2H)

ADP + Pi

CO2

ATP Mitochondrion

FIG. 19.8. Overview of energy transformations in oxidative phosphorylation. The electrochemical potential gradient across the mitochondrial membrane (⌬p) is represented by two components: the ⌬pH, the proton gradient; and ⌬␺, the membrane potential. The role of the electrochemical potential in oxidative phosphorylation is discussed in more depth in Chapter 21.

processes. The amount of energy available from a fuel is equivalent to the amount of heat that is generated when a fuel is burned. To conserve this energy for the generation of ATP, the process of cellular respiration transforms the energy from the chemical bonds of fuels into the reduction state of electron-accepting coenzymes: NAD⫹ and FAD (circle 1, Fig. 19.8). As these compounds transfer electrons to O2 in the electron-transport chain, most of this energy is transformed into an electrochemical gradient across the inner mitochondrial membrane (circle 2, Fig. 19.8). Much of the energy in the electrochemical gradient is used to regenerate ATP from ADP in oxidative phosphorylation (phosphorylation that requires O2). 1.

OXIDATION–REDUCTION REACTIONS

Oxidation–reduction reactions always involve a pair of chemicals: an electron donor, which is oxidized in the reactions; and an electron acceptor, which is reduced in the reaction. In fuel metabolism, the fuel donates electrons and is oxidized, and NAD⫹ and FAD accept electrons and are reduced. To remember this, think of the acronym LEO GER: Loss of Electrons ⫽ Oxidation; Gain of Electrons ⫽ Reduction. Compounds are oxidized in the body in essentially three ways: (1) the transfer of electrons from the compound as a hydrogen atom or a hydride ion, (2) the direct addition of oxygen from O2, and (3) the direct donation of electrons (e.g., Fe 2⫹ → Fe3⫹) (see Chapter 5). Fuel oxidation involves the transfer of electrons as a hydrogen atom or a hydride ion and, thus, reduced compounds have more hydrogen relative to oxygen than the oxidized compounds. Consequently, aldehydes are more reduced than acids, and alcohols are more reduced than aldehydes. When is NAD⫹, rather than FAD, used in a particular oxidation–reduction reaction? It depends on the chemical properties of the electron donor and the enzyme that catalyzes the reaction. In oxidation reactions, NAD⫹ accepts two electrons as a hydride ion to form NADH, and a proton (H⫹) is released into the medium (Fig. 19.9). It is generally used for metabolic reactions involving oxidation of alcohols and aldehydes. In contrast, FAD accepts two electrons as hydrogen atoms, which are donated singly from separate atoms (e.g., formation of a double bond or a disulfide) (Fig. 19.10).

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H ••

347

O

–

C NH2 +

N

O –

O

P

O

O

CH2 H

H

Nicotinamide H

HO

H

OH NH2

O

C N

C

N

C

N

CH HC –

O

P

O

N O

CH2

O H

H

H

HO

H

OR

NAD+ R=H O NADP+ R= P

O–

O–

FIG. 19.9. Reduction of NAD⫹ and NADP⫹. These structurally related coenzymes are reduced by accepting two electrons as H⫺, the hydride ion.

As the reduced coenzymes donate these electrons to O2 through the electrontransport chain, they are reoxidized. The energy derived from reoxidation of NADH and FAD(2H) is available for the generation of ATP by oxidative phosphorylation. In our analogy of ATP as a currency, the reduced coenzymes are our “paychecks” for oxidizing fuels. Because our cells spend ATP so fast, we must immediately convert our paychecks into ATP cash.

•

H

O

H H3C H3C H

N

C

N

N

N H C

O •

H

CH2

NH2

H C OH Riboflavin

N

H C OH –

H C OH

O

CH2

P

O

O

O O

P

N

H

–

N O H2C

N

H

O

O H H HO

H

H

OH

FIG. 19.10. Reduction of FAD. FAD accepts two electrons as two hydrogen atoms and is reduced. The reduced coenzyme is denoted in this text as FAD(2H) because it often accepts a total of two electrons one at a time, never going to the fully reduced form—FADH2. Flavin mononucleotide (FMN) consists of riboflavin with one phosphate group attached.

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Otto Shape decided to lose weight by decreasing his intake of fat and alcohol (ethanol) and increasing his intake of carbohydrates. Compare the structure of ethanol with that of glucose and fatty acids. Based on their oxidation states, which compound provides the most energy (calories) per gram?

HOH2C

(HC

OH)4

O C H

Glucose CH3CH2OH Ethanol O CH3

(CH2)16

C

A fatty acid

OH

2.

REDUCTION POTENTIAL

Each oxidation–reduction reaction makes or takes a fixed amount of energy, (⌬G0⬘), which is directly proportional to the ⌬E0⬘ (the difference in reduction potentials of the oxidation–reduction pair). The reduction potential of a compound, E0⬘, is a measure in volts of the energy change when that compound accepts electrons (becomes reduced); ⫺⌬E0⬘ is the energy change when the compound donates electrons (becomes oxidized). E0⬘ can be considered an expression of the willingness of the compound to accept electrons. Some examples of reduction potentials are shown in Table 19.4. Oxygen, which is the best electron acceptor, has the largest positive reduction potential (i.e., is the most willing to accept electrons and be reduced). As a consequence, the transfer of electrons from all compounds to O2 is energetically favorable and occurs with energy release. The more negative the reduction potential of a compound, the greater is the energy available for ATP generation when that compound passes its electrons to oxygen. The ⌬G0⬘ for transfer of electrons from NADH to O2 is greater than the transfer from FAD(2H) to O2 (see the reduction potential values for NADH and FAD[2H] in Table 19.4). Thus, the energy available for ATP synthesis from NADH is approximately ⫺53 kcal, and approximately ⫺41 kcal from the FAD-containing flavoproteins in the electron-transport chain. To calculate the free-energy change of an oxidation–reduction reaction, the reduction potential of the electron donor (NADH) is added to that of the acceptor (O2). The ⌬E0⬘ for the net reaction is calculated from the sum of the half-reactions. For NADH donation of electrons, it is ⫹0.320 V, opposite of that shown in Table 19.4 (remember, Table 19.4 shows the E0⬘ for accepting electrons), and for O2 acceptance it is ⫹0.816 V. The number of electrons being transferred is 2 (so, n ⫽ 2). The direct relationship between the energy changes in oxidation–reduction reactions and ⌬G0⬘ is expressed by the Nernst equation Equation 19.3: ⌬G0⬘ ⫽ ⫺n F ⌬E0⬘

where n is the number of electrons transferred and F is Faraday’s constant (23 kcal/ mol-V). Thus, a value of approximately ⫺53 kcal/mol is obtained for the energy available for ATP synthesis by transferring two electrons from NADH to oxygen. The ⌬E0⬘ for FAD(2H) to donate electrons to O2 is 1.016 V compared to a ⌬E0⬘ of 1.136 V for electron transfer from NADH to O2. 3.

CALORIC VALUES OF FUELS

The caloric value of a food is related directly to its oxidation state, which is a measure of ⌬G0⬘ for transfer of electrons from that fuel to O2. The electrons donated by the fuel are from its C–H and C–C bonds. Fatty acids such as palmitate [CH3(CH2)14COOH] have a caloric value of roughly 9 kcal/g. Glucose is already

Table 19.4 Reduction Potentials of Some Oxidation–Reduction Half-Reactions Reduction Half-Reactions ⫹

⫺

½O2 ⫹ 2H ⫹ 2e → H2O Cytochrome a-Fe3⫹ ⫹ 1e⫺ → cytochrome a-Fe2⫹ CoQ ⫹ 2H⫹ ⫹ 2e⫺ → CoQ-H2 Fumarate ⫹ 2H⫹ ⫹ 2e⫺ → succinate Oxalacetate ⫹ 2H⫹ ⫹ 2e⫺ → malate Acetaldehyde ⫹ 2H⫹ ⫹ 2e⫺ → ethanol Pyruvate ⫹ 2H⫹ ⫹ 2e⫺ → lactate Riboflavin ⫹ 2H⫹ ⫹ 2e⫺ → riboflavin-H2 FAD ⫹ 2H⫹ ⫹ 2e⫺ → FAD(2H) NAD⫹ ⫹ 2H⫹ ⫹ 2e⫺ → NADH ⫹ H⫹ Acetate ⫹ 2H⫹ ⫹ 2e⫺ → acetaldehyde

E 0⬘ at pH 7.0 0.816 0.290 0.060 0.030 ⫺0.102 ⫺0.163 ⫺0.190 ⫺0.200 ⫺0.219a ⫺0.320 ⫺0.468

a

This is the value for free FAD; when FAD is bound to a protein, its value can be altered in either direction.

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partially oxidized and has a caloric value of only about 4 kcal/g. The carbons, on average, contain fewer C–H bonds from which to donate electrons. The caloric value of a food is applicable in humans only if our cells have enzymes that can oxidize that fuel by transferring electrons from the fuel to NAD⫹, NADP⫹, or FAD. When we burn wood in a fireplace, electrons are transferred from cellulose and other carbohydrates to O2, releasing energy as heat. However, wood has no caloric content for humans; we cannot digest it and convert cellulose to a form that can be oxidized by our enzymes. Cholesterol, although it is a lipid, also has no caloric value for us because we cannot oxidize the carbons in its complex ring structure in reactions that generate NADH, FAD(2H), or NADPH.

B. NADPH in Oxidation–Reduction Reactions

NADP⫹ is similar to NAD⫹ and has the same reduction potential. However, NADP⫹ has an extra phosphate group on the ribose, which affects its enzyme binding (see Fig. 19.9). Consequently, most enzymes use either NAD⫹ or NADP⫹, but seldom both. In certain reactions, fuels are oxidized by transfer of electrons to NADP⫹ to form NADPH. For example, glucose 6-P dehydrogenase, in the pentose phosphate pathway, transfers electrons from glucose 6-P to NADP⫹ instead of NAD⫹. NADPH usually donates electrons to biosynthetic reactions such as fatty acid synthesis, and to detoxification reactions that use oxygen directly. Consequently, the energy in its reduction potential is usually used in energy-requiring reactions without first being converted to ATP currency.

In palmitate and other fatty acids, most carbons are more reduced than those in glucose or ethanol (more of the carbons have electrons in C–H bonds). Therefore, fatty acids have the greatest caloric content per gram, 9 kcal. In glucose, the carbons have already formed bonds with oxygen, and fewer electrons in C–H bonds are available to generate energy. Thus, the complete oxidation of glucose provides roughly 4 kcal/g. In ethanol, one carbon is a methyl group with C–H bonds, and one has an –OH group. Therefore, the oxidation state is intermediate between those of glucose and fatty acids, and ethanol thus has 7 kcal/g.

Glucose 2 ADP, P i

~P intermediates

C. Anaerobic Glycolysis Not all ATP is generated by fuel oxidation. In anaerobic glycolysis, glucose is degraded in reactions that form high-energy phosphorylated intermediates of the pathway (Fig. 19.11). These activated high-energy intermediates provide the energy for the generation of ATP from ADP without involving electron transfer to O2. Therefore, this pathway is called anaerobic glycolysis, and ATP is generated from substrate-level phosphorylation rather than from oxidative phosphorylation (see Chapter 22). Anaerobic glycolysis is a critical source of ATP for cells that have a decreased O2 supply, either because they are physiologically designed that way (e.g., cells in the kidney medulla, rapidly working muscle), or because their supply of O2 has been pathologically decreased (e.g., coronary artery disease).

349

2 ATP NADH Pyruvate

Lactate

Anaerobic glycolysis

FIG. 19.11. Anaerobic glycolysis. Phosphate is transferred from high-energy intermediates of the pathway to ADP. Because NADH from the pathway is reoxidized by reduction of pyruvate to lactate, no oxygen is required.

VI. OXYGENASES AND OXIDASES NOT INVOLVED IN ATP GENERATION Approximately 90% to 95% of the oxygen we consume is used by the terminal oxidase in the electron-transport chain for ATP generation via oxidative phosphorylation. The remainder of the O2 is used directly by oxygenases and other oxidases, enzymes that oxidize a compound in the body by transferring electrons directly to O2 (Fig. 19.12). The large positive reduction potential of O2 makes all of these reactions extremely favorable thermodynamically, but the electronic structure of O2 slows the speed of electron transfer. These enzymes, therefore, contain a metal ion that facilitates reduction of O2.

A. Oxidases Oxidases transfer electrons from the substrate to O2, which is reduced to water (H2O) or to hydrogen peroxide (H2O2). The terminal protein complex in the electrontransport chain, called cytochrome oxidase, is an oxidase because it accepts electrons donated to the chain by NADH and FAD(2H) and uses these to reduce O2 to H2O. Most of the other oxidases in the cell form H2O2 instead of H2O and are called peroxidases. Peroxidases are generally confined to peroxisomes to protect DNA and other cellular components from toxic free radicals (compounds containing single electrons in an outer orbital) generated by hydrogen peroxide.

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Oxidases O2 + 4e–, 4H+ O2 + SH2

2H2O S + H2O2

Mono-oxygenases O2 + S + Electron donor–XH2 H2O + Electron + donor–X

S

OH

Dioxygenases S

+ O2

SO2

FIG. 19.12. Oxidases and oxygenases. The fate of O2 is shown in red. S represents an organic substrate.

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NADPH + H+

NADP+

FAD FMN CYP-450 Fe-heme

RH, O2

ROH, H2O

FIG. 19.13. Cytochrome P450 monooxygenases. Electrons are donated by NADPH to O2 and the substrate. The flavin coenzymes FAD and FMN in one protein subunit participate in the transfer of single electrons to cytochrome P450, which is an Fe-heme–containing protein that absorbs light at a wavelength of 450 nm. The iron in the heme group aids in facilitating electron transfer to oxygen. The enzyme is embedded in a membrane, usually the endoplasmic reticulum.

B. Oxygenases Oxygenases, in contrast to oxidases, incorporate one or both of the atoms of oxygen into the organic substrate (see Fig. 19.12). Monooxygenases, enzymes that incorporate one atom of oxygen into the substrate and the other into H2O, are often named hydroxylases (e.g., phenylalanine hydroxylase, which adds a hydroxyl group to phenylalanine to form tyrosine) or mixed-function oxidases. Monooxygenases require an electron-donor substrate such as NADPH, a coenzyme such as FAD, which can transfer single electrons, and a metal or similar compound that can form a reactive oxygen complex (Fig. 19.13). They are usually found in the endoplasmic reticulum and occasionally in mitochondria. Dioxygenases, enzymes that incorporate both atoms of oxygen into the substrate, are used in the pathways for converting arachidonate into prostaglandins, thromboxanes, and leukotrienes.

VII. ENERGY BALANCE Our total energy expenditure is equivalent to our oxygen consumption (Fig. 19.14). The resting metabolic rate (energy expenditure of a person at rest, at 25°C, after an overnight fast) accounts for approximately 60% to 70% of our total energy expenditure and O2 consumption, and physical exercise accounts for the remainder. Of the resting metabolic rate, approximately 90% to 95% of O2 consumption is used by the mitochondrial electron-transport chain, and only 5% to 10% is required for nonmitochondrial oxidases and oxygenases and is not related to ATP synthesis. Approximately 20% to 30% of the energy from this mitochondrial O2 consumption is lost by proton leak back across the mitochondrial membrane, which dissipates the electrochemical gradient without ATP synthesis. The remainder of our O2 consumption is used for ATPases that maintain ion gradients and for biosynthetic pathways. ATP homeostasis refers to the ability of our cells to maintain constant levels of ATP despite fluctuations in the rate of use. Thus, increased use of ATP for exercise or biosynthetic reactions increases the rate of fuel oxidation. The major mechanism employed is feedback regulation; all of the pathways of fuel oxidation that lead to

Total oxygen consumption in the Standard state

Total mitochondrial oxygen consumption

Total ATP consumption in the Standard state Protein synthesis

Mitochondrial

Coupled to ATP synthesis

Na+K+ATPase Ca2+ATPase Gluconeogenesis Urea synthesis Myosin ATPase

Nonmitochondrial

Uncoupled by proton leak

Others (including RNA synthesis and substrate cycling)

FIG. 19.14. Estimated contribution of processes to energy use in standard state. (Reproduced, with permission, from Rolfe DFS, Brown GC. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol Rev. 1997;77:731–758.)

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351

generation of ATP are feedback-regulated by ATP levels, or by compounds related to the concentration of ATP. In general, the less ATP used, the less fuel will be oxidized to generate ATP. According to the first law of thermodynamics, the energy (in calories) in our consumed fuel can never be lost. Consumed fuel is either oxidized to meet the energy demands of the BMR ⫹ exercise, or it is stored as fat. Thus, an intake of calories in excess of those expended results in weight gain. The simple statement, “If you eat too much and don’t exercise, you will get fat,” is really a summary of the bioenergetics of the ATP–ADP cycle.

CLINICAL COMMENTS Otto Shape. Otto Shape visited his physician, who noted his increased weight. The physician recommended several diet modifications to Otto that would decrease the caloric content of his diet, and he pointed out the importance of exercise for weight reduction. He reminded Otto that the American Heart Association and the American Cancer Society recommended 45 to 60 minutes of moderate to vigorous exercise 5 to 7 days per week. He also reminded Otto that he would want to be a role model for his patients. Otto decided to begin an exercise regimen that included an hour of running each day. X. S. Teefore. Mr. Teefore exhibited the classical signs and symptoms of hyperthyroidism (increased secretion of the thyroid hormones T3 and T4; see Fig. 11.8 for the structure of T3), including a goiter (enlarged thyroid gland). T3 is the more active form of the hormone. T4 is synthesized and secreted in approximately 10 times greater amounts than T3. Liver and other cells contain an enzyme (a deiodinase) that removes one of the iodines from T4, converting it to T3. Thyroid function tests confirmed this diagnosis. Thyroid hormones (principally T3) modulate cellular energy production and utilization through their ability to increase the gene transcription (see Fig. 16.13) of many proteins involved in intermediary metabolism, including enzymes in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. They increase the rate of adenosine triphosphate (ATP) use by the Na⫹,K⫹ATPase and other enzymes. They also affect the efficiency of energy transformations, so that either more fuel must be oxidized to maintain a given level of ATP or more ATP must be expended to achieve the desired physiologic response. The loss of weight experienced by X. S. Teefore, in spite of a very good appetite, reflects his increased caloric requirements and less efficient use of fuels. The result is enhanced oxidation of adipose tissue stores as well as a catabolic effect on muscle and other protein-containing tissues. Through mechanisms that are not well understood, increased levels of thyroid hormone in the blood also increase the activity or “tone” of the sympathetic (adrenergic) nervous system. An activated sympathetic nervous system leads to a more rapid and forceful heartbeat (tachycardia and palpitations), increased nervousness (anxiety and insomnia), tremulousness (a sense of shakiness or jitteriness), and other symptoms. Cora Nari. Cora Nari was in left ventricular heart failure (LVF) when she presented to the hospital with her second heart attack in 8 months. The symptoms consistent with LVF were her rapid heart rate (104 beats/minute) and respiratory rate. On examining her lungs, her physician heard respiratory rales, caused by inspired air bubbling in fluid that had filled her lung air spaces secondary to LVF. This condition is referred to as congestive heart failure.

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Congestive heart failure occurs when the weakened pumping action of the ischemic left ventricular heart muscle leads to a reduced blood flow from the heart to the rest of the body. This leads to an increase in blood volume in the vessels that bring oxygenated blood from the lungs to the left side of the heart. The pressure inside these pulmonary vessels eventually reaches a critical level, greater than which water from the blood moves down a “pressure gradient” from the capillary lumen into alveolar air spaces of the lung (transudation). The patient experiences shortness of breath as the fluid in the air spaces interferes with oxygen exchange from the inspired air into arterial blood, causing hypoxia. The hypoxia then stimulates the respiratory center in the central nervous system, leading to a more rapid respiratory rate in an effort to increase the oxygen content of the blood. As the patient inhales deeply, the physician hears gurgling/crackling sounds (known as inspiratory rales) with a stethoscope placed over the posterior lung bases. These sounds represent the bubbling of inspired air as it enters the fluid-filled pulmonary alveolar air spaces.

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Cora’s rapid heart rate (tachycardia) resulted from a reduced capacity of her ischemic, failing left ventricular muscle to eject a normal amount of blood into the arteries leading away from the heart with each contraction. The resultant drop in intra-arterial pressure signaled a reflex response in the central nervous system that, in turn, caused an increase in heart rate in an attempt to bring the total amount of blood leaving the left ventricle each minute (the cardiac output) back toward a more appropriate level to maintain systemic blood pressure. Initial treatment of Cora’s congestive heart failure will include efforts to reduce the workload of the heart with diuretics and other “load reducers,” attempts to improve the force of left ventricular contraction with digitalis, and the administration of oxygen by nasal cannula to increase the oxygen levels to the viable heart tissue in the vicinity of the infarction.

BIOCHEMICAL COMMENTS Hypoxia Decreased mitochondrial electron transport chain Decreased ATP and adenine nucleotides Increased Na+

Increased Ca2+

Cellular swelling Increased plasma membrane permeability

Mitochondrial permeability transition

FIG. 19.15. Hypoxia, Ca2⫹, Na⫹, and cell death. Without an adequate O2 supply, decreased ATP synthesis from oxidative phosphorylation results in an increase of cytoplasmic Na⫹ and Ca2⫹ ions. Increased ion levels can trigger death cascades that involve increased permeability of the plasma membrane, loss of ion gradients, decreased cytosolic pH, mitochondrial Ca2⫹ overload, and a change in mitochondrial permeability called the mitochondrial permeability transition. The solid lines show the first sequence of events; the dashed lines show how these events feed back to accelerate the mitochondrial deterioration, making recovery of oxidative phosphorylation impossible.

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Active Transport and Cell Death. Most of us cannot remember when we first learned that we would die if we stopped breathing. However, exactly how cells die from a lack of oxygen is an intriguing question. Hypoxia leads to both physical and transcriptional changes. Pathologists generally describe two histologically distinct types of cell death: necrosis and apoptosis (programmed cell death). Cell death from a lack of O2, such as that occurs during a myocardial infarction, can be very rapid, and is considered necrosis. The lack of adenosine triphosphate (ATP) for the active transport of Na⫹ and Ca2⫹ triggers some of the death cascades that lead to necrosis (Fig. 19.15). The influx of Na⫹ and loss of the Na⫹ gradient across the plasma membrane is an early event accompanying ATP depletion during interruption of the O2 supply. One consequence of the increased intracellular Na⫹ concentration is that other transport processes driven by the Na⫹ gradient are impaired. For example, the Na⫹/H⫹ exchanger, which normally pumps out H⫹ generated from metabolism in exchange for extracellular Na⫹, can no longer function, and intracellular pH may drop. The increased intracellular H⫹ may impair ATP generation from anaerobic glycolysis. As a consequence of increased intracellular ion concentrations, water enters the cells and hydropic swelling occurs. Swelling is accompanied by the release of creatine kinase MB subunits, troponin I, and troponin C into the blood. These enzymes are measured in the blood as indicators of a myocardial infarction (see Chapters 6 and 7). Swelling is an early event and is considered a reversible stage of cell injury. Normally, intracellular Ca2⫹ concentration is carefully regulated to fluctuate at low levels (intracellular Ca2⫹ concentration is ⬍107 M, compared to approximately 103 M in extracellular fluid). Fluctuations of Ca2⫹ concentration at these low levels regulate myofibrillar contraction, energy metabolism, and other cellular processes. However, when Ca2⫹ concentration is increased greater than this normal range, it triggers cell death (necrosis). High Ca2⫹ concentrations activate a phospholipase that increases membrane permeability, resulting in further loss of ion gradients across the cell membrane. They also trigger opening of the mitochondrial permeability transition pore, which results in loss of mitochondrial function and further impairs oxidative phosphorylation. Intracellular Ca2⫹ levels may increase because of cell swelling, the lack of ATP for ATP-dependent Ca2⫹ pumps, or the loss of the Na⫹ gradient. Normally, Ca2⫹-ATPases located in the plasma membrane pump Ca2⫹ out of the cell. Ca2⫹-ATPases in the endoplasmic reticulum, and in the sarcoplasmic reticulum of heart and other muscles, sequester Ca2⫹ within the membranes, where it is

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353

bound by a low-affinity binding protein. Ca2⫹ is released from the sarcoplasmic reticulum in response to a nerve impulse, which signals contraction, and the increase of Ca2⫹ stimulates both muscle contraction and the oxidation of fuels. Within the heart, another Ca2⫹ transporter protein, the Na⫹/Ca2⫹ exchange transporter, coordinates the efflux of Ca2⫹ in exchange for Na⫹, so that Ca2⫹ is extruded with each contraction. Hypoxia also induces the transcription of genes in an attempt to compensate for the hypoxic conditions. A family of transcription factors, known as hypoxiainducible factors (HIFs), is activated under hypoxic conditions. These factors bind to hypoxia-responsive elements (promoter-proximal elements) in the regulatory region of target genes. More than 70 target genes are regulated by HIFs, including the gene for erythropoietin, which stimulates increased red blood cell production. Induction of these genes allows cells to adapt to and survive for some time under these hypoxic conditions. Key Concepts • • • • • •

• • • • • • •

•

Bioenergetics refers to cellular energy transformations. The high-energy phosphate bonds of ATP are a cell’s primary source of energy. ATP is generated through cellular respiration—the oxidation of fuels to carbon dioxide and water. ATP can also be generated, at reduced levels, via anaerobic glycolysis (in the absence of O2). The electrons captured from fuel oxidation generate NADH and FAD(2H), which are used to regenerate ATP via the process of oxidative phosphorylation. The energy available from ATP hydrolysis can be used for: Mechanical work (muscle contraction) Transport work (establishment of ion gradients across membranes) Biochemical work (energy-requiring chemical reactions, including detoxification reactions) Energy released from fuel oxidation that is not used for work is transformed into and released as heat. The many pathways of fuel oxidation are coordinately regulated to maintain ATP homeostasis. ⌬G0⬘ is the change in Gibbs free energy at pH 7.0 under standard conditions between the substrates and products of a reaction. Fuel oxidation has a negative ⌬G0⬘; the products formed have less chemical energy than the reactants (an exergonic reaction pathway). ATP synthesis has a positive ⌬G0⬘ and is endergonic; the reaction requires energy. Metabolic pathways have an overall negative ⌬G0⬘, which is obtained by summing all of the ⌬G0⬘ values for each reaction in the pathway. Oxidation–reduction reactions can be related to changes in free energy, the use of E0⬘, the chemical’s affinity for electrons. Compounds with higher E0⬘ values have greater affinity for electrons than those with lower E0⬘ values. Diseases discussed in this chapter are summarized in Table 19.5.

Table 19.5

Diseases Presented in this Chapter

Disease or Disorder

Environmental or Genetic

Obesity

Both

Hyperthyroidism

Environmental

Heart attack

Both

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Comments Understanding daily caloric needs can enable one to gain or lose weight through alterations in exercise and eating habits. Thyroid hormone is important in regulating energy metabolism; excessive T3 and T4 release enhance metabolism, leading to weight loss and a greater rate of heat production. The heart requires a constant level of energy, derived primarily from lactate, glucose, and fatty acids. This is necessary so that the rate of contraction can remain constant, or increase during appropriate periods. Interference of oxygen flow to certain areas of the heart will reduce energy generation, leading to a myocardial infarction.

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REVIEW QUESTIONS—CHAPTER 19 1.

The highest energy phosphate bond in ATP is located between which of the following groups: A. Adenosine and phosphate B. Ribose and phosphate C. Ribose and adenine D. Two hydroxyl groups in the ribose ring E. Two phosphate groups

2.

Which of the following bioenergetic terms or phrases is defined correctly? A. The first law of thermodynamics states that the universe tends toward a state of increased order. B. The second law of thermodynamics states that the total energy of a system remains constant. C. The change in enthalpy of a reaction is a measure of the total amount of heat that can be released from changes in the chemical bonds. D. ⌬G0⬘ of a reaction is the standard free-energy change measured at 37°C and a pH of 7.4. E. A high-energy bond is a bond that releases more than 3 kcal/mol of heat when it is hydrolyzed.

3.

Which statement best describes the direction a chemical reaction will follow? A. A reaction with positive free energy will proceed in a forward direction if the substrate concentration is raised high enough. B. Under standard conditions, a reaction will proceed in a forward direction if the free energy ⌬G0⬘ is positive.

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C. The direction of a reaction is independent of the initial substrate and product concentrations, because the direction is determined by the change in free energy. D. The concentration of all of the substrates must be higher than that of all of the products for the reaction to proceed in a forward direction. E. The enzyme for the reaction must be working at >50% of its maximum efficiency for the reaction to proceed in a forward direction. 4.

A patient, Mr. Perkins, has just suffered a heart attack. As a consequence, his heart will display which of the following changes? A. Increased intracellular O2 concentration B. Increased intracellular ATP concentration C. Increased intracellular H⫹ concentration D. Decreased intracellular Ca2⫹ concentration E. Decreased intracellular Na⫹ concentration

5.

Which of the following statements correctly describes reduction of one of the electron carriers, NAD⫹ or FAD? A. NAD⫹ accepts two electrons as hydrogen atoms to form NAD(2H). B. NAD⫹ accepts two electrons that are each donated from a separate atom of the substrate. C. NAD⫹ accepts two electrons as a hydride ion to form NADH. D. FAD releases a proton as it accepts two electrons. E. FAD must accept two electrons at a time.

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20

Tricarboxylic Acid Cycle

The tricarboxylic acid cycle (TCA cycle) accounts for more than two-thirds of the adenosine triphosphate (ATP) generated from fuel oxidation. The pathways for oxidation of fatty acids, glucose, amino acids, acetate, and ketone bodies all generate acetyl coenzyme A (acetyl-CoA), which is the substrate for the TCA cycle. As the activated two-carbon acetyl group is oxidized to two molecules of CO2, energy is conserved as NADH, FAD(2H), and guanosine triphosphate (GTP) (Fig. 20.1). NADH and FAD(2H) subsequently donate electrons to O2 via the electron-transport chain, with the generation of ATP from oxidative phosphorylation. Thus, the TCA cycle is central to energy generation from cellular respiration. Within the TCA cycle, the oxidative decarboxylation of ␣-ketoglutarate is catalyzed by the multisubunit ␣-ketoglutarate dehydrogenase complex, which contains the coenzymes thiamine pyrophosphate (TTP), lipoate, and flavin adenine dinucleotide (FAD). A similar complex, the pyruvate dehydrogenase complex (PDC), catalyzes the oxidation of pyruvate to acetyl-CoA, thereby providing a link between the pathways of glycolysis and the TCA cycle (see Fig. 20.1). The two-carbon acetyl group is the ultimate source of the electrons that are transferred to nicotinamide adenine dinucleotide (NAD⫹) and FAD and also the carbon in the two CO2 molecules that are produced. Oxaloacetate is used and regenerated in each turn of the cycle (see Fig. 20.1). However, when cells use intermediates of the TCA cycle for biosynthetic reactions, the carbons of oxaloacetate must be replaced by anaplerotic (filling-up) reactions, such as the pyruvate carboxylase reaction. The TCA cycle occurs in the mitochondrion, where its flux is tightly coordinated with the rate of the electron-transport chain and oxidative phosphorylation through feedback regulation that reflects the demand for ATP. The rate of the TCA cycle is increased when ATP utilization in the cell is increased through the response of several enzymes to adenosine diphosphate (ADP) levels, the NADH/NAD⫹ ratio, the rate of FAD(2H) oxidation, or the Ca2⫹ concentration. For example, isocitrate dehydrogenase is allosterically activated by ADP. There are two general consequences to impaired functioning of the TCA cycle: (1) an inability to generate ATP from fuel oxidation and (2) an accumulation of TCA cycle precursors. For example, inhibition of pyruvate oxidation in the TCA cycle results in its reduction to lactate, which can cause lactic acidosis. The most common situation leading to an impaired function of the TCA cycle is a relative lack of oxygen to accept electrons in the electron-transport chain.

355

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

Glucose

Fatty acids

Pyruvate

Ketone bodies

CO2

NADH + H+

Amino acids

Acetyl CoA

Acetate

CoASH

Oxaloacetate (4c)

Citrate (6c)

Malate (4c)

Isocitrate (6c)

Fumarate (4c) Tricarboxylic acid (TCA) cycle Succinate (4c)

NADH + H+

FAD (2H)

CO2

␣-Ketoglutarate (5c) GTP GDP

SuccinylCoA (4c)

NADH + H+ CO2

Net reaction 2CO2 + CoASH + 3NADH + 3H+ Acetyl CoA + 3NAD+ + FAD + FAD (2H) + GTP + GDP + Pi + 2H2O

FIG. 20.1. Summary of the tricarboxylic acid (TCA) cycle. The number of carbons in each intermediate of the cycle is indicated in parentheses by the name of the compound.

Confirmation of a suspected thiamine deficiency requires measuring thiamine levels. A standard assay for determining if thiamine levels are sufficient for metabolic processes is to use the enzyme transketolase, which requires thiamine pyrophosphate (TPP) for activity (see Chapter 29). Transketolase can be obtained from red blood cells, making sample collection relatively straightforward. The measurement of transketolase activity is done in the absence and presence of exogenous TPP. If the difference in activity levels is ⬎25%, then a thiamine deficiency is confirmed. Other more time-consuming assays for thiamine include high-performance liquid chromatography (HPLC) analysis to quantify the levels of both free thiamine and the active form, TPP, in biologic fluids, and microbiologic assays that measure bacterial cell growth in the presence of a sample that contains thiamine. Because the bacteria require thiamine for growth, samples with more thiamine will stimulate more cell growth than samples that contain lesser levels of the vitamin.

Lieberman_Ch20.indd 356

THE WAITING ROOM Otto Shape, a 26-year-old medical student, has faithfully followed his diet and aerobic exercise program of daily tennis and jogging (see Chapter 19). He has lost a total of 33 lb and is just 23 lb from his college weight of 154 lb. His exercise capacity has markedly improved; he can run for a longer time at a faster pace before noting shortness of breath or palpitations of his heart. Even his test scores in his medical school classes have improved. Ann O’Rexia suffers from anorexia nervosa (see Chapters 1, 3, and 9). In addition to a low body weight, decreased muscle mass, glycogen, and fat stores, she has iron deficiency anemia (see Chapter 16). She has started to gain weight and is trying a daily exercise program. However, she constantly feels weak and tired. When she walks, she feels pain in her calf muscles. On this visit to her nutritionist, they discuss the vitamin content of her diet and its role in energy metabolism. Al Martini has been hospitalized for congestive heart failure (see Chapter 8) and for head injuries sustained while driving under the influence of alcohol (Chapters 9 and 10). He completed an alcohol detoxification program, enrolled in a local Alcoholics Anonymous (AA) group, and began seeing a psychologist. During this time, his alcohol-related neurologic and cardiac manifestations of thiamine deficiency partially cleared. However, in spite of the support he was receiving, he began drinking excessive amounts of alcohol again while eating poorly. Three weeks later, he was readmitted with symptoms of “high-output” heart failure.

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CHAPTER 20 ■ TRICARBOXYLIC ACID CYCLE

I.

357

OVERVIEW OF THE TRICARBOXYLIC ACID CYCLE

The tricarboxylic acid (TCA) cycle is frequently called the Krebs cycle because Sir Hans Krebs first formulated its reactions into a cycle. It is also called the citric acid cycle because citrate was one of the first compounds known to participate. The most common name for this pathway, the tricarboxylic acid or TCA cycle, denotes the involvement of the tricarboxylates citrate and isocitrate. In order for the body to generate large amounts of adenosine triphosphate (ATP), the major pathways of fuel oxidation generate acetyl coenzyme A (acetylCoA), which is the substrate for the TCA cycle. In the first step of the TCA cycle, the acetyl portion of acetyl-CoA combines with the four-carbon intermediate oxaloacetate to form citrate (six carbons), which is rearranged to form isocitrate. In the next two oxidative decarboxylation reactions, electrons are transferred to nicotinamide adenine dinucleotide (NAD⫹) to form NADH, and two molecules of electron-depleted CO2 are released. Subsequently, a high-energy phosphate bond in guanosine triphosphate (GTP) is generated from substrate-level phosphorylation. In the remaining portion of the TCA cycle, succinate is oxidized to oxaloacetate with the generation of one FAD(2H) and one NADH. The net reaction of the TCA cycle, which is the sum of the equations for individual steps, shows that the two carbons of the acetyl group have been oxidized to two molecules of CO2, with conservation of energy as three molecules of NADH, one of FAD(2H), and one of GTP. The TCA cycle requires a large number of vitamins and minerals to function. These include niacin (NAD⫹), riboflavin (flavin adenine dinucleotide [FAD] and flavin mononucleotide [FMN]), pantothenic acid (coenzyme A), thiamine, Mg2⫹, Ca2⫹, Fe2⫹, and phosphate.

II. REACTIONS OF THE TRICARBOXYLIC ACID CYCLE

Lieberman_Ch20.indd 357

H O •• H C • • C ~ SCoA •• H ••

In the TCA cycle, the two-carbon acetyl group of acetyl-CoA is oxidized to two CO2 molecules (see Fig. 20.1). The function of the cycle is to conserve the energy from this oxidation, which it accomplishes principally by transferring electrons from intermediates of the cycle to NAD⫹ and FAD. The eight electrons donated by the acetyl group eventually end up in three molecules of NADH and one of FAD(2H) (Fig. 20.2). As a consequence, ATP can be generated from oxidative phosphorylation when NADH and FAD(2H) donate these electrons to O2 via the electron-transport chain. Initially, the acetyl group is incorporated into citrate, an intermediate of the TCA cycle (Fig. 20.3). As citrate progresses through the cycle to oxaloacetate, it is oxidized by four dehydrogenases (isocitrate dehydrogenase, ␣-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase), which remove electron-containing hydrogen or hydride atoms from a substrate and transfer them to electron-accepting coenzymes such as NAD⫹ or FAD. The isomerase aconitase rearranges electrons in citrate, thereby forming isocitrate to facilitate an electron transfer to NAD⫹. An iron cofactor in aconitase facilitates the isomerization. Although no O2 is introduced into the TCA cycle, the two molecules of CO2 produced have more oxygen than the acetyl group. These oxygen atoms are ultimately derived from the carbonyl group of acetyl-CoA, two molecules of water added by fumarase and citrate synthase and the PO42⫺ added to guanosine diphosphate (GDP). The overall yield of energy-containing compounds from the TCA cycle is three NADH, one FAD(2H), and one GTP. The high-energy phosphate bond of GTP is generated from substrate-level phosphorylation catalyzed by succinate thiokinase (succinyl-CoA synthetase). As the NADH and FAD(2H) are reoxidized in the electron-transport chain, approximately 2.5 ATP are generated for each NADH and 1.5 ATP for the FAD(2H). Consequently, the net energy yield from the TCA cycle and oxidative phosphorylation is about 10 high-energy phosphate bonds for each acetyl group oxidized.

Acetyl CoA

FIG. 20.2. The acetyl group of acetyl-CoA. Acetyl-CoA donates eight electrons to the TCA cycle, which are shown in red, and two carbons. The high-energy bond is shown by a ⬃. The acetyl group is the ultimate source of the carbons in the two molecules of CO2 that are produced and the source of electrons in the one molecule of FAD(2H) and three molecules of NADH, which have each accepted two electrons. However, the same carbon atoms and electrons that enter from one molecule of acetyl-CoA do not leave as CO2, NADH, or FAD(2H) within the same turn of the cycle.

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

CH3C COO C

Acetyl CoA

–

CoASH

O

–

COO Oxaloacetate

CH2

H2O

HO

COO– Aconitase

–

COO CH

C

CH2 COO Citrate

–

HO

COO–

Citrate synthase

CH2 Malate dehydrogenase

O SCoA

NADH + H+

NAD+

COO– CH2

CH2 H

C

COO

HO

C

H

–

COO Malate

COO– Isocitrate

ElectronH2O

transport

ATP

chain

Fumarase

COO–

NAD+

Oxidative phosphorylation

HC

H2O

CO2

NADH + H+

O2

COO–

CH

Isocitrate dehydrogenase

CH2 FAD(2H) FAD

COO–

Succinate dehydrogenase

CH2

NADH + H+

C NAD+

COO–

CH2 CoASH

CH2

CH2

–

Succinate thiokinase

CO2

CH2

COO Succinate

GDP + Pi GTP

C

O

COO– ␣–Ketoglutarate

CoASH O

␣-Ketoglutarate dehydrogenase

˜

COO– Fumarate

–

SCoA Succinyl CoA

FIG. 20.3. Reactions of the TCA cycle. The oxidation–reduction enzymes and coenzymes are shown in red. Entry of the two carbons of acetyl-CoA into the TCA cycle are indicated with the green box. The carbons released as CO2 are shown with yellow boxes.

A. Formation and Oxidation of Isocitrate Otto Shape’s exercise program increases his rate of ATP utilization and his rate of fuel oxidation in the TCA cycle. The TCA cycle generates NADH and FAD(2H), and the electron-transport chain transfers electrons from NADH and FAD(2H) to O2, thereby creating the electrochemical potential that drives ATP synthesis from ADP. As ATP is used in the cell, the rate of the electron-transport chain increases. The TCA cycle and other fuel oxidative pathways respond by increasing their rates of NADH and FAD(2H) production.

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The TCA cycle begins with condensation of the activated acetyl group and oxaloacetate to form the six-carbon intermediate citrate, a reaction that is catalyzed by the enzyme citrate synthase (see Fig. 20.3). Synthases, in general, catalyze the condensation of two organic molecules to form a carbon–carbon bond in the absence of high-energy phosphate bond energy. A synthetase catalyzes the same type of reaction, but requires high-energy phosphate bonds to complete the reaction. Because oxaloacetate is regenerated with each turn of the cycle, it is not really considered a substrate of the cycle or a source of electrons or carbon. In the next step of the TCA cycle, the hydroxyl (alcohol) group of citrate is moved to an adjacent carbon so that it can be oxidized to form a keto group. The isomerization of citrate to isocitrate is catalyzed by the enzyme aconitase, which is named for an intermediate of the reaction. The enzyme isocitrate dehydrogenase catalyzes the oxidation of the alcohol group and the subsequent cleavage

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359

of the carboxyl group to release CO2 (an oxidation followed by a decarboxylation) forming ␣-ketoglutarate.

B. ␣-Ketoglutarate to Succinyl Coenzyme A

The next step of the TCA cycle is the oxidative decarboxylation of ␣-ketoglutarate to succinyl-CoA, catalyzed by the ␣-ketoglutarate dehydrogenase complex (see Fig. 20.3). The dehydrogenase complex contains the coenzymes thiamine pyrophosphate (TPP), lipoic acid, and FAD. In this reaction, one of the carboxyl groups of ␣-ketoglutarate is released as CO2, and the adjacent keto group is oxidized to the level of an acid, which then combines with CoASH to form succinyl-CoA (see Fig. 20.3). Energy from the reaction is conserved principally in the reduction state of NADH, with a smaller amount present in the high-energy thioester bond of succinyl-CoA.

From Figure 20.3, which enzymes in the TCA cycle release CO2? How many moles of oxaloacetate are consumed in the TCA cycle for each mole of CO2 produced?

C. Generation of Guanosine Triphosphate Energy from the succinyl-CoA thioester bond is used to generate GTP from GDP and inorganic phosphate (Pi) in the reaction catalyzed by succinate thiokinase (also known as succinyl-CoA synthetase, for the reverse reaction) (see Fig. 20.3). This reaction is an example of substrate-level phosphorylation. By definition, substratelevel phosphorylation is the formation of a high-energy phosphate bond where none previously existed without the use of molecular O2 (in other words, not oxidative phosphorylation). The high-energy phosphate bond of GTP is energetically equivalent to that of ATP and can be used directly for energy-requiring reactions such as protein synthesis.

D. Oxidation of Succinate to Oxaloacetate Up to this stage of the TCA cycle, two carbons have been stripped of their available electrons and released as CO2. Two pairs of these electrons have been transferred to two NAD⫹, and one GTP has been generated. However, two additional pairs of electrons arising from acetyl-CoA still remain in the TCA cycle as part of succinate. The remaining steps of the TCA cycle transfer these two pairs of electrons to FAD and NAD⫹ and add H2O, thereby regenerating oxaloacetate. The sequence of reactions that converts succinate to oxaloacetate begins with the oxidation of succinate to fumarate (see Fig. 20.3). Single electrons are transferred from the two adjacent –CH2– methylene groups of succinate to an FAD bound to succinate dehydrogenase, thereby forming the double bond of fumarate. From the reduced enzyme-bound FAD, the electrons are passed into the electron-transport chain. An –OH– group and a proton from water add to the double bond of fumarate, converting it to malate. In the last reaction of the TCA cycle, the alcohol group of malate is oxidized to a keto group through the donation of electrons to NAD⫹. With regeneration of oxaloacetate, the TCA cycle is complete; the chemical bond energy, carbon, and electrons donated by the acetyl group have been converted to CO2, NADH, FAD(2H), GTP, and heat. The succinate-to-oxaloacetate sequence of reactions—oxidation through formation of a double bond, addition of water to the double bond, and oxidation of the resultant alcohol to a ketone—is found in many oxidative pathways in the cell, such as the pathways for the oxidation of fatty acids and oxidation of the branched-chain amino acids.

Ann O’Rexia has been malnourished for some time and has developed subclinical deficiencies of many vitamins, including riboflavin. The coenzymes FAD and FMN are synthesized from the vitamin riboflavin. Riboflavin is actively transported into cells where the enzyme flavokinase adds a phosphate to form FMN. FAD synthetase then adds AMP to form FAD. FAD is the major coenzyme in tissues and is generally found tightly bound to proteins, with about 10% being covalently bound. Its turnover in the body is very slow, and people can live for long periods on low intakes without displaying any signs of a riboflavin deficiency.

III. COENZYMES OF THE TRICARBOXYLIC ACID CYCLE The enzymes of the TCA cycle rely heavily on coenzymes for their catalytic function. Isocitrate dehydrogenase and malate dehydrogenase use NAD⫹ as a coenzyme, and succinate dehydrogenase uses FAD. Citrate synthase catalyzes a reaction that uses a CoA derivative, acetyl-CoA. The ␣-ketoglutarate dehydrogenase complex uses TPP, lipoate, and FAD as bound coenzymes, and NAD⫹ and CoASH as

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

1e–, H+

H

O CH3

N

CH3

N

NH

1e–, H+

O

N

1e–,

CH2

H+

Riboflavin

HCOH

FMN

Single electron

HCOH

CH3

N+

CH3

N

H

–

O

•

NH N

O

Single electron 1e–, H+

O

CH3

N

CH3

N

N H

NH

R

R

FADH•

FADH2

(half-reduced semiquinone)

(fully reduced)

O

HCOH CH2 FAD

O –

O

P

O

O –

O

P

NH2

O

N

N

O CH2 H

H

N

O H

N

H

OH OH Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)

FIG. 20.4. One-electron steps in the reduction of FAD. When FAD and FMN accept single electrons, they are converted to the half-reduced semiquinone, a semistable free-radical form. They can also accept two electrons to form the fully reduced form, FADH2. However, in most dehydrogenases, FADH2 is never formed. Instead, the first electron is shared with a group on the protein as the next electron is transferred. Therefore, in this text, overall acceptance of two electrons by FAD has been denoted by the more general abbreviation, FAD(2H).

Isocitrate dehydrogenase releases the first CO2, and ␣-ketoglutarate dehydrogenase releases the second CO2. There is no net consumption of oxaloacetate in the TCA cycle—the first step uses an oxaloacetate, and the last step produces one. The use and regeneration of oxaloacetate is the “cycle” part of the TCA cycle.

One of Otto Shape’s tennis partners told him that he had heard about a health food designed for athletes that contained succinate. The advertisement made the claim that succinate would provide an excellent source of energy during exercise because it could be metabolized directly without oxygen. Do you see anything wrong with this statement?

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substrates. Each of these coenzymes has unique structural features that enable it to fulfill its role in the TCA cycle.

A. Flavin Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide

Both FAD and NAD⫹ are electron-accepting coenzymes. Why is FAD used in some reactions and NAD⫹ in others? Their unique structural features enable FAD and NAD⫹ to act as electron acceptors in different types of reactions and to play different physiologic roles in the cell. FAD is able to accept single electrons (H•) and forms a half-reduced single-electron intermediate (Fig. 20.4). It thus participates in reactions in which single electrons are transferred independently from two different atoms, which occurs in double-bond formation (e.g., succinate to fumarate) and disulfide bond formation (e.g., lipoate to lipoate disulfide in the ␣-ketoglutarate dehydrogenase reaction). In contrast, NAD⫹ accepts a pair of electrons as the hydride ion (H⫺), which is attracted to the carbon opposite the positively charged pyridine ring (Fig. 20.5). This occurs, for example, in the oxidation of alcohols to ketones by malate dehydrogenase and isocitrate dehydrogenase. The nicotinamide ring accepts a hydride ion from the C–H bond, and the alcoholic hydrogen is released into the medium as a positively charged proton, H⫹. The free radical, single-electron forms of FAD are very reactive, and FADH can lose its electron through exposure to water or the initiation of chain reactions. As a consequence, FAD must remain very tightly, sometimes covalently, attached to its enzyme while it accepts and transfers electrons to another group bound on the

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COO–

COO–

CH2 H

H O

C

COO

C

–

O

O

– O COO

H

C NH2

C NH2

+

N

␣-Ketoglutarate

CH2

Isocitrate dehydrogenase

C H COO

CH2

CO2

–

••

Isocitrate

+

H+

N

R

R +

NAD

NADH

FIG. 20.5. Oxidation and decarboxylation of isocitrate. The alcohol group (C–OH) is oxidized to a ketone, with the C–H electrons donated to NAD⫹ as the hydride ion. Subsequent electron shifts in the pyridine ring remove the positive charge. The H of the ⫺OH group dissociates into water as a proton, H⫹. NAD⫹, the electron acceptor, is reduced.

enzyme (Fig. 20.6). Because FAD interacts with many functional groups on amino acid side chains in the active site, the E0⬘ for enzyme-bound FAD varies greatly and can be greater or much less than that of NAD⫹. In contrast, NAD⫹ and NADH are more like substrate and product than coenzymes. NADH plays a regulatory role in balancing energy metabolism that FAD(2H) cannot because FAD(2H) remains attached to its enzyme. Free NAD⫹ binds to a dehydrogenase and is reduced to NADH that is then released into the medium where it can bind and inhibit a different dehydrogenase. Consequently, oxidative enzymes are controlled by the NADH/NAD⫹ ratio and do not generate NADH faster than it can be reoxidized in the electron-transport chain. The regulation of the TCA cycle and other pathways of fuel oxidation by the NADH/NAD⫹ ratio is part of the mechanism for coordinating the rate of fuel oxidation to the rate of ATP use.

B. Role of Coenzyme A in the Tricarboxylic Acid Cycle CoASH, the acylation coenzyme, participates in reactions through the formation of a thioester bond between the sulfur (S) of CoASH and an acyl group (e.g., acetylCoA, succinyl-CoA) (Fig. 20.7). The complete structure of CoASH and its vitamin

A

O OAA

O CH3

C

~ SCoA

HO

Acetyl CoA

B O

O –

O

C

CH2

HS-CoA

CH2

Succinyl CoA

C~

Pi

C

CH2

C

O – O

Succinate

Fumarate

His-FAD Fe-S CoQ ETC acceptor

Inner mitochondrial membrane

CoQH2 Succinate dehydrogenase

FIG. 20.6. Succinate dehydrogenase contains covalently bound FAD. As a consequence, succinate dehydrogenase and similar flavoproteins reside in the inner mitochondrial membrane where they can transfer electrons directly into the electron-transport chain. The electrons are transferred from the covalently bound FAD to an Fe–S complex on the enzyme, and then to coenzyme Q in the electron-transport chain (see Chapter 21). Thus, FAD does not have to dissociate from the enzyme to transfer its electrons. All the other enzymes of the TCA cycle are found in the mitochondrial matrix.

C – O O Citrate GTP

CoASH

O

O –

SCoA

O–

CH2

Citrate synthase

GDP

C

The claim that succinate oxidation can produce energy without oxygen is wrong. It is probably based on the fact that succinate is oxidized to fumarate by the donation of electrons to FAD. However, ATP can be generated from this process only when these electrons are donated to oxygen in the electron-transport chain. The energy generated by the electron-transport chain is used for ATP synthesis in the process of oxidative phosphorylation. After the covalently bound FAD(2H) is oxidized back to FAD by the electron-transport chain, succinate dehydrogenase can oxidize another succinate molecule.

O

C

CH2

CH2

C O–

Succinate

FIG. 20.7. Utilization of the high-energy thioester bond of acyl-CoAs. Energy transformations are shown in red. A. The energy released by hydrolysis of the thioester bond of acetyl coenzyme A (acetyl-CoA) in the citrate synthase reaction contributes a large negative ⌬G0⬘ to the forward direction of the TCA cycle. B. The energy of the succinyl-CoA thioester bond is used for the synthesis of the high-energy phosphate bond of GTP.

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CoASH is synthesized from the vitamin pantothenate in a sequence of reactions that phosphorylate pantothenate, add the sulfhydryl portion of CoA from cysteine, and then add AMP and an additional phosphate group from ATP (see Fig. 8.12A). Pantothenate is widely distributed in foods (“pantos” means everywhere), so it is unlikely that Ann O’Rexia has developed a pantothenate deficiency. Although CoA is required in approximately 100 different reactions in mammalian cells, no Recommended Daily Allowance (RDA) has been established for pantothenate, in part, because indicators have not yet been found that specifically and sensitively reflect a deficiency of this vitamin in the human. The reported symptoms of pantothenate deficiency (fatigue, nausea, and loss of appetite) are characteristic of vitamin deficiencies in general.

precursor, pantothenate, is shown in Figure 8.12A. A thioester bond differs from a typical oxygen ester bond because S, unlike O, does not share its electrons and participate in resonance formations. One of the consequences of this feature of sulfur chemistry is that the carbonyl carbon, the ␣-carbon, and the ␤-carbon of the acyl group in a CoA thioester can be activated for participation in different types of reactions (e.g., in the citrate synthase reaction, the ␣-carbon methyl group is activated for condensation with oxaloacetate; see Figs. 20.3 and 20.7A). Another consequence is that the thioester bond is a high-energy bond that has a large negative ⌬G0⬘ of hydrolysis (approximately ⫺13 kcal/mol). The energy from cleavage of the high-energy thioester bonds of succinylCoA and acetyl-CoA is used in two different ways in the TCA cycle. When the succinyl-CoA thioester bond is cleaved by succinate thiokinase, the energy is used directly for activating an enzyme-bound phosphate that is transferred to GDP (see Fig. 20.7B). In contrast, when the thioester bond of acetyl-CoA is cleaved in the citrate synthase reaction, the energy is released, giving the reaction a large negative ⌬G0⬘ of ⫺7.7 kcal/mol. The large negative ⌬G0⬘ for citrate formation helps to keep the TCA cycle going in the forward direction.

C. The ␣-Keto Acid Dehydrogenase Complexes

The ␣-ketoglutarate dehydrogenase complex is one of a three-member family of similar ␣-keto acid dehydrogenase complexes. The other members of this family are the pyruvate dehydrogenase complex (PDC) and the branched-chain amino acid ␣-keto acid dehydrogenase complex. Each of these complexes is specific for a different ␣-keto acid structure. In the sequence of reactions catalyzed by the complexes, the ␣-keto acid is decarboxylated (i.e., releases the carboxyl group as CO2) (Fig. 20.8). The keto group is oxidized to the level of a carboxylic acid and then combined with CoASH to form an acyl-CoA thioester (e.g., succinyl-CoA). ␦ COO– ␥ CH2 ␤ CH2 ␣ C O COO

–

␣-Ketoglutarate NAD+ CoASH

Thiamine– P P lipoate FAD ␣-Ketoglutarate

CO2

dehydrogenase complex

NADH + H+

␦ COO– ␥ CH2 ␤ CH2 O

␣ C S CoA Succinyl CoA

FIG. 20.8. Oxidative decarboxylation of ␣-ketoglutarate. The ␣-ketoglutarate dehydrogenase complex oxidizes ␣-ketoglutarate to succinyl-CoA. The carboxyl group is released as CO2. The keto group on the ␣-carbon is oxidized and then forms the acyl-CoA thioester, succinyl-CoA. The ␣, ␤, ␥, and ␦ on succinyl-CoA refer to the sequence of atoms in ␣-ketoglutarate.

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363

OH R C

TPP

H

C

acid DH

1

R O

S

␣-Keto

CO2

E1

␣-Keto

COO–

acid DH

␣-Keto acid

TPP

FAD (2H) Dihydrolipoyl DH E3

S Lip

trans Ac

4

E2

trans Ac

trans Ac

3

2

Lip HS

O S C

FAD SH Lip SH

NAD+

5 NADH + H+

O R C

SCoA

CoASH R

FIG. 20.9. Mechanism of ␣-keto acid dehydrogenase complexes (including ␣-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and the branched-chain ␣-keto acid dehydrogenase complex). R represents the portion of the ␣-keto acid that begins with the ␤-carbon. In ␣-ketoglutarate, R is CH2–CH2–COOH. In pyruvate, R is ⫺CH3. The individual steps in the oxidative decarboxylation of ␣-keto acids are catalyzed by three different subunits: E1, ␣-keto acid decarboxylase (␣-ketoglutarate decarboxylase); E2, transacylase (transsuccinylase); and E3, dihydrolipoyl dehydrogenase. (1) Thiamine pyrophosphate (TPP) on E1 decarboxylates the ␣-keto acid and forms a covalent intermediate with the remaining portion. (2) The acyl portion of the ␣-keto acid is transferred by TPP on E1 to lipoate on E2, which is a transacylase. (3) E2 transfers the acyl group from lipoate to CoASH. Note how lipoate is reduced during this conversion. The lipoyl disulfide bond has been reduced to sulfhydral groups (dihydrolipoate). (4) E3, dihydrolipoyl dehydrogenase (DH) transfers the electrons from reduced lipoate to its tightly bound FAD molecule, thereby oxidizing lipoate back to its original disulfide form. (5) The electrons are then transferred from FAD(2H) to NAD⫹ to form NADH.

All of the ␣-keto acid dehydrogenase complexes are huge enzyme complexes composed of multiple subunits of three different enzymes, designated as E1, E2, and E3 (Fig. 20.9). E1 is an ␣-keto acid decarboxylase that contains TPP; it cleaves off the carboxyl group of the ␣-keto acid. E2 is a transacylase-containing lipoate; it transfers the acyl portion of the ␣-keto acid from thiamine to CoASH. E3 is dihydrolipoyl dehydrogenase, which contains FAD; it transfers electrons from reduced lipoate to NAD⫹. The collection of three enzyme activities into one huge complex enables the product of one enzyme to be transferred to the next enzyme without loss of energy. Complex formation also increases the rate of catalysis because the substrates for E2 and E3 remain bound to the enzyme complex. 1.

THIAMINE PYROPHOSPHATE IN THE ␣-KETOGLUTARATE DEHYDROGENASE COMPLEX

TPP is synthesized from the vitamin thiamine by the addition of pyrophosphate (see Fig. 8.11). The pyrophosphate group binds magnesium, which binds to amino acid side chains on the enzyme. This binding is relatively weak for a coenzyme, so thiamine turns over rapidly in the body, and a deficiency can develop rapidly in individuals who are on thiamine-free or low-thiamine diets. The general function of TPP is the cleavage of a carbon–carbon bond next to a keto group. In the ␣-ketoglutarate, pyruvate, and branched-chain ␣-keto acid dehydrogenase complexes, the functional carbon on the thiazole ring forms a covalent bond with the ␣-keto carbon, thereby cleaving the bond between the ␣-keto carbon and the adjacent carboxylic acid group (see Fig. 8.11 for the mechanism of this reaction). TPP is also a coenzyme for transketolase in the pentose phosphate pathway, where it similarly cleaves the carbon–carbon bond next to a keto group. In thiamine deficiency, ␣-ketoglutarate, pyruvate, and other ␣-keto acids accumulate in the blood.

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The E0⬘ for FAD accepting electrons is ⫺0.219 (see Table 19.4). The E0⬘ for NAD⫹ accepting electrons is ⫺0.32. Thus, transfer of electrons from FAD(2H) to NAD⫹ is energetically unfavorable. How do the ␣-keto acid dehydrogenase complexes allow this electron transfer to occur?

In Al Martini’s heart failure, which is caused by a dietary deficiency of the vitamin thiamine, pyruvate dehydrogenase, ␣-ketoglutarate dehydrogenase, and the branched-chain ␣-keto acid dehydrogenase complexes are less functional than normal. Because heart muscle, skeletal muscle, and nervous tissue have high rates of ATP production from the NADH produced by the oxidation of pyruvate to acetyl-CoA and of acetyl-CoA to CO2 in the TCA cycle, these tissues present with the most obvious signs of thiamine deficiency. In Western societies, gross thiamine deficiency is most often associated with alcoholism. The mechanism for active absorption of thiamine is strongly and directly inhibited by alcohol. Subclinical deficiency of thiamine from malnutrition or anorexia may be common in the general population and is usually associated with multiple vitamin deficiencies.

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The E0⬘ values were calculated in a test tube under standard conditions. When FAD is bound to an enzyme, as it is in the ␣-keto acid dehydrogenase complexes, amino acid side chains can alter its E0⬘ value. Thus, the transfer of electrons from the bound FAD(2H) to NAD⫹ in dihydrolipoyl dehydrogenase is actually energetically favorable.

O CH2

CH2 CH2 CH S

CH2

CH2

C N lysineH transacylase

CH2

S

enzyme

Lipoamide (oxidized) TPP intermediate

O CH2

CH2 CH2 CH HS

S

O

CH2

CH2

CH2

C N lysineH transacylase enzyme

C CH2 CH2 COO–

FIG. 20.10. Function of lipoate. Lipoate is attached to the ␧-amino group of a lysine side chain of the transacylase enzyme (E2). The oxidized lipoate disulfide form is reduced as it accepts the acyl group from thiamine pyrophosphate (TPP) attached to E1. The example shown is for the ␣-ketoglutarate dehydrogenase complex.

2.

Arsenic poisoning is caused by the presence of a large number of different arsenious compounds that are effective metabolic inhibitors. Acute accidental or intentional arsenic poisoning requires high doses and involves arsenate (AsO42⫺) and arsenite (AsO32⫺). Arsenite, which is 10 times more toxic than arsenate, binds to neighboring sulfhydryl groups, such as those in dihydrolipoate and in nearby cysteine pairs (vicinal) found in ␣-keto acid dehydrogenase complexes and in succinic dehydrogenase. Arsenate weakly inhibits enzymatic reactions involving phosphate, including the enzyme glyceraldehyde 3-P dehydrogenase in glycolysis (see Chapter 22). Thus, both aerobic and anaerobic ATP production can be inhibited. The low doses of arsenic compounds found in water supplies are a major public health concern but are associated with increased risk of cancer rather than direct toxicity.

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LIPOATE

Lipoate is a coenzyme found only in ␣-keto acid dehydrogenase complexes. It is synthesized in the human from carbohydrate and amino acids, and it does not require a vitamin precursor. Lipoate is attached to the transacylase enzyme through its carboxyl group, which is covalently bound to the terminal ⫺NH2 of a lysine in the protein (Fig. 20.10). At its functional end, lipoate contains a disulfide group that accepts electrons when it binds the acyl fragment of ␣-ketoglutarate. It can thus act like a long, flexible ⫺CH2– arm of the enzyme that reaches over to the decarboxylase to pick up the acyl fragment from thiamine and transfer it to the active site containing bound CoASH. It then swings over to dihydrolipoyl dehydrogenase to transfer electrons from the lipoyl sulfhydryl groups to FAD. 3.

FLAVIN ADENINE DINUCLEOTIDE AND DIHYDROLIPOYL DEHYDROGENASE

FAD on dihydrolipoyl dehydrogenase accepts electrons from the lipoyl sulfhydryl groups and transfers them to bound NAD⫹. FAD thus accepts and transfers electrons without leaving its binding site on the enzyme. The direction of the reaction is favored by interactions of FAD with groups on the enzyme, which change its reduction potential, and by the overall release of energy from cleavage and oxidation of ␣-ketoglutarate.

IV. ENERGETICS OF THE TRICARBOXYLIC ACID CYCLE Like all metabolic pathways, the TCA cycle operates with an overall net negative ⌬G0⬘ (Fig. 20.11). The conversion of substrates to products is, therefore, energetically favorable. However, some of the reactions, such as the malate dehydrogenase reaction, have a positive value. The net standard free-energy change for the TCA cycle, ⌬G0⬘, can be calculated from the sum of the ⌬G0⬘ values for the individual reactions. The ⌬G0⬘, ⫺13 kcal, is the amount of energy lost as heat. It can be considered the amount of energy spent to ensure that oxidation of the acetyl group to CO2 goes to completion. This value

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is surprisingly small. However, oxidation of NADH and FAD(2H) in the electrontransport chain helps to make acetyl oxidation more energetically favorable and pulls the TCA cycle forward.

A. Overall Efficiency of the Tricarboxylic Acid Cycle The reactions of the TCA cycle are extremely efficient in converting energy in the chemical bonds of the acetyl group to other forms. The total amount of energy available from the acetyl group is about 228 kcal/mol (the amount of energy that could be released from complete combustion of 1 mol of acetyl groups to CO2 in a bomb calorimeter). The products of the TCA cycle (NADH, FAD[2H], and GTP) contain about 207 kcal (Table 20.1). Thus, the TCA cycle reactions are able to conserve about 90% of the energy available from the oxidation of acetyl-CoA.

365

Table 20.1 Energy Yield of the Tricarboxylic Acid Cycle kcal/mol 3: NADH 3 ⫻ 53 1 FAD(2H) 1 GTP Sum

⫽ ⫽ ⫽ ⫽

159 41 7 207

The values given for energy yield from NADH and FAD(2H) are based on the equation ⌬G ⫽ ⫺n F ⌬E0⬘, explained in Chapter 19. GTP, guanosine triphosphate.

B. Thermodynamically and Kinetically Reversible and Irreversible Reactions

Three reactions in the TCA cycle have large negative values for ⌬G0⬘ that strongly favor the forward direction: the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and ␣-ketoglutarate dehydrogenase (see Fig. 20.11). Within the TCA cycle, these reactions are physiologically irreversible for two reasons: The products do not rise to high enough concentrations under physiologic conditions to overcome the large negative ⌬G0⬘ values; and the enzymes involved catalyze the reverse reaction very slowly. These reactions make the major contribution to the overall negative ⌬G0⬘ for the TCA cycle and keep it going in the forward direction. In contrast to these irreversible reactions, the reactions catalyzed by aconitase and malate dehydrogenase have a positive ⌬G0⬘ for the forward direction and are thermodynamically and kinetically reversible. Because aconitase is rapid in both directions, equilibrium values for the concentration ratio of products to substrates is maintained, and the concentration of citrate is about 20 times that of isocitrate. The accumulation of citrate instead of isocitrate facilitates transport of excess citrate to the cytosol, where it can provide a source of acetyl-CoA for pathways such as fatty acid and cholesterol synthesis. It also allows citrate to serve as an inhibitor of

Acetyl CoA CoA Oxaloacetate –7.7 kcal

NADH + H+

Citrate

NAD+ +7.1 kcal

+1.5 kcal Isocitrate

Malate

NAD+ H2O

–5.3 kcal

0 kcal

NADH + H+ CO2

␣-Ketoglutarate

Fumarate FAD(2H)

0 kcal

FAD Succinate

–0.7 kcal CoA

–8 kcal CoA

Succinyl CoA GTP Pi GDP

NAD+ NADH + H+ CO2

FIG. 20.11. Approximate ⌬G0⬘ values for the reactions in the TCA cycle, given for the forward direction. The reactions with large negative ⌬G0⬘ values are shown in red. See Chapter 19 for a discussion of the meaning of ⌬G0⬘ values.

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Otto Shape had difficulty losing weight because human fuel utilization is too efficient. His adipose tissue fatty acids are being converted to acetyl-CoA, which is being oxidized in the TCA cycle, thereby generating NADH and FAD(2H). The energy in these compounds is used for ATP synthesis from oxidative phosphorylation. If his fuel utilization were less efficient and his ATP yield were lower, he would have to oxidize much greater amounts of fat to get the ATP he needs for exercise.

citrate synthase when flux through isocitrate dehydrogenase is decreased. Likewise, the equilibrium constant of the malate dehydrogenase reaction favors the accumulation of malate over oxaloacetate, resulting in a low oxaloacetate concentration that is influenced by the NADH/NAD⫹ ratio. Thus, there is a net flux of oxaloacetate toward malate in the liver during fasting (as a result of fatty acid oxidation, which raises the NADH/NAD⫹ ratio), and malate can then be transported out of the mitochondria to provide a substrate for gluconeogenesis.

V. REGULATION OF THE TRICARBOXYLIC ACID CYCLE The oxidation of acetyl-CoA in the TCA cycle and the conservation of this energy as NADH and FAD(2H) is essential for generation of ATP in almost all tissues in the body. In spite of changes in the supply of fuels, type of fuels in the blood, or rate of ATP utilization, cells maintain ATP homeostasis (a constant level of ATP). The rate of the TCA cycle, like that of all fuel oxidation pathways, is principally regulated to correspond to the rate of the electron-transport chain, which is regulated by the ATP/ADP ratio and the rate of ATP utilization (see Chapter 21). The major sites of regulation are shown in Figure 20.12. Two major messengers feed information on the rate of ATP utilization back to the TCA cycle: (1) the phosphorylation state of ATP, as reflected in ATP and ADP levels; and (2) the reduction state of NAD⫹, as reflected in the ratio of NADH/ NAD⫹. Within the cell, even within the mitochondrion, the total adenine nucleotide

Fuel oxidation

Acetyl CoA CoA Oxaloacetate

–

Citrate

–

NADH

NAD+

NADH

Citrate

H+ + NADH malate dehydrogenase

Citrate synthase

NAD+

T Isocitrate

Malate

O2

Isocitrate dehydrogenase + – +

H2O Fumarate

␣-Ketoglutarate dehydrogenase

Electrontransport chain

FAD(2H) FAD

– +

Succinate

GTP Pi

ADP NADH Ca2+

C

H+ H+

H2O NAD+

ADP + Pi

NADH + H+

ATP

CO2

␣-Ketoglutarate

NADH Ca2+

Succinyl CoA

CoA

E

CoA NAD+ NADH + H+

CO2

GDP

FIG. 20.12. Major regulatory interactions in the TCA cycle. The rate of ATP hydrolysis controls the rate of ATP synthesis, which controls the rate of NADH oxidation in the electron-transport chain (ETC). All NADH and FAD(2H) produced by the cycle donate electrons to this chain (shown on the right). Thus, oxidation of acetyl-CoA in the TCA cycle can go only as fast as electrons from NADH enter the electron-transport chain, which is controlled by the ATP and ADP content of the cells. The ADP and NADH concentrations feed information on the rate of oxidative phosphorylation back to the TCA cycle. Isocitrate dehydrogenase (DH), ␣-ketoglutarate DH, and malate DH are inhibited by increased NADH concentration. The NADH/NAD⫹ ratio changes the concentration of oxaloacetate. Citrate is a product inhibitor of citrate synthase. ADP is an allosteric activator of isocitrate DH. During muscular contraction, increased Ca2⫹ concentrations activate isocitrate DH and ␣-ketoglutarate DH (as well as pyruvate DH).

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Table 20.2

367

Generalizations on the Regulation of Metabolic Pathways

1. Regulation matches function. The type of regulation use depends on the function of the pathway. Tissue-specific isozymes may allow the features of regulatory enzymes to match somewhat different functions of the pathway in different tissues. 2. Regulation of metabolic pathways occurs at rate-limiting steps, the slowest steps, in the pathway. These are reactions in which a small change of rate will affect the flux through the whole pathway. 3. Regulation usually occurs at the first committed step of a pathway or at metabolic branch points. In human cells, most pathways are interconnected with other pathways and have regulatory enzymes for every branch point. 4. Regulatory enzymes often catalyze physiologically irreversible reactions. These are also the steps that differ in biosynthetic and degradative pathways. 5. Many pathways have feedback regulation; that is, the end product of the pathway controls the rate of its own synthesis. Feedback regulation may involve inhibition of an early step in the pathway (feedback inhibition) or regulation of gene transcription. 6. Human cells use compartmentation to control access of substrate and activators or inhibitors to different enzymes. 7. Hormonal regulation integrates responses in pathways requiring more than one tissue. Hormones generally regulate fuel metabolism by: a. Changing the phosphorylation state of enzymes b. Changing the amount of enzyme present by changing its rate of synthesis (often induction or repression of mRNA synthesis) or degradation c. Changing the concentration of an activator or inhibitor

pool (AMP, ADP, plus ATP) and the total NAD pool (NAD⫹ plus NADH) are relatively constant. Thus, an increased rate of ATP utilization results in a small decrease of ATP concentration and an increase of ADP. Likewise, increased NADH oxidation to NAD⫹ by the electron-transport chain increases the rate of pathways that produce NADH. Under normal physiologic conditions, the TCA cycle and other oxidative pathways respond so rapidly to increased ATP demand that the ATP concentration does not change significantly.

A. Regulation of Citrate Synthase The principles of pathway regulation are summarized in Table 20.2. In pathways that are subject to feedback regulation, the first step of the pathway must be regulated so that precursors flow into alternative pathways if product is not needed. Citrate synthase, which is the first enzyme of the TCA cycle, is a simple enzyme that has no allosteric regulators. Its rate is controlled principally by the concentration of oxaloacetate, its substrate, and the concentration of citrate, a product inhibitor that is competitive with oxaloacetate (see Fig. 20.12). The malate–oxaloacetate equilibrium favors malate, so the oxaloacetate concentration is very low inside the mitochondrion and is below the apparent Km (see Chapter 9, Section I.A.4) of citrate synthase. When the NADH/NAD⫹ ratio decreases, the ratio of oxaloacetate to malate increases. When isocitrate dehydrogenase is activated, the concentration of citrate decreases, thus relieving the product inhibition of citrate synthase. Thus, both increased oxaloacetate and decreased citrate levels regulate the response of citrate synthase to conditions established by the electron-transport chain and oxidative phosphorylation. In the liver, the NADH/NAD⫹ ratio helps determine whether acetyl-CoA enters the TCA cycle or goes into the alternative pathway for ketone body synthesis.

As Otto Shape exercises, his myosin ATPase hydrolyzes ATP to provide the energy for movement of myofibrils. The decrease of ATP and increase of ADP stimulates the electron-transport chain to oxidize more NADH and FAD(2H). The TCA cycle is stimulated to provide more NADH and FAD(2H) to the electron-transport chain. The activation of the TCA cycle occurs through a decrease of the NADH/NAD⫹ ratio, an increase of ADP concentration, and an increase of Ca2⫹. Although regulation of the transcription of genes for TCA cycle enzymes is too slow to respond to changes of ATP demands during exercise, the number and size of mitochondria increase during training. Thus, Otto Shape is increasing his capacity for fuel oxidation as he trains.

B. Allosteric Regulation of Isocitrate Dehydrogenase Another generalization that can be made about regulation of metabolic pathways is that it occurs at the enzyme that catalyzes the rate-limiting (slowest) step in a pathway (see Table 20.2). Isocitrate dehydrogenase is considered one of the ratelimiting steps of the TCA cycle, and it is allosterically activated by ADP and inhibited by NADH (Fig. 20.13). In the absence of ADP, the enzyme exhibits positive cooperativity; as isocitrate binds to one subunit, other subunits are converted to an active conformation (see Chapter 9, Section III.A, on allosteric enzymes). In the presence of ADP, all of the subunits are in their active conformation, and isocitrate

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A +ADP, Km 0.1 mM No ADP Km 0.5 mM

v

binds more readily. Consequently, the apparent Km (the S0.5) shifts to a much lower value. Thus, at the concentration of isocitrate found in the mitochondrial matrix, a small change in the concentration of ADP can produce a large change in the rate of the isocitrate dehydrogenase reaction. Small changes in the concentration of the product, NADH, and of the cosubstrate, NAD⫹, also affect the rate of the enzyme more than they would a nonallosteric enzyme.

C. Regulation of ␣-Ketoglutarate Dehydrogenase

[Isocitrate]

B

v

6-fold activation

The ␣-ketoglutarate dehydrogenase complex, although it is not an allosteric enzyme, is product inhibited by NADH and succinyl-CoA and may also be inhibited by GTP (see Fig. 20.12). Thus, both ␣-ketoglutarate dehydrogenase and isocitrate dehydrogenase respond directly to changes in the relative levels of ADP and, hence, the rate at which NADH is oxidized by electron transport. Both of these enzymes are also activated by Ca⫹. In contracting heart muscle and possibly other muscle tissues, the release of Ca⫹ from the sarcoplasmic reticulum during muscle contraction may provide an additional activation of these enzymes when ATP is being rapidly hydrolyzed.

D. Regulation of Tricarboxylic Acid Cycle Intermediates No ADP [ADP]

C

Regulation of the TCA cycle serves two functions: (1) It ensures that NADH is generated fast enough to maintain ATP homeostasis, and (2) it regulates the concentration of TCA cycle intermediates. For example, in the liver, a decreased rate of isocitrate dehydrogenase increases citrate concentration, which stimulates citrate efflux to the cytosol. Several regulatory interactions occur in the TCA cycle, in addition to those mentioned previously, that control the levels of TCA intermediates and their flux into pathways that adjoin the TCA cycle.

VI. PRECURSORS OF ACETYL COENZYME A

v

[NADH]

FIG. 20.13. Allosteric regulation of isocitrate dehydrogenase (ICDH). Isocitrate, NAD⫹, and NADH bind in the active site; ADP and Ca2⫹ are activators and bind to separate allosteric sites. A. A graph of velocity versus isocitrate concentration shows positive cooperativity (sigmoid curve) in the absence of ADP. The allosteric activator ADP changes the curve into one closer to a rectangular hyperbola and decreases the Km (S0.5) for isocitrate. B. The allosteric activation by ADP is not an all-or-nothing response. The extent of activation by ADP depends on its concentration. C. Increases in the concentration of product, NADH, decrease the velocity of the enzyme through effects on the allosteric activation.

Compounds enter the TCA cycle as acetyl-CoA or as an intermediate that can be converted to malate or oxaloacetate. Compounds that enter as acetyl-CoA are oxidized to CO2. Compounds that enter as TCA cycle intermediates replenish intermediates that have been used in biosynthetic pathways, such as gluconeogenesis or heme synthesis, but cannot be fully oxidized to CO2.

A. Sources of Acetyl Coenzyme A Acetyl-CoA serves as a common point of convergence for the major pathways of fuel oxidation. It is generated directly from the ␤-oxidation of fatty acids and degradation of the ketone bodies ␤-hydroxybutyrate and acetoacetate (Fig. 20.14). It is also formed from acetate, which can arise from the diet or from ethanol oxidation. Glucose and other carbohydrates enter glycolysis, a pathway common to all cells, and are oxidized to pyruvate. The amino acids alanine and serine are also converted to pyruvate. Pyruvate is oxidized to acetyl-CoA by the PDC. Several amino acids such as leucine and isoleucine are also oxidized to acetyl-CoA. Thus, the final oxidation of acetyl-CoA to CO2 in the TCA cycle is the last step in all the major pathways of fuel oxidation.

B. Pyruvate Dehydrogenase Complex The PDC oxidizes pyruvate to acetyl-CoA, thus linking glycolysis and the TCA cycle. In the brain, which is dependent on the oxidation of glucose to CO2 to fulfill its ATP needs, regulation of the PDC is a life-and-death matter. 1.

STRUCTURE OF THE PYRUVATE DEHYDROGENASE COMPLEX

PDC belongs to the ␣-keto acid dehydrogenase complex family and thus shares structural and catalytic features with the ␣-ketoglutarate dehydrogenase complex and the branched-chain ␣-keto acid dehydrogenase complex (Fig. 20.15). It contains the same three basic types of catalytic subunits: (1) pyruvate decarboxylase

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COO–

O CH3 CH2

CH2

C

H

C

OH

HO

C

H

C

OH

CH2

H

C

OH

C

O

C

H

C

OH

CH3

C 6

CH2 COOH The fatty acid, palmitate

H3N

H O

CH2

+

OH

O

CH3 The ketone body, acetoacetate

CH2OH The sugar, glucose

C

H

Pyruvate

C

O – COO

Pyruvate CH3

O

CH3

369

The amino acid, alanine CH2OH CH3 Ethanol

NAD+ CoASH CO2 NADH + H+ CH3

Thiamine – P P lipoate FAD Pyruvate dehydrogenase complex O C ~ SCoA

Acetyl CoA

FIG. 20.15. PDC catalyzes oxidation of the ␣-keto acid pyruvate to acetyl-CoA.

O CH3

C

SCoA

FIG. 20.14. Origin of the acetyl group from various fuels. Acetyl coenzyme A (acetylCoA) is derived from the oxidation of fuels. The portions of fatty acids, ketone bodies, glucose, pyruvate, the amino acid alanine, and ethanol that are converted to the acetyl group of acetyl coenzyme A (acetyl-CoA) are shown in boxes.

subunits that bind TPP (E1), (2) transacetylase subunits that bind lipoate (E2), and (3) dihydrolipoyl dehydrogenase subunits that bind FAD (E3) (see Fig. 20.9). Although the E1 and E2 enzymes in PDC are relatively specific for pyruvate, the same dihydrolipoyl dehydrogenase participates in all of the ␣-keto acid dehydrogenase complexes. In addition to these three types of subunits, the PDC complex contains one additional subunit, an E3-binding protein (E3-BP). Each functional component of the PDC complex is present in multiple copies (e.g., bovine heart PDC has 30 subunits of E1, 60 subunits of E2, and 6 subunits each of E3 and E3-BP). The E1 enzyme is itself a tetramer of two different types of subunits, ␣ and ␤. 2.

REGULATION OF THE PYRUVATE DEHYDROGENASE COMPLEX

PDC activity is controlled principally through phosphorylation by pyruvate dehydrogenase kinase, which inhibits the enzyme and dephosphorylation by pyruvate dehydrogenase phosphatase, which activates it (Fig. 20.16). Pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase are regulatory subunits within the PDC complex and act only on the complex. PDC kinase transfers a phosphate from ATP to specific serine hydroxyl (ser-OH) groups on pyruvate decarboxylase (E1). PDC phosphatase removes these phosphate groups by hydrolysis. Phosphorylation of just one serine on the PDC E1 ␣-subunit can decrease its activity by ⬎99%. PDC kinase is present in complexes as tissue-specific isozymes that vary in their regulatory properties. PDC kinase is itself inhibited by ADP and pyruvate. Thus, when rapid ATP utilization results in an increase of ADP or when activation of glycolysis increases pyruvate levels, PDC kinase is inhibited and PDC remains in an active, nonphosphorylated form. PDC phosphatase requires Ca2⫹ for full activity. In the heart, increased intramitochondrial Ca2⫹ during rapid contraction activates the phosphatase, thereby increasing the amount of active, nonphosphorylated PDC. PDC is also regulated through inhibition by its products, acetyl-CoA and NADH. This inhibition is stronger than regular product inhibition because their binding to PDC stimulates its phosphorylation to the inactive form. The substrates of the enzyme, CoASH and NAD⫹, antagonize this product inhibition. Thus, when an ample supply of acetyl-CoA for the TCA cycle is already available from fatty acid oxidation, acetyl-CoA and NADH build up and dramatically decrease their own further synthesis by PDC.

Lieberman_Ch20.indd 369

Deficiencies of the PDC are among the most common inherited diseases leading to lactic acidemia and, similar to pyruvate carboxylase deficiency, are grouped into the category of Leigh disease (subacute necrotizing encephalopathy). In its severe form, PDC deficiency presents with overwhelming lactic acidosis at birth, with death in the neonatal period. In a second form of presentation, the lactic acidemia is moderate, but there is profound psychomotor retardation with increasing age. In many cases, concomitant damage to the brainstem and basal ganglia lead to death in infancy. The neurologic symptoms arise because the brain has a very limited ability to use fatty acids as a fuel and is, therefore, dependent on glucose metabolism for its energy supply. The most common PDC genetic defects are in the gene for the ␣-subunit of E1. The E1 ␣-gene is X-linked. Because of its importance in central nervous system metabolism, pyruvate dehydrogenase deficiency is a problem in both males and females, even if the female is a carrier. For this reason, it is classified as an X-linked dominant disorder.

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Pi PDC inactive ADP ADP Pyruvate Acetyl CoA NADH

– – + +

Phosphatase

Kinase

+

ATP

Ca2+

Pi PDC active

Pyruvate

+

–

CoASH NAD+

Acetyl CoA CO2

+

–

NADH

FIG. 20.16. Regulation of PDC. PDC kinase, a subunit of the enzyme, phosphorylates PDC at a specific serine residue, thereby converting PDC to an inactive form. The kinase is inhibited by adenosine diphosphate (ADP) and pyruvate. PDC phosphatase, another subunit of the enzyme, removes the phosphate, thereby activating PDC. The phosphatase is activated by Ca2⫹. When the substrates pyruvate and CoASH are bound to PDC, the kinase activity is inhibited and PDC is active. When the products acetyl-CoA and NADH bind to PDC, the kinase activity is stimulated, and the enzyme is phosphorylated to the inactive form. E1 and the kinase exist as tissue-specific isozymes with overlapping tissue specificity and somewhat different regulatory properties.

PDC can also be activated rapidly through a mechanism involving insulin, which plays a prominent role in adipocytes. In many tissues, insulin may, over time, slowly increase the amount of PDC present. The rate of other fuel oxidation pathways that feed into the TCA cycle is also increased when ATP utilization increases. Insulin, other hormones, and diet control the availability of fuels for these oxidative pathways.

VII. TCA CYCLE INTERMEDIATES AND ANAPLEROTIC REACTIONS A. TCA Cycle Intermediates Are Precursors for Biosynthetic Pathways The intermediates of the TCA cycle serve as precursors for a variety of different pathways present in different cell types (Fig. 20.17). This is particularly important in the central metabolic role of the liver. The TCA cycle in the liver is often called an “open cycle” because there is such a high efflux of intermediates. After a highcarbohydrate meal, citrate efflux and cleavage to acetyl-CoA provides acetyl units for cytosolic fatty acid synthesis. During fasting, gluconeogenic precursors are converted to malate, which leaves the mitochondria for cytosolic gluconeogenesis. The liver also uses TCA cycle intermediates to synthesize carbon skeletons of amino acids. Succinyl-CoA may be removed from the TCA cycle to form heme in cells of the liver and bone marrow. In the brain, ␣-ketoglutarate is converted to glutamate and then to ␥-aminobutyric acid (GABA), a neurotransmitter. In skeletal muscle, ␣-ketoglutarate is converted to glutamine, which is transported through the blood to other tissues. Pyruvate, citrate, ␣-ketoglutarate and malate, ADP, ATP, and phosphate (as well as many other compounds) have specific transporters in the inner mitochondrial membrane that transport compounds between the mitochondrial matrix and cytosol in exchange for a compound of similar charge. In contrast, CoASH, acetyl-CoA,

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Acetyl CoA Amino acid synthesis

Gluconeogenesis

Citrate

Fatty acid synthesis

␣-Ketoglutarate

Amino acid synthesis

Oxaloacetate

Malate

TCA cycle

Succinyl CoA

Neurotransmitter (brain)

Heme synthesis

FIG. 20.17. Efflux of intermediates from the tricarboxylic acid (TCA) cycle. In the liver, TCA cycle intermediates are continuously withdrawn into the pathways of fatty acid synthesis, amino acid synthesis, gluconeogenesis, and heme synthesis. In the brain, ␣-ketoglutarate is converted to glutamate and ␥-aminobutyric acid (GABA), both of which are neurotransmitters.

other CoA derivatives, NAD⫹ and NADH, and oxaloacetate are not transported at a metabolically significant rate. To obtain cytosolic acetyl-CoA, many cells transport citrate to the cytosol, where it is cleaved to acetyl-CoA and oxaloacetate by citrate lyase.

B. Anaplerotic Reactions Removal of any of the intermediates from the TCA cycle removes the four carbons that are used to regenerate oxaloacetate during each turn of the cycle. With depletion of oxaloacetate, it is impossible to continue oxidizing acetyl-CoA. To enable the TCA cycle to keep running, cells have to supply enough four-carbon intermediates from degradation of carbohydrate or certain amino acids to compensate for the rate of removal. Pathways or reactions that replenish the intermediates of the TCA cycle are referred to as anaplerotic (“filling up”). 1.

PYRUVATE CARBOXYLASE IS A MAJOR ANAPLEROTIC ENZYME

Pyruvate carboxylase is one of the major anaplerotic enzymes in the cell. It catalyzes the addition of CO2 to pyruvate to form oxaloacetate (Fig. 20.18). Like most carboxylases, pyruvate carboxylase contains biotin (a vitamin), which forms a covalent intermediate with CO2 in a reaction that requires ATP and Mg2⫹ (see Fig. 8.12). The activated CO2 is then transferred to pyruvate to form the carboxyl group of oxaloacetate. Pyruvate carboxylase is found in many tissues such as liver, brain, adipocytes, and fibroblasts where its function is anaplerotic. Its concentration is high in liver and kidney cortex where there is a continuous removal of oxaloacetate and malate from the TCA cycle to enter the gluconeogenic pathway. Pyruvate carboxylase is activated by acetyl-CoA and inhibited by high concentrations of many acyl-CoA derivatives. As the concentration of oxaloacetate is depleted through the efflux of TCA cycle intermediates, the rate of the citrate synthase reaction decreases and acetyl-CoA concentration rises. The acetyl-CoA then activates pyruvate carboxylase to synthesize more oxaloacetate. 2.

AMINO ACID DEGRADATION FORMS TCA CYCLE INTERMEDIATES

The pathways for oxidation of many amino acids convert their carbon skeletons into five- and four-carbon intermediates of the TCA cycle that can regenerate oxaloacetate

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COOH ATP +

– HCO3

+C

O

CH3 Pyruvate Pyruvate Biotin carboxylase + Acetyl CoA

COOH C

O + ADP + Pi

CH2 COO– Oxaloacetate

FIG. 20.18. Pyruvate carboxylase reaction. Pyruvate carboxylase adds a carboxyl group from bicarbonate (which is in equilibrium with CO2) to pyruvate to form oxaloacetate. Biotin is used to activate and transfer the CO2. The energy to form the covalent biotin–CO2 complex is provided by the high-energy phosphate bond of ATP, which is cleaved in the reaction. The enzyme is activated by acetyl coenzyme A (acetyl-CoA).

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Amino acids

Pyruvate Carbohydrates Fatty acids Amino acids

CO2 ATP

1 ADP + Pi

Acetyl CoA

Oxaloacetate

Citrate

Aspartate

5 Malate

Isocitrate

Amino acids

CO2

4 Amino acids

␣-Ketoglutarate

Fumarate

TA

Glutamate

2 GDH

CO2 Succinate

Succinyl CoA

NADH

3 Valine Isoleucine

Propionyl CoA

+

NH4

NAD+

Odd-chain fatty acids

FIG. 20.19. Major anaplerotic pathways of the tricarboxylic acid (TCA) cycle. (1) and (3) (red arrows) are the two major anaplerotic pathways. (1) Pyruvate carboxylase. (2) Glutamate is reversibly converted to ␣-ketoglutarate by transaminases (TA) and glutamate dehydrogenase (GDH) in many tissues. (3) The carbon skeletons of valine and isoleucine, a three-carbon unit from odd-chain fatty acid oxidation, and a number of other compounds enter the TCA cycle at the level of succinyl-CoA. Other amino acids are also degraded to fumarate (4) and oxaloacetate (5), principally in the liver.

Pyruvate carboxylase deficiency is one of the genetic diseases grouped together under the clinical manifestations of Leigh disease. In the mild form, the patient presents early in life with delayed development and a mild-to-moderate lactic acidemia. Patients who survive are severely mentally retarded, and there is a loss of cerebral neurons. In the brain, pyruvate carboxylase is present in the astrocytes, which use TCA cycle intermediates to synthesize glutamine. This pathway is essential for neuronal survival. The major cause of the lactic acidemia is that cells dependent on pyruvate carboxylase for an anaplerotic supply of oxaloacetate cannot oxidize pyruvate in the TCA cycle (because of low oxaloacetate levels), and the liver cannot convert pyruvate to glucose (because the pyruvate carboxylase reaction is required for this pathway to occur), so the excess pyruvate is converted to lactate.

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(Fig. 20.19). Alanine and serine carbons can enter through pyruvate carboxylase (see Fig. 20.19, circle 1). In all tissues with mitochondria (except for, surprisingly, the liver), oxidation of the two branched-chain amino acids isoleucine and valine to succinyl-CoA forms a major anaplerotic route (see Fig. 20.19, circle 3). In the liver, other compounds that form propionyl-CoA (e.g., methionine, threonine, and odd-chain-length or branched fatty acids) also enter the TCA cycle as succinyl-CoA. In most tissues, glutamine is taken up from the blood, converted to glutamate, and then oxidized to ␣-ketoglutarate, forming another major anaplerotic route (see Fig. 20.19, circle 2). However, the TCA cycle cannot be resupplied with intermediates by even-chain-length fatty acid oxidation or by ketone body oxidation, which forms only acetyl-CoA. In the TCA cycle, two carbons are lost from citrate before succinyl-CoA is formed, so there is no net conversion of acetyl carbon to oxaloacetate. CLINICAL COMMENTS Otto Shape. Otto Shape is experiencing the benefits of physical conditioning. A variety of functional adaptations in the heart, lungs, vascular system, and skeletal muscle occur in response to regular graded exercise. The pumping efficiency of the heart increases, allowing greater cardiac output with fewer beats per minute and at a lower rate of oxygen utilization. The lungs extract a greater percentage of oxygen from the inspired air, allowing fewer respirations

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373

per unit of activity. The vasodilatory capacity of the arterial beds in skeletal muscle increases, promoting greater delivery of oxygen and fuels to exercising muscle. Concurrently, the venous drainage capacity in muscle is enhanced, ensuring that lactic acid will not accumulate in contracting tissues. These adaptive changes in physiologic responses are accompanied by increases in the number, size, and activity of skeletal muscle mitochondria along with the content of TCA cycle enzymes and components of the electron-transport chain. These changes markedly enhance the oxidative capacity of exercising muscle. In skeletal muscle and other tissues, ATP is generated by anaerobic glycolysis when the rate of aerobic respiration is inadequate to meet the rate of ATP utilization. Under these circumstances, the rate of pyruvate production exceeds the cell’s capacity to oxidize NADH in the electron-transport chain and, hence, to oxidize pyruvate in the TCA cycle. The excess pyruvate is reduced to lactate. Because lactate is an acid, its accumulation affects the muscle and causes pain and swelling. Ann O’Rexia. Ann O’Rexia is experiencing fatigue for several reasons. She has iron deficiency anemia, which affects iron-containing hemoglobin in her red blood cells, iron in aconitase and succinic dehydrogenase, as well as iron in the heme proteins of the electron-transport chain. She may also be experiencing the consequences of multiple vitamin deficiencies, including thiamine, riboflavin, and niacin (the vitamin precursor of NAD⫹). It is less likely, but possible, that she also has subclinical deficiencies of pantothenate (the precursor of CoA) or biotin. As a result, Ann’s muscles must use glycolysis as their primary source of energy, which results in sore muscles. Riboflavin deficiency generally occurs in conjunction with other water-soluble vitamin deficiencies. The classic deficiency symptoms are cheilosis (inflammation of the corners of the mouth), glossitis (magenta tongue), and seborrheic (“greasy”) dermatitis. It is also characterized by sore throat, edema of the pharyngeal and oral mucus membranes, and normochromic, normocytic anemia associated with pure red cell cytoplasia of the bone marrow. However, it is not known whether the glossitis and dermatitis are actually due to multiple vitamin deficiencies. Riboflavin has a wide distribution in foods, and small amounts are present as coenzymes in most plant and animal tissues. Eggs, lean meats, milk, broccoli, and enriched breads and cereals are especially good sources. A portion of our niacin requirement can be met by synthesis from tryptophan. Meat (especially red meat), liver, legumes, milk, eggs, alfalfa, cereal grains, yeast, and fish are good sources of niacin and tryptophan. Al Martini. Al Martini presents a second time with an alcohol-related high-output form of heart failure that is sometimes referred to as wet beriberi or as the beriberi heart (see Chapter 9). The word wet refers to the fluid retention that may eventually occur when left ventricular contractility is so compromised that cardiac output, although initially relatively “high,” cannot meet the “demands” of the peripheral vascular beds, which have dilated in response to the thiamine deficiency. The cardiomyopathy is related directly to a reduction in the normal biochemical function of the vitamin thiamine in heart muscle. Inhibition of the ␣-keto acid dehydrogenase complexes causes accumulation of ␣-keto acids in heart muscle (and in blood), resulting in a chemically induced cardiomyopathy. Impairment of two other functions of thiamine may also contribute to the cardiomyopathy. Thiamine pyrophosphate (TPP) serves as the coenzyme for transketolase in the pentose phosphate pathway, and pentose phosphates accumulate in thiamine deficiency. In addition, thiamine triphosphate (a different coenzyme form) may function in Na⫹ conductance channels.

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Beriberi, now known to be caused by thiamine deficiency, was attributed to lack of a nitrogenous component in food by Takaki, a Japanese surgeon, in 1884. In 1890, Eijkman, a Dutch physician working in Java, noted that the polyneuritis associated with beriberi could be prevented by rice bran that had been removed during polishing. Thiamine is present in the bran portion of grains, and it is abundant in pork and legumes. In contrast to most vitamins, milk and milk products, seafood, fruits, and vegetables are not good sources of thiamine.

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Immediate treatment with large doses (50 to 100 mg) of intravenous thiamine may produce a measurable decrease in cardiac output and increase in peripheral vascular resistance as early as 30 minutes after the initial injection. Dietary supplementation of thiamine is not as effective because ethanol consumption interferes with thiamine absorption. Because ethanol also affects the absorption of most water-soluble vitamins or their conversion to the coenzyme form, Al Martini was also given a bolus containing a multivitamin supplement. BIOCHEMICAL COMMENTS

Matrix protein hsp 70

1 Cytosol

N

+++

TOM complex

OM IMS

2

+++ TIM complex

⌬␷

IM

––– ATP

ADP mt hsp 70

+++

N

ADP ATP

Matrix

+++ hsp 60

+++

ATP

N

3

ADP

N

Matrix processing protease +

N

FIG. 20.20. Model for the import of nuclearencoded proteins into the mitochondrial matrix. The matrix preprotein with its positively charged N-terminal presequence is shown in red. hsp, heat-shock protein; OM, outer mitochondrial membrane; IMS, intermembrane space; IM, inner mitochondrial membrane; TOM, translocases of the outer mitochondrial membrane; TIM, translocases of the inner mitochondrial membrane; mthsp70, mitochondrial heat-shock protein 70.

Lieberman_Ch20.indd 374

Compartmentation of Mitochondrial Enzymes. The mitochondrion forms a structural, functional, and regulatory compartment within the cell. The inner mitochondrial membrane is impermeable to anions and cations, and compounds can cross the membrane only on specific transport proteins. The enzymes of the tricarboxylic acid (TCA) cycle, therefore, have more direct access to products of the previous reaction in the pathway than they would if these products were able to diffuse throughout the cell. Complex formation between enzymes also restricts access to pathway intermediates. Malate dehydrogenase and citrate synthase may form a loosely associated complex. The multienzyme pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase complexes are examples of substrate channeling by tightly bound enzymes; only the transacylase enzyme has access to the thiamine-bound intermediate of the reaction, and only lipoamide dehydrogenase has access to reduced lipoic acid. Compartmentation plays an important role in regulation. The close association between the rate of the electron-transport chain and the rate of the TCA cycle is maintained by their mutual access to the same pool of NADH and NAD⫹ in the mitochondrial matrix. NAD⫹, NADH, CoASH, and acyl-CoA derivatives have no transport proteins and cannot cross the mitochondrial membrane. Thus, all of the dehydrogenases compete for the same NAD⫹ molecules and are inhibited when NADH rises. Likewise, accumulation of acyl-CoA derivatives (e.g., acetyl-CoA) within the mitochondrial matrix affects other CoA-using reactions, either by competing at the active site or by limiting CoASH availability. Import of Nuclear-Encoded Proteins. All mitochondrial matrix proteins, such as the TCA cycle enzymes, are encoded by the nuclear genome. They are imported into the mitochondrial matrix as unfolded proteins that are pushed and pulled through channels in the outer and inner mitochondrial membranes (Fig. 20.20). Proteins destined for the mitochondrial matrix have either a targeting N-terminal presequence of about 20 amino acids that includes several positively charged amino acid residues or an internal mitochondrial localizing signal. The mitochondrial matrix proteins are synthesized on free ribosomes in the cytosol and maintain an unfolded conformation by binding to hsp70 chaperonins. This basic presequence binds to a receptor in a translocases of the outer membrane (TOM) complex (see Fig. 20.20, step 1). The TOM complexes consist of channel proteins, assembly proteins, and receptor proteins with different specificities (e.g., TOM20 binds the matrix protein presequence). Negatively charged acidic residues on the receptors and in the channel pore assist in translocation of the matrix protein through the channel, presequence first. The matrix preprotein is translocated across the inner membrane through a translocases of the inner membrane (TIM) complex (see Fig. 20.20, step 2). Insertion of the preprotein into the TIM channel is driven by the potential difference across the membrane, ⌬␺. Mitochondrial hsp70 (mthsp70), which is bound to the matrix side of the TIM complex, binds the incoming preprotein and may “ratchet” it through the membrane. Adenosine triphosphate (ATP) is required for binding of mthsp70 to the TIM complex and again for the subsequent dissociation of the mthsp70 and

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the matrix preprotein. In the matrix, the preprotein may require another heat-shock protein, hsp60, for proper folding. The final step in the import process is cleavage of the signal sequence by a matrix-processing protease (see Fig. 20.20, step 3). Proteins of the inner mitochondrial membrane are imported through a similar process, using TOM and TIM complexes containing different protein components. Key Concepts • • • • • •

• • • •

The tricarboxylic acid (TCA) cycle accounts for more than two-thirds of the adenosine triphosphate (ATP) generated from fuel oxidation. All of the enzymes required for the TCA cycle are in the mitochondria. Acetyl coenzyme A (acetyl-CoA), generated from fuel oxidation, is the substrate for the TCA cycle. Acetyl-CoA, when oxidized via the cycle, generates CO2, reduced electron carriers, and guanosine triphosphate (GTP). The reduced electron carriers [NADH, FAD(2H)] donate electrons to O2 via the electron-transport chain, which leads to ATP generation from oxidative phosphorylation. The cycle requires several cofactors to function properly, some of which are derived from vitamins. These include thiamine pyrophosphate (TTP) (derived from vitamin B1), flavin adenine dinucleotide (FAD) (derived from vitamin B2, riboflavin), and coenzyme A (derived from pantothenic acid). Intermediates of the TCA cycle are used for many biosynthetic reactions and are replaced by anaplerotic (refilling) reactions within the cell. The cycle is carefully regulated within the mitochondria by energy and the levels of reduced electron carriers. As energy levels decrease, the rate of the cycle increases. Impaired functioning of the TCA cycle leads to an inability to generate ATP from fuel oxidation and an accumulation of TCA cycle precursors. Diseases discussed in this chapter are summarized in Table 20.3

Table 20.3

Diseases Discussed in Chapter 20

Disease or Disorder

Environmental or Genetic

Obesity

Both

Anorexia nervosa

Both

Congestive heart failure linked to alcoholism

Both

Arsenic poisoning

Environmental

Leigh disease (subacute necrotizing encephalopathy)

Genetic

Comments Increased physical activity without increasing caloric intake will lead to weight loss and increased exercise capacity. One effect of increased aerobic exercise is increasing the number and size of mitochondria in the muscle cells. Patients who have been malnourished for some time may exhibit subclinical deficiencies in many vitamins, including riboflavin and niacin, factors required for energy generation. Thiamine deficiency brought about by chronic alcohol ingestion leads to inefficient energy production by the heart and failure to adequately pump blood throughout the body. The vitamin B1 deficiency reduces the activity of pyruvate dehydrogenase and the TCA cycle, severely restricting ATP generation. Arsenite inhibits enzymes and cofactors with free adjacent sulfhydral groups (lipoic acid is a target of arsenite), whereas arsenate acts as a phosphate analog and inhibits substrate-level phosphorylation reactions. Deficiencies of the pyruvate dehydrogenase complex (PDC) as well as of pyruvate carboxylase are inherited disorders leading to lactic acidemia. In its most severe form, PDC deficiency presents with overwhelming lactic acidosis at birth, with death in the neonatal period. Even in less severe forms, neurologic symptoms arise due to the brain’s dependence on glucose metabolism for energy. The most common PDC deficiency is X-linked, in the ␣-subunit of the pyruvate decarboxylase (E1) subunit. Pyruvate carboxylase deficiency also leads to mental retardation.

TCA, tricarboxylic acid; ATP, adenosine triphosphate.

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REVIEW QUESTIONS—CHAPTER 20 1.

2.

3.

Which of the following coenzymes is unique to ␣-keto acid dehydrogenase complexes? A. NAD⫹ B. FAD C. GDP D. H2O E. Lipoic acid A patient diagnosed with thiamine deficiency exhibited fatigue and muscle cramps. The muscle cramps have been related to an accumulation of metabolic acids. Which of the following metabolic acids is most likely to accumulate in a thiamine deficiency? A. Isocitric acid B. Pyruvic acid C. Succinic acid D. Malic acid E. Oxaloacetic acid Succinate dehydrogenase differs from all other enzymes in the TCA cycle in that it is the only enzyme that displays which of the following characteristics? A. It is embedded in the inner mitochondrial membrane.

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B. C. D. E.

It is inhibited by NADH. It contains bound FAD. It contains Fe–S centers. It is regulated by a kinase.

4.

During exercise, stimulation of the TCA cycle results principally from which of the following? A. Allosteric activation of isocitrate dehydrogenase by increased NADH B. Allosteric activation of fumarase by increased ADP C. A rapid decrease in the concentration of four-carbon intermediates D. Product inhibition of citrate synthase E. Stimulation of the flux through a number of enzymes by a decreased NADH/NAD⫹ ratio

5.

Coenzyme A is synthesized from which of the following vitamins? A. Niacin B. Riboflavin C. Vitamin A D. Pantothenate E. Vitamin C

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21

Oxidative Phosphorylation and Mitochondrial Function

Energy from fuel oxidation is converted to the high-energy phosphate bonds of adenosine triphosphate (ATP) by the process of oxidative phosphorylation (OXPHOS). Most of the energy from oxidation of fuels in the tricarboxylic acid (TCA) cycle and other pathways is conserved in the form of the reduced electron-accepting coenzymes, NADH and FAD(2H). The electron-transport chain oxidizes NADH and FAD(2H) and donates the electrons to O2, which is reduced to H2O (Fig. 21.1). Energy from reduction of O2 is used for phosphorylation of adenosine diphosphate (ADP) to ATP by ATP synthase (F0F1 ATPase). The net yield of OXPHOS is approximately 2.5 mol of ATP per mole of NADH oxidized or 1.5 mol of ATP per mole of FAD(2H) oxidized. Chemiosmotic Model of ATP Synthesis. The chemiosmotic model explains how energy from transport of electrons to O2 is transformed into the high-energy phosphate bond of ATP (see Fig. 21.1). Basically, the electrontransport chain contains three large protein complexes (I, III, and IV) that span the inner mitochondrial membrane. As electrons pass through these complexes in a series of oxidation–reduction reactions, protons are transferred from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. The pumping of protons generates an electrochemical gradient (⌬p) across the membrane composed of the membrane potential and the proton gradient. ATP synthase contains a proton pore that spans the inner mitochondrial membrane and a catalytic headpiece that protrudes into the matrix. As protons are driven into the matrix through the pore, they change the conformation of the headpiece, which releases ATP from one site and catalyzes formation of ATP from ADP and inorganic phosphate (Pi) at another site. Deficiencies of Electron Transport. In cells, complete transfer of electrons from NADH and FAD(2H) through the chain to O2 is necessary for ATP generation. Impaired transfer through any complex can have pathologic consequences. Fatigue can result from iron deficiency anemia, which decreases Fe for Fe–S centers and cytochromes. Cytochrome c1 oxidase, which contains the O2-binding site, is inhibited by cyanide. Mitochondrial DNA (mtDNA), which is maternally inherited, encodes some of the subunits of the electrontransport chain complexes and ATP synthase. OXPHOS diseases are caused by mutations in nuclear DNA or mtDNA that decrease mitochondrial capacity for OXPHOS. Regulation of Oxidative Phosphorylation. The rate of the electrontransport chain is coupled to the rate of ATP synthesis by the transmembrane electrochemical gradient. As ATP is used for energy-requiring processes and ADP levels increase, proton influx through the ATP synthase pore generates more ATP, and the electron-transport chain responds to restore ⌬p. In uncoupling, protons return to the matrix by a mechanism that bypasses the ATP synthase pore, and the energy is released as heat. Proton leakage, chemical 377

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Electrochemical potential

Cytochrome c 4H+

Intermembrane space

4H+

nH+

+ + + Δp

Inner membrane Mitochondrial matrix

2H+

c CoQ

– – – NADH + H+

NAD+

2H+ + 1 2O2

Coenzyme Q

H2O

NADH:CoQ oxidoreductase

Cytochrome b-c1 complex

Cytochrome oxidase

Complex I

Complex III

Complex IV

ADP ATP + Pi ATP synthase

FIG. 21.1. Oxidative phosphorylation. Red arrows show the path of electron transport from NADH to O2. As electrons pass through the chain, protons are pumped from the mitochondrial matrix to the intermembrane space, thereby establishing an electrochemical potential gradient, ⌬p, across the inner mitochondrial membrane. The positive and negative charges on the membrane denote the membrane potential (⌬␺). ⌬p drives protons into the matrix through a pore in ATP synthase, which uses the energy to form ATP from ADP and Pi.

uncouplers, and regulated uncoupling proteins increase our metabolic rate and heat generation. Mitochondria and Cell Death. Although OXPHOS is a mitochondrial process, most ATP use occurs outside of the mitochondrion. ATP synthesized from OXPHOS is actively transported from the matrix to the intermembrane space by adenine nucleotide translocase (ANT). Porins form voltage-dependent anion channels (VDACs) through the outer mitochondrial membrane for the diffusion of H2O, ATP metabolites, and other ions. Under certain types of stress, ANT, VDACs, and other proteins form a nonspecific open channel known as the mitochondrial permeability transition pore. This pore is associated with events that lead rapidly to necrotic cell death.

THE WAITING ROOM

Cora Nari is experiencing a second myocardial infarction. Ischemia (low blood flow) has caused hypoxia (low levels of oxygen) in the threatened area of her heart muscle, resulting in inadequate generation of ATP for the maintenance of low intracellular Na⫹ and Ca2⫹ levels (see Chapter 19). As a consequence, the myocardial cells in that specific location have become swollen and the cytosolic proteins creatine kinase (MB isoform) and troponin (heart isoform) have leaked into the blood. (See Ann Jeina, Chapters 6 and 7).

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Cora Nari was recovering uneventfully from her heart attack 1 month earlier (see Chapter 19) when she won the Georgia State Lottery. When she heard her number announced over television, she experienced crushing chest pain, grew short of breath, and passed out. She regained consciousness as she was being rushed to the hospital emergency room. On initial examination, her blood pressure was extremely high and her heart rhythm was irregular. Her blood levels of creatine kinase muscle–brain (CK-MB) and troponin I (TnI) were elevated. An electrocardiogram showed unequivocal evidence of severe lack of oxygen (ischemia) in the muscles of the anterior and lateral walls of her heart. Life-support measures including nasal oxygen were initiated. An intravenous drip of nitroprusside, a vasodilating agent, was started in an effort to reduce her hypertension. After her blood pressure was well controlled, and because the hospital did not have a cardiac catheterization laboratory, a decision was made to administer intravenous tissue plasminogen activator (tPA) in an attempt to break up any intracoronary artery blood clots in vessels supplying the ischemic myocardium (thrombolytic therapy).

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A 123I thyroid uptake and scan performed on X. S. Teefore confirmed that his hyperthyroidism was the result of Graves disease (see Chapter 19). Graves disease, also known as diffuse toxic goiter, is an autoimmune genetic disorder caused by the generation of human thyroid-stimulating immunoglobulins. These immunoglobulins stimulate growth of the thyroid gland (goiter) and excess secretion of the thyroid hormones, T3 and T4. Because heat production is increased under these circumstances, Mr. Teefore’s heat intolerance and sweating were growing worse with time. Ivy Sharer, an intravenous drug user, appeared to be responding well to her multidrug regimens to treat pulmonary tuberculosis and AIDS (see Chapters 11, 12, 15, and 16). In the past 6 weeks, however, she has developed increasing weakness in her extremities, to the point that she has difficulty carrying light objects or walking. Physical examination indicates a diffuse proximal and distal muscle weakness associated with muscle atrophy. The muscles are neither painful on motion nor tender to compression. The blood level of the muscle enzymes, creatine phosphokinase (CPK) and aldolase, are normal. An electromyogram (EMG) revealed a generalized reduction in the muscle action potentials, suggestive of a primary myopathic process. Proton spectroscopy of her brain and upper spinal cord showed no anatomic or biochemical abnormalities. The diffuse and progressive skeletal muscle weakness was out of proportion to that expected from her AIDS or her tuberculosis. This information led her physicians to consider the possibility that her skeletal muscle dysfunction might be drug induced.

I.

An electromyogram measures the electrical potential of muscle cells both at rest and while contracting. Electrodes are inserted through the skin and into the muscle, and baseline recordings (no contraction) are obtained, followed by measurements of electrical activity when the muscle contracts. The electrode is retracted in a small amount, and the measurements are repeated. This occurs for up to 10 to 20 measurements, thereby sampling many distinct areas of the muscle. Under normal conditions, muscles at rest will have minimal electrical activity, which increases significantly as the muscle contracts. Electromyograms that deviate from the norm suggest an underlying pathology interfering with membrane polarization–depolarization as the nerve cells instruct the muscle cells to contract.

OXIDATIVE PHOSPHORYLATION

Generation of ATP from oxidative phosphorylation requires an electron donor (NADH or FAD[2H]), an electron acceptor (O2), an intact inner mitochondrial membrane that is impermeable to protons, all the components of the electrontransport chain, and ATP synthase. It is regulated by the rate of ATP use. Most cells are dependent on oxidative phosphorylation for ATP homeostasis. During oxygen deprivation from ischemia (low blood flow), an inability to generate energy from the electron-transport chain results in increased permeability of this membrane and mitochondrial swelling. Mitochondrial swelling is a key element in the pathogenesis of irreversible cell injury leading to cell lysis and death (necrosis).

A. Overview of Oxidative Phosphorylation Our understanding of oxidative phosphorylation is based on the chemiosmotic hypothesis, which proposes that the energy for ATP synthesis is provided by an electrochemical gradient across the inner mitochondrial membrane. This electrochemical gradient is generated by the components of the electron-transport chain, which pump protons across the inner mitochondrial membrane as they sequentially accept and donate electrons (see Fig. 21.1). The final acceptor is O2, which is reduced to H2O. 1.

379

ELECTRON TRANSFER FROM NADH TO OXYGEN

In the electron-transport chain, electrons donated by NADH or FAD(2H) are passed sequentially through a series of electron carriers embedded in the inner mitochondrial membrane. Each of the components of the electron-transfer chain is reduced as it accepts an electron and then oxidized as it passes the electrons to the next member of the chain. From NADH, electrons are transferred sequentially through NADH:CoQ oxidoreductase (complex I), coenzyme Q (CoQ), the cytochrome b–c1 complex (complex III), cytochrome c, and finally, cytochrome c oxidase (complex IV). NADH:CoQ oxidoreductase, the cytochrome b–c1 complex, and cytochrome c oxidase are each multisubunit protein complexes that span the inner

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Arlyn Foma, who has a folliculartype non-Hodgkin lymphoma was being treated with the anthracycline drug doxorubicin (see Chapter 16). During the course of his treatment, he developed biventricular heart failure. Although doxorubicin is a highly effective anticancer agent against a wide variety of human tumors, its clinical use is limited by a specific, cumulative, dose-dependent cardiotoxicity. Impairment of mitochondrial function may play a major role in this toxicity. Doxorubicin binds to cardiolipin, a lipid component of the inner membrane of mitochondria, where it might directly affect components of oxidative phosphorylation. Doxorubicin inhibits succinate oxidation, inactivates cytochrome oxidase, interacts with CoQ, adversely affects ion pumps, and inhibits ATP synthase, resulting in decreased ATP levels and mildly swollen mitochondria. It decreases the ability of the mitochondria to sequester Ca2⫹ and increases free radicals (highly reactive single-electron forms), leading to damage of the mitochondrial membrane (see Chapter 24). It also might affect heart function indirectly through other mechanisms.

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Cytosolic side nH+ ++++++++ Δψ

Proton motive force

––––––––

H+ H + H + ΔpH H+

nH+ Matrix side

FIG. 21.2. Proton motive force (electrochemical gradient) across the inner mitochondrial membrane. The proton motive force consists of a membrane potential, ⌬␺, and a proton gradient, denoted by ⌬pH for the difference in pH across the membrane. The electrochemical potential is called the proton motive force because it represents the potential energy driving protons to return to the more negatively charged alkaline matrix.

mitochondrial membrane. CoQ is a lipid-soluble quinone that is not protein bound and is free to diffuse in the lipid membrane. It transports electrons from complex I to complex III and is an intrinsic part of the proton pump for each of these complexes. Cytochrome c is a small protein in the intermembrane space that transfers electrons from the b–c1 complex to cytochrome oxidase. The terminal complex, cytochrome c oxidase, contains the binding site for O2. As O2 accepts electrons from the chain, it is reduced to H2O. 2.

THE ELECTROCHEMICAL POTENTIAL GRADIENT

At each of the three large membrane-spanning complexes in the chain, electron transfer is accompanied by proton pumping across the membrane. There is an energy drop of approximately 16 kcal in reduction potential as electrons pass through each of these complexes, which provides the energy required to move protons against a concentration gradient. The membrane is impermeable to protons, so they cannot diffuse through the lipid bilayer back into the matrix. Thus, in actively respiring mitochondria, the intermembrane space and cytosol may be approximately 0.75 pH unit lower than the matrix. The transmembrane movement of protons generates an electrochemical gradient with two components: the membrane potential (the external face of the membrane is charged positive relative to the matrix side) and the proton gradient (the intermembrane space has a higher proton concentration and is, therefore, more acidic than the matrix) (Fig. 21.2). The electrochemical gradient is sometimes called the proton motive force because it is the energy that pushes the protons to reenter the matrix to equilibrate on both sides of the membrane. The protons are attracted to the more negatively charged matrix side of the membrane, where the pH is more alkaline. 3.

ADENOSINE TRIPHOSPHATE SYNTHASE

ATP synthase (F0F1 ATPase), the enzyme that generates ATP, is a multisubunit enzyme that contains an inner membrane portion (F0) and a stalk and headpiece (F1) that project into the matrix (Fig. 21.3). The 12 c-subunits in the membrane form a rotor that is attached to a central asymmetric shaft composed of the ␧- and ␥-subunits. The headpiece is composed of three ␣␤-subunit pairs. Each ␤-subunit Matrix

␦

␣ ␤

␤

␣

␣ ␤ F1 Headpiece

b2 H+

␥ ⑀ a

C5 C1 C 2 C3 C4

F0 Pore

H+ Cytoplasmic side

FIG. 21.3. ATP synthase (F0F1 ATPase). Note that the matrix side of the mitochondrial inner membrane is at the top of the figure.

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ATP release

2

ADP + Pi

ATP synthesis

P+

AD Pi

2.

ATP synthesis

Pi

NADH:COQ OXIDOREDUCTASE

NADH:CoQ oxidoreductase (also named NADH dehydrogenase) is an enormous 42-subunit complex that contains a binding site for NADH, several FMN and Fe–S center-binding proteins, and binding sites for CoQ (see Fig. 21.5). An FMN accepts two electrons from NADH and is able to pass single electrons to the Fe–S centers (Fig. 21.6). Fe–S centers, which are able to delocalize single electrons into large orbitals, transfer electrons to and from CoQ. Fe–S centers are also present in other enzyme systems, such as proteins within the cytochrome b–c1 complex, which transfer electrons to CoQ and in aconitase in the TCA cycle.

Energy

P+

1.

Pi

Electron transport to O2 occurs via a series of oxidation–reduction steps in which each successive component of the chain is reduced as it accepts electrons and oxidized as it passes electrons to the next component of the chain. The oxidation– reduction components of the chain include flavin mononucleotide (FMN), iron– sulfur (Fe–S) centers, CoQ, and Fe in cytochromes b, c1, c, a, and a3. Copper (Cu) is also a component of cytochromes a and a3 (Fig. 21.5). With the exception of CoQ, all of these electron acceptors are tightly bound to the protein subunits of the carriers. FMN, like flavin adenine dinucleotide (FAD), is synthesized from the vitamin riboflavin (see Fig. 19.10). The reduction potential of each complex of the chain is at a lower energy level than the previous complex, so energy is released as electrons pass through each complex. This energy is used to move protons against their concentration gradient, so they become concentrated on the cytosolic side of the inner membrane.

ADP + Pi

1

AD

B. Oxidation–Reduction Components of the Electron-Transport Chain

ATP

P+ AD

contains a catalytic site for ATP synthesis. The headpiece is held stationary by a ␦-subunit attached to a long b-subunit connected to a-subunit in the membrane. The influx of protons through the proton channel turns the rotor. The proton channel is formed by the c-subunit on one side and the a-subunit on the other side. Although the channel is continuous, it has two offset portions: one portion open directly to the intermembrane space and one portion open directly to the matrix. In the current model, each c-subunit contains a glutamyl carboxyl group that extends into the proton channel. Because this carboxyl group accepts a proton from the intermembrane space, the c-subunit rotates into the hydrophobic lipid membrane. The rotation exposes a different proton-containing c-subunit to the portion of the channel that is open directly to the matrix side. Because the matrix has a lower proton concentration, the glutamyl carboxylic acid group releases a proton into the matrix portion of the channel. Rotation is completed by an attraction between the negatively charged glutamyl residue and a positively charged arginyl group on the a-subunit. According to the binding-change mechanism, as the asymmetric shaft rotates to a new position, it forms different binding associations with the ␣␤-subunits (Fig. 21.4). The new position of the shaft alters the conformation of one ␤-subunit so that it releases a molecule of ATP and another subunit spontaneously catalyzes synthesis of ATP from inorganic phosphate, one proton, and adenosine diphosphate (ADP). Thus, energy from the electrochemical gradient is used to change the conformation of the ATP synthase subunits so that the newly synthesized ATP is released. Twelve c-subunits are hypothesized, and it takes 12 protons to complete one turn of the rotor and synthesize three ATPs.

381

ATP release

FIG. 21.4. Binding-change mechanism for ATP synthesis. The three ␣␤-subunit pairs of the ATP synthase headpiece have binding sites that can exist in three different conformations, depending on the position of the ␥-stalk subunit. (1) When ADP ⫹ Pi bind to an open site and the proton influx rotates the ␥-spindle (white arrow), the conformation of the subunits change and ATP is released from one site. (ATP dissociation is thus the energyrequiring step). Bound ADP and Pi combine to form ATP at another site. (2) As the ADP ⫹ Pi bind to the new open site, and the ␥-shaft rotates, the conformations of the sites change again, and ATP is released. ADP and Pi combine to form another ATP.

SUCCINATE DEHYDROGENASE AND OTHER FLAVOPROTEINS

In addition to NADH:CoQ oxidoreductase, succinate dehydrogenase and other flavoproteins in the inner mitochondrial membrane also pass electrons to CoQ (see Fig. 21.5). Succinate dehydrogenase is part of the TCA cycle and also a component

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

Intermembrane space 4H+

Glycerol 3-phosphate dehydrogenase

CoQH2

Fe-S I

FMN

CoQ Fe-S (FAD)

FAD

CuA

Cyt c1

Cyt a Cyt a3 CuB IV

Cyt b

III

Succinate

NADH:CoQ oxidoreductase

2H+ Cyt c

Fe-s

CoQH2

FAD

CoQ II Fe-S

NADH NAD+

4H+

1/2 O2 + 2H+ H2O

Succinate ETF: Q dehydrogenase oxidoreductase

Cytochrome b-c1 complex

Cytochrome c oxidase

Matrix

FIG. 21.5. Components of the electron-transport chain. NADH:CoQ oxidoreductase (complex I) spans the membrane and has a protonpumping mechanism involving CoQ. The electrons go from CoQ to cytochrome b–c1 complex (complex III); electron transfer does not involve complex II. Succinate dehydrogenase (complex II), glycerol-3-phosphate dehydrogenase, and ETF:Q oxidoreductase all transfer electrons to CoQ, but they do not span the membrane and do not have proton-pumping mechanisms. As CoQ accepts electrons and protons from the matrix side, it is converted to QH2. Electrons are transferred from complex III to complex IV (cytochrome c oxidase) by cytochrome c, a small cytochrome in the intermembrane space that has reversible binding sites on the b–c1 complex and cytochrome c oxidase.

of complex II of the electron-transport chain. ETF-CoQ oxidoreductase accepts electrons from ETF (electron-transferring flavoprotein), which acquires them from fatty acid oxidation and other pathways. Both of these flavoproteins have Fe–S centers. Glycerol-3-phosphate dehydrogenase is a flavoprotein that is part of a shuttle for reoxidizing cytosolic NADH. The free-energy drop in electron transfer between NADH and CoQ of approximately ⫺13 to ⫺14 kcal is able to support movement of four protons. However, the FAD in succinate dehydrogenase (as well as ETF-CoQ oxidoreductase and glycerol-3-phosphate dehydrogenase) is at roughly the same redox potential as CoQ, and no energy is released as they transfer electrons to CoQ. These proteins (complex II) do not span the membrane and consequently do not have a protonpumping mechanism.

Pr Cys S Fe

S Pr

Cys

S

Fe S Cys Pr

S

Fe

S S

Fe S

3.

Cys Pr

COENZYME Q

CoQ is the only component of the electron-transport chain that is not protein bound. The large hydrophobic side chain of 10 isoprenoid units (50 carbons) confers lipid solubility, and CoQ is able to diffuse through the lipids of the inner mitochondrial membrane (Fig. 21.7). When the oxidized quinone form accepts a single electron, it forms a free radical (a compound with a single electron in an orbital). The transfer of single electrons makes it the major site for generation of toxic oxygen free radicals in the body (see Chapter 24). The semiquinone can accept a second electron and two protons from the matrix side of the membrane to form the fully reduced quinone. The mobility of CoQ in the membrane, its ability to accept one or two electrons, and its ability to accept

FIG. 21.6. Fe4S4 centers. In Fe–S centers, the Fe is chelated to free sulfur (S) atoms and to cysteine sulfhydryl groups on proteins (indicated by Pr). Other Fe–S centers contain Fe2S2. The protein subunits are sometimes called non–heme iron proteins. When these proteins are treated with acid, the free sulfur produces hydrogen sulfide (H2S)—the familiar smell of rotten eggs.

O

OH

OH

e – + H+

O Fully oxidized or quinone form (Q)

e – + H+

•O

CH3O

CH3

CH3

CH3O

[CH2CH

CCH2]10H

OH – form –

Semiquinone (free radical, Q•)

Reduced or quinol form (dihydroquinol, QH2)

FIG. 21.7. Coenzyme Q contains a quinone with a long lipophilic side chain comprising 10 isoprenoid units (thus, it is sometimes called CoQ10). CoQ can accept one electron (e⫺) to become the half-reduced form or two e⫺ to become fully reduced.

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and donate protons enable it to participate in the proton pumps for both complexes I and III as it shuttles electrons between them (see Section I.C). CoQ is also called ubiquinone (the ubiquitous quinone) because quinones with similar structures are found in all plants and animals. 4.

CYTOCHROMES

The remaining components in the electron-transport chain are cytochromes (see Fig. 21.5). Each cytochrome is a protein that contains a bound heme (i.e., an Fe atom bound to a porphyrin nucleus similar in structure to the heme in hemoglobin) (Fig. 21.8). Because of differences in the protein component of the cytochromes and small differences in the heme structure, each heme has a different reduction potential. The cytochromes of the b–c1 complex have a higher energy level than those of cytochrome oxidase (a and a3). Thus, energy is released by electron transfer between complexes III and IV. The iron atoms in the cytochromes are in the Fe3⫹ state. As they accept an electron, they are reduced to Fe2⫹. As they are reoxidized to Fe3⫹, the electrons pass to the next component of the electron-transport chain. 5.

COPPER (CU⫹) AND THE REDUCTION OF OXYGEN

The last cytochrome complex is cytochrome oxidase, which passes electrons from cytochrome c to O2 (see Fig. 21.5). It contains cytochromes a and a3 and the oxygen-binding site. A whole oxygen molecule, O2, must accept four electrons to be reduced to two H2O molecules. Bound copper (Cu⫹) ions in the cytochrome oxidase complex facilitate the collection of the four electrons and the reduction of O2. Cytochrome oxidase has a much lower Km for O2 than myoglobin (the heme-containing intracellular oxygen carrier) or hemoglobin (the heme-containing oxygen transporter in the blood). Thus, O2 is “pulled” from the erythrocyte to myoglobin and from myoglobin to cytochrome oxidase, where it is reduced to H2O.

383

Although iron deficiency anemia is characterized by decreased levels of hemoglobin and other ironcontaining proteins in the blood, the ironcontaining cytochromes and Fe–S centers of the electron-transport chain in tissues such as skeletal muscle are affected as rapidly. Fatigue in iron deficiency anemia, in patients such as Ann O’Rexia (see Chapter 16), results, in part, from the lack of electron transport for ATP production.

The iron in the heme in hemoglobin, unlike the iron in the heme of cytochromes, never changes its oxidation state (it is Fe2⫹ in hemoglobin). If the iron in hemoglobin were to become oxidized (Fe3⫹), the oxygen-binding capacity of the molecule would be lost. What accounts for this difference in iron oxidation states between hemoglobin and cytochromes?

C. Pumping of Protons One of the tenets of the chemiosmotic theory is that energy from the oxidation– reduction reactions of the electron-transport chain is used to transport protons from

CH3 (CH2 CH3

CH

C

CH2)3 H

CH2

H C OH HC

O H –

COO

CH2

CH N

C

CH3

N Fe N

CH2

CH

N HC

CH2

CH CH2

CH3

CH2 COO–

FIG. 21.8. Heme A. Heme A is found in cytochromes a and a3. Cytochromes are proteins that contain a heme chelated with an iron atom. Hemes are derivatives of protoporphyrin IX. Each cytochrome has a heme with different modifications of the side chains (indicated with dashed lines), resulting in a slightly different reduction potential and, consequently, a different position in the sequence of electron transfer.

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Normally, the protein structures binding the heme either protect the iron from oxidation (such as the globin proteins) or allow oxidation to occur (such as happens in the cytochromes). However, in hemoglobin M, a rare hemoglobin variant found in the human population, a tyrosine is substituted for the histidine at position F8 in the normal hemoglobin A. This tyrosine stabilizes the Fe3⫹ form of heme, and these subunits cannot bind oxygen. This is a lethal condition if it is homozygous.

the matrix to the intermembrane space. This proton pumping is generally facilitated by the vectorial arrangement of the membrane-spanning complexes. Their structure allows them to pick up electrons and protons on one side of the membrane and release protons on the other side of the membrane as they transfer an electron to the next component of the chain. The direct physical link between proton movement and electron transfer can be illustrated by an examination of the Q cycle for the b–c1 complex (Fig. 21.9). The Q cycle involves a double cycle of CoQ reduction and oxidation. CoQ accepts two protons at the matrix side together with two electrons; it then releases protons into the intermembrane space while donating one electron back to another component of the cytochrome b–c1 complex and one to cytochrome c. The mechanism for pumping protons at the NADH:CoQ oxidoreductase complex is not well understood, but it involves a Q cycle in which the Fe–S centers and FMN might participate. However, transmembrane proton movement at cytochrome c oxidase probably involves direct transport of the proton through a series of bound water molecules or amino acid side chains in the protein, a mechanism that has been described as a proton wire. The significance of the direct link between the electron transfer and proton movement is that one cannot occur without the other. Thus, when protons are not being used for ATP synthesis, the proton gradient and the membrane potential buildup. This “proton back pressure” controls the rate of proton pumping, which controls electron transport and O2 consumption.

D. Energy Yield from the Electron-Transport Chain The overall free-energy release from oxidation of NADH by O2 is approximately ⫺53 kcal, and from FAD(2H) it is approximately ⫺41 kcal. This ⌬G0 is so negative that the chain is never reversible; we never synthesize oxygen from H2O. It is so negative that it drives NADH and FAD(2H) formation from the pathways of fuel oxidation, such as the TCA cycle and glycolysis, to completion.

Intermembrane space

2H+

2 2e–

2QH2

2e– 2 ISP

1

2Q

2

2 C1

C

2e–

2e– bL

3

Q-Pool

2e– bH

Q

4 e– – e

Q–• QH2 Matrix

Inner mitochondrial membrane 2H

+

FIG. 21.9. The proton motive Q cycle for the b–c1 complex. (1) From 2QH2, electrons go down two different paths: One path is through an Fe–S center protein (ISP) toward cytochrome c (red arrows). Another path is “backward” to one of the b cytochromes (dashed arrows). (2) Electrons are transferred from ISP through cytochrome c1. Cytochrome c, which is in the intermembrane space, binds to the b–c1 complex to accept an electron. (3) Returning electrons go through another b cytochrome and are directed toward the matrix. (4) At the matrix side, electrons and 2H⫹ are accepted by Q. Q, coenzyme Q; Q•⫺, CoQ semiquinone; QH2, CoQ hydroquinone.

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Overall, each NADH donates two electrons, equivalent to the reduction of one-half of an O2 molecule. A generally (but not universally) accepted estimate of the stoichiometry of ATP synthesis is that four protons are pumped at complex I, four protons at complex III, and two protons at complex IV. With four protons translocated for each ATP synthesized, an estimated 2.5 ATPs are formed for each NADH oxidized, and 1.5 ATPs are formed for each of the other FAD(2H)containing flavoproteins that donate electrons to CoQ. (This calculation neglects proton requirements for the transport of phosphate and substrates from the cytosol, as well as the basal proton leak.) Thus, only approximately 30% of the energy available from NADH and FAD(2H) oxidation by O2 is used for ATP synthesis. Some of the remaining energy in the electrochemical potential is used for the transport of anions and Ca2⫹ into the mitochondrion. The remainder of the energy is released as heat. Consequently, the electron-transport chain is also our major source of heat.

385

Cora Nari has a lack of oxygen in the anterior and lateral walls of her heart caused by severe ischemia (lack of blood flow), resulting from clots formed within certain coronary arteries at the site of ruptured atherosclerotic plaques. The limited availability of O2 to act as an electron acceptor will decrease proton pumping and generation of an electrochemical potential gradient across the inner mitochondrial membrane of ischemic cells. As a consequence, the rate of ATP generation in these specific areas of her heart will decrease, thereby triggering events that lead to irreversible cell injury.

E. Respiratory Chain Inhibition and Sequential Transfer In the cell, electron flow in the electron-transport chain must be sequential from NADH or a flavoprotein all the way to O2 to generate ATP (see Fig. 21.5). In the absence of O2, no ATP is generated from oxidative phosphorylation because electrons back up in the chain. Even complex I cannot pump protons to generate the electrochemical gradient because every molecule of CoQ already has electrons that it cannot pass down the chain without an O2 to accept them at the end. The action of the respiratory chain inhibitor cyanide, which binds to cytochrome oxidase, is similar to that of anoxia: It prevents proton pumping by all three complexes. Complete inhibition of the b–c1 complex prevents pumping at cytochrome oxidase because there is no donor of electrons; it prevents pumping at complex I because there is no electron acceptor. Although complete inhibition of any one complex inhibits proton pumping at all of the complexes, partial inhibition of proton pumping can occur when only a fraction of the molecules of a complex contains bound inhibitor. The partial inhibition results in a partial decrease of the maximal rate of ATP synthesis. Table 21.1 lists chemical inhibitors of oxidative phosphorylation and indicates the steps within either electron transport or ATP synthesis at which they act.

Intravenous nitroprusside rapidly lowers elevated blood pressure through its direct vasodilating action. Fortunately, it was required in Cora Nari’s case only for several hours. During prolonged infusions of 24 to 48 hours or more, nitroprusside is converted to cyanide, an inhibitor of the cytochrome c oxidase complex. Because small amounts of cyanide are detoxified in the liver by conversion to thiocyanate, which is excreted in the urine, the conversion of nitroprusside to cyanide can be monitored by following blood thiocyanate levels.

II. OXIDATIVE PHOSPHORYLATION DISEASES Clinical diseases involving components of oxidative phosphorylation (referred to as OXPHOS diseases) are among the most commonly encountered degenerative diseases. The clinical pathology may be caused by gene mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) that encodes proteins required for normal oxidative phosphorylation.

Table 21.1

Inhibitors of Oxidative Phosphorylation

Inhibitor

Site of Inhibition

Rotenone, Amytal Antimycin C Carbon monoxide (CO) Cyanide (CN) Atractyloside Oligomycin

Transfer of electrons from complex I to coenzyme Q Transfer of electrons from complex III to cytochrome c Transfer of electrons from complex IV to oxygen Transfer of electrons from complex IV to oxygen Inhibits the adenine nucleotide translocase (ANT) Inhibits proton flow through the F0 component of the ATP synthase An uncoupler; facilitates proton transfer across the inner mitochondrial membrane A potassium ionophore; facilitates potassium ion transfer across the inner mitochondrial membrane

Dinitrophenol Valinomycin ATP, adenosine triphosphate.

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Cyanide binds to the Fe3⫹ in the heme of the cytochrome aa3 component of cytochrome c oxidase and prevents electron transport to O2. Mitochondrial respiration and energy production cease, and cell death rapidly occurs. The central nervous system is the primary target for cyanide toxicity. Acute inhalation of high concentrations of cyanide (e.g., smoke inhalation during a fire) provokes a brief central nervous system stimulation followed rapidly by convulsion, coma, and death. Acute exposure to lower amounts can cause light-headedness, breathlessness, dizziness, numbness, and headaches. Cyanide is present in the air as hydrogen cyanide (HCN), in soil and water as cyanide salts (e.g., NaCN), and in foods as cyanoglycosides. Most of the cyanide in the air usually comes from automobile exhaust. Examples of populations with potentially high exposures include active and passive smokers, people who are exposed to house or other building fires, residents who live near cyanide- or thiocyanate-containing hazardous waste sites, and workers involved in several manufacturing processes (e.g., photography or pesticide application). Cyanoglycosides such as amygdalin are present in edible plants such as almonds, pits from stone fruits (e.g., apricots, peaches, plums, cherries), sorghum, cassava, soybeans, spinach, lima beans, sweet potatoes, maize, millet, sugar cane, and bamboo shoots. HCN is released from cyanoglycosides by ␤-glucosidases present in the plant or in intestinal bacteria. Small amounts are inactivated in the liver principally by rhodanase, HO CH2OH which converts it to thiocyanate. O In the United States, toxic amounts of cyanoglycosides have been ingested as ground apricot pits, either as a result of their promotion as a health food or as a treatOCH2 HO HO HO ment for cancer. The drug Laetrile (amygdalin) was used as a cancer therapeutic agent, O although it was banned in the United States because it was ineffective and potentially CN toxic. Commercial fruit juices made from unpitted fruit could provide toxic amounts of O C HO OH cyanide, particularly in infants or children. In countries in which cassava is a dietary H staple, improper processing results in retention of its high cyanide content at potentially toxic levels. Amygdalin, a cyanoglycoside

A. Mitochondrial DNA and Oxidative Phosphorylation Diseases

Decreased activity of the electrontransport chain can result from inhibitors as well as from mutations in mtDNA and nuclear DNA. Why does an impairment of the electron-transport chain result in lactic acidosis?

Oxidative phosphorylation (OXPHOS) is responsible for producing most of the ATP that our cells require. The genes responsible for the polypeptides that comprise the OXPHOS complexes within the mitochondria are located within either the nuclear DNA (nDNA) or the mitochondrial DNA (mtDNA). A broad spectrum of human disorders (the OXPHOS diseases) may result from genetic mutations or nongenetic alterations (spontaneous mutations) in either the nDNA or the mtDNA. Increasingly, such changes appear to be responsible for at least some aspects of common disorders, such as Parkinson disease, dilated and hypertrophic cardiomyopathies, diabetes mellitus, Alzheimer disease, depressive disorders, and a host of less well-known clinical entities.

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The mtDNA is a small, 16,569 nucleotide-pair, double-stranded, circular DNA. It encodes 13 subunits of the complexes involved in oxidative phosphorylation: 7 of the 42 subunits of complex I (NADH:CoQ oxidoreductase complex), 1 of the 11 subunits of complex III (cytochrome b–c1 complex), 3 of the 13 subunits of complex IV (cytochrome oxidase), and 2 subunits of the F0 portion ATP–synthase complex. In addition, mtDNA encodes the necessary components for translation of its mRNA: a large and small ribosomal RNA (rRNA) and 22 transfer RNAs (tRNAs). Mutations in mtDNA have been identified as deletions, duplications, or point mutations (Table 21.2). The genetics of mutations in mtDNA are defined by maternal inheritance, replicative segregation, threshold expression, a high mtDNA mutation rate, and the accumulation of somatic mutations with age. The maternal inheritance pattern reflects the exclusive transmission of mtDNA from the mother to her children. The egg contains approximately 300,000 molecules of mtDNA packaged into mitochondria. These are retained during fertilization, whereas those of the sperm do not enter the egg or are lost. Usually, some mitochondria are present that have the mutant mtDNA and some have normal (wild-type) DNA. As cells divide during mitosis and meiosis, mitochondria replicate by fission, but various amounts of mitochondria with mutant and wild-type DNA are distributed to each daughter cell (replicative segregation). Thus, any cell can have a mixture of mitochondria, each with mutant or wild-type mtDNAs (heteroplasmy). The mitotic and meiotic segregation of the heteroplasmic mtDNA mutation results in variable oxidative phosphorylation deficiencies between patients with the same mutation and even among a patient’s own tissues. The disease pathology usually becomes worse with age because a small amount of normal mitochondria might confer normal function and exercise capacity while the patient is young. As the patient ages, somatic (spontaneous) mutations in mtDNA accumulate from the generation of free radicals within the mitochondria (see Chapter 24). These mutations frequently become permanent, partly because

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Table 21.2 Syndrome

Examples of OXPHOS Diseases Arising from mtDNA Mutations Characteristic Symptoms

mtDNA Mutation

I. mtDNA rearrangements in which genes are deleted or duplicated Kearns-Sayre Onset before 20 years of age, Deletion of contiguous syndrome characterized by ophthalsegments of tRNA and moplegia, atypical retinitis OXPHOS polypeptides, pigmentosa, mitochondrial or duplication mutations myopathy, and one of the consisting of tandemly following: cardiac conduction arranged normal mtDNA defect, cerebellar syndrome, and an mtDNA with a or elevated CSF proteins deletion mutation Pearson syndrome Systemic disorder of oxidative Deletion of contiguous phosphorylation that segments of tRNA and predominantly affects OXPHOS polypeptides, or bone marrow duplication mutations consisting of tandemly arranged normal mtDNA and a mtDNA with a deletion mutation II. mtDNA point mutations in tRNA or ribosomal RNA genes MERRF (myoclonic Progressive myoclonic epilepsy and epilepsy, a mitochondrial ragged red fiber myopathy with ragged red disease) fibers, and a slowly progressive dementia. Onset of symptoms: late childhood to adult MELAS Progressive neurodegenerative (mitochondrial disease characterized by myopathy, strokelike episodes first encephalomyopathy, occurring between 5 and lactic acidosis, 15 years of age and a and strokelike mitochondrial myopathy episodes) III. mtDNA missense mutations in OXPHOS polypeptides Leigh disease Mean age of onset, 1.5–5 years; (subacute clinical manifestations include necrotizing optic atrophy, ophthalmoplegia, encephalopathy) nystagmus, respiratory abnormalities, ataxia, hypotonia, spasticity, and developmental delay or regression LHON (Leber Late onset, acute optic atrophy hereditary optic neuropathy)

tRNALys

80%–90% mutations in tRNALeu

7%–20% of cases have mutations in F0 subunits of F0F1 ATPase.

90% of European and Asian cases result from mutation in NADH dehydrogenase

mtDNA, mitochondrial DNA; CSF, cerebrospinal fluid; tRNA, transfer RNA; OXPHOS, oxidative phosphorylation; rRNA, ribosomal RNA.

mtDNA does not have access to the same repair mechanisms available for nDNA (high mutation rate). Even in normal individuals, somatic mutations result in a decline of oxidative phosphorylation capacity with age (accumulation of somatic mutations with age). At some stage, the ATP-generating capacity of a tissue falls below the tissue-specific threshold for normal function (threshold expression). In general, symptoms of these defects appear in one or more of the tissues with the highest ATP demands: nervous tissue, heart, skeletal muscle, and kidney.

387

A patient experienced spontaneous muscle jerking (myoclonus) in her midteens, and her condition progressed over 10 years to include debilitating myoclonus, neurosensory hearing loss, dementia, hypoventilation, and mild cardiomyopathy. Energy metabolism was affected in the central nervous system, heart, and skeletal muscle, resulting in lactic acidosis. A history indicated that the patient’s mother, her grandmother, and two maternal aunts had symptoms involving either nervous or muscular tissue (clearly a case of maternal inheritance). However, no other relatives had identical symptoms. The symptoms and history of the patient are those of myoclonic epileptic ragged red fiber disease (MERRF). The affected tissues (central nervous system and muscle) are two of the tissues with the highest ATP requirements. Most cases of MERRF are caused by a point mutation in mitochondrial tRNALys (mtRNALys). The mitochondria, obtained by muscle biopsy, are enlarged and show abnormal patterns of cristae. The muscle tissue also shows ragged red fibers.

The effect of inhibition of electron transport is an impaired oxidation of pyruvate, fatty acids, and other fuels. In many cases, the inhibition of mitochondrial electron transport results in higher than normal levels of lactate and pyruvate in the blood and an increased lactate:pyruvate ratio. NADH oxidation requires the complete transfer of electrons from NADH to O2, and a defect anywhere along the chain will result in the accumulation of NADH and a decrease of NAD⫹. The increase in NADH/NAD⫹ inhibits pyruvate dehydrogenase and causes the accumulation of pyruvate. It also increases the conversion of pyruvate to lactate, and elevated levels of lactate appear in the blood. A large number of genetic defects of the proteins in respiratory chain complexes have, therefore, been classified together as “congenital lactic acidosis.”

B. Other Genetic Disorders of Oxidative Phosphorylation Genetic mutations also have been reported for mitochondrial proteins that are encoded by nDNA. Most of the estimated 1,000 proteins required for oxidative phosphorylation are encoded by nDNA, whereas mtDNA encodes only 13 subunits of the oxidative phosphorylation complexes (including ATP synthase). Nuclear DNA encodes the additional 70 or more subunits of the oxidative phosphorylation complexes, as well as the adenine nucleotide translocase (ANT) and other anion translocators. Coordinate regulation of expression of nDNA and mtDNA,

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importation of proteins into the mitochondria, assembly of the complexes, and regulation of mitochondrial fission are nuclear encoded. The nuclear respiratory factors (NRF-1 and NRF-2) are nuclear transcription factors that bind to and activate promoter regions of the nuclear genes that encode subunits of the respiratory chain complexes, including cytochrome c. They also activate the transcription of the nuclear gene for the mitochondrial transcription factor (mTF)-A. The protein product of this gene translocates into the mitochondrial matrix, where it stimulates transcription and replication of the mitochondrial genome. Nuclear DNA mutations differ from mtDNA mutations in several important respects. These mutations do not show a pattern of maternal inheritance but are usually autosomal recessive. The mutations are uniformly distributed to daughter cells and therefore are expressed in all tissues that contain the allele for a particular tissue-specific isoform. However, phenotypic expression will still be most apparent in tissues with high ATP requirements.

III. COUPLING OF ELECTRON TRANSPORT AND ADENOSINE TRIPHOSPHATE SYNTHESIS How does shivering generate heat?

The electrochemical gradient couples the rate of the electron-transport chain to the rate of ATP synthesis. Because electron flow requires proton pumping, electron flow cannot occur faster than protons are used for ATP synthesis (coupled oxidative phosphorylation) or returned to the matrix by a mechanism that short-circuits the ATP synthase pore (uncoupling).

A. Regulation through Coupling

NADH

5

e–

NAD+ H+

3

O2 H2O

4

ADP + Pi

1

ATP Matrix

H+ 2 Cytosolic side

FIG. 21.10. The concentration of ADP (or the phosphate potential, [ATP]/[ADP][Pi]) controls the rate of oxygen consumption. (1) ADP is phosphorylated to ATP by ATP synthase. (2) The release of the ATP requires proton flow through ATP synthase into the matrix. (3) The use of protons from the intermembrane space for ATP synthesis decreases the proton gradient. (4) As a result, the electron-transport chain pumps more protons, and oxygen is reduced to H2O. (5) As NADH donates electrons to the electron-transport chain, NAD⫹ is regenerated and returns to the TCA cycle or other NADH-producing pathways.

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As ATP chemical bond energy is used by energy-requiring reactions, ADP and Pi concentrations increase. The more ADP is present to bind to the ATP synthase, the greater will be proton flow through the ATP synthase pore, from the intermembrane space to the matrix. Thus, as ADP levels rise, proton influx increases, and the electrochemical gradient decreases (Fig. 21.10). The proton pumps of the electron-transport chain respond with increased proton pumping and electron flow to maintain the electrochemical gradient. The result is increased O2 consumption. The increased oxidation of NADH in the electron-transport chain and the increased concentration of ADP stimulate the pathways of fuel oxidation, such as the TCA cycle, to supply more NADH and FAD(2H) to the electron-transport chain. For example, during exercise we use more ATP for muscle contraction, consume more oxygen, oxidize more fuel (which means burn more calories), and generate more heat from the electron-transport chain. If we rest, and the rate of ATP use decreases, proton influx decreases, the electrochemical gradient increases, and proton “back pressure” decreases the rate of the electron-transport chain. NADH and FAD(2H) cannot be oxidized as rapidly in the electron-transport chain, and consequently, their buildup inhibits the enzymes that generate them. The system is poised to maintain very high levels of ATP at all times. In most tissues, the rate of ATP use is nearly constant over time. However, in skeletal muscles, the rates of ATP hydrolysis change dramatically as the muscle goes from rest to rapid contraction. Even under these circumstances, ATP concentration decreases by only approximately 20% because it is so rapidly regenerated. In the heart, Ca2⫹ activation of TCA cycle enzymes provides an extra push to NADH generation, so that neither ATP nor NADH levels fall as ATP demand is increased. The electron-transport chain has a very high capacity and can respond very rapidly to any increase in ATP use.

B. Uncoupling Adenosine Triphosphate Synthesis from Electron Transport When protons leak back into the matrix without going through the ATP synthase pore, they dissipate the electrochemical gradient across the membrane without

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generating ATP. This phenomenon is called uncoupling oxidative phosphorylation. It occurs with chemical compounds, known as uncouplers, and it occurs physiologically with uncoupling proteins that form proton conductance channels through the membrane. Uncoupling of oxidative phosphorylation results in increased oxygen consumption and heat production as electron flow and proton pumping attempt to maintain the electrochemical gradient. 1.

CHEMICAL UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION

389

Shivering results from muscular contraction, which increases the rate of ATP hydrolysis. As a consequence of proton entry for ATP synthesis, the electrontransport chain is stimulated. Oxygen consumption increases, as does the amount of energy lost as heat by the electron-transport chain.

Chemical uncouplers, also known as proton ionophores, are lipid-soluble compounds that rapidly transport protons from the cytosolic to the matrix side of the inner mitochondrial membrane (Fig. 21.11). Because the proton concentration is higher in the intermembrane space than in the matrix, uncouplers pick up protons from the intermembrane space. Their lipid solubility enables them to diffuse through the inner mitochondrial membrane while carrying protons and to release these protons on the matrix side. The rapid influx of protons dissipates the electrochemical potential gradient; therefore, the mitochondria are unable to synthesize ATP. Eventually, mitochondrial integrity and function are lost. 2.

UNCOUPLING PROTEINS AND THERMOGENESIS

Uncoupling proteins (UCPs) form channels through the inner mitochondrial membrane that are able to conduct protons from the intermembrane space to the matrix, thereby short-circuiting ATP synthase. UCP1 (thermogenin) is associated with heat production in brown adipose tissue. The major function of brown adipose tissue is nonshivering thermogenesis, whereas the major function of white adipose tissue is the storage of triacylglycerols in white lipid droplets. The brown color arises from the large number of mitochondria that participate. Human infants, who have little voluntary control over their environment and may kick their blankets off at night, have brown fat deposits along the neck, the breastplate, between the scapulae, and around the kidneys to protect them from cold. However, there is very little brown fat in most adults. In response to cold, sympathetic nerve endings release norepinephrine, which activates a lipase in brown adipose tissue that releases fatty acids from triacylglycerols (Fig. 21.12). Fatty acids serve as a fuel for the tissue (i.e., are oxidized to generate the electrochemical potential gradient and ATP) and participate directly in the proton

Matrix H+ NO2

H+ H+

HO NO2 H+ High [H+] causes outside protons to bond to DNP molecules

H+ NO2 –

O NO2

Low [H+] inside causes protons to dissociate from DNP molecules

Inner mitochondrial membrane

FIG. 21.11. Dinitrophenol (DNP) is lipid-soluble and can, therefore, diffuse across the membrane. It has a dissociable proton with a pKa near 7.2. Thus, in the intermembrane space where [H⫹] is high (pH low), DNP picks up a proton, which it carries across the membrane. At the lower proton concentration of the matrix, the H⫹ dissociates. As a consequence, cells cannot maintain their electrochemical gradient or synthesize ATP. DNP was once recommended in the United States as a weight-loss drug, based on the principle that decreased [ATP] and increased electron transport stimulate fuel oxidation. However, several deaths resulted from its use.

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A skeletal muscle biopsy performed on Ivy Sharer indicated proliferation of subsarcolemmal mitochondria with degeneration of muscle fibers (ragged red fibers) in approximately 55% of the total fibers observed. An analysis of mitochondrial DNA (mtDNA) indicated no genetic mutations but did show a moderate quantitative depletion of mtDNA. Ivy Sharer’s AIDS was being treated with zidovudine (azidothymidine [AZT]), which also can act as an inhibitor of the mtDNA polymerase (polymerase ␥). A review of the drug’s potential adverse effects showed that, rarely, it may cause varying degrees of mtDNA depletion in different tissues, including skeletal muscle. The depletion may cause a severe mitochondrial myopathy, including “ragged red fiber” accumulation within the skeletal muscle cells associated with ultrastructural abnormalities in their mitochondria.

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Hypothalamus Cold Sympathetic nerve Heat

Norepinephrine

Triglyceride

H+

Thermogenin

Fatty acid

Respiratory chain

O2

H2O

Mitochondrion

Brown fat cell

FIG. 21.12. Brown fat is a tissue specialized for nonshivering thermogenesis. Cold or excessive food intake stimulates the release of norepinephrine from the sympathetic nerve endings. As a result, a lipase is activated that releases fatty acids for oxidation. The proton conductance protein, thermogenin, is activated, and protons are brought into the matrix. This stimulates the electron-transport chain, which increases its rate of NADH and FAD(2H) oxidation and produces more heat.

Salicylate, which is a degradation product of aspirin in humans, is lipid-soluble and has a dissociable proton. In high concentrations, as in salicylate poisoning, salicylate is able to partially uncouple mitochondria. The decline of ATP concentration in the cell and consequent increase of AMP in the cytosol stimulates glycolysis. The overstimulation of the glycolytic pathway (see Chapter 22) results in increased levels of lactic acid in the blood and a metabolic acidosis. Fortunately, Dennis Veere did not develop this consequence of aspirin poisoning (see Chapter 4).

conductance channel by activating UCP1 along with reduced CoQ. When UCP1 is activated by fatty acids, it transports protons from the cytosolic side of the inner mitochondrial membrane back into the mitochondrial matrix without ATP generation. Thus, it partially uncouples oxidative phosphorylation and generates additional heat. The uncoupling proteins exist as a family of proteins: UCP1 (thermogenin) is expressed in brown adipose tissue, UCP2 is found in most cells, UCP3 is found principally in skeletal muscle, and UCP4 and UCP5 are found in the nervous system. These are highly regulated proteins that, when activated, increase the amount of energy from fuel oxidation that is being released as heat. However, recent data indicate that this may not be the primary role of UCP2 and UCP3. It has been hypothesized that UCP3 acts as a transport protein to remove fatty acid anions and lipid peroxides from the mitochondria, thereby reducing the risk of forming oxygen free radicals (see Chapter 24) and thus decreasing the occurrence of mitochondrial and cell injury. 3.

PROTON LEAK AND RESTING METABOLIC RATE

A low level of proton leak across the inner mitochondrial membrane occurs in our mitochondria all the time, and our mitochondria, thus, are normally partially uncoupled. It has been estimated that more than 20% of our resting metabolic rate is the energy expended to maintain the electrochemical gradient dissipated by our basal proton leak (also referred to as global proton leak). Some of the proton leak results from permeability of the membrane associated with proteins embedded in the lipid bilayer. An unknown amount may result from uncoupling proteins.

IV. TRANSPORT THROUGH INNER AND OUTER MITOCHONDRIAL MEMBRANES Most of the newly synthesized ATP that is released into the mitochondrial matrix must be transported out of the mitochondria, where it is used for energy-requiring processes such as active ion transport, muscle contraction, or biosynthetic reactions. Likewise, ADP, phosphate, pyruvate, and other metabolites must be transported into the matrix. This requires transport of compounds through both the inner and outer mitochondrial membranes.

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391

A. Transport through the Inner Mitochondrial Membrane The inner mitochondrial membrane forms a tight permeability barrier to all polar molecules, including ATP; ADP; Pi; anions such as pyruvate; and cations such as Ca2⫹, H⫹, and K⫹. Yet, the process of oxidative phosphorylation depends on rapid and continuous transport of many of these molecules across the inner mitochondrial membrane (Fig. 21.13). Ions and other polar molecules are transported across the inner mitochondrial membrane by specific protein translocases that nearly balance charge during the transport process. Most of the exchange transport is a form of active transport that generally uses energy from the electrochemical potential gradient, either the membrane potential or the proton gradient. ATP–ADP translocase (also called ANT, for adenine nucleotide translocase) transports ATP formed in the mitochondrial matrix to the intermembrane space in a specific 1:1 exchange for ADP produced from energy-requiring reactions outside of the mitochondria (see Fig. 21.13). Because ATP contains four negative charges and ADP contains only three, the exchange is promoted by the electrochemical potential gradient because the net effect is the transport of one negative charge from the matrix to the cytosol. Similar antiports exist for most metabolic anions. In contrast, inorganic phosphate and pyruvate are transported into the mitochondrial matrix on specific transporters called symports together with a proton. A specific transport protein for Ca2⫹ uptake, called the Ca2⫹ uniporter, is driven by the electrochemical potential gradient, which is negatively charged on the matrix side of the membrane relative to the cytosolic side. Other transporters include the dicarboxylate transporter (phosphate–malate exchange), the tricarboxylate transporter (citrate–malate exchange), the aspartate–glutamate transporter, and the malate–␣-ketoglutarate transporter.

HK ADP

Pi, pyruvate ANT O

ATP ––––

O2

HO

H+ H+

bra ne

ADP + Pi

–

O– Citrate

H+ Symport

em

ATP

+

P O–

Mitochondrial matrix

H+

ane mbr me

–––– Proton gradient

ter Ou AC

Membrane potential + ++++

VD

Electrochemical potential gradient

ATP – – – Antiport ADP ATP–ADP translocase

m er Inn

––

++

Ca2+

H+

COO– C

O

Symport CH3

FIG. 21.13. Transport of compounds across the inner and outer mitochondrial membranes. The electrochemical potential gradient drives the transport of ions across the inner mitochondrial membrane on specific translocases. Each translocase is composed of specific membranespanning helices that bind only specific compounds (adenine nucleotide translocase [ANT]). In contrast, the outer membrane contains relatively large unspecific pores called voltage-dependent anion channels (VDACs) through which a wide range of ions diffuse. These bind cytosolic proteins such as hexokinase (HK), which enables HK to have access to newly exported ATP.

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Investigators reported finding antibodies against cardiac ATP–ADP translocase in an individual who died of a viral cardiomyopathy. How could these antibodies result in death?

B. Transport through the Outer Mitochondrial Membrane Whereas the inner mitochondrial membrane is highly impermeable, the outer mitochondrial membrane is permeable to compounds with a molecular weight up to approximately 6,000 Da because it contains large nonspecific pores called voltagedependent anion channels (VDACs) that are formed by mitochondrial porins (see Fig. 21.13). Unlike most transport proteins, which are membrane-spanning helices with specific binding sites, VDACs are composed of porin homodimers that form a ␤-barrel with a relatively large nonspecific water-filled pore through the center. These channels are “open” at low transmembrane potential, with a preference for anions such as phosphate, chloride, pyruvate, citrate, and adenine nucleotides. VDACs thus facilitate translocation of these anions between the intermembrane space and the cytosol. Several cytosolic kinases, such as the hexokinase that initiates glycolysis, bind to the cytosolic side of the channel where they have ready access to newly synthesized ATP.

C. The Mitochondrial Permeability Transition Pore Outer mitochondrial membrane

Inner mitochondrial membrane Bax 2+

Ca Pi ROS ⌬p

+ –

ANT

V D A C

–

ATP [H+]

CD Bcl-2

Inter membrane space

FIG. 21.14. The mitochondrial permeability transition pore (MPTP). In the MPTP, ANT is thought to complex with VDAC. The conformation of ANT is regulated by cyclophilin D (CD), and Ca2⫹. The change to an open pore is activated by Ca2⫹, depletion of adenine nucleotides, and reactive oxygen species (ROS) that alter SH groups. It is inhibited by the electrochemical potential gradient (⌬p), by cytosolic ATP, and by a low cytosolic pH. VDACs bind several proteins, including Bcl-2 and Bax, which regulate apoptosis. Binding of proapoptotic members of the Bcl-2 family to VDAC may change the permeability of the outer membrane so as to either favor or block events leading to apoptosis.

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The mitochondrial permeability transition involves the opening of a large nonspecific pore (called MPTP, the mitochondrial permeability transition pore) through the inner mitochondrial membrane and outer membranes at sites where they form a junction (Fig. 21.14). In one model of the MPTP, the basic components of the pore are ANT, the VDAC, and cyclophilin D (CD) (which is a cis-trans isomerase for the proline peptide bond). Normally, ANT is a closed pore that functions specifically in a 1:1 exchange of matrix ATP for ADP in the intermembrane space. However, increased mitochondrial matrix Ca2⫹, excess phosphate, or reactive oxygen species (ROS), which form oxygen or oxygen–nitrogen radicals, can activate opening of the pore. Conversely, ATP on the cytosolic side of the pore (and possibly a pH ⬍7.0) and a membrane potential across the inner membrane protect against pore opening. Opening of the MPTP can be triggered by ischemia (hypoxia), which results in a temporary lack of O2 for maintaining the proton gradient and ATP synthesis. When the proton gradient is not being generated by the electron-transport chain, ATP synthase runs backward and hydrolyzes ATP in an attempt to restore the gradient, thus rapidly depleting cellular levels of ATP. As ATP is hydrolyzed to ADP, the ADP is converted to adenine, and the nucleotide pool is no longer able to protect against pore opening. This can lead to a downward spiral of cellular events. A lack of ATP for maintaining the low intracellular Ca2⫹ can contribute to pore opening. When the MPTP opens, protons flood in and maintaining a proton gradient becomes impossible. Anions and cations enter the matrix, mitochondrial swelling ensues, and the mitochondria become irreversibly damaged. The result is cell lysis and death (necrosis). CLINICAL COMMENTS Cora Nari. Thrombolysis stimulated by intravenous recombinant tissue plasminogen activator (tPA) restored O2 to Cora Nari’s heart muscle and successfully decreased the extent of ischemic damage. The rationale for the use of tPA within 4 to 6 hours after the onset of a myocardial infarction relates to the function of the normal intrinsic fibrinolytic system (see Chapter 45). This system is designed to dissolve unwanted intravascular clots through the action of the enzyme plasmin, a protease that digests the fibrin matrix within the clot. tPA stimulates the conversion of plasminogen to its active form, plasmin. The result is a lysis of the thrombus and improved blood flow through the previously obstructed vessel, allowing fuels and oxygen to reach the heart cells. The human tPA protein administered to Mrs. Nari is produced by recombinant DNA technology (see Chapter 17). This treatment rapidly restored oxygen supply to her heart.

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X. S. Teefore. Mr. Teefore could be treated with antithyroid drugs, by subtotal resection of the thyroid gland, or with radioactive iodine. Successful treatment normalizes thyroid hormone secretion, and all of the signs, symptoms, and metabolic alterations of hyperthyroidism quickly subside. Ivy Sharer. In the case of Ivy Sharer, a diffuse myopathic process was superimposed on her AIDS and her pulmonary tuberculosis, either of which could have caused progressive weakness. In addition, she could have been suffering from a congenital mtDNA myopathy, symptomatic only as she ages. A systematic diagnostic process, however, finally led her physician to conclude that her myopathy was caused by a disorder of oxidative phosphorylation induced by her treatment with zidovudine (AZT). Fortunately, when AZT was discontinued, Ivy’s myopathic symptoms gradually subsided. A repeat skeletal muscle biopsy performed 4 months later showed that her skeletal muscle cell mtDNA had been restored to normal and that she had experienced a reversible drug-induced disorder of oxidative phosphorylation. BIOCHEMICAL COMMENTS Mitochondria and Apoptosis. The loss of mitochondrial integrity is a major route initiating apoptosis (see Chapter 18, Section V). The intermembrane space contains procaspases 2, 3, and 9, which are proteolytic enzymes that are in the zymogen form (i.e., they must be proteolytically cleaved to be active). It also contains apoptosis-initiating factor (AIF) and caspase-activated DNAase (CAD). AIF has a nuclear-targeting sequence and is transported into the nucleus under appropriate conditions. Once AIF is inside the nucleus, it initiates chromatin condensation and degradation. Cytochrome c, which is loosely bound to the inner mitochondrial membrane, may also enter the intermembrane space when the electrochemical potential gradient is lost. The release of cytochrome c and the other proteins into the cytosol initiates apoptosis (see Chapter 18). What is the trigger for the release of cytochrome c and the other proteins from the mitochondria? The VDAC pore is not large enough to allow the passage of proteins. Several theories have been proposed, each supported and contradicted by experimental evidence. One is that Bax (a member of the Bcl-2 family of proteins that forms an ion channel in the outer mitochondrial membrane) allows the entry of ions into the intermembrane space, causing swelling of this space and rupture of the outer mitochondrial membrane. Another theory is that Bax and VDAC (which is known to bind Bax and other Bcl-2 family members) combine to form an extremely large pore, much larger than is formed by either alone. Finally, it is possible that the MPTP or ANT participate in rupture of the outer membrane, but that they close in a way that still provides the energy for apoptosis.

393

As ATP is hydrolyzed during muscular contraction, ADP is formed. This ADP must exchange into the mitochondria on ATP–ADP translocase to be converted back to ATP. Inhibition of ATP–ADP translocase results in rapid depletion of the cytosol ATP levels and loss of cardiac contractility.

As infusion of tPA lysed the clot blocking blood flow to Cora Nari’s heart, oxygenated blood was reintroduced into the ischemic heart. Although oxygen may rapidly restore the capacity to generate ATP, it often increases cell death, a phenomenon called ischemia–reperfusion injury. During ischemia, several factors may protect heart cells against irreversible injury and cell death until oxygen is reintroduced. The stimulation of anaerobic glycolysis in the cytosol generates ATP without oxygen because glucose is converted to lactic acid. Lactic acid decreases cytosolic pH. Both cytosolic ATP and a lowering of the pH protect against opening of the MPTP. In addition, Ca2⫹ uptake by mitochondria requires a membrane potential, and it is matrix Ca2⫹ that activates opening of the MPTP. Thus, depending on the severity of the ischemic insult, the MPTP may not open, or may open and reseal, until oxygen is reintroduced. Then, depending on the sequence of events, reestablishment of the proton gradient, mitochondrial uptake of Ca2⫹, or an increase of pH above 7.0 may activate the MPTP before the cell has recovered. In addition, the reintroduction of O2 generates oxygen free radicals, particularly through free radical forms of CoQ in the electron-transport chain. These also may open the MPTP. The role of free radicals in ischemia–reperfusion injury is discussed in more detail in Chapter 24.

Key Concepts • • • • • •

The reduced cofactors generated during fuel oxidation donate their electrons to the mitochondrial electron-transport chain. The electron-transport chain transfers the electrons to O2, which is reduced to water. As electrons travel through the electron-transport chain, protons are transferred from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. The asymmetric distribution of protons across the inner mitochondrial membrane generates an electrochemical gradient across the membrane. The electrochemical gradient consists of a change in pH (⌬pH) across the membrane and also a difference in charge (⌬␺) across the membrane. Proton entry into the mitochondrial matrix is energetically favorable and drives the synthesis of ATP via the ATP synthase.

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In addition to increased transcription of genes that encode TCA cycle enzymes and certain other enzymes of fuel oxidation, thyroid hormones increase the level of UCP2 and UCP3. In hyperthyroidism, the efficiency with which energy is derived from the oxidation of these fuels is significantly less than normal. As a consequence of the increased rate of the electron-transport chain, hyperthyroidism results in increased heat production. Patients with hyperthyroidism, such as X. S. Teefore, complain of constantly feeling hot and sweaty.

• • • • •

•

Respiration (oxygen consumption) is normally coupled to ATP synthesis; if one process is inhibited, the other is also inhibited. Uncouplers allow respiration to continue in the absence of ATP synthesis because the energy inherent in the proton gradient is released as heat. OXPHOS diseases are caused by mutations in either nuclear or mitochondrial DNA that lead to a decrease in mitochondrial capacity for synthesizing ATP via oxidative phosphorylation. Because the inner mitochondrial membrane is impermeable to virtually all biochemical compounds, transport systems exist to allow entry and exit of appropriate metabolites. Under appropriate stress, mitochondria will generate a nonspecific channel across both the inner and outer membranes that is known as the mitochondrial permeability transition pore. The opening of the pore is associated with events that lead to necrotic cell death. Diseases discussed in this chapter are summarized in Table 21.3.

Table 21.3

Diseases Discussed in Chapter 21

Disease or Disorder

Environmental or Genetic

Comments

Myocardial infarction

Both

Hyperthyroidism

Both

AIDS treatment

Environmental

Iron deficiency anemia

Environmental

Cyanide poisoning

Environmental

Mitochondrial disorders

Genetic

The lack of oxygen in the anterior and lateral walls of the heart is caused by severe ischemia because of clots formed within certain coronary arteries at the site of ruptured atherosclerotic plaques. The limited availability of oxygen to act as an electron acceptor decreases the proton motive force across the inner mitochondrial membrane of ischemic cells. This leads to reduced ATP generation, triggering events that lead to irreversible cell injury. Graves disease is an autoimmune genetic disorder caused by the generation of human thyroid stimulating immunoglobulins. These immunoglobulins stimulate growth of the thyroid gland and excess secretion of the thyroid hormones T3 and T4. AZT, a component of AIDS treatment cocktails, can act as an inhibitor of mitochondrial DNA polymerase. Under rare conditions, it can lead to a depletion of mitochondrial DNA in cells, leading to a severe mitochondrial myopathy. Lack of iron for heme synthesis, leading to reduced oxygen delivery to cells, and reduced iron in the electron-transfer chain, leading to muscle weakness. Cyanide binds to the Fe3⫹ in the heme of cytochrome aa3, a component of cytochrome oxidase. Mitochondrial respiration and energy production cease, and cell death rapidly occurs. Many types of mutations, leading to altered mitochondrial function, and reduced energy production, due to mutations in the mitochondrial DNA. See Table 21.2 for a full listing of these disorders.

ATP, adenosine triphosphate; AZT, azidothymidine.

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REVIEW QUESTIONS—CHAPTER 21 1.

2.

3.

Consider the following experiment. Carefully isolated liver mitochondria are incubated in the presence of a limiting amount of malate. Three minutes after adding the substrate, cyanide is added, and the reaction is allowed to proceed for another 7 minutes. At this point, which of the following components of the electron-transfer chain will be in an oxidized state? A. Complex I B. Complex II C. Complex III D. Coenzyme Q E. Cytochrome C Consider the following experiment. Carefully isolated liver mitochondria are placed in a weakly buffered solution. Malate is added as an energy source, and an increase in oxygen consumption confirms that the electron-transport chain is functioning properly within these organelles. Valinomycin and potassium are then added to the mitochondrial suspension. Valinomycin is a drug that allows potassium ions to freely cross the inner mitochondrial membrane. What is the effect of valinomycin on the proton motive force that had been generated by the oxidation of malate? A. The proton motive force will be reduced to a value of zero. B. There will be no change in the proton motive force. C. The proton motive force will be increased. D. The proton motive force will be decreased but to a value greater than zero. E. The proton motive force will be decreased to a value less than zero. Dinitrophenol acts as an uncoupler of oxidative phosphorylation by which of the following mechanisms? A. Activating the H⫹-ATPase B. Activating coenzyme Q

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C. Blocking proton transport across the inner mitochondrial membrane D. Allowing for proton exchange across the inner mitochondrial membrane E. Enhancing oxygen transport across the inner mitochondrial membrane 4.

A 25-year-old woman presents with chronic fatigue. A series of blood tests is ordered, and the results suggest that her red blood cell count is low because of iron deficiency anemia. Such a deficiency would lead to fatigue because of which of the following? A. Her decrease in Fe–S centers is impairing the transfer of electrons in the electron-transport chain. B. She is not producing enough H2O in the electrontransport chain, leading to dehydration, which has resulted in fatigue. C. Iron forms a chelate with NADH and FAD(2H) that is necessary for them to donate their electrons to the electron-transport chain. D. Iron acts as a cofactor for ␣-ketoglutarate DH in the TCA cycle, a reaction required for the flow of electrons through the electron-transport chain. E. Iron accompanies the protons that are pumped from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. Without iron, the proton gradient cannot be maintained to produce adequate ATP.

5.

Which of the following would be expected for a patient with an OXPHOS disease? A. A high ATP:ADP ratio in the mitochondria B. A high NADH:NAD⫹ ratio in the mitochondria C. A deletion on the X chromosome D. A high activity of complex II of the electrontransport chain E. A defect in the integrity of the inner mitochondrial membrane

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22

Generation of Adenosine Triphosphate from Glucose: Glycolysis

Glucose ATP

hexokinase

Glucose 6-P

Fructose 6-P ATP

Phosphofructokinase-1

Fructose-1,6-bis P 2 NADH 4 ATP Pyruvate Shuttle system Pyruvate Acetyl CoA TCA cycle

H+ O2 CO2 H2O NADH ADP + Pi ATP

H+ Electrontransport chain

ATP synthase Mitochondrion

H+

FIG. 22.1. Overview of glycolysis and the TCA cycle.

Glucose is the universal fuel for human cells. Every cell type in the human is able to generate adenosine triphosphate (ATP) from glycolysis, the pathway in which glucose is oxidized and cleaved to form pyruvate. The importance of glycolysis in our fuel economy is related to the availability of glucose in the blood, as well as the ability of glycolysis to generate ATP in both the presence and absence of O2. Glucose is the major sugar in our diet and the sugar that circulates in the blood to ensure that all cells have a continuous fuel supply. The brain uses glucose almost exclusively as a fuel. Glycolysis begins with the phosphorylation of glucose to glucose 6-phosphate (glucose 6-P) by hexokinase. In subsequent steps of the pathway, one glucose 6-P molecule is oxidized to two pyruvate molecules with generation of two molecules of nicotinamide adenine dinucleotide (NADH) (Fig. 22.1). A net generation of two molecules of ATP occurs through direct transfer of highenergy phosphate from intermediates of the pathway to adenosine diphosphate (ADP) (substrate-level phosphorylation). Glycolysis occurs in the cytosol and generates cytosolic NADH. Because NADH cannot cross the inner mitochondrial membrane, its reducing equivalents are transferred to the electron-transport chain by either the malate– aspartate shuttle or the glycerol 3-phosphate shuttle (see Fig. 22.1). Pyruvate is then oxidized completely to CO2 by pyruvate dehydrogenase and the tricarboxylic acid (TCA) cycle. Complete aerobic oxidation of glucose to CO2 can generate approximately 30 to 32 mol of ATP per mole of glucose. When cells have a limited supply of oxygen (e.g., the kidney medulla), or few or no mitochondria (e.g., the red cell), or greatly increased demands for ATP (e.g., skeletal muscle during high-intensity exercise), they rely on anaerobic glycolysis for generation of ATP. In anaerobic glycolysis, lactate dehydrogenase oxidizes the NADH generated from glycolysis by reducing pyruvate to lactate (Fig. 22.2). Because O2 is not required to reoxidize the NADH, the pathway is referred to as anaerobic. The energy yield from anaerobic glycolysis (2 mol ATP per mole of glucose) is much lower than the yield from aerobic oxidation. The lactate (lactic acid) is released into the blood. Under pathologic conditions that cause hypoxia, tissues may generate enough lactic acid to cause lactic acidemia. In each cell, glycolysis is regulated to ensure that ATP homeostasis is maintained, without using more glucose than necessary. In most cell types, hexokinase, the first enzyme of glycolysis, is inhibited by glucose 6-P (see Fig. 22.1). Thus, glucose is not taken up and phosphorylated by a cell unless glucose 6-P enters a metabolic pathway, such as glycolysis or glycogen synthesis. The control of glucose 6-P entry into glycolysis occurs at phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of the pathway.

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Glucose

PFK-1 is allosterically inhibited by ATP and allosterically activated by adenosine monophosphate (AMP). AMP increases in the cytosol as ATP is hydrolyzed by energy-requiring reactions. Glycolysis has functions in addition to ATP production. For example, in liver and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosynthesis. Glycolysis also provides precursors for the synthesis of compounds such as amino acids and five-carbon sugar phosphates.

THE WAITING ROOM

ATP NADH Pyruvate NADH Lactate Anaerobic glycolysis

Acetyl CoA TCA cycle

FIG. 22.2. Anaerobic glycolysis (shown in red). The conversion of glucose to lactate generates 2 ATP molecules from substratelevel phosphorylation. Because there is no net generation of NADH, there is no need for O2, and thus the pathway is anaerobic.

Lopa Fusor is a 68-year-old woman who is admitted to the hospital emergency department with very low blood pressure (80/40 mm Hg) caused by an acute hemorrhage from a previously diagnosed ulcer of the stomach. Lopa’s bleeding stomach ulcer has reduced her effective blood volume severely enough to compromise her ability to perfuse (deliver blood to) her tissues. She is also known to have chronic obstructive pulmonary disease (COPD) as a result of 42 years of smoking two packs of cigarettes per day. Her respiratory rate is rapid and labored, her skin is cold and clammy, and her lips are slightly blue (cyanotic). She appears anxious and moderately confused. As appropriate emergency measures are taken to stabilize her and elevate her blood pressure, blood is sent for immediate blood typing and crossmatching so that blood transfusions can be started. A battery of laboratory tests is ordered, including venous hemoglobin, hematocrit, lactate levels, and an arterial blood gas, which includes an arterial pH, partial pressures of oxygen (PO2) and carbon dioxide (PCO2), bicarbonate, and oxygen saturation. Results show that the hemorrhaging and COPD have resulted in hypoxemia, with decreased oxygen delivery to her tissues, and both a respiratory and metabolic acidosis. Otto Shape, a 26-year-old medical student, had gained weight during his first sedentary year in medical school. During his second year, he began watching his diet, jogging for an hour four times each week, and playing tennis twice a week. He has decided to compete in a 5-km race. To prepare for the race, he begins training with wind sprints, bouts of alternately running and walking. Ivan Applebod is a 56-year-old morbidly obese accountant (see Chapters 1 through 3). He decided to see his dentist because he felt excruciating pain in his teeth when he ate ice cream. He really likes sweets and keeps hard candy in his pocket. The dentist noted from Mr. Applebod’s history that he had numerous cavities as a child in his baby teeth. At this visit, the dentist found cavities in two of Mr. Applebod’s teeth.

I.

GLYCOLYSIS

Glycolysis is one of the principal pathways for generating ATP in cells and is present in all cell types. The central role of glycolysis in fuel metabolism is related to its ability to generate ATP with, and without, oxygen. The oxidation of glucose to pyruvate generates ATP from substrate-level phosphorylation (the transfer of phosphate from high-energy intermediates of the pathway to ADP) and NADH. Subsequently, the pyruvate may be oxidized to CO2 in the TCA cycle and ATP generated from electron transfer to oxygen in oxidative phosphorylation. However, if the pyruvate

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The hematocrit (the percentage of the volume of blood occupied by packed red blood cells) and hemoglobin content (grams of hemoglobin in 100 mL of blood) are measured to determine whether the oxygen-carrying capacity of the blood is adequate. Both values can be decreased by conditions that interfere with erythropoiesis (the synthesis of red blood cells in bone marrow), such as iron deficiency. They also can be decreased during chronic bleeding but not during immediate acute hemorrhage, if interstitial fluid replaces the lost blood volume and dilutes out the red blood cells. The PCO2 and PO2 are the partial pressures of carbon dioxide and oxygen, respectively, in the blood. The PO2 and oxygen saturation determine whether adequate oxygen is available for tissues. Measurement of the PCO2 and bicarbonate can distinguish between a metabolic and a respiratory acidosis (see Chapter 4).

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Glucose Phase I: Preparative phase

ATP ATP

Fructose 1,6-bis-P

2 Triose phosphates

Phase II: ATP–generating phase

2 NADH 2 ATP 2 ATP 2 Pyruvate

FIG. 22.3. Phases of the glycolytic pathway.

and NADH from glycolysis are converted to lactate (anaerobic glycolysis), ATP can be generated in the absence of oxygen, via substrate-level phosphorylation. Glucose is readily available from our diet, internal glycogen stores, and the blood. Carbohydrate provides 50% or more of the calories in most diets, and glucose is the major carbohydrate. Other dietary sugars, such as fructose and galactose, are oxidized by conversion to intermediates of glycolysis. Glucose is stored in cells as glycogen, which can provide an internal source of fuel for glycolysis in emergency situations (e.g., decreased supply of fuels and oxygen during ischemia, a low blood flow). Insulin and other hormones maintain blood glucose in a relatively constant range (glucose homeostasis), thereby ensuring that glucose is always available to cells that depend on glycolysis for generation of ATP. After a high-carbohydrate meal, glucose is the major fuel for almost all tissues. Exceptions include intestinal mucosal cells, which transport glucose from the gut into the blood, and cells in the proximal convoluted tubule of the kidney, which return glucose from the renal filtrate to the blood. During fasting, the brain continues to oxidize glucose because it has a limited capacity for the oxidation of fatty acids or other fuels. Cells also continue to use glucose for the portion of their ATP generation that must be met by anaerobic glycolysis, due to either a limited oxygen supply or a limited capacity for oxidative phosphorylation (e.g., the red blood cell). In addition to serving as an anaerobic and aerobic source of ATP, glycolysis is an anabolic pathway that provides biosynthetic precursors. For example, in liver and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosynthesis. Glycolysis also provides precursors for the synthesis of compounds such as amino acids and ribose 5-phosphate, the precursor of nucleotides. The integration of glycolysis with other anabolic pathways is discussed in Chapter 36.

A. The Reactions of Glycolysis

CH2OH O H

H

HO OH H

H OH OH

Glucose ATP ADP

Hexokinase glucokinase (liver)

2–

CH2OPO3 O H

1.

H

HO OH H

H OH OH

Glucose 6-P

Other Glycolysis pathways

Pentose phosphate pathway

Glycogen synthesis

FIG. 22.4. Glucose 6-phosphate metabolism.

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The glycolytic pathway, which cleaves 1 mol of glucose to 2 mol of the threecarbon compound pyruvate, consists of a preparative phase and an ATP-generating phase. In the initial preparative phase of glycolysis, glucose is phosphorylated twice by ATP and cleaved into two triose phosphates (Fig. 22.3). The ATP expenditure in the beginning of the preparative phase is sometimes called “priming the pump,” because this initial utilization of 2 mol of ATP per mole of glucose results in the production of 4 mol of ATP per mole of glucose in the ATP-generating phase. In the ATP-generating phase, glyceraldehyde 3-phosphate (glyceraldehyde 3-P) (a triose phosphate) is oxidized by NAD⫹ and phosphorylated using inorganic phosphate. The high-energy phosphate bond generated in this step is transferred to ADP to form ATP. The remaining phosphate is also rearranged to form another high-energy phosphate bond that is transferred to ADP. Because 2 mol of triose phosphate were formed, the yield from the ATP-generating phase is 4 mol of ATP and 2 mol of NADH. The result is a net yield of 2 mol of ATP, 2 mol of NADH, and 2 mol of pyruvate per mole of glucose. CONVERSION OF GLUCOSE TO GLUCOSE 6-PHOSPHATE

Glucose metabolism begins with transfer of a phosphate from ATP to glucose to form glucose 6-P (Fig. 22.4). Phosphorylation of glucose commits it to metabolism within the cell because glucose 6-P cannot be transported back across the plasma membrane. The phosphorylation reaction is irreversible under physiologic conditions because the reaction has a high-negative ⌬G0⬘. Phosphorylation does not, however, commit glucose to glycolysis. Glucose 6-P is a branch point in carbohydrate metabolism. It is a precursor for almost every pathway that uses glucose, including glycolysis, the pentose phosphate pathway, and glycogen synthesis. From the opposite point of view, it also can be generated from other pathways of carbohydrate metabolism, such as glycogenolysis

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(breakdown of glycogen), the pentose phosphate pathway, and gluconeogenesis (the synthesis of glucose from noncarbohydrate sources). Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a family of tissue-specific isoenzymes that differ in their kinetic properties. The isoenzyme found in liver and ␤-cells of the pancreas has a much higher Km than other hexokinases and is called glucokinase. In many cells, some of the hexokinase is bound to porins in the outer mitochondrial membrane (voltage-dependent anion channels; see Chapter 21), which gives these enzymes first access to newly synthesized ATP as it exits the mitochondria. 2.

CONVERSION OF GLUCOSE 6-PHOSPHATE TO THE TRIOSE PHOSPHATES

In the remainder of the preparative phase of glycolysis, glucose 6-P is isomerized to fructose 6-phosphate (fructose 6-P), again phosphorylated, and subsequently cleaved into two three-carbon fragments (Fig. 22.5). The isomerization, which positions a keto group next to carbon 3, is essential for the subsequent cleavage of the bond between carbons 3 and 4. The next step of glycolysis, phosphorylation of fructose 6-P to fructose 1,6-bisphosphate (fructose 1,6-bisP) by phosphofructokinase-1 (PFK-1), is generally considered the first committed step of the pathway. This phosphorylation requires ATP and is thermodynamically and kinetically irreversible. Therefore, PFK-1 irrevocably commits glucose to the glycolytic pathway. PFK-1 is a regulated enzyme in cells, and its regulation controls the entry of glucose into glycolysis. Like hexokinase, it exists as tissue-specific isoenzymes whose regulatory properties match variations in the role of glycolysis in different tissues. Fructose 1,6-bisP is cleaved into two phosphorylated three-carbon compounds (triose phosphates) by aldolase (see Fig. 22.5). Dihydroxyacetone phosphate (DHAP) is isomerized to glyceraldehyde 3-P, which is a triose phosphate. Aldolase is named for the mechanism of the forward reaction, which is an aldol cleavage, and the mechanism of the reverse reaction, which is an aldol condensation. The enzyme exists as tissue-specific isoenzymes, which all catalyze the cleavage of fructose 1,6-bisP but differ in their specificities for fructose 1-P. The enzyme uses a lysine residue at the active site to form a covalent bond with the substrate during the course of the reaction. Inability to form this covalent linkage inactivates the enzyme. Thus, at this point in glycolysis, for every mole of glucose that enters the pathway, 2 mol of glyceraldehyde 3-P are produced and continue through the pathway. 3.

OXIDATION AND SUBSTRATE-LEVEL PHOSPHORYLATION

In the next part of the glycolytic pathway, glyceraldehyde 3-P is oxidized and phosphorylated so that subsequent intermediates of glycolysis can donate phosphate to ADP to generate ATP. The first reaction in this sequence, catalyzed by glyceraldehyde 3-P dehydrogenase, is really the key to the pathway (see Fig. 22.5). This enzyme oxidizes the aldehyde group of glyceraldehyde 3-P to an enzyme-bound carboxyl group and transfers the electrons to NAD⫹ to form NADH. The oxidation step is dependent on a cysteine residue at the active site of the enzyme, which forms a high-energy thioester bond during the course of the reaction. The high-energy intermediate immediately accepts an inorganic phosphate to form the high-energy acyl phosphate bond in 1,3-bisphosphoglycerate, releasing the product from the cysteine residue on the enzyme. This high-energy phosphate bond is the start of substrate-level phosphorylation (the formation of a high-energy phosphate bond where none previously existed, without the use of oxygen). In the next reaction, the phosphate in this bond is transferred to ADP to form ATP by phosphoglycerate kinase. The energy of the acyl phosphate bond is high enough (⬃10 kcal/mol) so that transfer to ADP is an energetically favorable process. Another product of this reaction is 3-phosphoglycerate.

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O

O H

C

H

C

OH

HO

C

H

H

C

OH

H

C

OH

ATP

ADP

hexokinase (glucokinase in liver)

H

C

H

C

OH

HO

C

H

CH2OH

H

C

OH

H

C

OH

C

O

HO

C

H

H

C

OH

H

C

OH

phosphoglucose isomerase

2–

2– CH2OPO3

CH2OH

CH2OPO3

Glucose 6-phosphate

D-Glucose

Portion isomerized from aldehyde to keto sugar

Fructose 6-phosphate ATP phosphofructokinase–1 2–

CH2OPO3

ADP

C

O

2–

CH2OPO3 Aldol cleavage

C

O

HO

C

H

H

C

OH

H

C

OH

CH2OH Dihydroxyacetone phosphate

aldolase

triose phosphate isomerase

O 2–

CH2OPO3

H

C

H

C

OH 2–

Fructose 1,6-bisphosphate

CH2OPO3

Glyceraldehyde 3-phosphate Pi

glyceraldehyde 3-phosphate dehydrogenase

NAD+ NADH + H+ High energy acyl-phosphate H

O 2–

C ~ OPO3 C

OH 2–

CH2OPO3

1,3-Bisphosphoglycerate ADP High energy enolic phosphate

ATP

O

O C

O

C

O

–

CH3 Pyruvate

ATP ADP

C O

O –

H2O 2–

C ~ OPO3 pyruvate kinase

CH2 Phosphoenolpyruvate

phosphoglycerate kinase

H enolase

O –

C

O

C

OPO3

2–

CH2OH 2-Phosphoglycerate

H phosphoglycero– mutase

–

C

O

C

OH 2–

CH2OPO3

3-Phosphoglycerate

FIG. 22.5. Reactions of glycolysis. High-energy phosphates are indicated by the red squiggles.

To transfer the remaining low-energy phosphoester on 3-phosphoglycerate to ADP, it must be converted into a high-energy bond. This conversion is accomplished by moving the phosphate to the second carbon (forming 2-phosphoglycerate) and then removing water to form phosphoenolpyruvate (PEP). The enolphosphate bond is a high-energy bond (its hydrolysis releases approximately 14 kcal/mol of energy),

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so the transfer of phosphate to ADP by pyruvate kinase is energetically favorable (see Fig. 22.5). This final reaction converts PEP to pyruvate. 4.

SUMMARY OF THE GLYCOLYTIC PATHWAY

The overall net reaction in the glycolytic pathway is Glucose ⫹ 2 NAD⫹ ⫹ 2 Pi ⫹ 2 ADP → 2 pyruvate ⫹ 2 NADH ⫹ 4 H⫹ ⫹ 2 ATP ⫹ 2 H2O

The pathway occurs with an overall negative ⌬G0⬘ of approximately ⫺22 kcal/mol. Therefore, it cannot be reversed without the expenditure of energy.

B. Oxidative Fates of Pyruvate and Nicotinamide Adenine Dinucleotide The NADH produced from glycolysis must be continuously reoxidized back to NAD⫹ to provide an electron acceptor for the glyceraldehyde 3-P dehydrogenase reaction and prevent product inhibition. Without oxidation of this NADH, glycolysis cannot continue. There are two alternate routes for oxidation of cytosolic NADH (Fig. 22.6). One route is aerobic, involving shuttles that transfer reducing equivalents across the mitochondrial membrane and ultimately to the electron-transport chain and oxygen (see Fig. 22.6A). The other route is anaerobic (without the use of oxygen). In anaerobic glycolysis, NADH is reoxidized in the cytosol by lactate dehydrogenase (LDH), which reduces pyruvate to lactate (see Fig. 22.6B). The fate of pyruvate depends on the route used for NADH oxidation. If NADH is reoxidized in a shuttle system, pyruvate can be used for other pathways, one of which is oxidation to acetyl coenzyme A (acetyl-CoA) and entry into the TCA cycle for complete oxidation. Alternatively, in anaerobic glycolysis, pyruvate is reduced to lactate and diverted away from other potential pathways. Thus, the use of the shuttle systems allows for more ATP to be generated than by anaerobic glycolysis, by both oxidizing the cytoplasmically derived NADH in the electron-transport chain and by allowing pyruvate to be oxidized completely to CO2. The reason that shuttles are required for the oxidation of cytosolic NADH by the electron-transport chain is that the inner mitochondrial membrane is impermeable to NADH, and no transport protein exists that can translocate NADH across this membrane directly. Consequently, NADH is reoxidized to NAD⫹ in the cytosol by

A. Aerobic glycolysis

The confusion experienced by Lopa Fusor in the emergency department is caused by an inadequate delivery of oxygen to the brain. Neurons have very high ATP requirements, and most of this ATP is provided by aerobic oxidation of glucose to pyruvate in glycolysis and by pyruvate oxidation to CO2 in the TCA cycle. The brain has little or no capacity to oxidize fatty acids so its glucose consumption is high (approximately 125 to 150 g/day in the adult). Its oxygen demands are also high. If cerebral oxygen supply were completely interrupted, the brain would last only 10 seconds. The only reason that consciousness lasts longer during anoxia or asphyxia is that there is still some oxygen in the lungs and in circulating blood. A decrease of blood flow to approximately one-half of the normal rate results in a loss of consciousness.

B. Anaerobic glycolysis

Glucose 2 ADP + 2 Pi

Glucose 2 NAD+ XH2

2 ATP

2 NADH + 2H+ 2 Pyruvate

X

2 ADP + 2 Pi

Glycerol 3-P and Malate–aspartate shuttles

2 NAD+

2 ATP

Electron-transport chain

2 NADH + 2H+ 2 Pyruvate 2 Lactate Lactate dehydrogenase

Acetyl CoA NADH TCA cycle

CO2

O2

H2O ADP + Pi

FAD(2H)

H+

ATP Mitochondrion

FIG. 22.6. Alternate fates of pyruvate. A. The pyruvate produced by glycolysis enters mitochondria and is oxidized to CO2 and H2O. The reducing equivalents in NADH enter mitochondria via a shuttle system. B. Pyruvate is reduced to lactate in the cytosol, thereby using the reducing equivalents in NADH.

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Glucose

a reaction that transfers the electrons to DHAP in the glycerol 3-phosphate (glycerol 3-P) shuttle and oxaloacetate in the malate–aspartate shuttle. The NAD⫹ that is formed in the cytosol returns to glycolysis, whereas glycerol 3-P or malate carries the reducing equivalents that are ultimately transferred across the inner mitochondrial membrane. Thus, these shuttles transfer electrons and not NADH per se.

Pyruvate

NAD+

NADH +

H+

Cytosolic glycerol 3-P dehydrogenase

Glycerol 3-P

Dihydroxyacetone-P

1.

Mitochondrial glycerol 3-P dehydrogenase

Inner mitochondrial membrane FAD

GLYCEROL 3-PHOSPHATE SHUTTLE

The glycerol 3-P shuttle is the major shuttle in most tissues. In this shuttle, cytosolic NAD⫹ is regenerated by cytoplasmic glycerol 3-P dehydrogenase, which transfers electrons from NADH to DHAP to form glycerol 3-P (Fig. 22.7). Glycerol 3-P then diffuses through the outer mitochondrial membrane to the inner mitochondrial membrane, where the electrons are donated to a membrane-bound flavin adenine dinucleotide (FAD)–containing glycerophosphate dehydrogenase. This enzyme, like succinate dehydrogenase, ultimately donates electrons to coenzyme Q (CoQ), resulting in an energy yield of approximately 1.5 ATP from oxidative phosphorylation. DHAP returns to the cytosol to continue the shuttle. The sum of the reactions in this shuttle system is simply

FAD(2H)

Electron-transport chain

FIG. 22.7. Glycerol 3-P shuttle. Because NAD⫹ and NADH cannot cross the mitochondrial membrane, shuttles transfer the reducing equivalents into mitochondria. DHAP is reduced to glycerol 3-P by cytosolic glycerol 3-P dehydrogenase, using cytosolic NADH produced in glycolysis. Glycerol 3-P then reacts in the inner mitochondrial membrane with mitochondrial glycerol 3-P dehydrogenase, which transfers the electrons to FAD and regenerates DHAP, which returns to the cytosol. The electron-transport chain transfers the electrons to O2, which generates approximately 1.5 ATP molecules for each FAD(2H) that is oxidized.

NADHcytosol ⫹ H⫹ ⫹ FADmitochondria → NAD⫹cytosol ⫹ FAD(2H)mitochondria

2.

MALATE–ASPARTATE SHUTTLE

Many tissues contain both the glycerol 3-P shuttle and the malate–aspartate shuttle. In the malate–aspartate shuttle (Fig. 22.8), cytosolic NAD⫹ is regenerated by cytosolic malate dehydrogenase, which transfers electrons from NADH to cytosolic oxaloacetate to form malate. Malate is transported across the inner mitochondrial membrane by a specific translocase, which exchanges malate for ␣-ketoglutarate. In the matrix, malate is oxidized back to oxaloacetate by mitochondrial malate dehydrogenase, and NADH is generated. This NADH can donate electrons to the electron-transport chain with generation of approximately 2.5 mol of ATP per mole of NADH. The newly formed oxaloacetate cannot pass back through the inner mitochondrial membrane under physiologic conditions, so aspartate is used to return the oxaloacetate carbon skeleton to the cytosol. In the matrix, transamination reactions transfer an amino group to oxaloacetate to form aspartate, which is transported out to the cytosol (using an aspartate–glutamate exchange translocase) and converted back to oxaloacetate through another transamination reaction. The sum of all the reactions of this shuttle system is simply NADHcytosol ⫹ NAD⫹matrix → NAD⫹cytosol ⫹ NADHmatrix

Cytosol

Mitochondrion

Glucose 2 NAD+

Malate

2 NADH

Oxaloacetate

2 Pyruvate

Malate

NAD+

Oxaloacetate

NADH

␣-KG

␣-KG

Glutamate

Glutamate

TA

TA

Aspartate

Electrontransport chain

Aspartate

Inner mitochondrial membrane

FIG. 22.8. Malate–aspartate shuttle. NADH produced by glycolysis reduces oxaloacetate (OAA) to malate, which crosses the mitochondrial membrane and is reoxidized to OAA. The mitochondrial NADH donates electrons to the electron-transport chain, with 2.5 ATP molecules generated for each NADH. To complete the shuttle, OAA must return to the cytosol, although it cannot be transported directly on a translocase. Instead, it is transaminated to aspartate, which is then transported out to the cytosol, where it is transaminated back to OAA. The translocators exchange compounds in such a way that the shuttle is completely balanced. TA, transamination reaction; ␣-KG, ␣-ketoglutarate.

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C. Anaerobic Glycolysis When the oxidative capacity of a cell is limited (e.g., in the red blood cell, which has no mitochondria), the pyruvate and NADH produced from glycolysis cannot be oxidized aerobically. The NADH is therefore oxidized to NAD⫹ in the cytosol by reduction of pyruvate to lactate. This reaction is catalyzed by LDH (Fig. 22.9). The net reaction for anaerobic glycolysis is Glucose ⫹ 2 ADP ⫹ 2 Pi → 2 lactate ⫹ 2 ATP ⫹ 2 H2O ⫹ 2 H⫹

1.

ENERGY YIELD OF AEROBIC VERSUS ANAEROBIC GLYCOLYSIS

In both aerobic and anaerobic glycolysis, each mole of glucose generates 2 mol of ATP, 2 mol of NADH, and 2 mol of pyruvate. The energy yield from anaerobic glycolysis (1 mol of glucose to 2 mol of lactate) is only 2 mol of ATP per mole of glucose, as the NADH is recycled to NAD⫹ by reducing pyruvate to lactate. Neither the NADH nor the pyruvate produced is thus used for further energy generation. However, when oxygen is available and cytosolic NADH can be oxidized via a shuttle system, pyruvate can also enter the mitochondria and be completely oxidized to CO2 via pyruvate dehydrogenase (PDH) and the TCA cycle. The oxidation of pyruvate via this route generates roughly 12.5 mol of ATP per mole of pyruvate. If the cytosolic NADH is oxidized by the glycerol 3-P shuttle, approximately 1.5 mol of ATP are produced per NADH. If, instead, the NADH is oxidized by the malate–aspartate shuttle, approximately 2.5 mol are produced. Thus, the two NADH molecules produced during glycolysis can lead to 3 to 5 mol of ATP being produced, depending on which shuttle system is used to transfer the reducing equivalents. Because each pyruvate produced can give rise to 12.5 mol of ATP, altogether, 30 to 32 mol of ATP can be produced from 1 mol of glucose oxidized to carbon dioxide. To produce the same amount of ATP per unit time from anaerobic glycolysis as from the complete aerobic oxidation of glucose to CO2, anaerobic glycolysis must occur approximately 15 times faster and use approximately 15 times more glucose. Cells achieve this high rate of glycolysis by expressing high levels of glycolytic enzymes. In certain skeletal muscles and in most cells during hypoxic crises, high rates of glycolysis are associated with rapid degradation of internal glycogen stores to supply the required glucose 6-P. 2.

Glycolysis NADH + H+

O C

O

C

O

NAD+

–

CH3 Pyruvate

O C

Lactate dehydrogenase

H C

O– OH

CH3 Lactate

FIG. 22.9. Lactate dehydrogenase reaction. Pyruvate, which may be produced by glycolysis, is reduced to lactate. The reaction, which occurs in the cytosol, requires NADH and is catalyzed by lactate dehydrogenase. This reaction is readily reversible.

What are the energy-generating steps as pyruvate is completely oxidized to carbon dioxide to generate 12.5 molecules of ATP per pyruvate?

ACID PRODUCTION IN ANAEROBIC GLYCOLYSIS

Anaerobic glycolysis results in acid production in the form of H⫹. Glycolysis forms pyruvic acid, which is reduced to lactic acid. At an intracellular pH of 7.35, lactic acid dissociates to form the carboxylate anion, lactate, and H⫹ (the pKa for lactic acid is 3.85). Lactate and the H⫹ are both transported out of the cell into interstitial fluid by a transporter on the plasma membrane and eventually diffuse into the blood. If the amount of lactate generated exceeds the buffering capacity of the blood, the pH drops below the normal range, resulting in lactic acidosis (see Chapter 4). 3.

403

TISSUES DEPENDENT ON ANAEROBIC GLYCOLYSIS

Many tissues, including red and white blood cells, the kidney medulla, the tissues of the eye, and skeletal muscles, rely on anaerobic glycolysis for at least a portion of their ATP requirements (Table 22.1). Tissues (or cells) that are heavily dependent on anaerobic glycolysis usually have a low ATP demand, high levels of glycolytic enzymes, and few capillaries, such that oxygen must diffuse over a greater distance to reach target cells. The lack of mitochondria, or the increased rate of glycolysis, is often related to some aspect of cell function. For example, the mature red blood cell has no mitochondria because oxidative metabolism might interfere with its function in transporting oxygen bound to hemoglobin. Some of the lactic acid generated by anaerobic glycolysis in skin is secreted in sweat, where it acts as an antibacterial

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The dental caries in Ivan Applebod’s mouth were caused principally by the low pH generated from lactic acid production by oral bacteria. Below a pH of 5.5, decalcification of tooth enamel and dentine occurs. Lactobacilli and S. mutans are major contributors to this process because almost all of their energy is derived from the conversion of glucose or fructose to lactic acid, and they are able to grow well at the low pH generated by this process. Mr. Applebod’s dentist explained that bacteria in his dental plaque could convert all the sugar in his candy into acid in less than 20 minutes. The acid is buffered by bicarbonate and other buffers in saliva, but saliva production decreases in the evening. Thus, the acid could dissolve the hydroxyapatite in his tooth enamel during the night.

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In the complete oxidation of pyruvate to carbon dioxide, four steps generate NADH (pyruvate dehydrogenase, isocitrate dehydrogenase, ␣-ketoglutarate dehydrogenase, and malate dehydrogenase). One step generates FAD(2H) (succinate dehydrogenase) and one substrate-level phosphorylation (succinate thiokinase). Thus, because each NADH generates 2.5 ATP molecules, the overall contribution by NADH is 10 ATP molecules. The FAD(2H) generates an additional 1.5 ATP molecules, and the substrate-level phosphorylation provides one more. Therefore, 10 ⫹ 1.5 ⫹ 1 ⫽ 12.5 molecules of ATP. In response to the hypoxemia caused by Lopa Fusor’s COPD, she has increased hypoxia-inducible factor-1 (HIF-1) in her tissues. HIF-1 is a gene transcription factor found in tissues throughout the body (including the brain, heart, kidneys, lungs, liver, pancreas, skeletal muscles, and white blood cells) that plays a homeostatic role in coordinating tissue responses to hypoxia. Each tissue will respond with a subset of the following changes. HIF-1 increases transcription of the genes for many of the glycolytic enzymes, including PFK-1, enolase, phosphoglycerate kinase, and lactate dehydrogenase. HIF-1 also increases synthesis of several proteins that enhance oxygen delivery to tissues, including erythropoietin, which increases the generation of red blood cells in bone marrow; vascular endothelial growth factor, which regulates angiogenesis (formation of blood vessels); and inducible nitric oxide synthase, which synthesizes nitric oxide, a vasodilator. As a consequence, Mrs. Fusor was able to maintain hematocrit and hemoglobin levels that were on the high side of the normal range, and her tissues had an increased capacity for anaerobic glycolysis.

Table 22.1 Major Tissue Sites of Lactate Production in a Resting Man (An Average 70-kg Man Consumes about 300 g of Carbohydrate per Day) Daily Lactate Production (g/d) Total lactate production Red blood cells Skin Brain Skeletal muscles Renal medulla Intestinal mucosa Other tissues

115 29 20 17 16 15 8 10

agent. Many large tumors use anaerobic glycolysis for ATP production and lack capillaries in their core. In tissues with some mitochondria, both aerobic and anaerobic glycolysis occur simultaneously. The relative proportion of the two pathways depends on the mitochondrial oxidative capacity of the tissue and its oxygen supply and may vary among cell types within the same tissue because of cell distance from the capillaries. When a cell’s energy demand exceeds the capacity of the rate of the electron-transport chain and oxidative phosphorylation to produce ATP, glycolysis is activated, and the increased NADH/NAD⫹ ratio directs excess pyruvate into lactate. Because under these conditions—the PDH, the TCA cycle, and the electrontransport chain are operating as fast as they can—anaerobic glycolysis is meeting the need for additional ATP. 4.

FATE OF LACTATE

Lactate released from cells that undergo anaerobic glycolysis is taken up by other tissues (primarily the liver, heart, and skeletal muscle) and oxidized back to pyruvate. In the liver, the pyruvate is used to synthesize glucose (gluconeogenesis), which is returned to the blood. The cycling of lactate and glucose between peripheral tissues and liver is called the Cori cycle (Fig. 22.10). In many other tissues, lactate is oxidized to pyruvate, which is then oxidized to CO2 in the TCA cycle. Although the equilibrium of the LDH reaction favors lactate production, flux occurs in the opposite direction if NADH is being rapidly oxidized in the electron-transport chain (or is being used for gluconeogenesis): Lactate ⫹ NAD⫹ → pyruvate ⫹ NADH ⫹ H⫹

The heart, with its huge mitochondrial content and oxidative capacity, is able to use lactate released from other tissues as a fuel. During exercise such as bicycle riding, lactate released into the blood from skeletal muscles in the leg might be used by Cori Cycle

RBC Liver

Glucose

Glucose

Glucose

6ATP Gluconeogenesis

Blood

Glycolysis 2 ATP

2 Lactate

2 Lactate

2 Lactate

FIG. 22.10. The Cori cycle. Glucose, produced in the liver by gluconeogenesis, is converted to lactate by glycolysis in muscles, red blood cells, and many other cells. Lactate returns to the liver and is reconverted to glucose by gluconeogenesis.

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resting skeletal muscles in the arm. In the brain, glial cells and astrocytes produce lactate, which is used by neurons or released into the blood. LDH is a tetramer composed of A-subunits (also called M-subunits, for skeletal muscle form) and B-subunits (also called H-subunits, for heart). Different tissues produce different amounts of the two subunits, which then combine randomly to form five different tetramers (M4, M3H1, M2H2, M1H3, and H4). These isoenzymes differ only slightly in their properties, but the kinetic properties of the M4 form facilitate conversion of pyruvate to lactate in skeletal muscle, whereas the kinetic properties of H4 form facilitate conversion of lactate to pyruvate in the heart for energy generation.

The tissues of the eye are also partially dependent on anaerobic glycolysis. Vitreous body

Ciliary body Iris

Retina

Lens Pupil Cornea

II. OTHER FUNCTIONS OF GLYCOLYSIS Glycolysis, in addition to providing ATP, generates precursors for biosynthetic pathways (Fig. 22.11). Intermediates of the pathway can be converted to ribose 5-phosphate, the sugar incorporated into nucleotides such as ATP. Other sugars, such as UDP-glucose, mannose, and sialic acid, are also formed from intermediates of glycolysis. Serine is synthesized from 3-phosphoglycerate, and alanine from pyruvate. The backbone of triacylglycerols, glycerol 3-P, is derived from DHAP in the glycolytic pathway. The liver is the major site of biosynthetic reactions in the body. In addition to those pathways mentioned previously, the liver synthesizes fatty acids from the pyruvate generated by glycolysis. It also synthesizes glucose from lactate, glycerol 3-P, and amino acids in the gluconeogenic pathway, which is basically a reversal of glycolysis. Consequently, in liver, many of the glycolytic enzymes exist as isoenzymes with properties suited for these functions. The bisphosphoglycerate shunt is a “side reaction” of the glycolytic pathway in which 1,3-bisphosphoglycerate is converted to 2,3-bisphosphoglycerate (2,3BPG). Red blood cells form 2,3-BPG to serve as an allosteric inhibitor of oxygen Glucose

Five-carbon sugars

Glucose 6-P

405

Fovea centralis

Aqueous humor Ciliary muscle

Choroid Sclera

The eye contains cells that transmit or focus light, and these cells, therefore, cannot be filled with opaque structures such as mitochondria or densely packed capillary beds. The corneal epithelium generates most of its ATP aerobically from its few mitochondria but still metabolizes some glucose anaerobically. Oxygen is supplied by diffusion from the air. The lens of the eye is composed of fibers that must remain birefringent to transmit and focus light, so mitochondria are nearly absent. The small amount of ATP required (principally for ion balance) can readily be generated from anaerobic glycolysis even though the energy yield is low. The lens is able to pick up glucose and release lactate into the vitreous body and aqueous humor. It does not need oxygen and has no use for capillaries.

Glycerol-P 1,3-bis-Phosphoglycerate

Triglyceride Fatty acids

2,3--bis-Phosphoglycerate Serine

3-Phosphoglycerate

Alanine

Pyruvate

Acetyl CoA TCA cycle

Glutamate and other amino acids

FIG. 22.11. Biosynthetic functions of glycolysis. Compounds formed from intermediates of glycolysis are shown in the boxes. These pathways are discussed in later chapters. Dotted lines indicate that more than one step is required for the conversion shown in the figure.

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binding to heme (see Chapter 44). 2,3-BPG reenters the glycolytic pathway via dephosphorylation to 3-phosphoglycerate. It also functions as a coenzyme in the conversion of 3-phosphoglycerate to 2-phosphoglycerate by the glycolytic enzyme phosphoglyceromutase. Because 2,3-BPG is not depleted by its role in this catalytic process, most cells need only very small amounts.

III. REGULATION OF GLYCOLYSIS BY THE NEED FOR ADENOSINE TRIPHOSPHATE One of the major functions of glycolysis is the generation of ATP, so the pathway is regulated to maintain ATP homeostasis in all cells. PFK-1 and PDH, which links glycolysis and the TCA cycle, are both major regulatory sites that respond to feedback indicators of the rate of ATP utilization (Fig. 22.12). The supply of glucose 6-P for glycolysis is tissue-dependent and can be regulated at the steps of glucose Glucose – hexokinase

Glucose-6-P

Fructose-6-P ATP

+ –

phosphofructokinase-1 AMP, F-2,6-bisP ATP, citrate ADP Fructose-1,6-bis P Glyceraldehyde-3-P Pi NAD+ NADH + H+ 1,3-Bisphosphoglycerate ATP

PEP pyruvate kinase + –

NAD+

F-1,6-bisP ATP

ATP

NADH

Lactate

Pyruvate

Pyruvate NAD+ NADH

pyruvate dehydrogenase

Acetyl CoA

+ –

ADP, Ca2+ NADH, Acetyl CoA

Mitochondrion

FIG. 22.12. Major sites of regulation in the glycolytic pathway. Hexokinase and phosphofructokinase-1 are the major regulatory enzymes in skeletal muscle. The activity of pyruvate dehydrogenase in the mitochondrion determines whether pyruvate is converted to lactate or to acetyl coenzyme A (acetyl-CoA). The regulation shown for pyruvate kinase occurs only for the liver (L) isoenzyme.

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A. Relationships among ATP, ADP, and AMP Concentrations The AMP levels within the cytosol provide a better indicator of the rate of ATP utilization than the ATP concentration itself (Fig. 22.13). The concentration of AMP in the cytosol is determined by the equilibrium position of the adenylate kinase reaction, which catalyzes the following reaction: 2 ADP ↔ AMP ⫹ ATP

The equilibrium is such that hydrolysis of ATP to ADP in energy-requiring reactions increases both the ADP and AMP contents of the cytosol. However, ATP is present in much higher quantities than AMP or ADP, so a small decrease of ATP concentration in the cytosol causes a much larger percentage increase in the small AMP pool. In skeletal muscles, for instance, ATP levels are approximately 5 mM and decrease by no more than 20% during strenuous exercise (see Fig. 22.13). At the same time, ADP levels may increase by 50%, and AMP levels, which are in the micromolar range, may increase by 300%. AMP activates several metabolic pathways, including glycolysis, glycogenolysis, and fatty acid oxidation (particularly in muscle tissues), to ensure that ATP homeostasis is maintained.

5

ATP Rest Exercise

4 Concentration (mM)

transport into cells, glycogenolysis (the degradation of glycogen to form glucose), or the rate of glucose phosphorylation by hexokinase isoenzymes. Other regulatory mechanisms integrate the ATP-generating role of glycolysis with its anabolic roles. All of the regulatory enzymes of glycolysis exist as tissue-specific isoenzymes, which alter the regulation of the pathway to match variations in conditions and needs in different tissues. For example, in the liver, an isoenzyme of pyruvate kinase introduces an additional regulatory site in glycolysis that contributes to the inhibition of glycolysis when the reverse pathway, gluconeogenesis, is activated.

407

3

2 ADP 1

AMP

0

FIG. 22.13. Changes in ATP, ADP, and AMP concentrations in skeletal muscles during exercise. The concentration of ATP decreases by only approximately 20% during exercise, and the concentration of ADP rises. The concentration of AMP, produced by the adenylate kinase reaction, increases manyfold and serves as a sensitive indicator of decreasing ATP levels.

B. Regulation of Hexokinases Hexokinases exist as tissue-specific isoenzymes whose regulatory properties reflect the role of glycolysis in different tissues. In most tissues, hexokinase is a low-Km enzyme with a high affinity for glucose (see Chapter 9). It is inhibited by physiologic concentrations of its product, glucose 6-P (see Fig. 22.12). If glucose 6-P does not enter glycolysis or another pathway, it accumulates and decreases the activity of hexokinase. In the liver, the isoenzyme glucokinase is a high-Km enzyme that is not readily inhibited by glucose 6-P. Thus, glycolysis can continue in the liver even when energy levels are high, so that anabolic pathways, such as the synthesis of the major energy storage compounds, glycogen, and fatty acids, can occur.

C. Regulation of Phosphofructokinase-1 PFK-1 is the rate-limiting enzyme of glycolysis and controls the rate of glucose 6-P entry into glycolysis in most tissues. PFK-1 is an allosteric enzyme that has a total of six binding sites: two are for substrates (MgATP and fructose 6-P) and four are allosteric regulatory sites (see Fig. 22.12). The allosteric regulatory sites occupy a physically different domain on the enzyme than the catalytic site. When an allosteric effector binds, it changes the conformation at the active site and may activate or inhibit the enzyme (see also Chapter 9). The allosteric sites for PFK-1 include an inhibitory site for MgATP, an inhibitory site for citrate and other anions, an allosteric activation site for AMP, and an allosteric activation site for fructose 2,6-bisphosphate (fructose 2,6-bisP) and other bisphosphates. Several different tissue-specific isoforms of PFK-1 are affected in different ways by the concentration of these substrates and allosteric effectors but all contain these four allosteric sites. Three different types of PFK-1 isoenzyme subunits exist: M (muscle), L (liver), and C (other tissues). The three subunits show variable expression in different tissues, with some tissues having more than one type. For example, mature human

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A

muscle expresses only the M-subunit, the liver expresses principally the L-subunit, and erythrocytes express both the M- and the L-subunits. The C-subunit is present in highest levels in platelets, placenta, kidney, and fibroblasts but is relatively common to most tissues. Both the M- and L-subunits are sensitive to AMP and ATP regulation, but the C-subunits are much less so. Active PFK-1 is a tetramer, composed of four subunits. Within muscle, the M4 form predominates; but within tissues that express multiple isoenzymes of PFK-1, heterotetramers can form that have full activity.

1.0 + AMP or fructose-2,6-bis-P v V max

1. 1

2

3

4

5

Fructose 6-P (mM)

B 1.0 + AMP or fructose 2,6-bis-P v V max

2.

2

4

6

8

10

ATP (mM)

FIG. 22.14. Regulation of PFK-1 by AMP, ATP, and fructose 2,6-bisP. A. AMP and fructose 2,6-bisP activate PFK-1. B. ATP, as a substrate, increases the rate of the reaction at low concentrations but allosterically inhibits the enzyme at high concentrations.

ALLOSTERIC REGULATION OF PFK-1 BY AMP AND ATP

ATP binds to two different sites on the enzyme: the substrate-binding site and an allosteric inhibitory site. Under physiologic conditions in the cell, the ATP concentration is usually high enough to saturate the substrate-binding site and inhibit the enzyme by binding to the ATP allosteric site. This effect of ATP is opposed by AMP, which binds to a separate allosteric activator site (Fig. 22.14). For most of the PFK-1 isoenzymes, the binding of AMP increases the affinity of the enzyme for fructose 6-P (e.g., it shifts the kinetic curve to the left). Thus, increases in AMP concentration can greatly increase the rate of the enzyme (see Fig. 22.14), particularly when fructose 6-P concentrations are low. REGULATION OF PFK-1 BY FRUCTOSE 2,6-BISPHOSPHATE

Fructose 2,6-bisP is also an allosteric activator of PFK-1 that opposes ATP inhibition. Its effect on the rate of activity of PFK-1 is qualitatively similar to that of AMP, but it has a separate binding site. Fructose 2,6-bisP is not an intermediate of glycolysis but is synthesized by an enzyme that phosphorylates fructose 6-P at the 2-position. The enzyme is therefore named phosphofructokinase-2 (PFK-2); it is a bifunctional enzyme with two separate domains: a kinase domain and a phosphatase domain. At the kinase domain, fructose 6-P is phosphorylated to fructose 2,6-bisP; and at the phosphatase domain, fructose 2,6-bisP is hydrolyzed back to fructose 6-P. PFK-2 is regulated through changes in the ratio of activity of the two domains. For example, in skeletal muscles, high concentrations of fructose 6-P activate the kinase and inhibit the phosphatase, thereby increasing the concentration of fructose 2,6-bisP and activating glycolysis. PFK-2 can also be regulated through phosphorylation by serine–threonine protein kinases. The liver isoenzyme contains a phosphorylation site near the amino terminal that decreases the activity of the kinase and increases the phosphatase activity. This site is phosphorylated by the cyclic adenosine monophosphate (cAMP)–dependent protein kinase (protein kinase A) and is responsible for decreased levels of liver fructose 2,6-bisP during fasting conditions (as modulated by circulating glucagon levels, which is discussed in detail in Chapters 26 and 31). The cardiac isoenzyme contains a phosphorylation site near the carboxy terminal that can be phosphorylated in response to adrenergic activators of contraction (such as norepinephrine) and by increased AMP levels. Phosphorylation at this site

Otto Shape has started high-intensity exercise that will increase the production of lactate in his exercising skeletal muscles. In skeletal muscles, the amount of aerobic versus anaerobic glycolysis that occurs varies with the intensity of the exercise, with the duration of the exercise, with the type of skeletal muscle fiber involved, and with the level of training. Human skeletal muscles are usually combinations of type I fibers (called fast glycolytic fibers or white muscle fibers) and type IIb fibers (called slow oxidative fibers or red muscle fibers). The designation “fast” or “slow” refers to the fibers’ rate of shortening, which is determined by the level of the isoenzyme of myosin ATPase present. Compared with glycolytic fibers, oxidative fibers have a higher content of mitochondria and myoglobin, which gives them a red color. The gastrocnemius, a muscle in the leg used for running, has a high content of type IIb fibers. However, these fibers will still produce lactate during sprints when the ATP demand exceeds their oxidative capacity.

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increases the kinase activity and increases fructose 2,6-bisP levels, thereby contributing to the activation of glycolysis. 3.

ALLOSTERIC INHIBITION OF PFK-1 AT THE CITRATE SITE

The function of the citrate-anion allosteric site is to integrate glycolysis with other pathways. For example, the inhibition of PFK-1 by citrate may play a role in decreasing glycolytic flux in the heart during the oxidation of fatty acids.

D. Regulation of Pyruvate Kinase Pyruvate kinase exists as tissue-specific isoenzymes, designated as R (red blood cells), L (liver), and M1/M2 (muscle and other tissues). The M1 form present in brain, heart, and muscle contains no allosteric sites, and pyruvate kinase does not contribute to the regulation of glycolysis in these tissues (these tissues also do not undergo significant gluconeogenesis). However, the liver isoenzyme can be inhibited through phosphorylation by the cAMP-dependent protein kinase and by several allosteric effectors that contribute to the inhibition of glycolysis during fasting conditions. These allosteric effectors include activation by fructose 1,6-bisP, which ties the rate of pyruvate kinase to that of PFK-1, and inhibition by ATP, which signifies high energy levels.

409

Under ischemic conditions, AMP levels within the heart increase rapidly because of the lack of ATP production via oxidative phosphorylation. The increase in AMP levels activates the AMPactivated protein kinase, which phosphorylates the heart isoenzyme of PFK-2 to activate its kinase activity. This results in increased levels of fructose 2,6-bisP, which activates PFK-1 along with AMP so that the rate of glycolysis can increase to compensate for the lack of ATP production via aerobic means.

E. Pyruvate Dehydrogenase Regulation and Glycolysis PDH is also regulated principally by the rate of ATP utilization (see Chapter 20) through rapid phosphorylation to an inactive form. Thus, in a normally respiring cell, with an adequate supply of O2, glycolysis and the TCA cycle are activated together, and glucose can be completely oxidized to CO2. However, when tissues do not have an adequate supply of O2 to meet their ATP demands, the increased NADH/NAD⫹ ratio inhibits PDH, but AMP activates glycolysis. A proportion of the pyruvate is then reduced to lactate to allow glycolysis to continue.

IV. LACTIC ACIDEMIA Lactate production is a normal part of metabolism. In the absence of disease, elevated lactate levels in the blood are associated with anaerobic glycolysis during exercise. In lactic acidosis, lactic acid accumulates in blood to levels that significantly affect the pH (lactate levels ⬎5 mM and a decrease of blood pH below 7.2). Lactic acidosis generally results from a greatly increased NADH/NAD⫹ ratio in tissues (Fig. 22.15). The increased NADH concentration prevents pyruvate oxidation in the TCA cycle and directs pyruvate to lactate. To compensate for the decreased ATP production from oxidative metabolism, PFK-1, and therefore the entire glycolytic pathway, is activated. For example, consumption of large amounts of alcohol, which is rapidly oxidized in the liver and increases NADH levels, can result in lactic acidosis. Hypoxia in any tissue increases lactate production as cells attempt to compensate for a lack of O2 for oxidative phosphorylation. Several other problems that interfere with either the electron-transport chain or pyruvate oxidation in the TCA cycle result in lactic acidemia (see Fig. 22.15). For

During Cora Nari’s myocardial infarction (see Chapter 20), the ischemic area in her heart had a limited supply of oxygen and blood-borne fuels. The absence of oxygen for oxidative phosphorylation would decrease the levels of ATP and increase those of AMP, an activator of PFK-1 and the AMP-dependent protein kinase, resulting in a compensatory increase of anaerobic glycolysis and lactate production. However, obstruction of a vessel leading to her heart would decrease lactate removal, resulting in a decrease of intracellular pH. Under these conditions, at very low pH levels, glycolysis is inhibited and unable to compensate for the lack of oxidative phosphorylation.

Several methods can be used to determine lactate levels in blood. Two of the most commonly used enzymatic methods are given. The first is the conversion of lactate to pyruvate (which also converts NAD⫹ to NADH) in the presence of lactate dehydrogenase. Because NADH has considerable light absorption at 340 nm (and NAD⫹ does not), one can follow the increase in absorbance at this wavelength as the reaction proceeds and determine the levels of lactate that were initially present in the sample. To ensure that all of the lactate is measured, hydrazine is added to the reaction; hydrazine reacts with pyruvate to remove the product of the LDH reaction, which forces the reaction to go to completion. The second enzymatic procedure that is commonly used employs lactate oxidase, which converts lactate, in the presence of oxygen, to pyruvate and hydrogen peroxide. In this case, a second enzymatic reaction measures the amount of hydrogen peroxide produced (which removes the product of the lactate oxidase reaction, ensuring completion of the reaction). This second reaction uses peroxidase and a chromagen, which is converted to a colored product as the hydrogen peroxide is removed. The amount of colored product produced allows lactate levels to be determined accurately. Both procedures have been automated for use in the clinical laboratory.

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Decreased oxidation of NADH and FAD(2H) in the ET chain results in pyruvate lactate and fatty acids triglyceride

Glucose NAD+

Fatty acids

NADH

Glycerol-P

Pyruvate NADH

LDH

Triglyceride Fatty acyl carnitine

NAD+ Pyruvate

Lactate

PDH

Fatty acyl CoA

NADH

NADH, FAD(2H)

Acetyl CoA ATP CO2

ADP OAA

TCA cycle

Deficiencies or inhibition of TCA cycle enzymes (nuclear encoded) inhibit acetyl CoA oxidation, leading to increased pyruvate and lactate formation

ADP F0F1–ATPase

NADH

ATP O2

FAD SDH

H2O

Cytochrome oxidase Complex IV

Cyt c

Cu, Fe

Cytochrome b-c, Complex III

CoQ

FAD FaCoA-DH

Fe–S FMN

Fe

Anoxia, ischemia, cyanide, CO poisoning and other interruptions of the ET chain prevent electron flow and ATP synthesis, so glycolysis operates anaerobically to produce ATP, and lactate is formed

NADH:CoQ oxidoreductase Complex I

Genetic defects in proteins encoded by mtDNA (some subunits of Complexes I, III, IV and F0F1–ATPase) decrease electron transport and ATP synthesis, so glycolysis operates anaerobically to produce ATP, and lactate is formed

FIG. 22.15. Pathways leading to lactic acidemia.

Lopa Fusor had a decreased arterial PO2 and elevated arterial PCO2 caused by underperfusion of her lungs. The elevated CO2 content resulted in an increase of H2CO3 and acidity of the blood (see Chapter 4). The decreased O2 delivery to tissues resulted in increased lactate production from anaerobic glycolysis and an elevation of serum lactate to 10 times normal levels. The reduction in her arterial pH to 7.18 (reference range, 7.35 to 7.45), therefore, resulted from both a mild respiratory acidosis (elevated PCO2) and a more profound metabolic acidosis (elevated serum lactate level).

Lieberman_Ch22.indd 410

example, oxidative phosphorylation diseases (inherited deficiencies in subunits of complexes in the electron-transport chain, such as myoclonic epilepsy with ragged red fibers [MERFF]) increase the NADH/NAD⫹ ratio and inhibit PDH (see Chapter 21). Pyruvate accumulates and is converted to lactate to allow glycolytic ATP production to proceed. Similarly, impaired PDH activity from an inherited deficiency of E1 (the decarboxylase subunit of the complex), or from severe thiamine deficiency, increases blood lactate levels (see Chapter 20). Pyruvate carboxylase deficiency also can result in lactic acidosis (see Chapter 20), also because of an accumulation of pyruvate. Lactic acidosis can also result from inhibition of lactate utilization in gluconeogenesis (e.g., hereditary fructose intolerance, which is caused by a defective aldolase gene). If other pathways that use glucose 6-P are blocked, glucose 6-P can be shunted into glycolysis and lactate production (e.g., glucose 6-P deficiency). CLINICAL COMMENTS Lopa Fusor. Lopa Fusor was admitted to the hospital with severe hypotension caused by an acute hemorrhage. Her plasma lactic acid level was elevated and her arterial pH was low. The underlying mechanism for Ms. Fusor’s derangement in acid–base balance is a severe reduction in the amount of oxygen delivered to her tissues for cellular respiration (hypoxemia). Several

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411

concurrent processes contributed to this lack of oxygen. The first was her severely reduced blood pressure caused by a brisk hemorrhage from a bleeding gastric ulcer. The blood loss led to hypoperfusion and therefore reduced delivery of oxygen to her tissues. The marked reduction in the number of red blood cells in her circulation caused by blood loss further compromised oxygen delivery. Her preexisting chronic obstructive pulmonary disease (COPD) added to her hypoxemia by decreasing her ventilation and therefore the transfer of oxygen to her blood (low PO2). In addition, her COPD led to retention of carbon dioxide (high PCO2), which caused a respiratory acidosis because the retained CO2 interacted with water to form carbonic acid (H2CO3), which dissociates to H⫹ and bicarbonate. Otto Shape. In skeletal muscles, lactate production occurs when the need for ATP exceeds the capacity of the mitochondria for oxidative phosphorylation. Thus, increased lactate production accompanies an increased rate of the TCA cycle. The extent to which skeletal muscles use aerobic versus anaerobic glycolysis to supply ATP varies with the intensity of exercise. During low-intensity exercise, the rate of ATP use is lower, and fibers can generate this ATP from oxidative phosphorylation, with the complete oxidation of glucose to CO2. However, when Otto Shape sprints, a high-intensity exercise, the ATP demand exceeds the rate at which the electron-transport chain and the TCA cycle can generate ATP from oxidative phosphorylation. The increased AMP level signals the need for additional ATP and stimulates PFK-1. The NADH/NAD⫹ ratio directs the increase in pyruvate production toward lactate. The fall in pH causes muscle fatigue and pain. As Otto trains, the amounts of mitochondria and myoglobin in his skeletal muscle fibers increase, and these fibers rely less on anaerobic glycolysis. Ivan Applebod. Ivan Applebod had two sites of dental caries: one on a smooth surface and one in a fissure. The decreased pH resulting from lactic acid production by lactobacilli, which grow anaerobically within the fissure, is a major cause of fissure caries. Streptococcus mutans plays a major role in smooth-surface caries because it secretes dextran, an insoluble polysaccharide, which forms the base for plaque. S. mutans contains dextransucrase, a glucosyltransferase that transfers glucosyl units from dietary sucrose (the glucose–fructose disaccharide in sugar and sweets) to form the ␣(1→6) and ␣(1→3) linkages between the glucosyl units in dextran (Fig. 22.16). Dextransucrase is specific for sucrose and does not catalyze the polymerization of free glucose or glucose from other disaccharides or polysaccharides. Thus, sucrose is responsible for the cariogenic potential of candy. The sticky, water-insoluble dextran mediates the attachment of S. mutans and other bacteria to the tooth surface. This also keeps the acids produced from these bacteria close to the enamel surface. Fructose from sucrose is converted to intermediates of glycolysis and is rapidly metabolized to lactic acid. Other bacteria present in the plaque produce different acids from anaerobic metabolism, such as acetic acid and formic acid. The decrease in pH that results initiates demineralization of the hydroxyapatite of the tooth enamel. Ivan Applebod’s caries in his baby teeth could have been caused by sucking on bottles containing fruit juice. The sugar in fruit juice is also sucrose, and babies who fall asleep with a bottle of fruit juice in their mouth may develop caries. Rapid decay of these baby teeth can harm the development of their permanent teeth.

O H2C O

O (␣1,6)-bond H2C O

O H2C

CH2OH O

O

O

O n

(␣1,3)-bond

H2C O

O H2C

CH2OH

O

O

O

O n

FIG. 22.16. General structure of dextran. Glucosyl residues are linked by ␣-1,3-; ␣-1,6-; and some ␣-1,4-bonds.

BIOCHEMICAL COMMENTS The Mechanism of Glyceraldehyde 3-Phosphate Dehydrogenase. How is the first high-energy bond created in the glycolytic pathway? This is the work of the glyceraldehyde 3-P dehydrogenase reaction, which converts glyceraldehyde 3-P to 1,3-bisphosphoglycerate. This reaction

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+ H+ NAD+ H H

1

C

O

C

OH

H H

OH C S C

OH

Cys

NADH O C ~S

2 H

CH2O P

CH2O P

C

Cys

OH

CH2O P

Glyceraldehyde 3-P NAD+

3

NAD+

NADH H S

Cys NAD+

O C ~O P H

C

OH

CH2O P

4

O C ~S

Pi H

C

Cys

OH

CH2O P

FIG. 22.17. Mechanism of the glyceraldehyde 3-phosphate dehydrogenase reaction. (1) The enzyme forms a covalent linkage with the substrate, using a cysteine group at the active site. The enzyme also contains bound NAD⫹ close to the active site. (2) The substrate is oxidized, forming a high-energy thioester linkage (in red) and NADH. (3) NADH has a low affinity for the enzyme and is replaced by a new molecule of NAD⫹. (4) Inorganic phosphate attacks the thioester linkage, releasing the product 1,3-bisphosphoglycerate and regenerating the active enzyme in a form ready to initiate another reaction.

can be considered to be two separate half-reactions: the first being the oxidation of glyceraldehyde 3-P to 3-phosphoglycerate, and the second being the addition of inorganic phosphate to 3-phosphoglycerate to produce 1,3-bisphosphoglycerate. The ⌬G0⬘ for the first reaction is approximately ⫺12 kcal/mol; for the second reaction, it is approximately ⫹12 kcal/mol. Thus, although the first half-reaction is extremely favorable, the second half-reaction is unfavorable and does not proceed under cellular conditions. So how does the enzyme help this reaction to proceed? This is accomplished through the enzyme forming a covalent bond with the substrate, using an essential cysteine residue at the active site to form a high-energy thioester linkage during the course of the reaction (Fig. 22.17). Thus, the energy that would be released as heat in the oxidation of glyceraldehyde 3-P to 3-phosphoglycerate is conserved in the thioester linkage that is formed (such that the ⌬G0⬘ of the formation of the thioester intermediate from glyceraldehyde 3-P is close to zero). Then, replacement of the sulfur with inorganic phosphate to form the final product, 1,3-bisphosphoglycerate, is relatively straightforward, as the ⌬G0⬘ for that conversion is also close to zero, and the acylphosphate bond retains the energy from the oxidation of the aldehyde. This is one example of how covalent catalysis by an enzyme can result in the conservation of energy between different bond types.

Key Concepts • • • • •

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Glycolysis is the pathway in which glucose is oxidized and cleaved to form pyruvate. The enzymes of glycolysis are in the cytosol. Glucose is the major sugar in our diet; all cells can use glucose for energy. Glycolysis generates two molecules of ATP through substrate-level phosphorylation, and two molecules of NADH. The cytosolic NADH generated via glycolysis transfers its reducing equivalents to mitochondrial NAD⫹ via shuttle systems across the inner mitochondrial membrane.

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Table 22.2

Diseases Discussed in Chapter 22

Disease or Disorder

Environmental Genetic or

Chronic obstructive pulmonary disease

Both

Obesity

Both

Dental caries

Environmental

Lactic acidemia

Both

• • • • • • •

413

Comments Can lead to inefficient energy production in the nervous systems due to reduced oxygen delivery to the tissue Lactate production via anaerobic glycolysis in the muscle occurs during vigorous exercise Effects of carbohydrate metabolism on oral flora and acid production Elevated lactic acid due to mutations in a variety of enzymes involved in carbohydrate and energy metabolism

The pyruvate generated during glycolysis can enter the mitochondria and be oxidized completely to CO2 by PDH and the TCA cycle. Anaerobic glycolysis generates energy in cells with a limited supply of oxygen or few mitochondria. Under anaerobic conditions, pyruvate is reduced to lactate by NADH, thereby regenerating the NAD⫹ required for glycolysis to continue. Glycolysis is regulated to ensure that ATP homeostasis is maintained. The key regulated enzymes of glycolysis are hexokinase, phosphofructokinase-1, and pyruvate kinase. Mutations in the TCA cycle, or proteins involved in oxidative phosphorylation, can lead to an accumulation of pyruvate, which will lead to lactic acidemia. Diseases discussed in this chapter are summarized in Table 22.2.

REVIEW QUESTIONS—CHAPTER 22 1.

A major role of glycolysis is which of the following? A. To synthesize glucose B. To generate energy C. To produce FAD(2H) D. To synthesize glycogen E. To use ATP to generate heat

2.

Starting with glyceraldehyde 3-phosphate and synthesizing one molecule of pyruvate, the net yield of ATP and NADH would be which of the following? A. 1 ATP, 1 NADH B. 1 ATP, 2 NADH C. 1 ATP, 4 NADH D. 2 ATP, 1 NADH E. 2 ATP, 2 NADH F. 2 ATP, 4 NADH G. 3 ATP, 1 NADH H. 3 ATP, 2 NADH I. 3 ATP, 4 NADH

3.

When glycogen is degraded, glucose 1-phosphate is formed. Glucose 1-phosphate can then be isomerized to glucose 6-phosphate. Starting with glucose 1-phosphate and ending with two molecules of pyruvate, what is the net yield of glycolysis in terms of ATP and NADH formed? A. 1 ATP, 1 NADH B. 1 ATP, 2 NADH

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C. D. E. F. G. H. I.

1 ATP, 3 NADH 2 ATP, 1 NADH 2 ATP, 2 NADH 2 ATP, 3 NADH 3 ATP, 1 NADH 3 ATP, 2 NADH 3 ATP, 3 NADH

4.

Which of the following statements correctly describes an aspect of glycolysis? A. ATP is formed by oxidative phosphorylation. B. Two molecules of ATP are used in the beginning of the pathway. C. Pyruvate kinase is the rate-limiting enzyme. D. One molecule of pyruvate and three molecules of CO2 are formed from the oxidation of one glucose molecule. E. The reactions take place in the matrix of the mitochondria.

5.

How many moles of ATP are generated by the complete aerobic oxidation of 1 mol of glucose to 6 mol of CO2? A. 2 to 4 B. 10 to 12 C. 18 to 22 D. 30 to 32 E. 60 to 64

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23

Oxidation of Fatty Acids and Ketone Bodies Fatty acids are a major fuel for humans and supply our energy needs between meals and during periods of increased demand, such as exercise. During overnight fasting, fatty acids become the major fuel for cardiac muscle, skeletal muscle, and liver. The liver converts fatty acids to ketone bodies (acetoacetate and ␤-hydroxybutyrate), which also serve as major fuels for tissues (e.g., the gut). The brain, which does not have a significant capacity for fatty acid oxidation, can use ketone bodies as a fuel during prolonged fasting. The route of metabolism for a fatty acid depends somewhat on its chain length. Fatty acids are generally classified as very long-chain-length fatty acids (C20), long-chain fatty acids (C12 to C20), medium-chain fatty acids (C6 to C12), and short-chain fatty acids (C4). Adenosine Triphosphate (ATP) is generated from oxidation of fatty acids in the pathway of ␤-oxidation. Between meals and during overnight fasting, long-chain fatty acids are released from adipose tissue triacylglycerols. They circulate through blood bound to albumin (Fig. 23.1). In cells, they are converted to fatty acyl coenzyme A (fatty acyl-CoA) derivatives by acyl-CoA synthetases. The activated acyl group is transported into the mitochondrial matrix bound to carnitine, where fatty acyl-CoA is regenerated. In the pathway of ␤-oxidation, the fatty acyl group is sequentially oxidized to yield FAD(2H), NADH, and acetyl-CoA. Subsequent oxidation of NADH and FAD(2H) in the electron-transport chain, and oxidation of acetyl-CoA to CO2 in the tricarboxylic acid (TCA) cycle, generates ATP from oxidative phosphorylation. Many fatty acids have structures that require variations of this basic pattern. Long-chain fatty acids that are unsaturated fatty acids generally require additional isomerization and oxidation–reduction reactions to rearrange their double bonds during ␤-oxidation. Metabolism of water-soluble mediumchain-length fatty acids does not require carnitine and occurs only in the liver. Odd-chain-length fatty acids undergo ␤-oxidation to the terminal threecarbon propionyl-CoA, which enters the TCA cycle as succinyl-CoA. Fatty acids that do not readily undergo mitochondrial ␤-oxidation are oxidized first by alternate routes that convert them to a more suitable substrates or to urinary excretion products. Excess fatty acids may undergo microsomal ␻-oxidation, which converts them to dicarboxylic acids that appear in urine. Very long-chain fatty acids (both straight-chain and branched fatty acids such as phytanic acid) are whittled down to size in peroxisomes. Peroxisomal ␣- and ␤-oxidation generates hydrogen peroxide (H2O2), NADH, acetyl-CoA, or propionyl-CoA and a short- to medium-chain-length acyl-CoA. The acylCoA products are transferred to mitochondria to complete their metabolism. In the liver, much of the acetyl-CoA generated from fatty acid oxidation is converted to the ketone bodies, acetoacetate and ␤-hydroxybutyrate, which enter the blood (see Fig. 23.1). In other tissues, these ketone bodies are converted to acetyl-CoA, which is oxidized in the TCA cycle. The liver synthesizes ketone bodies but cannot use them as a fuel.

414

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415

Long-chain Fatty acid-albumin

1 ATP CoA

Fatty acid– binding proteins

Plasma membrane

2 Fatty acyl CoA Outer mitochondrial membrane

Carnitine CoA

3

Fatty acyl carnitine Inner mitochondrial membrane

Carnitine CoA Fatty acyl CoA

␤-Oxidation spiral 4

FAD (2H) NADH

5

Acetyl CoA

(Liver) Ketone bodies

TCA cycle

2CO2

NADH, FAD (2H), GTP

FIG. 23.1. Overview of mitochondrial long-chain fatty acid metabolism. (1) Fatty acid– binding proteins (FaBP) transport fatty acids across the plasma membrane and bind them in the cytosol. (2) Fatty acyl-CoA synthetase activates fatty acids to fatty acyl-CoAs. (3) Carnitine transports the activated fatty acyl group into mitochondria. (4) ␤-Oxidation generates NADH, FAD(2H), and acetyl-CoA. (5) In the liver, acetyl-CoA is converted to ketone bodies.

The rate of fatty acid oxidation is linked to the rate of NADH, FAD(2H), and acetyl-CoA oxidation and, thus, to the rate of oxidative phosphorylation and ATP use. Additional regulation occurs through malonyl-CoA, which inhibits formation of the fatty acyl carnitine derivatives. Fatty acids and ketone bodies are used as fuel when their level increases in the blood, which is determined by hormonal regulation of adipose tissue lipolysis.

THE WAITING ROOM Otto Shape was disappointed that he did not place in his 5-km race and decided that short-distance running is probably not right for him. After careful consideration, he decides to train for the marathon by running 12 miles three times per week. He is now 13 lb over his ideal weight, and he plans on losing this weight while studying for his pharmacology finals. He considers a variety of dietary supplements to increase his endurance and selects one that contains carnitine, coenzyme Q (CoQ), pantothenate, riboflavin, and creatine.

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The liver transaminases measured in the blood are aspartate aminotransferase (AST), which was previously designated serum glutamateoxaloacetate transaminase (SGOT), and alanine aminotransferase (ALT), which was previously designated serum glutamate pyruvate transaminase (SGPT). Elevation of these enzymes in the serum reflects damage of the liver cell plasma membrane. Transaminases catalyze the transfer of the nitrogen group of an amino acid to an acceptor ␣-keto acid. For AST, aspartate donates the nitrogen to ␣-ketoglutarate, forming oxaloacetate (the corresponding ␣-keto acid to aspartate) and glutamate. For ALT, alanine donates the nitrogen to ␣-ketoglutarate, forming pyruvate and glutamate. Transaminase activity is measured using a coupled reaction: Both oxaloacetate and pyruvate can be reduced by NADH to form, respectively—malate and lactate—in the presence of the appropriate secondary enzyme (malate dehydrogenase and lactate dehydrogenase). Thus, automated procedures that follow the loss of NADH in the reaction mix (by measuring the decrease in absorbance at 340 nm) can be used to measure the activity of AST and ALT in serum samples.

Lofata Burne is a 16-year-old girl. Since age 14 months, she has experienced recurrent episodes of profound fatigue associated with vomiting and increased perspiration, which required hospitalization. These episodes occurred only if she fasted for more than 8 hours. Because her mother gave her food late at night and woke her early in the morning for breakfast, Lofata’s physical and mental development had progressed normally. On the day of admission for this episode, Lofata had missed breakfast, and by noon, she was extremely fatigued, nauseated, sweaty, and limp. She was unable to hold any food in her stomach and was rushed to the hospital, where an infusion of glucose was started intravenously. Her symptoms responded dramatically to this therapy. Her initial serum glucose level was low at 38 mg/dL (reference range for fasting serum glucose levels, 70 to 100 mg/dL). Her blood urea nitrogen (BUN) level was slightly elevated at 26 mg/dL (reference range, 8 to 25 mg/dL) because of vomiting, which led to a degree of dehydration. Her blood levels of liver transaminases were slightly elevated, although her liver was not palpably enlarged. Despite elevated levels of free fatty acids (4.3 mM) in the blood, blood ketone bodies were lower than normal. Di Abietes, a 27-year-old woman with type 1 diabetes mellitus, had been admitted to the hospital in a ketoacidotic coma a year ago (see Chapter 4). She had been feeling drowsy and had been vomiting for 24 hours before that admission. At the time of admission, she was clinically dehydrated, her blood pressure was low, and her breathing was deep and rapid (Kussmaul breathing). Her pulse was rapid, and her breath had the odor of acetone. Her arterial blood pH was 7.08 (reference range, pH 7.36 to 7.44), and her blood ketone body levels were 15 mM (normal is approximately 0.2 mM for a person on a normal diet).

I.

During Otto Shape’s distance running (a moderate-intensity exercise), decreases in insulin and increases in insulin counterregulatory hormones, such as epinephrine and norepinephrine, increase adipose tissue lipolysis. Thus, his muscles are being provided with a supply of fatty acids in the blood that can be used as a fuel.

Lofata Burne developed symptoms during fasting when adipose tissue lipolysis was elevated. Under these circumstances, muscle tissue, liver, and many other tissues are oxidizing fatty acids as a fuel. After overnight fasting, approximately 60% to 70% of our energy supply is derived from the oxidation of fatty acids.

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FATTY ACIDS AS FUELS

The fatty acids oxidized as fuels are principally long-chain fatty acids released from adipose tissue triacylglycerol stores between meals, during overnight fasting, and during periods of increased fuel demand (e.g., during exercise). Adipose tissue triacylglycerols are derived from two sources: dietary lipids and triacylglycerols synthesized in the liver. The major fatty acids oxidized are the long-chain fatty acids, palmitate, oleate, and stearate, because they are highest in dietary lipids and are also synthesized in the human. Between meals, a decreased insulin level and increased levels of insulin counterregulatory hormones (e.g., glucagon) activate lipolysis, and free fatty acids are transported to tissues bound to serum albumin. Within tissues, energy is derived from oxidation of fatty acids to acetyl-CoA in the pathway of ␤-oxidation. Most of the enzymes involved in fatty acid oxidation are present as two or three isoenzymes, which have different but overlapping specificities for the chain length of the fatty acid. Metabolism of unsaturated fatty acids, odd-chain-length fatty acids, and medium-chain-length fatty acids requires variations of this basic pattern. The acetyl-CoA produced from fatty acid oxidation is principally oxidized in the tricarboxylic acid (TCA) cycle or converted to ketone bodies in the liver.

A. Characteristics of Fatty Acids Used as Fuels Fat constitutes approximately 38% of the calories in the average North American diet. Of this, more than 95% of the calories are present as triacylglycerols (three fatty acids esterified to a glycerol backbone). During ingestion and absorption, dietary triacylglycerols are broken down into their constituents and then reassembled for transport to adipose tissue in chylomicrons (see Chapter 2). Thus, the fatty acid composition of adipose triacylglycerols varies with the type of food consumed.

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417

The most common dietary fatty acids are the saturated long-chain fatty acids palmitate (C16) and stearate (C18), the monounsaturated fatty acid oleate (C18:1), and the polyunsaturated essential fatty acid linoleate (C18:2) (to review fatty acid nomenclature, consult Chapter 5). Animal fat contains, principally, saturated and monounsaturated long-chain fatty acids, whereas vegetable oils contain linoleate and some longer chain and polyunsaturated fatty acids. They also contain smaller amounts of branched-chain and odd-chain-length fatty acids. Medium-chain-length fatty acids are present principally in dairy fat (e.g., milk, butter), maternal milk, and vegetable oils. Adipose tissue triacylglycerols also contain fatty acids synthesized in the liver, principally from excess calories ingested as glucose. The pathway of fatty acid synthesis generates palmitate, which can be elongated to form stearate or can be unsaturated to form oleate. These fatty acids are assembled into triacylglycerols and transported to adipose tissues as the lipoprotein very low-density lipoprotein (VLDL).

B. Transport and Activation of Long-Chain Fatty Acids Long-chain fatty acids are hydrophobic and, therefore, water insoluble. In addition, they are toxic to cells because they can disrupt the hydrophobic bonding between amino acid side chains in proteins. Consequently, they are transported in the blood and in cells bound to proteins. 1.

CELLULAR UPTAKE OF LONG-CHAIN FATTY ACIDS

During fasting and other conditions of metabolic need, long-chain fatty acids are released from adipose tissue triacylglycerols by lipases. They travel in the blood bound in the hydrophobic binding pocket of albumin: the major serum protein (see Fig. 23.1). Fatty acids enter cells both by a saturable transport process and by diffusion through the lipid plasma membrane. A fatty acid–binding protein in the plasma membrane facilitates transport. An additional fatty acid–binding protein binds the fatty acid intracellularly and may facilitate its transport to the mitochondrion. The free fatty acid concentration in cells is, therefore, extremely low. 2.

ACTIVATION OF LONG-CHAIN FATTY ACIDS

Fatty acids must be activated to acyl-CoA derivatives before they can participate in ␤-oxidation and other metabolic pathways (Fig. 23.2). The process of activation involves an acyl-CoA synthetase (also called a thiokinase) that uses ATP energy to form the fatty acyl-CoA thioester bond. In this reaction, the ␤-bond of ATP is cleaved to form a fatty acyl adenosine monophosphate (AMP) intermediate and pyrophosphate (PPi). Subsequent cleavage of PPi helps to drive the reaction. The acyl-CoA synthetase that activates long-chain fatty acids—12 to 20 carbons in length—is present in three locations in the cell: the endoplasmic reticulum, outer mitochondrial membranes, and peroxisomal membranes (Table 23.1). This enzyme has no activity toward C22 or longer fatty acids, and has little activity below C12. In contrast, the synthetase for activation of very long-chain fatty acids is present in peroxisomes, and the medium-chain-length fatty acid–activating enzyme is present only in the mitochondrial matrix of liver and kidney cells. 3.

FATES OF FATTY ACYL -COAS

Fatty acyl-CoA formation, like the phosphorylation of glucose, is a prerequisite to metabolism of the fatty acid in the cell (Fig. 23.3). The multiple locations of the long-chain acyl-CoA synthetase reflect the location of different metabolic routes taken by fatty acyl-CoA derivatives in the cell (e.g., triacylglycerol and phospholipid synthesis in the endoplasmic reticulum, oxidation and plasmalogen synthe-

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

O ATP

–

O

O

P

P

O

O

O

–

O –

O

Fatty acid

O–

O

P O

Adenosine

–

R C O Fatty acyl CoA synthetase

O Fatty acyl AMP (enzyme-bound)

R

C

••

O

CoASH Fatty acyl CoA synthetase

Fatty acyl CoA

R

O

O O

P O

Adenosine

–

O

–

+ O P O P O– O–

O–

Pyrophosphate AMP

O C~SCoA

Inorganic pyrophosphatase

2Pi

FIG. 23.2. Activation of a fatty acid by a fatty acyl-CoA synthetase. The fatty acid is activated by reacting with ATP to form a high-energy fatty acyl AMP and pyrophosphate. The AMP is then exchanged for CoA. Pyrophosphate is cleaved by a pyrophosphatase. Table 23.1 Enzymes

Chain-Length Specificity of Fatty Acid Activation and Oxidation

Enzyme Acyl-CoA synthetases Very long chain Long chain

Energy ␤-Oxidation ketogenesis

Membrane lipids Phospholipids Sphingolipids

Storage Triacylglycerols

FIG. 23.3. Major metabolic routes for longchain fatty acyl-CoAs. Fatty acids are activated to acyl-CoA compounds for degradation in mitochondrial ␤-oxidation or incorporation into triacylglycerols or membrane lipids. When ␤-oxidation is blocked through an inherited enzyme deficiency or metabolic regulation, excess fatty acids are diverted into triacylglycerol synthesis.

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14–26 12–20

Medium chain

6–12

Acetyl

2–4

Acyltransferases CPTI

Fatty acyl CoA

Chain Length

Medium chain (octanoylcarnitine transferase) Carnitine:acetyl transferase Acyl-CoA dehydrogenases VLCAD LCAD MCAD SCAD Other enzymes Enoyl-CoA hydratase, short-chain L-3-Hydroxyacyl-CoA dehydrogenase, short-chain Acetoacetyl-CoA thiolase Trifunctional protein

12–16 6–12 2

14–20 12–18

Comments Found only in peroxisomes. Enzyme present in membranes of endoplasmic reticulum, mitochondria, and peroxisomes to facilitate different metabolic routes of acyl-CoAs. Exists as many variants, present only in mitochondrial matrix of kidney and liver. Also involved in xenobiotic metabolism. Present in cytoplasm and possibly mitochondrial matrix. Although maximum activity is for fatty acids 12–16 carbons long, it also acts on many smaller acyl-CoA derivatives. Substrate is medium-chain acyl-CoA derivatives generated during peroxisoma oxidation. High level in skeletal muscle and heart to facilitate use of acetate as a fuel. Present in inner mitochondrial membrane. Members of same enzyme family, which also includes acyl-CoA dehydrogenases for carbon skeleton of branched-chain amino acids.

4–12 4–6 4 4–16 4 12–16

Also called crotonase. Activity decreases with increasing chain length. Activity decreases with increasing chain length. Specific for acetoacetyl-CoA. Complex of long-chain enoyl hydratase, acyl-CoA dehydrogenase, and a thiolase with broad specificity. Most active with longer chains.

CPTI, carnitine palmitoyl transferase I; VLCAD, very long-chain acyl-CoA dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium acyl-CoA dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase deficiency.

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CHAPTER 23 ■ OXIDATION OF FATTY ACIDS AND KETONE BODIES

sis in the peroxisome, ␤-oxidation in mitochondria). In the liver and some other tissues, fatty acids that are not being used for energy generation are reincorporated (reesterified) into triacylglycerols. 4.

TRANSPORT OF LONG-CHAIN FATTY ACIDS INTO MITOCHONDRIA

Carnitine serves as the carrier that transports activated long-chain fatty acyl groups across the inner mitochondrial membrane (Fig. 23.4). Carnitine acyl transferases are able to reversibly transfer an activated fatty acyl group from CoA to the hydroxyl group of carnitine to form an acylcarnitine ester. The reaction is reversible, so the fatty acyl-CoA derivative can be regenerated from the carnitine ester. Carnitine palmitoyl transferase I (CPTI; also called carnitine acyltransferase I, CATI), the enzyme that transfers long-chain fatty acyl groups from CoA to carnitine, is located on the outer mitochondrial membrane (Fig. 23.5). Fatty acylcarnitine crosses the inner mitochondrial membrane with the aid of a translocase. The fatty acyl group is transferred back to CoA by a second enzyme, carnitine palmitoyl transferase II (CPTII or CATII). The carnitine released in this reaction returns to the cytosolic side of the mitochondrial membrane by the same translocase that brings fatty acylcarnitine to the matrix side. Long-chain fatty acyl-CoA, now located within the mitochondrial matrix, is a substrate for ␤-oxidation. Carnitine is obtained from the diet or synthesized from the side chain of lysine by a pathway that begins in skeletal muscle and is completed in the liver. The reactions use S-adenosylmethionine to donate methyl groups, and vitamin C (ascorbic acid) is also required for these reactions. Skeletal muscles have a high-affinity uptake system for carnitine, and most of the carnitine in the body is stored in skeletal muscle.

ATP + CoA Fatty acid

Cytosol

AMP + PPi

Fatty acyl CoA Carnitine: palmitoyltransferase I (CPT I)

Acyl CoA synthetase

Outer mitochondrial membrane CoA

Fatty acyl CoA

Fatty acylcarnitine

Carnitine Carnitine: palmitoyltransferase II

Carnitine: acylcarnitine translocase

Matrix

(CPT II)

Inner mitochondrial membrane

419

CH3 CH3 O CH3

(CH2)n C

+

N

CH3

CH2 O

CH CH2 COO–

Fatty acylcarnitine

FIG. 23.4. Structure of fatty acylcarnitine. Carnitine palmitoyl transferases catalyze the reversible transfer of a long-chain fatty acyl group from the fatty acyl-CoA to the hydroxyl group of carnitine. The atoms in the green box originate from the fatty acyl-CoA.

Several inherited diseases in the metabolism of carnitine or acylcarnitines have been described. These include defects in the following enzymes or systems: the transporter for carnitine uptake into muscle, CPTI, carnitine acylcarnitine translocase, and CPTII. Classical CPTII deficiency, the most common of these diseases, is characterized by adolescent-to-adult onset of recurrent episodes of acute myoglobinuria precipitated by prolonged exercise or fasting. During these episodes, the patient is weak and may be somewhat hypoglycemic with diminished ketosis (hypoketosis), but metabolic decompensation is not severe. Lipid deposits are found in skeletal muscles. Both creatine phosphokinase (CPK) and long-chain acylcarnitines are elevated in the blood. CPTII levels in fibroblasts are approximately 25% of normal. The remaining CPTII activity probably accounts for the mild effect on liver metabolism. In contrast, when CPTII deficiency presents in infants, CPTII levels are 10% of normal, the hypoglycemia and hypoketosis are severe, hepatomegaly occurs from the triacylglycerol deposits, and cardiomyopathy is also present.

CoA Fatty acylcarnitine Carnitine

Fatty acyl CoA

␤-Oxidation FIG. 23.5. Transport of long-chain fatty acids into mitochondria. The fatty acyl-CoA crosses the outer mitochondrial membrane. Carnitine palmitoyl transferase I in the outer mitochondrial membrane transfers the fatty acyl group to carnitine and releases CoASH. The fatty acyl carnitine is translocated into the mitochondrial matrix as carnitine moves out. Carnitine palmitoyl transferase II on the inner mitochondrial membrane transfers the fatty acyl group back to CoASH to form fatty acyl-CoA in the matrix.

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Otto Shape’s power supplement contains carnitine. However, his body can synthesize enough carnitine to meet his needs, and his diet contains carnitine. Carnitine deficiency has been found only in infants fed a soy-based formula that was not supplemented with carnitine. His other supplements likewise probably provide no benefit but are designed to facilitate fatty acid oxidation during exercise. Riboflavin is the vitamin precursor of FAD, which is required for acyl-CoA dehydrogenases and electron-transfer flavoproteins (ETFs). Coenzyme Q (CoQ) is synthesized in the body, but it is the recipient in the electron-transport chain for electrons passed from complexes I and II and the ETFs. Some reports suggest that supplementation with pantothenate—the precursor of CoA— improves performance.

COASH

␣ O

H3C

C. ␤-Oxidation of Long-Chain Fatty Acids

The oxidation of fatty acids to acetyl-CoA in the ␤-oxidation spiral conserves energy as FAD(2H) and NADH. FAD(2H) and NADH are oxidized in the electrontransport chain, generating ATP from oxidative phosphorylation. Acetyl-CoA is oxidized in the TCA cycle or converted to ketone bodies. 1.

THE ␤-OXIDATION SPIRAL

The fatty acid ␤-oxidation pathway sequentially cleaves the fatty acyl group into two-carbon acetyl-CoA units, beginning with the carboxyl end attached to CoA (Fig. 23.6). Before cleavage, the ␤-carbon is oxidized to a keto group in two reactions that generate NADH and FAD(2H); thus, the pathway is called ␤-oxidation. As each acetyl group is released, the cycle of ␤-oxidation and cleavage begins again, but each time the fatty acyl group is two carbons shorter. There are four types of reactions in the ␤-oxidation pathway (Fig. 23.7). In the first step, a double bond is formed between the ␤- and ␣-carbons by an acyl-CoA dehydrogenase that transfers electrons to FAD. The double bond is in the trans configuration (a 2-trans double bond). In the next step, an OH from water is added to the ␤-carbon, and an H from water is added to the ␣-carbon. The enzyme is called an enoyl hydratase (hydratases add the elements of water, and “-ene” in a name denotes a double bond). In the third step of ␤-oxidation, the hydroxyl group on the

Mitochondrial matrix

␤

CH2

CH2

C~ SCoA

Fatty acyl CoA

FAD

1

Palmitoyl CoA

Acyl CoA dehydrogenase

H3C

O

␣

CH2

[total C = n]

C~ SCoA

␤

CH3

O CH2

CH3

C~ SCoA

~1.5 ATP

FAD (2H)

␤

O CH

CH

C~ SCoA

trans ⌬2 Fatty enoyl CoA

+ O Six repetitions of the ␤-oxidation spiral

Enoyl CoA hydratase

Acetyl CoA

7 Acetyl CoA

FIG. 23.6. Overview of ␤-oxidation. Oxidation at the ␤-carbon is followed by cleavage of the ␣–␤ bond, releasing acetyl-CoA and a fatty acylCoA that is two carbons shorter than the original. The carbons cleaved to form acetyl-CoA are shown in red. Successive spirals of ␤-oxidation completely cleave an even-chain fatty acyl-CoA to acetyl-CoA.

H2O

2

CH3 C~ SCoA

␤-Oxidation Spiral

CH2

CH3

␤

OH CH

CH2

C~ SCoA

L-␤-Hydroxy acyl CoA

NAD+

3 ␤-Hydroxy acyl CoA dehydrogenase

CH3

O

CH2

␤

NADH + H+ O C

~2.5 ATP

O CH2

C~ SCoA

␤-Keto acyl CoA

CoASH

4 ␤-Keto thiolase

O CH3 [total C = (n – 2)]

CH2 C ~ SCoA + CH3 Fatty acyl CoA

O C~ SCoA Acetyl CoA

FIG. 23.7. Steps of ␤-oxidation. The four steps are repeated until an even-chain fatty acid is completely converted to acetyl-CoA. The FAD(2H) and NADH are reoxidized by the electron-transport chain—producing ATP.

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␤-carbon is oxidized to a ketone by a hydroxyacyl-CoA dehydrogenase. In this reaction, as in the conversion of most alcohols to ketones, the electrons are transferred to NAD to form NADH. In the last reaction of the sequence, the bond between the ␤- and ␣-carbons is cleaved by a reaction that attaches Coenzyme A (CoASH) to the ␤-carbon, and acetyl-CoA is released. This is a thiolytic reaction (lysis refers to breakage of the bond, and thio refers to the sulfur) catalyzed by enzymes called ␤-ketothiolases. The release of two carbons from the carboxyl end of the original fatty acyl-CoA produces acetyl-CoA and a fatty acyl-CoA that is two carbons shorter than the original. It is of interest to note that the ␤-oxidation spiral uses the same reaction types seen in the TCA cycle in the conversion of succinate to oxaloacetate. The shortened fatty acyl-CoA repeats these four steps until all of its carbons are converted to acetyl-CoA. ␤-Oxidation is thus a spiral rather than a cycle. In the last spiral, cleavage of the four-carbon fatty acyl-CoA (butyryl-CoA) produces two acetyl-CoAs. Thus, an even-chain fatty acid such as palmitoyl-CoA, which has 16 carbons, is cleaved seven times, producing 7 FAD(2H)s, 7 NADHs, and 8 acetyl-CoAs. 2.

CH2

H C

H C

Palmitoyl CoA

Palmitoloyl CoA

FAD Acyl CoA DH

FAD (2H) Acyl CoA DH

FAD (2H) ETF

FAD ETF

FAD ETF • QO

FAD (2H) ETF • QO

CoQH2

CoQ

ENERGY YIELD OF ␤-OXIDATION

Like the FAD in all flavoproteins, FAD(2H) bound to the acyl-CoA dehydrogenases is oxidized back to FAD without dissociating from the protein (Fig. 23.8). Electron-transfer flavoproteins (ETF) in the mitochondrial matrix accept electrons from the enzyme-bound FAD(2H) and transfer these electrons to ETF–coenzyme Q (CoQ) oxidoreductase (ETF-QO) in the inner mitochondrial membrane. ETF-QO, also a flavoprotein, transfers the electrons to CoQ in the electron-transport chain. Oxidative phosphorylation thus generates approximately 1.5 ATP for each FAD(2H) produced in the ␤-oxidation spiral. The total energy yield from the oxidation of 1 mol of palmityl-CoA to 8 mol of acetyl-CoA is 28 mol of ATP: 1.5 for each of the 7 FAD(2H), and 2.5 for each of the 7 NADH. To calculate the energy yield from oxidation of 1 mol of palmitate, two ATP need to be subtracted from the total because two high-energy phosphate bonds are cleaved when palmitate is activated to palmityl-CoA. 3.

CH2

421

CHAIN-LENGTH SPECIFICITY IN ␤-OXIDATION

Electron-transport chain

FIG. 23.8. Transfer of electrons from acylCoA dehydrogenase to the electron-transport chain. An FAD is tightly bound to each protein in these three electron-transfer reactions. ETF, electron-transfer flavoprotein; ETF-QO, electron-transferring flavoprotein–coenzyme Q (CoQ) oxidoreductase.

What is the total ATP yield for the oxidation of 1 mol of palmitic acid to carbon dioxide and water?

The four reactions of ␤-oxidation are catalyzed by sets of enzymes that are each specific for fatty acids with different chain lengths (see Table 23.1). The acyl-CoA dehydrogenases, which catalyze the first step of the pathway, are part of an enzyme family that has four different ranges of specificity. The subsequent steps of the spiral use enzymes that are specific for long- or short-chain enoyl-CoAs. Although these enzymes are structurally distinct, their specificities overlap to some extent. As the fatty acyl chains are shortened by consecutive cleavage of two acetyl units, they are transferred from enzymes that act on longer chains to those that act on shorter chains. Medium- or short-chain fatty acyl-CoA that may be formed from dietary fatty acids, or transferred from peroxisomes, enters the spiral at the enzyme that is most active for fatty acids of its chain length. 4.

OXIDATION OF UNSATURATED FATTY ACIDS

Approximately one-half of the fatty acids in the human diet are unsaturated, containing cis double bonds, with oleate (C18:1, 9) and linoleate (18:2, 9,12) being the most common. In ␤-oxidation of saturated fatty acids, a trans double bond is created between the second and third (␣- and ␤-) carbons. For unsaturated fatty acids to undergo the ␤-oxidation spiral, their cis double bonds must be isomerized to trans double bonds that will end up between the second and third carbons during ␤-oxidation, or the double bond must be reduced. The process is illustrated for the polyunsaturated fatty acid linoleate in Figure 23.9. Linoleate is obtained from the diet and cannot be synthesized in the human; thus, it is considered an essential fatty acid. Therefore, only that portion of linoleate that is not needed for other

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Palmitic acid is 16 carbons long with no double bonds, so it requires seven oxidation spirals to be completely converted to acetyl-CoA. After seven spirals, there are 7 FAD(2H)s, 7 NADHs, and 8 acetyl-CoAs. Each NADH yields 2.5 ATP, each FAD(2H) yields 1.5 ATP, and each acetyl-CoA yields 10 ATP as it is processed around the TCA cycle. This then yields 17.5  10.5  80  108 ATP. However, activation of palmitic acid to palmityl-CoA requires two high-energy bonds, so the net yield is 108  2 or 106 mol of ATP.

12

9 1

18

C

␤-Oxidation (three spirals) 4

Linoleolyl CoA cis-⌬9, cis-⌬12 SCoA O

3 Acetyl CoA 3

O C

2

cis-⌬3, cis-⌬6

SCoA

Enoyl CoA isomerase 4

2

1

C

3

SCoA

trans-⌬2, cis-⌬6

O One spiral of ␤-oxidation and the first step of the second spiral 5

4

Acetyl CoA

2

SCoA

1

C

3

trans-⌬2, cis-⌬4

O NADPH + H+

After reviewing Lofata Burne’s previous hospital records, a specialist suspected that Lofata’s medical problems were caused by a disorder in fatty acid metabolism. A battery of tests showed that Lofata’s blood contained elevated levels of several partially oxidized medium-chain fatty acids, such as octanoic acid (8:0) and 4-decenoic acid (10:1, 4). A urine specimen showed an increase in organic acid metabolites of medium-chain fatty acids containing 6 to 10 carbons, including medium-chain acylcarnitine derivatives. The profile of acylcarnitine species in the urine was characteristic of a genetically determined medium-chain acylCoA dehydrogenase (MCAD) deficiency. In this disease, long-chain fatty acids are metabolized by ␤-oxidation to a medium-chain-length acylCoA, such as octanoyl-CoA. Because further oxidation of this compound is blocked in MCAD deficiency, the medium-chain acyl group is transferred back to carnitine. These acylcarnitines are water-soluble and appear in blood and urine. The specific enzyme deficiency was demonstrated in cultured fibroblasts from Lofata’s skin as well as in her circulating monocytic leukocytes. In long-chain acyl-CoA dehydrogenase (LCAD) deficiency, fatty acylcarnitines accumulate in the blood. Those containing 14 carbons predominate. However, these do not appear in the urine.

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2,4-Dienoyl CoA reductase

5

NADP+ 1

3 4

O C

2

trans-⌬3

SCoA

Enoyl CoA isomerase 5

1

3 4

2

O C

trans-⌬2

SCoA

␤-Oxidation (four spirals) 5 Acetyl CoA

FIG. 23.9. Oxidation of linoleate. After three spirals of ␤-oxidation (dashed lines), there is now a 3,4-cis double bond and a 6,7-cis double bond. The 3,4-cis double bond is isomerized to a 2,3-trans double bond, which is in the proper configuration for the normal enzymes to act. One spiral of ␤-oxidation occurs, plus the first step of a second spiral. A reductase that uses NADPH now converts these two double bonds (between carbons 2 and 3 and between carbons 4 and 5) to one double bond between carbons 3 and 4 in a trans configuration. The isomerase (which can act on double bonds that are in either the cis or the trans configuration) moves this double bond to the 2,3-trans position, and ␤-oxidation can resume.

processes will be oxidized. Linoleate undergoes ␤-oxidation until one double bond is between carbons 3 and 4 near the carboxyl end of the fatty acyl chain and the other is between carbons 6 and 7. An isomerase moves the double bond from the 3,4-position so that it is trans and in the 2,3-position, and ␤-oxidation continues. When a conjugated pair of double bonds is formed (two double bonds separated by one single bond) at positions 2 and 4, an NADPH-dependent reductase reduces the pair to one trans double bond at position 3. Then isomerization and ␤-oxidation resume.

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CHAPTER 23 ■ OXIDATION OF FATTY ACIDS AND KETONE BODIES

423

O

␻

C~ SCoA

O CH3 CH2

C~ SCoA

Propionyl CoA

O CH3 C~ SCoA Acetyl CoA

FIG. 23.10. Formation of propionyl-CoA from odd-chain fatty acids. Successive spirals of ␤-oxidation cleave each of the bonds marked with dashed lines, producing acetyl-CoA except for the three carbons at the ␻-end, which produce propionyl-CoA.

In oleate (C18:1,  ), there is only one double bond between carbons 9 and 10. It is handled by an isomerization reaction similar to that shown for the double bond at position 9 of linoleate. 9

5.

H

H

H

C

C

H

H

O C SCoA

Propionyl CoA HCO3

ODD-CHAIN-LENGTH FATTY ACIDS

Fatty acids that contain an odd number of carbon atoms undergo ␤-oxidation, producing acetyl-CoA until the last spiral—when five carbons remain in the fatty acylCoA. In this case, cleavage by thiolase produces acetyl-CoA and a three-carbon fatty acyl-CoA, propionyl-CoA (Fig. 23.10). Carboxylation of propionyl-CoA yields methylmalonyl-CoA, which is ultimately converted to succinyl-CoA in a vitamin B12–dependent reaction (Fig. 23.11). Propionyl-CoA also arises from the oxidation of branched-chain amino acids. The propionyl-CoA to succinyl-CoA pathway is a major anaplerotic route for the TCA cycle and is used in the degradation of valine, isoleucine, and several other compounds. In the liver, this route provides precursors of oxaloacetate, which is converted to glucose. Thus, this small proportion of the odd-carbonnumber fatty acid chain can be converted to glucose. In contrast, the acetyl-CoA formed from ␤-oxidation of even-chain-number fatty acids in the liver either enters the TCA cycle, where it is principally oxidized to CO2, or it is converted to ketone bodies.

D. Oxidation of Medium-Chain-Length Fatty Acids Dietary medium-chain-length fatty acids are more water-soluble than long-chain fatty acids and are not stored in adipose triacylglycerol. After a meal, they enter the blood and pass into the portal vein to the liver. In the liver, they enter the mitochondrial matrix by the monocarboxylate transporter and are activated to acylCoA derivatives in the mitochondrial matrix (see Fig. 23.1). Medium-chain-length acyl-CoAs, such as long-chain acyl-CoAs, are oxidized to acetyl-CoA via the ␤-oxidation spiral. Medium-chain acyl-CoAs also can arise from the peroxisomal oxidation pathway. The medium-chain-length acyl-CoA synthetase has a broad range of specificity for compounds of approximately the same size that contain a carboxyl group, such as drugs (salicylate—from aspirin metabolism—and valproate, which is used to treat epileptic seizures) or benzoate, a common component of plants. Once the drug-CoA derivative is formed, the carboxyl group is conjugated with glycine to form a urinary excretion product. With certain disorders of fatty acid oxidation, medium- and short-chain fatty acylglycines may appear in the urine, together with acylcarnitines or dicarboxylic acids. Octanoylglycine, for example, will appear in the urine of a patient with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

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–

ATP Propionyl CoA carboxylase

Biotin ADP + Pi

H

H

H

C

C

H

O C SCoA

C –

O

O

D-Methylmalonyl CoA Methylmalonyl CoA epimerase

H

H

H

C

C

H

C

O

O C O

–

SCoA

L-Methylmalonyl CoA Methylmalonyl CoA mutase

H

Coenzyme B12

H

H

C

C

C

H

O C –

O

O

SCoA

Succinyl CoA

FIG. 23.11. Conversion of propionyl-CoA to succinyl-CoA. Succinyl-CoA, an intermediate of the TCA cycle, can form malate, which can be converted to glucose in the liver through the process of gluconeogenesis. Certain amino acids also form glucose by this route (see Chapter 39).

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

The unripe fruit of the ackee tree produces a toxin—hypoglycin— which causes a condition known as Jamaican vomiting sickness. The victims of the toxin are usually unwary children who eat this unripe fruit and develop a severe hypoglycemia, which is often fatal. Although hypoglycin causes hypoglycemia, it acts by inhibiting an acyl-CoA dehydrogenase involved in ␤-oxidation that has specificity for short- and medium-chain fatty acids. Because more glucose must be oxidized to compensate for the decreased ability of fatty acids to serve as fuel, blood glucose levels may fall to extremely low levels. Fatty acid levels, however, rise because of decreased ␤-oxidation. Because of the increased fatty acid levels, ␻-oxidation increases, and dicarboxylic acids are excreted in the urine. The diminished capacity to oxidize fatty acids in liver mitochondria results in decreased levels of acetyl-CoA, the substrate for ketone body synthesis.

As Otto Shape runs, his skeletal muscles increase their use of ATP and their rate of fuel oxidation. Fatty acid oxidation is accelerated by the increased rate of the electron-transport chain. As ATP is used and AMP increases, the AMP-PK acts to facilitate fuel use and maintain ATP homeostasis. Phosphorylation of acetyl-CoA carboxylase results in a decreased level of malonyl-CoA and increased activity of carnitine palmitoylCoA transferase I. At the same time, AMP-PK facilitates the recruitment of glucose transporters into the plasma membrane of skeletal muscle, thereby increasing the rate of glucose uptake. AMP and hormonal signals also increase the supply of glucose 6-phosphate from glycogenolysis. Thus, his muscles are supplied with more fuel, and all the oxidative pathways are accelerated.

E. Regulation of ␤-Oxidation Fatty acids are used as fuels principally when they are released from adipose tissue triacylglycerols in response to hormones that signal fasting or increased demand. Many tissues, such as muscle and kidney, oxidize fatty acids completely to CO2 and H2O. In these tissues, the acetyl-CoA produced by ␤-oxidation enters the TCA cycle. The FAD(2H) and the NADH from ␤-oxidation and the TCA cycle are reoxidized by the electron-transport chain, and ATP is generated. The process of ␤-oxidation is regulated by the cells’ requirements for energy (i.e., by the levels of ATP and NADH), because fatty acids cannot be oxidized any faster than NADH, and FAD(2H) are reoxidized in the electrontransport chain. Fatty acid oxidation also may be restricted by the mitochondrial CoASH pool size. Acetyl CoASH units must enter the TCA cycle or another metabolic pathway to regenerate CoASH required for formation of the fatty acyl-CoA derivative from fatty acyl carnitine. An additional type of regulation occurs at CPTI. CPTI is inhibited by malonylCoA, which is synthesized in the cytosol of many tissues by acetyl-CoA carboxylase (Fig. 23.12). Acetyl-CoA carboxylase is regulated by several different mechanisms, some of which are tissue dependent. In skeletal muscles and liver, it is inhibited when it is phosphorylated by the AMP-activated protein kinase (AMP-PK). Thus, during exercise—when AMP levels increase—AMP-PK is activated, and phosphorylates acetyl-CoA carboxylase, which becomes inactive. Consequently, malonyl-CoA levels decrease, CPTI is activated, and the ␤-oxidation of fatty acids is able to restore ATP homeostasis and decrease AMP levels. In liver, in addition to the negative regulation by the AMP-PK, acetyl-CoA carboxylase is activated by insulin-dependent mechanisms leading to elevated cytoplasmic citrate, an allosteric activator, which promotes the conversion of malonyl-CoA to palmitate in the fatty acid synthesis pathway. Thus, in the liver, malonyl-CoA inhibition of CPTI prevents newly synthesized fatty acids from being oxidized. ␤-Oxidation is strictly an aerobic pathway, dependent on oxygen, a good blood supply, and adequate levels of mitochondria. Tissues that lack mitochondria, such as red blood cells, cannot oxidize fatty acids by ␤-oxidation. Fatty acids also do not serve as a significant fuel for the brain. They are not used by adipocytes, whose function is to store triacylglycerols to provide a fuel for other tissues. Those tissues that do not use fatty acids as a fuel, or use them only to a limited extent, are able to use ketone bodies instead.

1 Fatty acid

ATP ADP

–

3 Electrontransport chain

–

AMP-PK (muscle, liver) – 2 Acetyl CoA Malonyl CoA Acetyl CoA Fatty acyl carnitine carboxylase + Insulin (liver) NADH – FAD (2H) ␤-Oxidation Fatty acyl CoA

Acetyl CoA

FIG. 23.12. Regulation of ␤-oxidation. (1) Hormones control the supply of fatty acids in the blood. (2) Carnitine palmitoyl transferase I is inhibited by malonyl-CoA, which is synthesized by acetyl-CoA carboxylase (ACC). AMP-PK is the AMP-activated protein kinase. (3) The rate of ATP use controls the rate of the electron-transport chain, which regulates the oxidative enzymes of ␤-oxidation and the TCA cycle.

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425

II. ALTERNATIVE ROUTES OF FATTY ACID OXIDATION

Fatty acids that are not readily oxidized by the enzymes of ␤-oxidation enter alternative pathways of oxidation, including peroxisomal ␤- and ␣-oxidation and microsomal ␻-oxidation. The function of these pathways is to convert as much as possible the unusual fatty acids to compounds that can be used as fuels or biosynthetic precursors, and to convert the remainder to compounds that can be excreted in bile or urine. During prolonged fasting, fatty acids released from adipose triacylglycerols may enter the ␻-oxidation or peroxisomal ␤-oxidation pathway, even though they have a normal composition. These pathways not only use fatty acids, they act on xenobiotic (a term used to cover all organic compounds that are foreign to an organism) carboxylic acids that are large hydrophobic molecules resembling fatty acids.

A. Peroxisomal Oxidation of Fatty Acids A small proportion of our diet consists of very long-chain fatty acids (20 or more carbons) or branched-chain fatty acids arising from degradative products of chlorophyll. Very long-chain fatty acid synthesis also occurs within the body, especially in cells of the brain and nervous system, which incorporate them into the sphingolipids of myelin. These fatty acids are oxidized by peroxisomal ␤- and ␣-oxidation pathways, which are essentially chain-shortening pathways. O

1.

VERY LONG-CHAIN FATTY ACIDS

Very long-chain fatty acids of 24 to 26 carbons are oxidized exclusively in peroxisomes by a sequence of reactions similar to mitochondrial ␤-oxidation in that they generate acetyl-CoA and NADH. However, the peroxisomal oxidation of straightchain fatty acids stops when the chain reaches 4 to 6 carbons in length. Some of the long-chain fatty acids also may be oxidized by this route. The long-chain fatty acyl-CoA synthetase is present in the peroxisomal membrane, and the acyl-CoA derivatives enter the peroxisome by a transporter that does not require carnitine. The first enzyme of peroxisomal ␤-oxidation is an oxidase, which donates electrons directly to molecular oxygen and produces hydrogen peroxide (H2O2) (Fig. 23.13). (In contrast, the first enzyme of mitochondrial ␤-oxidation is a dehydrogenase that contains FAD and transfers the electrons to the electron-transport chain via ETF.) Thus, the first enzyme of peroxisomal oxidation is not linked to energy production. The three remaining steps of ␤-oxidation are catalyzed by enoyl-CoA hydratase, hydroxyacylCoA dehydrogenase, and thiolase—enzymes with activities similar to those found in mitochondrial ␤-oxidation but coded for by different genes. Thus, one NADH and one acetyl-CoA are generated for each turn of the spiral. The peroxisomal ␤-oxidation spiral continues generating acetyl-CoA until a medium-chain acyl-CoA, which may be as short as butyryl-CoA, is produced (Fig. 23.14). Within the peroxisome, the acetyl groups can be transferred from CoA to carnitine by an acetylcarnitine transferase, or they can enter the cytosol. A similar reaction converts medium-chain-length acyl-CoAs and the short-chain butyrylCoA to acyl carnitine derivatives. These acylcarnitines diffuse from the peroxisome to the mitochondria, pass through the outer mitochondrial membrane, and are transported through the inner mitochondrial membrane via the carnitine translocase system. They are converted back to acyl-CoAs by carnitine:acyltransferases appropriate for their chain length and enter the normal pathways for ␤-oxidation and acetyl-CoA metabolism. The electrons from NADH and acetyl-CoA can also pass from the peroxisome to the cytosol. The export of NADH-containing electrons occurs through use of a shuttle system similar to those described for NADH electron transfer into the mitochondria.

Lieberman_Ch23.indd 425

R

CH2 CH2 C S-CoA FAD FADH2 H R C

H2O2 O2

O C C H

S-CoA

FIG. 23.13. Oxidation of fatty acids in peroxisomes. The first step of ␤-oxidation is catalyzed by an FAD-containing oxidase. The electrons are transferred from FAD(2H) to O2, which is reduced to hydrogen peroxide (H2O2).

Several inherited deficiencies of peroxisomal enzymes have been described. Zellweger syndrome, which results from defective peroxisomal biogenesis, leads to complex developmental and metabolic phenotypes that affect, principally, the liver and the brain. One of the metabolic characteristics of these diseases is an elevation of C26:0 and C26:1 fatty acid levels in plasma. Refsum disease is caused by a deficiency in a single peroxisomal enzyme, the phytanoylCoA hydroxylase that carries out ␣-oxidation of phytanic acid. Symptoms include retinitis pigmentosa, cerebellar ataxia, and chronic polyneuropathy. Because phytanic acid is obtained solely from the diet, placing patients on a low-phytanic acid diet has resulted in marked improvement.

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VLCFA

Outer mitochondrial membrane

VLCFA CoA

Inner mitochondrial membrane CoASH Carnitine CAT

VLACS VLCFA CoA (H2O2)n

C P T 1

(Acetyl CoA)n

(NADH)n CAT

SCFA CoA MCFA CoA

Acetylcarnitine

Acetyl CoA TCA cycle

Acetylcarnitine

NADH CO2, H2O

CAC

MCFA CoA SCFA CoA

COT SCFA-carnitine MCFA-carnitine

SCFA-carnitine MCFA-carnitine n turns of ␤-oxidation

Further

CPT II

Peroxisome

␤-oxidation

Mitochondrion

FIG. 23.14. Chain shortening by peroxisomal ␤-oxidation. Very long-chain fatty acyl-CoAs and some long-chain fatty acyl-CoAs are oxidized in peroxisomes through n cycles of ␤-oxidation to the stage of a short- to medium-chain fatty acyl-CoA. These short- to medium-chain fatty acylCoAs are converted to carnitine derivatives by COT or CAT in the peroxisomes. In the mitochondria, SCFA-carnitine is converted back to acylCoA derivatives by either CPTII or CAT. VLCFA, very long-chain fatty acyl; VLACS, very long-chain acyl-CoA synthetase; MCFA, medium-chain fatty acyl; SCFA, short-chain fatty acyl; CAT, carnitine acetyltransferase; COT, carnitine octanoyltransferase; CAC, carnitine acylcarnitine carrier; CPTI, carnitine palmitoyltransferase I; CPTII, carnitine palmityltransferase II; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.

Peroxisomes are present in almost every cell type and contain many degradative enzymes, in addition to fatty acyl-CoA oxidase, that generate hydrogen peroxide. H2O2 can generate toxic free radicals. Thus, these enzymes are confined to peroxisomes, where the H2O2 can be neutralized by the free-radical defense enzyme, catalase. Catalase converts H2O2 to water and O2 (see Chapter 24). 2.

␤-Oxidation CH3

CH3

CH3

CH3

COO–

CH3

␣-Oxidation FIG. 23.15. Oxidation of phytanic acid. A peroxisomal ␣-hydroxylase oxidizes the ␣-carbon, and its subsequent oxidation to a carboxyl group releases the carboxyl carbon as CO2. Subsequent spirals of peroxisomal ␤-oxidation alternately release propionyl and acetyl-CoA. At a chain length of approximately eight carbons, the remaining branched fatty acid is transferred to mitochondria as a medium-chain carnitine derivative.

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LONG-CHAIN BRANCHED-CHAIN FATTY ACIDS

Two of the most common branched-chain fatty acids in the diet are phytanic acid and pristanic acid, which are degradation products of chlorophyll and, thus, are consumed in green vegetables. Animals do not synthesize branched-chain fatty acids. These two multimethylated fatty acids are oxidized in peroxisomes to the level of a branched C8 fatty acid, which is then transferred to mitochondria. The pathway thus is similar to that for the oxidation of straight very long-chain fatty acids. Phytanic acid, a multimethylated C20 fatty acid, is first oxidized to pristanic acid using the ␣-oxidation pathway (Fig. 23.15). Phytanic acid hydroxylase introduces a hydroxyl group on the ␣-carbon, which is then oxidized to a carboxyl group with release of the original carboxyl group as CO2. By shortening the fatty acid by one carbon, the methyl groups will appear on the ␣-carbon rather than the ␤-carbon during the ␤-oxidation spiral and, therefore, can no longer interfere with oxidation of the ␤-carbon. Peroxisomal ␤-oxidation thus can proceed normally, releasing propionyl-CoA and acetyl-CoA with alternate turns of the spiral. When a medium-chain length of approximately eight carbons is reached, the fatty acid is transferred to the mitochondrion as a carnitine derivative and ␤-oxidation is resumed.

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427

O CH3

O–

(CH2)n C

␻ O HO

–

CH2

O

(CH2)n

C O–

O

O

C (CH2)n

C O–

␻-Oxidation of fatty acids converts them to dicarboxylic acids.

FIG. 23.16.

B. ␻-Oxidation of Fatty Acids

Fatty acids also may be oxidized at the ␻-carbon of the chain (the terminal methyl group) by enzymes in the endoplasmic reticulum (Fig. 23.16). The ␻-methyl group is first oxidized to an alcohol by an enzyme that uses cytochrome P450, molecular oxygen, and NADPH. Dehydrogenases convert the alcohol group to a carboxylic acid. The dicarboxylic acids produced by ␻-oxidation can undergo ␤-oxidation, forming compounds with 6 to 10 carbons that are water-soluble. Such compounds may then enter the blood, be oxidized as medium-chain fatty acids, or be excreted in urine as medium-chain dicarboxylic acids. The pathways of peroxisomal ␣- and ␤-oxidation, and microsomal ␻-oxidation, are not feedback regulated. These pathways function to decrease levels of waterinsoluble fatty acids or of xenobiotic compounds with a fatty acid–like structure that would become toxic to cells at high concentrations. Thus, their rate is regulated by the availability of substrate.

III. METABOLISM OF KETONE BODIES Overall, fatty acids released from adipose triacylglycerols serve as the major fuel for the body during fasting. These fatty acids are completely oxidized to CO2 and H2O by some tissues. In the liver, much of the acetyl-CoA generated from ␤-oxidation of fatty acids is used for synthesis of the ketone bodies acetoacetate and ␤-hydroxybutyrate, which enter the blood (Fig. 23.17). In skeletal muscles and other tissues, these ketone bodies are converted back to acetyl-CoA, which is oxidized in the TCA cycle with generation of ATP. An alternate fate of acetoacetate in tissues is the formation of cytosolic acetyl-CoA.

Normally, ␻-oxidation is a minor process. However, in conditions that interfere with ␤-oxidation (such as carnitine deficiency or deficiency in an enzyme of ␤-oxidation), ␻-oxidation produces dicarboxylic acids in increased amounts. These dicarboxylic acids are excreted in the urine. Lofata Burne was excreting dicarboxylic acids in her urine, particularly adipic acid (which has six carbons) and suberic acid (which has eight carbons): 

OOC—CH2—CH2—CH2—CH2—COO OOC–CH2—CH2—CH2—CH2—CH2—CH2—COO



Adipic acid Suberic acid

Octanoylglycine was also found in the urine.

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

Fatty acid

Liver

␤-Oxidation Acetyl CoA

Acetoacetate

␤-Hydroxybutyrate

Ketone bodies

Acetoacetate

␤-Hydroxybutyrate

CO2 + H2O

Muscle

FIG. 23.17. The ketone bodies, acetoacetate and ␤-hydroxybutyrate, are synthesized in the liver. Their principal fate is conversion back to acetyl-CoA and oxidation in the TCA cycle in other tissues.

A. Synthesis of Ketone Bodies In the liver, ketone bodies are synthesized in the mitochondrial matrix from acetylCoA generated from fatty acid oxidation (Fig. 23.18). The thiolase reaction of fatty acid oxidation, which converts one molecule of acetoacetyl-CoA to two molecules of acetyl-CoA, is a reversible reaction, although formation of acetoacetyl-CoA is not the favored direction. Thus, when acetyl-CoA levels are high, it can generate acetoacetyl-CoA for ketone body synthesis. The acetoacetyl-CoA will react with acetyl-CoA to produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The enzyme that catalyzes this reaction is HMG-CoA synthase. In the next reaction of the pathway, HMG-CoA lyase catalyzes the cleavage of HMG-CoA to form acetylCoA and acetoacetate. Acetoacetate can enter the blood directly or can be reduced by ␤-hydroxybutyrate dehydrogenase to ␤-hydroxybutyrate, which enters the blood (see Fig. 23.18). This dehydrogenase reaction is readily reversible and interconverts these two ketone bodies, which exist in an equilibrium ratio determined by the NADH/NAD ratio of the mitochondrial matrix. Under normal conditions, the ratio of ␤-hydroxybutyrate to acetoacetate in the blood is approximately 1:1. An alternative fate of acetoacetate is spontaneous decarboxylation—a nonenzymatic reaction that cleaves acetoacetate into CO2 and acetone (see Fig. 23.18). Because acetone is volatile, it is expired by the lungs. A small amount of acetone may be further metabolized in the body.

