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A

Dietary Reference Intakes (DRI)

NOTE: For all nutrients, values for infants are AI. Dashes indicate that values have not been determined. aThe water AI includes drinking water, water in beverages, and wa- ter in foods; in general, drinking water and other beverages contrib- ute about 70 to 80 percent, and foods, the remainder. Conversion factors: 1 L = 33.8 fluid oz; 1 L = 1.06 qt; 1 cup = 8 fluid oz. bThe Estimated Energy Requirement (EER) represents the average dietary energy intake that will maintain energy balance in a healthy person of a given gender, age, weight, height, and physical activity level. The values listed are based on an “active” person at the refer- ence height and weight and at the midpoint ages for each group

until age 19. Chapter 8 provides equations and tables to determine estimated energy requirements. cThe linolenic acid referred to in this table and text is the omega-3 fatty acid known as alpha-linolenic acid. dThe values listed are based on reference body weights. eAssumed to be from human milk. fAssumed to be from human milk and complementary foods and beverages. This includes approximately 0.6 L (∼21⁄2 cups) as total fluid including formula, juices, and drinking water. gFor energy, the age groups for young children are 1–2 years and 3–8 years.

hFor males, subtract 10 kcalories per day for each year of age above 19. iBecause weight need not change as adults age if activity is main- tained, reference weights for adults 19 through 30 years are applied to all adult age groups. jFor females, subtract 7 kcalories per day for each year of age above 19.

SOURCE: Adapted from the Dietary Reference Intakes series, National Academies Press. Copyright 1997, 1998, 2000, 2001, 2002, 2004, 2005, 2011 by the National Academies of Sciences.

Age (yr)

Males

0–0.5 — 62 (24) 6 (13) 0.7e 570 60 — 31 4.4 0.5 9.1 1.52

0.5–1 — 71 (28) 9 (20) 0.8f 743 95 — 30 4.6 0.5 11 1.20

1–3g — 86 (34) 12 (27) 1.3 1046 130 19 — 7 0.7 13 1.05

4–8g 15.3 115 (45) 20 (44) 1.7 1742 130 25 — 10 0.9 19 0.95

9–13 17.2 144 (57) 36 (79) 2.4 2279 130 31 — 12 1.2 34 0.95

14–18 20.5 174 (68) 61 (134) 3.3 3152 130 38 — 16 1.6 52 0.85

19–30 22.5 177 (70) 70 (154) 3.7 3067h 130 38 — 17 1.6 56 0.80

31–50 22.5i 177 (70)i 70 (154)i 3.7 3067h 130 38 — 17 1.6 56 0.80

>50 22.5i 177 (70)i 70 (154)i 3.7 3067h 130 30 — 14 1.6 56 0.80

Females

0–0.5 — 62 (24) 6 (13) 0.7e 520 60 — 31 4.4 0.5 9.1 1.52

0.5–1 — 71 (28) 9 (20) 0.8f 676 95 — 30 4.6 0.5 11 1.20

1–3g — 86 (34) 12 (27) 1.3 992 130 19 — 7 0.7 13 1.05

4–8g 15.3 115 (45) 20 (44) 1.7 1642 130 25 — 10 0.9 19 0.95

9–13 17.4 144 (57) 37 (81) 2.1 2071 130 26 — 10 1.0 34 0.95

14–18 20.4 163 (64) 54 (119) 2.3 2368 130 26 — 11 1.1 46 0.85

19–30 21.5 163 (64) 57 (126) 2.7 2403j 130 25 — 12 1.1 46 0.80

31–50 21.5i 163 (64)i 57 (126)i 2.7 2403j 130 25 — 12 1.1 46 0.80

>50 21.5i 163 (64)i 57 (126)i 2.7 2403j 130 21 — 11 1.1 46 0.80

Pregnancy

1st trimester 3.0 +0 175 28 — 13 1.4 46 0.80

2nd trimester 3.0 +340 175 28 — 13 1.4 71 1.10

3rd trimester 3.0 +452 175 28 — 13 1.4 71 1.10

Lactation

1st 6 months 3.8 +330 210 29 — 13 1.3 71 1.30

2nd 6 months 3.8 +400 210 29 — 13 1.3 71 1.30

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The Dietary Reference Intakes (DRI) include two sets of values that serve as goals for nutrient intake—Recommended Dietary Allowances (RDA) and Adequate Intakes (AI). The RDA reflect the average daily amount of a nutrient considered adequate to meet the needs of most healthy people. If there is insufficient evidence to determine an RDA, an AI is set. AI are more ten­ tative than RDA, but both may be used as goals for nutrient intakes. (Chapter 9 provides more details.)

In addition to the values that serve as goals for nutrient in­ takes (presented in the tables on these two pages), the DRI in­ clude a set of values called Tolerable Upper Intake Levels (UL). The UL represent the maximum amount of a nutrient that ap­ pears safe for most healthy people to consume on a regular ba­ sis. Turn the page for a listing of the UL for selected vitamins and minerals.

Estimated Energy Requirements (EER), Recommended Dietary Allowances (RDA), and Adequate Intakes (AI) for Water, Energy, and the Energy Nutrients

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-202

B

Infants 0–0.5 0.2 0.3 2 5 1.7 0.1 65 0.4 125 40 400 400 (10 µg) 4 2.0 0.5–1 0.3 0.4 4 6 1.8 0.3 80 0.5 150 50 500 400 (10 µg) 5 2.5 Children 1–3 0.5 0.5 6 8 2 0.5 150 0.9 200 15 300 600 (15 µg) 6 30 4–8 0.6 0.6 8 12 3 0.6 200 1.2 250 25 400 600 (15 µg) 7 55 Males 9–13 0.9 0.9 12 20 4 1.0 300 1.8 375 45 600 600 (15 µg) 11 60 14–18 1.2 1.3 16 25 5 1.3 400 2.4 550 75 900 600 (15 µg) 15 75 19–30 1.2 1.3 16 30 5 1.3 400 2.4 550 90 900 600 (15 µg) 15 120 31–50 1.2 1.3 16 30 5 1.3 400 2.4 550 90 900 600 (15 µg) 15 120 51–70 1.2 1.3 16 30 5 1.7 400 2.4 550 90 900 600 (15 µg) 15 120 >70 1.2 1.3 16 30 5 1.7 400 2.4 550 90 900 800 (20 µg) 15 120 Females 9–13 0.9 0.9 12 20 4 1.0 300 1.8 375 45 600 600 (15 µg) 11 60 14–18 1.0 1.0 14 25 5 1.2 400 2.4 400 65 700 600 (15 µg) 15 75 19–30 1.1 1.1 14 30 5 1.3 400 2.4 425 75 700 600 (15 µg) 15 90 31–50 1.1 1.1 14 30 5 1.3 400 2.4 425 75 700 600 (15 µg) 15 90 51–70 1.1 1.1 14 30 5 1.5 400 2.4 425 75 700 600 (15 µg) 15 90 >70 1.1 1.1 14 30 5 1.5 400 2.4 425 75 700 800 (20 µg) 15 90 Pregnancy ≤18 1.4 1.4 18 30 6 1.9 600 2.6 450 80 750 600 (15 µg) 15 75 19–30 1.4 1.4 18 30 6 1.9 600 2.6 450 85 770 600 (15 µg) 15 90 31–50 1.4 1.4 18 30 6 1.9 600 2.6 450 85 770 600 (15 µg) 15 90 Lactation ≤18 1.4 1.6 17 35 7 2.0 500 2.8 550 115 1200 600 (15 µg) 19 75 19–30 1.4 1.6 17 35 7 2.0 500 2.8 550 120 1300 600 (15 µg) 19 90 31–50 1.4 1.6 17 35 7 2.0 500 2.8 550 120 1300 600 (15 µg) 19 90

NOTE: For all nutrients, values for infants are AI. a Niacin recommendations are expressed as niacin equivalents (NE), except for recommendations for infants younger than 6 months, which are expressed as preformed niacin. bFolate recommendations are expressed as dietary folate equivalents (DFE).

cVitamin A recommendations are expressed as retinol activity equivalents (RAE). d Vitamin D recommendations are expressed as cholecalciferol and assume an absence of adequate exposure to sunlight. eVitamin E recommendations are expressed as α-tocopherol.

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Recommended Dietary Allowances (RDA) and Adequate Intakes (AI) for Vitamins

Age (yr)

Infants 0–0.5 120 180 400 200 100 30 0.27 2 110 15 200 0.003 0.01 0.2 2 0.5–1 370 570 700 260 275 75 11 3 130 20 220 0.6 0.5 5.5 3 Children 1–3 1000 1500 3000 700 460 80 7 3 90 20 340 1.2 0.7 11 17 4–8 1200 1900 3800 1000 500 130 10 5 90 30 440 1.5 1.0 15 22 Males 9–13 1500 2300 4500 1300 1250 240 8 8 120 40 700 1.9 2 25 34 14–18 1500 2300 4700 1300 1250 410 11 11 150 55 890 2.2 3 35 43 19–30 1500 2300 4700 1000 700 400 8 11 150 55 900 2.3 4 35 45 31–50 1500 2300 4700 1000 700 420 8 11 150 55 900 2.3 4 35 45 51–70 1300 2000 4700 1000 700 420 8 11 150 55 900 2.3 4 30 45 >70 1200 1800 4700 1200 700 420 8 11 150 55 900 2.3 4 30 45 Females 9–13 1500 2300 4500 1300 1250 240 8 8 120 40 700 1.6 2 21 34 14–18 1500 2300 4700 1300 1250 360 15 9 150 55 890 1.6 3 24 43 19–30 1500 2300 4700 1000 700 310 18 8 150 55 900 1.8 3 25 45 31–50 1500 2300 4700 1000 700 320 18 8 150 55 900 1.8 3 25 45 51–70 1300 2000 4700 1200 700 320 8 8 150 55 900 1.8 3 20 45 >70 1200 1800 4700 1200 700 320 8 8 150 55 900 1.8 3 20 45 Pregnancy ≤18 1500 2300 4700 1300 1250 400 27 12 220 60 1000 2.0 3 29 50 19–30 1500 2300 4700 1000 700 350 27 11 220 60 1000 2.0 3 30 50 31–50 1500 2300 4700 1000 700 360 27 11 220 60 1000 2.0 3 30 50 Lactation ≤18 1500 2300 5100 1300 1250 360 10 13 290 70 1300 2.6 3 44 50 19–30 1500 2300 5100 1000 700 310 9 12 290 70 1300 2.6 3 45 50 31–50 1500 2300 5100 1000 700 320 9 12 290 70 1300 2.6 3 45 50 NOTE: For all nutrients, values for infants are AI.

Recommended Dietary Allowances (RDA) and Adequate Intakes (AI) for Minerals

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Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-202

C

Infants

0–0.5 — — — — — 600 1000 (25 µg) —

0.5–1 — — — — — 600 1500 (38 µg) —

Children

1–3 10 30 300 1000 400 600 2500 (63 µg) 200

4–8 15 40 400 1000 650 900 3000 (75 µg) 300

9–13 20 60 600 2000 1200 1700 4000 (100 µg) 600

Adolescents

14–18 30 80 800 3000 1800 2800 4000 (100 µg) 800

Adults

19–70 35 100 1000 3500 2000 3000 4000 (100 µg) 1000

>70 35 100 1000 3500 2000 3000 4000 (100 µg) 1000

Pregnancy

≤18 30 80 800 3000 1800 2800 4000 (100 µg) 800

19–50 35 100 1000 3500 2000 3000 4000 (100 µg) 1000

Lactation

≤18 30 80 800 3000 1800 2800 4000 (100 µg) 800

19–50 35 100 1000 3500 2000 3000 4000 (100 µg) 1000

Infants

0–0.5 — — 1000 — — 40 4 — 45 — — 0.7 — — — —

0.5–1 — — 1500 — — 40 5 — 60 — — 0.9 — — — —

Children

1–3 1500 2300 2500 3000 65 40 7 200 90 1000 2 1.3 300 3 0.2 —

4–8 1900 2900 2500 3000 110 40 12 300 150 3000 3 2.2 600 6 0.3 —

9–13 2200 3400 3000 4000 350 40 23 600 280 5000 6 10 1100 11 0.6 —

Adolescents

14–18 2300 3600 3000 4000 350 45 34 900 400 8000 9 10 1700 17 1.0 —

Adults

19–50 2300 3600 2500 4000 350 45 40 1100 400 10,000 11 10 2000 20 1.0 1.8

51–70 2300 3600 2000 4000 350 45 40 1100 400 10,000 11 10 2000 20 1.0 1.8

>70 2300 3600 2000 3000 350 45 40 1100 400 10,000 11 10 2000 20 1.0 1.8

Pregnancy

≤18 2300 3600 3000 3500 350 45 34 900 400 8000 9 10 1700 17 1.0 —

19–50 2300 3600 2500 3500 350 45 40 1100 400 10,000 11 10 2000 20 1.0 —

Lactation

≤18 2300 3600 3000 4000 350 45 34 900 400 8000 9 10 1700 17 1.0 —

19–50 2300 3600 2500 4000 350 45 40 1100 400 10,000 11 10 2000 20 1.0 —

aThe UL for niacin and folate apply to synthetic forms obtained from supplements, fortified foods, or a combination of the two. bThe UL for vitamin A applies to the preformed vitamin only.

Tolerable Upper Intake Levels (UL) for Vitamins

dThe UL for magnesium applies to synthetic forms obtained from supplements or drugs only. NOTE: An Upper Limit was not established for vitamins and minerals not listed and for those age groups listed with a dash (—) because of a lack of data, not because these nutrients are safe to consume at any level of intake. All nutrients can have adverse effects when intakes are excessive.

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Tolerable Upper Intake Levels (UL) for Minerals

cThe UL for vitamin E applies to any form of supplemental α-tocopherol, fortified foods, or a combination of the two.

SOURCE: Adapted with permission from the Dietary Reference Intakes series, National Academies Press. Copyright 1997, 1998, 2000, 2001, 2002, 2005, 2011 by the National Academies of Sciences.

Age (yr)

Age (yr)

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ADVANCED NUTRITION AND HUMAN METABOLISM

SEVENTH EDITION

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Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-202

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ADVANCED NUTRITION AND HUMAN METABOLISM

SEVENTH EDITION

Sareen S. Gropper FLORIDA ATLANTIC UNIVERSITY

AUBURN UNIVERSITY (PROFESSOR EMERITUS)

Jack L. Smith UNIVERSITY OF DELAWARE

Timothy P. Carr UNIVERSITY OF NEBRASKA-LINCOLN

Australia • Brazil • Mexico • Singapore • United Kingdom • United States

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Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-202

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To my children Michelle and Michael, and to my husband, Daniel, for their ongoing encouragement, support, faith, and love and to the students who continue to impress and inspire me.

Sareen Gropper

To my wife, Carol, for her continued support, constant inspiration, and assistance in the preparation of this book.

Jack Smith

To my family—Rebecca, Erin, and Marion—for their unwavering support and to the many students who have made my career so enjoyable.

Tim Carr

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Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-202

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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vii

BRIEF CONTENTS

Preface xvii

SECTION I Cells and Their Nourishment 1 The Cell: A Microcosm of Life 1 2 The Digestive System: Mechanism for Nourishing the Body 29

SECTION II Macronutrients and Their Metabolism 3 Carbohydrates 61 4 Fiber 107 5 Lipids 125 6 Protein 175 7 Integration and Regulation of Metabolism and the Impact

of Exercise 245 8 Energy Expenditure, Body Composition, and Healthy Weight 273

SECTION III The Regulatory Nutrients 9 Water-Soluble Vitamins 299 10 Fat-Soluble Vitamins 369 11 Major Minerals 425 12 Water and Electrolytes 455 13 Essential Trace and Ultratrace Minerals 479 14 Nonessential Trace and Ultratrace Minerals 543

Glossary 557 Index 563

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ix

CONTENTS

Preface xvii

SECTION I Cells and Their Nourishment

CHAPTER 1 The Cell: A Microcosm of Life 1 Components of Cells 1 Plasma Membrane 1 Cytoplasmic Matrix 4 Mitochondrion 4 Nucleus 6 Endoplasmic Reticulum and Golgi Apparatus 10 Lysosomes and Peroxisomes 11 Selected Cellular Proteins 11 Receptors 11 Catalytic Proteins (Enzymes) 13 Apoptosis 16 Biological Energy 17 Energy Release and Consumption in Chemical

Reactions 18 Expressions of Energy 18 The Role of High-Energy Phosphate in Energy

Storage 21 Coupled Reactions in the Transfer of Energy 21 Reduction Potentials 23 Summary 24 PERSPECTIVE Nutritional Genomics: A New Perspective on Food by Ruth DeBusk, PhD, RD 26

CHAPTER 2 The Digestive System: Mechanism for Nourishing the Body 29 The Structures of the Digestive Tract And the Digestive and Absorptive Processes 29 The Oral Cavity 32 The Esophagus 33 The Stomach 35 The Small Intestine 40 The Accessory Organs 43 The Absorptive Process 49 The Colon (Large Intestine) 51

Coordination and Regulation of the Digestive Process 55 Neural Regulation 55 Regulatory Peptides 55 Summary 58 PERSPECTIVE The Nutritional Impact of Roux-En-Y Gastric Bypass, A Surgical Approach for the Treatment of Obesity 59

SECTION II Macronutrients and Their Metabolism

CHAPTER 3 Carbohydrates 61 Overview of Structural Features 61 Simple Carbohydrates 62 Monosaccharides 62 Disaccharides 65 Complex Carbohydrates 66 Oligosaccharides 66 Polysaccharides 66 Digestion 67 Digestion of Polysaccharides 68 Digestion of Disaccharides 68 Absorption, Transport, and Distribution 68 Intestinal Absorption

of Glucose and Galactose 68 Intestinal Absorption of Fructose 71 Post-Absorption Facilitated Transport 71 Glucose Transporters 71 Glucose Entry into Interstitial Fluid 74 Maintenance of Blood Glucose

Concentration 75 Glycemic Response to Carbohydrates 75 Glycemic Index and Glycemic Load 75 Integrated Metabolism in Tissues 77 Glycogenesis 77 Glycogenolysis 80 Glycolysis 81 The Tricarboxylic Acid Cycle 84 Formation of ATP 87 The Pentose Phosphate Pathway (Hexose

Monophosphate Shunt) 94

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x CO N T E N T S

Gluconeogenesis 95 Regulation of Metabolism 98 Allosteric Enzyme Modulation 98 Covalent Regulation 99 Genetic Regulation 99 Directional Shifts

in Reversible Reactions 99 Metabolic Control of Glycolysis and

Gluconeogenesis 100 Summary 101 PERSPECTIVE What Carbohydrates Do Americans Eat? 104

CHAPTER 4 Fiber 107 Definitions 107 Fiber and Plants 108 Chemistry and Characteristics of Fiber 108 Cellulose 108 Hemicellulose 111 Pectins 111 Lignin 111 Gums 111 b-Glucans 111 Fructans 112 Resistant Starch 112 Mucilages (Psyllium) 112 Polydextrose and Polyols 113 Resistant Dextrins 113 Chitin and Chitosan 113 Selected Properties of Fiber and Their Physiological Impact 113 Solubility in Water 114 Viscosity and Gel Formation 114 Fermentability 115 Health Benefits of Fiber 115 Cardiovascular Disease 115 Diabetes Mellitus 117 Appetite and/or Satiety and Weight Control 117 Gastrointestinal Disorders 117 Food Labels and Health Claims 119 Recommended Fiber Intake 119 Summary 120 PERSPECTIVE The Flavonoids: Roles in Health and Disease Prevention 122

CHAPTER 5 Lipids 125 Structure and Biological Importance 126 Fatty Acids 126 Triacylglycerols (Triglycerides) 130 Phospholipids 131 Sphingolipids 133 Sterols 133

Dietary Sources 136 Recommended Intakes 138 Digestion 138 Triacylglycerol Digestion 139 Phospholipid Digestion 140 Cholesterol Ester Digestion 140 Absorption 141 Fatty Acid, Monoacylglycerol, and

Lysophospholipid Absorption 141 Cholesterol Absorption 142 Lipid Release into Circulation 143 Transport and Storage 143 Lipoprotein Structure 143 Lipoprotein Metabolism 145 Lipids, Lipoproteins, and Cardiovascular Disease Risk 151 Cholesterol 152 Saturated and Unsaturated Fatty Acids 152 Trans Fatty Acids 153 Lipoprotein(a) 153 Apolipoprotein E 153 Integrated Metabolism in Tissues 154 Catabolism of Triacylglycerols and Fatty Acids 154 Formation of Ketone Bodies 157 Synthesis of Fatty Acids 158 Synthesis of Triacylglycerols and Phospholipids 163 Synthesis, Catabolism,

and Whole-Body Balance of Cholesterol 163 Regulation of Lipid Metabolism 165 Fatty Acids 165 Cholesterol 166 Brown Fat Thermogenesis 166 Ethyl Alcohol: Metabolism and Biochemical Impact 167 The Alcohol Dehydrogenase (ADH) Pathway 167 The Microsomal Ethanol Oxidizing System

(MEOS) 168 The Catalase System 169 Alcoholism: Biochemical

and Metabolic Alterations 169 Alcohol in Moderation: The Brighter Side 170 Summary 171 PERSPECTIVE The Role of Lipoproteins and Inflammation in Atherosclerosis 173

CHAPTER 6 Protein 175 Amino Acid Classification 175 Structure 175 Net Electrical Charge 176 Polarity 177 Essentiality 178 Sources of Amino Acids 178

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CO N T E N T S xi

Digestion 179 Stomach 179 Small Intestine 180 Absorption 181 Intestinal Cell Absorption 181 Extraintestinal Cell Absorption 184 Amino Acid Catabolism 184 Transamination of Amino Acids 186 Deamination of Amino Acids 187 Disposal of Ammonia 187 Carbon Skeleton/α-Keto Acid Uses 189 Hepatic Catabolism and Uses of Aromatic

Amino Acids 190 Hepatic Catabolism and Uses of Sulfur (S)–Containing

Amino Acids 194 Hepatic Catabolism and Uses of Branched-Chain

Amino Acids 196 Hepatic Catabolism and Uses of Basic Amino Acids 197 Hepatic Catabolism and Uses of Other Selected

Amino Acids 199 Protein Synthesis 201 Slow versus Fast Proteins 201 Plant versus Animal Proteins 201 Hormonal Effects 201 Amino Acids, Intracellular Signaling, and mTOR 202 Protein Intake, Distribution and Quantity at Meals 202 Protein Structure and Organization 203

Functional Roles of Proteins and Nitrogen-Containing Nonprotein Compounds 204 Catalysts 204 Messengers 206 Structural Elements 206 Buffers 206 Fluid Balancers 206 Immunoprotectors 207 Transporters 207 Acute-Phase Responders 208 Other Roles 208 Nitrogen-Containing Nonprotein Compounds 209 Interorgan “Flow” of Amino Acids and Organ-Specific Metabolism 218 Intestinal Cell Amino Acid Metabolism 218 Amino Acids in the Plasma 220 Glutamine and the Muscle, Intestine, Liver, and

Kidneys 220 Alanine and the Liver and Muscle 221 Skeletal Muscle Use of Amino Acids 222 Amino Acid Metabolism in the Kidneys 225 Brain and Accessory Tissues and Amino Acids 227 Catabolism of Tissue Proteins and Protein Turnover 229

Lysosomal Degradation (also called the Autophagic Lysosome Pathway) 230

Proteasomal Degradation (also called the Ubiquitin Proteasomal Pathway) 230

Changes in Body Mass With Age 231 Protein Quality and Protein and Amino Acid Needs 233 Evaluation of Protein Quality 233 Protein Information on Food Labels 236 Assessing Protein and Amino Acid Needs 236 Recommended Protein and Amino Acid Intakes 237 Protein Deficiency/Malnutrition 239 Summary 239 PERSPECTIVE Stress and Inflammation: Impact on Protein 241

CHAPTER 7 Integration and Regulation of Metabolism and the Impact of Exercise 245 Energy Homeostasis in the Cell 245 Regulatory Enzymes 247 Integration of Carbohydrate, Lipid, and Protein Metabolism 249 Interconversion of Fuel Molecules 249 Energy Distribution among Tissues 251 The Fed-Fast Cycle 255 The Fed State 255 The Postabsorptive State 256 The Fasting State 258 The Starvation State 259 Hormonal Regulation of Metabolism 261 Insulin 262 Glucagon 263 Epinephrine 263 Cortisol 263 Growth Hormone 263 Exercise and Nutrition 264 Muscle Function 264 Energy Sources in Resting Muscle 265 Muscle ATP Production during Exercise 265 Fuel Sources during Exercise 267 Summary 270 PERSPECTIVE The Role of Dietary Supplements in Sports Nutrition By Karsten Koehler, PhD 271

CHAPTER 8 Energy Expenditure, Body Composition and Healthy Weight 273 Measuring Energy Expenditure 273 Direct Calorimetry 273 Indirect Calorimetry 274 Doubly Labeled Water 276

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xii CO N T E N T S

Components of Energy Expenditure 276 Basal and Resting Metabolic Rate 277 Energy Expenditure of Physical Activity 278 Thermic Effect of Food 279 Thermoregulation 279 Body Weight: What Should We Weigh? 280 Ideal Body Weight Formulas 280 Body Mass Index 280 Measuring Body Composition 283 Field Methods 283 Laboratory Methods 284 Regulation of Energy Balance and Body Weight 286 Hormonal Influences 286 Intestinal Microbiota 289 Environmental Chemicals 289 Lifestyle Influences 289 Health Implications of Altered Body Weight 290 Metabolic Syndrome 290 Insulin Resistance 291 Weight-Loss Methods 291 Summary 292 PERSPECTIVE Eating disorders 294

SECTION III The Regulatory Nutrients

CHAPTER 9 Water-Soluble Vitamins 299 Vitamin C (Ascorbic Acid) 303 Sources 304 Digestion, Absorption, Transport, and Storage 305 Functions and Mechanisms of Action 305 Interactions with Other Nutrients 310 Metabolism and Excretion 310 Recommended Dietary Allowance 310 Deficiency: Scurvy 310 Toxicity 311 Assessment of Nutriture 312 Thiamin (Vitamin B1) 312 Sources 313 Digestion, Absorption, Transport, and Storage 313 Functions and Mechanisms of Action 314 Metabolism and Excretion 318 Recommended Dietary Allowance 318 Deficiency: Beriberi 318 Toxicity 319 Assessment of Nutriture 319 Riboflavin (Vitamin B2) 320 Sources 321 Digestion, Absorption, Transport, and Storage 321 Functions and Mechanisms of Action 322 Metabolism and Excretion 323

Recommended Dietary Allowance 324 Deficiency: Ariboflavinosis 324 Toxicity 324 Assessment of Nutriture 324 Niacin (Vitamin B3) 325 Sources 325 Digestion, Absorption, Transport, and Storage 326 Functions and Mechanisms of Action 327 Metabolism and Excretion 329 Recommended Dietary Allowance 329 Deficiency: Pellagra 329 Toxicity 329 Assessment of Nutriture 330 Pantothenic Acid 330 Sources 330 Digestion, Absorption, Transport, and Storage 332 Functions and Mechanisms of Action 332 Metabolism and Excretion 334 Adequate Intake 334 Deficiency: Burning Foot Syndrome 334 Toxicity 335 Assessment of Nutriture 335 Biotin 335 Sources 335 Digestion, Absorption, Transport, and Storage 336 Functions and Mechanisms of Action 336 Metabolism and Excretion 339 Adequate Intake 340 Deficiency 340 Toxicity 340 Assessment of Nutriture 340 Folate 341 Sources 341 Digestion, Absorption, Transport, and Storage 343 Functions and Mechanisms of Action 344 Interactions with Other Nutrients 348 Metabolism and Excretion 349 Recommended Dietary Allowance 349 Deficiency: Megaloblastic Macrocytic Anemia 349 Toxicity 351 Assessment of Nutriture 351 Vitamin B12 (Cobalamin) 352 Sources 352 Digestion, Absorption, Transport, and Storage 353 Functions and Mechanisms of Action 354 Metabolism and Excretion 356 Recommended Dietary Allowance 356 Deficiency: Megaloblastic Macrocytic Anemia 356 Toxicity 357 Assessment of Nutriture 357 Vitamin B6 358 Sources 359 Digestion, Absorption, Transport, and Storage 359 Functions and Mechanisms of Action 360

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CO N T E N T S xiii

Metabolism and Excretion 363 Recommended Dietary Allowance 363 Deficiency 363 Toxicity 364 Assessment of Nutriture 364 PERSPECTIVE Genetics and Nutrition: The Effect on Folic Acid Needs and Risk of Chronic Disease by Dr. Rita M. Johnson 365

CHAPTER 10 Fat-Soluble Vitamins 369 Vitamin A and Carotenoids 370 Sources 371 Digestion and Absorption 373 Transport, Metabolism, and Storage 376 Functions and Mechanisms of Action 378 Interactions with Other Nutrients 385 Metabolism and Excretion 386 Recommended Dietary Allowance 386 Deficiency 387 Toxicity 387 Assessment of Nutriture 388 Vitamin D 389 Sources 389 Absorption 391 Transport, Metabolism, and Storage 391 Functions and Mechanisms of Action 393 Interactions with Other Nutrients 398 Metabolism and Excretion 398 Recommended Dietary Allowance 398 Deficiency: Rickets and Osteomalacia 398 Toxicity 399 Assessment of Nutriture 400 Vitamin E 401 Sources 402 Digestion and Absorption 403 Transport, Metabolism, and Storage 403 Functions and Mechanisms of Action 403 Interactions with Other Nutrients 406 Metabolism and Excretion 407 Recommended Dietary Allowance 407 Deficiency 407 Toxicity 407 Assessment of Nutriture 407 Vitamin K 408 Sources 409 Absorption 409 Transport, Metabolism, and Storage 409 Functions and Mechanisms of Action 410 Interactions with Other Nutrients 413 Metabolism and Excretion 413 Adequate Intake 413 Deficiency 414 Toxicity 414 Assessment of Nutriture 414

PERSPECTIVE Antioxidant Nutrients, Reactive Species, and Disease 416

CHAPTER 11 Major Minerals 425 Calcium 426 Sources 426 Digestion, Absorption, and Transport 427 Regulation and Homeostasis 429 Functions and Mechanisms of Action 432 Interactions with Other Nutrients 435 Excretion 436 Recommended Dietary Allowance 436 Deficiency 436 Toxicity 437 Assessment of Nutriture 438 Phosphorus 439 Sources 439 Digestion, Absorption, and Transport 439 Regulation and Homeostasis 440 Functions and Mechanisms of Action 441 Excretion 443 Recommended Dietary Allowance 444 Deficiency 444 Toxicity 444 Assessment of Nutriture 445 Magnesium 445 Sources 445 Digestion, Absorption, and Transport 446 Regulation and Homeostasis 447 Functions and Mechanisms of Action 447 Interactions with Other Nutrients 448 Excretion 449 Recommended Dietary Allowance 449 Deficiency 449 Toxicity 451 Assessment of Nutriture 451 PERSPECTIVE Osteoporosis and Diet 452

CHAPTER 12 Water and Electrolytes 455 Water Functions 455 Body Water Content and Distribution 455 Water Losses, Sources, and Absorption 456 Recommended Water Intake 457 Water (Fluid) and Sodium Balance 457 Osmotic Pressure 457 Hydrostatic (Fluid/Capillary) Pressure 459 Colloidal Osmotic Pressure 459 Extracellular Fluid Volume and Osmolarity and

Hormonal Controls 459 Sodium 463 Sources 463

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xiv CO N T E N T S

Digestion, Absorption, Transport, and Storage 510 Functions and Mechanisms of Action 513 Interactions with Other Nutrients 515 Excretion 515 Recommended Dietary Allowance 516 Deficiency 516 Toxicity 517 Assessment of Nutriture 517 Selenium 518 Sources 518 Digestion, Absorption, Transport, and Storage 519 Metabolism 520 Functions and Mechanisms of Action 520 Interactions with Other Nutrients 524 Excretion 524 Recommended Dietary Allowance 524 Deficiency 524 Toxicity 525 Assessment of Nutriture 525 Chromium 525 Sources 526 Digestion, Absorption, Transport, and Storage 526 Functions and Mechanisms of Action 526 Excretion 527 Adequate Intake 527 Deficiency 528 Toxicity 528 Assessment of Nutriture 528 Iodine 528 Sources 528 Digestion, Absorption, Transport, and Storage 529 Functions and Mechanisms of Action 530 Interactions with Other Nutrients 531 Excretion 532 Recommended Dietary Allowance 532 Deficiency 532 Toxicity 533 Assessment of Nutriture 533 Manganese 534 Sources 534 Digestion, Absorption, Transport, and Storage 534 Functions and Mechanisms of Action 535 Interactions with Other Nutrients 536 Excretion 536 Adequate Intake 536 Deficiency 536 Toxicity 536 Assessment of Nutriture 536 Molybdenum 537 Sources 537 Digestion, Absorption, Transport, and Storage 537 Functions and Mechanisms of Action 537 Interactions with Other Nutrients 539 Excretion 539

Absorption and Transport 464 Functions and Interactions with Other Nutrients 464 Excretion 464 Adequate Intake, Deficiency, Toxicity, and

Assessment of Nutriture 465 Potassium 466 Sources 466 Absorption, Secretion, and Transport 466 Functions and Interactions with Other Nutrients 467 Excretion 467 Adequate Intake, Deficiency, Toxicity, and

Assessment of Nutriture 467 Chloride 468 Sources 468 Absorption, Secretion, and Transport 468 Functions 469 Excretion 469 Adequate Intake, Deficiency, Toxicity, and

Assessment of Nutriture 469 Acid-Base Balance: Control of Hydrogen Ion Concentration 469 Chemical Buffer Systems 470 Respiratory Regulation 472 Renal Regulation 472 Summary 474 PERSPECTIVE Macrominerals and Hypertension 476

CHAPTER 13 Essential Trace and Ultratrace Minerals 479 Iron 479 Sources 480 Digestion, Absorption, Transport, and Storage 482 Functions and Mechanisms of Action 490 Turnover 494 Interactions with Other Nutrients 495 Excretion 495 Recommended Dietary Allowance 496 Deficiency 496 Toxicity 497 Assessment of Nutriture 498 Zinc 499 Sources 499 Digestion, Absorption, Transport, and Storage 500 Functions and Mechanisms of Action 504 Interactions with Other Nutrients 507 Excretion 507 Recommended Dietary Allowance 508 Deficiency 508 Toxicity 508 Assessment of Nutriture 508 Copper 509 Sources 509

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CO N T E N T S xv

Recommended Dietary Allowance 540 Deficiency 540 Toxicity 540 Assessment of Nutriture 540 PERSPECTIVE Nutrient–Drug Interactions 541

CHAPTER 14 Nonessential Trace and Ultratrace Minerals 543 Fluoride 543 Sources 543 Absorption, Transport, Storage, and Excretion 545 Functions and Deficiency 545 Recommended Intake, Toxicity, and Assessment

of Nutriture 545 Arsenic 546 Sources 546 Absorption, Transport, Storage, and Excretion 547 Functions and Deficiency 548 Recommended Intake, Toxicity, and Assessment

of Nutriture 548 Boron 549 Sources 549 Absorption, Transport, Storage, and Excretion 549 Functions and Deficiency 549 Recommended Intake, Toxicity, and Assessment

of Nutriture 549

Nickel 550 Sources 550 Absorption, Transport, Storage, and Excretion 550 Functions and Deficiency 550 Recommended Intake, Toxicity, and Assessment

of Nutriture 551 Silicon 551 Sources 551 Absorption, Transport, Storage, and Excretion 552 Functions and Deficiency 552 Recommended Intake, Toxicity, and Assessment

of Nutriture 552 Vanadium 552 Sources 553 Absorption, Transport, Storage, and Excretion 553 Functions and Deficiency 553 Recommended Intake, Toxicity, and Assessment

of Nutriture 554 Cobalt 554 PERSPECTIVE No, Silver is not Another Essential Ultratrace Mineral: Tips To Identifying Bogus Claims About Dietary Supplements 555

Glossary 557 Index 563

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xvii

PREFACE

S ince the first edition was published in 1990, much has changed in the science of nutrition. But the purpose of the text—to provide thorough coverage of normal metabolism for upper-division nutrition students—remains the same. We continue to strive for a level of detail and scope of material that satisfy the needs of both instructors and students. With each succeeding edition, we have responded to suggestions from instructors, content reviewers, and stu- dents that have improved the text by enhancing the clarity of the material and by ensuring accuracy. In addition, we have included the latest and most pertinent nutrition sci- ence available to provide future nutrition professionals with the fundamental information vital to their careers and to provide the basis for assimilating new scientific discoveries.

Just as the body of information on nutrition science has increased, so has the team of authors working on this text. Dr. James Groff and Dr. Sara Hunt coauthored the first edi- tion. In subsequent editions, Dr. Sareen Gropper became a coauthor as Dr. Hunt entered retirement. In the fourth edi- tion, Dr. Jack L. Smith joined the author team now led by Dr. Gropper. In this seventh edition, Dr. Tim Carr has provided additional expertise and co-authorship on several chapters.

NEW TO THIS EDITION

All chapters of the seventh edition have been updated, and many feature new or enhanced tables and illustra- tions. The organization of the content among the chap- ters has remained similar to the sixth edition.

Chapter 1 The Cell: A Microcosm of Life

● expanded the discussion of the components of the cytoskeleton

● elaborated on the mechanisms of apoptosis ● condensed and focused chapter content

Chapter 2 The Digestive System: Mechanism for Nourishing the Body

● expanded coverage of saliva, the regulation of gastric se- cretions and motility, and the roles of colonic microflora

● added information on tight junctions

● incorporated discussion of disorders causing malfunc- tion of the gastrointestinal tract from the Perspective into the Chapter

● included new figures to enhance presentation of hy- drochloric acid secretion and hepatic physiology

● added a new Perspective addressing the nutritional impact of gastric bypass surgery

Chapter 3 Carbohydrates

● added a new Perspective on the trends in carbohydrate in- take in the United States over the past several decades

● expanded coverage on dextrins in the food supply and their digestion and metabolism

● added information on glycolysis and updated the rel- evant figures

● expanded coverage on the role of insulin and added a new figure on insulin signaling

Chapter 4 Fiber

● added new fiber definitions ● refocused and condensed the discussion of the proper-

ties of fiber to reflect current trends ● added 2015 Dietary Guidelines recommendations re-

lated to dietary patterns

Chapter 5 Lipids

● provided more thorough coverage on lipid digestion and absorption

● added a new figure depicting the major fat sources in the American diet

● expanded coverage on cholesterol, phytosterols, phos- pholipids, and sphingolipids

● added a new section on dietary sources of lipids and recommended intake

● reorganized and expanded the section on lipid trans- port and metabolism

● added a new figure depicting ethanol oxidation in the liver

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xviii P R E FAC E

Chapter 9 Water-Soluble Vitamins

● provided new tables addressing the water-soluble vita- min contents of foods

● added information on the amounts and forms of the vitamins used in supplements

● included new tables addressing common manifestations of water-soluble vitamin deficiencies and an overview of water-soluble vitamin absorption and storage

● expanded coverage of the metabolic roles of thiamin, niacin, and pantothenic acid

● added sections on selected pharmacological uses of the vitamins

● updated and expanded coverage of water soluble vita- min deficiencies including those at risk for deficiency and the treatment of deficiencies

Chapter 10 Fat-Soluble Vitamins

● added tables to more thoroughly cover the fat-soluble vitamin content of foods

● included a new table addressing common manifestations of fat-soluble vitamin deficiencies

● expanded the coverage of fat-soluble vitamin deficiencies including those at risk of deficiencies and the treatment of deficiencies

Chapter 11 Major Minerals

● provided more thorough coverage of the major min- eral contents of foods

● added information on the amounts and forms of the major minerals used in supplements

● expanded information on the manifestations associated with deficiencies of the major minerals and treatment of deficiencies

● added to the Perspective information on new tools used in assessing risk of osteoporosis

Chapter 12 Water and Electrolytes

● added new sections addressing water sources, absorp- tion, and recommendations

● reorganized and expanded the discussion of water and sodium balance as well as acid base balance

● elaborated on the roles of the kidneys in maintaining fluid and sodium balance as well as acid base balance

● included several new figures depicting the role of the kidneys and hormones in maintaining fluid and sodium balance

Chapter 6 Protein

● expanded the section on protein synthesis to include amino acid signaling, mTOR, and distribution of pro- tein intake

● added information on new methodology used to eval- uate protein quality

● provided a more detailed discussion of protein malnu- trition and its diagnosis

● redirected the Perspective to address the impact of stress and inflammation on protein

Chapter 7 Integration and Regulation of Metabolism and the Impact of Exercise

● added a new Perspective on sports nutrition and supplementation

● expanded coverage on the distribution of fuel mol- ecules and tissue-specific energy utilization

● added new section on muscle function and energy requirements during exercise, including new and up- dated figures

● expanded coverage on regulatory hormones ● expanded coverage on the fed-fast cycle, including

new and updated figures

Chapter 8 Energy Expenditure, Body Composition, and Healthy Weight

● reorganized the chapter sections to improve flow and readability

● added a new figure depicting obesity prevalence in the United States

● added a new table describing the Institute of Medi- cine’s Physical Activity Level (PAL) categories

● updated photographs illustrating methods of assessing body composition

● added new tables summarizing ideal body weight for- mulas and body mass index categories

● reorganized and updated the discussion on field and labo- ratory methods used to measure body composition

● provided more thorough coverage of factors regulating energy balance and body weight

● added a new section on the health implications of al- tered body weight

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P R E FAC E xix

ORGANIZATION

Each of the 14 chapters begins with a topic outline, fol- lowed by a brief introduction to the chapter’s subject matter. These features are followed in order by the chap- ter text, a brief summary that ties together the ideas presented in the chapter (in Chapters 1–8 and 12), a ref- erence list, and a Perspective with its own reference list.

The text is divided into three sections. Section I (Chap- ters 1 and 2) focuses on cell structure, gastrointestinal tract anatomy, and function with respect to digestion and absorption.

Section II (Chapters 3–8) discusses metabolism of the macronutrients. This section reviews primary met- abolic pathways for carbohydrates, lipids, and proteins, emphasizing those reactions particularly relevant to issues of health. Since most of the body’s energy pro- duction is associated with glycolysis or the tricarbox- ylic acid cycle by the way of the electron transport chain and oxidative phosphorylation, the carbohy- drates chapter (Chapter 3) covers these aspects of en- ergy transformation. We include a separate chapter (Chapter 4) on fiber. The metabolism of alcohol, which contributes to the caloric intake of many people, is dis- cussed within the lipids chapter (Chapter 5). Alcohol’s chemical structure more closely resembles that of car- bohydrates, but its metabolism is more similar to that of lipids. Chapter 7 discusses the interrelationships among the metabolic pathways that are common to the macronutrients. This chapter also includes a discus- sion of the regulation of the metabolic pathways and a description of the metabolic dynamics of the fed-fast cycle, along with a presentation of the effects of physi- cal exertion on the body’s metabolic pathways. Chapter 8 focuses on energy expenditure, energy balance, and healthy weight and also includes a brief discussion of measuring body composition and the health implica- tions of altered body weight.

Section III (Chapters 9–14) concerns those nutri- ents considered regulatory in nature: the water- and fat- soluble vitamins and the minerals, including the major minerals, trace minerals, and ultratrace minerals. These chapters cover nutrient features such as digestion, ab- sorption, transport, function, metabolism, excretion, deficiency, toxicity, and assessment of nutriture, as well as the latest Recommended Dietary Allowances or Ad- equate Intakes for each nutrient. Information about the major minerals has been split into two chapters: Chapter 11 addresses calcium, phosphorus, and magnesium, and Chapter 12 discusses sodium, potassium, and chloride. Chapter 12 integrates coverage of the maintenance of the body’s homeostatic environment—including discus- sions of body fluids, electrolyte balance, and pH mainte- nance—with the presentation of the electrolytes.

Chapter 13 Essential Trace and Ultratrace Minerals

● provided more thorough coverage of the trace and ul- tratrace mineral contents of foods including the addi- tion of new tables

● added information on the forms and amounts of trace minerals found in supplements

● added new sections on the pharmacological uses of minerals as appropriate

● expanded the discussion of the regulation of body and cellular iron along with a new figure showing controls on hepcidin

● included more information on manifestations of trace mineral deficiencies and their treatment

Chapter 14 Nonessential Trace and Ultratrace Minerals

● expanded the discussion of the roles of fluoride ● added information about the arsenic content of foods

and arsenic toxicity

PRESENTATION

The presentation of the text is designed to make the book easy for the reader to use. The second color draws atten- tion to important elements in the text, tables, and figures and helps generate reader interest. The Perspectives pro- vide applications of the information in the chapter text.

Because this book focuses on normal human nutrition and physiological function, it is an effective resource for students majoring in either nutrition sciences or dietetics. Intended for a course in advanced nutrition, the text pre- sumes a sound background in the biological sciences. At the same time, however, it provides a review of the basic sci- ences—particularly biochemistry and physiology, which are important to understanding the material. This text applies biochemistry to nutrient use from consumption through digestion, absorption, distribution, and cellular metabo- lism, making it a valuable reference for health care provid- ers. Health practitioners may use it as a resource to refresh their memories with regard to metabolic and physiological interrelationships and to obtain a concise update on current concepts related to human nutrition.

We continue to present nutrition as the science that inte- grates life processes from the molecular to the cellular level and on through the multisystem operation of the whole or- ganism. Our primary goal is to give a comprehensive picture of cell reactions at the tissue, organ, and system levels. Subject matter has been selected for its relevance to meeting this goal.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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xx P R E FAC E

We are indebted to the efforts of Chimborazo Publishing, Inc., who managed the creation of the instructor supplements, including the testbank, instructor’s manual, and lecture tools.

We owe special thanks to the reviewers whose thoughtful comments, criticisms, and suggestions were in- dispensable in shaping this text.

Seventh Edition Reviewers Michael E. Bizeau, Metropolitan State University of

Denver Janet Colson, Middle Tennessee State University Michael Crosier, Framingham State University J. Andrew Doyle, Georgia State University Elizabeth A. Kirk, Bastyr University Kevin L. Schalinske, Iowa State University Long Wang, California State University, Long Beach

Sixth Edition Reviewers Jodee L. Dorsey, Florida State University Jennifer Hemphill, Florida State University Elizabeth A. Kirk, Bastyr University and University of

Washington Steven E. Nizielski, Grand Valley State University Scott K. Reaves, California Polytechnic State University Karla P. Shelnutt, University of Florida

Fifth Edition Reviewers Richard C. Baybutt, Kansas State University Patricia B. Brevard, James Madison University Marie A. Caudill, California State Polytechnic University,

Pomona Prithiva Chanmugam, Louisiana State University Michele M. Doucette, Georgia State University Michael A. Dunn, University of Hawaii at Mānoa Steve Hertzler, Ohio State University Steven Nizielski, Grand Valley State University Kimberli Pike, Ball State University William R. Proulx, SUNY Oneonta Scott K. Reaves, California State University, San Luis

Obispo Donato F. Romagnolo, University of Arizona, Tucson James H. Swain, Case Western Reserve University

SUPPLEMENTARY MATERIAL

New to this edition, MindTap is a digital learning plat- form that works alongside your campus LMS to deliver course curriculum across the range of electronic devices in your life. MindTap is built on an “app” model, allowing enhanced digital collaboration and delivery of engaging content across a spectrum of Cengage and non-Cengage resources. Additionally, to enhance teaching and learn- ing from the textbook, the Instructor Companion Site provides instructors with book-specific lecture and class tools, such as PowerPoint® presentations, images, the in- structor’s manual, videos, and more, all available online via www.cengage.com/login. Lastly, Cengage Learning Testing Powered by Cognero is a flexible online system that allows the instructor to author, edit, and manage test bank content from multiple Cengage Learning solutions.

ACKNOWLEDGMENTS

Although this textbook represents countless hours of work by the authors, it is also the work of many other hardworking individuals. We cannot possibly list every- one who has helped, but we would like to call attention to a few individuals who have played particularly important roles. We thank our undergraduate and graduate nutri- tion students for their ongoing feedback. We thank the product manager, Krista Mastroianni; our content devel- oper, Kellie Petruzzelli; our art director, Michael Cook; our marketing manager, Ana Albinson; our content proj- ect manager, Carol Samet; and our permissions analysts, Christine Myaskovsky and Erika Mugavin. We extend special thanks to our production team and our copy edi- tor, Laura Specht Patchkofsky.

We appreciate the work of two additional contributors, who provided Perspectives published in previous editions as well as this edition of the text: Ruth M. DeBusk, Ph.D., R.D., for writing the Perspective “Nutritional Genomics: Another Perspective on Food,” and Rita M. Johnson, Ph.D., R.D., F.A.D.A., for the Perspective “Genetics and Nutrition: The Effect on Folic Acid Needs and Risk of Chronic Disease.” We also are very grateful for the writing contribution of Karsten Koehler, Ph.D. for their Perspective “The Role of Dietary Supplements in Sports Nutrition.”

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1

C ELLS ARE THE VERY ESSENCE OF LIFE. Cells may be defined as the basic living, structural, and functional units of the human body. They vary greatly in size, chemical composition, and function, but each one is a remarkable miniaturization of human life. Cells move, grow, ingest “food,” excrete wastes, react to their environment, and reproduce. This chapter provides a brief review of the basics of a cell, including cellular components, biological energy, and an overview of a cell’s natural life span.

Cells of multicellular organisms are called eukaryotic cells (from the Greek eu meaning “true,” and karyon meaning “nucleus”). Eukaryotic cells evolved from simpler, more primitive cells called prokaryotic cells (from the Greek meaning “before nucleus”). One distinguishing feature between the two cell types is that eukaryotic cells possess a defined nucleus, whereas prokaryotic cells do not. Also, eukaryotic cells are larger and much more complex structurally and functionally than their ancestors. Because this text addresses human metabolism and nutrition, all descriptions of cellular structure and function in this and subsequent chapters pertain to eukaryotic cells.

While specialization among cells is necessary for life, cells, in general, have certain basic similarities. All human cells have a plasma membrane and a nucleus (or have had a nucleus), and most contain an endoplasmic reticulum, Golgi apparatus, and mitochondria. For convenience of discussion, a “typical cell” is presented (Figure 1.1) to enable the identification of the various organelles and their functions, which characterize cellular life. Our discussion begins with the plasma membrane which forms the outer boundary of the cell, and then moves inward to examine the organelles found within the cell.

COMPONENTS OF CELLS

Plasma Membrane The plasma membrane is a sheet-like structure that encapsulates and surrounds the cell, allowing it to exist as a distinct unit. The plasma membrane, like other membranes within the cell, has distinct structural characteristics and functions.

● Plasma membranes are asymmetrical, with different inside and outside “faces.” ● Plasma membranes are not static, but are fluid structures.

Plasma membranes are composed primarily of proteins, cholesterol, and phospholipids. Phospholipids, shown in Figure 1.2, provide both a hydrophobic and a hydrophilic moiety that allows them to spontaneously form bimolecular sheets, called lipid bilayers, in aqueous environments like the human body.

THE CELL: A MICROCOSM OF LIFE

1

COMPONENTS OF CELLS Plasma Membrane Cytoplasmic Matrix Mitochondrion Nucleus Endoplasmic Reticulum and Golgi Apparatus Lysosomes and Peroxisomes

SELECTED CELLULAR PROTEINS Receptors Catalytic Proteins (Enzymes)

APOPTOSIS

BIOLOGICAL ENERGY Energy Release and Consumption in Chemical Reactions Expressions of Energy The Role of High-Energy Phosphate in Energy Storage Coupled Reactions in the Transfer of Energy Reduction Potentials

SUMMARY

P E R S P E C T I V E

NUTRITIONAL GENOMICS: ANOTHER PERSPECTIVE ON FOOD BY RUTH DEBUSK, PhD, RD

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2 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

Both integral and peripheral proteins are found interspersed with the plasma membrane’s lipid bilayer (Figure 1.3). These proteins are responsible for several membrane functions including mediating information transfer (as receptors), transporting ions and molecules (as channels, carriers, gates, and pumps), acting as cell adhesion molecules, and speeding up metabolic activities (as enzymes). Integral proteins are attached and embedded in the membrane through hydrophobic interactions; they are often transmembrane, spanning the entire structure. Peripheral proteins, in contrast, are associated with membranes through ionic interactions and are located on or near the membrane surface. Peripheral proteins may be attached to integral membrane proteins either directly or through intermediate proteins. Many of these membrane proteins have either lipid or carbohydrate attachments.

Carbohydrates are present in plasma membranes as glycolipids and glycoproteins. While some carbohydrate is found in all membranes, most of the glycolipids and glycoproteins of the cell are associated with the plasma membrane. The carbohydrate moiety of the membrane glycoproteins and glycolipids provides asymmetry to the membrane because the oligosaccharide side chains are located exclusively on the membrane layer facing the cell’s

It is this lipid bilayer that determines the structure of the plasma membrane. The fatty acid portion (hydrocarbon chain) of the phospholipids forms the hydrophobic (water- fearing) core of the membrane bilayer; it also inhibits many water-soluble compounds from passing into the cell and helps to retain water-soluble substances within the cell. The glycerol and phosphate-containing portions (polar head) of the phospholipid are hydrophilic (i.e., polar, water loving) and thus are oriented toward the cell’s aqueous environments found both outside the cell and in the cell cytosol.

Another important membrane lipid is cholesterol (Figure 1.3). Cholesterol influences the fluidity and thus permeability of membranes, affecting what may pass into and out of the cell; membranes with higher levels of cholesterol are less fluid. Within the membrane, cholesterol’s hydrocarbon side chain associates with the fatty acid/hydrocarbon chain portion of the phospholipids and cholesterol’s hydroxyl groups are positioned close to the polar head groups of the phospholipids. Cholesterol’s rigid planar steroid rings are positioned so as to interact with and stabilize the regions of the hydrocarbon chains closest to the polar head groups of the phospholipids. The rest of the hydrocarbon chain remains flexible and fluid.

Figure 1.1 Three-dimensional depiction of a typical mammalian liver cell. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Nucleolus

Plasma membrane

Nuclear membrane Smooth endoplasmic reticulum

Nuclear membrane pore

Rough endoplasmic reticulum

Lysosome

Mitochondrion

Cytosol

Filamentous cytoskeleton (microtubules)

Golgi apparatus

Nucleus

The nuclear membrane (or nuclear envelope) with its pores makes

communication possible between the nucleus and the

cytoplasmic matrix.

Endoplasmic reticulum provides continuity between the nuclear envelope, the Golgi apparatus, and the plasma membrane.

Rough endoplasmic reticulum A series of membrane sacks that contain ribosomes that build and process proteins.

The cytosol is the gel-like substance inside cells. Cytosol contains cell organelles, protein, electrolytes, and other molecules.

The Golgi apparatus is a series of membrane sacks that process and package proteins

after they leave the rough endoplasmic reticulum.

The nucleus contains the DNA in the cell. Molecules of DNA provide coded instructions used for protein synthesis.

Cell membrane or plasma membrane Cells are surrounded by a phospholipid bilayer that

contains embedded proteins, carbohydrates, and lipids. Membrane proteins act as receptors sensitive to external

stimuli and channels that regulate the movement of substances into and out of the cell.

Smooth endoplasmic reticulum Region of the endoplasmic reticulum involved in lipid synthesis. Smooth endoplasmic reticula do not have ribosomes and are not involved in protein synthesis.

Contains digestive enzymes that break up proteins, lipids, and nucleic acids. They also remove and recycle waste products.

Organelles that produce most of the energy (ATP) used by cells.

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 3

Figure 1.3 Fluid model of cell membrane. Lipids and proteins are mobile and can move laterally in the membrane.

Outside of Cell

Inside of Cell

Oligosaccharide side chain Glycolipid

Glycocalyx

Integral proteins

Cholesterol

Peripheral protein

Phospholipid membrane

Cholesterol enhances the mechanical stability and regulates membrane fluidity.

Part of transport system allowing specific water-soluble substances to pass through the membrane

Hydrophobic portion of cell membrane inhibits passage of water-soluble substances into and out of the cell.

Figure 1.2 Lipid bilayer structure of biological membranes.

Extracellular membrane proteins

Phospholipid bilayer

Intracellular space

Cytosol

Plasma membranes are made of a bilayer of phospholipids with proteins and cholesterol (not shown)

Hydrophobic fatty acids make up the interior portion of the plasma membrane

Hydrophilic polar head groups point toward hydrophilic environments

outer surface (and not toward the cytosol). In plasma membranes, these outer sugar residues form what is called the glycocalyx, the layer of carbohydrate on the cell’s outer surface. On the membranes of the organelles within the cell, however, the oligosaccharides are directed inward.

The plasma membrane glycoproteins may serve as the receptors for hormones, certain nutrients, and other substances that influence cellular function. Glycoproteins also may help regulate the intracellular communication necessary for cell growth and functions. Intracellular

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4 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

movement of organelles and the assembly of cellular components (such as spindle fibers for mitosis). Flagella and cilia also rely on microtubules for movement.

Intermediate filaments, about 10 nm in diameter, are a heterogeneous group of fibers that are dynamic, undergoing constant assembly and disassembly, controlled in part by phosphorylation and dephosphorylation. Intermediate filaments (Figure 1.4) provide mechanical strength to cells that are subjected to physical stress, such as neurons, muscle cells, and epithelial cells lining body cavities.

Microfilaments, the thinnest (about 4–6 nm in diameter) of the fibers making up the cytoskeleton, are long, linear, solid fibers made up of actin. Microfilaments, like the other fibers, polymerize and unpolymerize according to the needs of the cells. Microfilaments provide scaffolding or tracks for various cell functions. Microfilaments interact with microtubules to facilitate the movement of cellular organelles and vesicles, and their interactions with intermediate filaments are thought to enable communication from extracellular stimuli to organelles within the cytosol.

Structural Arrangement The structural arrangement within the cell influences metabolic pathways. The fluid portion of the matrix contains small molecules such as glucose, amino acids, oxygen, and carbon dioxide. This aqueous part of the cell is in contact with the cytoskeleton over a very broad surface area, and enables enzymes that are associated with the polymeric lattice to be in close proximity to their substrate molecules in the aqueous portion. Furthermore, the enzymes that catalyze the reactions of many metabolic pathways are oriented sequentially so that the product of one reaction is released in close proximity to the next enzyme for which it is a substrate; this enhances the velocity of the overall metabolic pathway. Such an arrangement exists among the enzymes that participate in glycolysis. Some other metabolic pathways that occur in the cytoplasmic matrix and that might be similarly affected include the hexose monophosphate shunt ( pentose phosphate pathway), glycogenesis, glycogenolysis, and fatty acid synthesis. The cytoplasmic matrix of eukaryotic cells contains a number of organelles, enclosed in bilayer membranes and described briefly in the following sections.

Mitochondrion The mitochondria are the primary sites of oxygen use in cells and are responsible for most of the metabolic energy (ATP) produced in cells. All cells in the body, with the exception of the erythrocyte, possess mitochondria. The erythrocyte disposes of its mitochondria and nucleus during the maturation process and then must depend solely on energy produced through anaerobic mechanisms,

communication occurs through pathways that convert information from one part of a cell to another in response to external stimuli. Generally, it involves the passage of chemical messengers from organelle to organelle or within the lipid bilayers of membranes. Intracellular communication is examined more closely in the “Receptors and Intracellular Signaling” section of this chapter.

Membranes are not structurally distinct from the aqueous compartments of the cell they surround. For example, the cytosol (or cytoplasm), which is the aqueous, gel-like, transparent substance, fills the cell and, together with a system of filaments, connects the various membranes of the cell. This interconnection creates a structure that makes it possible for a signal generated at one part of the cell to be transmitted quickly and efficiently to other regions of the cell.

Cytoplasmic Matrix The cytoplasmic (or cytosolic) matrix consists of a system of filaments or fibers (referred to as the cytoskeleton) that is found within the cytosol (Figures 1.1 and 1.4). The cyto- skeleton provides cells with:

● structural support, which defines the cell’s shape and helps to maintain its function

● a framework for positioning the various organelles (such as microvilli, which are extensions of intestinal cells)

● a network to direct the movement of materials and organelles within the cells

● a means of independent locomotion for specialized cells (such as sperm, white blood cells, and fibroblasts)

● a pathway for intercellular communication among cel- lular components (vital for cell activation and survival)

● possible transfer of RNA and DNA [1].

The cytoskeleton is made up of three groups of fibers: microtubules, intermediate filaments, and microfilaments.

Microtubules, Intermediate Filaments and Microfilaments Microtubules are hollow (with about a 24 nm outer diameter), relatively rigid tubular structures (Figure 1.4). They consist of primarily two proteins—a-tubulin and b-tubulin—which form heterodimers that polymerize end-to-end. Microtubules, once formed, can be further lengthened at one end by the addition of more dimers; the other end, however, may undergo disassembly. Micro- tubules interact with a number of intracellular com- ponents, including proteins. They provide mechanical support, like a platform or scaffold, to influence cell shape. They also provide a structure for the intracellular

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 5

enzymes in the mitochondrial matrix. The details of this process are described more fully in Chapter 3. Briefly, the mitochondria carry out the flow of electrons through the electron transport chain. This electron flow is strongly exothermic, and the energy released is used in part for ATP synthesis, an endothermic process. Molecular oxygen is ultimately, but indirectly, the oxidizing agent in these

primarily glycolysis. The mitochondria in different tissues vary according to the function of the tissue. In muscle, for example, the mitochondria are held tightly among the fibers of the contractile system. In the liver, however, the mitochondria have fewer restraints and move freely through the cytoplasmic matrix.

Mitochondrial Membrane The mitochondrion consists of a matrix or interior space surrounded by a double membrane (Figures 1.5 and 1.6). The mitochondrial outer membrane is relatively porous, whereas the inner membrane is selectively permeable, serving as a barrier between the cytoplasmic matrix and the mitochondrial matrix. The inner membrane has many invaginations, called the cristae, which increase its surface area, and has all the components of the electron transport chain embedded within it.

The electron transport (respiratory) chain is central to the process of oxidative phosphorylation, the mechanism by which most cellular ATP is produced. The components of the electron transport chain carry electrons and hydrogens during the catalytic oxidation of nutrients by

Figure 1.4 The cytoskeleton (microtrabecular lattice) provides a structure for cell organelles, microvilli (as found in intestinal mucosa cells), and large molecules. The cytosol is shown at about 300,000 times its actual size and was derived from hundreds of images of cultured cells viewed in a high-voltage electron microscope. Source: Adapted from Porter and Tucker, “The Ground Substance of the Cell,” 1981, Scientific American. Used by permission of Nelson Prentiss.

Endoplasmic reticulum

Ribosome

Plasma membrane

Mitochondrion

Polyribosome

Plasma membrane

Intermediate f ilaments

Microtubule

Microtrabeculae suspend the endoplasmic reticulum, mitochondria, and the microtubules.

The polyribosomes are located at the junctions of

the microtrabecular lattice.

Figure 1.5 The mitochondrion.

Ribosome

DNA Outer membrane

Matrix space

Inner membrane

Cristae

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6 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

into existing mitochondria. The genes contained in mitochondrial DNA, unlike those in the nucleus, are inherited only from the mother and code primarily for proteins needed for normal mitochondrial function and for ATP production. Several diseases—such as cytochrome c oxidase deficiency (also called complex IV deficiency), Leigh syndrome, and Kearns-Sayre syndrome—result from mutations in mitochondrial genes.

Nucleus The nucleus (see Figure 1.1) is the largest of the organelles within the cell. Because of its DNA content, the nucleus initiates and regulates most cellular activities. Surrounding the nucleus is the nuclear envelope, a dynamic structure composed of an inner and an outer membrane. The dynamic nature of these membranes makes communication possible between the nucleus and the cytoplasmic matrix and allows a continuous channel between the nucleus and the endoplasmic reticulum. At various intervals the two membranes of the nuclear envelope fuse, creating pores in the envelope. Clusters of proteins on the outer nuclear membrane serve as microtubule organization centers (MTOCs); these centers function to begin polymerizing and organizing the microtubules during mitosis. Within the nucleus, a matrix exists to facilitate nuclear functions.

reactions. The function of the electron transport chain is to couple the energy released by nutrient oxidation to the formation of ATP. The chain components are precisely positioned within the inner mitochondrial membrane, an important feature of the mitochondria, because it brings the products released in the matrix into close proximity with molecular oxygen. Figure 1.6 shows the flow of major reactants into and out of the mitochondrion.

Mitochondrial Matrix Among the metabolic enzyme systems functioning in the mitochondrial matrix are those that catalyze the reactions of the tricarboxylic cycle (TCA cycle; Chapter 3) and fatty acid oxidation (Chapter 5). Other enzymes are involved in the oxidative decarboxylation and carboxylation of pyru- vate (Chapter 3) and in certain reactions of amino acid metabolism (Chapter 6).

Mitochondria are capable of both fission and fusion, depending on the needs of the cell. They reproduce by dividing in two. Although the nucleus contains most of the cell’s deoxyribonucleic acid (DNA), the mitochondrial matrix contains a small amount of DNA and a few ribosomes, enabling limited synthesis of protein within the mitochondrion. Most mitochondrial enzymes are coded by nuclear DNA, synthesized on the rough endoplasmic reticulum (RER) in the cytosol, and then incorporated

Figure 1.6 Overview of a cross section of the mitochondria.

Pyruvate

The electron transport chain is positioned on the inner

membrane, and is central to oxidative phosphorylation.

Fatty acids

Pyruvate

Acetyl-CoA

TCA cycle

NADH

Outer membrane is relatively porous.

CO2

CO2

O2 O2 H2O

ADP 1

ATP

ATP

e

Fatty acids

ADP 1

PP

H1 H1 H1

Inner membrane is selectively porous.

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 7

These phases, together with replication, are reviewed briefly in this chapter, but the scope of this subject is large; interested readers should consult a current cell biology text or comprehensive biochemistry text for a more thorough description of protein biosynthesis.

Nucleic Acids Nucleic acids (DNA and RNA) are macromolecules formed from repeating units called nucleotides, sometimes referred to as nucleotide bases or just bases. Structurally, they consist of a nitrogenous core (either purine or pyrimidine), a pentose sugar (ribose in RNA, deoxyribose in DNA), and phosphate. Five different nucleotides are contained in the structures of nucleic acids: adenylic acid and guanylic acid are purines, and cytidylic acid, uridylic acid, and thymidylic acid are pyrimidines. The nucleotides are more commonly referred to by their nitrogenous base core only—namely, adenine, guanine, cytosine, uracil, and thymine, respectively. For convenience, particularly in describing the sequence of the polymeric nucleotides in a nucleic acid, the single-letter abbreviations are most often used. Adenine (A), guanine (G), and cytosine (C) are com- mon to both DNA and RNA, whereas uracil (U) is unique to RNA and thymine (T) is found only in DNA. When two

The nucleus (or nuclear matrix) contains substances such as minerals needed for nuclear function and molecules of DNA. DNA encodes the cell’s genetic information plus all the enzymes needed for its duplication. DNA is found wrapped around proteins called histones, and organized into structures called chromatin. Long strands of DNA and histones are known as chromosomes. Also within the nucleus is the nucleolus, a nonmembranebound structure, containing ribosomal RNA (rRNA), proteins, and DNA; it is the site of rRNA transcription and processing, and of ribosome assembly/synthesis.

Encoded within the nuclear DNA are thousands of genes that direct the synthesis of proteins. Each gene codes for a single specific protein. The cell genome is the entire set of genetic information, that is, all of the DNA within the cell. Barring mutations that may arise in the DNA, daughter cells, produced from a parent cell by mitosis, possess the identical genomic makeup of the parent cell. The process of DNA replication enables the DNA to be precisely copied at the time of mitosis.

After the cell receives a signal that protein synthesis is needed, protein biosynthesis occurs in phases referred to as transcription, translation, and elongation (Figure 1.7). Each phase requires DNA activity, RNA activity, or both.

Figure 1.7 Steps of protein synthesis. (1) Signals that protein synthesis needs to occur. (2) Transcription: The DNA molecule (gene) synthesizes the corresponding mRNA. (3) Translation: The corresponding mRNA molecule binds to a ribosome and directs protein synthesis based on the codon for each amino acid and the appropriate tRNA. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

mRNA subunits

tRNA subunits

amino acids

Key

Cell membrane

Cytosol

Cell membrane

Nucleus

❶ Cell signaling Cell signaling communicates the need to synthesize a protein to the nucleus.

❸ Nucleus

DNA

mRNA strand

Ribosome

tRNA subunit

Amino acid

mRNA strand

Polypeptide strand

❷ Transcription Transcription of a gene in the nucleus results in the synthesis of a strand of mRNA.

❸ Translation and Elongation The mRNA strand leaves the nucleus, binds to ribosomes, and directs protein translation with the help of tRNA subunits and their associated amino acids. This elongation process results in the production of a polypeptide strand.

Cytosol

Cytosol

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8 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

strands of nucleic acids interact with each other—as occurs in replication, transcription, and translation—bases in one strand pair specifically with bases in the second strand: A always pairs with T or U, and G pairs with C, in what is called complementary base pairing.

The nucleotides are connected by phosphates esterified to hydroxyl groups on the pentose—that is, deoxyribose or ribose—component of the nucleotide. The carbon atoms of the pentoses are assigned prime (′) numbers for identification. The phosphate group connects the 3′ carbon of one nucleotide with the 5′ carbon of the next nucleotide in the sequence. The 3′ carbon of the latter nucleotide in turn is connected to the 5′ carbon of the next nucleotide in the sequence, and so on. Therefore, nucleotides are attached to each other by 3′, 5′ diester bonds. The ends of a nucleic acid chain are called either the free 3′ end or the free 5′ end, meaning that the hydroxyl groups at those positions are not attached by phosphate to another nucleotide.

Cell Replication Cell replication involves the synthesis of daughter DNA molecules that are identical to the parental DNA. At cell division, the cell must copy its genome with a high degree of fidelity. Each strand of the DNA molecule acts as a template for synthesizing a new strand (Figure 1.8). The DNA molecule consists of two large strands of nucleic acid that are intertwined to form a double helix. During cell division the two unravel, with each form- ing a template for synthesizing a new strand through complementary base pairing. Incoming nucleotide bases first pair with their complementary bases in the template and then are connected through phosphate diester bonds by the enzyme DNA polymerase. The end result of the replication process is two new DNA chains that join with the two chains from the parent molecule to produce two new DNA molecules. Each new DNA molecule is therefore identical in base sequence to the parent, and each new cell of a tissue consequently carries within its nucleus identical information to direct its functioning. The two strands in the DNA double helix are antiparallel, which means that the free 5′ end of one strand is con- nected to the free 3′ end of the other. With this process, a cell is able to copy or replicate its genes before it passes them on to the daughter cell. Although errors sometimes occur during replication, mechanisms exist that correct or repair mismatched or damaged DNA.

Transcription Transcription is the process by which the genetic informa- tion (through the sequence of base pairs) in a single strand of DNA makes a specific sequence of bases in a messenger RNA (mRNA) chain (see Figure 1.7). A single strand of DNA can make many copies of the corresponding mRNA,

Figure 1.8 DNA replication.

Emerging progeny DNA

A T

G C

T A

A

G C

AT

GC

A T

G C

C G

A

A

G C

A T

GC

GC

GC CG

AT

A T

AT

GC

AT

A T

T

GC

AT

AT

G C

AT

AT

G C

Old OldNew

New

New

Old Old

Base pairing

The original DNA molecule

unravels so new identical DNA

molecules can be

synthesized.

During translation the double helix of DNA makes new strands by base pairing.

The two new DNA molecules contain an old strand and a new strand.

which become multiple templates for the assembly of a specific protein. This process multiplies the information contained in the DNA to produce many corresponding protein molecules. Transcription may require transcrip- tion factors, discussed under the subsection “Control of Gene Expression.”

Transcription proceeds continuously throughout the entire life cycle of the cell. In the process, various sections of the DNA molecule unravel, and one strand—called the sense strand—serves as the template for synthesizing mRNA. Sequences of DNA known as promoters allow genes to be turned “off ” or “on” and can initiate transcription; this promoter is usually found near (upstream) of the gene. The genetic code (gene) of the DNA is transcribed into mRNA through complementary base pairing, as in DNA replication, except that the purine adenine (A) pairs with the pyrimidine uracil (U) instead of with thymine (T). Genes are composed of critically sequenced base pairs along the entire length of the DNA strand that is being transcribed. A gene, on average, is just over 1,000 base pairs in length, compared with the nearly 5 million

3(5 10 )6 base pair length of typical chromosomal DNA chains. Although these figures provide a rough estimate of the number of genes per transcribed DNA chain, not all the base pairs of a gene are transcribed into functional mRNA.

Many genes for specific proteins are located on regions of the DNA nucleotide sequences that are not adjacent

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 9

At this point, the process stops, signaled by a “nonsense” codon that does not code for any amino acid. The completed protein dissociates from the mRNA. After translation, the newly synthesized protein may require some chemical, structural, or spatial ( three-dimensional) modification to attain its active form.

Post-translational modifications of proteins may involve, for example, the covalent addition of functional groups or the cleavage of a portion of the protein. Common modifications include phosphorylation as well as glycosylation, ubiquitination, methylation, and acetylation, among others. An example of protein modifications involving proteolytic cleavage is that needed to convert zymogens, such as those involved in protein digestion, to active enzymes.

Control of Gene Expression Each cell in the body contains a complete set of genes. Only a portion of the genes are expressed in special- ized cells of a given organ. The regulation of gene expression occurs primarily at three different levels. (1) Transcription-level control mechanisms determine if a particular gene can be transcribed. Transcriptional control is accomplished by large numbers of proteins (called transcriptional factors) that bind to the DNA at a site other than the one involved in serving as a template for the mRNA. These transcriptional factors can enhance, inhibit, or, in some cases, alter the frequency (number of times transcription occurs within a speci- fied time span) of the gene’s transcription. Several hor- mones, such as insulin, thyroid hormone, glucagon, and glucocorticoids, as well as nutrients, such as vitamins A and D, can alter the transcription of DNA by bind- ing along with transcription-factor proteins to DNA. (2) Processing-level control mechanisms determine the path by which mRNA can be translated into a polypep- tide. This mechanism of regulating gene expression is based on the splicing of RNA molecules, thus making it possible for one gene to code for two associated proteins. (3) Translation-level control mechanisms determine whether a particular mRNA is actually translated and, if so, how often and for how long. The translation-level control mechanism can involve the localization of the mRNA in a particular part of the cell or organ. It can also operate through interactions between specific mRNAs and various small RNA strands present within the cyto- sol. MicroRNAs (abbreviated miRNA) are small non- coding RNAs that silence gene expression by binding to mRNA to inhibit its translation and/or promote its deg- radation. For more detailed information on the control of gene expression and its relationship to disease, which is vastly more complex than has been presented here, the reader is referred to a recent textbook on molecular biology and biochemistry or cell biology.

to each other. Those regions that are part of the gene but do not code for a protein product are called introns (intervening sequences), and have to be removed from the mRNA before it is translated into protein (see the “Translation” section of this chapter). Enzymes excise the introns from the newly formed mRNA, and the ends of the functional, active mRNA segments are spliced together in a process called post-transcriptional processing. The gene segments that get both transcribed and translated into the protein product are called exons (expressed sequences).

Translation Translation is the process by which genetic information in an mRNA molecule is turned into the sequence of amino acids in the protein. After the mRNA is synthesized in the nucleus (see Figure 1.7), the mRNA is exported into the cytoplasmic matrix, where it is attached to ribosomal RNA (rRNA) of the ribosomes of the rough endoplasmic reticulum (RER) or to the free- standing polyribosomes (also called polysomes). On the ribosomes, the transcribed genetic code in the mRNA is used to bring amino acids into a specific sequence that produces the specified protein.

The genetic code for specifying the amino acid sequence of a protein resides in the mRNA in the form of three-base sequences called codons. Each codon codes for a single amino acid. Although a given amino acid may have several codons (e.g., the codons CUU, CUC, CUA, and CUG all code for the amino acid leucine), codons can code for only one amino acid. Each amino acid has one or more transfer RNAs (tRNAs), which deliver the amino acid to the mRNA for peptide synthesis. The three-base sequences of the tRNA attach to the codons by complementary base pairing.

Amino acids are first activated by ATP at their carboxyl end and then transferred to their specific tRNAs that bear the anticodon complementary to each amino acid’s codon. For example, because codons that code for leucine are sequenced CUU, CUC, CUA, or CUG, the only tRNAs to which an activated leucine can be attached would need to have the anticodon sequence GAA, GAG, GAU, or GAC. The tRNAs then bring the amino acids to the mRNA situated at the protein synthesis site on the ribosomes. After the amino acids are positioned according to codon–anticodon association, peptide bonds are formed between the aligned amino acids in a process called elongation (see Figure 1.7). Elongation extends the polypeptide chain of the protein product by translation. Each incoming amino acid is connected to the end of the growing peptide chain with a free carboxyl group (C-terminal end) by formation of further peptide bonds. New amino acids are incorporated until all the codons (corresponding to one completed protein or polypeptide chain) of the mRNA have been translated.

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10 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

as “stacks” because of this arrangement. Tubular networks are present at either end of the Golgi stacks.

● The cis-Golgi network is a compartment that accepts newly synthesized proteins coming from the ER.

● The trans-Golgi network is the exit site of the Golgi apparatus. It sorts proteins for delivery to their next destination.

Proteins destined for the Golgi apparatus form within the RER. Once they are transferred to the Golgi apparatus, additional molecules (such as carbohydrates or lipids) can be added to them there. The Golgi apparatus is the site for membrane differentiation and the development of surface specificity. For example, the polysaccharide moieties of mucopolysaccharides and of the membrane glycoproteins are synthesized and attached to the protein during its passage through the Golgi apparatus. Such an arrangement allows for the continual replacement of cellular membranes, including the plasma membrane.

The ER is a quality-control organelle in that it prevents proteins that have not achieved their normal tertiary or quaternary structure from reaching the cell surface. The ER can retrieve or retain proteins destined for residency within the ER, or it can target proteins for delivery to the cis-Golgi compartment. Retrieved or exported protein “cargo” is coated with protein complexes called coatomers, abbreviated COPs (coat proteins). Some coatomers are structurally similar to the clathrin coat of endocytic vesicles and are described later in this chapter. The choice of what is retrieved or retained by the ER and what is exported to the Golgi apparatus is mediated by signals that are inherent in the terminal amino acid sequences of the proteins in question. Certain amino acid sequences of cargo proteins are thought to interact specifically with certain coatomers.

The membrane-bound compartments of the ER and the Golgi apparatus are interconnected by transport vesicles, in which cargo proteins are moved from compartment to compartment. The vesicles leaving a compartment are formed by a budding and pinching off of the compartment membrane, and the vesicles then fuse with the membrane of the target compartment.

Secretion of products such as proteins from the cell can be either constitutive or regulated. If secretion follows a constitutive course, the secretion rate remains relatively constant, uninfluenced by external regulation. Regulated secretion, as the name implies, is affected by regulatory factors, and therefore its rate is changeable.

Among the more interesting areas of biomolecular research has been determining how newly synthesized proteins find their way from the ribosomes to their intended destinations. While proteins synthesized on the free polyribosomes remain within the cell to perform their specific structural, digestive, regulatory, or other functions, other proteins are destined elsewhere. At the time of synthesis, signal sequences direct proteins to

Endoplasmic Reticulum and Golgi Apparatus The endoplasmic reticulum (ER) is an extensive network of membranous channels pervading throughout the cytosol and providing continuity among the nuclear envelope, the Golgi apparatus, and the plasma membrane (see Figure 1.1). This structure, therefore, is a mechanism for communication from the innermost part of the cell to its exterior. In the laboratory, however, the ER cannot be separated from the cell as an isolated entity; during mechanical homogenization, the structure is disrupted and reforms into small spherical particles called microsomes.

The ER is classified as either rough (granular) or smooth (agranular). The granularity or lack of granularity is determined by the presence or absence of ribosomes. Rough endoplasmic reticulum (RER), so named because it is studded with ribosomes, abounds in cells where protein synthesis is a primary function. Smooth endoplasmic reticulum (SER) is found in most cells; however, because it is the site of synthesis for a variety of lipids, it is more abundant in cells that synthesize steroid hormones (e.g., within the adrenal cortex and gonads) and in liver cells, which synthesize fat transport molecules (the lipoproteins). In skeletal muscle, the smooth endoplasmic reticulum is called sarcoplasmic reticulum and is the site of the calcium ion pump, a necessity for the contractile process.