B. Oxidation of Ketone Bodies as Fuels

Acetoacetate and ␤-hydroxybutyrate can be oxidized as fuels in most tissues, including skeletal muscle, brain, certain cells of the kidney, and cells of the intestinal mucosa. Cells transport both acetoacetate and ␤-hydroxybutyrate from the circulating blood into the cytosol and into the mitochondrial matrix. Here, ␤-hydroxybutyrate is oxidized back to acetoacetate by ␤-hydroxybutyrate

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O CH3

429

O

C~ SCoA

+

CH3

Thiolase

C~ SCoA

2 Acetyl CoA

Co-ASH O C

CH3

CH2

~

C

Acetoacetyl CoA

O

S-CoA O CH3

HMG CoA synthase

Co-ASH OH

CH3

C~ SCoA

C

O CH2

C

O–

CH2

~

C

3-Hydroxy-3-methyl glutaryl CoA (HMG CoA)

O

S-CoA HMG CoA lyase

Acetyl CoA O

CH3 D-␤-Hydroxybutyrate dehydrogenase

C

OH CH

CH2

C

NADH + H+ NAD+

CH3

O O–

Acetoacetate Spontaneous

CO2 O

O CH2

C

O– D-␤-Hydroxybutyrate

CH3

C

CH3

Acetone

FIG. 23.18. Synthesis of the ketone bodies acetoacetate, ␤-hydroxybutyrate, and acetone. The portion of HMG-CoA shown in the tinted box is released as acetyl-CoA, and the remainder of the molecule forms acetoacetate. Acetoacetate is reduced to ␤-hydroxybutyrate or decarboxylated to acetone. Note that the dehydrogenase that interconverts acetoacetate and ␤-hydroxybutyrate is specific for the D-isomer. Thus, it differs from the dehydrogenases of ␤-oxidation, which act on 3-hydroxyacyl-CoA derivatives and is specific for the L-isomer.

dehydrogenase. This reaction produces NADH. Subsequent steps convert acetoacetate to acetyl-CoA (Fig. 23.19). In mitochondria, acetoacetate is activated to acetoacetyl-CoA by succinyl-CoA: acetoacetate-CoA transferase. As the name suggests, CoA is transferred from succinyl-CoA—a TCA-cycle intermediate—to acetoacetate. Although the liver produces ketone bodies, it does not use them, because this thiotransferase enzyme is not present in sufficient quantity. One molecule of acetoacetyl-CoA is cleaved to two molecules of acetyl-CoA by acetoacetyl-CoA thiolase, the same enzyme as is involved in ␤-oxidation. The principal fate of this acetyl-CoA is oxidation in the TCA cycle.

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Ketogenic diets, which are high-fat diets with a 3:1 ratio of lipid to carbohydrate, are being used to reduce the frequency of epileptic seizures in children. The reason for its effectiveness in the treatment of epilepsy is not known. Ketogenic diets are also used to treat children with pyruvate dehydrogenase deficiency. Ketone bodies can be used as a fuel by the brain in the absence of pyruvate dehydrogenase. They also can provide a source of cytosolic acetyl-CoA for acetylcholine synthesis. They often contain medium-chain triglycerides, which induce ketosis more effectively than long-chain triglycerides.

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430

OH CH3

C

O CH2

C O–

H

D-␤-Hydroxybutyrate NAD+

D-␤-Hydroxybutyrate dehyrdogenase

NADH + H+

O CH3

C

O CH2

C

C. Alternative Pathways of Ketone Body Metabolism

–

O Acetoacetate

Succinyl CoA

Succinyl CoA: acetoacetate CoA transferase

Succinate

O CH3

C

O CH2

C SCoA

Acetoacetyl CoA CoASH Thiolase

O CH3

O

+

C

CH3

SCoA

The energy yield from oxidation of acetoacetate is equivalent to the yield for oxidation of two molecules of acetyl-CoA in the TCA cycle (20 ATP) minus the energy for activation of acetoacetate (1 ATP). The energy of activation is calculated at one high-energy phosphate bond, because succinyl-CoA is normally converted to succinate in the TCA cycle, with generation of one molecule of guanosine triphosphate (GTP) (the energy equivalent of ATP). However, when the high-energy thioester bond of succinyl-CoA is transferred to acetoacetate, succinate is produced without the generation of this GTP. Oxidation of ␤-hydroxybutyrate generates one additional NADH. Therefore, the net energy yield from one molecule of ␤-hydroxybutyrate is approximately 21.5 molecules of ATP.

C SCoA

2 Acetyl CoA

FIG. 23.19. Oxidation of ketone bodies. ␤-Hydroxybutyrate is oxidized to acetoacetate, which is activated by accepting a CoA group from succinyl-CoA. Acetoacetyl-CoA is cleaved to two acetyl-CoA, which enter the TCA cycle and are oxidized.

Although fatty acid oxidation is usually the major source of ketone bodies, they also can be generated from the catabolism of certain amino acids: leucine, isoleucine, lysine, tryptophan, phenylalanine, and tyrosine. These amino acids are called ketogenic amino acids because their carbon skeleton is catabolized to acetyl-CoA or acetoacetyl-CoA, which may enter the pathway of ketone body synthesis in liver. Leucine and isoleucine also form acetyl-CoA and acetoacetyl-CoA in other tissues as well as the liver. Acetoacetate can be activated to acetoacetyl-CoA in the cytosol by an enzyme similar to the acyl-CoA synthetases. This acetoacetyl-CoA can be used directly in cholesterol synthesis. It also can be cleaved to two molecules of acetyl-CoA by a cytosolic thiolase. Cytosolic acetyl-CoA is required for processes such as acetylcholine synthesis in neuronal cells.

IV. THE ROLE OF FATTY ACIDS AND KETONE BODIES IN FUEL HOMEOSTASIS Fatty acids are used as fuels whenever fatty acid levels are elevated in the blood— that is, during fasting and starvation; because of a high-fat, low-carbohydrate diet; or during long-term low- to mild-intensity exercise. Under these conditions, a decrease in insulin and increased levels of glucagon, epinephrine, or other hormones stimulate adipose tissue lipolysis. Fatty acids begin to increase in the blood approximately 3 to 4 hours after a meal and progressively increase with time of fasting up to approximately 2 to 3 days (Fig. 23.20). In the liver, the rate of ketone body synthesis increases as the supply of fatty acids increases. However, the blood level of ketone bodies continues to increase, presumably because their utilization by skeletal muscles decreases. After 2 to 3 days of starvation, ketone bodies rise to a level in the blood that enables them to enter brain cells, where they are oxidized, thereby reducing the amount of glucose required by the brain. During prolonged fasting, they may supply as much as two-thirds of the energy requirements of the brain. The reduction in glucose requirements spares skeletal muscle protein, which is a major source of amino acid precursors for hepatic glucose synthesis from gluconeogenesis.

A. Preferential Use of Fatty Acids As fatty acids increase in the blood, they are used by skeletal muscles and certain other tissues in preference to glucose. Fatty acid oxidation generates NADH and FAD(2H) through both ␤-oxidation and the TCA cycle, resulting in relatively high NADH/ NAD ratios, acetyl-CoA concentrations, and ATP/ADP or ATP/AMP levels. In skeletal muscles, the AMP-PK (see Section I.E) adjusts the concentration of malonyl-CoA so that CPTI and ␤-oxidation operate at a rate that is able to sustain ATP homeostasis. With adequate levels of ATP obtained from fatty acid (or ketone body) oxidation, the rate of glycolysis is decreased. The activity of the regulatory enzymes in glycolysis and the TCA cycle (pyruvate dehydrogenase and phosphofructokinase-1 [PFK-1])

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Blood glucose and ketones (mmol/L)

6.0 ␤-Hydroxybutyrate

5.0

Glucose

4.0 3.0 2.0

Free fatty acids

1.0

Acetoacetate

0 0

10

20

30

40

Days of fasting

FIG. 23.20. Levels of ketone bodies in the blood at various times during fasting. Glucose levels remain relatively constant, as do levels of fatty acids. Ketone body levels, however, increase markedly, rising to levels at which they can be used by the brain and other nervous tissue. (From Cahill GF Jr, Aoki TT. How metabolism affects clinical problems. Med Times. 1970;98[10]:106.)

are decreased by the changes in concentration of their allosteric regulators (ADP—an activator of PDH—decreases in concentration; NADH and acetyl-CoA—inhibitors of PDH—are increased in concentration under these conditions; and ATP and citrate— inhibitors of PFK-1—are increased in concentration). As a consequence, glucose 6-phosphate (glucose 6-P) accumulates. Glucose 6-P inhibits hexokinase, thereby decreasing the rate of entry of glucose into glycolysis and its uptake from the blood. In skeletal muscles, this pattern of fuel metabolism is facilitated by the decrease in insulin concentration (see Chapter 36). Preferential use of fatty acids does not, however, restrict the ability of glycolysis to respond to an increase in AMP or ADP levels, such as what might occur during exercise or oxygen limitation.

B. Tissues that Use Ketone Bodies Skeletal muscles, the heart, the liver, and many other tissues use fatty acids as their major fuel during fasting and under other conditions that increase fatty acids in the blood. However, several other tissues (or cell types), such as the brain, use ketone bodies to a greater extent. For example, cells of the intestinal mucosa, which transport fatty acids from the intestine to the blood, use ketone bodies and amino acids rather than fatty acids during starvation. Adipocytes, which store fatty acids in triacylglycerols, do not use fatty acids as a fuel during fasting but can use ketone bodies. Ketone bodies cross the placenta and can be used by the fetus. Almost all tissues and cell types, with the exception of liver and red blood cells, are able to use ketone bodies as fuel.

431

Children are more prone to ketosis than adults are, because their bodies enter the fasting state more rapidly. Their bodies use more energy per unit mass (because their muscle-to-adipose tissue ratio is higher), and liver glycogen stores are depleted faster (the ratio of their brain mass to liver mass is higher). In children, blood ketone body levels reach 2 mM in 24 hours; in adults, it takes more than 3 days to reach this level. Mild pediatric infections that cause anorexia and vomiting are the most common causes of ketosis in children. Mild ketosis is observed in children after prolonged exercise, perhaps attributable to an abrupt decrease in muscular use of fatty acids liberated during exercise. The liver then oxidizes these fatty acids and produces ketone bodies.

The level of total ketone bodies in Di Abietes’ blood greatly exceeds normal fasting levels and the mild ketosis produced during exercise. In a person on a normal mealtime schedule, total blood ketone bodies rarely exceed 0.2 mM. During prolonged fasting, they may rise to 4 to 5 mM. Levels 7 mM are considered evidence of ketoacidosis, because the acid produced must reach this level to exceed the bicarbonate buffer system in the blood and compensatory respiration (Kussmaul respiration) (see Chapter 4).

C. Regulation of Ketone Body Synthesis Several events, in addition to the increased supply of fatty acids from adipose triacylglycerols, promote hepatic ketone body synthesis during fasting. The decreased insulin/glucagon ratio results in inhibition of acetyl-CoA carboxylase and decreased malonyl-CoA levels, which activates CPTI, thereby allowing fatty acyl-CoA to enter the pathway of ␤-oxidation (Fig. 23.21). When oxidation of fatty acyl-CoA to acetylCoA generates enough NADH and FAD(2H) to supply the ATP needs of the liver, acetyl-CoA is diverted from the TCA cycle into ketogenesis and oxaloacetate in the TCA cycle is diverted toward malate and into glucose synthesis (gluconeogenesis). This pattern is regulated by the NADH/NAD ratio, which is relatively high during ␤-oxidation. As the length of time of fasting continues, increased transcription of

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Why can red blood cells not use ketone bodies for energy?

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

Red blood cells lack mitochondria, which is the site of ketone body use.

1

Fatty acids

2

CPTI ( Malonyl CoA) FA-carnitine

FA-CoA

–

FAD (2H)

3

ATP

␤-Oxidation

NADH

5 Acetyl CoA

4

Acetoacetyl CoA

Ketone bodies

Oxaloacetate NADH NAD+

Citrate Malate

Gluconeogenesis TCA cycle

FIG. 23.21. Regulation of ketone body synthesis. (1) The supply of fatty acids is increased. (2) The malonyl-CoA inhibition of CPTI is lifted by inactivation of acetyl-CoA carboxylase. (3) ␤-Oxidation supplies NADH and FAD(2H), which are used by the electron-transport chain for oxidative phosphorylation. As ATP levels increase, less NADH is oxidized, and the NADH/NAD ratio is increased. (4) Oxaloacetate is converted into malate because of the high NADH levels, and the malate enters the cytoplasm for gluconeogenesis. (5) Acetyl-CoA is diverted from the TCA cycle into ketogenesis, in part because of low oxaloacetate levels, which reduces the rate of the citrate synthase reaction.

the gene for mitochondrial HMG-CoA synthase facilitates high rates of ketone body production. Although the liver has been described as “altruistic” because it provides ketone bodies for other tissues, it is simply getting rid of fuel that it does not need.

CLINICAL COMMENTS Otto Shape. As Otto Shape runs, he increases the rate at which his muscles oxidize all fuels. The increased rate of ATP use stimulates the electrontransport chain, which oxidizes NADH and FAD(2H) much faster, thereby increasing the rate at which fatty acids are oxidized. During exercise, he also uses muscle glycogen stores, which contribute glucose to glycolysis. In some of the fibers, the glucose is used anaerobically, thereby producing lactate. Some of the lactate will be used by his heart, and some will be taken up by the liver to be converted to glucose. As he trains, he increases his mitochondrial capacity, as well as his oxygen delivery, resulting in an increased ability to oxidize fatty acids and ketone bodies. As he runs, he increases fatty acid release from adipose tissue triacylglycerols. In the liver, fatty acids are being converted to ketone bodies, providing his muscles with another fuel. As a consequence, he experiences mild ketosis after his 12-mile run. More than 25 enzymes and specific transport proteins participate in mitochondrial fatty acid metabolism. At least 15 of these have been implicated in inherited diseases in the human.

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Lofata Burne. Recently, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, the cause of Lofata Burne’s problems, has emerged as one of the most common of the inborn errors of metabolism, with a carrier frequency ranging from 1 in 40 in northern European populations to 1 in 100 in Asians. Overall, the predicted disease frequency for MCAD deficiency is 1 in 15,000 persons.

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MCAD deficiency is an autosomal recessive disorder caused by the substitution of a T for an A at position 985 of the MCAD gene. This mutation causes a lysine to replace a glutamate residue in the protein, resulting in the production of an unstable dehydrogenase. The most frequent manifestation of MCAD deficiency is intermittent hypoketotic hypoglycemia during fasting (low levels of ketone bodies and low levels of glucose in the blood). Fatty acids normally would be oxidized to CO2 and H2O under these conditions. In MCAD deficiency, however, fatty acids are oxidized only until they reach medium-chain length. As a result, the body must rely more on oxidation of blood glucose to meet its energy needs. However, hepatic gluconeogenesis appears to be impaired in MCAD. Inhibition of gluconeogenesis may be caused by the lack of hepatic fatty acid oxidation to supply the energy required for gluconeogenesis, or by the accumulation of unoxidized fatty acid metabolites that inhibit gluconeogenic enzymes. As a consequence, liver glycogen stores are depleted more rapidly, and hypoglycemia results. The decrease in hepatic fatty acid oxidation results in less acetyl-CoA for ketone body synthesis and, consequently, a hypoketotic hypoglycemia develops. Some of the symptoms once ascribed to hypoglycemia are now believed to be caused by the accumulation of toxic fatty acid intermediates, especially in patients with only mild reductions in blood glucose levels. Lofata Burne’s mild elevation in the blood of liver transaminases may reflect an infiltration of her liver cells with unoxidized medium-chain fatty acids. The management of MCAD-deficient patients includes the intake of a relatively high-carbohydrate diet and the avoidance of prolonged fasting.

433

LCAD deficiency, a reduced activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase, is a rare disorder with a poor outcome. LCAD activity is contained within the mitochondrial trifunctional protein, which is a hetero-octamer of four ␣- and four ␤-subunits. This complex catalyzes three steps in the oxidation of long-chain fatty acids: the long-chain enoyl-CoA hydratase activity, the LCAD activity, and the long-chain ketothiolase activity. Mutations in LCAD are located in the -subunit. Although an intact protein is produced, LCAD activity is significantly reduced, and the other two activities of the complex are reduced approximately 40%. Generalized mitochondrial trifunctional protein deficiency, with a significant reduction in all three activities, can result from mutations in either the ␣- or ␤-chain. Patients with generalized trifunctional protein deficiency exhibit a wide clinical spectrum of disease—from severe to mild.

Di Abietes. A 26-year-old woman with type 1 diabetes mellitus, was admitted to the hospital in diabetic ketoacidosis. In this complication of diabetes mellitus, an acute deficiency of insulin, coupled with a relative excess of glucagon, results in a rapid mobilization of fuel stores from muscle (amino acids) and adipose tissue (fatty acids). Some of the amino acids are converted to glucose, and fatty acids are converted to ketones (acetoacetate, ␤-hydroxybutyrate, and acetone). The high glucagon:insulin ratio promotes the hepatic production of ketones. In response to the metabolic “stress,” the levels of insulin-antagonistic hormones, such as catecholamines, glucocorticoids, and growth hormone are increased in the blood. The insulin deficiency further reduces the peripheral use of glucose and ketones. Because of this interrelated dysmetabolism, plasma glucose levels reach 500 mg/dL (27.8 mmol/L) or more (normal fasting levels are 70 to 100 mg/dL or 3.9 to 5.5 mmol/L), and plasma ketones rise to levels of 8 to 15 mmol/L or more (normal is in the range of 0.2 to 2 mmol/L, depending on the fed state of the individual). The increased glucose presented to the renal glomeruli induces an osmotic diuresis, which further depletes intravascular volume, further reducing the renal excretion of hydrogen ions and glucose. As a result, the metabolic acidosis worsens, and the hyperosmolarity of the blood increases, at times exceeding 330 mOsm/kg (normal is in the range of 285 to 295 mOsm/kg). The severity of the hyperosmolar state correlates closely with the degree of central nervous system dysfunction and may end in coma and even death if left untreated. BIOCHEMICAL COMMENTS Acetylation and Regulation of Fatty Acid Oxidation. Previously in Chapter 16, acetylation of histones was described as a mechanism of regulating gene expression. Histone acetyltransferases (HATs) would catalyze histone acetylation on lysine side chains, leading to histone dissociation from the DNA. This freed the DNA to bind factors important for transcription to occur. Recent work has indicated that acetylation of long-chain

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acyl-CoA dehydrogenase (LCAD) regulates the activity of the enzyme and offers more complexity to the regulation of fatty acid oxidation. Acetylation of LCAD reduces enzymatic activity, whereas deacetylation will restore activity. A family of proteins known as the sirtuins (SIRT) are NAD-dependent protein deacetylases. There are seven forms in humans, designated SIRT1 through SIRT7, all of which affect certain areas of metabolism. The sirtuins catalyze the reaction shown in Figure 23.22, in which NAD is split into nicotinamide and 2-O-acetyl-ADPribose, and the protein target is deacetylated (the acetate group is transferred from the target to the 2 -hydroxy group of the ribose attached to the nicotinamide in NAD). Sirtuin 3 is localized to the mitochondrial matrix, and recent evidence indicates that it is a key regulator for fatty acid oxidation within the mitochondria. Using mice as a model system, it was demonstrated that SIRT3 expression is upregulated during fasting in the liver. A primary target of SIRT3 deacetylation activity is LCAD, which is hyperacetylated on lysine-42. When hyperacetylated, LCAD activity is

O

O C

N

NH2

C

NH2

O –

O

P

O

CH2

N

N+

O

nicotinamide +

O –

O

HO

O

P

O

OH

CH2

OH

O

NH2 N

O

N O

–

O

P

O

CH2

N

O

O

OH

C

CH3

N

O –

O

HO

OH

P

O

CH2

A

O

O

A = Adenine

H2O HO

OH

2-O-acetyl adenosine diphosphate ribose

NH O

C

H

C

NH

(CH2)4

H

O

N

C

NH

acetyl lysine substrate

CH3

O

C

H

C

H (CH2)4

NH

N

H

H

+

Deacetylated substrate

FIG. 23.22. Generalized sirtuin reaction.

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435

reduced. Because LCAD will initiate fatty acid oxidation, regulating LCAD activity will regulate overall use of long-chain fatty acids by liver mitochondria. Fasting will upregulate the sirtuins leading to activation of LCAD, and increased fatty acid oxidation by the liver. Mice lacking SIRT3 activity were deficient in fatty acid oxidation during fasting conditions, accumulating long-chain intermediates and triglyceride in the liver. Human LCAD was also demonstrated to be acetylated and SIRT3 would deacetylate the enzyme in a test tube. The recent finding of acetylation as a regulatory tool for fatty acid oxidation provides another avenue for complex regulation between various aspects of metabolism and provides fertile ground for further research in this area. Key Concepts • • • • • • • •

• • • • • •

Fatty acids are a major fuel for humans. During overnight fasting, fatty acids become the major fuel for cardiac muscle, skeletal muscle, and liver. The nervous system has a limited ability to use fatty acids directly as fuel. The liver converts fatty acids to ketone bodies, which can be used by the nervous system as a fuel during prolonged periods of fasting. Fatty acids are released from adipose tissue triacylglycerols under appropriate hormonal stimulation. In cells, fatty acids are activated to fatty acyl-CoA derivatives by acyl-CoA synthetases. Acyl-CoAs are transported into the mitochondria for oxidation via carnitine. ATP is generated from fatty acids by the pathway of ␤-oxidation. In ␤-oxidation, the fatty acyl group is sequentially oxidized to yield FAD(2H), NADH, and acetylCoA. Although the reactions are similar, enzyme specificity is determined by the acyl chain length of the substrate. Unsaturated and odd-chain-length fatty acids require additional reactions for their metabolism. ␤-Oxidation is regulated by the levels of FAD(2H), NADH, and acetyl-CoA. The entry of fatty acids into mitochondria is regulated by malonyl-CoA levels. Alternative pathways for very long-chain and branched-chain fatty acid oxidation occur within peroxisomes. If ␤-oxidation is impaired, other pathways of oxidation will be used, such as ␣- and ␻-oxidation. Table 23.2 summarizes the diseases discussed in this chapter.

Table 23.2

Diseases Discussed in Chapter 23

Disease or Disorder

Environmental or Genetic

Obesity

Both

MCAD deficiency

Genetic

Type 1 diabetes

Environmental

Carnitine deficiency

Both

Zellweger syndrome

Genetic

LCAD deficiency

Genetic

Jamaican vomiting disorder

Environmental

Comments The contribution of fatty acids to overall energy metabolism and energy storage. Lack of medium-chain acyl-CoA dehydrogenase activity, leading to hypoglycemia and reduced ketone body formation under fasting conditions. Ketoacidosis; overproduction of ketone bodies because of lack of insulin and metabolic dysregulation in the liver. A primary carnitine deficiency is the lack of a membrane transporter for carnitine; a secondary carnitine deficiency is because of other metabolic disorders. A defect in peroxisome biogenesis, leading to a lack of peroxisomes, inability to synthesize plasmalogens, or oxidize very long-chain fatty acids. A lack of long-chain acyl-CoA dehydrogenase activity, leading to hypoglycemia. Inhibition of carnitine activity by hypoglycin; can lead to death because of severe hypoglycemia.

MCAD, medium acyl-CoA dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase.

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REVIEW QUESTIONS—CHAPTER 23 1.

2.

A lack of the enzyme ETF-QO oxidoreductase leads to death. This is caused by which of the following reasons? A. The energy yield from glucose use is dramatically reduced. B. The energy yield from alcohol use is dramatically reduced. C. The energy yield from ketone body use is dramatically reduced. D. The energy yield from fatty acid use is dramatically reduced. E. The energy yield from glycogen use is dramatically reduced.

C. Oxidation, hydration, reduction, carbon–carbon bond breaking D. Oxidation, dehydration, reduction, oxidation, carbon– carbon bond breaking E. Reduction, hydration, oxidation, carbon–carbon bond breaking 4.

The ATP yield from the complete oxidation of 1 mol of a C18:0 fatty acid to carbon dioxide and water would be closest to which one of the following? A. 105 B. 115 C. 120 D. 125 E. 130

A. B. C. D.

Fatty acid oxidation is increased. Ketone body synthesis is increased. Blood glucose levels are increased. Levels of dicarboxylic acids in the blood are increased. E. Levels of very long-chain fatty acids in the blood are increased. 5.

3.

The oxidation of fatty acids is best described by which of the following sets of reactions? A. Oxidation, hydration, oxidation, carbon–carbon bond breaking B. Oxidation, dehydration, oxidation, carbon–carbon bond breaking

Lieberman_Ch23.indd 436

An individual with a deficiency of an enzyme in the pathway for carnitine synthesis is not eating adequate amounts of carnitine in the diet. Which of the following effects would you expect during fasting as compared with an individual with an adequate intake and synthesis of carnitine?

In which one of the following periods will fatty acids be the major source of fuel for the tissues of the body? A. B. C. D. E.

Immediately after breakfast Minutes after a snack Immediately after dinner While running the first mile of a marathon While running the last mile of a marathon

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24

Oxygen Toxicity and Free Radical Injury

O2 is both essential to human life and toxic. We are dependent on O2 for oxidation reactions in the pathways of adenosine triphosphate (ATP) generation, detoxification, and biosynthesis. However, when O2 accepts single electrons, it is transformed into highly reactive oxygen radicals that damage cellular lipids, proteins, and DNA. Damage by reactive oxygen radicals contributes to cellular death and degeneration in a wide range of diseases (Table 24.1). Radicals are compounds that contain a single electron, usually at an outside orbital. Oxygen is a biradical, a molecule that has two unpaired electrons in separate orbitals (Fig. 24.1). Through several enzymatic and nonenzymatic processes that routinely occur in cells, O2 accepts single electrons to form reactive oxygen species (ROS). ROS are highly reactive oxygen radicals or compounds that are readily converted in cells to these reactive radicals. The ROS formed by reduction of O2 are the radical superoxide (O2ⴚ), the nonradical hydrogen peroxide (H2O2), and the hydroxyl radical (OH•). ROS may be generated nonenzymatically or enzymatically as accidental by-products or major products of reactions. Superoxide may be generated nonenzymatically from coenzyme Q (CoQ) or from metal-containing enzymes (e.g., cytochrome P450, xanthine oxidase, and NADPH oxidase). The highly toxic hydroxyl radical is formed nonenzymatically from superoxide in the presence of Fe2ⴙ or Cuⴙ by the Fenton reaction and from hydrogen peroxide in the Haber-Weiss reaction. Oxygen radicals and their derivatives can be deadly to cells. The hydroxyl radical causes oxidative damage to proteins and DNA. It also forms lipid peroxides and malondialdehyde from membrane lipids containing polyunsaturated fatty acids. In some cases, free radical damage is the direct cause of a disease state (e.g., tissue damage initiated by exposure to ionizing radiation). In neurodegenerative diseases, such as Parkinson disease, or in ischemia-reperfusion injury, ROS may perpetuate the cellular damage caused by another process. Oxygen radicals are joined in their destructive damage by the free radical nitric oxide (NO) and the ROS hypochlorous acid (HOCl). NO combines with O2 or superoxide to form reactive nitrogen–oxygen species (RNOS), such as the nonradical peroxynitrite or the radical nitrogen dioxide. RNOS are Table 24.1

Oxygen is a biradical O2 that forms –

ROS

O2 H2O2 OH•

FIG. 24.1. O2 is a biradical. It has two antibonding electrons with parallel spins, (parallel arrows). It has a tendency to form toxic ROS, such as superoxide (O2⫺), the nonradical hydrogen peroxide (H2O2), and the hydroxyl radical (OH•).

Some Disease States Associated with Free Radical Injury

Atherogenesis Emphysema bronchitis Duchenne-type muscular dystrophy Pregnancy/preeclampsia Retrolental fibroplasia Cervical cancer Alcohol-induced liver disease Hemodialysis Diabetes Acute renal failure Aging

Cerebrovascular disorders Ischemia/reperfusion injury Neurodegenerative disorders Amyotrophic lateral sclerosis (Lou Gehrig disease) Alzheimer disease Down syndrome Ischemia-reperfusion injury following stroke OXPHOS diseases (mitochondrial DNA disorders) Multiple sclerosis Parkinson disease

437

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present in the environment (e.g., cigarette smoke) and are generated in cells. During phagocytosis of invading microorganisms, cells of the immune system produce O2⫺, HOCl, and NO through the actions of NADPH oxidase, myeloperoxidase, and inducible nitric oxide synthase, respectively. In addition to killing phagocytosed invading microorganisms, these toxic metabolites may damage surrounding tissue components. Cells protect themselves against damage by ROS and other radicals through repair processes, compartmentalization of free radical production, defense enzymes, and endogenous and exogenous antioxidants (free radical scavengers). The defense enzyme superoxide dismutase (SOD) removes the superoxide free radical. Catalase and glutathione peroxidase remove hydrogen peroxide and lipid peroxides. Vitamin E, vitamin C, and plant flavonoids act as antioxidants. Oxidative stress occurs when the rate of ROS generation exceeds the capacity of the cell for their removal (Fig. 24.2).

Cell defenses: antioxidants enzymes ROS RNOS

Oxidative stress

FIG. 24.2. Oxidative stress. Oxidative stress occurs when the rate of ROS and RNOS production overbalances the rate of their removal by cellular defense mechanisms. These defense mechanisms include a number of enzymes and antioxidants. Antioxidants usually react nonenzymatically with ROS.

The basal ganglia are part of a neuronal feedback loop that modulates and integrates the flow of information from the cerebral cortex to the motor neurons of the spinal cord. The neostriatum is the major input structure from the cerebral cortex. The substantia nigra pars compacta consists of neurons that provide integrative input to the neostriatum through pigmented neurons that use dopamine as a neurotransmitter (the nigrastriatal pathway). Integrated information feeds back to the basal ganglia and to the cerebral cortex to control voluntary movement. In Parkinson disease, a decrease in the amount of dopamine reaching the basal ganglia results in the movement disorder. In ventricular fibrillation, rapid premature beats from an irritative focus in ventricular muscle occur in runs of varying duration. Persistent fibrillation compromises cardiac output, leading to death. This arrythmia can result from severe ischemia (lack of blood flow) in the ventricular muscle of the heart caused by clots forming at the site of a ruptured atherosclerotic plaque. However, Cora Nari’s rapid beats began during the infusion of tissue plasminogen activator (TPA) as the clot was lysed. Thus, they probably resulted from reperfusing a previously ischemic area of her heart with oxygenated blood. This phenomenon is known as ischemia-reperfusion injury, and it is caused by cytotoxic ROS derived from oxygen in the blood that reperfuses previously hypoxic cells. Ischemic-reperfusion injury also may occur when tissue oxygenation is interrupted during surgery or transplantation.

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THE WAITING ROOM Two years ago, Les Dopaman, a 62-year-old man, noted an increasing tremor of his right hand when sitting quietly (resting tremor). The tremor disappeared if he actively used this hand to do purposeful movement. As this symptom progressed, he also complained of stiffness in his muscles that slowed his movements (bradykinesia). His wife noticed a change in his gait; he had begun taking short, shuffling steps and leaned forward as he walked (postural imbalance). He often appeared to be staring ahead with a rather immobile facial expression. She noted a tremor of his eyelids when he was asleep and, recently, a tremor of his legs when he was at rest. Because of these progressive symptoms and some subtle personality changes (anxiety and emotional lability), she convinced Les to see their family doctor. The doctor suspected that her patient probably had primary or idiopathic parkinsonism (Parkinson disease) and referred Mr. Dopaman to a neurologist. In Parkinson disease, neurons of the substantia nigra pars compacta, containing the pigment melanin and the neurotransmitter dopamine, degenerate. Cora Nari had done well since the successful lysis of blood clots in her coronary arteries with the use of intravenous recombinant tissue plasminogen activator (TPA) (see Chapters 19 and 21). This therapy quickly relieved the crushing chest pain (angina) she experienced when she won the lottery. At her first office visit after her discharge from the hospital, Cora’s cardiologist told her she had developed multiple premature contractions of the ventricular muscle of her heart as the clots were being lysed. This process could have led to a lifethreatening arrhythmia known as ventricular fibrillation. However, Cora’s arrhythmia responded quickly to pharmacologic suppression and did not recur during the remainder of her hospitalization.

I.

O2 AND THE GENERATION OF REACTIVE OXYGEN SPECIES

The generation of reactive oxygen species (ROS) from O2 in our cells is a natural, everyday occurrence. ROS are formed as accidental products of nonenzymatic and enzymatic reactions. Occasionally, they are deliberately synthesized

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CHAPTER 24 ■ OXYGEN TOXICITY AND FREE RADICAL INJURY

in enzyme-catalyzed reactions. Ultraviolet radiation and pollutants in the air can increase formation of toxic oxygen-containing compounds.

A. The Radical Nature of O2 A radical, by definition, is a molecule that has a single unpaired electron in an orbital. A free radical is a radical that is capable of independent existence. (Radicals formed in an enzyme active site during a reaction, for example, are not considered free radicals unless they can dissociate from the protein to interact with other molecules.) Radicals are highly reactive and initiate chain reactions by extracting an electron from a neighboring molecule to complete their own orbitals. Although the transition metals (e.g., Fe, Cu, Mo) have single electrons in orbitals, they are not usually considered free radicals because they are relatively stable, do not initiate chain reactions, and are bound to proteins in the cell. The oxygen molecule is a biradical, which means it has two single electrons in different orbitals. These electrons cannot both travel in the same orbital because they have parallel spins (they spin in the same direction). Although oxygen is very reactive from a thermodynamic standpoint, its single electrons cannot react rapidly with the paired electrons found in the covalent bonds of organic molecules. As a consequence, O2 reacts slowly through the acceptance of single electrons in reactions that require a catalyst (such as a metal-containing enzyme). Because the two unpaired electrons in oxygen have the same (parallel) spin, they are called antibonding electrons. In contrast, carbon–carbon and carbon–hydrogen bonds each contain two electrons, which have antiparallel spins and form a thermodynamically stable pair. As a consequence, O2 cannot readily oxidize a covalent bond because one of its electrons would have to flip its spin around to make new pairs. The difficulty in changing spins is called spin restriction. Without spin restriction, organic life forms could not have developed in the oxygen atmosphere on earth because they would be spontaneously oxidized by O2. O2 is capable of accepting a total of four electrons, which reduces it to water (Fig. 24.3). When O2 accepts one electron, superoxide is formed. Superoxide is still a radical because it has one unpaired electron remaining. This reaction is not thermodynamically favorable and requires a moderately strong reducing agent that can donate single electrons (e.g., the radical form of coenzyme Q [CoQH•] in the electron-transport chain). When superoxide accepts an electron, it is reduced to hydrogen peroxide, which is not a radical. The hydroxyl radical is formed in the next one-electron reduction step in the reduction sequence. Finally, acceptance of the last electron reduces the hydroxyl radical to H2O.

B. Characteristics of Reactive Oxygen Species ROS are oxygen-containing compounds that are highly reactive free radicals or compounds that are readily converted to these oxygen free radicals in the cell. The major oxygen metabolites produced by one-electron reduction of oxygen (superoxide, hydrogen peroxide, and the hydroxyl radical) are classified as ROS (Table 24.2). Reactive free radicals extract electrons (usually as hydrogen atoms) from other compounds to complete their own orbitals, thereby initiating free radical chain reactions. The hydroxyl radical is probably the most potent of the ROS. It initiates chain reactions that form lipid peroxides and organic radicals and adds directly to compounds. The superoxide anion is also highly reactive, but it has limited lipid solubility and cannot diffuse far. However, it can generate the more reactive hydroxyl and hydroperoxy radicals by reacting nonenzymatically with hydrogen peroxide in the Haber-Weiss reaction (Fig. 24.4). Hydrogen peroxide is not actually a radical; it is a weak oxidizing agent that is classified as a ROS because it can generate the hydroxyl radical (OH•). Transition metals, such as Fe2⫹ or Cu⫹, catalyze formation of the hydroxyl radical from

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439

Catecholamine (epinephrine, norepinephrine, dopamine) measurements use either serum or a 24-hour urine collection as samples for assay. After appropriate removal of cells and/or particulate matter, the sample is placed over an ion-exchange high-pressure liquid chromatography (HPLC) column and the eluate from the column is analyzed by sensitive electrochemical detection. Through comparison with retention times of standard catecholamines on the column, the various catecholamine species can be clearly resolved from each other. The electrochemical detection uses electrodes that are oxidized by the samples, and the amplitude current that is generated via the redox reaction allows one to determine the concentration of catecholamines in the specimen.

O2 Oxygen e– O2 – Superoxide e–, 2H+

H2O2 Hydrogen peroxide e–, H+ H2O + OH• Hydroxyl radical e–, H+ H2O

FIG. 24.3. Reduction of oxygen by four one-electron steps. The four one-electron reduction steps for O2 progressively generate superoxide, hydrogen peroxide (H2O2), and the hydroxyl radical plus water. Superoxide is sometimes written O2⫺ to better illustrate its single unpaired electron. H2O2, the halfreduced form of O2, has accepted two electrons and is therefore not an oxygen radical.

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Table 24.2

Reactive Oxygen Species and Reactive Nitrogen–Oxygen Species

Reactive Species

Properties ⫺

Superoxide anion (O2 ) Hydrogen peroxide (H2O2) Hydroxyl radical (OH•) RO•, R•, R–S organic radicals RCOO• peroxyl radical Hypochlorous acid (HOCl)

O2↓↑ Singlet oxygen

Nitric oxide (NO)

Peroxynitrite (ONOO⫺) The Haber-Weiss reaction –

+

O2

H2O2

Superoxide

Hydrogen peroxide H+

O2

+

•OH

+

H2O

Oxygen

Water

Hydroxyl radical

The Fenton reaction H2O2 Hydrogen peroxide Fe2+ Fe3+ •OH Hydroxyl radical

+

Produced by the electron-transport chain and at other sites. Cannot diffuse far from the site of origin. Generates other ROS. Not a free radical but can generate free radicals by reaction with a transition metal (e.g., Fe2⫹). Can diffuse into and through cell membranes. The most reactive species in attacking biologic molecules. Produced from H2O2 in the Fenton reaction in the presence of Fe2⫹ or Cu⫹. Organic free radicals (R denotes the remainder of the compound). Produced from ROH, RH (e.g., at the carbon of a double bond in a fatty acid) or RSH OH• attack. An organic peroxyl radical, such as occurs during lipid degradation (also denoted LOO•). Produced in neutrophils during the respiratory burst to destroy invading organisms. Toxicity is through halogenation and oxidation reactions. Attacking species is OCl⫺. Oxygen with antiparallel spins. Produced at high oxygen tensions from absorption of UV light. Decays so fast that it is probably not a significant in vivo source of toxicity. RNOS. A free radical produced endogenously by nitric oxide synthase. Binds to metal ions. Combines with O2 or other oxygen-containing radicals to produce additional RNOS. RNOS. A strong oxidizing agent that is not a free radical. It can generate nitrogen dioxide (NO2), which is a radical.

OH– Hydroxide ion

hydrogen peroxide in the nonenzymatic Fenton reaction (see Fig. 24.4.). Because hydrogen peroxide is lipid soluble, it can diffuse through membranes and generate OH• at localized Fe2⫹- or Cu⫹-containing sites, such as the electron-transport chain within the mitochondria. Hydrogen peroxide is also the precursor of hypochlorous acid (HOCl), a powerful oxidizing agent that is produced endogenously and enzymatically by phagocytic cells. To decrease occurrence of the Fenton reaction, accessibility to transition metals, such as Fe2⫹ and Cu⫹, are highly restricted in cells or in the body as a whole. Events that release iron from cellular storage sites, such as a crushing injury, are associated with increased free radical injury. Organic radicals are generated when superoxide or the hydroxyl radical indiscriminately extract electrons from other molecules. Organic peroxy radicals are intermediates of chain reactions, such as lipid peroxidation. Other organic radicals, such as the ethoxy radical, are intermediates of enzymatic reactions that escape into solution (see Table 24.2). An additional group of oxygen-containing radicals, termed reactive nitrogen– oxygen species (RNOS), contains nitrogen as well as oxygen. These radicals are derived principally from the free radical nitric oxide (NO), which is produced endogenously by the enzyme nitric oxide synthase. Nitric oxide combines with O2 or superoxide to produce additional RNOS.

C. Major Sources of Primary Reactive Oxygen Species in the Cell FIG. 24.4. Generation of the hydroxyl radical by the nonenzymatic Haber-Weiss and Fenton reactions. In the simplified versions of these reactions shown here, the transfer of single electrons generates the hydroxyl radical. ROS are shown in the boxes. In addition to Fe2⫹, Cu⫹ and many other metals can also serve as single-electron donors in the Fenton reaction.

Lieberman_Ch24.indd 440

ROS are constantly being formed in the cell; approximately 3% to 5% of the oxygen we consume is converted to oxygen free radicals. Some are produced as accidental by-products of normal enzymatic reactions that escape from the active site of metal-containing enzymes during oxidation reactions. Others, such as hydrogen peroxide, are physiologic products of oxidases in peroxisomes. Deliberate production of toxic free radicals occur during the inflammatory response. Drugs, natural radiation, air pollutants, and other chemicals also can increase formation of free radicals in cells.

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CHAPTER 24 ■ OXYGEN TOXICITY AND FREE RADICAL INJURY

1.

One of the major sites of superoxide generation is coenzyme Q (CoQ) in the mitochondrial electron-transport chain (Fig. 24.5). The one-electron reduced form of CoQ (CoQH•) is free within the membrane and can accidentally transfer an electron to dissolved O2, thereby forming superoxide. In contrast, when O2 binds to cytochrome oxidase and accepts electrons, none of the O2 radical intermediates are released from the enzyme, and no ROS are generated.

O2

2.

O2

OXIDASES, OXYGENASES, AND PEROXIDASES

Most of the oxidases, peroxidases, and oxygenases in the cell bind O2 and transfer single electrons to it via a metal. Free radical intermediates of these reactions may be accidentally released before the reduction is complete. Cytochrome P450 enzymes are a major source of free radicals “leaked” from reactions. Because these enzymes catalyze reactions in which single electrons are transferred to O2 and an organic substrate, the possibility of accidentally generating and releasing free radical intermediates is high (see Chapters 19 and 25). Induction of P450 enzymes by alcohol, drugs, or chemical toxicants leads to increased cellular injury. As an example, carbon tetrachloride (CCl4), which is used as a solvent in the dry cleaning industry, is converted by cytochrome P450 to a highly reactive free radical that has caused hepatocellular necrosis in workers in that industry. When the enzyme-bound CCl4 accepts an electron, it dissociates into CCl3• and Cl. The CCl3• radical, which cannot continue through the P450 reaction sequence, “leaks” from the enzyme active site and initiates chain reactions in the surrounding polyunsaturated lipids of the endoplasmic reticulum. These reactions spread into the plasma membrane and to proteins, eventually resulting in cell swelling, accumulation of lipids, and cell death. When substrates for cytochrome P450 enzymes are not present, its potential for destructive damage is diminished by repression of gene transcription. Hydrogen peroxide and lipid peroxides are generated enzymatically as major reaction products by several oxidases present in peroxisomes, mitochondria, and the endoplasmic reticulum. For example, monoamine oxidase, which oxidatively degrades the neurotransmitter dopamine, generates H2O2 at the mitochondrial membrane of certain neurons. Peroxisomal fatty acid oxidase generates H2O2 rather than FAD(2H) during the oxidation of very long-chain fatty acids (see Chapter 23). Xanthine oxidase, an enzyme of purine degradation that can reduce O2 to O2⫺or H2O2 in the cytosol, is thought to be a major contributor to ischemia-reperfusion injury, especially in intestinal mucosal and endothelial cells. Lipid peroxides are also formed enzymatically as intermediates in the pathways for synthesis of many eicosanoids, including leukotrienes and prostaglandins. 3.

NAD+

NADH

COENZYME Q GENERATES SUPEROXIDE

441

NADH dehydrogenase

FMN/Fe–S

CoQH•

CoQ

–

Fe–S Cytochrome b–c1, Fe-H Fe-H c O2 H2O

Fe-H–Cu Cytochrome aa3

FIG. 24.5. Generation of superoxide by CoQ in the electron-transport chain. In the process of transporting electrons to O2, some of the electrons escape when CoQH• accidentally interacts with O2 to form superoxide. Fe-H represents the Fe-heme center of the cytochromes. With insufficient oxygen, Cora Nari’s ischemic heart muscle mitochondria were unable to maintain cellular adenosine triphosphate (ATP) levels, resulting in high intracellular Na⫹ and Ca2⫹ levels. The reduced state of the electron carriers in the absence of oxygen and loss of mitochondrial ion gradients or membrane integrity leads to increased superoxide production once oxygen becomes available during reperfusion. The damage can be self-perpetuating, especially if iron bound to components of the electron-transport chain becomes available for the Fenton reaction or the mitochondrial permeability transition is activated.

IONIZING RADIATION

Cosmic rays that continuously bombard the earth, radioactive chemicals, and X-rays are forms of ionizing radiation. Ionizing radiation has a high enough energy level that it can split water into hydroxyl and hydrogen radicals, thus leading to radiation damage to the skin, mutations, cancer, and cell death (Fig. 24.6). It also may generate organic radicals through direct collision with organic cellular components.

II. OXYGEN RADICAL REACTIONS WITH CELLULAR COMPONENTS Oxygen radicals produce cellular dysfunction by reacting with lipids, proteins, carbohydrates, and DNA to extract electrons (summarized in Fig. 24.7). Evidence of free radical damage has been described in ⬎100 disease states. In some of these diseases, free radical damage is the primary cause of the disease; in others, it enhances complications of the disease.

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Les Dopaman, who is in the early stages of Parkinson disease, is treated with a monoamine oxidase B inhibitor. Monoamine oxidase is a copper-containing enzyme that inactivates dopamine in neurons, producing hydrogen peroxide (H2O2). The drug was originally administered to inhibit dopamine degradation. However, current theory suggests that the effectiveness of the drug is also related to a decrease of free radical formation within the cells of the basal ganglia. The dopaminergic neurons involved are particularly susceptible to the cytotoxic effects of ROS and reactive nitrogen– oxygen species (RNOS) that may arise from H2O2.

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H2O Ionizing radiation

h␯

•OH Hydroxyl radical

FIG. 24.6. radiation.

+

H• Hydrogen atom

Generation of free radicals from

Mitochondrial damage Membrane damage

The appearance of lipofuscin granules in many tissues increases during aging. The pigment lipofuscin (from the Greek lipos, for lipids, and the Latin fuscus, for dark) consists of a heterogeneous mixture of cross-linked polymerized lipids and protein formed by reactions between amino acid residues and lipid peroxidation products such as malondialdehyde. These cross-linked products are probably derived from peroxidatively damaged cell organelles that were autophagocytized by lysosomes but could not be digested. When these dark pigments appear on the skin of the hands in aged individuals, they are referred to as “liver spots,” a traditional hallmark of aging. In Les Dopaman and other patients with Parkinson disease, lipofuscin appears as Lewy bodies in degenerating neurons.

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SER RER

DNA damage

Production of ROS by xanthine oxidase in endothelial cells may be enhanced during ischemiareperfusion in Cora Nari’s heart. In undamaged tissues, xanthine oxidase exists as a dehydrogenase that uses NAD⫹ rather than O2 as an electron acceptor in the pathway for degradation of purines (hypoxanthine → xanthine → uric acid; see Chapter 41). When O2 levels decrease, phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) decreases, and degradation of ADP and adenine through xanthine oxidase increases. In the process, xanthine dehydrogenase is converted to an oxidase. As long as O2 levels are below the high K m of the enzyme for O2, little damage is done. However, during reperfusion, when O2 levels return to normal, xanthine oxidase generates H2O2 and O2⫺ at the site of injury.

Respiratory enzymes

Protein damage

Nucleus (DNA)

DNA

–

O2 OH•

H2O Na+

Cell swelling

Ca2+ Increased permeability Massive influx of Ca2+

Lipid peroxidation

FIG. 24.7. Free radical–mediated cellular injury. Superoxide and the hydroxyl radical initiate lipid peroxidation in the cellular, mitochondrial, nuclear, and endoplasmic reticulum membranes. The increase in cellular permeability results in an influx of Ca2⫹, which causes further mitochondrial damage. The cysteine sulfhydryl groups and other amino acid residues on proteins are oxidized and degraded. Nuclear and mitochondrial DNA can be oxidized, resulting in strand breaks and other types of damage. RNOS (NO, NO2, and peroxynitrite) have similar effects.

A. Membrane Attack: Formation of Lipid and Lipid Peroxy Radicals Chain reactions that form lipid free radicals and lipid peroxides in membranes make a major contribution to ROS-induced injury (Fig. 24.8). An initiator (such as a hydroxyl radical produced locally in the Fenton reaction) begins the chain reaction. It extracts a hydrogen atom, preferably from the double bond of a polyunsaturated fatty acid in a membrane lipid. The chain reaction is propagated when O2 adds to form lipid peroxyl radicals and lipid peroxides. Eventually, lipid degradation occurs, forming such products as malondialdehyde (from fatty acids with three or more double bonds) and ethane and pentane (from the ␻-terminal carbons of three- and six-carbon fatty acids, respectively). Malondialdehyde appears in the blood and urine and is used as an indicator of free radical damage. Peroxidation of lipid molecules invariably changes or damages lipid molecular structure. In addition to the self-destructive nature of membrane lipid peroxidation, the aldehydes that are formed can cross-link proteins. When the damaged lipids are the constituents of biologic membranes, the cohesive lipid bilayer arrangement and stable structural organization is disrupted (see Fig. 24.7). Disruption of mitochondrial membrane integrity may result in further free radical production.

B. Proteins and Peptides In proteins, the amino acids proline, histidine, arginine, cysteine, and methionine are particularly susceptible to hydroxyl-radical attack and oxidative damage. As a consequence of oxidative damage, the protein may fragment or residues cross-link with other residues. Free radical attack on protein cysteine residues can result in

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cross-linking and formation of aggregates that prevents their degradation. However, oxidative damage increases the susceptibility of other proteins to proteolytic digestion. Free radical attack and oxidation of the cysteine sulfhydryl residues of the tripeptide glutathione (␥-glutamylcysteinylglycine; see Section V.A.3) increases oxidative damage throughout the cell. Glutathione is a major component of cellular defense against free radical injury, and its oxidation reduces its protective effects.

443

Evidence of protein damage shows up in many diseases, particularly those associated with aging. In patients with cataracts, proteins in the lens of the eye exhibit free radical damage and contain methionine sulfoxide residues and tryptophan degradation products.

A. Initiation LH + •OH

L• + HOH

y

•

x

L•

B. Propagation L•

LOO•

+ O2

LOO•

LOOH + L•

+ LH • O O

y

x

LOO• H O O

y

x

Lipid peroxide LOOH

C. Degradation y

O

+ O

x

O O H

Malondialdehyde Degraded lipid peroxide

D. Termination LOO•

+ L•

LOOH + LH or

L• + Vit Ered

LH

+

Vit E•

Vit E• +

LH

+

Vit EOX

L•

FIG. 24.8. Lipid peroxidation: a free radical chain reaction. A. Lipid peroxidation is initiated by a hydroxyl or other radical that extracts a hydrogen atom from a polyunsaturated lipid (LH), thereby forming a lipid radical (L•). B. The free radical chain reaction is propagated by reaction with O2, forming the lipid peroxy radical (LOO•) and lipid peroxide (LOOH). C. Rearrangements of the single electron result in degradation of the lipid. Malondialdehyde, one of the compounds formed, is soluble and appears in blood. D. The chain reaction can be terminated by reduced vitamin E and other lipid-soluble antioxidants that donate single electrons. Two subsequent reduction steps form a stable, oxidized antioxidant.

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

C. DNA

O C

N

N

N H

HN H2N

Oxygen-derived free radicals are also a major source of DNA damage. Approximately 20 types of oxidatively altered DNA molecules have been identified. The nonspecific binding of Fe2⫹ to DNA facilitates localized production of the hydroxyl radical, which can cause base alterations in the DNA (Fig. 24.9). It also can attack the deoxyribose backbone and cause strand breaks. This DNA damage can be repaired to some extent by the cell (see Chapter 12) or minimized by apoptosis of the cell.

Guanine •OH

III. NITRIC OXIDE AND REACTIVE NITROGEN–OXYGEN SPECIES

O C

N

N

N H

HN

OH H2N

8-Hydroxyguanine

FIG. 24.9. Conversion of guanine to 8-hydroxyguanine by the hydroxy radical. The amount of 8-hydroxyguanosine present in cells can be used to estimate the amount of oxidative damage they have sustained. The addition of the hydroxyl group to guanine allows it to mispair with A residues at a low frequency, leading to the creation of a daughter molecule with an A–T base pair in this position.

Nitroglycerin, in tablet form, is often given to patients with coronary artery disease who experience ischemiainduced chest pain (angina). The nitroglycerin decomposes in the blood, forming nitric oxide (NO), a potent vasodilator, which increases blood flow to the heart and relieves the angina.

Arginine NADPH O2

A. Nitric Oxide Synthase At low concentrations, NO serves as a neurotransmitter or as a hormone. It is synthesized from arginine by NO synthases (Fig. 24.10). As a gas, it is able to diffuse through water and lipid membranes and into target cells. In the target cell, it exerts its physiologic effects by high-affinity binding to Fe-heme in the enzyme guanylyl cyclase, thereby activating a signal transduction cascade. However, NO is rapidly inactivated by nonspecific binding to many molecules, and therefore, cells that produce NO need to be close to the target cells. The body has three different tissue-specific isoforms of NO synthase, each encoded by a different gene: neuronal nitric oxide synthase (nNOS, isoform I), inducible nitric oxide synthase (iNOS, isoform II), and endothelial nitric oxide synthase (eNOS, isoform III). nNOS and eNOS are tightly regulated by Ca2⫹ concentration to produce the small amounts of NO required for its role as a neurotransmitter and hormone. In contrast, iNOS is present in many cells of the immune system and cell types with a similar lineage, such as macrophages and brain astroglia. This isoenzyme of NO synthase is regulated principally by induction of gene transcription and not by changes in Ca2⫹ concentration. It produces high and toxic levels of NO to assist in killing invading microorganisms. It is these very high levels of NO that are associated with generation of RNOS and NO toxicity.

B. Nitric Oxide Toxicity

NO synthase (Fe-Heme, FAD, FMN, BH4)

NO Nitric oxide

NADP+ Citrulline

FIG. 24.10. Nitric oxide synthase (NOS) synthesizes the free radical NO. Like cytochrome P450 enzymes, NOS uses Fe-heme, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) to transfer single electrons from NADPH to O2. NOS also requires the cofactor tetrahydrobiopterin (BH4).

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NO is an oxygen-containing free radical that, like O2, is both essential to life and toxic. NO has a single electron and therefore binds to other compounds that contain single electrons, such as Fe3⫹. As a gas, NO diffuses through the cytosol and lipid membranes and into cells. At low concentrations, it functions physiologically as a neurotransmitter and as a hormone that causes vasodilation. At high concentrations, however, it combines with O2 or with superoxide to form additional reactive and toxic species that contain both nitrogen and oxygen (RNOS). RNOS are involved in neurodegenerative diseases, such as Parkinson disease, and in chronic inflammatory diseases, such as rheumatoid arthritis.

The toxic actions of NO can be divided into two categories: direct toxic effects resulting from binding to Fe-containing proteins and indirect effects mediated by compounds formed when NO combines with O2 or with superoxide to form RNOS. 1.

DIRECT TOXIC EFFECTS OF NITRIC OXIDE

NO, as a radical, exerts direct toxic effects by combining with Fe-containing compounds that also have single electrons. Major destructive sites of attack include Fe–S centers (e.g., electron-transport chain complexes I through III, aconitase) and Fe-heme proteins (e.g., hemoglobin and electron-transport chain cytochromes). However, there is usually little damage because NO is present in low concentrations and Fe-heme compounds are present in excess capacity. NO can cause serious damage, however, through direct inhibition of respiration in

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cells that are already compromised through oxidative phosphorylation diseases or ischemia. 2.

REACTIVE NITROGEN–OXYGEN SPECIES TOXICITY

When NO is present in very high concentrations (e.g., during inflammation), it combines nonenzymatically with superoxide to form peroxynitrite (ONOO⫺) or with O2 to form N2O3 (Fig. 24.11). Peroxynitrite, although it is not a free radical, is a strong oxidizing agent that is stable and directly toxic. It can diffuse through the cell and lipid membranes to interact with a wide range of targets, including the methionine side chain in proteins and –SH groups (e.g., Fe–S centers in the electron-transport chain). It also breaks down to form additional RNOS, including the free radical nitrogen dioxide (NO2), which is an effective initiator of lipid peroxidation. Peroxynitrite products also react (nitration) with aromatic rings, forming compounds such as nitrotyrosine or nitroguanosine. N2O3, which can be derived from either NO2 or nitrite, is the agent of nitrosative stress, and it nitrosylates sulfhydryl and similarly reactive groups in the cell. Nitrosylation usually interferes with the proper functioning of the protein or lipid that has been modified. Thus, RNOS can do as much oxidative and free radical damage as non– nitrogen-containing ROS as well as nitrating and nitrosylating compounds. The result is widespread and includes inhibition of a large number of enzymes, mitochondrial lipid peroxidation, inhibition of the electron-transport chain and energy depletion, single-stranded or double-stranded breaks in DNA, and modification of bases in DNA.

Arginine Nitric oxide synthase

NO• O2 NO•

NO• 2 NO2

Nitric oxide (free radical)

Citrulline

N2O3 Nitrogen trioxide (nitrosating agent)

O2 – NO• ONOO–

NO2–

Peroxynitrite (strong oxidizing agent)

Nitrite

Physiologic pH

H+

HONO2

FORMS OF RNOS

Diet, Intestinal bacteria

Peroxynitrous acid

NO3– Nitrate ion (safe)

NO2+

•OH Hydroxyl radical +

Nitronium ion (nitrating agent)

NO2•

OH– +

Nitrogen dioxide (free radical)

Smog Organic smoke Cigarettes

FIG. 24.11. Formation of RNOS from nitric oxide. RNOS are shown in red. The type of damage caused by each RNOS is shown in parentheses. Of all the nitrogen–oxygen-containing compounds shown, only nitrate is relatively nontoxic. NO2 is one of the toxic agents present in smog, automobile exhaust, gas ranges, pilot lights, cigarette smoke, and smoke from forest fires or burning buildings.

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In patients with chronic granulomatous disease, phagocytes have genetic defects in NADPH oxidase. NADPH oxidase, of which there are several isozymes, generally has six different subunits (two in the cell membrane, ␣ and ␤, which together form flavoprotein b558, and four recruited from the cytosol, which include the small GTPase Rac, as well as a complex of p47phox, p67phox, and p40phox), and the genetic defect may be in any of four of the genes that encode these subunits. The membrane catalytic ␤-subunit of NADPH oxidase is a 91-kDa flavocytochrome glycoprotein. It transfers electrons from bound NADPH to flavin adenine dinucleotide (FAD), which transfers them to the Fe-heme components. The membranous ␣-subunit (p22) is required for stabilization. The cytosolic proteins are required for assembly of the complex. The 91-kDa ␤-subunit is affected most often in X-linked chronic granulatomous disease, whereas the ␣-subunit is affected in a rare autosomal recessive form. The cytosolic subunits p47 and p67 are affected most often in patients with the autosomal recessive form of granulomatous disease. In addition to their enhanced susceptibility to bacterial and fungal infections, these patients suffer from an apparent dysregulation of normal inflammatory responses.

IV. FORMATION OF FREE RADICALS DURING PHAGOCYTOSIS AND INFLAMMATION In response to infectious agents and other stimuli, phagocytic cells of the immune system (neutrophils, eosinophils, and monocytes/macrophages) exhibit a rapid consumption of O2 called the respiratory burst. The respiratory burst is a major source of superoxide, H2O2, the hydroxyl radical, HOCl, and RNOS. The generation of free radicals is part of the human antimicrobial defense system and is intended to destroy invading microorganisms, tumor cells, and other cells targeted for removal.

A. NADPH Oxidase The respiratory burst results from the activity of NADPH oxidase, which catalyzes the transfer of an electron from NADPH to O2 to form superoxide (Fig. 24.12). NADPH oxidase is assembled from cytosolic as well as membranous proteins recruited into the phagolysosome membrane as it surrounds an invading microorganism. Superoxide is released into the intramembranous space of the phagolysosome, where it is generally converted to H2O2 and other ROS that are effective against bacteria and fungal pathogens. H2O2 is formed by superoxide dismutase (SOD), which may come from the phagocytic cell or from the invading microorganism.

B. Myeloperoxidase and Hypochlorous Acid The formation of HOCl from H2O2 is catalyzed by myeloperoxidase, a hemecontaining enzyme that is present only in phagocytic cells of the immune system (predominantly neutrophils). The HOCl rapidly dissociates and loses a proton. Myeloperoxidase

Dissociation:

H2O2 ⫹ Cl⫺ ⫹ H⫹ → HOCl ⫹ H2O → ⫺OCl ⫹ H⫹ ⫹ H2O

Myeloperoxidase contains two Fe-hemelike centers, which gives it the green color seen in pus. Hypochlorous acid is a powerful toxin that destroys bacteria within seconds through halogenation and oxidation reactions. It oxidizes many

NADPH

O2

1

NADPH oxidase

–

NADP+

O2

NO

2 Bacterium

6 H2O2

HOCl

iNOS

5

3

Fe2+

Cl–

Fe3+

myeloperoxidase

4

OH•

ONOO– Bacterium

Invagination of neutrophil's cytoplasmic membrane

FIG. 24.12. Production of reactive oxygen species during the phagocytic respiratory burst by activated neutrophils. 1. Activation of NADPH oxidase on the outer side of the plasma membrane initiates the respiratory burst with the generation of superoxide. During phagocytosis, the plasma membrane invaginates, so superoxide is released into the vacuole space. 2. Superoxide (either spontaneously or enzymatically via SOD) generates H2O2. 3. Granules containing myeloperoxidase are secreted into the phagosome where myeloperoxidase generates HOCl and other halides. 4. H2O2 can also generate the hydroxyl radical from the Fenton reaction. 5. Inducible nitric oxide synthase may be activated and generate NO. 6. Nitric oxide combines with superoxide to form peroxynitrite, which may generate additional RNOS. The result is an attack on the membranes and other components of phagocytosed cells and eventual lysis. The whole process is referred to as the respiratory burst because it lasts only 30 to 60 minutes and consumes O2.

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Fe- and S-containing groups (e.g., sulfhydryl groups, Fe–S centers, ferredoxin, heme proteins, methionine), oxidatively decarboxylates and deaminates proteins, and cleaves peptide bonds. Aerobic bacteria under attack rapidly lose membrane transport, possibly because of damage to ATP synthase or electron-transport chain components (which reside in the plasma membrane of bacteria).

C. Reactive Nitrogen–Oxygen Species and Inflammation When human neutrophils of the immune system are activated to produce NO, NADPH oxidase is also activated. NO reacts rapidly with superoxide to generate peroxynitrite, which forms additional RNOS. NO also may be released into the surrounding medium to combine with superoxide in target cells. In several disease states, free radical release by neutrophils or macrophages during an inflammation contributes to injury in the surrounding tissues. During stroke or myocardial infarction, phagocytic cells that move into the ischemic area to remove dead cells may increase the area and extent of damage. The self-perpetuating mechanism of radical release by neutrophils during inflammation and immune-complex formation may explain some of the features of chronic inflammation in patients with rheumatoid arthritis. As a result of free radical release, the immunoglobulin G (IgG) proteins present in the synovial fluid are partially oxidized, which improves their binding with the rheumatoid antibody. This binding, in turn, stimulates the neutrophils to release more free radicals.

V. CELLULAR DEFENSES AGAINST OXYGEN TOXICITY

447

During Cora Nari’s myocardial ischemia (decreased blood flow), the ability of her heart to generate ATP from oxidative phosphorylation was compromised. The damage appeared to accelerate when oxygen was first reintroduced (reperfused) into the tissue. During ischemia, coenzyme Q (CoQ) and the other single-electron components of the electron-transport chain become saturated with electrons. When oxygen is reintroduced (reperfusion), electron donation to O2 to form superoxide is increased. The increase of superoxide results in enhanced formation of hydrogen peroxide and the hydroxyl radical. Macrophages in the area clean up cell debris from ischemic injury and produce nitric oxide, which may further damage mitochondria by generating RNOS that attack Fe–S centers and cytochromes in the electron-transport chain membrane lipids. Thus, the RNOS may increase the infarct size.

Our defenses against oxygen toxicity fall into the categories of antioxidant defense enzymes, dietary and endogenous antioxidants (free radical scavengers), cellular compartmentation, metal sequestration, and repair of damaged cellular components. The antioxidant defense enzymes react with ROS and cellular products of free radical chain reactions to convert them to nontoxic products. Dietary antioxidants, such as vitamin E and flavonoids, and endogenous antioxidants, such as urate, can terminate free radical chain reactions. Defense through compartmentation refers to separation of species and sites involved in ROS generation from the rest of the cell (Fig. 24.13). For example, many of the enzymes that produce hydrogen peroxide Fe sequestration Hemosiderin

Ferritin

H2O2

catalase

Peroxisomes

SOD

Cytoplasm GSH –

O2

SOD Compartmentation

Lipid bilayer of all cellular membranes

Mitochondrion glutathione peroxidase

Vitamin E + β-carotene

SOD + glutatathione peroxidase + GSH

FIG. 24.13. Compartmentization of free radical defenses. Various defenses against ROS are found in the various subcellular compartments of the cell. The location of free radical defense enzymes (shown in red) matches the type and amount of ROS generated in each subcellular compartment. The highest activities of these enzymes are found in the liver, adrenal gland, and kidney where mitochondrial and peroxisomal contents are high, and cytochrome P450 enzymes are found in abundance in the smooth endoplasmic reticulum. The enzymes SOD and glutathione peroxidase are present as isozymes in the various compartments. Another form of compartmentization involves the sequestration of Fe, which is stored as mobilizable Fe in ferritin. Excess Fe is stored in nonmobilizable hemosiderin deposits. Glutathione (GSH) is a nonenzymatic antioxidant.

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SECTION IV ■ FUEL OXIDATION AND THE GENERATION OF ADENOSINE TRIPHOSPHATE

A

are sequestered in peroxisomes with a high content of antioxidant enzymes. Metals are bound to a wide range of proteins within the blood and in cells, preventing their participation in the Fenton reaction. Iron, for example, is tightly bound to its storage protein, ferritin, and cannot react with hydrogen peroxide. Repair mechanisms for DNA and for removal of oxidized fatty acids from membrane lipids are available to the cell. Oxidized amino acids on proteins are continuously repaired through protein degradation and resynthesis of new proteins.

2 O2– Superoxide

2H+ Superoxide dismutase

O2 H2O2

A. Antioxidant Scavenging Enzymes

Hydrogen peroxide

The enzymatic defense against ROS includes SOD, catalase, and glutathione peroxidase.

B

1.

2 H2O2 Hydrogen peroxide Catalase (peroxisomes)

2 H2O + O2

FIG. 24.14. A. Superoxide dismutase converts superoxide to hydrogen peroxide, which is nontoxic unless it is converted to other ROS. B. Catalase reduces hydrogen peroxide. (ROS is shown in a yellow box.) The intracellular form of the Cu⫹–Zn2⫹ superoxide dismutase is encoded by the superoxide dismutase 1 (SOD1) gene. To date, 58 mutations in this gene have been discovered in individuals affected by familial amyotrophic lateral sclerosis (ALS, Lou Gehrig disease). How a mutation in this gene leads to the symptoms of this disease has yet to be understood. It is important to note that only 5% to 10% of the total cases of diagnosed ALS are caused by the familial form. Recent work has indicated that mutations in enzymes involved in RNA processing also lead to familial and sporadic ALS. A

HS GSH

CH2

3.

GLUTATHIONE PEROXIDASE AND GLUTATHIONE REDUCTASE

Glutathione (␥-glutamylcysteinylglycine) is one of the body’s principal means of protecting against oxidative damage (see also Chapter 29). Glutathione is a tripeptide composed of glutamate, cysteine, and glycine, with the amino group of cysteine joined in peptide linkage to the ␥-carboxyl group of glutamate (Fig. 24.15). In reactions that are catalyzed by glutathione peroxidases, the reactive sulfhydryl groups reduce hydrogen peroxide to water and lipid peroxides to nontoxic C

GSH + HSG O

HN

H2O2

Glutathione peroxidase

Glutathione peroxidase

CH2

NADPH H+

GSSG

CH2

Glutathione reductase

GSSG

2H2O O

NADP+

2 GSH

H2O2 Cysteine

CH

C

CATALASE

Hydrogen peroxide, once formed, must be reduced to water to prevent it from forming the hydroxyl radical in the Fenton reaction or the Haber-Weiss reaction (see Fig. 24.4). One of the enzymes that is capable of reducing hydrogen peroxide is catalase (see Fig. 24.14B). Catalase is found principally in peroxisomes and, to a lesser extent, in the cytosol and microsomal fraction of the cell. The highest activities are found in tissues with a high peroxisomal content (kidney and liver). In cells of the immune system, catalase serves to protect the cell against its own respiratory burst.

Glycine

HN C

2.

B

COO– CH2

SUPEROXIDE DISMUTASE

Conversion of superoxide anion to hydrogen peroxide and O2 (dismutation) by SOD is often called the primary defense against oxidative stress because superoxide is such a strong initiator of chain reactions (Fig. 24.14A). SOD exists as three isoenzyme forms: a Cu⫹–Zn2⫹ form present in the cytosol, a Mn2⫹ form present in mitochondria and a Cu⫹–Zn2⫹ form found extracellularly. The activity of Cu⫹– Zn2⫹ SOD is increased by chemicals or conditions (such as hyperbaric oxygen) that increase the production of superoxide.

Pentose phosphate pathway

2 H2O

Glutathione disulfide Glutamate

HCNH3+ COO–

FIG. 24.15. Glutathione peroxidase reduces hydrogen peroxide to water. A. The structure of glutathione. The sulfhydryl group of glutathione, which is oxidized to a disulfide, is shown in red. B. Glutathione peroxidase transfers electrons from GSH to hydrogen peroxide. C. Glutathione redox cycle. Glutathione reductase regenerates reduced glutathione. (ROS is shown in the yellow box.)

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alcohols. In these reactions, two glutathione molecules are oxidized to form a single molecule, glutathione disulfide. The sulfhydryl groups are also oxidized in nonenzymatic chain-terminating reactions with organic radicals. Glutathione peroxidases exist as a family of selenium enzymes with somewhat different properties and tissue locations. Within cells, they are found principally in the cytosol and mitochondria and are the major means for removing H2O2 produced outside of peroxisomes. They contribute to our dietary requirement for selenium and account for the protective effect of selenium in the prevention of free radical injury. Once oxidized glutathione (GSSG) is formed, it must be reduced back to the sulfhydryl form by glutathione reductase in a redox cycle (see Fig. 24.15C). Glutathione reductase contains a flavin adenine dinucleotide (FAD) and catalyzes transfer of electrons from NADPH to the disulfide bond of GSSG. NADPH is thus essential for protection against free radical injury. The major source of NADPH for this reaction is the pentose phosphate pathway (see Chapter 29).

B. Nonenzymatic Antioxidants (Free Radical Scavengers)

449

Why does the cell need a high content of SOD in mitochondria?

Premature infants with low levels of lung surfactant (see Chapter 33) require oxygen therapy. The level of oxygen must be closely monitored to prevent ROS-induced retinopathy and subsequent blindness (the retinopathy of prematurity) and to prevent bronchial pulmonary dysplasia. The tendency for these complications to develop is enhanced by the possibility of low levels of SOD and vitamin E in the premature infant.

Free radical scavengers convert free radicals to a nonradical, nontoxic form in nonenzymatic reactions. Most free radical scavengers are antioxidants, compounds that neutralize free radicals by donating a hydrogen atom (with its one electron) to the radical. Antioxidants therefore reduce free radicals and are themselves oxidized in the reaction. Dietary free radical scavengers (e.g., vitamin E, ascorbic acid, carotenoids, flavonoids) as well as endogenously produced free radical scavengers (e.g., urate, melatonin) have a common structural feature, a conjugated double bond system that may be an aromatic ring. 1.

VITAMIN E

Vitamin E (␣-tocopherol), the most widely distributed antioxidant in nature, is a lipid-soluble antioxidant vitamin that functions principally to protect against lipid peroxidation in membranes (see Fig. 24.13). Vitamin E comprises several tocopherols that differ in their methylation pattern. Among these, ␣-tocopherol is the most potent antioxidant and is present in the largest amounts in our diet (Fig. 24.16). Vitamin E is an efficient antioxidant and nonenzymatic terminator of free radical chain reactions, and it has little pro-oxidant activity. When vitamin E donates an electron to a lipid peroxy radical, it is converted to a free radical form that is stabilized by resonance. If this free radical form were to act as a pro-oxidant and abstract an electron from a polyunsaturated lipid, it would be oxidizing that lipid and actually propagate the free radical chain reaction. The chemistry of vitamin E is such that it has a much greater tendency to donate a second electron and go to the fully oxidized form. 2.

ASCORBIC ACID

Although ascorbate (vitamin C) is an oxidation–reduction coenzyme that functions in collagen synthesis and other reactions, it also plays a role in free radical defense. Reduced ascorbate can regenerate the reduced form of vitamin E by donating electrons in a redox cycle (Fig. 24.17). It is water-soluble and circulates unbound in blood and extracellular fluid, where it has access to the lipid-soluble vitamin E present in membranes and lipoprotein particles. 3.

CAROTENOIDS

Carotenoids is a term applied to ␤-carotene (the precursor of vitamin A) and similar compounds with functional oxygen-containing substituents on the rings, such as zeaxanthin and lutein (Fig. 24.18). These compounds can exert antioxidant effects as well as quench singlet O2 (singlet oxygen is a highly ROS in which there are no unpaired electrons in the outer orbitals, but there is one orbital that is completely empty). Epidemiologic studies have shown a correlation between diets that are high in fruits and vegetables and health benefits, leading to the hypothesis that

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Selenium (Se) is present in human proteins principally as selenocysteine (cysteine with the sulfur group replaced by Se, abbreviated sec). This amino acid functions in catalysis and has been found in 11 or more human enzymes, including the 4 enzymes of the glutathione peroxidase family. Selenium is supplied in the diet as selenomethionine from plants (methionine with the Se replacing the sulfur), selenocysteine from animal foods, and inorganic selenium. Se from all of these sources can be converted to selenophosphate. Selenophosphate reacts with a unique tRNA-containing bound serine to form a selenocysteine-tRNA, which incorporates selenocysteine into the appropriate protein as it is being synthesized. Se homeostasis in the body is controlled principally through regulation of its secretion as methylated Se. The current dietary requirement is approximately 70 ␮g/day for adult males and 55 ␮g/day for females. Deficiency symptoms reflect diminished antioxidant defenses and include symptoms of vitamin E deficiency.

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Mitochondria are major sites for generation of superoxide from the interaction of CoQ and O2. The Mn2⫹ superoxide dismutase present in mitochondria is not regulated through induction/repression of gene transcription, presumably because the rate of superoxide generation is always high. Mitochondria also have a high content of glutathione and glutathione peroxidase, and can thus convert H2O2 to H2O and prevent lipid peroxidation.

CH3 HO H3C

Phytyl

O CH3

␣-Tocopherol LOO• LOOH CH3

Vitamin E is found in the diet in the lipid fractions of some vegetable oils and in liver, egg yolks, and cereals. It is absorbed together with lipids, and fat malabsorption results in symptomatic deficiencies. Vitamin E circulates in the blood in lipoprotein particles. Its deficiency causes neurologic symptoms, probably because the polyunsaturated lipids in myelin and other membranes of the nervous system are particularly sensitive to free radical injury. Epidemiologic evidence suggests that individuals with a higher intake of foods containing vitamin E, ␤-carotene, and vitamin C have a somewhat lower risk of cancer and certain other ROS-related diseases than do individuals on diets that are deficient in these vitamins. However, studies in which well-nourished populations were given supplements of these antioxidant vitamins found either no effects or harmful effects compared with the beneficial effects from eating foods containing a wide variety of antioxidant compounds. Of the pure chemical supplements tested, there is evidence only for the efficacy of vitamin E. In two clinical trials, ␤-carotene (or ␤-carotene ⫹ vitamin A) was associated with a higher incidence of lung cancer among smokers and higher mortality rates. In one study, vitamin E intake was associated with a higher incidence of hemorrhagic stroke (possibly because of vitamin K mimicry).

HO

H3C

O 4 3

6

O

1 2

–

O

Tocopheryl radical LOO•

CH3 O H3C

L-Ascorbate

Phytyl

O O CH3 O L H2O LOOH

OH

CH3 O H3C

Phytyl

O CH3

Tocopheryl quinone

FIG. 24.16. Vitamin E (␣-tocopherol) terminates free radical lipid peroxidation by donating single electrons to lipid peroxyl radicals (LOO•) to form the more stable lipid peroxide, LOOH. In so doing, the ␣-tocopherol is converted to the fully oxidized tocopheryl quinone.

HO

HO

H O

O

–H

+e–

–e–

H

+

+H+ OH

Phytyl

O CH3

–e–

H 5

HO

•O

O

O +e–

O OH

O

Ascorbyl radical

O OH O Dehydro-L-ascorbic acid

FIG. 24.17. L-Ascorbate (the reduced form) donates single electrons to free radicals or disulfides in two steps as it is oxidized to dehydroL-ascorbic acid. Its principal role in free radical defense is probably regeneration of vitamin E. However, it also may react with superoxide, hydrogen peroxide, hypochlorite, the hydroxyl and peroxyl radicals, and NO2.

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␤-Carotene

Macular carotenoids Zeaxanthin

OH

HO Lutein

OH

HO

FIG. 24.18. Carotenoids are compounds related in structure to ␤-carotene. Lutein and zeaxanthin (the macular carotenoids) are analogs that contain hydroxyl groups.

carotenoids might slow the progression of cancer, atherosclerosis, and other degenerative diseases by acting as chain-breaking antioxidants. However, in clinical trials, ␤-carotene supplements had either no effect or an undesirable effect. Its ineffectiveness may be caused by the pro-oxidant activity of the free radical form. In contrast, epidemiologic studies relating the intake of lutein and zeaxanthin with decreased incidence of age-related macular degeneration have received progressive support. These two carotenoids are concentrated in the macula (the central portion of the retina) and are called the macular carotenoids. 4.

OTHER DIETARY ANTIOXIDANTS

Flavonoids are a group of structurally similar compounds that contain two spatially separate aromatic rings and are found in red wine, green tea, chocolate, and other plant-derived foods (Fig. 24.19). Flavonoids have been hypothesized to contribute to our free radical defenses in several ways. Some flavonoids inhibit enzymes responsible for superoxide anion production, such as xanthine oxidase. Others efficiently chelate Fe and Cu, making it impossible for these metals to participate in the Fenton OH OH O

HO

OH OH

O A flavonoid

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States among persons older than 50 years of age, and it affects 1.7 million people worldwide. In AMD, visual loss is related to oxidative damage to the retinal pigment epithelium (RPE) and the choriocapillaris epithelium. The photoreceptor/retinal pigment complex is exposed to sunlight, it is bathed in near-arterial levels of oxygen, and the membranes contain high concentrations of polyunsaturated fatty acids, all of which are conducive to oxidative damage. Lipofuscin granules, which accumulate in the RPE throughout life, may serve as photosensitizers, initiating damage by absorbing blue light and generating singlet oxygen (an energetically excited form of oxygen) that forms other radicals. Dark sunglasses are protective. Epidemiologic studies showed that the intake of lutein and zeaxanthin in dark green leafy vegetables (e.g., spinach, collard greens) also may be protective. Lutein and zeaxanthin accumulate in the macula and protect against free radical damage by absorbing blue light and quenching singlet oxygen.

FIG. 24.19. The flavonoid quercetin. All flavonoids have the same ring structure, shown in red. They differ in ring substituents (=O, –OH, and –OCH3). Quercetin is effective in Fe chelation and antioxidant activity. It is widely distributed in fruits (principally in the skins) and in vegetables (e.g., onions).

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O HN

N

O

N H

OH N H Uric acid

H O CH3O

CH2

CH2

N C

CH3

N H Melatonin

FIG. 24.20. Endogenous antioxidants. Uric acid and melatonin both act to successively neutralize several molecules of ROS.

reaction. They also may act as free radical scavengers by donating electrons to superoxide or lipid peroxy radicals or stabilize free radicals by complexing with them. It is difficult to tell how much dietary flavonoids contribute to our free radical defense system; they have a high pro-oxidant activity and are poorly absorbed. Nonetheless, we generally consume large amounts of flavonoids (approximately 800 mg/day), and there is evidence that they can contribute to the maintenance of vitamin E as an antioxidant.

Dopamine inactivation

1

MAO

H2O2

O2 Fe2+

2 O2–

•OH

NO RNOS

3 Lipid peroxidation Protein oxidation DNA strand breaks

4

Lipofuscin

Neuronal degeneration

Reduced dopamine release

FIG. 24.21. A model for the role of ROS and RNOS in neuronal degradation in Parkinson disease. 1. Dopamine levels are reduced by monoamine oxidase, which generates H2O2. 2. Superoxide also can be produced by mitochondria, which superoxide dismutase (SOD) will convert to H2O2. Iron levels increase, which allows the Fenton reaction to proceed, generating hydroxyl radicals. 3. NO, produced by inducible nitric oxide synthase, reacts with superoxide to form RNOS. 4. The RNOS and hydroxyl radical lead to radical chain reactions that result in lipid peroxidation, protein oxidation, the formation of lipofuscin, and neuronal degeneration. The end result is a reduced production and release of dopamine, which leads to the clinical symptoms observed.

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5.

ENDOGENOUS ANTIOXIDANTS

Several compounds that are synthesized endogenously for other functions, or as urinary excretion products, also function nonenzymatically as free radical antioxidants. Uric acid is formed from the degradation of purines and is released into extracellular fluids, including blood, saliva, and lung lining fluid (Fig. 24.20). Together with protein thiols, it accounts for the major free radical–trapping capacity of plasma. It is particularly important in the upper airways where there are few other antioxidants. It can directly scavenge hydroxyl radicals, oxyheme oxidants formed between the reaction of hemoglobin and peroxy radicals, and peroxyl radicals themselves. Having acted as a scavenger, uric acid produces a range of oxidation products that are subsequently excreted. Melatonin, which is a secretory product of the pineal gland, is a neurohormone that functions in regulation of our circadian rhythm, light–dark signal transduction, and sleep induction. In addition to these receptor-mediated functions, it functions as a nonenzymatic free radical scavenger that donates an electron (as hydrogen) to “neutralize” free radicals. It also can react with ROS and RNOS to form additional products, thereby undergoing suicidal transformations. Its effectiveness is related to both its lack of pro-oxidant activity and its joint hydrophilic/hydrophobic nature, which allows it to pass through membranes and the blood–brain barrier. CLINICAL COMMENTS Les Dopaman has “primary” parkinsonism. The pathogenesis of this disease is not well established and may be multifactorial (Fig. 24.21). Recent work has identified several genes, which, when mutated and inactive, lead to rare familial Parkinson disease. The major clinical disturbances in Parkinson disease are a result of dopamine depletion in the neostriatum, resulting from degeneration of dopaminergic neurons whose cell bodies reside in the substantia nigra pars compacta. The decrease in dopamine production is the result of severe degeneration of these

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nigrostriatal neurons. Although the agent that initiates the disease is unknown, various studies support a role for free radicals in Parkinson disease (mitochondrial dysfunction), along with alterations in the ubiquitin–proteasome pathway of protein degradation. Within these neurons, dopamine turnover is increased, dopamine levels are lower, glutathione is decreased, and lipofuscin (Lewy bodies) is increased. Iron levels are higher, and ferritin, the storage form of iron, is lower. Furthermore, the disease is mimicked by the compound 1-methyl-4-phenylpyridinium (MPP⫹), an inhibitor of NADH:CoQ oxidoreductase that increases superoxide production in these neurons and decreases ATP production. Analysis of mitochondria from patients with Parkinson disease indicates a 30% to 40% reduction in complex I activity. The reduced ATP levels may affect the ubiquitin–proteasome pathway negatively, reducing protein degradation and linking these two pathways, which can lead to Parkinson disease. Even so, it is not known whether oxidative stress makes a primary or secondary contribution to the disease process. Drug therapy is based on the severity of the disease. Several options are available. In the early phases of the disease, a monoamine oxidase B inhibitor can be used that inhibits dopamine degradation and decreases hydrogen peroxide formation. In later stages of the disease, patients are treated with levodopa (L-dopa), a precursor of dopamine, sometimes in combination with the monoamine oxidase B inhibitor. Cora Nari experienced angina caused by severe ischemia in the ventricular muscle of her heart. The ischemia was caused by clots that formed at the site of atherosclerotic plaques within the lumen of the coronary arteries. When tissue plasminogen activator (TPA) was infused to dissolve the clots, the ischemic area of her heart was reperfused with oxygenated blood, resulting in ischemia-reperfusion injury. In her case, the reperfusion injury resulted in ventricular fibrillation. During ischemia, several events occur simultaneously in cardiomyocytes. A decreased O2 supply results in decreased ATP generation from mitochondrial oxidative phosphorylation and inhibition of cardiac muscle contraction. As a consequence, cytosolic adenosine monophosphate (AMP) concentration increases, activating anaerobic glycolysis and lactic acid production. If ATP levels are inadequate to maintain Na⫹,K⫹-ATPase activity, intracellular Na⫹ increases, resulting in cellular swelling, a further increase in H⫹ concentration, and increases of cytosolic and subsequently mitochondrial Ca2⫹ levels. The decrease in ATP and increase in Ca2⫹ may open the mitochondrial permeability transition pore, resulting in permanent inhibition of oxidative phosphorylation. Damage to lipid membranes is further enhanced by Ca2⫹ activation of phospholipases. Reperfusion with O2 allows recovery of oxidative phosphorylation, provided that the mitochondrial membrane has maintained some integrity and the mitochondrial transition pore can close. However, it also increases generation of free radicals. The transfer of electrons from CoQ• to O2 to generate superoxide is increased. Endothelial production of superoxide by xanthine oxidase also may increase. These radicals may go on to form the hydroxyl radical, which can enhance the damage to components of the electron-transport chain and mitochondrial lipids, as well as activate the mitochondrial permeability transition. As macrophages move into the area to clean up cellular debris, they may generate NO and superoxide, thus introducing peroxynitrite and other free radicals into the area. Depending on the route and timing involved, the acute results may be cell death through necrosis, with slower cell death through apoptosis in the surrounding tissue. Currently, an intense study of ischemic insults to various animal organs is underway in an effort to discover ways of preventing reperfusion injury. These include methods designed to increase endogenous antioxidant activity, to reduce the generation of free radicals, and, finally, to develop exogenous antioxidants that, when administered before reperfusion, would prevent its injurious effects.

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Preconditioning tissues to hypoxia is also a viable option to reducing reperfusion injury. Each of these approaches has met with some success, but their clinical application awaits further refinement. With the growing number of invasive procedures aimed at restoring arterial blood flow through partially obstructed coronary vessels, such as clot lysis, balloon or laser angioplasty, and coronary artery bypass grafting, development of methods to prevent ischemia-reperfusion injury will become increasingly urgent. In Cora Nari’s case, oxygen was restored before permanent impairment of oxidative phosphorylation had occurred and the stage of irreversible injury was reached. However, reintroduction of oxygen did induce ventricular fibrillation, from which she recovered. BIOCHEMICAL COMMENTS Protection against Ozone in Lung Lining Fluid. The lung lining fluid, a thin fluid layer extending from the nasal cavity to the most distal lung alveoli, protects the epithelial cells lining our airways from ozone and other pollutants. Although ozone is not a radical species, many of its toxic effects are mediated through generation of the classical reactive oxygen species (ROS), as well as generation of aldehydes and ozonides. Polyunsaturated fatty acids represent the primary target for ozone, and peroxidation of membrane lipids is the most important mechanism of ozone-induced injury. However, ozone also oxidizes proteins. Although most individuals are able to protect against small amounts of ozone in the atmosphere, even slightly elevated ozone concentrations produce respiratory symptoms in 10% to 20% of the healthy population. The lung lining fluid has two phases: a gel phase that traps microorganisms and large particles and a soluble phase containing various ROS defense mechanisms that prevent pollutants from reaching the underlying lung epithelial cells (Fig. 24.22). When the ozone level of inspired air is low, ozone is neutralized principally by uric acid (UA) present in the fluid lining the nasal cavity. In the proximal and distal regions of the respiratory tract, glutathione (GSH) and

OZONE

Mucus Lung lining fluid

GSH

AA

UA

ROS Neutrophil

Protein

␣-TOC GSH-Px EC-SOD

Lipid

CHO

Secondary oxidants

Epithelial cell

Blood capillary

FIG. 24.22. Protection against ozone in the lung lining fluid. GSH, glutathione; AA, ascorbic acid (vitamin C); UA, uric acid; CHO, carbohydrate; ␣-TOC, vitamin E; GSH-Px, glutathione peroxidase; EC-SOD, extracellular superoxide dismutase.

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ascorbic acid (AA), in addition to UA, react directly with ozone. Ozone that escapes this antioxidant screen may react directly with proteins, lipids, and carbohydrates (CHO–) to generate secondary oxidants, such as lipid peroxides, that can initiate chain reactions. A second layer of defense protects against these oxidation and peroxidation products: ␣-tocopherol (vitamin E) and glutathione react directly with lipid radicals, glutathione peroxidase reacts with hydrogen peroxide and lipid peroxides, and extracellular superoxide dismutase (EC-SOD) converts superoxide to hydrogen peroxide. However, oxidative stress may still overwhelm even this extensive defense network because ozone also promotes neutrophil migration into the lung lining fluid. Once they are activated, the neutrophils produce a second wave of ROS (superoxide, hypochlorous acid [HOCl], and nitric oxide [NO]), which can lead to oxidative damage of the epithelial cells lining the lung. Key Concepts • • • • • • • •

• • • •

Oxygen radical generation contributes to cellular death and degeneration in various diseases. Radical damage occurs via electron extraction from a biologic molecule, creating a chain reaction of radical propagation. Reactive oxygen species (ROS) include superoxide, hydrogen peroxide, and the hydroxyl radical. ROS can be produced either enzymatically or nonenzymatically. ROS cause damage by oxidatively damaging DNA, proteins, and lipids, leading to mutations and cell death. Other radical species include nitric oxide (NO) and hypochlorous acid (HOCl). NO reacts with oxygen or superoxide to form a family of reactive nitrogen–oxygen species (RNOS). The immune response normally produces radical species (superoxide, HOCl, NO) to destroy invading microorganisms. Escape of radicals from the immune cells during this protective event can damage surrounding tissues. Cellular defense mechanisms against radical damage include defense enzymes, antioxidants, and compartmentalization of free radicals. Cellular defense enzymes include superoxide dismutase, catalase, and glutathione peroxidase. Antioxidants include vitamins E and C and plant flavonoids. Diseases discussed in this chapter are summarized in Table 24.3.

Table 24.3

Diseases Discussed in Chapter 24

Disease or Disorder

Environmental or Genetic

Free radical disease Parkinson disease

Both

Myocardial infarction

Both

Chronic granulomatous disease

Genetic

Respiratory distress syndrome Amyotrophic lateral sclerosis (ALS)

Both

Age-related macular degeneration

Both

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Environmental

Both

Comments Damage caused to proteins and lipids caused by free radical generation may lead to cellular dysfunction. Inability to convert tyrosine to DOPA; DOPA treatment can temporarily reverse tremors and other symptoms. Further damage to the heart muscle can occur because of free radical generation after oxygen is reintroduced to the cells, which were temporarily ischemic, a process known as ischemic reperfusion injury. This disorder occurs because of a reduced activity of NADPH oxidase, leading to a reduction in the oxidative burst by neutrophils, coupled with a dysregulated immune response to bacteria and fungi. Either mutations in surfactant or lack of surfactant production in newborns; lungs have difficulty inflating and compressing. The genetic form of ALS is caused by mutations in superoxide dismutase, leading to difficulty in disposing of superoxide radicals, leading to cell damage caused by excessive ROS. Oxidative damage occurs in the retinal pigment epithelium, leading to first, reduced vision, and second, to blindness.

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REVIEW QUESTIONS—CHAPTER 24 1.

2.

3.

Which of the following vitamins or enzymes is unable to protect against free radical damage? A. ␤-Carotene B. Glutathione peroxidase C. Superoxide dismutase D. Vitamin B6 E. Vitamin C F. Vitamin E Superoxide dismutase catalyzes which of the following reactions? A. O2⫺ ⫹ e⫺ ⫹ 2H⫹ → H2O2 B. 2O2⫺ ⫹ 2H⫹ → H2O2 ⫹ O2 C. O2⫺ ⫹ HO• ⫹ H⫹ → CO2 ⫹ H2O D. H2O2 ⫹ O2 → 4H2O E. O2⫺ ⫹ H2O2 ⫹ H⫹ → 2H2O ⫹ O2 The mechanism of vitamin E as an antioxidant is best described by which of the following? A. Vitamin E binds to free radicals and sequesters them from the contents of the cell. B. Vitamin E participates in the oxidation of the radicals. C. Vitamin E participates in the reduction of the radicals.

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D. Vitamin E forms a covalent bond with the radicals, thereby stabilizing the radical state. E. Vitamin E inhibits enzymes that produce free radicals. 4.

An accumulation of hydrogen peroxide in a cellular compartment can be converted to dangerous radical forms in the presence of which metal? A. Selenium B. Iron C. Manganese D. Magnesium E. Molybdenum

5.

The level of oxidative damage to mitochondrial DNA is ten times greater than that to nuclear DNA. This could be caused, in part, by which of the following? A. Superoxide dismutase is present in the mitochondria. B. The nucleus lacks glutathione. C. The nuclear membrane presents a barrier to reactive oxygen species. D. The mitochondrial membrane is permeable to reactive oxygen species. E. Mitochondrial DNA lacks histones.

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25

Metabolism of Ethanol

Ethanol is a dietary fuel that is metabolized to acetate principally in the liver with the generation of NADH. The principal route for metabolism of ethanol is through hepatic alcohol dehydrogenases (ADHs), which oxidize ethanol to acetaldehyde in the cytosol (Fig. 25.1). Acetaldehyde is further oxidized by acetaldehyde dehydrogenases to acetate, principally in mitochondria. Acetaldehyde, which is toxic, also may enter the blood. NADH produced by these reactions is used for adenosine triphosphate (ATP) generation through oxidative phosphorylation. Most of the acetate enters the blood and is taken up by skeletal muscles and other tissues, where it is activated to acetyl coenzyme A (acetyl-CoA) and is oxidized in the tricarboxylic acid (TCA) cycle. Approximately 10% to 20% of ingested ethanol is oxidized through a microsomal ethanol oxidizing system (MEOS), comprising cytochrome P450 enzymes in the endoplasmic reticulum (especially CYP2E1). CYP2E1 has a high Km for ethanol and is inducible by ethanol. Therefore, the proportion of ethanol metabolized through this route is greater at high ethanol concentrations and greater after chronic consumption of ethanol. Acute effects of alcohol ingestion arise principally from the generation of NADH, which greatly increases the NADH/NADⴙ ratio of the liver. As a consequence, fatty acid oxidation is inhibited, and ketogenesis may occur. The elevated NADH/NADⴙ ratio may also cause lactic acidosis and inhibit gluconeogenesis. Ethanol metabolism may result in alchohol-induced liver disease, including hepatic steatosis (fatty liver), alcohol-induced hepatitis, and cirrhosis. The principal toxic products of ethanol metabolism include acetaldehyde and free radicals. Acetaldehyde forms adducts with proteins and other compounds. The hydroxyethyl radical produced by the MEOS and other radicals produced during inflammation cause irreversible damage to the liver. Many other tissues are adversely affected by ethanol, acetaldehyde, or by the consequences of hepatic dysmetabolism and injury. Genetic polymorphisms in the enzymes of ethanol metabolism may be responsible for individual variations in the development of alcoholism or the development of liver cirrhosis.

457

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ADH Acetaldehyde O CH3 C H NAD+

ALDH

NADH + H+

Acetate O CH3 C OH

Ethanol CH3CH2OH

+ NADH NAD + +H

Liver

Muscle ACS Acetyl CoA TCA cycle

Acetate

Blood

Acetate

Acetaldehyde

FAD (2H)

CO2

3NADH, 3H+

FIG. 25.1. The major route for metabolism of ethanol and use of acetate by the muscle. ADH, alcohol dehydrogenase; ALDH, acetaldehyde dehydrogenase; ACS, acetyl-CoA synthetase.

THE WAITING ROOM A dietary history for Ivan Applebod showed that he had continued his habit of drinking scotch and soda each evening while watching TV, but he did not add the ethanol calories to his dietary intake. He justifies this calculation on the basis of a comment he heard on a radio program that calories from alcohol ingestion “don’t count” because they are empty calories that do not cause weight gain.

The anion gap is calculated by subtracting the sum of the value for serum chloride and for the serum HCO3⫺ content from the serum sodium concentration. If the gap is greater than normal, it suggests that acids such as the ketone bodies acetoacetate and ␤-hydroxybutyrate are present in the blood in increased amounts.

Al Martini was found lying semiconscious at the bottom of the stairs by his landlady when she returned from an overnight visit with friends. His face had multiple bruises and his right forearm was grotesquely angulated. Nonbloody dried vomitus stained his clothing. Mr. Martini was rushed by ambulance to the emergency room at the nearest hospital. In addition to multiple bruises and the compound fracture of his right forearm, he had deep and rapid (Kussmaul) respirations and was moderately dehydrated. Initial laboratory studies showed a relatively large anion gap of 34 mmol/L (reference range, 9 to 15 mmol/L). An arterial blood gas analysis (which measures pH in addition to the levels of dissolved O2 and CO2) confirmed the presence of a metabolic acidosis. Mr. Martini’s blood alcohol level was only slightly elevated. His serum glucose was 68 mg/dL (low normal). Jean Ann Tonich, a 46-year-old commercial artist, recently lost her job because of absenteeism. Her husband of 24 years had left her 10 months earlier. She complains of loss of appetite, fatigue, muscle weakness, and emotional depression. She has had occasional pain in the area of her liver, at times accompanied by nausea and vomiting. On physical examination, she appears disheveled and pale. The physician notes tenderness to light percussion over her liver and detects a small amount of ascites (fluid within the peritoneal cavity around the abdominal organs). The lower edge of her liver is palpable about 2 in below the lower margin of her right rib cage, suggesting liver enlargement. The edge feels somewhat more firm and nodular than normal. Jean Ann’s spleen is not palpably enlarged. There is a suggestion of mild

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jaundice (yellow discoloration of her skin and mucus membranes). No obvious neurologic or cognitive abnormalities are present. After detecting a hint of alcohol on Jean Ann’s breath, the physician questions her about possible alcohol abuse, which she denies. With more intensive questioning, however, Jean Ann admits that for the last 5 or 6 years, she has been drinking gin on a daily basis (approximately 4 to 5 drinks or 68 to 85 g ethanol) and eating infrequently. Laboratory tests showed that her serum ethanol level on the initial office visit was 245 mg/dL (0.245%). A serum ethanol level ⬎150 mg/dL (0.15%) is considered indicative of inebriation.

I.

ETHANOL METABOLISM

Ethanol is a small molecule that is both lipid- and water-soluble. It is therefore readily absorbed from the intestine by passive diffusion. A small percentage of ingested ethanol (0% to 5%) enters the gastric mucosal cells of the upper gastrointestinal (GI) tract (tongue, mouth, esophagus, and stomach), where it is metabolized. The remainder enters the blood. Of this, 85% to 98% is metabolized in the liver, and only 2% to 10% is excreted through the lungs or kidneys. The major route of ethanol metabolism in the liver is through liver alcohol dehydrogenase (ADH), a cytosolic enzyme that oxidizes ethanol to acetaldehyde with reduction of NAD⫹ to NADH (Fig. 25.2). If it is not removed by metabolism, acetaldehyde exerts toxic actions in the liver and can enter the blood and exert toxic effects in other tissues. Approximately 90% of the acetaldehyde that is generated is further metabolized to acetate in the liver. The major enzyme involved is a low Km mitochondrial acetaldehyde dehydrogenase (ALDH), which oxidizes acetaldehyde to acetate with generation of NADH (see Fig. 25.2). Acetate, which has no toxic effects, may be activated to acetyl coenzyme A (acetyl-CoA) in the liver (where it can enter either the tricarboxylic acid [TCA] cycle or the pathway for fatty acid synthesis). However, most of the acetate that is generated enters the blood and is activated to acetyl-CoA in skeletal muscles and other tissues (see Fig. 25.1). Acetate is generally considered nontoxic and is a normal constituent of the diet. The other principal route of ethanol oxidation in the liver is the microsomal ethanol oxidizing system (MEOS), which also oxidizes ethanol to acetaldehyde (Fig. 25.3). The principal microsomal enzyme involved is a cytochrome P450 mixed-function oxidase isozyme (CYP2E1), which uses NADPH as an additional electron donor and O2 as an electron acceptor. This route accounts for only 10% to 20% of ethanol oxidation in a moderate drinker. Each of the enzymes involved in ethanol metabolism (ADH, ALDH, and CYP2E1) exists as a family of isoenzymes. Individual variations in the quantity of these isoenzymes influence several factors such as the rate of ethanol clearance from the blood, the degree of inebriation exhibited by an individual, and differences in individual susceptibility to the development of alcohol-induced liver disease.

A. Alcohol Dehydrogenase ADH exists as a family of isoenzymes with varying specificity for chain length of the alcohol substrate (Table 25.1). Ethanol is a small molecule that does not exhibit much in the way of unique structural characteristics and, at high concentrations, is nonspecifically metabolized by many members of the ADH family. The ADHs that exhibit the highest specificity for ethanol are members of the ADH1 family. We have three genes for this family of ADHs, each of which exists as allelic variants (polymorphisms). The ADH1 alcohol dehydrogenases are present in high quantities in the liver, representing approximately 3% of all soluble protein. These ADHs, commonly referred

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Jaundice is a yellow discoloration involving the sclerae (the “whites” of the eyes) and skin. It is caused by the deposition of bilirubin, a yellow degradation product of heme. Bilirubin accumulates in the blood under conditions of liver injury, bile duct obstruction, and excessive degradation of heme. Jean Ann Tonich’s admitted ethanol consumption exceeds the definition of moderate drinking. Moderate drinking is now defined as not more than two drinks per day for men, but only one drink per day for women. A drink is defined as 12 oz of regular beer, 5 oz of wine, or 1.5 oz distilled spirits (80 proof).

CH3CH2OH Ethanol NAD+ ADH NADH + H+ O CH3 C

H

Acetaldehyde NAD+ ALDH NADH + H+ O CH3 C

O–

Acetate

FIG. 25.2. The pathway of ethanol metabolism. ADH, alcohol dehydrogenase; ALDH, acetaldehyde dehydrogenase.

CH3CH2OH Ethanol M E O S

NADPH + H+ + O2 NADP+ + 2H2O O

ER

CH3 C

H

Acetaldehyde

FIG. 25.3. The reaction catalyzed by the MEOS (which includes CYP2E1) in the endoplasmic reticulum (ER).

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Table 25.1 Major Isozymes of Medium-Chain-Length Alcohol Dehydrogenases Gene

Subunit

Tissue Distribution

Properties

ADH1A ADH1B ADH1C

␣ ␤ ␥

Km of 0.02 to 5 mM for ethanol. Active only with ethanol. High tissue capacity.

ADH2

␲

ADH3

␹

Most abundant in liver and adrenal glands. Much lower levels in kidney, lung, colon, small intestine, eye, ovary, blood vessels. None in brain or heart. Primarily liver, lower levels in GI tract. Ubiquitously expressed, but at higher levels in liver. The only isozyme present in germinal cells.

ADH4

␴

Present in highest levels in upper GI tract, gingiva and mouth, esophagus, down to the stomach. Not present in liver.

Km of 23 mM for ethanol. Relatively inactive toward ethanol (Km ⫽ 3400 mM). Active mainly toward long-chain alcohols, and ␻-OH fatty acids. Km of 58 mM. It is the most active of medium-chain alcohol dehydrogenases toward the substrate retinal.

GI, gastrointestinal.

to collectively as liver alcohol dehydrogenase, have a low Km for ethanol between 0.02 and 5 mM (high affinities). Thus, the liver is the major site of ethanol metabolism and the major site at which the toxic metabolite acetaldehyde is generated. Although the ADH4 and ADH2 enzymes make minor contributions to ethanol metabolism, they may contribute to its toxic effects. Ethanol concentrations can be quite high in the upper GI tract (e.g., beer is approximately 0.8 M ethanol), and acetaldehyde generated here by ADH4 enzymes (gastric ADH) might contribute to the risk for cancer associated with heavy drinking. Class II ADH genes are expressed primarily in the liver and at lower levels in the lower GI tract. The human has at least seven and possibly more genes that code for specific isoenzymes of medium-chain-length ADHs, the major enzyme responsible for the oxidation of ethanol to acetaldehyde in the human. These different ADHs have an approximately 60% to 70% identity and are assumed to have arisen from a common ancestral gene similar to the ADH3 isoenzyme many millions of years ago. The ADH1 alcohol dehydrogenases (ADH1A, ADH1B, and ADH1C) are all present in high concentration in the liver, and they have relatively high affinity and capacity for ethanol at low concentrations. (These properties are quantitatively reflected by their low Km, a parameter discussed in Chapter 9). They have a 90% to 94% sequence identity and are able to form both homodimers and heterodimers among themselves (e.g., ␤␤ or ␤␥). However, none of the ADH1s can form dimers with an ADH from ADH2, ADH3, or ADH4. The three genes for class I ADH are arranged in tandem, head to tail, on chromosome 4. The genes for the other classes of ADH are also on chromosome 4 in nearby locations.

B. Acetaldehyde Dehydrogenases Acetaldehyde is oxidized to acetate with the generation of NADH by acetaldehyde dehydrogenases (see Fig. 25.2). More than 80% of acetaldehyde oxidation in the human liver is normally catalyzed by mitochondrial acetaldehyde dehydrogenase (ALDH2), which has a high affinity for acetaldehyde (Km of 0.2 ␮M) and is highly specific. However, individuals with a common allelic variant of ALDH2 (designated as ALDH2*2) have a greatly decreased capacity for acetaldehyde metabolism due to an increased Km (46 ␮M) and a decreased Vmax (0.017 units/mg vs. 0.60 units/mg). Most of the remainder of acetaldehyde oxidation occurs through a cytosolic acetaldehyde dehydrogenase (ALDH1). Additional aldehyde dehydrogenases act on a variety of organic alcohols, toxins, and pollutants.

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The accumulation of acetaldehyde causes nausea and vomiting, and therefore, inactive acetaldehyde dehydrogenases are associated with a distaste for alcoholic beverages and protection against alcoholism. In one of the common allelic variants of ALDH2 (ALDH2*2), a single substitution increases the Km for acetaldehyde 23-fold (lowers the affinity) and decreases the Vmax 35-fold resulting in a very inactive enzyme. Homozygosity for the ALDH2*2 allele affords absolute protection against alcoholism; no individual with this genotype has been found among alcoholics. Alcoholics are frequently treated with acetaldehyde dehydrogenase inhibitors (e.g., disulfiram) to help them abstain from alcohol intake. Unfortunately, alcoholics who continue to drink while taking this drug are exposed to the toxic effects of elevated acetaldehyde levels. O

C. Fate of Acetate Metabolism of acetate requires activation to acetyl-CoA by acetyl-CoA synthetase in a reaction similar to that catalyzed by fatty acyl-CoA synthetases (Fig. 25.4). In liver, the principal isoform of acetyl-CoA synthetase (ACS I) is a cytosolic enzyme that generates acetyl-CoA for the cytosolic pathways of cholesterol and fatty acid synthesis. Acetate entry into these pathways is under regulatory control by mechanisms involving cholesterol or insulin. Thus, most of the acetate generated enters the blood. Acetate is taken up and oxidized by other tissues, notably heart and skeletal muscle, which have a high concentration of the mitochondrial acetyl-CoA synthetase isoform (ACS II). This enzyme is present in the mitochondrial matrix. It therefore generates acetyl-CoA that can enter the TCA cycle directly and be oxidized to CO2.

CH3

C

O– Acetate

Acetyl CoA synthetase

CoASH + ATP AMP + PPi

CH3

C

O SCoA

Acetyl CoA

FIG. 25.4. acetyl-CoA.

The activation of acetate to

D. The Microsomal Ethanol Oxidizing System Ethanol is also oxidized to acetaldehyde in the liver by the MEOS, which comprises members of the cytochrome P450 superfamily of enzymes. Ethanol and NADPH both donate electrons in the reaction, which reduces O2 to 2H2O (Fig. 25.5). The cytochrome P450 enzymes all have two major catalytic protein components: an electron-donating reductase system that transfers electrons from NADPH (cytochrome P450 reductase) and a cytochrome P450. The cytochrome P450 protein contains the binding sites for O2 and the substrate (e.g., ethanol) and carries out the reaction. The enzymes are present in the endoplasmic reticulum, which on isolation from disrupted cells forms a membrane fraction after centrifugation that was formerly called microsomes by biochemists. 1.

CYP2E1

The MEOS is part of the superfamily of cytochrome P450 enzymes, all of which catalyze similar oxidative reactions. Within the superfamily, at least 10 distinct gene families are found in mammals. More than 100 different cytochrome P450 isozymes exist within these 10-gene families. Each isoenzyme has a distinct classification according to its structural relationship with other isoenzymes. The isoenzyme that has the highest activity toward ethanol is called CYP2E1. CYP represents cytochrome P450. In CYP2E1, the “2” refers to the gene family (isozymes with ⬎40% amino acid sequence identity), the “E” to the subfamily (isozymes with ⬎55% sequence identity), and the “1” refers to the individual enzymes within this subfamily. A great deal of overlapping specificity exists among the various P450 isoenzymes, and ethanol is also oxidized by several other P450 isoenzymes. MEOS refers to the combined ethanol-oxidizing activity of all the P450 enzymes. CYP2E1 has a much higher Km for ethanol than the ADH1 alcohol dehydrogenases (11 mM [51 mg/dL], compared with 0.02 to 5 mM [0.09 to 22.5 mg/dL]). Thus, a greater proportion of ingested ethanol is metabolized through CYP2E1 at high levels of ethanol consumption than at low levels.

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NADP+, H+

NADPH FAD e– e–

RH

O2

ROH, H2O

FMN Fe-heme

Cytochrome Cytochrome P450 reductase P450

FIG. 25.5. General structure of cytochrome P450 enzymes. O2 binds to the P450 Fe-heme in the active site and is activated to a reactive form by accepting electrons. The electrons are donated by the cytochrome P450 reductase, which contains a flavin adenine dinucleotide (FAD) plus a flavin mononucleotide (FMN) or Fe–S center to facilitate the transfer of single electrons from NADPH to O2. The P450 enzymes involved in steroidogenesis have a somewhat different structure. For CYP2E1, RH is ethanol (CH3CH2OH) and ROH is acetaldehyde (CH3CHO).

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ADH1A and ADH1C are present as functional polymorphisms that differ in their properties. Genetic polymorphisms for ADH partially account for the observed differences in ethanol elimination rates among various individuals or populations. Although susceptibility to alcoholism is a complex function of genetics and socioeconomic factors, possession of the ADH1B*2 allele, which encodes a relatively fast ADH (high Vmax), is associated with a decreased susceptibility to alcoholism—presumably because of nausea and flushing caused by acetaldehyde accumulation (because the aldehyde dehydrogenase gene cannot keep up with the amount of acetaldehyde produced). This particular allele has a relatively high frequency in the East Asian population and a low frequency among white Europeans. In contrast, the ADH1B*1/1B*1 genotype (homozygous for allele 1 of the ADH1B gene) is a risk factor for the development of Wernicke-Korsakoff syndrome, a neuropsychiatric syndrome that is commonly associated with alcoholism.

As blood ethanol concentration rises above 18 mM (the legal intoxication limit is now defined as 0.08% in most states of the United States, which is approximately 18 mM), the brain and central nervous system are affected. Induction of CYP2E1 increases the rate of ethanol clearance from the blood, thereby contributing to increased alcohol tolerance. However, the apparent ability of a chronic alcoholic to drink without appearing inebriated is partly a learned behavior.

2.

INDUCTION OF P450 ENZYMES

The P450 enzymes are inducible both by their most specific substrate and by substrates for some of the other cytochrome P450 enzymes. Chronic consumption of ethanol increases hepatic CYP2E1 levels approximately five-fold to ten-fold. However, it also causes a twofold to fourfold increase in some of the other P450s from the same subfamily, from different subfamilies, and even from different gene families. The endoplasmic reticulum undergoes proliferation, with a general increase in the content of microsomal enzymes, including those that are not involved directly in ethanol metabolism. The increase in CYP2E1 with ethanol consumption occurs through transcriptional, posttranscriptional, and posttranslational regulation. Increased levels of mRNA resulting from induction of gene transcription or stabilization of message are found in actively drinking patients. The protein is also stabilized against degradation. In general, the mechanism for induction of P450 enzymes by their substrates occurs through the binding of the substrate (or related compound) to an intracellular receptor protein, followed by binding of the activated receptor to a response element in the target gene. Ethanol induction of CYP2E1 appears to act via stabilization of the protein and protection against degradation (an increased half-life for the synthesized protein). Although induction of CYP2E1 increases ethanol clearance from the blood, it has negative consequences. Acetaldehyde may be produced faster than it can be metabolized by ALDH, thereby increasing the risk of hepatic injury. An increased amount of acetaldehyde can enter the blood and can damage other tissues. In addition, cytochrome P450 enzymes are capable of generating free radicals, which also may lead to increased hepatic injury and cirrhosis (see Chapter 24). Overlapping specificity in the catalytic activity of P450 enzymes and in their inducers is responsible for several types of drug interactions. For example, phenobarbital, a barbiturate long used as a sleeping pill or for treatment of epilepsy, is converted to an inactive metabolite by cytochrome P450 monooxygenases CYP2B1 and CYP2B2. After treatment with phenobarbital, CYP2B2 is increased 50- to 100-fold. Individuals who take phenobarbital for prolonged periods develop a drug tolerance as CYP2B2 is induced, and the drug is metabolized to an inactive metabolite more rapidly. Consequently, these individuals use progressively higher doses of phenobarbital. Ethanol is an inhibitor of the phenobarbital-oxidizing P450 system. When large amounts of ethanol are consumed, the inactivation of phenobarbital is directly or indirectly inhibited. Therefore, when high doses of phenobarbital and ethanol are consumed at the same time, toxic levels of the barbiturate can accumulate in the blood.

E. Variations in the Pattern of Ethanol Metabolism The routes and rates of ethanol oxidation vary from individual to individual. Differences in ethanol metabolism may influence whether an individual becomes a chronic alcoholic, develops alcohol-induced liver disease, or develops other diseases associated with increased alcohol consumption (such as hepatocarcinogenesis, lung cancer, or breast cancer). Factors that determine the rate and route of ethanol oxidation in individuals include • Genotype—Polymorphic forms of ADHs and ALDHs can greatly affect the rate of ethanol oxidation and the accumulation of acetaldehyde. CYP2E1 activity may vary as much as 20-fold among individuals, partly because of differences in the inducibility of different allelic variants. • Drinking history—The level of gastric ADH decreases and CYP2E1 increases with the progression from a naive, to a moderate, and to a heavy and chronic consumer of alcohol.

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• Gender—Blood levels of ethanol after consuming a drink are normally higher for women than for men, partly because of lower levels of gastric ADH activity in women. After chronic consumption of ethanol, gastric ADH decreases in both men and women but the gender differences become even greater. Gender differences in blood alcohol levels also occur because women are normally smaller. Furthermore, in women, alcohol is distributed in a 12% smaller water space because a woman’s body composition consists of more fat and less water than that of a man. • Quantity—The amount of ethanol an individual consumes over a short period of time determines its metabolic route. Small amounts of ethanol are metabolized most efficiently through the low-Km pathway of ADH1 genes and ALDH2 genes. Little accumulation of NADH occurs to inhibit ethanol metabolism via these dehydrogenases. However, when higher amounts of ethanol are consumed within a short period, a disproportionately greater amount is metabolized through the MEOS. The MEOS, which has a much higher Km for ethanol, functions principally at high concentrations of ethanol. A higher activity of the MEOS may be expected to correlate with a tendency to develop alcoholinduced liver disease because both acetaldehyde and free radical levels will be increased.

F. The Energy Yield of Ethanol Oxidation The adenosine triphosphate (ATP) yield from ethanol oxidation to acetate varies with the route of ethanol metabolism. If ethanol is oxidized by the major route of cytosolic ADH and mitochondrial ALDH, one cytosolic and one mitochondrial NADH are generated with a maximum yield of 5 ATP. Oxidation of acetyl-CoA in the TCA cycle and the electron-transport chain leads to the generation of 10 highenergy phosphate bonds. However, activation of acetate to acetyl-CoA requires two high-energy phosphate bonds (one in the cleavage of ATP to adenosine monophosphate (AMP)⫹ pyrophosphate and one in the cleavage of pyrophosphate to phosphate), which must be subtracted. Thus, the maximum total energy yield is 13 mol of ATP per mole of ethanol. In contrast, oxidation of ethanol to acetaldehyde by CYP2E1 consumes energy in the form of NADPH, which is equivalent to 2.5 ATP. Thus, for every mole of ethanol metabolized by this route, only a maximum of 8.0 mol of ATP can be generated (10 ATP from acetyl-CoA oxidation through the TCA cycle, minus 2 for acetate activation; the NADH generated by aldehyde dehydrogenase is balanced by the loss of NADPH in the MEOS step).

II. TOXIC EFFECTS OF ETHANOL METABOLISM Alcohol-induced liver disease, a common and sometimes fatal consequence of chronic ethanol abuse, may manifest itself in three forms: fatty liver, alcoholinduced hepatitis, and cirrhosis. Each may occur alone, or they may be present in any combination in a given patient. Alcohol-induced cirrhosis is discovered in up to 9% of all autopsies performed in the United States, with a peak incidence in patients 40 to 55 years of age. However, ethanol ingestion also has acute effects on liver metabolism including inhibition of fatty acid oxidation and stimulation of triacylglycerol synthesis leading to a fatty liver. It also can result in ketoacidosis or lactic acidosis and cause hypoglycemia or hyperglycemia, depending on the dietary state. These effects are considered reversible. In contrast, acetaldehyde and free radicals generated from ethanol metabolism can result in alcohol-induced hepatitis, a condition in which the liver is inflamed and cells become necrotic and die. Diffuse damage to hepatocytes results in cirrhosis, characterized by fibrosis (scarring), disturbance of the normal architecture and blood flow, loss of liver function, and, ultimately, hepatic failure.

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At Ivan Applebod’s low level of ethanol consumption, ethanol is oxidized to acetate via ADH and ALDH in the liver and the acetate is activated to acetyl-CoA and oxidized to CO2 in skeletal muscle and other tissues. The overall energy yield of 13 ATP per ethanol molecule accounts for the caloric value of ethanol, approximately 7 kcal/g. However, chronic consumption of substantial amounts of alcohol does not have the effect on body weight expected from the caloric intake. This is partly attributable to induction of MEOS, resulting in a proportionately greater metabolism of ethanol through the MEOS with its lower energy yield (only approximately 8 ATP). In general, weight loss diets recommend no, or low, alcohol consumption because ethanol calories are “empty” in the sense that alcoholic beverages are generally low in vitamins, essential amino acids, and other required nutrients but not empty of calories.

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A. Acute Effects of Ethanol Arising from the Increased NADH/ NADⴙ Ratio Many of the acute effects of ethanol ingestion arise from the increased NADH/ NAD⫹ ratio in the liver (Fig. 25.6). At lower levels of ethanol intake, the rate of ethanol oxidation is regulated by the supply of ethanol (usually determined by how much ethanol we consume) and the rate at which NADH is reoxidized in the electron-transport chain. NADH is not a very effective product inhibitor of ADH or ALDH, and there is no other feedback regulation by ATP, ADP, or AMP. As a consequence, NADH generated in the cytosol and mitochondria tends to accumulate, increasing the NADH/NAD⫹ ratio to high levels (see Fig. 25.6, circle 1). The increase is even greater as the mitochondria become damaged from acetaldehyde or free radical injury.

NADPH

9

Interference, inhibition of drug metabolism

MEOS

Ethanol ADH

1

Acetaldehyde (toxin) NADH

ALDH NADH

H+ H+

e t c

TCA cycle

4

Glucose Gluconeogenesis

NADH FAD (2H)

Glycolysis DHAP

NAD+ Lactate

NADH Oxaloacetate

5

Ketone bodies β-oxidation

Ketoacidosis

2

Acetyl CoA NAD+

Glycerol 3-phosphate

Pyruvate

6

Malate

Acetyl CoA

NADH

NADH

Acetate (blood)

Acetate

8 Glycerol Alanine and other gluconeogenic precursors

3 Fatty acyl CoA

Fatty acids

ER Triacylglycerols Fatty steatosis VLDL

Purines

7 Lactate acidemia

Uric acid

Hypoglycemia

Hyperlipidemia

– Gout

Urine

FIG. 25.6. Acute effects of ethanol metabolism on lipid metabolism in the liver. (1) Metabolism of ethanol generates a high NADH/NAD⫹ ratio. (2) The high NADH/NAD⫹ ratio inhibits fatty acid oxidation and the TCA cycle resulting in accumulation of fatty acids. (3) Fatty acids are reesterified to glycerol 3-phosphate by acyltransferases in the endoplasmic reticulum. Glycerol 3-phosphate levels are increased because a high NADH/NAD⫹ ratio favors its formation from dihydroxyacetone phosphate (an intermediate of glycolysis). Ethanol-stimulated increases of endoplasmic reticulum enzymes also favor triacylglycerol formation. (4) NADH generated from ethanol oxidation can meet the requirements of the cell for ATP generation from oxidative phosphorylation. Thus, acetyl-CoA oxidation in the TCA cycle is inhibited. (5) The high NADH/ NAD⫹ ratio shifts oxaloacetate (OAA) toward malate, and acetyl-CoA is directed into ketone-body synthesis. Options (6) through (8) are discussed in the text.

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1.

CHANGES IN FATTY ACID METABOLISM

2.

ALCOHOL-INDUCED KETOACIDOSIS

465

The high NADH/NAD⫹ ratio generated from ethanol oxidation inhibits the oxidation of fatty acids, which accumulate in the liver (see Fig. 25.6, circles 2 and 3) These fatty acids are reesterified into triacylglycerols by combining with glycerol 3-phosphate (glycerol 3-P). The increased NADH/NAD⫹ ratio increases the availability of glycerol 3-P by promoting its synthesis from intermediates of glycolysis. The triacylglycerols are incorporated into very low-density lipoproteins (VLDLs) that accumulate in the liver and enter the blood resulting in an ethanol-induced hyperlipidemia. Although just a few drinks may result in hepatic fat accumulation, chronic consumption of alcohol greatly enhances the development of a fatty liver. Reesterification of fatty acids into triacylglycerols by fatty acyl transferases in the endoplasmic reticulum is enhanced (see Fig. 25.6). Because the transferases are microsomal enzymes, they are induced by ethanol consumption just as the MEOS is induced. The result is a fatty liver (hepatic steatosis). The source of the fatty acids can be dietary fat, fatty acids synthesized in the liver, or fatty acids released from adipose tissue stores. Adipose tissue lipolysis increases after ethanol consumption, possibly because of a release of epinephrine.

Fatty acids that are oxidized are converted to acetyl-CoA and subsequently to ketone bodies (acetoacetate and ␤-hydroxybutyrate). Enough NADH is generated from oxidation of ethanol and fatty acids that there is no need to oxidize acetyl-CoA in the TCA cycle. The very high NADH/NAD⫹ ratio shifts the oxaloacetate (OAA) in the TCA cycle to malate, leaving the OAA levels too low for citrate synthase to synhesize citrate (see Fig. 25.6, circle 4). The acetyl-CoA enters the pathway for ketone body synthesis instead of the TCA cycle. Although ketone bodies are being produced at a high rate, their metabolism in other tissues is restricted by the supply of acetate, which is the preferred fuel. Thus, the blood concentration of ketone bodies may be much higher than is found under normal fasting conditions. 3.

Al Martini’s admitting physician suspected an alcohol-induced ketoacidosis superimposed on a starvation ketoacidosis. Tests showed that his plasmafree fatty acid level was elevated, and his plasma ␤-hydroxybutyrate level was 40 times the upper limit of normal. The increased NADH/ NAD⫹ ratio from ethanol consumption inhibited the TCA cycle and shifted acetyl-CoA from fatty acid oxidation into the pathway of ketone-body synthesis.

LACTIC ACIDOSIS, HYPERURICEMIA, AND HYPOGLYCEMIA

Another consequence of the very high NADH/NAD⫹ ratio is that the balance in the lactate dehydrogenase reaction is shifted toward lactate, resulting in a lactic acidosis (see Fig. 25.6, circle 6). The elevation of blood lactate may decrease excretion of uric acid (see Fig. 25.6, circle 7) by the kidney. Consequently, patients with gout (which results from precipitated uric acid crystals in the joints) are advised not to drink excessive amounts of ethanol. Increased degradation of purines also may contribute to hyperuricemia. The increased NADH/NAD⫹ ratio also can cause hypoglycemia in a fasting individual who has been drinking and is dependent on gluconeogenesis to maintain blood glucose levels (Fig. 25.6, circles 6 and 8). Alanine and lactate are major gluconeogenic precursors that enter gluconeogenesis as pyruvate. The high NADH/ NAD⫹ ratio shifts the lactate dehydrogenase equilibrium to lactate, so that pyruvate formed from alanine is converted to lactate and cannot enter gluconeogenesis. The high NADH/NAD⫹ ratio also prevents other major gluconeogenic precursors, such as OAA and glycerol, from entering the gluconeogenic pathway. In contrast, ethanol consumption with a meal may result in a transient hyperglycemia, possibly because the high NADH/NAD⫹ ratio inhibits glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step.

B. Acetaldehyde Toxicity Many of the toxic effects of chronic ethanol consumption result from accumulation of acetaldehyde, which is produced from ethanol by both ADHs and the MEOS.

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H2O Oxidized glutathione

Lipid peroxidation Toxic radicals (ROS)

3 NADPH

+

NAD

Binding to glutathione

–

1 Binding to amino acids

2

+

Proteins (clotting factors) Binding to microtubules

Proteins

– Impaired protein secretion

MEOS

ADH

Ethanol

NADP

Amino acids

Acetaldehyde

NADH

Acetaldehyde Acetaldehyde

4 e t c

Freeradical injury Release of enzymes ALT and AST

6

NADH

Acetate

Acetate

–

Fatty acids Glycerol 3-phosphate

5

Swelling H2O

Triacylglycerols

VLDL

VLDL

Protein and lipid accumulation due to impaired secretion H2O

FIG. 25.7. The development of alcohol-induced hepatitis. (1) Acetaldehyde-adduct formation decreases protein synthesis and impairs protein secretion. (2) Free radical injury results partly from acetaldehyde-adduct formation with glutathione. (3) Induction of the MEOS increases formation of free radicals, which leads to lipid peroxidation and cell damage. (4) Mitochondrial damage inhibits the electron transport chain, which decreases acetaldehyde oxidation. (5) Microtubule damage increases VLDL and protein accumulation. (6) Cell damage leads to release of the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Acetaldehyde accumulates in the liver and is released into the blood after heavy doses of ethanol (Fig. 25.7). It is highly reactive and binds covalently to amino groups, sulfhydryl groups, nucleotides, and phospholipids to form “adducts.” 1.

ACETALDEHYDE AND ALCOHOL-INDUCED HEPATITIS

One of the results of acetaldehyde-adduct formation with amino acids is a general decrease in hepatic protein synthesis (see Fig. 25.7, circle 1). Calmodulin, ribonuclease, and tubulin are some of the proteins affected. Proteins in the heart and other tissues also may be affected by acetaldehyde that appears in the blood. As a consequence of forming acetaldehyde adducts of tubulin, there is a diminished secretion of serum proteins and VLDL from the liver. The liver synthesizes many blood proteins, including serum albumin, blood coagulation factors, and transport proteins for vitamins, steroids, and iron. These proteins accumulate in the liver together with lipid. The accumulation of proteins results in an influx of water (see Fig. 25.7, circle 6) within the hepatocytes and a swelling of the liver that contributes to portal hypertension and a disruption of hepatic architecture. 2.

ACETALDEHYDE AND FREE RADICAL DAMAGE

Acetaldehyde-adduct formation enhances free radical damage. Acetaldehyde binds directly to glutathione and diminishes its ability to protect against H2O2 and prevent lipid peroxidation (see Fig. 25.7, circle 2). It also binds to free radical defense enzymes.

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Damage to mitochondria from acetaldehyde and free radicals perpetuates a cycle of toxicity (see Fig. 25.7, circles 3 and 4). With chronic consumption of ethanol, mitochondria become damaged, the rate of electron transport is inhibited, and oxidative phosphorylation tends to become uncoupled. Fatty acid oxidation is decreased even further, thereby enhancing lipid accumulation (see Fig. 25.7, circle 5). The mitochondrial changes further impair mitochondrial acetaldehyde oxidation, thereby initiating a cycle of progressively increasing acetaldehyde damage.

C. Ethanol and Free Radical Formation Increased oxidative stress in the liver during chronic ethanol intoxication arises from increased production of free radicals, principally by CYP2E1. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the reductase and heme in the cytochrome P450 system transfer single electrons, thus operating through a mechanism that can generate free radicals. The hydroxyethyl radical (CH3CH2O•) is produced during ethanol metabolism and can be released as a free radical. Induction of CYP2E1 as well as other cytochrome P450 enzymes can increase the generation of free radicals from drug metabolism and from the activation of toxins and carcinogens (see Fig. 25.7, circle 3). These effects are enhanced by acetaldehyde-adduct damage. Phospholipids, the major lipid in cellular membranes, are a primary target of peroxidation caused by free radical release. Peroxidation of lipids in the inner mitochondrial membrane may contribute to the inhibition of electron transport and uncoupling of mitochondria, leading to inflammation and cellular necrosis. Induction of CYP2E1 and other P450 cytochromes also increases formation of other radicals and the activation of hepatocarcinogens.

D. Hepatic Cirrhosis and Loss of Liver Function Liver injury is irreversible at the stage that hepatic cirrhosis develops. Initially, the liver may be enlarged, full of fat, crossed with collagen fibers (fibrosis), and have nodules of regenerating hepatocytes ballooning between the fibers. As liver function is lost, the liver becomes shrunken (Laennec cirrhosis). During the development of cirrhosis, many of the normal metabolic functions of the liver are lost, including biosynthetic and detoxification pathways. Synthesis of blood proteins including blood coagulation factors and serum albumin is decreased. The capacity to incorporate amino groups into urea is decreased, resulting in the accumulation of toxic levels of ammonia in the blood. Conjugation and excretion of the yellow pigment bilirubin (a product of heme degradation) is diminished, and bilirubin accumulates in the blood. It is deposited in many tissues, including the skin and sclerae of the eyes, causing the patient to become visibly yellow. Such a patient is said to be jaundiced. CLINICAL COMMENTS Ivan Applebod. When ethanol consumption is low (⬍15% of the calories in the diet), it is used efficiently to produce adenosine triphosphate (ATP), thereby contributing to Ivan Applebod’s weight gain. However, in individuals with chronic consumption of large amounts of ethanol, the caloric content of ethanol is not converted to ATP as effectively. Some of the factors that may contribute to this decreased efficiency include mitochondrial damage (inhibition of oxidative phosphorylation and uncoupling) resulting in the loss of calories as heat, increased recycling of metabolites such as ketone bodies, and inhibition of the normal pathways of fatty acid and glucose oxidation. In addition, heavier drinkers metabolize an increased amount of alcohol through the microsomal ethanol oxidizing system (MEOS), which generates less ATP.

467

The noncaloric effect of heavy and chronic ethanol ingestion that led Ivan Applebod to believe ethanol has no calories may be partly attributable to uncoupling of oxidative phosphorylation. The hepatic mitochondria from tissues of chronic alcoholics may be partially uncoupled and unable to maintain the transmembrane proton gradient necessary for normal rates of ATP synthesis. Consequently, a greater proportion of the energy in ethanol is converted to heat. Metabolic disturbances such as the loss of ketone bodies in urine or futile cycling of glucose also might contribute to a diminished energy value for ethanol.

Because of the possibility of mild alcoholic hepatitis and perhaps chronic alcohol-induced cirrhosis, the physician ordered liver function studies on Jean Ann Tonich. The tests indicated an alanine aminotransferase (ALT) level of 46 U/L (reference range, 5 to 30 U/L) and an aspartate aminotransferase (AST) level of 98 U/L (reference range, 10 to 30 U/L). The concentration of these enzymes is high in hepatocytes. When hepatocellular membranes are damaged in any way, these enzymes are released into the blood. Jean Ann’s serum alkaline phosphatase level was 195 U/L (reference range, 56 to 155 U/L for an adult female). The serum total bilirubin level was 2.4 mg/dL (reference range, 0.2 to 1.0 mg/L). These tests show impaired capacity for normal liver function. Her blood hemoglobin and hematocrit levels were slightly below the normal range, consistent with a toxic effect of ethanol on red blood cell production by bone marrow. Serum folate and vitamin B12 levels were also slightly suppressed. Folate is dependent on the liver for its activation and recovery from the enterohepatic circulation. Vitamin B12 is dependent on the liver for synthesis of its blood carrier proteins. Thus, Jean Ann Tonich shows many of the consequences of hepatic damage.

Al Martini. Al Martini was suffering from acute effects of high ethanol ingestion in the absence of food intake. Both heavy ethanol consumption and low caloric intake increase adipose tissue lipolysis and elevate

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In liver fibrosis, disruption of the normal liver architecture, including sinusoids, impairs blood from the portal vein. Increased portal vein pressure (portal hypertension) causes capillaries to anastomose (to meet and unite or run into each other) and form thin-walled dilated esophageal venous conduits known as esophageal varices. When these burst, there is hemorrhaging into the GI tract. The bleeding can be very profuse because of the high venous pressure within these varices in addition to the adverse effect of impaired hepatic function on the production of blood-clotting proteins.

Although the full spectrum of alcohol-induced liver disease may be present in a well-nourished individual, the presence of nutritional deficiencies enhances the progression of the disease. Ethanol creates nutritional deficiencies in several different ways. The ingestion of ethanol reduces the GI absorption of foods that contain essential nutrients including vitamins, essential fatty acids, and essential amino acids. For example, ethanol interferes with absorption of folate, thiamine, and other nutrients. Secondary malabsorption can occur through GI complications, pancreatic insufficiency, and impaired hepatic metabolism or impaired hepatic storage of nutrients such as vitamin A. Changes in the level of transport proteins produced by the liver also strongly affect nutrient status.

blood fatty acids. As a consequence of his elevated hepatic NADH/NAD⫹ ratio, acetyl coenzyme A (acetyl-CoA) produced from fatty acid oxidation was diverted from the tricarboxylic acid (TCA) cycle into the pathway of ketone body synthesis. Because his skeletal muscles were using acetate as a fuel, ketone body use was diminished, resulting in ketoacidosis. Al’s moderately low blood glucose level also suggests that his high hepatic NADH level prevented pyruvate and glycerol from entering the gluconeogenic pathway. Pyruvate is diverted to lactate, which may have contributed to his metabolic acidosis and anion gap. Rehydration with intravenous fluids containing glucose and potassium was initiated. Al’s initial potassium level was low, possibly secondary to vomiting. An orthopedic surgeon was consulted regarding the compound fracture of his right forearm. Jean Ann Tonich. Jean Ann Tonich’s signs and symptoms as well as her laboratory profile were consistent with the presence of mild reversible alcohol-induced hepatocellular inflammation (alcohol-induced hepatitis) superimposed on a degree of irreversible scarring of liver tissues known as chronic alcoholic (Laennec) cirrhosis of the liver. The chronic inflammatory process associated with long-term ethanol abuse in patients such as Jean Ann Tonich is accompanied by increases in the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Her elevated bilirubin and alkaline phosphatase levels in the blood were consistent with hepatic damage. Her values for ALT and AST were significantly below those seen in acute viral hepatitis. In addition, the ratio of the absolute values for serum ALT and AST often differ in the two diseases tending to be ⬎1 in acute viral hepatitis and ⬍1 in chronic alcohol-induced cirrhosis. The reason for the difference in ratio of enzyme activities released is not fully understood, but a lower level of ALT in the serum may be attributable to an alcohol-induced deficiency of pyridoxal phosphate. In addition, serologic tests for viral hepatitis were nonreactive. Her serum folate and vitamin B12 were also slightly suppressed indicating impaired nutritional status. Jean Ann was strongly cautioned to abstain from alcohol immediately and to improve her nutritional status. In addition, she was referred to the hospital drug and alcohol rehabilitation unit for appropriate psychologic therapy and supportive social counseling. The physician also arranged for a follow-up office visit in 2 weeks.

BIOCHEMICAL COMMENTS Fibrosis in Chronic Alcohol-Induced Liver Disease. Fibrosis is the excessive accumulation of connective tissue in parenchymal organs. In the liver, it is a frequent event following a repeated or chronic insult of sufficient intensity (such as chronic ethanol intoxication or infection by a hepatitis virus) to trigger a “wound healing–like” reaction. Regardless of the insult, the events are similar: An overproduction of extracellular matrix components occurs, with the tendency to progress to sclerosis, accompanied by a degenerative alteration in the composition of matrix components (Table 25.2). Some individuals (⬍20% of those who chronically consume alcohol) go on to develop cirrhosis. The development of hepatic fibrosis after ethanol consumption is related to stimulation of the mitogenic development of stellate (Ito) cells into myofibroblasts, and stimulation of the production of collagen type I and fibronectin by these cells. The stellate cells are perisinusoidal cells lodged in the space of Disse that produce extracellular matrix protein. Normally, the space of Disse contains basement membrane–like collagen (collagen type IV) and laminin. As the stellate cells are activated, they change from a resting cell filled with lipids and vitamin A to one that proliferates, loses its vitamin A content, and secretes large quantities of extracellular matrix components.

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Table 25.2

469

Hepatic Injury

Stage of Injury

Main Features

Fibrosis: increase of connective tissue

Accumulation of both fibrillar and basement membrane–like collagens Increase of laminin and fibronectin Thickening of connective tissue septae Capillary formation within the sinusoids Decrease of hyaluronic acid and heparan sulfate proteoglycans Increase of chondroitin sulfate proteoglycans Progressive fragmentation and disappearance of elastic fibers Distortion of sinusoidal architecture and parenchymal damage Whole liver heavily distorted by thick bands of collagen surrounding nodules of hepatocytes with regenerative foci

Sclerosis: aging of fibrotic tissue

Cirrhosis: end-stage process of liver fibrotic degeneration

One of the initial events in the activation and proliferation of stellate cells is the activation of Kupffer cells, which are macrophages that reside in the liver sinusoids (Fig. 25.8). The Kupffer cells are probably activated by a product of the damaged hepatocytes, such as necrotic debris, iron, reactive oxygen species (ROS), acetaldehyde, or aldehyde products of lipid peroxidation. Kupffer cells also may produce acetaldehyde from ethanol internally through their own microsomal ethanol oxidizing system (MEOS) pathway. Activated Kupffer cells produce several products that contribute to activation of stellate cells. They generate additional ROS through NADPH oxidase during the oxidative burst and reactive nitrogen–oxygen species (RNOS) through inducible NO synthase (see Chapter 24). In addition, they secrete an impressive array of growth factors such as cytokines, chemokines, prostaglandins, and other reactive molecules. The cytokine transforming growth factor-␤1 (TGF-␤1), produced by both Kupffer cells and sinusoidal endothelial cells, is a major player in the activation of stellate cells. Once they are activated, the stellate cells produce collagen and proteases leading to an enhanced fibrotic network within the liver. Stellate cells may also be partially activated by hepatocyte release of ROS and acetaldehyde directly, with the involvement of Kuppfer cells.

Hepatocyte

Acetaldehyde Kupffer cell

Acetaldehydeprotein adducts Lipid peroxidation products

Activated Kupffer cell

TGF-β Stellate cell (Vitamin A)

Respiratory burst ROS NO Stimulated stellate cell

Extracellular matrix Collagen

Metallo Proteases

FIBROSIS

FIG. 25.8. Proposed model for the development of hepatic fibrosis involving hepatocytes, Kupffer cells, and stellate (Ito) cells. ROS, reactive oxygen species; NO, nitric oxide; TGF-␤1, transforming growth factor ␤1.

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Table 25.3

Diseases Discussed in Chapter 25

Disease or Disorder

Environmental or Genetic

Obesity

Both

Alcoholism

Both

Jaundice

Environmental

Liver fibrosis

Environmental

Comments Ethanol is a nutrient, and its caloric content can contribute to obesity. Alcohol addiction (alcoholism) may occur, leading to damage of internal organs by acetaldehyde production. Altered liver function leads to a reduced ability to conjugate and solubilize bilirubin, which leads to bilirubin deposition in the eyes and skin, giving them a yellow pallor. Jaundice is an indication of liver disease. Excessive damage to liver due to alcohol metabolism, particularly acetaldehyde accumulation, leading to extensive collagen secretion and loss of liver function.

Key Concepts • • • • •

•

• •

Ethanol metabolism occurs primarily in the liver, via a two-step oxidation sequence to acetate plus NADH. Acetate is activated to acetyl coenzyme A (acetyl-CoA) for energy generation by most tissues of the body. The alcohol dehydrogenase (ADH) family of enzymes catalyzes the first step of alcohol oxidation. The aldehyde dehydrogenase family of enzymes catalyzes the second step of the pathway. When ethanol levels are high, the microsomal ethanol oxidizing system (MEOS), consisting of CYP2E1, is induced. Acute effects of ethanol ingestion are due to the elevated NADH/NAD⫹ ratio, which leads to the following: Inhibition of fatty acid oxidation Ketogenesis Hyperlipidemia Inhibition of gluconeogenesis Lactic acidosis Hyperuricemia Chronic effects of ethanol ingestion include the following: Hepatic steatosis (fatty acid accumulation within the liver) Hepatitis Fibrosis (excessive collagen production within the liver) Cirrhois (eventual liver death) The chronic effects of ethanol are caused by acetaldehyde and reactive oxygen species (ROS) production during ethanol metabolism. The diseases discussed in this chapter are summarized in Table 25.3.

REVIEW QUESTIONS—CHAPTER 25 1.

2.

The fate of acetate, the product of ethanol metabolism, is which of the following? A. It is taken up by other tissues and activated to acetylCoA. B. It is toxic to the tissues of the body and can lead to hepatic necrosis. C. It is excreted in bile. D. It enters the TCA cycle directly to be oxidized. E. It is converted into NADH by ADH. Which of the following would be expected to occur after acute alcohol ingestion? A. The activation of fatty acid oxidation B. Lactic acidosis C. The inhibition of ketogenesis D. An increase in the NAD⫹/NADH ratio E. An increase in gluconeogenesis

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3.

A chronic alcoholic is in treatment for alcohol abuse. The drug disulfiram is prescribed for the patient. This drug deters the consumption of alcohol by which of the following mechanisms? A. Inhibiting the absorption of ethanol so that an individual cannot become intoxicated, regardless of how much he or she drinks B. Inhibiting the conversion of ethanol to acetaldehyde, which cause the excretion of unmetabolized ethanol C. Blocking the conversion of acetaldehyde to acetate, which causes the accumulation of acetaldehyde D. Activating the excessive metabolism of ethanol to acetate, which causes inebriation with consumption of a small amount of alcohol E. Preventing the excretion of acetate, which causes nausea and vomiting

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CHAPTER 25 ■ METABOLISM OF ETHANOL

4.

Induction of CYP2E1 would result in which of the following? A. A decreased clearance of ethanol from the blood B. A decrease in the rate of acetaldehyde production C. A low possibility of the generation of free radicals D. Protection from hepatic damage E. An increase of one’s alcohol tolerance level

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5.

471

Which one of the following consequences of chronic alcohol consumption is irreversible? A. Inhibition of fatty acid oxidation B. Activation of triacylglycerol synthesis C. Ketoacidosis D. Lactic acidosis E. Liver cirrhosis

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SECTION FIVE

Carbohydrate Metabolism

G

lucose is central to all of metabolism. It is the universal fuel for human cells and the source of carbon for the synthesis of most other compounds. Every human cell type uses glucose to obtain energy. The release of insulin and glucagon by the pancreas aids in the body’s use and storage of glucose. Other dietary sugars (mainly fructose and galactose) are converted to glucose or to intermediates of glucose metabolism. Glucose is the precursor for the synthesis of an array of other sugars that are required for the production of specialized compounds, such as lactose, cell surface antigens, nucleotides, or glycosaminoglycans. Glucose is also the fundamental precursor of noncarbohydrate compounds; it can be converted to lipids (including fatty acids, cholesterol, and steroid hormones), amino acids, and nucleic acids. Only those compounds that are synthesized from vitamins, essential amino acids, and essential fatty acids cannot be synthesized from glucose in humans. More than 40% of the calories in the typical diet in the United States are obtained from starch, sucrose, and lactose. These dietary carbohydrates are converted to glucose, galactose, and fructose in the digestive tract (Fig. V.1). Monosaccharides are absorbed from the intestine, enter the blood, and travel to the tissues, where they are metabolized. After glucose is transported into cells, it is phosphorylated by a hexokinase to form glucose 6-phosphate. Glucose 6-phosphate can then enter several metabolic pathways. The three that are common to all cell types are glycolysis, the pentose phosphate pathway, and glycogen synthesis (Fig. V.2). In tissues, fructose and galactose are converted to intermediates of glucose metabolism. Thus, the fate of these sugars parallels that of glucose (Fig. V.3). The major fate of glucose 6-phosphate is oxidation via the pathway of glycolysis (see Chapter 22), which provides a source of adenosine triphosphate (ATP) for all cell types. Cells that lack mitochondria cannot oxidize other fuels. They produce ATP from anaerobic glycolysis (the conversion of glucose to lactic acid). Cells that contain mitochondria oxidize glucose to CO2 and H2O via glycolysis and the

Blood

Starch Lactose

Sucrose

Fructose Glucose Intestine

Galactose

FIG. V.1. Overview of carbohydrate digestion. The major carbohydrates of the diet (starch, lactose, and sucrose) are digested to produce monosaccharides (glucose, fructose, and galactose), which enter the blood.

Glucose Glycogen

Glycogen synthesis

Glucose 1phosphate

Glucose-6-P

Pentose phosphate pathway

Pentose phosphates

Glycolysis

Pyruvate

FIG. V.2.

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Major pathways of glucose metabolism.

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Glucose

Glucose Glycogen ATP Glucose 1-phosphate

Glucose-6-P

Pyruvate

ATP

Galactose

Lactate

TCA cycle NADH FAD(2H) CO2

FIG. V.4. Conversion of glucose to lactate or to CO2. Acetyl-CoA, acetyl coenzyme A; ETC, electron-transport chain.

Pyruvate

tricarboxylic acid (TCA) cycle (Fig. V.4). Some tissues, such as the brain, depend on the oxidation of glucose to CO2 and H2O for energy because they have a limited capacity to use other fuels. Glucose produces the intermediates of glycolysis and the TCA cycle that are used for the synthesis of amino acids and both the glycerol and fatty acid moieties of triacylglycerols (Fig. V.5). Another important fate of glucose 6-phosphate is oxidation via the pentose phosphate pathway, which generates NADPH. The reducing equivalents of NADPH are used for biosynthetic reactions and for the prevention of oxidative damage to cells (see Chapter 24). In this pathway, glucose undergoes oxidation and decarboxylation to five-carbon sugars (pentoses), which may reenter the glycolytic pathway. They also may be used for nucleotide synthesis (Fig. V.6). There are also nonoxidative reactions, which can convert six- and five-carbon sugars.

Oxidative Reactions

Glucose Glucose

Glycine

TG

Cysteine Alanine

Biosynthesis NADPH

Glycerol phosphate

Serine

Pyruvate

Fructose

FIG. V.3. Overview of fructose and galactose metabolism. Fructose and galactose are converted to intermediates of glucose metabolism.

Acetyl CoA

E T C

Glucose 6-phosphate

Prevention of oxidative damage

Glucose 6-phosphate Pentose phosphates

Fatty acids Nucleotides

Acetyl CoA OAA TCA cycle

Pyruvate

Nonoxidative Reactions Glucose Glucose 6-phosphate

Glutamate and other amino acids

FIG. V.5. Conversion of glucose to amino acids and to the glycerol and fatty acid (FA) moieties of triacylglycerols (TG). Acetyl-CoA, acetyl coenzyme A; OAA, oxaloacetate; TCA, tricarboxylic acid.

Fructose 6-phosphate Pentose phosphates Glyceraldehyde 3-phosphate Nucleotides Pyruvate

474

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FIG. V.6. Overview of the pentose phosphate pathway. The oxidative reactions generate both NADPH and pentose phosphates. The nonoxidative reactions generate only pentose phosphates.

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Glucose

Glucuronides

Glucose 6-phosphate

Glucose 1-phosphate Glycogen

UDP-glucuronate UDP-glucose

Glycoproteins Glycolipids Proteoglycans

UDP-galactose Glucose

Lactose

FIG. V.7. Products derived from UDP-glucose.

Glucose 6-phosphate is also converted to UDP (uridine diphosphate)-glucose, which has many functions in the cell (Fig. V.7). The major fate of UDP-glucose is the synthesis of glycogen, the storage polymer of glucose. Although most cells have glycogen to provide emergency supplies of glucose, the largest stores are in muscle and liver. Muscle glycogen is used to generate ATP during muscle contraction. Liver glycogen is used to maintain blood glucose during fasting and during exercise or periods of enhanced need. UDP-glucose is also used for the formation of other sugars, and galactose and glucose are interconverted while attached to UDP. UDP-galactose is used for lactose synthesis in the mammary gland. In the liver, UDP-glucose is oxidized to UDP-glucuronate, which is used to convert bilirubin and other toxic compounds to glucuronides for excretion (see Fig. V.7). Nucleotide sugars are also used for the synthesis of proteoglycans, glycoproteins, and glycolipids (see Fig. V.7). Proteoglycans are major carbohydrate components of the extracellular matrix, cartilage, and extracellular fluids (such as the synovial fluid of joints), and they are discussed in more detail in Chapter 49. Most extracellular proteins are glycoproteins; that is, they contain covalently attached carbohydrates. For both cell membrane glycoproteins and glycolipids, the carbohydrate portion extends into the extracellular space. All cells are continuously supplied with glucose under normal circumstances; the body maintains a relatively narrow range of glucose concentration in the blood (approximately 80 to 100 mg/dL) in spite of the changes in dietary supply and tissue demand as we sleep and exercise. This process is called glucose homeostasis. Low blood glucose levels (hypoglycemia) are prevented by a release of glucose from the large glycogen stores in the liver (glycogenolysis); by synthesis of glucose from lactate, glycerol, and amino acids in the liver (gluconeogenesis) (Fig. V.8); and to a limited extent by a release of fatty acids from adipose tissue stores (lipolysis) to provide an alternate fuel when glucose is in short supply. High blood glucose levels (hyperglycemia) are prevented both by the conversion of glucose to glycogen and by its conversion to triacylglycerols in liver and adipose tissue. Thus, the pathways for glucose use as a fuel cannot

Blood Glycogen Glucose

Glycogenolysis

Glucose 1-phosphate Glucose-6-P Gluconeogenesis

Glycerol 3-phosphate

Glycerol PEP Alanine Pyruvate

Lactate

OAA TCA cycle

FIG. V.8. Production of blood glucose from glycogen (by glycogenolysis) and from alanine, lactate, and glycerol (by gluconeogenesis). PEP, phosphoenolpyruvate; OAA, oxaloacetate; TCA, tricarboxylic acid. 475

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Glucose Glycoproteins Glycolipids Proteoglycans

Glucuronides

Glycogen

UDP-glucuronate

UDP-glucose Glucose 1phosphate

Galactose

Glucose 6-phosphate

UDP-galactose

Pentose phosphate

Glucose

Fructose

Lactose

DHAP

Amino acids

Glycerol 3-phosphate

PEP

Alanine

Glycerol

TG

Pyruvate

Lactate FA

Acetyl CoA OAA TCA cycle CO2 Glutamate and other amino acids

FIG. V.9. Overview of the major pathways of glucose metabolism. Pathways for production of blood glucose are shown by dashed lines. Acetyl-CoA, acetyl coenzyme A; DHAP, dihydroxyacetone phosphate; FA, fatty acids; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid; TG, triacylglycerols.

be considered as totally separate from pathways involving amino acid and fatty acid metabolism (Fig. V.9). Intertissue balance in the use and storage of glucose during fasting and feeding is accomplished principally by the actions of the hormones of metabolic homeostasis—insulin and glucagon (Fig. V.10). However, cortisol, epinephrine, norepinephrine, and other hormones are also involved in intertissue adjustments of supply and demand in response to changes of physiologic state.

Glucagon release

Blood glucose

Insulin release

Glycogenolysis

Glycogen synthesis

Gluconeogenesis

Fatty acid synthesis

Lipolysis

Triglyceride synthesis

Liver glycolysis

Liver glycolysis

FIG. V.10. Pathways regulated by the release of glucagon (in response to a lowering of blood glucose levels) and insulin (released in response to an elevation of blood glucose levels). Tissue-specific differences occur in the response to these hormones, as detailed in the chapters of this section. 476

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26

Basic Concepts in the Regulation of Fuel Metabolism by Insulin, Glucagon, and Other Hormones

All cells use adenosine triphosphate (ATP) continuously and require a constant supply of fuels to provide energy for ATP generation. Insulin and glucagon are the two major hormones that regulate fuel mobilization and storage. Their function is to ensure that cells have a constant source of glucose, fatty acids, and amino acids for ATP generation and for cellular maintenance (Fig. 26.1). Because most tissues are partially or totally dependent on glucose for generation of ATP and for production of precursors of other pathways, insulin and glucagon maintain blood glucose levels near 80 to 100 mg/dL (90 mg/dL is the same as 5 mM) despite the fact that carbohydrate intake varies considerably over the course of a day. The maintenance of constant blood glucose levels (glucose homeostasis) requires insulin and glucagon to regulate carbohydrate, lipid, and amino acid metabolism in accordance with the needs and capacities of individual tissues. Basically, the dietary intake of all fuels in excess of immediate need is stored, and the appropriate fuel is mobilized when a demand occurs. For example, when dietary glucose is not available to cells in sufficient quantities, fatty acids are mobilized and used by skeletal muscle as a fuel (see Chapters 2 and 23). Under these circumstances, the liver can also convert fatty acids to ketone bodies that can be used by the brain. The fatty acids that are mobilized under these conditions spare glucose for use by the brain and other glucose-dependent tissues (such as red blood cells). Insulin and glucagon are important for the regulation of fuel storage and fuel mobilization (Fig. 26.2). Insulin, released from the ␤-cells of the pancreas in response to carbohydrate ingestion, promotes glucose utilization as a fuel and glucose storage as fat and glycogen. Insulin, therefore, is a major anabolic hormone. In addition to its storage function, insulin increases protein synthesis and cell growth. Blood insulin levels decrease as glucose is taken up by tissues and used. Glucagon, the major insulin counterregulatory hormone, is decreased in response to a carbohydrate meal and elevated during fasting. Its concentration in the blood increases as circulating levels of glucose fall, a response that promotes glucose production via glycogenolysis (glycogen degradation) and gluconeogenesis (glucose synthesis from amino acids and other noncarbohydrate precursors). Increased levels of circulating glucagon relative to insulin also stimulate the mobilization of fatty acids from adipose tissue. Epinephrine (the fight-or-flight hormone) and cortisol (a glucocorticoid released from the adrenal cortex in response to fasting and chronic stress) have effects on fuel metabolism that oppose those of insulin. Therefore, epinephrine and cortisol are considered to be insulin counterregulatory hormones. Insulin and glucagon are polypeptide hormones synthesized as prohormones in the pancreatic ␤- and ␣-cells, respectively. Proinsulin is

Brain [ATP]

Glucose

Liver Ketone bodies

Fatty acids

Adipocyte

[ATP]

Skeletal muscle

FIG. 26.1. Maintenance of fuel supplies to tissues. Glucagon release activates the pathways shown.

477

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478

SECTION V ■ CARBOHYDRATE METABOLISM

A

Glucose

cleaved into mature insulin and a connection peptide (C-peptide) in storage vesicles and precipitated with Zn2⫹. Insulin secretion is regulated principally by changes in blood glucose levels. Glucagon is also synthesized as a prohormone and cleaved into mature glucagon within storage vesicles. Its release is regulated principally through changes in the level of glucose and insulin bathing the ␣-cells located in the pancreatic islets of Langerhans. Glucagon exerts its effects on cells by binding to a receptor located on the plasma membrane of target cells for this hormone. The binding to these specific receptors by glucagon stimulates the synthesis of the intracellular second messenger, cyclic adenosine monophosphate (cAMP) (Fig. 26.3). cAMP activates protein kinase A (PKA), which phosphorylates key regulatory enzymes, thereby activating some while inhibiting others. Insulin, on the other hand, promotes the dephosphorylation of these key enzymes, leading to their activation or deactivation, depending on the enzyme. Changes of cAMP levels also induce or repress the synthesis of several enzymes. Insulin binds to a receptor on the cell surface of insulin-sensitive tissues and initiates a cascade of intracellular events that differs from those stimulated by glucagon. Insulin binding activates both autophosphorylation of the receptor and the phosphorylation of other enzymes by the receptor’s tyrosine kinase domain (see Chapter 11, Section III.B.3). The complete routes for signal transduction between this point and the final effects of insulin on the regulatory enzymes of fuel metabolism have not yet been fully established.

Insulin

Liver

Triglyceride synthesis Glycogen synthesis Active glycolysis

B Liver

Glucose Glucagon Epinephrine Glycogen degradation Gluconeogenesis

FIG. 26.2. Insulin and the insulin counterregulatory hormones. A. Insulin promotes glucose storage, as triglyceride (TG) or glycogen. B. Glucagon and epinephrine promote glucose release from the liver, activating glycogenolysis and gluconeogenesis. Cortisol will stimulate both glycogen synthesis and gluconeogenesis.

THE WAITING ROOM Pancreas Low blood glucose Glucagon

Cell membrane

Receptor

Cytosol Second messenger (cAMP)

Cellular response (activation of protein kinase A)

FIG. 26.3. Cellular response to glucagon, which is released from the pancreas in response to a decrease in blood glucose levels.

Ann Sulin returned to her physician for her monthly office visit. She has been seeing her physician for over a year because of obesity and elevated blood glucose levels. She still weighed 198 lb despite her insistence that she had adhered strictly to her hypocaloric diet. Her 2-hour postprandial blood glucose level was 180 mg/dL (reference range, 80 to 140 mg/dL). Bea Selmass is a 46-year-old woman who 6 months earlier began noting episodes of fatigue and confusion as she finished her daily prebreakfast jog. These episodes were occasionally accompanied by blurred vision and an unusually urgent sense of hunger. The ingestion of food relieved all of her symptoms within 25 to 30 minutes. In the last month, these attacks have occurred more frequently throughout the day, and she has learned to reduce their severity by eating between meals. As a result of this increase in caloric intake, she has recently gained 8 lb. A random serum glucose level done at 4:30 PM during her first office visit was subnormal at 46 mg/dL. Her physician, suspecting she had a form of fasting hypoglycemia, ordered a series of fasting serum glucose levels. In addition, he asked Bea to keep a careful diary of all of the symptoms that she experienced when her attacks were most severe.

I.

METABOLIC HOMEOSTASIS

Living cells require a constant source of fuels from which to derive adenosine triphosphate (ATP) for the maintenance of normal cell function and growth. Therefore, a balance must be achieved among carbohydrate, fat, and protein intake;

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CHAPTER 26 ■ BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM

their rates of oxidation; and their rates of storage when they are present in excess of immediate need. Alternatively, when the demands for these substrates increase, the rate of mobilization from storage sites and the rate of their de novo synthesis also require balanced regulation. The control of the balance between substrate need and substrate availability is referred to as metabolic homeostasis (Fig. 26.4). The intertissue integration required for metabolic homeostasis is achieved in three principal ways: 1. The concentration of nutrients or metabolites in the blood affects the rate at which they are used or stored in different tissues. 2. Hormones carry messages to their individual target tissues about the physiologic state of the body and the current level of nutrient supply or demand. 3. The central nervous system uses neural signals to control tissue metabolism, either directly or through the release of hormones. Fatty acids provide an example of the influence that the level of a compound in the blood has on its own rate of metabolism. The concentration of fatty acids in the blood is the major factor determining whether skeletal muscles will use fatty acids or glucose as a fuel (see Chapter 23). In contrast, hormones are (by definition) intravascular carriers of messages between their sites of synthesis and their target tissues. Epinephrine, for example, is a flight-or-fight hormone that in times of stress signals an immediate need for increased fuel availability. Its level is regulated principally through the activation of the sympathetic nervous system. Insulin and glucagon, however, are the two major hormones that regulate fuel storage and mobilization (see Fig. 26.2). Insulin is the major anabolic hormone of the body. It promotes the storage of fuels and the use of fuels for growth. Glucagon is the major hormone of fuel mobilization (Fig. 26.5). These hormones fluctuate continuously in response to our daily eating pattern. Glucose has a special role in metabolic homeostasis. Many tissues (e.g., the brain, red blood cells, kidney medulla, exercising skeletal muscle) depend on glycolysis for all or a part of their energy needs. As a consequence, these tissues require uninterrupted access to glucose to meet their rapid rate of ATP use. In the adult, a minimum of 190 g glucose is required per day, approximately 150 g for the brain and 40 g for other tissues. Significant decreases of blood glucose lower than 60 mg/dL limit

Insulin Blood fuel

Dietary Fuels: • Carbohydrate • Fat • Protein

Blood fuel

Fuel availability

479

Tissue needs

• Blood level of nutrient • Hormone level • Nerve impulse

FIG. 26.4. Metabolic homeostasis. The balance between fuel availability and the needs of tissues for different fuels is achieved by three types of messages: the level of the fuel or nutrients in the blood, the level of one of the hormones of metabolic homeostasis, or the nerve impulses that affect tissue metabolism or the release of hormones.

Fuel stores +

Growth

Neuronal signals

Glucagon +

Stress hormones

Blood fuel

Fuel utilization ATP Cell function

FIG. 26.5. Signals that regulate metabolic homeostasis. The major stress hormones are epinephrine and cortisol.

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SECTION V ■ CARBOHYDRATE METABOLISM

Hyperglycemia may cause a constellation of symptoms such as polyuria and subsequent polydipsia (increased thirst). These symptoms are caused by the osmotic diuresis that results from the high levels of glucose being excreted into the urine. This diuretic effect may decrease the effective plasma volume, thereby further increasing blood glucose levels. If this dehydration becomes severe, it may lead to cerebral dysfunction and even coma. The inability to move glucose into cells necessitates the oxidation of lipids as an alternative fuel. As a result, adipose stores are used, and a patient with poorly controlled diabetes mellitus loses weight in spite of a good appetite. Extremely high levels of serum glucose can cause nonketotic hyperosmolar coma in patients with type 2 diabetes mellitus (hyperosmolar hyperglycemic state [HHS] or nonketotic hyperglycemia). Such patients usually have sufficient insulin responsiveness to block fatty acid release and ketone body formation, but they are unable to significantly stimulate glucose entry into peripheral tissues. The severely elevated levels of glucose in the blood compared with those inside the cell lead to an osmotic effect that causes water to leave the cells and enter the blood. Chronic hyperglycemia also produces pathologic effects through the nonenzymatic glycosylation of a variety of proteins. Hemoglobin A (HbA), one of the proteins that becomes glycosylated, forms HbA1c (see Chapter 7). Ann Sulin’s high levels of HbA1c (14% of the total HbA, compared with the reference range of 4.7% to 6.4%) indicate that her blood glucose has been significantly elevated over the last 12 to 14 weeks, the halflife of hemoglobin in the bloodstream. All membrane and serum proteins exposed to high levels of glucose in the blood or interstitial fluid are candidates for nonenzymatic glycosylation. This process distorts protein structure and slows protein degradation, which leads to an accumulation of these products in various organs, thereby adversely affecting organ function. These events contribute to the long-term microvascular and macrovascular complications of diabetes mellitus, which include diabetic retinopathy, nephropathy, and neuropathy (microvascular), in addition to coronary artery, cerebral artery, peripheral artery disease, and atherosclerosis (macrovascular).

glucose metabolism in the brain and may elicit hypoglycemic symptoms (as experienced by Bea Selmass), presumably because the overall process of glucose flux through the blood–brain barrier, into the interstitial fluid, and subsequently into the neuronal cells is slow at low blood glucose levels because of the Km values of the glucose transporters required for this to occur (see Chapter 27). The continuous efflux of fuels from their storage depots during exercise, for example, is necessitated by the high amounts of fuel required to meet the need for ATP under these conditions. Disastrous results would occur if even a day’s supply of glucose, amino acids, and fatty acids could not enter cells normally and were instead left circulating in the blood. Glucose and amino acids would be at such high concentrations in the circulation that the hyperosmolar effect would cause progressively severe neurologic deficits and even coma. The concentration of glucose and amino acids would rise above the renal tubular threshold for these substances (the maximal concentration in the blood at which the kidney can completely resorb metabolites), and some of these compounds would be wasted as they spilled over into the urine. Nonenzymatic glycosylation of proteins would increase at higher blood glucose levels, altering the function of tissues in which these proteins reside. Triacylglycerols, present primarily in chylomicrons and very low-density lipoproteins (VLDLs), would rise in the blood, increasing the likelihood of atherosclerotic vascular disease. These potential metabolic derangements emphasize the need to maintain a normal balance between fuel storage and fuel use.

II. MAJOR HORMONES OF METABOLIC HOMEOSTASIS Insulin is the major anabolic hormone that promotes the storage of nutrients: glucose storage as glycogen in liver and muscle, conversion of glucose to triacylglycerols in liver and their storage in adipose tissue, and amino acid uptake and protein synthesis in skeletal muscle (Fig. 26.6). Insulin also increases the synthesis of albumin and other proteins by the liver. Insulin promotes the use of glucose as a fuel by facilitating its transport into muscle and adipose tissue. At the same time, insulin acts to inhibit fuel mobilization. Glucagon acts to maintain fuel availability in the absence of dietary glucose by stimulating the release of glucose from liver glycogen (see Chapter 28);

Liver

Glycogen –

+

+

–

Protein +

Glucose Fatty acids Amino acids

VLDL

Glucose +

Fatty acids –

+

+

Protein +

CO2 Glycogen

Skeletal muscle

Triacylglycerols

Adipocyte

FIG. 26.6. Major sites of insulin action on fuel metabolism. VLDL, very low-density lipoprotein; 䊝, stimulated by insulin; 䊞, inhibited by insulin.

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CHAPTER 26 ■ BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM

–

Highcarbohydrate meal

Liver

Glycogen

481

+

Glucose

mg/dL

120 –

+

Fatty acids

100 Glucose 80

Amino acids 120

µU/mL

Glucose

Fatty acids +

Triacylglycerols

Fatty acids

No effect

80

Insulin

40 0

Skeletal muscle 120 pg/mL

Adipocyte

FIG. 26.7. Major sites of glucagon action in fuel metabolism. 䊝, pathways stimulated by glucagon; 䊞, pathways inhibited by glucagon.

Glucagon 110 100 90 60

0

60

120

180

240

Minutes

by stimulating gluconeogenesis from lactate, glycerol, and amino acids (see Chapter 31); and, in conjunction with decreased insulin, by mobilizing fatty acids from adipose triacylglycerols to provide an alternate source of fuel (see Chapter 23 and Fig. 26.7). Its sites of action are principally the liver and adipose tissue; it has no influence on skeletal muscle metabolism because muscle cells lack glucagon receptors. The message carried by glucagon is that “glucose is gone”; that is, the current supply of glucose is inadequate to meet the immediate fuel requirements of the body. The release of insulin from the ␤-cells of the pancreas is dictated primarily by the level of glucose in the blood bathing the ␤-cells in the islets of Langerhans. The highest levels of insulin occur approximately 30 to 45 minutes after a highcarbohydrate meal (Fig. 26.8). They return to basal levels as the blood glucose concentration falls, approximately 120 minutes after the meal. The release of glucagon from the ␣-cells of the pancreas, conversely, is controlled principally through a reduction of glucose and/or a rise in the concentration of insulin in the blood bathing the ␣-cells in the pancreas. Therefore, the lowest levels of glucagon occur after a high-carbohydrate meal. Because all of the effects of glucagon are opposed by insulin, the simultaneous stimulation of insulin release and suppression of glucagon secretion by a high-carbohydrate meal provides integrated control of carbohydrate, fat, and protein metabolism. Insulin and glucagon are not the only regulators of fuel metabolism. The intertissue balance between the use and storage of glucose, fat, and protein is also accomplished by the circulating levels of metabolites in the blood, by neuronal signals, and by the other hormones of metabolic homeostasis (epinephrine, norepinephrine, cortisol, etc.) (Table 26.1). These hormones oppose the actions of insulin by mobilizing fuels. Like glucagon, they are insulin counterregulatory hormones (Fig. 26.9). Of all these hormones, only insulin and glucagon are synthesized and released in direct response to changing levels of fuels in the blood. The release of cortisol, epinephrine, and norepinephrine is mediated by neuronal signals. Rising levels of the insulin counterregulatory hormones in the blood reflect, for the most part, a current increase in the demand for fuel.

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FIG. 26.8. Blood glucose, insulin, and glucagon levels after a high-carbohydrate meal. Bea Selmass’s studies confirmed that her fasting serum glucose levels were below normal. She continued to experience the fatigue, confusion, and blurred vision she had described on her first office visit. These symptoms are referred to as the neuroglycopenic manifestations of severe hypoglycemia (neurologic symptoms resulting from an inadequate supply of glucose to the brain for the generation of ATP). Bea also noted the symptoms that are part of the adrenergic response to hypoglycemic stress. Stimulation of the sympathetic nervous system (because of the low levels of glucose reaching the brain) results in the release of epinephrine, a stress hormone, from the adrenal medulla. Elevated epinephrine levels cause tachycardia (rapid heart beat), palpitations, anxiety, tremulousness, pallor, and sweating. In addition to the symptoms described by Bea Selmass, individuals may experience confusion, light-headedness, headache, aberrant behavior, blurred vision, loss of consciousness, or seizures. When severe and prolonged, death may occur. Ms. Selmass’s doctor explained that the general diagnosis of “fasting” hypoglycemia was now established and that a specific cause for this disorder must be found.

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SECTION V ■ CARBOHYDRATE METABOLISM

Table 26.1 Hormones

Physiologic Actions of Insulin and Insulin Counterregulatory

Hormone

Function

Major Metabolic Pathways Affected

Insulin

Promotes fuel storage after a meal Promotes growth

Glucagon

Mobilizes fuels

Stimulates glucose storage as glycogen (muscle and liver) Stimulates fatty acid synthesis and storage after a high-carbohydrate meal Stimulates amino acid uptake and protein synthesis Activates gluconeogenesis and glycogenolysis (liver) during fasting Activates fatty acid release from adipose tissue Stimulates glucose production from glycogen (muscle and liver) Stimulates fatty acid release from adipose tissue Stimulates amino acid mobilization from muscle protein Stimulates gluconeogenesis to produce glucose for liver glycogen synthesis Stimulates fatty acid release from adipose tissue

Epinephrine

Cortisol

Maintains blood glucose levels during fasting Mobilizes fuels during acute stress Provides for changing requirements over the long-term

Low Blood Glucose

Hypothalamic regulatory center Pituitary ACTH Autonomic nervous system

␣-Cells Cortex Medulla Adrenal

Cortisol

Epinephrine

Pancreas

Norepinephrine

Glucagon

FIG. 26.9. Major insulin counterregulatory hormones. The stress of a low blood glucose level mediates the release of the major insulin counterregulatory hormones through neuronal signals. Hypoglycemia is one of the stress signals that stimulates the release of cortisol, epinephrine, and norepinephrine. Adrenocorticotropic hormone (ACTH) is released from the pituitary and stimulates the release of cortisol (a glucocorticoid) from the adrenal cortex. Neuronal signals stimulate the release of epinephrine from the adrenal medulla and norepinephrine from nerve endings. Neuronal signals also play a minor role in the release of glucagon. Although norepinephrine has counterregulatory actions, it is not a major counterregulatory hormone.

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III. SYNTHESIS AND RELEASE OF INSULIN AND GLUCAGON A. Endocrine Pancreas

483

The message that insulin carries to tissues is that glucose is plentiful and can be used as an immediate fuel or can be converted to storage forms such as triacylglycerol in adipocytes or glycogen in liver and muscle. Because insulin stimulates the uptake of glucose into tissues where it may be immediately oxidized or stored for later oxidation, this regulatory hormone lowers blood glucose levels. Therefore, one of the possible causes of Bea Selmass’s hypoglycemia is an insulinoma, a tumor that produces excessive insulin. Whenever an endocrine gland continues to release its hormone in spite of the presence of signals that normally would suppress its secretion, this persistent inappropriate release is said to be “autonomous.” Secretory neoplasms of endocrine glands generally produce their hormonal product autonomously in a chronic fashion.

Insulin and glucagon are synthesized in different cell types of the endocrine pancreas, which consists of microscopic clusters of small glands, the islets of Langerhans, scattered among the cells of the exocrine pancreas. The ␣-cells secrete glucagon and the ␤-cells secrete insulin into the hepatic portal vein via the pancreatic veins.

B. Synthesis and Secretion of Insulin Insulin is a polypeptide hormone. The active form of insulin is composed of two polypeptide chains (the A-chain and the B-chain) linked by two interchain disulfide bonds. The A-chain has an additional intrachain disulfide bond (Fig. 26.10). Insulin, like many other polypeptide hormones, is synthesized as a preprohormone that is converted in the rough endoplasmic reticulum (RER) to proinsulin. The “pre” sequence, a short hydrophobic signal sequence at the N-terminal end, is cleaved as it enters the lumen of the RER. Proinsulin folds into the proper conformation, and disulfide bonds are formed between the cysteine residues. It is then transported in microvesicles to the Golgi complex. It leaves the Golgi complex in storage vesicles, where a protease removes the biologically inactive “connecting peptide” (C-peptide) and a few small remnants, resulting in the formation of biologically active insulin (see Fig. 26.10). Zinc ions are also transported in these storage vesicles. Cleavage of the C-peptide decreases the solubility of the resulting insulin, which then coprecipitates with zinc. Exocytosis of the insulin storage vesicles from the cytosol of the ␤-cell into the blood is stimulated by rising levels of glucose in the blood bathing the ␤-cells.

20

Ala Leu Glu

Leu Ser Gly Ala Gly Pro Pro Gln Gly Gly Leu

Gly

C-Peptide

Leu

Glu Val

Gly

Gln

Ser 31

Gly

Leu

Val

Gln

Lys

Gln

Arg

Leu

Gly

Asp

Ile Val

NH2

Asn

Glu

Phe

S Cys

Val

A-Chain

21

S

Gln Ser Ile Cys Ser Leu Tyr

Arg

Leu

S

Thr Lys

10

S

Insulin

Gly His 10

Thr

S

Tyr Phe

B-Chain Leu

30

Pro

Cys Ser

Arg

Asn Glu

Thr

Gln

Glu 1

Tyr

S

Cys

Asn

Leu

Ala

Cys Gln

His

Glu

COOH

Glu Val Glu Gly Ala Leu Tyr Leu Val Cys 20

Arg

Gly

Phe

FIG. 26.10. Cleavage of proinsulin to insulin. Proinsulin is converted to insulin by proteolytic cleavage, which removes the C-peptide and a few additional amino acid residues. Cleavage occurs at the arrows. (From Murray RK, Granner DK, Mayer PA, et al. Harper’s Biochemistry. 23rd ed. Stanford, CT: Appleton & Lange; 1993:560.)

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SECTION V ■ CARBOHYDRATE METABOLISM

Autonomous hypersecretion of insulin from a suspected pancreatic ␤-cell tumor (an insulinoma) can be demonstrated in several ways. The simplest test is to draw blood for the measurement of both glucose and insulin simultaneously, at a time when the patient is spontaneously experiencing the characteristic adrenergic or neuroglycopenic symptoms of hypoglycemia. During such a test, Bea Selmass’s glucose levels fell to 45 mg/dL (normal, 80 to 100 mg/dL), and her ratio of insulin to glucose was far higher than normal. The elevated insulin levels markedly increased glucose uptake by the peripheral tissues, resulting in a dramatic lowering of blood glucose levels. In normal individuals, as blood glucose levels drop, insulin levels also drop. Insulin levels were determined by radioimmunoassay (see the Biochemical Comments in Chapter 43 for a description of this method).

Di Abietes has type 1 diabetes mellitus, formerly known as insulin-dependent diabetes mellitus (IDDM). This metabolic disorder is usually caused by antibody-mediated (autoimmune) destruction of the ␤-cells of the pancreas. Susceptibility to type 1 diabetes mellitus is, in part, conferred by a genetic defect in the human leukocyte antigen (HLA) region of ␤-cells that codes for the major histocompatibility complex II (MHC II). This protein presents an intracellular antigen to the cell surface for “self-recognition” by the cells involved in the immune response. Because of this defective protein, a cell-mediated immune response leads to varying degrees of ␤-cell destruction and eventually to dependence on exogenous insulin administration to control the levels of glucose in the blood.

Lieberman_Ch26.indd 484

Ca2+ +

⌬␷

3

[Ca2+]

K+

4 Fusion and exocytosis

Glucose

5

–

Insulin

2 Glycolysis 1 TCA cycle Oxidative phosphorylation

ATP

␤-Cell FIG. 26.11. Release of insulin by the ␤-cells. Details are given in the text.

Glucose enters the ␤-cell via specific glucose transporter proteins known as GLUT 2 (see Chapter 27). Glucose is phosphorylated through the action of glucokinase to form glucose 6-phosphate, which is metabolized through glycolysis, the TCA cycle, and oxidative phosphorylation. These reactions result in an increase in ATP levels within the ␤-cell (circle 1 in Fig. 26.11). As the ␤-cell [ATP]/ [ADP] ratio increases, the activity of a membrane-bound, ATP-dependent K⫹ channel (K⫹ATP) is inhibited (i.e., the channel is closed) (circle 2 in Fig. 26.11). The closing of this channel leads to a membrane depolarization (circle 3 in Fig. 26.11), which activates a voltage-gated Ca2⫹ channel that allows Ca2⫹ to enter the ␤-cell such that intracellular Ca2⫹ levels increase significantly (circle 4 in Fig. 26.11). The increase in intracellular Ca2⫹ stimulates the fusion of insulin containing exocytotic vesicles with the plasma membrane, resulting in insulin secretion (circle 5 in Fig. 26.11). Thus, an increase in glucose levels within the ␤-cells initiates insulin release. Other intracellular metabolites, particularly NADPH, have been proposed to play important roles in insulin release in response to glucose. This will be discussed further in later chapters.

C. Stimulation and Inhibition of Insulin Release The release of insulin occurs within minutes after the pancreas is exposed to a high glucose concentration. The threshold for insulin release is approximately 80 mg glucose/dL. Above 80 mg/dL, the rate of insulin release is not an all-or-nothing response but is proportional to the glucose concentration up to approximately 300 mg/dL. As insulin is secreted, the synthesis of new insulin molecules is stimulated, so that secretion is maintained until blood glucose levels fall. Insulin is rapidly removed from the circulation and degraded by the liver (and, to a lesser extent, by kidney and skeletal muscle), so blood insulin levels decrease rapidly once the rate of secretion slows. Several factors other than the blood glucose concentration can modulate insulin release (Table 26.2). The pancreatic islets are innervated by the autonomic nervous system, including a branch of the vagus nerve. These neural signals help to coordinate insulin release with the secretory signals initiated by the ingestion of fuels. However, signals from the central nervous system are not required for insulin secretion. Certain amino acids also can stimulate insulin secretion, although the amount of insulin released during a high-protein meal is very much lower than that released by a high-carbohydrate meal. Gastric inhibitory polypeptide (GIP) and

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CHAPTER 26 ■ BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM

Table 26.2

Regulators of Insulin Release

Regulator

Effect

Major regulators Glucose Minor regulators Amino acids Neural input Gut hormonesa Epinephrine (adrenergic)

⫹ ⫹ ⫹ ⫹ ⫺

⫹, stimulates; ⫺, inhibits. a Gut hormones that regulate fuel metabolism are discussed in Chapter 43.

glucagon-like peptide 1 (GLP-1), gut hormones released after the ingestion of food, also aid in the onset of insulin release. Epinephrine, secreted in response to fasting, stress, trauma, and vigorous exercise, decreases the release of insulin. Epinephrine release signals energy use, which indicates that less insulin needs to be secreted, as insulin stimulates energy storage.

D. Synthesis and Secretion of Glucagon

Glucagon, a polypeptide hormone, is synthesized in the ␣-cells of the pancreas by cleavage of the much larger preproglucagon, a 160–amino acid peptide. Like insulin, preproglucagon is produced on the RER and is converted to proglucagon as it enters the lumen of the endoplasmic reticulum. Proteolytic cleavage at various sites produces the mature 29–amino acid glucagon (molecular weight 3,500 Da) and larger glucagon-containing fragments (named GLP-1 and GLP-2). Glucagon is rapidly metabolized, primarily in the liver and kidneys. Its plasma half-life is only about 3 to 5 minutes. Glucagon secretion is regulated principally by circulating levels of glucose and insulin. Increasing levels of each inhibit glucagon release. Glucose probably has both a direct suppressive effect on secretion of glucagon from the ␣-cell as well as an indirect effect, the latter being mediated by its ability to stimulate the release of insulin. The direction of blood flow in the islets of the pancreas carries insulin from the ␤-cells in the center of the islets to the peripheral ␣-cells, where it suppresses glucagon secretion. Conversely, certain hormones stimulate glucagon secretion. Among these are the catecholamines (including epinephrine) and cortisol (Table 26.3). Many amino acids also stimulate glucagon release (Fig. 26.12). Thus, the high levels of glucagon that would be expected in the fasting state do not decrease after a high-protein meal. In fact, glucagon levels may increase, stimulating gluconeogenesis in the absence of dietary glucose. The relative amounts of insulin and glucagon in the blood after a mixed meal depend on the composition of the meal because glucose stimulates insulin release and amino acids stimulate glucagon release.

Table 26.3

Regulators of Glucagon Release

Regulator Major regulators Glucose Insulin Amino acids Minor regulators Cortisol Neural (stress) Epinephrine ⫹, stimulates; ⫺, inhibits.

Lieberman_Ch26.indd 485

Effect ⫺ ⫺ ⫹ ⫹ ⫹ ⫹

485

A form of diabetes known as maturityonset diabetes of the young (MODY) results from mutations in either pancreatic glucokinase or specific nuclear transcription factors. MODY type 2 is caused by a glucokinase mutation that results in an enzyme with reduced activity because of either an elevated Km for glucose or a reduced Vmax for the reaction. Because insulin release depends on normal glucose metabolism within the ␤-cell that yields a critical [ATP]/[ADP] ratio in the ␤-cell, individuals with this glucokinase mutation cannot significantly metabolize glucose unless glucose levels are higher than normal. Thus, although these patients can release insulin, they do so at higher than normal glucose levels and are, therefore, almost always in a hyperglycemic state. Interestingly, however, these patients are somewhat resistant to the long-term complications of chronic hyperglycemia. The mechanism for this seeming resistance is not well understood. Neonatal diabetes is an inherited disorder in which newborns develop diabetes within the first 3 months of life. The diabetes may be permanent, requiring life-long insulin treatment, or transient. The most common mutation leading to permanent neonatal diabetes is in the KCNJ11 gene, which encodes a subunit of the K⫹ATP channel in various tissues, including the pancreas. This is an activating mutation, which keeps the K⫹ATP channel open, and less susceptible to ATP inhibition. If the K⫹ATP channel cannot be closed, activation of the Ca2⫹ channel will not occur, and insulin secretion will be impaired. Ann Sulin is taking a sulfonylurea compound known as glipizide to treat her diabetes. The sulfonylureas act on the K⫹ATP channels on the surface of the pancreatic ␤-cells. The K⫹ATP channels contain pore-forming subunits (encoded by the KCNJ11 gene) and regulatory subunits (the subunit to which sulfonylurea compounds bind, encoded by the SUR1 gene). The binding of the drug to the sulfonylurea receptor closes K⫹ channels (as do elevated ATP levels), which, in turn, increases Ca2⫹ movement into the interior of the ␤-cell. This influx of calcium modulates the interaction of the insulin storage vesicles with the plasma membrane of the ␤-cell, resulting in the release of insulin into the circulation. Recently, a patient was discovered to have an activating mutation in the SUR1 gene (which would make it difficult to close the K⫹ATP channel), and, among other symptoms, the patient displayed neonatal diabetes.

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Highprotein meal Glucose (mg/dL)

Measurements of proinsulin and the connecting peptide between the ␣and ␤-chains of insulin (C-peptide) in Bea Selmass’s blood during her hospital fast provided confirmation that she had an insulinoma. Insulin and C-peptide are secreted in approximately equal proportions from the ␤-cell, but C-peptide is not cleared from the blood as rapidly as insulin. Therefore, it provides a reasonably accurate estimate of the rate of insulin secretion. Plasma C-peptide measurements are also potentially useful in treating patients with diabetes mellitus because they provide a way to estimate the degree of endogenous insulin secretion in patients who are receiving exogenous insulin, which lacks the C-peptide.

Nitrogen 90 85

6 7 8

Glucose 20 Insulin 10

200 Glucagon

180

Insulin (␮U/mL) ␣-Amino nitrogen (mg/dL)

SECTION V ■ CARBOHYDRATE METABOLISM

Glucagon (pg/mL)

486

160 140 120 100 –60

0

60

120 180 240

Minutes

Patients with type 1 diabetes mellitus, such as Di Abietes, have almost undetectable levels of insulin in their blood. Patients with type 2 diabetes mellitus, such as Ann Sulin, conversely, have normal or even elevated levels of insulin in their blood; however, the level of insulin in their blood is inappropriately low relative to their elevated blood glucose concentration. In type 2 diabetes mellitus, skeletal muscle, liver, and other tissues exhibit a resistance to the actions of insulin. As a result, insulin has a smaller than normal effect on glucose and fat metabolism in such patients. Levels of insulin in the blood must be higher than normal to maintain normal blood glucose levels. In the early stages of type 2 diabetes mellitus, these compensatory adjustments in insulin release may keep the blood glucose levels near the normal range. Over time, as the ␤-cells’ capacity to secrete high levels of insulin declines, blood glucose levels increase, and exogenous insulin becomes necessary.

Lieberman_Ch26.indd 486

FIG. 26.12. Release of insulin and glucagon in response to a high-protein meal. This figure shows the increase in the release of insulin and glucagon into the blood after an overnight fast followed by the ingestion of 100 g protein (equivalent to a slice of roast beef). Insulin levels do not increase nearly as much as they do after a high-carbohydrate meal (see Fig. 26.8). The levels of glucagon, however, significantly increase above those present in the fasting state.

However, amino acids also induce insulin secretion, but not to the same extent that glucose does. Although this may seem paradoxical, it actually makes good sense. Insulin release stimulates amino acid uptake by tissues and enhances protein synthesis. However, because glucagon levels also increase in response to a protein meal, and the critical factor is the insulin-to-glucagon ratio, sufficient glucagon is released that gluconeogenesis is enhanced (at the expense of protein synthesis), and the amino acids that are taken up by the tissues serve as a substrate for gluconeogenesis. The synthesis of glycogen and TGs is also reduced when glucagon levels in the blood rise. In fasting subjects, the average level of immunoreactive glucagon in the blood is 75 pg/mL and does not vary as much as insulin during the daily fasting–feeding cycle. However, only 30% to 40% of the measured immunoreactive glucagon is mature pancreatic glucagon. The rest is composed of larger immunoreactive fragments that are also produced in the pancreas or in the intestinal L-cells.

IV. MECHANISMS OF HORMONE ACTION For a hormone to affect the flux of substrates through a metabolic pathway, it must be able to change the rate at which that pathway proceeds by increasing or decreasing the rate of the slowest step(s). Either directly or indirectly, hormones affect the activity of specific enzymes or transport proteins that regulate the flux through a pathway. Thus, ultimately, the hormone must either cause the amount of the substrate for the enzyme to increase (if substrate supply is a ratelimiting factor), change the conformation at the active site by phosphorylating the enzyme, change the concentration of an allosteric effector of the enzyme, or change the amount of the protein by inducing or repressing its synthesis or by changing its turnover rate or location. Insulin, glucagon, and other hormones

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use all of these regulatory mechanisms to determine the rate of flux in metabolic pathways. The effects mediated by phosphorylation or changes in the kinetic properties of an enzyme occur rapidly within minutes. In contrast, it may take hours for induction or repression of enzyme synthesis to change the amount of an enzyme in the cell. The details of hormone action were described in Chapter 11 and are only summarized here.

A. Signal Transduction by Hormones that Bind to Plasma Membrane Receptors Hormones initiate their actions on target cells by binding to specific receptors or binding proteins. In the case of polypeptide hormones (such as insulin and glucagon) and catecholamines (epinephrine and norepinephrine), the action of the hormone is mediated through binding to a specific receptor on the plasma membrane (see Chapter 11, Section III). The first message of the hormone is transmitted to intracellular enzymes by the activated receptor and an intracellular second messenger; the hormone does not need to enter the cell to exert its effects. (In contrast, steroid hormones such as cortisol and the thyroid hormone triiodothyronine [T3] enter the cytosol and eventually move into the cell nucleus to exert their effects.) The mechanism by which the message carried by the hormone that ultimately affects the rate of the regulatory enzyme in the target cell is called signal transduction. The three basic types of signal transduction for hormones binding to receptors on the plasma membrane are (1) receptor coupling to adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP); (2) receptor kinase activity; and (3) receptor coupling to hydrolysis of phosphatidylinositol bisphosphate (PIP2). The hormones of metabolic homeostasis each use one of these mechanisms to carry out their physiologic effect. In addition, some hormones and neurotransmitters act through receptor coupling to gated ion channels (described in Chapter 11). 1.

SIGNAL TRANSDUCTION BY INSULIN

Insulin initiates its action by binding to a receptor on the plasma membrane of insulin’s many target cells (see Fig. 11.13). The insulin receptor has two types of subunits: the ␣-subunits to which insulin binds, and the ␤-subunits, which span the membrane and protrude into the cytosol. The cytosolic portion of the ␤-subunit has tyrosine kinase activity. On binding of insulin, the tyrosine kinase phosphorylates tyrosine residues on the ␤-subunit (autophosphorylation), as well as on several other enzymes within the cytosol. A principal substrate for phosphorylation by the receptor, insulin receptor substrate 1 (IRS-1), then recognizes and binds to various signal transduction proteins in regions referred to as SH2 domains. IRS-1 is involved in many of the physiologic responses to insulin through complex mechanisms that are the subject of intensive investigation. The basic tissue-specific cellular responses to insulin, however, can be grouped into five major categories: (1) insulin reverses glucagon-stimulated phosphorylation, (2) insulin works through a phosphorylation cascade that stimulates the phosphorylation of several enzymes, (3) insulin induces and represses the synthesis of specific enzymes, (4) insulin acts as a growth factor and has a general stimulatory effect on protein synthesis, and (5) insulin stimulates glucose and amino acid transport into cells (see Fig. V.10 in the introduction to Section Five). Several mechanisms have been proposed for the action of insulin in reversing glucagon-stimulated phosphorylation of the enzymes of carbohydrate metabolism. From the student’s point of view, the ability of insulin to reverse glucagon-stimulated phosphorylation occurs as if it were lowering cAMP and stimulating phosphatases that could remove those phosphates added by protein kinase A (PKA). In reality, the mechanism is more complex and still not fully understood.

Lieberman_Ch26.indd 487

487

The physiologic importance of insulin’s usual action of mediating the suppressive effect of glucose on glucagon secretion is apparent in patients with types 1 and 2 diabetes mellitus. Despite the presence of hyperglycemia, glucagon levels in such patients initially remain elevated (near fasting levels), either because of the absence of insulin’s suppressive effect or because of the resistance of the ␣-cells to insulin’s suppressive effect even in the face of adequate insulin levels in type 2 patients. Thus, these patients have inappropriately high glucagon levels, leading to the suggestion that diabetes mellitus is actually a “bihormonal” disorder.

During the “stress” of hypoglycemia, the autonomic nervous system stimulates the pancreas to secrete glucagon, which tends to restore the serum glucose level to normal. The increased activity of the adrenergic nervous system (through epinephrine) also alerts a patient, such as Bea Selmass, to the presence of increasingly severe hypoglycemia. Hopefully, this will induce the patient to ingest simple sugars or other carbohydrates, which, in turn, will also increase glucose levels in the blood. Bea Selmass gained 8 lb before resection of her pancreatic insulin-secreting adenoma through this mechanism.

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SECTION V ■ CARBOHYDRATE METABOLISM

2.

Phosphodiesterase is inhibited by methylxanthines, a class of compounds that includes caffeine. Would the effect of a methylxanthine on fuel metabolism be similar to fasting or to a highcarbohydrate meal?

Lieberman_Ch26.indd 488

SIGNAL TRANSDUCTION BY GLUCAGON

The pathway for signal transduction by glucagon is one that is common to several hormones; the glucagon receptor is coupled to adenylate cyclase and cAMP production (see Fig. 11.10). Glucagon, through G-proteins, activates the membrane-bound adenylate cyclase, increasing the synthesis of the intracellular second messenger 3⬘,5⬘-cyclic AMP (cAMP) (see Fig. 11.18). cAMP activates PKA (cAMP-dependent protein kinase), which changes the activity of enzymes by phosphorylating them at specific serine residues. Phosphorylation activates some enzymes and inhibits others. The G-proteins, which couple the glucagon receptor to adenylate cyclase, are proteins in the plasma membrane that bind guanosine triphosphate (GTP) and have dissociable subunits that interact with both the receptor and adenylate cyclase. In the absence of glucagon, the stimulatory Gs-protein complex binds guanosine diphosphate (GDP) but cannot bind to the unoccupied receptor or adenylate cyclase (see Fig. 11.17). Once glucagon binds to the receptor, the receptor also binds the Gs-complex, which then releases GDP and binds GTP. The ␣-subunit then dissociates from the ␤y-subunits and binds to adenylate cyclase, thereby activating it. As the GTP on the ␣-subunit is hydrolyzed to GDP, the subunit dissociates and recomplexes with the ␤- and y-subunits. Only continued occupancy of the glucagon receptor can keep adenylate cyclase active. Although glucagon works by activating adenylate cyclase, a few hormones inhibit adenylate cyclase. In this case, the inhibitory G-protein complex is called a Gi-complex. cAMP is the intracellular second messenger for several hormones that regulate fuel metabolism. The specificity of the physiologic response to each hormone results from the presence of specific receptors for that hormone in target tissues. For example, glucagon activates glucose production from glycogen in liver but not in skeletal muscle because glucagon receptors are present in liver but absent in skeletal muscle. However, skeletal muscle has adenylate cyclase, cAMP, and PKA, which can be activated by epinephrine binding to the ␤2-receptors in the membranes of muscle cells. Liver cells also have epinephrine receptors. cAMP is very rapidly degraded to AMP by a membrane-bound phosphodiesterase. The concentration of cAMP is thus very low in the cell, so changes in its concentration can occur rapidly in response to changes in the rate of synthesis. The amount of cAMP present at any time is a direct reflection of hormone binding and the activity of adenylate cyclase. It is not affected by ATP, ADP (adenosine diphosphate), or AMP levels in the cell. cAMP transmits the hormone signal to the cell by activating PKA (cAMPdependent protein kinase). As cAMP binds to the regulatory subunits of PKA, these subunits dissociate from the catalytic subunits, which are thereby activated (see Chapter 9, Fig. 9.10). Activated PKA phosphorylates serine residues of key regulatory enzymes in the pathways of carbohydrate and fat metabolism. Some enzymes are activated and others are inhibited by this change in phosphorylation state. The message of the hormone is terminated by the action of semispecific protein phosphatases that remove phosphate groups from the enzymes. The activity of the protein phosphatases is also controlled through hormonal regulation. Changes in the phosphorylation state of proteins that bind to cAMP response elements (CREs) in the promoter region of genes contribute to the regulation of gene transcription by several cAMP-coupled hormones (see Chapter 16). For instance, cAMP response element binding (CREB) protein is directly phosphorylated by PKA, a step essential for the initiation of transcription. Phosphorylation at other sites on CREB, by a variety of kinases, also may play a role in regulating transcription. The mechanism for signal transduction by glucagon illustrates some of the important principles of hormonal signaling mechanisms. The first principle is that specificity of action in tissues is conferred by the receptor on a target cell for

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CHAPTER 26 ■ BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM

glucagon. In general, the major actions of glucagon occur in liver, adipose tissue, and certain cells of the kidney that contain glucagon receptors. The second principle is that signal transduction involves amplification of the first message. Glucagon and other hormones are present in the blood in very low concentrations. However, these minute concentrations of hormone are adequate to initiate a cellular response because the binding of one molecule of glucagon to one receptor ultimately activates many PKA molecules, each of which phosphorylates hundreds of downstream enzymes. The third principle involves integration of metabolic responses. For instance, the glucagon-stimulated phosphorylation of enzymes simultaneously activates glycogen degradation, inhibits glycogen synthesis, and inhibits glycolysis in the liver (see Fig. V.10 in the introduction to Section Five). The fourth principle involves augmentation and antagonism of signals. An example of augmentation involves the actions of glucagon and epinephrine (which is released during exercise). Although these hormones bind to different receptors, each can increase cAMP and stimulate glycogen degradation. The fifth principle is that of rapid signal termination. In the case of glucagon, both the termination of the Gs-protein activation and the rapid degradation of cAMP contribute to signal termination.

B. Signal Transduction by Cortisol and Other Hormones that Interact with Intracellular Receptors Signal transduction by the glucocorticoid cortisol and other steroids that have glucocorticoid activity and by thyroid hormone involves hormone binding to intracellular (cytosolic) receptors or binding proteins, after which this hormone-binding protein complex, if not already in the nucleus, moves into the nucleus, where it interacts with chromatin. This interaction changes the rate of gene transcription in the target cells (see Chapter 16). The cellular responses to these hormones continue as long as the target cell is exposed to the specific hormones. Thus, disorders that cause a chronic excess in their secretion result in an equally persistent influence on fuel metabolism. For example, chronic stress such as that seen in prolonged sepsis may lead to varying degrees of glucose intolerance if high levels of epinephrine and cortisol persist. The effects of cortisol on gene transcription are usually synergistic to those of certain other hormones. For instance, the rates of gene transcription for some of the enzymes in the pathway for glucose synthesis from amino acids (gluconeogenesis) are induced by glucagon as well as by cortisol.

489

Inhibition of phosphodiesterase by methylxanthine would increase cAMP and have the same effects on fuel metabolism as would an increase of glucagon and epinephrine, as in the fasted state. Increased fuel mobilization would occur through glycogenolysis (the release of glucose from glycogen) and through lipolysis (the release of fatty acids from triacylglycerols).

Ann O’Rexia, to stay thin, frequently fasts for prolonged periods, but she jogs every morning (see Chapter 2). The release of epinephrine and norepinephrine and the increase of glucagon and fall of insulin during her exercise provide coordinated and augmented signals that stimulate the release of fuels above the fasting levels. Fuel mobilization will occur, of course, only as long as she has fuel stored as triacylglycerols.

C. Signal Transduction by Epinephrine and Norepinephrine Epinephrine and norepinephrine are catecholamines (Fig. 26.13). They can act as neurotransmitters or as hormones. A neurotransmitter allows a neural signal to be transmitted across the juncture or synapse between the nerve terminal of a proximal nerve axon and the cell body of a distal neuron. A hormone, conversely, is released into the blood and travels in the circulation to interact with specific receptors on the plasma membrane or cytosol of cells of the target organ. The general effect of these catecholamines is to prepare us for fight or flight. Under these acutely stressful circumstances, these “stress” hormones increase fuel mobilization, cardiac output, blood flow, and so on, which enables us to meet these stresses. The catecholamines bind to adrenergic receptors (the term adrenergic refers to nerve cells or fibers that are part of the involuntary or autonomic nervous system, a system that employs norepinephrine as a neurotransmitter). There are nine different types of adrenergic receptors: ␣1A, ␣1B, ␣1D, ␣2A, ␣2B, ␣2C, ␤1, ␤2, and ␤3. Only the three ␤- and ␣1-receptors are discussed here. The three ␤-receptors work through the adenylate cyclase–cAMP system, activating a Gs-protein, which activates adenylate cyclase, and eventually PKA. The ␤1-receptor is the major adrenergic receptor in the human heart and is primarily stimulated by norepinephrine. On activation, the ␤1-receptor increases the rate of muscle

Lieberman_Ch26.indd 489

HO HO

H O

H CH3

C

C NH

H Epinephrine

HO HO

H

H O

H

C

C NH2

H H Norepinephrine

FIG. 26.13. Structure of epinephrine and norepinephrine. Epinephrine and norepinephrine are synthesized from tyrosine and act as both hormones and neurotransmitters. They are catecholamines, the term catechol referring to a ring structure containing two hydroxyl groups.

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490

SECTION V ■ CARBOHYDRATE METABOLISM

Ann Sulin, a patient with type 2 diabetes mellitus, is experiencing insulin resistance. Her levels of circulating insulin are normal to high, although inappropriately low for her elevated level of blood glucose. However, her insulin target cells, such as muscle and fat, do not respond as those of a nondiabetic subject would to this level of insulin. For most type 2 patients, the site of insulin resistance is subsequent to binding of insulin to its receptor; that is, the number of receptors and their affinity for insulin is near normal. However, the binding of insulin at these receptors does not elicit most of the normal intracellular effects of insulin discussed earlier. Consequently, there is little stimulation of glucose metabolism and storage after a high-carbohydrate meal and little inhibition of hepatic gluconeogenesis.

contraction, in part because of PKA-mediated phosphorylation of phospholamban (see Chapter 47). The ␤2-receptor is present in liver, skeletal muscle, and other tissues and is involved in the mobilization of fuels (such as the generation of glucose through glycogenolysis). It also mediates vascular, bronchial, and uterine smooth muscle contraction. Epinephrine is a much more potent agonist for this receptor than norepinephrine, whose major action is neurotransmission. The ␤3-receptor is found predominantly in adipose tissue and to a lesser extent in skeletal muscle. Activation of this receptor stimulates fatty acid oxidation and thermogenesis, and agonists for this receptor may prove to be beneficial weight-loss agents. The a1-receptors, which are postsynaptic receptors, mediate vascular and smooth muscle contraction. They work through the PIP2 system (see Chapter 11, Section III.B.2) via activation of a Gq-protein, and phospholipase C-␤. This receptor also mediates glycogenolysis in liver. CLINICAL COMMENTS Ann Sulin. Ann Sulin has type 2 diabetes mellitus (formerly called non–insulin-dependent diabetes mellitus), whereas Di Abietes has type 1 diabetes mellitus (formerly designated insulin-dependent diabetes mellitus [IDDM]). Although the pathogenesis differs for these major forms of diabetes mellitus, both cause varying degrees of hyperglycemia. In type 1 diabetes mellitus, the pancreatic ␤-cells are gradually destroyed by antibodies directed at a variety of proteins within the ␤-cells. As insulin secretory capacity by the ␤-cells gradually diminishes below a critical level, the symptoms of chronic hyperglycemia develop rapidly. In type 2 diabetes mellitus, these symptoms develop more subtly and gradually over the course of months or years. Eighty-five percent or more of type 2 patients are obese and, like Ivan Applebod, have a high waist–hip ratio with regard to adipose tissue disposition. This abnormal distribution of fat in the visceral (peri-intestinal) adipocytes is associated with reduced sensitivity of fat cells, muscle cells, and liver cells to the actions of insulin outlined previously. This insulin resistance can be diminished through weight loss, specifically in the visceral depots. The development of type 2 diabetes, coupled with obesity and high blood pressure, can lead to the metabolic syndrome, a common clinical entity that is discussed in more detail in the next section of the text. Bea Selmass. Bea Selmass underwent a high-resolution ultrasonographic (ultrasound) study of her upper abdomen, which showed a 2.6-cm mass in the midportion of her pancreas. With this finding, her physicians decided that further noninvasive studies would not be necessary before surgical exploration of her upper abdomen was performed. At the time of surgery, a yellow-white 2.8-cm mass consisting primarily of insulin-rich ␤-cells was resected from her pancreas. No cytologic changes of malignancy were seen on microscopic examination of the surgical specimen, and no gross evidence of malignant behavior by the tumor (such as local metastases) was found. Bea had an uneventful postoperative recovery and no longer experienced the signs and symptoms of insulininduced hypoglycemia. BIOCHEMICAL COMMENTS Actions of Insulin. One of the important cellular responses to insulin is the reversal of glucagon-stimulated phosphorylation of enzymes. Mechanisms proposed for this action include the inhibition of adenylate cyclase, a reduction of cAMP levels, the stimulation of phosphodiesterase, the production of a specific protein (insulin factor), the release of a second messenger from

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491

a bound glycosylated phosphatidylinositol, and the phosphorylation of enzymes at a site that antagonizes protein kinase A phosphorylation. Not all of these physiologic actions of insulin occur in each of the insulin-sensitive organs of the body. Insulin is also able to antagonize the actions of glucagon at the level of specific induction or repression of key regulatory enzymes of carbohydrate metabolism. For instance, the rate of synthesis of messenger RNA (mRNA) for phosphoenolpyruvate carboxykinase, a key enzyme of the gluconeogenic pathway, is increased severalfold by glucagon (via cAMP) and decreased by insulin. Thus, all of the effects of glucagon, even the induction of certain enzymes, can be reversed by insulin. This antagonism is exerted through an insulin-sensitive hormone response element (IRE) in the promoter region of the genes. Insulin causes repression of the synthesis of enzymes that are induced by glucagon. The general stimulation of protein synthesis by insulin (its mitogenic or growthpromoting effect) appears to occur through a general increase in rates of mRNA translation for a broad spectrum of structural proteins. These actions result from a phosphorylation cascade initiated by autophosphorylation of the insulin receptor and ending in the phosphorylation of subunits of proteins that bind to and inhibit eukaryotic protein synthesis initiation factors (eIFs). When phosphorylated, the inhibitory proteins are released from the eIFs, allowing translation of mRNA to be stimulated. In this respect, the actions of insulin are similar to those of other hormones that act as growth factors and that also have receptors with tyrosine kinase activity. In addition to signal transduction, activation of the insulin receptor mediates the internalization of receptor-bound insulin molecules, increasing their subsequent degradation. Although unoccupied receptors can be internalized and eventually recycled to the plasma membrane, the receptor can be irreversibly degraded after prolonged occupation by insulin. The result of this process, referred to as receptor downregulation, is an attenuation of the insulin signal. The physiologic importance of receptor internalization on insulin sensitivity is poorly understood but could lead eventually to chronic hyperglycemia.

Key Concepts • •

•

•

• •

• • • • •

Glucose homeostasis is directed toward the maintenance of constant blood glucose levels. Insulin and glucagon are the two major hormones that regulate the balance between fuel mobilization and storage. They maintain blood glucose levels near 80 to 100 mg/dL despite varying levels of carbohydrate intake during the day. If dietary intake of all fuels is in excess of immediate caloric requirements, those fuels are stored as either glycogen or fat. Conversely, stored fuels are mobilized when demand exceeds the quantity of calories ingested. Insulin is released in response to carbohydrate ingestion and promotes glucose utilization as a fuel and glucose storage as fat and glycogen. Insulin secretion is regulated principally by blood glucose levels. Glucagon promotes glucose production via glycogenolysis (glycogen degradation) and gluconeogenesis (glucose synthesis from amino acids and other noncarbohydrate precursors). Glucagon release is regulated principally through suppression by rising levels of glucose and rising levels of insulin. Glucagon levels decrease in response to a carbohydrate meal and increase during fasting. Increased levels of glucagon relative to insulin stimulate the release of fatty acids from adipose tissue. Glucagon acts by binding to a receptor on the cell surface, which stimulates the synthesis of the intracellular second messenger, cAMP. cAMP activates protein kinase A, which phosphorylates key regulatory enzymes, activating some and inhibiting others. Insulin acts via a receptor tyrosine kinase and leads to the dephosphorylation of the key enzymes phosphorylated in response to glucagon. Hormones that antagonize insulin action, known as insulin counterregulatory hormones, include glucagon, epinephrine, and cortisol. Diseases discussed in this chapter are summarized in Table 26.4.

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Table 26.4

Diseases Discussed in Chapter 26

Disease or Disorder

Environmental or Genetic

Type 2 diabetes

Both

Emergence of insulin resistance, due to a wide variety of causes; tissues do not respond to insulin as they normally would.

Insulinoma

Environmental

Periodic release of insulin from a tumor of the ␤-cells, leading to hypoglycemic symptoms, which are accompanied by excessive appetite and weight gain.

Hyperglycemia

Both

Constantly elevated levels of glucose in the circulation due to a wide variety of causes. Hyperglycemia leads to protein glycation and potential loss of protein function in a variety of tissues.

Type 1 diabetes

Both

No production of insulin by the ␤-cells due to an autoimmune destruction of the ␤-cells. Hyperglycemia and ketoacidosis may result from the lack of insulin.

Maturity-onset diabetes of the young

Both

Form of diabetes caused by specific mutations, such as a mutation in pancreatic glucokinase, which alters the set point for insulin release from the ␤-cells.

Neonatal diabetes

Genetic

One cause of neonatal diabetes is a mutation in a subunit of the potassium channel in various tissues. Such a mutation in the pancreas leads to permanent opening of the potassium channel, keeping intracellular calcium levels low, and difficulty in releasing insulin from the ␤-cells.

Comments

REVIEW QUESTIONS—CHAPTER 26 1.

A. B. C. D. E. 2.

Increased glucagon release from the pancreas Decreased glucagon release from the pancreas High blood glucose levels Low blood glucose levels Elevated ketone body levels

4.

Which one of the following organs has the highest demand for glucose as a fuel? A. Brain B. Muscle (skeletal) C. Heart D. Liver E. Pancreas

5.

Glucagon release does not alter muscle metabolism because of which of the following? A. Muscle cells lack adenylate cyclase. B. Muscle cells lack protein kinase A. C. Muscle cells lack G-proteins. D. Muscle cells lack guanosine triphosphate (GTP). E. Muscle cells lack the glucagon receptor.

Caffeine is a potent inhibitor of the enzyme cAMP phosphodiesterase. Which of the following consequences would you expect to occur in the liver after drinking two cups of strong espresso coffee? A. B. C. D. E.

3.

lead to maturity-onset diabetes of the young (MODY) because of which of the following within the pancreatic ␤-cell? A. A reduced ability to raise cAMP levels B. A reduced ability to raise ATP levels C. A reduced ability to stimulate gene transcription D. A reduced ability to activate glycogen degradation E. A reduced ability to raise intracellular lactate levels

A patient with type 1 diabetes mellitus takes an insulin injection before eating dinner but then gets distracted and does not eat. Approximately 3 hours later, the patient becomes shaky, sweaty, and confused. These symptoms have occurred because of which of the following?

A prolonged response to insulin A prolonged response to glucagon An inhibition of protein kinase A An enhancement of glycolytic activity A reduced rate of glucose export to the circulation

Assume that an increase in blood glucose concentration from 5 to 10 mM would result in insulin release by the pancreas. A mutation in pancreatic glucokinase can

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27

Digestion, Absorption, and Transport of Carbohydrates

Carbohydrates are the largest source of dietary calories for most of the world’s population. The major carbohydrates in the US diet are starch, lactose, and sucrose. The starches amylose and amylopectin are polysaccharides composed of hundreds to millions of glucosyl units linked together through ␣-1,4- and ␣-1,6glycosidic bonds (Fig. 27.1). Lactose is a disaccharide composed of glucose and galactose, linked together through a ␤-1,4-glycosidic bond. Sucrose is a disaccharide composed of glucose and fructose, linked through an ␣-1,2-glycosidic bond. The digestive processes convert all of these dietary carbohydrates to their constituent monosaccharides by hydrolyzing glycosidic bonds between the sugars. The digestion of starch begins in the mouth (Fig. 27.2). The salivary gland releases ␣-amylase, which converts starch to smaller polysaccharides called ␣-dextrins. Salivary ␣-amylase is inactivated by the acidity of the stomach (HCl). Pancreatic ␣-amylase and bicarbonate are secreted by the exocrine pancreas into the lumen of the small intestine, where bicarbonate neutralizes the gastric secretions. Pancreatic ␣-amylase continues the digestion of ␣-dextrins, converting them to disaccharides (maltose), trisaccharides (maltotriose), and oligosaccharides called limit dextrins. Limit dextrins usually contain four to nine glucosyl residues and an isomaltose branch (two glucosyl residues attached through an ␣-1,6-glycosidic bond). The digestion of the disaccharides lactose and sucrose, as well as further digestion of maltose, maltotriose, and limit dextrins, occurs through disaccharidases attached to the membrane surface of the brush border (microvilli) of intestinal epithelial cells. Glucoamylase hydrolyzes the ␣-1,4-bonds of dextrins. The sucrase–isomaltase complex hydrolyzes sucrose, most of maltose, and almost all of the isomaltose formed by glucoamylase from limit dextrins. Lactase-glycosylceramidase (␤-glycosidase) hydrolyzes the ␤-glycosidic bonds in lactose and glycolipids. A fourth disaccharidase complex, trehalase, hydrolyzes the bond (an ␣-1,1-glycosidic bond) between two glucosyl units in the sugar trehalose. The monosaccharides produced by these hydrolases (glucose, fructose, and galactose) are then transported into the intestinal epithelial cells. Dietary fiber, composed principally of polysaccharides, cannot be digested by human enzymes in the intestinal tract. In the colon, dietary fiber and other nondigested carbohydrates may be converted to gases (H2, CO2, and methane) and short-chain fatty acids (principally acetic acid, propionic acid, and butyric acid) by bacteria in the colon. Glucose, galactose, and fructose formed by the digestive enzymes are transported into the absorptive epithelial cells of the small intestine by protein-mediated Na⫹-dependent active transport and facilitative diffusion. Monosaccharides are transported from these cells into the blood and circulate to the liver and peripheral tissues where they are taken up by facilitative transporters. Facilitative transport of glucose across epithelial cells and other cell membranes is mediated by a family of tissue-specific glucose transport proteins (GLUT 1 to GLUT 5). The type of transporter found in each cell reflects the role of glucose metabolism in that cell.

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SECTION V ■ CARBOHYDRATE METABOLISM

CH2OH O

CH2OH O

OH

O

OH

O

OH

α1,4

O OH

n

Amylose

CH2OH O O

CH2OH O

OH

OH

O OH

HO

CH2OH O

OH

O

O α1,6 CH2

O

OH

O OH

O OH

n

Amylopectin

HO

CH2OH O OH

CH2OH O O

OH

OH

β1,4 OH

OH Glucose

Galactose

Lactose

CH2OH O Glucose HO

OH OH

HOCH2 Fructose

O α1,2 O HO

HO

CH2OH

Sucrose FIG. 27.1. The structures of common dietary carbohydrates. For disaccharides and higher, the sugars are linked through glycosidic bonds between the anomeric carbon of one sugar and a hydroxyl group on another sugar. The glycosidic bond may be either ␣- or ␤-, depending on its position above or below the plane of the sugar containing the anomeric carbon. (See Chapter 5, Section II.A, to review terms used in the description of sugars.) The starch amylose is a polysaccharide of glucose residues linked with ␣-1,4-glycosidic bonds. Amylopectin is amylose with the addition of ␣-1,6-glycosidic branch points. Dietary sugars may be monosaccharides (single sugar residues), disaccharides (two sugar residues), oligosaccharides (several sugar residues), or polysaccharides (hundreds of sugar residues). For clarity, the hydrogen atoms are not shown in the figure.

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Starch Lactose Sucrose

495

Salivary salivary ␣–amylase

Sucrose Lactose

␣-Dextrins

Stomach Pancreas

␣-Amylase HCO3 – Tri- and oligosaccharides Maltose, Isomaltose

Maltase isomaltase

Sucrose Lactose

Small intestine

Glucose

Sucrase

Glucose Fructose

Lactase

Glucose Galactose

Fiber

Colon

Feces

FIG. 27.2. Overview of carbohydrate digestion. Digestion of the carbohydrates occurs first, followed by absorption of monosaccharides. Subsequent metabolic reactions occur after the sugars are absorbed.

THE WAITING ROOM Deria Voider is a 20-year-old exchange student from Nigeria who has noted gastrointestinal bloating, abdominal cramps, and intermittent diarrhea ever since arriving in the United States 6 months ago. A careful history shows that these symptoms occur most commonly about 45 minutes to 1 hour after eating breakfast but may occur after other meals as well. Dairy products, which were not a part of Deria’s diet in Nigeria, were identified as the probable offending agent because her gastrointestinal symptoms disappeared when milk and milk products were eliminated from her diet.

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Ann Sulin’s fasting and postprandial blood glucose levels are frequently above the normal range in spite of good compliance with insulin therapy. Her physician has referred her to a dietician skilled in training diabetic patients in the successful application of an appropriate American Diabetes Association diet. As part of the program, Ms. Sulin is asked to incorporate foods containing fiber into her diet, such as whole grains (e.g., wheat, oats, corn), legumes (e.g., peas, beans, lentils), tubers (e.g., potatoes, peanuts), and fruits. The dietary sugar in fruit juice and other sweets is sucrose, a disaccharide composed of glucose and fructose joined through their anomeric carbons. Nona Melos’s symptoms of pain and abdominal distension are caused by an inability to digest sucrose or absorb fructose, which are converted to gas by colonic bacteria. Nona’s stool sample had a pH of 5 and gave a positive test for sugar. The possibility of carbohydrate malabsorption was considered, and a hydrogen breath test was recommended.

Nona Melos is a 7-month-old baby girl, the second child born to unrelated parents. Her mother had a healthy, full-term pregnancy, and Nona’s birth weight was normal. She did not respond well to breastfeeding and was changed entirely to a formula based on cows’ milk at 4 weeks. Between 7 and 12 weeks of age, she was admitted to the hospital twice with a history o f screaming after feeding, but she was discharged after observation without a specific diagnosis. Elimination of cows’ milk from her diet did not relieve her symptoms; Nona’s mother reported that the screaming bouts were worse after Nona drank juice and that Nona frequently had gas and a distended abdomen. At 7 months, she was still thriving (weight ⬎97th percentile) with no abnormal findings on physical examination. A stool sample was taken.

I.

DIETARY CARBOHYDRATES

Carbohydrates are the largest source of calories in the average US diet and usually constitute 40% to 45% of our caloric intake. The plant starches amylopectin and amylose, which are present in grains, tubers, and vegetables, constitute approximately 50% to 60% of the carbohydrate calories consumed. These starches are polysaccharides, containing 10,000 to 1 million glucosyl units. In amylose, the glucosyl residues form a straight chain linked via ␣-1,4-glycosidic bonds; in amylopectin, the ␣-1,4-chains contain branches connected via ␣-1,6glycosidic bonds (see Fig. 27.1). The other major sugar found in fruits and vegetables is sucrose, a disaccharide of glucose and fructose (see Fig. 27.1). Sucrose and small amounts of the monosaccharides glucose and fructose are the major natural sweeteners found in fruit, honey, and vegetables. Dietary fiber, the part of the diet that cannot be digested by human enzymes of the intestinal tract, is also composed principally of plant polysaccharides and a polymer called lignan. Most foods derived from animals, such as meat or fish, contain very little carbohydrate except for small amounts of glycogen (which has a structure similar to amylopectin) and glycolipids. The major dietary carbohydrate of animal origin is lactose, a disaccharide composed of glucose and galactose that is found exclusively in milk and milk products (see Fig. 27.1). Sweeteners, in the form of sucrose and high-fructose corn syrup (starch, partially hydrolyzed and isomerized to fructose), also appear in the diet as additives to processed foods. On average, a person in the United States consumes 65 lb of added sucrose and 40 lb of high-fructose corn syrup solids per year. Although all cells require glucose for metabolic functions, neither glucose nor other sugars are specifically required in the diet. Glucose can be synthesized from many amino acids found in dietary protein. Fructose, galactose, xylulose, and all the other sugars required for metabolic processes in the human can be synthesized from glucose.

II. DIGESTION OF DIETARY CARBOHYDRATES In the digestive tract, dietary polysaccharides and disaccharides are converted to monosaccharides by glycosidases, enzymes that hydrolyze the glycosidic bonds

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Starch blockers were marketed many years ago as a means of losing weight without having to exercise or reduce your daily caloric intake. Starch blockers were based on a protein found in beans, which blocked the action of amylase. Thus, as the advertisements proclaimed, one could eat a large amount of starch during a meal, and as long as you took the starch blocker, the starch would pass through the digestive track without being metabolized. Unfortunately, this was too good to be true, and starch blockers were never shown to be effective in aiding weight loss. This was probably because of a combination of factors, such as inactivation of the inhibitor by the low pH in the stomach, and an excess of amylase activity as compared with the amount of starch blocker ingested. Recently, this issue has been revisited, as a starch blocker from wheat has been developed that may work as advertised, although much more work is required to determine whether this amylase inhibitor will be safe and effective in humans. Additionally, newer (and improved) preparations of the bean extract are also being readvertised.

between the sugars. All of these enzymes exhibit some specificity for the sugar, the glycosidic bond (␣- or ␤), and the number of saccharide units in the chain. The monosaccharides formed by glycosidases are transported across the intestinal mucosal cells into the interstitial fluid and subsequently enter the bloodstream. Undigested carbohydrates enter the colon, where they may be fermented by bacteria.

A. Salivary and Pancreatic ␣-Amylase The digestion of starch (amylopectin and amylose) begins in the mouth, where chewing mixes the food with saliva. The salivary glands secrete approximately 1 L of liquid per day into the mouth, containing salivary ␣-amylase and other components. ␣-Amylase is an endoglucosidase, which means that it hydrolyzes internal ␣-1,4-bonds between glucosyl residues at random intervals in the polysaccharide chains (Fig. 27.3). The shortened polysaccharide chains that are formed are called ␣-dextrins. Salivary ␣-amylase is largely inactivated by the acidity of the stomach contents, which contain HCl secreted by the parietal cells. The acidic gastric juice enters the duodenum, the upper part of the small intestine, where digestion continues. Secretions from the exocrine pancreas (approximately 1.5 L/day) flow down the pancreatic duct and also enter the duodenum. These secretions contain bicarbonate (HCO3⫺), which neutralizes the acidic pH of stomach contents, and digestive enzymes, including pancreatic ␣-amylase. Pancreatic ␣-amylase continues to hydrolyze the starches and glycogen, forming the disaccharide maltose, the trisaccharide maltotriose, and oligosaccharides. These oligosaccharides, called limit dextrins, are usually four to nine glucosyl units long

O

O O

O O

O O

O

HO

O

O

O

497

O

O

O

Starch

O

O

O

O

O

O

O O

O

Salivary and pancreatic ␣-amylase H O O HO

O O

O

O OH

O

O

O

O

O

O

O

O

O

HO

OH

Isomaltose

Maltose O

O O

O O

O HO

O O

O O

O OH

Trisaccharides (and larger oligosaccharides)

HO

O

O O

O

OH

␣-Dextrins (oligosaccharides with ␣-1,6-branches)

FIG. 27.3. Action of salivary and pancreatic ␣-amylases.

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SECTION V ■ CARBOHYDRATE METABOLISM

Amylase activity in the gut is abundant and is not normally rate limiting for the process of digestion. Alcohol-induced pancreatitis or surgical removal of part of the pancreas can decrease pancreatic secretion. Pancreatic exocrine secretion into the intestine also can be decreased as a result of cystic fibrosis (as in Sissy Fibrosa, see Chapter 18) in which mucus blocks the pancreatic duct, which eventually degenerates. However, pancreatic exocrine secretion can be decreased to 10% of normal and still not affect the rate of starch digestion because amylases are secreted in the saliva and pancreatic fluid in excessive amounts. In contrast, protein and fat digestion are more strongly affected in cystic fibrosis.

A Villi

and contain one or more ␣-1,6-branches. The two glucosyl residues that contain the ␣-1,6-glycosidic bond eventually become the disaccharide isomaltose, but ␣-amylase does not cleave these branched oligosaccharides all the way down to isomaltose. ␣-Amylase has no activity toward sugar-containing polymers other than glucose linked by ␣-1,4-bonds. ␣-Amylase displays no activity toward the ␣-1,6-bond at branch points and has little activity for the ␣-1,4-bond at the nonreducing end of a chain.

B. Disaccharidases of the Intestinal Brush Border Membrane The dietary disaccharides lactose and sucrose, as well as the products of starch digestion, are converted to monosaccharides by glycosidases attached to the membrane in the brush border of absorptive cells (Fig. 27.4). The different glycosidase activities are found in four glycoproteins: glucoamylase, the sucrase–isomaltase complex, the smaller glycoprotein trehalase, and lactaseglucosylceramidase (Table 27.1). These glycosidases are collectively called the small intestinal disaccharidases, although glucoamylase is really an oligosaccharidase. 1.

Mucosa Submucosa

GLUCOAMYLASE

Glucoamylase and the sucrase–isomaltase complex have similar structures and exhibit a great deal of sequence homogeneity (Fig. 27.5). A membrane-spanning domain near the N-terminal attaches the protein to the luminal membrane. The long

Table 27.1

B

Blood and lymph vessels

The Different Forms of the Brush Border Glycosidases

Complex

Catalytic Sites

Principal Activities

␤-Glucoamylase

␣-Glucosidase

Split ␣-1,4-glycosidic bonds between glucosyl units, beginning sequentially with the residue at the tail end (nonreducing end) of the chain. This is an exoglycosidase. Substrates include amylose, amylopectin, glycogen, and maltose. Same as above but with slightly different specificities and affinities for the substrates. Splits sucrose, maltose, and maltotriose. Splits ␣-1,-6-bonds in several limit dextrins, as well as the ␣-1,4-bonds in maltose and maltotriose. Splits ␤-glycosidic bonds between (phlorizin hydrolase) glucose or galactose and hydrophobic residues, such as the glycolipids glucosylceramide and galactosylceramide. Splits the ␤-1,4-bond between glucose and galactose. To a lesser extent also splits the ␤-1,4-bond between some cellulose disaccharides. Splits bond in trehalose, which is two glucosyl units linked ␣-1,1 through their anomeric carbons.

Absorptive and goblet cells ␤-Glucosidase

Sucrase–isomaltase

C

Sucrase–maltase Isomaltase–maltase

Nutrients Brush border (contains transport and digestive complexes)

␤-Glycosidase

Glucosyl–ceramidase

Absorptive cell Lactase

Basement membrane Capillary

FIG. 27.4. Location of disaccharidase complexes in intestinal villi.

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Trehalase

Trehalase

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CHAPTER 27 ■ DIGESTION, ABSORPTION, AND TRANSPORT OF CARBOHYDRATES

Can the glycosidic bonds of the structure shown hereafter be hydrolyzed by ␣-amylose?

C

Sucrase

CH2OH

CH2OH

O

C

499

O O

OH

N

O

OH

OH

OH

n

Isomaltase

Connecting segment (stalk)

Transmembrane segment N Sucrase– isomaltase

Cytoplasmic domain

FIG. 27.5. The major portion of the sucrase–isomaltase complex, containing the catalytic sites, protrudes from the absorptive cells into the lumen of the intestine. Other domains of the protein form a connecting segment (stalk) and an anchoring segment that extends through the membrane into the cell. The complex is synthesized as a single polypeptide chain that is split into its two enzyme subunits extracellularly. Each subunit is a domain with a catalytic site (distinct sucrase–maltase and isomaltase–maltase sites). In spite of their maltase activity, these catalytic sites are often called just sucrase and isomaltase.

Acarbose is an FDA-approved drug that blocks the activities of pancreatic ␣-amylase and brush border ␣-glucosidases (with specificity for glucose). The drug is produced from a microorganism and is a unique tetrasaccharide. Acarbose is given to patients with type 2 diabetes, with the purpose of reducing the rate at which ingested carbohydrate reaches the bloodstream after a meal. This is one approach to better control blood glucose levels in such patients. Weight loss has not been associated with use of this drug, but flatulence and diarrhea (due to colonic bacterial metabolism of the sugars) are side effects of taking this drug.

polypeptide chain forms two globular domains, each with a catalytic site. In glucoamylase, the two catalytic sites have similar activities, with only small differences in substrate specificity. The protein is heavily glycosylated with oligosaccharides that protect it from digestive proteases. Glucoamylase is an exoglucosidase that is specific for the ␣-1,4-bonds between glucosyl residues (Fig. 27.6). It begins at the nonreducing end of a polysaccharide or limit dextrin and sequentially hydrolyzes the bonds to release glucose monosaccharides. It will digest a limit dextrin down to isomaltose, the glucosyl disaccharide with an ␣-1,6-branch, which is subsequently hydrolyzed principally by the isomaltase activity in the sucrase–isomaltase complex. 2.

Maltose

α-1,4 bond O HO

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OH

maltase activity

SUCRASE–ISOMALTASE COMPLEX

The structure of the sucrase–isomaltase complex is very similar to that of glucoamylase, and these two proteins have a high degree of sequence homology. However, after the single polypeptide chain of sucrase–isomaltase is inserted through the membrane and the protein protrudes into the intestinal lumen, an intestinal protease clips it into two separate subunits that remain attached to each other. Each subunit has a catalytic site that differs in substrate specificity from the other through noncovalent interactions. The sucrase–maltase site accounts for approximately 100% of the intestine’s ability to hydrolyze sucrose in addition to maltase activity; the isomaltase–maltase site accounts for almost all of the

O O

1 O

O HO

2

O

O O

reducing end

Maltotriose

FIG. 27.6. Glucoamylase activity. Glucoamylase is an ␣-1,4-exoglycosidase that initiates cleavage at the nonreducing end of the sugar. Thus, for maltotriose, the bond labeled 1 is hydrolyzed first, which then allows the bond at position 2 to be the next one hydrolyzed.

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SECTION V ■ CARBOHYDRATE METABOLISM

No. This polysaccharide is cellulose, which contains ␤-1,4-glycosidic bonds. Pancreatic and salivary ␣-amylase cleave only ␣-1,4-bonds between glucosyl units. HO

3.

O O

α-1,6 bond O

HO

HO

OH isomaltase activity

O O

O

O

OH

O

HO

FIG. 27.7. Isomaltase activity. Arrows indicate the ␣-1,6-bonds that are cleaved. Individuals with genetic deficiencies of the sucrase–isomaltase complex show symptoms of sucrose intolerance but are able to digest normal amounts of starch in a meal without problems. The maltase activity in the glucoamylase complex, and residual activity in the sucrase–isomaltase complex (which is normally present in excess of need), is apparently sufficient to digest normal amounts of dietary starch. Which of the bonds in the structure shown hereafter are hydrolyzed by the sucrase–isomaltase complex? Which by glucoamylase? 1

O

HO

O

O

2 O

O HO

O

O O

O

3

4

5

CH2OH O 5 4

HO

O O

HO

TREHALASE

Trehalase is only half as long as the other disaccharidases and has only one catalytic site. It hydrolyzes the glycosidic bond in trehalose, a disaccharide composed of two glucosyl units linked by an ␣-bond between their anomeric carbons (Fig. 27.8). Trehalose, which is found in insects, algae, mushrooms, and other fungi, is not currently a major dietary component in the United States. However, unwitting consumption of trehalose can cause nausea, vomiting, and other symptoms of severe gastrointestinal distress if consumed by an individual deficient in the enzyme. Trehalase deficiency was discovered when a woman became very sick after eating mushrooms and was initially thought to have ␣-amanitin poisoning. 4. ␤-GLYCOSIDASE COMPLEX (LACTASE-GLUCOSYLCERAMIDASE)

The ␤-glycosidase complex is another large glycoprotein found in the brush border that has two catalytic sites extending in the lumen of the intestine. However, its primary structure is very different from that of the other enzymes, and it is attached to the membrane through its carboxyl end by a phosphatidylglycan anchor (see Fig. 10.6). The lactase catalytic site hydrolyzes the ␤-bond connecting glucose and galactose in lactose (a ␤-galactosidase activity; Fig. 27.9). The major activity of the other catalytic site in humans is the ␤-bond between glucose or galactose and ceramide in glycolipids (this catalytic site is sometimes called phlorizin hydrolase, named for its ability to hydrolyze an artificial substrate). 5.

LOCATION WITHIN THE INTESTINE

The production of maltose, maltotriose, and limit dextrins by pancreatic ␣-amylase occurs in the duodenum, the most proximal portion of the small intestine. Sucrase–isomaltase activity is highest in the jejunum, where the enzymes can hydrolyze sucrose and the products of starch digestion. ␤-Glycosidase activity is also highest in the jejenum. Glucoamylase activity increases progressively along the length of the small intestine, and its activity is highest in the ileum. Thus, it presents a final opportunity for digestion of starch oligomers that have escaped amylase and disaccharidase activities at the more proximal regions of the intestine.

Trehalose

6

H

intestine’s ability to hydrolyze ␣-1,6-bonds (Fig. 27.7), in addition to maltase activity. Together, these sites account for approximately 80% of the maltase activity of the small intestine. The remainder of the maltase activity is found in the glucoamylase complex.

H H

H

3

H

H 2

OH 1

1

OH

2

O

OH

Glucose Trehalase activity

OH 3 6

HOH2C O

5

4

OH

CH2OH O HO H

β-1,4 bond O

H Glucose

FIG. 27.8. Trehalose. This disaccharide contains two glucose moieties linked by an unusual bond that joins their anomeric carbons. It is cleaved by trehalase.

Lieberman_Ch27.indd 500

Lactose

H

H

H

OH

H

H

OH

Galactose

lactase

CH2OH O OH H OH

H H

H

OH

Glucose

FIG. 27.9. Lactase activity. Lactase is a ␤-galactosidase. It cleaves the ␤-galactoside lactose, the major sugar in milk, forming galactose and glucose.

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C. Metabolism of Sugars by Colonic Bacteria

Bonds (1) and (3) would first be hydrolyzed by glucoamylase. Bond (2) requires isomaltase. Bonds (4) and (5) can then be hydrolyzed by the sucrase– isomaltase complex, or by the glucoamylase complex, all of which can convert maltotriose and maltose to glucose.

Not all of the starch ingested as part of foods is normally digested in the small intestine (Fig. 27.10). Starches that are high in amylose or are less well hydrated (e.g., starch in dried beans) are resistant to digestion and enter the colon. Dietary fiber and undigested sugars also enter the colon. Here, colonic bacteria rapidly metabolize the saccharides, forming gases, short-chain fatty acids, and lactate. The major short-chain fatty acids formed are acetic acid (two carbons), propionic acid (three carbons), and butyric acid (four carbons). The short-chain fatty acids are absorbed by the colonic mucosal cells and can provide a substantial source of energy for these cells. The major gases formed are hydrogen gas (H2), carbon dioxide (CO2), and methane (CH4). These gases are released through the colon, resulting

CH2OH

CH2OH

O

H

H O

OH H

CH2OH

O O

OH

β(1 4)

H OH Cellulose

O

H H

OH

β-1,4-linked glucose

O

OH

H

H

OH

n

O CH2OH O HO H

COOH O

O

H HOH2C

OH OH

H

H

OH

H HOH2C

H

O

OH H OH

HO

HO H H OH

H

H

H OH

H

β-D-Xylose

α-L-Arabinose

H

H

OH

COCH3 O HO H OH

H O

H

OH

H

OH

N C CH3 OH H H N-AcetylMethylated galactosamine galacturonic acid

OH

H

Galacturonic acid

• Found in hemicelluloses, gums and mucilages • Components of pectin

Galactose CH2OH O HOO2SO

CH2OH O

H OH H

H

H

HO

OH

H OH

H

H

OH

Galactose-4-SO4 • Component of carrageenan

CH2OH

H O

CH CH

OH CH2OH O

CH2 O

H HO OH H

H

O

OH

OH H

Sucrose

CH2OH

H OH

OCH3 H

OH Phenyl propane derivatives • Found in lignin

Raffinose FIG. 27.10.

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Some indigestible carbohydrates. These compounds are components of dietary fiber.

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Nona Melos was given a hydrogen breath test, a test measuring the amount of hydrogen gas released after consuming a test dose of sugar. In this test, the patient breathes into a portable meter or a collecting bag attached to a nonportable device. The larger, nonportable devices measure the hydrogen in the breath via gas chromatography. The portable devices measure the hydrogen gas produced using hydrogenspecific electrodes and measuring a current that is created when hydrogen comes into contact with the electrode. The association of Nona’s symptoms with her ingestion of fruit juices suggests that she might have a problem resulting from low sucrase activity or an inability to absorb fructose. Her ability to thrive and her adequate weight gain suggest that any deficiencies of the sucrase–isomaltase complex must be partial and do not result in a functionally important reduction in maltase activity (maltase activity is also present in the glucoamylase complex). Her urine tested negative for sugar, suggesting the problem is in digestion or absorption, because only sugars that are absorbed and enter the blood can be found in urine. The basis of the hydrogen breath test is that if a sugar is not absorbed, it is metabolized in the intestinal lumen by bacteria that produce various gases, including hydrogen. The test is often accompanied by measurements of the amount of sugar that appear in the blood or feces and acidity of the feces.

in flatulence, or in the breath. Incomplete products of digestion in the intestines increase the retention of water in the colon, resulting in diarrhea.

D. Lactose Intolerance Lactose intolerance refers to a condition of pain, nausea, and flatulence after the ingestion of foods containing lactose, most notably dairy products. Although lactose intolerance is often caused by low levels of lactase, it also can be caused by intestinal injury (defined subsequently). 1.

NONPERSISTANT AND PERSISTANT LACTASE

Lactase activity increases in the human from about 6 to 8 weeks of gestation, and it rises during the late gestational period (27 to 32 weeks) through full term. It remains high for about 1 month after birth and then begins to decline. For most of the world’s population, lactase activity decreases to adult levels at approximately 5 to 7 years of age. Adult levels are less than 10% of those present in infants. These populations have adult hypolactasia (formerly called adult lactase deficiency) and exhibit the lactase nonpersistence phenotype. In people who are derived mainly from Western Northern Europeans, and milk-dependent nomadic tribes of Saharan Africa, the levels of lactase remain at or only slightly below infant levels throughout adulthood (lactase persistence phenotype). Thus, adult hypolactasia is the normal condition for most of the world’s population (Table 27.2). In contrast, congenital lactase deficiency is a severe autosomal recessive inherited disease in which lactase activity is significantly reduced, or totally absent. The disorder presents as soon as the newborn is fed breast milk or lactose-containing formula, resulting in watery diarrhea, weight loss, and dehydration. Treatment consists of removal of lactose from the diet, which allows for normal growth and development to occur. 2.

INTESTINAL INJURY

Intestinal diseases that injure the absorptive cells of the intestinal villi diminish lactase activity along the intestine, producing a condition known as secondary lactase deficiency. Kwashiorkor (protein malnutrition), colitis, gastroenteritis, tropical and nontropical sprue, and excessive alcohol consumption fall into this category. These diseases also affect other disaccharidases, but sucrase, maltase, isomaltase, and glucoamylase activities are usually present at such excessive levels that there are no pathologic effects. Lactase is usually the first activity lost and the last to recover.

III. DIETARY FIBER Dietary fiber is the portion of the diet that is resistant to digestion by human digestive enzymes. It consists principally of plant materials that are polysaccharide

Table 27.2

Prevalence of Late-Onset Lactase Deficiency

Group US population Asians American Indians (Oklahoma) Black Americans Mexican Americans White Americans Other populations Ibo, Yoruba (Nigeria) Italians Aborigines (Australia) Greeks Danes Dutch

Prevalence (%) 100 95 81 56 24 89 71 67 53 3 0

Reproduced with permission from Büller HA, Grand RJ. Lactose intolerance. Annu Rev Med. 1990;41: 141–148. Copyright 1990 by Annual Reviews, Inc.

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Table 27.3

Types of Fiber in the Diet

Classical Nomenclature Insoluble fiber Cellulose

Hemicelluloses Lignin

Classes of Compounds

Dietary Sources

Polysaccharide composed of glucosyl residues linked ␤-1,4

Whole wheat flour, unprocessed bran, cabbage, peas, green beans, wax beans, broccoli, brussel sprouts, cucumber with skin, green peppers, apples, carrots Polymers of arabinoxylans Bran cereals, whole grains, or galactomannans brussel sprouts, mustard beans, beet root Noncarbohydrate, Bran cereals, unprocessed polymeric derivatives bran, strawberries, eggplant of phenylpropane peas, green beans, radishes

Water-soluble fiber (or dispersible) Pectic substances Galactouranans, Squash, apples, citrus fruits arabinogalactans, ␤-glucans, arabinoxylans Gums Galactomannans, Oatmeal, dried beans, arabinogalactans cauliflower, green beans, cabbage, carrots, dried peas, potatoes, strawberries Mucilages Wide range of branched and Flax seed, psyllium, mustard substituted galactans seed

derivatives and lignan (see Fig. 27.10). The components of fiber are often divided into the categories of soluble and insoluble fiber, according to their ability to dissolve in water. Insoluble fiber consists of three major categories: cellulose, hemicellulose, and lignins. Soluble fiber categories include pectins, mucilages, and gums (Table 27.3). Although human enzymes cannot digest fiber, the bacterial flora in the normal human gut may metabolize the more soluble dietary fibers to gases and short-chain fatty acids, much as they do undigested starch and sugars. Some of these fatty acids may be absorbed and used by the colonic epithelial cells of the gut, and some may travel to the liver through the hepatic portal vein. We may obtain as much as 10% of our total calories from compounds produced by bacterial digestion of substances in our digestive tract. In 2005, the Committee on Dietary Reference Intakes issued new guidelines for fiber ingestion—anywhere from 25 to 38 g/day, depending on the age and sex of the individual. It was also recommended that 14 g of fiber should accompany every 1,000 cal ingested. No distinction was made between soluble and insoluble fibers. Adult males between the ages of 19 and 49 years require 38 g of fiber per day. Males age 50 years or more are recommended to consume 30 g of fiber per day. Females from ages 4 to 8 years require 25 g/day; from ages 9 to 18 years, 26 g/day; and from ages 19 to 50 years, 25 g/day. Women older than 50 years of age

Lactose (1 glass of milk, about 200 mL) Lactasedeficient cells

Intestinal lumen Gas

Bacterial fermentation Lactic acid Osmotic effect H2O

Fluid load (1,000 mL) Distention of gut walls

Peristalsis

Malabsorption Fats, Proteins, Drugs

Watery diarrhea (1L extracellular liquid lost per 9 g lactose in 1 glass of milk)

Lactose intolerance can either be the result of a primary deficiency of lactase production in the small bowel (as is the case for Deria Voider), or it can be secondary to an injury to the intestinal mucosa, where lactase is normally produced. The lactose that is not absorbed is converted by colonic bacteria to lactic acid, methane gas (CH4), and H2 gas (see accompanying figure). The osmotic effect of the lactose and lactic acid in the bowel lumen is responsible for the diarrhea that is often seen as part of this syndrome. Similar symptoms can result from sensitivity to milk proteins (milk intolerance) or from the malabsorption of other dietary sugars. In adults suspected of having a lactase deficiency, the diagnosis is usually made inferentially when avoidance of all dairy products results in relief of symptoms and a rechallenge with these foods reproduces the characteristic syndrome. If the results of these measures are equivocal, however, the malabsorption of lactose can be determined more specifically by measuring the H2 content of the patient’s breath after a test dose of lactose has been consumed. Deria Voider’s symptoms did not appear if she took available over-the-counter tablets containing lactase when she ate dairy products.

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Beans, peas, soybeans, and other leguminous plants contain oligosaccharides with (1,6)-linked galactose residues that cannot be hydrolyzed for absorption, including sucrose with one, two, or three galactose residues attached (see Fig. 27.10). What is the fate of these polysaccharides in the intestine?

are recommended to consume 21 g of fiber per day. These numbers are increased during pregnancy and lactation. One beneficial effect of fiber is seen in diverticular disease in which sacs or pouches may develop in the colon because of a weakening of the muscle and submucosal structures. Fiber is thought to “soften” the stool, thereby reducing pressure on the colonic wall and enhancing expulsion of feces. Certain types of soluble fiber have been associated with disease prevention. For example, pectins may lower blood cholesterol levels by binding bile acids. ␤-Glucan (obtained from oats) has also been shown, in some studies, to reduce cholesterol levels through a reduction in bile acid resorption in the intestine (see Chapter 34). Pectins also may have a beneficial effect in the diet of individuals with diabetes mellitus by slowing the rate of absorption of simple sugars and preventing high blood glucose levels after meals. However, each of the beneficial effects that have been related to “fiber” is relatively specific for the type of fiber and the physical form of the food that contains the fiber. This factor, along with many others, has made it difficult to obtain conclusive results from studies of the effects of fiber on human health.

IV. ABSORPTION OF SUGARS

The dietician explained to Ann Sulin the rationale for a person with diabetes to follow an American Diabetes Association diet plan. It is important for Ann to add a variety of fibers to her diet. The gel-forming, water-retaining pectins and gums delay gastric emptying and retard the rate of absorption of disaccharides and monosaccharides, thus reducing the rate at which blood glucose levels rise. The glycemic index of foods also needs to be considered for appropriate maintenance of blood glucose levels in persons with diabetes. Consumption of a low-glycemic-index diet results in a lower rise in blood glucose levels after eating, which can be more easily controlled by exogenous insulin. For example, Ms. Sulin is advised to eat pasta and rice (glycemic indices of 67 and 65, respectively) instead of potatoes (glycemic index of 80 to 120, depending on the method of preparation) and to incorporate breakfast cereals composed of wheat bran, barley, and oats into her morning routine.

Lieberman_Ch27.indd 504

Once the carbohydrates have been split into monosaccharides, the sugars are transported across the intestinal epithelial cells and into the blood for distribution to all tissues. Not all complex carbohydrates are digested at the same rate within the intestine, and some carbohydrate sources lead to a near-immediate rise in blood glucose levels after ingestion, whereas others slowly raise blood glucose levels over an extended period after ingestion. The glycemic index of a food is an indication of how rapidly blood glucose levels rise after consumption. Glucose and maltose have the highest glycemic indices (142, with white bread defined as an index of 100). Table 27.4 indicates the glycemic index for a variety of food types. Although there is no need to memorize this table, note that cornflakes and potatoes have high glycemic indices, whereas yogurt and skim milk have particularly low glycemic indices. The glycemic response to ingested foods depends not only on the glycemic index of the foods but also on the fiber and fat content of the food as well as its method of preparation. Highly glycemic carbohydrates can be consumed before and after exercise because their metabolism results in a rapid entry of glucose into the blood, where it is then immediately available for use by muscle cells. Low-glycemic carbohydrates enter the circulation slowly and can be used to best advantage if

Table 27.4 Glycemic Indices of Selected Foods, with Values Adjusted to White Bread of 100 Breads Whole wheat Pumpernickel (whole-grain rye) Pasta Spaghetti, white, boiled Cereal grains Barley (pearled) Rice (instant, boiled 1 min) Rice, polished (boiled 10–25 min) Sweet corn Breakfast cereals All bran Cornflakes Muesli Cookies Oatmeal Plain water crackers Root vegetables Potatoes (instant) Potato (new, white, boiled) Potato chips Yam

Legumes 100 88 67 36 65 81 80 74 121 96 78 100 120 80 77 74

Baked beans (canned) Butter beans Garden peas (frozen) Kidney beans (dried) Kidney beans (canned) Peanuts Fruit Apple Apple juice Orange Raisins Sugars Fructose Glucose Lactose Sucrose Dairy products Ice cream Whole milk Skim milk Yogurt

70 46 85 43 74 15 52 45 59 93 27 142 57 83 69 44 46 52

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consumed before exercise, such that as exercise progresses, glucose is slowly being absorbed from the intestine into the circulation in which it can be used to maintain blood glucose levels during the exercise period.

A. Absorption by the Intestinal Epithelium Glucose is transported through the absorptive cells of the intestine by facilitated diffusion and by Na⫹-dependent facilitated transport. (See Chapter 10 for a description of transport mechanisms.) The glucose molecule is extremely polar and cannot diffuse through the hydrophobic phospholipid bilayer of the cell membrane. Each hydroxyl group of the glucose molecule forms at least two hydrogen bonds with water molecules, and random movement would require energy to dislodge the polar hydroxyl groups from their hydrogen bonds and to disrupt the van der Waals forces between the hydrocarbon tails of the fatty acids in the membrane phospholipid. Glucose, therefore, enters the absorptive cells by binding to transport proteins, membrane-spanning proteins that bind the glucose molecule on one side of the membrane and release it on the opposite side (Fig. 27.11). Two types of glucose transport proteins are present in the intestinal absorptive cells: the Na⫹-dependent glucose transporters and the facilitative glucose transporters (Fig. 27.12). 1.

⫹

505

These sugars are not digested well by the human intestine but form good sources of energy for the bacteria of the gut. These bacteria convert the sugars to H2, lactic acid, and short-chain fatty acids. The amount of gas released after a meal containing beans is especially notorious.

NAⴙ-DEPENDENT TRANSPORTERS

Na -dependent glucose transporters, which are located on the luminal side of the absorptive cells, enable these cells to concentrate glucose from the intestinal lumen. CH2OH O

HO

HO

OH

OH

O Cell membrane

I

CH2OH O

HO

O

OH OH

HO

I

O HO

CH2OH O HO

OH

OH

I

O

I HO

CH2OH O

= Ligand (glucose) HO

OH

OH

FIG. 27.11. Facilitative transport. Transport of glucose occurs without rotation of the glucose molecule. Multiple groups on the protein bind the hydroxyl groups of glucose and close behind it as it is released into the cell (i.e., the transporter acts like a “gated pore”). O, outside; I, inside.

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SECTION V ■ CARBOHYDRATE METABOLISM

Lumen Na+ Fructose

Glucose

Galactose

Mucosal side

Brush border

Intestinal epithelium

ATP Fructose Glucose

Na+

Galactose

Serosal side

3 Na+ 2 K+ ADP + Pi

3 Na+ 2 K+

To capillaries

, Na+-glucose cotransporters

, Facilitated glucose transporters

, Na+,K+-ATPase

FIG. 27.12. Na⫹-dependent and facilitative transporters in the intestinal epithelial cells. Both glucose and fructose are transported by the facilitated glucose transporters on the luminal and serosal sides of the absorptive cells. Glucose and galactose are transported by the Na⫹-glucose cotransporters on the luminal (mucosal) side of the absorptive cells.

A low intracellular Na⫹ concentration is maintained by a Na⫹,K⫹-ATPase on the serosal (blood) side of the cell that uses the energy from adenosine triphosphate (ATP) cleavage to pump Na⫹ out of the cell into the blood. Thus, the transport of glucose from a low concentration in the lumen to a high concentration in the cell is promoted by the cotransport of Na⫹ from a high concentration in the lumen to a low concentration in the cell (secondary active transport). 2.

FACILITATIVE GLUCOSE TRANSPORTERS

3.

GALACTOSE AND FRUCTOSE ABSORPTION THROUGH GLUCOSE TRANSPORTERS

Facilitative glucose transporters, which do not bind Na⫹, are located on the serosal side of the cells. Glucose moves via the facilitative transporters from the high concentration inside the cell to the lower concentration in the blood without the expenditure of energy. In addition to the Na⫹-dependent glucose transporters, facilitative transporters for glucose also exist on the luminal side of the absorptive cells. The best characterized facilitative glucose transporters found in the plasma membranes of cells (referred to as GLUT 1 to GLUT 5) are described in Table 27.5. One common structural theme to these proteins is that they all contain 12 membrane-spanning domains. Note that the sodium-linked transporter on the luminal side of the intestinal epithelial cell is not a member of the GLUT family. The epithelial cells of the kidney, which reabsorb glucose from the lumen of the renal tubule back into the blood, have Na⫹-dependent glucose transporters similar to those of intestinal epithelial cells. They are thus also able to transport glucose against its concentration gradient. Other types of cells use mainly facilitative glucose transporters that carry glucose down its concentration gradient.

Galactose is absorbed through the same mechanisms as glucose. It enters the absorptive cells on the luminal side via the Na⫹-dependent glucose transporters and

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507

Table 27.5 Properties of the GLUT 1 to GLUT 5 Isoforms of the Glucose Transport Proteins Transporter

Tissue Distribution

Comments

GLUT 1

Human erythrocyte Blood–brain barrier Blood–retinal barrier Blood–placental barrier Blood–testis barrier Liver Kidney Pancreatic ␤-cell Serosal surface of intestinal mucosa cells Brain (neurons)

Expressed in cell types with barrier functions; a high-affinity glucose transport system

GLUT 2

GLUT 3 GLUT 4

Adipose tissue Skeletal muscle Heart muscle

GLUT 5

Intestinal epithelium Spermatozoa

A high-capacity, low-affinity transporter May be used as the glucose sensor in the pancreas Major transporter in the central nervous system; a high-affinity system Insulin-sensitive transporter. In the presence of insulin, the number of GLUT 4 transporters increases on the cell surface; a high-affinity system This is actually a fructose transporter

Genetic techniques have identified additional GLUT transporters (GLUT 6 to GLUT 12), but the roles of these transporters have not yet been fully described.

facilitative glucose transporters and is transported through the serosal side on the facilitative glucose transporters. Fructose both enters and leaves absorptive epithelial cells by facilitated diffusion, apparently via transport proteins that are part of the GLUT family. The transporter on the luminal side has been identified as GLUT 5. Although this transporter can transport glucose, it has a much higher activity with fructose (see Fig. 27.12). Other fructose transport proteins also may be present. For reasons yet unknown, fructose is absorbed at a much more rapid rate when it is ingested as sucrose than when it is ingested as a monosaccharide.

B. Transport of Monosaccharides into Tissues The properties of the GLUT transport proteins differ among tissues, reflecting the function of glucose metabolism in each tissue. In most cell types, the rate of glucose transport across the cell membrane is not rate limiting for glucose metabolism. This is because the isoform of transporter present in these cell types has a relatively low Km for glucose (i.e., a low concentration of glucose will result in half the maximal rate of glucose transport) or is present in relatively high concentration in the cell membrane so that the intracellular glucose concentration reflects that in the blood. Because the hexokinase isozyme present in these cells has an even lower Km for glucose (0.05 to 0.10 mM), variations in blood glucose levels do not affect the intracellular rate of glucose phosphorylation. However, in several tissues, the rate of transport becomes rate limiting when the serum level of glucose is low or when low levels of insulin signal the absence of dietary glucose. The erythrocyte (red blood cell) is an example of a tissue in which glucose transport is not rate limiting. Although the glucose transporter (GLUT 1) has a Km of 1 to 7 mM, it is present in extremely high concentrations, constituting approximately 5% of all membrane proteins. Consequently, as the blood glucose levels fall from a postprandial level of 140 mg/dL (7.5 mM) to the normal fasting level of 80 mg/dL (4.5 mM), or even the hypoglycemic level of 40 mg/dL (2.2 mM), the supply of glucose is still adequate for the rates at which glycolysis and the pentose phosphate pathway operate. In the liver, the Km for the glucose transporter (GLUT 2) is relatively high compared with that of other tissues, probably 15 mM or higher. This is in keeping with

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SECTION V ■ CARBOHYDRATE METABOLISM

Cell membrane Glucose transporter Insulin Receptor

+

the liver’s role as the organ that maintains blood glucose levels. Thus, the liver will convert glucose into other energy storage molecules only when blood glucose levels are high, such as the time immediately after ingesting a meal. In muscle and adipose tissue, the transport of glucose is greatly stimulated by insulin. The mechanism involves the recruitment of glucose transporters (specifically, GLUT 4) from intracellular vesicles into the plasma membrane (Fig. 27.13). In adipose tissue, the stimulation of glucose transport across the plasma membrane by insulin increases its availability for the synthesis of fatty acids and glycerol from the glycolytic pathway. In skeletal muscle, the stimulation of glucose transport by insulin increases its availability for glycolysis and glycogen synthesis.

V. GLUCOSE TRANSPORT THROUGH THE BLOOD–BRAIN BARRIER AND INTO NEURONS

G

G

G

G

G

G

G , Glucose

, Glucose transporters (GLUT4)

FIG. 27.13. Stimulation by insulin of glucose transport into muscle and adipose cells. Binding of insulin to its cell membrane receptor causes vesicles containing glucose transport proteins to move from inside the cell to the cell membrane.

A hypoglycemic response is elicited by a decrease of blood glucose concentration to some point between 18 and 54 mg/dL (1 and 3 mM). The hypoglycemic response is a result of a decreased supply of glucose to the brain and starts with lightheadedness and dizziness and may progress to coma. The slow rate of transport of glucose through the blood–brain barrier (from the blood into the cerebrospinal fluid) at low levels of glucose is thought to be responsible for this neuroglycopenic response. Glucose transport from the cerebrospinal fluid across the plasma membranes of neurons is rapid and is not rate limiting for ATP generation from glycolysis. In the brain, the endothelial cells of the capillaries have extremely tight junctions, and glucose must pass from the blood into the extracellular cerebrospinal fluid by GLUT 1 transporters in the endothelial cell membranes (Fig. 27.14) and then through the basement membrane. Measurements of the overall process of glucose transport from the blood into the brain (mediated by GLUT 3 on neural cells) show a Km,app of 7 to 11 mM and a maximal velocity not much greater than the rate of glucose utilization by the brain. Thus, decreases of blood glucose below Neural

Non-neural Inside of capillary

G

5

3

2

Endothelial cells

G

3

5

1

2 1

4 Cerebrospinal fluid

Interstitial fluid

1

Tight junctions between endothelial cells

1

No tight junctions

2

Narrow intercellular space

2

Sometimes wide intercellular gaps

3

Lack of pinocytosis

3

Pinocytosis

4

Continuous basement membrane

4

Discontinuous basement membrane

5

Glucose transporters in both membranes

5

Glucose can diffuse between cells and into interstitial fluid

FIG. 27.14. Glucose transport through the capillary endothelium in neural and nonneural tissues. Characteristics of transport in each type of tissue are listed by numbers that refer to the numbers in the drawing. G, glucose.

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509

the fasting level of 80 to 90 mg/dL (approximately 5 mM) are likely to significantly affect the rate of glucose metabolism in the brain because of reduced glucose transport into the brain.

CLINICAL COMMENTS Deria Voider. One of five Americans experiences some form of gastrointestinal discomfort from 30 minutes to 12 hours after ingesting lactose-rich foods. Most become symptomatic when they consume more than 25 g lactose at one time (e.g., 1 pint of milk or its equivalent). Deria Voider’s symptoms were caused by her “new” diet in this country, which included a glass of milk in addition to the milk she used on her cereal with breakfast each morning. Management of lactose intolerance includes a reduction or avoidance of lactosecontaining foods, depending on the severity of the deficiency of intestinal lactase. Hard cheeses (cheddar, swiss, Jarlsberg) are low in lactose and may be tolerated by patients with only moderate lactase deficiency. Yogurt with “live and active cultures” printed on the package contains bacteria that release free lactases when the bacteria are lysed by gastric acid and proteolytic enzymes. The free lactases then digest the lactose. Commercially available milk products that have been hydrolyzed with a lactase enzyme provide a 70% reduction in total lactose content, which may be adequate to prevent digestive symptoms in mildly affected patients. Tablets and capsules containing lactase are also available and should be taken half an hour before meals. Many adults who have a lactase deficiency develop the ability to ingest small amounts of lactose in dairy products without experiencing symptoms. This adaptation probably involves an increase in the population of colonic bacteria that can cleave lactose and not a recovery or induction of human lactase synthesis. For many individuals, dairy products are the major dietary source of calcium, and their complete elimination from the diet can lead to osteoporosis. Lactose, however, is used as a “filler” or carrying agent in more than 1,000 prescription and over-thecounter drugs in this country. People with lactose intolerance often unwittingly ingest lactose with their medications. Ann Sulin. Poorly controlled diabetic patients such as Ann Sulin frequently have elevations in serum glucose levels (hyperglycemia). This is often attributable to a lack of circulating, active insulin, which normally stimulates glucose uptake (through the recruitment of GLUT 4 transporters from the endoplasmic reticulum to the plasma membrane) by the peripheral tissues (heart, muscle, and adipose tissue). Without uptake by these tissues, glucose tends to accumulate within the bloodstream, leading to hyperglycemia. Nona Melos. The large amount of H2 produced on fructose ingestion suggested that Nona Melos’s problem was one of a deficiency in fructose transport into the absorptive cells of the intestinal villi. If fructose were being absorbed properly, the fructose would not have traveled to the colonic bacteria, which metabolized the fructose to generate the hydrogen gas. To confirm the diagnosis, a jejunal biopsy was taken; lactase, sucrase, maltase, and trehalase activities were normal in the jejunal cells. The tissue was also tested for the enzymes of fructose metabolism; these were in the normal range as well. Although Nona had no sugar in her urine, malabsorption of disaccharides can result in their appearance in the urine if damage to the intestinal mucosal cells allows their passage into the interstitial fluid. When Nona was placed on a diet free of fruit juices and other foods containing fructose, she did well and could tolerate small amounts of pure sucrose.

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More than 50% of the adult population is estimated to be unable to absorb fructose in high doses (50 g), and more than 10% cannot completely absorb 25 g fructose. These individuals, like those with other disorders of fructose metabolism, must avoid fruits and other foods that contain high concentrations of fructose.

BIOCHEMICAL COMMENTS Cholera. Cholera is an acute watery diarrheal disorder caused by the water-borne, gram-negative bacterium Vibrio cholerae. It is a disease of antiquity; descriptions of epidemics of the disease date to before 500 BC. During epidemics, the infection is spread by large numbers of Vibrio that enter water sources from the voluminous liquid stools and contaminate the environment, particularly in areas of extreme poverty, where plumbing and modern wastedisposal systems are primitive or nonexistent. Dennis Veere experienced cholera after eating contaminated shellfish (see Chapter 10). After being ingested, the V. cholerae organisms attach to the brush border of the intestinal epithelium and secrete an exotoxin that binds irreversibly to a specific chemical receptor (GM1 ganglioside) on the cell surface. This exotoxin catalyzes an adenosine diphosphate (ADP)-ribosylation reaction that increases adenylate cyclase activity and thus cyclic adenosine monophosphate (cAMP) levels in the enterocyte. As a result, the normal absorption of sodium, anions, and water from the gut lumen into the intestinal cell is markedly diminished. The exotoxin also stimulates the crypt cells to secrete chloride, accompanied by cations and water, from the bloodstream into the lumen of the gut. The resulting loss of solute-rich diarrheal fluid may, in severe cases, exceed 1 L/hour, leading to rapid dehydration and even death. The therapeutic approach to cholera takes advantage of the fact that the Na⫹dependent transporters for glucose and amino acids are not affected by the cholera exotoxin. As a result, coadministration of glucose and Na⫹ by mouth results in the uptake of glucose and Na⫹, accompanied by chloride and water, thereby partially correcting the ion deficits and fluid loss. Amino acids and small peptides are also absorbed by Na⫹-dependent cotransport involving transport proteins distinct from the Na⫹-dependent glucose transporters. Therefore, addition of protein to the glucose–sodium replacement solution enhances its effectiveness and markedly decreases the severity of the diarrhea. Adjunctive antibiotic therapy also shortens the diarrheal phase of cholera but does not decrease the need for the oral replacement therapy outlined earlier.

Key Concepts • • • • • • • • • • •

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The major carbohydrates in the US diet are starch, lactose, and sucrose. Starch is a polysaccharide composed of many glucose units linked together through ␣-1,4- and ␣-1,6-glycosidic bonds. Lactose is a disaccharide composed of glucose and galactose. Sucrose is a disaccharide composed of glucose and fructose. Digestion converts all dietary carbohydrates to their respective monosaccharides. Amylase digests starch; it is found in the saliva and pancreas, which releases it into the lumen of the small intestine. Intestinal epithelial cells contain disaccharidases, which cleave lactose, sucrose, and digestion products of starch into monosaccharides. Dietary fiber is composed of polysaccharides that cannot be digested by human enzymes. Monosaccharides are transported into the absorptive intestinal epithelial cells via active transport systems. Monosaccharides released into the blood via the intestinal epithelial cells are recovered by tissues that use facilitative transporters. Diseases discussed in this chapter are summarized in Table 27.6.

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Table 27.6

511

Diseases Discussed in Chapter 27

Disease or Disorder

Environmental or Genetic

Lactose intolerance

Both

Type 2 diabetes

Both

Fructose malabsorption

Genetic

Cholera

Environmental

Comments Reduced levels of lactase on the intestinal epithelial cell surface lead to reduced lactose digestion in the intestinal lumen, providing substrate for flora in the large intestine. Metabolism of the lactose by these bacteria leads to the generation of organic acids and gases. Diets consisting of low glycemic index carbohydrates will be beneficial in controlling the rise in blood glucose levels after eating. Inability to absorb fructose in the small intestine, leading to colonic bacteria metabolism of fructose and the generation of organic acids and gases. Increased cAMP levels in the intestinal epithelial cells lead to inhibition of ion transport and significant water extrusion from the affected cells, leading to severe diarrhea.

REVIEW QUESTIONS—CHAPTER 27 1.

2.

3.

C. Muscle D. Red blood cells E. Pancreas

The facilitative transporter that is most responsible for transporting fructose from the blood into cells is which of the following? A. GLUT 1 B. GLUT 2 C. GLUT 3 D. GLUT 4 E. GLUT 5

4.

An alcoholic patient developed pancreatitis that affected his exocrine pancreatic function. He exhibited discomfort after eating a high-carbohydrate meal. The patient most likely had a reduced ability to digest which of the following? A. Starch B. Lactose C. Fiber D. Sucrose E. Maltose

After digestion of a piece of cake that contains flour, milk, and sucrose as its primary ingredients, the major carbohydrate products that enter the blood are which of the following? A. Glucose B. Fructose and galactose C. Galactose and glucose D. Fructose and glucose E. Glucose, galactose, and fructose

5.

A patient has a genetic defect that causes intestinal epithelial cells to produce disaccharidases of much lower activity than normal. Compared with a normal person, after eating a bowl of milk and oatmeal sweetened with table sugar, this patient will exhibit higher levels of which of the following? A. Maltose, sucrose, and lactose in the stool B. Starch in the stool C. Galactose and fructose in the blood D. Glycogen in the muscles E. Insulin in the blood

A type 1 diabetic neglects to take his insulin injections while on a weekend vacation. Cells of which tissue will be most greatly affected by this mistake? A. Brain B. Liver

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Formation and Degradation of Glycogen Glycogen is the storage form of glucose found in most types of cells. It is composed of glucosyl units linked by ␣-1,4-glycosidic bonds, with ␣-1,6-branches occurring roughly every 8 to 10 glucosyl units (Fig. 28.1). The liver and skeletal muscle contains the largest glycogen stores. The formation of glycogen from glucose is an energy-requiring pathway that begins—like most of glucose metabolism—with the phosphorylation of glucose to glucose 6-phosphate (G6P). Glycogen synthesis from G6P involves the formation of uridine diphosphate glucose (UDP-G) and the transfer of glucosyl units from UDP-G to the ends of the glycogen chains by the enzyme glycogen synthase. Once the chains reach approximately 11 glucosyl units, a branching enzyme moves 6 to 8 units to form an ␣-1,6-branch. Glycogenolysis, the pathway for glycogen degradation, is not the reverse of the biosynthetic pathway. The degradative enzyme glycogen phosphorylase removes glucosyl units one at a time from the ends of the glycogen chains, converting them to glucose 1-phosphate without resynthesizing UDP-G or UTP. A debranching enzyme removes the glucosyl residues near each branch point. Liver glycogen serves as a source of blood glucose. To generate glucose, the glucose 1-phosphate produced from glycogen degradation is converted to G6P. Glucose 6-phosphatase, an enzyme found only in liver and kidney, converts G6P to free glucose, which then enters the blood. Glycogen synthesis and degradation are regulated in liver by hormonalchanges that signal the need for blood glucose (see Chapter 26). The body maintains fasting blood glucose levels at approximately 80 mg/dL to ensure that the brain and other tissues that are dependent on glucose for the generation of adenosine triphosphate (ATP) have a continuous supply. The lack of dietary glucose, signaled by a decrease of the insulin/glucagon ratio, activates liver glycogenolysis and inhibits glycogen synthesis. Epinephrine, which signals an increased use of blood glucose and other fuels for exercise or emergency situations, also activates liver glycogenolysis. The hormones that regulate liver glycogen metabolism work principally through changes in the phosphorylation state of glycogen synthase in the biosynthetic pathway and glycogen phosphorylase in the degradative pathway. In skeletal muscle, glycogen supplies G6P for ATP synthesis in the glycolytic pathway. Muscle glycogen phosphorylase is stimulated during exercise by the increase of adenosine monophosphate (AMP), an allosteric activator of the enzyme, and also by phosphorylation. The phosphorylation is stimulated by calcium released during contraction and by epinephrine—the fight-or-flight hormone. Glycogen synthesis is activated in resting muscles by the elevation of insulin after carbohydrate ingestion. The neonate must rapidly adapt to an intermittent fuel supply after birth. Once the umbilical cord is clamped, the supply of glucose from the maternal circulation is interrupted. The combined effect of epinephrine and glucagon on the liver glycogen stores of the neonate rapidly restore glucose levels to normal.

512

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513

CH

2 OH

O

O

OH

CH

2 OH

O OH

OH

OH O

bonds CH2OH O OH

Glucose residue linked α-1,6

CH2OH O

CH2 O O

OH Glucose residue linked α-1,4

α-1,6-Glycosidic bond

α-1,4-Glycosidic

O

O

OH

O OH

OH OH

Reducing end attached to glycogenin Nonreducing ends

FIG. 28.1. Glycogen structure. Glycogen is composed of glucosyl units linked by ␣-1,4-glycosidic bonds and ␣-1,6-glycosidic bonds. The branches occur more frequently in the center of the molecule, and less frequently in the periphery. The anomeric carbon that is not attached to another glucosyl residue (the reducing end) is attached to the protein glycogenin by a glycosidic bond. The hydrogen atoms have been omitted from this figure for clarity.

THE WAITING ROOM A newborn baby girl, Getta Carbo, was born after a 38-week gestation. Her mother, a 36-year-old woman, developed a significant viral infection that resulted in a severe loss of appetite and recurrent vomiting in the month preceding delivery. Fetal bradycardia (slower than normal fetal heart rate) was detected with each uterine contraction of labor—a sign of possible fetal distress. At birth, Getta was cyanotic (a bluish discoloration caused by a lack of adequate oxygenation of tissues) and limp. She responded to several minutes of assisted ventilation. Her Apgar score of 3 was low at 1 minute after birth, but improved to a score of 7 at 5 minutes. The Apgar score is an objective estimate of the overall condition of the newborn, determined at both 1 and 5 minutes after birth. The best score is 10 (normal in all respects). Physical examination in the nursery at 10 minutes showed a thin, malnourished female newborn. Her body temperature was slightly low, her heart rate was rapid, and her respiratory rate of 35 breaths per minute was elevated. Getta’s birth weight was only 2,100 g, compared with a normal value of 3,300 g. Her length was 47 cm, and her head circumference was 33 cm (low normal). The laboratory reported that Getta’s serum glucose level when she was unresponsive was 14 mg/dL. A glucose value 40 mg/dL (2.5 mM) is considered to be abnormal in newborn infants. At 5 hours of age, she was apneic (not breathing) and unresponsive. Ventilatory resuscitation was initiated and a cannula is placed in the umbilical vein. Blood for a glucose level was drawn through this cannula, and 5 mL of a 20% glucose solution was injected. Getta slowly responded to this therapy.

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Jim Bodie’s treadmill exercise and most other types of moderate exercise involving whole-body movement (running, skiing, dancing, tennis) increase the use of blood glucose and other fuels by skeletal muscles. The blood glucose is normally supplied by the stimulation of liver glycogenolysis and gluconeogenesis.

Jim Bodie, a 19-year-old body builder, was rushed to the hospital emergency room in a coma. One-half hour earlier, his mother had heard a loud crashing sound in the basement, where he had been lifting weights and completing his daily workout on the treadmill. She found her son on the floor having severe jerking movements of all muscles (a grand mal seizure). In the emergency room, the doctors learned that despite the objections of his family and friends, Jim regularly used androgens and other anabolic steroids in an effort to bulk up his muscle mass. On initial physical examination, he was comatose with occasional involuntary jerking movements of his extremities. Foamy saliva dripped from his mouth. He had bitten his tongue and had lost bowel and bladder control at the height of the seizure. The laboratory reported a serum glucose level of 18 mg/dL (extremely low). The intravenous infusion of 5% glucose (5 g of glucose per 100 mL of solution), which had been started earlier, was increased to 10%. In addition, 50 g of glucose was given over 30 seconds through the intravenous tubing.

I. Muscle Glycogen Glucose 1-phosphate

Glucose 6-phosphate Glycolysis ATP

Lactate CO2

STRUCTURE OF GLYCOGEN

Glycogen, the storage form of glucose, is a branched glucose polysaccharide composed of chains of glucosyl units linked by ␣-1,4-glycosidic bonds with ␣-1,6-branches, every 8 to 10 residues (see Fig. 28.1). In a molecule of this highly branched structure, only one glucosyl residue has an anomeric carbon that is not linked to another glucose residue. This anomeric carbon at the beginning of the chain is attached to the protein glycogenin. The other ends of the chains are called nonreducing ends (see Chapter 5). The branched structure permits rapid degradation and rapid synthesis of glycogen because enzymes can work on several chains simultaneously from the multiple nonreducing ends. Glycogen is present in tissues as polymers of very high molecular weight (107 to 8 10 Da) collected together in glycogen particles. The enzymes involved in glycogen synthesis and degradation, and some of the regulatory enzymes are bound to the surface of the glycogen particles.

II. FUNCTION OF GLYCOGEN IN SKELETAL MUSCLE AND LIVER

Glycogen

Liver

Glucose 1-phosphate Glucose 6-phosphate Glucose 6-phosphatase

Gluconeogenesis

Glucose

Blood glucose

FIG. 28.2. Glycogenolysis in skeletal muscle and liver. Glycogen stores serve different functions in muscle cells and liver. In the muscle and most other cell types, glycogen stores serve as a fuel source for the generation of ATP. In the liver, glycogen stores serve as a source of blood glucose.

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Glycogen is found in most cell types, where it serves as a reservoir of glucosyl units for adenosine triphosphate (ATP) generation from glycolysis. Glycogen is degraded mainly to glucose 1-phosphate, which is converted to glucose 6-phosphate (G6P). In skeletal muscle and other cell types, the G6P enters the glycolytic pathway (Fig. 28.2). Glycogen is an extremely important fuel source for skeletal muscle when ATP demands are high and when G6P is used rapidly in anaerobic glycolysis. In many other cell types, the small glycogen reservoir serves a similar purpose; it is an emergency fuel source that supplies glucose for the generation of ATP in the absence of oxygen or during restricted blood flow. In general, glycogenolysis and glycolysis are activated together in these cells. Glycogen serves a very different purpose in liver than in skeletal muscle and other tissues (see Fig. 28.2). Liver glycogen is the first and immediate source of glucose for the maintenance of blood glucose levels. In the liver, the G6P that is generated from glycogen degradation is hydrolyzed to glucose by glucose 6-phosphatase—an enzyme that is present only in the liver and kidneys. Glycogen degradation thus provides a readily mobilized source of blood glucose as dietary glucose decreases, or as exercise increases, the use of blood glucose by muscles.

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515

The pathways of glycogenolysis and gluconeogenesis in the liver both supply blood glucose, and consequently, these two pathways are activated together by glucagon. Gluconeogenesis, the synthesis of glucose from amino acids and other gluconeogenic precursors (discussed in detail in Chapter 31), also forms G6P, so that glucose 6-phosphatase serves as a “gateway” to the blood for both pathways (see Fig. 28.2). Regulation of glycogen synthesis serves to prevent futile cycling and waste of ATP. Futile cycling refers to a situation in which a substrate is converted to a product through one pathway, and the product is converted back to the substrate through another pathway. Because the biosynthetic pathway is energyrequiring, futile cycling results in a waste of high-energy phosphate bonds. Thus, glycogen synthesis is activated when glycogen degradation is inhibited and vice versa.

III. SYNTHESIS AND DEGRADATION OF GLYCOGEN Glycogen synthesis, like almost all the pathways of glucose metabolism, begins with the phosphorylation of glucose to G6P by hexokinase or, in the liver, glucokinase (Fig. 28.3). G6P is the precursor of glycolysis, the pentose phosphate pathway, and pathways for the synthesis of other sugars. In the pathway for glycogen synthesis, G6P is converted to glucose 1-phosphate by phosphoglucomutase— a reversible reaction.

Glycogen degradation

Glycogen

Glycogen synthesis Glycogen synthase 4:6-Transferase (branching enzyme)

Debrancher enzyme

D1

S3

Glycogen primer

Glucose (small amount)

UDP-G

Other pathways

UDP-glucose pyrophosphorylase

Glycogen phosphorylase

UTP

S2 Glucose 1-phosphate Phosphoglucomutase

D2 Glucose 6-phosphate Glucose 6phosphatase (liver only)

Pi

Hexokinase glucokinase (liver)

Glucose

ATP

Glycolysis Pentose phosphate pathway Other pathways

S1

Cell membrane Glucose

FIG. 28.3. Scheme of glycogen synthesis and degradation. (S1) G6P is formed from glucose by hexokinase in most cells, and glucokinase in the liver. It is a metabolic branch point for the pathways of glycolysis, the pentose phosphate pathway, and glycogen synthesis. (S2) UDP-glucose (UDP-G) is synthesized from glucose 1-phosphate. UDP-G is the branch point for glycogen synthesis and other pathways that require the addition of carbohydrate units. (S3) Glycogen synthesis is catalyzed by glycogen synthase and the branching enzyme. (D1) Glycogen degradation is catalyzed by glycogen phosphorylase and a debrancher enzyme. (D2) Glucose 6-phosphatase in the liver (and, to a small extent, the kidney) generates free glucose from G6P.

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O HOCH2

HOCH2 H

HO OH

H O P

H

OH

C

O

O H H

O

O

O–

+

UTP

HN

H H

H

HO OH

H O P

–

H

OH

O

O

O O

–

P

O C O CH2

–

N

O

CH

+

PPI

O

H H

Glucose 1-phosphate

CH

HO

H

H

OH

Uridine diphosphate glucose (UDP-glucose)

FIG. 28.4. Formation of UDP-glucose. The high-energy phosphate bond of UTP provides the energy for the formation of a high-energy bond in UDP-glucose. Pyrophosphate (PPi), released by the reaction, is cleaved to two inorganic phosphate (Pi).

Glucose residue linked ␣-1,4

Glucose residue linked ␣-1,6

Glycogen core

A. Glycogen Synthesis

UDP-glucose UDP

Glycogen synthase

Glycogen core 6 UDP-glucose 6 UDP

Glycogen synthase

Glycogen core 4:6-Transferase (branching enzyme)

Glycogen core UDP-glucose

Glycogen synthase

Continue with glycogen synthesis at all nonreducing ends

FIG. 28.5. details.

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Glycogen is both formed from and degraded to glucose 1-phosphate, but the biosynthetic and degradative pathways are separated and involve different enzymes (see Fig. 28.3). The biosynthetic pathway is an energy-requiring pathway; high-energy phosphate from UTP is used to activate the glucosyl residues to uridine diphosphate glucose (UDP-G) (Fig. 28.4). In the degradative pathway, the glycosidic bonds between the glucosyl residues in glycogen are simply cleaved by the addition of phosphate to produce glucose 1-phosphate (or water to produce free glucose), and UDP-G is not resynthesized. The existence of separate pathways for the formation and degradation of important compounds is a common theme in metabolism. Because the synthesis and degradation pathways use different enzymes, one can be activated while the other is inhibited.

Glycogen synthesis. See text for

Glycogen synthesis requires the formation of ␣-1,4-glycosidic bonds to link glucosyl residues in long chains and the formation of an ␣-1,6-branch every 8 to 10 residues (Fig. 28.5). Most of glycogen synthesis occurs through the lengthening of the polysaccharide chains of a preexisting glycogen molecule (a glycogen primer) in which the reducing end of the glycogen is attached to the protein glycogenin. To lengthen the glycogen chains, glucosyl residues are added from UDP-G to the nonreducing ends of the chain by glycogen synthase. The anomeric carbon of each glucosyl residue is attached in an ␣-1,4-glycosidic bond to the hydroxyl on carbon 4 of the terminal glucosyl residue. When the chain reaches approximately 11 residues in length, a 6- to 8-residue piece is cleaved by amylo4,6-transferase (also known as branching enzyme) and reattached to a glucosyl unit by an ␣-1,6-bond. Both chains continue to lengthen until they are long enough to produce two new branches. This process continues, producing highly branched molecules. Glycogen synthase, the enzyme that attaches the glucosyl residues in ␣-1,4-glycosidic bonds is the regulated step in the pathway. Branching of glycogen serves two major roles: increased sites for synthesis and degradation, and enhancing the solubility of the molecule. The synthesis of new glycogen primer molecules also occurs. Glycogenin, the protein to which glycogen is attached, glycosylates itself (autoglycosylation) by attaching the glucosyl residue of UDP-G to the hydroxyl side chain of a serine residue in the protein. The protein then extends the carbohydrate chain (using UDP-G as the substrate) until the glucosyl chain is long enough to serve as a substrate for glycogen synthase.

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Glucose residue linked ␣-1,6

Glucose residue linked ␣-1,4

Glycogen core 8 Pi Glycogen phosphorylase

8 Glucose 1-phosphate ( )

4:4-Transferase

Glycogen core ␣-1,6-Glucosidase 1 Glucose ( ) Glycogen core Glycogen phosphorylase

Degradation continues

FIG. 28.6.

Glycogen degradation. See text for details.

B. Degradation of Glycogen Glycogen is degraded by two enzymes: glycogen phosphorylase and the debrancher enzyme (Fig. 28.6). Glycogen degradation is a phosphorolysis reaction (breaking of a bond using a phosphate ion as a nucleophile). Enzymes that catalyze phosphorolysis reactions are named phosphorylase. Because more than one type of phosphorylase exists, the substrate usually is included in the name of the enzyme, such as glycogen phosphorylase or purine nucleoside phosphorylase. The enzyme glycogen phosphorylase starts at the nonreducing end of a chain and successively cleaves glucosyl residues by adding phosphate to the anomeric carbon of the terminal glycosidic bond, thereby releasing glucose 1-phosphate and producing a free 4-hydroxyl group on the glucose residue now at the end of the glycogen chain. However, glycogen phosphorylase cannot act on the glycosidic bonds of the four glucosyl residues closest to a branch point because the branching chain sterically hinders a proper fit into the catalytic site of the enzyme. The debrancher enzyme, which catalyzes the removal of the four residues closest to the branch point, has two catalytic activities: it acts as a transferase and as an ␣-1,6glucosidase. As a transferase, the debrancher first removes a unit containing three glucose residues and adds it to the end of a longer chain by an ␣-1,4-glycosidic bond. The one glucosyl residue remaining at the ␣-1,6-branch is hydrolyzed by the amylo-1,6-glucosidase activity of the debrancher, resulting in the release of free glucose. Thus, one glucose and approximately seven to nine glucose 1-phosphate residues are released for every branch point. Some degradation of glycogen also occurs within lysosomes when glycogen particles become surrounded by membranes that then fuse with the lysosomal membranes. A lysosomal glucosidase hydrolyzes this glycogen to glucose.

IV. DISORDERS OF GLYCOGEN METABOLISM A series of inborn errors of metabolism—the glycogen storage diseases—results from deficiencies in the enzymes of glycogenolysis (Table 28.1). The diseases are labeled I through X, and O. Several disorders have different subtypes as

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To confirm a diagnosis of phosphorylase deficiency (in either muscle or liver) a biopsy must be obtained, followed by a sensitive assay for phosphorylase activity within the biopsied tissue. There are several procedures to do this. The first is to incubate glycogen, inorganic phosphate, and a sample of the extracted biopsy tissue. If phosphorylase activity is present, glucose 1-phosphate will be produced. One can determine the amount of glucose 1-phosphate produced by converting it to G6P, using the enzyme phosphoglucomutase. The G6P levels are then measured by the conversion of G6P to 6-phosphogluconate using the enzyme G6P dehydrogenase. G6P dehydrogenase requires NADP, generating NADPH during the course of the reaction. The formation of NADPH can be followed spectrophotometrically as the absorbance at 340 nm will increase as NADPH is produced, and the increase in NADPH levels will be directly proportional to the amount of glucose 1-phosphate produced by phosphorylase. A second assay to measure phosphorylase activity uses radioactive glycogen as a substrate. The labeled glycogen is incubated with inorganic phosphate and sample extract, generating labeled glucose 1-phosphate. The radioactive glucose 1-phosphate is then separated from the glycogen and measured. A variation of this method is to use 32P-labeled inorganic phosphate, and measure the radioactive glucose 1-phosphate produced. A third assay for phosphorylase activity is to measure the reverse reaction (glycogen synthesis). Under appropriate conditions, the phosphorylase reaction can go backward in which the glucose residue from glucose 1-phosphate is added to an existing glycogen chain, releasing Pi. The phosphate produced using this method is then measured in a sensitive spectrophotometric assay.

A genetic defect of lysosomal glucosidase called type II glycogen storage disease or Pompe disease, leads to the accumulation of glycogen particles in large, membrane-enclosed residual bodies, which disrupt the function of liver and muscle cells. Children with this disease usually die of heart failure at a few months of age. Why do you think that a genetic deficiency in muscle glycogen phosphorylase (McArdle disease) is a mere inconvenience, whereas a deficiency of liver glycogen phosphorylase (Hers disease) can be lethal?

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Table 28.1 Glycogen Storage Diseases Type

Enzyme Affected

Primary Organ Involved

Manifestationsa

O

Glycogen synthase

Liver

Ib

Glucose 6-phosphatase (Von Gierkes disease) Lysosomal ␣-glucosidase (Pompe disease): may see clinical symptoms in childhood, juvenile, or adult life stages, depending on the nature of the mutation

Liver

Amylo-1,6-glucosidase (debrancher): form IIIa is the liver and muscle enzymes; form IIIb is a liver-specific form; and form IIIc is a muscle-specific form Amylo-4,6-glucosidase (branching enzyme) (Andersen disease)

Liver, skeletal muscle, heart

Hypoglycemia, hyperketonemia, failure to thrive, early death Enlarged liver and kidney, growth failure, severe fasting hypoglycemia, acidosis, lipemia, thrombocyte dysfunction Infantile form: early-onset progressive muscle hypotonia, cardiac failure, death before age 2 years Juvenile form: later-onset myopathy with variable cardiac involvement Adult form: limb-girdle muscular dystrophy-like features. Glycogen deposits accumulate in lysosomes Fasting hypoglycemia; hepatomegaly in infancy in some myopathic features. Glycogen deposits have short outer branches.

Muscle glycogen phosphorylase (McArdles disease) (expressed as either adult or infantile form) Liver glycogen phosphorylase (Hers disease) and its activating system (includes mutations in hepatic phosphorylase kinase and hepatic PKA) Phosphofructokinase-1 (Tarui syndrome) GLUT 2 (glucose/galactose transporter); Fanconi-Bickel syndrome

Skeletal muscle

II

III

IV V VIc

VII XI

All organs with lysosomes

Liver

Hepatosplenomegaly; symptoms may arise from a hepatic reaction to the presence of a foreign body (glycogen with long outer branches). Usually fatal. Exercise-induced muscular pain, cramps, and progressive weakness, sometimes with myoglobinuria

Liver

Hepatomegaly, mild hypoglycemia; good prognosis

Muscle, red blood cells Intestine, pancreas, kidney, liver

As in type V; in addition, enzymopathic hemolysis Glycogen accumulation in liver and kidney; rickets, growth retardation, glucosuria.

a

All of these diseases except type O are characterized by increased glycogen deposits. Glucose 6-phosphatase is composed of several subunits that also transport glucose, glucose 6-phosphate, phosphate, and PPi across the endoplasmic reticulum membranes. Therefore, there are several subtypes of this disease corresponding to defects in the different subunits. Type Ia is a lack of glucose 6-phosphatase activity; type Ib is a lack of glucose-6-phosphate translocase activity; type Ic is a lack of phosphotranslocase activity; type Id is a lack of glucose translocase activity. c Glycogen storage diseases IX (hepatic phosphorylase kinase) and X (hepatic protein kinase A [PKA]) have been reclassified to VI, which now refers to the hepatic glycogen phosphorylase activating system. Sources: Parker PH, Ballew M,Greene HL. Nutritional management of glycogen storage disease. Annu Rev Nutr. 1993;13:83–109. Copyright 1993 by Annual Reviews, Inc.; Shin YS. Glycogen storage disease: clinical, biochemical, and molecular heterogeneity. Semin Pediatr Neurol. 2006;13:115–120; Ozen H. Glycogen storage diseases: new perspectives. World J Gastroenterol. 2007;13:2541–2553. b

indicated in the note of Table 28.1. Muscle glycogen phosphorylase, the key regulatory enzyme of glycogen degradation, is genetically different from liver glycogen phosphorylase, and thus a person may have a defect in one and not the other.

Muscle glycogen is used within the muscle to support exercise. Thus, an individual with McArdle disease (type V glycogen storage disease) experiences no symptoms except unusual fatigue and muscle cramps during exercise. These symptoms may be accompanied by myoglobinuria and release of muscle creatine kinase into the blood. Liver glycogen is the first reservoir for the support of blood glucose levels, and a deficiency in glycogen phosphorylase or any of the other enzymes of liver glycogen degradation can result in fasting hypoglycemia. The hypoglycemia is usually mild because patients can still synthesize glucose from gluconeogenesis (see Table 28.1).

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V. REGULATION OF GLYCOGEN SYNTHESIS AND DEGRADATION The regulation of glycogen synthesis in different tissues matches the function of glycogen in each tissue. Liver glycogen serves principally for the support of blood glucose during fasting or during extreme need (e.g., exercise), and the degradative and biosynthetic pathways are regulated principally by changes in the insulin/glucagon ratio and by blood glucose levels, which reflect the availability of dietary glucose (Table 28.2). Degradation of liver glycogen is also activated by epinephrine, which is released in response to exercise, hypoglycemia, or other stress situations in which there is an immediate demand for blood glucose. In contrast, in skeletal muscles, glycogen is a reservoir of glucosyl units for the generation of ATP from glycolysis and glucose oxidation. As a consequence, muscle glycogenolysis is regulated principally by adenosine monophosphate (AMP), which signals a lack of ATP, and by Ca2 released during contraction. Epinephrine, which is released in response to exercise and other stress situations, also activates skeletal muscle glycogenolysis. The glycogen stores of resting muscle decrease very little during fasting.

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Regulation of Liver and Muscle Glycogen Storesa

State Liver Fasting Carbohydrate meal

Exercise and stress

Regulators

Response of Tissue

Blood: Glucagon ↑ Insulin ↓ Tissue: cAMP ↑ Blood: Glucagon ↓ Insulin ↑ Glucose ↑ Tissue: cAMP ↓ Glucose ↑ Blood: Epinephrine ↑ Tissue: cAMP ↑ Ca2-calmodulin ↑

Glycogen degradation ↑ Glycogen synthesis ↓ Glycogen degradation ↓ Glycogen synthesis ↑

Glycogen degradation ↑ Glycogen synthesis ↓

Muscle Fasting (rest)

Blood: Insulin ↓

Glycogen synthesis ↓ Glucose transport ↓

Carbohydrate meal (rest)

Blood: Insulin ↑

Exercise

Blood: Epinephrine ↑ Tissue: AMP ↓ Ca2-calmodulin ↑ cAMP ↑

Glycogen synthesis ↑ Glucose transport ↑ Glycogen synthesis ↓ Glycogen degradation ↓ Glycolysis ↑

↑, increased compared with other physiologic states; ↓, decreased compared with other physiologic states. a

A. Regulation of Glycogen Metabolism in Liver Liver glycogen is synthesized after a carbohydrate meal when blood glucose levels are elevated and are degraded as blood glucose levels decrease. When an individual eats a carbohydrate-containing meal, blood glucose levels immediately increase, insulin levels increase, and glucagon levels decrease (see Fig. 26.8). The increase of blood glucose levels and the rise of the insulin/glucagon ratio inhibit glycogen degradation and stimulate glycogen synthesis. The immediate increased transport of glucose into peripheral tissues, and storage of blood glucose as glycogen, helps to bring circulating blood glucose levels back to the normal 80- to 100-mg/dL range of the fasted state. As the length of time after a carbohydrate-containing meal increases, insulin levels decrease, and glucagon levels increase. The fall of the insulin/glucagon ratio results in inhibition of the biosynthetic pathway and activation of the degradative pathway. As a result, liver glycogen is rapidly degraded to glucose, which is released into the blood. Although glycogenolysis and gluconeogenesis are activated together by the same regulatory mechanisms, glycogenolysis responds more rapidly with a greater outpouring of glucose. A substantial proportion of liver glycogen is degraded within the first few hours after eating (Table 28.3). The rate of glycogenolysis is fairly constant for the first 22 hours, but in a prolonged fast, the rate decreases significantly as the liver glycogen supplies dwindle. Liver glycogen stores are therefore a rapidly rebuilt and degraded store of glucose, ever responsive to small and rapid changes of blood glucose levels.