Ribosomes associated with RER are composed of ribosomal RNA and structural protein. All proteins to be secreted (or excreted) from the cell or destined to be incorporated into an organelle membrane in the cell are synthesized on the RER. The clusters of ribosomes (i.e., polyribosomes or polysomes) that are freestanding in the cytosol are also the synthesis site for some proteins. All proteins synthesized in polyribosomes in the cytosol remain within the cytoplasmic matrix or are incorporated into an organelle.

Located on the RER of liver cells is a system of enzymes important in metabolizing many different drugs. This enzyme complex consists of a family of cytochromes called the P450 system that functions along with other enzymes. The P450 system is particularly active in oxidizing drugs, but because its action results in the simultaneous oxidation of other compounds as well, the system is collectively referred to as the mixed-function oxidase system. Lipophilic substances—such as steroid hormones and numerous drugs—can be made hydrophilic by oxidation, reduction, or hydrolysis, to enable their excretion in the bile or urine. This system is discussed further in Chapter 5.

The Golgi apparatus functions closely with the ER in trafficking and sorting proteins that are synthesized in the cell; it is particularly prominent in neurons and secretory cells. It consists of four to eight membrane- enclosed, flattened cisternae that are stacked in parallel (see Figure 1.1). The Golgi cisternae are often referred to

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 11

Receptors Receptors are highly specific proteins located in the plasma membrane and facing the exterior of the cell. Bound to the outer surface of these specific proteins are oligosaccharide chains, which are believed to act as recognition markers. Mem- brane receptors act as attachment sites for specific external stimuli such as hormones, growth factors, antibodies, lipopro- teins, and certain nutrients (examples are shown in Figures 1.9 and 1.10). These molecular stimuli, which bind specifically to receptors, are called ligands. Receptors are also located on the membranes of cell organelles; less is known about these receptors, but they appear to be glycoproteins necessary for correctly positioning newly synthesized cellular proteins.

their appropriate target compartment. These targeting sequences, located at the N-terminus of the protein, are generally cleaved (though not always) when the protein reaches its destination. Interaction between the signal sequences and specific receptors located on the various membranes permits the protein to enter its designated membrane or become incorporated into the designated organelle. It is believed that in at least some cases, diseases result not just from the synthesis of enzymes that are inactive or deficient, but also result from the synthesis of proteins that fail to reach their correct destination [2].

Lysosomes and Peroxisomes Lysosomes and peroxisomes are cell organelles packed with enzymes. Whereas the lysosomes (see Figure 1.1) serve as the cell’s digestive system, the peroxisomes perform some specific oxidative catabolic reactions. Lyso- somes are found in all cells with the exception of red blood cells but in varying numbers. Approximately 36 enzymes capable of degrading substances such as proteins, polysac- charides, nucleic acids, and phospholipids are held within the confines of a single thick lysosomal membrane. The membrane surrounding these catabolic enzymes has the capacity for selective fusion with other vesicles so that catabolism (or degradation) may occur as necessary. Further information on the role of lysosomes in protein and cell turnover is provided in Chapter 6.

Peroxisomes are small intracellular enzyme-containing organelles surrounded by a single membrane. They are believed to originate by “budding” from the smooth endoplasmic reticulum. The peroxisomes are similar to the lysosomes; however, rather than having digestive action, the peroxisomal enzymes are catabolic oxidative enzymes. Very-long-chain fatty acids are oxidized in peroxisomes, while most other fatty acids are oxidized in the mitochondrial matrix. Peroxisomes are also the site for certain reactions of amino acid catabolism and for the oxidation of ethanol to acetaldehyde. Hydrogen peroxide H2O2 is often produced within peroxisomes; this peroxisomal segregation from other cell parts is helpful given the reactive and destructive nature of H2O2 to cell components. The presence of the enzyme catalase within peroxisomes is also helpful for H2O2 degradation into water and molecular oxygen.

SELECTED CELLULAR PROTEINS

Two roles of cellular proteins are discussed; these roles include receptors, that is, proteins that modify the cell’s response to its environment, and enzymes, that is, pro- teins serving as catalysts for biochemical reactions within cells. The reader is directed to Chapter 6 for information on other roles of proteins in the body. Figure 1.9 An example of an internal chemical signal by a second messenger.

cAMP

Hormone

Adenyl cyclase

G-protein

Receptor

γ β α

Inactive adenyl cyclase

G-protein

Receptor

γ β

γ α

β

γ α

β

α

P

GTP GDP

GTP

GDP

GDP

ATP

❶ The hormone attaches to the receptor molecule.

The receptor has a G-protein (a protein with GTP or GDP

attached to it) attached.

➌ When a hormone attaches to the receptor, the GDP is converted to

GTP and a portion of the G-protein attaches to

adenyl cyclase, activating it. The activated adenyl cyclase reacts with

ATP to form cAMP.

➍ The G-protein functions as a GTPase. When GTP is

converted to GDP, the fragment of G-protein

moves back to the receptor.

➎ Adenyl cyclase is inactivated and the

receptor loses the hormone.

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12 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

5′-cyclic adenosine monophosphate (cyclic adenosine monophosphate [AMP], or cAMP). It is formed from adenosine triphosphate (ATP) by the enzyme adenyl cyclase. Cyclic AMP is frequently referred to as the second messenger in the stimulation of target cells by hormones. Figure 1.9 presents a model for the ligand-binding action of receptors, which leads to production of the internal signal cAMP. As shown in the figure, the stimulated receptor reacts with guanosine triphosphate (GTP)– binding protein (G-protein), which activates adenyl cyclase, triggering production of cAMP from ATP. G-protein is a trimer with three subunits (designated a, b, and g). The a-subunit binds with GDP or GTP and has GTPase activity. Attachment of a hormone to the receptor stim- ulates the exchange of GDP for GTP. The GTP binding causes the trimers to disassociate and the a unit to asso- ciate with an effector protein, adenyl cyclase. A single hormone-binding site can produce many cAMP molecules.

The mechanism of action of cAMP signaling within the cell is complex, but it can be viewed briefly as follows: cAMP is an activator of protein kinases. Protein kinases are enzymes that phosphorylate (add phosphate groups to) other enzymes and, in doing so, generally convert the enzymes from inactive forms into active forms. Protein kinases that can be activated by cAMP contain two subunits: one catalytic and one regulatory. In the inactive form of the kinase, the two subunits are bound in such a way that the catalytic portion of the molecule is inhibited sterically by the presence of the regulatory subunit. Phosphorylation of the enzyme by cAMP causes the subunits to dissociate, thereby freeing the catalytic subunit, which regains its full catalytic capacity. As protein kinases serve to phosphorylate proteins and generally activate them, phosphatases work in opposition in order to remove phosphate groups from proteins and inactivate them. Thus, together the protein kinases and phosphatases function to turn on and off enzymes.

Many intracellular chemical messengers are known other than those cited as examples in this section. Listed here, along with cAMP, are several additional examples:

● cyclic AMP (cAMP) ● cyclic GMP (cGMP) ● 1Ca2

● inositol triphosphate ● diacyl glycerol ● fructose-2,6-bisphosphate.

Receptors That Function as Ion Channels Receptors can also act as ion channels. In some cases, the binding of the ligand to its receptor causes a voltage change, which then becomes the signal for a cellular response. Such is the case when the neurotransmitter acetylcholine is the stimulus. The receptor for acetylcholine appears to

Although most receptor proteins are probably integral membrane proteins, some may be peripheral. In addition, receptor proteins can vary widely in their composition and mechanism of action. Although the composition and mechanism of action of many receptors have not yet been determined, at least three distinct types of receptors are known to exist and are listed and described hereafter:

● those that generate internal chemical signals ● those that function as ion channels ● those that internalize stimuli.

Receptors That Generate Internal Chemical Signals Upon interaction between some receptors and ligands, an internal chemical signal is generated to affect inter- nal cellular processes. The internal chemical signal most often produced by a stimulus–receptor interaction is 3′,

Figure 1.10 Internalization of a stimulus into a cell via its receptor.

Ligand

Mobile receptors

Nucleus

Lysosome

Endosome

Clathrin-coated pit

Clathrin-coated vesicle

Clathrin

Ligand binds with its receptor on the cell membrane.

Ligand and receptor move into a clathrin-coated pit.

Pit closes of f and forms a clathrin-coated vesicle.

The vesicle forms an endosome.

Ligand can be used by the cell or undergo lysosomal degradation.

Receptor is recycled to the surface of the cell membrane.

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 13

located on the brush border membrane of the epithelial cells lining the small intestine. Other enzymes that are components of the cellular membranes and most enzymes associated with organelle membranes are found on the inner membrane surface. For example, the enzymes of the electron transport chain are located within the inner membrane of the mitochondria.

Enzymes have an “active site” where they bind with a substrate. The functional activity of some enzymes, however, depends not only on the enzyme’s protein portion, but also on a nonprotein prosthetic group or coenzyme/ cofactor. Many of the B-vitamins serve as coenzymes and several minerals—such as Mg, Zn, Cu, Mn, and Fe—serve as inorganic prosthetic groups (or cofactors) for enzymes.

An enzyme’s active site possesses high specificity. This means that a substrate must “fit” perfectly into the specific contours of the enzyme’s active site so that the reacting parts of the substrate are in close proximity to the reacting parts of the enzyme. The most common analogy used to describe this is a lock and key. The concept of interlocking pieces of a puzzle has also been used to convey that the substrate and enzyme must fit. The enzyme’s specificity can come from the reactive groups of its amino acids as part of the amino acid sequence or primary structure. The specificity may also originate from the three- dimensional or tertiary structure of the enzyme. Mutations in genes that alter a protein’s amino acid composition can result in changes in enzyme structure and/or its active site and thus affect its ability to bind to its substrate(s). Such defects can lead to inborn errors (genetic disorders) of metabolism such as phenylketonuria (PKU).

The velocity of an enzyme-catalyzed reaction (the number of molecules of substrate reacted on in a specified time) increases if all of the active sites on the enzyme are “filled” with substrate. As the concentration of the substrate increases, the number of molecules of substrate available to the enzyme increases. This increases the number of substrate molecules acted on by the enzyme-catalyzed reaction and is said to increase the rate of the reaction. However, this relationship applies only to a concentration of substrate that is less than the concentration that “saturates” the enzyme. At saturation levels of substrate, the enzyme functions at its maximum velocity V( )max , and the occurrence of a still higher concentration of substrate cannot increase the velocity further.

The velocity of a chemical reaction is defined by an equilibrium constant. For enzyme-catalyzed reactions this equilibrium constant is known as Km, or the Michaelis constant. Km is a useful parameter that aids in establishing how enzymes react in the living cell. Km represents the concentration of a substrate that is found in an occurring reaction when the reaction is at one-half its maximum velocity. If an enzyme has a high Km value, then an abundance of substrate must be present to raise the rate of reaction to half its maximum velocity; in other words,

function as an ion channel in response to voltage change. Stimulation by acetylcholine signals the channels to open, allowing sodium 1(Na ) ions to pass through an otherwise impermeable membrane.

Receptors That Internalize Stimuli The internalization of a stimulus into a fibroblast by way of its receptor is illustrated in Figure 1.10. Recep- tors that perform in such a manner exist for a variety of biologically active molecules, including several hormones. Low-density lipoproteins (LDLs) are taken up by certain cells in much the same fashion (see Chapter 5), except that their receptors, rather than being mobile, are already clustered in coated pits. These pits, vesicles formed from the plasma membrane, are coated with several proteins, among which clathrin is primary. A coated pit containing the receptor with its ligand soon loses the clathrin coating and forms a smooth-walled vesicle. This vesicle delivers the ligand into the cell and then is recycled, along with the receptor, into the plasma membrane. If the endocytotic process is for scavenging, the ligand (perhaps a protein) is not used by the cell but instead undergoes lysosomal degradation, as shown in Figure 1.10 and exemplified by the endocytosis of LDL.

Receptors’ Role in Homeostasis The cells of every organ in the body have specialized recep- tors that respond to changes in external conditions. The reaction of a fibroblast to changes in blood glucose level is a good example of cellular adjustment to the existing environment that is made possible through receptor pro- teins. When blood glucose levels are low, the hormone epinephrine is released by the adrenal medulla. Epineph- rine attaches to and activates its receptor protein on the fibroblast, thereby causing it to stimulate G-protein and adenyl cyclase, which catalyzes the formation of cAMP from ATP. Then cAMP initiates a series of enzyme phos- phorylation modifications, as described earlier in this section, which ultimately generate glucose 1-phosphate for use by the fibroblast. In contrast, when blood glucose levels are elevated, the hormone insulin is secreted by the b-cells of the pancreas and reacts with receptors on the fibroblast membrane. Insulin facilitates glucose entry by increasing the number of cell membrane glucose receptors, which transport glucose in the cell. (Glucose transporters are covered in Chapter 3.)

Catalytic Proteins (Enzymes) Enzymes, which are found in all cellular compartments, are catalysts that take part in a reaction but are not part of the final product of that reaction. Some enzymes function externally (such as within the digestive tract); examples include some digestive enzymes, such as isomaltase, lac- tase, sucrase, maltase, and some peptidases, which are

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14 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

reversible, whereas others are unidirectional. Although some reactions in almost any pathway are reversible, it is important to understand that removal of one of the products (by that product reacting to produce the next compound in the pathway) drives the reaction toward forming more of that product. Removing (or using) the product, then, becomes the driving force that causes reactions to proceed primarily in the desired direction.

Regulation An important aspect of nutritional biochemistry is the regulation of metabolic pathways. Anabolic (synthetic) and catabolic (oxidative) reactions must be kept in a bal- ance appropriate for life (and perhaps growth). Regulation primarily involves the adjustment of the catalytic activity of certain participating enzymes. This enzyme regulation occurs through three major mechanisms:

● covalent modification of enzymes (also referred to as post-translational modification)

● modulation of allosteric enzymes ● increase in enzyme concentration by induction

(synthesis of more enzyme).

Covalent Modification With the first of these mechanisms, covalent modification, the enzyme is inactive until a post- translational modification is made. This is usually achieved by the addition or hydrolytic removal of phosphate groups to or from the enzyme, as previously discussed in the subsection “Receptors That Generate Internal Chemical Signals.” One example of covalent modification of enzymes is the regulation of glycogenesis (synthesis of glycogen from glucose) and glycogenolysis (breakdown of glycogen to glucose) (see Chapter 3). Another covalent modification involves cleavage; for example, some enzymes (like those secreted into the digestive tract to digest proteins) are syn- thesized as inactive proenzymes (also called zymogens). To activate the proenzyme (make it a functional enzyme), a portion of the proenzyme is hydrolyzed.

Allosteric Enzyme Modulation A second regulatory mechanism is that exerted by certain unique enzymes called allosteric enzymes. The term allosteric refers to the fact that these enzymes possess an allosteric or specific “other” site besides the catalytic site. Specific compounds, called modulators, can bind to these allo- steric sites and profoundly influence the activity of these regulatory enzymes. Modulators may be positive (i.e., causing an increase in enzyme activity), or they may exert a negative effect (i.e., inhibit activity). Modulating substances are believed to alter the activity of the allosteric enzyme by changing the conformation (three-dimen- sional structure) of the polypeptide chain or chains of the enzyme, thereby altering the binding of its catalytic site with the intended substrate. Negative modulators are

the enzyme has a low affinity for its substrate and it takes more substrate to react with the active site of the enzyme. An example of an enzyme with a high Km is glucokinase, found in the liver cells. Because glucose can diffuse freely into the liver, the fact that glucokinase has a high Km is very important to blood glucose regulation. If glucokinase had a low K /highm affinity for glucose, too much glucose would be removed from the blood during periods of fasting. Glucokinase (with its high Km but low affinity) can still convert excess glucose to glucose phosphate when the glucose load is high—for example, following a high- carbohydrate meal; however, the liver glucokinase does not function at its maximum velocity when glucose levels are in the normal range. The enzyme thus protects against high cellular concentrations of glucose.

The nature of enzyme catalysis can be described by the following reactions:

↔ (reversible reaction)

1 2Enzyme (E) substrate (S) E S complex

The substrate activated by combination with the enzyme is converted into an enzyme–product (E–P) complex through rearrangement of the substrate’s ions and atoms:

E–S E−P E–P E + P

The product is released, and the enzyme is free to react with more of the substrate.

Reversibility Most biochemical reactions are reversible, meaning that the same enzyme catalyzes a reaction in both directions. The extent to which a reaction can proceed in a reverse direction depends on several factors, the most important of which are the relative concentrations of substrate (reactant) and product and the differences in energy content between reactant and product. In instances when a large disparity in either energy content or concentration exists between reactant and product, the reaction can proceed in only one direction. Such a reaction is unidirectional rather than reversible. This topic is discussed later in this chapter. In unidirectional reactions, the same enzyme cannot cata- lyze in both directions. Instead, a different enzyme is required to catalyze the reverse direction of the reaction. Comparing glycolysis (the oxidation of glucose) with gluconeogenesis (the synthesis of glucose) allows us to see how unidirectional reactions may be reversed by introduc- ing a different enzyme.

Simultaneous reactions, catalyzed by various multienzyme systems or pathways, constitute cellular metabolism. Enzymes are compartmentalized within the cell and function in sequential chains. An example of a multienzyme system is the TCA cycle located in the mitochondrial matrix. Each sequential reaction is catalyzed by a different enzyme, and some reactions are

↔ →

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 15

targeted for regulation essentially catalyze unidirectional reactions. In every metabolic pathway, at least one reaction is essentially irreversible, exergonic, and enzyme limited. That is, the rate of the reaction is limited only by the activity of the enzyme catalyzing it. Such enzymes are frequently called the regulatory enzymes, capable of being stimulated or suppressed by one of the mechanisms described. Logically, an enzyme catalyzing a reaction reversibly at near equilibrium in the cell cannot be a regulatory enzyme because its up- or downregulation would affect its forward and reverse activities equally. This effect, in turn, would not accomplish the purpose of regulation, which is to stimulate the rate of the metabolic pathway in one direction to exceed the rate of the pathway in the reverse direction.

Examples of Enzyme Types Enzymes participating in cellular reactions are located throughout the cell in both the cytoplasmic matrix and the various organelles. The location of specific enzymes depends on the site of the metabolic pathways or metabolic reactions in which those enzymes participate. Enzyme classification, therefore, is based on the type of reaction catalyzed by the various enzymes. Enzymes fall within six general classifications:

● Oxidoreductases (dehydrogenases, reductases, oxidases, peroxidases, hydroxylases, and oxygenases) are enzymes that catalyze all reactions in which one com- pound is oxidized and another is reduced. Examples of oxidoreductases are the enzymes found in the electron transport chain located on the inner membrane of the mitochondria. Other examples are the cytochrome P450 enzymes located on the ER of liver cells.

● Transferases are enzymes that catalyze reactions not involving oxidation and reduction in which a functional group is transferred from one substrate to another. Included in this group of enzymes are transketolase, transaldolase, transmethylase, and the transaminases. The transaminases (a-amino transferases), which figure so prominently in protein metabolism, are located primarily in the mitochondrial matrix.

● Hydrolases (esterases, amidases, peptidases, phos- phatases, and glycosidases) are enzymes that catalyze cleavage of bonds between carbon atoms and some other kind of atom by adding water. Digestive enzymes fall within this classification, as do those enzymes contained within lysosomes.

● Lyases (decarboxylases, aldolases, synthetases, cleavage enzymes, deaminases, nucleotide cyclases, hydrases or hydratases, and dehydratases) are enzymes that catalyze cleavage of carbon-carbon, carbon-sulfur, and certain carbon-nitrogen bonds (peptide bonds excluded) with- out hydrolysis or oxidation-reduction. Citrate lyase, which frees acetyl-CoA for fatty acid synthesis in the

often the end products of a sequence of reactions. As an end product accumulates above a certain critical concen- tration, it can inhibit, through an allosteric enzyme, its own further production.

An excellent example of an allosteric enzyme is phosphofructokinase in the glycolytic pathway. Glycolysis gives rise to pyruvate, which is decarboxylated and oxidized to acetyl-CoA, which enters the mitochondrion and is further oxidized by the TCA cycle by combining with oxaloacetate to form citrate. Citrate is a negative modulator of phosphofructokinase. Therefore, an accumulation of citrate in the cell matrix causes the glycolytic pathway to be inhibited by regulating phosphofructokinase. In contrast, an accumulation of AMP or adenosine diphosphate (ADP), which indicates that ATP is depleted, signals the need for additional energy in the cell in the form of ATP. AMP or ADP therefore modulates phosphofructokinase positively. The result is an active glycolytic pathway that ultimately leads to the formation of more ATP through the TCA cycle–electron transport chain connection.

Allosteric mechanisms of regulation are considered to be of one of two types. In one type, the K series, the Km is affected, which alters the binding of the substrate to the enzyme. If the allosteric effect is positive, the enzyme can become “saturated” at a lower concentration. The other type of allosteric regulation, called the V series, increases the maximum velocity of the enzymatic reaction. If the allosteric effector is an inhibitor, the maximum velocity (V )m of the reaction will be decreased.

Induction The third mechanism of enzyme regulation, enzyme induction, creates changes in the concentration of certain inducible enzymes by increasing enzyme synthesis. Inducible enzymes are adaptive, meaning that they are synthesized at rates dictated by cellular circumstances. In contrast, constitutive enzymes, which are synthesized at a relatively constant rate, are uninfluenced by external stim- uli. Induction usually occurs through the action of certain hormones, such as the steroid hormones and the thyroid hormones, and is exerted through changes in the expres- sion of genes encoding the enzymes. Dietary changes can elicit the induction of some enzymes necessary to cope with the changing nutrient load. This regulatory mecha- nism is relatively slow, however, compared to the first two mechanisms, which exert their effects in terms of seconds or minutes.

The reverse of induction is the blockage of enzyme synthesis by blocking the formation of the mRNA of specific enzymes. This regulation of translation is one of the means by which small molecules, reacting with cellular proteins, can exert their effect on enzyme concentration and the activity of metabolic pathways.

Specific examples of enzyme regulation are described in subsequent chapters addressing nutrient metabolism. It should be noted at this point, however, that enzymes

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16 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

structural integrity of the plasma membrane. Membrane failure can also arise from mechanical disruption, such as would be caused by a viral attack on the cell. Damage to the plasma membrane is manifested as leakiness and eventual cell death, allowing an unimpeded passage of substances, including enzymes, from intracellular to extracellular compartments such as the blood.

Factors contributing to cellular damage and resulting in abnormal egress of cellular enzymes include, for example, hypoxia (inadequate oxygen supply), tissue necrosis and ischemia (impaired blood flow to a tissue or part of a tissue that in turn deprives affected cells of oxygen and nutrients), and damage from viral attack or organic chemicals such as alcohol and organophosphorus pesticide. Increased production of enzymes and other substances can also cause a spike in its serum concentration. Cancers affecting certain tissues can cause such increases. Substances that occur in body fluids as a result of malignant disease are called tumor markers. A tumor marker may be produced by the tumor itself or by the host, in response to a tumor. In addition to enzymes and isozymes, other forms of tumor markers include hormones, oncofetal protein antigens such as carcinoembryonic antigen (CEA), and products of oncogenes. Oncogenes are mutated genes that encode abnormal, mitosis-signaling proteins, which, in turn, can promote unregulated cell division.

Increases in blood serum concentrations of cellular enzymes can be indicators of even minor cellular damage because the intracellular concentration of enzymes is hundreds or thousands of times greater than in blood. However, not all intracellular enzymes are valuable in diagnosing damage to the cells in which they are contained. Several conditions must be met for the enzyme to be suitably diagnostic:

● The enzyme must have a sufficiently high degree of organ or tissue specificity.

● A steep concentration gradient of enzyme activity must exist between the interior and exterior of the cells under normal conditions. This makes small increases in serum activity detectible (assuming the laboratory assay is sensitive).

● The enzyme must function in the cytosol of the cell so that it leaks out whenever the plasma membrane suffers significant damage.

● The enzyme must be stable for a reasonable time period in the vascular compartment.

APOPTOSIS

Dying is said to be a normal part of living. So it is with the cell. Like every living thing, a cell has a well-defined life span, after which its structural and functional integrity

cytosol, is a good example of an enzyme belonging to this classification.

● Isomerases (racemases, epimerases, and mutases) are enzymes that catalyze the interconversion of optical or geometric isomers. Phosphohexose isomerase, which converts glucose-6-phosphate to fructose-6-phosphate in glycolysis (occurring in the cytosol), exemplifies this particular class of enzyme.

● Ligases are enzymes that catalyze the formation of bonds between carbon and a variety of other atoms, including oxygen, sulfur, and nitrogen. Forming bonds catalyzed by ligases requires energy that usually is pro- vided by hydrolysis of ATP. An example of a ligase is acetyl-CoA carboxylase, which initiates fatty acid syn- thesis in the cytosol. Through the action of acetyl-CoA carboxylase, a bicarbonate ion (HCO )32 is attached to acetyl-CoA to form malonyl-CoA, the initial com- pound formed in the synthesis of fatty acids.

Clinical Applications of Cellular Enzymes Enzymes in the body are synthesized intracellularly, and most of them function within the cell in which they were formed. Variations in amino acid sequence are not uncom- mon among some enzymes that catalyze the same reaction but are found in different tissues (such as the liver, muscle, and heart); such enzymes may be referred to as isozymes (or isoenzymes or protein isomers). Once made, some enzymes are secreted in an inactive form and are rendered active in the extracellular fluids where they function. Those that function in the blood are called plasma-specific enzymes.

Diagnostic enzymology focuses on intracellular enzymes, which, because of a problem within the cell structure, escape from the cell and ultimately express their activity in the serum. By measuring the serum activity of these released enzymes, both the site and often the extent of the cellular damage may be determined. If the site of the damage is to be determined with reasonable accuracy, the enzyme being measured must exhibit a relatively high degree of organ or tissue specificity. For instance, lactate dehydrogenase (LDH) is an enzyme that is widely distributed among cells such as the heart, liver, skeletal muscle, lymph nodes, erythrocytes, and platelets. Elevated serum levels of LDH do not have diagnostic value until the enzyme is separated into its five different isozyme forms and each is measured individually. Each isozyme is organ specific. The amount of elevation of the isozyme from the heart is an indication of the extent of tissue damage following, for example, a heart attack.

Intracellular enzymes are normally retained within the cell where they are produced by the plasma membrane. The plasma membrane is metabolically active, and its integrity depends on the local environment. Any process, for example, that impairs the cell’s use of nutrients can compromise the

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 17

Apoptosis can also be triggered by cytotoxic T-lymphocytes and natural killer (NK) cells. Natural killer cells, for example, release (from cytosolic granules) granzymes and a protein called perforin, which create pores in the membranes of cells targeted for destruction such as those that are stressed, infected, or malignant. Caspases also can be involved in this process.

The removal of a dead cell’s contents occurs without any of its contents escaping into the extracellular fluid. Thus, apoptosis does not trigger autoimmunity. However, defects in the apoptotic process may increase susceptibility to autoimmune diseases. Studies are ongoing to determine if specific human autoimmune diseases are related to such defects.

In contrast to apoptosis, which is programmed and characterized by cell shrinkage followed by cell breakup, the cell death process of oncosis (from onksos, meaning “swelling”) results from cell injury and is characterized by cellular swelling, along with swelling of the mitochondrial, nucleus and cytosol, and cytosol vacuolization.

Because cell death can be activated by specific genes, the expression of these genes must be tightly controlled to avoid inappropriate cell death. Interestingly, many of the proteins released in the process of apoptosis are found in the mitochondria or in its outer membrane space. Most have a specific role there and only when they are released into the cytosol do they have a role in apoptosis. The BCL-2 family is one key group of proteins involved in the regulation of the mitochondrial intrinsic apoptotic pathway by promoting or inhibiting mitochondrial outer membrane permeability. Examples of some antiapoptotic factors include Bc1-xL and Bcl-2, which protect the cell against apoptotic stimuli. Heat shock proteins also may attenuate apoptosis. Nutrients including vitamins A and D also exhibit roles in cell proliferation, differentiation, and growth, and sphingolipids are involved in survival along with cell growth, adhesion, and motility.

The study of how cell death can be controlled has important implications since the dysregulation of apoptosis is thought to be involved in the pathophysiology of numerous diseases. If cell death is prevented, then a transformed cell can continue to grow (rather than be destroyed) and promote oncogenesis (the formation of a tumor). Research in the area of apoptosis abounds; a few reviews [4–9] are listed for the interested reader.

BIOLOGICAL ENERGY

The previous sections of this chapter provide some descrip- tive insight into the makeup of a cell, how it reproduces, and how large and small molecules are synthesized within a cell or move in or out of a cell. All of these activities require energy. The cell obtains this energy from small

diminishes and it is removed from the body. Many terms have been used to describe naturally occurring cell death. It is now most commonly referred to as programmed cell death, to distinguish it from pathological cell death, which is not part of the normal physiological process. The term describing programmed cell death is apoptosis, a word borrowed from the Greek meaning to “fall out.”

Cells are constantly turned over in the body. For instance, 1010 neutrophils (a type of white blood cell) die and are replaced each day [3]. As cells die, they are replaced by new cells that are continuously being formed through cell mitosis. However, both daughter cells formed in the mitotic process do not always enjoy the full life span of the parent. If they did, the number of cells, and consequently tissue mass, could increase inordinately. Therefore, one of the two cells produced by mitosis generally is programmed to die before its sister. In fact, most dying cells are already doomed at the time they are formed. Those targeted for death are usually smaller than their surviving sisters, and their degradation begins even before the mitosis generating them is complete. The processes of cell division and cell death must be carefully regulated to generate the proper number of cells during development. Once cells mature, the appropriate number of cells must be maintained.

Apoptotic cell death (and cell survival) is brought about by several mechanisms. An intracellular (or intrinsic) pathway can be triggered by several different stimuli such as irreparable DNA damage and hypoxia, among others. Upon stimulation, proapoptotic factors (such as Bax, Bad, Bid, Noxa, and PUMA) are released into the cytosol from the mitochondria secondary to increased outer mitochondrial membrane permeability. Activation of mitochondrial death signaling occurs via the release of cytochrome c (among other cytotoxic proteins) into the cytosol. The binding of cytochrome c to apoptotic protein activating factor (Apaf-1) with involvement from caspase-9 and ATP leads to the formation of a multiprotein complex called an apoptosome. The apoptosome facilitates the recruitment and activation of other selected caspases (proteases with cysteine at their active sites) including caspase-3 and caspase-7. While the exact sequence of events leading to cell death is unclear, it is thought to involve the production of reactive oxygen species among other substances that induce structural alterations to the cell and its components, resulting in its death.

The extracellular (extrinsic) pathway for apoptosis is triggered when specific ligands such as molecules that belong to the tumor necrosis factor (TNF) family of cytokines bind to cell surface death receptors and generate apoptotic signaling. TNFs act through a series of protein–protein interactions that ultimately activate several caspases including caspase-3 and caspase-7 to induce cell death.

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18 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

Expressions of Energy Units of Energy The unit of energy used throughout this text is the calorie, abbreviated cal. In the expression of the higher caloric val- ues encountered in nutrition, the unit kilocalories (kcal) is often used: 1kcal = 1,000 cal. The international scientific community and many scientific journals use another unit of energy, called the joule (J) or the kilojoule (kJ). Calories can easily be converted to joules by the factor 4.18:

1 cal = 4.18 J, or 1 kcal = 4.18 kJ

To help you become familiar with both terms, this text primarily uses calories or kilocalories, followed by the cor- responding values in joules or kilojoules in parentheses; joules and kilojoules are sometimes used in scientific publications.

Free Energy The potential energy inherent in the chemical bonds of nutrients is released if the molecules undergo oxidation either through combustion or through oxidation within the cell. This energy is defined as free energy if, on its release, it is capable of doing work at constant tempera- ture and pressure—a condition that is met within the cell. In equations, G is used as an abbreviation for free energy and ΔG for the change in free energy.

CO2 and H O2 are the products of the complete oxidation of organic molecules containing only carbon, hydrogen, and oxygen, and they have an inherent free energy. The energy released in the course of oxidation of the organic molecules is in the form of either heat or chemical energy. The products have less free energy than do the original reactants. Because energy is neither created nor lost during the reaction, the total energy remains constant. Thus, the difference between the free energy in the products and that in the reactants in a given chemical reaction is a useful parameter for estimating the tendency for that reaction to occur. This difference is symbolized as follows:

Gproducts − Greactants = ΔG of the reaction

where G is free energy and Δ is a symbol signifying change.

molecules transformed (oxidized) to provide heat and chemical energy. The small molecules that are constantly required are supplied by the nutrients in food. The next section covers some basics of energy needs in the cell.

Most of the processes that sustain life involve energy. Some processes use energy, and others release it. The term energy conjures an image of physical “vim and vigor,” the fast runner or the weightlifter straining to lift hundreds of pounds. This notion of energy is accurate insofar as the contraction of muscle fibers associated with mechanical work is an energy-demanding process, requiring adenosine triphosphate (ATP), the major storage form of molecular energy in the cell. Beyond the ATP required for physical exertion, the living body has other, equally important, requirements for energy, including:

● the biosynthetic (anabolic) systems by which substances can be formed from simpler precursors

● active transport systems by which compounds or ions can be moved across membranes against a concentra- tion gradient

● the transfer of genetic information.

This section addresses the key role of energy transformation and heat production in using nutrients and sustaining life.

Energy Release and Consumption in Chemical Reactions Energy used by the body is ultimately derived from the energy contained in the macronutrients— carbohydrate, fat, and protein (and alcohol). If this energy is released, it may simply be expressed as heat, as would occur in the combustion of flammable sub- stances, or be preserved in the form of other chemical energy. Energy cannot be created or destroyed; it can only be transformed. Burning a molecule of glucose outside the body liberates heat, along with CO2 and H O2 as products of combustion, as shown:

→1 1 1C H O 6O 6CO 6H O heat6 12 6 2 2 2

The metabolism of glucose to the same CO2 and H O2 within the cell is nearly identical to that of simple combustion. The difference is that in metabolic oxidation a significant portion of the released energy is salvaged as chemical energy in the form of new, high-energy bonds. These bonds represent a usable source of energy for driving energy-requiring processes. Such stored energy is generally contained in phosphate anhydride bonds, chiefly those of ATP (Figure 1.11). The analogy between the combustion and the metabolic oxidation of a typical nutrient (palmitic acid) is illustrated in Figure 1.12. The metabolic oxidation illustrated releases 59% of the heat produced by the combustion and conserves about 40% of the chemical energy.

Figure 1.11 Adenosine triphosphate (ATP).

ADENOSINE RIBOSE PHOS PHOS PHOS

Anhydride bonds, which release a

large amount of energy when

hydrolyzed.

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 19

A and B, as illustrated in Figure 1.13. As the boulder descends to level B from level A, energy capable of doing work is liberated, and the change in free energy is a negative value. The reverse reaction, moving the boulder uphill to level A from level B, necessitates an input of energy, or an endothermic process, and the change is a positive value. The quantity of energy released in the downhill reaction is precisely the same as the quantity of energy required for the reverse (uphill) reaction—only the sign of ΔG changes.

Activation Energy Although exothermic reactions are favored over endothermic reactions in that they require no external energy input, they do not occur spontaneously. If they did, no energy-producing nutrients or fuels would exist throughout the universe because they would all have

Exothermic and Endothermic Reactions If the G value of the reactants is greater than the G value of the products, as in the case of the oxidation reaction, the reaction is said to be exothermic, or energy releas- ing, and the change in G (ΔG) is negative. In contrast, a positive ΔG indicates that the G value of the products is greater than that of the reactants, indicating that energy must be supplied to the system to convert the reactants into the higher-energy products. Such a reaction is called endothermic, or energy requiring.

Exothermic and endothermic reactions are sometimes referred to as downhill and uphill reactions, respectively, terms that help create an image of energy input and release. The free energy levels of reactants and products in a typical exothermic, or downhill, reaction can be likened to a boulder on a hillside that can occupy two positions,

Figure 1.12 A comparison of the simple combustion and the metabolic oxidation of the fatty acid palmitate.

CH3 (CH2)14 COOH 1 23O2 1 130ADP 1 130P

16CO2 1 16H2O 1 HEAT (2,340 kcal)

Palmitic acid

Simple combustion

16CO2 1 16H2O 1 130ATP 1 HEAT (1,384 kcal) Cellular oxidation

Approximately 40% of the energy released by metabolic oxidation is salvaged as ATP, with the remainder released in the form of heat.

The energy liberated from combustion assumes the form of heat only.

Figure 1.13 The uphill–downhill concept illustrating energy-releasing and energy-demanding processes.

Activation energy is the amount of

energy required to increase the energy

level to its transitional state.

An example of activation energy moves the boulder up the hill to a point from which it can “fall” down the hill.

A

B

Exotherm ic 2

Endotherm ic 1 G

G

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20 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

A: 5eqK [B]/[A]. The [] signify the concentration. If the denominator ([A]) is very small, dividing it into a much larger number results in Keq being large. [A] will be small if most of A (the reactant) is converted to the product B. In other words, Keq increases in value when the concentration of A decreases and that of B increases. If Keq has a value greater than 1, substance B is formed from substance A, whereas a value of Keq less than 1 indicates that at equilibrium A will be formed from B. An equilibrium constant equal to 1 indicates that no bias exists for either reaction. The Keq of a reaction can be used to calculate the standard free energy change of the reaction.

Standard Free Energy Change To compare the energy released or consumed in differ- ent reactions, it is convenient to define the free energy at standard conditions. Standard conditions are defined precisely: a temperature of 258C (298 K); a pressure of 1.0 atm (atmosphere); and the presence of both the reactants and the products at their standard concentrations, namely 1.0 mol/L. The standard free energy change ΔG 0 (the super- script zero designates standard conditions) for a chemical reaction is a constant for that particular reaction. The ΔG 0 is defined as the difference between the free energy content of the reactants and the free energy content of the products under standard conditions. Under such conditions, ΔG 0 is mathematically related to Keq by the equation

ΔG0= −2.3 RT log Keq

where R is the gas constant (1.987 cal/mol) and T is the absolute temperature, 298 K in this case. The factors 2.3, R, and T are constants, and their product is equal to –2.3(1.987)(298), or –1,362 cal/mol. The equation there- fore simplifies to

ΔG0 = −1,362 log Keq

This topic is important in understanding the energetics of metabolic pathways, but you should refer to a bio chemistry textbook for additional information on this subject.

Equilibrium Constant and Standard Free Energy Change The equilibrium constant of a reaction determines the sign and magnitude of the standard free energy change. For example, referring once again to the A → B reaction, the logarithm of a Keq value greater than 1.0 will be posi- tive, and because it is multiplied by a negative number, the sign of ΔG 0 will be negative. We have established that the reaction A → B is energetically favored if ΔG 0 is nega- tive. Conversely, the log of a Keq value less than 1.0 would be negative, and when multiplied by a negative number the sign of ΔG 0 would be positive. The ΔG 0 in this case indicates that the formation of A from B (B → A) is favored in the equilibrium.

transformed spontaneously to their lower energy level. A certain amount of energy must be introduced into reactant molecules to activate them to their transi- tion state, a higher energy level or barrier at which the exothermic conversion to products can indeed take place. The energy that must be imposed on the system to raise the reactants to their transition state is called the activation energy. Refer again to the boulder-and-hillside analogy in Figure 1.13. The boulder does not spontaneously descend until the required activation energy can dislodge it from its resting place to the brink of the slope.

Cellular Energy The cell derives its energy from a series of chemical reactions, each of which exhibits a free energy change. The reactions occur sequentially as nutrients are systematically oxidized ultimately to CO2 and H O2 . Nearly all the reactions in the cell are catalyzed by enzymes. Within a given cata- bolic pathway—for example, the oxidation of glucose to CO2 and H O2 —some reactions may be energy consuming (have a 1ΔG for the reaction). However, energy-releasing (those with a 2ΔG) reactions are favored, so the net energy transformation for the entire pathway has a 2ΔG and is exothermic.

Reversibility of Chemical Reactions Most cellular reactions are reversible, meaning that an enzyme (E) that can catalyze the conversion of hypotheti- cal substance A into substance B can also catalyze the reverse reaction, as shown:

Using the A, B interconversion as an example, let us review the concept of reversibility of a chemical reaction. In the presence of the specific enzyme E, substance A is converted to substance B. Initially, the reaction is unidirectional because only A is present. However, because the enzyme is also capable of converting substance B to substance A, the reverse reaction becomes significant as the concentration of B increases. From the moment the reaction is initiated, the amount of A decreases, while the amount of B increases to the point at which the rate of the two reactions becomes equal. At that point, the concentrations of A and B no longer change, and the system is said to be in equilibrium. Enzymes are only catalysts and do not change the equilibrium of the reaction. This concept is discussed more fully later. Whether the A → B reaction or the B → A reaction is energetically favored is indicated by the relative concentrations of A and B at equilibrium.

The equilibrium between reactants and products can be defined in mathematical terms and is called the equilibrium constant (K )eq . Keq is simply the ratio of the equilibrium concentration of product B to that of reactant

EA B

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 21

The Role of High-Energy Phosphate in Energy Storage The preceding section addressed the fundamental prin- ciple of free energy changes in chemical reactions and the fact that the cell obtains this chemical free energy through the catabolism of nutrient molecules. It also stated that this energy must somehow be used to drive the various energy- requiring processes and anabolic reactions so important in normal cell function. This section explains how ATP can be used as a universal source of energy to drive reactions. Examples of very-high-energy phosphate compounds are shown in Figure 1.15. Phosphoenolpyruvate and 1,3-bisphosphoglycerate are components of the oxidative pathway of glucose (Chapter 3), and creatine phosphate (also called phosphocreatine) is a storage form of high- energy phosphate available to replenish ATP in muscle. The hydrolysis of the phosphate anhydride bonds of ATP can liberate the stored chemical energy when needed. ATP thus can be thought of as an energy reservoir, serving as the major linking intermediate between energy-releasing and energy-demanding chemical reactions in the cell. In nearly all cases, the energy stored in ATP is released by the enzymatic hydrolysis of the anhydride bond connecting the b- and g-phosphates in the molecule (see Figure 1.11). The products of this hydrolysis are adenosine diphosphate (ADP) and inorganic phosphate (P )i . In certain instances, the free phosphate group is transferred to various accep- tors, a reaction that activates the acceptors to higher energy levels. The involvement of ATP as a link between the energy-releasing and energy-requiring cellular reac- tions and processes is summarized in Figure 1.16.

Coupled Reactions in the Transfer of Energy Some reactions require energy, and others yield energy. The coupling of these reactions makes it possible for a path- way to continue. The oxidation of glucose in the glycolysis pathway demonstrates the importance of coupled reactions in metabolism. An understanding of how

Standard pH For most compartments in the body, the pH is near neutral; for biochemical reactions, a standard pH value of 7 is adopted by convention. For human nutrition, the standard free energy change of reactions is designated ΔG 0 ′ This book uses this notation.

Nonstandard Physiological Conditions Physiologically standard conditions do not often exist. The difference between standard conditions and nonstandard conditions can explain why a reac- tion having a positive ΔG0′ can proceed exothermically (−ΔG0 ) in the cell. For example, consider the reaction catalyzed by the enzyme triosephosphate isomerase (TPI) shown in Figure 1.14. This particular reaction occurs in the glycolytic pathway through which glucose is converted to pyruvate. (The chemical structures and the pathway are discussed in detail in Chapter 3.) In the glycolytic pathway, the enzyme aldolase produces 1 mol each of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P) from 1mol of fructose-1,6-bisphosphate. Let us focus on the reaction that TPI catalyzes, which is an isomerization between the two products of the aldolase reaction. As explained in Chapter 3, only the G-3-P is further degraded in the subsequent reactions of glycolysis. This circumstance results in a substantially lower concentration of the G-3-P metabolite than of DHAP.

For this reaction, two important conditions within the cell deviate from “standard conditions”: namely, the temperature is the temperature of the body, ~378C (310 K), and neither the G-3-P nor DHAP are at 1.0 mol/L concentrations. The value of ΔG0 ′ for the reaction DHAP (reactant) → G-3-P (product) is 11,830 cal/mol (17,657 J/mol), indicating that under standard conditions the formation of DHAP is preferred over the formation of G-3-P. If we assume that the cellular concentration of DHAP is 50 times that of G-3-P because G-3-P is further metabolized, ΔG0 for the reaction is calculated to be equal to –577 cal/mol (–2,414 J/mol). The negative ΔG 0 shows that the reaction to form G-3-P is favored, as shown, despite the positive ΔG 0 for this reaction.

Figure 1.14 Example of a shift in the equilibrium by changing from standard conditions to physiological conditions.

Fructose-1,6-bisphosphate

Adolase

Dihydroxyacetone phosphate (DHAP)

Triosephosphate isomerase (TPI)Favored understandard conditions

Favored under physiological conditions

Glycerol-3-phosphate (G-3-P)

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22 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

carrier of phosphate groups. ADP can accept the phosphate groups from high-energy phosphate donor molecules and then, as ATP, transfer them to lower-energy receptor molecules. Two examples of this transfer are shown in Figure 1.17. By receiving the phosphate groups, the acceptor molecules become activated to a higher energy level, from which they can undergo subsequent reactions such as entering the glycolysis pathway. The end result is the transfer of chemical energy from donor molecules through ATP to receptor molecules. The second example is the release of a Pi group from creatine phosphate; this Pi joins with ADP forming ATP. Creatine phosphate serves as a ready reservoir to renew ATP levels quickly, particularly in muscle.

If a given quantity of energy is released in an exothermic reaction, the same amount of energy must be added to the system for that reaction to be driven in the reverse direction. For example, hydrolysis of the phosphate- ester bond of glucose-6-phosphate liberates 3,300 cal/mol (13.8 kJ/mol) of energy, and the reverse reaction, in

chemical energy is transformed from macronutrients (the carbohydrate, protein, fat, and alcohol in food) to storage forms (such as ATP), and how the stored energy is used to synthesize needed compounds for the body is fundamental to the study of human nutrition. These top- ics are covered in this section as well as throughout this book. The ΔG 0′ value for the phosphate bond hydrolysis of ATP is intermediate between those of certain high- energy phosphate compounds and compounds that pos- sess relatively low-energy phosphate esters. ATP’s central position on the energy scale lets it serve as an intermediate

Figure 1.15 Examples of very-high-energy phosphate compounds.

Phosphoenolpyruvate

CH2

O P

O

O2

C O2

COO2

1,3-bisphosphoglycerate

O P

O

O2CHHO

C O2

O

O

O2

O PCH2 O 2

Creatine phosphate

NH P

O

O2

C O2

1NH2

N CH2H3C COO 2

These compounds can phosphorylate ADP to make ATP.

High-energy phosphate bonds contain more energy than of ATP.

Figure 1.16 An illustration of how ATP is generated from the coupling of ADP and phosphate through the oxidative catabolism of nutrients and how it in turn is used for energy-requiring processes.

O2 CO2

H2O

ATPEnergy-releasingcatabolism

Muscular contraction (mechanical work)

Biosynthesis Anabolism

(chemical work)

Active transport (osmotic work)

Energy-requiring processes

Heat

Nutrients

ADP 1 Pi

Figure 1.17 Examples of high-energy phosphate bonds being transferred.

ADP Creatine phosphate

G 09 5 24,000 cal/mol 5 212.55 kJ/mol

ATP Creatine(b)

ATP Glucose

G 09 5 23,000 cal/mol 5 216.74 kJ/mol

ADP Glucose-6-phosphate(a)

The transfer of high-energy phosphate bond to glucose to activate it so it can enter the oxidative pathway.

When energy is needed, creatine phosphate is broken apart to release creatine and phosphate. The phosphate joins with ADP to produce and replenish ATP.

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 23

which the phosphate is added to glucose to form glucose- 6-phosphate, necessitates the input of 3,300 cal/mol (13.8 kJ/mol). These reactions can be expressed in terms of their standard free energy changes as shown in Figure 1.18. To phosphorylate glucose, the reaction must be coupled with the hydrolysis of ATP, which provides the necessary energy. The additional energy from the reaction is dissipated as heat.

The addition of phosphate to a molecule is called a phosphorylation reaction. It generally is accomplished by the enzymatic transfer of the terminal phosphate group of ATP to the molecule, rather than by the addition of free phosphate, as suggested in Figure 1.18. The reverse reaction is hypothetical, designed only to illustrate the energy requirement for phosphorylation of the glucose molecule. In fact, the enzymatic phosphorylation of glucose by ATP is the first reaction glucose undergoes upon entering the cell. This reaction promotes glucose to a higher energy level, from which it may be indirectly incorporated into glycogen as stored carbohydrate or systematically oxidized for energy. Phosphorylation therefore can be viewed as occurring in two reaction steps: (1) hydrolysis of ATP to ADP and phosphate and (2) addition of the phosphate to the substrate (glucose) molecule. A net energy change for the two reactions coupled together is shown in Figure 1.18. The net ΔG0′ for the coupled reaction is –4,000 cal/mol (16.7 kJ/mol).

The significance of these coupled reactions cannot be overstated. They show that even though energy is consumed in the endothermic formation of glucose-6- phosphate from glucose and phosphate, the energy released by the ATP hydrolysis is sufficient to force (or drive) the endothermic reaction that “costs” only 3,300 cal/mol. The coupled reactions result in 4,000 cal/mol (16.7 kJ/mol) left over. The reaction is catalyzed by the enzyme hexokinase or glucokinase, both of which hydrolyze the ATP and transfer the phosphate group to glucose. The enzyme brings the ATP and the glucose into close proximity, reducing the activation energy of the reactants and facilitating the phosphate group transfer. The overall reaction, which results in activating glucose at the expense of ATP, is energetically favorable, as evidenced by its high, negative standard free energy change.

Reduction Potentials As we will see when we discuss the formation of ATP in Chapter 3, ATP is formed in the electron transport chain after the macronutrients are oxidized. To better understand these oxidations and reductions, you need to understand reduction potentials. The energy to synthesize ATP becomes available following a sequence of individual reduction-oxidation (redox) reactions along the electron transport chain, with each component having a character- istic ability to donate and accept electrons. The released energy is used in part to synthesize ATP from ADP and phosphate. The tendency of a compound to donate and to receive electrons is expressed in terms of its standard reduction potential, E0′. The more negative the values of E0′ are, the greater the ability of the compound to donate electrons, whereas increasingly positive values signify an increasing tendency to accept electrons. The reducing capacity of a compound (its tendency to donate 1H and electrons) can be expressed by the E0′ value of its half- reaction, also called the compound’s electromotive potential.

Free energy changes accompany the transfer of electrons between electron donor–acceptor pairs of compounds and are related to the measurable electromotive force of the electron flow. Remember that in electron transfer, an electron donor reduces the acceptor, and in the process the electron donor becomes oxidized. Consequently, the acceptor, as it is reduced, oxidizes the donor. The quantity of energy released is directly proportional to the difference in the standard reduction potentials, D 99E0 , between the partners of the redox pair. The free energy of a redox reaction and the D 9E0 of the interacting compounds are related by the expression

MH NAD+

M + H+NADH

Figure 1.18 Exothermic reactions.

Forward reaction favored

G-6-P 1 ADPGlucose 1 ATP G 09 5 24,000 cal/mol (216.7 kJ/mol)

Coupled reaction favored

The hydrolysis of ATP to ADP and Pi has a large negative G 09 and is favored. The reverse reaction occurs with the electron transport chain to provide the energy needed.

The coupled reaction phosphorylating glucose and hydrolyzing ATP is energetically favored, with a negative G 09 of 4,000 cal/mol.

Glucose 1 PiG-6-P G 09 5 23,300 cal/mol (213.8 kJ/mol)

Glucose 1 PiG-6-P G 09 5 13,300 cal/mol (113.8 kJ/mol)

The hydrolysis of glucose-6-phosphate (G-6-P) to glucose and Pi has a negative G 09 and is favored. The reverse reaction is not energetically favored.

ADP 1 PiATP G 09 5 27,300 cal/mol (230.54 kJ/mol)

ADP 1 PiATP G 09 5 17,300 cal/mol (130.54 kJ/mol)

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24 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

D D5 29 9F0 0G n E

where D 9G0 is the standard free energy change in calories, n is the number of electrons transferred, and F is a constant called the Faraday (23,062 cal absolute volt equivalent).

An example of a reduction-oxidation reaction that occurs within the electron transport system is the transfer of hydrogen atoms and electrons from NADH through the flavin mononucleotide (FMN)–linked enzyme NADH dehydrogenase to oxidized coenzyme Q (CoQ). The half- reactions and 9E0 values for each of these reactions follow:

NADH + H+ →

1 1 1

5 2

1 1

51

1 1 1 2

9

1 2

9

E e

E

NADH H NAD 2H 2e 0.32volt

CoQH CoQ 2H 2 0.04 volt

0

2

0

NAD++ 2H+ + 2e−

E0′ = −0.32 volt

CoQH2 →

1 1 1

5 2

1 1

51

1 1 1 2

9

1 2

9

E e

E

NADH H NAD 2H 2e 0.32volt

CoQH CoQ 2H 2 0.04 volt

0

2

0

CoQ + 2H++ 2e−

E0′ = +0.04 volt

Because the NAD1 system has a relatively more negative 9E0 value than the CoQ system, NAD1 has a greater reduc-

ing potential than the CoQ system because electrons tend

to flow toward the system with the more positive 9E0 . The reduction of CoQ by NADH therefore is predictable, and the coupled reaction, linked by the FMN of NADH dehy- drogenase, can be written as follows:

ΔE0′ = 0.36 volt

Inserting this value for D 9E0 into the energy equation gives

D 5 2 5 2 9G 2(23,062)(0.36) 16,604 cal/mol0

The amount of energy liberated from this single reduction-oxidation reaction within the electron transport chain therefore is more than enough to phosphorylate ADP to ATP, which, as you will recall, requires about 7,300 cal/mol (35.7 kJ).

FMNH2

E09 5 10.04 volt

NAD1 CoQ

FMNNADH 1 H1 CoQH2

E09 5 20.32 volt

SUMMARY

This brief journey through the cell—beginning with its outer surface, the plasma membrane, and moving into its innermost part, where the nucleus is located—provides a view of how this living entity functions. Characteristics of the cell that seem particularly notable are as follows:

● The flexibility of the plasma membrane in adjusting or reacting to its environment while protecting the cell as it monitors what may pass into or out of the cell. Promi- nent in the membrane’s reaction to its environment are the receptor proteins, which are synthesized on the rough endoplasmic reticulum and moved through the Golgi apparatus to their intended site on the plasma membrane.

● The communication among the various components of the cell made possible through the cytosol, with its microtrabecular network, and also through the endo- plasmic reticulum and Golgi apparatus. The networking is such that communications flow not only among com- ponents within the cell but also between the nucleus and the plasma membrane.

● The efficient division of labor among the cell compo- nents (organelles). Each component has its own specific functions to perform, with little overlap. Furthermore, much evidence is accumulating to support the concept of an “assembly line” not only in oxidative phosphoryla- tion on the inner membrane of the mitochondrion but also in other metabolic pathways, wherever they occur.

● The superb management exercised by the nucleus to ensure that all the needed proteins are synthesized. The proteins needed as recognition markers, recep- tors, transport vehicles, and enzymes are available and located in the appropriate place in the cell as needed.

● The fact that, like all living things, cells must die a natu- ral death. This programmed process is called apoptosis, a particularly attractive focus of current research.

Despite the efficiency of the cell, it is still not a totally self-sufficient unit. Its continued operation is contingent on receiving appropriate and sufficient nutrients. Nutri- ents needed include not only those that can be used to produce energy, ATP, but also those stored as chemical energy. Most of the stored chemical energy is needed to maintain normal body temperature (released as heat energy). About 40% of the stored energy is conserved in the form of high-energy phosphate bonds, princi- pally ATP. The ATP can, in turn, activate various sub- strates by phosphorylation to higher energy levels from which they can undergo metabolism by specific enzymes. The exothermic hydrolysis of the ATP phosphate is suf- ficient to drive the endothermic phosphorylation, thereby completing the energy transfer from nutrient to metabolite. The oxidative pathways for the macronutrients (carbohy- drate, fat, and protein) and alcohol provide a continuous flow of energy for maintaining heat and replenishing ATP. The cell also needs nutrients required as building blocks

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 25

1. Belting M, Wittrup A. Nanotubes, exosomes, and nucleic acid- binding peptides provide novel mechanisms of intercellular commu- nication in eukaryotic cells: implications in health and disease. J Cell Biol. 2008; 183:1187–91.

2. Mihara K. Cell biology: moving inside membranes. Nature. 2003; 424:505–6.

3. Nagata, S. Apoptosis and autoimmune disease. Ann NY Acad Sci. 2010; 10–16.

4. Westhoff M, Bruhl O, Nonnenmach L, Karpel-Massler G, Debatin K. Killing me softly – Future challenges in apoptosis research. Int J Mol Sci. 2014; 15:3746–67.

5. Mukhopadhyay S, Panda PK, Sinha N, Das DN, Bhutia SK. Autoph- agy and apoptosis: where do they meet? Apoptosis. 2014; 19:555–66.

6. Liu J, Lin M, Yu J, et al. Targeting apoptotic and autophagic pathways for cancer therapeutics. J Canlet. 2011; 300:105–14.

7. Mevorach D, Trahtemberg U, Krispin A, et al. What do we mean when we write “senescence,” “apoptosis,” “necrosis,” or “clearance of dying cells”? Ann NY Acad Sci. 2010; 1209:1–9.

8. Noy N. Between death and survival: retinoic acid in regulation of apoptosis. Ann Rev Nutr. 2010; 30:201–17.

References Cited

for structural macromolecules. In addition, the cell must have an adequate supply of the so-called regulatory nutri- ents (i.e., vitamins, minerals, and water).

With a view of the structure of the “typical cell,” the division of labor among cellular component parts,

Suggested Readings

Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004; 116:281–97.

Remely M, Stefanska B, Lovrecic L, Magnet U, Haslberger AG. Nutriepig- enomics: the role of nutrition in epigenetic control of human diseases. Curr Opin Clin Nutr Metab Care. 2015; 18:328–33.

Web Sites

www.genome.gov/10001772 All About the Human Genome Project (HGP) National Human Genome Project

and the location within the cell where many of the key metabolic reactions necessary to continue life take place, we can now consider in subsequent chapters how the cell receives its nourishment and how the nutrients are metabolized.

9. Nair-Shalliker V, Fenech M, Forder PM, Clements MS, Armstrong BK. Sunlight and vitamin D affect DNA damage, cell division and cell death in human lymphocytes: a cross sectional study in South Australia. Mutagenesis. 2012; 27:609–14.

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26

variants to trigger dysfunction or disease. The expectation is that, as was seen with the INS and MTHFR gene variants, the presence of a variant potentially alters the individual’s goodness-of-fit with his or her environment compared with someone who does not have the variant. This information can provide the clinician with clues as to the individual’s genetic susceptibilities that increase the risk of disease and which foods and other environmental factors to avoid.

Examples abound of gene variants that convey a latent genetic susceptibility to developing a disease state that manifests only upon exposure to a specific food. Food allergies, intolerances, and sensitivities provide interesting examples. Immunoglobulin E (IgE)–mediated food allergy is one such example in which a genetic susceptibility lies dormant until triggered by the interaction with an offending food. With appropriate nutrigenetic testing, it is possible to know in advance that a person is at risk of potentially deadly anaphylaxis from certain foods. Nutrigenetics can also help to eliminate the trial-and-error aspects of food intolerances. Lactose intolerance is a case in point. Whether the production of lactase, the enzyme responsible for digesting the milk sugar lactose, persists beyond childhood is genetically determined and varies by population. For Caucasians of northern European descent, lactase persistence and lifelong lactose tolerance is the norm and results from a single nucleotide change in the lactase (LCT) gene [1]. It is presumed that other food intoler- ances are also genetically based and that their early detection will enable diets to be tailored to the individual’s metabolic capabilities.

Celiac disease is similarly genetically determined but envi- ronmentally triggered, can manifest at any stage in the life cycle, and is characterized by inflammation and an immune response following ingestion of gluten from wheat, barley, and rye. This disorder is estimated to occur at a frequency of 1 in 133–200 individuals, depending on the study population [2– 5]. Of the gene variants associated with the development of celiac disease, changes in the HLA-DQ2 and HLA-DQ8 genes have been identified as being necessary but not sufficient for the development of the disorder [6]. Being able to detect these variants prenatally or at least early in the postnatal period prevents an infant from developing the disease. Failure to recognize celiac susceptibility and prevent its occurrence through lifelong adherence to a gluten-free diet can result in severe digestive tract inflammation, intestinal tract malignan- cies, malabsorption, and, ultimately, severe malnutrition.

Nutrigenomics is another subdiscipline of nutritional genomics. This subdiscipline is concerned with identifying

Nutritional genomics is concerned with gene–environment interactions. This emerging discipline uses genetic technology to study the mechanisms by which genes and environmental factors communicate and the functional con- sequences of such interactions. A major focus of research is the influence of these interactions on human health. Among the anticipated successes that will flow from nutritional genomics research is the ability to identify effective approaches for the management and prevention of diet-related disease.

One fundamental biological principle underlying gene– environment interactions is critically important to the functional ability, and thereby health, of living organisms: The information contained within a gene, when translated into the amino acid sequence of a protein, is directly related to the functional capacity of the organism. For example, the gene INS encodes the information needed to make the protein hormone insulin. Once synthesized, insulin plays a key role in the entry of glucose into muscle cells, where it can supply cellular energy. In the absence of insulin or in the presence of an insulin protein whose function is impaired, glucose is not able to enter certain cells as needed and diabetes results.

In a second example, the enzyme 5,10-methylene tetra- hydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofo- late, a coenzyme form of the B vitamin folate. This enzyme is encoded by the MTHFR gene. In individuals with a variation in this gene, the activity of the enzyme is impaired and the dietary requirement for folate is elevated as compared with the recommended Dietary Reference Intake level. By supply- ing higher levels of folate in the diet, food is able to “rescue” an individual from his or her genetic limitation in the MTHFR gene. Thus food, in addition to providing gustatory and social pleasure, is a powerful environmental factor in terms of com- municating with the genetic material and influencing biologi- cal responses.

NUTRIGENETICS, NUTRIGENOMICS, AND NUTRITIONAL EPIGENETICS

Nutritional genomics encompasses the subdisciplines of nutrigenetics, nutrigenomics, and nutritional epigenetics. The previously mentioned INS and MTHFR examples describe the biological outcomes of a change in the deoxyribonucleic acid (DNA) (a “gene variant”) and are examples of nutrigenetics. This subdiscipline is concerned with detecting gene variants within an individual, discovering their effect on function, and identifying which environmental factors interact with those

environmental factors that have an effect on the expression of genes, identifying which genes respond to which environmental factors, defining the mechanisms involved, and determining useful health-related applications of these interactions. Nutrigenomics is of interest from a diet–disease perspective because it holds the promise of using food in a targeted fashion, beyond food’s ability to supply the raw materials for cellular function. If, for example, an individual has a susceptibility to chronic inflammation, the clinician may recommend that he or she eat a diet that supplies suf- ficient omega-3 fatty acids to reduce the expression of genes that code for inflammatory cytokines, thereby blunting the inflammatory response.

The GST gene that encodes glutathione-S-tranferase, an enzyme that functions in the Phase II biotransformation of lipid-soluble toxins into water-soluble forms that can be excreted, exemplifies both nutrigenetics and nutrigenom- ics. Individuals whose genome includes a variant in the GST gene will be impaired in their ability to protect against toxins and their detrimental effects. The GST variant is an example of nutrigenetics, in that it illustrates the effect of having an impaired Phase II enzyme and its consequences to biotrans- formation. Impaired biotransformation can lead to disease for an individual regularly exposed to an environment with an elevated level of toxic chemicals.

The GST example also serves as an example of nutrig- enomics. In humans there are two additional GST genes that encode similar enzymes that can compensate for the faulty gene. The expression of these additional GST genes can be switched on by glucosinolates, sulfur-containing metabolites formed from the digestion of cruciferous vegetables such as broccoli and other members of the cabbage family. Nutrige- nomics researchers are interested in identifying environmental factors that can increase the expression of other genes that can circumvent the limitation caused by a particular gene variant, such as those seen with the faulty GST gene. In this case, food is the environmental factor and glucosinolates are the bio- active components within food that can communicate with the genetic material and influence gene expression. Similarly Suhr and colleagues [7] have identified several phytonutrients from food that confer chemoprotection by controlling gene expression. Additional discussion of the role of bioactives in influencing gene expression can be found in the “Bioactive Food Components” section.

A third subdiscipline of nutritional genomics is nutritional epigenetics, which represents yet another mechanism for reg- ulating gene expression. Epigenetics is the study of changes in

NUTRITIONAL GENOMICS: ANOTHER PERSPECTIVE ON FOOD BY RUTH DEBUSK, PhD, RD

P E R S P E C T I V E

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C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE 27

gene expression that do not involve changes in the nucleotide sequence of DNA. Instead, chemical “tags” that can affect gene expression are added (to the DNA or to the histone proteins associated with DNA). One common type of epigenetic regulation of gene expression involves opening and closing DNA to control its accessibility to being transcribed. DNA that is tightly compacted is not available to be transcribed and, thus, expressed. The addition and removal of acetyl groups from the histone proteins that aid DNA in condensing and decondensing is a common mechanism for controlling gene expression. A second mechanism, the addition and removal of methyl groups to cytosine-containing nucleotides within the DNA sequence, can similarly control gene expression. The presence of methyl groups typically silences gene expression. In both instances, the ultimate source of these tags—that is, the acetyl and methyl groups—is the diet. The end result is the regulation of gene expression, which makes nutritional epigenetics yet another mechanism for fine-tuning the control of this process. Nutritional epigenetics is particularly important during development, during cellular differentiation, and in maintaining the distinct pattern of gene expression that characterizes the myocyte in contrast to the hepatocyte, for example.

Furthermore, a cell’s pattern of tags is heritable and can be passed to subsequent generations. From the work of Wolff and colleagues [8], Waterland and Jirtle [9], and Waterland [10], it is clear that diet plays a major role in epigenetic patterning. Although there are currently more questions than answers concerning epigenetics and its associated mechanisms and consequences, nutrition can be expected to factor prominently and will be an important determinant in the ability to reach one’s genetic potential and optimal health [11,12].

Nutritional genomics is an emerging field and considerable research is needed to generate well- documented associations among genes, diseases, and food before this field will fully make its impact. Once the research foundation is in place, the expectation is that nutri- tional genomics will be the source of effective therapeutic approaches to diet-related disease.

GENETIC VARIATION AND FUNCTION

As described previously, faulty INS and MTHFR genes have functional consequences for the individual. These detri- mental variations in the INS and MTHFR genes come about through changes (mutations) in the nucleotide sequence of the DNA over evolutionary time and are called gene variants. The vast majority of gene variants result from a change in one nucleotide subunit of DNA. When a change in a single nucleotide occurs frequently in a population, it is referred to as a “single-nucleotide polymorphism” or “SNP” (pro- nounced “snip”).

It is estimated that SNPs comprise approximately 10% of the human genome. SNPs are the basis for the uniqueness of each individual, and some result in differences in observable

traits, such as hair color, eye color, or stature. The majority influence metabolic processes critical to the workings of the trillions of cells that comprise the human body and provide its functional abilities. When a SNP in the DNA results in a change in the amino acid structure of the encoded protein such that the folding of the protein is altered in a way that negatively influences its function, the potential exists for dysfunction or disease to result. Although such changes are technically muta- tions (any change in the DNA sequence is a mutation), the term gene variant is typically used for those mutations whose impact on function is not sufficiently detrimental to cause disease by themselves. Such mutations are “silent” in their effect on func- tion until they interact with one or more environmental fac- tors. Thus it is not the existence of a change in the DNA but the impact of that change on function that is consequential.

Once a person’s variants are known, a well-documented association has been demonstrated between the variant and a disease, and the mechanism by which the dysfunction is triggered has been identified, then developing a therapeu- tic strategy for countering the negative effect on function becomes possible. For example, the VDR gene codes for the vitamin D receptor that is needed for cells to absorb vitamin D. If one has a variant in the VDR gene that impairs the absorp- tion of vitamin D, a therapeutic intervention might include increased exposure to sunlight, increased intake of vitamin D– containing foods, a vitamin D dietary supplement, or a combination of these approaches. Clinicians then have an effective approach for countering the genetic limitations of that individual to prevent or at least limit the severity of the dysfunction associated with particular gene variants.

Gene variants are detected using well-established genetic technology. Because each cell with a nucleus contains a full complement of the individual’s genetic material, multiple sources of DNA samples exist, from white blood cells to secre- tions to swabs taken from the inside of the cheek. Swabbing the cheek is a noninvasive method that is increasingly used to obtain DNA for genetic testing. In the laboratory the DNA is extracted and amplified, and specific “probe” sequences with fluorescent dye attached are used to query whether a sample from an individual contains a particular gene variant. If it does, the fluorescent probe will bind to the sample DNA and can then be detected.

BIOACTIVE FOOD COMPONENTS

Bioactive food components were introduced in the discussion of nutrigenomics. Of keen interest to researchers are the mech- anisms by which food influences gene expression. Lipophilic, small-molecular-weight molecules such as essential fatty acids, vitamin A, and steroid molecules are able to traverse the cellular and nuclear membranes. They subsequently interact with DNA by means of transcription factors, specialized proteins that bind to DNA in one region of the protein and in a second region are able to bind small-molecular-weight ligands. Many of these ligands originate with the diet and are capable of bind- ing to one or more transcription factors and influencing gene

expression. Expression may be activated or silenced fully or partially to meet the ever-changing needs of the cells.

Bioactives that are either too large or too hydrophilic to pass through the lipid bilayer of the cellular and nuclear membranes communicate with the cell by interacting with cell surface receptors. Binding to the receptors triggers signal transduction, a cascade of events that typically leads to the translocation of a transcription factor to the nucleus, where it can then bind DNA and turn gene expression on or off, as appropriate.

The identification and isolation of bioactive food com- ponents is an active area of study within nutrigenomics. Bioactives may be traditional nutrients, such as vitamins or essential fatty acids, or nontraditional nutrients, such as the phytonutrients epigallocatechin-3-O-gallate from green tea, lycopene from tomatoes, and resveratrol from purple grape juice. Bioactive food components may also be potential toxins that enter the food supply inadvertently. In addition to the example of glucosinolates in cruciferous vegetables discussed previously, another bioactive food component that has positive implications for many inflammatory dis- ease states is derived from linolenic acid, an essential fatty acid of the omega-3 class. This bioactive can modulate the expression of genes that promote inflammation, such as the PPARG (peroxisome proliferator-activated receptor gamma), IL1 (interleukin 1), IL6 (interleukin 6), and COX2 (cyclooxygen- ase-2) genes [13,14].

The communication between bioactive food components and the genetic material is an intricate web of events by which cells adjust to the state of their environment. As the fund of knowledge about which bioactives affect which genes and influence which functions accumulates, diet therapy is expected to become increasingly effective because it will become possible to select specific foods to target particular mechanisms. Furthermore, expect to see a movement away from general nutrient recommendations intended for the “average” person and toward recommendations personalized for the individual.

References Cited

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4. Ludvigsson JF, Montgomery SM, Ekbom A, et al. Small-intestinal histopathology and mortality risk in celiac disease. JAMA. 2009; 302:1171–78.

5. Rubio-Tapia A, Kyle RA, Kaplan EL, et al. Increased prevalence and mortality in undiagnosed celiac disease. Gastroenterology. 2009; 137:88–93.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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28 C H A P T E R 1 • THE CELL: A MICROCOSM OF LIFE

6. Romanos J, van Diemen CC, Nolte IM, et al. Analysis of HLA and non-HLA alleles can identify individuals at high risk for celiac disease. Gastroenterology. 2009; 137:834–40.

7. SurhYJ, KunduJK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med. 2008; 74:1526–39.

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9. Waterland RA, Jirtlem RL. Early nutrition, epigenetic changes at transposons and imprinted genes, and enhanced susceptibility to adult chronic diseases. Nutrition. 2004; 20:63–68.

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11. McKay JA, Mathers JC. Diet induced epigenetic changes and their implications for health. Acta Physiol (Oxf). 2011; 202:103–18.

12. Stover PJ, Caudill MA. Genetic and epigenetic contributions to human nutrition and health: managing genome-diet interactions. J Am Diet Assoc. 2008; 108:1480–87.

13. Massaro M, Scoditti E, Carluccio MA, et al. Omega-3 fatty acids, inflammation and angiogenesis: nutrigenomics effects as an explanation for anti- atherogenic and anti-inflammatory effects of fish and fish oils. J Nutrigenet Nutrigenomics. 2008; 1:4–23.

14. Wall R, Ross RP, Fitzgerald GF, et al. Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr Rev. 2010; 68:280–89.

Suggested Readings

Corella D, Ordovas JM. Nutrigenomics in cardiovascular medicine. Circ Cardiovasc Genet. 2009; 2:637–51.

Fenech M, El-Sohemy A, Cahill L, et al. Nutrigenetics and nutrigenomics: viewpoints on the current status and applications in nutrition research and practice. Nutrigenet Nutrigenomics. 2011; 4:69–89.

Kussmann M, Krause L, Siffert W. Nutrigenomics: where are we with genetic and epigenetic markers for dis- position and susceptibility? Nutr Rev. 2010 Nov; 68(suppl 1):S38–47.

McKay JA, Mathers JC. Diet induced epigenetic changes and their implications for health. Acta Physiol (Oxf ). 2011; 202:103–18.

Ordovás JM, Robertson R, Cléirigh EN. Gene-gene and gene- environment interactions defining lipid-related traits. Curr Opin Lipidol. 2011 Apr; 22:129–36.

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Smith CE, Ordovás JM. Fatty acid interactions with genetic polymorphisms for cardiovascular disease. Curr Opin Clin Nutr Metab Care. 2010; 13:139–44.

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29

N UTRITION INCLUDES THE SCIENCE OF NOURISHMENT. Ingestion of foods and beverages provides the body with at least one, if not more, of the nutrients needed to nourish the body. The body needs six classes of nutrients: carbohydrate, lipid, protein, vitamins, minerals, and water. For the body to use the carbohydrate, lipid, protein, and some vitamins and minerals found in foods, the food must first be digested—in other words, the food first must be broken down mechanically and chemically. This process of digestion occurs in the digestive tract and, once complete, yields nutrients ready for absorption and use by the body.

THE STRUCTURES OF THE DIGESTIVE TRACT AND THE DIGESTIVE AND ABSORPTIVE PROCESSES

The digestive tract, approximately 16 feet in length, includes organs that comprise the gastrointestinal (GI) tract (also called the alimentary canal or gut) as well as three accessory organs. The main structures of the digestive tract include the oral cavity, esophagus, and stomach (collectively referred to as the upper diges- tive tract), and the small and large intestines (called the lower digestive tract). The accessory organs include the pancreas, liver, and gallbladder. The accessory organs provide or store secretions that ultimately are delivered to the lumen (interior passageway) of the digestive tract and aid in the digestive and absorp- tive processes. Figure 2.1 illustrates the digestive tract and accessory organs. Figure 2.2 provides a cross-sectional view of the gastrointestinal tract that shows the lumen and the four main tunics, or layers, of the gastrointestinal tract:

● the mucosa ● the submucosa ● the muscularis externa ● the serosa.

This first layer, the mucosa, is the innermost layer, and is made of three sublayers: the mucosal membrane, the lamina propria, and the muscularis mucosa. The mucosa acts as a membrane, consists of epithelial cells that line the lumen of the gastrointestinal tract, and is the inner surface layer that is in contact with the food (and its nutrients) that we eat. In the small intestine, this layer is arranged differently than in other sections of the digestive tract (as discussed under “Structural Aspects, Secretions, and the Digestive Processes of the Small Intestine”). Both exocrine and endocrine cells are found among

THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

2

THE STRUCTURES OF THE DIGESTIVE TRACT AND THE DIGESTIVE AND ABSORPTIVE PROCESSES

The Oral Cavity The Esophagus The Stomach The Small Intestine The Accessory Organs The Absorptive Process The Colon (Large Intestine)

COORDINATION AND REGULATION OF THE DIGESTIVE PROCESS

Neural Regulation Regulatory Peptides

SUMMARY

P E R S P E C T I V E

THE NUTRITIONAL IMPACT OF ROUX-EN-Y GASTRIC BYPASS, A SURGICAL APPROACH FOR THE TREATMENT OF OBESITY

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30 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

in part, gastrointestinal secretions and local blood flow. The lymphoid tissue in the submucosa is similar to that found in the mucosa and protects the body against ingested foreign substances. The submucosa connects the first mucosal layer of the gastrointestinal tract to the muscularis externa, or third layer of the gastrointestinal tract.

The muscularis externa contains inner circular and outer longitudinal smooth muscles that surround (lie on top of) the submucosa and facilitate motility. This layer also includes the myenteric plexus, or plexus of Auerbach, which lies between the circular and the longitudinal muscles. This plexus controls the frequency and strength of contractions of the muscularis to regulate gastrointestinal motility.

The outermost layer, the serosa (sometimes called the adventitia) consists of relatively flat mesothelial cells that

the epithelial cells of the mucosa. The exocrine cells secrete a variety of enzymes and juices into the lumen of the gastrointestinal tract, and the endocrine (also called enteroendocrine) cells secrete various hormones into the blood. The lamina propria, another sublayer, lies adjacent to the epithelium and consists of primarily connective tissue and lymphoid tissue. This lymphoid tissue contains a number of cells, especially macrophages and lymphocytes, which provide protection against microorganisms. The third sublayer of the mucosa, the muscularis mucosa, is made up of a thin layer of smooth muscle.

Next to the mucosa is the submucosa. The submucosa, the second tunic or layer, is made up of connective tissue, blood and lymphatic vessels, more lymphoid tissue, and a network of nerves called the submucosal plexus, or plexus of Meissner. This plexus (plexus means network) controls,

Figure 2.1 The digestive tract and its accessory organs. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Organs of the gastrointestinal tract

Accessory organs

Salivary glands—release a mixture of water, mucus, and

enzymes

Liver—produces bile, an important secretion needed

for lipid digestion

Gallbladder—stores and releases bile, needed for

lipid digestion

Pancreas—releases pancreatic juice that

neutralizes chyme and contains enzymes needed for carbohydrate, protein,

and lipid digestion

Oral cavity—mechanical breakdown, moistening, and mixing of food with saliva

Pharynx—propels food from the back of the oral cavity into the esophagus

Esophagus—transports food from the pharynx to the stomach

Stomach—muscular contractions mix food with acid and enzymes, causing the chemical and physical breakdown of food into chyme

Small intestine—major site of enzymatic digestion and nutrient absorption

Large intestine—receives and prepares undigested food to be eliminated from the body as feces

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 31

Atrophy of these mucosa and submucosa layers can result in bacterial translocation from the intestine into the blood, leading to sepsis (infection). Within these layers of the digestive tract, immunoprotection is provided by leukocytes, especially T- and B-lymphocytes; plasma cells; natural killer (NK) cells; macrophages; microfold (M) cells; and dendritic cells, among others. Many of these cells are found in Peyer’s patches, which are aggregates of lymphoid tissue, usually present in a single layer, in the mucosa and submucosa. The plasma cells produce secretory IgA, which binds antigens ingested with foods, inhibits the growth of pathogenic bacteria, and inhibits bacterial translocation. Tissue macrophages secrete cytokines, which exhibit a variety of immunoprotective effects to defend against foreign substances. The M-cells are antigen-presenting cells; these M-cells pass or transport foreign antigens to the

produce small amounts of lubricating fluids. For many areas of the digestive tract, this layer is continuous with the peritoneum. The peritoneum is a membrane with two layers within the abdominal cavity. In the abdominal cavity, the visceral peritoneum surrounds the stomach and intestine, and the parietal peritoneum lines the pelvic cavity walls. These membranes are somewhat permeable and highly vascularized. Between the two membranes is the peritoneal cavity. The selective permeability and the rich blood supply of peritoneal membranes allow the peritoneal cavity to be used in dialysis, an ultrafiltration process used to treat kidney failure.

Immune system protection is located throughout the gastrointestinal tract (and called gut-associated lymphoid tissue or GALT), especially the mucosa and submucosa layers of the small intestine (and sometimes called mucosa-associated lymphoid tissue or MALT).