Table 28.3

Effect of Fasting on Liver Glycogen Content in the Human

Length of Fast (h)

Glycogen Content (␮mol/g)

Rate of Glycogenolysis (␮mol/kg/min)

0 2 4 24 64

300 260 216 42 16

— 4.3 4.3 1.7 0.3

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Maternal blood glucose readily crosses the placenta to enter the fetal circulation. During the last 9 or 10 weeks of gestation, glycogen formed from maternal glucose is deposited in the fetal liver under the influence of the insulin-dominated hormonal milieu of that period. At birth, maternal glucose supplies cease, causing a temporary physiologic drop in glucose levels in the newborn’s blood, even in normal healthy infants. This drop serves as one of the signals for glucagon release from the newborn’s pancreas, which in turn stimulates glycogenolysis. As a result, the glucose levels in the newborn return to normal. Healthy full-term babies have adequate stores of liver glycogen to survive short periods (12 hours) of caloric deprivation provided other aspects of fuel metabolism are normal. Because Getta Carbo’s mother was markedly anorexic during the critical period when the fetal liver is normally synthesizing glycogen from glucose supplied in the maternal blood, Getta’s liver glycogen stores were lower than normal. Thus, because fetal glycogen is the major source of fuel for the newborn in the early hours of life, Getta became profoundly hypoglycemic within 5 hours of birth because of her low levels of stored carbohydrate. 10 Plasma glucose (mmol/L)

Table 28.2

519

3.3

Normal range 2.2 mmol/L

1.5

Hypoglycemia 1

2

3

Hour after birth

Plasma glucose levels in the neonate. (From Mehta A. Prevention and management of neonatal hypoglycemia. Arch Dis Child Fetal Neonatal Ed. 1994;70:F54–F59.) The normal range of blood glucose levels in the neonate lies between the two purple lines. The dark blue area represents the range of hypoglycemia in the neonate that should be treated. Treatment of neonates with blood glucose levels that fall within the dashed red box—the zone of clinical uncertainty—is controversial. The units of plasma glucose are given in mmol/L. Both mg/dL (mg/100 mL) and mmol/L are used clinically for the values of blood glucose; 80 mg/ dL glucose is equivalent to 5 mmol/L (5 mM).

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A patient was diagnosed as an infant with type III glycogen storage disease, a deficiency of debrancher enzyme (see Table 28.1). The patient had hepatomegaly (an enlarged liver) and experienced bouts of mild hypoglycemia. To diagnose the disease, glycogen was obtained from the patient’s liver by biopsy after the patient had fasted overnight and compared with normal glycogen. The glycogen samples were treated with a preparation of commercial glycogen phosphorylase and commercial debrancher enzyme. The amounts of glucose 1-phosphate and glucose produced in the assay were then measured. The ratio of glucose 1-phosphate to glucose for the normal glycogen sample was 9:1, and the ratio for the patient was 3:1. Can you explain these results?

A

ATP P

Glycogen phosphorylase b (inactive)

B

Glycogen phosphorylase a (active) ATP P

Glycogen synthase I (or a) (active)

Glycogen synthase D (or b) (inactive)

FIG. 28.7. The conversion of active and inactive forms of glycogen phosphorylase (A) and glycogen synthase (B). Note how the nomenclature changes depending on the phosphorylation and activity state of the enzyme.

1.

NOMENCLATURE CONCERNS WITH ENZYMES THAT METABOLIZE GLYCOGEN

Both glycogen phosphorylase and glycogen synthase will be covalently modified to regulate their activity (Fig. 28.7). When it is activated by covalent modification, glycogen phosphorylase is referred to as glycogen phosphorylase a (remember a for active); when the covalent modification is removed and the enzyme is inactive, it is referred to as glycogen phosphorylase b. Glycogen synthase, when it is not covalently modified, is active and can be designated as glycogen synthase a or glycogen synthase I (the I stands for independent of modifiers for activity). When glycogen synthase is covalently modified, it is inactive in the form of glycogen synthase b or glycogen synthase D (for dependent on a modifier for activity). 2.

REGULATION OF LIVER GLYCOGEN METABOLISM BY INSULIN AND GLUCAGON

Insulin and glucagon regulate liver glycogen metabolism by changing the phosphorylation state of glycogen phosphorylase in the degradative pathway and glycogen synthase in the biosynthetic pathway. An increase of glucagon and decrease of insulin during the fasting state initiates a cyclic adenosine monophosphate (cAMP)-directed phosphorylation cascade, which results in the phosphorylation of glycogen phosphorylase to an active enzyme and the phosphorylation of glycogen synthase to an inactive enzyme (Fig. 28.8). As a consequence, glycogen degradation is stimulated, and glycogen synthesis is inhibited. 3.

GLUCAGON ACTIVATES A PHOSPHORYLATION CASCADE THAT CONVERTS GLYCOGEN PHOSPHORYLASE B TO GLYCOGEN PHOSPHORYLASE A

Glucagon regulates glycogen metabolism through its intracellular second messenger cAMP and protein kinase A (PKA) (see Chapter 26). Glucagon, by binding to its cell membrane receptor, transmits a signal through G proteins that activates adenylate cyclase, causing cAMP levels to increase (see Fig. 28.8). cAMP binds to the regulatory subunits of PKA, which dissociate from the catalytic subunits. The catalytic subunits of PKA are activated by the dissociation and phosphorylate the enzyme phosphorylase kinase—activating it. Phosphorylase kinase is the protein kinase that converts the inactive liver glycogen phosphorylase b conformer to the active glycogen phosphorylase a conformer by transferring a phosphate from ATP to a specific serine residue on the phosphorylase subunits. Because of the activation of glycogen phosphorylase, glycogenolysis is stimulated.

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Glucagon (liver only)

Epinephrine

+

Glucose

+

Cell membrane Cytoplasm

521

Adenylate cyclase

Gprotein

GTP +

Phosphodiesterase

1 AMP

ATP

Glucose

cAMP Protein kinase A (inactive) Pi Protein phosphatase

Phosphorylase kinase (inactive)

Pi

ATP

ADP

Glycogen synthase– P (inactive)

Active protein kinase A

ADP

Glycogen phosphorylase a (active) P

Glucose 1-phosphate Protein phosphatase

5 ATP Glycogen synthase (active)

ADP

4 Glycogen phosphorylase b (inactive)

Glucose 6-phosphate

Regulatory subunit-cAMP

ATP

3 Phosphorylase kinase– P (active)

2

Glucokinase

Glycogen Pi

Pi

UDP-glucose

6 Glucose 1-phosphate

Protein phosphatase

Glucose 6-phosphate 6Liver Glucose phosphatase

Blood glucose

FIG. 28.8. Regulation of glycogen synthesis and degradation in the liver. 1. Glucagon binding to the serpentine glucagon receptor or epinephrine binding to a serpentine ␤-receptor in the liver activates adenylate cyclase via G proteins, which synthesizes cAMP from ATP. 2. cAMP binds to PKA (cAMP-dependent protein kinase), thereby activating the catalytic subunits. 3. PKA activates phosphorylase kinase by phosphorylation. 4. Phosphorylase kinase adds a phosphate to specific serine residues on glycogen phosphorylase b, thereby converting it to the active glycogen phosphorylase a. 5. PKA also phosphorylates glycogen synthase thereby decreasing its activity. 6. Because of the inhibition of glycogen synthase and the activation of glycogen phosphorylase, glycogen is degraded to glucose 1-phosphate. The red dashed lines denote reactions that are decreased in the livers of fasting individuals.

4.

INHIBITION OF GLYCOGEN SYNTHASE BY GLUCAGONDIRECTED PHOSPHORYLATION

When glycogen degradation is activated by the cAMP-stimulated phosphorylation cascade, glycogen synthesis is simultaneously inhibited. The enzyme glycogen synthase is also phosphorylated by PKA, but this phosphorylation results in a less active form, glycogen synthase b. The phosphorylation of glycogen synthase is far more complex than glycogen phosphorylase. Glycogen synthase has multiple phosphorylation sites and is acted on by up to 10 different protein kinases. Phosphorylation by PKA does not, by itself, inactivate glycogen synthase. Instead, phosphorylation by PKA facilitates the subsequent addition of phosphate groups by other kinases, and these inactivate the enzyme. A term that has been applied to changes of activity resulting from multiple phosphorylations is hierarchical or synergistic phosphorylation—the phosphorylation of one site makes another site more reactive and easier to phosphorylate by a different protein kinase.

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With a deficiency of debrancher enzyme but normal levels of glycogen phosphorylase, the glycogen chains of the patient could be degraded in vivo only to within four residues of the branch point. When the glycogen samples were treated with the commercial preparation containing normal enzymes, one glucose residue was released for each ␣-1,6-branch. However, in the patient’s glycogen sample, with the short outer branches, three glucose 1-phosphates and one glucose residue were obtained for each ␣-1,6-branch. Normal glycogen has 8 to 10 glucosyl residues per branch and thus gives a ratio of approximately 9 mmol glucose 1-phosphate to 1 mmol glucose.

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Most of the enzymes that are regulated by phosphorylation have multiple phosphorylation sites. Glycogen phosphorylase, which has only one serine per subunit and can be phosphorylated only by phosphorylase kinase, is the exception. For some enzymes, the phosphorylation sites are antagonistic, and phosphorylation initiated by one hormone counteracts the effects of other hormones. For other enzymes, the phosphorylation sites are synergistic, and phosphorylation at one site stimulated by one hormone can act synergistically with phosphorylation at another site. 5.

Most of the enzymes that are regulated by phosphorylation also can be converted to the active conformation by allosteric effectors. Glycogen synthase b, the less active form of glycogen synthase, can be activated by the accumulation of G6P higher than physiologic levels. The activation of glycogen synthase by G6P may be important in individuals with glucose 6-phosphatase deficiency, a disorder known as type I or von Gierke glycogen storage disease (see Table 28.1). When G6P produced from gluconeogenesis accumulates in the liver, it activates glycogen synthesis even though the individual may be hypoglycemic and have low insulin levels. Glucose 1-phosphate is also elevated, resulting in the inhibition of glycogen phosphorylase. As a consequence, large glycogen deposits accumulate and hepatomegaly occurs. An inability of liver and muscle to store glucose as glycogen contributes to the hyperglycemia in patients such as Di Abietes, with type 1 diabetes mellitus and in patients such as Ann Sulin, with type 2 diabetes mellitus. The absence of insulin in type 1 diabetes mellitus patients and the high levels of glucagon result in decreased activity of glycogen synthase. Glycogen synthesis in skeletal muscles of type 1 patients is also limited by the lack of insulin-stimulated glucose transport. Insulin resistance in type 2 patients has the same effect. An injection of insulin suppresses glucagon release and alters the insulin/glucagon ratio. The result is rapid uptake of glucose into skeletal muscle and rapid conversion of glucose to glycogen in skeletal muscle and liver.

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REGULATION BY PROTEIN PHOSPHATASES

At the same time that PKA and phosphorylase kinase are adding phosphate groups to enzymes, the protein phosphatases that remove this phosphate are inhibited. Protein phosphatases remove the phosphate groups, bound to serine or other residues of enzymes, by hydrolysis. Hepatic protein-phosphatase-1 (PP-1), one of the major protein phosphatases involved in glycogen metabolism, removes phosphate groups from phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. During fasting, hepatic PP-1 is inactivated by several mechanisms. One is dissociation from the glycogen particle, such that the substrates are no longer available to the phosphatase. The second is the binding of inhibitor proteins, such as the protein called inhibitor-1, which, when phosphorylated by a glucagon (or epinephrine)-directed mechanism, binds to and inhibits phosphatase action. Insulin indirectly activates hepatic PP-1 through its own signal transduction cascade initiated at the insulin receptor tyrosine kinase. PP-1 will bind to proteins that target the phosphatase to glycogen particles. There are four such targeting proteins: GM, GL, R6, and R5/PTG (protein targeting to glycogen). GM is found in the heart and skeletal muscle, GL is found primarily in the liver, whereas R5/PTG and R6 are found in most tissues. The targeting subunits all bind to the same hydrophobic site on PP-1, leading to just one targeting subunit bound per PP-1 molecule. The targeting subunits allow for compartmentalized activation of PP-1 under the appropriate conditions, whereas other tissues or cellular compartments may still express an inhibited PP-1. Regulation of the phosphatase involves complex interactions between the target enzymes, the targeting subunits, the phosphatase, and protein inhibitor 1 and will not be considered further. 6.

INSULIN IN LIVER GLYCOGEN METABOLISM

Insulin is antagonistic to glucagon in the degradation and synthesis of glycogen. The glucose level in the blood is the signal that controls the secretion of insulin and glucagon. Glucose stimulates insulin release and suppresses glucagon release; after a high-carbohydrate meal, one increases whereas the other decreases. However, insulin levels in the blood change to a greater degree with the fasting–feeding cycle than do the glucagon levels, and thus insulin is considered the principal regulator of glycogen synthesis and degradation. The role of insulin in glycogen metabolism is often overlooked because the mechanisms by which insulin reverses all of the effects of glucagon on individual metabolic enzymes is still under investigation. In addition to the activation of hepatic PP-1 through the insulin-receptor tyrosine kinase phosphorylation cascade, insulin may activate the phosphodiesterase that converts cAMP to AMP, thereby decreasing cAMP levels and inactivating PKA. Regardless of the mechanisms involved, insulin is able to reverse all of the effects of glucagon and is the most important hormonal regulator of blood glucose levels. 7.

BLOOD GLUCOSE LEVELS AND GLYCOGEN SYNTHESIS AND DEGRADATION

When an individual eats a high-carbohydrate meal, glycogen degradation immediately stops. Although the changes in insulin and glucagon levels are relatively rapid (10 to 15 minutes), the direct inhibitory effect of rising glucose levels on glycogen degradation is even more rapid. Glucose, as an allosteric effector, inhibits liver glycogen phosphorylase a by stimulating dephosphorylation of this enzyme. As insulin

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levels rise and glucagon levels fall, cAMP levels decrease and PKA reassociates with its inhibitory subunits and becomes inactive. The protein phosphatases are activated, and glycogen phosphorylase a and glycogen synthase b are dephosphorylated. The collective result of these effects is rapid inhibition of glycogen degradation and rapid activation of glycogen synthesis. 8.

EPINEPHRINE AND CALCIUM IN THE REGULATION OF LIVER GLYCOGEN In the neonate, the release of epinephrine during labor and birth normally contributes to restoring blood glucose levels. Unfortunately, Getta Carbo did not have adequate liver glycogen stores to support a rise in her blood glucose levels.

Epinephrine, the fight-or-flight hormone, is released from the adrenal medulla in response to neural signals reflecting an increased demand for glucose. To flee from a dangerous situation, skeletal muscles use increased amounts of blood glucose to generate ATP. As a result, liver glycogenolysis must be stimulated. In the liver, epinephrine stimulates glycogenolysis through two different types of receptors: the ␣- and ␤-agonist receptors. A. Epinephrine Acting at ␤-Receptors

Epinephrine, acting at the ␤-receptors, transmits a signal through G proteins to adenylate cyclase, which increases cAMP levels and activates PKA. Therefore, regulation of glycogen degradation and synthesis in liver by epinephrine and glucagon are similar (see Fig. 28.8). B. Epinephrine Acting at ␣-Receptors

Epinephrine also binds to ␣-receptors in the hepatocyte. This binding activates glycogenolysis and inhibits glycogen synthesis principally by increasing the Ca2 levels in the liver. The effects of epinephrine at the ␣-agonist receptor are mediated by the phosphatidylinositol bisphosphate (PIP2)-Ca2 signal transduction system, one of the principal intracellular second messenger systems employed by many hormones (Fig. 28.9) (see Chapter 11).

␣-Agonist receptor

Epinephrine Phospholipase C

1

Protein kinase C

Extracellular

+

Cell membrane

+

G GTP

PIP2

DAG

Cytoplasm

+

2 IP3 +

Endoplasmic reticulum

+

Ca2+

Calmodulindependent protein kinase

4

Glycogen synthase (inactive)

P P P

Glycogen synthase (active)

Ca2+-calmodulin

5 3 +

Phosphorylase kinase

Glycogen phosphorylase a (active)

P

Glycogen phosphorylase b (inactive)

FIG. 28.9. Regulation of glycogen synthesis and degradation by epinephrine and Ca2. 1. The effect of epinephrine binding to ␣-agonist receptors in the liver transmits a signal via G proteins to phospholipase C, which hydrolyzes PIP2 to DAG and IP3. 2. IP3 stimulates the release of Ca2 from the endoplasmic reticulum. 3. Ca2 binds to the modifier protein calmodulin, which activates calmodulin-dependent protein kinase and phosphorylase kinase. Both Ca2 and DAG activate protein kinase C. 4. These three kinases phosphorylate glycogen synthase at different sites and decrease its activity. 5. Phosphorylase kinase phosphorylates glycogen phosphorylase b to the active form. It therefore activates glycogenolysis as well as inhibiting glycogen synthesis.

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Jim Bodie gradually regained consciousness with continued infusions of high-concentration glucose titrated to keep his serum glucose level between 120 and 160 mg/dL. Although he remained somnolent and moderately confused over the next 12 hours, he was eventually able to tell his physicians that he had self-injected approximately 80 U of regular (short-acting) insulin every 6 hours while eating a high-carbohydrate diet for the last 2 days preceding his seizure. Normal subjects under basal conditions secrete an average of 40 U of insulin daily. He had last injected insulin just before exercising. An article in a body-building magazine that he had read recently cited the anabolic effects of insulin on increasing muscle mass. He had purchased the insulin and necessary syringes from the same underground drug source from whom he regularly bought his anabolic steroids. Normally, muscle glycogenolysis supplies the glucose required for the kinds of high-intensity exercise that require anaerobic glycolysis, such as weightlifting. Jim’s treadmill exercise also uses blood glucose, which is supplied by liver glycogenolysis. The high serum insulin levels, resulting from the injection he gave himself just before his workout, activated both glucose transport into skeletal muscle and glycogen synthesis while inhibiting glycogen degradation. His exercise, which would continue to use blood glucose, could normally be supported by breakdown of liver glycogen. However, glycogen synthesis in his liver was activated and glycogen degradation was inhibited by the insulin injection.

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In the PIP2-Ca2 signal transduction system, the signal is transferred from the epinephrine receptor to membrane-bound phospholipase C by G proteins. Phospholipase C hydrolyzes PIP2 to form diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 stimulates the release of Ca2 from the endoplasmic reticulum. Ca2 and DAG activate protein kinase C. The amount of calcium bound to one of the calcium-binding proteins—calmodulin—is also increased. Calcium/calmodulin associates as a subunit with several enzymes and modifies their activities. It binds to inactive phosphorylase kinase, thereby partially activating this enzyme. (The fully activated enzyme is both bound to the calcium/ calmodulin subunit and phosphorylated.) Phosphorylase kinase then phosphorylates glycogen phosphorylase b, thereby activating glycogen degradation. Calcium/ calmodulin is also a modifier protein that activates one of the glycogen synthase kinases (calcium/calmodulin synthase kinase). Protein kinase C, calcium/calmodulin synthase kinase, and phosphorylase kinase all phosphorylate glycogen synthase at different serine residues on the enzyme, thereby inhibiting glycogen synthase and thus glycogen synthesis. The effect of epinephrine in the liver therefore enhances or is synergistic with the effects of glucagon. Epinephrine release during bouts of hypoglycemia or during exercise can stimulate hepatic glycogenolysis and inhibit glycogen synthesis very rapidly.

B. Regulation of Glycogen Synthesis and Degradation in Skeletal Muscle The regulation of glycogenolysis in skeletal muscle is related to the availability of ATP for muscular contraction. Skeletal muscle glycogen produces glucose 1-phosphate and a small amount of free glucose. Glucose 1-phosphate is converted to G6P, which is committed to the glycolytic pathway; the absence of glucose 6-phosphatase in skeletal muscle prevents conversion of the glucosyl units from glycogen to blood glucose. Skeletal muscle glycogen is therefore degraded only when the demand for ATP generation from glycolysis is high. The highest demands occur during anaerobic glycolysis, which requires more moles of glucose for each ATP produced than oxidation of glucose to CO2 (see Chapter 22). Anaerobic glycolysis occurs in tissues that have fewer mitochondria, a higher content of glycolytic enzymes, and higher levels of glycogen, or fast-twitch glycolytic fibers. It occurs most frequently at the onset of exercise—before vasodilation occurs to bring in blood-borne fuels. The regulation of skeletal muscle glycogen degradation therefore must respond very rapidly to the need for ATP—indicated by the increase in AMP. The regulation of skeletal muscle glycogen synthesis and degradation differs from that in liver in several important respects: (1) glucagon has no effect on muscle, and thus glycogen levels in muscle do not vary with the fasting/feeding state; (2) AMP is an allosteric activator of the muscle isozyme of glycogen phosphorylase, but not liver glycogen phosphorylase (Fig. 28.10); (3) the effects of Ca2 in muscle result principally from the release of Ca2 from the sarcoplasmic reticulum after neural stimulation, and not from epinephrine-stimulated uptake; (4) glucose is not a physiologic inhibitor of glycogen phosphorylase a in muscle; (5) glycogen is a stronger feedback inhibitor of muscle glycogen synthase than of liver glycogen synthase, resulting in a smaller amount of stored glycogen per gram weight of muscle tissue. However, the effects of epinephrine-stimulated phosphorylation by PKA on skeletal muscle glycogen degradation and glycogen synthesis are similar to those occurring in liver (see Fig. 28.8). Muscle glycogen phosphorylase is a genetically distinct isoenzyme of liver glycogen phosphorylase and contains an amino acid sequence that has a purine nucleotide-binding site. When AMP binds to this site, it changes the conformation at the catalytic site to a structure very similar to that in the phosphorylated enzyme (see Chapter 9, Fig. 9.9). Thus, hydrolysis of ATP to ADP and the consequent

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525

Epinephrine Nerve impulse Sarcoplasmic Ca2+ reticulum

cAMP

3

2

Ca2+

Protein kinase A +

ATP Myosin ATPase

Ca2+-calmodulin

ADP Muscle contraction

Phosphorylase kinase

Adenylate kinase

P

AMP

1

+

+

Glycogen phosphorylase b

P

Glycogen phosphorylase a

Pi

FIG. 28.10. Activation of muscle glycogen phosphorylase during exercise. Glycogenolysis in skeletal muscle is initiated by muscle contraction, neural impulses, and epinephrine. 1. AMP produced from the degradation of ATP during muscular contraction allosterically activates glycogen phosphorylase b. 2. The neural impulses that initiate contraction release Ca2 from the sarcoplasmic reticulum. The Ca2 binds to calmodulin, which is a modifier protein that activates phosphorylase kinase. 3. Phosphorylase kinase is also activated through phosphorylation by PKA. The formation of cAMP and the resultant activation of PKA are initiated by the binding of epinephrine to plasma membrane receptors.

increase of AMP generated by adenylate kinase during muscular contraction can directly stimulate glycogenolysis to provide fuel for the glycolytic pathway. AMP also stimulates glycolysis by activating phosphofructokinase-1, so this one effector activates both glycogenolysis and glycolysis. The activation of the calcium/ calmodulin subunit of phosphorylase kinase by the Ca2 released from the sarcoplasmic reticulum during muscle contraction also provides a direct and rapid means of stimulating glycogen degradation. CLINICAL COMMENTS Getta Carbo. Getta Carbo’s hypoglycemia illustrates the importance of glycogen stores in the neonate. At birth, the fetus must make two major adjustments in the way fuels are used: It must adapt to using a greater variety of fuels than were available in utero, and it must adjust to intermittent feeding. In utero, the fetus receives a relatively constant supply of glucose from the maternal circulation through the placenta, producing a level of glucose in the fetus that approximates 75% of maternal blood levels. With regard to the hormonal regulation of fuel use in utero, fetal tissues function in an environment dominated by insulin, which promotes growth. During the last 10 weeks of gestation, this hormonal milieu leads to glycogen formation and storage. At birth, the infant’s diet changes to one containing greater amounts of fat and lactose (galactose and glucose in equal ratio), presented at intervals rather than in a constant fashion. At the same time, the neonate’s need for glucose is relatively larger than that of the adult because the newborn’s ratio of brain to liver weight is greater. Thus, the infant has even greater difficulty maintaining glucose homeostasis than the adult. At the moment that the umbilical cord is clamped, the normal neonate is faced with a metabolic problem: The high insulin levels of late fetal existence must

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be quickly reversed to prevent hypoglycemia. This reversal is accomplished through the secretion of the counterregulatory hormones: epinephrine and glucagon. Glucagon release is triggered by the normal decline of blood glucose after birth. The neural response that stimulates the release of both glucagon and epinephrine is activated by the anoxia, cord clamping, and tactile stimulation that are part of a normal delivery. These responses have been referred to as the “normal sensor function” of the neonate. Within 3 to 4 hours of birth, these counterregulatory hormones reestablish normal serum glucose levels in the newborn’s blood through their glycogenolytic and gluconeogenic actions. The failure of Getta’s normal “sensor function” was partly caused by maternal malnutrition, which resulted in an inadequate deposition of glycogen in Getta’s liver before birth. The consequence was a serious degree of postnatal hypoglycemia. The ability to maintain glucose homeostasis during the first few days of life also depends on the activation of gluconeogenesis and the mobilization of fatty acids. Fatty acid oxidation in the liver not only promotes gluconeogenesis (see Chapter 31), but also generates ketone bodies. The neonatal brain has an enhanced capacity to use ketone bodies relative to that of infants (4-fold) and adults (40-fold). This ability is consistent with the relatively high fat content of breast milk. Jim Bodie. Jim Bodie attempted to build up his muscle mass with androgens and with insulin. The anabolic (nitrogen-retaining) effects of androgens on skeletal muscle cells enhance muscle mass by increasing amino acid flux into muscle and by stimulating protein synthesis. Exogenous insulin has the potential to increase muscle mass by similar actions and also by increasing the content of muscle glycogen. The most serious side effect of exogenous insulin administration is the development of severe hypoglycemia, such as what occurred in Jim’s case. The immediate adverse effect relates to an inadequate flow of fuel (glucose) to the metabolizing brain. When hypoglycemia is extreme, the patient may suffer a seizure and, if the hypoglycemia worsens, irreversible brain damage may occur. If prolonged, the patient will lapse into a coma and die.

BIOCHEMICAL COMMENTS Glycogen Synthase Kinase-3 (GSK-3). It should be clear that the regulation of glycogen metabolism is quite complex. Recent work has indicated that certain enzymes involved in regulating glycogen synthase activity also have far-reaching effects on other aspects of cell metabolism, such as cell structure, motility, growth, and survival. The best example of this is GSK-3. The enzyme was first identified as being an inhibitor of glycogen synthase. GSK-3 refers to two isozymes: GSK3␣ and GSK3␤. GSK-3 has been identified as a kinase that can phosphorylate >40 different proteins, including a large number of transcription factors. GSK-3 activity is also self-regulated by phosphorylation. GSK-3 activity is reduced by phosphorylation of a serine residue near its amino terminal. Protein kinase A (PKA), Akt, and protein kinase C can all catalyze this inhibitory phosphorylation event. GSK-3 is most active on protein substrates that have already been phosphorylated by other kinases (the substrates are said to be primed for further phosphorylation events). For example, GSK-3 will add phosphates to glycogen synthase, but only after glycogen synthase had been phosphorylated by PKA. GSK-3 binds to several proteins that sequester GSK-3 in certain pathways. This includes the wnt signaling pathway, disruption of which is a significant

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component of colon cancer, the patched–smoothened signal transduction pathway (see Chapter 18), and the phosphorylation of microtubule-associated proteins, which leads to altered cell motility. Activation of GSK-3 has also been linked to apoptosis, although GSK-3 activity is also required for cell survival. One of the effects of insulin is to phosphorylate GSK-3 via activation of Akt, rendering the GSK-3 inactive. Loss of activity of GSK-3 will lead to activation of glycogen synthase activity and the pathways of energy storage. In animal models of type 2 diabetes, there is a loss of inhibitory control of GSK-3, leading to greaterthan-normal GSK-3 activity, which antagonizes insulin action (promoting insulin resistance, a hallmark of type 2 diabetes). Studies in rats have shown that inhibitors of GSK-3 lowered blood glucose levels and stimulated glucose transport into muscles of insulin-resistant animals. Inappropriate stimulation of GSK-3 has also been implicated in Alzheimer disease. Current research concerning GSK-3 is geared toward understanding all of the roles of GSK-3 in cell growth and survival, and to decipher its actions in the multitude of multiprotein complexes with which it is associated. Inhibitors of GSK-3 are being examined as possible agents to treat diabetes, but interpretation of results is difficult due to the multitude of roles that GSK-3 plays within cells. It is possible that in the future, such drugs will be available to treat type 2 diabetes. Key Concepts • • •

•

• • • • • • • •

Glycogen is the storage form of glucose, composed of glucosyl units linked by ␣-1,4-glycosidic bonds with ␣-1,6-branches occurring about every 8 to 10 glucosyl units. Glycogen synthesis requires energy. Glycogen synthase transfers a glucosyl residue from the activated intermediate UDP-G to the nonreducing ends of existing glycogen chains during glycogen synthesis. The branching enzyme creates ␣-1,6-linkages in the glycogen chain. Glycogenolysis is the degradation of glycogen. Glycogen phosphorylase catalyzes a phosphorolysis reaction, using exogenous Pi to break ␣-1,4-linkages at the ends of glycogen chains, releasing glucose 1-phosphate. The debranching enzyme hydrolyzes the ␣-1,6-linkages in glycogen, releasing free glucose. Liver glycogen supplies blood glucose. Glycogen synthesis and degradation are regulated in the liver by hormonal changes that signify either a deficiency of or an excess of blood glucose. Lack of dietary glucose, signaled by a decrease of the insulin/glucagon ratio, activates liver glycogenolysis and inhibits glycogen synthesis. Epinephrine also activates liver glycogenolysis. Glucagon and epinephrine release lead to phosphorylation of glycogen synthase (inactivating it) and glycogen phosphorylase (activating it). Glycogenolysis in muscle supplies G6P for ATP synthesis in the glycolytic pathway. Muscle glycogen phosphorylase is allosterically activated by AMP, as well as by phosphorylation. Increases in sarcoplasmic Ca2 stimulates phosphorylation of muscle glycogen phosphorylase. Diseases discussed in this chapter are summarized in Table 28.4.

Table 28.4.

Diseases Discussed in Chapter 28

Disease or Disorder

Environmental or Genetic

Newborn hypoglycemia

Environmental

Insulin overdose

Environmental

Glycogen storage diseases

Genetic

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Comments Poor maternal nutrition may lead to inadequate glycogen levels in the newborn resulting in hypoglycemia during the early fasting period after birth. Insulin taken without carbohydrate ingestion will lead to severe hypoglycemia due to stimulation of glucose uptake by peripheral tissues, leading to insufficient glucose in the circulation for proper functioning of the nervous system. These have been summarized in Table 28.1. Affect storage and use of glycogen with different levels of severity from mild to fatal.

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REVIEW QUESTIONS—CHAPTER 28 1.

2.

3.

The degradation of glycogen normally produces which of the following? A. More glucose than glucose 1-phosphate B. More glucose 1-phosphate than glucose C. Equal amounts of glucose and glucose 1-phosphate D. Neither glucose or glucose 1-phosphate E. Only glucose 1-phosphate A patient has large deposits of liver glycogen, which after an overnight fast, had shorter-than-normal branches. This abnormality could be caused by a defective form of which one of the following proteins or activities? A. Glycogen phosphorylase B. Glucagon receptor C. Glycogenin D. Amylo-1,6-glucosidase E. Amylo-4,6-transferase An adolescent patient with a deficiency of muscle phosphorylase was examined while exercising his or her forearm by squeezing a rubber ball. Compared with a normal person performing the same exercise, this patient would exhibit which of the following? A. Exercise for a longer time without fatigue. B. Have increased glucose levels in blood drawn from his or her forearm. C. Have decreased lactate levels in blood drawn from his or her forearm.

Lieberman_CH28.indd 528

D. Have lower levels of glycogen in biopsy specimens from his or her forearm muscle. E. Hyperglycemia 4.

In a glucose tolerance test, an individual in the basal metabolic state ingests a large amount of glucose. If the individual is normal, this ingestion should result in which of the following? A. An enhanced glycogen synthase activity in the liver B. An increased ratio of glycogen phosphorylase a to glycogen phosphorylase b in the liver C. An increased rate of lactate formation by red blood cells D. An inhibition of PP-1 activity in the liver E. An increase of cAMP levels in the liver

5.

Consider a person with type 1 diabetes who has neglected to take insulin for the past 72 hours and has not eaten much as well. Which of the following best describes the activity level of hepatic enzymes involved in glycogen metabolism under these conditions?

A. B. C. D. E. F.

Glycogen Synthase

Phosphorylase Kinase

Glycogen Phosphorylase

Active Active Active Inactive Inactive Inactive

Active Active Inactive Inactive Active Active

Active Inactive Inactive Inactive Inactive Active

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29

Pathways of Sugar Metabolism: Pentose Phosphate Pathway, Fructose, and Galactose Metabolism

Glucose is at the center of carbohydrate metabolism and is the major dietary sugar. Other sugars in the diet are converted to intermediates of glucose metabolism, and their fates parallel that of glucose. When carbohydrates other than glucose are required for the synthesis of diverse compounds such as lactose, glycoproteins, or glycolipids, they are synthesized from glucose. Fructose, the second most common sugar in the adult diet, is ingested principally as the monosaccharide or as part of sucrose (Fig. 29.1). It is metabolized principally in the liver (and to a lesser extent in the small intestine and kidney) by phosphorylation at the 1-position to form fructose 1-phosphate (fructose 1-P), followed by conversion to intermediates of the glycolytic pathway. The major products of its metabolism in liver are, therefore, the same as for glucose (including lactate, blood glucose, and glycogen). Essential fructosuria (fructokinase deficiency) and hereditary fructose intolerance (HFI) (a deficiency of the fructose 1-P cleavage by aldolase B) are inherited disorders of fructose metabolism. Fructose synthesis from glucose in the polyol pathway occurs in seminal vesicles and other tissues. Aldose reductase converts glucose to the sugar alcohol sorbitol (a polyol), which is then oxidized to fructose. In the lens of the eye, elevated levels of sorbitol in diabetes mellitus may contribute to formation of cataracts. Galactose is ingested principally as lactose, which is converted to galactose and glucose in the intestine. Galactose is converted to glucose principally in the liver. It is phosphorylated to galactose 1-phosphate (galactose 1-P) by galactokinase and activated to a uridine diphosphate (UDP)-sugar by galactosyl uridylyltransferase. The metabolic pathway subsequently generates glucose 1-phosphate (glucose 1-P). Classical galactosemia, a deficiency of galactosyl uridylyltransferase, results in the accumulation of galactose 1-P in the liver and the inhibition of hepatic glycogen metabolism and other pathways that require UDP-sugars. Cataracts can occur from accumulation of galactose in the blood, which is converted to galactitol (the sugar alcohol of galactose) in the lens of the eye. The pentose phosphate pathway (also known as the hexose monophosphate shunt [HMP shunt]) consists of both oxidative and nonoxidative components (Fig. 29.2). In the oxidative pathway, glucose 6-phosphate (glucose 6-P) is oxidized to ribulose 5-phosphate (ribulose 5-P), CO2, and NADPH. Ribulose 5-P, a pentose, can be converted to ribose 5-phosphate (ribose 5-P) for nucleotide biosynthesis. The NADPH is used for reductive pathways, such as fatty acid biosynthesis, detoxification of drugs by monooxygenases, and the glutathione defense system against injury by reactive oxygen species (ROS). In the nonoxidative phase of the pathway, ribulose 5-P is converted to ribose 5-P and to intermediates of the glycolytic pathway. This portion of the pathway is reversible; therefore, ribose 5-P can also be formed from

Dietary sucrose Glucose Polyol pathway

Dietary fructose

1 CH OH 2 2

HO H H

3 4 5 6

C

O

C

H

C

OH

C

OH

CH2OH

Intermediates of glycolysis

FIG. 29.1. Fructose. The sugar fructose is found in the diet as the free sugar in foods such as honey or as a component of the disaccharide sucrose in fruits and sweets. It also can be synthesized from glucose via the polyol pathway. In the lens of the eye, the polyol pathway contributes to the formation of cataracts. Fructose is metabolized by conversion to intermediates of glycolysis.

529

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SECTION V ■ CARBOHYDRATE METABOLISM

Fatty acid synthesis

Glucose +

2 NADP

Glucose 6-phosphate

Glutathione reduction

2 NADPH

Oxidative

CO2

Other reactions. such as detoxification

Ribulose 5-phosphate Xylulose 5-phosphate

Fructose 6-phosphate Non-oxidative

Glyceraldehyde 3-phosphate NADH ATP

Ribose 5-phosphate

Nucleotide biosynthesis

Pyruvate Glycolysis

The pentose phosphate pathway

FIG. 29.2. Overview of the pentose phosphate pathway. The pentose phosphate pathway generates NADPH for reactions that require reducing equivalents (electrons) or ribose 5-P for nucleotide biosynthesis. Glucose 6-P is a substrate for both the pentose phosphate pathway and glycolysis. The five-carbon sugar intermediates of the pentose phosphate pathway are reversibly interconverted to intermediates of glycolysis. The portion of glycolysis that is not part of the pentose phosphate pathway is shown in red.

intermediates of glycolysis. One of the enzymes involved in these sugar interconversions, transketolase, uses thiamine pyrophosphate as a coenzyme. The sugars produced by the pentose phosphate pathway enter glycolysis as fructose 6-phosphate (fructose 6-P) and glyceraldehyde 3-phosphate (glyceraldehyde 3-P), and their further metabolism in the glycolytic pathway generates NADH, adenosine triphosphate (ATP), and pyruvate. The overall equation for the conversion of glucose 6-P to fructose 6-P and glyceraldehyde 3-P through both the oxidative and nonoxidative reactions of the pentose phosphate pathway is 3 Glucose 6-P ⫹ 6 NADP⫹ → 3 CO2 ⫹ 6 NADPH ⫹ 6 H⫹ ⫹ 2 fructose 6-P ⫹ glyceraldehyde 3-P

THE WAITING ROOM Candice Sucher is an 18-year-old girl who presented to her physician for a precollege physical examination. While taking her medical history, the doctor learned that she carefully avoided eating all fruits and any foods that contained table sugar. She related that from a very early age, she had learned that these foods caused severe weakness and symptoms suggestive of low blood sugar, such as tremulousness and sweating. Her medical history also indicated that her

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mother had described her as having been a very irritable baby who often cried incessantly, especially after meals, and vomited frequently. At these times, Candice’s abdomen had become distended, and she became drowsy and apathetic. Her mother had intuitively eliminated certain foods from Candice’s diet after which the severity and frequency of these symptoms diminished. Erin Galway is a 3-week-old female infant who began vomiting 3 days after birth, usually within 30 minutes after breastfeeding. Her abdomen became distended at these times, and she became irritable and cried frequently. When her mother noted that the whites of Erin’s eyes were yellow, she took her to a pediatrician. The doctor agreed that Erin was slightly jaundiced. He also noted an enlargement of her liver and questioned the possibility of early cataract formation in the lenses of Erin’s eyes. He ordered liver and kidney function tests and did two separate dipstick urine tests in his office, one designed to measure only glucose in the urine and the other capable of detecting any of the reducing sugars. Al Martini developed a fever of 101.5° F on the second day of his hospitalization for acute alcoholism. One of his physicians noticed that one of the lacerations on Mr. Martini’s arm was red and swollen with some pus drainage. The pus was cultured and gram-positive cocci were found and identified as Staphylococcus aureus. Because his landlady stated that he had an allergy to penicillin and the concern over methicillin-resistant S. aureus, Al was started on a course of the antibiotic combination of trimethoprim and sulfamethoxazole (TMP/ sulfa). To his landlady’s knowledge, he had never been treated with a sulfa drug previously. On the third day of therapy with TMP/sulfa for his infection, Mr. Martini was slightly jaundiced. His hemoglobin level had fallen by 3.5 g/dL from its value at admission, and his urine was red-brown because of the presence of free hemoglobin. Mr. Martini had apparently suffered acute hemolysis (lysis or destruction of some of his red blood cells) induced by his infection and exposure to the sulfa drug.

I.

FRUCTOSE

Fructose is found in the diet as a component of sucrose in fruit, as a free sugar in honey, and in high-fructose corn syrup (see Fig. 29.1). Fructose enters epithelial cells and other types of cells by facilitated diffusion on the GLUT 5 transporter. It is metabolized to intermediates of glycolysis. Problems with fructose absorption and metabolism are relatively more common than with other sugars.

A. Fructose Metabolism Fructose is metabolized by conversion to glyceraldehyde 3-phosphate (glyceraldehyde 3-P) and dihydroxyacetone phosphate, which are intermediates of glycolysis (Fig. 29.3). The steps parallel those of glycolysis. The first step in the metabolism of fructose, as with glucose, is phosphorylation. Fructokinase, the major kinase involved, phosphorylates fructose in the 1-position. Fructokinase has a high Vmax and rapidly phosphorylates fructose as it enters the cell. The fructose 1-phosphate (fructose 1-P) formed is not an intermediate of glycolysis but rather is cleaved by aldolase B to dihydroxyacetone phosphate (an intermediate of glycolysis) and glyceraldehyde. Glyceraldehyde is then phosphorylated to glyceraldehyde 3-P by triose kinase. Dihydroxyacetone phosphate and glyceraldehyde 3-P are intermediates of the glycolytic pathway and can proceed through it to pyruvate, the tricarboxylic acid (TCA) cycle, and fatty acid synthesis. Alternatively, these intermediates can also be converted to glucose by gluconeogenesis. In other words, the fate of fructose parallels that of glucose.

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When individuals with defects of aldolase B ingest fructose, the extremely high levels of fructose 1-phosphate (fructose 1-P) that accumulate in the liver and kidney cause several adverse effects. Hypoglycemia results from inhibition of glycogenolysis and gluconeogenesis. Glycogen phosphorylase (and possibly phosphoglucomutase and other enzymes of glycogen metabolism) are inhibited by the accumulated fructose 1-P. Aldolase B is required for glucose synthesis from glyceraldehyde 3-phosphate (glyceraldehyde 3-P) and dihydroxyacetone phosphate, and its low activity in aldolase B-deficient individuals is further decreased by the accumulated fructose 1-P. The inhibition of gluconeogenesis results in lactic acidosis. The accumulation of fructose 1-P also substantially depletes the intracellular phosphate pools. The fructokinase reaction uses adenosine triphosphate (ATP) at a rapid rate such that the mitochondria regenerate ATP rapidly, which leads to a drop in free phosphate levels. The low levels of phosphate release inhibition of adenosine monophosphate (AMP) deaminase, which converts AMP to inosine monophosphate (IMP). The nitrogenous base of IMP (hypoxanthine) is degraded to uric acid. The lack of phosphate and depletion of adenine nucleotides lead to a loss of ATP, further contributing to the inhibition of biosynthetic pathways, including gluconeogenesis.

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Essential fructosuria is a rare and benign genetic disorder caused by a deficiency of the enzyme fructokinase. Why is this disease benign, when a deficiency of aldolase B (hereditary fructose intolerance [HFI]) can be fatal?

Fructose

Glucose

ATP

Glycogen

ATP fructokinase

ADP

hexokinase

ADP

Fructose-1-P

Glucose-6-P

Glucose-1-P

aldolase B

Dihydroxyacetone-P Glyceraldehyde

Fructose-6-P Fructose-1,6-BP

ATP

aldolase B (liver) aldolase A (muscle)

triose kinase

ADP Glyceraldehyde-3-P

Dihydroxyacetone-P

Glyceraldehyde-3-P

O Lactate

H

C

H

C

OH

HO

C

H

H

C

OH

H

C

OH

CH2OH D-glucose NADPH + H+

Pyruvate Fatty acids TCA cycle

FIG. 29.3. Fructose metabolism. The pathway for the conversion of fructose to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (glyceraldehyde 3-P) is shown in red. These two compounds are intermediates of glycolysis and are converted in the liver principally to glucose, glycogen, or fatty acids. In the liver, aldolase B cleaves both fructose 1-phosphate (fructose 1-P) in the pathway for fructose metabolism and fructose 1,6-bisphosphate in the pathway for glycolysis.

Aldose reductase

NADP+ CH2OH H

C

OH

HO

C

H

H

C

OH

H

C

OH

CH2OH Sorbitol (polyol) NAD+ Sorbitol dehydrogenase

NADH + H+ CH2OH

The metabolism of fructose occurs principally in the liver and to a lesser extent in the small intestinal mucosa and proximal epithelium of the renal tubule because these tissues have both fructokinase and aldolase B. Aldolase exists as several isoforms: aldolases A, B, C, and fetal aldolase. Although all of these aldolase isoforms can cleave fructose 1,6-bisphosphate, the intermediate of glycolysis, only aldolase B can also cleave fructose 1-P. Aldolase A, present in muscle and most other tissues, and aldolase C, present in brain, have almost no ability to cleave fructose 1-P. Fetal aldolase, present in the liver before birth, is similar to aldolase C. Aldolase B is the rate-limiting enzyme of fructose metabolism, although it is not a rate-limiting enzyme of glycolysis. It has a much lower affinity for fructose l-P than fructose 1,6-bisphosphate and is very slow at physiologic levels of fructose 1-P. As a consequence, after ingesting a high dose of fructose, normal individuals accumulate fructose 1-P in the liver while it is slowly converted to glycolytic intermediates. Individuals with hereditary fructose intolerance (HFI) (a deficiency of aldolase B) accumulate much higher amounts of fructose 1-P in their livers. Other tissues also have the capacity to metabolize fructose but do so much more slowly. The hexokinase isoforms present in muscle, adipose tissue, and other tissues can convert fructose to fructose 6-phosphate (fructose 6-P) but react so much more efficiently with glucose. As a result, fructose phosphorylation is very slow in the presence of physiologic levels of intracellular glucose and glucose 6-phosphate (glucose 6-P).

C

O

HO

C

H

H

C

OH

B. Synthesis of Fructose in the Polyol Pathway

H

C

OH

Fructose can be synthesized from glucose in the polyol pathway. The polyol pathway is named for the first step of the pathway in which sugars are reduced to the sugar alcohol by the enzyme aldose reductase (Fig. 29.4). Glucose is reduced to the sugar alcohol sorbitol, and sorbitol is then oxidized to fructose. This pathway is present in seminal vesicles, which synthesize fructose for the seminal fluid. Spermatozoa use fructose as a major fuel source while in the seminal fluid and then

CH2OH D-fructose

FIG. 29.4. The polyol pathway converts glucose to fructose.

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CHAPTER 29 ■ PATHWAYS OF SUGAR METABOLISM

switch to glucose once in the female reproductive tract. Use of fructose is thought to prevent acrosomal breakdown of the plasma membrane (and consequent activation) while the spermatozoa are still in the seminal fluid. The polyol pathway is present in many tissues but its function in all tissues is not understood. Aldose reductase is relatively nonspecific, and its major function may be the metabolism of an aldehyde sugar other than glucose. The activity of this enzyme can lead to major problems in the lens of the eye where it is responsible for the production of sorbitol from glucose and galactitol from galactose. When the concentration of glucose or galactose is elevated in the blood, their respective sugar alcohols are synthesized in the lens more rapidly than they are removed, resulting in increased osmotic pressure within the lens.

II. GALACTOSE METABOLISM—METABOLISM TO GLUCOSE 1-PHOSPHATE Dietary galactose is metabolized principally by phosphorylation to galactose 1-phosphate (galactose 1-P), and then conversion to uridine diphosphate (UDP)galactose and glucose 1-phosphate (glucose 1-P) (Fig. 29.5). The phosphorylation of galactose, again an important first step in the pathway, is carried out by a specific kinase, galactokinase. The formation of UDP-galactose is accomplished by attack of the phosphate oxygen on galactose 1-P on the ␣-phosphate of UDP-glucose, releasing glucose 1-P while forming UDP-galactose. The enzyme that catalyzes this reaction is galactose l-P uridylyltransferase. The UDP-galactose is then converted to UDP-glucose by the reversible UDP-glucose epimerase (the configuration of the hydroxyl group on carbon 4 is reversed in this reaction). The net result of this sequence of reactions is that galactose is converted to glucose 1-P at the expense of one high-energy bond of adenosine triphosphate (ATP). The sum of these reactions is indicated in the following equations: (1) Galactose ⫹ ATP

→ Galactose 1-P ⫹ ADP

Galactokinase

(2) Galactose 1-P ⫹ UDP-glucose (3) UDP-galactose

Galactose 1–P Uridylytransferase

→ UDP-galactose ⫹ glucose 1-P

→ UDP-glucose

UDP-glucose epimerase

Net equation: Galactose ⫹ ATP → Glucose 1-P ⫹ ADP

533

In essential fructosuria, fructose cannot be converted to fructose 1-phosphate (fructose 1-P). This condition is benign because no toxic metabolites of fructose accumulate in the liver, and the patient remains nearly asymptomatic. Some of the ingested fructose is slowly phosphorylated by hexokinase in nonhepatic tissues and metabolized by glycolysis, and some appears in the urine. There is no renal threshold for fructose; the appearance of fructose in the urine (fructosuria) does not require a high fructose concentration in the blood. Hereditary fructose intolerance (HFI) conversely results in the accumulation of fructose 1-P and fructose. By inhibiting glycogenolysis and gluconeogenesis, the high levels of fructose 1-P caused the hypoglycemia that Candice Sucher experienced as an infant when she became apathetic and drowsy and as an adult when she experienced sweating and tremulousness.

The accumulation of sorbitol in muscle and nerve tissues may contribute to the peripheral neuropathy characteristic of patients with poorly controlled diabetes mellitus. This is one of the reasons it is so important for Di Abietes (who has type 1 diabetes mellitus) and Ann Sulin (who has type 2 diabetes mellitus) to achieve good glycemic control.

Nonclassical galactosemia Classical galactosemia

Galactose ATP Galactokinase

ADP Galactose 1-phosphate UDPglucose Epimerase

Galactose 1-phosphate uridylyltransferase

Glucose 1-phosphate

UDPgalactose Glucose 6-phosphate (Liver)

Glycolysis (other tissues)

Glucose

FIG. 29.5. Metabolism of galactose. Galactose is phosphorylated to galactose 1-phosphate (galactose 1-P) by galactokinase. Galactose 1-P reacts with UDP-glucose to release glucose 1-P. Galactose, thus, can be converted to blood glucose, enter glycolysis, or enter any of the metabolic routes of glucose. In classical galactosemia, a deficiency of galactose 1-P uridylyltransferase (shown in green) results in the accumulation of galactose 1-P in tissues and the appearance of galactose in the blood and urine. In nonclassical galactosemia, a deficiency of galactokinase (shown in red) results in the accumulation of galactose.

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The accumulation of sugars and sugar alcohols in the lens of patients with hyperglycemia (e.g., diabetes mellitus) results in the formation of cataracts. Glucose levels are elevated and increase the synthesis of sorbitol and fructose. As a consequence, a high osmotic pressure is created in the lens. The high glucose and fructose levels also result in nonenzymatic glycosylation of lens proteins. The result of the increased osmotic pressure and the glycosylation of the lens protein is an opaque cloudiness of the lens known as a cataract. Erin Galway seemed to have an early cataract, probably caused by the accumulation of galactose and its sugar alcohol galactitol.

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One of the most serious problems of classical galactosemia is irreversible mental retardation. Realizing this problem, Erin Galway’s physician wanted to begin immediate dietary therapy. A test that measures galactose 1-phosphate (galactose 1-P) uridylyltransferase in erythrocytes was ordered. This test is an enzymatic assay that mixes the unknown sample (in this case, a lysate from red blood cells, which contain the enzyme) with galactose 1-P, uridine diphosphate (UDP)-glucose and NADP⫹ in the presence of excess phosphoglucomutase and glucose-6-phosphate dehydrogenase. As the uridylyltransferase converts galactose 1-P to UDP-galactose and glucose 1-phosphate (glucose 1-P), the glucose 1-P is rapidly converted to glucose 6-phosphate (glucose 6-P) by phosphoglucomutase. The glucose 6-P is then converted to 6-phosphogluconate and NADPH by glucose-6-phosphate dehydrogenase. The resulting increase in absorbance at 340 nm allows a determination of the initial uridylyltransferase activity. The enzyme activity in Erin Galway’s red blood cells was virtually absent, confirming the diagnosis of classical galactosemia.

The enzymes for galactose conversion to glucose 1-P are present in many tissues, including the adult erythrocyte, fibroblasts, and fetal tissues. The liver has high activity of these enzymes and can convert dietary galactose to blood glucose and glycogen. The fate of dietary galactose like that of fructose, therefore, parallels that of glucose. The ability to metabolize galactose is even greater in infants than in adults. Newborn infants ingest up to 1 g of galactose per kilogram per feeding (as lactose). Yet the rate of metabolism is so high that the blood level in the systemic circulation is less than 3 mg/dL, and none of the galactose is lost in the urine.

III. THE PENTOSE PHOSPHATE PATHWAY The pentose phosphate pathway is essentially a scenic bypass route around the first stage of glycolysis that generates NADPH and ribose 5-phosphate (ribose 5-P) (as well as other pentose sugars). Glucose 6-P is the common precursor for both pathways. The oxidative first stage of the pentose phosphate pathway generates 2 moles of NADPH per mole of glucose 6-P oxidized. The second stage of the pentose phosphate pathway generates ribose 5-P and converts unused intermediates to fructose 6-P and glyceraldehyde 3-P in the glycolytic pathway (see Fig. 29.2). All cells require NADPH for reductive detoxification, and most cells require ribose 5-P for nucleotide synthesis. Consequently, the pathway is present in all cells. The enzymes reside in the cytosol as do the enzymes of glycolysis.

A. Oxidative Phase of the Pentose Phosphate Pathway 1.

NADPH PRODUCTION

In the oxidative, first phase of the pentose phosphate pathway, glucose 6-P undergoes and oxidation and decarboxylation to a pentose sugar, ribulose 5-phosphate (ribulose 5-P) (Fig. 29.6). The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, oxidizes the aldehyde at carbon 1 and reduces NADP⫹ to NADPH. The gluconolactone that is formed is rapidly hydrolyzed to 6-phosphogluconate, a sugar acid with a carboxylic acid group at carbon 1. The next oxidation step releases this carboxyl group as CO2, with the electrons being transferred to NADP⫹. This reaction is mechanistically very similar to the one catalyzed by isocitrate dehydrogenase in the TCA cycle. Thus, 2 moles of NADPH per mole of glucose 6-P are formed from this portion of the pathway. NADPH, rather than NADH, is generally used in the cell in pathways that require the input of electrons for reductive reactions because the ratio of NADPH/ NADP⫹ is much greater than the NADH/NAD⫹ ratio. The NADH generated from fuel oxidation is rapidly oxidized back to NAD⫹ by NADH dehydrogenase in the electron-transport chain, so the level of NADH in the cell is very low. NADPH can be generated from several reactions in the liver and other tissues but not in red blood cells. For example, in tissues with mitochondria, an energyrequiring transhydrogenase located near the complexes of the electron-transport chain can transfer reducing equivalents from NADH to NADP⫹ to generate NADPH. NADPH, however, cannot be oxidized directly by the electron-transport chain, and the ratio of NADPH to NADP⫹ in cells is >1. The reduction potential of NADPH, therefore, can contribute to the energy needed for biosynthetic processes and provide a constant source of reducing power for detoxification reactions. 2.

RIBOSE 5-PHOSPHATE FROM THE OXIDATIVE ARM OF THE PATHWAY

To generate ribose 5-P from the oxidative pathway, the ribulose 5-P formed from the action of the two oxidative steps is isomerized to produce ribose 5-P (a ketoseto-aldose conversion, similar to fructose 6-P being isomerized to glucose 6-P;

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see Section III.B.1). The ribose 5-P can then enter the pathway for nucleotide synthesis if needed, or it can be converted to glycolytic intermediates, as described below (Section III.B) for the nonoxidative phase of the pentose phosphate pathway. The pathway through which the ribose 5-P travels is determined by the needs of the cell at the time of its synthesis.

B. The Nonoxidative Phase of the Pentose Phosphate Pathway The nonoxidative reactions of this pathway are reversible reactions that allow intermediates of glycolysis (specifically, glyceraldehyde 3-P and fructose 6-P) to be converted to five-carbon sugars (such as ribose 5-P), and vice versa. The needs of the cell determine which direction this pathway proceeds. If the cell has produced ribose 5-P but does not need to synthesize nucleotides, then the ribose 5-P is converted to glycolytic intermediates. If the cell still requires NADPH, the ribose 5-P is converted back into glucose 6-P using nonoxidative reactions (see the following sections). And finally, if the cell already has a high level of NADPH but needs to produce nucleotides, the oxidative reactions of the pentose phosphate pathway are inhibited, and the glycolytic intermediates fructose 6-P and glyceraldehyde 3-P are used to produce the five-carbon sugars using exclusively the nonoxidative phase of the pentose phosphate pathway. 1.

CONVERSION OF RIBOSE 5-PHOSPHATE TO GLYCOLYTIC INTERMEDIATES

The nonoxidative portion of the pentose phosphate pathway consists of a series of rearrangement and transfer reactions that first convert ribulose 5-P to ribose 5-P and xylulose 5-phosphate (xylulose 5-P), and then the ribose 5-P and xylulose 5-P

H O C H

C

OH

HO

C

H

H

C

OH

H

C

OH

CH2OPO3 2– Glucose 6-phosphate NADP+

Glucose 6-phosphate dehydrogenase

NADPH + H+

O C H

C

OH

HO

C

H

H

C

OH

H

C

6-Phosphoglucono␦-lactone H2O H+

O C

O–

C

OH

HO

C

H

H

C

OH

H

C

OH

H

CH2OPO3 2– 6-Phosphogluconate NADP+ 6-Phosphogluconate dehydrogenase

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NADPH + H+ CO2

CH2OH

H

The transketolase activity of red blood cells is used to measure thiamine nutritional status and diagnose the presence of thiamine deficiency. The activity of transketolase is measured in the presence and absence of added thiamine pyrophosphate. If the thiamine intake of a patient is adequate, the addition of thiamine pyrophosphate does not increase the activity of transketolase because it already contains bound thiamine pyrophosphate. If the patient is thiamine-deficient, transketolase activity will be low, and adding thiamine pyrophosphate will greatly stimulate the reaction. Al Martini was diagnosed in Chapter 19 as having beriberi heart disease resulting from thiamine deficiency. The diagnosis was based on laboratory tests confirming the thiamine deficiency.

O

CH2OPO3 2–

Gluconolactonase

Erin Galway’s urine was negative for glucose when measured with the glucose oxidase strip but was positive for the presence of a reducing sugar. The reducing sugar was identified as galactose. Her liver function tests showed an increase in serum bilirubin and in several liver enzymes. Albumin was present in her urine. These findings and the clinical history increased her physician’s suspicion that Erin had classical galactosemia. Classical galactosemia is caused by a deficiency of galactose 1-phosphate (galactose 1-P) uridylyltransferase. In this disease, galactose 1-P accumulates in tissues, and galactose is elevated in the blood and urine. This condition differs from the rarer deficiency of galactokinase (nonclassical galactosemia) in which galactosemia and galactosuria occur but galactose 1-P is not formed. Both enzyme defects result in cataracts from galactitol formation by aldose reductase in the polyol pathway. Aldose reductase has a relatively high Km for galactose, approximately 12 to 20 mM, so galactitol is formed only in galactosemic patients who have eaten galactose. Galactitol is not further metabolized and diffuses out of the lens very slowly. Thus, hypergalactosemia is even more likely to cause cataracts than hyperglycemia. Erin Galway, although she is only 3 weeks old, appeared to have early cataracts forming in the lens of her eyes.

535

H

C

O

C

OH

C

OH

CH2OPO3 2– Ribulose 5-phosphate

FIG. 29.6. Oxidative portion of the pentose phosphate pathway. Carbon 1 of glucose 6-P is oxidized to an acid and then released as CO2 in an oxidation followed by a decarboxylation reaction. Each of the oxidation steps generates a NADPH.

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SECTION V ■ CARBOHYDRATE METABOLISM

How does the net energy yield from the metabolism of 3 mol of glucose 6-phosphate (glucose 6-P) through the pentose phosphate pathway to pyruvate compare with the yield of 3 mol of glucose 6-P through glycolysis?

H O C H

C

OH

H

C

OH

H

C

OH

CH2OPO3 2– Ribose 5-phosphate Isomerase

CH2OH

HO H

C

O

C

H

CH2OH C

O

OH

H

C

OH

CH2OPO3 2–

H

C

OH

C

Xylulose 5-phosphate

+

CH2OPO3 2– Ribulose 5-phosphate

H O Epimerase

C H

C

OH

CH2OH

H

C

OH

C

O

H

C

OH

HO

C

H

H

C

OH

CH2OPO3

2–

Ribose 5-phosphate

CH2OPO3 2– Xylulose 5-phosphate

Thiamine pyrophosphate

Transketolase

H O

FIG. 29.7. Ribulose 5-phosphate (ribulose 5-P) is epimerized (to xylulose 5-phosphate [xylulose 5-P], shown in red) and isomerized (to ribose 5-phosphate [ribose 5-P], shown in the yellow box).

C H

C

OH

CH2OPO3 2– Glyceraldehyde 3-phosphate

+ CH2OH C

O

HO

C

H

H

C

OH

H

C

OH

H

C

OH

CH2OPO3 2– Sedoheptulose 7-phosphate

FIG. 29.8. Two-carbon unit transferred by transketolase. Transketolase cleaves the bond next to the keto group and transfers the two-carbon keto fragment to an aldehyde. Thiamine pyrophosphate carries the two-carbon fragment, forming a covalent bond with the carbon of the keto group.

Lieberman_Ch29.indd 536

are converted to intermediates of the glycolytic pathway. The enzymes involved are epimerase, isomerase, transketolase, and transaldolase. The epimerase and isomerase convert ribulose 5-P to two other five-carbon sugars (Fig. 29.7). The isomerase converts ribulose 5-P to ribose 5-P. The epimerase changes the stereochemical position of one hydroxyl group (at carbon 3), converting ribulose 5-P to xylulose 5-P. Transketolase transfers two-carbon fragments of keto sugars (sugars with a keto group at carbon 2) to other sugars. Transketolase picks up a two-carbon fragment from xylulose 5-P by cleaving the carbon–carbon bond between the keto group and the adjacent carbon, thereby releasing glyceraldehyde 3-P (Fig. 29.8). The twocarbon fragment is covalently bound to thiamine pyrophosphate, which transfers it to the aldehyde carbon of another sugar, forming a new ketose. The role of thiamine pyrophosphate here is, thus, very similar to its role in the oxidative decarboxylation of pyruvate and ␣-ketoglutarate (see Chapter 20, Section I.B). Two reactions in the pentose phosphate pathway use transketolase. In the first, the two-carbon keto fragment from xylulose 5-P is transferred to ribose 5-P to form sedoheptulose 7-phosphate (sedoheptulose 7-P); and in the other, a two-carbon keto fragment (usually derived from xylulose 5-P) is transferred to erythrose 4-phosphate (erythrose 4-P) to form fructose 6-P. Transaldolase transfers a three-carbon keto fragment from sedoheptulose 7-P to glyceraldehyde 3-P to form erythrose 4-P and fructose 6-P (Fig. 29.9). The aldol

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CHAPTER 29 ■ PATHWAYS OF SUGAR METABOLISM

cleavage occurs between the two hydroxyl carbons adjacent to the keto group (on carbons 3 and 4 of the sugar). This reaction is similar to the aldolase reaction in glycolysis, and the enzyme uses an active amino group from the side chain of lysine to catalyze the reaction. The net result of the metabolism of 3 mol of ribulose 5-P in the pentose phosphate pathway is the formation of 2 mol of fructose 6-P and 1 mol of glyceraldehyde 3-P, which then continue through the glycolytic pathway with the production of NADH, ATP, and pyruvate. Because the pentose phosphate pathway begins with glucose 6-P and feeds back into the glycolytic pathway, it is sometimes called the hexose monophosphate (HMP) shunt (a shunt or a pathway for glucose 6-P). The reaction sequence starting from glucose 6-P, involving both the oxidative and nonoxidative phases of the pathway, is shown in Figure 29.10. 2.

The net energy yield from 3 mol of glucose 6-P metabolized through the pentose phosphate pathway and then the last portion of the glycolytic pathway is 6 mol of NADPH, 3 mol of CO2, 5 mol of NADH, 8 mol of ATP, and 5 mol of pyruvate. In contrast, the metabolism of 3 mol of glucose 6-P through glycolysis is 6 mol of NADH, 9 mol of ATP, and 6 mol of pyruvate.

GENERATION OF RIBOSE 5-PHOSPHATE FROM INTERMEDIATES OF GLYCOLYSIS

The reactions catalyzed by the epimerase, isomerase, transketolase, and transaldolase are all reversible reactions under physiologic conditions. Thus, ribose 5-P required for purine and pyrimidine synthesis can be generated from intermediates of the glycolytic pathway, as well as from the oxidative phase of the pentose phosphate pathway. The sequence of reactions that generates ribose 5-P from intermediates of glycolysis is as follows: (1) Fructose 6-P ⫹ glyceraldehyde 3-P (2) Erythrose 4-P ⫹ fructose 6-P

Transketolase

Transaldolase

(3) Sedoheptulose 7-P ⫹ glyceraldehyde 3-P

Erythrose 4-P ⫹ xylulose 5-P

Sedoheptulose 7-P ⫹ glyceraldehyde 3-P Transketolase

(4) 2 Xylulose 5-P

Epimerase

2 Ribulose 5-P

(5) 2 Ribulose 5-P

Isomerase

2 Ribose 5-P

CH2OH C

O

HO

C

H

H

C

OH

H

C

OH

H

C

OH

CH2OPO3 2– Sedoheptulose 7-phosphate

+

Ribose 5-P ⫹ xylulose 5-P

H O C H

Net equation: 2 Fructose 6-P ⫹ glyceraldehyde 3-P ↔ 3 Ribose 5-P

C. Role of the Pentose Phosphate Pathway in Generation of NADPH In general, the oxidative phase of the pentose phosphate pathway is the major source of NADPH in cells. NADPH provides the reducing equivalents for biosynthetic reactions and for oxidation–reduction reactions involved in protection against the toxicity of reactive oxygen species (ROS) (see Chapter 24). The glutathionemediated defense against oxidative stress is common to all cell types (including red blood cells), and the requirement for NADPH to maintain levels of reduced glutathione (GSH) probably accounts for the universal distribution of the pentose phosphate pathway among different types of cells. Figure 29.11 illustrates the importance of this pathway in maintaining the membrane integrity of the red blood

537

C

OH

CH2OPO3 2– Glyceraldehyde 3-phosphate Transaldolase

H O C H

C

OH

H

C

OH

CH2OPO3 2– Erythrose 4-phosphate

+ CH2OH

Doctors suspected that the underlying factor in the destruction of Al Martini’s red blood cells was an X-linked defect in the gene that codes for glucose-6-phosphate dehydrogenase. The red blood cell is dependent on this enzyme for a source of NADPH to maintain reduced levels of glutathione, one of its major defenses against oxidative stress (see Chapter 24). Glucose-6-phosphate dehydrogenase deficiency is the most common known enzymopathy, affecting approximately 7% of the world’s population and about 2% of the US population. Most glucose-6-phosphate dehydrogenase–deficient individuals are asymptomatic but can undergo an episode of hemolytic anemia if they are exposed to certain drugs, to certain types of infections, or if they ingest fava beans. When questioned, Al Martini replied that he did not know what a fava bean was and had no idea whether he was sensitive to them.

Lieberman_Ch29.indd 537

C

O

HO

C

H

H

C

OH

H

C

OH

CH2OPO3 2– Fructose 6-phosphate

FIG. 29.9. Transaldolase transfers a threecarbon fragment that contains an alcohol group next to a keto group.

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SECTION V ■ CARBOHYDRATE METABOLISM

Oxidative reactions 6 NADPH

3 CO2

3 Glucose 6-phosphate

3 Ribulose 5-phosphate Isomerase

Epimerase

Xylulose 5-phosphate

Ribose 5-phosphate

Transaldolase

Fructose 6-phosphate

Xylulose 5-phosphate

Nucleotide biosynthesis

Transketolase

Glyceraldehyde 3-phosphate

Epimerase

Nonoxidative reactions

Sedoheptulose 7-phosphate Erythrose 4-phosphate

Fructose 6-phosphate

Transketolase

Glyceraldehyde 3-phosphate

Glycolysis

FIG. 29.10. A balanced sequence of reactions in the pentose phosphate pathway. The interconversion of sugars in the pentose phosphate pathway results in conversion of 3 glucose 6-P to 6 NADPH, 3 CO2, 2 fructose 6-phosphate (fructose 6-P), and 1 glyceraldehyde 3-P.

Glucose Glucose 6-phosphate dehydrogenase deficiency

Glucose

Oxidant stress • Infections • Certain drugs • Fava beans

Glucose 6-phosphate

1

Erythrocyte

2

Hemolysis

4 Glucose 6-phosphate

Glucose 6-phosphate

NADP+

Glucose 6-phosphate dehydrogenase

Glycolysis 2 ATP

2 Pyruvate

6-Phosphogluconate NADH

Pentose phosphate pathway

2 GSH

3 Glutathione reductase

5

(ROS)

Glutathione peroxidase

GS-SG

NADPH + H+

HO•

H2O2

Heinz bodies

2 H2O

Met Hb

O2

–

Oxy Hb

2 Lactate

FIG. 29.11. Hemolysis caused by reactive oxygen species (ROS). (1) Maintenance of the integrity of the erythrocyte membrane depends on its ability to generate ATP and NADH from glycolysis. (2) NADPH is generated by the pentose phosphate pathway. (3) NADPH is used for the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Glutathione is necessary for the removal of H2O2 and lipid peroxides generated by ROS. (4) In the erythrocytes of healthy individuals, the continuous generation of superoxide ion from the nonenzymatic oxidation of hemoglobin provides a source of ROS. The glutathione defense system is compromised by glucose-6-phosphate dehydrogenase deficiency, infections, certain drugs, and the purine glycosides of fava beans. (5) As a consequence, Heinz bodies, aggregates of cross-linked hemoglobin, form on the cell membranes and subject the cell to mechanical stress as it tries to go through small capillaries. The action of the ROS on the cell membrane as well as mechanical stress from the lack of deformability result in hemolysis.

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cells. NADPH is also used for anabolic pathways, such as fatty acid synthesis, cholesterol synthesis, and fatty acid chain elongation (Table 29.1). It is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs. The highest concentrations of glucose-6-phosphate dehydrogenase are found in phagocytic cells where NADPH oxidase uses NADPH to form superoxide from molecular oxygen. The superoxide then generates hydrogen peroxide, which kills the microorganisms taken up by the phagocytic cells (see Chapter 24). The entry of glucose 6-P into the pentose phosphate pathway is controlled by the cellular concentration of NADPH. NADPH is a strong product inhibitor of glucose6-phosphate dehydrogenase, the first enzyme of the pathway. As NADPH is oxidized in other pathways, the product inhibition of glucose-6-phosphate dehydrogenase is relieved, and the rate of the enzyme is accelerated to produce more NADPH. In the liver, the synthesis of fatty acids from glucose is a major route of NADPH reoxidation. The synthesis of liver glucose-6-phosphate dehydrogenase, like the key enzymes of glycolysis and fatty acid synthesis, is induced by the increased insulin:glucagon ratio after a high-carbohydrate meal. A summary of the possible routes that glucose 6-P may follow using the pentose phosphate pathway is presented in Table 29.2.

Table 29.1 NADPH

539

Pathways that Require

Detoxification • Reduction of oxidized glutathione • Cytochrome P450 monooxygenases Reductive synthesis • Fatty acid synthesis • Fatty acid chain elongation • Cholesterol synthesis • Neurotransmitter synthesis • Deoxynucleotide synthesis • Superoxide synthesis

CLINICAL COMMENTS Candice Sucher. Hereditary fructose intolerance (HFI) is caused by a low level of fructose 1-phosphate (fructose 1-P) aldolase activity in aldolase B, an isozyme of fructose 1,6-bisphosphate aldolase that is also capable of cleaving fructose 1-P. In persons of European descent, the most common defect is a single missense mutation in exon 5 (G → C), resulting in an amino acid substitution (Ala → Pro). As a result of this substitution, a catalytically impaired aldolase B is synthesized in abundance. The exact prevalence of HFI in the United States is not established but is approximately 1 per 15,000 to 25,000 population. The disease is transmitted by an autosomal recessive inheritance pattern. When affected patients like Candice Sucher ingest fructose, fructose is converted to fructose 1-P. Because of the deficiency of aldolase B, fructose 1-P cannot be further metabolized to dihydroxyacetone phosphate and glyceraldehyde and accumulates in those tissues that have fructokinase (liver, kidney, and small intestine). Fructose is detected in the urine with the reducing sugar test (see the methods comment in Chapter 5). A DNA screening test (based on the generation of a new restriction site by the mutation) now provides a safe method to confirm a diagnosis of HFI.

Table 29.2 Cellular Needs Dictate the Direction of the Pentose Phosphate Pathway Reactions Cellular Need

Direction of Pathway

NADPH only

Oxidative reactions produce NADPH; nonoxidative reactions convert ribulose 5-P to glucose 6-P to produce more NADPH. Oxidative reactions produce NADPH and ribulose 5-P; the isomerase converts ribulose 5-P to ribose 5-P. Only the nonoxidative reactions. High NADPH inhibits glucose-6phosphate dehydrogenase, so transketolase and transaldolase are used to convert fructose 6-P and glyceraldehyde 3-P to ribose 5-P. Both the oxidative and nonoxidative reactions are used. The oxidative reactions generate NADPH and ribulose 5-P. The nonoxidative reactions convert the ribulose 5-P to fructose 6-P and glyceraldehyde 3-P, and glycolysis converts these intermediates to pyruvate.

NADPH ⫹ ribose 5-P Ribose 5-P only NADPH and pyruvate

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SECTION V ■ CARBOHYDRATE METABOLISM

In infants and small children, the major symptoms include poor feeding, vomiting, intestinal discomfort, and failure to thrive. The greater the ingestion of dietary fructose, the more severe is the clinical reaction. The result of prolonged ingestion of fructose is ultrastructural changes in the liver and kidney that result in hepatic and renal failure. HFI is usually a disease of infancy because adults with fructose intolerance who have survived avoid the ingestion of fruits, table sugar, and other sweets. Before the metabolic toxicity of fructose was appreciated, substitution of fructose for glucose in intravenous solutions and of fructose for sucrose in enteral tube feeding or diabetic diets was frequently recommended. (Enteral tube feeding refers to tubes placed into the gut; parenteral tube feeding refers to tubes placed into a vein, feeding intravenously.) Administration of intravenous fructose to patients with diabetes mellitus or other forms of insulin resistance avoided the hyperglycemia found with intravenous glucose, possibly because fructose metabolism in the liver bypasses the insulin-regulated step at phosphofructokinase-1 (PFK-1). However, because of the unregulated flow of fructose through glycolysis, intravenous fructose feeding frequently resulted in lactic acidosis (see Fig. 29.3). In addition, the fructokinase reaction is very rapid, and tissues became depleted of adenosine triphosphate (ATP) and phosphate when large quantities of fructose were metabolized over a short period. This led to cell death. Fructose is less toxic in the diet or in enteral feeding because of the relatively slow rate of fructose absorption. Erin Galway. Erin Galway has galactosemia, which is caused by a deficiency of galactose 1-phosphate (galactose 1-P) uridylyltransferase; it is one of the most common genetic diseases. Galactosemia is an autosomal recessive disorder of galactose metabolism that occurs in about 1 in 60,000 newborns. All of the states in the United States screen newborns for this disease because failure to begin immediate treatment results in mental retardation. Failure to thrive is the most common initial clinical symptom. Vomiting or diarrhea occurs in most patients, usually starting within a few days of beginning milk ingestion. Signs of deranged liver function, jaundice or hepatomegaly, are present almost as frequently after the first week of life. The jaundice of intrinsic liver disease may be accentuated by the severe hemolysis in some patients. Cataracts have been observed within a few days of birth. Management of patients requires eliminating galactose from the diet. Failure to eliminate this sugar results in progressive liver failure and death. In infants, artificial milk made from casein or soybean hydrolysate is used. Al Martini. Al Martini’s pus culture sent on the second day of his admission for acute alcoholism grew out Staphylococcus aureus. This organism has become resistant to a variety of antibiotics, so TMP/sulfa treatment was initiated. Unfortunately, it appeared that Mr. Martini had suffered an acute hemolysis (lysis or destruction of some of his red blood cells), probably induced by exposure to the sulfa drug and his infection to S. aureus. The hemoglobin that escaped from the lysed red blood cells was filtered by his kidneys and appeared in his urine. By mechanisms that are not fully delineated, certain drugs (such as sulfa drugs and antimalarials), a variety of infectious agents, and exposure to fava beans can cause red blood cell destruction in individuals with a genetic deficiency of glucose6-phosphate dehydrogenase. Presumably, these patients cannot generate enough reduced NADPH to defend against the reactive oxygen species (ROS). Although erythrocytes lack most of the other enzymatic sources of NADPH for the glutathione antioxidant system, they do have the defense mechanisms provided by the antioxidant vitamins E and C and catalase. Thus, individuals who are not totally

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541

deficient in glucose-6-phosphate dehydrogenase remain asymptomatic unless an additional oxidative stress such as an infection generates additional oxygen radicals. Some drugs, such as the antimalarial primaquine and the sulfonamide that Mr. Martini is taking, affect the ability of red blood cells to defend against oxidative stress. Fava beans, which look like fat string beans and are sometimes called broad beans, contain the purine glycosides vicine and isouramil. These compounds react with glutathione. It has been suggested that cellular levels of reduced glutathione (GSH) decrease to such an extent that critical sulfhydryl groups in some key proteins cannot be maintained in reduced form. The highest prevalence rates for glucose-6-phosphate dehydrogenase deficiency are found in tropical Africa and Asia, in some areas of the Middle East and the Mediterranean, and in Papua New Guinea. The geographic distribution of this deficiency is similar to that of sickle cell trait and is probably also related to the relative resistance it confers against the malaria parasite. Because individuals with this deficiency are asymptomatic unless they are exposed to an “oxidant challenge,” the clinical course of the hemolytic anemia is usually self-limited if the causative agent is removed. However, genetic polymorphism accounts for a substantial variability in the severity of the disease. Severely affected patients may have a chronic hemolytic anemia and other sequelae even without known exposure to drugs, infection, and other causative factors. In such patients, neonatal jaundice is also common and can be severe enough to cause death.

BIOCHEMICAL COMMENTS Xylulose 5-phosphate (xylulose 5-P), in addition to its key role as an intermediate in the nonoxidative reactions of the hexose monophosphate (HMP) shunt pathway, also has an important role in regulating gene transcription. When excess carbohydrate is present in the diet, the liver has the responsibility for converting the excess carbohydrate into lipid (triacylglycerol; see Chapter 32) for storage. The large influx of glucose into the liver leads to an increase in xylulose 5-P levels. Xylulose 5-P activates protein phosphatase 2A (PP2A), which dephosphorylates, among several proteins, a cytoplasmic transcription factor, ChREBP (carbohydrate response element-binding protein). The dephosphorylation of ChREBP leads to its nuclear translocation where it binds to appropriate promoter elements and increases the transcription of its target genes. Such genes include, within the liver, the enzyme responsible for the terminal step of glycolysis, pyruvate kinase, and four enzymes involved in the synthesis of fatty acids (malic enzyme, citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase). This promotes fatty acid biosynthesis, triglyceride synthesis, and the export of the triglycerides from the liver in the form of very low-density lipoproteins (VLDLs). The transcription factor ChREBP is phosphorylated either by protein kinase A (which is activated by glucagon or epinephrine, signaling a need to oxidize fatty acids, and not to synthesize them) or an adenosine monophosphate (AMP)activated protein kinase (indicating low energy levels, and a need for fatty acid oxidation). The AMP-activated protein kinase is discussed in greater detail in Section VI of this book. When it is phosphorylated, ChREBP leaves the nucleus and associates with scaffolding proteins in the cytoplasm where it waits to be activated via dephosphorylation. Thus, regulation of ChREBP is related to the feeding and fasting cycles and results in alterations in enzyme levels that support the fed state. This cycle, of which xylulose 5-P is a key regulator, is just one of the many mechanisms established to coordinate carbohydrate and fatty acid metabolism. You will learn more of these mechanisms in the next section of this book.

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SECTION V ■ CARBOHYDRATE METABOLISM

Table 29.3

Diseases Discussed in Chapter 29

Disease or Disorder

Environmental or Genetic

Hereditary fructose intolerance

Genetic

Galactosemia

Genetic

Glucose-6-phosphate dehydrogenase deficiency

Genetic, X-linked

Comments Lack of aldolase B, leading to an accumulation of fructose 1-phosphate (fructose 1-P) after fructose ingestion. The increased levels of fructose 1-P interfere with glycogen metabolism and can lead to hypoglycemia. Mutations in either galactokinase or galactose 1-phosphate (galactose 1-P) uridylyltransferase, leading to elevated galactose and/or galactose 1-P levels. This can lead to cataract formation (high galactose) and mental retardation (elevated galactose 1-P levels) if not treated early in life. Lack of glucose-6-phosphate dehydrogenase activity leads to hemolytic anemia in the presence of strong oxidizing agents.

Key Concepts •

•

• • •

• •

Fructose is ingested principally as the monosaccharide or as part of sucrose. Fructose metabolism generates fructose 1-phosphate (fructose 1-P), which is then converted to intermediates of the glycolytic pathway. Galactose is ingested principally as lactose, which is converted to glucose and galactose in the intestine. Galactose metabolism generates, first, galactose 1-phosphate (galactose 1-P), which is converted to uridine diphosphate (UDP)-galactose. The end product is glucose 1-phosphate (glucose 1-P), which is isomerized to glucose 6-phosphate (glucose 6-P), which then enters glycolysis. The energy yield through glycolysis for both fructose and galactose is the same as for glucose metabolism. The pentose phosphate pathway consists of both oxidative and nonoxidative reactions. The oxidative steps of the pentose phosphate pathway generate NADPH and ribulose 5-phosphate (ribulose 5-P) from glucose 6-P. Ribulose 5-P is converted to ribose 5-phosphate (ribose 5-P) for nucleotide biosynthesis. NADPH is used as reducing power for biosynthetic pathways. The nonoxidative steps of the pentose phosphate pathway reversibly convert five-carbon sugars to fructose 6-phosphate (fructose 6-P) and glyceraldehyde 3-phosphate (glyceraldehyde 3-P). Table 29.3 summarizes diseases discussed in this chapter.

REVIEW QUESTIONS—CHAPTER 29 1.

Hereditary fructose intolerance (HFI) is a rare recessive genetic disease that is most commonly caused by a mutation in exon 5 of the aldolase B gene. The mutation fortuitously creates a new ahaII recognition sequence. To test for the mutation, DNA was extracted from a wife, husband, and their two children, Jack and Jill. The DNA for exon 5 of the aldolase B gene was amplified by polymerase chain reaction (PCR), cleaved with ahaII, treated with alkali, subjected to electrophoresis on an agarose gel, and stained with a dye that binds to DNA. Which of the following conclusions can be made from the data presented? A. Both of the children have the disease. B. Neither of the children has the disease. C. Jill has the disease, Jack does not. D. Jack has the disease, Jill does not. E. There is not enough information to make a determination.

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306 bp

183 bp

123 bp

Wife

Husband

Jack

Jill

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CHAPTER 29 ■ PATHWAYS OF SUGAR METABOLISM

2.

3.

On examining the gel himself, the husband became concerned that he might not be the biologic father of one or both of the children. From the pattern on the gel, you can reasonably conclude which of the following? A. He is probably not Jill’s father. B. He is probably not Jack’s father. C. He could be the father of both children. D. He is probably not the father of either child. E. There is not enough information to make a determination. An alcoholic is brought to the emergency room in a hypoglycemic coma. Because alcoholics are frequently malnourished, which of the following enzymes can be used to test for a thiamine deficiency? A. Aldolase B. Transaldolase C. Transketolase D. Glucose-6-phosphate dehydrogenase E. UDP-galactose epimerase

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543

4.

Intravenous fructose feeding can lead to lactic acidosis caused by which of the following? A. Bypassing the regulated pyruvate kinase step B. Bypassing the regulated phosphofructokinase-1 (PFK-1) step C. Allosterically activating aldolase B D. Allosterically activating lactate dehydrogenase E. Increasing the [ATP]:[ADP] ratio in liver

5.

The polyol pathway of sorbitol production and the hexose monophosphate (HMP) shunt pathway are linked by which of the following? A. The HMP shunt produces 6-phosphogluconate, an intermediate in the polyol pathway. B. The HMP shunt produces NADPH, which is required for the polyol pathway. C. The HMP shunt produces ribitol, an intermediate of the polyol pathway. D. Both pathways use glucose as the starting material. E. Both pathways use fructose as the starting material.

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30

Synthesis of Glycosides, Lactose, Glycoproteins, and Glycolipids Many of the pathways for interconversion of sugars or the formation of sugar derivatives use activated sugars attached to nucleotides. Both uridine diphosphate (UDP)-glucose and UDP-galactose are used for glycosyltransferase reactions in many systems. Lactose, for example, is synthesized from UDPgalactose and glucose in the mammary gland. UDP-glucose also can be oxidized to form UDP-glucuronate, which is used to form glucuronide derivatives of bilirubin and xenobiotic compounds. Glucuronide derivatives are generally more readily excreted in urine or bile than the parent compound. In addition to serving as fuel, carbohydrates are often found in glycoproteins (carbohydrate chains attached to proteins) and glycolipids (carbohydrate chains attached to lipids). Nucleotide sugars are used to donate sugar residues for the formation of the glycosidic bonds in both glycoproteins and glycolipids. These carbohydrate groups have many different types of functions. Glycoproteins contain short chains of carbohydrates (oligosaccharides) that are usually branched. These oligosaccharides are generally composed of glucose, galactose, and their amino derivatives. In addition, mannose, L-fucose, and N-acetylneuraminic acid (NANA) are frequently present. The carbohydrate chains grow by the sequential addition of sugars to a serine or threonine residue of the protein. Nucleotide sugars are the precursors. Branched carbohydrate chains also may be attached to the amide nitrogen of asparagine in the protein. In this case, the chains are synthesized on dolicholphosphate and subsequently transferred to the protein. Glycoproteins are found in mucus, in the blood, in compartments within the cell (such as lysosomes), in the extracellular matrix, and embedded in the cell membrane with the carbohydrate portion extending into the extracellular space. Glycolipids belong to the class of sphingolipids. They are synthesized from nucleotide sugars that add monosaccharides sequentially to the hydroxymethyl group of the lipid ceramide (related to sphingosine). They often contain branches of NANA produced from cytidine monophosphate (CMP)-NANA. They are found in the cell membrane with the carbohydrate portion extruding from the cell surface. These carbohydrates, as well as some of the carbohydrates of glycoproteins, serve as cell recognition factors.

544

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CHAPTER 30 ■ SYNTHESIS OF GLYCOSIDES, LACTOSE, GLYCOPROTEINS, AND GLYCOLIPIDS

THE WAITING ROOM To help support herself through medical school, Erna Nemdy works evenings in a hospital blood bank. She is responsible for ensuring that compatible donor blood is available to patients who need blood transfusions. As part of her training, Erna has learned that the external surfaces of all blood cells contain large numbers of antigenic determinants. These determinants are often glycoproteins or glycolipids that differ from one individual to another. As a result, all blood transfusions expose the recipient to many foreign immunogens. Most of these, fortunately, do not induce antibodies or they induce antibodies that elicit little or no immunologic response. For routine blood transfusions, therefore, tests are performed only for the presence of antigens that determine whether the patient’s blood type is A, B, AB, or O, and Rh(D)-positive or -negative.

545

Blood typing in a clinical lab uses antibodies that recognize either the A antigen, the B antigen, or the Rh(D) antigen. Each antigen is distinctive, in part, because of the different carbohydrate chains attached to the protein. The blood sample is mixed with each antibody individually. If cell clumping (agglutination) occurs, the red blood cells are expressing the carbohydrate that is recognized by the antibody (recall from Chapter 7 that antibodies are bivalent; the agglutination occurs because one arm of the antibody binds to antigen on one cell, while the other arm binds to antigen on a second cell, thereby bringing the cells together). If neither the A nor B antibodies causes agglutination, the blood type is O, indicating a lack of either antigen.

Jay Sakz’s psychomotor development has become progressively more abnormal (see Chapter 15). At 2 years of age, he is obviously mentally retarded and nearly blind. His muscle weakness has progressed to the point that he cannot sit up or even crawl. As the result of a weak cough reflex, he is unable to clear his normal respiratory secretions and has had recurrent respiratory infections.

I.

INTERCONVERSIONS INVOLVING NUCLEOTIDE SUGARS

Activated sugars attached to nucleotides are converted to other sugars, oxidized to sugar acids, and joined to proteins, lipids, or other sugars through glycosidic bonds.

A. Reactions of UDP-Glucose Uridine diphosphate (UDP)-glucose is an activated sugar nucleotide that is a precursor of glycogen and lactose, UDP-glucuronate and glucuronides, and the carbohydrate chains in proteoglycans, glycoproteins, and glycolipids (Fig. 30.1). Both proteoglycans and glycosaminoglycans are discussed further in Chapter 49.

Glucose

Glycogen

Glucose 6-phosphate

Glucose 1-phosphate

Proteoglycans, Glycoproteins, Glycolipids

UDP-glucose

UDP-glucuronate

UTP UDP-galactose Lactose Glucose

FIG. 30.1. An overview of UDP-glucose metabolism. The activated glucose moiety of UDP-glucose can be attached by a glycosidic bond to other sugars, as in glycogen or the sugar oligosaccharide and polysaccharide side chains of proteoglycans, glycoproteins, and glycolipids. UDP-glucose also can be oxidized to UDP-glucuronate or epimerized to UDP-galactose, a precursor of lactose.

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SECTION V ■ CARBOHYDRATE METABOLISM

In the synthesis of many of the carbohydrate portions of these compounds, a sugar is transferred from the nucleotide sugar to an alcohol or other nucleophilic group to form a glycosidic bond (Fig. 30.2). The use of UDP as a leaving group in this reaction provides the energy for formation of the new bond. The enzymes that form glycosidic bonds are sugar transferases (e.g., glycogen synthase is a glucosyltransferase). Transferases are also involved in the formation of the glycosidic bonds in bilirubin glucuronides, proteoglycans, and lactose.

UDP-glucose Protein–OH Glycosyltransferase

UDP CH2OH O H H OH H HO O

C

H

H

OH

O NH

CH2

CH C

O

Glycosylated protein

FIG. 30.2. Glycosyltransferases. These enzymes transfer sugars from nucleotide sugars to nucleophilic amino acid residues on proteins, such as the hydroxyl group of serine or the amide group of asparagine. Other transferases transfer specific sugars from a nucleotide sugar to a hydroxyl group of other sugars. The bond formed between the anomeric carbon of the sugar and the nucleophilic group of another compound is a glycosidic bond.

What is the difference in structure between 6-phosphogluconate and glucuronic acid?

A failure of the liver to transport, store, or conjugate bilirubin results in the accumulation of unconjugated bilirubin in the blood. Jaundice (or icterus), the yellowish tinge to the skin and the whites of the eyes (sclera) experienced by Erin Galway, occurs when plasma becomes supersaturated with bilirubin (2 to 2.5 mg/dL) and the excess diffuses into tissues. When bilirubin levels are measured in the blood (see Chapter 6), one can measure either indirect bilirubin (this is the nonconjugated form of bilirubin, which is bound to albumin), direct bilirubin (the conjugated, water-soluble form), or total bilirubin (the sum of the direct and indirect levels). If total bilirubin levels are high, then a determination of direct and indirect bilirubin is needed to appropriately determine a cause for the elevation of total bilirubin.

B. UDP-Glucuronate: A Source of Negative Charges One of the major routes of UDP-glucose metabolism is the formation of UDPglucuronate, which serves as a precursor of other sugars and of glucuronides (Fig. 30.3). Glucuronate is formed by the oxidation of the alcohol on carbon 6 of glucose to an acid (through two oxidation states) by a NAD-dependent dehydrogenase (Fig. 30.4). Glucuronate is also present in the diet and can be formed from the degradation of inositol (the sugar alcohol that forms inositol trisphosphate[IP3]), an intracellular second messenger for many hormones.

C. Formation of Glucuronides The function of glucuronate in the excretion of bilirubin, drugs, xenobiotics, and other compounds containing a hydroxyl group is to add negative charges and increase their solubility. Bilirubin is a degradation product of heme that is formed in the reticuloendothelial system and is only slightly soluble in plasma. It is transported to the liver bound to albumin. In the liver, glucuronate residues are transferred from UDP-glucuronate to two carboxyl groups on bilirubin, sequentially forming bilirubin monoglucuronide and bilirubin diglucuronide, the “conjugated” forms of bilirubin (Fig. 30.5). The more soluble bilirubin diglucuronide (as compared with unconjugated bilirubin) is then actively transported into the bile for excretion. Many xenobiotics, drugs, steroids, and other compounds with hydroxyl groups and low solubility in water are converted to glucuronides in a similar fashion by glucuronyltransferases present in the endoplasmic reticulum (ER) and cytoplasm of the liver and kidney (Table 30.1). This is one of the major conjugation pathways for excretion of these compounds.

Bilirubin diglucuronide UDP-glucose

Bilirubin

UDP-glucuronate

Proteoglycans, glycoproteins

Glucuronides

Iduronate (GAGs)

Steroids Drugs Xenobiotics Bilirubin

OH

UDP-xylose (GAGs)

FIG. 30.3. Metabolic routes of UDP-glucuronate. UDP-glucuronate is formed from UDPglucose (shown in black). Glucuronate from UDP-glucuronate is incorporated into glycosaminoglycans (GAGs), where certain of the glucuronate residues are converted to iduronate (see Chapter 49). UDP-glucuronate is a precursor of UDP-xylose, another sugar residue incorporated into glycosaminoglycans. Glucuronate is also transferred to the carboxyl groups of bilirubin or the alcohol groups of steroids, drugs, and xenobiotics to form glucuronides. The “-ide” in the name glucuronide denotes that these compounds are glycosides. Xenobiotics are pharmacologically, endocrinologically, or toxicologically active substances that are not produced endogenously and, therefore, are foreign to an organism. Drugs are examples of xenobiotics.

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COO

O HO

CH3

O O

C

CH2

CH3

CH2 –

HO

O O

CH2

CH2

OH

CH2 NH CH

CH3 CH2

CH

N

2NAD+ UDP-glucose dehydrogenase

2NADH + 2H+

OH Bilirubin

Bilirubin diglucuronide FIG. 30.5. Formation of bilirubin diglucuronide. A glycosidic bond is formed between the anomeric hydroxyl of glucuronate and the carboxylate groups of bilirubin. The addition of the hydrophilic carbohydrate group and the negatively charged carboxyl group of the glucuronide increases the water solubility of the conjugated bilirubin and allows the otherwise insoluble bilirubin to be excreted in the urine or bile. The hydrogen atoms on the sugars have been omitted from the figure for clarity.

HO

D. Synthesis of UDP-Galactose and Lactose from Glucose Lactose is synthesized from UDP-galactose and glucose (Fig. 30.6). However, galactose is not required in the diet for lactose synthesis because galactose can be synthesized from glucose. 1.

CONVERSION OF GLUCOSE TO GALACTOSE

Galactose and glucose are epimers; they differ only in the stereochemical position of one hydroxyl group, at carbon 4. Thus, the formation of UDP-galactose from UDP-glucose is an epimerization (Fig. 30.7). The epimerase does not actually transfer the hydroxyl group; it oxidizes the hydroxyl to a ketone by transferring electrons to NAD, and then donates electrons back to re-form the alcohol group on the other side of the carbon. 2.

LACTOSE SYNTHESIS

Lactose is unique in that it is synthesized only in the mammary gland of the adult female for short periods during lactation. Lactose synthase, an enzyme

Table 30.1 Examples of Compounds Degraded and Excreted as Urinary Glucuronides Estrogen (female sex hormone) Progesterone (steroid hormone) Triiodothyronine (thyroid hormone) Acetylaminofluorene (xenobiotic carcinogen) Meprobamate (drug for sleep) Morphine (painkiller)

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O – O O

OH

O

UDP

OH UDP-Glucuronate UDP-glucuronate transferase (microsomal)

C

Glucuronate, once formed, can reenter the pathways of glucose metabolism through reactions that eventually convert it to D-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. In most mammals other than humans, an intermediate of this pathway is the precursor of ascorbic acid (vitamin C). Humans, however, are deficient in this pathway and cannot synthesize vitamin C.

UDP

OH

C Glucuronates

O

UDP-Glucose

CH3

C

OH OH

HO

NH

CH2

OH COO

N

CH CH

OH

O

CH2OH O

OH

–

547

HO

ROH (xenobiotics, drugs or other OH)

O – O O

O

R + UDP

OH OH

Glucuronide Bile or urine

FIG. 30.4. Formation of glucuronate and glucuronides. A glycosidic bond is formed between the anomeric hydroxyl of glucuronate (at carbon 1) and the hydroxyl group of a nonpolar compound. The negatively charged carboxyl group of the glucuronate increases the water solubility and allows otherwise nonpolar compounds to be excreted in the urine or bile. The hydrogen atoms have been omitted from the figure for clarity. 6-Phosphogluconate is produced by the first oxidative reaction in the pentose phosphate pathway, in which carbon 1 of glucose is oxidized to a carboxylate. In contrast, glucuronic acid is oxidized at carbon 6 to the carboxylate form. High concentrations of galactose 1-phosphate inhibit phosphoglucomutase, the enzyme that converts glucose 6-phosphate to glucose 1-phosphate. How can this inhibition account for the hypoglycemia and jaundice that accompany galactose1-phosphate uridylyltransferase deficiency?

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Many (60%) full-term newborns develop jaundice, termed neonatal jaundice. This is usually caused by an increased destruction of red blood cells after birth (the fetus has an unusually large number of red blood cells) and an immature bilirubin conjugating system in the liver. This leads to elevated levels of nonconjugated bilirubin, which is deposited in hydrophobic (fat) environments. If bilirubin levels reach a certain threshold at the age of 48 hours, the newborn is a candidate for phototherapy, in which the child is placed under lamps that emit light between the wavelengths of 425 and 475 nm. Bilirubin absorbs this light, undergoes chemical changes, and becomes more watersoluble. Usually, within a week of birth, the newborn’s liver can handle the load generated from red blood cell turnover. The inhibition of phosphoglucomutase by galactose 1-phosphate results in hypoglycemia by interfering with both the formation of uridine diphosphate (UDP)-glucose (the glycogen precursor) and the degradation of glycogen back to glucose 6-phosphate. Ninety percent of glycogen degradation leads to glucose 1-phosphate, which can only be converted to glucose 6-phosphate by phosphoglucomutase. When phosphoglucomutase activity is inhibited, less glucose 6-phosphate production occurs, and hence, less glucose is available for export. Thus, the stored glycogen is only approximately 10% efficient in raising blood glucose levels, and hypoglycemia results. UDP-glucose levels are reduced because glucose 1-phosphate is required to synthesize UDP-glucose, and in the absence of phosphoglucomutase activity, glucose 6-phosphate (derived from either the glucokinase reaction or gluconeogenesis) cannot be converted to glucose 1-phosphate. This prevents the formation of UDP-glucuronate, which is necessary to convert bilirubin to the diglucuronide form for transport into the bile. Bilirubin accumulates in tissues, giving them a yellow color (jaundice).

Glucose 1-phosphate UTP PPi UDP-glucose Epimerase

UDP-galactose D-glucose Lactose synthase (acceptor) (galactosyltransferase + ␣-lactalbumin) UDP

Lactose CH2OH O HO

CH2OH O OH O

OH

OH

OH

OH

FIG. 30.6. Lactose synthesis. Lactose is a disaccharide composed of galactose and glucose. UDP-galactose for the synthesis of lactose in the mammary gland is usually formed from the epimerization of UDP-glucose. Lactose synthase catalyzes the attack of the C4 alcohol group of glucose on the anomeric carbon of the galactose, releasing UDP and forming a glycosidic bond. Lactose synthase is composed of a galactosyltransferase and ␣-lactalbumin, which is a regulatory subunit.

present in the ER of the lactating mammary gland, catalyzes the last step in lactose biosynthesis: the transfer of galactose from UDP-galactose to glucose (see Fig. 30.6). Lactose synthase has two protein subunits: a galactosyltransferase and ␣-lactalbumin. ␣-Lactalbumin is a modifier protein synthesized after parturition (childbirth) in response to the hormone prolactin. This enzyme subunit lowers the Km of the galactosyltransferase for glucose from 1,200 to 1 mM, thereby increasing

CH2OH O HO

OH

O

UDP

OH UDP-Glucose UDP-glucose 4-epimerase (NAD+)

CH2OH O HO OH

O

UDP

OH

A pregnant woman who was extremely lactose-intolerant asked her physician if she would still be able to breastfeed her infant even though she could not drink milk or dairy products. What advice should she be given?

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UDP-Galactose

FIG. 30.7. Epimerization of UDP-glucose to UDP-galactose. The epimerization of glucose to galactose occurs on UDP-sugars. The epimerase uses NAD to oxidize the alcohol to a ketone, and then reduces the ketone back to an alcohol. The reaction is reversible; glucose being converted to galactose forms galactose for lactose synthesis, and galactose being converted to glucose is part of the pathway for the metabolism of dietary galactose. The hydrogen atoms have been omitted for clarity.

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the rate of lactose synthesis. In the absence of ␣-lactalbumin, galactosyltransferase transfers galactosyl units to glycoproteins.

E. Formation of Sugars for Glycolipid and Glycoprotein Synthesis The transferases that produce the oligosaccharide and polysaccharide side chains of glycolipids and attach sugar residues to proteins are specific for the sugar moiety and for the donating nucleotide (e.g., UDP, cytidine monophosphate [CMP], or guanosine diphosphate [GDP]). Some of the sugar nucleotides used for glycoprotein, proteoglycan (see Chapter 49), and glycolipid formation are listed in Table 30.2. They include the derivatives of glucose and galactose that we have already discussed, as well as acetylated amino sugars and derivatives of mannose. The reason for the large variety of sugars attached to proteins and lipids is that they have relatively specific and different functions, such as targeting a protein toward a membrane; providing recognition sites on the cell surface for other cells, hormones, or viruses or acting as lubricants or molecular sieves (see Chapter 49). The pathways for use and formation of many of these sugars are summarized in Figure 30.8. Note that many of the steps are reversible, so that glucose and other dietary sugars enter a common pool from which the diverse sugars can be formed. The amino sugars are all derived from glucosamine 6-phosphate. To synthesize glucosamine 6-phosphate, an amino group is transferred from the amide of glutamine to fructose 6-phosphate (Fig. 30.9). Amino sugars, such as glucosamine, can then be N-acetylated by an acetyltransferase. N-acetyltransferases are present in the ER and cytosol and provide another means of chemically modifying sugars, metabolites, drugs, and xenobiotic compounds. Individuals may vary greatly in their capacity for acetylation reactions. Mannose is found in the diet in small amounts. Like galactose, it is an epimer of glucose, and mannose and glucose are interconverted by epimerization reactions at carbon 2. The interconversion can take place either at the level of fructose 6-phosphate to mannose 6-phosphate or at the level of the derivatized sugars (see Fig. 30.8). N-acetylmannosamine is the precursor of N-acetylneuraminic acid (NANA, a sialic acid) and GDP-mannose is the precursor of GDP-fucose (see Fig. 30.8). The negative charge on NANA is obtained by the addition of a threecarbon carboxyl moiety from phosphoenolpyruvate.

549

Although the lactose in dairy products is a major source of galactose, the ingestion of lactose is not required for lactation. UDP-galactose in the mammary gland is derived principally from the epimerization of glucose. Dairy products are, however, a major dietary source of Ca2, so breastfeeding mothers need increased Ca2 from another source. Table 30.2 Examples of Sugar Nucleotides That Are Precursors for Transferase Reactions UDP-glucose UDP-galactose UDP-glucuronic acid UDP-xylose UDP-N-acetylglucosamine UDP-N-acetylgalactosamine CMP-N-acetylneuraminic acid GDP-fucose GDP-mannose UDP, uridine diphosphate; CMP, cytidine monophosphate; GDP, guanosine diphosphate.

II. GLYCOPROTEINS A. Structure and Function Glycoproteins contain short carbohydrate chains covalently linked to either serine/threonine or asparagine residues in the protein. These oligosaccharide chains are often branched and they do not contain repeating disaccharides (Fig. 30.10). Most proteins in the blood are glycoproteins. They serve as hormones, antibodies, enzymes (including those of the blood clotting cascade), and as structural components of the extracellular matrix. Collagen contains galactosyl units and disaccharides composed of galactosyl-glucose attached to hydroxylysine residues (see Chapter 49). The secretions of mucus-producing cells, such as salivary mucin, are glycoproteins (Fig. 30.11). Although most glycoproteins are secreted from cells, some are segregated in lysosomes, where they serve as the lysosomal enzymes that degrade various types of cellular and extracellular material. Other glycoproteins are produced like secretory proteins, but hydrophobic regions of the protein remain attached to the cell membrane, and the carbohydrate portion extends into the extracellular space (Fig. 30.12) (also see Chapter 15, Section I). These glycoproteins serve as receptors for compounds such as hormones, as transport proteins, and as cell attachment and cell–cell recognition sites. Bacteria and viruses also bind to these sites.

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By identifying the nature of antigenic determinants on the surface of the donor’s red blood cells, Erna Nemdy is able to classify the donor’s blood as belonging to certain specific blood groups. These antigenic determinants are located in the oligosaccharides of the glycoproteins and glycolipids of the cell membranes. The most important blood group in humans is the ABO group, which comprises two antigens: A and B. Individuals with the A antigen on their cells belong to blood group A, those with B antigen belong to group B, and those with both A and B antigens belong to group AB. The absence of both the A and B antigens results in blood type O (see Fig. 30.17).

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SECTION V ■ CARBOHYDRATE METABOLISM

Glucose

Glucose 6-phosphate

UDP-Glucuronic acid Glucose 1-phosphate

Iduronic acid Glycosaminoglycans

UDP-Glucose UDP-Xylose UDP-Galactose

Mannose

Fructose 6-phosphate

Mannose 6-phosphate

Glutamine

Glutamate Glucosamine 6-phosphate

Galactose 1-phosphate Galactose

Mannose 1-phosphate UTP Glucosamine-1-P

GDP-Mannose

Glycoproteins (Asn-linked)

Glycolipids Glycoproteins

GDP-4-Keto-6 deoxymannose

GDP-Fucose

UDP-Glucosamine

Fucose 1-phosphate

Glycosaminoglycans (e.g., heparin)

Acetyl-CoA N-Acetylglucosamine 6-phosphate

UDPglucose

N-Acetylglucosamine

Fucose

N-Acetylglucosamine 1-phosphate

Glycosaminoglycans (hyaluronic acid), glycoproteins

UDP-N-Acetylglucosamine

N-Acetylmannosamine

N-Acetylmannosamine 6-phosphate Phosphoenolpyruvate

Glycosaminoglycans (chondroitins), glycoproteins

UDP-N-Acetylgalactosamine

N-Acetylneuraminic acid 9-phosphate

N-Acetylgalactosamine 1-phosphate

N-Acetylneuraminic acid (Sialic acid)

Galactosamine

CMP-N-Acetylneuraminic acid

Gangliosides, glycoproteins

FIG. 30.8. Pathways for the interconversion of sugars. All of the different sugars found in glycosaminoglycans, gangliosides, and other compounds in the body can be synthesized from glucose. Dietary glucose, fructose, galactose, mannose, and other sugars enter a common pool from which other sugars are derived. The activated sugar is transferred from the nucleotide sugar, shown in orange boxes, to form a glycosidic bond with another sugar or amino acid residue. The green box next to each nucleotide sugar lists some of the compounds that contain the sugar. Iduronic acid, in the upper right corner of the diagram, is formed only after glucuronic acid is incorporated into a glycosaminoglycan (which is discussed in more detail in Chapter 49).

B. Synthesis The protein portion of glycoproteins is synthesized on the ER. The carbohydrate chains are attached to the protein in the lumen of the ER and the Golgi complex. In some cases, the initial sugar is added to a serine or a threonine residue in the protein, and the carbohydrate chain is extended by the sequential addition of sugar residues to the nonreducing end. As seen in Table 30.2, UDP-sugars are the precursors for

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Fructose-6-P NANA

NANA

Gal

Gal

GlcNAc

GlcNAc

Man

Glutamine O ( C NH2 ) Glutamate O ( C O– )

Man Glucosamine-6-P

Man

GlcNAc

Acetyl CoA N-acetyltransferase

GlcNAc CoASH Fuc

GlcNAc Asn

CH2O P O

Protein chain HO

FIG. 30.10. An example of a branched glycoprotein. NANA, N-acetylneuraminic acid; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose.

OH

OH O

N H

C CH3

N-Acetylglucosamine-6-P

the addition of four of the seven sugars that are usually found in glycoproteins— glucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. GDP-sugars are the precursors for the addition of mannose and L-fucose, and CMP-NANA is the precursor for NANA. Dolichol phosphate (Fig. 30.13) (which is synthesized from isoprene units, as discussed in Chapter 34) is involved in transferring branched sugar chains to the amide nitrogen of asparagine residues. Sugars are removed and added as the glycoprotein moves from the ER through the Golgi complex (Fig. 30.14). As discussed in Chapter 10, the carbohydrate chain is used as a targeting marker for lysosomal enzymes. I-cell (inclusion cell) disease is a rare condition in which lysosomal enzymes lack the mannose phosphate marker that targets them to lysosomes. The enzyme that is deficient in I-cell disease is a phosphotransferase located in the Golgi apparatus (Fig. 30.15). The phosphotransferase has the unique ability

FIG. 30.9. The formation of N-acetylglucosamine 6-phosphate. The amino sugar is formed by a transfer of the amino group from the amide of glutamine to a carbon of the sugar. The amino group is acetylated by the transfer of an acetyl group from acetyl-CoA. The hydrogen atoms from the sugar have been omitted for clarity.

–

H

O

H

–

–

–

H H

H

O

–

–

O

H –

–

H – –

–

H

O

–

Salivary mucin –

= Sialic acid = N-acetylglucosamine

FIG. 30.11. Structure of salivary mucin. The sugars form hydrogen bonds with water. Sialic acid provides a negatively charged carboxylate group. The protein is extremely large, and the negatively charged sialic acids extend the carbohydrate chains (by charge repulsion) so the molecules occupy a large space. All of the salivary glycoproteins contain O-linked sugars. NANA is a sialic acid.

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SECTION V ■ CARBOHYDRATE METABOLISM

Protein

Membrane protein

Carbohydrate

Secreted protein Secretory vesicle

Lysosomal enzyme Secretory vesicle

Lysosome

Mannose-P Golgi complex

Mannose-P receptor

FIG. 30.12. Route from the Golgi complex to the final destination for lysosomal enzymes, cell membrane proteins, and secreted proteins, which include glycoproteins and proteoglycans.

to recognize lysosomal proteins because of their three-dimensional structure, such that they can all be appropriately tagged for transport to the lysosomes. Consequently, as a result of the lack of mannose phosphate, lysosomal enzymes are secreted from the cells. Because lysosomes lack their normal complement of enzymes, undegraded molecules accumulate within membranes inside these cells, forming inclusion bodies.

III. GLYCOLIPIDS A. Function and Structure Glycolipids are derivatives of the lipid sphingosine. These sphingolipids include the cerebrosides and the gangliosides (Fig. 30.16; see also Chapter 5, Fig. 5.22). They contain ceramide, with carbohydrate moieties attached to its hydroxymethyl group. Glycolipids are involved in intercellular communication. Oligosaccharides of identical composition are present in both the glycolipids and glycoproteins associated with the cell membrane, where they serve as cell recognition factors. For example, carbohydrate residues in these oligosaccharides are the antigens of the ABO blood group substances (Fig. 30.17).

B. Synthesis Cerebrosides are synthesized from ceramide and UDP-glucose or UDP-galactose. They contain a single sugar (a monosaccharide). Gangliosides contain oligosaccharides produced from UDP-sugars and CMP-NANA, which is the precursor for

O –

O

H

O

P

O –

O

P

O

O

–

CH2

CH2

C

CH3 CH2

CH3

CH2

CH

C

CH3 CH2

CH2

CH

C

CH3

n

FIG. 30.13. Structure of dolichol phosphate. In humans, the isoprene unit (in brackets) is repeated approximately 17 times (n  ⬃17).

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A

553

= Dolichol = N-acetylglucosamine = Mannose = Glucose P = Phosphate

P

P

P

P

P

P

P

P

P

UDP

GDP

GDP

UDP

UMP

UDP

GDP

GDP

UDP

UDP

B Rough endoplasmic reticulum

1

2

3

4

P P

Dolichol Golgi complex

cis

medial

5

6

7

8

UDP

UDP

9

O-Mannose UDP-NAcGlc

GDP

phosphotransferase (defective in I-cell disease)

UMP Exit trans

10

UDP

11

CMP

O-Mannose 6-phosphate-1-NAcGlc H2O N-acetylglucosaminidase

N-AcGlc

FIG. 30.14. Action of dolichol phosphate in synthesizing the high-mannose form of oligosaccharides (A) and the processing of these carbohydrate groups (B). Transfer of the branched oligosaccharide from dolichol phosphate to a protein in the lumen of the rough endoplasmic reticulum (RER) (step 1) and processing of the oligosaccharide (steps 2–11). Steps 1 through 4 occur in the RER. The glycoprotein is transferred in vesicles to the Golgi complex, where further modifications of the oligosaccharides occur (steps 5–11). (B modified with permission from Kornfeld R, Kornfeld S. Assembly of aspargine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. Copyright 1985 by Annual Reviews, Inc.)

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O-Mannose 6-phosphate

FIG. 30.15. Synthesis of mannose 6-phosphate on the oligosaccharide of lysosomal proteins. The pathway for phosphorylating a mannose residue within the protein-attached oligosaccharide requires two steps. The first is a transfer of N-acetylglucosamine phosphate to the mannose residue, and the second is the release of N-acetylglucosamine from the intermediate product, leaving the phosphate behind on the mannose residue.

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SECTION V ■ CARBOHYDRATE METABOLISM

Blood Type

Galactose

Ceramide

O

Galactocerebroside

Glc

Ceramide

Gal

Type O

Gal

GalNAc

O

O

O

R

O H substance

Fuc

NANA

O GlcNAc

Ganglioside

CH2OH H C

Type A

O GalNAc

NH

H C

OH

C

O Gal

O

O

CH

(CH2)n

CH

CH3

O

O GlcNAc

O

R

O

O GlcNAc

O

R

O

Fuc

(CH2)12

O Type B

CH3

Gal

O Gal

O

Ceramide

FIG. 30.16. Structures of cerebrosides and gangliosides. In these glycolipids, sugars are attached to ceramide (shown below the glycolipids). The boxed portion of ceramide is sphingosine, from which the name “sphingolipids” is derived.

O

O

O

Fuc

FIG. 30.17. Structures of the blood group substances. Note that these structures are the same except that type A has N-acetylgalactosamine (GalNAc) at the nonreducing end, type B has galactose (Gal), and type O has neither. R is either a protein or the lipid ceramide. Each antigenic determinant is boxed. Fuc, fucose; GlcNAc, N-acetylglucosamine; Gal, galactose.

the NANA residues that branch from the linear chain. The synthesis of the sphingolipids is described in more detail in Chapter 33. Defects in the degradation of sphingolipids lead to the sphingolipidoses (also known as the gangliosidoses), as outlined in Table 30.3. Sphingolipids are produced in the Golgi complex. Their lipid component becomes part of the membrane of the secretory vesicle that buds from the trans face of the Golgi. After the vesicle membrane fuses with the cell membrane, the lipid component of the glycolipid remains in the outer layer of the cell membrane,

Table 30.3

Defective Enzymes in the Gangliosidoses

Disease

Enzyme Deficiency

Fucosidosis

␣-Fucosidase

Accumulated Lipid

Cer–Glc–Gal–GalNAc–Gal:Fuc H-isoantigen Cer–Glc–Gal(NeuAc)–GalNAc:Gal Generalized gangliosidosis GM1-␤-galactosidase GM1 ganglioside Tay-Sachs disease Hexosaminidase A Cer–Glc–Gal(NeuAc):GalNAc GM2 ganglioside Tay-Sachs variant or Hexosaminidase A Cer–Glc–Gal–Gal:GalNAc Sandhoff disease and B globoside plus GM2 ganglioside Fabry disease ␣-Galactosidase Cer–Glc–Gal:Gal globotriaosylceramide Ceramide lactoside lipidosis Ceramide lactosidase Cer–Glc:Gal ceramide lactoside (B-galactosidase) Metachromatic Arylsulfatase A Cer–Gal:OSO33– sulfogalactosylceramide leukodystrophy Krabbe disease ␤-Galactosidase Cer:Gal galactosylceramide Gaucher disease ␤-Glucosidase Cer:Glc glucosylceramide Niemann-Pick disease Sphingomyelinase Cer:P–choline sphingomyelin Farber disease Ceramidase Acyl:sphingosine ceramide NeuAc, N-acetylneuraminic acid; Cer, ceramide; Glc, glucose; Gal, galactose; Fuc, fucose. The colon indicates the bond that cannot be broken due to the enzyme deficiency associated with the disease.

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and the carbohydrate component extends into the extracellular space. Sometimes, the carbohydrate component is used as a recognition signal for foreign proteins; for example, cholera toxin (which affected Dennis Veere; see Chapter 10) binds to the carbohydrate portion of the GM1 ganglioside to allow its catalytic subunit to enter the cell. CLINICAL COMMENTS Erna Nemdy. During her stint in the hospital blood bank, Erna Nemdy learned that the importance of the ABO blood group system in transfusion therapy is based on two principles (Table 30.4): (1) Antibodies to A and to B antigens occur naturally in the blood serum of persons whose red blood cell surfaces lack the corresponding antigen (i.e., individuals with A antigens on their red blood cells have B antibodies in their serum and vice versa). These antibodies may arise as a result of previous exposure to cross-reacting antigens in bacteria and foods or to blood transfusions. (2) Antibodies to A and B are usually present in high titers and are capable of activating the entire complement system. As a result, these antibodies may cause intravascular destruction of a large number of incompatible red blood cells given inadvertently during a blood transfusion. Individuals with type AB blood have both A and B antigens and do not produce antibodies to either. Hence, they are “universal” recipients. They can safely receive red blood cells from individuals of A, B, AB, or O blood type. (However, they cannot safely receive serum from these individuals because it contains antibodies to A or B antigens.) Those with type O blood do not have either antigen. They are “universal” donors; that is, their red cells can safely be infused into type A, B, O, or AB individuals. (However, their serum contains antibodies to both A and B antigens and cannot be given safely to recipients with those antigens.) The second important red blood cell group is the Rh group. It is important because one of its antigenic determinants, the D antigen, is a very potent immunogen, stimulating the production of a large number of antibodies. The unique carbohydrate composition of the glycoproteins that constitute the antigenic determinants on red blood cells in part contributes to the relative immunogenicity of the A, B, and Rh(D) red blood cell groups in human blood. Jay Sakz. Tay-Sachs disease, the problem afflicting Jay Sakz, is an autosomal recessive disorder that is rare in the general population (1 in 300,000 births), but its prevalence in Jews of Eastern European extraction (who make up 90% of the Jewish population in the United States) is much higher (1 in 3,600 births). One in 28 Ashkenazi Jews carries this defective gene. Its presence can be discovered by measuring the tissue level of the protein produced by the gene (hexosaminidase A) or by recombinant DNA techniques. Skin fibroblasts of concerned couples planning a family are frequently used for these tests. Carriers of the affected gene have a reduced but functional level of this enzyme that normally hydrolyzes a specific bond between an N-acetyl-D-galactosamine and a D-galactose residue in the polar head of the ganglioside. No effective therapy is available. Enzyme replacement has met with little success because of the difficulties in getting the enzyme across the blood–brain barrier.

Table 30.4

The blood group substances are oligosaccharide components of glycolipids and glycoproteins found in most cell membranes. Those located on red blood cells have been studied extensively. A single genetic locus with two alleles determines an individual’s blood type. These genes encode glycosyltransferases involved in the synthesis of the oligosaccharides of the blood group substances. Most individuals can synthesize the H substance, an oligosaccharide that contains a fucose linked to a galactose at the nonreducing end of the blood group substance (see Fig. 30.17). Type A individuals produce an N-acetylgalactosamine transferase (encoded by the A gene) that attaches N-acetylgalactosamine to the galactose residue of the H substance. Type B individuals produce a galactosyltransferase (encoded by the B gene) that links galactose to the galactose residue of the H substance. Type AB individuals have both alleles and produce both transferases. Thus, some of the oligosaccharides of their blood group substances contain N-acetylgalactosamine and some contain galactose. Type O individuals produce a defective transferase, and, therefore, they do not attach either N-acetylgalactosamine or galactose to the H substance. Thus, individuals of blood type O have only the H substance. Erna Nemdy determined that a patient’s blood type was AB. The new surgical resident was eager to give this patient a blood transfusion and, because AB blood is rare and an adequate amount was not available in the blood bank, he requested type A blood. Should Erna give him type A blood for his patient?

Characteristics of the ABO Blood Groups

Red Cell Type

O

A

B

AB

Possible genotypes Antibodies in serum Frequency (in Caucasians) Can accept blood types

OO Anti-A and -B 45% O

AA or AO Anti-B 40% A, O

BB or BO Anti-A 10% B, O

AB None 5% A, B, AB, O

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The patient could safely receive type A blood cells from another person because he has both A and B antigens on his own cells and does not have antibodies in his serum to either type A or B cells. However, he should not be given type A serum (or type A whole blood) because type A serum contains antibodies to type B antigens, which are present on his cells.

Tay-Sachs disease, which is one of the gangliosidoses, is a multifaceted disorder. The sphingolipidoses, which include Fabry and Gaucher diseases, affect mainly the brain, the skin, and the reticuloendothelial system (e.g., liver and spleen). In these diseases, complex lipids accumulate. Each of these lipids contains a ceramide as part of its structure (see Table 30.3). The rate at which the lipid is synthesized is normal. However, the lysosomal enzyme required to degrade it is not very active, either because it is made in deficient quantities as a result of a mutation in a gene that specifically codes for the enzyme or because a critical protein required to activate the enzyme is deficient. Because the lipid cannot be degraded, it accumulates and causes degeneration of the affected tissues, with progressive malfunction such as the psychomotor deficits that occur as a result of the central nervous system involvement seen in most of these storage diseases. BIOCHEMICAL COMMENTS Biochemistry of Tay-Sachs Disease. Hexosaminidase A, the defective enzyme in Tay-Sachs disease, is actually composed of two subunits, an ␣- and a ␤-chain. The exact stoichiometry of the active enzyme is unknown, but it may be ␣2␤2. The ␣-subunit is coded for by the HexA gene, whereas the ␤-subunit is coded for by the HexB gene. In Tay-Sachs disease, the ␣-subunit is defective, and hexosaminidase A activity is lost. However, the ␤-subunit can form active tetramers in the absence of the ␣-subunit, and this activity, named hexosaminidase B, which cleaves the glycolipid globoside, retains activity in children with Tay-Sachs disease. Thus, children with Tay-Sachs disease accumulate the ganglioside GM2 but not globoside (Fig. 30.18). Mutation of the HexB gene, and production of a defective ␤-subunit, leads to inactivation of both hexosaminidase A and B activity. Such a mutation leads to Sandhoff disease. Both activities are lost because both activities require a functional ␤-subunit. The clinical course of this disease is similar to Tay-Sachs but with an accelerated timetable because of the initial accumulation of both GM2 and globoside in the lysosomes. A third type of mutation also can lead to disease symptoms similar to those of TaySachs disease. Children were identified with Tay-Sachs symptoms, but when both hexosaminidase A and B activities were measured in a test tube, they were normal. This disease, ultimately named Sandhoff activator disease, is caused by a mutation in a protein that is needed to activate hexosaminidaseA activity. In the absence of Sandhoff activator protein +

hexosaminidase A (α2β2)

Block in Sandhoff disease Block in Tay-Sachs disease GM2

ceramide

glc

gal

NAcGal

GM3

ceramide

glc

gal Sialic acid

Sialic acid hexosaminidase A or B (β4)

Block in Sandhoff disease Globoside ceramide glc gal gal

NAcGal

ceramide glc gal gal

FIG. 30.18. Substrate specificities of hexosaminidase A, B, and the function of the activator protein. Defects in the ␤-subunit inactivate both HexA and HexBactivities, leading to GM2 and globoside accumulation. A defect in Sandhoff activator protein also leads to GM2 accumulation, as HexA activity is reduced. Defects in the ␣-subunit inactivate only HexA activity, such that HexB activity toward globoside is unaffected. Glc, glucose; Gal, galactose; NAcGal, N-acetylgalactosamine.

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557

the activator, hexosaminidase A activity is minimal, and GM2 initially accumulates in lysosomes. This mutation has no effect on hexosaminidase B activity. When a glycolipid cannot be degraded because of an enzymatic mutation, it accumulates in residual bodies (vacuoles that contain material that lysosomal enzymes cannot digest). Normal cells contain a small number of residual bodies, but in diseases of lysosomal enzymes, large numbers of residual bodies accumulate within the cell, eventually interfering with normal cell function. In 70% of the cases of Tay-Sachs disease in persons of Ashkenazi Jewish background, exon 11 of the gene for the ␣-chain of hexosaminidase A contains a mutation. The normal gene sequence encodes a protein with the amino acids Arg–Ile–Ser–Tyr–Gly–Pro–Asp in this region, as shown in the following: •

•

10

20

5' CGTATATCCTATGGCCCTGAC Arg Ile Ser Tyr Gly Pro Asp

The mutant DNA sequence for this area is •

10

•

20

5' CGTATATCTATCCTATGGCCCTGAC Arg Ile Ser Ile Leu Trp Pro Stop

A four-base insertion (underlined) occurs in the mutated gene, which alters the reading frame of the protein and also introduces a premature stop codon farther down the protein, so no functional ␣-subunit can be produced. Key Concepts • • • •

• • •

• • • •

Reactions between sugars or the formation of sugar derivatives use sugars activated by attachment to nucleotides (a nucleotide sugar). UDP-glucose and UDP-galactose are substrates for many glycosyltransferase reactions. Lactose is formed from UDP-galactose and glucose. UDP-glucose is oxidized to UDP-glucuronate, which forms glucuronide derivatives of various hydrophobic compounds, making them more readily excreted in urine or bile than the parent compound. Glycoproteins and glycolipids contain various types of carbohydrate residues. The carbohydrates in glycoproteins can be either O-linked or N-linked and are synthesized in the endoplasmic reticulum and the Golgi apparatus. For O-linked carbohydrates, the carbohydrates are added sequentially (via nucleotide sugar precursors), beginning with a sugar linked to the hydroxyl group of the amino acid side chains of serine or threonine. For N-linked carbohydrates, the branched carbohydrate chain is first synthesized on dolichol phosphate and then transferred to the amide nitrogen of an asparagine residue of the protein. Glycolipids belong to the class of sphingolipids that add carbohydrate groups to the base ceramide one at a time from nucleotide sugars. Defects in the degradation of glycosphingolipids lead to a class of lysosomal diseases known as the sphingolipidoses. Table 30.5 summarizes the diseases discussed in this chapter.

Table 30.5

Diseases Discussed in Chapter 30

Disease or Disorder

Environmental or Genetic

Blood transfusions

Both

Tay-Sachs disease

Genetic

Jaundice

Both

Sphingolipidoses

Genetic

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Comments Blood typing is dependent on antigens on the cell surface, particularly the carbohydrate content of the antigen Lack of hexosaminidase A activity, leading to an accumulation of GM2 in the lysosomes Lack of ability to conjugate bilirubin with glucuronic acid in the liver Defects in ganglioside and sphingolipid degradation, as summarized in Table 30.3

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REVIEW QUESTIONS—CHAPTER 30 1.

Which of the following best describes a mother with galactosemia caused by a deficiency of galactose-1-phosphate uridylyltransferase? A. She can convert galactose to UDP-galactose for lactose synthesis during lactation. B. She can form galactose 1-phosphate from galactose. C. She can use galactose as a precursor to glucose production. D. She can use galactose to produce glycogen. E. She will have lower than normal levels of serum galactose after drinking milk.

2.

The immediate carbohydrate precursors for glycolipid and glycoprotein synthesis are which of the following? A. Sugar phosphates B. Sugar acids C. Sugar alcohols D. Nucleotide sugars E. Acylsugars

3.

A newborn is diagnosed with neonatal jaundice. In this patient, the bilirubin produced lacks which of the following carbohydrates? A. Glucose B. Gluconate

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C. Glucuronate D. Galactose E. Galactitol 4.

The nitrogen donor for the formation of amino sugars is which of the following? A. Ammonia B. Asparagine C. Glutamine D. Adenine E. Dolichol

5.

Which of the following glycolipids would accumulate in a patient with Sandhoff disease? A. GM1 B. Lactosylceramide C. Globoside D. Glucocerebroside E. GM3

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31

Gluconeogenesis and Maintenance of Blood Glucose Levels

During fasting, many of the reactions of glycolysis are reversed as the liver produces glucose to maintain blood glucose levels. This process of glucose production is called gluconeogenesis. Gluconeogenesis, which occurs primarily in the liver, is the pathway for the synthesis of glucose from compounds other than carbohydrates. In humans, the major precursors of glucose are lactate, glycerol, and amino acids, particularly alanine. Except for three key sequences, the reactions of gluconeogenesis are reversals of the steps of glycolysis (Fig. 31.1). The sequences of gluconeogenesis that do not use enzymes of glycolysis involve the irreversible, regulated steps of glycolysis. These three sequences are the conversion of (1) pyruvate to phosphoenolpyruvate (PEP), (2) fructose 1,6-bisphosphate to fructose 6-phosphate, and (3) glucose 6-phosphate to glucose. Some tissues of the body such as the brain and red blood cells cannot synthesize glucose on their own but depend on glucose for energy. On a long-term basis, most tissues also require glucose for other functions, such as synthesis of the ribose moiety of nucleotides or the carbohydrate portion of glycoproteins and glycolipids. Therefore, to survive, humans must have mechanisms for maintaining blood glucose levels. After a meal containing carbohydrates, blood glucose levels rise (Fig. 31.2). Some of the glucose from the diet is stored in the liver as glycogen. After 2 or 3 hours of fasting, this glycogen begins to be degraded by the process of glycogenolysis, and glucose is released into the blood. As glycogen stores decrease, adipose triacylglycerols are also degraded, providing fatty acids as an alternative fuel and glycerol for the synthesis of glucose by gluconeogenesis. Amino acids are also released from the muscle to serve as gluconeogenic precursors. During an overnight fast, blood glucose levels are maintained by both glycogenolysis and gluconeogenesis. However, after approximately 30 hours of fasting, liver glycogen stores are mostly depleted. Subsequently, gluconeogenesis is the only source of blood glucose. Changes in the metabolism of glucose that occur during the switch from the fed to the fasting state are regulated by the hormones insulin and glucagon. Insulin is elevated in the fed state, and glucagon is elevated during fasting. Insulin stimulates the transport of glucose into certain cells such as those in muscle and adipose tissue. Insulin also alters the activity of key enzymes that regulate metabolism, stimulating the storage of fuels. Glucagon counters the effects of insulin, stimulating the release of stored fuels and the conversion of lactate, amino acids, and glycerol to glucose. Blood glucose levels are maintained not only during fasting but also during exercise when muscle cells take up glucose from the blood and oxidize it for energy. During exercise, the liver supplies glucose to the blood by the processes of glycogenolysis and gluconeogenesis.

559

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Glycolysis

Gluconeogenesis

Glucose

Glucose

ATP

Pi

glucose 6phosphatase

glucokinase

ADP Glucose-6-P

Glucose-6-P

Fructose-6-P

Fructose-6-P

ATP phosphofructokinase-1

ADP

Fructose-1,6-P

Glyceraldehyde-3-P

Pi

fructose 1,6 bisphosphatase

Fructose-1,6-P

DHAP

DHAP

Glyceraldehyde-3-P

NAD+

NADH

NAD+

NADH

NAD+

NADH

1,3-Bisphosphoglycerate

Glycerol-3-P

ADP

ADP

ATP

ATP

3-Phosphoglycerate

Glycerol

2-Phosphoglycerate

Phosphoenolpyruvate ADP

Phosphoenolpyruvate PEP carboxykinase

pyruvate kinase

ATP

GTP OAA

Pyruvate NADH

GDP

pyruvate dehydrogenase

Acetyl CoA TCA cycle

NAD+

Fatty acids

Pyruvate

pyruvate carboxylase

OAA TCA cycle

Lactate Alanine, Amino acids

Amino acids Lactate

ATP

FIG. 31.1. Glycolysis and gluconeogenesis in the liver. The gluconeogenic pathway is almost the reverse of the glycolytic pathway, except for three reaction sequences. At these three steps, the reactions are catalyzed by different enzymes. The energy requirements of these reactions differ, and one pathway can be activated while the other is inhibited. The steps for which enzyme names are indicated are the irreversible steps of those pathways. All other steps are reversible, although for clarity, this is not indicated in the figure.

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Fed

Dietary carbohydrate

THE WAITING ROOM Al Martini, a known alcoholic, was brought to the emergency room by his landlady, who stated that he had been drinking heavily for the past week. During this time, his appetite had gradually diminished, and he had not eaten any food for the past 3 days. He was confused, combative, tremulous, and sweating profusely. His speech was slurred. His heart rate was rapid (110 beats/ minute). As his blood pressure was being determined, he had a grand mal seizure. His blood glucose drawn just before the onset of the seizure was 28 mg/dL or 1.6 mM (reference range for overnight fasting blood glucose, 80 to 100 mg/dL or 4.4 to 5.6 mM). His blood ethanol level drawn at the same time was 295 mg/dL (intoxication level, i.e., “confused” stage, 150 to 300 mg/dL). Emma Wheezer presented to the emergency room 3 days after being discharged from the hospital following a 10-day admission for severe refractory bronchial asthma. She required high-dose intravenous methylprednisolone (a synthetic anti-inflammatory glucocorticoid) for the first 8 days of her stay. After 2 additional days on oral prednisone, she was discharged on substantial pharmacologic doses of this steroid and instructed to return to her physician’s office in 5 days. She presented now with marked polyuria (increased urination), polydipsia (increased thirst), and muscle weakness. Her blood glucose was 275 mg/dL or 15 mM (reference range, 80 to 100 mg/dL or 4.4 to 5.6 mM).

561

Glucose

Gut

Fasting Glycogen

Brain

Glycerol Amino acids

RBC

Lactate Glucose

Liver

Other tissues

Starved Glucose

Brain

Glycerol Amino acids

Liver

RBC Lactate Glucose

Other tissues

FIG. 31.2. Sources of blood glucose in the fed, fasting, and starved states. RBC, red blood cells.

Di Abietes could not remember whether she had taken her 6:00 PM insulin dose, when, in fact, she had done so. Unfortunately, she decided to give herself her evening dose (for the second time). When she did not respond to her alarm clock at 6:00 AM the following morning, her roommate tried unsuccessfully to awaken her. The roommate called an ambulance, and Di was rushed to the hospital emergency room in a coma. Her pulse and blood pressure at admission were normal. Her skin was flushed and slightly moist. Her respirations were slightly slow. Ann O’Rexia continues to resist efforts on the part of her psychiatrist and family physician to convince her to increase her caloric intake. Her body weight varies between 97 and 99 lb, far below the desirable weight for a woman who is 5 ft 7 in tall. In spite of her severe diet, her fasting blood glucose levels range from 55 to 70 mg/dL. She denies having any hypoglycemic symptoms. Otto Shape has complied with his calorie-restricted diet and aerobic exercise program. He has lost another 7 lb and is closing in on his goal of weighing 154 lb. He notes increasing energy during the day and remains alert during lectures and assimilates the lecture material noticeably better than he did before starting his weight loss and exercise program. He jogs for 45 minutes each morning before breakfast.

I.

GLUCOSE METABOLISM IN THE LIVER

Glucose serves as a fuel for most tissues of the body. It is the major fuel for certain tissues such as the brain and red blood cells. After a meal, food is the source of blood glucose. The liver oxidizes glucose and stores the excess as glycogen. The liver also uses the pathway of glycolysis to convert glucose to pyruvate, which provides carbon for the synthesis of fatty acids. Glycerol 3-phosphate, produced from glycolytic intermediates, combines with fatty acids to form triacylglycerols, which

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The measurement of ketones in the blood and urine can indicate the level of starvation or the presence of diabetic ketoacidosis (DKA). There are several methods to detect ketones. One is the use of reagent strips for urine (based on the reaction of sodium nitroprusside with acetoacetate), but this method does not detect the major blood ketone (␤-hydroxybutyrate). A cyclic enzymatic method has been developed to overcome this, in which blood or plasma samples are incubated with acetoacetate decarboxylase, which removes all acetoacetate from the sample (converting it to acetone and carbon dioxide). Once this has been accomplished, ␤-hydroxybutyrate dehydrogenase is then incubated with the sample, along with thio-NAD⫹. The thio-NAD⫹ is converted to thio-NADH, generating a colored product and acetoacetate. Thio-NADH absorbs light at 405 nm, in the visible range, as compared to NADH, which absorbs at 340 nm, in the UV range. The use of thio-NAD⫹ allows clinical laboratory instrumentation to be used. The acetoacetate is then recycled back to ␤-hydroxybutyrate, in which NADH is converted to NAD⫹. The ␤-hydroxybutyrate produced is then cycled back to acetoacetate, generating more thio-NADH. The cycling enhances the sensitivity of the assay. Once equilibrium is reached, one can calculate from the change in absorbance per minute the concentration of the ␤-hydroxybutyrate in the sample.

are secreted into the blood in very low-density lipoproteins (VLDLs; explained further in Chapter 32). During fasting, the liver releases glucose into the blood so that glucose-dependent tissues do not suffer from a lack of energy. Two mechanisms are involved in this process: glycogenolysis and gluconeogenesis. Hormones, particularly insulin and glucagon, dictate whether glucose flows through glycolysis or whether the reactions are reversed and glucose is produced via gluconeogenesis.

II. GLUCONEOGENESIS Gluconeogenesis, the process by which glucose is synthesized from noncarbohydrate precursors, occurs mainly in the liver under fasting conditions. Under the more extreme conditions of starvation, the kidney cortex also may produce glucose. For the most part, the glucose produced by the kidney cortex is used by the kidney medulla, but some may enter the bloodstream. Starting with pyruvate, most of the steps of gluconeogenesis are simply reversals of those of glycolysis (Fig. 31.3). In fact, these pathways differ at only three points. Enzymes involved in catalyzing these steps are regulated so that either glycolysis or gluconeogenesis predominates, depending on physiologic conditions. Most of the steps of gluconeogenesis use the same enzymes that catalyze the process of glycolysis. The flow of carbon, of course, is in the reverse direction.

Glucose Pi glucose 6-phosphatase

Glucose 6-phosphate

Fructose 6-phosphate Pi fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphate

Dihydroxyacetone-P

Glycerol

Glyceraldehyde-3-P

Glycerol-3-P

Phosphoenolpyruvate phosphoenolpyruvate carboxykinase

Amino acids

TCA cycle

Oxaloacetate

Amino acids Alanine

pyruvate carboxylase

Pyruvate Lactate

FIG. 31.3. Key reactions of gluconeogenesis. The precursors are amino acids (particularly alanine), lactate, and glycerol. Heavy red arrows indicate steps that differ from those of glycolysis.

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Three reaction sequences of gluconeogenesis differ from the corresponding steps of glycolysis. They involve the conversion of pyruvate to phosphoenolpyruvate (PEP) and the reactions that remove phosphate from fructose 1,6-bisphosphate to form fructose 6-phosphate and from glucose 6-phosphate to form glucose (see Fig. 31.3). The conversion of pyruvate to PEP is catalyzed during gluconeogenesis by a series of enzymes instead of the single enzyme used for glycolysis. The reactions that remove phosphate from fructose 1,6-bisphosphate and from glucose 6-phosphate each use single enzymes that differ from the corresponding enzymes of glycolysis. Although phosphate is added during glycolysis by kinases, which use adenosine triphosphate (ATP), it is removed during gluconeogenesis by phosphatases that release inorganic phosphate (Pi) via hydrolysis reactions.

A. Precursors for Gluconeogenesis The three major carbon sources for gluconeogenesis in humans are lactate, glycerol, and amino acids, particularly alanine. Lactate is produced by anaerobic glycolysis in tissues such as exercising muscle or red blood cells, as well as by adipocytes during the fed state. Glycerol is released from adipose stores of triacylglycerol, and amino acids come mainly from amino acid pools in muscle where they may be obtained by degradation of muscle protein. Alanine, the major gluconeogenic amino acid, is produced in the muscle from other amino acids and from glucose (see Chapter 38). Because ethanol metabolism only gives rise to acetyl coenzyme A (acetyl-CoA), the carbons of ethanol cannot be used for gluconeogenesis.

B. Formation of Gluconeogenic Intermediates from Carbon Sources

563

Comatose patients in DKA have the smell of acetone (a derivative of the ketone body acetoacetate) on their breath. In addition, patients with DKA have deep, relatively rapid respirations typical of acidotic patients (Kussmaul respirations). These respirations result from an acidosis-induced stimulation of the respiratory center in the brain. More CO2 is exhaled in an attempt to reduce the amount of acid in the body: H⫹ ⫹ HCO3 → H2CO3 → H2O ⫹ CO2 (exhaled). The severe hyperglycemia of DKA also causes osmotic diuresis (i.e., glucose entering the urine carries an iso-osmotic amount of water with it), which, in turn, causes a contraction of blood volume. Volume depletion may be aggravated by vomiting, which is common in patients with DKA. DKA may cause dehydration (dry mucous membranes and loss of skin turgor), low blood pressure, and a rapid heartbeat. These respiratory and hemodynamic alterations are not seen in patients with hypoglycemic coma. The flushed, wet skin of hypoglycemic coma is in contrast to the dry skin observed in DKA.

The carbon sources for gluconeogenesis form pyruvate, intermediates of the tricarboxylic acid (TCA) cycle or intermediates that are common to both glycolysis and gluconeogenesis. 1.

LACTATE, AMINO ACIDS, AND GLYCEROL

Pyruvate is produced in the liver from the gluconeogenic precursors lactate and alanine. Lactate dehydrogenase oxidizes lactate to pyruvate, generating NADH (see Fig. 31.4A), and alanine aminotransferase converts alanine to pyruvate (see Fig. 31.4B). Although alanine is the major gluconeogenic amino acid, other amino acids such as serine serve as carbon sources for the synthesis of glucose because they also form pyruvate, the substrate for the initial step in the process. Some amino acids form intermediates of the TCA cycle (see Chapter 20), which can enter the gluconeogenic pathway.

Diabetes mellitus (DM) should be suspected if a venous plasma glucose level drawn regardless of when food was last eaten (a “random” sample of blood glucose) is “unequivocally elevated” (i.e., ⬎200 mg/dL), particularly in a patient who manifests the classic signs and symptoms of chronic hyperglycemia (polydipsia, polyuria, blurred vision, headaches, rapid weight loss, sometimes accompanied by nausea and vomiting). To confirm the diagnosis, the patient should fast overnight (10 to 16 hours), and the blood glucose measurement should be repeated. Values of ⬍100 mg/dL are considered normal. Values greater than or equal to 126 mg/dL are indicative of DM. The level of glycosylated hemoglobin (HbA1c) can also be measured to make the diagnosis, and if greater than 6.5% is diagnostic for DM. The levels of HbA1c can also gauge the extent of hyperglycemia over the past 4 to 8 weeks and is used to guide treatment. Values of fasting blood glucose between 100 and 125 mg/dL are designated impaired fasting glucose (IFG) (or prediabetes), and further testing should be performed to determine whether these individuals will eventually develop overt DM. Individuals with blood glucose levels in this range have been defined as “prediabetes.” The determination that fasting blood glucose levels of 126 mg/ L or a percentage of glycosylated hemoglobin of greater than 6.5% diagnostic for DM is based on data indicating that at these levels of glucose or glycosylated hemoglobin, patients begin to develop complications of DM, specifically retinopathy. The renal tubular transport maximum in the average healthy subject is such that glucose will not appear in the urine until the blood glucose level is ⬎180 mg/dL. As a result, reagent tapes (Tes-Tape or Dextrostix) designed to detect the presence of glucose in the urine are not sensitive enough to establish a diagnosis of early DM.

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A

lactate dehydrogenase

CH3 H

C

CH3

OH

C

–

COO

NAD+

–

COO

NADH + H+

Lactate

B

Pyruvate

alanine aminotransferase

CH3

H

C

+ NH3

CH3 C

–

COO

α-kg

glutamate

Alanine

C

O

O

COO– Pyruvate CH2OH

CH2OH CH2OH

HO

C

ATP

ADP

HO

H

CH2

H

CH2OH

C

O

NAD+ NADH + H+ –

P O

glycerol kinase

Glycerol

O

CH2

O –

C

glycerol 3-phosphate dehydrogenase

Glycerol 3-phosphate

O

O O

O–

P O

–

Dihydroxyacetone phosphate

FIG. 31.4. Metabolism of gluconeogenic precursors. A. Conversion of lactate to pyruvate. B. Conversion of alanine to pyruvate. In this reaction, alanine aminotransferase transfers the amino group of alanine to ␣-ketoglutarate (␣-kg) to form glutamate. The coenzyme for this reaction, pyridoxal phosphate, accepts and donates the amino group. C. Conversion of glycerol to dihydroxyacetone phosphate (DHAP).

Glucocorticoids are naturally occurring steroid hormones. In humans, the major glucocorticoid is cortisol. Glucocorticoids are produced in the adrenal cortex in response to various types of stress (see Chapter 43). One of their actions is to stimulate the degradation of muscle protein. Thus, increased amounts of amino acids become available as substrates for gluconeogenesis. Emma Wheezer noted muscle weakness, a result of the muscle-degrading action of the synthetic glucocorticoid prednisone, which she was taking for its anti-inflammatory effects.

In a fatty acid with 19 carbons, how many carbons (and which ones) have the capability to form glucose?

The carbons of glycerol are gluconeogenic because they form dihydroxyacetone phosphate (DHAP), a glycolytic intermediate (see Fig. 31.4C). 2.

PROPIONATE

Fatty acids with an odd number of carbon atoms, which are obtained mainly from vegetables in the diet, produce propionyl-CoA from the three carbons at the ␻-end of the chain (see Chapter 23). These carbons are relatively minor precursors of glucose in humans. Propionyl-CoA is converted to methylmalonyl-CoA, which is rearranged to form succinyl-CoA, a four-carbon intermediate of the TCA cycle that can be used for gluconeogenesis. The remaining carbons of an odd-chain fatty acid form acetyl-CoA, from which no net synthesis of glucose occurs. In some species, propionate is a major source of carbon for gluconeogenesis. Ruminants can produce massive amounts of glucose from propionate. In cows, the cellulose in grass is converted to propionate by bacteria in the rumen. This substrate is then used to generate more than 5 lb of glucose each day by the process of gluconeogenesis. ␤-Oxidation of fatty acids produces acetyl-CoA. Because the pyruvate dehydrogenase reaction is thermodynamically and kinetically irreversible, acetyl-CoA does not form pyruvate for gluconeogenesis. Therefore, if acetyl-CoA is to produce glucose, it must enter the TCA cycle and be converted to malate. For every two carbons of acetyl-CoA that are converted to malate, two carbons are released as CO2: one in the reaction catalyzed by isocitrate dehydrogenase and the other in the reaction catalyzed by ␣-ketoglutarate dehydrogenase. Therefore, there is no net synthesis of glucose from acetyl-CoA.

C. Pathway of Gluconeogenesis Gluconeogenesis occurs by a pathway that reverses many, but not all, of the steps of glycolysis. 1.

CONVERSION OF PYRUVATE TO PHOSPHOENOLPYRUVATE

In glycolysis, PEP is converted to pyruvate by pyruvate kinase. In gluconeogenesis, a series of steps is required to accomplish the reversal of this reaction (Fig. 31.5).

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565

Glucose PEP Cytosol

4 Phosphoenolpyruvate carboxykinase

CO2 GDP

+

Glucagon via cAMP

Pyruvate kinase (PK)

ADP

GTP

Inactive PK– P

ATP

Alanine

OAA

Pyruvate

1

NADH +

NAD

Lactate NADH

NAD+

Asp

Malate 3

Adipose TG

Pyruvate 2 Pyruvate carboxylase

CO2

+

Biotin ATP

Asp

OAA 1

FA –

ADP Pi NADH

2

Glucagon via cAMP

NADH Pyruvate dehydrogenase

FA

+

NAD + Activated by – Inhibited by

Inducible enzyme Inactive enzyme

Malate

Mitochondrion

Acetyl CoA

Ketone bodies

OAA exits from the mitochondrion either as 1 aspartate or 2 malate

FIG. 31.5. Conversion of pyruvate to PEP in the liver. Follow the shaded circled numbers on the diagram, starting with the precursors alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. In the cytoplasm, the malate or aspartate is converted back into oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase converts it to PEP (circle 4). The PEP formed is not converted to pyruvate because pyruvate kinase has been inactivated by phosphorylation by the cAMP-dependent protein kinase under these conditions. The white circled numbers are alternative routes for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA, oxaloacetate; FA, fatty acid; TG, triacylglycerol.

Pyruvate is carboxylated by pyruvate carboxylase to form oxaloacetate. This enzyme, which requires biotin, is the catalyst of an anaplerotic (refilling) reaction of the TCA cycle (see Chapter 20). In gluconeogenesis, this reaction replenishes the oxaloacetate that is used for the synthesis of glucose (Fig. 31.6). The CO2 that was added to pyruvate to form oxaloacetate is released in the reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), which generates PEP (Fig. 31.7A). For this reaction, guanosine triphosphate (GTP) provides a source of energy as well as the phosphate group of PEP. Pyruvate carboxylase is found in mitochondria. In various species, PEPCK is located either in the cytosol or in mitochondria, or it is distributed between these two compartments. In humans, the enzyme is distributed about equally in each compartment. Oxaloacetate, generated from pyruvate by pyruvate carboxylase or from amino acids that form intermediates of the TCA cycle, does not readily cross the mito-

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Only the three carbons at the ␻-end of an odd-chain fatty acid that form propionyl-CoA are converted to glucose. The remaining 16 carbons of a fatty acid with 19 carbons form acetyl-CoA, which does not form any net glucose.

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566

SECTION V ■ CARBOHYDRATE METABOLISM

A

COO– CH3 C

ATP

CO2 O

ADP + Pi

Biotin

–

COO

pyruvate carboxylase

Pyruvate

CH2 C

GTP

CH2 O

COO

C

–

O

CO2

GDP

CH2 C

Phosphoenolpyruvate carboxykinase

–

COO

Oxaloacetate

FIG. 31.6. loacetate.

COO–

Oxaloacetate

O O

COO–

P

O–

O–

Phosphoenolpyruvate

Conversion of pyruvate to oxaB

NAD+ Malate

Excessive ethanol metabolism blocks the production of gluconeogenic precursors. Cells have limited amounts of NAD, which exist either as NAD⫹ or as NADH. As the levels of NADH rise, those of NAD⫹ fall, and the ratio of the concentrations of NADH and NAD⫹ ([NADH]/[NAD⫹]) increases. In the presence of ethanol, which is very rapidly oxidized in the liver, the [NADH]/ [NAD⫹] ratio is much higher than it is in the normal fasting liver. High levels of NADH drive the lactate dehydrogenase reaction toward lactate. Therefore, lactate cannot enter the gluconeogenic pathway, and pyruvate that is generated from alanine is converted to lactate. Because glycerol is oxidized by NAD⫹ during its conversion to DHAP, the conversion of glycerol to glucose is also inhibited when NADH levels are elevated. Consequently, the major precursors lactate, alanine, and glycerol are not used for gluconeogenesis under conditions in which alcohol metabolism is high. CH3

CH2

OH

Ethanol

NADH

+ H+

O CH3

CH

Acetaldehyde NAD+

O CH3

C OH

Acetate

Lieberman_CH31.indd 566

+ H+

Oxaloacetate

Aspartate

␣-Ketoglutarate

Oxaloacetate

Glutamate

FIG. 31.7. Generation of PEP from gluconeogenic precursors. A. Conversion of oxaloacetate to PEP using PEP carboxykinase. B. Interconversion of oxaloacetate and malate. C. Transamination of aspartate to form oxaloacetate. Note that the cytosolic reaction is the reverse of the mitochondrial reaction as shown in Figure 31.5.

chondrial membrane. It is either decarboxylated to form PEP by the mitochondrial PEPCK or it is converted to malate or aspartate (see Figs. 31.7B and C). The conversion of oxaloacetate to malate requires NADH. PEP, malate, and aspartate can be transported into the cytosol. After malate or aspartate traverses the mitochondrial membrane (acting as a carrier of oxaloacetate) and enters the cytosol, it is reconverted to oxaloacetate by reversal of the reactions given previously (see Figs. 31.7B and C). The conversion of malate to oxaloacetate generates NADH. Whether oxaloacetate is transported across the mitochondrial membrane as malate or aspartate depends on the need for reducing equivalents in the cytosol. NADH is required to reduce 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate during gluconeogenesis. Oxaloacetate, produced from malate or aspartate in the cytosol, is converted to PEP by the cytosolic PEPCK (see Fig. 31.7A). CONVERSION OF PHOSPHOENOLPYRUVATE TO FRUCTOSE 1,6-BISPHOSPHATE

The remaining steps of gluconeogenesis occur in the cytosol (Fig. 31.8). Starting with PEP as a substrate, the steps of glycolysis are reversed to form glyceraldehyde 3-phosphate. For every two molecules of glyceraldehyde 3-phosphate that are formed, one is converted to DHAP. These two triose phosphates, DHAP and glyceraldehyde 3-phosphate, condense to form fructose 1,6-bisphosphate by a reversal of the aldolase reaction. Because glycerol forms DHAP, it enters the gluconeogenic pathway at this level. 3.

NADH

Malate dehydrogenase

C

2.

NAD+

NADH + H+

CONVERSION OF FRUCTOSE 1,6-BISPHOSPHATE TO FRUCTOSE 6-PHOSPHATE

The enzyme fructose 1,6-bisphosphatase releases Pi from fructose 1,6-bisphosphate to form fructose 6-phosphate. This is not a reversal of the phosphofructokinase-1 (PFK-1) reaction; ATP is not produced when the phosphate is removed from the 1-position of fructose 1,6-bisphosphate because that is a low-energy phosphate bond. Rather, Pi is released in this hydrolysis reaction. In the next reaction of

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Glucose

567

Pi

ATP

Glucose 6-phosphatase

Glucokinase

ADP Glucose 6-phosphate

Fructose 6-phosphate Phosphofructokinase 1

Pi

ATP Fructose 1,6-bisphosphatase

–

ATP Low AMP and F-2,6-P

(Endoplasmic reticulum)

ADP

Fructose 1,6-bisphosphate

Dihydroxyacetone phosphate

Glyceraldehyde 3-phosphate Pi NAD+

NADH NADH NAD+ Glycerol kinase

Glycerol ATP ADP

Cytosol

1,3-Bisphosphoglycerate ADP

Glycerol 3-phosphate

ATP 3-Phosphoglycerate

2-Phosphoglycerate – Inhibited by

Inducible enzyme Inactive enzyme

H2O Phosphoenolpyruvate (PEP)

FIG. 31.8. Conversion of phosphoenolpyruvate and glycerol to glucose. Gluconeogenic reactions are indicated by the red arrows. Glucokinase is inactive because of the low levels of glucose in the cell (below the Km for glucokinase), whereas PFK-1 is inactive because of the low concentration of the allosteric activators AMP and fructose 2,6-bisphosphate, coupled to high concentrations of ATP, an allosteric inhibitor.

gluconeogenesis, fructose 6-phosphate is converted to glucose 6-phosphate by the same isomerase used in glycolysis (phosphoglucoisomerase). 4.

CONVERSION OF GLUCOSE 6-PHOSPHATE TO GLUCOSE

Glucose 6-phosphatase hydrolyzes Pi from glucose 6-phosphate, and free glucose is released into the blood. As with fructose 1,6-bisphosphatase, this is not a reversal of the glucokinase reaction because the phosphate bond in glucose 6-phosphate is a low-energy bond, and ATP is not generated at this step. Glucose 6-phosphatase is located in the membrane of the endoplasmic reticulum. It is used not only in gluconeogenesis but also to produce blood glucose from the breakdown of liver glycogen.

D. Regulation of Gluconeogenesis Although gluconeogenesis occurs during fasting, it is also stimulated during prolonged exercise, by a high-protein diet, and under conditions of stress. The factors

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Al Martini had not eaten for 3 days, so he had no dietary source of glucose, and his liver glycogen stores were essentially depleted. He was solely dependent on gluconeogenesis to maintain his blood glucose levels. One of the consequences of ethanol ingestion and the subsequent rise in NADH levels is that the major carbon sources for gluconeogenesis cannot readily be converted to glucose. For example, amino acids that form intermediates of the tricarboxylic acid (TCA) cycle are converted to malate, which enters the cytosol and is converted to oxaloacetate, which proceeds through gluconeogenesis to form glucose. When excessive amounts of ethanol are ingested, elevated NADH levels inhibit the conversion of malate to oxaloacetate in the cytosol. Therefore, carbons from amino acids that form intermediates of the TCA cycle cannot be converted to glucose as readily. As a result of the excessive NADH formed during his alcoholic binges, Mr. Martini became hypoglycemic. His blood glucose level was 28 mg/dL.

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SECTION V ■ CARBOHYDRATE METABOLISM

that promote the overall flow of carbon from pyruvate to glucose include the availability of substrate and changes in the activity or amount of certain key enzymes of glycolysis and gluconeogenesis. 1.

AVAILABILITY OF SUBSTRATE

Gluconeogenesis is stimulated by the flow of its major substrates from peripheral tissues to the liver. Glycerol is released from adipose tissue whenever the levels of insulin are low and the levels of glucagon or the “stress” hormones, epinephrine and cortisol (a glucocorticoid), are elevated in the blood (see Chapter 26). Lactate is produced by muscle during exercise and by red blood cells. Amino acids are released from muscle whenever insulin is low or when cortisol is elevated. Amino acids are also available for gluconeogenesis when the dietary intake of protein is high and intake of carbohydrate is low. 2.

ACTIVITY OR AMOUNT OF KEY ENZYMES

Three sequences in the pathway of gluconeogenesis are regulated: 1. Pyruvate → PEP 2. Fructose 1,6-bisphosphate → fructose 6-phosphate 3. Glucose 6-phosphate → glucose These steps correspond to those in glycolysis that are catalyzed by regulatory enzymes. The enzymes involved in these steps of gluconeogenesis differ from those that catalyze the reverse reactions in glycolysis. The net flow of carbon whether from glucose to pyruvate (glycolysis) or from pyruvate to glucose (gluconeogenesis) depends on the relative activity or amount of these glycolytic or gluconeogenic enzymes (Fig. 31.9 and Table 31.1). 3.

CONVERSION OF PYRUVATE TO PHOSPHOENOLPYRUVATE

Pyruvate, a key substrate for gluconeogenesis, is derived from lactate and amino acids, particularly alanine. Pyruvate is not converted to acetyl-CoA under conditions that favor gluconeogenesis because pyruvate dehydrogenase is relatively inactive. Instead, pyruvate is converted to oxaloacetate by pyruvate carboxylase. Subsequently, oxaloacetate is converted to PEP by PEPCK. Because of the activity state of the enzymes discussed in subsequent sections, PEP reverses the steps of glycolysis, ultimately forming glucose. Pyruvate dehydrogenase is inactive. Under conditions of fasting, insulin levels are low, and glucagon levels are elevated. Consequently, fatty acids and glycerol are released from the triacylglycerol stores of adipose tissue. Fatty acids travel to the liver where they undergo ␤-oxidation producing acetyl-CoA, NADH, and ATP. As a consequence, the concentration of adenosine diphosphate (ADP) decreases. These changes result in the phosphorylation of pyruvate dehydrogenase to the inactive form. Therefore, pyruvate is not converted to acetyl-CoA (see Chapter 20). Pyruvate carboxylase is active. Acetyl-CoA, which is produced by oxidation of fatty acids, activates pyruvate carboxylase. Therefore, pyruvate, derived from lactate or alanine, is converted to oxaloacetate. Acetyl-CoA levels, then, reciprocally regulate pyruvate dehydrogenase and pyruvate carboxylase. High levels of acetyl-CoA inhibit pyruvate dehydrogenase while activating pyruvate carboxylase. Phosphoenolpyruvate carboxykinase is induced. Oxaloacetate produces PEP in a reaction catalyzed by PEPCK. Cytosolic PEPCK is an inducible enzyme, which means that the quantity of the enzyme in the cell increases because of increased transcription of its gene and increased translation of its mRNA. The major inducer is cyclic adenosine monophosphate (cAMP), which is increased by hormones that activate adenylate cyclase. Adenylate cyclase produces cAMP from ATP. Glucagon

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Glycolysis

569

Gluconeogenesis Glucose

Glucokinase (high Km)

Glucose 6-phosphatase

Glucose 6-phosphate

Fructose 6-phosphate F-2,6-P

Phosphofructokinase-1 +

Fructose 1,6-bisphosphatase –

Fructose 1,6-bisphosphate

Dihydroxyacetone phosphate

Glyceraldehyde 3-phosphate

Phosphoenolpyruvate –

cAMP

Pyruvate kinase– P (inactive)

Pyruvate kinase (active)

Pi

Phosphoenolpyruvate carboxykinase

Oxaloacetate

Pyruvate

Pyruvate carboxylase + Acetyl CoA

FIG. 31.9. Liver enzymes involved in regulating the substrate cycles of glycolysis and gluconeogenesis. Heavy arrows indicate the three substrate cycles. F-2,6-P, fructose 2,6-bisphosphate; 䊝, activated by; 䊞, inhibited by; , inducible enzyme.

Table 31.1 Regulation of Enzymes of Glycolysis and Gluconeogenesis in Liver Enzyme A. Glycolytic enzymes Pyruvate kinase

Phosphofructokinase-1 Glucokinase B. Gluconeogenic enzymes Pyruvate carboxylase Phosphoenolpyruvate carboxykinase Glucose 6-phosphatase Fructose 1,6-bisphosphatase

Mechanism Activated by F 1,6-BP Inhibited by ATP, alanine Inhibited by phosphorylation (glucagon and epinephrine lead to an increase in cAMP levels, which activates protein kinase A) Activated by F 2,6-BP, AMP Inhibited by ATP, citrate High Km for glucose Induced by insulin Activated by acetyl-CoA Induced (increase in gene transcription) by glucagon, epinephrine, glucocorticoids Repressed by insulin Induced (increase in gene transcription) during fasting Inhibited by F 2,6-BP, AMP Induced (increase in gene transcription) during fasting

F 1,6-BP, fructose 1,6-bisphosphate; F 2,6-BP, fructose 2,6-bisphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; AMP, adenosine monophosphate.

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SECTION V ■ CARBOHYDRATE METABOLISM

The mechanism of action of steroid hormones on glucose homeostasis differs from that of glucagon or epinephrine (see Chapters 16 and 26). Glucocorticoids are steroid hormones that stimulate gluconeogenesis, in part, because they induce the synthesis of PEPCK. Emma Wheezer had elevated levels of blood glucose because she was being treated with large pharmacologic doses of dexamethasone, a potent synthetic glucocorticoid.

is the hormone that causes cAMP to rise during fasting, whereas epinephrine acts during exercise or stress. cAMP activates protein kinase A, which phosphorylates a set of specific transcription factors (cAMP-response element binding protein, CREB) that stimulate transcription of the PEPCK gene (see Chapter 16 and Fig. 16.17). Increased synthesis of mRNA for PEPCK results in increased synthesis of the enzyme. Cortisol, the major human glucocorticoid, also induces PEPCK but through a different regulatory site on the PEPCK promoter. Pyruvate kinase is inactive. When glucagon is elevated, liver pyruvate kinase is phosphorylated and inactivated by a mechanism that involves cAMP and protein kinase A. Therefore, PEP is not reconverted to pyruvate. Rather, it continues along the pathway of gluconeogenesis. If PEP were reconverted to pyruvate, these substrates would simply cycle, causing a net loss of energy with no net generation of useful products. The inactivation of pyruvate kinase prevents such futile cycling and promotes the net synthesis of glucose. 4.

CONVERSION OF FRUCTOSE 1,6-BISPHOSPHATE TO FRUCTOSE 6-PHOSPHATE

The carbons of PEP reverse the steps of glycolysis, forming fructose 1,6-bisphosphate. Fructose 1,6-bisphosphatase acts on this bisphosphate to release Pi and produce fructose 6-phosphate. A futile substrate cycle is prevented at this step because, under conditions that favor gluconeogenesis, the concentrations of the compounds that activate the glycolytic enzyme PFK-1 are low. These same compounds, fructose 2,6-bisphosphate (whose levels are regulated by insulin and glucagon) and AMP, are allosteric inhibitors of fructose 1,6-bisphosphatase. When the concentrations of these allosteric effectors are low, PFK-1 is less active, fructose 1,6-bisphosphatase is more active, and the net flow of carbon is toward fructose 6-phosphate and, thus, toward glucose. The synthesis of fructose 1,6-bisphosphatase is also induced during fasting.

Glycogenolysis

Blood

5.

Glucose

Glucose 6-phosphatase catalyzes the conversion of glucose 6-phosphate to glucose, which is released from the liver cell (Fig. 31.10). The glycolytic enzyme glucokinase, which catalyzes the reverse reaction, is relatively inactive during gluconeogenesis. Glucokinase, which has a high S0.5 (Km) for glucose (see Chapter 9, Fig. 9.3), is not very active during fasting because the blood glucose level is lower (approximately 5 mM) than the S0.5 of the enzyme. Glucokinase is also an inducible enzyme. The concentration of the enzyme increases in the fed state, when blood glucose and insulin levels are elevated, and decreases in the fasting state, when glucose and insulin are low.

Gluconeogenesis G-1-P

G-6-P

G-6-P

Glucose

+

Glucose

Pi ER

Pi Liver cell

Transport proteins Glucose 6-phosphatase

FIG. 31.10. Location and function of glucose 6-phosphatase. Glucose 6-phosphate travels on a transporter into the endoplasmic reticulum (ER), where it is hydrolyzed by glucose 6-phosphatase to glucose and inorganic phosphate (Pi). These products travel back to the cytosol on transporters.

Lieberman_CH31.indd 570

CONVERSION OF GLUCOSE 6-PHOSPHATE TO GLUCOSE

E. Energy Is Required for the Synthesis of Glucose During the gluconeogenic reactions, 6 mol of high-energy phosphate bonds are cleaved. Two mol of pyruvate are required for the synthesis of 1 mol of glucose. As 2 mol of pyruvate are carboxylated by pyruvate carboxylase, 2 mol of ATP are hydrolyzed. PEPCK requires 2 mol of GTP (the equivalent of 2 mol of ATP) to convert 2 mol of oxaloacetate to 2 mol of PEP. An additional 2 mol of ATP are used when 2 mol of 3-phosphoglycerate are phosphorylated, forming 2 mol of 1,3-bisphosphoglycerate. Energy in the form of reducing equivalents (NADH) is also required for the conversion of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. Under fasting conditions, the energy required for gluconeogenesis is obtained from ␤-oxidation of fatty acids. Defects in fatty acid oxidation can lead to hypoglycemia because of reduced fatty acid–derived energy production within the liver. Glucose 6-phosphatase is used in both glycogenolysis and gluconeogenesis to produce free glucose from glucose 6-phosphate.

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III. CHANGES IN BLOOD GLUCOSE LEVELS AFTER A MEAL

Table 31.2

mg/dL 225

12.0

200 Blood glucose (mM)

The metabolic transitions that occur as a person eats a meal and progresses through the various stages of fasting have been described in detail in previous chapters. This chapter summarizes the concepts presented in these previous chapters. Because a thorough understanding of these concepts is so critical to medicine, a summary is not only warranted but is also essential. After a high-carbohydrate meal, blood glucose rises from a fasting level of approximately 80 to 100 mg/dL (⬃5 mM) to a level of approximately 120 to 140 mg/dL (8 mM) within a period of 30 minutes to 1 hour (Fig. 31.11). The concentration of glucose in the blood then begins to decrease, returning to the fasting range by approximately 2 hours after the meal (see also Chapter 26). Blood glucose levels increase as dietary glucose is digested and absorbed. The values go no higher than approximately 140 mg/dL in a normal, healthy person because tissues take up glucose from the blood, storing it for subsequent use and oxidizing it for energy. After the meal is digested and absorbed, blood glucose levels decline because cells continue to metabolize glucose. If blood glucose levels continued to rise after a meal, the high concentration of glucose would cause the release of water from tissues as a result of the osmotic effect of glucose. Tissues would become dehydrated, and their function would be affected. If hyperglycemia becomes severe, a hyperosmolar coma could result from dehydration of the brain. Conversely, if blood glucose levels continued to drop after a meal, tissues that depend on glucose would suffer from a lack of energy. If blood glucose levels dropped abruptly, the brain would not be able to produce an adequate amount of ATP. Light-headedness and dizziness would result, followed by drowsiness and, eventually, coma. Red blood cells would not be able to produce enough ATP to maintain the integrity of their membranes. Hemolysis of these cells would decrease the transport of oxygen to the tissues of the body. Eventually, all tissues that rely on oxygen for energy production would fail to perform their normal functions. If the problem were severe enough, death could result. Devastating consequences of glucose excess or insufficiency are normally avoided because the body is able to regulate its blood glucose levels. As the concentration of blood glucose approaches the normal fasting range of 80 to 100 mg/dL roughly 2 hours after a meal, the process of glycogenolysis is activated in the liver. Liver glycogen is the primary source of blood glucose during the first few hours of fasting. Subsequently, gluconeogenesis begins to play a role as an additional source of blood glucose. The carbon for gluconeogenesis, a process that occurs in the liver, is supplied by other tissues. Exercising muscle and red blood cells provide lactate through glycolysis; muscle also provides amino acids by degradation of protein; and glycerol is released from adipose tissue as triacylglycerol stores are mobilized. Even during a prolonged fast, blood glucose levels do not decrease dramatically. After 5 to 6 weeks of starvation, blood glucose levels decrease to only approximately 65 mg/dL (Table 31.2).

571

10.0

175 150

8.0

125 6.0

100 75

4.0

50 2.0 0 0

25 1

2

Time (h)

FIG. 31.11. Blood glucose concentrations at various times after a meal.

Blood Glucose Levels at Various Stages of Fasting

Stage of Fasting Glucose, 700 g/d IV Fasting, 12 h Starvation, 3 d Starvation, 5–6 wk

Glucose (mg/dL) 100 80 70 65

IV, intravenous. Source: Ruderman NB, Aoki TT, Cahill GF Jr. Gluconeogenesis and its disorders in man. In: Hanson RW, Mehlman MA, eds. Gluconeogenesis: Its Regulation in Mammalian Species. New York, NY: John Wiley & Sons; 1976:517.

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SECTION V ■ CARBOHYDRATE METABOLISM

When Di Abietes inadvertently injected an excessive amount of insulin, she caused an acute reduction in her blood glucose levels, which dropped acutely 4 to 5 hours later while she was asleep. Had she been awake, she would have first experienced symptoms caused by hypoglycemia-induced hyperactivity of her sympathetic nervous system (e.g., sweating, tremulousness, palpitations). Eventually, as her hypoglycemia became more profound, she would have experienced symptoms of “neuroglycopenia” (inadequate glucose supply to the brain), such as confusion, speech disturbances, emotional instability, possible seizure activity, and, finally, coma. She reached this neuroglycopenic stage of hypoglycemia while sleeping and thus could not be aroused at 6:00 AM. Ann O’ Rexia, whose intake of glucose and of glucose precursors has been severely restricted, has not developed any of these manifestations. Her lack of hypoglycemic symptoms can be explained by the very gradual reduction of her blood glucose levels as a consequence of near starvation and her ability to maintain blood glucose levels within an acceptable fasting range through hepatic gluconeogenesis. In addition, lipolysis of adipose triacylglycerols produces fatty acids, which are used as fuel and converted to ketone bodies by the liver. The oxidation of fatty acids and ketone bodies by the brain and muscle reduces the need for blood glucose. In Di Abietes’ case, the excessive dose of insulin inhibited lipolysis and ketone body synthesis, so these alternative fuels were not available to spare blood glucose. The rapidity with which hypoglycemia was induced could not be compensated for quickly enough by hepatic gluconeogenesis, which was inhibited by the insulin, and hypoglycemia ensued. An immediate fingerstick revealed that Di’s capillary blood glucose level was ⬍20 mg/dL. An intravenous infusion of a 50% solution of glucose was started, and her blood glucose level was determined frequently. When Di regained consciousness, the intravenous solution was eventually changed to 10% glucose. After 6 hours, her blood glucose levels stayed in the upper normal range, and she was able to tolerate oral feedings. She was transferred to the metabolic unit for overnight monitoring. By the next morning, her previous diabetes treatment regimen could be reestablished. The reasons that she had developed hypoglycemic coma were explained to Di, and she was discharged to the care of her family doctor.

A. Blood Glucose Levels in the Fed State The major factors involved in regulating blood glucose levels are the blood glucose concentration itself and hormones, particularly insulin and glucagon. As blood glucose levels rise after a meal, the increased glucose concentration stimulates the ␤-cells of the pancreas to release insulin (Fig. 31.12). Certain amino acids, particularly arginine and leucine, also stimulate insulin release from the pancreas. Blood levels of glucagon, which is secreted by the ␣-cells of the pancreas, may increase or decrease, depending on the content of the meal. Glucagon levels decrease in response to a high-carbohydrate meal, but they increase in response to a high-protein meal. After a typical mixed meal containing carbohydrate, protein, and fat, glucagon levels remain relatively constant, whereas insulin levels increase. 1.

FATE OF DIETARY GLUCOSE IN THE LIVER

After a meal, the liver oxidizes glucose to meet its immediate energy needs. Any excess glucose is converted to stored fuels. Glycogen is synthesized and stored in the liver, and glucose is converted to fatty acids and to the glycerol moiety that reacts with the fatty acids to produce triacylglycerols. These triacylglycerols are packaged in VLDL and transported to adipose tissue where the fatty acids are stored in adipose triacylglycerols. The VLDL can also deliver triglycerides (fatty acids) to the muscle for immediate oxidation, if required. Regulatory mechanisms control the conversion of glucose to stored fuels. As the concentration of glucose increases in the hepatic portal vein, the concentration of glucose in the liver may increase from the fasting level of 80 to 100 mg/dL (⬃5 mM) to a concentration of 180 to 360 mg/dL (10 to 20 mM). Consequently, the velocity of the glucokinase reaction increases because this enzyme has a high S0.5 (Km) for glucose (Fig. 31.13). Glucokinase is also induced by a highcarbohydrate diet; the quantity of the enzyme increases in response to elevated insulin levels. Insulin promotes the storage of glucose as glycogen by countering the effects of glucagon-stimulated phosphorylation. The response to insulin activates the phosphatases that dephosphorylate glycogen synthase (which leads to glycogen synthase activation) and glycogen phosphorylase (which leads to inhibition of the enzyme)

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Glucose meal

1.0

Glucokinase

0

0

Insulin

S 0.5

5 10 15 [Glucose] (mM)

100 50

20

FIG. 31.13. Velocity of the glucokinase reaction.

(Fig. 31.14A). Insulin also promotes the synthesis of the triacylglycerols that are released from the liver into the blood as VLDL. The regulatory mechanisms for this process are described in Chapter 33.

70 50 30 0

1

2

3

Hours

FATE OF DIETARY GLUCOSE IN PERIPHERAL TISSUES

RETURN OF BLOOD GLUCOSE TO FASTING LEVELS

After a meal has been digested and absorbed, blood glucose levels reach a peak and then begin to decline. The uptake of dietary glucose by cells particularly those in the liver, muscle, and adipose tissue lowers blood glucose levels. By 2 hours after a meal, blood glucose levels return to the normal fasting level of ⬍100 mg/dL.

Insulin

(μU/mL)

Glucose (mg/dL)

Protein meal

Glucagon (pg/mL)

Almost every cell in the body oxidizes glucose for energy. Certain critical tissues, particularly the brain, other nervous system tissues, and red blood cells depend especially on glucose for their energy supply. The brain requires approximately 150 g of glucose per day. In addition, approximately 40 g of glucose per day is required by other glucose-dependent tissues. Furthermore, all tissues require glucose for the pentose phosphate pathway, and many tissues use glucose for synthesis of glycoproteins and other carbohydrate-containing compounds. Insulin stimulates the transport of glucose into adipose and muscle cells by promoting the recruitment of glucose transporters to the cell membrane (see Fig. 31.14C). Other tissues such as the liver, brain, and red blood cells have a different type of glucose transporter that is not significantly affected by insulin. In muscle, glycogen is synthesized after a meal by a mechanism similar to that in the liver (see Fig. 31.14B). Metabolic differences exist between these tissues (see Chapter 28), but, in essence, insulin stimulates glycogen synthesis in resting muscle as it does in the liver. A key difference between muscle and liver is that insulin greatly stimulates the transport of glucose into muscle cells but only slightly stimulates its transport into liver cells. 3.

(μU/mL)

Km

2.

200 100

0.5

Glucagon (pg/mL)

vi V max

Glucose (mg/dL)

Hexokinase I

200 100

20 10

150 100 50 0

1

2

3

Hours

FIG. 31.12. Blood glucose, insulin, and glucagon levels after a high-carbohydrate meal and after a high-protein meal.

B. Blood Glucose Levels in the Fasting State 1.

CHANGES IN INSULIN AND GLUCAGON LEVELS

During fasting, as blood glucose levels decrease, insulin levels decrease and glucagon levels rise. These hormonal changes cause the liver to degrade glycogen by the process of glycogenolysis and to produce glucose by the process of gluconeogenesis so that blood glucose levels are maintained. 2.

STIMULATION OF GLYCOGENOLYSIS

Within a few hours after a high-carbohydrate meal, glucagon levels begin to rise. Glucagon binds to cell surface receptors and activates adenylate cyclase, causing cAMP levels in liver cells to rise (Fig. 31.15). cAMP activates protein kinase A,

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574

SECTION V ■ CARBOHYDRATE METABOLISM

Glucagon

A

+

Glucose

Glycerol-3-P FA TG

+

VLDL

Glycogen

adenylate cyclase

Cell membrane

+

–

VLDL

1 Liver

ATP

cAMP

Protein kinase A (inactive)

B

Regulatory subunit-cAMP

2

Glucose

ADP +

Phosphorylase kinase (inactive)

Glucose +

4

ATP Active protein kinase A

3

Glycogen

ADP

Glycogen

5 C

Glucose

VLDL

Phosphorylase b (inactive)

+

+

ATP

Pi ADP

Phosphorylase a (active) P

6

Glucose 1-phosphate

FA

DHAP

Blood glucose Glycerol-3-P

Pyruvate +

FA

CO2 + H2O

Glucose 6-phosphate Liver

Glucose +

ATP Glycogen synthase (active)

Phosphorylase kinase– P (active)

Muscle

Glycogen synthase– P (inactive)

Triacylglycerol

Adipose cell

FIG. 31.14. Glucose metabolism in various tissues. A. Effect of insulin on glycogen synthesis and degradation and on very lowdensity lipoprotein (VLDL) synthesis in the liver. B. Glucose metabolism in resting muscle in the fed state. Transport of glucose into cells and synthesis of glycogen are stimulated by insulin. C. Glucose metabolism in adipose tissue in the fed state. FA, fatty acids; DHAP, dihydroxyacetone phosphate; TG, triacylglycerol; 䊝, stimulated by insulin; 䊞, inhibited by insulin.

FIG. 31.15. Regulation of glycogenolysis in the liver by glucagon. (1) Glucagon binding to its receptor leads to activation of adenylate cyclase, which leads to an increase in cAMP levels. (2) cAMP binds to the regulatory subunits of protein kinase A, thereby activating the catalytic subunit. (3) Active protein kinase A phosphorylates and activates phosphorylase kinase, while simultaneously inactivating glycogen synthase (4). (5) Active phosphorylase kinase converts glycogen phosphorylase b to glycogen phosphorylase a. (6) Phosphorylase a degrades glycogen, producing glucose 1-phosphate, which is converted to glucose 6-phosphate, then glucose, for export into the blood.

which phosphorylates and inactivates glycogen synthase. Therefore, glycogen synthesis decreases. At the same time, protein kinase A stimulates glycogen degradation by a twostep mechanism. Protein kinase A phosphorylates and activates phosphorylase kinase. This enzyme, in turn, phosphorylates and activates glycogen phosphorylase. Glycogen phosphorylase catalyzes the phosphorolysis of glycogen, producing glucose 1-phosphate, which is converted to glucose 6-phosphate. Dephosphorylation of glucose 6-phosphate by glucose 6-phosphatase produces free glucose, which then enters the blood. 3.

STIMULATION OF GLUCONEOGENESIS

By 4 hours after a meal, the liver is supplying glucose to the blood not only by the process of glycogenolysis but also by the process of gluconeogenesis. Hormonal changes cause peripheral tissues to release precursors that provide carbon for gluconeogenesis, specifically lactate, amino acids, and glycerol. Regulatory mechanisms promote the conversion of gluconeogenic precursors to glucose (Fig. 31.16). These mechanisms prevent the occurrence of potential futile

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cycles, which would continuously convert substrates to products while consuming energy but producing no useful result. These regulatory mechanisms inactivate the glycolytic enzymes pyruvate kinase, PFK-1, and glucokinase during fasting and promote the flow of carbon to glucose via gluconeogenesis. These mechanisms operate at the three steps where glycolysis and gluconeogenesis differ. 1. Pyruvate (derived from lactate and alanine) is converted by the gluconeogenic pathway to PEP. PEP is not reconverted to pyruvate (a potentially futile cycle) because glucagon-stimulated phosphorylation inactivates pyruvate kinase. Therefore, PEP reverses the steps of glycolysis and forms fructose 1,6-bisphosphate. 2. Fructose 1,6-bisphosphate is converted to fructose 6-phosphate by a bisphosphatase. Because the glycolytic enzyme PFK-1 is relatively inactive mainly as a result of low fructose 2,6-bisphosphate levels, fructose 6-phosphate is not converted back to fructose 1,6-bisphosphate, and a second potential futile cycle is avoided. The low fructose 2,6-bisphosphate levels are attributable in part to the phosphorylation of phosphofructokinase-2 by protein kinase A, which has been activated in response to glucagon. Fructose 6-phosphate is converted to glucose 6-phosphate. 3. Glucose 6-phosphate is dephosphorylated by glucose 6-phosphatase, forming free glucose. Because glucokinase has a high S0.5 (Km) for glucose and glucose concentrations are relatively low in liver cells during fasting, glucose is released into the blood. Therefore, the third potential futile cycle does not occur. 4. Enzymes that participate in gluconeogenesis, but not in glycolysis, are active under fasting conditions. Pyruvate carboxylase is activated by acetyl-CoA, derived from oxidation of fatty acids. PEPCK, fructose 1,6-bisphosphatase, and glucose 6-phosphatase are induced; that is, the quantity of the enzymes increases. Fructose 1,6-bisphosphatase is also active because levels of fructose 2,6-bisphosphate, an inhibitor of the enzyme, are low. 4.

STIMULATION OF LIPOLYSIS

The hormonal changes that occur during fasting stimulate the breakdown of adipose triacylglycerols (see Chapters 3, 33, and 43). Consequently, fatty acids and glycerol are released into the blood (Fig. 31.17). Glycerol serves as a source of carbon for gluconeogenesis. Fatty acids become the major fuel of the body and are oxidized to CO2 and H2O by various tissues, which enable these tissues to decrease their use of glucose. Fatty acids are also oxidized to acetyl-CoA in the liver to provide energy for gluconeogenesis. In a prolonged fast, acetyl-CoA is converted to ketone bodies, which enter the blood and serve as an additional fuel source for the muscle and the brain.

C. Blood Glucose Levels during Prolonged Fasting (Starvation) During prolonged fasting, several changes occur in fuel use. These changes cause tissues to use less glucose than they use during a brief fast and to use predominantly fuels derived from adipose triacylglycerols (i.e., fatty acids and their derivatives, the ketone bodies). Therefore, blood glucose levels do not decrease drastically. In fact, even after 5 to 6 weeks of starvation, blood glucose levels are still in the range of 65 mg/dL (Fig. 31.18; see Table 31.2). The major change that occurs in starvation is a dramatic elevation of blood ketone body levels after 3 to 5 days of fasting (see Fig. 31.18). At these levels, the brain and other nervous tissues begin to use ketone bodies and, consequently, they oxidize less glucose, requiring roughly one-third as much glucose (approximately 40 g/day) as under normal dietary conditions. As a result of reduced glucose use, the rate of gluconeogenesis in the liver decreases, as does the production of urea (see Fig. 31.18). Because in this stage of starvation, amino acids obtained from the

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Plasma Glucose

Transporter

Glucose G-6-Pase

GK

Glucose-6-P

Fructose-6-P kinase/pase

F-1,6-Pase –

F-2,6-BP

PFK-1 +

F-1,6-BP

Triose phosphates

PEP PEPCK OAA

PK

Pyruvate

Lactate

+

cAMP

PK– P (inactive)

Alanine

FIG. 31.16. Regulation of gluconeogenesis (red arrows) in the liver. GK, glucokinase; G-6-Pase, glucose 6-phosphatase; PK, pyruvate kinase; OAA, oxaloacetate; PEPCK, phosphoenolpyruvate carboxykinase; F-1,6-Pase, fructose 1,6-bisphosphatase; F-2,6-BP, fructose 2,6-bisphosphate; PFK-1, phosphofructokinase-1.

The pathophysiology leading to an elevation of blood glucose after a meal differs between patients with type 1 diabetes mellitus and those with type 2 diabetes mellitus. Di Abietes, who has type 1 disease, cannot secrete insulin adequately in response to a meal because of a defect in the ␤-cells of her pancreas. Ann Sulin, however, has type 2 disease. In this form of the disorder, the cause of glucose intolerance is more complex, involving at least a delay in the release of relatively appropriate amounts of insulin after a meal combined with a degree of resistance to the actions of insulin in skeletal muscle and adipocytes. Excessive hepatic gluconeogenesis occurs even though blood glucose levels are elevated.

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SECTION V ■ CARBOHYDRATE METABOLISM

Blood

Glycogen

Glucose

1

Liver

Insulin

Acetyl CoA

3

2 Glucose

Brain

Glucose

Glucagon

12

CO2

[ATP] FA

Acetyl CoA

11

7

Glycerol

TCA

KB Lactate

[ATP]

4 RBC Lactate

Urea

10 Adipose

9

KB

5 TG

Kidney

AA FA

8 6

AA Acetyl CoA

Protein Urine

TCA

Muscle

CO2

[ATP]

FIG. 31.17. Tissue interrelationships during fasting. (1) Blood glucose levels drop, decreasing insulin and raising blood glucagon levels. (2) Glycogenolysis is induced in the liver to raise blood glucose levels. (3) The brain uses the glucose released by the liver, as do the red blood cells (4). (5) Adipose tissues are induced to release free fatty acids and glycerol from stored triglycerides. (6) The muscle and liver use fatty acids for energy. (7) The liver converts fatty acid–derived acetyl-CoA to ketone bodies for export, which the muscles (8) and brain can use for energy. (9) Protein turnover is induced in muscle, and amino acids leave the muscle and travel to the liver for use as gluconeogenic precursors. (10) The high rate of amino acid metabolism in the liver generates urea, which travels to the kidney for excretion. (11) Red blood cells produce lactate, which returns to the liver as a substrate for gluconeogenesis. (12) The glycerol released from adipose tissue is used by the liver for gluconeogenesis. KB, ketone bodies; FA, fatty acids; AA, amino acids; TG, triacylglycerols.

degradation of existing proteins are the major gluconeogenic precursor; reducing glucose requirements in tissues reduces the rate of protein degradation and hence the rate of urea formation. Protein from muscle and other tissues is, therefore, spared because there is less need for amino acids for gluconeogenesis. Body protein, particularly muscle protein, is not primarily a storage form of fuel in the same sense as glycogen or triacylglycerol; proteins have many functions besides fuel storage. For example, proteins function as enzymes, as structural proteins, and in muscle contraction. If tissue protein is degraded to too great an extent, body function can be severely compromised. If starvation continues and no other problems such as infections occur, a starving individual usually dies because of severe protein loss that causes malfunction of major organs, such as the heart. Therefore, the increase in ketone body levels that results in the sparing of body protein allows individuals to survive for extended periods without ingesting food.

D. Summary of Sources of Blood Glucose Immediately after a meal, dietary carbohydrates serve as the major source of blood glucose (Fig. 31.19). As blood glucose levels return to the fasting range within

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577

␤-Hydroxybutyrate and Acetoacetate

Blood glucose and ketone concentration (mM)

7.0

6.0

5.0

NH4 + Total urinary nitrogen (g/d) Glucose 4.0 20

4.0

3.0

3.0 Urinary ammonia

2.0

2.0

10

Free fatty acids 1.0

1.0 Total urinary nitrogen

0

0

10

20

30

40

Days of fasting

FIG. 31.18. Changes in blood fuels during fasting. The units for fatty acids, glucose, and ketone bodies are millimolar (on left) and for urinary nitrogen and ammonia are grams per day (on right). (Modified from Linder MC, ed. Nutritional Biochemistry and Metabolism with Clinical Applications. 2nd ed. Norwalk, CT: Appleton & Lange; 1991:103. Copyright 1991 Appleton & Lange.)

Glucose oxidized (g/h)

2 hours after a meal, glycogenolysis is stimulated and begins to supply glucose to the blood. Subsequently, glucose is also produced by gluconeogenesis. During a 12-hour fast, glycogenolysis is the major source of blood glucose. Thus, it is the major pathway by which glucose is produced in the basal state (after a 12-hour fast). However, by approximately 16 hours of fasting, glycogenolysis and gluconeogenesis contribute equally to the maintenance of blood glucose. By 30 hours after a meal, liver glycogen stores are substantially depleted. Subsequently, gluconeogenesis is the primary source of blood glucose.

40

Ingested glucose

20 Glycogenolysis

Fed

Gluconeogenesis

8 16 24

2 8 16 24 32 40

Hours

Days

Fasting

Starved

FIG. 31.19. Sources of blood glucose in fed, fasting, and starved states. Note that the scale changes from hours to days. (From Ruderman NB, Aoki TT, Cahill GF Jr. In: Hanson RW, Mehlman MA, eds. Gluconeogenesis: Its Regulation in Mammalian Species. New York, NY: John Wiley & Sons; 1976:518. Copyright 1976 John Wiley & Sons.)

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SECTION V ■ CARBOHYDRATE METABOLISM

Otto Shape is able to jog for 45 minutes before eating breakfast without developing symptoms of hypoglycemia in spite of enhanced glucose use by skeletal muscle during exercise. He maintains his blood glucose level in an adequate range through hepatic glycogenolysis and gluconeogenesis.

The mechanisms that cause fats to be used as the major fuel and that allow blood glucose levels to be maintained during periods of food deprivation result in the conservation of body protein and, consequently, permit survival during prolonged fasting for periods often exceeding 1 month or more.

E. Blood Glucose Levels during Exercise During exercise, mechanisms very similar to those that are used during fasting operate to maintain blood glucose levels. The liver maintains blood glucose levels through both glucagon- and epinephrine-induced glycogenolysis and gluconeogenesis. The use of fuels by muscle during exercise, including the uptake and use of blood glucose, is discussed in Chapter 47. Remember that muscle glycogen is not used to maintain blood glucose levels; muscle cells lack glucose 6-phosphatase, so glucose cannot be produced from glucose 6-phosphate for export. CLINICAL COMMENTS Al Martini. The chronic excessive ingestion of ethanol concurrent with a recent reduction in nutrient intake caused Al Martini’s blood glucose level to decrease to 28 mg/dL. This degree of hypoglycemia caused the release of several “counterregulatory” hormones into the blood, including glucagon, growth hormone, cortisol, and epinephrine (adrenaline). Some of the patient’s signs and symptoms are primarily the result of an increase in adrenergic nervous system activity after a rapid decrease in blood glucose. The subsequent increase in epinephrine levels in the blood leads to tremulousness, excessive sweating, and rapid heart rate. Other manifestations arise when the brain has insufficient glucose, hence the term “neuroglycopenic symptoms.” Mr. Martini was confused, combative, had slurred speech, and eventually had a grand mal seizure. If he was not treated quickly by intravenous glucose administration, Mr. Martini might have lapsed into a coma. Permanent neurologic deficits and even death may result if severe hypoglycemia is not corrected within 6 to 10 hours. Emma Wheezer. The elevation in blood glucose that occurred in Emma Wheezer’s case was primarily a consequence of the large pharmacologic doses of glucocorticoid that she received in an effort to reduce the intrabronchial inflammatory reaction characteristic of asthmatic bronchospasm. Although the development of hyperglycemia in this case could be classified as a “secondary” form of diabetes mellitus (caused by the activation of liver glucose export by the drug), most patients treated with glucocorticoids do not develop glucose intolerance. Ms. Wheezer, therefore, may have a predisposition to the eventual development of “primary” diabetes mellitus. In hyperglycemia, increased amounts of glucose enter the urine, causing large amounts of water to be excreted. This “osmotic diuresis” is responsible for the increased volume of urine (polyuria) noted by the patient. Because of increased urinary water loss, the effective circulating blood volume is reduced. Therefore, less blood reaches volume-sensitive receptors in the central nervous system, which then trigger the sensation of thirst, causing increased drinking activity (polydipsia). A diabetic diet and the tapering of her steroid dose over a period of several weeks gradually returned Ms. Wheezer’s blood glucose level to the normal range. Di Abietes and Ann Sulin. Chronically elevated levels of glucose in the blood may contribute to the development of the microvascular complications of diabetes mellitus, such as diabetic retinal damage, kidney damage, and nerve damage, as well as macrovascular complications such as cerebrovascular, peripheral vascular, and coronary vascular insufficiency. The precise mechanism by which long-term hyperglycemia induces these vascular changes is not fully established.

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One postulated mechanism proposes that nonenzymatic glycation (glycosylation) of proteins in vascular tissue alters the structure and functions of these proteins. A protein that is exposed to chronically increased levels of glucose will covalently bind glucose, a process called glycation or glycosylation. This process is not regulated by enzymes (see Chapter 9). These nonenzymatically glycated proteins slowly form cross-linked protein adducts (often called advanced glycosylation end products) within the microvasculature and macrovasculature. By cross-linking vascular matrix proteins and plasma proteins, chronic hyperglycemia may cause narrowing of the luminal diameter of the microvessels in the retina (causing diabetic retinopathy), the renal glomeruli (causing diabetic nephropathy), and the microvessels supplying peripheral and autonomic nerve fibers (causing diabetic neuropathy). The same process has been postulated to accelerate atherosclerotic change in the macrovasculature, particularly in the brain (causing strokes), the coronary arteries (causing heart attacks), and the peripheral arteries (causing peripheral arterial insufficiency and possibly gangrene). The abnormal lipid metabolism associated with poorly controlled diabetes mellitus also may contribute to the accelerated atherosclerosis associated with this metabolic disorder (see Chapters 33 and 34). Until recently, it was argued that meticulous control of blood glucose levels in a patient with diabetes would not necessarily prevent or even slow these complications of chronic hyperglycemia. The results of the Diabetes Control and Complications Trial, followed by the United Kingdom Prospective Diabetes study, however, suggest that maintaining long-term tightly controlled blood glucose levels in patients with diabetes slows the progress of unregulated glycation of proteins as well as corrects their dyslipidemia. In this way, careful control may favorably affect the course of the microvascular complications of diabetes mellitus in patients such as Di Abietes and Ann Sulin.

A

Glucose Plants

Animals CO2

B

Glucose

Amino acids

C

Lipids

Other sugars

Glucose

Glycogen

D

Glucose Pyruvate Lactate

E

Glucose Pyruvate Alanine

BIOCHEMICAL COMMENTS Glucose Production. Plants are the ultimate source of the earth’s supply of glucose. Plants produce glucose from atmospheric CO2 by the process of photosynthesis (Fig. 31.20A). In contrast to plants, humans cannot synthesize glucose by the fixation of CO2. Although we have a process called gluconeogenesis, the term may really be a misnomer. Glucose is not generated anew by gluconeogenesis; compounds produced from glucose are simply recycled to glucose. We obtain glucose from the plants, including bacteria, that we eat and, to some extent, from animals in our food supply. We use this glucose both as a fuel and as a source of carbon for the synthesis of fatty acids, amino acids, and other sugars (see Fig. 31.20B). We store glucose as glycogen, which, along with gluconeogenesis, provides glucose when needed for energy (see Fig. 31.20C). Lactate, one of the carbon sources for gluconeogenesis, is actually produced from glucose by tissues that obtain energy by oxidizing glucose to pyruvate through glycolysis. The pyruvate is then reduced to lactate, released into the bloodstream, and reconverted to glucose by the process of gluconeogenesis in the liver. This process is known as the Cori cycle (Fig. 31.20D). Carbons of alanine, another carbon source for gluconeogenesis, may be produced from glucose. In muscle, glucose is converted via glycolysis to pyruvate and transaminated to alanine. Alanine from muscle is recycled to glucose in the liver. This process is known as the glucose–alanine cycle (Fig. 31.20E). Glucose also may be used to produce nonessential amino acids other than alanine, which are subsequently reconverted to glucose in the liver by gluconeogenesis. Even the essential amino acids that we obtain from dietary proteins are synthesized in plants and bacteria using glucose as the major source of carbon. Therefore, all amino acids

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F

Glucose

Glycerol Fatty acids

Dihydroxyacetone phosphate Glycerol

Fatty acids

Triacylglycerol

FIG. 31.20. A–F. Recycling of glucose.

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SECTION V ■ CARBOHYDRATE METABOLISM

Table 31.3

Diseases Discussed in Chapter 31

Disease or Disorder

Environmental or Genetic

Ethanol-induced hypoglycemia

Environmental

Asthma

Environmental

Insulin overdose

Environmental

Anorexia

Both

Weight loss

Environmental

Diabetic ketoacidosis

Environmental

Comments Ethanol, combined with poor nutrition, leads to hypoglycemia caused by excessive ethanol metabolism altering the NADH/NAD⫹ ratio in the liver. A treatment to reduce bronchoconstriction is inhalation/administration of glucocorticoids. Such treatments stimulate gluconeogenesis and can lead to hyperglycemia. Hypoglycemia as a result of insulin overdose caused by insulin stimulation of glucose transport into muscle and fat cells. The use of ketone bodies as an alternative energy source during prolonged fasting preserves muscle protein as reduced levels of glucose are now required by the nervous system. Maintenance of blood glucose levels during dieting occurs caused by glycogen release. Excessive production of ketone bodies in a patient with type 1 diabetes whose insulin levels are too low, coupled with hyperglycemia; rarely observed in type 2 diabetes.

that are converted to glucose in humans, including the essential amino acids, were originally synthesized from glucose. The production of glucose from glycerol, the third major source of carbon for gluconeogenesis, is also a recycling process. Glycerol is derived from glucose via the DHAP intermediate of glycolysis. Fatty acids are then esterified to the glycerol and stored as triacylglycerol. When these fatty acids are released from the triacylglycerol, the glycerol moiety can travel to the liver and be reconverted to glucose (see Fig. 31.20F). Key Concepts • • •

•

•

The process of glucose production is termed gluconeogenesis. Gluconeogenesis occurs primarily in the liver. The major precursors for glucose production are lactate, glycerol, and amino acids. The gluconeogenic pathway uses the reversible reactions of glycolysis, plus additional reactions to bypass the irreversible steps. Pyruvate carboxylase (pyruvate to oxaloacetate) and phosphoenolpyruvate carboxykinase (PEPCK, oxaloacetate to phosphoenolpyruvate [PEP]) bypass the pyruvate kinase step. Fructose 1,6-bisphosphatase (fructose 1,6-bisphosphate to fructose 6-phosphate) bypasses the phosphofructokinase-1 (PFK-1) step. Glucose 6-phosphatase (glucose 6-phosphate to glucose) bypasses the glucokinase step. Gluconeogenesis and glycogenolysis are carefully regulated so that blood glucose levels can be maintained at a constant level during fasting. The regulation of triglyceride metabolism is also linked to the regulation of blood glucose levels. Table 31.3 summarizes the diseases discussed in this chapter.

REVIEW QUESTIONS—CHAPTER 31 1.

A common intermediate in the conversion of glycerol and lactate to glucose is which of the following? A. Pyruvate B. Oxaloacetate C. Malate D. Glucose 6-phosphate E. Phosphoenolpyruvate

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2.

A patient presented with a bacterial infection that produced an endotoxin that inhibits phosphoenolpyruvate carboxykinase (PEPCK). In this patient, then, under these conditions, glucose production from which of the following precursors would be inhibited? A. Alanine B. Glycerol

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C. Even-chain-number fatty acids D. Phosphoenolpyruvate E. Galactose 3.

Which of the following statements best describes glucagon? A. It acts as an anabolic hormone. B. It acts on skeletal muscle, liver, and adipose tissue. C. It acts primarily on the liver and adipose tissue. D. Its concentration in the blood increases after a highcarbohydrate meal. E. Its concentration increases in the blood when insulin levels increase.

4.

Which of the following is most likely to occur in a normal individual after ingesting a high-carbohydrate meal? A. Only insulin levels decrease. B. Only insulin levels increase.

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C. Only glucagon levels increase. D. Both insulin and glucagon levels decrease. E. Both insulin and glucagon levels increase. 5.

A patient arrives at the hospital in an ambulance. He or she is currently in a coma. Before lapsing into the coma, his or her symptoms included vomiting, dehydration, low blood pressure, and a rapid heartbeat. He or she also had relatively rapid respirations, resulting in more carbon dioxide being exhaled. These symptoms are consistent with which of the following conditions? A. The patient lacks a pancreas. B. Ketoalkalosis C. Hypoglycemic coma D. Diabetic ketoacidosis E. Insulin shock in a patient with diabetes

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SECTION SIX

Lipid Metabolism

M

ost of the lipids found in the body fall into the categories of fatty acids and triacylglycerols; glycerophospholipids and sphingolipids; eicosanoids; cholesterol, bile salts, and steroid hormones; and fat-soluble vitamins. These lipids have very diverse chemical structures and functions. However, they are related by a common property—their relative insolubility in water. Fatty acids, which are stored as triacylglycerols, serve as fuels, providing the body with its major source of energy (Fig. VI.1). Glycerophospholipids and sphingolipids, which contain esterified fatty acids, are found in membranes and in blood lipoproteins at the interfaces between the lipid components of these structures and the surrounding water. These membrane lipids form hydrophobic barriers between subcellular compartments and between cellular constituents and the extracellular milieu. Polyunsaturated fatty acids containing 20 carbons form the eicosanoids, which regulate many cellular processes (Fig. VI.2). Cholesterol adds stability to the phospholipid bilayer of membranes. It serves as the precursor of the bile salts, detergentlike compounds that function in the process of lipid digestion and absorption (Fig. VI.3). Cholesterol also serves as the precursor of the steroid hormones, which have many actions, including the regulation of metabolism, growth, and reproduction. The fat-soluble vitamins are lipids that are involved in such varied functions as vision, growth, and differentiation (vitamin A), blood clotting (vitamin K), prevention of oxidative damage to cells (vitamin E), and calcium metabolism (vitamin D). Triacylglycerols, the major dietary lipids, are digested in the lumen of the intestine (Fig. VI.4). The initial digestive products, free fatty acids and 2-monoacylglycerol, are reconverted to triacylglycerols in intestinal epithelial cells, packaged in lipoproteins known as chylomicrons (so they can safely enter the circulation) and secreted into the lymph. Ultimately, chylomicrons enter the blood, serving as one of the major blood lipoproteins. Very low-density lipoprotein (VLDL) is produced in the liver, mainly from dietary carbohydrate. Lipogenesis is an insulin-stimulated process through which glucose is converted to fatty acids, which are subsequently esterified to glycerol to form the triacylglycerols that are packaged in VLDL and secreted from the liver. Thus, chylomicrons primarily transport dietary lipids, and VLDL transports endogenously synthesized lipids. The triacylglycerols of chylomicrons and VLDL are digested by lipoprotein lipase (LPL), an enzyme found attached to capillary endothelial cells (see Fig. VI.4). The fatty acids that are released are taken up by muscle and many other tissues and oxidized to CO2 and water to produce energy (see Chapter 23). After a meal, these fatty acids are taken up by adipose tissue and stored as triacylglycerols. LPL converts chylomicrons to chylomicron remnants and VLDL to intermediatedensity lipoprotein (IDL). These products, which have relatively low triacylglycerol content, are taken up by the liver by the process of endocytosis and degraded by lysosomal action. IDL may also be converted to low-density lipoprotein (LDL) by further digestion of triacylglycerol. Endocytosis of LDL occurs in peripheral tissues as well as the liver (Table VI.1) and is the major means of cholesterol transport and delivery to peripheral tissues.

Lieberman_CH32.indd 583

Gllucose Glu Glucos Glucose ucose cose ose

Fatty acids Oxidatio O Ox xida xi dat ati tion on CO C O2 + H2O

Pho Pho Ph h hos hospholipids osp os sph sp pho ph ho ollip olip oli ol ip piid pid pi ds ds and an and nd sph ssphingolipi sp sphi phin phi hing hin ing ngo ng goli gol go olip oli lip ipids ipi ip s

Tri Tria Triacylglycerol riacylglycerol iacylglycerol acylglycerol cylglycero cylglycerol l l lglyce lglycerol l ((ad dipose tissue storres))

FIG. VI.1. Summary of fatty acid metabolism.

Arachidonic A Ar rachidonic achidonic chidonic hidonic idonic donic o aci a ac acid id ((or o E or EPA) EPA A) A)

Leu eukotriene euk ukkotr uko ko kot otr otri otrie trie rie rien ien iene ene enes ne es e s

Thromboxane T Throm Thro hrom hr hro hrom omb om mbo mb box bo oxa ox xan xa ane an nes ne Prostaglandins rostaglandins ostaglandins ostaglandi ostaglandin os tag gla d di s

FIG. VI.2. Summary of eicosanoid synthesis. EPA, eicosapentaenoic acid.

Acetyl ce y Co CoA

Cholesterrol

Membranes M emb mb mbr brrane bra bran ran ane nes es s

Ste St Ste teroid eroid ro roi oid id dh hormo ho o ones Bilill salts Bile B ltl s

FIG. VI.3. Summary of cholesterol metabolism.

583

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Blood Table VI.1

Blood Lipoproteins

Chylomicrons • Produced in intestinal epithelial cells from dietary fat • Carries triacylglycerol in blood VLDL (very low-density lipoprotein) • Produced in liver mainly from dietary carbohydrate • Carries triacylglycerol in blood IDL (intermediate-density lipoprotein) • Produced in blood (remnant of VLDL after triacylglycerol digestion) • Endocytosed by liver or converted to LDL LDL (low-density lipoprotein) • Produced in blood (remnant of IDL after triacylglycerol digestion; end product of VLDL) • Contains high concentration of cholesterol and cholesterol esters • Endocytosed by liver and peripheral tissues HDL (high-density lipoprotein) • Produced in liver and intestine • Exchanges proteins and lipids with other lipoproteins • Functions in the return of cholesterol from peripheral tissues to the liver

Glucose

Lipid (TG)

Glycerol 3phosphate Chylomicrons

TG

Lymph

FA CoA

VLDL

Liver

TG Chylomicrons 2-MG + FA

L TG P L

Muscle CO2 + H2 O

TG FA

Peripheral tissues TG

Adipose Small intestine

Fed state

Capillary wall

FIG. VI.4. Overview of triacylglycerol metabolism in the fed state. TG, triacylglycerol; 2-MG, 2-monoacylglycerol; FA, fatty acid; circled TG, triacylglycerols of VLDL and chylomicrons; LPL, lipoprotein lipase.

The principal function of high-density lipoprotein (HDL) is to transport excess cholesterol obtained from peripheral tissues to the liver and to exchange proteins and lipids with chylomicrons and VLDL. The protein exchange converts “nascent” particles to “mature” particles. During fasting, fatty acids and glycerol are released from adipose triacylglycerol stores (Fig. VI.5). The glycerol travels to the liver and is used for gluconeogenesis. Only the liver contains glycerol kinase, which is required for glycerol metabolism. The fatty acids form complexes with albumin in the blood and are taken up by muscle, kidney, and other tissues, where adenosine triphosphate (ATP) is generated by their oxidation to CO2 and water. Liver also converts some of the carbon to ketone bodies, which are released into the blood. Ketone bodies are oxidized for energy in muscle, kidney, and other tissues during fasting, and in the brain during prolonged starvation (see Chapter 23). Blood Glucose

Glucose

Liver Glycerol Ketone bodies Triacylglycerols

Adipose

Ketone bodies

Fatty acids

Fasting Acetyl CoA CO2 + H2 O

Muscle 584 FIG. VI.5. Overview of triacylglycerol metabolism during fasting.

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32

Digestion and Transport of Dietary Lipids

Triacylglycerols are the major fat in the human diet, consisting of three fatty acids esterified to a glycerol backbone. Limited digestion of these lipids occurs in the mouth (lingual lipase) and stomach (gastric lipase) because of the low solubility of the substrate. In the intestine, however, the fats are emulsified by bile salts that are released from the gallbladder. This increases the available surface area of the lipids for pancreatic lipase and colipase to bind and to digest the triglycerides. Degradation products are free fatty acids and 2-monoacylglycerol. When partially digested food enters the intestine, the hormone cholecystokinin is secreted by the intestine, which signals the gallbladder to contract and release bile acids, and the pancreas to release digestive enzymes. In addition to triacylglycerols, phospholipids, cholesterol, and cholesterol esters (cholesterol esterified to fatty acids) are present in the foods we eat. Phospholipids are hydrolyzed in the intestinal lumen by phospholipase A2, and cholesterol esters are hydrolyzed by cholesterol esterase. Both of these enzymes are secreted from the pancreas. The products of enzymatic digestion (free fatty acids, glycerol, lysophospholipids, cholesterol) form micelles with bile acids in the intestinal lumen. The micelles interact with the enterocyte membrane and allow diffusion of the lipid-soluble components across the enterocyte membrane into the cell. The bile acids, however, do not enter the enterocyte at this time. They remain in the intestinal lumen, travel farther down, and are then reabsorbed and sent back to the liver by the enterohepatic circulation. This allows the bile salts to be used multiple times in fat digestion. The intestinal epithelial cells resynthesize triacylglycerol from free fatty acids and 2-monoacylglycerol and package them with a protein, apolipoprotein B-48, phospholipids, and cholesterol esters into a soluble lipoprotein particle known as a chylomicron. The chylomicrons are secreted into the lymph and eventually end up in the circulation where they can distribute dietary lipids to all tissues of the body. Once they are in the circulation, the newly released (“nascent”) chylomicrons interact with another lipoprotein particle, high-density lipoprotein (HDL) and acquire two apoproteins from HDL, apoprotein CII (apoCII) and apoprotein E (apoE). This converts the nascent chylomicron to a “mature” chylomicron. The apoCII on the mature chylomicron activates the enzyme lipoprotein lipase (LPL), which is located on the inner surface of the capillary endothelial cells of muscle and adipose tissue. The LPL digests the triglyceride in the chylomicron, producing free fatty acids and glycerol. The fatty acids enter the adjacent organs either for energy production (muscle) or fat storage (adipocyte). The glycerol that is released is metabolized in the liver. As the chylomicron loses triglyceride, its density increases and it becomes a chylomicron remnant, which is taken up by the liver by receptors that recognize apoE. In the liver, the chylomicron remnant is degraded into its component parts for further disposition by the liver.

The lymph system is a network of vessels that surround interstitial cavities in the body. Cells secrete various compounds into the lymph, and the lymph vessels transport these fluids away from the interstitial spaces in the body tissues and into the bloodstream. In the case of the intestinal lymph system, the lymph enters the bloodstream through the thoracic duct. These vessels are designed so that under normal conditions the contents of the blood cannot enter the lymphatic system. The lymph fluid is similar in composition to that of the blood but lacks the cells found in blood. 585

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586

SECTION VI ■ LIPID METABOLISM

THE WAITING ROOM Amylase is produced only in the salivary glands and the acinar cells of the pancreas. Thus, elevated serum amylase can be a sign of pancreatitis. Salivary gland lesions, such as mumps, can also increase serum amylase levels. Amylase is the enzyme that digests starch, and several automated assays can detect its activity in the serum or urine. A recent method uses an enzyme-coupled system linked to an increase in ultraviolet light absorption at 340 nm. Maltopentose (five glucose residues linked by ␣-[1,4]-bonds) is the artificial substrate, and amylase cleaves this substrate to maltose and maltotriose (two and three linked sugars, respectively). ␣-Glucosidase, which is added to the reaction mixture, cleaves the products of amylase digestion to free glucose. Once free glucose is formed, hexokinase, which has also been added to the assay mixture, converts the glucose-to-glucose 6-phosphate, and then glucose-6-phosphate dehydrogenase (another component of the assay mixture) converts glucose 6-phosphate to 6-phosphogluconate, with the production of NADPH from NADP⫹. By measuring the change in absorbance at 340 nm, it can be determined how much NADPH is produced, which can then be converted into units of amylase activity for the production of glucose. As an alternative, chromogenic assays have also been developed. One of these assays uses a starch substrate to which a chromogenic dye has been attached, which renders the starch– dye complex insoluble. As amylase cleaves the starch substrate, the smaller particles produced are soluble, which leads to the chromogen being solubilized, and a change in the color of the solution.

I.

Currently, 38% of the calories (kcal) in the typical US diet come from fat. The content of fat in the diet increased from the early 1900s until the 1960s, and then decreased as we became aware of the unhealthy effects of a high-fat diet. According to current recommendations, fat should provide no more than 30% of the total calories of a healthy diet.

Triacylglycerols are the major fat in the human diet because they are the major storage lipid in the plants and animals that constitute our food supply. Triacylglycerols contain a glycerol backbone to which three fatty acids are esterified (Fig. 32.1). The main route for digestion of triacylglycerols involves hydrolysis to fatty acids and 2-monoacylglycerol in the lumen of the intestine. However, the route depends to some extent on the chain length of the fatty acids. Lingual and gastric lipases are produced by cells at the back of the tongue and in the stomach, respectively. These lipases preferentially hydrolyze short- and medium-chain fatty acids (containing 12 or fewer carbon atoms) from dietary triacylglycerols. Therefore, they are most active in infants and young children who drink relatively large quantities of cow’s milk, which contains triacylglycerols with a high percentage of short- and mediumchain fatty acids.

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Will Sichel has had several episodes of mild back and lower extremity pain over the last year, probably caused by minor sickle cell crises. He then developed severe right upper abdominal pain radiating to his lower right chest and his right flank 36 hours before being admitted to the emergency room. He states that the pain is not like his usual crisis pain. Intractable vomiting began 12 hours after the onset of these new symptoms. He reports that his urine is the color of iced tea and his stool now has a light clay color. On physical examination, his body temperature is slightly elevated, and his heart rate is rapid. The whites of his eyes (the sclerae) are obviously jaundiced (or icteric, a yellow discoloration caused by the accumulation of bilirubin pigment). He is exquisitely tender to pressure over his right upper abdomen. The emergency room physician suspects that Will is not in sickle cell crisis but instead has either acute cholecystitis (gallbladder inflammation) or a gallstone lodged in his common bile duct, causing cholestasis (the inability of the bile from the liver to reach his small intestine). His hemoglobin level is low at 7.6 mg/dL (reference range, 12 to 16 mg/dL) but unchanged from his baseline 3 months earlier. His serum total bilirubin level is 3.2 mg/dL (reference range, 0.2 to 1.0 mg/dL), and his direct (conjugated) bilirubin level is 0.9 mg/dL (reference range, 0 to 0.2 mg/dL). Intravenous fluids were started; he was not allowed to take anything by mouth; a nasogastric tube was passed and placed on constant suction; and symptomatic therapy was started for pain and nausea. He was sent for an ultrasonographic (ultrasound) study of his upper abdomen. Al Martini has continued to abuse alcohol and to eat poorly. After a particularly heavy intake of vodka, a steady severe pain began in his upper mid abdomen. This pain spread to the left upper quadrant and eventually radiated to his mid back. He began vomiting nonbloody material and was brought to the hospital emergency room with fever, a rapid heartbeat, and a mild reduction in blood pressure. On physical examination, he was dehydrated and tender to pressure over the upper abdomen. His vomitus and stool were both negative for occult blood. Blood samples were sent to the laboratory for a variety of hematologic and chemical tests, including a measurement of serum amylase and lipase, digestive enzymes that are normally secreted from the exocrine pancreas through the pancreatic ducts into the lumen of the small intestine.

DIGESTION OF TRIACYLGLYCEROLS

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CHAPTER 32 ■ DIGESTION AND TRANSPORT OF DIETARY LIPIDS

O 1

O CH3

(CH2)7

CH

CH

(CH2)7

C

O

2 3

CH2

O

CH CH2

C

(CH2)14 CH3

O O

C

(CH2)16 CH3

FIG. 32.1. Structure of a triacylglycerol. The glycerol moiety is highlighted, and its carbons are numbered.

A. Action of Bile Salts Dietary fat leaves the stomach and enters the small intestine where it is emulsified (suspended in small particles in the aqueous environment) by bile salts (Fig. 32.2). The bile salts are amphipathic compounds (containing both hydrophobic and hydrophilic components), synthesized in the liver (see Chapter 34 for the pathway) and secreted via the gallbladder into the intestinal lumen. The contraction of the gallbladder and secretion of pancreatic enzymes are stimulated by the gut hormone cholecystokinin, which is secreted by the intestinal cells when stomach contents enter the intestine. Bile salts act as detergents, binding to the globules of dietary fat as they are broken up by the peristaltic action of the intestinal muscle. This emulsified fat, which has an increased surface area compared with unemulsified fat, is attacked by digestive enzymes from the pancreas (Fig. 32.3).

B. Action of Pancreatic Lipase The major enzyme that digests dietary triacylglycerols is a lipase produced in the pancreas. Pancreatic lipase is secreted along with another protein, colipase, in response to the release of cholecystokinin from the intestine. The peptide hormone secretin is also released by the small intestine in response to acidic materials (such as the partially digested materials from the stomach, which contains HCl) entering the duodenum. Secretin signals the liver, pancreas, and certain intestinal cells to secrete bicarbonate. Bicarbonate raises the pH of the contents of the intestinal lumen into a range (pH ⬃6) that is optimal for the action of all of the digestive enzymes of the intestine. Bile salts inhibit pancreatic lipase activity by coating the substrate and not allowing the enzyme access to the substrate. The colipase binds to the dietary fat and to the lipase, relieving the bile salt inhibition and allowing triglyceride to enter the active site of the lipase. This enhances lipase activity. Pancreatic lipase hydrolyzes fatty acids of all chain lengths from positions 1 and 3 of the glycerol moiety of the triacylglycerol, producing free fatty acids and 2-monoacylglycerol—that is,

587

The mammary gland produces milk, which is the major source of nutrients for the breastfed human infant. The fatty acid composition of human milk varies, depending on the diet of the mother. However, long-chain fatty acids predominate, particularly palmitic, oleic, and linoleic acids. Although the amount of fat contained in human milk and cow’s milk is similar, cow’s milk contains more short- and medium-chain fatty acids and does not contain the long-chain, polyunsaturated fatty acids found in human milk that are important in brain development. Although the concentrations of pancreatic lipase and bile salts are low in the intestinal lumen of the newborn infant, the fat of human milk is still readily absorbed. This is true because lingual and gastric lipases produced by the infant partially compensate for the lower levels of pancreatic lipase. The human mammary gland also produces lipases that enter the milk. One of these lipases, which require lower levels of bile salts than pancreatic lipase, is not inactivated by stomach acid and functions in the intestine for several hours.

Al Martini’s serum levels of pancreatic amylase (which digests dietary starch) and pancreatic lipase were elevated, a finding consistent with a diagnosis of acute pancreatitis. The elevated levels of these enzymes in the blood are the result of their escape from the inflamed exocrine cells of the pancreas into the surrounding pancreatic veins. The cause of this inflammatory pancreatic process in this case was related to the toxic effect of acute and chronic excessive alcohol ingestion.

O C O– HO

CH3

CH3

HO

OH Cholate

FIG. 32.2. Structure of a bile salt. The bile salts are derived from cholesterol and retain the cholesterol ring structure. They differ from cholesterol in that the rings in bile salts contain more hydroxyl groups and a polar side chain and lack a C5–C6 double bond.

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SECTION VI ■ LIPID METABOLISM

Triacylglycerol (TG) Gallbladder

Bile salts (bs)

HCO3– Pancreas

lipase colipase

bs Blood bs

bs TG Chylomicrons

bs

bs

colipase lipase

Small intestine

FA +

O R CO

Lymph OH

OH 2-Monoacylglycerol (2-MG) bs bs Micelle

bs FA 2-MG bs bs

Chylomicrons

Nascent chylomicrons FA apoB48

O R2C O

O OCR1

2–MG

bs (Ileum)

O OCR3

Triacylglycerol

PhosphoTG lipids

bs

FIG. 32.3. Digestion of triacylglycerols in the intestinal lumen. FA, fatty acid.

H2O FA3

R2C

O O

O OCR1

OH Diacylglycerol

II. ABSORPTION OF DIETARY LIPIDS

H2O

The fatty acids and 2-monoacylglycerols produced by digestion are packaged into micelles, tiny microdroplets that are emulsified by bile salts (see Fig. 32.3). For bile salt micelles to form, the concentration of bile salts in the intestinal lumen must reach 5 to 15 mM (the critical micelle concentration, CMC). Below this concentration, the bile salts are soluble; above it, micelles will form. Other dietary lipids, such as cholesterol, lysophospholipids, and fat-soluble vitamins, are also packaged in these micelles. The micelles travel through a layer of water (the unstirred water layer) to the microvilli on the surface of the intestinal epithelial cells, where the fatty acids, 2-monoacylglycerols, and other dietary lipids are absorbed, but the bile salts are left behind in the lumen of the gut. The bile salts are extensively resorbed when they reach the ileum. Greater than 95% of the bile salts are recirculated, traveling through the enterohepatic

FA1 O R2C O

OH

OH 2-Monoacylglycerol

FIG. 32.4. Action of pancreatic lipase. Fatty acids (FAs) are cleaved from positions 1 and 3 of the triacylglycerol, and a monoacylglycerol with a fatty acid at position 2 is produced.

Lieberman_CH32.indd 588

glycerol with a fatty acid esterified at position 2 (Fig. 32.4). The pancreas also produces esterases that remove fatty acids from compounds (such as cholesterol esters) and phospholipase A2 (which is released in its zymogen form and is activated by trypsin) that digests phospholipids to a free fatty acid and a lysophospholipid (Fig. 32.5).

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CHAPTER 32 ■ DIGESTION AND TRANSPORT OF DIETARY LIPIDS

A

R

C

R

O O

R2

O O H2C O C O CH H2C

O

C

R2

R1

O P

O O–

Cholesterol esterase

Cholesterol ester

B

C

O

C

Cholesterol

O O–

Phospholipase A2

X

HO

O H2C

C

C

H

O

H2C

O

HO

O–

P

R1

O

X

O–

Phospholipid

FIG. 32.5.

O

Lysophospholipid

Action of pancreatic esterases (A) and phospholipase A2 (B).

circulation to the liver, which secretes them into the bile for storage in the gallbladder and ejection into the intestinal lumen during another digestive cycle (Fig. 32.6). Short- and medium-chain fatty acids (C4 to C12) do not require bile salts for their absorption. They are absorbed directly into intestinal epithelial cells. Because they do not need to be packaged to increase their solubility, they enter the portal blood (rather than the lymph) and are transported to the liver bound to serum albumin.

589

In patients such as Will Sichel who have severe and recurrent episodes of increased red blood cell destruction (hemolytic anemia), greater than normal amounts of the red cell pigment heme must be processed by the liver and spleen. In these organs, heme (derived from hemoglobin) is degraded to bilirubin, which is excreted by the liver in the bile. If large quantities of bilirubin are presented to the liver as a consequence of acute hemolysis, the capacity of the liver to conjugate it, that is, convert it to the water-soluble bilirubin diglucuronide, can be overwhelmed. As a result, a greater percentage of the bilirubin entering the hepatic biliary ducts in patients with hemolysis is in the less water-soluble forms. In the gallbladder, these relatively insoluble particles tend to precipitate as gallstones that are rich in calcium bilirubinate. In some patients, one or more stones may leave the gallbladder through the cystic duct and enter the common bile duct. Most pass harmlessly into the small intestine and are later excreted in the stool. Larger stones, however, may become entrapped in the lumen of the common bile duct, where they cause varying degrees of obstruction to bile flow (cholestasis) with associated ductal spasm, producing pain. If adequate amounts of bile salts do not enter the intestinal lumen, dietary fats cannot readily be emulsified and digested.

Liver

Bile salts

Pancreas

Stomach

Gallbladder Common bile duct Enterohepatic circulation carrying bile salts Ileum 95%

5% Feces

FIG. 32.6. Recycling of bile salts. Bile salts are synthesized in the liver, stored in the gallbladder, secreted into the small intestine, resorbed in the ileum, and returned to the liver via the enterohepatic circulation. Under normal circumstances, 5% or less of luminal bile acids are excreted in the stool.

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When he was finally able to tolerate a full diet, Al Martini’s stools became bulky, glistening, yellow-brown, and foul smelling. They floated on the surface of the toilet water. What caused this problem?

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590

SECTION VI ■ LIPID METABOLISM

Activation of fatty acids CoASH

ATP FA

FA-AMP

FA CoA AMP

Triacylglycerol synthesis

R2C

O O

OH

FA1CoA R2C

OH

CoASH

2-Monoacylglycerol

O O

O OCR1

FA3CoA R2C

OH

CoASH

Diacylglycerol

O O

O OCR1

Apoprotein B48

O OCR3

Other lipids

Nascent chylomicrons

Triacylglycerol

FIG. 32.7. Resynthesis of triacylglycerols in intestinal epithelial cells. Fatty acids (FA), produced by digestion, are activated in intestinal epithelial cells and then esterified to the 2-monoacylglycerol produced by digestion. The triacylglycerols are packaged in nascent chylomicrons and secreted into the lymph.

Al Martini’s stool changes are characteristic of steatorrhea (fat-laden stools caused by malabsorption of dietary fats), in this case caused by a lack of pancreatic secretions, particularly pancreatic lipase, which normally digests dietary fat. Steatorrhea also may be caused by insufficient production or secretion of bile salts. Therefore, Will Sichel might also develop this condition.

Because the fat-soluble vitamins (A, D, E, and K) are absorbed from micelles along with the long-chain fatty acids and 2-monoacylglycerols, prolonged obstruction of the duct that carries exocrine secretions from the pancreas and the gallbladder into the intestine (via the common duct) could lead to a deficiency of these metabolically important substances. If the obstruction of Will Sichel’s common duct continues, he will eventually suffer from a fat-soluble vitamin deficiency.

III. SYNTHESIS OF CHYLOMICRONS Within the intestinal epithelial cells, the fatty acids and 2-monoacylglycerols are condensed by enzymatic reactions in the smooth endoplasmic reticulum to form triacylglycerols. The fatty acids are activated to fatty acyl-coenzyme A (fatty acyl-CoA) by the same process used for activation of fatty acids before ␤-oxidation (see Chapter 23). A fatty acyl-CoA then reacts with a 2-monoacylglycerol to form a diacylglycerol, which reacts with another fatty acyl-CoA to form a triacylglycerol (Fig. 32.7). The reactions for triacylglycerol synthesis in intestinal cells differ from those in liver and adipose cells in that 2-monoacylglycerol is an intermediate in triacylglycerol synthesis in intestinal cells, whereas phosphatidic acid is the necessary intermediate in other tissues. Triacylglycerols are transported in lipoprotein particles because they are insoluble in water. If triacylglycerols entered the blood directly, they would coalesce, impeding blood flow. Intestinal cells package triacylglycerols together with proteins and phospholipids in chylomicrons, which are lipoprotein particles that do not readily coalesce in aqueous solutions (Fig. 32.8). Chylomicrons also contain

Cholesterol

Phospholipid Cholesterol ester

Peripheral apoprotein

Cholesterol

Monolayer of mainly amphipathic lipids

Triacylglycerol Apoprotein B-100

Core of mainly nonpolar lipids

FIG. 32.8. Example of the structure of a blood lipoprotein. VLDL is depicted. Lipoproteins contain phospholipids and proteins on the surface, with their hydrophilic regions interacting with water. Hydrophobic molecules are in the interior of the lipoprotein. The hydroxyl group of cholesterol is near the surface. In cholesterol esters, the hydroxyl group is esterified to a fatty acid. Cholesterol esters are found in the interior of lipoproteins and are synthesized by reaction of cholesterol with an activated fatty acid (see Chapter 33).

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CHAPTER 32 ■ DIGESTION AND TRANSPORT OF DIETARY LIPIDS

100 Percentage of total weight

Intestinal lumen Brush border villae

RER (ApoB-48)

SER (TG)

Chylomicrons

TG 80 60 40 20

CE

Protein C

PL

0

FIG. 32.9. Composition of a typical chylomicron. Although the composition varies to some extent, the major component is triacylglycerol (TG). C, cholesterol; CE, cholesterol ester; PL, phospholipid.

Golgi complex

Nucleus Nascent chylomicrons

Lymph

FIG. 32.10. Formation and secretion of nascent chylomicrons. The triacylglycerol is produced in the smooth endoplasmic reticulum (SER) of intestinal epithelial cells from the digestive products, fatty acids, and 2-monoacylglycerols. The protein is synthesized in the rough endoplasmic reticulum (RER). The major apoprotein in chylomicrons is B-48. Assembly of the lipoproteins occurs in both the ER and the Golgi complex.

cholesterol and fat-soluble vitamins, but their major component is triglyceride derived from the diet (Fig. 32.9). The protein constituents of the lipoproteins are known as apoproteins. The major apoprotein associated with chylomicrons as they leave the intestinal cells is B-48 (Fig. 32.10). The B-48 apoprotein is structurally and genetically related to the B-100 apoprotein synthesized in the liver that serves as a major protein of another lipid carrier, very low-density lipoprotein (VLDL). These two apoproteins are encoded by the same gene. In the intestine, the primary transcript of this gene undergoes RNA editing (Fig. 32.11, and see Chapter 16). A stop codon is generated that causes a protein to be produced in the intestine that is 48% of the size of the protein produced in the liver—hence, the designations B-48 and B-100. The protein component of the lipoproteins is synthesized on the rough endoplasmic reticulum (RER). Lipids, which are synthesized in the smooth endoplasmic reticulum, are complexed with the proteins to form the chylomicrons (see Fig. 32.10).

IV. TRANSPORT OF DIETARY LIPIDS IN THE BLOOD By the process of exocytosis, nascent chylomicrons are secreted by the intestinal epithelial cells into the chyle of the lymphatic system and enter the blood through the thoracic duct. Nascent chylomicrons begin to enter the blood within 1 to 2 hours after the start of a meal; as the meal is digested and absorbed, they continue to enter the blood for many hours. Initially, the particles are called nascent (newborn) chylomicrons. As they accept proteins from high-density lipoprotein (HDL) within the lymph and the blood, they become “mature” chylomicrons.

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Olestra is an artificial fat substitute designed to allow individuals to obtain the taste and food consistency of fat without the calories from fat. The structure of olestra is shown and consists of a sucrose molecule to which fatty acids are esterified to the hydroxyl groups.

OR

OR OR

O

OR OR

O RO

O RO

RO

Olestra = octa-acyl sucrose R = fatty acyl group

The fatty acids attached to sucrose are resistant to hydrolysis by pancreatic lipase, so olestra passes through the intestine intact and is eliminated in the feces. As a result, no useful calories can be obtained through the metabolism of olestra, although in the mouth, the sucrose portion of the molecule imparts a sweet taste. Because olestra can pass through the digestive system unimpeded, it can also carry with it essential fat-soluble vitamins. Therefore, foods prepared with olestra are supplemented with these vitamins.

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SECTION VI ■ LIPID METABOLISM

Because of their high triacylglycerol content, chylomicrons are the least dense of the blood lipoproteins. When blood is collected from patients with certain types of hyperlipoproteinemias (high concentrations of lipoproteins in the blood) in which chylomicron levels are elevated, and the blood is allowed to stand in the refrigerator overnight, the chylomicrons float to the top of the liquid and coalesce, forming a creamy layer.

B-apoprotein gene C Liver

mRNA 5'

Transcription and RNA editing

C

3'

U

5'

3'

(Stop codon) Translation ApoB-100 N

C

N

4,536 amino acids

One manner in which individuals can lose weight is to inhibit the activity of pancreatic lipase. This results in reduced fat digestion and absorption and a reduced caloric yield from the diet. The drug orlistat is a chemically synthesized derivative of lipstatin, a natural lipase inhibitor found in certain bacteria. The drug works in the intestinal lumen and forms a covalent bond with the active-site serine residues of both gastric and pancreatic lipase, thereby inhibiting their activities. Nondigested triglycerides are not absorbed by the intestine and are eliminated in the feces. Under normal use of the drug, approximately 30% of dietary fat absorption is inhibited. Because excessive nondigested fat in the intestines can lead to gastrointestinal distress related to excessive intestinal gas formation, individuals who take this drug need to follow a diet with reduced daily intake of fat, which should be evenly distributed among the meals of the day.

Intestine

C ApoB-48 2 ,152 amino acids

FIG. 32.11. B apoprotein gene. The gene, located on chromosome 2, is transcribed and translated in liver to produce apoB-100, which is 4,536 amino acids in length (one of the longest single-polypeptide chains). In intestinal cells, RNA editing converts a cytosine (C) to a uracil (U) via deamination, producing a stop codon. Consequently, the B apoprotein of intestinal cells (apoB-48) contains only 2,152 amino acids. ApoB-48 is 48% of the size of apoB-100.

HDL transfers proteins to the nascent chylomicrons, particularly apoE and apoCII (Fig. 32.12). ApoE is recognized by membrane receptors, particularly those on the surface of liver cells, allowing apoE-bearing lipoproteins to enter these cells by endocytosis for subsequent digestion by lysosomes. ApoCII acts as an activator of lipoprotein lipase (LPL), the enzyme on capillary endothelial cells, primarily within muscle and adipose tissue, which digests the triacylglycerols of the chylomicrons and VLDL in the blood.

V. FATE OF CHYLOMICRONS The triacylglycerols of the chylomicrons are digested by LPL attached to the proteoglycans in the basement membranes of endothelial cells that line the capillary

Blood HDL ApoCII ApoA ApoE

ApoB-48 Nascent chylomicron

HDL

Ap o I CI

ApoB-48 Mature chylomicron o Ap

E

FIG. 32.12. Transfer of proteins from HDL to chylomicrons. Newly synthesized chylomicrons (nascent chylomicrons) mature as they receive apoproteins CII and E from HDL. HDL functions in the transfer of these apoproteins and also in transfer of cholesterol from peripheral tissues to the liver (see Table VI.1 in the introduction to Section Six).

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593

Lymph Chylomicrons

Capillary walls

Blood Chylomicrons

Chylomicrons

Intestinal epithelial cell

Lysosomes

Liver

Endocytic vesicle FA Cholesterol Amino acids Glycerol

Chylo- L micron P TG L CII

FA

CO2 + H2 O Muscle

Chylomicron remnants

FA + Glycerol

Receptors

FA

TG Stores

Adipose tissue

FIG. 32.13. Fate of chylomicrons. Nascent chylomicrons are synthesized in intestinal epithelial cells, secreted into the lymph, pass into the blood, and become mature chylomicrons (see Fig. 32.10). On capillary walls in adipose tissue and muscle, lipoprotein lipase (LPL) activated by apoCII digests the triacylglycerols (TGs) of chylomicrons to fatty acids and glycerol. Fatty acids (FAs) are oxidized in muscle or stored in adipose cells as triacylglycerols. The remnants of the chylomicrons are taken up by the liver by receptor-mediated endocytosis (through recognition of apoE on the remnant). Lysosomal enzymes within the hepatocyte digest the remnants, releasing the products into the cytosol.

walls (Fig. 32.13). LPL is produced by adipose cells, muscle cells (particularly cardiac muscle), and cells of the lactating mammary gland. The isozyme synthesized in adipose cells has a higher Km than the isozyme synthesized in muscle cells. Therefore, adipose LPL is more active after a meal, when chylomicrons levels are elevated in the blood. Insulin stimulates the synthesis and secretion of adipose LPL so that after a meal, when triglyceride levels increase in circulation, LPL has been upregulated (through insulin release) to facilitate the hydrolysis of fatty acids from the triglyceride. The fatty acids released from triacylglycerols by LPL are not very soluble in water. They become soluble in blood by forming complexes with the protein albumin. The major fate of the fatty acids is storage as triacylglycerol in adipose tissue. However, these fatty acids also may be oxidized for energy in muscle and other tissues (see Fig. 32.13). The LPL in the capillaries of muscle cells has a lower Km than adipose LPL. Thus, muscle cells can obtain fatty acids from blood lipoproteins whenever they are needed for energy, even if the concentration of the lipoproteins is low. The glycerol released from chylomicron triacylglycerols by LPL may be used for triacylglycerol synthesis in the liver in the fed state. The portion of a chylomicron that remains in the blood after LPL action is known as a chylomicron remnant. The remnant has lost many of the apoC molecules bound to the mature chylomicron, which exposes apoE. This remnant binds to receptors on hepatocytes (the major cells of the liver), which recognize apoE, and is taken up by the process of endocytosis. Lysosomes fuse with the endocytic vesicles, and the chylomicron remnants are degraded by lysosomal enzymes. The products of lysosomal digestion (e.g., fatty acids, amino acids, glycerol, cholesterol, phosphate) can be reused by the cell.

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Heparin is a complex polysaccharide that is a component of proteoglycans (see Chapter 49). Isolated heparin is frequently used as an anticoagulant because it binds to antithrombin III (ATIII), and the activated ATIII then binds factors necessary for clotting and inhibits them from working. As LPL is bound to the capillary endothelium through binding to proteoglycans, heparin also can bind to LPL and dislodge it from the capillary wall. This leads to loss of LPL activity and an increase of triglyceride content in the blood.

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CLINICAL COMMENTS Will Sichel. The upper abdominal ultrasound study showed a large gallstone lodged in Will Sichel’s common bile duct with dilation of this duct proximal to the stone. Will was scheduled for endoscopic retrograde cholangiopancreatography (ERCP). (An ERCP involves cannulation of the common bile duct—and, if necessary, the pancreatic duct—through a tube placed through the mouth and stomach and into the upper small intestine.) With this technique, a stone can be snared in the common duct and removed to relieve an obstruction. If common duct obstruction is severe enough, bilirubin flows back into the venous blood draining from the liver. As a consequence, serum bilirubin levels, particularly the indirect (unconjugated) fraction, increase. Tissues such as the sclerae of the eye take up this pigment, which causes them to become yellow (jaundiced, icteric). Will Sichel’s condition was severe enough to cause jaundice by this mechanism. Al Martini. Alcohol excess may produce proteinaceous plugs in the small pancreatic ducts, causing back pressure injury and autodigestion of the pancreatic acini drained by these obstructed channels. This process causes one form of acute pancreatitis. Al Martini had an episode of acute alcoholinduced pancreatitis superimposed on a more chronic alcohol-related inflammatory process in the pancreas—in other words, chronic pancreatitis. As a result of decreased secretion of pancreatic lipase through the pancreatic ducts and into the lumen of the small intestine, dietary fat was not absorbed at a normal rate, and steatorrhea (fat-rich stools) occurred. If abstinence from alcohol does not allow adequate recovery of the enzymatic secretory function of the pancreas, Mr. Martini will have to take a commercial preparation of pancreatic enzymes with meals that contain even minimal amounts of fat.

BIOCHEMICAL COMMENTS Microsomal Triglyceride Transfer Protein. The assembly of chylomicrons within the endoplasmic reticulum (ER) of the enterocyte requires the activity of microsomal triglyceride transfer protein (MTP). The protein is a dimer of two nonidentical subunits. The smaller subunit (57 kDa) is protein disulfide isomerase (PDI; see Chapter 7, Section IX.A), whereas the larger subunit (97 kDa) contains the triglyceride transfer activity. MTP accelerates the transport of triglycerides, cholesterol esters, and phospholipids across membranes of subcellular organelles. The role of PDI in this complex is not known; the disulfide isomerase activity of this subunit is not needed for triglyceride transport to occur. The lack of triglyceride transfer activity leads to the disease abetalipoproteinemia. This disease affects both chylomicron assembly in the intestine and very low-density lipoprotein (VLDL) assembly in the liver. Both particles require a B apoprotein for their assembly (apoB-48 for chylomicrons, apoB-100 for VLDL), and MTP binds to the B apoproteins. For both chylomicron and VLDL assembly, a small apoB-containing particle is first produced within the lumen of the ER. The appropriate apoB is made on the rough endoplasmic reticulum (RER) and is inserted into the ER lumen during its synthesis (see Chapter 15, Section IX). As the protein is being translated, lipid (a small amount of triglyceride) begins to associate with the protein, and the lipid association is catalyzed by MTP. This leads to the generation of small apoB-containing particles; these particles are not formed in patients with abetalipoproteinemia. Thus, it appears that MTP activity is necessary to transfer triacylglycerol formed within the ER to the apoB protein. The second stage of particle assembly is the fusion of the initial apoB particle

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595

ER Lumen ApoB-48

ApoB particle MTP LIPID

Ribosome

MTP

Larger ApoB particle

To Golgi for maturation and secretion

TG Cytoplasm

FIG. 32.14. A model of microsomal triglyceride transfer protein (MTP) action. MTP is required to transfer lipid to apoB-48 as it is synthesized and to transfer lipid from the cytoplasm to the lumen of the endoplasmic reticulum.

with triacylglycerol droplets within the ER. The role of MTP in this second step is still under investigation; it may be required for the transfer of triacylglycerol from the cytoplasm to the lumen of the ER to form this lipid droplet. These steps are illustrated in Figure 32.14. The symptoms of abetalipoproteinemia include lipid malabsorption (and its accompanying symptoms such as steatorrhea and vomiting), which can result in caloric deficiencies and weight loss. Because lipid-soluble vitamin distribution occurs through chylomicron circulation, signs and symptoms of deficiencies in the lipid-soluble vitamins may be seen in these patients. MTP inhibitors have been investigated and studied for their effect on circulating lipid and cholesterol levels. Although the inhibitors discovered to date are effective in lowering circulating lipid levels, they also initiate severe hepatic steatosis (fatty liver), an unacceptable complication that could lead to liver failure. The steatosis comes about by an accumulation of triglyceride in the liver because of the inability to form VLDL and export the triglyceride from the liver. The accumulation of triglyceride within hepatocytes will eventually interfere with hepatic function and structure. Current research for MTP inhibitors is aimed toward reducing the severity of fat accumulation in the liver (e.g., by specifically targeting the intestinal MTP without affecting the hepatic MTP). Key Concepts • • • • • • • •

• • •

Triacylglycerols are the major fat source in the human diet. Lipases (lingual lipase in the saliva and gastric lipase in the stomach) perform limited digestion of triacylglycerol before food enters the intestine. As food enters the intestine, cholecystokinin is released, which signals the gallbladder to release bile acids and the exocrine pancreas to release digestive enzymes. Within the intestine, bile salts emulsify fats, which increase their accessibility to pancreatic lipase and colipase. Triacylglycerols are degraded to form free fatty acids and 2-monoacylgylcerol by pancreatic lipase and colipase. Dietary phospholipids are hydrolyzed by pancreatic phospholipase A2 in the intestine. Dietary cholesterol esters (cholesterol esterified to a fatty acid) are hydrolyzed by pancreatic cholesterol esterase in the intestine. Micelles, consisting of bile acids and the products of fat digestion, form within the intestinal lumen and interact with the enterocyte membrane. Lipid-soluble components diffuse from the micelle into the cell. Bile salts are resorbed farther down the intestinal tract and returned to the liver by the enterohepatic circulation. The intestinal epithelial cells resynthesize triacylglycerol and package them into nascent chylomicrons for release into the circulation. Once they are in the circulation, the nascent chylomicrons interact with HDL particles and acquire two additional protein components: apoCII and apoE.

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SECTION VI ■ LIPID METABOLISM

Table 32.1

Diseases Discussed in Chapter 32

Disease or Disorder

Environmental or Genetic

Sickle cell disease

Genetic

Alcoholism

Both

•

•

• •

Comments Cholecystitis may result as a consequence of sickle cell disease because of increased red cell destruction in the spleen, and an inability of the liver to conjugate all of the bilirubin resulting from heme degradation. Pancreatitis may result from chronic alcohol abuse, leading to malabsorption problems within the intestine.

ApoCII activates lipoprotein lipase on capillary endothelium of muscle and adipose tissue, which digests the triglycerides in the chylomicron. The fatty acids released from the chylomicron enter the muscle for energy production or the fat cell for storage. The glycerol released is metabolized only in the liver. As the chylomicron loses triglyceride, its density increases, and it becomes a chylomicron remnant. Chylomicron remnants are removed from circulation by the liver through specific binding of the remnant to apoE receptors on the liver membrane. Once it is in the liver, the remnant is degraded, and the lipids are recycled. Table 32.1 summarizes the diseases discussed in this chapter.

REVIEW QUESTIONS—CHAPTER 32 1.

The most abundant component of chylomicrons is which of the following? A. apoB-48 B. Triglyceride C. Phospholipid D. Cholesterol E. Cholesterol ester

2.

The conversion of nascent chylomicrons to mature chylomicrons requires which of the following? A. Bile salts B. 2-Monoacylglycerol C. Lipoprotein lipase D. High-density lipoprotein E. Lymphatic system

3.

The apoproteins B-48 and B-100 are similar with respect to which of the following? A. They are synthesized from the same gene. B. They are derived by alternative spicing of the same hnRNA. C. apoB-48 is a proteolytic product of apoB-100. D. Both are found in mature chylomicrons. E. Both are found in very low-density lipoproteins.

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4.

Bile salts must reach a particular concentration within the intestinal lumen before they are effective agents for lipid digestion. This is because of which of the following? A. The bile salt concentration must be equal to the triglyceride concentration. B. The bile salt solubility in the lumen is a critical factor. C. The ability of bile salts to bind lipase is concentration dependent. D. The bile salts cannot be reabsorbed in the ileum until they reach a certain concentration. E. The bile salts do not activate lipase until they reach a particular concentration.

5.

Type III hyperlipidemia is caused by a deficiency of apoprotein E. Analysis of the serum of patients with this disorder would exhibit which of the following? A. An absence of chylomicrons after eating B. Higher than normal levels of VLDL after eating C. Normal triglyceride levels D. Elevated triglyceride levels E. Lower than normal triglyceride levels

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33

Synthesis of Fatty Acids, Triacylglycerols, and the Major Membrane Lipids

Fatty acids are synthesized mainly in the liver in humans, with dietary glucose serving as the major source of carbon. Glucose is converted through glycolysis to pyruvate, which enters the mitochondrion and forms both acetyl coenzyme A (acetyl-CoA) and oxaloacetate (Fig. 33.1). These two compounds condense, forming citrate. Citrate is transported to the cytosol, where it is cleaved to form acetyl-CoA, the source of carbon for the reactions that occur on the fatty acid synthase complex. The key regulatory enzyme for the process, acetyl-CoA carboxylase, produces malonyl-CoA from acetyl-CoA. The growing fatty acid chain, attached to the fatty acid synthase complex in the cytosol, is elongated by the sequential addition of two-carbon units provided by malonyl-CoA. NADPH, produced by the pentose phosphate pathway and the malic enzyme, provides reducing equivalents. When the growing fatty acid chain is 16 carbons in length, it is released as palmitate. After activation to a CoA derivative, palmitate can be elongated and desaturated to produce a series of fatty acids. Fatty acids, produced in cells or obtained from the diet, are used by various tissues for the synthesis of triacylglycerols (the major storage form of fuel) and the glycerophospholipids and sphingolipids (the major components of cell membranes). In the liver, triacylglycerols are produced from fatty acyl-CoA and glycerol 3-phosphate. Phosphatidic acid serves as an intermediate in this pathway. The triacylglycerols are not stored in the liver but rather packaged with apoproteins and other lipids in very low-density lipoprotein (VLDL) and secreted into the blood (see Fig. 33.1). In the capillaries of various tissues (particularly adipose tissue, muscle, and the lactating mammary gland), lipoprotein lipase (LPL) digests the triacylglycerols of VLDL, forming fatty acids and glycerol (Fig. 33.2). The glycerol travels to the liver where it is used. Some of the fatty acids are oxidized by muscle and other tissues. After a meal, however, most of the fatty acids are converted to triacylglycerols in adipose cells, where they are stored. These fatty acids are released during fasting and serve as the predominant fuel for the body. Glycerophospholipids are also synthesized from fatty acyl-CoA, which forms esters with glycerol 3-phosphate, producing phosphatidic acid. Various head groups are added to carbon 3 of the glycerol-3-phosphate moiety of phosphatidic acid, generating amphipathic compounds such as phosphatidylcholine, phosphatidylinositol, and cardiolipin (Fig. 33.3A). In the formation of plasmalogens and platelet-activating factor (PAF), a long-chain fatty alcohol forms an ether with carbon 1, replacing the fatty acyl ester (see Fig. 33.3B). Cleavage of phospholipids is catalyzed by phospholipases found in cell membranes, lysosomes, and pancreatic juice. Sphingolipids, which are prevalent in membranes and the myelin sheath of the central nervous system, are built on serine rather than glycerol. In the synthesis of sphingolipids, serine and palmitoyl-CoA condense, forming a

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SECTION VI ■ LIPID METABOLISM

Glucose Liver TG Glycolysis

Apoproteins

Glycerol 3-phosphate FACoA DHAP

VLDL

Palmitate NADP+

Pyruvate

Fatty acid synthase

Blood

NADPH Malonyl CoA

Pyruvate

OAA

Other lipids

Acetyl CoA carboxylase

Acetyl CoA

Citrate

OAA

Acetyl CoA

Citrate

FIG. 33.1. Lipogenesis, the synthesis of triacylglycerols (TGs) from glucose. In humans, the synthesis of fatty acids from glucose occurs mainly in the liver. Fatty acids (FAs) are converted to TG, packaged in VLDL, and secreted into the blood. OAA, oxaloacetate.

compound that is related to sphingosine. Reduction of this compound, followed by addition of a second fatty acid in amide linkage, produces ceramide. Carbohydrate groups attach to ceramide, forming glycolipids such as the cerebrosides, globosides, and gangliosides (see Fig. 33.3C). The addition of phosphocholine to ceramide produces sphingomyelin. These sphingolipids are degraded by lysosomal enzymes.

Glucose

VLDL TG

VLDL

FACoA Glycerol 3-phosphate

Liver

VLDL– TG

L P L

CO2 + H2 O

Muscle Glycerol

FA TG

Adipose

FIG. 33.2. Fate of VLDL triacylglycerol (TG). The TG of VLDL, produced in the liver, is digested by lipoprotein lipase (LPL), present on the endothelial cells lining the capillaries in adipose and skeletal muscle tissue. Fatty acids are released and either oxidized or stored in tissues as TG. Glycerol is used by the liver because hepatocytes contain glycerol kinase. FA, fatty acid (or fatty acyl group).

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A

599

O Fatty acid 1

O C Glycerol

O Fatty acid 2

O C O O P

O

Head group

O–

B

H

H

O C

C

Hydrocarbon tail

Glycerol

O Fatty acid

O C O O P

O

Head group

O–

Sphingosine

C O H N

Fatty acid

C O

O P

O

Choline

O–

Sphingosine

Sphingomyelin

O H N

O

Fatty acid

C

Carbohydrate Glycolipid

FIG. 33.3. A. General structure of a glycerophospholipid. The fatty acids are joined by ester bonds to the glycerol moiety. Various combinations of fatty acids may be present. The fatty acid at carbon 2 of the glycerol is usually unsaturated. The head group is the group attached to the phosphate on position 3 of the glycerol moiety. Choline is the most common head group, but ethanolamine, serine, inositol, or phosphatidylglycerol also may be present. The phosphate group is negatively charged, and the head group may carry a positive charge (choline and ethanolamine), or both a positive and a negative charge (serine). The inositol may be phosphorylated and thus negatively charged. B. General structure of a plasmalogen. Carbon 1 of glycerol is joined to a long-chain fatty alcohol by an ether linkage. The fatty alcohol group has a double bond between carbons 1 and 2. The head group is usually ethanolamine or choline. C. General structures of the sphingolipids. The “backbone” is sphingosine rather than glycerol. Ceramide is sphingosine with a fatty acid joined to its amino group by an amide linkage. Sphingomyelin contains phosphocholine, whereas glycolipids contain carbohydrate groups.

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SECTION VI ■ LIPID METABOLISM

THE WAITING ROOM

The dietician did a careful analysis of Percy Veere’s diet, which was indeed low in fat, adequate in protein, but excessive in carbohydrates, especially refined sugars. Percy’s total caloric intake averaged about 430 kilocalories (kcal) a day in excess of his isocaloric requirements. This excess carbohydrate was being converted to fats, accounting for Percy’s weight gain. A new diet with a total caloric content that would prevent further gain in weight was prescribed.

Cholesterol determinations in serum use a sequence of enzyme-coupled reactions. Cholesteryl esterase is used to release the fatty acids esterified to circulating cholesterol, producing free cholesterol. The second enzyme in the sequence, cholesterol oxidase, oxidizes cholesterol and reduces oxygen to form hydrogen peroxide. Horseradish peroxidase is then used to catalyze the conversion of a colorless dye to a colored dye via an oxidation–reduction reaction using the electrons from hydrogen peroxide. The intensity of the color obtained is directly proportional to the level of cholesterol in the sample. Serum triglycerides can also be determined by enzymatic means. The serum sample is treated with a bacterial lipase, which cleaves triglyceride into three fatty acids plus glycerol. The glycerol is converted to glycerol 3-phosphate by glycerol kinase, and glycerophosphate oxidase will generate dihydroxyacetone phosphate and hydrogen peroxide. As with the cholesterol determination, the hydrogen peroxide is used as a reducing agent to convert a colorless dye to a colored dye, and the intensity of color produced is directly proportional to the amount of triglyceride in the sample. Because there is always free glycerol in serum samples, a blank is also run in which the lipase step is omitted. The blank value obtained is subtracted from the sample run in the presence of lipase.

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Percy Veere’s mental depression slowly responded to antidepressant medication, to the therapy sessions with his psychiatrist, and to frequent visits from an old high school sweetheart whose husband had died several years earlier. While he was hospitalized for malnutrition, Mr. Veere’s appetite returned. By the time of discharge, he had gained back 8 of the 22 lb he had lost and weighed 133 lb. During the next few months, Mr. Veere developed a craving for “sweet foods” such as the candy he bought and shared with his new friend. After 6 months of this high-carbohydrate courtship, Percy had gained another 22 lb and now weighed 155 lb, 8 lb more than he weighed when his depression began. He became concerned about the possibility that he would soon be overweight and consulted his dietitian, explaining that he had faithfully followed his low-fat diet but had “gone overboard” with carbohydrates. He asked whether it was possible to become fat without eating fat. Cora Nari’s hypertension and heart failure have been well controlled on medication, and she has lost 10 lb since she had her recent heart attack. Her fasting serum lipid profile before discharge from the hospital indicated a significantly elevated serum low-density lipoprotein (LDL) cholesterol level of 175 mg/dL (recommended level for a patient with known coronary artery disease is 100 mg/dL or less), a serum triacylglycerol level of 280 mg/dL (reference range, 60 to 150 mg/dL), and a serum high-density lipoprotein (HDL) cholesterol level of 34 mg/dL (reference range, ⬎50 mg/dL for healthy women). While she was still in the hospital, she was asked to obtain the most recent serum lipid profiles of her older brother and her younger sister, both of whom were experiencing chest pain. Her brother’s profile showed normal triacylglycerols, moderately elevated LDL cholesterol, and significantly suppressed HDL cholesterol levels. Her sister’s profile showed only hypertriglyceridemia (high blood triacylglycerols). Colleen Lakker was born 6 weeks prematurely. She appeared normal until about 30 minutes after delivery, when her respirations became rapid at 64 breaths per minute with audible respiratory grunting. The spaces between her ribs (intercostal spaces) retracted inward with each inspiration, and her lips and fingers became cyanotic from a lack of oxygen in her arterial blood. An arterial blood sample indicated a low partial pressure of oxygen (PO2) and a slightly elevated partial pressure of carbon dioxide (PCO2). The arterial pH was somewhat suppressed, in part, from an accumulation of lactic acid secondary to the hypoxemia (a low level of oxygen in her blood). A chest radiograph showed a fine reticular granularity of the lung tissue, especially in the left lower lobe area. From these clinical data, a diagnosis of respiratory distress syndrome (RDS), also known as hyaline membrane disease, was made. Colleen was immediately transferred to the neonatal intensive care unit, where, with intensive respiration therapy, she slowly improved.

I.

FATTY ACID SYNTHESIS

Fatty acids are synthesized whenever an excess of calories is ingested. The major source of carbon for the synthesis of fatty acids is dietary carbohydrate. An excess of dietary protein also can result in an increase in fatty acid synthesis. In this case, the carbon source is amino acids that can be converted to acetyl-CoA or

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

tricarboxylic acid (TCA) cycle intermediates (see Chapter 39). Fatty acid synthesis occurs primarily in the liver in humans, although it can also occur, to a lesser extent, in adipose tissue. When an excess of dietary carbohydrate is consumed, glucose is converted to acetyl-CoA, which provides the two-carbon units that condense in a series of reactions on the fatty acid synthase complex, producing palmitate (see Fig. 33.1). Palmitate is then converted to other fatty acids. The fatty acid synthase complex is located in the cytosol and, therefore, it uses cytosolic acetyl-CoA.

Glucose Glycolysis Pyruvate

Pyruvate Pyruvate carboxylase

OAA

A. Conversion of Glucose to Cytosolic Acetyl-CoA The pathway for the synthesis of cytosolic acetyl-CoA from glucose begins with glycolysis, which converts glucose to pyruvate in the cytosol (Fig. 33.4). Pyruvate enters mitochondria, where it is converted to acetyl-CoA by pyruvate dehydrogenase and to oxaloacetate by pyruvate carboxylase. The pathway that pyruvate follows is dictated by the acetyl-CoA levels in the mitochondria. When acetyl-CoA levels are high, pyruvate dehydrogenase is inhibited and pyruvate carboxylase activity is stimulated. As oxaloacetate levels increase because of the activity of pyruvate carboxylase, oxaloacetate condenses with acetyl-CoA to form citrate. This condensation reduces the acetyl-CoA levels, which leads to the activation of pyruvate dehydrogenase and inhibition of pyruvate carboxylase. Through such reciprocal regulation, citrate can be continuously synthesized and transported across the inner mitochondrial membrane. In the cytosol, citrate is cleaved by citrate lyase to re-form acetyl-CoA and oxaloacetate. This circuitous route is required because pyruvate dehydrogenase, the enzyme that converts pyruvate to acetyl-CoA, is found only in mitochondria and because acetyl-CoA cannot directly cross the mitochondrial membrane. The NADPH required for fatty acid synthesis is generated by the pentose phosphate pathway (see Chapter 29) and from recycling of the oxaloacetate produced by citrate lyase (Fig. 33.5). Oxaloacetate is converted back to pyruvate in two steps: the reduction of oxaloacetate to malate by NAD⫹-dependent malate dehydrogenase and the oxidation and decarboxylation of malate to pyruvate by an NADP⫹-dependent malate dehydrogenase (malic enzyme) (Fig. 33.6). The pyruvate formed by malic enzyme is reconverted to citrate. The NADPH that is generated by malic enzyme, along with the NADPH generated by glucose6-phosphate and gluconate-6-phosphate dehydrogenases in the pentose phosphate pathway, is used for the reduction reactions that occur on the fatty acid synthase complex (Fig. 33.7).

Pyruvate dehydrogenase

OAA Acetyl CoA

Acetyl CoA

Citrate lyase

Citrate

Citrate

FIG. 33.4. Conversion of glucose to cytosolic acetyl-CoA. OAA, oxaloacetate.

Glucose CO2

NADPH NADP+

Pyruvate

Malic enzyme

Malate Pyruvate

Cytosolic malate dehydrogenase

COO–

NAD+ NADH H

OAA

Acetyl CoA

Citrate

Citrate lyase

OAA

Acetyl CoA

ADP + Pi

Citrate ATP

FIG. 33.5. Fate of citrate in the cytosol. Citrate lyase is also called citrate cleavage enzyme. OAA, oxaloacetate; ↑, inducible enzyme.

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NADP+ CO2 NADPH

CH2 C

OH –

Malic enzyme

CH3 C

O

COO

COO–

Malate

Pyruvate

FIG. 33.6. Reaction catalyzed by malic enzyme. This enzyme is also called the decarboxylating or NADP-dependent malate dehydrogenase.

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SECTION VI ■ LIPID METABOLISM

Glucose G-6-P NADP+ Pentose-P Glycolysis pathway F-6-P

F-1,6-BP NADPH Glyceraldehyde-3-P

DHAP

Pyruvate

NADP+ malic enzyme

Malate

Pyruvate

OAA

Acetyl CoA

OAA Acetyl CoA

Citrate

Citrate

FIG. 33.7. Sources of NADPH for fatty acid synthesis. NADPH is produced by the pentose phosphate pathway and by malic enzyme. F-1,6-BP, fructose 1,6-bisphosphate; F-6-P, fructose 6-phosphate; G-6-P, glucose 6-phosphate; OAA, oxaloacetate.

O C ~ SCoA

CH3

Acetyl CoA CO2

ATP

Biotin Acetyl CoA carboxylase

O –

O

C

ADP + Pi

O CH2

C ~ SCoA

Malonyl CoA

FIG. 33.8. Reaction catalyzed by acetylCoA carboxylase. CO2 is covalently attached to biotin, which is linked by an amide bond to the ⑀-amino group of a lysine residue of the enzyme. Hydrolysis of ATP is required for the attachment of CO2 to biotin.

Lieberman_CH33.indd 602

The generation of cytosolic acetyl-CoA from pyruvate is stimulated by elevation of the insulin/glucagon ratio after a carbohydrate meal. Insulin activates pyruvate dehydrogenase by stimulating the phosphatase that dephosphorylates the enzyme to an active form (see Chapter 20). The synthesis of malic enzyme, glucose6-phosphate dehydrogenase, and citrate lyase is induced by the high insulin/ glucagon ratio. The ability of citrate to accumulate, and to leave the mitochondrial matrix for the synthesis of fatty acids, is attributable to the allosteric inhibition of isocitrate dehydrogenase by high energy levels within the matrix under these conditions. The concerted regulation of glycolysis and fatty acid synthesis is described in Chapter 36.

B. Conversion of Acetyl-CoA to Malonyl-CoA Cytosolic acetyl-CoA is converted to malonyl-CoA, which serves as the immediate donor of the two-carbon units that are added to the growing fatty acid chain on the fatty acid synthase complex. To synthesize malonyl-CoA, acetyl-CoA carboxylase adds a carboxyl group to acetyl-CoA in a reaction that requires biotin and adenosine triphosphate (ATP) (Fig. 33.8). Acetyl-CoA carboxylase is the rate-limiting enzyme of fatty acid synthesis. Its activity is regulated by phosphorylation, allosteric modification, and induction/ repression of its synthesis (Fig. 33.9). Citrate allosterically activates acetyl-CoA carboxylase by causing the individual enzyme molecules (each composed of four subunits) to polymerize. Palmitoyl-CoA, produced from palmitate (the end product of fatty acid synthase activity), inhibits acetyl-CoA carboxylase. Phosphorylation by an adenosine monophosphate (AMP)-activated protein kinase inhibits the enzyme in the fasting state when energy levels are low. AMP is a much more sensitive indicator of low energy levels because of the adenylate kinase reaction. The [AMP]/[ATP] ratio is proportional to the square of the [ADP]/[ATP] ratio, so a fivefold change in adenosine diphosphate (ADP) levels corresponds to a 25-fold

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

603

Glucose

Citrate Insulin +

Phosphatase

Acetyl CoA

Pi +

Acetyl CoA carboxylase–P (inactive)

Acetyl CoA carboxylase –

ADP

ATP

AMP-activated protein kinase

Malonyl CoA

Palmitate

Palmitoyl CoA

FIG. 33.9. Regulation of acetyl-CoA carboxylase. This enzyme is regulated allosterically, both positively and negatively, by phosphorylation ( P ) and dephosphorylation, and by diet-induced induction ( ). It is active in the dephosphorylated state when citrate causes it to polymerize. Dephosphorylation is catalyzed by an insulin-stimulated phosphatase. Low energy levels, via activation of the AMP-activated protein kinase, cause the enzyme to be phosphorylated and inactivated. The ultimate product of fatty acid synthesis, palmitate, is converted to its CoA derivative, palmitoyl-CoA, which inhibits the enzyme. A high-calorie diet increases the rate of transcription of the gene for acetyl-CoA carboxylase, whereas a low-calorie diet reduces transcription of this gene.

ACP CH2 O –

O

P

O

O

change in AMP levels. Acetyl-CoA carboxylase is activated by dephosphorylation in the fed state when energy and insulin levels are high. A high insulin/glucagon ratio also results in induction of the synthesis of both acetyl-CoA carboxylase and the next enzyme in the pathway, fatty acid synthase.

C. Fatty Acid Synthase Complex As an overview, fatty acid synthase sequentially adds two-carbon units from malonyl-CoA to the growing fatty acyl chain to form palmitate. After the addition of each two-carbon unit, the growing chain undergoes two reduction reactions that require NADPH. Fatty acid synthase is a large enzyme composed of two identical subunits, which each have seven catalytic activities and an acyl carrier protein (ACP) segment in a continuous polypeptide chain. The ACP segment contains a phosphopantetheine residue that is derived from the cleavage of coenzyme A (Fig. 33.10). The two dimers associate in a head-to-tail arrangement, so that the phosphopantetheinyl sulfhydryl group on one subunit and a cysteinyl sulfhydryl group on another subunit are closely aligned. In the initial step of fatty acid synthesis, an acetyl moiety is transferred from acetyl-CoA to the ACP phosphopantetheinyl sulfhydryl group of one subunit and then to the cysteinyl sulfhydryl group of the other subunit. The malonyl moiety from malonyl-CoA then attaches to the ACP phosphopantetheinyl sulfhydryl group of the first subunit. The acetyl and malonyl moieties condense, with the release of the malonyl carboxyl group as CO2. A four-carbon ␤-keto acyl chain is now attached to the ACP phosphopantetheinyl sulfhydryl group (Fig. 33.11).

Lieberman_CH33.indd 603

CH2 CH3 C

CH3

CHOH Pantothenic acid

C

O

HN CH2 CH2 C

O

HN CH2 CH2 SH

Malonyl CoA

FIG. 33.10. Phosphopantetheinyl residue of the fatty acid synthase complex. The portion derived from the vitamin, pantothenic acid, is indicated. Phosphopantetheine is covalently linked to a serine residue of the acyl carrier protein (ACP) segment of the enzyme. The sulfhydryl group reacts with malonyl-CoA to form a thioester during the synthesis of fatty acids.

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604

SECTION VI ■ LIPID METABOLISM

FAS P SCoA C

SH

S

O

O

C ωCH

CH2

3

COO– Malonyl CoA P

FAS

S

S H

C

S

O

C

CH2 C

O

O

C

O

ωCH

CH2

␻CH

S

Malonyl and acetyl groups attached to different subunits of fatty acid synthase

P

3

COO–

3

NADPH + H+ NADP+

FAS P S

P S

SH

O

O

C ωCH

CH2

O

C

S

C

3

Condensation produces a β-ketoacyl group

–

COO

CH2 HCOH ␻CH

CO2

3

FAS H2O

P S

S H

C P

O

CH2

S

SH

C

C

O

ωCH

O

3

CH CH ␻CH

3

NADPH + H+

FIG. 33.11. Addition of a two-carbon unit to an acetyl group on fatty acid synthase. The malonyl group attaches to the phosphopantetheinyl residue (P) of the ACP of the fatty acid synthase. The acetyl group, which is attached to a cysteinyl sulfhydryl group, condenses with the malonyl group. CO2 is released, and a 3-ketoacyl group is formed. The carbon that eventually forms the ␻-methyl group of palmitate is labeled ␻.

NADP+

P S

SH

C

O

CH2 CH2 ␻CH

3

FIG. 33.12. Reduction of a ␤-ketoacyl group on the fatty acid synthase complex. NADPH is the reducing agent.

Lieberman_CH33.indd 604

A series of three reactions first reduces the four-carbon keto group to an alcohol, then removes water to form a double bond, and lastly reduces the double bond (Fig. 33.12). NADPH provides the reducing equivalents for these reactions. The net result is that the original acetyl group is elongated by two carbons. The four-carbon fatty acyl chain is then transferred to the cysteinyl sulfhydryl group and subsequently condenses with a malonyl group. This sequence of reactions is repeated until the chain is 16 carbons in length. At this point, hydrolysis occurs, and palmitate is released (Fig. 33.13). Palmitate is elongated and desaturated to produce a series of fatty acids. In the liver, palmitate and other newly synthesized fatty acids are converted to triacylglycerols that are packaged into very low-density lipoprotein (VLDL) for secretion.

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

FA synthase

1 s

Cy

P SH

P

P SH

S C

P

SH

SH

S

O

S O

C

␻CH

␻CH

3

3

CO2

C

S O

C

O

␻CH

CH2

C

NADPH + H+

P S C

O

S H

NADP+

3

CH2

3

COO–

O CH3

2

O

C ␻CH

P

3

SCoA

S

Acetyl CoA

ATP

CO2

O

ADP + Pi

C

CH2

Biotin

C SCoA

O

CH2

COO–

acetyl CoA carboxylase

SH

HCOH

Malonyl CoA

␻CH

3

Palmitate (C16)

4 H2O

2 NADP+ P

5

S

SH

C

O

2 NADPH

4 H2O

NADP+

3

CO2 P

2

S

SH

C

O

P

1

S C

S O

CH2

CH2

CH2

CH2

C

COO–

O

CH2

CH2

CH2

CH2

␻CH

3

P SH

C

O

CH2 CH2 ␻CH

3

P S

5

S

C

O

CH2 CH2 ␻CH

3

NADPH + H+

SH

C

O

CH2 CH2 ␻CH

3

P S C

O

S H

CH CH ␻CH

3

␻CH

3

FIG. 33.13. Synthesis of palmitate on the fatty acid synthase complex. Initially, acetyl-CoA adds to the synthase. It provides the ␻-methyl group of palmitate. Malonyl-CoA provides the two-carbon units that are added to the growing fatty acyl chain. The addition and reduction steps are repeated until palmitate is produced. (1) Transfer of the malonyl group to the phosphopantetheinyl residue. (2) Condensation of the malonyl and fatty acyl groups. (3) Reduction of the ␤-ketoacyl group. (4) Dehydration. (5) Reduction of the double bond. P, a phosphopantetheinyl group attached to the fatty acid synthase complex; Cys-SH, a cysteinyl residue on a different subunit of the fatty acid synthase.

In the liver, the oxidation of newly synthesized fatty acids back to acetylCoA via the mitochondrial ␤-oxidation pathway is prevented by malonyl-CoA. Carnitine: palmitoyl transferase I, the enzyme involved in the transport of longchain fatty acids into mitochondria (see Chapter 23), is inhibited by malony-CoA (Fig. 33.14). Malonyl-CoA levels are elevated when acetyl-CoA carboxylase is activated, and thus fatty acid oxidation is inhibited while fatty acid synthesis is proceeding. This inhibition prevents the occurrence of a futile cycle.

Where does the methyl group of the first acetyl-CoA that binds to fatty acid synthase appear in palmitate, the final product?

D. Elongation of Fatty Acids After synthesis on the fatty acid synthase complex, palmitate is activated, forming palmitoyl-CoA. Palmitoyl-CoA and other activated long-chain fatty acids can be elongated, two carbons at a time, by a series of reactions that occur in the endoplasmic reticulum (Fig. 33.15). Malonyl-CoA serves as the donor of the two-carbon units, and NADPH provides the reducing equivalents. The series of elongation reactions resemble those of fatty acid synthesis except that the fatty acyl chain is attached to coenzyme A rather than to the phosphopantetheinyl residue of an ACP. The major elongation reaction that occurs in the body involves the conversion of

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SECTION VI ■ LIPID METABOLISM

FACoA

SCoA O

C

Palmitate

CH2 COO Malonyl CoA

FA synthase

SCoA C

O

FACoA Carnitine

(CH2)14 ␻CH

CO2

3

CoASH

Palmitoyl CoA SCoA C

CPTI

–

Malonyl CoA

FA-carnitine CPTII

Acetyl CoA

O

CH2 C

FACoA

CoASH

O

(CH2)14 ␻CH

␤-Oxidation

3

NADPH NADP+ SCoA C

O

FIG. 33.14. Inhibition of carnitine: palmitoyl transferase (CPTI, also called carnitine: acyl transferase I) by malonyl-CoA. During fatty acid synthesis, malonyl-CoA levels are high. This compound inhibits CPTI, which is involved in the transport of long-chain fatty acids into mitochondria for ␤-oxidation. This mechanism prevents newly synthesized fatty acids from undergoing immediate oxidation.

CH2 H C OH (CH2)14 ␻CH

palmitoyl-CoA (C16) to stearyl-CoA (C18). Very long-chain fatty acids (C22 to C24) are also produced, particularly in the brain.

3

E. Desaturation of Fatty Acids H2O SCoA C

O

CH CH (CH2)14 ␻CH

3

NADPH NADP+ SCoA C

O

CH2 CH2 (CH2)14 ␻CH

3

Stearoyl CoA

FIG. 33.15. Elongation of long-chain fatty acids in the endoplasmic reticulum. The example shown is palmitoyl-CoA being extended to stearoyl-CoA.

Lieberman_CH33.indd 606

Desaturation of fatty acids involves a process that requires molecular oxygen (O2), NADH, and cytochrome b5. The reaction, which occurs in the endoplasmic reticulum, results in the oxidation of both the fatty acid and NADH (Fig. 33.16). The most common desaturation reactions involve the placement of a double bond between carbons 9 and 10 in the conversion of palmitic acid to palmitoleic acid (16:1, ⌬9) and the conversion of stearic acid to oleic acid (18:1, ⌬9). Other positions that can be desaturated in humans include carbons 5 and 6. Polyunsaturated fatty acids with double bonds, three carbons from the methyl end (␻3 fatty acids) and six carbons from the methyl end (␻6 fatty acids), are required for the synthesis of eicosanoids (see Chapter 35). Because humans cannot synthesize these fatty acids de novo (i.e., from glucose via palmitate), they must be present in the diet or the diet must contain other fatty acids that can be converted to these fatty acids. We obtain ␻6 and ␻3 polyunsaturated fatty acids mainly from dietary plant oils that contain the ␻6 fatty acid linoleic acid (18:2, ⌬9,12) and the ␻3 fatty acid ␣-linolenic acid (18:3, ⌬9,12,15). Linoleic and linolenic acids are thus considered essential fatty acids for the human diet. In the body, linoleic acid can be converted by elongation and desaturation reactions to arachidonic acid (20:4, ⌬5,8,11,14), which is used for the synthesis of the major class of human prostaglandins and other eicosanoids (Fig. 33.17). Elongation and desaturation of ␣-linolenic acid produces eicosapentaenoic acid (EPA; 20:5, ⌬5,8,11,14,17), which is the precursor of a different class of eicosanoids (see Chapter 35). Plants are able to introduce double bonds into fatty acids in the region between C10 and the ␻-end and, therefore, can synthesize ␻3 and ␻6 polyunsaturated fatty acids. Fish oils also contain ␻3 and ␻6 fatty acids, particularly EPA

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

607

O CH3

(CH2)n

CH2

CH2

(CH2)m

+ O2 + 2H+

C SCoA

Saturated fatty acyl CoA fatty acyl CoA desaturase

O CH3

(CH2)n

CH

CH

(CH2)m

C

2 Cyt b5 (Fe2+)

Cyt b5 reductase (FAD)

NADH + H+

2 Cyt b5 (Fe3+)

Cyt b5 reductase (FADH2)

NAD+

2 H2O SCoA

Monosaturated fatty acyl CoA

FIG. 33.16. Desaturation of fatty acids. The process occurs in the endoplasmic reticulum and uses molecular oxygen. Both the fatty acid and NADH are oxidized. Human desaturases cannot introduce double bonds between carbon 9 and the methyl end. Therefore, m is equal to or less than 7.

(␻3, 20:5, ⌬5,8,11,14,17) and docosahexaenoic acid (DHA; ␻3, 22:6, ⌬4,7,10,13,16,19). Fish obtain these fatty acids by eating phytoplankton (plants that float in water). Arachidonic acid is listed in some textbooks as an essential fatty acid. Although it is an ␻6 fatty acid, it is not essential in the diet if linoleic acid is present because arachidonic acid can be synthesized from dietary linoleic acid (see Fig. 33.17).

12

O

9

C ~ SCoA

18

Diet

The methyl group of acetyl-CoA becomes the ␻-carbon (the terminal methyl group) of palmitate. Each new two-carbon unit is added to the carboxyl end of the growing fatty acyl chain (see Fig. 33.11).

Linoleoyl CoA (⌬9,12-octadecadienoyl CoA) O2 + NADH + H+

⌬6-Desaturase

2H2O + NAD+ 12

9

6

18

C ~ SCoA O

␥-Linolenoyl CoA (⌬6,9,12-octadecatrienoyl CoA) Malonyl CoA Elongation 14

11

8

20

C ~ SCoA O

Dihomo-␥-linolenoyl CoA (⌬8,11,14-eicosatrienoyl CoA) O2 + NADH + H+

⌬5-Desaturase

2 H2O + NAD+

O 14 20

11

8

5

C ~ SCoA

Arachidonyl CoA (⌬5,8,11,14-eicosatetraenoyl CoA)

FIG. 33.17. Conversion of linoleic acid to arachidonic acid. Dietary linoleic acid (as linoleoyl-CoA) is desaturated at carbon 6, elongated by two carbons, and then desaturated at carbon 5 to produce arachidonyl-CoA.

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SECTION VI ■ LIPID METABOLISM

The gene for glycerol kinase is located on the X chromosome close to the DMD gene (which codes for dystrophin) and the NROB1 gene (which codes for a protein designated as DAX1). DAX1 is critical for the development of the adrenal glands, pituitary, hypothalamus, and gonads. Complex glycerol kinase deficiency results from a contiguous deletion of the X chromosome, which deletes all or part of the glycerol kinase gene along with the NROB1 gene and/or the DMD gene. The patient exhibits adrenal insufficiency, hyperglycerolemia, and, if the DMD gene is deleted, Duchenne muscular dystrophy. Abetalipoproteinemia, which is caused by a lack of microsomal triglyceride transfer protein (MTP; see Chapter 32) activity, results in an inability to assemble both chylomicrons in the intestine and VLDL particles in the liver. Why do some alcoholics have high VLDL levels?

The fact that several different abnormal lipoprotein profiles were found in Cora Nari and her siblings, and that each had evidence of coronary artery disease, suggests that Cora has familial combined hyperlipidemia (FCH). This diagnostic impression is further supported by the finding that Cora’s profile of lipid abnormalities appeared to change somewhat from one determination to the next, a characteristic of FCH. This hereditary disorder of lipid metabolism is believed to be quite common, with an estimated prevalence of about 1 per 100 population. The mechanisms for FCH are incompletely understood but may involve, in part, a genetically determined increase in the production of apoprotein B-100. As a result, packaging of VLDL is increased and blood VLDL levels may be elevated. Depending on the efficiency of lipolysis of VLDL by LPL, VLDL levels may be normal and LDL levels may be elevated, or both VLDL and LDL levels may be high. In addition, the phenotypic expression of FCH in any given family member may be determined by the degree of associated obesity, the diet, the use of specific drugs, or other factors that change over time. Additionally, FCH may be a multigenic trait, and even though the disease appears as an autosomal dominant trait in pedigree analysis, no genes have yet been definitively linked to this condition.

Lieberman_CH33.indd 608

The essential fatty acid linoleic acid is required in the diet for at least two reasons: (1) It serves as a precursor of arachidonic acid from which eicosanoids are produced. (2) It covalently binds another fatty acid attached to cerebrosides in the skin, forming an unusual lipid (acylglucosylceramide) that helps to make the skin impermeable to water. This function of linoleic acid may help to explain the red, scaly dermatitis and other skin problems associated with a dietary deficiency of essential fatty acids.

II. SYNTHESIS OF TRIACYLGLYCEROLS AND VERY LOW-DENSITY LIPOPROTEIN PARTICLES In liver and adipose tissue, triacylglycerols are produced by a pathway that contains a phosphatidic acid intermediate (Fig. 33.18). Phosphatidic acid is also the precursor of the glycerolipids found in cell membranes and the blood lipoproteins. The sources of glycerol 3-phosphate, which provides the glycerol moiety for triacylglycerol synthesis, differ in liver and adipose tissue. In liver, glycerol 3-phosphate is produced from the phosphorylation of glycerol by glycerol kinase or from the reduction of dihydroxyacetone phosphate (DHAP) derived from glycolysis. White adipose tissue lacks glycerol kinase and can produce glycerol 3-phosphate only from glucose via DHAP. Thus, adipose tissue can store fatty acids only when glycolysis is activated, that is, in the fed state. In both adipose tissue and liver, triacylglycerols are produced by a pathway in which glycerol 3-phosphate reacts with fatty acyl-CoA to form phosphatidic acid. Dephosphorylation of phosphatidic acid produces diacylglycerol. Another fatty acyl-CoA reacts with the diacylglycerol to form a triacylglycerol (see Fig. 33.18). The triacylglycerol, which is produced in the smooth endoplasmic reticulum of the liver, is packaged with cholesterol, phospholipids, and proteins (synthesized in the rough endoplasmic reticulum) to form VLDL (Fig. 33.19, see Fig. 32.8). The microsomal triglyceride transfer protein (MTP), which is required for chylomicron assembly, is also required for VLDL assembly. The major protein of VLDL is apoB-100. There is one long apoB-100 molecule wound through the surface of each VLDL particle. ApoB-100 is encoded by the same gene as the apoB-48 of chylomicrons, but it is a longer protein (see Fig. 32.11). In intestinal cells, RNA editing produces a smaller messenger RNA (mRNA) and thus a shorter protein, apoB-48. VLDL is processed in the Golgi complex and secreted into the blood by the liver (Figs. 33.20 and 33.21). The fatty acid residues of the triacylglycerols ultimately are stored in the triacylglycerols of adipose cells. Note that, in comparison to chylomicrons (see Chapter 32), VLDL particles are denser because they contain a lower percentage of triglyceride than do the chylomicrons. Similar to chylomicrons, VLDL particles are first synthesized in a nascent form, and on entering the circulation, they acquire apoproteins CII and E from HDL particles to become mature VLDL particles.

III. FATE OF THE VERY LOW-DENSITY LIPOPROTEIN TRIGLYCERIDE Lipoprotein lipase (LPL), which is attached to the basement membrane proteoglycans of capillary endothelial cells, cleaves the triacylglycerols in both VLDL and chylomicrons, forming fatty acids and glycerol. Apoprotein CII, which these lipoproteins obtain from HDL, activates LPL. The low Km of the muscle LPL isozyme permits muscle to use the fatty acids of chylomicrons and VLDL as a source of fuel even when the blood concentration of these lipoproteins is very low. The LPL isozyme in adipose tissue has a high Km and is most active after

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

Liver

Liver and adipose tissue

Glycerol

Glucose ATP DHAP

ADP

Glycerol kinase

NADH NAD+ Glycerol 3-phosphate

In alcoholism, NADH levels in the liver are elevated (see Chapter 25). High levels of NADH inhibit the oxidation of fatty acids. Therefore, fatty acids, mobilized from adipose tissue, are reesterified to glycerol 3-phosphate in the liver, forming triacylglycerols, which are packaged into VLDL and secreted into the blood. Elevated VLDL is frequently associated with chronic alcoholism. As alcohol-induced liver disease progresses, the ability to secrete the triacylglycerols is diminished, resulting in a fatty liver.

FA1CoA

FA2CoA O

R2C

O O

O

CR1 O

O

P

O–

O– Phosphatidic acid Pi O

R2C

O O

O CR1

OH Diacylglycerol FA3CoA O

R2C

O O

O CR1 O O CR3

Triacylglycerol Liver Adipose stores

FIG. 33.18. Synthesis of triacylglycerol in liver and adipose tissue. Glycerol 3-phosphate is produced from glucose in both tissues. It is also produced from glycerol in liver but not in adipose tissue, which lacks glycerol kinase. The steps from glycerol 3-phosphate are the same in the two tissues. FA, fatty acyl group.

a meal, when blood levels of chylomicrons and VLDL are elevated. The fate of the VLDL particle after triglyceride has been removed by LPL is the generation of an IDL (intermediate-density lipoprotein) particle, which can further lose triglyceride to become an LDL particle. The fate of the IDL and LDL particles is discussed in Chapter 34.

Lieberman_CH33.indd 609

Percent of total weight

Blood VLDL

100

VLDL

80 60

TG

40 PL

20 Protein C

CE

0

FIG. 33.19. Composition of a typical VLDL particle. The major component is triacylglycerol (TG). C, cholesterol; CE, cholesterol ester; PL, phospholipid.

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610

SECTION VI ■ LIPID METABOLISM

Glucose Glucose

Liver NADP+

G-6-P Pentose-P pathway

Glycolysis

TG Glycerol-3-P

F-6-P

FACoA

ApoB-100

Other lipids

F-1,6-BP Palmitate Glyceraldehyde-3-P

DHAP

VLDL

NADPH

Nucleus

fatty acid synthase

NADP+

Pyruvate

1

Blood

Malate Malonyl CoA Pyruvate

RER

2

OAA

OAA

Acetyl CoA

Citrate

Acetyl CoA

Citrate

3 FIG. 33.20. Synthesis of VLDL from glucose in the liver. G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-bisphosphate; FA, fatty acyl group; TG, triacylglycerol.

Golgi complex Secretory vesicle

IV. STORAGE OF TRIACYLGLYCEROLS IN ADIPOSE TISSUE After a meal, the triacylglycerol stores of adipose tissue increase (Fig. 33.22). Adipose cells synthesize LPL and secrete it into the capillaries of adipose tissue when the insulin/glucagon ratio is elevated. This enzyme digests the triacylglycerols of both chylomicrons and VLDL. The fatty acids enter adipose cells and are

Liver cell VLDL Phospholipid

Fed state

Cholesterol

TG Glucose Glucose Blood

+

DHAP

Insulin Chylomicrons

+

Remnants Apoprotein B-100

VLDL

Triacylglycerol

TG +

IDL

FIG. 33.21. Synthesis, processing, and secretion of VLDL. Proteins synthesized on the rough endoplasmic reticulum (RER) (circle 1) are packaged with triacylglycerols in the ER and Golgi complex to form VLDL (circle 2). VLDL are transported to the cell membrane in secretory vesicles (circle 3) and secreted by exocytosis. Red circles represent VLDL particles. An enlarged VLDL particle is depicted at the bottom of the figure.

Lieberman_CH33.indd 610

LDL

CII

L P L

Glycerol 3phosphate LPL

FA Liver

Glycerol

FACoA FA

Adipose cell

FIG. 33.22. Conversion of the fatty acid (FA) from the triacylglycerols (TG) of chylomicrons and VLDL to the TG stored in adipose cells. Note that insulin stimulates both the transport of glucose into adipose cells and the synthesis and secretion of lipoprotein lipase (LPL) from the cells. Glucose provides the glycerol 3-phosphate for TG synthesis. Apoprotein CII activates LPL.

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

activated, forming fatty acyl-CoA, which reacts with glycerol 3-phosphate to form triacylglycerol by the same pathway used in the liver (see Fig. 33.18). Because adipose tissue lacks glycerol kinase and cannot use the glycerol produced by LPL, the glycerol travels through the blood to the liver, which uses it for the synthesis of triacylglycerol. In adipose cells, under fed conditions, glycerol 3-phosphate is derived from glucose. In addition to stimulating the synthesis and release of LPL, insulin stimulates glucose metabolism in adipose cells. Insulin leads to the activation of the glycolytic enzyme phosphofructokinase-1 by the activation of the kinase activity of phosphofructokinase-2, which increases fructose 2,6-bisphosphate levels. Insulin also stimulates the dephosphorylation of pyruvate dehydrogenase, so that the pyruvate produced by glycolysis can be oxidized in the TCA cycle. Furthermore, insulin stimulates the conversion of glucose to fatty acids in adipose cells, although the liver is the major site of fatty acid synthesis in humans.

V. RELEASE OF FATTY ACIDS FROM ADIPOSE TRIACYLGLYCEROLS During fasting, the decrease of insulin and the increase of glucagon cause cyclic adenosine monophosphate (cAMP) levels to rise in adipose cells, stimulating lipolysis (Fig. 33.23). Protein kinase A phosphorylates hormone-sensitive lipase to produce a more active form of the enzyme. Hormone-sensitive lipase, also known as adipose triacylglycerol lipase, cleaves a fatty acid from a triacylglycerol. Subsequently, other lipases complete the process of lipolysis, and fatty acids and glycerol are released into the blood. Simultaneously, to regulate the amount of fatty acids released into circulation, triglyceride synthesis occurs along with glyceroneogenesis (see Section VI). The fatty acids, which travel in the blood complexed with albumin, enter cells of muscle and other tissues, where they are oxidized to CO2 and water to produce energy. During prolonged fasting, acetyl-CoA produced by ␤-oxidation of fatty

611

Fatty acids for VLDL synthesis in the liver may be obtained from the blood or they may be synthesized from glucose. In a healthy individual, the major source of the fatty acids of VLDL triacylglycerol is excess dietary glucose. In individuals with diabetes mellitus, fatty acids mobilized from adipose triacylglycerols in excess of the oxidative capacity of tissues are a major source of the fatty acids reesterified in liver to VLDL triacylglycerol. These individuals frequently have elevated levels of blood triacylglycerols.

Because the fatty acids of adipose triacylglycerols come both from chylomicrons and VLDL, we produce our major fat stores both from dietary fat (which produces chylomicrons) and dietary sugar (which produces VLDL). An excess of dietary protein also can be used to produce the fatty acids for VLDL synthesis. The dietician carefully explained to Percy Veere that we can become fat from eating excess fat, excess sugar, or excess protein.

In some cases of hyperlipidemia, LPL is defective. If a blood lipid profile is performed on patients with an LPL deficiency, which lipids will be elevated?

Fasted state Lipase (inactive)

TG

Blood Protein kinase A

Hormonesensitive lipase– P (active)

+

cAMP +

Low insulin/high glucagon

ATP Other lipases

FA FA FA Glycerol

FA FA FA Glycerol

Adipose cell

FIG. 33.23. Mobilization of adipose triacylglycerol (TG). In the fasted state, when insulin levels are low and glucagon is elevated, intracellular cyclic adenosine monophosphate (cAMP) increases and activates protein kinase A, which phosphorylates hormone-sensitive lipase (HSL). Phosphorylated HSL is active and initiates the breakdown of adipose TG. Reesterification of fatty acids does occur, along with glyceroneogenesis, in the fasted state to regulate the release of fatty acids from the adipocyte. HSL is also called triacylglycerol lipase. FA, fatty acid.

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SECTION VI ■ LIPID METABOLISM

Individuals with a defective LPL have high blood triacylglycerol levels. Their levels of chylomicrons and VLDL, which contain large amounts of triacylglycerols, are elevated because they are not digested at the normal rate by LPL.

acids in the liver is converted to ketone bodies, which are released into the blood. The glycerol derived from lipolysis in adipose cells is used by the liver during fasting as a source of carbon for gluconeogenesis.

VI. REGULATION OF FATTY ACID RELEASE BY GLYCERONEOGENESIS Recently, another important mechanism that regulates free fatty acid release from the adipocyte has been described. Fatty acids within the adipocyte are also used to resynthesize triglyceride as a mechanism to limit fatty acid release into the circulation. Resynthesis of triglycerides (which is occurring at the same time that hormone-sensitive lipase is active) depends on an adequate supply of glycerol 3-phosphate, which is in turn dependent on adipose glyceroneogenesis (synthesis of new glycerol). Thus, within the adipocyte, hormone-sensitive lipase initiates the conversion of triglyceride to glycerol and three free fatty acids. The glycerol is sent to the liver for gluconeogenesis; however, within the adipocyte, glycerol 3-phosphate is synthesized from the carbons of amino acids, lactate, or pyruvate (Fig. 33.24). The key enzyme that allows this to occur is phosphoenolpyruvate carboxykinase (PEPCK), which is induced in the adipocyte by elevated cAMP levels (through the cAMP response element binding protein [CREB] family of transcription factors). Elevated adipocyte cAMP levels occur in response to glucagon or epinephrine, two signals that indicate the adipocyte should release fatty acids into the circulation. The pyruvate formed in the adipocyte from lactate is converted to oxaloacetate by pyruvate carboxylase. Amino acid carbons that are metabolized to TCA cycle intermediates are also converted to oxaloacetate. The oxaloacetate is converted to phosphoenolpyruvate (PEP) via the induced PEPCK, and the PEP follows the gluconeogenic pathway to DHAP, which is then reduced to glycerol 3-phosphate. The glycerol 3-phosphate is used to reesterify free fatty acids before they leave the adipocyte, thus modulating free fatty acid release by the fat cells. It has been demonstrated that as much as 30% to 40% of the released fatty acids are reesterified to triglyceride without ever leaving the adipocyte.

Adipocyte: Fasting Conditions 40% HSL TG

60% FFA

To tissues

Glycerol

To liver

Glycerol 3-phosphate NAD+

DHAP

Pi

G3P

NAD+

NADH 2PG

NADH 1,3 BPG

3PG

ATP ADP PEP GDP GTP CO2

PEPCK

OAA

Lactate Pyruvate Amino acids

FIG. 33.24. Recycling of free fatty acids and the regeneration of glycerol 3-phosphate within the adipocyte under fasting conditions. OAA, oxaloacetate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; G3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; TG, triglyceride; HSL, hormone-sensitive lipase; FFA, free fatty acids; PEPCK, phosphoenolpyruvate carboxykinase.

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613

Data to support the important role of glyceroneogenesis in modulating fatty acid release from the adipocyte have been obtained in mice. Mice that have been engineered so that they no longer express adipocyte PEPCK have reduced triglyceride levels and increased levels of serum free fatty acids (if PEPCK is not expressed, glyceroneogenesis will not occur, the levels of reesterification will be low, so increased levels of fatty acids will be released into the circulation). Mice that have been engineered to overproduce PEPCK have dysregulated glyceroneogenesis (too much reesterification) and are obese, with reduced free fatty acid release upon hormone-sensitive lipase activation. The role of glyceroneogenesis in overall lipid metabolism, and type 2 diabetes, is currently a subject of intense investigation.

VII. METABOLISM OF GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS Fatty acids, obtained from the diet or synthesized from glucose, are the precursors of glycerophospholipids and of sphingolipids (Fig. 33.25). These lipids are major components of cellular membranes. Glycerophospholipids are also components of blood lipoproteins, bile, and lung surfactant. They are the source of the polyunsaturated fatty acids, particularly arachidonic acid, that serve as precursors of the eicosanoids (e.g., prostaglandins, thromboxanes, leukotrienes; see Chapter 35). Ether glycerophospholipids differ from other glycerophospholipids in that the alkyl or alkenyl chain (an alkyl chain with a double bond) is joined to carbon 1 of the glycerol moiety by an ether rather than an ester bond. Examples of ether lipids are the plasmalogens and platelet-activating factor (PAF). Sphingolipids are particularly important in signal transduction and in forming the myelin sheath surrounding nerves in the central nervous system. In glycerolipids and ether glycerolipids, glycerol serves as the backbone to which fatty acids and other substituents are attached. Sphingosine, derived from serine, provides the backbone for sphingolipids.

Glycerolipids

Phospholipids

Adipose stores Blood lipoproteins

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol bisphosphate (PIP2) Phosphatidylglycerol Cardiolipin

Fatty acid

Fatty acid

P

Head group

Glycolipids

Sphingomyelin

Cerebrosides Sulfatides Globosides Gangliosides

Ether Glycero

Fatty acid

Sphingophospholipids

Plasmalogens Platelet activating factor

Fatty acid Glycerol

Glycerol

Fatty acid

Ether glycerolipids

Fatty acid

P

Head group

Fatty acid

P

Head group

Sphingosine

Glycerophospholipids

Sphingosine

Triacylglycerols

Sphingolipids

Fatty acid

Carbohydrate

FIG. 33.25. Types of glycerolipids and sphingolipids. Glycerolipids contain glycerol, and sphingolipids contain sphingosine. The category of phospholipids overlaps both glycerolipids and sphingolipids. The head groups include choline, ethanolamine, serine, inositol, glycerol, and phosphatidylglycerol. The carbohydrates are monosaccharides (which may be sulfated), oligosaccharides, and oligosaccharides with branches of N-acetylneuraminic acid. P, phosphate.

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SECTION VI ■ LIPID METABOLISM

A. Synthesis of Phospholipids Containing Glycerol 1.

Phosphatidylcholine (lecithin) is not required in the diet because it can be synthesized in the body. The components of phosphatidylcholine (including choline) all can be produced, as shown in Figure 33.27. A pathway for de novo choline synthesis from glucose exists, but the rate of synthesis is inadequate to provide for the necessary amounts of choline. Thus, choline has been classified as an essential nutrient, with an adequate intake (AI) of 425 mg/day in women and 550 mg/day in men. Because choline is widely distributed in the food supply, primarily in phosphatidylcholine (lecithin), deficiencies have not been observed in humans on a normal diet. Deficiencies may occur, however, in patients on total parenteral nutrition (TPN), that is, supported solely by intravenous feeding. The fatty livers that have been observed in these patients probably result from a decreased ability to synthesize phospholipids for VLDL formation.

GLYCEROPHOSPHOLIPIDS

The initial steps in the synthesis of glycerophospholipids are similar to those of triacylglycerol synthesis. Glycerol 3-phosphate reacts with two activated fatty acids to form phosphatidic acid. Two different mechanisms are then used to add a head group to the molecule (Fig. 33.26). A head group is a chemical group, such as choline or serine, attached to carbon 3 of a glycerol moiety that contains hydrophobic groups, usually fatty acids, at positions 1 and 2. Head groups are hydrophilic, either charged or polar. The head groups all contain a free hydroxyl group, which is used to link to the phosphate on carbon 3 of the glycerol backbone. In the first mechanism, phosphatidic acid is cleaved by a phosphatase to form diacylglycerol (DAG). DAG then reacts with an activated head group. In the synthesis of phosphatidylcholine, the head group choline is activated by combining with cytidine triphosphate (CTP) to form cytidine diphosphate (CDP)-choline (Fig. 33.27). Phosphocholine is then transferred to carbon 3 of DAG, and cytidine monophosphate (CMP) is released. Phosphatidylethanolamine is produced by a similar reaction involving CDP-ethanolamine. Various types of interconversions occur among these phospholipids (see Fig. 33.27). Phosphatidylserine is produced by a reaction in which the ethanolamine moiety of phosphatidylethanolamine is exchanged for serine. Phosphatidylserine can be converted back to phosphatidylethanolamine by a decarboxylation reaction. Phosphatidylethanolamine can be methylated to form phosphatidylcholine (see Chapter 40). In the second mechanism for the synthesis of glycerolipids, phosphatidic acid reacts with CTP to form CDP-DAG (Fig. 33.28). This compound can react with phosphatidylglycerol (which itself is formed from the condensation of CDP-DAG and glycerol 3-phosphate) to produce cardiolipin or with inositol to produce phosphatidylinositol. Cardiolipin is a component of the inner mitochondrial membrane. Phosphatidylinositol can be phosphorylated to form phosphatidylinositol 4,5-bisphosphate (PIP2), which is a component of cell membranes. In response to signals such as the binding of hormones to membrane receptors, PIP2 can be cleaved to form the second messengers DAG and inositol triphosphate (IP3) (see Chapter 11). 2.

ETHER GLYCEROLIPIDS

The ether glycerolipids are synthesized from the glycolytic intermediate DHAP. A fatty acyl-CoA reacts with carbon 1 of DHAP, forming an ester (Fig. 33.29). This

Phosphatidic acid Phosphorylated Head group

1

2 Pi

CTP Diacylglycerol CDP-Head group CMP Glycerophospholipid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine

CTP

PPi

CDP–Diacylglycerol Head group CMP Glycerophospholipid Phosphatidylinositol Cardiolipin Phosphatidylglycerol

FIG. 33.26. Strategies for addition of the head group to form glycerophospholipids. In both cases, cytidine triphosphate (CTP) is used to drive the reaction.

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CHAPTER 33 ■ SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS

O

R2

O

CH2

C O

CH

C R1

O

CH2OH Diacylglycerol

CDP-ethanolamine

CDP-Choline

CMP

CMP

O O R2

C O

1 CH 2 2

C R1

O

CH2

O

P

O

O

3 SAM Ethanolamine

O

CH

3

O

R2

2 3

CH2NH3

O

2

C O

+

CH2

1 CH

C R1 Choline

O

CH CH2

O

–

P

O

CH2

CH2

CH3 +

–

O

O

Phosphatidylethanolamine

N

CH3

CH3

Phosphatidycholine

Serine

CO2

Ethanolamine O O R2

C O

1 CH 2 2

O

3

O

+

O

CH

CH2

Serine

C R1

NH3

P

O

CH2

COO–

CH

O– Phosphatidylserine

FIG. 33.27. Synthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The multiple pathways reflect the importance of phospholipids in membrane structure. For example, phosphatidylcholine (PC) can be synthesized from dietary choline when it is available. If choline is not available, PC can be made from dietary carbohydrate, although the amount synthesized is inadequate to prevent choline deficiency. SAM is S-adenosylmethionine, a methyl group donor for many biochemical reactions (see Chapter 40).

Phosphatidic acid CTP CDP-diacylglycerol Phosphatidylglycerol

Inositol CMP

CMP

Cardiolipin O

O

R2

O

CH2

C O

C

O

CH2

CH2

C R1 H

O

H O

P

Phosphatidylinositol (PI) –

O

C

O

OH

CH2

O

P O R4

O O C

H O

CH2

O

C

C

O

CH2

O R3

R2

C O

1 CH

2

2 3

O

CH2

–

C R1 OH

O

CH O

2

P

O H –

O

O

OH 3

H H

1

4

H H

6

Phosphatidylglycerol

OH

OH OH 5

H

Inositol Kinase

Diphosphatidylglycerol (cardiolipin)

Phosphatidylinositol bisphosphate (PIP2)

FIG. 33.28. Synthesis of cardiolipin and phosphatidylinositol.

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SECTION VI ■ LIPID METABOLISM

O O R

R1 CH2 CH2

DHAP

C ˜ SCoA 2 NADPH

C ˜ SCoA

O CH2

O

CH2

OH

O

C O CH2

R1 CH2

C R

O

P O– O– O

R

C

O– CH2 C

O

O

CH2

O

R2

C

O

C

R1

O

O–

P O

CH2

CH2

O

Reduction of C2 to an alcohol, addition of a fatty acid and dephosphorylation O

CH2

–

CH2

CH2

R1

CH2

CH2

R1 Alkyl group

H

CH2

OH

CH2

O

CDP-ethanolamine O R2

C

O

C

H

CH2

O O

P

Ethanolamine –

O

NADPH O2 O R2

C

CH2 O

C

O

H

CH2

CH

CH

R1 Alkenyl group

O O

P

Ethanolamine –

O

Ethanolamine plasmalogen

FIG. 33.29. Synthesis of a plasmalogen.

fatty acyl group is exchanged for a fatty alcohol, produced by reduction of a fatty acid. Thus, the ether linkage is formed. Then the keto group on carbon 2 of the DHAP moiety is reduced and esterified to a fatty acid. Addition of the head group proceeds by a series of reactions analogous to those for synthesis of phosphatidylcholine. Formation of a double bond between carbons 1 and 2 of the alkyl group produces a plasmalogen. Ethanolamine plasmalogen is found in myelin and choline plasmalogen in heart muscle. PAF is similar to choline plasmalogen except that an acetyl group replaces the fatty acyl group at carbon 2 of the glycerol moiety, and the alkyl group on carbon 1 is saturated. PAF is released from phagocytic blood cells in response to various stimuli. It causes platelet aggregation, edema, and hypotension, and it is involved in the allergic response. Plasmalogen synthesis occurs within peroxisomes, and in individuals with Zellweger syndrome (a defect in peroxisome biogenesis), plasmalogen synthesis is compromised. If it is severe enough, this syndrome leads to death at an early age.

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617

The respiratory distress syndrome (RDS) of a premature infant such as Colleen Lakker is, in part, related to a deficiency in the synthesis of a substance known as lung surfactant. The major constituents of surfactant are dipalmitoylphosphatidylcholine, phosphatidylglycerol, apoproteins (surfactant proteins: Sp-A, Sp-B, and Sp-C), and cholesterol. O H2C O

O CH3

(CH2)14

C

O

CH

H2C O

C

(CH2)14

CH3

O P

CH3 O

CH2

CH2

+

O–

N

CH3

CH3

Dipalmitoylphosphatidylcholine, the major component of lung surfactant

These components of lung surfactant normally contribute to a reduction in the surface tension within the air spaces (alveoli) of the lung, preventing their collapse. The premature infant has not yet begun to produce adequate amounts of lung surfactant. Inflated terminal sac (alveolus) Without lung surfactant, sac collapses. Ten times the normal pressure is needed for re-inflation.

Expiration

Inspiration

Lung surfactant reduces the surface tension of water (fluid) lining the surface of the alveolar sac, preventing collapse.

Less pressure is needed to re-inflate sac when surfactant is present.

The effect of lung surfactant

B. Degradation of Glycerophospholipids Phospholipases located in cell membranes or in lysosomes degrade glycerophospholipids. Phospholipase A1 removes the fatty acyl group on carbon 1 of the glycerol moiety, and phospholipase A2 removes the fatty acid on carbon 2 (Fig. 33.30). The C2 fatty acid in cell membrane phospholipids is usually an unsaturated fatty acid, which is frequently arachidonic acid. It is removed in response to signals for the synthesis of eicosanoids. The bond joining carbon 3 Phospholipase A1 O 1

CH2

O

C O

Phospholipase C

2

CH

3

CH2

O

Phospholipase A2

O O

P O

C

O

Head group

–

Phospholipase D

FIG. 33.30.

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Bonds cleaved by phospholipases.

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SECTION VI ■ LIPID METABOLISM

CH2OH +

HC

NH3

COO– Serine

+ CH3

O

(CH2)14

C ˜ SCoA

Palmitoyl CoA HSCoA, CO2

PLP CH2OH

H

C

NH2

C

O

From serine

C. Sphingolipids

CH2 CH2

From palmitate

(CH2)12 CH3 NADPH

Reduction to form dihydrosphingosine

NADP+

CH2OH H

C

NH2

H

C

OH

CH2 CH2 (CH2)12 CH3 FACoA

Addition of a fatty acyl group CH2OH

H

C

H

C

NH OH

C

O

CH2

(CH2)n

CH2

CH3

It has become increasingly apparent in recent years that adipose tissue does more than just store triglyceride; it is also an active endocrine organ that secretes a variety of factors to regulate both glucose and fat metabolism. Two of the best characterized factors are leptin and adiponectin.

CH3 FAD FADH2 CH2OH C

H

C

Oxidation

A. Leptin

NH OH

C

O

CH

(CH2)n

CH

CH3

(CH2)12 CH3 Ceramide

FIG. 33.31. Synthesis of ceramide. The changes that occur in each reaction are highlighted. PLP, pyridoxal phosphate.

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Sphingolipids serve in intercellular communication and as the antigenic determinants of the ABO blood groups. Some are used as receptors by viruses and bacterial toxins, although it is unlikely that this was the purpose for which they originally evolved. Before the functions of the sphingolipids were elucidated, these compounds appeared to be inscrutable riddles. They were, therefore, named for the Sphinx of Thebes, who killed passersby who could not solve her riddle. The synthesis of sphingolipids begins with the formation of ceramide (Fig. 33.31). Serine and palmitoyl-CoA condense to form a product that is reduced. A very longchain fatty acid (usually containing 22 carbons) forms an amide with the amino group, a double bond is generated, and ceramide is formed. Ceramide reacts with phosphatidylcholine to form sphingomyelin, a component of the myelin sheath (Fig. 33.32) and the only sphingosine-based phospholipid. Ceramide also reacts with uridine diphosphate (UDP)-sugars to form cerebrosides, which contain a single monosaccharide, usually galactose or glucose. Galactocerebroside may react with 3⬘-phosphoadenosine 5⬘-phosphosulfate (PAPS, an active sulfate donor; Fig. 33.33) to form sulfatides, the major sulfolipids of the brain. Additional sugars may be added to ceramide to form globosides, and gangliosides are produced by the addition of N-acetylneuraminic acid (NANA) as branches from the oligosaccharide chains (see Fig. 33.32 and Chapter 30). Sphingolipids are degraded by lysosomal enzymes (see Chapter 30). Deficiencies of these enzymes result in a group of lysosomal storage diseases known as the sphingolipidoses.

VIII. THE ADIPOCYTE AS AN ENDOCRINE ORGAN

(CH2)12

H

of the glycerol moiety to phosphate is cleaved by phospholipase C. Hormonal stimuli activate phospholipase C, which hydrolyzes PIP2 to produce the second messengers DAG and IP3. The bond between the phosphate and the head group is cleaved by phospholipase D, producing phosphatidic acid and the free alcohol of the head group. Phospholipase A2 provides the major repair mechanism for membrane lipids damaged by oxidative free radical reactions. Arachidonic acid, which is a polyunsaturated fatty acid, can be peroxidatively cleaved in free radical reactions to malondialdehyde and other products. Phospholipase A2 recognizes the distortion of membrane structure caused by the partially degraded fatty acid and removes it. Acyltransferases then add back a new arachidonic acid molecule.

Leptin was initially discovered in an obese mouse model as a circulating factor that, when added to a genetically obese mouse (ob/ob), resulted in a loss of weight. Leptin binds to a receptor that is linked to janus kinase (JAK) (see Chapter 11), so leptin’s signal is transmitted by variations in the activity of the signal transducer and activator transcription (STAT) factors. Leptin is released from adipocytes as their triglyceride levels increase and binds to receptors in the hypothalamus, which leads to the release of neuropeptides that signal a cessation of eating (anorexigenic factors). Giving leptin to leptin-deficient patients will result in a weight loss, but administering leptin to obese patients does not have the same effect. It is believed that the lack of a leptin effect is caused by the development of leptin resistance in many obese patients. Leptin resistance could result from the constant stimulation

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O Ceramide

O

P

OCH2

CH2

CH3 CH3 CH3

+

N

O–

Phosphatidylcholine

619

Sphingomyelin

DAG CH2OH H

C

H

C

NH C

OH

S

O

CH

(CH2)n

CH

CH3

PAP

UDP-Galactose Ceramide

Ceramide

Sulfatide

Gal

Galactocerebroside Ceramide

(CH2)12

Glc

Gal

Globoside

UDP-Glucose

CH3

–

Gal 3 SO3

Ceramide Glucocerebroside UDP-sugars CMP-NANA

Ceramide

Glc Gal GalNAc NANA

Ganglioside

FIG. 33.32. Synthesis of sphingolipids from ceramide. Phosphocholine or sugars add to the hydroxymethyl group of ceramide (in yellow box) to form sphingomyelins, cerebrosides, sulfatides, globosides, and gangliosides. The ganglioside shown in the figure is GM2. Gal, galactose; Glc, glucose; GalNAc, N-acetylgalactosamine; NANA, N-acetylneuraminic acid.

of the leptin receptors in obese individuals, leading to receptor desensitization. Another possibility is leptin-induced synthesis of factors that block leptin-induced signal transduction. As an example, leptin induces the synthesis of SOCS3 (suppressor of cytokine signaling-3), a factor that antagonizes STAT activation. Long-term leptin stimulation may lead to constant expression of SOCS3, which would result in a diminished cellular response to leptin. O– –

O3S

O

P

O

CH2 O

Ad

O HO

OH ATP ADP

O– –

O3S O P O –

O

O

CH2 O

Ad

–

O P

O

OH

O 3'-Phosphoadenosine 5'-phosphosulfate (PAPS-"active sulfate")

FIG. 33.33. The synthesis of 3⬘-phosphoadenosine 5⬘-phosphosulfate (PAPS), an active sulfate donor. PAPS donates sulfate groups to cerebrosides to form sulfatides and is also involved in glycosaminoglycan biosynthesis (see Chapter 49). Ad, adenine.

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SECTION VI ■ LIPID METABOLISM

B. Adiponectin Adiponectin is the most abundantly secreted hormone from the adipocyte. Unlike leptin, adiponectin secretion is reduced as the adipocyte gets larger. The reduced secretion of adiponectin may be linked to the development of insulin resistance in obesity (reduced cellular responses to insulin; see the Biochemical Comments for a further discussion of insulin resistance). Adiponectin will bind to either of two receptors (AdipoR1 and AdipoR2), which initiate a signal transduction cascade resulting in the activation of the AMP-activated protein kinase (AMPK) and activation of the nuclear transcription factor PPAR-␣ (peroxisome proliferatoractivated receptor-␣). Within the muscle, activation of AMPK leads to enhanced fatty acid oxidation and glucose uptake. Within the liver, activation of AMPK also leads to enhanced fatty acid oxidation, as opposed to synthesis. AMPK activation in liver and muscle, then, leads to a reduction of blood glucose levels and free fatty acids. Recall that as the adipocytes increase in size, less adiponectin is released; therefore, as obesity occurs, it is more difficult for circulating fatty acids and glucose to be used by the tissues. This contributes, in part, to the elevated glucose and fat levels seen in the circulation of obese patients (the insulin resistance syndrome). Activation of PPAR-␣ (see Chapter 46 for more details) leads to enhanced fatty acid oxidation by the liver and muscle. PPAR-␣ is the target of the fibrate group of lipid-lowering drugs. PPAR-␣ activation leads to increased transcription of genes involved in fatty acid transport, energy uncoupling, and fatty acid oxidation (for further information on the action of fibrates, see the Biochemical Comments in Chapter 34). The thiazolidinedione group of antidiabetic drugs (such as pioglitazone) is used to control type 2 diabetes. These drugs bind to and activate PPAR-␥ in adipose tissues and lead, in part, to increased adiponectin synthesis and release, which aids in reducing circulating fat and glucose levels.

CLINICAL COMMENTS Percy Veere. If Percy Veere had continued to eat a hypercaloric diet rich in carbohydrates, he would have become obese. In an effort to define obesity, it has been agreed internationally that the ratio of the patient’s body weight in kilograms to his or her height in meters squared (W/H2) is the most useful and reproducible measure. This ratio is referred to as the body mass index (BMI). Normal men and women fall into the range of 18.5 to 25. Percy’s current value is 21.3 and rising. It is estimated that more than 30% of adults in the United States have a BMI of ⬎30, with more than 60% exhibiting a BMI of ⬎25. For individuals with a BMI of ⬎27, which is quite close to a weight 20% above the “ideal” or desirable weight, an attempt at weight loss should be strongly advised. The idea that obesity is a benign condition unless it is accompanied by other risk factors for cardiovascular disease is disputed by several long-term, properly controlled prospective studies. These studies show that obesity is an independent risk factor not only for heart attacks and strokes, but also for the development of insulin resistance, type 2 diabetes mellitus, hypertension, and gallbladder disease. Percy did not want to become overweight and decided to follow his new diet faithfully. Cora Nari. Because Cora Nari’s lipid profile indicated an elevation in both serum triacylglycerols and LDL cholesterol, she was classified as having a combined hyperlipidemia. The dissimilarities in the lipid profiles of Cora and her two siblings, both of whom were experiencing anginal chest

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pain, are characteristic of the multigenic syndrome referred to as familial combined hyperlipidemia (FCH). Approximately 1% of the North American population has FCH. It is the most common cause of coronary artery disease in the United States. In contrast to patients with familial hypercholesterolemia (FH), patients with FCH do not have fatty deposits within the skin or tendons (xanthomas) (see Chapter 34). In FCH, coronary artery disease usually appears by the fifth decade of life. Treatment of FCH includes restriction of dietary fat. Patients who do not respond adequately to dietary therapy are treated with antilipidemic drugs. Selection of the appropriate antilipidemic drugs depends on the specific phenotypic expression of the patients’ multigenic disease as manifested by their particular serum lipid profile. In Cora’s case, a decrease in both serum triacylglycerols and LDL cholesterol must be achieved. If possible, her serum HDL cholesterol level should also be raised to a level ⬎50 mg/dL. To accomplish these therapeutic goals, her physician initially prescribed a statin (pravastatin) and fast-release nicotinic acid (niacin) because these agents have the potential to lower serum triacylglycerol levels and cause a reciprocal rise in serum HDL cholesterol levels, as well as complementing the statin in lowering serum total and LDL cholesterol levels. The mechanisms suggested for niacin’s triacylglycerol-lowering action include enhancement of the action of LPL, inhibition of lipolysis in adipose tissue, and a decrease in esterification of triacylglycerols in the liver (see Chapter 34, Table 34.5). The mechanism by which niacin lowers the serum total and LDL cholesterol levels is related to the decrease in hepatic production of VLDL. When the level of VLDL in the circulation decreases, the production of its daughter particles, IDL and LDL, also decreases. Cora found niacin’s side effects of flushing and itching to be intolerable so the drug was discontinued. Statins, such as pravastatin, inhibit cholesterol synthesis by inhibiting the activity of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the pathway (see Chapter 34). After 3 months of therapy, pravastatin decreased Cora’s LDL cholesterol from a pretreatment level of 175 mg/dL to 122 mg/dL (still higher than the recommended treatment goal of 100 mg/dL or less in a patient with established coronary artery disease). Her fasting serum triacylglycerol concentration was decreased from a pretreatment level of 280 mg/dL to 178 mg/ dL (a treatment goal for serum triacylglycerol when the pretreatment level is ⬍500 mg/dL has not been established). In patients with diabetes, the goal is to bring the triacylglycerol level to 150 mg/dL or less.

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Concentration (mg/100 mL)

Colleen Lakker. Colleen Lakker suffered from respiratory distress syndrome (RDS), which is a major cause of death in the newborn. RDS is preventable if prematurity can be avoided by appropriate management of high-risk pregnancy and labor. Before delivery, the obstetrician must attempt to predict and possibly treat pulmonary prematurity in utero. For example, estimation of fetal head circumference by ultrasonography, monitoring for fetal arterial oxygen saturation, and determination of the ratio of the concentrations of phosphatidylcholine (lecithin) and that of sphingomyelin in the amniotic fluid may help to identify premature infants who are predisposed to RDS (Fig. 33.34). The administration of synthetic corticosteroids 48 to 72 hours before delivery of a fetus of ⬍33 weeks of gestation in women who have toxemia of pregnancy, diabetes mellitus, or chronic renal disease may reduce the incidence or mortality of RDS by stimulating fetal synthesis of lung surfactant. The administration of one dose of surfactant into the trachea of the premature infant immediately after birth may transiently improve respiratory function but does not improve overall mortality. In Colleen’s case, intensive therapy allowed her to survive this acute respiratory complication of prematurity.

22 20

621

Amniotic fluid

18 16 Phosphatidyl choline

14 12 10 8

Sphingomyelin

6 4 2 0

18 20 22 24 26 28 30 32 34 36 38 Term

Gestation (wk)

FIG. 33.34. Comparison of phosphatidylcholine and sphingomyelin in amniotic fluid. Phosphatidylcholine is the major lipid in lung surfactant. The concentration of phosphatidylcholine relative to sphingomyelin rises at 35 weeks of gestation, indicating pulmonary maturity.

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BIOCHEMICAL COMMENTS Metabolic Syndrome. Obesity is a relatively modern problem brought about by an excess of nutrients and reduced physical activity. As individuals become obese, adipocyte function, in terms of its biochemical and endocrine roles, is altered. Adiponectin levels fall, and with it, reduced fatty acid oxidation occurs in tissues. The release of free fatty acids is also increased in large adipocytes, presumably because of the high concentration of substrate (triglyceride), even if hormone-sensitive lipase is not activated. This is coupled with a deficiency of perilipins in obese individuals. Perilipins are adipocyte phosphoproteins that bind to triacylglycerol droplets and regulate the accessibility of the triglyceride to the lipases. A decrease in perilipin synthesis leads to an enhanced basal rate of lipolysis. Fat cells begin to proliferate early in life, starting in the third trimester of gestation. Proliferation essentially ceases before puberty, and thereafter fat cells change mainly in size. However, some increase in the number of fat cells can occur in adulthood if preadipocytes are induced to proliferate by growth factors and changes in the nutritional state. Weight reduction results in a decrease in the size of fat cells rather than a decrease in number. After weight loss, the amount of LPL, an enzyme involved in the transfer of fatty acids from blood triacylglycerols to the triacylglycerol stores of adipocytes, increases. In addition, the amount of mRNA for LPL also increases. All of these factors suggest that individuals who become obese, particularly those who do so early in life, will have difficulty losing weight and maintaining a lower body adipose mass. Signals that initiate or inhibit feeding are extremely complex and include psychologic and hormonal factors as well as neurotransmitter activity. These signals are integrated and relayed through the hypothalamus. Destruction of specific regions of the hypothalamus can lead to overeating and obesity or to anorexia and weight loss. Overeating and obesity are associated with damage to the ventromedial or the paraventricular nucleus, whereas weight loss and anorexia are related to damage to more lateral hypothalamic regions. Compounds that act as satiety signals have been identified in brain tissue and include leptin and glucagonlike peptide-1 (GLP-1). Appetite suppressors developed from compounds such as these may be used in the future for the treatment of obesity. Increased circulating levels of nonesterified (or free) fatty acids (NEFAs) are observed in obesity and is associated with insulin resistance. Insulin resistance is also a hallmark of type 2 diabetes. There are several theories about why increased NEFA promote insulin resistance. One will be presented here, along with the effects of NEFA on insulin release from the pancreas. As NEFA levels in the circulation rise, muscle begins to use predominantly NEFA as an energy source. This reduces muscle glucose metabolism, as a result of the buildup of acetyl-CoA in the mitochondria, export of citrate to the cytoplasm, and inhibition of PFK-1. Because glucose is not being metabolized, its uptake by muscle is reduced. As muscle is the predominant tissue that takes up glucose in response to insulin, impaired glucose uptake (resulting from fat oxidation) is manifested as a sign of insulin resistance. NEFA are also postulated to interfere with pancreatic ␤-cell secretion of insulin, further contributing to insulin resistance (see the following text for more on this topic). Obesity, insulin resistance, and altered blood lipid levels are the start of a syndrome known as metabolic syndrome. Metabolic syndrome is diagnosed based on the recommendations of the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III), with minor modifications. For a diagnosis of metabolic syndrome, at least three of the following components should be evident: • • • • •

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Increased waist circumference (40 in or more for men, 35 in or more for women) Elevated triglycerides (ⱖ150 mg/dL) Reduced HDL (⬍40 mg/dL for men, ⬍50 mg/dL for women) Elevated blood pressure (ⱖ130/85 mm Hg) Elevated fasting glucose (ⱖ100 mg/dL)

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Individuals with metabolic syndrome are at increased risk for type 2 diabetes and cardiovascular disease. Treatment, in addition to lifestyle changes to reduce weight, increase exercise, and change diet, will be discussed further in Chapter 34. A characteristic of the metabolic syndrome is insulin resistance. Part of this resistance is caused by altered insulin release from the ␤-cells of the pancreas under hyperlipidemic conditions. To understand how this occurs, it is necessary to revisit normal glucose-stimulated insulin secretion (see Fig. 26.11). Glucose is metabolized in the pancreatic ␤-cell to generate ATP, which closes ATP-sensitive K⫹ channels, which leads to a membrane depolarization, which activates voltage-gated Ca2⫹ channels in the membrane. The corresponding increase in intracellular calcium levels leads to stimulation of the exocytosis of insulin-containing vesicles. However, the process is more complicated than this and is coupled to pyruvate cycling within the ␤-cell and the generation of NADPH. The exact role of NADPH in stimulating insulin release has not yet been elucidated. Islet cells express pyruvate carboxylase, but very low levels of PEPCK. As can be seen in Figure 33.35, NADPH is generated in the cytosol of the islets cells by malic enzyme and the cytosolic isozyme of isocitrate dehydrogenase, which uses NADP⫹ instead of NAD⫹, as the mitochondrial enzyme does. Thus, under normal conditions, glucose is metabolized to pyruvate, and the pyruvate enters the mitochondrion. Some of the pyruvate is converted to acetylCoA to generate energy; some of the pyruvate is converted to oxaloacetate. The oxaloacetate generated can be converted to malate and exported to the cytoplasm, where it is recycled to pyruvate by malic enzyme, generating NADPH. Alternatively, the oxaloacetate and acetyl-CoA generated within the mitochondria can condense and form citrate, isocitrate, and ␣-ketoglutarate, all of which can leave the mitochondria and enter the cytosol. Cytosolic isocitrate is oxidized to ␣-ketoglutarate, generating NADPH. Cytosolic citrate is split by citrate lyase to acetyl-CoA and oxaloacetate, and the oxaloacetate is reduced to malate and cycled to pyruvate, generating more NADPH. The cytosolic acetyl-CoA is used for limited fatty acid production in the islet cell. The elevated cytosolic NADPH then aids, in an unknown manner, in the release of insulin from the ␤-cell.

Cytosol

Mitochondrion

Cytosol

Glucose Pyruvate Pyruvate

Malic enzyme

Pyruvate Pyruvate carboxylase

PDH

NADPH

Malate Fatty acids

Acetyl CoA Malic enzyme

NADPH

OAA OAA

Malate

Acetyl CoA Citrate

Malate

Fumarate

Isocitrate

Citrate

Isocitrate Isocitrate dehydrogenase

α-KG

Succinate

NADPH

α-KG

Succinyl CoA

FIG. 33.35. Generation of NADPH via pyruvate cycling in islet cells in response to glucose. Details are provided in the text. (Adapted from Muoio DM, Newgard CB. Obesityrelated derangements in metabolic regulation. Annu Rev Biochem. 2006;75:367–401.)

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So what goes wrong when ␤-cells are chronically exposed to high levels of NEFA in the circulation? The ␤-cell begins to oxidize the fatty acids, which dramatically raises the acetyl-CoA levels in the ␤-cell mitochondria. This leads to the activation of pyruvate carboxylase and enhanced pyruvate cycling, with significant increases in resting NADPH levels. This then leads to blunted increases in NADPH levels when glucose levels increase, as pyruvate cycling is already maximal because of the activation of pyruvate carboxylase. Thus, the ␤-cell releases less insulin in response to the increase in blood glucose levels, thereby contributing further to hyperglycemia that was initiated by the resistance to insulin’s action in peripheral tissues. Key Concepts • • • •

• • • •

• • • • • •

Fatty acids are synthesized mainly in the liver, primarily from glucose. Glucose is converted to pyruvate via glycolysis, which enters the mitochondrion and forms both acetyl-CoA and oxaloacetate, which then forms citrate. The newly synthesized citrate is transported to the cytosol, where it is cleaved to form acetyl-CoA, which is the source of carbons for fatty acid biosynthesis. Two enzymes, acetyl-CoA carboxylase (the key regulatory step) and fatty acid synthase, produce palmitic acid (16 carbons, no double bonds) from acetyl-CoA. After activation to palmitoyl-CoA, the fatty acid can be elongated or desaturated (adding double bonds) by enzymes in the endoplasmic reticulum. Fatty acids are used to produce triacylglycerols (for energy storage) and glycerophospholipids and sphingolipids (for structural components of cell membranes). Liver-derived triacylglycerol is packaged with various apoproteins and secreted into the circulation as VLDL. As with dietary chylomicrons, LPL in the capillaries of adipose tissue, muscle, and the lactating mammary gland digests the triacylglycerol of VLDL, forming fatty acids and glycerol. Glycerophospholipids, synthesized from fatty acyl-CoA and glycerol 3-phosphate, are all derived from phosphatidic acid. Various head groups are added to phosphatidic acid to form the mature glycerophospholipids. Phospholipid degradation is catalyzed by phospholipases. Sphingolipids are synthesized from sphingosine, which is derived from palmitoyl-CoA and serine. Glycolipids, such as cerebrosides, globosides, and gangliosides, are sphingolipids. The sole sphingosine-based phospholipid is sphingomyelin. The adipocyte is an active endocrine organ, producing adipokines that help to regulate appetite and adipocyte size. Metabolic syndrome refers to a clustering of a variety of metabolic abnormalities that together dramatically increase the risk of type 2 diabetes and cardiovascular disease. Diseases discussed in this chapter are summarized in Table 33.1.

Table 33.1

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Diseases Discussed in Chapter 33

Disease or Disorder

Environmental or Genetic

Obesity

Both

Heart disease, familial combined hyperlipidemia (FCH)

Both

Respiratory distress syndrome

Both

Abetalipoproteinemia

Genetic

Comments Weight gain will occur from excessive calorie consumption: fat can be derived from carbohydrates, protein, and triglyceride in the diet FCH, leading to elevated cholesterol and triglyceride levels in the serum. Levels of lipid in the blood, and symptoms displayed by patients, will vary from patient to patient. Inability of lungs to properly expand and contract due to lack of surfactant, a complex mixture of lipids and apoproteins Lack of microsomal triglyceride transport protein, leading to reduced production of VLDL and chylomicrons within the liver and intestine, respectively

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REVIEW QUESTIONS—CHAPTER 33 1.

2.

3.

Which of the following is involved in the synthesis of triacylglycerols in adipose tissue? A. Fatty acids obtained from chylomicrons and VLDL B. Glycerol 3-phosphate derived from blood glycerol C. 2-Monoacylglycerol as an obligatory intermediate D. LPL to catalyze the formation of ester bonds E. Acetoacetyl-CoA as an obligatory intermediate A molecule of palmitic acid, attached to carbon 1 of the glycerol moiety of a triacylglycerol, is ingested and digested. The fatty acid is stored in a fat cell and ultimately is oxidized to carbon dioxide and water in a muscle cell. Choose the molecular complex in the blood in which the palmitate residue is carried from the lumen of the gut to the surface of the gut epithelial cell. A. VLDL B. Chylomicron C. Fatty acid–albumin complex D. Bile salt micelle E. LDL A patient with hyperlipoproteinemia would be most likely to benefit from a low-carbohydrate diet if the lipoproteins that are elevated in blood are which of the following?

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A. B. C. D. E.

Chylomicrons VLDL HDL LDL IDL

4.

Which of the following is a characteristic of sphingosine? A. It is converted to ceramide by reacting with a UDPsugar. B. It contains a glycerol moiety. C. It is synthesized from palmitoyl-CoA and serine. D. It is a precursor of cardiolipin. E. It is only synthesized in neuronal cells.

5.

Newly synthesized fatty acids are not immediately degraded because of which of the following? A. Tissues that synthesize fatty acids do not contain the enzymes that degrade fatty acids. B. High NADPH levels inhibit ␤-oxidation. C. In the presence of insulin, the key fatty acid degrading enzyme is not induced. D. Newly synthesized fatty acids cannot be converted to their CoA derivatives. E. Transport of fatty acids into mitochondria is inhibited under conditions in which fatty acids are being synthesized.

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34

626

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Cholesterol Absorption, Synthesis, Metabolism, and Fate Cholesterol is one of the most well-recognized molecules in human biology, in part because of the direct relationship between its concentrations in blood and tissues and the development of atherosclerotic vascular disease. Cholesterol, which is transported in the blood in lipoproteins because of its absolute insolubility in water, serves as a stabilizing component of cell membranes and as a precursor of the bile salts and steroid hormones. Precursors of cholesterol are converted to ubiquinone, dolichol, and, in the skin, to cholecalciferol, the active form of vitamin D. As a major component of blood lipoproteins, cholesterol can appear in its free, unesterified form in the outer shell of these macromolecules and as cholesterol esters in the lipoprotein core. Cholesterol is obtained from the diet or synthesized by a pathway that occurs in most cells of the body, but to a greater extent in cells of the liver and intestine. The precursor for cholesterol synthesis is acetyl coenzyme A (acetyl-CoA), which can be produced from glucose, fatty acids, or amino acids. Two molecules of acetyl-CoA form acetoacetyl-CoA, which condenses with another molecule of acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA). Reduction of HMG-CoA produces mevalonate. This reaction, catalyzed by HMG-CoA reductase, is the major rate-limiting step of cholesterol synthesis. Mevalonate produces isoprene units that condense, eventually forming squalene. Cyclization of squalene produces the steroid ring system, and several subsequent reactions generate cholesterol. The adrenal cortex and the gonads also synthesize cholesterol in significant amounts and use it as a precursor for steroid hormone synthesis. Cholesterol is packaged in chylomicrons in the intestine and in very lowdensity lipoprotein (VLDL) in the liver. It is transported in the blood in these lipoprotein particles, which also transport triacylglycerols. As the triacylglycerols of the blood lipoproteins are digested by lipoprotein lipase, chylomicrons are converted to chylomicron remnants, and VLDL is converted to intermediate-density lipoprotein (IDL) and subsequently to low-density lipoprotein (LDL). These products return to the liver, where they bind to receptors in cell membranes and are taken up by endocytosis and digested by lysosomal enzymes. LDL is also endocytosed by nonhepatic (peripheral) tissues. Cholesterol and other products of lysosomal digestion are released into the cellular pools. The liver uses this recycled cholesterol, and the cholesterol that is synthesized from acetyl-CoA, to produce VLDL and to synthesize bile salts. Intracellular cholesterol obtained from blood lipoproteins decreases the synthesis of cholesterol within cells, stimulates the storage of cholesterol as cholesterol esters, and decreases the synthesis of LDL receptors. LDL receptors are found on the surface of the cells and bind various classes of lipoproteins before endocytosis. Although high-density lipoprotein (HDL) contains triacylglycerols and cholesterol, its function is very different from that of the chylomicrons and VLDL, which transport triacylglycerols. HDL exchanges proteins and lipids with the other lipoproteins in the blood. HDL transfers apolipoprotein E (apo E) and

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apolipoprotein CII (apo CII) to chylomicrons and VLDL. After digestion of the VLDL triacylglycerols, apo E and apo CII are transferred back to HDL. In addition, HDL obtains cholesterol from other lipoproteins and from cell membranes and converts it to cholesterol esters by the lecithin-cholesterol acyltransferase (LCAT) reaction. Then HDL either directly transports cholesterol and cholesterol esters to the liver or transfers cholesterol esters to other lipoproteins via the cholesterol ester transfer protein (CETP). Ultimately, lipoprotein particles carry the cholesterol and cholesterol esters to the liver, where endocytosis and lysosomal digestion occur. Thus, reverse cholesterol transport (i.e., the return of cholesterol to the liver) is a major function of HDL. Elevated levels of cholesterol in the blood are associated with the formation of atherosclerotic plaques that can occlude blood vessels, causing heart attacks and strokes. Although high levels of LDL cholesterol are especially atherogenic, high levels of HDL cholesterol are protective because HDL particles are involved in the process of removing cholesterol from tissues, such as the lining cells of vessels, and returning it to the liver. Bile salts, which are produced in the liver from cholesterol obtained from the blood lipoproteins or synthesized from acetyl-CoA, are secreted into the bile. They are stored in the gallbladder and released into the intestine during a meal. The bile salts emulsify dietary triacylglycerols, thus aiding in digestion. The digestive products are absorbed by intestinal epithelial cells from bile salt micelles, tiny microdroplets that contain bile salts at their water interface. After the contents of the micelles are absorbed, most of the bile salts travel to the ileum, where they are resorbed and recycled by the liver. Less than 5% of the bile salts that enter the lumen of the small intestine are eventually excreted in the feces. Although the fecal excretion of bile salts is relatively low, it is a major means by which the body disposes of the steroid nucleus of cholesterol. Because the ring structure of cholesterol cannot be degraded in the body, it is excreted mainly in the bile as free cholesterol and bile salts. The steroid hormones, derived from cholesterol, include the adrenal cortical hormones (e.g., cortisol, aldosterone, and the adrenal sex steroids dehydroepiandrosterone [DHEA] and androstenedione) and the gonadal hormones (e.g., the ovarian and testicular sex steroids, such as testosterone and estrogen).

THE WAITING ROOM At his next office visit, Ivan Applebod’s case was reviewed by his physician. Mr. Applebod has several of the major risk factors for coronary heart disease (CHD). These include a sedentary lifestyle, marked obesity, hypertension, hyperlipidemia, and early type 2 diabetes. Unfortunately, he has not followed his doctor’s advice with regard to a diabetic diet designed to effect a significant loss of weight, nor has he followed an aerobic exercise program. As a consequence, his weight has gone from 270 to 281 lb. After a 14-hour fast, his serum glucose is now 214 mg/dL (normal, ⬍100 mg/dL), and his serum total cholesterol level is 314 mg/dL (desired level is ⱕ200 mg/dL). His serum triacylglycerol level is 295 mg/dL (desired level is ⱕ150 mg/dL), and his serum high-density lipoprotein (HDL) cholesterol is 24 mg/dL (desired level is ⱖ40 mg/dL for a man). His calculated serum low-density lipoprotein (LDL) cholesterol level is 231 mg/dL (desired level for a person with two or more risk factors for CHD is ⱕ130 mg/dL, unless

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Until recently, the concentration of low-density lipoprotein (LDL) cholesterol could only be directly determined by sophisticated laboratory techniques not available for routine clinical use. As a consequence, the LDL cholesterol concentration in the blood was derived indirectly by using the Friedewald formula: the sum of the high-density lipoprotein (HDL) cholesterol level and the triacylglycerol (TG) level divided by 5 (which gives an estimate of the VLDL cholesterol level) subtracted from the total cholesterol level: LDL cholesterol ⫽ total cholesterol ⫺ [HDL cholesterol ⫹ (TG/5)] This equation yields inaccurate LDL cholesterol levels 15% to 20% of the time and fails completely when serum triacylglycerol levels are ⬎400 mg/dL. A recently developed test called LDL direct isolates LDL cholesterol by using a special immunoseparation reagent. Not only is this direct assay for LDL cholesterol more accurate than the indirect Friedewald calculation, it also is not affected by mildly to moderately elevated serum triacylglycerol levels and can be used for a patient who has not fasted. It does not require the expense of determining serum total cholesterol, HDL cholesterol, and triacylglycerol levels.

Table 34.1 ATP III: LDL-C Goals and Cut Points for Therapy in Different Risk Categories

Risk Category CHD or CHD risk equivalents (10-year risk ⬎20%) 2⫹ risk factors (10-year risk ⱕ20%) 0–1 risk factor

LDL Goal (mg/dL)

LDL Level at Which to Initiate Therapeutic Lifestyle Changes (mg/dL)

LDL Level at Which to Consider Drug Therapy (mg/dL)

⬍100

ⱖ100

ⱖ130 (100–129: drug optional)

⬍130

ⱖ130

⬍160

ⱖ160

10-year risk 10%–20%: ⱖ130 10-year risk ⬍10%: ⱖ160 ⱖ190 (160–189: LDLlowering drug optional)

LDL-C, low-density lipoprotein cholesterol; CHD, coronary heart disease. These values are expected to be updated in the fall of 2011. See http://www.nhlbi.nih.gov/guidelines/cholesterol/atp4/index.htm Source: Executive summary of the National Cholesterol Education Program. Third report of the National Cholesterol Education Programs (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Final report. Circulation. 2002;106:3145–3457.

one of the risk factors is diabetes mellitus, in which case the LDL cholesterol level should be ⬍100 mg/dL). Mr. Applebod exhibits sufficient criteria to be classified as having metabolic syndrome. Ann Jeina was carefully followed by her physician after she survived her heart attack. Before she was discharged from the hospital, after a 14-hour fast, her serum triacylglycerol level was 158 mg/dL (slightly above the upper range of normal), and her HDL cholesterol level was low at 32 mg/dL (normal for women is ⱖ50 mg/dL). Her serum total cholesterol level was elevated at 420 mg/dL (reference range is ⱕ200 mg/dL for a woman with known CHD). From these values, her LDL cholesterol level was calculated to be 356 mg/dL (desirable level for a person with established heart disease is ⬍100 mg/dL). Both of Ms. Jeina’s younger brothers had “very high” serum cholesterol levels, and both had suffered heart attacks in their midforties. With this information, a tentative diagnosis of familial hypercholesterolemia, type IIA, was made, and the patient was started on a step I diet as recommended by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III. This panel recommends that decisions with regard to when dietary and drug therapy should be initiated based on the serum LDL cholesterol level, as detailed in Table 34.1. Because a step I diet (Table 34.2) usually lowers serum total and LDL cholesterol levels by no more than 15%, Ms. Jeina was also started on a potent statin, atorvastatin, as she had already experienced a myocardial infarction, and her LDL is greater than 30 points above the recommended level. Table 34.2

Dietary Therapy for Elevated Blood Cholesterol

Nutrient b

Cholesterol Total fat Saturated fat Polyunsaturated fat Monounsaturated fat Carbohydrates Protein Calories

Step I Diet

Step II Dieta

⬍300 mg/d ⱕ30%b 8%–10% ⱕ10% ⱕ15% ⱖ55% ⬃15% To achieve and maintain desirable body weight

⬍200 mg/d 30% ⬍7% ⱕ10% ⱕ15% ⱖ55% ⬃15%

a

The step II diet is applied if 3 months on the step I diet has failed to reduce blood cholesterol to the desired level (see Table 34.1). b Except for the values given in milligram per day, all values are percentage of total calories eaten daily. Source: Based on National Cholesterol Education Program. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA. 1993;269(23):3015–3023.

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Vera Leizd is a 34-year-old woman in whom pubertal changes began at age 12, leading to the development of normal secondary sexual characteristics and the onset of menses at age 13. Her menstrual periods occurred on a monthly basis over the next 7 years, but the flow was scant. At age 20, she noted a gradual increase in her intermenstrual interval from her normal of 28 days to 32 to 38 days. The volume of her menstrual flow also gradually diminished. After 7 months, her menstrual periods ceased. She complained of increasing oiliness of her skin, the appearance of acnelike lesions on her face and upper back, and the appearance of short, dark terminal hairs on the mustache and sideburn areas of her face. The amount of extremity hair also increased, and she noticed a disturbing loss of hair from her scalp.

I.

INTESTINAL ABSORPTION OF CHOLESTEROL

Cholesterol absorption by intestinal cells is a key regulatory point in human sterol metabolism because it ultimately determines what percentage of the 1,000 mg of biliary cholesterol produced by the liver each day, and what percentage of the 300 mg of dietary cholesterol entering the gut per day is eventually absorbed into the blood. In normal subjects, approximately 55% of this intestinal pool enters the blood through the enterocyte each day. The details of cholesterol absorption from dietary sources were outlined in Chapter 32. Although the absorption of cholesterol from the intestinal lumen is a diffusioncontrolled process, there is also a mechanism to remove unwanted or excessive cholesterol and plant sterols from the enterocyte. The transport of sterols out of the enterocyte and into the gut lumen is related to the products of genes that code for the adenosine triphosphate (ATP)-binding cassette (ABC) protein family, specifically ABCG5 and ABCG8. These proteins couple ATP hydrolysis to the transport of unwanted or excessive cholesterol and plant sterols (phytosterols) from the enterocyte back into the gut lumen. Another member of the ABC family, ABCA1, is required for reverse cholesterol transport and the biogenesis of HDL. Cholesterol cannot be metabolized to carbon dioxide and water and is, therefore, eliminated from the body principally in the feces as unreabsorbed sterols and bile acids. ABC protein expression increases the amount of sterols present in the gut lumen, with the potential to increase elimination of the sterols into the feces. Patients with a condition known as phytosterolemia (a rare autosomal recessive disease, also known as sitosterolemia) have a defect in the function of either ABCG5 or ABCG8 in the enterocytes, which leads to accumulation of cholesterol and phytosterols within these cells. These eventually reach the bloodstream, markedly elevating the level of cholesterol and phytosterol in the blood. This accounts for the increased cardiovascular morbidity in individuals with this disorder. From these experiments of nature, it is clear that agents that either amplify the expression of the ABC proteins within enterocytes or that block cholesterol absorption from the lumen have therapeutic potential in the treatment of patients with hypercholesterolemia. Ezetimibe is a compound that is structurally different from the sterols. Its primary action in lowering serum cholesterol levels is to block cholesterol absorption through a specific but as yet poorly characterized cholesterol absorption mechanism in the brush border of enterocytes. The target of ezetimibe is the Niemann–Pick C1-like 1 protein (NPC1L1), which is believed to transport cholesterol into cells via an absorptive endocytotic mechanism, involving the protein clathrin. The reduction of cholesterol absorption from the intestinal lumen has been shown to reduce blood levels of LDL cholesterol, particularly when used with a drug that also blocks endogenous cholesterol synthesis.

What effect would be predicted for ezetimibe on endogenous cholesterol synthesis?

Cholesterol is an alicyclic compound whose basic structure includes the perhydrocyclopentanophenanthrene nucleus containing four fused rings (Fig. 34.1). In its “free” form, the cholesterol molecule contains 27 carbon atoms, a simple hydroxyl

Lieberman_CH34.indd 629

13

11 1

9

C

10

2

16

D 14

15

8

A 3

II. CHOLESTEROL SYNTHESIS

17

12

B 7

5 4

6

FIG. 34.1. The basic ring structure of sterols; the perhydrocyclopentanophenanthrene nucleus. Each ring is labeled A, B, C, or D.

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If less cholesterol is obtained from diet, then cellular cholesterol synthesis would be upregulated. Thus, ezetimibe has a better chance of reducing whole body cholesterol levels when endogenous cholesterol synthesis is also inhibited, than in the absence of such inhibition.

21

22

24

26 25

20 23 18

17 27

19

3

HO

FIG. 34.2. The structure of cholesterol.

O CH3 C SCoA Acetyl CoA O CH3

C

SCoA

CoA-SH O

O CH3

C

CH2

C

SCoA

Acetoacetyl CoA O HMG-CoA synthase

CH3

C

SCoA

CoA-SH O C

O–

CH2 CH3 C

OH

CH2

␤-hydroxy␤-methylglutaryl CoA (HMG-CoA)

C O

SCoA 2NADPH + 2H+

HMG-CoA reductase

2NADP+ CoA-SH O C

O–

CH2 CH3 C

OH

CH2 CH2OH Mevalonate

FIG. 34.3. The conversion of three molecules of acetyl-CoA to mevalonic acid.

Ann Jeina’s serum total and LDL cholesterol levels improved only modestly after 3 months on a step I diet and the statin. Three additional months on a more severe low-fat diet (step II diet) brought little further improvement.

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group at C3, a double bond between C5 and C6, an eight-membered hydrocarbon chain attached to carbon 17 in the D-ring, a methyl group (carbon 19) attached to carbon 10, and a second methyl group (carbon 18) attached to carbon 13 (Fig. 34.2). Approximately one-third of plasma cholesterol exists in the free (or unesterified) form. The remaining two-thirds exist as cholesterol esters in which a longchain fatty acid (usually linoleic acid) is attached by ester linkage to the hydroxyl group at C3 of the A-ring. The proportions of free and esterified cholesterol in the blood can be measured using methods such as high-performance liquid chromatography (HPLC). The structure of cholesterol suggests that its synthesis involves multimolecular interactions and significant reducing power. All 27 carbons are derived from one precursor, acetyl-CoA. Acetyl-CoA can be obtained from several sources, including the ␤-oxidation of fatty acids, the oxidation of ketogenic amino acids such as leucine and lysine, and the pyruvate dehydrogenase reaction. The reducing power for cholesterol synthesis is supplied in the form of NADPH. The latter is provided by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the hexose monophosphate shunt pathway (see Chapter 29). Cholesterol synthesis occurs in the cytosol, requiring hydrolysis of high-energy thioester bonds of acetyl-CoA and phosphoanhydride bonds of ATP. Its synthesis occurs in four stages.

A. Stage 1: Synthesis of Mevalonate from Acetyl-CoA The first stage of cholesterol synthesis leads to the production of the intermediate mevalonate (Fig. 34.3). The synthesis of mevalonate is the committed, rate-limiting step in cholesterol formation. In this cytoplasmic pathway, two molecules of acetylCoA condense, forming acetoacetyl-CoA, which then condenses with a third molecule of acetyl-CoA to yield the six-carbon compound ␤-hydroxy-␤-methylglutaryl-CoA (HMG-CoA). The HMG-CoA synthase in this reaction is present in the cytosol and is distinct from the mitochondrial HMG-CoA synthase that catalyses HMG-CoA synthesis involved in production of ketone bodies. The committed step and major point of regulation of cholesterol synthesis in stage 1 involves reduction of HMG-CoA to mevalonate, a reaction that is catalyzed by HMG-CoA reductase, an enzyme embedded in the membrane of the endoplasmic reticulum. HMG-CoA reductase contains eight membrane-spanning domains, and the amino-terminal domain, which faces the cytoplasm, contains the enzymatic activity. The reducing equivalents for this reaction are donated by two molecules of NADPH. The regulation of the activity of HMGCoA reductase is controlled in multiple ways. 1.

TRANSCRIPTIONAL CONTROL

The rate of synthesis of HMG-CoA reductase messenger RNA (mRNA) is controlled by one of the family of sterol-regulatory element-binding proteins (SREBPs)

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631

A +

SREBP

NH3

Degradation

Cholesterol SCAP

SCAP

SREBP

SREBP

+

S1P ER membrane

Gene transcription

S2P

Golgi membrane

C

B HMG-CoA reductase

SRE

DNA-binding domain

DNA-binding domain

+

Sterols

Proteolysis, degradation

AMP-activated protein kinase kinase ATP

AMP-activated protein kinase (inactive)

Glucagon Sterols

ADP

AMP

+

AMP-activated protein kinase (active)

+

ATP ER membrane

P ADP

HMG-CoA reductase (active)

HMG-CoA reductase (inactive)

P

Insulin Pi

Phosphatase

+

FIG. 34.4. Regulation of HMG-CoA reductase activity. See text for details and abbreviations. A. Transcriptional control. B. Regulation by proteolysis. C. Regulation by phosphorylation.

(Fig. 34.4A). These transcription factors belong to the basic helix–loop–helix leucine zipper (bHLH-Zip) family of transcription factors that directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triacylglycerols, and phospholipids as well as the production of the NADPH cofactors required to synthesize these molecules. SREBPs specifically enhance transcription of the HMG-CoA reductase gene by binding to the sterol-regulatory element (SRE) upstream of the gene. When bound, the rate of transcription is increased. SREBPs, after synthesis, are integral proteins of the endoplasmic reticulum (ER). The SREBP is bound to SCAP (SREBP cleavage-activating protein) in the ER membrane when cholesterol levels are high. When cholesterol levels drop, the sterol leaves its SCAP-binding site, and the SREBP:SCAP complex is transported to the Golgi apparatus. Within the Golgi, two proteolytic cleavages occur (via the site 1 [S1P] and site 2 [S2P] proteases), which release the N-terminal transcription factor domain from the Golgi membrane. Once released, the active amino terminal component travels to the nucleus to bind to SREs. The soluble SREBPs are rapidly turned over and need to be continuously produced to stimulate reductase mRNA transcription effectively. When cytoplasmic sterol levels rise, the sterols bind to SCAP and prevent translocation of the complex to the Golgi, leading to a decrease in transcription of the reductase gene and thus less reductase protein being produced. 2.

PROTEOLYTIC DEGRADATION OF HMG-COA REDUCTASE

Rising levels of cholesterol and bile salts in cells that synthesize these molecules also may cause a change in the oligomerization state of the membrane domain of HMG-CoA

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reductase, rendering the enzyme more susceptible to proteolysis (see Fig. 34.4B). This, in turn, decreases its activity. The membrane domains of HMG-CoA reductase contain sterol-sensing regions, which are similar to those in SCAP. 3.

REGULATION BY COVALENT MODIFICATION

In addition to the inductive and repressive influences we have mentioned, the activity of the reductase is also regulated by phosphorylation and dephosphorylation (see Fig. 34.4C). Elevated glucagon levels increase phosphorylation of the enzyme, thereby inactivating it, whereas hyperinsulinemia increases the activity of the reductase by activating phosphatases, which dephosphorylate the reductase. Increased levels of intracellular sterols also may increase phosphorylation of HMG-CoA reductase, thereby reducing its activity as well (feedback suppression). Thyroid hormone also increases enzyme activity, whereas glucocorticoids decrease its activity. The enzyme that phosphorylates HMG-CoA reductase is the adenosine monophosphate (AMP)-activated protein kinase, which itself is regulated by phosphorylation by one of several AMP-activated protein kinase kinases (one of which is LKB1, described further in the following text). Thus, cholesterol synthesis decreases when ATP levels are low and increases when ATP levels are high, similar to what occurs with fatty acid synthesis (recall that acetyl-CoA carboxylase is also phosphorylated and inhibited by the AMP-activated protein kinase). The need for ATP in cholesterol biosynthesis will be evident as the further reactions of the pathway are discussed.

B. Stage 2: Conversion of Mevalonate to Two Activated Isoprenes In the second stage of cholesterol synthesis, three phosphate groups are transferred from three molecules of ATP to mevalonate (Fig. 34.5). The purpose of these phosphate transfers is to activate both carbon 5 and the hydroxyl group on carbon 3 for further reactions in which these groups will participate. The phosphate group attached to the C3 hydroxyl group of mevalonate in the 3-phospho-5-pyrophosphomevalonate intermediate is removed along with the carboxyl group on C1. This produces a double bond in the five-carbon product, ⌬3-isopentenyl pyrophosphate, the first of two activated isoprenes that are necessary for the synthesis of cholesterol. The second activated isoprene is formed when ⌬3-isopentenyl pyrophosphate is isomerized to dimethylallyl pyrophosphate (see Fig. 34.5). Isoprenes, in addition to being used for cholesterol biosynthesis, are also used in the synthesis of coenzyme Q and dolichol.

C. Stage 3: Condensation of Six Activated Five-Carbon Isoprenes to Form the 30-Carbon Squalene The next stage in the biosynthesis of cholesterol involves the head-to-tail condensation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The “head” in this case refers to the end of the molecule to which pyrophosphate is linked. In this reaction, the pyrophosphate group of dimethylallyl pyrophosphate is displaced, and a 10-carbon chain, known as geranyl pyrophosphate, is generated (Fig. 34.6). Geranyl pyrophosphate then undergoes another head-to-tail condensation with isopentenyl pyrophosphate, resulting in the formation of the 15-carbon intermediate, farnesyl pyrophosphate. After this, two molecules of farnesyl pyrophosphate undergo a head-to-head fusion, and both pyrophosphate groups are removed to form squalene, a compound that was first isolated from the liver of sharks (genus Squalus). Squalene contains 30 carbons (24 in the main chain and 6 in the methyl group branches; see Fig. 34.6). Geranyl pyrophosphate and farnesyl pyrophosphate are key components in cholesterol biosynthesis, and both farnesyl and geranyl groups can form covalent bonds with proteins, particularly the G proteins and certain proto-oncogene products involved in signal transduction. These hydrophobic groups anchor the proteins in the cell membrane.

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O –O

633

CH3

C

CH2

C

CH2

CH2OH

OH Mevalonate ATP ADP O –O

CH3

C

CH2

C

O CH2

CH2

O

P O– O–

OH ATP ADP O –O

CH3

C

CH2

C

CH2

CH2

O

O

O

P O

P

O– OH 5-pyrophosphate mevalonate

O–

O–

ATP ADP O –O

CH3

C

CH2

C

CH2

CH2

O

O –O

O

O

P O

P O–

O–

O–

P O O–

3-phospho 5-pyrophosphate mevalonate CO2 Pi CH3 CH2

C

CH2

CH2

O

O

O

P O

P

O–

O– O– Δ3-isopentenyl pyrophosphate

CH3 CH3

C

CH

CH2

O

O

O

P O

P

O–

O–

O–

Dimethylallyl pyrophosphate

FIG. 34.5. The formation of activated isoprene units (⌬3-isopentenyl pyrophosphate and dimethylallyl pyrophosphate) from mevalonic acid. Note the large ATP requirement for these steps.

D. Stage 4: Conversion of Squalene to the Four-Ring Steroid Nucleus The enzyme squalene monooxygenase adds a single oxygen atom from O2 to the end of the squalene molecule, forming an epoxide. NADPH then reduces the other oxygen atom of O2 to H2O. The unsaturated carbons of the squalene 2,3-epoxide are aligned in a way that allows conversion of the linear squalene epoxide into a cyclic structure. The cyclization leads to the formation of lanosterol, a sterol with the fourring structure characteristic of the steroid nucleus. A series of complex reactions,

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SECTION VI ■ LIPID METABOLISM

O

O O

P O

P

O–

O–

O O–

+

O

P

O O

O– Δ3-isopentenyl

Dimethylallyl pyrophosphate

P

O–

O–

pyrophosphate

PPi O O

O

P

O

O–

P O– O–

Geranyl pyrophosphate O PPi

Squalene monooxygenase

3

NADPH + H+ O2 H2O NADP+

2

P

O

O–

Squalene

O

P O

P O–

O–

O–

O

O O

O

P

O–

O–

Farnesyl pyrophosphate NADPH + H+ NADP+

Farnesyl pyrophosphate 2PPi

Squalene

O Squalene 2,3-epoxide Cyclase (2 steps)

FIG. 34.6. The formation of squalene from six isoprene units. The activation of the isoprene units drives their condensation to form geranyl pyrophosphate, farnesyl pyrophosphate, and squalene.

containing many steps and elucidated in the late 1950s, leads to the formation of cholesterol (Fig. 34.7).

III. SEVERAL FATES OF CHOLESTEROL

HO Lanosterol Many reactions

HO

Cholesterol

FIG. 34.7. The conversion of squalene to cholesterol. Squalene is shown in a different conformation than in Figure 34.6 to indicate better how the cyclization reaction occurs.

Lieberman_CH34.indd 634

Almost all mammalian cells are capable of producing cholesterol. Most of the biosynthesis of cholesterol occurs within liver cells, although the gut, the adrenal cortex, and the gonads (as well as the placenta in pregnant women) also produce significant quantities of the sterol. A fraction of hepatic cholesterol is used for the synthesis of hepatic membranes, but the bulk of synthesized cholesterol is secreted from the hepatocyte as one of three moieties: cholesterol esters, biliary cholesterol (cholesterol found in the bile), or bile acids. Cholesterol ester production in the liver is catalyzed by acyl-CoA–cholesterol acyl transferase (ACAT). ACAT catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group on carbon 3 of cholesterol (Fig. 34.8). Cholesterol esters are more hydrophobic than is free cholesterol. The liver packages some of the esterified cholesterol into the hollow core of lipoproteins, primarily VLDL. VLDL is secreted from the hepatocyte into the blood and transports the cholesterol esters (triacylglycerols, phospholipids, apoproteins, etc.) to the tissues that require greater amounts of cholesterol than they can synthesize de novo. These tissues then use the cholesterol for the synthesis

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of membranes, for the formation of steroid hormones, and for the biosynthesis of vitamin D. The residual cholesterol esters not used in these ways are stored in the liver for later use. The hepatic cholesterol pool serves as a source of cholesterol for the synthesis of the relatively hydrophilic bile acids and their salts (see Chapter 32). These derivatives of cholesterol are very effective detergents because they contain both polar and nonpolar regions. They are introduced into the biliary ducts of the liver. They are stored and concentrated in the gallbladder and later discharged into the gut in response to the ingestion of food. They aid in the digestion of intraluminal lipids by forming micelles with them, which increases the surface area of lipids exposed to the digestive action of intraluminal lipases. Free cholesterol also enters the gut lumen via the biliary tract (approximately 1,000 mg daily, which mixes with 300 mg of dietary cholesterol to form an intestinal pool, roughly 55% of which is resorbed by the enterocytes and enters the bloodstream daily). On a low-cholesterol diet, the liver synthesizes approximately 800 mg of cholesterol per day to replace bile salts and cholesterol lost from the enterohepatic circulation into the feces. Conversely, a greater intake of dietary cholesterol suppresses the rate of hepatic cholesterol synthesis (feedback repression).

IV. SYNTHESIS OF BILE SALTS A. Conversion of Cholesterol to Cholic Acid and Chenocholic Acid

635

HO Cholesterol ACAT

Fatty acyl-CoA CoA-SH

R

C

O O Cholesterol ester

FIG. 34.8. The ACAT reaction, producing cholesterol esters. ACAT, acyl-CoA–cholesterol acyl transferase.

Bile salts are synthesized in the liver from cholesterol by reactions that hydroxylate the steroid nucleus and cleave the side chain. In the first and rate-limiting reaction, an ␣-hydroxyl group is added to carbon 7 (on the ␣ side of the B-ring). The activity of the 7-␣-hydroxylase that catalyzes this step is decreased by an increase in bile salt concentration (Fig. 34.9). In subsequent steps, the double bond in the B-ring is reduced, and an additional hydroxylation may occur. Two different sets of compounds are produced. One set has ␣-hydroxyl groups at positions 3, 7, and 12 and produces the cholic acid series

H3C CH3

HO NADP+ + H+

Cholesterol Cytochrome P450 (Fe2+) O2 + H+ 7␣–hydroxylase

NADPH

Cytochrome P450 (Fe3+)

H2O

H3C CH3

HO

OH 7␣-Hydroxycholesterol

FIG. 34.9. The reaction catalyzed by 7-␣-hydroxylase. An ␣-hydroxyl group is formed at position 7 of cholesterol. This reaction, which is inhibited by bile salts, is the rate-limiting step in bile salt synthesis.

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SECTION VI ■ LIPID METABOLISM

Liver Cholesterol 7α-hydroxylase

–

Bile acids

7α-Hydroxycholesterol Reduction, hydroxylation, and conversion of hydroxyls to α 3α, 7α-Diol

3α, 7α, 12α,-Triol Oxidation of side chain COO–

HO

OH

Chenodeoxycholic acid

COO–

HO

HO

OH Cholic acid

FIG. 34.10. Synthesis of bile salts. Two sets of bile salts are generated: one with ␣-hydroxyl groups at positions 3 and 7 (the chenocholate series) and the other with ␣-hydroxyls at positions 3, 7, and 12 (the cholate series). Note how bile salt accumulation will inhibit the initial step of the pathway, catalyzed by 7-␣-hydroxylase.

of bile salts. The other set has ␣-hydroxyl groups only at positions 3 and 7 and produces the chenodeoxycholic acid series (also known as the chenocholic acid series, Fig. 34.10). Three carbons are removed from the side chain by an oxidation reaction. The remaining five-carbon fragment attached to the ring structure contains a carboxyl group (see Fig. 34.10). The pKa of the bile acids is approximately 6. Therefore, in the contents of the intestinal lumen, which normally have a pH of 6, approximately 50% of the molecules are present in the protonated form and 50% are ionized, which form bile salts. (The terms bile acids and bile salts are often used interchangeably, but bile salts actually refers to the ionized form of the molecules.)

B. Conjugation of Bile Salts The carboxyl group at the end of the side chain of the bile salts is activated by a reaction that requires ATP and coenzyme A. The CoA derivatives can react with either glycine or taurine (which is derived from cysteine), forming amides that are known as conjugated bile salts (Fig. 34.11). In glycocholic acid and glycochenodeoxycholic acid, the bile acids are conjugated with glycine. These compounds have a pKa of approximately 4, so compared to their unconjugated forms, a higher percentage of the molecules is present in the ionized form at the pH of the intestine. The taurine conjugates, taurocholic and taurochenodeoxycholic acid, all have a pKa of approximately 2. Therefore, compared with the glycoconjugates, an even greater percentage of the molecules of these conjugates are ionized in the lumen of the gut.

V. FATE OF THE BILE SALTS The bile salts are produced in the liver and secreted into the bile (Fig. 34.12). They are stored in the gallbladder and released into the intestine during a meal, where they serve as detergents that aid in the digestion of dietary lipids (see Chapter 32). Intestinal bacteria deconjugate and dehydroxylate the bile salts, removing the glycine and taurine residues and the hydroxyl group at position 7. The bile salts that lack a hydroxyl group at position 7 are called secondary bile salts. The deconjugated and dehydroxylated bile salts are less soluble and, therefore, are less readily resorbed

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637

Cholic acid (pK ~ 6) ATP CoASH AMP + Pi O C

SCoA

OH CH3 CH3

OH

HO

Cholyl CoA

+

H3N CH2

CH2

+

SO3–

H3N CH2 CoASH

Taurine

CoASH

C CH3

N H

SO3–

C HO CH3

CH3

HO

Glycine O

O HO

COO–

N H

COO–

CH3

OH

OH

HO

Taurocholic acid pK˜2

Glycocholic acid pK˜4

FIG. 34.11. Conjugation of bile salts. Conjugation lowers the pKa of the bile salts, making them better detergents; that is, they are more ionized in the contents of the intestinal lumen (pH 艐 6) than are the unconjugated bile salts (pKa 艐 6). The reactions are the same for the chenocholic acid series of bile salts.

Liver (synthesizes 0.2–0.6 g/day and recycles >95%) Secondary bile salts are reconjugated, but not rehydroxylated

Liver Cholesterol

Enterohepatic circulation

Bile salts

Gallbladder

Fat digestion Intestine

Bile salts reabsorbed (12–32 g/day) and returned to liver for recycling > 95% efficiency

Pool of bile salts = 2–4 g (recycles 6–8 times/day) Bacteria in gut deconjugate and dehydroxylate bile salts < 5% Feces (0.2–0.6 g/day)

FIG. 34.12. Overview of bile salt metabolism.

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SECTION VI ■ LIPID METABOLISM

from the intestinal lumen than the bile salts that have not been subjected to bacterial action (Fig. 34.13). Lithocholic acid, a secondary bile salt that has a hydroxyl group only at position 3, is the least soluble bile salt. Its major fate is excretion. Greater than 95% of the bile salts are resorbed in the ileum and return to the liver via the enterohepatic circulation (via the portal vein; see Fig. 34.12). The secondary

Primary bile salts COO– OH CH3 12

CH3 3

7

OH

HO

Cholic acid COO– CH3 12

CH3 3

7

OH

HO

Chenodeoxycholic acid

Secondary bile salts COO– OH CH3 12

CH3 3

7

HO Deoxycholic acid COO– CH3 12

CH3 3

7

HO Lithocholic acid

FIG. 34.13. Structures of the primary and secondary bile salts. Primary bile salts form conjugates with taurine or glycine in the liver. After secretion into the intestine, they may be deconjugated and dehydroxylated by the bacterial flora, forming secondary bile salts. Note that dehydroxylation occurs at position 7, forming the deoxy family of bile salts. Dehydroxylation at position 12 also leads to excretion of the bile salt.

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639

bile salts may be reconjugated in the liver, but they are not rehydroxylated. The bile salts are recycled by the liver, which secretes them into the bile. This enterohepatic recirculation of bile salts is extremely efficient. Less than 5% of the bile salts entering the gut are excreted in the feces each day. Because the steroid nucleus cannot be degraded in the body, the excretion of bile salts serves as a major route for removal of the steroid nucleus and thus of cholesterol from the body.

VI. TRANSPORT OF CHOLESTEROL BY THE BLOOD LIPOPROTEINS Because they are hydrophobic and essentially insoluble in the water of the blood, cholesterol and cholesterol esters, like triacylglycerols and phospholipids, must be transported through the bloodstream packaged as lipoproteins. These macromolecules are water-soluble. Each lipoprotein particle is composed of a core of hydrophobic lipids such as cholesterol esters and triacylglycerols surrounded by a shell of polar lipids (the phospholipids), which allows a hydration shell to form around the lipoprotein (see Fig. 32.8). This occurs when the positive charge of the nitrogen atom of the phospholipid (phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine) forms an ionic bond with the negatively charged hydroxyl ion of the environment. In addition, the shell contains a variety of apoproteins that also increase the water solubility of the lipoprotein. Free cholesterol molecules are dispersed throughout the lipoprotein shell to stabilize it in a way that allows it to maintain its spherical shape. The major carriers of lipids are chylomicrons (see Chapter 32), VLDL, and HDL. Metabolism of VLDL leads to IDL and LDL. Metabolism of chylomicrons leads to formation of chylomicron remnants. Through this carrier mechanism, lipids leave their tissue of origin, enter the bloodstream, and are transported to the tissues, where their components are either used in synthetic or oxidative process or stored for later use. The apoproteins (“apo” describes the protein within the shell of the particle in its lipid-free form) not only add to the hydrophilicity and structural stability of the particle, they have other functions as well: (1) They activate certain enzymes required for normal lipoprotein metabolism and (2) they act as ligands on the surface of the lipoprotein that target specific receptors on peripheral tissues that require lipoprotein delivery for their innate cellular functions. Ten principal apoproteins have been characterized. Their tissue sources, molecular mass, distribution within lipoproteins, and metabolic functions are shown in Table 34.3. The lipoproteins themselves are distributed among eight major classes. Some of their characteristics are shown in Table 34.4. Each class of lipoprotein has a specific function determined by its apolipoprotein content, its tissue of origin, and the proportion of the macromolecule made up of triacylglycerols, cholesterol esters, free cholesterol, and phospholipids (see Tables 34.3 and 34.4).

A. The Chylomicrons Chylomicrons are the largest of the lipoproteins and the least dense because of their rich triacylglycerol content. They are synthesized from dietary lipids (the “exogenous” lipoprotein pathway) within the epithelial cells of the small intestine and then secreted into the lymphatic vessels draining the gut (see Fig. 32.13). They enter the bloodstream via the left subclavian vein. The major apolipoproteins of chylomicrons are apolipoprotein B48 (apoB48), apoCII, and apoE (see Table 34.3). The apoCII activates lipoprotein lipase (LPL), an enzyme that projects into the lumen of capillaries in adipose tissue, cardiac muscle, skeletal muscle, and the acinar cells of mammary tissue. This activation allows LPL to hydrolyze the chylomicrons, leading to the release of free fatty acids derived from core triacylglycerides of the lipoprotein into these target cells. The muscle cells then oxidize the fatty acids as fuel, whereas the adipocytes and mammary cells store them as triacylglycerols (fat) or, in the case

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SECTION VI ■ LIPID METABOLISM

Table 34.3

Characteristics of the Major Apoproteins

Apoprotein

Primary Tissue Source

ApoAI

Intestine, liver

ApoAII

Liver

ApoAIV

Molecular Mass (Daltons)

Lipoprotein Distribution

Metabolic Function

28,016

HDL (chylomicrons)

17,414

HDL (chylomicrons)

Intestine

46,465

HDL (chylomicrons)

ApoB48

Intestine

264,000

Chylomicrons

ApoB100

Liver

540,000

VLDL, IDL, LDL

ApoCI

Liver

6,630

Chylomicrons, VLDL, IDL, HDL

ApoCII

Liver

8,900

ApoCIII

Liver

8,800

Chylomicrons, VLDL, IDL, HDL Chylomicrons, VLDL, IDL, HDL

ApoE

Liver

34,145

Apo(a)

Liver

Activates LCAT; structural component of HDL Uncertain; may regulate transfer of apoproteins from HDL to other lipoprotein particles Uncertain; may be involved in assembly of HDL and chylomicrons Assembly and secretion of chylomicrons from small bowel VLDL assembly and secretion; structural protein of VLDL, IDL, and LDL; ligand for LDL receptor Unknown; may inhibit hepatic uptake of chylomicron and VLDL remnants Cofactor activator of lipoprotein lipase (LPL) Inhibitor of LPL; may inhibit hepatic uptake of chylomicrons and VLDL remnants Ligand for binding of several lipoproteins to the LDL receptor, to the LDL receptor-related protein (LRP), and possibly to a separate apoE receptor Unknown; consists of apoB100 linked by a disulfide bond to apoprotein(a)

Chylomicron remnants, VLDL, IDL, HDL

Lipoprotein “little” a (Lp[a])

HDL, high-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein.

Table 34.4

Characteristics of the Major Lipoproteins

Lipoprotein

Density Range (g/mL)

Particle Diameter (mm) Range

Electrophoretic Mobility

Chylomicrons Chylomicron remnants VLDL IDL

0.930 0.930–1.006 0.930–1.006 1.006–1.019

75–1200 30–80 30–80 25–35

Origin Slow pre-␤ Pre-␤ Slow pre-␤

LDL HDL2 HDL3 Lip(a)

1.019–1.063 1.063–1.125 1.125–1.210 1.050–1.120

18–25 9–12 5–9 25

␤ ␣ ␣ Pre-␤

Lipid (%)a TG

Chol

PL

80–95

2–7

3–9

55–80 20–50

5–15 20–40

10–20 15–25

5–15 5–10

40–50 15–25

20–25 20–30

Function Deliver dietary lipids Return dietary lipids to the liver Deliver endogenous lipids Return endogenous lipids to the liver; precursor of LDL Deliver cholesterol to cells Reverse cholesterol transport Reverse cholesterol transport

TG, triacylglycerol; Chol, the sum of free and esterified cholesterol; PL, phospholipid; VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. a The remaining percentage composition is composed of apoproteins.

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641

Capillary walls Blood Glucose FA Cholesterol Amino acids FA Pi Glycerol

TG

VLDL

VLDL

Lysosomes

L VLDL P TG L CII FA + Glycerol

IDL

Liver

IDL TG LDL Cholesterol Amino acids FA Pi Glycerol

Muscle CO2 + H2O FA Adipose tissue FA TG Stores

H T G L

LDL receptor Lysosomes

Macrophage Oxidized LDL Foam cell

Peripheral cells Intima of blood vessel

FIG. 34.14. Fate of VLDL. VLDL triacylglycerol (TG) is degraded by LPL, forming IDL. IDL can either be endocytosed by the liver through a receptor-mediated process or further digested, mainly by hepatic triacylglycerol lipase (HTGL), to form LDL. LDL may be endocytosed by receptor-mediated processes in the liver or in peripheral cells. LDL also may be oxidized and taken up by “scavenger” receptors on macrophages. The scavenger pathway plays a role in atherosclerosis. FA, fatty acids; Pi, inorganic phosphate.

of the lactating breast, use them for milk formation. The partially hydrolyzed chylomicrons remaining in the bloodstream (the chylomicron remnants), now partly depleted of their core triacylglycerols, have lost their apoCII but still retain their apoE and apoB48 proteins. Receptors in the plasma membranes of the liver cells bind to apoE on the surface of these remnants, allowing them to be taken up by the liver through a process of receptor-mediated endocytosis (see the following text).

B. Very Low-Density Lipoprotein If dietary intake of carbohydrates exceeds the immediate fuel requirements of the liver, the excess carbohydrates are converted to triacylglycerols, which, along with free and esterified cholesterol, phospholipids, and the major apoprotein apoB100 (see Table 34.3), are packaged to form nascent VLDL. These particles are then secreted from the liver (the “endogenous” pathway of lipoprotein metabolism) into the bloodstream (Fig. 34.14), where they accept apoCII and apoE from circulating HDL particles. This then forms the mature VLDL particle. The density, particle size, and lipid content of VLDL particles are given in Table 34.3. These particles are then transported from the hepatic veins to capillaries in skeletal and cardiac muscle and adipose tissue, as well as lactating mammary tissues, where LPL is activated by apoCII in the VLDL particles. The activated enzyme facilitates the hydrolysis of the triacylglycerol in VLDL, causing the release of fatty acids and glycerol from a portion of core triacylglycerols. These fatty acids are oxidized as fuel by muscle cells, used in the resynthesis of triacylglycerols in fat cells, and used for milk production in the lactating breast. The residual particles remaining in the bloodstream are called VLDL remnants. Approximately 50% of these remnants are taken up from the blood by liver cells through the binding of VLDL apoE to the hepatocyte plasma membrane apoE receptor, followed by endocytic internalization of the VLDL remnant.

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C. Intermediate-Density Lipoprotein and Low-Density Lipoprotein Approximately half of the VLDL remnants are not taken up by the liver but, instead, have additional core triacylglycerols removed to form IDL, a specialized class of VLDL remnants. With the removal of additional triacylglycerols from IDL through the action of hepatic triglyceride lipase within hepatic sinusoids, LDL is generated from IDL. As can be seen in Table 34.4, the LDL particles are rich in cholesterol and cholesterol esters. Approximately 60% of the LDL is transported back to the liver, where its apoB100 binds to specific apoB100 receptors in the liver cell plasma membranes, allowing particles to be endocytosed into the hepatocyte. The remaining 40% of LDL particles is carried to extrahepatic tissues such as adrenocortical and gonadal cells that also contain apoB100 receptors, allowing them to internalize the LDL particles and use their cholesterol for the synthesis of steroid hormones. Some of the cholesterol of the internalized LDL is used for membrane synthesis and vitamin D synthesis as well. If an excess of LDL particles is present in the blood, this specific receptor-mediated uptake of LDL by hepatic and nonhepatic tissue becomes saturated. The “excess” LDL particles are now more readily available for nonspecific uptake of LDL by macrophages (scavenger cells) present near the endothelial cells of arteries. This exposure of vascular endothelial cells to high levels of LDL is believed to induce an inflammatory response by these cells, a process that has been suggested to initiate the complex cascade of atherosclerosis discussed in following sections.

D. High-Density Lipoprotein The fourth class of lipoproteins is HDL, which plays several roles in whole-body lipid metabolism. 1.

SYNTHESIS OF HIGH-DENSITY LIPOPROTEIN

HDL particles can be created by several mechanisms. The first is synthesis of nascent HDL by the liver and intestine as a relatively small molecule whose shell, like that of other lipoproteins, contains phospholipids, free cholesterol, and a variety of apoproteins, predominant among which are apoAI, apoAII, apoCI, and apoCII (see Table 34.3). Very low levels of triacylglycerols or cholesterol esters are found in the hollow core of this early, or nascent, version of HDL. A second method for HDL generation is the budding of apoproteins from chylomicrons and VLDL particles as they are digested by LPL. The apoproteins (particularly apoAI) and shells can then accumulate more lipid, as described in the following text. A third method for HDL generation is free apoAI, which may be shed from other circulating lipoproteins. The apoAI acquires cholesterol and phospholipids from other lipoproteins and cell membranes, forming a nascentlike HDL particle within the circulation. 2.

MATURATION OF NASCENT HIGH-DENSITY LIPOPROTEIN

In the process of maturation, the nascent HDL particles accumulate phospholipids and cholesterol from cells lining the blood vessels. As the central hollow core of nascent HDL progressively fills with cholesterol esters, HDL takes on a more globular shape to eventually form the mature HDL particle. The transfer of lipids to nascent HDL does not require enzymatic activity. 3.

REVERSE CHOLESTEROL TRANSPORT

A major benefit of HDL particles derives from their ability to remove cholesterol from cholesterol-laden cells and to return the cholesterol to the liver, a process known as reverse cholesterol transport. This is particularly beneficial in vascular tissue; by reducing cellular cholesterol levels in the subintimal space, the likelihood that foam cells (lipid-laden macrophages that engulf oxidized LDL-cholesterol and

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represent an early stage in the development of atherosclerotic plaque) will form within the blood vessel wall is reduced. Reverse cholesterol transport requires a directional movement of cholesterol from the cell to the lipoprotein particle. Cells contain the protein ABCA1 (ATPbinding cassette protein 1) that uses ATP hydrolysis to move cholesterol from the inner leaflet of the membrane to the outer leaflet (similar to the efflux of phytosterols by ABCG5 and ABCG8, see Section I of this chapter). Once the cholesterol has reached the outer membrane leaflet, the HDL particle can accept it; but if the cholesterol is not modified within the HDL particle, the cholesterol can leave the particle by the same route that it entered. To trap the cholesterol within the HDL core, the HDL particle acquires the enzyme lecithin-cholesterol acyltransferase (LCAT) from the circulation (LCAT is synthesized and secreted by the liver). LCAT catalyzes the transfer of a fatty acid from the 2-position of lecithin (phosphatidylcholine) in the phospholipid shell of the particle to the 3-hydroxyl group of cholesterol, forming a cholesterol ester (Fig. 34.15). The cholesterol ester migrates to the core of the HDL particle and is no longer free to return to the cell. Elevated levels of lipoprotein-associated cholesterol in the blood, particularly that associated with LDL but also that in the more triacylglycerol-rich lipoproteins, are associated with the formation of cholesterol-rich atheromatous plaque in the vessel wall, leading eventually to diffuse atherosclerotic vascular disease

H

H C

643

Two genetically determined disorders, familial HDL deficiency and Tangier disease, result from mutations in the adenosine triphosphate (ATP)-binding cassette 1 (ABCA1) protein. Cholesterol-depleted HDL cannot transport free cholesterol from cells that lack the ability to express this protein. As a consequence, HDL is rapidly degraded. These disorders have established a role for ABCA1 protein in the regulation of HDL levels in the blood.

O O

C

R1 O

HC

O

C O

R2

HC H

O

P

O CH2CH2N(CH3)3

+

O– Lecithin (PC)

HO Cholesterol

LCAT

R2

C

O O Cholesterol ester

O

H HC

O

HC

OH

HC H

O

C

R1

O P

+

O CH2CH2N(CH3)3

O– Lysolecithin

FIG. 34.15. fatty acid.

Lieberman_CH34.indd 643

The reaction catalyzed by LCAT. R1, saturated fatty acid; R2, unsaturated

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Because Ann Jeina continued to experience intermittent chest pain in spite of good control of her hypertension and a 20-lb weight loss, her physician decided that in addition to seeing a cardiologist to further evaluate the chest pain, a second drug is needed to be added to her regimen to further lower her blood LDL cholesterol level. Consequently, treatment with ezetimibe, a drug that blocks cholesterol absorption from the intestine, was added to complement the atorvastatin Ann was already taking.

that may result in acute cardiovascular events, such as myocardial infarction, stroke, or symptomatic peripheral vascular insufficiency. High levels of HDL in the blood, therefore, are believed to be vasculoprotective because these high levels increase the rate of reverse cholesterol transport “away” from the blood vessels and “toward” the liver (“out of harm’s way”). 4.

FATE OF HIGH-DENSITY LIPOPROTEIN CHOLESTEROL

Mature HDL particles can bind to specific receptors on hepatocytes (such as the apoE receptor), but the primary means of clearance of HDL from the blood is through its uptake by the scavenger receptor SR-B1. This receptor is present on many cell types. It does not carry out endocytosis per se, but once the HDL particle is bound to the receptor, its cholesterol and cholesterol esters are transferred into the cells. When depleted of cholesterol and its esters, the HDL particle dissociates from the SR-B1 receptor and reenters the circulation. SR-B1 receptors can be upregulated in certain cell types that require cholesterol for biosynthetic purposes, such as the cells that produce the steroid hormones. The SR-B1 receptors are not downregulated when cholesterol levels are high. 5.

HIGH-DENSITY LIPOPROTEIN INTERACTIONS WITH OTHER PARTICLES

In addition to its ability to pick up cholesterol from cell membranes, HDL also exchanges apoproteins and lipids with other lipoproteins in the blood. For example, HDL transfers apoE and apoCII to chylomicrons and to VLDL. The apoCII stimulates the degradation of the triacylglycerols of chylomicrons and VLDL by activating LPL (Fig. 34.16). After digestion of the chylomicrons and the VLDL triacylglycerols, apoE and apoCII are transferred back to HDL. When HDL obtains free cholesterol from cell membranes, the free cholesterol is esterified at the third carbon of the A-ring via the LCAT reaction (see Fig. 34.14). From this point, HDL either transports the free cholesterol and cholesterol esters directly to the liver,

Liver Bile salts Lysosome action

Blood ApoB-48 Nascent chylomicron

HDL Cholesterol

HDL ApoC II ApoA

Glucose

ApoB-48

ApoE

ApoCII Chylomicron

ApoE ApoB-100 Nascent VLDL

LCAT

ApoB-100 IDL

ApoAI C CE HDL

LDL

CE

TG

VLDL

CETP

TG

C C

C

C

VLDL ApoCII ApoE Cell membrane

C Cell

FIG. 34.16. Functions and fate of HDL. Nascent HDL is synthesized in liver and intestinal cells. It exchanges proteins with chylomicrons and VLDL. HDL picks up cholesterol (C) from cell membranes. This cholesterol is converted to cholesterol ester (CE) by the LCAT reaction. HDL transfers CE to VLDL in exchange for triacylglycerol (TG). The cholesterol ester transfer protein (CETP) mediates this exchange.

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as described earlier, or by CETP to circulating triacylglycerol-rich lipoproteins such as VLDL and VLDL remnants (see Fig. 34.16). In exchange, triacylglycerols from the latter lipoproteins are transferred to HDL (Fig. 34.17). The greater the concentration of triacylglycerol-rich lipoproteins in the blood, the greater will be the rate of these exchanges. Thus, the CETP exchange pathway may explain the observation that whenever triacylglycerol-rich lipoproteins are present in the blood in high concentrations, the amount of cholesterol reaching the liver via cholesterolenriched VLDL and VLDL remnants increases, and a proportional reduction in the total amount of cholesterol and cholesterol esters that are transferred directly to the liver via HDL occurs. Mature HDL particles are designated as HDL3; after reverse cholesterol transport and the accumulation of cholesterol esters, they become the atherogenic protective form, HDL2. The CETP reaction then leads to the loss of cholesterol and gain of triacylglycerol, such that the particles become larger and eventually regenerate HDL3 particles (see Table 34.4). Hepatic lipase can then remove triacylglycerol from HDL3 particles to regenerate HDL2 particles.

VII. LIPOPROTEINS ENTER CELLS BY RECEPTOR-MEDIATED ENDOCYTOSIS

VLDL

CE TG

Cholesterol ester transfer protein (CETP)

CE TG

HDL

As stated earlier, each lipoprotein particle contains specific apoproteins on its surface that act as ligands for specific plasma membrane receptors on target tissues such as the liver, the adrenal cortex, the gonads, and other cells that require one or more of the components of the lipoproteins. With the exception of the scavenger receptor SR-B1, the interaction of ligand and receptor initiates the process of endocytosis shown for LDL in Figure 34.18. The receptors for LDL, for example, are found in specific areas of the plasma membrane of the target cell for circulating lipoproteins. These are known as coated pits, and they contain a unique protein LDL particle

645

ApoB-100 Cholesterol ester

LDL receptor

Receptor-mediated endocytosis

FIG. 34.17. Function of cholesterol ester transfer protein (CETP). CETP transfers cholesterol esters (CEs) from HDL to VLDL in exchange for triacylglycerol (TG).

The cholesterol ester transfer protein (CETP) reaction, under conditions of high levels of triglyceride-rich lipoproteins, generates elevated levels of HDL3, which are less atheroprotective than HDL2. CETP inhibitors are currently being evaluated as a means of increasing HDL2 levels, with limited success. Initial clinical trials of a promising drug were terminated early because of an increased incidence of cardiovascular events when the drug was given in combination with an inhibitor of hydroxymethylglutaryl-CoA (HMG-CoA) reductase. Different CETP inhibitors, however, are currently being examined as potential HDL2 elevating agents.

Endosome Golgi complex Lysosome

Cholesterol

Amino acids Fatty acids

LDL receptor synthesis

Cholesterol ester droplet Nucleus

FIG. 34.18.

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Endoplasmic reticulum

Cholesterol uptake by receptor-mediated endocytosis.

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SECTION VI ■ LIPID METABOLISM

called clathrin. The plasma membrane in the vicinity of the receptor–LDL complex invaginates and fuses to form an endocytic vesicle. These vesicles then fuse with lysosomes, acidic subcellular vesicles that contain several degradative enzymes. The cholesterol esters of LDL are hydrolyzed to form free cholesterol, which is rapidly reesterified through the action of ACAT. This rapid reesterification is necessary to avoid the damaging effect of high levels of free cholesterol on cellular membranes. The newly esterified cholesterol contains primarily oleate or palmitoleate (monounsaturated fatty acids), unlike those of the cholesterol esters in LDL, which are rich in linoleate, a polyunsaturated fatty acid. As is true for the synthesis and activity of HMG-CoA reductase, the synthesis of the LDL receptor itself is subject to feedback inhibition by increasing levels of cholesterol within the cell. One probable mechanism for this feedback regulation involves one or more of the SREBPs described earlier. These proteins or the cofactors that are required for the full expression of genes that code for the LDL receptor are also capable of sensing the concentration of sterols within the cell. When sterol levels are high, the process that leads to the binding of the SREBP to the SRE of these genes is suppressed (see Fig. 34.4A). The rate of synthesis from mRNA for the LDL receptor is diminished under these circumstances. This, in turn, appropriately reduces the amount of cholesterol that can enter these cholesterol-rich cells by receptor-mediated endocytosis (downregulation of receptor synthesis). When the intracellular levels of cholesterol decrease, these processes are reversed, and cells act to increase their cholesterol levels. Both synthesis of cholesterol from acetyl-CoA and synthesis of LDL receptors are stimulated. An increased number of receptors (upregulation of receptor synthesis) results in an increased uptake of LDL cholesterol from the blood, with a subsequent reduction of LDL-cholesterol levels. At the same time, the cellular cholesterol pool is replenished.

VIII. LIPOPROTEIN RECEPTORS The best characterized lipoprotein receptor, the LDL receptor, specifically recognizes apoB100 and apoE. Therefore, this receptor binds VLDL, IDL, and chylomicron remnants in addition to LDL. The binding reaction is characterized by its saturability and occurs with high affinity and a narrow range of specificity. Other receptors, such as the LDL receptor-related proteins (LRPs) and the macrophage scavenger receptor (notably types SR-A1 and SR-A2, which are located primarily near the endothelial surface of vascular endothelial cells) have broad specificity and bind many other ligands in addition to the blood lipoproteins.

A. The Low-Density Lipoprotein Receptor The LDL receptor has a mosaic structure encoded by a gene that was assembled by a process known as exon shuffling. The gene contains 18 exons and is >45 kilobases (kb) in length; it is located on the short arm of chromosome 19. The protein encoded by the gene is composed of six different regions (Fig. 34.19). The first region, at the amino terminus, contains the LDL-binding region, a cysteine-rich sequence of 40 residues. Acidic side chains in this region bind ionic calcium. When these side chains are protonated, calcium is released from its binding sites. This release leads to conformational changes that allow the LDL to dissociate from its receptor docking site. Disulfide bonds, formed from the cysteine residues, have a stabilizing influence on the structural integrity of this portion of the receptor. The second region of the receptor contains domains that are homologous with epidermal growth factor (EGF) as well as with a complex consisting of six repeats that resembles the blades of the transducin ␤-subunit, forming a propellerlike moiety. The third region of the LDL receptor contains a chain of N-linked oligosaccharides, whereas the fourth region contains a domain that is rich in serine and threonine and contains O-linked sugars. This region may have a role in physically

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647

N Ca2+

Region one LDL-binding domain

Region two Epidermal growth factor–like domain Transducin-␤-subunit–like domain

Region three N-linked oligosaccharide domain

Region four O-linked oligosaccharide domain Region five Transmembrane domain

C

Region six Intracellular (cytosolic) domain

FIG. 34.19. Structure of the LDL receptor. The protein has six major regions, which are described in this text.

extending the receptor away from the membrane so that the LDL-binding region is accessible to the LDL molecule. The fifth region contains 22 hydrophobic residues that constitute the membranespanning unit of the receptor, whereas the sixth region extends into the cytosol, where it regulates the interaction between the C-terminal domain of the LDL receptor and the clathrin-containing coated pit where the process of receptor-mediated endocytosis is initiated. The number of LDL receptors, the binding of LDL to its receptors, and the postreceptor binding process can be diminished for a variety of reasons, all of which may lead to an accumulation of LDL cholesterol in the blood and premature atherosclerosis. These abnormalities can result from mutations in one (heterozygous—seen in approximately 1 in 500 people) or both (homozygous—seen in about 1 in 1 million people) of the alleles for the LDL receptor (familial hypercholesterolemia). Heterozygotes produce approximately half of the normal complement of LDL receptors, whereas the homozygotes produce almost no LDL receptor protein (receptor-negative familial hypercholesterolemia). The latter have serum total cholesterol levels in the range of 500 to 800 mg/dL.

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SECTION VI ■ LIPID METABOLISM

Number of mutations

40

30

20 Promoter 10

0 5'

3'

Exon no. 1 Signal sequence

2 3

4

5 6

Ligand binding

7 8 9 10 11 12 13 14 EGF precursor homology

15

16 17 18

O-linked sugars

Cytoplasmic

Membranespanning

FIG. 34.20. Location of 353 point mutations and small deletions/insertions (⬍25 base pairs) in the LDL receptor gene in individuals with familial hypercholesterolemia (FH). Exons are shown as vertical boxes and introns as the lines connecting them. (From Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolaemia. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The Metabolic and Molecular Bases of Inherited Disease. Vol 3. 8th ed. New York, NY: McGraw-Hill; 2001:2863–2913.)

Ann Jeina’s blood lipid levels (in mg/ dL) were Triacylglycerol Total cholesterol HDL cholesterol LDL cholesterol

158 420 32 356

She was diagnosed as having familial hypercholesterolemia (FH), type IIA, which is caused by genetic defects in the gene that encodes the LDL receptor (see Fig. 34.20). As a result of the receptor defect, LDL cannot readily be taken up by cells, and its concentration in the blood is elevated. LDL particles contain a high percentage, by weight, of cholesterol and cholesterol esters, more than other blood lipoproteins. However, LDL triacylglycerol levels are low because LDL is produced by digestion of the triacylglycerols of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL). Therefore, individuals with type IIA hyperlipoproteinemia have very high blood cholesterol levels, but their levels of triacylglycerols may be in or near the normal range (see Table 34.4).

Lieberman_CH34.indd 648

The genetic mutations are mainly deletions, but insertions or duplications also occur, as well as missense and nonsense point mutations (Fig. 34.20). Four classes of mutations have been identified. The first class involves “null” alleles that either direct the synthesis of no protein at all or a protein that cannot be precipitated by antibodies to the LDL receptor. In the second class, the alleles encode proteins, but they cannot be transported to the cell surface. The third class of mutant alleles encodes proteins that reach the cell surface but cannot bind LDL normally. Finally, the fourth class encodes proteins that reach the surface and bind LDL but fail to cluster and internalize the LDL particles. The result of each of these mutations is that blood levels of LDL are elevated because cells cannot take up these particles at a normal rate.

B. Low-Density Lipoprotein Receptor-Related Protein The LRP is structurally related to the LDL receptor but recognizes a broader spectrum of ligands. In addition to lipoproteins, it binds the blood proteins ␣2-macroglobulin (a protein that inhibits blood proteases) and tissue plasminogen activator (TPA) and its inhibitors. The LRP receptor recognizes the apoE of lipoproteins and binds remnants produced by the digestion of the triacylglycerols of chylomicrons and VLDL by LPL. Thus, one of its functions is believed to be clearing these remnants from the blood. The LRP receptor is abundant in the cell membranes of the liver, brain, and placenta. In contrast to the LDL receptor, synthesis of the LRP receptor is not significantly affected by an increase in the intracellular concentration of cholesterol. However, insulin causes the number of these receptors on the cell surface to increase, consistent with the need to remove chylomicron remnants that otherwise would accumulate after eating a meal.

C. Macrophage Scavenger Receptor Some cells, particularly the phagocytic macrophages, have nonspecific receptors known as scavenger receptors that bind various types of molecules, including oxidatively modified LDL particles. There are several different types of scavenger

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receptors. SR-B1 is used primarily for HDL binding, whereas the scavenger receptors expressed on macrophages are SR-A1 and SR-A2. Modification of LDL frequently involves oxidative damage, particularly of polyunsaturated fatty acyl groups (see Chapter 24). In contrast to the LDL receptors, the scavenger receptors are not subject to downregulation. The continued presence of scavenger receptors in the cell membrane allows the cells to take up oxidatively modified LDL long after intracellular cholesterol levels are elevated. When the macrophages become engorged with lipid, they are called foam cells. An accumulation of these foam cells in the subendothelial space of blood vessels form the earliest gross evidence of a developing atherosclerotic plaque known as a fatty streak. The processes that cause oxidation of LDL involve superoxide radicals, nitric oxide, hydrogen peroxide, and other oxidants (see Chapter 24). Antioxidants such as vitamin E, ascorbic acid (vitamin C), and carotenoids may be involved in protecting LDL from oxidation.

IX. ANATOMIC AND BIOCHEMICAL ASPECTS OF ATHEROSCLEROSIS The normal artery is composed of three distinct layers (Fig. 34.21). That which is closest to the lumen of the vessel, the intima, is lined by a monolayer of endothelial cells that are bathed by the circulating blood. Just beneath these specialized cells lies the subintimal extracellular matrix, in which some vascular smooth muscle cells are embedded (the subintimal space). The middle layer, known as the tunica media, is separated from the intima by the internal elastic lamina. The tunica media contains lamellae of smooth muscle cells surrounded by an elastin- and collagenrich matrix. The external elastic lamina forms the border between the tunica media and the outermost layer, the adventitia. This layer contains nerve fibers and mast cells. It is the origin of the vasa vasorum, which supplies blood to the outer twothirds of the tunica media. The initial step in the development of an atherosclerotic lesion within the wall of an artery is the formation of a fatty streak. The fatty streak represents an accumulation of lipid-laden macrophages or foam cells in the subintimal space. These fatty streaks are visible as a yellow-white linear streak that bulges slightly into the lumen of the vessel. These streaks are initiated when one or more known vascular risk factors for atherosclerosis, all of which have the potential to injure the vascular endothelial cells, reach a critical threshold at the site of future lesions. Examples of such risk factors include elevated intra-arterial pressure (arterial hypertension); elevated circulating levels of various lipids such as LDL, chylomicron remnants, and VLDL remnants; or low levels of circulating HDL, cigarette smoking, chronic elevations Internal elastic lamina

Adventitia

FIG. 34.21.

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Ivan Applebod’s blood lipid levels were: Triacylglycerol Total cholesterol HDL cholesterol LDL cholesterol

295 314 24 231

The elevated serum levels of LDL cholesterol found in patients such as Ivan Applebod who have type 2 diabetes mellitus is multifactorial. One of the mechanisms responsible for this increase involves the presence of chronically elevated levels of glucose in the blood of poorly controlled diabetics. This prolonged hyperglycemia increases the rate of nonenzymatic attachment of glucose to various proteins in the body, a process referred to as glycation or glycosylation of proteins. Glycation may adversely affect the structure or the function of the protein involved. For example, glycation of the LDL receptor and of proteins in the LDL particle may interfere with the normal “fit” of LDL particles with their specific receptors. As a result, less circulating LDL is internalized into cells by receptor-mediated endocytosis, and the serum LDL cholesterol level rises. Additionally, because Mr. Applebod is obese, he exhibits higher than normal levels of circulating free fatty acids, which the liver uses to increase the synthesis of VLDL, leading to hypertriglyceridemia.

External elastic lamina

Lumen

Tunica media (vascular smooth muscle)

649

Endothelial cell

Subintimal extracellular matrix (subintimal space)

The different layers of the arterial wall.

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SECTION VI ■ LIPID METABOLISM

In addition to dietary therapy, aimed at reducing her blood cholesterol levels, Ann Jeina was treated with atorvastatin, a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor. The HMGCoA reductase inhibitors decrease the rate of synthesis of cholesterol in cells. As cellular cholesterol levels decrease, the synthesis of LDL receptors increases. As the number of receptors increase on the cell surface, the uptake of LDL is accelerated. Consequently, the blood level of LDL cholesterol decreases. Ann was also treated with ezetimibe, which blocks cholesterol absorption from the intestinal lumen.

High-density lipoprotein (HDL) is considered to be the “good cholesterol” because it accepts free cholesterol from peripheral tissues, such as cells in the walls of blood vessels. This cholesterol is converted to cholesterol ester, part of which is transferred to VLDL by cholesterol ester transfer protein (CETP) and returned to the liver by IDL and LDL. The remainder of the cholesterol is transferred directly as part of the HDL molecule to the liver. The liver reuses the cholesterol in the synthesis of VLDL, converts it to bile salts, or excretes it directly into the bile. HDL, therefore, tends to lower blood cholesterol levels. Lower blood cholesterol levels correlate with lower rates of death caused by atherosclerosis.

Lieberman_CH34.indd 650

in blood glucose levels, high circulating levels of the vasoconstricting octapeptide angiotensin II, and others. The resulting insult to endothelial cells may trigger these cells to secrete adhesion molecules that bind to circulating monocytes and markedly slow their rate of movement past the endothelium. When these monocytic cells are slowed enough, they accumulate and have access to the physical spaces that exist between endothelial cells. This accumulation of monocytic cells resembles the classical inflammatory response to injury. These changes have led to the suggestion that atherosclerosis is, in fact, an inflammatory disorder and, therefore, is one that might be prevented or attenuated through the use of anti-inflammatory agents such as acetylsalicylic acid (aspirin) and HMG-CoA reductase inhibitors (statins), which have been shown to suppress the inflammatory cascade as well as to inhibit the action of HMG-CoA reductase. The monocytic cells are transformed into macrophages that migrate through the spaces between endothelial cells. They enter the subintimal space under the influence of chemoattractant cytokines (e.g., chemokine macrophage chemoattractant protein I) secreted by vascular cells in response to exposure to oxidatively modified fatty acids within the lipoproteins. The macrophages can replicate and exhibit augmented expression of receptors that recognize oxidatively modified lipoproteins. Unlike the classic LDL receptors on liver and many nonhepatic cells, these macrophage-bound receptors are high-capacity, low-specificity receptors (scavenger receptors). They bind to and internalize oxidatively modified fatty acids within LDLs to become subintimal foam cells as described previously. As these foam cells accumulate, they deform the overlying endothelium, causing microscopic separations between endothelial cells, exposing these foam cells and the underlying extracellular matrix to the blood. These exposed areas serve as sites for platelet adhesion and aggregation. Activated platelets secrete cytokines that perpetuate this process and increase the potential for thrombus (clot) formation locally. As the evolving plaque matures, a fibrous cap forms over its expanding “roof,” which now bulges into the vascular lumen, thereby partially occluding it. Vascular smooth muscle cells now migrate from the tunica media to the subintimal space and secrete additional plaque matrix material. The smooth muscle cells also secrete metalloproteinases that thin the fibrous cap near its “elbow” at the periphery of the plaque. This thinning progresses until the fibrous cap ruptures, allowing the plaque contents to physically contact the procoagulant elements present within the circulation. This leads to acute thrombus formation. If this thrombus completely occludes the remaining lumen of the vessel, an infarction of tissues distal to the occlusion (i.e., an acute myocardial infarction) may occur (Fig. 34.22). Most plaques that rupture also contain focal areas of calcification, which appears to result from the induction of the same cluster of genes as those that promote the formation of bone. The inducers for this process include oxidized sterols as well as transforming growth factor ␤ (TGF-␤) derived from certain vascular cells. Finally, high intraluminal shear forces develop in these thinning or eroded areas of the plaque’s fibrous cap, inducing macrophages to secrete additional metalloproteinases that further degrade the arterial-fibrous cap matrix. This contributes further to plaque rupture and thrombus formation (see Fig. 34.22). The consequence is a macrovascular ischemic event such as an acute myocardial infarction (AMI) or an acute cerebrovascular accident (CVA).

X. STEROID HORMONES Cholesterol is the precursor of all five classes of steroid hormones: glucocorticoids, mineralocorticoids, androgens, estrogens, and progestins. These hormones are synthesized in the adrenal cortex, ovaries, testes, and ovarian corpus luteum. Steroid hormones are transported through the blood from their sites of synthesis to their target organs, where, because of their hydrophobicity, they cross the cell

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B

Healed fissure

A Atherosclerotic vessel

Plaque fissure with small thrombus

Buried thrombus

C

Atherosclerotic plaque

Mural Intraintimal intraluminal thrombus thrombus (nonocclusive)

D

Occlusive intraluminal thrombus

FIG. 34.22. Evolution of an atherosclerotic plaque. Plaque capsule eroded near the “elbow” of plaque creating an early plaque fissure (A), which may heal as plaque increases in size (B) or may grow as thrombus expands, having an intraluminal portion and an intraintimal portion (C). If the fissure is not properly sealed, the thrombus may grow and completely occlude the vessel lumen (D), causing an acute infarction of tissues downstream of the vessel occlusion.

membrane and bind to specific receptors in either the cytoplasm or the nucleus. The bound receptors then bind to DNA to regulate gene transcription (see Chapter 16, Section III.C.2, and Fig. 16.12). Because of their hydrophobicity, steroid hormones must be complexed with a serum protein. Serum albumin can act as a nonspecific carrier for the steroid hormones, but there are specific carriers as well. The cholesterol used for steroid hormone synthesis is synthesized in the tissues from acetyl-CoA, extracted from intracellular cholesterol ester pools, or taken up by the cell in the form of cholesterol-containing lipoproteins (either internalized by the LDL receptor or absorbed by the SR-B1 receptor). In general, glucocorticoids and progestins contain 21 carbons, androgens contain 19 carbons, and estrogens contain 18 carbons. The specific complement of enzymes present in the cells of an organ determines which hormones the organ can synthesize. The oxidative reactions that lead to the synthesis and secretion of glucocorticoids such as cortisol are stimulated by adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. The role of cortisol as a stress-released hormone is discussed in Chapter 43. Mineralocorticoids such as aldosterone are also synthesized in the adrenal cortex and are secreted in response to angiotensin II or III, rising potassium levels in the blood, or hyponatremia (low levels of sodium ions in the blood). Aldosterone stimulates sodium reuptake in the kidney, sweat glands, salivary glands, and other tissues, with a resulting increase in extracellular fluid volume and eventually in blood pressure. The angiotensins are produced in response to a reduction in extracellular fluid volume, which may occur as a result of such things as excessive sweating, persistent vomiting without sufficient rehydration, or bleeding without adequate replacement of blood.

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651

In patients such as Ann Jeina and Ivan Applebod, who have elevated levels of VLDL or LDL, HDL levels are often low. These patients are predisposed to atherosclerosis and suffer from a high incidence of heart attacks and strokes. Exercise and estrogen administration both increase HDL levels. This is one of the reasons exercise is often recommended to aid in the prevention or treatment of heart disease. Prior to menopause, the incidence of heart attacks is relatively low in women, but it rises after menopause and increases to the level found in men by the age of 65 or 70 years. Moderate consumption of ethanol (alcohol) has also been correlated with increased HDL levels. Recent studies suggest that the beneficial amount of ethanol may be quite low, about two small glasses of wine a day, and that beneficial effects ascribed to ethanol may result from other components of wine and alcoholic beverages. In spite of the evidence that postmenopausal hormone replacement therapy (HRT) decreases circulating levels of LDL and increases HDL levels, recent data suggest that HRT may actually increase the rate of atherosclerotic vascular disease in these women. As a result, the accepted indications for estrogen administration are now limited to intolerable “hot flashes” or vaginal dryness.

Lipoprotein(a) is essentially an LDL particle that is covalently bound to apoprotein(a). It is called “lipoprotein little a” to avoid confusion with the apoprotein A found in LDL. The structure of apoprotein(a) is very similar to that of plasminogen, a precursor of the protease plasmin that degrades fibrin, a major component of blood clots. Lipoprotein(a), however, cannot be converted to active plasmin. There are reports that high concentrations of lipoprotein(a) correlate with an increased risk of coronary artery disease, even in patients in whom the lipid profile is otherwise normal. The physiologic function of lipoprotein(a) remains elusive.

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SECTION VI ■ LIPID METABOLISM

Vera Leizd consulted her gynecologist, who confirmed that her problems were probably the result of an excess production of androgens (virilization) and ordered blood and urine studies to determine whether Vera’s adrenal cortices or her ovaries were causing her virilizing syndrome.

Androgens such as testosterone are synthesized in the Leydig cells of the testes and to a lesser extent in the ovary and are secreted in response to luteinizing hormone (LH) produced in the anterior pituitary gland. In men, testosterone is commonly converted to dihydrotestosterone (DHT), a higher affinity form of the hormone, within specific target tissues. This active form of the hormone stimulates the production of sperm proteins in Sertoli cells and the development of secondary sex characteristics. Estrogens such as 17-␤-estradiol are synthesized in the ovarian follicle and the corpus luteum, from which their secretion is stimulated by follicle-stimulating hormone (FSH) produced in the anterior pituitary gland. In women, 17-␤-estradiol feeds back negatively on the synthesis and secretion of the pituitary gonadotropins, such as FSH. Estrogen and progesterone prepare the uterine endometrium for implantation of the fertilized ovum, and among other actions promote differentiation of the mammary gland. Progestogens such as progesterone are synthesized in the corpus luteum, and their secretion is stimulated by LH. As mentioned, in concert with estradiol, progesterone prepares the uterine endometrium for implantation of the fertilized ovum and acts as a differentiation factor in mammary gland development. The biosynthesis of glucocorticoids and mineralocorticoids (in the adrenal cortex), and that of sex steroids (in the adrenal cortex and gonads), requires several distinct cytochrome P450 enzymes (see Chapter 24). These monooxygenases are involved in the transfer of electrons from NADPH through electron-transfer protein intermediates to molecular oxygen, which then oxidizes a variety of the ring carbons of cholesterol. Cholesterol is converted to progesterone in the first two steps of the synthesis of all steroid hormones. Cytochrome P450SCC side-chain cleavage enzyme (previously referred to as cholesterol desmolase and classified as CYP11A) is located in the mitochondrial inner membrane and removes six carbons from the side chain of cholesterol, forming pregnenolone, which has 21 carbons (Fig. 34.23). The next step, the conversion of pregnenolone to progesterone, is catalyzed by 3-␤-hydroxysteroid dehydrogenase, an enzyme that is not a member of the cytochrome P450 family. Other steroid hormones are produced from progesterone by reactions that involve the other members of the P450 family. These include the mitochondrial enzyme cytochrome P450C11 (CYP11B1), which catalyzes ␤-hydroxylation at carbon 11 and two ER enzymes: P450C17 (17-␣-hydroxylation, CYP17) and P450C21 (hydroxylation at carbon 21, CYP21). As the synthesis of the steroid hormones is discussed, notice how certain enzymes are used in more than one pathway. Defects in these enzymes lead to multiple abnormalities in steroid synthesis, which, in turn, results in a variety of abnormal phenotypes.

A. Synthesis of Cortisol The adrenocortical biosynthetic pathway that leads to cortisol synthesis occurs in the middle layer of the adrenal cortex, known as the zona fasciculata. Free cholesterol is transported by an intracellular carrier protein to the inner mitochondrial membrane of cells (Fig. 34.24), where the side chain is cleaved to form pregnenolone. Pregnenolone returns to the cytosol, where it forms progesterone. In the membranes of the endoplasmic reticulum, the enzyme P450C17 (CYP17) catalyzes the hydroxylation of C17 of progesterone or pregnenolone and can also catalyze the cleavage of the two-carbon side chain of these compounds at C17 (a C17–C20 lyase activity), which forms androstenedione from 17-␣-hydroxyprogesterone. These two separate functions of the same enzyme allow further steroid synthesis to proceed along two separate pathways: The 17-hydroxylated steroids that retain their side chains are precursors of cortisol (C21), whereas those from which the side chain was cleaved (C19 steroids)

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21

22 20

24

26

23

25

18 12 11

C

19 1 2 3

HO

A

27

17 13

D

14

16 15

9 10 5

4

B

8 7

6

Cholesterol (C27) P450SCC (CYPIIA) CH3 C

O P450* C17

17-␣-Hydroxy pregnenolone (C21)

(CYP17)

P450C17 (CYP17) HO

O

Pregnenolone (C21) (3-␤HSD)

3-␤-Hydroxy steroid dehydrogenase

CH3 C

O

HO DHEA (C19) 3-␤-Hydroxy steroid dehydrogenase

3-␤-Hydroxy steroid dehydrogenase

(3-␤HSD)

(3-␤HSD)

O

O

Progesterone (C21)

(CYP21)

P450* C17

P450* C21

(CYP17) P450C17

17-␣-Hydroxy progesterone (C21)

11-deoxycorticosterone (C21) (DOC)

(CYP17) O

P450* C11

P450* C21

(CYP11B1)

(CYP21)

Androstenedione (C19) C17 dehydrogenase

OH

Corticosterone (C21)

Aromatase

11-Deoxycortisol (C21)

(CYP19) P450* C11

Aldosterone

(CYP11B2) synthase

O

(CYP11B1)

Testosterone (C19) Aromatase

HO

O CH2OH HC C O

O

CH2OH C HO

O Aldosterone (C21)

(CYP19) O

O OH

HO Cortisol (C21)

OH

HO Estrone (C18)

Estradiol (C18)

FIG. 34.23. Synthesis of the steroid hormones. The rings of the precursor, cholesterol, are lettered. Dihydrotestosterone is produced from testosterone by reduction of the carbon–carbon double bond in ring A. DHEA, dehydroepiandrosterone. The dashed lines indicate alternative pathways to the major pathways indicated. The starred enzymes are those that may be defective in congenital adrenal hyperplasia. 653

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SECTION VI ■ LIPID METABOLISM

LDL ACTH Cortisol

R G

LDL receptor AC

ATP

Cholesterol ester

cAMP

lipase

Protein kinase A

Endoplasmic reticulum Progesterone 3

Cholesterol

Cholesterol 1 2

Pregnenolone

4 11-Deoxycortisol 5 Mitochondrion Cortisol

FIG. 34.24. Cellular route for cortisol synthesis. Cholesterol is synthesized from acetylCoA or derived from low-density lipoprotein (LDL), which is endocytosed and digested by lysosomal enzymes. Cholesterol is stored in cells of the adrenal cortex as cholesterol esters. ACTH signals the cell to convert cholesterol to cortisol and transmits its signal through a G protein–coupled receptor that results in the activation of adenylate cyclase, and, ultimately, PKA. (1) Cholesterol desmolase (P450SCC, involved in side-chain cleavage); (2) 3-␤-hydroxysteroid dehydrogenase; (3) 17-␣-hydroxylase (P450C17); (4) 21-hydroxylase (P450C21); (5) 11-␤-hydroxylase (P450C11).

are precursors of androgens (male sex hormones) and estrogens (female sex hormones). In the pathway of cortisol synthesis, the 17-hydroxylation of progesterone yields 17-␣-hydroxyprogesterone, which is then hydroxylated by the P450C21 (CYP21) complex at carbon 21 to form 11-deoxycortisol. Progesterone can also be hydroxylated directly by P450C21 within the ER to form deoxycorticosterone (DOC), which is a precursor of the mineralocorticoid, aldosterone (see Fig. 34.23). The final step in cortisol synthesis requires transport of 11-deoxycortisol back to the inner membrane of the mitochondria, where P450C11 (11-␤-hydroxylase, CYP11B1) catalyzes the ␤-hydroxylation of the substrate at carbon 21, in a reaction that requires molecular oxygen and electrons derived from NADPH, to form cortisol. The rate of biosynthesis of cortisol and other adrenal steroids depends on stimulation of the adrenal cortical cells by ACTH.

B. Synthesis of Aldosterone The synthesis of the potent mineralocorticoid aldosterone in the zona glomerulosa of the adrenal cortex also begins with the conversion of cholesterol to progesterone (see Figs. 34.23 and 34.24). Progesterone is then hydroxylated at C21, a reaction

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CHAPTER 34 ■ CHOLESTEROL ABSORPTION, SYNTHESIS, METABOLISM, AND FATE

catalyzed by P450C21 (CYP21), to yield DOC. The P450C11 (CYP11B1) enzyme system then catalyzes the reactions that convert DOC to corticosterone. The terminal steps in aldosterone synthesis, catalyzed by the P450 aldosterone system (CYP11B2), involve the oxidation of corticosterone to 18-hydroxycorticosterone, which is oxidized to aldosterone. The primary stimulus for aldosterone production is the octapeptide angiotensin II, although hyperkalemia (greater than normal levels of potassium in the blood) or hyponatremia (less than normal levels of sodium in the blood) may stimulate aldosterone synthesis directly as well. ACTH has a permissive action in aldosterone production. It allows cells to respond optimally to their primary stimulus, angiotensin II.

C. Synthesis of the Adrenal Androgens Adrenal androgen biosynthesis proceeds from cleavage of the 2-carbon side chain of 17-hydroxypregnenolone at C17 to form the 19-carbon adrenal androgen dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) in the zona reticulosum of the adrenal cortex (see Fig. 34.23). These compounds, which are weak androgens, represent a significant percentage of the total steroid production by the normal adrenal cortex and are the major androgens synthesized in the adrenal gland. Androstenedione, another weak adrenal androgen, is produced by oxidation of the ␤-hydroxy group to a carbonyl group by 3-␤-hydroxysteroid dehydrogenase. This androgen is converted to testosterone primarily in extra-adrenal tissues. Although the adrenal cortex makes very little estrogen, the weak adrenal androgens may be converted to estrogens in the peripheral tissues, particularly in adipose tissue (Fig. 34.25).

D. Synthesis of Testosterone Luteinizing hormone (LH) from the anterior pituitary stimulates the synthesis of testosterone and other androgens by the Leydig cells of the human testicle. In many ways, the pathways leading to androgen synthesis in the testicle are similar to those described for the adrenal cortex. In the human testicle, the predominant pathway leading to testosterone synthesis is through pregnenolone to 17-␣-hydroxypregnenolone to DHEA (the ⌬5 pathway), and then from DHEA to androstenedione, and from androstenedione to testosterone (see Fig. 34.23). As for all steroids, the rate-limiting step in testosterone production is the conversion of cholesterol to pregnenolone. LH controls the rate of side-chain cleavage from cholesterol at carbon 21 to form pregnenolone and thus regulates the rate of testosterone synthesis. In its target cells, the double bond in ring A of testosterone is reduced through the action of 5-␣-reductase, forming the active hormone DHT.

Hyperplasia or tumors of the adrenal cortex that produce excess aldosterone result in a condition known as primary aldosteronism, which is characterized by enhanced sodium and water retention, resulting in hypertension.

Although aldosterone is the major mineralocorticoid in humans, excessive production of a weaker mineralocorticoid, DOC, which occurs in patients with a deficiency of the 11-hydroxylase (the P450C11 enzyme, CYP11B1), may lead to clinical signs and symptoms of mineralocorticoid excess even though aldosterone secretion is suppressed in these patients.

Adrenal O

OH

O

O Dehydroepiandrosterone

Adipose tissue Estrogens

Androstenedione

Extra-adrenal tissues Testosterone

FIG. 34.25. Adrenal androgens. These weak androgens are converted to testosterone or estrogens in other tissues.

Congenital adrenal hyperplasia (CAH) is a group of diseases caused by a genetically determined deficiency in a variety of enzymes required for cortisol synthesis. The most common deficiency is that of 21-␣-hydroxylase (CYP21), the activity of which is necessary to convert progesterone to 11-deoxycorticosterone and 17-␣-hydroxyprogesterone to 11-deoxycortisol. Thus, this deficiency reduces both aldosterone and cortisol production without affecting androgen production. If the enzyme deficiency is severe, the precursors for aldosterone and cortisol production are shunted to androgen synthesis, producing an overabundance of androgens, which leads to prenatal masculinization in females and postnatal virilization in males. Another enzyme deficiency in this group of diseases is that of 11-␤-hydroxylase (CYP11B1), which results in the accumulation of 11-deoxycorticosterone. An excess of this mineralocorticoid leads to hypertension (through binding of 11-deoxycorticosterone to the aldosterone receptor). In this form of CAH, 11-deoxycortisol also accumulates, but its biologic activity is minimal and no specific clinical signs and symptoms result. The androgen pathway is unaffected, and the increased adrenocorticotropic hormone (ACTH) levels may increase the levels of adrenal androgens in the blood. A third possible enzyme deficiency is that of 17-␣-hydroxylase (CYP17). A defect in 17-␣-hydroxylase leads to aldosterone excess and hypertension; however, because adrenal androgen synthesis requires this enzyme, no virilization occurs in these patients.

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SECTION VI ■ LIPID METABOLISM

Biologically, the most potent circulating androgen is testosterone. Approximately 50% of the testosterone in the blood in a normal woman is produced equally in the ovaries and in the adrenal cortices. The remaining half is derived from ovarian and adrenal androstenedione, which, after secretion into the blood, is converted to testosterone in adipose tissue, muscle, liver, and skin. The adrenal cortex, however, is the major source of the relatively weak androgen dehydroepiandrosterone (DHEA). The serum concentration of its stable metabolite, DHEAS, is used as a measure of adrenal androgen production in hyperandrogenic patients with diffuse excessive growth of secondary sexual hair (e.g., facial hair as well as that in the axillae, the suprapubic area, the chest, and the upper extremities).

E. Synthesis of Estrogens and Progesterone

The results of the blood tests on Vera Leizd showed that her level of testosterone was normal but that her serum DHEAS level was significantly elevated. Which tissue was the most likely source of the androgens that caused Vera’s hirsutism (a male pattern of secondary sexual hair growth)?

XI. VITAMIN D SYNTHESIS

Rickets is a disorder of young children caused by a deficiency of vitamin D. Low levels of calcium and phosphorus in the blood are associated with skeletal deformities in these children.

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Ovarian production of estrogens, progestins (compounds related to progesterone), and androgens requires the activity of the cytochrome P450 family of oxidative enzymes used for the synthesis of other steroid hormones. Ovarian estrogens are C18 steroids with a phenolic hydroxyl group at C3 and either a hydroxyl group (estradiol) or a ketone group (estrone) at C17. Although the major steroidproducing compartments of the ovary (the granulosa cell, the theca cell, the stromal cell, and the cells of the corpus luteum) have all of the enzyme systems required for the synthesis of multiple steroids, the granulosa cells secrete primarily estrogens, the theca and stromal cells secrete primarily androgens, and the cells of the corpus luteum secrete primarily progesterone. The ovarian granulosa cell, in response to stimulation by FSH from the anterior pituitary gland and through the catalytic activity of P450 aromatase (CYP19), converts testosterone to estradiol, the predominant and most potent of the ovarian estrogens (see Fig. 34.23). Similarly, androstenedione is converted to estrone in the ovary, although the major site of estrone production from androstenedione occurs in extraovarian tissues, principally skeletal muscle and adipose tissue.

Vitamin D is unique in that it can be either obtained from the diet (as vitamin D2 or D3) or synthesized from a cholesterol precursor, a process that requires reactions in the skin, liver, and kidney. The calciferols, including several forms of vitamin D, are a family of steroids that affect calcium homeostasis (Fig. 34.26). Cholecalciferol (vitamin D3) requires ultraviolet light for its production from 7-dehydrocholesterol, which is present in cutaneous tissues (skin) in animals and available from ergosterol in plants. This irradiation cleaves the carbon–carbon bond at C9–C10, opening the B-ring to form cholecalciferol, an inactive precursor of 1,25-(OH)2-cholecalciferol (calcitriol). Calcitriol is the most potent biologically active form of vitamin D (see Fig. 34.26). The formation of calcitriol from cholecalciferol begins in the liver and ends in the kidney, where the pathway is regulated. In this activation process, carbon 25 of vitamin D2 or D3 is hydroxylated in the microsomes of the liver to form 25-hydroxycholecalciferol (calcidiol). Calcidiol circulates to the kidney bound to vitamin D–binding globulin (transcalciferin). In the proximal convoluted tubule of the kidney, a mixed-function oxidase, which requires molecular O2 and NADPH as cofactors, hydroxylates carbon 1 on the A-ring to form calcitriol. This step is tightly regulated and is the rate-limiting step in the production of the active hormone. The release of parathyroid hormone from the parathyroid gland results in activation of this last step of active hormone formation. 1,25-(OH)2D3 (calcitriol) is approximately 100 times more potent than 25-(OH)D3 in its actions, yet 25-(OH)D3 is present in the blood in a concentration that may be 100 times greater, which suggests that it may play some role in calcium and phosphorus homeostasis. The biologically active forms of vitamin D are sterol hormones and, like other steroids, diffuse passively through the plasma membrane. In the intestine, bone, and kidney, the sterol then moves into the nucleus and binds to specific vitamin D3 receptors. This complex activates genes that encode proteins mediating the action of active vitamin D3. In the intestinal mucosal cell, for example, transcription of genes that encode calcium-transporting proteins are activated. These proteins are capable of carrying Ca2⫹ (and phosphorus) absorbed from the gut lumen across the cell, making it available for eventual passage into the circulation.

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CLINICAL COMMENTS Ann Jeina. Ann Jeina is typical of patients with essentially normal serum triacylglycerol levels and elevated serum total cholesterol levels that are repeatedly in the upper 1% of the general population (e.g., 325 to 500 mg/dL). When similar lipid abnormalities are present in other family members in a pattern of autosomal dominant inheritance and no secondary causes for these lipid alterations (e.g., hypothyroidism) are present, the entity referred to as familial hypercholesterolemia (FH), type IIA, is the most likely cause of this hereditary dyslipidemia. FH is a genetic disorder caused by an abnormality in one or more alleles responsible for the formation or the functional integrity of high-affinity low-density lipoprotein (LDL) receptors on the plasma membrane of cells that normally initiate the internalization of circulating LDL and other blood lipoproteins. Heterozygotes for FH (1 in 500 of the population) have roughly one-half of the normal complement or functional capacity of such receptors, whereas homozygotes (1 in 1 million of the population) have essentially no functional LDL receptors. The rare patient with the homozygous form of FH has a more extreme elevation of serum total and LDL cholesterol than does the heterozygote and, as a result, has a more profound predisposition to premature coronary artery disease. Chronic hypercholesterolemia not only may cause the deposition of lipid within vascular tissues leading to atherosclerosis but also may cause the deposition of lipid within the skin and eye. When this occurs in the medial aspect of the upper and lower eyelids, it is referred to as xanthelasma. Similar deposits known as xanthomas may occur in the iris of the eye (arcus lipidalis) as well as the tendons of the hands (“knuckle pads”) and Achilles tendons. Although therapy aimed at inserting competent LDL receptor genes into the cells of patients with homozygous FH is being designed for the future, the current approach in the treatment of heterozygotes is to attempt to increase the rate of synthesis of LDL receptors in cells pharmacologically. Ann Jeina was treated with ezetimibe, a drug that blocks cholesterol absorption in the intestine, causing a portion of the dietary cholesterol to be carried into the feces rather than packaged into chylomicrons. This reduces the levels of chylomicronbased cholesterol and cholesterol delivered to the liver by chylomicron remnants. HMG-CoA reductase inhibitors, such as atorvastatin, which Ann is also taking, stimulate the synthesis of additional LDL receptors by inhibiting HMG-CoA reductase, the rate-limiting enzyme for cholesterol synthesis. The subsequent decline in the intracellular free cholesterol pool also stimulates the synthesis of additional LDL receptors. These additional receptors reduce circulating LDL-cholesterol levels by increasing receptor-mediated endocytosis of LDL particles. A combination of strict dietary and dual pharmacologic therapy, aimed at decreasing the cholesterol levels of the body, is usually quite effective in correcting the lipid abnormality and, hopefully, the associated risk of atherosclerotic cardiovascular disease in patients with heterozygous familial hypercholesterolemia. Ivan Applebod. Low-density lipoprotein (LDL) cholesterol is the primary target of cholesterol-lowering therapy because both epidemiologic and experimental evidence strongly suggest a benefit of lowering serum LDL cholesterol in the prevention of atherosclerotic cardiovascular disease. Similar evidence for raising subnormal levels of serum HDL cholesterol is less conclusive but adequate to support such efforts, particularly in high-risk patients, such as Ivan Applebod, who have multiple cardiovascular risk factors. For individuals whose LDL levels are less than 30 points from their target, the first-line therapy in this attempt is nonpharmacologic and includes such measures as increasing aerobic exercise, weight loss in overweight patients, avoidance of excessive alcohol intake,

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Vera Leizd’s hirsutism was most likely the result of a problem in her adrenal cortex that caused excessive production of DHEA. CH3 H C H3C

CH2

CH2

CH3 CH

CH2

CH3

H3C HO 7-Dehydrocholesterol Skin

+

UV light

CH3 H C H3C

CH2

CH2

CH3 CH

CH2

CH3

H2C HO Cholecalciferol (D3) Liver 25–Hydroxycholecalciferol Kidney

+

PTH

1-α-hydroxylase

CH3 H C H3C

CH2

CH3 C OH

25

CH2

CH2

CH3

CH2 1

HO

OH 1,25-Dihydroxycholecalciferol (1,25-(OH)2D3)

FIG. 34.26. Synthesis of active vitamin D. 1,25-(OH)2D3 is produced from 7-dehydrocholesterol, a precursor of cholesterol. In the skin, ultraviolet (UV) light produces cholecalciferol, which is hydroxylated at the 25-position in the liver and at the 1-position in the kidney to form the active hormone. PTH, parathyroid hormone.

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SECTION VI ■ LIPID METABOLISM

Ann Jeina was treated with a statin (atorvastatin) and ezetimibe. Ezetimibe reduces the percentage of absorption of free cholesterol present in the lumen of the gut and hence the amount of cholesterol available to the enterocyte to package into chylomicrons. This, in turn, reduces the amount of cholesterol returning to the liver in chylomicron remnants. The net result is a reduction in the cholesterol pool in hepatocytes. The latter induces the synthesis of an increased number of LDL receptors by the liver cells. As a consequence, the capacity of the liver to increase hepatic uptake of LDL from the circulation leads to a decrease in serum LDL levels.

Table 34.5

reducing the intake of refined sugars, and cessation of smoking. If these efforts fail, or if the existing LDL levels are greater than 30 points from their target, drug therapy to lower serum LDL cholesterol levels must be considered. So far, Mr. Applebod has failed in his attempts to diet and exercise. His LDL cholesterol level is 231 mg/dL. According to Table 34.1, he is a candidate for more stringent dietary therapy and for drug treatment. He should be given an HMG-CoA reductase inhibitor such as pravastatin or atorvastatin. Other lipid-lowering drugs such as the fibric acid derivatives and ezetimibe, which also decrease triacylglycerol levels and potentially increase HDL levels, could be considered (Table 34.5). A low daily dose of aspirin (81 mg) could also be prescribed (see Chapter 35). It is important to gain early control of Mr. Applebod’s metabolic syndrome before the effects of insulin resistance can no longer be reversed. Vera Leizd. Vera Leizd was born with a normal female genotype and phenotype, had normal female sexual development, spontaneous onset of puberty, and regular, although somewhat scanty, menses until the age of 20 years. At that point, she developed secondary amenorrhea (cessation of menses) and evidence of male hormone excess with early virilization (masculinization). The differential diagnosis included an ovarian versus an adrenocortical source of the excess androgenic steroids. A screening test to determine whether the adrenal cortex or the ovary is the source of excess male hormone involves measuring the concentration of dehydroepiandrosterone sulfate (DHEAS) in the patient’s plasma because the adrenal cortex makes most of the DHEA and the ovary makes little or none. Vera’s plasma DHEAS level was moderately elevated, identifying her adrenal cortices as the likely source of her virilizing syndrome. If the excess production of androgens is not the result of an adrenal tumor, but rather the result of a defect in the pathway for cortisol production, the simple treatment is to administer glucocorticoids by mouth. The rationale for such treatment can be better understood by reviewing Figure 34.23. If Vera Leizd has a genetically determined partial deficiency in the P450C11 enzyme system needed

Mechanism(s) of Action and Efficacy of Lipid-Lowering Agents Percentage Change in Serum Lipid Level (Monotherapy) Total

LDL

Agent

Mechanism of Action

Cholesterol

Cholesterol

Cholesterol

Triacylglycerols

Statins

Inhibits HMG-CoA reductase activity Increase fecal excretion of bile salts

↓ 15%–60%

↓ 20%–60%

↑ 5%–15%

↓ 10%–40%

↓ 15%–20%

↓ 10%–25%

↑ 3%–5%

Activates LPL; reduces hepatic production of VLDL; reduces catabolism of HDL Antagonizes PPAR␣, causing an increase in LPL activity, a decrease in apoprotein CIII production, and an increase in apoprotein AI production Reduces intestinal absorption of free cholesterol from the gut lumen

↓ 22%–25%

↓ 10%–25%

↑ 15%–35%

Variable, depending on pretreatment level of triacylglycerols (may increase) ↓ 20%–50%

↓ 12%–15%

Variable, depending on pretreatment levels of other lipids

↑ 5%–15%

↓ 20%–50%

↓ 10%–15%

↓ 15%–20%

↑ 1%–3%

↓ 5%–8% if triacylglycerols are high pretreatment

Bile acid resins

Niacin

Fibrates

Ezetimibe

HDL

LPL, lipoprotein lipase; LDL, low-density lipoprotein; HMG-CoA, hydroxymethylglutaryl-CoA; HDL, high-density lipoprotein; triacylglycerols, triglycerides; PPAR, peroxisome proliferator-activated receptor; VLDL, very low-density lipoprotein. Source: Adapted from National Cholesterol Education Program. Third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Final Report. Circulation. 2002;106:3143–3457.

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659

to convert 11-deoxycortisol to cortisol, her blood cortisol levels would fall. By virtue of the normal positive feedback mechanism, a subnormal level of cortisol in the blood would induce the anterior pituitary to make more ACTH. The latter would not only stimulate the cortisol pathway to increase cortisol synthesis to normal but, in the process, would also induce increased production of adrenal androgens such as DHEA and DHEAS. The increased levels of the adrenal androgens (although relatively weak androgens) would cause varying degrees of virilization, depending on the severity of the enzyme deficiency. The administration of a glucocorticoid by mouth would suppress the high level of secretion of ACTH from the anterior pituitary gland that occurs in response to the reduced levels of cortisol secreted from the adrenal cortex. Treatment with prednisone (a synthetic glucocorticoid), therefore, will prevent the ACTH-induced overproduction of adrenal androgens. However, when ACTH secretion returns to normal, endogenous cortisol synthesis falls below normal. The administered prednisone brings the net glucocorticoid activity in the blood back to physiologic levels. Vera’s adrenal androgen levels in the blood returned to normal after several weeks of therapy with prednisone (a synthetic glucocorticoid). As a result, her menses eventually resumed, and her virilizing features slowly resolved. Because Vera’s symptoms began in adult life, her genetically determined adrenal hyperplasia is referred to as a “nonclassic” or “atypical” form of the disorder. A more severe enzyme deficiency leads to the “classic” disease, which is associated with excessive fetal adrenal androgen production in utero and manifests at birth, often with ambiguous external genitalia and virilizing features in the female neonate.

BIOCHEMICAL COMMENTS Drugs used to treat certain aspects of the metabolic syndrome improve insulin sensitivity and regulate lipid levels through modulation of the pathways discussed in Chapters 32 through 34. These drugs work by modifying the regulatory pathways that have been discussed so far with regard to carbohydrate and lipid metabolism. Metformin. Metformin has been used for ⬎30 years as a treatment for type 2 diabetes. Metformin reduces blood glucose levels by inhibiting hepatic gluconeogenesis, which is active in these patients because of the liver’s resistance to the effects of insulin. Metformin also reduces lipid synthesis in the liver, which aids in modulating blood lipid levels in these patients. Metformin accomplishes its effects by activating the adenosine monophosphate (AMP)-activated protein kinase (AMPK). It does so through activation of an upstream protein kinase, LKB1, via an unknown mechanism. As discussed previously, AMPK, when active, phosphorylates and reduces the activity of acetyl-CoA carboxylase (required for fatty acid synthesis) and HMG-CoA reductase (reducing the biosynthesis of cholesterol). Activation of AMPK also activates glucose uptake by the muscle (see Chapter 47), which is significant for reducing circulating blood glucose levels. The activation of AMPK also leads to a cascade of transcriptional regulation that reduces the liver’s ability to undergo both gluconeogenesis and lipogenesis. Activated AMPK phosphorylates a coactivator of the CREB transcription factor named transducer of regulated CREB activity 2 (TORC2) (Fig. 34.27). When TORC2 is phosphorylated, it is sequestered in the cytoplasm, and CREB becomes very inefficient at transcribing a gene that is required to upregulate genes that code for the enzymes involved in gluconeogenesis. This important transcriptional coactivator is named peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC1␣). PGC1␣ participates in the transcriptional activation of key gluconeogenic enzymes, such as glucose 6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK). Thus, in

Lieberman_CH34.indd 659

The LKB1 protein is a tumor suppressor; loss of LKB1 activity leads to Peutz-Jeghers syndrome (PJS). PJS exhibits the early development of vascular polyps in the gastrointestinal tract and an increased incidence of carcinomas at a relatively young age. LKB1 regulates the activity of 14 kinases that include, and are similar to, the adenosine monophosphate (AMP)-activated protein kinase. Loss of the normal regulation of these kinases, due to the absence of LKB1 activity, significantly contributes to tumor formation.

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SECTION VI ■ LIPID METABOLISM

LKB1 Metformin AMPK

+

TORC2 AMPK

PO4

TORC2

3– +

CREB TORC2

PO43– Sequester in cytoplasm

CREB TORC2 PGC1␣ expression

Glucose export

Enhanced gluconeogenesis

Increased gluconeogenic gene expression Nuclear membrane

FIG. 34.27. Action of metformin on gluconeogenesis. Under normal conditions, increases of hepatic cyclic adenosine monophosphate (cAMP) levels (e.g., in response to glucagon) activate CREB, which, in combination with TORC2, leads to increased transcription of genes required for gluconeogenesis. Under conditions of insulin resistance, this pathway remains stimulated, even in the presence of insulin. Metformin stimulates the activation of AMPK, which phosphorylates TORC2 and sequesters it in the cytoplasm, thereby decreasing synthesis of gluconeogenic enzymes and reducing hepatic output of glucose. The abbreviations used are defined in this text.

the presence of metformin, hepatic gluconeogenesis is reduced and muscle uptake of blood glucose is enhanced, leading to stabilization of blood glucose levels. The physiologic regulators of LKB1 have yet to be identified. Activation of AMPK also inhibits liver lipogenesis. In addition to phosphorylating and inhibiting acetyl-CoA carboxylase activity (which reduces malonyl-CoA levels, leading to reduced fatty acid synthesis and enhanced fatty acid oxidation), AMPK activity decreases the transcription of key lipogenic enzymes, including fatty acid synthase and acetyl-CoA carboxylase. The reduced transcriptional activity is mediated via an AMPK inhibition of the transcription of sterol-regulatory element-binding protein 1 (SREBP-1), which in addition to regulating the transcription of HMG-CoA reductase, also regulates the transcription of other lipogenic enzymes. The AMPK is discussed in more detail in the Biochemical Comments section of Chapter 36. Fibrates. The fibrates are a class of drugs used to decrease lipid levels (principally triglycerides) in patients. A major target of the fibrates is peroxisome proliferator-activated receptor-␣ (PPAR␣). Fibrate binding to PPAR␣ activates this transcription factor, which then leads to the transcription of a multitude of genes that degrade lipids. These targets include the genes for fatty acid transport proteins (so there is an enhanced rate of fatty acid transport into cells), fatty acid translocase (to increase mitochondrial uptake of the fatty acids), long-chain fatty acyl-CoA synthetase (activation of the fatty acids in the cytoplasm), and carnitine palmitoyl transferase I (which enhances the uptake of fatty acids into the mitochondria). In addition, PPAR␣ activation enhances lipoprotein lipase (LPL) expression, represses apoCIII expression (apoCIII inhibits the apoCII activation of LPL), and stimulates apoAI and apoAII synthesis, the major proteins in HDL. These transcriptional changes all lead to enhanced fat use and a reduction of circulating particles. Thiazolidinediones. A third class of drugs used for the treatment of insulin resistance and type 2 diabetes mellitus is the thiazolidinediones (TZDs), which activate the peroxisome proliferator-activated receptor-␥ (PPAR␥) class of transcription factors. PPAR␥ is expressed primarily in adipose tissue. This transcription factor is responsible for activating the transcription of

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661

adiponectin (see Chapter 33, Section VIII.B), leading to increased circulating adiponectin levels. The increase in adiponectin reduces the fat content of the liver and enhances insulin sensitivity via an AMPK-dependent pathway. Thiazolidinediones also lead to a reduction in plasma free fatty acid levels, which leads to enhanced insulin sensitivity (see the Biochemical Comments in Chapter 33). Key Concepts • • • •

• • • • • •

• • •

• •

Cholesterol regulates membrane fluidity and is a precursor of bile salts, steroid hormones (such as estrogen and testosterone), and vitamin D. Cholesterol, because of its hydrophobic nature, is transported in the blood as a component of lipoproteins. Within the lipoproteins, cholesterol can appear in its unesterified form in the outer shell of the particle or as cholesterol esters in the core of the particle. De novo cholesterol synthesis requires acetyl-CoA as a precursor, which is initially converted to hydroxymethylglutaryl CoA (HMG-CoA). The cholesterol synthesized in this way is packaged, along with triglyceride, into VLDL in the liver and then released into circulation. The conversion of HMG-CoA to mevalonic acid, catalyzed by HMG-CoA reductase, is the regulated and rate-limiting step of cholesterol biosynthesis. In the circulation, the triglycerides in VLDL are digested by lipoprotein lipase, which converts the particle to IDL and then to LDL. IDL and LDL bind specifically to receptors on the liver cell, are internalized, and the particle components are recycled. A third lipoprotein particle, HDL, functions to transfer apolipoprotein E and apolipoprotein CII to nascent chylomicrons and nascent VLDL. HDL also participates in reverse cholesterol transport, the movement of cholesterol from cell membranes to the HDL particle, which returns the cholesterol to the liver. Atherosclerotic plaques are associated with elevated levels of blood cholesterol levels. High levels of LDL are more strongly associated with the generation of atherosclerotic plaques, whereas high levels of HDL are protective because of their participation in reverse cholesterol transport. Bile salts are required for fat emulsification and micelle formation in the small intestine. Bile salts are recycled via the enterohepatic circulation, forming the secondary bile acids in the process. The steroid hormones are derived from cholesterol, which is converted to pregnenolone, which is the precursor for the mineralocorticoids (such as aldosterone), the glucocorticoids (such as cortisol), and the sex steroids (such as testosterone and estrogen). Lipid-lowering drugs act on a variety of targets within liver, intestine, and adipocytes. Diseases discussed in this chapter are summarized in Table 34.6.

Table 34.6

Diseases Discussed in Chapter 34

Disease or Disorder

Environmental or Genetic

Hypercholesterolemia

Both

Familial hypercholesterolemia, type II

Genetic

Virilization

Both

Congenital adrenal hyperplasia (CAH)

Genetic

Rickets

Environmental

Lieberman_CH34.indd 661

Comments Defined by elevated levels of cholesterol in the blood, often leading to coronary artery disease. Defect in LDL receptor, leading to elevated cholesterol levels, and premature death caused by coronary artery disease. Excessive release of androgenic steroids due to a variety of causes. CAH is a constellation of disorders caused by mutations in enzymes required for cortisol synthesis. One potential consequence is excessive androgen synthesis, which may lead to prenatal masculinization of females. The different symptoms observed between patients are caused by different enzyme deficiencies in the patients. Due to a lack of vitamin D, calcium metabolism is altered, leading to skeletal deformities.

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SECTION VI ■ LIPID METABOLISM

REVIEW QUESTIONS—CHAPTER 34 1.

Which of the following steps in the biosynthesis of cholesterol is the committed rate-limiting step? A. The condensation of acetoacetyl-CoA with a molecule of acetyl-CoA to yield ␤-hydroxy-␤-methylglutarylCoA (HMG-CoA) B. The reduction of HMG-CoA to mevalonate C. The conversion of mevalonate to two activated isoprenes D. The formation of farnesyl pyrophosphate E. Condensation of six activated isoprene units to form squalene

2.

Considering the final steps in cholesterol biosynthesis, when squalene is eventually converted to lanosterol, which of the following statements is correct? A. All of the sterols have three fused rings (the steroid nucleus) and are alcohols with a hydroxyl group at C3. B. The action of squalene monooxygenase oxidizes C14 of the squalene chain, forming an epoxide. C. Squalene monooxygenase is considered a mixedfunction oxidase because it catalyzes a reaction in which only one of the oxygen atoms of O2 is incorporated into the organic substrate. D. Squalene monooxygenase uses reduced flavin nucleotides, such as FAD(2H), as the cosubstrate in the reaction. E. Squalene is joined at carbons 1 and 30 to form the fused-ring structure of sterols.

Lieberman_CH34.indd 662

3.

Of the major risk factors for the development of atherosclerotic cardiovascular disease (ASCVD), such as sedentary lifestyle, obesity, cigarette smoking, diabetes mellitus, hypertension, and hyperlipidemia, which one, if present, is the only risk factor in a given patient without a history of having had a myocardial infarction that requires that the therapeutic goal for the serum LDL cholesterol level be ⬍100 mg/dL? A. Obesity B. Cigarette smoking C. Diabetes mellitus D. Hypertension E. Sedentary lifestyle

4.

Which one of the following apoproteins acts as a cofactor activator of the enzyme lipoprotein lipase (LPL)? A. ApoCIII B. ApoCII C. ApoB100 D. ApoB48s E. ApoE

5.

Which one of the following sequences places the lipoproteins in the order of most dense to least dense? A. HDL/VLDL/chylomicrons/LDL B. HDL/LDL/VLDL/chylomicrons C. LDL/chylomicrons/HDL/VLDL D. VLDL/chylomicrons/LDL/HDL E. LDL/chylomicrons/VLDL/HDL

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35

Metabolism of the Eicosanoids

The eicosanoids, which include the prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), are among the most potent regulators of cellular function in nature and are produced by almost every cell in the body. They act mainly as “local” hormones, affecting the cells that produce them or neighboring cells of a different type. Eicosanoids participate in many processes in the body, particularly the inflammatory response that occurs after infection or injury. The inflammatory response is the sum of the body’s efforts to destroy invading organisms and to repair damage. It includes control of bleeding through the formation of blood clots. In the process of protecting the body from a variety of insults, the inflammatory response can produce symptoms such as pain, swelling, and fever. An exaggerated or inappropriate expression of the normal inflammatory response may occur in individuals who have allergic or hypersensitivity reactions. In addition to participating in the inflammatory response, eicosanoids also regulate smooth muscle contraction (particularly in the intestine and uterus). They increase water and sodium excretion by the kidney and are involved in regulating blood pressure. They frequently serve as modulators; some eicosanoids stimulate and others inhibit the same process. For example, some serve as constrictors and others as dilators of blood vessels. They are also involved in regulating bronchoconstriction and bronchodilation. Eicosanoids are derived from polyunsaturated fatty acids containing 20 carbon atoms, which are found in cell membranes esterified to membrane phospholipids. Arachidonic acid, derived from the diet or synthesized from linoleate, is the compound from which most eicosanoids are produced in the body. Compounds that serve as signals for eicosanoid production bind to cell membrane receptors and activate phospholipases that cleave the polyunsaturated fatty acids from cell membrane phospholipids (Fig. 35.1). Arachidonic acid is enzymatically metabolized by three major pathways. The two pathways that have been most thoroughly studied are the cyclooxygenase pathway (which produces prostaglandins and thromboxanes) and the lipoxygenase pathway (which produces leukotrienes). The cytochrome P450 pathway generates eicosanoids with less well-defined physiologic functions. Isoprostanes are a relatively new class of eicosanoids derived from nonenzymatic free radical–catalyzed reactions. The isoprostanes are similar to prostaglandins in structure and may play a role in inflammatory responses and free radical–mediated tissue injury. Isoprostane levels in blood are used to assess oxidative stress in patients. In brain tissue, arachidonic acid can be coupled to ethanolamine to generate anandamide. This compound can bind and activate cannabinoid receptors with actions similar to those of ⌬9-tetrahydrocannabinol (THC). Many eicosanoids have very short half-lives, in the range of a few minutes or less. They are rapidly inactivated and excreted. 663

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SECTION VI ■ LIPID METABOLISM

Diet (essential fatty acids) Linoleate

Arachidonic acid O O

OC

R1

O

P

R2 C O Choline

Membrane phospholipid Phospholipase A2 –

Epoxides

COO–

cyt P450

lipoxygenase

11

9

7

COO

O

Glucocorticoids

Arachidonic acid (C20:4,⌬5,8,11,14)

–

5 6

Cyclo-oxygenase

LTA4

14

–

Aspirin and other NSAIDs

Leukotrienes COOH

O O

OH

TXA2

O

COO–

O

PGH2

OH

Thromboxanes

PGE2

PGF2␣

PGA2

PGI2 (prostacyclin)

Prostaglandins

FIG. 35.1. Overview of eicosanoid metabolism. Eicosanoids are produced from fatty acids released from membrane phospholipids. In humans, arachidonic acid is the major precursor of the eicosanoids, which include the prostaglandins, leukotrienes, and thromboxanes. 䊞 “inhibits”; cyt, cytochrome; NSAIDs, nonsteroidal anti-inflammatory drugs.

THE WAITING ROOM Since her admission to the hospital for an acute myocardial infarction, Ann Jeina has been taking the cholesterol absorption blocking drug, ezetimibe and the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin, to lower her blood cholesterol levels (see Chapter 34). She also takes 81 mg of acetylsalicylic acid (ASA; aspirin) each day. At her most recent visit to her cardiologist, she asked whether she should continue to take aspirin because she no longer has any chest pain. She was told that the use of aspirin in her case was not to alleviate pain but to reduce the risk of a second heart attack and that she

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665

should continue to take this drug for the remainder of her life unless a complication such as gastrointestinal bleeding occurred as a result of its use. Emma Wheezer has done well regarding her respiratory function since her earlier hospitalization for an acute asthmatic attack (see Chapter 31). She has been maintained on two puffs of triamcinolone acetonide (a potent inhaled corticosteroid) two times per day and has not required systemic steroids for months. The glucose intolerance precipitated by high intravenous and oral doses of the synthetic glucocorticoid prednisone during her earlier hospitalization resolved after this drug was discontinued. She has come to her doctor now because she is concerned that the low-grade fever and cough she has developed over the last 36 hours may trigger another acute asthma attack.

I.

SOURCE OF THE EICOSANOIDS

The most abundant, and therefore the most common precursor of the eicosanoids (eicosa is the Greek word for 20) is arachidonic acid (eicosatetraenoic acid, ␻6,20:4, ⌬5,8,11,14), a polyunsaturated fatty acid with 20 carbons and four double bonds (see Fig. 35.1). It is esterified to phospholipids located in the lipid bilayer that constitutes the plasma membrane of the cell. Because arachidonic acid cannot be synthesized de novo in the body, the diet must contain arachidonic acid or other fatty acids from which arachidonic acid can be produced. The major dietary precursor for arachidonic acid synthesis is the essential fatty acid linoleate, which is present in plant oils (see Chapter 33). The composition of the diet affects the fatty acid content of membrane phospholipids. Individuals with a high content of saturated fatty acids in their diets have a high content of saturated fatty acids in their membrane lipids. Likewise, individuals with a high content of polyunsaturated fatty acids in their diets have a high content of polyunsaturated fatty acids in their membrane lipids. The arachidonic acid in membrane phospholipids is released from the lipid bilayer as a consequence of the activation of membrane-bound phospholipase A2 or C (see Chapter 33, Fig. 33.30, and Fig. 35.2). This activation occurs when a variety of stimuli (agonists) such as histamine or the cytokines interact with a specific plasma membrane receptor on the target cell surface. Phospholipase A2

Dietary deficiencies of essential fatty acids are rare. However, some cases have been reported in patients receiving total parenteral nutrition (TPN). Although the most obvious symptom is a red scaly dermatitis, deficiencies of essential fatty acids also result in a decreased availability of precursors for eicosanoid synthesis.

Stimulus Binding

Receptor

Phosphatidylinositol bisphosphate

Phosphatidylcholine Phospholipase A2

Ca2+

+

Arachidonic acid

1

+

2

Cell membrane

Phospholipase C

1,2-Diacylglycerol Diacylglycerol lipase

Arachidonic acid

Monoacylglycerol Monoacylglycerol lipase

Arachidonic acid

FIG. 35.2. Release of arachidonic acid from membrane lipids. The binding of a stimulus to its receptor activates pathway 1 or 2.

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SECTION VI ■ LIPID METABOLISM

Arachidonic acid Cyclo-oxygenase

PGG2

Prostaglandins

HPETE

Thromboxanes

FIG. 35.3.

Inflammation involving the mucosal and smooth muscle layers of the respiratory tract plays a major role in the development of acute asthmatic bronchospasm in patients such as Emma Wheezer. Prednisone and other potent glucocorticoids are capable of preventing or suppressing this inflammation. In part, the glucocorticoids act by inhibiting the recruitment of leukocytes and monocytes–macrophages into affected areas. They also limit the ability of these cells to elaborate a variety of chemotactic factors and other substances, such as certain eicosanoids, which enhance the inflammatory process. Glucocorticoids, for example, suppress the transcription and translation of the inducible form of the cyclo-oxygenase enzyme, COX-2. Glucocorticoids also induce the synthesis of a protein or family of proteins (lipocortins or macrocortins) that inhibit the activity of phospholipase A2. As a result, the synthesis of prostaglandins and leukotrienes is decreased and the inflammatory response in bronchial tissues is reduced (see Figs. 35.1 and 35.2).

X 9 10 11

7 8

12

Y

5 6

13

3 4

14 15 16

17

COO–

1 2

18

19

20

HO

FIG. 35.4. Structural features common to the biologically active prostaglandins. These compounds have 20 carbons, with a carboxyl group at carbon 1. Carbons 8 through 12 form a five-membered ring with substituents (usually a hydroxyl or keto group) at carbons 9 (X) and 11 (Y). Carbon 15 contains a hydroxyl group, and a trans double bond connects carbons 13 and 14. Double bonds also may be present between carbons 5 and 6 and between carbons 17 and 18 (see Fig. 35.6).

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Cytochrome P450

Lipoxygenase

Leukotrienes

HETE

Epoxides

Lipoxins

diHETE

HETE

Pathways for the metabolism of arachidonic acid.

is specific for the sn-2 position of phosphoacylglycerols, the site of attachment of arachidonic acid to the glycerol moiety. Phospholipase C, on the other hand, hydrolyzes phosphorylated inositol from the inositol glycerophospholipids, generating a diacylglycerol containing arachidonic acid. This arachidonic acid is subsequently released by the action of other lipases.

II. PATHWAYS FOR EICOSANOID SYNTHESIS After arachidonic acid is released into the cytosol, it is converted to eicosanoids by a variety of enzymes with activities that vary among tissues. This variation explains why some cells, such as those in the vascular endothelium, synthesize prostaglandins E2 and I2 (PGE2 and PGI2), whereas cells such as platelets synthesize primarily thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Three major pathways for the metabolism of arachidonic acid occur in various tissues (Fig. 35.3). The first of these, the cyclo-oxygenase pathway, leads to the synthesis of prostaglandins and thromboxanes. The second, the lipoxygenase pathway, yields the leukotrienes, HETEs, and lipoxins. The third pathway, catalyzed by the cytochrome P450 system, is responsible for the synthesis of the epoxides, HETEs, and diHETEs.

A. Cyclo-oxygenase Pathway: Synthesis of the Prostaglandins and Thromboxanes 1.

STRUCTURES OF THE PROSTAGLANDINS

Prostaglandins are fatty acids containing 20 carbon atoms, including an internal five-carbon ring. In addition to this ring, each of the biologically active prostaglandins has a hydroxyl group at carbon 15, a double bond between carbons 13 and 14, and various substituents on the ring (Fig. 35.4). The nomenclature for the prostaglandins (PGs) involve the assignment of a capital letter (PGE), an Arabic numeral subscript (PGE1), and for the PGF family, a Greek letter subscript (e.g., PGF2␣). The capital letter, in this case “F,” refers to the ring substituents shown in Figure 35.5. The subscript that follows the capital letter (PGF1) refers to the PG series 1, 2, or 3, determined by the number of unsaturated bonds present in the linear portion of the hydrocarbon chain (Fig. 35.6). It does not include double bonds in the internal ring. Prostaglandins of the 1 series have one double bond (between carbons 13 and 14). The 2 series has two double bonds (between carbons 13 and 14 and between carbons 5 and 6). The 3 series has three double bonds (between carbons 13 and 14, 5 and 6, and 17 and 18). The double bonds between carbons 13 and 14 are trans; the others are cis. The Greek letter subscript, found only in the F series, refers to the position of the hydroxyl group at carbon 9. This hydroxyl group exists primarily in the ␣-position, where it lies below the plane of the ring, as does the hydroxyl group at carbon 11 (see Figs. 35.4 and 35.5).

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CHAPTER 35 ■ METABOLISM OF THE EICOSANOIDS

O

OH R7

R7 R8

R8 O

PGA

PGD OH

O

R7

R7

R8

R8 OH

OH

PGE

PGF␣ R4 O

O

R7

O

R8

R8 OH

PGG or PGH

PGI

FIG. 35.5. Ring substituents of the prostaglandins (PGs). The letter after PG denotes the configuration of the ring and its substituents. R4, R7, and R8 represent the nonring portions of the molecule. R4 contains four carbons (including the carboxyl group). R7 and R8 contain seven and eight carbons, respectively, and correspond to the 1, 2, or 3 series shown in Figure 35.6. Note that the prostacyclins (PGI) contain two rings.

2.

STRUCTURE OF THE THROMBOXANES

The thromboxanes, derived from arachidonic acid via the cyclo-oxygenase pathway, closely resemble the prostaglandins in structure except that they contain a six-membered ring that includes an oxygen atom (Fig. 35.7). The most common thromboxane, TXA2, contains an additional oxygen atom attached to both carbon 9 and carbon 11 of the ring. The thromboxanes were named for their action in producing blood clots (thrombi). 3.

BIOSYNTHESIS OF THE PROSTAGLANDINS AND THROMBOXANES

Only the biosynthesis of those prostaglandins derived from arachidonic acid (e.g., the 2 series, such as PGE2, PGI2, TXA2) are described because those derived from eicosatrienoic acid (the 1 series) or from eicosapentaenoic acid (the 3 series) are present in very small amounts in humans on a normal diet. The biochemical reactions that lead to the synthesis of prostaglandins and thromboxanes are illustrated in Figure 35.8. The initial step, which is catalyzed by a cyclo-oxygenase, forms the five-membered ring and adds four atoms of oxygen (two between carbons 9 and 11, and two at carbon 15) to form the unstable endoperoxide, PGG2. The hydroperoxy group at carbon 15 is quickly reduced to a hydroxyl group by a peroxidase to form another endoperoxide, PGH2. The next step is tissue specific (see Fig. 35.8). Depending on the type of cell involved, PGH2 may be reduced to PGE2 or PGD2 by specific enzymes (PGE synthase and PGD synthase). PGE2 may be further reduced by PGE 9-ketoreductase to form PGF2␣. PGF2␣ also may be formed directly from PGH2 by the action of

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667

The measurement of prostaglandin levels, in plasma or urine, is best done by radioimmunoassay (see Chapter 43, Biochemical Comments). Antibodies specific for each prostaglandin or thromboxane form are commercially available, and through competition with a standard amount of antigen, one can determine the concentration of the metabolite in the biological fluid. Recently, a more sensitive technique has been developed that can assay prostaglandin levels as low as 40 pg/mL. This technique is called fluorescent polarization immunoassay (FPIA). The method is based on the properties of small, fluorescent molecules. Molecules that fluoresce absorb light at a particular wavelength and will emit light of a lower wavelength (the fluorescence). If one excites a small fluorophore with polarized light, the fluorescence will be polarized if the molecule rotates slowly; if the molecule rotates rapidly, the emitted light will not be polarized. If, then, the fluorophore is bound to a much larger molecule, such as an antibody, its rotation would be greatly diminished and the fluorescent signal emitted will be highly polarized. One can therefore measure the polarization of the emitted light as a function of how much fluorescent standard is bound to the antibody. So for these assays, a known amount of a fluorescent prostaglandin is incubated with the samples; if the sample contains nonfluorescent prostaglandin, it will compete for binding with the fluorescent prostaglandin, relegating some fluorescent prostaglandin to being nonbound. When the excitation light hits the sample, the amount of polarization will decrease in proportion to the amount of fluorescent prostaglandin displaced from the antibody. Through use of a standard curve, one can then calculate the level of prostaglandin in the sample to very low levels.

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SECTION VI ■ LIPID METABOLISM

8,11,14-Eicosatrienoic acid (dihomo-␥-linolenic acid) COOH

Series 1

C20: ⌬8,11,14 (precursor) ␻6

O

H

COOH 15

HO

H

13

H OH PGE1

Arachidonic acid COOH

2

C20: ⌬5,8,11,14, (precursor) ␻6

O

H

5

COOH

15

HO

H

13

H OH PGE2

Eicosapentaenoic acid COOH

3

C20: ⌬5,8,11,14,17 (precursor) ␻3

O

H

5

COOH

15

HO

H

13

H OH

17

PGE3

9

COOH

O 11

O

OH Thromboxane A2 (TXA2)

FIG. 35.7. The thromboxane ring. In contrast to the prostaglandins, which have a fivemembered carbon ring, thromboxanes have a six-membered ring (shown in red) containing an oxygen atom. Substituents are attached to the ring at carbons 9 and 11. In the case of TXA2 (shown previously), an oxygen atom connects carbons 9 and 11.

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FIG. 35.6. Prostaglandins of the 1, 2, and 3 series and their precursors. The numeral (as a subscript in the name of the compound) refers to the number of double bonds in the nonring portion of the prostaglandin. Trans double bonds are at position 13, and cis double bonds are at positions 5 and 17. The hydroxyl group at carbon 15 is required for biologic activity.

an endoperoxide reductase. Some of the major functions of the prostaglandins are listed in Table 35.1. PGH2 may be converted to the thromboxane TXA2, a reaction catalyzed by TXA synthase (see Fig. 35.8). This enzyme is present in high concentration in platelets. In the vascular endothelium, however, PGH2 is converted to the prostaglandin PGI2 (prostacyclin) by PGI synthase (see Fig. 35.8). TXA2 and PGI2 have important antagonistic biologic effects on vasomotor and smooth muscle tone and on platelet aggregation. Some of the known functions of the thromboxanes are listed in Table 35.2.

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669

Arachidonic acid 2O2

Cyclo-oxygenase

O O

8

9 11

COOH 15

12

OOH PGG2 2GSH Peroxidase

O

COOH

O PGD synthase

PGD2 PGF2␣

PGE 9-keto reductase

PGE synthase

GSSG

OH PGH2 PGI synthase

TXA synthase

PGE2 COOH O

O

OH

COOH

O

OH

Prostacyclin PGI2 • Produced by vascular endothelial cells • Inhibits platelet aggregation • Causes vasodilation

OH

Thromboxane TXA2 • Produced by platelets • Stimulates platelet aggregation • Causes vasoconstriction

FIG. 35.8. Formation of prostaglandins (including the prostacyclin PGI2) and thromboxane TXA2 from arachidonic acid. The conversion of arachidonic acid to PGH2 is catalyzed by a membrane-bound enzyme, prostaglandin endoperoxide synthase, which has cyclo-oxygenase and peroxidase activities. The reducing agent is glutathione (GSH), which is oxidized to form a disulfide between two glutathione molecules (GSSG).

In the 1990s, the cyclo-oxygenase enzyme was found to exist as two distinct isoforms, designated COX-1 and COX-2. COX-1 is regarded as a constitutive form of the enzyme, is widely expressed in almost all tissues, is the only form expressed in mature platelets, and is involved in the production of prostaglandins and thromboxanes for “normal” physiologic functions. COX-2 is an inducible form of the enzyme regulated by a variety of cytokines and growth factors. COX-2 messenger

Table 35.1

Some Functions of the Prostaglandins

PGI2, PGE2, PGD2

PGF2␣

Increase Vasodilation cAMP Decrease Platelet aggregation Leukocyte aggregation IL-1a and IL-2 T-cell proliferation Lymphocyte migration

Increases Vasoconstriction Bronchoconstriction Smooth muscle contraction

a

IL, interleukin, a cytokine that augments the activity of many cells in the immune system.

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Table 35.2 Some Functions of Thromboxane A2 Increases Vasoconstriction Platelet aggregation Lymphocyte proliferation Bronchoconstriction

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SECTION VI ■ LIPID METABOLISM

The predominant eicosanoid in platelets is TXA2, a potent vasoconstrictor and a stimulator of platelet aggregation. The latter action initiates thrombus formation at sites of vascular injury as well as in the vicinity of a ruptured atherosclerotic plaque in the lumen of vessels such as the coronary arteries. Such thrombi may cause sudden total occlusion of the vascular lumen, causing acute ischemic damage to tissues distal to the block (i.e., acute myocardial infarction). Aspirin, by covalently acetylating the active site of cyclo-oxygenase, blocks the production of TXA2 from its major precursor, arachidonic acid. By causing this mild hemostatic defect, low-dose aspirin has been shown to be effective in prevention of acute myocardial infarction (see Clinical Comments). For Ivan Applebod (who has symptoms of coronary heart disease), aspirin is used to prevent a first heart attack (primary prevention). For Ann Jeina and Cora Nari (who have already had heart attacks), aspirin is used to prevent a second heart attack (secondary prevention).

Diets that include cold-water fish (e.g., salmon, mackerel, brook trout, herring), with a high content of polyunsaturated fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) (see Chapter 33), result in a high content of these fatty acids in membrane phospholipids. It has been suggested that such diets are effective in preventing heart disease, in part because they lead to formation of more TXA3 relative to TXA2. TXA3 is less effective in stimulating platelet aggregation than its counterpart in the 2 series, TXA2.

RNA (mRNA) and protein levels are usually low in most healthy tissue but are expressed at high levels in inflamed tissue. Because of the importance of prostaglandins in mediating the inflammatory response, drugs that block prostaglandin production should provide relief from pain. The cyclo-oxygenase enzyme is inhibited by all nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid (aspirin). Aspirin transfers an acetyl group to the enzyme, irreversibly inactivating it (Fig. 35.9). Other NSAIDs (e.g., acetaminophen, ibuprofen) act as reversible inhibitors of cyclo-oxygenase. Acetaminophen is the major ingredient in Tylenol, and ibuprofen is the major ingredient in other NSAIDs such as Motrin, Nuprin, and Advil (see Fig. 35.9). Although they have some relative selectivity for inhibiting either COX-1 or COX-2, NSAIDs block the activity of both isoforms. These findings have provided the impetus for the development of selective COX-2 inhibitors, which are proposed to act as potent anti-inflammatory agents by inhibiting COX-2 activity, without the gastrointestinal (stomach ulcers) and antiplatelet side effects commonly associated with NSAID use. These adverse effects of NSAIDs are thought to be caused by COX-1 inhibition. Examples of these newer selective COX-2 inhibitors are celecoxib (Celebrex) and rofecoxib (Vioxx). 4.

INACTIVATION OF THE PROSTAGLANDINS AND THROMBOXANES

Prostaglandins and thromboxanes are rapidly inactivated. Their half-lives (t1/2) range from seconds to minutes. The prostaglandins are inactivated by oxidation of the 15-hydroxy group—which is critical for their activity—to a ketone. The double bond at carbon 13 is reduced. Subsequently, both ␤- and ␻-oxidation of the nonring portions occur, producing dicarboxylic acids that are excreted in the urine. Active TXA2 is rapidly metabolized to TXB2 by cleavage of the oxygen bridge between carbons 9 and 11 to form two hydroxyl groups. TXB2 has no biologic activity.

COO– O

O C

CH3

Acetylsalicylate (aspirin) O Ser

OH

Ser

Active cyclo-oxygenase

COO

O

C

CH3

Inactive cyclo-oxygenase

–

OH

Salicylate O HO

NH

C

CH3

Acetaminophen

H3C H3C

CH3 CH

CH2

CH

COOH

Ibuprofen

FIG. 35.9. Action of aspirin and other NSAIDs.

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CHAPTER 35 ■ METABOLISM OF THE EICOSANOIDS

Table 35.3

Properties of COX-1 and COX-2 COX-1

COX-2

Platelet aggregation, stomach cytoprotection

Inflammation, hyperalgesia

Response to: NSAIDs Steroids

Decreased activity No effect

COX-2 inhibitor

No effect

Decreased activity Decreased synthesis and activity Decreased activity

Primary function

NSAIDs, nonsteroidal anti-inflammatory drugs. Data adapted from Patrono and Baigent (see References) indicate that low-dose aspirin (50 to 100 mg/day) reduces platelet TXA2 levels by 97%, with no effect on whole body PGI2, thereby leading to cardioprotection. High-dose aspirin (650 to 1,300 mg/day), in addition to reducing TXA2 levels, also reduced PGI2 levels by 60% to 80%. COX-2 specific inhibitors, at high levels, had no effect on TXA2 levels but reduced whole body PGI2 levels by 60% to 80%; leading to an increased risk of myocardial infarction.

B. Lipoxygenase Pathway: Synthesis of the Leukotrienes, Hydroxyeicosatetraenoic, and Lipoxins In addition to serving as a substrate for the cyclo-oxygenase pathway, arachidonic acid also acts as a substrate for the lipoxygenase pathway. The lipoxygenase enzymes catalyze the incorporation of an oxygen molecule into a carbon of one of several double bonds of arachidonic acid, forming a hydroperoxy (—OOH) group at these positions. With this oxygenation, the double bond isomerizes to a position one carbon removed from the hydroperoxy group and is transformed from the cis to the trans configuration (Fig. 35.10). The unstable hydroperoxy group is then converted to the more stable hydroxy group. Lipoxygenases may act at carbons 5, 12, or 15. The type of lipoxygenase varies from tissue to tissue. For example, polymorphonuclear leukocytes contain primarily 5-lipoxygenase, platelets are rich in 12-lipoxygenase, and eosinophilic leukocytes contain primarily 15-lipoxygenase.

671

Although the COX-2 inhibitors did relieve the development of gastrointestinal ulcers in patients taking nonsteroidal anti-inflammatory drugs (NSAIDs), further studies indicated that specific COX-2 inhibitors may have a negative effect on cardiovascular function. Vioxx was withdrawn from the market by its manufacturer because of these negative patient studies. It has been postulated that long-term use of COX-2 inhibitors alter the balance of prostacyclin (antithrombotic, PGI2) and thromboxane (prothrombotic) because platelets, the major source of the thromboxanes, do not express COX-2 and thromboxane synthesis is not reduced with COX-2 inhibitors (see Fig. 35.8, indicating that PGI2 synthesis in the blood vessels will be decreased by COX-2 inhibitors). This will tilt the balance of the eicosanoids synthesized toward a thrombotic pathway. The COX-2 inhibitors that remain on the market must be used with caution, and they are contraindicated in patients with ischemic heart disease or stroke. Some properties of COX-1 and COX-2 are indicated in Table 35.3.

Arachidonic acid COOH 5-Lipoxygenase

15-Lipoxygenase 12-Lipoxygenase

OOH COOH

COOH

COOH

HOO HOO 5-HPETE

12-HPETE

15-HPETE

OH COOH

COOH

COOH

HO HO 5-HETE

12-HETE

15-HETE

FIG. 35.10. Action of lipoxygenases in the formation of HPETEs (hydroperoxyeicosatetraenoic acids) and HETEs (hydroxyeicosatetraenoic acids). Lipoxygenases add hydroperoxy groups at positions 5, 12, or 15 with rearrangement of the double bond. HPETEs are unstable and are rapidly reduced to form HETEs or are converted to leukotrienes and lipoxins (see Figs. 35.11 and 35.12).

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SECTION VI ■ LIPID METABOLISM

Arachidonic acid 5-Lipoxygenase 5

COOH

OOH 5-HPETE 11

9

7

O 6

COOH

5

14

GSH 11

9

7

LTA4

OH 6

OH

OH

COOH

5

COOH

S 14

␥-Glu Cys Gly

Glutathione

LTC4

LTB4 Glutamate LTD4 Glycine LTE4

FIG. 35.11. Formation of leukotrienes and their glutathione (GSH) derivatives. HPETE, hydroperoxyeicosatetraenoic acid; LT, leukotriene (thus, LTA4 is leukotriene A4).

1.

Arachidonic acid 15-Lipoxygenase

5-Lipoxygenase

Reductions

HO OH COOH

OH Lipoxin A4 (LXA4)

FIG. 35.12. Formation of the lipoxins. These compounds contain three hydroxyl groups.

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LEUKOTRIENE SYNTHESIS

As shown in Figure 35.11, the synthesis of the leukotrienes begins with the formation of hydroperoxyeicosatetraenoic acids (HPETEs). These products are either reduced to the corresponding hydroxy metabolites, HETEs (see Fig. 35.10), or are metabolized to form leukotrienes or lipoxins (see Figs. 35.11 and 35.12). The major leukotrienes are produced by 5-lipoxygenase. Leukotrienes are so named because they are synthesized in leukocytes and contain the typical triene structure, that is, three double bonds in series (in this case, at positions 7, 9, and 11) (see Fig. 35.11). In leukocytes and mast cells, 5-HPETE is converted to an epoxide, leukotriene A4 (LTA4). The subscript 4 refers to four double bonds in the leukotriene. Three of the double bonds (at carbons 7, 9, and 11) are conjugated, that is, they form a triene. Other functional leukotrienes are formed from LTA4 by one of two pathways. In the first, LTA4 is converted to LTB4, a 5,12-dihydroxy derivative. The second metabolic pathway involves the addition of reduced glutathione to carbon 6 to form LTC4, a reaction catalyzed by glutathione S-transferase. Glutamate is removed from the glutathione moiety of LTC4 through the action of ␥-glutamyl transpeptidase to form LTD4. A dipeptidase then cleaves the glycine residue from LTD4 to form LTE4 (see Fig. 35.11). The major functions of some of the leukotrienes are listed in Table 35.4. 2.

LIPOXIN SYNTHESIS AND ACTIONS

The lipoxins are formed through the action of 15-lipoxygenase followed by the action of 5-lipoxygenase on arachidonic acid. A series of reductions of the resultant hydroperoxy groups lead to the formation of trihydroxy derivatives of arachidonic acid known as the lipoxins (see Fig. 35.12). Lipoxins induce chemotaxis and stimulate superoxide anion production in leukocytes.

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CHAPTER 35 ■ METABOLISM OF THE EICOSANOIDS

C. Cytochrome P450 Pathway: Synthesis and Actions of Epoxides, Hydroxyeicosatetraenoics, and Diol Forms Hydroxyeicosatetraenoics A third mechanism for the oxygenation of arachidonic acid involves the cytochrome P450 pathway. The activity of the monooxygenases in this microsomal system yields epoxides, certain forms of HETEs (e.g., ␻-hydroxy derivatives), and diol forms (diHETEs) (Fig. 35.13). The biologic activities of these compounds include actions in ocular, vascular, endocrine, and renal systems. Some of these actions are attributed to inhibition of the Na⫹,K⫹-ATPase. The physiologic role of these compounds remains to be fully characterized.

D. Isoprostane Synthesis Isoprostanes are derived from arachidonic acid by lipid peroxidation, initiated by free radicals. There is no enzymatic mechanism for their production. Arachidonic acid, although still a component of a phospholipid, undergoes free radical damage, and then phospholipase A2 removes the altered eicosanoid from the phospholipid and releases it into circulation (Fig. 35.14). The level of isoprostanes in the urine can be used as a measure of the oxidative stress of a patient and is a useful biologic marker for patients suffering oxidative stress caused by a variety of disorders. Recall that oxidative stress results from reactive oxygen or nitrogen species that are not fully neutralized by antioxidants (see Chapter 24). The increase in reactive oxygen and nitrogen species results in protein, lipid, and nucleic acid modifications. Many neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, and Lou Gehrig disease, have increased oxidative stress in common. Surprisingly, these altered arachidonic acid molecules also have biologic activity when measured on cultured cells; it is not known whether intracellular levels reach high enough concentrations to elicit these biologic effects. The best studied isoprostane is similar to PGF2␣, and this molecule has similar effects on cultured cells as does PGF2␣ (see Table 35.1).

E. Endocannabinoid Synthesis Endocannabinoids are endogenous ligands for the cannabinoid receptors (CB1 and CB2), with effects primarily in the nervous system. Anandamide was the first such ligand to be isolated and identified. Anandamide is synthesized in neurons from phosphatidylethanolamine as outlined in Figure 35.15. The biosynthetic pathway is unique in transferring an arachidonic acid group from the 2-position to the free amino group on ethanolamine, and then using a unique phospholipase D to cleave the modified ethanolamine from the phospholipid. The synthesis of anandamide is regulated, in part, by agonists that cause calcium influx into nerve cells. Once anandamide is released, it acts as a retrograde messenger, binding to receptors on the presynaptic membrane that alter ion fluxes such that neurotransmitter release from the presynaptic neuron can be increased and an analgesic effect obtained. Anandamide is degraded by the enzyme fatty acid amide hydrolase, which splits anandamide to arachidonic acid and ethanolamine. The hydrolase enzyme is a current target of drug research because inhibiting the action of this enzyme will prolong the analgesic effects induced by anandamide.

673

Table 35.4 Some Functions of Leukotrienes LTB4 Increases Vascular permeability T-cell proliferation Leukocyte aggregation INF-␥ IL-1 IL-2 LTC4 and LTD4 Increase Bronchoconstriction Vascular permeability INF-␥ INF, interferon; IL, interleukin.

O COOH

Epoxide (5,6-EET) HO

OH COOH

Dihydroxide (5,6-diHETE)

FIG. 35.13. Examples of compounds produced from arachidonic acid by the cytochrome P450 pathway.

III. MECHANISM OF ACTION OF THE EICOSANOIDS The eicosanoids have a wide variety of physiologic effects, which are generally initiated through interaction of the eicosanoid with a specific receptor on the plasma membrane of a target cell (Table 35.5). This eicosanoid-receptor binding either activates the adenylate cyclase-cAMP-protein kinase A system (PGE, PGD, and

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SECTION VI ■ LIPID METABOLISM

O

O O

CH2

R1

C O

Membrane

O

CH2

R1

C O

Membrane

HC

O

C R2 O–

HC

O

C O

R2

HC H

O

P

HC H

O

P

OCH2CH2NH3

+

OX

O R2 = arachidonic acid

R2 = arachidonic acid TAE

O

Free radicals

C

O–

O– O CH2

O CH2 HC HC H

C O

O

O O

HC

O

H O

HC H

O

P

R1

C O

R* 2

P

OX

C

O

R1

O OCH2CH2

H N

C

O– N-acyl-phosphatidyl ethanolamine selective phospholipase D

O– Phospholipase A2

O O CH2 HC

O

C

HC

OH

HC H

O

O

OH O

P

P

O–

O–

OX

O C H N

+

O–

Lysophospholipid + Isoprostane OH

R1

C

Lysophosphatidic acid

R1

O HC H

O

CH2

Anandamide

O

OH C

OH

O–

Fatty acid amide hydrolase

O OH

FIG. 35.14. Generation of an isoprostane. Free radical damage to a phospholipid on the arachidonic acid residue at position 2 generates an isoprostane, which is then removed from the damaged phospholipid by phospholipase A2. The example of an isoprostane shown in this figure is just one of many that can be produced.

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C

Arachidonic acid

O–

+

H N H

CH2CH2OH

Ethanolamine

FIG. 35.15. Anandamide synthesis and degradation. TAE, transacylase.

PGI series) or causes an increase in the level of calcium in the cytosol of target cells (PGF2␣, TXA2, the endoperoxides, and the leukotrienes). In some systems, the eicosanoids appear to modulate the degree of activation of adenylate cyclase in response to other stimuli. In these instances, the eicosanoid may bind to a regulatory subunit of the guanosine triphosphate (GTP)-binding proteins (G proteins) within the plasma membrane of the target cell (see Chapter 11). If the eicosanoid binds to the stimulatory subunit, the effect of the stimulus is amplified. Conversely, if the eicosanoid binds to the inhibitory subunit, the cellular response to the stimulus is reduced. Through these influences on the activation of adenylate cyclase, eicosanoids contribute to the regulation of cell function.

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CHAPTER 35 ■ METABOLISM OF THE EICOSANOIDS

Table 35.5

Prostaglandin and Thromboxane Receptors

Receptor

Type of Ligand

G protein Coupled

DP CRTH2 EP1 EP2 EP3 EP4 FP IP TP

PGD series PGD series PGE series PGE series PGE series PGE series PGF series PGI series Thromboxane A

Yes Yes Yes Yes Yes Yes Yes Yes Yes

cAMP Response

Calcium Response

Increase Decrease None Increase Decrease Increase None Increase or decreasea Increase or decreaseb

Increase Increase Increase None None None Increase None Increase

a

Depends on the modification of the C-terminal of the receptor. Depends on cell type responding to the ligand.

b

Some of the biologic effects of certain eicosanoids occur as a result of a paracrine or autocrine action. One paracrine action is the contraction of vascular smooth muscle cells caused by TXA2 released from circulating platelets (vasoconstriction). An autocrine action of eicosanoids is exemplified by platelet aggregation induced by TXA2 produced by the platelets themselves. The eicosanoids influence the cellular function of almost every tissue of the body. Certain organ systems are affected to a greater degree than others. CLINICAL COMMENTS Ann Jeina, Cora Nari, and Ivan Applebod. In the presence of aspirin, cyclo-oxygenase is irreversibly inactivated by acetylation. New cyclo-oxygenase molecules are not produced in platelets because these cells have no nuclei and, therefore, cannot synthesize new mRNA. Thus, the inhibition of cyclo-oxygenase by aspirin persists for the life span of the platelet (7 to 10 days). When aspirin is taken daily at doses between 81 and 325 mg, new platelets are affected as they are generated. Higher doses do not improve efficacy but do increase side effects such as gastrointestinal bleeding and being bruised easily. Patients with established or suspected atherosclerotic coronary disease such as Ann Jeina, Cora Nari, and Ivan Applebod, benefit from the action of low-dose aspirin (approximately 81 mg/day), which produces a mild defect in hemostasis. This action of aspirin helps to prevent thrombus formation in the area of an atherosclerotic plaque at critical sites in the vascular tree.

675

Although our knowledge of the spectrum of biologic actions of the endogenous eicosanoids is incomplete, several actions are well enough established to allow their application in a variety of clinical situations or diseases. For example, drugs that are analogs of PGE1 and PGE2 suppress gastric ulceration in part by inhibiting secretion of hydrochloric acid in the mucosal cells of the stomach. Analogs of PGE1 are used in the treatment of sexual impotence. Men with certain forms of sexual impotence can selfinject this agent into the corpus cavernosum of the penis to induce immediate but temporary penile tumescence. The erection lasts for 1 to 3 hours. The stimulatory action of PGE2 and PGF2␣ on uterine muscle contraction has led to the use of these prostaglandins to induce labor and to control postpartum bleeding. PGE1 is also used as palliative therapy in neonates with congenital heart defects to maintain patency of the ductus arteriosus until surgery can be performed. Analogs of PGI2 have been shown to be effective in the treatment of primary pulmonary hypertension.

Emma Wheezer. Corticosteroids reduce inflammation, in part, through their inhibitory effect on phospholipase A2. In addition, suppression of COX-2 induction is now thought to be a primary anti-inflammatory mechanism of action for glucocorticoids. Despite the unquestionable value of glucocorticoid therapy in a variety of diseases associated with acute inflammation of tissues, the suppression of the inflammatory response with pharmacologic doses of corticosteroids is potentially hazardous. The sudden appearance of temporary glucose intolerance when Emma Wheezer was treated with large doses of prednisone—a gluconeogenic steroid (glucocorticoid)—is just one of the many potential adverse effects of this class of drugs when they are given systemically in pharmacologic doses more than an extended period. The inhaled steroids, conversely, have far fewer systemic side effects because their absorption across the bronchial mucosa into the circulation is very limited. This property allows them to be used more than prolonged periods in the treatment of asthma. The fact that inhalation allows direct delivery of the agent to the primary site of inflammation adds to their effectiveness in the treatment of these patients.

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SECTION VI ■ LIPID METABOLISM

Although they are uncomfortable, the pain, swelling, and fever that are part of the inflammatory response serve as an important warning sign that the host is threatened and that some specific counteractions must be taken against the offending agent or process. Although the use of anti-inflammatory drugs may bring welcome to symptomatic relief, their use may, in part, diminish the effectiveness of the host’s response to the inciting agent.

BIOCHEMICAL COMMENTS The Inflammatory Process. Inflammation is the response of the body to infection or injury, directed at destroying the infectious agents and repairing the damaged areas. It involves an increase of the blood supply to the affected region by means of vasodilation. The capillaries become more permeable so that fluid, large molecules, and white blood cells can cross, leaving the blood and entering the tissue. White blood cells (particularly neutrophils and monocytes) move by chemotaxis to the injured site. Redness (rubor), heat (calor), swelling (tumor), and pain (dolor) are associated with the inflammatory process. Redness and heat are caused by the increased blood flow. Swelling is caused by the increased movement of fluid and white blood cells into the area of inflammation. Pain is caused by the release of chemical compounds and the compression of nerves in the vicinity of the inflammation. The chemical mediators of inflammation usually are produced by activation of complement (a family of blood proteins that are cleaved to form active fragments) or of the blood clotting cascade (see Chapter 45). These processes cause the release of histamine from mast cells and the production of kinins by cleavage of kininogens. Among their other effects, both histamine and kinins increase vascular permeability. They stimulate the synthesis of eicosanoids that act on the motility and metabolism of white blood cells and cause the aggregation of platelets to arrest bleeding. Some of the prostaglandins act on thermoregulatory centers of the brain, producing fever. Cytokines are also released, which stimulate the proliferation of cells involved in the immune response.

Key Concepts • • • • • •

The eicosanoids (prostaglandins, thromboxanes, and leukotrienes) are potent regulators of cellular function and are derived from polyunsaturated fatty acids containing 20 carbon atoms. Eicosanoids are important in the inflammatory response, smooth muscle contraction, blood pressure regulation, and bronchoconstriction and bronchodilation. The prostaglandins and thromboxanes require cyclo-oxygenase activity to be synthesized, whereas the leukotrienes require lipoxygenase activity. Eicosanoids act by binding to specific membrane receptors, which, depending on the eicosanoid, alter either protein kinase A activity or calcium levels within the target cells. Cyclo-oxygenase is the target of nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, which covalently acetylates and inactivates the enzyme in platelets. Diseases discussed in this chapter are summarized in Table 35.6.

Table 35.6

Diseases Discussed in Chapter 35

Disease or Disorder

Environmental or Genetic

Cardiac (protection against future myocardial infarctions)

Environmental

Asthma

Environmental

Comments NSAIDs such as aspirin are used to block prostaglandin production via inhibition of cyclo-oxygenase. Low-dose aspirin provides potential protective effects for those with cardiovascular disease. The use of inhalants containing corticosteroids can control and reduce inflammation by inhibiting the recruitment of leukocytes and monocytes into affected areas. They also lead to a decrease in the synthesis of prostaglandins and leukotrienes.

NSAIDs, nonsteroidal anti-inflammatory drugs.

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677

REVIEW QUESTIONS—CHAPTER 35 1.

2.

3.

In humans, prostaglandins are derived primarily from which of the following? A. Glucose B. Acetyl-CoA C. Arachidonic acid D. Oleic acid E. Leukotrienes Aspirin will inhibit which of the following reaction pathways? A. Arachidonic acid → thromboxanes B. Arachidonic acid → leukotrienes C. Arachidonic acid → phospholipids D. Linoleic acid → arachidonic acid E. Acetyl-CoA → linoleic acid Which of the following drugs lead to the covalent modification and inactivation of both the COX-1 and COX-2 enzymes? A. Aspirin B. Tylenol C. Celebrex

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D. Vioxx E. Advil 4.

Thromboxane A2, which is found in high levels in platelets, aids in wound repair through induction of which of the following activities? A. Inhibits COX-2 gene expression B. Inhibits COX-1 gene expression C. Vasoconstriction D. Vasodilation E. Bronchodilation

5.

Certain prostaglandins, when binding to their receptor, induce an increase in intracellular calcium levels. The signal that leads to the elevation of intracellular calcium is initiated by which of the following enzymes? A. Protein kinase A B. Phospholipase C C. Phospholipase A2 D. Protein kinase C E. Cyclo-oxygenase

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36

Integration of Carbohydrate and Lipid Metabolism This chapter summarizes and integrates the major pathways for the use of carbohydrates and fats as fuels. We concentrate on reviewing the regulatory mechanisms that determine the flux of metabolites in the fed and fasting states, integrating the pathways that were described separately under carbohydrate and lipid metabolism. The next section of this book covers the mechanisms by which the pathways of nitrogen metabolism are coordinated with fat and carbohydrate metabolism. For the species to survive, it is necessary for us to store excess food when we eat and to use these stores when we are fasting. Regulatory mechanisms direct compounds through the pathways of metabolism involved in the storage and use of fuels. These mechanisms are controlled by hormones, by the concentration of available fuels, and by the energy needs of the body. Changes in hormone levels, in the concentration of fuels, and in energy requirements affect the activity of key enzymes in the major pathways of metabolism. Intracellular enzymes are generally regulated by activation and inhibition, by phosphorylation and dephosphorylation (or other covalent modifications), by induction and repression of synthesis, and by degradation. Activation and inhibition of enzymes cause immediate changes in metabolism. Phosphorylation and dephosphorylation of enzymes affect metabolism slightly less rapidly. Induction and repression of enzyme synthesis are much slower processes, usually affecting metabolic flux over a period of hours. Degradation of enzymes decreases the amount available to catalyze reactions. The pathways of metabolism have multiple control points and multiple regulators at each control point. The function of these complex mechanisms is to produce a graded response to a stimulus and to provide sensitivity to multiple stimuli so that an exact balance is maintained between flux through a given step (or series of steps) and the need or use for the product. Pyruvate dehydrogenase is an example of an enzyme that has multiple regulatory mechanisms. Regardless of insulin levels, the enzyme cannot become fully activated in the presence of products and absence of substrates. The major hormones that regulate the pathways of fuel metabolism are insulin and glucagon. In liver, all effects of glucagon are reversed by insulin, and some of the pathways that insulin activates are inhibited by glucagon. Thus, the pathways of carbohydrate and lipid metabolism are generally regulated by changes in the insulin/glucagon ratio. Although glycogen is a critical storage form of fuel because blood glucose levels must be carefully maintained, adipose triacylglycerols are quantitatively the major fuel store in humans. After a meal, both dietary glucose and fat are stored in adipose tissue as triacylglycerol. This fuel is released during fasting, when it provides the main source of energy for the tissues of the body. The length of time we can survive without food depends mainly on the size of our bodies’ fat stores.

678

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679

THE WAITING ROOM Within 2 months of her surgery to remove a benign insulin-secreting ␤-cell tumor of the pancreas, Bea Selmass (see Chapter 26) was again jogging lightly. She had lost the 8 lb that she had gained in the 6 weeks before her surgery. Because her hypoglycemic episodes were no longer occurring, she had no need to eat frequent carbohydrate snacks to prevent the adrenergic and neuroglycopenic symptoms that she had experienced when her tumor was secreting excessive amounts of insulin. A few months after her last hospitalization, Di Abietes was once again brought to the hospital emergency room in diabetic ketoacidosis (DKA). Blood samples for glucose and electrolytes were drawn repeatedly during the first 24 hours. The hospital laboratory reported that the serum in each of these specimens appeared opalescent rather than having its normal clear or transparent appearance. This opalescence results from light scattering caused by elevated levels of triacylglycerol-rich lipoproteins in the blood. When Ann Sulin initially presented with type 2 diabetes mellitus at age 39, she was approximately 30 lb higher than her ideal weight. Her high serum glucose levels were accompanied by abnormalities in her 14-hour fasting lipid profile. Her serum total cholesterol, low-density lipoprotein (LDL) cholesterol, and triacylglycerol levels were elevated and her serum high-density lipoprotein (HDL) cholesterol level was lower than the normal range. Glucose

I.

REGULATION OF CARBOHYDRATE AND LIPID METABOLISM IN THE FED STATE A. Mechanisms That Affect Glycogen and Triacylglycerol Synthesis in Liver After a meal, the liver synthesizes glycogen and triacylglycerol. The level of glycogen stored in the liver can increase from approximately 80 g after an overnight fast to a limit of approximately 200 to 300 g. Although the liver synthesizes triacylglycerol, it does not store this fuel but rather packages it in very low-density lipoprotein (VLDL) and secretes it in the blood. The fatty acids of the VLDL triacylglycerols secreted from the liver are stored as adipose triacylglycerols. Adipose tissue has an almost infinite capacity to store fat, limited mainly by the ability of the heart to pump blood through the capillaries of the expanding adipose mass. Although we store fat throughout our bodies, it tends to accumulate in places where it does not interfere too much with our mobility: in the abdomen, hips, thighs, and buttocks. Both the synthesis of liver glycogen and the conversion by the liver of dietary glucose to triacylglycerol (lipogenesis) are regulated by mechanisms involving key enzymes in these pathways. 1.

GLUCOKINASE

After a meal, glucose can be converted to glycogen or to triacylglycerol in the liver. For both processes, glucose is first converted to glucose 6-phosphate by glucokinase, a liver enzyme that has a high Km for glucose (Fig. 36.1). Because of the enzyme’s low affinity for glucose, this enzyme is most active in the fed state, when the concentration of glucose is particularly high because the hepatic portal vein carries digestive products directly from the intestine to the liver. Synthesis of glucokinase is also induced by insulin (which is elevated after a meal) and repressed by glucagon (which

Lieberman_CH36.indd 679

ATP

glucokinase

high Km

ADP Glucose-6-P

Fructose-6-P phosphofructokinase-1

AMP + , F-2,6-BP ATP – , Citrate –

+

,

Fructose-1,6-BP

Phosphoenolpyruvate

pyruvate kinase

–

phosphorylation (cAMP-dependent)

–

Alanine

+

F-1,6-P

Pyruvate

FIG. 36.1. Regulation of glucokinase, PFK-1, and pyruvate kinase in the liver. Fructose-1, 6-BP, fructose 1,6-bisphosphatase; fructose-2, 6-BP, fructose 2,6-bisphosphate.

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SECTION VI ■ LIPID METABOLISM

glycogen synthase– P (inactive) protein kinase A +

is elevated during fasting). In keeping with the liver’s function in maintaining blood glucose levels, this system is set up such that the liver can metabolize glucose only when sugar levels are high and not when sugar levels are low. protein phosphatase

ADP

+

Glucagon

ATP Pi

glycogen synthase (active)

Glycogen

Insulin

UTP

Glucose-6-P Glucose

FIG. 36.2. Regulation of glycogen synthase. This enzyme is phosphorylated by a series of kinases, which are initiated by the cAMPdependent protein kinase, under fasting conditions. It is dephosphorylated and active after a meal, and glycogen is stored. P , phosphate; 䊝, activated by.

P Pyruvate dehydrogenase inactive ADP

kinase

Phosphatase

+

Ca2+

Pi

ATP Pyruvate dehydrogenase active +

Pyruvate

–

CoASH NAD+

Acetyl CoA CO2

+

–

NADH

FIG. 36.3. Regulation of pyruvate dehydrogenase (PDH). A kinase associated with the PDH complex phosphorylates and inactivates the enzyme. 䊝, activated by; 䊞, inhibited by.

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PHOSPHOFRUCTOKINASE-1 AND PYRUVATE KINASE

For lipogenesis, glucose 6-phosphate is converted through glycolysis to pyruvate. Key enzymes that regulate this pathway in the liver are phosphofructokinase-1 (PFK-1) and pyruvate kinase. PFK-1 is allosterically activated in the fed state by fructose 2,6-bisphosphate and adenosine monophosphate (AMP) (see Fig. 36.1). Phosphofructokinase-2 (PFK-2), the enzyme that produces the activator fructose 2,6-bisphosphate, is dephosphorylated and the kinase activity is active after a meal (see Chapter 22). Pyruvate kinase is also activated by dephosphorylation, which is stimulated by the increase of the insulin/glucagon ratio in the fed state (see Fig. 36.1).

Glucose-1-P

–

GLYCOGEN SYNTHASE

In the conversion of glucose 6-phosphate to glycogen, the key regulatory enzyme is glycogen synthase. This enzyme is activated by the dephosphorylation that occurs when insulin is elevated and glucagon is decreased (Fig. 36.2) and by the increased level of glucose. 3.

UDP-Glucose PPi

ADP

2.

4.

PYRUVATE DEHYDROGENASE AND PYRUVATE CARBOXYLASE

The conversion of pyruvate to fatty acids requires a source of acetyl coenzyme A (acetyl-CoA) in the cytosol. Pyruvate can only be converted to acetyl-CoA in mitochondria, so it enters mitochondria and forms acetyl-CoA through the pyruvate dehydrogenase (PDH) reaction. This enzyme is dephosphorylated and most active when its supply of substrates and adenosine diphosphate (ADP) is high—its products are used and insulin is present (Fig. 36.3). Pyruvate is also converted to oxaloacetate. The enzyme that catalyzes this reaction, pyruvate carboxylase, is activated by acetyl-CoA. Because acetyl-CoA cannot cross the mitochondrial membrane directly to form fatty acids in the cytosol, it condenses with oxaloacetate, thus producing citrate. The citrate that is not required for tricarboxylic acid (TCA) cycle activity crosses the membrane and enters the cytosol. As fatty acids are produced under conditions of high energy, the high NADH/ NAD⫹ ratio in the mitochondria inhibits isocitrate dehydrogenase, which leads to citrate accumulation within the mitochondrial matrix. As the citrate accumulates, it is transported out into the cytosol to donate carbons for fatty acid synthesis. 5.

CITRATE LYASE, MALIC ENZYME, AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE

In the cytosol, citrate is cleaved by citrate lyase, an inducible enzyme, to form oxaloacetate and acetyl-CoA (Fig. 36.4). The acetyl-CoA is used for fatty acid biosynthesis and for cholesterol synthesis—pathways that are activated by insulin. Oxaloacetate is recycled to pyruvate via cytosolic malate dehydrogenase and malic enzyme, which is inducible. Malic enzyme generates NADPH for the reactions of the fatty acid synthase complex. NADPH is also produced by the two enzymes of the pentose phosphate pathway (see Chapter 29): glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase is also induced by insulin. 6.

ACETYL-CoA CARBOXYLASE

Acetyl-CoA is converted to malonyl-CoA, which provides the two-carbon units for elongation of the growing fatty acyl chain on the fatty acid synthase complex. Acetyl-CoA carboxylase, the enzyme that catalyzes the conversion of acetyl-CoA to malonyl-CoA, is controlled by three of the major mechanisms that regulate enzyme activity (Fig. 36.5). It is activated by citrate, which causes the enzyme to polymerize,

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681

Glucose NADP+

Glucose 6-phosphate

Palmitate

Glucose 6phosphate dehydrogenase FA synthase

CO2

NADPH NADP+ Malic enzyme

Pyruvate

Malate

Malonyl CoA NAD+

Pyruvate

NADH OAA

Acetyl CoA

Citrate lyase

Citrate

OAA

Acetyl CoA

ADP + Pi

Citrate ATP

FIG. 36.4. Regulation of citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and fatty acid synthase. Citrate lyase, which provides acetyl-CoA for fatty acid biosynthesis, the enzymes that provide NADPH (malic enzyme, glucose-6-phosphate dehydrogenase), as well as fatty acid synthase are inducible ↑. FA, fatty acid.

and inhibited by long-chain fatty acyl-CoA. A phosphatase stimulated by insulin activates the enzyme by dephosphorylation. The third means by which this enzyme is regulated is induction: the quantity of the enzyme increases in the fed state. Malonyl-CoA, the product of the acetyl-CoA carboxylase reaction, provides the carbons for the synthesis of palmitate on the fatty acid synthase complex. MalonylCoA also inhibits carnitine palmitoyl transferase I (CPTI, also known as carnitine acyltransferase I), the enzyme that prepares long-chain fatty acyl-CoA for transport Glucose

Citrate Insulin +

Phosphatase

Acetyl CoA

Pi +

Acetyl CoA carboxylase–P (inactive)

Acetyl CoA carboxylase –

ADP

ATP

AMP-activated protein kinase

Malonyl CoA

+

Low energy levels

Palmitate

Palmitoyl CoA

FIG. 36.5. Regulation of acetyl-CoA carboxylase (ACC). ACC is regulated by activation and inhibition, by phosphorylation (mediated by the AMP-activated protein kinase) and dephosphorylation (via an insulin-stimulated phosphatase), and by induction and repression. It is active in the fed state.

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FACoA

Palmitate

into mitochondria (Fig. 36.6). In the fed state, when acetyl-CoA carboxylase is active and malonyl-CoA levels are elevated, newly synthesized fatty acids are converted to triacylglycerols for storage, rather than being transported into mitochondria for oxidation and formation of ketone bodies. 7.

FA synthase

FACoA Carnitine CPTI

–

Malonyl CoA

FA-carnitine CPTII FACoA

Acetyl CoA

CoASH

␤-Oxidation

FIG. 36.6. Inhibition of transport of fatty acids (FAs) into mitochondria by malonyl-CoA. In the fed state, malonyl-CoA (the substrate for fatty acid synthesis produced by acetyl-CoA carboxylase) is elevated. It inhibits carnitine palmitoyl transferase I (CPTI), preventing the transport of long-chain fatty acids into mitochondria. Therefore, substrate is not available for ␤-oxidation and ketone body synthesis. The measurement of triglycerides in blood samples is performed using a coupled assay. The sample is incubated with a lipase that converts the triglyceride to glycerol and three fatty acids. The glycerol is then converted to glycerol 3-phosphate by glycerol kinase and ATP, and the glycerol 3-phosphate is then oxidized by a bacterial glycerol-3-phosphate dehydrogenase to produce DHAP and hydrogen peroxide. The hydrogen peroxide, in the presence of peroxidase, will oxidize a colorless substrate, which turns color when oxidized. Measurement of the color change is directly proportional to the amount of glycerol generated in the sample. Di Abietes has type 1 diabetes mellitus (insulin dependent), a disease associated with a severe deficiency or absence of insulin production by the ␤-cells of the pancreas. One of the effects of insulin is to stimulate production of LPL. Because of low insulin levels, Di Abietes tends to have low levels of this enzyme. Hydrolysis of the triacylglycerols in chylomicrons and in VLDL is decreased and hypertriglyceridemia results.

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FATTY ACID SYNTHASE COMPLEX

In a well-fed individual, the quantity of the fatty acid synthase complex is increased (see Fig. 36.4). The genes that produce this enzyme complex are induced by increases in the insulin/glucagon ratio. The amount of the complex increases slowly after a few days of a high-carbohydrate diet. Glucose-6-phosphate dehydrogenase which generates NADPH in the pentose phosphate pathway and malic enzyme which produces NADPH are also induced by the increase of insulin. The palmitate produced by the synthase complex is converted to palmitoyl-CoA and elongated and desaturated to form other fatty acyl-CoA molecules, which are converted to triacylglycerols. These triacylglycerols are packaged and secreted into the blood as VLDL.

B. Mechanisms That Affect the Fate of Chylomicrons and VLDL The lipoprotein triacylglycerols in chylomicrons and VLDL are hydrolyzed to fatty acids and glycerol by lipoprotein lipase (LPL), an enzyme attached to endothelial cells of capillaries in muscle and adipose tissue. The enzyme found in muscle, particularly heart muscle, has a low Km for these blood lipoproteins. Therefore, it acts even when these lipoproteins are present at very low concentrations in the blood. The fatty acids enter muscle cells and are oxidized for energy. The enzyme found in adipose tissue has a higher Km and is most active after a meal when blood lipoprotein levels are elevated.

C. Mechanisms That Affect Triacylglycerol Storage in Adipose Tissue Insulin stimulates adipose cells to synthesize and secrete LPL, which hydrolyzes the chylomicron and VLDL triacylglycerols. Apoprotein CII (apoCII), donated to chylomicrons and VLDL by HDL, activates LPL (Fig. 36.7). Fatty acids released from chylomicrons and VLDL by LPL are stored as triacylglycerols in adipose cells. The glycerol released by LPL is not used by adipose cells because they lack glycerol kinase. Glycerol can be used by liver cells, however, because these cells do contain glycerol kinase. In the fed state, liver cells convert

Fed state TG Glucose Glucose Blood

+

DHAP

Insulin Chylomicrons

+

Remnants VLDL

TG +

IDL LDL

CII

L P L

Glycerol 3phosphate LPL FACoA

FA Liver

Glycerol

FA Adipose cell

FIG. 36.7. Regulation of the storage of triacylglycerols (TGs) in adipose tissue. Insulin stimulates the secretion of LPL from adipose cells and the transport of glucose into these cells. ApoCII activates LPL. FA, fatty acids.

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glycerol to the glycerol moiety of the triacylglycerols of VLDL, which is secreted from the liver to distribute the newly synthesized triglycerides to the tissues. Insulin causes the number of glucose transporters in adipose cell membranes to increase. Glucose enters these cells and is oxidized, producing energy and providing the glycerol 3-phosphate moiety for triacylglycerol synthesis (via the dihydroxyacetone phosphate intermediate of glycolysis).

II. REGULATION OF CARBOHYDRATE AND LIPID METABOLISM DURING FASTING A. Mechanisms in Liver That Serve to Maintain Blood Glucose Levels During fasting, the insulin/glucagon ratio decreases. Liver glycogen is degraded to produce blood glucose because enzymes of glycogen degradation are activated by cAMP-directed phosphorylation (Fig. 36.8). Glucagon stimulates adenylate

Glucagon +

683

Why does Ann Sulin have a hypertriglyceridemia?

The 20% to 30% of patients with an insulinoma gain weight as part of their syndrome. Bea Selmass gained 8 lb 6 weeks before her surgery. Although she was primed by her high insulin levels both to store and to use fuel more efficiently, she would not have gained weight if she had not consumed more calories than what was required for her daily energy expenditure during her illness (see Chapter 1). Bea consumed extra carbohydrate calories—mostly as hard candies and table sugar—to avoid the symptoms of hypoglycemia.

G-protein +

Adenylate cyclase

Cell membrane

1 ATP

cAMP

Protein kinase A (inactive)

2

Regulatory subunit-cAMP ADP

Phosphorylase kinase (inactive)

4

ATP Active protein kinase A

3

Glycogen

5 Phosphorylase b (inactive)

ATP

ATP Glycogen synthase (active)

ADP Phosphorylase kinase– P (active)

Glycogen synthase– P (inactive)

Pi ADP

Phosphorylase a (active) P

6 Glucose 1phosphate

Glucose 6-phosphate Liver Blood glucose

FIG. 36.8. Regulation of the enzymes of glycogen degradation in the liver. (1) Glucagon (or epinephrine) binds to its cell membrane receptor, initially activating a G protein, which activates adenylate cyclase. (2) As cAMP levels rise, inhibitory subunits are removed from protein kinase A, which now phosphorylates phosphorylase kinase. (3) (4) The cAMP-dependent protein kinase also phosphorylates glycogen synthase, inactivating the enzyme. (5) Phosphorylated phosphorylase kinase phosphorylates glycogen phosphorylase. (6) Phosphorylated glycogen phosphorylase catalyzes the phosphorolysis of glycogen, producing glucose 1-phosphate. These events occur during fasting and produce glucose to maintain a relatively constant level of blood glucose.

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SECTION VI ■ LIPID METABOLISM

Ann Sulin has type 2 diabetes mellitus. She produces insulin, but her adipose tissue is partially resistant to its actions. Therefore, her adipose tissue does not produce as much LPL as a normal person, which is one of the reasons why VLDL and chylomicrons remain elevated in her blood.

Glycolysis

Gluconeogenesis Glucose

glucokinase (high Km)

glucose 6-phosphatase

Glucose 6-phosphate

Fructose 6-phosphate F-2,6-BP

phosphofructokinase-1 +

fructose 1,6-bisphosphatase –

Fructose 1,6-bisphosphate

Dihydroxyacetone phosphate

Glyceraldehyde 3-phosphate

Phosphoenolpyruvate +

cAMP

pyruvate kinase– P (inactive)

pyruvate kinase (active)

Pi

phosphoenolpyruvate carboxykinase

Oxaloacetate

Pyruvate

pyruvate carboxylase + Acetyl CoA

FIG. 36.9. Regulation of gluconeogenesis (red arrows) and glycolysis (black arrows) during fasting. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase are induced ↑. Fructose 1,6-bisphosphatase is also active because, during fasting, the level of its inhibitor, fructose 2,6-bisphosphate, is low. The corresponding enzymes of glycolysis are not very active during fasting. The rate of glucokinase is low because it has a high Km for glucose and the glucose concentration is low. PFK-1 is not very active because the concentration of its activator fructose 2,6-bisphosphate is low. Pyruvate kinase is inactivated by cAMP-mediated phosphorylation. F-2,6-BP, fructose 2,6-bisphosphate.

Di Abietes suffers from hyperglycemia because her insulin levels tend to be low and her glucagon levels tend to be high. Her muscle and adipose cells do not take up glucose at a normal rate, and she produces glucose by glycogenolysis and gluconeogenesis. As a result, her blood glucose levels are elevated. Ann Sulin is in a similar metabolic state. However, in her case, she produces insulin but her tissues are resistant to its actions.

Lieberman_CH36.indd 684

cyclase to produce cAMP, which activates protein kinase A. Protein kinase A phosphorylates phosphorylase kinase, which then phosphorylates and activates glycogen phosphorylase. Protein kinase A also phosphorylates but, in this case, inactivates glycogen synthase. Gluconeogenesis is stimulated because the synthesis of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase is induced and because there is an increased availability of precursors. Fructose 1,6-bisphosphatase is also activated because the levels of its inhibitor, fructose 2,6-bisphosphate, are low (Fig. 36.9). During fasting, the activities of the corresponding enzymes of glycolysis are decreased. Induction of enzyme synthesis requires activation of transcription factors. One of the factors that is activated is CREB, which stands for cAMP response element– binding protein. CREB is phosphorylated and activated by the cAMP-dependent

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685

Fasted state

TG

Hormonesensitive lipase (inactive)

Blood Protein kinase A

Hormonesensitive lipase– P (active)

+

cAMP +

Low insulin/high glucagon

ATP Other lipases

FA FA FA Glycerol

FA FA FA Glycerol

Adipose cell

FIG. 36.10. Regulation of hormone-sensitive lipase (HSL) in adipose tissue. During fasting, the glucagon/insulin ratio rises causing cAMP levels to be elevated. Protein kinase A is activated and phosphorylates HSL, activating this enzyme. HSL-P initiates the mobilization of adipose triacylglycerol by removing a fatty acid (FA). Other lipases then act, producing fatty acids and glycerol. Insulin stimulates the phosphatase that inactivates HSL in the fed state.

protein kinase, which itself is activated on glucagon or epinephrine stimulation. A second set of transcription factors activated are the C/EPBs (CCAAT enhancer– binding proteins), although the regulation of their activity is still under investigation and is quite complex.

B. Mechanisms That Affect Lipolysis in Adipose Tissue During fasting, as blood insulin levels fall and glucagon levels rise, the level of cAMP rises in adipose cells. Consequently, protein kinase A is activated and causes phosphorylation of hormone-sensitive lipase (HSL). The phosphorylated form of this enzyme is active and cleaves fatty acids from triacylglycerols (Fig. 36.10). Other hormones (e.g., epinephrine, adrenocorticotropic hormone [ACTH], growth hormone) also activate this enzyme (see Chapter 43). Glyceroneogenesis and resynthesis of triglyceride by the adipocyte regulate the rate of release of fatty acids during fasting.

C. Mechanisms That Affect Ketone Body Production by the Liver As fatty acids are released from adipose tissue during fasting, they travel in the blood complexed with albumin. These fatty acids are oxidized by various tissues, particularly muscle. In the liver, fatty acids are transported into mitochondria because acetyl-CoA carboxylase is inactive, malonyl-CoA levels are low, and CPTI is active (see Fig. 36.6). Acetyl-CoA, produced by ␤-oxidation, is converted to ketone bodies. Ketone bodies are used as an energy source by many tissues (Table 36.1) to spare the use of glucose and the necessity of degrading muscle protein to provide the precursors for gluconeogenesis. The high levels of acetyl-CoA in the liver (derived from fat oxidation) inhibit pyruvate dehydrogenase (which prevents pyruvate from being converted to acetyl-CoA) and activate pyruvate carboxylase, which produces oxaloacetate for gluconeogenesis. The oxaloacetate does not condense with acetyl-CoA to form citrate for two reasons. The first is that under these conditions (a high rate of fat oxidation in the liver mitochondria), energy levels in the mitochondrial matrix are high; that is, there are high levels of NADH

Lieberman_CH36.indd 685

Insulin normally inhibits lipolysis by decreasing the lipolytic activity of hormone-sensitive lipase (HSL) in the adipocyte. Individuals such as Di Abietes, who have a deficiency of insulin, have increased lipolysis and a subsequent increase in the concentration of free fatty acids in the blood. The liver, in turn, uses some of these fatty acids to synthesize triacylglycerols, which then are used in the hepatic production of VLDL. VLDL is not stored in the liver but is secreted into the blood, raising its serum concentration. Di also has low levels of LPL because of decreased insulin levels. Her hypertriglyceridemia is the result, therefore, of both overproduction of VLDL by the liver and decreased breakdown of VLDL triacylglycerol for storage in adipose cells. The serum begins to appear cloudy when the triacylglycerol level reaches 200 mg/dL. As the triacylglycerol level increases further, the degree of serum opalescence increases proportionately.

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SECTION VI ■ LIPID METABOLISM

Table 36.1

Fuel Use by Various Tissues during Starvation (Fasting)a

Tissue Nervous system Skeletal muscle Heart muscle Liver Intestinal epithelial cells Kidney

Glucose

Fatty Acids

Ketone Bodies

⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹

⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹

⫺Indicates no substrate utilization; ⫹, a low level of substrate utilization; ⫹⫹, a high level of substrate utilization.

a

and ATP present. The high NADH level inhibits isocitrate dehydrogenase. As a result, citrate accumulates and inhibits citrate synthase from producing more citrate. The second reason that citrate synthesis is depressed is that the high NADH/NAD⫹ ratio also diverts oxaloacetate into malate, so the malate can exit the mitochondria (via the malate/aspartate shuttle) for use in gluconeogenesis.

D. Regulation of the Use of Glucose and Fatty Acids by Muscle Because Di Abietes produces very little insulin, she is prone to developing ketoacidosis. When insulin levels are low, HSL of adipose tissue is very active, resulting in increased lipolysis. The fatty acids that are released travel to the liver, where they are converted to the triacylglycerols of VLDL. They also undergo ␤-oxidation and conversion to ketone bodies. If Di does not take exogenous insulin or if her insulin levels decrease abruptly for some physiologic reason, she may develop ketoacidosis (diabetic ketoacidosis [DKA]). In fact, she has had repeated bouts of DKA. For reasons that are not as well understood, individuals with type 2 diabetes mellitus such as Ann Sulin do not tend to develop ketoacidosis. One possible explanation is that the insulin resistance is tissue specific; the insulin sensitivity of adipocytes may be greater than that of muscle and liver. It has been suggested that the level of insulin required to suppress lipolysis is only 10% that required to enhance glucose use by muscle and adipocyte. Such a tissue-specific sensitivity would lead to less fatty acids being released from adipocytes in type 2 diabetes than in type 1 diabetes, although in both cases, the release of fatty acids would be greater than that of an individual without the disease. If, however, a person with type 2 diabetes has a precipitating event, such as the release of stress hormones (see Chapter 43), then ketoacidosis is more likely to be found as the stress hormones counteract the effects of insulin on the adipocyte.

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During exercise, the fuel that is used initially by muscle cells is muscle glycogen. As exercise continues and the blood supply to the tissue increases, glucose is taken up from the blood and is oxidized. Liver glycogenolysis and gluconeogenesis replenish the blood glucose supply. However, because insulin levels drop, the concentration of the GLUT4 glucose transporters in the membrane is reduced, thereby reducing glucose entry from the circulation into the muscle. Thus, as fatty acids become available because of increased lipolysis of adipose triacylglycerols, the exercising muscle begins to oxidize fatty acids. ␤-Oxidation produces NADH and acetyl-CoA, which slows the flow of carbon from glucose through the reaction catalyzed by pyruvate dehydrogenase (see Fig. 36.3). Thus, the oxidation of fatty acids provides a major portion of the increased demand for ATP generation and spares blood glucose.

III. THE IMPORTANCE OF AMP AND FRUCTOSE 2,6-BISPHOSPHATE The switch between catabolic and anabolic pathways is often regulated by the levels of AMP and fructose 2,6-bisphosphate in cells, particularly the liver. It is logical for AMP to be a critical regulator. Because a cell uses ATP in energyrequiring pathways, the levels of AMP accumulate more rapidly than that of ADP because of the adenylate kinase reaction (2 ADP → ATP and AMP). The rise in AMP levels then signals that more energy is required (usually through allosteric binding sites on enzymes and the activation of the AMP-activated protein kinase), and the cell switches to the activation of catabolic pathways. As AMP levels drop, and ATP levels rise, the anabolic pathways are activated to store the excess energy. The levels of fructose 2,6-bisphosphate are also critical in regulating glycolysis versus gluconeogenesis in the liver. Under conditions of high blood glucose and insulin release, fructose 2,6-bisphosphate levels are high because PFK-2 is in its activated state. The fructose 2,6-bisphosphate activates PFK-1 and inhibits fructose 1,6-bisphosphatase, thereby allowing glycolysis to proceed. When blood glucose levels are low, and glucagon is released, PFK-2 is phosphorylated by the cAMPdependent protein kinase and is inhibited, thereby lowering fructose 2,6-bisphosphate levels and inhibiting glycolysis, whereas favoring gluconeogenesis.

IV. GENERAL SUMMARY All of the material in this chapter was presented previously. However, because this information is so critical for understanding biochemistry in a way that will

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687

allow it to be used in interpreting clinical situations, it has been summarized in this chapter. In addition, the information presented previously under carbohydrate metabolism has been integrated with lipid metabolism. We have—for the most part—left out the role of allosteric modifiers and other regulatory mechanisms that finely coordinate these processes to an exquisite level. Because such details may be important for specific clinical situations, we hope this summary will serve as a framework to which the details can be fitted as students advance in their clinical studies. Figure 36.11 is a comprehensive figure, and Tables 36.2 and 36.3 provide a list of the major regulatory enzymes of carbohydrate and lipid metabolism in

Catecholamines

Glucagon

Insulin

Glucose

ATP GK (hi Km)

Glucose

Pi NADPH

ATP

cAMP

ATP ?

Ca2+

UTP

UDP-Glucose

G-6-P

G-1-P

Ribulose-5-P ATP PFK

+ +

F-6-P F2,6-BP AMP

+

PK +

GDP

Phos a

Lactate

NADH

NAD

NADPH KB

FACoA NADP

Pyruvate Insulin CO2 HMG-CoA PDH–P PDH – NADH Acetyl CoA +

–

+

+

NAD

Asp

Malate

Asp

OAA NADH

–

Malate

–

αKG

Glycerol

Malonyl CoA

Phospholipids Cholesterol

KB

FACoA Palmitate

+

+

Citrate

FADH2 FACoA

VLDL

Lysosome

LDL

Steroid hormones Bile acids

Insulin Cholesterol Bile salts HMG-CoA

NADPH Malonyl CoA

Malate +

AcC

OAA

+

Citrate Isocitrate

VLDL

TG

GTP

NADH

cAMP

IDL

CO2

PEP-CK

OAA

GS– P

+

+

Blood

Insulin

Glycogen

+ Insulin P PhK– P Pi Phos b Pi PhK + Insulin cAMP B Apoprotein PK– P Insulin

Pyruvate

Alanine

GS

UDP

Liver +

+

Glycerol PEP

Pi

Pi

Pi F2,6-BP – AMP cAMP

F1,6-BP

CO2

2Pi

PPi

Insulin AcC–P

cAMP

Acetyl CoA

Citrate

+

FA Adipose TG

Induction Repression

+ –

Activation Inhibition

FIG. 36.11. Regulation of carbohydrate and lipid metabolism in the liver. Solid red arrows indicate the flow of metabolites in the fed state. Solid black arrows indicate the flow during fasting. ACC, acetyl-CoA carboxylase; ␣KG, ␣-ketoglutarate; F1,6-BP, fructose 1,6-phosphate; F2,6-BP, fructose 2,6-bisphosphate; F-6-P, fructose 6-phosphate; G-6-P, glucose 6-phosphate; FA, fatty acid or fatty acyl group; GK, glucokinase; GS, glycogen synthase; OAA, oxaloacetate; circled P, phosphate group; PEP, phosphoenolpyruvate; PFK, phosphofructokinase-1; Phos, glycogen phosphorylase; PhK, phosphorylase kinase; PK, pyruvate kinase; TG, triacylglycerol.

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Table 36.2

Flowchart of Changes in Liver Metabolism

When blood sugar increases: Insulin is released, which leads to the dephosphorylation of: • PFK-2 (kinase activity now active) • Pyruvate kinase (now active) • Glycogen synthase (now active) • Phosphorylase kinase (now inactive) • Glycogen phosphorylase (now inactive) • Pyruvate dehydrogenase (now active) • Acetyl-CoA carboxylase (now active)

When blood sugar decreases: Glucagon is released, which leads to the phosphorylation of: • PFK-2 (phosphatase activity now active) • Pyruvate kinase (now inactive) • Glycogen synthase (now inactive) • Phosphorylase kinase (now active) • Glycogen phosphorylase (now active) • Pyruvate dehydrogenase (now inactive) • Acetyl-CoA carboxylase (now inactive)

Which leads to active • Glycolysis • Fatty acid synthesis • Glycogen synthesis

Which leads to active • Glycogenolysis • Fatty acid oxidation • Gluconeogenesis

Note. PKF-2, phosphofructokinase-2.

the liver, an order of the events that occur and the mechanisms by which they are controlled. This figure and tables should help students to integrate this mass of material. Now that many of the details of the pathways have been presented, it may be worthwhile to reread the first three chapters of this book. A student who understands Table 36.3 Regulation of Liver Enzymes Involved in Glycogen, Blood Glucose, and Triacylglycerol Synthesis and Degradation Enzyme

Liver Enzymes Regulated by Activation/Inhibition Activated by State in which Active

Phosphofructokinase-1 Pyruvate carboxylase Acetyl-CoA carboxylase Carnitine palmitoyl transferase I

Enzyme

Fructose 2,6-bisP, AMP Acetyl-CoA Citrate Loss of inhibitor (malonyl-CoA)

Fed Fed and fasting Fed Fasting

Liver Enzymes Regulated by Phosphorylation/Dephosphorylation Active form State in which Active

Glycogen synthase Phosphorylase kinase Glycogen phosphorylase Phosphofructokinase-2/fructose 2,6-bisphosphatase (acts as a kinase, increasing fructose 2,6-bisP levels) Phosphofructokinase-2/fructose 2,6-bisphosphatase (acts as a phosphatase, decreasing fructose 2,6-bisP levels)

Dephosphorylated Phosphorylated Phosphorylated Dephosphorylated

Fed Fasting Fasting Fed

Phosphorylated

Fasting

Pyruvate kinase Pyruvate dehydrogenase Acetyl-CoA carboxylase

Dephosphorylated Dephosphorylated Dephosphorylated

Fed Fed Fed

Enzyme

Liver Enzymes Regulated by Induction/Repression State in which Induced Process Affected

Glucokinase Citrate lyase Acetyl-CoA carboxylase Fatty acid synthase Malic enzyme Glucose-6-phosphate dehydrogenase Glucose 6-phosphatase Fructose 1,6-bisphosphatase Phosphoenolpyruvate carboxykinase

Fed Fed Fed Fed Fed Fed Fasted Fasted Fasted

Glucose → TG Glucose → TG Glucose → TG Glucose → TG Production of NADPH Production of NADPH Production of blood glucose Production of blood glucose Production of blood glucose

AMP, adenosine monophosphate; fructose 2,6-bisP, fructose 2,6-bisphosphate; TG, triacylglycerol.

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689

biochemistry within the context of fuel metabolism is in a very good position to solve clinical problems that involve metabolic derangements.

CLINICAL COMMENTS Bea Selmass. Bea Selmass’s younger sister was very concerned that Bea’s pancreatic tumor might be genetically determined or potentially malignant, so she accompanied Bea to her second postoperative visit to the endocrinologist. The doctor explained that insulinomas may be familial in up to 20% of cases and that in 10% of patients with insulinomas, additional secretory neoplasms may occur in the anterior pituitary or the parathyroid glands (a genetically determined syndrome known as multiple endocrine neoplasia, type I, or simply MEN I). Bea’s tumor showed no evidence of malignancy, and the histologic slides—although not always definitive—showed a benign-appearing process. The doctor was careful to explain, however, that close observation for recurrent hypoglycemia and for the signs and symptoms suggestive of other facets of MEN I would be necessary for the remainder of Bea’s and her immediate family’s life. Di Abietes. Diabetes mellitus is a well-accepted risk factor for the development of coronary artery disease; the risk is three to four times higher in the population with diabetes than in the population without diabetes. Although chronically elevated serum levels of chylomicrons and very low-density lipoprotein (VLDL) may contribute to this atherogenic predisposition, the premature vascular disease seen in Di Abietes and other patients with type 1 diabetes mellitus, as well as Ann Sulin and other patients with type 2 diabetes mellitus, is also related to other abnormalities in lipid metabolism. Among these are the increase in glycation (nonenzymatic attachment of glucose molecules to proteins) of low-density lipoprotein (LDL) apoproteins as well as glycation of the proteins of the LDL receptor, which occurs when serum glucose levels are chronically elevated. These glycations interfere with the normal interaction or “fit” of the circulating LDL particles with their specific receptors on cell membranes. As a consequence, the rate of uptake of circulating LDL by the normal target cells is diminished. The LDL particles, therefore, remain in the circulation and eventually bind nonspecifically to “scavenger” receptors located on macrophages adjacent to the endothelial surfaces of blood vessels—one of the early steps in the process of atherogenesis.

BIOCHEMICAL COMMENTS AMPK. The AMP-activated protein kinase (AMPK) is a pivotal regulatory molecule in the metabolism of carbohydrates and fats. The hepatic targets of AMPK include the following proteins: • Acetyl-CoA carboxylase (phosphorylation reduces activity, leading to reduced fatty acid synthesis) • eEF2 kinase (this protein is activated when phosphorylated, and it will lead to a reduction in protein synthesis) • Glycerol 3-phosphate acyltransferase (GPAT) (phosphorylation reduces activity, leading to reduced triglyceride synthesis) • HMG-CoA reductase (phosphorylation reduces activity, leading to reduced cholesterol synthesis) • Malonyl-CoA decarboxylase (MCD; active when phosphorylated, reduces malonyl CoA levels, allowing fatty acid oxidation to occur) • Mammalian target of rapamycin (mTOR) (reduced activity when phosphorylated, leading to reduced protein synthesis)

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SECTION VI ■ LIPID METABOLISM

• Tuberous sclerosis complex 2 (TSC2; increased activity when phosphorylated, leading to reduced protein synthesis) • Target of rapamycin complex 2 (TORC2) (the protein is sequestered in the cytoplasm when phosphorylated, leading to decreased expression—at the transcriptional level—of gluconeogenic enzymes) The overall effect of AMPK activation is reduced fatty acid and triglyceride synthesis (via effects on acetyl-CoA carboxylase, GPAT, and MCD), reduced cholesterol synthesis (via inhibition of HMG-CoA reductase), and reduced protein synthesis (via effects on mTOR and TSC2). There is a concommitant increase in fatty acid oxidation to raise ATP levels. Because the processes described previously are all highly energy-dependent, it makes sense to turn them off when energy levels are low, as exemplified by increased AMP levels. mTOR (a drug that is a potent immunosuppressant) is a protein kinase, which, when active, phosphorylates key proteins that regulate and initiate protein synthesis. AMPK phosphorylation of mTOR blocks the activation of mTOR. mTOR can be activated by TSC2 through a complex pathway involving the GTP-binding protein Rheb (ras homolog enriched in brain). The TSC complex (consisting of TSC1 and TSC2) acts as a GTPase activating protein for Rheb. Rheb-GTP activates mTOR, whereas Rheb-GDP does not. Phosphorylation of TSC2 by the AMPK activates the GTPase-activating activity of TSC2, leading to Rheb-GDP formation, and reduced mTOR activity. The reduced mTOR activity leads to a reduction of protein synthesis. mTOR also plays a critical role in transmitting the signal from the insulin receptor to an increase in protein synthesis within the cell. Insulin receptor activation leads to Akt activation (see Fig. 11.14). The protein kinase Akt (protein kinase B) will phosphorylate the TSC1/TSC2 complex and inactivate the GTPase activating component of the complex. Under these conditions, Rheb-GTP will be long lived and mTOR will be active, leading to an enhancement of protein synthesis in the cell. The interaction of TSC1/TSC2, mTOR, and its regulatory kinases is depicted in Figure 36.12. The AMPK can be activated in several ways, all of which depend on increased AMP levels within the cell. As the concentration of AMP increases, AMPK is activated by allosteric means, by phosphorylation by LKB1 (see Chapter 34), or by phosphorylation by a calmodulin kinase kinase. AMPK is inactivated by dephosphorylation by protein phosphatases or by a decrease in AMP levels. Small changes in intracellular AMP levels can have profound effects on AMPK activity because of these multiple regulatory pathways. AMPK is a heterotrimeric complex that consists of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥). The allosteric activation of AMPK occurs via AMP binding to the ␣-subunit; the phosphorylation activation of AMPK occurs via threonine phosphorylation on the ␣-subunit. Different tissues express different isoforms of the ␣-, ␤-, and ␥-subunits, giving rise to a wide variety of isozymes of AMPK in the different tissues. Key Concepts • • • • •

• •

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Three key controlling elements determine whether a fuel is metabolized or stored: hormones, concentration of available fuels, and energy needs of the body. Key intracellular enzymes are generally regulated by allosteric activation and inhibition, by covalent modification, by transcriptional control, and by degradation. Regulation is complex to allow sensitivity and feedback to multiple stimuli so that an exact balance can be maintained between synthesis of a product and need for the product. The insulin/glucagon ratio is primarily responsible for the hormonal regulation of carbohydrate and lipid metabolism. The key enzymes of glycolysis, fatty acid synthesis, fatty acid degradation, glycogen synthesis, glycogenolysis, and gluconeogenesis are all regulated in a coordinated manner, allowing the appropriate pathways to be activated and inhibited without the creation of futile cycles. This chapter summarizes—in one package—all of the major regulatory events discussed in the previous 17 chapters. Diseases discussed in this chapter are summarized in Table 36.4.

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691

Insulin

AMP ratio ATP

+ AMPK (active)

Akt (active)

+ TSC1/ TSC2 GDP

Rheb-GTP (active)

GTP

–

+

Rheb-GDP (inactive)

+ Pi

mTOR Complex

phosphorylate S6K1

4E-BPs

Activation of translation

FIG. 36.12. Central role of mTOR and the TSC1/TSC2 complex in regulating protein synthesis. The TSC1/TSC2 complex acts as a GTPase-activating protein for the G protein Rheb. When active, Rheb-GTP activates the mTOR kinase, leading to phosphorylation events that promote protein synthesis (S6K1 and 4E-BPs are phosphorylated). AMPK phosphorylation of the TSC1/TSC2 complex activates the complex such that the GTPase activity of Rheb is enhanced, leading to Rheb inactivation and loss of mTOR kinase activity. This will lead to a decrease in protein synthesis. Insulin binding to its receptor leads to the activation of the protein kinase Akt (see Fig. 11.14). Akt will phosphorylate the TSC1/TSC2 complex at sites distinct from AMPK, which inhibits the GTPase-activating activity of the complex. This allows Rheb-GTP to be active for extended periods of time, activating mTOR, and increasing protein synthesis in the cell in response to insulin.

Table 36.4

Diseases Discussed in Chapter 36

Disease or Disorder

Environmental or Genetic

Insulinoma

Environmental (10%–20% genetic) Environmental

Diabetic ketoacidosis

Type 2 diabetes

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Both

Comments A tumor of the pancreas leading to excessive, episodic insulin secretion, usually coupled with weight gain to avoid hypoglycemia. Diabetic ketoacidosis occurs due to an elevation of ketone body levels caused by reduced levels of insulin in a person with type 1 diabetes. Diabetic ketoacidosis is not normally seen in persons with type 2 diabetes because only a partial resistance of adipocytes to circulating insulin. Reduced ability of tissues to respond to insulin even though insulin is being produced. Different tissues may display a differential sensitivity to insulin, particularly fat cells as compared to muscle cells.

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SECTION VI ■ LIPID METABOLISM

REVIEW QUESTIONS—CHAPTER 36 1.

2.

3.

A 20-year-old woman with diabetes mellitus was admitted to the hospital in a semiconscious state with fever, nausea, and vomiting. Her breath smelled of acetone. A urine sample was strongly positive for ketone bodies. Which one of the following statements about this woman is correct? A. A blood glucose test will probably show that her blood glucose level is much lower than 80 mg/dL. B. An injection of insulin will decrease her ketone body production. C. She should be given a glucose infusion so she will regain consciousness. D. Glucagon should be administered to stimulate glycogenolysis and gluconeogenesis in the liver. E. The acetone was produced by decarboxylation of the ketone body ␤-hydroxybutyrate. A woman was told by her physician to go on a low-fat diet. She decided to continue to consume the same number of calories by increasing her carbohydrate intake while decreasing her fat intake. Which of the following blood lipoprotein levels would be decreased as a consequence of her diet? A. VLDL B. IDL C. HDL D. Chylomicrons E. HDL Assume that an individual has been eating excess calories daily such that he will gain weight. Under which of the following conditions will the person gain weight most rapidly? A. If all the excess calories are due to carbohydrate. B. If all the excess calories are due to triacylgycerol.

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C. If all the excess calories are split 50%/50% between carbohydrate and triacylgycerol. D. If all the excess calories are split 25%/75% between carbohydrate and triacylgycerol. E. It makes no difference what form the excess calories are in. 4.

A chronic alcoholic has been admitted to the hospital because of a severe hypoglycemic episode brought about by excessive alcohol consumption for the past 5 days. A blood lipid analysis indicates much higher than expected VLDL levels. The elevated VLDL is attributable to which of the following underlying cause? A. Alcohol-induced inhibition of lipoprotein lipase B. Elevated NADH levels in the liver C. Alcohol-induced transcription of the apo B-100 gene D. NADH activation of phosphoenolpyruvate carboxykinase E. Acetaldehyde induction of enzymes on the endoplasmic reticulum

5.

Certain patients with abetalipoproteinemia frequently have difficulty maintaining blood volume; their blood has trouble clotting. This symptom is attributable to which of the following? A. Inability to produce chylomicrons B. Inability to produce VLDL C. Inability to synthesize clotting factors D. Inability to synthesize fatty acids E. Inability to absorb short-chain fatty acids

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SECTION SEVEN

Nitrogen Metabolism

D

ietary proteins are the primary source of the nitrogen that is metabolized by the body (Fig. VII.1). Amino acids, produced by digestion of dietary proteins, are absorbed through intestinal epithelial cells and enter the blood. Various cells take up these amino acids, which enter the cellular pools. They are used for the synthesis of proteins and other nitrogen-containing compounds or they are oxidized for energy. Protein synthesis, the translation of mRNA on ribosomes (see Chapter 15), is a dynamic process. Within the body, proteins are constantly being synthesized and degraded, partially draining and then refilling the cellular amino acid pools. Compounds derived from amino acids include cellular proteins, hormones, neurotransmitters, creatine phosphate, the heme of hemoglobin and the cytochromes, the skin pigment melanin, and the purine and pyrimidine bases of nucleotides and nucleic acids. In fact, all of the nitrogen-containing compounds of the body are synthesized from amino acids. Many of these pathways are outlined in the following chapters. In addition to serving as the precursors for the nitrogen-containing compounds of the body and as the building blocks for protein synthesis, amino acids are also a source of energy. Amino acids are either oxidized directly or the amino acid carbons are converted to glucose and then oxidized or stored as glycogen. The amino acid carbons may also be converted to fatty acids and stored as adipose triacylglycerols. Glycogen and triacylglycerols are oxidized during periods of fasting. The liver is the major site of amino acid oxidation. However, most tissues can oxidize the branched-chain amino acids (leucine, isoleucine, and valine). Before the carbon skeletons of amino acids are oxidized, the nitrogen must be removed. Amino acid nitrogen forms ammonia, which is toxic to the body. In the liver, ammonia and the amino groups from amino acids are converted to urea, which is nontoxic, water-soluble, and readily excreted in the urine. The process by which urea is produced is known as the urea cycle. The liver is the organ responsible for producing urea. Branched-chain amino acids can be oxidized in many tissues, but the nitrogen must always travel to the liver for disposal. Although urea is the major nitrogenous excretory product of the body, nitrogen is also excreted in other compounds (Table VII.1). Uric acid is the degradation product of the purine bases, creatinine is produced from creatine phosphate, and ammonia is released from glutamine particularly by the kidney, where it helps to buffer the urine by reacting with protons to form ammonium ions (NH4⫹). These compounds are excreted mainly in the urine, but substantial amounts are also lost in the feces and through the skin. Small amounts of nitrogen-containing metabolites are formed from the degradation of neurotransmitters, hormones, and other specialized amino acid products and excreted in the urine. Some of these degradation products, such as bilirubin (formed from the degradation of heme), are excreted mainly in the feces. Eleven of the 20 amino acids used to form proteins are synthesized in the body if an adequate amount is not present in the diet (Table VII.2). Ten of these amino acids can be produced from glucose; the 11th, tyrosine, is synthesized from the essential amino acid phenylalanine. It should be noted that cysteine, one of the

Dietary proteins Digestion (stomach, intestine) Amino acids in blood Membrane Cell Amino acids

Proteins

N-containing compounds Carbon

Nitrogen O

CO2 + H2O Energy

H2N

C

NH2

Urea and other nitrogenous excretory products Urine

FIG. VII.1. Summary of amino acid metabolism. Dietary proteins are digested to amino acids in the stomach and intestine, which are absorbed by the intestinal epithelium, transferred to the circulation, and taken up by cells. Amino acids are used to synthesize proteins and other nitrogen-containing compounds. The carbon skeletons of amino acids are also oxidized for energy, and the nitrogen is converted to urea and other nitrogenous excretory products.

The healthy human adult is in nitrogen balance; in other words, the amount of nitrogen excreted each day (mainly in the urine) equals the amount consumed (mainly as dietary protein). Negative nitrogen balance occurs when the amount of nitrogen excreted is greater than the amount consumed, and positive nitrogen balance occurs when the amount excreted is less than that consumed (see Chapter 1). 693

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Table VII.1

Major Nitrogenous Urinary Excretory Products

Amount Excreted in Urine Per Daya Ureab NH4⫹ Creatinine Uric acid

12–20 g urea nitrogen (12,000–20,000 mg) 140–1,500 mg ammonia nitrogen Men 14–26 mg/kg Women 11–20 mg/kg 250–750 mg

a

The amounts are expressed in the units that are generally reported by clinical laboratories. Note that the amounts for creatinine and uric acid are for the whole compound, whereas those for urea and ammonia are for the nitrogen content. b Under normal circumstances, approximately 90% of the nitrogen excreted in the urine is in the form of urea. The exact amounts of each component vary, however, depending on dietary protein intake and physiologic state. For instance, NH4⫹ excretion increases during acidosis because the kidney secretes ammonia to bind protons in the urine.

10 amino acids produced from glucose, obtains its sulfur atom from the essential amino acid methionine. Certain amino acids are essential in humans. “Essential” means that the carbon skeleton cannot be synthesized and, therefore, these amino acids are required in the diet (Table VII.3). The essential amino acids are also called the indispensable amino acids. Arginine is essential during periods of growth; in adults it is no longer considered essential.

Table VII.3 the Diet

Amino Acids Essential in

Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Arginine (not required by the adult but required for growth)

Table VII.2

Amino Acids Synthesized in the Bodya

From Glucose

From an Essential Amino Acid

Alanine Arginine Asparagine Aspartate Cysteineb Glutamate Glutamine Glycine Proline Serine

Tyrosine (from phenylalanine)

a

These amino acids are called “nonessential” or “dispensable,” terms that refer to dietary requirements. Of course, within the body, they are necessary. We cannot survive without them. b Although the carbons of cysteine can be derived from glucose, its sulfur is obtained from the essential amino acid methionine.

694

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Glucose

Fatty acids

Glucose

Protein Blood

Glycogen

Fatty acids

Amino acids

Triacylglycerol

G-1-P

Amino acids

synthesis

G-6-P

Synthesis of important compounds

(N)

Amino acids

Cell

Pyruvate

Acetyl CoA

αKG Deamination (Ser, Thr, His)

Transamination Gut

Amino acids

OAA

Proteins

Citrate

Ketone bodies

GABA Glutathione Heme Nicotinamide (NAD+,NADP+) Serotonin Melatonin Norepinephrine Epinephrine Histamine Melanin Pyrimidines Purines Creatine-P Thyroxine Sphingosine

NADH Malate

ATP

electron transport chain

Uric acid Creatinine

Gln Glu α-Keto acids (carbon skeletons)

NH4+

NH4+ Urea

CO2

H2O O2 TCA cycle

To urine

Carbamoyl-P Ornithine Citrulline Asp

Urea cycle

Arg

Argininosuccinate

FIG. VII.2. Overview of nitrogen metabolism. The metabolism of nitrogen-containing compounds is shown on the right and that of glucose and fatty acids is shown on the left. This figure shows a hypothetical, composite cell. No single-cell type has all of these pathways. Many of the pathways shown are described in the next few chapters. ␣-KG, ␣-ketoglutarate; OAA, oxaloacetate; G-6-P, glucose 6-phosphate; G-1-P, glucose 1-phosphate.

After nitrogen is removed from amino acids, the carbon skeletons are oxidized (Fig. VII.2). Most of the carbons are converted to pyruvate, intermediates of the tricarboxylic acid (TCA) cycle, or to acetyl coenzyme A (acetyl-CoA). In the liver, particularly during fasting, these carbons may be converted to glucose or to ketone bodies and released into the blood. Other tissues then oxidize the glucose and ketone bodies. Ultimately, the carbons of the amino acids are converted to CO2 and H2O.

695

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37

Protein Digestion and Amino Acid Absorption Proteolytic enzymes (also called proteases) break down dietary proteins into their constituent amino acids in the stomach and the intestine. Many of these digestive proteases are synthesized as larger, inactive forms known as zymogens. After zymogens are secreted into the digestive tract, they are cleaved to produce the active proteases. In the stomach, pepsin begins the digestion of proteins by hydrolyzing them to smaller polypeptides. The contents of the stomach pass into the small intestine, where enzymes produced by the exocrine pancreas act. The pancreatic proteases (trypsin, chymotrypsin, elastase, and the carboxypeptidases) cleave the polypeptides into oligopeptides and amino acids. Further cleavage of the oligopeptides to amino acids is accomplished by enzymes produced by the intestinal epithelial cells. These enzymes include aminopeptidases located on the brush border and other peptidases located within the cells. Ultimately, the amino acids produced by protein digestion are absorbed through the intestinal epithelial cells and enter the blood. A large number of overlapping transport systems exist for amino acids in cells. Some systems contain facilitative transporters, whereas others express sodium-linked transporters, which allow the active transport of amino acids into cells. Defects in amino acid transport can lead to disease. Proteins are also continually synthesized and degraded (turnover) in cells. A wide variety of proteases in cells carry out this activity. Lysosomal proteases (cathepsins) degrade proteins that enter lysosomes. Cytoplasmic proteins targeted for turnover are covalently linked to the small protein ubiquitin, which then interacts with a large protein complex, the proteasome, to degrade the protein in an adenosine triphosphate (ATP)-dependent process. The amino acids released from proteins during turnover can then be used for the synthesis of new proteins or for energy generation.

696

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CHAPTER 37 ■ PROTEIN DIGESTION AND AMINO ACID ABSORPTION

THE WAITING ROOM Sissy Fibrosa, a young child with cystic fibrosis (see Chapter 17), has had repeated bouts of bronchitis caused by Pseudomonas aeruginosa. With each of these infections, her response to aerosolized antibiotics has been good. However, her malabsorption of food continues, resulting in foul-smelling, glistening, bulky stools. Her growth records show a slow decline. She is now in the 24th percentile for height and the 20th percentile for weight. She is often listless and irritable, and she tires easily. When her pediatrician discovered that her levels of the serum proteins albumin and prealbumin were low to low normal (indicating protein malnutrition), Sissy was given entericcoated microspheres of pancreatic enzymes. Almost immediately, the character of Sissy’s stools became more normal and she began gaining weight. Over the next 6 months, her growth curves showed improvement, and she seemed brighter, more active, and less irritable. For the first few months after a painful episode of renal colic, during which he passed a kidney stone (see Chapter 6), Cal Kulis had faithfully maintained a high daily fluid intake and had taken the medication required to increase the pH of his urine. Because he has cystinuria, these measures were necessary to increase the solubility of the large amounts of cystine present in his urine and thereby to prevent further formation of kidney stones (calculi). With time, however, he became increasingly complacent about his preventive program. After failing to take his medication for a month, he experienced another severe episode of renal colic with grossly bloody urine. Fortunately, he passed the stone spontaneously, after which he vowed to comply faithfully with his prescribed therapy. His mother heard that some dietary amino acids were not absorbed in patients with cystinuria and asked whether any dietary changes would reduce Cal’s chances of developing additional renal stones.

I.

Food

HCl

Stomach

Protein Pepsin

Pancreas

Peptides HCO3

–

Trypsinogen Chymotrypsinogen Proelastase Procarboxypeptidases A and B

Small intestine

PROTEIN DIGESTION

The digestion of protein begins in the stomach and is completed in the intestine (Fig. 37.1). The enzymes that digest proteins are produced as inactive precursors (zymogens) that are larger than the active enzymes. The inactive zymogens are secreted from the cells in which they are synthesized and enter the lumen of the digestive tract, where they are cleaved to smaller forms that have proteolytic activity (Fig. 37.2). These active enzymes have different specificities; no single enzyme can completely digest a protein. However, by acting in concert, they can digest dietary proteins to amino acids and small peptides, which are cleaved by peptidases associated with intestinal epithelial cells.

A. Digestion of Proteins in the Stomach Pepsinogen is secreted by the chief cells of the stomach. The gastric parietal cells secrete HCl. The acid in the stomach lumen alters the conformation of pepsinogen so that it can cleave itself, producing the active protease pepsin. Thus, the activation of pepsinogen is autocatalytic. Dietary proteins are denatured by the acid in the stomach. This inactivates the proteins and partially unfolds them so that they are better substrates for proteases. However, at the low pH of the stomach, pepsin is not denatured and acts as an endopeptidase, cleaving peptide bonds at various points within the protein chain.

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Aminopeptidases

Blood Di- and tripeptides + amino acids

Di- and tripeptidases

Amino acids

Amino acids

Intestinal epithelial cell

FIG. 37.1. Digestion of proteins. The proteolytic enzymes, trypsin, chymotrypsin, elastase, and the carboxypeptidases are produced as zymogens (the [pro] and [ogen], in red, accompanying the enzyme name) that are activated by cleavage after they enter the gastrointestinal lumen (see Fig. 37.2). Pepsinogen is produced within the stomach and is activated within the stomach (to pepsin) as the pH drops due to HCl secretion.

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SECTION VII ■ NITROGEN METABOLISM

Kwashiorkor, a common problem of children in third world countries, is caused by a deficiency of protein in a diet that is adequate in calories. Children with kwashiorkor suffer from muscle wasting and decreased concentration of plasma proteins, particularly albumin. The result is an increase in interstitial fluid that causes edema and a distended abdomen, which makes the children appear “plump” (see Chapter 44). The muscle wasting is caused by the lack of essential amino acids in the diet; existing proteins must be broken down to produce these amino acids for new protein synthesis. These problems may be compounded by a decreased ability to produce digestive enzymes and new intestinal epithelial cells because of a decreased availability of amino acids for the synthesis of new proteins.

Elastase is also found in neutrophils, which are white blood cells that engulf and destroy invading bacteria. Neutrophils frequently act in the lung, and elastase is sometimes released into the lung as the neutrophils work. In normal individuals, the released elastase is blocked from destroying lung cells by the action of circulating ␣-1-antitrypsin, a protease inhibitor that is synthesized and secreted by the liver. Certain individuals have a genetic mutation that leads to the production of an inactive ␣-1-antitrypsin protein (␣-1-antitrypsin deficiency). The lack of this enzyme activity leads to the development of emphysema caused by proteolytic destruction of lung cells, which results in a reduction in the expansion/contraction capability of the lungs. Deficiency of ␣-1-antitrypsin can be measured using a dried blood spot. The blood is solubilized using a buffer, and then various amounts of the blood sample are incubated with antibodies specific for ␣-1-antitrypsin. The antigen–antibody complexes formed will disperse light, and the extent of light scattering is proportional to the concentration of ␣-1-antitrypsin in the solution. This procedure is known as rate nephelometry. Obtaining blood samples from a finger prick and allowing the blood to dry on a card enables rapid screening of individuals for this disorder.

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Proenzymes (zymogens) Pepsinogen

Trypsinogen

Chymotrypsinogen

Proelastase

Procarboxypeptidases

Active enzymes H+

Enteropeptidase

Trypsin

Trypsin

Trypsin

Pepsin

Trypsin

Chymotrypsin

Elastase

Carboxypeptidases

FIG. 37.2. Activation of the gastric and pancreatic zymogens. Pepsinogen catalyzes its own cleavage as the pH of the stomach drops. Trypsinogen is cleaved by enteropeptidase in the intestine to form the active protease trypsin. Trypsin then plays a key role by catalyzing the cleavage and activation of the other pancreatic zymogens.

Although pepsin has fairly broad specificity, it tends to cleave peptide bonds in which the carboxyl group is provided by an aromatic or acidic amino acid (Fig. 37.3). Smaller peptides and some free amino acids are produced.

B. Digestion of Proteins by Enzymes from the Pancreas As the gastric contents empty into the intestine, they encounter the secretions from the exocrine pancreas. Recall that the exocrine pancreas secretes amylase for starch digestion, and lipase and colipase for dietary triacylglycerol digestion. Another pancreatic secretion is bicarbonate, which, in addition to neutralizing stomach acid, raises the pH so that the pancreatic proteases, which are also present in pancreatic secretions, can be active. As secreted, these pancreatic proteases are in the inactive proenzyme form (zymogens). Because the active forms of these enzymes can digest each other, it is important for their zymogen forms all to be activated within a short span of time. This is accomplished by the cleavage of trypsinogen to the active enzyme trypsin, which then cleaves the other pancreatic zymogens, producing their active forms (see Fig. 37.2). The zymogen trypsinogen is cleaved to form trypsin by enteropeptidase (a protease, formerly called enterokinase) secreted by the brush border cells of the small intestine. Trypsin catalyzes the cleavages that convert chymotrypsinogen to the active enzyme chymotrypsin, proelastase to elastase, and the procarboxypeptidases to the carboxypeptidases. Thus, trypsin plays a central role in digestion because it both cleaves dietary proteins and activates other digestive proteases produced by the pancreas. Trypsin, chymotrypsin, and elastase are serine proteases (see Chapter 9) that act as endopeptidases. Trypsin is the most specific of these enzymes, cleaving peptide bonds in which the carboxyl (carbonyl) group is provided by lysine or arginine (see Fig. 37.3). Chymotrypsin is less specific but favors residues that contain hydrophobic amino acids. Elastase cleaves not only elastin (for which it was named) but also other proteins at bonds in which the carboxyl group is contributed by amino acid residues with small side chains (alanine, glycine, and serine). The actions of these pancreatic endopeptidases continue the digestion of dietary proteins begun by pepsin in the stomach.

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CHAPTER 37 ■ PROTEIN DIGESTION AND AMINO ACID ABSORPTION

The smaller peptides formed by the action of trypsin, chymotrypsin, and elastase are attacked by exopeptidases, which are proteases that cleave one amino acid at a time from the end of the chain. Procarboxypeptidases, zymogens produced by the pancreas, are converted by trypsin to the active carboxypeptidases. These exopeptidases remove amino acids from the carboxyl ends of peptide chains. Carboxypeptidase A preferentially releases hydrophobic amino acids, whereas carboxypeptidase B releases basic amino acids (arginine and lysine).

+

NH3 Aminopeptidases

H

H

Trypsin

H

A. Cotransport of Na and Amino Acids

Intestinal lumen

Amino acid Na+ Brush border

Amino acid

C

O

Phe Tyr Glu Asp

C

R

Arg Lys

C

O

H

C

R

C

O

H

C

R

C

O

Phe Tyr Trp Leu Ala Gly Ser

NH

Carboxypeptidases

C

R

C

O

NH H

C

R

COO–

Carboxypeptidase A (hydrophobic) Carboxypeptidase B (Arg Lys)

FIG. 37.3. Action of the digestive proteases. Pepsin, trypsin, chymotrypsin, and elastase are endopeptidases; they hydrolyze peptide bonds within chains. The others are exopeptidases; aminopeptidases remove the amino acid at the N-terminus and the carboxypeptidases remove the amino acid at the C-terminus. For each proteolytic enzyme, the amino acid residues involved in the peptide bond that is cleaved are listed beside the R group to the right of the enzyme name.

Active transporter Na+

ADP +Pi

K+ +

K

Facilitated Amino transporter acid

Portal vein

FIG. 37.4. Transepithelial amino acid transport. Na⫹-dependent carriers transport both Na⫹ and an amino acid into the intestinal epithelial cell from the intestinal lumen. Na⫹ is pumped out on the serosal side (across the basolateral membrane) in exchange for K⫹ by the Na⫹,K⫹-ATPase. On the serosal side, the amino acid is carried by a facilitated transporter down to its concentration gradient into the blood. This process is an example of secondary active transport. Pi, inorganic phosphate.

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R

Na+

ATP

Serosal side

C

NH Elastase

C-terminus

Amino acids are absorbed from the lumen of the small intestine principally by semispecific Na⫹-dependent transport proteins in the luminal membrane of the intestinal cell brush border, similar to that already seen for carbohydrate transport (Fig. 37.4). The cotransport of Na⫹ and the amino acid from the outside of the apical membrane to the inside of the cell is driven by the low intracellular Na⫹ concentration. Low intracellular

O

NH Chymotrypsin

H

ⴙ

C

N-terminus

NH

II. ABSORPTION OF AMINO ACIDS

Amino acids are absorbed from the intestinal lumen through secondary active Na⫹dependent transport systems and through facilitated diffusion.

R

NH Pepsin

C. Digestion of Proteins by Enzymes from Intestinal Cells Exopeptidases produced by intestinal epithelial cells act within the brush border and also within the cell. Aminopeptidases, located on the brush border, cleave one amino acid at a time from the amino end of peptides. Intracellular peptidases act on small peptides that are absorbed by the cells. The concerted action of the proteolytic enzymes produced by cells of the stomach, pancreas, and intestine cleaves dietary proteins to amino acids. The digestive enzymes digest themselves as well as dietary protein. They also digest the intestinal cells that are regularly sloughed off into the lumen. These cells are replaced by cells that mature from precursor cells in the duodenal crypts. The amount of protein that is digested and absorbed each day from digestive juices and cells released into the intestinal lumen may be equal to or greater than the amount of protein consumed in the diet (50 to 100 g).

C

699

Patients with cystic fibrosis, such as Sissy Fibrosa, have a genetically determined defect in the function of the chloride channels. In the pancreatic secretory ducts, which carry pancreatic enzymes into the lumen of the small intestines, this defect causes inspissation (drying and thickening) of pancreatic exocrine secretions, leading eventually to obstruction of these ducts. One result of this problem is the inability of pancreatic enzymes to enter the intestinal lumen to digest dietary proteins.

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The pancreas synthesizes and stores the zymogens in secretory granules. The pancreas also synthesizes a secretory trypsin inhibitor. The need for the inhibitor is to block any trypsin activity that may occur from accidental trypsinogen activation. If the inhibitor were not present, trypsinogen activation would lead to the activation of all of the zymogens in the pancreas, which would lead to the digestion of intracellular pancreatic proteins. Such episodes can lead to pancreatitis.

Hartnup disease is another genetically determined and relatively rare autosomal recessive disorder. It is caused by a defect in the transport of neutral amino acids across both intestinal and renal epithelial cells (system B0, gene SLC6A19, where SLC refers to the solute carrier family of transport proteins, of which there are 55, with a total of 362 different genes). The signs and symptoms are caused, in part, by a deficiency of essential amino acids (see Clinical Comments). Cystinuria (system B0,⫹, genes involved are SLC7A9 and SLC3A1, as the transporter is a heteromeric complex) and Hartnup disease involve defects in two different transport proteins. In each case, the defect is present both in intestinal cells, causing malabsorption of the amino acids from the digestive products in the intestinal lumen, and in kidney tubular cells, causing a decreased resorption of these amino acids from the glomerular filtrate and an increased concentration of the amino acids in the urine.

Why do patients with cystinuria and Hartnup disease have hyperaminoaciduria without associated hyperaminoacidemia?

Na⫹ results from the pumping of Na⫹ out of the cell by a Na⫹,K⫹-ATPase on the serosal membrane. Thus, the primary transport mechanism is the creation of a sodium gradient; the secondary transport process is the coupling of amino acids to the influx of sodium. This mechanism allows the cells to concentrate amino acids from the intestinal lumen. The amino acids are then transported out of the cell into the interstitial fluid, principally by facilitated transporters in the serosal membrane (see Fig. 37.4). At least six different Na⫹-dependent amino acid carriers are located in the apical brush border membrane of the epithelial cells. These carriers have overlapping specificity for different amino acids. One carrier preferentially transports neutral amino acids, another transports proline and hydroxyproline, a third preferentially transports acidic amino acids, and a fourth transports basic amino acids (lysine, arginine, the urea-cycle intermediate ornithine) and cystine (two cysteine residues linked by a disulfide bond). In addition to these Na⫹-dependent carriers, some amino acids are transported across the luminal membrane by facilitated transport carriers. Most amino acids are transported by more than one transport system. As with glucose transport, the Na⫹-dependent carriers of the apical membrane of the intestinal epithelial cells are also present in the renal epithelium. However, different isozymes are present in the cell membranes of other tissues. Conversely, the facilitated transport carriers in the serosal membrane of the intestinal epithelia are similar to those found in other cell types in the body. During starvation, the intestinal epithelia, like these other cells, take up amino acids from the blood to use as an energy source. Thus, amino acid transport across the serosal membrane is bidirectional.

B. Transport of Amino Acids into Cells Amino acids that enter the blood are transported across cell membranes of the various tissues principally by Na⫹-dependent cotransporters and, to a lesser extent, by facilitated transporters (Table 37.1). In this respect, amino acid transport differs from glucose transport, which is Na⫹-dependent transport in the intestinal and renal epithelium but facilitated transport in other cell types. The Na⫹ dependence of amino acid transport in liver, muscle, and other tissues allows these cells to concentrate amino acids from the blood. These transport proteins have different genetic bases, amino acid compositions, and somewhat different specificities than those in the luminal membrane of intestinal epithelia. They also differ somewhat

Table 37.1

A Partial Listing of Amino Acid Transport Systemsa

System Name

Sodium Dependent?

A

Yes

ASC N

Yes Yes

L

No

B0,⫹

Yes

B0

Yes

XAG⫺

Yes

Zwitterionic amino acids (monoamino, monocarboxylic acid amino acids) Anionic amino acids (Asp, Glu)

Imino

Yes

Pro, hydroxyproline, Gly

Specificity

Tissues Expressed

Small and polar neutral amino acids (Ala, Ser, Gln, Gly, Pro, Cys, Asn, His, Met) Small amino acids (Ala, Ser, Cys) Gln, Asn, His

Many

Branched and aromatic amino acids (His, Met, Leu, Ile, Val, Phe, Tyr, Trp) Basic amino acids

Many Liver, basolateral membrane of kidney Manyb Intestine (brush border)c and kidney Intestine and kidneyd Intestine (brush border) and kidney Intestine (brush border) and kidney

a

Not all transport systems are listed. This transport system will be exploited in a treatment for PKU, see Chapter 39. This system is most likely defective in cystinuria. d This system is most likely defective in Hartnup disease. b c

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among tissues. For instance, the N-system for glutamine uptake is present in the liver but either is not present in other tissues or is present as an isoform with different properties. There is also some overlap in specificity of the transport proteins, with most amino acids being transported by more than one carrier.

III. PROTEIN TURNOVER AND REPLENISHMENT OF THE INTRACELLULAR AMINO ACID POOL The amino acid pool within cells is generated both from dietary amino acids and from the degradation of existing proteins within the cell. All proteins within cells have a half-life (t½), a time at which 50% of the protein that was synthesized at a particular time will have been degraded. Some proteins are inherently short-lived, with half-lives of 5 to 20 minutes. Other proteins are present for extended periods, with half-lives of many hours or even days. Thus, proteins are continuously being synthesized and degraded in the body, using a variety of enzyme systems to do so (Table 37.2). Examples of proteins that undergo extensive synthesis and degradation are hemoglobin, muscle proteins, digestive enzymes, and the proteins of cells sloughed off from the gastrointestinal tract. Hemoglobin is produced in reticulocytes and reconverted to amino acids by the phagocytic cells that remove mature red blood cells from the circulation on a daily basis. Muscle protein is degraded during periods of fasting, and the amino acids are used for gluconeogenesis. After ingestion of protein in the diet, muscle protein is resynthesized. Adults cannot increase the amount of muscle or other body proteins by eating an excess amount of protein. If dietary protein is consumed in excess of our needs, it is converted to glycogen and triacylglycerols, which are then stored. A large amount of protein is recycled daily in the form of digestive enzymes, which are themselves degraded by digestive proteases. In addition, approximately one-fourth of the cells lining the walls of the gastrointestinal tract are lost each day and replaced by newly synthesized cells. As cells leave the gastrointestinal wall, their proteins and other components are digested by enzymes in the lumen of the gut, and the products are absorbed. Additionally, red blood cells have a life span of about 120 days. Every day, 3 ⫻ 1011 red blood cells die and are phagocytosed. The hemoglobin in these cells is degraded to amino acids by lysosomal proteases, and their amino acids are reused in the synthesis of new proteins. Only approximately 6% (roughly 10 g) of the protein that enters the digestive tract (including dietary proteins, digestive enzymes, and the proteins in sloughed-off cells) is excreted in the feces each day. The remainder is recycled. Proteins are also recycled within cells. The differences in amino acid composition of the various proteins of the body, the vast range in turnover times (t½), and the recycling of amino acids are all important factors that help to determine the requirements for specific amino acids and total protein in the diet. The synthesis

Table 37.2

Proteases Involved in Protein Turnover/Degradation

Classification

Mechanism

Role

Cathepsins Caspases

Cysteine proteases Cysteine proteases, which cleave after aspartate Require zinc for catalysis

Lysosomal enzymes Apoptosis; activated from procaspases (see Chapter 18) Model extracellular matrix components; regulated by TIMPs (tissue inhibitors of matrix metalloproteinases) Protein turnover

Matrix metalloproteinases Proteasome Serine proteases Calpains

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Large complex that degrades ubiquitin-tagged proteins Active-site serine in a catalytic triad with histidine and aspartic acid Calcium-dependent cysteine proteases

Digestion and blood clotting; activated usually from zymogens (see Chapter 45) Many different cellular roles

701

Trace amounts of polypeptides pass into the blood. They may be transported through intestinal epithelial cells, probably by pinocytosis, or they may slip between the cells that line the gut wall. This process is particularly troublesome for premature infants because it can lead to allergies caused by proteins in their food.

Patients with cystinuria and Hartnup disease have defective transport proteins in both the intestine and the kidney. These patients do not absorb the affected amino acids at a normal rate from the digestive products in the intestinal lumen. They also do not readily resorb these amino acids from the glomerular filtrate into the blood. Therefore, they do not have hyperaminoacidemia (a high concentration in the blood). Normally, only a few percentage of the amino acids that enter the glomerular filtrate are excreted in the urine; most are resorbed. In these diseases, much larger amounts of the affected amino acids are excreted in the urine, resulting in hyperaminoaciduria.

Cal Kulis and other patients with cystinuria have a genetically determined defect in the transport of cystine and the basic amino acids—lysine, arginine, and ornithine—across the brush border membranes of cells in both their small intestine and renal tubules (system B0,⫹). However, they do not appear to have any symptoms of amino acid deficiency, in part because the amino acids cysteine (which is oxidized in blood and urine to form the disulfide cystine) and arginine can be synthesized in the body (i.e., they are “nonessential” amino acids). Ornithine (an amino acid that is not found in proteins but serves as an intermediate of the urea cycle) can also be synthesized. The most serious problem for these patients is the insolubility of cystine, which can form kidney stones that may lodge in the ureter, causing genitourinary bleeding and severe pain known as renal colic.

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of many enzymes is induced in response to physiologic demand (such as fasting or feeding). These enzymes are continuously being degraded. Intracellular proteins are also damaged by oxidation and other modifications that limit their function. Mechanisms for intracellular degradation of unnecessary or damaged proteins involve lysosomes and the ubiquitin/proteasome system.

A. Lysosomal Protein Turnover Lysosomes participate in the process of autophagy, in which intracellular components are surrounded by membranes that fuse with lysosomes and endocytosis (see Chapter 10). Autophagy is a complex regulated process in which cytoplasm is sequestered into vesicles and delivered to the lysosomes. Within the lysosomes, the cathepsin family of proteases degrades the ingested proteins to individual amino acids. The recycled amino acids can then leave the lysosome and rejoin the intracellular amino acid pool. Although the details of how autophagy is induced are still being unraveled, starvation of a cell is a trigger to induce this process. This allows old proteins to be recycled and the newly released amino acids to be used for new protein synthesis, to enable the cell to survive starvation conditions. The mammalian target of rapamycin (mTOR) (see Chapter 36) plays a key role in regulating autophagy, as outlined in Figure 37.5.

Low energy high AMP; low ATP

+

LKB1

+ AMPK Activation of Akt by insulin

+

TSC1/ TSC2

–

– Rheb

+ mTOR

– Autophagy

FIG. 37.5. Overview of mTOR participation in autophagy. When active, the mTOR kinase will phosphorylate a protein crucial to the initiation of phagosome formation, which will inhibit autophagy. mTOR kinase is activated by Rheb-GTP (ras homolog enriched in brain, bound to GTP). Under low energy conditions (starvation), autophagy is favored as the AMPactivated protein kinase (AMPK) is activated, which phosphorylates the tuberous sclerosis complexes 1 and 2 (TSC1/TSC2) activating its Rheb-GTPase activating activity. This leads to the inactivation of Rheb (Rheb-GTP is converted to the inactive Rheb-GDP), which leads to the inactivation of mTOR. When inactive, mTOR does not block autophagy, and self-proteolysis is favored, as is an inhibition of protein synthesis. In the presence of growth factors that activate the Akt kinase, Akt phosphorylates the TSC1/TSC2 complex at a site distinct from the AMPK site. The phosphorylation event by Akt leads to an inhibition of the TSC1/TSC2 RhebGTPase activating activity, which allows Rheb to be active for extended periods. Active Rheb leads to activation of mTOR, inhibition of autophagy, and activation of protein synthesis.

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703

Protein targeted for destruction 19S Regulatory Particle

26S proteasome requires ATP for proteolysis

ADP ATP

Proteolytic residues

ACTIVE PROTEASOME COMPLEXES

Requires ATP for proteolysis

20S proteasome The 20S proteasome can have either one or two 19S regulatory particles associated with it

FIG. 37.6. The proteasome and cap proteins. The cap proteins (PA700 and PA28) regulate the activity of this proteolytic complex by recruiting to the complex the substrates for proteolysis. The ATP requirement is to unfold and denature the proteins targeted for destruction.

B. The Ubiquitin–Proteasome Pathway Ubiquitin is a small protein (76 amino acids) that is highly conserved. Its amino acid sequence in yeast and humans differs by only three residues. Ubiquitin targets intracellular proteins for degradation by covalently binding to the ␧-amino group of lysine residues. This is accomplished by a three-enzyme system that adds ubiquitin to proteins targeted for degradation. Often, the target protein is polyubiquitinylated, a process in which additional ubiquitin molecules are added to previous ubiquitin molecules, forming a long ubiquitin tail on the target protein. After polyubiquitinylation is complete, the targeted protein is released from the three-enzyme complex and is directed to the proteasome via a variety of mechanisms. A protease complex, known as the proteasome, then degrades the targeted protein, releasing intact ubiquitin that can again mark other proteins for degradation (Fig. 37.6). The basic proteasome is a cylindrical 20S protein complex with multiple internal proteolytic sites. Adenosine triphosphate (ATP) hydrolysis is used both to unfold the tagged protein and to push the protein into the core of the cylinder. The complex is regulated by 19S regulatory particles (cap complexes), which bind the ubiquinylated protein (a step that requires ATP) and deliver them to the complex. After the target protein is degraded, the ubiquitin is released intact and recycled. The resulting amino acids join the intracellular pool of free amino acids. Many proteins that contain regions rich in the amino acids proline (P), glutamate (E), serine (S), and threonine (T) have short half-lives. These regions are known as PEST sequences, based on the one-letter abbreviations used for these amino acids. Most of the proteins that contain PEST sequences are hydrolyzed by the ubiquitin– proteasome system. CLINICAL COMMENTS Sissy Fibrosa. Sissy Fibrosa’s growth and weight curves were both subnormal until her pediatrician added pancreatic enzyme supplements to her treatment plan. These supplements digest dietary protein, releasing essential and other amino acids from the dietary protein, which are then absorbed by the epithelial cells of Sissy’s small intestine, through which they are transported into the blood. A discernable improvement in Sissy’s body weight and growth curves was noted within months of starting this therapy.

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Besides the proportions of essential amino acids present in various foods, the quality of a dietary protein is also determined by the rate at which it is digested and, in a more general way, by its capacity to contribute to the growth of the infant. In this regard, the proteins in foods of animal origin are more digestible than are those derived from plants. For example, the digestibility of proteins in eggs is approximately 97%; that of meats, poultry, and fish is 85% to 100%; and that from wheat, soybeans, and other legumes ranges from 75% to 90%. The official daily dietary “protein requirement” accepted by the United States and Canadian government is 0.8 g of protein per kilogram of desirable body weight for adults (approximately 56 g for an adult man and 44 g for an adult woman). On an average weight basis, the requirement per kilogram is much greater for infants and children. This fact underscores the importance of improving Sissy Fibrosa’s protein digestion to optimize her potential for normal growth and development. Cal Kulis. In patients with cystinuria, such as Cal Kulis, the inability to absorb cystine and basic amino acids from the gut and the increased loss of these amino acids in the urine may be expected to cause a deficiency of these compounds in the blood. However, because three of these amino acids can be synthesized in the body (i.e., they are nonessential amino acids), their concentrations in the plasma remain normal, and clinical manifestations of a deficiency state do not develop. It is not clear why symptoms related to a lysine deficiency have not been observed. In a disorder that was first observed in the Hartnup family and bears their name, the intestinal and renal transport defect involves the neutral amino acids (monoamine, monocarboxylic acids), including several essential amino acids (isoleucine, leucine, phenylalanine, threonine, tryptophan, and valine) as well as certain nonessential amino acids (alanine, serine, and tyrosine). A reduction in the availability of these essential amino acids may be expected to cause a variety of clinical disorders. Yet children with the Hartnup disorder identified by routine newborn urine screening almost always remain clinically normal. However, some patients with the Hartnup biochemical phenotype eventually develop pellagra-like manifestations, which usually include a photosensitivity rash, ataxia, and neuropsychiatric symptoms. Pellagra results from a dietary deficiency of the vitamin niacin or the essential amino acid tryptophan, which are both precursors for the nicotinamide moiety of NAD and NADP. In asymptomatic patients, the transport abnormality may be incomplete and so subtle as to allow no phenotypic expression of Hartnup disease. These patients also may be capable of absorbing some small peptides that contain the neutral amino acids. The only rational treatment for patients who have pellagra-like symptoms is to administer niacin (nicotinic acid) in oral doses up to 300 mg/day. Although the rash, ataxia, and neuropsychiatric manifestations of niacin deficiency may disappear, the hyperaminoaciduria and intestinal transport defect do not respond to this therapy. In addition to niacin, a high-protein diet may benefit some patients.

BIOCHEMICAL COMMENTS The ␥-Glutamyl Cycle. The ␥-glutamyl cycle is necessary for the synthesis of glutathione, a compound that protects cells from oxidative damage (see Chapter 24). When it was first discovered, the cycle was thought to be important in amino acid transport, but its involvement in such transport is now thought to be limited to salvage of cysteine. The enzymes of the cycle are present in many tissues, although certain tissues lack one or more of the enzymes of the cycle. The entire cycle is presented in Figure 37.7. In this case, the extracellular amino acid reacts with glutathione (␥-glutamyl-cysteinyl-glycine) in a reaction catalyzed by a transpeptidase present in the cell membrane. A ␥-glutamyl amino acid is

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ATP

ADP + Pi

Amino acid

705

Glutathione

␥-Glutamylcysteine

␥-Glutamyl

Glycine

transpeptidase

ADP + Pi Cysteinylglycine ATP

Cysteine

␥-Glutamylamino acid 5-Oxoprolinase

5-Oxoproline

Amino acid

ATP

Glutamate

ADP + Pi

FIG. 37.7. The ␥-glutamyl cycle. In cells of the intestine and kidney, amino acids can be transported across the cell membrane by reacting with glutathione (␥-glutamyl-cysteinyl-glycine) to form a ␥-glutamyl amino acid. The amino acid is released into the cell, and glutathione is resynthesized. However, the major role of this cycle is glutathione synthesis because many tissues lack the transpeptidase and 5-oxoprolinase activities. Pi, inorganic phosphate.

formed, which travels across the cell membrane and releases the amino acid into the cell. The other products of these two reactions are reconverted to glutathione. The reactions that convert glutamate to glutathione in the ␥-glutamyl cycle are the same reactions as those required for the synthesis of glutathione. The enzymes for glutathione synthesis, but not the transpeptidase, are found in most tissues. The oxoprolinase is also missing from many tissues, so the major role of this pathway is one of glutathione synthesis from glutamate, cysteine, and glycine. The transpeptidase is the only protease in the cell that can break the ␥-glutamyl linkage in glutathione. Glutathione is also involved in reducing compounds such as hydrogen peroxide (see Chapter 21). It also protects cells, particularly erythrocytes, from oxidative damage through formation of oxidized glutathione—two glutathione residues connected by a disulfide bond (see Chapter 24). Key Concepts • • • • • • • • • • • • •

Proteases (proteolytic enzymes) break down dietary proteins into peptides and then their constituent amino acids in the stomach and intestine. Pepsin initiates protein breakdown in the stomach. Upon entering the small intestine, inactive zymogens secreted from the pancreas are activated to continue protein digestion. Enzymes produced by the intestinal epithelial cells are also required to fully degrade proteins. The amino acids generated by proteolysis in the intestinal lumen are transported into the intestinal epithelial cells, from which they enter the circulation for use by the tissues. Transport systems for amino acids are similar to transport systems for monosaccharides; both facilitative and active transport systems exist. There are several overlapping transport systems for amino acids in cells. Protein degradation (turnover) occurs continuously in all cells. Proteins can be degraded by lysosomal enzymes (cathepsins). Proteins are also targeted for destruction by being covalently linked to the small protein ubiquitin. The ubiquitin-tagged proteins interact with the proteasome, a large complex that degrades proteins to small peptides in an ATP-dependent process. Amino acids released from proteins during turnover can be used for the synthesis of new proteins, for energy generation, or for gluconeogenesis. Diseases discussed in this chapter are summarized in Table 37.3.

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Table 37.3

Diseases Discussed in Chapter 37

Disease or Disorder

Genetic or Environmental

Cystic fibrosis

Genetic

Cystinuria

Genetic

Kwashiorkor

Environmental

Hartnup disease

Genetic

Comments Patients with cystic fibrosis often experience a blockage of the pancreatic duct, which necessitates oral ingestion of digestive enzymes for appropriate nutrient degradation and absorption. A mutation in a membrane transport protein (B0⫹) for basic amino acids, including cystine, which is expressed in kidney and intestine. Kidney stones may develop due to this disorder. Protein–calorie malnutrition (diet is adequate in calories but lacking in protein), leading to excessive protein degradation in the extremities and edema. A mutation in a membrane transport protein (B0) for neutral amino acids, including tryptophan, which is expressed in both the kidney and intestine. Some patients may develop pellagra-like symptoms due to the lack of tryptophan.

REVIEW QUESTIONS—CHAPTER 37

1.

2.

An individual with a deficiency in the conversion of trypsinogen to trypsin would be expected to experience a more detrimental effect on protein digestion than an individual who was defective in any of the other digestive proteases. This is a result of which of the following? A. Trypsin has a greater and wider range of substrates on which to act. B. Trypsin activates pepsinogen, so digestion can begin in the stomach. C. Trypsin activates the other zymogens that are secreted by the pancreas. D. Trypsin activates enteropeptidase, which is needed to activate the other pancreatic zymogens. E. Trypsin inhibits intestinal motility, so the substrates can be hydrolyzed for longer periods. An individual has been shown to have a deficiency in an intestinal epithelial cell amino acid transport system for leucine. However, the individual shows no symptoms of amino acid deficiency. This could be due to which of the following? A. The body synthesizes leucine to compensate for the transport defect. B. The kidney reabsorbs leucine and sends it to other tissues. C. There are multiple transport systems for leucine. D. Isoleucine takes the place of leucine in proteins. E. Leucine is not necessary for bulk protein synthesis.

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3.

Kwashiorkor can result from which of the following? A. Consuming a calorie-deficient diet that is also deficient in protein. B. Consuming a calorie-adequate diet that is deficient in carbohydrates. C. Consuming a calorie-adequate diet that is deficient in fatty acids. D. Consuming a calorie-adequate diet that is deficient in proteins. E. Consuming a calorie-deficient diet that is primarily proteins.

4.

Which of the following enzymes is activated through an autocatalytic process? A. Enteropeptidase B. Trypsinogen C. Pepsinogen D. Aminopeptidase E. Proelastase

5.

Children with kwashiorkor usually have a fatty liver. This is due to which of the following? A. The high-fat content of their diet B. The high-carbohydrate content of their diet C. The high-protein content of their diet D. The lack of substrates for gluconeogenesis in the liver E. The lack of substrates for protein synthesis in the liver F. The lack of substrates for glycogen synthesis in the liver

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38

Fate of Amino Acid Nitrogen: Urea Cycle

In comparison with carbohydrate and lipid metabolism, the metabolism of amino acids is complex. We must be concerned not only with the fate of the carbon atoms of the amino acids but also with the fate of the nitrogen. During their metabolism, amino acids travel in the blood from one tissue to another. Ultimately, most of the nitrogen is converted to urea in the liver and the carbons are oxidized to CO2 and H2O by several tissues (Fig. 38.1). After a meal that contains protein, amino acids released by digestion (see Chapter 37) pass from the gut through the hepatic portal vein to the liver (see Fig. 38.2A). In a normal diet containing 60 to 100 g protein, most of the amino acids are used for the synthesis of proteins in the liver and in other tissues. Excess amino acids may be converted to glucose or triacylglycerol. During fasting, muscle protein is cleaved to amino acids. Some of the amino acids are partially oxidized to produce energy (see Fig. 38.2B). Portions of these amino acids are converted to alanine and glutamine, which, along with other amino acids, are released into the blood. Glutamine is oxidized by various tissues, including the lymphocytes, gut, and kidney, which convert some of the carbons and nitrogen to alanine. Alanine and other amino acids travel to the liver, where the carbons are converted to glucose and ketone bodies, and the nitrogen is converted to urea, which is excreted by the kidneys. Glucose, produced by gluconeogenesis, is subsequently oxidized to CO2 and H2O by many tissues, and ketone bodies are oxidized by tissues such as muscle and kidney. Several enzymes are important in the process of interconverting amino acids and in removing nitrogen so that the carbon skeletons can be oxidized. These include dehydratases, transaminases, glutamate dehydrogenases, glutaminases, and deaminases. The conversion of amino acid nitrogen to urea occurs mainly in the liver. Urea is formed in the urea cycle from NH4⫹, bicarbonate, and the nitrogen of aspartate (see Fig. 38.1). Initially, NH4⫹, bicarbonate, and adenosine triphosphate (ATP) react to produce carbamoyl phosphate, which reacts with ornithine to form citrulline. Aspartate then reacts with citrulline to form argininosuccinate, which releases fumarate, forming arginine. Finally, arginase cleaves arginine to release urea and regenerate ornithine. The cycle is regulated in a feed-forward manner, such that when amino acid degradation is occurring, the rate of the cycle is increased.

Amino acids Nitrogen COO– CH2 H

C

Carbon

Storage CO2,H2O

NH4+ CO2

Energy

NH2

COO– Aspartate

Urea cycle

O H2N

C

NH2

Urea

FIG. 38.1. Fate of amino acid carbons and nitrogen. Amino acid carbon can be used either for energy storage (glycogen, fatty acids) or for energy. Amino acid nitrogen is used for urea synthesis. One nitrogen of urea comes from NH4⫹, the other from aspartate.

707

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A

Dietary protein

Amino acids Proteins Essential nitrogencontaining compounds

Gut

Proteins Glucose

CO2

Cells

Excess amino acids

Fed

Glycogen

Triacylglycerols

Liver Blood

VLDL

B Kidney NH3

Fasting

NH4 +

Urine

Alanine

Muscle Protein

Alanine Glutamine

Gut

Amino acids TCA

Liver Urea

Urine

Alanine Glucose Other amino acids

Ketone bodies

Muscle, Kidney

Brain, RBCs CO2 + H2O

Lactate

FIG. 38.2. Roles of various tissues in amino acid metabolism. A. In the fed state, amino acids released by digestion of dietary proteins travel through the hepatic portal vein to the liver, where they are used for the synthesis of proteins, particularly the blood proteins, such as serum albumin. The carbon skeletons of excess amino acids are converted to glucose or to triacylglycerols. The latter are then packaged and secreted in very low-density lipoproteins (VLDLs). The glucose produced from amino acids in the fed state is stored as glycogen or released into the blood if blood glucose levels are low. Amino acids that pass through the liver are converted to proteins in cells of other tissues. B. During fasting, amino acids are released from muscle protein. Some enter the blood directly. Others are partially oxidized and the nitrogen is stored in the form of alanine and glutamine, which enter the blood. In the kidney, glutamine releases ammonia into the urine and is converted to alanine and serine. In the cells of the gut, glutamine is converted to alanine. Alanine (the major gluconeogenic amino acid) and other amino acids enter the liver, where their nitrogen is converted to urea, which is excreted in the urine, and their carbons are converted to glucose and ketone bodies, which are oxidized by various tissues for energy. RBCs, red blood cells; TCA, tricarboxylic acid.

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CHAPTER 38 ■ FATE OF AMINO ACID NITROGEN: UREA CYCLE

A

␣-Keto acid1

Amino acid1

Percy Veere and his high school friend decided to take a Caribbean cruise, during which they sampled the cuisine of many of the islands on their itinerary. One month after their return to the United States, Percy complained of severe malaise, loss of appetite, nausea, vomiting, and arthralgias (joint pains). He had a low-grade fever and noted a persistent and increasing pain in the area of his liver. His friend noted a yellow discoloration of the whites of Percy’s eyes and skin. Percy’s urine turned the color of iced tea, and his stool became a light-clay color. His doctor found his liver to be enlarged and tender. Liver function tests were ordered. Serologic testing for viral hepatitis types B, C, and D were nonreactive, but fecal studies showed “shedding” of hepatitis virus type A. Tests for antibodies to antigens of the hepatitis A virus (anti-HAV) in the serum were positive for the immunoglobulin M type. A diagnosis of acute viral hepatitis type A was made, probably contracted from virus-contaminated food Percy had eaten while on his cruise. His physician explained that there was no specific treatment for type A viral hepatitis but recommended symptomatic and supportive care and prevention of transmission to others by the fecal–oral route. Percy took acetaminophen three to four times a day for fever and arthralgias throughout his illness.

I. FATE OF AMINO ACID NITROGEN A. Transamination Reactions Transamination is the major process for removing nitrogen from amino acids. In most instances, the nitrogen is transferred as an amino group from the original amino acid to ␣-ketoglutarate, forming glutamate, whereas the original amino acid is converted to its corresponding ␣-keto acid (Fig. 38.3). For example, the amino acid aspartate can be transaminated to form its corresponding ␣-keto acid, oxaloacetate. In the process, the amino group is transferred to ␣-ketoglutarate, which is converted to its corresponding amino acid, glutamate. All amino acids except lysine and threonine have the ability to undergo transamination reactions. The enzymes that catalyze these reactions are known as transaminases or aminotransferases. For most of these reactions, ␣-ketoglutarate and glutamate serve as one of the ␣-keto acid–amino acid pairs. Pyridoxal phosphate is the required cofactor for these reactions. Pyridoxal phosphate is derived from vitamin B6 (pyridoxine), as shown in Figure 38.4. Overall, in a transamination reaction, an amino group from one amino acid becomes the amino group of a second amino acid. Because these reactions are readily reversible, they can be used to remove nitrogen from amino acids or to transfer nitrogen to ␣-keto acids to form amino acids. Thus, they are involved both in amino acid degradation and in amino acid synthesis.

PLP

␣-Keto acid2

THE WAITING ROOM B

COO +

H3N

C

Amino acid2

–

COO

H

C

CH2

–

O

CH2 –

COO–

COO Aspartate

Oxaloacetate PLP

COO– C

O

CH2

COO– +

H 3N

C

H

CH2

CH2

CH2 –

COO

␣-Ketoglutarate

COO– Glutamate

FIG. 38.3. Transamination. The amino group from one amino acid is transferred to another. Pairs of amino acids and their corresponding ␣-keto acids are involved in these reactions. ␣-Ketoglutarate and glutamate are usually one of the pairs. The reactions, which are readily reversible, use pyridoxal phosphate (PLP) as a cofactor. The enzymes are called transaminases or aminotransferases. A. A generalized reaction. B. The aspartate transaminase reaction.

B. Removal of Amino Acid Nitrogen as Ammonia Cells in the body and bacteria in the gut release the nitrogen of certain amino acids as ammonia or ammonium ion (NH4⫹) (Fig. 38.5). Because these two forms of nitrogen can be interconverted, the terms are sometimes used interchangeably. The ammonium ion releases a proton to form ammonia by a reaction with a pKa of 9.3 (Fig. 38.6). Therefore, at physiologic pH, the equilibrium favors NH4⫹ by a factor of approximately 100/1 (see Chapter 4, the Henderson–Hasselbalch equation).