Figure 2.2 The sublayers of the small intestine. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Lymph vessel

Vein

Artery

Nerve

Serosa • Connective tissue • Outer cover that protects the GI tract

Muscularis externa • Two layers of smooth muscles—longitudinal muscle and circular muscle • Responsible for GI motility Submucosa

• Connective tissue • Contains blood vessels, lymphatic vessels, nerves, and lymphoid tissue

Mucosa • Innermost mucous membrane layer • Produces and releases secretions needed for digestion • Lymphoid tissue protects the body

Lumen

Circular muscle

Longitudinal muscle

Notice that the muscle fibers run in different directions, which influences muscular movements of the GI tract.

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32 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

these parts of the digestive tract. Other sections include information on the structures and roles of the pancreas, liver, and gallbladder, and the roles of a variety of enzymes. Table 2.1 provides an overview of some of the enzymes and zymogens (also referred to as proenzymes or inactive enzymes, which must be altered to function as an enzyme) that participate in digesting the nutrients in foods.

The Oral Cavity The mouth and pharynx (or throat) constitute the oral cavity and provide the entryway to the digestive tract. On entering the mouth, food is chewed by the actions of the teeth and jaw muscles and is made ready for swallowing by mixing with secretions (saliva) released from the sali- vary glands. Three pairs of small, bilateral saliva-secreting

Peyer’s patches or lymphocytes, which in turn mount an immune response. After processing the foreign antigens, some of these lymphocytes are released from the Peyer’s patches and enter circulation to augment the immune response. Dendritic cells, a type of macrophage, also are found in the gastrointestinal tract. Dendritic cells destroy foreign substances and then serve as antigen-presenting cells to stimulate lymphocyte activity and proliferation. The processing and presentation of antigens by antigen- presenting cells further triggers recognition of antigens by other parts of the immune system as “safe” or “harmful.”

The digestive process begins in the oral cavity and proceeds sequentially through the esophagus, stomach, small intestine, and finally into the colon (large intestine). The next subsections of this chapter describe the structures and digestive processes that occur in each of

Enzyme or Zymogen/Enzyme Site of Secretion Preferred Substrate(s) Primary Site of Action

Salivary a-amylase Mouth a (1-4) bonds in starch, dextrins Mouth, stomach

Lingual lipase Mouth Triacylglycerol Mouth, stomach

Pepsinogen/pepsin Stomach Carboxyl end of phe, tyr, trp, met, leu, glu, asp* Stomach

Gastric lipase Stomach Triacylglycerol (mostly medium chain) Stomach

Trypsinogen/trypsin Pancreas Carboxyl end of lys, arg* Small intestine

Chymotrypsinogen/chymotrypsin Pancreas Carboxyl end of phe, tyr, trp, met, asn, his* Small intestine

Procarboxypeptidase/carboxypeptidase A Pancreas C-terminal neutral amino acids Small intestine

Carboxypeptidase B Pancreas C-terminal basic amino acids Small intestine

Proelastase/elastase Pancreas Fibrous connective tissue proteins—elastin Small intestine

Collagenase Pancreas Collagen Small intestine

Ribonuclease Pancreas Ribonucleic acids Small intestine

Deoxyribonuclease Pancreas Deoxyribonucleic acids Small intestine

Pancreatic a-amylase Pancreas a (1-4) bonds, in starch, maltotriose Small intestine

Pancreatic lipase and colipase Pancreas Triacylglycerol Small intestine

Phospholipase Pancreas Lecithin and other phospholipids Small intestine

Cholesterol esterase Pancreas Cholesterol esters Small intestine

Retinyl ester hydrolase Pancreas Retinyl esters Small intestine

Amino peptidases Small intestine N-terminal amino acids Small intestine

Dipeptidases Small intestine Dipeptides Small intestine

Nucleotidase Small intestine Nucleotides Small intestine

Nucleosidase Small intestine Nucleosides Small intestine

Alkaline phosphatase Small intestine Organic phosphates Small intestine

Monoglyceride lipase Small intestine Monoglycerides Small intestine

Alpha dextrinase or isomaltase Small intestine a (1-6) bonds in dextrins, oligosaccharides Small intestine

Glucoamylase, glucosidase, and sucrase Small intestine a (1-4) bonds in maltose, maltotriose Small intestine

Trehalase Small intestine Trehalose Small intestine

Disaccharidases Small intestine Small intestine

Sucrase Sucrose

Maltase Maltose

Lactase Lactose

* Amino acid abbreviations: phe, phenylalanine; tyr, tyrosine; trp, tryptophan; met, methionine; leu, leucine; glu, glutamic acid; asp, aspartic acid; lys, lysine; arg, arginine; asn, asparagine; and his, histidine.

Table 2.1 Digestive Enzymes and Their Actions

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 33

disease and Sjögren’s syndrome, among others. Insufficient saliva production not only causes the mouth and throat to become dry, but also impairs swallowing and diminishes the cleansing of our teeth and gums from food residue, acids, and old epithelial cells that have been shed from the oral mucosa. Dental caries and gum disease result if preventative care is not taken. Saliva substitutes and stimulants to increase saliva production can be helpful for some with xerostomia.

The Esophagus From the mouth, food, now mixed with saliva and called a bolus, is passed through the pharynx into the esopha- gus. The esophagus is about 10 inches long and close to an inch (2 cm) in diameter (see Figure 2.1). The passage of the bolus of food from the oral cavity into the esophagus constitutes swallowing. Swallowing, which can be divided into several stages (voluntary, pharyngeal, and esopha- geal), is a reflex response initiated by a voluntary action and regulated by the swallowing center in the medulla of the brain. To swallow food, the esophageal sphincter relaxes, allowing the esophagus to open. Food then passes into the esophagus. Simultaneously, the larynx (part of the respiratory tract) moves upward, inducing the epiglottis to shift over the glottis. The closure of the glottis is important in keeping food from entering the trachea, which leads to the lungs. Once food is in the esophagus, the larynx shifts downward to allow the glottis to reopen.

When the bolus of food moves into and down the esophagus, both the striated (voluntary) muscle of the upper portion of the esophagus and the smooth (involuntary) muscle of the distal portion are stretched and stimulated by the nervous system. The result is peristalsis, a progressive wave-like motion that moves the bolus through the esophagus into the stomach in usually less than 10 seconds. While the swallowing of food triggers the primary peristaltic wave, secondary waves (through the activation of stretch receptors in the esophagus) also may be initiated if, for example, food gets lodged in the esophagus. Peristalsis occurs throughout the digestive tract from the esophagus to the colon and propels the contents in the lumen distally.

At the lower (distal) end of the esophagus, just above the juncture with the stomach, lies the gastroesophageal sphincter, also called the lower esophageal sphincter (Figure 2.4). Calling it a sphincter may be a misnomer because no consensus exists about whether this particular muscle area is sufficiently hypertrophied to constitute a true sphincter. Several sphincters or valves, which are circular muscles, are located throughout the digestive tract; these sphincters allow food to pass from one section of the gastrointestinal tract to another. On swallowing, the gastroesophageal

salivary glands—the parotid, the submandibular, and the sublingual—are distributed throughout the lining of the oral cavity, along the jaw from the base of the ear to the chin (Figure 2.3). Secretions (about 1–2 L/day) from these glands constitute saliva, which is made up of mostly water (99.5%) along with proteins (enzymes, mucus, antiviral/anti- bacterial proteins), electrolytes (sodium, potassium, chlo- ride), and some solutes (urea, phosphates, bicarbonate). The water in saliva helps dissolve foods. The principal enzyme of saliva is salivary a–amylase (also called ptyalin; see Table 2.1). This enzyme hydrolyzes internal a (1-4) bonds within starch. A second digestive enzyme, lingual lipase, is produced by lingual serous glands on the tongue and in the back of the mouth. This enzyme hydrolyzes dietary triacylglycerols (tri- glycerides) primarily after food has been swallowed and is in the stomach. The enzyme’s activity diminishes with age and is limited by the coalescing of the fats within the stomach. Lingual lipase activity is most helpful in infants, enhancing the digestion of triacylglycerols in milk. Mucus in the saliva lubricates food and coats and protects the oral mucosa. Some of the antibacterial and antiviral proteins in saliva include the antibody IgA (immunoglobulin A) and the enzyme lyso- zyme, which lyses (destroys) the cell walls of some bacteria. An R-protein in saliva functions in the stomach to enhance the absorption of vitamin B12. Bicarbonate in saliva assists in neutralizing acids in consumed foods and acids produced by bacteria inhabiting the oral cavity. The pH of saliva is about 7.

Saliva is released into the oral cavity 24 hours per day. Basal, or resting, secretion rates (when we are not eating) are about 0.3–0.5 mL/minute, and with food consumption, saliva secretion rates usually increase to about 2 mL/minute. Insufficient saliva production results in xerostomia (dry mouth), and may occur with the use of some medications, cancer-associated radiation and chemotherapies, as well as disorders such as Parkinson’s

Figure 2.3 Secretions of the oral cavity.

Mouth Salivary glands

Parotid Sublingual Submandibular/ submaxillary

Pharynx

Esophagus Saliva containing Water Electrolytes Mucus Enzymes* Antibacterial and antiviral proteins R-protein Solutes

*Main enzyme in saliva is salivary amylase, which hydrolyzes α (1-4) bonds in starch.

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34 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

Figure 2.4 Structure of the stomach including a gastric gland and its secretions. Source: Adapted from Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning. Dr. Fred Hossler/Visuals Unlimited

Lower esophageal or gastroesophageal sphincter— regulates the flow of food from the esophagus into the stomach

The stomach has 3 layers of muscle—

longitudinal, circular, and diagonal. Forceful contractions of these

muscles enable food to mix with gastric juice to

form chyme.

Pyloric sphincter— regulates the flow of chyme from the stomach into the upper or proximal small intestine (duodenum)

Rugae— The lining of the stomach

has many folds called rugae. As the stomach fills

with food, these folds flatten, allowing the walls of the stomach to expand.

Cardia

Antrum

Body

Smooth muscle layer

Fundus

Entrance

Entrance to gastric pits, which contain cells that produce gastric juice

Longitudinal Circular

Diagonal

Greater curvature Pacemaker

Gastric mucosal barrier

Lymphatic vessel

Diagonal muscle

Circular muscle

Longitudinal muscle

Serosa

Artery and vein

Muscularis

Mucosa

Submucosa

Mucus-secreting neck cells on the surface of the gastric pit produce an alkaline mucus that forms the gastric mucosal barrier. This protects the mucosal lining from the acidity of the gastric juice.

Parietal (oxyntic) cells produce hydrochloric acid (HCI) and intrinsic factor, which is needed for the absorption of vitamin B12.

Enteroendocrine G-cells produce the hormone gastrin, which stimulates parietal and chief cells.

Chief (peptic or zymogenic) cells produce enzymes needed for protein and fat digestion.

Gastric pit

sphincter pressure drops. This drop in gastroesophageal sphincter pressure relaxes (opens) the sphincter so that food may pass from the esophagus into the stomach.

Multiple mechanisms, including neural and hormonal, regulate gastroesophageal sphincter pressure. The musculature of the gastroesophageal sphincter has a

tonic pressure that is normally higher than the intragastric pressure (the pressure within the stomach). This high tonic pressure keeps the sphincter closed. Keeping this sphincter closed is important because it prevents gastroesophageal reflux (the movement of substances from the stomach back into the esophagus).

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 35

proximal section of the small intestine. The stomach con- tains four main regions (shown in Figure 2.4):

● the cardia region lies below the gastroesophageal sphincter and receives the swallowed food (bolus) from the esophagus.

● the fundus lies adjacent or lateral to and above the cardia.

● the body, the large central region, serves primarily as the reservoir for swallowed food and is the main production site for gastric juice.

● the antrum or pyloric portion, the lower (distal) one- third of the stomach, provides strong peristalsis to grind and mix food with the gastric juices (which forms chyme, a thick, semiliquid mass of partially digested food) and to empty the chyme into the duodenum.

The stomach’s circular, longitudinal, and oblique smooth muscles enable the mixing of the food with gastric juices, including its acid and enzymes. The volume of the stomach when empty (resting) is about 50 mL (~2 oz), but on being filled it can expand to accommodate from 1 L to approximately 1.5 L (~37–52 oz). When the stomach is empty, folds (called rugae) present in all but the antrum section are visible; however, as we eat and the stomach fills, the rugae disappear. Receptive relaxation allows gastric expansion with food intake with minimal impact on intragastric pressure unless food intake exceeds the stomach’s volume capacity.

Gastric juices, which are produced in significant quantities by glands found within the gastric mucosa and submucosa, facilitate the digestion of nutrients within the chyme. These glands include:

● the cardiac glands, found in a narrow rim at the juncture of the esophagus and the stomach

● the oxyntic glands, found in the fundus and body ● the pyloric glands, located primarily in the antrum.

Several cell types, which secrete different substances, are found within gastric glands, as shown in Figure 2.4. Some of the cells and their secretions that are found in a gastric oxyntic gland include:

● neck (mucous) cells, which secrete mucus ● parietal (oxyntic) cells, which secrete hydrochloric acid

and intrinsic factor ● chief (peptic) cells, which secrete pepsinogen and gastric

lipase ● enteroendocrine cells, which secrete a variety of

hormones.

Unlike the oxyntic glands, the cardiac glands contain no parietal cells and the pyloric glands contain no chief cells.

Selected Disorders of the Esophagus A person experiencing gastroesophageal reflux feels a burning sensation (known as heartburn or pyrosis) in the midchest. The burning usually occurs after eating, and may last for several hours. Repeated episodes may be diagnosed as gastroesophageal reflux disease (abbreviated GERD), also called acid reflux disease. Because of the low (acidic) pH of gastric (stomach) juices and because the esophageal mucosa does not have the same protective lay- ers as does the gastric mucosa, significant damage to the esophagus may occur with chronic acid reflux including edema (swelling); tissue erosion and ulceration; blood vessel (usually capillary) damage; spasms; and fibrotic tis- sue formation, which can cause a narrowing (stricture) within the esophagus. Additional symptoms may include a chronic cough, excessive belching, and/or a sour taste in the mouth. While medications to neutralize the acid and/ or to reduce acid production are important to promote healing, some dietary changes can also help. To minimize reductions in sphincter pressure, high-fat foods as well as chocolate, nicotine, alcohol, and carminatives (volatile oil extracts of plants, most often oils of spearmint and pepper- mint) should be avoided. Substances that increase gastric acid production (such as alcohol, excessive calcium, and decaffeinated and caffeinated coffee and tea) also should be avoided. Because citrus products and other acidic foods or beverages, as well as spices such as red and black pep- per, nutmeg, cloves, and chili powder, can directly irritate inflamed tissues, avoidance of these substances is also encouraged. Additional suggestions include: (1) Eating smaller (versus larger) meals and drinking fluids between meals (versus with meals), since large gastric volume may promote reflux; (2) Losing weight (if overweight or obese) and avoiding tight-fitting clothes, since these may directly increase gastric pressure; and (3) Avoiding lying down, lifting, or bending for at least 2 hours after eating, since such actions place gastric contents nearer to the sphinc- ter and may promote reflux. A discussion of some of the medications used in the management of gastroesophageal reflux disease as well as ulcers is presented in the section “Selected Disorders of the Stomach.” Surgical treatment of chronic acid reflux that has not responded to medications and dietary changes usually involves fundoplication, a pro- cedure in which a portion of the stomach (the fundus) is wrapped around the sphincter (and thus tightens it).

The Stomach Once the bolus of food has passed through the gastro- esophageal sphincter, it enters the stomach, a J-shaped sac-like organ located on the left side of the abdomen under the diaphragm. The stomach extends from the gas- troesophageal sphincter to the duodenum, the upper or

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36 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

The high concentration of hydrochloric acid in the gastric juice is responsible for its low pH, about 2. The pH value is the negative logarithm of the hydrogen ion concentration. The lower the pH is, the more acidic the solution is. Figure 2.6 shows the approximate pH values of body fluids and, for comparison, some other compounds and beverages. Notice that the pH of orange juice (and typically of all fruit juices) is higher than that of gastric juice. Thus, drinking such juices does not lower the gastric pH.

In addition to creating an acid environment, hydrochloric acid has several other functions in gastric juice, including:

● converting or activating the zymogen pepsinogen to form pepsin (needed for protein digestion)

The main constituents of gastric juice produced by the different cells of these gastric glands include water, electrolytes, hydrochloric acid, enzymes, mucus, and intrinsic factor. About 2 L (usual range 1–3 L) of this juice are secreted each day. The next section describes some of these constituents: hydrochloric acid, enzymes, and mucus. A discussion of intrinsic factor, which is found in gastric juice and needed for vitamin B12 absorption, is provided in Chapter 9.

Gastric Juice Gastric juice contains an abundance of hydrochloric acid, which is secreted as separate hydrogen ions (H+) and chlo- ride ions (Cl−) from parietal cells into the lumen of the stomach. The mechanism by which hydrochloric acid is secreted is shown and described in Figure 2.5.

Figure 2.5 Mechanism of HCl secretion. Source: Adapted from Sherwood, Human Physiology, 9/e. © Cengage Learning.

Cl–

Gastric lumenPlasma

H+ H+

K+

K+

CO2 CO2 + H2O H2CO3

Carbonic acid

Cellular metabolism

Carbonic anhydrase

Parietal cell

Cl–

HCO3 –

Cl–

Membrane key

= Active transport

= Secondary active transport

= Passive diffusion

ATP

Parietal cells actively secrete hydrogen (H+) and chloride (Cl-) by two different transport systems. A hydrogen (proton) potassium ATPase exchange system (H+, K+-ATPase), also referred to as a proton pump, secretes hydrogens (protons) into the lumen in exchange for potassium ions (K+) with each ATP molecule hydrolyzed.

Following the active exchange, the potassium ions typically diffuse out of the parietal cells and back into the lumen.

The hydrogen arises, along with bicarbonate, from the dissociation of carbonic acid (H2CO3). The carbonic acid is generated within the parietal cell from carbonic anhydrase, an enzyme found in high concentrations within parietal cells, using water and carbon dioxide. The water and carbon dioxide are produced within the cell from normal metabolism; the carbon dioxide also may arise in the cell following diffusion from the plasma.

The chloride ions needed to form hydrochloric acid arise initially from the plasma from which they are transported by a secondary active transport system in exchange for bicarbonate into the parietal cells. This antiporter carries simultaneously the bicarbonate down its concentration gradient into the plasma and the chloride against its concentration gradient into the parietal cell.

From the parietal cells, the chloride ions then diffuse out via a chloride channel into the gastric lumen joining the hydrogen ions to generate hydrochloric acid.

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 37

Pepsin functions as a protease, an enzyme that hydrolyzes proteins. Specifically, pepsin is an endo- peptidase, meaning that it hydrolyzes interior peptide bonds within proteins. Optimal pepsin activity occurs at about pH 3.5. Another enzyme made by gastric chief cells is gastric lipase. Gastric lipase hydrolyzes fatty acids from glycerol’s third carbon in triacylglycerols. This enzyme is thought to be responsible for up to about 20% of lipid digestion. The salivary a-amylase found in gastric juice originates from the salivary glands of the mouth. This enzyme, which hydrolyzes starch, retains some activity in the stomach until it is inactivated by the low pH of gastric juice. Additional information about pepsin and salivary a-amylase can be found in Chapters 6 and 3, respectively. Gastric lipase is discussed further in Chapter 5.

Gastric juice also contains mucus, which is secreted both by neck (mucous) cells in gastric glands and by mucosal epithelial cells; these epithelial cells also release bicarbonate (HCO )32 . Mucus composition varies depending on its location in the digestive tract, but it generally consists of a network of different glycoproteins called mucins. Most mucins bind water and are gel-forming and thus provide lubrication and protection. In the stomach, mucus both coats the gastric contents as well as forms a layer about 2 mm thick on the gastric mucosal membrane to coat and protect it. Embedded within this gastric mucus layer is bicarbonate creating a local pH of about 6–7 versus the very acidic pH of about 2 in the gastric lumen. Production and release of mucus within the stomach is enhanced by prostaglandins, vagal nerve stimulation, acetylcholine, and various hormones. Substances that inhibit or diminish mucus secretion increase the risk for ulcer formation.

Regulation of Gastric Secretions The regulation of gastric secretions can be divided into three phases based on events occurring before food reaches the stomach, once food is in the stomach, and after food has left the stomach. Multiple mechanisms, both neural and chemical, influence each of the three phases; some of the many hormones and peptides that are involved are shown in Figure 2.7 and are presented later in the chapter in Table 2.2.

In the cephalic (first) phase, eating or tasting food, as well as thinking about, seeing, and/or smelling food, stimulates gastric secretions. Vagal stimulation of primarily the submucosal plexus promotes the secretion of the neurotransmitter acetylcholine and enhances the release of the hormone gastrin from G cells. Acetylcholine and gastrin both trigger the release of the paracrine histamine by mast cells and enterochromaffin-like cells in the gastric glands. Each stimulates hydrochloric acid secretion by parietal cells—histamine binds to H2 receptors, gastrin binds to gastrin receptors, and acetylcholine acts on muscarinic receptors on the parietal cells. Additionally, acetylcholine stimulates the chief cells, promoting enzyme release.

● denaturing proteins (i.e., destructing or “uncoiling” the tertiary and secondary protein structures to expose the protein’s interior peptide bonds so pepsin can perform its enzymatic functions)

● releasing various nutrients such as minerals from organic complexes so absorption can occur

● acting as a bactericide agent (needed to kill bacteria ingested along with food).

Three enzymes (see Table 2.1) are found in gastric juice. The enzyme pepsin is secreted into gastric juice initially as a zymogen called pepsinogen. Specifically, pepsinogen is secreted in granules into the gastric lumen by chief cells when they are stimulated by acetylcholine and/or acid. Pepsinogen is then converted (activated) to pepsin, an active enzyme, by hydrochloric acid or the presence of previously formed pepsin in the gastric lumen.

Acid or pepsin

Pepsinogen Pepsin

Figure 2.6 Approximate pHs of selected body fluids, compounds, and beverages.

14

13

12

11

10

9

8

7

6

5

4

3

2

1

0

Ammonia

Baking soda

Bile Pancreatic juice Intestinal juice Blood Milk Saliva

pH scale

Basic

Acidic

Neutral

Urine

Coffee

Orange juice

Vinegar

Lemon juice Gastric juice

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38 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

secretions as well as reduce peristalsis in the antrum, and slow gastric emptying to “finish up” digestive actions in the stomach, but simultaneously these hormones also promote digestive processes within the small intestine. Other hormones that play lesser roles in diminishing gastric acid production include glucose-dependent insulinotrophic peptide and peptide YY, the paracrine glucagon-like peptides, and the neurocrine vasoactive intestinal polypeptide.

Regulation of Gastric Motility and Gastric Emptying Peristalsis occurring in the stomach is strongest in the lower body and antral sections. The peristaltic waves pro- pel the digestive contents through the stomach as well as through most of the other portions of the digestive tract. Additionally, in the antrum, retropulsion pushes the chyme back and forth between peristaltic contractions to help grind and liquify food particles. Another means of motil- ity present in the stomach is a basic electrical rhythm that is initiated by the interstitial cells of Cajal (also referred to as pacemaker cells), found in the outer circular muscles (muscularis externa) near the myenteric plexus in the body of the stomach at the greater curvature. The pacemaker cells in the stomach generate wave-like signals (or slow- wave potentials) at a rate of about three per minute that move from the fundus toward the pyloric sphincter and help to coordinate peristalsis and other motor activity.

Gastric emptying is affected by factors both in the stomach and duodenum. In the antrum of the stomach,

The second, or gastric, phase occurs when ingested food reaches the stomach. Distension of the stomach (identified by stretch receptors in the stomach layers) along with the presence of protein and some other consumed substances, especially caffeine and alcohol, in the stomach enhance gastric secretions in this phase. The ability of proteins, primarily those that have been digested into small peptides and/or amino acids, to enhance gastric secretions occurs through multiple pathways including, for example, stimulating chemoreceptors that initiate submucosal plexus nerve activity; promoting gastrin release; and activating the parasympathetic nervous system, which further enhances vagal activity to the stomach.

The third, or intestinal, phase of gastric secretions occurs after food has left the stomach and has entered the duodenum. In this phase, a reduction in chyme volume in the stomach and a reduction in the pH of gastric juice (to 105 microbes/mL. Bacterial overgrowth in the small intestine induces deficiencies of

nutrients, such as vitamin B12 and iron, which the bacteria use for their own growth. Additionally, the bacteria may induce deficiencies of thiamin and fat-soluble vitamins. Fat-soluble vitamin deficiencies occur with bacterial deconjugation of bile that is needed for fat and fat-soluble vitamin absorption. Thiamin can be destroyed in the small intestine from thiaminases released by the bacteria.

The Colon (Large Intestine) Once through the ileocecal sphincter, materials move into the cecum, the right side of the colon, and then move sequentially through the ascending, transverse, descending, and sigmoid sections (Figure 2.18). The colon in its entirety is almost 5 feet long and is larger in diameter (about 3 inches) than the small intestine (about 1½ inches), thus explaining the terminology distinction (large versus small) between the two intestines.

Rather than being a part of the entire wall of the digestive tract, as it is in the upper digestive tract, the longitudinal muscle in the colon is gathered into three muscular bands or strips called teniae (also spelled taenia or teneae) coli that extend throughout most of the colon. The length of the teniae coli is smaller than that of the underlying circular muscles and mucosa, which causes the underlying layers to form pouches called haustra.

On initially entering the colon, the contents are still quite fluid. Contraction of the musculature of the large intestine is coordinated so as to mix the intestinal contents and to keep material in the proximal (ascending) colon a sufficient length of time for absorption of nutrients to occur. The proximal colonic mucosal cells typically absorb sodium, chloride, and water. About 90–95% of the water and sodium entering the colon each day is absorbed. Colonic absorption of sodium, which occurs by active transport and which enhances water absorption, is influenced by a number of factors, including hormones. Antidiuretic hormone (also called vasopressin) secreted from the pituitary gland, for example, decreases sodium absorption, whereas glucocorticoids like cortisol secreted

Figure 2.18 The colon. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Descending colon

Sigmoid colon

Transverse colon

Ascending colon

Ileocecal sphincter

Cecum

Appendix Rectum

Anal canal

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52 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

from the adrenal gland and mineralocorticoids such as aldosterone secreted from the adrenal gland increase sodium absorption in the colon. Further information on water and electrolyte absorption is found in Chapter 12.

Colonic Secretions and Motility and Their Regulation Secretions into the lumen of the colon are few, but present. Goblet cells secrete mucus. Mucus acts as a lubricant for fecal matter and protects the colonic mucosal cells. The mucus, present in a double layer, lies between the colonic mucosal cells and the bacteria that reside in the colon and thus help reduce the likelihood of bacterial translocation. Bicarbonate is also secreted into the lumen in exchange for chloride, which is absorbed. Bicarbonate provides an alkaline environment that helps neutralize acids produced by colonic anaerobic bacteria.

Haustral contractions, characterized as oscillating contractions of the circular muscles, provide one form of motility within the large intestine. These contractions are regulated in part by the basic electrical rhythm of the colon’s smooth muscle layer and occur at a rate of about two to six contractions per minute. Peristalsis provides minimal motility in the colon. Instead, more vigorous mass-action peristaltic-like contractions (i.e., contractions of large sections of smooth muscle within the colon) promote movement of material from one section of the colon to the next toward the rectum. Neural reflexes also affect motility. For example, the gastrocolic reflex, which occurs in response to gastrin and enteric nervous system activity, promotes contractions within the distal colon and rectum to promote defecation.

The end result of the passage of material through the colon, which usually takes about 12–72 hours, is that the unabsorbed materials are progressively dehydrated. Typically, the approximately 500 mL to 1 L of materials that enter the large intestine each day is reduced to about 150–200 g of defecated material. This fecal material is about 75% water and 25% solids. Fecal solids usually include sloughed gastrointestinal cells, digestive juice constituents, fiber, small amounts of unabsorbed fat and bile, and bacteria. The bacteria may account for about 30% of dry fecal weight.

Colonic Bacteria The trillions of microorganisms (which can weigh up to 5 lbs) that are living in the gastrointestinal tract make up our gut microbiota (or microflora). These microorgan- isms include both gram-negative and gram-positive bac- terial strains, representing over 1,000 species. Although intestinal bacterial counts in the large intestine have been reported to be as high as 1012 per gram of gastrointesti- nal tract contents, bacteria are found throughout the gas- trointestinal tract. The mouth contains mostly anaerobic

bacteria. The stomach contains few bacteria because of its low pH, but some more acid-resistant bacteria that are present include lactobacilli and streptococci. The proxi- mal small intestine contains both aerobes and facultative anaerobes. Most bacteria found in the ileum and large intestine are anaerobes, including bacteroides, lactobacilli, and clostridia. Other examples of bacteria that inhabit the large intestine are bifidobacteria, methanogens, eubacteria, and streptococci. Anaerobic species are thought to out- number aerobic species by at least 10-fold, but the exact composition of the microflora is affected by a variety of factors such as substrate availability, pH, medications, and diet, among others.

Bacteria gain nutrients for their own growth from undigested and/or unabsorbed food residues in the intestines. Enzymes synthesized by the bacteria but lacking in humans allow for the digestion of many nutrients to generate substrates for bacterial energy production and to attain, for example, carbon atoms necessary for bacterial maintenance and/or growth. Starch that has not undergone hydrolysis by pancreatic amylase, for example, may be used by gram-negative bacteroides and by gram-positive bifidobacteria or eubacteria. Mucins found in mucus secretions of the gastrointestinal tract may be broken down and used by bacteria such as bacteroides, bifidobacteria, and clostridia. Digestive enzymes themselves may even serve as substrates for bacteria such as bacteroides and clostridia. In addition, sugar alcohols such as sorbitol and xylitol; disaccharides such as lactose; and some fibers may be degraded by selected bacteria in the colon.

Many products are generated from the bacterial use of undigested and unabsorbed materials in the colon. Several B-vitamins as well as vitamin K are produced by bacteria in the colon and may be absorbed to varying degrees. Some particularly beneficial acids that are produced during carbohydrate fermentation (an anaerobic process by which bacteria break down substances, primarily carbohydrate and protein) by specific strains of bacteria include lactic acid and three short-chain fatty acids— acetic acid, butyric acid, and propionic acid. These short- chain fatty acids provide many benefits to the host, as shown in part in Figure 2.19, and more specifically listed hereafter.

● Acidify the luminal environment. The presence of short-chain fatty acids in the colon decreases the pH within the lumen of the colon. This more acidic envi- ronment has several positive effects. (1) With the more acidic pH, free bile acids become less soluble and the activity of bacterial 7 a dehydroxylase diminishes (opti- mal pH ~ 6–6.5) resulting in decreased conversion of primary bile acids to secondary (more harmful) bile acids. (2) With the lower pH, calcium, released with fiber degradation, binds to and promotes the excre- tion of bile acids (and thus prevents their conversion

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 53

to secondary bile acids). (3) The lower pH favors the growth of beneficial lactobacilli and bifidobacteria and inhibits the growth of pH-sensitive pathogenic bacte- ria. (4) The acidic environment enhances the produc- tion of mucin, which forms part of the physical barrier overlying intestinal cells. This increased mucin con- tent provides a greater physical barrier and decreases the likelihood of pathogenic bacterial colonization as well as bacterial translocation. (5) The low pH may improve the absorption of minerals released during fermentation.

● Serve as signaling molecules by interacting with recep- tors on enteroendocrine cells that mediate the synthe- sis of hormones and peptides and by effects on histone acetylation involved in gene expression.

● Exhibit trophic effects, specifically stimulating prolif- eration and growth and maintaining the integrity (pre- venting atrophy) of the colonic mucosal cells.

● Improve colonic and splanchnic blood flow. Short- chain fatty acids are thought to directly affect smooth muscle as well as to interact with the enteric nervous system. This improved blood flow enhances both the delivery of nutrients to the colon and the transport of nutrients from the colon to the liver. (Note that the term

splanchnic generally refers to organs in the abdominal cavity such as the liver, spleen, stomach, and intestines.)

● Increase water and sodium absorption in the colon. The absorption of the short-chain fatty acids in turn stimulates water and sodium absorption into the mucosal cells of the colon.

● Provide energy and serve as substrates for use within cells. Over 95% of the short-chain fatty acids are absorbed and utilized by the body. Butyric acid serves as a major energy source for colonic mucosal cells. In fact, butyric acid is thought to supply colonic cells with over two-thirds of their energy needs. Absorbed propionic acid and acetic acid are transported via the portal vein to the liver. In the liver, propionic acid is largely metabo- lized along with small amounts of acetic acid. Much of the propionic acid is converted to succinyl-CoA, which may be used by the liver for glucose or energy produc- tion. Propionic acid also may alter cholesterol metabo- lism. Most of the acetic acid passes through the liver and is used by other tissues, including skeletal and cardiac muscle and the kidneys and brain. Acetic acid may be used for the synthesis of cholesterol and fatty acids. Short-chain fatty acids also may impact glycogenolysis and play a role in insulin release and/or sensitivity.

Figure 2.19 Some benefits from the presence of bacteria in the large intestine.

Improve some nutrient

absorption

Increase bile acid excretion

Decrease secondary bile acid formation

Enhance mucosal barrier protection

Inhibit growth and adhesion of pathogens

Increase growth of health-promoting

bacterial populations

Acidify lumen of the colon

Intestinal bacteria

Fermentation of nutrients and food substances

Short-chain fatty acid production

Provide energy and serve as substrates

for body cells

Stimulate the immune system

Produce vitamins and other

modulatory factors

Promote excretion of

harmful substances

Enhance fecal bulk

Enhance host’s immune

function

Enhance production of antimicrobial

substances

Alter intestinal bacterial

populations

Exhibit trophic effects on

mucosal cells

Serve as signaling

molecules

Alter metabolic

profile

Inhibit tumor formation

Improve colonic and splanchnic

blood f low

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54 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

● May inhibit tumors. In vitro studies suggest short- chain fatty acids promote apoptosis and promote the arrest of growth and differentiation in tumor cell lines.

● Stimulate the immune system by enhancing the pro- duction of macrophages, T-helper lymphocytes, leuko- cytes, antibodies, and cytokines and improving antibody response.

As can be gleaned from this list, the short-chain fatty acids generated by bacteria in the gastrointestinal tract play several important roles. The bacteria themselves also provide direct benefits and augment some of the benefits attained from the short-chain fatty acids. Some examples of healthful actions of bacteria include the ability to:

● Enhance the host’s immune defense system by increas- ing secretory IgA production, tightening the mucosal barrier, enhancing cytokine responses, enhancing phagocytic activity, and producing antimicrobial sub- stances such as bacteriocin.

● Displace, exclude, or antagonize pathogenic bacteria from colonizing, for example, by competing for attach- ment sites on the intestinal mucosa, by strengthening the mucosal barrier to normalize intestinal permeability and to prevent pathogenic bacterial translocation, and by producing substances like biosurfactants that reduce adhesion of pathogens to the mucosa.

● Scavenge, sequester, transform, and/or promote the excretion of harmful/carcinogenic substances such as bile acids, nitrosamines, heterocyclic amines, and mutagenic compounds. Moreover, some bacteria, such as Lactobacillus acidophilus, may be able to inhibit the production of carcinogenic compounds.

● Enhance fecal bulk and dilute fecal contents to mini- mize exposure with colonic mucosal cells.

Another possible role of the microbiota is in energy metabolism and thus regulation of body weight. Data are limited at present but some studies suggest products generated by colonic microbes may exert signals that influence brain activity, including effects on appetite regulation and energy metabolism.

A less desirable result of the presence of colonic bacteria is gas production, although swallowed air also contributes to this problem. Several different gases are generated by these bacteria, including methane (CH4), hydrogen (H2), hydrogen sulfide (H2S), and carbon dioxide (CO2). One estimate suggests that colonic bacterial fermentation of about 10 g of carbohydrate can generate several liters of hydrogen gas. While much of the hydrogen and other gases that are generated can be used by other bacteria in the colon, gases that are not used are excreted.

Measurement of hydrogen gas produced by bacteria is used as a basis to diagnose lactose intolerance, a condition in which the enzyme lactase is not made in sufficient

quantities to digest the disaccharide lactose. Lactose intolerance is fairly common among adults, especially those of African American, Native American, and Asian heritage. When a person with lactose intolerance ingests the carbohydrate lactose (e.g., by drinking milk), the undigested lactose enters the colon and is fermented by colonic bacteria. These colonic bacteria, upon fermenting the lactose, produce more hydrogen gas than usual. Much of this hydrogen gas made by the bacteria is absorbed by the body and then exhaled in the breath. In fact, to diagnose lactose intolerance, a person may be asked to consume about 50 g of lactose and have their breath analyzed for hydrogen gas for the next several hours. Generally, if the person is lactose intolerant, hydrogen gas excretion in the breath increases for about 1–1½ hours after lactose is consumed. An absence of an increase in breath hydrogen gas concentrations suggests adequate lactose digestion. Symptoms of lactose intolerance include bloating, gas, and abdominal pain.

Other products are made as bacteria degrade amino acids in the colon. For example, bacterial degradation of the branched-chain amino acids generates the branched- chain fatty acids isobutyric acid and isovaleric acid. Deamination (removal of the amino group) of aromatic amino acids yields phenolic compounds. Amines such as histamine result from bacterial decarboxylation of amino acids such as histidine. Ammonia is generated by bacterial deamination of amino acids as well as by bacterial urease action on urea that has been secreted into the gastrointestinal tract from the blood. The ammonia can be absorbed by the colon and circulated to the liver, where it can be reused to synthesize urea or amino acids. About 25%, or 8 g, of the body’s urea may be handled in this fashion. This process must be controlled in people with liver disease (cirrhosis) as high amounts of ammonia in the blood are thought to contribute to the development of hepatic encephalopathy (coma). Uric acid and creatinine may also be released into the digestive tract and metabolized by colonic bacteria.

Intestinal Conditions and Probiotics Imbalances in the number and composition of gut microbiota have been linked with a number of conditions like inflammatory bowel diseases (Crohn’s disease and ulcerative colitis), colon cancer, rheumatoid arthritis, and diabetes, among others, and have prompted increased therapeutic use of probiotics (pro means “life” in Greek) and prebiotics. Probiotics are live microorganisms (i.e., active cultures of specific strains of bacteria) that when administered in ade- quate amounts confer health benefits to its hosts. Prebiotics (discussed in more detail in Chapter 4) are substances that are not digested by human digestive enzymes but confer health benefits to the host by acting as substrates for the growth and/or activity of one or more species of healthful bacteria in the colon.

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 55

The most common probiotic bacteria are lactic acid bacteria, usually strains of Lactobacillus and Bifidobacterium genera. To be considered a probiotic, the product must contain 100 million live active bacteria per gram. At present, probiotics are mostly consumed as yogurt with live cultures and as fermented or cultured milk and milk products (such as buttermilk and kefir). In the United States, yogurt is often fermented by Lactobacillus bulgaricus and Streptococcus thermophilus, and milk is usually fermented by L. acidophilus and L. casei. Other bacteria used to manufacture dairy products include Leuconostoc esntheroides, L. mesenteroides, and Lactococcus lactis. Other food sources of probiotics include miso, tempeh, and some soy beverages/products.

Consumption of probiotics has been shown to improve symptoms of irritable bowel syndrome and inflammatory bowel diseases as well as several types of diarrhea. To be effective, probiotics usually need to contain 1–10 billion colony-forming units (CFUs) per dose, with doses given once or twice daily or sometimes a few times per week. Tolerance is typically satisfactory; however, bacterial sepsis (infection) is possible, especially in those with impaired immune function (immunosuppression), intestinal tract dysfunction (characterized by increased gastrointestinal permeability or a defective barrier), or other chronic health conditions such as diabetes mellitus, cancer, abscesses, and organ transplant.

COORDINATION AND REGULATION OF THE DIGESTIVE PROCESS

The central nervous system, which comprises the brain and spinal cord, affects the body via efferent neurons. Efferent neurons to skeletal muscles make up the somatic division, and efferent neurons to the internal organs rep- resent the autonomic division of the nervous system. The autonomic division can be divided into the sympathetic and the parasympathetic nervous systems.

Neural Regulation The autonomic division communicates with the diges- tive organs directly, but it can also communicate with the digestive tract’s own (local) nervous system. Generally, the sympathetic system decreases or slows down digestive tract motility and secretions, while the parasympathetic nervous system stimulates the digestive tract, promoting motility (such as peristalsis), gastrointestinal reflexes, and the secre- tion of hormones and enzymes. The parasympathetic sys- tem interacts with the digestive tract primarily through the vagus nerve.

The digestive system’s local nervous system is known as the enteric nervous system or the intrinsic nerve plexuses and includes about 100 million neurons and their processes embedded in the layers of the gastrointestinal tract beginning in the esophagus and extending to the anus. The enteric nervous system consists of two neuronal networks or plexuses: the myenteric or Auerbach plexus and the submucosal or Meissner plexus. Sensory information is received by the enteric nervous system in part from different receptors within the gastrointestinal tract layers; these receptors monitor “local” conditions within the digestive organs. Mechanoreceptors detect distension or pressure in the gastrointestinal tract walls. Chemoreceptors monitor changes in chemical composition, and osmoreceptors detect changes in the osmolarity, such as that of chyme. Receipt of this sensory information by the enteric nervous system results in changes in the digestive tract’s smooth muscle functions (affecting motility) and/or changes to specific cells and glands (affecting the release of enzymes and hormones). Neural reflexes also may result from the stimulation of these receptors, as discussed in the next paragraph. The myenteric plexus, which lies between the circular and longitudinal smooth muscles of the digestive tract, generally controls motility, and when this plexus is stimulated, gastrointestinal activity generally increases. The submucosal plexus typically controls the release of secretions and affects local blood flow. Some of the many neurotransmitters released by the enteric nervous system are acetylcholine, 5-hydroxytryptamine (serotonin), norepinephrine, gamma aminobutyric acid (GABA), vasoactive intestinal polypeptide, and nitric oxide.

Neural reflexes also occur within the digestive tract to effect changes in secretions, blood flow, and/or motility. For example, with the ileogastric reflex, gastric motility is inhibited when the ileum becomes distended. This action allows more time for the contents of the lower small intestine, the ileum, to be emptied before more chyme is released from the stomach into the upper small intestine. With the gastroileal reflex, ileal motility is stimulated when gastric motility and secretions increase. This neural reflex promotes overall motility within the stomach and small intestine. Other reflexes also affect the intestines. For example, with the colonoileal reflex, stimulation of receptors within the colon in turn inhibits the emptying of the contents from the ileum into the colon. Such actions slow down overall motility in these organs. Similar actions occur with the intestinointestinal reflex, which diminishes intestinal motility when a segment of the intestine is overdistended.

Regulatory Peptides Factors influencing digestion and absorption are coor- dinated, in part, by a group of gastrointestinal tract mol- ecules called regulatory peptides or, more specifically,

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56 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

gastrointestinal hormones and neuropeptides. More than 100 regulatory peptides are thought to affect gastrointes- tinal functions. These peptides are released by endocrine cells within the digestive tract or its accessory organs, by enteric nerves, or both. These enteroendocrine cells, which are often identified by letters (e.g., G cells, S cells, I cells, etc.), are found throughout the digestive system. Most of the regulatory peptides released by these cells work in an endo- crine manner, being released into the blood in response to specific stimuli and traveling to region(s) of the digestive tract and/or its accessory organs to evoke changes. A few regulatory peptides, however, work in a paracrine manner, being released into the local area where they diffuse through extracellular spaces to evoke changes in target tissues.

Regulatory peptides affect a variety of digestive functions, such as gastrointestinal tract motility, cell growth, and the secretion of digestive enzymes, electrolytes, and water. Most, but not all, have multiple actions; some are strictly inhibitory or stimulatory, whereas some mediate both types of responses. Many of the functions of regulatory peptides have been addressed to varying degrees in the sections on regulation of gastric and intestinal secretions and motility. Table 2.2 summarizes some of the functions of a few of these peptides, while more detailed information is provided hereafter.

● Gastrin, secreted into the blood primarily by G cells in the antrum of the stomach and proximal small intes- tine, acts mainly in the stomach to stimulate the release

of hydrochloric acid and pepsin, and to a lesser extent to stimulate gastric motility and emptying. Gastrin also stimulates the release of histamine, which further induces gastric acid release, and has trophic actions (stimulates cell growth) on gastric and intestinal mucosa. Gastrin release is stimulated mainly by gastric distension and the presence of protein digestion products in the stomach, as well as by the release of gastrin-releasing polypeptide by the vagus nerve. Gastrin secretion is inhibited by the presence of acid in the antrum and by the release of somatostatin.

● Cholecystokinin (CCK), secreted into the blood by I cells of the proximal small intestine and by enteric nerves in the distal ileum and colon, principally stimu- lates pancreatic acinar secretory cells to release digestive enzymes into the duodenum. It also has trophic actions on the pancreas and stimulates gallbladder contraction and the relaxation of the sphincter of Oddi to facilitate the release of bile into the duodenum. Lesser roles of cholecystokinin include decreasing gastric emptying and gastric acid secretion. Cholecystokinin release is stimulated by the presence of protein digestion prod- ucts and fat in the duodenum, which is logical given the hormone’s actions on the pancreas, but its release dimin- ishes as nutrients are absorbed or moved into more dis- tal sections of the digestive tract. In neurons in the brain, cholecystokinin is thought to influence the perception of appetite, among other processes.

Hormone/Peptide Main Production Sites Selected Function(s)

Gastrin Stomach and small intestine Stimulates gastric acid secretion Stimulates pepsinogen secretion

Cholecystokinin Small intestine and enteric nerves Stimulates gallbladder contraction Stimulates sphincter of Oddi relaxation Stimulates pancreatic enzyme secretion

Secretin Small intestine Stimulates pancreas juice secretion Diminishes gastric emptying Diminishes gastric acid secretion

Motilin Stomach and intestines Stimulates gastric and intestinal motility between meals

Glucose-dependent insulinotropic peptide Small intestine Stimulates insulin secretion May diminish gastric acid secretion

Peptide YY Small and large intestines Diminishes gastric acid secretion Diminishes gastric emptying

Somatostatin Pancreas, stomach, and small intestine Diminishes gastric acid secretion Diminishes gastric emptying Diminishes pancreatic enzyme secretions Inhibits gallbladder contraction

Glucagon-like peptides Small and large intestines Stimulates insulin secretion Reduces digestive tract motility Reduces gastric secretions

Pancreatic polypeptide Pancreas Decreases gastric emptying Reduces pancreatic exocrine secretions

Table 2.2 Selected Regulatory Hormones/Peptides of the Gastrointestinal Tract, Their Main Production Site(s), and Selected Digestive Tract Functions

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C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY 57

● Secretin is secreted into the blood by S cells in the proximal small intestine in response to the presence of unneutralized acidic chyme and the products of protein digestion in the duodenum. Secretin acts primarily on pancreatic duct cells, stimulating the release of pancre- atic juice rich in bicarbonate. The presence of this bicar- bonate in the duodenum in turn neutralizes the acidic chyme and serves as feedback control. Secretin also exhibits trophic action on the pancreas and decreases gastric acid secretion and gastric emptying.

● Peptide YY (PYY), secreted into the blood by L cells of the ileum and colon, decreases appetite as well as decreases gastric acid secretion and gastric emptying. Its release is stimulated by the presence of fat in the small intestine.

● Motilin, secreted by M cells in the stomach, small intes- tine, and colon, controls the MMC, promoting gastric emptying and stimulating motility in the intestines between meals. Its release is stimulated by acetylcholine and serotonin. Acetylcholine is released by nerves. Sero- tonin is released both from nerves and from enterochro- maffin-like cells within the gastrointestinal tract.

Four paracrines affecting the digestive tract are somatostatin, histamine, glucagon-like peptides, and insulin-like growth factor-1.

● Somatostatin, synthesized by pancreatic d (D) cells as well as cells in the antrum and duodenum, inhibits gastrin release, and thus inhibits gastric acid secretion, through effects on parietal and enterochromaffin-like cells. Somatostatin also suppresses the actions of gas- trin, glucose-dependent insulinotropic peptide, secretin, vasoactive intestinal polypeptide, and motilin. Further actions include inhibition of gastric emptying, pancre- atic exocrine secretions, and gallbladder contraction. Release of the somatostatin is promoted by a drop, below about 2, in the pH of gastric juice.

● Histamine, secreted by mast cells and enterochromaf- fin-like cells in the stomach, stimulates parietal cells to secrete hydrochloric acid. Histamine release is stimu- lated by both gastrin and acetylcholine.

● Glucagon-like peptides, secreted by L cells of the dis- tal small intestine and colon and by the nervous sys- tem, primarily stimulate the pancreas to release insulin and inhibit glucagon secretion. The peptides also may decrease appetite and diminish gastric emptying, gastric secretions, and intestinal motility. Release of the pep- tides occurs with the presence of nutrients in the lumen of the small intestine.

● Insulin-like growth factor-1, also secreted by endocrine cells of the gastrointestinal tract, increases proliferation of the gastrointestinal tract. Its release is stimulated by the presence of nutrients in the digestive tract.

Of the following neurocrine peptides involved with digestive tract functions, vasoactive intestinal polypeptide has one of the larger roles. Vasoactive intestinal polypeptide (VIP) is present in gastrointestinal tract nerves and the central nervous system, and may also be present in the blood. The peptide is thought to stimulate intestinal and pancreatic secretions, relax intestinal smooth muscle including most gastrointestinal sphincters, and inhibit gastric acid secretion. Another neuropeptide called neurotensin is produced by both neurons and N cells of the small intestine (especially the ileum), but its exact physiological role in the digestive process at normal circulating concentrations is unclear. The peptide, however, is known to have multiple actions in the brain.

Two hormones exhibiting lesser direct effects on the digestive tract but impacting nutrient utilization include glucose-dependent insulinotropic peptide (previously called gastric inhibitory peptide) and amylin. Glucose- dependent insulinotropic peptide (GIP), a peptide produced by K cells of the duodenum and jejunum, primarily functions to stimulate insulin release by the pancreatic beta cells. The hormone also may inhibit gastric acid secretion. Amylin, a hormone that is cosecreted with the insulin by pancreatic beta cells, functions to inhibit glucagon secretion as well as gastric emptying. Insulin’s role in promoting glucose uptake, along with the role of another pancreatic hormone glucagon, is discussed in detail in Chapter 3.

In addition to direct effects on the digestive tract and effects on nutrient utilization, other hormones affect appetite. While a discussion of appetite regulation is beyond the scope of this chapter, information of a few appetite-regulating hormones is presented here as well as in Chapter 8. Ghrelin, a peptide secreted primarily from endocrine cells of the stomach, acts on the hypothalamus to stimulate food intake. Plasma concentrations of ghrelin typically rise before eating (e.g., a fasting situation) and decrease immediately after eating, especially carbohydrates. Two other appetite-enhancing peptides include neuropeptide Y (NPY) and agouti- related protein (AGRP). Leptin, secreted mainly by white adipose tissue in proportion to fat stores, suppresses food intake. Leptin’s activity occurs at least in part in conjunction with a-melanocyte-stimulating hormone (a-MSH), which stimulates MC4 receptors, primarily in the hypothalamus. Another hormone suppressing food intake in conjunction with leptin is corticotropin- releasing hormone (CRH).

A review of these regulatory peptides clearly shows that these various mediators of the digestive processes work in concert to stimulate and inhibit food intake as needed and to coordinate the movement of digestive tract contents and the breakdown of the nutrients within the digestive tract.

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58 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

SUMMARY

Examining the various mechanisms in the gastro-intestinal tract that allow food to be ingested, digested, and absorbed, and its residue to then be excreted reveals the complexity of the digestion and absorption processes. Normal digestion and absorption of nutrients depend not only on a healthy digestive tract but also on integration of the digestive system with the nervous, endocrine, and cir- culatory systems.

The many factors that influence digestion and absorp- tion—including dispersion and mixing of ingested food, quantity and composition of gastrointestinal secretions, enterocyte integrity, the expanse of intestinal absorptive area, and the transit time of intestinal contents—must be coordinated so that the body can be nourished without dis- rupting the homeostasis of body fluids. Much of the coor- dination required is provided by regulatory peptides, some of which are provided by the nervous system as well as by the endocrine cells of the gastrointestinal tract.

Although the basic structure of the digestive tract— which consists of the mucosa, submucosa, muscula- ris externa, and serosa—remains the same throughout, structural modifications enable various segments of the

gastrointestinal tract to perform more specific functions. Gastric glands that underlie the gastric mucosa secrete flu- ids and compounds necessary for the stomach’s digestive functions. Other particularly noteworthy features are the villi and the microvilli, which dramatically increase the sur- face area exposed to the contents of the intestinal lumen. This enlarged surface area helps maximize absorption, not only of ingested nutrients but also of endogenous secretions released into the gastrointestinal tract.

Study of the digestive system makes abundantly clear the fact that a person’s adequate nourishment, and there- fore his or her health, depends in large measure on a normally functioning gastrointestinal tract. Particularly crucial to nourishment and health is a normally func- tioning small intestine because that is where the greatest amount of digestion and absorption occurs. Later chapters of this book expand on digestion and absorption of indi- vidual nutrients.

Web Site

www.nlm.nih.gov/research/visible/visible_human.html The Visible Human Project

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59

usually in liquid form, are permitted; such restrictions make ingestion of recommended amounts of nutrients, especially protein, challenging. Protein intakes of 1.1–1.5 g/kg per ideal body weight or in total amounts ranging from about 60 to 120 g daily are recommended for bariatric surgical patients. Additionally, supplemental leucine (which has been shown to promote protein synthesis) for protein-malnourished bariatric patients also has been recommended. Postsurgical monitoring should include regularly scheduled measure- ments of muscle strength and muscle mass, which are often negatively impacted with poor protein status. Some physical symptoms suggesting protein deficiency may include brittle hair and alopecia (hair loss), generalized edema (swelling), and asthenia (weakness).

Several vitamin deficiencies occur among bariatric surgi- cal patients. Of the water-soluble vitamins, deficiencies of thiamin, vitamin B12, and folate are common. Thiamin defi- ciency occurs with excessive or recurrent vomiting (emesis), which is often present, as well as from reductions in thiamin intake and absorption (normally from the proximal small intestine). Treatment of thiamin deficiency characterized by neurologic symptoms may require parenteral administration of the vitamin. In the absence of neurologic symptoms, oral thiamin supplementation in doses of about 50–100 mg/day is usually recommended to attain or maintain thiamin status.

stomach pouch and jejunum. A large section of the stomach and the duodenum are surgically stapled and bypassed (Figure 1).

RYGB is the most common bariatric procedure performed in the United States. Yet, it is not without complications, both medical and nutritional. Macronutrient and micronutrient deficiencies occur following RYGB. Some result from poor compliance to postsurgical nutritional treatment plans, while many others occur due to RYGB-induced modifications to the digestive tract. Some of the surgically induced alterations most impacting digestion and absorption include reducing the size of the stomach, shortening the length of the small intestine in contact with nutrients, and disrupting the normal continuity of the digestive system and its accessory organs (affecting bile release and pancreatic secretions). Additionally, bacterial overgrowth in the “bypassed section” of the small intestine can promote deficiencies of some nutrients. This per- spective focuses on some of the most prevalent nutritional consequences associated with RYGB.

Of the macronutrients, protein deficiency occurs rather frequently. It typically results from inadequate protein intake, reduced gastric acid secretion (which normally facilitates pro- tein denaturation and pepsinogen activation in the stomach to facilitate protein digestion), insufficient amino acid absorp- tion (reduced absorptive surface), and extreme weight loss. For several weeks post-op, only very small amounts of foods,

Obesity is a national epidemic in the United States and one of the most prevalent health conditions worldwide, with close to 2 billion people classified as either overweight or obese. While changes in lifestyle, including diet and physical activity, and, if needed, pharmacological intervention, are the preferred treatment approaches for obesity, obese individu- als who meet selected criteria and who are at increased risk of obesity-related mortality may be candidates for bariatric surgery.

Bariatric surgical options can be classified as restrictive, malabsorptive, or both. Restrictive procedures, such as gas- tric banding and sleeve gastrectomy, reduce the size of the stomach by up to 85%, which thus limits gastric volume and food intake. The digestive tract, however, remains intact with these restrictive procedures. Malabsorptive procedures reduce nutrient absorption. Bariatric surgical procedures, such as biliopancreatic diversion with or without duodenal switch and Roux-en-Y gastric bypass (RYGB), are both restrictive and malabsorptive, reducing the size of the stomach as well as altering intestinal tract continuity. Specifically, with RYGB, the proximal and distal portions of the stomach are surgically separated and a small gastric pouch is created. A loop of the jejunum (referred to as the Roux limb) is attached to the gas- tric pouch with shorter Roux limbs resulting in greater post-op malabsorption. A biliopancreatic limb is attached to the Roux limb at a site distal to the anastomosis (attachment) of the

THE NUTRITIONAL IMPACT OF ROUX-EN-Y GASTRIC BYPASS, A SURGICAL APPROACH FOR THE TREATMENT OF OBESITY

P E R S P E C T I V E

Figure 1 The anatomy of a Roux-en-Y gastric bypass.

Esophagus

Proximal pouch of stomach

Intestinal roux limb

Duodenum

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60 C H A P T E R 2 • THE DIGESTIVE SYSTEM: MECHANISM FOR NOURISHING THE BODY

zinc deficiency include skin lesions, poor wound healing, and hair loss (alopecia). Plasma or blood cell zinc concentrations also decrease with deficiency, along with 24-hour urinary zinc excretion. Practice guidelines suggest oral supplemen- tation providing about 10–40 mg elemental zinc per day to treat deficiency; prolonged intakes in amounts higher than 40 mg per day can induce copper deficiency or impair cop- per status. Providing 1–2 mg of elemental copper with such zinc supplementation is suggested to minimize this interac- tion. However, dosages of 2–5 mg (sometimes higher) of elemental copper per day (given in divided doses) for up to 3 months may be needed to correct copper deficiency and replenish stores. In some cases, intravenous infusion of cop- per may be initially needed prior to oral supplementation. Serum copper and ceruloplasmin concentrations, which are reduced with deficiency, can be used to assess copper. Copper deficiency is also characterized by neutropenia, thrombocy- topenia, hypochromic anemia, decreased erythropoiesis, and neurologic dysfunction.

Bariatric surgery is an effective treatment for obesity and many of its comorbidities. Yet, as can be gleaned from this perspective, the RYGB procedure is not without nutritional consequences. This perspective has reviewed some of the more prevalent nutritional complications associated with RYGB. The articles at the end of this Perspective provide additional information on the complications associated with bariatric surgeries.

Suggested Readings

Dykstra MA, Switzer NJ, Sherman V, Karmali S, Birch DW. Roux-en-Y gastric bypass: How and why it fails? Surgery Curr Res. 2014; 4:165–8.

Handzlik-Orlik G, Holecki M, Orlik B, Wylezol M, Dulawa J. Nutrition management of the post-bariatric surgery patient. Nutr Clin Prac. 2014; 29:718–39.

Mohammad AE, Elrazek AA, Elbanna AEM, Bilasy SE. Medical management of patients after bariatric surgery: Principles and guidelines. World J Gastrointest Surg. 2014; 6:220–8.

Soenen S, Rayner CK, Jones KL, Morowitz M. The ageing gas- trointestinal tract. Curr Opin Clin Nutr Metab Care. 2016; 19:12–8.

Stein J, Stier C, Raab H, Weiner R. The nutritional and phar- macological consequences of obesity surgery. Aliment Pharmacol Ther. 2014; 40:582–609.

Thompson KL. Nutrition support for the critically ill, post- bariatric surgery patient. Top Clin Nutr. 2014; 29:98–112.

(75 µg) to 10,000 IU (250 µg) (but sometimes higher doses) per day for several months or until serum 25-hydroxyvitamin D concentrations exceed 30 ng/mL. Vitamin A deficiency is typically characterized by low serum retinol and vision/ ophthalmological problems. Symptomatic vitamin A defi- ciency is usually treated with oral supplements, providing 5,000–25,000 IU vitamin A per day, and may be needed for 6–12 months to correct the deficit. Short-term treatment with larger doses has also been used with severe vitamin A deficiency.

Of the minerals, calcium, iron, zinc, and copper defi- ciencies are commonly reported. Calcium is best absorbed from a slightly acidic environment in the proximal small intestine and requires adequate vitamin D status; however, these conditions do not exist following RYGB. General practice guidelines suggest up to 2 g of elemental calcium along with vitamin D supplements daily for those who have had RYGB.

Iron deficiency is one of the most well-studied and documented deficiencies in those who have had RYGB. Aforementioned reductions in acid production and rerouting of the proximal intestine represent surgical-induced changes contributing to the deficiency. Inflammation, which may be present with obesity, also diminishes intestinal iron absorp- tion. Finally, iron intake is often poor because meat (a good source of iron) is frequently not tolerated. Deficiency is usu- ally detected by evaluation of biochemical indices such as low serum ferritin, increased serum soluble transferrin recep- tors, low transferrin saturation, elevated total iron-binding capacity, low serum iron, and low mean cell volume (MCV). MCV, however, may be normal with the copresence of vitamin B12 and folate deficiencies, and ferritin concentrations may be elevated in the presence of inflammation. While treatment of deficiency often requires intravenously administered iron, oral doses (in amounts up to 300 mg) may be tried initially. Typi- cally lower doses of iron taken orally a couple of times per day are better tolerated (less side effects) than higher doses taken less frequently. Ingestion of foods rich in vitamin C along with the iron supplements is normally recommended to facilitate iron absorption.

Zinc and copper deficiencies have also been documented in bariatric surgery patients. Poor dietary intake of foods rich in these trace minerals and reductions in gastric acid contribute to the deficiencies. Additionally, both nutrients, like calcium and iron, are better absorbed from a slightly acidic environ- ment in the proximal small intestine. Classic symptoms of

Vitamin B12 deficiency results from several surgery- induced problems, especially insufficient intrinsic factor. Intrinsic factor is made by parietal cells in the stomach and binds to the vitamin in the duodenum so it can be absorbed in the ileum. However, with RYGB, secretions from gastric parietal cells are reduced, so little intrinsic factor is released. Additionally, hydrochloric acid in the stomach is needed to help release the vitamin from foods, but as with intrinsic factor, the amount of acid produced after RYGB is often not sufficient to facilitate this release. A third factor contributing to deficiency is inadequate intake. Finally, should bacterial overgrowth occur, the bacteria use the vitamin for their own growth needs and thus limit the vitamin’s availability. Because most individuals have large stores of vitamin B12, deficiency symptoms (i.e., neurological problems, cogni- tive dysfunction, and macrocytic anemia, among others) may not appear for some time. Treatment of a vitamin B12 deficiency generally requires injections of the vitamin, but because about 1–3% of vitamin B12 may be absorbed without intrinsic factor, oral ingestion of high doses of the vitamin (about 1,000–2,000 µg/day) or vitamin B12 nasal sprays can sometimes correct the deficiency.

Folate deficiency may result from inadequate dietary intake and/or from insufficient absorption of folate due to surgery-induced changes in the intestinal continuity. Oral, supplemental folate in amounts of 800–1,000 μg per day for several months is usually needed to treat the deficiency.

Fat malabsorption occurs in RYGP primarily if the com- mon channel below the biliopancreatic and Roux limb anastomosis is too short (i.e., less than about 100 cm). Fat malabsorption in turn leads to malabsorption and deficien- cies of the fat-soluble vitamins. Insufficient bile (which is no longer directed into the duodenum through the sphincter of Oddi) and the bypassing of much of the jejunum, where most fat-soluble vitamins are absorbed also contribute to the malabsorption. Vitamin D problems also occur with obesity because the greater amounts of subcutaneous fat that are present store more of the vitamin and don’t release (mobilize) the vitamin into the blood as quickly when intake is insuf- ficient. Of the fat-soluble vitamins, deficiencies of vitamins D and A are common, although physical signs of a vitamin D deficiency are not usually present. Low serum 25-hydroxyvi- tamin D concentrations, especially if coupled with high serum parathyroid hormone concentrations, suggest impaired vitamin D status. Treatment requires oral supple- ments of the vitamin in amounts ranging from about 3,000 IU

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61

T HE MAJOR SOURCE OF ENERGY FUEL in the average human diet is car-bohydrate, supplying half or more of the total caloric intake. Roughly half of dietary carbohydrate is in the form of polysaccharides such as starches and dextrins, derived largely from cereal grains and vegetables. The remaining half is supplied as simple sugars, the most abundant being sucrose, followed by lactose, maltose, glucose, and fructose.

OVERVIEW OF STRUCTURAL FEATURES

Carbohydrates are polyhydroxy aldehydes or ketones, or substances that pro- duce these compounds when hydrolyzed. They are constructed from carbon, oxygen, and hydrogen atoms that occur in a proportion that approximates that of a “hydrate of carbon,” CH O2 , accounting for the term carbohydrate. Carbo- hydrates comprise two major classes: simple carbohydrates and complex car- bohydrates. Simple carbohydrates include monosaccharides and disaccharides. Complex carbohydrates include oligosaccharides containing 3–10 saccharide units and polysaccharides containing more than 10 units (Figure 3.1).

Simple Carbohydrates

● Monosaccharides are structurally the simplest form of carbohydrate in that they cannot be reduced in size to smaller carbohydrate units by hydrolysis. Monosaccharides are called simple sugars and are sometimes referred to as monosaccharide units or residues. The most abundant monosaccharide in nature—and certainly the most important nutritionally—is the six-carbon sugar glucose.

● Disaccharides consist of two monosaccharide units joined by covalent bonds. Within this group, sucrose, consisting of one glucose and one fructose residue, is nutritionally the most significant, furnishing approx- imately one-third of total dietary carbohydrate in an average Western diet.

Complex Carbohydrates

● Oligosaccharides consist of short chains of monosaccharide units that are also joined by covalent bonds. The number of units is designated by a prefix (tri-, tetra-, penta-, and so on), followed by the word saccharide. Some oligo- saccharides known as the “flatulent sugars” are found naturally in legumes and grains, whereas other oligosaccharides called dextrins are often added

CARBOHYDRATES3

OVERVIEW OF STRUCTURAL FEATURES

SIMPLE CARBOHYDRATES Monosaccharides Disaccharides

COMPLEX CARBOHYDRATES Oligosaccharides Polysaccharides

DIGESTION Digestion of Polysaccharides Digestion of Disaccharides

ABSORPTION, TRANSPORT, AND DISTRIBUTION Intestinal Absorption of Glucose and Galactose Intestinal Absorption of Fructose Post-Absorption Facilitated Transport Glucose Transporters Glucose Entry into Interstitial Fluid Maintenance of Blood Glucose Concentration

GLYCEMIC RESPONSE TO CARBOHYDRATES Glycemic Index and Glycemic Load

INTEGRATED METABOLISM IN TISSUES Glycogenesis Glycogenolysis Glycolysis The Tricarboxylic Acid Cycle Formation of ATP The Pentose Phosphate Pathway (Hexose Monophosphate Shunt) Gluconeogenesis

REGULATION OF METABOLISM Allosteric Enzyme Modulation Covalent Regulation Genetic Regulation Directional Shifts in Reversible Reactions Metabolic Control of Glycolysis and Gluconeogenesis

SUMMARY

P E R S P E C T I V E

WHAT CARBOHYDRATES DO AMERICANS EAT?

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62 C H A P T E R 3 • CARbOHYdRAtES

systems are stereospecific. For a more extensive discussion, refer to a general biochemistry text.

Many organic substances, including carbohydrates, are optically active: If plane-polarized light is passed through a solution of the substances, the plane of light is rotated to the right (for dextrorotatory substances) or to the left (for levorotatory ones). The direction and extent of the rotation are characteristic of a particular compound and depend on the substance’s concentration and temperature and the wavelength of the light. The right or left direction of light rotation is expressed as + (dextrorotatory) or − (levorotatory), and the number of angular degrees indicates the extent of rotation.

Optical activity is attributed to the presence of one or more asymmetrical or chiral carbon atoms in the molecule. Chiral carbon atoms have four different atoms or groups covalently attached to them. Aldoses with at least three carbon atoms and ketoses with at least four carbons have a chiral carbon atom. Because different groups are attached, it is not possible to move any two atoms or groups to other positions and rotate the new structure so that it can be superimposed on the original. Instead, when two of these molecules are side by side, repositioning groups in one creates a pair of molecules that are mirror images of each other. The molecules are said to be enantiomers, a special class within a broader family of compounds called stereoisomers. Diastereomers, another type of stereoisomers, are compounds having two or more chiral carbon atoms that have the same four groups attached but are not mirror images of each other.

If an asymmetrical substance rotates the plane of polarized light a certain number of degrees to the right, its enantiomer rotates the light the same number of degrees to the left. Enantiomers exist in D or L orientation, and if

to food and beverage products to improve their texture, appearance, and nutritional value.

● Polysaccharides are long chains of monosaccharide units that may number from several into the hundreds or even thousands. The major polysaccharides of interest in nutrition are glycogen, found in certain animal tissues, and starch and cellulose, both of plant origin. All of these polysaccharides consist of only glucose units.

SIMPLE CARBOHYDRATES

Monosaccharides As monosaccharides occur in nature or arise as intermedi- ate products in digestion, they contain from three to seven carbon atoms and accordingly are termed trioses, tetroses, pentoses, hexoses, and heptoses. They cannot be further broken down with mild hydrolytic conditions, only with strong chemical oxidizing agents. In addition to hydroxyl groups, these compounds possess a functional carbonyl group, C5O, that can be either an aldehyde or a ketone. Hence, they are further designated as aldoses, sugars hav- ing an aldehyde group, and ketoses, sugars possessing a ketone group. These two classifications together with the number of carbon atoms describe a particular monosac- charide. For example, a five-carbon sugar having a ketone group is a ketopentose; a six-carbon aldehyde-possessing sugar is an aldohexose, and so forth.

Stereoisomerism: Chiral Carbons A brief discussion of stereoisomerism—the occurrence of a molecule in different spatial configurations—as it relates to carbohydrates is provided here because most biological

Figure 3.1 Classification of carbohydrates. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Monosaccharides (1 sugar unit)

Disaccharides (2 sugar units)

Glucose Fructose Galactose Lactose Sucrose

Glucose GlucoseGalactose

Glucose Fructose

Glucose Glucose

Maltose StarchDextrins Glycogen Dietary f iber

Carbohydrates

Trehalose

Glucose Galactose Fructose Glucose

Oligosaccharides (3–10 sugar units)

Polysaccharides (>10 sugar units)

Raf finose Stachyose Verbascose

Complex carbohydratesSimple carbohydrates

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C H A P T E R 3 • CARbOHYdRAtES 63

anomeric carbon, the carbon atom comprising the carbonyl function. Notice that the anomeric carbon is number 1 in the aldose (glucose) and number 2 in the ketose (fructose). This is due to the fact that the carbon bearing the most important functional group is given the lowest number possible.

Ring Structures In solution, the monosaccharides do not exist in an open- chain form. They do not undergo reactions characteris- tic of true aldehydes and ketones. Instead, the molecules form a cyclic ring structure through a reaction between the carbonyl group and a hydroxyl group. If the cyclized sugar contains an aldehyde, it is called a hemiacetal; if the sugar contains a keto group, it is called a hemiketal. This formation of the cyclic structures forms an additional chi- ral carbon. Therefore, the participating groups within a monosaccharide are the aldehyde or ketone of the ano- meric carbon atom and the alcohol group attached to the highest-numbered chiral carbon atom, as illustrated in Table 3.1 using the examples of D-glucose, D-galactose, and D-fructose. The formation of the hemiacetal or hemiketal produces a new chiral center at the anomeric carbon, des- ignated by an asterisk in the structures in Table 3.1, and therefore the bond direction of the newly formed hydroxyl becomes significant. In the Fisher projections shown, the anomeric hydroxyls are arbitrarily positioned to the right, resulting in an alpha (a) configuration. If the anomeric hydroxyl were directed to the left, the structure would be in a beta (b) configuration. Cyclization to the hemiacetal or hemiketal can produce either the a- or the b-isomer. In aqueous solution, an equilibrium mixture of the a-, b-, and open isomers exists, with the concentration of the b form roughly twice that of the a form. In essence, the a-hemiacetal can change to the open structure and again form a ring with either the a or the b configuration.

a compound is structurally D, its enantiomer is L. The D or L designation does not predict the direction of rotation of plane-polarized light, but rather is simply a structural analogy to the reference compound glyceraldehyde. Glyceraldehyde’s D and L forms are, by convention, drawn as shown in Figure 3.2. Note that in the D configuration the —OH on the chiral carbon points to the right, and in the L configuration, to the left. Remember, these forms are not superimposable.

Monosaccharides with more than three carbons have more than one chiral center. In such cases, the highest- numbered chiral carbon indicates whether the molecule is of the D or the L configuration. Monosaccharides of the D configuration are much more important nutritionally than their L isomers because D isomers exist as such in dietary carbohydrate and are metabolized specifically in that form. The reason for this specificity is that the enzymes involved in carbohydrate digestion and metabolism are stereospecific for D sugars, meaning that they react only with D sugars and are inactive toward L forms. The D and L forms of glucose and fructose are shown in Figure 3.3. Note that all of the —OH groups of the stereoisomers are flipped to the opposite side.

In Figure 3.3 the structures of glucose and fructose are shown as open-chain models, in which the carbonyl (aldehyde or ketone) functions are free. The monosaccharides generally do not exist in open-chain form, as explained later, but they are shown that way here to clarify the D-L concept and to illustrate the

Figure 3.2 Structural formulas of the D and L configurations of glyceraldehyde.

HC

HC

O

OH

H2C H2COH

D-glyceraldehyde

HC

CH

O

HO

OH

L-glyceraldehyde

Figure 3.3 Structural (open-chain) models of the D and L forms of the monosaccharides glucose and fructose.

6CH2OH

HO—3C—H

D-glucose

H—2C—OH

H—4C—OH

H—5C—OH

1CH2OH

6CH2OH

HO—3C—H

D-fructose

2C O

H—4C—OH

H—5C—OH

CH2OH

H—C—OH

L-glucose

HO—C—H

HO—C—H

HO—C—H

CH2OH

HO—C—H

HO—C—H

CH2OH

H—C—OH

L-fructose

C O

Highest-numbered chiral carbon

H1C O HC O

Carbon number Aldehyde group Ketone group

Anomeric carbon

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64 C H A P T E R 3 • CARbOHYdRAtES

1CH O

H—2C—OH

H—4C—OH α-D-glucose

HO—3CH

6CH2OH

H—5C—OH

OH

H1C

HO—3C—H O

H—2C—OH

6CH2OH

H—4C—OH

H—5C

6CH2OH

5

4 1

3 2HO OH OH

OH

O

*

*

*

6

5

4 1

3 2

O

*

1CH O

H—2C—OH

α-D-galactose HO—3C—H

HO—4C—H

6CH2OH

H—5C—OH

HO 1CH

HO—3C—H

HO—4C—H

O

H—2C—OH

6CH2OH

H—5C

6CH2OH

5

4 1

3 2

HO OH

OH

OH

O

*

*

*

6

5

4 1

3 2

O

*

1CH2OH

2C O

β-D-fructose HO—3C—H

H—4C—OH

6CH2OH

H—5C—OH

1CH2OH

HO—3CH

H—4C—OH O

HO—2C*

6CH2OH

H—5C

6CH2OH *

CH2OH OH

* Anomeric carbon.

HO

OH *

O

5

4 3

2

1

*

O

5

4 3

2

1

6

Stereoisomerism among the monosaccharides, and also among other nutrients such as amino acids and lipids, has important metabolic implications because of the stereospecificity of certain metabolic enzymes. An interesting example of stereospecificity is the action of the digestive enzyme a-amylase, which hydrolyzes polyglucose molecules such as starches, in which the glucose units are connected through an a-linkage. Cellulose is also a polymer of glucose, but one in which the monomeric glucose residues are connected by b-linkages, and it is resistant to the a-amylase hydrolysis present in the human digestive system.

Haworth Models The structures of the cyclized monosaccharides are more conveniently and accurately represented by Haworth mod- els. In such models the carbons and oxygen comprising the

five- or six-membered ring are depicted as lying in a hori- zontal plane, with the hydroxyl groups pointing down or up from the plane. Those groups directed to the right in the open-chain structure point down in the Haworth model, and those directed to the left point up. Table 3.1 shows the structural relationship of simple projection and Haworth formulas for the major naturally occurring hexoses: glu- cose, galactose, and fructose. Remember that in solution the cyclic monosaccharides open and close to form an equilibrium between the a and the b forms. Regardless of how the cyclic structure is written, the molecule exists in both forms in solution unless the anomeric carbon has formed a chemical bond and is no longer able to open and close. The different ways of drawing the structures are pre- sented here because all are used in the nutrition literature. Chemists often portray the structures to show the true

Table 3.1 Various Structural Representations among the Hexoses: Glucose, Galactose, and Fructose

Hexose Fisher Projection Cyclized Fisher Projection Haworth Simplified Haworth

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C H A P T E R 3 • CARbOHYdRAtES 65

Disaccharides Disaccharides contain two monosaccharide units attached to one another through acetal bonds. Acetal bonds, also called glycosidic bonds because they occur in the special case of carbohydrate structures, are formed between a hydroxyl group of one monosaccharide unit and a hydroxyl group of a second monosaccharide, with the elimination of one molecule of water. The glycosidic bonds generally involve the hydroxyl group on the anomeric carbon of one member of the pair of monosaccharides and the hydroxyl group on carbon 4 or 6 of the second member. Furthermore, the glycosidic bond can be a or b in orientation, depending on whether the anomeric hydroxyl group was a or b before the glycosidic bond was formed and on the specificity of the enzymatic reaction catalyzing their formation. Specific gly- cosidic bonds therefore may be designated a(1-4), b(1-4), a(1-6), and so on. Disaccharides are major energy-supply- ing nutrients in the diet. The most common disaccharides in the diet are maltose, lactose, and sucrose (Figure 3.5).

Maltose Maltose is formed primarily from the partial hydrolysis of starch and therefore is found in malt beverages such as beer and malt liquors. It consists of two glucose units linked through an a(1-4) glycosidic bond. The glucose unit on the right in Figure 3.5 is shown with the anomeric car- bon in the b position (thus called b-maltose).

Lactose Lactose is found naturally only in milk and milk products. It is composed of galactose linked by a b(1-4) glycosidic bond to glucose. The anomeric carbon of glucose is in the a position (Figure 3.5).

Sucrose Sucrose (cane sugar, beet sugar) is the most widely distrib- uted of the disaccharides and is the most commonly used natural sweetener. It is composed of glucose and fructose and is structurally unique in that its glycosidic bond involves the anomeric hydroxyl of both residues. The linkage is a with respect to the glucose residue and b with respect to the fructose residue (Figure 3.5). Because it has no free hemiac- etal or hemiketal function, sucrose is not a reducing sugar.

bond angles. The structures can be shown in a boat con- figuration or a chair configuration. Additional information can be obtained from a biochemistry textbook.

Pentoses Compared to the hexoses, pentose sugars furnish little dietary energy because relatively few are available in the diet. However, they are readily synthesized in the cell from hexose precursors and are incorporated into meta- bolically important compounds. The aldopentose ribose, for example, is a constituent of key nucleotides such as the adenosine phosphates—adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine mono- phosphate (AMP), cyclic adenosine monophosphate (cAMP), and the nicotinamide adenine dinucleotides ( 1NAD , 1NADP ). Ribose and its deoxygenated form, deoxyribose, are part of the structures of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), respectively. Ribitol, a reduction product of ribose, is a constituent of the vitamin riboflavin and of the flavin coenzymes: flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). The structural formulas of ribose, deoxyribose, and ribitol are depicted in Figure 3.4.

Amino and Acid Derivatives Amino sugars, including glucosamine and galactosamine, occur in oligosaccharides and polysaccharides such as chitin and chrondroitin. The amino sugars have an amino group replacing the 2OH on C2. Monosaccharides such as glucose can be enzymatically oxidized to glucuronic acid. Glucuronic acid is part of the glucuronic acid path- way (discussed later in this chapter) and is found in many glycoproteins, covered in Chapter 6.

Reducing Sugars Monosaccharides that are cyclized into hemiacetals or hemiketals are sometimes called reducing sugars because they are capable of reducing other substances, such as the copper ion (from 1Cu2 to 1Cu ). This property is useful in identifying which end of a polysaccharide chain has the monosaccharide unit that can open and close. This role of reducing sugars is discussed in more detail in the section on polysaccharides.