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SECTION VII ■ NITROGEN METABOLISM

CH2OH CH2OH

HO CH3

N H+ Pyridoxine (vitamin B6)

Brain Aspartate Purine nucleotide cycle

Muscle Fumarate

Aspartate Purine nucleotide cycle Fumarate

NAD+ NADH + H+

GDH

CHO CH2OH

HO

␣-Ketoglutarate

Glutamate

NAD+ (NADP+)

PLP

Serine

Pyruvate

NADH (NADPH) Threonine

CH3

N H+ Pyridoxaldehyde ATP

NH+4 Glutamine

Glutamate

Asparagine

Aspartate

PLP

␣-Ketobutyrate Histidine

Urocanate

ADP Urea

CHO CH2 P

HO CH3

Amino acids

N H+

Bacteria Various products

Gut

Pyridoxal phosphate (PLP)

FIG. 38.4. Activation of vitamin B6 to pyridoxal phosphate. The two steps are an oxidation and a phosphorylation.

FIG. 38.5. Summary of the sources of NH4⫹ for the urea cycle. All of the reactions are irreversible except that of glutamate dehydrogenase (GDH). Only the dehydratase reactions, which produce NH4⫹ from serine and threonine, require pyridoxal phosphate as a cofactor. The reactions that are not shown occurring in the muscle or the gut can all occur in the liver, where the NH4⫹ generated can be converted to urea. The purine nucleotide cycle of the brain and muscle is described further in Chapter 41. PLP, pyridoxal phosphate.

However, it is important to note that NH3 is also present in the body because this is the form that can cross cell membranes. For example, NH3 passes into the urine from kidney tubule cells and decreases the acidity of the urine by binding protons, forming NH4⫹. Once the NH4⫹ is formed, the compound can no longer freely diffuse across membranes. Glutamate can be oxidatively deaminated by a reaction catalyzed by glutamate dehydrogenase that produces ammonium ion and ␣-ketoglutarate (Fig. 38.7). Either

COO– +

H3N HOH + NH4

+

NH3 + H+ pKa = 9.3

FIG. 38.6. Formation of ammonia from an ammonium ion. Because the pKa is 9.3, the concentration of NH4⫹ at physiologic pH is almost 100 times that of NH3.

Lieberman_CH38.indd 710

C

H

–

COO NAD(P)+

NAD(P)H

CH2

O

CH2 NH+4

CH2 COO

C

–

Glutamate

Glutamate dehydrogenase

+

H+

CH2 COO–

␣-Ketoglutarate

FIG. 38.7. Reaction catalyzed by glutamate dehydrogenase. This reaction is readily reversible and can use either NAD⫹ or NADP⫹ as a cofactor. The oxygen on ␣-ketoglutarate is derived from H2O.

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CHAPTER 38 ■ FATE OF AMINO ACID NITROGEN: UREA CYCLE

NAD⫹ or NADP⫹ can serve as the cofactor. This reaction, which occurs in the mitochondria of most cells, is readily reversible; it can incorporate ammonia into glutamate or release ammonia from glutamate. Glutamate can collect nitrogen from other amino acids as a consequence of transamination reactions and then release ammonia through the glutamate dehydrogenase reaction. This process provides one source of the ammonia that enters the urea cycle. Glutamate dehydrogenase is one of three mammalian enzymes that can “fix” ammonia into organic molecules. The other two are glutamine synthetase and carbamoyl phosphate synthetase I (CPSI). In addition to glutamate, several amino acids release their nitrogen as NH4⫹ (see Fig. 38.5). Histidine may be directly deaminated to form NH4⫹ and urocanate. The deaminations of serine and threonine are dehydration reactions that require pyridoxal phosphate and are catalyzed by serine dehydratase. Serine forms pyruvate, and threonine forms ␣-ketobutyrate. In both cases, NH4⫹ is released. Glutamine and asparagine contain R-group amides that may be released as NH4⫹ by deamidation. Asparagine is deamidated by asparaginase, yielding aspartate and NH4⫹. Glutaminase acts on glutamine, forming glutamate and NH4⫹. The glutaminase reaction is particularly important in the kidney, where the ammonium ion produced is excreted directly into the urine, where it forms salts with metabolic acids, facilitating their removal in the urine. In muscle and brain, but not in liver, the purine nucleotide cycle allows NH4⫹ to be released from amino acids (see Fig. 38.5). Nitrogen is collected by glutamate from other amino acids by means of transamination reactions. Glutamate then transfers its amino group to oxaloacetate to form aspartate, which supplies nitrogen to the purine nucleotide cycle (see Chapter 41). The reactions of the cycle release fumarate and NH4⫹. The ammonium ion formed can leave the muscle in the form of glutamine. In summary, NH4⫹ that enters the urea cycle is produced in the body by deamination or deamidation of amino acids (see Fig. 38.5). A significant amount of NH4⫹ is also produced by bacteria that live in the lumen of the intestinal tract. This ammonium ion enters the hepatic portal vein and travels to the liver.

711

Percy Veere’s laboratory studies showed that his serum alanine transaminase (ALT) level was 675 U/L (reference range, 5 to 30 U), and his serum aspartate transaminase (AST) level was 601 U/L (reference range, 10 to 30 U). His serum alkaline phosphatase level was 284 U/L (reference range for an adult male, 40 to 125 U), and his serum total bilirubin was 9.6 mg/dL (reference range, 0.2 to 1.0 mg/dL). Bilirubin is a degradation product of heme, as described in Chapter 44. Cellular enzymes such as AST, ALT, and alkaline phosphatase leak into the blood through the membranes of hepatic cells that have been damaged as a result of the inflammatory process. In acute viral hepatitis, the serum ALT level is often elevated to a greater extent than the serum AST level. Alkaline phosphatase, which is present on membranes between liver cells and the bile duct, is also elevated in the blood in acute viral hepatitis. The rise in serum total bilirubin occurs as a result of the inability of the infected liver to conjugate bilirubin and of a partial or complete occlusion of the hepatic biliary drainage ducts caused by inflammatory swelling within the liver. In fulminant hepatic failure, the serum bilirubin level may exceed 20 mg/dL, a poor prognostic sign.

C. Role of Glutamate in the Metabolism of Amino Acid Nitrogen Glutamate plays a pivotal role in the metabolism of amino acids. It is involved in both synthesis and degradation. Glutamate provides nitrogen for amino acid synthesis (Fig. 38.8). In this process, glutamate obtains its nitrogen either from other amino acids by transamination ␣-Ketoglutarate Transamination PLP

GDH

Glutamate

␣-Keto acids PLP Amino acids

Vitamin B6 deficiency symptoms include dermatitis, a microcytic, hypochromic anemia, weakness, irritability, and, in some cases, convulsions. Xanthurenic acid (a degradation product of tryptophan) and other compounds appear in the urine because of an inability to metabolize amino acids completely. A decreased ability to synthesize heme from glycine may cause the microcytic anemia (see Chapter 44), and decreased decarboxylation of amino acids to form neurotransmitters (see Chapter 48) may explain the convulsions. Although vitamin B6 is required for a large number of reactions involved in amino acid metabolism, it is also required for the glycogen phosphorylase reaction (see Chapter 28).

␣-Ketoglutarate FIG. 38.8. Role of glutamate in amino acid synthesis. Glutamate transfers nitrogen by means of transamination reactions to ␣-keto acids to form amino acids. This nitrogen is either obtained by glutamate from transamination of other amino acids or from NH4⫹ by means of the glutamate dehydrogenase (GDH) reaction. PLP, pyridoxal phosphate, the active form of vitamin B6 (pyridoxine).

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SECTION VII ■ NITROGEN METABOLISM

Amino acids

␣-Ketoglutarate

Transamination

␣-Keto acids

GDH

Glutamate

Transamination

␣-Ketoglutarate

Oxaloacetate

Other reactions

NH4+ Urea Urea cycle

Aspartate

FIG. 38.9. Role of glutamate in urea production. Glutamate collects nitrogen from other amino acids by transamination reactions. This nitrogen can be released as NH4⫹ by glutamate dehydrogenase (GDH). NH4⫹ is also produced by other reactions (see Fig. 38.5). NH4⫹ provides one of the nitrogens for urea synthesis. The other nitrogen comes from aspartate, which is obtained from glutamate by transamination of oxaloacetate.

reactions or from NH4⫹ by the glutamate dehydrogenase reaction. Transamination reactions then serve to transfer amino groups from glutamate to ␣-keto acids to produce their corresponding amino acids. When amino acids are degraded and urea is formed, glutamate collects nitrogen from other amino acids by transamination reactions. Some of this nitrogen is released as ammonia by the glutamate dehydrogenase reaction, but much larger amounts of ammonia are produced from the other sources shown in Figure 38.5. NH4⫹ is one of the two forms in which nitrogen enters the urea cycle (Fig. 38.9). The second form of nitrogen for urea synthesis is provided by aspartate (see Fig. 38.9). Glutamate can be the source of the nitrogen. Glutamate transfers its amino group to oxaloacetate, and aspartate and ␣-ketoglutarate are formed.

D. Role of Alanine and Glutamine in Transporting Amino Acid Nitrogen to the Liver Protein turnover and amino acid degradation occur in all tissues; however, the ureacycle enzymes are active primarily in the liver (the intestine expresses low levels of activity of these enzymes; see Chapter 42). Thus, a mechanism needs to be in place to transport amino acid nitrogen to the liver. Alanine and glutamine are the major carriers of nitrogen in the blood. Alanine is exported primarily by muscle. Because muscle is metabolizing glucose through glycolysis, pyruvate is available in the muscle. The pyruvate is transaminated by glutamate to form alanine, which travels to the liver (Fig. 38.10). The glutamate is formed by transamination of an amino

Muscle Amino acid1

␣-KG

␣-Keto acid1 Glutamate Glucose

Glycolysis

Liver

Alanine Alanine Pyruvate

Carbon

Nitrogen

Glucose

Urea Urine

FIG. 38.10. The glucose/alanine cycle. Within the muscle, amino acid degradation leads to the transfer of nitrogens to ␣-ketoglutarate (␣-KG) and pyruvate. The alanine formed travels to the liver, where the carbons of alanine are used for gluconeogenesis and the alanine nitrogen is used for urea biosynthesis. This could occur during exercise when the muscle uses blood-borne glucose (see Chapter 47).

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Muscle, peripheral tissues NH4+

␣-KG

Glutamate

NADPH NADP+

Liver

Glutamine

NH4+

NH4+

Glutamine

Glutamate ATP

713

Urea

ADP, Pi

+

NH4

␣-KG

Urine

FIG. 38.11. Synthesis of glutamine in peripheral tissues and its transport to the liver. Within the liver, glutaminase converts glutamine to glutamate. Note how ␣-ketoglutarate can accept two molecules of ammonia to form glutamine. ␣-KG, ␣-ketoglutarate; GDH, glutamate dehydrogenase; Pi, inorganic phosphate.

acid that is being degraded. Upon arriving at the liver, alanine is transaminated to pyruvate, and the nitrogen is used for urea synthesis. The pyruvate formed is used for gluconeogenesis, and the glucose is exported to the muscle for use as energy. This cycle of moving carbons and nitrogen between the muscle and liver is known as the glucose/alanine cycle. Glutamine is synthesized from glutamate by the fixation of ammonia, requiring energy (adenosine triphosphate [ATP]) and the enzyme glutamine synthetase (Fig. 38.11), which is a cytoplasmic enzyme found in all cells. In the liver, glutamine synthetase is located in cells surrounding the portal vein. Its major role is to convert any ammonia that has escaped from urea production into glutamine, so that free ammonia does not leave the liver and enters the circulation. Under conditions of rapid amino acid degradation within a tissue, so that ammonia levels increase, the glutamate that has been formed from transamination reactions accepts another nitrogen molecule to form glutamine. The glutamine travels to the liver, kidney, or intestines, where glutaminase (see Fig. 38.11) removes the amide nitrogen to form glutamate plus ammonia. In the kidney, the release of ammonia, and the formation of ammonium ion, serves to form salts with metabolic acids in the urine. In the intestine, the glutamine is used as a fuel (see Chapter 42). In the liver, the ammonia is used for urea biosynthesis.

II. UREA CYCLE The normal human adult is in nitrogen balance; that is, the amount of nitrogen ingested each day, mainly in the form of dietary protein, is equal to the amount of nitrogen excreted. The major nitrogenous excretory product is urea, which exits from the body in the urine. This innocuous compound, produced mainly in the liver by the urea cycle, serves as the disposal form of ammonia, which is toxic, particularly to the brain and central nervous system. Normally, little ammonia (or NH4⫹) is present in the blood. The concentration ranges between 30 and 60 ␮M. Ammonia is rapidly removed from the blood and converted to urea by the liver. Nitrogen travels in the blood mainly in amino acids, particularly alanine and glutamine. The urea cycle was proposed in 1932 by Hans Krebs and a medical student, Kurt Henseleit, based on their laboratory observations. It was originally called the Krebs–Henseleit cycle. Subsequently, Krebs used this concept of metabolic cycling to explain a second process that also bears his name, the Krebs (or tricarboxylic acid [TCA]) cycle.

A. Reactions of the Urea Cycle

Nitrogen enters the urea cycle as NH4⫹ and aspartate (Fig. 38.12). NH4⫹ forms carbamoyl phosphate, which reacts with ornithine to form citrulline. Ornithine is

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Percy Veere’s symptoms and laboratory abnormalities did not slowly subside over the next 6 weeks as they usually do in uncomplicated viral hepatitis A infections. Instead, his serum total bilirubin, ALT, AST, and alkaline phosphatase levels increased further. His vomiting became intractable, and his friend noted jerking motions of his arms (asterixis), facial grimacing, restlessness, slowed mentation, and slight disorientation. He was admitted to the hospital with a diagnosis of hepatic failure with incipient hepatic encephalopathy (brain dysfunction caused by accumulation of various toxins in the blood), a rare complication of acute type A viral hepatitis alone. The possibility of a superimposed acute hepatic toxicity caused by the use of acetaminophen was considered.

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714

SECTION VII ■ NITROGEN METABOLISM

Mitochondrion CO2 + H2O

Cytosol

Urine

HCO3– + NH4 +

NH2 Urea C

NH2

O NH2

2 ATP

2 ADP + Pi

Arginase

C O

P

H O–

C

H

C

Ornithine transcarbamoylase

C CH2

C

O CH2

NH

C

NH

CH2

NH2

C

COOH

H

Argininosuccinate synthetase

Citrulline

COOH H2N

C

ATP

COOH NH

CH CH2 COOH

CH2

3

NH2

COOH

Citrulline

NH

CH2

CH2 H

COOH Fumarate

NH O

CH2

CH2 C

Argininosuccinate lyase

NH2

CH2

H

CH

4

Ornithine

NH2

Pi

COOH HC

NH2

COOH

2

NH2

Arginine

CH2

COOH

C

COOH

CH2

NH2

Ornithine

O– Carbamoyl phosphate

H

CH2NH2

CH2

H2N

CH2 CH2

CH2

O

NH

NH

CH2

CH2NH2

1

O

C

H2O

5

Carbamoyl phosphate synthetase I (CPSI)

C

NH2

COOH Argininosuccinate

AMP + PPi

H

CH2 COOH Aspartate

FIG. 38.12.

Urea cycle. The steps of the cycle are numbered 1 through 5. Pi, inorganic phosphate.

the compound that both initiates and is regenerated by the cycle (similar to oxaloacetate in the TCA cycle). Aspartate reacts with citrulline, eventually donating its nitrogen for urea formation. Arginine is formed in two successive steps. Cleavage of arginine by arginase releases urea and regenerates ornithine. 1.

SYNTHESIS OF CARBAMOYL PHOSPHATE

In the first step of the urea cycle, NH4⫹, bicarbonate, and ATP react to form carbamoyl phosphate (see Fig. 38.12). The cleavage of two molecules of ATP is required to form the high-energy phosphate bond of carbamoyl phosphate. CPSI, the enzyme that catalyzes this first step of the urea cycle, is found mainly in mitochondria of the liver and intestine. The Roman numeral suggests that another carbamoyl phosphate synthetase exists, and indeed, CPSII, located in the cytosol, produces carbamoyl phosphate for pyrimidine biosynthesis, using nitrogen from glutamine (see Chapter 41).

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2.

PRODUCTION OF ARGININE BY THE UREA CYCLE

Carbamoyl phosphate reacts with ornithine to form citrulline (see Fig. 38.12). The high-energy phosphate bond of carbamoyl phosphate provides the energy required for this reaction, which occurs in mitochondria and is catalyzed by ornithine transcarbamoylase (OTC). The product citrulline is transported across the mitochondrial membranes in exchange for cytoplasmic ornithine and enters the cytosol. The carrier for this transport reaction catalyzes an electroneutral exchange of the two compounds. In the cytosol, citrulline reacts with aspartate, the second source of nitrogen for urea synthesis, to produce argininosuccinate (see Fig. 38.12). This reaction, catalyzed by argininosuccinate synthetase, is driven by the hydrolysis of ATP to adenosine monophosphate (AMP) and pyrophosphate. Aspartate is produced by transamination of oxaloacetate. Argininosuccinate is cleaved by argininosuccinate lyase to form fumarate and arginine (see Fig. 38.12). Fumarate is produced from the carbons of argininosuccinate provided by aspartate. Fumarate is converted to malate (using cytoplasmic fumarase), which is used either for the synthesis of glucose by the gluconeogenic pathway or for the regeneration of oxaloacetate by cytoplasmic reactions similar to those observed in the TCA cycle (Fig. 38.13). The oxaloacetate that is formed is transaminated to generate the aspartate that carries nitrogen into the urea cycle. Thus, the carbons of fumarate can be recycled to aspartate. 3.

715

When ornithine transcarbamoylase (OTC) is deficient, the carbamoyl phosphate that normally would enter the urea cycle accumulates and floods the pathway for pyrimidine biosynthesis. Under these conditions, excess orotic acid (orotate), an intermediate in pyrimidine biosynthesis, is excreted in the urine. It produces no ill effects but is indicative of a problem in the urea cycle.

Carbamoyl phosphate Pathway when OTC is defective Orotate

Pyrimidines

Urine

CLEAVAGE OF ARGININE TO PRODUCE UREA

Arginine, which contains nitrogens derived from NH4⫹ and aspartate, is cleaved by arginase, producing urea and regenerating ornithine (see Fig. 38.12). Urea is produced from the guanidinium group on the side chain of arginine. The portion of arginine originally derived from ornithine is reconverted to ornithine. The reactions by which citrulline is converted to arginine and arginine is cleaved to produce urea occur in the cytosol. Ornithine, the other product of the arginase reaction, is transported into the mitochondrion in exchange for citrulline, where it can react with carbamoyl phosphate, initiating another round of the cycle.

B. Origin of Ornithine Ornithine is an amino acid. However, it is not incorporated into proteins during the process of protein synthesis because no genetic codon exists for this amino

H2O

Fumarate

Arginine Urea

Malate

Ornithine

NAD+

Argininosuccinate

NADH

Carbamoyl phosphate

Oxaloacetate Glutamate

␣-Ketoglutarate

Aspartate

Citrulline

FIG. 38.13. The Krebs bi-cycle, indicating the common steps between the TCA and urea cycles. All reactions shown occur in the cytoplasm except for the synthesis of citrulline, which occurs in the mitochondria.

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SECTION VII ■ NITROGEN METABOLISM

NH3+

O

CH2

C

CH2

CH2 CH2

CH2 H

C

+

NH3

C

O

C

C O–

O

O

–

O–

O

␣-Ketoglutarate

Ornithine PLP

Ornithine aminotransferase

O C

H

O C

H

CH2

CH2

CH2

CH2

C

NH3+

H

C O

O–

C

NH3+

C O–

Glutamate semialdehyde

O

O–

Glutamate

FIG. 38.14. The ornithine aminotransferase reaction. This is a reversible reaction that depends on pyridoxal phosphate, which normally favors ornithine degradation. PLP, pyridoxal phosphate.

The precise pathogenesis of the central nervous system (CNS) signs and symptoms that accompany liver failure (hepatic encephalopathy) in patients such as Percy Veere is not completely understood. These changes are, however, attributable in part to toxic materials that are derived from the metabolism of nitrogenous substrates by bacteria in the gut that circulate to the liver in the portal vein. These materials “bypass” their normal metabolism by the liver cells, however, because the acute inflammatory process of viral hepatitis severely limits the ability of liver cells to degrade these compounds to harmless metabolites. As a result, these toxins are “shunted” into the hepatic veins unaltered and eventually reach the brain through the systemic circulation (“portal–systemic encephalopathy”).

acid. Although ornithine is normally regenerated by the urea cycle (as one of the products of the arginase reaction), ornithine also can be synthesized de novo if needed. The reaction is an unusual transamination reaction catalyzed by ornithine aminotransferase under specific conditions in the intestine (Fig. 38.14). The usual direction of this reaction is the formation of glutamate semialdehyde, which is the first step of the degradation pathway for ornithine.

C. Regulation of the Urea Cycle The human liver has a vast capacity to convert amino acid nitrogen to urea, thereby preventing toxic effects from ammonia, which would otherwise accumulate. In general, the urea cycle is regulated by substrate availability; the higher the rate of ammonia production, the higher is the rate of urea formation. Regulation by substrate availability is a general characteristic of disposal pathways, such as the urea cycle, which remove toxic compounds from the body. This is a type of “feed-forward” regulation, in contrast to the “feedback” regulation characteristic of pathways that produce functional end products. Two other types of regulation control the urea cycle: allosteric activation of CPSI by N-acetylglutamate (NAG) and induction/repression of the synthesis of urea-cycle enzymes. NAG is formed specifically to activate CPSI; it has no other known function in mammals. The synthesis of NAG from acetyl coenzyme A (acetyl-CoA) and glutamate is stimulated by arginine (Fig. 38.15). Thus, as arginine levels increase within the liver, two important reactions are stimulated. The first is the synthesis of NAG, which increases the rate at which carbamoyl phosphate is produced. The second is to produce more ornithine (via the arginase reaction), so that the cycle can operate more rapidly. The induction of urea-cycle enzymes occurs in response to conditions that require increased protein metabolism, such as a high-protein diet or prolonged fasting. In both of these physiologic states, as amino acid carbon is converted to glucose, amino acid nitrogen is converted to urea. The induction of the synthesis of urea-cycle enzymes under these conditions occurs even though the uninduced enzyme levels are far in excess of the capacity required. The ability of a high-protein diet to increase urea-cycle enzyme levels is another type of “feed-forward” regulation.

Arginine CO2 + NH4 + –

COO

(CH2)2 +

CH

NH3 –

–

COO

CH3

+

C

O

S

+

2 ATP

(CH2)2

O

CH

NH C –

COO

CoA

COO

Glutamate

Acetyl CoA

N-Acetylglutamate

+

CH3

Carbamoyl phosphate synthetase (CPSI)

2 ADP + Pi

O

O

H2N C

O P

O

–

O– Carbamoyl phosphate

FIG. 38.15. Activation of carbamoyl phosphate synthetase I (CPSI). Arginine stimulates the synthesis of N-acetylglutamate, which activates CPSI. Pi, inorganic phosphate.

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CHAPTER 38 ■ FATE OF AMINO ACID NITROGEN: UREA CYCLE

Nitrogen excretion (g/d)

16 Other nitrogenous products

14 12

Urea-N

10 8 6 4 2 0

IV Glucose (700 g/d)

IV Glucose (150 g/d)

12 h

3d

"Fed"

5–6 wk

Fasting

FIG. 38.16. Nitrogen excretion during fasting. Human subjects were initially given intravenous (IV) glucose as indicated, then fasted. Total nitrogen excretion was measured as well as the nitrogen in urea (brown area). (Based on Ruderman NB, Aoki TT, Cahill GF Jr. Gluconeogenesis and its disorders in man. In: Hanson RW, Mehlman MA, eds. Gluconeogenesis: Its Regulation in Mammalian Species. New York, NY: John Wiley & Sons; 1976:518.)

D. Function of the Urea Cycle during Fasting During fasting, the liver maintains blood glucose levels. Amino acids from muscle protein are a major carbon source for the production of glucose by the pathway of gluconeogenesis. As amino acid carbons are converted to glucose, the nitrogens are converted to urea. Thus, the urinary excretion of urea is high during fasting (Fig. 38.16). As fasting progresses, however, the brain begins to use ketone bodies, sparing blood glucose. Less muscle protein is cleaved to provide amino acids for gluconeogenesis, and decreased production of glucose from amino acids is accompanied by decreased production of urea (see Chapter 31). The major amino acid substrate for gluconeogenesis is alanine, which is synthesized in peripheral tissues to act as a nitrogen carrier (see Fig. 38.10). Glucagon release, which is expected during fasting, stimulates alanine transport into the liver by activating the transcription of transport systems for alanine. Two molecules of alanine are required to generate one molecule of glucose. The nitrogen from the two molecules of alanine is converted to one molecule of urea (Fig. 38.17).

COO H2N

C

␣-Ketoglutarate

–

H

NAD+

1

NH2

2

NADH

Glutamate

CH3 L-alanine

NH4+

C

O

NH2 Urea

Oxaloacetate Glucose

Pyruvate

␣-Ketoglutarate

3 Aspartate

FIG. 38.17. Conversion of alanine to glucose and urea. (1) Alanine, the key gluconeogenic amino acid, is transaminated to form pyruvate, which is converted to glucose. The nitrogen, now in glutamate, can be released as NH4⫹ (2) or transferred to oxaloacetate to form aspartate (3). NH4⫹ and aspartate enter the urea cycle, which produces urea. In summary, the carbons of alanine form glucose, and the nitrogens form urea. Two molecules of alanine are required to produce one molecule of glucose and one molecule of urea.

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717

NH4⫹ is one of the toxins that results from the degradation of urea or proteins by intestinal bacteria and is not metabolized by the infected liver. The subsequent elevation of ammonia concentrations in the fluid bathing the brain causes depletion of tricarboxylic acid (TCA) cycle intermediates and adenosine triphosphate (ATP) in the central nervous system. ␣-Ketoglutarate, a TCA cycle intermediate, combines with ammonia to form glutamate in a reaction catalyzed by glutamate dehydrogenase. Glutamate subsequently reacts with ammonia to form glutamine. The absolute level of ammonia and its metabolites, such as glutamine, in the blood or cerebrospinal fluid in patients with hepatic encephalopathy correlates only roughly with the presence or severity of the neurologic signs and symptoms. ␥-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the brain, is also produced in the gut lumen and is shunted into the systemic circulation in increased amounts in patients with hepatic failure. In addition, other compounds (such as aromatic amino acids, false neurotransmitters, and certain short-chain fatty acids) bypass liver metabolism and accumulate in the systemic circulation, adversely affecting central nervous system function. Their relative importance in the pathogenesis of hepatic encephalopathy remains to be determined.

In addition to producing urea, the reactions of the urea cycle also serve as the pathway for the biosynthesis of arginine. Therefore, this amino acid is not required in the diet of the adult; however, it is required in the diet for growth. Urea is not cleaved by human enzymes. However, bacteria, including those in the human digestive tract, can cleave urea to ammonia and CO2 using the enzyme urease. Urease is not produced by humans. To some extent, humans excrete urea into the gut and saliva. Intestinal bacteria convert urea to ammonia. This ammonia, as well as ammonia produced by other bacterial reactions in the gut, is absorbed into the hepatic portal vein. It is normally extracted by the liver and converted to urea. Approximately onefourth of the total urea released by the liver each day is recycled by bacteria.

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Blood ammonia levels can be determined in several ways. One is to use an ammonia-specific electrode. Another is to use enzyme-coupled reactions that result in either a color change or an absorbance change. One enzyme-coupled system takes advantage of glutamate dehydrogenase. The unknown sample is incubated with glutamate dehydrogenase, ␣-ketoglutarate, and NADPH. If ammonia is present, glutamate will be produced, as will NADP⫹. As NADPH is converted to NADP⫹, the absorbance of light at 340 nm will decrease, and because one NADP⫹ is formed per ammonia molecule used, the concentration of ammonia can be determined. Blood urea nitrogen (BUN) is a measurement for the urea content of the blood. The key to measuring BUN is to split urea into carbon dioxide and two ammonia molecules by the bacterial enzyme urease. The ammonia levels are then determined as described previously, or via a colorimetric assay based on pH indicator dyes. As ammonia is generated, it binds protons, forming ammonium ions, which raises the pH. The extent of the pH change will be proportional to the amount of ammonia generated. Once BUN is determined, the urea concentration can be determined (in milligrams per deciliter) by multiplying the nitrogen value by 2.14.

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E. Disorders of the Urea Cycle Disorders of the urea cycle are dangerous because of the accumulation of ammonia in the circulation. Ammonia is toxic to the nervous system, and its concentration in the body must be carefully controlled. Under normal conditions, free ammonia is rapidly fixed into either ␣-ketoglutarate (by glutamate dehydrogenase, to form glutamate) or glutamate (by glutamine synthetase, to form glutamine). The glutamine can be used by many tissues, including the liver; the glutamate donates nitrogens to pyruvate to form alanine, which travels to the liver. Within the liver, as the nitrogens are removed from their carriers, CPSI fixes the ammonia into carbamoyl phosphate to initiate the urea cycle. However, when a urea-cycle enzyme is defective, the cycle is interrupted, which leads to an accumulation of urea-cycle intermediates before the block. Because of the block in the urea cycle, glutamine levels increase in the circulation, and because ␣-ketoglutarate is no longer being regenerated by removal of nitrogen from glutamine, the ␣-ketoglutarate levels are too low to fix more free ammonia, leading to elevated ammonia levels in the blood. Therefore, defects in any urea-cycle enzyme lead to elevated glutamine and ammonia levels in the circulation. However, the extent of the elevation depends on which enzyme is defective; see Question 1 at the end of this chapter. Ammonia toxicity will lead to brain swelling due, in part, to an osmotic imbalance caused by high levels of both ammonia and glutamine in the astrocytes. As ammonia levels increase in the astrocytes, more glutamine is produced (via glutamine synthetase), which only exacerbates the osmotic imbalance. The ammonia levels inhibit glutaminase, leading to glutamine elevation. Additionally, high levels of glutamine alter the permeability of the mitochondrial membrane, leading to an opening of the mitochondrial permeability transition pore, which leads to cell death (see Chapter 21). Another toxic effect of ammonia is a lowering of glutamate levels (due to the high activity of the glutamine synthetase reaction). Glutamate is a neurotransmitter, and glutamatergic neurotransmission is impaired, causing brain dysfunction. As glutamate is one of the excitatory neurotransmitters, the absence of glutamate neurotransmission can result in lethargy and reduced nervous system activity. The most common urea-cycle defect is OTC deficiency, which is an X-linked disorder. This disorder occurs with a frequency of between 1 in 20,000 and 1 in 80,000 live births. The reason for the range of values is that there is a late-onset form of OTC deficiency that may be underrepresented in the data used to determine the frequency of the disorder in the population. The major clinical problem in treating patients with urea-cycle defects is reducing the effects of excessive blood ammonia on the nervous system. High ammonia levels can lead to irreversible neuronal damage and mental retardation. So how is hyperammonemia treated? The key to treating patients with urea-cycle defects is to diagnose the disease early and then treat aggressively with compounds that can aid in nitrogen removal. Lowprotein diets are essential to reduce the potential for excessive amino acid degradation. If the enzyme defect in the urea cycle comes after the synthesis of argininosuccinate, massive arginine supplementation has proved beneficial. Once argininosuccinate has been synthesized, the two nitrogen molecules destined for excretion have been incorporated into the substrate; the problem is that ornithine cannot be regenerated. If ornithine could be replenished to allow the cycle to continue, argininosuccinate could be used as the carrier for nitrogen excretion from the body. Thus, ingesting large levels of arginine leads to ornithine production by the arginase reaction, and nitrogen excretion via argininosuccinate in the urine can be enhanced. Arginine therapy will not work for enzyme defects that exist in steps before the synthesis of argininosuccinate. For these disorders, drugs are used that form conjugates with amino acids. The conjugated amino acids are excreted, and the body then has to use its nitrogen to resynthesize the excreted amino acid. The two compounds most frequently used are benzoic acid and phenylbutyrate. (The active component

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719

−SCoA

A

ATP AMP + PPi (activation)

C O−

O

C

O

SCoA O H

−O

C

C H (Glycine) NH3+ −SCoA

C O

C

SCoA

O

NH

Hippuric acid (excreted)

CH2 C O

O−

O

B

CH2CH2CH2

C

O CH2

O−

C

O−

␤-Oxidation Phenylbutyrate O CH2

C

SCoA

Phenylacetate CoAS− ATP AMP + PPi

O C

NH2

CH2 CH2 H

C

NH3+

HSCoA

C O

O−

O

(Glutamine) CH2

C

O H NH C

CH2CH2

C

NH2

C O

O−

Phenylacetylglutamine (excreted)

FIG. 38.18. The metabolism of benzoic acid (A) and phenylbutyrate (B), two agents used to reduce nitrogen levels in patients with urea-cycle defects.

of phenylbutyrate is phenylacetate, its oxidation product. Phenylacetate has a bad odor, which makes it difficult to take orally.) As indicated in Figure 38.18A, benzoic acid, after activation, reacts with glycine to form hippuric acid, which is excreted. As glycine is synthesized from serine, the body now uses nitrogens to synthesize serine, so more glycine can be produced. Phenylacetate (see Fig. 38.18B) forms a conjugate with glutamine, which is excreted. This conjugate removes two nitrogens per molecule and requires the body to resynthesize glutamine from glucose, thereby using another two nitrogen molecules. Urea-cycle defects are excellent candidates for treatment by gene therapy. This is because the defect has to be repaired in only one cell type (in this case, the

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SECTION VII ■ NITROGEN METABOLISM

hepatocyte), which makes it easier to target the vector carrying the replacement gene. Preliminary gene therapy experiments had been carried out on individuals with OTC deficiency (the most common inherited defect of the urea cycle), but the experiments came to a halt when one of the patients died of a severe immunologic reaction to the vector used to deliver the gene. This incident has placed gene replacement therapy in the United States “on hold” for the foreseeable future. CLINICAL COMMENTS Percy Veere. The two most serious complications of acute viral hepatitis found in patients such as Percy Veere are massive hepatic necrosis leading to fulminant liver failure and the eventual development of chronic hepatitis. Both complications are rare in acute viral hepatitis type A, however, suggesting that acetaminophen toxicity may have contributed to Percy’s otherwise unexpectedly severe hepatocellular dysfunction and early hepatic encephalopathy. Fortunately, bed rest, rehydration, parenteral nutrition, and therapy directed at decreasing the production of toxins that result from bacterial degradation of nitrogenous substrates in the gut lumen (e.g., administration of lactulose, which reduces gut ammonia levels by a variety of mechanisms; the use of enemas and certain antibiotics, such as rifaximin, to decrease the intestinal flora, a low-protein diet) prevented Percy from progressing to the later stages of hepatic encephalopathy. As with most patients who survive an episode of fulminant hepatic failure, recovery to his previous state of health occurred over the next 3 months. Percy’s liver function studies returned to normal, and a follow-up liver biopsy showed no histologic abnormalities. BIOCHEMICAL COMMENTS Pyridoxal Phosphate. Pyridoxal phosphate is a key coenzyme for amino acid metabolism. This cofactor participates in a wide variety of reactions involving amino acids, such as transamination, deamination, decarboxylation, ␤-elimination, racemization, and ␥-elimination. The type of reaction that takes place is dictated by the amino acid substrate and the enzymes that catalyze the reaction. All amino acid reactions that require pyridoxal phosphate occur with the amino group of the amino acid bonded covalently to the aldehyde carbon of the coenzyme (Fig. 38.19). As an example of a mechanism through which pyridoxal

Decarboxylation

Racemization H H

H

H

C C

C

␥-Elimination

Y X

N

␤-Elimination O P O–

O–

Amino acid

Transamination

O –

O C

C O

H C H N

H OH

Pyridoxal phosphate

CH3

FIG. 38.19. Pyridoxal phosphate attached covalently to an amino acid substrate. The arrows indicate which bonds are broken for the various types of reactions in which pyridoxal phosphate is involved. X and Y represent chemical leaving groups that may be present on the amino acid (such as the hydroxyl group on serine or threonine).

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R2 H

C

R1 COO

–

H2N

H Enzyme-(lys)

Amino acid2 Enzyme-(lys)

O P

NH2

R2 C

N

C

COO–

H2N C H O–

H2C

4 H

Amino acid1 Enzyme – (lys)

+

N

CH3

–

R1 H

Enzyme-bound pyridoxal phosphate

N

O P

+

H2C O P

CH3

H

OH +

H

R1 COO

–

C

N

O P

–

CH OH

N

COO

N

CH H2C

CH3

N

R2 C

COO–

HC OH

N

C N

HC H2C

NH2

1

H COO

721

H2C NH2

CH3

CH2

3

H

O P

O P

R2 C

COO

–

O

␣-Keto acid2

2

O–

H2C

H2O

+

N

CH3

H Pyridoxamine phosphate

OH N

CH3

H H2O

R1 C

COO–

O

␣-Keto acid1

FIG. 38.20. Function of pyridoxal phosphate (PLP) in transamination reactions. The order in which the reactions occur is 1 to 4. Pyridoxal phosphate (enzyme-bound) reacts with amino acid1, forming a Schiff base (a carbon–nitrogen double bond). After a shift of the double bond, ␣-keto acid1 is released through hydrolysis of the Schiff base, and pyridoxamine phosphate is produced. Pyridoxamine phosphate then forms a Schiff base with ␣-keto acid2. After the double bond shifts, amino acid2 is released through hydrolysis of the Schiff base and enzyme-bound pyridoxal phosphate is regenerated. The net result is that the amino group from amino acid1 is transferred to ␣-keto acid2.

phosphate aids catalysis, Figure 38.20 indicates how a transamination reaction occurs. The other reaction types that require pyridoxal phosphate follow similar mechanisms. Key Concepts • • • • • •

Amino acid catabolism generates urea, which is a nontoxic carrier of nitrogen atoms. Urea synthesis occurs in the liver. The amino acids alanine and glutamine carry amino acid nitrogen from peripheral tissues to the liver. Key enzymes involved in nitrogen disposal are transaminases, glutamate dehydrogenase, and glutaminase. The urea cycle consists of four steps and incorporates a nitrogen from ammonia and one from aspartate into urea. Disorders of the urea cycle lead to hyperammonemia, a condition that is toxic to nervous system health and development. Diseases discussed in this chapter are summarized in Table 38.1.

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Table 38.1

Diseases Discussed in Chapter 38

Disease or Disorder

Genetic or Environmental

Viral hepatitis Pyridoxamine deficiency

Environmental Environmental

Hepatic encephalopathy

Environmental

Ammonia toxicity

Both

Ornithine transcarbamoylase deficiency CPSI deficiency, argininosuccinate synthetase deficiency, argininosuccinate lyase deficiency, and arginase deficiency

Genetic All genetic

Comments Infection of the liver by viral hepatitis may lead to liver failure. The lack of vitamin B6 affects many systems, such as heme synthesis, glycogen phosphorylase activity, and neurotransmitter synthesis, leading to possibly dementia, dermatitis, anemia, weakness, and convulsions. Liver failure leading to brain dysfunction due to the liver’s inability to rid the body of toxins, including ammonia Ammonia accumulation interferes with energy production and neurotransmitter synthesis in the brain, altering brain function. Most common urea-cycle defect, leading to elevated blood ammonia and orotic acid levels, and will lead to mental impairment if not treated. Mutations in urea cycle enzymes, leading to various degrees of hyperammonemia and inability to synthesize urea. Can be distinguished by the type of urea cycle intermediates that accumulate in the blood.

CPSI, carbamoyl phosphate synthetase I.

REVIEW QUESTIONS—CHAPTER 38 1.

Deficiency diseases have been described that involve each of the five enzymes of the urea cycle. Clinical manifestations may appear in the neonatal period. Infants with defects in the first four enzymes usually appear normal at birth, but after 24 hours, progressively develop lethargy, hypothermia, and apnea. They have high blood ammonia levels, and the brain becomes swollen. One possible explanation for the swelling is the osmotic effect of the accumulation of glutamine in the brain produced by the reactions of ammonia with ␣-ketoglutarate and glutamate. Arginase deficiency is not as severe as deficiencies of the other urea-cycle enzymes. Given the following information about five newborn infants (identified as I through V) who appeared normal at birth but developed hyperammonemia after 24 hours, determine which urea-cycle enzyme might be defective in each case (for each infant, choose from the same five answers, lettered A through E). All infants had low levels of blood urea nitrogen (BUN). (Normal citrulline levels are 10 to 20 ␮M.) Urine Orotate

Blood Citrulline

Blood Arginine

Blood NH3

I II III

Low — —

Low High (⬎1,000 ␮M) —

Low Low High

IV V

High —

Low High (200 ␮M)

Low Low

High High Moderately high High High

Infant

—, Value not determined; low, below normal; high, above normal.

A. B. C. D. E.

Carbamoyl phosphate synthetase I Ornithine transcarbamoylase Argininosuccinate synthetase Argininosuccinate lyase Arginase

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2.

The nitrogens in urea are derived directly from which of the following compounds? A. Ornithine and carbamoyl phosphate B. Ornithine and aspartate C. Ornithine and glutamate D. Carbamoyl phosphate and aspartate E. Carbamoyl phosphate and glutamine F. Aspartate and glutamine

3.

Which one of the following enzymes can fix ammonia into an organic molecule? A. Alanine–pyruvate aminotransferase B. Glutaminase C. Glutamate dehydrogenase D. Arginase E. Argininosuccinate synthetase

4.

Pyridoxal phosphate, which is required for transaminations, is also required for which of the following pathways? A. Glycolysis B. Gluconeogenesis C. Glycogenolysis D. The TCA cycle E. Fatty acid oxidation

5.

The major regulated step of the urea cycle is which one of the following? A. Carbamoyl phosphate synthetase I B. Ornithine transcarbamoylase C. Argininosuccinate synthetase D. Argininosuccinate lyase E. Arginase

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39

Synthesis and Degradation of Amino Acids

Because each of the 20 common amino acids has a unique structure, their metabolic pathways differ. Despite this, some generalities do apply to both the synthesis and degradation of all amino acids. These are summarized in the following sections. Because several amino acid pathways are clinically relevant, we present most of the diverse pathways that occur in humans. However, we will be as succinct as possible. Important Coenzymes. Pyridoxal phosphate (derived from vitamin B6) is the quintessential coenzyme of amino acid metabolism. In degradation, it is involved in the removal of amino groups, principally through transamination reactions and in donation of amino groups for various amino acid biosynthetic pathways. It is also required for certain reactions that involve the carbon skeleton of amino acids. Tetrahydrofolate (FH4) is a coenzyme that is used to transfer one-carbon groups at various oxidation states. FH4 is used in both amino acid degradation (e.g., serine and histidine) and biosynthesis (e.g., glycine). Tetrahydrobiopterin (BH4) is a cofactor that is required for ring hydroxylation reactions (e.g., phenylalanine to tyrosine). Synthesis of the Amino Acids. Eleven of the 20 common amino acids can be synthesized in the body (Fig. 39.1). The other nine are considered “essential” and must be obtained from the diet. Almost all of the amino acids that can be synthesized by humans are amino acids used for the synthesis of additional nitrogen-containing compounds. Examples include glycine, which is used for porphyrin and purine synthesis; glutamate, which is required for neurotransmitter and purine synthesis; and aspartate, which is required for both purine and pyrimidine biosynthesis. Nine of the 11 “nonessential” amino acids can be produced from glucose plus, of course, a source of nitrogen such as another amino acid or ammonia. The other two nonessential amino acids, tyrosine and cysteine, require an essential amino acid for their synthesis (phenylalanine for tyrosine, methionine for cysteine). The carbons for cysteine synthesis come from glucose; the methionine donates only the sulfur. The carbon skeletons of the 10 nonessential amino acids derived from glucose are produced from intermediates of glycolysis and the tricarboxylic acid (TCA) cycle (see Fig. 39.1). Four amino acids (serine, glycine, cysteine, and alanine) are produced from glucose through components of the glycolytic pathway. TCA cycle intermediates (which can be produced from glucose) provide carbon for synthesis of the six remaining nonessential amino acids. ␣-Ketoglutarate is the precursor for the synthesis of glutamate, glutamine, proline, and arginine. Oxaloacetate provides carbon for the synthesis of aspartate and asparagine. Regulation of the biosynthesis of individual amino acids can be quite complex, but the overriding feature is that the pathways are feedback-regulated such that as the concentration of free amino acid increases, a key biosynthetic enzyme is allosterically or transcriptionally inhibited. Amino acid levels, however, are always maintained at a level such that the aminoacyl-tRNA synthetases can remain active, and protein synthesis can continue.

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SECTION VII ■ NITROGEN METABOLISM

Glucose Glycine Methionine (S)

Phosphoglycerate Serine

Asparagine

Pyruvate

Glutamine

Aspartate

TA

Oxaloacetate

TA

Cysteine

Alanine

Acetyl CoA

Phenylalanine

Tyrosine

Citrate Glutamine

Isocitrate

␣-Ketoglutarate

TA

GDH

Glutamate

Glutamate semialdehyde Proline

Arginine

FIG. 39.1. Overview of the synthesis of the nonessential amino acids. The carbons of 10 amino acids may be produced from glucose through intermediates of glycolysis or the TCA cycle. The 11th nonessential amino acid, tyrosine, is synthesized by hydroxylation of the essential amino acid phenylalanine. Only the sulfur of cysteine comes from the essential amino acid methionine; its carbons and nitrogen come from serine. Transamination (TA) reactions involve pyridoxal phosphate (PLP) and another amino acid/␣-keto acid pair.

Degradation of Amino Acids. The degradation pathways for amino acids are, in general, distinct from biosynthetic pathways. This allows for separate regulation of the anabolic and catabolic pathways. Because protein is a fuel, almost every amino acid has a degradative pathway that can generate NADH, which is used as an electron source for oxidative phosphorylation. However, the energy-generating pathway may involve direct oxidation, oxidation in the TCA cycle, conversion to glucose and then oxidation, or conversion to ketone bodies, which are then oxidized. The fate of the carbons of the amino acids depends on the physiologic state of the individual and the tissue in which the degradation occurs. For example, in the liver during fasting, the carbon skeletons of the amino acids produce glucose, ketone bodies, and CO2. In the fed state, the liver can convert intermediates of amino acid metabolism to glycogen and triacylglycerols. Thus, the fate of the carbons of the amino acids parallels that of glucose and fatty acids. The liver is the only tissue that has all of the pathways of amino acid synthesis and degradation. As amino acids are degraded, their carbons are converted to (1) CO2, (2) compounds that produce glucose in the liver (pyruvate and the TCA cycle intermediates ␣-ketoglutarate, succinyl coenzyme A [succinyl-CoA], fumarate, and oxaloacetate), and (3) ketone bodies or their precursors (acetoacetate and acetyl-CoA) (Fig. 39.2). For simplicity, amino acids are considered to be glucogenic if their carbon skeletons can be converted to a precursor of glucose and ketogenic if their carbon skeletons can be converted directly to acetyl-CoA or acetoacetate. Some amino acids contain carbons that produce a glucose precursor and other carbons that produce acetyl-CoA or acetoacetate. These amino acids are both glucogenic and ketogenic. The amino acids that are synthesized from intermediates of glycolysis (serine, alanine, and cysteine) plus certain amino acids (threonine, glycine,

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725

A Tryptophan Threonine

Alanine Serine Cysteine

Glycine

Alanine

Blood

Pyruvate Acetyl CoA Arginine Histidine Glutamine Proline

Oxaloacetate

Muscle Gut Kidney

Aspartate Asparagine Glutamate

Glucose

Liver

TCA cycle

Malate

␣-Ketoglutarate

Fumarate

Succinyl CoA

Aspartate Tyrosine Phenylanine

Methylmalonyl CoA Valine Threonine Isoleucine Methionine

Propionyl CoA

B Leucine

Acetyl CoA + Acetoacetyl CoA

Threonine Lysine Isoleucine Tryptophan

HMG CoA Acetoacetate (ketone bodies)

Phenylalanine, Tyrosine

FIG. 39.2. Degradation of amino acids. A. Amino acids that produce pyruvate or intermediates of the TCA cycle. These amino acids are considered glucogenic because they can produce glucose in the liver. The fumarate group of amino acids produces cytoplasmic fumarate. Potential mechanisms whereby the cytoplasmic fumarate can be oxidized are presented in Section III.C.1. B. Amino acids that produce acetyl-CoA or ketone bodies. These amino acids are considered ketogenic.

and tryptophan) produce pyruvate when they are degraded. The amino acids synthesized from TCA cycle intermediates (aspartate, asparagine, glutamate, glutamine, proline, and arginine) are reconverted to these intermediates during degradation. Histidine is converted to glutamate and then to the TCA cycle intermediate ␣-ketoglutarate. Methionine, threonine, valine, and isoleucine form succinyl CoA, and phenylalanine (after conversion to tyrosine) forms fumarate. Because pyruvate and the TCA cycle intermediates can produce glucose in the liver, these amino acids are glucogenic. Some amino acids with carbons that produce glucose also contain other carbons that produce ketone bodies. Tryptophan, isoleucine, and threonine produce acetyl-CoA, and phenylalanine and tyrosine produce acetoacetate. These amino acids are both glucogenic and ketogenic. Two of the essential amino acids (lysine and leucine) are strictly ketogenic. They do not produce glucose, only acetoacetate and acetyl-CoA.

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THE WAITING ROOM All states have enacted legislation requiring that newborns be screened for various metabolic abnormalities, of which phenylketonuria (PKU) is one. A common screening procedure is the Guthrie bacterial inhibition assay. In this assay, spores of the organism Bacillus subtilis are plated on an agar plate containing ␤2-thienylalanine, an inhibitor of B. subtilis growth. The blood sample obtained from the infant (in the form of a dried blood spot on a filter disk) is placed on the agar plate. If the phenylalanine content of the blood is greater than 2 to 4 mg/dL, the phenylalanine will counteract the effects of the ␤2-thienylalanine, and bacterial growth will occur. An alternative assay is a microfluorometric determination of phenylalanine, via its incorporation into a ninhydrin–copper complex with the dipeptide L-leucyl-L-alanine. Positive results with either assay then require verification and actual measurement of phenylalanine levels, using either high-performance liquid chromatography (HPLC) separation of the components of the blood or enzymatic and fluorometric assays.

Piquet Yuria, a 4-month-old female infant, emigrated from the Soviet Union with her French mother and Russian father 1 month ago. She was normal at birth but in the last several weeks was less than normally attentive to her surroundings. Her psychomotor maturation seemed to be delayed, and a tremor of her extremities had recently appeared. When her mother found her having gross twitching movements in her crib, she brought the infant to the hospital emergency room. A pediatrician examined Piquet and immediately noted a musty odor from the baby’s wet diaper. A drop of her blood was obtained from a heel prick and used to perform a Guthrie bacterial inhibition assay using a special type of filter paper. This screening procedure was positive for an excess of phenylalanine in Piquet’s blood. Homer Sistine, a 14-year-old boy, had a sudden grand mal seizure (with jerking movements of the torso and head) in his eighth-grade classroom. The school physician noted mild weakness of the muscles of the left side of Homer’s face and of his left arm and leg. Homer was hospitalized with a tentative diagnosis of a cerebrovascular accident involving the right cerebral hemisphere, which presumably triggered the seizure. Homer’s past medical history was normal, except for slight mental retardation requiring placement in a special education group. He also had a downward partial dislocation of the lenses of both eyes, for which he had had a surgical procedure (a peripheral iridectomy). Homer’s left-sided neurologic deficits cleared spontaneously within 3 days, but a computerized axial tomogram (CAT) showed changes consistent with a small infarction (a damaged area caused by a temporary or permanent loss of adequate arterial blood flow) in the right cerebral hemisphere. A neurologist noted that Homer had a slight waddling gait, which his mother said began several years earlier and was progressing with time. Further studies confirmed the decreased mineralization (decreased calcification) of the skeleton (called osteopenia if mild and osteoporosis if more severe) and high methionine and homocysteine but low cystine levels in the blood. All of this information, plus the increased length of the long bones of Homer’s extremities and a slight curvature of his spine (scoliosis), caused his physician to suspect that Homer might have an inborn error of metabolism.

I.

THE ROLE OF COFACTORS IN AMINO ACID METABOLISM

Amino acid metabolism requires the participation of three important cofactors. Pyridoxal phosphate (PLP) is the quintessential coenzyme of amino acid metabolism (see Biochemical Comments in Chapter 38). The coenzyme tetrahydrofolate (FH4) is required in certain amino acid pathways to either accept or donate a one-carbon group. The carbon can be in various states of oxidation. Chapter 40 describes the reactions of FH4 in much more detail. The coenzyme tetrahydrobiopterin (BH4) is required for ring hydroxylations. The reactions involve molecular oxygen, and one atom of oxygen is incorporated into the product. The second is found in water (see Chapter 24). BH4 is important for the synthesis of tyrosine and neurotransmitters (see Chapter 48).

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II. AMINO ACIDS DERIVED FROM INTERMEDIATES OF GLYCOLYSIS Four amino acids are synthesized from intermediates of glycolysis: serine, glycine, cysteine, and alanine. Serine, which produces glycine and cysteine, is synthesized from 3-phosphoglycerate; and alanine is formed by transamination of pyruvate, the product of glycolysis (Fig. 39.3). When these amino acids are degraded, their carbon atoms are converted to pyruvate or to intermediates of the glycolytic/gluconeogenic pathway and, therefore, can produce glucose or be oxidized to CO2.

Glucose

Glycine

3-Phosphoglycerate

Serine

2-Phosphoglycerate Cysteine

A. Serine

Pyruvate

In the biosynthesis of serine from glucose, 3-phosphoglycerate is first oxidized to a 2-keto compound (3-phosphohydroxypyruvate), which is then transaminated to form phosphoserine (Fig. 39.4). Phosphoserine phosphatase removes the phosphate, forming serine. The major sites of serine synthesis are the liver and kidney. Serine can be used by many tissues and is generally degraded by transamination to hydroxypyruvate, followed by reduction and phosphorylation to form 2-phosphoglycerate, an intermediate of glycolysis that forms phosphoenolpyruvate (PEP) and, subsequently, pyruvate. Serine also can undergo ␤-elimination of its hydroxyl group, catalyzed by serine dehydratase, to form pyruvate directly.

Glucose

Glycolysis

P

CH2 O HO

C

Alanine

FIG. 39.3. Amino acids derived from intermediates of glycolysis. These amino acids can be synthesized from glucose. Their carbons can be reconverted to glucose in the liver.

CH2OH

H

COO

SO4 2–

O

P

–

C

H

COO

3-Phosphoglycerate

PEP

Pyruvate

–

2-Phosphoglycerate

NAD+

ADP 3-Phosphoglycerate dehydrogenase

NADH

P

CH2 O C

ATP COO

O

COO

H C

–

–

OH

CH2OH

3-Phosphohydroxypyruvate

Glycerate NAD+

Glutamate PLP

␣-Ketoglutarate

NADH P

CH2 O H

C

NH3

COO

CH2OH

+

C

–

O

COO

3-Phospho-L-serine Phosphoserine phosphatase

–

Hydroxypyruvate

–

PLP Alanine CH2

Pi H

C

OH

NH3

Pyruvate

+

COO– Serine

FIG. 39.4. The major pathway for serine synthesis from glucose is on the left, and for serine degradation on the right. Serine levels are maintained because serine causes repression ( ) of 3-phosphoglycerate dehydrogenase synthesis. Serine also inhibits (䊞) phosphoserine phosphatase. PEP, phosphoenolpyruvate; Pi, inorganic phosphate; PLP, pyridoxal phosphate.

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Regulatory mechanisms maintain serine levels in the body. When serine levels fall, serine synthesis is increased by induction of 3-phosphoglycerate dehydrogenase and by release of the feedback inhibition of phosphoserine phosphatase (caused by higher levels of serine). When serine levels rise, synthesis of serine decreases because synthesis of the dehydrogenase is repressed and the phosphatase is inhibited (see Fig. 39.4).

B. Glycine Glycine can be synthesized from serine and, to a minor extent, threonine. The major route from serine is by a reversible reaction that involves FH4 and PLP (Fig. 39.5). Tetrahydrofolate is a coenzyme that transfers one-carbon groups at different levels of oxidation. It is derived from the vitamin folate and is discussed in more detail in Chapter 40. The minor pathway for glycine production involves threonine degradation (this is an aldolase-like reaction because threonine contains a hydroxyl group located two carbons from the carbonyl group). The conversion of glycine to glyoxylate by the enzyme D-amino acid oxidase is a degradative pathway of glycine that is clinically relevant. Once glyoxylate is formed, it can be oxidized to oxalate, which is sparingly soluble and tends to precipitate in kidney tubules, leading to formation of kidney stones. Approximately 40% of oxalate formation in the liver comes from glycine metabolism. Dietary oxalate accumulation has been estimated to be a low contributor to excreted oxalate in the urine because of poor absorption of oxalate in the intestine. Although glyoxalate can be transaminated back to glycine, this is not really considered a biosynthetic route for “new” glycine because the primary route for glyoxylate formation is from glycine oxidation. Generation of energy from glycine occurs through a dehydrogenase (glycine cleavage enzyme) that oxidizes glycine to CO2, ammonia, and a carbon that is donated to FH4.

Oxalate, produced from glycine or obtained from the diet, forms precipitates with calcium. Kidney stones (renal calculi) are often composed of calcium oxalate. A lack of the transaminase that can convert glyoxylate to glycine (see Fig. 39.5) leads to the disease primary oxaluria type I (PH 1). This disease has a consequence of renal failure attributable to excessive accumulation of oxalate in the kidney.

CH3 H H

O

C

C

C

OH

NH4

+

O–

Threonine O PLP

CH3 C

Serine hydroxymethyl transferase

PLP

Serine FH4

H2C

N5,N10–CH2–FH4

FH4

NH3

+

NAD+

NH4

D-amino acid

COO–

+

H

C

COO Transaminase

Pyruvate

Alanine

O2

O

oxidase

COO– Glycine

H2O2

NADH Glycine

NH4

+ cleavage

enzyme

COO– Oxalate

–

TPP

Glyoxylate

–

COO C

N5,N10–CH2–FH4

CO2

H

O2

O

CO2 COO– H C OH

CH2

C

O

CH2

CH2

COO–

CH2

␣-Keto-

COO

glutarate

␣-Hydroxy␤-ketoadipate

CO2 + H2O

–

FIG. 39.5. Metabolism of glycine. Glycine can be synthesized from serine (major route) or threonine. Glycine forms serine or CO2 and NH4⫹ by reactions that require tetrahydrofolate (FH4). Glycine also forms glyoxylate, which is converted to oxalate or to CO2 and H2O. N 5, N10–CH2–FH4, N 5, N10-methylene tetrahydrofolate (see Chapter 40); TPP, thiamine pyrophosphate.

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729

Methionine SH CH2 CH2 −

OOC

CH

+

CH2OH

H C NH3

+

COO− Homocysteine

NH3

Serine

PLP cystathionine –

β-synthase

H2O −

OOC

CH

CH2

S

+

NH3

CH2 CH2

Succinyl CoA

+

H C NH3 COO−

L-Methylmalonyl

CoA

D-Methylmalonyl

CoA

Cystathionine H2O cystathionase

PLP

NH4+ −

OOC

α-Ketobutyrate

CH

CH2

Propionyl CoA

SH

+

NH3

Cysteine O2 –

OOC

CH

CH2

−

SO2

+

NH3

Cysteine sulfinic acid α-Ketoglutarate PLP Glutamate Pyruvate 2−

SO3 Sulfite 2−

SO4 ATP Sulfate

PAPS

Urine

FIG. 39.6. Synthesis and degradation of cysteine. Cysteine is synthesized from the carbons and nitrogen of serine and the sulfur of homocysteine (which is derived from methionine). During the degradation of cysteine, the sulfur is converted to sulfate and either excreted in the urine or converted to PAPS (universal sulfate donor; 3⬘-phosphoadenosine 5⬘-phosphosulfate), and the carbons are converted to pyruvate. PLP, pyridoxal phosphate.

C. Cysteine The carbons and nitrogen for cysteine synthesis are provided by serine, and the sulfur is provided by methionine (Fig. 39.6). Serine reacts with homocysteine (which is produced from methionine) to form cystathionine. This reaction is catalyzed by cystathionine ␤-synthase. Cleavage of cystathionine by cystathionase

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Cystathioninuria, the presence of cystathionine in the urine, is relatively common in premature infants. As they mature, cystathionase levels rise, and the levels of cystathionine in the urine decrease. In adults, a genetic deficiency of cystathionase causes cystathioninuria. Individuals with a genetically normal cystathionase can also develop cystathioninuria from a dietary deficiency of pyridoxine (vitamin B6) because cystathionase requires the cofactor pyridoxal phosphate (PLP). No characteristic clinical abnormalities have been observed in individuals with cystathionase deficiency, and it is probably a benign disorder.

Cystinuria and cystinosis are disorders involving two different transport proteins for cystine, the disulfide formed from two molecules of cysteine. Cystinuria is caused by a defect in the transport protein that carries cystine, lysine, arginine, and ornithine into intestinal epithelial cells and that permits resorption of these amino acids by renal tubular cells. Cystine, which is not very soluble in the urine, forms renal calculi (stones). Cal Kulis, a patient with cystinuria, developed cystine stones (see Chapter 37). Cystinosis is a rare disorder caused by a defective carrier that normally transports cystine across the lysosomal membrane from lysosomal vesicles to the cytosol. Cystine accumulates in the lysosomes in many tissues and forms crystals, leading to a depletion of intracellular cysteine levels. Children with this disorder develop renal failure by 6 to 12 years of age, through a mechanism that has not yet been fully elucidated.

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produces cysteine and ␣-ketobutyrate, which forms succinyl-CoA via propionylCoA. Both cystathionine ␤-synthase (␤-elimination) and cystathionase (␥-elimination) require PLP. Cysteine inhibits cystathionine ␤-synthase and, therefore, regulates its own production to adjust for the dietary supply of cysteine. Because cysteine derives its sulfur from the essential amino acid methionine, cysteine becomes essential if the supply of methionine is inadequate for cysteine synthesis. Conversely, an adequate dietary source of cysteine “spares” methionine; that is, it decreases the amount that must be degraded to produce cysteine. When cysteine is degraded, the nitrogen is converted to urea, the carbons to pyruvate, and the sulfur to sulfate, which has two potential fates (see Fig. 39.6; see also Chapter 43). Sulfate generation, in an aqueous medium, is essentially generating sulfuric acid, and both the acid and sulfate needs to be disposed of in the urine. Sulfate is also used in most cells to generate an activated form of sulfate known as PAPS (3⬘-phosphoadenosine 5⬘-phosphosulfate), which is used as a sulfate donor in modifying carbohydrates or amino acids in various structures (glycosaminoglycans) and proteins in the body. The conversion of methionine to homocysteine and homocysteine to cysteine is the major degradative route for these two amino acids. Because this is the only degradative route for homocysteine, vitamin B6 deficiency or congenital cystathionine ␤-synthase deficiency can result in homocystinemia, which has been reported to be associated with cardiovascular disease.

D. Alanine Alanine is produced from pyruvate by a transamination reaction catalyzed by alanine aminotransaminase (ALT) and may be converted back to pyruvate by a reversal of the same reaction (see Fig. 39.3). Alanine is the major gluconeogenic amino acid because it is produced in many tissues for the transport of nitrogen to the liver.

III. AMINO ACIDS RELATED TO TCA CYCLE INTERMEDIATES Two groups of amino acids are synthesized from TCA cycle intermediates; one group from ␣-ketoglutarate and one from oxaloacetate (see Fig. 39.1). During degradation, four groups of amino acids are converted to the TCA cycle intermediates: ␣-ketoglutarate, oxaloacetate, succinyl-CoA, and fumarate (see Fig. 39.2A).

A. Amino Acids Related through ␣-Ketoglutarate/Glutamate 1.

GLUTAMATE

The five carbons of glutamate are derived from ␣-ketoglutarate either by transamination or by the glutamate dehydrogenase reaction (see Chapter 38). Because ␣-ketoglutarate can be synthesized from glucose, all of the carbons of glutamate can be obtained from glucose (see Fig. 39.1). When glutamate is degraded, it is likewise converted back to ␣-ketoglutarate either by transamination or by glutamate dehydrogenase. In the liver, ␣-ketoglutarate leads to the formation of malate, which produces glucose via gluconeogenesis. Thus, glutamate can be derived from glucose and reconverted to glucose (Fig. 39.7). Glutamate is used for the synthesis of several other amino acids (glutamine, proline, ornithine, and arginine) (see Fig. 39.7) and for providing the glutamyl moiety of glutathione (␥-glutamyl-cysteinyl-glycine; see Biochemical Comments in Chapter 37). Glutathione is an important antioxidant, as has been described previously (see Chapter 24).

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Glucose

Histidine

␣-Ketoglutarate

Formiminoglutamate (FIGLU)

Homocysteine is oxidized to a disulfide—homocystine. To indicate that both forms are being considered, the term “homocyst(e)ine” is used. Homocysteine

Glutamine

–

–

COO

Glutamate +

Glutamate semialdehyde

H 3N

Ornithine

Proline

COO +

H3N

CH

CH

CH2

CH2

CH2

CH2

SH

S

SH

S

CH2

CH2

Urea Arginase (Liver)

CH2

Arginine

FIG. 39.7. Amino acids related through glutamate. These amino acids contain carbons that can be reconverted to glutamate, which can be converted to glucose in the liver. All of these amino acids except histidine can be synthesized from glucose.

2.

GLUTAMINE

Glutamine is produced from glutamate by glutamine synthetase, which adds NH4⫹ to the carboxyl group of the side chain, forming an amide (Fig. 39.8). This is one of only three human enzymes that can fix free ammonia into an organic molecule; the other two are glutamate dehydrogenase and carbamoyl phosphate synthetase I (see Chapter 38). Glutamine is reconverted to glutamate by

COO– CH2 CH2 H C NH3 + COO– Glutamate NH4+

NH4+

ATP

H

C

CH2 +

NH3

COO– Homocysteine

H

C

+

NH3

COO– Homocystine

Because a colorimetric screening test for urinary homocystine was positive, the doctor ordered several biochemical studies on Homer Sistine’s serum, which included tests for methionine, homocyst(e)ine (both free and protein-bound), cystine, vitamin B12, and folate. The level of homocystine in a 24-hour urine collection was also measured. The results were as follows: The serum methionine level was 980 ␮M (reference range, ⬍30 ␮M); serum homocyst(e)ine (both free and protein-bound) was markedly elevated; cystine was not detected in the serum; and the serum B12 and folate levels were normal. A 24-hour urine homocystine level was elevated. Based on these measurements, Homer Sistine’s doctor concluded that Homer had homocystinuria caused by an enzyme deficiency. What was the rationale for this conclusion?

Glutaminase

Glutamine synthetase

ADP + Pi

H2O NH2 C

O

CH2 CH2 H C NH3+ COO– Glutamine

FIG. 39.8. Synthesis and degradation of glutamine. Different enzymes catalyze the addition and the removal of the amide nitrogen of glutamine. Pi, inorganic phosphate.

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If the blood levels of methionine and homocysteine are very elevated and cystine is low, cystathionine ␤-synthase could be defective, but a cystathionase deficiency is also a possibility. With a deficiency of either of these enzymes, cysteine could not be synthesized, and levels of homocysteine would rise. Homocysteine would be converted to methionine by reactions that require vitamin B12 and tetrahydrofolate (FH4) (see Chapter 40). In addition, it would be oxidized to homocystine, which would appear in the urine. The levels of cysteine (measured as its oxidation product cystine) would be low. A measurement of serum cystathionine levels would help to distinguish between a cystathionase or cystathionine ␤-synthase deficiency. +

NH3 –

COO CH2 CH2 CH

COO–

a different enzyme, glutaminase, which is particularly important in the kidney. The ammonia it produces enters the urine and can be used as an expendable cation to aid in the excretion of metabolic acids (NH3 ⫹ H⫹ → NH4⫹), as discussed in Chapter 38. 3.

PROLINE

In the synthesis of proline, glutamate is first phosphorylated and then converted to glutamate 5-semialdehyde by reduction of the side-chain carboxyl group to an aldehyde (Fig. 39.9). This semialdehyde spontaneously cyclizes (forming an internal Schiff base between the aldehyde and the ␣-amino group). Reduction of this cyclic compound yields proline. Hydroxyproline is formed only after proline has been incorporated into collagen (see Chapter 49) by the prolyl hydroxylase system, which uses molecular oxygen, iron, ␣-ketoglutarate, and ascorbic acid (vitamin C). Proline is converted back to glutamate semialdehyde, which is oxidized to form glutamate. The synthesis and degradation of proline use different enzymes even though the intermediates are the same. Hydroxyproline, however, has an entirely different degradative pathway (not shown). The presence of the hydroxyl group in hydroxyproline allows an aldolase-like reaction to occur once the ring has been hydrolyzed, which is not possible with proline.

Glutamate ATP ADP + Pi

1

NADPH + H+

NADH + H+

4

NAD+

NADP+ +

NH3 – H C CH2 CH2 CH COO

O Glutamate semialdehyde Spontaneous cyclization CH2

H2C

–

+ CH COO N H ⌬1-Pyrroline 5-carboxylate

HC

NADPH + H+

2

3 FAD

HISTIDINE

Although histidine cannot be synthesized in humans, five of its carbons form glutamate when it is degraded. In a series of steps, histidine is converted to formiminoglutamate (FIGLU). The subsequent reactions transfer one carbon of FIGLU to the FH4 pool (see Chapter 40) and release NH4⫹ and glutamate (Fig. 39.10).

CH2

H2C H2C

ARGININE

Arginine is synthesized from glutamate via glutamate semialdehyde, which is transaminated to form ornithine, an intermediate of the urea cycle (see Fig. 38.14). This activity (ornithine aminotransferase) appears to be greatest in the epithelial cells of the small intestine (see Chapter 42). The reactions of the urea cycle then produce arginine. However, the quantities of arginine generated by the urea cycle are adequate only for the adult and are insufficient to support growth. Therefore, during periods of growth, arginine becomes an essential amino acid. It is important to realize that if arginine is used for protein synthesis, the levels of ornithine will drop, thereby slowing the urea cycle. This will stimulate the formation of ornithine from glutamate. Arginine is cleaved by arginase to form urea and ornithine. If ornithine is present in amounts in excess of those required for the urea cycle, it is transaminated to glutamate semialdehyde, which is reduced to glutamate. The conversion of an aldehyde to a primary amine is a unique form of a transamination reaction and requires PLP. 5.

FAD(2H)

NADP+

+

N H2

CH COO–

Proline

FIG. 39.9. Synthesis and degradation of proline. Reactions 1, 3, and 4 occur in mitochondria. Reaction 2 occurs in the cytosol. Synthesis and degradation involve different enzymes. The cyclization reaction (formation of a Schiff base) is nonenzymatic; that is, it is spontaneous. Pi, inorganic phosphate.

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4.

B. Amino Acids Related to Oxaloacetate (Aspartate and Asparagine) Aspartate is produced by transamination of oxaloacetate. This reaction is readily reversible, so aspartate can be reconverted to oxaloacetate (Fig. 39.11). Asparagine is formed from aspartate by a reaction in which glutamine provides the nitrogen for formation of the amide group. Thus, this reaction differs from the synthesis of glutamine from glutamate, in which NH4⫹ provides the nitrogen. However, the reaction catalyzed by asparaginase, which hydrolyzes asparagine to NH4⫹ and aspartate, is analogous to the reaction catalyzed by glutaminase.

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C. Amino Acids That Form Fumarate 1.

ASPARTATE

Although the major route for aspartate degradation involves its conversion to oxaloacetate, carbons from aspartate can form fumarate in the urea cycle (see Chapter 38). This reaction generates cytosolic fumarate, which must be converted to malate (using cytoplasmic fumarase) for transport into the mitochondria for oxidative or anaplerotic purposes. An analogous sequence of reactions occurs in the purine nucleotide cycle. Aspartate reacts with inosine monophosphate (IMP) to form an intermediate (adenylosuccinate) that is then cleaved, forming adenosine monophosphate (AMP) and fumarate (see Chapter 41).

Certain types of tumor cells, particularly leukemic cells, require asparagine for their growth. Therefore, asparaginase has been used as an antitumor agent. It acts by converting asparagine to aspartate in the blood, decreasing the amount of asparagine available for tumor cell growth. Asparaginase has been used for more than 30 years to treat acute lymphoblastic leukemia.

Diet

2.

PHENYLALANINE AND TYROSINE

Phenylalanine is converted to tyrosine by a hydroxylation reaction. Tyrosine, produced from phenylalanine or obtained from the diet, is oxidized, ultimately forming acetoacetate and fumarate. The oxidative steps required to reach this point are, surprisingly, not energy generating. The conversion of fumarate to malate, followed by the action of malic enzyme, allows the carbons to be used for gluconeogenesis. The conversion of phenylalanine to tyrosine and the production of acetoacetate are considered further in Section IV of this chapter.

NH3 +

N

N Histidine Histidase

CH

D. Amino Acids That Form Succinyl-CoA The essential amino acids methionine, valine, isoleucine, and threonine are degraded to form propionyl-CoA. The conversion of propionyl-CoA to succinylCoA is common to their degradative pathways. Propionyl-CoA is also generated from the oxidation of odd-chain fatty acids.

N

FH4 Glutamate

Transamination PLP

N 5-Formimino-FH4

COO– CH2

NH4 +

NH3

+

N 5,N10-Methenyl-FH4

COO– Aspartate

H2O NH4

Asparagine synthetase

+

Asparaginase

H2O

O NH2

CH2 H C

–

COO

N-Formiminoglutamate (FIGLU)

Oxaloacetate

C

CH2

NH

O

COO–

AMP + PPi

CH2

CH

CH2

Glutamate

COO

NH

COO–

ATP Glutamine

–

CH

Urocanate

OOC CH

H C

NH4 +

N

–

C

COO–

CH2 CH

N10-Formyl-FH4

FIG. 39.10. Degradation of histidine. The highlighted portion of histidine forms glutamate. The remainder of the molecule provides one carbon for the tetrahydrofolate (FH4) pool (see Chapter 40) and releases NH4⫹.

NH3 + –

COO Asparagine

FIG. 39.11. Synthesis and degradation of aspartate and asparagine. Note that the amide nitrogen of asparagine is derived from glutamine. (The amide nitrogen of glutamine comes from NH4⫹; see Fig. 38.11.)

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A biopsy specimen from Homer Sistine’s liver was sent to the hospital’s biochemistry research laboratory for enzyme assays. Cystathionine ␤-synthase activity was reported to be 7% of that found in normal liver.

Homocystinuria is caused by deficiencies in the enzymes cystathionine ␤-synthase and cystathionase as well as by deficiencies of methyltetrahydrofolate (CH3-FH4) or of methyl-B12. The deficiencies of CH3-FH4 or of methyl-B12 are caused by either an inadequate dietary intake of folate or vitamin B12 or by defective enzymes involved in joining methyl groups to tetrahydrofolate (FH4), transferring methyl groups from FH4 to B12, or passing them from B12 to homocysteine to form methionine (see Chapter 40). Is Homer Sistine’s homocystinuria caused by any of these problems?

Propionyl-CoA is carboxylated in a reaction that requires biotin and forms The D-methylmalonyl-CoA is racemized to L-methylmalonyl-CoA, which is rearranged in a vitamin B12-requiring reaction to produce succinyl-CoA, a TCA cycle intermediate (see Fig. 23.11). D-methylmalonyl-CoA.

1.

METHIONINE

Methionine is converted to S-adenosylmethionine (SAM), which donates its methyl group to other compounds to form S-adenosylhomocysteine (SAH). SAH is then converted to homocysteine (Fig. 39.12). Methionine can be regenerated from homocysteine by a reaction that requires both FH4 and vitamin B12 (a topic that is considered in more detail in Chapter 40). Alternatively, by reactions that require PLP, homocysteine can provide the sulfur needed for the synthesis of cysteine (see Fig. 39.6). Carbons of homocysteine are then metabolized to ␣-ketobutyrate, which undergoes oxidative decarboxylation to propionyl-CoA. The propionyl-CoA is then converted to succinyl-CoA (see Fig. 39.12). 2.

THREONINE

In humans, threonine is primarily degraded by a PLP-requiring dehydratase to ammonia and ␣-ketobutyrate, which subsequently undergoes oxidative decarboxylation to form propionyl-CoA, just as in the case for methionine (see Fig. 39.12).

Methionine N5

CH3

FH4

B12

FH4

B12 CH3

SAM

Homocysteine Serine PLP

“CH3” donated S-adenosyl homocysteine

Cystathionine Cysteine

PLP

␣-Ketobutyrate

Threonine NH3

CO2 Propionyl CoA CO2

Biotin

Isoleucine Acetyl CoA Valine

D-methylmalonyl CoA

L-methylmalonyl CoA Vitamin B12 Succinyl CoA

TCA cycle Glucose

FIG. 39.12. Conversion of amino acids to succinyl-CoA. The amino acids methionine, threonine, isoleucine, and valine—all of which form succinyl-CoA via methylmalonylCoA—are essential in the diet. The carbons of serine are converted to cysteine and do not form succinyl-CoA by this pathway. PLP, pyridoxal phosphate; SAM, S-adenosylmethionine; B12-CH3, methylcobalamin; N5-CH3-FH4, N5-methyltetrahydrofolate.

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3.

VALINE AND ISOLEUCINE

The branched-chain amino acids (valine, isoleucine, and leucine) are universal fuels, and the degradation of these amino acids occurs at low levels in the mitochondria of most tissues, but the muscle carries out the highest level of branched-chain amino acid oxidation. The branched-chain amino acids make up almost 25% of the content of the average protein, so their use as fuel is quite significant. The degradative pathway for valine and isoleucine has two major functions, the first being energy generation and the second to provide precursors to replenish TCA cycle intermediates (anaplerosis). Valine and isoleucine, two of the three branched-chain amino acids, contain carbons that form succinylCoA. The initial step in the degradation of the branched-chain amino acids is a transamination reaction. Although the enzyme that catalyzes this reaction is present in most tissues, the level of activity varies from tissue to tissue. However, its activity is particularly high in muscle. In the second step of the degradative pathway, the ␣-keto analogs of these amino acids undergo oxidative decarboxylation by the ␣-keto acid dehydrogenase complex in a reaction similar in its mechanism and cofactor requirements to pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase (see Chapter 20). As with the first enzyme of the pathway, the highest level of activity for this dehydrogenase is found in muscle tissue. Subsequently, the pathways for degradation of these amino acids follow parallel routes (Fig. 39.13). The steps are analogous to those for ␤-oxidation of fatty acids, so NADH and FAD(2H) are generated for energy production.

Leucine

␣-Keto-␤-methylvalerate

␣-Ketoisocaproate

Transamination

␣-Ketoisovalerate Oxidative decarboxylation (␣-keto acid dehydrogenase)

CO2

CO2

CO2

NADH

NADH

NADH

2-Methylbutyryl CoA

Isobutyryl CoA

FAD(2H) CO2

2NADH Acetyl CoA

CO2

Defective in maple syrup urine disease

Thiamine deficiency will lead to an accumulation of ␣-keto acids in the blood because of an inability of pyruvate dehydrogenase, ␣-ketoglutarate dehydrogenase, and branched-chain ␣-keto acid dehydrogenase to catalyze their reactions (see Chapter 8). Al Martini had a thiamine deficiency resulting from his chronic alcoholism. His ketoacidosis resulted partly from the accumulation of these ␣-keto acids in his blood and partly from the accumulation of ketone bodies used for energy production.

Isovaleryl CoA

FAD(2H)

2 NADH

Homer Sistine’s methionine levels are elevated, and his vitamin B12 and folate levels are normal. Therefore, he does not have a deficiency of dietary folate or vitamin B12 or of the enzymes that transfer methyl groups from tetrahydrofolate to homocysteine to form methionine. In these cases, homocysteine levels are elevated but methionine levels are low.

What compounds form succinylCoA via propionyl-CoA and methylmalonyl-CoA?

Isoleucine

Valine

735

Propionyl CoA

HMG CoA

Acetoacetate

CO2

D-methylmalonyl CoA L-methylmalonyl CoA Succinyl CoA

Ketogenic

Gluconeogenic

FIG. 39.13. Degradation of the branched-chain amino acids. Valine forms propionylCoA. Isoleucine forms propionyl-CoA and acetyl-CoA. Leucine forms acetoacetate and acetyl-CoA.

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In maple syrup urine disease, the branched-chain ␣-keto acid dehydrogenase that oxidatively decarboxylates the branched-chain amino acids is defective. As a result, the branched-chain amino acids and their ␣-keto analogs (produced by transamination) accumulate. They appear in the urine, giving it the odor of maple syrup or burnt sugar. The accumulation of ␣-keto analogs leads to neurologic complications. This condition is difficult to treat by dietary restriction because abnormalities in the metabolism of three essential amino acids contribute to the disease. In addition to methionine, threonine, isoleucine, and valine (see Fig. 39.14), the last three carbons at the ␻-end of odd-chain fatty acids form succinyl-CoA by this route (see Chapter 23). Alcaptonuria occurs when homogentisate, an intermediate in tyrosine metabolism, cannot be further oxidized because the next enzyme in the pathway, homogentisate oxidase, is defective. Homogentisate accumulates and auto-oxidizes, forming a dark pigment, which discolors the urine and stains the diapers of affected infants. Later in life, the chronic accumulation of this pigment in cartilage may cause arthritic joint pain.

Valine and isoleucine are converted to succinyl-CoA (see Fig. 39.12). Isoleucine also forms acetyl-CoA. Leucine, the third branched-chain amino acid, does not produce succinyl-CoA. It forms acetoacetate and acetyl-CoA and is strictly ketogenic.

IV. AMINO ACIDS THAT FORM ACETYL CoA AND ACETOACETATE Seven amino acids produce acetyl-CoA or acetoacetate and, therefore, are categorized as ketogenic. Of these, isoleucine, threonine, and the aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are converted to compounds that produce both glucose and acetyl-CoA or acetoacetate (Fig. 39.14). Leucine and lysine do not produce glucose; they produce acetyl-CoA and acetoacetate.

A. Phenylalanine and Tyrosine Phenylalanine is converted to tyrosine, which undergoes oxidative degradation (Fig. 39.15). The last step in the pathway produces both fumarate and the ketone body acetoacetate. Deficiencies of different enzymes in the pathway result in phenylketonuria, tyrosinemia, and alcaptonuria. Phenylalanine is hydroxylated to form tyrosine by a mixed-function oxidase, phenylalanine hydroxylase (PAH), which requires molecular oxygen and BH4 (Fig. 39.16). The cofactor BH4 is converted to quinonoid dihydrobiopterin by this reaction. BH4 is not synthesized from a vitamin; it can be

Phenylalanine

Tyrosine Tryptophan Homogentisic acid Formate

Alanine

Fumarate

TCA

Threonine Glucose

Pyruvate Glucose

Acetyl CoA

Nicotinamide moiety of NAD, NADP

Lysine

Acetoacetate

Leucine

Isoleucine Succinyl CoA

Glucose

FIG. 39.14. Ketogenic amino acids. Some of these amino acids (tryptophan, phenylalanine, and tyrosine) also contain carbons that can form glucose. Leucine and lysine are strictly ketogenic; they do not form glucose.

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CHAPTER 39 ■ SYNTHESIS AND DEGRADATION OF AMINO ACIDS

+

NH3 CH2 C

COO

CH

–

C Phenylalanine

PKU

Phenylalanine hydroxylase +

NH3 CH2

HO C

CH

COO

–

C Tyrosine

Tyrosinemia II

Tyrosine aminotransferase

PLP O HO

CH2 C

C

COO–

C p-Hydroxyphenylpyruvate CO2 OH C

CH2

COO–

Homogentisate

Alcaptonuria

Tyrosinemia I

Homogentisate oxidase

Fumarylacetoacetate hydrolase

O OOC

CH

CH COO

Fumarate

–

CH3

– C CH2 COO

Acetoacetate

FIG. 39.15. Degradation of phenylalanine and tyrosine. The carboxyl carbon forms CO2, and the other carbons form fumarate or acetoacetate as indicated. Deficiencies of enzymes (dark bars) result in the indicated diseases. PKU, phenylketonuria; PLP, pyridoxal phosphate.

synthesized in the body from guanosine triphosphate (GTP). However, as is the case with other cofactors, the body contains limited amounts. Therefore, dihydrobiopterin must be reconverted to BH4 for the reaction to continue to produce tyrosine.

B. Tryptophan Tryptophan is oxidized to produce alanine (from the nonring carbons), formate, and acetyl-CoA. Tryptophan is, therefore, both glucogenic and ketogenic (Fig. 39.17). NAD⫹ and NADP⫹ can be produced from the ring structure of tryptophan. Therefore, tryptophan “spares” the dietary requirement for niacin. The higher the dietary levels of tryptophan, the lower are the levels of niacin required to prevent symptoms of deficiency.

Lieberman_CH39.indd 737

Transient tyrosinemia is frequently observed in newborn infants, especially those that are premature. For the most part, the condition appears to be benign, and dietary restriction of protein returns plasma tyrosine levels to normal. The biochemical defect is most likely a low level, attributable to immaturity, of 4-hydroxyphenylpyruvate dioxygenase. Because this enzyme requires ascorbate, ascorbate supplementation also aids in reducing circulating tyrosine levels. Other types of tyrosinemia are related to specific enzyme defects (see Fig. 39.15). Tyrosinemia II is caused by a genetic deficiency of tyrosine aminotransferase (TAT) and may lead to lesions of the eye and skin as well as neurologic problems. Patients are treated with a low-tyrosine, low-phenylalanine diet. Tyrosinemia I (also called tyrosinosis) is caused by a genetic deficiency of fumarylacetoacetate hydrolase. The acute form is associated with liver failure, a cabbage-like body odor, and death within the first year of life.

C

HO

–

737

A small subset of patients with hyperphenylalaninemia shows an appropriate reduction in plasma phenylalanine levels with dietary restriction of this amino acid; however, these patients still develop progressive neurologic symptoms and seizures and usually die within the first 2 years of life (“malignant” hyperphenylalaninemia). These infants exhibit normal phenylalanine hydroxylase (PAH) activity but have a deficiency in dihydropteridine reductase (DHPR), an enzyme required for the regeneration of tetrahydrobiopterin (BH4), a cofactor of PAH (see Fig. 39.16). Less frequently, DHPR activity is normal but a defect in the biosynthesis of BH4 exists. In either case, dietary therapy corrects the hyperphenylalaninemia. However, BH4 is also a cofactor for two other hydroxylations required in the synthesis of neurotransmitters in the brain: the hydroxylation of tryptophan to 5-hydroxytryptophan and of tyrosine to L-dopa (see Chapter 48). It has been suggested that the resulting deficit in central nervous system neurotransmitter activity is, at least in part, responsible for the neurologic manifestations and eventual death of these patients.

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SECTION VII ■ NITROGEN METABOLISM

GTP

biosynthesis +

NAD+

N

H 2N

H N H

HN O

N

H2N

H N H

COO–

CH2 CH

H

Phenylalanine

H CH CH CH3 O2

OH OH Tetrahydrobiopterin (BH4)

Dihydropteridine reductase

NADH + H+

N H

NH3

Phenylalanine hydroxylase

H2O H +

N N

H CH CH CH3

NH3 COO–

CH2 CH

HO

O

OH OH Quinonoid dihydrobiopterin (BH2)

Tyrosine

FIG. 39.16. Hydroxylation of phenylalanine. Phenylalanine hydroxylase (PAH) is a mixed-function oxidase; that is, molecular oxygen (O2) donates one atom to water and one to the product, tyrosine. The cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2) and must be reduced back to BH4 for PAH to continue forming tyrosine. BH4 is synthesized in the body from GTP. PKU results from deficiencies of PAH (the classic form), dihydropteridine reductase, or enzymes in the biosynthetic pathway for BH4.

+

NH3 CH2 CH N

COO–

C Tryptophan

Kynurenine

HCOO– Formate

PLP Kynurenine

hydroxylase

If the dietary levels of niacin and tryptophan are insufficient, the condition known as pellagra results. The symptoms of pellagra are dermatitis, diarrhea, dementia, and, finally, death. In addition, abnormal metabolism of tryptophan occurs in a vitamin B6 deficiency. Kynurenine intermediates in tryptophan degradation cannot be cleaved because kynureninase requires PLP derived from vitamin B6. Consequently, these intermediates enter a minor pathway for tryptophan metabolism that produces xanthurenic acid, which is excreted in the urine.

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Xanthurenic acid and other urinary metabolites

+

NH3 CH3 CH

–

COO

Alanine CO2 Nicotinamide moiety of NAD and NADP

Acetyl CoA

FIG. 39.17. Degradation of tryptophan. One of the ring carbons (in red) produces formate. The nonring portion (indicated by the box) forms alanine. Kynurenine is an intermediate, which can be converted to several urinary excretion products (e.g., xanthurenate), degraded to CO2 and acetyl-CoA, or converted to the nicotinamide moiety of NAD and NADP, which also can be formed from the vitamin niacin. PLP, pyridoxal phosphate.

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C. Threonine, Isoleucine, Leucine, and Lysine As discussed previously, the major route of threonine degradation in humans is by threonine dehydratase (see Section III.D.2). In a minor pathway, threonine degradation by threonine aldolase produces glycine and acetyl-CoA in the liver. Isoleucine produces both succinyl-CoA and acetyl-CoA (see Section III.D.3). Leucine is purely ketogenic and produces hydroxymethylglutaryl CoA (HMG-CoA), which is cleaved to form acetyl-CoA and the ketone body acetoacetate (see Figs. 39.13 and 39.14). Most of the tissues in which it is oxidized can use ketone bodies, with the exception of the liver. As with valine and isoleucine, leucine is a universal fuel, with its primary metabolism occurring in muscle. Lysine cannot be directly transaminated at either of its two amino groups. Lysine is degraded by a complex pathway in which saccharopine, ␣-ketoadipate, and crotonyl-CoA are intermediates. During the degradation pathway, NADH and FADH2 are generated for energy. Ultimately, lysine generates acetyl-CoA (see Fig. 39.14) and is strictly ketogenic.

On more definitive testing of Piquet Yuria’s blood, the plasma level of phenylalanine was elevated at 25 mg/dL (reference range, ⬍1.2). Several phenyl ketones and other products of phenylalanine metabolism, which give the urine a characteristic odor, were found in significant quantities in the baby’s urine. +

NH3 COO–

CH2 CH

Phenylalanine Transamination O CH2 C

COO–

Phenylpyruvate

CLINICAL COMMENTS Piquet Yuria. The overall incidence of PKU, leading to hyperphenylalaninemia, is approximately 100 per million births, with a wide geographic and ethnic variation. PKU occurs by autosomal recessive transmission of a defective phenylalanine hydroxylase (PAH) gene, causing accumulation of phenylalanine in the blood that is much higher than the normal concentration in young children and adults (⬍1 to 2 mg/dL). In the newborn, the upper limit of normal is almost twice this value. Values greater than 20 mg/dL are usually found in patients, such as Piquet, who have “classic” PKU. Patients with classic PKU usually appear normal at birth. If the disease is not recognized and treated within the first month of life, the infant gradually develops varying degrees of irreversible mental retardation (IQ scores frequently less than 50), delayed psychomotor maturation, tremors, seizures, eczema, and hyperactivity. The neurologic sequelae may result in part from the competitive interaction of phenylalanine with brain amino acid transport systems and inhibition of neurotransmitter synthesis. A successful dietary therapy for PKU, in addition to phenylalanine reduction, is supplementation of the diet with large, neutral amino acids (such as tryptophan, tyrosine, histidine, and leucine). These large, neutral amino acids share a transport system (the L system) with phenylalanine, and can overcome the high levels of phenylalanine in the blood, allowing the neurotransmitter precursors to enter the nervous system. If the disease is not recognized in time, the biochemical alterations lead to impaired myelin synthesis and delayed neuronal development, which result in the clinical picture in patients such as Piquet. Because of the simplicity of the test for PKU (elevated phenylalanine levels in the blood), all newborns in the United States are required to have a PKU test at birth. Early detection of the disease can lead to early treatment, and the neurologic consequences of the disease can be bypassed. To restrict dietary levels of phenylalanine, special semisynthetic preparations such as Lofenalac or PKU aid are used in the United States. Use of these preparations reduces dietary intake of phenylalanine to 250 to 500 mg/day while maintaining normal intake of all other dietary nutrients. Although it is generally agreed that scrupulous adherence to this regimen is mandatory for the first decade of life, less consensus exists regarding its indefinite use. Evidence suggests, however, that without lifelong compliance with dietary restriction of phenylalanine, even adults

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CO2 CH2 COO– Phenylacetate OH CH2 CH COO– Phenyllactate

A liver biopsy was sent to the special chemistry research laboratory, where it was determined that the level of activity of PAH in Piquet’s blood was less than 1% of that found in normal infants. A diagnosis of “classic” PKU was made. Until gene therapy allows substitution of the defective PAH gene with its normal counterpart in vivo, the mainstay of therapy in classic PKU is to maintain levels of phenylalanine in the blood between 3 and 12 mg/dL through dietary restriction of this essential amino acid.

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SECTION VII ■ NITROGEN METABOLISM

will develop at least neurologic sequelae of PKU. A pregnant woman with PKU must be particularly careful to maintain satisfactory plasma levels of phenylalanine throughout gestation to avoid the adverse effects of hyperphenylalaninemia on the fetus. The use of glycomacropeptide from whey is a promising development in PKU treatment. This protein—when pure—contains no phenylalanine, but as isolated has a very low phenylalanine content, while providing adequate levels of the other amino acids. The use of glycomacropeptide in PKU diet preparation is expanding and provides alternatives to the dietary therapy not previously available. Piquet’s parents were given thorough dietary instruction, which they followed carefully. Although her pediatrician was not optimistic, it was hoped that the damage done to her nervous system before dietary therapy was minimal and that her subsequent psychomotor development would allow her to lead a relatively normal life. The pathologic findings that underlie the clinical features manifested by Homer Sistine are presumed (but not proved) to be the consequence of chronic elevations of homocysteine (and perhaps other compounds, e.g., methionine) in the blood and tissues. The zonular fibers that normally hold the lens of the eye in place become frayed and break, causing dislocation of the lens. The skeleton reveals a loss of bone ground substance (i.e., osteoporosis), which may explain the curvature of the spine. The elongation of the long bones beyond their normal genetically determined length leads to tall stature. Animal experiments suggest that increased concentrations of homocysteine and methionine in the brain may trap adenosine as S-adenosylhomocysteine, diminishing adenosine levels. Because adenosine normally acts as a central nervous system depressant, its deficiency may be associated with a lowering of the seizure threshold as well as a reduction in cognitive function.

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Homer Sistine. The most characteristic biochemical features of the disorder that affects Homer Sistine, a cystathionine ␤-synthase deficiency, are the presence of an accumulation of both homocyst(e)ine and methionine in the blood. Because renal tubular reabsorption of methionine is highly efficient, this amino acid may not appear in the urine. Homocystine, the disulfide of homocysteine, is less efficiently reabsorbed, and amounts in excess of 1 mmol may be excreted in the urine each day. In the type of homocystinuria in which the patient is deficient in cystathione ␤-synthase, the elevation in serum methionine levels is presumed to be the result of enhanced rates of conversion of homocysteine to methionine because of increased availability of homocysteine (see Fig. 39.12). In type II and type III homocystinuria, in which there is a deficiency in the synthesis of methyl cobalamin and of N 5-methyltetrahydrofolate, respectively (both required for the methylation of homocysteine to form methionine), serum homocysteine levels are elevated but serum methionine levels are low (see Fig. 39.12). Acute vascular events are common in these patients. Thrombi (blood clots) and emboli (clots that have broken off and traveled to a distant site in the vascular system) have been reported in almost every major artery and vein as well as in smaller vessels. These clots result in infarcts in vital organs such as the liver, the myocardium (heart muscle), the lungs, the kidneys, and many other tissues. Although increased serum levels of homocysteine have been implicated in enhanced platelet aggregation and damage to vascular endothelial cells (leading to clotting and accelerated atherosclerosis), no generally accepted mechanism for these vascular events has yet emerged. Treatment is directed toward early reduction of the elevated levels of homocysteine and methionine in the blood. In addition to a diet that is low in methionine, very high oral doses of pyridoxine (vitamin B6) have significantly decreased the plasma levels of homocysteine and methionine in some patients with cystathionine ␤-synthase deficiency. (Genetically determined “responders” to pyridoxine treatment make up approximately 50% of type I homocystinurics.) Pyridoxal phosphate (PLP) serves as a cofactor for cystathionine ␤-synthase; however, the molecular properties of the defective enzyme that confer the responsiveness to vitamin B6 therapy are not known. The terms hypermethioninemia, homocystinuria (or -emia), and cystathioninuria (or -emia) designate biochemical abnormalities and are not specific clinical diseases. Each may be caused by more than one specific genetic defect. For example, at least seven distinct genetic alterations can cause increased excretion of homocystine in the urine. A deficiency of cystathionine ␤-synthase is the most common cause of homocystinuria; more than 600 such proven cases have been studied.

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CHAPTER 39 ■ SYNTHESIS AND DEGRADATION OF AMINO ACIDS

Table 39.1

741

Genetic Disorders of Amino Acid Metabolism

Amino Acid Degradation Pathway

Missing Enzyme

Product That Accumulates

Disease

Symptoms

Phenylalanine hydroxylase Dihydropteridine reductase Homogentisate oxidase Fumarylacetoacetate hydrolase Tyrosine aminotransferase Cystathionase Cystathionine ␤-synthase

Phenylalanine Phenylalanine Homogentisic acid Fumarylacetoacetate Tyrosine Cystathionine Homocysteine

PKU (classical) PKU (nonclassical) Alcaptonuria Tyrosinemia I Tyrosinemia II Cystathioninuria Homocysteinemia

Glycine

Glycine transaminase

Glyoxylate

Primary oxaluria type I

Branched-chain amino acids (leucine, isoleucine, valine)

Branched-chain ␣-keto acid dehydrogenase

␣-Keto acids of the branched chain amino acids

Maple syrup urine disease

Mental retardation Mental retardation Black urine, arthritis Liver failure, early death Neurologic defects Benign Cardiovascular complications and neurologic problems Renal failure because of stone formation Mental retardation

Phenylalanine

Tyrosine Methionine

BIOCHEMICAL COMMENTS Phenylketonuria. Many enzyme deficiency diseases have been discovered that affect the pathways of amino acid metabolism. These deficiency diseases have helped researchers to elucidate the pathways in humans, in whom experimental manipulation is, at best, unethical. These spontaneous mutations (“experiments” of nature), although devastating to patients, have resulted in an understanding of these diseases that now permit treatment of inborn errors of metabolism that were once considered to be untreatable. Classic phenylketonuria (PKU) is caused by mutations in the gene located on chromosome 12 that encodes the enzyme phenylalanine hydroxylase (PAH). This enzyme normally catalyzes the hydroxylation of phenylalanine to tyrosine, the rate-limiting step in the major pathway by which phenylalanine is catabolized. In early experiments, sequence analysis of mutant clones indicated a single base substitution in the gene, with a G-to-A transition at the canonical 5⬘ donor splice site of intron 12 and expression of a truncated unstable protein product. This protein lacked the C-terminal region, a structural change that yielded less than 1% of the normal activity of PAH. Since these initial studies, DNA analysis has shown more than 100 mutations (missense, nonsense, insertions, and deletions) in the PAH gene, associated with PKU and non-PKU hyperphenylalaninemia. That PKU is a heterogeneous phenotype is supported by studies measuring PAH activity in needle biopsy samples taken from the livers of a large group of patients with varying degrees of hyperphenylalaninemia. PAH activity varied from less than 1% of normal in patients with classic PKU to up to 35% of normal in those with a non-PKU form of hyperphenylalaninemia (such as a defect in BH4 production; see Chapter 48). The genetic diseases affecting amino acid degradation that have been discussed in this chapter are summarized in Table 39.1. This is just a partial listing of disorders in amino acid metabolism; there are many others that are less common and were not discussed in this chapter. Key Concepts • •

Humans can synthesize only 11 of the 20 amino acids required for protein synthesis; the other 9 are considered to be essential amino acids in the diet. Amino acid metabolism uses, to a large extent, the cofactors pyridoxal phosphate, tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4). Pyridoxal phosphate is required primarily for transamination reactions. BH4 is required for ring hydroxylation reactions. FH4 is required for one-carbon metabolism and is discussed further in Chapter 40.

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•

• •

•

The nonessential amino acids can be synthesized from glycolytic intermediates (serine, glycine, cysteine, and pyruvate), TCA cycle intermediates (aspartate, asparagine, glutamate, glutamine, proline, arginine, and ornithine), or from existing amino acids (tyrosine from phenylalanine). When amino acids are degraded, the nitrogen is converted to urea, and the carbon skeletons are classified as either glucogenic (a precursor of glucose) or ketogenic (a precursor of ketone bodies). Defects in amino acid degradation pathways can lead to disease. Glycine degradation can lead to oxalate production, which may lead to one class of kidney stone formation. Defects in methionine degradation can lead to hyperhomocysteinemia, which has been linked to blood-clotting disorders and heart disease. A defect in branched-chain amino acid degradation leads to maple syrup urine disease, which has severe neurologic consequences. Defects in phenylalanine and tyrosine degradation lead to phenylketonuria (PKU), alcaptonuria, and albinism. Table 39.2 summarizes, in a slightly different format than Table 39.1, the diseases discussed in this chapter.

Table 39.2

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Diseases Discussed in Chapter 39

Disease or Disorder

Environmental or Genetic

PKU

Genetic

Alcaptonuria

Genetic

Tyrosinemia

Genetic

Cystathioninuria

Genetic

Homocysteinemia

Genetic

Primary oxaluria type I

Genetic

Maple syrup urine disease

Genetic

Cystinosis

Genetic

Thiamine deficiency

Environmental

Comments Classical PKU is caused by a defect in phenylalanine hydroxylase whereas nonclassical PKU is caused by a defect in dihydropteridine reductase (or an inability to synthesize tetrahydrobiopterin). Both forms of PKU will lead to mental retardation if treatment is not initiated at an early age. Alcaptonuria is caused by a defect in homogentisate oxidase, leading to an accumulation of homogentisic acid. Arthritis may develop later in life. Tyrosinemia type I is a defect in fumarylacetoacetate hydrolase, leading to liver failure and early death. Tyrosinemia type II is a defect in tyrosine aminotransferase, leading to neurologic defects. Defect in cystathionase, leading to an accumulation of cystathionine. No major complications result from this mutation. A defect in cystathionine ␤-synthase leads to accumulation of homocysteine, which can result in cardiologic and neurologic complications in the patient. Defect in glycine transaminase leading to oxalate accumulate, and renal failure because of stone formation within the kidney. A defect in the branched-chain ␣-keto acid dehydrogenase, leading to an accumulation of the ␣-keto acids of the branched-chain amino acids, resulting in mental retardation. A defect in the transport protein that carries cystine across lysosomal membranes. Cystine accumulates in lysosomes, interfering with and ultimately destroying their function. A thiamine deficiency leads to accumulation of ␣-keto acids because the enzymes that catalyze oxidative decarboxylation reactions will not function in the absence of this vitamin. This will interfere with energy production, and lead to ketoacidosis.

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CHAPTER 39 ■ SYNTHESIS AND DEGRADATION OF AMINO ACIDS

743

REVIEW QUESTIONS—CHAPTER 39 1.

2.

3.

If an individual has a vitamin B6 deficiency, which of the following amino acids could still be synthesized and be considered nonessential? A. Tyrosine B. Serine C. Alanine D. Cysteine E. Aspartate The degradation of amino acids can be classified into families, which are named after the end product of the degradative pathway. Which of the following is such an end product? A. Citrate B. Glyceraldehyde 3-phosphate C. Fructose 6-phosphate D. Malate E. Succinyl-CoA A newborn infant has elevated levels of phenylalanine and phenylpyruvate in her blood. Which of the following enzymes might be deficient in this baby?

Lieberman_CH39.indd 743

A. B. C. D. E.

Phenylalanine dehydrogenase Phenylalanine oxidase Dihydropteridine reductase Tyrosine hydroxylase Tetrahydrofolate synthase

4.

Pyridoxal phosphate is required for which of the following reaction pathways or individual reactions? A. Phenylalanine → tyrosine B. Methionine → cysteine ⫹ ␣-ketobutyrate C. Propionyl-CoA → succinyl-CoA D. Pyruvate → acetyl-CoA E. Glucose → glycogen

5.

A folic acid deficiency would interfere with the synthesis of which of the following amino acids from the indicated precursors? A. Aspartate from oxaloacetate and glutamate B. Glutamate from glucose and ammonia C. Glycine from glucose and alanine D. Proline from glutamate E. Serine from glucose and alanine

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40

Tetrahydrofolate, Vitamin B12, and S-Adenosylmethionine

Serine Glycine Histidine Formaldehyde Formate Tetrahydrofolate (FH4)

Sources of one-carbon units

Formyl

FH4 • C

Methylene

The one-carbon pool

Methyl Precursors

Products

dTMP Serine Purines B12 • CH3

Products after receiving carbon

FIG. 40.1. Overview of the one-carbon pool. FH4 • C indicates tetrahydrofolate (FH4) containing a one-carbon unit that is at the formyl, methylene, or methyl level of oxidation (see Fig. 40.3). The origin of the carbons is indicated, as are the final products after a onecarbon transfer.

Groups that contain a single carbon atom can be transferred from one compound to another. These carbon atoms may be in several different oxidation states. The most oxidized form, CO2, is transferred by biotin. One-carbon groups at lower levels of oxidation than CO2 are transferred by reactions involving tetrahydrofolate (FH4), vitamin B12, and S-adenosylmethionine (SAM). Tetrahydrofolate. Tetrahydrofolate, which is produced from the vitamin folate, is the primary one-carbon carrier in the body. This vitamin obtains one-carbon units from serine, glycine, histidine, formaldehyde, and formate (Fig. 40.1). While these carbons are attached to FH4, they can be either oxidized or reduced. As a result, folate can exist in a variety of chemical forms. Once a carbon has been reduced to the methyl level (methyl-FH4), however, it cannot be reoxidized. Collectively, these one-carbon groups attached to their carrier FH4 are known as the one-carbon pool. The term “folate” is used to represent a water-soluble B-complex vitamin that functions in transferring single-carbon groups at various stages of oxidation. The one-carbon groups carried by FH4 are used for many biosynthetic reactions. For example, one-carbon units are transferred to the pyrimidine base of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), to the amino acid glycine to form serine, to precursors of the purine bases to produce carbons C2 and C8 of the purine ring, and to vitamin B12. Vitamin B12. Vitamin B12 is involved in two reactions in the body. It participates in the rearrangement of the methyl group of L-methylmalonyl coenzyme A (L-methylmalonyl-CoA) to form succinyl-CoA, and it transfers a methyl group, obtained from FH4, to homocysteine, forming methionine. S-adenosylmethionine. SAM, produced from methionine and adenosine triphosphate (ATP), transfers the methyl group to precursors that form several compounds, including creatine, phosphatidylcholine, epinephrine, melatonin, methylated nucleotides, and methylated DNA. Methionine metabolism is very dependent on both FH4 and vitamin B12. Homocysteine is derived from methionine metabolism and can be converted back into methionine by using both methyl-FH4 and vitamin B12. This is the only reaction in which methyl-FH4 can donate the methyl group. If the enzyme that catalyzes this reaction is defective, or if vitamin B12 or FH4 levels are insufficient, homocysteine will accumulate. Elevated homocysteine levels have been linked to cardiovascular and neurologic disease. A vitamin B12 deficiency can be brought about by the lack of intrinsic factor, a gastric protein required for the absorption of dietary B12. A consequence of vitamin B12 deficiency is the accumulation of methyl-FH4 and a decrease in other folate derivatives. This is known as the methyl-trap hypothesis, in which,

744

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CHAPTER 40 ■ TETRAHYDROFOLATE, VITAMIN B12, AND S-ADENOSYLMETHIONINE

because of the B12 deficiency, most of the carbons in the FH4 pool are trapped in the methyl-FH4 form, which is the most stable. The carbons cannot be released from the folate because the one reaction in which they participate cannot occur because of the B12 deficiency. This leads to a functional folate deficiency, even though total levels of folate are normal. A folate deficiency (whether functional or actual) leads to megaloblastic anemia caused by an inability of blood cell precursors to synthesize DNA and, therefore, to divide. This leads to large, partially replicated cells being released into the blood to attempt to replenish the cells that have died. Folate deficiencies also have been linked to an increased incidence of neural tube defects, such as spina bifida, in mothers who become pregnant while folate deficient.

THE WAITING ROOM After resection of the cancer in his large intestine and completion of a course of postoperative chemotherapy with 5-fluorouracil (5-FU), Colin Tuma returned to his gastroenterologist for a routine follow-up colonoscopy. His colon was completely normal, with excellent healing at the site of the anastomosis. His physician expressed great optimism about a possible cure of Colin’s previous malignancy but cautioned him about the need for regular colonoscopic examinations over the next few years. The 5-FU treatment had also reduced the growth of the metastatic tumors so they may be able to be resected in the future. Bea Twelvlow, a 75-year-old woman, went to see her physician because of numbness and tingling in her legs. A diet history indicated a normal and healthy diet, but Bea was not taking any supplemental vitamin pills. Laboratory results indicated a slight elevation of methylmalonic acid, and this led the physician to suspect a vitamin B12 deficiency. Direct measurement of serum B12 levels did indicate a deficiency, but the results of a Schilling test were normal. The initial laboratory profile, determined when Jean Ann Tonich first presented to her physician with evidence of early alcohol-induced hepatitis, included a hematologic analysis that showed that Jean Ann was anemic. Her hemoglobin was 11 g/dL (reference range, 12 to 16 g/dL for an adult woman). The erythrocyte (red blood cell) count was 3.6 million cells/mm3 (reference range, 4.0 to 5.2 million cells/mm3 for an adult woman). The average volume of her red blood cells (mean corpuscular volume or MCV) was 108 fL (reference range, 80 to 100 fL; 1 fL ⫽ 10–12 mL), and the hematology laboratory reported a striking variation in the size and shape of the red blood cells in a smear of her peripheral blood (see Chapter 44). The nuclei of the circulating granulocytic leukocytes had increased nuclear segmentation (polysegmented neutrophils). Because these findings are suggestive of a macrocytic anemia (in which blood cells are larger than normal), measurements of serum folate and vitamin B12 (cobalamin) levels were ordered.

I. TETRAHYDROFOLATE A. Structure and Forms of Tetrahydrofolate Folates exist in many chemical forms. The coenzyme form that functions in accepting one-carbon groups is tetrahydrofolate polyglutamate (Fig. 40.2), generally referred to as just tetrahydrofolate or FH4. It has three major structural components,

Lieberman_CH40.indd 745

745

The Schilling test involves the patient ingesting radioactive (60Co) crystalline vitamin B12, after which a 24-hour urine sample is collected. The radioactivity in the urine sample is compared with the input radioactivity, and the difference represents the amount of B12 not absorbed through the digestive tract. Such tests can distinguish between problems in removing B12 from bound dietary proteins or if the deficiency is caused by a lack of intrinsic factor or other proteins involved in transporting B12 throughout the body. Another method to determine if intrinsic factor activity is reduced is to determine the levels of antiintrinsic antibodies in the blood. Autoantibodies toward intrinsic factor commonly develop in individuals with lack of intrinsic factor activity, and the levels of such antibodies can be determined in an enzyme-linked immunosorbent assay (ELISA) using recombinant human intrinsic factor bound to plastic wells as the antigen.

Folate deficiencies occur frequently in chronic alcoholics. Several factors are involved: inadequate dietary intake of folate; direct damage to intestinal cells and brush border enzymes, which interferes with absorption of dietary folate; a defect in the enterohepatic circulation, which reduces the absorption of folate; liver damage that causes decreased hepatic production of plasma proteins; and interference with kidney reabsorption of folate. Because of the possibility of a direct toxic effect of alcohol on hematopoietic tissues in Jean Ann Tonich, a bone marrow aspirate was performed. The aspirate contained a greater than normal number of red and white blood cell precursors, most of which were larger than normal. The large red blood cells are called megaloblasts. These hematopoietic precursor cells, when exposed to too little folate and/or vitamin B12, show slowed cell division, but cytoplasmic development occurs at a normal rate. Hence, the megaloblastic cells tend to be large, with an increased ratio of RNA to DNA. Megaloblastic erythroid progenitors are usually destroyed in the bone marrow (although some reach the circulation). Thus, marrow cellularity is often increased but production of red blood cells is decreased, a condition called ineffective erythropoiesis. Therefore, Jean Ann has megaloblastic anemia, characteristic of folate or B12 deficiency.

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Jean Ann Tonich’s serum folic acid level was 3.1 ng/mL (reference range, 6 to 15 ng/mL), and her serum B12 level was 154 pg/mL (reference range, 150 to 750 pg/mL). Her serum iron level was normal. It was clear, therefore, that Jean Ann’s megaloblastic anemia was caused by a folate deficiency (although her B12 levels were in the low range of normal). The management of a pure folate deficiency in an alcoholic patient includes cessation of alcohol intake and a diet that is rich in folate.

Pteridine ring

PABA

OH N 5

N

8

H2N

N

6 7

9

CH2

O C

H N

COO C

–

H

10

CH2

N

CH2

Folate (F)

COO–

n

NADPH Dihydrofolate reductase

NADP+

OH N 5

N

8

H2N

Sulfa drugs, which are used to treat certain bacterial infections, are analogs of para-aminobenzoic acid. They prevent growth and cell division in bacteria by interfering with the synthesis of folate. Because we cannot synthesize folate, sulfa drugs do not affect human cells in this way.

H N

Glutamate

N

6 7

N H H

9

CH2 H

H N R 10

Dihydrofolate (FH2)

NADPH Dihydrofolate reductase

OH

5

N H2N

8

N

NADP+

H N H 6 7

N H H

9

CH2 H

H N R 10

Tetrahydrofolate (FH4)

The current U.S. Recommended Dietary Allowance (RDA) for folate equivalents is approximately 400 ␮g for adult men and women. In addition to being prevalent in green leafy vegetables, other good sources of this vitamin are liver, yeast, legumes, and some fruits. Protracted cooking of these foods, however, can destroy up to 90% of their folate content. A standard U.S. diet provides 50 to 500 ␮g of absorbable folate each day. Folate deficiency in pregnant women, especially during the month before conception and the month after, increases the risk of neural tube defects, such as spina bifida, in the fetus. To reduce the potential risk of neural tube defects for women who are capable of becoming pregnant, the recommendation is to take 400 ␮g of folic acid daily in a multivitamin pill. If the women have a history of having a child with a neural tube defect, this amount is increased to 4,000 ␮g/day for the month before and the month after conception. Flourcontaining products in the United States are now supplemented with folate to reduce the risk of neural tube defects in newborns.

Lieberman_CH40.indd 746

FIG. 40.2. Reduction of folate to tetrahydrofolate (FH4). The same enzyme, dihydrofolate reductase (DHFR), catalyzes both reactions. Multiple glutamate residues are added within cells (n ⬃ 5). Plants can synthesize folate but humans cannot. Therefore, folate is a dietary requirement. R is the portion of the folate molecule shown to the right of N10. The different precursors of FH4 are indicated in the figure. PABA, para-aminobenzoic acid.

a bicyclic pteridine ring, para-aminobenzoic acid, and a polyglutamate tail consisting of several glutamate residues joined in amide linkage. The one-carbon group that is accepted by the coenzyme and then transferred to another compound is bound to N5, to N10, or both. Different forms of folate may differ in the oxidation state of the one-carbon group, in the number of glutamate residues attached, or in the degree of oxidation of the pteridine ring. When the term “folate” or “folic acid” is applied to a specific chemical form, it is the most oxidized form of the pteridine ring (see Fig. 40.2). Folate is reduced to dihydrofolate (FH2) and then to FH4 by dihydrofolate reductase (DHFR) present in cells. Reduction is the favored direction of the reaction; therefore, most of the folate present in the body is present as the reduced coenzyme form, FH4.

B. The Vitamin Folate Folates are synthesized in bacteria and higher plants and ingested in green leafy vegetables, fruits, and legumes in the diet. The vitamin was named for its presence in green, leafy vegetables (foliage). Most of the dietary folate derived from natural food sources is present in the reduced coenzyme form. However, vitamin supplements and fortified foods contain principally the oxidized form of the pteridine ring.

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CHAPTER 40 ■ TETRAHYDROFOLATE, VITAMIN B12, AND S-ADENOSYLMETHIONINE

As dietary folates pass into the proximal third of the small intestine, folate conjugases in the brush border of the lumen cleave off glutamate residues to produce the monoglutamate form of folate, which is then absorbed (see Fig. 40.2, upper structure, when n ⫽ 1). Within the intestinal cells, folate is converted principally to N5-methyl-FH4, which enters the portal vein and goes to the liver. Smaller amounts of other forms of folate also follow this route. The liver, which stores half of the body’s folate, takes up much of the folate from the portal circulation; uptake may be through active transport or receptor-mediated endocytosis. Within the liver, FH4 is reconjugated to the polyglutamate form before being used in reactions. A small amount of the folate is partially degraded, and the components enter the urine. A relatively large portion of the folate enters the bile and is subsequently reabsorbed (very similar to the fate of bile salts in the enterohepatic circulation). N5-Methyl-FH4, the major form of folate in the blood, is loosely bound to plasma proteins, particularly serum albumin.

C. Oxidation and Reduction of the One-Carbon Groups of Tetrahydrofolate

One-carbon groups transferred by FH4 are attached either to N 5 or N10, or they form a bridge between N 5 and N10. The collection of one-carbon groups attached to FH4 is known as the one-carbon pool. While they are attached to FH4, these one-carbon units can be oxidized and reduced (Fig. 40.3). Thus, reactions that require a carbon in a particular oxidation state may use carbon from the one-carbon pool that was donated in a different oxidation state. The individual steps for reduction of the one-carbon group are shown in Figure 40.3. The most oxidized form is N10-formyl-FH4. The most reduced form is N 5-methyl-FH4. Once the methyl group is formed, it is not readily reoxidized back to N 5,N10-methylene-FH4, and thus N 5-methyl-FH4 tends to accumulate in the cell.

D. Sources of One-Carbon Groups Carried by FH4

Carbon sources for the one-carbon pool include serine, glycine, formaldehyde, histidine, and formate (Fig. 40.4). These donors transfer the carbons to folate in different oxidation states. Serine is the major carbon source of one-carbon groups in the human. Its hydroxymethyl group is transferred to FH4 in a reversible reaction, catalyzed by the enzyme serine hydroxymethyltransferase. This reaction produces glycine and N 5,N10-methylene-FH4. Because serine can be synthesized from 3-phosphoglycerate, an intermediate of glycolysis, dietary carbohydrate can serve as a source of carbon for the one-carbon pool. The glycine that is produced may be further degraded by donation of a carbon to folate. Additional donors that form N 5,N10-methylene-FH4 are listed in Table 40.1. Histidine and formate provide examples of compounds that donate carbon in different oxidation levels (see Fig. 40.4). Degradation of histidine produces formiminoglutamate (FIGLU), which reacts with FH4 to donate a carbon and nitrogen (generating N 5-formimino-FH4), thereby releasing glutamate. Formate, produced from tryptophan oxidation, can react with FH4 and generate N10-formyl-FH4, the most oxidized folate derivative.

E. Recipients of One-Carbon Groups The one-carbon groups on FH4 may be oxidized or reduced (see Fig. 40.3) and then transferred to other compounds (see Fig. 40.4 and Table 40.1). Transfers of this sort are involved in the synthesis of glycine from serine, the synthesis of the base thymine required for DNA synthesis, the purine bases required for both DNA and RNA synthesis, and the transfer of methyl groups to vitamin B12. Because the conversion of serine to glycine is readily reversible, glycine can be converted to serine by drawing carbon from the one-carbon pool.

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Hereditary folate malabsorption is a rare disease caused by a mutation in a proton-coupled folate transporter (PCFT, gene SLC46A1). Loss of PCFT activity in the intestinal proximal jejunum and duodenum lead to systemic folate deficiency. Newborns begin to exhibit symptoms of folate deficiency after a few months of life, after the folate obtained from the mother has been excreted. The children develop anemia, diarrhea, and become immunocompromised (because of the reduction in blood cell differentiation). High-dose oral folate can be used to treat the patients, as another folate transporter (the reduced folate carrier, gene SLC19A1) can absorb sufficient folate when folate is present at high concentrations. A deficiency of folate results in the accumulation of formiminoglutamate (FIGLU), which is excreted in the urine. A histidine load test can be used to detect folate deficiency. Patients are given a test dose of histidine (a histidine load), and the amount of FIGLU that appears in the urine is measured and compared to normal values. Values greater than normal indicate folate deficiency. The concentration of folate in serum can also be determined by a microbiologic test. Certain strains of bacteria require folate for growth, and if these bacteria are plated on a medium that lacks folate, they will not grow. Folate levels can be quantitated by preparing a standard curve using plates containing different levels of folate and determining the level of growth of the bacteria at that folate level. The unknown sample is then tested for bacterial growth and the extent of growth compared to the standard curve to determine the amount of folate in the unknown sample. Other tests for folate include measuring the binding of folate to certain proteins and use of immunologic reagents that specifically bind to and identify folate. Tetrahydrofolate (FH4) is required for the synthesis of deoxythymidine monophosphate (dTMP) and the purine bases used to produce the precursors for DNA replication. Therefore, FH4 is required for cell division. Blockage of the synthesis of thymine and the purine bases, either by a dietary deficiency of folate or by drugs that interfere with folate metabolism, results in a decreased rate of cell division and growth.

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SECTION VII ■ NITROGEN METABOLISM

A

OH

H N H 5

N H2N

B

8

N

H N R

9

6

CH2 10

7

Tetrahydrofolate (FH4)

N H H

H H

N H 5

6

FH4

N

9

CH2

10

Formate + ATP ADP + Pi H

HCO

N H 5

6

N

9

CH2

N10-formyl-FH4

10

H+ H2O CH N 5

N

10

H

N5,N10-methenyl-FH4

CH2

6

9

NADPH NADP+ CH2 N 5

N

10

H

N5,N10-methylene-FH4

CH2

6

9

NADH NAD+ CH3

H

N H 5

6

9

CH2

N 10

N5-methyl-FH4

FIG. 40.3. One-carbon units attached to tetrahydrofolate (FH4). A. The active form of FH4. For definition of R, see Figure 40.2. B. Interconversions of one-carbon units of FH4. Only the portion of FH4 from N 5 to N10 is shown, which is indicated by the green box in A. After a formyl group forms a bridge between N 5 and N10, two reductions can occur. Note that N 5-methyl-FH4 cannot be reoxidized. The most oxidized form of FH4 is at the top of the figure, whereas the most reduced form is at the bottom.

The nucleotide deoxythymidine monophosphate (dTMP) is produced from deoxyuridine monophosphate (dUMP) by a reaction in which dUMP is methylated to form dTMP (Fig. 40.5). The source of carbon is N 5,N10-methylene-FH4. Two hydrogen atoms from FH4 are used to reduce the donated carbon to the methyl level. Consequently, FH2 is produced. Reduction of FH2 by NADPH in a reaction catalyzed by DHFR regenerates FH4. This is the only reaction involving FH4 in which

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CHAPTER 40 ■ TETRAHYDROFOLATE, VITAMIN B12, AND S-ADENOSYLMETHIONINE

Histidine

Tryptophan

3 Sources of one-carbon groups

Serine

Glycine

1

2

NAD(P)+

dUMP

4

NH4+

NAD(P)H

N5,N10-Methylene FH4 Donation of oxidized carbon groups

HCOOH (formate)

N5-Formimino FH4

N5,N10-Methenyl FH4

N10-Formyl FH4

NADH

6

5

7

NAD+

dTMP FH2

N5-Methyl FH4

NADPH

Donation of methyl group NADP+

B12 FH4

Purine biosynthesis

Adenosine

Homocysteine

S-adenosyl homocysteine

R CH3

Methionine

S-adenosyl methionine (SAM, methyl donor)

R

8

FH4

ATP

PPi, Pi

FIG. 40.4. Sources of carbon (reactions 1 to 4) for the tetrahydrofolate (FH4) pool and the recipients of carbon (reactions 5 to 8) from the pool. See Figure 40.3 to view the FH4 derivatives involved in each reaction.

the folate group is oxidized as the one-carbon group is donated to the recipient. Recall that DHFR is also required to reduce the oxidized form of the vitamin, which is obtained from the diet (see Fig. 40.2). Thus, DHFR is essential for regenerating FH4 both in the tissues and from the diet. These reactions contribute to the effect of folate deficiency on DNA synthesis because dTMP is required only for the synthesis of DNA. During the synthesis of the purine bases, carbons 2 and 8 are obtained from the one-carbon pool (see Chapter 41). N10-formyl-FH4 provides both carbons. Folate deficiency also hinders these reactions, contributing to an inability to replicate DNA because of the lack of precursors. After the carbon group carried by FH4 is reduced to the methyl level, it is transferred to vitamin B12. This is the only reaction through which the methyl group can leave FH4 (recall that the reaction that creates N 5-methyl-FH4 is not reversible).

Table 40.1

One-Carbon Pool: Sources and Recipients of Carbon

a

Source

Form of One-Carbon Donor Produced b

Recipient

Final Product

N10-formyl-FH4 N 5, N10-methylene-FH4

Purine precursor dUMP Glycine

Purine (C2 and C8) dTMP Serine

Vitamin B12

Methylcobalamin

Choline

N 5-methyl-FH4 N 5-formimino-FH4 is converted to N 5, N10-methenyl-FH4 Betaine

Homocysteine

Methionine

SAM

Methionine and dimethylglycine N-methylglycine (sarcosine)

Formate Serine Glycine Formaldehyde N 5, N10-methylene-FH4 Histidine

Glycine (there are many others; see Fig. 40.9B)

dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; SAM, S-adenosylmethionine. a The major source of carbon is serine. b The carbon unit attached to FH4 can be oxidized and reduced (see Fig. 40.3). At the methyl level, reoxidation does not occur.

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Individuals with non-Hodgkin lymphoma receive several drugs to treat the tumor, including methotrexate. The structure of methotrexate is shown in the following: NH2

5

N H2N

N 8

N

6 7

9

CH2

CH3

O

N

C

H N

COO C

–

H

10

N

CH2 CH2 COO–

Methotrexate

What compound does methotrexate resemble?

A better understanding of the structure and function of the purine and pyrimidine bases and of folate metabolism led to the development of compounds having antimetabolic and antifolate action useful for treatment of neoplastic disease. For example, Colin Tuma was successfully treated for colon cancer with 5-fluorouracil (5-FU) (see Chapter 12 and Fig. 40.5). 5-FU is a pyrimidine analog, which is converted in cells to the nucleotide fluorodeoxyuridylate (FdUMP). FdUMP causes a “thymineless death,” especially for tumor cells that have a rapid turnover rate. It prevents the growth of cancer cells by blocking the thymidylate synthase reaction, that is, the conversion of deoxyuridine monophosphate (dUMP) to dTMP.

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SECTION VII ■ NITROGEN METABOLISM

Methotrexate has the same structure as folate except that it has an amino group on C4 and a methyl group on N10. Anticancer drugs such as methotrexate are folate analogs that act by inhibiting dihydrofolate reductase (DHFR), thereby preventing the conversion of FH2 to tetrahydrofolate (FH4) (see Fig. 40.5). Thus, the cellular pools of FH4 are not replenished, and reactions that require FH4 cannot proceed. Jean Ann Tonich’s megaloblastic anemia was treated, in part, with folate supplements (see Clinical Comments section). Within 48 hours of the initiation of folate therapy, megaloblastic or “ineffective” erythropoiesis usually subsides, and effective erythropoiesis begins. Megaloblastic anemia is caused by a decrease in the synthesis of thymine and the purine bases. These deficiencies lead to an inability of hematopoietic (and other) cells to synthesize DNA and, therefore, to divide. Their persistently thwarted attempts at normal DNA replication, DNA repair, and cell division produce abnormally large cells (called megaloblasts) with abundant cytoplasm capable of protein synthesis but with clumping and fragmentation of nuclear chromatin (see Chapter 44). Some of these large cells, although immature, are released early from the marrow in an attempt to compensate for the anemia. Thus, peripheral blood smears also contain megaloblasts. Many of the large immature cells, however, are destroyed in the marrow and never reach the circulation.

5-Fluorouracil

O HN O

Thymidylate

N

O

O

N5,N10Methylene FH4

Deoxyribose-P dUMP

CH3

HN

synthase

N

Deoxyribose-P dTMP

FH2 Dihydrofolate

Glycine

Methotrexate NADPH

Serine

Dihydrofolate reductase

FH4

NADP+

FIG. 40.5. Transfer of a one-carbon unit from N 5,N10-methylene-FH4 to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP). FH4 (tetrahydrofolate) is oxidized to FH2 (dihydrofolate) in this reaction. FH2 is reduced to FH4 by dihydrofolate reductase (DHFR) and FH4 is converted to N 5,N10-methylene-FH4 using serine as a carbon donor. Shaded bars indicate the steps at which the antimetabolites 5-fluorouracil (5-FU) and methotrexate act. 5-FU inhibits thymidylate synthase. Methotrexate inhibits DHFR.

II. VITAMIN B12 A. Structure and Forms of Vitamin B12

The structure of vitamin B12 (also known as cobalamin) is complex (Fig. 40.6). It contains a corrin ring, which is similar to the porphyrin ring found in heme. The corrin ring differs from heme, however, in that two of the four pyrrole rings are joined directly rather than by a methylene bridge. Its most unusual feature is the presence of cobalt, coordinated with the corrin ring (similar to the iron coordinated with the porphyrin ring). This cobalt can form a bond with a carbon atom. In the body, it reacts with the carbon of a methyl group, forming methylcobalamin, or with the 5⬘-carbon of 5⬘-deoxyadenosine, forming 5⬘-deoxyadenosylcobalamin (note that in this case, the “deoxy” designation refers to the 5⬘-carbon, not the 2⬘-carbon as is the case in the sugar found in DNA). The form of B12 found in vitamin supplements is cyanocobalamin in which a CN group is linked to the cobalt.

B. Absorption and Transport of Vitamin B12

The average daily diet in Western countries contains 5 to 30 ␮g of vitamin B12, of which 1 to 5 ␮g is absorbed into the blood. (The Recommended Dietary Allowance [RDA] is 2.4 ␮g/day.) Total body content of this vitamin in an adult is approximately 2 to 5 mg, of which 1 mg is present in the liver. As a result, a dietary deficiency of B12 is uncommon and is observed only after several years on a diet that is deficient in this vitamin. In spite of Jean Ann Tonich’s relatively malnourished state because of her chronic alcoholism, her serum cobalamin level was still within the low-to-normal range. If her undernourished state had continued, a cobalamin deficiency would eventually have developed.

Lieberman_CH40.indd 750

Although vitamin B12 is produced by bacteria, it cannot be synthesized by higher plants or animals. The major source of vitamin B12 is dietary meat, eggs, dairy products, fish, poultry, and seafood. The animals that serve as the source of these foods obtain B12 mainly from the bacteria in their food supply. The absorption of B12 from the diet is a complex process (Fig. 40.7). Ingested B12 can exist in two forms, either free or bound to dietary proteins. If free, the B12 binds to proteins known as R-binders (haptocorrins, also known as transcobalamin I), which are secreted by the salivary glands and the gastric mucosa, in either the saliva or the stomach. If the ingested B12 is bound to proteins, it must be released from the proteins by the action of digestive proteases in both the stomach and small intestine. Once the B12 is released from its bound protein, it binds to the haptocorrins. In the small intestine, the pancreatic proteases digest the haptocorrins, and the released B12 then binds to intrinsic factor, a glycoprotein secreted by the parietal cells of the stomach when food enters the stomach. The intrinsic factor-B12 complex attaches to specific receptors in the terminal segment of the small intestine known as the ileum, after which the complex is internalized.

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CHAPTER 40 ■ TETRAHYDROFOLATE, VITAMIN B12, AND S-ADENOSYLMETHIONINE

O C NH2

NH2 C O

CH2

CH3

CH2

CH2

X

H

CH3

O

H O C H2N

CH2 C NH2 CH3

CH2

CH2

N

N

CH3

N

H

Co3+ N

O

CH3

CH2 C NH2 H

CH3 CH3

H O C H2N

CH2

CH2

CH3

CH2

CH2 C O CH3

N

NH

CH3

N

CH2 HC CH3 O O H

O H OH H

751

Pernicious anemia, a deficiency of intrinsic factor, is a relatively common problem that is caused by malabsorption of dietary cobalamin. It may result from an inherited defect that leads to decreased ability of gastric parietal cells to synthesize intrinsic factor or from partial resection of the stomach or of the ileum. Production of intrinsic factor often declines with age and may be low in elderly individuals. An alternative circumstance that leads to the development of a B12 deficiency is pancreatic insufficiency or a high intestinal pH, which results from too little acid being produced by the stomach. Both of these conditions prevent the degradation of the R-binder–B12 complex; as a result, B12 is not released from the R-binder protein and, therefore, cannot bind to intrinsic factor. The protein receptor for the B12-intrinsic factor complex is named cubilin, and the internalization of the B12-intrinsic factor-cubilin complex requires the activity of a transmembrane protein named amnionless. Congenital malabsorption of B12 can also arise from inherited mutations in either cubilin or amnionless.

P O– O

CH2OH H

FIG. 40.6. Vitamin B12. X, 5⬘-deoxyadenosine in deoxyadenosylcobalamin; X ⫽ CH3 in methylcobalamin; X ⫽ CN in cyanocobalamin (the commercial form found in vitamin tablets).

The B12 within the enterocyte complexes with transcobalamin II and then released into circulation. The transcobalamin II-B12 complex delivers B12 to the tissues, which contain specific receptors for this complex. The liver takes up approximately 50% of the vitamin B12, and the remainder is transported to other tissues. The amount of the vitamin stored in the liver is large enough that 3 to 6 years pass before symptoms of a dietary deficiency occur.

How should vitamin B12 be administered to a patient with pernicious anemia?

C. Functions of Vitamin B12

Vitamin B12 is involved in two reactions in the body: the transfer of a methyl group from N 5-methyl-FH4 to homocysteine to form methionine and the rearrangement of L-methylmalonyl-CoA to form succinyl-CoA (Fig. 40.8). FH4 receives a one-carbon group from serine or from other sources. This carbon is reduced to the methyl level and transferred to vitamin B12, forming methyl-B12 (or methylcobalamin). Methylcobalamin transfers the methyl group to homocysteine, which is converted to methionine by the enzyme methionine synthase. Methionine can then be activated to S-adenosylmethionine (SAM) to transfer the methyl group to other compounds (Fig. 40.9). Vitamin B12 also participates in the conversion of L-methylmalonyl-CoA to succinyl-CoA. In this case, the active form of the coenzyme is 5⬘-deoxyadenosylcobalamin.

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SECTION VII ■ NITROGEN METABOLISM

Because the problem in pernicious anemia is a lack of intrinsic factor, which results in an inability to absorb vitamin B12 from the gastrointestinal tract, B12 cannot be administered orally to treat this condition. In the past, it was usually given by injection. An effective nasal spray containing B12 has recently been marketed, however, and its use precludes the need for lifelong injections of this vitamin.

Dietary B12 (

) Stomach Gastric mucosa R-binders Parietal cells Intrinsic factor

Pancreas Proteases

A

Liver

SH ~50%

CH2

B12

CH2 H

C

+

NH3

Transcobalamin II

COO–

Blood

Homocysteine Ileum

B12 • CH3

Methylcobalamin

B12

~50%

Other tissues

FIG. 40.7. Absorption, transport, and storage of vitamin B12. Dietary B12 binds to R-binders (haptocorrins) in the stomach and travels to the intestine, where the R-binders are destroyed by pancreatic proteases. The freed B12 then binds to intrinsic factor (IF). B12 is absorbed in the ileum and carried by proteins named transcobalamins (TC) to the liver, where B12 is stored.

CH3 S CH2 CH2 H

C

+

NH3

COO– Methionine

B H

–

H

COO

C

C H

H

C ~ SCoA

III. S-ADENOSYLMETHIONINE

O Methylmalonyl CoA B12

Adenosyl cobalamin

COO– H

C

H

H

C

H

C ~ SCoA O Succinyl CoA

FIG. 40.8. The two reactions involving vitamin B12 in humans.

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This reaction is part of the metabolic route for the conversion of carbons from valine, isoleucine, threonine, and the last three carbons of odd-chain fatty acids, all of which form propionyl-CoA, to the tricarboxylic acid (TCA) cycle intermediate succinyl-CoA (see Chapter 39).

SAM participates in the synthesis of many compounds that contain methyl groups. It is used in reactions that add methyl groups to either oxygen or nitrogen atoms in the acceptor (contrast that to folate derivatives, which can add one-carbon groups to sulfur or to carbon). As examples, SAM is required for the conversion of phosphatidylethanolamine to phosphatidylcholine, guanidinoacetate to creatine, norepinephrine to epinephrine, acetylserotonin to melatonin, and nucleotides to methylated nucleotides (see Fig. 40.9B). It is also required for the inactivation of catecholamines and serotonin (see Chapter 48). More than 35 reactions in humans require methyl donation from SAM. SAM is synthesized from methionine and adenosine triphosphate (ATP). As with the activation of vitamin B12, ATP donates the adenosine. With the transfer of its methyl group, SAM forms S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed to form homocysteine and adenosine. Methionine, required for the synthesis of SAM, is obtained from the diet or produced from homocysteine, which accepts a methyl group from vitamin B12 (see Fig. 40.9A). Thus, the methyl group of methionine is regenerated. The portion

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A

Methionine

FH4 • CH3

ATP 3Pi

B12

S-adenosylmethionine (SAM) Precursor CH3 –product FH4

B12 • CH3

S-adenosylhomocysteine (SAH) Homocysteine

B

Precursor

Adenosine

Methylated product SAM

Norepinephrine

Epinephrine SAM Creatine

Guanidinoacetate SAM Nucleotides

Methylated nucleotides SAM

Phosphatidylethanolamine

Phosphatidylcholine SAM

Acetylserotonin

Melatonin

FIG. 40.9. Relationships among tetrahydrofolate (FH4), B12, and S-adenosymelthionine (SAM). A. Overall scheme. B. Some specific reactions that require SAM.

of methionine that is essential in the diet is the homocysteine moiety. If we had an adequate dietary source of homocysteine, methionine would not be required in the diet. However, there is no good dietary source of homocysteine, whereas methionine is plentiful in the diet. Homocysteine provides the sulfur atom for the synthesis of cysteine (see Chapter 39). In this case, homocysteine reacts with serine to form cystathionine, which is cleaved, yielding cysteine and ␣-ketobutyrate. The first reaction in this sequence is inhibited by cysteine. Thus, methionine, via homocysteine, is not used for cysteine synthesis unless the levels of cysteine in the body are lower than required for its metabolic functions. An adequate dietary supply of cysteine, therefore, can “spare” (or reduce) the dietary requirement for methionine.

753

There are two major clinical manifestations of cobalamin (B12) deficiency. One such presentation is hematopoietic (caused by the adverse effects of a B12 deficiency on folate metabolism), and the other is neurologic (caused by hypomethylation in the nervous system). The hemopoietic problems associated with a B12 deficiency are identical to those observed in a folate deficiency and, in fact, result from a folate deficiency secondary to (i.e., caused by) the B12 deficiency (i.e., the methyl-trap hypothesis). As the tetrahydrofolate (FH4) pool is exhausted, deficiencies of the FH4 derivatives needed for purine and dTMP biosynthesis develop, leading to the characteristic megaloblastic anemia. The classical clinical presentation of the neurologic dysfunction associated with a B12 deficiency includes symmetric numbness and tingling of the hands and feet, diminishing vibratory and position sense, and progression to a spastic gait disturbance. The patient may become somnolent or may become extremely irritable (“megaloblastic madness”). Eventually, blind spots in the central portions of the visual fields develop, accompanied by alterations in gustatory (taste) and olfactory (smell) function. This is believed to be caused by hypomethylation within the nervous system brought about by an inability to recycle homocysteine to methionine and from there to S-adenosylmethionine (SAM). The latter is the required methyl donor in these reactions. The nervous system lacks the betaine pathway of methionine regeneration (see Fig. 40.10) and is dependent on the B12 system. With a B12 deficiency, this pathway is inoperable in the nervous system.

IV. RELATIONSHIPS AMONG FOLATE, VITAMIN B12, AND S-ADENOSYLMETHIONINE A. The Methyl-Trap Hypothesis If one analyzes the flow of carbon in the folate cycle, the equilibrium lies in the direction of the N 5-methyl-FH4 form. This appears to be the most stable form of carbon attached to the vitamin. However, in only one reaction can the methyl group be removed from N 5-methyl-FH4, and that is the methionine synthase reaction, which requires vitamin B12. Thus, if vitamin B12 is deficient, or if the methionine synthase enzyme is defective, N 5-methyl-FH4 accumulates. Eventually, most folate forms in the body become “trapped” in the N 5-methyl form. A functional folate deficiency results because the carbons cannot be removed from the folate. The appearance of a functional folate deficiency caused by a lack of vitamin B12 is known as the methyl-trap hypothesis, and its clinical implications are discussed in following sections.

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Many health food stores now sell SAMe, a stabilized version of SAM. SAMe has been hypothesized to relieve depression because the synthesis of certain neurotransmitters requires methylation by SAM (see Chapter 48). This has led to the hypothesis that by increasing SAM levels in the nervous system, the biosynthesis of these neurotransmitters will be accelerated. This in turn might alleviate the feelings of depression. There have been reports in the literature indicating that this may occur, but its efficacy as an antidepressant must be confirmed. The major questions that must be addressed include the stability of SAMe in the digestive system and the level of uptake of SAMe by cells of the nervous system.

B. Hyperhomocysteinemia Elevated homocysteine levels have been linked to cardiovascular and neurologic disease. Homocysteine levels can accumulate in several ways, which are related to both folic acid and vitamin B12 metabolism. Homocysteine is derived from SAM, which arises when SAM donates a methyl group (Fig. 40.10). Because SAM is frequently donating methyl groups, there is constant production of SAH, which leads to constant production of homocysteine. Recall from Chapter 39 that homocysteine has two biochemical fates. The homocysteine produced can be either remethylated to methionine or condensed with serine to form cystathionine. There are two routes to methionine production. The major one is methylation by N 5-methyl-FH4, which requires vitamin B12. The liver also contains a second pathway in which betaine (a degradation product of choline) can donate a methyl group to homocysteine to form methionine, but this is a minor pathway. The conversion of homocysteine to cystathionine requires pyridoxal phosphate (PLP). Thus, if an individual is deficient in vitamin B12, the conversion of homocysteine to methionine by the major route is inhibited. This directs homocysteine to produce cystathionine, which eventually produces cysteine. As cysteine levels accumulate, the enzyme that makes cystathionine undergoes feedback inhibition, and that pathway is also inhibited (see Fig. 40.10). This, overall, leads to accumulation of homocysteine, which is released into the blood. Homocysteine also accumulates in the blood if a mutation is present in the enzyme that converts N 5,N10-methylene-FH4 to N 5-methyl-FH4. When this occurs, the levels of N 5-methyl-FH4 are too low to allow homocysteine to be converted to methionine. The loss of this pathway, coupled with the feedback inhibition by cysteine on cystathionine formation, also leads to elevated homocysteine levels in the blood. A third way in which serum homocysteine levels can be elevated is by a mutated cystathionine ␤-synthase or a deficiency in vitamin B6, the required cofactor for that enzyme. These defects block the ability of homocysteine to be converted to cystathionine, and the homocysteine that does accumulate cannot

N 5,N 10-methylene-FH4

NADH

Glycine Serine

2

FH4

1

NAD+ N 5-methyl-FH4

ATP Methionine

B12

Homocysteine

Dimethyl glycine Betaine

PPi, Pi

SAM R R CH3

S-adenosyl homocysteine

B6

3

Adenosine Serine

–

Cystathionine B6

␣-Ketobutyrate, NH3

Cysteine

FIG. 40.10. Reaction pathways that involve homocysteine. Defects in numbered enzymes (1, methionine synthase; 2, N 5,N10-methylene-FH4 reductase; 3, cystathionine ␤-synthase) lead to elevated homocysteine. Recall that as cysteine accumulates, there is feedback inhibition on cystathionine ␤-synthase to stop further cysteine production.

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755

all be accommodated by conversion to methionine. Thus, an accumulation of homocysteine results.

C. Neural Tube Defects Folate deficiency during pregnancy has been associated with an increased risk of neural tube defects in the developing fetus. This risk is significantly reduced if women take folic acid supplements periconceptually. The link between folate deficiency and neural tube defects was first observed in women with hyperhomocysteinemia brought about by a thermolabile variant of N 5,N10-methyleneFH4 reductase. This form of the enzyme, which results from a single nucleotide change (C to T) in position 677 of the gene that encodes the protein, is less active at body temperature than at lower temperatures. This results in a reduced level of N 5-methyl-FH4 being generated and, therefore, an increase in the levels of homocysteine. Along with the elevated homocysteine, the women were also folate deficient. The folate deficiency and the subsequent inhibition of DNA synthesis leads to neural tube defects. The elevated homocysteine is one indication that such a deficit is present. These findings have led to the recommendation that women considering getting pregnant begin taking folate supplements before conception occurs, and for at least 1 month after conception. The US Department of Agriculture has, in fact, mandated that folate be added to flour-containing products in the United States.

D. Folate Deficiencies and DNA Synthesis Folate deficiencies result in decreased availability of the deoxythymidine and purine nucleotides that serve as precursors for DNA synthesis. The decreased concentrations of these precursors affect not only the DNA synthesis that occurs during replication before cell division, but also the DNA synthesis that occurs as a step in the processes that repair damaged DNA. Decreased methylation of dUMP to form dTMP, a reaction that requires N 5,N10-methylene-FH4 as a coenzyme (see Fig. 40.5), leads to an increase in the intracellular dUTP/deoxythymidine triphosphate (dTTP) ratio. This change causes a significant increase in the incorporation of uracil into DNA. Although much of this uracil can be removed by DNA-repair enzymes, the lack of available dTTP blocks the step of DNA repair that is catalyzed by DNA polymerase. The result is fragmentation of DNA as well as blockade of normal DNA replication. These abnormal nuclear processes are responsible for the clumping and polysegmentation seen in the nuclei of neutrophilic leukocytes in the bone marrow and in the peripheral blood of patients with megaloblastic anemia caused either by a primary folate deficiency or one that is secondary to B12 deficiency. The abnormalities in DNA synthesis and repair lead to an irreversible loss of the capacity for cell division and eventually to cell death.

V. CHOLINE AND ONE-CARBON METABOLISM Other compounds involved in one-carbon metabolism are derived from degradation products of choline. Choline, an essential component of certain phospholipids, is oxidized to form betaine aldehyde, which is further oxidized to betaine (trimethylglycine). In the liver, betaine can donate a methyl group to homocysteine to form methionine and dimethyl glycine. This allows the liver to have two routes for homocysteine conversion to methionine. This is in contrast to the nervous system, which only expresses the primary B12-requiring pathway. Under conditions in which SAM accumulates, glycine can be methylated to form sarcosine (N-methyl glycine). This route is used when methionine levels are high and excess methionine needs to be metabolized (Fig. 40.11).

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SECTION VII ■ NITROGEN METABOLISM

HO

CH2

+

CH2

N

NAD+

Choline

O –O

O

(CH3)3 NADH

C

CH2

+

N

O

(CH3)3

H

NAD+

CH3 C

CH2

+N

–O

CH3 Betaine

S

CH2

CH2

CH2

CH2

C

+ NH3

COO– Homocysteine

H

C

C

CH2

CH2

+

N

(CH3)3

Betaine

N

CH3 CH3

Dimethylglycine

CH3 SH

C

NADH

O

CH3

H

–O

+

NH3

COO– Methionine

FIG. 40.11. Choline and one-carbon metabolism.

CLINICAL COMMENTS Jean Ann Tonich. Jean Ann Tonich developed a folate deficiency and is on the verge of developing a cobalamin (vitamin B12) deficiency as a consequence of prolonged, moderately severe malnutrition related to chronic alcoholism. Before folate therapy is started, the physician must ascertain that the megaloblastic anemia is not caused by a pure B12 deficiency or a combined deficiency of folate and B12. If folate is given without cobalamin to a B12-deficient patient, the drug only partially corrects the megaloblastic anemia because it will “bypass” the methyl folate trap and provide adequate tetrahydrofolate (FH4) coenzyme for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and for a resurgence of purine synthesis. As a result, normal DNA synthesis, DNA repair, and cell division occur. However, the neurologic syndrome resulting from hypomethylation in nervous tissue may progress unless the physician realizes that B12 supplementation is required. In Jean Ann’s case, in which the serum B12 concentration was borderline low and in which the dietary history supported the possibility of a B12 deficiency, a combination of folate and B12 supplements is required to avoid this potential therapeutic trap. Colin Tuma. Colin Tuma continued to do well and returned faithfully for his regular colonoscopic examinations. Bea Twelvlow. Bea Twelvlow was diagnosed with an inability to absorb dietary B12 but not crystalline B12 (the Schilling test results were normal). One of the consequences of aging is a reduced acid production by the gastric mucosa (atrophic gastritis), which limits the ability of pepsin to work on dietary protein. Reduced pepsin efficiency then reduces the amount of bound B12 released from dietary protein as a result of which the B12 is not available for absorption. Because Bea absorbs crystalline B12 without a problem, her condition can be easily treated by taking vitamin B12 supplements orally.

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757

BIOCHEMICAL COMMENTS A potential mechanism relating to folate deficiencies and neural tube defects. As indicated in Section IV.C of this chapter, neural tube defects in newborns have been associated with folate deficiency during pregnancy. Although the mechanism leading to neural tube defects is vague, new research has indicated that the induction of micro RNAs (miRNAs) may play a role in altering the normal developmental pattern of neural tube closure. Under conditions of a folate deficiency, hypomethylation occurs in the nervous system, affecting membrane phospholipid biosynthesis (such as phosphatidylcholine), myelin basic protein (see Chapter 48), and neurotransmitter biosynthesis (see Chapter 48). The reduced levels of neurotransmitters may interfere with normal gene expression during embryogenesis. DNA methylation is also reduced because of reduced S-adenosylmethionine (SAM) levels when folate is limiting. Hypomethylation is also the result of increased levels of S-adenosylhomocysteine (SAH), which accumulates during a folate deficiency. SAH will inhibit DNA methyltransferase enzymes by tightly binding to the enzyme and preventing the normal substrate, SAM, from binding to the enzyme. The enzymes affinity for SAH is higher than that of SAM, contributing to the hypomethylation observed. MiRNA genes are frequently near CpG islands in DNA, and it has been predicted that alterations in cytosine methylation may be a means of regulating miRNA expression. Experimentally, a cell line in which two DNA methyltransferase genes were knocked out (inactivated) resulted in a significant reduction in global genomic methylation and the differential expression of 13 miRNAs (seven of those genes were overexpressed, whereas the other 6 displayed a reduction in expression). A similar result was obtained with another cell line that was placed in folate-deficient media; global hypomethylation and alterations in miRNA expression were observed. As an example, miR-222 was identified as a potential miRNA that is upregulated under conditions of folate deprivation. A predicted target of miR-222 is the DNMT1 gene (a DNA methyltransferase), whose activity is critical for the maintenance of methylation patterns in DNA. Overexpression of miR-222 would reduce DNMT1 gene expression, thereby altering methylation patterns in the cell. A reduction of DNMT1 activity has been shown to increase the expression of several genes, including ␤-catenin (see Chapter 18). This will lead to enhanced cell proliferation and inhibition of differentiation in the nervous system (all leading to a failure to close the neural tube). Such studies as those described previously are in their infancy but produced a promising start for unraveling the effects of DNA methylation and miRNA expression on cell growth and differentiation in the nervous system.

Key Concepts •

• • • • •

One-carbon groups at lower oxidation states than carbon dioxide (which is carried by biotin) are transferred by reactions that involve tetrahydrofolate (FH4), vitamin B12, and S-adenosylmethionine (SAM). FH4 is produced from the vitamin folate and obtains one-carbon units from serine, glycine, histidine, formaldehyde, and formic acid. The carbon attached to FH4 can be oxidized or reduced, thus producing several different forms of FH4. However, once a carbon has been reduced to the methyl level, it cannot be reoxidized. The carbons attached to FH4 are known collectively as the one-carbon pool. The carbons carried by folate are used in a limited number of biochemical reactions, but they are very important in forming deoxythymidine monophosphate (dTMP) and the purine rings. Vitamin B12 participates in two reactions in the body: conversion of L-methylmalonyl-CoA to succinyl-CoA and conversion of homocysteine to methionine.

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Table 40.2

Diseases Discussed in Chapter 40

Disease or Disorder

Environmental or Genetic

Colon cancer

Both

Pernicious anemia

Both

Alcohol-induced megaloblastic anemia

Environmental

Neural tube defects

Both

Comments Colon cancer can be treated by drugs, which block the action of thymidylate synthase, blocking DNA synthesis by reducing the supply of dTTP. Pernicious anemia is caused by the lack of intrinsic factor, which leads to a B12 deficiency. The B12 deficiency indirectly interferes with DNA synthesis. In cells of the erythroid lineage, cell size increases without cell division leading to megaloblastic anemia. Alcohol-induced malnutrition that can lead to folate and/or B12 deficiencies. The folate and/or B12 deficiency will lead to the development of megaloblastic anemia. A lack of folate derivatives leads to reduced methylation in the nervous system, altering gene expression and increasing the risk of neural tube defects.

dTTP, deoxythymidine triphosphate.

• •

•

SAM, formed from adenosine triphosphate (ATP) and methionine, transfers the methyl group to precursors of a variety of methylated compounds. Both vitamin B12 and methyl-FH4 are required in methionine metabolism; a deficiency of vitamin B12 leads to overproduction and trapping of folate in the methyl form, leading to a functional folate deficiency. Such deficiencies can lead to Megaloblastic anemia Neural tube defects in newborns Diseases discussed in this chapter are summarized in Table 40.2.

REVIEW QUESTIONS—CHAPTER 40 1.

2.

3.

Which of the following reactions requires N 5,N10methylene-FH4 as a carbon donor? A. Homocysteine to methionine B. Serine to glycine C. Betaine to dimethylglycine D. dUMP to dTMP E. The de novo biosynthesis of the purine ring Propionic acid accumulation from amino acid degradation will result from a deficiency of which of the following vitamins? A. Vitamin B6 B. Biotin C. Folic acid D. Vitamin B12 E. Vitamin B1 F. Vitamin B2 A dietary vitamin B12 deficiency can result from which of the following? A. Excessive intrinsic factor production by the gastric parietal cells B. Eating a diet that is high in animal protein

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C. Pancreatic insufficiency D. Increased absorption of folic acid E. Inability to conjugate the vitamin with glutamic acid 4.

Which of the following forms of tetrahydrofolate is required for the synthesis of methionine from homocysteine? A. N 5,N10-methylene tetrahydrofolate B. N 5-methyl tetrahydrofolate C. N 5,N10-methenyl tetrahydrofolate D. N10-formyl tetrahydrofolate E. N 5-formimino tetrahydrofolate

5.

An alternative method to methylate homocysteine to form methionine is which of the following? A. Using glycine and FH4 as the methyl donor B. Using dimethylglycine as the methyl donor C. Using choline as the methyl donor D. Using sarcosine as the methyl donor E. Using betaine as the methyl donor

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41

Purine and Pyrimidine Metabolism

Purines and pyrimidines are required for synthesizing nucleotides and nucleic acids. These molecules can be synthesized either from scratch, de novo, or salvaged from existing bases. Dietary uptake of purine and pyrimidine bases is low because most of the ingested nucleic acids are metabolized by the intestinal epithelial cells. The de novo pathway of purine synthesis is complex, consisting of 11 steps, and requiring six molecules of adenosine triphosphate (ATP) for every purine synthesized. The precursors that donate components to produce purine nucleotides include glycine, ribose 5-phosphate, glutamine, aspartate, carbon dioxide, and N10-formyl-FH4 (Fig. 41.1). Purines are synthesized as ribonucleotides, with the initial purine synthesized being inosine monophosphate (IMP). Adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are each derived from IMP in two-step reaction pathways. The purine nucleotide salvage pathway allows free purine bases to be converted into nucleotides, nucleotides into nucleosides, and nucleosides into free bases. Enzymes included in this pathway are AMP and adenosine deaminase, adenosine kinase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase (APRT), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mutations in a number of these enzymes lead to serious diseases. Deficiencies in purine nucleoside phosphorylase and adenosine deaminase lead to immunodeficiency disorders. A deficiency in HGPRT leads to Lesch–Nyhan syndrome. The purine nucleotide cycle, in which aspartate carbons are converted to fumarate to replenish tricarboxylic acid (TCA) cycle intermediates in working muscle, and the aspartate nitrogen is released as ammonia, uses components of the purine nucleotide salvage pathway. Pyrimidine bases are first synthesized as the free base and then converted to a nucleotide. Aspartate and carbamoyl phosphate form all components of the pyrimidine ring. Ribose 5-phosphate, which is converted to phosphoribosyl pyrophosphate (PRPP), is required to donate the sugar phosphate to form a nucleotide. The first pyrimidine nucleotide produced is orotate monophosphate (OMP). The OMP is converted to uridine monophosphate (UMP), which becomes the precursor for both cytidine triphosphate (CTP) and deoxythymidine monophosphate (dTMP) production. The formation of deoxyribonucleotides requires ribonucleotide reductase activity, which catalyzes the reduction of ribose on nucleotide diphosphate substrates to 2⬘-deoxyribose. Substrates for the enzyme include adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and uridine diphosphate (UDP). Regulation of the enzyme is complex. There are two major allosteric sites. One controls the overall activity of the enzyme, whereas the other determines the substrate specificity of the enzyme. All deoxyribonucleotides are synthesized using this one enzyme. The regulation of de novo purine nucleotide biosynthesis occurs at four points in the pathway. The enzymes PRPP synthetase, amidophosphoribosyl transferase, IMP dehydrogenase, and adenylosuccinate synthetase are regulated by allosteric modifiers, as they occur at key branch points through the

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CO2 Aspartate (N) N10-formylFH4

N1

6 5

2 3

4

N

Glutamine (amide N)

Glycine N 7

N10-formylFH4 N Glutamine RP (amide N) 8

9

FIG. 41.1. Origin of the atoms of the purine base. FH4, tetrahydrofolate; RP, ribose 5⬘-phosphate.

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SECTION VII ■ NITROGEN METABOLISM

pathway. Pyrimidine synthesis is regulated at the first committed step, which is the synthesis of cytoplasmic carbamoyl phosphate, by the enzyme carbamoyl phosphate synthetase II (CPSII). Purines, when degraded, cannot generate energy, nor can the purine ring be substantially modified. The end product of purine ring degradation is uric acid, which is excreted in the urine. Uric acid has limited solubility, and if it were to accumulate, uric acid crystals would precipitate in tissues of the body that have a reduced temperature (such as the big toe). This condition of acute painful inflammation of specific soft tissues and joints is called gout. Pyrimidines, when degraded, however, give rise to water-soluble compounds such as urea, carbon dioxide, and water and do not lead to a disease state if pyrimidine catabolism is increased.

THE WAITING ROOM The initial acute inflammatory process that caused Lotta Topaigne to experience a painful attack of gouty arthritis responded quickly to colchicine therapy (see Chapter 10). Several weeks after the inflammatory signs and symptoms in her right great toe subsided, Lotta was placed on allopurinol, a drug that reduces uric acid synthesis. Her serum uric acid level gradually fell from a pretreatment level of 9.2 mg/dL into the normal range (2.5 to 8.0 mg/dL). She remained free of gouty symptoms when she returned to her physician for a follow-up office visit.

I.

PURINES AND PYRIMIDINES

As has been seen in previous chapters, nucleotides serve numerous functions in different reaction pathways. For example, nucleotides are the activated precursors of DNA and RNA. Nucleotides form the structural moieties of many coenzymes (examples include NADH, FAD, and coenzyme A). Nucleotides are critical elements in energy metabolism (adenosine triphosphate [ATP], guanosine triphosphate [GTP]). Nucleotide derivatives are frequently activated intermediates in many biosynthetic pathways. For example, uridine diphosphate (UDP)-glucose and cytidine diphosphate (CDP)-diacylglycerol are precursors of glycogen and phosphoglycerides, respectively. S-Adenosylmethionine carries an activated methyl group. In addition, nucleotides act as second messengers in intracellular signaling (e.g., cyclic adenosine monophosphate [cAMP], cyclic guanosine monophosphate [cGMP]). Finally, nucleotides and nucleosides act as metabolic allosteric regulators. Think about all of the enzymes that have been studied that are regulated by levels of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP). Dietary uptake of purine and pyrimidine bases is minimal. The diet contains nucleic acids and the exocrine pancreas secretes deoxyribonuclease and ribonuclease, along with the proteolytic and lipolytic enzymes. This enables digested nucleic acids to be converted to nucleotides. The intestinal epithelial cells contain alkaline phosphatase activity, which converts nucleotides to nucleosides. Other enzymes within the epithelial cells tend to metabolize the nucleosides to uric acid (which is released into the circulation) or to salvage them for their own needs. Approximately 5% of ingested nucleotides make it into the circulation, either as the free base or as a nucleoside. Because of the minimal dietary uptake of these important molecules, de novo synthesis of purines and pyrimidines is required.

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II. PURINE BIOSYNTHESIS

Ribose 5-phosphate + ATP

The purine bases are produced de novo by pathways that use amino acids as precursors and produce nucleotides. Most de novo synthesis occurs in the liver (Fig. 41.2), and the nitrogenous bases and nucleosides are then transported to other tissues by red blood cells. The brain also synthesizes significant amounts of nucleotides. Because the de novo pathway requires at least six high-energy bonds per purine produced, a salvage pathway, which is used by many cell types, can convert free bases and nucleosides to nucleotides.

Glutamine + PRPP

Glycine (+ ATP) N10-formyl-FH4 (C8) Glutamine (N3) (+ ATP) (+ ATP to close ring)

A. De Novo Synthesis of the Purine Nucleotides 1.

761

SYNTHESIS OF INOSINE MONOPHOSPHATE

As purines are built on a ribose base (see Fig. 41.2), an activated form of ribose is used to initiate the purine biosynthetic pathway. 5-Phosphoribosyl-1-pyrophosphate (PRPP) is the activated source of the ribose moiety. It is synthesized from ATP and ribose 5⬘-phosphate (Fig. 41.3), which is produced from glucose through the pentose phosphate pathway (see Chapter 29). The enzyme that catalyzes this reaction, PRPP synthetase, is a regulated enzyme (see Section II.A.5); however, this step is not the committed step of purine biosynthesis. PRPP has many other uses, which are described as the chapter progresses. In the first committed step of the purine biosynthetic pathway, PRPP reacts with glutamine to form 5-phosphoribosyl 1-amine (Fig. 41.4). This reaction, which produces nitrogen 9 of the purine ring, is catalyzed by glutamine phosphoribosyl amidotransferase, a highly regulated enzyme. In the next step of the pathway, the entire glycine molecule is added to the growing precursor. Glycine provides carbons 4 and 5 and nitrogen 7 of the purine ring (Fig. 41.5). This step requires energy in the form of ATP. Subsequently, carbon 8 is provided by N10-formyl-FH4, nitrogen 3 by glutamine, carbon 6 by CO2, nitrogen 1 by aspartate, and carbon 2 by N10-formyltetrahydrofolate (see Fig. 41.1). Note that six high-energy bonds of ATP are required (starting with ribose 5-phosphate) to synthesize the first purine nucleotide, inosine monophosphate (IMP). This nucleotide contains the base hypoxanthine joined by an N-glycosidic bond from nitrogen 9 of the purine ring to carbon 1 of the ribose (Fig. 41.6). Hypoxanthine is not found in DNA, but it is the precursor for the other purine bases.

CO2 (C6) Aspartate (N1) (+ ATP) N10-formyl-FH4 (C2) GTP Aspartate

IMP ATP Glutamine

AMP

GMP

ADP

GDP

GTP RNA ATP

RR

RR

dGDP dGTP dADP

DNA dATP

FIG. 41.2. Overview of purine production, starting with glutamine, ribose 5-phosphate, and ATP. The steps that require ATP are also indicated in this figure. FH4, tetrahydrofolate; PRPP, 5-phosphoribosyl-1-pyrophosphate; RR, ribonucleotide reductase.

O –

O

O CH2

P O–

O H H OH

H H OH

OH

Ribose 5-phosphate ATP PRPP synthetase

AMP O –

O

O CH2

P O

O H

–

H H OH

H O OH

O P

O O

–

O

O–

P –

O

5-Phosphoribosyl 1-pyrophosphate (PRPP)

FIG. 41.3. Synthesis of PRPP. Ribose 5-phosphate is produced from glucose by the pentose phosphate pathway.

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SECTION VII ■ NITROGEN METABOLISM

P

O

O CH2 H H

H H O

OH

P

O

P

OH

PRPP H2O Glutamine phosphoribosyl amidotransferase

P

O CH2

O

H H

P

OH

H 2C

Phosphoribosylglycinamide synthetase

O C

ADP + Pi

O–

NH+3

O C

NH

O

O H2C H H

H H

OH

OH

Glycinamide ribosyl 5-phosphate

FIG. 41.5. Incorporation of glycine into the purine precursor. The ATP is required for the condensation of the glycine carboxylic acid group with the 1⬘-amino group of phosphoribosyl 1-amine.

O C

HC

C

N

H

H

N

O CH2

H H OH

O

OH

Inosine monophosphate (IMP)

FIG. 41.6. Structure of inosine monophosphate (IMP). The base is hypoxanthine.

Lieberman_CH41.indd 762

NH+3 H H OH

5-Phosphoribosyl 1-amine

FIG. 41.4. The first step in purine biosynthesis. The purine base is built on the ribose moiety. The availability of the substrate PRPP is a major determinant of the rate of this reaction.

However, hypoxanthine is found in the anticodon of transfer RNA (tRNA) molecules (see Chapter 15) and is a critical component for allowing wobble base pairs to form. 2.

SYNTHESIS OF ADENOSINE MONOPHOSPHATE

IMP serves as the branch point from which both adenine and guanine nucleotides can be produced (see Fig. 41.2). AMP is derived from IMP in two steps (Fig. 41.7). In the first step, aspartate is added to IMP to form adenylosuccinate, a reaction similar to the one catalyzed by argininosuccinate synthetase in the urea cycle. Note that this reaction requires a high-energy bond, donated by GTP. Fumarate is then released from the adenylosuccinate by the enzyme adenylosuccinase to form AMP. This is similar to the aspartate-to-fumarate conversion seen in the urea cycle (see Chapter 38). In both cases, aspartate donates a nitrogen to the product, while the carbons of aspartate are released as fumarate. 3.

SYNTHESIS OF GUANOSINE MONOPHOSPHATE

Guanosine monophosphate (GMP) is also synthesized from IMP in two steps (Fig. 41.8). In the first step, the hypoxanthine base is oxidized by IMP dehydrogenase to produce the base xanthine and the nucleotide xanthosine monophosphate (XMP). Glutamine then donates the amide nitrogen to XMP to form GMP in a reaction that is catalyzed by GMP synthetase. This second reaction requires energy in the form of ATP.

N

HN

CH

P

OH

NH+3

O

Glycine

H2C

P

O CH2 H H

5-Phosphoribosyl 1-amine ATP

Glutamate PPi

NH+3 H H

OH

Glutamine

4.

PHOSPHORYLATION OF AMP AND GMP

AMP and GMP can be phosphorylated to the diphosphate and triphosphate levels. The production of nucleoside diphosphates requires specific nucleoside monophosphate kinases, whereas the production of nucleoside triphosphates requires nucleoside diphosphate kinases, which are active with a wide range of nucleoside diphosphates. The purine nucleoside triphosphates are also used for energy-requiring processes in the cell and also as precursors for RNA synthesis (see Fig. 41.2). 5.

REGULATION OF PURINE SYNTHESIS

Regulation of purine synthesis occurs at several sites (Fig. 41.9). Four key enzymes are regulated: PRPP synthetase, amidophosphoribosyl transferase, adenylosuccinate synthetase, and IMP dehydrogenase. The first two enzymes regulate IMP synthesis; the last two regulate the production of AMP and GMP, respectively.

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CHAPTER 41 ■ PURINE AND PYRIMIDINE METABOLISM

O

O N

HN

N

HN

N

N

N

N

R5P IMP

R5P IMP

O C

NAD+ + H2O

O–

CH2

GTP

H C NH3+ C

GDP, Pi

O

O

H O C C CH2 C O– NH N N

O

O

Gln GMP synthetase

Glutamate O–

CH

AMP, PPi O

CH O–

O Fumarate

N N

N

N

HN HN H

N

N

R5P GMP

NH2 N

R5P

ATP

O

C

N

N H XMP

R5P Adenylosuccinate

C

N

HN

N

N

IMP dehydrogenase

NADH + H+

O–

Aspartate

–O

763

FIG. 41.8. The conversion of IMP to GMP. Note that ATP is required for the synthesis of GMP.

R5P AMP

FIG. 41.7. The conversion of IMP to AMP. Note that GTP is required for the synthesis of AMP. R5P, ribose 5-phosphate.

A primary site of regulation is the synthesis of PRPP. PRPP synthetase is negatively affected by guanosine diphosphate (GDP) and, at a distinct allosteric site, by ADP. Thus, the simultaneous binding of an oxypurine (e.g., GDP) and an aminopurine (e.g., ADP) can occur, with the result being a synergistic inhibition of the enzyme. This enzyme is not the committed step of purine biosynthesis; PRPP is also used in pyrimidine synthesis and both the purine and pyrimidine salvage pathways. The committed step of purine synthesis is the formation of 5-phosphoribosyl 1-amine by glutamine phosphoribosyl amidotransferase. This enzyme is strongly inhibited by GMP and AMP (the end products of the purine biosynthetic pathway). The enzyme is also inhibited by the corresponding nucleoside diphosphates and triphosphates, but under cellular conditions, these compounds probably do not play a central role in regulation. The active enzyme is a monomer of 133,000 Da but is converted to an inactive dimer (270,000 Da) by binding of the end products. Cellular concentrations of PRPP and glutamine are usually below their Km for glutamine phosphoribosyl amidotransferase. Thus, any situation that leads to an increase in their concentration can lead to an increase in de novo purine biosynthesis.

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Ribose 5-phosphate PRPP synthetase –

–

5-Phosphoribosyl 1-pyrophosphate (PRPP) Glutamine phosphoribosyl amidotransferase –

–

5-Phosphoribosyl 1-amine

IMP

IMP dehydrogenase –

XMP

Adenylosuccinate synthetase –

Adenylosuccinate

GMP

AMP

GDP

ADP

GTP

ATP

FIG. 41.9. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric sites: one for ADP, the other for GDP. Glutamine phosphoribosyl amidotransferase contains adenine nucleotide– and guanine nucleotide–binding sites; the monophosphates are the most important, although the diphosphates and triphosphates will also bind to and inhibit the enzyme. Adenylosuccinate synthetase is inhibited by AMP; IMP dehydrogenase is inhibited by GMP.

The enzymes that convert IMP to XMP and adenylosuccinate are both regulated. GMP inhibits the activity of IMP dehydrogenase, and AMP inhibits adenylosuccinate synthetase. Note that the synthesis of AMP is dependent on GTP (of which GMP is a precursor), whereas the synthesis of GMP is dependent on ATP (which is made from AMP). This serves as a type of positive regulatory mechanism to balance the pools of these precursors: When the levels of ATP are high, GMP will be made; when the levels of GTP are high, AMP synthesis will take place. GMP and AMP act as negative effectors at these branch points, a classic example of feedback inhibition.

B. Purine Salvage Pathways

A deficiency in purine nucleoside phosphorylase activity leads to an immune disorder in which T-cell immunity is compromised. B-cell immunity, conversely, may be only slightly compromised or even normal. Children who lack this activity have recurrent infections and more than half display neurologic complications. Symptoms of the disorder first appear at between 6 months and 4 years of age.

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Most of the de novo synthesis of the bases of nucleotides occurs in the liver and to some extent in the brain, neutrophils, and other cells of the immune system. Within the liver, nucleotides can be converted to nucleosides or free bases, which can be transported to other tissues via the red blood cells in the circulation. In addition, the small amounts of dietary bases or nucleosides that are absorbed also enter cells in this form. Thus, most cells can salvage these bases to generate nucleotides for RNA and DNA synthesis. For certain cell types such as lymphocytes, the salvage of bases is the major form of nucleotide generation. The overall picture of salvage is shown in Figure 41.10. The pathways allow free bases, nucleosides, and nucleotides to be easily interconverted. The major enzymes required are purine nucleoside phosphorylase, phosphoribosyl transferases, and deaminases. Purine nucleoside phosphorylase catalyzes a phosphorolysis reaction of the N-glycosidic bond that attaches the base to the sugar moiety in the nucleosides guanosine and inosine (Fig. 41.11). Thus, guanosine and inosine are converted to

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CHAPTER 41 ■ PURINE AND PYRIMIDINE METABOLISM

Free Bases Adenine

Nucleotides APRT

PRPP

Nucleosides 5'-nucleotidase

AMP PPi

Pi

AMP deaminase

HGPRT

PRPP

adenosine deaminase

adenosine kinase

ADP

NH3 Hypoxanthine

Adenosine

ATP

NH3

5'-nucleotidase

IMP PPi

Inosine

Pi

Ribose 1-Phosphate purine nucleoside phosphorylase

Guanine

HGPRT

PRPP

GMP

5'-nucleotidase

PPi Ribose 1-Phosphate

Guanosine

Adenosine deaminase activity is measured by coupling the deamination of adenosine (to inosine) with purine nucleoside phosphorylase, which will generate hypoxanthine and ribose 1-phosphate from inosine. The hypoxanthine generated then reacts with xanthine oxidase, generating uric acid and hydrogen peroxide. The hydrogen peroxide produced is then measured in the presence of peroxidase and a colorless dye. The oxidation of the colorless dye creates a colored dye, and the intensity of the color (which is directly proportional to the amount of inosine produced) can be determined spectrophotometrically.

Pi

purine nucleoside phosphorylase

FIG. 41.10. Salvage of bases. The purine bases hypoxanthine and guanine react with PRPP to form the nucleotides inosine and guanosine monophosphate, respectively. The enzyme that catalyzes the reaction is hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Adenine forms AMP in a reaction that is catalyzed by adenine phosphoribosyltransferase (APRT). Nucleotides are converted to nucleosides by 5⬘-nucleotidase. Free bases are generated from nucleosides by purine nucleoside phosphorylase (although adenosine is not a substrate of this enzyme). Deamination of the base adenine occurs with AMP and adenosine deaminase. Of the purines, only adenosine can be phosphorylated directly back to a nucleotide by adenosine kinase.

guanine and hypoxanthine, respectively, along with ribose 1-phosphate. The ribose 1-phosphate can be isomerized to ribose 5-phosphate, and the free bases then salvaged or degraded, depending on cellular needs. The phosphoribosyl transferase enzymes catalyze the addition of a ribose 5-phosphate group from PRPP to a free base, generating a nucleotide and pyrophosphate (Fig. 41.12). Two enzymes do this for purine metabolism: adenine phosphoribosyl transferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The reactions they catalyze are the same, differing only in their substrate specificity. Adenosine and AMP can be deaminated by adenosine deaminase and AMP deaminase, respectively, to form inosine and IMP (see Fig. 41.10). Adenosine is also the only nucleoside to be directly phosphorylated to a nucleotide by adenosine kinase. Guanosine and inosine must be converted to free bases by purine nucleoside phosphorylase before they can be converted to nucleotides by HGPRT. A portion of the salvage pathway that is important in muscle is the purine nucleotide cycle (Fig. 41.13). The net effect of these reactions is the deamination of aspartate to fumarate (as AMP is synthesized from IMP and then deaminated back to IMP by AMP deaminase). Under conditions in which the muscle must generate energy, the fumarate derived from the purine nucleotide cycle is used anaplerotically to replenish tricarboxylic acid (TCA) cycle intermediates and to allow the cycle to operate at high speed. Deficiencies in enzymes of this cycle lead to muscle fatigue during exercise.

III. SYNTHESIS OF THE PYRIMIDINE NUCLEOTIDES A. De Novo Pathways In the synthesis of the pyrimidine nucleotides, the base is synthesized first, and then it is attached to the ribose 5⬘-phosphate moiety (Fig. 41.14). The origin of the atoms

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765

Lesch–Nyhan syndrome is caused by a defective hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (see Fig. 41.12). In this condition, purine bases cannot be salvaged. Instead, they are degraded, forming excessive amounts of uric acid. Individuals with the severe form of this syndrome suffer from mental retardation. They are also prone to chewing off their fingers and performing other acts of self-mutilation.

HO

Purine base

CH2 O

OH

OH

Purine nucleoside phosphorylase

HO

Pi

CH2 O O O P O– OH

OH

O–

Ribose 1-phosphate + Free purine base (hypoxanthine or guanine)

FIG. 41.11. The purine nucleoside phosphorylase reaction, which converts guanosine or inosine to ribose 1-phosphate plus the free base guanine or hypoxanthine.

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SECTION VII ■ NITROGEN METABOLISM

O –

O

O

O

O CH2

P

H

O

–

H H OH

H O

P

OH

O

O O

–

P

O–

O–

5-Phosphoribosyl 1-pyrophosphate (PRPP) Phosphoribosyltransferase

Base

PPi O –

O

P O–

O

O CH2

Base

H H

H H

OH

OH

Nucleotide

FIG. 41.12. The phosphoribosyltransferase reaction. APRT uses the free base adenine; HGPRT can use either hypoxanthine or guanine as a substrate.

of the ring (aspartate and carbamoyl phosphate, which is derived from carbon dioxide and glutamine) is shown in Figure 41.15. In the initial reaction of the pathway, glutamine combines with bicarbonate and ATP to form carbamoyl phosphate. This reaction is analogous to the first reaction of the urea cycle, except that it uses glutamine as the source of the nitrogen (rather than ammonia) and it occurs in the cytosol (rather than in mitochondria). The reaction is catalyzed by carbamoyl phosphate synthetase II (CPSII), which is the regulated step of the pathway. The analogous reaction in urea synthesis is catalyzed by carbamoyl phosphate synthetase I (CPSI), which is activated by N-acetylglutamate. The similarities and differences between these two carbamoyl phosphate synthetase enzymes are described in Table 41.1. In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction that is catalyzed by aspartate transcarbamoylase (Fig. 41.16). The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase), which is oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase. The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing

Adenylosuccinate (AS) AS synthetase

GDP, Pi

AS lyase

Fumarate Aspartate

GTP IMP

AMP

AMP deaminase

NH3

FIG. 41.13. The purine nucleotide cycle. Using a combination of biosynthetic and salvage enzymes, the net effect is the conversion of aspartate to fumarate plus ammonia, with the fumarate playing an anaplerotic role in the muscle. AS, adenylosuccinate.

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Glutamine + CO2 + 2ATP CPS-II UTP –

+

Glutamine (amide N) CO2

PRPP

Carbamoyl phosphate Aspartate

N3 2

767

Aspartate

4 5 1

6

N

FIG. 41.15. The origin of the atoms in the pyrimidine ring.

Orotate PRPP

CO2 UMP RR UDP

dUDP

UTP PI

Glutamine

RNA

NH4+

CTP

dUMP CDP

dCMP

FH2

RR

dCTP

dCDP

N5,N10-Methylene-FH4

dTMP

DNA dTTP

dTDP

FIG. 41.14. Synthesis of the pyrimidine bases. CPS-II, carbamoyl phosphate synthetase II; FH2 and FH4, forms of folate; RR, ribonucleotide reductase; 䊝, stimulated by; 䊞, inhibited by.

orotidine 5⬘-phosphate, which is decarboxylated by orotidylic acid decarboxylase to form uridine monophosphate (UMP) (see Fig. 41.16). In mammals, the first three enzymes of the pathway (CPSII, aspartate transcarbamoylase, and dihydroorotase) are located on the same polypeptide, designated as CAD. The last two enzymes of the pathway are similarly located on a polypeptide known as UMP synthase (the orotate phosphoribosyl transferase and orotidylic acid decarboxylase activities). UMP is phosphorylated to UTP. An amino group, derived from the amide of glutamine, is added to carbon 4 to produce cytidine triphosphate (CTP) by the enzyme CTP synthetase (this reaction cannot occur at the nucleotide monophosphate level). UTP and CTP are precursors for the synthesis of RNA (see Fig. 41.14). The synthesis of thymidine triphosphate (TTP) is described in Section IV.

Table 41.1 Comparison of Carbamoyl Phosphate Synthetases (CPSI and CPSII) Pathway Source of nitrogen Location Activator Inhibitor

CPSI

CPSII

Urea cycle NH4⫹ Mitochondria N-Acetylglutamate —

Pyrimidine biosynthesis Glutamine Cytosol PRPP UTP

PRPP, phosphoribosyl pyrophosphate; UTP, uridine triphosphate.

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SECTION VII ■ NITROGEN METABOLISM

–

O

B. Salvage of Pyrimidine Bases

O C

CH2 CH

+H N 3

COO–

Aspartate H2N C O O P Carbamoyl phosphate

Pi

–

O

O C

CH2

H2N O

CH

C N H

–

COO

Carbamoyl aspartate

O HN O

N H

COO

–

C. Regulation of De Novo Pyrimidine Synthesis

Orotic acid (orotate) PRPP

Orotate phosphoribosyltransferase

PPi

O HN O

–

N

Pyrimidine bases are normally salvaged by a two-step route. First, a relatively nonspecific pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their respective nucleosides (Fig. 41.17). Notice that the preferred direction for this reaction is the reverse phosphorylase reaction, in which phosphate is released and is not being used as a nucleophile to release the pyrimidine base from the nucleoside. The more specific nucleoside kinases then react with the nucleosides, forming nucleotides (Table 41.2). As with purines, further phosphorylation is carried out by increasingly more specific kinases. The nucleoside phosphorylase–nucleoside kinase route for synthesis of pyrimidine nucleoside monophosphates is relatively inefficient for salvage of pyrimidine bases because of the very low concentration of the bases in plasma and tissues. Pyrimidine phosphorylase can use all of the pyrimidines but has a preference for uracil and is sometimes called uridine phosphorylase. The phosphorylase uses cytosine fairly well but has a very, very low affinity for thymine; therefore, a ribonucleoside containing thymine is almost never made in vivo. A second phosphorylase, thymidine phosphorylase, has a much higher affinity for thymine and adds a deoxyribose residue (see Fig. 41.17). Of the various ribonucleosides and deoxyribonucleoside kinases, one that merits special mention is thymidine kinase (TK). This enzyme is allosterically inhibited by deoxythymidine triphosphate (dTTP). Activity of TK in a given cell is closely related to the proliferative state of that cell. During the cell cycle, the activity of TK rises dramatically as cells enter S-phase, and in general, rapidly dividing cells have high levels of this enzyme. Radiolabeled thymidine is widely used for isotopic labeling of DNA, for example, in radioautographic investigations or to estimate rates of intracellular DNA synthesis.

COO

R-5- P

The regulated step of pyrimidine synthesis in humans is CPSII. The enzyme is inhibited by UTP and activated by PRPP (see Fig. 41.14). Thus, as pyrimidines decrease in concentration (as indicated by UTP levels), CPSII is activated and pyrimidines are synthesized. The activity is also regulated by the cell cycle. As cells approach S-phase, CPSII becomes more sensitive to PRPP activation and less sensitive to UTP inhibition. At the end of S-phase, the inhibition by UTP is more pronounced, and the activation by PRPP is reduced. These changes in the allosteric properties of CPSII are related to its phosphorylation state. Phosphorylation of the enzyme at a specific site by a MAP kinase leads to a more easily activated enzyme. Phosphorylation at a second site by the cAMP-dependent protein kinase leads to a more easily inhibited enzyme.

OMP

IV. THE PRODUCTION OF DEOXYRIBONUCLEOTIDES

Orotidine 5'-P decarboxylase

CO2

O HN 3 2

O

4

5

1 6

N R-5- P UMP

Block in hereditary orotic aciduria

FIG. 41.16. Conversion of carbamoyl phosphate and aspartate to UMP. The defective enzymes in hereditary orotic aciduria are indicated by the dark bar.

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For DNA synthesis to occur, the ribose moiety must be reduced to deoxyribose (Fig. 41.18). This reduction occurs at the diphosphate level and is catalyzed by ribonucleotide reductase, which requires the protein thioredoxin. The deoxyribonucleoside diphosphates can be phosphorylated to the triphosphate level and used as precursors for DNA synthesis (see Figs. 41.2 and 41.14). Table 41.2 Salvage Reactions for Conversion of Pyrimidine Nucleosides to Nucleotides Enzyme

Reaction

Uridine-cytidine kinase

Uridine ⫹ ATP → UMP ⫹ ADP Cytidine ⫹ ATP → CMP ⫹ ADP Deoxythymidine ⫹ ATP → dTMP ⫹ ADP Deoxycytidine ⫹ ATP → dCMP ⫹ ADP

Deoxythymidine kinase Deoxycytidine kinase

ATP, adenosine triphosphate; UMP, uridine monophosphate; ADP, adenosine diphosphate; CMP, cytidine monophosphate; dTMP, deoxythymidine monophosphate; dCMP, deoxycytidine monophosphate.

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The regulation of ribonucleotide reductase is quite complex. The enzyme contains two allosteric sites: one controlling the activity of the enzyme and the other controlling the substrate specificity of the enzyme. ATP bound to the activity site activates the enzyme; deoxyadenosine triphosphate (dATP) bound to this site inhibits the enzyme. Substrate specificity is more complex. ATP bound to the substrate site activates the reduction of pyrimidines (CDP and UDP) to form deoxycytidine diphosphate (dCDP) and deoxyuridine diphosphate (dUDP). The dUDP is not used for DNA synthesis; rather, it is used to produce deoxythymidine monophosphate (dTMP) (see the following discussions). Once dTMP is produced, it is phosphorylated to dTTP, which then binds to the substrate site and induces the reduction of GDP. As dGTP accumulates, it replaces dTTP in the substrate site and allows ADP to be reduced to deoxyadenosine diphosphate (dADP). This leads to the accumulation of dATP, which inhibits the overall activity of the enzyme. These allosteric changes are summarized in Table 41.3. dUDP can be dephosphorylated to form deoxyuridine monophosphate (dUMP), or, alternatively, deoxycytidine monophosphate (dCMP) can be deaminated to form dUMP. Methylene tetrahydrofolate transfers a methyl group to dUMP to form dTMP (see Fig. 40.5). Phosphorylation reactions produce dTTP, a precursor for DNA synthesis and a regulator of ribonucleotide reductase.

V. DEGRADATION OF PURINE AND PYRIMIDINE BASES A. Purine Bases The degradation of the purine nucleotides (AMP and GMP) occurs mainly in the liver (Fig. 41.19). Salvage enzymes are used for most of these reactions. AMP is first deaminated to produce IMP (AMP deaminase). Then IMP and GMP are dephosphorylated (5⬘-nucleotidase), and the ribose is cleaved from the base by purine nucleoside phosphorylase. Hypoxanthine, the base produced by cleavage of IMP, is converted by xanthine oxidase to xanthine, and guanine is deaminated by the enzyme guanase to produce xanthine. The pathways for the degradation of adenine and guanine merge at this point. Xanthine is converted by xanthine oxidase to uric acid, which is excreted in the urine. Xanthine oxidase is a molybdenum-requiring enzyme

O P

O

P

Base

O H H

NDP

H H

HO

OH

SH

NADP+

thioredoxin

SH Thioredoxin reductase

ribonucleotide reductase

Free Bases Uracil or Cytosine

Thymine

Nucleoside

Ribose 1-phosphate

Pi

Uridine or Cytidine

Deoxyribose 1-phosphate Thymidine Pi

FIG. 41.17. Salvage reactions for pyrimidine nucleoside production. Thymine phosphorylase uses deoxyribose 1-phosphate as a substrate, so ribothymidine is rarely formed.

In hereditary orotic aciduria, orotic acid is excreted in the urine because the enzymes that convert it to uridine monophosphate, orotate phosphoribosyltransferase and orotidine 5⬘-phosphate decarboxylase, are defective (see Fig. 41.16). Pyrimidines cannot be synthesized and, therefore, normal growth does not occur. Oral administration of uridine is used to treat this condition. Uridine, which is converted to uridine monophosphate (UMP), bypasses the metabolic block and provides the body with a source of pyrimidines, as both cytidine triphosphate (CTP) and deoxythymidine monophosphate (dTMP) can be produced from UMP.

When ornithine transcarbamoylase is deficient (urea-cycle disorder), excess carbamoyl phosphate from the mitochondria leaks into the cytoplasm. The elevated levels of cytoplasmic carbamoyl phosphate lead to pyrimidine production, as the regulated step of the pathway, the reaction catalyzed by carbamoyl phosphate synthetase II, is being bypassed. Thus, orotic aciduria results.

S

NADPH

thioredoxin

S O P

O

P

H H dNDP

Base

O

HO

H H H

FIG. 41.18. Reduction of ribose to deoxyribose. Reduction occurs at the nucleoside diphosphate level. A ribonucleoside diphosphate (NDP) is converted to a deoxyribonucleoside diphosphate (dNDP). Thioredoxin is oxidized to a disulfide, which must be reduced for the reaction to continue producing dNDP.

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SECTION VII ■ NITROGEN METABOLISM

Gout is caused by excessive uric acid levels in the blood and tissues. To determine whether a person with gout has developed this problem because of overproduction of purine nucleotides or because of a decreased ability to excrete uric acid, an oral dose of a 15N-labeled amino acid is sometimes used. Which amino acid would be most appropriate to use for this purpose?

Table 41.3

Effectors of Ribonucleotide Reductase Activity

Uric acid has a pK of 5.4. It is ionized in the body to form urate. Urate is not very soluble in an aqueous environment. The quantity in normal human blood is very close to the solubility constant.

that uses molecular oxygen and produces hydrogen peroxide (H2O2). Another form of xanthine oxidase uses NAD⫹ as the electron acceptor (see Chapter 24). Note how little energy is derived from the degradation of the purine ring. Thus, it is to the cell’s advantage to recycle and salvage the ring because it costs energy to produce and not much is obtained in return.

Preferred Substrate

Effector Bound to Overall Activity Site

Effector Bound to Substrate Specificity Site

None CDP UDP GDP ADP

dATP ATP ATP ATP ATP

Any nucleotide ATP or dATP ATP or dATP dTTP dGTP

CDP, cytidine diphosphate; UDP, uridine diphosphate; GDP, guanosine diphosphate; ADP, adenosine diphosphate; dATP, deoxyadenosine triphosphate; ATP, adenosine triphosphate; dTTP, deoxythymidine triphosphate; dGTP, deoxyguanosine triphosphate.

O AMP

N

HN

H H2N

NH4

N

N

+

RP GMP

IMP Pi

Pi

Inosine Pi

Guanosine Pi O

Ribose 1phosphate N

HN

O

Ribose 1phosphate N

HN H

H2N

N

H

N H

N

Guanine

N H

Hypoxanthine O2

Allopurinol + NH4

Xanthine oxidase

O

H2O2

N

HN

H N N H H Xanthine O2 Xanthine oxidase Allopurinol O

H2O2 O HN

N O

O

N N H H Uric acid

–

pKa = 5.4

Urine

FIG. 41.19. Degradation of the purine bases. The reactions inhibited by allopurinol are indicated. A second form of xanthine oxidase exists that uses NAD⫹ instead of O2 as the electron acceptor.

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B. Pyrimidine Bases The pyrimidine nucleotides are dephosphorylated, and the nucleosides are cleaved to produce ribose 1-phosphate and the free pyrimidine bases cytosine, uracil, and thymine. Cytosine is deaminated, forming uracil, which is converted to CO2, NH4⫹, and ␤-alanine. Thymine is converted to CO2, NH4⫹, and ␤-aminoisobutyrate (Fig. 41.20). These products of pyrimidine degradation are excreted in the urine or converted to CO2, H2O, and NH4⫹ (which forms urea). They do not cause any problems for the body, in contrast to urate, which is produced from the purines and can precipitate, causing gout. As with the purine degradation pathway, little energy can be generated by pyrimidine degradation. CLINICAL COMMENTS Lotta Topaigne. Hyperuricemia in Lotta Topaigne’s case arose as a consequence of overproduction of uric acid. Treatment with allopurinol not only inhibits xanthine oxidase, lowering the formation of uric acid with an increase in the excretion of hypoxanthine and xanthine, but also decreases the overall synthesis of purine nucleotides. Hypoxanthine and xanthine produced by purine degradation are salvaged (i.e., converted to nucleotides) by a process that requires the consumption of phosphoribosyl pyrophosphate (PRPP). PRPP is a substrate for the glutamine phosphoribosyl amidotransferase reaction that initiates purine biosynthesis. Because the normal cellular levels of PRPP and glutamine are below the Km of the enzyme, changes in the level of either substrate can accelerate or reduce the rate of the reaction. Therefore, decreased levels of PRPP cause decreased synthesis of purine nucleotides. BIOCHEMICAL COMMENTS Adenosine Deaminase Deficiency. A deficiency in adenosine deaminase activity leads to severe combined immunodeficiency disease, or SCID. In the severe form of combined immunodeficiency, both T cells (which provide cell-based immunity, see Chapter 44) and B cells (which produce antibodies) are deficient, leaving the individual without a functional immune system. Children born with this disorder do not develop a mature thymus gland and suffer from many opportunistic infections because of their lack of a functional immune system. Death results if the child is not placed in a sterile environment. Administration of polyethylene glycol–modified adenosine deaminase has been successful in treating the disorder, and the ADA gene was the first to be used in gene therapy in treating the disorder. The question that remains, however, is that even though all the cells of the body lack ADA activity, why are the immune cells specifically targeted for destruction? The specific immune disorder is not caused by any defect in purine salvage pathways, as children with Lesch–Nyhan syndrome have a functional immune system, although there are other major problems in those children. This suggests that perhaps the accumulation of precursors to ADA lead to toxic effects. Three hypotheses have been proposed and are outlined in the following section. In the absence of ADA activity, both adenosine and deoxyadenosine accumulate. When deoxyadenosine accumulates, adenosine kinase can convert it to dAMP. Other kinases then allow deoxyadenosine triphosphate (dATP) to accumulate in the lymphocytes. Why specifically the lymphocytes? The other cells of the body secrete the deoxyadenosine they cannot use, and it accumulates in the circulation. As the lymphocytes are present in the circulation, they tend to accumulate this compound more so than cells that are not constantly present in the blood. As dATP accumulates, ribonucleotide reductase becomes inhibited, and the cells can no

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The entire glycine molecule is incorporated into the precursor of the purine nucleotides. The nitrogen of this glycine also appears in uric acid, the product of purine degradation. 15N-labeled glycine could be used, therefore, to determine whether purines are being overproduced.

Normally, as cells die, their purine nucleotides are degraded to hypoxanthine and xanthine, which are converted to uric acid by xanthine oxidase (see Fig. 41.19). Allopurinol (a structural analog of hypoxanthine) is a substrate for xanthine oxidase. It is converted to oxypurinol (also called alloxanthine), which remains tightly bound to the enzyme, preventing further catalytic activity (see Fig. 8.18). Thus, allopurinol is a suicide inhibitor. It reduces the production of uric acid and hence its concentration in the blood and tissues (e.g., the synovial lining of the joints in Lotta Topaigne’s great toe). Xanthine and hypoxanthine accumulate, and urate levels decrease. Overall, the amount of purine being degraded is spread over three products rather than appearing in only one. Therefore, none of the compounds exceeds its solubility constant, precipitation does not occur, and the symptoms of gout gradually subside.

O H2N

CH2

CH2

C

␤-Alanine H +

H3N

CH2

C

O–

O C

O– CH3 ␤-Aminoisobutyrate

FIG. 41.20. Water-soluble end products of pyrimidine degradation.

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Once nucleotide biosynthesis and salvage was understood at the pathway level, it was quickly realized that one way to inhibit cell proliferation would be to block purine or pyrimidine synthesis. Thus, drugs were developed that would interfere with a cell’s ability to generate precursors for DNA synthesis, thereby inhibiting cell growth. This is particularly important for cancer cells, which have lost their normal growth-regulatory properties. Such drugs have been introduced previously with a number of different patients. Colin Tuma was treated with 5-fluorouracil, which inhibits thymidylate synthase (dUMPto-TMP synthesis). Arlyn Foma was treated with a combination of drugs (R-CHOP) for his leukemia. These drugs all interfere with DNA synthesis via a variety of mechanisms. Mannie Weitzels was treated with hydroxyurea to block ribonucleotide reductase activity, with the goal of inhibiting DNA synthesis in the leukemic cells. Development of these drugs would not have been possible without an understanding of the biochemistry of both purine and pyrimidine salvage and synthesis and DNA replication. Such drugs also affect rapidly dividing normal cells, which brings about several side effects of chemotherapeutic regimens.

Table 41.4

Gene Disorders in Purine and Pyrimidine Metabolism

Disease

Gene Defect

Metabolite That Accumulates

Clinical Symptoms

Gout Severe combined immunodeficiency disease (SCID)

Multiple causes Adenosine deaminase (purine salvage pathway)

Uric acid Deoxyadenosine and derivatives thereof

Immunodeficiency disease

Purine nucleoside phosphorylase

Purine nucleosides

Lesch–Nyhan syndrome

Hypoxanthine-guanine phosphoribosyltransferase UMP synthase

Purines, uric acid

Painful joints Loss of immune system function, including absence of T and B cells Partial loss of immune system; no T cells, but B cells are present Mental retardation, self-mutilation

Hereditary orotic aciduria

Orotic acid

Growth retardation

UMP, uridine monophosphate.

longer produce deoxyribonucleotides for DNA synthesis. Thus, when cells are supposed to grow and differentiate in response to cytokines, they cannot, and they die. A second hypothesis suggests that the accumulation of deoxyadenosine in lymphocytes leads to an inhibition of S-adenosylhomocysteine hydrolase, the enzyme that converts S-adenosylhomocysteine to homocysteine and adenosine. This leads to hypomethylation in the cell and an accumulation of S-adenosylhomocysteine. S-Adenosylhomocysteine accumulation has been linked to the triggering of apoptosis. The third hypothesis suggested is that elevated adenosine levels lead to inappropriate activation of adenosine receptors. Adenosine is also a signaling molecule, and stimulation of the adenosine receptors results in activation of protein kinase A and elevated cAMP levels in thymocytes. Elevated levels of cAMP in these cells trigger both apoptosis and developmental arrest of the cell. Although it is still not clear which potential mechanism best explains the arrested development of immune cells, it is clear that elevated levels of adenosine and deoxyadenosine are toxic. The biochemical disorders of purine and pyrimidine metabolism discussed in this chapter are summarized in Table 41.4. Key Concepts • • • • • • • • • • • •

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Purine and pyrimidine nucleotides can both be synthesized from scratch (de novo) or salvaged from existing bases. De novo purine synthesis is complex, requiring 11 steps and six molecules of ATP for every purine synthesized. Purines are initially synthesized in the ribonucleotide form. The precursors for de novo purine synthesis are glycine, ribose 5-phosphate, glutamine, aspartate, carbon dioxide, and N10-formyl-FH4. The initial purine ribonucleotide synthesized is inosine monophosphate (IMP). AMP and GMP are each derived from IMP. Since de novo purine synthesis requires a large amount of energy, purine nucleotide salvage pathways exist such that free purine bases can be converted to nucleotides. Mutations in purine salvage enzymes are associated with severe diseases, such as Lesch–Nyhan syndrome and severe combined immunodeficiency disease (SCID). Pyrimidine bases are initially synthesized as the free base and then converted to nucleotides. Aspartate and cytoplasmic carbamoyl phosphate are the precursors for pyrimidine ring synthesis. The initial pyrimidine nucleotide synthesized is orotate monophosphate, which is converted to uridine monophosphate. The other pyrimidine nucleotides are derived from a uracil-containing intermediate. Deoxyribonucleotides are derived by reduction of ribonucleotides, as catalyzed by ribonucleotide reductase. The regulation of ribonucleotide reductase is complex. Degradation of purine containing nucleotides results in production of uric acid, which is eliminated in the urine. Elevated uric acid levels in the blood lead to gout. Diseases discussed in this chapter are summarized in Table 41.5.

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Table 41.5

773

Diseases Discussed in Chapter 41

Disease or Disorder

Environmental or Genetic

Gout

Both

PNP (purine nucleoside phosphorylase) deficiency

Genetic

Lesch–Nyhan syndrome (lack of hypoxanthine-guanine phosphoribosyl transferase activity)

Genetic

Hereditary orotic aciduria

Genetic

Adenosine deaminase deficiency

Genetic

Cancer

Both

Comments Painful joints caused by the precipitation of uric acid in the blood. A defect in a purine salvage enzyme, leading to a loss of T-cell function, and a partial immunodeficiency disease. The loss of HGPRT activity leads to the accumulation of purines and uric acid, with mental retardation and self-mutilation resulting in severe cases. A defect in UMP synthase, leading to orotic acid accumulation and growth retardation. The loss of adenosine deaminase activity leads to SCID (severe combined immunodeficiency disease), with a loss of both T- and B-cell function. The use of drugs that interfere with DNA replication will destroy rapidly dividing cells at a faster rate than normal cells.

HGPRT, hypoxanthine-guanine phosphoribosyltransferase; UMP, uridine monophosphate.

REVIEW QUESTIONS—CHAPTER 41 1.

Similarities between carbamoyl phosphate synthetase I and carbamoyl phosphate synthetase II include which one of the following? A. Carbon source B. Intracellular location C. Nitrogen source D. Regulation by N-acetyl glutamate E. Regulation by UMP

2.

Gout can result from a reduction in activity of which one of the following enzymes? A. Glutamine phosphoribosyl amidotransferase B. Glucose 6-phosphatase C. Glucose-6-phosphate dehydrogenase D. PRPP synthetase E. Purine nucleoside phosphorylase

3.

Lesch–Nyhan syndrome is due to an inability to catalyze which of the following reactions? A. Adenine to AMP B. Adenosine to AMP C. Guanine to GMP D. Guanosine to GMP E. Thymine to TMP F. Thymidine to TMP

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4.

Allopurinol can be used to treat gout because of its ability to inhibit which one of the following reactions? A. AMP to XMP B. Xanthine to uric acid C. Inosine to hypoxanthine D. IMP to XMP E. XMP to GMP

5.

The regulation of ribonucleotide reductase is quite complex. Assuming that an enzyme deficiency leads to highly elevated levels of dGTP, what effect would you predict on the reduction of ribonucleotides to deoxyribonucleotides under these conditions? A. Elevated levels of dCDP will be produced. B. The formation of dADP will be favored. C. AMP will begin to be reduced. D. Reduced thioredoxin will become rate-limiting, thereby reducing the activity of ribonucleotide reductase. E. Deoxy-GTP will bind to the overall activity site and inhibit the functioning of the enzyme.

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42

Intertissue Relationships in the Metabolism of Amino Acids

Protein Amino acids

Skeletal muscle

Glutamine

Macrophages Lymphocytes Fibroblasts

Kidney

NH4

Liver

+

Actute-phase proteins Urea

FIG. 42.1. Amino acid flux in sepsis and trauma. In sepsis and traumatic injury, glutamine and other amino acids are released from skeletal muscle for uptake by tissues involved in the immune response and tissue repair, such as macrophages, lymphocytes, fibroblasts, and the liver. Nitrogen excretion as urea and NH4⫹ results in negative nitrogen balance.

The body maintains a relatively large free amino acid pool in the blood, even during fasting. As a result, tissues have continuous access to individual amino acids for the synthesis of proteins and essential amino acid derivatives, such as neurotransmitters. The amino acid pool also provides the liver with amino acid substrates for gluconeogenesis and provides several other cell types with a source of fuel. The free amino acid pool is derived from dietary amino acids and the turnover of proteins in the body. During an overnight fast and during hypercatabolic states, degradation of labile protein particularly that in skeletal muscle is the major source of free amino acids. The liver is the major site of amino acid metabolism in the body and the major site of urea synthesis. The liver is also the major site of amino acid degradation. Hepatocytes partially oxidize most amino acids, converting the carbon skeleton to glucose, ketone bodies, or CO2. Because ammonia is toxic, the liver converts most of the nitrogen from amino acid degradation to urea, which is excreted in the urine. The nitrogen derived from amino acid catabolism in other tissues is transported to the liver as alanine or glutamine and converted to urea. The branched-chain amino acids, or BCAAs (valine, isoleucine, and leucine), are oxidized principally in skeletal muscle and other tissues and not in the liver. In skeletal muscle, the carbon skeletons and some of the nitrogen are converted to glutamine, which is released into the blood. The remainder of the nitrogen is incorporated into alanine, which is taken up by the liver and converted to urea and glucose. The formation and release of glutamine from skeletal muscle and other tissues serves several functions. In the kidney, the NH4⫹ carried by glutamine is excreted into the urine. This process removes protons formed during fuel oxidation and helps to maintain the body’s pH, especially during metabolic acidosis. Glutamine also provides a fuel for the kidney and gut. In rapidly dividing cells (e.g., lymphocytes and macrophages), glutamine is required as a fuel, as a nitrogen donor for biosynthetic reactions, and as a substrate for protein synthesis. During conditions of sepsis (the presence of various pathogenic organisms or their toxins in the blood or tissues), trauma, injury, or burns, the body enters a catabolic state characterized by a negative nitrogen balance (Fig. 42.1). Increased net protein degradation in skeletal muscle increases the availability of glutamine and other amino acids for cell division and protein synthesis in cells involved in the immune response and wound healing. In these conditions, increased release of glucocorticoids from the adrenal cortex stimulates proteolysis.

774

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775

THE WAITING ROOM Katta Bolic, a 62-year-old homeless woman, was found by a neighborhood child who heard Katta’s moans coming from an abandoned building. The child’s mother called the police who took Katta to the hospital emergency room. The patient was semicomatose, incontinent of urine, and her clothes were stained with vomitus. She had a fever of 103°F, was trembling uncontrollably, appeared to be severely dehydrated, and had marked muscle wasting. Her heart rate was very rapid, and her blood pressure was low (85/46 mm Hg). Her abdomen was distended and without bowel sounds. She responded to moderate pressure on her abdomen with moaning and grimacing. Blood was sent for a broad laboratory profile, and cultures of her urine and blood were taken. Intravenous saline with glucose, thiamine and folate, and parenteral broad-spectrum antibiotics were begun. Radiography performed after her vital signs were stabilized suggested a bowel perforation. These findings were compatible with a diagnosis of a ruptured viscus (e.g., an infected colonic diverticulum that perforated, allowing colonic bacteria to infect the tissues of the peritoneal cavity, causing peritonitis). Further studies confirmed that a diverticulum had ruptured, and appropriate surgery was performed. All of the blood cultures grew out Escherichia coli, indicating that Katta also had a gram-negative infection of her blood (septicemia) that had been seeded by the proliferating organisms in her peritoneal cavity. Intensive fluid and electrolyte therapy and antibiotic coverage were continued. The medical team (surgeons, internists, and nutritionists) began developing a complex therapeutic plan to reverse Katta’s severely catabolic state.

I.

MAINTENANCE OF THE FREE AMINO ACID POOL IN BLOOD

The body maintains a relatively large free amino acid pool in the blood, even in the absence of an intake of dietary protein. The large free amino acid pool ensures the continuous availability of individual amino acids to tissues for the synthesis of proteins, neurotransmitters, and other nitrogen-containing compounds (Fig. 42.2). In a normal, well-fed, healthy individual, approximately 300 to 600 g of body protein is degraded per day. At the same time, roughly 100 g of protein is consumed in the diet per day, which adds additional amino acids. From this pool, tissues use amino acids for the continuous synthesis of new proteins (300 to 600 g) to replace those degraded. The continuous turnover of proteins in the body makes the complete complement of amino acids available for the synthesis of new and different proteins, such as antibodies. Protein turnover allows shifts in the quantities of different proteins produced in tissues in response to changes in physiologic state and continuously removes modified or damaged proteins. It also provides a complete pool of specific amino acids that can be used as oxidizable substrates; precursors for gluconeogenesis and for heme, creatine phosphate, purine, pyrimidine, and neurotransmitter synthesis; for ammoniagenesis to maintain blood pH levels; and for numerous other functions. The concentration of free amino acids in the blood is not nearly as rigidly controlled as blood glucose levels. The free amino acid pool in the blood is only a small part (0.5%) of the total amino acid pool in whole-body protein. Because of the large skeletal muscle mass, approximately 80% of the body’s total protein is in skeletal muscle. Consequently, the concentration of individual amino acids in the blood is strongly affected by the rates of protein synthesis and degradation in skeletal muscle, as well as the rate of uptake and utilization of individual amino acids for metabolism in liver and other tissues. For the most

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SECTION VII ■ NITROGEN METABOLISM

Synthesis of new proteins Dietary protein

1

3

Endogenous protein

4

Blood Amino acids

2

Urinary metabolites

5

6 Urea (urine)

Purines, pyrimidines, heme, neurotransmitters, hormones, and other functional nitrogen products

ATP C

N

CO2

8 NH4

+

7

Glucose

ATP Glycogen

Lipid Dietary glucose

FIG. 42.2. Maintenance of the blood amino acid pool. Dietary protein (1) and degradation of endogenous protein (2) provide a source of essential amino acids (those that cannot be synthesized in the human). (3) The synthesis of new protein is the major use of amino acids from the free amino acid pool. (4) Compounds synthesized from amino acid precursors are essential for physiologic functions. Many of these compounds are degraded to nitrogencontaining urinary metabolites and do not return to the free amino acid pool. (5) In tissues, the nitrogen is removed from amino acids by transamination and deamination reactions. (6) The nitrogen from amino acid degradation appears in the urine primarily as urea or NH4⫹, the ammonium ion. Ammonia excretion is necessary to maintain the pH of the blood. (7) Amino acids are used as fuels either directly or after being converted to glucose by gluconeogenesis. (8) Some amino acids can be synthesized in humans provided that glucose and a nitrogen source are available.

part, changes in the rate of protein synthesis and degradation take place over a span of hours.

A. Interorgan Flux of Amino Acids in the Postabsorptive State What changes in hormone levels and fuel metabolism occur during an overnight fast?

The fasting state provides an example of the interorgan flux of amino acids necessary to maintain the free amino acid pool in the blood and supply tissues with their required amino acids (Fig. 42.3). During an overnight fast, protein synthesis in the liver and other tissues continues but at a diminished rate compared with the postprandial state. Net degradation of labile protein occurs in skeletal muscle (which contains the body’s largest protein mass) and other tissues. 1.

RELEASE OF AMINO ACIDS FROM SKELETAL MUSCLE DURING FASTING

The efflux of amino acids from skeletal muscle supports the essential amino acid pool in the blood (see Fig. 42.3). Skeletal muscle oxidizes the branched-chain amino acids (BCAAs) (valine, leucine, isoleucine) to produce energy and glutamine. The amino groups of the BCAAs and of aspartate and glutamate are transferred out of skeletal muscle in alanine and glutamine. Alanine and glutamine account for approximately 50% of the total ␣-amino nitrogen released by skeletal muscle (Fig. 42.4). The release of amino acids from skeletal muscle is stimulated during an overnight fast by the decrease of insulin and increase of glucocorticoid levels in the blood (see Chapters 31 and 43). Insulin promotes the uptake of amino acids and the general synthesis of proteins. The mechanisms for the stimulation of protein synthesis in human skeletal muscle are not all known but probably include an activation of the A system for amino acid transport (a modest effect), a general effect on initiation of translation, and an inhibition of lysosomal proteolysis. The fall of blood insulin levels during an overnight fast results in net proteolysis

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777

Kidney NH3

Brain

NH4

Glutamine

+

Urea Valine, Isoleucine

Gut

Alanine

Alanine

Lactate

Glutamine BCAAs

Skeletal muscle

Liver Cells of the immune system

Lactate

Urea Glucose

Amino acids

␣-Keto acids Alanine

FIG. 42.3. Interorgan amino acid exchange after an overnight fast. After an overnight fast (the postabsorptive state), the use of amino acids for protein synthesis, for fuels, and for the synthesis of essential functional compounds continues. The free amino acid pool is supported largely by net degradation of skeletal muscle protein. Glutamine and alanine serve as amino-group carriers from skeletal muscle to other tissues. Glutamine brings NH4⫹ to the kidney for the excretion of protons and serves as a fuel for the kidney, gut, and cells of the immune system. Alanine transfers amino groups from the skeletal muscle, the kidney, and the gut to the liver, where they are converted to urea for excretion. The brain continues to use amino acids for neurotransmitter synthesis.

Amino acid release from human forearm Composition of average protein

Total % of amino acids

25 20 15 10 5 0

Alanine Glutamine

Branchedchain amino acids

FIG. 42.4. Amino acid release from skeletal muscle. The arteriovenous difference (the concentration in arterial blood minus the concentration in venous blood) across the human forearm has been measured for many amino acids. This graph compares the amount of alanine, glutamine, and branched-chain amino acids (BCAAs) released with their composition in the average protein. Alanine and glutamine represent a much higher percentage of total nitrogen released than originally present in the degraded protein, evidence that they are being synthesized in the skeletal muscle. The BCAAs (leucine, valine, and isoleucine) are released in much lower amounts than those present in the degraded protein, evidence that they are being catabolized. Aspartate and glutamate also contribute nitrogen to the formation of alanine and glutamine.

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The hormonal changes that occur during an overnight fast include a decrease of blood insulin levels and an increase of glucagon relative to levels after a high-carbohydrate meal. Glucocorticoid levels also increase in the blood. These hormones coordinate the changes of fat, carbohydrate, and amino acid metabolism. Fatty acids are released from adipose triacylglycerols and are used as the major fuel by heart, skeletal muscle, liver, and other tissues. The liver converts some of the fatty acids to ketone bodies. Liver glycogen stores are diminished and gluconeogenesis becomes the major support of blood glucose levels for glucose-dependent tissues. The major precursors of gluconeogenesis include amino acids released from skeletal muscle, lactate, and glycerol.

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SECTION VII ■ NITROGEN METABOLISM

and release of amino acids. As glucocorticoid release from the adrenal cortex increases, induction of ubiquitin synthesis and increase of ubiquitin-dependent proteolysis also occur.

Glucose

Gluconeogenesis

+

Glucagon

2.

Carbon skeletons NH4 +

Urea cycle

Urea

Amino acid degradation +

Glucagon

Alanine, other amino acids

FIG. 42.5. Hormonal regulation of hepatic amino acid metabolism in the postabsorptive state. 䊝, glucagon-mediated activation of enzymes or proteins; , induction of enzyme synthesis mediated by glucagon and glucocorticoids. Induction of urea cycle enzymes occurs both during fasting and after a highprotein meal. Because many individuals in the United States normally have a high-protein diet, the levels of urea cycle enzymes may not fluctuate to any great extent.

The body normally produces approximately 1 mmol of protons per kilogram of body weight per day. Nevertheless, the pH of the blood and extracellular fluid is normally maintained between 7.36 and 7.44. The narrow range is maintained principally by the bicarbonate (HCO3⫺), phosphate (HPO4⫺), and hemoglobin buffering systems and by the excretion of an amount of acid equal to that produced. The excretion of protons by the kidney regenerates bicarbonate, which can be reclaimed from the glomerular filtrate. The acids are produced from normal fuel metabolism. The major acid is carbonic acid, which is formed from water and CO2 produced in the tricarboxylic acid (TCA) cycle and other oxidative pathways. The oxidation of sulfur-containing amino acids (methionine and cysteine) ultimately produces sulfuric acid (H2SO4), which dissociates into 2H⫹ ⫹ SO42⫺, and the protons and sulfate are excreted. The hydrolysis of phosphate esters produces the equivalent of phosphoric acid. What other acids produced during metabolism appear in the blood?

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AMINO ACID METABOLISM IN LIVER DURING FASTING

The major site of alanine uptake is the liver, which disposes of the amino nitrogen by incorporating it into urea (see Fig. 42.3). The liver also extracts free amino acids, ␣-keto acids, and some glutamine from the blood. Alanine and other amino acids are oxidized and their carbon skeletons converted principally to glucose. Glucagon and glucocorticoids stimulate the uptake of amino acids into liver and increase gluconeogenesis and ureagenesis (Fig. 42.5). Alanine transport into the liver, in particular, is enhanced by glucagon. The induction of the synthesis of gluconeogenic enzymes by glucagon and glucocorticoids during the overnight fast correlates with an induction of many of the enzymes of amino acid degradation (e.g., tyrosine aminotransferase) and an induction of urea cycle enzymes (see Chapter 38). Urea synthesis also increases because of the increased supply of NH4⫹ from amino acid degradation in the liver. 3.

METABOLISM OF AMINO ACIDS IN OTHER TISSUES DURING FASTING

Glucose, produced by the liver, is used for energy by the brain and other glucosedependent tissues, such as erythrocytes. The muscle, under conditions of exercise, when the adenosine monophosphate (AMP)-activated protein kinase is active, also oxidizes some of this glucose to pyruvate, which is used for the carbon skeleton of alanine (the glucose–alanine cycle; see Chapter 38). Glutamine is generated in skeletal muscle from the oxidation of BCAAs and by the lungs and brain for the removal of NH4⫹ formed from amino acid catabolism or entering from the blood. The kidney, the gut, and the cells with rapid turnover rates such as those of the immune system are the major sites of glutamine uptake (see Fig. 42.3). Glutamine serves as a fuel for these tissues, as a nitrogen donor for purine synthesis, and as a substrate for ammoniagenesis in the kidney. Much of the unused nitrogen from glutamine is transferred to pyruvate to form alanine in these tissues. Alanine then carries the unused nitrogen back to the liver. The brain is glucose dependent but, like many cells in the body, can use BCAAs for energy. The BCAAs also provide a source of nitrogen for neurotransmitter synthesis during fasting. Other amino acids released from skeletal muscle protein degradation also serve as precursors of neurotransmitters.

B. Principles Governing Amino Acid Flux between Tissues The pattern of interorgan flux of amino acids is strongly affected by conditions that change the supply of fuels (e.g., the overnight fast, a mixed meal, a high-protein meal) and by conditions that increase the demand for amino acids (metabolic acidosis, surgical stress, traumatic injury, burns, wound healing, and sepsis). The flux of amino acid carbon and nitrogen in these different conditions is dictated by several considerations. 1. Ammonia (NH3) is toxic. Consequently, it is transported between tissues as alanine or glutamine. Alanine is the principal carrier of amino acid nitrogen from other tissues back to the liver, where the nitrogen is converted to urea and subsequently excreted into the urine by the kidneys. The amount of urea synthesized is proportional to the amount of amino acid carbon that is being oxidized as a fuel. The differences in amino acid metabolism between tissues are dictated by the types and amounts of different enzyme and transport proteins present in each tissue and the ability of each tissue to respond to different regulatory messages (hormones and neural signals).

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2. The pool of glutamine in the blood serves several essential metabolic functions (Table 42.1). It provides ammonia for excretion of protons in the urine as NH4⫹. It serves as a fuel for the gut, the kidney, and the cells of the immune system. Glutamine is also required by the cells of the immune system and other rapidly dividing cells in which its amide group serves as the source of nitrogen for biosynthetic reactions. In the brain, the formation of glutamine from glutamate and NH4⫹ provides a means of removing ammonia and of transporting glutamate between different cell types within the brain. The use of the blood glutamine pool is prioritized. During metabolic acidosis, the kidney becomes the predominant site of glutamine uptake at the expense of glutamine use in other tissues. Conversely, during sepsis, in the absence of acidosis, cells involved in the immune response (macrophages, hepatocytes) become the preferential sites of glutamine uptake. 3. BCAAs (valine, leucine, and isoleucine) form a significant portion of the composition of the average protein and can be converted to tricarboxylic acid (TCA) cycle intermediates and used as fuels by almost all tissues. Valine and isoleucine are also the major precursors of glutamine. Except for the BCAAs and alanine, aspartate, and glutamate, the catabolism of amino acids occurs principally in the liver. The ability to convert four-carbon intermediates of the TCA cycle to pyruvate is required for oxidation of both BCAAs and glutamine. This sequence of reactions requires phosphoenolpyruvate (PEP) carboxykinase or decarboxylating malate dehydrogenase (malic enzyme). Most tissues have one, or both, of these enzymes. 4. Amino acids are major gluconeogenic substrates, and most of the energy obtained from their oxidation is derived from oxidation of the glucose formed from their carbon skeletons. A much smaller percentage of amino acid carbon is converted to acetyl coenzyme A (acetyl-CoA) or to ketone bodies and oxidized. The use of amino acids for glucose synthesis for the brain and other glucoserequiring tissues is subject to the hormonal regulatory mechanisms of glucose homeostasis (see Chapters 31 and 36). 5. The relative rates of protein synthesis and degradation (protein turnover) determine the size of the free amino acid pools available for the synthesis of new proteins and for other essential functions. For example, the synthesis of new proteins to mount an immune response is supported by the net degradation of other proteins in the body.

II. UTILIZATION OF AMINO ACIDS IN INDIVIDUAL TISSUES

Katta Bolic was in a severe stage of negative nitrogen balance on admission, which was caused by both her malnourished state and her intra-abdominal infection complicated by sepsis. The physiologic response to advanced catabolic status includes a degradation of muscle protein with the release of amino acids into the blood. This release is coupled with increased uptake of amino acids for acute-phase protein synthesis by the liver (systemic response) and other cells involved in the immune response to general and severe infection. Table 42.1

Functions of Glutamine

Protein synthesis Ammoniagenesis for proton excretion Nitrogen donor for synthesis of purines, pyrimidines, NAD⫹, amino sugars, asparagine, and other compounds Glutamate donor for synthesis of glutathione, GABA, ornithine, arginine, proline, and other compounds GABA, ␥-aminobutyric acid.

Lactic acid is produced from glucose and amino acid metabolism. The ketone bodies (acetoacetate and ␤-hydroxybutyrate) produced during fatty acid oxidation are also acids. Many ␣-keto acids, formed from transamination reactions, are also found in the blood.

Because tissues differ in their physiologic functions, they have different amino acid requirements and contribute differently to whole-body nitrogen metabolism. However, all tissues share a common requirement for essential amino acids for protein synthesis, and protein turnover is an ongoing process in all cells.

A. The Kidney One of the primary roles of amino acid nitrogen is to provide ammonia in the kidney for the excretion of protons in the urine. NH4⫹ is released from glutamine by glutaminase and by glutamate dehydrogenase, resulting in the formation of ␣-ketoglutarate (Fig. 42.6). ␣-Ketoglutarate is used as a fuel by the kidney and is oxidized to CO2, converted to glucose for use in cells in the renal medulla or converted to alanine to return ammonia to the liver for urea synthesis. 1.

USE OF GLUTAMINE NITROGEN TO BUFFER URINE

The rate of glutamine uptake from the blood and its use by the kidney depends principally on the amount of acid that must be excreted to maintain a normal pH

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SECTION VII ■ NITROGEN METABOLISM

Table 42.2

Arterial glutamine

Glutamine

Bicarbonate

Glutaminase

(renal vein) Glutamate Glutamate dehydrogenase

NH4 +

␣-Ketoglutarate

Glucose

CO2

Urine

FIG. 42.6. Renal glutamine metabolism. Renal tubule cells preferentially oxidize glutamine. During metabolic acidosis, it is the major fuel for the kidney. Conversion of glutamine to ␣-ketoglutarate generates NH4⫹. Ammonium ion excretion helps to buffer systemic acidemia.

Excretion of Compounds in the Urine

Component

g/24 h

Nitrogen (mmol)

H2O SO42⫺ PO42⫺ K⫹ Urea Creatinine Uric acid NH4⫹

1,000 2–5 2–5 1–2 12–20 1–1.8 0.2–0.8 0.2–1 (up to 10 in acidosis)

— — — — 400–650 25–50 4–16 11–55 (up to 550 in acidosis)

in the blood. In metabolic acidosis, the excretion of NH4⫹ by the kidney increases severalfold (Table 42.2). Because glutamine nitrogen provides approximately two-thirds of the NH4⫹ excreted by the kidney, glutamine uptake by the kidney also increases. Renal glutamine use for proton excretion takes precedence over the requirements of other tissues for glutamine. Ammonia increases proton excretion by providing a buffer for protons that are transported into the renal tubular fluid (which is transformed into urine as it passes through the tubules of the kidney) (Fig. 42.7). Specific transporters in the membranes of the renal tubular cells transport protons from these cells into the tubular lumen in exchange for Na⫹. The protons in the tubular fluid are buffered by phosphate, by bicarbonate, and by ammonia. Ammonia (NH3), which is uncharged, enters the urine by free diffusion through the cell membrane. As it combines with a proton in the fluid, it forms ammonium ion (NH4⫹), which cannot be transported back into the cells and is excreted in the urine. 2.

GLUTAMINE AS A FUEL FOR THE KIDNEY

Glutamine is used as a fuel by the kidney in the normal fed state and, to a greater extent, during fasting and metabolic acidosis (Table 42.3). The carbon skeleton forms ␣-ketoglutarate, which is oxidized to CO2, converted to glucose or released as the carbon skeleton of serine or alanine (Fig. 42.8). ␣-Ketoglutarate can be converted to oxaloacetate by TCA cycle reactions, and oxaloacetate is converted to PEP by PEP carboxykinase. PEP can then be converted to pyruvate and subsequently acetyl-CoA, alanine, serine, or glucose. The glucose is used principally by the cells of the renal medulla, which have a relatively high dependence on anaerobic glycolysis because of their lower oxygen supply and mitochondrial capacity. The lactate released from anaerobic glycolysis in these cells is taken up and oxidized in the renal cortical cells, which have a higher mitochondrial capacity and a greater blood supply.

B. Skeletal Muscle Skeletal muscle, because of its large mass, is a major site of protein synthesis and degradation in the human. After a high-protein meal, insulin promotes the uptake of certain amino acids and stimulates net protein synthesis. The insulin stimulation of protein synthesis is dependent on an adequate supply of amino acids to undergo protein synthesis. During fasting and other catabolic states, a net degradation of skeletal muscle protein and release of amino acids occur (see Fig. 42.3). The net degradation of protein affects functional proteins, such as myosin, which are sacrificed to meet more urgent demands for amino acids in other tissues. During sepsis, degradation of skeletal muscle protein is stimulated by the glucocorticoid cortisol. The effect of cortisol is exerted through the activation of ubiquitin-dependent proteolysis. During fasting, the decrease of blood insulin levels and the increase of blood cortisol levels increase net protein degradation.

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CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

781

Glomerulus

Capillary

Glomerular filtrate in the renal tubule lumen: H2O, urea, – SO42–, PO43–, HCO3, sugars,amino acids

Renal tubule cell H+ NH4+

NH3 H+

H+ Na+

NH4+

NH3

NH3

free diffusion

H2O

CO2 H2CO3

HCO3–

HCO3–

H+

PO43–

H+ Na

+

HPO42–

To portal vein

To urine

FIG. 42.7. Ammonia excretion by the kidney. Ammonia increases proton excretion by combining with a proton to form ammonium ion in the renal tubular fluid, which is transformed into urine as it passes through the tubules of the kidney. As blood is filtered in the capillary bed of the glomerulus, urea, sugars, amino acids, ions, and H2O enter the renal tubular fluid (glomerular filtrate). As this fluid passes through a progression of tubules (the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule, and the collecting duct) on its way to becoming urine, various components are reabsorbed or added to the filtrate by the epithelial cells lining the tubules. Specific transporters in the membranes of the renal tubule cells transport protons into the tubule lumen in exchange for Na⫹ so that the glomerular filtrate becomes more acidic as it is transformed into urine. The protons in the tubule fluid are buffered by phosphate, by bicarbonate, and by NH3. The ammonia, which is uncharged, is able to diffuse through the membrane of the renal tubule cells into the urine. As it combines with a proton in the urine, it forms NH4⫹, which cannot be transported back into the cells. The removal of protons as NH4⫹ decreases the requirement for bicarbonate excretion to buffer the urine.

Skeletal muscle is a major site of glutamine synthesis, thereby satisfying the demand for glutamine during the postabsorptive state, during metabolic acidosis, and during septic stress and trauma. The carbon skeleton and nitrogen of glutamine are derived principally from the metabolism of BCAAs. Amino acid degradation in skeletal muscle is also accompanied by the formation of alanine, which transfers amino groups from skeletal muscle to the liver in the glucose–alanine cycle. Table 42.3

Major Fuel Sources for the Kidney Percentage of Total CO2 Formed in Different Physiologic States

Fuel Lactate Glucosea Fatty acids Glutamine

Normal

Acidosis

Fasted

45 25 15 15

20 20 20 40

15 0 60 25

a

Glucose used in the renal medulla is produced in the renal cortex.

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782

SECTION VII ■ NITROGEN METABOLISM

Glutamine NH4 +

Glucose Gluconeogenesis

Glutamate GDH

NH4 +

␣-Ketoglutarate CO2

(ATP) TCA cycle OAA

Alanine

Malate PEPCK

PEP Glu

CO2

Fatty acids

CO2

Serine

TA

CO2

Acetyl CoA

Pyruvate

Pyruvate Lactate

␣-KG TA

Alanine

Glucose Glycolysis

FIG. 42.8. Metabolism of glutamine and other fuels in the kidney. To completely oxidize glutamate carbon to CO2, it must enter the tricarboxylic acid (TCA) cycle as acetyl-CoA. Carbon entering the TCA cycle as ␣-ketoglutarate (␣-KG) exits as oxaloacetate and is converted to phosphoenolpyruvate (PEP) by PEP carboxykinase. PEP is converted to pyruvate, which may be oxidized to acetyl-CoA. PEP also can be converted to serine, glucose, or alanine. GDH, glutamate dehydrogenase; OAA, oxaloacetate; PEPCK, phosphoenolpyruvate carboxykinase; TA, transaminase.

1.

When the carbon skeleton of alanine is derived from glucose, the efflux of alanine from skeletal muscle and its uptake by liver provide no net transfer of amino acid carbon to the liver for gluconeogenesis. However, some of the alanine carbon is derived from sources other than glucose. Which amino acids can provide carbon for alanine formation? (Hint: See Fig. 42.9.)

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OXIDATION OF BRANCHED-CHAIN AMINO ACIDS IN SKELETAL MUSCLE

The BCAAs play a special role in muscle and most other tissues because they are the major amino acids that can be oxidized in tissues other than the liver. However, all tissues can interconvert amino acids and TCA cycle intermediates through transaminase reactions, that is, alanine ↔ pyruvate, aspartate ↔ oxaloacetate, and ␣-ketoglutarate ↔ glutamate. The first step of the pathway, transamination of the BCAAs to ␣-keto acids, occurs principally in brain, heart, kidney, and skeletal muscles. These tissues have a high content of BCAA transaminase relative to the low levels in liver. The ␣-keto acids of the BCAAs are then either released into the blood and taken up by liver or oxidized to CO2 or glutamine within the muscle or other tissue (Fig. 42.9). They can be oxidized by all tissues that contain mitochondria. The oxidative pathways of the BCAAs convert the carbon skeleton to either succinyl-CoA or acetyl-CoA (see Chapter 39 and Fig. 42.9). The pathways generate NADH and FAD(2H) for adenosine triphosphate (ATP) synthesis before the conversion of carbon into intermediates of the TCA cycle, thus providing the muscle with energy without loss of carbon as CO2. Leucine is “ketogenic” in that it is converted to acetyl-CoA and acetoacetate. Skeletal muscle, adipocytes, and most other tissues are able to use these products and, therefore, oxidize leucine directly to CO2. The portion of isoleucine that is converted to acetylCoA is also oxidized directly to CO2. For the portion of valine and isoleucine that enters the TCA cycle as succinyl-CoA to be completely oxidized to CO2, it must first be converted to acetyl-CoA. To form acetyl-CoA, succinyl-CoA is oxidized to malate in the TCA cycle, and malate is then converted to pyruvate by malic enzyme (malate ⫹ NADP⫹ → pyruvate ⫹ NADPH ⫹ H⫹) (see Fig. 42.9). Pyruvate can then be oxidized to acetyl-CoA. Alternatively, pyruvate can form alanine or lactate.

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CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

Leucine

Isoleucine TA

1 Fatty acids

1

α-Keto acid

TA

α-Keto acid

3

NADH

3

NADH

NADH

FAD(2H)

FAD(2H)

FAD(2H) HMG CoA Acetoacetate

Glucose Acetyl CoA TA Pyruvate Alanine

NADPH

Isocitrate

NADH

2 Malate

TCA cycle

NADH

NADH

FAD (2H)

CO2

α-KG

FAD(2H) NADH

Succinyl CoA

TA

CO2 Glutamate NH3

3 α-Keto acid 1

TA

Isoleucine

Some of the alanine released from skeletal muscle is derived directly from protein degradation. The carbon skeletons of valine, isoleucine, aspartate, and glutamate, which are converted to malate and oxaloacetate in the TCA cycle, can be converted to pyruvate and subsequently transaminated to alanine. The extent to which these amino acids contribute carbon to alanine efflux differs between different types of muscles in the human. These amino acids also may contribute to alanine efflux from the gut.

Citrate

OAA

NADP+

783

3

NADH FAD (2H) (ATP generation)

Glutamine

α-Keto acid 1

TA Valine

FIG. 42.9. Metabolism of the carbon skeletons of BCAAs in skeletal muscle. (1) The first step in the metabolism of BCAAs is transamination (TA). (2) Carbon from valine and isoleucine enters the tricarboxylic acid (TCA) cycle as succinyl-CoA and is converted to pyruvate by decarboxylating malate dehydrogenase (malic enzyme). (3) The oxidative pathways generate NADH and FAD(2H) even before the carbon skeleton enters the TCA cycle. The rate-limiting enzyme in the oxidative pathways is the ␣-keto acid dehydrogenase complex. The carbon skeleton also can be converted to glutamate and alanine, shown in red.

2.

CONVERSION OF BRANCHED-CHAIN AMINO ACIDS TO GLUTAMINE

The major route of valine and isoleucine catabolism in skeletal muscle is to enter the TCA cycle as succinyl-CoA and exit as ␣-ketoglutarate to provide the carbon skeleton for glutamine formation (see Fig. 42.9). Some of the glutamine and CO2 that is formed from net protein degradation in skeletal muscle may also arise from the carbon skeletons of aspartate and glutamate. These amino acids are transaminated and become part of the pool of four-carbon intermediates of the TCA cycle. Glutamine nitrogen is derived principally from the BCAAs (Fig. 42.10). The ␣-amino group arises from transamination reactions that form glutamate from ␣-ketoglutarate, and the amide nitrogen is formed from the addition of free ammonia to glutamate by glutamine synthetase. Free ammonia in skeletal muscle arises principally from the deamination of glutamate by glutamate dehydrogenase or from the purine nucleotide cycle.

Lieberman_CH42.indd 783

The purine nucleotide cycle is found in skeletal muscle and brain but is absent in the liver and in many other tissues. One of its functions in skeletal muscle is to respond to the rapid use of adenosine triphosphate (ATP) during exercise. During exercise, the rapid hydrolysis of ATP increases adenosine monophosphate (AMP) levels, resulting in an activation of AMP deaminase (see Fig. 42.11). As a consequence, the cellular concentration of inosine monophosphate (IMP) increases and ammonia is generated. IMP, like AMP, activates muscle glycogen phosphorylase during exercise (see Chapter 22). The ammonia that is generated may help to buffer the increased lactic acid production that occurs in skeletal muscles during strenuous exercise.

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SECTION VII ■ NITROGEN METABOLISM

+

BCAA-(NH3) ␣-KG

+

Glu-(NH3) Glutamine synthetase

Branchedchain ␣-Keto acid

Glutamate dehydrogenase

( + H+, Glucocorticoids)

+

Glu-(NH3) OAA TA

+

NH4

␣-KG +

Asp-(NH3)

Purine nucleotide cycle

H2O O Glutamine (C NH2 ) +

NH3

FIG. 42.10. Formation of glutamine from the amino groups of BCAAs. The BCAAs are first transaminated with ␣-ketoglutarate to form glutamate and the branched-chain ␣-keto acids. The glutamate nitrogen can then follow either of two paths leading to glutamine formation. ␣-KG, ␣-ketoglutarate; OAA, oxaloacetate; TA, transamination.

In the purine nucleotide cycle (Fig. 42.11), the deamination of AMP to inosine monophosphate (IMP) releases NH4⫹. AMP is resynthesized with amino groups provided from aspartate. The aspartate amino groups can arise from the BCAAs through transamination reactions. The fumarate can be used to replenish TCA cycle intermediates (see Fig. 41.13).

ATP Exercise

Pi

ADP Adenylate kinase

3.

AMP

AMP deaminase

Fumarate

NH3 Adenylosuccinate

IMP

Asp ( NH3 )

THE GLUCOSE–ALANINE CYCLE

The nitrogen arising from the oxidation of BCAAs in skeletal muscle can also be transferred back to the liver as alanine in the glucose–alanine cycle (Fig. 42.12; see also Fig. 38.10). The amino group of the BCAAs is first transferred to ␣-ketoglutarate to form glutamate and then transferred to pyruvate to form alanine by sequential transamination reactions. The pyruvate arises principally from glucose via the glycolytic pathway. The alanine released from skeletal muscle is taken up principally by the liver, where the amino group is incorporated into urea, and the carbon skeleton can be converted back to glucose through gluconeogenesis. Although

BCAAs ( NH3 )

FIG. 42.11 Purine nucleotide cycle. In skeletal muscle, the purine nucleotide cycle can convert the amino groups of the branchedchain acids (BCAAs) to NH3, which is incorporated into glutamine. Compare this to Figure 41.13, in which the fumarate generated is used in an anaplerotic role in the muscle.

Glucose Glucose

Muscle Glucose

Liver Pyruvate

Pyruvate

Blood

FIG. 42.12. Glucose–alanine cycle. The pathway for transfer of the amino groups from BCAAs in skeletal muscle to urea in the liver is shown in red.

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CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

Postprandial state Lumen of gut

785

Postabsorptive state Intestinal epithelial cell

Glutamine

Glutamine

Glutamine

NH4+ Glutamate NH4+

GDH

Blood

NH4+ Citrulline, ornithine

Pyruvate TA

Glucose Alanine

␣-KG

BCAAs Aspartate Glutamate

CO2

TCA Malate Lactate

Acetyl CoA Pyruvate

Ketone bodies BCAAs

FIG. 42.13. Amino acid metabolism in the gut. The pathways of glutamine metabolism in the gut are the same whether it is supplied by the diet (postprandial state) or from the blood (postabsorptive state). Cells of the gut also metabolize aspartate, glutamate, and BCAAs. Glucose is converted principally to the carbon skeleton of alanine. ␣-KG, ␣-ketoglutarate; GDH, glutamate dehydrogenase; TA, transaminase.

the amount of alanine formed varies with dietary intake and physiologic state, the transport of nitrogen from skeletal muscle to liver as alanine occurs almost continuously throughout our daily fasting–feeding cycle.

C. THE GUT Amino acids are an important fuel for the intestinal mucosal cells after a protein-containing meal and in catabolic states such as fasting or surgical trauma (Fig. 42.13). During fasting, glutamine is one of the major amino acids used by the gut. The principal fates of glutamine carbon in the gut are oxidation to CO2 and conversion to the carbon skeletons of lactate, citrulline, and ornithine. The gut also oxidizes BCAAs. Nitrogen derived from amino acid degradation is converted to citrulline, alanine, NH4⫹, and other compounds that are released into the blood and taken up by the liver. Although most of the carbon in this alanine is derived from glucose, the oxidation of glucose to CO2 is not a major fuel pathway for the gut. Fatty acids are also not a significant source of fuel for the intestinal mucosal cells, although they do use ketone bodies. Even though the liver is the organ that generates urea, the intestine also contains the enzymes for the urea cycle (including carbamoyl synthetase I). However, within the intestine, the Vmax values for argininosuccinate synthetase and argininosuccinate lyase are very low, suggesting that the primary role of the urea cycle enzymes in the gut is to produce citrulline from the carbons of glutamine (glutamine → glutamate → glutamate semialdehyde → ornithine → citrulline). The citrulline is released in the circulation for use by the liver. After a protein meal, dietary glutamine is a major fuel for the gut, and the products of glutamine metabolism are similar to those seen in the postabsorptive state. The gut also uses dietary aspartate and glutamate, which enter the TCA cycle. Colonocytes (the cells of the colon) also use short-chain fatty acids, derived from bacterial action in the lumen.

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SECTION VII ■ NITROGEN METABOLISM

The importance of the gut in whole-body nitrogen metabolism arises from the high rate of division and death of intestinal mucosal cells and the need to continuously provide these cells with amino acids to sustain the high rates of protein synthesis needed for cellular division. Not only are these cells important for the uptake of nutrients, they maintain a barrier against invading bacteria from the gut lumen and are, therefore, part of our passive defense system. As a result of these important functions, the intestinal mucosal cells are supplied with the amino acids required for protein synthesis and fuel oxidation at the expense of the more expendable skeletal muscle protein. However, glutamine use by the gut is diminished by metabolic acidosis compared with the postabsorptive or postprandial state. During metabolic acidosis, the uptake of glutamine by the kidney is increased and blood glutamine levels decrease. As a consequence, the gut takes up less glutamine.

D. The Liver The liver is the major site of amino acid metabolism. It is the major site of amino acid catabolism and converts most of the carbon in amino acids to intermediates of the TCA cycle or the glycolytic pathway (which can be converted to glucose or oxidized to CO2) or to acetyl-CoA and ketone bodies. The liver is also the major site for urea synthesis. It can take up both glutamine and alanine and convert the nitrogen to urea for disposal (see Chapter 38). Other pathways in the liver give it an unusually high amino acid requirement. The liver synthesizes plasma proteins, such as serum albumin, transferrin, and the proteins of the blood-coagulation cascade. It is a major site for the synthesis of nonessential amino acids, the conjugation of xenobiotic compounds with glycine, the synthesis of heme and purine nucleotides, and the synthesis of glutathione.

E. Brain and Nervous Tissue 1.

THE AMINO ACID POOL AND NEUROTRANSMITTER SYNTHESIS

A major function of amino acid metabolism in neural tissue is the synthesis of neurotransmitters. More than 40 compounds are believed to function as neurotransmitters, and all of these contain nitrogen derived from precursor amino acids. They include amino acids, which are themselves neurotransmitters (e.g., glutamate, glycine), the catecholamines derived from tyrosine (dopamine, epinephrine, and norepinephrine), serotonin (derived from tryptophan), ␥-aminobutyric acid (GABA, derived from glutamate), acetylcholine (derived from choline synthesized in the liver and acetyl-CoA), and many peptides. In general, neurotransmitters are formed in the presynaptic terminals of axons and stored in vesicles until they are released by a transient change in electrochemical potential along the axon. Subsequent catabolism of some of the neurotransmitters results in the formation of a urinary excretion product. The rapid metabolism of neurotransmitters requires the continuous availability of a precursor pool of amino acids for de novo neurotransmitter synthesis (see Chapter 48). 2.

METABOLISM OF GLUTAMINE IN THE BRAIN

The brain is a net glutamine producer, owing principally to the presence of glutamine synthetase in astroglial cells (see Chapter 48). Glutamate and aspartate are synthesized in these cells, using amino groups donated by the BCAAs (principally valine) and TCA cycle intermediates formed from glucose and from the carbon skeletons of BCAAs (Fig. 42.14). The glutamate is converted to glutamine by glutamine synthetase, which incorporates NH4⫹ released from deamination of amino acids and deamination of AMP in the purine nucleotide cycle in the brain. This glutamine may efflux from the brain, carrying excess NH4⫹ into the blood, or serve as a precursor of glutamate in neuronal cells.

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CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

Blood BCAAs

Blood– brain barrier

Astroglial cell

␣-KG

BCAAs

Glutamate

BCKA NH4

+

NH3

NH3

NH4

+

Neurons

Purine nucleotide cycle

Glutamine synthetase

Glutamine

787

GABA NH4+

Glutamine

CO2

Glutamate

FIG. 42.14. Role of glutamine in the brain. Glutamine serves as a nitrogen transporter in the brain for the synthesis of many different neurotransmitters. Different neurons convert glutamine to ␥-aminobutyric acid (GABA) or to glutamate. Glutamine also transports excess NH4⫹ from the brain into the blood. ␣-KG, ␣-ketoglutarate; BCKA, branched-chain keto acids.

Glutamine synthesized in the astroglial cells is a precursor of glutamate (an excitatory neurotransmitter) and GABA (an inhibitory neurotransmitter) in the neuronal cells (see Fig. 42.14). It is converted to glutamate by a neuronal glutaminase isozyme. In GABAergic neurons, glutamate is then decarboxylated to GABA, which is released during excitation of the neuron. GABA is one of the neurotransmitters that is recycled; a transaminase converts it to succinaldehyde, which is then oxidized to succinate. Succinate enters the TCA cycle.

III. CHANGES IN AMINO ACID METABOLISM WITH DIETARY AND PHYSIOLOGIC STATE The rate and pattern of amino acid use by different tissues change with dietary and physiologic state. Two such states, the postprandial period following a high-protein meal and the hypercatabolic state produced by sepsis or surgical trauma, differ from the postabsorptive state with respect to the availability of amino acids and other fuels and the levels of different hormones in the blood. As a result, the pattern of amino acid use is somewhat different.

A. High-Protein Meal After the ingestion of a high-protein meal, the gut and the liver use most of the absorbed amino acids (Fig. 42.15). Glutamate and aspartate are used as fuels by the gut, and very little enters the portal vein. The gut also may use some BCAAs. The liver takes up 60% to 70% of the amino acids present in the portal vein. These amino acids, for the most part, are converted to glucose in the gluconeogenic pathway. After a pure protein meal, the increased levels of dietary amino acids reaching the pancreas stimulate the release of glucagon above fasting levels, thereby increasing amino acid uptake into the liver and stimulating gluconeogenesis. Insulin release is also stimulated, but not nearly to the levels found after a high-carbohydrate meal. In general, the insulin released after a high-protein meal is sufficiently high that the uptake of BCAAs into skeletal muscle and net protein synthesis is stimulated, but gluconeogenesis in the liver is not inhibited. The higher the carbohydrate content of the meal, the higher is the insulin/glucagon ratio and the greater the shift of amino acids away from gluconeogenesis into biosynthetic pathways in the liver such as the synthesis of plasma proteins. Most of the amino acid nitrogen entering the peripheral circulation after a highprotein meal or a mixed meal is present as BCAAs. Because the liver has low levels of transaminases for these amino acids, it cannot oxidize them to a significant

Lieberman_CH42.indd 787

During hyperammonemia, ammonia (NH3) can diffuse into the brain from the blood. The ammonia is able to inhibit the neural isozyme of glutaminase, thereby decreasing additional ammonia formation in the brain and inhibiting the formation of glutamate and its subsequent metabolism to ␥-aminobutyric acid (GABA). This effect of ammonia might contribute to the lethargy associated with the hyperammonemia found in patients with hepatic disease. The levels of transthyretin (binds to vitamin A and thyroid hormones in the blood) and serum albumin in the blood may be used as indicators of the degree of protein malnutrition. In the absence of hepatic disease, decreased levels of these proteins in the blood indicate insufficient availability of amino acids to the liver for synthesis of serum proteins. The levels of transthyretin can be determined by immunologic means. The levels of albumin are rapidly determined using a hydrophobic dye-binding assay and determining the amount of colored dye bound to albumin. The dye that is used binds specifically to the hydrophobic pocket of albumin and not to other proteins in the serum.

In what ways does liver metabolism after a high-protein meal resemble liver metabolism in the fasting state?

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SECTION VII ■ NITROGEN METABOLISM

Both of these dietary states are characterized by an elevation of glucagon. Glucagon stimulates amino acid transport into the liver, stimulates gluconeogenesis through decreasing levels of fructose 2,6-bisphosphate, and induces the synthesis of enzymes in the urea cycle, the gluconeogenic pathway, and the pathways for degradation of some of the amino acids.

Skeletal muscle Protein synthesis +

Insulin TCA

[ATP]

Alanine

Glutamine

BCAAs Other amino acids

Protein

Liver +

Aspartate, glutamate, glutamine, BCAAs

Lactate, citrulline, NH3

Gut

Amino acid Gluconeodegradation genesis Urea cycle

[ATP] CO2

Glucagon

Glucose

Urea

TCA [ATP]

CO2

Brain

FIG. 42.15. Flux of amino acids after a high-protein meal.

extent, and they enter the systemic circulation. The BCAAs are slowly taken up by skeletal muscle and other tissues. These peripheral nonhepatic tissues use the amino acids derived from the diet principally for net protein synthesis. High-protein, low-carbohydrate diets are based on the premise that ingesting high-protein, low-carbohydrate meals will keep circulating insulin levels low, so that energy storage is not induced, and glucagon release will point the insulin/glucagon ratio to energy mobilization, particularly fatty acid release from the adipocyte and oxidation by the tissues. The lack of energy storage coupled with the loss of fat leads to weight loss.

B. Hypercatabolic States

The degree of the body’s hypercatabolic response depends on the severity and duration of the trauma or stress. After an uncomplicated surgical procedure in an otherwise healthy patient, the net negative nitrogen balance may be limited to about 1 week. The mild nitrogen losses are usually reversed by dietary protein supplementation as the patient recovers. With more severe traumatic injury or septic stress, the body may catabolize body protein and adipose tissue lipids for a prolonged period, and the negative nitrogen balance may not be corrected for weeks.

Lieberman_CH42.indd 788

Surgery, trauma, burns, and septic stress are examples of hypercatabolic states characterized by increased fuel use and a negative nitrogen balance (Fig. 42.16). The mobilization of body protein, fat, and carbohydrate stores serves to maintain normal tissue function in the presence of a limited dietary intake, as well as to support the energy and amino acid requirements for the immune response and wound healing. The negative nitrogen balance that occurs in these hypercatabolic states results from accelerated protein turnover and an increased rate of net protein degradation, primarily in skeletal muscle. The catabolic state of sepsis (acute, generalized, febrile infection) is one of enhanced mobilization of fuels and amino acids to provide the energy and precursors required by cells of the immune system, host defense mechanisms, and wound healing. The amino acids must provide the substrates for new protein synthesis and cell division. Glucose synthesis and release are enhanced to provide fuel for these cells, and the patient may become mildly hyperglycemic. In these hypercatabolic states, skeletal muscle protein synthesis decreases and protein degradation increases. Oxidation of BCAAs is increased, and glutamine production enhanced. Amino acid uptake is diminished. Cortisol is the major hormonal mediator of these responses, although certain cytokines may also have direct effects on skeletal muscle metabolism. As occurs during fasting and metabolic

01/09/12 9:32 PM

Cumulative nitrogen balance (g total)

Daily nitrogen balance (g/d)

Temperature (°F)

CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

Inoculation 106 104 102 100 98

789

Exposure

5 0 –5 –10 –15

Tularemia

Sandfly fever

Pair-fed nonexposed controls

10 0 –10 –30 –50 –70 –90 0

5

10

15

20

0

5

10

15

20

Days after exposure

FIG. 42.16. Negative nitrogen balance during infection. The effects of experimentally induced infections on nitrogen balance were determined in human volunteers. After inoculation with sandfly fever, increased amino acid catabolism produced a negative nitrogen balance. A few days after exposure, the daily nitrogen balance became positive, until the volunteers returned to their original state. Experiments with patients exposed to tularemia showed that the negative nitrogen balance was much larger than could be expected from a decreased appetite alone. Volunteers who ate the same amount of food as the infected individuals (pair-fed nonexposed controls) had a much smaller cumulative negative nitrogen balance than the infected volunteers. (From Beisel WR. Magnitude of the host nutritional responses to infection. Am J Clin Nutr. 1977;30:1236–1247. Copyright 1977 American Society for Clinical Nutrition.)

acidosis, increased levels of cortisol stimulate ubiquitin-mediated proteolysis, induce the synthesis of glutamine synthetase, and enhance release of amino acids and glutamine from the muscle cells. The amino acids released from skeletal muscle during periods of hypercatabolic stress are used in a prioritized manner, with the cellular components of the immune system receiving top priority. For example, the uptake of amino acids by the liver for the synthesis of acute-phase proteins, which are part of the immune system, is greatly increased. Conversely, during the early phase of the acute response, the synthesis of other plasma proteins (e.g., albumin) is decreased. The increased availability of amino acids and the increased cortisol levels also stimulate gluconeogenesis, thereby providing fuel for the glucose-dependent cells of the immune system (e.g., lymphocytes). An increase of urea synthesis accompanies the acceleration of amino acid degradation. The increased efflux of glutamine from skeletal muscle during sepsis serves several functions (see Fig. 42.1). It provides the rapidly dividing cells of the immune system with an energy source. Glutamine is available as a nitrogen donor for purine synthesis, for NAD⫹ synthesis (to convert nicotinic acid to nicotinamide), and for other biosynthetic functions that are essential to growth and division of the cells. Increased production of metabolic acids may accompany stress such as sepsis, so there is increased use of glutamine by the kidney. Under the influence of elevated levels of glucocorticoids, epinephrine, and glucagon, fatty acids are mobilized from adipose tissue to provide alternate fuels

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Katta Bolic’s severe negative nitrogen balance was caused by both her malnourished state and her intra-abdominal infection complicated by sepsis. The systemic and diverse responses the body makes to insults such as an acute febrile illness are termed the acute-phase response. An early event in this response is the stimulation of phagocytic activity (see Fig. 42.17). Stimulated macrophages release cytokines, which are regulatory proteins that stimulate the release of cortisol, insulin, and growth hormone. Cytokines also directly mediate the acute-phase response of the liver and skeletal muscle to sepsis.

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for other tissues and spare glucose. Under these conditions, fatty acids are the major energy source for skeletal muscle, and glucose uptake is decreased. These changes may lead to mild hyperglycemia. CLINICAL COMMENTS Katta Bolic. The clinician can determine whether a patient such as Katta Bolic is mounting an acute-phase response to some insult, however subtle, by determining whether several unique acute-phase proteins are being secreted by the liver. C-reactive protein, so named because of its ability to interact with the C-polysaccharide of pneumococci, and serum amyloid A protein, a precursor of the amyloid fibril found in secondary amyloidosis, are elevated in patients who are undergoing the acute-phase response and as compared with healthy individuals. Other proteins normally found in the blood of healthy individuals are present in increased concentrations in patients undergoing an acute-phase response. These include haptoglobin, certain protease inhibitors, complement components, ceruloplasmin, and fibrinogen. The elevated concentration of these proteins in the blood increases the erythrocyte sedimentation rate (ESR), another laboratory measure of the presence of an acute-phase response. To determine the ESR, the patient’s blood is placed vertically in a small-bore glass tube. The speed with which the red blood cells sediment toward the bottom of the tube depends on what percentage of the red blood cells clump together and thereby become more dense. The degree of clumping is directly correlated with the presence of one or more of the acute-phase proteins listed previously. These proteins interfere with what is known as the zeta-potential of the red blood cells, which normally prevents the red blood cells from clumping. Because many different proteins can individually alter the zeta-potential, the ESR is a nonspecific test for the presence of acute inflammation. The weight loss often noted in septic patients is caused primarily by a loss of appetite resulting from the effect of certain cytokines on the medullary appetite center. Other causes include increased energy expenditure due to fever and enhanced muscle proteolysis. BIOCHEMICAL COMMENTS Amino acid metabolism in trauma and sepsis. After a catabolic insult such as injury, trauma, infection, or cancer, the interorgan flow of glutamine and fuels is altered dramatically. Teleologically, the changes in metabolism that occur give first priority to cells that are part of the immune system. Evidence suggests that the changes in glutamine and fuel metabolism are mediated by the insulin counterregulatory hormones, such as cortisol and epinephrine, and several different cytokines (see Chapter 11 for a review of cytokines). Cytokines appear to play a more important role than hormones during sepsis, although they exert their effects, in part, through hormones (Fig. 42.17). Although cytokines can be released from a variety of cells, macrophages are the principal source during trauma and sepsis. Two cytokines that are important in sepsis are interleukin 1 (IL-1) and tumor necrosis factor (TNF). IL-1 and TNF affect amino acid metabolism both through regulation of the release of glucocorticoids and through direct effects on tissues. Although cytokines are generally considered to be paracrine, with their effects being exerted over cells in the immediate vicinity, TNF and IL-1 increase in the blood during sepsis. Other cytokines, such as IL-6, also may be involved. During sepsis, TNF, IL-1, and possibly other cytokines, bacterial products, or mediators act on the brain to stimulate the release of glucocorticoids from the adrenal cortex (a process mediated by adrenocorticotropic hormone [ACTH]), epinephrine

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791

Sepsis Brain Bacterial products CRF

Macrophage

ACTH TNF, IL-1

Pancreas Glucagon

Adrenal medulla

Adrenal cortex

TNF, IL-1, IL-6 Glucocorticoids

Insulin Epinephrine

Liver

Glucocorticoids Amino acids, alanine

Muscle Glutamine Amino acid uptake Protein synthesis Protein breakdown

Alanine

Gut

Amino acid uptake Protein synthesis Acute-phase protein synthesis

FIG. 42.17. Cytokines and hormones mediate amino acid flux during sepsis. Bacterial products act on macrophages to stimulate the release of cytokines and on the brain to stimulate the sympathoadrenal response. The result is a stimulation of the release of the insulin counterregulatory hormones, epinephrine, glucagon, and glucocorticoids. The glucocorticoid cortisol may be the principal mediator of net muscle protein degradation during sepsis. Hepatic protein synthesis, particularly that of acute-phase proteins, is stimulated by both cortisol and cytokines. Amino acid metabolism in the gut is also probably affected by glucocorticoids and cytokines. Because of the release of the counterregulatory hormones, muscle and other tissues become resistant to insulin action, as indicated by the bar on the figure. (Adapted with permission from Fischer JE, Hasselgren PO. Cytokines and glucocorticoids in the regulation of the hepatoskeletal muscle axis in sepsis. Am J Surg. 1991;161:266–271.)

from the adrenal medulla, and both insulin and glucagon from the pancreas. Although insulin is elevated during sepsis, the tissues exhibit an insulin resistance that is similar to that of the patient with non–insulin-dependent diabetes mellitus, possibly resulting from the elevated levels of the insulin counterregulatory hormones (glucocorticoids, epinephrine, and glucagon). Changes in the rate of acute-phase protein synthesis are mediated, at least in part, by effects of TNF, IL-1, and IL-6 on the synthesis of groups of proteins in the liver. The molecular mechanism whereby sepsis/trauma leads to reduced muscle protein synthesis involves, in part, mammalian target of rapamycin (mTOR; see Chapter 36, Figure 36.12). Sepsis reduces translational efficiency, primarily at the translation initiation step. Initiation is regulated by, and requires, specific eukaryotic initiation factors (eIFs, see Chapter 15). eIF4E is a multifactor complex required to assemble the charged initiator tRNAiMet– 40S small ribosomeal subunit complex onto the capped mRNA. The activity of eIF4E is regulated by the eIF4E binding protein, 4E-BP1. When eIF4E is bound to 4E-BP1, translation initiation is reduced. mTOR, when active, will phosphorylate 4E-BP1, causing the binding protein to dissociate from the eIF4E complex, thereby stimulating translation initiation. Sepsis, and/or release of TNF-␣, leads to the inactivation of mTOR and reduced phosphorylation of 4E-BP1, resulting in a decrease in translation. The reduced translation, coupled with enhanced protein turnover, allows the muscle to export amino acids for use by other tissues.

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Hypercatabolic states may be accompanied by varying degrees of insulin resistance caused, in part, by the release of counterregulatory hormones into the blood. Thus, patients with diabetes mellitus may require higher levels of exogenous insulin during sepsis.

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Table 42.4

Diseases Discussed in Chapter 42

Disease or Disorder

Environmental of Genetic

Catabolic state

Environmental

Hyperammonemia

Both

Comments The body adapts to a catabolic state by degrading proteins to enhance survival. This change in metabolism is initiated through the stress response, as mediated by cortisol, epinephrine, and norepinephrine, among other signaling molecules Hyperammonemia can result from mutations in urea cycle enzymes or due to a failing liver as caused by a variety of conditions (one of which is alcohol abuse over many years).

Key Concepts • • • • • •

•

•

•

The body maintains a large free amino acid pool in the blood, even during fasting, allowing tissues continuous access to these building blocks. Amino acids are used for gluconeogenesis by the liver as a fuel source for the gut and as neurotransmitter precursors in the nervous system. They are also required by all organs for protein synthesis. During an overnight fast and during hypercatabolic states, degradation of labile protein (primarily from skeletal muscle) is the major source of free amino acids. The liver is the major site for urea synthesis. Nitrogen from other tissues travels to the liver in the form of glutamine and alanine. Branched-chain amino acids (BCAAs) are oxidized primarily in the skeletal muscle. Glutamine in the blood serves several roles: The kidney uses the ammonium ion carried by glutamine for excretion in the urine to act as a buffer against acidotic conditions. The kidney and the gut use glutamine as a fuel source. All tissues use glutamine for protein synthesis. The body can enter a catabolic state, characterized by negative nitrogen balance, under the following conditions: Sepsis (the presence of various pathogenic organisms or their toxins in the blood or tissues) Trauma Injury Burns The negative nitrogen balance results from increased net protein degradation in skeletal muscle brought about by the release of glucocorticoids. The released amino acids are used for protein synthesis and cell division in cells involved in the immune response and wound healing. Diseases discussed in this chapter are summarized in Table 42.4.

REVIEW QUESTIONS—CHAPTER 42 1.

Which of the profiles indicated in the following would occur within 2 hours after eating a meal that was very high in protein and low in carbohydrates? Blood Glucagon Levels

Liver Gluconeogenesis

BCAA Oxidation in Muscle

↓ ↑ ↓ ↑ ↓ ↑ ↓ ↑

↓ ↓ ↑ ↑ ↓ ↓ ↑ ↑

↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓

A. B. C. D. E. F. G. H.

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2.

The gut uses glutamine as an energy source, but it can also secrete citrulline, synthesized from the carbons of glutamine. Which of the following compounds is an obligatory intermediate in this conversion (consider only the carbon atoms of glutamine while answering this question)? A. Aspartate B. Succinyl-CoA C. Glutamate D. Serine E. Fumarate

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CHAPTER 42 ■ INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS

3.

4.

The signal that indicates to muscle that protein degradation needs to be initiated is which of the following? A. Insulin B. Glucagon C. Epinephrine D. Cortisol E. Glucose The skeletal muscles convert branched-chain amino acid (BCAA) carbons to glutamine for export to the rest of the body. An obligatory intermediate, which carries carbons originally from the BCAAs, in the conversion of BCAAs to glutamine, is which of the following? A. Urea B. Pyruvate

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C. Lactate D. Isocitrate E. Phosphoenolpyruvate 5.

An individual in sepsis will display which of the following metabolic patterns?

A. B. C. D. E. F.

Nitrogen Balance

Gluconeogenesis

Fatty Acid Oxidation

Positive Negative Positive Negative Positive Negative

↑ ↑ ↑ ↑ ↓ ↓

↓ ↑ ↓ ↓ ↑ ↓

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SECTION EIGHT

Tissue Metabolism

A

lthough many of the pathways described previously are present in all tissues of the body, many tissues also carry out specialized functions and contain unique biochemical pathways. This section describes a number of such tissues and, in some cases, how the tissues interact with the rest of the body to coordinate their functions. Previous chapters have focused primarily on insulin and glucagon as the major mediators for regulating metabolic pathways; however, a large number of other hormones also regulate the storage and use of metabolic fuels (see Chapter 43). These hormones primarily counteract the effects of insulin and are called counterregulatory hormones. They include growth hormone, thyroid hormone, glucocorticoids such as cortisol, small peptides such as the somatostatins, and small molecules such as the catecholamines. Growth hormone works, in part, by inducing the synthesis of the insulin-like growth factors. These hormones can exert their effects rapidly (through covalent modification of selected enzymes) or long term (through alterations in the rate of synthesis of selected enzymes). The interplay of these hormones with insulin and glucagon is discussed, as are the synthesis, secretion, and conditions leading to secretion of each hormone. The proteins and cells in the blood form their own tissue system (see Chapter 44). All blood cells are derived from a common precursor—the stem cell—in the bone marrow. Different cytokine signals trigger differentiation of a particular blood cell lineage. For example, when oxygen supply to the tissues is decreased, the kidney responds by releasing erythropoietin. This hormone specifically stimulates the production of red blood cells. Red blood cells have limited metabolic functions, owing to their lack of internal organelles. Their main function is to deliver oxygen to the tissues through the binding of oxygen to hemoglobin. When the number of red blood cells is decreased, that, by definition, represents anemia. This can be attributable to many causes, including nutritional deficiencies or mutations (hereditary anemias). The morphology of the red blood cell can sometimes aid in distinguishing the various types of anemia. Red blood cell metabolism is geared toward preserving the ability of these cells to transport oxygen, as well as to regulate oxygen binding to hemoglobin. Glycolysis provides energy and NADH to protect the oxidation state of the heme-bound iron. The hexose monophosphate shunt pathway generates NADPH to protect red blood cell membranes from oxidation. Heme synthesis, which uses succinyl coenzyme A (succinyl-CoA) and glycine for all of the carbon and nitrogen atoms in the structure, occurs in the precursors of red blood cells. Inherited defects in heme synthesis leads to a class of diseases known as the porphyrias. Because red blood cells normally pass through the very narrow capillaries, their membranes must be easily deformable. This deformability is, in part, attributable to the complex cytoskeletal structure that surrounds the erythrocyte. Mutations in these structural proteins can lead to less deformable cells, which are more easily lysed as they circulate in the bloodstream. This, in turn, can result in hemolytic anemia. Among other functions, the hematologic system is responsible for hemostasis as well as for maintaining a constant blood volume (see Chapter 45). A tear in the wall of a vessel can lead to blood loss, which, when extensive, can be fatal. Repairing vessel damage, whether internal or external, is accomplished by a complicated series of zymogen activations of circulating blood proteins resulting in the formation of a

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fibrin clot (the coagulation cascade). Platelets play a critical role in hemostasis not only through their release of procoagulants but through their ability to form aggregates within the thrombus (clot) as well. Clots function as a plug, allowing defects or rents in vessel walls to repair and thereby preventing further blood loss. Conversely, inappropriate or accelerated clot formation in the lumen of vessels that supply blood to vital organs or tissues can cause an intraluminal obstruction to flow that may lead to an acute cerebral or myocardial infarction. Because clotting must be tightly controlled, intricate mechanisms exist that regulate this important hematologic function. The liver is an altruistic organ that provides multiple services for other tissues (see Chapter 46). It supplies glucose and ketone bodies to the rest of the body when fuel stores are limited. It disposes of ammonia as urea when amino acid degradation occurs. It is the site of detoxification of xenobiotics, and it synthesizes many of the proteins found in the blood. The liver synthesizes triacylglycerols and cholesterol and distributes them to other tissues in the form of very low-density lipoprotein (VLDL). The liver also synthesizes bile acids for fat digestion in the intestine. The liver recycles cholesterol and triglyceride through its uptake of intermediate-density lipoprotein (IDL), chylomicron and VLDL remnants, and low-density lipoprotein (LDL) particles. Because of its protective nature and its strategic location between the gut and the systemic circulation, the liver has “first crack” at all compounds that enter the blood through the enterohepatic circulation. Thus, xenobiotic compounds can be detoxified as they enter the liver, before they have a chance to reach other tissues. Muscle cells contain unique pathways that allow them to store energy as creatine phosphate and to regulate closely their use of fatty acids as an energy source (see Chapter 47). The adenosine monophosphate (AMP)-activated protein kinase is an important regulator of muscle energy metabolism. Muscle is composed of different types of contractile fibers that derive their energy from different sources. For example, the slow-twitch type I fibers use oxidative energy pathways, whereas the type II fast-twitch fibers use the glycolytic pathway for their energy requirements. The nervous system consists of various cell types that are functionally interconnected so as to allow efficient signal transmission throughout the system (see Chapter 48). The cells of the central nervous system are protected from potentially toxic compounds by the blood–brain barrier, which restricts entry of potentially toxic compounds into the nervous system (ammonia, however, is a notable exception). The brain cells communicate with each other and with other organs through the synthesis of neurotransmitters and neuropeptides. Many of the neurotransmitters are derived from amino acids, most of which are synthesized within the nerve cells. Because the pathways of amino acid and neurotransmitter biosynthesis require cofactors (such as pyridoxal phosphate, thiamine pyrophosphate, and vitamin B12), deficiencies of these cofactors can lead to neuropathies (dysfunction of specific neurons in the nervous system). Because of the restrictions imposed by the blood–brain barrier, the brain also must synthesize its own lipids. An adequate supply of lipids is vital to the central nervous system because lipids are constituents of the myelin sheath that surrounds the neurons and allows them to conduct impulses normally. The neurodegenerative disorders, such as multiple sclerosis, are a consequence of varying degrees of demyelination of the neurons. Connective tissue, which consists primarily of fibroblasts, produces extracellular matrix materials that surround cells and tissues, determining their appropriate position within the organ (see Chapter 49). These materials include structural proteins (collagen and elastin), adhesive proteins (fibronectin), and glycosaminoglycans (heparan sulfate and chondroitin sulfate). The unique structures of the proteins and carbohydrates in the extracellular matrix allow tissues and organs to carry out their many functions. Loss of these supportive and barrier functions of connective tissue sometimes lead to significant clinical consequences, such as those that result from the microvascular alterations that lead to blindness or renal failure, or peripheral neuropathies in patients with diabetes mellitus.

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43

Actions of Hormones That Regulate Fuel Metabolism

Many hormones affect fuel metabolism, including those that regulate appetite as well as those that influence absorption, transport, and oxidation of foodstuffs. The major hormones that influence nutrient metabolism and their actions on muscle, liver, and adipose tissue are listed in Table 43.1. Insulin is the major anabolic hormone. It promotes the storage of nutrients as glycogen in liver and muscle and as triacylglycerols in adipose tissue. It also stimulates the synthesis of proteins in tissues such as muscle. At the same time, insulin acts to inhibit fuel mobilization. Glucagon is the major counterregulatory hormone. The term “counterregulatory” means that its actions are generally opposed to those of insulin (contrainsular). The major action of glucagon is to mobilize fuel reserves by stimulating glycogenolysis and gluconeogenesis. These actions ensure that glucose will be available to glucose-dependent tissues between meals. Epinephrine, norepinephrine, cortisol, somatostatin, and growth hormone (GH) also have contrainsular activity. Thyroid hormone also must be classified as an insulin-counterregulatory hormone because it increases the rate of fuel consumption and also increases the sensitivity of the target cells to other insulin-counterregulatory hormones. Insulin and the counterregulatory hormones exert two types of metabolic regulation (see Chapter 26). The first type of control occurs within minutes to hours of the hormone–receptor interaction and usually results from changes in the catalytic activity or kinetics of key preexisting enzymes caused by phosphorylation or dephosphorylation of these enzymes. The second type of control involves regulation of the synthesis of key enzymes by mechanisms that stimulate or inhibit transcription and translation of messenger RNA (mRNA). These processes are slow and require hours to days.

797

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Muscle Glucose Uptake

↑

—

Thyroid hormone

Somatostatin

b

—

—

↑

↑

↑

—

↑

↑

↑

—

↑

↑↑ ↑↑

↓↓

Ketogenesis

↓↓

Glucose Output

—

↑

↑ (mainly permissive) ↑

↑

↑

↓↓

Liver Gluconeogenesis

—

—

—

↑↑ (initial) —

↑↑

↓↓

Glycogenolysis

—

—

—

↑

↓

↓

↑

Glycogenesis

—

—

↑

↑

—

—

↑

Protein Synthesis

—

—

—

—

—

—

↑↑

Adipose Fat Synthesis

↑ (mainly permissive) ↑ (permissive) —

↑ (permissive)

↑ (at large doses) ↑↑

↓↓

Tissue Lipolysis

b

Hormones with actions that are generally opposed to those of insulin. Somatostatin’s effects on metabolism are indirect via suppression of secretion of insulin, glucagon, growth hormone, and thyroid hormone and by effects on gastric acid secretion, gastric emptying time, and pancreatic exocrine secretion (see text). ↑↑, pronounced increased effect; ↑, moderate increased effect; ↓, moderate decreased effect; —, no effect.

a

↓ (weakly)

↓ (weakly)

Growth hormone

—

↑

↓

↓

—

↑

↑

— ↓

—

—

—

Epinephrine and norepinephrine Glucocorticoid

—

↑↑

↑↑

Protein Synthesis

↑↑

Glucose Use

Major Hormones That Regulate Fuel Metabolism

Anabolic hormones Insulin Counterregulatory hormonesa Glucagon

Hormones

Table 43.1

798

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799

THE WAITING ROOM Otto Shape, now a third-year medical student, was assigned to do a history and physical examination on a newly admitted 47-year-old patient named Corti Solemia. Mr. Solemia had consulted his physician for increasing weakness and fatigue and was found to have a severely elevated serum glucose level. While he was examining the patient, Otto noted marked redness of Mr. Solemia’s facial skin, as well as reddish purple stripes (striae) in the skin of his lower abdomen and thighs. The patient’s body fat was unusually distributed in that it appeared to be excessively deposited centrally in his face, neck, upper back, chest, and abdomen, whereas the distal portions of his arms and legs appeared to be almost devoid of fat. Mr. Solemia’s skin appeared thinned and large bruises were present over many areas of his body for which Mr. Solemia had no explanation. The neurologic examination showed severe muscle weakness especially in the proximal arms and legs, where the muscles seemed to have atrophied. Sam Atotrope, a 42-year-old jeweler, noted increasingly severe headaches behind his eyes that were sometimes associated with a “flash of light” in his visual field. At times his vision seemed blurred, making it difficult to perform some of the intricate work required of a jeweler. He consulted his ophthalmologist, who was impressed with the striking change in Sam’s facial features that had occurred since he last saw the patient 5 years earlier. The normal skin creases in Sam’s face had grown much deeper, his skin appeared to be thickened, his nose and lips appeared more bulbous, and his jaw seemed more prominent. The doctor also noted that Sam’s hands appeared bulky, and his voice had deepened. An eye examination showed that Sam’s optic nerves appeared slightly atrophied and there was a loss of the upper outer quadrants of his visual fields.

I.

PHYSIOLOGIC EFFECTS OF INSULIN

The effects of insulin on fuel metabolism and substrate flux were considered in many of the earlier chapters of this book, particularly in Chapter 26. Insulin stimulates the storage of glycogen in liver and muscle and the synthesis of fatty acids and triacylglycerols and their storage in adipose tissue. In addition, insulin stimulates the synthesis in various tissues of ⬎50 proteins, some of which contribute to the growth of the organism. In fact, it is difficult to separate the effects of insulin on cell growth from those of a family of proteins known as the somatomedins or the insulin-like growth factors I and II (IGF-I and IGF-II) (see Section III.B.6). Finally, insulin has paracrine actions within the pancreatic islet cells. When insulin is released from the ␤-cells, it suppresses glucagon release from the ␣-cells.

The measurement of hormone levels in blood is best performed using immunologic reagents that specifically recognize the hormone being measured. Such techniques are further described in the Biochemical Comments of this chapter.

II. PHYSIOLOGIC EFFECTS OF GLUCAGON Glucagon is one of several counterregulatory (contrainsular) hormones. It is synthesized as part of a large precursor protein called proglucagon. Proglucagon is produced in the ␣-cells of the islets of Langerhans in the pancreas and in the L-cells of the intestine. It contains several peptides linked in tandem: glicentin-related peptide, glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2). Proteolytic cleavage of proglucagon releases various combinations of its constituent peptides. Glucagon is cleaved from proglucagon in the pancreas and constitutes 30% to 40% of the immunoreactive glucagon in the blood. The remaining immunoreactivity is caused by other cleavage products of proglucagon released

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800

SECTION VIII ■ TISSUE METABOLISM

from the pancreas and the intestine. Pancreatic glucagon has a plasma half-life of 3 to 6 minutes and is removed mainly by the liver and kidney. Glucagon promotes glycogenolysis, gluconeogenesis, and ketogenesis by stimulating the generation of cyclic adenosine monophosphate (cAMP) in target cells. The liver is the major target organ for glucagon, in part because the concentrations of this hormone bathing the liver cells in the portal blood are higher than in the peripheral circulation. Levels of glucagon in the portal vein may reach concentrations as high as 500 pg/mL. Finally, glucagon stimulates insulin release from the ␤-cells of the pancreas. Whether this is a paracrine effect or an endocrine effect has not been established. The pattern of blood flow in the pancreatic islet cells is believed to bathe the ␤-cells first and then the ␣-cells. Therefore, the ␤-cells may influence ␣-cell function by an endocrine mechanism, whereas the influence of ␣-cell hormone on ␤-cell function is more likely to be paracrine.

III. PHYSIOLOGIC EFFECTS OF OTHER COUNTERREGULATORY HORMONES A. Somatostatin 1.

BIOCHEMISTRY

Preprosomatostatin, a 116-amino acid peptide, is encoded by a gene located on the long arm of chromosome 3. Somatostatin (SS-14), a cyclic peptide with a molecular weight of 1,600 Da, is produced from the 14 amino acids at the C-terminus of this precursor molecule. SS-14 was first isolated from the hypothalamus and named for its ability to inhibit the release of growth hormone (GH, somatotropin) from the anterior pituitary. It also inhibits the release of insulin. In addition to the hypothalamus, somatostatin is also secreted from the D-cells (␦-cells) of the pancreatic islets, many areas of the central nervous system (CNS) outside of the hypothalamus, and gastric and duodenal mucosal cells. SS-14 predominates in the CNS and is the sole form secreted by the ␦-cells of the pancreas. In the gut, however, prosomatostatin (SS-28), which has 14 additional amino acids extending from the C-terminal portion of the precursor, makes up 70% to 75% of the immunoreactivity (the amount of hormone that reacts with antibodies to SS-14). The prohormone SS-28 is 7 to 10 times more potent in inhibiting the release of GH and insulin than SS-14. 2. Tolbutamide, a sulfonylurea drug that increases insulin secretion, also increases the secretion of pancreatic somatostatin.

SECRETION OF SOMATOSTATIN

The secretagogues for somatostatin are similar to those that cause secretion of insulin. The metabolites that increase somatostatin release include glucose, arginine, and leucine. The hormones that stimulate somatostatin secretion include glucagon, vasoactive intestinal polypeptide (VIP), and cholecystokinin (CCK). Insulin, however, does not influence somatostatin secretion directly. 3.

PHYSIOLOGIC EFFECTS OF SOMATOSTATIN

Five somatostatin receptors have been identified and characterized, all of which are members of the G protein coupled-receptor superfamily. Four of the five receptors do not distinguish between SS-14 and SS-28. Somatostatin binds to its plasma membrane receptors on target cells. These “activated” receptors interact with a variety of intracellular signaling pathways, depending on the cell type expressing the receptor, and which somatostatin receptor is being expressed. These pathways include inactivation of adenylate cyclase (via an inhibitory G protein), regulation of phosphotyrosine phosphatases and mitogen-activated protein (MAP) kinases, and alterations of intracellular ion concentrations (calcium [Ca2⫹] and potassium). The inactivation of adenylate cyclases reduces the production of cAMP, and protein kinase A is not activated. This inhibitory effect suppresses secretion of GH and thyroid-stimulating hormone (TSH) from the anterior pituitary gland as well as the secretion of insulin and glucagon from the pancreatic islets. If one were

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CHAPTER 43 ■ ACTIONS OF HORMONES THAT REGULATE FUEL METABOLISM

to summarize the action of somatostatin in one phrase, it would be “somatostatin inhibits the secretion of many other hormones.” As such, it acts to regulate the effects of those other hormones. In addition to these effects on hormones that regulate fuel metabolism, somatostatin also reduces nutrient absorption from the gut by prolonging gastric emptying time (through a decrease in the secretion of gastrin, which reduces gastric acid secretion), by diminishing pancreatic exocrine secretions (i.e., digestive enzymes, bicarbonate, and water), and by decreasing visceral blood flow. Thus, somatostatin exerts a broad, albeit indirect, influence on nutrient absorption and, therefore, on the use of fuels. Somatostatin and its synthetic analogs are used clinically to treat a variety of secretory neoplasms such as GH-secreting tumors of the pituitary. Such tumors can cause gigantism if GH is secreted in excess before the closure of the growth centers of the ends of long bones or acromegaly if excess GH is chronically secreted after the closure of these centers (as in Sam Atotrope).

B. Growth Hormone 1.

BIOCHEMISTRY

GH is a polypeptide that, as its name implies, stimulates growth. Many of its effects are mediated by insulin-like growth factors (IGFs, also known as somatomedins) that are produced by cells in response to the binding of GH to its cell membrane receptors (see Section III.B.6). However, GH also has direct effects on fuel metabolism. Human GH is a water-soluble 22-kDa polypeptide with a plasma half-life of 20 to 50 minutes. It is composed of a single chain of 191 amino acids having two intramolecular disulfide bonds (Fig. 43.1). The gene for GH is located on chromosome 17. It is secreted by the somatotroph cells (the cells that synthesize and release GH) in the lateral areas of the anterior pituitary. GH is structurally related to human prolactin and to human chorionic somatomammotropin (hCS) from the placenta—a polypeptide that stimulates growth of the developing fetus. Yet the hCS peptide has only 0.1% of the growth-inducing potency of GH. GH is the most abundant trophic hormone in the anterior pituitary, being present in concentrations of 5 to 15 mg/g of tissue. The other anterior pituitary hormones are present in quantities of micrograms per gram of tissue. The actions of GH can be classified as those that occur as a consequence of the hormone’s direct effect on target cells and those that occur indirectly through the ability of GH to generate other factors, particularly IGF-I. The IGF-I–independent actions of GH are exerted primarily in hepatocytes. GH administration is followed by an early increase in the synthesis of 8 to 10 proteins, among which are IGF-I, ␣2-macroglobulin, and the serine protease inhibitors Spi 2.1 and Spi 2.3. Expression of the gene for ornithine decarboxylase, an enzyme that is active in polyamine synthesis (and, therefore, in the regulation of cell proliferation), is also significantly increased by GH.

100

N 1

In addition to its effects on normal GH secretion, somatostatin also suppresses the pathologic increase in GH that occurs in acromegaly (caused by a GH-secreting pituitary tumor), diabetes mellitus, and carcinoid tumors (tumors that secrete serotonin). Somatostatin also suppresses the basal secretion of TSH, thyrotropin-releasing hormone (TRH), insulin, and glucagon. The hormone also has a suppressive effect on a wide variety of nonendocrine secretions. The major limitation in the clinical use of native somatostatin is its short half-life of less than 3 minutes in the circulation. Analogs of native somatostatin, however, have been developed that are resistant to degradation and, therefore, have a longer half-life. One such analog is octreotide, an octapeptide variant of somatostatin with a half-life of approximately 110 minutes.

A magnetic resonance imaging (MRI) scan of Sam Atotrope’s brain showed a macroadenoma (a tumor ⬎10 mm in diameter) in the pituitary gland, with superior extension that compressed the optic nerve as it crossed above the sella turcica, causing his visual problems. The skeletal and visceral changes noted by the ophthalmologist are characteristic of acromegalic patients with chronically elevated serum levels of GH and IGF-I. Therapeutic alternatives for acromegaly caused by a GH-secreting tumor of the anterior pituitary gland include surgery if the mass is amenable or lifelong medical therapy with a somatostatin analog, such as octreotide; or a dopamine agonist that inhibits the secretion of GH, such as cabergoline; or a GH-receptor antagonist, such as pegvisomant. Another therapeutic option is stereotactic radiation therapy. If the excessive secretion of GH is controlled successfully, some of the visceral or soft tissue changes of acromegaly may slowly subside to varying degrees. The skeletal changes, however, cannot be reversed.

150

C

191

50

Human growth hormone

FIG. 43.1. Structure of human growth hormone. (From Murray RK, Granner DK, Mayes PA, et al. Harper’s Biochemistry. 23rd ed. Stamford, CT: Appleton & Lange; 1993.)

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The ophthalmologist ordered a morning fasting serum GH level on Sam Atotrope, which was elevated at 56 ng/mL (normal, 0 to 5 ng/mL), as well as a determination of circulating insulin-like growth factor I (IGF-I) levels.

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Sam Atotrope was given an oral dose of 100 g glucose syrup. This dose would suppress serum GH levels to ⬍2 ng/mL in normal subjects, but not in patients with acromegaly who have an autonomously secreting pituitary tumor making GH. Because Sam’s serum GH level was 43 ng/mL after the oral glucose load, a diagnosis of acromegaly was made. The patient was referred to an endocrinologist for further evaluation.

GH stimulates IGF-I gene expression not only in the liver but in several extrahepatic tissues as well. In acromegalics, rising levels of IGF-I cause a gradual generalized increase in skeletal, muscular, and visceral growth. As a consequence, a diffuse increase occurs in the bulk of all tissues (“megaly” means enlargement) especially in the “acral” (most peripheral) tissues of the body such as the face, the hands, and the feet; hence the term “acromegaly.” Sam Atotrope’s coarse facial features and bulky hands are typical of patients with acromegaly.

Muscle and adipocyte cell membranes contain GH receptors that mediate direct, rapid metabolic effects on glucose and amino acid transport as well as on lipolysis. These receptors use associated cytoplasmic tyrosine kinases for signal transduction (such as the janus kinases; see Chapter 11, Section III.C). STAT (signal transducer and activator of transcription) transcription factors are activated and, depending on the tissue, the MAP kinase pathway and/or the AKT pathway is also activated. For example, in adipose tissue, GH has acute insulin-like effects followed by increased lipolysis, inhibition of lipoprotein lipase, stimulation of hormone-sensitive lipase, decreased glucose transport, and decreased lipogenesis. In muscle, GH causes increased amino acid transport, increased nitrogen retention, increased fat-free (lean) tissue, and increased energy expenditure. GH also has growth-promoting effects. GH receptors are present on a variety of tissues in which GH increases IGF-I gene expression. The subsequent rise in IGF-I levels contributes to cell multiplication and differentiation by autocrine or paracrine mechanisms. These, in turn, lead to skeletal, muscular, and visceral growth. These actions are accompanied by a direct anabolic influence of GH on protein metabolism with a diversion of amino acids from oxidation to protein synthesis and a shift to a positive nitrogen balance. 2.

CONTROL OF SECRETION OF GROWTH HORMONE

Although the regulation of GH secretion is complex, the major influence is hormonal (Fig. 43.2). The pulsatile pattern of GH secretion reflects the interplay of two hypothalamic regulatory peptides. Release is stimulated by growth hormone-releasing hormone (GHRH, also called somatocrinin). The structure of GHRH was identified in 1982 (Fig. 43.3). It exists as both a 40- and a 44-amino acid peptide encoded on chromosome 20 and produced exclusively in cells of the arcuate nucleus. Its C-terminal leucine residue is amidated. Full biologic activity of this releasing hormone resides in the first 29 amino acids of the N-terminal portion of the molecule. GHRH interacts with specific receptors on the plasma membranes of the somatotrophs. The intracellular signaling mechanisms that result in GH synthesis and release appear to be multiple, as cAMP and calcium-calmodulin both stimulate GH release. Conversely, GH secretion is suppressed by growth hormone release–inhibiting hormone (GHRIH, the same as somatostatin, which has already been discussed). In addition, IGF-I, produced primarily in the liver in response to the action of GH on hepatocytes, feeds back negatively on the somatotrophs to limit GH secretion. Other physiologic factors (e.g., exercise and sleep) and many pathologic factors also control its release (Table 43.2). In addition, GH release is modulated by plasma levels of all of the metabolic fuels, including proteins, fats, and carbohydrates. A rising level of glucose in the blood normally suppresses GH release, whereas hypoglycemia increases GH secretion in normal subjects. Amino acids, such as arginine, stimulate release of GH when their concentrations rise in the blood. Rising levels of fatty acids may blunt the GH response to arginine or a rapidly dropping blood glucose level. However, prolonged fasting, in which fatty acids are mobilized in an effort to spare protein, is associated with a rise in GH secretion. Some of the physiologic, pharmacologic, and pathologic influences on GH secretion are given in Table 43.2. These modulators of GH secretion provide the basis for clinical suppression and stimulation tests in patients suspected of having excessive or deficient GH secretion. 3.

EFFECTS OF GROWTH HORMONE ON ENERGY METABOLISM

GH affects the uptake and oxidation of fuels in adipose tissue, muscle, and liver and indirectly influences energy metabolism through its actions on the islet cells of the pancreas. In summary, GH increases the availability of fatty acids, which are oxidized for energy. This and other effects of GH spare glucose and protein; that is, GH indirectly decreases the oxidation of glucose and amino acids (Fig. 43.4).

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4.

EFFECTS OF GROWTH HORMONE ON ADIPOSE TISSUE

GH increases the sensitivity of the adipocyte to the lipolytic action of the catecholamines and decreases its sensitivity to the lipogenic action of insulin. These actions lead to the release of free fatty acids and glycerol into the blood to be metabolized by the liver. GH also decreases esterification of fatty acids, thereby reducing triacylglycerol synthesis within the fat cell. Recent evidence suggests that GH may impair glucose uptake by both fat and muscle cells by a postreceptor inhibition of insulin action. As a result of the metabolic effects of GH, the clinical course of acromegaly (increased GH secretion) may be complicated by impaired glucose tolerance or even overt diabetes mellitus. 5.

EFFECTS OF GROWTH HORMONE ON MUSCLE

The lipolytic effects of GH increase free fatty acid levels in the blood that bathes muscle. These fatty acids are used preferentially as fuel, indirectly suppressing glucose uptake by muscle cells. Through the effects on glucose uptake, the rate of glycolysis is proportionately reduced. GH increases the transport of amino acids into muscle cells, providing substrate for protein synthesis. Through a separate mechanism, GH increases the synthesis of DNA and RNA. The positive effect on nitrogen balance is reinforced by the proteinsparing effect of GH-induced lipolysis that makes fatty acids available to muscle as an alternative fuel source. 6.

EFFECTS OF GROWTH HORMONE ON THE LIVER

When plasma insulin levels are low, as in the fasting state, GH enhances fatty acid oxidation to acetyl coenzyme A (acetyl-CoA). This effect, in concert with the increased flow of fatty acids from adipose tissue, enhances ketogenesis. The increased amount of glycerol reaching the liver as a consequence of enhanced lipolysis acts as a substrate for gluconeogenesis. Hepatic glycogen synthesis is also stimulated by GH in part because of the increased gluconeogenesis in the liver. Finally, glucose metabolism is suppressed by GH at several steps in the glycolytic pathway. +

H3N

Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Ser

Ser Met Ile Asp Gln Leu Leu Lys Arg Ala

Leu

Blood glucose Blood amino acids Sleep rhythms

+

Stress Exercise

+

Brain

+

+

Hypothalamus –

+

GHRH

Somatostatin

+

–

Anterior pituitary Growth hormone

–

Growth hormone IGF-I

+

IGF-I

IGF-I

Liver and other cells

FIG. 43.2. Control of GH secretion. Various factors stimulate the release of GHRH from the hypothalamus. The hypothalamus also releases somatostatin in response to other stimuli. GHRH stimulates and somatostatin inhibits the release of growth hormone from the anterior pituitary. GH causes the release of IGF-I from liver and other tissues. IGF-I inhibits GHRH release and stimulates somatostatin release.

Arg Gln Gln Gly

O

Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu

C NH2

Growth hormone– releasing hormone (GHRH)

S

S

Ala Gly Cys Lys

Thr Asn

Phe Phe Trp Lys Thr

Ser Cys

Phe

Growth hormone release– inhibiting hormone (GHRIH) (Somatostatin)

FIG. 43.3. Structures of growth hormone-releasing hormone (GHRH) and growth hormone release–inhibiting hormone (GHRIH, the same as somatostatin). GHRH has an amide at the C-terminal (in box).

Lieberman_CH43.indd 803

While Sam Atotrope was trying to decide which of the major alternatives for the treatment of his growth hormone (GH)–secreting pituitary tumor to choose, he noted progressive fatigue and the onset of increasing urinary frequency associated with a marked increase in thirst. In addition, he had lost 4 lb over the course of the last 6 weeks in spite of a good appetite. His physician suspected that Mr. Atotrope had developed diabetes mellitus, perhaps related to the chronic hypersecretion of GH. This suspicion was confirmed when Sam’s serum glucose level—drawn before breakfast—was reported to be 236 mg/dL.

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SECTION VIII ■ TISSUE METABOLISM

Table 43.2 Physiologic

Pharmacologic

Pathologic

Some Factors Affecting Growth Hormone Secretion Stimulate

Suppress

Low blood glucose after meals High blood amino acids after meals Exercise Sleep Stress GHRH Estrogens ␣-Adrenergic agonists ␤-Adrenergic antagonists Dopamine agonists Serotonin precursors K⫹ infusion Starvation Anorexia nervosa Ectopic GHRH production Acromegaly Chronic renal failure Hypoglycemia

High blood glucose after meals High blood fatty acids

Somatostatin Progesterone ␣-Adrenergic antagonists ␤-Adrenergic agonists Dopamine antagonists Growth hormone and IGF-I Obesity Hypothyroidism Hyperthyroidism

GHRH, growth hormone-releasing hormone; IGF-I, insulin-like growth factor I.