Figure 3.4 Structural formulas of the pentoses ribose and deoxyribose and of the alcohol ribitol.

HO—H2C

H H H H

OH OH

OHO

b-D-ribose

CH2OH

CH2OH

H—C—OH

H—C—OH

H—C—OH

Ribitol

5

4

3 2

1 1

HO—H2C

H H H H

H

OHO 5

4

3 2

No hydroxyl group

A reduction product of ribose

b-D-2-deoxyribose

OH

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66 C H A P T E R 3 • CARbOHYdRAtES

composed of a single type of monomeric unit, it is called a homopolysaccharide. If two or more different types of monosaccharides make up its structure, it is called a heteropolysaccharide. Both types exist in nature; how- ever, homopolysaccharides are of far greater importance in nutrition because of their abundance in many natural foods. The polyglucoses starch and glycogen, for example, are the major storage forms of carbohydrate in plant and animal tissues, respectively. Polyglucoses range in molecu- lar weight from a few thousand to 500,000.

The reducing property of a saccharide is useful in describing polysaccharide structure by enabling one end of a linear polysaccharide to be distinguished from the other. In a polyglucose chain, for example, the glucose residue at one end of the chain has a hemiacetal group because its anomeric carbon atom is not involved in acetal bonding to another glucose residue. The residue at the other end of the chain is not in hemiacetal form because it is attached by acetal bonding to the next residue in the chain. A linear polyglucose molecule therefore has a reducing end (the hemiacetal end) and a nonreducing end (at which no hemiacetal exists). This notation is useful in designating at which end of a polysaccharide certain enzymatic reactions occur.

Starch The most common digestible polysaccharide in plants is starch. Its two forms, amylose and amylopectin, are both polymers of D-glucose. The amylose molecule is a linear, unbranched chain in which the glucose residues are attached solely through a(1-4) glycosidic bonds. In water, amylose chains adopt a helical conformation, as shown in Figure 3.6a. Amylopectin, on the other hand, is a branched-chain polymer, with branch points occurring through a(1-6) bonds, as illustrated in Figure 3.6b. Both amylose and amylopectin occur in cereal grains, potatoes, legumes, and other vegetables. Amylose contributes about 15–20%, and amylopectin 80–85%, of the total starch con- tent of these foods.

Trehalose Another disaccharide, trehalose, is found naturally in fungi (mushrooms) and in other foods of the plant kingdom. Tre- halose is an a(1-1) linkage of two D-glucose molecules. It is a nonreducing sugar. Since trehalose is digested slowly, pro- vokes a low glycemic response, and possesses different physi- cal and chemical properties from other sugars, it has become an ingredient in processed foods in Japan and other countries. It has been granted Generally Recognized as Safe (GRAS) sta- tus by the U.S. Food and Drug Administration (FDA) [1,2].

COMPLEX CARBOHYDRATES

Oligosaccharides Raffinose (a trisaccharide), stachyose (a tetrasaccharide), and verbascose (a pentasaccharide) are made up of glu- cose, galactose, and fructose and are found in beans, peas, bran, and whole grains. Human digestive enzymes do not hydrolyze them, but the bacteria within the intestine can digest them, producing gases that cause flatulence. Dex- trins are a category of oligosaccharides composed entirely of glucose and used as an additive in foods, pharmaceu- ticals, and nutritional supplements. Dextrins are made from starch, which is hydrolyzed under controlled con- ditions to produce glucose chains of desired lengths. The shorter-chain dextrins (3–20 glucose units) are used most frequently for food and drug applications and will be listed on product labels as maltodextrin, corn syrup solids, or hydrolyzed corn starch. Note that commonly used dex- trins will be categorized as either oligo- or polysaccharides, depending on the specific chain length.

Polysaccharides The glycosidic bonding of monosaccharide residues may be repeated many times to form high-molecular-weight polymers called polysaccharides. If the structure is

Figure 3.5 Common disaccharides.

CH2OH

H H

H

H

HO OH

OH

O

O

β-Maltose

Glucose Glucose

Glucose Glucose

Fructose

Galactose

CH2OH

H

H

H

OH

OH

OH

O

CH2OH

H H

H

H

HO

OH

OH

O O

α-Lactose

H

OH

CH2OH

H

H

HH

OH OH

O

H

O

Sucrose

H

OH

CH2OH

H

H

OH

OH

HO

HO H2C

H CH2OH H

OH

OH

H

O

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C H A P T E R 3 • CARbOHYdRAtES 67

by mammalian digestive enzymes, it is defined as a dietary fiber and is not considered an energy source. However, colonic bacteria can digest it, resulting in several digestion products including short-chain fatty acids that provide energy to the body and play important roles in the gastro- intestinal tract. A more extensive discussion of fiber and short-chain fatty acids is presented in Chapter 4.

DIGESTION

Polysaccharides and disaccharides are the most abundant carbohydrates in the food supply, although some free glu- cose and fructose are present in honey, certain fruits and vegetables, “invert” sugar used in confections, and high- fructose corn syrup. Before dietary carbohydrates can be used by the body’s cells, they must first be absorbed from the gastrointestinal (GI) tract into the bloodstream, a pro- cess normally restricted to monosaccharides—the form of carbohydrates enterocytes can absorb. Poly-, tri-, and disaccharides therefore must be hydrolyzed. The hydro- lytic enzymes involved are collectively called glycosidases or, alternatively, carbohydrases.

Glycogen The major form of stored carbohydrate in animal tis- sues is glycogen, which is localized primarily in liver and skeletal muscle. Glycogen is similar to amylopectin, but more highly branched (Figure 3.6c). The glucose residues within glycogen serve as a readily available source of glu- cose. When dictated by the body’s energy demands, glu- cose residues are sequentially removed enzymatically from the nonreducing ends of the glycogen chains and enter energy-releasing pathways of metabolism. This process, called glycogenolysis, is discussed later in this chapter. The high degree of branching in glycogen and amylopec- tin offers a distinct metabolic advantage because it presents a large number of nonreducing ends from which glucose residues can be cleaved.

Cellulose Cellulose is the major component of cell walls in plants and, like the starches, a homopolysaccharide of glucose. It differs from the starches in that the glycosidic bonds connecting the residues are b(1-4), rendering the molecule resistant to the digestive enzyme a-amylase, which is stereospecific to favor a(1-4) linkages. Because cellulose is not digestible

Figure 3.6 Structure of starches and glycogen. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Amylose (a)

Amylopectin (b)

A branch point

α(1-6)

α(1-4)

Amylopectin consists of glucose molecules bonded together in a highly branched arrangement.

Amylose is a linear chain of glucose molecules bonded together by α(1-4) glycosidic bonds.

Amylopectin has both α(1-4) glycosidic bonds and α(1-6) glycosidic bonds. In amylopectin, α(1-6) glycosidic bonds occur at branch points.

Glycogen (c)

Glycogen is a highly branched arrangement of glucose molecules consisting of both α(1-4) glycosidic bonds and α(1-6) glycosidic bonds.

There are many more branch points in glycogen than in amylopectin.

Enzymes can hydrolyze many glucose molecules simultaneously for a quick release of glucose.

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68 C H A P T E R 3 • CARbOHYdRAtES

the enterocytes are lactase, sucrase, maltase, and treha- lase. Lactase catalyzes the cleavage of lactose to equimolar amounts of galactose and glucose. As was pointed out ear- lier, lactose has a b(1–4) linkage, and lactase is stereospe- cific for this b linkage. Lactase activity is high in infants, but in most mammals, including humans, it decreases a few years after weaning. This diminishing activity can lead to lactose malabsorption and intolerance. The frequency of lactose intolerance in human populations varies widely depending on geography, race, and ethnicity. The highest frequency is seen in Native Americans and in people of Asian, African, and Middle Eastern descent. The lowest frequency is seen in white individuals originating from northern European countries. Many lactose-free prod- ucts are available for individuals with lactose intolerance. Additionally, lactase can be added directly to regular milk products to hydrolyze the lactose.

Sucrase hydrolyzes sucrose to yield one glucose and one fructose residue. Maltase hydrolyzes maltose to yield two glucose units. Trehalase is a brush border disaccharidase that hydrolyzes the a(1-1) glycosidic bonds of trehalose to yield two molecules of glucose.

In summary, nearly all dietary starches and disaccharides ultimately are hydrolyzed completely by specific glycosidases to their constituent monosaccharide units. Monosaccharides, together with small amounts of remaining disaccharides, can then be absorbed by the intestinal mucosal cells.

ABSORPTION, TRANSPORT, AND DISTRIBUTION

The wall of the small intestine is composed of absorptive enterocytes that line projections called villi that extend into the lumen. On the surface of the lumen side, the absorptive cells have hairlike projections called micro- villi (the brush border). The anatomic advantage of the villi–microvilli structure (shown in Figures 2.9 and 2.10) is that it presents an enormous surface area to the intestinal contents, thereby facilitating absorption. The absorptive capacity of the human intestine has been estimated to amount to about 5,400 g/day for glucose and 4,800 g/day for fructose—a capacity that would never be reached in a normal diet. Digestion and absorption of carbohydrates are so efficient that nearly all monosaccharides are usually absorbed by the end of the jejunum.

Intestinal Absorption of Glucose and Galactose After carbohydrate digestion, glucose and galactose are absorbed into the enterocyte by the same mechanisms involving both active transport and facilitated transport.

Digestion of Polysaccharides The digestion of polysaccharides (starches) starts in the mouth. The key enzyme is salivary a-amylase, a glycosidase that specifically hydrolyzes a(1-4) glycosidic linkages. The b(1-4) bonds of cellulose, the b(1-4) bonds of lactose, and the a(1-6) linkages that form branch points in the starch amylopectin are resistant to this enzyme. Given the short period of time that food is in the mouth before being swal- lowed, this phase of digestion produces few mono- or disac- charides. However, the salivary amylase action continues in the stomach until the gastric acid penetrates the food bolus and lowers the pH sufficiently to inactivate the enzyme.

The starches move into the duodenum and jejunum, where they are acted upon by pancreatic a-amylase. The presence of pancreatic bicarbonate in the duodenum elevates the pH to a level favorable for enzymatic function. The a-amylase hydrolyzes a(1-4) glycosidic bonds in both amylose and amylopectin to produce oligosaccharides (also called dextrins or limit dextrins), maltose, and maltotriose (Figure 3.7). The branched oligosaccharides, trisaccharide maltotriose, and maltose are further digested by specific enzymes in the brush border. a-amylase can further break down the oligosaccharides to maltose and maltotriose. The partially hydrolyzed amylopectin is not fully digested by a-amylase; this enzyme’s action stops several residues short of the a(1-6) bonds, leaving a limit dextrin. These limit dextrins are acted on by a-dextrinase (also called isomaltase), which is attached to the brush border membrane. This enzyme contains two polypeptides with two active sites, one with specificity to a(1-4) linkages and the other with specificity to both a(1-4) and a(1-6) linkages. a-dextrinase is the only intestinal enzyme that will hydrolyze a(1-6) glycosidic bonds. Glucose is released from limit dextrins by the combined action of a-dextrinase and other brush border enzymes (Figure 3.7).

A portion of the starch of beans and certain vegetables and other resistant starches are not fully digested (see also Chapter 4). This is partially due to the accessibility of the food to the enzyme and partially related to naturally occurring amylase inhibitors in some foods. a-amylase inhibitors can be used to impede digestion of dietary starch and thus reduce nutrient absorption as a means to combat the overweight and obesity problem [3].

Digestion of Disaccharides Virtually no digestion of disaccharides or small oligo- saccharides occurs in the mouth, stomach, or lumen of the small intestine. Digestion takes place almost entirely within the microvilli (the brush border) of the upper small intestine via disaccharidase activity, and the resulting monosaccharides immediately enter the enterocytes with the facilitation of specific transporters (discussed later) (Figures 2.10 and 2.17). Among the enzymes located on

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C H A P T E R 3 • CARbOHYdRAtES 69

Figure 3.7 Starch digestion. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

A. Digestion of amylose and amylopectin in the mouth

B. There is no digestion of amylose and amylopectin in the stomach

C. Digestion of amylose and amylopectin in the small intestine

D. Digestion of amylose and amylopectin on the brush border of the small intestine

Glucose

Amylose

Dextrins

Dextrins

Amylopectin

No further digestion

No further digestion

Dextrins

Maltose

Pancreatic α-amylase

Maltose

Glucose

Maltase

Maltose

Glucose

Maltase Limit dextrins

Glucose

α-dextrinase (isomaltase)

Dextrins

Maltose, maltotriose, and limit dextrins

Pancreatic α-amylase

Salivary α-amylase

α-amylase

Amylose: Salivary glands release salivary α-amylase, which hydrolyzes α(1-4) glycosidic bonds in amylose, forming dextrins.

Amylopectin: Salivary glands release salivary α-amylase, which hydrolyzes α(1-4) glycosidic bonds in amylopectin, forming dextrins.

Amylopectin: Acidity of gastric juice destroys the enzymatic activity of salivary α-amylase. The dextrins pass unchanged into the small intestine.

Amylopectin: The pancreas releases pancreatic α-amylase, which hydrolyzes α(1-4) glycosidic bonds to produce limit dextrins, maltotriose, isomaltose, and maltose. Hydrolysis stops four residues away from the α(1-6) bond.

Amylopectin: Maltose, maltotriose, and isomaltose are further hydrolyzed in the brush border by the enzyme maltase or α-dextrinase to glucose. α-dextrinase is the sole carbohydrase capable of hydrolysing α(1-6) glycosidic bonds.

Amylose: Acidity of gastric juice destroys the enzymatic activity of α-amylase. The dextrins pass unchanged into the small intestine.

Amylose: The pancreas releases pancreatic α-amylase, which hydrolyzes α(1-4) glycosidic bonds, into the small intestine. Dextrins are broken down into maltose.

Amylose: Maltose is hydrolyzed by maltase, a brush border enzyme, forming free glucose.

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70 C H A P T E R 3 • CARbOHYdRAtES

high-capacity transporter that facilitates the exit of glucose and other hexoses from the enterocyte into the underlying capillaries for delivery into the hepatic portal vein.

1Na that has entered the cell is “pumped” back out by the energy-requiring Na1/K1-ATPase located in the basolateral membrane. Na1/K1-ATPase works by first combining with ATP in the presence of Na1 on the inner surface of the cell membrane. The enzyme then is phosphorylated by the breakdown of ATP to adenosine diphosphate (ADP) and consequently is able to move three

1Na out of the enterocyte. On the outer surface of the cell membrane, the ATPase becomes dephosphorylated by hydrolysis in the presence of K1 and then is able to return two 1K into the cell. The term pump is used because the Na and K ions are both transported across the membrane against their concentration gradients. The overall process of glucose transport into the enterocyte via SGLT1 is considered active transport because of the involvement of

1 1Na /K -ATPase at the basolateral membrane. The activity of the 1 1Na /K -ATPase is responsible for most of the active transport in the body and is the major energy demand of the body at rest.

Facilitated Transport Some glucose (and galactose) can be absorbed into the enterocyte independent of SGLT1 and thus without the input of energy. When glucose concentration in the

The relative contribution of active transport versus facili- tated transport depends on the amount of carbohydrate consumed; facilitated transport participates to a greater extent following a large carbohydrate meal.

Active Transport The active transport mechanism for glucose and galactose absorption into enterocytes requires energy as ATP and the involvement of a specific transporter protein, designated sodium-glucose transporter 1 (SGLT1) (Figure 3.8a). The SGLT1 is positioned on the intestinal lumen side of the enterocyte (in the brush border membrane) and simulta- neously transports one molecule of glucose (or galactose) and two molecules of 1Na in the same direction and is thus a symporter. A mutation in the SGLT1 gene is associated with glucose-galactose malabsorption. The SGLT1 protein has two binding sites: One binds 1Na and the other binds glucose. The glucose binding site is not available unless the transport protein has already bound 1Na . The attach- ment of 1Na to the carrier increases the transport protein’s affinity for glucose. Sodium is moving down a concentra- tion gradient because the intracellular concentration of

1Na is low. After 1Na and glucose are transported into the enterocyte, they are released from SGLT1. As the intra- cellular concentration increases, glucose binds to another transporter in the basolateral membrane, designated glu- cose transporter type 2 (GLUT2). GLUT2 is a low-affinity,

Figure 3.8 Transport of monosaccharides into enterocytes. (a) Active transport of glucose and galactose requiring ATP and Na1. (b) Facilitated transport of glucose and galactose into the enterocyte by GLUT2 when the intestinal lumen glucose levels are high; glucose and galactose may also exit the cell with assistance from GLUT2. (c) Fructose entering the enterocyte via transport facilitated by GLUT5 and leaving the cell via transport facilitated by GLUT2.

Microvillus

(a) (b) (c)

GLUT2

GLUT5

GLUT2

GLUT2

SGLT1

SGLT1 symporter simultaneously transports

glucose and sodium into the cell through

the cell membrane.

Intestinal epithelium

Sodium is moved from the epithelium cell into

the bloodstream by a Na+/K+ATPase and

K+ is moved from the blood back into the cell.

Lumen of gut

Blood

ATP

Na+

K+

ADP + P

Na+

Na+

Glucose, galactose, and fructose leave the cell facilitated by GLUT 2.

Fructose

Glucose/ galactose

Glucose/ galactose

Glucose/ galactose

Glucose/ galactose

Glucose/ galactose

Fructose

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C H A P T E R 3 • CARbOHYdRAtES 71

increases in the presence of high levels of glucose (as is present in sucrose and high-fructose corn syrup); this may relate to the presence of GLUT2 in the brush border membrane [8]. The Perspective at the end of this chapter discusses the trends in carbohydrate intake over the past several decades and the major food sources that deliver the glucose and fructose to the enterocyte for absorption.

Post-Absorption Facilitated Transport Following the intestinal absorption of glucose, galactose, and fructose, they enter the hepatic portal vein, where they are carried directly to the liver. Essentially all of the galac- tose and fructose is taken up by the liver through specific facilitated transporters and metabolized, whereas only 30–40% of glucose is taken up by the liver, with the majority passing through into the systemic circulation. This explains why glucose, but not galactose or fructose, is found in the peripheral blood and why the latter sugars are not directly subject to the strict hormonal regulation that is such an important part of glucose homeostasis. Galactose is largely converted glucose derivatives and stored as liver glycogen through pathways described later in this chapter. In con- trast, the majority of fructose enters an alternative pathway and is catabolized for energy according to the liver’s energy demand. If an occasional meal is high in fructose, and the liver’s energy needs have been met, excess fructose is con- verted to triacylglycerol and transported out of the liver for distribution to muscle and adipose tissue. However, diets chronically high in fructose can cause hyperlipidemia and triacylglycerol accumulation in the liver.

Glucose is nutritionally the most abundant mono- saccharide because it is the exclusive constituent of starch and also occurs in each of three major disaccharides (Figure 3.1). The portion of dietary glucose taken up by the liver can be used for energy, stored as glycogen, or returned to the blood during nonfed periods by pathways described in Chapter 7. The remainder of the glucose passes into the systemic blood supply and is then distributed among other tissues, such as muscle, kidneys, brain, and adipose tissue. Glucose enters the cells in these organs by facilitated transport. In skeletal muscle and adipose tissue the process is insulin dependent, whereas in the liver, kidneys, brain, erythrocytes (red blood cells), and other tissues it is insulin independent. Because of the nutritional importance of glucose, the facilitated transport process by which it enters the cells of certain organs and tissues warrants a closer look. The following section explores the process in greater detail.

Glucose Transporters Glucose is effectively used by a wide variety of cell types under normal conditions, and its concentration in the blood must be precisely controlled. Glucose plays a central

intestinal mucosa is high, such as after the ingestion of a large carbohydrate-containing meal, glucose is transported into the enterocyte by GLUT2 in the brush border mem- brane [4]. When large amounts of glucose enter the entero- cyte, intracellular GLUT2 is translocated to the brush border membrane by the movement of the cytoskeleton and the contraction of myosin. After high-carbohydrate meals, more glucose is transported into the enterocyte by facilitated transport than by active transport via SGLT1.

Rising levels of blood glucose triggers insulin secretion, causing GLUT2 to be translocated from the brush border membrane back to intracellular vesicles. While GLUT2 is not directly dependent on insulin for facilitated transport, this indirect effect of insulin results in reduced intestinal glucose absorption when blood glucose levels are high. In insulin-resistant individuals or those with type 2 diabetes, GLUT2 is resistant to the effect of insulin, and the GLUT2 remains in the brush border membrane. The result is that glucose continues to be absorbed at a higher rate [4,5]. The role of insulin in metabolic regulation is discussed in detail later in this chapter and in Chapters 7 and 8.

Intestinal Absorption of Fructose The primary mechanism for fructose transport into the enterocyte is via a specific facilitative transporter, GLUT5 (Figure 3.8c). GLUT5 has a high affinity for fructose and is not influenced by the presence of glucose [6]. Fructose absorption does not require energy (i.e., 1Na -ATP-dependent transport by SGLT1). The rate of uptake of fructose is much slower than that of both glucose and galactose but is increased when GLUT2 is present in the brush border membrane of the enterocyte, as discussed previously. As the intracellular concentration increases, fructose is trans- ported from the enterocyte into the hepatic portal vein by GLUT2 in the basolateral membrane, the same transporter that moves glucose out of the cell. The facilitative trans- port process can proceed only down a concentration gra- dient. At typical dietary intakes, there is no fructose in the systemic circulation due to efficient removal by the liver, where it is phosphorylated and trapped in the hepatocytes. Although fructose is absorbed more slowly than glucose or galactose, which are actively absorbed, it is absorbed faster than sugar alcohols such as sorbitol and xylitol, which are absorbed purely by passive diffusion.

Many individuals (about 60%) cannot completely absorb fructose when consumed in large amounts, ranging from 20 to 50 g [7]. Those with limited absorption who ingest large amounts of fructose experience intestinal pain, gas, and diarrhea, symptomatic of malabsorption. This level of intake is readily achievable for individuals who consume 25–30 ounces of a carbonated beverage sweetened with either sugar or high-fructose corn syrup. It has been observed that the threshold of fructose that can be consumed before malabsorption symptoms develop

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72 C H A P T E R 3 • CARbOHYdRAtES

In its simplest form, a transporter protein: ● has a specific combining site for the molecule being

transported ● undergoes a conformational change upon binding the

molecule, allowing the molecule to be translocated to the other side of the membrane and released

● has the ability to reverse the conformational changes without the molecule being bound to the transporter so that the process can be repeated.

This section focuses on the family of glucose transport proteins (GLUTs). Of the 14 GLUT isoforms that have been identified, only those that have been well studied and shown to have a major role in glucose metabolism are summarized in Table 3.2, though all 14 are described briefly. All cells express at least one GLUT isoform on their plasma membrane. The different isoforms have distinct tissue distributions and biochemical properties, and they contribute to the precise disposal of glucose according to varying physiological conditions. Redundancy of GLUT proteins in cells throughout the body helps to ensure the uptake and use of glucose as a critical fuel source under a variety of physiological conditions. Major characteristics of each of the GLUTs are: [9]

● GLUT1 was the first GLUT identified and the most intensely studied. As the most ubiquitously expressed GLUT, GLUT1 is responsible for the basic supply of glu- cose to erythrocytes, endothelial cells of the brain, and most fetal tissue. It supplies the glucose to the develop- ing central nervous system during embryogenesis.

● GLUT2 is a low-affinity, high-capacity transporter with predominant expression in the b-cells of the pancreas, liver, small intestine, and kidney. As discussed previ- ously, GLUT2 is involved in the transport of glucose and fructose from enterocytes into the portal blood, and when the concentration of glucose in the intestinal

role in metabolism and cellular homeostasis. Most cells in the body are dependent upon a continuous supply of glucose to supply energy in the form of ATP. The signs and symptoms associated with diabetes mellitus are a graphic example of the consequences of a disturbance in glucose homeostasis.

The cellular uptake of glucose requires that it cross the plasma membrane of the cell. The highly polar glucose molecule cannot move across the cellular membrane by simple diffusion because it cannot pass through the nonpolar matrix of the lipid bilayer. For glucose to be used by cells, an efficient transport system for moving the molecule into and out of cells is essential. In certain cells, such as epithelial cells of the small intestine and renal tubule, glucose crosses the plasma membrane against a concentration gradient by active transport, pumped by an

1 1Na /K -ATPase symport system (SGLT1), as described previously. However, not all cells require an energy- dependent transporter for glucose uptake. A family of protein carriers functions in the facilitated transport of glucose and other monosaccharides, and is called glucose transporters, abbreviated GLUT.

A total of 14 glucose transport proteins have been identified, along with the genes that code for them. The genome project has aided in this identification because, considered collectively, all transport proteins share a structure in common and have similar sequences in the genes that code for them. About 28% of the amino acid sequences are common within the family of transport proteins. Each GLUT is an integral protein, penetrating and spanning the lipid bilayer of the plasma membrane. Twelve transmembrane a-helix segments are present in each of the transporters. Figure 3.9 shows a typical transporter, which is oriented so that hydrophilic regions of the protein chain protrude into the extracellular and cytoplasmic media, while the hydrophobic regions traverse the membrane, juxtaposed with the membrane’s lipid matrix.

Figure 3.9 A model for the structural orientation of the glucose transporter.

Outside

Inside

Some helices form a hydrophobic pocket.

The loops on the extracellular and cytoplasmic sides of the membrane are primarily hydrophilic.

Components of the transmembrane channel.

The transmembrane segments consist largely of hydrophobic amino acids.

H3N +

COO2

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C H A P T E R 3 • CARbOHYdRAtES 73

spleen, brown adipose tissue, and lung. This GLUT has been studied mostly in animal models.

● GLUT9 is primarily detected in the liver and kidney, with lower levels found in the small intestine, placenta, lung, and leukocytes. The metabolic importance of GLUT9 is not fully understood. In addition to a high affinity for glucose and fructose, GLUT9’s primary func- tion appears to be the transport of uric acid.

● GLUT10 is present in the heart, lung, brain, liver, skeletal muscle, pancreas, placenta, and kidney. Its physiological role in humans is under investigation and is not fully understood at this time.

● GLUT11 has been cloned based on genomic information and is expressed in three isoforms. Its physiological role in humans has not been identified.

● GLUT12 has been identified in the skeletal muscle, heart, small intestine, and prostrate. The amount of GLUT12 found in the cellular membrane is increased by insulin (similar to GLUT4) in the normal indi- vidual, but not under conditions of obesity and type 2 diabetes that cause resistant insulin receptors. GLUT12 does not appear to be stored in storage ves- icles and does not undergo cycling between storage vesicles and the membrane like GLUT4 does. The affinity of GLUT12 for glucose is unknown at this time, but it does transfer glucose, so there is some degree of affinity.

● GLUT13 is highly expressed in certain regions of the brain and to a lower extent in adipose tissue and the kid- ney. This GLUT has no carbohydrate transport activity. It appears to transport hydrogen ions (protons; H1) and inositol. Its physiological role is not completely under- stood at this time.

● GLUT14 is similar (if not identical) to GLUT3 and pos- sibly serves as a backup for that GLUT. It is expressed in the testis.

The current knowledge of GLUTs and their physiological actions has been acquired using molecular biology techniques. The genome project identified genes that had a high degree of similarity to the GLUTs, providing a reason and the tools to look for them in various tissues. The protein molecules were then cloned. Another technique that has been used, called the knockout mouse, blocks the expression of the specific gene under study. With this technique it is possible to determine what effect the absence of the GLUT has on the animal, and hence to learn more of its function.

Role of Insulin Insulin and GLUT4 play extremely important roles in the uptake of glucose in muscle and adipose tissue, especially following a carbohydrate-rich meal. The sequence of events involving insulin and GLUT4

lumen is high, it transports glucose and fructose into the enterocyte. The rate of transport is highly dependent upon the blood glucose concentration. In the pancreas, GLUT2 appears to be the sensitive indicator of blood glucose levels and is involved in the release of insulin from the b-cells. High insulin levels cause GLUT2 to leave the plasma membrane of the enterocyte and return to storage vesicles.

● GLUT3 is a high-affinity glucose transporter with pre- dominant expression in those tissues that are highly dependent upon glucose, such as the brain and neurons. It is also expressed in cells and tissues that have a high requirement for glucose such as spermatozoa, the pla- centa, and preimplantation embryos. Some data suggest that a possible dysregulation of GLUT3 might lead to glu- cose deficits in the brain and thus to dyslexia in children.

● GLUT4 is the primary means by which insulin regu- lates the cellular uptake of glucose in muscle and adi- pose tissue. Other cells and tissues such as the liver, kidneys, erythrocytes, and brain do not express GLUT4 and therefore are not dependent upon insulin for glu- cose uptake. One of the actions of insulin is to cause the translocation of GLUT4 from GLUT4 storage vesicles (GSV; discussed in the next section).

● GLUT5 is specific for the transport of fructose and will not transport glucose. It is expressed primarily in the small intestine, but to a lesser degree in kidney, brain, skeletal muscle, and adipose tissue also.

● GLUT6 (which was formerly designated GLUT9) is expressed primarily in the brain, spleen, and peripheral leukocytes. It appears to transport hexoses only at higher concentrations.

● GLUT7 has been found in the small intestine and colon. It has a high affinity for glucose and fructose but does not bind galactose or xylose.

● GLUT8 (formerly GLUTXI) is expressed mainly in the testis with lower levels in the brain, adrenal gland, liver,

Transporter Protein Substrates Major Sites of Expression

GLUT1 Glucose, galactose, mannose, glucosamine

Erythrocytes, central nervous system, blood– brain barrier, placenta, fetal tissues in general

GLUT2 Glucose, galactose, fructose, mannose, glucosamine

Liver, b-cells of pancreas, kidney, small intestine

GLUT3 Glucose, galactose, mannose, xylose, dehydroascorbic acid

Brain (neurons), spermatozoa, placenta, preimplantation embryos

GLUT4 (insulin dependent)

Glucose, glucosamine, dehydroascorbic acid

Muscle, heart, brown and white adipocytes

GLUT5 Fructose, but not glucose Intestine, kidney, brain, skeletal muscle, adipose tissue

Table 3.2 Glucose Transporters (GLUT)

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74 C H A P T E R 3 • CARbOHYdRAtES

❸  An interaction between GSVs and the plasma mem- brane occurs, mediated by a tethering complex; this is a step called tethering.

❹  The GSVs dock with the plasma membrane in prepara- tion for fusion.

❺  The lipid bilayers of the GSVs and plasma membrane fuse.

❻  Endocytosis—the GLUT4 becomes part of the plasma membrane and is available for transporting glucose into the cell.

In the presence of insulin, GLUT4 cycles continuously through the endosomal system. In insulin-resistant states or at low insulin levels, the GLUT4 stays in the GSVs and its presence in the cell membrane is reduced. Interestingly, exercise causes similar translocation of GLUT4 from the GSVs to the cell membrane, as well as increased GLUT4 expression [10].

Glucose Entry into Interstitial Fluid The endothelial tissue of which blood vessel walls are constructed is freely permeable to glucose. Some tissues, most notably the brain, possess an additional layer of epi- thelial tissue between the blood vessel and the cells of the brain. Unlike the endothelium, epithelial layers are not readily permeable to many substrates, and the passage of metabolites, such as glucose, through them requires

are critical to normalizing blood glucose and thus preventing hyperglycemia. When blood glucose levels are elevated, insulin is released by the b-cells of the pancreas into the bloodstream, where it circulates and binds with specific insulin receptors on cell membranes. Insulin binding causes GLUT4 to translocate to the cell surface, but binding also results in other important cellular responses as depicted in Figure 3.10.

GLUT4 is an insulin-responsive transporter that is synthesized on the ribosomes of the rough endoplasmic reticulum and then transferred to the Golgi apparatus, where it is packaged into GLUT4 storage vesicles (GSVs). Binding of insulin to its receptor causes the GSV to translocate to the cell membrane. Key to the ability of insulin to bind to the receptor site on the cell membranes of skeletal muscle, cardiac muscle, or adipose tissue cells are the activation of phosphatidylinositol-3-kinase and the cascading reactions that follow. This activity is discussed more fully in Chapter 7. The net result of insulin’s effects on the cell membrane is to cause translocation of GLUT4 to the cell membrane; this process can be described simply as follows:

❶  The biosynthesis of GLUT4 and its storage in GSVs are stimulated.

❷  The GSVs are transported to the cell membrane by ele- ments of the cytoskeleton including the microtubules and actin.

Figure 3.10 Insulin signaling pathways and the translocation of GLUT4. Abbreviations: GRB2, growth factor receptor binding protein-2; IRS1, insulin receptor substrate 1; PI3K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol-3,4,5- trisphosphate; PDK1, PIP3-dependent kinase 1; PKB, protein kinase B (also called Akt). Source: Adapted from Augstin R., Life, 2010;62:315– 33, Figure 3B.

Insulin

α-chain

β-chain

P P

IRS1

PI3K

PDK1 GRB2

MAP kinase pathway

Protein synthesis Fatty acid synthesis Lipolysis Gluconeogenesis Glycogenesis

Metabolic pathways

Cell membrane

Insulin receptor

Cell proliferation Cell dif ferentiation Mitosis Apoptosis

GLUT4 remains in the storage

vesicle until insulin signals its

translocation to the plasma

membrane. The release of insulin causes GLUT4 to

move back into storage vesicles. GLUT4 storage

vesicles

GLUT4

Insulin binds to its receptor in response to rising blood glucose.

PKB/Akt

PIP3

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C H A P T E R 3 • CARbOHYdRAtES 75

Glycemic Index and Glycemic Load The glycemic index is an alternative way to classify dietary carbohydrates. It has been suggested that the glycemic index (GI) and glycemic load (GL) offer a means to exam- ine the relative risks of diets designed to prevent coronary heart disease (CHD) and obesity.

The effect that carbohydrate-containing foods have on blood glucose concentrations is called the glycemic response to the food. Some foods that are rapidly digested and absorbed (high-GI foods) cause a rapid rise in blood glucose levels that, because of the effect of insulin released in response, can subsequently lead to a rapid fall even below the fasting level. Other foods cause a slower and more extended rise with a lower peak level of glucose and insulin and a gradual fall (low-GI foods). The glycemic index concept was developed to provide a numerical value to represent the effect of a particular food on blood glucose levels. It provides a quantitative comparison between foods. The glycemic index is defined as the increase in blood glucose level above the baseline level (fasting level) during a 2-hour period following the consumption of a defined amount of carbohydrate (usually 50 g) compared with the same amount of carbohydrate in a reference food. A related quantitative measure, the glycemic load, considers both the quantity and the quality of the carbohydrate in a food. The glycemic load equals the glycemic index times the grams of carbohydrate in a typical portion of the food. A food’s GI and GL can be quite different; for example, the carbohydrate in carrots has a high GI score, but the GL for carrots is low because a half-cup serving of carrots contains only 6.13 g of carbohydrate. The higher the GL, the greater the expected elevation in blood glucose and the insulinogenic effect of the food.

Some studies of GI values have used glucose as the reference food, while others used white bread. The reference food is assigned a score of 100. In practice, the glycemic index is measured by determining the elevation of blood glucose for 2 hours following ingestion and plotting the values against time. The area under the curve for the test food is divided by the area under the curve for the reference food, and the result is multiplied by 100 (Figure 3.11). If glucose is used as the reference food and assigned a glycemic index of 100, white bread has a GI of about 71. When white bread is used as the reference, some foods will have a glycemic index of greater than 100.

There are many potential criticisms of the use of GI and GL for labeling purposes, foremost the wide variation of GI values for apparently similar foods and between laboratories. Factors that may cause this variation include the amount of carbohydrate in the meal, composition of the meal (particularly fiber, protein, and fat), previous meal composition, physical activity level of the subjects, choice of the reference food, and glucose tolerance of the subjects [11,12]. The variations observed could also

active transport or facilitated diffusion. For this reason, the epithelium is called the blood–tissue barrier of the body. Among the blood–tissue barriers studied—including those of the brain, cerebrospinal fluid, retina, testes, and placenta—GLUT1 appears to be the prime isoform for cross-barrier glucose transport, though other GLUTs appear to be involved (see Table 3.2).

Maintenance of Blood Glucose Concentration Maintenance of normal blood glucose concentration is an important homeostatic function and is a major function of the small intestine, liver, kidneys, skeletal muscle, and adipose tissue. Regulation is the net effect of the organs’ metabolic processes that remove glucose from or return glucose to the blood. These pathways, which are examined in detail in the section “Integrated Metabolism in Tissues,” are hormonally influenced, primarily by the antagonistic pancreatic hormones insulin and glucagon and to a lesser extent by the glucocorticoid hormones of the adrenal cor- tex. The rise in blood glucose following the ingestion of carbohydrate, for example, triggers the release of insulin while reducing the secretion of glucagon. Insulin is the main hormone that lowers blood glucose levels and is the primary anabolic hormone. Insulin stimulates the cellular uptake of glucose, amino acids, and lipid, which leads to their conversion to storage forms in muscle and adipose tissue. The storage form for glucose, glycogen, is synthe- sized through the process called glycogenesis. Glucagon, the primary catabolic hormone that has opposite effects of insulin, increases the breakdown of liver glycogen by a process called glycogenolysis. (Glucagon also increases the breakdown of lipid stored in adipose tissue and inhibits the synthesis of proteins, as discussed in later chapters.) Addi- tional mechanisms to increase blood glucose levels include an increase in the secretion of glucocorticoid hormones, primarily cortisol. Glucocorticoids cause increased activity of hepatic gluconeogenesis, a process of glucose synthesis described in detail in a later section of this chapter.

GLYCEMIC RESPONSE TO CARBOHYDRATES

The rate at which glucose is absorbed from the intestinal tract appears to be an important parameter in controlling the homeostasis of blood glucose, insulin release, obesity, and possibly weight loss, and has led to the concepts of glycemic index (GI) and glycemic load (GL). Persistently elevated blood glucose and insulin levels are also linked with obesity and the development of chronic diseases. The role of these factors in the development of insulin resis- tance and type 2 diabetes is covered in Chapters 7 and 8.

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76 C H A P T E R 3 • CARbOHYdRAtES

reflect real differences among samples of the same food due to factors such as its food form, ripeness, location of growth, and variety. For example, the glycemic index for a baked russet potato is 76.5 and for an instant mashed potato is 87.7 (using glucose as the reference food) [13]. Even the temperature of the food can make a difference:

Figure 3.11 Blood glucose changes following carbohydrate intake (glycemic index). Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Low-glycemic index response

0 1 2 3 4 5

Hours after eating (graph b)

Blood glucose

Normal level

90

100

110

120

130

140

80 Normal level

High-glycemic index response

0 1 2 3 4 5

Hours after eating (graph a)

Bl oo

d gl

uc os

e (m

g/ dL

)

90

100

110

120

130

140

80

Bl oo

d gl

uc os

e (m

g/ dL

)

Blood glucose

❶ The elevation in blood glucose level above the baseline following consumption of a high- glycemic index food or 50 g of glucose in a reference food (glucose or white bread). The glycemic index of the reference food is by def inition equal to 100 (graph a).

❷ The elevation of blood glucose levels above the baseline following the intake of 50 g of glucose in a low-glycemic index food (graph b).

❸ The glycemic index is calculated by dividing the area under the curve for the test food by the area under the curve for the reference food and multiplying the result by 100.

Calculation of Glycemic Index

Glycemic Index

Food Tested 5White Bread 100 5Glucose 100

White bread1 100 71

Baked russet potato1 107.7 76.5

Instant mashed potatoes1 123.5 87.7

Boiled red potato (hot)1 125.9 89.4

Boiled red potato (cold)1 79.2 56.2

Bran muffin2 85 60

Coca Cola2 90 63

Apple juice, unsweetened2 57 40

Tomato juice2 54 38

Bagel2 103 72

Whole-meal rye bread2 89 62

Rye-kernel bread2 (pumpernickel) 58 41

Whole-wheat bread2 74 52

All-Bran cereal2 54 38

Cheerios2 106 74

Corn Flakes2 116 81

Raisin Bran2 87 61

Sweet corn2 86 60

Couscous2 81 61

Rice2 73 51

Brown rice2 72 50

Ice cream2 89 62

Soy milk2 63 44

Raw apple2 57 40

Banana2 73 51

Orange2 69 48

Raw pineapple2 94 66

Baked beans2 57 40

Dried beans2 52 36

Kidney beans2 33 23

Lentils2 40 28

Spaghetti, durum wheat (boiled)2 91 64

Spaghetti, whole meal (boiled)2 32 46

Sucrose2 83 58

1 Source for data: Fernandes G, Velangi A, Wolever TM. Glycemic index of potatoes commonly consumed in North America. J Am Diet Assoc. 2005; 105:557–62. 2 Source for data: Foster-Powell K, Holt SH, Brand-Miller JC. International table of glycemic index and glycemic load values: 2002. Am J ClinNutr. 2002; 76:5-76.

Table 3.3 Glycemic Index of Common Foods with White Bread and Glucose Used as the Reference Food

A boiled red potato eaten hot (with the starch gelatinized) has a glycemic index of 89.4, but the same potato eaten cooler (with the starch back to a crystalline structure) has a glycemic index of 56.2 (Table 3.3).

Glycemic index and glycemic load have proven useful in evaluating the risk of developing chronic disease and obesity. Long-term consumption of a diet with a relatively high GL is associated with an increased risk of obesity,

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C H A P T E R 3 • CARbOHYdRAtES 77

of pyruvic acid, lactate instead of lactic acid). Because of the central role of glucose in carbohydrate nutrition, its metabolic fate is featured here. The entry of fructose and galactose into the metabolic pathways is introduced later in the discussion.

Glycogenesis The term glycogenesis refers to the pathway by which glucose ultimately is converted into its storage form glycogen—a process vital to ensuring a reserve of quick energy. The major sites of glycogen synthesis and storage are the liver and skeletal muscle, while a small amount of glycogen is found in the kidneys and heart, among other tissues. Glycogen accounts for as much as 7% of the weight of the liver, particularly following a high-carbohydrate meal. Liver glycogen can be broken down to glucose and reenter the bloodstream. Therefore, it plays an impor- tant role in maintaining blood glucose homeostasis. The other major site of glycogen storage is skeletal muscle. In human skeletal muscle, glycogen generally accounts for a little less than 1% of the weight of the tissue. Although the concentration of glycogen in the liver is greater, muscle stores account for most of the body’s glycogen because the muscle makes up a much greater portion of the body’s weight. The liver can store approximately 100 g of glyco- gen, whereas muscle can store about 500 g. The glycogen stores in muscle are an energy source within that mus- cle fiber and cannot directly contribute to blood glucose levels. (Muscle lacks the enzyme that converts the phos- phorylated glucose back to free glucose.) We discuss how metabolic products of glucose can return to the liver and be converted to glucose.

The initial part of the glycogenic pathway is illustrated in Figure 3.13. Glucose is first phosphorylated upon entering the cell, producing glucose-6-phosphate. In muscle and other nonhepatic cells, the enzyme catalyzing this phosphate transfer from ATP is hexokinase, a mixture of hexokinase isozymes type 1 and 2. The properties of

type 2 diabetes, and cardiovascular diseases [14–16]. The literature suggests that the longer and higher the elevation of blood glucose and insulin, the greater the risk of developing chronic diseases and obesity.

Many published tables provide the glycemic index for different foods. The most complete is an international table [17]. Selected examples from this publication have been reproduced in Table 3.3 along with the glycemic index of potatoes. Remember that the food products differ in different regions of the world. The glycemic indices listed in Table 3.3 are intended to be used to show trends, not to prepare diets.

INTEGRATED METABOLISM IN TISSUES

The metabolic fate of the monosaccharides, especially glu- cose, depends to a great extent on the body’s energy needs. This section covers the individual pathways of carbohy- drate metabolism. The following section addresses the ways metabolism is regulated, including covalent modifi- cations, allosteric mechanisms, substrate-level regulation, induction, post-translational modification, and translo- cation. Several terms used in carbohydrate metabolism sound and appear to be similar but are in fact quite differ- ent. The metabolic pathways of carbohydrate metabolism are listed below:

● Glycogenesis: The synthesis of glycogen ● Glycogenolysis: The breakdown of glycogen ● Glycolysis: The oxidation of glucose to pyruvate ● Gluconeogenesis: The synthesis of glucose from non-

carbohydrate sources ● Pentose phosphate pathway (hexose monophosphate

shunt): The production of five-carbon monosaccharides (pentoses) and nicotinamide adenine dinucleotide phos- phate (NADPH)

● Tricarboxylic acid (TCA) cycle: The oxidation of acetyl-CoA to yield CO2 and high-energy electrons.

An integrated overview of these pathways is given in Figure 3.12. The metabolism of glycogen is covered first, followed by the energy-producing pathways (glycolysis and the TCA cycle). A detailed review of the pathways’ intermediary metabolites and sites of regulation is provided in the sections that follow. The detailed pathways with the names of the chemicals and their structures are shown in the later figures. These are followed with a discussion of the individual reactions and additional comments that are particularly significant from a nutritional standpoint. It is important to recognize that under physiological conditions, many of these molecules exist as conjugated bases and are named accordingly (e.g., pyruvate instead

Figure 3.12 Integrated overview of carbohydrate metabolic pathways.

Galactose

Galactose

Glycogenesis

Glycogenolysis

Glycogen

Lactate

Fructose

Glycolysis

Gluconeogenesis

PyruvateGlucose

Pentose phosphate pathway (hexose monophosphate shunt)

Noncarbohydrate sources

TCA cycle

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78 C H A P T E R 3 • CARbOHYdRAtES

Figure 3.13 (a) Reactions of glycogenesis, by which the formation of glycogen from glucose occurs. (b) The primer function of glycogenin. The glucosyl transferase activity of glycogenin catalyzes the attachment to itself of from two to seven glucose residues transferred from UDP-glucose. The letter n represents an unspecified number of UDP-glucose molecules.

Cell membrane

a-D-glucose

O

Glucose-6-P

Phosphoglucomutase

Gluconeogenic precursors

Glycogenin primer

Glycogen (unbranched)

Glycogen (branched) (a)

O O P

Glucose-1-P

O

O

O P P U

ATP

UTP

UDP-glucose

PPi

ADP

Glucosyl transferase (glycogenin)

nUDP-Glu

Glycogenin Glu Glu Glu Glu

(b)

nUDP

Glycogen synthase and

branching enzyme

Glycogen is formed from gluconeogenic precursors in addition to blood glucose.

Uridine triphosphate (UTP) reacts with G-1-P to form an activated compound.

The dephosphorylated form of glycogen synthase is more active than the phosphorylated form. Insulin facilitates dephosphorylation. This is the primary target of insulin’s stimulatory effect on glycogenesis.

Hexokinase in muscle. Glucokinase in liver.

O P

Hexokinase (Types 1 and 2) Glucokinase (Hexokinase Type 4)

Located in muscle, brain, and adipose tissue

Located in liver and pancreas

Allosterically inhibited by glucose-6-P (its product)

Not inhibited by glucose-6-P

Low Km; function at maximum velocity at fasting blood glucose concentrations

High Km; functions at maximum velocity only when glucose levels are high (such as following a high-carbohydrate meal)

Not induced by insulin in normal individuals

Induced by insulin in normal individuals

Not induced by insulin in insulin- resistant individuals

Not induced by insulin in insulin-resistant individuals

Table 3.4 Properties of Hexokinase and Glucokinasethis enzyme are shown in Table 3.4. Muscle hexokinase is an allosteric enzyme that is negatively modulated by the product of the reaction, glucose-6-phosphate. This means that when the muscle cell has adequate glucose- 6-phosphate, the entry of additional glucose into the cell is slowed. Muscle hexokinase has a low Km, which means it can function at maximum velocity when blood glucose levels are at normal (fasting) levels.

Glucose phosphorylation in the liver is catalyzed primarily by a hexokinase isozyme called glucokinase (sometimes called hexokinase 4). Although the reaction product, glucose-6-phosphate, is the same as in other tissues, interesting differences distinguish glucokinase from hexokinase (Table 3.4). For example, muscle hexokinase is allosterically inhibited by glucose-6-phosphate, whereas liver glucokinase is not. This characteristic allows excess glucose entering the liver cell to be phosphorylated quickly and encourages glucose entry when blood glucose levels are elevated. Also, glucokinase has a much higher Km than hexokinase, meaning that it can convert glucose to

its phosphate form at a higher velocity should the blood concentration of glucose rise significantly, particularly after a carbohydrate-rich meal.

Glycogenesis is initiated by the presence of glucose- 6-phosphate. The phosphorylation of glucose as it enters the liver cell keeps the level of free glucose low, which

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C H A P T E R 3 • CARbOHYdRAtES 79

of glycogen synthase. The glycogen synthase reaction is the primary target of insulin’s stimulatory effect on glycogenesis.

When six or seven glucose molecules are added to the glycogen chain, the branching enzyme transfers them to a C—6—OH group (Figure 3.14). Glycogen synthase cannot form the a(1-6) bonds of the branch points. This action is left to the amylo(1-4 →1-6)-transglycosylase or branching enzyme, which transfers a seven-residue oligosaccharide segment from the end of the main glycogen chain to carbon number 6 hydroxyl groups. Branching within the glycogen molecule is important because it increases the molecule’s solubility and compactness. Branching also

enhances the entry of glucose into the liver cell due to the concentration gradient between the blood and the liver cell interior. Therefore, the liver has the capacity to reduce blood glucose concentration when it becomes high. Remember, the liver is not dependent upon insulin for glucose transport into the cell, but glucokinase is inducible by insulin. Insulin blood levels are increased by elevated blood glucose levels. Glucokinase activity is below normal in people with type 1 diabetes mellitus because they have very low insulin levels, and the glucokinase is therefore not induced. In type 2 diabetes the glucokinase is not induced by the insulin-resistant membrane receptors. In either case, the low glucokinase activity contributes to the liver cell’s inability to rapidly take up and metabolize glucose, which results even though GLUT2 of the liver is not regulated by insulin.

Newly synthesized glucose via gluconeogenesis provides another source of glucose-6-phosphate that can be used for glycogen synthesis in the liver, even when there is an abundance of glucose following a carbohydrate-rich meal. As discussed in detail later in this chapter, gluconeogenesis produces glucose-6-phosphate from noncarbohydrate sources including lactate, a by-product of glycolysis in red blood cells and muscle. While it may seem paradoxical for both gluconeogenesis and glycogenesis to function simultaneously, gluconeogenesis provides about one-third of the glucose-6-phosphate used for glycogen synthesis in the liver.

The next step in glycogenesis is the transfer of phosphate from carbon 6 of the glucose molecule to carbon 1 in a reaction catalyzed by the enzyme phosphoglucomutase (Figure 3.13). Nucleoside triphosphates other than ATP sometimes function as activating substances in intermediary metabolism. In the next reaction of glycogenesis, energy derived from the hydrolysis of the a-b-phosphate anhydride bond of uridine triphosphate (UTP to UMP) allows the resulting uridine monophosphate to be coupled to the glucose-1-phosphate to form uridine diphosphate-glucose (UDP-glucose). Glucose is incorporated into glycogen as UDP-glucose. The reaction is catalyzed by glycogen synthase and requires some preformed glycogen as a primer, to which the incoming glucose units can be attached. The initial glycogen is formed by binding a glucose residue to a tyrosine residue of a protein called glycogenin. In this case, glycogenin acts as the primer. Additional glucose residues are attached by glycogen synthase to form chains of up to eight units. The role of glycogenin in glycogenesis has been reviewed [18]. In muscle the protein remains in the core of the glycogen molecule, but in the liver more glycogen molecules than glycogenin molecules are present, so the glycogen must separate from the protein. Glycogen synthase exists in an active (dephosphorylated) form and a less active (phosphorylated) form. Insulin facilitates glycogen synthesis by stimulating the dephosphorylation

Figure 3.14 Formation of glycogen branches by the branching enzyme.

(1-4)-terminal chains of glycogen

O

H O

O

O

O

O

O

O

O

O

O O

O O

O O

O

O O

O O

O O

O O

O O

O

O

O

O

O

O

OO

O

O

O

O

O O O

O

O

O

O

O

O O

O

O O

O

O

O

O

O

O

O O

O

O O

O

O

Branching enzyme cuts here…

…and transfers a seven-residue terminal segment to a C–6–OH group

O H

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O O

O O

O

O

O

O

O O O

O

O

O

O

O

O

O O O

O

O O

O

O

O

H O

O

O O

O

O

O

O

O

O

O

O

O

O

Seven glucose residues

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80 C H A P T E R 3 • CARbOHYdRAtES

Figure 3.15 The reactions of glycogenolysis, by which glucose residues are sequentially removed from the nonreducing ends of glycogen segments.

makes available many nonreducing ends of chains from which glucose residues can be cleaved rapidly and used for energy, in the process known as glycogenolysis and described in the following section. The overall pathway of glycogenesis, like most synthetic pathways, consumes energy because an ATP and a UTP are consumed for each molecule of glucose introduced.

Glycogenolysis The potential energy of glycogen is contained within the glucose residues that make up its structure. In accordance with the body’s energy demands, the residues can be sys- tematically cleaved one at a time from the nonreducing ends of the glycogen branches and routed through energy- releasing pathways. The breakdown of glycogen into indi- vidual glucose units, in the form of glucose-1-phosphate, is called glycogenolysis and is catalyzed by the enzyme gly- cogen phosphorylase. The steps involved in glycogenolysis are shown in Figure 3.15.

Although glycogen phosphorylase cleaves a(1-4) glycosidic bonds, it cannot hydrolyze a(1-6) bonds. Phosphorylase acts repetitively along linear portions of the glycogen molecule until it reaches a point four glucose residues away from an a(1-6) branch point. Here the degradation process stops, resuming only after an enzyme called the debranching enzyme cleaves the a(1-6) bond at the branch point.

At times of heightened glycogenolytic activity, the formation of increased amounts of glucose-1-phosphate shifts the phosphoglucomutase reaction toward production

of the 6-phosphate isomer. In the liver (and kidneys), glucose-6-phosphate can enter into the oxidative pathway for glucose (glycolysis) or become free glucose. The conversion of glucose-6-phosphate to free glucose requires the action of glucose-6-phosphatase. This enzyme is not expressed in muscle cells or adipocytes. Therefore, free glucose can be formed only from liver or kidney glycogen and transported through the bloodstream to other tissues for oxidation.

Like its counterpart glycogenesis, glycogenolysis is highly regulated. Its catalyzing enzyme, phosphorylase, is regulated by both covalent and allosteric mechanisms. The regulation is different for the phosphorylation isozymes in muscle than in liver. The muscle and liver isozymes fulfill different physiological purposes: In muscle, the glucose is released from glycogen to provide glucose for energy within the cell, whereas in the liver the glucose is released to provide blood glucose. As phosphorylase is activated for glycogen phosphorylation, glycogen synthase is inhibited.

Glycogenolysis Regulation Covalent Regulation Covalent regulation of phosphorylase is enhanced by glucagon and the catecholamines, epineph- rine and norepinephrine. These hormones cause a covalent modification of phosphorylase by converting it to an active form through the second messenger cAMP, which regu- lates the phosphorylation site of the enzymes involved, as discussed in Chapter 1. These hormones bind to a receptor on the cell membrane that causes adenyl cyclase to be acti- vated to produce cAMP. The cAMP causes inactive phos- phorylase kinase to become active by phosphorylating it. The active phosphorylase kinase plus ATP converts inactive

Nonreducing end of glycogen chain

Phosphoglucomutase

OH

CH2OH

OH

O

OHO

OH

HPO4

CH2OH

OH

O

O

Glucose-1-P Residual glycogen

chain

OH

1

CH2OH

OH

O

O PHO

Glucose-6-P

OH

CH2O P

OH

O

HO OH

Glucose

Glycogen phosphorylase

OH

CH2OH

OH

O

HO OH

HO

OH

CH2OH

OH

O

O

Pi

Liver and kidney

Muscle and liver

Glycolysis

Phosphorylated bond

If G-6-P levels are elevated

22

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C H A P T E R 3 • CARbOHYdRAtES 81

(nonphosphorylated) phosphorylase b to active (phosphor- ylated) phosphorylase a. The phosphorylated phosphory- lase is less sensitive to the allosteric activation discussed later in this chapter. Phosphorylase a can be converted back to the inactive form, phosphorylase b, by phosphoprotein phosphatase 1 (PP-1). A Nobel Prize was awarded for elu- cidating this pathway (Figure 3.16).

Allosteric Activation The allosteric activation of phosphor- ylase b is carried out by AMP to convert it to the active phosphorylase a. When energy levels are low, cellular ATP has been hydrolyzed to AMP, more energy is needed, and the phosphorylase a releases glucose-1-phosphate. The AMP binds to an allosteric site on phosphorylase b, which increases the binding of the glycogen (see Figure 1.10). This allosteric site can also bind ATP, which is an allosteric inhibitor of the enzyme. Glucose-6-phosphate and caffeine are also allosteric inhibitors of the enzyme.

Muscle Phosphorylase The muscle and liver phosphorylase are isozymes. The muscle enzyme releases glucose-1-phos- phate, which can be converted to glucose-6-phosphate that enters into the glycolysis pathway to provide energy for the cell. Muscle phosphorylase is more sensitive to intracellular ligands such as AMP for activation. The muscle enzyme is inhibited by metabolites, ATP, glucose-6-phosphate, and glu- cose. During times of stress the hormones epinephrine and norepinephrine stimulate cAMP synthesis and along with PP-1 covalently modify phosphorylase to the active form. Nervous stimulation and Ca2+ ions have the same effect.

Liver Phosphorylase Liver phosphorylase is less sensitive to intracellular ligands. It shows a weak increase in activity in the presence of AMP (10–20%) and is insensitive to inhi- bition by ATP or glucose-6-phosphate. Liver phosphorylase is regulated by hormonal controls such as glucagon.

Glycolysis Glycolysis is the pathway by which glucose is degraded into two 3-carbon units, pyruvate. From pyruvate, the metabolic course depends largely on the availability of reducing units in the cytosol, which is dependent upon the availability of oxygen within the cell. Glycolysis can func- tion under either aerobic or anaerobic conditions. Under anaerobic conditions or in a situation without sufficient reducing equivalents due to either the lack of oxygen or high cellular metabolism, pyruvate is converted to lactate. Under otherwise normal conditions, the conversion to lactate occurs mainly in times of strenuous exercise when the demand for oxygen by the working muscles exceeds that which is available. Lactate produced under anaerobic conditions can also diffuse from the muscle to the blood- stream and be carried to the liver for conversion to glu- cose. Under these anaerobic conditions, glycolysis releases a small amount of usable energy that can help sustain the muscles even in a state of oxygen debt. Providing this energy is the major function of the anaerobic pathway of glucose to lactate. Anaerobic glycolysis is the sole source of energy for erythrocytes because these cells do not contain mitochondria. Both the brain and gastrointestinal tract also produce much of their energy from glycolysis.

Under aerobic conditions, pyruvate can be transported into the mitochondria and participate in the TCA cycle, in which it becomes completely oxidized to CO2 and H2O. Complete oxidation is accompanied by the release of relatively large amounts of energy, much of which is captured in ATP molecules by the mechanism of oxidative phosphorylation. The glycolytic enzymes function within the cytosol of the cell, but the enzymes catalyzing the TCA cycle reactions are located within the mitochondrion. Therefore, pyruvate must enter the mitochondrion for complete oxidation. Glycolysis followed by TCA cycle activity (aerobic catabolism of glucose) demands an ample

Figure 3.16 An overview of the regulation of glycogen phosphorylase. It is positively regulated covalently by cAMP and positively allosterically regulated by AMP. It is negatively regulated by ATP and glucose-6-P, which cause shifts in the equilibrium between the inactive and active (“b”) forms.

Phosphorylase b (active)

(allosteric regulation)

Pi

Pi

ATP

cAMP

Phosphorylase b (inactive)

(covalent regulation)Phosphoprotein phosphatase (PP-1)

Phosphorylase b kinase

Stimulated by hormones glucagon and epinephrine and cAMP, the second messenger

Allosterically regulated positively by AMP and negatively by ATP and G-6-P

Phosphorylase a (active)

Glycogen Glucose-1-phosphate

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82 C H A P T E R 3 • CARbOHYdRAtES

modulated (by allosteric mechanisms) by ATP and citrate (a product of the TCA cycle and an indication that energy needs are met). The inhibition by ATP is reversed by AMP, an indication that the cell needs more energy. There is a relationship among the levels of ATP, ADP, and AMP. They are interconverted by the reaction:

ADP ADP ATP AMP↔1 1

This reaction is catalyzed by adenylate kinase. When the reaction reaches equilibrium the quantity of ADP is about 10% of that of ATP, and AMP levels are less than 1% of those of ATP. Small changes in ATP are amplified in changes in AMP. The regulation of phosphofructokinase reactions is modulated by the relative amounts of ATP and AMP.

Phosphofructokinase is also regulated by fructose- 2,6-bisphosphate, which is a potent allosteric activator that increases the affinity of the enzyme for its substrate, fructose-6-phosphate. Levels of fructose-2,6-bisphosphate are controlled by the enzyme phosphofructokinase-2. This enzyme is induced by glucagon and is different from the phosphofructokinase in the glycolytic pathway. Other activities of this enzyme are discussed in the gluconeogenesis section of this chapter.

❹  Fructose bisphosphate aldolase (or simply aldolase) cleaves fructose-1,6-bisphosphate, a hexose, into two triose phosphates, glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHAP). The remain- ing steps in glycolysis involve three-carbon units rather than six-carbon units.

❺  The isomers G-3-P and DHAP are interconverted by the enzyme triose phosphate isomerase. In an isolated system, the equilibrium favors DHAP formation. In the cellular environment, however, it is shifted com- pletely toward producing G-3-P because this metabo- lite is continuously removed from the equilibrium by the subsequent reaction ❻.

❻  In this reaction, G-3-P is oxidized to a carboxylic acid, while inorganic phosphate is incorporated as a carbox- ylic phosphoric anhydride bond (a high-energy com- pound). The enzyme is glyceraldehyde-3-phosphate dehydrogenase, which uses 1NAD as its hydrogen- accepting cosubstrate. Under aerobic conditions, the NADH formed is reoxidized to 1NAD by O2 through the electron transport chain in the mitochondria, as explained in the next section. The reason why O2 is not necessary to sustain the reaction of converting G-3-P to 1,3-bisphosphoglycerate is that under anaerobic condi- tions the 1NAD consumed is restored by a subsequent reaction converting pyruvate to lactate (see reaction ⓫).

❼  This reaction, catalyzed by phosphoglycerate kinase, exemplifies a substrate-level phosphorylation of ADP. A more detailed review of substrate-level phosphorylation, by which ATP is formed from ADP by the transfer of a phosphate from a high-energy donor molecule, is covered

supply of oxygen, a condition that generally is met in normal, resting mammalian cells. In a normal, aerobic situation, complete oxidation of pyruvate generally occurs, with only a small amount of lactate being formed. The primary importance of glycolysis in energy metabolism, therefore, is in providing the initial sequence of reactions (to pyruvate) necessary for the complete oxidation of glucose by the TCA cycle, which supplies relatively large quantities of ATP.

Nearly all cells conduct glycolysis, but most of the energy derived from dietary carbohydrates is stored and/ or utilized by the liver, muscle, and adipose tissue, which together constitute a major portion of total body mass. The brain is an extravagant consumer of carbohydrate energy, but lacks the ability to store it. In cells that lack mitochondria, such as erythrocytes, the pathway of glycolysis is the sole provider of ATP by the mechanism of substrate-level phosphorylation of ADP, discussed later in this chapter. The pathway of glycolysis, under both aerobic and anaerobic conditions, is summarized in Figure 3.17. While the figure illustrates how each monosaccharide enters the glycolytic pathway, including glucose from glycogen breakdown, each possibility may not be present in every cell. Following are comments on selected reactions (the numbers correspond to the numbers in Figure 3.17).

❶  The hexokinase/glucokinase reaction consumes 1 mol ATP/mol glucose. The properties of these enzymes were covered in Table 3.4. Glucokinase is present in the liver and pancreas. Hexokinase is located in muscle, adipose tissue, and the brain. As discussed earlier, the hexokinase in muscle has a low Km, which means it can function at maximum velocity at normal blood glucose levels. When the muscle cell accumulates glucose-6-phosphate, the hexokinase is inhibited. Liver glucokinase, in contrast, has a high Km, which means it requires a high concentration of glucose in blood to function at maxi- mum velocity. The liver does not remove large quantities of glucose from blood unless blood glucose is elevated. Glucokinase in the liver is induced by insulin. Phos- phorylating glucose serves to “prime” the glycolytic pathway by trapping glucose in the cell and energizing the molecule for subsequent reactions.

❷  Phosphoglucose isomerase (also called glucose phosphate isomerase) catalyzes movement of the carbonyl group from the first carbon (glucose) to the second carbon (fructose). This is an interconversion of isomers—glucose- 6-phosphate to fructose-6-phosphate—and is reversible.

❸  Phosphofructokinase catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate using an ATP. The term bis means that the two phos- phates are on different carbons. The phosphofructo- kinase reaction is an important regulatory site. This irreversible step commits the cell to metabolize glu- cose rather than converting it to another sugar or stor- ing it as glycogen. Phosphofructokinase is negatively

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C H A P T E R 3 • CARbOHYdRAtES 83

Figure 3.17 Glycolysis, indicating the mode of entry of glucose, fructose, glycogen, and galactose into glycolysis. Hydroxyl groups on ring structures are indicated by a line pointing above or below the ring.

Cell membrane Galactose

Galactose-1-P

Glucose

ATP ADP

Glucose-6-P

Fructose-6-P

Glucose-1-P

Pentose phosphate pathway

Glycogen

Fructose

⓲O O P

CH

O

O P

Mitochondrial oxidation

(aerobic)

(anaerobic)

OOP

Fructose 1,6 bis P OOP PO

ATP

ADP

ATP

ADP

ATP ADP

Fructose-1-P

DHAP G-3-P

CH2

CH2 OH

PO

OC

OC

COO–

CH3

COO–

CH2

C O P

COO–

CH3

CH OH

1,3 bis P glycerate

3- P glycerate

2- P glycerate

PEP

Pyruvate Lactate

~

COO–

CH2

CH OH

CH2

CH OH

C O

O

P~

PO

PO

COO–

CH2

CH

OH

PO

OHCH

CH2 PO

Phosphorylation consumes 1 mol ATP/mol glucose

Isomerization catalyzed by phosphoglucose isomerase

Irreversible, highly regulated (allosteric) reaction; controls the rate of glycolysis

Cleavage of a hexose into two trioses ❹

DHAP = dihydroxyacetone phosphate and G-3-P = glyceraldehyde-3-phosphate; reaction shifts to G-3-P because it is removed

Two moles of substrate per mole of glucose❻

Substrate-level phosphorylation❼

An isomerization❽

Dehydration produces another high-energy phosphate bond

Substrate-level phosphorylation❿

Regenerates NAD+ under anaerobic conditions⓫

Hexokinase in muscle, kidney, and other nonhepatic tissues

Fructokinase in liver only⓭

Dietary galactose is phosphorylated and isomerized to Glu-1-P

–⓮ ⓯

Pentose phosphate pathway (hexose monophosphate shunt)

Glycogenesis⓱

Glycogenolysis⓲

AT P

AD P

NADH + H+ NAD+

NAD+ Pi

O

NADH + H+

ADP

ATP

ADP

ATP

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84 C H A P T E R 3 • CARbOHYdRAtES

in the “Substrate-Level Phosphorylation” section. Two moles of ATP are synthesized because glucose (a hexose) yields two trioses. This reaction replaces the two ATPs used to prime glycolysis. Under conditions of high ATP (which means low ADP) the reaction can be reversed.

❽  Phosphoglycerate mutase catalyzes the transfer of the phosphate group from the number 3 carbon to the number 2 carbon of the glycerate molecule.

❾  Dehydration of 2-phosphoglycerate by the enzyme eno- lase introduces a double bond that imparts high energy to the phosphate bond.

❿  Phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalyzed by pyruvate kinase to yield pyruvate. This is the second site of sub- strate-level phosphorylation of ADP in the glycolytic pathway to make two ATPs. Two ATPs were used to prime glycolysis, two were produced in reaction ❼, and two were produced in this reaction, for a net gain of two ATPs to this point. The hexose has now been split into two 3-carbon units. Phosphoenolpyruvate kinase is a highly regulated enzyme. It is activated allo- sterically by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. In the liver it is also regulated covalently by glucagon through the cAMP mechanism discussed earlier, which transfers a phosphoryl group from ATP. The phosphorylated enzyme is more sensitive to inhibition by ATP.

⓫  The lactate dehydrogenase reaction reduces pyruvate to lactate while oxidizing NADH to 1NAD . The 1NAD formed in the reaction can replace the 1NAD consumed earlier under anaerobic conditions. This reaction is most active in situations of oxygen debt, as occurs in prolonged muscular activity. Under aerobic conditions, pyruvate enters the mitochondrion for complete oxidation via TCA cycle. A third important option available to pyruvate is its conversion to the amino acid alanine by amino trans- ferase, a reaction by which pyruvate acquires an amino group from the amino acid glutamate (Chapter 6). The alternate pathways for pyruvate, together with the fact that pyruvate is also the product of the catabolism of various amino acids, makes pyruvate an important link between protein (amino acid) and carbohydrate metabolism.

⓬  This reaction occurs in muscle, kidneys, and other non- hepatic tissues where fructose is phosphorylated by hexo- kinase to form fructose-6-phosphate. This is a relatively unimportant reaction because the liver clears nearly all of the dietary fructose when first absorbed from the gas- trointestinal tract. The hexokinase reaction is slow and occurs only in the presence of high levels of blood fruc- tose, a situation that is rarely encountered in humans.

⓭  Fructokinase is abundant in the liver and catalyzes the conversion of fructose to fructose-1-phosphate. This

reaction is important when fructose is consumed because nearly all dietary fructose enters the hepatocyte on the first pass. Following a carbohydrate-rich meal, fructose is committed to glycolysis rather than being converted to glucose and stored as glycogen. Because fructose-1-phos- phate enters glycolysis at reaction ❹—and because reac- tion ❸ is irreversible and stimulated by insulin following a meal—fructose in the liver follows a one-way trip to becoming pyruvate (and possibly lactate).

⓮– ⓯ Like glucose and fructose, galactose is first phosphorylated (by galactokinase) to form galactose- 1-phosphate, which occurs primarily in the liver when first absorbed from the gastrointestinal tract. The galactose-1-phosphate is converted to UDP-galactose, which is converted to UDP-glucose by subsequent reac- tions. The result of these reactions is the production of glucose-1-phosphate. When dietary galactose is accom- panied by comparatively large amounts of glucose, the glucose-1-phosphate from galactose is driven mostly toward glycogenesis as the flow of glucose-1-phosphate from glucose pushes the reaction toward glycogenesis.

⓰  This is the point where glucose-6-phosphate enters into a pathway called the pentose phosphate pathway (hex- ose monophosphate shunt), which is discussed later in this chapter.

⓱  Glycogenesis is the conversion of glucose-1-phosphate into glycogen and occurs primarily in the liver and skel- etal muscle. Glycogenesis is stimulated by insulin fol- lowing a carbohydrate-rich meal (see Chapter 7).

⓲  Glycogenolysis is the hydrolysis of glycogen into indi- vidual glucose-1-phosphate units. In skeletal muscle, the liberated glucose-1-phosphate enters glycolysis for energy utilization. In the liver, glucose-1-phosphate can enter glycolysis or be converted to free glucose for release into the system circulation.

The Tricarboxylic Acid Cycle The tricarboxylic acid (TCA) cycle, also called the Krebs cycle or the citric acid cycle, is central to energy metab- olism in the body. Under aerobic conditions, the TCA cycle is the final pathway by which fuel molecules—car- bohydrates, fatty acids, and amino acids—are completely oxidized to CO2 so that their energy is released and transferred to ATP molecules. Greater than 90% of food energy captured and used in the human body involves the TCA cycle in conjunction with oxidative phosphoryla- tion (see Figure 1.7). The enzymes of the TCA cycle are located in the mitochondrial matrix and work in concert with “energy carriers” that shuttle high-energy electrons released by the TCA cycle to the electron transport chain located in the inner mitochondrial membrane. These so- called energy carriers, NADH and FADH2, are formed by

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C H A P T E R 3 • CARbOHYdRAtES 85

amounts of NADH and FADH as acetyl-CoA continually feeds into the cycle. Following are comments on the indi- vidual reactions (Figure 3.18):

❶  The formation of citrate from oxaloacetate and acetyl- CoA is catalyzed by the enzyme citrate synthase. The reac- tion is regulated negatively by NADH and succinyl-CoA.

❷  The isomerization of citrate to isocitrate involves cis aconitate as an intermediate. The isomerization, cata- lyzed by aconitase, results in the repositioning of the —OH group onto an adjacent carbon.

❸  Catalyzed by the enzyme isocitrate dehydrogenase, this is the first of four dehydrogenation reactions within the cycle. The main products are NADH, CO2, and a-ketoglutarate. The reaction is positively modulated by ADP and negatively modulated by ATP and NADH.

❹  Decarboxylation and dehydrogenation of a-ketoglutarate is mechanistically identical to the pyruvate dehy- drogenase complex reaction in its multienzyme– multicofactor requirement. The main products are NADH, CO2, and succinyl-CoA.

❺  Succinyl-CoA contains a high-energy thioester bond that is hydrolyzed by the enzyme succinyl-CoA syn- thetase (also called succinyl thiokinase) and that releases sufficient energy to drive the phosphorylation of guanosine diphosphate (GDP) by inorganic phos- phate. The resulting guanosine triphosphate (GTP) can transfer its phosphate to ADP to make ATP in a reaction catalyzed by the enzyme nucleoside diphos- phate kinase. This reaction is another example of ATP production through substrate-level phosphorylation.

❻  The succinate dehydrogenase reaction yields fumarate and uses FAD instead of 1NAD as a proton and elec- tron acceptor. Succinate dehydrogenase is bound in the inner membrane of the mitochondria. Other TCA enzymes are found in the mitochondrial matrix.

❼  Fumarase incorporates the elements of H O2 across the double bond of fumarate to form malate.

❽  The conversion of malate to oxaloacetate completes the cycle. 1NAD acts as the proton and electron acceptor in this dehydrogenation reaction, which is catalyzed by malate dehydrogenase.

Oxaloacetate and Tricarboxylic Acid Cycle Intermediates To keep the TCA cycle functioning, oxaloacetate and/or other TCA cycle intermediates that can form oxaloacetate must be replenished in the cycle. Oxaloacetate, fumarate, succinyl-CoA, and a-ketoglutarate can all be formed from certain amino acids, but the single most important mecha- nism for ensuring an ample supply of oxaloacetate is the reaction that forms oxaloacetate (four carbons) directly

reduction (accept electrons) when fuel molecules are oxi- dized (lose electrons). In this way, the main function of the TCA cycle is to generate high-energy electrons that power the synthesis of ATP via oxidative phosphorylation. The TCA cycle is shown in Figure 3.18.

The primary molecule entering the TCA cycle is acetyl- CoA. Therefore, fuel molecules must first be transported into mitochondria and converted to acetyl-CoA for complete oxidation. In the case of carbohydrate, glycolysis in the cytosol produces pyruvate, which is transported into mitochondria and converted acetyl-CoA (discussed in the next section). Fatty acids and amino acids are also transported into mitochondria and converted to acetyl-CoA (discussed in later chapters).

Conversion of Pyruvate to Acetyl-CoA Conversion of pyruvate to acetyl-CoA is accomplished in the mitochondrial matrix by a multienzyme complex called the pyruvate dehydrogenase complex (PDC). This multienzyme system is made up of three enzymes: pyru- vate dehydrogenase, dihydrolipoamide acetyltransferase, and dihydrolipoamide dehydrogenase. Several cofac- tors are required for the reaction, including coenzyme A (CoA), thiamin pyrophosphate, 1Mg2 , 1NAD , FAD, and lipoic acid. Four vitamins, therefore, are necessary for the activity of the complex: pantothenic acid (a compo- nent of CoA), thiamin, niacin, and riboflavin. The role of these vitamins and others as precursors of coenzymes is discussed in Chapter 9. The net effect of the complex is decarboxylation, producing CO2, and dehydrogenation of pyruvate, with 1NAD serving as the terminal acceptor of a hydride ion (one proton and two electrons). The active sites of the three enzymes are packed closely together, which allows the passing of the product of one reaction to the next enzyme. This reaction yields energy because the oxidation of NADH produces ATP by oxidative phos- phorylation. The reaction is regulated allosterically: nega- tively by ATP, acetyl-CoA, and NADH, and positively by NAD1 and ADP. The PDC is also regulated covalently: a Mg21-dependent enzyme, pyruvate dehydrogenase kinase, phosphorylates the complex when NADH and acetyl-CoA levels rise. Reactivation of the PDC occurs by the enzyme pyruvate dehydrogenase phosphatase, which removes the phosphate. Insulin and Ca21 ions activate the kinase to activate the PDC.

Release of High-Energy Electrons The condensation of acetyl-CoA with oxaloacetate initiates the TCA cycle reactions. Note that the TCA cycle itself does not directly generate much ATP. Rather, it gener- ates high-energy electrons that are transferred to 1NAD and FAD, thus yielding NADH and FADH, respectively. Because the reactions are cyclic, oxaloacetate is regener- ated after one trip through the cycle, so a relatively small number of oxaloacetate molecules can generate large

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86 C H A P T E R 3 • CARbOHYdRAtES

Figure 3.18 The tricarboxylic acid (TCA) cycle.

Isocitrate

H2C COO –

HC COO–

HC

OH

COO–

C

O

Acetyl-CoA

C S

O

Pyruvate

CH3C

H3C

H2C

C

O

O–

OFrom glycolysis

Pyruvate dehydrogenase

From β-oxidation of fatty acids

Citrate synthase

Oxaloacetate COO–

COO–

COO– COO–

H2C COO –

H2C COO –

H2C COO –

HO C

Aconitase

Citrate

Isocitrate dehydrogenase

α-Ketoglutarate α-Ketoglutarate dehydrogenase

C

C

SCoA

Succinyl-CoA

O

O

Nucleoside diphosphate

kinase

Succinyl-CoA synthetase

Succinate

FADH2

FAD Succinate

dehydrogenase

C

C

Fumarate

H

H

H C

HO

H2C COO –

H2C

H2C

COO–

H2C

H2C

COO–

COO–

H2C COO –

Malate

Fumarase

Malate dehydrogenase

TRICARBOXYLIC ACID CYCLE

(citric acid cycle, Krebs cycle, TCA cycle)

H2O

COO–

–OOC

P

ATP CO2

CO2

GTP GDP

ADP

CoASH

NADH + H+

NAD+

NAD+

NAD+

NAD+

CoASH

CoASH

CO2

CoA

NADH + H+

NADH + H+

NADH + H+

Transports pyruvate into the mitochondria as acetyl-CoA and produces a NADH

H2O

Acetyl-CoA adds two carbons to oxaloacetate to start the cycle.

Isomerization takes place by removing H2O and then adding it back.

A CO2 is lost and a NADH is produced.

Another CO2 is lost and another NADH is produced.

A substrate-level phosphorylation.

FAD+ is reduced to form FADH2.

Add H2O across the double bond.

Third NADH produced in the TCA cycle. One FADH2 and one NADH produced in the conversion of pyruvate to acetyl-CoA.

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C H A P T E R 3 • CARbOHYdRAtES 87

resulting FADH2 transfers its electrons directly to the electron transport chain, producing 1.5 ATPs per mole of NADH (Figure 3.20). This shuttle is not reversible.

Malate–Aspartate Shuttle System The most active shut- tle compound, malate, is freely permeable to the inner mitochondrial membrane. Oxaloacetate from the cytosol is reduced by the NADH to form malate and 1NAD . The malate is oxidized by the enzyme malate dehydrogenase to oxaloacetate in the matrix of mitochondria, producing NADH that enters the electron transport chain and generates 2.5 ATPs per mole of NADH. The oxaloacetate undergoes transamination by aspartate amino transferase to form aspar- tate, which is freely permeable to the inner membrane and can move back out into the cytosol. The effect is that reduc- ing equivalents of NADH are transferred into mitochondria, even though the inner mitochondrial membrane is imperme- able to NADH itself (Figure 3.21). This shuttle is reversible.