A major effect of GH on liver is to stimulate production and release of IGFs. The IGFs are also known as somatomedins. The two somatomedins in humans share structural homologies with proinsulin, and both have substantial insulinlike growth activity—hence the designations insulin-like growth factor I (human IGF-I, or somatomedin-C) and insulin-like growth factor II (human IGF-II, or somatomedin-A). IGF-I is a single chain basic peptide that has 70 amino acids, and IGF-II is slightly acidic with 67 amino acids. These two peptides are identical to insulin in half of their residues. In addition, they contain a structural domain that is homologous to the C-peptide of proinsulin. A broad spectrum of normal cells respond to high doses of insulin by increasing thymidine uptake and initiating cell propagation. In most instances, IGF-I

Hypothalamus

Pituitary GHRH

Somatostatin

+

–

Growth hormone

Liver • IGF-I Gluconeogenesis Glycogen synthesis

Growth plate • Growth

Adipose tissue • Lipolysis

Muscle Glucose uptake Protein synthesis

FIG. 43.4. Anabolic effects of growth hormone on various tissues.

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CHAPTER 43 ■ ACTIONS OF HORMONES THAT REGULATE FUEL METABOLISM

causes the same response as insulin in these cells but at significantly smaller, more physiologic concentrations. Thus, the IGFs are more potent than insulin in their growth-promoting actions. Evidence suggests that the IGFs exert their effects through either an endocrine or a paracrine/autocrine mechanism. IGF-I appears to stimulate cell propagation and growth by binding to specific IGF-I receptors on the plasma membrane of target cells, rather than binding to GH receptors (Fig. 43.5). Like insulin, the intracellular portion of the plasma membrane receptor for IGF-I (but not IGF-II) has intrinsic tyrosine kinase activity. The fact that the receptors for insulin and several other growth factors have intrinsic tyrosine kinase activity indicates that tyrosine phosphorylation initiates the process of cellular replication and growth. Subsequently, a chain of kinases is activated, which include several proto-oncogene products (see Chapters 11 and 18). Most cells of the body have mRNA for IGF, but the liver has the greatest concentration of these messages, followed by kidney and heart. The synthesis of IGF-I is regulated, for the most part, by GH whereas hepatic production of IGF-II is independent of GH levels in the blood.

Aminergic neurons

Hypothalamus

GHRH

Somatotroph

1.

SYNTHESIS OF THE CATECHOLAMINES

Tyrosine is the precursor of the catecholamines. The pathway for the biosynthesis of these molecules is described in Chapter 48. 2. SECRETION OF THE CATECHOLAMINES

Secretion of epinephrine and norepinephrine from the adrenal medulla is stimulated by a variety of stresses, including pain, hemorrhage, exercise, hypoglycemia, and hypoxia. Release is mediated by stress-induced transmission of nerve impulses emanating from adrenergic nuclei in the hypothalamus. These impulses stimulate the release of the neurotransmitter acetylcholine from preganglionic neurons that innervate the adrenomedullary cells. Acetylcholine depolarizes the plasma membranes of these cells, allowing the rapid entry of extracellular Ca2⫹ into the cytosol. Ca2⫹ stimulates the synthesis and release of epinephrine and norepinephrine from the chromaffin granules into the extracellular space by exocytosis. 3.

PHYSIOLOGIC EFFECTS OF EPINEPHRINE AND NOREPINEPHRINE

The catecholamines act through two major types of receptors on the plasma membrane of target cells: the ␣-adrenergic and the ␤-adrenergic receptors (see Chapter 26, Section IV.C). The actions of epinephrine and norepinephrine in the liver, the adipocyte, the skeletal muscle cell, and the ␣- and ␤-cells of the pancreas directly influence fuel metabolism (Fig. 43.6). These catecholamines are counterregulatory hormones that have metabolic effects directed toward mobilization of fuels from their storage sites for oxidation by cells to meet the increased energy requirements of acute and

Lieberman_CH43.indd 805

–

GH Liver cell

GH receptor

IGF synthesis IGF

C. Catecholamines (Epinephrine, Norepinephrine, Dopamine) The catecholamines belong to a family of bioamines and are secretory products of the sympathoadrenal system. They are required for the body to adapt to a great variety of acute and chronic stresses. Epinephrine (80% to 85% of stored catecholamines) is synthesized primarily in the cells of the adrenal medulla, whereas norepinephrine (15% to 20% of stored catecholamines) is synthesized and stored not only in the adrenal medulla but also in various areas of the CNS and in the nerve endings of the adrenergic nervous system. Dopamine, another catecholamine, acts primarily as a neurotransmitter and has little effect on fuel metabolism. The first total chemical synthesis of epinephrine was accomplished by Stolz et al. in 1904. In 1950, Sutherland was the first to demonstrate that epinephrine (and glucagon) induces glycogenolysis. This marked the beginning of our understanding of the molecular mechanisms through which hormones act.

GHRIH

+

Tyrosine kinase

Other tissues

Growth, Sulfation of bone

IGF receptor Protein– P Mitogenic response Growth

FIG. 43.5. Production and action of IGFs. The hypothalamus produces GHRH, which stimulates somatotrophs in the anterior pituitary to release GH. GHRIH inhibits GH release. GH binds to cell surface receptors and stimulates IGF production and release by liver and other tissues. IGF binds to cell surface receptors and stimulates the phosphorylation of proteins that lead to cell division and growth.

High levels of circulating IGF-I have been linked to the development of breast, prostate, colon, and lung cancer. Additionally, experimental modulation of IGF-I receptor activity can alter the growth of different types of tumor cells. Current research is aimed at targeting the interaction of IGF-I and its receptor to reduce tumor cell proliferation.

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In patients suspected of having a neoplasm of the adrenal medulla that is secreting excessive quantities of epinephrine or norepinephrine (a pheochromocytoma), either the catecholamines themselves (epinephrine, norepinephrine, and dopamine) or their metabolites (the metanephrines and vanillylmandelic acid [VMA]) may be measured in a 24-hour urine collection, or the level of catecholamines in the blood may be measured. A patient who has consistently elevated levels in the blood or urine should be considered to have a pheochromocytoma, particularly if the patient has signs and symptoms of catecholamine excess such as excessive sweating, palpitations, tremulousness, and hypertension.

chronic stress. They simultaneously suppress insulin secretion, which ensures that fuel fluxes will continue in the direction of fuel use rather than storage as long as the stressful stimulus persists. In addition, norepinephrine works as a neurotransmitter and affects the sympathetic nervous system in the heart, lungs, blood vessels, bladder, gut, and other organs. These effects of catecholamines on the heart and blood vessels increase cardiac output and systemic blood pressure, hemodynamic changes that facilitate the delivery of blood-borne fuels to metabolically active tissues. Epinephrine has a short half-life in the blood and, to be effective pharmacologically, it must be administered parenterally. It may be used clinically to support the beating of the heart, to dilate inflamed bronchial muscles, and even to decrease bleeding from organs during surgery. 4.

METABOLISM AND INACTIVATION OF CATECHOLAMINES

Catecholamines have a relatively low affinity for both ␣- and ␤-receptors. After binding, the catecholamine disassociates from its receptor quickly, causing the duration of the biologic response to be brief. The free hormone is degraded in several ways as outlined in Chapter 48.

D. Glucocorticoids 1. A relatively rare form of secondary hypertension (high blood pressure) is caused by a catecholaminesecreting neoplasm of the adrenal medulla, known as a pheochromocytoma. Patients with this kind of tumor periodically secrete large amounts of epinephrine and norepinephrine into the bloodstream. Symptoms related to this secretion include a sudden and often severe increase in blood pressure, heart palpitations, a throbbing headache, and inappropriate and diffuse sweating. In addition, chronic hypersecretion of these catecholamines may lead to impaired glucose tolerance or even overt diabetes mellitus. Describe the actions of these hormones that lead to the significant rise in glucose levels.

BIOCHEMISTRY

Cortisol (hydrocortisone) is the major physiologic glucocorticoid (GC) in humans, although corticosterone also has some GC activity. GCs, such as cortisol, were

Pancreas ␣-cell

␤-cell –

+

Epi

Insulin

Epi

Glucagon

Liver Glycogen +

Glucose

Epi Glucose

+

Epi

Glycerol Epi

+

FA

Epi +

Glycogen TG

Adipose

Pyruvate and lactate

Muscle

FIG. 43.6. Effects of epinephrine on fuel metabolism and pancreatic endocrine function. Epinephrine (Epi) stimulates glycogen breakdown in muscle and liver, gluconeogenesis in liver, and lipolysis in adipose tissue. Epi further reinforces these effects because it increases the secretion of glucagon, a hormone that shares many of the same effects as epinephrine. Epi also inhibits insulin release while stimulating glucagon release from the pancreas. FA, fatty acid; TG, triglyceride.

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named for their ability to raise blood glucose levels. These steroids are among the “counterregulatory” hormones that protect the body from insulin-induced hypoglycemia. The biosynthesis of steroid hormones and their basic mechanism of action has been described in Chapters 16 and 34. 2.

SECRETION OF GLUCOCORTICOIDS

The synthesis and secretion of cortisol are controlled by a cascade of neural and endocrine signals linked in tandem in the cerebrocortical–hypothalamic– pituitary–adrenocortical axis. Cerebrocortical signals to the midbrain are initiated in the cerebral cortex by “stressful” signals such as pain, hypoglycemia, hemorrhage, and exercise (Fig. 43.7). These nonspecific “stresses” elicit the production of monoamines in the cell bodies of neurons of the midbrain. Those that stimulate the synthesis and release of corticotropin-releasing hormone (CRH) are acetylcholine and serotonin. These neurotransmitters then induce the production of CRH by neurons originating in the paraventricular nucleus. These neurons discharge CRH into the hypothalamico-hypophyseal portal blood. CRH is delivered through these portal vessels to specific receptors on the cell membrane of the adrenocorticotropic hormone (ACTH)-secreting cells of the anterior pituitary gland (corticotrophs). This hormone–receptor interaction causes ACTH to be released into the general circulation, eventually to interact with specific receptors for ACTH on the plasma membranes of cells in the zona fasciculata and zona reticulosum of the adrenal cortex. The major trophic influence of ACTH on cortisol synthesis is at the level of the conversion of cholesterol to pregnenolone, from which the adrenal steroid hormones are derived (see Chapter 34 for the biosynthesis of the steroid hormones). Cortisol is secreted from the adrenal cortex in response to ACTH. The concentration of free (unbound) cortisol that bathes the CRH-producing cells of the hypothalamus and the ACTH-producing cells of the anterior pituitary acts as a negative feedback signal that has a regulatory influence on the release of CRH and ACTH (see Fig. 43.7). High cortisol levels in the blood suppress CRH and ACTH secretion, and low cortisol levels stimulate secretion. In severe stress, however, the negative feedback signal on ACTH secretion exerted by high cortisol levels in the blood is overridden by the stress-induced activity of the higher portions of the axis (see Fig. 43.7). The effects of GCs on fuel metabolism in liver, skeletal muscle, and adipose tissue are outlined in Table 43.1 and in Figure 43.8. Their effects on other tissues are diverse and, in many instances, essential for life. Some of the nonmetabolic actions of GCs are listed in Table 43.3.

The catecholamines are counterregulatory hormones that mobilize fuels from their storage sites for oxidation in target cells to meet the increased energy requirements that occur during acute or chronic stress, or in this case, the release of catecholamines by a tumor in the adrenal medulla. These actions provide the liver, for example, with increased levels of substrate needed for gluconeogenesis. Although in normal individuals, most of the glucose generated through this mechanism is oxidized—blood glucose levels rise in the process. In addition, the catecholamines suppress insulin secretion to ensure that fuels will continue to flow in the direction of use rather than storage under these circumstances. Hence, blood glucose levels may rise in patients who have a pheochromocytoma. Hemorrhage Emotions Exercise Hypoglycemia Pain Infections

Cold exposure

Trauma

Sleep

Toxins Hypothalamus Acetylcholine, serotonin

CRH –

Pituitary

+

3.

EFFECTS OF GLUCOCORTICOIDS

GCs have diverse actions that affect most tissues of the body. At first glance, some of these effects may appear to be contradictory (such as inhibition of glucose uptake by certain tissues), but taken together they promote survival in times of stress. In many tissues, GCs inhibit DNA, RNA, and protein synthesis and stimulate the degradation of these macromolecules. In response to chronic stress, GCs act to make fuels available, so that when the acute alarm sounds and epinephrine is released, the organism can fight or flee. When GCs are elevated, glucose uptake by the cells of many tissues is inhibited, lipolysis occurs in peripheral adipose tissue, and proteolysis occurs in skin, lymphoid cells, and muscle. The fatty acids that are released are oxidized by the liver for energy, and the glycerol and amino acids serve in the liver as substrates for the production of glucose, which is converted to glycogen and stored. The alarm signal of epinephrine stimulates liver glycogen breakdown, making glucose available as fuel to combat the acute stress. The mechanism by which GCs exert these effects involves binding of the steroid to intracellular receptors, interaction of the steroid–receptor complex with

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–

Cortisol

ACTH

+

Adrenal gland

FIG. 43.7. Regulation of cortisol secretion. Various factors act on the hypothalamus to stimulate the release of CRH. CRH stimulates the release of ACTH from the anterior pituitary, which stimulates the release of cortisol from the adrenal cortex. Cortisol inhibits the release of CRH and ACTH via negative feedback loops.

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When Otto Shape was writing his list of differential diagnoses to explain the clinical presentation of Corti Solemia, he suddenly thought of a relatively rare endocrine disorder that could explain all of the presenting signs and symptoms. He made a provisional diagnosis of excessive secretion of cortisol secondary to an excess secretion of ACTH (Cushing “disease”) or by a primary increase of cortisol production by an adrenocortical tumor (Cushing syndrome). Otto suggested that resting, fasting plasma cortisol and ACTH levels be measured at 8:00 the next morning. These studies showed that Mr. Solemia’s morning plasma ACTH and cortisol levels were both significantly higher than the reference range. Therefore, Otto concluded that Mr. Solemia probably had a tumor that was producing ACTH autonomously (i.e., not subject to normal feedback inhibition by cortisol). The high plasma levels of ACTH were stimulating the adrenal cortex to produce excessive amounts of cortisol. Additional laboratory and imaging studies indicated that the hypercortisolemia was caused by a benign ACTH-secreting adenoma of the anterior pituitary gland (Cushing “disease”).

Liver

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+

GC

+

+

GC

Epi

GC Glucose

AA

Glycerol

Precursors PEPCK

Glycogen storage

Glycogen

Glucose

AA

FA

+

+

TG

GC

Muscle

Adipose

Lipolysis Glucose utilization

Protein degradation Protein synthesis Glucose utilization

FIG. 43.8. Effects of GCs on fuel metabolism. GCs stimulate lipolysis in adipose tissue and the release of amino acids from muscle protein. In liver, GCs stimulate gluconeogenesis and the synthesis of glycogen. The breakdown of liver glycogen is stimulated by epinephrine. AA, amino acid; Epi, epinephrine; PEPCK, phosphoenolpyruvate carboxykinase; TG, triglyceride.

GC response elements on DNA, transcription of genes, and synthesis of specific proteins (see Chapter 16, Section III.C.2). In some cases, the specific proteins responsible for the GC effect are known (e.g., the induction of phosphoenolpyruvate carboxykinase that stimulates gluconeogenesis). In other cases, the proteins responsible for the GC effect have not yet been identified. Table 43.3

Otto Shape was now able to explain the mechanism for most of Corti Solemia’s signs and symptoms. For example, Otto knew the metabolic explanation for the patient’s hyperglycemia. Some of Mr. Solemia’s muscle wasting and weakness were caused by the catabolic effect of hypercortisolemia on protein stores, such as those in skeletal muscle, to provide amino acids as precursors for gluconeogenesis. This catabolic action also resulted in the degradation of elastin, a major supportive protein of the skin, as well as an increased fragility of the walls of the capillaries of the cutaneous tissues. These changes resulted in the easy bruisability and the torn subcutaneous tissues of the lower abdomen, which resulted in red striae or stripes. The plethora (redness) of Mr. Solemia’s facial skin was also caused in part by the thinning of the skin as well as by a cortisol-induced increase in the bone marrow production of red blood cells, which enhanced the “redness” of the subcutaneous tissues.

Gluconeogenesis

Some Nonmetabolic Physiologic Actions of Glucocorticoids

On electrolyte and water balance: Increase sodium and water retention (1/3,000 the potency of aldosterone) Increase renal glomerular filtration rate to maintain water excretion rate On cardiovascular system: Indirect effect of glucocorticoid actions on sodium and water metabolism (above) Maintain volume of microcirculation to tissues (cardiac output) Maintain normal vasomotor response to vasoconstricting agents On skeletal muscle: Maintain muscle function by providing normal microcirculation to muscle Influence muscle mass by enhancing protein catabolism and suppressing protein synthesis On central nervous system: Indirect Maintain normal cerebral microcirculation Direct Influence mood, behavior Influence sensitivity of special senses to stimuli Suppress CRH, ACTH, and ADH secretion On formed elements in blood: Increase red blood cell mass and granulocyte proliferation Decrease lymphocyte, monocyte, and basophil proliferation Anti-inflammatory actions: Inhibit early inflammatory process (i.e., edema, fibrin deposition, capillary dilation, leukocyte migration, and phagocytic action) Inhibit late inflammatory process (proliferation of capillaries and fibroblasts, deposition of collagen, and later scar formation) Immune-suppressant actions (of questionable significance at physiologic levels): Prevent manifestations of humoral and cellular immunity Interfere with production of cytokines needed for immune competence via cell-to-cell communication CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone.

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CHAPTER 43 ■ ACTIONS OF HORMONES THAT REGULATE FUEL METABOLISM

E. Thyroid Hormone 1.

BIOCHEMISTRY

The secretory products of the thyroid acinar cells are tetra-iodothyronine (thyroxine, T4) and triiodothyronine (T3). Their structures are shown in Figure 43.9. The basic steps in the synthesis of T3 and T4 in these cells involve the transport or trapping of iodide from the blood into the thyroid acinar cell against an electrochemical gradient; the oxidation of iodide to form an iodinating species; the iodination of tyrosyl residues on the protein, thyroglobulin (Tgb), to form iodotyrosines; and the coupling of residues of monoiodotyrosine and diiodotyrosine in Tgb to form residues of T3 and T4 (Fig. 43.10). Proteolytic cleavage of Tgb releases free T3 and T4. The steps in thyroid hormone synthesis are stimulated by TSH, a glycoprotein produced by the anterior pituitary. Approximately 35% of T4 is deiodinated at the 5⬘-position to form T3, and 43% is deiodinated at the 5⬘-position to form the inactive “reverse” T3. Further deiodination or oxidative deamination leads to formation of compounds that have no biologic activity. Iodide transport from the blood into the thyroid acinar cell is accomplished through an energy-requiring, iodide-trapping mechanism that requires symport with sodium (Na⫹). The sodium–iodide symporter (NIS, encoded by the SLC5A5 gene) is driven by the electrochemical gradient across the membrane that is established by the Na⫹,K⫹-ATPase. For each iodide anion transported across the membrane, two Na⫹ ions are cotransported to facilitate and drive the translocation of the ions. Loss of NIS activity leads to congenital iodide transport defect. The rate of iodide transport is influenced by the absolute concentration of iodide within the thyroid cell. An internal autoregulatory mechanism decreases transport of iodide into the cell when the intracellular iodide concentration exceeds a certain threshold and increases transport when intracellular iodide falls lower than this threshold level. The iodide-concentrating or trapping process in the plasma membrane of thyroid acinar cells create iodide levels within the thyroid cell that are several hundredfold greater than those in the blood, depending on the current size of the total body iodide pool and the present need for new hormone synthesis. The oxidation of intracellular iodide is catalyzed by thyroid peroxidase (located at the apical border of the thyroid acinar cell) in what may be a two-electron oxidation step forming I⫹ (iodinium ion). I⫹ may react with a tyrosine residue in the protein Tgb to form a tyrosine quinoid and then a 3⬘-monoiodotyrosine (MIT) residue. It has been suggested that a second iodide is added to the ring by similar mechanisms to form a 3,5-diiodotyrosine (DIT) residue. Because iodide is added to these organic compounds, iodination is also referred to as the “organification of iodide.”

I

809

If Corti Solemia’s problem had been caused by a neoplasm of the adrenal cortex, what would his levels of blood ACTH and cortisol have been?

Once iodide anion enters the thyroid follicular cell, it must be transported across the apical membrane to react with thyroid peroxidase and thyroglobulin (Tgb). The apical iodide transporter is pendrin (encoded by the SLC26A4 gene), and mutations in pendrin lead to Pendred syndrome. Children with this syndrome display a total loss of hearing, goiter (swelling of the thyroid gland), and metabolic defects in iodide organification.

I

HO

O

I

CH2

CH

COOH

NH2

I

3,5,3',5'-Tetra-iodothyronine (T4)

I

I

HO

O

CH2

I

CH

COOH

NH2

3,5,3'-Tri-iodothyronine (T3)

FIG. 43.9.

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Thyroid hormones T3 and T4.

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SECTION VIII ■ TISSUE METABOLISM

Tgb with Tyr residues

Tgb with DIT

Tgb with T4

I

OH

OH

H2O2

I

I

Colloid

Iodination

I+

I

OH

I O

Coupling

I

OH

OH

I

I

PD Exocytosis

Pinocytosis

Tgb with T4 (+ T3)

Lysosomes Tgb Digestion by lysosomal proteases Protein synthesis

I– Thyroid follicular cell

RER Blood

Tgb with T4 (+ T3)

Iodine pump

I–

T 3 T4

NIS T3 T4

FIG. 43.10. Synthesis of the thyroid hormones (T3 and T4). The protein thyroglobulin (Tgb) is synthesized in thyroid follicular cells and secreted into the colloid. Iodination and coupling of tyrosine residues in Tgb produce T3 and T4 residues, which are released from Tgb by pinocytosis (endocytosis) and lysosomal action. The coupling of a monoiodotyrosine (MIT) with a diiodotyrosine (DIT) to form triiodothyronine (T3) is not depicted here. RER, rough endoplasmic reticulum; NIS, sodium–iodide symporter; PD, pendrin.

If Corti Solemia’s problem had resulted from primary hypersecretion of cortisol by a neoplasm of the adrenal cortex, his blood cortisol levels would have been elevated. The cortisol would have acted on the CRH-producing cells of the hypothalamus and the ACTH-secreting cells of the anterior pituitary by a negative feedback mechanism to decrease ACTH levels in the blood. Because his cortisol and ACTH levels were both high, Mr. Solemia’s tumor was most likely in the pituitary gland or possibly in neoplastic extrapituitary tissue that was secreting ACTH “ectopically.” (“Ectopic” means that the tumor or neoplasm is producing and secreting a substance that is not ordinarily made or secreted by the tissue from which the tumor developed.) Mr. Solemia’s tumor was in the anterior pituitary, not in an extrapituitary ACTH-producing site.

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The biosynthesis of thyroid hormone proceeds with the coupling of an MIT and a DIT residue to form a T3 residue or of two DIT residues to form a T4 residue. T3 and T4 are stored in the thyroid follicle as amino acid residues in Tgb. Under most circumstances, the T4/T3 ratio in Tgb is approximately 13:1. Normally, the thyroid gland secretes 80 to 100 ␮g of T4 and approximately 5 ␮g of T3 per day. The additional 22 to 25 ␮g of T3 “produced” daily is the result of the deiodination of the 5⬘-carbon of T4 in peripheral tissues. T3 is believed to be the predominant biologically active form of thyroid hormone in the body. The thyroid gland is unique in that it has the capacity to store large amounts of hormone as amino acid residues in Tgb within its colloid space. This storage accounts for the low overall turnover rate of T3 and T4 in the body. The plasma half-life of T4 is approximately 7 days, and that of T3 is 1 to 1.5 days. These relatively long plasma half-lives result from binding of T3 and T4 to several transport proteins in the blood. Of these transport proteins, thyroidbinding globulin (TBG) has the highest affinity for these hormones and carries approximately 70% of bound T3 and T4. Only 0.03% of total T4 and 0.3% of total T3 in the blood are in the unbound state. This free fraction of hormone has biologic activity because it is the only form that is capable of diffusing across target cell membranes to interact with intracellular receptors. The transport proteins, therefore, serve as a large reservoir of hormone that can release additional free hormone as the metabolic need arises. The thyroid hormones are degraded in liver, kidney, muscle, and other tissues by deiodination, which produces compounds with no biologic activity.

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2.

SECRETION OF THYROID HORMONE

The release of T3 and T4 from Tgb is controlled by TSH from the anterior pituitary. TSH stimulates the endocytosis of Tgb to form endocytic vesicles within the thyroid acinar cells (see Fig. 43.10). Lysosomes fuse with these vesicles, and lysosomal proteases hydrolyze Tgb, releasing free T4 and T3 into the blood in a 10:1 ratio. In various tissues, T4 is deiodinated, forming T3, which is the active form of the hormone. TSH is synthesized in the thyrotropic cells of the anterior pituitary. Its secretion is regulated primarily by a balance between the stimulatory action of hypothalamic thyrotropin-releasing hormone (TRH) and the inhibitory (negative feedback) influence of thyroid hormone (primarily T3) at levels higher than a critical threshold in the blood bathing the pituitary thyrotrophs. TSH secretion occurs in a circadian pattern, a surge beginning late in the afternoon and peaking before the onset of sleep. In addition, TSH is secreted in a pulsatile fashion, with intervals of 2 to 6 hours between peaks. TSH stimulates all phases of thyroid hormone synthesis by the thyroid gland, including iodide trapping from the plasma, organification of iodide, coupling of MIT and DIT, endocytosis of Tgb, and proteolysis of Tgb to release T3 and T4 (see Fig. 43.10). In addition, the vascularity of the thyroid gland increases as TSH stimulates hypertrophy and hyperplasia of the thyroid acinar cells. The predominant mechanism of action of TSH is mediated by binding of TSH to its G protein coupled-receptor on the plasma membrane of the thyroid acinar cell, leading to an increase in the concentration of cytosolic cAMP (through G␣s) and Ca2⫹ (through G␣q). The increase in Ca2⫹ is brought about by activation of phospholipase C, eventually leading to the activation of the MAP kinase pathway. The large protein Tgb, which contains T3 and T4 in peptide linkage, is stored extracellularly in the colloid that fills the central space of each thyroid follicle. Each of the biochemical reactions that leads to the release and eventual secretion of T3 and T4, such as those that lead to their formation in Tgbs, is TSH-dependent. Rising levels of serum TSH stimulate the endocytosis of stored Tgb into the thyroid acinar cell. Lysosomal enzymes then cleave T3 and T4 from Tgb. T3 and T4 are secreted into the bloodstream in response to rising levels of TSH. As the free T3 level in the blood bathing the thyrotrophs of the anterior pituitary gland rises, the feedback loop is closed. Secretion of TSH is inhibited until the free T3 levels in the systemic circulation fall just below a critical level, which once again signals the release of TSH. This feedback mechanism ensures an uninterrupted supply of biologically active free T3 in the blood (Fig. 43.11). High levels of T3 also inhibit the release of TRH from the hypothalamus. 3.

811

The “central” deposition of fat in patients, such as Corti Solemia, with Cushing “disease” or syndrome is not readily explained because GCs actually cause lipolysis in adipose tissue. The increased appetite caused by an excess of GC and the lipogenic effects of the hyperinsulinemia that accompanies the GC-induced chronic increase in blood glucose levels have been suggested as possible causes. Why the fat is deposited centrally under these circumstances, however, is not understood. This central deposition leads to the development of a large fat pad at the center of the upper back (“buffalo hump”), to accumulation of fat in the cheeks and jaws (“moon facies”) and neck area, as well as a marked increase in abdominal fat. Simultaneously, there is a loss of adipose and muscle tissue below the elbows and knees, exaggerating the appearance of “central obesity” in Cushing “disease” or syndrome.

In areas of the world in which the soil is deficient in iodide, hypothyroidism is common. The thyroid gland enlarges (forms a goiter) in an attempt to produce more thyroid hormone. In the United States, table salt (NaCl) enriched with iodide (iodized salt) is used to prevent hypothyroidism caused by iodine deficiency.

PHYSIOLOGIC EFFECTS OF THYROID HORMONE

Only those physiologic actions of thyroid hormone that influence fuel metabolism are considered here. It is important to stress the term “physiologic” because the effects of supraphysiologic concentrations of thyroid hormone on fuel metabolism may not be simple extensions of their physiologic effects. For example, when T3 is present in excess, it has severe catabolic effects that increase the flow of amino acids from muscle into the blood and eventually to the liver. In general, the following comments apply to the effects of thyroid hormone on energy metabolism in individuals who have normal thyroid hormone levels in their blood. i.

Effects of Thyroid Hormone on the Liver

Several of the actions of thyroid hormone affect carbohydrate and lipid metabolism in the liver. Thyroid hormone increases glycolysis and cholesterol synthesis and increases the conversion of cholesterol to bile salts. Through its action of increasing

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SECTION VIII ■ TISSUE METABOLISM

Hypothalamus TRH – +

Pituitary

the sensitivity of the hepatocyte to the gluconeogenic and glycogenolytic actions of epinephrine, T3 indirectly increases hepatic glucose production (permissive or facilitatory action). Because of its ability to sensitize the adipocyte to the lipolytic action of epinephrine, T3 increases the flow of fatty acids to the liver and thereby indirectly increases hepatic triacylglycerol synthesis. The concurrent increase in the flow of glycerol to the liver (as a result of increased lipolysis) further enhances hepatic gluconeogenesis. ii.

TSH –

T3

iii. Effects of Thyroid Hormone on Muscle

T3 T4

Liver and other cells

+

Thyroid

FIG. 43.11. Feedback regulation of thyroid hormone levels. TRH from the hypothalamus stimulates the release of TSH from the anterior pituitary, which stimulates the release of T3 and T4 from the thyroid. T4 is converted to T3 in the liver and other cells. T3 inhibits the release of TSH from the anterior pituitary and of TRH from the hypothalamus.

A patient presents with the following clinical and laboratory profile: The serum free and total T3 and T4 and the serum TSH levels are elevated, but the patient has symptoms of mild hypothyroidism, including a diffuse, palpable goiter. What single abnormality in the pituitary–thyroid–thyroid hormone target cell axis would explain all of these findings?

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Effects of Thyroid Hormone on the Adipocyte

T3 has an amplifying or facilitatory effect on the lipolytic action of epinephrine on fat cells. Yet thyroid hormone has a bipolar effect on lipid storage because it increases the availability of glucose to the fat cells, which serves as a precursor for fatty acid and glycerol 3-phosphate synthesis. The major determinant of the rate of lipogenesis, however, is not T3, but rather the amount of glucose and insulin available to the adipocyte for triacylglycerol synthesis. In physiologic concentrations, T3 increases glucose uptake by muscle cells. It also stimulates protein synthesis and, therefore, growth of muscle, through its stimulatory actions on gene expression. In physiologic concentrations, thyroid hormone sensitizes the muscle cell to the glycogenolytic actions of epinephrine. Glycolysis in muscle is increased by this action of T3. iv. Effects of Thyroid Hormone on the Pancreas

Thyroid hormone increases the sensitivity of the ␤-cells of the pancreas to those stimuli that normally promote insulin release and is required for optimal insulin secretion. 4.

CALORIGENIC EFFECTS OF THYROID HORMONE

The oxidation of fuels convert approximately 25% of the potential energy present in the foods ingested by humans to adenosine triphosphate (ATP). This relative inefficiency of the human “engine” leads to the production of heat as a consequence of fuel use. This inefficiency, in part, allows homeothermic animals to maintain a constant body temperature in spite of rapidly changing environmental conditions. The acute response to cold exposure is shivering, which is probably secondary to increased activity of the sympathetic nervous system in response to this “stressful” stimulus. Thyroid hormone participates in this acute response by sensitizing the sympathetic nervous system to the stimulatory effect of cold exposure. The ability of T3 to increase heat production is related to its effects on the pathways of fuel oxidation, which both generate ATP and release energy as heat. The effects of T3 on the sympathetic nervous system increases the release of norepinephrine. Norepinephrine stimulates the uncoupling protein thermogenin in brown adipose tissue (BAT), resulting in increased heat production from the uncoupling of oxidative phosphorylation (see Chapter 21). Very little residual brown fat persists in normal adult human beings, however. Norepinephrine also increases the permeability of BAT and skeletal muscle to Na⫹. Because an increase of intracellular Na⫹ is potentially toxic to cells, Na⫹,K⫹-ATPase is stimulated to transport Na⫹ out of the cell in exchange for K⫹. The increased hydrolysis of ATP by Na⫹,K⫹-ATPase stimulates the oxidation of fuels and the regeneration of more ATP and heat from oxidative phosphorylation. Over a longer time course, thyroid hormone also increases the level of Na⫹,K⫹ATPase and many of the enzymes of fuel oxidation. Because even at normal room temperature ATP use by Na⫹,K⫹-ATPase accounts for 20% or more of our basal metabolic rate (BMR), changes in its activity can cause relatively large increases in heat production.

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CHAPTER 43 ■ ACTIONS OF HORMONES THAT REGULATE FUEL METABOLISM

Thyroid hormone also may increase heat production by stimulating ATP use in futile cycles (in which reversible ATP-consuming conversions of substrate to product and back to substrate use fuels and, therefore, produce heat).

F. Gastrointestinal-Derived Hormones That Affect Fuel Metabolism In addition to insulin and the counterregulatory hormones discussed so far, a variety of peptides synthesized in the endocrine cells of the pancreatic islets, or the cells of the enteric nervous system, or the endocrine cells of the stomach, small bowel and large bowel, as well as certain cells of the central and peripheral nervous system influence fuel metabolism directly. Some of these peptides and their tissues of origin, their actions on fuel metabolism, and the factors that stimulate (or suppress) their secretion are listed in Table 43.4. In addition to these peptides, others such as gastrin, motilin, pancreatic polypeptide (PP), peptide YY (PYY), and secretin may also influence fuel metabolism by indirect effects on the synthesis or secretion of insulin or the counterregulatory hormones (Table 43.5). For example, gastrin induces gastric acid secretion, which ultimately affects nutrient absorption and metabolism. Motilin, secreted by enteroendocrine M-cells of the proximal small bowel, stimulates gastric and pancreatic enzyme secretion, which in turn influences nutrient digestion. PP from the pancreatic islets reduces gastric emptying and slows upper intestinal motility. PYY from the ␣-cells in the mature pancreatic islets inhibits gastric acid secretion. Finally, secretin, produced by the enteroendocrine S-cells in the proximal small bowel, regulates pancreatic enzyme secretion and inhibits gastrin release and secretion of gastric acid. Although these “gut” hormones do not influence fuel metabolism directly, they have a significant impact on how ingested nutrients are digested and prepared for absorption. If digestion or absorption of fuels is altered through a disturbance in the delicate interplay among all of the peptides, fuel metabolism will be altered as well. Several of these gastrointestinal peptides, such as GLP-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP), do not act as direct insulin secretagogues when blood glucose levels are normal but do so after a meal large enough to cause an increase in the blood glucose concentration. The release of these peptides may explain why the modest postprandial increase in serum glucose that is seen in normal subjects has a relatively robust stimulatory effect on insulin release, whereas a similar glucose concentration in vitro elicits a significantly smaller increase in insulin secretion. Likewise, this effect (certain factors potentiating insulin release), known as the “incretin effect,” could account for the greater ␤-cell response seen after an oral glucose load as opposed to that seen after the administration of glucose intravenously. This phenomenon is estimated to account for approximately 50% to 70% of the total insulin secreted after oral glucose administration. It is clear then that the gastrointestinal tract plays a critical role in peripheral energy homeostasis through its ability to influence the digestion, absorption, and assimilation of ingested nutrients. Importantly, the incretin hormones also regulate the amount of nutrients ingested through their central action as satiety signals. In Table 43.6, the actions of GLP-1 and GIP on key target organs that are important for the control of glucose homeostasis are given. Both GLP-1 and GIP enhance the synthesis and release of insulin as well as exerting a positive influence on the survival of pancreatic islet cells. In addition, GLP-1 contributes to the regulation of glucose homeostasis by inhibiting the secretion of glucagon from the ␣-cells of the pancreas as well as by slowing the rate of gastric emptying. GIP, but not GLP-1, interacts with GIP receptors on adipocytes, an interaction that is coupled to energy storage. Figure 43.12 summarizes the key effects of GLP-1 and GIP on energy metabolism.

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813

A generalized (i.e., involving all of the target cells for thyroid hormone in the body) but incomplete resistance of cells to the actions of thyroid hormone could explain the profile of the patient. In Refetoff disorder, a mutation in the portion of the gene that encodes the ligand-binding domain of the ␤-subunit of the thyroid hormone–receptor protein (expressed in all thyroid hormone responsive cells) causes a relative resistance to the suppressive action of thyroid hormone on the secretion of TSH by the thyrotrophs of the anterior pituitary gland. Therefore, the gland releases more TSH than normal into the blood. The elevated level of TSH causes an enlargement of the thyroid gland (goiter) as well as an increase in the secretion of thyroid hormone into the blood. As a result, the serum levels of both T3 and T4 rise in the blood. The increase in the secretion of thyroid hormone may or may not be adequate to fully compensate for the relative resistance of the peripheral tissues to thyroid hormone. If the compensatory increase in the secretion of thyroid hormone is inadequate, the patient may develop the signs and symptoms of hypothyroidism.

In hypothyroid patients, insulin release may be suboptimal, although glucose intolerance on this basis alone is uncommon. In hyperthyroidism, the degradation and the clearance of insulin are increased. These effects, plus the increased demand for insulin caused by the changes in glucose metabolism, may lead to varying degrees of glucose intolerance in these patients (a condition called metathyroid diabetes mellitus). A patient with uncomplicated hyperthyroidism, however, rarely develops significant diabetes mellitus.

Ghrelin, a hormone identified as a growth hormone secretagogue, has recently been linked to appetite stimulation. The mechanism whereby this occur is through the activation of the AMPactivated protein kinase in the hypothalamus. The activation of this kinase leads to the release of neuropeptide Y, which increases appetite. Research geared toward interrupting the ghrelin/ghrelin receptor signaling system is increasing to develop new antiobesity agents.

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Table 43.4

Gastrointestinal-Derived Hormones That Affect Fuel Metabolism Directly

Hormone

Primary Cell/Tissue of Origin

Actions

Secretory Stimuli (and Inhibitors)

Amylin

Pancreatic ␤-cell, endocrine cells of stomach and small intestine

Co-secreted with insulin in response to oral nutrients

CGRP

Enteric neurons and enteroendocrine cells of the rectum Nervous system, pituitary, neurons of gut, pancreas, thyroid, and adrenal gland Neuroendocrine K-cells of duodenum and proximal jejunum

1. Inhibits arginine-stimulated and postprandial glucagon secretion 2. Inhbits insulin secretion Inhibits insulin secretion

Primary counter-regulatory hormone that restores glucose levels in hypoglycemic state (increases glycogenolysis and gluconeogenesis as well as protein– lipid flux in liver and muscle) 1. Enhances glucose disposal after meals by inhibiting glucagon secretion and stimulating insulin secretion 2. Acts through second messengers in ␤-cells to increase sensitivity of these cells to glucose (an incretin) Stimulates intestinal hexose transport Inhibits glucose-stimulated insulin secretion

Neural and humoral factors released in response to hypoglycemia

Galanin

GIP

GRP

Enteric nervous system and pancreas

Ghrelin

Central nervous system, stomach, small intestine, and colon Pancreatic ␣-cell, central nervous system

Glucagon

Oral glucose intake and gastric acid secretion Inhibits secretion of insulin, somatostatin, Intestinal distension enteroglucagon, pancreatic polypeptide, and others 1. Increases insulin release via an Oral nutrient ingestion, especially “incretin” effect long-chain fatty acids 2. Regulates glucose and lipid metabolism Stimulates release of cholecystokinin; GIP, gastrin, glucagon, GLP-1, GLP-2, and somatostatin Stimulates growth hormone release Fasting

GLP-1

Enteroendocrine L-cells in ileum, colon, and central nervous system

GLP-2 Neuropeptide Y

Same as for GLP-1 Central and peripheral nervous system, pancreatic islet cells

NT

Small intestine N-cells (especially ileum), In brain, modulates dopamine neuroenteric nervous system, adrenal gland, transmission and anterior pituitary pancreas secretions Brain, lung, and enteric nervous system Stimulates insulin and catecholamine release Central nervous system, pancreatic 1. Inhibits secretion of insulin, glucagon ␦-cells, and enteroendocrine ␦-cells and PP (islets), and gastrin, secretin, GLP-1, and GLP-2 (in gut) 2. Reduces carbohydrate absorption from gut lumen

PACAP Somatostatin

VIP

Widely expressed in the central and peripheral nervous systems

May regulate release of insulin and pancreatic glucagon

1. 2. 3. 4.

Oral nutrient ingestion Vagus nerve GRP and GIP Somatostatin inhibits secretion

Same as GLP-1 Oral nutrient ingestion and activation of sympathetic nervous system 1. Luminal lipid nutrients 2. GRP 3. Somatostatin inhibits secretion Activation of central nervous system 1. 2. 3. 4. 5. 6. 1. 2.

Luminal nutrients GLP-1 GIP PACAP VIP ␤-Adrenergic stimulation Mechanical stimulation of gut Activation of central and peripheral nervous systems

CGRP, calcitonin gene-related peptide; GIP, gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide; GRP, gastrin-releasing peptide; GLP-1, glucagon-like peptide-1; GLP-2, glucagon-like peptide-2; NT, neurotensin; PACAP, pituitary adenylate cyclase activating peptide; PP, pancreatic polypeptide; VIP, vasoactive intestinal peptide.

As the biological effects of the incretins were being discovered, it was hypothesized that agents which would increase the levels of the incretins, or increase their half-life in circulation, may provide an effective means of treating type 2 diabetes by increasing insulin secretion from the pancreas. The half-lives of GIP and GLP-1 in the circulation are on the order of 2.5 minutes. The protease DPP-4 (dipeptidyl protease 4) found on the surface of kidneys, intestine, liver, and many other tissues is responsible for inactivating GIP and GLP-1. If one wants to increase the efficacy of the incretins, synthetic incretin mimetics with longer half-lives could be developed, along with drugs which inhibit DPP-4, thereby increasing the serum half-lives of GIP and GLP-1. Such drugs have been developed and are used for the treatment of type 2 diabetes

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Table 43.5

Gastrointestinal-Derived Hormones That Affect Fuel Metabolism Indirectly

Hormone

Primary Cell/Tissue of Origin

Actions

Secretory Stimuli (and Inhibitors)

CCK

Enteroendocrine I-cells, enteric nerves, others

1. Oral nutrient ingestion 2. GGRP and bombesin from gut

Gastrin

Enteroendocrine G-cells of the stomach, duodenal bulb, and other cells

1. Inhibits proximal gastric motility 2. Increases antral and pyloric contractions 3. Regulates nutrient-stimulated enzyme secretion and gallbladder contraction 4. Increases postprandial satiety Induces gastric acid secretion

Motilin

Enteroendocrine M-cells in upper small bowel and other cells

PP

1. Induces phase III contractions in stomach 2. Stimulates gastric secretion and pancreatic enzyme secretion 3. Induces gallbladder contraction Endocrine cells in periphery of 1. Reduces CCK-mediated gastric acid secretion islets in the head of the pancreas 2. Increases intestinal transit time (slows motility)

PYY

Enteroendocrine cells, developing pancreas; alpha cells in mature islets

Secretin

Enteroendocrine S-cells in upper small bowel

Tachykinins Neurons localized in the submucous and myenteric plexuses; enterochromaffin cells in gut epithelium TRH Enteric nervous system, colon, Gcells of stomach, and pancreatic ␤-cells

1. Inhibits both gastric acid secretion and gastric motility 2. Slows intestinal motility 3. Inhibits pancreatic exocrine secretion 1. Stimulates pancreatic and biliary bicarbonate and water secretion 2. Regulates pancreatic enzyme secretion 3. Inhibits postprandial gastric emptying, gastrin release, and gastric acid secretion 1. Regulates vasomotor and gastrointestinal smooth muscle contraction 2. Mucus secretion and water absorption 1. Suppresses hormone-stimulated gastric acid secretion 2. Inhibits cholesterol synthesis within the intestinal mucosa

1. Luminal contents, especially aromatic amino acids, calcium, coffee, and ethanol 2. Vagus nerve stimulation; activation of ␤-adrenergic and GABA neurons 3. Somatostatin inhibits secretion 1. Duodenal alkalinization 2. Gastric distension 3. Secretion suppressed by nutrients in duodenum Stimulated by intraluminal nutrients, hypoglycemia, and vagal nerve stimulation 1. Oral nutrient ingestion 2. Bile acids and fatty acids 3. Amino acids in colon 1. Gastric acid, bile salts, fatty acids, peptides, and ethanol 2. Somatostatin inhibits secretion

Direct and indirect activation of neurons in submucosa and myenteric plexuses in gut epithelium In the stomach, histamine and serotonin stimulate secretion

CCK, cholecystokinin; GGRP, gastric gastrin releasing peptide; GABA, ␥-aminobutyric acid; PP, pancreatic polypeptide; PYY, peptide YY; TRH, thyrotropin-releasing hormone.

Table 43.6

Actions of GLP-1 and GIP Relevant to Glucose Control

Pancreas Stimulates glucose-dependent insulin release Increase insulin biosynthesis Inhibits glucagon secretion Stimulates somatostatin secretion Induces ␤-cell proliferation Inhibits ␤-cell apoptosis Gastrointestinal tract Inhibits gastric emptying Inhibits gastric acid secretion Central nervous system Inhibits food and water intake Promotes satiety and weight loss Cardiovascular system Improves cardiovascular function after ischemia Adipose tissue Insulin-like lipogenic actions Lipid storage

GLP-1

GIP

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫺ ⫺ ⫹ ⫹

⫹ ⫹

⫺ ⫹

⫹ ⫹

⫺ ⫺

⫹

⫺

⫺ ⫺

⫹ ⫹

GLP-1, glucagon-like peptide-1; GIP, gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide.

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SECTION VIII ■ TISSUE METABOLISM

Islet neogenesis

Adipocyte

Increased lipolysis Increased fatty acid synthesis

Intestines Pancreas ␤-cell GIP

CCK

Pancreas GLP-1

␤-cell

␣-cell

Stomach Gastrin Ghrelin

Brain

Decreased gastric emptying

Decreased glucagon secretion

Increased insulin secretion and biosynthesis Increased ␤-cell proliferation and survival

Increased nausea Increased satiety Decreased food intake Decreased body weight

FIG. 43.12. Actions of selected peptides on vital tissues involved in glucose homeostasis. Both GLP-1 and GIP increase insulin secretion and ␤-cell survival. GLP-1 has additional actions related to glucose metabolism. In contrast, gastrin and CCK do not acutely regulate plasma glucose levels, but appear to increase ␤-cell proliferation.

mellitus. The first are potent GLP-1 receptor agonists. Exendin-4 or exenatide (Byetta) isolated from the venom of a lizard—Heloderma suspectum—was the first drug approved for such treatment. This agonist of the GLP-1 receptor must be administered subcutaneously, but because of its relative resistance to enzymatic cleavage by dipeptidyl peptidase 4 (unlike native GLP-1, which is rapidly cleaved by this enzyme), its biologic half-life in the plasma allows it to be administered only twice daily. DPP-4 cleaves GLP-1 after amino acid 2 (alanine) and breaks the alanine–glutamate peptide bond at that position. Exenatide has a glycine–glutamate sequence at amino acids 2 and 3, rendering this peptide more resistant to DPP-4 action than GLP-1. A second GLP-1 receptor agonist is liraglutide, which is a modified version of GLP-1. Liraglutide has a substitution at position 34 of the peptide, of an arginine for a lysine (K34R), along with the addition of a palmitate at position K26 (covalently linked to the lysine side chain). Addition of the fatty acid to the peptide allows liraglutide to bind to albumin in the circulation, protecting it from DPP-4, and allowing just a single daily dosing. The second class of agents (first marketed in October 2006 as sitagliptin [Januvia]) are orally administered inhibitors of DPP-4. Through this action, sitagliptin slows the rate of catalytic cleavage of GIP and GLP-1 by DPP-4 and, therefore, prolongs their half-lives in the blood, allowing sitagliptin to be administered just twice daily. The contrasting actions of GLP-1 receptor agonists and the DPP-4 inhibitors are listed in Table 43.7. Since the introduction of sitagliptin, other DPP-4 inhibitors have been introduced, including alogliptin, saxagliptin, and vildagliptin. Only sitagliptin and saxagliptin are approved by the FDA for use in the United States. Early estimates of their glucose-lowering efficacy in patients with type 2 diabetes mellitus suggests that the drugs which boost incretin action lowers the blood hemoglobin A1c level to approximately the same extent as does the other currently available oral antidiabetic agents (see the Biochemical Comments in Chapter 34) such as the sulfonylureas, metformin, and the thiazolidinediones (e.g., rosiglitazone [Avandia] and pioglitazone [Actos]).

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Table 43.7 Similarities and Differences in GLP-1 Receptor Agonists and DPP-4 Inhibitors Characteristic

GLP-1 Receptor Agonists

DPP-4 Inhibitors

Administration GLP-1 concentrations Increased insulin secretion Reduced glucagon secretion Activation of portal glucose sensor Gastric emptying inhibited Weight loss Loss of appetite, nausea Proliferation of ␤-cells Potential immunogenicity

Injection Pharmacologic Yes Yes No Yes Yes Yes Yes Yes

Oral Physiologic Yes Yes Yes No No No Yes No

GLP-1, glucagon-like peptide-1; DPP-4, dipeptidyl peptidase 4.

G. Neural Factors That Control Secretion of Insulin and Counterregulatory Hormones Although a full treatment is beyond the scope of this section, the gastrointestinal neuroendocrine system is described briefly here with regard to its effects on fuel metabolism. The pancreatic islet cells are innervated by both the adrenergic and the cholinergic limbs of the autonomic nervous system. Although stimulation of both the sympathetic and the parasympathetic systems increases glucagon secretion, insulin secretion is increased by vagus nerve fibers and suppressed by sympathetic fibers via the ␣-adrenoreceptors. Evidence also suggests that the sympathetic nervous system regulates pancreatic ␤-cell function indirectly through stimulation or suppression of the secretion of somatostatin, ␤2-adrenergic receptor number, and the neuropeptides—neuropeptide Y and galanin. A tightly controlled interaction between the hormonal and neural factors that control nutrient metabolism is necessary to maintain normal fuel and, hence, energy homeostasis.

H. The Endocannabinoid System and Energy Homeostasis The discovery of the endocannabinoid system (ECS) dates to 4,000 years ago, when the psychotropic and therapeutic properties of the plant Cannabis sativa were first described in India. Research into the functional characteristics of the ECS led to the characterization of ⌬9-tetrahydrocannabinol (⌬9-THC), the major psychoactive substance in marijuana (an exogenous cannabinoid). With this discovery, the biochemical and physiologic properties of this novel endogenous signaling system were described. The ECS warrants further discussion here because of its many influences on energy homeostasis (see Chapter 35 for the biosynthesis of endogenous endocannabinoids). Exogenous ligands (e.g., ⌬9-THC) and endogenous ligands (e.g., anandamide or AEA, and 2-arachidonoylglycerol or 2-AG) interact with two basic receptors of the seven-transmembrane G protein–coupled family of receptors. Only the cannabinoid receptor 1 (CB1) receptor will be discussed. The CB2 receptor is found primarily in cells related to immune function and does not affect energy homeostasis. The CB1 receptor is expressed in many tissues, including adipose tissue, muscle, liver, gastrointestinal tract, pancreas, and the CNS. The functions of the ECS in the brain influence energy metabolism via two major mechanisms. The first mechanism involves the ability of the cannabinoids to modulate signaling from hypothalamic nuclei to most of the trophic hormone-producing cells of the anterior pituitary gland. For example, these ligands modulate the rate of secretion of GH, ACTH, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and TSH. All of these pituitary trophic hormones, in turn, trigger the release of hormones from peripheral endocrine organs (e.g., IGF-I, cortisol, sex steroids, and thyroid hormone, respectively), all of which directly or indirectly influence nutrient intake and energy use or storage (see earlier text).

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To establish the diagnosis of a secretory tumor of an endocrine gland, one must first demonstrate that basal serum levels of the hormone in question are regularly elevated. More important, one must show that the hypersecretion of the hormone (and hence its elevated level in the peripheral blood) cannot be adequately inhibited by “maneuvers” that are known to suppress secretion from a normally functioning gland (i.e., one must show that the hypersecretion is “autonomous”). To ensure that both the basal and the postsuppression levels of the specific hormone to be tested will reflect the true secretory rate of the suspected endocrine tumor, all of the known factors that can stimulate the synthesis of the hormone must be eliminated. For GH, for example, the secretagogues (stimulants to secretion) include nutritional factors; the patient’s level of activity, consciousness, and stress; and certain drugs. GH secretion is stimulated by a high-protein meal or by a low level of fatty acids or glucose in the blood. Vigorous exercise; stage III to IV sleep; psychologic and physical stress; and levodopa, clonidine, and estrogens also increase GH release. The suppression test used to demonstrate the autonomous hypersecretion of GH involves giving the patient an oral glucose load and measuring GH levels subsequently. A sudden rise in blood glucose suppresses serum GH to 2 ng/mL or less in normal subjects, but not in patients with active acromegaly. If one attempts to demonstrate autonomous hypersecretion of GH in a patient suspected of having acromegaly, therefore, before drawing the blood for both the basal (pre-glucose load) serum GH level and the post-glucose load serum GH level, one must be certain that the patient has not eaten for 6 to 8 hours, has not done vigorous exercise for at least 4 hours, remains fully awake during the entire testing period (in a nonstressed state to the extent possible), and has not taken any drugs known to increase GH secretion for at least 1 week. Under these carefully controlled circumstances, if both the basal and postsuppression serum levels of the suspect hormone are elevated, one can conclude that autonomous hypersecretion is probably present. At this point, localization procedures (such as an MRI of the pituitary gland in an acromegalic suspect) are performed to further confirm the diagnosis.

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Endocannabinoid receptors have also been found in the thyroid gland and in adrenal cortical cells of rodents, but their importance in energy homeostasis remains to be elucidated in these animals. The second “central” energy-related response to CB1 receptor activation is to induce hyperphagia, particularly for highly palatable foods even in satiated animals (increase in incentive value of food regardless of the quality of the macronutrient) (the “incentive hypothesis”). Importantly, this orexigenic action of the endocannabinoids is fully inhibited if the animals are given CB1-specific antagonists prior to receiving the endocannabinoid. In this regard, CB1 receptor blockade has also been shown to decrease the “reward” potential of addictive drugs. The most significant reward pathway appears to be part of the mesolimbic-dopaminergic system in the brain. It has been demonstrated that inhibitors of the enzyme fatty acid synthase have significant anorexigenic actions. Because CB1 receptor activation modulates the fatty acid synthase pathway in the hypothalamus, the inhibition of hypothalamic expression by the CB1 receptor antagonist rimonabant (Acomplia) may explain, in part, the anorexigenic properties of the endocannabinoid antagonists in general. In addition to these “central” effects of the exogenous and endogenous cannabinoids on energy homeostasis, there are “peripheral” effects on metabolic functions as well. The administration of CB1 receptor antagonists is associated with reduction in body weight that persists beyond the duration of the anorexigenic action of the agent. To explain this phenomenon, a direct stimulating effect of CB1 receptor antagonists on energy expenditure has been suggested. The expression of CB1 receptors in adipose tissue and the capacity of CB1 receptor antagonists to inhibit cannabinoid-induced lipogenesis suggests that the ECS also operates via peripheral pathways that, when activated, alter energy balance as well. More specifically, CB1 receptor antagonists appear to reduce adipose mass. This lipolytic action is the result of induction of enzymes involved in ␤-oxidation of fatty acids as well as enzymes of the tricarboxylic acid (TCA) cycle. Glucose homeostasis has also been shown to be more tightly regulated in that CB1 receptor antagonists increase the expression of the insulin-responsive glucose transporter 4 (GLUT 4), suggesting an antidiabetogenic action of these antagonists. All of this is supported by data showing that the chronic administration of CB1 receptor antagonists increases the level of adiponectin in the blood of rodents and, in addition, increases the production of uncoupling proteins (UCPs) (e.g., UCP-1 and UCP-3 mRNA in BAT of mice). The latter suggests that CB1 receptor blockade may stimulate thermogenesis and increase oxygen consumption in adipocytes. Taken together, these findings suggest that the ECS plays a major role in the regulation of energy homeostasis in fat tissue. Hepatocytes contain about twice the amount of 2-AG as the cells of other peripheral organs, suggesting that liver cells are also targets of the cannabinoids. For example, the cannabinoid receptor agonist H4210 stimulates the expression of genes that code for lipogenic enzymes such as lipogenic transcription factor SREBP-1c and its targets, acetyl-CoA carboxylase-1 and fatty acid synthase. It is likely, therefore, that blockade of the hepatic ECS protects liver cells from the damaging effect of a chronically high-fat diet (e.g., hepatic steatosis). Finally, evidence suggests that the ECS in gastrointestinal cells may modulate nutrient intake and thereby affect energy homeostasis. The concentration of AEA in intestinal cells increases sharply when caloric intake is significantly restricted. This increase, together with that which occurs in the CNS, may synergize to increase appetite under these experimental conditions. These findings suggest that cannabinoid antagonists may have therapeutic value in the treatment of obesity. SR141716, also known as rimonabant (Acomplia) has been studied in multicenter phase III trials to determine its efficacy as an anorexiant. Approval by the U.S. Food and Drug Administration for the marketing

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of rimonabant was not forthcoming, however, because the advisory committee considering rimonabant has advised against its marketing due to reports of psychiatric side effects in patients treated with this agent. CLINICAL COMMENTS Corti Solemia. One of the functions of cortisol is to prepare the body to deal with periods of stress. In response to cortisol, the body re-sorts its fuel stores so that they can rapidly be made available for the “fight-orflight” response to the alarm signal sounded by epinephrine. Cortisol causes gluconeogenic substrates to move from peripheral tissues to the liver, where they are converted to glucose and stored as glycogen. The release of epinephrine stimulates the breakdown of glycogen, increasing the supply of glucose to the blood. Thus, fuel becomes available for muscle to fight or flee. Cushing “disease,” the cause of Corti Solemia’s current problems, results from prolonged hypersecretion of ACTH from a benign pituitary tumor. ACTH stimulates the adrenal cortex to produce cortisol, and blood levels of this steroid hormone rise. Other nonpituitary causes of Cushing syndrome, however, include a primary tumor of the adrenal cortex secreting excessive amounts of cortisol directly into the bloodstream. This disorder also can result from the release of ACTH from secretory nonendocrine, nonpituitary neoplasms (“ectopic” ACTH syndrome). Cushing syndrome is often caused by excessive doses of synthetic GCs used to treat a variety of disorders because of their potent anti-inflammatory effects (iatrogenic Cushing syndrome). Sam Atotrope. The diabetogenic potential of chronically elevated GH levels in the blood is manifested by the significant incidence of diabetes mellitus (25%) and impaired glucose tolerance (33%) in patients with acromegaly such as Sam Atotrope. Under normal circumstances, however, physiologic concentrations of GH (as well as of cortisol and thyroid hormone) have a facilitatory or permissive effect on the quantity of insulin released in response to hyperglycemia and other insulin secretagogues. This “proinsular” effect is probably intended to act as a “brake” to dampen any potentially excessive “contrainsular” effects that increments in GH and the other counterregulatory hormones exerted. BIOCHEMICAL COMMENTS Radioimmunoassays. Most hormones are present in body fluids in picomolar to nanomolar amounts, requiring highly sensitive assays to determine their concentration in the blood or urine. Radioimmunoassays (RIAs), developed in the 1960s, use an antibody, generated in animals, against a specific antigen (the hormone to be measured). Determining the concentration of the hormone in the sample involves incubating the plasma or urine sample with the antibody and then quantifying the level of antigen–antibody complex formed during the incubation by one of several techniques. The classic RIA uses very high-affinity antibodies, which have been fixed (immobilized) on the inner surface of a test tube, a Teflon bead, or a magnetized particle. A standard curve is prepared, using a set amount of the antibody and various known concentrations of the unlabeled hormone to be measured. In addition to a known concentration of the unlabeled hormone, each tube contains the same small, carefully measured amount of radiolabeled hormone. The labeled hormone and the unlabeled hormone compete for binding to the antibody. The higher the amount of unlabeled hormone in the sample, the less radiolabeled

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Radioactive T4 bound to antibody (%)

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60 50 40 30 20 10 0

0.005

0.01

0.015

0.02

Nonradioactive T4 (␮g)

FIG. 43.13. Standard curve for a radioimmunoassay. A constant amount of radioactive T4 is added to a series of tubes, each of which contains a different amount of nonradioactive T4. The amount of radioactive hormone that binds to an antibody that is specific for the hormone is measured and plotted against the nonradioactive hormone concentration. When more nonradioactive T4 is present in the tube, less radioactive T4 binds to the antibody.

hormone is bound. A standard curve is plotted (Fig. 43.13). The unknown sample from the patient’s blood or urine, containing the unlabeled hormone to be measured, is incubated with the immobilized antibody in the presence of the same small, carefully measured amount of radiolabeled hormone. The amount of radiolabeled hormone bound to the antibody is determined, and the standard curve is used to quantitate the amount of unlabeled hormone in the patient sample. The same principle is used in immunoradiometric assays (IRMAs), but with this technique, the antibody, rather than the antigen to be measured, is radiolabeled. The sensitivity of RIAs can be enhanced using a “sandwich technique.” This method uses two different monoclonal antibodies (antibodies generated by a single clone of plasma cells rather than multiple clones), each of which recognizes a different specific portion of the hormone’s structure. The first antibody, attached to a solid support matrix such as a plastic culture dish, binds the hormone to be assayed. After exposure of the patient sample to this first antibody, the excess plasma is washed away, and the second antibody (which is radiolabeled) is then incubated with the first antibody–hormone complex. The amount of binding of the second (labeled) antibody to the first complex is proportional to the concentration of the hormone in the sample. The sandwich technique can be improved even further if the second antibody is attached to an enzyme, such as alkaline phosphatase. The enzyme rapidly converts an added colorless substrate into a colored product or a nonfluorescent substrate into a highly fluorescent product. These changes can be quantitated if the degree of change in color or fluorescence is proportional to the amount of hormone present in the patient sample. Less than a nanogram (10⫺9 g) of a protein can be measured by such an enzyme-linked immunosorbent assay (ELISA).

Key Concepts • • • •

• •

• •

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Insulin is the major anabolic hormone of the body. Hormones that counteract the action of insulin are known as counterregulatory (contrainsular) hormones. Glucagon is the major counterregulatory hormone. Other contrainsular hormones are Epinephrine Norepinephrine Cortisol Somatostatin Growth hormone Thyroid hormone Somatostatin inhibits insulin secretion, as well as the secretion of a large number of other hormones. Growth hormone (GH) exhibits a wide variety of effects. GH increases lipolysis in adipose tissue, which increases the availability of fatty acids for oxidation, thereby reducing the oxidation of glucose and amino acids. GH increases amino acid uptake into muscle cells, thereby increasing muscle protein synthesis. GH stimulates gluconeogenesis (from amino acid substrates) and glycogen production in the liver. The catecholamines have metabolic effects directed toward mobilization of fuels from their storage sites for oxidation by cells while simultaneously suppressing insulin secretion. Cortisol (a glucocorticoid) promotes survival in times of stress, primarily via alteration of gene expression. ATP-requiring processes such as DNA, RNA, and protein synthesis are inhibited. ■ Fuels are made available. ■ Fat-cell lipolysis is stimulated. ■ Muscle proteolysis is stimulated. ■ Glucose uptake by many tissues is inhibited to provide the nervous system with the glucose. The liver uses the carbons of the amino acids for gluconeogenesis and glycogen storage.

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Table 43.8

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Diseases Discussed in Chapter 43

Disease or Disorder

Environmental or Genetic

Hypercortisolemia

Both

Acromegaly

Environmental

Cushing disease

Environmental

Cushing syndrome

Environmental or genetic (MEN) Environmental

Hypothyroidism

Comments Excessive cortisol secretion, leading to inappropriate catabolic responses Excessive secretion of growth hormone, most often caused by ACTH- or GH-secreting neoplasms A pituitary adenoma leading to excessive secretion of ACTH, which leads to excessive cortisol secretion. Exposure to high levels of cortisol for prolonged periods with its subsequent effects Reduced secretion of thyroid hormone, weight gain

ACTH, adrenocorticotropic hormone; GH, growth hormone; MEN, multiple endocrine neoplasia 1.

• •

•

• • • • • • •

Thyroid hormone secretion is regulated by thyroid-stimulating hormone (TSH) and thyrotropinreleasing hormone (TRH). Thyroid hormone effects in the liver include: Increases in glycolysis and cholesterol synthesis Increase in the synthesis of bile salts Increase in triglyceride synthesis Thyroid hormone effects on fat cells include: Increased lipolysis Increased glycerol release to the liver Thyroid hormone also stimulates heat production via a variety of mechanisms. The intestine and stomach (the gut) also secrete a variety of factors that affect fuel metabolism by working with (or against) the other hormones already described. The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are synthesized in specialized cells of the gastrointestinal tract. GLP-1 and GIP influence nutrient homeostasis by increasing insulin release from the pancreatic ␤-cells in a glucose-dependent manner. Incretin action facilitates the uptake of glucose by muscle tissue and by the liver while simultaneously suppressing glucagon secretion by the ␣-cells of the pancreas. The incretins also increase the levels of cAMP in the islets leading to expansion of ␤-cell mass and resistance to ␤-cell apoptosis. Diseases discussed in this chapter are summarized in Table 43.8.

REVIEW QUESTIONS—CHAPTER 43 1.

As a third-year medical student, you examine your first patient. You find that he is 52 years old, has a round face, acne, and a large hump of fat on the back of his neck. He complains that he is too weak to “mow his lawn.” His fasting blood glucose level is 170 mg/dL (reference range, 80 to 100 mg/dL). His plasma cortisol levels are 62 ␮g/mL (reference range, 3 to 31␮g/mL). His plasma ACTH levels are 0 pg/mL (reference range, 0 to 100 pg/mL). Based on the information given, if the patient’s problem is attributable to a single cause, the most likely diagnosis is which of the following? A. Non–insulin-dependent diabetes mellitus B. Insulin-dependent diabetes mellitus C. A secretory tumor of the anterior pituitary

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D. A secretory tumor of the posterior pituitary E. A secretory tumor of the adrenal cortex 2.

A woman was scheduled for a growth hormone suppression test. If each of the following events occurred the morning of the test, which one of the events would be most likely to cause a decrease in growth hormone levels? A. She ate four large doughnuts for breakfast. B. She was on estrogen replacement therapy and took her tablets after breakfast. C. While unlocking her car, she was chased by the neighbor’s vicious dog. D. She fell asleep at the start of the test and slept soundly until it was completed 1.5 hours later. E. She forgot to eat breakfast before the test.

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3.

A dietary deficiency of iodine will lead to which of the following? A. A direct effect on the synthesis of thyroglobulin on ribosomes B. An increased secretion of TSH C. Decreased production of TRH D. Increased heat production E. Weight loss

4.

A woman whose thyroid gland was surgically removed was treated with 0.10 mg of thyroxine daily (tablet form). After 3 months of treatment, serial serum TSH levels ranged between 10 and 15 mIU/mL (reference range, 0.3 to 5.0 mIU/mL). She complained of fatigue, weight gain, and hoarseness. Her dose of thyroid hormone should be adjusted in which direction? A. More thyroid hormone should be given B. Less thyroid hormone should be given C. No adjustment is needed

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5.

A patient complains of nervousness, palpitations, sweating, and weight loss without loss of appetite, and has a goiter. Suspecting a defect in thyroid function, the physician orders a total serum T4. The test is performed by radioimmunoassay. The standard curve for the assay, which measures T4 in 0.1 mL serum, is shown in Figure 43.13. Normal levels of T4 ⫽ 4 to 10 ␮g/dL. In an assay of 0.1 mL of the patient’s serum, 15% of the radioactive T4 was bound by the antibody. According to the radioimmunoassay, the approximate blood level of T4 is which of the following? A. 0.015 ␮g/dL B. 0.15 ␮g/dL C. 15 ␮g/dL D. 20 ␮g/dL E. 30 ␮g/dL

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44

The Biochemistry of Erythrocytes and Other Blood Cells

The cells of the blood are classified as erythrocytes, leukocytes, or thrombocytes. The erythrocytes (red blood cells) carry oxygen to the tissues and are the most numerous cells in the blood. The leukocytes (white blood cells) are involved in defense against infection, and the thrombocytes (platelets) function in blood clotting. All of the cells in the blood can be generated from hematopoietic stem cells in the bone marrow on demand. For example, in response to infection, leukocytes secrete cytokines called interleukins that stimulate the production of additional leukocytes to fight the infection. Decreased supply of oxygen to the tissues signals the kidney to release erythropoietin, a hormone that stimulates the production of red blood cells. The red blood cell has limited metabolic function, owing to its lack of internal organelles. Glycolysis is the main energy-generating pathway, with lactate production regenerating NAD⫹ for glycolysis to continue. The NADH produced in glycolysis is also used to reduce the ferric form of hemoglobin, methemoglobin, to the normal ferrous state. Glycolysis also leads to a side pathway in which 2,3-bisphosphoglycerate is produced, which is a major allosteric effector for oxygen binding to hemoglobin. The hexose monophosphate shunt pathway generates NADPH to protect red blood cell membrane lipids and proteins from oxidation, through regeneration of reduced glutathione. Heme synthesis occurs in the precursors of red blood cells and is a complex pathway that originates from succinyl coenzyme A (succinyl-CoA) and glycine. Mutations in any of the steps of heme synthesis lead to a group of diseases known collectively as porphyrias. The red blood cell membrane must be highly deformable to allow it to travel throughout the capillary system in the body. This is because of a complex cytoskeletal structure that consists of the major proteins spectrin, ankyrin, and band 3 protein. Mutations in these proteins lead to improper formation of the membrane cytoskeleton, ultimately resulting in malformed red blood cells, spherocytes, in the circulation. Spherocytes have a shortened life span, leading to loss of blood cells. When the body does not have sufficient red blood cells, the patient is said to be anemic. Anemia can result from many causes. Nutritional deficiencies of iron, folate, or vitamin B12 prevent the formation of adequate numbers of red blood cells. Mutations in the genes that encode red blood cell metabolic enzymes, membrane structural proteins, and globins cause hereditary anemias. The appearance of red blood cells on a blood smear frequently provides clues to the cause of an anemia. Because the mutations that give rise to hereditary anemias also provide some protection against malaria, hereditary anemias are some of the most common genetic diseases known. The human alters globin gene expression during development, a process known as hemoglobin switching. The switch between expression of one gene to another is regulated by transcription factor binding to the promoter regions of these genes. Current research is attempting to reactivate fetal hemoglobin genes to combat sickle cell disease and thalassemia. 823

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THE WAITING ROOM Anne Niemick, who has ␤⫹-thalassemia, complains of pain in her lower spine (see Chapters 14 and 15). A quantitative computed tomogram (CT) of the vertebral bodies of the lumbar spine shows evidence of an area of early spinal cord compression in the upper lumbar region. She is suffering from severe anemia, resulting in stimulation of production of red blood cell precursors (the erythroid mass) from the stem cells in her bone marrow. This expansion of marrow volume causes osteoporosis leading to compression fractures in the lumbar spine, which, in turn, causes pain. Local irradiation to reduce the marrow volume in the lumbar spine is considered, as is a program of regular blood transfusions to maintain the oxygen-carrying capacity of circulating red blood cells. The results of special studies related to the genetic defect underlying her thalassemia are pending, although preliminary studies have shown that she has elevated levels of fetal hemoglobin, which, in part, moderates the manifestations of her disease. Anne Niemick’s parents have returned to the clinic to discuss the results of these tests. Spiro Site is a 21-year-old college student who complains of feeling tired all the time. Two years previously he had had gallstones removed, which consisted mostly of bilirubin. His spleen is palpable, and jaundice (icterus) is evidenced by yellowing of the whites of his eyes. His hemoglobin is low (8 g/dL; reference value, 13.5 to 17.5 g/dL). A blood smear showed dark, rounded, abnormally small red blood cells called spherocytes as well as an increase in the number of circulating immature red blood cells known as reticulocytes.

I.

CELLS OF THE BLOOD

The blood, together with the bone marrow, makes up the organ system that makes a significant contribution to achieving homeostasis, the maintenance of the normal composition of the body’s internal environment. Blood can be considered a liquid tissue consisting of water, proteins, and specialized cells. The most abundant cells in the blood are the erythrocytes or red blood cells, which transport oxygen to the tissues and contribute to buffering of the blood through the binding of protons by hemoglobin (see Section IV of this chapter, and the material in Chapter 4, Section IV.B, and Chapter 7, Section VII). Red blood cells lose all internal organelles during the process of differentiation. The white blood cells (leukocytes) are nucleated cells present in blood that function in the defense against infection. The platelets (thrombocytes), which contain cytoplasmic organelles but no nucleus, are involved in the control of bleeding by contributing to normal thrombus (clot) formation within the lumen of the blood vessel. The average concentration of these cells in the blood of normal individuals is presented in Table 44.1.

Table 44.1 Normal Values of Blood Cell Concentrations in Adults 3

Cell Type

Mean (cells/mm )

Erythrocytes

5.2 ⫻ 106 (men); 4.6 ⫻ 106 (women) 4,300 2,700 500 230 40

Neutrophils Lymphocytes Monocytes Eosinophils Basophils

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A. Classification and Functions of Leukocytes and Thrombocytes The leukocytes can be classified either as polymorphonuclear leukocytes (granulocytes) or mononuclear leukocytes, depending on the morphology of the nucleus in these cells. The mononuclear leukocyte has a rounded nucleus, whereas the polymorphonuclear leukocytes have a multilobed nucleus. 1.

THE GRANULOCYTES

The granulocytes, so named because of the presence of secretory granules visible on staining, are the neutrophils, eosinophils, and basophils. When these

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cells are activated in response to chemical stimuli, the vesicle membranes fuse with the cell plasma membrane, resulting in the release of the granule contents (degranulation). The granules contain many cell-signaling molecules that mediate inflammatory processes. The granulocytes, in addition to displaying segmented nuclei (are polymorphonuclear), can be distinguished from each other by their staining properties (caused by different granular contents) in standard hematologic blood smears: neutrophils stain pink, eosinophils stain red, and basophils stain blue. Neutrophils are phagocytic cells that migrate rapidly to areas of infection or tissue damage. As part of the response to acute infection, neutrophils engulf foreign bodies and destroy them, in part, by initiating the respiratory burst (see Chapter 24). The respiratory burst creates oxygen radicals that rapidly destroy the foreign material found at the site of infection. A primary function of eosinophils is to fight viral infections (eosinophils release RNase from their granules), to remove fibrin during inflammation, and to protect against parasites such as worms. The eosinophilic granules are lysosomes containing hydrolytic enzymes and cationic proteins, which are toxic to parasitic worms. Eosinophils have also been implicated in asthma and allergic responses, as well as antigen presentation to T cells. Elucidating the function of eosinophils is currently an active area of research. Basophils, the least abundant of the leukocytes, participate in hypersensitivity reactions such as allergic responses. Histamine, produced by the decarboxylation of histidine, is stored in the secretory granules of basophils. Release of histamine during basophil activation stimulates smooth muscle cell contraction and increases vascular permeability. The granules also contain enzymes such as proteases, ␤-glucuronidase, and lysophospholipase. These enzymes degrade microbial structures and assist in the remodeling of damaged tissue. 2.

MONONUCLEAR LEUKOCYTES

The mononuclear leukocytes consist of various classes of lymphocytes and the monocytes. Lymphocytes are small, round cells that were originally identified in lymph fluid. These cells have a high ratio of nuclear volume to cytoplasmic volume and are the primary antigen (foreign body)-recognizing cells. There are three major types of lymphocytes: T cells, B cells, and NK cells. The precursors of T cells (thymus-derived lymphocytes) are produced in the bone marrow and then migrate to the thymus, where they mature before being released to the circulation. Several subclasses of T cells exist. These subclasses are identified by different surface membrane proteins, the presence of which correlate with the function of the subclass. Lymphocytes that mature in the bone marrow are the B cells, which secrete antibodies in response to antigen binding. The third class of lymphocytes is the natural killer cells (NK cells), which target virally infected and malignant cells for destruction. Circulatory monocytes are the precursors of tissue macrophages. Macrophages (“large eaters”) are phagocytic cells that enter inflammatory sites and consume microorganisms, and necrotic host cell debris left behind by granulocyte attack of the foreign material. Macrophages in the spleen play an important role in maintaining the oxygen-delivering capabilities of the blood by removing damaged red blood cells that have a reduced oxygen-carrying capacity. 3.

THE THROMBOCYTES

Platelets are heavily granulated disc-like cells that aid in intravascular clotting. Like the erythrocyte, platelets lack a nucleus. Their function is discussed in the following chapter. Platelets arise by budding of the cytoplasm of megakaryocytes, multinucleated cells that reside in the bone marrow.

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Table 44.2 Normal Hemoglobin Levels in Blood (g/dL)

Table 44.3 Classification of the Anemias on the Basis of Red Cell Morphology

Adult Males Females Children Newborns 3–12 mo 1 y to puberty

Red Cell Morphology

Functional Deficit

Possible Causes

13.5–17.5 11.5–15.5

Microcytic, hypochromic

Impaired hemoglobin synthesis

15.0–21.0 9.5–12.5 11.0–13.5

Macrocytic, normochromic

Impaired DNA synthesis

Normocytic, normochromic

Red cell loss

Iron deficiency, mutation leading to thalassemia, lead poisoning Vitamin B12 or folic acid deficiency, erythroleukemia Acute bleeding, sickle cell disease, red cell metabolic defects, red cell membrane defects

B. Anemia

A complete blood count (CBC) is ordered when a physician suspects a problem in the cellular composition of a patient’s blood. The cells within the collected blood are counted and typed using an automated analyzer based on flow cytometry (counting cells one at a time as they flow through a detector). As each cell flows through the machine, a laser shines light at the cell, which leads to predictable light scattering and absorbance depending on the cell type. Based on the light-scattering and absorption pattern, the machine keeps track of the results of each cell that flows through the machine, leading to a very accurate count of each cell type present in the sample. The data from this analysis will include the total number of red blood cells per liter, the amount of hemoglobin in the red blood cells (in grams per liter), the hematocrit (the fraction of whole blood that consists of red blood cells), the mean corpuscular volume, the total number of white blood cells, as well as a count of the different types of white blood cells (neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

Lieberman_CH44.indd 826

The major function of erythrocytes is to deliver oxygen to the tissues. To do this, a sufficient concentration of hemoglobin in the red blood cells is necessary for efficient oxygen delivery to occur. When the hemoglobin concentration falls below normal values (Table 44.2), the patient is classified as anemic. Anemias can be categorized based on red cell size and hemoglobin concentration. Red blood cells can be of normal size (normocytic), small (microcytic), or large (macrocytic). Cells containing a normal hemoglobin concentration are termed normochromic; those with decreased concentration are hypochromic. This classification system provides important diagnostic tools (Table 44.3) that enable one to properly classify, diagnose, and treat the anemia. Other measurements used to classify the type of anemia present include the mean corpuscular volume (MCV) and the mean corpuscular hemoglobin concentration (MCHC). The MCV is the average volume of the red blood cell, expressed in femtoliters (10⫺15 L). Normal MCV values range from 80 to 100 fL. The MCHC is the average concentration of hemoglobin in each individual erythrocyte, expressed in grams per liter. The normal range is 32 to 37 g/L; a value of ⬍32 g/L indicates hypochromic cells. Thus, microcytic, hypochromic red blood cells have an MCV of ⬍80 fl and an MCHC of ⬍32 g/L. Macrocytic, normochromic cells have an MCV of ⬎100 fl, with an MCHC between 32 and 37 g/L.

II. ERYTHROCYTE METABOLISM A. The Mature Erythrocyte To understand how the erythrocyte can carry out its major function, a discussion of erythrocyte metabolism is required. Mature erythrocytes contain no intracellular organelles, so the metabolic enzymes of the red blood cell are limited to those found in the cytoplasm. In addition to hemoglobin, the cytosol of the red blood cell contains enzymes necessary for the prevention and repair of damage done by reactive oxygen species (see Chapter 24) and the generation of energy (Fig. 44.1). Erythrocytes can only generate adenosine triphosphate (ATP) by glycolysis (see Chapter 22). The ATP is used for ion transport across the cell membrane (primarily Na⫹, K⫹, and Ca⫹), the phosphorylation of membrane proteins, and the priming reactions of glycolysis. Erythrocyte glycolysis also uses the Rapoport–Luebering shunt to generate 2,3-bisphosphoglycerate (2,3-BPG). Red cells contain 4 to 5 mM 2,3-BPG, compared with trace amounts in other cells. The trace amounts of 2,3-BPG found in cells other than erythrocytes is required for the phosphoglycerate mutase reaction of glycolysis, in which 3-phosphoglycerate is isomerized to 2-phosphoglycerate. As the 2,3-BPG is regenerated during each reaction cycle, it is required in only catalytic amounts. As discussed in more detail in Section IV, 2,3-BPG is a modulator of oxygen binding to hemoglobin that stabilizes the deoxy form of hemoglobin, thereby facilitating the release of oxygen to the tissues. To bind oxygen, the iron of hemoglobin must be in the ferrous (⫹2) state. Reactive oxygen species can oxidize the iron to the ferric (⫹3) state, producing

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Oxidizing agent

Destroyed oxidizing agent

Reduced glutathione Glucose ADP ATP Glucose-6-P

NADP+

827

Oxidized glutathione

NADPH 5-carbon sugars HMP shunt

Fructose-6-P ADP ATP Fructose 1,6 BP DHAP

Fe3+-hemoglobin 2+-hemoglobin

Fe

Reduced cytochrome b5

Glyceraldehyde-3-P NAD+

Pi

NADH mutase 2,3 BPG 1,3 bisphosphoglycerate cytochrome b5 ADP Rapoportreductase ATP Luebering shunt 3-phosphoglycerate

Oxidized cytochrome b5

phosphatase

2-phosphoglycerate

PEP ADP ATP Pyruvate NADH NAD+ Lactate

FIG. 44.1. Overview of erythrocyte metabolism. Glycolysis is the major pathway, with branches for the hexose monophosphate shunt (for protection against oxidizing agents) and the Rapoport–Luebering shunt (which generates 2,3-bisphosphoglycerate, which moderates oxygen binding to hemoglobin). The NADH generated from glycolysis can be used to reduce methemoglobin (Fe3⫹) to normal hemoglobin (Fe2⫹), or to convert pyruvate to lactate, so that NAD⫹ can be regenerated and used for glycolysis. Pathways that are unique to the erythrocyte are indicated in red. See text for abbreviations.

methemoglobin. Some of the NADH produced by glycolysis is used to regenerate hemoglobin from methemoglobin by the NADH-cytochrome b5 methemoglobin reductase system. Cytochrome b5 reduces the Fe3⫹ of methemoglobin. The oxidized cytochrome b5 is then reduced by a flavin-containing enzyme, cytochrome b5 reductase (also called methemoglobin reductase), using NADH as the reducing agent. Approximately 5% to 10% of the glucose metabolized by red blood cells is used to generate NADPH by way of the hexose monophosphate shunt. The NADPH is used to maintain glutathione in the reduced state. The glutathione cycle is the red blood cell’s chief defense against damage to proteins and lipids by reactive oxygen species (see Chapter 24). The enzyme that catalyzes the first step of the hexose monophosphate shunt is glucose-6-phosphate dehydrogenase (G6PD). The lifetime of the red blood cell correlates with G6PD activity. Lacking ribosomes, the red blood cell cannot

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An inherited deficiency in pyruvate kinase leads to hemolytic anemia (an anemia caused by the destruction of red blood cells; hemoglobin values typically drop to 4 to 10 g/dL in this condition, with normal values being 13.5 to 17.5 in males, or 11.5 to 15.5 in females). Because the amount of adenosine triphosphate (ATP) formed from glycolysis is decreased by 50%, red blood cell ion transporters cannot function effectively. The red blood cells tend to gain Ca2⫹ and lose K⫹ and water. The water loss increases the intracellular hemoglobin concentration. With the increase in intracellular hemoglobin concentration, the internal viscosity of the cell is increased to the point that the cell becomes rigid and, therefore, more susceptible to damage by shear forces in the circulation. Once they are damaged, the red blood cells are removed from circulation, leading to the anemia. However, the effects of the anemia are frequently moderated by the twofold to threefold elevation in 2,3-bisphosphoglycerate (2,3-BPG) concentration that results from the blockage of the conversion of phosphoenolpyruvate to pyruvate. Because 2,3-BPG binding to hemoglobin decreases the affinity of hemoglobin for oxygen, the red blood cells that remain in circulation are highly efficient in releasing their bound oxygen to the tissues.

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SECTION VIII ■ TISSUE METABOLISM

Congenital methemoglobinemia, the presence of excess methemoglobin, is found in people with an enzymatic deficiency in cytochrome b5 reductase or in people who have inherited hemoglobin M. In hemoglobin M, a single amino acid substitution in the heme-binding pocket stabilizes the ferric (Fe3⫹) oxygen. Individuals with congenital methemoglobinemia appear cyanotic but have few clinical problems. Methemoglobinemia can be acquired by ingestion of certain oxidants such as nitrites, quinones, aniline, and sulfonamides. Acquired methemoglobinemia can be treated by the administration of reducing agents, such as ascorbic acid or methylene blue.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in humans, probably, in part, because individuals with G6PD deficiency are resistant to malaria. The resistance to malaria counterbalances the deleterious effects of the deficiency. G6PDdeficient red cells have a shorter life span and are more likely to lyse under conditions of oxidative stress. When soldiers during the Korean War were given the antimalarial drug primaquine prophylactically, approximately 10% of the soldiers of African ancestry developed spontaneous anemia. Because the gene for G6PD is found on the X chromosome, these men had only one copy of a variant G6PD gene.

All known glucose-6-phosphate dehydrogenase (G6PD) variant genes contain small in-frame deletions or missense mutations. The corresponding proteins, therefore, have decreased stability or lowered activity, leading to a reduced half-life or life span for the red cell. No mutations have been found that result in complete absence of G6PD. Based on studies with knockout mice, those mutations would be expected to result in embryonic lethality.

Pyridoxine (vitamin B6) deficiencies are often associated with a microcytic, hypochromic anemia. Why would a B6 deficiency result in small (microcytic), pale (hypochromic) red blood cells?

Lieberman_CH44.indd 828

CH2 CH3

CH

HC

CH N

CH3

CH3

2+

N Fe N −

OOC

CH2

CH2

CH

N

CH2

CH

HC CH2

CH3

CH2 COO−

FIG. 44.2. Structure of heme. The side chains can be abbreviated as MVMVMPPM. M, methyl (MCH3); P, propionyl (MCH2MCH2MCOO⫺); V, vinyl (MCHBCH2).

synthesize new G6PD protein. Consequently, as the G6PD activity decreases, oxidative damage accumulates, leading to lysis of the erythrocyte. When red blood cell lysis (hemolysis) substantially exceeds the normal rate of red blood cell production, the number of erythrocytes in the blood drops below normal values, leading to hemolytic anemia.

B. The Erythrocyte Precursor Cells and Heme Synthesis 1.

HEME STRUCTURE

Heme consists of a porphyrin ring coordinated with an atom of iron (Fig. 44.2). Four pyrrole rings are joined by methenyl bridges (–CH–) to form the porphyrin ring (see Fig. 7.12). Eight side chains serve as substituents on the porphyrin ring, two on each pyrrole. These side chains may be acetyl (A), propionyl (P), methyl (M), or vinyl (V) groups. In heme, the order of these groups is M V M V M P P M. This order, in which the position of the methyl group is reversed on the fourth ring, is characteristic of the porphyrins of the type III series, the most abundant in nature. Heme is the most common porphyrin found in the body. It is complexed with proteins to form hemoglobin, myoglobin, and the cytochromes (see Chapters 7 and 21), including cytochrome P450 (see Chapter 24). 2.

SYNTHESIS OF HEME

Heme is synthesized from glycine and succinyl coenzyme A (succinyl-CoA) (Fig. 44.3), which condense in the initial reaction to form ␦-aminolevulinic acid (␦-ALA) (Fig. 44.4). The enzyme that catalyzes this reaction, ␦-ALA synthase, requires the participation of pyridoxal phosphate, as the reaction is an amino acid decarboxylation reaction (glycine is decarboxylated; see Chapter 39). The next reaction of heme synthesis is catalyzed by ␦-ALA dehydratase, in which two molecules of ␦-ALA condense to form the pyrrole, porphobilinogen (Fig. 44.5). Four of these pyrrole rings condense to form a linear chain and then a series of porphyrinogens. The side chains of these porphyrinogens initially contain acetyl (A) and propionyl (P) groups. The acetyl groups are decarboxylated to form methyl groups. Then the first two propionyl side chains are decarboxylated and oxidized to vinyl groups, forming a protoporphyrinogen. The methylene bridges are subsequently oxidized to form protoporphyrin IX (see Fig. 44.3). Heme is red and is responsible for the color of red blood cells and of muscles that contain a large number of mitochondria. In the final step of the pathway, iron (as Fe2⫹) is incorporated into protoporphyrin IX in a reaction catalyzed by ferrochelatase (also known as heme synthase).

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CHAPTER 44 ■ THE BIOCHEMISTRY OF ERYTHROCYTES AND OTHER BLOOD CELLS

Succinyl CoA + glycine

–

␦-Aminolevulinic acid synthase

␦-Aminolevulinic acid (␦-ALA) ␦-Aminolevulinic acid dehydratase N

Porphyrias

␦-ALA dehydratase porphyria

Acute intermittent porphyria

Hydroxymethylbilane Uroporphyrinogen III cosynthase

Congenital erythropoietic porphyria

Uroporphyrinogen III N

N

In a B6 deficiency, the rate of heme production is slow because the first reaction in heme synthesis requires pyridoxal phosphate (see Fig. 44.4). Thus, less heme is synthesized, causing red blood cells to be small and pale. Iron stores are usually elevated.

Porphobilinogen Porphobilinogen deaminase

N

829

Uroporphyrinogen decarboxylase

N

General Coproporphyrinogen III structure of Coproporphyrinogen porphyrinogens oxidase

Porphyria cutanea tarda

␦-Aminolevulinic acid (␦-ALA) dehydratase, which contains zinc, and ferrochelatase are inactivated by lead. Thus, in lead poisoning, ␦-ALA and protoporphyrin IX accumulate, and the production of heme is decreased. Anemia results from a lack of hemoglobin, and energy production decreases because of the lack of cytochromes for the electron-transport chain.

Hereditary coproporphyria

Protoporphyrinogen IX Protoporphyrinogen oxidase

Variegate porphyria

Protoporphyrin IX Fe2+

Ferrochelatase

Erythropoietic protoporphyria –

COO

Heme

FIG. 44.3. Synthesis of heme. To produce one molecule of heme, eight molecules each of glycine and succinyl-CoA are required. A series of porphyrinogens is generated in sequence. Finally, iron is added to produce heme. Heme regulates its own production by repressing the synthesis of ␦-aminolevulinic acid (␦-ALA) synthase (↓) and by directly inhibiting the activity of this enzyme (⫺). Deficiencies of enzymes in the pathway result in a series of diseases known as porphyrias (listed on the right, beside the deficient enzyme).

CH2 CH2 C

O

SCoA Succinyl CoA

+ 3.

SOURCE OF IRON

Iron, which is obtained from the diet, has a U.S. Recommended Dietary Allowance (RDA) of 10 mg for men and postmenopausal women, and 15 mg for premenopausal women. The average U.S. diet contains 10 to 50 mg of iron. However, only 10% to 15% is normally absorbed, and iron deficiencies are fairly common. The iron in meats is in the form of heme, which is readily absorbed. The nonheme iron in plants is not as readily absorbed, in part, because plants often contain oxalates, phytates, tannins, and other phenolic compounds that chelate or form insoluble precipitates with iron, preventing its absorption. Conversely, vitamin C (ascorbic acid) increases the uptake of nonheme iron from the digestive tract. The uptake of iron is also increased in times of need by mechanisms that are not yet understood. Iron is absorbed in the ferrous (Fe2⫹) state (Fig. 44.6), but is oxidized to the ferric state by a ferroxidase known as ceruloplasmin (a copper-containing enzyme) for transport through the body. Because free iron is toxic, it is usually found in the body bound to proteins (see Fig. 44.6). Iron is carried in the blood (as Fe3⫹) by the protein apotransferrin, with which it forms a complex known as transferrin. Transferrin is usually only one-third saturated with iron. The total iron-binding capacity of blood, due mainly to its content of transferrin, is approximately 300 ␮g/dL. Transferrin, with bound iron, binds to the transferrin receptor on the cell surface and the complex is internalized into the cell. The internalized membrane develops into an endosome, with a slightly

Lieberman_CH44.indd 829

+

H2C

NH3

COO– Glycine δ-ALA synthase

PLP CO2

CoAS– COO– CH2 CH2 C H2C

O +

NH3

δ-Aminolevulinic acid (δ-ALA) FIG. 44.4. Synthesis of ␦-aminolevulinic acid (␦-ALA). The atoms in red in ␦-aminolevulinic acid are derived from glycine. PLP, pyridoxal phosphate.

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SECTION VIII ■ TISSUE METABOLISM

COO–

Porphyrias are a group of rare inherited disorders resulting from deficiencies of enzymes in the pathway for heme biosynthesis (see Fig. 44.3). Intermediates of the pathway accumulate and may have toxic effects on the nervous system that cause neuropsychiatric symptoms. When porphyrinogens accumulate, they may be converted by light to porphyrins, which react with molecular oxygen to form oxygen radicals. These radicals may cause severe damage to the skin. Thus, individuals with excessive production of porphyrins are photosensitive. The scarring and increased growth of facial hair seen in some porphyrias may have contributed to the rise of the werewolf legends.

COO–

CH2

CH2

CH2

CH2

O C

C

H C

O

H

H

CH2

NH

NH2

2 ␦-ALA ␦-ALA dehydratase

2H2O COO–

COO–

CH2

CH2

CH2

C

C

C

CH

CH2 NH2

N H

Porphobilinogen (a pyrrole)

FIG. 44.5. Two molecules of ␦-ALA condense to form porphobilinogen.

Bone Erythropoiesis

Dietary iron

Transferrin

Many tissues Cytochromes Iron-enzymes Myoglobin

Blood loss • Bleeding • Menstruation

RBC Hemoglobin

Phagocytosis

Liver

RE cells Ferritin (Fe3+)

Hemosiderin

Ferritin (Fe3+) Serum ferritin

Bile (Fe) Fe2+

Transferrin

Hemosiderin

Transferrin

Intestinal epithelial cell Fe2+

Transferrin (Fe3+) Ferroxidase (ceruloplasmin)

(

+

10–15% absorbed by vitamin C)

Feces

Urine

Sweat

Skin desquamation

Iron loss

Feces

FIG. 44.6. Iron metabolism. Iron is absorbed from the diet, transported in the blood by transferrin, stored in ferritin, and used for the synthesis of cytochromes, iron-containing enzymes, hemoglobin, and myoglobin. It is lost from the body with bleeding and sloughed-off cells, sweat, urine, and feces. Hemosiderin is the protein in which excess iron is stored. Small amounts of ferritin enter the blood and can be used to measure the adequacy of iron stores. RE, reticuloendothelial.

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CHAPTER 44 ■ THE BIOCHEMISTRY OF ERYTHROCYTES AND OTHER BLOOD CELLS

acidic pH. The iron (in the ferrous form) is transported out of the endosome into the cytoplasm via the divalent metal ion transporter 1 (DMT-1). Once in the cytoplasm, the iron is oxidized and binds to ferritin for long-term storage. Storage of iron occurs in most cells but especially those of the liver, spleen, and bone marrow. In these cells, the storage protein, apoferritin, forms a complex with iron (Fe3⫹) known as ferritin. Normally, ferritin is present in the blood in small amounts. The level increases, however, as iron stores increase. Therefore, the amount of ferritin in the blood is the most sensitive indicator of the amount of iron in the body’s stores. Iron can be drawn from ferritin stores, transported in the blood as transferrin, and taken up via receptor-mediated endocytosis by cells that require iron (e.g., by reticulocytes that are synthesizing hemoglobin). When excess iron is absorbed from the diet, it is stored as hemosiderin, a form of ferritin complexed with additional iron that cannot be readily mobilized. 4.

831

An inherited mutation in SLC11A2 (the gene encoding divalent metal ion transporter 1 [DMT-1]) leads to an iron deficiency anemia, as indicated by a refractory hypochromic microcytic anemia. The iron is trapped in endosomal vesicles and cannot be released to bind to ferritin, or used in other necessary biosynthetic reactions. This leads to reduced heme synthesis, reduced globin synthesis, and anemia.

REGULATION OF HEME SYNTHESIS

Heme regulates its own synthesis by mechanisms that affect the first enzyme in the pathway, ␦-ALA synthase (see Fig. 44.3). Heme represses the synthesis of this enzyme, and also directly inhibits the activity of the enzyme (an allosteric modifier). Thus, heme is synthesized when heme levels fall. As heme levels rise, the rate of heme synthesis decreases. Heme also regulates the synthesis of hemoglobin by stimulating synthesis of the protein globin. Heme maintains the ribosomal initiation complex for globin synthesis in an active state (see Chapter 15). 5.

DEGRADATION OF HEME

Heme is degraded to form bilirubin, which is conjugated with glucuronic acid and excreted in the bile (Fig. 44.7). Although heme from cytochromes and myoglobin

RBC Hemoglobin

120 days Myoglobin Cytochromes Fe2+

Globin

Heme

Bilirubin

CO

Bilirubin-albumin Albumin

Urobilinogen Feces

UDP-glucuronate

Bilirubin diglucuronide

Urine

Amino acids R E S

BLOOD L I V E R

Bile Bacteria

Stercobilin

FIG. 44.7. Overview of heme degradation. Heme is degraded to bilirubin, carried in the blood by albumin, conjugated to form the diglucuronide in the liver, and excreted in the bile. The iron is returned to the body’s iron stores. RBC, red blood cells; RES, reticuloendothelial system.

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SECTION VIII ■ TISSUE METABOLISM

M

V

Bridge cleaved

N

M

M 2+

N Fe N P

V

N

P

M

Heme O2 CO, Fe 2+

Heme oxygenase

M

The iron lost by adult men (approximately 1 mg/day) by desquamation of the skin and in bile, feces, urine, and sweat is replaced by iron absorbed from the diet. Men are not as likely to suffer from iron deficiencies as premenopausal adult women, who also lose iron during menstruation and who must supply iron to meet the needs of a growing fetus during pregnancy. If a man eating a Western diet has iron deficiency anemia, his physician should suspect bleeding from the gastrointestinal tract as a result of ulcers or colon cancer.

O

V

M

N H

P

P

N H

M

M

N

V

O

N H

Biliverdin IX␣ NADPH Biliverdin reductase

M

O

V

N H

M

P

N H

NADP+ P

M

N H

H

M

V

N H

O

Bilirubin IX␣

FIG. 44.8. Conversion of heme to bilirubin. A methylene bridge in heme is cleaved, releasing carbon monoxide (CO) and iron. Then the center methylene bridge is reduced.

Drugs, such as phenobarbital, induce enzymes of the drug-metabolizing systems of the endoplasmic reticulum that contain cytochrome P450. Because heme is used for synthesis of cytochrome P450, free heme levels fall and ␦-aminolevulinic acid (␦-ALA) synthase is induced to increase the rate of heme synthesis.

In an iron deficiency, what characteristics will blood exhibit?

Lieberman_CH44.indd 832

also undergoes conversion to bilirubin, the major source of this bile pigment is hemoglobin. After red blood cells reach the end of their life span (approximately 120 days), they are phagocytosed by cells of the reticuloendothelial system. Globin is cleaved to its constituent amino acids, and iron is returned to the body’s iron stores. Heme is oxidized and cleaved to produce carbon monoxide and biliverdin (Fig. 44.8). Biliverdin is reduced to bilirubin, which is transported to the liver complexed with serum albumin. In the liver, bilirubin is converted to a more water-soluble compound by reacting with UDP-glucuronate to form bilirubin monoglucuronide, which is converted to the diglucuronide (see Fig. 30.5). This conjugated form of bilirubin is excreted into the bile. In the intestine, bacteria deconjugate bilirubin diglucuronide and convert the bilirubin to urobilinogens (see Fig. 44.7). Some urobilinogen is absorbed into the blood and excreted in the urine. However, most of the urobilinogen is oxidized to urobilins, such as stercobilin, and excreted in the feces. These pigments give feces their brown color.

III. THE RED BLOOD CELL MEMBRANE Under the microscope, the red blood cell appears to be a red disc with a pale central area (biconcave disc) (Fig. 44.9). The biconcave disc shape (as opposed

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CHAPTER 44 ■ THE BIOCHEMISTRY OF ERYTHROCYTES AND OTHER BLOOD CELLS

to a spherical shape) serves to facilitate gas exchange across the cell membrane. The membrane proteins that maintain the shape of the red blood cell also allow the red blood cell to traverse the capillaries with very small luminal diameters to deliver oxygen to the tissues. The interior diameters of many capillaries are smaller than the approximately 7.5-␮m diameter of the red cell. Furthermore, in passing through the kidney, red blood cells traverse hypertonic areas that are up to six times the normal isotonicity, and back again, causing the red cell to shrink and expand during its travels. The spleen is the organ responsible for determining the viability of the red blood cells. Erythrocytes pass through the spleen 120 times per day. The elliptical passageways through the spleen are approximately 3 ␮m in diameter, and normal red cells traverse them in approximately 30 seconds. Thus, to survive in the circulation, the red cell must be highly deformable. Damaged red cells that are no longer deformable become trapped in the passages in the spleen, where they are destroyed by macrophages. The reason for the erythrocyte’s deformability lies in its shape and in the organization of the proteins that make up the red blood cell membrane. The surface area of the red cell is approximately 140 ␮m2, which is greater than the surface of a sphere needed to enclose the contents of the red cell (98 ␮m2). The presence of this extra membrane and the cytoskeleton that supports it allows the red cell to be stretched and deformed by mechanical stresses as the cell passes through narrow vascular beds. On the cytoplasmic side of the membrane, proteins form a two-dimensional lattice that gives the red cell its flexibility (Fig. 44.10). The major proteins are spectrin, actin, band 4.1, band 4.2, and ankyrin. Spectrin, the major

A Band 3 protein

Glycophorin A

4.2

Glycophorin C

4.1

833

A

B 5 ␮m

FIG. 44.9. The shape of the red blood cell. A. Wright-stained cells, displaying the pale staining in the center. B. Scanning electron micrograph, showing the biconcave disc structure of the cells. The stacks of erythrocytes in this preparation (collected from a blood tube) are not unusual. These photographs were obtained, with permission, from Cohen BJ, Wood DL. Memmler’s the Human Body in Health and Disease. 9th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2000 (Panel A) and Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th ed. New York, NY: Garland Science; 2002:600 (Panel B).

Actin

Ankyrin 4.1

␣-Spectrin

␤-Spectrin

Iron deficiency will result in a microcytic, hypochromic anemia. Red blood cells will be small and pale. In contrast to a vitamin B6 deficiency, which also results in a microcytic, hypochromic anemia, iron stores are low in an iron deficiency anemia.

B

Band 4.1

Band 3 protein Actin

Ankyrin

Spectrin dimer

FIG. 44.10. A generalized view of the erythrocyte cytoskeleton. A. The major protein, spectrin, is linked to the plasma membrane either through interactions with ankyrin and band 3, or with actin, band 4.1, and glycophorin. Other proteins in this complex, not shown, are tropomyosin and adducin. B. A view from inside the cell, looking up at the cytoskeleton. This view displays the cross-linking of the spectrin dimers to actin and band 3 anchor sites.

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SECTION VIII ■ TISSUE METABOLISM

Defects in erythrocyte cytoskeletal proteins lead to hemolytic anemia. Shear stresses in the circulation result in the loss of pieces of the red cell membrane. As the membrane is lost, the red blood cell becomes more spherical and loses its deformability. As these cells become more spherical, they are more likely to lyse in response to mechanical stresses in the circulation, or to be trapped and destroyed in the spleen.

IV. AGENTS THAT AFFECT OXYGEN BINDING

Hb + O2

HbO2

protein, is a heterodimer composed of ␣- and ␤-subunits wound around each other. The dimers self-associate at the heads. At the opposite end of the spectrin dimers, actin and band 4.1 bind near to each other. Multiple spectrins can bind to each actin filament, resulting in a branched membrane cytoskeleton. The spectrin cytoskeleton is connected to the membrane lipid bilayer by ankyrin, which interacts with ␤-spectrin and the integral membrane protein, band 3. Band 4.2 helps to stabilize this connection. Band 4.1 anchors the spectrin skeleton with the membrane by binding the integral membrane protein glycophorin C and the actin complex, which has bound multiple spectrin dimers. When the red blood cell is subjected to mechanical stress, the spectrin network rearranges. Some spectrin molecules become uncoiled and extended; others become compressed, thereby changing the shape of the cell, but not its surface area. The mature erythrocyte cannot synthesize new membrane proteins or lipids. However, membrane lipids can be freely exchanged with circulating lipoprotein lipids. The glutathione system protects the proteins and lipids from oxidative damage. The unusual names for some erythrocyte membrane proteins, such as band 4.1, arose through analysis of red blood cell membranes by polyacrylamide gel electrophoresis. The stained bands observed in the gel were numbered according to molecular weight (band 1, band 2, etc.), and as functions were assigned to the proteins, more common names were assigned to the proteins (e.g., spectrin is actually band 1).

The major agents that affect oxygen binding to hemoglobin are shown in Figure 44.11.

1 Hydrogen ions

A. 2,3-Bisphosphoglycerate

2 2,3-Bisphosphoglycerate 3 Covalent binding of CO2

FIG. 44.11. Agents that affect oxygen binding by hemoglobin. Binding of hydrogen ions, 2,3-bisphosphoglycerate, and carbon dioxide to hemoglobin decrease its affinity for oxygen.

2,3-Bisphosphoglycerate (2,3-BPG) is formed in red blood cells from the glycolytic intermediate 1,3-bisphosphoglycerate, as indicated in Figure 44.1. 2,3-BPG binds to hemoglobin in the central cavity formed by the four subunits, increasing the energy required for the conformational changes that facilitate the binding of oxygen. Thus, 2,3-BPG lowers the affinity of hemoglobin for oxygen. Therefore, oxygen is less readily bound (i.e., is more readily released in tissues) when hemoglobin contains 2,3-BPG.

B. Proton Binding (Bohr Effect) 100

Tissues

Lungs

80 % Saturation

7.6

7.2 6.8 pH

60 Hb 40 20

0 40

80

120

pO2

FIG. 44.12. Effect of pH on oxygen saturation curves. As the pH decreases, the affinity of hemoglobin for oxygen decreases, producing the Bohr effect.

Lieberman_CH44.indd 834

The binding of protons by hemoglobin lowers its affinity for oxygen (Fig. 44.12), contributing to a phenomenon known as the Bohr effect (Fig. 44.13). The pH of the blood decreases as it enters the tissues (and the proton concentration rises) because the CO2 produced by metabolism is converted to carbonic acid by the reaction catalyzed by carbonic anhydrase in red blood cells. Dissociation of carbonic acid produces protons that react with several amino acid residues in hemoglobin, causing conformational changes that promote the release of oxygen. In the lungs, this process is reversed. Oxygen binds to hemoglobin, causing a release of protons, which combine with bicarbonate to form carbonic acid. This decrease of protons causes the pH of the blood to rise. Carbonic anhydrase cleaves the carbonic acid to H2O and CO2, and the CO2 is exhaled. Thus, in tissues in which the pH of the blood is low because of the CO2 produced by metabolism, oxygen is released from hemoglobin. In the lungs, where the pH of the blood is higher because CO2 is being exhaled, oxygen binds to hemoglobin.

C. Carbon Dioxide Although most of the CO2 produced by metabolism in the tissues is carried to the lungs as bicarbonate, some of the CO2 is covalently bound to hemoglobin (Fig. 44.14). In the tissues, CO2 forms carbamate adducts with the N-terminal amino groups of

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A RBC Tissues

CO2 Carbonic anhydrase

H2O

H2CO3

–

HCO3 H+ HbO2 HHb O2

Tissues

B RBC Exhaled

CO2 H2O

Carbonic anhydrase

H2CO3

–

HCO3 H+ HbO2 HHb O2

Lungs

FIG. 44.13. Effect of H⫹ on oxygen binding by hemoglobin (Hb). A. In the tissues, CO2 is released. In the red blood cell, this CO2 forms carbonic acid, which releases protons. The protons bind to Hb, causing it to release oxygen to the tissues. B. In the lungs, the reactions are reversed. O2 binds to protonated Hb, causing the release of protons. They bind to bicarbonate (HCO3⫺), forming carbonic acid, which is cleaved to water and CO2, which is exhaled. RBC, red blood cell.

Hb

V. HEMATOPOIESIS The various types of cells (lineages) that make up the blood are constantly being produced in the bone marrow. All cell lineages are descended from hematopoietic stem cells, cells that are renewable throughout the life of the host. The population of hematopoietic stem cells is quite small. Estimates vary between 1 and 10 per 105 bone marrow cells. In the presence of the appropriate signals, hematopoietic

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CO2

Hemoglobin

Hb

deoxyhemoglobin and stabilizes the deoxy conformation. In the lungs, where the PO2 is high, oxygen binds to hemoglobin and this bound CO2 is released.

+

NH3 +

N

COO–

+ H+

H Carbamate of hemoglobin

FIG. 44.14. Binding of CO2 to hemoglobin. CO2 forms carbamates with the N-terminal amino groups of hemoglobin (Hb) chains. Approximately 15% of the CO2 in blood is carried to the lungs bound to Hb. The reaction releases protons, which contribute to the Bohr effect. The overall effect is stabilization of the deoxy form of hemoglobin.

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SECTION VIII ■ TISSUE METABOLISM

Populations of hematopoietic cells enriched with stem cells can be isolated by fluorescence-activated cell sorting, based on the expression of specific cell-surface markers. Increasing the population of stem cells in cells used for a bone marrow transplantation increases the chances of success of the transplantation.

stem cells proliferate, differentiate, and mature into any of the types of cells that make up the blood (Fig. 44.15). Hematopoietic differentiation is hierarchical. The number of fates a developing blood cell may adopt becomes progressively restricted. Hematopoietic progenitors are designated colony-forming unit–lineage, or colony-forming unit–erythroid (CFU-E). Progenitors that form very large colonies are termed burst-forming units.

A. Cytokines and Hematopoiesis Developing progenitor cells in the marrow grow in proximity with marrow stromal cells. These include fibroblasts, endothelial cells, adipocytes, and macrophages.

Selfrenewal

Pluripotent stem cell

CFU-GEMM (mixed myeloid progenitor cell)

BFU-EMeg

BFU-E

CFU-Meg

Lymphoid stem cell

CFU-Ba

CFU-GMEo

CFU-GM

NK-precursor

CFU-Eo

B-lymphocytes

T-lymphocytes

NK-cell

Monocyte

CFU-E

Megakaryoctye

Macrophage

Basophil

Platelets

Red blood cells

Neutrophil

Eosinophil

FIG. 44.15. The hematopoietic tree. All blood cells arise from the self-renewing pluripotent stem cell. Different cytokines are required at each step for these events to occur. BFU, burst-forming unit; CFU, colony-forming unit.

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The stromal cells form an extracellular matrix and secrete growth factors that regulate hematopoietic development. The hematopoietic growth factors have multiple effects. An individual growth factor may stimulate proliferation, differentiation, and maturation of the progenitor cells and also may prevent apoptosis. These factors also may activate various functions within the mature cell. Some hematopoietic growth factors act on multiple lineages, whereas others have more limited targets. Most hematopoietic growth factors are recognized by receptors belonging to the cytokine receptor superfamily. Binding of ligand to receptor results in receptor aggregation, which induces phosphorylation of Janus kinases (JAKs). The JAKs are a family of cytoplasmic tyrosine kinases that are active when phosphorylated (see Chapter 11, Section III.C, and Fig. 11.15). The activated JAKs then phosphorylate the cytokine receptor. Phosphorylation of the receptor creates docking regions where additional signal transduction molecules bind, including members of the signal transducer and activator of transcription (STAT) family of transcription factors. The JAKs phosphorylate the STATs, which dimerize and translocate to the nucleus, where they activate target genes. Additional signal transduction proteins bind to the phosphorylated cytokine receptor, leading to activation of the Ras/Raf/MAP kinase pathways. Other pathways are also activated, some of which lead to an inhibition of apoptosis (see Chapter 18). The response to cytokine binding is usually transient because the cell contains multiple negative regulators of cytokine signaling. The family of silencer of cytokine signaling (SOCS) proteins is induced by cytokine binding. One member of the family binds to the phosphorylated receptor and prevents the docking of signal transduction proteins. Other SOCS proteins bind to JAKs and inhibit them. Whether SOCS inhibition of JAKs is a consequence of steric inhibition or whether SOCS recruit phosphatases that then dephosphorylate the JAKs (Fig. 44.16) is uncertain. SHP-1 is a tyrosine phosphatase found primarily in hematopoietic cells that is necessary for proper development of myeloid and lymphoid lineages. Its function is to dephosphorylate JAK2, thereby inactivating it. STATs are also inactivated. The protein inhibitors of activated STAT (PIAS) family of proteins bind to phosphorylated STATs and prevent their dimerization

837

Leukemias, malignancies of the blood, arise when a differentiating hematopoietic cell does not complete its developmental program but remains in an immature, proliferative state. Leukemias have been found in every hematopoietic lineage.

In X-linked severe combined immunodeficiency (SCID) disease, the most common form of SCID, circulating T lymphocytes are not formed, and B lymphocytes are not active. The affected gene encodes the ␥-chain of the interleukin-2 receptor. Mutant receptors are unable to activate Janus kinase 3 (JAK3), and the cells are unresponsive to the cytokines that stimulate growth and differentiation. Recall also that adenosine deaminase deficiency (see Chapter 41), which is not X-linked, also leads to a form of SCID, but for different reasons.

GF

GF

GF JAK P

JAK

JAK

1

P

JAK P P

STAT P P

–

JAK P STATP

3

JAK P P STA T

5

STAT

STAT P P STAT Nucleus

2

4

SOCS

Transcription

FIG. 44.16. Cytokine signaling through the JAK/STAT pathway. (1) Cytokine binding to receptors initiates dimerization and activation of the JAK kinase, which phosphorylates the receptor on tyrosine residues. (2) STAT proteins bind to the activated receptors and are themselves phosphorylated. (3) Phosphorylated STAT proteins dimerize, travel to the nucleus, and initiate gene transcription. (4) One family of proteins whose synthesis is stimulated by STATs is the SOCS (suppressor of cytokine signaling) family, which inhibits further activation of STAT proteins (5) by a variety of mechanisms.

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SECTION VIII ■ TISSUE METABOLISM

Families have been identified whose members have a mutant erythropoietin (EPO) receptor that is unable to bind SHP-1. Erythropoietin is the hematopoietic cytokine that stimulates production of red blood cells. Individuals with the mutant EPO receptor have a higher than normal percentage of red blood cells in the circulation because the mutant EPO receptor cannot be deactivated by SHP-1. Erythropoietin causes sustained activation of Janus kinase 2 (JAK2) and STAT 5 in these cases.

Perturbed JAK/STAT signaling is associated with development of lymphoid and myeloid leukemias, severe congenital neutropenia (a condition in which levels of circulating neutrophils are severely reduced), and Fanconi anemia, which is characterized by bone marrow failure and increased susceptibility to malignancy.

A complication of sickle cell disease is an increased formation of gallstones. A sickle cell crisis accompanied by the intravascular destruction of red blood cells (hemolysis) experienced by patients with sickle cell disease, such as Will Sichel, increases the amount of unconjugated bilirubin that is transported to the liver. If the concentration of this unconjugated bilirubin exceeds the capacity of the hepatocytes to conjugate it to the more soluble diglucuronide through interaction with hepatic UDP-glucuronate, both the total and the unconjugated bilirubin levels in the blood increase. More unconjugated bilirubin is then secreted by the liver into the bile. The increase in unconjugated bilirubin (which is not very water-soluble) results in its precipitation within the gallbladder lumen, leading to the formation of pigmented (calcium bilirubinate) gallstones.

or promote the dissociation of STAT dimers. STATs also may be inactivated by dephosphorylation, although the specific phosphatases have not yet been identified, or by targeting activated STATs for proteolytic degradation.

B. Erythropoiesis The production of red cells is regulated by the demands of oxygen delivery to the tissues. In response to reduced tissue oxygenation, the kidney releases the hormone erythropoietin, which stimulates the multiplication and maturation of erythroid progenitors. The progression along the erythroid pathway begins with the stem cell and passes through the mixed myeloid progenitor cell (CFU-GEMM, colonyforming unit–granulocyte, erythroid, monocyte, megakaryocyte), burst-forming unit–erythroid (BFU-E), CFU-E, and to the first recognizable red cell precursor, the normoblast. Each normoblast undergoes four more cycles of cell division. During these four cycles, the nucleus becomes smaller and more condensed. After the last division, the nucleus is extruded. The red cell at this state is called a reticulocyte. Reticulocytes still retain ribosomes and messenger RNA (mRNA) and are capable of synthesizing hemoglobin. They are released from the bone marrow and circulate for 1 to 2 days. Reticulocytes mature in the spleen, where the ribosomes and mRNA are lost (Fig. 44.17).

C. Nutritional Anemias Each person produces approximately 1012 red blood cells per day. Because so many cells must be produced, nutritional deficiencies in iron, vitamin B12, and folate prevent adequate red blood cell formation. The physical appearance of the cells in the case of a nutritional anemia frequently provides a clue as to the nature of the deficiency. In the case of iron deficiency, the cells are smaller and paler than normal. The lack of iron results in decreased heme synthesis, which in turn affects globin synthesis. Maturing red cells following their normal developmental program divide until their hemoglobin has reached the appropriate concentration. Iron (and hemoglobin)-deficient developing red blood cells continue dividing past their normal stopping point, resulting in small (microcytic) red cells. The cells are also pale—because of the lack of hemoglobin—compared with normal cells (thus, a pale, microcytic anemia results). Bone marrow Stem cells

CFU-GEMM

BFU-EMeg

BFU-E

+

CFU-E

Pronormoblast +

+

Reticulocyte

Erythropoietin Circulating red cells O2 sensor

Oxygen delivery

Kidney

FIG. 44.17. Erythropoietin stimulation of erythrocyte maturation. The abbreviations are described in the text.

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Deficiencies of folate or vitamin B12 can cause megaloblastic anemia, in which the cells are larger than normal. Folate and B12 are required for DNA synthesis (see Chapters 40 and 41). When these vitamins are deficient, DNA replication and nuclear division do not keep pace with the maturation of the cytoplasm. Consequently, the nucleus is extruded before the requisite number of cell divisions has taken place, and the cell volume is greater than it should be, and fewer blood cells are produced.

VI. HEMOGLOBINOPATHIES, HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN, AND HEMOGLOBIN SWITCHING A. Hemoglobinopathies: Disorders in the Structure or Amount of the Globin Chains More than 700 different mutant hemoglobins have been discovered. Most arise from a single base substitution, resulting in a single amino acid replacement. Many have been discovered during population screenings and are not clinically significant. However, in patients with hemoglobin S (HbS, sickle cell anemia), the most common hemoglobin mutation, the amino acid substitution has a devastating effect in the homozygote (see Will Sichel in Chapter 6). Another common hemoglobin variant, hemoglobin C (HbC), results from a glu-to-lys replacement in the same position as the HbS mutation. This mutation has two effects. It promotes water loss from the cell by activating the K⫹ transporter by an unknown mechanism, resulting in a higher than normal concentration of hemoglobin within the cell. The amino acid replacement also substantially lowers the hemoglobin solubility in the homozygote, resulting in a tendency of the mutant hemoglobin to precipitate within the red cell, although, unlike sickle cells, the cell does not become deformed. Homozygotes for the HbC mutation have a mild hemolytic anemia. Heterozygous individuals are clinically unaffected.

839

Hemoglobin C (HbC) is found in high frequency in West Africa, in regions with a high frequency of hemoglobin S (HbS). Consequently, compound heterozygotes for HbS and HbC are not uncommon both in some African regions and among African Americans. HbS/HbC individuals have significantly more hematopathology than individuals with sickle cell trait (adult hemoglobin [HbA]/ HbS). Polymerization of deoxygenated HbS is dependent on the HbS concentration within the cell. The presence of HbC in the compound heterozygote increases the HbS concentration by stimulating K⫹ and water efflux from the cell. Because the HbC globin tends to precipitate, the proportion of HbS tends to be higher in HbS/HbC cells than in the cells of individuals with sickle cell trait (HbS/HbA). The way in which multiple mutations ameliorate or exacerbate hematologic diseases has provided insights into the molecular mechanisms of hemoglobin function and developmental regulation.

B. Thalassemias

For optimum function, the hemoglobin ␣- and ␤-globin chains must have the proper structure and be synthesized in a 1:1 ratio. A large excess of one subunit over the other results in the class of diseases called thalassemias. These anemias are clinically very heterogeneous, as they can arise by multiple mechanisms. Like sickle cell anemia, the thalassemia mutations provide resistance to malaria in the heterozygous state. Hemoglobin single amino acid replacement mutations that give rise to a globin subunit of decreased stability is one mechanism by which thalassemia arises. More common, however, are mutations that result in decreased synthesis of one subunit. The ␣-thalassemias usually arise from complete gene deletions. Two copies of the ␣-globin gene are found on each chromosome 16, for a total of four ␣-globin genes per precursor cell. If one copy of the gene is deleted, the size and hemoglobin concentration of the individual red blood cells is minimally reduced. If two copies are deleted, the red blood cells are of decreased size (microcytic) and reduced hemoglobin concentration (hypochromic). However, the individual is usually not anemic. The loss of three ␣-globin genes causes a moderately severe microcytic hypochromic anemia (hemoglobin, 7 to 10 g/dL) with splenomegaly (enlarged spleen). The absence of four ␣-globin genes (hydrops fetalis) is usually fatal in utero. As discussed in Chapter 14, ␤-thalassemia is a very heterogeneous genetic disease. Insufficient ␤-globin synthesis can result from deletions, promoter mutations, and splice-junction mutations. Heterozygotes for ␤⫹ (some globin chain synthesis) or ␤-null (␤0, no globin chain synthesis) are generally asymptomatic, although they typically have microcytic, hypochromic red blood cells and may have mild anemia. ␤⫹/␤⫹ homozygotes have anemia of variable severity, ␤⫹/␤0 compound heterozygotes tend to be more severely affected, and ␤0/␤0 homozygotes have severe disease. In general, diseases of ␤-chain deficiency are more severe than

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There are two ways in which an individual might have two ␣-globin genes deleted. In one case, one copy of chromosome 16 might have both ␣-globin genes deleted, whereas the other copy had two functional ␣-globin genes. In the second case, both chromosomes might have lost one of their two copies of the ␣-globin gene. The former possibility is more common among Asians, the latter among Africans.

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SECTION VIII ■ TISSUE METABOLISM

diseases of ␣-chain deficiency. Excess ␤-chains form a homotetramer, hemoglobin H (HbH), which is ineffective for delivering oxygen to the tissues because of its high oxygen affinity. As red blood cells age, HbH precipitates in the cells, forming inclusion bodies. Red blood cells with inclusion bodies have shortened life spans because they are more likely to be trapped and destroyed in the spleen. Excess ␣-chains are unable to form a stable tetramer. However, excess ␣-chains precipitate in erythrocytes at every developmental stage. The ␣-chain precipitation in erythroid precursors results in their widespread destruction, a process called ineffective erythropoiesis. The precipitated ␣-chains also damage red blood cell membranes through the heme-facilitated lipid oxidation by reactive oxygen species. Both lipids and proteins, particularly band 4.1, are damaged. The difference in amino acid composition between the ␤-chains of HbA and the ␥-chains of fetal hemoglobin (HbF) results in structural changes that cause HbF to have a lower affinity for 2,3-bisphosphoglycerate (2,3-BPG) than adult hemoglobin (HbA) and thus a greater affinity for oxygen. Therefore, the oxygen released from the mother’s hemoglobin (HbA) is readily bound by HbF in the fetus. Thus, the transfer of oxygen from the mother to the fetus is facilitated by the structural difference between the hemoglobin molecule of the mother and that of the fetus.

C. Hereditary Persistence of Fetal Hemoglobin Fetal hemoglobin (HbF), the predominant hemoglobin of the fetal period, consists of two ␣-chains and two ␥-chains, whereas adult hemoglobin (HbA) consists of two ␣- and two ␤-chains. The process that regulates the conversion of HbF to HbA is called hemoglobin switching. Hemoglobin switching is not 100%; most individuals continue to produce a small amount of HbF throughout life. However, some people, who are clinically normal, produce abnormally high levels (up to 100%) of HbF in place of HbA. Patients with hemoglobinopathies such as ␤-thalassemia or sickle cell anemia frequently have less severe illnesses if their levels of HbF are elevated. One goal of much research on hemoglobin switching is to discover a way to reactivate transcription of the ␥-globin genes to compensate for defective ␤-globin synthesis. Individuals who express HbF past birth have hereditary persistence of fetal hemoglobin (HPFH). 1.

NONDELETION FORMS OF HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

The nondeletion forms of HPFH are those that derive from point mutations in the A␥ and G␥ promoters. When these mutations are found with sickle cell or ␤-thalassemia mutations, they have an ameliorating effect on the disease because of the increased production of ␥-chains. 2.

DELETION FORMS OF HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

In deletion HPFH, both the entire ␦- and ␤-genes have been deleted from one copy of chromosome 11 and only HbF can be produced. In some individuals, the fetal globins remain activated after birth, and enough HbF is produced that the individual is clinically normal. Other individuals with similar deletions that remove the entire ␦- and ␤-genes do not produce enough fetal hemoglobin to compensate for the deletion and are considered to have ␦0␤0-thalassemia. The difference between these two outcomes is believed to be the site at which the deletions end within the ␤-globin gene cluster. In deletion HPFH, powerful enhancer sequences 3⬘ of the ␤-globin gene are resituated because of the deletion so that they activate the ␥-promoters. In individuals with ␦0␤0-thalassemia, the enhancer sequences have not been relocated so that they can interact with the ␥-promoters.

D. Hemoglobin Switching: A Developmental Process Controlled by Transcription Factors In humans, embryonic megaloblasts (the embryonic red blood cell is large and is termed a “blast” because it retains its nucleus) are first produced in the yolk sac approximately 15 days after fertilization. After 6 weeks, the site of erythropoiesis shifts to the liver. The liver and to a lesser extent the spleen are the major sites of fetal erythropoiesis. In the last few weeks before birth, the bone marrow begins producing red blood cells. By 8 to 10 weeks after birth, the bone marrow is the sole site of erythrocyte production. The composition of the hemoglobin also changes with

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841

development because both the ␣-globin locus and the ␤-globin locus have multiple genes that are differentially expressed during development (Fig. 44.18).

E. Structure and Transcriptional Regulation of the ␣- and ␤-Globin Gene Loci

The ␣-globin locus on chromosome 16 contains the embryonic ␨ (zeta) gene and two copies of the ␣-gene, ␣2 and ␣1. The ␤-globin locus on chromosome 11 contains the embryonic ␧-gene; two copies of the fetal ␤-globin gene, G␥ and A␥ (which differ by one amino acid); and two adult genes, ␦ and ␤. The order of the genes along the chromosome parallels the order of expression of the genes during development (see Fig. 44.18). The embryonic hemoglobins are ␨2␧2 (Gower 1), ␨2␥2 (Portland), and ␣2␥2 (Gower 2). Fetal hemoglobin is predominantly ␣2G␥2. The major adult species is ␣2␤2 (hemoglobin A); the minor adult species is ␣2␦2 (hemoglobin A2). The fetal hemoglobin found in adult cells is ␣2A␥2. The timing of hemoglobin switching is controlled by a developmental clock that is not significantly altered by environmental conditions and is related to changes in expression of specific transcription factors. Premature newborns convert from HbF to HbA on schedule with their gestational ages.

A

Chromosome 16

␨

HS40 5'

␣2

␣1

3'

Chromosome 11

⑀

LCR 5'

G␥

A␥

␦

␤

3'

Embryo: ␨2⑀2 = Gower 1

␨2␥2 = Portland ␣2⑀2 = Gower 2 Fetus: ␣2␥2 = HbF Adult: ␣2␥2 = HbF

␣2␦2 = A2 ␣2␤2 = A

% of total globin synthesis

B 50

␣

␥

␤

25

⑀ ␨

␦

0 0

6

18 30 Prenatal age (wk)

6 Birth

18 30 Postnatal age (wk)

42

FIG. 44.18. Globin gene clusters and expression during development. A. The globin gene clusters with the ␣-genes on chromosome 16 and the ␤-genes on chromosome 11. LCR, locus control region. B. The switching of globin chain synthesis during development.

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SECTION VIII ■ TISSUE METABOLISM

CLINICAL COMMENTS Spiro Site. Spiro Site’s red blood cells are deficient in spectrin. This deficiency impairs the ability of his erythrocytes to maintain the redundant surface area necessary to maintain deformability. Mechanical stresses in the circulation cause progressive loss of pieces of membrane. As membrane components are lost, Spiro Site’s red blood cells become spherical and unable to deform. His spleen is enlarged because of the large number of red blood cells that have become trapped within it. His erythrocytes are lysed by mechanical stresses in the circulation and by macrophages in the spleen. Consequently, this hemolytic process results in anemia. His gallstones were the result of the large amounts of bilirubin that were produced and stored in the gallbladder as a result of the hemolysis. The abnormally rounded red cells seen on a blood smear are characteristic of hereditary spherocytosis. Mutations in the genes for ankyrin, ␤-spectrin, or band 3 account for three quarters of the cases of hereditary spherocytosis, whereas mutations in the genes for ␣-spectrin or band 4.2 account for the remainder. The result of defective synthesis of any of the membrane cytoskeletal proteins results in improper formation of the membrane cytoskeleton. Excess membrane proteins are catabolized, resulting in a net deficiency of spectrin. Spiro Site underwent a splenectomy. Because the spleen was the major site of destruction of his red blood cells, his anemia improved significantly after surgery. He was discharged with the recommendation to take a folate supplement daily. It was explained to Mr. Site that because the spleen plays a major role in protection against certain bacterial agents, he would require immunizations against Pneumococcus, Meningococcus, and Haemophilus influenzae type b. Anne Niemick. Anne Niemick was found to be a compound heterozygote for mutations in the ␤-globin gene. On one gene, a mutation in position 6 of intron 1 converted a T to a C. The presence of this mutation, for unknown reasons, raises HbF production. The other ␤-globin gene had a mutation in position 110 of exon 1 (a C-to-T mutation). Both ␤-globin chains have reduced activity, but combined with the increased expression of HbF, the result is a ␤⫹-thalassemia.

BIOCHEMICAL COMMENTS Control of Hemoglobin Switching. How is hemoglobin switching controlled? Although there are still many unanswered questions, some of the molecular mechanisms have been identified. The ␣-globin locus covers ⬃100 kb (kilobases). The major regulatory element, HS40, is a nucleasesensitive region of DNA that lies 5⬘ of the ␨-gene (see Fig. 44.18). HS40 acts as an erythroid-specific enhancer that interacts with the upstream regulatory regions of the ␨- and ␣-genes, and stimulates their transcription. The region immediately 5⬘ of the ␨-gene contains the regulatory sequences responsible for silencing ␨-gene transcription. However, the exact sequences and transcription factors responsible for this silencing have not yet been identified. Even after silencing, low levels of ␨-gene transcripts are still produced after the embryonic period; however, they are not translated. This is because both the ␨-globin and ␣-globin transcripts have regions that bind to a messenger ribonucleoprotein (mRNP) stability-determining complex. Binding to this complex prevents the messenger RNA (mRNA) from being degraded. The ␣-globin messenger RNA has a much higher affinity for the mRNP than the ␨-globin message, which leads to the ␨-globin message being rapidly degraded.

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843

The ␤-globin locus covers ~100 kb. From 5 to 25 kb upstream of the ␧-gene is the locus control region (LCR), containing five DNAse hypersensitive sites. The LCR is necessary for the function of the ␤-globin locus. It maintains the chromatin of the entire locus in an active configuration and acts as an enhancer and entry point for the factors that transcribe the genes of the ␤-globin locus. One model of the control of hemoglobin switching postulates that proteins bound at the promoters of the ␧-, ␥-, and ␤-globin genes compete to interact with the enhancers of the LCR. Each gene in the ␤-globin locus has individual regulatory elements—a promoter, silencers, or enhancers that control its developmental regulation. The promoters that control the ␥- and ␤-globin genes have been intensively studied because of their clinical relevance. The ␧-globin gene, like the ␨-globin gene, has silencers in the 5⬘-regulatory region. Binding of proteins to these regions turns off the ␧-gene. The proximal region of the ␥-globin gene promoter has multiple transcription factor–binding sites (Fig. 44.19). Many hereditary persistence of fetal hemoglobin (HPFH) mutations map to these transcription factor–binding sites, either by destroying a site or by creating a new one, but the exact mechanisms are still not understood. Two sites that appear to be significant in the control of hemoglobin switching are the stage-selector protein-binding (SSP) site and the CAAT box region. When the SSP complex is bound to the promoter, the ␥-globin gene has a competitive advantage over the ␤-globin promoter for interaction with the LCR. A second transcription factor, Sp1, also binds at the SSP-binding site, where it may act as a repressor, and competition between these two protein complexes for the SSP-binding site helps to determine the activity of the ␥-globin gene. A similar

A. CDP

GATA GATA –175

CP1 CP1 CAAT CAAT –115

–85

SSP TATA

Transcription Start Site

–30

BCL11A

B.

GATA1 FOG1

LCR 5'

ε

Gγ

NURD

Aγ

δ

β

3'

Chromosome 11

FIG. 44.19. A. The ␥-globin gene promoter, indicating some of the transcription factor– binding sites associated with hereditary persistence of fetal hemoglobin. B. The ␤-globin gene locus. When BCL11A is expressed, the interaction between the LCR and the ␥-gene promoter is blocked, turning off ␥-gene expression. NURD is a chromatin remodeling complex containing histone deacetylase activity.

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mechanism appears to be operating at the CAAT box. CP1, thought to be a transcription activator, binds at the CAAT box. CAAT displacement protein (CDP) is a repressor that binds at the CAAT site and displaces CP1. Part of the mechanism of hemoglobin switching appears to be the binding of repressors at the ␤-globin and ␥-globin upstream regulatory regions. The ␤-globin gene also has binding sites for multiple transcription factors in its regulatory regions. Mutations that affect binding of transcription factors can produce thalassemia by reducing the activity of the ␤-globin promoter. There is also an enhancer 3⬘ of the poly A signal that seems to be required for stage-specific activation of the ␤-globin promoter. Further insights into the control of hemoglobin switching indicate that the transcription factor BCL11A is a strong repressor of ␥-globin gene expression. BCL11A interacts with a variety of other transcription factors (GATA-1, FOG1, and the NURD repressor complex [nucleosome remodeling and histone deacetylase]) to repress ␥-globin expression. This appears to be caused by BCL11A interfering with LCR interactions with the ␥-globin gene promoter. Experiments that reduce, or eliminate, BCL11A expression lead to an increase in ␥-globin synthesis. BCL11A expression is regulated by the transcription factor KLF1, which is essential for ␤-globin expression. KLF1 increases BCL11A expression, which blocks ␥-globin gene expression, while KFL1 stimulates ␤-globin gene expression. These recent results suggest that drugs, which interfere with KLF1 action, would enhance and activate fetal globin gene expression in individuals with either ␤-thalassemia or sickle cell disease (see Fig. 44.19B). Key Concepts • •

• •

•

•

•

Lieberman_CH44.indd 844

The blood contains a wide variety of distinct cell types, each of whose function is necessary for maintaining the body’s internal environment. Erythrocytes transport oxygen throughout the body and return carbon dioxide back to the lung. Erythrocytes lack nuclei and carry out limited metabolic reactions. ■ Glycolysis provides energy and NADH. ■ The NADH maintains the iron in hemoglobin in the ferrous state. ■ The HMP shunt provides NADPH to regenerate reduced glutathione to protect the membrane from oxidative damage. ■ 1,3-Bisphosphoglycerate is converted to 2,3-bisphosphoglycerate as a by-product of glycolysis to regulate oxygen binding to hemoglobin. Heme synthesis occurs in the erythrocyte precursor, using succinyl-CoA and glycine. Inherited defects in heme synthesis lead to porphyrias. Iron, a critical part of heme, is carried throughout the body on protein carriers because free iron is toxic. The erythrocyte membrane is flexible as a result of its unique cytoskeletal structure, which allows erythrocytes to deform in order to travel through narrow capillaries. Oxygen binding to hemoglobin in the erythrocyte is modulated by a variety of factors. ■ 2,3-Bisphosphoglycerate stabilizes the deoxy form of hemoglobin. ■ Proton binding to hemoglobin stabilizes the deoxy form of hemoglobin (the Bohr effect). ■ Carbon dioxide links covalently to the amino termini of the four globin chains in a hemoglobin molecule, further stabilizing the deoxy form of hemoglobin. Hematopoiesis is the generation of the unique blood cell types from a single precursor stem cell in the bone marrow. Polymorphonuclear leukocytes consist of a variety of cell types that release chemical signals when activated (granulocytes), phagocytose foreign bodies (neutrophils), destroy parasites (eosinophils), and are involved in the allergic response (basophils). Mononuclear leukocytes include the lymphocytes (necessary for the immune response) and monocytes (which develop into macrophages, which engulf debris left behind after granulocytes attack foreign material). A wide variety of mutations can lead to alterations in hemoglobin function (hemoglobinopathies): Sickle cell anemia Thalassemias Hereditary persistence of fetal hemoglobin (hemoglobin switching and its regulation) Diseases discussed in this chapter are summarized in Table 44.4.

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CHAPTER 44 ■ THE BIOCHEMISTRY OF ERYTHROCYTES AND OTHER BLOOD CELLS

Table 44.4

845

Diseases Discussed in Chapter 44

Disease or Disorder

Environmental or Genetic

Thalassemias

Genetic

Pyruvate kinase deficiency

Genetic

Congenital methemoglobinemia

Genetic

Glucose-6-phosphate dehydrogenase deficiency Porphyrias

Genetic Genetic

Iron deficiency

Both

X-linked severe-combined immunodeficiency syndrome Defective erythropoietin receptor

Genetic

Genetic

Hemoglobin C

Genetic

Hereditary persistence of fetal hemoglobin

Genetic

Spherocytosis

Genetic

Comments Unbalanced synthesis of alpha and beta chains of hemoglobin, leading to anemia. Red cell hemolysis, leading to fewer red cells. An increase in 2,3-BPG levels often masks the effects of the anemia. Oxidation of the iron in heme to the ferric state, which will not bind oxygen, although many individuals with this disorder are asymptomatic. Affects red blood cell membrane stability through an inability to protect membrane proteins and lipids against oxidation. Inherited defects in almost any step of heme synthesis leading to a series of diseases with different symptoms and outcomes. Reduced iron leads to reduced heme synthesis and reduced oxygen delivery to the tissues. Loss of a cytokine receptor subunit, leading to a complete loss of B- and T-cell maturation and proliferation, and no functional immune system. Red blood cell formation is reduced under conditions in which red blood cell production should be increased (such as reduced oxygen delivery to the tissues). A point mutation in hemoglobin leading to a lysine for a glutamic acid at position 6 of the beta chain, leading to hemolytic anemia in the homozygous state. Mutations in promoter and enhancer regions leading to misexpression of the globin gamma gene, and constant expression of the gene Mutations in any of several red blood cell membrane proteins (such as spectrin), leading to instability of the red cells, destruction of the red blood cells, and an anemia.

2,3-BPG, 2,3-bisphosphoglycerate.

REVIEW QUESTIONS—CHAPTER 44 1.

2.

A compensatory mechanism to allow adequate oxygen delivery to the tissues at high altitudes, where oxygen concentrations are low, is which of the following? A. An increase in 2,3-bisphosphoglycerate synthesis by the red cell B. A decrease in 2,3-bisphosphoglycerate synthesis by the red cell C. An increase in hemoglobin synthesis by the red cell D. A decrease in hemoglobin synthesis by the red cell E. Decreasing the blood pH A 2-year-old boy of normal weight and height is brought to a clinic because of excessive fatigue. Blood work indicates anemia, with microcytic hypochromic red cells. The boy lives in a 100-year-old apartment building and has been caught ingesting paint chips. His parents indicate that the child eats a healthy diet and takes a Flintstones vitamin

Lieberman_CH44.indd 845

supplement every day. His anemia is most likely attributable to a deficiency in which of the following? A. Iron B. Vitamin B12 C. Folate D. Heme E. Vitamin B6 3.

Drugs are being developed that will induce the transcription of globin genes, which are normally silent in patients affected with sickle cell disease. A good target gene for such therapy in this disease would be which of the following? A. The ␣1-gene B. The ␣2-gene C. The ␥-gene D. The ␤-gene E. The ␨-gene

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SECTION VIII ■ TISSUE METABOLISM

4.

A mature blood cell that lacks a nucleus is which of the following? A. Lymphocyte B. Basophil C. Eosinophil D. Platelet E. Neutrophil

5.

A family has two children, one with a mild case of thalassemia and a second with a severe case of thalassemia, requiring frequent blood transfusions as part of the treatment plan. One parent is of Mediterranean descent, the other is of Asian descent. Neither parent exhibits clinical signs of thalassemia. Both children express 20% of the expected level of ␤-globin; the more severely affected child expresses normal levels of ␣-globin, whereas the less severely affected child expresses only 50% of the normal

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levels of ␣-globin. Why is the child who has a deficiency in ␣-globin expression less severely affected? A. Thalassemia is caused by a mutation in the ␣-gene, and the more severely affected child expresses more of it. B. The less severely affected child must be synthesizing the ␨-gene to make up for the deficiency in ␣-chain synthesis. C. The more severely affected child also has HPFH. D. The more severely affected child produces more inactive globin tetramers than the less severely affected child. E. Thalassemia is caused by an iron deficiency, and when the child is synthesizing normal levels of ␣-globin, there is insufficient iron to populate all of the heme molecules synthesized.

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45

Blood Plasma Proteins, Coagulation, and Fibrinolysis

The blood is the body’s main transport system. Although the transport and delivery of oxygen to the cells of the tissues is carried out by specialized cells, other vital components such as nutrients, metabolites, electrolytes, and hormones are all carried in the noncellular fraction of the blood, the plasma. Some components, such as glucose, are dissolved in the plasma; others, for example, lipids and steroid hormones, are bound to carrier proteins for transport. The osmotic pressure exerted by the plasma proteins regulates the distribution of water between the blood and the tissues. Plasma proteins in conjunction with platelets maintain the integrity of the circulatory system through the process of clotting. Blood normally circulates through endothelium-lined blood vessels. When a blood vessel is severed, a blood clot (called a thrombus, which is formed by the process of thrombosis) forms as part of hemostasis, the physiologic response that stops bleeding. Blood clots also form to repair damage to the endothelial lining in which components of the subendothelial layer become accessible to plasma proteins. In hemostasis and thrombosis, a primary hemostatic plug, consisting of aggregated platelets and a fibrin clot, forms at the site of injury. Platelets are attached to the subendothelial layer of the vessel principally through the protein von Willebrand factor (vWF) and are activated to bind fibrinogen. Fibrinogen binds the platelets to each other to form a platelet aggregate. The platelet aggregate is rapidly covered with layers of fibrin, which are formed from fibrinogen by the proteolytic enzyme thrombin. Injury to the endothelium and exposure of tissue factor on the subendothelial layer to plasma proteins also activate the blood coagulation cascade, which ultimately activates thrombin and factor XIII. Factor XIII cross-links strands of polymerized fibrin monomers to form a stable clot (the secondary hemostatic plug). The blood coagulation cascade consists of a series of enzymes (such as factor X), which are inactive until they are proteolytically cleaved by the preceding enzyme in the cascade. Other proteins (factor V and factor VIII) serve as binding proteins, which assemble factor complexes at the site of injury. Ca2⫹ and ␥-carboxyglutamate residues in the proteins (formed by a vitamin K–dependent process in the liver) attach the factor complexes to phospholipids exposed on platelet membranes. Consequently, thrombus formation is rapidly accelerated and localized to the site of injury. Regulatory mechanisms within the blood coagulation cascade and antifibrinolytic mechanisms prevent random coagulation within blood vessels that might obstruct blood flow. Impairments in these mechanisms lead to thrombosis.

847

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SECTION VIII ■ TISSUE METABOLISM

THE WAITING ROOM There are two basic assays for measuring the activity of factor VIII in a blood sample: the first is a functional (clotting) assay and the second is a coupled (to factor X activation) enzyme system leading to the cleavage of a chromogenic substrate, generating a colored product. For the functional assay, a sample of the patient’s plasma is added to a factor VIIIdeficient sample of plasma (which can be obtained commercially), and the time to generate clot formation is determined. Although clot formation is the end product of this assay, one is measuring the eventual activation of factor II to IIa, which allows clot formation to initiate. In the automated, chromogenic assay, a sample of the unknown is mixed with purified factor IXa, Ca2⫹, phospholipid vesicles, the chromogenic substrate, and purified factor X. If the unknown sample contains factor VIII, the factors VIII and IXa will synergize and activate factor X to Xa. The factor Xa will cleave the chromogenic substrate, forming a colored product, whose concentration can be determined spectrophotometrically. Comparison to a standard curve of factor VIII addition enables an accurate estimate of the level of active factor VIII in the sample.

In cases of severe protein malnutrition (kwashiorkor), the concentration of the plasma proteins decreases as a result of which the osmotic pressure of the blood decreases. As a result, fluid is not drawn back to the blood and instead accumulates in the interstitial space (edema). The distended bellies of famine victims are the result of fluid accumulation in the extravascular tissues because of the severely decreased concentration of plasma proteins, particularly albumin. Albumin synthesis decreases fairly early under conditions of protein malnutrition.

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Sloe Klotter, a 6-month-old male infant, was brought to his pediatrician’s office with a painful, expanding mass in his right upper thigh that was first noted just hours after he fell down three uncarpeted steps in his home. The child appeared to be in severe distress. A radiograph showed no fractures, but a soft tissue swelling, consistent with a hematoma (bleeding into the tissues), was noted. Sloe’s mother related that soon after he began to crawl, his knees occasionally became swollen and seemed painful. The pediatrician suspected a disorder of coagulation. A screening coagulation profile suggested a possible deficiency of factor VIII, a protein involved in the formation of blood clots. Sloe’s plasma factor VIII level was found to be only 3% of the average level found in normal subjects. A diagnosis of hemophilia A was made.

I.

PLASMA PROTEINS MAINTAIN PROPER DISTRIBUTION OF WATER BETWEEN BLOOD AND TISSUES

When cells are removed from the blood, the remaining plasma is composed of water, nutrients, metabolites, hormones, electrolytes, and proteins. Plasma has essentially the same electrolyte composition as other extracellular fluids and constitutes approximately 25% of the body’s total extracellular fluid. The plasma proteins serve several functions, including maintaining the proper distribution of water between the blood and the tissues; transporting nutrients, metabolites, and hormones throughout the body; defending against infection; and maintaining the integrity of the circulation through clotting. Many diseases alter the amounts of plasma proteins produced and hence their concentration in the blood. These changes can be determined by electrophoresis of plasma proteins over the course of a disease.

A. Body Fluid Maintenance between Tissues and Blood As the arterial blood enters the capillaries, fluid moves from the intravascular space into the interstitial space (that surrounding the capillaries) because of what are known as Starling forces. The hydrostatic pressure in the arteriolar end of the capillaries (⬃37 mm Hg) exceeds the sum of the tissue pressure (⬃1 mm Hg) and the osmotic pressure of the plasma proteins (⬃25 mm Hg). Thus, water tends to leave the capillaries and enter extravascular spaces. At the venous end of the capillaries, the hydrostatic pressure falls to approximately 17 mm Hg while the osmotic pressure and the tissue pressure remain constant, resulting in movement of fluid back from the extravascular (interstitial) spaces and into the blood. Thus, most of the force bringing water back from the tissues into the plasma is the osmotic pressure mediated by the presence of proteins in the plasma.

B. The Major Serum Protein, Albumin As indicated in Table 45.1, the liver produces several transport or binding proteins and secretes them into the blood. The major protein synthesized is albumin, which constitutes approximately 60% of the total plasma protein, but because of its relatively small size (69 kDa), it is thought to contribute 70% to 80% of the total osmotic pressure of the plasma. Albumin, like most plasma proteins, is a glycoprotein and is a carrier of free fatty acids, calcium, zinc, steroid hormones, copper, and bilirubin. Many drugs also bind to albumin, which may have important pharmacologic implications. For example, when a drug binds to albumin, such binding will

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

Table 45.1

Specific Plasma-Binding Proteins Synthesized in the Liver

Albumin Ceruloplasmin Corticosteroid-binding globulin Haptoglobin Lipoproteins Retinol-binding protein Sex hormone–binding globulin Transferrin Transthyretin

Binds free fatty acids, calcium, zinc, steroid hormones, copper, bilirubin Binds copper; appears to be more important as a copper storage pool than as a transport protein; integrates iron and copper homeostasis Binds cortisol Binds extracorpuscular heme Transport cholesterol and fatty acids Binds vitamin A Binds estradiol and testosterone Transports iron Binds thyroxine (T4); also forms a complex with retinol-binding protein

likely lower the effective concentration of that drug and may lengthen its lifetime in the circulation. Drug dosimetry may need to be recalculated if a patient’s plasma protein concentration is abnormal.

II. THE PLASMA CONTAINS PROTEINS THAT AID IN IMMUNE DEFENSE Two different sets of proteins aid in the immune response: the immunoglobulins and the complement proteins. The immunoglobulins are secreted by a subset of differentiated B lymphocytes termed plasma cells and bind antigens at binding sites formed by the hypervariable regions of the proteins (see Chapter 7). Once the antibody–antigen complex is formed, it must be cleared from the circulation. The complement system participates in this function. The complement system consisting of approximately 20 proteins becomes activated in either of two ways: The first is interaction with antigen–antibody complexes, and the second, specific for bacterial infections, is through interaction of bacterial cell polysaccharides with complement protein C3b. Activation of the complement system by either trigger results in a proteolytic activation cascade of the proteins of the complement system, resulting in the release of biologically active peptides or polypeptide fragments. These peptides mediate the inflammatory response, attract phagocytic cells to the area, initiate degranulation of granulocytes, and promote clearance of antigen–antibody complexes. Protease inhibitors in plasma serve to carefully control the inflammatory response. Activated neutrophils release lysosomal proteases from their granules that can attack elastin, the various types of collagen, and other extracellular matrix proteins. The plasma proteins ␣1-antiproteinase (␣1-antitrypsin) and ␣2-macroglobulin limit proteolytic damage by forming noncovalent complexes with the proteases, thereby inactivating them. However, the product of neutrophil myeloperoxidase, HOCl, inactivates the protease inhibitors, thereby ensuring that the proteases are active at the site of infection.

III. PLASMA PROTEINS MAINTAIN THE INTEGRITY OF THE CIRCULATORY SYSTEM Blood is lost from the circulation when the endothelial lining of the blood vessels is damaged and the vessel wall is partly or wholly severed. When this occurs, the subendothelial cell layer is exposed, consisting of the basement membrane of the endothelial cells and smooth muscle cells. In response to the damage, a barrier (the hemostatic plug, a fibrin clot) initiated by platelet binding to the damaged area is formed at the site of injury. Regulatory mechanisms limit clot formation to the site of injury and control its size and stability. As the vessel heals, the clot is degraded (fibrinolysis). Plasma proteins are required for these processes to occur.

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In spite of the importance of albumin in the maintenance of osmotic pressure in the blood, individuals who lack albumin (analbuminemia) have only moderate edema. Apparently, the concentration of other plasma proteins is increased to compensate for the lack of albumin. The frequency of analbuminemia is less than one per million individuals.

␣1-Antiproteinase (AAP) is the main serine protease inhibitor of human plasma. Individuals with a point mutation in exon 5 of the AAP gene, which results in a single amino acid substitution in the protein, have diminished secretion of AAP from the liver. These individuals are at increased risk for developing emphysema. When neutrophils degranulate in the lungs as part of the body’s defense against microorganisms, insufficient levels of AAP are present to neutralize the elastase and other proteases released. The excess proteolytic activity damages lung tissue, leading to loss of alveolar elasticity and emphysema. Methionine 358 of AAP is necessary for AAP binding to the proteases. Oxidation of this methionine destroys AAP’s protease-binding capacity. Cigarette smoke oxidizes Met-358, thereby increasing the risk of emphysema.

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SECTION VIII ■ TISSUE METABOLISM

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which antibodies to platelet glycoproteins are produced. Antibody binding to platelets results in their clearance by the spleen. An early symptom of the disorder is the appearance of small red spots on the skin (petechial hemorrhages) caused by blood leakage from capillaries. Minor damage to vascular endothelial cells is constantly being caused by mechanical forces related to blood flow. In patients with ITP, few platelets are available to repair the damage.

von Willebrand factor (vWF) is a large multimeric glycoprotein with a subunit molecular weight of 220,000 Da. Its size in the circulation ranges between 500 and 20,000 kDa, and its role in circulation is to stabilize factor VIII and protect it from degradation. The high-molecular-weight (HMW) forms are concentrated in the endothelium of blood vessels and are released in response to stress hormones and endothelial damage. HMW vWF released by the endothelium is cleaved by a specific metalloprotease in the serum, reducing the size of the circulating vWF. Large vWF multimers are more effective at forming complexes with platelets than are small vWF multimers. vWF deficiency is the most common cause of inherited bleeding disorders. Both platelet adherence and the clotting cascade are affected because levels of factor VIII are low. In the absence of vWF, factor VIII is rapidly cleared from the system. The vWF gene is large, covering approximately 180 kilobases and contains 52 exons. Multiple mutations are known with varying clinical presentations.

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A. Formation of the Hemostatic Plug 1.

THE PLATELET

Platelets are nonnucleated cells in the blood whose major role is to form mechanical plugs at the site of vessel injury and to secrete regulators of the clotting process and vascular repair. In the absence of platelets, leakage of blood from rents in small vessels is common. Platelets are derived from megakaryocytes in the bone marrow. Megakaryocytes differentiate from the hematopoietic stem cell. As the megakaryocyte matures, it undergoes synchronous nuclear replication without cellular division to form a cell with a multilobed nucleus and enlarged cytoplasm. At approximately the eight-nucleus stage, the cytoplasm becomes granular, and the platelets are budded off the cytoplasm. A single megakaryocyte gives rise to approximately 4,000 platelets. In the nonactivated platelet (a platelet that is not involved in forming a clot), the plasma membrane invaginates extensively into the interior of the cell, forming an open membrane (canalicular) system. Because the plasma membrane contains receptors and phospholipids that accelerate the clotting process, the canalicular structure substantially increases the membrane surface area that is potentially available for clotting reactions. The platelet interior contains microfilaments and an extensive actin/myosin system. Platelet activation in response to endothelial injury causes Ca2⫹-dependent changes in the contractile elements, which, in turn, substantially change the architecture of the plasma membrane. Long pseudopodia are generated, increasing the surface area of the membrane as clot formation is initiated. Platelets contain three types of granules. The first are electron-dense granules, which contain calcium, adenosine diphosphate (ADP), adenosine triphosphate (ATP), and serotonin. The second type of granule is the ␣-granule, which contains a heparin antagonist (heparin interferes with blood clotting; see the Biochemical Comments section), platelet-derived growth factor, ␤-thromboglobulin, fibrinogen, von Willebrand factor (vWF), and other clotting factors. The third type of granule is the lysosomal granule, which contains hydrolytic enzymes. During activation, the contents of these granules, which modulate platelet aggregation and clotting, are secreted. 2.

PLATELET ACTIVATION

Three fundamental mechanisms are involved in platelet function during blood coagulation: adhesion, aggregation, and secretion. Adhesion sets off a series of reactions termed platelet activation, which leads to platelet aggregation and secretion of platelet granule contents. The adhesion step refers primarily to the platelet-subendothelial interaction that occurs when platelets initially adhere to the sites of blood vessel injury (Fig. 45.1). Blood vessel injury exposes collagen, subendothelial matrix-bound vWF, and other matrix components. vWF is a protein synthesized in endothelial cells and megakaryocytes and is located in the subendothelial matrix, in specific platelet granules, and in the circulation bound to factor VIII. The platelet cell membrane contains glycoproteins (Gps) that bind to collagen and to vWF, causing the platelet to adhere to the subendothelium. Binding to collagen by GpIa (integrin ␣2␤1) causes the platelet to change its shape from a flat disc to a spherical cell. The latter extrudes long pseudopods, which promote platelet/ platelet interactions. Binding of subendothelial vWF by GpIb causes changes in the platelet membrane that expose GpIIb-IIIa (integrin ␣IIb␤3)-binding sites to fibrinogen and vWF. The initial adherence of platelets sets off a series of reactions (platelet activation) that result in more platelets being recruited and aggregated at the site of injury. After initial adherence, some of the platelets release the contents of their dense granules and their ␣-granules, with ADP release being of particular

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

GPIb

GPIIb GPIIIa

851

GPIa Platelet membrane

2 Adhesion

VWF

3

1

Exposed by initial adhesion events

Adhesion

Subendothelial collagen

FIG. 45.1. Adhesion of platelets to the subendothelial cell layer. (1) GpIa initially binds to the exposed collagen, which results in changes in the three-dimensional configuration of the complex, allowing GpIb to bind to von Willebrand factor (vWF) (2). (3) This second binding event exposes the GpIIb-GpIIIa complex, which also can bind to vWF and fibrinogen.

importance because ADP is a potent platelet activator. ADP released from the platelets and from damaged red blood cells binds to a receptor on the platelet membrane, which leads to the further unmasking of GpIIb-IIIa-binding sites. Aggregation of platelets cannot take place without ADP stimulation because ADP induces swelling of the activated platelets, promoting platelet/platelet contact and adherence. Fibrinogen is a protein that circulates in the blood and is also found in platelet granules. It consists of two triple helices held together with disulfide bonds (Fig. 45.2). Binding of fibrinogen to activated platelets is necessary for aggregation, providing, in part, the mechanism by which platelets adhere to one another. Cleavage of fibrinogen by thrombin (a protease that is activated by the coagulation cascade) produces fibrin monomers that polymerize and, together with platelets, form a “soft clot.” Thrombin itself is a potent activator of platelets, through binding to a specific receptor on the platelet surface.

Defects in GpIb cause a bleeding disorder known as Bernard–Soulier syndrome. Platelet aggregation is affected because of the inability of GpIb to adhere to subendothelial vWF.

B. The Blood Coagulation Cascade Thrombus (clot) formation is enhanced by thrombin activation, which is mediated by the complex interaction that constitutes the blood coagulation cascade. This cascade (Fig. 45.3) consists primarily of proteins that serve as enzymes or cofactors, which function to accelerate thrombin formation and localize it at the site of injury. These proteins are listed in Table 45.2. All of these proteins are present in the plasma as proproteins (zymogens). These precursor proteins are activated by cleavage of the polypeptide chain at one or more sites. The key to successful and appropriate thrombus formation is the regulation of the proteases that activate these zymogens. The proenzymes (factors VII, IX, X, XI, and prothrombin) are serine proteases that, when activated by cleavage, cleave the next proenzyme in the cascade. Because of the sequential activation, great acceleration and amplification of the response is achieved. That cleavage and activation have occurred is indicated by the addition of an “a” to the name of the proenzyme (e.g., factor IX is cleaved to form the active factor IXa). The cofactor proteins (tissue factor, factors V and VIII) serve as binding sites for other factors. Tissue factor is not related structurally to the other blood coagulation cofactors and is an integral membrane protein that does not require cleavage for active function. Factors V and VIII serve as procofactors, which, when activated by cleavage, function as binding sites for other factors. Two additional proteins that are considered part of the blood coagulation cascade, protein S and protein C, are regulatory proteins. Only protein C is

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Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by the formation in the circulation of microclots (microthrombi) consisting of aggregated platelets. The microthrombi collect in the microvasculature and damage red blood cells, resulting in hemolytic anemia. They also damage vascular endothelium, exposing collagen and releasing HMW vWF promoting more platelet aggregation. The subsequent depletion of platelets renders the patient susceptible to internal hemorrhage. Mortality in untreated TTP can approach 90%. Familial TTP is associated with mutations in the vWF-specific metalloprotease, although not all individuals with defective protease develop TTP. Sporadic cases of TTP are associated with the development of an antibody to the metalloprotease.

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SECTION VIII ■ TISSUE METABOLISM

A

Fibrinogen –

B

–

–

Site of thrombin attack

–

Fibrinogen H2O Thrombin

–

–

Fibrinopeptides

Fibrin monomer Aggregation

Soft clot of fibrin

FIG. 45.2. Cleavage of fibrinogen results in clot formation. A. Fibrinogen, the precursor protein of fibrin, is formed from two triple helices joined together at their N-terminal ends. The ␣,␤-peptides are held together by disulfide bonds, and the ␥-peptides are joined to each other by disulfide bonds. The terminal ␣,␤-peptide regions, shown in red, contain negatively charged glutamate and aspartate residues that repel each other and prevent aggregation. B. Thrombin, a serine protease, cleaves the terminal portions of fibrinogen that contain negative charges. The fibrin monomers can then aggregate and form a “soft” clot. The soft clot is subsequently cross-linked by another enzyme.

regulated by proteolytic cleavage, and when it is activated, it is itself a serine protease. Additionally, in response to collagen and thrombin, platelets release vasoconstrictors. Serotonin is released from the dense granules of the platelets, and the synthesis of thromboxane A2 is stimulated. This reduces blood flow to the damaged area. Platelet-derived growth factor, which stimulates proliferation of vascular cells, is also released into the environment surrounding the damage.

C. The Process of Blood Coagulation Activation of the blood coagulation cascade is triggered by the reaction of plasma proteins with the subendothelium at the same time that platelets are adhering to the subendothelial layer. Historically, two different pathways were discovered, one dependent on external stimuli (such as blunt trauma, which initiates the extrinsic

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

Intrinsic pathway

853

Extrinsic pathway Trauma

XI

XIa PL, Ca IX

VIIIa

PL, Ca

PL, Ca X V

VII

Tissue factor (III)

IXa VIII

PL, Ca

VIIa

X

Xa Va PL, Ca II Prothrombin

IIa Thrombin

I Fibrinogen XIII

Ia Fibrin aggregate (soft clot) XIIIa Cross-linked clot (hard clot)

FIG. 45.3. The blood coagulation cascade. Activation of clot formation occurs through interlocking pathways, termed the intrinsic and extrinsic pathways. The intrinsic pathway is activated when factor XI is converted to factor XIa by thrombin. The extrinsic pathway (external damage, such as a cut) is activated by tissue factor. The reactions designated by “PL, Ca” are occurring through cofactors bound to phospholipids (PLs) on the platelet and blood vessel endothelial cell surface in a Ca2⫹-coordination complex. Factors VIIa, IXa, Xa, XIa and thrombin are serine proteases. Note the positive feedback regulation of thrombin on the activation of proteases earlier in the cascade sequence (indicated by the dashed lines).

Table 45.2

Proteins of Blood Coagulation

Factor Coagulation factors I II III IV V VII VIII IX X XI XIII Regulatory proteins Thrombomodulin Protein C Protein S

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Descriptive Name

Function/Active Form

Fibrinogen Prothrombin Tissue factor Ca2⫹ Proaccelerin, labile factor Proconvertin Antihemophilia factor A Antihemophilia factor B, Christmas factor Stuart-Prower factor Plasma thromboplastin antecedent Fibrin-stabilizing factor

Fibrin Serine protease Receptor and cofactor Cofactor Cofactor Serine protease Cofactor Serine protease Serine protease Serine protease Ca2⫹-dependent transglutaminase

Endothelial cell receptor, binds thrombin Activated by thrombomodulin-bound thrombin; protease cofactor; binds activated protein C

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SECTION VIII ■ TISSUE METABOLISM

The use of an active-site serine to cleave a peptide bond is common to a variety of enzymes referred to as serine proteases. Serine proteases are essential for activating the formation of a blood clot from fibrin. Fibrin and many of the other proteins involved in blood coagulation are present in the blood as inactive precursors or zymogens, which must be activated by proteolytic cleavage. Thrombin, the serine protease that converts fibrinogen to fibrin, has the same aspartate–histidine–serine catalytic triad found in chymotrypsin and trypsin. Thrombin is activated by proteolytic cleavage of its precursor protein, prothrombin. The sequence of proteolytic cleavages leading to thrombin activation requires factor VIII, the blood clotting protein that is deficient in Sloe Klotter.

pathway) and one using internal stimuli (the intrinsic pathway). As our understanding of blood clotting has expanded, it has become obvious that these distinctions are no longer correct because there is overlap between the pathways, but the terms have persisted in the description of the pathways. In the case of external trauma, damaged tissues present tissue factor to the blood at the injured site, thereby triggering the extrinsic phase of blood coagulation. Circulating factor VII binds to tissue factor, which autocatalyzes its own activation to factor VIIa. Factor VIIa then activates factor X (to Xa) in the extrinsic pathway and factor IX (to IXa) in the intrinsic pathway. Factor IXa, as part of the intrinsic pathway, also activates factor X. Therefore, activation of both the extrinsic and intrinsic pathways results in the conversion of factor X to factor Xa. All of these conversions require access to membranes and calcium; the platelet membrane, which had adhered to the damaged site, is used as a scaffold for the activation reactions to occur. The ␥-carboxylated clotting proteins are chelated to membrane surfaces via electrostatic interactions with calcium and negatively charged phospholipids of the platelet membrane. The protein cofactors VIIIa and Va serve as sites for assembling enzyme–cofactor complexes on the platelet surface, thereby accelerating and localizing the reaction. The result is thrombin formation, which augments its own formation by converting factors V, VIII, and XI into activated cofactors and stimulating platelet degranulation. The initial activation of prothrombin is slow because the activator cofactors, VIIIa and Va, are present only in small amounts. However, once a small amount of thrombin is activated, it accelerates its own production by cleaving factors V and VIII to their active forms. Note that these factors are in the intrinsic pathway. The intrinsic pathway is thought to sustain the coagulation response initiated by the extrinsic pathway. The major substrate of thrombin is fibrinogen, which is hydrolyzed to form fibrin monomers that undergo spontaneous polymerization to form the fibrin clot. This is considered a “soft” clot because the fibrin monomers are not cross-linked. Cross-linking requires factor XIIIa, which is activated by thrombin cleavage of factor XIII. 1.

CROSS-LINKING OF FIBRIN

Factor XIIIa catalyzes a transamidation reaction between glutamine and lysine side chains on adjacent fibrin monomers. The covalent cross-linking takes place in three dimensions, creating a strong network of fibers that is resistant to mechanical and proteolytic damage. This network of fibrin fibers traps the aggregated platelets and other cells, forming the clot that plugs the vent in the vascular wall (Fig. 45.4). Factor XIIIa is the only enzyme in the blood coagulation cascade that is not a serine protease.

lys

lys

CH2

CH2

CH2

CH2

CH2

CH2

2.

CH2

CH2

In several of the essential steps in the blood coagulation cascade, the activated protease is bound in a complex attached to the surfaces of the platelets that have aggregated at the site of injury. Factors VII, IX, X, and prothrombin contain a domain in which one or more glutamate residues are carboxylated to ␥-carboxyglutamate in a reaction that requires vitamin K (Fig. 45.5). Prothrombin and factor X both contain 10 or more ␥-carboxyglutamate residues that bind Ca2⫹. The Ca2⫹ forms a coordination complex with the negatively charged platelet membrane phospholipids and the ␥-carboxyglutamates, thereby localizing the complex assembly and thrombin formation to the platelet surface. Cofactor Va contains a binding site for both factor Xa and prothrombin, the zymogen substrate of factor Xa. Upon binding to the factor Va–platelet complex, prothrombin undergoes a conformational change, rendering it more susceptible to enzymatic cleavage. Binding of factor Xa to the factor Va–prothrombin–platelet complex allows the prothrombin-to-thrombin conversion. Complex assembly

XIIIa

NH2 + O

NH NH3

NH2 C CH2 CH2

O

C CH2 CH2 Gln

Gln

FIG. 45.4. The transamidation reaction catalyzed by factor XIIIa, transglutaminase. This reaction cross-links fibrin monomers, allowing “hard” clots to form.

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FACTOR COMPLEXES

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

A

Vitamin K1 (Phylloquinone) CH3

O CH3

R R

CH

CH2

C

CH2

855

CH3 (CH2

CH2

CH

CH2)3

H

Vitamin K2 (Menaquinone)

O CH3 R

(CH2

CH

C

CH2)8

H

Vitamin K3 (Menadione) R H

B CH2 CH2

CH2 Prozymogen

C O– O Glutamate residue

C

Carboxylated zymogen

CO2 Vitamin K–dependent

O

carboxylase

Vitamin KH2 (hydroquinone)

C

H C O

O– O–

O2

Vitamin K epoxide O

OH

CH3

CH3

O R

R O

OH

O CH3

R2

R1H2 R1

Vitamin K R2H2 reductase

R O

Vitamin K epoxide reductase

FIG. 45.5. A. Structures of vitamin K derivatives. Phylloquinone is found in green leaves, and intestinal bacteria synthesize menaquinone. Humans convert menadione to a vitamin K active form. B. Vitamin K-dependent formation of ␥-carboxyglutamate residues. Thrombin, factor VII, factor IX, and factor X are bound to their phospholipid activation sites on cell membranes by Ca2⫹. The vitamin K-dependent carboxylase, which adds the extra carboxyl group, uses a reduced form of vitamin K (KH2) as the electron donor and converts vitamin K to an epoxide. Vitamin K epoxide is reduced, in two steps, back to its active form by the enzymes vitamin K epoxide reductase and vitamin K reductase.

accelerates the rate of this conversion 10,000- to 15,000-fold as compared with noncomplex formation. Factor VIIIa forms a similar type of complex on the surface of activated platelets but binds factor IXa and its zymogen substrate, factor X. Tissue factor works slightly different because it is an integral membrane protein. However, once it is exposed by injury, it also binds factor VIIa and initiates complex formation. Complex assembly has two physiologically important consequences. First, it enhances the rate of thrombin formation by as much as several hundred thousandfold, enabling the clot to form rapidly enough to preserve hemostasis. Second, such explosive thrombin formation is localized to the site of vascular injury at which the negatively charged phospholipids are exposed. From our knowledge of the location of such phospholipids in cellular and subcellular organelle membranes, these surface-binding sites are only exposed at an injury site in which cell rupture exposes the internal membrane surface (recall that certain phospholipids face only the cytoplasm; if these lipids are now exposed to the environment, substantial cell damage must have occurred).

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SECTION VIII ■ TISSUE METABOLISM

O

O

OH CH2 O

CH3 Warfarin

Warfarin is a commonly used rat poison and thus is occasionally encountered in emergency departments in cases of accidental poisoning. It is effective as a rat poison because it takes many hours to develop pathologic symptoms, which allows one poisoned trap to kill more than one rat.

Deficiency in the amount or functionality of protein C or protein S increases the risk of venous thromboembolism. Individuals who are homozygous for these mutations do not survive the neonatal period unless they are given replacement therapy.

VITAMIN K REQUIREMENT FOR BLOOD COAGULATION

The formation of the ␥-carboxyglutamate residues on blood coagulation factors takes place in the hepatocyte before release of the protein. Within the hepatocyte, vitamin K (which is present in the quinone form) is reduced to form vitamin KH2 by a microsomal quinone reductase (see Fig. 45.5). Vitamin KH2 is a cofactor for carboxylases that add a carboxyl group to the appropriate glutamate residues in the proenzyme to form the carboxylated proenzyme (e.g., prothrombin). In the same reaction, vitamin K is converted to vitamin K epoxide. To recover active vitamin KH2, vitamin K is first reduced to the quinone form by vitamin K epoxide reductase and then to the active hydroquinone form.

D. Regulation through Feedback Amplification and Inhibition Once the formation of the clot (thrombus) begins, clot formation is accelerated in an almost explosive manner by several processes that are collectively termed feedback amplification. 1.

CH

C

3.

THE ROLE OF THROMBIN IN REGULATION

Thrombin has both a prothrombotic regulatory role (feedback amplification) and an antithrombotic regulatory role (feedback inhibition). The prothrombotic action is initiated when thrombin stimulates its own formation by activating factors V, VIII, and XI, thereby accelerating the rate of clot formation (see Fig. 45.3). Thrombin also promotes clot formation by activating platelet aggregation, stimulating the release of factor VIII from vWF and cleaving factor XIII to factor XIIIa. Antithrombotic effects of thrombin result from its binding to an endothelial cell receptor called thrombomodulin (Fig. 45.6). Thrombomodulin abolishes the clotting function of thrombin and allows thrombin to activate protein C, which has anticoagulant effects. 2.

PROTEINS S AND C

Protein C and its cofactor protein S serve to suppress the activity of the coagulation cascade. After activation, protein C forms a complex with protein S. Protein S anchors the activated protein C (APC) complex to the clot through Ca2⫹/␥-carboxyglutamate binding to platelet phospholipids. The APC destroys the active blood coagulation cofactors factor VIIIa and Va by proteolytic cleavage, decreasing the production of thrombin. The APC also stimulates endothelial cells to increase secretion of the prostaglandin I2 (PGI2), which reduces platelet aggregation.

Activated protein C–protein S complex destroys Protein S Factors Va and VIIIa Ca2+ Activated protein C Thrombin Protein C othel E nd

ial cell membrane

PL

su rfa ce

Warfarin (Coumadin) is a slow- and long-acting blood anticoagulant with a structure resembling that of vitamin K. The structural similarity allows the compound to compete with vitamin K and prevent ␥-carboxylation of glutamate residues in factors II, VII, IX, X, and proteins C and S. The noncarboxylated blood clotting protein precursors increase in both the blood and plasma, but they are unable to promote blood coagulation because they cannot bind calcium and thus cannot bind to their phospholipid membrane sites of activation.

Pla tele t

856

Thrombomodulin

FIG. 45.6. Antithrombotic effects of thrombin. Thrombin, bound to thrombomodulin on the endothelial cell surface, activates protein C. Activated protein C (APC) in complex with protein S binds to the platelet membrane, and the activated complex destroys factors Va and VIIIa, thereby inhibiting the coagulation cascade.

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3.

SERPINS

Many proteases of the blood coagulation enzyme system are serine proteases. Because uncontrolled proteolytic activity would be destructive, modulating mechanisms control and limit intravascular proteolysis. The serine protease inhibitors (serpins) are a group of naturally occurring inhibitory proteins that are present in the plasma at high concentration (approximately 10% of the plasma proteins are serpins). At least eight major inhibitors have been found that share a common mechanism of action and inhibit proteases involved in coagulation and clot dissolution (fibrinolysis). Each inhibitor possesses a reactive site that appears to be an ideal substrate for a specific serine protease and thus acts as a trap for that protease. The bound serine protease attacks a peptide bond located at a critical amino acid residue within the serpin and forms a tight enzyme–inhibitor complex. The activity of thrombin is controlled by the serpin antithrombin III (ATIII). Regulation of blood coagulation at the level of thrombin is critical because this enzyme affects the pathways of both coagulation and fibrinolysis (see Section III.F). One molecule of ATIII irreversibly inactivates one molecule of thrombin through reaction of an arginine residue in ATIII with the active-site serine residue of thrombin. ATIII–thrombin complex formation is markedly enhanced in the presence of heparin. Heparin is a glycosaminoglycan (see Chapter 49) found in the secretory granules of mast cells and in the loose connective tissue around small vascular beds. Heparin binds to lysyl residues on ATIII and dramatically accelerates its rate of binding to thrombin. This is because of an allosteric alteration in ATIII such that the position of the critical arginine residue of ATIII is more readily available for interaction with thrombin. The formation of the ATIII–thrombin complex releases the heparin molecule so that it can be reused and, therefore, the function of heparin is catalytic. Thrombin that is attached to a surface, for example, to thrombomodulin on the endothelial cell membrane, is no longer participating in clot formation and is not readily attacked by ATIII or the ATIII–heparin complex. The ATIII–heparin complex also can inactivate the serine protease factors XIIIa, XIa, IXa, and Xa but has no effect on factor VIIa or APC.

857

In European populations, a point mutation in the factor V gene (factor V Leiden) causes the replacement of an arginine with a glutamine in the preferred site for cleavage by activated protein C (APC), rendering factor Va Leiden resistant to APC. Heterozygous individuals have a sixfold to eightfold increased risk of deep vein thromboses, and homozygous individuals have a 30- to 140-fold increased risk. The factor V Leiden mutation does not appear to be associated with increased risk of arterial thrombosis, such as myocardial infarction, except in young women who smoke. Genetic studies suggest that the factor V Leiden mutation arose after the separation of the European, Asian, and African populations. The frequency of this variant indicates that it conferred some selective advantage at one time. In the developed world, inherited APC resistance is the most prevalent risk factor for familial thrombotic disease.

E. Thromboresistance of Vascular Endothelium Endothelial cells of blood vessels provide a selectively permeable barrier between the circulating blood and the tissues. The normal endothelial cell lining neither activates coagulation nor supports platelet adhesion; thus, it is called a nonthrombogenic surface. The thromboresistance of normal vascular endothelium is contributed to by several properties. Endothelial cells are highly negatively charged, a feature that may repel the negatively charged platelets. Endothelial cells synthesize PGI2 and nitric oxide, vasodilators, and powerful inhibitors of platelet aggregation. PGI2 synthesis is stimulated by thrombin, epinephrine, and local vascular injury. Endothelial cells also synthesize two cofactors that each inhibits the action of thrombin, thrombomodulin, and heparan sulfate. Heparan sulfate is a glycosaminoglycan similar to heparin that potentiates ATIII but not as efficiently. The inactivation of thrombin is accelerated by heparan sulfate present on the endothelial cell surface. Thus, the intact endothelium has the capability of modifying thrombin action and inhibiting platelet aggregation.

F. Fibrinolysis After successful formation of a hemostatic plug, further propagation of the clot must be prevented. This is accomplished in part by switching off blood coagulation and in part by turning on fibrinolysis. Fibrinolysis involves the degradation of fibrin in a clot by plasmin, a serine protease that is formed from its zymogen, plasminogen. Plasminogen is a circulating serum protein that has a high affinity for fibrin, promoting the incorporation of plasminogen in the developing clot. The activity of plasminogen is mediated by proteins known as plasminogen activators.

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SECTION VIII ■ TISSUE METABOLISM

Both streptokinase and tissue plasminogen activator (tPA) have been approved for the treatment of myocardial infarction. The rationale is that the enzyme will lead to clot dissolution, thereby restoring blood flow to the heart. The use of both drugs reduces mortality. Although there are more side effects associated with the use of streptokinase, it is substantially cheaper than tPA.

The conversion of plasminogen to plasmin by plasminogen activators can occur both in the liquid phase of the blood and at the clot surface; however, the latter process is by far more efficient. APC, in addition to turning off the blood coagulation cascade, also stimulates the release of plasminogen activator from tissues (tPA, tissue plasminogen activator) and simultaneously inactivates an inhibitor of plasminogen activator, PAI-1. Plasminogen activator release can lead to plasmin formation in the circulation. However, the circulating plasmin is rapidly inactivated by binding with ␣2-antiplasmin, a circulating protease inhibitor. Clot-bound plasmin is not readily inactivated by ␣2-antiplasmin. Thus, plasminogen binding to fibrin facilitates its activation to plasmin, protects it from blood serpins, and localizes it on the fibrin substrate for subsequent efficient proteolytic attack. This mechanism allows for dissolution of fibrin in pathologic thrombi or oversized hemostatic plugs and at the same time prevents degradation of fibrinogen in the circulating blood. Two endogenous plasminogen activators are most important; both are synthesized in a variety of cells. tPA is produced chiefly by the vascular endothelial cells, has a high binding affinity for fibrin, and plays a major role in fibrinolysis. Single-chain urokinase (U-PA) is synthesized in most cells and tissues and has a moderate affinity for fibrin. Streptokinase, the bacterial exogenous plasminogen activator from ␤-hemolytic streptococci, is not an enzyme but an allosteric modifier of human plasminogen that allows an autocatalytic reaction such that plasmin is formed. In vivo, physical stress, hypoxia, and large numbers of low-molecularweight (LMW) organic compounds promote increased synthesis and release of tPA and U-PA from tissues into the blood. The balance between release of plasminogen activators, the availability of fibrin, and inhibitors of the activators and plasmin determine regulation of the fibrinolytic response, as indicated in Figure 45.7.

G. Regulation of Fibrinolysis Antiactivators regulate interaction of plasminogen in blood with plasminogen activators in a dynamic equilibrium. Even if minute amounts of plasmin are generated (e.g., after release of vascular plasminogen activator after stress), the enzyme is probably inactivated by antiplasmin. Upon activation of the blood coagulation system, a fibrin clot is formed, which not only strongly binds tPA and plasminogen from blood but also accelerates the rate of plasmin activation. The clot-bound plasmin is protected from inhibitors while attached to fibrin. The enzyme is inactivated by ␣2-antiplasmin and ␣2-macroglobulin after proteolytic dissolution of fibrin and its liberation into the liquid phase of blood. Thus, the fibrin network catalyzes both initiation and regulation of fibrinolysis.

streptokinase

Plasminogen +

tPA PAI-1 U-PA

streptokinaseplasminogen complex

– +

Plasmin –

Fibrin

α2-antiplasmin

Fibrin degradation products

FIG. 45.7. Regulation of plasmin activation. Plasminogen can be activated by either tissue plasminogen activator (tPA) or single-chain urokinase (U-PA) (䊝). PAI-1 blocks tPA action (䊞). Streptokinase binding to plasminogen allows autocatalysis to form plasmin. Circulating ␣2-antiplasmin blocks (䊞) the activity of any soluble plasmin that may be in the blood.

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

859

CLINICAL COMMENTS Sloe Klotter. Sloe Klotter has hemophilia A, the most frequently encountered serious disorder of blood coagulation in humans, occurring in 1 in every 10,000 males. The disease is transmitted with an X-linked pattern of inheritance. The most common manifestations of hemophilia A are those caused by bleeding into soft tissues (hematomas) such as muscle or into body spaces such as the peritoneal cavity, joint spaces, or the lumen of the gastrointestinal tract. When bleeding occurs repeatedly into joints (hemarthrosis), the joint may eventually become deformed and immobile. In the past, bleeding episodes have been managed primarily by administration of factor VIII, sometimes referred to as antihemophilia cofactor. Unfortunately, the concentration of factor VIII in plasma is quite low (0.3 nM, compared with 8,800 nM for fibrinogen), requiring that it be prepared from multiple human donors. Before donor screening and virus inactivation procedures during preparation essentially eliminated transmission with blood transfusions, ⬎50% of patients with hemophilia treated with factor VIII during the 1980s in Western Europe or North America became infected with HIV and hepatitis C. Recombinant factor VIII is now available for clinical use and mitigates the threat of HIV/hepatitis C transmission in these patients.

Another X-linked bleeding disorder is hemophilia B, which is caused by mutations in the gene for factor IX. Lack of factor IX activity leads to an inability to convert prothrombin to thrombin and impaired clotting.

BIOCHEMICAL COMMENTS Drugs That Inhibit Blood Coagulation. Several drugs have been developed that inhibit the blood coagulation cascade. Such drugs are useful in cases in which patients develop spontaneous thrombi, which, if left untreated, would result in a fatal pulmonary embolism. There are three major classes of such drugs: the heparins, vitamin K antagonists, and specific inhibitors of thrombin. Heparin binds to and activates antithrombin III (ATIII), which leads to thrombin inactivation. ATIII also blocks the activity of factors VIIIa, IXa, Xa, and XIa. Heparin can be administered in either of two forms: unfractionated or highmolecular-weight (HMW) heparin and fractionated or LMW heparin. HMW heparin is a heterogenous mixture of glycosaminoglycans, with an average chain length of 45 monosaccharides with an average molecular weight of 15 kDa (the range is 3 to 30 kDa). LMW heparins are fragments of HMW heparin, containing fewer than 18 monosaccharides with an average molecular weight of 4 to 5 kDa. HMW heparin binds to plasma proteins and cell surfaces in addition to its prime target, ATIII. Because different individuals synthesize different levels of plasma proteins, the use of this form of heparin as an anticoagulant requires constant monitoring of the patient to ensure that the correct dosage has been given so that spontaneous thrombi do not develop but not so much that spontaneous bleeding occurs. LMW heparin has fewer nonspecific interactions than HMW heparin, and its effects are easier to predict on patients, so constant monitoring is not required. A major complication of heparin therapy is heparin-induced thrombocytopenia (HIT, excessive blood clotting with a reduction in the number of circulating platelets). This unexpected result of heparin treatment is caused by heparin binding to a platelet protein, platelet factor 4 (PF4), which induces a conformational change in PF4 such that the immune system believes the complex is foreign. Thus, antibodies are developed against the heparin–PF4 complex. When the antibodies bind to the platelets, the platelets become activated, and thrombi develop. Treatment consists of removing the heparin and using a different form of antithrombotic agent. The classic vitamin K antagonist is warfarin. Warfarin acts by blocking the vitamin K reductase enzymes required to regenerate active vitamin K

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SECTION VIII ■ TISSUE METABOLISM

A

HO

COO– O

OSO3– O OH

OSO3– O O

OH

O NH

OSO3–

OH

COO– OH

O

OSO3

O

OSO3–

NH

–

OSO3– O

O

OSO3–

OCH3 NH OSO3–

B AT III

AT III

F

AT III

Xa

F

Xa

F

FIG. 45.8. A. Structure of fondaparinux. B. Mechanism of fondaparinux action. The drug (shown in blue) binds to ATIII, which induces a conformational change so that factor Xa can now bind to ATIII. Once factor Xa binds and is inactivated, the drug is released and can activate another molecule of ATIII.

(see Fig. 45.5). This results in reduced ␥-carboxylation of factors II, VII, IX, and X. In the absence of ␥-carboxylation, the factors cannot bind calcium nor form the complexes necessary for the coagulation cascade to be initiated. However, warfarin also blocks the activity of proteins S and C, so both blood clotting and the regulation of clotting are impaired by warfarin administration. Both heparin and warfarin therapy suffer from their lack of specificity, so drugs specific for single steps in the blood coagulation pathway have been sought and identified. Analysis of heparin potentiation of factor Xa binding to ATIII showed that a unique pentasaccharide sequence was required. An appropriate pentasaccharide, named fondaparinux, was developed that would specifically enhance ATIII interactions with factor Xa (Fig. 45.8). Fondaparinux stimulates the binding of ATIII to factor Xa by 300-fold and is specific for factor Xa inhibition. Fondaparinux does not affect thrombin or platelet activity, and it is not an activating agent of platelets. Because fondaparinux does not bind to PF4, HIT is not a complication with this therapy. Direct thrombin inhibitors are based on the hirudin molecules, which were initially discovered in leeches and other blood-sucking organisms. These organisms would not be able to feed if the blood clotted at the site of the puncture wound, so the organisms secrete thrombin inhibitors to prevent clotting from occurring. Hirudin treatment itself is dangerous in that formation of the hirudin–thrombin complex is irreversible, and use of the drug requires constant monitoring of the patient. Thus, to overcome this problem, rational drug design based on the hirudin structure was used, and a synthetic 20-amino acid peptide known as bivalirudin was synthesized. This agent has a high binding affinity and specificity for thrombin although its effects on thrombin are transient (not irreversible), making this a safer agent for long-term use. Key Concepts • • • •

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The plasma contains water, nutrients, metabolites, hormones, electrolytes, and proteins. Plasma proteins provide osmotic pressure to maintain fluid balance between the tissues and the blood. The plasma proteins provide transport for many molecules and also aid in immune defense. The plasma proteins, in association with platelets, maintain the integrity of the circulatory system. Platelets form mechanical plugs at the site of vessel injury and secrete regulators of the blood clotting process. Platelets become activated when bound to a site of injury, which leads to more platelet binding and aggregation at the injured site. Circulating fibrinogen also binds to the activated platelets at sites of injury.

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CHAPTER 45 ■ BLOOD PLASMA PROTEINS, COAGULATION, AND FIBRINOLYSIS

•

•

861

Clot formation is carefully regulated such that overclotting (thrombosis) or underclotting (bleeding) does not occur. The clotting cascade consists of a series of protease activation steps leading to the activation of thrombin, which converts fibrinogen to fibrin. Thrombin also activates transglutaminase, which cross-links the fibrin and leads to “hard clot” formation. Proteins S and C regulate the clotting cascade and are activated by thrombin. In order to form a clot, protein complexes must adhere to the activated platelets, which occurs via ␥-carboxyglutamate binding to calcium and platelet membranes. Serpins are serine protease inhibitors; the serpin antithrombin III (ATIII) aids in regulating blood coagulation by modulating thrombin activity. Heparin enhances the interaction of thrombin with ATIII. Plasmin, the active product of plasminogen, is the only protease that can dissolve fibrin clots. Hemophilia A is caused by a lack of factor VIII, an essential factor required for thrombin activation. Diseases discussed in this chapter are summarized in Table 45.3.

Table 45.3

Diseases Discussed in Chapter 45

Disease or Disorder

Environmental or Genetic

Comments

Factor V Leiden

Genetic

Factor V is altered such that it cannot be inactivated by activated protein C, leading to hypercoagulation and deep vein thrombosis (excessive clotting).

Hemophilia B

Genetic

Lack of factor IX in the blood clotting cascade such that clots cannot be formed.

Hemophilia A

Genetic

Lack of factor VIII in blood clotting cascade, leading to a bleeding disorder (reduced clot formation).

␣1-Antiproteinase deficiency

Genetic

Lack of ␣1-antitrypsin leads to elastase destruction of lung tissue, progressing to emphysema and chronic obstructive pulmonary disease (COPD).

Kwashiorkor

Environmental

Protein deficiency in a caloriesufficient diet leads to a reduction in circulating blood proteins, resulting in edema.

Thrombocytopenic purpura (TPP)

Both

Formation of microscopic thrombi, which lead to hemolysis and organ failure if untreated. The idiopathic type may be caused by autoimmune destruction of platelets.

vWF deficiency

Both

A defect in von Willebrand factor, a necessary factor for initiation of the blood clotting cascade. In its absence, clotting will not occur, and bleeding disorders will result.

Bernard-Soulier syndrome

Genetic

Lack of GpIb, the receptor for von Willebrand factor (vWF). Leads to lack of function of vWF and a bleeding disorder as the coagulation cascade cannot be fully initiated.

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SECTION VIII ■ TISSUE METABOLISM

REVIEW QUESTIONS—CHAPTER 45

1.

2.

3.

The edema observed in patients with noncalorie protein malnutrition is caused by which of the following? A. Loss of muscle mass B. Ingestion of excess carbohydrates C. Increased fluid uptake D. Reduced protein synthesis in the liver E. Increased production of ketone bodies A recent surgery patient receiving warfarin therapy was found to be bleeding internally. The clotting process is impaired in this patient primarily because of which of the following? A. Inability of the liver to synthesize clotting factors B. Specific inhibition of factor XIII activation C. Inability to form clotting factor complexes on membranes D. Reduction of plasma calcium levels E. Enhancement of protein C activity An inactivating mutation in which of the following proenzymes would be expected to lead to thrombosis, uncontrolled blood clotting? A. Factor XIII B. Prothrombin

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C. Protein C D. Factor VIII E. Tissue factor 4.

Classical hemophilia A results in an inability to directly activate which of the following factors? A. Factor II B. Factor IX C. Factor X D. Protein S E. Protein C

5.

Hemophilia B results in an inability to directly activate which of the following factors? A. Factor II B. Factor IX C. Factor X D. Protein S E. Protein C

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46

Liver Metabolism

The liver is strategically interposed between the general circulation and the digestive tract. It receives 20% to 25% of the volume of blood leaving the heart each minute (the cardiac output) through the portal vein (which delivers absorbed nutrients and other substances from the gastrointestinal tract to the liver) and through the hepatic artery (which delivers blood from the general circulation back to the liver). Potentially toxic agents absorbed from the gut or delivered to the liver by the hepatic artery must pass through this metabolically active organ before they can reach the other organs of the body. The liver’s relatively large size (approximately 3% of total body weight) allows extended residence time in the liver for nutrients to be properly metabolized as well as for potentially harmful substances to be detoxified and prepared for excretion into the urine or feces. Among other functions, therefore, the liver, along with the kidney and gut, is an excretory organ equipped with a broad spectrum of detoxifying mechanisms. It can, for example, carry out metabolic conversion pathways and use secretory systems that allow the excretion of potentially toxic compounds. Concurrently, the liver contains highly specific and selective transport mechanisms for essential nutrients that are required not only to sustain its own energy but also to provide physiologically important substrates for the systemic needs of the organism. In addition to the myriad of transport processes within the sinusoidal and canalicular plasma membrane sheets, intracellular hepatocytic transport systems exist in organelles such as endosomes, mitochondria, lysosomes, as well as the nucleus. The sequential transport steps carried out by these organelles include (1) uptake, (2) intracellular binding and sequestration, (3) metabolism, (4) sinusoidal secretion, and (5) biliary excretion. The rate of hepatobiliary transport is determined, in part, by the rate of activity of each of these steps. The overall transport rate is also determined by such factors as hepatic blood flow, plasma protein binding, and the rate of canalicular reabsorption. The various aspects of the major metabolic processes performed by the liver have been discussed in greater detail elsewhere in this book. These previous sources will be referred to as the broad spectrum of the liver’s contributions to overall health and diseases are described.

863

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THE WAITING ROOM Jean Ann Tonich’s family difficulties continued and in spite of a period of sobriety that lasted 6 months, she eventually started drinking increasing amounts of gin again in an effort to deal with her anxieties. Her appetite for food declined slowly as well. She gradually withdrew from much of the social support system that her doctors and friends had attempted to build during her efforts for rehabilitation. Upper mid-abdominal pain became almost constant, and she noted increasing girth and distention of her abdomen. Early one morning, she was awakened in excruciating pain in her upper abdomen. She vomited a dark-brown “coffee ground” material followed by copious amounts of bright red blood. She called a friend, who rushed her to the hospital emergency room. Amy Biasis, a 23-year-old missionary, was brought to the hospital emergency department complaining of the abrupt onset of fever, chills, and severe pain in the right upper quadrant of her abdomen. The pain was constant and radiated to her right shoulder top. She vomited undigested food twice in the hour before arriving at the emergency department. This did not relieve her pain. Her medical history indicated that, while serving as a missionary in western Belize, Central America, 2 months earlier, she had a 3-day illness that included fever, chills, and mild but persistent diarrhea. A friend of Amy’s there, a medical missionary, had given her an unidentified medication for 7 days. Amy’s diarrhea slowly resolved, and she felt well again until her current abdominal symptoms began. On physical examination, she appeared toxic and had a temperature of 101°F. She was sweating profusely. Her inferior-anterior liver margin was palpable three fingerbreadths below the right rib cage, suggestive of an enlarged liver. The liver edge was rounded and tender. Gentle fist percussion of the lower posterior right rib cage caused severe pain. Routine laboratory studies were ordered, and a computed tomogram (CT) of the upper abdomen was scheduled to be done immediately.

I.

Liver lobule To central hepatic vein

S i n u s o i d

Bile canniculus

Hepatocyte

Bile duct Endothelial cells

Portal vein

FIG. 46.1.

Hepatic artery

Schematic view of liver anatomy.

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LIVER ANATOMY

The human liver consists of two lobes, each containing multiple lobules and sinusoids. The liver receives 75% of its blood supply from the portal vein, which carries blood returning to the heart from the small intestine, stomach, pancreas, and spleen. The remaining 25% of the liver’s blood supply is arterial, carried to the liver by the hepatic artery. Blood from both the portal vein and hepatic artery empty into a common conduit, mixing their contents as they enter the liver sinusoids (Fig. 46.1). The sinusoids are expandable vascular channels that run through the hepatic lobules. They are lined with endothelial cells that have been described as “leaky” because, as blood flows through the sinusoids, the contents of the plasma have relatively free access to the hepatocytes, which are located on the other side of the endothelial cells. The liver is also an exocrine organ, secreting bile into the biliary drainage system. The hepatocytes secrete bile into the bile canniculus, whose contents flow parallel to that in the sinusoids but in the opposite direction. The canniculi empty into the bile ducts. The lumina of the bile ducts then fuse, forming the common bile duct. The common duct then releases bile into the duodenum. Some of the liver’s effluent is stored in the gallbladder and discharged into the duodenum postprandially to aid in digestion. The entire liver surface is covered by a capsule of connective tissue that branches and extends throughout the liver. This capsule provides support for the blood

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vessels, lymphatic vessels, and bile ducts that permeate the liver. In addition, this connective tissue sheet subdivides the liver lobes into the smaller lobules.

II. LIVER CELL TYPES The primary cell type of the liver is the hepatocyte. Hepatocytes, also known as hepatic parenchymal cells, form the liver lobules. Eighty percent of the liver volume is composed of hepatocytes, but only 60% of the total number of cells in the liver are hepatocytes. The other 40% of the cells are nonparenchymal cells and constitute the lining cells of the walls of the sinusoids. The lining cells comprise the endothelial cells, Kupffer cells, and hepatic stellate cells. In addition, intrahepatic lymphocytes, which include pit cells (liver-specific natural killer cells) are also present in the sinusoidal lining.

A. Hepatocytes The hepatocyte is the cell that carries out the many functions of the liver. Almost all pathways of metabolism are represented in the hepatocyte, and these pathways are controlled through the actions of hormones that bind to receptors located on the plasma membrane of their cells. Although hepatocytes are normally quiescent cells with low turnover and a long life span, they can be stimulated to grow if damage occurs to other cells in the liver. The liver mass has a relatively constant relationship to the total body mass of adult individuals. Deviation from the normal or optimal ratio (caused, for example, by a partial hepatectomy or significant hepatic cell death or injury) is rapidly corrected by hepatic growth caused by a proportional increase in hepatocyte replication.

The reports of Amy Biasis’s initial laboratory studies showed an elevation in her serum hepatic transaminases, her serum alkaline phosphatases, as well as her serum total bilirubin level.

B. Endothelial Cells The sinusoidal endothelial cells constitute the lining cells of the sinusoid. Unlike endothelial cells in other body tissues, these cells contain fenestrations with a mean diameter of 100 nm. They do not, therefore, form a tight basement membrane barrier between themselves and the hepatocytes. In this way, they allow free diffusion of small molecules to the hepatocytes but not of particles the size of chylomicrons (chylomicron remnants, however, which are smaller than chylomicrons, do have free passage to the hepatocyte). The endothelial cells are capable of endocytosing many ligands and also may secrete cytokines when appropriately stimulated. Because of their positioning, lack of tight junctions, and absence of a tight basement membrane, the liver endothelial cells do not present a significant barrier to the movement of the contents of the sinusoids into hepatocytes. Their fenestrations or pores further promote the free passage of blood components through this membrane into the liver parenchymal cells.

C. Kupffer Cells Kupffer cells are located within the sinusoidal lining. They contain almost onequarter of all the lysosomes of the liver. The Kupffer cells are tissue macrophages with both endocytotic and phagocytic capacity. They phagocytose many substances such as denatured albumin, bacteria, and immune complexes. They protect the liver from gut-derived particulate materials and bacterial products. On stimulation by immunomodulators, these cells secrete potent mediators of the inflammatory response and play a role in liver immune defense through the release of cytokines that lead to the inactivation of substances considered foreign to the organism. The Kupffer cells also remove damaged erythrocytes from the circulation.

D. Hepatic Stellate Cells The stellate cells are also called perisinusoidal or Ito cells. There are approximately 5 to 20 of these cells per 100 hepatocytes. The stellate cells are lipid-filled cells and also serve as the primary storage site for vitamin A. They also control the turnover

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The CT scan of Amy Biasis’s upper abdomen showed an elevated right hemidiaphragm as well as several cystic masses in her liver, the largest of which was located in the superior portion of the right lobe. Her clinical history as well as her history of possible exposure to various parasites while working in a part of Belize, Central America, that is known to practice substandard sanitation, prompted her physicians to order a titer of serum antibodies against the parasite Entamoeba histolytica, in addition to measuring serum antibodies against other invasive parasites.

of hepatic connective tissue and extracellular matrix and regulate the contractility of the sinusoids. When cirrhosis of the liver is present, the stellate cells are stimulated by various signals to increase their synthesis of extracellular matrix material. This, in turn, diffusely infiltrates the liver, eventually interfering with the function of the hepatocytes.

E. Pit Cells The hepatic pit cells, also known as liver-associated lymphocytes, are natural killer cells, which are a defense mechanism against the invasion of the liver by potentially toxic agents, such as tumor cells or viruses.

III. MAJOR FUNCTIONS OF THE LIVER A. The Liver Is a Central Receiving and Recycling Center for the Body The liver can carry out a multitude of biochemical reactions. This is necessary because of its role in constantly monitoring, recycling, modifying, and distributing all of the various compounds absorbed from the digestive tract and delivered to the liver. If any portion of an ingested compound is potentially useful to that organism, the liver retrieves this portion and converts it to a substrate that can be used by hepatic and nonhepatic cells. At the same time, the liver removes many of the toxic compounds that are ingested or produced in the body and targets them for excretion in the urine or in the bile. As mentioned previously, the liver receives nutrient-rich blood from the enteric circulation through the portal vein; thus, all of the compounds that enter the blood from the digestive tract pass through the liver on their way to other tissues. The enterohepatic circulation allows the liver first access to nutrients to fulfill specific functions (such as the synthesis of blood coagulation proteins, heme, purines, and pyrimidines) and first access to ingested toxic compounds (such as ethanol) and to such potentially harmful metabolic products (such as NH4⫹ produced from bacterial metabolism in the gut). In addition to the blood supply from the portal vein, the liver receives oxygenrich blood through the hepatic artery; this arterial blood mixes with the blood from the portal vein in the sinusoids. This unusual mixing process gives the liver access to various metabolites ingested and produced in the periphery and secreted into the peripheral circulation, such as glucose, individual amino acids, certain proteins, iron–transferrin complexes, and waste metabolites as well as potential toxins produced during substrate metabolism. As mentioned, fenestrations in the endothelial cells, combined with gaps between the cells, the lack of a basement membrane between the endothelial cells and the hepatocytes, and low portal blood pressure (which results in slow blood flow) contribute to the efficient exchange of compounds between sinusoidal blood and the hepatocyte and clearance of unwanted compounds from the blood. Thus, large molecules targeted for processing, such as serum proteins and chylomicron remnants, can be removed, degraded, and their components recycled by hepatocytes. Similarly, newly synthesized molecules, such as very low-density lipoprotein (VLDL) and serum proteins, can be easily secreted into the blood. In addition, the liver can convert all of the amino acids found in proteins into glucose, fatty acids, or ketone bodies. The secretion of VLDL by the liver not only delivers excess calories to adipose tissue for storage of fatty acids in triacylglycerol, it also delivers phospholipids and cholesterol to tissues that are in need of these compounds for synthesis of cell walls as well as other functions. The secretion of glycoproteins by the liver is accomplished through the liver’s gluconeogenic capacity and its access to a variety of dietary sugars to form the oligosaccharide chains, as well as its access to dietary amino acids with which it synthesizes proteins. Thus, the liver has the capacity to carry out a large number of biosynthetic reactions. It has the biochemical wherewithal to synthesize a myriad

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of compounds from a broad spectrum of precursors. At the same time, the liver metabolizes compounds into biochemically useful products. Alternatively, it has the ability to degrade and excrete those compounds presented to it that cannot be further used by the body. Each of the liver cells described contains specialized transport and uptake mechanisms for enzymes, infectious agents, drugs, and other xenobiotics that specifically target these substances to certain liver cell types. These are accomplished by linking these agents covalently by way of biodegradable bonds to their specific carrier. The latter then determines the particular fate of the drug by using specific cell recognition, uptake, transport, and biodegradation pathways.

B. Inactivation and Detoxification of Xenobiotic Compounds and Metabolites Xenobiotics are compounds that have no nutrient value (cannot be used by the body for energy requirements) and are potentially toxic. They are present as natural components of foods or they may be introduced into foods as additives or through processing. Pharmacologic and recreational drugs are also xenobiotic compounds. The liver is the principal site in the body for the degradation of these compounds. Because many of these substances are lipophilic, they are oxidized, hydroxylated, or hydrolyzed by enzymes in phase I reactions. Phase I reactions introduce or expose hydroxyl groups or other reactive sites that can be used for conjugation reactions (the phase II reactions). The conjugation reactions add a negatively charged group such as glycine or sulfate to the molecule. Many xenobiotic compounds are transformed through several different pathways. A general scheme of inactivation is shown in Figure 46.2. The conjugation and inactivation pathways are similar to those used by the liver to inactivate many of its own metabolic waste products. These pathways are intimately related to the biosynthetic cascades that exist in the liver. The liver can synthesize the precursors that are required for conjugation and inactivation reactions from other compounds. For example, sulfation is used by the liver to clear steroid hormones from the circulation. The sulfate used for this purpose can be obtained from the degradation of cysteine or methionine. The liver, kidney, and intestine are the major sites in the body for biotransformation of xenobiotic compounds. Many xenobiotic compounds contain aromatic rings (such as benzopyrene in tobacco smoke) or heterocyclic ring structures (such as the nitrogen-containing rings of nicotine or pyridoxine) that we are unable to degrade or recycle into useful components. These structures are hydrophobic, causing the molecules to be retained in adipose tissue unless they are sequestered by the liver, kidney, or intestine for biotransformation reactions. Sometimes, however, the phase I and II reactions backfire, and harmless hydrophobic molecules are converted to toxins or potent chemical carcinogens. 1.

CYTOCHROME P450 AND XENOBIOTIC METABOLISM

The toxification/detoxification of xenobiotics is accomplished through the activity of a group of enzymes with a broad spectrum of biologic activity. Some examples

RH Xenobiotic or waste metabolite in the diet or peripheral circulation

Phase I reactions

Phase II reactions

R OH Reduction Conjugation (primary Oxidation metabolite) Sulfation Hydroxylation Methylation Hydrolysis Glucuronidation

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A knowledge of functional characteristics of liver cells has been used to design diagnostic agents to determine the normalcy of specific biochemical pathways of the hepatocytes. These “tailor-made” pharmaceuticals can be designed to be taken up by one or more of the transport mechanisms available to the liver. For example, receptor-related endocytic processes can be used as targets to probe specific receptor-mediated transport functions of the liver cells. The asialoglycoprotein receptor, also known as the hepatic-binding protein, has been used in this diagnostic approach. The substrate 99Tcm-galactosyl-neoglycoalbumin (NGA) was developed as a specific ligand for selective uptake via this specific hepatic receptor. The timing and extent of the assimilation of this probe into the hepatocytes, as determined by imaging the liver at precise intervals after the administration of this isotope, yields an estimate of hepatic blood flow as well as the transport capacity of this specific hepatic transporter protein.

Antibody titers against Entamoeba histolytica by use of an enzyme immunoassay were strongly reactive in Amy Biasis’s blood. A diagnosis of amoebiasis was made. Her physicians started a nitroimidazole amoebicide (metronidazole) orally in a dose of 500 mg every 6 hours for 10 days. By the third day of treatment, Amy began to feel noticeably better. Her physicians told her that they expected a full clinical response in 95% of patients with amoebic liver abscesses treated in this way, although her multiple hepatic abscesses adversely affected her prognosis to a degree. After her response to oral therapy for the liver abcess, she was also treated with a “luminal agent” to clear the organism from her intestine.

O R

O

S

O–

O– Secondary metabolite, suitable for excretion

FIG. 46.2. General scheme of xenobiotic detoxification. The toxic compound RH has been converted to a sulfated derivative suitable for excretion.

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Table 46.1 Examples of Enzymes Used in Biotransformation of Xenobiotic Compounds Acetyltransferase Amidase-esterase Dehydrogenase Flavin-containing monooxygenase Glutathione S-transferase Methyltransferase Mixed-function oxidase Reductase Sulfotransferase UDP-glucosyltransferase UDP-glucuronosyltransferase UDP, uridine diphosphate.

NADP+, H+

NADPH FAD e– e–

RH O2

ROH, H2O

FMN Fe-heme

Cytochrome Cytochrome P450 reductase P450

FIG. 46.3. General structure of the P450 enzymes. O2 binds to the P450 Fe-heme in the active site and is activated to a reactive form by accepting electrons. The electrons are donated by the cytochrome P450 reductase, which contains an FAD plus an FMN or Fe–S center to facilitate the transfer of single electrons from NADPH to O2. The P450 enzymes involved in steroidogenesis have a somewhat different structure. For CYP2E1, RH is ethanol (CH3CH2OH), and ROH is acetaldehyde (CH3COH).

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of enzymes involved in xenobiotic transformation are described in Table 46.1. Of the wide variety of enzymes that are involved in xenobiotic metabolism, only the cytochrome P450–dependent monooxygenase system is discussed here. The cytochrome P450–dependent monooxygenase enzymes are determinants in oxidative, peroxidative, and reductive degradation of exogenous (chemicals, carcinogens, pollutants, etc.) and endogenous (steroids, prostaglandins, retinoids, etc.) substances. The key enzymatic constituents of this system are the flavoprotein NADPH-cytochrome P450 oxidoreductase and cytochrome P450 (Fig. 46.3). The latter is the terminal electron acceptor and substrate-binding site of the microsomal mixed-function oxidase complex, a very versatile catalytic system. The system got its name in 1962, when Omura and Sato found a pigment with unique spectral characteristics derived from liver microsomes of rabbits. When reduced and complexed with carbon monoxide, it exhibited a spectral absorbance maximum at 450 nm. The major role of the cytochrome P450 enzymes (see Chapter 25) is to oxidize substrates and introduce oxygen to the structure. Similar reactions can be carried out by other flavin monooxygenases that do not contain cytochrome P450. The human cytochrome P450 enzyme family contains 57 functional genes, which produce proteins with at least 40% sequence homology. These isozymes have different but overlapping specificities. The human enzymes are generally divided into nine major subfamilies, and each of these is further subdivided. For example, in the naming of the principal enzyme involved in the oxidation of ethanol to acetaldehyde, CYP2E1, the CYP denotes the cytochrome P450 family, the 2 denotes the subfamily, the E denotes ethanol, and the 1 denotes the specific isozyme. The CYP3A4 isoform accounts for 30% to 40% of CYP450 enzymes in the liver and 70% of cytochrome enzymes in gut wall enterocytes. It metabolizes the greatest number of drugs in humans. Specific drugs are substrates for CYP3A4. The concomitant ingestion of two CYP3A4 substrates could potentially induce competition for the binding site, which, in turn, could alter the blood levels of these two agents. The drug with the highest affinity for the enzyme will be preferentially metabolized, whereas the metabolism (and degradation) of the other drug will be reduced. The latter drug’s concentration in the blood will then rise. Moreover, many substances or drugs impair or inhibit the activity of the CYP3A4 enzyme, thereby impairing the body’s ability to metabolize a drug. Some of the lipid-lowering agents known as the statins (HMG-CoA reductase inhibitors) require CYP3A4 for degradation. Appropriate drug treatment and dosing takes into account the normal degradative pathway of the drug. However, grapefruit juice is a potent inhibitor of CYP3A4-mediated drug metabolism. Evidence suggests that if a statin is regularly taken with grapefruit juice, its level in the blood may increase as much as 15-fold. This marked increase in plasma concentration could increase the muscle and liver toxicity of the statin in question because side effects of the statins appear to be dose-related. The cytochrome P450 isozymes all have certain features in common: 1. They all contain cytochrome P450, oxidize the substrate, and reduce oxygen. 2. They all have a flavin-containing reductase subunit that uses NADPH, and not NADH, as a substrate. 3. They are all found in the smooth endoplasmic reticulum and are referred to as microsomal enzymes (e.g., CYP2E1 is also referred to as the microsomal ethanol-oxidizing system, MEOS). 4. They are all bound to the lipid portion of the membrane, probably to phosphatidylcholine. 5. They are all inducible by the presence of their own best substrate and somewhat less inducible by the substrates for other P450 isozymes. 6. They all generate a reactive free radical compound as an intermediate in the reaction.

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H H

H C

C

Cl

Phase I reaction CYP2E1

Phase II reactions

O CH2

C Cl H

Chloroethylene oxide

Vinyl chloride

Covalent binding to proteins, DNA; cell damage

FIG. 46.4.

2. i.

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H ClCH2

C

O Chloroacetaldehyde Glutathione S-transferase

Conjugation with glutathione, excretion

Detoxification of vinyl chloride.

EXAMPLES OF CYTOCHROME P450 DETOXIFICATION REACTIONS Vinyl Chloride

The detoxification of vinyl chloride provides an example of effective detoxification by a P450 isozyme (ethanol detoxification was discussed in Chapter 25). Vinyl chloride is used in the synthesis of plastics and can cause angiosarcoma in the liver of exposed workers. It is activated in a phase I reaction to a reactive epoxide by a hepatic P450 isozyme (CYP2E1), which can react with guanine in DNA or other cellular molecules. However, it also can be converted to chloroacetaldehyde, conjugated with reduced glutathione, and excreted in a series of phase II reactions (Fig. 46.4). ii.

Aflatoxin B1

Aflatoxin B1 is an example of a compound that is made more toxic by a cytochrome P450 reaction (CYP2A1). Current research suggests that ingested aflatoxin B1 in contaminated food (it is produced by a fungus [Aspergillus flavus] that grows on peanuts that may have been stored in damp conditions) is directly involved in hepatocarcinogenesis in humans by introducing a G → T mutation into the p53 gene. Aflatoxin is metabolically activated to its 8,9-epoxide by two different isozymes of cytochrome P450. The epoxide modifies DNA by forming covalent adducts with guanine residues. In addition, the epoxide can combine with lysine residues in proteins and thus is also a hepatotoxin. iii. Acetaminophen

Acetaminophen (Tylenol) is an example of a xenobiotic that is metabolized by the liver for safe excretion; however, it can be toxic if ingested in high doses. The pathways for acetaminophen metabolism are shown in Figure 46.5. As shown in the figure, acetaminophen can be glucuronylated or sulfated for safe excretion by the kidney. However, a cytochrome P450 enzyme produces the toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI), which can be excreted safely in the urine after conjugation with glutathione. NAPQI is a dangerous and unstable metabolite that can damage cellular proteins and lead to death of the hepatocyte. Under normal conditions, when acetaminophen is taken in the correct therapeutic amounts, ⬍10% of the drug forms NAPQI, an amount that can be readily handled by the glutathione detoxifying system (phase II reactions). However, when taken in doses that are potentially toxic, the sulfotransferase and glucuronyl transferase systems are overwhelmed, and more acetaminophen is metabolized through the NAPQI route. When this occurs, the levels of glutathione in the hepatocyte are insufficient to detoxify NAPQI, and hepatocyte death can result. The enzyme that produces NAPQI—CYP2E1—is induced by alcohol (see Chapter 25, MEOS). Thus, individuals who chronically abuse alcohol have increased sensitivity to acetaminophen toxicity because a higher percentage of

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H

O

H

N C CH3

Kidney, urine Glucuronate

UDP-glucuronyl transferase

O N C CH3

H

O N C CH3 Kidney, urine

Sulfo transferase

OH

SO4

Acetaminophen EtOH

H

N-acetyl cysteine

O N C CH3

+

+

CYP2E1

O N C CH3

H

O N C CH3

GSH SG OH Mercaptouric acid Kidney, urine

Glutathione S-transferase

Cell proteins

O NAPQI (N-acetyl-pbenzoquinoneimine) (toxic intermediate)

S-protein OH

FIG. 46.5. Pathways of acetaminophen detoxification. N-acetyl cysteine stimulates the production of glutathione, thereby reducing the levels of NAPQI, which can damage cellular proteins. Ethanol upregulates CYP2E1 activity (the MEOS).

acetaminophen metabolism is directed toward NAPQI, compared with an individual with low levels of CYP2E1. Therefore, even recommended therapeutic doses of acetaminophen can be toxic to these individuals. An effective treatment for acetaminophen poisoning involves the use of N-acetyl cysteine. This compound supplies cysteine as a precursor for increased glutathione production, which in turn enhances the phase II reactions, which reduces the levels of the toxic intermediate.

C. Regulation of Blood Glucose Levels One of the primary functions of the liver is to maintain blood glucose concentrations within the normal range. The manner in which the liver accomplishes this has been the subject of previous chapters (Chapters 26, 31, and 36). In brief, the pancreas monitors blood glucose levels and secretes insulin when blood glucose levels rise and glucagon when such levels decrease. These hormones initiate regulatory cascades that affect liver glycogenolysis, glycogen synthesis, glycolysis, and gluconeogenesis. In addition, sustained physiologic increases in growth hormone, cortisol, and catecholamine secretion help to sustain normal blood glucose levels during fasting (see Chapter 43). When blood glucose levels drop, glycolysis and glycogen synthesis are inhibited, and gluconeogenesis and glycogenolysis are activated. Concurrently, fatty acid oxidation is activated to provide energy for glucose synthesis. During an overnight fast, blood glucose levels are maintained primarily by glycogenolysis and, if gluconeogenesis is required, the energy (six ATP molecules are required to produce one molecule of glucose from two molecules of pyruvate) is obtained by fatty acid oxidation. On insulin release, the opposing pathways are activated such that excess fuels can be stored either as glycogen or fatty acids. The pathways are regulated by the activation or inhibition of two key kinases, the cyclic adenosine monophosphate (cAMP)–dependent protein kinase, and the AMP-activated protein kinase (see Fig. 36.11 for a review of these pathways). Recall that the liver can export glucose because it is one of only two tissues that express glucose 6-phosphatase.

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D. Synthesis and Export of Cholesterol and Triacylglycerol When food supplies are plentiful, hormonal activation leads to fatty acid, triacylglycerol, and cholesterol synthesis. High dietary intake and intestinal absorption of cholesterol compensatorily reduces the rate of hepatic cholesterol synthesis, in which case the liver acts as a recycling depot for sending excess dietary cholesterol to the peripheral tissue when needed as well as accepting cholesterol from these tissues when required. The pathways of cholesterol metabolism were discussed in Chapter 34.

E. Ammonia and the Urea Cycle The liver is the primary organ for synthesizing urea and thus is the central depot for the disposition of ammonia in the body. Ammonia groups travel to the liver on glutamine and alanine, and the liver converts these ammonia nitrogens to urea for excretion in the urine. The reactions of the urea cycle were discussed in Chapter 38. Table 46.2 lists some of the important nitrogen-containing compounds that are primarily synthesized or metabolized by the liver.

F. Formation of Ketone Bodies The liver is the only organ that can produce ketone bodies, yet it is one of the few that cannot use these molecules for energy production. Ketone bodies are produced when the rate of glucose synthesis is limited (i.e., substrates for gluconeogenesis are limited) and fatty acid oxidation is occurring rapidly. Ketone bodies can cross the

Table 46.2

Nitrogen-Containing Products Produced by the Liver

Product

Precursors

Tissues

Function

Creatine

Arginine, glycine, and SAM

Liver

Glutathione

Glutamate, cysteine, glycine

All tissues, but highest use in the liver

Purines

Glycine, glutamine, aspartate, carbon dioxide, tetrahydrofolate, PRPP

Liver, small amounts in brain and cells of the immune system

Pyrimidines

Aspartate, glutamine, carbon dioxide

Sialic acid (NANA), other amino sugars

Glutamine

Liver, small amounts in brain and cells of the immune system Most cells

Forms creatine phosphate in muscle for energy storage. Excreted as creatinine. Protection against free radical injury by reduction of hydrogen peroxide and lipid peroxides. In liver and kidney, forms mercapturic acids. Adenine and guanine nucleosides and nucleotides. DNA, RNA, and coenzymes and energy-transferring nucleotides Uracil, thymine, and cytosine

Sulfated compounds

Cysteine

Liver and kidney produce sulfate

Taurine Glycocholic acid, and glycochenodeoxycholic acid

Cysteine Glycine, bile salts

Liver Liver

Sphingosine

Serine and palmitoyl-CoA

Liver, brain, and other tissues

Heme

Glycine and succinyl-CoA

Liver, bone marrow cytochromes. Heme from bone marrow Liver, kidney

Glycine conjugates of Glycine, medium-size hydrophobic xenobiotic compounds carboxylic acids Niacin Tryptophan, glutamine One-carbon methyl donors for tetrahydrofolate and SAM

Glycine, serine, histidine, methionine

Liver Most cells, but highest in liver

In the liver, synthesis of oligosaccharide chains on secreted proteins. Most cells, glycoproteins, proteoglycans, and glycolipids Many cells use sulfate in blood for formation of PAPS, which transfers sulfate to proteoglycans, drugs, and xenobiotics. Conjugated bile salts Conjugated bile salts are excreted into the bile and assists in the absorption of lipids and fat-soluble vitamins through the formation of micelles. Precursor of sphingolipids found in myelin and other membranes. Heme from liver is incorporated into hemoglobin. Inactivation and targeting toward urinary excretion. NAD, NADP coenzymes for oxidation reactions Choline, phosphatidylcholine, purine and pyrimidine synthesis, inactivation of waste metabolites and xenobiotics through methylation

SAM, S-adenosyl methionine; NANA, N-acetylneuraminic acid; PRPP, 5⬘-phosphoribosyl 1⬘-pyrophosphate; PAPS, 3⬘-phosphoadenosine 5⬘-phosphosulfate.

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Cirrhosis of the liver results in portal hypertension, which, because of increasing back pressure into the esophageal veins, promotes the development of dilated thin-walled esophageal veins (varices). At the same time, synthesis of blood coagulation proteins by the liver and required vitamin K-dependent reactions are greatly diminished (resulting in a prolonged prothrombin time, which, in turn, increases clotting time). When the esophageal varices rupture, massive bleeding into the thoracic or abdominal cavity as well as the stomach may occur. Much of the protein content of the blood entering the gastrointestinal tract is metabolized by intestinal bacteria, releasing ammonium ion, which enters the portal vein. Because hepatocellular function has been compromised, the urea cycle capacity is inadequate, and the ammonium ion enters the peripheral circulation, thereby contributing to hepatic encephalopathy (brain toxicity caused by elevated ammonia levels).

blood–brain barrier and become a major fuel for the nervous system under conditions of starvation. Synthesis and metabolism of ketone bodies have been described in Chapter 23.

G. Nucleotide Biosynthesis The liver can synthesize and salvage all ribonucleotides and deoxyribonucleotides for other cells to use. Certain cells have lost the capacity to produce nucleotides de novo but can use the salvage pathways to convert free bases to nucleotides. The liver can secrete free bases into the circulation for these cells to use for this purpose. Nucleotide synthesis and degradation are discussed in Chapter 41.

H. Synthesis of Blood Proteins The liver is the primary site of the synthesis of circulating proteins such as albumin and the clotting factors. When liver protein synthesis is compromised, the protein levels in the blood are reduced. Hypoproteinemia may lead to edema because of a decrease in the protein-mediated osmotic pressure in the blood. This, in turn, causes plasma water to leave the circulation and enter (and expand) the interstitial space, causing edema. Most circulating plasma proteins are synthesized by the liver. Therefore, the hepatocyte has a well-developed endoplasmic reticulum, Golgi system, and cellular cytoskeleton, all of which function in the synthesis, processing, and secretion of proteins. The most abundant plasma protein produced by the liver is albumin, which represents 55% to 60% of the total plasma protein pool. Albumin serves as a carrier for a large number of hydrophobic compounds such as fatty acids, steroids, hydrophobic amino acids, vitamins, and pharmacologic agents. It is also an important osmotic regulator in the maintenance of normal plasma osmotic pressure. The other proteins synthesized by the liver are, for the most part, glycoproteins. They function in hemostasis, transport, protease inhibition, and ligand binding and as secretagogues for hormone release. The acute-phase proteins that are part of the immune response and the body’s response to many forms of “injury” are also synthesized in the liver. Table 46.3 lists some of the proteins and their functions.

I.

The Synthesis of Glycoproteins and Proteoglycans

The liver, because it is the site of synthesis of most of the blood proteins (including the glycoproteins), has a high requirement for the sugars that go into the oligosaccharide portion of glycoproteins (the synthesis of glycoproteins is discussed in Chapter 30). These include mannose, fructose, galactose, and amino sugars. One of the intriguing aspects of the hepatic biosynthetic pathways that use carbohydrate in the synthesis of these compounds is that the liver is not dependent on either dietary glucose or hepatic glucose to generate the precursor intermediates for these pathways. This is because the liver can generate carbohydrates from dietary amino acids (which enter gluconeogenesis generally as pyruvate or an intermediate of the tricarboxylic acid [TCA] cycle), lactate (generated from anaerobic glycolysis in other tissues), and glycerol (generated by the release of free fatty acids from the adipocyte). Of course, if dietary carbohydrate is available, the liver can use that source as well. Most of the sugars secreted by the liver are O-linked, that is, the carbohydrate is attached to the protein at its anomeric carbon through a glycosidic link to the Table 46.3

A Partial List of Proteins Synthesized in the Liver

Type of Protein

Examples

Blood coagulation

Blood coagulation factors: fibrinogen; prothrombin; factors V, VII, IX, and X. Also ␣2-macroglobulin Transferrin (iron), ceruloplasmin (copper), haptoglobin (heme), hemopexin (heme) Apoprotein B100, apoprotein A1 ␣1-Antitrypsin

Metal-binding proteins Lipid transport Protease inhibitor

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A. O-linked

873

B. N-linked

O HOCH2

CH2 O HO H

C

H O

H OH H

H

CH2

O

O OH H

O

FIG. 46.6.

H H

C CH2

O

CH NH

NH C

O

CH3

CH3 GalNAc

NH C

CH NH

NH C

O

O H H

Serine

GlcNAc

Asparagine

The general configuration of O-linked (A) and N-linked (B) glycoproteins.

–OH of a serine or a threonine residue. This is in contrast to the N-linked arrangement in which there is an N-glycosyl link to the amide nitrogen of an asparagine residue (Fig. 46.6). A particularly important O-linked sugar is N-acetylneuraminic acid (NANA or sialic acid), a nine-carbon sugar that is synthesized from fructose 6-phosphate and phosphoenolpyruvate (see Fig. 30.8). As circulating proteins age, NANA (sialic acid) residues are lost from the serum proteins. This change signals their removal from the circulation and their eventual degradation. An asialoglycoprotein receptor on the liver cell surface binds such proteins, and the receptor– ligand complex is endocytosed and transported to the lysosomes. The amino acids from the degraded protein are then recycled within the liver.

J. The Pentose Phosphate Pathway The major functions of the pentose phosphate pathway (see Chapter 29) are the generation of NADPH and five-carbon sugars. All cell types, including red blood cells, can carry out this pathway because they need to generate NADPH so that the activity of glutathione reductase, the enzyme that catalyzes the conversion of oxidized glutathione (GSSG) back to reduced glutathione (GSH), can be maintained. Without the activity of this enzyme, the protection against free radical injury is lost. All cells also need this pathway for the generation of ribose, especially those cells that are dividing rapidly or that have high rates of DNA synthesis. The liver has a much greater demand for NADPH than do most other organs. It uses NADPH for the biosynthesis of fatty acids and cholesterol, which the liver must make to produce phospholipids, and for the synthesis of VLDL and bile salts. It also uses NADPH for other biosynthetic reactions such as proline synthesis. NADPH is also used by mixed-function oxidases such as cytochrome P450 that are involved in the metabolism of xenobiotics and of a variety of pharmaceuticals. Because the liver participates in so many reactions that are capable of generating free radicals, the liver uses more glutathione and NADPH to maintain glutathione reductase and catalase activity than any other tissue. Consequently, the concentration of glucose-6-phosphate dehydrogenase (the rate-limiting and regulated enzyme in the pentose phosphate pathway) is high in the liver, and the rate of flux through this pathway may be as high as 30% of the rate of flux through glycolysis.

IV. FUELS FOR THE LIVER The reactions used to modify and inactivate dietary toxins and waste metabolites are energy-requiring, as are the reactions used by anabolic (biosynthetic) pathways such as gluconeogenesis and fatty acid synthesis. Thus, the liver has a high energy requirement and consumes approximately 20% of the total oxygen used by the body. The principal forms in which energy is supplied to these reactions are the

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high-energy phosphate bonds of adenosine triphosphate (ATP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), reduced NADPH, and acyl coenzyme A (acyl-CoA) thioesters. The energy for the formation of these compounds is obtained directly from oxidative metabolism, the TCA cycle, or the electron-transport chain and oxidative phosphorylation. After a mixed meal containing carbohydrate, the major fuels used by the liver are glucose, galactose, and fructose. If ethanol is consumed, the liver is the major site of ethanol oxidation, yielding principally acetate and then acetyl-CoA. During an overnight fast, fatty acids become the major fuel for the liver. They are oxidized to carbon dioxide or ketone bodies. The liver also can use all of the amino acids as fuels (although its use of branched-chain amino acids is small compared to the muscles), converting many of them to glucose. The urea cycle disposes of the ammonia that is generated from amino acid oxidation.

A. Carbohydrate Metabolism in the Liver After a carbohydrate-containing meal, glucose, galactose, and fructose enter the portal circulation and flow to the liver. This organ serves as the major site in the body for the use of dietary galactose and fructose. It metabolizes these compounds by converting them to glucose and intermediates of glycolysis. Their fate is essentially the same as that of glucose (Table 46.4).

B. Glucose as a Fuel The entry of glucose into the liver is dependent on a high concentration of glucose in the portal vein after a high-carbohydrate meal. Because the Km for both the glucose transporter (GLUT2) and glucokinase is so high (approximately 10 mM), glucose enters the liver principally after its concentration rises to 10 to 40 mM in the portal blood and not at the lower 5-mM concentration in the hepatic artery. The increase in insulin secretion that follows a high-carbohydrate meal promotes the conversion of glucose to glycogen. In addition, the rate of glycolysis is increased (phosphofructokinase-2 [PFK-2] kinase activity is active; thus phosphofructokinase-1 [PFK-1] is activated by fructose 2,6-bisphosphate) so that acetyl-CoA can be produced for fatty acid synthesis (acetyl-CoA carboxylase is activated by citrate; see Chapter 33). Thus, after a high-carbohydrate meal, the liver uses glucose as its major fuel while activating the pathways for glycogen and fatty acid synthesis. The rate of glucose use by the liver is determined, in part, by the level of activity of glucokinase. Glucokinase activity is regulated by a glucokinase regulatory protein (RP, Fig. 46.7), which is located in the nucleus. In the absence of glucose, glucokinase is partially sequestered within the nucleus, bound to RP in an inactive form. High concentrations of fructose 6-phosphate promote the interaction of glucokinase with RP, whereas high levels of either glucose or fructose 1-phosphate block glucokinase from binding to RP and promote the dissociation of the complex.

Table 46.4

Major Fates of Carbohydrates in the Liver

Storage as glycogen Glycolysis to pyruvate Followed by oxidation to carbon dioxide in the TCA cycle Precursors for the synthesis of glycerol 3-phosphate (the backbone of triacylglycerols and other glycolipids), sialic acid, and serine Entry into the TCA cycle and exit as citrate, followed by conversion to acetyl-CoA, malonyl-CoA, and entry into fatty acid synthesis and secretion as VLDL Synthesis of phospholipids and other lipids from triacylglycerols Conversion to mannose, sialic acid, and other sugars necessary for the synthesis of oligosaccharides for glycoproteins, including those secreted into blood. Synthesis of acid sugars for proteoglycan synthesis and formation of glucuronides Oxidation in the pentose phosphate pathway for the formation of NADPH (necessary for biosynthetic reactions such as fatty acid synthesis, glutathione reduction, and other NADPHutilizing detoxification reactions) TCA, tricarboxylic acid; VLDL, very low-density lipoprotein.

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A

875

B Glucose or fructose 1-phosphate GK

Nucleus RP

RP GK

GK

GK GK

(Inactive) (Active)

FIG. 46.7. Regulation of glucokinase (GK) by regulatory protein (RP). RP is localized to the nucleus, and in the absence of glucose or presence of fructose 6-phosphate, most glucokinase is translocated to the nucleus and binds RP. This leads to the formation of the inactive form of glucokinase. When glucose or fructose 1-phosphate levels rise, glucokinase is released from RP. It then translocates to the cytoplasm and actively converts glucose to glucose 6-phosphate.

Thus, as glucose levels rise in the cytoplasm and nucleus (e.g., because of increased blood glucose levels after a meal), there is a significant enhancement of glucose phosphorylation as glucokinase is released from the nucleus, travels to the cytoplasm, and phosphorylates glucose. The role of glucokinase RP is very complex. Mice that have been genetically engineered so that they do not express the RP (knockout mice) display reduced levels of total glucokinase activity in the liver. This is attributable to the finding that RP is important in the posttranscriptional processing of the mRNA for glucokinase. In the absence of RP, less glucokinase is produced. These mice, therefore, have no glucokinase in the nucleus, reduced cytoplasmic glucokinase content, and inefficient glucose phosphorylation in the liver when glucose levels rise. The major regulatory step for liver glycolysis is the PFK-1 step. Even under fasting conditions, the ATP concentration in the liver (approximately 2.5 mM) is sufficiently high to inhibit PFK-1 activity. Thus, liver glycolysis is basically controlled by modulating the levels of fructose 2,6-bisphosphate—the product of the PFK-2 reaction. As fructose 2,6-bisphosphate levels increase (which will occur in the presence of insulin), the rate of glycolysis increases; when glucagon levels increase and protein kinase A is activated so that PFK-2 is phosphorylated and its kinase activity inactive, glycolysis slows down and gluconeogenesis is enhanced (see Chapters 22 and 31).

Why would you expect fructose 1-phosphate levels to promote the dissociation of glucokinase from regulatory protein (RP)?

Glucokinase activators are being investigated as potential drugs to lower blood glucose levels in type 2 diabetes. The activators will enhance glucose metabolism in the pancreas, resulting in increased insulin release. These drugs are currently in clinical trials.

C. Lipid Metabolism Long-chain fatty acids are a major fuel for the liver during periods of fasting, when they are released from adipose tissue triacylglycerols and travel to the liver as fatty acids bound to albumin. In the liver, they bind to fatty acid–binding proteins and are then activated on the outer mitochondrial membrane, the peroxisomal membrane, and the smooth endoplasmic reticulum by fatty acyl-CoA synthetases. The fatty acyl group is transferred from CoA to carnitine for transport through the inner mitochondrial membrane, where it is reconverted back into fatty acyl-CoA and oxidized to acetyl-CoA in the ␤-oxidation spiral (see Chapter 23). The enzymes in the pathways of fatty acid activation and ␤-oxidation (the synthetases, the carnitine acyltransferases, and the dehydrogenases of ␤-oxidation) are somewhat specific for the length of the fatty acid carbon chain. The chain-length specificity is divided into enzymes for long-chain (C20 to approximately C12), medium-chain (approximately C12 to C4), and short-chain fatty acids (C4 to C2). The major lipids oxidized in the liver as fuels are the long-chain fatty acids (pal-

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Fructose 1-phosphate is produced from fructose metabolism. The major dietary source of fructose, the ingestion of which would lead to increased fructose 1-phosphate levels, is sucrose. Sucrose is a disaccharide of glucose and fructose. Thus, an elevation of fructose 1-phosphate usually indicates an elevation of glucose levels as well.

mitic, stearic, and oleic acids) because these are the lipids that are synthesized in the liver, are the major lipids ingested from meat or dairy sources, and are the major form of fatty acids present in adipose tissue triacylglycerols. The liver, as well as many other tissues, uses fatty acids as fuels when the concentration of the fatty acid–albumin complex is increased in the blood.

Medium-chain triglycerides (MCTs) are important components of nutritional supplements used in patients with digestive disorders. They, therefore, can be employed as an easily absorbed source of calories in patients who have a gastrointestinal (GI) disorder that may result in malabsorption of nutrients. These diseases include pancreatic insufficiency, intraluminal bile salt deficiency caused by cholestatic liver disease, biliary obstruction, ileal disease or resection, and disease that causes obstruction of intestinal lymphatics. Remember, however, that MCTs do not contain polyunsaturated fatty acids that can be used for synthesis of eicosanoids (see Chapter 35).

2.

Zellweger (cerebrohepatorenal) syndrome occurs in individuals with a rare inherited absence of peroxisomes in all tissues. Patients accumulate C26 to C38 polyenoic acids in brain tissue because of defective peroxisomal oxidation of the very long-chain fatty acids synthesized in the brain for myelin formation. In liver, bile acid and ether lipid synthesis are affected, as is the oxidation of very long-chain fatty acids.

3.

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1.

MEDIUM-CHAIN-LENGTH FATTY ACID OXIDATION

The liver and certain cells in the kidney are the major sites for the oxidation of medium-chain-length fatty acids. These fatty acids usually enter the diet of infants in maternal milk as medium-chain-length triacylglycerols (MCTs). In the intestine, the MCTs are hydrolyzed by gastric lipase, bile salt–dependent lipases, and pancreatic lipase more readily than long-chain triacylglycerols. In the enterocytes, they are neither reconverted to triacylglycerols nor incorporated into chylomicrons. Instead, they are released directly into the portal circulation (fatty acids of approximately eight-carbon chain lengths or less are water-soluble). In the liver, they diffuse through the inner mitochondrial membrane and are activated to acyl-CoA derivatives by medium-chain-length fatty acid–activating enzyme (MMFAE), a family of similar isozymes present only in liver and kidney. The medium-chain fatty acylCoA is then oxidized by the normal route beginning with medium-chain-length acyl-CoA dehydrogenase (MCAD; see Chapter 23). PEROXISOMAL OXIDATION OF VERY LONG-CHAIN FATTY ACIDS

Peroxisomes are present in greater numbers in the liver than in other tissues. Liver peroxisomes contain the enzymes for the oxidation of very long-chain fatty acids such as C24:0 and phytanic acid, for the cleavage of the cholesterol side chain necessary for the synthesis of bile salts, for a step in the biosynthesis of ether lipids, and for several steps in arachidonic acid metabolism. Peroxisomes also contain catalase and are capable of detoxifying hydrogen peroxide. Very long-chain fatty acids of C20 to C26 or greater are activated to CoA derivatives by very long-chain acyl-CoA synthetase present in the peroxisomal membrane. The very long-chain acyl-CoA derivatives are then oxidized in liver peroxisomes to the eight-carbon octanoyl-CoA level. In contrast to mitochondrial ␤-oxidation, the first enzyme in peroxisomal ␤-oxidation introduces a double bond and generates hydrogen peroxide instead of FAD(2H). The remainder of the cycle, however, remains the same, releasing NADH and acetyl-CoA. Peroxisomal catalase inactivates the hydrogen peroxide, and the acetyl-CoA can be used in biosynthetic pathways such as those of cholesterol and dolichol synthesis. The octanoyl-CoA that is the end product of peroxisomal oxidation leaves the peroxisomes and the octanoyl group is transferred through the inner mitochondrial membrane by medium-chain-length acylcarnitine transferase. In the mitochondria, it enters the regular ␤-oxidation pathway, beginning with MCAD. PEROXISOME PROLIFERATOR–ACTIVATED RECEPTORS

The peroxisome proliferator–activated receptors (PPARs) play an important role in liver metabolism. These receptors obtained their name from the finding that certain agonists were able to induce the proliferation of peroxisomes in the liver. These agonists included hypolipidemic agents, nonsteroidal anti-inflammatory agents, and environmental toxins. The receptors that bind these agents, the PPARs, are members of a nuclear receptor family and, when activated, stimulate new gene transcription. In the liver, the major form of PPAR regulates directly the activity of genes that are involved in fatty acid uptake and ␤- and ␻-oxidation of fatty acids. Although the roles of the PPARs have been discussed previously (see Section VI of this book), it is worth reviewing them here. There are three major PPAR isoforms: ␣, ␦/␤, and ␥. The major form found in the liver is the ␣-form. Fatty

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Table 46.5

Genes Regulated by Activation of PPAR␣

Fatty acid transport proteins (upregulated) The mitochondrial and peroxisomal enzymes of fatty acid oxidation (upregulated) Carnitine palmitoyl transferase I (upregulated) Lipoprotein lipase (upregulated) Apoproteins A1 and A2 (upregulated, leading to increased HDL production) Apoprotein CIII (downregulated) Acyl-Coenzyme A synthetase (upregulated) HDL, high-density lipoprotein.

acids are an endogenous ligand for PPAR␣, such that when the level of fatty acids in the circulation is increased (with a concurrent increase in the fatty acid content of hepatocytes), there is increased gene transcription for those proteins involved in regulating fatty acid metabolism (Table 46.5). Genetically altered mice have been generated that lack PPAR␣. These knockout mice exhibit no abnormal phenotype when they are fed a normal diet. When they are fasted, however, or when they are fed a high-fat diet, these mice develop severe fatty infiltration of the liver. The inability to increase the rate of fatty acid oxidation in this organ leads to excessive fatty acid buildup in the hepatocytes. It also leads to an insufficient energy supply with which to make glucose (leading to hypoglycemia) as well as an inability to produce ketone bodies. In normal fasted mice or mice fed a high-fat diet, fatty acids will eventually stimulate their own oxidation via peroxisome proliferation and by induction of other enzymes needed for their oxidation. The knockout mice cannot make these compensations. 4.

The fibrates (e.g., clofibrate) are a class of drugs that bind to PPARs to elicit changes in lipid metabolism. They are typically prescribed for individuals with elevated triglyceride levels because they increase the rate of triglyceride oxidation. This, in turn, leads to a reduction in serum triacylglycerol levels. Fibrates, through PPAR␣ stimulation, also suppress apoprotein CIII (apoCIII) synthesis and stimulate lipoprotein lipase (LPL) activity. ApoCIII normally inhibits LPL activity, so by reducing CIII synthesis overall, LPL activity is increased. ApoCIII also blocks apoprotein E (apoE) on intermediate-density lipoprotein (IDL) particles, causing the IDL particles to accumulate because they cannot be taken up by the apoE receptor in the liver. The suppression of apoCIII levels allows more IDL to be endocytosed, thereby also reducing circulating triacylglycerol levels.

XENOBIOTICS METABOLIZED AS FATTY ACIDS

The liver uses the pathways of fatty acid metabolism to detoxify very hydrophobic and lipid-soluble xenobiotics that, like fatty acids, either have carboxylic acid groups or can be metabolized to compounds that contain carboxylic acids. Benzoate and salicylate are examples of xenobiotics that are metabolized in this way. Benzoate is naturally present in plant foods and is added to foods such as sodas as a preservative. Its structure is similar to that of salicylic acid (which is derived from the degradation of aspirin). Salicylic acid and benzoate are similar in size to medium-chain–length fatty acids and are activated to an acyl-CoA derivative by MMFAE (Fig. 46.8). The acyl group is then conjugated with glycine, which targets the compound for urinary excretion. The glycine derivatives of salicylate and benzoate are called salicylurate and hippurate, respectively. Salicylurate is the major urinary metabolite of aspirin in humans. Benzoate has been administered to treat hyperammonemia associated with congenital defects because urinary hippurate excretion tends to lower the free ammonia pool. Aspirin cannot be used for this purpose because it is toxic in the large doses required. 5.

877

THE METABOLISM OF LIPIDS IN LIVER DISEASE

Chronic parenchymal liver disease is associated with relatively predictable changes in plasma lipids and lipoproteins. Some of these changes are related to a reduction in the activity of lecithin-cholesterol acyltransferase (LCAT). This plasma enzyme is synthesized and glycosylated in the liver; then enters the blood, where it catalyzes the transfer of a fatty acid from the 2-position of lecithin to the 3-␤-OH group of free cholesterol to produce cholesterol ester and lysolecithin. As expected, in severe parenchymal liver disease, in which LCAT activity is decreased, plasma levels of cholesterol ester are reduced and free cholesterol levels are normal or increased. Plasma triacylglycerols are normally cleared by peripheral lipases (lipoprotein lipase [LPL] and hepatic triglyceride lipase [HTGL]). Because the activities of both LPL and HTGL are reduced in patients with hepatocellular disease, a relatively high level of plasma triacylglycerols may be found in both acute and chronic hepatitis,

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Reye’s syndrome is characterized clinically by vomiting with signs of progressive central nervous system damage. In addition, there are signs of hepatic injury and hypoglycemia. There is mitochondrial dysfunction with decreased activity of hepatic mitochondrial enzymes. Hepatic coma may occur as serum ammonia levels rise. It is associated epidemiologically with the consumption of aspirin by children during a viral illness, but it may occur in the absence of exposure to salicylates. The incidence in the United States has decreased dramatically since the 1980s, when parents were made aware of the dangers of giving aspirin to children to reduce fever. Reye’s syndrome is not necessarily confined to children. In patients who die of this disease, the liver at autopsy shows swollen and disrupted mitochondria and extensive accumulation of lipid droplets with fatty vacuolization of cells in both the liver and the renal tubules.

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A O C O–

O

O C CH3

Aspirin O CoASH

C SCoA

OH Salicylate

B

O

O

C O–

OH ATP AMP, PPi

Glycine –SCoA

O C N CH2 C H O– OH Salicyluric acid

O C O–

Benzoate

O

O CoASH

C SCoA

ATP AMP + PPi

Glycine –SCoA

O C N CH2 C H O–

Hippuric acid

FIG. 46.8. Benzoate and salicylate metabolism.

The level of nonesterified fatty acid (NEFA) levels in blood samples can be determined using enzymecoupled reactions. The unknown sample is incubated with coenzyme A (CoA) and acylCoA synthetase, which adds the coenzyme to the nonesterified fatty acids, creating acylCoA. The acyl-CoA produced is oxidized by acyl-CoA oxidase, which generates hydrogen peroxide. The hydrogen peroxide is used as a source of electrons by peroxidase to reduce a chromogenic substrate, which produces a colored reaction product. The concentration of the reaction product is determined spectrophotometrically and is directly proportional to the level of NEFA in the sample.

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in patients with cirrhosis of the liver, and in patients with other diffuse hepatocellular disorders. With low LCAT activity and elevated triacylglycerol level described, low-density lipoprotein (LDL) particles have an abnormal composition. They are relatively triacylglycerol-rich and cholesterol ester–poor. High-density lipoprotein (HDL) metabolism may be abnormal in chronic liver disease as well. For example, because the conversion of HDL3 (less antiatherosclerotic) to HDL2 (more antiatherosclerotic) is catalyzed by LCAT, the reduced activity of LCAT in patients with cirrhosis leads to a decrease in the HDL2:HDL3 ratio. Conversely, the conversion of HDL2 to HDL3 requires hepatic lipases. If the activity of this lipase is reduced, one would expect an elevation in the HDL2:HDL3 ratio. Because the HDL2:HDL3 ratio is usually elevated in cirrhosis, the lipase deficiency appears to be the more dominant of the two mechanisms. These changes may result in an overall increase in serum total HDL levels. How this affects the efficiency of the reverse cholesterol transport mechanism and the predisposition to atherosclerosis is not fully understood. With regard to triacylglycerol levels in patients with severe parenchymal liver disease, the hepatic production of the triacylglycerol-rich, VLDL particle is impaired. Yet the total level of plasma triacylglycerols remains relatively normal because the LDL particle in such patients is triacylglycerol-rich, for reasons that have not been fully elucidated. Nonesterified fatty acid (NEFA) levels are elevated in patients with cirrhosis. This change might be expected because basal hepatic glucose output is low in these patients. As a result, more NEFA are presumably required (via increased lipolysis) to meet the fasting energy requirements of peripheral tissues.

D. Amino Acid Metabolism in the Liver The liver is the principal site of amino acid metabolism in humans. It essentially balances the free amino acid pool in the blood through the metabolism of amino acids supplied by the diet after a protein-containing meal and through metabolism

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879

of amino acids supplied principally by skeletal muscles during an overnight fast. In an adult who is no longer growing linearly, the total protein content of the body on a daily basis is approximately constant, so the net degradation of amino acids (either to other compounds or used for energy) is approximately equal to the amount consumed. The key points concerning hepatic amino acid metabolism are the following: 1. The liver contains all the pathways for catabolism of all of the amino acids (although its metabolism of the branched-chain amino acids is low) and can oxidize most of the carbon skeletons to carbon dioxide. A small proportion of the carbon skeletons are converted to ketone bodies. The liver also contains the pathways for converting amino acid carbon skeletons to glucose (gluconeogenesis) that can be released into the blood. 2. Because the liver is the principal site of amino acid catabolism, it also contains the urea cycle, the pathway that converts toxic ammonium ion to nontoxic urea. The urea is then excreted in the urine. 3. After a mixed or high-protein meal, the gut uses dietary aspartate, glutamate, and glutamine as fuel (during fasting, the gut uses glutamine from the blood as a major fuel). Thus, the ingested acidic amino acids do not enter the general circulation. The nitrogen from gut metabolism of these amino acids is passed to the liver as citrulline or ammonium ion via the portal vein. 4. The branched-chain amino acids (valine, leucine, and isoleucine) can be used as a fuel by most cell types, including cells of the gut and skeletal muscle. After a high-protein meal, most of the branched-chain amino acids are not oxidized by the liver (because of very low activity of the branched-chain amino acid transaminase) and instead enter the peripheral circulation to be used as a fuel by other tissues or for protein synthesis (these amino acids are essential amino acids). The liver does, however, take up whatever amino acids it needs to carry out its own protein synthesis. 5. Most tissues transfer the amino acid nitrogen to the liver to dispose of as urea. They, therefore, produce either alanine (from the pyruvate–glucose–alanine cycle, in skeletal muscle, kidney, and intestinal mucosa) or glutamine (skeletal muscle, lungs, neural tissues) or serine (kidney), which are released into the blood and taken up by the liver. 6. The liver uses amino acids for the synthesis of proteins that it requires as well as for the synthesis of proteins to be used elsewhere. For example, the liver uses the carbon skeletons and nitrogens of amino acids for the synthesis of nitrogencontaining compounds such as heme, purines, and pyrimidines. The amino acid precursors for these compounds are all nonessential because they can be synthesized in the liver.

E. Amino Acid Metabolism in Liver Disease The concentration of amino acids in the blood of patients with liver disease is often elevated. This change is, in part, attributable to a significantly increased rate of protein turnover (a general catabolic effect seen in severely ill patients) as well as to impaired amino acid uptake by the diseased liver. It is unlikely that the increased levels are caused by degradation of liver protein and the subsequent release of amino acids from the failing hepatocyte into the blood. This is true because the total protein content of the liver is only approximately 300 g. To account for the elevated amino acid levels in the blood, the entire protein content of the liver would have to be degraded within 6 to 8 hours to account for the increased protein turnover rates found. Because 18 to 20 times more protein is present in skeletal muscle (greater mass), the muscle is probably the major source of the elevated plasma levels of amino acids seen in catabolic states such as cirrhosis of the liver. In cirrhotic patients, such as Jean Ann Tonich, the fasting blood ␣-amino nitrogen level is elevated as a result of reduced clearance. Urea synthesis is reduced as well.

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Unlike Amy Biasis, whose hepatic amoebic disorder was more localized (abscesses), Jean Anne Tonich had a diffuse hepatic disease, known as alcohol-induced cirrhosis (referred to historically as Laennec’s cirrhosis). The latter is characterized by diffuse fine scarring, a fairly uniform loss of hepatic cells, and the formation of small regenerative nodules (sometimes referred to as micronodular cirrhosis). With continued alcohol intake, fibroblasts and activated stellate cells deposit collagen at the site of persistent injury. This leads to the formation of weblike septa of connective tissue in periportal and pericentral zones. These eventually connect portal triads and central veins. With further exposure to alcohol, the liver shrinks and then becomes nodular and firm as end-stage cirrhosis develops. Unless they are successfully weaned from alcohol, these patients eventually die of liver failure. Amy Biasis, however, can probably look forward to enjoying a normal liver function after successful amebicidal therapy without evidence of residual hepatic scarring.

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The plasma profile of amino acids in cirrhosis characteristically shows an elevation in aromatic amino acids, phenylalanine and tyrosine, and in free tryptophan and methionine. The latter changes may be caused by impaired hepatic use of these amino acids as well as by portosystemic shunting. Although the mechanism is not known, a reduction in fasting plasma levels of the branched-chain amino acids (BCAAs) is also seen in cirrhotic patients. These findings, however, must be interpreted with caution because most of the free amino acid pool in humans is found in the intracellular space. Therefore, changes seen in their plasma concentrations do not necessarily reflect their general metabolic fate. Yet the elevation in aromatic amino acids and the suppression of the level of BCAAs in the blood of cirrhotics have been implicated in the pathogenesis of hepatic encephalopathy.

V. DISEASES OF THE LIVER Diseases of the liver can be clinically and biochemically devastating because no other organ can compensate for the loss of the multitude of functions that the liver normally performs. Alcohol-induced liver disease has been discussed in Chapter 25. Several diseases can lead to hepatic fibrosis (see Biochemical Comments) and cirrhosis. When this occurs to a great enough extent, liver function becomes inadequate for life. Signs and symptoms of liver disease include elevated levels of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma (caused by hepatocyte injury or death with a consequent release of these enzymes into the blood), jaundice (an accumulation of bilirubin in the blood caused by inefficient bilirubin glucuronidation by the liver; see Chapter 45), increased clotting times (the liver has difficulty producing clotting factors for secretion), edema (reduced albumin synthesis by the liver leads to a reduction in osmotic pressure in the blood), and hepatic encephalopathy (reduced urea cycle activity leading to excessive levels of ammonia and other toxic compounds in the central nervous system). CLINICAL COMMENTS Jean Ann Tonich. Patients with cirrhosis of the liver who have no known genetic propensity to glucose intolerance, such as Jean Ann Tonich, tend to have higher blood glucose levels than do normal subjects in both fasting and fed states. The mechanisms that may increase glucose levels in the fasting state include a reduction in the metabolic clearance rate of glucose by 25% to 40% compared with normal subjects. This reduction in glucose clearance results, in part, from increased oxidation of fatty acids and ketone bodies and the consequent decrease in glucose oxidation by peripheral tissues in cirrhosis patients. This is suggested by the discovery that plasma nonesterified fatty acid (NEFA) levels are high in many patients with hepatocellular dysfunction, in part because of decreased hepatic clearance of NEFA and in part because of increased adipose tissue lipolysis. Another possible explanation for the reduction in whole-body glucose use in cirrhotic patients relates to the finding that ketone body production is increased in some patients with cirrhosis. This could lead to enhanced use of ketone bodies for fuel by the central nervous system in such patients, thereby reducing the need for glucose oxidation by the highly metabolically active brain. After glucose ingestion (fed state), many patients with liver disease have abnormally elevated blood glucose levels (“hepatogenous diabetes”). Using World Health Organization (WHO) criteria, 60% to 80% of cirrhotic patients have varying degrees of glucose intolerance, and overt diabetes mellitus occurs two to four times as often in cirrhotics than it does in subjects without liver disease. The proposed mechanisms include a degree of insulin resistance in peripheral tissues; however, as the cirrhotic process progresses, these patients develop a marked impairment of insulin secretion as well. Although the mechanisms are not well understood, this decrease in insulin secretion leads to increased hepatic glucose output (leading to

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fasting hyperglycemia) and reduced suppression of hepatic glucose output after meals, leading to postprandial hyperglycemia as well. If the patient has an underlying genetic predisposition to diabetes mellitus, the superimposition of the mechanisms outlined previously will lead to an earlier and more significant breakdown in glucose tolerance in these specific patients. BIOCHEMICAL COMMENTS Hepatic Fibrosis. Extensive and progressive fibrosis of the hepatic parenchyma leads to cirrhosis of the liver, a process that has many causes. The development of fibrosis requires the activities of hepatic stellate cells, cytokines, proteases, and protease inhibitors. A major change that occurs when fibrosis is initiated is that the normally “sparse” or “leaky” basement membrane between the endothelial cell and the hepatocyte is replaced with a high-density membrane containing fibrillar collagen. This occurs because of both increased synthesis of a different type of collagen than is normally produced and a reduction in the turnover rate of existing extracellular matrix components. The supportive tissues of the normal liver contain an extracellular matrix that, among other proteins, includes type IV collagen (which does not form fibers), glycoproteins, and proteoglycans. After a sustained insult to the liver, a threefold to eightfold increase occurs in extracellular matrix components, some of which contain fibril-producing collagen (types I and III), glycoproteins, and proteoglycans. The accumulation of these fibril-producing compounds leads to a loss of endothelial cell fenestrations and, therefore, a loss of the normal sievelike function of the basement membranes. These changes interfere with normal transmembrane metabolic exchanges between the blood and hepatocytes. The hepatic stellate cell is the source of the increased and abnormal collagen production. These cells are activated by growth factors whose secretion is induced by injury to the hepatocytes or endothelial cells. Growth factors involved in cellular activation include transforming growth factor ␤1 (TGF-␤1) (which is derived from the endothelial cells, Kupffer cells, and platelets) and platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) from platelets. The release of PDGF stimulates stellate cell proliferation and, in the process, increases their synthesis and release of extracellular matrix materials and remodeling enzymes. These enzymes include matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs, as well as converting (activating) enzymes. This cascade leads to the degradation of the normal extracellular matrix and replacement with a much denser and more rigid type of matrix material. These changes are in part the result of an increase in the activity of tissue inhibitors of MMPs for the new collagen relative to the original collagen in the extracellular matrix. One consequence of the increasing stiffness of the hepatic vascular channels through which hepatic blood must flow is a greater resistance to the free flow of blood through the liver as a whole. Resistance to intrahepatic blood flow is also increased by a loss of vascular endothelial cell fenestrations, loss of free space between the endothelial cells and the hepatocytes (space of Disse), and even loss of vascular channels per se. This increased vascular resistance leads to an elevation of intrasinusoidal fluid pressure. When this intrahepatic (portal) hypertension reaches a critical threshold, the shunting of portal blood away from the liver (portosystemic shunting) contributes further to hepatic dysfunction. If the portal hypertension cannot be reduced, portal blood will continue to bypass the liver and return to the heart through the normally low-pressure esophageal veins. When this increasing intraesophageal venous pressure becomes severe enough, the walls of these veins thin dramatically and expand to form varices, which may burst suddenly, causing life-threatening esophageal variceal hemorrhage. This is a potentially fatal complication of cirrhosis of the liver.

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Table 46.6 Disease or Disorder Liver failure Amoebiasis Cirrhois

Zellweger syndrome Reye’s syndrome

Diseases Discussed in Chapter 46 Genetic or Environmental Comments Both, though primarily environmental Environmental Environmental

Genetic Environmental

Destruction of hepatocyte function, leading to hyperammonemia, edema, and jaundice. Infection by amoeba, Entamoeba histolytica Destruction of hepatocytes via inappropriate collagen and other fibrous protein deposition within the liver, usually in response to environmental insult. Lack of functional peroxisomes in all cells of the liver, kidney, and brain. Both the brain and liver primarily affected, with progressive loss of nervous system function.

Key Concepts •

•

• • •

The liver consists of a variety of cell types, each with a different function. Hepatocytes carry out the bulk of the metabolic pathways of the liver. Endothelial cells line the sinusoids and release growth factors. Kupffer cells are tissue macrophages that protect the liver from gut-derived particulate materials and bacterial products. Stellate cells store vitamin A and regulate the contractility of the sinusoids. Pit cells are liver-associated lymphocytes, which act as a defense mechanism against potentially toxic agents. The liver is the body’s central receiving and recycling center: Inactivation and detoxification of xenobiotic compounds and metabolites via cytochrome P450 systems Regulation of blood glucose levels Synthesis and export of cholesterol and triglyceride via VLDL Synthesis and excretion of urea Formation of ketone bodies from fatty acid oxidation Nucleotide biosynthesis Synthesis of blood proteins The liver highly coordinates its use of fuels versus that of the body. This is, in part, controlled by modulation of the activity of PPAR␣. Liver disease affects amino acid, carbohydrate, and lipid metabolism, leading to abnormalities in virtually all aspects of metabolism. Diseases discussed in this chapter are summarized in Table 46.6.

REVIEW QUESTIONS—CHAPTER 46 1.

2.

Drinking grapefruit juice while taking statins can lead to potentially devastating side effects. This is caused by a component of grapefruit juice doing which of the following? A. Interfering with hepatic uptake of statins B. Accelerating the conversion of the statin to a more toxic form C. Inhibiting the inactivation of statins D. Upregulating the HMG-CoA reductase E. Downregulating the HMG-CoA reductase Which one of the following characteristics of cytochrome P450 enzymes is correct? A. They are all found in the Golgi apparatus and are referred to as microsomal enzymes.

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B. They all contain a flavin-containing reductase unit that uses NADH and not NADPH as a source of electrons. C. They are all inducible by oxygen, which binds to the iron of the cytochrome. D. They all oxidize the substrate on which they act. E. They all generate a free radical compound as a final product of the reaction. 3.

Fairly predictable changes occur in the various metabolic pathways of lipid metabolism in patients with moderately advanced hepatocellular disease. Which one of the following changes would you expect to see under these conditions? A. The activity of plasma lecithin cholesterol acyltransferase (LCAT) is increased.

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B. Serum cholesterol esters are increased. C. Hepatic triglyceride lipase (HTGL) activity is increased. D. Serum triacylglycerol levels are increased. E. Serum nonesterified fatty acid levels are decreased. 4.

After a 2-week alcoholic binge, Jean Ann Tonich ingested some Tylenol to help her with a severe headache. She took three times the suggested dose because of the severity of the pain. Within 24 hours, Jean Ann became very lethargic, vomited frequently, and developed severe abdominal pain. The symptoms Jean Ann is experiencing are attributable to a reaction to Tylenol because of which of the following? A. The hypoglycemia experienced by the patient B. Ethanol-induced inhibition of Tylenol metabolism C. The hyperglycemia experienced by the patient D. Ethanol-induced acceleration of Tylenol metabolism E. Acetaminophen inhibition of VLDL secretion by the liver

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5.

883

An individual displays impaired glucose tolerance; blood glucose levels remain elevated after a meal for a longer time than is normal, although they do eventually go down to fasting levels. The patient has a normal release of insulin from the pancreas in response to elevated blood glucose levels. Hepatocytes obtained from the patient display normal levels of insulin binding to its receptor, and normal activation of the intrinsic tyrosine kinase activity associated with the insulin receptor. Analysis of glucose 6-phosphate formation within the hepatocytes, however, indicates a much slower rate of formation than in hepatocytes obtained from a normal control. A possible mutation that could lead to these results is which of the following? A. A decrease in the Km of glucokinase B. An increase in the Vmax of glucokinase C. A nonfunctional glucokinase-regulatory protein D. An increase in hexokinase activity E. A decrease in hexokinase activity

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47

Metabolism of Muscle at Rest and during Exercise There are three types of muscle cells: smooth, skeletal, and cardiac. In all types of muscle, contraction occurs via an actin/myosin sliding filament system, which is regulated by oscillations in intracellular calcium levels. Muscle cells use stored glycogen and circulating glucose, fatty acids, and amino acids as energy sources. Muscle glycolysis is regulated differently from the liver, with the key difference being the regulation of phosphofructokinase-2 (PFK-2). Muscle PFK-2 is not inhibited by phosphorylation; cardiac PFK-2 is actually activated by phosphorylation by a number of protein kinases. Thus, under conditions in which liver PFK-2 is inactive, and glycolysis is running slowly, muscle glycolysis is either unaffected, or even stimulated, depending on the isoform of PFK-2 being expressed. Although muscle cells do not synthesize fatty acids, they do contain an isozyme of acetyl-CoA carboxylase (ACC-2) to regulate the rate of fatty acid oxidation. ACC-2 produces malonyl-CoA, which inhibits carnitine palmitoyl transferase I, thereby blocking fatty acid entry into the mitochondria. Muscle also contains malonyl-CoA decarboxylase, which catalyzes the conversion of malonyl-CoA to acetyl-CoA and carbon dioxide. Thus, both the synthesis and degradation of malonyl-CoA is carefully regulated in muscle cells to balance glucose and fatty acid oxidation. Both allosteric and covalent means of regulation are employed. Citrate activates ACC-2, and phosphorylation of ACC-2 by the adenosine monophosphate (AMP)-activated protein kinase inhibits ACC-2 activity. Phosphorylation of malonyl-CoA decarboxylase by the AMPactivated protein kinase activates the enzyme, further enhancing fatty acid oxidation when energy levels are low. Muscles use creatine phosphate to store high-energy bonds. Creatine is derived from arginine and glycine in the kidney, and the guanidinoacetate formed is methylated (using S-adenosylmethionine) in the liver to form creatine. The enzyme creatine phosphokinase (CPK) then catalyzes the reversible transfer of a high-energy phosphate from adenosine triphosphate (ATP) to creatine, forming creatine phosphate and adenosine diphosphate (ADP). Creatine phosphate is unstable and spontaneously cyclizes to form creatinine, which is excreted in the urine. The spontaneous production of creatinine occurs at a constant rate and is proportional to body muscle mass. Thus, the amount of creatinine excreted each day (the creatinine clearance rate) is constant and can be used as an indicator of the normalcy of the excretory function of the kidneys. Skeletal muscle cells can be subdivided into type I and type II fibers. Type I fibers are slow-twitch fibers that use primarily oxidative metabolism for energy, whereas the type II fibers (fast-twitch) use glycolysis as their primary energygenerating pathway. Glucose transport into muscle cells can be stimulated during exercise because of the activity of the AMP-activated protein kinase. Fatty acid uptake into exercising muscle is dependent on the levels of circulating fatty acids, which are increased by epinephrine release.

884

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THE WAITING ROOM Rena Felya, a 9-year-old girl, complained of a severe pain in her throat and difficulty in swallowing. She had chills, sweats, headache, and a fever of 102.4°F. When her symptoms persisted for several days, her mother took her to her pediatrician, who found diffuse erythema (redness) in her posterior pharynx (throat), with yellow exudates (patches) on her tonsils. Large, tender lymph nodes were present under her jaw on both sides of her neck. A throat culture was taken, and therapy with penicillin was begun. Although the sore throat and fever improved, 10 days after the onset of the original infection, Rena’s eyes and legs became swollen and her urine suddenly turned the color of Coca-Cola. Her blood pressure was elevated. Protein and red blood cells were found in her urine. Her serum creatinine level (see Chapter 3 for how creatinine is measured) was elevated at 1.8 mg/dL (reference range, 0.3 to 0.7 mg/dL for a child). Because the throat culture grew group A ␤-hemolytic streptococci, the doctor ordered a Streptozyme test. This test was positive for antibodies to streptolysin O and several other streptococcal antigens. As a result, a diagnosis of acute poststreptococcal glomerulonephritis was made. Supportive therapy, including bed rest and treatment for hypertension, was initiated.

I.

A. Skeletal muscle

Nuclei

B. Smooth muscle Nuclei

MUSCLE CELL TYPES

Muscle consists of three different types: skeletal, smooth, and cardiac (Fig. 47.1). The metabolism of each is similar, but the functions of the muscles are quite different.

A. Skeletal Muscle Skeletal muscles are those muscles that are attached to bone and facilitate the movement of the skeleton. Skeletal muscles are found in pairs, which are responsible for opposing, coordinated directions of motion on the skeleton. The muscles appear striated under the microscope and are controlled voluntarily (you think about moving a specific muscle group, and then it happens). Skeletal muscle cells are long, cylindrical fibers that run the length of the muscle. The fibers are multinucleated because of cell fusion during embryogenesis. The cell membrane surrounding the fibers is called the sarcolemma, and the sarcoplasm is the intracellular milieu, which contains the proteins, organelles, and contractile apparatus of the cell. The sarcoplasmic reticulum is analogous to the endoplasmic reticulum in other cell types and is an internal membrane system that runs throughout the length of the muscle fiber. Another membrane structure, the transverse tubules (T tubules), are thousands of invaginations of the sarcolemma that tunnel from the surface toward the center of the muscle fiber to make contact with the terminal cisterns of the sarcoplasmic reticulum. Because the T tubules are open to the outside of the muscle fiber and are filled with extracellular fluid, the muscle action potential that propagates along the surface of the muscle fiber’s sarcolemma travels into the T tubules and to the sarcoplasmic reticulum. The striations in skeletal muscle are attributable to the presence and organization of myofibrils in the cells. Myofibrils are threadlike structures consisting of thin and thick filaments. The contractile proteins actin and myosin are contained within the filaments—myosin in the thick filaments and actin in the thin filaments. The sliding of these filaments relative to each other, using myosin-catalyzed adenosine triphosphate (ATP) hydrolysis as an energy source, allows for the contraction and relaxation of the muscle (see Fig. 19.4).

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C. Cardiac muscle

Intercalated disks

FIG. 47.1. Structures of the three different muscle types. (Adapted from Junqueira LC, Carneiro J. Basic Histology, Text and Atlas. 10th ed. New York, NY: Lange, McGraw-Hill; 2003.)

Damaged muscle cells release myoglobin, which can be observed clinically as a reddish tint to the urine. The primary measurement for myoglobin is via immunoassays. The primary antibody is linked to an insoluble support, whereas the second antibody is linked to alkaline phosphatase. Using a fluorescent substrate for the phosphatase allows low levels of myoglobin to be detected. If myoglobin is high in the sera, it could be a sign of a myocardial infarction. If high in the urine, muscle damage and potential renal failure could be the culprits.

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Duchenne muscular dystrophy is caused by the absence of the protein dystrophin, which is a structural protein located in the sarcolemma. Dystrophin is required to maintain the integrity of the sarcolemma, and when it is absent, there is a loss of muscle function, caused by breakdown of the sarcolemma. The gene is X-linked, and mutations that lead to Duchenne’s muscular dystrophy generally result from large deletions of the gene, such that dystrophin is absent from the membrane. Becker muscular dystrophy, a milder form of disease, is caused by point mutations in the dystrophin gene. In Becker muscular dystrophy, dystrophin is present in the sarcolemma, but in a mutated form.

Table 47.1

Properties of Muscle Fiber Types Type II Fibers

Type I Fibers

Type IIa

Type IIb

Slow-twitch (slow speed of cont raction) Slow-oxidative (low glycogen content)

Intermediate-twitch (fast speed of contraction) Fast-oxidative glycolytic fibers (intermediate glycogen levels) Intermediate fiber diameter

Fast-twitch (fast speed of contraction) Fast-glycolytic (high glycogen content)

High myoglobin content (appear red) Small fiber diameter Increased concentration of capillaries surrounding muscle (greater oxygen delivery) High capacity for aerobic metabolism

High myoglobin content (appear red) Increased oxidative capacity on training Intermediate resistance to fatigue

High resistance to fatigue Used for prolonged, aerobic exercise

Low myoglobin content (appear white) Low mitochondrial content Limited aerobic metabolism Large fiber diameter More sensitive to fatigue compared with other fiber types Least efficient use of energy, primarily glycolytic Used for sprinting and resistance tasks

Muscle fibers can be classified as either fast twitch or slow twitch. The slowtwitch fibers, or type I fibers (also called slow-oxidative fibers), contain large amounts of mitochondria and myoglobin (giving them a red color), use respiration and oxidative phosphorylation for energy, and are relatively resistant to fatigue. Compared with fast-twitch fibers, their glycogen content is low. The slow-twitch fibers develop force slowly but maintain contractions longer than fast-twitch muscles. The fast-twitch fibers, or type II, can be subdivided as type IIa or type IIb. Type IIb fibers (also called fast-glycolytic fibers) have few mitochondria and low levels of myoglobin (hence, they appear white). They are rich in glycogen and use glycogenolysis and glycolysis as their primary energy source. These muscles are prone to fatigue because continued reliance on glycolysis to produce ATP leads to an increase in lactic acid levels, resulting in a drop in the intracellular pH. As the pH drops, the ability of the muscle to produce ATP also diminishes. However, fasttwitch muscle can develop greater force than slow-twitch muscle, so contractions occur more rapidly. Type IIa fibers (also called fast-oxidative glycolytic fibers) have properties of both type I and IIb fibers and thus display functional characteristics of both fiber types. The properties of types I, IIa, and IIb fibers are summarized in Table 47.1. Muscles are a mixture of the different fiber types, but depending on the function, a muscle may have a preponderance of one fiber type over another. Type I fibers are found in postural muscles such as the psoas in the back musculature or the soleus in the leg. The ratio of type I to type II varies with the muscle. The triceps, which functions phasically, has 32.6% type I, whereas the soleus, which functions tonically, has 87.7% type I. Type II fibers are more prevalent in the large muscles of the limbs that are responsible for sudden, powerful movements. Extraocular muscles would also have more of these fibers than type I.

B. Smooth Muscle Cells Smooth muscle cells are found in the digestive system, blood vessels, bladder, airways, and uterus. The cells have a spindle shape with a central nucleus (see Fig. 47.1B). The designation “smooth” refers to the fact that these cells, which contain a single nucleus, display no striations under the microscope. The contraction

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of smooth muscle is controlled involuntarily (the cells contract and relax without any conscious attempt to have them do so; examples of smooth muscle activity include moving food along the digestive tract, altering the diameter of the blood vessels, and expelling urine from the bladder). In contrast to skeletal muscle, these cells have the ability to maintain tension for extended periods, and do so efficiently, with a low use of energy.

C. Cardiac Muscle Cells The cardiac muscle cells are similar to skeletal muscle in that they are striated (contain fibers), but like smooth muscle cells, they are regulated involuntarily (we do not have to think about making our heart beat). The cells are quadrangular in shape (see Fig. 47.1C) and form a network with multiple other cells through tight membrane junctions and gap junctions. The multicellular contacts allow the cells to act as a common unit and to contract and relax synchronously. Cardiac muscle cells are designed for endurance and consistency. They depend on aerobic metabolism for their energy needs because they contain many mitochondria and very little glycogen. These cells thus generate only a small amount of their energy from glycolysis using glucose derived from glycogen. A reduced flow of oxygen-rich blood to the heart muscle may lead to a myocardial infarction (heart attack). The amount of ATP that can be generated by glycolysis alone is not sufficient to meet the energy requirements of the contracting heart.

II. NEURONAL SIGNALS TO MUSCLE For an extensive review of how muscle contracts or a detailed view of the signaling to allow muscle contraction, consult a medical physiology book. Only a brief overview is presented here. The nerve–muscle cell junction is called the neuromuscular junction (Fig. 47.2). When appropriately stimulated, the nerve cell releases acetylcholine at the junction, which binds to acetylcholine receptors on the muscle membrane. This binding stimulates the opening of sodium channels on the sarcolemma. The massive influx of sodium ions results in the generation of an action potential in the sarcolemma at the edges of the motor end plate of the neuromuscular junction. The action potential sweeps across the surface of the muscle fiber and down the transverse tubules to the sarcoplasmic reticulum, where it initiates the release of calcium

The ryanodine receptors are calcium release channels found in the endoplasmic reticulum and sarcoplasmic reticulum of muscle cells. One type of receptor can be activated by a depolarization signal (depolarization-induced calcium release). Another receptor type is activated by calcium ions (calcium-induced calcium release). The proteins received their name because they bind ryanodine, a toxin obtained from the stem and roots of the plant Ryania speciosa. Ryanodine inhibits sarcoplasmic reticulum calcium release and acts as a paralytic agent. It was first used commercially in insecticides.

Schwann cell Synaptic vesicles

Axon terminal

Synaptic cleft

Acetylcholine

Sarcolemma Voltage-gated Na+ channels

Acetylcholine receptor

Muscle cell

FIG. 47.2. The neuromuscular junction. When they are stimulated appropriately, the synaptic vesicles, containing acetylcholine, fuse with the axonal membrane and release acetylcholine into the synaptic cleft. The acetylcholine binds to its receptors on the muscle cells, which initiate signaling for muscle contraction.

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1 Synaptic

Acetylcholine

cleft

Sarcolemma

Na+ K+

2 Na+ SR

3

K+

Δψ

Ca2+

T-tubule

SR

4

Ca

2+

T-tubule

Ca2+ Sarcoplasm

FIG. 47.3. Events leading to sarcoplasmic reticulum calcium release in skeletal muscle. (1) Acetylcholine, released at the synaptic cleft, binds to acetylcholine receptors on the sarcolemma, leading to a change of conformation of the receptors so that they now act as an ion pore. This allows sodium to enter the cell and potassium to leave. (2) The membrane polarization that results from these ion movements is transmitted throughout the muscle fiber by the T-tubule system. (3) A receptor in the T tubules (the dihydropyridine receptor [DHPR]) is activated by membrane polarization (a voltage-gated activation) so that activated DHPR binds physically to and activates the ryanodine receptor in the sarcoplasmic reticulum (depolarization-induced calcium release). (4) The activation of the ryanodine receptor, which is a calcium channel, leads to calcium release from the sarcoplasmic reticulum (SR) into the sarcoplasm. In cardiac muscle, activation of DHPR leads to calcium release from the T tubules, and this small calcium release is responsible for the activation of the cardiac ryanodine receptor (calcium-induced calcium release) to release large amounts of calcium into the sarcoplasm. Acetylcholine levels in the neuromuscular junction are rapidly reduced by the enzyme acetylcholinesterase. A number of nerve gas poisons act to inhibit acetylcholinesterase (such as sarin and VX), so that muscles are continuously stimulated to contract. This leads to blurred vision, bronchoconstriction, seizures, respiratory arrest, and death. The poisons are covalent modifiers of acetylcholinesterase; therefore, recovery from exposure to such poisons requires the synthesis of new enzyme. A new generation of acetylcholinesterase inhibitors, which act reversibly (i.e., they do not form covalent bonds with the enzyme), is now being used to treat dementia, in particular dementia as brought about by Alzheimer disease.

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from its lumen, via the ryanodine receptor (Fig. 47.3). The calcium ion binds to troponin, resulting in a conformational change in the troponin–tropomyosin complexes so that they move away from the myosin-binding sites on the actin. When the binding site becomes available, the myosin head attaches to the myosin-binding site on the actin. The binding is followed by a conformational change (pivoting) in the myosin head, which shortens the sarcomere. After the pivoting, ATP binds the myosin head, which detaches from the actin and is available to bind another myosin-binding site on the actin. As long as calcium ion and ATP remain available, the myosin heads will repeat this cycle of attachment, pivoting, and detachment (Fig. 47.4). This movement requires ATP, and when ATP levels are low (such as occurs during ischemia), the ability of the muscle to relax or contract is compromised. As the calcium release channel closes, the calcium is pumped back into the sarcoplasmic reticulum against its concentration gradient using the energyrequiring protein SERCA (sarcoplasmic reticulum Ca2⫹ ATPase), and contraction stops. This basic process occurs in all muscle cell types, with some slight variations between cell types.

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A Z-line

Actin thin filament

Myosin thick filament

Z-line

Resting

Contracted

B 1. Troponin

Actin 4.

Ca2+

Tropomyosin Myosin head

ADP +Pi

Direction of movement of the actin

ADP +Pi

ADP +Pi

Ca2+ ADP +Pi

Myosin

5.

2.

Ca2+

Ca2+

Ca2+

Ca2+

ATP

ATP

Ca2+

Ca2+

Binding site ADP +Pi

ADP +Pi

3.

6.

ADP +Pi

ADP +Pi

ADP +Pi ATP

ADP +Pi ATP

FIG. 47.4. An overview of muscle contraction. A. Muscle contraction. During muscle contraction, the myosin head binds to the actin thin filament. Pivoting of the myosin head toward the center of the sarcomere pulls the Z-lines closer together, with subsequent shortening of the sarcomere. B. A closer look at myosin–actin interactions. (1) A resting sarcomere. The troponin–tropomyosin complex is blocking the myosin-binding sites on the actin. The myosin head is already energized to power a contraction. (2) Exposure of the active site. After calcium binding to troponin, a conformational change in the troponin molecule pulls the troponin away from the binding site. (3) Cross-bridge attachment. Once the binding sites on the actin are exposed, the myosin head binds to it. (4) Myosin head pivoting. After cross-bridge attachment, the energy stored in the myosin head is released, and the myosin head pivots toward the center of the sarcomere (power stroke). Now the ADP and phosphate bound to the myosin head are released. (5) Detachment of the crossbridge. Now a molecule of ATP binds to the myosin head with simultaneous detachment of the myosin head from the binding site on the actin molecule. (6) Reactivation of the myosin head. The ATPase activity of the myosin head hydrolyzes the ATP into ADP and phosphate. The energy released from the hydrolysis of this high-energy bond is used to reenergize the myosin head, and the entire cycle can be repeated as long as calcium is present and there are sufficient ATP reserves. 889

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III. GLYCOLYSIS AND FATTY ACID METABOLISM IN MUSCLE CELLS The pathways of glycolysis and fatty acid oxidation in muscle are the same as has been previously described (see Chapters 22 and 23). The difference between muscles and other tissues is how these pathways are regulated. Phosphofructokinase-2 (PFK-2) is negatively regulated by phosphorylation in the liver (the enzyme that catalyzes the phosphorylation is the cyclic adenosine monophosphate [cAMP]–dependent protein kinase). However, in skeletal muscle, PFK-2 is not regulated by phosphorylation. This is because the skeletal muscle isozyme of PFK-2 lacks the regulatory serine residue, which is phosphorylated in the liver. However, the cardiac isozyme of PFK-2 is phosphorylated and activated by a kinase cascade initiated by insulin. This allows the heart to activate glycolysis and to use blood glucose when blood glucose levels are elevated. The adenosine monophosphate (AMP)-activated protein kinase also activates cardiac PFK-2 (kinase activity) as a signal that energy is low. Fatty acid uptake by muscle requires the participation of fatty acid–binding proteins and the usual enzymes of fatty acid oxidation. Fatty acyl-CoA uptake into the mitochondria is controlled by malonyl-CoA, which is produced by an isozyme of acetyl-CoA carboxylase (ACC-2; the ACC-1 isozyme is found in liver and adipose tissue cytosol and is used for fatty acid biosynthesis). ACC-2 (a mitochondrial protein, linked to carnitine palmitoyl transferase I [CPTI] in the outer mitochondrial membrane) is inhibited by phosphorylation by the AMP-activated protein kinase (AMP-PK) so that when energy levels are low, the levels of malonyl-CoA drop, allowing fatty acid oxidation by the mitochondria. In addition, muscle cells also contain the enzyme malonyl-CoA decarboxylase, which is activated by phosphorylation by the AMP-PK. Malonyl-CoA decarboxylase converts malonyl-CoA to acetyl-CoA, thereby relieving the inhibition of CPTI and stimulating fatty acid oxidation (Fig. 47.5). Muscle cells do not synthesize fatty acids; the presence of acetyl-CoA carboxylase in muscle is exclusively for regulatory purposes. Mice that have been bred to lack ACC-2 have a 50% reduction of fat stores compared with control mice. This was shown to be attributable to a 30% increase in skeletal muscle fatty acid oxidation resulting from dysregulation of CPTI, brought about by the lack of malonyl-CoA inhibition of CPTI.

CPT-1 Mitochondrial matrix

Fatty-acyl CoA AMP

–

Acetyl CoA MCoADC

2

+

+

AMPPK

Malonyl CoA

1

3

ACC-2

–

Acetyl CoA

FIG. 47.5. Regulation of fatty acyl-CoA entry into muscle mitochondria. (1) Acetyl-CoA carboxylase-2 (ACC-2) converts acetyl-CoA to malonyl-CoA, which inhibits carnitine palmitoyl transferase I (CPTI), thereby blocking fatty acyl-CoA entry into the mitochondria. (2) However, as energy levels drop, AMP levels rise because of the activity of the adenylate kinase reaction. (3) The increase in AMP levels activates the AMP-activated protein kinase (AMP-PK), which phosphorylates and inactivates ACC-2, and also phosphorylates and activates malonyl-CoA decarboxylase (MCoADC). The decarboxylase converts malonylCoA to acetyl-CoA, thereby relieving the inhibition of CPTI and allowing fatty acyl-CoA entry into the mitochondria. This allows the muscle to generate ATP via the oxidation of fatty acids.

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IV. FUEL USE IN CARDIAC MUSCLE A. Normal Conditions The heart uses primarily fatty acids (60% to 80%), lactate, and glucose (20% to 40%) as its energy sources. Ninety-eight percent of cardiac ATP is generated by oxidative means; 2% is derived from glycolysis. The lactate used by the heart is taken up by a monocarboxylate transporter in the cell membrane that is also used for the transport of ketone bodies. However, ketone bodies are not a preferred fuel for the heart; the heart prefers to use fatty acids. Lactate is generated by red blood cells and working skeletal muscle. When the lactate is used by the heart, it is oxidized to carbon dioxide and water, following the pathway lactate to pyruvate, pyruvate to acetyl-CoA, acetyl-CoA oxidation in the tricarboxylic acid (TCA) cycle, and ATP synthesis through oxidative phosphorylation. An alternative fate for lactate is its use in the reactions of the Cori cycle in the liver. Glucose transport into the cardiocyte occurs via both GLUT1 and GLUT4 transporters, although approximately 90% of the transporters are GLUT4. Insulin stimulates an increase in the number of GLUT4 transporters in the cardiac cell membrane, as does myocardial ischemia. This ischemia-induced increase in GLUT4 transporter number is additive to the effect of insulin on the translocation of GLUT4 transporters to the plasma membrane. Fatty acid uptake into cardiac muscle is similar to that for other muscle cell types and requires fatty acid–binding proteins and carnitine palmitoyl transferase I for transfer into the mitochondria. Fatty acid oxidation in cardiac muscle cells is regulated by altering the activities of ACC-2 and malonyl-CoA decarboxylase. Under conditions in which ketone bodies are produced, fatty acid levels in the plasma are also elevated. Because the heart preferentially burns fatty acids as a fuel rather than the ketone bodies produced by the liver, the ketone bodies are spared for use by the nervous system.

B. Ischemic Conditions When blood flow to the heart is interrupted, the heart switches to anaerobic metabolism. The rate of glycolysis increases, but the accumulation of protons (via lactate formation) is detrimental to the heart. Ischemia also increases the levels of free fatty acids in the blood and, surprisingly, when oxygen is reintroduced to the heart, the high rate of fatty acid oxidation in the heart is detrimental to the recovery of the damaged heart cells. Fatty acid oxidation occurs so rapidly that NADH accumulates in the mitochondria, leading to a reduced rate of NADH shuttle activity, an increased cytoplasmic NADH level, and lactate formation, which generates more protons. In addition, fatty acid oxidation increases the levels of mitochondrial acetyl-CoA, which inhibits pyruvate dehydrogenase, leading to cytoplasmic pyruvate accumulation and lactate production. As lactate production increases and the intracellular pH of the heart drops, it is more difficult to maintain ion gradients across the sarcolemma. ATP hydrolysis is required to repair these gradients, which are essential for heart function. However, the use of ATP for gradient repair reduces the amount of ATP available for the heart to use in contraction, which, in turn, compromises the ability of the heart to recover from the ischemic event.

A new class of drugs, known as partial fatty acid oxidation (pFOX) inhibitors, is being developed to reduce the extensive fatty acid oxidation in heart after an ischemic episode. The reduction in fatty acid oxidation induced by the drug will allow glucose oxidation to occur and reduce lactate buildup in the damaged heart muscle. Other possible targets of such drugs, which have yet to be developed, include ACC-2, malonylCoA decarboxylase, and carnitine palmitoyl transferase I.

V. FUEL USE IN SKELETAL MUSCLE Skeletal muscles use many fuels to generate ATP. The most abundant immediate source of ATP is creatine phosphate. ATP also can be generated from glycogen stores, either anaerobically (generating lactate) or aerobically, in which case pyruvate is converted to acetyl-CoA for oxidation via the TCA cycle. All human skeletal muscles have some mitochondria and thus are capable of fatty acid and ketone body oxidation. Skeletal muscles are also capable of completely oxidizing the carbon skeletons of alanine, aspartate, glutamate, valine, leucine, and isoleucine, but not other amino acids. Each of these fuel oxidation pathways plays a particular role in skeletal muscle metabolism.

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SECTION VIII ■ TISSUE METABOLISM

O

H2N

NH

ATP

ADP

C

H O P~ N

NH C

O

N

CH3

N

Creatine (phospho) kinase (CPK or CK)

CH2 C O

–

–

CH3

CH2 C

OH

HO

O

Creatine phosphate

Creatine

FIG. 47.6. The creatine phosphokinase reaction. The high-energy bond is the unusual nitrogen–phosphate bond, as indicated by the red squiggle.

A. Adenosine Triphosphate and Creatine Phosphate ATP is not a good choice as a molecule to store in quantity for energy reserves. Many reactions are allosterically activated or inhibited by ATP levels, especially those that generate energy. Muscle cells solve this problem by storing high-energy phosphate bonds in the form of creatine phosphate. When energy is required, creatine phosphate donates a phosphate to adenosine diphosphate (ADP) to regenerate ATP for muscle contraction (Fig. 47.6). Creatine synthesis begins in the kidney and is completed in the liver. In the kidney, glycine combines with arginine to form guanidinoacetate. In this reaction, the guanidinium group of arginine (the group that also forms urea) is transferred to glycine, and the remainder of the arginine molecule is released as ornithine. Guanidinoacetate then travels to the liver, where it is methylated by S-adenosylmethionine to form creatine (Fig. 47.7).

NH2 NH C NH

Arginine

(CH2)3 H NH2

C

NH2 NH C

NH2

NH

COOH

CH2

CH2

C O

OH

NH2 Ornithine

Glycine

H

(CH2)3 C

NH2

C O

OH

Guanidinoacetate

COOH

SAM

Kidney

Liver

S-adenosyl homocysteine NH2 NH C

To:

Brain Heart Skeletal muscle

N

CH3

CH2 C O

OH

Creatine

FIG. 47.7. The synthesis of creatine from arginine, glycine, and S-adenosylmethionine. Synthesis originates in the kidney and is completed in the liver.

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–

–

O

O

H – O P~ N

–

NH

O P OH Pi

C

O

893

N

H N

O CH3

CH2

Spontaneous cyclization (nonenzymatic)

C

NH

O C CH2

N

CH3

C HO

O

Creatine phosphate

Creatinine

Muscle and Brain FIG. 47.8.

The spontaneous production of creatinine from creatine phosphate.

The creatine formed is released from the liver and travels through the bloodstream to other tissues, particularly brain, heart, and skeletal muscle, where it reacts with ATP to form the high-energy compound creatine phosphate (see Fig. 47.6). This reaction, catalyzed by creatine phosphokinase (CK, also abbreviated as CPK), is reversible. Therefore, cells can use creatine phosphate to regenerate ATP. Creatine phosphate serves as a small reservoir of high-energy phosphate that can readily regenerate ATP from ADP. As a result, it plays a particularly important role in muscle during exercise. It also carries high-energy phosphate from mitochondria, where ATP is synthesized, to myosin filaments, where ATP is used for muscle contraction. Creatine phosphate is an unstable compound. It spontaneously cyclizes, forming creatinine (Fig. 47.8). Creatinine cannot be further metabolized and is excreted in the urine. The amount of creatinine excreted each day is constant and depends on body muscle mass. Therefore, it can be used as a gauge for determining the amounts of other compounds excreted in the urine and as an indicator of renal excretory function. The daily volume of urine is determined by such factors as the volume of blood reaching the renal glomeruli and the amount of renal tubular fluid reabsorbed from the tubular urine back into the interstitial space of the kidneys over time. At any given moment, the concentration of a compound in a single urine specimen does not give a good indication of the total amount that is being excreted on a daily basis. However, if the concentration of the compound is divided by the concentration of creatinine, the result provides a better indication of the true excretion rate.

B. Fuel Use at Rest Muscle fuel use at rest is dependent on the serum levels of glucose, amino acids, and fatty acids. If blood glucose and amino acids are elevated, glucose will be converted to glycogen, and branched-chain amino acid metabolism will be high. Fatty acids will be used for acetyl-CoA production and will satisfy the energy needs of the muscle under these conditions. There is a balance between glucose oxidation and fatty acid oxidation, which is regulated by citrate. When the muscle cell has adequate energy, citrate leaves the mitochondria and activates ACC-2, which produces malonyl-CoA. The malonylCoA inhibits carnitine palmitoyl transferase-1, thereby reducing fatty acid oxidation by the muscle. Malonyl-CoA decarboxylase is also inactive because the AMPPK is not active in the fed state. Thus, the muscle regulates its oxidation of glucose and fatty acids in part through monitoring of cytoplasmic citrate levels.

C. Fuel Use during Starvation As blood glucose levels drop, insulin levels drop. This reduces the levels of GLUT4 transporters in the muscle membrane, and glucose use by muscle drops significantly. This conserves glucose for use by the nervous system and red blood

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Each kidney normally contains approximately 1 million glomerular units. Each unit is supplied by arterial blood via the renal arteries and acts as a “filter.” Metabolites such as creatinine leave the blood by passing through pores or channels in the glomerular capillaries and enter the fluid within the proximal kidney tubule for eventual excretion in the urine. When they are functionally intact, these glomerular tissues are impermeable to all but the smallest of proteins. When they are acutely inflamed, however, this barrier function is lost to varying degrees, and albumin and other proteins may appear in the urine. The marked inflammatory changes in the glomerular capillaries that accompany poststreptococcal glomerulonephritis significantly reduce the flow of blood to the filtering surfaces of these vessels. As a result, creatinine, urea, and other circulating metabolites that are filtered into the urine at a normal rate (the glomerular filtration rate [GFR]) in the absence of kidney disease now fail to reach the filters, and, therefore, they accumulate in the plasma. These changes explain Rena Felya’s laboratory profile during her acute inflammatory glomerular disease (glomerulonephritis). In most patients, prognosis is excellent, although in some patients recovery may not occur. Such patients may progress to chronic renal insufficiency and even renal failure.