Formation of ATP The majority of energy-requiring reactions in the body depend on ATP as a cosubstrate to furnish the energy that drives the reaction. Thus, ATP acts as the main energy currency and must be continually synthesized from the energy provided by macronutrients. In the case of car- bohydrates, glycolysis, the TCA cycle, and the electron transport chain work together to synthesize ATP from the complete oxidation of the starting substrate (glucose, fructose, and galactose). The anaerobic steps that occur in the cytosol (glycolysis) are able to synthesis a small number of ATP, whereas the majority of ATP are synthesized in the

from pyruvate (three carbons) by the addition of CO2. This reaction, shown in Figure 3.19, is catalyzed by pyruvate carboxylase. The “uphill” incorporation of CO2 is accom- plished at the expense of ATP, and the reaction requires the participation of biotin (see Chapter 9). The conversion of pyruvate to oxaloacetate is called an anaplerotic (replen- ishing) process because of its role in restoring oxaloacetate to the cycle. Interestingly, pyruvate carboxylase is regulated positively by acetyl-CoA, thereby ensuring oxaloacetate formation in response to increasing levels of acetyl-CoA.

NADH from Glycolysis: The Shuttle Systems NADH produced in the cytosol during glycolysis (the glyceraldehyde-3-phosphate dehydrogenase reaction) is unable to directly participate in oxidative phosphorylation because the inner mitochondrial membrane is imperme- able to NADH. Under anaerobic conditions, NADH in the cytosol is used in the lactate dehydrogenase reduction of pyruvate to lactate, thereby becoming reoxidized to

1NAD without involving oxygen. In this manner, 1NAD is restored to sustain the glyceraldehyde-3-phosphate dehy- drogenase reaction, allowing the production of lactate to continue in the absence of oxygen.

When the supply of oxygen is adequate to allow total oxidation of incoming glucose, the production of pyruvate and NADH from glycolysis is accelerated and lactate is not formed. In this situation, the reducing equivalents of NADH (the protons and electrons) are transported from the cytosol to the mitochondrial matrix by two separate shuttle systems. These shuttle systems are specific to certain tissues. The glycerol-3-phosphate shuttle functions in the brain and skeletal muscle, whereas the more active malate– aspartate shuttle functions in the liver, kidney, and heart.

Glycerol-3-Phosphate Shuttle System NADH in the cyto- sol transfers its reducing equivalents to dihydroxyacetone phosphate, forming glycerol-3-phosphate that freely dif- fuses across the outer mitochondrial membrane. The reaction is catalyzed by the cytosolic isoform of glycerol- 3-phosphate dehydrogenase. The reducing equivalents of glycerol-3-phosphate are then transferred to FAD that is associated with a membrane-bound isoform of glycerol-3-phosphate dehydrogenase located on the outer face of the inner mitochondrial membrane. Finally, the

Figure 3.19 The reaction by which oxaloacetate is formed directly from pyruvate.

O

CH3—C—COO 2

ATP ADP 1 Pi

Pyruvate

COO2

CH2 C—COO 2

Oxaloacetate

CO2

Supplies oxaloacetate to keep the TCA cycle running

O

Figure 3.20 Glycerol-3-phosphate shuttle.

+ H+ Glycerol-

3-phosphate

Cytosol

Mitochondrial matrix

Inner mitochondrial membrane

Electron- transport chainFAD FADH2

NAD+ NADH

E E

Dihydroxyacetone phosphate

Cytosol NADH transfer to FADH2, which enters the electron transport chain yielding 1.5 ATPs.

A glycerophosphate in the cytosol and one in mitochondrial membrane has net ef fect of transfering cytosol NADH to membrane FADH2.

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88 C H A P T E R 3 • CARbOHYdRAtES

Figure 3.21 Malate–aspartate shuttle.

Malate

+ H+

Oxaloacetate

Inner mitochondrial

membrane

Malate

Cytosol Matrix

Oxaloacetate

Malate dehydrogenase

Malate dehydrogenase

AspartateAspartate

Glutamate Glutamate

Aspartate aminotransferase

Aspartate aminotransferase

Aspartate– glutamate

carrier

α-Ketoglutarate α-Ketoglutarate α-Ketoglutarate–

Malate carrier

NAD+ NAD+

NADH+ H+NADH

α-Ketoglurate and malate move freely across the inner mitochondrial membrane.

Oxidation/reduction of NAD+/NADH has net effect of moving NADH into mitochondria.

Aspartate moves freely across mitochondrial membrane.

mitochondria by oxidative phosphorylation. The large ionic gradient created by the release of high-energy electrons and protons via the TCA cycle is what powers the synthesis of ATP in oxidative phosphorylation. In contrast, some ATP are synthesized by direct phosphorylation involving high- energy phosphate donors, referred to as substrate-level phosphorylation.

Substrate-Level Phosphorylation Two reactions in glycolysis and one reaction in the TCA cycle produce ATP by substrate-level phosphorylation. Phosphorylation of ADP to form ATP is accomplished by phosphate donors having more energy than the amount needed ∆( 7,300 cal/mol or 35.7 kJ/mol)0GD 5 1 1 for the reaction.

Table 3.5 lists the standard free energy of hydrolysis of selected phosphate-containing compounds in both kcal and kJ. Phosphorylated molecules have a wide range of free energies of hydrolysis of their phosphate groups. Many of them release less energy than ATP, but some release more. Figure 1.15 gives the structures of phosphoenolpyruvate, 1,3-bisphosphoglycerate, and phosphocreatine, three compounds that have more free energy than ATP and are

capable of phosphorylating ADP. The ∆G0 of hydrolysis of the compounds, listed in Table 3.5, is called the phosphate group transfer potential and is a measure of the compounds’ capacities to donate phosphate groups to other substances. The more negative the transfer potential, the more potent the phosphate-donating power. Therefore, a compound that releases more energy on hydrolysis of its phosphate can transfer that phosphate to an acceptor molecule having a relatively more positive transfer potential. For this transfer to occur in actuality, however, there must be a specific enzyme to catalyze the transfer. A phosphate

Compound DG 0(cal) DG 0(kJ)

Phosphoenolpyruvate –14,800 –62.2

1,3-diphosphoglycerate –11,800 –49.6

Phosphocreatine –10,300 –43.3

ATP –7,300 –35.7

Glucose-1-phosphate –5,000 –21.0

Adenosine monophosphate (AMP) –3,400 –9.2

Glucose-6-phosphate –3,300 –13.9

Table 3.5 Free Energy of Hydrolysis (Phosphate Group Transfer Potential) of Some Phosphorylated Compounds

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C H A P T E R 3 • CARbOHYdRAtES 89

to molecular oxygen, which becomes reduced to H O2 in the process. The compounds participating in this sequen- tial reduction-oxidation constitute the electron transport chain, also known as the respiratory chain because the elec- tron transfer is linked to the uptake of O2, which is made available to the tissues by respiration. The energy provided by the electron flow allows the protons to be translocated from the mitochondrial matrix to the space between the inner and outer membranes, which creates an energy gradi- ent that powers the phosphorylation of ADP to form ATP. The term oxidative phosphorylation is a descriptive blend of simultaneous processes involving electron transport, trans- location of protons, oxidation of a metabolite by oxygen, and the phosphorylation of ADP to make ATP.

Cellular oxidation of a compound can occur by several different reactions: the addition of oxygen, the removal of electrons, and the removal of protons and electrons together (as hydrogen atoms or hydride ions). All of these reactions are catalyzed by enzymes collectively termed oxidoreductases. Among these, the dehydrogenases remove protons and electrons from nutrient metabolites and are particularly important in energy transformation. The protons and electrons removed from metabolites by dehydrogenases generally produce NADH or FADH2, which are either already in or shuttled into the mitochondria and move along the electron transport chain.

After oxidation of substrate molecules by a dehydrogenase enzyme, the protons and electrons are transferred to a cosubstrate, such as the vitamin-derived nicotinamide adenine dinucleotide 1(NAD ) or flavin adenine dinucleotide (FAD). The structures of both the oxidized and reduced forms of these cosubstrates are shown in Figures 3.23 and 3.24. After accepting protons and electrons from reactions of the TCA cycle, NADH

group can be enzymatically transferred from ATP to glucose, a transfer that can be predicted from Table 3.5. It can also be predicted from Table 3.5 that compounds with a more negative phosphate group transfer potential than ATP can transfer phosphate to ADP, forming ATP. This kind of reaction does, in fact, occur in the hexokinase/ glucokinase reactions. The phosphorylation of ADP by phosphocreatine represents an important mode for ATP formation in muscle, and the reaction exemplifies a substrate-level phosphorylation (Figure 3.22).

Biological Oxidation and the Electron Transport Chain The majority of ATP synthesized in mitochondria begins with the oxidation of fuel molecules and the release of electrons and protons by the TCA cycle. The electrons and protons are delivered (by NADH and FADH2) to the inner mitochondrial membrane where the electrons are passed through a series of intermediate compounds and ultimately

Figure 3.23 Nicotinamide adenine dinucleotide 1(NAD ) and its reduced form (NADH).

NH2

N N

N N O

2O—P O

O

—C—NH2

N1

O

O

H H

* OH NAD1

OH

* P added on this OH group for NADP.

CH2O

H H

OH OH

O

—C—NH2

N

R NADH

OH H

Site of oxidation and reduction

2O—P—O—CH2

Figure 3.22 (a) Example of high- energy phosphate bond being transferred from high-energy compound phosphocreatine to form ATP. (b) The transfer of the high-energy phosphate bond to a compound that becomes activated, allowing it to enter into the glycolytic pathway.

ADP Phosphocreatine

G 09 5 23,000 cal/mol

ATP Creatine(a)

ATP Glucose

G 09 5 24,000 cal/mol

ADP Glucose-6-phosphate(b)

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90 C H A P T E R 3 • CARbOHYdRAtES

Figure 3.24 Flavin adenine dinucleotide (FAD) and its reduced form (FADH )2 . 5 1R ribitol phosphate AMP.

O

O FAD

R

Reduction takes place

N

N

N

NH

CH3

CH3

O

O FADH2

R

N

N H

H N

NH

CH3

CH3

Protons and electrons from reactions of the TCA cycle attach to the nitrogens in the box.

molecules smaller than 10 kilodaltons, and the inner mem- brane, which has very limited permeability. Remember that the enzymes of the TCA cycle, except for succinyl- CoA synthetase and those involved in fatty acid oxidation (discussed in Chapter 5), are located in the matrix of the mitochondria. The translocation of protons 1(H ) from within the matrix to the intermembrane space (the space between the cristae and outer membrane) provides much of the energy that drives the phosphorylation of ADP to make ATP. The electron transport chain starts with NADH or FADH2, whether it is shuttled in from the cytosol, as dis- cussed previously, or produced within the mitochondria.

Components of the Electron Transport Chain and Oxidative Phosphorylation Glycolysis produces cytoplasmic NADH and FADH2, and their shuttling into the mitochondria has already been dis- cussed (see Figures 3.20 and 3.21). Figure 3.26 presents a highly simplified overview of the electron transport chain. As indicated, the reactions actually take place in four distinct complexes of associated proteins and enzymes, which can be isolated and purified. Complex I, NADH- coenzyme Q (CoQ) reductase, accepts electrons from NADH that resulted from glycolysis, the TCA cycle, and fatty acid oxidation. Complex II, succinate CoQ dehydro- genase, includes the membrane-bound succinate dehy- drogenase that is part of the TCA cycle. Both Complex I and II produce CoQH2. CoQH2 is the substrate for Complex III, CoQ–cytochrome c reductase. Complex IV is cytochrome oxidase. It is responsible for reducing molecu- lar oxygen to form H2O. The complexes work indepen- dently and are connected by mobile acceptors of electrons,

Figure 3.25 The sequential arrangement of the components of the electron transport chain, showing its division into four complexes, I, II, III, and IV. Coenzyme Q (ubiquinone) is shared by complexes I, II, and III. Cyt c is shared by complexes III and IV.

NADH 1 H+

NAD+ FMN

FMN

FMNH2 Fe-S

O

CoQ

COQH2

CoQ

Fe-S

Cyt b Fe-S Cyt c1 Cyt c Cyt a–a3

H2O

½ O2

FAD FADH2

I

II

III

IV

4H+ 4H+ 2H+

Mobile electron carrier, CoQ and

cytochrome c

Fe+2

Fe+2

Fe+3

Fe+3

Fe+2

Fe+3

Fe+3

Fe+2

Fe+2

Fe+3

Fe+3

Fe+2

and FADH2 move to the inner mitochondrial membrane to initiate the electron transport chain. The sequential arrangement of reactions in the electron transport chain is shown in Figure 3.25. Dashed lines outline the four complexes. Either NADH or FADH2 is the initial electron donor for the electron transport chain.

Anatomical Site for the Electron Transport Chain The structure of the mitochondrion is illustrated in Figures 1.6 and 1.7. Refer to Chapter 1 for a descrip- tion of the outer membrane, which is permeable to most

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C H A P T E R 3 • CARbOHYdRAtES 91

CoQ and cytochrome c. Each complex is discussed briefly here. For a more detailed explanation, consult a general biochemistry textbook.

Complex I: NADH–Coenzyme Q Oxidoreductase Complex I— also known as NADH dehydrogenase—transfers a pair of electrons from NADH to CoQ. The structures of the oxi- dized and reduced forms of CoQ are shown in Figure 3.27. Complex I is made of many polypeptide chains, a molecule of FMN, and several Fe-S clusters, along with additional iron molecules. The iron molecules bind with the sulfur- containing amino acid cysteine. The iron transfers one electron at a time, cycling between Fe21 and Fe31. CoQ is a highly hydrophobic compound and it diffuses freely in the hydrophobic core of the inner membrane. The result of the multistep reaction is the transfer of electrons and protons from NADH to CoQ to form first CoQ hydroqui- none and then CoQH2 and actively transfer protons from the matrix side of the inner mitochondrial membrane to the intermembrane space. The importance of the buildup of protons in the intermembrane space is discussed in the following sections. The oxidation of NADH through the

electron transport chain results in the synthesis of approxi- mately 2.5 ATP molecules.

Complex II: Succinate Dehydrogenase Complex II is the succinate dehydrogenase enzyme, which is the only TCA cycle enzyme that is an integral part of the inner mito- chondrial membrane. Beside the succinate dehydroge- nase, Complex II contains a FAD protein and Fe-S clusters (similar to those discussed previously). When succinate is converted to fumarate in the TCA cycle, FAD is reduced to FADH2. The FADH2 is oxidized with one electron transfer through the Fe-S centers to reduce CoQ to CoQH2. Unlike in Complex I, the protons released from FADH2 are not transported to the intermembrane space. The oxidation of FADH2 through the electron transport chain results in the formation of approximately 1.5 molecules of ATP.

Complex III: Coenzyme Q–Cytochrome c Oxidoreductase Reduced CoQ passes its electrons to cytochrome c in the third complex of the electron transport chain in a pathway known as the Q cycle. The complex contains three differ- ent cytochromes and Fe-S protein. The cytochromes con- tain heme molecules with an iron molecule in the center. The iron in the center of the cytochromes is oxidized and reduced as electrons flow through. Electrons pass through the Q cycle in two phases. In the first phase a CoQH2 (ubiquinol) passes one electron to form the semiquinone (one of the two hydroquinones oxidized), then another electron and proton are transferred to the semiquinone to produce oxidized CoQ (quinone), releasing four protons to the intermembrane space. The electrons are then trans- ferred to cytochrome c1 (and associated cytochromes), and CoQ picks up two protons from the matrix, resulting in the reduction of a CoQ to CoQH2. This means that two turns of the CoQ cycle result in the oxidation of 2CoQH2 to CoQ, the release of 4 1H in the intermembrane space, and the reduction of one CoQ to CoQH2. Like CoQ, cyto- chrome c is a mobile carrier. This characteristic means that cytochrome c is able to migrate along the membrane. Cytochrome c associates loosely with the inner mitochon- drial membrane on the matrix side of the membrane.

Figure 3.26 Schematic of electron transport modules connecting through coenzyme Q.

Complex I

NADH dehydrogenase

Complex II

Succinate dehydrogenase FAD Fe-S centers

NADH-CoQ oxidoreductase Fe-S centers Complex III

Cytochrome c

Complex IV

Cytochrome c oxidase

Cu ions CoQ-cytochrome c

oxidase CoQ/CoQH2

H2O 1

2O2/

Figure 3.27 Oxidized and reduced forms of coenzyme Q, or ubiquinone. The subscript n indicates the number of isoprenoid units in the side chain (most commonly 10). A one-electron transfer results in the formation of a semiquinone with only one of the quinone groups reduced.

O

O

CoQ (ubiquinone) (oxidized)

CH3

OH

OH

CH3—O

CH3—O

CH3—O

CH3—O

—CH3

CoQH2 (ubiquinol) (reduced)

CH3—CH3 —(CH2—CH C—CH2)nH

—(CH2—CH C—CH2)nH

The groups in the boxes function in the transfer of H+

and electrons.

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92 C H A P T E R 3 • CARbOHYdRAtES

Phosphorylation of ADP to Form ATP The intimate association of energy release with oxidation is exemplified by the oxidation of glucose to CO2 plus water and energy, discussed earlier in this chapter. Glycolysis occurs in the cytosol; the TCA cycle, electron transport, proton translocation, and oxidative phosphorylation occur in the mitochondria. It has already been established that the complete oxidation of 1 glucose yields either 30 or 32 ATPs. The complete biological oxidation of 1 mol of glu- cose yields approximately 700 kcal (or 2,937 kJ). The stan- dard free energy for the hydrolysis of ATP that has been used throughout this chapter is 7.3 kcal (30.5 kJ). How- ever, standard conditions are at a concentration of 1 mol/L, whereas the concentration of ATP within the cell is closer

It can then pass its electrons on to cytochrome c oxidase in Complex IV, which is discussed next.

Complex IV: Cytochrome c Oxidase Complex IV is called cytochrome c oxidase. It accepts electrons from cyto- chrome c and catalyzes a four-electron reduction of oxygen to form water. This reaction is the final one in the oxida- tion of the energy-providing nutrients (carbohydrate, fat, protein, and alcohol) to produce usable chemical energy in the form of ATP. The structure of cytochrome c oxidase is known; it is made up of multiple subunits. Some of the subunits are encoded from nuclear DNA and some from mitochondrial DNA. These latter proteins contain iron and copper. These metal ions cycle between their oxidized (Fe , Cu )3 21 1 and reduced (Fe , Cu )2 11 1 states. Cytochrome c oxidase also contains two cytochromes, cytochrome a and cytochrome a3, which contain different heme moieties. Protons are transported to the intermembrane space.

Electron transport can carry on without phosphorylation, but the phosphorylation of ADP to form ATP (discussed in the next section) is dependent upon electron transport, with the transport terminating as molecular oxygen is reduced to H2O. A schematic of the inner mitochondrial membrane showing the four complexes of the electron transport chain is shown in Figure 3.28. The free energy change at various sites within the electron transport chain is shown in Table 3.6.

Figure 3.28 An illustration of oxidative phosphorylation coupled with ATP synthase. Energy from electron transport pumps protons into the intermembrane space from the matrix against a concentration gradient. The protons move back into the matrix through channels in the F F0 1 ATP-synthase aggregate.

ATPADP 1 Pi

Intermembrane space

Matrix

4H+ 4H+

2H+

H+

I III IV

Cyt c

H2O ½O2 1 2H +FAD

ATP-synthase

FADH2

NAD+NADH 1 H+

II

F0

F1

As electrons pass through the electron transport chain, H+ are translocated to the intermembrane space, creating an ionic gradient that powers ATP-synthase.

Conformational changes of the enzyme protein result in ATP

synthesis and movement of H+ back into the mitochondrial matrix.

Reaction DG˚’ (cal/mol) ADP Phosphorylation Site?

NAD1 FMN –922 No

FMN CoQ –15,682 Yes

CoQ cyt b –1,380 No

Cyt b cyt c1 –7,380 Yes

Cyt c1 cyt c –922 No

Cyt c cyt a –1,845 No

Cyt a O1 2 2 –24,450 Yes

Table 3.6 Free Energy Changes at Various Sites within the Electron Transport Chain Showing Phosphorylation Sites

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C H A P T E R 3 • CARbOHYdRAtES 93

ATP Synthase Figure 3.28 illustrates electron transport, proton translocation, and oxidative phosphorylation. The disparity in both the proton concentration and electrical charge on either side of the inner membrane of the mito- chondria has already been discussed. It is this proton gradi- ent that provides the energy for ATP synthesis that occurs with the aid of ATP synthase. ATP synthase is made up of two main components, F0 and F1, each with multiple subunits. F0 is fixed in the membrane and F1 sticks out of the membrane into the mitochondrial matrix. Respiratory stalks extend from the cristae. If these stalks are removed, electron transport can proceed, but phosphorylation of ADP does not occur. Some of the subunits of F1 are capable of rotating and have sites that bind ATP, ADP, and Pi. They also contain channels that allow proton movement through the membrane. For each pair of electrons traversing Com- plex IV, the rotating subunits of F1 can complete one rota- tion and produce three ATPs. At the same time, protons from the intermembrane space are moved back into the matrix from the intermembrane space. The number of pro- tons moved back depends on the number of subunits in the rotating stalk (this can vary between 10 and 15), result- ing in 3–5 protons per ATP formed moving back into the matrix. The return flow of protons furnishes the energy necessary for the synthesis of ATP from ADP and Pi.

ATP is synthesized in mitochondria but must be moved to the cytosol to supply energy for the cell. There is an ATP- ADP translocase that shuttles ATP out of the mitochondria and ADP in. With the shuttle, the equivalent of one proton is moved from the cytosol to the mitochondrial matrix. Since the synthesis of one of ATP involves the movement of three protons from the cytosol to the matrix, with the translocase activity about four protons total are moved back into the matrix.

ATPs Produced by Complete Glucose Oxidation The complete oxidation of glucose to CO2 and H O2 can be shown by this equation:

C H O 6 O 6 CO 6 H O energy6 12 6 2 2 2→1 1 1

Complete oxidation is achieved by the combined reaction sequences of the glycolytic and TCA cycle pathways. The energy-conserving steps yield a net of two ATPs by substrate-level reactions in the glycolytic pathway and two ATPs (or one ATP and one GTP) by substrate- level reactions in the TCA cycle. In addition, there are three NADH and one FADH2 produced from each acetyl-CoA that goes through the TCA cycle. Two acetyl-CoAs are produced from each molecule of glucose, which releases two molecules of CO2 and two NADH. In summary, one molecule of glucose produces:

● 6 molecules of CO2 (released) ● 4 ATP ● 10 NADH ● 2 FADH2.

to 1–5 mmol/L. The free energy of hydrolysis at this con- centration is closer to 12 kcal (50 kJ). The free energies of other compounds with high phosphate transport potential such as phosphoenolpyruvate, 1,3-bisphosphoglycerate, and phosphocreatine are also increased proportionally. It is more straightforward to use standard free energy in talking about these reactions. However, to determine the energy efficiency of the biological oxidation of glucose, the free energy of ATP under biological conditions must be considered. In living cells, 32 mol of ATP capture 384 kcal (32 3 12). The efficiency is therefore 384/700 3 100 or about 54% [19]. The remaining energy is released as heat. This is an efficient process as engines go.

The previous discussion on electron transport focused on the translocation of protons 1(H ) from the matrix to the intermembrane space. This translocation is vital to the phosphorylation of ADP to form ATP. The translocation of protons requires energy but in return creates a pool of potential energy. The generally accepted mechanism for the synthesis of ATP was first proposed by Peter Mitchell in 1961. He proposed that the energy stored in the difference in the concentration of 1H between the matrix of the mitochondria and the intermembrane space was the driving force for coupled ATP formation. This proposal was called the chemiosmotic hypothesis. We examine its main points to support our understanding of the coupling of phosphorylation with the electron transport chain. A recent review presents current research about proton translocation [20].

Translocation of 1H To determine if the pH gradient and electrical charge difference are sufficient to provide the energy for ATP synthesis, we must examine the number of 1H translocated at each complex. Direct measurements have been difficult and disagreement exists among exerts, but the consensus is that for every 2 electrons that pass through Complex I (NADH dehydrogenase) and Com- plex III, 4 1H are translocated by each complex, for a total of 8. For Complex IV an additional 2 1H are translocated by each pair of electrons passing through the complex. No protons are translocated in Complex II. This means that for every NADH oxidized to water, a total of 10 protons are translocated from the matrix to the intermembrane space. The electrical charge across the inner membrane changes because of the positively charged protons in the intermem- brane space, a difference estimated to be approximately 0.18 volts. It is also assumed that the pH difference between the mitochondrial matrix and the inner membrane is one unit. Using these assumptions, the free energy available is –94.49 kcal/mol (–23.3 kJ/mol). This is the potential free energy available to move protons back into the matrix of the mitochondria and at the same time couple phosphorylation of ADP to ATP with electron transport. Paul Boyer and John Walker shared the 1997 Nobel Prize for chemistry for their work on ATP synthase. A review of Paul Boyer’s research on ATP synthase sums up several decades of work [21].

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94 C H A P T E R 3 • CARbOHYdRAtES

The NADH and FADH2 are in the matrix of the mitochondria and are oxidized by the electron transport chain and oxidative phosphorylation to ultimately produce ATP. By convention, it has been assumed in the past that three ATPs are formed by oxidative phosphorylation from NADH, and two ATPs are formed from FADH2. As previously discussed, the actual number of ATPs formed from NADH is closer to 2.5; for FADH2, it is 1.5. If the integers (3/2) are used for the number of ATPs produced from NADH/FADH2, a total of 38 mol of ATP are formed. If we accept the 2.5/1.5 ratio, 32 mol of ATP are produced from each mol of glucose. Oxidative phosphorylation is only active under aerobic conditions. Under anaerobic conditions, only two ATPs are produced from each glucose at substrate level.

The actual number of ATPs formed aerobically from glucose varies because of the two different shuttle mechanisms that transport the electrons from NADH produced by the glycolytic pathway into the mitochondria. One mechanism, the glycerol-3-phosphate shuttle system, transfers the electrons to FADH2 and therefore yields only 1.5 ATPs. The other shuttle system, the malate–aspartate shuttle, transfers the electrons to NADH inside the mitochondria and yields 2.5 ATPs.

The conversion of the chemical energy of carbohydrates to form ATP is an integral part of carbohydrate metabolism. The next sections cover other aspects of carbohydrate metabolism. Comprehensive reviews of electron transport, proton translocation, and oxidative phosphorylation are available to the interested reader [22–24].

The Pentose Phosphate Pathway (Hexose Monophosphate Shunt) The pentose phosphate pathway (also called the hexose monophosphate shunt) is one of the pathways that is avail- able to glucose in the cytosol and is shown in Figure 3.29. It generates important intermediates not produced in other pathways. The pentose phosphate pathway has two important products:

● pentose phosphates, necessary for the synthesis of the nucleic acids found in DNA and RNA and for other nucleotides (see Figure 3.4)

● the reduced cosubstrate NADPH, used for important metabolic functions, including the biosynthesis of fatty acids (Chapter 5), the maintenance of reducing sub- strates in red blood cells necessary to ensure the func- tional integrity of the cells, and drug metabolism in the liver.

The cells of some tissues have a high demand for NADPH, particularly those that are active in the synthesis of fatty acids, such as cells of the mammary gland, adipose tissue, adrenal cortex, and liver. These tissues predictably

engage the entire pentose phosphate pathway, recycling pentose phosphates back to glucose-6-phosphate to repeat the cycle and ensure an ample supply of NADPH. The pathway reactions that include the dehydrogenase reactions and therefore the formation of NADPH from 1NADP are called the oxidative reactions of the pathway. This segment of the pathway is illustrated on the left in Figure 3.29. The pentose phosphate pathway also synthesizes three-, four-, five-, six-, and seven-carbon sugars.

This pathway begins by oxidizing glucose-6- phosphate in two consecutive dehydrogenase reactions catalyzed by glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD). Both reactions require NADP1 as cosubstrate, accounting for the formation of NADPH as a reduction product. The first reaction (G-6-PD) is irreversible and highly regulated. It is strongly inhibited by the cosubstrate NADPH and fatty acid CoAs. Pentose phosphate formation is achieved by the decarboxylation of 6-phosphogluconate to form the pentose phosphate, ribulose 5-phosphate, which in turn is isomerized to its aldose isomer, ribose 5-phosphate.

Pentose phosphates can subsequently be “recycled” back to hexose phosphates through the transketolase and transaldolase reactions illustrated in Figure 3.29. This recycling of pentose phosphates to hexose phosphates therefore does not produce pentoses, but it does ensure generous production of NADPH as the cycle repeats.

The re-formation of glucose-6-phosphate from the pentose phosphates, through reactions catalyzed by transketolase, transaldolase, and hexose phosphate isomerase, is called the nonoxidative reactions of the pathway and is shown on the right in Figure 3.29. Transketolase and transaldolase enzymes catalyze complex reactions in which three-, four-, five-, six-, and seven-carbon phosphate sugars are interconverted. These reactions are detailed in most comprehensive biochemistry texts.

The reversibility of the transketolase and transaldolase reactions allows hexose phosphates to be converted directly into pentose phosphates, bypassing the oxidative reactions. Therefore, cells that undergo a more rapid rate of replication and that consequently have a greater need for pentose phosphates for nucleic acid synthesis can produce these products in this manner.

The pathway’s activity is low in skeletal muscle because of the limited demand for NADPH (fatty acid synthesis) in this tissue and also because of muscle’s reliance on glucose and fatty acids for energy metabolism. Glucose- 6-phosphate can be used for either glycolysis or for the pentose phosphate pathway. The choice is made based upon the cell’s needs for energy (by assessing the ATP/ ADP ratio) or for biosynthesis (by assessing the NADP1/ NADPH ratio). The level of NADPH is generally much higher than that of 1NADP .

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C H A P T E R 3 • CARbOHYdRAtES 95

Figure 3.29 The pentose phosphate pathway (hexose monophosphate shunt), showing the oxidative stage (left side of diagram) and the nonoxidative stage (right side of diagram). Abbreviations: G-6-PD, glucose-6-phosphate dehydrogenase; 6-PGD, 6-phosphogluconate dehydrogenase.

Glucose-6-phosphate

O PCH2

O PCH2

OH

OH

OH HO

O

Oxidative stage

Hexose phosphate isomerase

D-ribulose 5-phosphate

Phosphopentose isomerase

D-xylulose 5-phosphate

Fructose-6-phosphate

Glyceraldehyde- 3-phosphate

Transketolase

Transaldolase

Transketolase

P O CH2 OHCH2

OH

OH

HO

O

Nonoxidative stage

(G-6-PD)

NADPH 1 H1

NADP1

6-phosphoglucono-lactone

6-phosphogluconate

O PCH2

O

OH

OH HO

O

Gluconolactonase

6-PGD

CO2

NADP1

NADPH 1 H1

COO2

CH OH

CH OH

CH OH

CHHO

O PCH2

CH2 OH

CH OH

CH OH

C O

O PCH2

CH OH

CH O

O PCH2

CH2 OH

CHHO

CH OH

C O

D-ribose 5-phosphate

O PCH2

CH OH

CH OH

CH OH

CH O

Epime rase

Reversible

Gluconeogenesis Glucose is an essential nutrient for most cells. The brain and other tissues of the central nervous system (CNS) and red blood cells are particularly dependent upon glu- cose as a nutrient. When dietary intake of carbohydrate is reduced and blood glucose concentration declines, hormones including glucagon trigger accelerated glu- cose synthesis from noncarbohydrate sources in a pro- cess called gluconeogenesis. Lactate, glycerol (a product

of triacylglycerol hydrolysis), and certain amino acids represent important noncarbohydrate sources. The liver is the major site of this activity, although under cer- tain circumstances, such as prolonged starvation, the kidneys become increasingly important in gluconeo- genesis. Most of the glucose formed by the liver and the kidneys is released into the blood to maintain blood glucose levels.

Many steps in gluconeogenesis are the reverse of glycolysis. Gluconeogenesis synthesizes glucose and

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96 C H A P T E R 3 • CARbOHYdRAtES

consumes ATP and 1NAD rather than producing ATP and NADH. Most of the cytoplasmic enzymes involved in glycolysis, which is the conversion of glucose to pyruvate, catalyze their reactions reversibly and therefore provide the means for also converting pyruvate to glucose. When the cell is oxidizing glucose for energy, however, it does not need to make glucose from gluconeogenesis. Both glycolysis and gluconeogenesis must be regulated, and it is the nonreversible reactions that are regulated. Three reactions in the glycolytic sequence are highly exergonic, highly regulated, and not reversible: those catalyzed by the enzymes glucokinase (hexokinase), phosphofructokinase, and pyruvate kinase (sites 1, 3, and 10 in Figure 3.17). All of these reactions involve ATP and are unidirectional by virtue of the high, negative free energy change of the reactions. Therefore, the process of gluconeogenesis requires that these reactions be either bypassed or circumvented by other enzyme systems. The presence or absence of these enzymes determines whether a certain organ or tissue

is capable of conducting gluconeogenesis. As shown in Figure 3.30, the glucokinase and phosphofructokinase reactions can be bypassed by specific phosphatases (glucose-6-phosphatase and fructose-1,6-bisphosphatase, respectively) that remove phosphate groups by hydrolysis.

The bypass of the pyruvate kinase reaction involves the formation of oxaloacetate as an intermediate. Mitochondrial pyruvate can be converted to oxaloacetate by pyruvate carboxylase, a reaction that was discussed earlier as an anaplerotic process. Oxaloacetate, in turn, can be decarboxylated and phosphorylated to phosphoenolpyruvate (PEP) by PEP carboxykinase, thereby completing the bypass of the pyruvate kinase reaction. However, the PEP carboxykinase reaction is a cytosolic reaction and therefore oxaloacetate must leave the mitochondrion to be acted upon by the enzyme. Because the mitochondrial membrane is impermeable to oxaloacetate, it must first be converted to either malate (by malate dehydrogenase) or aspartate (by transamination

Figure 3.30 The principal regulatory mechanisms in glycolysis and gluconeogenesis. Nonreversible reactions of glycolysis and gluconeogenesis showing regulated steps. Inhibitors are indicated by minus signs and activators by plus signs. Source: Garrett & Grisham, Biochemistry, 4th Edition. © Cengage Learning.

To bloodstream

Glucose

Glucose-6-phosphate

Fructose-6-phosphate

Fructose-1,6-bisphosphate

Phosphoenolpyruvate

Oxaloacetate

Pyruvate

Glucose-6-phosphataseGlucokinase or Hexokinase

Fructose-1,6-bisphosphatasePhosphofructokinase

Phosphoenolpyruvate carboxykinase

Pyruvate kinase

Pyruvate carboxylase

Glucose -6-phosphate

Fructose-2,6-bisphosphate AMP ATP Citrate

F-1,6-BP Acetyl-CoA ATP Alanine cAMP-dependent phosphorylation

[Glucose-6-phosphate] (substrate-level

control)

F-2,6-BP AMP

Acetyl-CoA

Regulation of glycolysis

Regulation of gluconeogenesis

+ + – –

+ – – – –

– –

Citrate +

+

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C H A P T E R 3 • CARbOHYdRAtES 97

with glutamate; see Chapter 6), both of which freely traverse the mitochondrial membrane. This mechanism is similar to the malate–aspartate shuttle previously discussed. In the cytosol, the malate or aspartate can be converted back to oxaloacetate by malate dehydrogenase or aspartate aminotransferase (glutamate oxaloacetate transaminase), respectively.

Noncarbohydrate Sources Amino Acid Utilization The conversion of pyruvate to oxa- loacetate in the initial steps of gluconeogenesis allows for the carbon skeletons of various amino acids to enter the gluconeogenic pathway. Such amino acids accordingly are called glucogenic. Glucogenic amino acids can be catabo- lized to pyruvate or oxaloacetate when metabolic conditions favor glucose synthesis. Furthermore, since some amino

acids can convert to various TCA cycle intermediates— and because the intermediates can leave the mitochondrion in the form of malate or aspartate—utilization of TCA cycle intermediates represents another way that amino acids can be converted to glucose. Reactions showing the entry of noncarbohydrate substances into the gluconeogenic system are shown in Figure 3.31.

Lactate Utilization Lactate is produced by red blood cells continuously and by skeletal muscle during strenuous phys- ical exertion. The majority of lactate produced is released into the blood, where it travels to the liver for conversion to glucose via gluconeogenesis. The newly made glucose can, in turn, be released into the blood. Recall that muscle cells lack glucose-6-phosphatase and cannot produce free glu- cose from noncarbohydrate sources. Thus, the liver is able

Figure 3.31 The reactions of gluconeogenesis, showing the bypass of the unidirectional pyruvate kinase reaction and the entry into the pathway of noncarbohydrate substances such as glycerol, lactate, and amino acids. Abbreviations: G6P, glucose-6- phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3- phosphate; BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate. Source: Garrett & Grisham, Biochemistry, 4th Edition. © Cengage Learning.

Glycerol

DHAP G3P

BPG Cytosol3PG

2PG

P y r u v a t e

k i n a s e

F6P

FBP

G6P Glucose

Lactate

Oxaloacetate

Oxaloacetate

Pyruvate

Pyruvate

Malate

Malate

Mitochondrion

PEP

Fumarate

Succinate

Succinyl-CoA

a-ketoglutarate Amino acids

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98 C H A P T E R 3 • CARbOHYdRAtES

to prevent the accumulation of lactate while replenishing blood glucose. This is an important relationship between muscle and liver, especially during strenuous (anaerobic) physical activity when blood glucose is being used, at least in part, to fuel muscle by glycolysis that produces lactate. The ability of the liver to convert muscle-derived lactate to glucose, and for muscle to take up that glucose and use it in glycolysis, constitutes the Cori cycle.

Glycerol Utilization The hydrolysis of triacylglycerols stored in adipose tissue produces fatty acids and glycerol (discussed further in Chapter 5). Fatty acids are a rich source of energy that provides fuel for muscle and other tissues by being catabolized to acetyl-CoA for entry into the TCA cycle (see Figure 3.18). The remaining glycerol molecule is released from adipose tissue into the blood, where is travels to the liver for conversion to glucose via gluconeogenesis.

Fatty acids, in contrast with glycerol and other noncarbohydrate sources, cannot be used to make glucose because humans lack the necessary enzymes to convert acetyl-CoA to pyruvate or any intermediate along the gluconeogenic pathway. It is conceivable that after fatty acid–derived acetyl-CoA enters the TCA cycle, diverting oxaloacetate or other TCA-cycle intermediates into gluconeogenesis would reflect an indirect contribution of fatty acids to glucose synthesis. This scenario does occur to a very limited extent, which explains why trace amounts of carbon atoms from fatty acids are found in newly synthesized glucose. However, such a pathway is insignificant and unsustainable because depletion of oxaloacetate prevents the TCA cycle from continuing. Another interesting scenario by which fatty acids might provide carbon skeletons for gluconeogenesis is based on computer modeling of all possible enzyme systems and pathways present in humans [25]. The model suggests that when fatty acids are used for ketone body synthesis during carbohydrate deficit (see Chapter 5), the by-product acetone can be used to make pyruvate, which can enter the gluconeogenic pathway. While theoretically possible, additional metabolic research is needed to confirm whether this pathway is a quantitatively important source of glucose.

REGULATION OF METABOLISM

This section focuses on the general mechanisms used for regulation and then describes the regulation of glycolysis and gluconeogenesis as a more detailed example. The regu- latory mechanisms for the other pathways are similar. The TCA cycle is the most prolific producer of ATP through oxidative phosphorylation and uses acetyl-CoA produced from glucose, fatty acids, and certain amino acids. The regulation of these pathways is covered in the appropriate chapters.

The purpose of regulation of glycolysis and gluconeogenesis is to maintain homeostasis. The reactions of metabolism are altered to meet the nutritional and biochemical demands of the body. An excellent example of metabolic regulation is the reciprocal regulation of the glycolysis (catabolic) pathways and the gluconeogenic (anabolic) pathways. The glycolytic conversion of glucose to pyruvate liberates energy, whereas the reversal of the process from pyruvate to glucose (gluconeogenesis) consumes energy. The pyruvate kinase bypass in itself is energetically expensive, considering that 1 mol of ATP and 1 mol of GTP must be expended in converting intramitochondrial pyruvate to extramitochondrial PEP (Figure 3.31). It follows that among the factors that regulate the glycolysis/gluconeogenesis activity ratio is the body’s need for energy. In a broad sense, regulation is achieved by four mechanisms:

● negative or positive modulation of allosteric enzymes by effector compounds

● hormonal activation by covalent modification or induc- tion of specific enzymes

● directional shifts in reversible reactions by changes in reactant or product concentrations

● translocation of enzymes within the cell (covered in Chapter 1).

The concept of enzyme regulation was covered in Chapter 1, but a brief discussion of the principles is included here, with the regulation of carbohydrate metabolism in mind. This is followed by a more detailed examination of the regulation of glycolysis and gluconeogenesis.

Allosteric Enzyme Modulation Allosteric mechanisms can stimulate or suppress the enzymatic activity of a pathway. An allosteric, or regula- tory, enzyme is said to be positively or negatively modu- lated. Modulators, which are usually compounds within the pathway, generally act by altering the conformational structure of the allosteric enzyme. Allosteric enzymes catalyze unidirectional, or nonreversible, reactions. The modulators of the enzymes of the unidirectional reactions must either stimulate or suppress a reaction in one direc- tion only. General examples of allosteric modulators are presented in the following sections.

AMP, ADP, and ATP as Allosteric Modulators An indication of the energy status of a cell and an impor- tant regulatory factor in energy metabolism is the ratio of the cellular concentrations of ADP (or AMP) to ATP. The usual breakdown product of ATP is ADP, but as ADP increases in concentration, some of it becomes enzymati- cally converted to AMP (to produce an ATP). Therefore, ADP and/or AMP accumulation can signify an excessive use of ATP and its depletion.

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C H A P T E R 3 • CARbOHYdRAtES 99

AMP, ADP, and ATP all act as modulators of certain allosteric enzymes, but the effect of AMP or ADP opposes that of ATP. For example, if ATP is abundant and ADP is scarce, additional energy is not needed. Energy-releasing (ATP-producing) pathways are negatively modulated, reducing the production of additional ATP. The reverse is also true; an increase in AMP (or ADP) concentration conversely signifies a depletion of ATP and the need to produce more of this energy source. In such a case, AMP or ADP can positively modulate allosteric enzymes of the energy-releasing pathways.

Two examples of positive modulation by AMP are its ability to cause a shift from the inactive form of phosphorylase b to an active form in glycogenolysis and the activation of phosphofructokinase in the glycolytic pathway, discussed in the next paragraph. Increased levels of AMP are accompanied by an enhanced activity of either of these reactions that encourages glucose catabolism. The resulting shift in metabolic direction, as signaled by the AMP buildup, causes the release of energy as glucose is metabolized and helps restore depleted ATP stores.

Phosphofructokinase is modulated positively by AMP and ADP and negatively by ATP. As the store of ATP increases, slowing of the glycolytic pathway is called for. Phosphofructokinase is an extremely important rate- controlling allosteric enzyme and is modulated by a variety of substances. Its regulatory function has already been described in Chapter 1.

Other regulatory enzymes in carbohydrate metabolism that are modulated by ATP—all negatively—are pyruvate dehydrogenase complex, citrate synthase, and isocitrate dehydrogenase. Pyruvate dehydrogenase complex is positively modulated by AMP, and citrate synthase and isocitrate dehydrogenase are positively modulated by ADP.

Regulatory Effect of 1NADH/ NAD and 1NADPH/NADP Another example of allosteric mechanisms is the ratio of NADH to 1NAD . NADH and 1NAD can regulate their own formation through negative modulation. NADH is a product of glycolysis. Its buildup would indicate the path- way is not needed to produce additional ATP. If 1NAD accumulates, the oxidative step in glycolysis would be favored. In the fasted state, the liver typically has a high

1NAD /NADH ratio (about 700, meaning that the level of NADH is low) and it produces more glucose than it needs through gluconeogenesis, releasing the glucose into the blood. In contrast, muscle will be actively cataboliz- ing glucose, and its 1NAD /NADH ratio will be lower and will favor lactate production. Dehydrogenase reactions, which involve the interconversion of the reduced and oxi- dized forms of the cosubstrate, are reversible. If metabolic conditions cause either NADH or 1NAD to accumulate, the equilibrium is shifted to return the ratio to normal. Pyruvate dehydrogenase complex is positively modulated

by 1NAD , whereas pyruvate kinase, citrate synthase, and a-ketoglutarate dehydrogenase are negatively modulated by NADH.

Whether the pentose phosphate pathway makes pentoses or NADPH is dependent upon the level of NADPH and

1NADP . Glucose-6-phosphate dehydrogenase is inhibited by high levels of NADPH and acetyl-CoA, which would indicate that demands for lipid biosynthesis are met. If the NADPH levels drop, the pathway can produce ribose. If the cell has more ribose than needed, the pathway follows the reaction on the right side of Figure 3.29 and makes more glucose and more NADPH.

Covalent Regulation Covalent modification is another mechanism of enzyme (resulting in pathway) regulation. This involves the bind- ing or unbinding of a group by a covalent bond and is one of the mechanisms by which hormones can exert their action. Examples include the covalent regulation of glyco- gen synthase and glycogen phosphorylase, enzymes dis- cussed in the sections on glycogenesis and glycogenolysis, respectively.

Phosphorylation inactivates glycogen synthase, whereas dephosphorylation activates it. In contrast, phosphorylation activates glycogen phosphorylase, and dephosphorylation inactivates it. These actions can be controlled by the actions of glucagon and epinephrine. Both hormones function by the phosphorylation of pathway enzymes through the second messenger cAMP.

Genetic Regulation Another important example of enzyme regulation is through genetic control. The abundance of an enzyme can be either induced or suppressed. Such a change might arise through a prolonged shift in the dietary intake of certain nutrients. Induction stimulates transcription of new mes- senger RNA, programmed to produce the enzyme.

Specific hormones can influence (induce or suppress) the expression of a gene. One of the actions of certain hormones such as cortisol is to stimulate protein breakdown and decrease protein synthesis in skeletal muscle. In the liver, cortisol stimulates glycogen synthesis and gluconeogenesis by increasing the expression of several genes that encode for enzymes of the gluconeogenic pathway.

Directional Shifts in Reversible Reactions Another control mechanism for pathways is based on enzyme kinetics, the concentration of the reactants and prod- ucts in the cell. Most enzymes catalyze reactions reversibly,

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100 C H A P T E R 3 • CARbOHYdRAtES

and the preferred direction in which a reversible reaction is proceeding at a particular moment is largely dependent upon the relative concentration of each reactant and product. An increasing concentration of one of the reactants drives or forces the reaction toward forming the other.

This concept is exemplified by the phosphoglucomutase reaction, which interconverts glucose-6-phosphate and glucose-1-phosphate and which functions in the pathways of glycogenesis and glycogenolysis (see Figures 3.13 and 3.15). At times of heightened glycogenolytic activity (rapid breakdown of glycogen), glucose-1-phosphate concentration rises sharply, driving the reaction toward the formation of glucose-6-phosphate. With the body at rest, gluconeogenesis and glycogenesis are accelerated, increasing the concentration of glucose-6-phosphate. This increase in turn shifts the phosphoglucomutase reaction toward the formation of glucose-1-phosphate and ultimately glycogen.

Metabolic Control of Glycolysis and Gluconeogenesis Most enzymatic reactions are reversible, depending on their free energy. Yet certainly in a given cell and generally in the cells of a particular organ the pathways are going in only one direction at a given time. The previous sections reviewed the different methods the body uses for controlling metabolic pathways. Glycolysis and gluconeogenesis provide examples of these control mechanisms in action. Figure 3.30 shows the reactions in both pathways that are under metabolic control by the mechanisms discussed, with the regulation of glycoly- sis on the left and that of gluconeogenesis on the right. The modulators that are activators are indicated by a plus sign, and those that are inhibitors by a minus sign.

The end result of gluconeogenesis is the formation of glucose, the molecule with which glycolysis begins. It is also true that the end product of glycolysis is pyruvate, and pyruvate is the first reactant of gluconeogenesis. As was pointed out earlier, however, gluconeogenesis is not simply the reversal of glycolysis. These two pathways are controlled reciprocally. Which of the two pathways is active at a given time depends on the energy status of the cell. In glycolysis there are three regulated enzymes, all of which catalyze exergonic reactions: hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase. These three reactions are replaced in the gluconeogenic pathway with those catalyzed by glucose-6-phosphatase; fructose-1,6-bisphosphatase; and the pyruvate carboxylase- PEP-carboxylase pair. The control of these reactions is considered for each pathway. The fate of pyruvate is strongly dependent upon acetyl-CoA levels. Acetyl-CoA inhibits the glycolytic enzyme pyruvate kinase allosterically and activates pyruvate carboxylase. This latter enzyme is found only in the mitochondria and is part of the gluconeogenic pathway that transfers mitochondrial pyruvate to PEP. If the

TCA cycle is not active (adequate cellular ATP), the pyruvate is converted to glucose via gluconeogenesis.

In gluconeogenesis, glucose-6-phosphatase is controlled by the level of substrate. Because the Km for this enzyme is much higher than the level of glucose-6-phosphate that is normally present, the reaction proceeds very slowly unless a high concentration of this substrate accumulates. A buildup of glucose-6-phosphate is needed to activate the gluconeogenesis pathway.

Another control point for gluconeogenesis is the enzyme fructose-1,6-bisphosphatase, which is allosterically inhibited by AMP and activated by citrate. The effects of AMP and citrate on this enzyme are the opposite in glycolysis. When AMP levels are low (which means ATP is adequate) the gluconeogenesis pathway is active and glycolysis is reduced. Another allosteric regulator of fructose-1,6-bisphosphatase is fructose-2,6-bisphosphate. The levels of fructose-2,6-bisphosphate are controlled by the enzyme phosphofructokinase-2 (PFK-2). This enzyme is different than the phosphofructokinase of the glycolytic pathway. Fructose-6-phosphate (the substrate of phosphofructokinase of glycolysis) activates PFK-2, which would inhibit gluconeogenesis.

Another means of control for these two pathways is the level of enzymes. In glycolysis, glucokinase, phosphofructokinase, and pyruvate kinase are inducible enzymes, meaning that their concentrations can rise and fall in response to molecular signals such as a sustained change in the concentration of a certain metabolite. In the gluconeogenic pathway glucose-6-phosphatase, fructose bisphosphatase, PEP carboxykinase, and pyruvate carboxylase are inducible. The other enzymes of both pathways are constitutive (Chapter 1), meaning that their rate of synthesis is constant. Glucagon and glucocorticoid hormones are known to stimulate gluconeogenesis by inducing the key gluconeogenic enzymes to form, and insulin may stimulate glycolysis by inducing increased synthesis of key glycolytic enzymes.

The interrelationship among pathways of carbohydrate metabolism is exemplified by the regulation of blood glucose concentration. The integration of the pathways, a topic of Chapter 7, is best understood after metabolism of lipids and amino acids has been discussed (Chapters 5 and 6). Largely through the opposing effects of insulin and glucagon, the fasting serum glucose level normally is maintained within the approximate range of 80–100 mg/dL (4.5–5.5 mmol/L). Whenever blood glucose levels are excessive or sustained at high levels because insulin is insufficient, other insulin-independent pathways of carbohydrate metabolism for lowering blood glucose become increasingly active. Such insulin-independent pathways are indicated in Figure 3.32. The overactivity of these pathways in certain tissues is believed to be partly responsible for the clinical manifestations of type 1 diabetes mellitus.

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C H A P T E R 3 • CARbOHYdRAtES 101

Figure 3.32 Insulin-independent and -dependent pathways of glucose metabolism.

Glucuronates UDP-glucuronates UDP-glucose Glycogen

Insulin-independent pathways

Insulin-dependent pathways

Glycogenesis

(polyol pathway) Fructose Sorbitol Glucose Glucose-6-P

Pentose phosphate pathway (hexose monophosphate shunt)

Proteoglycans Glucosamine 6-P Fructose-6-P Glycolysis and

oxidation

SUMMARY

This chapter has dealt with a subject of vital importance in nutrition: the release and conversion of the energy contained within nutrient molecules into ATP energy usable by the body. It examines an important food source of that energy, carbohydrates. The major sources of dietary carbohydrate are the starches and the disaccharides. In the course of digestion, these are hydrolyzed by specific gly- cosidases to their component monosaccharides, which are absorbed into the intestine cell by active and facilita- tive transport. Practically all fructose and galactose is transported to the liver to be metabolized. Some glucose is transported to the liver, while the majority of glucose is transported into cells of various tissues, passing through the cells’ outer membrane by facilitative transport by way of transporters. Different tissues use different GLUTs that are part of the family of glucose transporters. The GLUT4 that transports glucose into muscle and adipose tissue is stimulated by insulin. Insulin translocates the preformed GLUT4 from intracellular vesicles to the cell membrane. In the cells, monosaccharides are first phosphorylated at the expense of ATP and then can follow any of several integrated pathways of metabolism. In muscle, brain, and adipose tissue, glucose is phosphorylated by hexokinase (types 1 and 2). In the liver, glucose is phosphorylated by an isoenzyme of hexokinase called glucokinase; fructose is phosphorylated mainly by fructokinase; and galactose is phosphorylated by galactokinase.

During times of energy excess, cellular glucose and cer- tain metabolites can be converted to glycogen, primarily in liver and skeletal muscle. Liver glycogen is mostly made from dietary and circulating glucose, while about one- third of the glucose-6-phosphate converted to glycogen is derived from gluconeogenesis (lactate, pyruvate, and TCA cycle intermediates). When energy is needed cellular glu- cose can be routed through the energy-releasing pathways of glycolysis and the TCA cycle for ATP production. Gly- colytic reactions convert glucose (or glucose residues from glycogen) to pyruvate. From pyruvate, either an aerobic

course (complete oxidation in the TCA cycle) or an anaero- bic course (to lactate) can be followed. Nearly all the energy formed by the oxidation of carbohydrates to CO2 and H O2 is released via the TCA cycle, as reduced coenzymes are oxidized by mitochondrial electron transport. On complete oxidation, approximately 40% of this energy is retained in the high-energy phosphate bonds of ATP. The remaining energy supplies heat to the body.

Noncarbohydrate substances derived from the other major nutrients—including lactate from red blood cells and muscle, glycerol from triacylglycerols, and certain amino acids—can be converted to glucose or glycogen by the pathways of gluconeogenesis. The basic carbon skeleton of fatty acids (metabolized to acetyl-CoA units) cannot be converted to a net synthesis of glucose, but some of the car- bons from fatty acids find their way into the carbohydrate molecule due to small amounts of TCA cycle intermedi- ates being used in gluconeogenesis. In gluconeogenesis, the reactions are basically the reversible reactions of gly- colysis, shifted toward glucose synthesis in accordance with reduced energy demand by the body. Three kinase reactions occurring in glycolysis are not reversible, however, requir- ing the involvement of different enzymes and pathways to circumvent those reactions in the process of gluconeogene- sis. Muscle glycogen provides a source of glucose for energy only for muscle fibers in which it is stored because muscle lacks the enzyme glucose-6-phosphatase, which forms free glucose from glucose-6-phosphate. Glucose-6-phosphatase is active in the liver, however, which means that the liver can release free glucose from its glycogen stores into the circulation for maintaining blood glucose and for use by other tissues. The Cori cycle describes the liver’s uptake and gluconeogenic conversion of muscle-produced lactate to glucose.

A metabolic pathway is regulated according to the body’s need for energy or for maintaining homeostatic cellular concentrations of certain metabolites. Regulation is exerted mainly through hormones, through substrate

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102 C H A P T E R 3 • CARbOHYdRAtES

2. the TCA cycle and oxidative phosphorylation, by which high-energy electrons derived from food molecules are passed through the electron transport chain in mito- chondria, creating an energy gradient used to phos- phorylate ADP to form ATP.

Oxidative phosphorylation is the major route for ATP production. Electron flow in the electron transport chain is from reduced cosubstrates to molecular oxygen. Molecular oxygen becomes the ultimate oxidizing agent and becomes H O2 in the process. The downhill flow of electrons and pro- ton translocation generate sufficient energy to affect oxida- tive phosphorylation at multiple sites along the chain. The energy from this process that is not conserved as chemical energy (ATP) is given off as heat. About 60% of the energy assumes the form of heat.

Carbohydrate metabolism, including the energy- releasing, systematic oxidation of glucose to CO2 and H O2 , exemplifies reactions of substrate-level and oxidative phosphorylation. Similar energy transfer happens with the fatty acid and amino acid pathways whenever a dehydra- tion reaction occurs.

The pentose phosphate pathway generates important intermediates not produced in other pathways of the body, such as pentose phosphates for RNA and DNA synthesis and NADPH, which is used in the synthesis of fatty acids and in drug metabolism.

This chapter provides examples of the regulation of metabolism, an important topic in nutrition. This topic is revisited several times in Chapters 7 and 8. Understanding the integration of metabolism and the control of energy balance is important. Much of the effects of exercise, dis- ease, weight loss, and weight gain can be explained with these principles.

concentrations (which can affect the velocity of enzyme reactions), and through allosteric enzymes that can be modulated negatively or positively by certain pathway products.

In Chapters 5 and 6, we see that fatty acids and the car- bon skeleton of various amino acids also are ultimately oxidized through the TCA cycle. The amino acids that do become TCA cycle intermediates, however, may not be completely oxidized to CO2 and H O2 , but instead may leave the cycle to be converted to glucose or glycogen (by gluconeogenesis) should dietary intake of carbohydrate be low. The glycerol portion of triacylglycerols enters the gly- colytic pathway at the level of dihydroxyacetone phosphate, from which point it can be oxidized for energy or used to synthesize glucose or glycogen. The fatty acids from tria- cylglycerols enter the TCA cycle as acetyl-CoA, which is oxidized to CO2 and H O2 but cannot contribute carbon for the net synthesis of glucose. This topic is considered further in Chapter 5.

These examples of the entrance of noncarbohydrate substances into the pathways discussed in this chapter are cited here to remind the reader that these pathways are not singularly committed to carbohydrate metabolism. Rather, they must be thought of as common ground for the inter- conversion and oxidation of fats and proteins as well as carbohydrate. Maintaining this broad perspective will be essential when we move on to Chapters 5 and 6, which examine the metabolism of lipids and proteins, respectively.

Much of the energy needs of the body are met by the production and utilization of ATP. ATP can be generated by two distinct mechanisms:

1. the transfer of a phosphate group from compounds with a very-high-energy phosphate transfer potential to ADP, a process called substrate-level phosphorylation

References Cited

1. Richards AB, Krakowka S, Dexter LB, et al. Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food Chem Toxic. 2002; 40:871–98.

2. van Can JGP, Ijzerman TH, van Loon LJC, et al. Reduced glycaemic and insulinaemic responses following trehalose ingestion: implica- tions for postprandial substrate use. Brit J Nutr. 2009; 102:1395–99.

3. Barrett ML, Udani JK. A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): A review of clinical studies on weight loss and glycemic control. Nutr J. 2011; 10:24.

4. Kellett GL, Brot-Laroche E, Mace OJ, Leturque A. Sugar absorption in the intestine: the role of GLUT2. Annu Rev Nutr. 2008; 28:35–54.

5. Kellett GL, Brot-Laroche E. Apical GLUT2: a major pathway of intes- tinal sugar absorption. Diabetes. 2005; 54:3056–62.

6. Augustin R. The protein family of glucose transport facilitators: it is not only about glucose after all. Life. 2010; 62:315–33.

7. Riby J, Fujisawa T, Kretchmer N. Fructose absorption. Am J Clin Nutr. 1993; 58(suppl 5):S748–53.

8. Jones HF, Butler RN, Brooks DA. Intestinal fructose transport and malabsorption in humans. Am J Physiol Gastrointest Liver Physiol. 2011; 300:G202–06.

9. Thorens B, Mueckler M. Glucose transporters in the 21st century. Am J Physiol Endocrinol Metab. 2010; 298:E141–45.

10. Richter EA, Hargreaves M. Exercise, GLUT4, and skeletal muscle glu- cose uptake. Physiol Rev. 2013; 93:993–1017.

11. Aziz A. The glycemic index: methodological aspects related to the interpretation of health effects and to regulatory labeling. J AOAC Intl. 2009; 92:879–87.

12. Venn BJ, Green TJ. Glycemic index and glycemic load: measurement issues and their effect on diet-disease relationships. Eur J Clin Nutr. 2007; 61:S122–31.

13. Fernandes G, Velangi A, Wolever TM. Glycemic index of potatoes commonly consumed in North America. J Am Diet Assoc. 2005; 105:557–62.

14. Schwingshackl L, Hoffmann G. Long-term effects of low glycemic index/load vs. high glycemic index/load diets on parameters of obesity and obesity-associated risks: A systematic review and meta- analysis. Nutr Metab Cardiovasc Dis. 2013; 23:699–706.

15. Greenwood DC, Threapleton DE, Evans CEL, et al. Glycemic index, glycemic load, carbohydrates, and type 2 diabetes. Diabetes Care. 2013; 36:4166–71.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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C H A P T E R 3 • CARbOHYdRAtES 103

16. Mirrahimi A, Chiavaroli L, Srichaikul K, et al. The role of glycemic index and glycemic load in cardiovascular disease and its risk factors: a review of the recent literature. Curr Atheroscler Rep. 2014; 16:381.

17. Foster-Powell K, Holt SHA, Brand-Miller JC. International table of glycemic index and glycemic load values: 2002. Am J Clin Nutr. 2002; 76:5–56.

18. Smythe C, Cohen P. The discovery of glycogenin and the prim- ing mechanism for glycogen biosynthesis. Eur J Biochem. 1991; 200:625–31.

19. Garrett RH, Grisham CM. Biochemistry. 4th ed. Belmont, CA: Thomson Brooks/Cole Publishers. 2010.

20. Buchbinder JL, Bath BL, Fletterick RJ. Structural relationships among regulated and unregulated phosphorylases. Annu Rev Biophys Bio- mol Struct. 2001; 30:191–209.

21. Hosler J, Ferguson-Miller S, Mills D. Energy transduction: proton transfer through the respiratory complexes. Annu Rev Biochem. 2006; 75:165–87.

22. Boyer P. The ATP synthase-A splendid molecular machine. Annu Rev Biochem. 1997; 66:717–49.

23. Tyler D. ATP synthesis in mitochondria. In: The Mitochondrion in Health and Disease. New York: VCH Publishers, Inc. 1992. pp. 353–402.

24. Hatefi Y. The mitochondrial electron transport and oxidative phos- phorylation system. Annu Rev Biochem. 1985; 54:1015–69.

25. Kaleta C, de Figueiredo LF, Werner S, et al. In silico evidence for glu- coneogenesis from fatty acids in humans. PLoS Comput Biol. 2011; 7(7):e1002116.

Web Sites

www.nlm.nih.gov National Library of Medicine

www.medscape.com WebMD. Provides specialty information and education for physicians and other health professionals.

www.cdc.gov Centers for Disease Control and Prevention

www.ama-assn.org American Medical Association

http://vcell.ndsu.edu/animations/ A series of “Virtual Cell” animations demonstrating electron transport chain, ATP synthesis, insulin signaling, and other biological processes funded by the National Science Foundation. Other animations are applicable to other chapters and are a good resource.

www.hopkinsmedicine.org Johns Hopkins School of Medicine

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104

system—may overestimate actual intake by individuals due to inclusion of nonedible food portions and food lost through waste and spoilage in the home and marketing system. Con- sequently, the USDA provides loss-adjusted food availability data to more closely reflect actual intake [7].

Documenting food intake by direct or indirect methods is just the first step in learning what nutrients we consume. Con- verting food intake into nutrient intake requires knowledge about the chemical composition of every food consumed. The Agricultural Research Service of the USDA maintains the most comprehensive system for collecting and disseminating food composition data. Information contained in the USDA National Nutrient Database for Standard Reference provides the basis for nearly all public and commercial nutrient databases and food composition tables used in the United States and several for- eign countries [8]. The Nutrient Database contains information for approximately 150 components of food, including essential and nonessential nutrients, for over 8,600 foods. The database is constantly being updated and expanded as new information becomes available. The data comes from academic research, the food industry, government laboratories, and independent food-testing laboratories. Values in the database may also be based on calculations using appropriate algorithms, factors, or recipes.

Valuable information regarding food and nutrient intake can be obtained by combining the food availability and

Although a seemingly simple question, measuring the food and nutrients we eat is a difficult task. Several methods, based primarily on self-reported data, have been used to directly assess the amount of food consumed by indi- viduals [1]. The accuracy of such methods depends entirely on the ability of subjects to know the foods they are eating; to know portion size and record the amount of each food; to record every food and beverage consumed; and to be truth- ful. In view of these requirements, it is easy to see why direct methods frequently result in underreporting of intake, par- ticularly by subjects with elevated body mass index [2–5]. The National Health and Nutrition Examination Surveys (NHANES), funded and managed by the Centers for Disease Control and Prevention, have been ongoing since the 1960s and represent the most widely used dataset for estimating food intake using direct assessment [6].

A different approach to estimating food intake is to mea- sure the amount of food available for human consumption in the United States. The total amount available of each food category is then divided by the total population for each year and expressed on a per capita basis. The U.S. Department of Agriculture has been reporting such data since 1909, which is useful for determining food consumption trends because they are a proxy for actual food intake. Food availability data— sometimes called food “disappearance” because the data reflect available food that “disappears” into the food marketing

nutrient composition data. Each database is freely accessible and can be downloaded for combining, although the USDA has already done much of the work for us. Spreadsheets con- taining the combined data, called the Nutrient Content of the U.S. Food Supply, are also available for downloading [9]. With this arsenal of data, one can choose to examine the type and amount of food consumed, their nutrient composition, or the amount of nutrients consumed by major food groups.

CARBOHYDRATES IN THE FOOD SUPPLY

Examining the USDA data for the years 1970 to 2010 reveals many things. First, carbohydrates are the most abundant macronutrient (by weight) in the food supply and contribute most of the total energy in the American diet (as shown in Figure 1). Second, during this 40-year period, carbohydrate availability increased about 10% [10]. This period of time is significant because obesity prevalence in children and adults increased in parallel [11]. It is tempting to blame the increase in carbohydrate intake for the increase in obesity prevalence, but one should be cautious in assuming a direct causal relationship on the basis of correlations alone without further research.

A third observation gleaned from the USDA data for the year 2010 is that most of the carbohydrate was provided by grain products (42%) and sugar and sweeteners (35%). The remaining contributors of carbohydrate were comparatively

WHAT CARBOHYDRATES DO AMERICANS EAT?

P E R S P E C T I V E

Figure 1 Per capita availability of macronutrients from all food sources in the U.S. food supply.

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C H A P T E R 3 • CARbOHYdRAtES 105

availability and nutrient composition databases. This approach offers the advantage of examining trends over time (decades), and it avoids the difficulties of surveying individuals directly. Using the loss-adjusted USDA data also allows us to deter- mine the food sources of all major nutrients and the amounts available on a per capita basis. In this Perspective, examination of the food sources and amounts of carbohydrate in the U.S. food supply reveals the following:

● Carbohydrates are the most abundant macronutrient in the food supply and provide the majority of dietary energy

● Grain products are the primary source of dietary carbohydrate, followed by sugar and sweeteners

● Between 1970 and 2010, the availability of carbohydrate from all food sources increased 10%

● Between 1970 and 2010, the availability of carbohydrate from grain products increased 24%

● Between 1970 and 2010, the availability of carbohydrate from sugar and sweeteners increased 1%

● The sugar and sweeteners category has not significantly increased because HFCS have merely replaced sugar

● Upon digestion, food carbohydrates yield four times more glucose than fructose

● Between 1970 and 2010, the availability of glucose from all food sources increased 13%

● Between 1970 and 2010, the availability of fructose from all food sources did not change.

Conclusions from these findings are best made when the entire picture is considered. Misinterpretations can easily be made when only a portion of the findings are used. For exam- ple, the use of HFCS has significantly increased since 1970,

to a lesser extent, galactose if dairy products are consumed). Starch yields exclusively glucose; sugar and HFCS yield equal amounts of glucose and fructose; dairy products containing lactose yield glucose and galactose; and some foods provide glucose and fructose as monosaccharides. In view of the heightened awareness of fructose as a potential contributor to obesity-related diseases [12], it is useful to express the USDA food availability data in terms of the component monosac- charides resulting from carbohydrate digestion. Making such a calculation, as shown in Figure 3, allows us to examine the amounts of glucose, fructose, and galactose available for absorption from all digestible carbohydrates consumed.

Figure 3 can be interpreted as the “carbohydrate” line from Figure 1, broken down into its monosaccharide units. Viewing the data this way clearly shows glucose, not fructose, is the most abundant saccharide provided by food carbohydrates. Recall that the major food source of carbohydrates is grain products that contribute only glucose when starch is digested. The second most abundant food source of carbohydrates, sugars and sweeteners, contributes equal amounts of glucose and fructose, while fruits and vegetables contribute smaller amounts of both glucose and fructose. Thus, every food cate- gory that contains carbohydrate contributes glucose, resulting in four times more glucose than fructose in the food supply.

Another important observation from Figure 3 is the change that occurred in monosaccharide availability between 1970 and 2010. The overall trend in glucose availability increased 13%, whereas the overall trend in fructose availability did not change during the 40-year period [10]. Once again, these facts contra- dict information found in the lay press and on social media that incorrectly emphasize an increase in fructose when the spot- light should be focused on the significant increase in glucose.

Conclusion Measuring food and nutrient intake of Americans can be accomplished by indirect methods using the USDA food

minor and included vegetables (7%), fruits (6%), and dairy products (6%). Moreover, since 1970 the carbohydrate contributed by grain products increased 24%, whereas the contribution from sugar and sweeteners as a group increased only about 1% [9]. Many consumers may be confused by this outcome because the facts contradict the avalanche of misin- formation in the lay press and on social media claiming that intake of sugar and sweeteners has skyrocketed—a conclu- sion that is clearly not supported by data.

Confusion may also stem from the widespread misunder- standing of sugar and sweeteners in the food supply. As illus- trated in Figure 2, sugar (sucrose) was the primary sweetening agent used in 1970. High-fructose corn syrups (HFCS) were introduced after 1970 as a sugar alternative because of lower cost and desirable functional properties. Two major types of HFCS are used by food manufacturers, HFCS-42 and HFCS-55. The saccharide composition of HFCS-42 is 42% fructose, 52% glucose, and 6% other saccharides, whereas the saccharide composition of HFCS-55 is 55% fructose, 41% glucose, and 4% other saccharides. Food manufacturers generally use HFCS-42 as a sweetening agent in dry products such as cereals and baked goods. HFCS-55 is used mainly in beverages such as fruit juices and soft drinks. When present together in the U.S. food supply, the two HFCS contribute about equal proportions of fructose and glucose, which is identical to the saccharide composition of sugar (50% fructose, 50% glucose). So while it is true HFCS have significantly increased in the food supply since 1970, the availability of sugar and sweeteners combined has changed very little, due to the replacement of sugar with HFCS. Consum- ers can be easily misled when they hear only the HFCS story.

GLUCOSE VERSUS FRUCTOSE

All digestible carbohydrates in the food supply, irrespective of the food source, must be broken down to their monosaccha- ride units for absorption into the body. Nature provides foods that, when digested, yield mostly glucose and fructose (and,

Figure 2 Per capita availability of carbohydrates from sugar and high-fructose corn syrups (HFCS) in the U.S. food supply.

Sugar HFCS-42 HFCS-55

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106 C H A P T E R 3 • CARbOHYdRAtES

8. U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 27, May 2015. http://ndb .nal.usda.gov/ Accessed 10/25/2015.

9. U.S. Department of Agriculture, Center for Nutrition Policy and Promotion. Nutrient content of the US food supply. http://www.cnpp.usda.gov/USfoodsupply Accessed 10/25/2015.

10. Carden TJ, Carr TP. Food availability of glucose and fat, but not fructose, increased in the US between 1970 and 2009: analysis of the USDA food availability data system. Nutr J. 2013; 12:130.

11. Centers for Disease Control and Prevention, National Center for Health Statistics. Health, United States, 2013: with special feature on prescription drugs. Hyattsville, Maryland. 2014.

12. Rippe JM, Angelopoulos TJ. Fructose-containing sugars and cardiovascular disease. Adv Nutr. 2015; 6:430-9.

Nutrition Examination Survey: Underreporting of energy intake. Am J Clin Nutr. 1997; 65 (4 Suppl):1203S–1209S.

3. Rennie KL, Coward A, Jebb SA. Estimating under- reporting of energy intake in dietary surveys using an individualised method. Brit J Nutr. 2007; 97:1169–76.

4. Poslusna K, Ruprich J, de Vries JHM, et al.. Misreporting of energy and micronutrient intake estimated by food records and 24 hour recalls, control and adjustment methods in practice. Brit J Nutr. 2009; 101(Suppl. 2): S73–S85.

5. Stice E, Palmrose CA, Burger KS. Elevated BMI and male sex are associated with greater underreporting of caloric intake as assessed by doubly labeled water. J Nutr. 2015; 145:2412–8.

6. Centers for Disease Control and Prevention, National Center for Health Statistics. National Health and Nutrition Examination Survey. http://www.cdc.gov/ nchs/nhanes.htm Accessed 10/25/2015.

7. U.S. Department of Agriculture, Economic Research Service. Food availability (per capita) data system. http://www.ers.usda.gov/data-products/food -availability-(per-capita)-data-system/.aspx Accessed 10/25/2015.

leading some to conclude that HFCS (and the fructose they contribute) are the cause of obesity and metabolic diseases. However, when one considers the entire picture, the increased use of HFCS has mirrored the decline in sugar usage, resulting in virtually no change in the amount of saccharides contrib- uted by the combined sugar and sweeteners group. Also, the glucose-to-fructose ratio in sugar and the HFCS together is approximately the same and has not changed since 1970, so fructose availability has remained relatively unchanged.

Perhaps the most important conclusion from the USDA data should focus on glucose as the major saccharide con- tributed by carbohydrates in the U.S. food supply. Significantly more glucose compared to fructose is available for intestinal absorption as a result of eating a typical American diet. Furthermore, the overall trend in glucose availability has increased since 1970, due mainly to increased availability of grain products. When addressing the dietary factors that contribute to obesity, it is logical to focus attention on all car- bohydrates, the main energy source from food, to control total energy intake. The USDA data indicate that glucose from starch in grain products is the major carbohydrate that contributes to total energy intake.

References Cited

1. Thompson FE, Byers T. Dietary assessment resource manual. J Nutr. 1994; 124:2245S–2317S.

2. Briefel RR, Sempos CT, McDowell MA, et al. Dietary methods research in the third National Health and

Figure 3 Per capita availability of monosaccharides from digestible carbohydrates in the U.S. food supply.

0

50

100

150

200

250

1970 1975 1980 1985 1990 1995 2000 2005 2010

M on

os ac

ch ar

id e

Av ai

la bi

lit y

(g ra

m s/

da y)

Year

Glucose Fructose Galactose

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107

F IBER not only enhances the health of the gastrointestinal tract but fiber-rich foods play key roles in the prevention and management of several diseases. The 2015 Dietary Guidelines Advisory Committee for the Dietary Guidelines for Americans has labeled fiber as a “nutrient of pub- lic health concern,” a designation based on findings that fiber intakes are low among most Americans and that fiber is important for health [1]. The varied health benefits of fiber are related to the fact that fiber is not a single entity or even a group of chemically related compounds, but instead consists of multiple different components with distinctive characteristics. This chapter addresses definitions, chemistries, properties, sources, health benefits, allowed health claims, food labels, and recommended intake of fiber.

DEFINITIONS

With the publication of the 2002 Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Protein, and Amino Acids by the National Academy of Sciences Food and Nutrition Board, definitions for dietary, functional, and total fiber were established. Dietary fiber refers to nondigestible (by human digestive enzymes) carbohydrates and lignin that are intact and intrinsic in plants [2]. Dietary fibers, listed later in Figure 4.2, include cellulose, hemicellulose, pectins, lignin, gums, b-glucans, fructans, and resistant starches [2]. Functional fiber consists of isolated, extracted, or manufactured nondigestible carbohydrates that have been shown to have beneficial physiological effects in humans [2]; they are usually added to foods as well as found in supplements. All dietary fibers are functional fibers with the exceptions of hemicellulose, fructans, and lignin (the Food and Nutrition Board stated that fructans and lignin require additional studies showing beneficial physiological effects in humans to be classified as functional fibers) [2]. Psyllium, a mucilage, is considered a functional fiber [2]. Chitin and chitosan, polydextrose and polyols, and resistant dextrins require additional studies showing positive physiological effects in humans to be con- sidered functional fibers [2]. The term total fiber refers to dietary fiber present within the food plus functional fiber that has been added to the food.

In 2009, a branch of the World Health Organization adopted another definition of dietary fiber: Carbohydrate polymers with 10 or more monomeric units (i.e., monosaccharides), which are not hydrolyzed by human digestive enzymes and (a) are in foods (intrinsic and intact), or (b) have been extracted from food and have physiological benefits to health, or (c) are synthetic or modified and have physiological benefits to health [3]. Not included under this definition are oligosaccharides with degrees of polymerization between 3 and 9 (i.e., oligosaccharides containing chains of three to nine

FIBER4

DEFINITIONS

FIBER AND PLANTS

CHEMISTRY AND CHARACTERISTICS OF FIBER Cellulose Hemicellulose Pectins Lignin Gums β-Glucans Fructans Resistant Starch Mucilages (Psyllium) Polydextrose and Polyols Resistant Dextrins Chitin and Chitosan

SELECTED PROPERTIES OF FIBER AND THEIR PHYSIOLOGICAL IMPACT Solubility in Water Viscosity and Gel Formation Fermentability

HEALTH BENEFITS OF FIBER Cardiovascular Disease Diabetes Mellitus Appetite and/or Satiety and Weight Control Gastrointestinal Disorders

FOOD LABELS AND HEALTH CLAIMS

RECOMMENDED FIBER INTAKE

SUMMARY

P E R S P E C T I V E

THE FLAVONOIDS: ROLES IN HEALTH AND DISEASE PREVENTION

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108 C H A P T E R 4 • FIBER

cellulose and pectin as well as some hemicellulose and, in selected fruits, some fructans and lesser amounts of other fibers. Legumes are also fiber-rich, containing cellulose, hemicellulose, pectins, gums, galactooligosaccharides, and resistant starches, among others. This next section reviews the chemistry and characteristics of fibers. Figure 4.2 shows these fibers and selected characteristics of the fibers; these characteristics and their impact on physiological processes and health are discussed in later sections of the chapter.

CHEMISTRY AND CHARACTERISTICS OF FIBER

Cellulose Cellulose (Figure 4.3a), a dietary fiber and functional fiber, is a long, linear polymer (a high-molecular- weight substance made up of a repeating chain) of up to 10,000 b (1-4)–linked glucose units. Hydrogen bonding between sugar residues in adjacent, parallel-running cel- lulose chains imparts a three-dimensional structure to cellulose. Being a large, linear, neutrally charged molecule, cellulose is water insoluble, although it can be modified chemically (e.g., carboxymethyl cellulose, methylcel- lulose, and hydroxypropyl methylcellulose) for use as a food additive and this modified form may be more water soluble and a little more fermentable by colonic bacteria than naturally occurring cellulose. Cellulose that is found naturally in foods is not typically degraded by colonic bacteria. This nonfermentable characteristic of cellulose helps promote laxation. Examples of some cellulose-rich foods include whole grains, bran, legumes, peas, nuts, root vegetables, vegetables of the cabbage family, seeds (mainly the outer covering), and apples. Purified, pow- dered cellulose (usually isolated from wood) and modified cellulose are added to foods, for example, as a thickening or texturing agent or to prevent caking or syneresis (leak- age of liquid). Some examples of foods to which cellulose or a modified form of cellulose is added include breads, cake mixes, sauces, sandwich spreads, dips, frozen meat products (e.g., chicken nuggets), and fruit juice mixes.

monosaccharides) such as some fructooligosaccharides and galactooligosaccharides [3]. The term fiber that appears on food labels reflects dietary fiber, but includes oligosaccharides [4].

FIBER AND PLANTS

Fiber is found in plant foods. Figure 4.1 shows the anatomy of a wheat plant. The endosperm of the plant contains mostly starch along with small quantities of fiber (mainly cellulose, hemicellulose, and resistant starch). The germ layer is rich pri- marily in some vitamins, minerals, and essential fatty acids, but also contains small amounts of fiber (mainly cellulose, lignin, and fructans). It is the bran component of cereals that contains the most fiber (over 95%). The outer bran layer of cereals consists of primary and secondary cell walls. These walls are fiber-rich, containing strands of cellulose arranged within a matrix of other fibers, especially hemicellulose and pectins, but also lesser amounts of fructans, resistant starch, and b-glucans. Other substances such as suberin ( consisting of various phenolic compounds, long-chain alcohols, and polymeric esters of fatty acids), cutin (also made of polymeric esters of fatty acids that is secreted onto the plant surface), and waxes (complex hydrophobic, hydrocarbon compounds that coat the plant’s external surfaces) are also components of the cell wall but do not contribute to the fiber content. Additional fibers may also be found within plants, but these vary with the plant species, the part of the plant (leaf, root, or stem), and the plant’s maturity.

Whole-grain cereals and grain products provide cellulose, hemicellulose, lignin, some gums, b-glucans, some galactooligosaccharides (mainly raffinose and stachyose), and some fructans. Of the cereals, rye and barley typically contain more fiber than other grains. Fruits and vegetables provide almost equal quantities (~30%) of

Figure 4.1 The partial anatomy of a wheat plant.

Endosperm

Germ

A wheat kernel

Husk (chaf f )

Bran layers

Kernel

Stem

Root

Figure 4.2 Dietary fibers and some of their selected properties.

Dietary Fibers

Lignin

Cellulose

Hemicellulose*

Pectins*

β-glucans

Gums

Fructans

Resistant starches

Soluble Dietary Fibers Fructans Pectins* β–glucans Gums (guar) Psyllium**

Fermentable

Viscous gel- forming

Insoluble

Nonfermentable

* Some are more soluble than others ** Not as soluble as others listed

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C H A P T E R 4 • FIBER 109

Figure 4.3 Chemical structures of dietary fibers and some functional fibers.

OH H H

O

O

CH2OH

CH2OH

H

H OH

H

OH H

O

CH2OH

H

H OH

(a) Cellulose

H

O

H

OH H

O

CH2OH

H

H OH

H

OO

H

OH

O

CH2OH

OH

OH

O

CH2OH

OH

(f) -glucan (from oats)

O OH

O

CH2OH

OH

O OH

O

CH2OH

OH

O

OH H H, OH

O

H

H

H

D-xylose

OH

(b) Hemicellulose (major component sugars)

Backbone chain

H

HO OH

OH H, OH

O

H

H

D-mannose

H

H

HO OH H

H, OH

O

CH2OH

H

H

D-galactose

OH

H

HO

CO2H

OH H H H, OH

O

H

H

H

L-arabinose

OH

Side chains

H

HO

OH H, OH

O

H

H

4-O-methyl-D-glucuronic acid

OH

H

CH3O OH H

H, OH

O

CH2OH

H

H

D-galactose

OH

H

HO

(c) Pectin

OH

O

C—OCH3

OH

O

O

(d) Phenols in lignin

OCH3

Trans-coniferyl

CH CHCH2OH

—GALP—GALP—GALP—GALP—

HO

OH

O

C—OH

OH

OH

O

C—OCH3

OH

O

O

O

O

OH

O

C—OH

OH

O

O

(e) Gum arabic X

X—GALP

GA

4 1 4 1 4 1

3

1

3

1

X

X

X—GALP

GA

X

Trans-p-coumaryl

CH CHCH2OH

HO

OCH3

Trans-sinapyl

X: L-rhamnopyranose or L-arabinofuranose GALP: galactopyranose GA: glucuronic acid

CH CHCH2OH

HO

CH3O

O

OH

O

CH2OH

OH

O O

β

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110 C H A P T E R 4 • FIBER

O

O

CH2OH

OH

OH

OH

O

CH2

OH

(i) Raf f inose

O HO

O H

HO

HO

HO

HO

OH

O

OH

(j) Stachyose

(k) Verbascose

galactose

galactose

galactose

glucose fructose

CH2OH

CH2OH

CH2OH

O CH2

O CH2

OH

O

OH glucose

HO

OH

O

OH

HO

O HO

O

OH fructose

CH2OH

CH2OH

HO

OH

O

OH

HO

galactose galactose 2

CH2

OH

O

OH glucose

HO

CH2

OH

O

OH

O HO

O

HO fructose

CH2OH

HOCH2

HOCH2

O O

nHO

CH2 HO

O

O

HO

HO

O

O

HO OH

OH

O

(g) Inulin

HO CH2

HO CH2

HO CH2

HO CH2

HO

HO

O

nHO

CH2

CH2

OH

HO

O

O

HO

CH2

HO

O

O

(h) Fructooligosaccharide

HO CH2

HO CH2

HO CH2

Figure 4.3 (Continued)

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C H A P T E R 4 • FIBER 111

Lignin Lignin is a highly branched polymer of phenol units (versus sugars) with strong intramolecular bonding. The primary phenols that compose lignin include trans-coniferyl, trans- sinapyl, and trans-p-coumaryl, shown in Figure 4.3d. Lignin provides structural support in plant cell walls. It is found in the bran layer of cereals and in the stems and seeds of fruits and vegetables. Lignin is insoluble in water, has hydrophobic binding capacity, and is generally not fermented by colonic bacteria. Lignin is a dietary fiber and may serve as a functional fiber. Foods high in lignin include wheat, rye, mature root vegetables such as carrots, flaxseed, and fruits with edible seeds such as many berries.

Gums Gums, also called hydrocolloids, are secreted at the site of plant injury by specialized secretory cells and can be exuded from plants (i.e., forced out of plant tissues). Gums that originate as tree exudates include gum arabic, gum karaya, and gum ghatti; gum tragacanth is a shrub exudate. Gums are often highly branched and are composed of a variety of sugars and sugar derivatives. Gum arabic, shown in Figure 4.3e,for example, contains a main galactose back- bone joined by b (1-3) linkages and b (1-6) linkages with side chains of galactose, arabinose, rhamnose, glucuronic acid, or methylglucuronic acid. The nonreducing ends ter- minate with a rhamnopyrosyl unit. Of the tree exudates, gum arabic is most commonly used as a food additive to promote gelling, thickening, and stabilizing. It is found in candies such as caramels, gumdrops, and toffees, as well as in other assorted products.

Guar gum and locust bean gum (also called carob gum) are made from the ground endosperm of guar seeds and locust bean seeds, respectively. These water-soluble gums consist mostly of galactomannans, which contain a mannose backbone in 1-4 linkages and in a 2:1 or 4:1 ratio with galactose present in the side chains. Guar galactomannans have more branches than locust bean galactomannans. Both guar gum and locust bean gum are added as a thickening agent and water-binding agent (among other roles) to products such as bakery goods, sauces, dairy products, ice creams, dips, and salad dressings. Gums are also found naturally in foods such as oatmeal, barley, and legumes. Gums are dietary and functional fibers. They are water soluble, fermentable by colonic bacteria, and some (like guar gum) form viscous gels.

b-Glucans b-glucans (Figure 4.3f) are homopolymers of glucose units, but are smaller in size and contain different linkages than cellulose. Oat b-glucan consists of a chain of glucoses joined mostly in b (1-4) linkages but also some b (1-3) linkages. b-glucans are water soluble, highly fermentable by colonic

Hemicellulose Hemicellulose, another dietary fiber, consists of a heterogeneous group of polysaccharides. These polysaccharides vary among plants and within a plant depending on location. One example of a hemicel- lulose structure is b (1-4)–linked D-xylopyranose units with branches of 4-O-methyl D-glucopyranose uronic acids linked by a (1-2) bonds or with branches of L-arabinofuranosyl units linked by a (1-3) bonds. Hemicelluloses contain both hexoses and pentoses in their backbone and branched side chains. The b (1-4)– linked sugars in the backbone, which form a basis for hemicellulose classification, usually include the pentose xylose and hexoses such as mannose and galactose, while sugars such as arabinose, glucuronic acid, and galac- tose, among others, are found in the side chains. Some of these sugars are shown in Figure 4.3b. The sugars in the side chains confer important characteristics on the hemicellulose. For example, hemicelluloses that con- tain acids in their side chains are slightly charged and more water soluble, while other hemicelluloses are water insoluble. Similarly, fermentability of the hemicelluloses by intestinal bacteria is also influenced by these sugars and their positions. For example, hexose and uronic acid components of hemicellulose are more accessible to bacterial enzymes and thus more fermentable than are the other hemicellulose sugars. Foods that are relatively high in hemicellulose include whole grains as well as nuts, legumes, and some vegetables and fruits.

Pectins Pectins, a dietary and functional fiber, represent another family of heterogeneous polysaccharides found in plant cell walls, intercellular regions of plants, and in the outer skin and rind of some fruits and vegetables. Galacturonic acid is a primary constituent of pectin’s backbone and is found as an unbranched chain of a (1-4)–linked D-galacturonic acid units, as shown in Figure 4.3c. Chains of pentoses (xylose and arabinose) and hexoses (galactose, rhamnose, and fucose) are attached to pectin’s backbone. Rich sources of pectins include many fruits—apples, berries, apricots, cherries, grapes, and citrus fruits—as well as legumes, nuts, and some vegetables. In some fruits, pectin is broken down as the fruit ripens and becomes softer. Commercially, pectins are usually extracted from citrus peel or apples and may be added to products, such as fruit strips, fruit juices, and icing, among others. In jellies and jams, pectin is used to promote gelling. Pectin is added to some enteral nutri- tion products used for tube feeding to provide a source of fiber in the diet. Pectins are water soluble and have a high ion-binding potential. In the digestive tract, pectins form viscous gels and are almost completely fermented by bacteria in the colon.

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112 C H A P T E R 4 • FIBER

is physically inaccessible to digestion due to its location within a section of the plant’s structure. Food sources of RS1 include whole or partially milled grains and seeds. Resistant starch–designated RS2 represents starch that resists digestion because it is tightly packaged inside of granules within foods. The tight packaging is associ- ated with the linear structure of amylose (a component of starch along with amylopectin), and is especially prevalent in some “raw or uncooked” plant foods such as unripe (green) bananas, potatoes, and some legumes and maize. The heating of foods with these starches, how- ever, gelatinizes the starch and increases its ability to be digested. Both RS1 and RS2 are dietary fibers.

Another resistant starch, RS3, is called retrograde starch or amylose. It is formed with moist-heat cooking and then cooling of starch that has gelatinized. This cooking and then cooling alters the starch to make it more resistant to digestion. Examples of foods rich in RS3 include cooked and cooled potatoes, rice, pasta, bread, and some corn. Lastly, RS4 results from chemical modifications of starch (usually isolated from corn). Examples of modifications include the formation of starch esters or cross-bonded starches, which retard the ability of the starch to swell during cooking and thus keep it in a more granular form that resists digestion. This type of resistant starch is found in some corn-based products. RS3 and RS4 are functional fibers, and both may be partially fermented by colonic bacteria. RS3 may also stimulate the growth of healthful bacteria in the colon, and may improve the glycemic response following carbohydrate ingestion. Americans are thought to consume up to about 10 g of resistant starch daily. Consumption of up to 20 g of resistant starch has been recommended to obtain health benefits.

Mucilages (Psyllium) Mucilages are plant polysaccharides with a structure simi- lar to gums. Mucilages are found in the seeds of a variety of plants, including flax and psyllium, among others. Psyl- lium, from the husk of psyllium seeds (also called plantago or fleas seed), contains several polysaccharides, including arabinoxylan, which has a xylose backbone and arabinose side chains. Psyllium is fairly soluble in water, containing about 70–80% water-soluble polysaccharides and 20–30% water-insoluble polysaccharides. Products to which psyl- lium has been added have high water-binding capacities and form viscous gels in the digestive tract. Psyllium is added to Metamucil® for its laxative properties as well as other products to promote reductions in serum lipids. The Food and Drug Administration permits a health claim for psyllium with consumption of 10.2 g (providing 7 g of viscous fiber) resulting in significant reductions in serum LDL cholesterol [5]. Foods containing psyllium that bear a health claim are required to state on the label that the

bacteria, and form viscous gels within the digestive tract. b-glucans are found in relatively high amounts in two grains, oats (oat bran, rolled oats, and whole oat flour) and barley (whole grain and dry milled). b-glucans extracted from cere- als are used commercially as a functional fiber because of their effectiveness in reducing serum cholesterol and moderating blood glucose concentrations. The Food and Drug Admin- istration permits a health claim for b-glucans describing reductions in serum LDL cholesterol resulting from the daily consumption of $ 3 g of b-glucans from oats [5].

Fructans Fructans, sometimes called polyfructose, include inulin, oligofructose, and fructooligosaccharides. Fructans are chemically composed of fructose units in chains of varying length. Inulin consists of a b (2-1)–linked fructose chain that contains from 2 to about 60 units (usually at least 10), with a glucose molecule at the end of the fructose chain linked by an a (1-2) bond (Figure 4.3g). Oligofructose is similar in structure to inulin but generally contains less than 10 fructose units. Inulin and oligofructose are dietary fibers. Fructooligosaccharides are a functional fiber formed from the partial hydrolysis of inulin or synthesized from sucrose by adding fructose; they typically contain about two to four or five fructose units, and may or may not contain an end glucose molecule (Figure 4.3h). Fructans, especially fructooligosaccharides and oligofruc- tose, are water soluble and highly fermentable by colonic bacteria, but do not form viscous gels in the digestive tract. Fructooligosaccharides and inulin also function as prebi- otics, promoting the growth of healthful bifidobacteria.

Fructans (mainly inulin) are found naturally in some plants. The most common food sources of inulin include chicory, asparagus, leeks, onions, garlic, Jerusalem artichoke, tomatoes, and bananas; fresh artichoke, for example, contains about 5.8 g per 100 g, and minced dried onion flakes provide 4 g per 100 g [6]. Wheat, barley, and rye also contain some fructans. Fructans are also added to some foods. Oligofructose is commonly used, for example, in cereals, fruit preparations for yogurt, dairy products, and frozen desserts. Inulin is used to replace fat in fillings, table spreads, dairy products, dressings, and frozen desserts, to name a few examples. Both inulin and fructooligosaccharides are found in supplements (such as fiber gummy supplements), and fructooligosaccharides are also added to foods. Americans are thought to consume up to about 4 g of fructans each day from foods.

Resistant Starch Resistant starch (RS) is starch that cannot be or is not easily enzymatically digested. There are four main types (numbered 1 to 4) of resistant starch. RS1 is starch that

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C H A P T E R 4 • FIBER 113

Chitosan is a deacetylated form of chitin. Both chitin and chitosan have high molecular weights, are insoluble in water, and can adsorb (interact or complex with) dietary lipids, primarily unesterified cholesterol and phospholipids, and promote their excretion in the feces. Modified forms of chitin and chitosan have been designed for nutraceutical and functional food applications. The Food and Nutrition Board of the National Academy of Sciences designated chitin and chitosan as functional fibers pending the results of additional studies showing physiological effects in humans. Table 4.1 lists some food sources of fiber.

SELECTED PROPERTIES OF FIBER AND THEIR PHYSIOLOGICAL IMPACT

The physiological effects and ultimately the health benefits of fiber vary based on certain characteristics of fiber, most notably viscosity and fermentability, but also to a lesser extent based on solubility and chain length (longer versus shorter chain). Shorter-chain fibers include fructooligosacharides (see section on fructans) and galactooligosaccharides (also called galactans). This latter group includes sugars such as raffinose, stachyose, and verbascose. Raffinose is a trisaccharide of fructose, glucose, and galactose (Figure 4.3i). Stachyose is a tetrasaccharide of fructose, glucose, and galactose to which another galactose is linked (Figure 4.3j). Verbascose is an oligosaccharide containing fructose, glucose, and three galactose molecules (Figure 4.3k). Galactooligosaccharides are found naturally in human milk and in peas (field peas, chickpeas, and green peas), lentils, and beans (such as soy,

food should be eaten with at least a full glass of liquid and that choking may result if the product is not ingested with enough liquid [5]. In addition, the label should state that the food should not be eaten if a person has difficulty swallowing [5].

Polydextrose and Polyols Polydextrose is a polysaccharide consisting of glucose and sorbitol units that have been polymerized at high temperatures and under a partial vacuum. Polydextrose, available commercially, is added to foods as a bulking agent or as a sugar substitute. Polyols are hydrogenated carbohydrates or sugar alcohols and are used commercially to replace sugars in some foods; they do not, however, raise blood glucose concentra- tions to the same extent as sucrose and some other naturally occurring sugars. Examples of polyols include polyglycitol, sorbitol, xylitol, maltitol, mannitol, and isomalt. Polyglycitol and malitol, for example, are found in syrups; others are found in mints and gums. Polyols are also found naturally in some fruits like apples, watermelon, plums, peaches, and pears, to name a few. Polyols absorb water in the colon and contribute to laxation. Both polyols and polydextrose can be partially fermented by colonic bacteria and may enhance the growth of healthful bacteria. The Food and Nutrition Board of the National Academy of Sciences with the 2002 publication of dietary reference intakes for fiber designated polydextrose and polyols as functional fibers pending the results of additional studies showing physiological effects in humans [2].

Resistant Dextrins Resistant dextrins, also called resistant maltodextrins, are generated by heating and enzymatically treating (with amylase) starch, usually cornstarch or wheat starch. Resistant dextrins chemically consist of glucose polymers containing a variety of glucosidic bonds. The resistant dex- trin wheat dextrin is added to foods as well as found as a dietary supplement. Wheat dextrin is water soluble and fermentable by colonic bacteria; it also has been shown to enhance the growth of healthful bacteria in the colon. With the 2002 publication of Dietary Reference Intakes for fiber, the Food and Nutrition Board of the National Acad- emy of Sciences designated resistant dextrins as functional fibers pending the results of additional studies showing physiological effects in humans [2].

Chitin and Chitosan Chitin is a straight-chain polymer containing b (1-4)–linked glucose units, similar in structure to cellulose, but with an N-acetyl amino group substituted for the hydroxyl group at carbon 2 of glucose. Chitin is a component of the exoskeleton of insects and is found in the shells of crabs, shrimp, and lobsters.

Type of Fiber Examples of Food Sources

Cellulose All plant foods, especially wheat bran, legumes, nuts, peas, root vegetables (such as carrots), vegetables of the cabbage family, celery, broccoli, coverings of seeds, and apples

Hemicellulose Whole grains, especially bran, nuts, and legumes

Lignin Whole grains, especially wheat bran, mature root vegetables (such as carrots), fruits with edible seeds (such as strawberries), and broccoli (especially the stalk)

Pectins Citrus fruits, strawberries, apples, raspberries, legumes, nuts, some vegetables (such as carrots), and oat products

Gums Oatmeal, barley, and legumes

b-glucans Oat products and barley

Resistant starches RS1: partially milled grains and seeds; RS2: unripe (green) bananas, legumes, raw potato, and high-amylose corn; RS3: rice, pasta, cold cooked potatoes, and high-amylose corn

Fructans Chicory, asparagus, onion, garlic, artichoke, tomatoes, bananas, rye, and barley

Chitosan, chitin Shells of crab, shrimp, and lobster

Table 4.1 Food Sources of Fiber

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114 C H A P T E R 4 • FIBER

effects in turn positively impact blood glucose and lipid concentrations. In contrast, insoluble fibers were generally accepted to decrease (speed up) intestinal transit time and increase fecal weight to positively impact laxation. However, it is now known that not all soluble fibers alter nutrient absorption, and that insoluble fibers have varied effects on fecal weight. With these observations, the focus has shifted away from classifications based on solubility/ insolubility and more toward viscosity and gel formation.

Viscosity and Gel Formation Viscosity is related to fiber’s ability both to bind or hold water (think of fiber as a dry sponge that hydrates or soaks up water and digestive juices as it moves through the diges- tive tract) and to form a gel (think of freshly made Jello® as it is starting to “set”) within the digestive tract. Most fibers are strongly hydrophilic (water loving) with water-holding capacity influenced by chemical structure, particle size, pH, and electrolyte concentration. But, while most fibers hold water to some extent, not all fibers form a viscous gel when interacting with fluids within the digestive tract. It is this viscosity property of fiber, as well as another property, fermentability, that is most associated with health benefits as discussed in the next sections of this chapter.

Viscous gel-forming fibers include mainly pectins, b-glucans, mucilages (e.g., psyllium), and gums (mainly guar gum). These fibers, upon absorbing in some cases up to several times their weight in water, produce a viscous, gelatinous mass within the digestive tract. Ingesting foods rich in these gel-forming fibers is associated with:

● increased gastric distension, delayed gastric emptying, and longer intestinal transit time, which slows down the diges- tive process and may increase satiety (feelings of fullness)

● reduced nutrient digestion as the viscous gel traps nutri- ents (especially glucose and lipids) and interferes with their ability to interact with the digestive enzymes

● reduced micelle formation as the viscous gel traps bile (needed for micelle formation) and reduces lipid absorp- tion and enterohepatic recirculation of bile

● decreased convective movement of nutrients (especially amino acid and fatty acids) within the intestinal lumen. Convective currents, induced by peristaltic movements, bring nutrients from the lumen to the intestinal cell’s brush border membrane for absorption

● decreased nutrient (especially glucose and lipids) dif- fusion rates through a thickened, unstirred water layer that has become viscous and more “resistant” to nutrient movement (needed for absorption).

It is through these various actions that viscous, gel-forming fibers reduce the absorption of glucose and lipids such as cholesterol. Additionally, bile acids get

mung, lima, snap, northern, and navy, among others). Galactooligosaccharides, like fructooligosaccharides, are not digestible by human digestive enzymes, but are highly soluble and fermentable by colonic bacteria.

In contrast to the shorter-chain galacto- and fructooligosaccharides, the longer-chain fibers vary in degree of solubility and fermentability, and thus are sometimes subdivided into four groups: (1) soluble and highly fermentable, (2) intermediately soluble and fermentable, (3) insoluble and slowly fermentable, and (4) insoluble and nonfermentable. This section discusses the solubility, viscosity, and fermentability of fibers. However, as you read about these characteristics and their effects on the physiological processes, remember that we eat foods containing a mixture of dietary fiber, not foods with just cellulose, hemicellulose, pectins, gums, and so forth. Thus, the described effects on the body processes are more variable and are not as straightforward as presented in this chapter.

Solubility in Water One approach to classifying fiber that has been used for decades is based on fiber’s solubility or insolubility. Water-soluble fibers are those that dissolve in hot water, whereas insoluble fibers do not dissolve in hot water. Shorter-chain water-soluble fibers include both fructooli- gosaccharides and galactooligosaccharides. Longer-chain water-soluble fibers include pectins, gums (mainly guar), inulin, and resistant starches, as well as the resistant dextrin wheat dextrin. Fibers that are intermediately soluble include psyllium, b-glucans, and some hemicelluloses and pectins. Foods typically rich in water-soluble fibers include legumes, oats, barley, rye, chia, flaxseeds, most fruits (espe- cially berries, bananas, apples, pears, plums, and prunes), some vegetables (carrots, broccoli, artichokes, and onions), and cooked and cooled pasta, rice, and potatoes.

Insoluble fibers include mainly cellulose, lignin, and some hemicelluloses, and to a lesser extent some pectins, some resistant starches, chitosan, and chitin. Examples of foods rich in insoluble fiber include whole-grain products, bran, legumes, nuts, seeds, some vegetables (such as cauliflower, zucchini, celery, and green beans), and some fruits. Generally, vegetables and most grain products contain more insoluble fibers than soluble fibers. Fruits tend to be higher in soluble fibers, which are found in the fruit’s pulp and skin; the skin of fruit, however, also provides some insoluble fibers.

This solubility/insolubility approach to classifying fibers, which has been used as a basis for some observed biomarkers and health outcomes, is now considered, because of inconsistent findings, to be of less significance. For example, soluble fibers were generally accepted to delay gastric emptying, increase intestinal transit time (slower movement), and decrease nutrient absorption. These

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C H A P T E R 4 • FIBER 115

fermentation by intestinal microorganisms belonging to the human microbiota. Third, the ingredient must selectively stimulate the growth and/or activity of health- promoting intestinal bacteria. The main bacterial species associated with health and well-being in humans are those of the Bifidobacterium and Lactobacillus genera. The benefits from ingesting prebiotics relate most directly to their stimulation of the growth and/or activity of bacteria in the colon and to the bacteria-generated short-chain fatty acids from fiber fermentation. Food ingredients meeting the criteria for prebiotics include fructans, lactulose (at sublaxative doses), and galactooligosaccharides. Several other fibers (such as wheat dextrins, aracia gum, and polydextrose) also have been shown to stimulate the growth of various species of healthful bacteria or have been shown to provide health benefits through the production of short- chain fatty acids. The amounts of the various prebiotics that need to be ingested to promote desirable effects vary. Similarly, the side effects from prebiotic use (which may include excessive gas, abdominal bloating, cramping, and osmotic diarrhea) also vary with the amount and type of fiber consumed. Generally, shorter-chain fibers such as the oligosaccharides produce side effects at lower intakes than longer-chain fibers. The use of prebiotics has been shown to be helpful in the prevention of some types of diarrhea. This next section of the chapter addresses some of the health benefits and proposed mechanisms of action of fiber. The benefits of the presence of healthful bacteria in the colon and from the short-chain fatty acids that are generated from colonic bacteria are discussed in Chapter 2. Figure 4.4 reviews some of the physiological effects on the digestive tract from the consumption of fiber.

HEALTH BENEFITS OF FIBER

Several systematic reviews and meta-analyses have been conducted examining relationships between fiber intake and/or the intake of foods rich in fiber (most commonly whole grains, fruits, and vegetables) and specific diseases. Positive outcomes are reported, especially for cardiovascular disease but also for health, with inverse relationships between dietary fiber intake and overall mortality shown in both men and women. The roles of fiber in four areas—cardiovascular disease; diabetes; appetite, satiety, and weight control; and selected gastro- intestinal disorders—are reviewed briefly hereafter.

Cardiovascular Disease Studies examining fiber intake consistently report that ingestion of diets high in fiber is associated with a reduced risk of death from cardiovascular disease. Consistent evidence has also been reported for inverse relationships

trapped within the viscous gel to limit micelle formation that is needed for fat absorption; this further contributes to favorable effects on blood lipids. Other effects of gel formation on nutrients, such as on the bioavailability of carotenoids, vary with the food matrix and the fiber.

Fermentability Fiber reaches the colon undigested by human digestive enzymes. Colonic bacteria then ferment (degrade to v arying degrees) this undigested mass. Fibers that are not typically fermented include principally the water-insoluble fibers—cellulose and lignin, along with some hemicellu- loses and some resistant starches like RS1. Fibers that are not fermented are beneficial in promoting laxation and thus treating constipation primarily by increasing fecal bulk or volume, also called stool bulk or weight. The role of nonfermentable fibers in laxation is discussed further in the section on “Health Benefits of Fiber.”

The fermentation of fibers occurs mainly in the proximal (upper) colon—that is, by the cecum and in the ascending region of the colon—and diminishes as the undigested mass moves through the transverse and descending sections of the colon. The shorter-chain fibers, fructooligosaccharides and galactooligosaccharides, are rapidly and almost completely fermented by bacteria in the colon. The longer-chain fermentable fibers (that are also soluble) include pectins, inulin, resistant starch, and gums (guar). Fermentable fibers that are of intermediate solubility include b-glucans and psyllium. Some insoluble fibers that are more slowly fermentable include some lignin and hemicelluloses. Additionally, the resistant dextrin wheat dextrin and polydextrose are fermentable.

Fermentation of fiber by colonic bacteria provides energy and substances for microbial growth as well as products such as short-chain fatty acids (discussed in Chapter 2) that may be used by the human host. The amount of energy realized to the host depends mostly upon the amount and type of fiber that is ingested and the short-chain fatty acids that are produced, but is usually estimated at about 1.5–2.5 kcal/g. While fermentable fibers do not contribute substantially to fecal bulk (as do the nonfermentable fibers), fermentable fibers increase fecal bacteria mass, and the increased bacteria mass in the feces in turn attracts water to enhance stool size. Some fermentable fibers also function in the colon as prebiotics.

Prebiotics are substances that are not digested by human digestive enzymes but provide health benefits to the host by acting as substrates for the growth and/or activity of one or more species of healthful bacteria in the colon. For a fiber, or other substance, to be considered as a prebiotic, three criteria must be satisfied. First, the ingredient must be able to resist digestion by human enzymes and absorption. Second, the ingredient must serve as a substrate for

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116 C H A P T E R 4 • FIBER

barley-based cereals. It is important to also note that consumption of plant foods provides phytosterols and phytostanols, which in amounts ranging from about 1.6 to 3 g/day has been shown to decrease total and LDL serum cholesterol concentrations.

Several modes of action are thought to promote fiber’s hypercholesterolemic effects. These actions include reductions in cholesterol absorption and bile reabsorption and subsequent changes in hepatic cholesterol metabolism and clearance of lipoproteins. Basically, viscous gel- forming fibers trap bile acids and cholesterol within a gelatinous mass to limit micelle formation and absorption and thus enhance their excretion in the feces. The decrease in dietary cholesterol absorption and the decrease in bile acids returned to the liver (enterohepatic recirculation) necessitate the use of cholesterol that is in the body for synthesis of new bile acids. Additionally, changes in cholesterol metabolism and in lipoprotein clearance from the blood (associated with the upregulation of LDL receptors) result to also lower serum cholesterol concentrations. Moreover, cholesterol synthesis appears to be inhibited by fiber-induced shifts in bile acid production from cholic acid toward chenodeoxycholic acid and by propionic acid (the mechanisms are not known); these changes then positively impact serum cholesterol concentrations. Finally, reductions in hepatic triglycerides and fatty acid synthesis also may be contributing to the observed effects.

between intake of fruits and vegetables (primarily greater than five servings per day) and heart attack and stroke, and between intake of whole grains and heart disease.

Studies focusing on the effects of fiber or diets rich in fiber on heart disease risk factors, usually serum cholesterol concentrations, have also typically been favorable. Lower serum total and LDL cholesterol concentrations (and in some studies lower serum triglyceride concentrations) have been demonstrated with ingestion of several viscous gel-forming fibers, especially pectins, b-glucans, psyllium, and guar gum, but also to lesser degrees with ingestion of other fibers including resistant dextrins, methylcellulose, inulin, and fructooligosaccharides. The most well-studied cholesterol-lowering high-fiber foods/fibers are b-glucan from barley and oats, as well as psyllium. In fact, each of these has been studied sufficiently to have health claims.

Quantities of fiber needed to lower serum lipid concentrations vary; effective LDL-cholesterol lowering quantities for pectin range from about 12 to 24 g, for guar gum about 9–30 g, for barley b-glucan and methylcellulose about 5 g, and for psyllium and oat b-glucan about 6 g [7]. Ingestion of 60 g wheat dextrin, a resistant dextrin, also has been shown to reduce serum total cholesterol concentrations. To consume from foods the amount of fiber necessary to lower serum lipids, one would need to ingest, for example, about 6–10 servings per day of soluble fiber–rich fruits and vegetables, or about 2–3 servings per day of legumes or oat- or

Figure 4.4 Selected gastrointestinal responses to fiber ingestion.

Viscous gel-forming f ibers

Non- or less fermentable f ibers

Fermentable f ibers

Blunted glycemic response Slower rise in blood glucose Reduced insulin secretion

Lower serum cholesterol May lower serum triglycerides

Reduced nutrient absorption and bile acid reabsorption

Gastric distension Delayed gastric emptying Longer intestinal transit time

Reduced nutrient digestion

Increased water holding

Greater frequency of defecation

Growth of bacterial populations

Short-chain fatty acid production

Increased fecal mass

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C H A P T E R 4 • FIBER 117

such as ghrelin, glucagon like peptide-1, peptide YY, and cholecystokinin. Additionally, however, the consumption of nonviscous fibers such as galactooligosaccharides has been shown to reduce appetite; such actions have been attributed to changes in metabolism, gut microbiota, and gastrointestinal tract peptides. As expected from the diversity of polysaccharides, fiber’s effects on sati- ety and appetite vary with the type, amount, and form ( supplement or food) of fiber consumed, among other factors. And, while some studies have reported reduced energy intakes and weight loss on high-fiber diets, others have not. The 2015 Dietary Guidelines Advisory Com- mittee does not mention fiber in relation to body weight; but states that there is moderate evidence that dietary patterns higher in fruits, vegetables, and whole grains (i.e., high-fiber foods), that include seafood and legumes, that are moderate in dairy products (low-fat and nonfat) and alcohol; that are lower in red and processed meats, and that are low in sugar-sweetened beverages and refined grains are associated with favorable outcomes related to healthy body weight (including lower body mass index, waist circumferences, or percent body fat) or risk of obesity [1].

Gastrointestinal Disorders Fiber intake has been linked with several gastrointestinal conditions. Three disorders, constipation, diverticular disease, and colon cancer, that have been associated with low fiber consumption, and one condition, irritable bowel syndrome, that has been linked with the consumption of specific fibers are discussed.

Constipation is characterized by long transit time, difficult stool expulsion, low stool output, and incomplete rectal emptying. Increasing consumption of fiber through supplements or fiber-rich foods can improve constipation. While all fibers are beneficial, nonfermentable or less fermentable fibers (such as psyllium, cellulose, inulin, etc.) tend to increase fecal bulk to a greater extent than more fermentable fibers. Fecal bulk is especially affected by water-holding capacity and particle size. Nonfermentable fibers, such as those found in wheat bran, are highly effective in laxation because they can absorb several times their weight of water, thereby increasing fecal volume. This larger fecal volume decreases intraluminal pressure, and provides for a greater frequency of defecation (i.e., reduced/quicker intestinal transit time). Particle size also plays a role, with, for example, larger or coarser bran having the ability to hold more water (than smaller or finer bran) and thus providing greater fecal volume. Several products on the market designed to help individuals with constipation contain fiber. Fiberall® and Metamucil®, for example, contain psyllium. Benefiber® contains wheat dextrin, and FiberChoice® contains inulin. Orafti® contains fructooligosaccharides, and Citrucel® contains

Diets rich in high-fiber foods and the consumption of functional fibers like psyllium have also been associated with both lower systolic and diastolic blood pressure readings and with reductions in blood pressure among those with hypertension, another risk factor for heart disease. The 2015 Dietary Guidelines Advisory Committee concluded that there was consistent and strong evidence that dietary patterns that are lower in saturated fat, cholesterol, and sodium, and richer in fiber, potassium, and unsaturated fats are beneficial for reducing cardiovascular disease [1].

Diabetes Mellitus Inverse associations between dietary fiber intake (as well as high intakes of fruits, vegetables, and complex carbohydrates) and risk of developing type 2 diabetes have been demon- strated in several studies. Consumption of diets high in fiber has also been generally associated with improved glycemic control (also referred to as blunting the glycemic response) in individuals with diabetes and prediabetes. Specifically, the ingestion of fiber supplements or foods rich in viscous gel-forming fibers improves glycemic con- trol largely through reduced rates of glucose absorption and insulin secretion. Reductions in insulin secretion are thought to result at least in part both from slower glucose absorption into the blood as well as from altered secretion of gastrointestinal tract regulatory peptides such as glucagon- like peptides and glucose-dependent insulinotropic peptide, which influence glucagon and insulin secretion and gastrointestinal tract motility. Changes in glycogen catabolism and the resulting release of glucose into the blood also may be influenced by short-chain fatty acids that are produced with fiber fermentation in the colon. Improvements in glycemic control are usually observed with fiber intakes of at least 30 g per day, although fiber supplementation in doses of at least 20 g may also be ben- eficial. The 2015 Dietary Guidelines Advisory Committee states that there is moderate evidence that dietary patterns higher in fruits, vegetables, and whole grains (note these are fiber-rich foods) and lower in red and processed meats, high-fat dairy, refined grains, and sweets/sugar-sweetened beverages reduce the risk of developing type 2 diabetes [1].

Appetite and/or Satiety and Weight Control Fiber-rich foods, versus low-fiber foods, tend to have a lower energy density and a higher volume, which can pro- mote satiety. Satiety also may result from ingestion of foods containing viscous gel-forming fibers due to fiber-induced delays in gastric emptying and/or alterations in the release of digestive tract hormones known to modulate appetite

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118 C H A P T E R 4 • FIBER

studies evaluating the appropriateness of different biomarkers, fiber types, form, and dosages, among other factors, on the incidence of colon cancer are needed. At present, the American Cancer Society, as well as several other organizations charged with improving health, recommends eating fiber-rich foods from several food groups, especially fruits, vegetables, and whole grains, to reduce the risk of cancer. The 2015 Dietary Guidelines Advisory Committee does not single out fiber, but states that there is moderate evidence for an inverse relationship between dietary patterns that are higher in fruits, vegetables, legumes, whole grains (i.e., fiber-rich foods), lean meats/seafood, and low-fat dairy; moderate in alcohol; and low in red and/or processed meats, saturated fat, and sodas/sweets relative to other dietary patterns and risk of colon/rectal cancer [1].

Whereas the aforementioned health conditions have been linked with inadequate fiber intake, irritable bowel syndrome is a condition that has been associated with the ingestion of selected fibers, including some that act as prebiotics. The classic symptoms of this condition— bloating, gas (flatulence), abdominal cramping, and diarrhea or constipation or a mixed bowel pattern—often occur after eating. Causative agents triggering these symptoms in susceptible individuals typically include several short-chain, highly fermentable fibers including fructooligosaccharides and galactooligosaccharides, as well as polyols. In addition to these fibers, the monosaccharide fructose and the disaccharide lactose (along with wheat bran) also may trigger symptoms. These substances have been coined FODMAP—fermentable, oligo-, di-, monosaccharides, and polyols—and restriction of foods rich in these carbohydrates is purported to help alleviate symptoms of irritable bowel syndrome.

The low-FODMAP diet consists of an extensive list of “foods to avoid.” For example, to minimize fructose consumption, one must limit foods with added high- fructose corn syrup, which include many beverages along with sauces and condiments (barbecue sauce, ketchup, syrups, etc.), along with foods naturally rich in fructose such as agave, honey, and many fruits. Fructans (which must be limited) are found in many vegetables. To avoid galactooligosaccharides, one must minimize intakes of most legumes and peas. Lactose is found primarily in dairy products. Polyols are found in chewing gums and mints as well as some fruits. Variable benefits from these dietary restrictions on observed symptoms have been reported, but long-term efficacy data for the low-FODMAP diet are needed. Similarly, the effectiveness of fiber in treating irritable bowel syndrome symptoms has been examined in systematic reviews with generally mixed results. Of the various fibers, psyllium appears to be relatively helpful in improving some symptoms, especially in those with constipation versus those with diarrhea.

methylcellulose. Increasing fiber intake to at least 20 g per day is generally recommended to help treat constipation.

Another gastrointestinal tract disorder that has been linked to diets low in fiber is diverticular disease, which is characterized by the presence of diverticula in the colon. Diverticula, protruding or bulging pouches of the wall of the colon, are thought to form when the colon’s wall weakens. This weakening is theorized to result in chronic constipation associated with low fecal bulk and straining to pass hard fecal matter. (The straining increases the pressure inside the colon and weakens its walls.) When fecal matter becomes trapped in the diverticula, the pouches become inflamed (called diverticulitis) and the person experiences pain and sometimes fever, diarrhea, gastrointestinal bleeding, and infection. Diets high in fibers that increase stool weight, as discussed in the preceding section on constipation, reduce straining and the likelihood of fecal matter becoming trapped in the diverticula. However, whether a high-fiber diet reduces the likelihood of formation of new diverticula once the condition has developed is unclear [8]. Recommendations for fiber intake for those with diverticular disease are the same as those recommended for all Americans, about 20–35 g per day.

Epidemiologic studies, meta-analyses, and prospective studies have shown that diets high in fruits, vegetables, and whole grains are linked with lower risk of colon cancer. Fiber intake also has been linked with a lower risk of colorectal cancer as well as some other cancers (breast, esophageal, etc.) in some, but not all, studies. Yet, the mechanism and whether it is fiber and/or another constituent in high-fiber foods contributing to the link are not clear. Many mechanisms have been suggested to explain how fiber may be preventive against colon cancer, including (1) adsorbing and promoting the excretion of primary bile acids, thereby decreasing their free concentration and availability for conversion to more harmful (carcinogenic) secondary bile acids; (2) adsorbing procarcinogens and carcinogens and/or diluting intestinal contents to minimize carcinogenic compound interactions with colonic mucosal cells; and (3) reducing colonic transit time, which in turn decreases the time during which toxins can be synthesized and in which they are in contact with the colonic mucosa cells. Other indirect effects of fiber also have been speculated as helpful to reduce colon cancer risk. For example, the short-chain fatty acids that are generated during fiber fermentation in the colon decrease the pH within the lumen of the colon; this acidification in turn confers several benefits to the host, as described in Chapter 2. Unfortunately, intervention study results have not been positive. For example, the Polyp Prevention Trial, which examined the effect of a high-fiber, low-fat diet on the recurrence of adenoma (a marker of colorectal cancer), failed to show benefits. Researchers agree that additional

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C H A P T E R 4 • FIBER 119

assuming each serving of fruits, vegetables, and whole grains provides 2 g of dietary fiber and each serving of legumes contributing 5 g of dietary fiber. To complete the estimation, fiber from any consumed fiber supplements and from the ingestion of any high-fiber cereals or other products should be added to the total.

The MyPlate guidelines from the U.S. Department of Agriculture do not provide recommendations on individual nutrient intakes like fiber, but instead focus on consuming foods from within and across food groups to meet nutrient needs. MyPlate suggests that adults consume at least 2 cups of fruits, 2½ cups of vegetables, and a minimum of 3 oz of whole grains per day, with additional recommendations to include at least 1½ cups of beans and peas weekly [9]; exact recommended amounts vary with a person’s gender, age, and total energy needs.

No Tolerable Upper Intake Level for dietary fiber or functional fiber has been established [2]. Tolerance to fiber intake varies from person to person, and problems associated with the use of supplements vary with the type and dose of fiber ingested. Generally, supplements containing fibers that are very rapidly fermented are associated with more undesirable side effects than those that are more slowly or not fermented. The most common complaints with fiber “over” consumption include abdominal discomfort, bloating, gas, and altered stool output; however, gastrointestinal tract tolerance generally improves over time.

Reduced absorption of some minerals also has been purported as an adverse effect of ingesting too much dietary fiber. And, while this may be a problem in individuals consuming fiber in quantities well in excess of recommended amounts, it is not likely that healthy adults consuming recommended amounts of fiber will develop mineral deficiencies. The proposed “problem” is thought to occur because of the adsorption of some divalent minerals (including calcium, magnesium, zinc, and iron) to some fibers (like those containing uronic acid, such as hemicellulose, pectins, and gums, as well as with lignin, which has both carboxyl and hydroxyl groups).

FOOD LABELS AND HEALTH CLAIMS

Nutrient recommendations for fiber, as well as for other nutrients, are found on the Nutrition Facts panel on food labels. The recommendation for fiber provided on food panel labels is 25 g of dietary fiber for a 2,000-kcal diet. Some food labels also provide information on quantities of soluble and insoluble fibers in the product. For exam- ple, the label on a box of cereal might show that a serving (1 cup) provides 7 g of dietary fiber, with 6 g listed as insol- uble and 1 g listed as soluble.

Based on the total amount of dietary fiber provided by a serving of the food, food labels may state that the food is an “excellent” or “good” source of fiber. Foods claiming to be an “excellent source of fiber” by the manufacturer must provide at least 20% of recommendations in a serving—that is, 0.20 3 25 g, or 5 g of fiber. Foods may be considered a “good source of fiber” if they provide 10% of recommendations or 2.5 g of fiber/serving.

The Food and Drug Administration (FDA) has approved several fiber-related health claims [5]. The claims typically focus on consumption of fiber-rich foods such as fruits, vegetables, and whole grains coupled with consumption of a low-fat diet, as shown below.

● Diets low in fat and rich in high-fiber foods (or rich in fruits and vegetables) may reduce the risk of certain cancers.

● Diets low in saturated fat (or low in fat) and rich in soluble fiber (or rich in whole oats and psyllium seed husk) may reduce the risk of heart disease.

● Diets low in total fat, saturated fat, and cholesterol and rich in whole grains and other plant foods may help reduce the risk of heart disease.

RECOMMENDED FIBER INTAKE

Recommendations for increasing the amount of fiber in the U.S. diet have come from several government agencies and private organizations, each with a concern for improv- ing the health of Americans. The Dietary Guidelines sug- gest that Americans ingest 14 g of fiber per 1,000 kcal. In 2002, the National Academy of Sciences Food and Nutri- tion Board established Dietary Reference Intakes, specifi- cally Adequate Intakes, for fiber. These recommendations, shown in Table 4.2, were established based on amounts of fiber shown to protect against heart disease [2]. Unfortu- nately, most Americans fail to meet recommendations, with intakes often reaching only about 18 g of fiber per day [2].

Table 4.3 shows the dietary fiber content of selected foods. General estimates of fiber intake can be calculated

Population Group Age (years) Total Fiber (g)

Men 19–50 $ 51

38 31

Women 19–50 $ 51

25 21

Children 1–3 4–8

19 25

Girls 9–18 26

Boys 9–13 14–18

31 38

Table 4.2 Recommended Fiber Intakes [2]

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120 C H A P T E R 4 • FIBER

Food Group Soluble Fiber

(g / 100 g) Insoluble Fiber

(g / 100 g) Total

Vegetables (cooked)

Asparagus 2.0

Broccoli 1.85 2.81 4.66

Carrots 1.58 2.29 3.87

Cauliflower 0.70 3.50 4.20

Corn 2.0

Lettuce (raw) 1.3

Mushrooms 2.4

Potato baked

With skin 0.61 1.70 2.31

Boiled, no skin 0.99 1.06 2.05

Grain and Grain Products

Rice

White 0.3

Brown 1.8

Couscous 2.8

Bread

White 2.4

Whole grain 6.8

Crackers (wheat) 10.6

Cereals (cold)

All Bran® 29.3

Raisin Bran® 11.1

Corn Flakes® 2.5

Cheerios® 10

Food Group Soluble Fiber

(g / 100 g) Insoluble Fiber

(g / 100 g) Total

Fruits (raw)

Apple with skin 0.70 2.00 2.70

Banana 0.58 1.21 1.79

Grapes 0.24 0.36 0.60

Mango 0.69 1.08 1.76

Orange 1.37 0.99 2.35

Peach with skin 1.31 1.54 2.85

Pear with skin 0.92 2.25 3.16

Pineapple 0.04 1.42 1.46

Plum with skin 1.12 1.76 2.88

Strawberries 0.60 1.70 2.30

Watermelon 0.13 0.27 0.40

Legumes/Beans (cooked)

Black 8.7

Kidney 1.36 5.77 7.13

Lima 1.02 4.21 5.23

Navy 10.5

Pinto 0.99 5.66 6.65

Nuts

Almonds 12.3

Cashews 3.2

Pecans 9.6

Peanuts 8.1

Walnuts 6.7

Table 4.3 Dietary Fiber Content of Selected Foods* [10,11]

SUMMARY

The physiological effects of fiber in the gastrointestinal tract are as varied as the number of fiber components and their physiochemical properties. Two important char- acteristics related to health are viscosity/gel formation and fermentability. These characteristics not only impact digestive tract function and health but also affect risk fac- tors for disease, especially heart disease and diabetes. To obtain fiber through the diet, food sources of fiber need to be varied, ideally within and across all plant-based food groups including whole-grain cereals and cereal products, legumes, nuts, seeds, fruits, and vegetables.

1. Report of the Dietary Guidelines Advisory Committee for the Dietary Guidelines for Americans 2015. http://www.health.gov/ dietaryguidelines/2015-scientific-report/

2. Food and Nutrition Board. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Protein and Amino Acids. Washington DC: National Academy of Sciences, 2002.

3. Jones JM. CODEX-aligned dietary fiber definitions help to bridge the “fiber gap”. Nutr J. 2014;13:34. doi:10.1186/1475-2891-13-34.

4. Slavin J. Fiber and prebiotics: mechanisms and health benefits. Nutri- ents. 2013;5: 1417–35.

5. Food and Drug Administration. Guidance for Industry: a Food Labeling Guide. http://www.fda.gov/Food/GuidanceRegulation/

* Soluble and insoluble fiber contents provided when available.

Yet, countering these possible effects are studies that show that fermentation of these fibers and the resulting acidic environment enhance the release of minerals from fiber and promote mineral absorption from the colon. Maillard products are also mentioned in the scientific literature as having mineral binding potential. These products contain

enzyme-resistant linkages between the amino group of amino acids, especially lysine, and the carbonyl group of reducing sugars, which have formed during cooking, particularly in baking and frying foods. Yet, as with fiber ingestion, mineral deficiencies are not thought to be likely from the ingestion of Maillard products.

References Cited

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C H A P T E R 4 • FIBER 121

Suggested Readings

Asano T, McLeod RS. Dietary fibre for the prevention of colorectal ade- noma and carcinomas. Cochrane Database Syst Rev. 2002; CD003430.

Ben Q, Sun Y, Chai R, Qian A, Xu B, Yuan Y. Dietary fiber intake reduces risk for colorectal adenoma: a meta-analysis. Gastroenterology. 2014; 146:689–99.

Eswaran S, Muir J, Chey WD. Fiber and functional gastrointestinal dis- orders. Am J Gastroenterol. 2013; 108:718–27.

Evert AB, Boucher JL, Cypress M, et al. Nutrition therapy recommenda- tions for the management of adults with diabetes. Diabetes Care. 2014 37:S120–43.

GuidanceDocumentsRegulatoryInformation/LabelingNutrition/ ucm064919.htm

6. Hogarth AJ, Hunter DE, Jacobs WA, et al. Ion chromatographic deter- mination of three fructooligosaccharide oligomers in prepared and preserved foods. J Agric Food Chem. 2000; 48:5326–30.

7. Anderson JW, Baird P, Davis RH, et al. Health benefits of dietary fiber. Nutr Rev. 2009; 67:188–205.

8. Slavin JL. Position of the American Dietetic Association: health impli- cations of dietary fiber. J Am Diet Assoc. 2008; 108:1716–31.

9. U.S. Department of Agriculture MyPlate. U.S. Department of Agri- culture. http://www.choosemyplate.gov/

10. Li BW, Andrews KW, Pehrsson PR. Individual sugars, soluble and insoluble dietary fiber contents of 70 high consumption foods. J Food Comp & Anal. 2002; 15: 715–23.

11. U.S. Department of Agriculture Nutrient Data Laboratory. www.nal .usda.gov/fnic/foodcomp/search

Fukuda S, Ohno H. Gut microbiome and metabolic diseases. Semin Immunopathol. 2014; 36:103–14.

Jones JM. Dietary fiber future directions: Integrating new definitions and findings to inform nutrition research and communication. Adv Nutr. 2013; 4:8–15.

Kanmani P, Kumar RS, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Pro- biotics and its functionally valuable products: a review. Crit Rev Food Sci and Nutr. 2013; 53:641–58.

Kunzmann AT, Coleman HG, Huang W, Kitahara CM, Cantwell MM, Berndt SI. Dietary fiber intake and risk of colorectal cancer and inci- dent and recurrent adenoma in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Am J Clin Nutr. 2015; 102:881–90.

Kushi LH, Doyle C, McCullough M, et al. The American Cancer Society 2012 Guidelines on nutrition and physical activity for cancer preven- tion: reducing the risk of cancer with healthy food choices and physi- cal activity. Cancer J Clin. 2012; 62:30–67.

Quiros-Sauceda AE, Palafox-Carlos H, Sayago-Ayerdi SG, Ayala-Zavala JF, Bello-Perez LA, Alvarez-Parrilla E, de la Rosa LA, Gonzalez- Cordova AF, Gonzalez-Aguilar GA. Dietary fiber and phenolic com- pounds as possible functional ingredients: interactions and possible effect after ingestion. Food Funct. 2014; 5:1063–72.

Sanchez D, Miguel M, Aleixandre A. Dietary fiber, gut peptides, and adi- pocytokines. J Med Food. 2012; 15:223–30.

Slavin JL, Lloyd B. Health benefits of fruits and vegetables. Adv Nutr. 2012; 3:506–16.

Walsh CJ, Guinane CM, O’Toole PW, Cotter PD. Beneficial modulation of the gut microbiota. FEBS Letters. 2014; 588:4120–30.

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122 C H A P T E R 4 • FIBER

Flavonoid Subclass Common Phytochemicals Main Sources

Flavonols Quercetin, kaempferol, myricetin, and isorhamnetin Onions, tea, olives, kale, leafy lettuce, cranberries, tomatoes, cherries, apples, applesauce, turnip greens, endive, ginkgo biloba, chili peppers, chives, and celery

Flavanols Catechins, epicatechin, and epigallo-catechin-3-gallate Green tea, pears, grapes, wine, berries, apples, applesauce, apple juice, cocoa and cocoa products

Derived tannins Theaflavins, theorubigins, and theabrownins Fermented teas (black and oolong)

Condensed tannins/ proanthocyanidins

Procyanidins, prodelphinidins, and propelargonidin Cocoa, cocoa products, stone fruits, grapes, wine, strawberries, cranberries, legumes, cinnamon, beer, and barley

Flavones Apigenin and luteolin Parsley, thyme, celery, celery seed, oregano, and hot peppers

Flavanones Hesperetin, naringenin, and eriodictyol Citrus fruits and juices

Anthocyanins Cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin

Berries, cherries, bananas, plums, oranges, grapes, and red wine

Isoflavones Genistein, daidzein, equol, and Glycitein Legumes, especially soybeans and soy foods—soynuts, soy milk, tofu, miso, soy sauce, and edamame

Table 1 Flavonoid Subclasses, Common Phytochemicals, and Their Sources

Quercetin is among the more well studied of the flavonoids, and exhibits several biological actions helpful in the preven- tion of cardiovascular disease and some of its risk factors like hypertension. Kaempferol and myricetin also have some antihypertensive and antiathlerosclerotic properties.

Flavanols Flavanols, also called flavan-3-ols, are another subclass of flavonoids, and can be further categorized based on chemical structure. Monomer forms are called catechins, and condensed or polymerized forms are called proanthocyanidins or tannins. Some of the food sources containing these flavanols are listed in Table 1. Catechins may help reduce the risk of hypertension and cardiovascular disease. Of the proanthocyanidins in foods, procyanidin is one of the most common, and studies suggest it may be beneficial in preventing heart disease and cancer. Flavanol intake has been estimated at 50–100 mg/day in the United States [1].

Flavones and Flavanones Another category of flavonoids are the flavones, which include luteolin and apigenin. Only a few foods, listed in Table 1, have been identified as good sources of flavones. In comparison with the other flavonoids, not as much research has been conducted on these phytochemicals.

The flavanones also consist of just a few compounds, primarily narigenin, hesperetin, and eriodictyol, and are found mostly in citrus fruits and their juices. A glass of fruit juice is thought to provide 40–140 mg of flavanone glycosides [2]. Both hesperetin and its glycoside (mean- ing attached to a sugar) form hesperidin are found in relatively high amounts in oranges, and exhibit several biological properties that are thought to aid in the

Chapter 4 described fiber and some of its characteristics that make it important in the diet. However, other substances in plant foods are also of significance. These substances are known as phytochemicals, a group of compounds that are biologically active in the body. Of the thousands of phytochemicals, polyphenolic phytochemicals (also referred to as polyphenols, meaning they contain more than one phenol unit), make up the largest group.

The polyphenols include more than 8,000 compounds, and can be divided into a variety of classes. One of the largest of these classes is the flavonoids, which include a group of over 4,000 plant metabolites. This Perspective reviews some of the more ubiquitous flavonoids in foods and their potential roles in maintaining health and pre- venting disease.

FLAVONOIDS

The flavonoids are organic, bioactive, polyphenolic secondary metabolites that occur in small quantities in a wide variety of plants (especially fruits, vegetables, nuts, seeds, herbs, spices, and tea). The flavonoids of dietary significance can be divided, based on functional groups attached to the common flavone backbone, into six subclasses—flavonols, flavanols, flavones, flavanones, anthocyanins, and isoflavones. The flavone and flavonols are subclasses, however, and are sometimes grouped together and referred to as 4-oxoflavonoids. Table 1 provides a list of these flavonoid subclasses along with major food sources.

Flavonols The flavonol subclass includes two main compounds— quercetin and kaempferol, but also myricetin and isorham- nectin. These flavonols are widely found in foods (Table 1).

prevention of both cardiovascular disease and cancer. Naringenin also demonstrates activities that are anti- inflammatory and antiatherogenic [3].

Anthocyanins Anthocyanins are pigments found mostly in the skin of plants, and thus provide color (usually red, blue, or purple) to many fruits and vegetables. Major food sources include blueberries, strawberries, raspberries, red grapes, and blackberries, among others listed in Table 1. A 100 g serving of berries can provide up to 500 mg of anthocyanins [2].

Anthocyanins are found free (unattached) as well as attached to sugars (anthocyanidin glycosides) or acyl groups in foods. Of the dozens of anthocyanidins, the six most commonly found include cyanidin, delphinidin, petunidin, peonidin, pelargonidin, and malvidin. Consumption of anthocyanins and/or foods rich in these flavonoids has been suggested to benefit the heart, eyes (vision), and nerves, and may also be protective against cancer and diabetes [4].

Isoflavones A final category of flavonoids is the isoflavones; the two main isoflavones are genistein and daidzein. They are found mostly in soybeans and soy products, as presented in Table 1. Isofla- vones, along with lignans (found in seeds, whole grains, nuts, and some fruits and vegetables) and coumestans (found in broccoli and sprouts) are phytoestrogens; they are structur- ally similar to estrogen in that the phenol ring can bind to estrogen receptors on some body tissues. Soy products have been marketed for use by women during perimenopause to help alleviate some of the side effects of diminished natural estrogen in the body.

THE FLAVONOIDS: ROLES IN HEALTH AND DISEASE PREVENTION

P E R S P E C T I V E

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C H A P T E R 4 • FIBER 123

Phytochemical Class Common Phytochemicals Sources

Carotenoids b-carotene, a-carotene, lutein, and lycopene Tomatoes, pumpkins, squash, carrots, watermelon, papayas, guavas

Terpenes Limonene and carvone Citrus fruits, cherries, ginkgo biloba

Organosulphides Diallyl sulphide, allyl methyl sulphide, and S-allylcysteine Garlic, onions, leeks, cruciferous vegetables (broccoli, cabbage, Brussels sprouts, mustard, watercress)

Phenolic acids Hydroxycinnamic acids: caffeic acid, ferulic acid, chlorogenic acid, and neochlorogenic curcumin

Coffee, blueberries, cherries, pears, apples, oranges, grapefruit, tomatoes, kiwi, plums, white potatoes

Phenolic acids Hydroxybenzoic acids: ellagic and gallic acids Grapes, grape juice, red wine, tea, raspberries, strawberries, nuts

Lignans Secoisolariciresinol and matairesinol Berries, flaxseeds, sesame seeds, legumes, nuts, broccoli, cabbage, kale, rye bread

Saponins Panaxadiol and panaxatriol Alfalfa sprouts, potatoes, tomatoes, ginseng

Phytosterols b-sitosterol, campesterol, and stigmasterol Vegetable oils (soy, rapeseed, corn, sunflower)

Glucosinolates Glucobrassicin, gluconapin, sinigrin, and glucoiberin Cruciferous vegetables (see organosulphides)

Isothiocyanates Allylisothiocyanates and indoles Cruciferous vegetables (see organosulphides)

Table 2 Phytochemicals and Their Sources

Other Phytochemicals These flavonoids are among the thousands of phyto- chemicals found in foods. Some additional classes and examples of phytochemicals within each class, along with some food sources are listed in Table 2. Within each of these classes are phytochemicals with a wide range of biological actions that also are thought to help protect against disease and maintain health.

Although Tables 1 and 2 provide examples of foods con- taining different phytochemicals, note that most plant foods contain multiple phytochemicals. Tomatoes, for example, may contain as many as 10,000 different phytochemicals, and tea provides several flavonoids, flavonols, flavanols, and proan- thocyanidins, along with other phytochemicals. Phytochemi- cal contents further vary based on the plant species, its stage of ripeness, and the methods used for storing and processing the plant as well as with the climate or environmental condi- tions in which the plant was grown.

An Overview of Flavonoid Digestion, Absorption, and Metabolism Most phytochemicals are found in foods in a variety of forms, and these forms influence the digestion and the rate and extent of absorption of the phytochemical. Polyphenols in foods may exist free (unattached) or in some cases as a glyco- side conjugate (also called a glycone). The names of the con- jugated and unconjugated forms differ slightly; for example, the flavanone hesperidin is conjugated to sugar, and its free/ unconjugated form is known as hesperetin.

In some cases, the glycoside forms of the flavonoids must be digested to aglycones (unconjugated forms) before being absorbed. Other phytochemicals do not require extensive digestion, and may be more directly absorbed from the small intestine (and to a small extent the stomach). Glycosylated quercetin, for example, may be absorbed directly or hydro- lyzed first by β-glycosidase. Many other digestive enzymes in the small intestine also assist in the cleavage of sugars (and other functional groups) bound to the flavonoids to enable absorption. The method of absorption of most flavonoids is

thought to involve carriers; however, the absorptive processes have not been clearly elucidated.

Some flavonoids are neither digested nor absorbed in the upper digestive tract, but instead undergo degradation by colonic microflora. The bacteria hydrolyze the glycosides, (as well as other attached functional groups such as glucuro- nides, sulfates, amides, lactones, etc.) generating metabolites that may be absorbed or that exert effects on the body from within the colon. Lignans, for example, are metabolized by colonic bacteria to the metabolites enterodiol and enterolac- tone, which are then absorbed. These enterolignans exhibit weak estrogenic activity and/or antiestrogenic effects upon binding to estrogen receptors on various body tissues. Bacteria in the colon also utilize anthocyanin glycosides, deglycosyl- ating them to aglycones, which are then further degraded. The extent of absorption of the products generated from the actions of the bacteria is not well established.

Once absorbed, most flavonoid metabolites are conju- gated in the cells of the small intestine and then enter portal blood for transport to the liver. Some metabolites, however, efflux from the enterocyte back into the lumen of the small intestine via adenosine-binding cassette (ABC) transporters. Those flavonoid metabolites that enter portal blood are taken up largely by the liver where they undergo further metabo- lism, especially conjugation with methyl or sulfate groups, or glucuronic acid. These conjugated metabolites are then released into systemic circulation bound to plasma proteins like albumin. The amount of the metabolites present in the plasma varies considerably with the type of flavonoid con- sumed, the food source, and the amount ingested; little is known about the metabolism of all the different polyphenols in the body, and thus about what metabolites are present in the plasma after consumption of a specific polyphenol.

FLAVONOIDS AND HEALTH AND DISEASE PREVENTION

Diets rich in plant foods (whole grains, legumes, nuts, seeds, vegetables, and fruits) are typically associated with reduc- tions in the risk of various diseases or conditions, especially

cardiovascular disease, but also to a lesser extent some cancers, neurodegenerative conditions, and osteoporotic fractures, among others. Diets rich in plant foods, as we now know, are also rich in flavonoids and other phytochemicals. Dietary flavonoid intakes and/or specific flavonoids have been shown in some studies to be beneficial in disease prevention, primarily reducing cardiovascular disease risk and mortality, nonfatal events, and all-cause mortality [5–8].

Flavonoids exhibit a broad spectrum of biological activities that affect a variety of metabolic processes that may be related to the development of diseases. Several fla- vonoids provide cardioprotective effects with antioxidant and anti-inflammatory functions, vasodilatory effects (blood vessel relaxation), antiplatelet adhesion, and anticoagulant effects. Quercetin, a well-studied flavonol, for example, exhibits direct antioxidant functions (scavenging free radi- cals), activates signaling pathways, inhibits inflammation, and promotes vascular relaxation [3-9]. Kaempferol also has antihypertensive actions via enhancing endothelium vasore- laxation and protecting against endothelial damage [3-9]. Myricetin, another flavonol, also demonstrates antiplatelet, antihypertensive, and antiatherosclerotic properties. Catechins (monomeric flavanols) are also anti-inflammatory, and some isoflavones exhibit cholesterol-lowering effects that may be protective against heart disease.

It is a variety of actions of several flavonoids that are also thought to help in the prevention of some cancers. Some of these actions include antioxidant and anti-inflammatory functions, antiangiogenesis actions, and antiproliferative and apoptotic effects on tumor cells. The catechins (flavanols), for example, target signaling pathways to inhibit the growth of some cancers and promote apoptosis [5,9-11]. The flavonol quercetin exhibits direct antioxidant functions (scavenging free radicals), has apoptotic effects, and activates signaling pathways, which may be beneficial in cancer prevention [3,5,9-11]. The flavonol myricetin also demonstrates proper- ties that may reduce the development of some cancers. Addi- tionally, the isoflavone genestein, lignans, glucosinolates, isothiocyanates, terpenes, and some phenolic acids such

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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124 C H A P T E R 4 • FIBER

Suggested Readings

Balentine DA, Dwyer JT, Erdman JW, Ferruzzi MG, Gaine PC, Harnly JM, Kwik-Uribe CL. Recommendations on reporting requirements for flavonoids in research. Am J Clin Nutr. 2015; doi10.3945/ajcn.113.071274.

Bhagwat S, Haytowitz DB, Holden JM. USDA database for the flavonoid content of selected foods. Release 3.1. Beltsville, MD: USDA. 2013.

Bohn T. Dietary factors affecting polyphenol bioavailability. Nutr Rev. 2014;72:429–52. http://www.ars.usda.gov/ Services/docs.htm?docid56231

Fang J. Bioavailability of anthocyanins. Drug Metab Rev. 2014; 46:508–520.

Gonzalez-Abuin N, Pinent M, Casanova-Marti A, Arola L, Blay M, Ardevol A. Procyanidins and their healthy protective effects against type 2 diabetes. Curr Med Chem. 2015; 22:39–50.

Gupta C, Prakash D. Phytonutrients as therapeutic agents. J Complement Integr Med. 2014; 11:151–69.

Howes MR, Simmonds MSJ. The role of phytochemicals as micronutrients in health and disease. Curr Opin Clin Nutr Metab Care. 2014; 17:558–66.

Murphy MM, Barraj LM, Herman D, Bi X, Cheatham R, Randolph RK. Phytonutrient intake by adults in the United States in relation to fruit and vegetable consumption. J Acad Nutr Diet. 2012; 112:222–9.

Myers G, Prince RL, Kerr DA, Devine A, Woodman RJ, Lewis JR, Hodgson JM. Tea and flavonoid intake predict osteoporotic fracture risk in elderly Australian women: a prospective study. Am J Clin Nutr. 2015; 102:958–65.

Peluso I, Miglio C, Morabito G, Ioannone F, Serafini M. Flavonoids and immune function in human: A systematic review. Crit Rev Food Sci Nutr. 2015; 55:383–95.

Roohbakhsh A, Parhiz H, Soltani F, Rezaee R, Iranshahi M. Molecular mechanisms behind the biological effects of hesperidin and hesperetin for the prevention of cancer and cardiovascular diseases. Life Sciences. 2015; 124:64–74.

Sebastian RS, Enns CW, Goldman JD, Martin CL, Steinfeldt LC, Murayi T, Moshfegh AJ. A new database facilitates characterization of flavonoid intake, sources, and positive associations with diet quality among US adults. J Nutr. 2015; 145:1239–48.

Sesso HD, Gaziano JM, Liu S, Buring JE. Flavonoid intake and the risk of cardiovascular disease in women. Am J Clin Nutr. 2003; 77:1400–8.

Singh BN, Singh HB, Singh A, Naqvi AH, Singh BR. Dietary phytochemicals alter epigenetic events and signaling pathways for inhibition of metastasis cascade. Cancer Metastasis Rev. 2014; 33:41–85.

as hydroxycinnamic acid have been shown to inhibit tumor formation and/or proliferation.

Inflammation is thought to contribute to the develop- ment of some neurodegenerative conditions. Flavonoids are thought to suppress neuroinflammation, as well as target signaling pathways and enhance cerebrovascular blood flow to improve cognitive function [10-12]. Diets high in flavonoids are thus thought to reduce age-associated cognitive impair- ments and/or cognitive decline.

Many of the demonstrated actions of flavonoids have been studied in vitro, in cultured cells, or in isolated tissues using spe- cific glycosides or aglycone forms of the various phytochemicals. The forms of the polyphenolic phytochemicals used in the studies, however, have not been consistently the same as the forms in which the polyphenolic phytochemicals are found in the body. Moreover, the amounts or concentrations of the phytochemi- cals used in the studies have often been much higher than the amounts of the phytochemicals found naturally in the body. Differences in the metabolism of the thousands of phytochemi- cals in the body also complicate the interpretation of research studies and the ability to make recommendations. While more prospective studies are being conducted in humans, the results of such studies are typically mixed. To date, study findings most support the role of flavonoids in reducing the risk of cardiovas- cular diseases and/or its risk factors; however, more prospective, randomized controlled trials are needed to further examine the effectiveness of flavonoids in the prevention of neurodegenera- tive conditions and diseases such as cancers and diabetes. See Suggested Readings for more information on phytochemicals, including specific mechanisms by which the various flavonoids and other phytochemicals are thought to function.

5. Tena JD, Burgos-Moron E, Calderon-Montano J, Sanz I, Sainz J, Lopez-Lazaro M. Consumption of the dietary flavonoids quercetin, luteolin and kaempferol and overall risk of cancer — A review and meta- analysis of the epidemiological data. WebmedCentral 2013 (May);article ID WMC004264 at www .webmedcentral.com

6. Ponzo V, Goitre I, Fadda M, Gambino R, Francesco AD, Soldati L, Gentile L, Magistroni P, Cassader M, Bo S. Dietary flavonoid intake and cardiovascular disease risk: a population-based cohort study. J Transl Med. 2015; 13:218–30.

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8. Zamora-Ros R, Rabassa M, Cherubini A, Urpi-Sarda M, Bandinelli S, Ferrucci L, Andres-Lacueva A. High concentrations of a urinary biomarker of polyphenol intake are associated with decrease mortality in older adults. J Nutr. 2013; 143:1445–50.

9. Miles SL, McFarland M, Niles RM. Molecular and physiological actions of quercetin: need for clinical trials to assess its benefits in human disease. Nutr Rev. 2014; 72:720–34.

10. Boreddy SR, Srivastava SK. Pancreatic cancer chemoprevention by phytochemicals. Cancer Letters. 2013; 334:86–94.

11. Song NR, Lee KW, Lee HJ. Molecular targets of dietary phytochemicals for human chronic diseases: cancer, obesity, and alzheimer’s disease. J Food Drug Anal. 2012;20:342–5.

12. Rodriguez-Mateos A, Vauzour D, Krueger CG, Shanmu- ganayagam D, Reed J, Calani L, Mena P, Rio DD, Crozier A. Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update. Arch Toxicol. 2014; 88:1803–53.

1. Schroeter H, Heiss C, Spencer JPE, Keen CL, Lupton JR, Schmitz HH. Recommending flavanols and procyanidins for cardiovascular health: current knowledge and future needs. Mol Aspect Med. 2010; 31:546–57.

2. Manach C, Scalbert A, Morand C, et al. Polyphenols: food sources and bioavailability. Am J Clin Nutr. 2004; 79:727–47.

3. Salvamani S, Gunasekaran B, Shaharuddin NA, Ahmad SA, Shukor MY. Antiatherosclerotic effects of plant flavonoids. Biomed Res Internl. 2014; doi. org/10.1155/2014/480258.

4. Pojer E, Mattivi F, Johnson D, Stockley CS. The case for anthocyanin consumption to promote human health: a review. Compr Rev Food Sci Food Safety. 2013; 12:483–508.

References Cited

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125

T HE PROPERTY THAT SETS LIPIDS apart from other major nutrients is their solubility in organic solvents such as ether, chloroform, and acetone. If lipids are defined according to this property, as is generally the case, many diverse molecules fit the criteria and are thus considered lipids. Unlike carbohydrates and proteins, classifying lipids on the basis of solubility covers a broad range of molecules with diverse structural and functional properties. Such biological diversity is a benefit to plants and animals due to the many roles lipids play. As body fat, lipids serve as a depot of stored energy, provide protection to internal organs, and insulate against heat loss. Lipids also form the basis of cellular membranes, steroid hormones, bile acids, eicosanoids, and other signaling molecules. The roles of the fat-soluble vitamins are discussed in Chapter 10.

The diversity of lipids poses a challenge in creating a classification system beyond their solubility property. A traditional way of classifying lipids is based on how many products result from hydrolysis: “simple” lipids are those yielding two types of products on hydrolysis, whereas “complex” lipids yield three or more products. An alternative way of classifying lipids is based on the products of synthesis. In this system, lipids are defined as molecules arising from two distinct pathways that produce fatty acids (and their derivatives) or sterols (and their derivatives). Neither system is entirely adequate for the study of nutrition in which emphasis is placed on the structure and function of lipids. Consequently, the lipids discussed in this chapter are limited to those most relevant to human nutrition and are organized by their structural and functional similarities:

● Fatty acids

● Triacylglycerols, diacylglycerols, and monoacylglycerols

● Phospholipids

● Sphingolipids

● Sterols (cholesterol, bile acids, and phytosterols).

This chapter also describes lipoproteins—complexes of lipids and proteins— that allow lipids to be transported in the aqueous environment of the blood. Finally, this chapter discusses the metabolism of ethyl alcohol. Although not a lipid, ethyl alcohol is a common dietary component and is catabolized similarly to lipids.

Lipids5 STRUCTURE AND BIOLOGICAL IMPORTANCE Fatty Acids Triacylglycerols (Triglycerides) Phospholipids Sphingolipids Sterols

DIETARY SOURCES Recommended Intakes

DIGESTION Triacylglycerol Digestion Phospholipid Digestion Cholesterol Ester Digestion

ABSORPTION Fatty Acid, Monoacylglycerol, and Lysophospholipid

Absorption Cholesterol Absorption Lipid Release into Circulation

TRANSPORT AND STORAGE Lipoprotein Structure Lipoprotein Metabolism

LIPIDS, LIPOPROTEINS, AND CARDIOVASCULAR DISEASE RISK Cholesterol Saturated and Unsaturated Fatty Acids Trans Fatty Acids Lipoprotein(a) Apolipoprotein E

INTEGRATED METABOLISM IN TISSUES Catabolism of Triacylglycerols and Fatty Acids Formation of Ketone Bodies Synthesis of Fatty Acids Synthesis of Triacylglycerols and Phospholipids Synthesis, Catabolism, and Whole-Body Balance

of Cholesterol

REGULATION OF LIPID METABOLISM Fatty Acids Cholesterol

BROWN FAT THERMOGENESIS

ETHYL ALCOHOL: METABOLISM AND BIOCHEMICAL IMPACT The Alcohol Dehydrogenase (ADH) Pathway The Microsomal Ethanol Oxidizing System (MEOS) The Catalase System Alcoholism: Biochemical and Metabolic Alterations Alcohol in Moderation: The Brighter Side

SUMMARY

P E R S P E C T I V E

THE ROLE OF LIPOPROTEINS AND INFLAMMATION IN ATHEROSCLEROSIS

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126 C h a p t e r 5 • LIPIDS

2 or more carbon-carbon double bonds). PUFAs of nutritional interest may have as many as 6 double bonds. Where a carbon-carbon double bond exists, there is an opportunity for either cis or trans geometric isomerism that significantly affects the molecular configuration and functionality of the molecule. The cis isomer results in folding and bending of the molecule into a U-like orientation, whereas the trans form has the effect of extending the molecule into a linear shape similar to that of saturated fatty acids. The more carbon-carbon cis double bonds occurring within a chain, the more pronounced is the bending effect. The degree of bending plays an important role in the structure and function of cell membranes. The structures in Figure 5.1 illustrate saturation and unsaturation in an 18-carbon fatty acid and show how cis or trans isomerization affects the molecular configuration.

Most naturally occurring unsaturated fatty acids are of the cis configuration, although the trans form does appear in some natural plant oils, in dairy products, and lamb

StrUCtUre aND BIOLOGICaL IMpOrtaNCe

Fatty Acids As a class, fatty acids are the simplest of the lipids. They are composed of a hydrocarbon chain with a methyl group at one end and a carboxylic acid group at the other. There- fore, fatty acids have a polar, hydrophilic end and a nonpo- lar, hydrophobic end that is insoluble in water (Figure 5.1). Fatty acids exist alone or as components of the more com- plex lipids, discussed in later sections. They are of vital importance as an energy nutrient, furnishing most of the calories derived from dietary fat.

The lengths of the hydrocarbon chains of fatty acids found in foods and body tissues vary from 4 to about 24 carbon atoms, although the most common fatty acids in nature are 18 carbons. The fatty acids may be saturated (SFA), monounsaturated (MUFA, possessing 1 carbon- carbon double bond), or polyunsaturated (PUFA, having

Figure 5.1 Structures of selected fatty acids.

CH3 CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

OH

C

O

Hydrophobic

Stearic acid

Hydrophilic

Carboxylic acid end

Methyl end

CH3 CH2 CH2

CH2 CH2

CH2 C

CH2 CH2

CH2 CH2

C

H

H

H H

CH2

CH2 CH2

CH2 CH2

OH

C

O

O

Hydrophobic

Hydrophobic Hydrophilic

Elaidic acid (trans form)

Hydrophilic

Carboxylic acid end

Methyl end

CH3

CH2

CH2

C C

H2C

CH2—CH2

CH2—CH2

CH2—CH2

CH2—CH2

CH2—CH2

CH2—C—OH

Oleic acid (cis form)

Carboxylic acid end

Methyl end

Cis form results in folding back and

kinking of the molecule into a U-like orientation.

Trans fatty acids have the effect of extending

the molecule into a linear shape similar to

saturated fatty acids.

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C h a p t e r 5 • LIPIDS 127

example, the notation 18:2 Δ9,12 describes linoleic acid. The first number, 18 in this case, represents the number of carbon atoms; the number following the colon refers to the total number of double bonds present; and the superscript numbers following the delta symbol designate the carbon atoms at which the double bonds begin, counting from the carboxyl end of the fatty acid.

A second commonly used system of notation locates the position of double bonds on carbon atoms counted from the methyl, or omega (v), end of the hydrocarbon chain. For instance, the notation for linoleic acid would be 18:2 v-6. Substitution of the omega symbol with the letter n has been popularized. Using this designation, the notation for linoleic acid would be expressed as 18:2 n-6. In this system, the total number of carbon atoms in the chain is given by the first number, the number of double bonds is given by the number following the colon, and the location (carbon atom number) of the first double bond counting from the methyl end is given by the number following v- or n-. This system of notation takes into account the fact that double bonds in a fatty acid are always positioned so that they are separated by three carbons. Thus, if you know the total number of double bonds and the location of the first relative to either the methyl or carboxylic end, you can determine the locations of the remaining double bonds.

Figure 5.2 demonstrates the designation of linoleic acid using each of the two systems: 18:2 9, 12D9,12 (delta) or 18:2 v-6 or 18:2 n-6 (omega). The fatty acid a-linolenic acid, which contains three double bonds, is identified as 18:3 9, 12, 15D9,12,15 or 18:3 v-3 or 18:3 n-3.

Table 5.1 lists some naturally occurring fatty acids and their dietary sources. For unsaturated fatty acids, the table shows the Δ and v system designations, and commonly used abbreviations. The list includes only those fatty acids with chain lengths of 14 or more carbon atoms because these fatty acids are most important both nutritionally and functionally. For example, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2) together

and beef fat as a result of biohydrogenation by ruminant bacteria. Trans fatty acids may also be commercially produced as a result of partial hydrogenation. Trans fatty acids resulting from biohydrogenation contain the trans double bond mostly at the Δ11 carbon, whereas commercial hydrogenation produces a normal distribution of trans isomers with the peak around the Δ10 isomer. Partial hydrogenation, a process commonly used in making frying oils and commercial food products, is designed to solidify vegetable oils at room temperature. Double bonds of cis orientation that are not reduced in the process undergo an isomeric rearrangement to the trans form, which is energetically more stable. The hydrogenation of the fatty acids (as part of triacylglycerols) changes the melting point, giving the product a higher degree of plasticity (spreadability) and hardness so that it remains solid at room temperature, which are desirable to both the consumer and the food manufacturer. Partially hydrogenated oils have often been used as frying oils to enhance their stability at frying temperatures. Higher frying temperatures reduce the uptake of fat during cooking. Due to health concerns, the availability of trans fatty acids from partially hydrogenated oils in the U.S. food supply has significantly declined in recent years and is currently estimated to be 1.3 g per person per day [1]. The role of trans fatty acids in the etiology of cardiovascular disease (CVD) is discussed in the section “Lipids, Lipoproteins, and Cardiovascular Disease Risk” in this chapter.