Muscle and brain cells contain large amounts of creatine phosphokinase (CK), and damage to these cells causes the enzyme to leak into the blood. Serum CK is measured to diagnose and evaluate patients who have had strokes and heart attacks. The presence of 5% or more of the CK in the blood as the muscle isoform is indicative of a heart attack (see Chapters 8 and 9).

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cells. In cardiac muscle, PFK-2 is phosphorylated and activated by insulin. The lack of insulin results in a reduced use of glucose by these cells as well. Pyruvate dehydrogenase is inhibited by the high levels of acetyl-CoA and NADH being produced by fatty acid oxidation. Fatty acids become the muscle’s preferred fuel under starvation conditions. The AMP-PK is active because of lower than normal ATP levels, ACC-2 is inhibited, and malonyl-CoA decarboxylase is activated, thereby retaining full activity of CPTI. The lack of glucose reduces the glycolytic rate, and glycogen synthesis does not occur because of the inactivation of glycogen synthase by epinephrinestimulated phosphorylation. Recall that in prolonged starvation, muscle proteolysis is induced (in part by cortisol release) for gluconeogenesis by the liver. This does not, however, alter the use of fatty acids by the muscle for its own energy needs under these conditions.

D. Fuel Use during Exercise The rate of ATP use in skeletal muscle during exercise can be as much as 100 times greater than that in resting skeletal muscles; thus, the pathways of fuel oxidation must be rapidly activated during exercise to respond to the much greater demand for ATP. ATP and creatine phosphate would be rapidly used up if they were not continuously regenerated. The synthesis of ATP occurs from glycolysis (either aerobic or anaerobic) and oxidative phosphorylation (which requires a constant supply of oxygen). Anaerobic glycolysis is especially important as a source of ATP in three conditions. The first is during the initial period of exercise, before exercise-stimulated increase in blood flow and substrate and oxygen delivery begin, allowing aerobic processes to occur. The second condition in which anaerobic glycolysis is important is exercise by muscle containing predominately fast-twitch glycolytic muscle fibers because these fibers have low oxidative capacity and generate most of their ATP through glycolysis. The third condition is during strenuous activity, when the ATP demand exceeds the oxidative capacity of the tissue, and the increased ATP demand is met by anaerobic glycolysis. 1.

ANAEROBIC GLYCOLYSIS AT THE ONSET OF EXERCISE

During rest, most of the ATP required in all types of muscle fibers is obtained from aerobic metabolism. However, as soon as exercise begins, the demand for ATP increases. The amount of ATP present in skeletal muscle could sustain exercise for only 1.2 seconds if it were not regenerated, and the amount of phosphocreatine could sustain exercise for only 9 seconds if it were not regenerated. It takes longer than 1 minute for the blood supply to exercising muscle to increase significantly as a result of vasodilation and, therefore, oxidative metabolism of blood-borne glucose and fatty acids cannot increase rapidly at the onset of exercise. Thus, for the first few minutes of exercise, the conversion of glycogen to lactate provides a considerable portion of the ATP requirement. 2.

ANAEROBIC GLYCOLYSIS IN TYPE IIB FAST-TWITCH GLYCOLYTIC FIBERS

Although humans have no muscles that consist entirely of fast-twitch fibers, many other animals do. Examples are white abdominal muscles of fish and the pectoral muscles of game birds (turkey white meat). These muscles contract rapidly and vigorously (“fast twitch” refers to the time to peak tension), but only for short periods. Thus, they are used for activities such as flight by birds and for sprinting and weight lifting by humans. In such muscles, the glycolytic capacity is high because the enzymes of glycolysis are present in large amounts (thus, the overall Vmax [maximum velocity] is large). The levels of hexokinase, however, are low, so very little circulating glucose

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895

is used. The low levels of hexokinase in fast-twitch glycolytic fibers prevent the muscle from drawing on blood glucose to meet this high demand for ATP, thus avoiding hypoglycemia. Glucose 6-phosphate, formed from glycogenolysis, further inhibits hexokinase. The tissues rely on endogenous fuel stores (glycogen and creatine phosphate) to generate ATP, following the pathway of glycogen breakdown to glucose 1-phosphate, the conversion of glucose 1-phosphate to glucose 6-phosphate, and the metabolism of glucose 6-phosphate to lactate. Thus, anaerobic glycolysis is the main source of ATP during exercise of these muscle fibers. 3.

ANAEROBIC GLYCOLYSIS FROM GLYCOGEN

Glycogenolysis and glycolysis during exercise are activated together because both phosphofructokinase-1 (PFK-1) (the rate-limiting enzyme of glycolysis) and glycogen phosphorylase b (the inhibited form of glycogen phosphorylase) are allosterically activated by AMP. AMP is an ideal activator because its concentration is normally kept low by the adenylate kinase (also called myokinase in muscle) equilibrium (2ADP ↔ AMP ⫹ ATP). Thus, whenever ATP levels decrease slightly, the AMP concentration increases manyfold (Fig. 47.9). Starting from a molecule of glucose 1-phosphate derived from glycogenolysis, three ATP molecules are produced in anaerobic glycolysis, as compared with 31 to 33 molecules of ATP in aerobic glycolysis. To compensate for the low ATP yield of anaerobic glycolysis, fast-twitch glycolytic fibers have a much higher content of glycolytic enzymes, and the rate of glucose 6-phosphate use is more than 12 times as fast as in slow-twitch fibers. Muscle fatigue during exercise generally results from a lowering of the pH of the tissue to approximately 6.4. Both aerobic and anaerobic metabolism lowers the pH. Both the lowering of pH and lactate production can cause pain. Metabolic fatigue also can occur once muscle glycogen is depleted. Muscle glycogen stores are used up in ⬍2 minutes of anaerobic exercise. If you do pushups, you can prove this to yourself. The muscles used in push-ups, a high-strength exercise, are principally fast-twitch glycolytic fibers. Time yourself from the start of your push-ups. No matter how well you have trained, you probably cannot do push-ups for as long as 2 minutes. Furthermore, you will feel the pain as the muscle pH drops as lactate production continues. The regulation of muscle glycogen metabolism is complex. Recall that glycogen degradation in muscle is not sensitive to glucagon (muscles lack glucagon receptors), so there is little change in muscle glycogen stores during overnight fasting or long-term fasting, if the individual remains at rest. Glycogen synthase is inhibited during exercise but can be activated in resting muscle by the release of insulin after a high-carbohydrate meal. Unlike the liver form of glycogen phosphorylase, the muscle isozyme contains an allosteric site for AMP binding. When AMP binds to

Glycogen + Phosphorylase b

Glucose 1-phosphate 2ADP

Adenylate kinase

ATP +

AMP

Muscle contraction

Glucose 6-phosphate + PFK-1

Pyruvate ADP

Pi

FIG. 47.9. Activation of muscle glycogenolysis and glycolysis by AMP. As muscle contracts, ATP is converted to ADP and inorganic phosphate (Pi). In the adenylate kinase reaction, two ADP molecules react to form ATP and AMP. The ATP is used for contraction. As AMP accumulates, it activates glycogenolysis and glycolysis.

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SECTION VIII ■ TISSUE METABOLISM

muscle glycogen phosphorylase b, the enzyme is activated even though it is not phosphorylated. Thus, as muscle begins to work and the myosin-ATPase hydrolyzes existing ATP stores to ADP, AMP begins to accumulate (because of the adenylate kinase reaction), and glycogen degradation is enhanced. The activation of muscle glycogen phosphorylase b is further enhanced by the release of Ca2⫹ from the sarcoplasmic reticulum, which occurs when muscles are stimulated to contract. The increase in sarcoplasmic Ca2⫹ also leads to the allosteric activation of glycogen phosphorylase kinase (through binding to the calmodulin subunit of the enzyme), which phosphorylates muscle glycogen phosphorylase b, fully activating it. And, finally, during intense exercise, epinephrine release stimulates the activation of adenylate cyclase in muscle cells, thereby activating the cAMP-dependent protein kinase (see Fig. 28.10). Protein kinase A phosphorylates and fully activates glycogen phosphorylase kinase so that continued activation of muscle glycogen phosphorylase can occur. The hormonal signal is slower than the initial activation events triggered by AMP and calcium (Fig. 47.10).

Epinephrine +

adenylate cyclase

Cell membrane

1 ATP

cAMP

Protein kinase (inactive)

Regulatory subunit-cAMP

2

ADP Phosphorylase kinase (inactive)

Ca2+–calmodulin

4

ATP active protein kinase A +

3 ADP

Glycogen

5 Phosphorylase b (inactive) +

AMP

ATP Glycogen synthase (active)

Phosphorylase kinase– P (active)

ATP

Glycogen synthase– P (inactive)

Pi ADP

Phosphorylase a (active) P

6

Glucose 1-phosphate

Glucose 6-phosphate Muscle Lactate or CO2 + H2O

FIG. 47.10. Stimulation of glycogenolysis in muscle by epinephrine. (1) Epinephrine binding to its receptor leads to the activation of adenylate cyclase, which increases cAMP levels. (2) cAMP binds to the regulatory subunits of protein kinase A, thereby activating the catalytic subunits. (3) Active protein kinase A phosphorylates and activates phosphorylase kinase. Phosphorylase kinase also can be activated partially by the Ca2⫹–calmodulin complex as Ca2⫹ levels increase as muscles contract. (4) Protein kinase A phosphorylates and inactivates glycogen synthase. (5) Active phosphorylase kinase converts glycogen phosphorylase b to glycogen phosphorylase a. (6) Glycogen degradation forms glucose 1-phosphate, which is converted to glucose 6-phosphate, which enters the glycolytic pathway for energy production.

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4.

ANAEROBIC GLYCOLYSIS DURING HIGH-INTENSITY EXERCISE

Once exercise begins, the electron-transport chain, the TCA cycle, and fatty acid oxidation are activated by the increase of ADP and the decrease of ATP. Pyruvate dehydrogenase remains in the active, nonphosphorylated state as long as NADH can be reoxidized in the electron-transport chain and acetyl-CoA can enter the TCA cycle. However, even though mitochondrial metabolism is working at its maximum capacity, additional ATP may be needed for very strenuous, high-intensity exercise. When this occurs, ATP is not being produced rapidly enough to meet the muscle’s needs, and AMP begins to accumulate. Increased AMP levels activate PFK-1 and glycogenolysis, thereby providing additional ATP from anaerobic glycolysis (the additional pyruvate produced does not enter the mitochondria but rather is converted to lactate so that glycolysis can continue). Thus, under these conditions, most of the pyruvate formed by glycolysis enters the TCA cycle, whereas the remainder is reduced to lactate to regenerate NAD⫹ for continued use in glycolysis. 5.

897

If Otto Shape runs at a pace at which his muscles require approximately 500 kcal/hour, how long could he run on the amount of glucose that is present in circulating blood? Assume that the blood volume is 5 L.

FATE OF LACTATE RELEASED DURING EXERCISE

The lactate that is released from skeletal muscles during exercise can be used by resting skeletal muscles or by the heart, a muscle with a large amount of mitochondria and very high oxidative capacity. In such muscles, the NADH/NAD⫹ ratio is lower than in exercising skeletal muscle, and the lactate dehydrogenase reaction proceeds in the direction of pyruvate formation. The pyruvate that is generated is then converted to acetyl-CoA and oxidized in the TCA cycle, producing energy by oxidative phosphorylation. The second potential fate of lactate is that it will return to the liver through the Cori cycle, where it is converted to glucose (see Fig. 22.10).

VI. MILD- AND MODERATE-INTENSITY LONG-TERM EXERCISE A. Lactate Release Decreases with Duration of Exercise Mild-to-moderate intensity exercise can be performed for longer periods than can high-intensity exercise. This is because of the aerobic oxidation of glucose and fatty acids, which generates more energy per fuel molecule than anaerobic metabolism, and which also produces lactic acid at a slower rate than anaerobic metabolism. Thus, during mild- and moderate-intensity exercise, the release of lactate diminishes as the aerobic metabolism of glucose and fatty acids becomes predominant.

B. Blood Glucose as a Fuel At any given time during fasting, the blood contains only approximately 5 g of glucose, enough to support a person running at a moderate pace for a few minutes. Therefore, the blood glucose supply must be constantly replenished. The liver performs this function by processes similar to those used during fasting. The liver produces glucose by breaking down its own glycogen stores and by gluconeogenesis. The major source of carbon for gluconeogenesis during exercise is, of course, lactate, produced by the exercising muscle, but amino acids and glycerol are also used (Fig. 47.11). Epinephrine released during exercise stimulates liver glycogenolysis and gluconeogenesis by causing cAMP levels to increase. During long periods of exercise, blood glucose levels are maintained by the liver through hepatic glycogenolysis and gluconeogenesis. The amount of glucose that the liver must export is greatest at higher workloads, in which case the muscle is using a greater proportion of the glucose for anaerobic metabolism. With increasing duration of exercise, an increasing proportion of blood glucose is supplied by gluconeogenesis. For up to 40 minutes of mild exercise, glycogenolysis is mainly responsible for the glucose output of the liver. However, after 40 to 240 minutes of exercise, the total glucose output of the liver decreases. This is caused by the increased use of fatty acids, which are being released from adipose tissue triacylglycerols (stimulated by epinephrine release). Glucose uptake by the muscle is stimulated by the increase

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2.0 Glycogen

mmol/min

1.5

1.0

0.5

25%

Pyruvate Glycerol Amino acids Lactate

Pyruvate Glycerol Amino acids

23%

Lactate

40 min Basal

45%

240 min Exercise

FIG. 47.11. Production of blood glucose by the liver from various precursors during rest and during prolonged exercise. The green area represents the contribution of liver glycogen to blood glucose, and the lighter area represents the contribution of gluconeogenesis. (From Wahren J, et al. In: Howald H, Poortmans JR, eds. Metabolic Adaptation to Prolonged Physical Exercise. Cambridge, MA: Birkhauser; 1973:148.) Remember from Chapter 1 that a food calorie (cal) is equivalent to 1 kcal of energy. One gram of glucose can give rise to 4 kcal of energy, so at a rate of consumption of 500 kcal/hour we have (500 kcal/h) ⫻ (1 g glucose/4 kcal energy) ⫻ (1 h/60 min) ⫽ 2 g glucose/min Thus, Otto must use 2 g of glucose per minute to run at his current pace. In the fasting state, blood glucose levels are approximately 90 mg/dL, or 900 mg/L. Because blood volume is estimated at 5 L, Otto has 4.5 g glucose available. If it is not replenished, that amount of glucose will support only 2.5 minutes of running at 2 g glucose per minute.

in AMP levels and the activation of the AMP-activated protein kinase, which stimulates the translocation of GLUT4 transporters to the muscle membrane. The hormonal changes that direct the increased hepatic glycogenolysis, hepatic gluconeogenesis, and adipose tissue lipolysis include a decrease in insulin and an increase in glucagon, epinephrine, and norepinephrine. Plasma levels of growth hormone, cortisol, and thyroid-stimulating hormone (TSH) also increase and may contribute to fuel mobilization as well (see Chapter 43). The activation of hepatic glycogenolysis occurs through glucagon and epinephrine release. Hepatic gluconeogenesis is activated by the increased supply of precursors (lactate, glycerol, amino acids, and pyruvate), the induction of gluconeogenic enzymes by glucagon and cortisol (this occurs only during prolonged exercise), and the increased supply of fatty acids to provide the ATP and NADH needed for gluconeogenesis and the regulation of gluconeogenic enzymes.

C. Free Fatty Acids as a Source of Adenosine Triphosphate The longer the duration of the exercise, the greater the reliance of the muscle on free fatty acids for the generation of ATP (Fig. 47.12). Because ATP generation from free fatty acids depends on mitochondria and oxidative phosphorylation, longdistance running uses muscles that are principally slow-twitch oxidative fibers, such as the gastrocnemius. It is also important to realize that resting skeletal muscle uses free fatty acids as a principal fuel. At almost anytime except the postprandial state (right after eating), free fatty acids are the preferred fuel for skeletal muscle. The preferential use of fatty acids over glucose as a fuel in skeletal muscle depends on the following factors: 1. The availability of free fatty acids in the blood, which depends on their release from adipose tissue triacylglycerols by hormone-sensitive lipase. During prolonged exercise, the small decrease of insulin and increases of glucagon, epinephrine and norepinephrine, cortisol, and possibly growth hormone all activate adipocyte tissue lipolysis.

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899

Fuel as % of total O2 uptake

100 Muscle glycogen 75 Blood-borne fatty acids 50 Blood-borne glucose 25 Exhaustion 0

1

2

3

4

Hours

FIG. 47.12. Fuels used during exercise. The pattern of fuel use changes with the duration of the exercise. (From Felig P, Baxter JD, Broadus AE, et al. Endocrinology & Metabolism. New York, NY: McGraw-Hill; 1981:796.)

2. Inhibition of glycolysis by products of fatty acid oxidation. Pyruvate dehydrogenase activity is inhibited by acetyl-CoA, NADH, and ATP, all of which are elevated as fatty acid oxidation proceeds. As AMP levels drop and ATP levels rise, PFK-1 activity is decreased (see Chapter 22). 3. Glucose transport may be reduced during long-term exercise. Glucose transport into skeletal muscles via the GLUT4 transporter is greatly activated by either insulin or exercise. During long-term exercise, the effect of falling insulin levels or increased fatty acid levels may counteract the stimulation of glucose transport by the exercise itself. 4. Oxidation of ketone bodies also increases during exercise. Their use as a fuel is dependent on their rate of production by the liver. Ketone bodies are never, however, a major fuel for skeletal muscle (muscles prefer free fatty acids). 5. Acetyl-CoA carboxylase (isozyme ACC-2) must be inactivated for the muscle to use fatty acids. This occurs as the AMP-PK is activated and phosphorylates ACC-2, rendering it inactive, and activates malonyl-CoA decarboxylase, to reduce malonyl-CoA levels and allow full activity of CPTI.

D. Branched-Chain Amino Acids Branched-chain amino acid oxidation has been estimated to supply a maximum of 20% of the ATP supply of resting muscle. Oxidation of branched-chain amino acids in muscle serves two functions. The first is the generation of ATP, and the second is the synthesis of glutamine, which effluxes from the muscle. The highest rates of branched-chain amino acid oxidation occur under conditions of acidosis, in which there is a higher demand for glutamine to transfer ammonia to the kidney and to buffer the urine as ammonium ion during proton excretion. Recall that glutamine synthesis occurs from the carbon skeletons of branched-chain amino acid oxidation (valine and isoleucine) after the initial five steps of the oxidative pathway.

E. The Purine Nucleotide Cycle Exercise increases the activity of the purine nucleotide cycle, which converts aspartate to fumarate plus ammonia (see Fig. 41.13). The ammonia is used to buffer the proton production and lactate production from glycolysis, and the fumarate is recycled and can form glutamine.

F. Acetate Acetate is an excellent fuel for skeletal muscle. It is treated by the muscle as a very short-chain fatty acid. It is activated to acetyl-CoA in the cytosol and then transferred into the mitochondria via acetylcarnitine transferase, an isozyme of carnitine

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SECTION VIII ■ TISSUE METABOLISM

palmitoyl transferase. Sources of acetate include the diet (vinegar is acetic acid) and acetate produced in the liver from alcohol metabolism. Certain commercial power bars for athletes contain acetate.

VII. METABOLIC EFFECTS OF TRAINING ON MUSCLE METABOLISM The effect of training depends, to some extent, on the type of training. In general, training increases the muscle glycogen stores and increases the number and size of mitochondria. The fibers thus increase their capacity for generation of ATP from oxidative metabolism and their ability to use fatty acids as a fuel. The winners in marathon races seem to use muscle glycogen more efficiently than others. Training to improve strength, power, and endurance of muscle performance is called resistance training. Its goal is to increase the size of the muscle fibers (hypertrophy of the muscle). Muscle fibers can develop a maximal force of 3 to 4 kg/cm2 of muscle area. Thus, if we could increase our muscle size from 80 to 120 cm2, the maximal resistance that could be lifted would increase from 240 to 360 kg. Hypertrophy occurs by increased protein synthesis in the muscle and a reduction in existing protein turnover. CLINICAL COMMENTS Rena Felya. Poststreptococcal glomerulonephritis (PSGN) may follow pharyngeal or cutaneous infection with one of a limited number of “nephritogenic” strains of group A ␤-hemolytic streptococci. The pathogenesis of PSGN involves a host immune (antibody) response to one or more of the enzymes secreted by the bacterial cells. The antigen–antibody complexes are deposited on the tissues of glomerular units, causing a local acute inflammatory response. Hypertension may occur as a consequence of sodium and water retention caused by an inability of the inflamed glomerular units to filter sodium and water into the urine. Proteinuria is usually mild if the immune response is self-limited. Overall, one of the most useful clinical indicators of glomerular filtration rate in both health and disease is the serum creatinine concentration. The endogenous production of creatinine, which averages approximately 15 mg/kg of body weight per day, is correlated with muscle mass and, therefore, tends to be constant for a given individual if renal function is normal. Any rise in serum creatinine in a patient such as Rena Felya, therefore, can be assumed to result from decreased excretion of this metabolite into the urine. The extent of the rise in the blood is related directly to the severity of the pathologic process involving the glomerular units in the kidneys. BIOCHEMICAL COMMENTS The SERCA Pump. The SERCA pump is a transmembrane protein of 110 kDa that is present in several different isoforms throughout the body. Three genes encode SERCA proteins: designated SERCA1, SERCA2, and SERCA3. The SERCA1 gene produces two alternatively spliced transcripts: SERCA1a and SERCA1b. SERCA1b is expressed in the fetal and neonatal fasttwitch skeletal muscles and is replaced by SERCA1a in adult fast-twitch muscles. The SERCA2 gene also undergoes alternative splicing, producing the SERCA2a and SERCA2b isoforms. The SERCA2b isoform is expressed in all cell types and is associated with inositol trisphosphate (IP3)-regulated calcium stores. SERCA2a is the primary isoform expressed in cardiac tissue. SERCA3 produces at least five different alternatively spliced isoforms, which are specifically expressed in different tissues. SERCA2a plays an important role in cardiac contraction and relaxation. Contraction is initiated by the release of calcium from intracellular stores, whereas relaxation occurs as the calcium is resequestered in the sarcoplasmic reticulum, in part mediated by the SERCA2a protein. The SERCA2a pump is regulated, in part,

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CHAPTER 47 ■ METABOLISM OF MUSCLE AT REST AND DURING EXERCISE

901

by its association with the protein phospholamban (PLN). PLN is a pentameric molecule consisting of five identical subunits of molecular weight 22,000 Da. PLN associates with SERCA2a in the sarcoplasmic reticulum and reduces its pumping activity. Because new contractions cannot occur until cytosolic calcium has been resequestered into the sarcoplasmic reticulum, a reduction in SERCA2a activity increases the relaxation time. However, when called on, the heart can increase its rate of contractions by inhibiting the activity of phospholamban. This is accomplished by phosphorylation of phospholamban by protein kinase A (PKA). Epinephrine release stimulates the heart to beat faster. This occurs through epinephrine binding to its receptor, activating a G protein, which leads to adenylate cyclase activation, elevation of cAMP levels, and activation of protein kinase A. PKA phosphorylates PLN, thereby reducing its association with SERCA2a and relieving the inhibition of pumping activity. This results in reduced relaxation times and more frequent contractions. Mutations in PLN lead to cardiomyopathies, primarily an autosomal dominant form of dilated cardiomyopathy. This particular mutation substitutes an arginine in place of cysteine at position 9 in PLN, which forms an inactive complex with PKA and blocks PKA phosphorylation of PLN. Individuals with this form of PLN develop cardiomyopathy in their teens. In this condition, the cardiac muscle does not pump well (because of the constant inhibition of SERCA2a) and becomes enlarged (dilated). Because of the poor pumping action of the heart, fluid can build up in the lungs. The pulmonary congestion results in a sense of breathlessness (left heart failure). Eventually, progressive left heart failure leads to fluid accumulation in other tissues and organs of the body, such as the legs and ankles (right heart failure). Key Concepts •

• • • •

•

Muscle comprises three different types: skeletal, smooth, and cardiac. Skeletal muscle facilitates movement of the skeleton. ■ Slow-twitch fibers contain large amounts of mitochondria and myoglobin, and generate energy primarily via oxidative means. ■ Fast-twitch fibers have few mitochondria, low levels of myoglobin, and are rich in glycogen. These fibers generate energy primarily via glycolysis. Smooth muscle cells display no striations and aid in maintaining the shape and movement of the blood vessels, airways, uterus, and digestive systems. Cardiac muscle cells contain striations but are regulated involuntarily. They use aerobic metabolism, oxidizing fatty acids, glucose, and lactate, and they contain many mitochondria, with very little glycogen. Acetylcholine release at the neuromuscular junction leads to muscle contraction. Fatty acid oxidation in muscle is controlled by the levels of malonyl-CoA produced by a musclespecific isozyme of acetyl-CoA carboxylase (ACC-2). Skeletal muscle uses many fuels to generate ATP, storing excess high-energy phosphate bonds as creatine phosphate. Muscle fuel use is regulated carefully. At rest, the muscle uses what is available in the blood (glucose, amino acids, fatty acids). During starvation, fatty acids are the preferred energy source (even over ketone bodies). During exercise, stored glycogen, blood glucose, and blood fatty acids are the primary sources of energy for the skeletal muscles. Diseases discussed in this chapter are summarized in Table 47.2.

Table 47.2

Diseases Discussed in Chapter 47

Disease or Disorder

Genetic or Environmental

Renal failure

Both

The lack of kidney function can lead to encephalopathy due to the buildup of toxic metabolites in the blood.

Duchenne muscular dystrophy

Genetic

The lack of dystrophin, due to deletions in the DMD gene on the X chromosome, lead to muscle dysfunction at an early age.

Comments

DMD, Duchenne muscular dystrophy.

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REVIEW QUESTIONS—CHAPTER 47 1.

2.

3.

The process of stretching before exercise has which of the following biochemical benefits? A. Stimulates the release of epinephrine B. Activates glycolysis in the liver C. Increases blood flow to the muscles D. Activates glycolysis in the muscles E. Stimulates glycogenolysis in the liver The major metabolic fuel for participating in a prolonged aerobic exercise event is which of the following? A. Liver glycogen B. Muscle glycogen C. Brain glycogen D. Adipose triacylglycerol E. Red blood cell–produced lactate A 24-hour urine collection showed that an individual’s excretion of creatinine was much lower than normal. Decreased excretion of creatinine could be caused by which of the following? A. A decreased dietary intake of creatine B. A higher than normal muscle mass resulting from weight lifting C. A genetic defect in the enzyme that converts creatine phosphate to creatinine

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D. Kidney failure E. A vegetarian diet 4.

In the biosynthetic pathways for the synthesis of heme, creatine, and guanine, which one of the following amino acids directly provides carbon atoms that appear in the final product? A. Serine B. Aspartate C. Cysteine D. Glutamate E. Glycine

5.

In skeletal muscle, increased hydrolysis of ATP during muscular contraction leads to which of the following? A. A decrease in the rate of palmitate oxidation to acetyl-CoA B. A decrease in the rate of NADH oxidation by the electron-transport chain C. Activation of PFK-1 D. An increase in the proton gradient across the inner mitochondrial membrane E. Activation of glycogen synthase

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48

Metabolism of the Nervous System

The nervous system consists of various cell types. The most abundant cell in the nervous system is the glial cell, which consists of astrocytes and oligodendrocytes in the central nervous system, and Schwann cells in the peripheral nervous system. These cells provide support for the neurons and synthesize the protective myelin sheath that surrounds the axons emanating from the neurons. Microglial cells in the nervous system act as immune cells, destroying and ingesting foreign organisms that enter the nervous system. The interface between the brain parenchyma and the cerebrospinal fluid compartment is formed by the ependymal cells, which line the cavities of the brain and spinal cord. These cells use their cilia to allow for the circulation of the cerebrospinal fluid (CSF), which bathes the cells of the central nervous system. The cells of the brain are separated from free contact with the rest of the body by the blood–brain barrier. The capillaries of the brain exhibit features, such as tight endothelial cell junctions, that restrict their permeability to metabolites in the blood. This protects the brain from compounds that might be toxic or otherwise interfere with nerve impulse transmission. It also affects the entry of precursors for brain metabolic pathways such as fuel metabolism and neurotransmitter synthesis. Neurotransmitters can be divided structurally into two categories: small nitrogen-containing neurotransmitters and neuropeptides. The small nitrogen-containing neurotransmitters are generally synthesized in the presynaptic terminal from amino acids and intermediates of glycolysis and the tricarboxylic acid (TCA) cycle. They are retained in storage vesicles until the neuron is depolarized. The catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine) are derived from tyrosine. Serotonin is synthesized from tryptophan. Acetylcholine is synthesized from choline, which can be supplied from the diet or is synthesized and stored as part of phosphatidylcholine. Glutamate and its neurotransmitter derivative, ␥-aminobutyric acid (GABA), are derived from ␣-ketoglutarate in the TCA cycle. Glycine is synthesized in the brain from serine. The synthesis of the neurotransmitters is regulated to correspond to the rate of depolarization of the individual neurons. Several cofactors are required for the synthesis of neurotransmitters, and deficiencies of pyridoxal phosphate, thiamine pyrophosphate, and vitamin B12 result in a variety of neurologic dysfunctions. Brain metabolism has a high requirement for glucose and oxygen. Deficiencies of either (hypoglycemia or hypoxia) affect brain function because they influence adenosine triphosphate (ATP) production and the supply of precursors for neurotransmitter synthesis. Ischemia elicits a condition in which increased calcium levels, swelling, glutamate excitotoxicity, and nitric oxide generation affect brain function and can lead to a stroke. The generation of free radicals and abnormalities in nitric oxide production are important players in the pathogenesis of a variety of neurodegenerative diseases. Because of the restrictions posed by the blood–brain barrier to the entry of a variety of substances into the central nervous system, the brain generally synthesizes and degrades its own lipids. Essential fatty acids can enter the brain but the

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more common fatty acids do not. The turnover of lipids at the synaptic membrane is very rapid, and the neuron must replace those lipids lost during exocytosis. The glial cells produce the myelin sheath, which is composed primarily of lipids. These lipids are of a different composition than those of the neuronal cells. Because there is considerable lipid synthesis and turnover in the brain, this organ is sensitive to disorders of peroxisomal function (Refsum disease; interference in very long-chain fatty acid oxidation and ␣-oxidation) and lysosomal diseases (mucopolysaccharidoses; inability to degrade complex lipids and glycolipids).

THE WAITING ROOM Katie Colamin, a 34-year-old dress designer, developed alarming palpitations of her heart while bending forward to pick up her cat. She also developed a pounding headache and sweated profusely. After 5 to 10 minutes, these symptoms subsided. One week later, her aerobic exercise instructor, a registered nurse, noted that Katie grew very pale and was tremulous during exercise. The instructor took Katie’s blood pressure, which was 220 mm Hg systolic (normal, up to 120 at rest) and 132 mm Hg diastolic (normal, up to 80 at rest). Within 15 minutes, Katie recovered, and her blood pressure returned to normal. The instructor told Katie to see her physician the next day. The doctor told Katie that her symptom complex coupled with severe hypertension strongly suggested the presence of a tumor in the medulla of one of her adrenal glands (a pheochromocytoma) that was episodically secreting large amounts of catecholamines, such as norepinephrine (noradrenaline) and epinephrine (adrenaline). Her blood pressure was normal until moderate pressure to the left of her umbilicus caused Katie to suddenly develop a typical attack, and her blood pressure rose rapidly. In addition to ordering several biochemical tests, Katie also was immediately scheduled for a magnetic resonance imaging (MRI) study of her abdomen and pelvis. The MRI showed a 3.5 ⫻ 2.8 ⫻ 2.6 cm mass in the left adrenal gland, with imaging characteristics typical of a pheochromocytoma. Ivan Applebod’s brother, Evan Applebod, is 6 ft tall and weighed 425 lb (BMI ⫽ 57.6 kg/m2). He had only been successful in losing weight once in his life: in 1977, when he had lost 30 lb through a combination of diet and exercise with the support of a registered dietian. However, he quickly regained this weight over the next year. Evan’s weight was not usually a concern for him, but in 1997, he became worried when it became difficult for him to take walks or go fishing because of joint pain in his knees. He was also suffering from symptoms suggestive of a peripheral neuropathy, manifesting primarily as tingling in his legs. In 1997, Evan approached his physician desperate for help to lose weight. The physician placed Evan on a new drug, Redux, which had just been approved for use as a weight-loss agent, and a low-fat, low-calorie diet along with physical therapy to help increase his activity level. In 4 months, Evan’s weight dropped from 425 to 375 lb, his total cholesterol dropped from 250 to 185 mmol/L, and his serum triglycerides dropped from 375 to 130 mmol/L. However, Redux was withdrawn from the market by its manufacturer late in 1997 because of its toxicity. Evan was then placed on Prozac, a drug used primarily as an antidepressant and less commonly as an appetite suppressant.

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I.

905

CELL TYPES OF THE NERVOUS SYSTEM

The nervous system consists of neurons, the cells that transmit signals, and supporting cells, the neuroglia. The neuroglia consists of oligodendrocytes and astrocytes (known collectively as glial cells), microglial cells, ependymal cells, and Schwann cells. The neuroglia are designed to support and sustain the neurons and do so by surrounding neurons and holding them in place, supplying nutrients and oxygen to the neurons, insulating neurons so their electrical signals are more rapidly propagated, and cleaning up any debris that enters the nervous system. The central nervous system (CNS) consists of the brain and spinal cord. This system integrates all signals emanating from the peripheral nervous system (PNS). The PNS is composed of all neurons that lie outside the CNS.

A. Neurons Neurons consist of a cell body (soma) from which long (axons) and short (dendrites) extensions protrude. Dendrites receive information from the axons of other neurons, whereas the axons transmit information to other neurons. The axon–dendrite connection is known as a synapse (Fig. 48.1). Most neurons contain multiple dendrites, each of which can receive signals from multiple axons. This configuration allows a single neuron to integrate information from multiple sources. Although neurons also contain just one axon, most axons branch extensively and distribute information to multiple targets (divergence). The neurons transmit signals by changes in the electrical potential across their membrane. Signaling across a synapse requires the release of neurotransmitters that, when bound to their specific receptors, initiate an electrical signal in the receiving or target cell. Neurons are terminally differentiated cells and, as such, have little capability for division. As a result, injured neurons have a limited capacity to repair themselves and frequently undergo apoptosis (programmed cell death) when damaged.

B. Neuroglial Cells 1.

ASTROCYTES

The astrocytes are found in the CNS and are star-shaped cells that provide physical and nutritional support for neurons. During development of the CNS, the astrocytes guide neuronal migration to their final adult position and form a matrix that keeps neurons in place. These cells serve several functions, including phagocytosing debris left behind by cells, providing lactate (from glucose metabolism) as a carbon

Cell body (soma) Neuron

Axon

Synapse Dendrite Soma

FIG. 48.1. A neuron consists of a cell body (soma) with short extensions (dendrites) and a long extension (axon). The axon–dendrite interface is the synapse. A soma may receive input from multiple axons.

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source for the neurons, and controlling the brain extracellular ionic environment. Astrocytes help to regulate the content of the extracellular fluid (ECF) by taking up, processing, and metabolizing nutrients and waste products. 2.

OLIGODENDROCYTES

Oligodendrocytes provide the myelin sheath that surrounds the axon, acting as “insulation” for many of the neurons in the CNS. The myelin sheath is the lipid– protein covering of the axons (see Section V.B for a description of the composition and synthesis of the myelin sheath). Oligodendrocytes can form myelin sheaths around multiple neurons in the CNS by sending out processes that bind to the axons on target neurons. The speed with which a neuron conducts its electric signal (action potential) is directly proportional to the degree of myelination. Oligodendrocytes, along with the astrocytes, form a supporting matrix for the neurons. Oligodendrocytes have a limited capacity for mitosis, and if damaged, do not replicate. If this occurs, demyelination of the axons may occur, resulting in abnormalities in signal conduction along that axon (see Biochemical Comments). 3.

SCHWANN CELLS

Schwann cells are the supporting cells of the PNS. Like oligodendrocytes, Schwann cells form myelin sheaths around the axons, but unlike the oligodendrocytes, Schwann cells myelinate only one axon. Schwann cells also clean up cellular debris in the PNS. The Schwann cells also provide a means for peripheral axons to regenerate if damaged. There is a synergistic interaction among the Schwann cells, secreted growth factors, and the axon that allows damaged axons to reconnect to the appropriate target axon. 4.

MICROGLIAL CELLS

The microglial cells are the smallest glial cells in the nervous system. They serve as immunologically responsive cells that function similarly to the action of macrophages in the circulation. Microglial cells destroy invading microorganisms and phagocytose cellular debris. 5.

EPENDYMAL CELLS

The ependymal cells are ciliated cells that line the cavities (ventricles) of the CNS and the spinal cord. In some areas of the brain, the ependymal cells are functionally specialized to elaborate and secrete cerebrospinal fluid (CSF) into the ventricular system. The beating of the ependymal cilia allow for efficient circulation of the CSF throughout the CNS. The CSF acts as both a shock absorber protecting the CNS from mechanical trauma and as a system for the removal of metabolic wastes. The CSF can be aspirated from the spinal canal and analyzed to determine whether disorders of CNS function, with their characteristic CSF changes, are present. For many years, it had been believed that damaged neurons in the CNS could not regenerate because it was thought that there are no pluripotent stem cells (cells that can differentiate into various cell types found in the CNS) in the CNS. However, recent data suggest that cells found within the ependymal layer can act as neural stem cells, which under appropriate stimulation can regenerate neurons. Such a finding opens up a large number of potential treatments for diseases that alter neuronal cell function.

II. THE BLOOD–BRAIN BARRIER A. Capillary Structure In the capillary beds of most organs, rapid passage of molecules occurs from the blood through the endothelial wall of the capillaries into the interstitial fluid. Thus, the composition of interstitial fluid resembles that of blood, and specific receptors or transporters in the plasma membrane of the cells being bathed by the interstitial fluid may interact directly with amino acids, hormones, or other compounds from

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the blood. In the brain, transcapillary movement of substrates in the peripheral circulation into the brain is highly restricted by the blood–brain barrier. This barrier limits the accessibility of blood-borne toxins and other potentially harmful compounds to the neurons of the CNS. The blood–brain barrier begins with the endothelial cells that form the inner lining of the vessels supplying blood to the CNS (Fig. 48.2). Unlike the endothelial cells of other organs, these cells are joined by tight junctions that do not permit the movement of polar molecules from the blood into the interstitial fluid bathing the neurons. They also lack mechanisms for transendothelial transport that are present in other capillaries of the body. These mechanisms include fenestrations (“windows” or pores that span the endothelial lining and permit the rapid movement of molecules across membranes) or transpinocytosis (vesicular transport from one side of the endothelial cell to another). The endothelial cells serve actively, as well as passively, to protect the brain. Because they contain a variety of drug-metabolizing enzyme systems similar to the drug-metabolizing enzymes found in the liver, the endothelial cells can metabolize neurotransmitters and toxic chemicals and, therefore, form an enzymatic barrier to entry of these potentially harmful substances into the brain. They actively pump hydrophobic molecules that diffuse into endothelial cells back into the blood (especially xenobiotics) with P-glycoproteins, which act as transmembranous, ATP-dependent efflux pumps. Although lipophilic substances, water, oxygen, and carbon dioxide can readily cross the blood–brain barrier by passive diffusion, other molecules depend on specific transport systems. Differential transporters on the luminal and abluminal endothelial membranes can transport compounds into, as well as out of, the brain. Further protection against the free entry of blood-borne compounds into the CNS is provided by a continuous collagen-containing basement membrane that completely surrounds the capillaries. The basement membrane appears to be surrounded by the foot processes of astrocytes. Thus, compounds must pass through endothelial cell membranes, the enzymatic barrier in the endothelial cells, the basement membrane, and possibly additional cellular barriers formed by the astrocytes to reach the neurons in the brain.

907

Inside of capillary

3

1

2

4 5 1

Tight junctions between endothelial cells

2

Narrow intercellular spaces

3

Lack of pinocytosis

4

Continuous basement membrane

5

Astrocyte extension

FIG. 48.2. The blood–brain barrier. Compounds in the blood cannot pass freely into the brain; they must traverse the endothelial cells, basement membrane, and astrocytes using specific carriers to gain access to the brain. Very lipophilic molecules may pass through all of these membranes in the absence of a carrier.

B. Transport through the Blood–Brain Barrier Many nonpolar substances, such as drugs and inert gases, probably diffuse through the endothelial cell membranes. A large number of other compounds are transported through the endothelial capillaries by facilitative transport, whereas others, such as nonessential fatty acids, cannot cross the blood–brain barrier. Essential fatty acids, however, are transported across the barrier. 1.

FUELS

Glucose, which is the principal fuel of the brain, is transported through both endothelial membranes by facilitated diffusion via the GLUT-1 transporter (see Fig. 27.14). GLUT-3 transporters present on the neurons then allow the neurons to transport the glucose from the ECF. Glial cells express GLUT-1 transporters. Although the rate of glucose transport into the ECF normally exceeds the rate required for energy metabolism by the brain, glucose transport may become ratelimiting as blood glucose levels fall lower than the normal range. Thus, individuals begin to experience hypoglycemic symptoms at approximately 60 mg/dL, as the glucose levels are reduced to the Km, or lower than the Km values of the GLUT-1 transporters in the endothelial cells of the barrier. Monocarboxylic acids, including L-lactate, acetate, pyruvate, and the ketone bodies acetoacetate and ␤-hydroxybutyrate, are transported by a separate stereospecific system that is slower than the transport system for glucose. During starvation, when the level of ketone bodies in the blood is elevated, this transporter is

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Several disorders of glucose transport across the blood–brain barrier are known. The most common of these is facilitated glucose transporter protein type 1 (GLUT-1) deficiency syndrome. In this disorder, GLUT-1 transporters are impaired, which results in a low glucose concentration in the CSF (a condition known as hypoglycorrhachia). A diagnostic indication of this disorder is that in normal blood glucose levels, the ratio of CSF glucose to blood glucose levels is ⬍0.4. Clinical features are variable but include seizures, developmental delay, and a complex motor disorder. These symptoms are the result of inadequate glucose levels in the brain. The disorder can be treated by prescribing a ketogenic diet (high-fat, low-carbohydrate). This will force the patient to produce ketone bodies, which are easily transported into the CNS and can spare the brain’s requirement for glucose as an energy source.

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The finding that the large neutral amino acids (LNAAs) have a common carrier system across the blood–brain barrier suggests that if one amino acid is in excess, it can, by competitive inhibition, result in lower transport of the other amino acids. This suggests that the mental retardation that results from untreated phenylketonuria (PKU) and maple syrup urine disease (see Chapter 39) may be attributable to the high levels of either phenylalanine or branchedchain amino acids in the blood. These high levels overwhelm the LNAA carrier, so excessive levels of the damaging amino acid enter the central nervous system (CNS). In support of this theory is the finding that treatment of PKU patients with large doses of LNAAs that lack phenylalanine resulted in a decrease of phenylalanine levels in the cerebrospinal fluid (CSF) and brain, with an improvement in the patients’ cognitive functions as well.

upregulated. Ketone bodies are important fuels for the brain of both adults and neonates during prolonged starvation (⬎48 hours). 2.

AMINO ACIDS AND VITAMINS

Large neutral amino acids (LNAAs), such as phenylalanine, leucine, tyrosine, isoleucine, valine, tryptophan, methionine, and histidine, enter the CSF rapidly via a single amino acid transporter (L [leucine preferring]-system amino acid transporter). Many of these compounds are essential in the diet and must be imported for protein synthesis or as precursors of neurotransmitters. Because a single transporter is involved, these amino acids compete with each other for transport into the brain. The entry of small neutral amino acids, such as alanine, glycine, proline, and ␥-aminobutyric acid (GABA), is markedly restricted because their influx could dramatically change the content of neurotransmitters (see Section III). They are synthesized in the brain, and some are transported out of the CNS and into the blood via the A (alanine-preferring)-system carrier. Vitamins have specific transporters through the blood–brain barrier, just as they do in most tissues. 3.

RECEPTOR-MEDIATED TRANSCYTOSIS

Certain proteins such as insulin, transferrin, and insulin-like growth factors cross the blood–brain barrier by receptor-mediated transcytosis. Once the protein binds to its membrane receptor, the membrane containing the receptor–protein complex is endocytosed into the endothelial cell to form a vesicle. It is released on the other side of the endothelial cell. Absorptive-mediated transcytosis also can occur. This differs from receptor-mediated transcytosis in that the protein binds nonspecifically to the membrane and not to a distinct receptor.

III. SYNTHESIS OF SMALL NITROGEN-CONTAINING NEUROTRANSMITTERS Molecules that serve as neurotransmitters fall into two basic structural categories: (1) small nitrogen-containing molecules and (2) neuropeptides. The major small nitrogen-containing molecule neurotransmitters include glutamate, GABA, glycine, acetylcholine, dopamine, norepinephrine, serotonin, and histamine. Additional neurotransmitters that fall into this category include epinephrine, aspartate, and nitric oxide. In general, each neuron synthesizes only those neurotransmitters that it uses for transmission through a synapse or to another cell. The neuronal tracts are often identified by their neurotransmitter; for example, a dopaminergic tract synthesizes and releases the neurotransmitter dopamine. Neuropeptides are usually small peptides that are synthesized and processed in the CNS. Some of these peptides have targets within the CNS (such as endorphins, which bind to opioid receptors and block pain signals), whereas others are released into the circulation to bind to receptors on other organs (such as growth hormone and thyroid-stimulating hormone). Many neuropeptides are synthesized as a larger precursor, which is then cleaved to produce the active peptides. Until recently, the assumption was that a neuron synthesized and released only a single neurotransmitter. More recent evidence suggests that a neuron may contain (1) more than one small-molecule neurotransmitter, (2) more than one neuropeptide neurotransmitter, or (3) both types of neurotransmitters. The differential release of the various neurotransmitters is the result of the neuron altering its frequency and pattern of firing.

A. General Features of Neurotransmitter Synthesis Several features are common to the synthesis, secretion, and metabolism of most small nitrogen-containing neurotransmitters (Table 48.1). Most of these neurotransmitters are synthesized from amino acids, intermediates of glycolysis and the TCA cycle, and O2 in the cytoplasm of the presynaptic terminal. The rate of synthesis is generally regulated to correspond to the rate of firing of the neuron. Once they are synthesized,

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CHAPTER 48 ■ METABOLISM OF THE NERVOUS SYSTEM

Table 48.1

909

Features Common to Neurotransmittersa

• Synthesis from amino acid and common metabolic precursors usually occurs in the cytoplasm of the presynaptic nerve terminal. The synthetic enzymes are transported by fast axonal transport from the cell body, where they are synthesized, to the presynaptic terminal. • The synthesis of the neurotransmitter is regulated to correspond to the rate of firing of the neuron, both acutely and through long-term enhancement of synaptic transmission. • The neurotransmitter is actively taken up into storage vesicles in the presynaptic terminal. • The neurotransmitter acts at a receptor on the postsynaptic membrane. • The action of the neurotransmitter is terminated through reuptake into the presynaptic terminal, diffusion away from the synapse, or enzymatic inactivation. The enzymatic inactivation may occur in the postsynaptic terminal, the presynaptic terminal, or an adjacent astrocyte or microglial cell. • The blood–brain barrier affects the supply of precursors for neurotransmitter synthesis. Not all neurotransmitters exhibit all of these features. Nitric oxide is an exception to most of these generalities. Some neurotransmitters (epinephrine, serotonin, and histamine) are also secreted by cells other than neurons. Their synthesis and secretion by nonneuronal cells follow other principles. a

the neurotransmitters are transported into storage vesicles by an ATP-requiring pump linked with the proton gradient. Release from the storage vesicle is triggered by the nerve impulse that depolarizes the postsynaptic membrane and causes an influx of Ca2⫹ ions through voltage-gated calcium channels. The influx of Ca2⫹ promotes fusion of the vesicle with the synaptic membrane and release of the neurotransmitter into the synaptic cleft. The transmission across the synapse is completed by binding of the neurotransmitter to a receptor on the postsynaptic membrane (Fig. 48.3). The action of the neurotransmitter is terminated through reuptake into the presynaptic terminal, uptake into glial cells, diffusion away from the synapse, or enzymatic inactivation. The enzymatic inactivation may occur in the postsynaptic terminal, the presynaptic terminal, or an adjacent astrocyte microglia cell or in endothelial cells in the brain capillaries. Not all neurotransmitters exhibit all of these features. Nitric oxide, because it is a gas, is an exception to most of these generalities. Some neurotransmitters are synthesized and secreted by both neurons and other cells (e.g., epinephrine, serotonin, histamine).

FIG. 48.3.

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Drugs have been developed that block neurotransmitter uptake into storage vesicles. Reserpine, which blocks catecholamine uptake into vesicles, had been used as an antihypertensive and antiepileptic drug for many years, but it was noted that a small percentage of patients on the drug became depressed and even suicidal. Animals treated with reserpine showed signs of lethargy and poor appetite, similar to depression in humans. Thus, a link was forged between monoamine release and depression in humans.

Action of neurotransmitters.

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Phenylalanine BH4

phenylalanine hydroxylase

BH2 Albinism NH+3

HO

O–

CH CH2

tyrosine hydroxylase

(Cu2+-dependent)

C

Dopa

Melanins

O L–Tyrosine

Melanocytes

BH4

tyrosine hydroxylase

BH2

OH NH+3

HO

O–

CH CH2

C O

Dopa dopa decarboxylase

PLP CO2

OH Neurons

HO CH2 CH2

NH+3

Dopamine Adrenal medulla

O2 dopamine

β-hydroxylase

Cu2+ Vitamin C

OH HO CH2 CH

NH+3

OH Norepinephrine phenylethanolamine N-methyltransferase

S-Adenosylmethionine S-Adenosylhomocysteine

OH HO CH2 CH

+

CH3

NH2

OH Epinephrine

FIG. 48.4. The pathways of catecholamine and melanin biosynthesis. The dark boxes indicate the enzymes, which, when defective, lead to albinism. BH4, tetrahydrobiopterin; PLP, pyridoxal phosphate; Dopa, dihydroxyphenylalanine.

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911

B. Dopamine, Norepinephrine, and Epinephrine 1.

SYNTHESIS OF THE CATECHOLAMINE NEUROTRANSMITTERS

The three neurotransmitters dopamine, norepinephrine, and epinephrine are synthesized in a common pathway from the amino acid L-tyrosine. Tyrosine is supplied in the diet or is synthesized in the liver from the essential amino acid phenylalanine by phenylalanine hydroxylase (see Chapter 39). The pathway of catecholamine biosynthesis is shown in Figure 48.4. The first and rate-limiting step in the synthesis of these neurotransmitters from tyrosine is the hydroxylation of the tyrosine ring by tyrosine hydroxylase, a tetrahydrobiopterin (BH4)-requiring enzyme. The product formed is dihydroxyphenylalanine (Dopa). The phenyl ring with two adjacent –OH groups is a catechol, and hence dopamine, norepinephrine, and epinephrine are called catecholamines. The second step in catecholamine synthesis is the decarboxylation of dopa to form dopamine. This reaction, like many decarboxylation reactions of amino acids, requires pyridoxal phosphate. Dopaminergic neurons (neurons that use dopamine as a neurotransmitter) stop the synthesis at this point because these neurons do not synthesize the enzymes required for the subsequent steps. Neurons that secrete norepinephrine synthesize it from dopamine in a hydroxylation reaction catalyzed by dopamine ␤-hydroxylase (DBH). This enzyme is present only within the storage vesicles of these cells. Like tyrosine hydroxylase, it is a mixed-function oxidase that requires an electron donor. Ascorbic acid (vitamin C) serves as the electron donor and is oxidized in the reaction. Copper (Cu2⫹) is a bound cofactor that is required for the electron transfer. Although the adrenal medulla is the major site of epinephrine synthesis, epinephrine is also synthesized in a few neurons that use it as a neurotransmitter. These neurons contain the above pathway for norepinephrine synthesis and, in addition, contain the enzyme that transfers a methyl group from S-adenosylmethionine (SAM) to norepinephrine to form epinephrine. Thus, epinephrine synthesis is dependent on the presence of adequate levels of vitamin B12 and folate in order to produce the SAM (see Chapter 40). 2.

Storage vesicle

ATP Chromogranins NT+ DBH

H+ ATP

H+

VMAT2

H+ ADP

V-ATPase

NT+

FIG. 48.5. Transport of catecholamines into storage vesicles. This is a secondary active transport based on the generation of a proton gradient across the vesicular membrane. DBH, dopamine ␤-hydroxylase; NT⫹, positively charged neurotransmitter (catecholamine); V-ATPase, vesicular ATPase; VMAT2, vesicle monoamine transporter 2.

STORAGE AND RELEASE OF CATECHOLAMINES

Ordinarily, only low concentrations of catecholamines are free in the cytosol, whereas high concentrations are found within the storage vesicles. Conversion of tyrosine to L-dopa and that of L-dopa to dopamine occurs in the cytosol. Dopamine is then taken up into the storage vesicles. In norepinephrine-containing neurons, the final ␤-hydroxylation reaction occurs in the vesicles. The catecholamines are transported into vesicles by the protein VMAT2 (vesicle monoamine transporter 2) (Fig. 48.5). The vesicle transporters contain 12 transmembrane domains and are homologous to a family of bacterial drug-resistant transporters, including P-glycoprotein. The mechanism that concentrates the catecholamines in the storage vesicles is an ATP-dependent process linked to a proton pump (secondary active transport). Protons are pumped into the vesicles by a vesicular ATPase (V-ATPase). The protons then exchange for the positively charged catecholamine via the transporter VMAT2. The influx of the catecholamine is thus driven by the H⫹ gradient across the vesicular membrane. The intravesicular concentration of catecholamines is approximately 0.5 M, roughly 100 times the cytosolic concentration. In the vesicles, the catecholamines exist in a complex with ATP and acidic proteins known as chromogranins. The vesicles play a dual role: They maintain a ready supply of catecholamines at the nerve terminal that is available for immediate release, and they mediate the process of release. When an action potential reaches the nerve terminal, Ca2⫹ channels open, allowing an influx of Ca2⫹, which promotes the fusion of vesicles with the neuronal membrane. The vesicles then discharge their soluble contents, including the neurotransmitters, ATP, chromogranins, and DBH, into the extraneuronal space

Lieberman_CH48.indd 911

Chromogranins are required for the biogenesis of the secretory vesicles. When chromogranins are released from the vesicles, they can be proteolytically clipped to form bioactive peptides. Elevated levels of chromogranins in the circulation may be found in patients who have neuroendocrine tumors, such as pheochromocytomas.

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SECTION VIII ■ TISSUE METABOLISM

In albinism, either the copperdependent tyrosine hydroxylase of melanocytes (which is distinct from the tyrosine hydroxylase found in the adrenal medulla) or other enzymes that convert tyrosine to melanins may be defective. Individuals with albinism suffer from a lack of pigment in the skin, hair, and eyes, and they are sensitive to sunlight.

Tyramine is a degradation product of tyrosine that can lead to headaches, palpitations, nausea and vomiting, and elevated blood pressure if it is present in large quantities. Tyramine leads to norepinephrine release, which binds to its receptors, stimulating them. Tyramine is inactivated by MAO-A, but if a person is taking a MAO inhibitor, foods containing tyramine should be avoided.

by the process of exocytosis. In some cases, the catecholamines affect other neurons. In other cases, they circulate in the blood and initiate responses in peripheral tissues. 3.

INACTIVATION AND DEGRADATION OF CATECHOLAMINE NEUROTRANSMITTERS

The action of catecholamines is terminated through reuptake into the presynaptic terminal and diffusion away from the synapse. Degradative enzymes are present in the presynaptic terminal and in adjacent cells, including glial cells and endothelial cells. Two of the major reactions in the process of inactivation and degradation of catecholamines are catalyzed by monoamine oxidase (MAO) and catechol-Omethyltransferase (COMT). MAO is present on the outer mitochondrial membrane of many cells and oxidizes the carbon containing the amino group to an aldehyde, thereby releasing ammonium ion (Fig. 48.6). In the presynaptic terminal, MAO inactivates catecholamines that are not protected in storage vesicles. (Thus, drugs that deplete storage vesicles indirectly increase catecholamine degradation.) There are two isoforms of MAO with different specificities of action: MAO-A preferentially deaminates norepinephrine and serotonin, whereas MAO-B acts on a wide spectrum of phenylethylamines (“phenylethyl” refers to a –CH2– group linked to a phenyl ring). MAO in the liver and other sites protects against the ingestion of dietary biogenic amines such as the tyramine found in many cheeses.

HO

OH +

HO

CH CH2 NH3 Norepinephrine

MAO +

NH4

Katie Colamin’s doctor ordered plasma fractionated metanephrine (methylated norepinephrine) levels and also had Katie collect a 24-hour urine specimen for the determination of catecholamines and their degradation products (in particular, metanephrine). All of these tests showed unequivocal elevations of these compounds in Katie’s blood and urine. Katie was placed on phenoxybenzamine, an ␣1- and ␣2-adrenergic receptor antagonist that blocks the pharmacologic effect of the elevated catecholamines on these receptors. Once stable on the ␣-blockers, Katie was started on an agent that blocked ␤-adrenergic receptors (propranolol). After ruling out evidence to suggest metastatic disease to the liver or other organs (in case Katie’s tumor was malignant), the doctor referred Katie to a surgeon with extensive experience in adrenal surgery. Katie’s doctor also ordered diagnostic studies to rule out multiple endocrine neoplasm (MEN) syndrome. The tests for the presence of MEN were negative.

Lieberman_CH48.indd 912

HO HO

OH

O

CH CH

SAH CH3O

OH

HO

CH CH2 NH3

Oxidation HO HO

COMT

SAM

NH4

+

MAO

+

OH CH COO–

COMT

Oxidation

SAM SAH

CH3O

OH

HO

CH COO–

3-Methoxy-4-hydroxymandelic acid (vanillylmandelic acid, VMA)

FIG. 48.6. Inactivation of catecholamines. Methylation and oxidation may occur in any order. Methylated and oxidized derivatives of norepinephrine and epinephrine are produced, and 3-methoxy-4-hydroxymandelic acid is the final product. These compounds are excreted in the urine. COMT, catechol-O-methyltransferase; MAO, monoamine oxidase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

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CHAPTER 48 ■ METABOLISM OF THE NERVOUS SYSTEM

COMT is also found in many cells, including erythrocytes. It works on a broad spectrum of extraneuronal catechols and those that have diffused away from the synapse. COMT transfers a methyl group from SAM to a hydroxyl group on the catecholamine or its degradation product (see Fig. 48.6). Because the inactivation reaction requires SAM, it is indirectly dependent on vitamin B12 and folate. The action of MAO and COMT can occur in almost any order, resulting in a large number of degradation products and intermediates, many of which appear in the urine. Cerebrospinal homovanillylmandelic acid (HVA) is an indicator of dopamine degradation. Its concentration is decreased in the brain of patients with Parkinson disease. 4.

REGULATION OF TYROSINE HYDROXYLASE

Efficient regulatory mechanisms coordinate the synthesis of catecholamine neurotransmitters with the rate of firing. Tyrosine hydroxylase, the first committed step and rate-limiting enzyme in the pathway, is regulated by feedback inhibition that is coordinated with depolarization of the nerve terminal. Tyrosine hydroxylase is inhibited by free cytosolic catecholamines that compete at the binding site on the enzyme for the pterin cofactor (BH4; see Chapter 39). Depolarization of the nerve terminal activates tyrosine hydroxylase. Depolarization also activates several protein kinases (including protein kinase C, protein kinase A [the cAMP-dependent protein kinase], and CAM kinases [Ca2⫹-calmodulin– dependent kinases]) that phosphorylate tyrosine hydroxylase. These activation steps result in an enzyme that binds BH4 more tightly, making it less sensitive to endproduct inhibition. In addition to these short-term regulatory processes, a long-term process involves alterations in the amounts of tyrosine hydroxylase and dopamine ␤-hydroxylase present in nerve terminals. When sympathetic neuronal activity is increased for a prolonged period, the amounts of mRNA that code for tyrosine hydroxylase and dopamine ␤-hydroxylase are increased in the neuronal perikarya (the cell body of the neuron). The increased gene transcription may be the result of phosphorylation of CREB (cAMP response element–binding protein; see Chapter 26) by protein kinase A or by other protein kinases. CREB then binds to the CRE (cAMP response element) in the promoter region of the gene (similar to the mechanism for the induction of gluconeogenic enzymes in the liver). The newly synthesized enzyme molecules are then transported down the axon to the nerve terminals. The concentration of dopamine decarboxylase in the terminal does not appear to change in response to neuronal activity.

C. Metabolism of Serotonin The pathway for the synthesis of serotonin from tryptophan is very similar to the pathway for the synthesis of norepinephrine from tyrosine (Fig. 48.7). The first enzyme of the pathway, tryptophan hydroxylase, uses an enzymatic mechanism similar to that of tyrosine and phenylalanine hydroxylase and requires BH4 to hydroxylate the ring structure of tryptophan. The second step of the pathway is a decarboxylation reaction catalyzed by the same enzyme that decarboxylates Dopa. Serotonin, like the catecholamine neurotransmitters, can be inactivated by MAO. The neurotransmitter melatonin is also synthesized from tryptophan (see Fig. 48.7). Melatonin is produced in the pineal gland in response to the light–dark cycle, its level in the blood is rising in a dark environment. It is probably through melatonin that the pineal gland conveys information about light–dark cycles to the body, organizing seasonal and circadian rhythms. Melatonin also may be involved in regulating reproductive functions.

913

In addition to the catecholamines, serotonin is also inactivated by monoamine oxidase (MAO). The activity of several antipsychotic drugs is based on inhibiting MAO. The first generation of drugs (exemplified by iproniazid, which was originally developed as an antituberculosis drug and was found to induce mood swings in patients) were irreversible inhibitors of both the A and B forms of MAO. Although they did reduce the severity of depression (by maintaining higher levels of serotonin), these drugs suffered from the “cheese” effect. Cheese and other foods that are processed over long periods (such as red wine) contain tyramine, a degradation product of tyrosine. Usually, tyramine is inactivated by MAO-A, but if an individual is taking a MAO inhibitor, tyramine level increases. Tyramine induces the release of norepinephrine from storage vesicles, which leads to potentially life-threatening hypertensive episodes. When it was realized that MAO existed in two forms, selective irreversible inhibitors were developed; examples include clorgyline for MAO-A and deprenyl for MAO-B. Deprenyl has been used to treat Parkinson disease (which is caused by a lack of dopamine, which is also inactivated by MAO). Deprenyl, however, is not an antidepressant. Clorgyline is an antidepressant but suffers from the “cheese” effect. This led to the development of the third generation of MAO inhibitors, which are reversible inhibitors of the enzyme, as typified by moclobemide. Moclobemide is a specific, reversible inhibitor of MAO-A and is effective as an antidepressant. More important, because of the reversible nature of the drug, the “cheese” effect is not observed because as tyramine levels increase, they displace the drug from MAO, and the tyramine is safely inactivated. NH2

OH Tyramine

D. Metabolism of Histamine Within the brain, histamine is produced both by mast cells and by certain neuronal fibers. Mast cells are a family of bone marrow–derived secretory cells that store and

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SECTION VIII ■ TISSUE METABOLISM

A potential complication of using heparin as an anticoagulant is the generation of anti-heparin antibodies in the patients. The production of such antibodies leads to the patient developing heparin-induced thrombocytopenia (HIT). The anti-heparin antibodies bind to the heparin platelet factor 4 complex and also to the platelet surface. The antibody-induced binding leads to platelet activation, serotonin release, and thrombin activation. The platelet number drops (the thrombocytopenia), but thrombus formation (caused by thrombin activation) increases. To determine if a patient has developed HIT, the serotonin-release assay has emerged as the gold standard. Platelets store serotonin in granules, and release the serotonin upon activation. Anti-heparin antibody sensitive platelets are obtained from donors and incubated with radioactively labeled serotonin in order for the platelets to incorporate the serotonin. The treated platelets are then incubated with two samples of the patient’s sera: one with a high level of exogenous heparin and the second with a low level of heparin. Serotonin release is then measured by determining the percentage of total radioactivity released by the platelets into the assay buffer. A positive result is obtained if, when using a low concentration of heparin, greater than 20% of the radioactivity incorporated into the platelets is released, and when using the high concentration of heparin, less than 20% of the incorporated radioactivity is released. The rationale behind the use of high heparin levels is to titrate out the antibody in the sera such that it cannot bind to the platelet cell surface, and to demonstrate that the release is caused by antibody binding to platelets.

+

CH NH3

CH2

Nicotinamide moiety of NAD(P)

COO– N H Tryptophan O2

BH4

H2O

BH2

HO

Tryptophan hydroxylase

CH2

+

CH NH3 COO–

N H 5-Hydroxytryptophan CO2 dopa decarboxylase

PLP

HO

CH2

CH2

+

NH3

MAO-A NH3

N H

HO

CH2

Serotonin Acetyl CoA

CH2

H

5-Hydroxyindoleacetaldehyde

O CH2

C

N

CoASH HO

O

NH C

NAD+

CH3

NADH N H

O HO

CH2

N-acetyl Serotonin SAM

O CH2

O–

N H

SAH CH3O

C

CH2

NH C

CH3

5-Hydroxyindole acetic acid

N H Melatonin

FIG. 48.7. Synthesis and inactivation of serotonin. The catecholamines exert their physiologic and pharmacologic effects by circulating in the bloodstream to target cells whose plasma membranes contain catecholamine receptors. This interaction initiates a biochemical cascade leading to responses that are specific for different types of cells. Patients such as Katie Colamin experience palpitations, excessive sweating, hypertensive headaches, and a host of other symptoms when a catecholamine-producing tumor of the adrenal medulla suddenly secretes supraphysiologic amounts of epinephrine and/ or norepinephrine into the venous blood draining the neoplasm.

Lieberman_CH48.indd 914

release high concentrations of histamine. They are prevalent in the thalamus, hypothalamus, dura mater, leptomeninges, and choroid plexus. Histaminergic neuronal cell bodies in humans are found in the tuberomammillary nucleus of the posterior basal hypothalamus. The fibers project into nearly all areas of the CNS, including the cerebral cortex, the brainstem, and the spinal cord. Histamine is synthesized from histidine in a single enzymatic step. The enzyme histidine decarboxylase requires pyridoxal phosphate, and its mechanism is very similar to that of dopa decarboxylase (Fig. 48.8). Like other neurotransmitters, newly synthesized neuronal histamine is stored in the nerve terminal vesicles. Depolarization of nerve terminals activates the exocytotic release of histamine by a voltage-dependent as well as a calcium-dependent mechanism. Once it is released from neurons, histamine is thought to activate both postsynaptic and presynaptic receptors. Unlike other neurotransmitters, histamine does

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CHAPTER 48 ■ METABOLISM OF THE NERVOUS SYSTEM

H C

CH2 +

HN

CO2

N

4

+

NH3

CH2

N

Brain

CH2

CH2

+

NH3

N N CH3 SAM SAH Histamine methyl transferase Methylhistamine

Diamine oxidase

CH2 HN

O

NH3

Histamine

Peripheral + tissues NH

–

PLP Histidine decarboxylase CH2

HN

O C

+

NH4

C

O H

N

MAO-B

CH2

CH3

N

C

O H

N

Imidazole acetaldehyde NAD+

NAD+

NADH

HN

NADH

O CH2 N

Imidazole acetic acid

C

O

O CH2 C

–

CH3

N

O–

N

Methylimidazole acetic acid

FIG. 48.8. Synthesis and inactivation of histamine; note the different pathways for brain and peripheral tissues. SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

not appear to be recycled into the presynaptic terminal to any great extent. However, astrocytes have a specific high-affinity uptake system for histamine and may be the major sites of the inactivation and degradation of this monoamine. The first step in the inactivation of histamine in the brain is methylation (see Fig. 48.8). The enzyme histamine methyltransferase transfers a methyl group from SAM to a nitrogen ring of histamine to form methylhistamine. The second step is oxidation by MAO-B, followed by an additional oxidation step. In peripheral tissues, histamine undergoes deamination by diamine oxidase, followed by oxidation to a carboxylic acid (see Fig. 48.8).

E. Acetylcholine 1.

915

Evan Applebod was placed on Redux, which increased the secretion of serotonin. Serotonin has been implicated in many processes, including mood control and appetite regulation. Serotonin agonists are thought to exert their hypophagic actions via stimulation of receptors located on proopiomelanocortin (POMC) containing neurons within the arcuate (ARC) nucleus of the hypothalamus. When serotonin levels are high, satiety results; when serotonin levels are low, increased appetite or depression or both can occur. Thus, drugs that can increase serotonin levels may be able to control appetite and depression. Redux was a second-generation drug developed from fenfluramine, a known appetite suppressant. When it was first used, fenfluramine could not be resolved between two distinct optical isomers (d versus l). The l-isomer induced sleepiness, so to counteract this effect; fenfluramine was often given with phentermine, which elevated norepinephrine levels to counteract the drowsiness (the combination of drugs was known as fen/phen). Once the two isomers of fenfluramine could be resolved, dexfenfluramine (Redux) was developed. Because levels of serotonin have been linked to mood, many antidepressant drugs were developed that affect serotonin levels. The first of these is the MAO inhibitors; the second class is the tricyclics; and the third class is known as selective serotonin reuptake inhibitors (SSRIs). The SSRIs block reuptake of serotonin from the synapse, leading to an elevated response to serotonin. Redux not only acted as an SSRI but also increased the secretion of serotonin, leading to elevated levels of this compound in the synapse. None of the other drugs that affect serotonin levels has this effect.

SYNTHESIS

The synthesis of acetylcholine from acetyl coenzyme A (acetyl-CoA) and choline is catalyzed by the enzyme choline acetyltransferase (ChAT) (Fig. 48.9). This synthetic step occurs in the presynaptic terminal. The compound is stored in vesicles and later released through calcium-mediated exocytosis. Choline is taken up by the presynaptic terminal from the blood via a low-affinity transport system (high Km) and from the synaptic cleft via a high-affinity transport mechanism (low Km). It is also derived from the hydrolysis of phosphatidylcholine (and possibly sphingomyelin) in membrane lipids. Thus, membrane lipids may form a storage site for choline, and their hydrolysis, with the subsequent release of choline, is highly regulated. Choline is a common component of the diet but also can be synthesized in the human as part of the pathway for the synthesis of phospholipids (see Chapter 33). The only route for choline synthesis is via the sequential addition of three methyl

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SECTION VIII ■ TISSUE METABOLISM

Histamine elicits several effects on different tissues. Histamine is the major mediator of the allergic response, and when it is released from mast cells (a type of white blood cell found in tissues), it leads to vasodilation and an increase in the permeability of blood vessel walls. This leads to the allergic symptoms of a runny nose and watery eyes. When histamine is released in the lungs, the airways constrict in an attempt to reduce the intake of the allergic material. The ultimate result of this, however, is bronchospasm, which can lead to difficulty in breathing. In the brain, histamine is an excitatory neurotransmitter. Antihistamines block histamine from binding to its receptor. In the tissues, this counteracts histamine’s effect on vasodilation and blood vessel wall permeability, but in the brain, the effect is to cause drowsiness. The new generation of “nondrowsy” antihistamines have been modified so that they cannot pass through the blood–brain barrier. Thus, the effects on the peripheral tissues are retained, with no effect on CNS histamine response.

It is believed that the vitamin B12 requirement for choline synthesis contributes to the neurologic symptoms of vitamin B12 deficiency. The methyl groups for choline synthesis are donated by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) in the reaction. Recall that formation of SAM through recycling of homocysteine requires both tetrahydrofolate and vitamin B12 (unless extraordinary amounts of methionine are available to bypass the B12-dependent methionine synthase step).

CH3

C

O SCoA + HO

CH3 CH2

CH2

+

N

CH3

CH3 Choline acetyltransferase

CoA

CH3

CH3

O C O

CH2

CH2

+

N

CH3

CH3 Acetylcholine Acetylcholinesterase

CH3

O C O– +

HO

Acetic acid

CH2CH2

+

N

(CH3)3

Choline

FIG. 48.9. Acetylcholine synthesis and degradation.

groups from SAM to the ethanolamine portion of phosphatidylethanolamine to form phosphatidylcholine. Phosphatidylcholine is subsequently hydrolyzed to release choline or phosphocholine. Conversion of phosphatidylethanolamine to phosphatidylcholine occurs in many tissues, including liver and brain. This conversion is folate-dependent and vitamin B12–dependent. The acetyl group used for acetylcholine synthesis is derived principally from glucose oxidation to pyruvate and decarboxylation of pyruvate to form acetyl-CoA via the pyruvate dehydrogenase reaction. This is because neuronal tissues have only a limited capacity to oxidize fatty acids to acetyl-CoA, so glucose oxidation is the major source of acetyl groups. Pyruvate dehydrogenase is found only in mitochondria. The acetyl group is probably transported to the cytoplasm as part of citrate, which is then cleaved in the cytosol to form acetyl-CoA and oxaloacetate. 2.

INACTIVATION OF ACETYLCHOLINE

Acetylcholine is inactivated by acetylcholinesterase, which is a serine esterase that forms a covalent bond with the acetyl group. The enzyme is inhibited by a wide range of compounds (pharmacologic agents and neurotoxins) that form a covalent bond with this reactive serine group. Neurotoxins such as Sarin (the gas used in Japanese subways by a terrorist group) and the nerve gas in the movie The Rock work through this mechanism. Acetylcholine is the major neurotransmitter at the neuromuscular junctions; inability to inactivate this molecule leads to constant activation of the nerve–muscle synapses, a condition that leads to varying degrees of paralysis.

F. Glutamate and ␥-Aminobutyric Acid 1.

SYNTHESIS OF GLUTAMATE

Glutamate functions as an excitatory neurotransmitter within the central nervous system, leading to the depolarization of neurons. Within nerve terminals, glutamate is generally synthesized de novo from glucose rather than taken up from the blood because its plasma concentration is low and it does not readily cross the blood– brain barrier. Glutamate is synthesized primarily from the TCA cycle intermediate ␣-ketoglutarate (Fig. 48.10). This process can occur via either of two routes. The first is via the enzyme glutamate dehydrogenase, which reduces ␣-ketoglutarate to glutamate, thereby incorporating free ammonia into the carbon backbone. The ammonia pool is provided by amino acid/neurotransmitter degradation or by diffusion of ammonia across the blood–brain barrier. The second route is through transamination reactions

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CHAPTER 48 ■ METABOLISM OF THE NERVOUS SYSTEM

O C

O

–

COO

+

H C

C

NH3

CH2

CH2

CH2 COO

NH2

–

COO

O

CH2

C O

–

C

CH2 –

COO–

␣-KG NH3

␣-KG

NH3 NADH

Glutaminase

O

CH2

GDH

PLP

NAD+

AA

␣-Keto acid –

COO +

H3N

C

O

H

C

CH2

H

C

CH2

CH2

–

PLP

CH2

COO

Glutamate CO2

C O O Succinate

PLP

–

+

H3N

␣-KG

CH2

O–

CH2

CH2

COO–

CO2

TCA cycle reactions AcCoA

CH2

GABA shunt

CH2 COO

O

–

␥-Aminobutyric acid (GABA)

917

The supply of choline in the brain can become rate-limiting for acetylcholine synthesis, and supplementation of the diet with lecithin (phosphatidylcholine) has been used to increase brain acetylcholine in patients suffering from tardive dyskinesia (often persistent involuntary movements of the facial muscles and tongue). The neonate has a very high demand for choline, for both brain lipid synthesis (phosphatidylcholine and sphingomyelin) and acetylcholine biosynthesis. High levels of phosphatidylcholine in maternal milk and a high activity of a high-affinity transport system through the blood–brain barrier for choline in the neonate help to maintain brain choline concentrations. The fetus also has a high demand for choline, and there is a highaffinity transport system for choline across the placenta. The choline is derived from maternal stores, maternal diet, and synthesis primarily in the maternal liver. Because choline synthesis is dependent on folate and vitamin B12, the high fetal demand may contribute to the increased maternal requirement for both vitamins during pregnancy. An inherited pyruvate dehydrogenase deficiency, a thiamine deficiency, or hypoxia deprives the brain of a source of acetyl-CoA for acetylcholine synthesis as well as a source of acetyl-CoA for adenosine triphosphate (ATP) generation from the TCA cycle.

FIG. 48.10. Synthesis of glutamate and GABA and the GABA shunt. ␣-KG, ␣-ketoglutarate; GDH, glutamate dehydrogenase; PLP, pyridoxal phosphate.

in which an amino group is transferred from other amino acids to ␣-ketoglutarate to form glutamate. Glutamate also can be synthesized from glutamine, using glutaminase. The glutamine is derived from glial cells as described in Section III.F.2. Like other neurotransmitters, glutamate is stored in vesicles, and its release is Ca2⫹-dependent. It is removed from the synaptic cleft by high-affinity uptake systems in nerve terminals and glial cells. 2.

␥-AMINOBUTYRIC ACID

GABA is the major inhibitory neurotransmitter in the central nervous system. Its functional significance is far-reaching, and altered GABAergic function plays a role in many neurologic and psychiatric disorders. GABA is synthesized by the decarboxylation of glutamate (see Fig. 48.10) in a single step catalyzed by the enzyme glutamic acid decarboxylase (GAD). GABA is recycled in the central nervous system by a series of reactions called the GABA shunt, which conserves glutamate and GABA (see Fig. 48.10). Much of the uptake of GABA occurs in glial cells. The GABA shunt in glial cells produces glutamate, which is converted to glutamine and transported out of the glial cells to neurons, where it is converted back to glutamate. Glutamine thus serves as a transporter of glutamate between cells in the CNS (see Chapter 42). Glial cells lack GAD and cannot synthesize GABA.

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Tiagabine is a drug that inhibits the reuptake of GABA from the synapse, and it has been used to treat epilepsy as well as other convulsant disorders. Because GABA is an inhibitory neurotransmitter in the brain, its prolonged presence can block neurotransmission by other agents, thereby reducing the frequency of convulsions.

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SECTION VIII ■ TISSUE METABOLISM

918

Stimulator molecule

Nerve impulse

NO

NO

NO NO

2.

Guanylate cyclase

+

cGMP Relaxation

FIG. 48.11. Action of nitric oxide (NO) in vasodilation. The synthesis of NO occurs in response to a stimulator binding to a receptor on some cells or to a nerve impulse in neurons. NO enters smooth muscle cells, stimulating guanylate cyclase to produce cGMP, which causes smooth muscle cell relaxation. When the smooth muscle cells relax, blood vessels dilate.

Retrograde

A

NT

B

NT

GLYCINE

3.

CONVERSION OF ARGININE TO NITRIC OXIDE

Nitric oxide (NO) is a biologic messenger in a variety of physiologic responses, including vasodilation, neurotransmission, and the ability of the immune system to kill tumor cells and parasites. NO is synthesized from arginine in a reaction catalyzed by NO synthase (see Fig. 24.10). NO synthase exists as tissue-specific forms of two families of enzymes. The form present in macrophages is responsible for overproduction of NO, leading to its cytotoxic actions on parasites and tumor cells. The enzyme present in nervous tissue, vascular endothelium, platelets, and other tissues is responsible for the physiologic responses to NO such as vasodilation and neural transmission. In target cells, NO activates a soluble guanylate cyclase, which results in increased cellular levels of cGMP (3⬘,5⬘-cyclic GMP) (Fig. 48.11). In smooth muscle cells, cGMP, like cAMP, activates one or more protein kinases, which are responsible for the relaxation of smooth muscle and the subsequent dilation of vessels. NO stimulates penile erection by acting as a neurotransmitter, stimulating smooth muscle relaxation that permits the corpus cavernosum to fill with blood. Nitric oxide can readily cross cell membranes because it is a gas. As a result, its effect may not necessarily be limited to the neuron that synthesizes it (Fig. 48.12). There is ample evidence that NO may function as a retrograde messenger that can influence neurotransmitter release from the presynaptic terminal after diffusing from the postsynaptic neuron (where it is synthesized). There is also evidence supporting retrograde messenger roles for both arachidonic acid and carbon monoxide in the CNS.

IV. METABOLIC ENCEPHALOPATHIES AND NEUROPATHIES NO

C

D

FIG. 48.12. NO as a retrograde messenger. Cell A releases a neurotransmitter, which stimulates cell C to produce NO. NO, being a gas, can diffuse back and regulate cell A’s production and release of neurotransmitters. NO can also diffuse to cell B and stimulate cell B to produce a different neurotransmitter to elicit a response from cell D. NT, neurotransmitter.

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ASPARTATE

Glycine is the major inhibitory neurotransmitter in the spinal cord. Most of the glycine in neurons is synthesized de novo within the nerve terminal from serine by the enzyme serine hydroxymethyltransferase, which requires folic acid. Serine, in turn, is synthesized from the intermediate 3-phosphoglycerate in the glycolytic pathway. The action of glycine is probably terminated via uptake by a high-affinity transporter.

GTP Smooth muscle cell

1.

Aspartate, like glutamate, is an excitatory neurotransmitter, but it functions in far fewer pathways. It is synthesized from the TCA cycle intermediate oxaloacetate via transamination reactions. Like glutamate synthesis, aspartate synthesis uses oxaloacetate that must be replaced through anaplerotic reactions. Aspartate cannot pass through the blood–brain barrier.

Arg O2 +

Arg O2

+

G. Other Amino Acid Neurotransmitters

The brain has an absolute dependence on the blood for its supply of glucose and oxygen. It uses approximately 20% of the oxygen supply of the body. During the developmental period and during prolonged fasting, ketone bodies can be used as a fuel, but they cannot totally substitute for glucose. Glucose is converted to pyruvate in glycolysis, and the pyruvate is oxidized in the TCA cycle. Anaerobic glycolysis, with a yield of two molecules of ATP per molecule of glucose, cannot sustain the ATP requirement of the brain, which can be provided only by the complete oxidation of glucose to CO2, which yields approximately 32 ATP per glucose. However, during periods of mild hypoglycemia or mild hypoxia, decreased neurotransmitter synthesis contributes as much, if not more, to the development of symptoms as does an absolute deficiency of ATP for energy needs.

A. Hypoglycemic Encephalopathy Hypoglycemia is sometimes encountered in medical conditions such as malignancies producing insulin, insulin-like growth factors, or chronic alcoholism. Early

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clinical signs in hypoglycemia reflect the appearance of physiologic protective mechanisms initiated by hypothalamic sensory nuclei such as sweating, palpitations, anxiety, and hunger. If these symptoms are ignored, they proceed to a more serious CNS disorder, progressing through confusion and lethargy to seizures and eventually coma. Prolonged hypoglycemia can lead to irreversible brain damage. During the progression of hypoglycemic encephalopathy, as blood glucose falls lower than 2.5 mM (45 mg/dL), the brain attempts to use internal substrates such as glutamate and TCA cycle intermediates as fuels. Because the pool size of these substrates is quite small, they are quickly depleted. If blood glucose levels continue to fall lower than 1 mM (18 mg/dL), ATP levels become depleted. As the blood glucose drops from 2.5 to 2.0 mM (45 to 36 mg/dL, before electroencephalographic [EEG] changes are observed), the symptoms appear to arise from decreased synthesis of neurotransmitters in particular regions of the brain rather than a global energy deficit. Figure 48.13 summarizes the relationship between the oxidation of glucose in glycolysis and the provision of precursors for the synthesis of neurotransmitters in different types of neurons. As hypoglycemia progresses lower than 1 mM (18 mg/dL) and high-energy phosphate levels are depleted, the EEG becomes isoelectric, and neuronal cell death ensues. As is the case in some other metabolic encephalopathies, cell death is not global in distribution; rather, certain brain structures, in particular hippocampal and cortical structures, are selectively vulnerable to hypoglycemic insult. Pathophysiologic mechanisms responsible for neuronal cell death in hypoglycemia include the involvement of glutamate excitotoxicity. Glutamate excitotoxicity occurs when the cellular energy reserves are depleted. The failure of the energydependent reuptake pumps results in a buildup of glutamate in the synaptic cleft and overstimulation of the postsynaptic glutamate receptors. The prolonged glutamate receptor activation leads to prolonged opening of the receptor ion channel and the influx of lethal amounts of Ca2⫹ ion, which can activate cytotoxic intracellular pathways in the postsynaptic neuron. Glucose

Fructose 1,6-bisphosphate

3-Phosphoglycerate

Serine

FH4

Glycine

Pyruvate

Pyruvate

TCA cycle OAA

␣-KG

Mitochondrion Aspartate

Glutamate

GABA

FIG. 48.13. Glucose metabolism leading to the synthesis of the neurotransmitters glycine, aspartate, glutamate, and GABA. As blood glucose levels drop and brain glucose levels diminish, synthesis of these neurotransmitters may be compromised. ␣-KG, ␣-ketoglutarate.

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B. Hypoxic Encephalopathy Experimental studies with human volunteers show that cerebral energy metabolism remains normal when mild to moderate hypoxia (partial pressure of oxygen, or PaO2 ⫽ 25 to 40 mm Hg) results in severe cognitive dysfunction. The diminished cognitive function is believed to result from impaired neurotransmitter synthesis. In mild hypoxia, cerebral blood flow increases to maintain oxygen delivery to the brain. In addition, anaerobic glycolysis is accelerated, resulting in maintenance of ATP levels. This occurs, however, at the expense of an increase of lactate production and a fall of pH. Acute hypoxia (PaO2 ⱕ20 mm Hg) generally results in a coma. Hypoxia can result from insufficient oxygen reaching the blood (e.g., at high altitudes), severe anemia (e.g., iron deficiency), or a direct insult to the oxygenutilizing capacity of the brain (e.g., cyanide poisoning). All forms of hypoxia result in diminished neurotransmitter synthesis. Inhibition of pyruvate dehydrogenase diminishes acetylcholine synthesis, which is acutely sensitive to hypoxia. Glutamate and GABA synthesis, which depend on a functioning TCA cycle, are decreased as a result of elevated NADH levels, which inhibit TCA cycle enzymes. NADH levels are increased when oxygen is unavailable to accept electrons from the electron-transport chain and NADH cannot be readily converted back into NAD⫹. Even the synthesis of catecholamine neurotransmitters may be decreased because the hydroxylase reactions require O2.

C. Relationship between Glutamate Synthesis and the Anaplerotic Pathways of Pyruvate Carboxylase and Methylmalonyl-CoA Mutase

Synthesis of glutamate removes ␣-ketoglutarate from the TCA cycle, thereby decreasing the regeneration of oxaloacetate in the TCA cycle. Because oxaloacetate is necessary for the oxidation of acetyl-CoA, oxaloacetate must be replaced by anaplerotic reactions. There are two major types of anaplerotic reactions: (1) pyruvate carboxylase and (2) the degradative pathway of the branched-chain amino acids, valine and isoleucine, which contribute succinyl-CoA to the TCA cycle. This pathway uses vitamin B12 (but not folate) in the reaction catalyzed by methylmalonyl-CoA mutase.

V. LIPID SYNTHESIS IN THE BRAIN AND PERIPHERAL NERVOUS SYSTEM Several features of lipid synthesis and degradation in the nervous system distinguish it from most other tissues. The first is that the portion of the neuronal cell membrane involved in synaptic transmission has a unique role and a unique composition. At the presynaptic terminal, the lipid composition changes rapidly as storage vesicles containing the neurotransmitter fuse with the cell membrane and release their contents. Portions of the membrane are also lost as endocytotic vesicles. On the postsynaptic terminal, the membrane contains the receptors for the neurotransmitter as well as a high concentration of membrane signaling components such as phosphatidylinositol. A second important feature of brain lipid metabolism is that the blood–brain barrier restricts the entry of nonessential fatty acids, such as palmitate, that are released from adipose tissue or present in the diet. Conversely, essential fatty acids are taken up by the brain. Because of these considerations, the brain is constantly synthesizing those lipids (cholesterol, fatty acids, glycosphingolipids, and phospholipids) which it needs for various neurologic functions. Neuronal signaling also requires that nonneuronal glial cells synthesize myelin, a multilayered membrane that surrounds the axons of many neurons. Myelin is lipid-rich and has a different lipid composition than the neuronal membranes. The white matter in the brain contains significantly more myelin than the gray matter; it is the presence of

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myelin sheaths that is responsible for the characteristic color differences that exist between the two types of brain tissue.

A. Brain Lipid Synthesis and Oxidation Because the blood–brain barrier significantly inhibits the entry of certain fatty acids and lipids into the CNS, virtually all lipids found there must be synthesized within the CNS. The exceptions are the essential fatty acids (linoleic and linolenic acid) that do enter the brain where they are elongated or further desaturated. The uptake of fatty acids into the CNS is insufficient to meet the energy demands of the CNS—hence the requirement for aerobic glucose metabolism. Thus, cholesterol, glycerol, sphingolipids, glycosphingolipids, and cerebrosides are all synthesized using pathways discussed previously in this text. Of particular note is that very long-chain fatty acids are synthesized in the brain, where they play a major role in myelin formation. Oxidation and turnover of brain lipids occurs as described previously (see Chapter 23). Peroxisomal fatty acid oxidation is important in the brain because the brain contains very long-chain fatty acids and phytanic acid (from the diet), both of which are oxidized in the peroxisomes by ␣-oxidation. Thus, disorders that affect peroxisome biogenesis (such as Refsum disease) severely affect brain cells because of the inability to metabolize both branched-chain and very long-chain fatty acids. If there is a disorder in which the degradation of glycosphingolipids or mucopolysaccharides is impaired, lysosomes in brain cells become engorged with partially digested glycolipids, leading to varying degrees of neurologic dysfunction.

B. Myelin Synthesis A rapid rate of nerve conduction in the peripheral and central motor nerves depends on the formation of myelin, a multilayered lipid and protein structure that is formed from the plasma membrane of glial cells. In the peripheral nervous system, the Schwann cell is responsible for myelinating one portion of an axon of one nerve cell. The Schwann cell does this by wrapping itself around the axon multiple times so that a multilayered sheath of membrane surrounds the axon. In the central nervous system, the oligodendrocyte is responsible for myelination. Unlike the Schwann cell, oligodendrocytes can myelinate portions of numerous axons (up to 40) and do so by extending a thin process that wraps around the axon multiple times. Thus, CNS axons are surrounded only by the membranes of oligodendrocytes, whereas axons in the PNS are surrounded by the entire Schwann cell. A generalized view of myelination is depicted in Figure 48.14. To maintain the myelin structure, the oligodendrocyte synthesizes four times its own weight in lipids per day. 1.

MYELIN LIPIDS

As the plasma membrane of the glial cell is converted into myelin, the lipid composition of the brain changes (Table 48.2). The lipid-to-protein ratio is greatly increased, as is the content of sphingolipids. The myelin is a tightly packed structure, and there are significant hydrophobic interactions between the lipids and proteins to allow this to occur. Cerebrosides constitute approximately 16% of total myelin lipid and are almost completely absent from other cell-type membrane lipids. The predominant cerebroside, galactosylcerebroside, has a single sugar attached to the hydroxyl group of the sphingosine. In contrast, sphingomyelin, which one might guess is the predominant lipid of myelin, is present in roughly the same low concentration in all membranes. Galactocerebrosides pack more tightly together than phosphatidylcholine; the sugar, although polar, carries no positively charged amino group or negatively charged phosphate. The brain synthesizes very long-chain fatty acids (⬎20 carbons long); these long uncharged side chains develop strong hydrophobic associations, allowing close packing of the myelin sheath. The high cholesterol content of the membrane also contributes to the tight packing, although the myelin proteins are also required to complete the tightness of the packing process.

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Lipid bilayer

Extracellular space

+ + +

+ + +

+ + +

+ + +

Layers of myelin

+ + +

+ + +

Schwann cell nucleus

+ + +

Axon

+ + +

+ + +

+ + +

Intracellular space + + +

Myelin basic protein (MBP)

Proteolipid protein

FIG. 48.14. A composite diagram indicating a Schwann cell that has wrapped around a portion of an axon forming the myelin sheath. The expansion represents a portion of the myelin sheath. CNS myelin is shown, although it is similar to PNS myelin except that Po replaces proteolipid protein. Recall that there are multiple layers of membrane surrounding the axon; proteolipid protein protrudes into the extracellular space and aids in compaction of the membranes through hydrophobic interactions. MBPs help to stabilize the structure from within the membrane.

2.

MYELIN STRUCTURAL PROTEINS

The layers of myelin are held together by protein–lipid and protein–protein interactions, and any disruption can lead to demyelination of the membrane (see Biochemical Comments). Although numerous proteins are found in both the CNS and PNS, only the major proteins are discussed here. The major proteins in the CNS and PNS are different. In the CNS, two proteins constitute between 60% and 80% of the total proteins—proteolipid protein and myelin basic proteins (MBPs). The proteolipid protein is a very hydrophobic protein that forms large aggregates in aqueous solution and is relatively resistant to proteolysis. Its molecular weight, based on sequence analysis, is 30,000 Da. Proteolipid protein is highly conserved in sequence among species. Its role is thought to be one of promoting the formation and stabilization of the multilayered myelin structure.

Table 48.2

Protein and Lipid Composition of CNS Myelin and Human Brain

Substancea Protein Lipid Cholesterol Cerebroside Sulfatide Total galactolipid Ethanolamine phosphatide Phosphatidylcholine Sphingomyelin Phosphatidylserine Phosphatidylinositol Plasmalogen Total phospholipids

Myelin

White Matter

30.0 70.0 27.7 22.7 3.8 27.5 15.6 11.2 7.9 4.8 0.6 12.3 43.1

39.0 54.9 27.5 19.8 5.4 26.4 14.9 12.8 7.7 7.9 0.9 11.2 45.9

Gray Matter 55.3 32.7 22.0 5.4 1.7 7.3 22.7 26.7 6.9 8.7 2.7 8.8 69.5

Protein and lipid figures in percentage dry weight; all others in percentage total lipid weight. Data from Norton W. In: Siegel GJ, Albers RW, Agranoff BW, Katzman R, eds. Basic Neurochemistry. 3rd ed. Boston, MA: Little, Brown and Company; 1981:77. a

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The MBPs are a family of proteins. Unlike proteolipid protein, MBPs are easily extracted from the membrane and are soluble in aqueous solution. The major MBP has no tertiary structure and has a molecular weight of 15,000 Da. MBP is located on the cytoplasmic face of myelin membranes. Antibodies directed against MBPs elicit experimental allergic encephalomyelitis (EAE), which has become a model system for understanding multiple sclerosis—a demyelinating disease. A model of how proteolipid protein and MBPs aid in stabilizing myelin is shown in Figure 48.14. In the PNS, the major myelin protein is Po, a glycoprotein that accounts for ⬎50% of the PNS myelin protein content. The molecular weight of Po is 30,000 Da, the same as proteolipid protein. Po is thought to play a similar structural role in maintaining myelin structure as proteolipid protein does in the CNS. Myelin basic proteins are also found in the PNS, with some similarities and differences to the MBPs found in the CNS. The major PNS-specific MBP has been designated as P2.

CLINICAL COMMENTS Katie Colamin. Catecholamines affect nearly every tissue and organ in the body. Their integrated release from nerve endings of the sympathetic (adrenergic) nervous system plays a critical role in the reflex responses we make to sudden changes in our internal and external environment. For example, under stress, catecholamines appropriately increase heart rate, blood pressure, myocardial (heart muscle) contractility, and conduction velocity of the heart. Episodic, inappropriate secretion of catecholamines in supraphysiologic amounts, such as those occurs in patients with pheochromocytomas, like Katie Colamin, causes an often acute and alarming array of symptoms and signs of a hyperadrenergic state. Most of the signs and symptoms related to catecholamine excess can be masked by phenoxybenzamine, a long-acting ␣1- and ␣2-adrenergic receptor antagonist, combined with a ␤1- and ␤2-adrenergic receptor blocker such as propranolol. Pharmacologic therapy alone is reserved for patients with inoperable pheochromocytomas (e.g., patients with malignant tumors with metastases and patients with severe heart disease). Because of the sudden, unpredictable, and sometimes life-threatening discharges of large amounts of catecholamines from these tumors, definitive therapy involves surgical resection of the neoplasm(s) after appropriate preoperative preparation of the patient with the agents mentioned previously. Katie’s tumor was resected without intraoperative or postoperative complications. After surgery, she remained free of symptoms, and her blood pressure decreased to normal levels. Evan Applebod. Evan Applebod, after stopping Redux, was placed on Prozac, an antidepressant that acts as a selective serotonin reuptake inhibitor (SSRI) but does not lead to increased synthesis or secretion of serotonin, as did dexfenfluramine in Redux. Thus, the mechanism of action of these two drugs is different, even if the end result (elevated levels of serotonin) is the same. Unfortunately, Prozac did not work as well for Mr. Applebod as did Redux, and he regained his 50 lb within 1 year after switching medications. Redux was withdrawn from the market by its manufacturer because of reports of heart valve abnormalities in a small percentage of patients who had taken either phen/fen (fenfluramine and phentermine) or Redux. Since then, the U.S. Food and Drug Administration (FDA) has banned the use of Redux for weight loss because of the undesirable side effects. Mr. Applebod now has several other options. Other medical treatments include orlistat, a partial inhibitor of dietary fatty acid absorption from the gastrointestinal tract; phentermine alone (described previously); and several medications used to treat other conditions, including diabetes, seizures, and depression, that also lead to weight loss. These latter agents are not FDA approved

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for weight loss, and thus Evan’s physician did not want to try any more medications with him. Instead, the physician referred Evan for bariatric surgery.

BIOCHEMICAL COMMENTS Demyelinating Diseases of the CNS. The importance of myelin in nerve transmission is underscored by the wide variety of demyelinating diseases, all of which lead to neurologic symptoms. The best known disease in this class is multiple sclerosis (MS). MS can be a progressive disease of the CNS in which demyelination of CNS neurons is the key anatomic and pathologic finding. The cause of MS has yet to be determined, although it is believed that an event occurs that triggers the formation of autoimmune antibodies directed against components of the nervous system. This event could be a bacterial or viral infection that stimulates the immune system to fight off the invaders. Unfortunately, this stimulus may also trigger the autoimmune response that leads to the antibody-mediated demyelinating process. The unusual geographic distribution of MS is of interest. Patients are concentrated in northern and southern latitudes, yet its incidence is almost nil at the equator. Clinical presentation of MS varies widely. Most commonly, it is a mild disease that has few or no obvious clinical manifestations. At the other end of the spectrum is a rapidly progressive and fatal disease. The most well-known presentation is the relapsing–remitting type. In this type, early in the course of the disease, the natural history is one of exacerbations followed by remission. Eventually, the CNS cannot repair the damage that has accumulated through the years, and remissions occur less and less frequently. Available treatments for MS target the relapsing–remitting type of disease. The primary injury to the CNS in MS is the loss of myelin in the white matter, which interferes with nerve conduction along the demyelinated area (the insulator is lost). The CNS compensates by stimulating the oligodendrocyte to remyelinate the damaged axon, and when this occurs, remission is achieved. Often, remyelination leads to a slowing in conduction velocity because of a reduced myelin thickness (speed is proportional to myelin thickness) or a shortening of the internodal distances (the action potential has to be propagated more times). Eventually, when it becomes too difficult to remyelinate large areas of the CNS, the neuron adapts by upregulating and redistributing along its membrane ion pumps to allow nerve conductance along demyelinated axons. Eventually, this adaptation also fails and the disease progresses. Treatment of MS is now based on blocking the action of the immune system. Because antibodies directed against cellular components appear to be responsible for the progression of the disease (regardless of how the autoantibodies were first generated), agents that interfere with immune responses have had various levels of success in keeping patients in remission for extended periods. Other demyelinating diseases also exist, and their cause is much more straightforward. These are relatively rare disorders. In all of these diseases, there is no fully effective treatment for the patient. Inherited mutations in Po (the major PNS myelin protein) leads to a version of Charcot–Marie–Tooth polyneuropathy syndrome. The inheritance pattern for this disease is autosomal dominant, indicating that the expression of one mutated allele leads to expression of the disease. Mutations in proteolipid protein (the major myelin protein in the CNS) lead to Pelizaeus–Merzbacher disease and X-linked spastic paraplegia type 2 disease. These diseases display a wide range of phenotypes, from a lack of motor development and early death (most severe) to mild gait disturbances. The phenotype displayed depends on the precise location of the mutation within the protein. An altered function of either Po or proteolipid protein leads to demyelination and its subsequent clinical manifestations.

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Key Concepts •

•

•

•

•

The nervous system consists of a variety of cell types with different functions. Neurons transmit and receive signals from other neurons at synaptic junctions. Astrocytes, found in the central nervous system, provide physical and nutritional support for the neurons. Oligodendrocytes provide the myelin sheath that coats the axon, providing insulation for the electric signal that is propagated along the axon. ■ Myelin has a lipid composition that is distinct from that of cellular membranes. ■ A lack of myelin leads to demyelinating diseases as a result of impaired signal transmission across the axon. Schwann cells are the supporting cells (and myelin-producing cells) of the peripheral nervous system. Microglial cells destroy invading microorganisms and phagocytose cellular debris. Ependymal cells line the cavities of the CNS and spinal cord. The brain is protected against blood-borne toxic agents by the blood–brain barrier. Glucose, amino acids, vitamins, ketone bodies, and essential fatty acids (but not other fatty acids) can all be transported across the blood–brain barrier. Proteins such as insulin can cross the blood–brain barrier by receptor-mediated transcytosis. Neurotransmitters are synthesized primarily from amino acids in the nervous system; others are derived from intermediates of glycolysis and the TCA cycle. Neurotransmitters are synthesized in the cytoplasm of the presynaptic terminal, and then transported into storage vesicles for release upon receiving the appropriate signal. Neurotransmitter action is terminated by reuptake into the presynaptic terminal or diffusion away from the synapse or by enzymatic inactivation. Monoamine oxidase is a key enzyme for the inactivation of the catecholamines and serotonin. An encephalopathy will develop if the nervous system cannot generate sufficient ATP. Hypoglycemic encephalopathy (lack of glucose to the brain) Hypoxic encephalopathy (lack of oxygen to the brain) Diseases discussed in this chapter are summarized in Table 48.3.

Table 48.3

Diseases Discussed in Chapter 48

Disease or Disorder

Genetic or Environmental

Albinism

Genetic

Hypercholesterolemia

Both

Multiple sclerosis

Environmental

Pheochromocytoma

Environmental

Facilitated glucose transporter protein type 1 deficiency syndrome Tyramine poisoning

Genetic

Depression

Both

Appetite suppression

Both

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Environmental

Comments A lack of melanoctye tyrosinase leads to the inability to produce Dopa, a required precursor to melanin production, such that pigment formation is inhibited. Elevated cholesterol levels in the blood may be regulated by appropriate pharmacologic agents. An autoimmune induced loss of myelin sheath formation around neurons. Tumor of the adrenal gland leading to episodic and excessive epinephrine and norepinephrine release. Infantile seizures related to low glucose levels in the nervous system (low glucose in the cerebrospinal fluid). Tyramine, a compound found in aged cheeses, for example, is degraded by monoamine oxidase. In the presence of monoamine oxidase inhibitors, tyramine levels can accumulate triggering the release of high levels of norepinephrine, leading to a hypertensive crisis. Drugs used to elevate serotonin levels may alleviate depression and may also lead to appetite suppression A variety of drugs have been used to treat obesity, although many have side effects which need to be carefully monitored.

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REVIEW QUESTIONS—CHAPTER 48 1.

2.

3.

A patient with a tumor of the adrenal medulla experienced palpitations, excessive sweating, and hypertensive headaches. His urine contained increased amounts of vanillylmandelic acid. His symptoms are probably caused by an overproduction of which of the following? A. Acetylcholine B. Norepinephrine and epinephrine C. Dopa and serotonin D. Histamine E. Melatonin The two lipids found in highest concentration in myelin are which of the following? A. Cholesterol and cerebrosides such as galactosylceramide B. Cholesterol and phosphatidylcholine C. Galactosylceramide sulfatide and sphingomyelin D. Plasmalogens and sphingomyelin E. Triacylglycerols and lecithin Myelin basic protein can best be described by which of the following? A. It is synthesized in Schwann cells, but not in oligodendrocytes. B. It is a transmembrane protein found only in peripheral myelin. C. It attaches the two extracellular leaflets together in central myelin. D. It contains basic amino acid residues that bind the negatively charged extracellular sides of the myelin membrane together.

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E. It contains lysine and arginine residues that binds the negatively charged intracellular sides of the myelin membrane together. 4.

A patient presented with dysmorphia and cerebellar degeneration. Analysis of his blood indicated elevated levels of phytanic acid and very long-chain fatty acids, but no elevation of palmitate. His symptoms are consistent with a defect in an enzyme involved in which of the following? A. ␣-Oxidation B. Mitochondrial ␤-oxidation C. Transport of enzymes into lysosomes D. Degradation of mucopolysaccharides E. Elongation of fatty acids

5.

One of the presenting symptoms of vitamin B6 deficiency is dementia. This may result from an inability to synthesize serotonin, norepinephrine, histamine, and ␥-aminobutyric acid (GABA) from their respective amino acid precursors. This is because vitamin B6 is required for which type of reaction? A. Hydroxylation B. Transamination C. Deamination D. Decarboxylation E. Oxidation

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49

The Extracellular Matrix and Connective Tissue

Many of the cells in tissues are embedded in an extracellular matrix that fills the spaces between cells and binds cells and tissue together. In so doing, the extracellular matrix aids in determining the shape of tissues as well as the nature of the partitioning between tissue types. In the skin, loose connective tissue beneath epithelial cell layers consists of an extracellular matrix in which fibroblasts, blood vessels, and other components are distributed (Fig. 49.1). Other types of connective tissue, such as tendon and cartilage, consist largely of extracellular matrix, which is principally responsible for their structure and function. This matrix also forms the sheetlike basal laminae, or basement membranes, on which layers of epithelial cells rest, and which act as supportive tissue for muscle cells, adipose cells, and peripheral nerves. Basic components of the extracellular matrix include fibrous structural proteins, such as collagens, proteoglycans containing long glycosaminoglycan chains attached to a protein backbone, and adhesion proteins linking components of the matrix to each other and to cells. These fibrous structural proteins are composed of repeating elements that form a linear structure. Collagens, elastin, and laminin are the principal structural proteins of connective tissue. Proteoglycans consist of a core protein covalently attached to many long, linear chains of glycosaminoglycans, which contain repeating disaccharide units. The repeating disaccharides usually contain a hexosamine and a uronic acid, and these sugars are frequently sulfated. Synthesis of the proteoglycans starts with the attachment of a sugar to a serine, threonine, or asparagine residue of the protein. Additional sugars, donated by UDP-sugar precursors, add sequentially to the nonreducing end of the molecule. Proteoglycans, such as glycoproteins and glycolipids, are synthesized in the endoplasmic reticulum (ER) and the Golgi complex. The glycosaminoglycan chains of proteoglycans are degraded by lysosomal enzymes that cleave one sugar at a time from the nonreducing end of the chain. An inability to degrade proteoglycans leads to a set of diseases known as the mucopolysaccharidoses. Adhesion proteins, such as fibronectin and laminin, are extracellular glycoproteins that contain separate distinct binding domains for proteoglycans, collagen, and fibrin. These domains allow these adhesion proteins to bind the various components of the extracellular matrix. They also contain specific binding domains for specific cell surface receptors known as integrins. These integrins bind to fibronectin on the external surface, span the plasma membrane of cells, and adhere to proteins, which in turn bind to the intracellular actin filaments of the cytoskeleton. Integrins also provide a mechanism for signaling between cells via both internal signals and through signals generated via the extracellular matrix. Cell movement within the extracellular matrix requires remodeling of the various components of the matrix. This is accomplished by a variety

Epithelial cell layer C o nt ni es cs tu ie v e

Basal lamina Collagen Elastic fibers

Proteoglycan

FIG. 49.1. An overview of connective tissue extracellular matrix. Supporting the epithelial cell layer is a basal lamina, beneath which are collagen, elastic fibers, and proteoglycans. The cell types present in connective tissue, such as fibroblasts and macrophages, have been removed from the diagram for clarity.

927

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of matrix metalloproteinases (MMPs) and regulators of the MMPs, tissue inhibitors of matrix metalloproteinases (TIMPs). Dysregulation of this delicate balance of the regulators of cell movement allows cancer cells to travel to other parts of the body (metastasize) as well as to spread locally to contiguous tissues.

THE WAITING ROOM Sis Lupus (first introduced in Chapter 14) noted a moderate reduction in pain and swelling in the joints of her fingers while she was taking her immunosuppressant medication. At her next checkup, her rheumatologist described to Sis the underlying inflammatory tissue changes that her systemic lupus erythematosus (SLE) was causing in the joint tissues. Ann Sulin complained of a declining appetite for food as well as severe weakness and fatigue. The reduction in her kidneys’ ability to maintain normal daily total urinary net acid excretion contributed to her worsening metabolic acidosis. This, plus her declining ability to excrete nitrogenous waste products such as creatinine and urea into her urine (“azotemia”) were responsible for many of her symptoms. Her serum creatinine level was rising steadily. As it approached a level of 5 mg/dL, she developed a litany of complaints caused by the multisystem dysfunction associated with her worsening metabolic acidosis and retention of nitrogenous waste products (“uremia”). Her physicians discussed the need to consider peritoneal dialysis or hemodialysis.

I. COMPOSITION OF THE EXTRACELLULAR MATRIX A. Fibrous Proteins 1.

FIG. 49.2.

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The triple helix of collagen.

COLLAGEN

Collagen, a family of fibrous proteins, is produced by a variety of cell types but principally by fibroblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells. Type I collagen, collagen(I), the most abundant protein in mammals, is a fibrous protein that is the major component of connective tissue. It is found in the extracellular matrix (ECM) of loose connective tissue, bone, tendons, skin, blood vessels, and the cornea of the eye. Collagen(I) contains approximately 33% glycine and 21% proline and hydroxyproline. Hydroxyproline is an amino acid produced by posttranslational modification of peptidyl proline residues. Procollagen(I), the precursor of collagen(I), is a triple helix composed of three polypeptide (pro-␣) chains that are twisted around each other, forming a ropelike structure. Polymerization of collagen(I) molecules forms collagen fibrils, which provide great tensile strength to connective tissues (Fig. 49.2). The individual polypeptide chains each contain approximately 1,000 amino acid residues. The three polypeptide chains of the triple helix are linked by interchain hydrogen bonds. Each turn of the triple helix contains three amino acid residues, such that every third amino acid is in close contact with the other two strands in the center of the structure. Only glycine, which lacks a side chain, can fit in this position, and indeed, every third amino acid residue of collagen is glycine. Thus, collagen is a polymer of (Gly-X-Y) repeats, where Y is frequently proline or hydroxyproline and X is any other amino acid found in collagen.

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CHAPTER 49 ■ THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE

O N

CH

H2C

O

C

CH2

N

+

Prolyl hydroxylase

O2

H

CH

H2C

Ascorbate

C H

CH2

CO2

H

CH CH2 CH2 CH2

+ Succinate

OH

4-Hydroxyproline residue O

H

C

C

Proline residue

N

929

O

C

+

Lysyl hydroxylase Ascorbate

O2

CO2

CH2 + NH 3

Lysine residue

N

CH

H

CH2

C

+ Succinate

CH2 OH

CH CH2 + NH

3

5-Hydroxylysine residue

FIG. 49.3. Hydroxylation of proline and lysine residues in collagen. Proline and lysine residues within the collagen chains are hydroxylated by reactions that require vitamin C.

Procollagen(I) is an example of a protein that undergoes extensive posttranslational modifications. Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues. These reactions occur after the protein has been synthesized (Fig. 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes prolyl hydroxylase and lysyl hydroxylase. Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose–glucose). The role of carbohydrates in collagen structure is still controversial. In the absence of vitamin C (scurvy), the melting temperature of collagen drops from 42°C to 24°C because of the loss of interstrand hydrogen bond formation, which is in turn caused by the lack of hydroxyproline residues. The side chains of lysine residues also may be oxidized to form the aldehyde— allysine. These aldehyde residues produce covalent cross-links between collagen molecules (Fig. 49.4). An allysine residue on one collagen molecule reacts with the amino group of a lysine residue on another molecule, forming a covalent Schiff base that is converted to more stable covalent cross-links. Aldol condensation also may occur between two allysine residues, which forms the structure lysinonorleucine. i.

Types of Collagen

At least 28 different types of collagen have been characterized (Table 49.1). Although each type of collagen is found only in particular locations in the body, more than one type may be present in the ECM at a given location. The various types of collagen can be classified as fibril-forming (types I, II, III, V, XI, XXIV, and XXVII), network-forming (types IV, VIII, and X), those that associate with fibril surfaces (types IX, XII, XIV, XXI, and XXII), those that are transmembrane proteins (types XIII, XVII, XXIII, and XXV), endostatin-forming (types XV and XVIII), and those that form periodic beaded filaments (type VI). All collagens contain three polypeptide chains with at least one stretch of triple helix. The non–triple helical domains can be short (as in the fibril-forming

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Protein

930

SECTION VIII ■ TISSUE METABOLISM

CH2

Table 49.1

+

CH2

NH3

Lysine residue

A

O2

Lysyl oxidase

NH3 + OH–

Types of Collagen

Collagen Type

Gene

Structural Details

Localization

I II III

Col1A1–Col1A2 Col2A1 Col3A1

Fibrils Fibrils Fibrils

IV

Col4A1–Col4A6

V

Col5A1–Col5A3

Nonfibrillar, mesh collagen Small fibers, N-terminal globular domains

VI

Col6A1–Col6A3

VII

Col7A1

Microfibrils, with both N- and C-terminal globular domains An anchoring collagen

VIII

Col8A1–Col8A2

Nonfibrillar, mesh collagen

IX

Col9A1–Col9A3

FACIT; N-terminal globular domain

X

Col10A1

XI XII

Col11A1–Col11A3 Col12A1

Nonfibrillar, mesh collagen, with C-terminal globular domain Small fibers FACIT

Skin, tendon, bone, cornea Cartilage, vitreous humor Skin, muscle, associates with type I collagen All basal laminae (basement membranes) Associates with type I collagen in most interstitial tissues Associates with type I collagen in most interstitial tissues Epithelial cells; dermal– epidermal junction Cornea, some endothelial cells Associates with type II collagen in cartilage and vitreous humor Growth plate, hypertrophic and mineralizing cartilage

XIII XIV XV

Col13A1 Col14A1 Col15A1

XVI XVII XVIII XIX XXI

Col16A1 Col17A1 Col18A1 Col19A1 Col21A1

Transmembrane collagen FACIT Endostatin-forming collagen Other Transmembrane collagen Endostatin forming Other FACIT

XXII XXIII

Col22A1 Col23A1

FACIT Transmembrane collagen

XXIV XXV XXVI XXVII

Col24A1 Col25A1 EMID2 Col27A1

Fibrils Transmembrane collagen Other Fibrils

XXVIII

Col28A1

Other

O CH2

+

C

H 2N

CH2

CH2

H Allysine residue

Second lysine residue

B

H2O

CH2

CH

N

CH2

CH2

Schiff base

C

O CH2

HO

+

C

C

H

CH

H

Allysine (aldo form)

Allysine (enol form)

Aldol condensation

H CH2

O C

HO CH

CH

H

O C

CH

C

Lysinonorleucine

FIG. 49.4. Formation of cross-links in collagen. A. Lysine residues are oxidized to allysine (an aldehyde). Allysine may react with an unmodified lysine residue to form a Schiff base (B), or two allysine residues may undergo an aldol condensation (C).

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Ubiquitous Epidermal cell surface Endothelial cells Ubiquitous Heart, skeletal muscle, stomach, kidney, placenta Tissue junctions Lung, skin, cornea, brain, kidney, tendon Bone Brain Mesenchymal cells Stomach, lung, gonads, skin, tooth Nervous system

See the text for descriptions of the differences in types of collagen. Type XX collagen is not present in humans. FACIT, fibril-associated collagens with interrupted triple helices.

H2O

CH2

Cartilage, vitreous humor Interacts with types I and II collagen in soft tissues Cell surfaces, epithelial cells Soft tissue Endothelial cells

collagens), or they can be rather large, such that the triple helix is actually a minor component of the overall structure (examples are collagen types XII and XIV). The fibril-associated collagens with interrupted triple helices (FACITs, collagen types IX, XII, and XIV) collagen types associate with fibrillar collagens, without themselves forming fibers. The endostatin-forming collagens are cleaved at their C-terminus to form endostatin, an inhibitor of angiogenesis. The network-forming collagens (type IV) form a meshlike structure because of large (approximately 230 amino acids) noncollagenous domains at the carboxy terminus (Fig. 49.5). And finally, several collagen types are actually transmembrane proteins (XIII, XVII, XXIII, and XXV) found on epithelial or epidermal cell surfaces, which play a role in several cellular processes, including adhesion of components of the ECM to cells embedded within it. Type XXV collagen has been associated with the neuronal plaques, which develop during Alzheimer disease. Types I, II, and III collagens form fibrils that assemble into large insoluble fibers. The fibrils (see in the following text) are strengthened through covalent

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A. Protomer Carboxy terminus Amino terminus

B. Dimer Carboxy-terminal hexamer (NC1 domain)

C. Type IV collagen tetramer

Aggregation at amino termini (7S domain)

D. Suprastructure NC1 hexamer 7S domain

FIG. 49.5. Type IV collagen contains a globular carboxy-terminal domain (A), which forms tropocollagen dimers (hexamers of collagen, B). Four dimers associate at the aminoterminal domains to form a 7S domain (C), and the tetramers form a lattice (D), which provides structural support to the basal lamina.

cross-links between lysine residues on adjacent fibrils. The arrangement of the fibrils gives individual tissues their distinct characteristics. Tendons, which attach muscles to bones, contain collagen fibrils aligned parallel to the long axis of the tendon, thus giving the tendon tremendous tensile strength. The types of collagen that do not form fibrils perform a series of distinct roles. Fibril-associated collagens bind to the surface of collagen fibrils and link them to other matrix-forming components. The transmembrane collagens form anchoring fibrils that link components of the extracellular matrix to underlying connective tissue. The network-forming collagens (type IV) form a flexible collagen that is part of the basement membrane and basal lamina that surround many cells. ii.

Synthesis and Secretion of Collagen

Collagen is synthesized within the endoplasmic reticulum as a precursor known as preprocollagen. The presequence acts as the signal sequence for the protein and is

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Endostatins block angiogenesis (new blood vessel formation) by inhibiting endothelial cell migration. Because endothelial cell migration and proliferation are required to form new blood vessels, inhibiting this action blocks angiogenesis. Tumor growth is dependent on a blood supply, inhibiting angiogenesis can reduce tumor cell proliferation. One type of osteogenesis imperfecta (OI) is caused by a mutation in a gene that codes for collagen. The phenotype of affected individuals varies greatly, depending on the location and type of mutation. See the Biochemical Comments for more information concerning this type of OI.

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Table 49.2

Steps in Collagen Biosynthesis

Location

Process

Rough endoplasmic reticulum Lumen of ER

Synthesis of preprocollagen; insertion of the procollagen molecule into the lumen of the ER Hydroxylation of proline and lysine residues; glycosylation of selected hydroxylysine residues Self-assembly of the tropocollagen molecule, initiated by disulfide bond formation in the carboxy-terminal extensions; triple helix formation Procollagen prepared for secretion from cell Cleavage of the propeptides, removing the amino- and carboxy-terminal extensions, and self-assembly of the collagen molecules into fibrils and then fibers

Lumen of ER and Golgi apparatus Secretory vesicle Extracellular

ER, endoplasmic reticulum.

cleaved, forming procollagen within the endoplasmic reticulum. From there, it is transported to the Golgi apparatus (Table 49.2). Three procollagen molecules associate through formation of interstrand and intrastrand disulfide bonds at the carboxy terminus; once these disulfides are formed, the three molecules can align properly to initiate formation of the triple helix. The triple helix forms from the carboxy end toward the amino end, forming tropocollagen. The tropocollagen contains a triple helical segment between two globular ends: the amino- and carboxy-terminal extensions. The tropocollagen is secreted from the cell, the extensions are removed using extracellular proteases, and the mature collagen takes its place within the ECM. The individual fibrils of collagen line up in a highly ordered fashion to form the collagen fiber. 2.

ELASTIN

Elastin is the major protein found in elastic fibers, which are located in the ECM of connective tissue of smooth muscle cells, endothelial and microvascular cells, chondrocytes, and fibroblasts. Elastic fibers allow tissues to expand and contract; this is of particular importance to blood vessels, which must deform and reform repeatedly in response to the changes in intravascular pressure that occur with the contraction of the left ventricle of the heart. It is also important for the lungs, which stretch each time a breath is inhaled and return to their original shape with each exhalation. In addition to elastin, the elastic fibers contain microfibrils, which are composed of several acidic glycoproteins, the major ones being fibrillin-1 and fibrillin-2. i.

Supravalvular aortic stenosis (SVAS) results from an insufficiency of elastin in the vessel wall, leading to a narrowing of the large elastic arteries. Current theory suggests that the levels of elastin in the vessel walls may regulate the number of smooth muscle cell rings that encircle the vessel. If the levels of elastin are low, smooth muscle hypertrophy results, leading to a narrowing and stenosis of the artery.

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Tropoelastin

Elastin has a highly cross-linked, insoluble, amorphous structure. Its precursor, tropoelastin, is a molecule of high solubility, which is synthesized on the rough endoplasmic reticulum (RER) for eventual secretion. Tropoelastin contains two types of alternating domains. The first domain consists of a hydrophilic sequence that is rich in lysine and alanine residues. The second domain consists of a hydrophobic sequence that is rich in valine, proline, and glycine, which frequently occur in repeats of VPGVG or VGGVG. The protein contains approximately 16 regions of each domain, alternating throughout the protein (Fig. 49.6). Upon secretion from the cell, the tropoelastin is aligned with the microfibrils, and lysyl oxidase initiates the reactions that cross-link elastin molecules, using lysine residues within the hydrophilic alternating domains in the proteins. This cross-linking reaction is the same as that which occurs in collagen. In this reaction, two, three, or four lysine residues are cross-linked to form a stable structure. The net result of the cross-linking is the generation of a fibrous mesh that encircles the cells.

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Hydrophilic C-terminus

Signal peptide

Hydrophilic exon 26a (for alternative splicing)

Hydrophilic cross-linking exons

FIG. 49.6.

ii.

3'-Untranslated region

Hydrophobic exons

The cDNA structure of elastin, indicating the repeating cross-linking and hydrophobic domains.

Elastic Properties of Elastin

Elastic fibers have the ability to stretch and then to re-form without requiring an obvious energy source to do so. The mechanism by which this stretching and relaxing actively occurs is still controversial but does relate to the basic principles of protein folding described in Chapter 7. When the elastic fibers are stretched (such as when a breath is taken in and the lung fills up with air), the amorphous elastin structure is stretched. This stretching exposes the repeating hydrophobic regions of the molecule to the aqueous environment. This, in turn, leads to a decrease in the entropy of water because water molecules need to rearrange to form cages about each hydrophobic domain. When this stretching force within the lung is removed (e.g., when the subject exhales), the elastin takes on its original structure because of the increase in entropy that occurs because the water no longer needs to form cages about hydrophobic domains. Thus, the hydrophobic effect is the primary force that allows this stretched structure to re-form. Elastin is inherently stable, with a halflife of up to 70 years. 3.

LAMININ

After type IV collagen, laminin is the most abundant protein in basal laminae. Laminin provides additional structural support for the tissues through its ability to bind to type IV collagen, to other molecules present in the ECM, and to cell surfaceassociated proteins (the integrins; see Section I.D). i.

Laminin is a heterotrimeric protein that is shaped, for the most part, like a cross (Fig. 49.7). The trimer is composed of ␣-, ␤-, and ␥-subunits. There are five possible ␣-proteins (designated ␣1 through ␣5), three different versions of the ␤-subunit (␤1 through ␤3), and three different ␥-forms (␥1 through ␥3). Thus, there is a potential for the formation of as many as 45 different combinations of these three subunits. However, only 18 have been discovered. Laminin 111, composed of ␣1␤1␥1, is typical of this class of proteins. The major feature of the laminin structure is a coiled ␣-helix, which joins the three subunits together and forms a rigid rod. All three chains have extensions at the amino-terminal end. Only the ␣-chain has a significant carboxy-terminal extension past the rodlike structure. It is the laminin extensions that allow laminin to bind to other components within the ECM and to provide stability for the structure. Components of the ECM that are bound by laminin include collagen, sulfated lipids, and proteoglycans. ii.

HNH

Laminin Structure

Laminin Biosynthesis

Like other secreted proteins, laminin is synthesized with a leader sequence that targets the three chains to the endoplasmic reticulum. Chain association occurs

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Gobular domains H HN

H NH

Disulfide bonds

HOOC

Coiled coil; rigid rods

COOH

COOH

FIG. 49.7. The structure of laminin.

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SECTION VIII ■ TISSUE METABOLISM

Defects in the structures of laminin 5 or laminin 6 (proteins that contribute to the cohesion of the dermis and epidermis) lead to the disorder referred to as junctional epidermolysis bullosa (JEB). In this disorder, there can be severe spontaneous blistering of the skin and mucous membranes. A severe form of the disease, JEB gravis, is often fatal early in life. Death occurs as a result of epithelial blistering of the respiratory, digestive, and genitourinary systems. Congenital muscular dystrophy (CMD) results from a defect in laminin 2, which is a component of the bridge that links the muscle cell cytoskeleton to the extracellular matrix. Lack of this bridge triggers muscle cell apoptosis, which results in weakened muscles.

The ECM is not simply a glue that holds cells together; it also serves to keep cells from moving to other locations and to prevent large molecules and other particles, such as microorganisms, from reaching contiguous and distant cells. This confining property of the matrix is medically important. For example, infections spread, in part, because the infectious agent alters the “containing” capacity of the ECM. Cancer cells that metastasize (migrate to other tissues) can do so only by altering the integrity of the matrix. Diseases such as rheumatoid arthritis (an autoimmune destruction of articular and periarticular tissues) and osteoarthritis (degenerative joint disease often associated with aging) involve damage to the functional capacity of the matrix. Alterations in the structural characteristics of the matrix of the renal glomerulus may allow proteins to be excreted into the urine, an indication of inexorable decline in renal function. Genetic defects may cause components of the matrix to be structurally and functionally abnormal, resulting in connective tissue disorders such as the Ehlers–Danlos syndrome (caused by several mutations that affect specific collagen genes) and Marfan syndrome (a defect in the protein fibrillin, in which ⬎330 different mutations, many of which give rise to different phenotypes, have been identified). Deficiencies of lysosomal enzymes involved in normal degradation of molecules of the matrix result in diseases such as the mucopolysaccharidoses.

Lieberman_CH49.indd 934

within the Golgi apparatus before secretion from the cell. After laminin is secreted by the cell, the amino-terminal extensions promote self-association as well as the binding to other ECM components. Disulfide linkages are formed to stabilize the trimer, but there is much less posttranslational processing of laminin than there is of collagen and elastin.

B. Proteoglycans The fibrous structural proteins of the ECM are embedded in gels formed from proteoglycans. Proteoglycans consist of polysaccharides called glycosaminoglycans (GAGs) linked to a core protein. The GAGs are composed of repeating units of disaccharides (Fig. 49.8). One sugar of the disaccharide is either N-acetylglucosamine or N-acetylgalactosamine, and the second is usually acidic (either glucuronic acid or iduronic acid). These sugars are modified by the addition of sulfate groups to the parent sugar. A proteoglycan may contain ⬎100 GAG chains and consist of up to 95% carbohydrate by weight. The negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules, thereby creating a hydrated gel. The gel provides a flexible mechanical support for the ECM. The gel also acts as a filter that allows the diffusion of ions (e.g., Ca2⫹), H2O, and other small molecules but slows diffusion of proteins and movement of cells. The gel also acts as a lubricant. Hyaluronan is the only GAG that occurs as a single long polysaccharide chain and is the only GAG that is not sulfated. 1.

STRUCTURE AND FUNCTION OF THE PROTEOGLYCANS

Proteoglycans are found in interstitial connective tissues—for example, the synovial fluid of joints, the vitreous humor of the eye, arterial walls, bone, cartilage, and cornea. They are major components of the ECM in these tissues. The proteoglycans interact with a variety of proteins in the matrix, such as collagen and elastin, fibronectin (which is involved in cell adhesion and migration), and laminin. Proteoglycans are proteins that contain many chains of GAGs (formerly called mucopolysaccharides). After synthesis, proteoglycans are secreted from cells; thus, they function extracellularly. Because the long, negatively charged glycosaminoglycan chains repel each other, the proteoglycans occupy a very large space and act as “molecular sieves,” determining which substances enter or leave cells (Table 49.3). Their properties also give resilience and a degree of flexibility to substances such as cartilage, permitting compression and re-expansion of the molecule to occur. At least seven types of glycosaminoglycans exist, which differ in the monosaccharides present in their repeating disaccharide units: chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronic acid, and keratan sulfates I and II. Except for hyaluronic acid, the glycosaminoglycans are linked to proteins, usually attached covalently to serine or threonine residues (Fig. 49.9). Keratan sulfate I is attached to asparagine. 2.

SYNTHESIS OF THE PROTEOGLYCANS

The protein component of the proteoglycans is synthesized on the ER. It enters the lumen of this organelle, where the initial glycosylations occur. UDP-sugars serve as the precursors that add sugar units, one at a time, first to the protein and then to the nonreducing end of the growing carbohydrate chain (Fig. 49.10). Glycosylation occurs initially in the lumen of the ER and subsequently in the Golgi complex. Glycosyltransferases, the enzymes that add sugars to the chain, are specific for the sugar being added, the type of linkage that is formed, and the sugars already present in the chain. Once the initial sugars are attached to the protein, the alternating action of two glycosyltransferases adds the sugars of the repeating disaccharide to the growing glycosaminoglycan chain. Sulfation occurs after addition of the sugar. The 3⬘-phosphoadenosine 5⬘-phosphosulfate (PAPS), also called active sulfate,

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935

Hyaluronate COO–

CH2OH O H H

O H H OH

H H O

H

OH

Glucuronic acid

H H

HO H

β(1 3)

O

NHCOCH3

N-Acetylglucosamine

Chondroitin 6-sulfate COO

–

–

CH2OSO3 O HO H

O H H

O

OH

H H

H

OH

Glucuronic acid

H

O

H H H

β(1 3)

NHCOCH3

N-Acetylgalactosamine 6-sulfate

Heparin –

H O

CH2OSO3 O H H H

H COO –

O

O

OH

H H

OH

H

H

OSO3–

H

NHSO3–

α(1 4)

Iduronic acid

N-sulfoD-Glucosamine6-sulfate

Keratan sulfate CH2OH O HO H H H

–

CH2OSO3 O H

O

O

H H

OH

H H

OH

H

NHCOCH3

Galactose

β(1 4)

N-Acetylglucosamine 4-sulfate

Dermatan sulfate –

H O H COO–

O

OH

H H

H

OH

Iduronic acid

O3S CH2OH O O H H H

β(1 3)

O

H H NHCOCH3

N-Acetylgalactosamine 6-sulfate

FIG. 49.8. Repeating disaccharides of some glycosaminoglycans. These repeating disaccharides usually contain an N-acetylated sugar and a uronic acid, which usually is glucuronic acid or iduronic acid. Sulfate groups are often present and are included in the sugar names in this figure. Iduronic acid and glucuronic acid are epimers at position 5 of the sugar.

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SECTION VIII ■ TISSUE METABOLISM

Table 49.3 Some Specific Functions of the Glycosaminoglycans and Proteoglycans Glycosaminoglycan

Function

Hyaluronic acid

Cell migration in: Embryogenesis Morphogenesis Wound healing Formation of bone, cartilage, cornea Transparency of cornea Transparency of cornea Binds LDL to plasma walls Anticoagulant (binds antithrombin III) Causes release of lipoprotein lipase from capillary walls Component of skin fibroblasts and aortic wall; commonly found on cell surfaces

Chondroitin sulfate proteoglycans Keratan sulfate proteoglycans Dermatan sulfate proteoglycans Heparin Heparan sulfate (syndecan) LDL, low-density lipoprotein.

The functional properties of a normal joint depend, in part, on the presence of a soft, well-lubricated, deformable, and compressible layer of cartilaginous tissue covering the ends of the long bones that constitute the joint. Bone Synovial lining Cartilage Calcified cartilage Capsule

Synovial cavity

In Sis Lupus’ case, the pathologic process that characterizes SLE disrupted the structural and functional integrity of her articular (joint) cartilage.

provides the sulfate groups (see Fig. 33.33). An epimerase converts glucuronic acid residues to iduronic acid residues. After synthesis, the proteoglycan is secreted from the cell. Its structure resembles a bottlebrush, with many glycosaminoglycan chains extending from the core protein (Fig. 49.11). The proteoglycans may form large aggregates, noncovalently attached by a “link” protein to hyaluronic acid (Fig. 49.12). The proteoglycans interact with the adhesion protein, fibronectin, which is attached to the cell membrane protein integrin. Cross-linked fibers of collagen also associate with this complex, forming the ECM (Fig. 49.13). The long polysaccharide side chains of the proteoglycans in cartilage contain many anionic groups. This high concentration of negative charges attracts cations that create a high osmotic pressure within cartilage, drawing water into this specialized connective tissue and placing the collagen network under tension. At equilibrium, the resulting tension balances the swelling pressure caused by the proteoglycans. The complementary roles of this macromolecular organization give cartilage its resilience. Cartilage can thus withstand the compressive load of weight bearing and then re-expand to its previous dimensions when that load is relieved. 3.

DEGRADATION OF PROTEOGLYCANS

Lysosomal enzymes degrade proteoglycans, glycoproteins, and glycolipids, which are brought into the cell by the process of endocytosis. Lysosomes fuse with the

Core protein Glycosaminoglycan

B

Link trisaccharide

Galactose

A n

Galactose

Xylose

O

N

H

CH2

C

H Serine

O

C

Uronic acid N-acetylated sugar

FIG. 49.9. Attachment of glycosaminoglycans to proteins. The sugars are linked to a serine or threonine residue of the protein. A and B represent the sugars of the repeating disaccharide.

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n

B

937

A

PAP 8 1

PAPS

7 6

B

A

UDP

UDP

UDP

UDP

2

UDP

7

A

UDP

UDP A

B

6

1 2 3 4 5 6 7 8

A

UDP

Xyl-transferase Gal-transferase I Gal-transferase II GlcUA-transferase I GalNAc-transferase I GlcUA-transferase II GalNAc-transferase II Sulfotransferase

3

UDP

UDP

B

UDP A

UDP

UDP UDP A

Protein core Xylose Galactose N-acetylgalactosamine Glucuronic acid Sulfate

4

B B 5

FIG. 49.10. Synthesis of chondroitin sulfate. Sugars are added to the protein one at a time, with UDP-sugars serving as the precursors. Initially, a xylose residue is added to a serine in the protein. Then two galactose residues are added, followed by a glucuronic acid (GlcUA) and an N-acetylglucosamine (GalNAc). Subsequent additions occur by the alternating action of two enzymes that produce the repeating disaccharide units. One enzyme ➅ adds GlcUA residues, and the other ➆ adds GalNAc. As the chain grows, sulfate groups are added by phosphoadenosine phosphosulfate (PAPS). (Modified from Roden L. In: Fishman WH, ed. Metabolic Conjugation and Metabolic Hydrolysis. Vol. II. Orlando, FL: Academic Press; 1970:401.)

– –

–

–

–

n –

– n

–

–

–

–

– n

– –

–

– –

–

–

–

–

Chondroitin sulfate

– n

– –

–

–

–

–

Protein

– n

Core protein

Repeating disaccharide

Link proteins

FIG. 49.11. “Bottlebrush” structure of a proteoglycan with a magnified segment.

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Keratan sulfate

Hyaluronic acid

FIG. 49.12. Proteoglycan aggregate.

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SECTION VIII ■ TISSUE METABOLISM

Collagen

Proteoglycan

Fibronectin

Cell membrane Integrin

FIG. 49.13. Interactions between the cell membrane and the components of the extracellular matrix.

Table 49.4

Defective Enzymes in the Mucopolysaccharidoses

Disease

Enzyme Deficiency

Accumulated Products

Hunter Hurler ⫹ Scheie Maroteaux–Lamy Mucolipidosis VII Sanfilippo A Sanfilippo B Sanfilippo D

Iduronate sulfatase ␣-L-iduronidase N-acetylgalactosamine sulfatase ␤-Glucuronidase Heparan sulfamidase N-acetylglucosaminidase N-acetylglucosamine 6-sulfatase

Heparan sulfate, dermatan sulfate Heparan sulfate, dermatan sulfate Dermatan sulfate Heparan sulfate, dermatan sulfate Heparan sulfate Heparan sulfate Heparin sulfate

These disorders share many clinical features, although there are significant variations between disorders, and even within a single disorder, based on the amount of residual activity remaining. In most cases, multiple organ systems are affected (with bone and cartilage being a primary target). For some disorders, there is significant neuronal involvement, leading to mental retardation.

endocytic vesicles, and lysosomal proteases digest the protein component. The carbohydrate component is degraded by lysosomal glycosidases. Lysosomes contain both endoglycosidases and exoglycosidases. The endoglycosidases cleave the chains into shorter oligosaccharides. Then exoglycosidases, specific for each type of linkage, remove the sugar residues one at a time from the nonreducing ends. Deficiencies of lysosomal glycosidases cause partially degraded carbohydrates from proteoglycans, glycoproteins, and glycolipids to accumulate within membrane-enclosed vesicles inside cells. These “residual bodies” can cause marked enlargement of the organ, with impairment of its function. In the clinical disorders known as the mucopolysaccharidoses (caused by accumulation of partially degraded glycosaminoglycans), deformities of the skeleton may occur (Table 49.4). Mental retardation often accompanies these skeletal changes.

II. INTEGRINS Integrins are the major cellular receptors for ECM proteins and provide a link between the internal cytoskeleton of cells (primarily the actin microfilament system) and extracellular proteins, such as fibronectin, collagen, and laminin. Integrins consist of an ␣- and a ␤-subunit. There are 18 distinct ␣- and 10 distinct ␤-gene products. Twenty-four unique ␣-/␤-dimers have been discovered. Mice have been genetically engineered to be unable to express many of the integrin genes (one gene at a time), and the phenotypes of these knockout mice vary from embryonic lethality (the ␣5 gene is an example) to virtually no observable defects (as exemplified by the ␣1 gene). In addition to anchoring the cell’s cytoskeleton to the ECM, thereby providing a stable environment in which the cell can reside, the integrins are also involved in a wide variety of cell signaling options. Certain integrins, such as those associated with white blood cells, are normally inactive because the white cell must circulate freely in the bloodstream. However, if an infection occurs, cells located in the area of the infection release cytokines, which activate the integrins on the white blood cells, allowing them to bind to vascular endothelial cells (leukocyte adhesion) at the site of infection. Leukocyte adhesion deficiency (LAD) is a genetic disorder that results from mutations in the ␤2-integrin such that leukocytes cannot be recruited to the sites of infection. Conversely, drugs are now being developed to block either the ␤2- or ␣4-integrins (on lymphocytes) to treat inflammatory and autoimmune disorders by interfering with the normal white cell response to cytokines. Integrins can be activated by “inside-out” mechanisms, whereby intracellular signaling events activate the molecule, or by “outside-in” mechanisms, in which a binding event with the extracellular portion of the molecule initiates intracellular signaling events. For those integrins that bind cells to ECM components, activation of specific integrins can result in migration of the affected cell through the ECM. This mechanism is operative during growth, during cellular differentiation, and in the process of metastasis of malignant cells to neighboring tissues.

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III. ADHESION PROTEINS Adhesion proteins are found in the ECM and link integrins to ECM components. Adhesion proteins, of which fibronectin is a prime example, are large multidomain proteins that allow binding to many different components simultaneously. In addition to integrin-binding sites, fibronectin contains binding sites for collagen and glycosaminoglycans. As the integrin molecule is bound to intracellular cytoskeletal proteins, the adhesion proteins provide a bridge between the actin cytoskeleton of the cell and the cells’ position within the ECM. Loss of adhesion protein capability can lead to either physiologic or abnormal cell movement. Alternative splicing of fibronectin allows many different forms of this adhesion protein to be expressed, including a soluble form (versus cell-associated forms), which is found in the plasma. The metabolic significance of these products remains to be determined. Fibronectin was first discovered as a large, external transformation-sensitive (LETS) protein, which was lost when fibroblasts were transformed into tumor cells. Many tumor cells secrete less than normal amounts of adhesion protein material, which allows for more movement within the extracellular milieu. This, in turn, increases the potential for the tumor cells to leave their original location and take root at another location within the body (metastasis).

IV. MATRIX METALLOPROTEINASES The ECM contains a series of proteases known as the matrix metalloproteinases or MMPs. These are zinc-containing proteases that use the zinc to appropriately position water to participate in the proteolytic reaction. Over 20 different types of human MMPs exist, and they cleave all proteins found in the ECM, including collagen and laminin. Because MMPs degrade ECM components, their expression is important to allow cell migration and tissue remodeling during growth and differentiation. In addition, many growth factors bind to ECM components and, as bound components, do not exhibit their normal growth-promoting activity. Destruction of the ECM by the MMPs releases these growth factors, allowing them to bind to cell surface receptors to initiate growth of tissues. Thus, coordinated expression of the MMPs is required for appropriate cell movement and growth. Cancer cells that metastasize require extensive ECM remodeling and usually use MMP activity to spread throughout the body. A propeptide is present in newly synthesized MMPs that contains a critical cysteine residue. The cysteine residue in the propeptide binds to the zinc atom at the active site of the protease and prevents the propeptide from exhibiting proteolytic activity. Removal of the propeptide is required to activate the MMPs. Once they are activated, certain MMPs can activate other forms of MMP. Regulation of MMP activity is quite complex. These regulatory processes include transcriptional regulation, proteolytic activation, inhibition by the circulating protein ␣2-macroglobulin, and regulation by a class of inhibitors known as tissue inhibitors of metalloproteinases or TIMPs. It is important that the synthesis of TIMPs and MMPs be coordinately regulated because dissociation of their expression can facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis. CLINICAL COMMENTS Sis Lupus. Articular cartilage is a living tissue with a turnover time determined by a balance between the rate of its synthesis and that of its degradation (Fig. 49.14). The chondrocytes that are embedded in the matrix of intra-articular cartilage participate in both its synthesis and its enzymatic degradation. The latter occurs as a result of cleavage of proteoglycan aggregates by enzymes produced and secreted by the chondrocytes.

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The movement of tumor cells from its tissue of origin (metastasis) through the blood or lymph system, and colonization of a target tissue, requires degradation of the extracellular matrix to allow for cell movement. This is accomplished by a family of proteins known as matrix metalloproteinases (MMPs). The MMPs degrade specific extracellular matrix components (such as collagen or elastin), thereby allowing cells access through this compartment. One assay for determining if MMPs are present in a biological sample is the gelatin zymography assay; a newer, more sensitive assay is based on fluorescence resonance energy transfer (FRET). In the zymography assay polyacrylamide gels containing the protein gelatin are prepared, and the enzyme samples are run through the gel, in the presence of SDS. After the gel has run, enzyme activity is reconstituted by substituting Triton X-100 for the SDS. An assay buffer is then placed over the gel, which is left overnight. During this part of the procedure, if a lane on the gel contained MMP activity, the MMP would be digesting the gelatin in the area of the gel where the MMP resided. After the activity phase is complete, the gel is developed with Coomassie stain, which binds to the proteins in the gel, including the gelatin. A positive result would appear as white bands on a blue background. The white bands are caused by the absence of gelatin in that region of the gel, as the MMPs present at that region have digested the gelatin such that Coomassie stain has nothing to bind to in that area of the gel. The FRET assay uses a peptide substrate that contains both a fluorophore and a quencher in close proximity. When excited, the quencher blocks fluorescence emittance from the fluorophore because of the close proximity of the two molecules on the peptide. After MMP treatment, however, the peptide is cleaved between the flurophore and quencher, such that the quencher is no longer in close proximity to the fluorophore. This results in a strong fluorescence emittance. Thus, fluorescence intensity will increase as the peptide is cleaved in the presence of the MMP. This is a very sensitive assay, detecting subnanogram levels of a wide variety of members of the MMP family.

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Pericellular matrix

Intercellular matrix

Synovial fluid

Chondrocyte Biosynthesis

t1 2 = 100-800 d

t 1 2 = 4–30 d

Degradation products Lysosomal degradation

FIG. 49.14. Synthesis and degradation of proteoglycans by chondrocytes. (From Cohen RD, et al. The Metabolic Basis of Acquired Disease. Vol. 2. London: Bailliere Tindall; 1990:1859.)

In systemic lupus erythematosus (SLE), the condition that affects Sis Lupus, this delicate balance is disrupted in favor of enzymatic degradation, leading to dissolution of articular cartilage and, with it, the loss of its critical cushioning functions. The underlying mechanisms responsible for this process in SLE involves an autoimmune induced inflammation. In this sense, SLE is an “autoimmune” disease because antibodies are produced by the host that attack “self” proteins. This process excites the local release of cytokines such as interleukin-1 (IL-1), which increases the proteolytic activity of the chondrocytes, causing further loss of articular proteins such as the proteoglycans. The associated inflammatory cascade is responsible for Sis Lupus’ joint pain. Ann Sulin. The microvascular complications of both type 1 and type 2 diabetes mellitus involve the small vessels of the retina (diabetic retinopathy), the renal glomerular capillaries (diabetic nephropathy), and the vessels that supply blood to the peripheral nerves (autonomic neuropathy). The lack of adequate control of Ann Sulin’s diabetic state over many years caused a progressive loss of the filtering function of the approximately 1.5 million glomerular capillary-mesangial units that are present in her kidneys. Chronic hyperglycemia is postulated to be a major metabolic initiator or inducer of diabetic microvascular disease, including those renal glomerular changes that often lead to end-stage renal disease (“glucose toxicity”). For a comprehensive review of the four postulated molecular mechanisms by which chronic hyperglycemia causes these vascular derangements, the reader is referred to an excellent review by Sheetz and King (see the online Suggested Readings). Regardless of which of the postulated mechanisms (increased flux through the aldose reductase or polyol pathway [see Chapter 30], the generation of

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advanced glycosylation end products [AGEs], the generation of reactive oxygen intermediates [see Chapter 24], or excessive activation of protein kinase C [see Chapter 18]) will eventually be shown to be the predominant causative mechanism, each can lead to the production of critical intracellular and extracellular signaling molecules (e.g., cytokines). These, in turn, can cause pathologic changes within the glomerular filtration apparatus that reduces renal function. These changes include increased synthesis of type IV collagen, fibronectin, and some of the proteoglycans, causing the glomerular basement membrane (GBM) (Fig. 49.15) to become diffusely thickened throughout the glomerular capillary network. This membrane thickening alters certain specific filtration properties of the GBM, preventing some of the metabolites that normally enter the urine from the glomerular capillary blood (via the fenestrated capillary endothelium) from doing so (a decline in glomerular filtration rate [GFR]). As a result, these potentially toxic substances accumulate in the blood and contribute to the overall clinical presentation of advancing uremia. In spite of the thickening of the GBM, this membrane becomes “leaky” for some macromolecules (e.g., albumin) that normally do not enter the urine from the glomerular capillaries (microalbuminuria). Suggested mechanisms for this increased permeability or leakiness include reduced synthesis of the specific proteoglycan, heparan sulfate, as well as increased basement membrane production of vascular endothelium growth factor (VEGF), a known angiogenic and permeability factor, and expansion of the extracellular matrix in the mesangium. The mesangium consists of specialized tissue containing collagen, proteoglycans, and other macromolecules that surround the glomerular capillaries and that, through its gel-like and sieving properties, determine, in part, the glomerular capillary hydraulic filtration pressure as well as the functional status of the capillary endothelium– mesangial glomerular basement membrane filtration apparatus (see Fig. 49.15). As the mesangial tissue expands, the efficiency of glomerular filtration diminishes proportionately. The cause of these mesangial changes is, in part, the consequence of increased expression of certain growth factors, especially transforming growth

Glomerulus Capillary loops

Fenestrated capillary endothelium Urinary space

Capillary lumen

Capillary lumen

Parietal epithelium (Bowman capsule)

Proximal tubule Bowman space (urinary space)

Urine

Mesangial cells

Mesangial matrix

Glomerular basement membrane Bowman space (urinary space) (proximal-most part of a nephron)

Capillary lumen

Capillary lumen

FIG. 49.15. A cross section of a normal renal glomerulus showing four capillary tufts delivering blood to the glomerulus for filtration across the fenestrated capillary endothelium, then through the glomerular basement membrane into the Bowman space to form urine. The urine then enters the proximal tubule of the nephron. This filtration removes potentially toxic metabolic end products from the blood. The mesangium, by contracting and expanding, controls the efficiency of these filtering and excretory functions by regulating the hydraulic filtration pressures in the glomerulus. An intact basement membrane must be present to maintain the integrity of the filtering process.

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factor ␤ (TGF-␤) and connective tissue growth factor (CTGF). Future therapeutic approaches in patients with early diabetic nephropathy may include the use of antibodies that neutralize TGF-␤. BIOCHEMICAL COMMENTS

Osteogenesis imperfecta (OI) can occur due to mutations in genes other than collagen. Mutations in CRTAP (cartilage associated protein) or LEPRE1 (prolyl 3-hydroxylase 1, or PH3-1) lead to defective collagen fibers being produced. CRTAP forms a complex with PH3-1 and cyclophilin to hydroxylate a specific proline residue in types I and II collagen. Failure to hydroxylate this proline residue leads to unstable collagen and moderate to severe forms of OI. The pattern of inheritance for both CRTAP and LEPRE1 mutations is autosomal recessive.

Osteogenesis Imperfecta. Osteogenesis imperfecta (OI) is a heterogenous group of diseases that have in common a defect in collagen production. This defect can be either of two types: the first type is associated with a reduction in the synthesis of normal collagen (resulting from a gene deletion or splice-site mutation). The second type is associated with the synthesis of a mutated form of collagen. Most of the mutations have a dominant-negative effect, leading to an autosomal dominant mode of transmission. In the second type of OI, many of the known mutations involve substitutions of another amino acid for glycine. This results in an unstable collagen molecule because glycine is the only amino acid that can fit between the other two chains within the triple helix of collagen. If the mutation is near the carboxy-terminal end of the molecule, the phenotype of the disease is usually more severe than if the mutation is near the amino-terminal end (recall that triple helix formation proceeds from the carboxy- to the amino-terminal end of the molecule). Of interest are mutations that replace glycine with either serine or cysteine. Such mutations are more stable than expected because of the hydrogen bonding capabilities of serine and the ability of cysteine to form disulfide bonds. Both would aid in preventing the strands of the triple helix from unwinding. Children with OI can be treated with a class of compounds known as bisphosphonates, which consist of two phosphates linked by a carbon or nitrogen bridge (thus, they are analogs of pyrophosphate, in which the two phosphates are linked by oxygen). Normal bone remodeling is the result of a coordinated “coupling” between osteoclast activity (cells that resorb bone) and osteoblast activity (cells that form bone). In OI, bone resorption outpaces bone formation because osteoclast activity is enhanced (perhaps because of the reduced levels of normal collagen present to act as nucleating sites for bone formation). This leads to a net loss of bone mass and fragility of the skeleton. Bisphosphonates inhibit osteoclast action with the potential to increase bone mass and its tensile strength. Key Concepts • • • • • •

• • • • •

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The extracellular matrix (ECM) consists of fibrous structural proteins, proteoglycans, and adhesion proteins. The ECM provides support to the tissues and restricts movement of cells. Collagen is the most abundant fibrous protein, and it consists of a triple helix stabilized by hydrogen bonds and intramolecular cross-links. There are more than 25 different types of collagen. Elastin is the major protein found in elastic fibers, and it is responsible for the contractility exhibited by these fibers. Laminin provides structural support to tissues via binding to various components of the ECM. Proteoglycans consist of polysaccharides (glycosaminoglycans) bound to a core protein. The polysaccharides are usually a repeating disaccharide unit containing negative charges. Because of charge repulsion, the proteoglycans form a hydrated gel that provides flexible mechanical support to the ECM. Integrins are cellular membrane receptors for ECM proteins, and they link the cellular cytoskeleton to extracellular proteins. Integrins are also signaling proteins when they are bound to appropriate components. Adhesion proteins link the integrins to ECM components. Matrix metalloproteinases are the only proteases that can degrade ECM components, and they are carefully regulated by the tissue inhibitors of matrix metalloproteinases (TIMPS). Diseases discussed in this chapter are summarized in Table 49.5.

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Table 49.5

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Diseases Discussed in Chapter 49

Disease or Disorder

Genetic or Environmental

Lupus

Environmental

Type 2 diabetes

Both

Osteogenesis imperfecta

Genetic

Supravalvular aortic stenosis (Williams syndrome) Junctional epidermolysis bullosa

Genetic

Mucopolysaccharidoses

Genetic

Genetic

Comments Alterations in cell matrix components caused by an autoimmune induced trigger Cell matrix interactions can be altered because of elevated glucose levels and nonenzymatic glycosylation Inherited mutations in collagen genes that disrupt the function of the altered collagen An inherited mutation in the elastin gene leading to abnormal heart function A blistering skin condition caused by a mutation in either one form of collagen or laminin Defects in the breakdown of mucopolysaccharides, found primarily in the extracellular matrix. See Table 49.4 for more details on these diseases.

REVIEW QUESTIONS—CHAPTER 49 1.

2.

Individuals who develop scurvy suffer from sore and bleeding gums and loss of teeth. This is a result, in part, of the synthesis of a defective collagen molecule. The step that is affected in collagen biosynthesis attributable to scurvy is which of the following? A. The formation of disulfide bonds, which initiates tropocollagen formation B. The formation of lysyl cross-links between collagen molecules C. Secretion of tropocollagen into the extracellular matrix D. The formation of collagen fibrils E. The hydroxylation of proline residues, which stabilizes the collagen structure The underlying mechanism that allows elastin to exhibit elastic properties (expand and contract) is which of the following? A. Proteolysis during expansion, and resynthesis during contraction B. Breaking of disulfide bonds during expansion, and re-formation of these bonds during contraction C. A decrease in entropy during expansion, and an increase in entropy during contraction D. The breaking of salt bridges during expansion, and re-formation of the salt bridges during contraction E. Hydroxylation of elastin during expansion, and decarboxylation of elastin during contraction

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3.

The underlying mechanism by which glycosaminoglycans allow for the formation of a gel-like substance in the extracellular matrix occur by which of the following? A. Charge attraction between glycosaminoglycan chains B. Charge repulsion between glycosaminoglycan chains C. Hydrogen bonding between glycosaminoglycan chains D. Covalent cross-linking between glycosaminoglycan chains E. Hydroxylation of adjacent glycosaminoglycan chains

4.

The movement of tumor cells from their site of origin to other locations within the body requires the activity of which of the following proteins? A. Collagen B. Laminin C. Proteoglycans D. Elastin E. Matrix metalloproteinases

5.

Fibronectin is frequently absent in malignant fibroblast cells. One of the major functions of fibronectin is which of the following? A. To inhibit the action of matrix metalloproteinases B. To coordinate collagen deposition within the extracellular matrix C. To fix the position of cells within the extracellular matrix D. To regulate glycosaminoglycan production E. To extend glycosaminoglycan chains using nucleotide sugars

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Answers to Review Questions protein, 150 ⫻ 4 ⫽ 600 kcal; fat, 95 ⫻ 9 ⫽ 855 kcal; alcohol, 45 ⫻ 7 ⫽ 315 kcal) (thus, A is incorrect). His fat intake was 21% (855 ⫼ 4,110) of his total caloric intake. His alcohol intake was 7.7% (315 ⫼ 4,110) (thus, C is incorrect). His protein intake was well above the RDA of 0.8 g/kg body weight (thus, D is incorrect). His RMR is roughly 24 kcal/day/kg body weight or 2,880 kcal/day (it will actually be less because he is obese and has a greater proportion of metabolically less active tissue than the average 70-kg man). His daily energy expenditure is about 3,744 kcal/day (1.3 ⫻ 2,880) or less. Thus, his intake is greater than his expenditure, and he is in positive caloric balance and is gaining weight (thus, E is incorrect).

CHAPTER 1 1.

2.

3.

4.

5.

The answer is B. In the process of respiration, O2 is consumed and fuels are oxidized to CO2 and H2O. The energy from the oxidation reactions is used to generate ATP from ADP and Pi. However, a small amount of energy is also released as heat (thus, C is incorrect). Although fuels can be stored as triacylglycerols, this is not part of respiration (thus, A is incorrect). Respiration is a catabolic pathway (fuels are degraded), as opposed to an anabolic pathway (compounds combine to make larger molecules) (thus, E is incorrect). The answer is A. Mrs. Jones’s diet lacks grain products, fruits, and vegetables, all of which are good sources of vitamin C. Her diet is adequate in protein, as eggs, milk, cheese, and cream contain significant levels of protein. Her calcium levels should be fine because of the milk, cream, and cheese in her diet. Vitamin B12 is derived from foods of animal origin such as eggs, milk, and cheese. As the patient’s weight has been stable for a year, her diet contains sufficient calories to allow her to maintain this weight, which is in the normal range for a patient who is 5 ft 4 in tall, as her BMI is 21.5. The answer is A. The resting metabolic rate (RMR) is the calories being expended by a recently awakened resting person who has fasted for 12 to 18 hours and whose body temperature is at 20°C. It is equivalent to the energy expenditure of our major organs and resting skeletal muscle. Women generally have a lower RMR per kilogram body weight because more of their body weight is usually metabolically less active adipose tissue. Children have a higher RMR per kilogram body weight because more of their body weight is metabolically active organs such as brain. The RMR increases in a cold environment because more energy is being expended to generate heat. The RMR is not equivalent to our daily energy expenditure (DEE), which includes RMR, physical activity, and diet-induced thermogenesis. The answer is D. The Recommended Daily Allowance (RDA) of a nutrient is determined from the Estimated Average Requirement (EAR) ⫹ 2 standard deviations (SD) of the mean, and should meet the needs for 97% to 98% of the healthy population. It is, therefore, a reasonable goal for the intake of a healthy individual. The EAR is the amount that prevents development of established signs of deficiency in 50% of the healthy population. Although data with laboratory animals have been used to establish deficiency symptoms, RDAs are based on data collected on nutrient ingestion by humans. The answer is B. The recommended total fat intake is less than 30% of total calories. His total caloric consumption was 4,110 kcal/day (carbohydrate, 4 ⫻ 585 ⫽ 2,340 kcal;

CHAPTER 2 1.

2.

3.

The answer is E. During digestion of a mixed meal, starch and other carbohydrates, proteins, and dietary triacylglycerols are broken into their monomeric units (carbohydrates into simple monosaccharides, protein into amino acids, triacylglycerols into fatty acids and glycerol). Glucose is the principal sugar in dietary carbohydrates, and thus it increases in the blood. Amino acids and monosaccharides enter the portal vein and go to the liver first. After digestion of fats and absorption of the fatty acids, most fatty acids are converted back into triacylglycerols and subsequently into chylomicrons by intestinal cells. Chylomicrons go through lymphatic vessels and then blood, principally to adipose tissue. The answer is C. After a high-carbohydrate meal, glucose is the major fuel for most tissues, including skeletal muscle, adipose tissue, and liver. The increase in blood glucose levels stimulates the release of insulin, not glucagon. Insulin stimulates the transport of glucose in skeletal muscle and adipose tissue, not brain. Liver, not skeletal muscle, converts glucose to fatty acids. Although the red blood cell uses glucose as its only fuel at all times, it generates ATP from conversion of glucose to lactate, not CO2. The answer is C. In the fed state, insulin will be released because of the increase in blood glucose levels. Insulin will act on muscle cells to increase glucose uptake in the muscle. Insulin will also stimulate the liver to synthesize both glycogen and fatty acids, which leads to enhanced triglyceride synthesis and very low-density lipoprotein (VLDL) production to deliver the fatty acids to other tissues of the body. Insulin will stimulate glucose uptake in fat cells, but does not stimulate fatty acid synthesis in the fat cells (i.e., unique to the liver), but will lead to enhanced triglyceride synthesis in the fat cells.

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4.

5.

Answer is B. Chylomicrons are the lipoprotein particles formed in intestinal epithelial cells from dietary fats, and they contain principally triacylglycerols formed from components of dietary triacylglycerols. A decreased intake of calories in general would include a decreased consumption of fat, carbohydrate, and protein, which might not lower chylomicron levels. Dietary cholesterol, although found in chylomicrons, is not their principal component. The answer is A. The patient’s BMI is in the obese range, with large abdominal fat deposits. He needs to decrease his intake of total calories because an excess of calories ingested as carbohydrate, fat, or protein results in deposition of triacylglycerols in adipose tissue. If he keeps his total caloric intake the same, substitution of one type of food for another will help very little with weight loss. (However, a decreased intake of fat may be advisable for other reasons). Limited food diets, such as the ice cream and sherry diet, or a high protein diet of shrimp, work if they decrease appetite and, therefore, ingestion of total calories.

3.

4.

CHAPTER 3 1.

2.

The answer is A. By 24 hours after a meal, hepatic (liver) gluconeogenesis is the major source of blood glucose because hepatic glycogen stores have been nearly depleted. Muscle and other tissues lack an enzyme necessary to convert glycogen or amino acids to glucose (thus, B is incorrect). The liver is the only significant source of blood glucose. Glucose is synthesized in the liver from amino acids (provided by protein degradation), from glycerol (provided by hydrolysis of triacylglycerols in adipose tissue), and from lactate (provided by anaerobic glycolysis in red blood cells and other tissues). Glucose cannot be synthesized from fatty acids or ketone bodies (thus, D and E are incorrect). The answer is A. The liver will produce ketone bodies when fatty acid oxidation is increased, which occurs when glucagon is the predominant hormone (glucagon leads to fatty acid release from the fat cells, for oxidation in the liver and muscle). This would be the case in an individual who cannot produce insulin and is not taking insulin injections. However, in this situation, the ketone bodies are not being used by the nervous system (brain) because of the high levels of glucose in the circulation. This leads to severely elevated ketone levels due to nonuse. The glucose is high because, in the absence of insulin, muscle and fat cells are not using the glucose in circulation as an energy source. Recall, although the liver produces ketone bodies, it lacks a necessary enzyme to use ketone bodies as an energy source. There is no relation between blood urea nitrogen levels and the rate of ketone body

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production. The muscle reduces its use of ketone bodies under these conditions, but not of fatty acids. The answer is C. The major change during prolonged fasting is that as muscles decrease their use of ketone bodies, ketone bodies increase enormously in the blood and are used by brain as a fuel. However, even during starvation, glucose is still required by the brain, which cannot oxidize fatty acids to an appreciable extent (thus, D is incorrect). Red blood cells can use only glucose as a fuel (thus, B is incorrect). Because the brain, red blood cells, and certain other tissues are glucose dependent, the liver continues to synthesize glucose, and blood glucose levels are maintained at only slightly less than fasting levels (thus, A is incorrect). Adipose tissue stores (approximately 135,000 kcal) are not depleted in a well-nourished individual after 1 week of fasting (thus, E is incorrect). The answer is A. Decreased serum albumin could have several causes, including hepatic disease that decreases the ability of the liver to synthesize serum proteins, protein malnutrition, marasmus, or diseases that affect the ability of the intestine to digest protein and absorb the amino acids. However, his BMI is in the healthy weight range (thus, B and D are incorrect). His normal CHI indicates that he has not lost muscle mass and is, therefore, not suffering from protein malnutrition (thus, B, C, D, and E are incorrect). The answer is C. His protein intake of 150 kcal is about 37 g protein (150 kcal ⫼ 4 kcal/g ⫽ 37 g) below the Recommended Dietary Allowance (RDA) of 0.8 g protein per kilogram body weight (thus, A is incorrect, as Otto weighs approximately 88 kg). His carbohydrate intake of 150 kcal is below the glucose requirements of his brain and red blood cells (about 150 g/day; see Chapter 2) (thus, B is incorrect). Therefore, he will be breaking down muscle protein to synthesize glucose for the brain and other glucose-dependent tissues and adipose tissue mass to supply fatty acids for muscle and tissues able to oxidize fatty acids. Because he will be breaking down muscle protein to amino acids and converting the nitrogen from both these amino acids and his dietary amino acids to urea, his nitrogen excretion will be greater than his intake and he will be in negative nitrogen balance (thus, D is incorrect). It is unlikely that he will develop hypoglycemia while he is able to supply gluconeogenic precursors.

CHAPTER 4 1.

The answer is A. The pH is the negative log of the hydrogen ion concentration, [H⫹]. Thus, at a pH of 7.5, [H⫹] is 10⫺7.5; and at pH 6.5, it is 10⫺6.5. The [H⫹] has changed by a factor of 10⫺6.5/10⫺7.5, which is 101 or 10. Any decrease of 1 pH unit is a 10-fold increase of [H⫹] or a 10-fold decrease of [OH⫺]. A shift in the concentration

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2.

3.

4.

5.

ANSWERS TO REVIEW QUESTIONS

of buffer anions has definitely occurred, but the change in pH reflects the increase in hydrogen ion concentration in excess of that absorbed by buffers. The answer is C. To solve this problem, one first needs to calculate the concentration of the conjugate base and acid of the buffer at pH 7.4. Using the Henderson-Hasselbalch equation, one can calculate that the concentration of the conjugate base is 0.061 M and that of the acid is 0.039 M. At this point, the reaction occurs, generating 0.01 M protons (10 ␮mol/mL is a concentration of 10 mM, or 0.01 M). As the protons are generated, they will combine with the conjugate base, producing the acid. This will change the concentration of conjugate base to 0.051 M, and the concentration of the acid to 0.049 M. Plugging those values into the Henderson-Hasselbalch equation leads to a pH value of 7.22. Recall, if the concentration of the conjugate base equaled the concentration of the acid, the pH would be the pK value (in this case 7.2). Because protons are being generated, the pH will drop, not rise, so answer (A) cannot be correct. The answer is B. NH3 is a weak base, which associates with a proton to produce the ammonium ion (NH3 ⫹ H⫹ → NH4⫹), which has a pKa of 9.5. Thus, at pH 7.4, most of the ammonia will be present as ammonium ion. The absorption of hydrogen ions will tend to increase, not decrease, the pH of the blood (thus, A is incorrect). With the decrease of hydrogen ions, carbonic acid will dissociate to produce more bicarbonate (H2CO3 → HCO3⫺ ⫹ H⫹) and more CO2 will go toward carbonic acid (thus, C is incorrect). Kussmaul respiration, an increased expiration of CO2, occurs under an acidosis, the opposite condition. The answer is C. Vomiting expels the strong acid gastric acid (HCl). As cells in the stomach secrete more HCl, they draw on H⫹ ions in interstitial fluid and blood, thereby tending to increase blood pH and cause an alkalosis. The other conditions tend to produce an acidosis (thus, A, B, D, and E are incorrect). Lactic acid is a weak acid secreted into the blood by muscles during exercise. A patient with increased ketone body production can exhibit a fall of pH because the ketone bodies acetoacetate and ␤-hydroxybutyrate are dissociated acids. As bicarbonate in the intestinal lumen is lost in the watery diarrhea, more bicarbonate is secreted by intestinal cells. As intestinal cells produce bicarbonate, more H⫹ is also generated (H2CO3 → HCO3⫺ ⫹ H⫹). While the bicarbonate produced by these cells is released into the intestinal lumen, the protons accumulate in blood, resulting in an acidosis. Hypercatabolism (see E), an increased rate of catabolism, generates additional CO2, which produces more acid (CO2 ⫹ H2O → H2CO3 → HCO3⫺ ⫹ H⫹). The answer is B. Methylmalonic acid contains two carboxylic acid groups and is, therefore, a weak acid. According to the Henderson-Hasselbalch equation, the carboxylic acid groups are 50% dissociated at their pKa (thus,

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C is incorrect). Carboxylic acid groups usually have a pKa between 2 and 5, so this acid would be nearly fully dissociated at a blood pH of 7.4 and cannot buffer intracellularly at neutral pH (thus, D and E are incorrect). (Although you do not need to know anything about methylmalonate to answer this question, methylmalonate is an organic acid generated in patients with a problem in metabolism of methylmalonyl coenzyme A, such as a deficiency in vitamin B12. Its acidity may contribute to the development of symptoms involving the nervous system. Its appearance in the urine can be classified as an organic aciduria.)

CHAPTER 5 1.

2.

3.

4.

The answer is D. Water-soluble compounds are polar (contain an uneven distribution of charge) so that the more positive portion of the molecule hydrogen-bonds (shares electrons) with the oxygen of water, and the more negative portion of the molecule hydrogen-bonds with the hydrogens of water. Water-soluble compounds need not contain a full negative or positive charge (thus, B and C are incorrect) and generally contain oxygen or nitrogen, in addition to carbon and hydrogen (see A). Large C–H portions of a molecule, such as an aromatic ring, are nonpolar and contribute to the insolubility of the molecule in water (see E). The answer is E. The compound contains an ⫺OH group, which should appear in the name as an “-ol” or a “hydroxyl-” group. All answers fit this criterion. The structure also contains a carboxylate group (⫺COO⫺), which should appear in the name as an “-ate” or “acid.” Only D and E fit this criterion. Counting backward from the carboxylate group (carbon 1), the second carbon is ␣, the third carbon is ␤, and the fourth carbon, containing the hydroxyl group, is ␥. Thus, the compound is ␥-hydroxybutyrate. A, B, and C can also be eliminated because “meth-” denotes a single carbon, “eth-” denotes two carbons, and the “-ene” in ethylene denotes a double bond. The answer is B. The term “glycosidic bond” refers to a covalent bond formed between the anomeric carbon of one sugar, when it is in a ring form, and a hydroxyl group or nitrogen of another compound (see Fig. 5.16) (thus, A, D, and E are incorrect). Disaccharides can be linked through their anomeric carbons, but not polysaccharides, because there would be no anomeric carbon left to form a link with the next sugar in the chain (thus, C is incorrect). The answer is A. Triglycerides (triacylglycerols) are composed of a glycerol backbone to which three fatty acids (“tri-”) are attached (see Fig. 5.18). The carboxylic acid group of each fatty acid forms an ester with one of the three ⫺OH groups on glycerol; thus, the fatty acid is an “acyl” group. Sphingolipids, in contrast, contain one fatty acyl group and one group derived from a fatty acid, attached to a group derived from serine (see Fig. 5.20).

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5.

The answer is B. Sphingolipids contain a ceramide group, which is sphingosine with an attached fatty acid. They do not contain a glycerol moiety (thus, A is incorrect). However, different sphingolipids have different substituents on the ⫺CH2OH group of ceramide. For example, sphingomyelin contains phosphorylcholine, and gangliosides contain NANA (thus, C and D are incorrect). No known sphingolipids contain a steroid (E).

CHAPTER 6 1.

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The answer is D. In hydrogen bonds, a hydrogen atom is shared between two electron-rich groups, such as oxygen or nitrogen. Thus, the nitrogen in the peptide bond may share its hydrogen with the oxygen in an aspartate carboxyl group, which will be negatively charged at a pH of 7.4. Leucine is nonpolar and cannot participate in hydrogen bonding (thus, A is incorrect), and aspartyl and glutamyl residues are both negatively charged (thus, B is incorrect). Bonds between two fully charged groups are electrostatic bonds, not hydrogen bonds (thus, C is incorrect). Sulfhydryl groups do not participate in hydrogen bonding. The answer is A. The peptide backbone contains only carbon and nitrogen atoms linked covalently in a chain. The amide nitrogen of one amino acid (N) is covalently attached to the ␣-carbon of the same amino acid (C), which is covalently linked to the carboxyl carbon (C) of that amino acid, which forms a peptide bond with the nitrogen of the next amino acid (N). The answer is A. Arginine is a basic amino acid that has a positively charged side chain at neutral pH. It can, therefore, form tight electrostatic bonds with negatively charged asp and glu side chains in insulin. The ␣-carboxylic acid groups at the N-terminals of proteins are bound through peptide bonds (thus, B is incorrect). The arginine side chain is not hydrophobic, and it cannot form disulfide bonds because it has no sulfhydryl group (thus, C and D are incorrect). Its basic group is an ureido group that cannot form peptide bonds (see E). The answer is B. Only these amino acids have side chain hydroxyl groups. As a general rule, serine–threonine protein kinases form one group of protein kinases, and tyrosine protein kinases form another. The answer is A. Adding a phosphate group to the serine side chain adds negative charges to the side chain, allowing ionic interactions to develop. These new ionic interactions allow the protein to change shape and to alter its activity. Substituting a glutamate for the serine adds a negative charge to this location within the protein, which may participate in ionic interactions and lead to a shape change in the protein. None of the other suggested mutations (serine to either threonine [which adds a hydroxyl group, just as

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serine has], to tyrosine [again, another hydroxyl containing amino acid side chain], to lysine [adding a positive charge rather than a negative charge], or to leucine [a totally hydrophobic side chain]) will lead to the insertion of negative charges at this location in the protein.

CHAPTER 7 1.

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The answer is B. The regular repeating structure of an ␣-helix is possible because it is formed by hydrogen bonds within the peptide backbone of a single strand. Thus, ␣-helices can be formed from a variety of primary structures. However, proline cannot accommodate the bends for an ␣-helix because the atoms involved in the peptide backbone are part of a ring structure, and glycine cannot provide the space filling required for a stable structure. The answer is C. Globular proteins fold into a spherelike structure with their hydrophilic residues on the outside to interact with water (hence, A is incorrect) and their hydrophobic residues on the inside away from water (thus, B is incorrect). Secondary structures are formed by hydrogen bonding, not hydrophobic bonding (thus, D is incorrect). Disulfide bonds are rare in globular proteins and are not needed to maintain a stable structure (thus, E is incorrect). The answer is D. When an ␣-helix is formed, all side chains face the outside of the helix, which is the hydrophobic environment of the lipid membrane. Thus, the side chains should be hydrophobic. Of the amino acids listed, only leucine fits the bill (see Chapter 6). Proline interrupts helical structure because the imino bond of its ring structure does not allow the polypeptide backbone to form the angles required for ␣-helical formation. The answer is A. The characteristic staining of amyloid arises from fibrils of ␤-pleated sheet structure perpendicular to the axis of the fiber (thus, B, C, and D are incorrect). The native conformation of a protein is generally the most stable and lowest energy conformation, and the lower its energy state, the more readily a protein folds into its native conformation and the less likely it will assume the insoluble ␤-pleated sheet structure of amyloid (thus, E is incorrect). The answer is A. The protein hydrolyzes ATP, which is a characteristic of the actin fold. None of the other folds described will hydrolyze ATP.

CHAPTER 8 1.

The answer is B. In most reactions, the substrate binds to the enzyme before its reaction with the coenzyme occurs. Thus, the substrate may bind but it cannot react with the coenzyme to form the transition-state complex.

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Each coenzyme carries out a single type of reaction, so no other coenzyme can substitute (thus, C is incorrect). The three-dimensional geometry of the reaction is so specific that functional groups on amino acid side chains cannot substitute (thus, D is incorrect). Free coenzymes are not very reactive because amino acid side chains in the active site are required to activate the coenzyme or the reactants (thus, E is incorrect). However, increasing the supply of vitamins to increase the amount of coenzyme bound to the enzyme can sometimes help. The answer is D. The patient was diagnosed with maturity-onset diabetes of the young (MODY) caused by this mutation. In glucokinase, binding of glucose normally causes a huge conformational change in the actin fold that creates the binding site for ATP. Although proline and leucine are both nonpolar amino acids, B is incorrect—proline creates kinks in helices and thus would be expected to disturb the large conformational change required (see Chapter 7). In general, binding of the first substrate to an enzyme creates conformational changes that increases the binding of the second substrate or brings functional groups into position for further steps in the reaction. Thus, a mutation need not be in the active site to impair the reaction, and A is incorrect. It would probably take more energy to fold the enzyme into the form required for the transition-state complex, and fewer molecules would acquire the energy necessary (thus, C is incorrect). The active site lacks the functional groups required for an alternate mechanism (thus, E is incorrect). The answer is A. When the pKa of an ionizable group is below the pH value, the group will be deprotonated. When the pKa of an ionizable group is above the pH value, the group will be protonated. Thus, at pH 5.2, glutamate 35 (with a pKa of 5.9, which is greater than 5.2) will remain protonated, and aspartate 52 (with a pKa of 4.5, which is less than 5.2) will be ionized (because the side chain carries a negative charge when deprotonated). Therefore, E35 is protonated, and D52 is ionized. There is sufficient information presented to answer this question. The answer is A. The glucose residue from UDP-glucose is being transferred to the alcohol group of another compound. In isomerization reactions, groups are transferred within the same molecule (thus, B is incorrect). There is no cleavage or synthesis of carbon–carbon bonds (thus, C and D are incorrect). Oxidation–reduction has not occurred because no hydrogens or oxygen atoms have been removed or added in the conversion of substrate to product. The answer is C. Transfer of a carbohydrate residue from one molecule to another is a glycosyltransferase reaction. Kinases transfer phosphate groups, dehydrogenases transfer electrons as hydrogen atoms or hydride ions, transaminases transfer amino groups, and isomerases transfer atoms within the same molecule.

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CHAPTER 9 1.

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The answer is B. The rate of the reaction is directly proportional to the proportion of enzyme molecules that contain bound substrate. Thus, it is at 50% of its maximal rate when 50% of the molecules contain bound substrate (thus, A, C, and D are incorrect). The rate of the reaction is directly proportional to the amount of enzyme present, which is incorporated into the term Vmax (where Vmax ⫽ k[total enzyme]) (thus, E is incorrect). The answer is A. The patient’s enzyme has a lower Km than the normal enzyme and, therefore, requires a lower glucose concentration to reach ½Vmax. Thus, the mutation may have increased the affinity of the enzyme for glucose, but it has greatly decreased the subsequent steps of the reaction leading to formation of the transition-state complex, and thus Vmax is much slower. The difference in Vmax is so great that the patient’s enzyme is much slower whether you are above or below its Km for glucose. You can test this by substituting 2 mM glucose and 4 mM glucose into the Michaelis-Menten equation, v ⫽ Vmax S/(Km ⫹ S) for the patient’s enzyme and for the normal enzyme. The values are 0.0095 U/mg and 0.0129 U/mg for the patient’s enzyme versus 23.2 U/mg and 37.2 U/mg for the normal enzyme, respectively (thus, B and C are incorrect). At near-saturating glucose concentrations, both enzymes will be near Vmax, which is equal to kcat times the enzyme concentration. Thus, it will take nearly 500 times as much of the patient’s enzyme to achieve the normal rate (93 ⫼ 0.2), and so C is incorrect. E is incorrect because rates change most as you decrease substrate concentration below the Km. Thus, the enzyme with the highest Km will show the largest changes in rate. The answer is B. Ethanol has a structure very similar to methanol (a structural analog) and thus can be expected to compete with methanol at its substrate-binding site. This inhibition is competitive with respect to methanol and, therefore, Vmax for methanol will not be altered and ethanol inhibition can be overcome by high concentrations of methanol (thus, A, C, and D are incorrect). E is illogical because the substrate methanol stays in the same binding site as it is converted to its product, formaldehyde. The answer is A. Allosteric activators will shift the sigmoidal kinetic curve for the enzyme to the left, thereby reducing the Km,app (so one-half maximal velocity will be reached at a lower substrate concentration), without affecting the maximal velocity (although in some cases Vmax can also be increased). Allosteric inhibitors will shift the curve to the right, increasing the Km,app and, sometimes, also decreasing the Vmax. The answer is C. The most effective regulation should be a feed-forward type of regulation in which the toxin activates the pathway. One of the most common ways this occurs is through the toxin acting to increase the amount

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ANSWERS TO REVIEW QUESTIONS

of enzyme by increasing transcription of its gene. A and B describe mechanisms of feedback regulation, in which the end product of the pathway decreases its own rate of synthesis and are, therefore, incorrect. D is incorrect because a high Km for the toxin might prevent the enzyme from working effectively at low toxin concentrations, although it would allow the enzyme to respond to increases of toxin concentration. It would do little good for the toxin to allosterically activate any enzyme but the rate-limiting enzyme (thus, E is incorrect).

concentrate protons within the lysosome, which generates the acidic environment required for enzyme activity. Answer B is, therefore, incorrect because lysosomes are acidic compared to the cytoplasm, not basic. ATP is not required to activate, regulate, or as a cofactor, for any lysosomal hydrolase.

CHAPTER 11 1.

CHAPTER 10 1.

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The answer is D. Phosphatidylserine, the only lipid with a net negative charge at neutral pH, is located in the inner leaflet, where it contributes to the more negatively charged intracellular side of the membrane. The membrane is composed principally of phospholipids and cholesterol (thus, A is incorrect). Phospholipids are amphipathic (contain polar and nonpolar ends, see Chapter 5), with their polar head groups extending into the aqueous medium inside and outside of the cell. The fatty acyl groups of the two layers face each other on the inside of the bilayer, and the polar oligosaccharides groups of glycoproteins and glycolipids extend into the aqueous medium (thus, B, C, and E are incorrect). The answer is E. The transmembrane regions are ␣-helices with hydrophobic amino acid side chains binding to membrane lipids. The hydrophobic interactions hinder their extraction (thus, A and D are incorrect). Because they are not easily extracted, they are classified as integral proteins (thus, B is incorrect). The carboxy and amino terminals of transmembrane proteins extend into the aqueous intracellular and extracellular medium and thus need to contain many hydrophilic residues (thus, C is incorrect). The answer is A. Most of fuel oxidation and ATP generation occurs in the mitochondrion. Although some may also occur in the cytosol, the amount is much smaller in most cells. The answer is A. Channels that open in response to a change in ion concentration across the membrane (which results in a change in membrane potential, or voltage, across the membrane) are known as voltage-gated channels. The calcium influx is not passive diffusion, as a carrier is required (the channel). This is not an active transport process, as calcium is flowing down its concentration gradient, and the cell is not concentrating calcium within it. A ligand-gated channel opens when a particular ligand binds to it, not when the membrane potential changes. There is no phosphorylation event described in the opening of this calcium channel, so it is not an example of a phosphorylation-gated channel. The answer is A. ATP is required by the vesicular ATPase, which uses the energy of ATP hydrolysis to

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2.

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The answer is C. Each chemical messenger has a specific protein receptor in a target cell that will generally bind only that messenger, and only that cell responds to the message. Only endocrine messengers reach their target cells through the blood (thus, A is incorrect). Many chemical messengers have paracrine actions. Messengers are secreted by only one type of cell (thus, B is incorrect). Many messengers bind to extracellular domains of plasma membrane receptors and do not enter the cells (thus, D is incorrect). No chemical messengers are metabolized to intracellular second messengers (thus, E is incorrect). The answer is D. Only lipophilic chemical messengers are able to diffuse into cells and bind to transcriptionfactor receptors. Because they are water insoluble, they must be transported in the blood bound to a protein. Cytokines, polypeptide hormones, and small-molecule neurotransmitters are not lipophilic (thus, A and B are incorrect). Because they work through regulation of gene transcription, their effects are relatively slow (thus, C is incorrect). Like all messengers, they are secreted in response to a specific stimulus (thus, E is incorrect). The answer is D. Parathyroid hormone is a polypeptide hormone and thus must bind to a plasma membrane receptor instead of an intracellular receptor (thus, A and B are incorrect). Hormones that bind to plasma membrane receptors do not need to enter the cell to transmit their signals (thus, C is incorrect). Heptahelical receptors work through heterotrimeric G proteins that have an ␣-subunit, and tyrosine kinase receptors work through monomeric G proteins that have no subunits (thus, D is correct and E is incorrect). The answer is C. G␣s normally activates adenylyl cyclase to generate cyclic adenosine monophosphate (cAMP) in response to parathyroid hormone. Because the patient has end-organ unresponsiveness, he or she must have a deficiency in the signaling pathway (thus, A is incorrect) and cAMP will be decreased. Decreased GTPase activity will increase binding to adenylyl cyclase and increase responsiveness (thus, B is incorrect). Neither IP3 nor phosphatidylinositol 3,4,5-trisphosphate are involved in signal transduction by the G␣s subunit. The answer is A. GRB2 binding to the receptor requires phosphotyrosine residues on the receptor. GRB2 normally binds to the receptor through its SH2 domains, which

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ANSWERS TO REVIEW QUESTIONS

recognize phosphotyrosine residues on the receptor. In the absence of these phosphotyrosine residues, GRB2 would not be able to bind to the receptor. Growth factor binding to the receptor does not require phosphotyrosine residues. It is the binding of the growth factor to the receptor, through a conformational change in the receptor that activates the intrinsic tyrosine kinase of the receptor, which then leads to autophosphorylation of the receptor. The lack of intracellular tyrosine residues on the receptor will not alter the events initiated by the conformational change of the receptor. Dimerization of receptors occurs upon binding growth factor and is not dependent on the activation of the tyrosine kinase activity, nor of autophosphorylation of the receptor.

CHAPTER 13 1.

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CHAPTER 12 1.

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The answer is D. The complementary strand must run in the opposite direction, so the 5⬘-end must base-pair with the G at the 3⬘-end of the given strand. Therefore, the 5⬘-end of the complementary strand must be C. G would then base-pair to C, A to T, and T to A. B is incorrect because the bases do not base-pair with each other with the sequences indicated, C is incorrect because U is not found in DNA, and E is incorrect because it has the wrong polarity (the 5⬘-T in answer E would not base-pair with the 3⬘-G in the given sequence). The answer is C. The RNA strand must be complementary to the DNA strand, and A in DNA base-pairs with U in RNA, whereas T in DNA base-pairs with A in RNA, G in DNA base-pairs with C in RNA, and C in DNA base-pairs with G in RNA. A and E are incorrect because they contain T, which is found in DNA, not RNA. B is incorrect because the base-pairing rules are broken when the strands are aligned in antiparallel fashion. D is incorrect because the polarity of the strand is incorrect (if one were to switch the 5⬘- and 3⬘-ends, the answer would be correct). The answer is D. The phosphate is in an ester bond to two ribose groups, generating the phosphodiester bond in the DNA backbone. None of the other interactions is correct. The answer is E. There are four possible bases at each position in the molecule, so as a total there are 48 possibilities. This comes out to 65,536 possible DNA sequences for a molecule that is only 8 bases long. Because the sequence of one strand in double-stranded DNA dictates the sequence of the other strand, we do not need to consider the second strand in this calculation. The answer is B. The backbone is composed of the phosphates and deoxyribose in phosphodiester linkages. The bases are internal to the backbone, base-paired to bases in the complementary strand, and forming stacking interactions within the double helix.

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The answer is B. The 3⬘-to-5⬘ exonuclease activity is required for proofreading (check the base just inserted, and if it is incorrect, remove it), and reverse transcriptase does not have this activity, whereas pol ␦ does. Both reverse transcriptase and pol ␦ synthesize DNA in the 5⬘-to-3⬘ directions (all DNA polymerases do this), and both follow standard Watson–Crick base-pairing rules (A with T or U, G with C). Neither polymerase can synthesize DNA in the wrong direction (3⬘-to-5⬘) nor insert inosine into a growing DNA chain. Thus, the only difference between the two polymerases is answer B. The answer is D. In 50 seconds, each replication origin will have synthesized 100 kb of DNA (50 in each direction). Because there are 10 origins, 10 ⫻ 100 will yield the 1,000 kb needed to replicate the DNA. The first origin will be 50 kb from one end, and the remaining nine origins will each be 100 kb apart. The answer is E. The role of the primer is to provide a free 3⬘-OH group for DNA polymerase to add the next nucleotide and form a phosphodiester bond. When DNA repair occurs, one of the remaining bases in the DNA will have a free 3⬘-OH, which DNA polymerase I will use to begin extension of the DNA. The answer is A. Helicase aids in unwinding DNA strands, along with DNA gyrase, so that the replication fork can keep moving. The 3⬘-to-5⬘ exonuclease activity is required for proofreading by DNA polymerases, DNA ligase connects a 3⬘-OH with an adjacent 5⬘-phosphate, primase lays down the RNA primer, and AP endonuclease looks for apurinic or apyrimidinic sites (without a purine or pyrimidine base, respectively) to remove them from the DNA strand. The answer is B. Xeroderma pigmentosum is a set of diseases all related to an inability to repair thymine dimers, leading to an inability to excise UV-damaged DNA. It does not affect bypass polymerases, which can synthesize across the damaged region, sometimes making mutations in its path. The primase gene, or mismatch repair, is not involved in excising thymine dimers. Proofreading ability of DNA polymerases is likewise not involved in this process.

CHAPTER 14 1.

The answer is B. The transcript that is produced is copied from the DNA template strand, which must be of the opposite orientation from the transcript. So the 5⬘-end of the template strand should base-pair with the 3⬘-end of the transcript, or the G. Thus, CGTACGGAT would basepair with the transcript and would represent the template strand.

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ANSWERS TO REVIEW QUESTIONS

2.

3.

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5.

The answer is A. Amanda Tin weighs 50 kg, and if 1 mg/kg body weight is the LD50, then for Amanda, 5 mg of toxin would bring her to the LD50. Because one mushroom contains 7 mg of the toxin, ingesting just one mushroom could be fatal. The answer is C. Enhancer sequences can be thousands of bases away from the basal promoter and still stimulate transcription of the gene. This is accomplished by looping of the DNA so that the proteins binding to the enhancer sequence (transactivators) can also bind to proteins bound to the promoter (coactivators). A promoter-proximal element is a DNA sequence nearby the promoter that can bind transcription factors that aid in recruiting RNA polymerase to the promoter region. The answer is D. Both prokaryotes and eukaryotes require RNA polymerase binding to an upstream promoter element. Answer A applies only to eukaryotes; prokaryote mRNA is not capped, nor does it contain a poly(A) tail. Prokaryotes have no nucleus, thus the 5⬘-end of an mRNA is immediately available for ribosome binding and initiation of translation (thus, B is incorrect). Answer C is incorrect overall; RNA synthesis, like DNA synthesis, is always in the 5⬘-to-3⬘ direction. Answer E is incorrect because introns are present only in eukaryotic genes. The answer is A. A ␤-thalassemia refers to a condition in which the ␣-chain of globin is produced in excess of the ␤-chain. The greater the ratio of ␣- to ␤-chain, the more severe the disease. Patients are usually asymptomatic at a ratio of 2:1, but once the ratio is greater than 2:1, symptoms will become evident. The reduction in ␤-globin synthesis can come about because of splicing mutations, promoter mutations, or point mutations within the coding regions of the ␤-globin gene.

3.

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CHAPTER 15 1.

2.

The answer is A. It is important to note that the question is asking about prokaryotic mechanisms. In prokaryotes, there is no nucleus (thus, E cannot be correct), and translation begins before transcription is terminated (coupled translation–transcription, thus B is incorrect). Therefore, before the ribosome can move from one codon to the next (translocation), or the protein synthesis machinery terminates (via termination factors), the initiating tRNA must bind and align with the mRNA to initiate translation, indicating that answer A is the first step of the choices listed that must occur. The answer is C. Because the extract contains all normal components, the Cys-tRNA charged with alanine will compete with Cys-tRNA charged with cysteine for binding to the cysteine codons. Thus, the protein will have some alanines put in the place of cysteine, leading to a deficiency of cysteine residues. Answer A is incorrect

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because the tRNA recognizes the cysteine codon, not the alanine codon. Answer B is incorrect because of the competition mentioned previously. The answer is C. There are 64 triplet codes, and only 20 amino acids, so some amino acids have more than one codon that codes for them. The other answers do not address this issue. The answer is A. In order to answer this question, one first needs to find the start codon: AUG in mRNA, ATG in DNA. Bases 3 to 5 in the sequence contain this element. Next, the sequence needs to specify a phenylalanine residue near the amino terminus, which is UUU or UUC in mRNA or TTT or TTG in DNA, but these sequences need to be in frame with the initiating methionine codon. The last element to look for is the absence of a premature stop codon, as this protein is 300 amino acids long. There is no stop sequence in the remaining bases of this piece of DNA. For answer B, the last three bases of this sequence are TGA, which in mRNA would be UGA, and this is an in-frame stop signal. This sequence would not give rise to a protein that contained 300 amino acids. For answer C, the TTT in that sequence (bases 10 to 12) are not in frame with the initiating methionine, indicating that there is no phenylalanine near the amino terminus in the protein encoded by this sequence. For answers D and E, there are no ATG sequences, indicating that the initiating methionine is absent, and that this stretch of DNA cannot code for the amino terminal end of a protein. The answer is D. Because each codon is three bases long, there are four possible bases that can fill each codon position (A, T, C, or G). That means there are 4 ⫻ 4 ⫻ 4 possible combinations of bases for a total of 64 codons. There are fewer than 64 aminoacyl tRNA synthetases because one tRNA recognizes more than one codon (as a result of wobble); thus, A is incorrect. Not all bases participate in wobble (G can base-pair with U, and C, A, or U can basepair with I, but those are the only combinations possible), and only one reading frame is used for each message produced (thus, B and C are incorrect). The rate of protein synthesis is not dependent on the number of codons, but rather on how rapidly the components required to synthesize proteins can be brought to the ribosomes and how much RNA is present to be translated.

CHAPTER 16 1.

The answer is E. Many prokaryotic genes are organized into operons, in which one polycistronic mRNA contains the translational start and stop sites for several related genes. Although each gene within the mRNA can be read from a different reading frame, the reading frame is always consistent within each gene (thus, A is incorrect). Redundancy in codon/tRNA interactions has nothing

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ANSWERS TO REVIEW QUESTIONS

to do with multiple cistrons within an mRNA (thus, B is incorrect). Operator sequences are in DNA and initiate transcription, not translation (thus, C is incorrect). Alternative splicing occurs only in eukaryotes (which have introns), not in prokaryotes (thus, D is incorrect). The answer is D. In order to transcribe the lac operon, the repressor protein (lac I gene product) must bind allolactose and leave the operator region, and the cAMP–CRP complex must bind to the promoter in order for RNA polymerase to bind. Of the choices offered, only raising cAMP levels can allow transcription of the operon when both lactose and glucose are high. Raising cAMP, even though glucose is present, will allow the cAMP–CRP complex to bind and recruit RNA polymerase. Answers that call for mutations in the repressor (answers A and E) will not affect binding of cAMP–CRP. Mutations in the DNA (answers B and C) do not allow CRP binding in the absence of cAMP. The answer is A. The repressor will bind to the operator and block transcription of all genes in the operon unless prevented by the inducer allolactose. If the repressor has lost its affinity for the inducer, it cannot dissociate from the operator and the genes in the operon will not be expressed (thus, E is incorrect). If the repressor has lost its affinity for the operator (answer B), then the operon would be expressed constitutively. Because the question states that there is a mutation in the I (repressor) gene, answer D is incorrect, and mutations in the I gene do not affect transacting factors from binding to the promoter, although the only other one for the lac operon is the CRP. The answer is B. The sequence, if read 5⬘ to 3⬘, is identical to the complementary sequence read 5⬘ to 3⬘. None of the other sequences fits this pattern. The answer is A. All DNA-binding proteins contain an ␣-helix that binds to the major or minor groove in DNA. These proteins do not recognize RNA molecules (thus, B is incorrect), nor do they form bonds between the peptide backbone and the DNA backbone (thus, C is incorrect; if this was correct, how could there be any specificity in protein binding to DNA?). Only zinc fingers contain zinc, and dimers are formed by hydrogen bonding—not by disulfide linkages.

3.

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CHAPTER 18 1.

CHAPTER 17 1.

2.

The answer is B. All DNA fragments are negatively charged and will migrate toward the positive charge. The only difference between the fragments is their size, and the smaller fragments will move faster than the larger fragments because of their ability to squeeze through the gel at a faster rate. The answer is E. The enzyme recognizes six bases, and the probability that the correct base is in each position is 1 in 4, so the overall probability is (¼)6, or 1 in 4,096 bases.

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The answer is B. The Sanger technique requires both deoxyribonucleotides and dideoxyribonucleotides, and a template DNA. It does not use Taq polymerase (which is for PCR), nor does it need reverse transcriptase (which is required for producing DNA from RNA). The answer is A. PCR experiments, using primers that flank the repeat area, can determine the number of repeats in a gene as compared to a gene with no or few repeats (the PCR product would be larger for a region containing multiple repeats as compared to a region with few repeats). Similarly, using restriction endonuclease recognition sites, which flank the repeat, one will see restriction fragment length polymorphisms, the length of the restriction fragment being dependent on the number of repeats in the gene. SNP analysis, however, examines single nucleotide polymorphisms, not multiple triplet repeats, and would not be a suitable method for determining a region of the genome, which contained multiple triplet nucleotide repeats. Most individuals will have a certain number of repeats, and PCR and RFLP will enable expanded repeat regions to be distinguished from small repeat regions relatively easily. The answer is C. A Northern blot allows one to determine which genes are being transcribed in a tissue at the time of mRNA isolation. The mRNA is run on a gel, transferred to filter paper, and then analyzed with a probe. If albumin is being transcribed, then a probe for albumin should give a positive result in the Northern blot. A library screening will not indicate if a particular gene is being transcribed, nor will a Southern blot. Those techniques will only allow one to determine that the gene is present in the genome. A Western blot analyzes protein content, not mRNA content. Analysis of VNTRs does not provide information about whether a gene is transcribed.

2.

The answer is C. The ras oncogene has a point mutation in codon 12 (position 35 of the DNA chain) in which T replaces G. This changes the codon from one that specifies glycine to one that specifies valine. Thus, there is a single amino acid change in the proto-oncogene (a valine for a glycine) that changes ras to an oncogene. The answer is C. Ras, when it is oncogenic, has lost its GTPase activity and thus remains active for a longer time. Answer A is incorrect because GAP proteins activate the GTPase activity of Ras, and this mutation would make Ras less active. cAMP does not interact directly with Ras (thus, B is incorrect), and if Ras could no longer bind GTP, it would not be active (hence, D is also incorrect). Ras is not phosphorylated by the MAP kinase (thus, E is incorrect).

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ANSWERS TO REVIEW QUESTIONS

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The answer is B. Retroviral oncogenes were originally obtained from genes on a host’s chromosome. All retroviruses do not contain oncogenes (thus, A is incorrect); proto-oncogenes are found in host cells, not viruses (thus, C is incorrect); the oncogenes that lead to human disease are very similar to those that are mutated in animals (thus, D is incorrect); and oncogenes are a misexpressed or mutated version of normal cellular genes, not viral genes (thus, E is incorrect). The answer is D. When p53 increases in response to DNA damage, it acts as a transcription factor and induces the transcription of p21, which blocks phosphorylation of Rb. Rb then stays bound to transcription factor E2F, and E2F cannot induce the genes required for the G1-to-S transition. p53 does not induce transcription of either cyclin D or cdk4 (thus, A and B are incorrect), nor does p53 bind to E2F (thus, C is incorrect), nor does p53 contain kinase activity (thus, E is incorrect). The answer is B. Tumor-suppressor genes balance cell growth and quiescence. When they are not expressed (via loss-of-function mutations), the balance shifts to cell proliferation and tumorigenesis (thus, A is incorrect). Answer C is incorrect because tumor-suppressor genes do not act on viral genes, answer D is incorrect because tumorsuppressor genes are not specifically targeted to just one aspect of the cell cycle, and answer E is incorrect because a loss of expression of tumor-suppressor genes leads to tumor formation, not expression of these genes.

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CHAPTER 19 1.

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The answer is E. Both of the high-energy phosphate bonds in ATP are located between phosphate groups (both the ␣- and ␤-phosphates, and the ␤- and ␥-phosphates). The phosphate bond between the ␣-phosphate and ribose (or adenosine) is not a high-energy bond (thus, A and B are incorrect), and there is no phosphate between the ribose and adenine, or two hydroxyl groups in the ribose ring; therefore, answers C and D are incorrect. The answer is C. The change in enthalpy, ⌬H, is the total amount of heat that can be released in a reaction. The first law of thermodynamics states that the total energy of a system remains constant, and the second law of thermodynamics states the universe tends toward a state of disorder (thus, A and B are incorrect). Answer D is incorrect because ⌬G0⬘ is the standard free energy change measured at 25°C and a pH of 7. Answer E is incorrect because a high-energy bond releases more than about 7 kcal/mol of heat when it is hydrolyzed. The definition of a high-energy bond is based on the hydrolysis of one of the high-energy bonds of ATP. The answer is A. The concentration of the substrates and products influence the direction of a reaction. Answer B is

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incorrect because reactions with a positive free energy, at 1 M concentrations of substrate and product, will proceed in the reverse direction. Answer C is incorrect because substrate and product concentrations do influence the free energy of a reaction. Answer D is incorrect because the free energy must be considered (in addition to the substrate and product concentrations) to determine the direction of a reaction. Answer E is false; an enzyme’s efficiency does not influence the direction of a reaction. The answer is C. A heart attack results in decreased pumping of blood and thus a decreased oxygen supply to the heart (thus, A is incorrect). The lack of oxygen leads to a lack of ATP (thus, B is incorrect) because of an inability to perform oxidative phosphorylation. The lack of ATP impairs the working of Na⫹, K⫹-ATPase, which pumps sodium out of the cell in exchange for potassium. Therefore, intracellular levels of sodium will increase as Na⫹ enters the cell through other transport mechanisms (thus, E is incorrect). The high intracellular sodium concentration then blocks the functioning of the Na⫹/H⫹ antiporter (which sends protons out of the cell in exchange for sodium. Because intracellular sodium is high, the driving force for this reaction is lost), which leads to increased intracellular H⫹ or a lower intracellular pH (thus, C is correct). The intracellular pH also decreases because of glycolysis in the absence of oxygen, which produces lactic acid. The loss of the sodium gradient, coupled with the lack of ATP, leads to increased calcium in the cell (thus, D is incorrect) because of an inability to pump calcium out. The answer is C. NAD⫹ accepts two electrons as hydride ions to form NADH (thus, A and B are incorrect). Answers D and E are incorrect because FAD can accept two single electrons from separate atoms, together with protons, or can accept a pair of electrons.

CHAPTER 20 1.

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The answer is E. The ␣-keto acid dehydrogenase complex uses thiamine pyrophosphate, lipoic acid, FAD, NAD⫹, and CoASH. Of these, only lipoate is used by no other enzyme and is unique to the ␣-keto acid dehydrogenase complexes. Many dehydrogenases use NAD⫹ as a coenzyme (such as malate dehydrogenase) or FAD (such as succinate dehydrogenase) (thus, A and B are incorrect). Answer C is incorrect because GDP is not a coenzyme for ␣-keto acid dehydrogenase complexes. Answer D is incorrect because H2O is not a coenzyme. The answer is B. Thiamine pyrophosphate is the coenzyme for the ␣-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes. With these complexes inactive, pyruvic acid and ␣-ketoglutaric acid accumulate and dissociate to generate the anion and H⫹. As ␣-ketoglutarate is not listed as an answer, the only possible answer is pyruvate.

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The answer is A. Succinate dehydrogenase is the only TCA cycle enzyme located in the inner mitochondrial membrane. The other enzymes are in the mitochondrial matrix. Answer B is incorrect because succinate dehydrogenase is not regulated by NADH. Answer C is incorrect because ␣-ketoglutarate dehydrogenase also contains a bound FAD (the difference is that the FAD[2H] in ␣-ketoglutarate dehydrogenase donates its electrons to NAD⫹, whereas the FAD[2H] in succinate dehydrogenase donates its electrons directly to the electron-transfer chain). Answer D is incorrect because both succinate dehydrogenase and aconitase have Fe–S centers. Answer E is incorrect because succinate dehydrogenase is not regulated by a kinase. Kinases regulate enzymes by phosphorylation (e.g., the regulation of pyruvate dehydrogenase occurs through reversible phosphorylation). The answer is E. NADH decreases during exercise to generate energy for the exercise (if it were increased, it would inhibit the cycle and slow it down); thus the NADH/NAD⫹ ratio is decreased; and the lack of NADH activates flux through isocitrate dehydrogenase, ␣-ketoglutarate dehydrogenase, and malate dehydrogenase. Isocitrate dehydrogenase is inhibited by NADH; so, answer A is not correct. Fumarase is not regulated, thus answer B is incorrect. The four-carbon intermediates of the cycle are regenerated during each turn of the cycle, so their concentrations do not decrease (thus, C is incorrect). Product inhibition of citrate synthase would slow the cycle and not generate more energy; hence, D is incorrect. The answer is D. Pantothenate is the vitamin precursor of coenzyme A. Niacin is the vitamin precursor of NAD, and riboflavin is the vitamin precursor of FAD and FMN. Vitamins A and C are used with only minor modifications, if any.

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CHAPTER 21 1.

The answer is B. For a component to be in the oxidized state, it must have donated, or never received, electrons. Complex II will metabolize succinate to produce fumarate (generating FAD[2H]), but no succinate is available in this experiment. Thus, complex II never sees any electrons and is always in an oxidized state. The substrate malate is oxidized to oxaloacetate, generating NADH, which donates electrons to complex I of the electron-transport chain. These electrons are transferred to coenzyme Q, which donates electrons to complex III, to cytochrome c, and then to complex IV. Cyanide will block the transfer of electrons from complex IV to oxygen, so all previous complexes containing electrons will be backed up and the electrons will be “stuck” in the complexes, making these components reduced. Thus, answers A and C through E must be incorrect.

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The answer is D. The proton motive force consists of two components: a ⌬pH and a ⌬␺ (electrical component). The addition of valinomycin and potassium will destroy the electrical component but not the pH component. Thus, the proton motive force will decrease but will still be greater than zero. Thus, the other answers are all incorrect. The answer is D. Dinitrophenol equilibrates the proton concentration across the inner mitochondrial membrane, thereby destroying the proton motive force. Thus, none of the other answers is correct. The answer is A. A deficiency of Fe–S centers in the electron-transport chain would impair the transfer of electrons down the chain and reduce ATP production by oxidative phosphorylation. Answer B is incorrect because the decreased production of water from the electron-transport chain is not of sufficient magnitude to cause her to become dehydrated. Answer C is incorrect because iron does not form a chelate with NADH and FAD(2H). Answer D is incorrect because iron is not a cofactor for ␣-ketoglutarate dehydrogenase. Answer E is incorrect because iron does not accompany the protons that make up the proton gradient. The answer is B. NADH would not be reoxidized as efficiently by the electron-transport chain, and the NADH/ NAD⫹ ratio would increase. Answer A is incorrect because ATP would not be produced at a high rate. Therefore, ADP would build up, and the ATP:ADP ratio would be low. Answer C is incorrect because OXPHOS diseases can be caused by mutations in nuclear or mitochondrial DNA, and not all OXPHOS proteins are encoded by the X chromosome. Answer D is incorrect because, depending on the nature of the mutation, the activity of complex II of the electron-transport chain might be normal or decreased, but there is no reason to expect increased activity. Answer E is incorrect because the integrity of the inner mitochondrial membrane would not necessarily be affected. It could be, but it would not be expected for all patients with OXPHOS disorders.

CHAPTER 22 1.

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The answer is B. The major roles of glycolysis are to generate energy and to produce precursors for other biosynthetic pathways. Gluconeogenesis is the pathway that generates glucose (thus, A is incorrect), FAD(2H) is produced in the mitochondria by a variety of reactions but not glycolysis (thus, C is incorrect), glycogen synthesis occurs under conditions in which glycolysis is inhibited (thus, D is incorrect), and glycolysis does not hydrolyze ATP to generate heat (i.e., nonshivering thermogenesis; thus, E is incorrect). The answer is D. By starting with glyceraldehyde 3-phosphate, the energy-requiring steps of glycolysis are bypassed. Thus, as glyceraldehyde 3-phosphate is

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ANSWERS TO REVIEW QUESTIONS

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converted to pyruvate, two molecules of ATP will be produced (at the phosphoglycerate kinase and pyruvate kinase steps) and one molecule of NADH will be produced (at the glyceraldehyde-3-phosphate dehydrogenase step). The answer is H. Glucose 1-phosphate is isomerized to glucose 6-phosphate, which then enters glycolysis. This skips the hexokinase step, which uses 1 ATP. Thus, starting from glucose 1-phosphate, one would get the normal 2 ATP and 2 NADH, but with one less ATP used, for a total yield of 3 ATP and 2 NADH. The answer is B. The pathway consumes 2 ATP at the beginning of the pathway and produces 4 ATP at the end of the pathway for each molecule of glucose. Therefore, the net energy production is 2 ATP for each molecule of glucose. Glycolysis synthesizes ATP via substrate-level phosphorylation, not oxidative phosphorylation (thus, A is incorrect) and synthesizes two molecules of pyruvate in the process (thus, D is incorrect). The pathway is cytosolic (thus, D is incorrect), and the rate-limiting step is the one catalyzed by PFK-1 (thus, C is incorrect). The answer is D. Each mole of glucose produces 2 mol of pyruvate, 2 mol of ATP, and 2 mol of NADH during glycolysis. As each pyruvate is converted to acetyl-CoA, 1 NADH is produced. Each acetyl-CoA enters the TCA cycle to produce 1 GTP, 3 NADH, and 1 FAD(2H). Therefore, the oxidation of 1 mol of glucose would yield a total of 2 mol of ATP and 2 mol of NADH from glycolytic reactions; 2 mol of NADH from the conversion of 2 mol of pyruvate to 2 mol of acetyl-CoA; and 6 mol of NADH, 2 mol of GTP, and 2 mol of FAD(2H) from the oxidation of 2 mol of acetylCoA in the TCA cycle. Each GTP has the same energy as 1 ATP. Each mitochondrial NADH is oxidized to produce 2.5 ATP. Each cytoplasmic NADH can generate either 1.5 ATP (using the glycerol phosphate shuttle) or 2.5 ATP (using the malate–aspartate shuttle). Each mitochondrial FAD(2H) is oxidized to produce 1.5 ATP. Therefore, the total ATP yield from the oxidation of 1 mol of glucose is 30 to 32 mol of ATP (30 mol if the glycerol phosphate shuttle is used; 32 mol if the malate–aspartate shuttle is used).

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The answer is C. An 18-carbon saturated fatty acid would require eight spirals of fatty acid oxidation, which yields 8 NADH, 8 FAD(2H), and 9 acyl-CoA. As each NADH gives rise to 2.5 ATP, and each FAD(2H) gives rise to 1.5 ATP, the reduced cofactors will give rise to 32 ATP. Each acyl-CoA gives rise to 10 ATP, for a total of 90 ATP. This then yields 122 ATP, but we must subtract 2 ATP for the activation step, at which two highenergy bonds are broken. Thus, the net yield is 120 ATPs for each molecule of fatty acid oxidized. The answer is A. Fatty acid oxidation is initiated by the acyl-CoA dehydrogenase (an oxidation step), followed by hydration of the double bond formed in the first step, followed by the hydroxyacyl-CoA dehydrogenase step (another oxidation), and then attack of the ␤-carbonyl by CoA, breaking a carbon–carbon bond (the thiolase step). The answer is D. A lack of carnitine would lead to an inability to transport fatty acyl-CoAs into the mitochondria. This would lead to a decrease in fatty acid oxidation (thus, A is incorrect), a decrease in ketone body production because fatty acids cannot be oxidized (thus, B is incorrect), a decrease in blood glucose levels because gluconeogenesis is impaired as a result of a lack of energy (thus, C is incorrect), and no increase in the levels of very long-chain fatty acids because these are initially oxidized in the peroxisomes and do not require carnitine for entry into that organelle (thus, E is incorrect). The ␻-oxidation system, which creates dicarboxylic acids, is found in the endoplasmic reticulum, and as the concentration of fatty acyl-CoAs increase in tissues, they will be oxidized by this alternative pathway. The answer is E. Fatty acids are the major fuel for the body during prolonged exercise and fasting. Answers A, B, and C are incorrect because glucose would be the major fuel after eating. Answer D is incorrect because, at the start of exercise, muscle glycogen and gluconeogenesis are being used as the major source of fuel.

CHAPTER 24 CHAPTER 23 1.

The answer is D. The ETF:CoQ oxidoreductase is required to transfer the electrons from the FAD(2H) of the acyl-CoA dehydrogenase to coenzyme Q. When the oxidoreductase is missing, the electrons cannot be transferred, and the acyl-CoA dehydrogenase cannot continue to oxidize fatty acids because it has a reduced cofactor instead of an oxidized cofactor. During times of fasting, when fatty acids are the primary energy source, no energy will be forthcoming, gluconeogenesis is shut down, and death may result. The lack of this enzyme does not affect the other pathways listed as potential answers.

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The answer is D. Vitamin B6 is a water-soluble vitamin that is important for amino acid and glycogen metabolism but has no role in protecting against free radical damage. Ascorbate (vitamin C), vitamin E, and ␤-carotene can all react with free radicals to terminate chain propagation, whereas superoxide dismutase uses the superoxide radical as a substrate and converts it to hydrogen peroxide, and glutathione peroxidase removes hydrogen peroxide from the cell, converting it to water. The answer is B. Superoxide dismutase combines two superoxide radicals to produce hydrogen peroxide and molecular oxygen. None of the other reactions is correct.

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The answer is C. Vitamin E donates an electron and proton to the radical, thereby converting the radical to a stable form (LOO• → LOOH). The vitamin thus prevents the free radical from oxidizing another compound by extracting an H from that compound and propagating a free radical chain reaction. The radical form of vitamin E generated is relatively stable and actually donates another electron and proton to a second free radical, forming oxidized vitamin E. The answer is B. The Fenton reaction is the nonenzymatic donation of an electron from Fe2⫹ to H2O2 to produce Fe3⫹, the hydroxyl radical, and hydroxide ion. Only Fe2⫹ or Cu1⫹ can be used in this reaction; thus, the other answers are incorrect. The answer is E. Histones coat nuclear DNA and protect it from damage by radicals. Mitochondrial DNA lacks histones, so when radicals are formed, the DNA can be easily oxidized. Answers A and B are nonsensical; superoxide dismutase reduces radical concentrations, so the fact that it is present in the mitochondria should help to protect the DNA from damage, not enhance it; and glutathione also protects against radical damage, and if the nucleus lacks it, then one would expect higher levels of nuclear DNA damage, not reduced levels. Reactive oxygen species can diffuse across membranes, so answers C and D are incorrect. Other factors that increase mitochondrial DNA damage relative to nuclear DNA are the proximity of mitochondrial DNA to the membrane, and the fact that most radical species are formed from coenzyme Q, which is found within the mitochondria.

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CHAPTER 25 1.

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The answer is A. Acetate is converted to acetyl-CoA by other tissues so that it can enter the TCA cycle to generate ATP. Answer B is incorrect because acetaldehyde, not acetate, is toxic to cells. Answer C is incorrect because acetate is excreted by the lung and kidney, and not in the bile. Answer D is incorrect because acetate cannot enter the TCA cycle directly. It must be converted to acetylCoA first. Answer E is incorrect because alcohol dehydrogenase converts ethanol into acetaldehyde. It does not convert acetate into NADH. The answer is B. There is an increase in the NADH/ NAD⫹ ratio because NADH is produced by the conversion of ethanol to acetate (thus, D is incorrect). The increased ratio of NADH/NAD⫹ favors the conversion of gluconeogenic precursors (such as lactate and oxaloacetate) to their reduced counterparts (lactate and malate, respectively), in order to generate NAD⫹ for ethanol metabolism. This reduces the concentration of gluconeogenic precursors, slows down gluconeogenesis (thus, E is incorrect), and can lead to lactic acidosis. Answer A

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is incorrect because the increase of NADH inhibits fatty acid oxidation. Answer C is incorrect because ketogenesis increases as a result of the increase of NADH. NADH inhibits key enzymes of the TCA cycle, thereby diverting acetyl-CoA from the TCA cycle and toward ketone body synthesis. The answer is C. Disulfiram blocks the conversion of acetaldehyde to acetate. The accumulation of acetaldehyde is toxic and causes vomiting and nausea. Answers A and B are incorrect because disulfiram would not interfere with the absorption of ethanol or the first step of its metabolism. Answer D is incorrect because an acetaldehyde dehydrogenase inhibitor (such as disulfiram) would inhibit the conversion of ethanol to acetate, not increase the rate of the conversion. Answer E is incorrect because disulfiram does not interfere with the excretion of acetate, nor does acetate accumulation lead to nausea and vomiting. The answer is E. An increase in the concentration of CYP2E1 (the MEOS system) would result in an increase of ethanol metabolism and clearance from the blood (thus, A is incorrect). An increased rate of acetaldehyde production would result (thus, B is incorrect). The increase in CYP2E1 would cause an increase in the probability of producing free radicals (thus, C is incorrect). Answer D is incorrect because hepatic damage would be more likely to occur because there is an increase of free radical production. Answer E is correct because the increased clearance rate of ethanol from the blood results in a higher alcohol tolerance level. The answer is E. Liver cirrhosis is irreversible. It is an end-stage process of liver fibrosis. Answers A, B, C, and D are all consequences of liver disease, but they are all reversible. Therefore, E is the only answer that is correct.

CHAPTER 26 1.

The answer is D. Once insulin is injected, glucose transport into the peripheral tissues will be enhanced. If the patient does not eat, the normal fasting level of glucose will drop even further because of the injection of insulin, which increases the movement of glucose into muscle and fat cells. The patient becomes hypoglycemic, as a result of which epinephrine is released from the adrenal medulla. This, in turn, leads to the signs and symptoms associated with high levels of epinephrine in the blood. Answers A and B are incorrect because as glucose levels drop, glucagon will be released from the pancreas to raise blood glucose levels, which would alleviate the symptoms. Answer E is incorrect because ketone body production does not produce hypoglycemic symptoms, nor would they be significantly elevated only a few hours after the insulin shock the patient is experiencing.

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ANSWERS TO REVIEW QUESTIONS

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The answer is B. When glucagon binds to its receptor, the enzyme adenylate cyclase is eventually activated (through the action of G proteins), which raises cAMP levels in the cell. The cAMP phosphodiesterase opposes this rise in cAMP, and hydrolyzes cAMP to 5⬘-AMP. If the phosphodiesterase is inhibited by caffeine, cAMP levels would stay elevated for an extended period, enhancing the glucagon response. The glucagon response in liver is to export glucose (thus, E is incorrect) and to inhibit glycolysis (thus, D is incorrect). cAMP activates protein kinase A, making answer C incorrect as well. The effect of insulin is to reduce cAMP levels (thus, A is incorrect). The answer is B. Insulin release is dependent on an increase in the [ATP]/[ADP] ratio within the pancreatic ␤-cell. In MODY, the mutation in glucokinase results in a less active glucokinase at glucose concentrations that normally stimulate insulin release. Thus, higher concentrations of glucose are required to stimulate glycolysis and the tricarboxylic acid (TCA) cycle to effectively raise the ratio of ATP to ADP. Answer A is incorrect because cAMP levels are not related to the mechanism of insulin release. Answer C is incorrect because, initially, transcription is not involved, as insulin release is caused by exocytosis of preformed insulin in secretory vesicles. Answer D is incorrect because the pancreas will not degrade glycogen under conditions of high blood glucose, and answer E is incorrect because lactate does not play in role in stimulating insulin release. The answer is A. The brain requires glucose because fatty acids cannot readily cross the blood–brain barrier to enter neuronal cells. Thus, glucose production is maintained at an adequate level to allow the brain to continue to burn glucose for its energy needs. The other organs listed as possible answers can switch to the use of alternative fuel sources (lactate, fatty acids, amino acids) and are not as dependent on glucose for their energy requirements as is the brain. The answer is E. Muscle does not express glucagon receptors, so they are refractory to glucagons’ actions. Muscle does, however, contain GTP (made via the TCA cycle), G proteins, protein kinase A, and adenylate cyclase (epinephrine stimulation of muscle cells raises cAMP levels and activates protein kinase A).

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CHAPTER 27 1.

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The answer is E. The GLUT 5 transporter has a much higher affinity for fructose than glucose and is the facilitator of choice for fructose uptake by cells. The other GLUT transporters do not transport fructose to any significant extent. The answer is A. The pancreas produces ␣-amylase, which digests starch in the intestinal lumen. If pancreatic ␣-amylase cannot enter the lumen because of pancreatitis,

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the starch will not be digested to a significant extent. (The salivary ␣-amylase begins the process, but only for the time during which the food is in the mouth, as the acidic conditions of the stomach destroy the salivary activity.) The discomfort arises from the bacteria in the intestine, digesting the starch and producing acids and gases. Lactose, sucrose, and maltose are all disaccharides that would be cleaved by the intestinal disaccharidases located on the brush border of the intestinal epithelial cells (thus, B, D, and E are incorrect). These activities might be slightly reduced, as the pancreas would also have difficulty excreting bicarbonate to the intestine, and the low pH of the stomach contents might reduce the activity of these enzymes. However, these enzymes are present in excess and will eventually digest the disaccharides. Fiber cannot be digested by human enzymes, so answer C is incorrect. The answer is C. Insulin is required to stimulate glucose transport into muscle and fat cells but not into brain, liver, pancreas, or red blood cells. Thus, muscle would be feeling the effects of glucose deprivation and would be unable to replenish its own glycogen supplies as a result of its inability to extract blood glucose, even though blood glucose levels would be high. The answer is E. Flour contains starch, which will lead to glucose production in the intestine. Milk contains lactose, a disaccharide of glucose and galactose, which will be split by lactase in the small intestine. Sucrose is a disaccharide of glucose and fructose, which is split by sucrase in the small intestine. Thus, glucose, galactose, and fructose will all be available in the lumen of the small intestine for transport through the intestinal epithelial cells and into the circulation. The answer is A. Salivary and pancreatic ␣-amylase will partially digest starch to glucose, but maltose and disaccharides will pass through the intestine and exit with the stool, as a result of the limited activity of the brush border enzymes. Because the amylase enzymes are working, there will only be normal levels of starch in the stool (thus, B is incorrect). Not all available glucose is entering the blood, so less insulin will be released by the pancreas (thus, E is incorrect), which will lead to less glucose uptake by the muscles and less glycogen production (thus, D is incorrect). Because neither lactose nor sucrose can be digested to a large extent in the intestinal lumen under these conditions, it would be difficult to have elevated levels of galactose or fructose in the blood (thus, C is incorrect).

CHAPTER 28 1.

The answer is B. Glycogen phosphorylase produces glucose 1-phosphate; the debranching enzyme hydrolyzes branch points and thus releases free glucose. Ninety

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ANSWERS TO REVIEW QUESTIONS

percent of the glycogen contains ␣-1,4-bonds and only 10% are ␣-1,6-bonds, so more glucose 1-phosphate will be produced than glucose. The answer is D. If, after fasting, the branches were shorter than normal, glycogen phosphorylase must be functional and capable of being activated by glucagon (thus, A and B are incorrect). The branching enzyme (amylo-4,6-transferase) is also normal because branch points are present within the glycogen (thus, E is incorrect). Because glycogen is also present, glycogenin is present in order to build the carbohydrate chains, indicating that C is incorrect. If the debranching activity is abnormal (the amylo-1,6-glucosidase), glycogen phosphorylase would break the glycogen down up to four residues from branch points and would then stop. With no debranching activity, the resultant glycogen would contain the normal number of branches, but the branched chains would be shorter than normal. The answer is C. The patient has McArdle disease, a glycogen storage disease caused by a deficiency of muscle glycogen phosphorylase. Because he or she cannot degrade glycogen to produce energy for muscle contraction, he or she becomes fatigued more readily than a normal person (thus, A is incorrect), the glycogen levels in her muscle will be higher than normal as a result of the inability to degrade them (thus, D is incorrect), and his or her blood lactate levels will be lower because of the lack of glucose for entry into glycolysis. He or she will, however, draw on the glucose in his or her circulation for energy, so his or her forearm blood glucose levels will be decreased (thus, B is incorrect), and because the liver is not affected, blood glucose levels can be maintained by liver glycogenolysis (thus, E is incorrect). The answer is A. After ingestion of glucose, insulin levels rise, cAMP levels within the cell drop (thus, E is incorrect), and protein-phosphatase-I is activated (thus, D is incorrect). Glycogen phosphorylase a is converted to glycogen phosphorylase b by the phosphatase (thus, B is incorrect), and glycogen synthase is activated by the phosphatase. Red blood cells continue to use glucose at their normal rate, thus lactate formation will remain the same (thus, C is incorrect). The answer is F. In the absence of insulin, glucagonstimulated activities predominate. This leads to the activation of protein kinase A, the phosphorylation and inactivation of glycogen synthase, the phosphorylation and activation of phosphorylase kinase, and the phosphorylation and activation of glycogen phosphorylase.

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CHAPTER 29 1.

The answer is D. The aldolase B gene has two alleles. One or both may have mutations. Because HFI is recessive, both alleles must be mutated for the disease to be

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expressed. Examination of the gel shows that the normal gene is cleaved by ahaII to produce a 306-bp (base pair) restriction fragment. When a mutation creates a new ahaII site within the gene, this 306-bp fragment is cleaved into two fragments of 183 and 123 bp (which together contain 306 bp). The husband and Jill, thus, are carriers. They have one normal allele that produces the 306-bp fragment and one that has an additional ahaII site, which is cleaved to yield the two fragments of 183 and 123 bp. The wife and Jack have the disease. Both of their alleles have the additional ahaII site and produce only 183- and 123-bp fragments. The answer is C. This man could be the father of both children. He could provide either a normal gene (which produces a 306-bp ahaII fragment) or a mutant gene (which produces 183- and 123-bp ahaII fragments) to his offspring. This mother could provide only the mutant gene (of which she has two copies). Jill is a carrier. She received the mutant gene from her mother and could have received the normal gene from this man. Jack has the disease. He received one mutant gene from his mother and another from his father. The answer is C. Transketolase requires thiamine pyrophosphate as a cofactor, whereas none of the other enzymes listed does. Thus, if an individual has a thiamine deficiency, transketolase activity as isolated from a patient’s blood cells will be enhanced by the addition of thiamine; in well-nourished individuals, the addition of thiamine will not enhance transketolase activity. The answer is B. Fructose is converted to fructose 1-phosphate by fructokinase, and aldolase B in the liver splits the fructose 1-P into glyceraldehyde and dihydroxyacetone phosphate. Thus, the major regulated step of glycolysis, PFK-1, is bypassed and PEP is rapidly produced. As the [PEP] increases, pyruvate kinase produces pyruvate. As the glyceraldehyde-3-phosphate dehydrogenase reaction is proceeding rapidly (remember that fructokinase is a high Vmax enzyme, so there is a lot of substrate proceeding through the glycolytic pathway), the intracellular [NADH]/[NAD⫹] ratio is high, and the pyruvate produced is converted to lactate in order to regenerate NAD⫹. Thus, the pyruvate kinase step is not bypassed (thus, A is incorrect). Neither aldolase B nor lactate dehydrogenase is allosterically regulated (thus, C and D are incorrect), and even though the [ATP]:[ADP] ratio is high in the liver under these conditions, the ratio does not affect lactate formation (thus, E is incorrect). The answer is B. Reduction of sugar aldehydes to alcohols requires NADPH, which is generated primarily by the pentose phosphate pathway. 6-Phosphogluconate is not a polyol (thus, A is incorrect; 6-phosphogluconate is glucose oxidized at position 1 to form a carboxylic acid); ribitol is not a product of the pentose phosphate pathway (thus, B is incorrect; ribulose 5-phosphate is a product of

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ANSWERS TO REVIEW QUESTIONS

the pentose phosphate pathway); the HMP shunt uses glucose 6-phosphate as the starting material, not free glucose as in the sorbitol pathway (thus, D and E are incorrect).

CHAPTER 30 1.

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The answer is B. Galactose metabolism requires the phosphorylation of galactose to galactose 1-phosphate, which is then converted to UDP-galactose (which is the step that is defective in the patient), and then epimerized to UDP-glucose. Although the mother cannot convert galactose to lactose because of the enzyme deficiency, she can make UDP-glucose from glucose 6-phosphate, and once she has made UDP-glucose, she can epimerize it to form UDP-galactose and can synthesize lactose (thus, A is incorrect). However, because of her enzyme deficiency, the mother cannot convert galactose 1-phosphate to UDPgalactose or UDP-glucose, so the dietary galactose cannot be used for glycogen synthesis or glucose production (thus, C and D are incorrect). After ingesting milk, the galactose levels will be elevated in the serum because of the metabolic block in the cells (thus, E is incorrect). The answer is D. Nucleotide sugars, such as UDPglucose, UDP-galactose, and CMP-sialic acid, donate sugars to the growing carbohydrate chain. The other activated sugars listed do not contribute to this synthesis. The answer is C. Bilirubin is conjugated with glucuronic acid residues to enhance its solubility. Glucuronic acid is glucose oxidized at position 6; gluconic acid is glucose oxidized at position 1 and is generated by the HMP shunt pathway. The answer is C. Glutamine donates the amide nitrogen to fructose 6-phosphate to form glucosamine 6-phosphate. None of the other nitrogen-containing compounds (A, B, and D) donate their nitrogen to carbohydrates. Dolichol contains no nitrogens and is the carrier for carbohydrate chain synthesis of N-linked glycoproteins. The answer is C. Sandhoff disease is a deficiency of both hexosaminidase A and B activity, resulting from loss of the ␤-subunit activity of these enzymes. The degradative step at which amino sugars need to be removed from the glycolipids would be defective, such that globoside and GM2 accumulate in this disease. The other answers are incorrect; GM1 does contain an amino sugar, but it is converted to GM2 before the block is apparent.

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CHAPTER 31 1.

The answer is D. Glycerol is converted to glycerol 3-phosphate, which is oxidized to form glyceraldehyde 3-phosphate. The glyceraldehyde 3-phosphate formed then follows the gluconeogenic pathway to glucose. Lactate is converted to pyruvate, which is then carboxylated to form

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oxaloacetate. The oxaloacetate is decarboxylated to form phosphoenolpyruvate and then run through gluconeogenesis to glucose. Because glycerol enters the gluconeogenic pathway at the glyceraldehyde 3-phosphate step and lactate at the PEP step, the only compounds in common between these two starting points are the steps from glyceraldehyde 3-phosphate to glucose. Of the choices listed in the question, only glucose 6-phosphate is in that part of the pathway. The answer is A. PEPCK converts oxaloacetate to phosphoenolpyruvate. In combination with pyruvate carboxylase, it is used to bypass the pyruvate kinase reaction. Thus, compounds that enter gluconeogenesis between PEP and OAA (such as lactate, alanine, or any TCA cycle intermediate) must use PEPCK to produce PEP. Glycerol enters gluconeogenesis as glyceraldehyde 3-phosphate, bypassing the PEPCK step. Galactose is converted to glucose 1-phosphate, then glucose 6-phosphate, also bypassing the PEPCK step. Even-chain fatty acids can only give rise to acetyl-CoA, which cannot be used to synthesize glucose. The answer is C. Glucagon counteracts insulin’s action, in that it is a catabolic hormone (thus, A is incorrect), and its levels decrease when insulin levels increase (thus, E is incorrect). As insulin levels rise after a meal, glucagon levels will decrease, so D is incorrect. The muscle lacks glucagon receptors and cannot respond to glucagon, hence B is incorrect. The answer is B. High blood glucose levels signal the release of insulin from the pancreas; glucagon levels either stay the same or decrease slightly. The answer is D. The hyperglycemia in an untreated diabetic creates osmotic diuresis, which means that excessive water is lost through urination. This can lead to a contraction of blood volume, leading to low blood pressure and a rapid heart beat. It also leads to dehydration. The rapid respirations result from acidosis-induced stimulation of the respiratory center of the brain, in order to reduce the amount of acid in the blood. Ketone bodies have accumulated, leading to diabetic ketoacidosis (thus, B is incorrect). A patient in a hypoglycemic coma (which can be caused by excessive insulin administration) does not exhibit dehydration, low blood pressure, or rapid respirations; in fact, the patient will sweat profusely as a result of epinephrine release (thus, C and E are incorrect). Answer A is incorrect because lack of a pancreas would be fatal.

CHAPTER 32 1.

The answer is B. Chylomicrons transport dietary lipids, and ⬎80% of the chylomicron is triglyceride. All other components are present at ⬍10%, hence all other answers are incorrect.

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The answer is D. High-density lipoproteins transfer apoproteins CII and E to nascent chylomicrons to convert them to mature chylomicrons. Bile salts are required to emulsify dietary lipid, 2-monoacylglycerol is a digestion product of pancreatic lipase, lipoprotein lipase digests triglyceride from mature chylomicrons, and the lymphatic system delivers the nascent chylomicrons to the bloodstream. The answer is A. Both apoB-48 and apoB-100 are derived from the same gene and from the same mRNA (there is no difference in splicing between the two, thus B is incorrect). However, RNA editing introduces a stop codon in the message such that B-48 stops protein synthesis approximately 48% along the message. Thus, proteolytic cleavage is not correct. B-48 is found only in chylomicrons, and B-100 is found only in VLDL particles. The answer is B. The bile salts must be above their critical micelle concentration to form micelles with the components of lipase digestion, fatty acids, and 2-monoacylgycerol. In the absence of micelle formation, lipid absorption would not occur. The critical micelle concentration is independent of triglyceride concentration (thus, A is incorrect). Bile salts do not bind or activate lipase (thus, C and E are incorrect). The absorption of bile salts in the ileum is not related to digestion (thus, D is incorrect). The answer is D. Nascent chylomicrons would be synthesized, which can only acquire apo CII from HDL (thus, A is incorrect). The chylomicrons would be degraded in part by lipoprotein lipase, leading to chylomicron remnant formation. However, the chylomicron remnants would remain in circulation because of the lack of apo E (thus, B is incorrect). Because these remnant particles still contain a fair amount of triglyceride, serum triglyceride levels will be elevated (thus, C and E are incorrect).

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CHAPTER 34 1.

CHAPTER 33 1.

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The answer is A. Fatty acids, cleaved from the triacylglycerols of blood lipoproteins by the action of lipoprotein lipase, are taken up by adipose cells and react with coenzyme A to form fatty acyl-CoA. Glucose is converted via dihydroxyacetone phosphate to glycerol 3-phosphate, which reacts with fatty acyl-CoA to form phosphatidic acid (adipose tissue lacks glycerol kinase, so it cannot use glycerol directly). After inorganic phosphate is released from phosphatidic acid, the resulting diacylglycerol reacts with another fatty acyl-CoA to form a triacylglycerol, which is stored in adipose cells (2-monoacylglycerol is an intermediate of triglyceride synthesis only in the intestine, not in adipose tissue). The answer is D. The triacylglycerol is degraded by pancreatic lipase, which releases the fatty acids at positions 1 and 3. The fatty acids released are then transported to

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the cell surface in a bile salt micelle. The only exception is short-chain fatty acids (shorter than palmitic acid), which can diffuse to the cell surface and enter the intestinal cell in the absence of micelle formation. The answer is B. Dietary carbohydrate is converted to lipid in the liver and exported via VLDL. Thus, a lowcarbohydrate diet will reduce VLDL formation and reduce the hyperlipoproteinemia. The answer is C. Sphingosine is derived from the condensation of palmitoyl-CoA and serine. It is converted to ceramide via reaction with a fatty acyl-CoA, not a UDP-sugar. There is no glycerol in this structure, and cardiolipin is derived from phosphatidic acid and phosphatidylglycerol. All cells synthesize sphingosine; its synthesis is not restricted to neuronal cells. The answer is E. When fatty acids are being synthesized, malonyl-CoA accumulates, which inhibits carnitine palmitoyl transferase I. This blocks fatty acid entry into the mitochondrion for oxidation. Many tissues both synthesize and degrade fatty acids (such as liver and muscle; thus, A is incorrect). NADPH blocks the glucose6-phosphate dehydrogenase reaction but not fatty acid oxidation (thus, B is incorrect). Insulin has no effect on the synthesis of the enzymes involved in fatty acid degradation (unlike the effect of insulin on the induction of enzymes involved in fatty acid synthesis; thus, C is incorrect). Finally, newly synthesized fatty acids are converted to their CoA derivatives for elongation and desaturation (thus, D is incorrect).

2.

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The answer is B. The reduction of HMG-CoA to mevalonate is catalyzed by HMG-CoA reductase, which is an integral enzyme in the smooth endoplasmic reticulum. The activity of HMG-CoA reductase is carefully controlled and is the rate-limiting step for de novo cholesterol biosynthesis. The other steps listed, although they are on the pathway to cholesterol, are not catalyzed by regulated enzymes. The answer is C. The squalene monooxygenase reaction uses one of the oxygen atoms of O2 to form an epoxide at one end of the squalene molecule, which requires NADPH as a cosubstrate of the reaction. All sterols have four fused rings (thus, A is incorrect). Because the epoxide is formed at one end of the squalene molecule, answer B is incorrect. Answer D is incorrect because NADPH provides the reducing power, not FAD(2H); and answer E is incorrect because carbons 1 and 30 remain independent during the cyclization of squalene. The answer is C. The presence of chronic hyperglycemia (usually accompanied by high levels of free fatty acids in the blood) causes diffuse multiorgan toxic effects

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ANSWERS TO REVIEW QUESTIONS

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(“glucose toxicity” and “lipotoxicity”), to the extent that it raises the risk for a future atherosclerotic event to a level equal to that posed by a history of the patient having already suffered such an event in the past. The other risk factors, in the absence of a previous myocardial infarction, do not require the suggested lower limits for circulating cholesterol levels. The answer is B. ApoCIII appears to inhibit the activation of LPL. ApoE acts as a ligand in binding several lipoproteins to the LDL receptor, the LDL receptor-related protein (LRP), and possibly to a separate apoE receptor. ApoB48 is required for the normal assembly and secretion of chylomicrons from the small bowel, whereas apoB100 is required in the liver for the assembly and secretion of VLDL. ApoCII is the activator of LPL. The answer is B. Because chylomicrons contain the most triacylglycerol, they are the least dense of the blood lipoproteins. Because VLDL contains more protein, it is denser than chylomicrons. Because LDL is produced by degradation of the triacylglycerol in VLDL, LDL is denser than VLDL. HDL is the densest of the blood lipoproteins. It has the most protein and the least triacylglycerol.

CHAPTER 35 1.

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The answer is C. Most prostaglandins are synthesized from arachidonic acid (cis-⌬5,8,11,14 C20:4), which is derived from the essential fatty acid, linoleic acid (cis⌬9,12 C18:2). Glucose, oleic acid, or acetyl-CoA cannot give rise to linoleic or arachidonic acid, as mammals cannot introduce double bonds six carbons from the ␻-end of a fatty acid. Leukotrienes are also derived from arachidonic acid, but they are not precursors of prostaglandins; they follow a different pathway. The answer is A. Arachidonic acid is produced from linoleic acid (an essential fatty acid) by a series of elongation and desaturation reactions. Arachidonic acid is stored in membrane phospholipids, released, and oxidized by a cyclooxygenase (which is inhibited by aspirin) in the first step of the biosynthesis of prostaglandins, prostacyclins, and thromboxanes. Leukotrienes require the enzyme lipoxygenase, rather than cyclooxygenase, for their synthesis from arachidonic acid. The answer is A. Aspirin leads to the acetylation of COX-1 and COX-2, which inhibits the enzymes. Tylenol contains acetaminophen, which is a competitive inhibitor of both COX-1 and COX-2, but acetaminophen does not attach covalently to the enzymes. Advil contains ibuprofen, which is another competitive inhibitor of the COX enzymes. Vioxx and Celebrex contain inhibitors that are specific for COX-2, which is the form of cyclooxygenase that is induced during inflammation. Vioxx and Celebrex do not inhibit COX-1 activity.

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The answer is C. Release of TXA2 by platelets at wounds, while enhancing platelet aggregation (see Chapter 45), also induces vasoconstriction, reducing blood flow to the wounded area. TXA2 has no effect on either COX-1 or COX-2 activity and does induce bronchial constriction, but not bronchial dilation. The answer is B. Phospholipase C is activated by a Gq protein, which is coupled to the prostaglandin receptor. Activation of phospholipase C leads to the hydrolysis of phosphatidylinositol bisphosphate (PIP2) to produce diacylglycerol and inositol trisphosphate (IP3). The IP3 leads to the increase in intracellular calcium levels (see Chapter 11). Activation of the protein kinase A pathway, via a G protein and adenylate cyclase, does not lead to a rise in intracellular calcium levels. Protein kinase C is activated by diacylglycerol, one of the products of PIP2 hydrolysis, but protein kinase C is not involved in the increase in intracellular calcium levels. Phospholipase A2 releases arachidonic acid from phospholipids, but this response is not required when prostaglandins bind to their receptors.

CHAPTER 36 1.

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The answer is B. The acetone on the woman’s breath (which is produced by decarboxylation of acetoacetate; thus, E is incorrect) and the ketones in her urine indicate that she is in diabetic ketoacidosis. This is caused by low insulin levels, so her blood glucose levels are high because the glucose is not being taken up by the peripheral tissues (thus, A and C are incorrect). An insulin injection will reduce her blood glucose levels and decrease the release of fatty acids from adipose triglycerides. Consequently, ketone body production will decrease. Glucagon injections would just exacerbate the woman’s current condition (thus, D is incorrect). The answer is D. Chylomicrons are blood lipoproteins produced from dietary fat. VLDL is produced mainly from dietary carbohydrate. IDL and LDL are produced from VLDL. HDL does not transport triacylglycerol to the tissues. The answer is B. Consider the energy required to convert dietary carbohydrates to triacylglycerol. Some ATP is generated from glycolysis and the pyruvate dehydrogenase reaction, but energy is also lost as the fatty acids are synthesized (the synthesis of each malonyl-CoA requires ATP, and the reduction steps require two molecules of NADPH). Dietary fat, however, only requires activation and attachment to glycerol; the fatty acid chain does not need to be synthesized. Therefore, it requires less energy to package dietary fat into chylomicrons than it does to convert dietary carbohydrate into fatty acids for incorporation into VLDL. Thus, weight gain will be more rapid if

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all the excess calories are derived from fat as opposed to carbohydrates. The answer is B. Metabolism of ethanol leads to production of NADH in the liver, which will inhibit fatty acid oxidation in the liver. Because the patient has not eaten for 5 days, the insulin/glucagon ratio is low, hormone-sensitive lipase is activated, and fatty acids are being released by the adipocyte and taken up by the muscle and liver. However, because the liver NADH levels are high as a result of ethanol metabolism, the fatty acids received from the adipocyte are repackaged into triacylglycerol (the high NADH promotes the conversion of dihydroxyacetone phosphate to glycerol 3-phosphate as well) and secreted from the liver in the form of VLDL. None of the other answers is a correct statement. The answer is A. The clotting problems are caused by a lack of vitamin K, a lipid-soluble vitamin. Vitamin K is absorbed from the diet in mixed micelles and packaged with chylomicrons for delivery to the other tissues. Individuals with abetalipoproteinemia lack the microsomal triglyceride transfer protein and cannot produce chylomicrons effectively, thus vitamin K deficiency can result. Such patients also cannot produce VLDL, but lipidsoluble vitamin distribution does not depend on VLDL particles, only on chylomicrons. The other answers are all incorrect statements.

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CHAPTER 38 1.

CHAPTER 37 1.

2.

The answer is C. Trypsinogen, which is secreted by the intestine, is activated by enteropeptidase, a protein found in the intestine (thus, D is backward and incorrect). Once trypsin is formed, it activates all of the other zymogens secreted by the pancreas. Trypsin does not activate pepsinogen (thus, B is incorrect) because pepsinogen is found in the stomach and autocatalyzes its own activation when the pH drops as a result of acid secretion. Trypsin has no effect on intestinal motility (hence, E is incorrect) and also does not have a much broader base of substrates than any other protease (trypsin cleaves on the carboxy side of basic side chains, lysine, and arginine; thus, A is incorrect). The answer is C. Leucine can be transported by several different amino acid systems. Leucine is an essential amino acid, so the body cannot synthesize it (thus, A is incorrect). If the intestine cannot absorb leucine, then the kidneys do not have a chance to reabsorb it, so B is incorrect. Leucine and isoleucine have different structures and cannot substitute for each other in all positions within a protein, thus D is incorrect. Leucine is an important component of proteins and is required for protein synthesis; hence, E is incorrect.

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The answer is D. Kwashiorkor is a disease that results from eating a calorie-adequate diet that lacks protein. None of the other answers is correct. The answer is C. Pepsinogen, under acidic conditions, autocatalyzes its conversion to pepsin in the stomach. Both enteropeptidase and aminopeptidases are synthesized in active form by the intestine (thus, A and D are incorrect). Enteropeptidase activates trypsinogen (thus, B is incorrect), which then activates proelastase (thus, E is incorrect). The answer is E. Because of the lack of protein in the diet, protein synthesis in the liver is impaired (lack of essential amino acids). The liver can still synthesize fatty acids from carbohydrate or fat sources, but very lowdensity lipoprotein (VLDL) particles cannot be assembled because of the shortage of apoprotein B-100. Thus, the fatty acids remain in the liver, leading to a fatty liver. None of the other answers explains this finding.

2.

Infant I has a defect in carbamoyl phosphate synthetase I (CPSI; answer A), and infant IV has a defect in ornithine transcarbamoylase (OTC; answer B). Infants with high ammonia, low arginine, and low citrulline levels must have a defect in a urea-cycle enzyme before the step that produces citrulline, that is, CPSI or OTC. If CPSI is functional and carbamoyl phosphate is produced but cannot be further metabolized, more than the normal amount is diverted to the pathway for pyrimidine synthesis and the intermediate orotate appears in the urine. Therefore, infant I has a defect in CPSI (citrulline is low and less than the normal amount of orotate is in the urine). Infant IV has an OTC defect; carbamoyl phosphate is produced, but it cannot be converted to citrulline, so citrulline is low and orotate is present in the urine. Infants II and V have high levels of citrulline but low levels of arginine. Therefore, they cannot produce arginine from citrulline. Argininosuccinate synthetase or argininosuccinate lyase is defective. The very elevated citrulline levels in infant II suggest that the block is in argininosuccinate synthetase (answer C). In infant V, citrulline levels are more moderately elevated, suggesting that citrulline can be converted to argininosuccinate and that the defect is in argininosuccinate lyase (answer D). Thus, the accumulated intermediates of the urea cycle are distributed between argininosuccinate and citrulline (both of which can be excreted in the urine). The high levels of arginine and more moderate hyperammonemia in infant III suggest that, in this case, the defect is in arginase (answer E). The answer is D. The nitrogens in urea are derived from carbamoyl phosphate and aspartate directly during one turn of the cycle. The nitrogen in the side chain of

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ANSWERS TO REVIEW QUESTIONS

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ornithine is never incorporated into urea because it stays with ornithine (thus, A, B, and C are incorrect). Glutamine does not donate a nitrogen directly during the urea cycle, so E and F are also incorrect. The answer is C. Glutamate dehydrogenase fixes ammonia into ␣-ketoglutarate, generating glutamate, in a reversible reaction that also requires NAD(P)H. Alaninepyruvate aminotransferase catalyzes the transfer of nitrogen from alanine to an ␣-keto acid acceptor but does not use ammonia as a substrate. Glutaminase converts glutamine to glutamate and ammonia, but the reaction is not reversible. Arginase splits arginine into urea and ornithine, and argininosuccinate synthetase forms argininosuccinate from citrulline and aspirate. The only other two enzymes that can fix ammonia into an organic compound are carbamoyl phosphate synthetase I and glutamine synthetase. The answer is C. Glycogen phosphorylase requires pyridoxal phosphate to break the ␣-1,4-linkages in glycogen. None of the other pathways listed contains an enzyme that requires this cofactor. The answer is A. CPSI is the major regulated step, being activated by N-acetylglutamate, the synthesis of which is stimulated by arginine. None of the other urea-cycle enzymes is regulated allosterically.

3-phosphoglycerate and alanine. Folic acid is not needed to synthesize aspartate (from oxaloacetate by transamination), glutamate (from ␣-ketoglutarate by transamination), proline (from glutamate by a series of steps that do not require folic acid), or serine (from 3-phosphoglycerate, with no one-carbon metabolism needed).

CHAPTER 40 1.

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CHAPTER 39 1.

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The answer is A. Tyrosine is derived from phenylalanine, which requires BH4 but not vitamin B6. Vitamin B6 is required in the synthesis of serine (transamination), alanine (another transamination), cysteine (␤-elimination, ␤-addition, ␤-elimination), and aspartate (transamination). The answer is E. The other end products are acetoacetate, acetyl-CoA, fumarate, oxaloacetate, ␣-ketoglutarate, and pyruvate. The answer is C. The classical form of PKU, a deficiency of phenylalanine hydroxylase, results in elevations of phenylalanine and phenylpyruvate. However, this enzyme is not a choice. In the nonclassical variant of PKU, there is a problem in either synthesizing or regenerating BH4. The enzyme that converts BH2 to BH4 is dihydropteridine reductase. The answer is B. The reaction pathway in which methionine goes to cysteine and ␣-ketobutyrate requires pyridoxal phosphate at two steps: the cystathionine ␤-synthase reaction and the cystathionase reaction. Phenylalanine to tyrosine requires BH4; propionyl-CoA to succinyl-CoA requires B12; pyruvate to acetyl-CoA requires thiamine pyrophosphate, lipoic acid, NAD, FAD, and coenzyme A; and glucose to glycogen does not require a cofactor. The answer is C. Folic acid is required for the conversion of serine to glycine and the serine is produced from

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5.

The answer is D. The homocysteine-to-methionine reaction requires N5-methyl-FH4; serine to glycine requires free FH4 and generates N5,N10-methylene-FH4; betaine donates a methyl group to homocysteine to form methionine without the participation of FH4; and the purine ring requires N10-formyl-FH4 in its biosynthesis. The answer is B. Propionic acid is derived from an accumulation of propionyl-CoA. The normal pathway for the degradation of propionyl-CoA is, first, a biotin-dependent carboxylation to D-methylmalonyl-CoA, racemization to L-methylmalonyl-CoA, and then, the B12-dependent rearrangement to succinyl-CoA. The answer is C. An inability to hydrolyze dietary protein and release bound B12 will lead to a deficiency. Pancreatic insufficiency will result in reduction of proteases in the pancreas and reduced ability to release B12 from protein. Both R-binders and intrinsic factor are required for appropriate B12 absorption; animal protein contains high levels of B12, and glutamic acid is conjugated to folic acid, not to B12. Folic acid absorption does not affect B12 absorption. The answer is B. The only three forms of folate that transfer carbons are the N5-methyl-FH4 form, the N5,N10methylene-FH4 form, and the N10-formyl form. None of the other forms participates in reactions in which the carbon is transferred. It is the N5-methyl form that transfers the methyl group to form methionine from homocysteine. The answer is E. Choline, derived from phosphatidylcholine, is converted to betaine (trimethylglycine). Betaine can donate a methyl group to homocysteine to form methionine plus dimethylglycine. Sarcosine is N-methylglycine, which is formed when excess SAM methylates glycine, but it is not used as a methyl donor in this reaction.

CHAPTER 41 1.

The answer is A. Both carbamoyl phosphate synthetase (CPS) I and II use carbon dioxide as the carbon source in the production of carbamoyl phosphate. CPSI is located in the mitochondria, whereas CPSII is in the cytoplasm (thus, B is incorrect). CPSI can fix ammonia; CPSII requires glutamine as the nitrogen source (thus, C is incorrect).

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N-acetylglutamate activates CPSI; CPSII is activated by PRPP (thus, D is incorrect). UMP inhibits CPSII, but has no effect on CPSI (thus, E is also incorrect). The answer is B. A lack of glucose 6-phosphatase activity (von Gierke disease) leads to an accumulation of glucose 6-phosphate, which leads to an increase in ribose 5-phosphate levels, and then an increase in PRPP levels. As PRPP levels rise, purine synthesis is stimulated, leading to excessive levels of purines in the blood. The degradation of the extra purines leads to uric acid production and gout. A loss of either PRPP synthetase activity or glutamine phosphoribosyl aminotransferase activity would lead to reduced purine synthesis and hypouricemia (thus, A and D are incorrect). A lack of glucose-6-phosphate dehydrogenase would hinder ribose 5-phosphate production and thus would not lead to excessive purine synthesis. A lack of purine nucleoside phosphorylase would hinder the salvage pathway, leading to an accumulation of nucleosides. Purine nucleoside phosphorylase activity is required to synthesize uric acid, so in the absence of this enzyme, less uric acid would be produced (thus, E is incorrect). The answer is C. The enzyme defect in Lesch–Nyhan syndrome involves hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This enzyme converts the free base to a nucleotide—specifically, guanine to GMP and hypoxanthine to IMP. Adenine phosphoribosyltransferase (APRT) converts adenine to AMP (thus, A is incorrect). Adenosine kinase converts adenosine to AMP (thus, B is incorrect). There are no enzymes to convert guanosine to GMP or thymine to TMP (thus, D and E are incorrect). Pyrimidine nucleoside phosphorylase will convert thymine to thymidine but not to the nucleotide level. Thymidine kinase converts thymidine to TMP (thus, F is incorrect). The answer is B. Allopurinol inhibits the conversion of both hypoxanthine to xanthine and xanthine to uric acid. This occurs because both of those reactions are catalyzed by xanthine oxidase, the target of allopurinol. Answer A is incorrect because AMP is not converted directly to XMP (AMP, when degraded, is deaminated to form IMP, which loses its phosphate to become inosine, which undergoes phosphorolysis to generate hypoxanthine and ribose 1-phosphate). Answer C is incorrect because the inosine-to-hypoxanthine conversion, catalyzed by nucleoside phosphorylase, is not inhibited by allopurinol. Answer D is incorrect because the conversion of hypoxanthine to xanthine occurs at the free base level, not at the nucleotide level. Answer E is incorrect because GMP is converted first to guanosine (loss of phosphate), the guanosine is converted to guanine and ribose 1-phosphate, and the guanine is then converted to xanthine by guanase. The answer is B. If dGTP were to accumulate in cells, the dGTP would bind to the substrate specificity site of ribonucleotide reductase and direct the synthesis of dADP.

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This would lead to elevations of dATP levels, which would inhibit the activity of ribonucleotide reductase. The inhibition of ribonucleotide reductase leads to a cessation of cell proliferation, as the supply of deoxyribonucleotides for DNA synthesis become limiting. Answer A is incorrect because ATP would need to bind to the substrate specificity site to direct the synthesis of dCDP. That would not occur under these conditions of elevated dGTP levels. Answer C is incorrect because the enzyme works only on diphosphates; AMP would never be a substrate for this enzyme. Answer D is incorrect because the thioredoxin is always regenerated and does not become rate limiting for the reductase reaction. Answer E is incorrect because dGTP does not bind to the activity site of the reductase; only ATP (activator) or dATP (inhibitor) is capable of binding to the activity site.

CHAPTER 42 1.

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The answer is D. High levels of amino acids in the blood stimulate the pancreas to release glucagon (thus, A, C, E, and G are incorrect). Insulin is also released, but the glucagon/insulin ratio is such that the liver still uses the carbons of amino acids to synthesize glucose (thus, A, E, and F are incorrect). However, the insulin levels are high enough to stimulate BCAA uptake into the muscle for oxidation (thus, E, F, G, and H are incorrect). The answer is C. The pathway followed is glutamine to glutamate, to glutamate semialdehyde, to ornithine, and then, after condensation with carbamoyl phosphate, to citrulline. Aspartate, succinyl-CoA, serine, and fumarate are not part of this pathway. The answer is D. Cortisol is released during fasting and times of stress and signals muscle cells to initiate ubiquitin-mediated protein degradation. The other hormones listed do not have this effect on muscle protein metabolism. Insulin stimulates protein synthesis; glucagon has no effect on muscle because muscle has no glucagon receptors. Epinephrine initiates glycogen degradation but not protein degradation, and glucose is not a signaling molecule for muscle as it can be for the pancreas. The answer is D. Glutamine is derived from glutamate, which is formed from ␣-ketoglutarate. Only isoleucine and valine can give rise to glutamine because leucine is strictly ketogenic. These amino acids give rise to succinyl-CoA, which goes around the TCA cycle to form citrate (after condensing with acetyl-CoA), which then forms isocitrate (the correct answer), and the isocitrate is converted to ␣-ketoglutarate. Urea, pyruvate, phosphoenolpyruvate, and lactate are not required intermediates in the conversion of BCAA carbons to glutamine carbons.

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ANSWERS TO REVIEW QUESTIONS

5.

The answer is B. An individual in sepsis will be catabolic; protein degradation exceeds protein intake (leading to negative nitrogen balance; thus, A, C, and E are incorrect). The liver is synthesizing glucose from amino acid precursors to raise blood glucose levels for the immune cells and the nervous system (thus, gluconeogenesis is active, and E and F are incorrect). Fatty acid release and oxidation has also been stimulated to provide an energy source for the liver and skeletal muscle (indicating that C, D, and F are incorrect).

5.

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3.

4.

The answer is E. A tumor of the adrenal cortex is secreting excessive amounts of cortisol into the blood, which adversely affects glucose tolerance, suppresses pituitary secretion of ACTH, and, through chronic hypercortisolemia, causes the physical changes described in this patient. Uncomplicated non–insulin-dependent and insulin-dependent diabetes mellitus can be eliminated as possible diagnoses because they are not associated with elevated plasma cortisol levels and low plasma ACTH levels. In this patient, hyperglycemia resulted from the diabetogenic effects of chronic hypercortisolemia. An ACTH-secreting tumor of the anterior pituitary gland would cause hypercortisolemia, which, in turn, could adversely affect glucose tolerance, but in this case, the plasma ACTH levels would have been high rather than 0 pg/mL. The posterior pituitary gland secretes oxytocin and vasopressin, neither of which influences blood glucose, cortisol, nor ACTH levels. The answer is A. High blood glucose levels cause a decrease in growth hormone levels in the blood. This fact serves as the basis for the glucose suppression test for growth hormone. Answers B, C, and D all would cause growth hormone levels to increase, whereas answer E would have no effect on the test. The answer is B. When iodine is deficient in the diet, the thyroid does not make normal amounts of T3 and T4. Consequently, there is less feedback inhibition of TSH production and release; hence, an increased secretion of TSH would be observed. There is no direct effect on thyroglobulin synthesis (thus, A is incorrect). TRH is released by the hypothalamus to release TSH from the pituitary; a lack of thyroid hormone would increase production of TRH, not decrease it (thus, C is incorrect). An overproduction of thyroid hormone leads to increased heat production and weight loss; lack of thyroid hormone does not lead to these symptoms (thus, D and E are incorrect). The answer is A. The woman is experiencing hypothyroidism; TSH is elevated in an attempt to secrete more thyroid hormone, as the existing dose is too low to suppress TSH release.

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The answer is C. If 15% of the radioactive T4 is bound to antibody, the amount of T4 in 0.1 mL of the patient’s serum is 0.015 ␮g/0.1 mL or 15 ␮g/dL (1.0 dL ⫽ 100 mL).

CHAPTER 44 1.

CHAPTER 43 1.

965

2.

3.

4.

The answer is A. Increased 2,3-bisphosphoglycerate in the red cell will favor the deoxy conformation of hemoglobin and thus allow more oxygen to be released in the tissues. This is useful because the hemoglobin is not as saturated at high altitudes as at low elevations because of the lower concentration of oxygen at high altitudes. Answers C and D are incorrect because the red cells do not synthesize proteins. Answer E is incorrect because reducing the blood pH will not aid in oxygen delivery; the Bohr effect works best when tissue pH is lower than blood pH, in order to stabilize the deoxy form of hemoglobin. If the pH of both the blood and the tissue are the same, the Bohr effect will not be able to occur. The answer is D. The boy is suffering from lead poisoning, which interrupts heme synthesis at the ALAdehydratase and ferrochelatase steps. Without heme, the oxygen-carrying capability of blood is reduced, and the flow of electrons through the electron-transfer chain is reduced because of the lack of functional cytochromes. Together, these lead to an inability to generate energy, and fatigue results. Answer A is incorrect because although an iron deficiency would lead to the same symptoms, this would not be expected in the patient because of his daily vitamin uptake. Lead ingestion will not lead to an iron loss. Answers B and C are incorrect because vitamin B12 and folate deficiencies will lead to macrocytic anemia due to disruption of DNA synthesis. Answer E is incorrect because the ingestion of paint chips is unlikely to lead to a vitamin B6 deficiency in a child, particularly one who is taking daily vitamins. The answer is C. Turning on a gene that would provide a functional alternative to the ␤-gene would enable the defective ␤-protein to be bypassed. Only the ␥-chain can do this, but it is normally only found in fetal hemoglobin. The ␦-chain is also a ␤-replacement globin, but it was not listed as a potential answer. Answer D is incorrect because it is the ␤-chain that is mutated, and it is already being expressed. Unlike the ␣-gene, of which there are two copies per chromosome, there is only one copy of the ␤-gene per chromosome. The other genes listed (answers A, B, and E) are ␣-chain replacements, and expression of these genes will not alleviate the problem inherent in the ␤-gene. The answer is D. The only two types of blood cells that lack nuclei are the mature red blood cell and the platelet. Platelets arise from membrane budding from megakaryocytes and are essentially membrane sacs containing the

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ANSWERS TO REVIEW QUESTIONS

cytoplasmic contents of their precursor cell. All of the other cell types listed contain a nucleus. The answer is D. Thalassemias result from an imbalance in the synthesis of ␣- and ␤-chains. Excessive synthesis of ␣-chains results in their precipitation in developing red cells, which often kills the developing cell. The more severely affected child has an ␣/␤ ratio of 1:5, whereas the less severely affected child has a ratio of 1:2½. When ␤-chains are in excess, they form stable tetramers that bind but do not release oxygen, thus reducing the red cell’s ability to deliver oxygen. Thus, this difference in chain ratio makes an important difference in the functioning of the red cell.

CHAPTER 46 1.

2.

CHAPTER 45 1.

2.

3.

4.

5.

The answer is D. Under conditions of reduced protein ingestion, essential amino acids are scarce and the liver reduces protein synthesis, including circulating plasma proteins. The reduction of protein in the plasma results in a lower osmotic pressure, so excess fluid in the extravascular spaces cannot return to the blood and remains outside of the circulation, collecting in tissues. The answer is C. Warfarin inhibits the reduction of vitamin K epoxide, so active vitamin K levels decrease. The reduction in active vitamin K levels reduces the ␥-carboxylation of clotting factors. In the absence of ␥-carboxylation, the clotting factors cannot bind to calcium to form membrane-associated complexes with other clotting factors. Warfarin has no effect on the liver’s ability to synthesize the clotting factor (the synthesized factor is not modified), nor does warfarin specifically inhibit the activation of factor XIII. The inhibition is more global than just attacking one step in the coagulation cascade. Plasma calcium levels are not altered by warfarin, and protein C activity is actually decreased in the presence of warfarin because protein C is one of the proteins that is ␥-carboxylated in a vitamin K–dependent reaction. The answer is C. Activated protein C turns off the clotting cascade; in the absence of protein C, regulation of clotting is impaired, and clots can develop when not required. Mutations in any of the other answers listed would lead to excessive bleeding, as an essential component of the clotting cascade would be inactivated. The answer is C. Classical hemophilia is the absence of factor VIII, which is a necessary cofactor for the activation of factor X by factor IXa. Factor II is directly activated by factor Xa, and factor IX is directly activated by factor XIa. Proteins C and S are directly activated by thrombin, factor IIa. The answer is B. Hemophilia B is an inactivating mutation in factor IX, such that factor IXa cannot be formed. Factor XIa is formed but its substrate, factor IX, is defective, and a nonactive protein results.

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3.

4.

5.

The answer is C. Grapefruit juice contains a component that blocks CYP3A4 activity, which is the cytochrome P450 isozyme that converts statins to an inactive form. If the degradative enzyme is inhibited, statin levels rise above normal, accelerating their damage of muscle cells. Grapefruit juice does not affect hepatic uptake of the drug (thus, A is incorrect), nor is it accelerating statin metabolism (thus, B is also incorrect). Although HMG-CoA reductase is the target of the statins, grapefruit juice neither upregulates nor downregulates the amount of enzyme present in the cell (thus, D and E are incorrect). The answer is D. Cytochrome P450 enzymes oxidize their substrates, transferring the electrons to molecular oxygen to form water and a hydroxylated product. The enzymes require NADPH (thus, B is incorrect) and are located in the endoplasmic reticulum membrane (thus, A is incorrect). Oxygen does not induce all cytochrome P450 members (although it is a substrate for all these isozymes; thus, C is incorrect), and although these enzymes proceed through a free radical mechanism, the final products are not radicals (thus, E is incorrect). The answer is D. Hepatocellular disease reduces protein synthesis in the liver, which leads to reduced levels of both LCAT and hepatic triglyceride lipase being produced (thus, A and C are incorrect). Because LCAT activity is reduced, cholesterol ester formation in circulating particles is reduced (thus, B is incorrect). Because a diseased liver has trouble synthesizing glucose, fatty acid release from adipocytes is increased to provide energy (thus, E is incorrect). Serum triacylglycerol levels are increased as a result of the reduced hepatic triglyceride lipase activity; LPL activity is also reduced in liver disease. The answer is D. Ethanol induces the CYP2E1 system, which converts acetaminophen to NAPQI, a toxic intermediate. Under normal conditions (noninduced levels of CYP2E1), the conversion of acetaminophen to NAPQI results in low levels of NAPQI being produced, which can easily be detoxified. However, when CYP2E1 is induced, the excessive levels of NAPQI being produced when Tylenol is taken in greater than recommended amounts cannot be readily detoxified, and NAPQI binds to proteins and inactivates them, leading to hepatocyte death. The toxicity is neither related to blood glucose levels (thus, A and C are incorrect), nor to secretion of VLDL (thus, E is incorrect). Ethanol does not inhibit the detoxification of Tylenol per se, but rather accelerates one of its potential metabolic fates (thus, B is also incorrect). The answer is C. The glucokinase-regulatory protein regulates glucokinase expression at a posttranscriptional level. A lack of regulatory protein activity results in less glucokinase being present in the cell and a reduced overall rate of glucose phosphorylation by the liver. This results

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ANSWERS TO REVIEW QUESTIONS

in less circulating glucose being removed by the liver, and a longer clearance time for glucose levels to return to fasting levels. A decrease in the glucokinase Km, or an increase in the Vmax for glucokinase, would lead to the opposite effect, enhanced glucose phosphorylation by the liver, and an accelerated clearance from the circulation (thus, A and B are incorrect). The liver does not express hexokinase, so D and E are also incorrect.

phosphorylation of glycogen synthase as induced by epinephrine. Palmitate is oxidized and glycolysis increases because of the activation of PFK-1 by AMP. As ATP decreases, AMP rises as a result of the myokinase reaction (2ADP → ATP ⫹ AMP).

CHAPTER 48 1.

CHAPTER 47 1.

2.

3.

4.

5.

The answer is C. Stretching aids in stimulating blood flow to the muscles, which enhances oxidative muscle metabolism (by allowing for better oxygen delivery). Stretching, per se, does not stimulate epinephrine release (thus, A is incorrect), nor does it activate glycolysis in either the liver or muscle. The answer is D. Adipose triacylglycerol is the largest energy store in the body and is the predominant fuel in longterm aerobic exercise. During exercise, muscle glycogen is used for bursts of speed but not for long-term energy requirements. Liver glycogen is used to maintain blood glucose levels for use by the nervous system and as a supplement for use by the muscle when rapid speed is required; however, it is not designed to be a long-term energy source. The brain does not contain significant levels of glycogen, and lactic acid produced by the red blood cells is used as a gluconeogenic precursor by liver, but not as a fuel for muscle. The answer is D. Creatine is synthesized from glycine, arginine, and S-adenosylmethionine (thus, intake of dietary creatine is not relevant). In muscle, creatine is converted to creatine phosphate, which is nonenzymatically cyclized to form creatinine (thus, C is incorrect). The amount of creatinine excreted by the kidneys each day depends on body muscle mass but is constant for each individual (therefore, if there is an increase in body muscle mass, there would be an increase in creatinine excretion). In kidney failure, the excretion of creatinine into the urine is low, and an elevation of serum creatinine would be observed. The answer is E. Glycine is required for the synthesis of heme (combining with succinyl-CoA in the initial step), for the synthesis of purine rings (the entire glycine molecule is incorporated into adenine and guanine), and for creatine, where glycine reacts with arginine in the first step. The answer is C. A decrease in the concentration of ATP (which occurs as muscle contracts) stimulates processes that generate ATP. The proton gradient across the inner mitochondrial membrane decreases as protons enter the matrix via the ATPase to synthesize ATP; NADH oxidation by the electron-transport chain increases to reestablish the proton gradient; fuel use also increases to generate more ATP. Glycogen synthesis is inhibited because of

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967

2.

3.

4.

5.

The answer is B. The symptoms exhibited by the patient are caused by excessive release of epinephrine or norepinephrine. Vanillylmandelic acid is also the degradation product of norepinephrine; thus, these hormones are being overproduced. Acetylcholine degradation leads to the formation of acetic acid and choline, which are not observed (thus, A is incorrect). Although dopa degradation could lead to vanillylmandelic acid production, serotonin degradation does not (it leads to 5-hydroxyindoleacetic acid), and the symptoms exhibited by the patient are not consistent with dopa or serotonin overproduction (thus, C is incorrect). Histamine and melatonin also do not produce the symptoms exhibited by the patient (thus, D and E are incorrect). The answer is A. Myelin contains very high levels of cholesterol and cerebrosides, particularly galactosylcerebrosides. The answer is E. Myelin basic protein is a basic protein, indicating that it must contain a significant number of lysine and arginine residues. MBP is found on the intracellular side of the myelin membrane, and its role is to compact the membrane by binding to negative charges on both sides of it, thereby reducing the “width” of the membrane. Both Schwann cells and oligodendrocytes synthesize myelin (thus, A is incorrect). MBP is not a transmembrane protein (proteolipid protein in the CNS and Po in the PNS are, so B is incorrect), and because MBP is found intracellularly, answers C and D cannot be correct. The answer is A. The accumulation of both phytanic acid and very long-chain fatty acids indicates a problem in peroxisomal fatty acid oxidation, which is where ␣-oxidation occurs. Lysosomal transport is, therefore, not required to metabolize these fatty acids (thus, C is incorrect). The finding that palmitate levels are low indicates that ␤-oxidation is occurring; therefore, answer B is incorrect. The compounds that accumulate are not mucopolysaccharides nor is fatty acid elongation required in the metabolism of these compounds (thus, D and E are incorrect). The answer is D. Vitamin B6 participates in transamination and decarboxylation reactions (and indirectly in deamination reactions). The one common feature in the synthesis of serotonin, GABA, norepinephrine, and histamine is decarboxylation of an amino acid, which requires vitamin B6. The other reactions are not required in the biosynthesis of these neurotransmitters.

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ANSWERS TO REVIEW QUESTIONS

CHAPTER 49 1.

2.

The answer is E. Scurvy is caused by a deficiency of vitamin C. Vitamin C is a required cofactor for the hydroxylation of both proline and lysine residues in collagen. The hydroxyproline residues that are formed stabilize the triple helix through the formation of hydrogen bonds with other collagen molecules within the helix. The loss of this stabilizing force greatly reduces the strength of the collagen fibers. The hydroxylation of lysine allows carbohydrates to be attached to collagen, although the role of the carbohydrates is still unknown. Vitamin C is not required for disulfide bond formation, the formation of lysyl cross-links (that enzyme is lysyl oxidase), secretion of tropocollagen, or the formation of collagen fibrils. Thus, all the other choices are incorrect. The answer is C. When elastin expands as a result of outside forces (such as the respiratory muscles causing the lungs to expand with air), hydrophobic regions of elastin are exposed to the aqueous environment, resulting in a decrease in the entropy of water. When the outside force is removed (by relaxation of the respiratory muscles), the driving force for contraction of elastin is an increase in the entropy of water, so that the hydrophobic residues of elastin are again shielded from the environment. The expansion and contraction of elastin does not involve covalent modifications (thus, A, B, and E are incorrect), nor does it involve extensive changes in salt bridge formation (thus, D is also incorrect).

Lieberman_Answers.indd 968

3.

4.

5.

The answer is B. Glycosaminoglycan chains contain negative charges, resulting from the presence of acidic sugars and the sulfated sugars in the molecule. Thus, in their characteristic bottleneck structure, the chains repel each other (thus, A is incorrect), yet they also attract positively charged cations and water into the spaces between the chains. The water forms hydrogen bonds with the sugars and a gel-like space is created. This gel acts as a diffusion sieve for materials that leave, or enter, this space. Hydroxylation and cross-linking of chains does not occur (thus, D and E are incorrect), nor does hydrogen bonding between chains (they are too far apart because of the charge repulsion, but they do form hydrogen bonds with water). The answer is E. In order for cells to migrate, they must free themselves from the extracellular matrix material, which requires remodeling of the matrix components. Because of the unique structural aspects of these components, only a small subset of proteases, the metalloproteinases, is capable of doing this. The other answers listed are all components of the matrix, which must be remodeled in order for cell migration to occur. The answer is C. Fibronectin binds to integrins on the cell surface, as well as to various extracellular matrix components (collagen and glycosaminoglycans). This binding fixes the position of the cell within the matrix. Loss of these binding components can lead to undesirable cell movement. Fibronectin plays no role in any of the other functions listed as possible answers.