Fatty Acid Nomenclature Two systems of notation have been developed to provide a shorthand way to indicate the chemical structure of a fatty acid. Both systems are used regularly and will be used interchangeably in the text for different purposes.

The delta (Δ) system of notation has been established to denote the chain length of the fatty acids and the number and position of any double bonds that may be present. For

Figure 5.2 The structure of linoleic acid, showing the two systems for nomenclature.

Linoleic acid CH3—(CH2)4—CH CH—CH2—CH CH—(CH2)7—COOH

912

Methyl end ω-6 (or n-6)

ω-9 (or n-9)

Carboxyl endΔ9Δ12

The delta (Δ) system counts from the carboxyl end.

The notation for linoleic acid is 18:2 Δ9,12.

The omega (ω) system counts from the methyl end. The notation for linoleic acid is 18:2 ω-6 or 18:2 n-6.

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128 C h a p t e r 5 • LIPIDS

Notation Common Name Formula Source*

Saturated Fatty Acids

14:0 Myristic acid CH (CH ) COOH3 2 122 2 Coconut and palm kernel oil, fish oils

16:0 Palmitic acid CH (CH ) COOH3 2 142 2 All animal and plant fats, notably palm oil

18:0 Stearic acid CH (CH ) COOH3 2 162 2 All animal and plant fats, notably cocoa butter

20:0 Arachidic acid CH (CH ) COOH3 2 182 2 Peanut oil, wild-caught salmon oil

24:0 Lignoceric acid CH (CH ) COOH3 2 222 2 Peanut oil

Unsaturated Fatty Acids

16:1 9D (n-7) Palmitoleic acid CH (CH ) CH CH (CH ) COOH3 2 5 2 72 2 5 2 2 Fish oils, poultry fat

18:1 9D (n-9) Oleic acid CH (CH ) CH CH (CH ) COOH3 2 7 2 72 2 5 2 2 All animal and plant fats

18:2 9 , 12D (n-6) Linoleic acid ) )( (CH CH CH CH CH CH CH CH COOH3 2 4 2 2 72 2 5 2 2 5 2 2 Most plant oils, poultry fat 18:3 9 , 12 , 15D (n-3) a-linolenic acid ) )( (CH CH CH CH CH COOH3 2 3 2 72 2 5 2 2 Linseed (flax), soybean, and canola oils 20:4 5, 8 , 11, 14D (n-6) Arachidonic acid CH (CH ) (CH CH CH) (CH ) COOH3 2 3 2 4 2 32 2 2 5 2 2 Fish oils

20:5 5, 8 , 11, 14 , 17D (n-3) Eicosapentaenoic acid )(CH CH CH CH (CH ) COOH3 2 5 2 32 2 5 2 2 Marine algae and fish that consume the algae 22:6 4 , 7 , 10 , 13, 16 , 19D (n-3) Docosahexaenoic acid )(CH CH CH CH (CH ) COOH3 2 6 2 22 2 5 2 2 Marine algae and fish that consume the algae

* Fats and oils in the food supply contain many types of fatty acids of varying proportions. The sources listed here indicate foods that are comparatively enriched in the specific fatty acid.

Table 5.1 Some Naturally Occurring Fatty Acids

account for about 90% of the fatty acids in the average U.S. diet. However, shorter-chain fatty acids do occur in nature and are present in the food supply. Butyric acid (4:0) and lauric acid (12:0), for instance, are abundant in milk fat and coconut oil, respectively.

Most fatty acids have an even number of carbon atoms. The reason for this will be evident in the discussion of fatty acid synthesis. Odd-number-carbon fatty acids occur naturally to some extent in some food sources. For example, certain fish, such as menhaden, mullet, and tuna, as well as the bacterium Euglena gracilis, contain compar- atively high concentrations of odd-numbered-carbon fatty acids.

Essential Fatty Acids If fat is entirely excluded from the diet of humans, a con- dition develops that is characterized by retarded growth, dermatitis, kidney lesions, and early death. Studies have shown that eating certain unsaturated fatty acids is effec- tive in curing the conditions related to the lack of these fatty acids. Two unsaturated fatty acids cannot be synthesized in the body and must be acquired in the diet from plant foods. The two essential fatty acids are linoleic acid (18:2 n-6) and a-linolenic acid (18:3 n-3). They are essential because humans lack enzymes called 12D and 15D desaturases, which incorporate double bonds at these positions. These enzymes are found only in plants. Humans are incapable of forming double bonds beyond the 9D carbon in the chain. If a 9,12D fatty acid is obtained from the diet, however, additional double bonds can be incorporated at 6D (desaturation). Fatty acid chains can also be elongated by the enzymatic addition of two carbon atoms at the carboxylic acid end

of the chain. These reactions are discussed further in the “Synthesis of Fatty Acids” section of this chapter.

In mammalian cells, linoleic acid can be converted to arachidonic acid (20:4 n-6) via the so-called “omega-6 pathway.” The intermediates in the desaturation and elongation pathway are:

linoleic acid (18:2 n-6)

linolenic acid (18:3 n-6)

eicosatriaenoic acid (20:3 n-6)

arachidonic acid (20:4 n-6)

g2

In a similar manner, a-linolenic acid can be converted to eicosapentaenoic acid (20:5 n-3) via the “omega-3 pathway.” Both arachidonic and eicosapentaenoic acid are metabolically significant because they are precursors of eicosanoids, important signaling molecules discussed later in this chapter. Linseed (flax) oil is particularly rich in a-linolenic acid, whereas fish oils are good sources of eicosapentaenoic acid and docosahexaenoic acid (22:6 n-3). The fatty acid composition of common fats and oils is given in Table 5.2. It is interesting to note that fatty acid composition in wild-caught salmon is different than farm- raised salmon, due to differences in the diets that these fish consume. Farm-raised salmon are often fed plant sources of protein and fat (corn or soybean meal) that influences their fatty acid composition.

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Table 5.2 Fatty Acid Com position of Fats and Oils

SFA M

UFA PUFA

Butyric

Caproic

Caprylic

Capric

Lauric

Myristic

Palmitic

Stearic

Arachidic

Palmitoleic

Oleic

Gadoleic

Linoleic

Arachadonic

a-Linolenic

Eicosapentaenoic

Docosapentaenoic

Docosahexaenoic

4:0 6:0

8:0 10:0

12:0 14:0

16:0 18:0

20:0 16:1 n-9

18:1 n-9

20:1 n-9

18:2 n-6

20:4 n-6

18:3 n-3

20:5 n-3

22:5 n-3

22:6 n-3

Plant Oils %

of Total Fat

Canola Oil 4.3

2.1 0.7

0.2 61.7

18.6 9.1

Cocoa Butter 0.1

25.4 33.2

0.2 32.6

2.8 0.1

Coconut Oil 0.8

8.2 5.6

43.7 16.8

8.4 2.5

5.9 1.7

Corn Oil 0.1

10.6 1.8

0.4 0.1

27.3 0.1

53.2 1.2

Cottonseed Oil 0.8

22.7 2.3

0.1 0.8

17.0 51.5

0.2

Flax (Linseed) Oil 0.1

5.1 3.4

0.1 18.3

14.2 53.4

Olive Oil 11.3

2.0 0.4

1.3 71.3

0.3 9.8

0.8

Palm Oil

0.1 1.0

43.5 4.3

0.3 36.6

0.1 9.1

0.2

Palm Kernel Oil

0.2 3.3

3.7 47.0

16.4 8.1

2.8 11.4

1.6

Peanut Oil 0.1

9.5 2.2

1.4 0.1

44.8 1.3

32.0

Saffl ow

er Oil 0.1

4.3 2.3

0.3 0.1

12.0 0.1

77.7 0.4

Soybean Oil 0.1

10.6 4.0

0.3 0.1

23.2 53.7

7.6

Sunflow er Oil

0.1 7.0

4.5 0.4

0.1 18.7

0.1 67.5

0.8

Anim al Fats

Beef Tallow 0.9

3.7 24.9

18.9 0.1

4.2 36.0

0.3 3.1

0.6

Chicken Fat 0.1

0.9 21.6

6.0 0.1

5.7 37.3

1.1 19.5

1.0

Lard (Pork Fat) 0.1

0.2 1.3

23.8 13.5

0.1 2.7

41.2 1.0

10.2 0.1

M ilk (Butter) Fat

3.2 1.9

1.1 2.5

2.8 10.0

26.2 12.1

0.1 2.2

25.0 2.2

1.5

Fish Oils

Herring Oil 0.2

7.2 11.7

0.8 9.6

12.0 13.6

1.1 0.3

0.8 6.3

0.6 4.2

M ackerel Oil

4.9 15.3

3.0 5.2

16.4 7.5

1.6 1.3

1.1 6.5

1.5 10.1

M enhaden Oil

8.0 15.1

3.8 10.5

14.5 1.3

2.2 1.2

1.5 13.2

4.9 8.6

Salm on Oil (w

ild) 2.1

10.0 3.3

4.0 21.3

3.5 2.7

4.2 4.7

5.1 4.5

17.6

Salm on Oil (farm

ed) 4.1

14.0 3.7

0.2 5.9

20.2 2.0

6.7 0.7

1.1 6.4

2.9 8.2

Source: U.S. Departm ent of Agriculture, National Nutrient Database for Standard Reference, Release 28.

129

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130 C h a p t e r 5 • LIPIDS

Figure 5.3 Linkage of fatty acids to glycerol to form a triacylglycerol. Chain length of fatty acid is (n 1 2). Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

H

H

H

H

C OH

OH

H

C HO

HO

HO

1

C OH C

Glycerol TriacylglycerolFatty acids

O

(CH2)n CH3

(CH2)n CH3

(CH2)n CH3

C

O

C

O

Triacylglycerol symbol

These fatty acids can be saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), or a combination.

3

2

1 H

C

H

C O

HO

C

H

H O

C

O

C

O

C

O

Fatty acid

Fatty acid

Fatty acid

Glycerol molecule

An ester bond

3

2

1

n-6 versus n-3 Fatty Acids It is estimated that our human ancestors consumed foods that provided equal amounts of n-6 and n-3 fatty acids. Today, the intake of n-3 fatty acids is quite low and over- whelmed by n-6 fatty acids in the diet, with linoleic acid providing 80–90% of all PUFA. This is due to the wide- spread use of plant oils, such as soybean oil, in the produc- tion of manufactured food products and in foodservice frying oils, coupled with the relatively low intake of fish and other n-3 fatty acid sources in Western diets. Assess- ing the metabolic impact of dietary n-6 and n-3 fatty acids is important in the field of nutrition. The disproportion- ate amounts of n-6 and n-3 fatty acids can have metabolic consequences that are discussed further in the “Synthesis of Fatty Acids” section of this chapter.

Triacylglycerols (Triglycerides) Most adipose tissue is composed of triacylglycerols, which represent a highly concentrated form of stored energy. (Triacylglycerols is the currently accepted name that has replaced the older name triglycerides.) Triacylglycerols also account for nearly 95% of dietary fat. Structurally, they are composed of a trihydroxyalcohol, glycerol, to which three fatty acids are attached by ester bonds, as shown in Figure 5.3; the formation of each of these ester bonds liberates a water molecule. The fatty acids may be all the same (a simple triacylglycerol) or different (a mixed tria- cylglycerol). The fatty acids in triacylglycerols can be all saturated, all monounsaturated, all polyunsaturated, or any combination. Triacylglycerols exist as fats (solid) or oils (liquid) at room temperature, depending on the nature of the component fatty acids. Triacylglycerols that contain a relatively high proportion of short-chain fatty acids or

unsaturated fatty acids tend to be liquid oils at room tem- perature, whereas those made up of saturated fatty acids of longer chain length have higher melting points and thus exist as solid fats.

Carbons 1 and 3 of glycerol are not the same when viewed in a three-dimensional model. Also, when different fatty acids are attached to the first and third carbons of glycerol, the second carbon becomes asymmetric. (See Chapter 3 for a discussion of stereoisomerism.) Enzymes of the body are able to distinguish between the three carbons of glycerol and are generally quite specific. This specificity is important in digesting and synthesizing triacylglycerols, as is discussed later in this chapter.

The specific glycerol hydroxyl group to which a certain fatty acid is attached is indicated by a numbering system for the three glycerol carbons, in much the same way as glyceraldehyde is numbered (see Figure 3.2). This system is complicated somewhat by the fact that the central carbon of the glycerol is asymmetrical when different fatty acids are esterified at the two end carbon atoms and therefore may exist in either the D or the L form. A system of nomenclature called stereospecific numbering (sn) has been adopted in which the glycerol is presented as shown in Figure 5.3, with the C-2 hydroxyl group oriented to the left (L) and the carbons numbered 1 through 3 beginning at the top.

Acylglycerols may be composed of glycerol esterified to a single fatty acid (a monoacylglycerol) or to two fatty acids (a diacylglycerol), with the fatty acids attached to any of the three carbons of glycerol. Though present in the body only in small amounts, the mono- and diacylglycerols are important intermediates in some metabolic reactions and may form the basis of other lipid classes. They are also used in processed foods, where they function as emulsifying agents.

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C h a p t e r 5 • LIPIDS 131

When triacylglycerols in adipose tissue are used for energy, the fatty acids are cleaved from glycerol by lipases and released from the cell in free (nonesterified) form. The free fatty acids bind to serum albumin and are transported to various tissues for oxidation via the tricarboxylic acid cycle (TCA cycle).

Phospholipids Phospholipids, as the name implies, are phosphate- containing lipids that form the structural basis of all cell membranes, including the membranes of organelles within the cell (see Figures 1.2 and 1.3). Because of their amphipathic properties, phospholipids are also critical components of plasma lipoproteins in which phospholipids, triacylglycerols, and other lipids form stable complexes, thus allowing them to be transported in the blood.

Glycerol forms the structural backbone of phos- pholipids. Fatty acids are esterified to the hydroxyl groups at the sn-1 and sn-2 positions of glycerol; a phosphate group is esterified at the sn-3 position and, in turn, a polar “head group” is esterified to the phosphate. A phospholipid molecule lacking the head group is known as phosphatidic acid (Figure 5.4). The conventional numbering of the glycerol carbon atoms is the same as that for triacylglycerols, provided the glycerol is written in the L configuration so that the sn-2 fatty acid constituent is directed to the left, as shown in Figure 5.4. The fatty acid portion of the molecule is hydrophobic, while the phosphate and the polar head group are hydrophilic, thus giving phospholipids their amphipathic property. Phospholipids generally have a saturated fatty acid at sn-1 and an unsaturated fatty acid at sn-2, although many fatty acid combinations are possible, resulting in a broad range of distinct phospholipids. Despite this fact, phospholipids are named according to the specific head group rather than their fatty acids. Common head group molecules are choline, ethanolamine, serine, and inositol, each possessing an alcohol group through which esterification to the phosphate takes place (Figure 5.4). The compounds are named as the phosphatidyl derivatives of the alcohols, as indicated in the figure. The most common phospholipid in mammal tissues is phosphatidylcholine, making up about half of the phospholipids in cell membranes, followed by phosphatidylethanolamine in terms of abundance. Food grade phosphatidylcholine (called lecithin) is produced commercially from egg yolks and soybeans and used as an emulsifier in the production of foods that contain both fat and water, such as margarine and chocolate. Phosphatidylserine and phosphatidylinositol are also found in cell membranes, but they serve important functions beyond membrane structure. Phosphatidylserine participates in apoptosis by attracting phagocytes during cellular degradation. Phosphatidylinositol participates in several cell functions, as described in the next section.

Diphosphatidylglycerol is another phospholipid found in several tissues of the body. It is also called cardiolipin and was originally identified within heart muscle (Figure 5.5). The structure of cardiolipin can be viewed as two phospholipid molecules that share a common head

Figure 5.4 Typical structure of phospholipids. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

1

H 1

2

3

C

H

C O

O H

C

H

HO OH

HO OH

OH

OH

C

O

C

O

P

O

O

Glycerol molecule

Phospholipid symbol

Polar head group

Polar head groups

CH2 CH2 N(CH3)3O

O

O

Phosphatidyl choline

Hydrophilic portion

Hydrophobic portion

Fatty acid

Fatty acid

Most cases a saturated fatty acid

Most cases an unsaturated fatty acid

CH2 CH2 NH3 Phosphatidyl ethanolamine

NH3CH2 CH Phosphatidyl serine

Phosphatidyl inositol

COO–

O

1

1

Figure 5.5 Structure of diphosphatidylglycerol (cardiolipin).

CH2 C

O

O R1

R1 = Fatty acid (typically saturated)

R2 = Fatty acid (typically polyunsaturated)CH C

O

O R2

CH C

O

O R2

CH2 C

O

O R1

CH2 P

O

O

O2

O CH2

CHOH

CH2 P

O

O

O2

O CH2

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132 C h a p t e r 5 • LIPIDS

group of glycerol. The overall structure therefore contains three glycerol molecules and four fatty acids. Cardiolipin is located exclusively in the inner membrane of mitochondria and attaches cytochrome c to the membrane.

The main structure of phospholipids described thus far involves ester bonds between the glycerol backbone and the fatty acids at the sn-1 and sn-2 positions. In some cases that linkage is an ether bond (—C—O—C—) or vinyl ether bond (—C—O—C5C—), resulting in the so-called “ether phospholipids.” Platelet-activating factor is perhaps the most studied ether phospholipid, having a fatty acid ether bond at sn-1, an acetate ester bond at sn-2, and phospho- choline as the head group at sn-3. Platelet-activating factor is an important signaling molecule that participates in several metabolic events including inflammation, platelet aggregation, and neural functions. Another well-known ether phospholipid is plasmalogen, which has a fatty acid vinyl ether bond at sn-1, a fatty acid ester bond at sn-2, and phospho-choline, ethanolamine, or serine as the head group at sn-3. Plasmalogens are found in many tissues, most notably the heart and brain. Choline plasmalogen constitutes up to 40% of all phospholipids in the heart, while ethanolamine plasmalogen makes up about 20% of phospholipids in the brain and is concentrated mostly in the myelin sheath.

Biological Roles of Phospholipids Phospholipids play several important roles in the body. Phospholipids are more polar than the triacylglycerols and sterols, and therefore tend to attract water molecules. Because of their amphipathic nature, phospholipids are found on the surface of blood-borne lipoprotein particles, thereby stabilizing the particles in the aqueous medium. Furthermore, phospholipids are important components

Figure 5.6 Inositol dual signaling system.

Phosphatidylinositol

Phosphorylation in plasma

membrane

2 ATPs

2 ADPs

H2O

Phosphatidylinositol-4,5-bisphosphate

Diacylglycerol Inositol-1,4,5-trisphosphate

Activation of protein kinase C

Release of intracellular Ca21

Enzyme activation

Enzyme activation

Other hormonal responses

Hydrolyzed by hormone-sensitive phospholipase C in plasma membrane to yield the inositol triphosphate

Ca21 is a second messenger causing other hormonal responses.

of cell and organelle membranes, where they form bilay- ers (see Chapter 1) and serve as a selective barrier for the passage of water-soluble and fat-soluble materials across the membrane. In addition to lending structural support to the membrane, they serve as a source of physiologically active compounds. We discuss later how arachidonate can be released on demand from membrane-bound phospha- tidylcholine and phosphatidylinositol when it is needed for synthesis of eicosanoids.

Phosphatidylinositol plays a specific role in anchoring membrane proteins when the proteins are covalently attached to lipids. Phosphatidylinositols anchor a wide variety of surface antigens and other surface enzymes in eukaryotic cells. In addition, certain hydrolytic products of phosphatidylinositol are active in intracellular signaling and act as second messengers in hormone function. An example of this role is the mechanism of action of insulin (discussed in Chapter 3). Phosphatidylinositol in the plasma membrane can be doubly phosphorylated by ATP, forming phosphatidylinositol-4,5-bisphosphate. Stimulation of the cell by certain hormones, such as insulin, activates a specific phospholipase C, which produces inositol-1,4,5-trisphosphate and diacylglycerol. Both of these products function as second messengers in cell signaling. Inositol-1,4,5-trisphosphate causes the release of Ca21 held within membrane-bound compartments of the cell, triggering the activation of a variety of Ca21-dependent enzymes and hormonal responses. Diacylglycerol binds to and activates an enzyme, protein kinase C, which transfers phosphate groups to several cytosolic proteins, thereby altering their enzymatic activities. This dual-signal hypothesis of phosphatidylinositol hydrolysis is represented in Figure 5.6 [2].

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C h a p t e r 5 • LIPIDS 133

Sterols Sterols are structurally quite different than the other lipid classes. They are characterized by having a four-ring steroid nucleus and at least one hydroxyl group, hence the name sterol (steroid alcohol). This section describes three cate- gories of sterols important to human nutrition: cholesterol, bile acids, and phytosterols.

Cholesterol Cholesterol is the most common sterol in humans. It can exist in free form, or the hydroxyl group at C-3 can be esterified with a fatty acid. The structure of choles- terol is shown in Figure 5.8, along with the numbering system for the carbons in the steroid nucleus and the side chain. Cholesterol is an important constituent of plasma membranes along with phospholipids due to its amphipathic nature. In free form, the hydroxyl group of cholesterol interacts with the phospholipid head group so that the hydrophobic side chain of cholesterol is ori- ented in parallel with the fatty acids of phospholipids (see Figure 1.3). Cholesterol constitutes nearly 25% of the lip- ids in plasma membranes of some nerve cells, but may be absent in intracellular membranes. Cells can regulate the amount of cholesterol in membranes by esterify- ing “excess” cholesterol with a fatty acid and storing the cholesterol esters in vesicles within the cytosol. When unesterified (free) cholesterol is needed, the cholesterol esters are hydrolyzed and free cholesterol is transported to the membrane.

Cholesterol serves as the precursor for many important steroids in the body, including the bile acids; steroid sex hormones such as estrogens, androgens, and progesterone; the adrenocortical hormones; and vitamin D (cholecalciferol). The major derivatives of cholesterol are shown in Figure 5.9. These steroids differ structurally from one another in the arrangement of double bonds in the ring system, the presence of carbonyl or hydroxyl groups, and the nature of the side chain at C-17. All of

Sphingolipids Sphingolipids are found in the plasma membrane of all cells, although their concentration is highest in cells of the central nervous system. Unlike the lipid classes dis- cussed thus far, sphingolipids are built on the amino alcohol sphingosine rather than glycerol as the structural backbone (Figure 5.7). All sphingolipids have a fatty acid attached to the amino group (R1 in the figure). The sim- plest sphingolipid is ceramide, in which the terminal hydroxyl has no other group attached. The other sphin- golipids build on ceramide, with substituent molecules attached to the terminal hydroxyl group (R 2 in the fig- ure). Sphingomyelin is formed when phosphocholine is added to ceramide. (Due to the presence of phosphate, sphingomyelin can also be considered a phospholipid, although it makes more sense to classify it primarily as a sphingolipid because of its structural similarity to other sphingolipids.) Sphingomyelin is particularly abundant in the myelin sheath of nerve tissues and thus important for nervous system function.

Cerebrosides are formed when a single sugar molecule, either galactose or glucose, attaches to the terminal hydroxyl group of ceramide. Galactocerebrosides are abundant in the myelin sheath of nerves and in brain tissue, particularly the white matter, whereas glucocerebrosides are found mainly in spleen and red blood cells. Cerebrosides are located on the plasma membranes where they serve a protective role, acting as an insulator and facilitator in the proper conduction of nervous impulses. Gangliosides resemble cerebrosides, except they have multiple sugar units linked to the terminal hydroxyl group of ceramide. In addition, gangliosides have a negatively charged sialic acid molecule attached to the oligosaccharide chain. Gangliosides are located on the outer surface of plasma membranes mainly in nerve tissue where they function as markers in cellular recognition and as receptors for certain hormones and toxins including the cholera toxin.

Figure 5.7 Structure of sphingolipids.

CH CH

OH

NH

O

The amino alcohol sphingosine (shaded area) forms the structural backbone of sphingolipids

CH3 (CH2)12 CH CH2CH R 2

R1 R1 R2

All sphingolipids have a fatty acid attached to the amino group

Attachment defines the type of sphingolipid

Ceramide Sphingomyelin Cerebroside Ganglioside

Fatty acid Fatty acid Fatty acid Fatty acid

H Phosphocholine Galactose or glucose Oligosaccharide

Sphingolipid

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134 C h a p t e r 5 • LIPIDS

Figure 5.8 Structure of a sterol, cholesterol, and a cholesterol ester. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

Steroid nucleus

Cholesterol

A cholesterol ester

H

C

H H

C

H

CH

H

H

C

H

C

H C C

H

H C

C C

H

H

H H

C

H

H

C H

H

C

H

H

C

H

C

C C

H H H

H

H

H

1

2

3

4 5

6 7

8

9 10

11 12

13

14 15

16 17

H

C

H

H H

H

C

C

CH3

CH3 CH3

H3C

H3C

H

H

H

C

C C

H

H H

C

H

H

H

C

C

C

C C

H

H

OO

C

H

C

H

H H

H

C

H

H

C

H

C

H

C C

H H H

C

H

H

C

H

C

C

Ester bond

Fatty acid

A cholesterol ester is an example of

a sterol ester.

CH3

H

H

H

C

C CH3C

H3C

H

C H

H H

C

H

H

H

C

C

C

C C

H

H

HO H

C

H

H H

H

C

H

H

H H

H

H

C

H

C

H

C C

H H H

C

C

H

H

C

CH3

H

CH3

C

C

C

The areas highlighted in red make this sterol a cholesterol molecule.

18

19

20

21 22 23 24 25 27

26

these structural modifications are mediated by enzymes that function as dehydrogenases, isomerases, hydroxylases, or desmolases. Desmolases remove or shorten the length of side chains on the steroid nucleus.

Bile Acids and Bile Salts As discussed in Chapter 2, bile acids and bile salts are critical components of bile that act as detergents in the small intestine to emulsify dietary lipids for digestion and

absorption. The liver synthesizes two bile acids, cholic acid and chenodeoxycholic acid, each of which is con- jugated with either glycine or taurine, resulting in four different primary bile salts (Figure 5.10). After the newly formed bile salts enter the small intestine via bile secre- tion, they are subject to dehydroxylation by bacteria, thus producing secondary bile salts. All of the bile salts can be reabsorbed into the enterohepatic circulation and returned to the liver. In this way, secondary bile salts,

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C h a p t e r 5 • LIPIDS 135

vegetables have low concentrations. Although cereal grains have only modest concentrations, humans consume large amounts of grain products, making them a quantitatively important source of phytosterols. Intake of total phytosterols from natural food sources is about 200–300 mg/day, with Asian and vegetarian diets providing significantly more [3]. The manufacture of foods and supplements enriched with phytosterols has increased in recent years because of their cholesterol- lowering properties. Phytosterol intake of 2 g/day results in blood cholesterol reductions of 10% or more. Approval to make health claims about phytosterols on food and supplement products has been granted by the U.S. Food and Drug Administration, the European Foods Safety Authority, and Health Canada. Because of their similarity to cholesterol, phytosterols have the ability to displace cholesterol from micelles that form during digestion, reducing the amount of cholesterol available for intestinal absorption.

while not directly synthesized by the liver, are present in gallbladder bile.

Phytosterols Plant cell membranes contain structural sterols in a man- ner similar to cholesterol in animal cells. These phytoster- ols are structurally similar to cholesterol, with only slight differences in the side chain (refer to Figure 5.8). Some phytosterols are actually stanols—meaning, the double bond between carbons 5 and 6 is eliminated by saturat- ing the molecule with hydrogen atoms. Strictly speaking, stanols are chemically different than sterols, but they are often counted together under the heading of phytosterols. Stanols constitute about 5–10% of total phytosterols pres- ent in nature. The hydroxyl group at C-3 of phytosterols can be esterified with a fatty acid.

Phytosterols are found throughout the food supply. Plant oils, legumes, nuts, and seeds have relatively high concentrations of phytosterols, while fruits and

Figure 5.9 The formation of physiologically important steroids from cholesterol. Only representative compounds from each category of steroid are shown.

Progesterone

C

CH3

HO

O

Cortisol

C O

CH2

OH

O

OH

O Testosterone

OH

O

Estradiol

OH

Sex glands

Adrenal glands

UV light

Liver

Cholesterol

7-dehydrocholesterol

HO

HO

Cholic acid HO

COOH

OH

HO Vitamin D3

HO

HO

Corticosteroid hormones

Sex hormones

Bile acids

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136 C h a p t e r 5 • LIPIDS

“Cooking Oils” category, which represents all uses of edible oils (mostly plant derived) in the United States, including those used in the food industry for the manufacture of food products; cooking and frying oils used by restaurants and other foodservice institutions; and salad and cooking oils used directly by consumers. The “Shortening” category represents solid fats (mostly plant derived) that are used for similar purposes as “Cooking Oils”. Figure 5.11 further shows that the food categories of animal origin contribute significant amounts of fat in the food supply. Note that “Butter” has been separated from “Dairy Products” to emphasize its individual contribution to overall fat intake. The “Other” category includes fat contributed by fruits, vegetables, fish, and specialty oils.

DIetarY SOUrCeS

Triacylglycerols—fats and oils—are ubiquitous in the food supply. They are found naturally in both plant and animal foods. They are also present in prepared and manufactured foods. Foods prepared in restaurants often contain high-fat ingredients and are cooked in oil; grocery stores are abun- dant with prepared and packaged foods containing fat; and many consumers use fats and oils when cooking at home. In order to track the amount and type of fat consumed in the United States, the Agricultural Research Service of the U.S. Department of Agriculture documents each year the major food groups that contribute fat to the food supply [4]. As indicated in Figure 5.11, the primary source of fat is the

Figure 5.10 The formation of glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate conjugated bile acids.

Cholic acid

Glycine+ or

HO

HO

3 7 OH

COO− Carboxyl end

Glycocholate Taurocholate

12

Chenodeoxycholic acid HO

3 7 OH

COO− Carboxyl end

12

Taurine

Glycine Taurine

Amino group

HO

HO

3

12

7 OH

C N H

O

HO

HO

3

12

7 OH

C N H

O

Glycochenodeoxycholate Taurochenodeoxycholate

3

12

7 OHHO

C N H

O

COO−

CH3

CH3

CH3

CH3

HO

CH3CH3

H3C

H3C

H3C

3

12

7 OH

C N H

O

SO3CH2CH2

COO−CH2

CH2

SO3CH2H3N +

Amino group

H3N + CH2

SO3CH2CH2

SO3CH2CH2

CH2

CH3

CH3

CH3

H3C

H3C

H3C

CH3

COO−

COO−

CH3

CH3 Amino group

H3N +

Amino group

H3N +CH2

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C h a p t e r 5 • LIPIDS 137

Figure 5.11 Dietary fat contribution from major food groups. Source: U.S. Department of Agriculture, Nutrient Content of the U.S. Food Supply, 2010.

0 5 10 15 20 25 30 35 40 45

Other

Eggs

Margarine

Grain Products

Butter

Lard, Tallow

Legumes, Nuts, Seeds

Poultry

Shortening

Dairy Products

Red Meats

Cooking Oils

Fat Intake (g/day per capita)

MUFA

SFA

PUFA

SFA MUFA n-6 PUFA

n-3 PUFA

grams

Bean burrito 1 each 4.3 2.3 3.5 0.6

Beef, top sirloin, broiled 3 oz 3.1 3.4 0.3 0.0

Beef, ribeye, broiled 3 oz 4.9 5.1 0.4 90% is oleic acid. Knowing the fatty acid composition of common fats and oils (Table 5.2) can help guide health care professionals and consumers in making well-informed decisions about dietary fats.

Although the information in Figure 5.11 shows only the major food groups, it is helpful to know the main contributors of fat when seeking to modify one’s fat intake. For example, if reduction in total fat intake is desired, focusing on all foods made with or cooked in “cooking oils” would be a good starting point. Reduction in red meats, dairy products, and foods made with shortening would also be advisable. If reducing SFA is the goal, the obvious targets are red meats and dairy products because of their relatively high proportion of SFA. However, products containing cooking oils and shortening should not be overlooked, particularly plant-derived solid fats that have relatively high proportions of SFA, such as palm kernel, palm, and coconut oils. Table 5.3 provides the fat content of foods commonly consumed in the United States. In updating the 2015 Dietary Guidelines for Americans, the Advisory Committee emphasized that “strong and consistent evidence from [randomized controlled trials] shows that replacing SFA with unsaturated fats, especially PUFA, significantly reduces total and LDL cholesterol . . . and reduces the risk of [cardiovascular disease] events and coronary mortality” [5]. These relationships are discussed later in the chapter.

The intake of trans fatty acid (1.3 g/day) is relatively minor, although they may be found in shortening, cooking oils, red meats (beef), dairy products, butter, margarine, and tallow. As mentioned earlier in the chapter, partially hydrogenated oils have been used extensively as frying oils

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138 C h a p t e r 5 • LIPIDS

Therefore, consumption of EPA and DHA (present in fatty fish) can avert deficiencies associated with low a-linolenic acid.

Trans fatty acids appear to provide no specific health benefits beyond providing energy. Therefore, no RDA or AI has been set. While it may seem tempting to establish an Upper Limit (UL) for trans fatty acids, they are unavoidable in the food supply and restricting intake of “natural” foods containing trans fatty acids, such as dairy products and meats, could have the unintended effect of creating deficiencies in other essential nutrients, notably high- quality protein and vitamins D and B12. Nevertheless, it is recommended that trans fatty acid consumption be as low as possible while consuming a nutritionally adequate diet.

The Dietary Guidelines for Americans recommend limiting SFA intake to 10% of total calories and that unsaturated fatty acids should be the primary source of dietary fat. Whether SFA are replaced by MUFA or PUFA depends on the dietary strategy employed, but in either case will likely result in health benefits. When SFA are replaced with PUFA, it is recommended that n-3 PUFA be selected to minimize the metabolic effects of “too much” n-6 linoleic acid. With regard to MUFA, much attention has focused on the so-called Mediterranean diet. Defining this diet has been challenging, but its general characteristics include high levels of MUFA intake (largely from olive oil) and significantly lower PUFA intake compared to the United States [6]. The Mediterranean diet also includes relatively high amounts of fiber and protein.

DIGeStION

Dietary lipids are hydrophobic and therefore pose a spe- cial problem to digestive enzymes. Like all proteins, diges- tive enzymes are hydrophilic and normally function in an aqueous environment. The dietary lipid targeted for diges- tion is emulsified by an efficient process, mediated mainly by bile salts. This emulsification greatly increases the sur- face area of the dietary lipid, consequently increasing the accessibility of the fat to digestive enzymes.

Triacylglycerols, phospholipids, cholesterol, and phytosterols provide the lipid component of the typical Western diet. Of these, triacylglycerols, commonly called fats or triglycerides, are by far the major contributor. The National Health and Nutrition Examination Survey (NHANES) for the years 2011–2012 found that males 20 years and over consume an average of 94 g/day and females 20 years and over consume an average of 67 g/day [7]. The intake of cholesterol is significantly less, estimated to be 338 and 229 mg/ day for the same groups, respectively [7]. Phytosterol intake is not tracked by NHANES, but, as mentioned earlier, it is about 200–300 mg/day, similar to cholesterol intake. Also not tracked by NHANES is phospholipid

and as an ingredient in manufactured products because of their superior functional properties that include greater shelf-life and stability at high frying temperatures. However, their use has significantly declined because of health concerns about trans fatty acids. Federal labeling requirements for packaged foods, laws restricting their use, and grassroots efforts have contributed to the decreased use of partially hydrogenated oils. Food labeling regulations currently require products to specify the amount of trans fatty acids per serving. Food processors want to be able to label their products as having “zero trans fat.” One strategy to accomplish this is to use a blend of natural oils containing a chain length and unsaturation level that provide the desired properties without any hydrogenation. For example, margarines that once contained hydrogenated oils may now contain a blend of soybean oil (liquid at room temperature) with palm and/ or palm kernel oil (solid at room temperature). Note that labeling regulations permit a food containing less than 0.5 g of trans fat per serving to be labeled as providing zero. Consuming several servings of foods labeled as containing zero trans fat can result in consuming more than the recommended level.

Recommended Intakes Recommendations regarding dietary fat and fatty acids have historically come from several governmental and nongovernmental (nonprofit) organizations, including the American Heart Association, the Institute of Medicine (IOM), the U.S. Department of Agriculture, and the U.S. Department of Health and Human Services. These orga- nizations work together in order to provide a cohesive message when making recommendations to the public.

The Food and Nutrition Board of the IOM has not established a Recommended Dietary Allowance (RDA) value for total fat intake. Adequate Intake (AI) levels have been established for infants, but not for adults or children over the age of 12 months (see inside front cover of the book). Rather than focusing on total fat, current recommendations and guidelines focus on specific fatty acids due to their individual effects associated with the prevention or promotion of disease.

AIs are established for the essential fatty acids, linoleic (18:2 n-6) and a-linolenic acid (18:3 n-3), at levels that prevent deficiency symptoms. The AI for a-linolenic acid is also set at levels believed to provide overall health benefits associated with the consumption of n-3 fatty acids (discussed later in the chapter). Table 5.2 shows flax (linseed) oil as having the highest percentage of a-linolenic acid among the common plant oils, followed by canola and soybean oils. a-Linolenic acid serves as a precursor for the highly unsaturated n-3 fatty acids (EPA and DHA), although the conversion efficiency of a-linolenic to EPA and DHA acid is very low in humans.

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C h a p t e r 5 • LIPIDS 139

of hormones of the enterogastrone family such as secretin, which inhibits gastric motility. Dietary fats therefore have a “high satiety value.”

The partially hydrolyzed lipid emulsion leaves the stomach and enters the duodenum as small lipid droplets. Further emulsification takes place because as mechanical shearing continues, it is complemented by bile salts that are released from the gallbladder as a result of stimulation by the hormone cholecystokinin (CCK). The small intestine has the capacity to digest a large quantity of triacylglycerols: up to 600 g with 95% efficiency [9]. Significant hydrolysis and absorption, especially of the long-chain fatty acids, require less acidity, appropriate lipases, more effective emulsifying agents (bile salts), and specialized absorptive cells. These conditions are provided in the lumen of the upper small intestine.

The pancreas simultaneously releases pancreatic lipase and bicarbonate, elevating the pH to a level suitable for pancreatic lipase activity. In combination with bile salts, the triacylglycerol breakdown products (free fatty acids and mono- and diacylglycerols) are themselves excellent emulsifying agents due to their amphipathic properties. Such molecules tend to arrange themselves on the surface of small fat particles, with their hydrophobic regions pointed inward and their hydrophilic regions turned outward toward the water phase. This chemical action, together with the help of peristaltic agitation, converts the fat into small droplets with a greatly increased surface area. The small droplets then can be readily acted upon by pancreatic lipase.

The action of pancreatic lipase on ingested triacylglycerols results in a complex mixture of diacylglycerols, monoacylglycerols, and free fatty acids. Its specificity is primarily toward sn-1-linked fatty acids and secondarily to sn-3 bonds. Therefore, the digestive action of pancreatic lipase progresses from triacylglycerols → 2,3-diacylglycerols and 1,2-diacylglycerols → 2-monoacylglycerols. Only a small percentage of the triacylglycerols is hydrolyzed totally to free glycerol. The complete hydrolysis of triacylglycerols that does occur probably follows the isomerization of the 2-monoacylglycerol to 1-monoacylglycerol, which is then hydrolyzed. Thus, the action of pancreatic lipase produces mostly 2-monoacylglycerols and free fatty acids that gradually shrink the size of the small fat droplet, finally resulting in bile salt–stabilized micelles. An overview of triacylglycerol digestion is summarized in Table 5.4.

An inhibitor of gastric and pancreatic lipase, orlistat, has been developed to reduce the absorption of dietary triacylglycerols. It is marketed both as Xenical, a prescription-only product, and Alli, an over-the-counter product. The rationale for use is that when the hydrolysis of triacylglycerols is restricted, less dietary fat will be absorbed, resulting in decreased caloric intake. Xenical inhibits the absorption about 30%, equivalent to a reduction of about 200 kcal from fat per day. As one might

intake, although it is estimated to be about 2–3 g/day. Digestive enzymes involved in breaking down dietary lipids in the gastrointestinal (GI) tract are esterases that cleave the fatty acid ester bonds within triacylglycerols (lipases), phospholipids (phospholipases), cholesterol esters (cholesterol esterase), and phytosterol esters (also cholesterol esterase).

Triacylglycerol Digestion Most dietary triacylglycerol digestion is completed in the lumen of the small intestine, although the process actually begins in the mouth and stomach with lingual lipase released by the serous gland, which lies beneath the tongue, and gastric lipase produced by the chief cells of the stomach. Basal secretion of these lipases apparently occurs continuously but can be stimulated by neural sympathetic agonists, high dietary fat, and sucking and swallowing. These lipases account for limited triacylglycerol digestion (10–30%) that occurs in the stomach. The lipase activity is made possible by the enzymes’ particularly high sta- bility at the low pH of the gastric juices. Gastric lipase readily penetrates milk fat globules without substrate stabilization by bile salts, a feature that makes it particu- larly important for fat digestion in the suckling infant, whose pancreatic function may not be fully developed. Both lingual and gastric lipases act preferentially on tria- cylglycerols containing medium- and short-chain fatty acids. They preferentially hydrolyze fatty acids at the sn-3 position, releasing a fatty acid and 1,2-diacylglycerols as products. This specificity again is advantageous for the suckling infant because in milk, triacylglycerols’ short- and medium-chain fatty acids are usually esterified at the sn-3 position [8]. Short- and medium-chain fatty acids are metabolized more directly than are long-chain fatty acids—that is, they can be absorbed into the blood and transported directly to the liver via the hepatic portal vein, as discussed later in this chapter. Commercially available high-energy formulas for preterm infants, which are rich in triacylglycerols containing short- and medium-chain fatty acids esterified at the sn-3 position, are designed to take advantage of the lipases’ specificity. These products supply ample energy to the premature infant in a small volume [8].

For dietary triacylglycerols to be hydrolyzed by lingual and gastric lipases in the stomach, some degree of emulsification must occur to expose a sufficient surface area of the substrate. Muscle contractions of the stomach and the squirting of the fat through a partially opened pyloric sphincter produce shear forces sufficient for emulsification. Also, potential emulsifiers in the acid milieu of the stomach include complex polysaccharides, phospholipids, and peptic digests of dietary proteins. The presence of undigested lipid in the stomach delays the rate at which the stomach contents empty, presumably by way

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140 C h a p t e r 5 • LIPIDS

of phospholipid, the bile releases significant amounts of phospholipid (specifically phosphatidylcholine) into the small intestine, perhaps five times more than the diet provides. Both dietary and biliary phospholipid is subject to hydrolysis by phospholipase A2 , which tar- gets the fatty acid at the sn-2 position of glycerol. The products of hydrolysis are lysophospholipid and a free fatty acid. These products, together with the products of triacylglycerol digestion and bile salts, incorporate into the resulting micelles for transport to the intestinal cell. Micelles that contain hydrolyzed lipids are nega- tively charged and have a much smaller diameter (~5 nm) than the unhydrolyzed precursor particles, allowing them access to the intramicrovillus spaces (50–100 nm) of the intestinal membrane.

Cholesterol Ester Digestion Some of the cholesterol present in food is esterified with a fatty acid. About 10% of the cholesterol in egg yolks is ester- ified, whereas about 50% in meat and poultry is esterified.

expect, the most frequently reported side effects of orlistat are gastrointestinal discomfort, fecal incontinence, and steatorrhea (presence of fat in feces).

The Role of Colipase Pancreatic lipase activation is complex, requiring the par- ticipation of the protein colipase, calcium ions, and bile salts. Colipase is formed by the hydrolytic activation by trypsin of procolipase, also of pancreatic origin. It con- tains approximately 100 amino acid residues and possesses distinctly hydrophobic regions that are believed to act as lipid-binding sites. Colipase has been shown to associate strongly with pancreatic lipase and therefore may act as an anchor, or linking point, for attachment of the enzyme to the bile salt–stabilized fat droplet.

Phospholipid Digestion Phospholipids are hydrolyzed by a specific esterase, phospholipase A2 , made and secreted by the pancreas. Recall from Chapter 2 that, in addition to dietary sources

Table 5.4 Overview of Triacylglycerol Digestion

H

H

H

Fatty acid

C

H

C

Fatty acid + H2O

+ Fatty acid

C

H

Fatty acid

H

H

H

Fatty acid

C

H

C

Fatty acid

C

H

OH

H

H

H

Fatty acid

C

H

C

Fatty acid + H2O

+ Fatty acid

C

H

Fatty acid

H

H

H

Fatty acid

C

H

C

Fatty acid

C

H

OH

H

H

H

Fatty acid

C

H

C

Fatty acid + H2O

+ 2 Fatty acids

C

H

Fatty acid

H

H

H

OH

C

H

C

Fatty acid

C

H

OH

Mouth

Stomach

Small intestine

Triacylglycerol

Triacylglycerols, diacylglycerols, and fatty acids

Triacylglycerol, diacylglycerol, and fatty acids

Emulsif ied triacylglycerols, diacylglycerols, and fatty acid micelles

Minor amount of digestion

Additional digestion

Phase I: Emulsif ication

Monoacylglycerols and fatty acids

Phase II: Enzymatic digestion

Lingual lipase produced in the salivary glands

Gastric lipase produced in the stomach

Bile; no lipase

Monoacylglycerol

Diacylglycerol

Diacylglycerol

Pancreatic lipase produced in the pancreas

Lingual lipase cleaves some fatty acids here.

Gastric lipase cleaves some fatty acids here.

Pancreatic lipase cleaves some fatty acids here.

Location Major Events Required Enzyme or Secretion Details

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C h a p t e r 5 • LIPIDS 141

and phytosterols, as well as fat-soluble vitamins. Stabilized by the polar bile salts, the micellar particles are sufficiently water soluble to penetrate the unstirred water layer that bathes the enterocytes of the small intestine. Micelles are small enough to interact with the microvilli at the brush border, whereupon their lipid contents move into the enterocytes. The term absorption refers to an overall process that includes the transport of digested lipids from the intestinal lumen across the brush border membrane; the reassembly of those lipids by esterification; and finally the release of the lipids into the circulation.

Fatty Acid, Monoacylglycerol, and Lysophospholipid Absorption The mechanism for moving fatty acids, monoacylglycerols, and lysophospholipids across the brush border membrane is not fully understood, although two general mechanisms

Cholesterol esters cannot be absorbed and therefore must be hydrolyzed to free cholesterol and free fatty acid to be incorporated into micelles for delivery to intestinal cells. Hydrolysis is achieved by cholesterol esterase, made and secreted by the pancreas. Free cholesterol from the diet (and from bile) requires no digestion and can directly incorporate in micelles. Cholesterol esterase also hydro- lyzes phytosterol esters consumed in the diet. As previously mentioned, free phytosterols can displace cholesterol from the micelle, resulting in less cholesterol being available for absorption. A summary of the digestion of lipids is shown in Figure 5.12.

aBSOrptION

Micelles contain the final digestion products from lipid hydrolysis, including free long-chain fatty acids, 2-monoacylglycerols, lysophospholipids, free cholesterol,

Figure 5.12 Summary of digestion and absorption of dietary lipids. Abbreviations: TAG, triacylglycerol; C, cholesterol; CE, cholesterol ester; PL, phospholipid; DAG, diacylglycerol; MAG, monoacylglycerol; FA, fatty acid; and α-GP, α-glycerolphosphate.

❷ Only TAG are acted upon in the stomach.

Lingual and gastric lipase hydrolyze medium- and

short-chain fatty acids from the sn-3 position to

yield DAG.

❸ TAG, DAG, C, CE, and PL enter the lumen

of the small intestine.

❹ These lipids along with bile salts form micelles and are acted upon by

intestinal and pancreatic enzymes.

❶ Dietary lipids include TAG, C, CE, and PL. These lipids enter the stomach largely intact.

❻ Glycerol, MAG, lysophospholipid, C, and long-chain FA are absorbed into the enterocyte with the aid of transfer proteins. These lipids may also move through the brush border membrane into the enterocyte by diffusion.

❼ In the enterocyte ER, glycerol is converted to α-GP. Additional α-GP is formed from glucose by glycolysis. α-GP, FA, MAG, and DAG are reformed to TAG. Lysophosphatides are re-esterified with FA to make PL. C is esterified to CE.

❺ Short- and medium-chain free fatty acids do not get incorporated into micelles for absorption into the intestinal cells

❽ The reformed lipids, along with apo-B48, form a chylomicron that leaves the enterocyte by exocytosis into the lymph, then empty into blood circulation. Other apolipoproteins are transferred to the chylomicrons from other lipoprotein complexes.

Chylomicron

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142 C h a p t e r 5 • LIPIDS

Cholesterol Absorption Cholesterol that enters the small intestine comes from two sources: the diet and bile. As previously mentioned, dietary intake of cholesterol is about 300 mg/day, whereas the bile contributes 800–1,400 mg/day. Because the majority of cholesterol available for absorption is of hepatic origin, the efficiency of absorption can affect how much choles- terol is retained in the body. Cholesterol not absorbed is excreted in the feces. Given that no oxidative pathway for cholesterol exists in humans, fecal excretion represents the primary catabolic route in which whole-body cholesterol homeostasis in maintained. Therefore, the efficiency of cho- lesterol absorption is a critical point of regulation and the target of drug and dietary therapies that block absorption and promote the removal of cholesterol from the body [11].

Cholesterol in the intestine must incorporate into micelles for delivery to the enterocyte. Uptake by the cell is mediated by a brush border protein called Niemann-Pick C1 like 1 (NPC1L1). Once inside the cell, cholesterol is carried through the cytosol by sterol carrier proteins. Cholesterol may incorporate into enterocyte membranes, although the majority is esterified in preparation for transport out of the cell as a component of large lipid-protein aggregates called chylomicrons. Cholesterol esterification is catalyzed by acyl-CoA:cholesterol acyltransferase 2 (ACAT2), which is required for chylomicron formation to occur.

Phytosterols are also transported into the intestinal cell by NPC1L1. Despite the ability of NPC1L1 to transport both cholesterol and phytosterols, essentially no phytosterols incorporate into chylomicrons or enter the circulation. This is due to the presence of two additional proteins— members of the ATP-binding cassette (ABC) transporter family called ABCG5 and ABCG8—that reside adjacent to NPC1L1 in the brush border membrane. The role of ABCG5 and ABCG8 is to redirect phytosterols back into the intestinal lumen immediately after being taken into the cell. ABCG5 and ABCG8 also redirect some cholesterol back into the intestinal lumen, so that the overall efficiency of cholesterol absorption is about 50–60% [11]. A rare autosomal recessive disorder called sitosterolemia can occur as a result of mutations in either ABCG5 or ABCG8, causing hyperabsorption of cholesterol and phytosterols.

Strategies to block cholesterol absorption date back to the 1950s when patients with elevated blood cholesterol were given a commercial preparation of phytosterols suspended in fruit-flavored syrup (marketed as Cytellin). Phytosterols are known to displace cholesterol from micelles and compete for binding to NCP1L1. The product had limited success and was largely replaced by powerful prescription drugs, including ezetimibe, which directly inhibits NPC1L1, resulting in significant reductions (about 18%) in blood cholesterol levels. For patients who cannot tolerate prescription drugs, foods and supplements enriched with phytosterols are increasingly available and effective at reducing blood cholesterol concentration by 10% or more [3].

have been suggested involving a protein-independent diffusion model and a protein-dependent model. Diffusion across the brush border membrane occurs when the concentration in the intestinal lumen exceeds that of the cell. Diffusion is made possible because of the simi- lar amphipathic nature of both digestion products and membrane lipids, which allows the fatty acids, monoac- ylglycerols, and lysophospholipids to associate with the membrane lipids as they pass into the cell interior. Pro- tein-dependent transport appears to involve transporters that are lipid specific.

An important protein transporter of fatty acids in enterocytes is called CD36, located on the brush border. CD36 is expressed in a variety of cells throughout the body where it serves as a binding site for other molecules in addition to fatty acids [10]. CD36 also transports monoacylglycerols into the cell, but whether it transports lysophospholipids is unknown. Well-defined transport proteins for lysophospholipids are known to exist in yeast, although the presence of similar proteins in mammalian cells, while assumed to exist, have not been reported in the scientific literature.

Other proteins implicated in fatty acid uptake into enterocytes are a family of proteins called the fatty acid transport proteins (FATP), particularly FATP4. However, unlike CD36, which functions on the brush border membrane, FATP4 resides primarily in intracellular membranes where it facilitates the attachment of coenzyme A to fatty acids already in the cell, thereby priming the fatty acids for synthesis of triacylglycerols, phospholipids, or cholesterol esters. Note that dietary lipids require digestion for transport across the brush border membrane, but in order to move them out of the enterocyte, the lipids need to be reassembled into triacylglycerols, phospholipids, or cholesterol esters. Consequently, FATP4 promotes the absorption of fatty acids by facilitating the flow of lipids through the enterocyte.

In the enterocyte the products of lipid digestion (free fatty acids, monoacylglycerols, and lysophospholipids) move to the endoplasmic reticulum where they are re-esterified. There appears to be specific transport proteins, called fatty acid binding proteins (FABP), that carry them in the aqueous cytosol. FABP were first discovered to carry fatty acids (hence the name), but have since been reported to transport lysophospholipids and monoacylglycerols [10]. Once in the endoplasmic reticulum, acyltransferases transfer the fatty acid– CoA molecules onto the monoacylglycerol and lysophospholipid to produce triacylglycerol and phos- pholipid, respectively. Note that triacylglycerols can also be synthesized from a-glycerophosphate in the enterocytes. This metabolite can be formed either from the phosphorylation of free glycerol or from reduction of dihydroxyacetone phosphate, an intermediate in the pathway of glycolysis (see Figure 3.17).

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C h a p t e r 5 • LIPIDS 143

medium-chain fatty acids are used clinically to treat patients with intestinal disorders because the medium- chain fatty acids can be absorbed directly to the portal blood without the need for chylomicron formation. Key features of intestinal digestion and absorption of lipids are depicted in Figure 5.12.

traNSpOrt aND StOraGe

Lipids are transported in the blood as components of highly organized lipid-protein complexes (or particles) called lipoproteins. Chylomicrons, as mentioned in the previous section, are a class of lipoproteins. The other classes are very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Each lipoprotein class participates in transport systems that can be defined as exogenous (dietary) lipid transport, endogenous lipid transport, and reverse cholesterol trans- port. In this section, the structure of lipoproteins is first described, followed by a discussion on the lipid transport systems and the central role of the liver.

Lipoprotein Structure All lipoproteins share similar structural features in which hydrophobic, nonpolar “neutral” lipids (triacylglycerols and cholesterol esters) reside in the spherical core, surrounded by a monolayer of amphipathic phospholipids and free cholesterol that partitions the neutral lipid from the aqueous environment. Added to the surface are proteins (called apolipoproteins or apoproteins) that impart structural sta- bility and functionality by serving as enzyme activators or ligands for cell receptors. The arrangement of the lipid and protein components of a typical lipoprotein is represented in Figure 5.13. The illustration depicts the apoproteins as being either peripheral (residing mostly on the external

Lipid Release into Circulation For dietary lipids to be fully absorbed into the circula- tion, they must first be packaged in a form that allows for transport in the aqueous bloodstream. Lipids that are re- esterified in the endoplasmic reticulum of the enterocytes are assembled into large lipid-protein aggregate structures called chylomicrons [12]. The formation of chylomicrons occurs in direct response to eating a fat-containing meal; therefore, the proportions of the various lipids in chylo- microns reflect that of the diet. Chylomicrons are spheri- cal particles containing mostly triacylglycerols and some cholesterol esters in the core (due to their hydrophobic- ity), with amphipathic phospholipids, free cholesterol, and protein on the surface. The main protein added to the particle surface is called apolipoprotein B-48 (apoB-48) that helps stabilize the triacylglycerol-rich chylomicron in the aqueous environment of the circulation. Chylomi- crons are released from the enterocytes by exocytosis into the lymphatic system, where they travel a few inches via the thoracic duct to the left subclavian vein, at which point they enter the systemic blood circulation. The metabolic advantage of first entering the lymphatic system is to bypass the liver, an organ that would have catabolized the chylomi- crons if they had entered the hepatic portal vein. This short detour around the liver allows chylomicrons to deliver their triacylglycerol cargo to other tissues such as muscle and adipose tissue (discussed in detail in the next section).

Medium-chain fatty acids (those containing 6–12 carbon atoms), if present in the diet, have the ability to pass from the enterocyte directly into the portal blood, where they bind to albumin and are transported directly to the liver. Most of the medium-chain fatty acids escape esterification in the enterocyte and enter the portal blood as free fatty acids. The different fates of the long- and medium-chain fatty acids result from the specificity of the acyl-CoA synthetase enzymes for long-chain fatty acids. Triacylglycerols containing

Figure 5.13 Generalized structure of a plasma lipoprotein.

Phospholipid

Cholesteryl ester

Triacylglycerol

Core of mainly nonpolar lipids

Monolayer of mainly polar lipids

Integral apoprotein (e.g., apoB)

Free cholesterol

Peripheral apoprotein (e.g., apoC)

Amphipathic phospholipids and free cholesterol form a monolayer surrounding nonpolar “neutral” lipids.

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144 C h a p t e r 5 • LIPIDS

class; for example, chylomicrons are defined by having apoB-48, apoA-1, apoC-2, apoE, and so on. A partial listing of the apoproteins—together with their molecular weight, the lipoprotein class with which they are associated, and their postulated physiological function—is found in Table 5.5.

surface of the lipoprotein) or integral (having multiple regions that span the phospholipid monolayer). The apo- proteins are abbreviated “apo” and are identified using letters and numbers. Each lipoprotein class will have a complement of apoproteins that is characteristic of that

Apolipoprotein Lipoprotein(s) Molecular Mass (Da) Additional Remarks

apoA-1 HDL, chylomicrons 28,000 Activator of lecithin: cholesterol acyltransferase (LCAT); ligand for HDL receptor

apoA-2 HDL, chylomicrons 17,000 Structure is two identical monomers joined by a disulfide bridge

apoA-4 Secreted with chylomicrons but transfers to HDL 46,000 Associated with the formation of triacylglycerol-rich lipoproteins; function unknown

apoB-100 LDL, VLDL, IDL 550,000 Synthesized in liver; ligand for LDL receptor

apoB-48 Chylomicrons, chylomicron remnants 260,000 Synthesized in intestine

apoC-1 VLDL, HDL, chylomicrons 7,600 Possible activator of LCAT

apoC-2 VLDL, HDL, chylomicrons 8,916 Activator of extrahepatic lipoprotein lipase

apoC-3 VLDL, HDL, chylomicrons 8,750 Several polymorphic forms depending on content of sialic acids

apoD Subfraction of HDL 20,000 Function unknown

apoE VLDL, HDL, chylomicrons, chylomicron remnants 34,000 Ligand for chylomicron remnant receptor

Table 5.5 Apolipoproteins of Human Plasma Lipoproteins

Figure 5.14 Lipid and protein of lipoprotein classes. Source: Beerman/McGuire, Nutritional Sciences, 1/e. © Cengage Learning.

82% 7% 2% 9%

52% 18% 22% 8%

31% 22% 29% 18%

9% 23% 47% 21%

3% 28% 19% 50%

Phospholipid

Cholesteryl ester

Cholesterol

Triacylglycerol

Triacylglycerol Phospholipid

Cholesterol Protein

Chylomicron

Protein

VLDL (Very-Low-Density Lipoprotein)

IDL (Intermediate-Density Lipoprotein)

LDL (Low-Density Lipoprotein)

HDL (High-Density Lipoprotein)

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C h a p t e r 5 • LIPIDS 145

involves VLDL, IDL, and LDL and refers to the transport of triacylglycerols from the liver to peripheral tissues for storage or energy utilization. This system operates con- tinuously to maintain proper balance of fatty acids and triacylglycerols that accumulate in the liver during normal metabolism. Reverse cholesterol transport involves HDL and refers to the ability of HDL to pick up excess choles- terol from peripheral tissues and deliver it to the liver for excretion from the body (via bile) or conversion to other important molecules (see Figure 5.9).

Exogenous Lipid Transport Immediately after a fat-containing meal, the exogenous (dietary) lipids are packaged into chylomicrons within the enterocyte and distributed to peripheral tissues, mainly muscle and adipose tissue. When chylomicrons are released from the enterocyte, they contain mostly triacylglycerols, reflecting the abundance of triacylglyc- erols in the diet. They also contain apoB-48 and apoA-1. The apoB-48 protein made by the intestine is related to apoB-100 (made by the liver), in that both arise from the same gene, although the intestinal cell contains a stop codon that results in a truncated protein that is 48% of the sequence of apoB-100. After chylomicrons enter the blood, they acquire more apoproteins (mainly apoE and apoC-2) from HDL as the lipoproteins interact in the circulation. The exogenous lipid transport system and chylomicron metabolism are illustrated in Figure 5.15.

In addition to the apoprotein composition, each lipoprotein class has its own characteristic lipid composition, physical properties, and metabolic function. Initially, lipoproteins were separated from serum by electrophoresis and therefore were named based on their movement in an electrical gradient. Later, they were separated by centrifugation and were named based on their density. These names persist even though other methods are often used for their separation. Lipoproteins with higher proportions of lipid have a lower density. The largest and least dense lipoproteins are the chylomicrons, having a high lipid:protein ratio. The smallest and most dense are HDL, having a low lipid:protein ratio. The relative percentage of lipids and protein in each lipoprotein class is shown in Figure 5.14.

Lipoprotein Metabolism The main function of lipoproteins is to transport lipids in the blood. Each lipoprotein class is specialized with regard to which lipids are transported, where the lipids are deliv- ered, and the lipoprotein’s metabolic fate after the job is completed. The exogenous lipid transport system involves chylomicrons and refers to the transport of dietary lipids, primarily triacylglycerols, from the intestine to peripheral tissues for storage or energy utilization. This system oper- ates only after a fat-containing meal. Chylomicrons disap- pear after all of the dietary triacylglycerols are delivered to target tissues. The endogenous lipid transport system

Figure 5.15 Exogenous lipid transport. Abbreviations: TAG, triacylglycerol; MAG, monoacylglycerols; PL, phospholipid; HL, hepatic lipase; LRP, LDL receptor-related protein; C, cholesterol and cholesterol esters. Source: Modified from Harpers Illustrated Biochemistry, Figure 25-3, page 221, by R. K. Murray, D. K. Rodwell, and W. Victor, 27th edition (Lange Medical Books/McGraw-Hill 2006).

TAG C

Chylomicron contains apoB-48 and apoA-1.

Apolipoproteins E and C-2 are transferred to the chylomicron from HDL.

❸ Chylomicrons deliver the TAG to tissues other than the liver, particularly adipose and muscle.

Adipose tissue and muscle cannot phosphorylate glycerol so they transfer it to the serum to be picked up by the liver or kidney.

When much of the TAG are transferred from the chylomicrons they become chylomicron remnants.

The chylomicron remnant transfers the apoA and apoC back to HDL.

The chylomicron remnant attaches to the liver binding site containing hepatic lipase, and the fatty acids, cholesterol, and cholesteryl esters are transferred to the liver.

Dietary TAG

Chylomicron

Lymphatics

apo B-48

TAG C

Small intestine

Liver

Cholesterol Fatty acids

apo A

apo A

apo A

Glycerol

Chylomicron remnant

HDL

Non-hepatic tissues

Fatty acids and MAG

apo B-48

apo B-48

Chylomicron

apo E

apo E

apo E

apo C

apo C

TAG C

PL C

LRP

ap oC

, ap oE

Lipoprotein lipase

apoA, apoC

HL

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146 C h a p t e r 5 • LIPIDS

the cells. As the chylomicron becomes depleted of its core triacylglycerols, the lipoprotein structure shrinks in size, yet retains the other lipids. About 80% of the chylomicron triacylglycerols are delivered to target tissues in this manner. Once depleted of its triacylglycerols, the chylomicron “remnant” particles separate from the cell surface and reenter the circulation. The chylomicron remnants may donate some of its apoproteins to HDL. The chylomicron remnants travel to the liver where a specific receptor recognizes their apoE component, enabling the uptake of the entire particle into hepatocytes. The receptor is called LDL receptor–related protein 1 (LRP1). A second receptor, syndecan-1, has recently been identified that also binds chylomicron remnants and removes them from the circulation [13]. In addition, lipoproteins that have apoE, such as chylomicron remnants, can bind to the LDL receptor (discussed in the next section) and be cleared from the circulation. Hepatic lipase is a key enzyme that hydrolyzes the triacylglycerols and phospholipids of the chylomicron remnants as they enter the hepatocyte.

The interaction of chylomicrons with adipocytes and subsequent lipid metabolism in the fed state is presented in Figure 5.16. Adipocytes are the major storage site

Chylomicrons enter the bloodstream at a relatively slow rate, which prevents excessive increases in blood triacylglycerol levels. Entry of chylomicrons into the blood can continue for up to 14 hours after consumption of a large meal rich in fat. Blood triacylglycerol concentration usually peaks 30 minutes to 3 hours after a meal and returns to near normal within 5–6 hours. These times can vary, however, depending on the stomach emptying time, which in turn depends on the size and composition of the meal. The presence of triacylglycerol-laden chylomicrons accounts for the turbidity (milky appearance) of postprandial plasma and can interfere with clinical readings when “fasting triglyceride” values are desired. Twelve hours of fasting is usually required to obtain true readings that are devoid of chylomicrons.

Circulating chylomicrons interact with tissues that express the enzyme lipoprotein lipase, primarily skeletal muscle, heart muscle, and adipose tissue (but not liver). This interaction occurs due to the presence of apoC-2, which activates the enzyme. Chylomicrons dock on the cell surface where lipoprotein lipase hydrolyzes the triacylglycerols, producing free fatty acids and 2-monoacylglycerols that are quickly taken up into

Figure 5.16 Lipid metabolism in the adipose cell following a meal. Abbreviations: CHYLM, chylomicron; DAG, diacylglycerol; MAG, monoacylglycerol; TAG, triacylglycerol; and FFA, free fatty acid.

Glucose is metabolized to make acetyl-CoA, which can be converted to fatty acids.

Lipoprotein lipase acts on TAG in chylomicrons (CHYLM) causing free fatty acids (FFA) and MAG to enter the adipocyte.

❸ Lipoprotein lipase acts on VLDL so FFA and MAG enter the cell.

❹ The pathways favor energy storage as TAG. Insulin stimulates lipogenesis by promoting entry of glucose into the cell and by inhibiting the hormone-sensitive lipase that hydrolyzes the stored TAG to FFA and glycerol.

Adipocyte

Glucose

Glycerol

GLU-6-P

Triose-P

Pyruvate

Acetyl-CoA

TCA cycle

Blood vessel

FFA DAG MAG

CHYLM

LPL TAG

LPL TAG

LPL TAG

VLDL

IDL

LDL

CR

Triacylglycerol pool

Fatty acid pool

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C h a p t e r 5 • LIPIDS 147

glucose, fructose, and amino acids. It can also utilize “preformed” lipids delivered to it as chylomicron remnants, LDL, and HDL. A third source of lipid for VLDL synthesis comes from free fatty acids bound to serum albumin that are taken up by the liver. The free fatty acids may be of dietary origin (absorbed directly into the portal blood) or from adipose tissue (released into the systemic circulation during lipolysis). Figure 5.17 depicts the interrelationships among the pathways of lipid, carbohydrate, and protein metabolism in the liver, illustrating how lipids from remnant particle uptake, albumin-bound free fatty acids, and nonlipid precursors can be converted to triacylglycerols and secreted as VLDL into the systemic circulation.

Glucose, fructose, and amino acids that enter the liver from the hepatic portal vein can be converted to fatty acids and incorporated into VLDL if in excess and other demands for these molecules are met. Excess glucose and fructose not used for energy via the TCA cycle results in an accumulation of acetyl-CoA, which can be used to synthesize fatty acids (see Chapter 3). The glycerol needed for triacylglycerols is made from triose phosphates such as glycerol-3-phosphate. Amino acids can serve as precursors for fatty acids because they can be metabolically converted to acetyl-CoA or pyruvate. The synthesis of fatty acids, triacylglycerols, and phospholipids is described in detail later in this chapter.

In addition to triacylglycerols, the liver processes phospholipids, cholesterol, and cholesterol esters. Phos- pholipids from chylomicron remnants can incorporate into cell membranes or be used in the assembly of VLDL. Cholesterol and cholesterol esters from chylomicron remnants may be used in several ways:

● converted to bile salts and secreted in the bile ● secreted directly into the bile as free cholesterol ● incorporated into cellular membranes as free cholesterol ● incorporated into VLDL and released into the blood.

VLDL are assembled in the liver from endogenous triacylglycerols in much the same way as chylomicrons are assembled in the enterocytes from dietary triacylglycerols. The lipids are carried to the endoplasmic reticulum, assembled into VLDL with its complement of apoproteins, and secreted from the cell by exocytosis. The main structural apoprotein on VLDL is apoB- 100; one molecule of apoB-100 is associated with each VLDL particle. Because of its large size, the apoB- 100 protein encircles the VLDL particle with several regions that anchor within the phospholipid monolayer. Newly secreted VLDL also contain apoC-1 and apoE. Circulating VLDL acquire apoC-2 and additional apoE

for triacylglycerol and the most likely target of chylomicrons following a fat-containing meal. Usually the amount of fat consumed by an individual in a single meal exceeds the immediate energy demands of tissues. Therefore most dietary triacylglycerol must be stored, at least temporarily, until needed when energy demand exceeds energy intake. Triacylglycerol is in a continuous state of turnover in adipocytes; that is, constant lipolysis (hydrolysis during energy needs) is countered by constant re-esterification to form triacylglycerols (storage during energy excess). These two processes are not simply forward and reverse directions of the same reactions but are different pathways involving different enzymes and substrates. In the fed state, metabolic pathways in adipocytes favor triacylglycerol synthesis, a process strongly influenced by insulin. Insulin increases the uptake of free fatty acids and monoacylglycerols in adipocytes by stimulating lipoprotein lipase. Insulin also accelerates the entry of glucose into adipocytes and its conversion to fatty acids. Glycolysis in adipocytes provides a source of glycerol- 3-phosphate for re-esterification with the fatty acids to form triacylglycerols. Absorbed monoacylglycerols also furnish the glycerol backbone for re-esterification. Insulin further exerts its lipogenic action on adipose by strongly inhibiting hormone-sensitive lipase, which hydrolyzes stored triacylglycerols, thus favoring triacylglycerol synthesis.

Endogenous Lipid Transport The endogenous lipid transport system begins and ends with the liver. In brief, hepatic triacylglycerols are pack- aged in VLDL and delivered to peripheral tissues in a man- ner similar to chylomicrons. After delivery, the leftover particles, referred to as LDL, are depleted of triacylglycerol but relatively enriched in cholesterol. As remnant parti- cles, LDL are removed from the circulation for catabolism by specific receptors on the plasma membrane of cells, primarily hepatocytes. If the LDL receptors are in short supply, LDL can accumulate in the blood, causing the con- centration of LDL-associated cholesterol to rise. The health implications of elevated LDL cholesterol concentration are discussed later in this chapter.

The synthesis and role of VLDL are discussed first. The liver has a limited capacity to store triacylglycerols and must continually move them out for transport to peripheral tissues where they can be stored or used for energy. The liver’s ability to synthesize and secrete triacylglycerols in VLDL helps to maintain the balance of energy-containing nutrients throughout the body. The liver is capable of synthesizing new fatty acids and triacylglycerols from nonlipid precursors such as

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148 C h a p t e r 5 • LIPIDS

Figure 5.17 Metabolism in the liver following a fatty meal. Abbreviations: CR, chylomicron remnant; ALB, albumin; FFA, free fatty acid; MAG, monoacylglycerol; DAG, diacylglycerol; C, cholesterol; CE, cholesterol ester; TAG, triacylglycerol.

DAG MAG

Dietary nutrients enter the liver through the portal vein. Glucose can be converted to glycogen or enter glycolysis.

Amino acids enter the amino acid pool and some are metabolized to produce pyruvate and oxaloacetate.

Serum FFA, bound to albumin, enter the fatty pool and are TAG.

❹ CR enter the hepatocyte by endocytosis, and are taken up by a lysosome. FFA, MAG, and C are released. The lipids are reformed to TAG and CE and packaged.

TAG, C, and PL are packaged with apolipoproteins and enter the circulation as VLDL.

VLDL deliver triacylglycerols to muscle and adipose tissue.

Dietary nutrients

To systemic

circulationGlucose

Glycogen

Glycerol

Hepatocyte

Apoprotein

GLU-6-P

Triose-P

Pyruvate

Acetyl-CoA

Oxaloacetate TCA cycle

Amino acids

ALB-FFA

NH3

NH3

Hepatic veins

Portal vein

VLDL VLDL

FFA

Phospholipid Cholesterol

Biliary excretion

Triacylglycerol pool

Fatty acid pool

❺ ❻

CRCR

from HDL. The main features of the endogenous lipid transport system are depicted in Figure 5.18.

By virtue of apoC-2 on its surface, VLDL bind to and interact with lipoprotein lipase on adipose and muscle cells in a manner similar to the binding and hydrolysis of triacylglycerols in chylomicrons. Within the muscle cell, the free fatty acids and monoacylglycerols from VLDL are primarily oxidized for energy, with only limited amounts resynthesized for storage as triacylglycerols. Endurance- trained muscle, however, does contain some triacylglycerol deposits. In adipose tissue, in contrast, the absorbed fatty acids are largely used to resynthesize triacylglycerols for storage. As the triacylglycerols are removed from VLDL, a smaller transient IDL particle is formed. A few IDL particles may separate from the cell and return to the circulation; however, most remain attached and the removal of triacylglycerols continues until a triacylglycerol- depleted LDL particle remains. As LDL particles shrink in size, they lose all of their apoproteins except apoB- 100. Several events can determine the size of LDL, their interaction with lipoprotein lipase, and other lipoproteins

with the intravascular space where exchange of lipids can occur. Clinical studies have indicated that small dense LDL are more atherogenic than larger LDL, which emphasizes the importance of having a more thorough analysis conducted on LDL subfractions in individuals who are at risk for cardiovascular disease. The LDL particles separate from the cell and enter the circulation with a significantly different lipid profile compared to VLDL. Whereas VLDL are rich in triacylglycerols, LDL are composed of the remaining lipids that were secreted by the liver in VLDL. The relative percentage of phospholipids, free cholesterol, and cholesterol esters in LDL are greater than VLDL (see Figure 5.14), making LDL the primary carrier of cholesterol in the bloodstream of most people. Furthermore, apoB-100 is the only remaining apoprotein on LDL (one molecule per LDL particle). It is imperative that LDL, as the major carrier of cholesterol, be removed from the blood to prevent the accumulation of LDL cholesterol.

Clearance of LDL from blood is accomplished by a cell surface receptor—the LDL receptor—that recognizes apoB-100 and binds LDL particles for uptake into

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C h a p t e r 5 • LIPIDS 149

The LDL receptor has been extensively studied since elevated serum LDL-cholesterol concentration is a known risk factor for cardiovascular disease (CVD). Factors that influence the number of receptors on the cell surface impact LDL-cholesterol concentration. Dietary components are known to strongly influence the number of LDL receptors: saturated and trans fatty acids decrease receptors, whereas soluble fiber and phytosterols increase receptors. In addition, obesity reduces the number of LDL receptors; therefore, obese individuals are less responsive to dietary interventions that normally improve serum cholesterol profiles [15]. Genetic studies have also identified naturally occurring mutations that result in abnormal LDL receptors that can cause dramatically elevated cholesterol levels, termed familial hypercholesterolemia. More recently, a hepatic protein called PCSK9 was discovered that binds to LDL receptors and disrupts the recycling mechanism that returns the receptors to the cell surface after internalization. Interestingly, people with a mutation in PCSK9 that disables its function have low LDL cholesterol concentration and have lower risk of developing CVD [14].

Reverse Cholesterol Transport Reverse cholesterol transport refers to the ability of cir- culating HDL to pick up excess cholesterol from periph- eral tissues and deliver it to the liver for excretion from the body via bile, as either free cholesterol or bile acids. While every cell in the body can synthesize cholesterol, mammals lack the oxidative enzymes necessary to degrade cholesterol. Therefore, the transport of cholesterol from

the cell [14]. ApoE also binds to the LDL receptor, so lipoproteins expressing apoE also have the potential to be cleared from the circulation via the LDL receptor. LDL binds to the LDL receptors on cell membranes with high affinity and specificity. The LDL receptors located on hepatocytes are particularly important, as they remove 70–80% of LDL from the circulation. Membrane-bound LDL is then internalized by endocytosis. The interaction between the receptors and apoB-100 is the key to the cell’s internalization of the LDL. Figure 5.19 depicts the fate of the LDL particle following its binding to the membrane receptor. The internalized LDL particle is carried to lysosomes, and the receptor is released and returns to the surface of the cell. In the lysosome the apoprotein and cholesterol ester components are hydrolyzed by lysosomal enzymes into amino acids, free fatty acids, and free cholesterol. The influx of free cholesterol exerts the following regulatory functions:

● The rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), is suppressed through decreased transcrip- tion of the reductase gene and the concomitant increased degradation of the enzyme.

● The enzyme regulating cholesterol esterification, acyl- CoA:cholesterol acyltransferase (ACAT), is activated, thus promoting cholesterol ester storage.

● The synthesis of LDL receptors is suppressed through decreased transcription of the receptor gene, thereby preventing further entry of LDL into the cell.

Figure 5.18 Endogenous lipid transport. Abbreviations: B-100, apolipoprotein B-100; E, apolipoprotein E; TAG, triacylglycerol; C, cholesterol and cholesterol esters; and PL, phospholipid. Source: Modified from Harpers Illustrated Biochemistry, Figure 25-4, page 222, by R. K. Murray, D. K. Rodwell, and W. Victor, 27th edition (Lange Medical Books/McGraw-Hill 2006).

Nascent VLDL

B-100

TAG C

Liver

Cholesterol

Fatty acids

❺ ❻

Nascent VLDL are made in the Golgi apparatus of the liver.

Additional apolipoproteins C and E are transferred from HDL.

❸ The fatty acids from triacylglycerols (TAG) are hydrolyzed by lipoprotein lipase found mainly in muscle and adipose tissue.

As the TAG is removed from the VLDL, the particle becomes smaller and becomes an IDL.

Further loss of TAG and it becomes a LDL.

LDL are taken up by LDL receptors found in the liver and non-hepatic tissue.

LDL

HDL

Non-hepatic tissues

Non-hepatic tissues

Fatty acids and MAG

Glycerol

apo E

apo E

apo E

apo C

B-100

IDL

B-100

TAG C

PL C

C

LDL receptor

ap oC

ap

oE

apo C

apo A

Lipoprotein lipase

TAG C

B-100

VLDL

apo E

apo C

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150 C h a p t e r 5 • LIPIDS

As nascent HDL acquire phospholipids and cholesterol, they also acquire an intravascular enzyme called lecithin:cholesterol acyltransferase (LCAT). This enzyme forms cholesterol esters by catalyzing the transfer of fatty acids (usually polyunsaturated) from the sn-2 position of phosphatidylcholine to free cholesterol within the HDL particle. Because the resulting cholesterol esters are nonpolar, they migrate to the core of the particle, forming mature HDL. The small spherical HDL can further interact with peripheral tissues as the apoA-1 binds to SR-B1 and yet another receptor, ABCG1. (Mature HDL bind to ABCG1, but not ABCA1. Both nascent and mature HDL bind to SR-B1.) Further binding to cell receptors and the continued action of LCAT causes HDL to grow in size. The accumulated cholesterol esters in HDL can be transferred to other lipoproteins through the action of cholesterol ester transfer protein (CETP). By distributing cholesterol esters to VLDL and LDL, cholesterol ester transfer protein helps to reduce the size of HDL so that interaction with cell surface receptors is optimized, thus increasing HDL’s ability to accept more cholesterol.

The final step in reverse cholesterol transport is the binding of HDL to SR-B1 receptors on the surface of hepatocytes. Two actions are possible: the cholesterol esters may be selectively deposited in the liver cells and the depleted HDL returned to the circulation, or the entire HDL particle may be internalized and degraded. Intracellular degradation of HDL occurs in lysosomes in a manner similar to the degradation of LDL (see Figure 5.18). The cholesterol esters are hydrolyzed by cholesterol ester hydrolase, and the free cholesterol can be secreted directly into bile or converted to bile salts and

peripheral cells to the liver for excretion is a critically important pathway for maintaining cholesterol homeo- stasis. Conversion of cholesterol to regulatory molecules (hormones) also occurs in the liver and other tissues, but this is quantitatively small compared to the amount excreted through the bile. The role of HDL in reverse cho- lesterol transport is shown in Figure 5.20.