01/09/12 9:36 PM

Patient Index Note: Page numbers followed by f denote figures. A Abietes, Dianne (Di) acetoacetate measurement in, 67 blood glucose in after a meal, 575 self-monitoring of, 55, 67 dehydration in, 52 diabetic coma in, 44 diabetic ketoacidosis in, 41, 55, 57, 416, 431, 433 mechanisms of, 679, 686 drowsiness and vomiting in, 416 E. coli urinary infection in, 210, 212 follow-up of, 55 glucose transport in, 159 glycemic control in, 533 glycogen in muscle synthesis of, 522 storage of, 522 glycosylated hemoglobin in, 89, 105, 108 hyperglycemia in, 183 mechanisms of, 684, 685 opalescent serum from, 679, 685 hypoglycemia in, from insulin overdose, 561, 572, 578 insulin levels in, blood, 486 ketones in, 67, 431 Kussmaul breathing in, 48, 51, 416, 431 lipid metabolism abnormalities in, 689 lipolysis on free fatty acids in, 685 microvascular complications in, 578–579 osmotic diuresis in, 44 partial pressure of CO2 in, 49 pathophysiology of, 484 pH of, blood, 49 polyuria in, 44 presentation and diagnosis of, 41, 50 rehydration of, 44 treatment of, 50–51, 303 Humalog (lispro insulin) in, 82f, 85 Humulin in, 85 urinary tract infection in, 223 vascular disease in, 689 Anoma, Melvin (Mel) biopsy of initial, 223 with mole recurrence, 329 clinical presentation and diagnosis of, 210 disease characteristics in, 312 hereditary risk factors for, 319 mole after treatment in, 311 pathophysiology of, 210 self-examination in, 329 smoking carcinogens on, 219 Applebod, Evan presentation and diagnosis of, 904 Prozac treatment of, 923 weight loss program for cholesterol reduction from, 904 drug options in, 923–924

Redux in, 904, 915 Redux withdrawal in, 923 Applebod, Ivan body weight classification of, 11, 17, 27 calories in diet of, 7, 8 ethanol in, 458, 463, 467 caries and cavities in, 397, 403, 411, 411f coronary heart disease, risk factors for, 627 daily energy expenditure of, 11 history for, 4 hypercholesterolemia in, 26, 27 HDL cholesterol with elevated VLDL or LDL in, 651 LDL cholesterol in, 649 lipid levels in, 649 hyperglycemia and diabetes mellitus type 2 in, 24, 27 hypertriglyceridemia in, 649 metabolic rate of basal, 11 resting, 9 metabolic syndrome in, 27, 627–628 treatment of aspirin in, 670, 675 diet and exercise in, 657–658 drugs in, 658 exercise program in, 27 weight loss program in, 17 weight gain calculation in, 14 Atotrope, Sam acromegaly in from growth hormone–secreting tumor, 801 treatment options for, 801 diabetes mellitus in, from growth hormone– secreting tumor, 803, 819 disease characteristics in, 801, 802 insulin-like growth factor I in, 801–802 presentation and diagnosis of, 799, 801–802 B Biasis, Amy abdominal cystic masses in, 866 amebiasis in, 866, 867 laboratory results in, 865 presentation and diagnosis of, 863 treatment of, 867 treatment outcomes in, 879 Bodie, Jim androgen and insulin use by, 526 insulin overdose of presentation and diagnosis of, 514, 526 treatment of, 524 Bolic, Katta acute-phase response in, 789, 790 negative nitrogen balance in, 779, 780 presentation and diagnosis of, 775 septicemia in, 775 weight loss in, 790

Burne, Lofata disease characteristics in, 422, 432–433 fasting symptoms in, 416 ␻-oxidation of fatty acids in, 427, 427f presentation and diagnosis of, 416, 422 C Carbo, Getta anorexia of mother of, 519 glycogen stores in, inadequate, 519, 523, 525–526 presentation and diagnosis of, 513 treatment of, 526 Colamin, Katie catecholamine in biochemical cascade of, 914 effects of, 923 fractionated metanephrine testing in, 912 presentation and diagnosis of, 904, 912 treatment of, 912 D Dopaman, Les Lewy bodies in, 442, 453 pathogenesis of, 452 pathophysiology of, 452–453 brain structures in, 438 reactive oxygen and nitrogen–oxygen species in, 452–453, 452f presentation and diagnosis of, 438, 452 F Felya, Rena disease characteristics in, 885, 893, 900 presentation and diagnosis of, 885 Fibrosa, Sissy bronchitis in, 697 chloride channel defect in, 699 DNA sequencing testing in, 300 family history of, 300 genetics of, 306, 699 growth and weight in, subnormal, 697, 703 pancreatic enzyme supplement treatment of, 697, 703 presentation and diagnosis of, 289 protein malnutrition in, 697 Foma, Arlyn drug use in, 266 microRNAs in, 283 pathophysiology of, 284 presentation and diagnosis of, 266 treatment of, 284, 379 combination chemotherapy in, 772 doxorubicin toxicity from, 379 interferon-␣ in, 279 Fusor, Lopa acid–base imbalance in, 410–411 arterial PO2 and PCO2 in, 410

969

Lieberman_Patient_Index.indd 969

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970

PATIENT INDEX

Fusor, Lopa (continued) chronic obstructive pulmonary disease in, 404, 411 confusion in, from brain hypoxia, 401 hypoxemia in, 404, 410–411 presentation and diagnosis of, 397, 410–411 respiratory acidosis in, 410 G Galway, Erin, 535 cataract in, early, 533 jaundice in, 531, 546 management of, 540 mental retardation prevention in, 534, 540 presentation and diagnostic testing for, 531 test results and diagnosis of, 535 I Itis, Newman presentation and diagnosis of, 194 treatment of, 194, 205, 206 azithromycin in, 257 chloramphenicol in, lack of, 257 follow-up after, 249 levofloxacin in, 256 streptomycin in, lack of, 255 J Jeina, Ann history and diagnosis of, 71–72, 107 hypercholesterolemia in familial hypercholesterolemia type II in, 648, 657 HDL cholesterol with elevated VLDL or LDL in, 651 LDL cholesterol level in, 630 treatment for, 664 atorvastatin in, 650, 657, 658 ezetimibe in, 644, 657, 658, 664 step 1 diet and statin in, 630 myocardial infarction in creatine kinase after, 81, 85–86, 86f, 99, 107 hospitalization for, 89 myoglobin release in, 99, 107 treatment for aspirin in, 664–665, 670, 675 K Klotter, Sloe factor VIII deficiency in, 848, 859 presentation and diagnosis of, 848 treatment of, 859 Kulis, Cal amino acid absorption problems in, 704 cystine stones and cystinuria in, 76, 85, 697, 730 cystine transport defect in, 701 Hartnup disease and, 704 history and diagnosis of, 71 treatment of cystinuria in, 85, 697 L Lakker, Colleen disease characteristics in, 600, 621, 621f presentation and diagnosis of, 600 Leizd, Vera disease characteristics in, 658–659 hirsutism etiology in, 656–657 presentation and diagnosis of, 629, 658 treatment rationale for, 658–659 virilization from excess androgens in, 652 Lloyd, Amy Bence-Jones proteins in urine in, 107 diagnosis of, 89, 107

Lieberman_Patient_Index.indd 970

kidney amyloid deposits in, 93 M protein in, 101 plasma cell dyscrasia in, 101 renal failure in, 107 treatment of, 107–108 Lupus, Sis articular cartilage disruption in, 928, 936, 936f, 939–940, 940f finger joint pain and swelling in, 928, 936f pathophysiology of, 238, 244 presentation and diagnosis of, 228, 238 treatment for, 244 M Martini, Al acetaldehyde in, 125 alcohol-induced ketoacidosis in, 465, 468 ethanol in on brain membrane fluidity, 156, 167 metabolism of, 136 glucose-6-phosphate dehydrogenase deficiency in, 537, 540 heart disease in beriberi heart disease in, 130, 373–374 Breathalyzer testing of, 136 heart failure from dietary deficiency in, 356, 363 hemolysis from infection and sulfa drug in, 531, 540–541 hypoglycemia in adrenergic response to, 578 from starvation with excess drinking, 561, 567, 578 Kussmaul respirations in, 458 liver damage in, 139, 167 MEOS, NADPH, and NADP⫹ in, 165, 167 metabolic acidosis in, 458 NADH/NAD⫹ ratio in, 465, 468 NADH on alcohol dehydrogenase in, 139 pancreatitis in pancreatic amylase and lipase in, 586, 587 pancreatitis in, 586, 587, 590, 594 steatorrhea from, 590 symptoms and diagnosis of, 113, 458 thiamine deficiency in, 113, 130, 363, 735 trimethoprim/sulfamethoxazole in allergy to, 540 for infection, 531 Melos, Nona fructose metabolism problems of, 496, 502, 509 hydrogen breath testing of, 502 presentation and diagnosis of, 496, 509 N Nari, Cora angina in, from severe ischemia, 453 congestive heart failure in, 351 hospital admission of, 337 hyperlipidemia in combined, 620 familial combined, 608, 620–621 lipoproteins in, 600 in second MI, 378 treatment of, 621 hypoxia in on ATP generation, 338, 385 superoxide production and damage from, 441 left ventricular heart failure in, 351–352 myocardial infarction in earlier, 337 glycolysis inhibition from, 409 ischemia damage in, 453

presentation and diagnosis of, in first MI, 337, 351 superoxide and reactive nitrogen–oxygen species with, 447 treatment of aspirin in, 670, 675 heart failure in, 352 ischemia–reperfusion injury in, 393, 442, 453–454 nitroprusside in, 385 premature ventricular contractions after, 438 in second MI, 378 tPA in, 378, 392, 438, 453 Nemdy, Erna blood bank work of, 545, 549, 555–556 immunization and of child, 249 diphtheria, of child, 258, 262 hepatitis B, 289, 303, 306 Niemick, Anne classification of, 260–261 fetal hemoglobin and severe anemia in, 824, 842 first follow-up of, 249 gene mutation in ␤-globin gene compound heterozygote in, 842 inherited alleles in, 243 pathophysiology of, 232, 237, 243, 252–253, 260–261 precursor cell differentiation in, 285 spinal cord compression in, lumbar, 824 O O’Rexia, Ann amenorrhea in, 31, 36 anemia in hypochromic, 285 iron deficiency, 266, 284–285, 383 cortisol and gluconeogenesis in, 178, 188 daily energy expenditure of, 11 diagnosis and referral of, 17–18, 36 diet of calories in, 6, 7 fasting in, 6, 7, 489 fatigue in, 356, 373 glucagon in, 173 glucokinase in, high-carbohydrate meal on, 138–139 glucose 6-phosphate, glycogen synthesis, and glycogenolysis in, 137, 173 history for, 4 hypoglycemia with starvation in, lack of, 561, 572 jogging in epinephrine and norepinephrine in stress response with, 175, 188 with fasting, 489 inadequate fuel stores for, 148 with increased eating, 172 muscle glycogen phosphorylase activation in, 142, 143f on physiology, 172 weight gain and, 136 malnutrition in protein-calorie, type III, 36–37 severe, and hospitalization, 31, 34 metabolic rate of basal, 11 resting, 9 PEP carboxykinase in, 280, 280f vitamin deficiencies in, 356 pantothenate, 362, 373 riboflavin, 359, 373

01/09/12 9:36 PM

PATIENT INDEX

weight in classification of, 11 gain of, 356 weight loss calculation in, 14 O’Tyne, Nick adenocarcinoma in, 312 brain metastasis in, 311, 328–329 clinical presentation and diagnosis of, 210 smoking and, 223, 327 treatment of, surgical, 210, 311, 328 S Sakz, Jay hexosaminidase A deficiency in, 261 presentation and diagnosis of, 249, 545 Selmass, Bea after pancreatic tumor surgery, 679 disease characteristics in, 483, 484, 486, 490 familial vs. multiple endocrine neoplasia in, 689 “fasting” hypoglycemia in, 481, 487 presentation and diagnosis of, 478, 481 proinsulin measurement in, 486 weight gain from, 683 Shape, Otto basal metabolic rate of, 337, 342 clinical presentation of, 337 daily energy requirement in, resting, 344 energy expenditure in, thermodynamics of, 342 exercise on blood glucose in before breakfast, 578 circulating glucose use while running in, 897–898 exercise program of, 351 on ATP utilization and TCA cycle, 358 fatty acid oxidation in, 424, 432 5-km race training in, 397 high-intensity exercise on lactate in, 408, 411, 432 improvements from, 356 ketosis after run in, 432 marathon training in, 415, 416 physical conditioning benefits in, 372–373 progress in, 561 supplements for, 415, 420 supplements for, succinate, 360–361 history for, 4 weight gain by, 342 weight goals of, 337 weight loss program for, 17 dietary calorie decrease in, 351 fat and alcohol reduction in, 348 improvements due to, 356 progress in, 561 Sharer, Ivy clinical course of, 194, 206 IV drug use in, 223 needle-sharing partner HIV testing and, 290, 293 night sweats in, continuing, 289 presentation and diagnosis of, 194 treatment of, 206 didanosine in, 213, 295 drugs in, 194, 195 multidrug therapy in, 223 muscle weakness from, 379, 393 noncompliance with, 210 RNA replication blocking by, 213 zidovudine in, 196, 196f zidovudine toxicity in, 389 tuberculosis in, 229 presentation and diagnosis of, 228, 236 treatment of, 229, 236, 243–244

Lieberman_Patient_Index.indd 971

Sichel, Carrie genetic counseling of, 306–307 testing of in fiancé, 303, 306–307 in patient, 289, 298 Sichel, Will bilirubin in, 72 gallstone disease in, 586, 589, 594, 838 heme processing in, 589 hemoglobin in polymerization of, 100 sickle, 203 structure of, 79 history and diagnosis of, 71 mutation in, 75 HbS variant in, 79 missense, 252 pathophysiology of, 291, 298 point, 291 restriction fragment analysis of, 291, 298 sickle cell crisis in initial, 89, 106–107, 107f readmission for, 89 steatorrhea in, 590 treatment of, 84–85 vaso-occlusion in, 84–85 vitamin deficiency potential in, 590 Sistine, Homer etiology in, 734–735 liver biopsy in, 734 pathologic findings and clinical features of, 740 presentation, testing, and diagnosis of, 726, 731 Site, Spiro disease characteristics in, 842 presentation and diagnosis of, 824 splenectomy in, 842 Solemia, Corti central fat deposition in, 811 disease characteristics in, 808, 819 presentation and diagnosis of, 799 tumor location in, 809–810 Sthenia, Mya pathophysiology of, 174 presentation and diagnosis of, 172, 187 Sucher, Candice hereditary fructose intolerance in, 533, 539–540 presentation of, 530–531 sugar and fruit intolerance of, 530–531 Sulin, Ann appetite loss in, 928 azotemia and uremia in, 928 diabetic ketoacidosis in, lack of, 686 diet for fiber in, 496, 504 glycemic index in, 504 glucose in blood, after a meal, 575 glycemic control in, 533 glycogen storage in, 522 hyperglycemia in, 478, 509 mechanisms of, 684 hypertriglyceridemia in, 682, 684 insulin levels in, blood, 486 insulin resistance in, 490 lipid metabolism abnormalities in, 689 lipoprotein lipase in, 682 microvascular complications in, 578–579, 940–942, 941f presentation and diagnosis of, 478, 679 sulfonylurea mechanism of action in, 485

971

T Teefore, X. S. ATP utilization and fuel oxidation in, 346 basal metabolic rate in, 344 diagnosis of, 379 effects of, 344 pathophysiology of, 351, 379 presentation and diagnosis of, 337, 351 treatment options for, 393 uncoupling proteins in, 394 Tim, Vicky DNA amplification and, 297 DNA fingerprinting of rape and murder suspects for, 289, 302, 302f, 307 Tin, Amanda mushrooms eaten by, 228, 230 presentation and diagnosis of, 228, 244 treatment and prognosis in, 244 Tonich, Jean Ann alcohol-induced cirrhosis in, 863, 867, 880 blood glucose and NEFA levels in, 880 hepatogenous diabetes in, 880–881 alcohol-induced hepatitis in, 468, 745 folate deficiency in, 746, 756 liver damage in, 467 jaundice from, 459 liver function test results with, 467 megaloblastic anemia in presentation and diagnosis of, 745–746 treatment of, 750 presentation and diagnosis of, 458–459 treatment outcomes in, expected, 879 vitamin B12 (cobalamin) in, 750, 756 Topaigne, Lotta acute gouty attack in, 167, 177, 771 gout in diagnosis of, 55, 67, 113 outcome of, 130 pathophysiology of, 65, 67, 771 hyperuricemia in, 771 monosodium urate crystals in deposition of, 65, 163 phagocytosis of, 163 treatment of, 130 allopurinol in, 113, 129, 167, 760, 771 colchicine in, 153, 166–167, 760 uric acid in, 66, 163 Tuma, Colin adenocarcinoma in, 312 liver metastases of, 311 presentation and diagnosis of, 194 ras proto-oncogene in, 312 treatment of chemotherapy in, 311 colonoscopies after, 329 5-fluorouracil in, 194, 204, 206, 745, 749, 772 leukopenia from, 206 tumor location in, 194, 206 tumor metastasis in, 206 Twelvlow, Bea presentation and diagnosis of, 745 vitamin B12 absorption problem in, 756 V Veere, Dennis “the Menace” aspirin poisoning in diagnosis of, 41, 46 metabolic acidosis in, 46 on mitochondria, 390 treatment of, 51 cholera in AB toxin cell surface binding sites in, 158 bacteria and pathogenesis of, 154

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972

PATIENT INDEX

Veere, Dennis “the Menace” (continued) CFTR channel activation in, 186 cholera toxin A on, 172, 186 diarrhea and dehydration in, 158, 160, 166, 168, 186 hypovolemic shock treatment for, 172 symptoms and diagnosis of, 153 treatment of, 188 malathion poisoning in acetylcholinesterase inhibition in, 127 presentation and chemistry of, 113, 113f treatment and prognosis in, 130 Veere, Percy alveolar hyperventilation in, 51 creatinine–height index of, 36 depression in dehydration with, 52 from malnutrition, 31 diet in craving for sweets in, 600 folate and B12 in, 16 high-carbohydrate, weight gain from, 600, 611 weight loss, 620

Lieberman_Patient_Index.indd 972

hepatitis A in ALT, AST, and alkaline phosphatase in, 711, 713 bilirubin in, 711 hepatic encephalopathy in, 713, 716 presentation and diagnosis of, 704 history for, 4 malnutrition in hospitalization for, 31, 33–34 iron deficiency anemia in, 15 protein-calorie, type I, 36 vitamin B12 deficiency in, 15 paresthesias in, 51 weight loss in, 15 Voider, Deria lactase deficiency and lactose intolerance of, 503, 509 management of, 509 presentation and diagnosis of, 495 W Weitzels, Mannie disease characteristics in, 284, 311, 315 karyotyping of, 311

Philadelphia chromosome in, 274, 284, 311, 315 presentation and diagnosis of, 266, 311 progenitor cells in, 285 treatment of, 284, 328 hydroxyurea in, 772 interferon-␣ in, 279 Wheezer, Emma diabetes mellitus predisposition in, 578 fever and cough in, 665 inflammation on, 666 presentation and diagnosis of, 561 treatment of high-dose glucocorticoids in, hyperglycemia from, 561, 564, 570, 578 inhaled steroids in, 675 triamcinolone acetonide in, 665 Y Yuria, Piquet definitive testing of, 739 disease characteristics in, 739–740 presentation and diagnosis of, 726

01/09/12 9:36 PM

Index Note: Page numbers followed by f denote figures; page numbers followed by t denote tables. A AB blood group characteristics of, 549, 554f, 555 transfusions for, 555–556 ABCA1 cholesterol transport by, 629, 643 mutations in, 643 Abdominal fat, 27 Abetalipoproteinemia, 594–595, 608, 624t A blood group, 549, 554f, 555 Abl proto-oncogene, 315 ABO blood groups, 549, 554f, 555, 555t Absorptive state, 21–26 amino acids in, 23f, 26 carbohydrates in, 21, 21f, 22 definition of, 21 digestion and absorption in, 22–23, 23f fats in, 21, 21f, 22–23, 23f glucose in, 23f, 24–26, 25f hormone levels in, 23–24, 23f lipoproteins in, 23f, 26 proteins in, 6f, 21, 21f, 22, 23f AB toxin, cholera, 158 Abzymes, 117 Acarbose, 499 Acetaldehyde, 130 from ethanol metabolism, 465–467, 466f liver injury in alcoholics from, 125, 167 on tubulin polymerization, 167 Acetaldehyde–adduct formation, 466, 466f, 467, 469 Acetaldehyde dehydrogenase (ALDH), 458f, 459, 459f, 460–461 Acetaminophen cytochrome P450 detoxification of, 869–870, 870f on prostaglandins, 670, 670f structure of, 670f Acetate in ethanol metabolism, fate of, 461, 461f as fuel in long-term exercise, 899–900 Acetic acid, titration curve for, 46–47, 47f Acetoacetate, 33, 33f, 779 alternative metabolism pathways for, 430 amino acid synthesis via, 736–739 (See also Amino acid synthesis, acetyl-CoA and acetoacetate in) oxidation of, 428–430, 430f synthesis of, 428, 428f, 429f Acetone, 33, 33f in ketoacidosis, 55 odor of, 55 synthesis of, 428, 429f Acetylation in fatty acid oxidation regulation, 433–435, 434f of histone, 272–273, 273f of long-chain acyl-CoA dehydrogenase, 433–435, 434f

Acetylcholine (ACh) at acetylcholine receptor, 173–174, 173f acetylcholinesterase on, 888 choline on synthesis of, 917 inactivation of, 916, 916f at muscarinic receptors, 174 in myasthenia gravis, 172 at neuromuscular junction, 887, 887f, 888 at nicotinic acetylcholine receptor, 174, 174f structure of, 175, 175f synthesis of, 915–916, 916f Acetylcholine (ACh) receptors in myasthenia gravis, 174 structure and function of, 173–174, 173f, 174f, 184 Acetylcholinesterase, 127, 128f, 916, 916f on acetylcholine at neuromuscular junction, 888 nerve gas targeting of, 888 Acetylcholinesterase inhibitors, 888, 916 Acetyl-CoA carboxylase (ACC) reaction of, 602, 602f regulation of, 602, 603f, 680–682, 681f, 682f Acetyl coenzyme A (acetyl-CoA), 5, 334–335, 335f, 564, 565 acetylcholine from, 915, 916f amino acid synthesis via, 736–739 (See also Amino acid synthesis, acetyl-CoA and acetoacetate in) cytosolic, from glucose, 601–602, 601f in fatty acid oxidation in fasting, 32f, 33 from glucose, cytosolic, 601–602, 601f high-energy thioester bond of, 361–362, 361f malonyl-CoA from, 602–603, 602f, 603f mevalonate synthesis from, 630–632, 630f, 631f from nitrogen metabolism, 695, 695f in TCA cycle, 357, 357f in TCA cycle, precursors of, 368–370 pyruvate dehydrogenase complex in, 363f, 368–370, 369f, 370f (See also Pyruvate dehydrogenase complex) sources of, 368, 369f N-Acetyl-glucosamine 6-phosphate synthesis, 549, 551f N-Acetylglucosamine phosphotransferase mutation, 260 Acetylsalicylate (acetylsalicylic acid), 46 on prostaglandins, 670, 670f toxicity of, 46 Acid, 44–46. See also specific acids conjugate, 45 definition of, 41, 44 dissociation of, 46, 46t equilibrium constant for dissociation of, 46 gluconic, 60, 61f inorganic, 41

pathways for metabolism of cyclo-oxygenase pathway in, 666–670 (See also Cyclo-oxygenase pathway) cytochrome P450 pathway in, 666f, 673, 673f lipoxygenase pathway in, 671–672, 671f, 672f strong, 45–46, 45t structure of, 665 undissociated, 41 uronic, 60 weak, 45–46, 45t Acid anhydride, 56f Acid–base catalysis, 119–120 Acidemia, lactic, 409–410, 410f, 413t Acid, metabolic, 41 buffers and, 47–50 bicarbonate buffer system in, 48 hydrochloric acid in, 50 hydrogen, ammonium, and phosphate ions in, urinary, 45t, 46f, 50 intracellular pH in, 45t, 49–50, 49f principles of, 47 red blood cell bicarbonate and hemoglobin in, 48–49, 49f sources of, 47 Acidosis lactic, 387t, 409–410, 410f, 464f, 465 metabolic, 46, 410, 458 respiratory, 410 Ackee tree, 424, 435t Acquired immunodeficiency syndrome (AIDS), 208t, 225t complications of, 246t in IV drug users, 223 multidrug therapy for, 244 retroviruses in, 206 treatment of AZT in, 394t multidrug, 223 tuberculosis with, 229, 236, 246t Western blot testing of, 293 Acromegaly, 821t from growth hormone–secreting tumor, 801 insulin-like growth factor I in, 801–802 treatment of, 801 ACTH syndrome, ectopic, 810, 819 Actin, 165, 885 Actin filaments, 166, 167f Actin fold, 94–95, 94f Action potential, 173 Activated intermediates, with high-energy bonds, 344–345 Activated protein C (APC) complex, 856, 856f, 857 Activation energy, 112, 116, 116f Activation-transfer coenzymes, 122–123, 122f, 124f

973

Lieberman_Subject_Index.indd 973

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974

INDEX

Activators, 274–276, 275f Activators, allosteric, 140–143, 141f Active sites, enzyme (catalytic), 112, 114, 114f Active transport, 158f, 341–342, 696 cell death and, 352–353, 352f energy and transporter proteins in, 160–161, 160f, 161f primary, 160 secondary, 160 Acute lymphoblastic leukemia, asparaginase for, 730 Acute-phase proteins, 872 Acute phase response, 789, 790 Acylcarnitine metabolism disorders, 419 Acyl-CoA-cholesterol acyl transferase (ACAT), 634, 635f Acyl-CoA synthetase, 417, 418f Acylglycerols, 62–63, 63f Acyl group, 55 Adaptation, regulation of gene expression for, 266 Adducts, acetaldehyde, 466, 466f, 467 Adenine, 195, 195f Adenocarcinoma, 312. See also specific types colon, 194, 208t intestinal, 331t lung, 331t Adenoma, 194 Adenomatous polyps, 194 Adenosine deaminase (ADA), 765, 765f Adenosine deaminase (ADA) deficiency, 771–772, 772t, 773t Adenosine deaminase (ADA) gene mutation, 304 Adenosine diphosphate (ADP) concentation vs. ATP and AMP, in glycolysis regulation, 407, 407f on oxygen consumption, 388, 388f Adenosine, inosine and inosine monophosphate from deamination of, 765, 765f Adenosine monophosphate (AMP) in anabolic vs. catabolic pathways, 686 vs. ATP and ADP concentration, in glycolysis regulation, 407, 407f in ischemia, 409 phosphorylation of, 761f, 762 synthesis of, from inosine monophosphate, 761f, 762, 763f Adenosine monophosphate (AMP)-activated protein kinase (AMPK) metformin on, 659–660, 660f thiazolidinediones on, 660 Adenosine phosphoribosyl transferase (APRT), 765, 766f Adenosine triphosphate (ATP) in exercise, long-term from branched-chain amino acids, 899, 899f from free fatty acids, 898–899, 899f from fuel oxidation, 4, 4f on glycolysis, 406–409 (See also Glycolysis) hexokinase in, 406f, 407 major sites of, 406–407, 406f phosphofructokinase-1 regulation in, 406f–408f, 407–409 allosteric, by AMP and ATP, 408, 408f by allosteric inhibition, at citrate site, 409 by fructose 2,6-bis-phosphate, 408–409 PFKI-1 in, 406f, 407–408 pyruvate dehydrogenase in, 409 pyruvate kinase in, 409 regulation of, vs. ADP and AMP, 407, 407f high-energy phosphate bonds in, 338, 338f, 344–345 homeostasis of, 345, 350–351

Lieberman_Subject_Index.indd 974

hydrolysis of, 338, 338f, 339–340 lack of, in cell death, 352, 352f in organs, 344, 344t in skeletal muscle generation of, 891 synthesis of, 892–893, 892f use of, 892–893, 892f, 893f synthesis of, electron transport coupling with, 388–390 regulation through, 388, 388f uncoupling of, 388–390, 389f, 390f Adenosine triphosphate (ATP)-binding cassette (ABC) protein (ABCA1) cholesterol transport by, 629, 643 mutations in, 643 S-Adenosylmethionine, 752–753, 753f Adenoviruses in gene therapy, 303–304, 305 in vectors, 305 Adenylate cyclase, in muscle, 896, 896f Adenylate kinase, 345 Adenylosuccinate synthetase, 764, 764f Adenylyl cyclase function of, 81 in heptahelical receptors, 185–186, 186f structure and isoforms of, 81, 81f Adequate Intake (AI), 10, 19 vs. Estimated Average Requirement, 10 for vitamins, 12, 13t–14t Adhesion proteins, 939 Adipocyte differentiation of, 280 as endocrine organ adiponectin in, 620 function of, 618 leptin in, 618–619 number and size of, 622 proliferation of, in early life, 622 thyroid hormone on, 812 Adiponectin, 620, 622 Adipose tissue in fasting state lipolysis of, 685, 685f prolonged, 34–35 fuel storage in, 10 function of, 1 glucose metabolism in, after a meal, 23f, 25–26 triacylglycerol storage regulation in, 682–683, 682f weight loss and, 10 Adipose triacylglycerol, 7, 7t, 33 ADP-ribosylating factor, 166 ADP-ribosyl transferases, in pathogenic bacteria, 83f, 84 Adrenal cortex hyperplasia of, 655 tumors of, 655, 809–810 Adrenal glands androgen synthesis in, 653f, 655, 655f hyperplasia of, 657–658 Adrenaline binding sites, 95–96, 96f Adrenal medulla neoplasms, hormone testing in, 806 Adrenergic receptors function of, 489 types of, 489–490 Adrenocorticotropic hormone (ACTH), 651, 807, 819 Adrenoleukodystrophy, 164 Adult-onset diabetes mellitus. See Diabetes mellitus type 2 Advanced glycosylation end products (AGEs), 105, 105f, 579

Aerobic glycolysis, 335 definition of, 335 energy yield of, vs. anaerobic glycolysis, 403 on pyruvate, 401, 401f Aflatoxin B1, cytochrome P450 detoxification of, 869 Age-related macular degeneration (AMD), 451, 455t AIDS. See Acquired immunodeficiency syndrome (AIDS) Akt, 182, 182f Alanine in ammonia transport, 778 chemical structure of, 73, 74f, 75t in gluconeogenesis during fasting, 717, 717f from intermediates of glycolysis, 727f, 730 in liver nitrogen transport to, 712–713, 712f production of, 563, 564f uptake of, 879 in skeletal muscle, sources of, 783 structure of, 64, 65f Alanine aminotransferase (ALT), 416, 730 in hepatitis, 711, 713 liver production of, 563, 564f Albinism, 912, 925t Albumin with kidney malfunction, 893 in kwashiorkor, 848 lack of, 849 Alcaptonuria, 736, 741t, 742t Alcohol. See Ethanol Alcohol dehydrogenase (ADH), 125, 126, 126f, 458f, 459–460, 459f, 460t genetic polymorphisms of, 462 metals in, 126, 126f NADH on, 139 Alcohol dehydrogenase 1A (ADH 1A), 462 Alcohol dehydrogenase 1C (ADH 1C), 462 Alcoholics (alcoholism), 150t, 169t, 470t, 596t acetaldehyde in liver injury in, 125 congestive heart failure in, 375t folate deficiency in, 745, 746 metabolic acidosis in, 458 moderate drinking in, 459 susceptibility to, 462 thiamine deficiency in, 123, 363, 735 VLDL in, high, 608, 609 Alcohol-induced cirrhosis, 467, 468 blood glucose and NEFA levels in, 880 hepatogenous diabetes in, 880–881 pathophysiology of, 879 presentation of, 864 Alcohol-induced hepatitis, 463, 466, 466f, 468, 745 Alcohol-induced ketoacidosis, 464f, 465 Alcohol-induced liver disease, 463, 468 chronic, liver fibrosis in, 468–469, 469f, 469t nutritional deficiencies from, 467–468 Alcohol-induced megaloblastic anemia, 745–746, 750, 758t Alcohol intoxication, legal limit of, 462 Alcohols, 55–56, 56f Aldehyde, 56f Aldohexose, 58, 58f Aldolase, 132f, 133, 532 Aldolase B, 532, 532f Aldose B defects in, fructose 1-P accumulation with, 531 structure of, 531 Aldosterone function of, 651 structure of, 177f Aldosteronism, primary, 655

01/09/12 9:36 PM

INDEX

Aldosugars, 54 Aliphatic compounds, 55, 55f Aliphatic, polar, uncharged amino acids, 74f, 75–76, 75t Alkalemia, from hyperventilation, 51 Alkaline phosphatase, in hepatitis, 711 Alkylating agents, 328 Alleles, 203, 203f Allele-specific oligonucleotide probes, 298 Allergic reactions, basophils in, 825 Allopurinol, 129, 129f, 771 Allosteric activators, 140–143, 141f Allosteric enzymes, 140–142, 141f Allosteric inhibitors, 140–143, 141f Allosteric regulation, of isocitrate dehydrogenase, 367–368, 367t, 368f Alloxanthine, 771 All-trans-retinoic acid, 177f ␣1-antiproteinase (AAP), 849 ␣1-antiproteinase (AAP) deficiency, 861t ␣-1-antitrypsin deficiency, 698 ␣2-antiplasmin, 858, 858f ␣2-macroglobulin, 858 ␣-amanitin, on RNA polymerase II, 230 ␣-amylase, 22, 497–498, 497f, 587 ␣-dextrins, 497, 497f ␣-globin gene deletion of, 839 structure and transcriptional regulation of locus of, 841, 841f ␣-helix, 91, 91f, 92f ␣-ketoacids, 779 ␣-ketoglutarate dehydrogenase, regulation of, 366f, 368 ␣-ketoglutarate/glutamate amino acids related through, 730–732 arginine in, 716f, 732 glutamate in, 724f, 730, 731f glutamine in, 731–732, 731f histidine in, 732, 733f proline in, 732, 732f succinyl coenzyme A from, 358f, 359 ␣-lactalbumin, 548–549, 548f ␣-linoleic acid, 11 ␣-linolenic acid, 11 ␣-thalassemias, 839 ␣-tocopherol, 450, 450f deficiency of, 13t, 14 as free radical scavenger, 447f, 449, 450, 450f Alu sequences, 242 Alveolar hyperventilation, 51 Alzheimer disease, acetylcholinesterase inhibitors for, 888 Amanita mushroom poisoning overview of, 246t on RNA polymerase II, 230 Amebiasis, 865–866 Amides formation of, 58f structure of, 56f Amines biogenic, 175, 175f structure of, 57–58, 58f Amino acid, 70–87 abbreviations for, 73, 74f absorption of, 699–701 Na⫹ and amino acid cotransport in, 699–700, 699f transport into cells in, 700–701, 700t acidic and basic, 74f, 75t, 76–78, 78f aliphatic, polar, uncharged, 74f, 75–76, 75t aromatic, 74f, 75, 75t blood–brain barrier transport of, 908

Lieberman_Subject_Index.indd 975

branched-chain (See Branched-chain amino acids (BCAAs)) as building blocks, 693 classification of, 70 degradation of, 724–725, 725f digestion of, rate of, 704 dissociation of, 70, 71f dissociation of side chains of, 77, 78f enzyme and protein databases of, 86 essential, 11 in body, 693–694, 694t in diet, 694, 694t in fed state, 21, 21f, 23f, 26 as fuels, 334f, 335 general properties of, 70, 71f in genetic code, 249–250, 250t hydropathic index of, 73, 75t ketogenic, 430, 736, 736f L- and D-, 72, 72f modified, 71 nitrogen fate in, 707–713 alanine and glutamine in nitrogen transport to liver in, 712–713, 712f, 713f glutamate role in in amino acid synthesis, 711–712, 711f in urea production, 710f, 712, 712f overview of, 707f removal as ammonia in, 709–711, 710f transamination reactions in, 709, 709f nonpolar aliphatic, 73–75, 74f, 75t hydrophobic, 70 oxidation of, 4–5, 4f, 371–372, 372f in proteins, 5, 6f regulatory modifications of, 83f, 84 sulfur-containing, 74f, 75t, 76 tissue utilization of, 779–787 by brain and nervous tissue, 786–787, 787f by gut, 785–786, 785f by kidney, 779–780, 780f–782f, 780t by liver, 786 by skeletal muscle, 780–785 (See also Skeletal muscle, amino acid utilization by) Amino acid carriers, Na⫹-dependent, 700 Amino acid metabolism, 693–695, 693f, 694t, 695t carbon fate in, 707f cofactors in, 726 excretory products of, 693, 694t function of, 779 genetic disorders of, 741t high-protein meal on, 787–788, 788f hypercatabolic states on, 774f, 788–790, 789f intertissue differences in, 778 in liver, 878–879 in liver disease, 879–880 pyridoxal phosphate in, 720–721, 720f, 721f in sepsis and trauma, 774, 774f by tissue, 708f in trauma and sepsis, 790–791, 791f Amino acids, free, 12 blood pool of, 775–779 interorgan flux of, in postabsorptive state, 776–778, 777f metabolism in liver during fasting in, 777f, 778, 778f metabolism in other tissues during fasting in, 778 release from skeletal muscle during fasting in, 776–778, 777f location and maintenance of, 775–776, 776f

975

size and function of, 775 tissue flux of, 778–779, 779t Amino acid structure, 54, 64–65, 65f, 70, 71f general, 71f, 72–73, 72f overview of, 70 peptide bonds in, 73, 73f primary basic, 71f, 72–73, 72f modified fatty acylation or prenylation in, 83f, 84 glycosylation in, 82–84, 83f posttranslational modification in, 82, 83f posttranslational modifications in, other, 83f, 84 regulatory modifications in, 83f, 84 in selenocysteine, 84, 84f variations in, 78–82 developmental, 80, 80f in hypervariable regions, 78 in noncritical (variant) regions, 78 polymorphisms in protein structure in, 78–79 protein families and superfamilies in, 79–80, 80f species variations in insulin in, 81–82, 81f tissue variations in, 81, 81f side chains in, 72 on catalysis, 121, 121t chemical properties of, 70 classification of, 73–78, 74f dissociation of, 77, 78f trans, 91, 92f substitutions in, 70 Amino acid synthesis acetyl-CoA and acetoacetate in, 736–739 isoleucine in, 738 leucine in, 735f, 736f, 738 lysine in, 736f, 738 overview of, 736, 736f phenylalanine and tyrosine in, 736–737, 737f, 738f threonine in, 738 tryptophan in, 737, 738f cofactors in, 726 from intermediates of glycolysis, 727–730 alanine in, 727f, 730 cysteine in, 729–730, 729f glycine in, 728, 728f overview of, 727, 727f serine in, 727–728, 727f overview of, 723, 724f TCA cycle intermediates and, 730–736 ␣-ketoglutarate/glutamate in, 730–732 arginine in, 716f, 732 glutamate in, 724f, 730, 731f glutamine in, 731–732, 731f histidine in, 732, 733f proline in, 732, 732f fumarate-forming aspartate, 733 phenylalanine and tyrosine, 733 oxaloacetate (aspartate and asparagine) in, 732, 733f succinyl-CoA–forming, 733–736 methionine in, 734, 734f overview of, 733–734 threonine in, 734, 734f valine and isoleucine in, 735–736, 735f

01/09/12 9:36 PM

976

INDEX

Amino acid transport systems for, 700–701, 700t transepithelial, 699–700, 699f Aminoacyl-tRNA definition of, 252 A site binding of, 256–257, 257f synthesis of, 252–253, 253f Aminoacyl-tRNA synthetases, 252–253, 253f Amino groups, 54, 56f Aminopeptidases, 699, 699f Amino sugars, 60, 60f, 871t Aminotransferases, 709 Ammonia, 335 from ammonium ion, 709, 710f blood, 713, 717 determination of, 718 breakdown of, 717 detoxification of, in liver, 871 excretion of, urinary, 693, 694t, 713, 780–781, 781t, 782f in hepatic encephalopathy, 717 intertissue transport of, 778 nitrogen removal as, 709–711, 710f Ammonia toxicity, 718, 722t, 792t on brain, 787 transport in, 778 Ammonium ion ammonia from, 709, 710f dissociation constant for, 51 Ammonium, urinary, buffering by, 45t, 50 Amnionless, 751 Amniotic fluid, phosphatidylcholine and sphingomyelin in, 621, 621f Amoebiasis, 882t AMP-activated protein kinase (AMPK), 620, 689–690, 691f, 796 AMPK. See AMP-activated protein kinase (AMPK) Amplification of DNA sequences, 295–297 DNA cloning in, 295, 296f libraries in, 295–297 polymerase chain reaction in, 297, 297f of genes, 274 of proto-oncogenes, 313 Amygdalin, 386 Amylase, 586. See also Glucoamylase ␣-, 497–498, 497f, 587 gut activity of, 498 urine activity of, assay of, 586 Amylase inhibitors, 497 Amylin, 814t Amylo-1,6-glucosidase deficiency, 518t Amylo-4,6-glucosidase deficiency, 518t Amyloid deposition, 93 Amyloidosis/AL, 110t Bence-Jones proteins in, 107 characteristics of, 107 pathophysiology of, 101 treatment of, 101, 107–108 Amylopectin dietary sources of, 496 structure of, 493, 494f, 495 Amylose dietary sources of, 496 structure of, 493, 494f, 495 Amyotrophic lateral sclerosis (ALS), 448, 455t Anabolic hormones, 798t Anabolic pathways, 1, 5 Anaerobic glycolysis, 397f, 403–405 acid production in, 403 ATP and energy from, 349, 349f definition of, 335, 335f, 397f

Lieberman_Subject_Index.indd 976

energy yield of, vs. aerobic glycolysis, 403 in exercise as ATP source, 894 from glycogen, 895–896, 895f in high-intensity exercise, 897 at onset, 894 in type IIb fast-twitch glycolytic fibers, 894–895 lactate fate (Cori cycle) in, 404–405, 404f lactic dehydrogenase reaction in, 403, 403f with mitochondria reduction, 524 overview of, 335, 335f on pyruvate, 401, 401f tissues dependent on, 403–404, 404t Analbuminemia, 849 Anandamide (AEA), 673, 674f, 817–818 Anaplerotic reactions, 124f, 371–372, 371f, 372f Andersen disease, 518t Androgen synthesis, 652, 653f, 655, 655f. See also specific androgens Android obesity, 27 Androstenedione synthesis of, 653f, 655, 655f testosterone from, 655, 655f, 656 Anemia, 826, 826t classification of, 826, 826t hemolytic, 795, 827 from erythrocyte cytoskeleton defects, 834 hereditary, 795 hypochromic, 285 iron-deficiency, 394t, 838 diet and, 15 from divalent metal ion transporter 1 gene mutation, 831 fatigue in, 383 in malnutrition, 13 microcytic, hypochromic anemia in, 833 presentation and diagnosis of, 266, 284–285 in women, 832 from lead poisoning, 829, 829f macrocytic, 745 megaloblastic, 13t, 745 alcohol-induced, 745–746, 750, 758t pathophysiology of, 750 treatment of, 750 from vitamin B12 deficiency, 745, 839 nutritional, 838–839 pernicious, 758t pathophysiology of, 751–752 vitamin B12 for, 751–752 sickle cell (See Sickle cell anemia [disease]) Angelman syndrome, 273, 286t Angina (pectoris), 337 definition of, 71 nitroglycerin for, 444 tissue plasminogen activator for, 438 Angiogenesis, endostatins on, 931 Angiotensin II, in aldosterone synthesis, 655 Anhydride synthesis, 57, 58f Animal fat, 417 Anion gap calculation, 458 Anions, 57, 58f Ankyrin, 833–834, 833f Annealing, 292 Anorexia nervosa, 19t, 150t, 189t, 286t, 375t, 580t ANT, 391, 391f Anterior pituitary tumor, 810 Anthropometric measurements, 27–28 Antiactivators, of fibrinolysis, 858 Antiapoptotic signals, by BCL-2 family, 324–325, 325f, 325t

Antibiotics. See also specific agents macrolide, 257 mechanisms of action of, 255, 256, 257 on protein synthesis, 261–262, 261t Antibodies, catalytic, 117 Antibonding electrons, 439 Anticoagulants, 856, 859–860, 860f. See also specific agents direct thrombin inhibitors (hirudin), 860 fondaparinux, 860, 860f heparins, 859 warfarin, 855f, 859–860 Antidepressant. See also specific agents on serotonin, 915 Antidiabetic agents. See also specific agents mechanism of action of, 620 Antihemophilia cofactor, 859 Antihistamines, 916 Antioxidant scavenging enzymes, 448–449, 448f Antioxidants, nonenzymatic, 449–452 ascorbic acid, 449, 450f carotenoids, 449–451, 451f endogenous (melatonin, uric acid), 452, 452f flavonoids, 451–452, 451f vitamin E, 447f, 449, 450, 450f Antiparallel DNA strands, 198, 198f Antiparallel strands, 92, 92f Antiports, 391, 391f Antisense strand, DNA, 230, 230f Antithrombin III (ATIII), 857 Aortic stenosis, supravalvular, 932, 943t APC, 322, 322f Apolipoprotein B-100, in familial combined hyperlipidemia, 608 Apoprotein, 99 functions of, 639, 640t HDL synthesis from, 642 in LDL, glycation of, in diabetes, 689 in lipoprotein, 639, 642, 644, 645 major, characteristics of, 639, 640t Apoprotein(a), 650 Apoprotein B-48, 591, 591f Apoprotein CIII (apoCII), fibrates on, 877 Apoptosis, 322–326 active transport and, 352–353, 352f cancer cell bypass of, 325–326, 325f definition and mechanisms of, 322–323 microRNAs and, 326 mitochondria and, 393 normal pathways to, 323–325 BCL-2 family proapoptotic and antiapoptotic signals in, 324–325, 325f, 325t caspases in, 323, 323f components in, 323, 323f death receptor pathway to, 323, 324f mitochondrial integrity pathway to, 324, 324f in normal processes, 323 regulators of, in signal transduction cascade, 316t Apoptosis-initiating factor (AIF), 393 Apoptosome, 324, 324f Apoptotic bodies, 323 Appetite suppression, 925t Aquaporins, 158 Arachidonic acid, 61, 63f, 176, 176f cleaving of, 618 in eicosanoid metabolism, 663, 664f, 665–666, 665f as essential fatty acid, 607 from linoleic acid, 606–608, 607 in membrane phospholipids, 665–666, 665f metabolism pathways for, 666, 666f

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INDEX

2-Arachidonoylglycerol (2-AG), 817–818 Arcus lipidalis, 657 Arf, 166 Arginine, 11, 694, 694t chemical structure of, 74f, 75t, 76–77 cleavage of, to urea, 714f, 715 degradation of, 716f, 732 dissociation of side chains of, 77, 78f nitric oxide from, 444f, 918, 918f production of, 714f, 715, 715f synthesis of, 716f, 717, 732 for urea cycle disorders, 718 Argininosuccinate deficiency of, 722t enzyme defect after synthesis of, 718 fumarate and arginine from, 714f, 715 in Krebs bi-cycle, 715, 715f production of, 714f, 715 in urea cycle, 714f, 715 Argininosuccinate synthetase, 714f, 715 Argininosuccinate synthetase deficiency, 718, 722t Argonaut, 283 Ariboflavinosis, 13t Arm muscle circumference (AMC), midarm, 27–28 Aromatic amino acids, 74f, 75, 75t Aromatic compounds, 55, 55f Arsenic poisoning, 364, 375t Arsenite, 364 Arteriovenous difference, 777f Artery, layers of, 649, 649f Arthritis, rheumatoid, 934 L-Ascorbate, 449, 450f Ascorbic acid, as free radical scavenger, 449, 450f Asialoglycoprotein receptor, 867 Asparaginase, 730 Asparagine chemical structure of, 74f, 75–76, 75t synthesis and degradation of, 732, 733f tumor cell requirement for, 730 Aspartate, 733 action of, 918 chemical structure and bonds of, 74f, 75t, 76 dissociation of side chains of, 77, 78f synthesis and degradation of, 732, 733f, 918 UMP from, 766–767, 768f Aspartate aminotransferase (AST), 416 Aspartate transaminase (AST), in hepatitis, 711, 713 Aspirin for coronary atherosclerosis, 675 as covalent inhibitor, 127 on cyclo-oxygenase, 675 in heart attack prevention, 670 on prostaglandins, 670, 670f Reye’s syndrome from, 877 on thromboxane A2 synthesis, 670 Aspirin poisoning diagnosis of, 41, 46 metabolic acidosis in, 46 on mitochondria, 390 treatment of, 51 Association constant (Ka), for binding site on protein, 97, 98 Asthma, 580t, 676t corticosteroids for, 675 inflammation in, 666 Astrocytes, 905–906 Atherosclerosis anatomic and biochemical aspects of, 649–650, 649f cholesterol elevation in, 643–644

Lieberman_Subject_Index.indd 977

cholesterol in, 16 plaque evolution in, 16, 650, 650f risk factors for, 649–650 Atherosclerotic plaques, 16 Atorvastatin, 650, 657, 658 ATP-ADP cycle, 336–345, 336f biochemical work in, 342–345 activated intermediates with high-energy bonds in, 344–345 additive ⌬G0 values in, 342–343, 343f, 343t substrate and product concentrations on ⌬G in, 340f, 343–344, 343t energy for work in, 337–340 basic principles of, 337–338 change in Gibbs free energy in, 338–339, 339t ⌬G0 in, 339–340 exothermic and endothermic reactions in, 339–341, 340f, 340t high-energy ATP phosphate bond in, 338, 338f thermodynamics in, 339, 339t energy transformations in for mechanical work, 341, 341f for transport work, 160f, 341–342 ATP–ADP cycle, 4, 4f ATP–ADP translocase, 391–393, 391f ATPase myosin, 341, 341f plasma and vesicular, 341–342 ATP-binding cassette (ABC) protein (ABCA1) cholesterol transport by, 629, 643 mutations in, 643 ATP homeostasis, 345, 350–351 ATP synthase in cellular respiration, 333 in cellular respiration, mitochondrial, 334, 334f in oxidative phosphorylation, 380–381, 380f AT-rich region, as recognition site, 233 Atropine, 174 Attenuation, of transcription, 270–271, 271f AUG start codon, 251, 251f Autocrine actions, of chemical messengers, 174, 175f Automated analyzer, 826 Autonomic neuropathy, 940 Autonomous hormone release, 483 Autophagosome, 163 Autophagy, 163, 702, 702f Autosomal chromosomes, 202 Axon, 905, 905f Azidothymidine (AZT, zidovudine), 196, 196f, 213 Azidothymidine (AZT, zidovudine) toxicity, 389, 393 Azithromycin, 205, 206, 257 Azotemia, with diabetes mellitus, 928 B B-48 apoprotein, 591, 591f Bacteria, 154. See also specific types and infections vs. human cells, 168–169 pathogenic, ADP-ribosyl transferases in, 83f, 84 in urine, 210 Bacterial artificial chromosomes (BACs), 296 Bacterial gene transcription, 233–234, 234f Bacteriophage, 195, 295, 296 Band 4.1, 833–834, 833f Band 4.2, 833–834, 833f B apoprotein gene, 592f

977

Basal factors, 232–233, 233f Basal ganglia, 438 Basal metabolic rate (BMR), 8, 8t, 9t, 344 Basal state, 31, 32f Basal transcription complex, 274, 275f Base, 45, 195, 195f, 195t conjugate, 41, 46 definition of, 41 nitrogenous, 65, 66f Base excision repair, 219, 220f Base pairing elimination of errors in, prokaryote, 212t, 213 in genetic code, 249, 250f, 250t mechanisms of, 196–198, 197f, 198f Base pairs, 194 Basic helix-loop-helix (bHLH), 278f, 279 B blood group, 549, 554f, 555 b-c1 complex, proton motive Q cycle for, 384, 384f B cells, 825 Bcl-2 mutation, 325 Bcl-2 proteins, 324–325, 325f, 325t Bcr-Abl fusion protein, 315, 327 Beans, indigestibility of, 504–505 Bence-Jones proteins, in amyloidosis/AL, 107 Bends, 92, 94f Benzene ring, 55, 55f Benzo[a]pyrene oxidation, 218–219, 218f Benzoate metabolism, 877, 878f Benzoic acid, for urea cycle disorders, 718–719, 719f Beriberi, 13t, 14, 373 Beriberi heart disease, 130, 133t, 373–374 Bernard–Soulier syndrome, 851, 861t ␤1-adrenergic receptors, 489–490 ␤2-adrenergic receptors binding sites in, 95–96, 96f location and functions of, 490 ␤3-adrenergic receptors, 490 ␤-barrel, 100, 102f ␤-catenins, 322, 322f, 329 ␤ cells defect in, in diabetes mellitus type 1, 484 insulin release by, 484, 484f ␤-globin gene compound heterozygote for mutations in, 842 in hemoglobin switching, 843–844 structure and transcriptional regulation of, 841, 841f ␤-glucoamylase, 498–501, 498t, 499f ␤-glycosidase complex, 498t, 500, 500f ␤-hydroxybutyrate, 33, 33f, 779 oxidation of, 428–430, 430f synthesis of, 428, 428f, 429f ␤-interferon for chronic myelogenous leukemia, 328 recombinant, 302 ␤-oxidation of fatty acids, regulation of, 424, 424f of long-chain fatty acids, 420–423 ␤-oxidation spiral in, 420–421, 420f chain length specificity in, 418t, 421 energy yield of, 421, 421f odd-chain-length fatty acids in, 423, 423f unsaturated fatty acids in, 421–423, 422f ␤-(pleated) sheets, 91–92, 92f, 93, 94f ␤-thalassemia, 228, 246t, 263t, 839–840 mutations in, 232, 243, 261, 261t homozygous, in splice-junction sequences, 237 point, at polyadenylation site, 237 pathophysiology of, 232 types of, 243

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978

INDEX

␤-turn, 92, 93f Betaine, 754 Bicarbonate as buffer, 41, 41f in red blood cells, 45t, 48–49, 49f Bicarbonate buffer system, 48, 48f Bicarbonate–carbonic acid buffer system, 47 Bidirectional replication, 210–211, 211f Bile acid resins, 658t Bile salts (bile acids), 22 cholesterol in, 54 conjugation of, 636, 637f enterohepatic recirculation of, 638–639 fate of, 636–639, 637f metabolism of, 636, 637f recycling of, 588–589, 589f resorption of, 588–589 structure of, 587, 587f in primary bile salts, 638f in secondary bile salts, 636–637, 638f synthesis of cholesterol to cholic acid and chenocholic acid, 635–636, 635f conjugation in, 636, 637f location of, 635 in triacylglycerol digestion, 587, 588f Bilirubin, 72, 546, 547f degradation of, 547f, 831f, 832 excess, gallstones from, 842 in hepatitis, 711 in jaundice, 546 in jaundice, neonatal, 548 measurement of, 72 in sickle cell anemia, 589 Bilirubin A, from heme, 831–832, 832f Bilirubin diglucuronide, 546, 547f Binding-change mechanism, 381, 381f Binding domain, 95–96, 96f Binding sites, 73 Biochemical work, 342–345 activated intermediates with high-energy bonds in, 344–345 additive ⌬G0 values in, 342–343, 343f, 343t substrate and product concentrations on ⌬G in, 340f, 343–344, 343t Biochemistry, 39 Bioenergetics, cellular, 336–353 active transport and cell death in, 352–353, 352f ATP-ADP cycle in, 336–345 (See also ATP-ADP cycle) ATP homeostasis in, 345, 350–351 definition of, 336 energy balance in, 350–351, 350f energy from fuel oxidation in, 345–349 (See also Oxidation, fuel) oxidases in, 349, 349f oxygenases in, 349f, 350f, 351 thermogenesis in, 345 Biogenic amines, 175, 175f Bioinformatics, 300 Biologic compounds, 55 Biosynthetic pathways, 1. See also specific pathways Biotin, 123, 124f deficiency of, 13t Recommended Dietary Allowance for, 13t Biradical, oxygen as, 439, 439f 2,3-Bis-phosphoglycerate (2,3-BPG) in erythrocyte metabolism, 826, 827f on oxygen binding to hemoglobin, 834, 834f 1,3-Bis-phosphoglycerate (1,3-BPG), high-energy phosphate bonds in, 345, 345f

Lieberman_Subject_Index.indd 978

Bis-phosphoglycerate shunt, 405–406, 405f Bisphosphonates for osteogenesis imperfecta, 942 structure and function of, 942 Bivalirudin, 860 Blast, 840 Blebs, 323 Blood–brain barrier, 796, 906–908 capillary structure of, 906–907, 907f transport through of amino aids and vitamins, 908 fuels in, 907–908 of glucose, 508–509, 508f receptor-mediated transcytosis in, 908 Blood cells, 824–826. See also specific types anemia and, 826, 826t concentrations of, normal, 824, 824t erythrocytes, 824, 826 leukocytes basophils, 825 classification of, 824 definition of, 824 eosinophils, 825 granulocytes, 824–825 mononuclear, 824, 825 morphonuclear, 824 thrombocytes, 825 Blood coagulation cascade, 851–852, 853f, 853t Blood gas analyzers, 41, 41f Blood groups characteristics of, 549, 554f, 555 glycolipid carbohydrates in, 158 transfusions for, 555–556 Blood group substances genetics of, 555 structures of, 554f, 555 Blood polypeptides, 701 Blood transfusions, 545, 557t Blood typing, 545, 549 Blood urea nitrogen (BUN) in blood and urine, 36 measurement of, 718 B lymphocytes, 285 Body fluids ions and electrolytes in, 44, 44t maintenance of, between tissues and blood, 848 Body mass index (BMI) definition of, 620 evaluation of, 12 formula for, 10 healthy, 10 measurement of, 27 Bohr effect, 834, 834f, 835f Bond. See also specific types carbon–carbon, 55, 56 carbon–nitrogen, 57, 57f carbon–oxygen, 57, 57f carbon–sulfur, 57, 57f chemical, energy for formation of, 342 disulfide, 70, 76, 76f electrostatic, 70, 76, 77f glycosidic, 54, 61, 62f high-energy, 344 (See also Adenosine triphosphate [ATP]; specific types) activated intermediates with, 344–345 phosphate, 338, 338f, 344–345, 345f hydrogen, 75, 75f in amino acids, 70 in water, 43, 43f water–polar molecules, 43, 44f hydrophobic, 75, 75f ionic, 70, 76, 77f

N-glycosidic, 61, 62f O-glycosidic, 61, 62f peptide, 70, 73, 73f, 256f, 257 polar, 57 scissile, 117, 117f, 118 Bone marrow transplantation, quantifying stem cells in, 836 Bradycardia, fetal, 513 Bradykinesia, 438 Brain amino acid utilization by, 786–787, 787f ammonia toxicity on, 787 glucose metabolism in, after a meal, 23f, 25 glucose transport through, 508–509, 508f glutamine metabolism in, 786–787, 787f lipid oxidation in, 920–921 lipid synthesis in, 796, 920–921 purine nucleotide cycle in, 783 Brain tumor, metastatic, 311 Branched-chain amino acids (BCAAs) in brain, 778 degradation of, 735–736, 735f functions of, 779 in gut, 785, 785f in liver, 879 in long-term exercise, 899, 899f metabolism of, 779, 879 in skeletal muscle conversion to glutamine of, 783–784, 783f, 784f glucose-alanine cycle in, 712f, 784–785, 784f glutamine synthesis in, 781 oxidation of, 776, 777f, 782, 783f protein synthesis and degradation in, 777f, 780 BRCA1/BRCA2 mutations, 315, 329–330 Breast cancer, hereditary, 224 Breast milk, human, 587 Breathalyzer test, 136 Brown fat, 389–390, 390f Brush border membrane intestinal disaccharidases, 498–500, 498f, 498t ␤-glycosidase complex in, 498t, 500, 500f glucoamylase in, 498–501, 498t, 499f location of, 498f, 500 sucrase-isomaltase complex in, 498t, 499–501, 499f, 500f trehalase in, 498, 498t, 500, 500f Buffers, 46–47, 47f definition of, 41 metabolic acids and, 47–50 bicarbonate and hemoglobin in red blood cells in, 48–49, 49f bicarbonate buffer system in, 48 hydrochloric acid in, 50 intracellular pH in, 45t, 49–50, 49f principles of, 47 urinary hydrogen, ammonium, and phosphate ions in, 45t, 46f, 50 Burkitt lymphoma, 314, 331t Busulfan, 328 Bypass polymerases, 215 C Ca2⫹, ATPase, 160–161 Ca2⫹ uniporter, 391 Cabergoline, 801 Cadherins, 322, 322f, 329 Caffeine, on fuel metabolism, 487–488 Ca⫹, increased intracellular, 352–353, 352f Calciferols, 656, 657f Calcitonin gene-related peptide (CGRP), 814t

01/09/12 9:36 PM

INDEX

Calcitriol synthesis, 656, 657f Calcium deficiency of, 15 dietary guidelines for, 16 function of, 14 on glycogen metabolism in liver, 521f, 523–524, 523f sarcoplasmic reticulum release of, 887–888, 888f Calcium–calmodulin, 144, 144f, 524, 525f Calcium-induced calcium release, 887 Calcium release channels, in ryanodine receptors, 887 Calculus (calculi). See Stones Calmodulin, 524, 525f Caloric balance, 10 Calorie (cal), 5 definition of, 898 in fuels, 345, 348–349 Calorimetry, indirect, 9 cAMP, 179, 179f, 185 epinephrine and glucagon on, 148 in signal transduction, 488 cAMP phosphodiesterase, 185–186, 186f Cancer, 192, 773t. See also specific types causes of, 310f, 311–312 definition of, 310 development of, 310, 310f doxorubicin for, 198 epidemiology of, 327, 327f on extracellular matrix, 934 gene expression alteration in, 273 mutations in, 310, 310f Cancer molecular biology, 310–331 apoptosis in, 322–326 (See also Apoptosis) differences in, 327 DNA damage in mutations in, 313–315 chemical and physical DNA alterations in, 313, 313f gain-of-function proto-oncogene mutations in, 313–314, 314f, 321 loss-of-function mutations in, 314 repair enzyme mutations in, 314–315 DNA repair mutations in in hereditary breast cancer, 329–330 in hereditary nonpolyposis colorectal cancer, 329 etiology in, 310f, 311–312 multiple mutations in, 326–327, 326f oncogenes in, 315–319 (See also Oncogenes) tumor suppressor genes in, 319–322 (See also Tumor suppressor genes) viruses in, 328 Cannabinoid antagonists, 818–819 Cannabinoid receptor 1 (CB1), 817–818 Cannabinoid receptor 2 (CB2), 817 Cap-binding complex, 254 Capillary beds, 906 Capping, mRNA transcript, 235–236, 236f Cap proteins, 703, 703f Carbamoyl phosphate synthesis of, 714, 714f UMP from, 766–767, 768f Carbamoyl phosphate synthetase I (CPSI) activation of, 716, 716f in pyrimidine nucleotide synthesis, 766, 767t in urea cycle, 711, 714, 714f, 716, 716f, 718 Carbamoyl phosphate synthetase I (CPSI) deficiency, 722t Carbamoyl phosphate synthetase II (CPSII) activation of, 714 in pyrimidine nucleotide synthesis, 766, 767f, 767t, 768

Lieberman_Subject_Index.indd 979

Carbohydrate. See also Glycogen absorption of, 22 caloric content of, 5, 5t classification of, 54 dietary, 11 absorption of, 22 digestion of, 22, 496–502 (See also Carbohydrate digestion) structure and sources of, 494f, 496 in fed state, 21, 21f, 22 indigestible, 501, 501f meal high in, on glucokinase, 138–139 oxidation of, 4–5, 4f structure of, 5, 5f, 54, 58–61 in glycosides, 61, 62f in monosaccharides (See Monosaccharide) structures of, 493, 494f Carbohydrate and lipid metabolism regulation, in fasting state, 683–686 adipose tissue lipolysis in, 685, 685f hepatic blood glucose maintenance in, 683–685, 683f, 684f hepatic ketone body production in, 682f, 685–686, 686t of liver enzymes, 688t muscle glucose and fatty acid use in, 680f, 686 overview and flowchart of, 687–698, 687f, 688t Carbohydrate and lipid metabolism regulation, in fed state, 679–683 chylomicron and VLDL fate in, 682 hepatic glycogen and triacylglycerol synthesis in, 679–682 acetyl-CoA carboxylase in, 680–682, 681f, 682f citrate lyase and malic enzyme in, 680, 681f fatty acid synthase complex in, 681f, 682 glucokinase in, 679–680, 679f glucose-6-phosphate dehydrogenase in, 680, 681f glycogen synthase in, 680, 680f malonyl-CoA in, 681–682, 682f overview of, 679 pyruvate dehydrogenase and pyruvate carboxylase in, 680, 680f of liver enzymes, 688t overview and flowchart of, 687–698, 687f, 688t triacylglycerol storage in adipose tissue in, 682–683 Carbohydrate digestion, 22, 473, 473f, 493, 495f, 496–502 ␣-amylase in, 497–498, 497f fiber and, 502–504, 503t glycosidases in, 496–497 of indigestible carbohydrates, 501, 501f intestinal brush border membrane disaccharidases in, 498–500, 498t ␤-glycosidase complex in, 498t, 500, 500f glucoamylase in, 498–501, 498t, 499f location of, 498f, 500 sucrase-isomaltase complex in, 498t, 499–501, 499f, 500f trehalase in, 498, 498t, 500, 500f lactose intolerance in, 502, 502t, 503f, 509, 511t overview of, 473, 473f, 493, 495f sugar absorption in, 504–508 (See also Sugar absorption) sugar metabolism in, by colonic bacteria, 501–502, 501f Carbohydrate malabsorption, 496

979

Carbohydrate metabolism digestion in (See Carbohydrate digestion) fructose and galactose metabolism in, 473, 474f glucagon release–regulated pathways in, 476, 476f glucose homeostasis in, 475–476 glucose metabolism pathways in, 475–476, 476f glucose to amino acids and triacylglycerol moieties in, 474, 474f glucose to lactate and CO2 in, 474, 474f glycogen synthesis in (See Glycogen synthesis [glycogenesis]) in liver, 874, 874t overview of, 473–476, 473f–476f pentose phosphate pathway in, 473, 473f, 474f TCA cycle in, 473–474, 474f UDP-glucose products in, 475, 475f Carbohydrate response element-binding protein (ChREBP), 541 Carbon, 55 Carbon–carbon bonds, 55, 56 Carbon–carbon groups, 55, 56 Carbon dioxide (CO2) as acid source, 41 from fuel oxidation, 3, 4, 4f on oxygen binding to hemoglobin, 834–835, 834f, 835f Carbon–hydrogen groups, 55 Carbonic acid, 48, 48f in bicarbonate–carbonic acid buffer system, 47 from fuel metabolism, 778 Carbonic anhydrase, 48–49, 49f Carbon–nitrogen bonds, 57, 57f Carbon–nitrogen groups, 56f Carbon–oxygen bonds, 57, 57f Carbon–oxygen groups, 56, 56f Carbon–sulfur bonds, 57, 57f Carbon–sulfur groups, 56f Carbon tetrachloride (CCl4), 441 Carbonyl group, 55 Carboxylases, 124f, 133 Carboxylate, 56 Carboxylate group, 56–57, 57f Carboxylations, 258 Carboxylic acid, 56f Carboxypeptidase A, 698f, 699, 699f Carboxypeptidase B, 698f, 699, 699f Carboxypeptidases, 699f Carcinogens, 218, 219, 312, 313, 314f Cardiac ischemia cardiac muscle fuel use in, 891 pathophysiology of, 393, 453 Cardiac muscle cells, 885f, 887 Cardiac protection, 676t Cardiolipin, 379 Cardiomyopathy from protein phospholamban mutation, 901 from thiamine deficiency, 373 Caries, dental, 403, 411, 411f, 413t Carnitine deficiency of, 420 as exercise supplement, 420 fatty acyl, 419, 419f function of, 419 inherited diseases in metabolism of, 419 sources of, 419 Carnitine acylcarnitine translocase deficiency, 419 Carnitine acyltransferase I (CATI), 419, 419f inhibition of, 605, 606f malonyl-CoA on, 681–682, 682f

01/09/12 9:36 PM

980

INDEX

Carnitine deficiency, 435t Carnitinepalmitoyl transferase I (CPTI), 419, 419f inhibition of, 605, 606f malonyl-CoA on, 681–682, 682f Carotenoid as free radical scavengers, 449–451, 451f macular, 451, 451f Caspase, 145, 323, 323f Caspase-activated DNase (CAD), 393 Cassava, 386 Catabolic pathways, 1, 5 Catabolic states, 792t Catabolite activator protein (CAP), 269–270, 270f Catalase, 440f, 448, 448f Catalysis. See also specific processes and substances acid–base, 119–120 covalent, 120 enzyme, 112, 113f functional groups in, 121–126 on amino acid side chains, 121, 121t coenzymes in, 121–126 metal ions in, 122f, 126, 126f noncatalytic roles of cofactors in, 126 nucleophilic, 118f–119f, 119 Catalytic antibodies, 117 Catalytic sites, active, 112 Catalytic triad, 119 Cataracts in diabetes, 533 free radical protein damage in, 443 Catechol, 489f Catecholamines, 910–913. See also Dopamine; Epinephrine; Norepinephrine biochemical cascade of, 914, 923 on fuel metabolism, 798t, 805–807, 806f (See also specific hormones) inactivation and degradation of, 912–913, 912f measurements of, 439 metabolism and inactivation of, 806 in pheochromocytoma, 923 physiologic actions of, 805–806, 806f storage and release of, 911–912, 911f synthesis of, 805, 910f, 911 tyrosine hydroxylase regulation in, 913 Catechol-O-methyltransferase (COMT), 912–913, 912f Catenins, 322, 322f Cathepsins, 162, 696, 701t, 702 CCDs, 67–68, 68f Cdk4 mutation, 329 cDNA library, 295–296 Cell adhesion, tumor suppressor genes on, 322, 322f Cell aging, telomeres in, 218 Cell biology, 152–169. See also specific components compartmentation in, 153–154, 153f components in, 153f cytoskeleton in, 165–167 actin filaments in, 166, 167f general structure of, 165 intermediate filaments in, 165, 167, 168f microtubules in, 165–166, 165f endoplasmic reticulum in, 164–165, 165f Golgi complex in, 165 lysosomes in, 161–163, 162f mitochondria in, 163–164, 163f nucleus in, 164, 164f organelles in, 154 peroxisomes in, 164 plasma membrane in, 154–161 (See also Plasma membrane) prokaryotes vs. eukaryotes in, 154

Lieberman_Subject_Index.indd 980

Cell cycle eukaryotic, 214–215, 215f G1/S transition in, 318, 318f oncogenes and, 215f, 317–319, 317f regulators of, in signal transduction cascade, 316t Cell cycling, frequency of, 215 Cell death active transport and, 352–353, 352f with hypoxia, on ATP generation, 338 programmed (See Apoptosis) Cell, human vs. bacteria, 168–169 Cell signaling, 171–189 chemical messengers in, 172–176 eicosanoids in, 64f, 176, 176f endocrine, paracrine, and autocrine actions of, 174, 175f in endocrine system, 82f, 175 general features of, 171f, 172–173 growth factors in, 176 in immune system, 175–176 in nervous system, 175, 175f in nicotinic acetylcholine receptor, 173–174, 173f, 174f guanylyl cyclase receptors in, 188, 188f intracellular transcription factor receptors in, 176–178 intracellular vs. plasma membrane receptors in, 176–177, 176f steroid hormone/thyroid hormone superfamily in, 176f, 177–178 plasma membrane receptors in, 178–179 signal termination in, 187, 187f signal transduction in, 179–187 changes in response to signals in, 187 by cytokine receptors (JAK-STAT proteins), 179f, 182–183, 183f heptahelical receptors in, 179, 179f, 184–187 adenylyl cyclase and cAMP phosphodiesterase in, 185–186, 186f heterotrimeric G-proteins in, 184, 185f, 185t names and general properties of, 96f, 179f, 184 phosphatidylinositol signaling by, 173f, 186–187 receptor serine–threonine kinases in, 179f, 183–184, 183f tyrosine kinase receptors in, 179–182, 179f (See also Tyrosine kinase receptors) Cellular bioenergetics. See Bioenergetics, cellular Cellular respiration, 333–334, 333f, 334f Cellulose, glycosidic bond hydrolysis in, 499–500 Central dogma, 207 Central nervous system demyelinating diseases of, 924 liver failure on, 716 Ceramide, 64, 64f derivatives of, 64, 64f sphingolipid synthesis from, 618, 619f structure of, 598, 598f synthesis of, 618, 618f Ceramide lactoside lipidosis, 554t c-erbB-2 gene, 315 Cerebrohepatorenal syndrome, 164, 425, 435t, 616, 876, 882t Cerebrosides, 922 structure and function of, 552, 554f synthesis of, 552

cGMP receptors, 188, 188f Chaperones, 258 Chaperonins, 104 Charcot–Marie–Tooth polyneuropathy syndrome, 924 Charges, partial, 57, 57f “Cheese” effect, 913 Cheilosis, 373 Chemical bonds. See Bond Chemical groups, 54. See also specific groups Chemical messengers, 172–176 eicosanoids in, 64f, 176, 176f endocrine, paracrine, and autocrine actions of, 174, 175f in endocrine system, 82f, 175 general features of, 171f, 172–173 growth factors in, 176 in immune system, 175–176 in nervous system, 175, 175f in nicotinic acetylcholine receptor, 173–174, 173f, 174f Chemical uncouplers, 389, 389f Chemiosmotic theory, 383–384 Chemokines, 175–176 Chemotherapy, 772. See also specific agents Chenocholic acid series, 636, 636f Chenodeoxycholic acid series, 636, 636f Chloramphenicol, 257 on protein synthesis, 261t, 262 Chloride dietary, in hypertension, 16 function of, 14 Chloride channel defect, in cystic fibrosis, 699 Chlorinated aromatic hydrocarbons, in environmental toxins, 67–68, 68f Chlorinated dibenzo-p-dioxins (CDDs), 67–68, 68f Cholecalciferol synthesis, 656, 657f Cholecystokinin (CCK), 815t Cholera, 154, 169t, 189t, 510, 511t AB toxin cell surface binding sites in, 158 Arf in, 166 bacteria and pathogenesis of, 154 CFTR channel activation in, 186 cholera toxin A on, 172, 186 hypovolemic shock treatment for, 172 pathophysiology of, 166 symptoms and diagnosis of, 153 treatment of, 188 Vibrio cholerae toxin in, 168 watery diarrhea and dehydration in, 158, 160, 166, 168, 186 Cholestasis, in sickle cell anemia, 589 Cholesterol. See also specific types absorption of, 623–624, 623f, 629 in atherosclerotic plaques, 16 chemistry of, 54 dietary, 16 guidelines for, 16 on hepatic synthesis of, 635 elevated, in atherosclerosis, 643–644 fates of, 634–635, 635f function of, 583 “good,” 650 high (See Hypercholesterolemia) liver pool of, 634–635 metabolism of, 583, 583f in plasma membrane, 156, 156f receptor-mediated endocytosis of, 645–646, 645f serum, enzymatic tests for, 600 solubility of, 66 steroid hormone synthesis from, 652, 653f

01/09/12 9:36 PM

INDEX

structure of, 64, 65f, 629–630, 629f, 630f synthesis and export of, in liver, 871 tissue use of, 634–635 Cholesterol ester transfer protein (CETP) reaction, 644f, 645, 645f, 650 Cholesterol synthesis, 629–634 ezetimibe on, 629–630 in liver, 871 stage 1: acetyl-CoA to mevalonate, 630–632, 630f, 631f stage 2: mevalonate to two activate isoprenes, 632, 633f stage 3: six activated five-carbon isoprenes to 30-carbon squalene, 632, 634f stage 4: squalene to four-ring steroid nucleus, 624f, 633–634 structure in, 629–630, 629f, 630f Cholesterol transport blood lipoproteins in, 639–645 apoproteins in, 639, 640t, 645 chylomicrons in, 593f, 639–641 high-density lipoproteins in, 640t, 642–645, 643f, 644f intermediate- and low-density lipoproteins in, 642 lipoprotein structure in, 590f, 639 mechanism of, 639 very low-density lipoproteins in, 640t, 641, 641f in liver, 871 reverse, 642–644, 643f Cholic acid solubility, 66 Choline on acetylcholine synthesis, 917 chemical structure of, 57, 57f, 58f deficiency of, 13t, 614 as essential nutrient, 614 one-carbon metabolism and, 755, 756f Recommended Dietary Allowance for, 13t sources of, 915–916 synthesis of, 614, 915–916 Choline acetyltransferase (ChAT), 915, 916f Chondrocytes, proteoglycans in, 939, 940f Chondroitin sulfate synthesis, 934–936, 937f, 938f Choriocapillaris epithelium, oxidative damage to, 451 ChREBP, 541 Chromagranins, 911, 911f Chromatin, 164, 164f, 194, 201, 201f, 272 Chromatin remodeling, 272–273, 273f Chromogenic assay, of factor VII activity, 848 Chromosomes autosomal, 202 homologous, 79, 202, 203f replication of ends of, 217–218, 217f, 218f structure and organization of, 79, 201–203 DNA molecule size in, 201 DNA packaging in, 201, 201f, 202f genes in, 203, 203f human genome in, 201–203, 202f translocations in, 221, 222, 222f Chromosome walking, 299 Chronic granulomatous disease, 446, 455t Chronic myelogenous leukemia (CML), 283, 285, 286t, 331t Bcl-2 mutation in, 325 karyotyping of, 311 pathophysiology of, 311 Philadelphia chromosome in, 274, 284, 311, 315 presentation and diagnosis of, 266, 311 treatment for, 327, 328

Lieberman_Subject_Index.indd 981

Chronic obstructive pulmonary disease (COPD), 397, 404, 411, 413t Chylomicrons, 21, 26, 584t, 593f, 639–641 characteristics of, 640t composition of, 590–591, 591f density of, 592 fate of, 592–593, 593f function of, 583 regulation of fate of, in fed state, 682 remnants of, 593 structure of, 590–591, 591f synthesis of, 583, 590–591, 590f, 591f Chymotrypsin, 75, 698, 698f, 699f actions of, 117, 117f catalytic mechanism of, 117–121 energy diagram with chymotrypsin in, 120–121, 120f fundamentals of, 117, 117f reaction in absence of enzyme in, 117 strategies in, 117–120 acyl-chymotrypsin intermediate hydrolysis in, 118f–119f, 120 acyl-enzyme intermediate in, 118f–119f, 119–120 specificity in, 118f–119f, 119 stages of, 117–119, 118f–119f catalytic mechanisms of, 117–121 definition of, 117 Chymotrypsinogen, 145, 697, 697f, 698f Cigarette smoking carcinogens in, 218–219 epidemiology of, 327 Ciprofloxacin, 212 Cirrhosis, 880, 882t alcohol-induced (Laennec), 467, 468 blood glucose and NEFA levels in, 880 hepatogenous diabetes in, 880–881 pathophysiology of, 879 presentation of, 864 amino acid metabolism in, 879–880 glucose levels in, 880 hepatic, 467, 468 insulin resistance in, 880–881 lipid metabolism in, 877–878 micronodular, 879 nonesterified fatty acids in, 880 portal hypertension from, 872 Cis-acting, 230 Cis-trans isomerase, 104 Cistron, 232, 232f in bacterial gene transcription, 233–234 definition of, 233 Citrate fate, in cytosol, 601, 601f Citrate lyase, 680, 681f Citrate synthase, 366f, 367, 367t Citric acid cycle. See Tricarboxylic acid (TCA) cycle Citrulline, 785, 785f Clarithromycin, 257 Clarke electrode, 42 Clathrin, 646 Clofibrate, 877 Clonal expansion, 312 Cloning DNA, 295, 296f positional, 299 Clorgyline, 913 Clostridium perfringens, 155 Clotting, 796 c-myc, 313, 314 CO2-releasing enzymes, in TCA cycle, 358f, 359–360 Coactivators, 233

981

Coagulation, blood, 852–856 activation of, 852 factor complexes in, 854–855, 855f fibrin cross-linking in, 854, 854f intrinsic and extrinsic pathways of, 852–854, 853f pathophysiology of, 854 proteins of, 853t vitamin K derivatives and, 854, 855f vitamin K requirement for, 855f, 856 Coagulation cascade, 795–796, 851–852, 853f, 853t CoASH, 361–362, 361f Cobalamin. See Vitamin B12 Cockayne syndrome, 224, 225t Coding strand, DNA, 230, 230f Codons, 230, 230f, 249, 250t degeneracy of genetic code for, 250, 250t wobble hypothesis and, 250, 250f Coenzyme, 114, 121–126 activation-transfer, 122–123, 122f, 124f binding on catalytic power of, 123 definition of, 121 oxidation-reduction, 123–126, 125f vitamins as, 121 Coenzyme A (CoA), 123, 124f Coenzyme Q (CoQ) in electron-transport chain, 382–383, 382f as exercise supplement, 420 superoxide from, 441, 441f Cofactors in amino acid metabolism, 726 noncatalytic roles of, 126 proteins in, 851 Coils, 93 Colchicine for gout, 153, 166–167 therapeutic index of, 167 on tubulin synthesis, 166, 167 Colic, renal, 701 Collagen, 928–932 cross-link formation in, 929, 930f hydroxylation in, 929, 929f procollagen(I) in, 928–929 secretion of, 932 structure of, 928, 928f synthesis defects in, 942 synthesis of, 928–929, 929f, 931–932, 932t type I-III, 928, 930t, 931, 931f type IV, 930, 930t, 931, 931f types of, 929–932, 930t Colon cancer (adenocarcinoma), 194, 758t 5-FU for, 204, 204f hereditary, mutations in, 322 steps in development of, 326–327, 326f Colonocytes, 785 Colony-stimulating factors (CSFs), 301–302 Coma diabetic ketone bodies in, 48 mechanisms of, 44 smell of, 563 nonketotic hyperosmolar, 480 Community-acquired pneumonia, 194 Compartmentation cell, 153–154 of free radical defenses, 447–448, 447f as oxygen toxicity defense, 447–448, 447f substrate channeling through, 147 Competitive inhibition, 139, 140f Complementary DNA, 223 Complete blood count (CBC), 826 Complex glycerol kinase deficiency, 608

01/09/12 9:36 PM

982

INDEX

Compounds, structures of, 54–68. See also specific compounds carbohydrates in, 54, 58–61 chlorinated aromatic hydrocarbon environmental toxins in, 67–68 free radicals in, 54, 66 functional groups in, 54, 55–57 lipids in, 54, 61–64 nitrogen-containing, 54, 64–66 Conditionally essential nutrients, 10 Conformational changes, enzyme regulation by, 140–145 allosteric enzymes in, 140–142, 141f covalent modification in, 142–143, 142f, 143f protein–protein interactions in, 143–145, 143f–145f proteolytic cleavage in, 145 Congenital adrenal hyperplasia (CAH), 655, 661t Congenital methemoglobinemia, 828, 845t Congenital muscular dystrophy (CMD), 934 Congestive heart failure, 351 with alcoholism, 375t with earlier heart attack, 337 hypoxia on ATP generation in, 338 left ventricular, 351–352 presentation and diagnosis of, 337, 351 treatment of, 352 Conjugate acid, 45 Conjugate base, 41, 46 Connective tissue, 796 Consensus sequence, 231, 231f, 237, 237f Constant (C) regions, immunoglobulin, 100–101, 103f Constants, thermodynamic, 339t Contraction, muscle, 888, 889f Contrainsular effect, 819 Cooperativity of hemoglobin, 108–110, 109f of O2 binding in hemoglobin, 100, 101f, 108–110, 109f positive, 100, 101f in substrate binding to allosteric enzymes, 140, 141f Copper (Cu⫹), in electron-transport chain, 382f, 383 CoQ10, 382, 382f Corepressor, 269, 269f Cori cycle, 404–405, 404f, 579, 579f Coronary artery disease risk factors, diabetes in, 689 Coronary heart disease risk factors, 627 Corrin ring, 750, 751f Cortical skeleton, 156f, 157 Corticosteroids. See also specific types for inflammation, 675 Corticotropin-releasing hormone (CRH), 807 Cortisol, 178, 564 on fuel metabolism, 798t, 806–808 biochemistry of, 806–807 effects of, 807–808, 808t secretion of, 798t, 807, 807f, 808f, 808t functions of, 819 on gluconeogenesis, 178 physiologic actions of, 481, 482f, 482t on signal transduction, 489 structure of, 177f synthesis of, 652–655, 653f, 654f Corynebacterium diphtheriae, 258 Cosmids, 295 Cotransporters, 160 Counseling, genetic, 303 Counterregulation, of opposing pathways, 147

Lieberman_Subject_Index.indd 982

Counterregulatory hormones, 795, 798t. See also specific hormones on fuel metabolism, 817 insulin, on fuel metabolism, 477, 478f, 481, 482t Coupling, regulation through, 388, 388f Covalent catalysis, 120 Covalent inhibitors, 127, 128f Covalent modification, conformational changes from, 142–143, 142f, 143f COX-1, 669–670, 671t COX-1 inhibitors, 670 COX-2, 669–670 glucocorticoids on, 666 properties of, 670, 671t COX-2 inhibitors, 670, 671 COX-2 inhibitors, selective, 670 C-peptide measurement, for diabetes, 486 CPTI deficiency, 419 CPTII deficiency, 419 C-reactive protein, 790 Creatine creatine phosphate from, 893, 893f synthesis of in kidney, 892–893, 892f in liver, 871t, 892–893, 892f Creatine kinase (CK) during/after myocardial infarction, 81, 85–86, 86f, 99 primary structure variation in, 70–71 Creatine phosphate high-energy phosphate bonds in, 345, 345f in skeletal muscle, 892–893, 892f, 893f Creatinine, 31, 893, 893f in blood, 36 glomerular filtration rate on, 900 in protein malnourished state, 36 in urine, 36, 693, 694t Creatinine–height index (CHI), 36 Creatinine phosphokinase (CK, CPK) from brain and muscle cells, 893 reaction of, 892, 892f serum, in stroke and heart attack, 893 Creutzfeldt-Jakob disease acquired, 105–106, 106f familial, 106 prion protein in, 157 Cristae, 163, 163f, 334f Critical micelle concentration (CMC), 588 Crossing over, 221 Cross-talk, hormone, 186 CRTAP mutation, 942 Cubilin, 751 Curare, 174 Cushing disease, 808, 811, 819, 821t Cushing syndrome, 808, 811, 821t Cyanide from nitroprusside, 385 poisoning with, 386, 394t Cyanoglycosides, 386 Cyanosis, at birth, 513 Cyclic adenosine monophosphate (cAMP), 179, 179f, 185 epinephrine and glucagon on, 148 in signal transduction, 488 Cyclic AMP response element–binding protein (CREB), 186, 279, 488 in gluconeogenesis, 684–685 phosphorylated, 280, 280f, 913 Cyclin on activators, 317 in oncogenesis, 317–319, 318f synthesis of, by cell cycle phase, 317, 317f Cyclin D, 318, 318f

Cyclin-dependent kinase inhibitors (CKIs) on cyclin-CDK activity, 317, 317f in oncogenesis, 317–319, 317f, 318f Cyclin-dependent kinases (CDKs) on activators, 317 in oncogenesis, 317–319, 318f Cyclo-oxygenase pathway, 666–670, 666f biosynthesis in, 667–670, 669f inactivation in, 670 prostaglandin structure in, 666, 666f–668f thromboxane structure in, 667, 668f Cyclophosphamide, 266, 328 CYP11B1, 652, 653f, 654, 655 CYP11B1 deficiency, 655 CYPE2E1 on ethanol, 461, 461f, 462 induction of, 462 Cystathionase deficiency, 730, 734 Cystathionine ␤ synthase, 730 Cystathionine ␤ synthase deficiency, 730, 734, 734f, 740 Cystathioninemia, 740 Cystathioninuria, 730, 740, 741t, 742t Cysteine, 11 chemical structure of, 74f, 75t, 76, 76f dissociation of side chains of, 77, 78f from intermediates of glycolysis, 729–730, 729f structure of, 70 sulfur in, 74f, 75t, 76 Cysteine proteases, 162 Cystic fibrosis, 160, 289, 308t, 706t carriers of, 301 CF gene base deletions in, 296 chloride channel defect in, 699 DNA sequencing in testing for, 300 DNA sequencing of, 295 epidemiology of, 306 genetics of, 306 mutations in, pathophysiology of, 306 presentation and diagnosis of, 289 small bowel obstruction in, 289 Cystic fibrosis transmembrane conductance regulator (CFTR), 158–160, 159f, 289 Cystic fibrosis transmembrane conductance regulator (CFTR) channel, 186 Cystine stones, 76, 730 Cystine transport defect, 701 Cystinosis, 730, 742t Cystinuria, 76, 87t, 706t, 730 cystine transport defect in, 701 definition and pathophysiology of, 85 hyperaminoaciduria without hyperaminoacidemia in, 700–701 pathophysiology of, 700, 701, 704 treatment of, 85 Cytidine triphosphate (CTP), high-energy phosphate bonds in, 344–345 Cytochrome a3, heme in, 382f, 383, 383f Cytochrome a, heme in, 382f, 383, 383f Cytochrome b5 reductase deficiency, 828 Cytochrome c, 324, 324f Cytochrome, in electron-transport chain, 382f, 383, 383f Cytochrome oxidase, 98, 382f, 383 Cytochrome P450–dependent mono-oxygenase system, 868, 868f Cytochrome P450 enzymes, 868, 868f. See also specific enzymes free radicals from, 441 in microsomal ethanol oxidizing system excess alcohol consumption on, 463 induction of, 462 structure and function of, 459, 461, 461f

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INDEX

Cytochrome P450 monooxygenases, 350, 350f Cytochrome P450 pathway, 666f, 673, 673f Cytochrome P450 system on acetaminophen, 869–870, 870f on aflatoxin B1, 869 on vinyl chloride, 869, 869f xenobiotic metabolism by, 867–868, 868f Cytokeratin filament, 168f Cytokine, 175 on hematopoiesis, 836–838, 837f in inflammatory process, 676 in sepsis, 790–791, 791f on stem cells, 285 Cytokine receptor signal transduction, 179f, 182–183, 183f Cytoplasmic dyneins, 165f, 166 Cytosine deamination of, 221 structure and nucleosides of, 195, 195f Cytoskeleton, 165–167 actin filaments in, 166, 167f general structure of, 165 intermediate filaments in, 165, 167, 168f microtubules in, 165–166, 165f D Daily energy expenditure (DEE), 8–10 basal metabolic rate in, 8, 8t, 9t calculations of, 8, 9t, 10 definition of, 8 diet-induced thermogenesis in, 9–10 healthy body weight in, 10 metabolism and inactivation of, 806 physical activity in, 9, 9t resting metabolic rate in, 8 weight gain and loss in, 10 D-amino acids, 72, 72f Dasatinib, 327 DAX1, 608 DDT, 67, 68f Death cell active transport and, 352–353, 352f with hypoxia, on ATP generation, 338 programmed (See Apoptosis) telomeres in, 218 by starvation, 35 Death receptor pathway, 323, 324f Death receptors, 323, 324f Debrancher enzyme, 517, 517f Decarboxylases, 133 Dedicated protein kinases, 142–143, 143f Degeneracy, genetic code, 250, 250t Dehydratases, 133 Dehydration, 51–52, 52f Dehydroepiandrosterone (DHEA), 653f, 655, 655f, 656 Dehydroepiandrosterone sulfate (DHEAS), 655–657 Dehydrogenases, 125 Deletion, 252, 274. See also specific types Deletion mutation, 251t, 252 ␦0␤0-thalassemia, 840 ␦-aminolevulinic acid (␦-ALA) porphobilinogen from, 828, 830f synthesis of, 828, 829f ␦-aminolevulinic acid (␦-ALA) dehydratase, 829, 829f ␦-aminolevulinic acid (␦-ALA) synthase, 828, 829f ⌬G, 339t additive values of, 342–343, 343f, 343t vs. ⌬G0, 343t, 344

Lieberman_Subject_Index.indd 983

in energy for work, 338–339, 339t general expression for, 340, 340t substrate and product concentrations on, 340f, 343–344, 343t

⌬G0 additive values of, 342–343, 343f, 343t vs. ⌬G, 343t, 344 in energy for work, 339–340 Dementia, acetylcholinesterase inhibitors for, 888 Demyelinating diseases, central nervous system, 924 Denaturation DNA, 200, 200f protein misfolding and prions in, 105–106, 106f nonenzymatic modification in, 105, 105f temperature, pH, and solvents in, 105 protein folding and, 105–106, 106f of RNA, 200, 200f Dendrite, 905, 905f Dental caries, 403, 411, 411f, 413t Deoxycholic acid, 638, 638f Deoxycorticosterone (DOC) excessive production of, 655 synthesis of, 653f, 654 Deoxyribonucleic acid. See DNA Deoxyribose, 195, 195f Deoxythymidine monophosphate (dTMP) synthesis, 747, 748, 749f, 750f Depolarization-induced calcium release, 887 Deprenyl, 913 Depression, 19t, 925t dehydration with, 52 from malnutrition, 31 SAMe for, 754 Dermatan sulfate, 934, 935f Dermatitis, seborrheic, 373 Destabilization, of developing product, 120 Detoxification pathways, 1 Dexamethasone, on glucose, 570 Dextran, 411, 411f Dextransucrase, 411, 411f Dextrins ␣-, 497, 497f limit, 497–498 Dextrose, 59 Diabetes mellitus (DM) as coronary artery disease risk factor, 689 diabetic nephropathy in, 941–942, 941f glucose levels in, 563 glucose transport in, 159 from growth hormone–secreting tumor, 803, 819 hepatogenous, 880–881 ketoacidosis in, 686 LDL apoprotein glycation in, 689 lipolysis in, 685 macrovascular complications of, 480 maturity-onset diabetes of the young, 485, 492t metathyroid, 813 microvascular complications of, 480, 578–579, 940–942, 941f neonatal, 485, 492t pathophysiology of, 563 sepsis on insulin resistance in, 791 triacylglycerols in, 611 urinary tract infection with, 223 Diabetes mellitus type 1, 53t, 68t, 87t, 110t, 435t, 492t coma in, 44 dehydration in, 52 diagnosis of, 41, 50 glycogen synthesis in skeletal muscles in, 522

983

glycosylated hemoglobin in, 105, 108 HLA defect in, 484 hyperglycemia mechanisms in, 182 insulin levels in, blood, 486 ketoacidosis in, 41, 55, 57, 416, 431, 433 Kussmaul breathing in, 49, 51 lipoprotein lipase in, 682 mechanisms of, 682 osmotic diuresis in, 44 partial pressure of CO2 in, 49 pH in, blood, 49 polyuria in, 44 rehydration in, 44 treatment of, 50–51 vs. type 2, 490 Diabetes mellitus type 2, 110t, 492t, 511t, 691t, 943t diagnosis of, 24 glucokinase activators for, 875 glycogen storage and, 522 insulin levels in, blood, 486 nonketotic hyperosmolar coma in, 480 obesity in, 490 vs. type 1, 490 waist–hip ratio in, 490 Diabetic coma ketone bodies in, 48 mechanisms of, 44 smell of, 563 Diabetic ketoacidosis (DKA) comatose patients in, smell of, 563 ketone measurement for, 562 overview of, 580t, 691t pathophysiology of, 42, 50, 55, 686 presentation and diagnosis of, 48, 67, 416, 431, 433, 679 Diabetic nephropathy, 579, 940–942, 941f Diabetic neuropathy, 579 Diabetic retinopathy, 579, 940 Dichloro-diphenyl-trichloroethane (DDT), 67, 68f Didanosine (ddI), 213, 295 Dideoxynucleosides, 213 Dietary deficiency, 121. See also specific substances and disorders Dietary fuels. See Fuel, dietary; specific fuels Dietary guidelines, 15–17 for alcohol, 16 for fats, 16 general recommendations in, 15 for proteins, 16 for vegetables, fruits and grains, 16 for vitamins and minerals, 16–17 Dietary Reference Intakes (DRIs), 18–19. See also specific substances Dietary requirements, 10–15. See also specific substances adequate intake in, 10 carbohydrates in, 11 conditionally essential nutrients in, 10 essential nutrients in, 10 fatty acids in, essential, 11 minerals in, 14–15, 15t protein in essential amino acids in, 11 nitrogen balance of, 12, 12t quantity and quality of, 11 Recommended Dietary Allowance in definition and overview of, 10–11, 18 for fiber, 503–504 for folate, 13t, 746 for protein, 704 for vitamins, 12–14, 13t–14t vitamins in, 12–14, 13t–14t water in, 15

01/09/12 9:36 PM

984

INDEX

Diet-induced thermogenesis (DIT), 9–10 Diet, ketogenic for epileptic seizures, 429 for pyruvate dehydrogenase deficiency, 429 Differentiation of adipocytes, 280 regulation of gene expression for, 266 Diffuse large B-cell lymphoma, 283 Diffuse toxic goiter. See Hyperthyroidism Diffusion facilitative, 158, 158f, 159f simple, 157–158, 158f Digestion and absorption of carbohydrates, 21, 21f, 22 of fats, 22–23 of proteins, 6f, 21, 21f, 22, 23f Digitalis, 352 Dihydrofolate reductase (DHR), 274 in dTMP to dUMP reaction, 748, 750f for folate reduction, 746, 746f, 749 methotrexate on, 750, 750f Dihydrolipoyl dehydrogenase, 364 Dihydropteridine reductase (DHPR) deficiency, 737, 738f Dihydropyridine receptor (DHPR), 888f Dihydroxide synthesis, 666f, 673, 673f Dihydroxyacetone phosphate (DHAP), 564, 564f Diisopropylphosphofluoridate (DFP), as covalent inhibitor, 127, 128f Dilated cardiomyopathy, from protein phospholamban mutation, 901 Dinitrophenol (DNP), 389, 389f Diols, in hydroxyeicosatetraenoics, 673, Dioxins, 67–68, 68f Dioxygenases, 349f, 350 Dipeptidyl protease 4 (DPP-4), 814, 816 Dipeptidyl protease 4 (DPP-4) inhibitors, 816, 817t Diphthamide, 258 Diphtheria, 258, 263t immunization against, 249, 257, 262 pathophysiology of, 262 Diphtheria toxin, 258 Diploid cells, 202, 202f, 240, 272 Dipolar molecule, 41 Direct thrombin inhibitors, 860 Disaccharidases, small intestinal, 498–500, 498f, 498t ␤-glycosidase complex in, 498t, 500, 500f glucoamylase in, 498–501, 498t, 499f location of, 498f, 500 sucrase-isomaltase complex in, 498t, 499–501, 499f, 500f trehalase in, 498, 498t, 500, 500f Disaccharides, 22, 61, 62f Dissociation. See also specific reactions of acids, 46, 46t of amino acids, 70, 71f of amino acid side chains, 77, 78f of water, 44, 44f Dissociation constant, 41 for ammonium ions, 51 for water, 45 Distal-promoter elements, 229 Disulfide, 56f Disulfide bonds, 70, 76, 76f Diuresis, osmotic, 44, 563 Divalent metal ion transporter 1 (DMT-1) function of, 831 gene defect in, 831 Divergent evolution, 79

Lieberman_Subject_Index.indd 984

DNA antisense strand of, 230, 230f cancer alterations in, 313, 313f chemical synthesis of, 291 coding (sense) strand of, 230, 230f denaturation of, 200, 200f detecting specific sequences of, 293, 293f in eukaryotes vs. prokaryotes, 240–243 size of, 240–242 diploid human cells in, 240 introns in, 241 repetitive sequences in, 241–242, 242f summary of differences in, 242–243, 243t folate deficiency on replication of, 749 in gene therapy, 304–305 melting of, 200 in mitochondria, 194–195 oxygen-derived free radicals on, 444, 444f packaging of, in chromosomes, 201, 201f, 202f production of, 761f, 767f, 768–769, 769f, 770t recombinant techniques of (See Recombinant DNA techniques) renaturation of, 200, 201f repetitive, 241–242, 242f in viruses, 195 DNA-binding protein structure, 275f–278f, 277–279 DNA chips, 300 DNA cloning, 295, 296f DNA fingerprinting, 299, 299f accuracy of, 307 forensic, 289, 302, 302f statistical issues in, 307 DNA fragments, obtaining chemical synthesis of DNA in, 291 restriction fragments in, 290–291, 290f, 290t, 291f reverse transcriptase in, 291 DNA glycosylases, 219–220 DNA gyrase, 211, 212 DNA ligase, 213, 214f, 217t DNA methylation, 273 DNA polymerases, 217t in eukaryotes, 215, 216t in prokaryotes, 212–213, 212f, 212t DNA polymorphisms definition and types of, 298 detection of, 298–300, 299f DNA fingerprinting and, 307 DNA regulatory sequences, 275, 275f DNA repair, 218–221 disease and, 224 disorders of, 224 enzyme mutations on, 314–315 mechanisms of base excision repair in, 219, 220f mismatch repair in, 220, 221f nucleotide excision repair in, 219, 220f steps in, 219, 220f transcription-coupled repair in, 221 mutagens and, 218–219, 218f, 219f DNA sequences amplification of, 295–297 DNA cloning in, 295, 296f libraries in, 295–297 polymerase chain reaction in, 297, 297f identification of, 292–295 detecting specific DNA sequences in, 293, 293f gel electrophoresis in, 292, 293f probes in, 292, 292f sequencing in, 293–295, 294f, 295f

DNA sequencing, 293–295, 294f, 295f DNA structure, 194–200 antiparallel strands in, 198, 198f base pairing in, 196–198, 197f, 198f bases in, 195, 195f, 195t determination of, 195–196, 195f–197f, 195t double helix in, 198–200, 199f, 200f location in, 164f, 194–195 polynucleotide chain in, 196, 197f replication in, 196–198, 198f Z, B, and A forms in, 200, 200f DNA synthesis, 209–218 in eukaryotes, 213–218 DNA polymerases in, 215, 216t eukaryotic cell cycle and, 214–215, 215f points of origin for replication in, 215, 216f vs. prokaryotes, 213–214 replication complex in, 214f, 215–217, 216f, 217t replication of ends of chromosomes in, 217–218, 217f, 218f overview of, 209, 209f in prokaryotes, 210–213 base-pairing error elimination in, 212t, 213 bidirectional replication in, 210–211, 211f DNA ligase in, 213, 214f DNA polymerase in, 212–213, 212f, 212t DNA synthesis at replication fork in, 213, 214f parental strand unwinding in, 211, 211f RNA primers in, 213, 214f semiconservative replication in, 209f, 211, 211f at replication fork, 213, 214f replication in, 209, 209f DNA template, 228, 230, 230f in mRNA synthesis, 235f, 237 in RNA synthesis, 234f DNA viruses, in cancer, 328 Docosahexaenoic acid (DHA), 11 Dolichol phosphate, 551, 552f, 553f Domain binding, 95–96, 96f structural, 93–94, 94f Dopa decarboxylase, 910f, 911, 913, 914f Dopamine, 910–913 chemical structure of, 57, 58f on fuel metabolism, 798t, 805–806, 806f inactivation and degradation of, 912–913, 912f measurements of, 439 secretion of, 805 storage and release of, 911–912, 911f synthesis of, 910f, 911 tyrosine hydroxylase regulation in, 913 Dopamine ␤-hydroxylase (DBH), 910f, 911, 911f Double helix, DNA, 198–200, 199f, 200f Double minutes, 274 Downregulation, receptor, 491 Downstream, 178 Doxorubicin, 266 for cancer, 198 cardiotoxicity of, 379 DPP-4, 814, 816 DPP-4 inhibitors, 816, 817t Drink, 459 Drinking, moderate, 459 D-sugars, 54, 58–59, 59f Duchenne muscular dystrophy, 886, 901t Duodenum, 500 Dyneins, 165f, 166

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INDEX

Dystrophin, 157 in Duchenne muscular dystrophy, 886 function of, 886 E E2 transcription factor (E2F), 318, 318f Early endosomes, 162 E-cadherin, 322, 322f Ectopic ACTH syndrome, 810, 819 Ectopic secretion, 810 Editing, RNA, 282, 282f Edrophonium chloride, 174 Ehlers–Danlos syndrome, 934 Eicosanoid, 11, 64f, 176, 176f. See also Leukotriene (LT); Prostaglandin (PG); Thromboxane (TX) autocrine, 675 definition of, 663 in inflammatory process, 676 mechanism of action of, 673–675 paracrine, 675 receptors for, 673, 676t source of, 665–666, 665f Eicosanoid metabolism, 663–673 overview of, 663, 664f source of eicosanoids in, 665–666, 665f synthesis pathways in, 583, 583f, 666–673 cyclo-oxygenase, 666–670, 666f (See also Cyclo-oxygenase pathway) cytochrome P450, 666f, 673, 673f endocannabinoid, 673, 674f isoprostane, 673, 674f lipoxygenase, 666f, 671–672, 671f–672f overview of, 666f polyunsaturated fatty acids in, 606 Eicosapentaenoic acid (EPA), 11 Elastase, 698, 698f, 699f Elastin, 932–933, 933f elastic properties of, 933 tropoelastin in, 932, 933f Electrochemical potential gradient, 380, 380f Electrolytes, 44, 44t. See also specific electrolytes Electromyogram, 379 Electron, antibonding, 439 Electron transfer, from NADH to oxygen, 379–380 Electron-transferring flavoprotein (ETF), 382 Electron transport ATP synthesis coupling with, 388–390 regulation through, 388, 388f uncoupling of, 388–390, 389f, 390f inhibition of, 387 Electron-transport chain, 333, 334f energy yield from, 384–385 oxidation–reduction components in, 381–383 coenzyme Q in, 382–383, 382f copper and oxygen reduction in, 382f, 383 cytochromes in, 382f, 383, 383f NADH:CoQ oxidoreductase in, 381, 382f overview of, 347f, 381, 382f succinate dehydrogenase and other flavoproteins in, 381–382, 382f Electrophiles, 126 Electrophoresis, 77–78 Electrophoresis, gel, 292, 293f Electrophoretic separation, 71 Electrostatic bonds, 70, 76, 77f Elongation, protein, 256–257, 256f, 257f Emphysema, from ␣-1-antitrypsin deficiency, 698 Enantiomers, 59, 59f Encephalomyelitis, experimental allergic, 923

Lieberman_Subject_Index.indd 985

Encephalopathy hypoglycemic, 918–919, 919f hypoxic, 920 metabolic, 918–920 Endergonic reactions, 340, 340f Endocannabinoid synthesis, 673, 674f Endocannabinoid system (ECS), 817–819 Endocrine gland secretory tumor, diagnosing, 818 Endocytosis, 157, 158f, 161 Endocytosis, receptor-mediated, 162–163 Endogenous antioxidants, 452, 452f Endoglycosidases, 938 Endopeptidases, 698, 699f Endoplasmic reticulum, 164–165, 165f Endosomes early, 162 recycling, 162 Endostatins, on angiogenesis, 931 Endothelial cells in blood–brain barrier, 907, 907f hepatic, 865 Endothermic reactions, 339–341, 340f, 340t End-replication problem, 217, 217f Energy endocannabinoid system on homeostasis of, 817–819 from fuel oxidation, 4, 4f transformations of in fuel metabolism, 333–334, 333f, 334f for mechanical work, 341, 341f for transport work, 160f, 341–342 for work, 337–340 basic principles of, 337–338 change in Gibbs free energy (⌬G) in, 338–339, 339t ⌬G0 in, 339–340 exothermic and endothermic reactions in, 339–341, 340f, 340t high-energy ATP phosphate bond in, 338, 338f thermodynamics in, 339, 339t Energy balance, 350–351, 350f Energy expenditure daily, 8–10 (See also Daily energy expenditure (DEE)) thermodynamics of, 342 Energy transfer from fuels, through oxidative phosphorylation, 345–349 caloric values of fuels in, 348–349 overview of, 345–346, 346f oxidation–reduction reactions in, 346–347, 347f reduction potential in, 348, 348t Enhancers, 230, 231f, 275, 275f Enoyl hydratase, 420 Enteral tube feeding, 540 Enterokinase, 698, 698f Enteropeptidase, 698, 698f Entropy, 340 Environmental toxins, chlorinated aromatic hydrocarbons in, 67–68, 68f Enzyme, 22, 112–133. See also specific enzymes allosteric, 140–142, 141f basic reactions of, 130–131 catalytic actions of, 112, 113f chymotrypsin catalytic mechanisms in, 117–121 (See also Chymotrypsin, catalytic mechanisms of) classes of, 131 databases of, 86 functional groups in catalysis and, 121–126 on amino acid side chains, 121, 121t coenzymes in, 121–126 (See also Coenzyme)

985

metal ions in, 122f, 126, 126f noncatalytic roles of cofactors in, 126 function of, 39 in Golgi, 162–163 hydrolases, 117f, 131 inhibitors of, 139–140 mechanism-based inhibitors in, 127–130 covalent, 127, 128f definition of, 127 heavy metals in, 130 transition-state analogs and compounds resembling intermediate reaction stages in, 127–129 allopurinol, 129, 129f penicillin, 127–129, 128f oxidoreductases, 125f, 126f, 129f, 131 pH and temperature in, optimal, 126–127, 127f reactions catalyzed by, 113–117 active site in, 114, 114f cell, 138–139 mechanisms of, 113 specificity in, 113–114, 114f, 115 steps of, 113 substrate-binding sites in, 115–117, 115f induced-fit model for, 114f, 115–116, 116f lock-and-key model for, 115, 115f transition-state complex in, 116–117, 116f restriction, 290–291, 290f, 290t, 291f synthesis of, regulated, 145 transferases, 114f, 124f, 131, 132f velocity and concentration of, 138 Enzyme-binding site, 112 Enzyme regulation, 135–150 by changes in amount of enzyme, 145 by conformational changes, 140–145 allosteric enzymes in, 140–142, 141f covalent modification in, 142–143, 142f, 143f protein–protein interactions in, 143–145, 143f–145f proteolytic cleavage in, 145 general overview of, 136–137 by metabolic pathway regulation counterregulation of opposing pathways in, 147 feedback regulation in, 146, 146f feed-forward regulation in, 147 levels of complexity in, 146f, 147 rate-limiting step in, 135f, 146, 146f substrate channeling through compartmentation in, 147 tissue isozymes of regulatory proteins in, 147 rate-limiting enzymes in, 135f, 136 by substrate and product concentration, 137–140 enzyme-catalyzed reactions in cell in, 138–139 reversible inhibition within active site in, 139–140, 140f velocity and substrate concentration in, 137–139, 137f, 138f Eosinophils, 825 Ependymal cells, 906 Epidermal growth factor (EGF), in hepatic fibrosis, 881 Epigenetics, 272 Epileptic seizures, ketogenic diets for, 429 Epimerases, 59 Epimers, 54, 59

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986

INDEX

Epinephrine, 188, 910–913 binding sites for, 95–96, 96f on cAMP, 148 chemical synthesis of, 805 on fuel metabolism, 798t, 805–807, 806f on glycogen metabolism in liver, 521f, 523–524, 523f on glycogenolysis in skeletal muscle, 896, 896f, 897 inactivation and degradation of, 912–913, 912f measurements of, 439 in metabolic homeostasis, 479, 479f metabolism and inactivation of, 806 on newborn blood glucose, 523, 526 physiologic actions of, 481, 482f, 482t, 805–806, 806f secretion of, 805 signal transduction by, 489–490, 489f storage and release of, 911–912, 911f in stress response, 175 structure of, 175, 175f, 489, 489f synthesis of, 489, 489f, 805, 910f, 911 tyrosine hydroxylase regulation in, 913 Epoxide synthesis, 666f, 673, 673f ␧-globin gene, 843 Epstein-Barr virus (EBV), in cancer, 328 Equilibrium constant (Ka) for binding site on protein, 97, 98 for dissociation of weak acid, 46 Equivalent units, 40t Erythrocyte, 824, 826 bicarbonate and hemoglobin in, 45t, 48–49, 49f classification of, 826 cytoskeleton defects in, 834 functions of, 826 glucose metabolism in, after a meal, 23f, 25 glucose transport into, 507 ketone bodies and, 431–432 membrane of, 156–157, 156f, 832–834, 833f metabolic functions of, 795 metabolism of, 795 precursor cells and heme synthesis in, 828–832 heme degradation in, 831–832, 831f, 832f heme structure in, 828, 828f heme synthesis in, 828, 829f heme synthesis in, regulation of, 829f, 831 iron source, metabolism, and storage in, 829–831, 830f in mature erythrocyte, 826–828, 827f production of, 838, 838f shape of, 832–833, 833f sickled, 107, 107f zeta-potential of, 790 Erythrocyte sedimentation rate (ESR), 790 Erythromycin, 257, 261t, 262 Erythropoiesis, 745, 838, 838f Erythropoietin, 301, 838, 838f Erythropoietin receptor defect, 845t Escherichia coli. See also Prokaryotes genome for, potential base pairs produced by, 241–242 structure of, 267, 267f Esophageal varices in cirrhosis with portal hypertension, 872 from ethanol, 468 Essential amino acids, 11 Essential fatty acids dietary, 11 dietary deficiencies of, 665

Lieberman_Subject_Index.indd 986

Essential fructosuria, 532–533 Essential nutrients, 10 Esters, 56f structure of, 56f synthesis of, 57, 58f Estimated Average Requirement (ERA), 10 Estradiol solubility of, 66 synthesis of, 652, 653f, 655f, 656 Estrogen on high-density lipoprotein, 650 synthesis of, 652, 653f, 656 Estrogen receptor, zinc fingers of, 277–278, 277f ETF-CoQ oxidoreductase, 382 Ethambutol, 244 Ethanol, 459 as antivitamin, 121 blood clearance of, 136 on brain membrane fluidity, 156 calories in, 5t, 7 with chronic high-level ingestion, 467 with low-level ingestion, 463 clinical laboratory analysis of, 126 dietary guidelines for, 16 as fuel, dietary, 5t, 7 on gluconeogenesis, 566, 567 on high-density lipoprotein, 650 on organ systems, 113 on phenobarbital-oxidizing P450 system, 462 in pregnancy, 16 structure of, 56f Ethanol-induced hyperglycemia, 580t Ethanol metabolism, 457–470 acetaldehyde dehydrogenase in, 458f, 459, 459f, 460–461 acetate fate in, 461, 461f alcohol dehydrogenase in, 125, 130, 458f, 459–460, 459f, 460t drinking history on, 462 energy (calories) in, 348–349, 463, 467 gender on, 463 genotype on, 462 major route for, 458f, 459, 459f microsomal ethanol oxidizing system in, 459, 459f, 461–462, 461f quantity consumed on, 463 toxic effects of, 54, 463–467 acetaldehyde in, 465–467, 466f alcohol-induced hepatitis in, 463, 466, 466f, 468 alcohol-induced liver disease in, 463, 468 esophageal varices in, 468 free radical formation in, 466f, 467 hepatic cirrhosis and liver function loss in, 467, 468 Laennec hepatitis in, 467, 468 liver fibrosis in, 468–469, 469f, 469t liver metabolisms in, 463 liver NADH/NAD⫹ ratio in, increased, 464–465, 464f nutritional deficiencies in, 467–468 Ether, 56f Ether glycerolipid synthesis, 614–617, 616f Euchromatin, 194, 272 Eukaryote complexity of, 271 definition of, 154 DNA synthesis in, 213–218 DNA polymerases in, 215, 216t eukaryotic cell cycle and, 214–215, 215f points of origin for replication in, 215, 216f vs. prokaryotes, 213–214

replication complex in, 214f, 215–217, 216f, 217t replication of ends of chromosomes in, 217–218, 217f, 218f genes of, 203 expression of, 271–272 transcription of, 234–240 (See also Transcription, of eukaryotic genes) Eukaryotic cell cycle, 214–215, 215f Evaporation, water loss via, 52, 52f Evolution, divergent, 79 Exenatide, 816 Exendin-2, 816 Exercise blood glucose with, 514 glucose in, 514, 578 on high-density lipoprotein, 650 high-intensity, ATP demand and lactate in, 408, 411 for hypercholesterolemia, 657 lactate in, muscle, 32 metabolic homeostasis in, 480 mild- and moderate-intensity long-term, 897–900 acetate in, 899–900 blood glucose as fuel in, 897–898, 898f branched-chain amino acids in, 899, 899f free fatty acids as ATP source in, 898–899, 899f lactate release in, duration on, 897 purine nucleotide cycle in, 766f, 899 skeletal muscle fuel use in, 894–897 anaerobic glycolysis in as ATP source, 894 from glycogen, 895–896, 895f with high-intensity exercise, 897 at onset, 894 in type IIb fast-twitch glycolytic fibers, 894–895 ATP use in, 894 lactate fate in, 897 Exercise supplements carnitine, 420 coenzyme Q, 420 pantothenate, 420 riboflavin, 420 Exergonic reactions, 340 Exocytosis, 161 Exoglycosidases, 938 Exons, 235f, 237, 237f, 238f Exon shuffling, 237, 646 Exopeptidases, 699 Exothermic reactions, 339–341, 340t Experimental allergic encephalomyelitis (EAE), 923 Expiration, water loss via, 52, 52f Extracellular fluid (ECF) in body, 42, 43f electrolytes in, 44, 44t Extracellular matrix (ECM) disease on integrity of, 934 fibrous proteins in, 928–934 collagen in, 928–932 (See also Collagen) elastin in, 932–933, 933f laminin in, 933–934, 933f functions of, 934 overview of, 927–928, 927f proteoglycans in, 934–938 (See also Proteoglycan) Eye, anaerobic glycolysis in, 405, 405f Ezetimibe on endogenous cholesterol synthesis, 629–630 mechanism of action and efficacy of, 629, 657, 658, 658t

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INDEX

F Fabry disease, 554t, 556 Facilitative diffusion, 158, 158f, 159f Facilitative glucose transporter, 159, 506, 507t Facilitative glucose transporter protein type 1 deficiency syndrome, 925t Facilitative transport, 505, 505f, 506f Facilitative transporter, 696 Factor complexes, 854–855, 855f Factor V, 851 Factor VII, 848 Factor VIII, 848, 851 Factor VIIIa complexes, 855 Factor VIII deficiency, 848, 859 Factor VIII, recombinant, 859 Factor V Leiden disease, 861t Factor V Leiden mutation, 857 Factor XIIIa, 854 FAD, reduction of, 346–347, 347f Familial adenomatous polyposis (FAP), mutations in, 322 Familial combined hyperlipidemia (FCH), 608, 620–621, 624t Familial Creutzfeldt-Jakob disease, 106 Familial high-density lipoprotein (HDL) deficiency, 643 Familial hypercholesterolemia (FH), 657 Alu sequences in, 242 vs. familial combined hyperlipidemia, 621 LDL receptor mutations in, 647, 648f Familial hypercholesterolemia (FH) type II, 648, 661t Familial thrombotic thrombocytopenic purpura, 851 Fanconi-Bickel syndrome, 518t Farber disease, 554t Farnesyl pyrophosphate, 632, 634f Fast glycolytic fibers, 408, 886, 886t Fasting, overnight amino acid metabolism in in liver, 777f, 778, 778f in other tissues, 778 in skeletal muscle, 776–778, 777f fatty acid oxidation after, 416 hormone and fuel metabolism changes in, 777 Fasting state, 30–37 adipose tissue in, 33, 33f basal state and, 31, 32f definition of, 30 fuel in, 3, 3f fuel oxidation in, 30 glucose in, 30, 573–577, 574f, 577f blood, maintenance of, 30, 31–32, 32f with prolonged fasting, 571t, 575–576, 575f sources of, 576–577, 577f stages of, 571, 571t on glycogen, liver, 32, 32f, 679 metabolic changes in with prolonged fasting adipose tissue in, 34–35 liver in, 34, 35f, 35t, 36f summary of, 32f, 33, 34t nitrogen excretion in, 717, 717f starvation in, 30 urea excretion in, 34, 36f Fat animal, 417 brown, 389–390, 390f Fat, body abdominal, 27 oxidation of, 334, 334f stored, 334 upper-body deposition of, 27 Fat cell. See Adipocyte

Lieberman_Subject_Index.indd 987

Fat, dietary. See also Triacylglycerol (TG) caloric content of, 5t, 6 digestion and absorption of, 22–23 in fed state, 21, 21f, 22–23, 23f as fuel store, 3, 611 function and storage of, 7, 7t guidelines for intake of, 16 oxidation of, 4–5, 4f structure of, 6, 6f in U.S. diet, 586 Fat-free mass (FFM), 9 Fatigue, exercise metabolic, 895 muscle, 895 Fatty acid, 54. See also specific types adipose triglycerides in, dietary, 416–417 in breast milk, 587 dietary deficiencies of, 665 energy in, 348–349 essential dietary, 11 dietary deficiencies of, 665 in fasting state muscle use of, 680f, 686 overnight, 777 free as ATP source in long-term exercise, 898–899, 899f recycling of, 612, 612f as fuel, 334, 416–417 function of, 583 glyceroneogenesis on release of, 612–613, 612f in homeostasis fuel, 427–428 metabolic, 479, 479f monounsaturated, 417 nonesterified, in obesity, 622 polyunsaturated, 54 dietary, 417 structure of, 6f, 583 saturated long-chain, 417 vs. unsaturated, 61, 63f short-chain, 501 structure of, 61–62, 63f trans-, 61, 63f uptake of in cardiac muscle, 891 in muscle, 890 xenobiotics metabolized as, 877 Fatty acid, long-chain, 417 ␤-oxidation of, 420–423 (See also Fatty acid oxidation) mitochondrial, inherited defects in, 432 transport and activation of, 417–419 (See also Fatty acid oxidation) Fatty acid metabolism, 583, 583f ethanol on, 464–465, 464f in muscle cells, 889, 889f Fatty acid oxidation, 4–5, 4f, 416–424 acetylation in regulation of, 433–435, 434f alternative routes of, 425–427 ␻-oxidation in, 427, 427f peroxisomal oxidation in of long-chain branched-chain fatty acids, 426, 426f of very-long-chain fatty acids, 425–426, 425f, 426f ␤-oxidation in, regulation of, 424, 424f ␤-oxidation of long-chain fatty acids in, 420–423 ␤-oxidation spiral in, 420–421, 420f chain length specificity in, 418t, 421

987

energy yield of, 421, 421f odd-chain-length fatty acids in, 423, 423f unsaturated fatty acids in, 421–423, 422f fatty acids as fuels in, 416–417 in long-chain branched-chain fatty acids, 426, 426f long-chain fatty acid transport and activation in, 417–419 activation in, 417, 418f, 418t cellular uptake in, 415f, 417 fatty acyl-CoA fates in, 417–419, 418f mitochondrial, 415f transport into mitochondria in, 419, 419f in medium-chain-length fatty acids, 415f, 423 mitochondrial, inherited defects in, 415f in very-long-chain fatty acids, 425–426, 425f, 426f Fatty acid synthase complex, 603–605, 603f–606f ␤-ketoacyl group reduction in, 604, 604f carnitine:palmitoyltransferase inhibition in, 605, 606f palmitate synthesis on, 604, 605f phosphopantetheinyl residue of, 603, 603f regulation of, 681f, 682 two-carbon unit addition to acetyl group on, 603, 604f Fatty acid synthase inhibitors, 818 Fatty acid synthesis, 600–608 acetyl-CoA to malonyl-CoA in, 602–603, 602f, 603f carbon source in, 600–601 desaturation in, 606–608, 607f elongation in, 605–606, 606f fatty acid synthase complex in, 603–605, 603f–606f glucose to cytosolic acetyl-CoA in, 601–602, 601f in liver and adipose tissue, 601 Fatty acylation, of amino acids, 83f, 84 Fatty acyl carnitine, 419, 419f Fatty acyl-CoA, fates of, 417–419, 418f Fatty acyl-CoA synthetases, 875 Fatty streak, 649 Fava bean allergy to, 537, 540–541 pathophysiology of, 541 Fe4S4 centers, 382, 382f Feces, water loss via, 52, 52f Fed state, 21–26 amino acids in, 23f, 26 carbohydrate and lipid metabolism regulation in, 679–683 (See also Carbohydrate and lipid metabolism regulation, in fed state) carbohydrates in, 21, 21f, 22 definition of, 21 digestion and absorption in, 22–23, 23f fats in, 21, 21f, 22–23, 23f glucose in, 23f, 24–26, 25f blood (plasma), 572–573, 573f–575f sources of, 561f, 576–577, 577f hormone levels in, 23–24, 23f lipoproteins in, 23f, 26 liver metabolism in, 687, 688t proteins in, 6f, 21, 21f, 22, 23f triacylglycerol metabolism in, 583, 584f Feedback thrombin amplification of, 853f, 856, 856f thrombin inhibition of, 853f, 856, 856f Feedback regulation, 146, 146f Feed-forward regulation, 147

01/09/12 9:36 PM

988

INDEX

Feminization, testicular, 277, 286t Fenfluramine, 915 Fen/phen, 915 Fenton reaction, 440, 440f Ferric iron measurement, 266 Ferritin stores, 831 Ferritin synthesis, translational regulation of, 282, 283f, 284f Ferrous iron, 266 Fetal alcohol syndrome (FAS), 16 Fetal hemoglobin (HbF) hereditary persistence of, 840, 845t structure and function of, 840 Fetal isoforms, 70 Fiber, dietary, 496, 502–504 in disease prevention, 504 recommended daily ingestion of, 503–504 types and components of, 501f, 503, 503t Fibrates, 660, 877 mechanism of action and efficacy of, 658t target of, 620 Fibril-associated collagens, 931 Fibril-associated collagens with interrupted triple helices (FACIT), 930 Fibrin clot, 849 Fibrin cross-linking, 854, 854f Fibrinogen, 145, 301, 851, 852f Fibrinolysis, 857 mechanisms and role of, 857–858 regulation of, 858 Fibronectin, 939 Fibrosis definition of, 468 liver, 468, 470t, 881 in alcohol-induced liver disease, 468–469, 469f, 469t definition of, 468 Fibrous proteins, 90, 928–934 collagen in, 928–932 (See also Collagen) elastin in, 932–933, 933f laminin in, 933–934, 933f Fingerprinting, DNA, 299, 299f accuracy of, 307 forensic, 289, 302, 302f statistical issues in, 307 First law of thermodynamics, 339t, 340 First messengers, 179 Fish, cold-water, 670 Fish oils, 11, 806–807 5'-terminal, 235, 236f 5'-to-3' direction mRNA capping in, 235, 235f in RNA transcript synthesis, 227, 229f, 230f Flavin adenine dinucleotide (FAD), 360, 364 ␣-keto acid dehydrogenase complexes on electron transfer in, 348t, 363–364 in succinate dehydrogenase, 360–361, 361f Flavin mononucleotide (FMN), 360 Flavonoids, as free radical scavengers, 451–452, 451f Flavoproteins, in electron-transport chain, 381–382, 382f Flipases, 156 Flow cytometry, 826 Fluids, body ions and electrolytes in, 44, 44t maintenance of, between tissues and blood, 848 Fluorescence resonance energy transfer (FRET), 939 Fluorescent-activated cell sorting, of hematopoietic stem cells, 836 Fluorescent polarization immunoassay (FPIA), 667

Lieberman_Subject_Index.indd 988

Fluoride, dietary, 17 Fluorodeoxyuridylate (FdUMP), 749 5-Fluorouracil (5-FU) for colon cancer metastases, 311 mechanisms of action of, 749 structure of, 204, 204f Foam cells, 649 Folate malabsorption of, hereditary, 747 reduction to tetrahydrofolate of, 745–746, 746f serum, microbiologic test of, 747 vitamin B12 and S-adenosylmethionine with folate deficiency in, 755, 757 hyperhomocysteinemia in, 754–755, 754f methyl-trap hypothesis of, 753 Folate deficiency, 13t in alcoholics, 745, 746 on DNA replication, 749 on DNA synthesis, 755 forminoglutamate accumulation in, 747 hypomethylation in nervous system in, 757 megaloblastic anemia from, 745, 746 miR-222 upregulation in, 757 neural tube defects from, 755, 757 from vitamin B12 deficiency, 753 Folate, dietary, 746–747, 746f food sources of, 13t, 16 Recommended Dietary Allowance for, 13t, 746 as vitamin, 746–747 Folate supplement for megaloblastic anemia, 750 without cobalamin, in vitamin B12-deficient patient, 756 Fold actin, 94–95, 94f globin, 98f, 99 in globular proteins, 94–95, 94f immunoglobulin, 100, 102f nucleotide-binding, 94f, 95 Folding, protein, 101–106 denaturation and, 105–106, 106f native conformation in, 101–103 primary structure in, 103–104, 104f Follicular lymphoma, 283, 286t Bcl-2 mutation in, 325 treatment of, doxorubicin cardiotoxicity in, 379 Fondaparinux, 860, 860f Food. See also specific foods as fuel, 1 thermic effect of, 9–10 Formate, one-carbon groups from, 747, 749f Formiminoglutamate (FIGLU) accumulation, with folate deficiency, 747 Fragile sites, 275 Fragile X disease (syndrome), 275, 286t Fragments, restriction, 290–291, 290f, 290t, 291f Frameshift mutations, 252, 252f Free amino acids, 12 blood pool of, 775–779 interorgan flux of, in postabsorptive state during fasting, 776–778, 777f metabolism in liver of, 777f, 778, 778f metabolism in other tissues of, 778 skeletal muscle release of, 776–778, 777f location and maintenance of, 775–776, 776f size and function of, 775 tissue flux of, 778–779, 779t

Free fatty acids as ATP source in long-term exercise, 898–899, 899f recycling of, 612, 612f Free radical, 54, 66 definition of, 439 from ethanol, 466f, 467 generation of (See Oxygen toxicity and free radical injury) structures of, 54, 66 Free radical disease, 455t Free radical injury diseases, 437, 437t Free radical scavengers, 449–452 ascorbic acid, 449, 450f carotenoids, 449–451, 451f endogenous (melatonin, uric acid), 452, 452f flavonoids, 451–452, 451f vitamin E, 447f, 449, 450, 450f Friedewald formula, 628 Fructokinase, 531 Fructokinase deficiency, 532–533 Fructose, 411 dietary sources of, 496, 529f, 531 glucose transporters for, 506–507, 506f metabolism of, 473, 474f, 529, 531–532, 532f problems digesting, 496, 509–510, 511t structure of, 58, 58f, 493, 494f, 529, 529f synthesis of, 529f synthesis of, in polyol pathway, 532–533, 532f Fructose 1,6-bisphosphatase, regulation of, 684, 684f Fructose-1-phosphate, on glucokinase dissociation from regulatory protein, 875–876 Fructose 2,6-bisphosphate in anabolic vs. catabolic pathways, 686 in hepatic glycolysis vs. gluconeogenesis, 686 Fructose intolerance, hereditary, 533, 539–540, 542t Fructosuria, essential, 532–533 Fruit dietary guidelines for, 16 malabsorption of, 496, 509–510, 511t Fuel caloric values of, 348–349 in fasting, 3, 3f metabolism of, 1–2 oxidation of, 4, 4f Fuel depots, 21 Fuel, dietary, 4–7. See also specific fuels calories and kilocalories in, 5 carbohydrates, 5, 5f ethanol, 5t, 7 fats, 6, 6f oxidation of, 5–6, 5f proteins, 5–6 Fuel homeostasis fatty acids in, 427–428 ketone bodies in, 427–432, 431f, 432f overview of, 430 regulation of synthesis of, 431–432, 432f tissues using, 431 Fuel metabolism, 24. See also specific types methylxanthines on, 488 Fuel metabolism regulation, 477–492. See also specific fuels; specific topics glucagon in endocrine pancreas in, 483 release of cellular response to, 478, 478f factors in, 485–486, 486t on pathway activation, 477, 477f synthesis and secretion of, 485–486, 485t

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INDEX

hormone signal transduction in, 486–490 by cortisol, 489 by epinephrine and norepinephrine, 489–490, 489f overview of, 486–487 by plasma membrane receptor–binding hormones, 487–489 glucagon, 144f, 179f, 185f, 186f, 488–489 insulin, 181f, 487 principles of, 487 hormones in, specific, 797–821 (See also Hormones regulating fuel metabolism) insulin in, 483–485 actions of, 490–491 endocrine pancreas in, 483 release stimulation/inhibition of, 484–486, 485t, 486f secretion of, 484, 484f synthesis of, 483, 483f metabolic homeostasis hormones in, 480–482 blood glucose, insulin, and glucagon levels in, 481, 481f circulating levels of metabolites in, 481 glucagon in, 480–481, 481f insulin counterregulatory hormones in, 477, 478f, 481, 482t insulin in, 477, 478f, 480, 480f metabolic homeostasis in, 478–480, 479f prohormones in, 477–478 Fuel oxidation. See Oxidation, fuel Fuel storage pathways, 1 Fuel stores, body. See also specific fuels definition and characteristics of, 7, 7t fat, 7, 7t glycogen, 7–8, 7t protein, 7t, 8 Fumarate, amino acids forming aspartate, 733 phenylalanine and tyrosine, 733 Functional assay, of factor VII activity, 848 Functional deficiency, 121 Functional groups, on biologic compounds, 54, 55–57. See also specific groups and compounds charge-carrying, 56–57, 57f in compound name, 55–56 definition of, 54, 55 major types of, 54, 55, 56f nomenclature for, 57, 58f oxidized and reduced, 56 polar bonds and partial charges in, 57, 57f reactivity of, 57, 57f of thiamine pyrophosphate, 122, 122f G G1 phase, 214, 215f G1/S transition, 318, 318f G2 phase, 215, 215f GABA shunt, 917, 917f G-actin folds, 94–95, 94f Gain-of-function proto-oncogene mutations, 313–314, 314f, 321 Galactitol, 535 Galactocerebrosides, 921 Galactose from glucose, 547, 548f glucose transporters for, 506–507, 506f metabolism of to glucose 1-phosphate, 533–534, 533f overview of, 473, 474f

Lieberman_Subject_Index.indd 989

Galactose 1-phosphate (galactose 1-P) deficiency of, 535 on phosphoglucomutase, 547–548 test for, 534 Galactose-1-phosphate uridylyltransferase deficiency, 547–548 Galactosemia, 542t cause of, 540 classical vs. nonclassical, 535 diagnosis of, 535 epidemiology of, 540 management of, 540 mental retardation from, 534, 540 Galanin, 814t, 817 Gallstones bilirubin backflow and jaundice in, 594 bilirubin excess in, 842 in sickle cell disease, 589, 838 ␥-aminobutyric acid (GABA), 917, 917f in hepatic failure, 717 structure of, 175, 175f ␥-globin gene, 843, 843f ␥-glutamyl cycle, 704–705, 705f ␥-interferon, for chronic myelogenous leukemia, 328 Gangliosides, 64, 64f, 261 defective enzymes in, 554, 554t structure and function of, 552, 554f synthesis of, 552–555 Gangliosidoses, 261, 554, 554t, 556, 557t Gas constant (R), 340 Gas gangrene, 155, 169t Gastric acid, 50 Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP), 813–816, 814t, 816f Gastrin, 813, 815t Gastrin-releasing peptide (GRP), 814t Gated channels, 158–159, 158f, 159f Gaucher disease, 554t, 556 GDP-sugars, 551, 553f GEF (guanine nucleotide exchange factor), 180, 180f Gelatin zymography assay, 939 Gel electrophoresis, 292, 293f Gene, 203, 203f inactive, 266 sequences of, 230, 230f, 231f structural, 267 Gene amplification, 274 Gene deletions, 252, 274. See also specific types Gene expression in cancer cells, 273 definition of, 265 Gene expression regulation for adaptation and differentiation, 266 by iron, 283f, 284f, 285 Gene expression regulation, in eukaryotes, 271–284 gene availability for transcription in, 272–274 chromatin remodeling in, 272–273, 273f diploid cells and, 272 DNA methylation in, 273 gene amplification in, 274 gene deletions in, 274 gene rearrangement in, 102f, 273–274, 274f at multiple levels, 272 overview of, 271–272 posttranscriptional RNA processing in alternative splicing and polyadenylation sites in, 281, 281f overview of, 281 RNA editing in, 282, 282f

989

at transcription level, 274–280 basal transcription complex in, 274, 275f DNA-binding protein structure in, 275f–278f, 277–279 enhancers in, 275, 275f gene-specific regulatory proteins in, 274–276, 275f multiple regulators of promoters in, 279–280, 280f steroid hormone/thyroid hormone receptor transcription factors in, 276–277, 276f, 277f transcription factor regulation in, 279 zinc-finger motifs in, 277–278, 277f, 278f at translation level, 282–284 initiation in, 282, 282f, 283f microRNAs in, 283–284, 283f transport and stability of mRNA in, 284, 284f Gene expression regulation, in prokaryotes, 267–271 RNA polymerase binding repressors in, 267–269 corepressors in, 269, 269f inducers in, 268–269, 268f mRNA in, 267, 267f operons in, 267, 267f RNA polymerase binding stimulation in, 269–270, 270f sigma factors in, 270 transcription attenuation in, 270–271, 271f General transcription factors, 232–233, 233f Gene rearrangement, 102f, 273–274, 274f Gene silencing, 303 Gene-specific regulatory proteins, 233, 274–276, 275f Gene-specific transcription factors, 233, 274–276, 275f Gene therapy, 303–305 adenoviral vectors for, 305 adenoviruses in, 303–304, 305 DNA alone or lipid-coated DNA in, 304–305 retroviruses in, 245f, 303, 304f technical problems in, 307 Genetic code, 70, 248–251, 250t amino acids in, 249–250, 250t base pairing in, 249, 250f, 250t breaking of, 249–250 degenerate but unambiguous properties of, 250, 250f, 250t nonoverlapping nature of, 251 universality of, 251 Genetic counseling, 303 Genetic rearrangements, 221–223 crossing over in, 221 fundamentals of, 102f, 221, 274f recombination in, general or homologous, 221–222, 222f translocations in, 221, 222, 222f transposable elements in, 221, 222–223, 223f Gene transcription in bacteria, 233–234, 234f in eukaryotes, 234–240 (See also Transcription, of eukaryotic genes) xylulose 5-phosphate in, 541 Genome definition of, 201 human, 201–203, 202f mapping of, 307 single nucleotide polymorphisms in, 307 size of, 307 Genomic imprinting, methylation in, 273

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990

INDEX

Genomic library, 295–296 Geranyl pyrophosphate, 632, 634f Germ cell lines, defective genes in, 307 Gerstmann-Straussler-Scheinker disease, 106 Ghrelin, 813, 814t Ghrelin receptor signaling system, 813 Gibbs free energy (G), 334, 338 availability of, 342 Gibbs free energy change (⌬G), 339t additive values of, 342–343, 343f, 343t vs. ⌬G0, 343t, 344 energy for work from, 338–339, 339t general expression for, 340, 340t substrate and product concentrations on, 340f, 343–344, 343t Gleevec, 327 Glial cells, 905–906 Glipizide, 485 Global proton leak, 390 Globin fold, 98f, 99 Globin, regulation of heme synthesis of, 255 Globular protein, 90 folds in, 94–95, 94f solubility of, 95 Glomerular basement membrane (GBM), in diabetic nephropathy, 940 Glomerular filtration rate (GFR), creatinine in, 900 Glomerulonephritis, poststreptococcal pathogenesis of, 900 pathophysiology of, 893 presentation and diagnosis of, 885 Glomerulus, 940, 940f Glossitis, 373 GLP-1, 813–816, 814t, 816f GLP-1 receptor antagonists, 816, 817t GLP-2, 814t Glucagon after a meal, 23–24, 23f on cAMP, 148 on fuel metabolism, 480–481, 480f, 481f, 798t endocrine pancreas in, 483 in liver, 520, 521f physiologic effects of, 799–800 release of cellular response to, 478, 478f factors in, 485–486, 486t on pathway activation, 477, 477f secretion of, 485–486, 485t synthesis of, 485–486, 485t, 799–800 on metabolic homeostasis, 478f, 479, 479f, 480–481, 481f, 814t physiologic actions of, 481, 482f, 482t release of, pathways regulated by, 476, 476f on signal transduction, 144f, 179f, 185f, 186f, 488–489 Glucagon-like peptide-1 (GLP-1), 813–816, 814t, 815t, 816f Glucagon-like peptide-2 (GLP-2), 814t Glucoamylase, 498–501, 498t, 499f Glucocorticoid-response element (GRE), 276f, 277 Glucocorticoids. See also specific types on fuel metabolism, 798t, 806–808 biochemistry of, 806–807 effects of, 807–808, 808t secretion of, 798t, 807, 807f, 808f, 808t on inflammation, 666 naturally occurring, 564 overnight fast on, 777 physiologic actions of metabolic, 798t nonmetabolic, 808t synthesis of, 652, 653f

Lieberman_Subject_Index.indd 990

Glucokinase, 147, 399 glucokinase regulatory protein on, 874–875, 875f glucose-binding site in, 115, 115f high-carbohydrate meal on, 138–139 in liver, 874–875, 875f in maturity onset diabetes of the young, 138 reaction catalyzed by, 114f regulation of, 679–680, 679f, 684f Glucokinase activators, for diabetes type 2, 875 Glucokinase regulatory protein, 874–875, 875f Gluconeogenesis, 562–570 cortisol on, 178 definition of, 515, 562 energy for, 570 in fasting, 31, 32–33, 32f alanine in, 717, 717f prolonged, 34, 35f, 36f gluconeogenic intermediates from carbon in, 563–564, 564f in liver, 514f, 515, 560f, 562 metformin on, 659–660, 660f pathway of, 564–567 fructose 1,6-bisphosphate to fructose 6-phosphate in, 566–567 glucose 6-phosphate to glucose in, 567 oxaloacetate to phosphoenolpyruvate in, 565, 566f phosphoenolpyruvate to fructose 1,6-bisphosphate in, 566, 567f pyruvate to oxaloacetate in, 565, 566f pyruvate to phosphoenolpyruvate in, 564–566, 565f, 566f in plants, 579, 579f precursors for, 563 regulation of, 567–570, 684, 684f fructose 1,6-bisphosphate to fructose 6-phosphate in, 570 glucose 6-phosphate to glucose in, 138f, 570, 570f key enzyme activity and amount in, 568, 569f, 569t pyruvate to phosphoenolpyruvate in, 568–570 substrate availability in, 568 steps and key reactions in, 560f, 562–563, 562f Gluconic acid, 60, 61f Glucosamine 6-phosphate, amino sugars from, 549, 551f Glucose, 5, 5f as allosteric effector, 522 from ATP (See Glycolysis) brain use of, 907 calories in, 898 dietary, 398 energy in, 348–349 in fasting state brain and erythrocyte use of, 778 muscle use of, 680f, 686 sources of, 561f in fed state, 21, 21f, 561f as fuel, 334f, 335, 398 (See also Glycolysis) in metabolic homeostasis, 479–480, 479f from nitrogen metabolism, 695, 695f oxidation of, 4–5, 4f recycling of, 579, 579f sources of, 561f in starved state, 561f stereospecificity of, 59 structure of, 493, 494f Glucose-6-phosphatase, 514, 515f, 570, 570f deficiency of, 518t regulation of, 684, 684f

Glucose-6-phosphate (G6P) glucose-1-phosphate from, 340–341, 340f, 343, 345 from glycogen, 514, 515f metabolism and feedback regulation of, 137 pentose phosphate pathway metabolism of, 536–537 structure and solubility of, 58f triose phosphate conversion of, 399, 400f Glucose-6-phosphate dehydrogenase (G6PD) gene mutations of, 828 in hexose monophosphate shunt, 827–828, 827f regulation of, 680, 681f Glucose-6-phosphate dehydrogenase (G6PD) deficiency, 537, 540–541, 542t, 828, 845t Glucose-alanine cycle, 579, 579f in skeletal muscle, 784–785, 784f in transporting amino acid nitrogen to liver, 712–713, 712f Glucose, blood (plasma) after a meal, 571–578 mechanisms of, 571 in type 1 vs. type 2 diabetes, 575 at various times, 571, 571f in exercise, 514, 578 in fasting state, 30, 573–575, 574f hepatic maintenance of, 683–685, 683f, 684f stages of, 571, 571t with starvation, 571t, 575–576, 575f in fed state, 572–573, 573f–575f as fuel in long-term exercise, 897–898, 898f at rest, 898f on fuel metabolism, 481, 481f on glycogen metabolism in liver, 522–523 liver regulation of, 870 on metabolic homeostasis, 481, 481f in neonate 1-3 hours after birth, 519, 519f epinephrine on, 523 normal ranges of, 519, 519f sources of, 576–578, 577f units for, 40 Glucose-dependent insulinotropic polypeptide (GIP), 813–816, 814t, 816f Glucose/galactose transporter deficiency, 518t Glucose homeostasis, 475–476 in newborn, 523, 526 steroid hormones on, 570 Glucose metabolism. See also Gluconeogenesis acetyl-CoA from, 601, 601f after a meal, 24–26 in adipose tissue, 23f, 25–26 in brain and neural tissues, 23f, 25 glycogen, triacylglycerols, and carbon dioxide from, 23f, 24 in muscle, 25–26, 25f in red blood cells, 23f, 25 amino acids and triacylglycerol moieties from, 474, 474f galactose from, 547, 548f glucose 6-phosphate conversion of, 398–399, 398f, 400f glycine from, 919, 919f hexokinase isozyme Km values for, 138, 138f lactate and CO2 from, 474, 474f in liver, 561–562, 874–875, 875f major pathways of, 475–476, 476f overview of, 473, 474f

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INDEX

Glucose synthesis. See Gluconeogenesis Glucose transport in cardiac muscle, 891 through blood–brain barrier and neurons, 508–509, 508f Glucose transport disorders, 907 Glucose transporter in blood–brain barrier and neurons, 508–509, 508f in intestinal epithelium, 505–507 facilitative, 159, 506, 507t for galactose and fructose, 506–507, 506f Na⫹-dependent, 505–506, 506f for monosaccharides, 507–508, 508f Glucose transporter protein (GLUT) family, 506–507, 507t Glucose transporter protein type 1 (GLUT-1), 907, 925t Glucose transporter protein type 1 (GLUT-1) deficiency syndrome, 907 Glucose transporter protein type 2 (GLUT-2) deficiency, 518t Glucose transporter protein type 3 (GLUT-3), 907 Glucosidosis, 554t Glucuronate, 546–547, 547f Glucuronic acid, 60, 61f, 546–547 Glucuronide, 546–547, 547f, 547t Glutamate amino acids related through, 730, 731f in amino acid synthesis, 711–712, 711f chemical structure and bonds of, 74f, 75t, 76 deamination of, 710–711, 710f degradation of, 724f, 730, 731f dissociation of side chains of, 77, 78f function of, 916 high levels of, 718 synthesis of, 724f, 730, 731f, 916–917, 917f in urea production, 710f, 712, 712f Glutamate dehydrogenase, 916 Glutamate dehydrogenase reaction, 710–711, 710f, 718 Glutamate-oxaloacetate transaminase, 131, 132f Glutamic acid decarboxylase (GAD), 917 Glutaminase, 713, 713f, 917, 917f Glutamine in ammonia transport, 778 chemical structure of, 74f, 75–76, 75t degradation of, 731–732, 731f functions of, 779, 779t gut use of, 785, 785f high levels of, 718 liver uptake of, 879 nitrogen transport to liver by, 712–713, 712f, 713f skeletal muscle synthesis of, 778 tissues using, 778 Glutamine metabolism in brain, 786–787, 787f in kidney as fuel, 780, 781t, 782f nitrogen as urine buffer in, 780–781, 781t, 782f overview of, 780, 781f Glutamine phosphoribosyl amidotransferase, 763, 764f Glutamine synthesis, 713, 713f, 731–732, 731f anaplerotic pathways of pyruvate carboxylase and methylmalonyl-CoA mutase and, 920 in skeletal muscle, 781 Glutamine synthetase, 711

Lieberman_Subject_Index.indd 991

Glutarate. See ␣-ketoglutarate/glutamate Glutathione, 448, 448f in mitochondria, 450 synthesis of, in liver, 871t Glutathione peroxidase, 448–449, 448f in mitochondria, 450 selenium in, 449 Glutathione reductase, 448–449, 448f Glycation, 579 of LDL apoproteins in, 689 of LDL receptor, 649 Glycemic indices, 504, 504t Glycemic response, 504–505 Glyceraldehyde D- and L-, 58–59, 59f structure of, 56 Glyceraldehyde-3-phosphate dehydrogenase reaction, 411–412, 412f Glycerol in gluconeogenesis, 563–564, 564f glucose from, 579f, 580 structure of, 56 Glycerol 3-phosphate, 608 Glycerol 3-phosphate dehydrogenase, 382 Glycerol 3-phosphate shuttle, 402, 402f Glycerolipid synthesis, ether, 614–617, 616f Glycerol kinase deficiency of, 608 gene for, 608 Glyceroneogenesis, on fatty acid release, 612–613, 612f Glycerophospholipid function of, 583, 613 structure of, 613, 613f types of, 613, 613f Glycerophospholipid metabolism, 613–618 degradation in, 617–618, 617f synthesis in, 614, 614f, 615f synthesis of phospholipids containing glycerol in ether glycerolipids, 614–617, 616f glycerophospholipids, 614, 614f, 615f Glycine chemical structure of, 73–75, 74f, 75t from glucose metabolism, 919, 919f in gout testing, 770–771 from intermediates of glycolysis, 728, 728f one-carbon groups from, 747, 749f synthesis and action of, 918 xenobiotic conjugates of, 871t Glycocalyx, 155, 157 Glycochenodeoxycholic acid synthesis, 871t Glycocholic acid synthesis, 871t Glycogen. See also Carbohydrate for anaerobic glycolysis in exercise, 895–896, 895f dietary sources of, 496 disorders of, 517–518, 518t energy from, 342 function and storage of, 7–8, 7t in liver, 475, 514, 514f, 518 in fasting overnight, 679 regulation of enzymes degrading, 683–684, 683f in fed state, 679–682 (See also Carbohydrate and lipid metabolism regulation, in fed state) muscle, 518 in skeletal muscle, 514, 514f structure of, 512, 513f, 514 Glycogenesis. See Glycogen synthesis (glycogenesis)

991

Glycogen metabolism biosynthetic pathways in, 516, 516f degradation in, 515–517, 515f, 517f, 518t, 619t from glucose, 23f, 24 Glycogen metabolism disorders glycogen storage disease type I, 522 glycogen storage disease type III, 518t, 520–521 Glycogen metabolism regulation, 518–525 of glycogenesis, 515, 515f, 518, 519t of glycogen stores, 519t in liver, 519–524, 519t blood glucose levels in, 522–523 enzyme nomenclature in, 520, 520f epinephrine and calcium in, 521f, 523–524, 523f fasting on, 481f, 518t, 519 glucagon in phosphorylase B to phosphorylase A conversion in, 520, 521f glycogen synthase inhibition in, 520–521 insulin and glucagon in, 520, 521f, 522 overview of, 481f, 518t, 519 protein phosphatase regulation in, 521 overview of, 518, 519t in pregnancy and neonates, 519, 519f in skeletal muscle, 524–525, 525f Glycogenolysis, 515, 515f, 517, 517f, 518, 519t Glycogen phosphorylase in muscle, 142, 143f, 144, 518, 524–525, 525f nomenclature for, 520, 520f Glycogen phosphorylase A, 520, 521f on glycogen metabolism, 520, 521f nomenclature for, 520, 520f Glycogen phosphorylase B, 520, 521f on glycogen metabolism, 520, 521f nomenclature for, 520, 520f Glycogen phosphorylase deficiency in liver, 518t in muscle, 517–518, 518t Glycogen storage diseases, 517–518, 518t Glycogen storage disease type I, 522 Glycogen storage disease type III, 518t, 520–521 Glycogen stores, 3, 7–8, 7t, 518 in diabetes mellitus, 522 in liver, 32, 32f, 518 after overnight fast, 777 in fasting state, 32, 32f in neonate, inadequate, 519, 523, 525–526 regulation of metabolism of, 519t Glycogen synthase, 131 inhibition of, by glucose-directed phosphorylation, 521–522 nomenclature for, 520, 520f regulation of, 680, 680f, 683f, 684 Glycogen synthase deficiency, 518t Glycogen synthase kinase-3 (GSK-3), 526–527 Glycogen synthesis (glycogenesis), 31, 32, 32f, 137 activated intermediates of, 343, 343f energetics of, 342, 343, 343f in liver, 514f, 515 overview of, 473, 473f, 475 regulation of, 515, 515f, 518, 519t in skeletal muscle, 514, 514f steps and intermediates in, 515–516, 515f, 516f Glycolipid, 54, 475, 475f, 552–555 structure and function of, 64f, 552, 554f synthesis of process of, 552–554 sugar formation for, 549, 549t, 550f, 551f

01/09/12 9:36 PM

992

INDEX

Glycolysis, 23f, 24, 396–413, 515 aerobic, 335 definition of, 335 energy yield of, vs. anaerobic glycolysis, 403 on pyruvate, 401, 401f amino acid synthesis from intermediates of, 727–730 alanine in, 727f, 730 cysteine in, 729–730, 729f glycine in, 728, 728f overview of, 727, 727f serine in, 727–728, 727f anaerobic (See Anaerobic glycolysis) biosynthetic functions of, 405, 405f bis-phosphoglycerate shunt in, 405–406, 405f glyceraldehyde-3-phosphate dehydrogenase reaction in, 411–412, 412f lactic acidemia and, 409–410, 410f in liver, 560f glucokinase in, 874–875, 875f glucokinase regulatory protein on, 874–875, 875f PFK-1 on, 874, 875 in muscle cells, 889 overview of, 396f, 397–398, 473–474, 473f, 474f pyruvate and nicotinamide adenine dinucleotide oxidative fates in, 401–402, 401f, 402f reactions of, 398–401 glucose 6-phosphate to triose phosphates, 399, 400f glucose to glucose 6-phosphate, 398–399, 398f, 400f glycolytic pathway phases, 40f, 398, 398f oxidation and substrate-level phosphorylation, 399–401, 400f summary of, 401 regulation of, by need for ATP, 406–409 ATP, ADP, and AMP concentration relationships in, 407, 407f hexokinase in, 406f, 407 major sites of, 406–407, 406f phosphofructokinase-1 regulation in, 406f–408f, 407–409 allosteric, by AMP and ATP, 408, 408f by allosteric inhibition, at citrate site, 409 by fructose 1,6-bisphosphate, 408–409 role of, 406f, 407 types of, 407–408 pyruvate dehydrogenase regulation and glycolysis in, 409 pyruvate kinase regulation in, 409 Glycolytic fibers, 408 Glycolytic pathway. See Glycolysis, reactions of Glycomacropeptide, 740 Glycophosphatidylinositol glycan (GPI) anchor, 157, 157f Glycoprotein, 54, 61, 475, 475f, 549–552 O-linked and N-linked structures of, 872–873, 873f structure and function of, 549, 551f, 552f synthesis of, 549t, 550–552, 552f, 553f in liver, 872–873, 873f sugar formation for, 549, 549t, 550f, 551f Glycosaminoglycan (GAG), 61, 934, 935f protein attachments of, 934, 936f structure of, 934, 935f types of, 934 Glycosidases, 496–497 Glycosidic bonds, 54, 61, 62f

Lieberman_Subject_Index.indd 992

Glycosphingolipid, 155 Glycosylated hemoglobin, with sustained hyperglycemia, 105, 108 Glycosylation, 108, 259, 579 of amino acids, 82–84, 83f of hemoglobin, in diabetes, 105, 108 Glycosyltransferases, 546, 546f Goiter, 811 Goiter, diffuse toxic. See Hyperthyroidism Golgi complex, 165, 259, 260f Golgi, enzymes in, 162–163 Gout, 68t, 133t, 169t, 772t, 773t acute phase of attack of, 167 diagnosis of, 55, 67, 113 outcome of, 130 pathophysiology of, 65, 67, 770 synovitis in, 163 treatment of, 130 allopurinol in, 113, 129, 167 colchicine in, 153, 166–167 G-protein heterotrimeric, 184, 185f, 185t in signal transduction, 488 small (monomeric), 144, 145f G protein-coupled receptors (GPCRs), 179, 179f, 184–187 adenylyl cyclase and cAMP phosphodiesterase in, 185–186, 186f heterotrimeric G-proteins in, 184, 185f, 185t names and general properties of, 96f, 179f, 184 phosphatidylinositol signaling by, 173f, 186–187 Grain, dietary guidelines for, 16 Gram-negative bacteria, 194 Gram-positive bacteria, 194 Gram stain, 194 Granulocytes, 824–825 Granulomatous disease, chronic, 446, 455t Graves disease. See Hyperthyroidism Grooves, in DNA, 198, 199f Group transfer reaction, 131, 132f Growth factor receptors, in signal transduction cascade, 315, 316t Growth factors, 176 hematopoietic, 301–302 in signal transduction cascade, 315, 316t Growth hormone (GH) acromegaly from tumor secreting, 801 on fuel metabolism, 798t, 801–805 in adipose tissue, 803 biochemistry of, 801–802, 801f control of secretion of, 802, 803f, 804t energy from, 802, 804f in liver, 803–805 in muscle, 803 production of, 301–302, 302f Growth hormone release–inhibiting hormone (GHRIH). See Somatostatin Growth hormone releasing hormone (GHRH), 802, 803f Growth hormone suppression test, 818 Growth-suppressor genes, 192 Guanine 8-hydroxyguanine from, 444, 444f structure and nucleosides of, 195, 195f Guanine nucleotide exchange factor (GEF), 180, 180f Guanosine cap, 227, 235–236, 235f, 236 Guanosine monophosphate (GMP) phosphorylation of, 761f, 762 synthesis of, 762, 763f

Guanosine triphosphate (GTP) generation of, 358f, 359 high-energy phosphate bonds in, 344–345 Guanylyl cyclase receptors, 188, 188t Guide strand, 283, 283f Gut, amino acids utilization by, 785–786, 785f Guthrie bacterial inhibition assay, 726 Gynecoid obesity, 27 H Haber-Weiss reaction, 439, 440f Hairpin loop, 93f Half-life, protein, 701 Haploid cells, 201–202 Haptocorrins, 750 Hartnup disease, 704, 706t cystinuria in, 701, 704 hyperaminoaciduria without hyperaminoacidemia in, 700–701 pathophysiology of, 700, 701, 704 pellagra-like symptoms in, 704 HbA1c, 105, 105f, 108 HbS/HbC individuals, 839 Heart, ATP transformation by, 338 Heart attack, 353t, 887 angina pectoris in, 337 clinical presentation of, 337 creatinine phosphokinase in, 893 Heart failure. See Congestive heart failure Heat from fuel oxidation, 4, 4f in inflammatory process, 676 Heat-shock protein (hsp) function of, 104 in protein folding, 104, 104f Heat-shock protein 70 (hsp70), 94 Heavy metals, in inhibitors, 130 Height measurement, 27 Helicases, 211, 211f, 217t Helix-loop-helix, 278f, 279 Helix-turn-helix, 278f, 279 Hemarthrosis, 859 Hematocrit, in diagnostic testing, 397 Hematomas, 859 Hematopoiesis, 835–839 cytokines and, 836–838, 837f erythropoiesis in, 838, 838f nutritional anemias in, 838–839 stem cells and hematopoietic tree in, 835–836, 836f Hematopoietic growth factors endogenous, 837, 837f therapeutic, 301–302 Hematopoietic stem cells, 835–836, 836f Heme, 383, 383f bilirubin A from, 831–832, 832f degradation of, 831–832, 831f, 832f on globin mRNA translation in, 282, 282f synthesis of, 255 oxygen binding and, 99–100, 99f structure of, 99, 828, 828f synthesis of, 795, 828, 829f in liver, 871t phenobarbital on, 832 regulation of, 829f, 831 Hemoglobin, 70, 97–100 agents affecting oxygen binding to 2,3-bis-phosphoglycerate, 834, 834f carbon dioxide, 834–835, 834f, 835f proton binding (Bohr effect), 834, 834f, 835f blood, measurement of, 228

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INDEX

as buffer, 41, 41f components of, 105f cooperativity of, 100, 101f, 108–110, 109f cooperativity of O2 binding in, 100, 101f, 108–110, 109f developmental variations in, 80, 80f fetal hereditary persistence of, 840 vs. maternal, 840 structure and function of, 840 glycosylation of, in diabetes, 105, 108 histidine of, proximal, 99f, 100 iron in, 15, 383–384, 383f vs. myoglobin structure, 80, 80f normal levels of, 826, 826t oxygen binding in, 100, 101f oxygen binding in, and heme, 99–100, 99f oxygen saturation curve for, 98, 98f oxygen saturation of, structure on, 97–98, 98f polymerization of, in sickle cell anemia, 100 polymorphisms of, 70 in red blood cells, 45t, 48–49, 49f sickle (HbS), 78, 203 structure–function relationships in, 97–100, 98f Hemoglobin C, 839, 845t Hemoglobin M, 384, 828 Hemoglobinopathies, 839 Hemoglobin S, 839 Hemoglobin switching, 840–841, 841f ␣- and ␤-globin genes in, 841, 841f control of, 842–844, 843f Hemolytic anemia, 795, 827, 834 Hemophilia A, 848, 859, 861t Hemophilia B, 861t Hemostasis, 795–796 Hemostatic plug formation of, 850–851, 851f role of, 849 Henderson–Hasselbalch equation, 46 for bicarbonate buffer system, 48 midpoint in, 47 for weak acid, 46 Heparin, 857, 859 anticoagulant actions of, 593 complications of, 914 naturally occurring, 593 structure of, 934, 935f Heparin-induced thrombocytopenia (HIT), 859, 914 Heparin sulfate, 857 Hepatic-binding protein, 867 Hepatic cirrhosis, 467, 468 Hepatic encephalopathy, 713, 716, 717, 722t Hepatic failure, 716, 882t Hepatic fibrosis, 468, 470t, 881 in alcohol-induced liver disease, 468–469, 469f, 469t definition of, 468 Hepatic protein-phosphatase-1 (PP-1), 522 Hepatic triglyceride lipase (HTGL), in liver disease, 877–878 Hepatitis alcohol-induced, 463, 466, 466f, 468, 745 viral, 722t acetaminophen toxicity with, 720 complications of, 720 symptoms and testing for, 704, 711, 713 Hepatitis B vaccination, 289, 303, 306 Hepatitis B virus cancer and, 328 infection with, 308t

Lieberman_Subject_Index.indd 993

Hepatitis C virus, in cancer, 328 Hepatocytes, 865 Hepatogenous diabetes, 880–881 Heptahelical receptors, 179, 179f, 184–187 adenylyl cyclase and cAMP phosphodiesterase in, 185–186, 186f heterotrimeric G-proteins in, 184, 185f, 185t names and general properties of, 96f, 179f, 184 phosphatidylinositol signaling by, 173f, 186–187 HER2 gene, 315 Herceptin, 315 Hereditary anemias, 795 Hereditary breast cancer BRCA1/BRCA2 genes in, 224, 225t DNA repair mutations in, 329–330 Hereditary folate malabsorption, 747 Hereditary fructose intolerance (HFI), 533, 539–540, 542t Hereditary nonpolyposis colorectal cancer (HNPCC), 221, 224, 225t, 329 Hereditary orotic aciduria, 768f, 769, 772t, 773t Hereditary persistence of fetal hemoglobin (HPFH), 840, 845t Hereditary spherocytosis, 842 Herpesvirus (HHV-8), in cancer, 328 Hers disease, 518t Heterochromatin, 194, 272 Heterocyclic ring structures, 54 Heterotrimeric G-proteins, 184, 185f, 185t HETE synthesis, 671, 671f Hexokinase, 147 function of, 399 glucose binding to, on conformation, 116, 116f regulation of, 406f, 407 Hexosaminidase A, 249, 261 Hexose monophosphate (HMP) shunt, 537, 795, 827–828, 827f High-density lipoprotein (HDL), 584t, 642–645 characteristics of, 640t estrogen on, 650 ethanol on, 650 exercise on, 650 fate of, 644, 644f functions of, 584, 644–645, 644f as “good” cholesterol, 650 interactions with other particles of, 644–645, 644f in liver disease, metabolism of, 878 low, with elevated VLDL or LDL, 650 maturation of, 642 in reverse cholesterol transport, 642–644, 643f synthesis of, 642 High-energy bonds, 344–345. See also specific types High-energy phosphate bonds. See also specific types activated intermediates with, 344–345 in ATP, 338, 338f, 344–345 (See also Adenosine triphosphate (ATP)) in 1,3-bis-phosphoglycerate, 345, 345f in creatine phosphate, 345, 345f in phosphoenolpyruvate, 345, 345f Highly variable regions, 299 High-molecular-weight heparin (HMWH), 859 Hippurate, 877 Hirsutism, 656–657 Hirudin, 860

993

Histamine, 825 in allergic response, 916 in inflammatory process, 676 metabolism of, 913–915, 915f tissue-specific effects of, 916 Histamine methyltransferase, 915 Histidine, 11 chemical structure of, 74f, 75t, 76 degradation of, 732, 733f dissociation of side chains of, 77, 78f isoelectric point and titration curve of, 76–77, 77f one-carbon groups from, 747, 749f proximal, 99f, 100 Histidine decarboxylase, 914, 915f Histidine load test, 747 Histone, 164 acetylation of, 272–273, 273f arginine and lysine in, 201–202 net charge of, 201–202 Histone acetyltransferases (HAT), 273, 433 Histone deacetylase (HDAT), 273, 273f HIV. See Acquired immunodeficiency syndrome (AIDS); Human immunodeficiency virus (HIV) HMG-CoA reductase, 630–632 proteolytic degradation of, 631–632 regulation of, 630–631, 631f structure and function of, 630 HMG-CoA reductase inhibitors, 650, 657, 658 for hyperlipidemia, 621 mechanism of action and efficacy of, 658t Hogness box, 231f, 232 Hogness–Goldberg box, 231f, 232 Holliday structure, 222 Holoprotein, 99 Homeostasis ATP, 345, 350–351 fuel (See Fuel homeostasis) glucose, 475–476 metabolic, 478–480, 479f Homocysteine in homocystinuria, 731–732 reaction pathways with, 753, 754–755, 754f Homocyst(e)ine, 731 Homocystinemia, 740, 741t, 742t Homocystinuria biochemical studies of, 731–732 definition of, 740 etiology of, 734–735 signs, symptoms and diagnosis of, 740 treatment of, 740 Homogentisate oxidase, defective, 736 Homologous chromosomes, 202, 203f Homologous recombination, 221–222, 222f Homovanillylmandelic acid (HVA) from catecholamines, 912f in Parkinson disease, 913 Hormone cross-talk, 186 Hormone receptors. See also specific receptors in signal transduction cascade, 316t Hormone replacement therapy (HRT), 650 Hormone-response elements in gene regulatory control region, 178, 276–277, 276f insulin-sensitive, 491 Hormones, 1–2. See also specific hormones after a meal, 23–24, 23f autonomous release of, 483–484 measurement of blood levels of, 799 in metabolic homeostasis, 479, 479f prohormones and, 477–478 Hormone-sensitive lipase (HSL), 685, 685f

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994

INDEX

Hormones regulating fuel metabolism, 477–492, 797–821 catecholamines (epinephrine, norepinephrine, dopamine), 798t, 805–806, 806f (See also specific hormones) endocannabinoid system and energy homeostasis in, 817–819 GI-derived peptides, 814–816, 816f (See also specific peptides) direct acting, 813, 814t indirect acting, 813, 815t glucagon, 798t endocrine pancreas in, 483 physiologic effects of, 799–800 release of, 485–486, 485t cellular response to, 478, 478f factors in, 485–486, 486t on pathway activation, 477, 477f synthesis of, 485–486, 485t, 799–800 glucocorticoids, 798t, 806–808 biochemistry of, 806–807 effects of, 807–808, 808f, 808t secretion of, 798t, 807, 807f, 808f, 808t growth hormone, 798t, 801–805 on adipose tissue, 803 biochemistry of, 801–802, 801f control of secretion of, 802, 803f, 804t on energy metabolism, 802, 804f on liver, 803–805 on muscle, 803 insulin, 483–485, 798t actions of, 490–491 endocrine pancreas in, 483 physiologic effects of, 799 release stimulation/inhibition of, 484–486, 485t, 486f secretion of, 484, 484f synthesis of, 483, 483f metabolic homeostasis hormones in, 480–482 blood glucose, insulin, and glucagon levels in, 481, 481f circulating levels of metabolites in, 481 glucagon in, 480–481, 481f insulin counterregulatory hormones in, 477, 478f, 481, 482t insulin in, 477, 478f, 480, 480f metabolic homeostasis in, 478–480, 479f neural factors controlling insulin and counterregulatory factors in, 817 prohormones in, 477–478 signal transduction in, 486–490 by binding to plasma membrane receptors, 487–489 glucagon in, 144f, 179f, 185f, 186f, 488–489 insulin in, 181f, 487 principles of, 487 by cortisol, 489 by epinephrine and norepinephrine, 489–490, 489f overview of, 486–487 somatostatin in, 798t biochemistry of, 800 physiologic effects of, 800–801 secretion of, 800 thyroid hormone in, 798t, 809–813 biochemistry and synthesis of, 809–810, 809f, 810f calorigenic effects of, 812–813 feedback regulation of, 811, 812f physiologic effects of, 811–812 secretion of, 810f, 811, 812f HPETEs, 671, 671f

Lieberman_Subject_Index.indd 994

HS40, 841f, 842 H substance, 555 Human chorionic somatomammotropin (hCS), 801 Human epidermal growth factor receptor gene (HER2, c-erbB-2), 315 Human genome, 201–203, 202f Human genome mapping, 307 Human immunodeficiency virus (HIV). See also Acquired immunodeficiency syndrome (AIDS) in cancer, 328 production of, 244–246, 245f replication rate of, 213 resistant strains of, 244 Human leukocyte antigen (HLA) defect, in diabetes mellitus type 1, 484 Human papillomavirus (HPV), in cancer, 328 Human T-lymphotropic virus-1 (HTLV-1), in cancer, 328 Hyaline membrane disease. See Respiratory distress syndrome (RDS) Hyaluronate, 934, 935f Hybridization, 291 of DNA, 200, 201f of RNA, 200, 201f Hydratase, 420 Hydration shells, 43, 44f Hydrochloric acid (HCl), stomach, 50 Hydrocortisone, on fuel metabolism, 798t, 806–808 biochemistry of, 806–807 effects of, 807–808, 808t secretion of, 798t, 807, 807f, 808f, 808t Hydrogen from indigestible carbohydrates, 501–502 urinary, buffering by, 45t, 50 Hydrogen bond, 75, 75f in amino acids, 70 in water, 43, 43f water–polar molecules, 43, 44f Hydrogen breath test, 502 Hydrogen cyanide (HCN), 386 Hydrogen ions, 41, 44–45, 44f Hydrogen peroxide, 439–441, 439f, 440f, 440t Hydrolases, lysosomal, 162, 162f Hydrolysis of acyl-chymotrypsin intermediate, 118f–119f, 120 of ATP, 338, 338f, 339–340 of chylomicrons and VLDL by lipoprotein lipase, 682 definition of, 117 of glycosidic bond, 499–500 Hydropathic index, 73, 75t Hydroperoxyeicosatetraenoic acid (HPETE) in leukotriene synthesis, 672, 672f synthesis of, 671, 671f Hydrophilic compounds, 57 Hydrophobic bond, 75, 75f Hydrophobic compounds, 57 Hydrophobic core, in globular proteins, 95 Hydrophobic effect, 57, 70 in globular protein solubility, 95 in secondary structure, 90, 90f in tertiary structure, 91 Hydrophobicity, 54 Hydroxyeicosatetraenoic acid (HETE) in leukotriene synthesis, 672, 672f synthesis of, 666f, 671, 671f, 673, 673f Hydroxyethyl radical, 467 8-Hydroxyguanine, from guanine, 444, 444f 11-Hydroxylase deficiency, 655 Hydroxylases, 131

Hydroxyl group, 55 Hydroxyl ion, 41 Hydroxyl radical, 439, 439f, 440f, 440t 4-Hydroxyphenylpyruvate dioxygenase defect, 737 Hydroxyproline, 928–929, 929f Hydroxyurea, 328 Hyperaminoaciduria, without hyperaminoacidemia, 700–701 Hyperammonemia, 787, 792t. See also Ammonia toxicity Hypercatabolic states, 792t amino acid metabolism in, 774f, 788–790, 789f insulin resistance in, 791 Hypercholesterolemia, 925t chronic, 657 diet and exercise for, 657 familial, 657 Alu sequences in, 242 vs. familial combined hyperlipidemia, 621 LDL receptor mutations in, 647, 648f type II, 648, 661t treatment of dietary, 628, 628t HMG-CoA reductase inhibitors of, 657 LDL cholesterol in, 657 Hypercortisolemia, 821t Hyperglycemia, 475, 492t chronic diabetic microvascular disease from, 940–941 diabetic nephropathy from, 941, 941f in diabetics, 478, 509 glycosylated hemoglobin HbA1c fraction in, 105, 108 diagnosis of, 24 ethanol-induced, 465, 580t nonketotic, 480 pathophysiology of, 480 symptoms of, 480 Hyperhomocysteinemia, 754–755, 754f Hyperkalemia, aldosterone synthesis in, 655 Hyperlipidemia, 26. See also Familial hypercholesterolemia (FH); Hypercholesterolemia defective LPL in, 611–612 familial combined, 608, 620–621, 624t Hyperlipoproteinemia, chylomicron levels in, 592 Hypermethioninemia, 740 Hyperosmolar hyperglycemic state (HHS), 480 Hyperphenylalaninemia, 737, 739–740 Hypersensitivity reactions, basophils in, 825 Hypertension dietary sodium and chloride in, 16 portal, 872 from poststreptococcal glomerulonephritis, 900 Hyperthyroidism, 8, 353t, 394t, 813 on ATP utilization and fuel oxidation, 346 on basal metabolic rate, 344 effects of, 344 pathophysiology of, 335, 344, 351 presentation and diagnosis of, 337, 351, 379 Hypertriglyceridemia, 649 in diabetes mellitus type 2, 683–684 VLDL synthesis in, 649 Hyperuricemia, 464f, 465, 771 Hypervariable regions, 78 Hyperventilation, 51, 53t Hypochlorous acid (HOCl), 440, 440t, 446–447, 446f

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INDEX

Hypochromic anemia, 285 Hypoglycemia, 475 diagnosis of, 481 from ethanol, 464f, 465 in galactose-1-phosphate uridylyltransferase deficiency, 547–548 from insulin overdose, 514, 516, 524 in neonate, 519, 523, 525–526 neuroglycopenic manifestations of, 481 newborn, 527t Hypoglycemic encephalopathy, 918–919, 919f Hypoglycin toxicity, 424, 435t Hypoglycorrhachia, 907 Hypoketosis, 419 Hypolactasia, adult, 502, 502t Hyponatremia, aldosterone synthesis in, 655 Hypoproteinemia, 872 Hypothalamus, T3 and T4 release from, 337 Hypothyroidism, 8, 811, 813, 821t Hypovolemia, 51–52, 52f Hypoxanthine, 761–762, 769 Hypoxanthine-guanine phosphoribosyl transferase (HGPRT), 765, 766f Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, 765, 766f, 772t, 773t Hypoxia on acetyl-CoA for acetylcholine synthesis, 917 on ATP generation, 338 brain, confusion from, 401 Ca2⫹, Na⫹, and cell death with, 338, 352–353, 352f etiology of, 920 in myocardial infarction, 378 on neurotransmitter synthesis, 920 Hypoxia-inducible factors (HIFs), 353 with chronic obstructive pulmonary disease, 404 Hypoxic encephalopathy, 920 I Ibuprofen on prostaglandins, 670, 670f structure of, 670f I-cell (inclusion cell) disease, 260, 263t, 551–552, 553f Icterus. See Jaundice Idiopathic thrombocytopenic purpura (ITP), 850 Immunization, decrease in, 258 Immunodeficiency disease, 772t Immunoglobulin classes of, 100 function of, 100 structure–function relationships in, 100–101, 102f, 103f Immunoglobulin D, 281, 281f Immunoglobulin fold, 100, 102f Immunoglobulin M, 281, 281f Immunoradiometric assays (IRMAs), 820 IMP dehydrogenase, 762, 763f, 764, 764f Inborn errors of metabolism, 517–518, 518t, 726 Incretin effect, 813 Indigestible carbohydrates, 501, 501f Indirect calorimetry, 9 Induced-fit model, 114f, 115–116, 116f Inducers, 268–269, 268f, 274–276, 275f Ineffective erythropoiesis, 745 Inflammation in asthma, 666 corticosteroids for, 675 free radical formation during, 447 Inflammatory process, 676

Lieberman_Subject_Index.indd 995

Inhibition. See also specific reactions competitive, 139, 140f noncompetitive, 139, 140f simple product, in metabolic pathways, 139–140 uncompetitive, 139, 140f Inhibitor-1, 522 Inhibitors. See also specific inhibitors allosteric, 140–143, 141f covalent, 127, 128f enzyme, 139–140 mechanism-based, 127–130 covalent, 127, 128f definition of, 127 heavy metals in, 130 transition-state analogs and compounds resembling intermediate reaction stages in, 127–129 allopurinol, 129, 129f penicillin, 127–129, 128f suicide, 129, 129f Initiation, of translation protein, 253–255, 254f, 255f, 255t regulation of, 282, 282f, 283f Inner membrane skeleton, 156f, 157 Inorganic acids, 41 Inosine monophosphate (IMP) structure of, 761–762, 762f synthesis of, 759f, 761–762, 761f, 762f Inositol 1,4,5-trisphosphate (IP3), 180, 180f Insertion mutation, 251t, 252 Inspiratory rales, 351 Insulin actions of, 490–491 autonomous hypersecretion of, 483–484 for body building, 514, 516, 524 bovine and porcine, vs. human, 82, 82f endocrine action of, 175 on fuel metabolism, 483–485, 798t actions of, 490–491 after a high-carbohydrate meal, 481, 481f after a high-protein meal, 486, 486f after a meal, 23–24, 23f in blood, 481, 481f control of release of, 481, 481f endocrine pancreas in, 483 functions and sites of action of, 480, 480f between meals, 416 overview of, 477, 478f physiologic effects of, 482t, 799 secretion of, 484, 484f stimulation/inhibition of release of insulin in, 484–486, 485t, 486f synthesis of, 483, 483f function of, 182 on gene expression, 280 on glucagon release, 522 on glucose blood, 483 suppressive action of, 487 transport of, 24, 508, 508f on glycogen metabolism, 520, 521f, 522 on insulin/glucagon ratio, 522 on lipolysis, 685 on metabolic homeostasis, 477, 478f–480f, 479, 480 overdose of, 514, 516, 524, 527t hypoglycemia from, 572, 580t physiologic actions of, 481, 482t production of, 301–302, 302f on protein synthesis, 255 on signal transduction, 181f, 487

995

structure of, 81–82 primary, species variations in, 81–82, 82f quaternary, 97 in synthetic insulin, amino acid position in, 85 synthesis of, 81–82 Insulin counterregulatory hormones. See also specific hormones on fuel metabolism, 477, 478f, 481, 482f, 482t physiologic actions of, 481, 482f, 482t Insulin-like growth factor (IGF), 801, 804–805 Insulin-like growth factor binding protein 3 (IGF-BP3), 320, 321f Insulin-like growth factor I (IGF-I), 804–805, 805f on growth hormone, 801–802 high circulating levels of in acromegaly, 801–802 cancers and, 805 Insulin-like growth factor II (IGF-II), 804–805, 805f Insulinoma, 486, 492t, 691t autonomous secretion in, 483 diagnosis of, 484 familial, 689 weight gain with, 683 Insulin receptor, 181–182, 181f, 182f Insulin receptor substrate (IRS), 181–182, 181f, 182f Insulin receptor substrate 1 (IRS-1), 487 Insulin resistance, 490 glycogen synthase kinase-3 in, 527 in hypercatabolic states, 791 in liver disease, 880–881 in metabolic syndrome, 484f, 623 Insulin resistance syndrome, 620 Insulin-sensitive hormone response element (IRE), 491 Integrins, 938 Intercellular signaling pathways, 1 Interferon for chronic myelogenous leukemia, 328 on JAK and STAT, 279 recombinant ␤-, 302 therapy with, 279 Interleukin-2 (IL-2), in sepsis, 790–791, 791f Interleukin-6 (IL-6), 328 Interleukin-10 (IL-10), 328 Interleukin, therapeutic, 302 Intermediate-density lipoprotein (IDL), 640t, 642 Intermediate filaments (IFs), 165, 167, 168f Interphase, 194 Intestinal adenocarcinoma, 331t Intestinal brush border membrane disaccharidases, 498–500, 498f, 498t. See also Small intestinal disaccharidases Intestinal epithelium, sugar absorption by, 505–507 facilitative transport in, 505, 505f, 506f glucose transporters in facilitative, 506, 507t of galactose and fructose, 506–507, 506f Na⫹-dependent, 505–506, 506f Intestinal lymph system, 585 Intestine, urea cycle enzymes in, 785 Intoxication, alcohol, legal limit of, 462 Intracellular fluid (ICF) in body, 42, 43f electrolytes in, 44, 44t Intracellular pH, buffers and, 45t, 49–50, 49f Intracellular receptors, vs. plasma membrane receptors, 176–177, 176f Intravenous drug users, AIDS in, 223

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996

INDEX

Intrinsic factor, 750 Intrinsic factor deficiency, 751–752 Intron, 235f, 237, 237f, 241 definition of, 234 removal of, in mRNA synthesis, 235f, 237, 237f, 238f Iodide, thyroid hormone on, 809 Iodide transporter, apical membrane, 809 Iodine deficiency, hypothyroidism and goiter from, 811 Ion channel receptors, 179, 179f Ionic bonds, 70, 76, 77f Ion, in body fluids, 44, 44t Ionizing radiation, free radical intermediates from, 441, 442f Ion product (Kw), for water, 45 Iproniazid, 913 Iron dietary guidelines for, 16, 829 dietary sources of, 829 on ferritin synthesis, 282, 283f, 284f function of, 15 in hemoglobin, 383–384, 383f measurement of, 266 metabolism of, 829–831, 830f source of, 829, 830f storage of, 831 on transcription, 283f, 284f, 285 vitamin C on uptake of, 829, 830f Iron deficiency, 845t Iron-deficiency anemia, 15, 394t, 838 diet and, 15 from divalent metal ion transporter 1 gene mutation, 831 fatigue in, 383 in malnutrition, 13 microcytic, hypochromic anemia in, 833 presentation and diagnosis of, 266, 284–285 in women, 832 Iron response element (IRE), 282, 283f, 284f, 285 Iron response-element binding protein (IRE-BP), 282, 283f, 284f Ischemia, cardiac cardiac muscle fuel use in, 891 pathophysiology of, 393, 453 Ischemia–reperfusion injury, 393, 442, 453–454 Islet cells innervation and control of secretions of, 817 NADPH from pyruvate cycling in, 623–624, 623f Islets of Langerhans, 483 Isocitrate, 358–359, 358f Isocitrate dehydrogenase, 367–368, 367t, 368f Isoelectric point (pI), 76–77, 77f Isoforms fetal and adult, 70 tissue-specific, 81, 81f Isoleucine degradation, 735–736, 735f, 738 Isomaltase, 499–500, 499f, 500f Isomerases, 133 Isoniazid, 244 Isoprene unit, 54 Isoprenyl unit, 55f, 64 Isoprostane synthesis, 673, 674f Isotonic saline, osmolality of, 45 Isozyme, 81, 81f hexokinase, Km values for, 138, 138f tissue, of regulatory proteins, 147 tissue-specific, 71 Ito cells, 865–866 in alcohol-induced liver disease, 468–469, 469f hepatic fibrosis on, 881

Lieberman_Subject_Index.indd 996

J Jaffé reaction, 31 JAK, 279 JAK/STAT pathway cytokine signaling through, 837, 837f disturbances in, 838 JAK-STAT proteins, signal transduction by, 179f, 182–183, 183f JAK-STAT receptors, 179, 179f Jamaican vomiting disorder, 424, 435t Janus kinase (JAK), 618, 837, 837f Jaundice, 459, 470t, 557t, 880 bilirubin in, 546 definition of, 546 in galactose-1-phosphate uridylyltransferase deficiency, 547–548 neonatal, bilirubin in, 548 JEB gravis, 934 Joint normal structure and function of, 936 systemic lupus erythematosus on, 936, 936f Junctional epidermolysis bullosa (JEB), 934, 943t K Kaposi sarcoma, 328 Karyotype analysis, 311 Kearns-Sayre syndrome, 387t Keratan sulfate, 934, 935f Ketoacidosis acetone in, 55 alcohol-induced, 464f, 465 definition of, 42 diabetic (See Diabetic ketoacidosis (DKA)) Ketogenic amino acids, 430, 736, 736f Ketogenic diets for epileptic seizures, 429 for pyruvate dehydrogenase deficiency, 429 Ketohexose, 58, 58f Ketone measurement of, 562 names and structures of, 55–56, 56f Ketone bodies, 31, 33, 33f, 41, 335, 779 in blood and urine, 36 in fuel homeostasis, 427–432, 431f, 432f overview of, 430 regulation of synthesis of, 431–432, 432f tissues using, 431 in ketoacidosis and diabetic coma, 48 metabolism of, 427–430 alternative pathways of, 430 overview of, 427 oxidation in, as fuels, 428–430, 430f in newborn, 526 from nitrogen metabolism, 695, 695f red blood cells and, 431–432 synthesis of, 428, 428f, 429f in liver, 55, 871–872 in liver, in fasting state, 682f, 685–686, 686t Ketone groups in acetoacetate and acetone, 56 structure of, 56f Ketosis, in children, 431 Keto sugars, 54 Kidney amino acid utilization by, 779–780, 780f–782f, 780t ammonia excretion by, 780–781, 781t, 782f fuel sources for, 780, 781t, 782f glomerular units in, 893 water loss via, 52, 52f Kilocalorie (kcal), 5

Kinase nonreceptor, 279 receptor, 279 Kinase-binding receptors, 179, 179f Kinase receptors, 179, 179f Kinesins, 165f, 166 Kinetic barriers, 104 Kinins, in inflammatory process, 676 Kozak sequence, 255, 255t Krabbe disease, 554t Krebs bi-cycle, 715, 715f Krebs cycle. See Tricarboxylic acid (TCA) cycle Krebs–Henseleit cycle. See Urea cycle Kupffer cells, 469, 469f, 865 Kussmaul breathing, 48, 416, 458, 563 Kwashiorkor, 13, 19t, 698, 706t, 848, 861t L lac operon, 268–270, 268f, 270f ␣-Lactalbumin, 548–549, 548f Lactase, 22 activity of, 498t, 500, 500f nonpersistent and persistent, 502 Lactase deficiency adult, 502, 502t congenital, 502 pathophysiology of, 503, 503f Lactase-glucosylceramidase, 498t, 500, 500f Lactate, 32 blood levels of, determining, 409 in cardiac muscle, 891 in exercise duration on release of, 897 fate of, 897 high-intensity, 408, 411 fate of, 404–405, 404f pyruvate from, 563, 564f Lactate dehydrogenase (LDH), 563, 564f 3D structure of, 93, 94f, 95 mechanism of action of, 125–126, 125f reaction for, 403, 403f Lactation, with lactose intolerance, 548–549 Lactic acid, 41, 403, 779 Lactic acidemia, 409–410, 410f, 413t Lactic acidosis, 409–410, 410f from ethanol, 464f, 465 in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes, 387t Lactic dehydrogenase (LDH) reaction for, 403, 403f structure of, 405 Lactobacilli, tooth, 403 Lactose, 5 dietary sources of, 496 as filler, 509 structure of, 493, 494f, 496 synthesis of, 547–549, 548f Lactose intolerance, 502, 502t, 511t incidence of, 509 lactation and, 548–549 management of, 509 pathophysiology of, 503, 503f Laennec’s cirrhosis. See Alcohol-induced cirrhosis Lagging strand in eukaryotes, 216, 216f in prokaryotes, 213, 214f Laminin biosynthesis of, 933–934 structural defects in, 934 structure of, 933, 933f

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INDEX

L-amino acids, 72, 72f Large, external transformation-sensitive (LETS) protein, 939 Large neutral amino acids (LNAAs), blood–brain barrier transport of, 908 L-Ascorbate, 449, 450f Laws of thermodynamics, 339t, 340 LCAD deficiency, 422, 433, 435t LDL apoprotein glycation, in diabetes, 689 LDL cholesterol, 16 LDL direct, 628 LDL receptor. See Low-density lipoprotein (LDL) receptor LDL receptor-related protein (LRP), 648 Leading strand in eukaryotes, 216, 216f in prokaryotes, 213, 214f Lead poisoning anemia in, 829, 829f pathophysiology of, 829, 829f Leber hereditary optic neuropathy (LHEN), 387t Lecithin, 63, 64f, 916 in amniotic fluid, 621, 621f dietary, 917 neonatal demand for, 917 in plasma membrane, 155, 155f synthesis of, 614, 614f Lecithin-cholesterol acyltransferase (LCAT) in liver disease, 877–878 synthesis, secretion, and actions of, 643, 643f, 644 Left ventricular heart failure, 351–352 Leigh disease, 369, 375t, 387t LEPRE1 mutation, 942 Leptin, 618–619 Lesch-Nyhan syndrome, 765, 766f, 772t, 773t Leucine degradation, 735f, 736f, 738 Leucine zippers, 278, 278f Leukemia acute lymphoblastic, asparaginase for, 730 asparaginase for, 730 chronic myelogenous, 283, 285, 286t, 331t Bcl-2 mutation in, 325 karyotyping of, 311 pathophysiology of, 311 Philadelphia chromosome in, 274, 284, 311, 315 presentation and diagnosis of, 266, 311 treatment for, 327, 328 hemorrhagic manifestations of, 311 pathophysiology of, 837 Leukocyte basophils, 825 classification of, 824 definition of, 824 eosinophils, 825 granulocytes, 824–825 mononuclear, 824, 825 morphonuclear, 824 Leukocyte adhesion deficiency (LAD), 938 Leukopenia, from 5-fluorouracil, 206 Leukotriene (LT) functions of, 673t gout pain from, 177 metabolism of, 664f (See also Eicosanoid metabolism) synthesis of, 672, 672f Levofloxacin, 256 Lewy bodies, 442, 453 LHON, 387t Libraries, 295–297 Li-Fraumeni syndrome, 320, 331t Ligand, 73

Lieberman_Subject_Index.indd 997

Ligand binding, quantification of, 97 Ligand-gated channels, 158–159 Ligases, 133 Lignan, 496 Limit dextrins, 497–498 LINEs, 242, 243 Lineweaver-Burk plots, 149, 149f Lineweaver-Burk transformation, 148–149, 148f Linoleate in arachidonic acid synthesis, 665 oxidation of, 421–422, 422f Linoleic acid, 606–608 arachidonic acid from, 606–608f, 607 Linolenic acid, 606 Lipase lipoprotein, 583, 584f pancreatic, 22–23, 587–588, 588f Lipid definition and classification of, 54 in myelin, 920–921 oxygen radical reactions with, 442, 442f, 443f in plasma membrane, 156–157, 156f structure of, 54, 61–64 acylglycerols in, 62–63, 63f fatty acids in, 61–62, 63f phosphoacylglycerols in, 63, 64f sphingolipids in, 64, 64f steroids in, 64, 65f Lipid-anchored proteins, 157 Lipid-coated DNA, in gene therapy, 304–305 Lipid, dietary absorption of, 588–589, 588f, 589f in breast milk, 587 chylomicrons in fate of, 592–593, 593f structure of, 590–591, 591f synthesis of, 590–591, 590f, 591f microsomal triglyceride transfer protein and, 594–595, 595f transport to blood of, 591–592, 592f triacylglycerols in digestion of, 586–588, 587f–589f resynthesis of, in intestinal epithelial cells, 590, 590f structure and function of, 586, 587f in U.S. diet, 586 Lipid-free radicals, formation of, 442, 443f Lipid-lowering agents, 658–659, 658t Lipid metabolism in liver, 875–878 enzymes in, 875 ethanol on, 464–465, 464f in liver disease, 877–878 long-chain-length fatty acids in, 875 medium-chain-length fatty acid oxidation in, 876 need for, 875–876 peroxisomal proliferator–activated receptors in, 876–877, 877t very long-chain-length fatty acid peroxisomal oxidation in, 876 xenobiotics metabolized as fatty acids in, 877 overview of, 583–584, 583f regulation of in fasting state, 683–686 (See also Carbohydrate and lipid metabolism regulation, in fasting state) in fed state, 679–683 (See also Carbohydrate and lipid metabolism regulation, in fed state) Lipid peroxidation, 442, 442f, 443f, 673, 674f

997

Lipid synthesis. See also specific lipids in brain and peripheral nervous system, 796, 920–923 distinguishing features of, 920–921 mechanisms of, 921 myelin synthesis in, 921–923, 922f, 922t Lipoate, 364, 364f Lipocortins, 666 Lipofuscin granules, 442 in age-related macular degeneration, 451 in Parkinson’s disease, 442, 453 Lipogenesis, 583 Lipolysis, 33 in diabetes mellitus, 685 in fasting state in adipose tissue, 685, 685f insulin on, 685 Lipoprotein. See also specific types classes and characteristics of, 639, 640t in fed state, 23f, 26 receptor-mediated endocytosis of, 645–646, 645f structure of, 590f, 639 Lipoprotein(a), 650 Lipoprotein, blood, 583, 584t cholesterol transport by, 639–645 apoproteins in, 639, 640t, 645 chylomicrons in, 593f, 639–641 high-density lipoproteins in, 640t, 642–645, 643f, 644f intermediate-density lipoproteins in, 642 lipoprotein structure in, 590f, 639 low-density lipoproteins in, 642 mechanism of, 639 very low-density lipoproteins in, 640t, 641, 641f structure of, 590, 590f Lipoprotein lipase (LPL), 583, 584f chylomicron and VLDL hydrolysis by, 682 defective, in hyperlipidemia, 611–612 in diabetes mellitus type 1, 682 VLDL triacylglycerol digestion by, 597, 598f on VLDL triglyceride, 608 Lipoprotein receptors, 646–649 low-density lipoprotein receptor in, 646–648 mutations in, 647–648, 648f structure of, 646–647, 647f low-density lipoprotein receptor-related protein in, 648 macrophage scavenger receptor in, 648–649 Lipoxin synthesis, 672, 672f Lipoxygenase, 671, 671f Lipoxygenase pathway, 666f, 671–672 arachidonic acid as substrate for, 671 hydroxyeicosatetraenoic acid synthesis in, 671, 671f leukotriene synthesis in, 672, 672f lipoxin synthesis in, 672, 672f lipoxygenase action in, 671, 671f Liraglutide, 816 Lispro amino acid position in, 85 genetic engineering of, 303 vs. normal human insulin, 303 Lithocholic acid, 638, 638f Liver, 796, 863–882 anatomy of, 864–865, 864f cell types in endothelial cells, 865 hepatocytes, 865 Kupffer cells, 865 pit cells, 866 stellate cells, 865–866 diagnostic agents for, 867

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998

INDEX

Liver (continued) ethanol on, 467, 468 in fasting state, 32 in fasting state, prolonged, 34, 35f, 35t, 36f regenerative capacity of, 215–216 thyroid hormone on, 811–812 Liver alcohol dehydrogenase, 130, 460 Liver-associated lymphocytes, 866 Liver disease, 880 alcohol-induced, 463, 468 chronic, liver fibrosis in, 468–469, 469f, 469t nutritional deficiencies from, 467–468 amino acid metabolism in, 879–880 insulin resistance in, 880–881 lipid metabolism in, 877–878 Liver failure, 716, 882t Liver fibrosis, 468, 470t, 881 in alcohol-induced liver disease, 468–469, 469f, 469t definition of, 468 Liver functions, 866–873 ammonia detoxification in, 871 fuels for, 873–880 amino acid metabolism in, 878–879 carbohydrate metabolism in, 874, 874t glucose metabolism in, 561–562, 874–875, 875f (See also Gluconeogenesis) lipids in, 875–878 (See also Lipid metabolism, in liver) need for, 873 sources of, 873–874 metabolism in of amino acid, 786 ethanol on, 463 in fed vs. fasting states, 687, 688t overview of, 863 pentose phosphate pathway in, 873 as receiving and recycling center, 866–867 regulation in of glucose, 870 of glycogen metabolism, 519–524, 519t (See also Glycogen metabolism regulation, in liver) synthesis in, 405 of binding proteins, 848–849, 849t of cholesterol and triacylglycerols, 871 of glucose, 560f, 562 of glycoprotein and proteoglycan, 872–873, 873f of ketone bodies, 682f, 685–686, 686t, 871–872 of nitrogen-containing compounds, 871, 871t of nucleotides, 872 processes of, 796 of proteins, 879 of proteins, blood, 872, 872t of urea, 871 xenobiotic and metabolite inactivation and detoxification in, 867–870 cytochrome P450 enzymes in, 868, 868f cytochrome P450 system in, 867–868, 868f on acetaminophen, 869–870, 870f on aflatoxin B1, 869 on vinyl chloride, 869, 869f enzymes in, 868t general scheme for, 867, 867f pathways and processes in, 867, 867f “Liver spots,” 442 Liver transaminases, 416

Lieberman_Subject_Index.indd 998

LKB1 protein, 659 LMO2 gene, 305 Load reducers, 352 Lock-and-key model, 115, 115f Locus control region (LCR), 843 Long-chain acyl CoA dehydrogenase (LCAD) acetylation, 433–435, 434f Long-chain acyl CoA dehydrogenase (LCAD) deficiency, 422, 433, 435t Long-chain branched-chain fatty acids, peroxisomal oxidation of, 426, 426f Long-chain fatty acids ␤-oxidation of, 420–423 ␤-oxidation spiral in, 420–421, 420f chain length specificity in, 418t, 421 energy yield of, 421, 421f odd-chain-length fatty acids in, 423, 423f unsaturated fatty acids in, 421–423, 422f mitochondrial, inherited defects in, 432 saturated, 417 transport and activation of, 417–419 activation in, 417, 418f, 418t cellular uptake in, 415f, 417 fatty acyl CoA fates in, 417–419, 418f mitochondrial, 415f saturated, 417 transport into mitochondria of, 419, 419f very long-chain, peroxisomal oxidation of, 425–426, 425f, 426f Long interspersed elements (LINEs), 242, 243 Loops, 92, 93f, 94f Loss-of-function mutations, 314 Lou Gehrig disease, 448, 455t Low-density lipoprotein (LDL), 16, 584t, 642 calculation of Friedewald formula in, 628 LDL direct test in, 628 characteristics of, 640t cholesterol and cholesterol esters in, 648 in diabetes type 2, 649 elevated low high-density lipoprotein levels in, 650 treatment of, 657–658 in fed state, 26 goals and cut points for, 628 Low-density lipoprotein (LDL) receptor, 646–648 in familial hypercholesterolemia, 242 function of, 242 mutations in, 647–648, 648f structure of, 646–647, 647f Low-density lipoprotein receptor-related protein (LRP), 648 Low-molecular-weight heparin (LMWH), 859 L-sugars, 54, 58–59, 59f Luminal agent, 867 Lung adenocarcinoma, 331t Lung cancer, 223, 225t Lung lining fluid definition of, 454 ozone protection in, 454–455, 454f phases of, 454, 454f Lung transudation, 351 Lupus, 238, 246t, 943t articular cartilage disruption in, 928, 936, 936f, 939–940, 940f DNase in, reduced, 244 on joints, 936, 936f pathophysiology of, 244 Lutein, 451, 451f Luteinizing hormone (LH), in testosterone synthesis, 655 Lymphocytes, liver-associated, 866

Lymphoma Burkitt, 314, 331t follicular, Bcl-2 mutation in, 325 non-Hodgkin chemotherapy for, 266, 274, 283 diffuse large B-cell, 283 follicular type, 283, 286t types of, 283 Lymph system, 585 Lysase, 132f, 133 Lysine chemical structure of, 74f, 75t, 76–77 degradation of, 736f, 738 dissociation of side chains of, 77, 78f Lysosomal enzyme defects, genetic, 162 Lysosomal enzymes, 549, 552f Lysosomal glucosidase deficiency, 517, 518t Lysosomal hydrolases, 162, 162f Lysosomal protein turnover, 702, 702f Lysosomal reactions, 162, 162f Lysosomal storage diseases, 162 Lysosomes, 161–163, 162f endoglycosidases in, 938 exoglycosidases in, 938 proteoglycan degradation by, 936–938 M M2 muscarinic receptors, 184 Macrocortins, 666 Macrocytic anemia, 745 Macrolide antibiotics, 257 Macrophages, 825 Macrophage scavenger receptor, 648–649 Macular carotenoids, 451, 451f Macular degeneration, age-related, 451, 455t Magnesium, 14–15 Major grooves, DNA, 198, 199f Malaria resistance in G6PD deficiency, 828 in sickle cell anemia, 79 Malate–aspartate shuttle, 402, 402f Malathion, 113, 113f Malathion poisoning, 133t acetylcholinesterase inhibition in, 127 presentation and chemistry of, 113, 113f treatment and prognosis in, 130 Malic enzyme reaction of, 601, 601f regulation of, 680, 681f Malignant, 194 Malignant melanoma, 225t, 331t hereditary risk factors in, 319 incidence of, 223 mutations in, 329 risk factors in, 319 Malignant neoplasm (tumor), 310, 312. See also specific neoplasms Malnutrition, 13, 36 depression from, 31 protein, 13 severe (kwashiorkor), 13, 91t, 698, 706t, 848 thryoid hormone and transthyretin in, 787 protein-calorie, 13, 31 vitamin, 13t–14t, 14 Malondialdehyde, 442, 443f Malonyl-CoA from acetyl-CoA, 602–603, 602f, 603f on carnitine:palmitoyltransferase I, 681–682, 682f in palmitate synthesis, 681–682, 682f Malonyl-CoA decarboxylase, 893 Maltopentose, 586

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INDEX

Mammalian target of rapamycin (TOR), 791 Mammary gland, 587 Mannose, 549, 550 interconversion of, 549, 550f structure and functions of, 549 Mannose-6-phosphate synthesis, 553f MAP kinase, in oncogenesis, 315–316, 317f MAPKKK, 180 Maple syrup urine disease, 736, 741t, 742t, 908 Marasmus, 13, 19t, 31 Marfan syndrome, 934 Markers, polymorphic, 299 Mast cells, histamine synthesis by, 913–914 Matrix metalloproteinases (MMP), 881, 939 Maturity-onset diabetes of the young (MODY), 485, 492t MCAD deficiency, 422, 423, 432–433, 435t McArdle disease, 517–518, 518t mdm2 gene, 320 Mean corpuscular hemoglobin concentration (MCHC), 826 Mean corpuscular volume (MCV), 826 Mechanical work, energy transformations for, 341, 341f Medium-chain acyl CoA dehydrogenase (MCAD) deficiency, 422, 423, 432–433, 435t Medium-chain-length fatty acid–activating enzyme (MMFAE), 876, 877 Medium-chain-length fatty acid oxidation, 415f, 423 Medium-chain-length triacylglycerols (triglycerides), 876 Megakaryocytes, 850 Megaloblast, 745, 750 Megaloblastic anemia, 13t, 745 alcohol-induced, 745–746, 750, 758t pathophysiology of, 750 treatment of, 750 from vitamin B12 deficiency, 745, 839 Megaloblastic madness, 753 Melanin synthesis, 910f Melanocyte function of, 312 tyrosine hydroxylase of, in albinism, 912 Melanoma, 218, 225t, 312 hereditary risk factors in, 319 incidence of, 223 malignant, 225t, 331t hereditary risk factors in, 319 incidence of, 223 mutations in, 329 risk factors in, 319 MELAS, 387t Melatonin, as antioxidant, 452, 452f Melting, DNA, 200 Membrane attack, by free radicals, 442, 442f, 443f Membrane, plasma, 154–161. See also Plasma membrane Membranes, 153–154 Menadione, 853f, 855f Menaquinone, 853f, 855f MEOS. See Microsomal ethanol oxidizing system (MEOS) MERRF, 387, 387t Mesangium, 941, 941f Messenger, chemical, 172–176. See also Chemical messengers; specific messengers Messenger RNA (mRNA), 145, 191 degradation of, regulation of, 284, 284f in E. coli, 267, 267f promoter regions of genes for, 231–233, 231f, 232f

Lieberman_Subject_Index.indd 999

reading frame of, 251, 251f stabilization of, 145 structure of, 204, 204f synthesis of, 235–237, 235f–238f intron removal in, 235f, 237, 237f, 238f overview of, 235, 235f poly(A) tail addition in, 236–237, 236f transcription and capping in, 235–236, 236f transport and stability of, 284, 284f Metabolic acid, 41 buffers and, 47–50 bicarbonate and hemoglobin in red blood cells in, 45t, 48–49, 49f bicarbonate buffer system in, 48, 48f, 49f hydrochloric acid in, 50 intracellular pH in, 45t, 49–50, 49f principles of, 47 sources of, 47 urinary hydrogen, ammonium, and phosphate ions in, 45t, 46f, 50 Metabolic acidosis, 46, 410, 458 Metabolic capacities, by tissue, 34t Metabolic diseases, 39. See also specific diseases Metabolic encephalopathies, 918–920 glutamine synthesis and anaplerotic pathways of pyruvate carboxylase and methylmalonyl-CoA mutase in, 920 hypoglycemic, 918–919, 919f hypoxic, 920 Metabolic fatigue, in exercise, 895 Metabolic homeostasis, 478–480, 479f Metabolic homeostasis hormones, 480–482. See also specific hormones glucagon in, 480–481, 481f glucose, insulin, and glucagon levels in, blood, 481, 481f insulin counterregulatory hormones in, 477, 478f, 481, 482t insulin in, 477, 478f, 480, 480f metabolites in, circulating levels of, 481 Metabolic rate, resting, 8, 9t, 390 Metabolic syndrome, 27, 490, 622–624, 623f drugs for, 659 fibrates, 660 metformin, 659–660, 660f thiazolidinediones, 660–661 insulin resistance in, 484f, 623 NADPH from islet cell pyruvate cycling in, 623–624, 623f signs and symptoms of, 627–628 Metabolism. See also specific types energy from, 4 fuel, 1–2, 2f, 24, 333–334, 333f, 334f (See also specific fuels) oxygen in, 3 thermodynamics of, 342 Metabolites. See also specific reactions and types blood and urine measurements of, 36 circulating, on metabolic homeostasis, 481 Metachromatic leukodystrophy, 554t Metal ions, in catalysis, 122f, 126, 126f Metanephrine, plasma fractionated, 912 Metaphase, 194 Metastasis, 192, 194, 312, 939 Metathyroid diabetes mellitus, 813 Metformin, 659–660, 660f Methanol toxicity, 54 Methemoglobinemia, congenital, 828, 845t Methionine chemical structure of, 74f, 75t, 76 degradation of, 734, 734f from homocysteine, 752f

999

in homocystinuria, 731–732 in S-adenosylmethionine synthesis, 752–753, 753f sulfur in, 74f, 75t, 76 Methotrexate, 274 mechanism of action of, 750, 750f structure of, 749–750, 749f Methylated norepinephrine, 912 Methylation DNA, 273 in genomic imprinting, 273 Methyl-B12 deficiency, 734 Methyl caps synthesis of, 235–236, 236f types of, 235, 235f Methylmalonyl-CoA mutase, 920 Methylprednisolone for asthma, 561 muscle weakness from, 561, 564 Methyltetrahydrofolate deficiency, 734 Methyl-trap hypothesis, 753 Methylxanthines, on fuel metabolism, 487–488 Metronidazole, 867 Mevalonate, from acetyl-CoA, 630–632, 630f, 631f Micelles, 64, 588, 588f Michaelis–Menten equation, 137–138, 137f Microarrays, 300 Microfilaments, 166, 168f Microglial cells, 906 Micronodular cirrhosis, 879 MicroRNAs (miRNAs), 303 apoptosis and, 326 definition and function of, 206, 229–230 gene expression regulation by, 283–284, 283f in non-Hodgkin’s lymphomas, 283 as proto-oncogenes, 315 Microsomal enzymes, 868 Microsomal ethanol oxidizing system (MEOS), 136, 459f, 868 alcoholism on, 148, 165, 463 compartmentation in, 147 cytochrome P450 enzymes in CYPE2E1 in, 459, 461–462 induction of, 462 structure and function of, 459, 461, 461f function of, 136 Microsomal triglyceride transfer protein (MTP), 594–595, 595f, 608 Microsomal triglyceride transfer protein (MTP) inhibitors, 595 Microsomes, 165, 461 Microtubules, 165–166, 165f Microvascular complications, of diabetes mellitus, 480, 578–579, 940–942, 941f Midarm anthropometry, 27 Midarm arm muscle circumference (MUAMC), 27–28 Milk, human breast, 587 Mineralocorticoid synthesis, 651, 652, 653f Minerals. See also specific minerals dietary, 14–15, 15t dietary guidelines for, 16–17 Minor grooves, in DNA, 198, 199f Mismatch repair, 220, 221f Missense mutations, 251t, 252 Mitochondria, 163–164, 163f apoptosis and, 393 compartmentation of enzymes of, 374 cyanide poisoning on, 386 DNA in, 194–195 doxorubicin on, 379 glutathione and glutathione peroxidase in, 450 oxidative metabolism in, 333–334, 334f

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1000

INDEX

Mitochondria (continued) OXPHOS complexes in, genes for, 386 reduced numbers of, anaerobic glycolysis with, 524 ribosomes in, 205 superoxide dismutase in, 449–450 Mitochondrial disorders, 394t Mitochondrial integrity pathway, to apoptosis, 324, 324f Mitochondrial membranes, 374 pyruvate transfer across, 164–165 transport through, 390–392 in inner mitochondrial membrane transport, 391, 391f mitochondrial permeability transition pore in, 392, 392f in outer mitochondrial membrane transport, 391f, 392 overview of, 390 Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), 387t Mitochondrial permeability transition pore (MPTP), 392, 392f, 393 Mitochondrial porins, 391f, 392 Mitosis, 214 Mixed-function oxidase, 736–737, 738f Mixed triacylglycerols, 63 Moclobemide, 913 Moderate drinking, 459 Modulator proteins calcium–calmodulin family of, 144, 144f definition of, 144 Molecular biology, 191. See also specific topics ethical issues in, 192 general techniques of, 288 overview of, 191–192, 191f recombinant DNA techniques in, 288–308 (See also Recombinant DNA techniques) Moles, 223, 312 Monoamine oxidase (MAO), 441, 912, 912f, 913 Monoamine oxidase (MAO) inhibitors, 913 Monocarboxylic acids, blood–brain barrier transport of, 907–908 Monomeric G proteins, 144, 145f Mononuclear leukocytes, 824, 825 Mono-oxygenases, 349f, 350 Monosaccharide, 22, 58–61 basic structure and classification of, 58, 58f D- and L- sugars in, 58–59, 59f ring structures in, 59, 60f stereoisomers and epimers in, 59, 59f, 60 substituted sugars in, 59–60, 60f transport into tissues of, 507–508, 508f Monosodium urate crystals. See also Gout deposition of, 65, 163 phagocytosis of, 163 Monounsaturated fatty acids, 417 Morbid obesity, 10 Morphonuclear leukocytes, 824 Motifs, 93 Motilin, 813, 815t M phase, 215, 215f M protein, 101 mRNA–protein product relationship, 251, 251f mTOR, 690, 691f, 702, 702f mTOR kinase, 702 Mucin, salivary, 549, 551f Mucopolysaccharides, 934, 935f Mucopolysaccharidoses, 934, 938, 938t, 943t Multimer, 96–97 Multiple endocrine neoplasia (MEN), 689

Lieberman_Subject_Index.indd 1000

Multiple sclerosis (MS), 924, 925t Multiplexing, 301 Multisubstrate reactions, 138 Muscarinic receptors, acetylcholine, 174, 184 Muscle, 884–901 contraction of, 888, 889f fatigue of, in exercise, 895 fuel use in cardiac muscle, 891 skeletal muscle, 891–897 (See also Skeletal muscle) glucose metabolism in, after a meal, 25–26, 25f neuronal signals to, 887–889, 887f in muscle contraction, 888, 889f neuromuscular junction in, 887–888, 887f sarcoplasmic reticulum calcium release in, 887–888, 888f overview of, 884 thyroid hormone on, 812 training on, 900 weakness of, from glucocorticoids, 561, 564 Muscle cells cardiac, 885f, 887 glycolysis and fatty acid metabolism in, 889, 889f pathways in, 796 skeletal, 885–886, 885f smooth, 885f, 886–887 Muscle fibers, 796 exercising, ATP use in, 341, 341f type I, 886, 886t type II, 886, 886t Muscular dystrophy congenital, 934 Duchenne, 886, 901t Mushroom poisoning, Amanita, 246t Mutagens, 218–219, 218f, 219f Mutarotases, 59 Mutarotation, 59, 60f Mutases, 133 Mutations, 70, 191. See also specific types in cancer, 192, 310, 310f deletion, 251t, 252 detection of allele-specific oligonucleotide probes in, 298 polymerase chain reaction in, 298–299 from repetitive DNA, 299 restriction fragment length polymorphisms in, 298 frameshift, 251t, 252, 252f gain-of-function proto-oncogene, 313–314, 314f, 321 insertion, 251t, 252 loss-of-function, 314 missense, 251t, 252 multiple, in cancer, 326–327, 326f nonsense, 251t, 252 point, 78–79, 251t, 252 in repair enzymes, 314–315 silent, 251t, 252 transforming, 312 types and effects of, 251–252, 251t Myasthenia gravis, 172, 174, 189t acetylcholine receptor in, 174 diagnosis of, 187 testing for, 174 Myelin lipids in, 920–921, 922t structural proteins in, 922, 923t synthesis of, 921–923, 922f, 922t Myelin basic proteins (MBPs), 922–923, 922f

Myelin sheaths, 906, 921, 922f Myeloperoxidase, 46f, 446 Myocardial infarction (MI), 81, 87t, 110t, 394t, 455t creatine kinase after, 81, 85–86, 86f, 99 myoglobin release in, 99 pathophysiology of, 335, 378 protection against, 676t treatment of, 352, 378 Myocardial ischemia pathophysiology of, 453 reactive nitrogen–oxygen species in, 447 superoxide and reactive nitrogen-oxygen species in, 447 Myoclonic epilepsy and ragged red fiber disease (MERRF), 387, 387t Myofibrils, 885 Myoglobin, 97–100 from damaged muscle, 885 immunoassay of, 885 in myocardial infarction, release of, 99 oxygen binding and heme in, 99–100, 99f oxygen saturation curve for, 98, 98f structure–function relationships in, 97–100, 98f structure of chemical, 79–80, 80f vs. hemoglobin, 80, 80f histidine in, proximal, 99f, 100 on O2 saturation, 97–98, 98f Myokinase, 895, 895f Myosin, 885 Myosin ATPase, 341, 341f N Na⫹ cotransport with amino acids of, 699–700, 699f increased intracellular, 352 N-acetyl-glucosamine 6-phosphate synthesis, 549, 551f N-acetylglucosamine phosphotransferase mutation, 260 N-acetylneuraminic acid (NANA) synthesis, 871t, 873 N-acetyl-p-benzoquinoneimine (NAPQI) detoxification, cytochrome P450, 869–870, 870f NaCl, in isotonic saline, 45 ⫹ NAD , 124–125, 124f NAD⫹-dependent protein deacetylases, 434 Na⫹-dependent amino acid carriers, 700 Na⫹-dependent amino acid transport, 696, 699–700, 699f Na⫹-dependent glucose transporters cholera and, 510 mechanism of action of, 505–506, 506f NADH, 139 on alcohol dehydrogenase, 139 electron transfer to oxygen from, 379–380 NADH:CoQ oxidoreductase, 381, 382f NADH dehydrogenase, 381, 382f NADH/NAD⫹ ratio, ethanol on, 464–465, 464f NAD, oxidative fates of, 401–402, 402f NADPH for fatty acid synthesis, sources of, 601, 602f liver demand for, 873 in oxidation–reduction reactions, 347f, 349 pathways requiring, 539t pentose phosphate pathway for generation of, 537–539, 538f from pyruvate cycling in islet cells, 623–624, 623f NADPH oxidase, 446, 446f

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INDEX

NADPH oxidase defects, genetic, 446 NADP⫹ reduction, 346–347, 347f NAD⫹ reduction, 346–347, 347f Na⫹-glucose transporter, 160, 161f Na⫹/H⫹ exchanger, 352 Na⫹,K⫹-ATPase, 160, 160f Native conformation, 104 Natural killer (NK) cells, 825 Necrotizing encephalopathy, subacute, 369, 375t, 387t Negative control, 268 Neonatal diabetes, 485, 492t Neonatal jaundice, 548 Neonate blood glucose in 1–3 hours after birth, 519, 519f epinephrine on, 523 normal ranges of, 519, 519f glycogen in inadequate stores of, 519, 523, 525–526 regulation of metabolism of, 519, 519f Neoplasm. See also specific types definition of, 194 malignant, 310, 312 Neostriatum, 438 Nephropathy, diabetic, 579, 940–942, 941f Nerve gas, on acetylcholinesterase, 888 Nervous system, 903–925 amino acid utilization by, 786–787, 787f brain lipid synthesis in, 920–923 chemical messengers in, 175, 175f glucose metabolism in, after a meal, 23f, 25 glucose transport through, 508–509, 508f pathways in, 796 small nitrogen-containing neurotransmitter synthesis in, 908–918 (See also Neurotransmitter synthesis, of small nitrogen-containing neurotransmitters) Network-forming collagens, 930–931, 931f Neural tube defects, 755, 757, 758t Neurocrine signaling, 174 Neuroendocrine signaling, 175 Neuroendocrine tumors, chromogranins in, 912 Neurofibromatosis (NF-1), 321, 331t Neurofibromin (NF-1) action of, 321, 321f mutation of, 321, 321f Neuroglial (glial) cells astrocytes, 905–906 ependymal cells, 906 microglial cells, 906 oligodendrocytes, 906 Schwann cells, 906 Neuromuscular junction, 887, 887f Neurons, 905, 905f ATP requirements of, 401 glucose transport into, 508–509, 508f Neuropathy diabetic, 579 peripheral, 908 Neuropeptides, 175, 908. See also specific peptides Neuropeptide Y, 814t, 817 Neurotensin (NT), 814t Neurotoxins, 916 Neurotransmitter, 489. See also specific types drugs blocking uptake of, 909 Neurotransmitter synthesis amino acid pool and, 786 hypoxia on, 920

Lieberman_Subject_Index.indd 1001

Neurotransmitter synthesis, of small nitrogencontaining neurotransmitters, 908–918 acetylcholine in inactivation of, 916, 916f synthesis of, 915–916, 916f action in, 909, 909f arginine to nitric oxide in, 918, 918f aspartate in, 918 catecholamines in, 910–913 inactivation and degradation of, 912–913, 912f storage and release of, 911–912, 911f synthesis of, 910f, 911 tyrosine hydroxylase regulation in, 913 ␥-aminobutyric acid in, 917, 917f general features of, 908–909, 909t glutamate in, 916–917, 917f glycine in, 918 histamine in, 913–915, 915f overview of, 908 serotonin in, 913, 914f Neutrophils, elastase in, 698 Nevi, 312 Newborn hypoglycemia, 527t NGA, 867 N-glycosidic bonds, 61, 62f Niacin deficiency of, 13t for hyperlipidemia, 621 mechanism of action and efficacy of, 658t Recommended Dietary Allowance for, 13t synthesis of, 871t Nicotinamide adenine dinucleotide (NAD), oxidative fates of, 401–402, 402f Nicotinic acetylcholine receptor, 184 acetylcholine at, 174, 174f structure and action of, 173–174, 173f, 174f Niemann–Pick C1-like 1 protein (NPC1L1), 629 Niemann-Pick disease, 554t Nigrastriatal pathway, 438 Nilotinib, 327 Nitric oxide (NO), 440, 440f from arginine, 444f, 918, 918f function of, 918 as retrograde messenger, 918, 918f toxicity of, 444–445, 445f Nitric oxide synthase (NOS), 444, 444f Nitrogen excretion of, in fasting, 717, 717f excretory products of, 693, 694t metabolism of, 693–695, 693f, 694t, 695t Nitrogen balance, 12, 12t, 693, 713 Nitrogen balance, negative, 779, 788, 789f Nitrogen-containing compounds, 54, 64–66. See also specific compounds amino acids in, 54, 64–65, 65f charges on, 56–57, 58f definition and classification of, 54, 64 ring structures in in nucleosides and nucleotides, 62f, 65 in purines, pyrimidines, and pyridines, 65, 66f in tautomers, 66, 66f synthesis of, 871, 871t Nitrogen dioxide (NO2), 445, 445f Nitrogenous bases, 65, 66f Nitroglycerin, 444 Nitroprusside cyanide from, 385 as vasodilator, 385 Nitrosamines, DNA mutations from, 313, 313f NK cells, 825

1001

N-linked glycoproteins, 872–873, 873f N-myc, 313 Nomenclature, for biologic compounds, 57, 58f Nonactivated platelet, 850 Noncompetitive inhibition, 139, 140f Noncritical regions, 78 Nonesterified fatty acids (NEFA) in cirrhosis, 880 enzyme-coupled reactions and, 877 in liver disease, 878 in obesity, 622 Non–heme iron proteins, 382, 382f Non-Hodgkin lymphoma chemotherapy for, 266, 274, 283 diffuse large B-cell, 283 follicular type, 283, 286t methotrexate for, 749–750 types of, 283 Non–insulin-dependent diabetes mellitus (NIDDM). See Diabetes mellitus type 2 Nonketotic hyperglycemia, 480 Nonketotic hyperosmolar coma, 480 Nonpolar, aliphatic amino acids, 73–75, 74f, 75t Nonreceptor kinases, 279 Nonreducing ends, 514 Nonsense mutations, 251t, 252 Nonsteroidal antiinflammatory drugs (NSAIDs), on prostaglandins, 670, 670f Nontemplate strand, DNA, 230, 230f Nonthrombogenic surface, 857 Norepinephrine, 910–913 on fuel metabolism, 798t, 805–807, 806f inactivation and degradation of, 806, 912–913, 912f measurements of, 439 metabolism of, 806 methylated, 912 physiologic actions of, 481, 482f, 805–806, 806f secretion of, 805 signal transduction by, 489–490, 489f storage and release of, 911–912, 911f in stress response, 175 structure and synthesis of, 489, 489f, 910f, 911 tyrosine hydroxylase regulation in, 913 Northern blots, 293, 294f NO synthase, 918 Nuclear-encoded proteins, mitochondrial matrix import of, 374–375, 374f Nuclear localization signal (NLS), 164 Nuclear receptors, 274–276, 275f Nuclear respiratory factors (NRF-/NRF-2), 388 Nucleic acid structure, 193–208. See also specific nucleic acids of chromosomes, 201–203 of DNA, 194–200 overview of, 193–194 of polynucleotides, 193, 193f of retroviruses, 206–207, 207f of RNA, 203–206 Nucleoid, 168 Nucleolus, 164, 164f Nucleophilic catalysis, 118f–119f, 119 Nucleosides, 195, 195f, 195t chemical structure of, 54 structure of, 62f, 65, 195, 196f Nucleoside triphosphates. See also specific types high-energy phosphate bonds in, 344–345 Nucleosome cores, 201, 202f Nucleotide-binding fold, 94f, 95 Nucleotide excision repair (NER), 219, 220f

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1002

INDEX

Nucleotide reverse transcriptase inhibitors (NRTIs), 213, 295 Nucleotides. See also specific types biosynthesis of, 872 in carbohydrate synthesis, 475, 475f chemical structure of, 54 function of, 760 structure of, 62f, 65, 193, 195, 196f Nucleotide sugar interconversions, 545–549 glucuronide formation in, 546–547, 547f, 547t lactose synthesis in, 547–549, 548f sugar formation for glycolipid and glycoprotein synthesis in, 549, 549t, 550f, 551f UDP-galactose synthesis in, 547, 548f UDP-glucose reactions in, 545–546, 545f UDP-glucuronate in, 546, 546f, 547f Nucleus, 164, 164f Nutrients. See also specific nutrients essential, 10 Nutritional anemias, 838–839 O Obesity, 19t, 353t, 375t, 413t, 435t, 470t, 624t android, 27 definition of, 10, 620 in diabetes mellitus type 2, 490 epidemiology of, 17, 620 etiology of, 622 gynecoid, 27 as modern problem, 622 morbid, 10 O blood group, 549, 554f, 555 Octanoylglycine, 423, 427 Octreotide, 801 O-glycosidic bonds, 61, 62f Oils fish, 11 plant, 11 Okazaki fragments in eukaryotes, 216–217 in prokaryotes, 213, 214f Oleate, 6f Oleic acid, 61–62, 63f Olestra, 591 Oligodendrocytes, 906 Oligonucleotides, 205, 291 Oligosaccharides, 22, 61, 62f O-linked glycoproteins, 872–873, 873f ␻-oxidation, of fatty acids, 427, 427f Oncogenes, 312, 315–319 cell cycle and, 215f, 317–319, 317f definition of, 321 pathophysiology of, 315 ras, 312 signal transduction cascade and, 315–317 growth factors and growth factor receptors in, 315 proto-oncogenes in, 315, 317f signal transduction proteins in, 180f, 315–316, 316t, 317f transcription factors in, 316–317, 317f Oncogenic virus, 314 One-carbon groups oxidation and reduction of, in tetrahydrofolate, 747, 748f recipients of, 746f, 747–749, 748f, 749t, 750f sources of, 747, 749f, 749t synthesis of, 871t One-carbon pool, 747, 748f overview of, 744, 744f sources and recipients of, 749t Operator, 268

Lieberman_Subject_Index.indd 1002

Operon, 232, 232f inducible, 268–269, 268f lac, 268–270, 268f, 270f in prokaryotes, 267, 267f repressible, 269, 269f trp, 269, 270–271, 271f Opposing pathways, counterregulation of, 147 Organelles, 154 Organic molecules, 54. See also specific molecules Organophosphate compounds, 113, 113f acetylcholinesterase inhibition by, 127, 128f poisoning with, 113 oriC, 210, 211f Orientation, 119 Orlistat, 592 Ornithine, 701 from arginase reaction, 715 origin of, 715–716, 716f Ornithine aminotransferase reaction, 716, 716f Ornithine transcarbamoylase (OTC) deficiency, 192, 722t gene therapy for, 720 incidence of, 718 late-onset, 718 orotic aciduria from, 769 pathophysiology of, 715 Orotate 5'-phosphate decarboxylase, defective, 768f, 769 Orotate phosphoribosyltransferase, defective, 769 Orotic acid (orotate), 715 Orotic aciduria hereditary, 768f, 769, 772t, 773t from ornithine transcarbamoylase deficiency, 769 Osmolality, 44 of isotonic saline, 45 of water, 44 Osmotic diuresis, 44, 563 Osmotic pressure, 44 Osteoarthritis, on extracellular matrix, 934 Osteogenesis imperfecta (OI), 931, 942, 943t Osteomalacia, 15, 19t Osteopenia, 726 Osteoporosis, 15, 19t, 726 Outpouches, 323 Overweight, 10 Oxaloacetate amino acids related to, 732, 733f succinate oxidation to, 358f, 359 Oxaluria, 728, 728f, 741t, 742t Oxidase, 131, 349, 349f free radical intermediates from, 441 mixed-function, 736–737, 738f Oxidation, 56. See also specific reactions and sites ␤-, 420–424 (See also ␤-oxidation) in mitochondria metabolism, 333–334, 334f substrate-level phosphorylation and, 399–401, 400f Oxidation, fuel, 1, 345–349. See also specific pathways anaerobic glycolysis in, 349, 349f dietary, 5–6, 5f energy transfer through oxidative phosphorylation in, 345–349 (See also Oxidative phosphorylation (OXPHOS)) in fasting state, 30, 335 mechanisms of, 333–334, 333f, 334f metabolic problems of, 335 NADPH in oxidation–reduction reactions in, 347f, 349 overview of, 333–334, 333f, 334f

principles of, 345 in skeletal muscle, 25–26, 25f Oxidation–reduction coenzymes in, 123–126, 125f in electron-transport chain, 381–383 coenzyme Q in, 382–383, 382f copper and oxygen reduction in, 382f, 383 cytochromes in, 382f, 383, 383f NADH:CoQ oxidoreductase in, 381, 382f overview of, 347f, 381, 382f succinate dehydrogenase and other flavoproteins in, 381–382, 382f NADPH in, 347f, 349 reactions of, 346–347, 347f, 349 Oxidative fibers, 408 Oxidative phosphorylation (OXPHOS), 5, 379–385 ATP from, 386 cyanide on, 385, 385t, 386 energy transfer from fuels through, 345–349 caloric values of fuels in, 348–349 overview of, 345–346, 346f oxidation–reduction reactions in, 346–347, 347f reduction potential in, 348, 348t energy yield from electron-transport chain in, 384–385 in heart, 338 inhibitors of, 385, 385t mitochondria genes in, 386 overview of, 378f, 379–381 ATP synthase in, 380–381, 380f binding-change mechanism in, 381, 381f electrochemical potential gradient in, 380, 380f electron transfer from NADH to oxygen in, 379–380 oxidation–reduction components of electrontransport chain in, 381–383 (See also Oxidation–reduction) proton pumping in, 383–384, 384f respiratory chain inhibition and sequential transfer in, 382f, 385 Oxidative phosphorylation (OXPHOS) disorders, 385–388 clinical pathology of, 385 mutations in gene, 386, 387t mitochondria DNA, 386–387, 387t mitochondria gene, 386–387, 387t nuclear DNA, 387–388 Oxidative stress, 438, 438f Oxidized functional groups, 56, 56f Oxidized sugars, 60–61, 61f Oxidoreductases, 131 Oxygen (O2) ADP concentration on consumption of, 388, 388f as biradical, 439, 439f electron transfer from NADH to, 379–380 in metabolism, 3 oxidase use of, 349, 349f oxygenase use of, 349f, 350 radical nature of, 439, 439f reduction of, 439, 439f singlet, 440t Oxygenases, 131, 349f, 350f, 351, 441 Oxygen binding heme and, 99–100, 99f in hemoglobin, 100, 101f to hemoglobin, agents on 2,3-bis-phosphoglycerate, 834, 834f carbon dioxide, 834–835, 834f, 835f proton binding (Bohr effect), 834, 834f, 835f

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INDEX

Oxygen therapy in premature infants, ROSinduced retinopathy from, 449 Oxygen toxicity and free radical injury, 437–455 cellular defenses against oxygen toxicity in, 447–452 antioxidant scavenging enzymes in, 448–449, 448f compartmentation in, 447–448, 447f nonenzymatic antioxidants (free radical scavengers) in, 449–452 ascorbic acid, 449, 450f carotenoids, 449–451, 451f endogenous, 452, 452f flavonoids, 451–452, 451f vitamin E, 447f, 449, 450, 450f free radical formation in phagocytosis and inflammation in, 446–447, 446f free radical injury in, diseases with, 437, 437t nitric oxide and reactive nitrogen–oxygen species in nitric oxide synthase in, 444, 444f nitric oxide toxicity in, 444–445, 445f reactive nitrogen–oxygen species characteristics in, 440, 440t O2 and reactive oxygen species generation in, 438–441 O2 as biradical in, 439, 439f overview of, 438–439 primary reactive oxygen species cell sources in, 440–441, 441f radical nature of O2 in, 439, 439f reactive oxygen species characteristics in, 439–440, 440f, 440t oxidative stress in, 438, 438f oxygen radical reactions with cellular components in, 441–444 DNA in, 444, 444f membrane attack in, 442, 442f, 443f overview of, 441, 442f proteins and peptides in, 442–443 ozone protection in, lung lining fluid in, 454–455, 454f Ozone, lung lining fluid protection against, 454–455, 454f P p16 mutation, 329 p53 mutation, 329 p53 protein on cell cycle and apoptosis, 320, 321f mutation of, in Li-Fraumeni syndrome, 320 Pain, in inflammatory process, 676 Palindrome, 278, 290–291 Palmitate methyl group of first acetyl-CoA in, 604f, 605, 607 structure of, 6f Palmitic acid, 61, 421–422 Palmitoleic acid, 61–62 Palmitoyl-CoA elongation, 605, 606f Pancreas thyroid hormone on, 812 zymogens in, 700 Pancrease, 483 Pancreatic ␣-amylase, 497–498, 497f, 587 Pancreatic enzymes. See also specific types protein digestion by, 698–699, 698f, 699f Pancreatic esterase, 588, 589f Pancreatic islet cells, control of secretions of, 817 Pancreatic lipase, 22–23, 587–588, 588f inhibition of, for weight loss, 592 in pancreatitis, 587 Pancreatic polypeptide (PP), 813, 815t

Lieberman_Subject_Index.indd 1003

Pancreatitis alcohol-induced, 498, 587, 590, 594 mechanisms of, 700 pancreatic amylase and lipase in, 587 Pantothenate deficiency of, 362 definition of, 373 as exercise supplement, 420 in TCA cycle, 124f, 361–362 Pantothenic acid, 13t Pantothenic acid deficiency, 13t Paracrine actions, of chemical messengers, 174, 175f Paralogs, 79 Parental strand unwinding, in prokaryotes, 211, 211f Parenteral tube feeding, 540 Paresthesias, 51 Parkinson disease, 455t homovanillylmandelic acid in, 913 Lewy bodies in, 442, 453 pathogenesis of, 452 pathophysiology of, 452–453 brain structures in, 438 reactive oxygen species and reactive nitrogen–oxygen species in, 452–453, 452f presentation and diagnosis of, 438 PARP-1 inhibitors, 330 Partial charges, 57, 57f Partial fatty acid oxidation (pFOX) inhibitors, 891 Partial pressure of CO2 (PaCO2), 48 Passive transport, 158, 158f, 159f patched coreceptor gene, 321, 322f, 327 patched/smoothened signaling system, 321, 322f, 327 PCO2 measurement, 42 Pearson syndrome, 387t Peas, indigestibility of, 504–505 Pegvisomant, 801 Pelizaeus–Merzbacher disease, 924 Pellagra, 13t, 704, 738 Pendred syndrome, 809 Pendrin, 809 Penicillin, 127–129, 128f Pentose phosphate pathway, 530f, 534–539, 539t balanced sequence of reactions in, 537, 538f in liver, 873 in NADPH generation, 537–539, 538f nonoxidative phase of, 535–537 overview of, 535 ribose-5-phosphate generation from intermediates of glycolysis in, 537 ribose-5-phosphate to glycolytic intermediates in, 535–537, 536f, 537f overview of, 530f oxidative phase of NADP production in, 534, 535f overview of, 530f, 534 ribose 5-phosphate from, 534–535 Pepsin, 22, 23f, 698, 698f, 699f Pepsinogen, 697, 698f Peptide backbone, 3D structure of, 90f, 91 Peptide bonds formation of, 256f, 257 in polypeptide chains, 70, 73, 73f Peptides. See also specific peptides oxygen radical reactions with, 442–443 Peptide YY (PYY), 813, 815t Peptidoglycan, 154 Peptidyltransferase, 204, 257 Perilipins, 622

1003

Peripheral membrane proteins, 157 Peripheral neuropathy, 908 Perisinusoidal cells, 865–866, 881 Pernicious anemia, 758t pathophysiology of, 751–752 vitamin B12 for, 751–752 Peroxidases, free radical intermediates from, 441 Peroxidation, lipid, 442, 442f, 443f Peroxisomal diseases, 164 Peroxisomal oxidation of long-chain branched-chain fatty acids, 426, 426f of very-long-chain fatty acids, 425–426, 425f, 426f Peroxisomal proliferator–activated receptors (PPARs), 876–877, 877t Peroxisome proliferator–activated receptor-␣ (PPAR␣), 660, 877 Peroxisome proliferator–activated receptor-␥ (PPAR␥), 660–661 Peroxisomes, 164 Peroxyl radical, 440t Peroxynitrite, 440t, 445, 445f Pertussis, 263t PEST sequences, 703 Peutz-Jeghers syndrome (PJS), 659 pH body, 778 for enzyme reactions, 126–127, 127f on hypothalamic respiratory center, 49 intracellular, buffers and, 45t, 49–50, 49f urinary, 45t, 50 of water, 41, 41f, 44–45 Phage, 195 Phagocytosis, 163, 446–447, 446f Phase I reaction, 867 Phase II reaction, 867 Phenobarbital, 462, 832 Phenoxybenzamine, for pheochromocytoma, 912, 912f Phentermine, 923 Phenylacetate, 719, 719f Phenylalanine, 733 bonds in, 75, 75f chemical structure of, 74f, 75, 75t degradation of, 736, 737f dietary restriction of, 739–740 hydroxylation of, 736–737, 738f Phenylalanine hydroxylase (PAH), 736–737, 738f, 910f, 911 Phenylbutyrate, for urea cycle disorders, 718–719, 719f Phenylethanol-amine N-methyltransferase, 910f Phenyl group, 55, 55f Phenylketonuria (PKU), 741, 741t, 742t mental retardation mechanisms in, 908 screening for, 726 Pheochromocytoma, 806–807, 925t catecholamine secretion in, 923 chromogranins in, 912 pathophysiology of, 908 presentation and diagnosis of, 908 Philadelphia chromosome Bcr-Abl protein in, 327 in chronic myelogenous leukemia, 274, 284, 311, 315 karyotype of, 311 Phlorizin hydrolase, 500 Phosphatase and tenin homolog (PTEN), 180 Phosphate bonds, high-energy, 344–345 activated intermediates with, 344–345 in ATP, 338, 338f (See also Adenosine triphosphate (ATP))

01/09/12 9:36 PM

1004

INDEX

Phosphate group, 56–57, 57f Phosphate ions, as buffer, 41, 41f, 45t, 50 Phosphatidic acid, 63, 64f Phosphatidylcholine, 63, 64f, 916 in amniotic fluid, 621, 621f dietary, 917 neonatal demand for, 917 in plasma membrane, 155, 155f synthesis of, 614, 614f Phosphatidylinositol, 155–156, 155f, 180 Phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-trisP), 180, 180f Phosphatidylinositol phosphates, in signal transduction, 180, 180f Phosphatidylinositol signaling, by heptahelical receptors, 173f, 186–187 Phosphatidylserine, 156, 323 Phosphoacylglycerols (phosphoglycerides), 54, 63, 64f 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) in proteoglycan synthesis, 934–936 synthesis of, 618, 619f Phosphodiesterase, methylxanthines on, 487 Phosphoenolpyruvate (PEP) on fatty acid release, 612, 612f high-energy phosphate bonds in, 345, 345f Phosphoenolpyruvate carboxykinase (PEPCK), 280, 280f induction of, 568–570 regulation of, 684f regulatory region of gene for, 279, 280f Phosphoenolpyruvate kinase (PEPK), 612–613, 612f Phosphoesters, 56f, 57, 58f Phosphofructokinase-1 (PFK-1) deficiency, 518t Phosphofructokinase-1 (PFK-1) regulation, 406f–408f, 407–409, 684f allosteric, by AMP and ATP, 408, 408f by allosteric inhibition, at citrate site, 409 by fructose 2,6-bis-phosphate, 408–409 role of, 406f, 407 types of, 407–408 Phosphofructokinase-2 (PFK-2), 889 Phosphoglucomutase (PGM) high galactose 1-phosphate concentrations on, 547–548 reaction catalyzed by, 339–340, 340f reversibility of reaction catalyzed by, 339, 340f, 344, 515 6-Phosphogluconate, 534, 546–547 Phospholipase bonds cleaved by, 617, 617f on glycerophospholipids, 617–618, 617f Phospholipase A2 in arachidonic acid release from membranes, 665–666, 665f synthesis and function of, 588, 589f Phospholipase C, 665f, 666 Phospholipids, plasma membrane, 155, 155f 5-Phosphoribosyl-1-pyrophosphate (PRPP) regulation of purine biosynthesis via, 761f, 762–763, 764f synthesis of, 761, 761f Phosphoribosyl pyrophosphate (PRPP), in gout, 771 Phosphoribosyl transferase enzymes, 765, 766f Phosphoric acid, from fuel metabolism, 778 Phosphorus, 14 Phosphorus deficiency, 15 Phosphorylase, 517, 517f. See also Glycogen phosphorylase Phosphorylase a, 683f Phosphorylase b, 683f

Lieberman_Subject_Index.indd 1004

Phosphorylase kinase, 520, 521f calcium/calmodulin on, 524, 525f regulation of, 683f, 684 Phosphorylation, 142, 142f oxidative (See Oxidative phosphorylation) substrate-level, 335, 359, 399–401, 400f Phosphorylation cascade, 180, 316, 317f Phosphorylation-gated channels, 158 Phosphoryl transfer reactions, 342–343, 343f, 343t Phylloquinone, 853f, 855f Physical activity. See also Skeletal muscle energy for, 9, 9t Phytanic acid oxidation of, 426, 426f in Refsum disease, 425 Phytosterolemia, 629 Pit cells, 866 Pituitary adenylate cyclase activating peptide (PACAP), 814t Pituitary gland tumors, ACTH and cortisol levels in, 810 Pituitary macroadenoma, 801 pKa, 41 Plant oils, 11 Plants, gluconeogenesis in, 579, 579f Plaques, atherosclerotic, 16 Plasma ATPase (P-ATPase), 341–342 Plasma-binding proteins, 848–849, 849t Plasma cell dyscrasia, 101 Plasmalogen, 164, 597, 598f Plasma membrane, 154–161 molecule transport across, 157–161 active transport in, 158f, 160–161, 160f, 161f endocytosis in, 157, 158f facilitative diffusion in, 158, 158f, 159f gated channels in, 158–159, 158f, 159f simple diffusion in, 157–158, 158f structure of, 154–157 fundamentals of, 155–156, 155f glycocalyx of, 155, 157 lipids in, 156–157, 156f proteins in, 156–157, 156f, 157f vesicular transport across, 161 Plasma membrane receptors, 176–179, 176f Plasma proteins. See Protein, plasma Plasmids, 195, 295 Plasmin, 301, 857, 858, 858f Plasminogen, 857–858 Plasminogen activators, 857–858, 858t Platelet-derived growth factor (PDGF), 176, 881 Platelet factor 4 (PF4), 859 Platelets, 825, 850 activation of, 850–851, 851f formation of, 850 in hemostasis, 796 nonactivated, 850 Pleckstrin homology (PH) domain, 181, 181f Pneumocystis, 263t Pneumonia, community-acquired, 194 Pneumonitis, 208t PNP, 773t PO2 measurement, 42 Point mutations, 78–79, 251t Points of origin for replication, eukaryote, 215, 216f Polar bonds, 57 Polar groups, 57 Polar organic molecules, 43 Polyadenylation signal (AAUAAA), 236–237, 236f Polyadenylation sites, 281, 281f Poly-ADP ribose polymerase (PARP-1) inhibitors, 330

Polycistronic mRNA, 267, 267f Polycistronic transcript, 232f, 233–234 Polydipsia, from glucocorticoids, 561 Polymerase chain reaction (PCR), 297–299, 297f Polymerases bypass, 215 DNA in eukaryotes, 215, 216t in prokaryotes, 212–213, 212f, 212t Polymorphic markers, 299 Polymorphisms, 70, 79 DNA definition and types of, 298 detection of, 298–300, 299f fingerprinting and, 307 restriction fragment length, 298 single nucleotide, 307 Polyneuritis, with beriberi, 373 Polynucleosome, 201, 202f Polynucleotide, 193, 193f Polynucleotide chain, DNA, 196, 197f Polyol pathway, fructose synthesis in, 532–533, 532f Polypeptides in blood, 701 chains of, 70 Polyps, adenomatous, 194 Polysaccharides, 61, 62f Polysaccharide starch, 5, 5f Polysomes, 258, 258f Poly(A) tail addition, in mRNA synthesis, 236–237, 236f Poly(A) tail synthesis, 236–237, 236f Polyubiquitinylation, 703 Polyunsaturated fatty acids, 54 dietary, 417 in eicosanoid synthesis, 606 source of, 606 structure of, 6f, 583 synthesis of, 606 Polyuria, 44, 561 Pompe disease, 162, 517, 518t Porins, 163 Porphobilinogen synthesis, 828, 830f Porphyria, 795, 845t Porphyrin, heme, 828, 828f Portal hypertension, 872 Portal–systemic encephalopathy, 716 Portosystemic shunting, 881 Positional cloning, 299 Positive control, 268 Positive cooperativity, 100, 101f Poststreptococcal glomerulonephritis (PSGN) kidney inflammation in, 893 pathogenesis of, 900 pathophysiology of, 893 presentation and diagnosis of, 885 Posttranscriptional RNA processing alternative splicing and polyadenylation sites in, 281, 281f overview of, 281 RNA editing in, 282, 282f Posttranslational modifications of amino acids, 71, 82–84, 83f in protein synthesis, 258–259, 259t Potassium, 14 Potocytosis, 161 Prader-Willi syndrome, 273, 286t Prednisone, for non-Hodgkin lymphoma, 266 Pregnancy alcohol consumption in, 16 glycogen metabolism regulation in, 519, 519f Premature ventricular contractions, 438

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INDEX

Pre-mRNA, 227, 234, 235, 235f Prenylation, of amino acids, 83f, 84 Preprocollagen, 932 Pressure-gated channels, 158 Pribnow box, 231–232, 231f Primary active transport, 160 Primary oxaluria type I (PH 1), 728, 728f, 741t, 742t Primary structure. See also specific substances of amino acids (See Amino acid structure, primary) in protein folding, 103–104, 104f of proteins, 70, 89f, 90 Primase, 217t Prime, 196f, 203 Prion, 105 Prion diseases, 105–106, 106f, 110t Prion protein, 157 Proapoptotic signals, BCL-2 family, 324–325, 325f, 325t Probes, 292, 292f, 298 Procarboxypeptidases, 697, 697f, 698, 698f, 699 Processing, protein, 258 Processivity, 212–213 Procollagen(I), 928–929 Product inhibition, in metabolic pathways, 139–140 Proelastase, 697, 697f, 698, 698f Proenzymes, 851 Progenitor cells, 285 Progesterone synthesis, 652, 653f, 656 Progestin synthesis, 653f, 656 Progestogen synthesis, 652, 653f Proglucagon, 799 Programmed cell death, 322–326, 352–353. See also Apoptosis Prohormones, 477–478 Proinsular effect, 819 Prokaryotes definition of, 154, 267 DNA synthesis in, 210–213 base-pairing error elimination in, 212t, 213 bidirectional replication in, 210–211, 211f DNA ligase in, 213, 214f DNA polymerase in, 212–213, 212f, 212t DNA synthesis at replication fork in, 213, 214f parental strand unwinding in, 211, 211f RNA primers in, 213, 214f semiconservative replication in, 209f, 211, 211f Escherichia coli as, 267, 267f gene expression regulation in, 267–271 corepressors in, 269, 269f inducers in, 268–269, 268f mRNA in, 267, 267f operons in, 267, 267f RNA polymerase binding repressors in, 267–269 corepressors in, 269, 269f inducers in, 268–269, 268f mRNA in, 267, 267f operons in, 267, 267f RNA polymerase binding stimulation in, 269–270, 270f sigma factors in, 270 transcription attenuation in, 270–271, 271f genes of, 203 Proliferating cell nuclear antigen (PCNA), 217t Proline chemical structure of, 73, 74f, 75t as helix breaker, 91 synthesis and degradation of, 732, 732f

Lieberman_Subject_Index.indd 1005

Promoter-proximal elements, 229, 231f, 232, 233, 239 Promoters (promoter regions) in eukaryotes, 231f, 232–233, 232f of mRNA genes, 231–233, 231f, 232f multiple regulators of, 279–280, 280f in prokaryotes, 231–232, 231f, 232f for RNA transcription, 227, 227f, 229, 230, 231f for tRNA transcription, 239, 240f Propionate, 564 Propionyl-CoA, 564, 565 from odd-chain fatty acids, 423, 423f synthesis of, 423, 423f, 733–734 Propionyl CoA-to-succinyl CoA pathway, 423, 423f Propranolol, for pheochromocytoma, 912 Prostacyclin (PGI2), 176, 176f Prostacyclin (PGI2) synthesis, 667–670, 669f Prostaglandin (PG). See also Eicosanoid drugs blocking, 670, 670f functions of, 668, 669t inactivation of, 670 metabolism of, 664f nomenclature for, 666, 666f–668f radioimmunoassay of levels of, 667 structure of, 666, 666f–668f synthesis of, 666–670 Prostaglandin E1 biosynthesis of, 667–668, 669f drug analogs of, 675 structure of, 666, 666f–668f Prostaglandin E2 biosynthesis of, 667–668, 669f drug analogs of, 675 structure of, 666, 666f–668f Prosthetic groups, 99 Proteases, 22, 23f, 701, 701t Proteasome, 305, 703, 703f Protein absorption of, 6f, 22, 23f acute-phase, 872 adhesion, 939 on coagulation cascade, 856, 856f definition of, 39 in fed state, 6f, 21, 21f, 22, 23f function and storage of, 7t, 8, 70 globular, 90 half-life of, 701 oxidation of, 4–5, 4f oxygen radical reactions with, 442–443 plasma-binding, 848–849, 849t in plasma membrane, 156–157, 156f, 157f recycling of, 701–702 regulatory gene-specific, 274–276, 275f tissue isozymes of, 147 subcellular/extracellular targeting of, 259–260, 259f, 260f synthesis of in liver, blood proteins in, 872 in skeletal muscle, 777f, 780 therapeutic production of complex human proteins, 301–302 insulin and growth hormone, 301–302, 302f tissue location of, 775 transmembrane, 90, 95–96, 96f in urine, detecting, 89 Protein C, 851–852, 856, 856f Protein-calorie malnutrition, 13, 31 Protein C deficiency, 856 Protein databases, 86 Protein degradation. See also specific proteins regulated, 145 in skeletal muscle, 777f, 780

1005

Protein denaturation misfolding and prions in, 105–106, 106f nonenzymatic modification in, 105, 105f temperature, pH, and solvents in, 105 Protein, dietary caloric content of, 5t, 6 essential amino acids in, 11 fat stores from, 611 guidelines for, 16 high-protein meal on amino acid metabolism and, 787–788, 788f nitrogen balance of, 12, 12t quantity and quality of, 11 recommended daily intake of, 704 Protein digestion, 6f, 22, 23f, 697–699 enzymes as zymogens in, 697, 697f, 698f by intestinal cell enzymes, 699 overview of, 697, 697f by pancreatic enzymes, 698–699, 698f, 699f rate of, 704 in stomach, 697–698 Protein families, 79–80 Protein, fibrous, 90, 928–934 collagen in, 928–932 (See also Collagen) elastin in, 932–933, 933f laminin in, 933–934, 933f Protein folding denaturation and, 105–106, 106f heat-shock proteins in, 104, 104f misfolding in, 105–106, 106f native conformation in, 101–103 primary structure in, 103–104, 104f Protein inhibitors of activated STAT (PIAS), 182 Protein kinase, 142, 142f Protein kinase A (PKA), 142–143, 143f activated, 279 cAMP on, 148 regulation of, 683f, 684 in signal transduction, 488–489 targets of, 186 Protein kinase B (Akt), 182, 182f Protein kinase, dedicated, 142–143, 143f Protein malnutrition, 13 severe (kwashiorkor), 13, 19t, 698, 706t, 848, 861t thyroid hormone and transthyretin in, 787 Protein phosphatase, 142, 142f, 521 Protein phospholamban (PLN), 901 Protein phospholamban (PLN) mutations, 901 Protein, plasma, 848–861 blood-tissue water distribution by albumin in, 848–849, 849t body fluid maintenance in, 848 in circulatory system integrity, 849–858 blood coagulation cascade in, 851–852, 853f, 853t blood coagulation process in, 852–856 (See also Coagulation, blood) blood loss and, 849 feedback amplification and inhibition in, 853f, 856–857, 856f fibrinolysis in, 857–858, 858f fibrinolysis regulation in, 858 hemostatic plug formation in, 850–851, 851f hemostatic plug role in, 849 plasmin activation in, regulation of, 858, 858f vascular endothelium thromboresistance in, 857 composition and function of, 848 in immune defense, 849 synthesis of, in liver, 872, 872t

01/09/12 9:36 PM

1006

INDEX

Protein–protein interactions, conformational changes from, 143–145, 143f–145f Protein S, 851 Protein S deficiency, 856 Protein structure, 5–6, 6f amino acids in (See Amino acid structure, primary) amino acid substitutions in, 70 primary, 70, 89f, 90 quaternary, 89f, 90, 96–97 secondary, 89f, 90 tertiary, 89f, 90 three-dimensional, 89f, 90 Protein, structure–function relationships in, 88–110 3D peptide backbone structure in, 90f, 91 3D structure in, 89f, 90 in immunoglobulins, 100–101, 102f, 103f ligand binding quantification in, 97 in myoglobin and hemoglobin, 97–100 cooperativity of O2 binding in hemoglobin in, 100, 101f, 108–110, 109f oxygen binding and heme in, 99–100, 99f structure on O2 saturation of, 97–98, 98f overview of, 88 protein folding in, 101–106 denaturation and, 105–106, 106f native conformation in, 101–103 primary structure in, 103–104, 104f quaternary structure of, 96–97 secondary structure of, 91–94 ␣-helix in, 91, 91f, 92f ␤-sheets in, 91–92, 92f definition of, 91 nonrepetitive, 92, 93f, 94f patterns of, 93, 94f tertiary structure of, 93–96 basic, 93, 94f globular protein folds in, 94–95, 94f globular protein solubility in, 95 structural domains in, 93–94, 94f in transmembrane proteins, 95–96, 96f Protein superfamilies, 79 Protein synthesis, 145, 248–263. See also specific proteins aminoacyl-tRNA formation in, 252–253, 252f, 253f antibiotics inhibiting, 261–262, 261t as dynamic process, 693 genetic code in, 248–251, 250f, 250t (See also Genetic code) in liver, 872, 872t mRNA–protein product relationship in, 251, 251f mutation types and effects in, 251–252, 251t polysomes in, 258, 258f posttranslational modifications in, 258–259, 259t processing in, 258 regulation of, 271–284 (See also Gene expression regulation, in eukaryotes) subcellular/extracellular targeting of, 259–260, 259f, 260f translation in, 253–258 (See also Translation) Protein turnover, 701–703, 701t, 775, 779 lysosomal, 702, 702f proteases in, 701, 701t protein recycling in, 701–702 ubiquitin–proteasome pathway in, 703, 703f Proteoglycan, 54, 60–61, 475, 475f, 934–938 in chondrocytes, 939, 940f degradation of, 936–937 secretion and aggregation of, 936, 937f, 938f structure and function of, 934, 935f, 936f, 936t synthesis of, 872–873, 934–936, 937f, 938f

Lieberman_Subject_Index.indd 1006

Proteolipid protein (PLP), 922, 922f Proteolysis, 33 Proteolytic cleavage, enzyme regulation by, 145 Proteomics, 305–306, 305f Proteosomes, 145 Proteus syndrome, 321 Prothrombin, 145, 854 Protomer, 96–97 Proton, 44, 44f Proton binding, 834, 834f, 835f Proton-coupled folate transporter (PCFT) mutation, 747 Proton ionophores, 389, 389f Proton leak global, 390 resting metabolic rate and, 390 Proton motive force, 380, 380f Proton motive Q cycle, for b-c1 complex, 384, 384f Proton pumping, in oxidative phosphorylation, 383–384, 384f Proton wire, 384 Proto-oncogenes, 192 Abl, 315 amplification of, 313 in cancer etiology, 310f, 312 classes of, 315, 316t gain-of-function mutations of, 313–314, 314f, 321 microRNA as, 315 pathophysiology of, 315 phosphorylation cascade in activation of, 316, 317f in signal transduction cascade, 315, 317f transposition and translocation of, 313, 314f Proximal histidine, 99f, 100 Proximity, 119 PRPP synthetase, 761f, 762–763, 764f PubMed, 86 Purine chemical structure of, 54 dietary, 760 function of, 760 salvage pathways for, 764–765, 765f structure of, 65, 66f synthesis of, 871t Purine bases degradation of, 769–770, 770f structure and nucleosides of, 195, 195f, 195t Purine biosynthesis, 761–765 de novo, 761–764 adenosine monophosphate and guanosine monophosphate phosphorylation in, 761f, 762 adenosine monophosphate synthesis from inosine monophosphate in, 761f, 762, 763f guanosine monophosphate synthesis in, 762, 763f inosine monophosphate synthesis in, 759f, 761–762, 761f, 762f regulation of, 762–764, 764f overview of, 761, 761f purine salvage pathways in, 764–765, 765f Purine metabolism, 759, 759f Purine nucleoside phosphorylase functions of, 764–765 reaction of, 764–765, 765f Purine nucleoside phosphorylase (PNP) deficiency, 764, 773t Purine nucleotide cycle, 783–784, 784f in long-term exercise, 766f, 899 in skeletal muscle, 783, 784f Pyrazinamide, 244

Pyridoxal phosphate (PLP), 123, 124f, 720–721, 720f, 721f in homocysteine conversion, 754 in transamination, 709, 709f Pyridoxine. See Vitamin B6 Pyrimidine antagonists of, 328 chemical structure of, 54 dietary, 760 function of, 760 salvage of bases in, 768, 768t, 769f structure of, 65, 66f synthesis of, 871t Pyrimidine bases degradation of, 771, 771f salvage of, 768, 768t, 769f structure and nucleosides of, 195, 195f, 195t Pyrimidine biosynthesis, 765–768 de novo pathways of, 765–767, 767f–768f, 767t pyrimidine base salvage in, 768, 768t, 769f pyrimidine ring atoms in, 766, 767f regulation of, 767f, 768 Pyrimidine dimer, 218, 219f Pyrimidine phosphorylase, 768 Pyruvate anaerobic glycolysis on, 401, 401f islet cell cycling of, NADPH from, 623–624, 623f oxidation to carbon dioxide of, 403–404 oxidative fates of, 401–402, 401f, 402f synthesis of in gluconeogenesis, 568 in liver, 563, 564f Pyruvate carboxylase active, 568 as anaplerotic enzyme, 124f, 371, 371f deficiency of, on TCA cycle, 372 glutamine synthesis and, 920 regulation of, 680, 680f, 684f Pyruvate dehydrogenase (PDH), 680, 916 glycolysis and, 409 inactive, 568 Pyruvate dehydrogenase complex (PDC), 368–370 function of, 368 genetic defects of, 369 regulation of, 369–370, 370f structure of, 363f, 368–369, 369f Pyruvate dehydrogenase (PDH) deficiency, 369 inherited, on acetyl-CoA for acetylcholine synthesis, 917 ketogenic diets for, 429 Pyruvate kinase inactive, 570 regulation of, 409, 684f Pyruvate kinase deficiency, 827, 845t Pyruvate transfer, across mitochondrial membrane, 164–165 Q Q cycle, for b-c1 complex, 384, 384f Quaternary amine, 56f Quaternary structure of insulin, 97 protein, 89f, 90, 96–97 in sickle cell anemia, 100 Quercetin, as free radical scavengers, 451–452, 451f R Radiation, ionizing, free radical intermediates from, 441, 442f Radical, 54, 66, 439. See also Free radical biradical, 439, 439f hydroxyethyl, 467

01/09/12 9:36 PM

INDEX

hydroxyl, 439, 439f, 440f, 440t oxygen as, 439, 439f peroxyl, 440t Radioimmunoassays (RIAs), 819–820, 820f Rales, inspiratory, 351 Rapoport–Luebering shunt, 826, 827f Ras-MAP kinase pathway, 180 ras mutation, 329 ras oncogene, 312 Ras protein regulators, 321, 321f Rate-limiting enzymes, 141 Rate-limiting step, 135f, 146, 146f Rate nephelometry, 698 Rat poison, warfarin in, 856 R-binders, 750 Reactive nitrogen–oxygen species (RNOS). See also specific types in alcohol-induced liver disease, 469, 469f characteristics of, 440, 440t formation of, 445, 445f inflammation and, 447 in myocardial ischemia damage, 447 Reactive oxygen species (ROS). See also specific types in alcohol-induced liver disease, 469, 469f characteristics of, 439–440, 440f, 440t definition of, 439 generation of, 438–441 O2 in as biradical, 437, 437f radical nature of, 439, 439f overview of, 438–439 reactive nitrogen–oxygen species characteristics and, 440, 440t hemolysis by, 537–539, 538f, 540 primary, cell sources of, 440–441, 441f retinopathy from, in premature infants, 449 Reactivity, functional group, 57, 58f Reannealing, DNA, 200 Rearrangement, gene, 102f, 273–274, 274f Receptor affinity, ligand, 98 Receptor downregulation, 491 Receptor kinases, 279 Receptor-mediated endocytosis, 161, 162–163, 645–646, 645f Receptor-mediated transcytosis, blood–brain barrier, 908 Receptors. See also specific receptors; specific types plasma membrane, 178–179 Receptor serine–threonine kinases, 179f, 183–184, 183f Recognition site, 253, 253f Recombinant ␤-interferon, 302 Recombinant DNA techniques, 288–308 amplifying DNA sequences in, 295–297 DNA cloning in, 295, 296f libraries in, 295–297 polymerase chain reaction in, 297, 297f development of, 290 in disease diagnosis, 298–300 DNA polymorphisms in, 298 polymorphism detection in, 298–300, 299f in disease prevention and treatment, 301–305 gene therapy in, 245f, 303–305, 304f genetic counseling in, 303 small, interfering RNA in, 283f, 302–303 therapeutic protein production in of complex human proteins, 301–302 of insulin and growth hormone, 301–302, 302f transgenic animals in, 305 vaccines in, 301

Lieberman_Subject_Index.indd 1007

human genome mapping in, 307 identifying DNA sequences in, 292–295 detecting specific DNA sequences in, 293, 293f DNA sequencing in, 293–295, 294f, 295f gel electrophoresis in, 292, 293f probes in, 292, 292f obtaining DNA fragments and gene copies in chemical synthesis of DNA in, 291 restriction fragments in, 290–291, 290f, 290t, 291f reverse transcriptase in, 291 proteomics in, 305–306, 305f Recombinant factor VIII, 859 Recombination, 191 Recombination, homologous, 221–222, 222f Recommended Dietary Allowance (RDA) definition and overview of, 10–11, 18 for fiber, 503–504 for folate, 13t, 746 for protein, 704 for vitamins, 12–14, 13t–14t Recycling endosomes, 162 Red blood cell (RBC). See Erythrocyte Red muscle fibers, 408 Redness, 676 Reduced functional groups, 56, 56f Reduced sugars, 60–61, 61f Reduction, 56. See also Oxidation–reduction; specific reactions of ␤-ketoacyl group of fatty acid synthase complex, 604, 604f FAD, 346–347, 347f of folate to tetrahydrofolate, 745–746, 746f NAD⫹, 346–347, 347f NADP⫹, 346–347, 347f of oxygen, 439, 439f Reduction potential, 348, 348t Redux, 904, 915 Refetoff disorder, 813 Refsum disease, 425 Regulation. See also specific processes feedback, 146, 146f feed-forward, 147 Regulatory enzyme, 135f, 146, 146f. See also specific enzymes and processes Regulatory protein (RP), 874–875, 875f fructose-1-phosphate on glucokinase dissociation from, 875–876 gene-specific, 274–276, 275f tissue isozymes of, 147 Renal colic, 701 Renal failure, 901t Renal glomerulus, 940, 940f Renal stones, cystine, 76, 730 Renaturation, DNA, 200 Repair enzymes, mutations in, 314–315 Repetitive DNA, 241, 242f, 299, 299f Replication, 191, 191f, 209, 209f bidirectional, 210–211, 211f DNA, 196–198, 198f semiconservative, 209f, 211, 211f Replication complex, 214f, 215–217, 216f, 217t Replication fork, 209f, 213, 214f Replicons, 215 Repressible operon, 269, 269f Repression, 269, 276 Repressors, 232, 274–276, 275f on RNA polymerase binding, 267–269, 268f corepressors in, 269, 269f inducers in, 268–269, 268f Reserpine, 909 Residual body, 163

1007

Resistance training, 900 Respiration, 4, 4f Respiration, cellular, 333–334, 333f, 334f Respiratory acidosis, 410 Respiratory burst, phagocytic, 446, 446f Respiratory center, pH on, 49 Respiratory chain inhibition, 382f, 385 Respiratory distress syndrome (RDS), 455t, 624t Respiratory quotient (RQ), 9 Resting energy expenditure (REE), 8 daily requirement for, 344 sources of, 344, 344t Resting metabolic rate (RMR), 8, 9t, 390 Restriction endonucleases, 290–291, 290f, 290t, 291f Restriction enzymes, 290–291, 290f, 290t, 291f Restriction fragment length polymorphisms (RFLPs), 298 Restriction fragments, 290–291, 290f, 290t, 291f Reticulocytes, 255 Retinal pigment epithelium (RPE), oxidative damage to, 451 Retinoblastoma cell cycle and oncogenes in, 318, 318f rb gene mutations, 319–320, 319f, 320f 9-cis Retinoic acid, 177f Retinoids, 175, 177f Retinol, 175 Retinopathy diabetic, 579, 940 of prematurity, 449 Retroposons, 223 Retroviruses, 206–207, 207f. See also specific types in cancer, 328 in gene therapy, 245f, 303, 304f Reverse cholesterol transport, 642–644, 643f Reverse transcriptase, 207, 207f, 223, 223f dideoxynucleotide affinity of, 295 DNA produced by, 291 drugs against, in HIV treatment, 246 in HIV production, 244–245, 245f Reye’s syndrome, 877, 882t Rheumatoid arthritis, 934 Rho factor, 233 Riboflavin deficiency of, 13t, 359, 373 as exercise supplement, 420 in foods, 373 Recommended Dietary Allowance for, 13t Ribonucleic acid. See RNA Ribonucleotide reductase effectors, 769, 770t Ribose, 195, 195f Ribosomal RNA (rRNA), 191, 205, 205f Ribosomal RNA (rRNA) transcription in eukaryotes, 237–238, 239f in prokaryotes, 234, 234f Ribosome mitochondrial, 205 synthesis of, 238, 239f Ribozyme, 204 Rickets, 14t, 656, 661t Rifampin, 229, 236, 244 Rimonabant, 818–819 Ring structures benzene, 55, 55f Corrin, 750, 751f heterocyclic, 54 monosaccharide, 59, 60f in nitrogen-containing compounds nucleosides and nucleotides, 62f, 65 purines, pyrimidines, and pyridines, 65, 66f, 766, 767f tautomers, 66, 66f

01/09/12 9:36 PM

1008

INDEX

Rituximab, 266 RNA, 191. See also specific types in eukaryotes vs. prokaryotes, 242–243, 243t hybridization of, 200, 201f in micro RNAs, 205–206 in oligonucleotides, 205 polycistronic, 267, 267f structure of, 203–206 general features of, 196f, 203–204, 203f in mRNA, 204, 204f in rRNA, 205, 205f in tRNA, 205, 206f types of, 204 in viruses, 195 RNA editing, 282, 282f RNA polymerase, 228–233 action of, 227f, 228–229, 229f definition of, 228 in eukaryotes, 229–230, 229t, 230f gene recognition by, 230 gene sequences and, 230, 230f, 231f promoter regions of mRNA genes and, 231–233, 231f, 232f strand discrimination at promoter by, 231 RNA polymerase II, ␣-amanitin on, 230 RNA polymerase, prokaryote in bacteria, 229 binding of repressors on, 267–269, 268f, 269f sigma factors on, 270 stimulation of, 269–270, 270f RNA primer, prokaryote, 213, 214f RNA processing, posttranscriptional alternative splicing and polyadenylation sites in, 281, 281f overview of, 281 RNA editing in, 282, 282f RNA retroviruses, in cancer, 328 RNase H, 213, 223f RNase P, 234 RNA synthesis, 227–246 bacterial gene transcription in, 233–234, 234f eukaryotic gene transcription in, 234–240 mRNA in, 235–237, 235f–238f overview of, 234 vs. prokaryotic gene, 234 rRNA in, 237–238, 239f tRNA in, 238–240, 240f, 241f eukaryotic vs. prokaryotic DNA and RNA in, 240–243 diploid human cells in, 240 introns in human genes in, 241 repetitive sequences in eukaryotic DNA in, 241–242, 242f summary of differences in, 242–243, 243t gene regions and, 227, 227f in HIV virus production, 244–246 RNA polymerases in, 228–233 (See also RNA polymerase) Rough endoplasmic reticulum (RER) protein synthesis of, 259–260, 259f, 260f structure and function of, 164–165, 165f R-Smad, 184 RXR receptor, 277, 277f Ryanodine, 887 Ryanodine receptor, 887, 887f, 888 S S-Adenosylmethionine (SAM), 752–753, 753f in choline synthesis, 916 homocysteine from, 754 one-carbon methyl donors for, synthesis of, 871t SAMe form of, 754

Lieberman_Subject_Index.indd 1008

Salicylate (salicylic acid) metabolism of, 877, 878f overdose/poisoning with, 41, 46, 51, 53t, 390 Salicylurate, 877 Salivary ␣-amylase, 497–498, 497f Salivary mucin, 549, 551f Salt bridges, 100 SAMe, 754 Sanger method, 294–296, 295f Sarcolemma, 885 Sarcoplasm, 885 Sarcoplasmic reticulum Ca21 ATPase (SERCA), 888 Sarcoplasmic reticulum, calcium release in, 887–888, 888f Sarin, 916 Saturated fatty acids, 61, 63f Saturated long-chain fatty acids, 417 Saturation kinetics of enzymes, 137–138, 137f of transporter proteins, 158, 159f Saxagliptin, 816 Scavenger receptors, 648–649, 650 Scavenging enzymes, antioxidant, 448–449, 448f Schilling test, 745 Schwann cell myelination, 921–923, 922f Schwann cells, 906 Scissile bond, 117, 117f Scurvy, 13t Seborrheic dermatitis, 373 Secondary active transport, 160 Secondary protein structure, 89f, 90 Secondary structure nonrepetitive, 92, 93f, 94f patterns of, 93, 94f Secondary structure, protein, 91–94 ␣-helix in, 91, 91f, 92f ␤-sheets in, 91–92, 92f definition of, 91 nonrepetitive secondary structures in, 92, 93f, 94f secondary structure patterns in, 93, 94f Second law of thermodynamics, 339t Second messengers, 179, 179f Secretagogue, 818 Secretin, 813, 815t Secretory tumor, endocrine gland, 818 Sedentary habits, 10 Sedimentation coefficient, 205 Seeding, amyloid, 93 Selective COX-2 inhibitors, 670 Selective serotonin reuptake inhibitors (SSRIs), 915 Selenium, 449 Selenocysteine, 84, 84f Selenophosphate, 449 Semiconservative replication, 209f, 211, 211f Semiquinone, 360, 360f Senescence, telomeres in, 218 Sense strand, DNA, 230, 230f Sepsis amino acid metabolism in, 774, 774f, 790–791, 791f cytokines and hormones in, 790, 791f hypercatabolic response in, 788–790, 789f on translational efficiency, 791 Sequencing, DNA, 293–295, 294f, 295f Sequential transfer, in oxidative phosphorylation, 382f, 385 SERCA, 888 SERCA pump, 900–901 Serine chemical structure of, 74f, 75–76, 75t from intermediates of glycolysis, 727–728, 727f

liver uptake of, 879 one-carbon groups from, 747, 749f Serine proteases, 854 Serine proteinase inhibitors, 857 Serine–threonine kinase receptors, 179f Serotonin actions of, 915 in appetite and weight loss, 915 metabolism of, 913, 914f monoamine oxidase activation of, 913 Redux on secretion of, 915 Serpentine (G protein–coupled) receptors, 179, 179f, 184–187 adenylyl cyclase and cAMP phosphodiesterase in, 185–186, 186f heterotrimeric G-proteins in, 184, 185f, 185t names and general properties of, 96f, 179f, 184 phosphatidylinositol signaling by, 173f, 186–187 Serpins, 857 Serum, 312 Serum amyloid A, 101 Serum glutamate-oxaloacetate transaminase (SGOT), 416 Serum glutamate pyruvate transaminase (SGPT), 416 Severe combined immunodeficiency disease (SCID), 771, 772t adenosine deaminase gene mutation in, 304 X-linked, 305, 837, 845t Severinghaus electrode, 42 SH2 domain, 180, 487 SH3 domain, 180 Shine–Dalgarno sequence, 255, 255t Shivering, heat from, 388–389 Short-chain fatty acids, 501 Short interspersed elements (SINEs), 242 Short tandem repeat (STR), 301 Sialic acid (NANA) synthesis, 871t, 873 Sickle cell anemia (disease), 87t, 110t, 308t, 596t bilirubin excess in, 589 carrier of, 203 gallstones in, 589, 838 heme processing in, 589 hemoglobin polymerization in, 100 inheritance of, 79 malaria protection in, 79 mutations in, 70, 75 consequence of, 298 direct test for, 298 missense, 252 point, 79, 291 quaternary structure in, 100 sickle hemoglobin in, 203 steatorrhea in, 590 variants of, 79 vaso-occlusion in, 84–85 Sickle cell anemia trait, testing for, 289 Sickle cell crisis, 89, 106–107, 107f, 838 Sickle cell hemoglobin, glutamate substitution for valine in, 78 Sickled red blood cells, 107, 107f Sickle hemoglobin (HbS), 203 Sigma (␴) factor, 229, 233, 270 Signaling, cell. See Cell signaling Signal-recognition particle (SRP), 259, 259f Signal sequences, 259, 259f Signal termination, 187, 187f Signal transducer and activator of transcription (STAT) factors, 182, 279 family of, 837, 837f leptin on, 618–619

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INDEX

Signal transduction, 179–187 by cytokine receptors, 179f, 182–183, 183f definition of, 487 heptahelical receptors in, 179, 179f, 184–187 adenylyl cyclase and cAMP phosphodiesterase in, 185–186, 186f heterotrimeric G-proteins in, 184, 185f, 185t names and general properties of, 96f, 179f, 184 phosphatidylinositol signaling by, 173f, 186–187 hormone, on fuel metabolism, 486–490 by cortisol, 489 by epinephrine and norepinephrine, 489–490, 489f overview of, 486–487 by plasma membrane receptor binding, 487–489 glucagon in, 144f, 179f, 185f, 186f, 488–489 insulin in, 181f, 487 principles of, 487 receptor serine–threonine kinases in, 179f, 183–184, 183f response to signals in, changes in, 187 types of, 487 tyrosine kinase receptors in, 179–182, 179f insulin receptor in, 181–182, 182f phosphatidylinositol phosphates in, 180, 180f Ras-MAP kinase pathway in, 180 structure of, 179, 179f Signal transduction cascade, oncogenes and, 315–317 growth factors and growth factor receptors in, 315, 316t proto-oncogenes in, 315, 317f signal transduction proteins in, 180f, 315–316, 316t, 317f transcription factors in, 316–317, 316t, 317f Signal transduction proteins, 180f, 315–316, 317f Silence of cytokine signaling (SOCS) proteins, 837, 837f Silencers, 275 Silencing, gene, 303 Silent mutations, 251t, 252 Simple diffusion, 157–158, 158f Simple product inhibition, in metabolic pathways, 139–140 SINEs, 242 Single-chain urokinase (scuPA), 858, 858f Single nucleotide polymorphisms (SNPs), 307 Singlet oxygen, 440t Sirtuins, 434–435, 434f Sister chromatids, 194 Sitagliptin, 816 Sitosterolemia, 629 Skeletal muscle, 885. See also Muscle amino acid utilization by, 780–785 branched-chain amino acid oxidation in, 782, 783f branched-chain amino acid to glutamine in, 783–784, 783f, 784f glucose-alanine cycle in, 712f, 784–785, 784f glutamine synthesis in, 781 protein synthesis and degradation in, 777f, 780 cells of, 885–886, 885f glycogen metabolism regulation in, 524–525, 525f oxidation of fuels in, 25–26, 25f purine nucleotide cycle in, 783, 784f

Lieberman_Subject_Index.indd 1009

Skeletal muscle fuel use, 891–897 ATP and creatine phosphate in, 892–893, 892f, 893f for ATP generation, 891 in exercise, 894–897 anaerobic glycolysis as ATP source in, 894 anaerobic glycolysis at onset of exercise in, 894 anaerobic glycolysis from glycogen in, 895–896, 895f anaerobic glycolysis in high-intensity exercise in, 897 anaerobic glycolysis in type IIb fast-twitch glycolytic fibers in, 894–895 ATP use in, 894 lactate fate in, 897 pathways of, general, 891 at rest, 893 in starvation, 893–894 Skin cancer, 219 Skinfold thickness (SFT), 27 SLC11A2 mutation, 831 Slot blotting, 298 Slow-oxidative fibers, 408, 886, 886t Smad proteins, 184 Small G-proteins, 144, 145f Small, interfering RNA, 283f, 302–303 Small intestinal disaccharidases, 498–500, 498f, 498t ␤-glycosidase complex in, 498t, 500, 500f glucoamylase in, 498–501, 498t, 499f location of, 498f, 500 sucrase-isomaltase complex in, 498t, 499–501, 499f, 500f trehalase in, 498, 498t, 500, 500f Small neutral amino acids (SNAAs), blood–brain barrier transport of, 908 Small nitrogen-containing neurotransmitter synthesis. See Neurotransmitter synthesis, of small nitrogencontaining neurotransmitters Small nuclear ribonucleoproteins (snRNPs, snurps), 205–206, 237, 238, 238f Smith proteins, 238 Smoking carcinogens in, 218–219 epidemiology of, 327 Smooth endoplasmic reticulum (SER), 164, 165f smoothened coreceptor gene, 321, 322f, 327 Smooth muscle cells, 885f, 886–887 snRNPs, 205–206, 237, 238f snurps, 205–206, 237, 238f Sodium dietary guidelines for, 16 in hypertension, 16 function of, 14 Sodium channel at neuromuscular junction, 887, 887f voltage-gated, 887, 887f Sodium-dependent amino acid transport, 696, 699–700, 699f Solenoid structures, 201 Solubility of globular protein, 95 in water, 57 Solvent, 41, 43, 44f Soma, 905, 905f Somatocrinin, 802, 803f Somatomedin, 804–805 Somatomedin-A, 804–805, 805f Somatomedin-C, 804–805, 805f

1009

Somatostatin, 814t on fuel metabolism, 798t biochemistry of, 800 physiologic effects of, 800–801 secretion of, 800 structure and function of, 802, 803f Somatotroph cells, 801 SOS protein, 180, 180f Southern blots, 293, 294f Soybeans, indigestibility of, 504–505 Spastic paraplegia type 2 disease, X-linked, 924 Specific dynamic action (SDA), 9 Specificity, enzyme, 113–114, 114f, 115 Specific transcription factors, 275, 275f Spectrin, 833–834, 833f deficiency of, 842 in red blood cells, 156f, 157 S phase, 214–215, 215f Spherocytosis, 842, 845t Sphingolipidoses, 554, 554t, 556, 557t, 618 Sphingolipids, 54, 64, 64f function of, 583, 613 metabolism of, 618, 618f, 619f structure of, 597, 598f, 613, 613f synthesis of, 554–555 types of, 613, 613f Sphingomyelin, 64, 64f, 921 in amniotic fluid, 621, 621f in plasma membrane, 155, 155f structure of, 598, 598f Sphingosine synthesis, 871t Spin restriction, 439 Splice junctions, 237, 237f Spliceosome, 237, 238f Splicing, alternative, 281, 281f Sprinting, ATP demand in, 408, 411 Squalene to cholesterol, 624f, 633–634 formation of, 632, 634f SR141716, 818–819 SREBPs (sterol-regulatory element-binding proteins), 630–631, 631f, 646 SREP cleavage-activating protein (SCAP), 631, 631f Stabilization of messenger RNA, 145 of transition-state complex, 120 Starch, 5, 5f, 22 Starch blockers, 497 Starling forces, 848 Starvation, 30 death by, 35 fuel use in by skeletal muscle, 893–894 by tissues, 685, 686t glucose in blood, 571t, 575–576, 575f sources of, 561f, 576–577, 577f Statins, 650, 657, 658 for hyperlipidemia, 621 mechanism of action and efficacy of, 658t STATs, 182, 279 Stearate, 6f Stearic acid, 61, 63f Steatorrhea in alcoholics, 590 in sickle cell disease, 590 Stellate cells, 865–866 in alcohol-induced liver disease, 468–469, 469f hepatic fibrosis on, 881 Stem cells, 795 hematopoietic, 835–836, 836f omnipotent, 285

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1010

INDEX

Stereoisomers, 54, 59, 59f, 60 Stereoisomers, monosaccharide, 58–59, 59f Steroid hormone. See also specific hormones adrenocorticotropic hormone on, 651 cholesterol in, 54 classes of, 650 on glucose homeostasis, 570 signal transduction by, 489 structure of, 64, 65f transport of, 650–651 Steroid hormone receptors, 276–277, 276f Steroid hormone receptor transcription factors, 276–277, 276f, 277f Steroid hormone synthesis, 650–656, 653f adrenal androgens, 653f, 655, 655f aldosterone, 653f, 654–655, 654f androgens, 652, 653f androstenedione, 653f, 655, 655f cholesterol, 652, 653f cortisol, 652–655, 653f, 654f estrogens and progesterone, 652, 653f, 656 glucocorticoids, 652, 653f mineralocorticoids, 651, 652, 653f progestogens, 652, 653f testosterone, 653f, 655 Steroid hormone/thyroid hormone superfamily receptors, 176f, 177–178 Steroid nucleus, 64, 65f Sterol, 629–630, 629f, 630f Sterol-regulatory element-binding proteins (SREBPs), 630–631, 631f, 646 Stones cystine, 76, 730 definition of, 71 gallstones bilirubin backflow and jaundice in, 594 bilirubin excess in, 842 in sickle cell disease, 589, 838 Streptococcus mutans, in dental caries, 403, 411 Streptokinase, 858, 858f Streptomycin, 255, 261, 261t Stress hormones, in metabolic homeostasis, 479, 479f Stringency, 291 Stroke, creatinine phosphokinase in, 893 Strong acids, 45–46, 45t Structural domains, 93–94, 94f Structural genes, 267 Structures, of major compounds, 54–68. See also specific compounds carbohydrates in, 54, 58–61 chlorinated aromatic hydrocarbon environmental toxins, 67–68 free radicals in, 54, 66 functional groups in, 54, 55–57 lipids in, 54, 61–64 nitrogen-containing, 54, 64–66 Subacute necrotizing encephalopathy, 369, 375t, 387t Substantia nigra pas compacta, 438 Substituted sugars, 59–60, 60f Substrate. See also specific substrates channeling through compartmentation of, 147 concentration and velocity in, 137–139, 137f, 138f enzyme concentration and, 138 hexokinase isozyme Km values for glucose in, 138, 138f Michaelis–Menten equation in, 137–138, 137f Substrate-binding sites, 112, 115–117, 115f induced-fit model for, 114f, 115–116, 116f lock-and-key model for, 115, 115f

Lieberman_Subject_Index.indd 1010

Substrate-level phosphorylation oxidation and, 399–401, 400f process of, 335, 359 Succinate as energy source, 360–361 oxidation to oxaloacetate of, 358f, 359 Succinate dehydrogenase in electron-transport chain, 381–382, 382f flavin adenine dinucleotide in, 360–361, 361f Succinyl-CoA from ␣-ketoglutarate reaction, 358f, 359 amino acid conversion to, 734, 734f amino acids forming, 733–735 methionine in, 734, 734f overview of, 733–734 threonine in, 734, 734f valine and isoleucine in, 735–736, 735f compounds forming, 735–736 Succinyl-CoA synthetase, 359 Sucrase, 22 Sucrase-isomaltase complex genetic deficiencies in, 500 structure and function of, 498t, 499–501, 499f, 500f Sucrose, 5, 496 dietary sources of, 496 structure of, 493, 494f, 496 Sugar, 54 amino, 60, 60f colonic bacteria metabolism of, 501–502, 501f D- and L-, 58–59, 59f dietary fat stores from, 611 refined, guidelines for, 16 for glycolipid and glycoprotein synthesis, 549, 549t, 550f, 551f hydrophilicity of, 57, 58f interconversion of, pathway for, 549, 550f oxidized and reduced, 60–61, 61f substituted, 59–60, 60f Sugar absorption, 504–508 glycemic indices in, 504, 504t glycemic response in, 504–505 by intestinal epithelium, 505–507 facilitative glucose transporters in, 506, 507t facilitative transport in, 505, 505f, 506f of galactose and fructose, via glucose transporters, 506–507, 506f Na⫹-dependent glucose transporters in, 505–506, 506f monosaccharide transport into tissues in, 507–508, 508f Sugar metabolism pathways, 529–542 for fructose, 531–533 dietary sources of, 529f, 531 metabolism of, 531–532, 532f structure of, 529f synthesis of, in polyol pathway, 532–533, 532f for galactose, 533–534, 533f NADPH in, 539, 539t pentose phosphate pathway in, 530f, 534–539 (See also Pentose phosphate pathway) xylulose 5-phosphate in gene transcription in, 541 Sugar nucleotide interconversions, 545–549 glucuronide formation in, 546–547, 547f, 547t lactose synthesis in, 547–549, 548f sugar formation for glycolipid and glycoprotein synthesis in, 549, 549t, 550f, 551f UDP-galactose synthesis in, 547, 548f

UDP-glucose reactions in, 545–546, 545f UDP-glucuronate in, 546, 546f, 547f Sugar nucleotides, as precursor for transferase reactions, 549, 549t L-Sugars, 54, 58–59, 59f Suicide inhibitor, 129, 129f Sulfa drugs, 745 Sulfated compound synthesis, 871t Sulfate group, 56–57, 57f Sulfhydryl group, 56f Sulfonylureas, 485 Sulfur, 15 Sulfur-containing amino acids, 74f, 75t, 76 Sulfuric acid, 41 in body, 46f, 50 from fuel metabolism, 778 Supercoil, 201 Superfamily, 79 Superoxide, 54, 66 from coenzyme Q, 441, 441f in myocardial ischemia damage, 447 Superoxide anion, 440t Superoxide dismutase (SOD), 448, 448f, 449–450 Superoxide dismutase 1 (SOD1) gene, 448 Suppressors of cytokine signaling (SOCS), 182 Supravalvular aortic stenosis (SVAS), 932, 943t SUR1 gene, 485 Surgery, hypercatabolic response in, 788–790, 789f Sweat, water loss via, 52, 52f Sweeteners, 496 Swelling cell, 352 in inflammatory process, 676 Symports, 160, 391, 391f Synapse, 905, 905f Syndrome X, 27 Synovitis, in gout, 163 Synthases, 133 Synthetases, 133 Systemic lupus erythematosus (SLE), 238, 246t, 943t articular cartilage disruption in, 928, 936, 936f, 939–940, 940f DNase in, reduced, 244 on joints, 936, 936f pathophysiology of, 244 T T3. See also Thyroid hormone (T3, T4) on energy production, 351 hypothalamus release of, 337 measurement of, 337 T4. See also Thyroid hormone (T3, T4) hypothalamus release of, 337 measurement of, 337 Tachycardia, in left ventricular heart failure, 352 Tachykinins, 815t Tangier disease, 643 Targeting sequences, 259, 259f Tarui syndrome, 518t Tat, 328 TATA box, 231–232, 231f, 274, 275f TATA box mutations, in thalassemia, 232 Taurine synthesis, 871t Taurochenodeoxycholic acid, 636 Taurocholic acid, 636, 637f Tautomers, 66, 66f Tay-Sachs disease, 162, 249, 261, 263t, 554t, 555–557, 557t TCA cycle. See Tricarboxylic acid (TCA) cycle T-cell immunity, purine nucleoside phosphorylase deficiency on, 764

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INDEX

T cells, 825 99 Tcm-galactosyl-neoglycoalbumin (NGA), 867 Telomerase, 217–218, 218f Telomere, 217–218 Temperature, for enzyme reactions, 127 5'-Terminal, 235, 236f Termination, in protein translation, 257–258 Termination signals, in bacterial gene transcription, 233 Tertiary protein structure, 89f, 90, 93–96 basic, 93, 94f globular protein folds in, 94–95, 94f globular protein solubility in, 95 structural domains in, 93–94, 94f in transmembrane proteins, 95–96, 96f Testicular feminization, 277, 286t Testosterone potency of, 656 synthesis of, 652, 653f, 655, 655f Tetracycline, on protein synthesis, 261, 261t Tetrahydrobiopterin (BH4), 726, 736–737, 738f Tetrahydrofolate (FH4), 726, 728, 745–749, 746f in deoxythymidine monophosphate synthesis, 747, 748, 749f, 750f one-carbon groups of oxidation and reduction of, 747, 748f recipients of, 746f, 747–749, 748f, 749t, 750f sources of, 747, 749f, 749t one-carbon methyl donors for, synthesis of, 871t one-carbon pool in, 744, 744f reduction of folate to, 745–746, 746f structure and forms of, 745–746, 746f vitamin folate in, 746–747, 746f Thalassemia, 228, 232, 243, 839–840, 845t anemia from mutations in, 243 ␤-thalassemia, 228, 246t, 263t mutations in, 232, 243, 261, 261t homozygous, 237 point, 237 pathophysiology of, 232 types of, 243 classification of, 243 Thalassemia intermedia, 253 Therapeutic protein production of complex human proteins, 301–302 of insulin and growth hormone, 301–302, 302f Thermal regulation, water in, 43 Thermic effect of food (TEF), 9–10 Thermodynamics of energy expenditure, 342 expressions, laws, and constants in, 339, 339t laws of, 339, 339t, 340 Thermogenesis, 345 adaptive, 342 diet-induced, 9–10 uncoupling proteins and, 389–390, 390f Thermogenin, 389–390, 390f Thermus aquaticus, 297 Thiamine beriberi and, 130, 373–374 deficiency of, 13t, 14 dietary sources of, 373 Recommended Dietary Allowance for, 13t Thiamine deficiency, 133t, 742t in alcoholics, 123, 363 ␣-keto acids from, 735 confirmation of, 356 heart failure from, 363 inherited, on acetyl-CoA, 917

Lieberman_Subject_Index.indd 1011

Thiamine pyrophosphate (TPP) on ␣-keto acid dehydrogenase complex, 122f, 363 functional groups of, 122, 122f in thiamine deficiency, 373 in thiamine deficiency testing, 356 Thiazolidinediones (TZDs), 660–661 Thick filaments, 885 Thin filaments, 885 Thioesters formation of, 57, 58f structure of, 56f Thiokinase, 417, 418f Thiolases, 133 3'-to-5' direction, 227, 229f, 230f Threonine chemical structure of, 74f, 75–76, 75t degradation of, 734, 734f, 738 Thrombin activation and formation of, 851, 853f, 854 antithrombotic effects of, 856, 856f fibrinogen cleavage by, 851, 852f function of, 854 on thrombosis, 853f, 856, 856f Thrombin inhibitor, direct, 860 Thrombocyte, 825 Thrombocytopenia, heparin-induced, 859, 914 Thrombocytopenic purpura (TTP), 851, 861t Thrombomodulin, 856, 856f Thromboresistance, of vascular endothelium, 857 Thrombotic thrombocytopenic purpura (TTP), 851, 861t Thromboxane (TX). See also Eicosanoid inactivation of, 670 metabolism of, 664f structure of, 667, 668f synthesis of, 667–670 Thromboxane A2 functions of, 669t in platelet aggregation and thrombus formation, 670 structure of, 667, 668f synthesis of, 667–670, 669f, 852 synthesis of, aspirin on, 670 Thromboxane A3, from cold-water fish, 670 Thymidine kinase, 768 Thymidine phosphorylase, 768, 769f Thymine nucleosides of, 195, 195f structure of, 195, 195f, 203, 203f Thymine dimer, 219f Thyroid function assessment, 337 Thyroid hormone (T3, T4) blood assay of, 787 in protein malnutrition, 787 on energy production, 351 on fuel metabolism, 798t, 809–813 biochemistry and synthesis of, 809–810, 809f, 810f calorigenic effects of, 812–813 feedback regulation of, 811, 812f physiologic effects of, 811–812 secretion of, 810f, 811, 812f structure of, 177f on uncoupling proteins, 394 Thyroid hormone receptor transcription factors, 276–277, 276f, 277f Thyroid peroxidase, 809 Thyroid-stimulating hormone (TSH), 337, 811 Thyrotropin-releasing hormone (TRH), 811, 815t Tiagabine, 917 TIM complex, 374–375, 374f

1011

Tissue factor, 851 Tissue inhibitors of metalloproteinases (TIMPs), 939 Tissue isozymes, of regulatory proteins, 147 Tissue metabolism, 795–796. See also specific tissues Tissue plasminogen activator (tPA), 301, 858, 858f for angina, 438 mechanism of action of, 392 Tissue-specific isoforms, 81, 81f Tissue-specific isozymes, 71 T lymphocytes, 285 TNM system, 329 ␣-Tocopherol, 450, 450f deficiency of, 13t, 14 as free radical scavenger, 447f, 449, 450, 450f Tolbutamide, 800 Tolerable Upper Intake Level (UL), 13t–14t, 14, 18 TOM complex, 374, 374f Topoisomerases, 201, 211, 211f, 217t Total body water, 42–43, 43f Total iron-binding capacity (TIBC), 266 Total parenteral nutrition (TPN) choline deficiency in, 614 essential fatty acid deficiencies in, 665 Training, physical metabolic effects on muscle metabolism of, 900 resistance, 900 Trans-acting, 230 Transactivators, 233, 274–276, 275f Transaldolase, 536, 537f Transaminase, 131, 132f, 416, 709 Transamination process of, 709, 709f pyridoxal phosphate in, 720–721, 720f, 721f Transcellular fluid, 42–43, 43f Transcobalamin I, 750 Transcobalamin II, 751 Transcription, 191, 191f, 249 attenuation of, 270–271, 271f of bacterial genes, 233–234, 234f definition of, 228 of eukaryotic genes, 234–240 mRNA in, 235–237, 235f–238f overview of, 234 vs. prokaryotic gene, 234 rRNA in, 237–238, 239f tRNA in, 238–240, 240f, 241f gene availability regulation in, 272–274 chromatin remodeling in, 272–273, 273f diploid cells and, 272 DNA methylation in, 273 gene amplification in, 274 gene deletions in, 274 gene rearrangement in, 102f, 273–274, 274f gene expression regulation in, 274–280 basal transcription complex in, 274, 275f DNA-binding protein structure in, 275f–278f, 277–279 enhancers in, 275, 275f gene-specific regulatory proteins in, 274–276, 275f multiple regulators of promoters in, 279–280, 280f transcription factors in regulation of, 279 steroid hormone/thyroid hormone receptor, 276–277, 276f, 277f zinc-finger motifs in, 277–278, 277f, 278f iron on, 283f, 284f, 285 of mRNA transcript, 235–236, 236f

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1012

INDEX

Transcription (continued) of ribosomal RNA in eukaryotes, 237–238, 239f in prokaryotes, 234, 234f of RNA in bacterial gene, 233–234, 234f in eukaryotic gene, 234–240 mRNA in, 235–237, 235f–238f overview of, 234 vs. prokaryotic gene, 234 rRNA in, 237–238, 239f tRNA in, 238–240, 240f, 241f promoters for, 227, 227f, 229, 230, 231f of transfer RNA in eukaryotes, 238–240, 240f, 241f in prokaryotes, 234, 234f promoters for, 239, 240f xylulose 5-phosphate in, 541 Transcription apparatus, 232–233, 233f Transcription associated factors (TAF), 233 Transcription complex, basal, 274, 275f Transcription-coupled repair, 221 Transcription factors. See also specific factors gene-specific, 274–276, 275f oncogenes and, 316–317, 316t, 317f regulation of, 279 steroid hormone receptor in, 276–277, 276f, 277f thyroid hormone receptor in, 276–277, 276f, 277f zinc-finger, 278, 278f Transcytosis, receptor-mediated, across blood– brain barrier, 908 Trans-fatty acids, 61, 63f Transfected cells, 295 Transferases, 131, 132 Transferrin, 829, 830f Transferrin receptor mRNA, on mRNA degradation, 284, 284f Transfer RNA (tRNA), 191 folding of, 239, 240f structure of, 205, 206f, 239, 240f transcription of in eukaryotes, 238–240, 240f, 241f in prokaryotes, 234, 234f Transfer, sequential, in oxidative phosphorylation, 382f, 385 Transformed cells, 295, 312 Transforming growth factor ␤ (TGF-␤), 179f, 183–184, 183f Transforming growth factor ␤1 (TGF-␤1), in alcohol-induced liver disease, 469, 469f Transfusion, blood, 557t Transgenic animals, 302, 305 Transient tyrosinemia, 737 Transition metals, 439–440, 440f Transition state, 112 Transition-state analogs, 127–129 allopurinol conversion to, 129, 129f penicillin as, 127–129, 128f Transition-state complex, 116–117, 116f, 120 Transketolase, 356, 536, 536f Translation, 191, 191f, 249, 253–258 elongation in, 256–257, 256f, 257f gene expression regulation in, 282–284 initiation of translation in, 282, 282f, 283f microRNAs in, 283–284, 283f initiation of, 253–255, 254f, 255f, 255t, 282, 282f, 283f modifications after, 258–259, 259t overview of, 253, 254f termination in, 257–258

Lieberman_Subject_Index.indd 1012

Translocation, 221, 222, 222f in chromosomes, 221, 222, 222f observation of, 274 in protein synthesis, 256f, 257 of proto-oncogenes, 313, 314f Transmembrane proteins, 90, 95–96, 96f Transmissible spongiform encephalopathies, 106 Transport active, 158f, 160–161, 160f, 161f, 341–342, 696 primary, 160 secondary, 160 of monosaccharides into tissues, 507–508, 508f passive, 158, 158f, 159f through mitochondrial membranes, 390–392 Transporters. See also specific types glucose for blood–brain barrier and neurons, 508–509, 508f facilitative, 506, 507t for galactose and fructose, 506–507, 506f for monosaccharides into tissues, 507–508, 508f Na⫹-dependent, 505–506, 506f proteins as facilitative diffusion via, 158, 158f, 159f mitochondrial membrane, 391, 391f sodium-linked, 696 Transport signaling pathways, 1 Transport work, energy transformations for, 160f, 341–342 Transposase, 223, 223f Transposition, of proto-oncogenes, 313, 314f Transposons, 221, 223, 223f Transthyretin, blood assay of, 787 in protein malnutrition, 787 Transudation, lung, 351 Trauma amino acid metabolism in, 774, 774f, 790–791, 791f hypercatabolic response in, 788–790, 789f Trehalase, 498, 498t, 500, 500f Triacylglycerol (TG), 54. See also Fat, dietary absorption of, 22–23, 23f in adipose tissue, 33, 334 function of, 7, 7t mobilization of, 611, 611f release from, 611–612, 611f storage in, 7, 7t, 609f, 610–611, 610f storage of, regulation of, 682–683, 682f blood high, with defective LPL, 612 measurement of, 682 in diet, 416–417 digestion of, 22–23, 23f, 586–588 bile salts in, 587, 587f, 588f pancreatic esterase in, 588, 589f pancreatic lipase in, 587–588, 588f phospholipase A2 in, 588, 589f export of, from liver, 871 in fed state, 21, 21f function of, 586 from glycogen, 23f, 24 in liver disease, 878 metabolism of in fasting, 583, 584f in fed state, 583, 584f mixed, 63 oxidation of, 4–5, 4f, 6, 334, 334f serum, enzymatic tests for, 600 stored, 334 structure of, 6, 6f, 55–56, 62–63, 586, 587f

synthesis of, 608, 609f, 679–682 (See also under Carbohydrate and lipid metabolism regulation, in fed state) from glucose, 597, 598f in liver, 871 resynthesis in intestinal epithelial cells in, 590, 590f transport of, 590–591, 590f Tricarboxylic acid (TCA) cycle, 355–375, 695, 695f acetyl coenzyme A precursors in, 23f, 24, 368–370 acetyl-CoA sources in, 368, 369f pyruvate dehydrogenase complex in, 363f, 368–370, 369f, 370f (See also Pyruvate dehydrogenase complex) anaplerotic reactions in, 124f, 371–372, 371f, 372f CO2-releasing enzymes in, 358f, 359–360 coenzymes of, 359–364 ␣-keto acid dehydrogenase complexes, 362–364, 362f–364f coenzyme A, 124f, 358f, 361–362, 361f flavin adenine dinucleotide, 360–361, 360f, 361f nicotinamide adenine dinucleotide, 361 overview of, 359–360 definition of, 5 energetics of, 364–366, 365f, 365t intermediate precursors for biosynthetic pathways in, 370–371 mitochondrial enzyme compartmentation and, 374 nuclear-encoded protein import and, 374–375, 374f overview of, 333–334, 334f, 356f, 357, 473–474, 474f pyruvate carboxylase deficiency and, 372 reactions of, 357–359 ␣-ketoglutarate to succinyl coenzyme A in, 358f, 359 fundamentals of, 357, 357f, 358f guanosine triphosphate generation in, 358f, 359 isocitrate formation and oxidation in, 358–359, 358f succinate oxidation to oxaloacetate in, 358f, 359 regulation of, 366–368, 366f, 367t allosteric regulation of isocitrate dehydrogenase in, 367–368, 367t, 368f ␣-ketoglutarate dehydrogenase in, 366f, 368 citrate synthase in, 366f, 367, 367t major interactions in, 366–367, 366f, 367t TCA cycle intermediates in, 368 Triglycerides. See Triacylglycerol (TG) Trimethoprim-sulfamethoxazole allergy, 531 Trisaccharide, 22 Tropoelastin, 932, 933f Troponin, 107 Troponin-I, 107 Troponin-T, 107 trp operon, 269, 270–271, 271f Trypsin, 697, 698, 698f, 699f Trypsinogen, 697, 697f, 698f Tryptophan bonds and polarity in, 75 chemical structure of, 74f, 75, 75t deficiency of, 738 degradation of, 737, 738f Tryptophan hydroxylase, 913, 914f TSH-releasing hormone (TSHRH), 337 T tubules, 885

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INDEX

Tube feeding enteral, 540 parenteral, 540 Tuberculosis with AIDS, 229, 236, 246t treatment of, 229, 236, 243–244 Tubulin, 165–166, 165f Tubulin synthesis, colchicine on, 166, 167 Tumor, 192. See also Cancer; specific types benign, 312 definition of, 312 malignant, 310, 312 metastasis of, 312 Tumor growth factor ␤1 (TGF-␤1), in hepatic fibrosis, 881 Tumor necrosis factor (TNF), 323, 790–791, 791f Tumor necrosis factor-1 (TNF-1) receptors, 323 Tumor suppressor, 321 Tumor suppressor genes, 192, 319–322 in cancer, 319t on cell adhesion, 322, 322f in direct regulation of cell cycle p53 in, 320, 321f retinoblastoma (rb) gene in, 319–320, 319f, 320f examples of, 319t function of, 319 on receptors and signal transduction patched and smoothened coreceptor genes in, 321, 322f, 327 Ras protein regulators in, 321, 321f Tunica media, 649, 649f Turns, 92, 93f Type 1 diabetes mellitus. See Diabetes mellitus type 1 Type 2 diabetes mellitus. See Diabetes mellitus type 2 Type I glycogen storage disease, 522 Type IIa muscle fibers, 886, 886t Type IIb muscle fibers, 886, 886t, 894–895 Type II glycogen storage disease, 517–518, 518t Type III glycogen storage disease, 518t, 520–521 Type I muscle fibers, 886, 886t Tyramine, 912, 925t foods with, MAO inhibitors and, 913 poisoning with, 925t structure of, 913f Tyrosine, 11, 733 in catecholamine synthesis, 910f, 911 chemical structure of, 74f, 75, 75t degradation of, 736, 737f dissociation of side chains of, 77, 78f Tyrosine aminotransferase (TAT) deficiency, 737 Tyrosine hydroxylase, 910f, 911 melanocyte, in albinism, 912 regulation of, 913 L-Tyrosine, in catecholamine synthesis, 910f, 911 Tyrosine kinase inhibitors, 327, 328 Tyrosine kinase receptors, 179–182, 179f insulin receptor in, 181–182, 181f, 182f phosphatidylinositol phosphates in signal transduction in, 180, 180f Ras-MAP kinase pathway in, 180 structure of, 179, 179f Tyrosinemia I, 737, 741t, 742t Tyrosinemia II, 737, 741t, 742t Tyrosinemia, transient, 737 Tyrosinosis. See Tyrosinemia I U Ubiquinone, 382–383, 382f Ubiquitin, 703, 703f Ubiquitin–proteasome pathway, 703, 703f

Lieberman_Subject_Index.indd 1013

UCP1, 389–390, 390f UDP-galactose, 475 UDP-galactose synthesis, 547, 548f UDP-glucose energy from, 343, 343f fate and products of, 475, 475f formation of, 516, 516f function of, 545 metabolism of, 545–546, 545f, 546f UDP-glucuronate, 546, 546f, 547f UDP-sugars, 549t, 550–551 Ulcer, bleeding stomach, 397 Uncompetitive inhibition, 139, 140f Uncouplers, chemical, 389, 389f Uncoupling proteins (UCPs) thermogenesis and, 389–390, 390f thyroid hormones on, 394 Underweight, 10 Undissociated acid, 41 Units of measurement, 40t Unsaturated fatty acids, 61, 63f Upstream, 178 Uracil, 203, 203f Urate microtubules in vesicular movement of crystals of, 166 solubility of, 770 Urea, 32, 32f in blood, 36 in fasting, excretion of, 34, 36f recycling of, 717 synthesis of glutamate in, 710f, 712, 712f in liver, 871 in urine, 36, 693, 694t Urea cycle, 693, 713–720 arginine biosynthesis in, 717 disorders of, 718–720 ammonia toxicity in, 718 arginine for, 718 benzoic acid and phenylbutyrate for, 718–719, 719f gene therapy for, 719–720 glutamate and glutamine excess in, 718 OTC deficiency in, 718, 720 in fasting, 717, 717f in liver, 879 nitrogen for, sources of, 710f ornithine origin in, 715–716, 716f reactions of, 713–715 arginine cleavage to produce urea in, 714f, 715 arginine production in, 714f, 715, 715f carbamoyl phosphate synthesis in, 714, 714f overview of, 713–714, 714f regulation of, 716, 716f Urea-cycle disorder. See Ornithine transcarbamoylase (OTC) deficiency Urea cycle enzymes, intestinal, 785 Urease, 717 Uremia, with diabetes mellitus type 2, 928 Uric acid, 66, 163 as antioxidant, 452, 452f in blood or urine, 153 excretion of, 66 pK of, 770 tautomers of, 66, 66f in urine, 693, 694t Uridine phosphorylase, 767f, 768 Uridine triphosphate (UTP), 344 Urinalysis, 210 Urinary pH, 45t, 50 Urinary tract infection, 223, 225t

1013

Urine bacteria in, 210 compounds excreted in, 780, 781t, 782f protein in, detecting, 89 water loss via, 52, 52f Urokinase, single-chain, 858, 858f Uronic acid, 60 V Vaccines hepatitis B, 289, 303, 306 recombinant DNA techniques for, 301 Valine chemical structure and bonds of, 76 degradation of, 735–736, 735f Variable number of tandem repeats (VNTR), 299, 299f Variable (V) regions, immunoglobulin, 100–101, 103f Variant regions, 78 Vascular endothelium, thromboresistance of, 857 Vasoactive intestinal peptide (VIP), 814t Vasoconstrictors, platelet, 852 Vaso-occlusion, in sickle cell anemia, 84–85 Vegans, vitamin deficiency in, 14 Vegetable oils, 417 Vegetables, dietary guidelines for, 16 Vegetarians, dietary protein for, 11 Velocity, substrate concentration and, 137–139, 137f, 138f enzyme concentration in, 138 hexokinase isozyme Km values for glucose in, 138, 138f Michaelis–Menten equation in, 137–138, 137f multisubstrate reactions in, 138 Ventricular fibrillation, 438 Very-long-chain fatty acid oxidation, peroxisomal, 425–426, 425f, 426f Very low-density lipoprotein (VLDL), 584t, 641, 641f characteristics of, 640t composition of particles of, 608, 609f fate of, 608–609, 610f, 640t, 641, 641f regulation of, in fed state, 682 triacylglycerol of, 597, 598f in fed state, 26 high in alcoholics, 608, 609 low HDL levels in, 650 liver packaging of, 634, 679 processing and secretion of, 608, 610f structure of, 590, 590f synthesis of, 583 in adipose tissue, 608, 610f in liver, 608, 610f, 611 Vesicle monoamine transporter 2 (VMAT2), 911, 911f Vesicles, in catecholamine transport, 911–912, 911f Vesicular ATPase (V-ATPase), 341–342 Vesicular transport, across plasma membrane, 161 Vibrio cholerae, 153–154, 510 Vincristine, 266 Vinyl chloride, cytochrome P450 on, 869, 869f Virilization, 652, 661t Virus in cancer, 328 DNA and RNA in, 195 oncogenic, 314 Vitamin blood–brain barrier transport of, 908 as coenzymes, 121 dietary, 12–14, 13t–14t dietary guidelines for, 16–17

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1014

INDEX

Vitamin (continued) ethanol on absorption of, 121 excessive intake of, 14 Recommended Dietary Allowance for, 12–14, 13t–14t Tolerable Upper Intake Level of, 13t–14t, 14, 18 Vitamin A, 13t Vitamin A deficiency, 13t Vitamin B6, 13t, 123, 124f Vitamin B6 deficiency, 13t, 711 cystathioninuria from, 730 microcytic, hypochromic anemia with, 828–829, 833 Vitamin B12, 750–752 absorption, transport, and storage of, 750–751, 752f in choline synthesis, 916 dietary guidelines for, 16, 750 food sources of, 13t, 16 functions of, 751–752, 752f for pernicious anemia, 751–752 Recommended Dietary Allowance for, 13t structure and forms of, 750, 751f Vitamin B12 deficiency, 13t, 14 choline in, 916 clinical presentations of, 753 development of, 750 megaloblastic anemia from, 745, 839 Vitamin C as free radical scavenger, 449, 450f on iron uptake, 829, 830f Recommended Dietary Allowance for, 13t Vitamin C deficiency, 13t Vitamin D Recommended Dietary Allowance for, 14t synthesis of, 656, 657f Vitamin D3, 177f Vitamin D deficiency, 13t Vitamin deficiencies, 13–14, 13t–14t Vitamin E (␣-tocopherol), 447f, 449, 450, 450f Vitamin E (␣-tocopherol) deficiency, 13t, 14 Vitamin K in blood coagulation, 855f, 856 derivatives of, 854, 855f Recommended Dietary Allowance for, 13t Vitamin K deficiency, 13t Vitamin KH2, 856 VLDL remnants, 641 VMAT2, 911, 911f Voltage-dependent anion channels (VDACs), 391f, 392, 393 Voltage-gated channels, 158 Voltage-gated Na⫹ channels, 887, 887f

Lieberman_Subject_Index.indd 1014

von Gierke (glycogen storage) disease, 518t, 522 von Willebrand factor (vWF), 850 von Willebrand factor (vWF) deficiency, 850, 861t W Waist circumference, 28 Waist-to-hip ratio, 28 Warfarin, 855f, 856, 859–860 Waste disposal pathways, 1 Water, 42–44 blood-tissue distribution of albumin in, 848–849, 849t body fluid maintenance in, 848 body, 41 total, 42–43, 43f weight of, 42, 43f dehydration and, 51–52, 52f dietary, 15 dipolar nature of, 43, 43f dissociation constant of, 45 dissociation of, 41, 44, 44f electrolytes in, 44, 44t from fuel oxidation, 3 gain of, 52, 52f hydrogen bonds in, 43, 43f hydrogen ions in, 44–45, 44f ion product of, 45 loss of, 51–52, 52f osmolality and movement of, 44 pH of, 41, 41f, 44–45 role of, 42 solubility in, 57 as solvent, 43, 44f in thermal regulation, 43 Weak acid, 45–46, 45t, 46 Weight, body. See also Body mass index (BMI) gain of, 10 healthy, 10 loss of, 10, 580t measurement of, 27 water in, 42, 43f Wernicke-Korsakoff syndrome, 462 Western blots, 293, 294f White blood cell (WBC). See Leukocyte White muscle fibers, 408 Williams syndrome, 932, 943t Wobble hypothesis, 250, 250f Work biochemical, 342–345 activated intermediates with high-energy bonds in, 344–345 additive ⌬G0 values in, 342–343, 343f, 343t substrate and product concentrations on ⌬G in, 340f, 343–344, 343t

energy for, 337–340 (See also ATP-ADP cycle) basic principle of, 337–338 change in Gibbs free energy (⌬G) in, 338–339, 339t ⌬G0 in, 339–340 exothermic and endothermic reactions in, 339–341, 340f, 340t high-energy ATP phosphate bond in, 338, 338f thermodynamics in, 339, 339t X Xanthelasma, 657 Xanthine oxidase, 441, 442, 769–770, 770f, 771 allopurinol as suicide inhibitor of, 129, 129f in ischemia–reperfusion injury, 442 Xanthoma, 657 Xanthurenic acid, in vitamin B6 deficiency, 711 Xenobiotic, 1, 3 biotransformation of, 867, 867f definition of, 867 dietary, 17 glycine conjugates of, synthesis of, 871t metabolism as fatty acids of, 877 Xeroderma pigmentosum, 219, 224, 225t, 233 Xerophthalmia, 13t X-linked severe combined immunodeficiency disease (X-SCID), 305, 837, 845t X-linked spastic paraplegia type 2 disease, 924 Xylulose 5-phosphate in gene transcription, 541 in hexose monophosphate shunt pathway, 530f, 535–537, 536f, 538f, 541 Y Yeast artificial chromosomes (YACs), 296 Z Zeaxanthin, 451, 451f Zellweger syndrome, 164, 425, 435t, 616, 876, 882t Zeta-potential, of red blood cells, 790 Zidovudine (ZDV), 196, 196f, 213 Zidovudine (ZDV) toxicity, 389, 393 Zinc-finger motifs, 277–278, 277f, 278f Zinc-finger transcription factors, 278, 278f Zwitterions, 70 Zymogens, 851 cleavage of, 697, 698f definition of, 145 in pancreas, 700 synthesis and secretion of, 145, 697, 697f

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