The process by which HDL collects cholesterol from peripheral tissues and transports it to the liver involves multiple cell surface receptors, intravascular enzymes, and transfer of lipids among circulating lipoproteins. One constant throughout the entire process is HDL’s main apoprotein, apoA-1. Unlike chylomicrons and VLDL, which are assembled into complete lipoproteins within the cell, HDL arise entirely within the intravascular space starting with lipid-free apoA-1. Molecules of apoA-1 are produced and secreted into the circulation by the liver and small intestine; apoA-1 released from chylomicrons and VLDL during triacylglycerol hydrolysis may also be used to create HDL. Nascent HDL are made when the lipid-free apoA-1 binds to the liver ABCA1 receptor and acquires phospholipids and free cholesterol from the hepatocyte. Nascent HDL are discoidal in shape due to the ability of the amphipathic lipids to form a bilayer. Additional phospholipids and cholesterol are acquired when nascent HDL interact with ABCA1 and another receptor, SR-B1, located in peripheral tissues such as muscle, adipose, and macrophages within coronary arteries. The ability of nascent HDL to accept cholesterol from macrophages benefits the cardiovascular system by reducing the amount of deposited cholesterol in the vascular endothelium, thus decreasing the risk of CVD (discussed in detail in the next section).

Figure 5.19 Sequential steps in endocytosis of LDL leading to synthesis and storage of cholesterol ester. Source: M. Brown, J. Goldstein, “Receptor mediated endocytosis: insights from the lipoprotein receptor system.” © 1986 The Noble Foundation. Used by courtesy of The Samuel Roberts Noble Foundation, Ardmore, OK.

❶ ➋

LDL particle with apoB attaches to the LDL receptor. Endocytosis of LDL particle and receptor. LDL particle fuses with lysosome. LDL receptor returns to the membrane surface. Proteins of LDL particle hydrolyzed to amino acids. Free cholesterol released from LDL particle. HMG-CoA reductase is involved in cholesterol synthesis. When excess cholesterol is present, synthesis of cholesterol and LDL receptors are inhibited. Cholesterol transferred to Golgi, esterif ied with ACAT, and stored in the cell.

LDL receptor

Cholesterol ester

Cholesterol

Amino acidsLysosome

Cholesterol esters

Protein

LDL

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C h a p t e r 5 • LIPIDS 151

and bone-forming cells. Further research will reveal its biological importance in these areas [17].

LIpIDS, LIpOprOteINS, aND CarDIOVaSCULar DISeaSe rISK

Atherosclerosis is a degenerative disease of the vascular endothelium. The principal players in the atherogenic process are cells of the immune system, which cause a pro-inflammatory environment, and lipids, primarily cholesterol and cholesterol esters. An early response to arterial endothelial cell injury is an increased adherence of monocytes and T lymphocytes to the area of the injury. Cytokines, protein products of the monocytes and lympho- cytes, mediate the atherogenic process by chemotactically attracting phagocytic cells to the area. Additional exposure to a high level of circulating LDL and the deposition and oxidative modification of cholesterol esters further pro- mote the inflammatory process. The process is marked

secreted (see Figure 5.10). This process is the major route by which cholesterol is eliminated from the body. The efficiency with which HDL accept and transport cholesterol is reflected in the distribution of HDL particle sizes that exist in the circulation. As large HDL represent the final stage just prior to delivery to the liver, a high proportion of large HDL are associated with decreased risk of CVD. A preponderance of small HDL, it is hypothesized, reflects inefficiencies in the ability of HDL to gather cholesterol esters for delivery to the liver. Small HDL are positively associated with CVD [16].

Additional functions of HDL have recently been suggested [17]. These include a role as an anti- inflammatory regulator through interactions with the vascular endothelium and circulating inflammatory cells. Some evidence supports the idea that HDL is an integral component of innate immunity. HDL has also been shown to have antiapoptotic functions for a number of cell types, including vascular endothelial and smooth muscle cells, some leukocytes, pancreatic b cells, cardiomyocytes,

Figure 5.20 Reverse cholesterol transport.

1 Lipid-free apoA-1 is secreted by the liver and intestine. It is also released from chylomicrons and VLDL during TAG hydrolysis.

ApoA-1 acquires PL and C from interaction with liver ABCA1, resulting in nascent HDL particles.

Nascent HDL acquire additional PL and C via ABCA1 and additional C via SR-B1 in peripheral tissues.

The enzyme LCAT, carried on HDL particles, esterif ies C to CE that migrate to the particle core.

The now spherical mature HDL continue to acquire PL and C via ABCG1 and C via SR-B1 in peripheral tissues.

LCAT continues to esterify C to CE, forming larger HDL.

Some CE are transferred to VLDL and LDL, mediated by CETP.

Liver SR-B1 binds HDL. CE may be selectively removed, or the HDL particle may be internalized and degraded.

TAG-rich lipoproteins

Non hepatic tissues

VLDL LDL

Larger HDL

PL C

CE

LCAT

apoA-1

PL C

CE

Mature HDL

LCAT

apoA-1

apoA-1 apoA-1

PL, C

Nascent (discoidal)

HDL

Small intestine

Biliary PL Biliary C Bile salts

Liver

SR-B1

SR-B1

SR-B1

ABCA1

ABCA1

ABCG1

CETP

➑ ❹

❻ ➐

PL C PL

C

PL C

PL C

C

C

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152 C h a p t e r 5 • LIPIDS

For example, immunological methods for quantifying apoB (the major LDL apoprotein) and apoA-1 (the primary HDL apoprotein) are now widely used. Ratios of apoA to apoB then serve as an indicator of CVD risk, with risk decreasing as the ratio decreases [18].

ApoB in the nonfasted serum includes both apoB-48, made in the enterocyte, and apoB-100, made in the hepatocyte; the majority is apoB-100 and is found in VLDL, IDL, and LDL. The total moles of apoB-100 present in the serum indicate the number of potentially atherogenic particles.

ApoA-1 is the major apolipoprotein in the HDL particles that are part of the reverse cholesterol transport system. HDL particles are antiatherogenic. They also have anti- inflammatory and antioxidant properties. Measurements of apoB and apoA should be made in fasting samples so that “contamination” of apoB-48 (chylomicrons) is avoided.

The impact of dietary cholesterol on serum cholesterol levels has been a controversial topic for many years. While there is definitive evidence that elevated LDL-C (and high LDL:HDL ratio) increases CVD risk, a link between dietary cholesterol and serum cholesterol has never been firmly established. The controversy started in 1968 when the American Heart Association announced a recommendation to limit cholesterol intake to less than 300 mg/day, focusing specifically on eggs (no more than three egg yolks per week) because of their high cholesterol content. Despite having weak evidence for making such a recommendation, and having no clear rationale for choosing 300 mg/day as the benchmark, the recommendation created a fear of dietary cholesterol that has persisted for nearly 50 years [19]. The preponderance of research, however, clearly indicates that dietary cholesterol has little or no impact on serum cholesterol. This is because compensatory mechanisms are engaged when cholesterol is consumed, such as increased biliary cholesterol excretion and the downregulation of cholesterol synthesis (discussed in the “Synthesis of Cholesterol” section later in this chapter). The American Heart Association and the 2015 Dietary Guidelines for Americans no longer recommend a restriction on cholesterol intake.

Saturated and Unsaturated Fatty Acids Extensive research has examined the effects of ingestion of dietary fats containing primarily saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyun- saturated fatty acids (PUFA), or trans fatty acids on total serum cholesterol or LDL-C levels as end points. The findings from older research studies generally led to the conclusions that SFA are hypercholesterolemic, PUFA are hypocholesterolemic, and MUFA are neutral (neither increasing nor lowering serum cholesterol). A compre- hensive review of the scientific literature linking diet and

by the uptake of LDL by phagocytic cells that become engorged with lipid, called foam cells. Phagocytic uptake is accelerated if the apoB-100 component of the LDL has been oxidized. The lipid-filled foam cells may then infil- trate the endothelium and develop into a fatty plaque. As lipid continues to accumulate within the plaque, the lumen of the blood vessel is progressively occluded. Atherosclero- sis was once considered to be a disease caused exclusively by dyslipidemia; however, atherosclerosis is now consid- ered to be a disease of both dyslipidemia and immune- system–induced inflammation. The Perspective at the end of this chapter discusses in detail the role of lipoproteins and inflammation in atherosclerosis development.

Ever since plaque was found to be composed chiefly of lipids, an enormous research effort has been underway to investigate the possible link between dietary lipids and the development of atherosclerosis. The presumed existence of such a link has come to be known as the lipid hypothesis, which maintains that dietary lipid intake can alter blood lipid levels, which in turn initiate or exacerbate atherogenesis. The next section contains a brief account of the involvement of certain dietary lipids, and of genetically acquired apolipoproteins, in atherogenesis.

Cholesterol At center stage in the lipid hypothesis controversy is cho- lesterol. The effects of dietary interventions designed to improve serum lipid profiles are often measured by the extent to which the interventions raise or lower serum cholesterol. This reasoning is justified in that cholesterol is a major component of atherogenic fatty plaque, and many studies have linked CVD risk to chronically elevated serum cholesterol levels. Receiving the greatest attention, however, is not so much the change in total cholesterol concentration but how the cholesterol is distributed between its two major transport lipoproteins, LDL and HDL. Because cholesterol is commonly and conveniently quantified in clinical laboratories, assays can be used to establish LDL:HDL ratios by measuring the amount of cholesterol in each of the lipoprotein classes. Assayed cholesterol associated with LDL is designated LDL-C by laboratory analysts, and cholesterol transported in HDL is designated HDL-C.

Because maintaining relatively low serum levels of LDL and relatively high levels of HDL (a low LDL:HDL ratio) is desirable, the concept of “good” and “bad” cholesterol emerged. The “good” form is the cholesterol associated with HDL, and the “bad” form is the cholesterol transported as LDL. It is important to understand, however, that cholesterol itself is not good or bad; rather, it serves as a proxy for the relative concentrations of LDL and HDL, ratios of which can indeed be good or bad. LDL:HDL ratios are, in fact, determined more reliably by measurements other than cholesterol content.

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C h a p t e r 5 • LIPIDS 153

of energy or more) overestimate the impact of trans fatty acids, given their low abundance in the food supply. Inter- pretation of results is also complicated by the uncertainty of knowing whether experimental outcomes were due to inclusion of trans fatty acids or the removal of displaced fatty acids. Despite these experimental shortcomings, the American Heart Association and the Dietary Guidelines for Americans recommend that trans fatty acid intake be as low as possible (zero intake, though advisable, is not con- sidered practical because of the small amount of natural trans fat in the food supply).

Lipoprotein(a) Lipoprotein(a) [Lp(a)] is composed of a low-density lipo- protein (LDL) particle containing apoB-100 and a cova- lently linked glycoprotein called apolipoprotein(a). The physiological function of the Lp(a) particle has not been identified, although it is associated with increased risk of CVD. Unlike other lipoprotein classes, the serum concen- tration of Lp(a) is genetically determined. It exhibits a very broad and skewed distribution in the population and is not influenced by dietary or other environmental factors. Apolipoprotein(a) has a strong structural homology (simi- lar amino acid sequence) with plasminogen. Plasminogen is the inactive precursor of the enzyme plasmin, which dissolves blood clots by its hydrolytic action on fibrin. Apolipoprotein(a) has several genetic isoforms that vary in size. The smaller molecular weight isoforms appear to be more pathogenic [24].

Apolipoprotein E Among the apolipoproteins already discussed in this chap- ter, apoE deserves special mention because of its multiple roles in lipid metabolism, neurobiology, and cellular func- tion. There are three isoforms of apoE in humans: apoE2, apoE3, and apoE4. One of the isoforms, apoE4, has been associated with CVD and Alzheimer’s disease.

A single individual inherits one apoE allele from each parent, thus various homozygous and heterozygous combinations are possible. Allele frequencies show nonrandom global distribution, with the frequency of apoE4 increasing as one moves north from the equator. The apoE4 frequency is higher in people of northern European descent (about 25%) and lower in Mediterranean and Asian populations (10 to 1; the GSH-to-GSSG ratio represents an indicator of the cell’s redox state. In fact, the ratio of GSH to GSSG is thought to be the most important regulator of the cellular redox potential.

Proteins also serve as receptors on cell membranes and can function in storage roles. For example, some minerals such as copper, iron, and zinc are stored in body tissues bound to proteins; these proteins are often called metalloproteins.

Many proteins in the body are conjugated proteins— that is, proteins that are joined to nonprotein components. Glycoproteins, one type of conjugated protein, represent a huge group of proteins with multiple functions. Glycoproteins consist of a protein covalently bound to a carbohydrate component. The carbohydrate in glycoproteins generally includes short chains of glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and acetylneuraminic (sialic) acid at the terminal end of the oligosaccharide chain. The carbohydrate portion of the glycoprotein can make up as much as 85% of the glycoprotein’s weight. The carbohydrate component is bound typically through an N-glycosidic linkage with asparagine’s amide group in its side chain or through an O-glycosidic linkage with the hydroxy group in serine’s or threonine’s side chain. Glycoproteins are found in the blood, on the outer surface of plasma membranes, and in association with the extracellular matrix (which surrounds and supports some body cells). In the extracellular matrix of bone, for example, glycoproteins play structural roles. Mucus, which is found in body secretions, is rich in glycoproteins. Mucus both lubricates and protects epithelial cells in the body. Some of the body’s hormones (such as thyrotropin) and blood proteins (such as transthyretin and immunoglobulins) are glycoproteins.

Another group of conjugated proteins is the proteoglycans, which are found in every tissue of the body. Most proteoglycans are associated with the extracellular matrix but also with other structural matrix components, where they form cross-links to enhance strength and resilience and to modulate adhesion and communication between cells and between the extracellular matrix and cells. Proteoglycans are macromolecules consisting of a core protein covalently conjugated, typically by O-glycosidic or N-glycosylamine linkages, to one or more glycosaminoglycans. Glycosaminoglycans consist of long chains of repeating disaccharides, comprise up to 95% of the weight of the proteoglycan, and are the main site of the proteoglycan that interacts with cell surface proteins or extracellular matrix proteins. Examples of glycosaminoglycans include hyaluronic acid (found in high concentrations in cartilage), chondroitin sulfate (found in high concentrations in bone and cartilage), keratan sulfate and dermatan sulfate (found in the cornea of

Nitrogen-Containing Nonprotein Compound

Constituent Amino Acids

Glutathione Cysteine, glycine, glutamate

Carnitine Lysine, methionine

Creatine Arginine, glycine, methionine

Carnosine Histidine, β-alanine

Choline Serine

Table 6.6 Sources of Nitrogen for Some Nitrogen-Containing Nonprotein Compounds

Figure 6.22 The structure of glutathione in its reduced form (GSH).

O O

O–

Glycine

SH

CH2

*

Cysteine Glutamate

–O C C N

O H

H

H

C

H

H

C

H

H

C C N C

O H H

Unusual peptide linkage

C C

NH3

H

*γ carbon

+

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210 C H A P T E R 6 • PROTEIN

Glutathione synthesis is sensitive to protein intake and pathological conditions. Hepatic, intestinal, and systemic GSH concentrations decline with poor protein intake as well as during inflammation and disease; this decline negatively impacts the body, necessitating strategies to enhance or at least maintain GSH concentrations. Glutathione is discussed further in the section on selenium in Chapter 13.

Carnitine Carnitine, another nitrogen-containing compound, is made (Figure 6.23) from the amino acid lysine that has been meth- ylated; the methyl groups are derived from S-adenosyl methi- onine (SAM), which is made in the body from the oxidation of the amino acid methionine. Following lysine methyla- tion, trimethyllysine undergoes hydroxylation at the 3 posi- tion to form 3-OH trimethyllysine. Hydroxytrimethyllysine is further metabolized to generate γ-butyrobetaine and

Glutathione is found in the cytosol of most cells, but small amounts also are found within cell organelles and in the plasma. Glutathione has several functions. It is a major antioxidant with the ability to scavenge free radicals (O2·• and OH· •), thereby protecting critical cell components including SH-containing proteins against oxidation. With the enzyme glutathione peroxidase, glutathione protects cells by reacting with hydrogen peroxides )(H O2 2 and lipid hydroperoxides (LOOHs) before they can cause damage. Glutathione also transports amino acids as part of the γ-glutamyl cycle (Figure 6.4) in some tissues. It participates in the synthesis of leukotriene (LT) C4, which mediates the body’s response to inflammation. Glutathione is also involved in the conversion of prostaglandin H2 to prostaglandins D2 and E2 by endoperoxide isomerase. Glutathione can conjugate with nitric oxide to form S-nitrosoglutathione.

Figure 6.23 Carnitine synthesis.

Dehydroascorbate Ascorbate ❶

Succinate CO2α-ketoglutarate

Trimethyl- lysine

hydroxylase Fe 3+Fe2+

NAD+

NADH

O2

Trimethyllysine

De hyd

roa sco

rba te

Asc orb

ate ❶

Suc cin

ate

CO 2

α-k eto

glu tar

ate

4-b uty

rob eta

ine

hyd rox

yla se

Fe 3+

Fe 2+

O 2

+N

CH2

H3C CH3

CH3

C

COO–

H +NH3

CH2

CH2

CH2

COOH

COOH

C O

(CH2)2

COOH

COOH

(CH2)2

Glycine

Serine hydroxymethyl transferase-PLP-dependent

3-OH trimethyllysine

+N

CH2

H3C CH3

CH3

C

COO–

H +NH3

CH2

CH2

HC OH

Carnitine

+N

CH2

H3C CH3

CH3

COO–

CH2

HC OH

4-butyrobetaine aldehyde

4-butyrobetaine

+N

CH2

H3C CH3

CH3

CH2

CH2

HC O

COO2

CH2 +NH3

❶ Ascorbate functions as a reducing agent in two reactions. In both reactions for carnitine synthesis, the vitamin is needed to reduce the iron atom that has been oxidized (Fe3+) in the reaction back to its reduced (Fe2+) state.

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C H A P T E R 6 • PROTEIN 211

Carnitine deficiency, though rare, results in impaired energy metabolism. Carnitine supplementation increases plasma and muscle carnitine concentrations, and has been beneficial for some people with specific cardiac problems and diabetes. Supplementation with carnitine does not, however, “burn fat” as suggested in some advertisements.

Creatine Creatine, a key component of the energy compound creatine phosphate, also called phosphocreatine, can be obtained from foods (primarily meat and fish) or synthesized from three amino acids in the body. Creatine synthesis, which is shown in Figure 6.24, begins first in the kidneys and requires arginine and glycine. The second step occurs in the liver and involves the methylation of guanidinoacetate using SAM (S-adenosyl methionine).

Once synthesized, creatine is released into the blood for transport to tissues. About 95% of creatine is in muscle, with the remaining 5% in organs such as the kidneys and brain. In tissues, creatine is found both in free form as creatine and in its phosphorylated form. The phosphorylation of creatine to form phosphocreatine is shown here:

Creatine kinase—Mg2+ Creatine Phosphocreatine

ATP ADP

subsequently carnitine. Iron, vitamin B6 (as PLP), vitamin C, and niacin (as 1NAD ) are needed for carnitine synthesis.

In addition to being synthesized in the liver and kidneys, carnitine is found in foods, especially meats such as beef and pork. In these foods, carnitine may be free or bound (as acylcarnitine) to long- or short-chain fatty acid esters. Carnitine from food or supplements is absorbed in the proximal small intestine by sodium-dependent active transport and passive diffusion; diffusion typically predominates with ingestion of supplements providing 0.5–6 g [16]. Approximately 54–87% of carnitine intake is absorbed. Intestinal absorption of carnitine is thought to be saturated with intakes of about 2 g [17]. Muscle represents the primary carnitine pool, although no carnitine is made there. Intramuscular concentrations of carnitine are generally 50 times greater than usual plasma concentrations. Carnitine homeostasis is maintained principally by the kidneys, with >90% of filtered carnitine and acylcarnitine being reabsorbed.

Carnitine, found in most body tissues, is needed for the transport of fatty acids, especially long-chain fatty acids, across the inner mitochondrial membrane for oxidation. The inner mitochondrial membrane is impermeable to long-chain (10 or more) fatty acyl-coenzyme (Co) As. This role of carnitine is discussed in more detail in Chapter 5. Carnitine is also needed for ketone catabolism for energy. Carnitine also forms acylcarnitines from short-chain acyl- CoAs. These acylcarnitines may serve to buffer the free CoA pool.

Figure 6.24 Creatine synthesis and use.

H3C—N

C

CH2

COO2

Creatine (found in muscle)

C C O

HN NH2H2N

ATP

Mg12

Creatine kinase

Ornithine

Guanidinoacetate

(kidney)

ADP 1

H3C—N

C

H

CH2

COO2

2

Phosphocreatine Creatinine (excreted in the

urine)

N—PO3

NH2—CH2—COOH H2N—C—NH—CH2—CH2—CH—COO 21

H

N

CH2CH3

N

H2N 1

1 H2N

C—NH—CH2—COOH

H2N

SAH

SAM

(liver)

(spontaneous)

H2OPi

Glycine Arginine 1NH3

1

NH2

❶ Arginine and glycine react to form guanidinoacetate by the action of L-arginine:glycine amidinotransferase. In this reaction, the guanidinium (also called the amidino) group of arginine is transferred to the amino group of glycine; the remainder of the arginine molecule is released as ornithine.

❷ Methylation of guanidinoacetate requires guanidinoacetate methyltransferase with SAM (S-adenosyl methionine) providing the methyl groups.

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212 C H A P T E R 6 • PROTEIN

Phosphocreatine functions as a “storehouse for high- energy phosphate.” In fact, over half of the creatine in muscle at rest is in the form of phosphocreatine. Phosphocreatine replenishes ATP in a muscle that is rapidly contracting. Remember, muscle contraction requires energy. This energy is obtained with the hydrolysis of ATP. However, the ATP in muscle can suffice for only a fraction of a second. Phosphocreatine, stored in the muscle and possessing a higher phosphate group transfer potential than ATP, can transfer a phosphoryl group to ADP, thereby forming ATP or assisting in ATP regeneration, providing energy for muscular activity. Creatine kinase, also called creatine phosphokinase (abbreviated CK or CPK), catalyzes the phosphate transfer in active muscle, as shown here.

Creatine kinase—Mg2+ Phosphocreatine Creatine

ADP ATP

Creatine kinase is made up of different subunits in different tissues. For example, in the heart, creatine kinase is made up of two subunits designated M and B. (The brain and muscle also have creatine kinase, but in these tissues the enzyme is made up of the BB and MM subunits, respectively.) Damage to the heart, as with a heart attack, causes the enzyme to “leak” out of the heart and reach elevated concentrations in the blood. Thus, an elevation in CK-MB in the blood along with other indicators is used to diagnose a heart attack. Similarly, damage to skeletal muscle, as may occur with trauma, results in elevations of CK-MM in the blood.

The availability of phosphocreatine and its use by muscle are thought to delay the breakdown of muscle glycogen stores, which upon further catabolism can also be used by muscle for energy. Creatine and creatine phosphate do not remain indefinitely in muscle; rather, both slowly but spontaneously cyclize (as shown in Figure 6.24) because of nonreversible, nonenzymatic dehydration. This cyclization of creatine and phosphocreatine forms creatinine. Once formed, creatinine leaves the muscle, passes across the glomerulus of the kidneys, and is excreted like other nitrogenous waste products (e.g., urea, ammonia, uric acid) in the urine. Creatinine clearance is sometimes used as a means of estimating kidney function. The urinary excretion of creatinine is used as an indicator of existing muscle mass, as discussed later in the section under “Skeletal Muscle” titled “Indicators of Muscle Mass and Muscle/Protein Catabolism.” Not all creatinine, however, gets excreted in the urine. Small amounts may be secreted into the gut and, like urea, metabolized by intestinal bacteria. The effects of creatine supplementation on athletic performance are discussed in the Perspective for Chapter 7.

Figure 6.25 Carnosine.

C H

HN N

O

H2N CH2 CH2 CH2CHC CNH CH

Carnosine Carnosine (also called β-alanyl histidine; Figure 6.25) is made from the amino acid histidine and β-alanine in an energy-dependent reaction catalyzed by carnosine synthetase. In the body, carnosine is synthesized and found largely in the cytosol of skeletal and cardiac muscle, but also in the brain, kidneys, and stomach. Related compounds include a methylated form of carnosine known as anserine (β-alanyl methylhistidine) and homocarnosine (γ-aminobutyryl histidine), among others. Carnosine is also found in foods, primarily meats, and may be digested into histidine and β-alanine in the intestine or possibly absorbed intact by peptide transporters. While not all of the functions of carnosine have been identified, some studies have shown that carnosine acts as both a buf- fer and an antioxidant within muscle cells; it may also reduce calcium needs for muscle contractility. The use of β-alanine supplements (about 3–6 g per day) increases muscle carnosine concentrations; the effects of supple- mentation on athletic performance are discussed in the Perspective for Chapter 7.

Choline Choline (Figure 6.26) is made in the body primarily in the liver through the methylation (involving S-adenosyl methionine, or SAM) of the phospholipid phosphatidylethanolamine when linked with the catabolism of phosphatidylcholine. The formation of phosphatidylserine from phosphatidylcholine involving the replacement of choline with serine by phosphatidylserine synthase 1 also releases choline for other use in the body.

In foods, choline is found free (unattached) in small amounts but is more commonly found in foods bound as part of phosphatidylcholine (also called lecithin) and sphingomyelin, among other forms. Foods rich in lecithin include eggs, meats (especially liver and other organ meats), shrimp, cod, salmon, wheat germ, and legumes such as soybeans and peanuts. Lecithin is also added to many foods as an emulsifier. Intake is estimated at about 6–10 g/day [18]. Pancreatic enzymes hydrolyze some choline from its bound forms. Free choline is absorbed in the small intestine by diffusion and carrier-mediated uptake and transported via the blood to tissues. Choline existing as phosphatidylcholine and sphingomyelin are incorporated into chylomicrons for transport to tissues. The liver and kidneys store choline to a limited extent.

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C H A P T E R 6 • PROTEIN 213

through dietary consumption of animal products and foods containing fats. A Tolerable Upper Intake Level of 3.5 g of choline daily also has been set [18]. The Tolerable Upper Intake Level represents the highest level of daily intake that is likely to pose no risks of adverse health effects to most people in the general population [18]. Adverse effects associated with ingestion of large doses of choline include excessive sweating, salivation, vomiting, and a fishy body odor. Intakes of 7.5 g of choline have caused small hypotensive effects [18].

Purine and Pyrimidine Bases Nitrogenous bases, along with a five-carbon sugar and phosphoric acid, are needed for the synthesis of two nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), in the body. It is amino acids that provide the source for the nitrogen in these bases. The nitrogenous bases can be divided into two categories: pyrimidines and purines. The pyrimidines are six-membered rings containing nitrogen atoms in positions 1 and 3. The pyrimidine bases include uracil, cytosine, and thymidine. Deoxycytidine and thymidine (also called deoxythymidine) are found in DNA. Cytidine and uridine are present in RNA. The purines are made up of two fused rings with nitrogen atoms in positions 1, 3, 7, and 9. The purine bases include adenine and gua- nine and are found in DNA as deoxyadenosine and deoxy- guanosine and in RNA as adenosine and guanosine. A brief review of purine and pyrimidine synthesis and degradation follows.

The synthesis of the nitrogen-containing bases used to make nucleic acids and nucleotides occurs for the most part de novo in the liver. The individual steps in pyrimidine synthesis are shown in Figure 6.27. First, synthesis of the pyrimidines uracil, cytosine, and thymine (or in nucleotide form UTP, CTP, and TTP, respectively) is initiated by the formation of carbamoyl phosphate from the amino acid glutamine, CO2 , and ATP. The enzyme carbamoyl phosphate synthetase II catalyzes this reaction in the cytosol and is distinct from carbamoyl phosphate synthetase I, which is needed in the initial step of urea synthesis and is found in the mitochondria. Second, carbamoyl phosphate reacts with the amino acid aspartate to form N-carbamoyl aspartate. Aspartate transcarbamoylase catalyzes the reaction, which is the committed step in pyrimidine biosynthesis. Following several additional reactions, detailed in Figure 6.27, uridine monophosphate (UMP) is synthesized. Defects in the activity of either OMP decarboxylase used to make UMP (reaction 6 in Figure 6.27) or orotate phosphoribosyl transferase (reaction 5 in Figure 6.27) result in the genetic disorder orotic aciduria. This condition is characterized by megaloblastic anemia, leukopenia, retarded growth, and the excretion of large amounts of orotic acid in the urine.

The interconversions among the pyrimidine nucleoside triphosphates are shown in Figure 6.28 and are discussed

Choline is a nitrogen-containing compound that is also often presented and/or discussed with the B vitamins, although it is not defined as a vitamin. It has several functions. Most choline is used to synthesize phosphatidylcholine and sphingomyelin, major components of cell membranes. Phosphatidylcholine also functions in intracellular signaling and in the secretion of very-low-density lipoproteins from the liver. Sphingomyelin is a component of myelin that functions as a sheath around nerves and is important in nerve conduction. Choline is also used in the formation of platelet aggregating factor and for the neurotransmitter acetylcholine. To be converted to acetylcholine, free choline crosses the blood–brain barrier and enters cerebral cells from the plasma through a specific choline transport system. Within the presynaptic terminal of the neuron, acetylcholine is formed by the action of choline acetyltransferase as follows:

1 1Choline acetyl-CoA Acetylcholine CoA→

The acetyl-CoA needed for the reaction is thought to arise from glucose metabolism by neural glycolysis and the action of the pyruvate dehydrogenase complex. Concentrations of choline in cholinergic neurons typi- cally are below the Km of choline acetyltransferase; thus, the enzyme normally is not saturated. Choline from acetylcholine can be reused following synaptic transmission; the enzyme acetylcholinesterase hydrolyzes the neurotransmitter. Phospholipases can also liberate choline from lecithin and sphingomyelin as needed.

Choline is oxidized in the liver and kidneys (see Figure 6.12). In the liver, choline oxidation generates betaine, which functions as a methyl donor in the generation of methionine from homocysteine. Further metabolism of betaine (also called trimethylglycine) generates dimethyl glycine (also called sarcosine), and subsequently glycine; the reactions require folate as tetrahydrofolate and generate another folate derivative, 5,10-methylene tetrahydrofolate. These reactions are shown in the section of Chapter 9 on folate (specifically, the amino acid metabolism of serine and glycine).

Experimental diets devoid of choline can decrease plasma choline and phosphatidylcholine concentrations. In some cases, insufficient dietary choline intakes promote muscle damage and the development of a fatty liver accompanied by altered liver enzymes and some hepatic necrosis. Low intakes of both choline and betaine have been associated with inflammation. Because de novo synthesis does not consistently meet the body’s needs for choline, the Food and Nutrition Board has suggested an Adequate Intake of 425 mg and 550 mg of choline daily for adult females and males, respectively [18]. Such intakes are easily obtained

Figure 6.26 Choline.

CH3

CH3

CH3 CH2 CH2OH +N

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214 C H A P T E R 6 • PROTEIN

phospholipid synthesis, and UTP is used to form activated intermediates in the metabolism of various sugars. Drugs used to treat cancer often target key enzymes needed for the synthesis of purines or pyrimidines, which are needed by both healthy and cancer cells to grow and multiply. The drug methotrexate, for example, inhibits dihydrofolate reductase activity and thereby decreases dTMP (and thus TTP) formation. Rapidly dividing cells such as cancer cells are more susceptible to the effects of these drugs.

The purine bases adenine and guanine (Figure 6.29) are synthesized de novo as nucleoside monophosphates by sequential addition of carbons and nitrogens to ribose-5-phosphate that has originated from the hexose monophosphate shunt. As shown in Figure 6.30, in the initial reaction, ribose 5-phosphate reacts with ATP to form 5-phosphoribosyl 1-pyrophosphate (PRPP). Glutamine then donates a nitrogen to form 5-phosphoribosylamine. This step represents the committed step in purine nucleotide synthesis. Next in a series of reactions, nitrogen and carbon atoms from glycine are added, formylation occurs by tetrahydrofolate, another nitrogen atom is donated by the amide group of glutamine, and ring closure occurs. Another set of reactions involving the addition of carbons from carbon dioxide and from 10-formyl THF (from folate) and a nitrogen from aspartate occurs. The net result of all of these reactions is the formation of

next. Once uridine monophosphate (UMP) is formed, it may react with other nucleoside di- and triphosphates. UMP can be converted to uridine diphosphate (UDP) utilizing ATP. UDP can be converted to uridine triphosphate (UTP) also using ATP, and UTP can be converted to cytosine triphosphate (CTP) using ATP and an amino group from glutamine. Alternately, UDP can be reduced to deoxy(d)UDP by ribonucleotide reductase; this reaction requires riboflavin as FADH2 and the protein thioredoxin. DeoxyUDP can then be converted to dUMP. The formation of deoxythymidine (also called thymidine) monophosphate (dTMP or TMP) from dUMP is catalyzed by thymidylate synthetase; the reaction requires folate as 5,10 methylene tetrahydrofolate and forms another folate derivative dihydrofolate (DHF). Dihydrofolate reductase is needed to convert DHF to tetrahydrofolate, which is then converted to 5,10 methylene tetrahydrofolate and thus allows for dTMP synthesis. DeoxyTMP can be phosphorylated to form deoxythymidine diphosphate (dTDP) and then phosphorylated again to produce deoxythymidine triphosphate (dTTP, or abbreviated TTP). Thus, through these reactions CTP, (d)TTP, and UTP have been generated and can be used for the synthesis of DNA and RNA. The pyrimidine ring structure and its sources of carbon and nitrogen atoms along with the structures of the pyrimidine bases are shown in Figure 6.29. CTP is also used in

Figure 6.27 The initial reactions of pyrimidine synthesis.

Glutamate

Carbamoyl PO4 synthetase II

2 ATP 2 ADP + Pi

+ CO2 Carbamoyl-PO4

Carbamoyl aspartate

Dihydroorotic acid

Orotic acid

Dihydroorotase

Dihydroorotate dehydrogenase

OMP decarboxylase

Orotate phosphoribosyl transferaseOrotidine 5-monophosphate

(OMP)

Uridine monophosphate

(UMP)

Aspartate

Aspartate transcarbamoylase

Pi

H2O

CoQ

CoQH2

PPi 5-phosphoribosyl 1-pyrophosphate

(PRPP) CO2

❺ ❻

❶ Carbamoyl phosphate (PO4) is made from glutamine and carbon dioxide (CO2). The enzyme carbamoyl PO4 synthetase II is found in cytosol and is dif ferent from the mitochondrial enzyme carbamoyl PO4 synthetase I involved in the urea cycle.

❷ Aspartate transcarbamoylase catalyzes the committed step in pyrimidine synthesis and converts carbamoyl phosphate to carbamoyl aspartate. Carbamoyl aspartate can only be used for pyrimidine synthesis.

–❸ Carbamoyl aspartate is converted to dihydroorotic acid, which is then converted to orotic acid (or orotate).❹

❺ Orotic acid is covalently bonded to 5-phosphoribosyl 1-pyrophosphate (which is made from ATP and ribose 5-phosphate) to form orotidine 5-monophosphate. Defects in the activity of this enzyme cause orotic acid to build up in body f luids and cause orotic aciduria.

❻ Decarboxylation of OMP produces UMP, which can be used to form the other pyrimidine nucleotides.

Glutamine

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C H A P T E R 6 • PROTEIN 215

two reactions are catalyzed by hypoxanthine–guanine phosphoribosyl transferase. Defects in this enzyme cause the disorder Lesch-Nylan syndrome, a genetic X-linked condition characterized most notably by self-mutilation, such as the biting off of one’s fingers, and premature death. Other symptoms include mental retardation and the accumulation of hypoxanthine, phosphoribosyl pyrophosphate, and uric acid in body fluids.

Degradation of pyrimidines involves the sequential hydrolysis of the nucleoside triphosphates to mononucleotides, nucleosides, and, finally, free bases. This process can be accomplished in most cells by lysosomal enzymes. During catabolism of pyrimidines, the ring is opened with the production of CO2 and ammonia from the carbamoyl portion of the molecule. The ammonia can be converted into urea and excreted. Malonyl-CoA and methylmalonyl-CoA, produced from the remainder of the ring, follow their normal metabolic pathways, thus requiring no special excretion route.

Purines (GMP and AMP) are progressively oxidized for degradation primarily in the liver, yielding xanthine, which is converted to uric acid for excretion (Figure 6.32).

a purine ring. The ring (Figure 6.29) is thus derived from components of several amino acids, including glutamine, glycine, and aspartate, as well as from folate and CO2.

The formation of purine nucleoside triphosphates for DNA and RNA synthesis is shown in Figure 6.31. Inosine monophosphate (IMP) is used to synthesize adenosine monophosphate (AMP) and guanosine monophosphate (GMP). AMP and GMP are phosphorylated to ADP and GDP, respectively, by ATP. The deoxyribotides are formed at the diphosphate level by converting ribose to deoxyribose, thereby producing dADP and dGDP. ADP can be phosphorylated to ATP by oxidative phosphorylation; the remaining nucleotides are phosphorylated to their triphosphate form by ATP.

Purine nucleotides can also be synthesized by the salvage pathway, which requires much less energy than de novo synthesis. In the salvage pathway, the purine base adenine reacts with 5-phosphoribosyl 1-pyrophosphate (PRPP) to form 1AMP PPi in a reaction catalyzed by adenine phosphoribosyl transferase. The purine guanine can also react with PRPP to form 1GMP PPi. Hypoxanthine can react with PRPP to form 1IMP PPi. These last

Figure 6.28 The formation of the pyrimidine nucleoside triphosphates UTP, CTP, and TTP for DNA and RNA synthesis.

Glutamine Serine

Glycine

Glutamate

❶ UMP reacts with ATP to generate uridine diphosphate (UDP).

❷ UDP is then converted to uridine triphosphate (UTP).

❸ UTP is used with the amino acid glutamine (Gln) to make cytosine triphosphate (CTP).

❹ UDP can be reduced using NADPH + H+ in a reaction that also involves ribof lavin and thioredoxin to form deoxyuridine diphosphate (dUDP).

❺ dUDP can be converted to deoxyuridine monophosphate (dUMP).

❻ dUMP can be converted to deoxythymidine monophosphate (also referred to as thymidine monophosphate and abbreviated dTMP or TMP, respectively) by the enzyme thymidylate synthetase. Folate as 5,10 methylene tetrahydrofolate (THF) provides a one-carbon unit to convert dUMP to dTMP. The dihydrofolate (DHF) that is formed must be converted back to THF for the cycle to continue. This reaction is catalyzed by DHF reductase, which is the target for the anti-cancer drug methotrexate.

❼ dTMP can be phosphorylated using ATP to form deoxythymidine diphosphate (dTDP).

❽ dTDP can be phosphorylated using ATP to form deoxythymidine triphosphate (dTTP), which is needed for DNA synthesis.

Uridine monophosphate (UMP)

Uridine diphosphate (UDP)

Deoxyuridine diphosphate (dUDP)

Deoxyuridine monophosphate (dUMP)

Uridine triphosphate (UTP)

Cytosine triphosphate (CTP)

(needed for DNA and RNA synthesis)

Deoxythymidine monophosphate (dTMP)

Deoxythymidine diphosphate (dTDP)

Deoxythymidine triphosphate (dTTP)

(needed for DNA synthesis)

Dihydrofolate (DHF)

DHF reductase

Serine hydroxymethyl transferase

5,10 methylene tetrahydrofolate

(THF)

Thymidylate synthetase

Reductase ATP

ADP

Kinase

ATP

THF

H2O

ADP Kinase

Kinase

NADPH + H+

NADPH + H+

NADP+

NADP+

H2O

Pi

ATP

ADP

ATP

ADP

CTP synthetase

Kinase

ATP

ADP

(needed for RNA synthesis)

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216 C H A P T E R 6 • PROTEIN

Figure 6.29 The pyrimidine and purine ring structures and the pyrimidine and purine bases. Cytosine, adenine, and guanine are found in both DNA and RNA. Thymine is found in DNA and uracil only in RNA.

Cytosine

Adenine Guanine

Thymine Uracil

A pyrimidine ring and its sources of carbon and nitrogen atoms

A purine ring and its sources of carbon and nitrogen atoms

C N

N HC

CH

CH

3 C

O

N N

NH2

N H

C

CH CH

CHCH

From aspartate

From aspartate

From aspartate

From glutamine

From carbon dioxide

From carbon dioxide

C

O

O

N H

C C

C CH3

HN

HN

C

O

O

N H

C

C N

NH2

N NH

HC

C

C CH

N C

N

N N C

C

C C

N C

N NH

C

C

C CH

N

O

H2N 3

1

1

9

From glycine

From glycine

From 10-formyl tetrahydrofolate

From 10-formyl tetrahydrofolate

From glutamine

From glutamine

7

Figure 6.30 Synthesis of inosine monophosphate (IMP), which is used to synthesize other purine nucleotides.

Glutamine

Glutamate

H2O ATP

(provides a pyroPO4 group to ribose 5-phosphate)

AMP

Aspartate

Ribose 5-phosphate (from the hexose monophosphate shunt pathway)

5-phosphoribosyl 1-pyrophosphate (PRPP)

Glutamine PRPP amidotransferase (committed step)

PPi

5-phosphoribosylaminoimidazole Inosine monophosphate

(IMP)

5-phosphoribosyl 5-amino 4-carboxyimidazole

5-phosphoribosyl 4-succinocarboxamide

5-aminoimidazole

5-phosphoribosyl 5-amino 4-imidazole- carboxamide (AICAR)

5-phosphoribosyl 5-formamido 4-imidazole-

carboxamide (FAICAR)

ATP

ADP + Pi

THF

10-formyl THF

Fumarate 5-phosphoribosylamine

5-phosphoribosylglycinamide (GAR)

5-phosphoribosyl formylglycinamide

5-phosphoribosyl formylglycinamidine

ATP

ATP

THF

ADP + Pi

ADP + Pi

Glycine

10-formyl tetra- hydrofolate (THF)

(FGAR)

Glutamine

Glutamate

CO2

H2O

Phosphoribosyl pyrophosphate synthetase

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C H A P T E R 6 • PROTEIN 217

Figure 6.31 The formation of purines and nucleoside triphosphates needed for DNA and RNA synthesis.

Inosine monophosphate (IMP)

GDP + Pi

Adenylosuccinate synthetase

Adenylosuccinate Xanthine monophosphate (XMP)

Guanosine monophosphate (GMP)

Guanosine diphosphate (GDP)

Adenosine monophosphate (AMP)

Adenosine diphosphate (ADP) Pi

ATP (needed for RNA synthesis)

Deoxy ADP

Deoxy ATP (dATP) (needed for DNA synthesis)

ATP

ADP

oxidative phosphorylation

Adenylosuccinate lyase

Fumarate

ATP

ATP

AMP + PPi

ADP

ATP

ADP

ATP

ADP

ATP

Deoxy GDP Guanosine triphosphate (GTP) (needed for RNA synthesis)

Deoxy GTP (dGTP) (needed for DNA synthesis)

ADP

H2O

H2O

NADH + H+ IMP dehydrogenase

Aspartate

Glutamine

Glutamate

NAD+

GTP

Figure 6.32 The degradation of the purines AMP and GMP generates uric acid.

Guanosine monophosphate (GMP)

Adenosine monophosphate (AMP)

Guanosine Inosine monophosphate (IMP)

Inosine

Hypoxanthine

Xanthine oxidoreductase

Xanthine oxidoreductase

Ribose 1-phosphate

Guanine

Xanthine

Uric acid (excreted in the urine)

+NH4

+NH4

H2O

Ribose 1-phosphate

Pi

Pi

Pi

Pi

❶❷

❷ ➌

➌➍

❻ Xanthine is converted to uric acid, which is excreted in the urine.

❺ Hypoxanthine is converted to xanthine.

❹ Guanine is deaminated to form xanthine.

❸ A ribose is removed from the inosine and guanosine to form hypoxanthine and guanine, respectively.

❷ IMP and GMP are dephosphorylated, generating inosine and guanosine, respectively.

❶ AMP is deaminated to produce IMP.

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218 C H A P T E R 6 • PROTEIN

Xanthine oxidoreductase, a molybdenum- and iron- dependent flavoenzyme, converts hypoxanthine (generated from AMP) to xanthine and also converts xanthine (made from both AMP and GMP) to uric acid. The oxidase form of the enzyme uses molecular oxygen and generates hydrogen peroxide, while the dehydrogenase form uses 1NAD+ and forms 1 1NADH H . The uric acid that is produced is normally excreted in the urine, although up to 200 mg also may be secreted into the digestive tract. In the disorder gout and in renal failure, uric acid accumulates in the body, causing painful joints among other problems. Allopurinol is one of several drugs used to treat gout; it works by binding to the enzyme to prevent its interaction with xanthine and hypoxanthine and thus diminish uric acid production. The oxidase (rather than the dehydrogenase) form of the enzyme predominates in several body tissues under conditions of oxygen deprivation (as with a heart attack). A problem in this situation is that when oxygen delivery relieves this deprivation, hydrogen peroxide and free radical production both increase and may further damage the injured tissues. Research involving introduction of enzymes and antioxidant nutrients to help minimize tissue damage with reoxygenation is ongoing.

INTERORGAN “FLOW” OF AMINO ACIDS AND ORGAN-SPECIFIC METABOLISM

While tissues and organs use amino acids to synthesize proteins and some nitrogen-containing compounds, the metabolism of the amino acids varies to some extent among the different organs. In many instances, the products generated from amino acid metabolism in one organ may be needed by another organ, creating a dependence between organs. This interdependence begins with the intestinal cells, which are the first cells of the body to receive dietary amino acids. The first part of this section covers amino acid metabolism by intestinal cells, followed by a discussion of amino acids in the plasma and then the specific roles that glutamine and alanine play among body tissues. Lastly, specific uses of amino acids by other selected tissues and organs such as skeletal muscle, the kid- neys, and the brain are presented.

Intestinal Cell Amino Acid Metabolism Intestinal cells use amino acids for energy production as well as for the synthesis of proteins and nitrogen-containing compounds. Some of the uses of amino acids in enterocytes include:

● structural proteins ● nucleotides

● apoproteins necessary for lipoprotein (chylomicron) formation

● new digestive enzymes ● hormones ● nitrogen-containing compounds.

Amino acids may be totally or partially metabolized within intestinal cells. It is estimated that the intestine (which represents about 3–6% of the body weight) uses 30–40% and splanchnic tissues use up to 50% of some of the essential amino acids absorbed from the diet [19]. Uses in the intestinal cells of individual amino acids vary. For example, the intestines appear to use up to about 90% of glutamate that is absorbed from the diet [19]. The next five subsections discuss the metabolism of glutamine, glutamate, aspartate, arginine, and methionine in intestinal cells. Figure 6.33 provides a partial overview of intestinal cell amino acid metabolism.

Intestinal Glutamine Metabolism Glutamine serves several roles in the intestines. It is degraded extensively by intestinal cells, providing a primary source of energy. It has also been shown to have trophic (growth) effects, stimulating gastrointestinal mucosa cell proliferation. Consequently, glutamine helps to prevent both atrophy of gut mucosa and bacterial translocation. In addition, glutamine has been shown to enhance the synthesis of heat shock proteins. It is also needed in large quantities along with threonine for the synthesis of mucins found in gastrointestinal tract mucus secretions. These roles of glutamine in the gastrointestinal tract have prompted several companies to enrich enteral and parenteral (intravenous) nutrition products with glutamine. When glutamine is provided through tube feedings, over 50% of glutamine is extracted by the splanchnic (visceral) bed. It is estimated that the human gastrointestinal tract uses up to 10 g of glutamine per day, and that the cells of the immune system use over 10 g per day. In addition to dietary glutamine, much of the body’s glutamine that is produced by the skeletal muscles (and to lesser extents by the lungs, brain, heart, and adipose tissue) is released and taken up, mostly by the intestinal cells.

Glutamine not used for energy production within the intestine also may be partially catabolized to generate ammonia and glutamate. The ammonia enters the portal blood for uptake by the liver or may be used within the intestinal cell for carbamoyl phosphate synthesis. The glutamate thus formed is discussed next.

Intestinal Glutamate Metabolism In the intestinal cell, glutamate arises directly from the diet or from glutamine metabolism. It is often transaminated with pyruvate to form α-ketoglutarate and alanine (Figure 6.33); the alanine typically enters portal blood for transport to the liver. Glutamate not used for alanine synthesis is often

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C H A P T E R 6 • PROTEIN 219

in intestinal cells by the action of carbamoyl phosphate synthetase I using ammonia )(NH3 , carbon dioxide )(CO2 or bicarbonate )( 2HCO3 , and 2ATP, as shown in Figure 6.33 and here:

NH3 + HCO3– + 2ATP Carbamoyl phosphate + 2ADP + Pi

The carbamoyl phosphate in turn is used along with orni- thine to synthesize citrulline in a reaction catalyzed by ornithine transcarbamoylase, as follows:

11Carbamoyl phosphate Ornithine Citrulline→

Citrulline that is made in the enterocytes is released into blood and then typically taken up, mostly by the kidneys, which use it for arginine synthesis (this pathway is referred to as the intestinal–renal axis of arginine synthesis and is not thought to be affected by dietary arginine ingestion). The liver may also take up the citrulline as needed for the urea cycle. Because of the role of the intestine in citrulline synthesis and the need for citrulline in arginine synthesis, arginine production can be impaired in individuals with intestinal injury. In such a situation, arginine becomes a conditionally essential amino acid and either arginine or citrulline must be supplemented in the diet.

Intestinal Methionine (and Cysteine) Metabolism Methionine also is metabolized by intestinal cells. Studies suggest that up to 52% of methionine intake is metabolized in the gut [5]. Cysteine, generated from methionine or obtained directly from the diet, is used in the intestinal cells to make glutathione. Alternately, cysteine is metabolized

used with glycine and cysteine to make glutathione, or it may be used to synthesize proline, as shown here:

The majority of proline synthesis is thought to occur through intestinal cell glutamate metabolism. Proline is then released into portal blood for delivery to the liver. Lastly, glutamate may be used along with aspartate to synthesize ornithine, which in turn may be released into portal blood or can be used to make citrulline (Figure 6.33). Thus, very little glutamate leaves the intestinal cell as glutamate and enters portal blood.

Intestinal Aspartate Metabolism In addition to metabolism of glutamine and glutamate, metabolism of aspartate from the diet generally occurs within intestinal cells. Aspartate most often undergoes transamination to generate oxaloacetate; aspartate’s amino group in turn is used to synthesize ornithine. Very little aspartate (like glutamate) leaves the intestinal cells as aspartate and is found in portal blood.

Intestinal Arginine Metabolism Arginine is also used by intestinal cells. Up to 40% of dietary arginine is oxidized in enterocytes, yielding citrul- line and urea [20]. Carbamoyl phosphate is synthesized

Glutamate Glutamate γ-semialdehyde

Pyrroline 5-carboxylate Proline

NADPH + H+ NADP+

Figure 6.33 A partial overview of amino acid metabolism in the intestinal cell.

Glutamine

Glutamate Glutamate γ-semialdehyde

α-ketoglutarate (TCA cycle—energy

production)

Ornithine

Oxaloacetate

Ammonia

(Ammonia) (Ammonia)

Aminotransferase

Amino transferase

H2O

Pyruvate

Glutaminase Glutamine synthetase

Synthase

NH3 NH3

2 ATP

2 ADP + Pi

Ornithine transcarbamoylase

Carbamoyl phosphate

NH3 CO2 or HCO3

Proline

Arginine

Enters portal blood

NADPH + H+

Oxidase

NADP+

Aspartate

Alanine

Enters portal blood

Pyrroline 5-carboxylate Citrulline Enters portal blood

Spontaneous

Glutamate also can be used in the intestine to make ornithine. Aspartate is also used in the reaction.

2

Glutamine degradation yields ammonia (which

can be used for the synthesis of carbamoyl

phosphate) and glutamate.

Glutamate may be transaminated to form

α-ketoglutarate and alanine, which

goes to the liver via portal blood.

Carbamoyl phosphate and ornithine are used to make citrulline, which enters portal blood and is taken up by the liver and kidneys.

Carbamoyl phosphate synthetase I

Urea

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220 C H A P T E R 6 • PROTEIN

with glutamate in an ATP-dependent reaction to form glutamine. It is estimated that the body produces 40–80 g glutamine per day. Ammonia is typically generated in these cells by amino acid deamination and deamidation. In muscle it also forms from AMP deamination; AMP is generated in the muscle with ATP degradation as occurs rapidly with exercise. Glutamate is formed in muscle and other cells from the transamination of the branched-chain amino acids with α-ketoglutarate to form branched-chain α-keto acids and glutamate, respectively. As shown in Figure 6.34, ammonia generated from AMP deamination combines with the glutamate to produce glutamine.

The glutamine that is formed in the muscle is released into the blood and transported for use by other tissues. Whereas the cells of the gastrointestinal tract as well as the immune system (such as lymphocytes, monocytes, and macrophages) rely on glutamine catabolism for energy production, glutamine in the liver and kidneys is utilized differently. In the absorptive state (or with alkalosis), liver glutaminase activity increases, yielding ammonia for the urea cycle. In an acidotic state, the use of glutamine for the urea cycle diminishes, and the liver releases glutamine into the blood for transport to and uptake by the kidneys for use in acid-base balance. In the renal tubular cells, glutamine is catabolized by glutaminase to yield ammonia and glutamate. The glutamate may be further catabolized by glutamate dehydrogenase to yield α-ketoglutarate plus another ammonia. Ammonia reacts with 1H to form an ammonium ion in the lumen of the kidney tubule; the ammonium ion is then excreted in the urine. Renal glutaminase activity and ammonia excretion increase with acidosis and decrease with alkalosis.

Glutamine use by cells increases dramatically with hypercatabolic conditions such as infection and trauma. In these conditions muscle glutamine release increases but cannot meet other cellular demands. Thus, glutamine “stores” can become depleted and some cell functions may become impaired. Remember, glutamine plays several roles that are especially critical with illness/injury. To briefly review, glutamine is used extensively by immune system cells. Glutamine promotes proliferation of these cells and glutamine metabolites are used directly by these cells, for example, for purine and pyrimidine synthesis. Purines and pyrimidines are required in large quantities by activated lymphocytes and macrophages. Expression of cell surface activation markers and production of cytokines such as interferon and tumor necrosis factor α by lymphocytes and lymphokine-activated killer cell activity also depend on glutamine. Furthermore, phagocytes require adequate glutamine availability. Glutamine also promotes the synthesis of heat shock/stress proteins, which help protect body cells. Glutamine prevents atrophy of the intestine, protects against intestinal bacterial translocation, and serves as the major substrate for energy production for intestinal cells. Finally, glutamine, along with alanine, uptake into cells

primarily (70–90%) to taurine, and to a lesser extent (10–30%) to pyruvate and sulfite [5]. These reactions can be reviewed in Figure 6.12.

Amino Acids in the Plasma After ingestion of a protein-containing meal, amino acid concentrations typically rise in the plasma for several hours, then return to basal concentrations. In basal situations and between meals, plasma amino acid concentrations are relatively stable and are species specific; however, absolute concentrations of specific amino acids in the plasma vary from person to person.

Amino acids circulating in the plasma and found within cells arise from digestion and absorption of dietary (exogenous) protein as well as from the breakdown of existing body (endogenous) tissues. These endogenous amino acids intermingle with exogenous amino acids to form a “pool” totaling about 150 g. The pool includes amino acids in the plasma as well as amino acids in the cytosol of body cells. Reuse of endogenous amino acids is thought to represent the primary source of amino acids for protein synthesis. Despite differences in protein intake and in degradation rates of tissue proteins, the pattern of the amino acids in the amino acid pool appears to remain relatively constant, although the pattern is quite different from that found in body proteins.

The total amount of the essential amino acids found in the pool is less than that of the nonessential amino acids. The essential amino acids found in greatest concentrations are lysine and threonine. Of the nonessential amino acids, those found in greatest concentrations are alanine, glutamate, aspartate, and glutamine. In fact, up to 80 g of glutamine can be found in the body’s amino acid pool.

Amino acids within the pool, regardless of source, are taken up by tissues and metabolized in response to various stimuli such as hormones and physiological state. Tissues extract amino acids for energy production or for the synthesis of nonessential amino acids, protein, nitrogen- containing nonprotein compounds, biogenic amines, neurotransmitters, neuropeptides, hormones, glucose, fatty acids, or ketones, depending on the nutritional status and hormonal environment.

Glutamine and the Muscle, Intestine, Liver, and Kidneys Glutamine has several major roles in the body, one of which is in ammonia transport. Whereas ammonia arising in the liver from amino acid reactions is typically shuttled into the urea cycle, this is not true in other tis- sues. In extrahepatic tissues, especially muscle but also the lungs, heart, brain, and adipose, glutamine synthetase catalyzes the utilization of ammonia or ammonium ions

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C H A P T E R 6 • PROTEIN 221

discussed in the previous section, transamination reactions in muscle generate glutamate, which is used, especially in a fed state/after eating, to synthesize glutamine for release into the blood. In several situations (between meals, with excessive glucose needs, with illness characterized by increased release of epinephrine and cortisol, or in situ- ations such as fasting marked by low hepatic glycogen stores and a glucagon-to-insulin ratio favoring glucagon), glutamate typically transfers its amino group to pyruvate, generated from glucose oxidation via glycolysis, to form α-ketoglutarate and alanine, respectively. Once made, the alanine is released from the muscle into the blood for travel to the liver. Within the liver, alanine undergoes transamination back to pyruvate, which is then used to remake glucose. The glutamate that is generated with transamination can undergo deamination to provide ammonia for urea synthesis. These reactions are known

promotes increases in cell volume with possible associated regulatory roles in intermediary metabolism. Glutamine supplementation, about 20–25 g/day, typically normalizes plasma glutamine concentrations and improves outcomes in critically ill patients. Administration of glutamine as a dipeptide (alanyl-glutamine or glycyl-glutamine) is needed in either an intravenous or enteral solution because the amino acid is not stable in aqueous solutions used in feeding. Dipeptidases on the surface epithelium of blood vessels are thought to hydrolyze the dipeptide so that the glutamine is available for use.

Alanine and the Liver and Muscle In addition to glutamine, the amino acid alanine is also important in the intertissue (between tissues) transfer of amino groups generated from amino acid catabolism. As

Figure 6.34 Some pathways of glutamine generation in muscle.

Valine, isoleucine, or leucine

Corresponding branched-chain

α-keto acid

α-ketoglutarate

Glutamate

α-keto- glutarate

Oxaloacetate

Aspartate

Adenylosuccinate

α-ketoglutarate

BCAA transaminase BCAA transaminase

NAD+

NADH

Glutamate dehydrogenase

Fumarate AMP AMP deaminase

NH3

IMP

Glutamate

Corresponding branched-chain

α-keto acid

α-ketoglutarate

H2O

ATP

Glutamine synthetase

Glutamine

ADP + Pi

Valine, isoleucine, or leucine

Muscle

❶ Glutamate is generated in muscle as branched- chain amino acids are transaminated with α-ketoglutarate.

❶ ❶

➋ Some glutamate is deaminated to yield α-ketoglutarate and ammonia.

➍ Glutamine synthetase catalyzes the formation of glutamine from ammonia and glutamate.

➌ Ammonia is also formed from AMP deaminase. AMP is generated in muscle from ATP degradation, which occurs at higher rates with exercise.

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222 C H A P T E R 6 • PROTEIN

net protein synthesis (i.e., protein synthesis is greater than protein degradation).

In a postabsorptive state such as between meals or in a fasting situation, the reverse is true. Protein degradation predominates over synthesis, and amino acids may be released into the blood for use by other tissues. While alanine is released in the greatest concentration, other amino acids (including phenylalanine, methionine, lysine, arginine, histidine, tyrosine, proline, tryptophan, threonine, and glycine) are released in lesser quantities.

Muscle protein degradation is also associated with exercise. Cortisol, secreted by the adrenal glands, in response to exercise-induced stress, promotes, in part, this muscle and amino acid catabolism (see “Hormonal Regulation of Metabolism” and “Exercise and Nutrition” sections in Chapter 7).

Like other tissues, muscles preferentially catabolize some amino acids more than others; six amino acids (aspartate, asparagine, glutamate, leucine, isoleucine,

as the glucose–alanine or alanine–glucose cycle and are shown in Figure 6.35. The glucose that is generated from the alanine is subsequently released into the blood, where it is available to be taken up and used by muscle. Muscle cells use the glucose through glycolysis and generate pyruvate. The formed pyruvate is again available for transamination to re-form alanine. This alanine–glucose cycle serves to transport nitrogen to the liver for conversion to urea while also allowing needed substrates to be regenerated.

Skeletal Muscle Use of Amino Acids About 40% of the body’s protein is found in muscle, and skeletal muscle mass represents about 43% of the body’s mass. Uptake of amino acids by the skeletal muscles read- ily occurs following ingestion of food, especially a mixed meal rich in protein. Exercise further encourages amino acid uptake by muscles (see “Exercise and Nutrition” sec- tion in Chapter 7). After eating, skeletal muscles exhibit

Figure 6.35 The alanine–glucose cycle: alanine generation in muscle, and glucose generation in the liver.

Glycogen Glucose 6-PO4

Glycolysis Gluconeogenesis

Glucose Glucose

Muscle Blood

Liver

Pyruvate Alanine Alanine Alanine Pyruvate

Glutamate α-ketoglutarate

Leucineα-ketoisocaproate

α-ketoglutarate Glutamate

Deaminated NH3

Urea

❶ ❷

Alanine is formed in muscle cells from transamination with glutamate (generated from leucine transamination) and from pyruvate (generated from glucose oxidation via glycolysis).

Alanine travels in the blood to the liver.

In the liver, alanine is transaminated with α-ketoglutarate to form pyruvate.❸

Pyruvate can be converted back to glucose in a series of reactions.❹

The glucose is released from the liver into the blood for uptake by tissues such as muscle, which use glucose for energy.❺

The glutamate formed in the liver can be deaminated to release ammonia; the ammonia is used in the liver for urea production.❻

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C H A P T E R 6 • PROTEIN 223

and valine) appear to be catabolized to greater extents in the skeletal muscle than other tissues. This use of amino acids by muscle as well as leucine’s role in promoting protein synthesis has prompted the consumption of branched-chain amino acid supplements by some athletes. The catabolism of the branched-chain amino acids (isoleucine, leucine, valine) is discussed in the following subsection and is shown in Figure 6.36.

Isoleucine, Leucine, and Valine Catabolism Muscle, as well as the heart, kidneys, diaphragm, adipose tissue, and other organs (except, for the most part, the liver), possesses branched-chain aminotransferases, located in both the cytosol and mitochondria and respon- sible for the transamination of all three branched-chain amino acids. Following transamination, the α-keto acids of the branched-chain amino acids either remain within

Figure 6.36 Branched-chain amino acid metabolism.

Isoleucine

Transaminase or transferase

α-methylbutyryl- CoA

α-keto β-methyl valerate

CO2

HCO3

Propionyl-CoA carboxylase-(biotin)

Methylmalonyl-CoA mutase-(vitamin B12)

Racemase

CoA

α-methyl β-hydroxy butyryl-CoA

α-methylacetoacetyl-CoA

BCKAD*

Tiglyl-CoA

FADH2

FAD+

NADH

NAD+

NADH

NAD

L-methylmalonyl-CoA**

Succinyl-CoA

H2O

H2O

D-methylmalonyl-CoA

Acetyl-CoAPropionyl-CoA

NAD+

NADH + H+

AMP + PPi

*Branched-chain α-keto acid dehydrogenase (BCKAD), requiring thiamin as TDP/TPP, niacin as NADH, and Mg2+ and CoA from pantothenate. **Common intermediate in the catabolism of methionine, threonine, isoleucine, and valine.

ATP

Leucine

Transferase or transaminase

NADH

NAD

HCO3

Isovaleryl-CoA

α-ketoisocaproate

CO2

Succinyl- CoA

CoA

CoA

Tholase

CoA

β-methylglutaconyl- CoA

β-hydroxy β-methylglutaryl-CoA

(HMG CoA)

BCKAD*

β-methylcrotonyl- CoA

FADH2

FAD+

Acetoacetyl-CoA

Acetoacetate

Valine

Transferase or transaminase

Isobutyryl-CoA

α-ketoisovalerate

CO2

CoA

β-hydroxyisobutyryl- CoA

β-hydroxyisobutyryl-CoA hydroxylase

β-hydroxyisobutyrate dehydrogenase

Methylmalonate semialdehyde

BCKAD*

Methylarylyl- CoA

FADH2

FAD+

β-hydroxyisobutyrate

CoA

NADH + H+

Succinate

NAD+

Succinyl-CoA

L-methylmalonyl- CoA**

Amino isobutyrate

NAD+ CoA

NADH + H+

H2O

H2O

H2O

β-hydroxyacyl-CoA dehydrogenase

Hydratase

Dehydrogenase

Acetyl-CoA acyl transferase

β-methylglutaconyl- CoA hydratase

β-methyl crotonyl- CoA carboxylase (biotin)

Isovaleryl-CoA dehydrogenase

HMG-CoA lyase

Transferase

Defect in this enzyme complex causes maple syrup urine disease.

Defect in this enzyme results in propionic acidemia.

Defect in this enzyme results in methylmalonic acidemia.

❶ ❶

Methylmalonyl-CoA mutase-(vitamin B12)

CO2

CO2

O2

β-hydroxy- β-methylbutyrate

(HMB)

HMB-CoA ATP

ADP + Pi

α-methylacyl-CoA dehydrogenase

Hydratase

Methylmalonic semialdehyde dehydrogenase

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224 C H A P T E R 6 • PROTEIN

muscle or may be transported (bound to albumin) in the blood to other tissues (including the liver) for use.

Further catabolism of the branched-chain α-keto acids occurs by decarboxylation in an irreversible reaction catalyzed by the branched-chain α-keto acid dehydrogenase (BCKAD) complex. BCKAD is a large multienzyme complex made up of three subunits: E1α, E1β, and E2. This enzyme complex is found in the mitochondria of many tissues, including liver, muscle, heart, kidneys, intestine, and brain. It is highly regulated through phosphorylation (inactivation) and dephosphorylation (activation) mechanisms involving kinase and phosphatase proteins that act on the E1α subunit and act through end-product inhibition. This enzyme complex operates in a fashion similar to the pyruvate dehydrogenase complex (see Chapter 3) in that it requires thiamin in its coenzyme form TDP, niacin as NADH, and 1Mg2 and CoA from pantothenic acid.

The details of the oxidation of the three branched- chain amino acids are shown in Figure 6.36. As with other amino acids, the complete oxidation of branched-chain amino acids yields products that are glucogenic and/or ketogenic. Valine oxidation yields succinyl-CoA, and is thus considered glucogenic. The end products of isoleucine catabolism are succinyl-CoA and acetyl-CoA, which are glucogenic and ketogenic, respectively. The complete oxidation of leucine results in acetyl-CoA and acetoacetate formation; acetoacetate may be further metabolized to form acetyl-CoA. Leucine is thus totally ketogenic.

Other common intermediates are formed during branched-chain amino acid oxidation. Isoleucine, for example, generates propionyl-CoA, which is a common intermediate in the degradative pathways of methionine and threonine. Valine catabolism generates methylmalonyl- CoA, a common intermediate in the degradative pathways of methionine, threonine, and isoleucine.

Leucine’s metabolism also generates β-hydroxy β-methylbutyrate (HMB) (Figure 6.36). HMB is important for the production of β-hydroxy β-methylglutaryl (HMG)- CoA, a precursor for de novo cholesterol synthesis in the muscle. It appears that with some illnesses and with muscle damage, HMG-CoA concentrations may be inadequate to support cholesterol synthesis. Supplementation with HMB, usually as calcium HMB monohydrate (about 3 g per day given in three 1-g doses), provides cells with a source of HMG-CoA to maintain cholesterol synthesis and thus cell function. In addition, HMB appears to attenuate both muscle proteolysis and depression of muscle protein synthesis to improve muscle mass. Atrophy of muscle with muscle damage or secondary to conditions such as cancer, sepsis, and acquired immune deficiency syndrome (AIDS), among others, is due primarily to the activity of the ubiquitin-proteasome pathway (see the “Catabolism of Tissue Proteins” section); HMB appears to inhibit this pathway as well as to stimulate, along with leucine,

protein synthesis through mTOR. HMB’s effects have been demonstrated in healthy individuals as well as in those with conditions typically associated with muscle loss such as cancer and AIDS.

Leucine is one of the few amino acids that is completely oxidized in the muscle for energy. Leucine is oxidized in a manner similar to fatty acids, and its oxidation results in the production of 1 mol of acetyl-CoA and 1 mol of acetoacetate. Complete oxidation of leucine generates more ATP molecules on a molar basis than complete oxidation of glucose. Leucine appears to be preferentially oxidized during fasting situations. During fasting, leucine concentrations rise in the blood and muscle, and the capacity of the muscle to degrade leucine increases concurrently. This rise in capacity supplies the muscle with the equivalent of 3 mol of acetyl-CoA per molecule of leucine oxidized; the acetyl-CoA produces energy for the muscle while simultaneously inhibiting the oxidation of pyruvate, which is derived from glucose oxidation via glycolysis. Pyruvate is then transaminated to alanine and transported via the blood to the liver (see the previous section “Alanine and the Liver and Muscle”).

Disorders of Isoleucine, Leucine, and Valine Metabolism Maple syrup urine disease (MSUD) results from genetic mutations in BCKAD complex activity. The condition affects about 1 in 225,000 individuals worldwide, but in the Mennonite population in the United States it impacts about 1 in 150. MSUD, if untreated, results in an accumulation of the branched-chain amino acids and their alpha ketoacids in the blood and body fluids. The condition is characterized by acidosis, vomiting, lethargy, and frequently coma and death. High plasma leucine concentrations (versus high plasma isoleucine and valine) are more neurotoxic, and thus one aspect of management involves maintaining plasma concentrations of especially leucine but also isoleucine and valine in the normal range. A diet restricted in leucine, isoleucine, and valine intakes is required; large doses of thiamin are also tried to see if supplementation enhances residual BCKDC activity (remember thiamin is a coenzyme for the BCKDC).

Defects in some enzymes required for leucine degradation also have been documented (see Figure 6.36). Defects in isovaleryl-CoA dehydrogenase result in isovaleric acidemia. Although fairly rare, it is one of the more prevalent disorders of leucine metabolism, affecting about 1 in 250,000 worldwide but about 1 in 62,000 in Germany. Defects in β-methyl crotonyl-CoA carboxylase cause β-methyl crotonylglycinuria. Impaired activity of β-methylglutaconyl-CoA hydratase causes β-methyl-glutaconic aciduria and altered activity of β-hydroxyl β-methylglutaryl (HMG)-CoA lyase causes β-hydroxyl β-methylglutaric aciduria. Each of these disorders results in the production and accumulation of numerous acids and other compounds in body fluids,

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C H A P T E R 6 • PROTEIN 225

causing acidosis, dehydration, neurological problems, seizures, coma, and mental retardation, among other problems. A leucine-restricted diet is typically prescribed for these conditions. In some cases, to prevent the accumulation of toxic compounds, supplements of carnitine and glycine may be useful. Dietary fat restriction is also needed for those with HMG-CoA lyase deficiency.

Defective propionyl-CoA carboxylase and methylmalonyl-CoA mutase activities result in propionic acidemia and methylmalonic acidemia, respectively. These enzymes in addition to affecting valine and isoleucine oxidation are also common to methionine and threonine catabolism. Refer back to the section “Disorders of Methionine Metabolism.”

Indicators of Muscle Mass and Muscle/Protein Catabolism While muscle proteolysis generates amino acids that are released into the plasma for circulation to and use by other tissues, changes in plasma amino acid concentrations do not reflect changes in muscle mass. Instead, two previously mentioned compounds, creatinine and 3-methylhistidine, are used as indicators of existing muscle mass and muscle degradation, respectively. Urinary creatinine excretion is used to assess muscle mass because creatinine is the degradation product of creatine, which constitutes a fairly standard proportion of muscle (approximately 0.3–0.5% of muscle mass by weight). Urinary creatinine excretion reflects about 1.7% of the total creatine pool per day and is expressed per 24 hours, as a coefficient based on weight or height; however, because of variation in muscle creatine content, urinary creatinine is not always an accurate indicator of muscle mass.

The urinary excretion of 3-methylhistidine is used as an indicator of muscle catabolism (degradation). As mentioned under the section on histidine in “Hepatic Catabolism and Uses of Basic Amino Acids,” the amino acid histidine is found in high concentrations as 3-methylhistidine in the muscle protein actin. Because 3-methylhistidine cannot be reused for protein synthesis following protein degradation and is excreted in the urine, its urinary excretion can be measured and serves as an indicator of muscle breakdown. A drawback to its use, though, is that actin is not found only in muscle but appears to occur in other body tissues, including the intestine and platelets, which have high turnover rates. Thus, urinary 3-methylhistidine excretion also may represent an index of protein breakdown for many nonmuscle tissues in the body.

Amino Acid Metabolism in the Kidneys The kidneys preferentially take up and metabolize a number of amino acids and nitrogen-containing compounds (Figure 6.37). The kidneys’ roles include:

● glutamine catabolism for acid-base balance ● glycine catabolism for acid-base balance ● serine synthesis from glycine ● arginine and glycine use to form guanidinoacetate for

creatine synthesis ● glutathione catabolism ● arginine synthesis from citrulline ● tyrosine synthesis from phenylalanine ● histidine generation from carnosine degradation.

In fact, the kidneys are considered to be the major site in the body for arginine, histidine, serine, and perhaps tyro- sine production [21].

Glutamine uptake by the kidneys has been estimated at 7–10 g per day [21] but uptake increases dramatically with acidosis, whereas glutamine uptake by the intestine, liver, and other organs diminishes. Especially in acidotic conditions, glutamine and then glutamate are deamidated and deaminated, respectively, in the kidneys, resulting in two ammonias. In the kidney’s tubular lumen, the ammonias combine with 1H ions and form ammonium ions, which are excreted in the urine. 1H ions enter the tubular lumen in exchange for 1Na . In the lumen, the 1H ions may also react with bicarbonate )( 2HCO3 to form water and carbon dioxide and with dibasic phosphate

)( 2HPO42 to form monobasic phosphate )( 2H PO2 4 . Glycine utilization by the kidneys under acidotic conditions is similar to glutamine utilization; glycine is degraded, forming ammonia and carbon dioxide. The ammonia then enters into the tubular lumen, where it reacts with

1H ions, forming ammonium ions that are excreted in the urine. The loss of the 1H from the body serves to increase blood pH from an acidotic state to a value ideally within the normal range of about 7.35–7.45.

Under healthy (nonacidotic) conditions, glycine is used by the kidneys (proximal tubule) for the synthesis of the amino acid serine. The kidneys also use glycine along with arginine for the synthesis of guanidinoacetate; this compound then travels to the liver, where it is used to generate creatine. The kidneys are thought to take up about 1.5 g of glycine per day [21]. Glycine, however, is also generated from glutathione catabolism in the proximal tubules of the kidneys.

Most arginine that is made in the body for tissue use is made in the kidneys from citrulline that was generated in the intestines and has been extracted from the blood; remember, the arginine made in the liver is immediately degraded to form urea and is thus not available to body tissues. It is estimated that the kidneys extract about 1.5 g of citrulline per day from the blood and release about 2–4 g of arginine daily [21].

Phenylalanine catabolism to tyrosine in the kidneys also has been demonstrated. It is estimated that the kidneys take up about 0.5–1 g of phenylalanine from the blood

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226 C H A P T E R 6 • PROTEIN

each day and releases about 1 g of tyrosine [21]. In addition to phenylalanine degradation, carnosine is oxidized by the kidneys, releasing histidine for use by other body tissues.

The kidneys also can generate glucose for the body. The kidneys, like the liver and to some extent like the small intestine, have the enzymes necessary for gluconeogenesis. See Chapter 3 for a detailed description of these reactions.

The role of the kidneys in nitrogen metabolism cannot be overemphasized. The organ is responsible for ridding the body of nitrogenous wastes that would otherwise accumulate in the blood plasma. Kidney glomeruli act as filters of blood plasma, and all the constituents in plasma, with the exception of plasma proteins, move into the filtrate. Essential nutrients such as sodium, amino

acids, and glucose are actively reabsorbed as the filtrate moves through the tubules. Many other substances are not actively reabsorbed and must either move along an electrical gradient or move osmotically with water to enter the tubular cells. The amount of these substances that enters the tubular cells, then, depends on how much water moves into the cells and how permeable the cells are to the specific substances. The cell membranes are relatively impermeable to urea and uric acid, and are particularly impermeable to creatinine, little to none of which is typically reabsorbed.

Nitrogenous wastes found in the urine are listed in Table 6.7. About 80% of nitrogen is lost in the urine as urea under normal conditions. In acidotic conditions, urinary urea nitrogen losses decrease and urinary excretion of

Figure 6.37 Amino acid metabolism in selected organs.

Glucose

Brain Blood

Serotonin

Dopamine

Norepinephrine

NH3

CO2

Kidney

NH3

Guanidino- acetate

Guanidino- acetate (to liver)

Citrulline

Citrulline

Arg

Carnosine β-ala

Muscle

α-keto acid

α-keto acid

oxalo- acetate

TCA α-keto-

glutarate

α-keto- glutarate

α-keto acid

Glucose Pyruvate

Acetyl-CoA

NH3

α-keto acids

α-keto- glutarate

Leu

Leu

Glu

Glu

Glu

Gln

Gln

Ala

Trp

Tyr

Gln Gln

Blood

Blood

Blood

GABA

Glu

His

Gln

Phe Phe

Tyr

TyrGln Asp

Asp

Arg

Ser

Ser

Gly

Gly

Pyruvate Oxaloacetate

α-keto- glutarate

Glu

AspAla

Ala

His

Ile Val

Ile Val

Ile Val

Leu

α-keto acids of

Ile and Val

Leu

Asp

ATP + CO2 + H2O

Ala

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C H A P T E R 6 • PROTEIN 227

ammonium ions rises. In addition to urea and ammonia, usual nitrogenous wastes found in the urine include creatinine and uric acid, with lesser or trace amounts of creatine ( 200 mg/L have been reported with severe sepsis and burns.

● Orosomucoid (α1-acid glycoprotein): a protein important in wound healing and immunomodulatory functions. Orosomucoid concentrations rise about two- to fivefold with inflammation.

● Serum amyloid A: a protein that displaces apoprotein A1 on high-density lipoproteins and facilitates cholesterol delivery to cells and cholesterol removal from damaged tissue. The protein also recruits immune cells (neutrophils and monocytes) to inflammatory sites and induces extracellular matrix degrading enzymes. Concentrations may rise 20- to 1,000-fold with inflammation.

● Fibrinogen: a protein that contributes to blood viscosity and can be converted to fibrin by thrombin to promote blood clotting; increased plasma fibrinogen may increase arterial thromboembolism (blood clot formation and dislodgement) risk.

Sepsis/trauma

Fatty acids

Blood

Muscle

Glycogenolysis

Amino acids Acute-phase protein synthesis

Gluconeogenesis

Cytokines Glucagon Catecholamines Cortisol Insulin

Glucose

Alanine Lactate

Glutamine Amino acids

Muscle Proteins Proteolysis

Immune cells

Other organs

Glycerol

Fatty acids

Pyruvate

LactateAdipose

Liver

Figure 2 Substrate utilization during metabolic stress with increased responses shown by heavier black arrows.

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C H A P T E R 6 • PROTEIN 243

● Fibrinonectin: a glycoprotein functioning in cell adhesion and wound healing.

● Haptoglobin: a protein that binds hemoglobin that has been released into the blood due to red blood cell hemolysis and inhibits microbial use of iron.

● Ceruloplasmin: a copper-containing protein with the ability to scavenge free radicals and with oxidase activity to promote iron oxidation and thus inhibit microbial iron use.

● α2 Macroglobulin: a protease inhibitor that, for example, inhibits blood coagulation and fibrinolysis.

In addition to the synthesis of these proteins, more metallothionein (a zinc-containing protein) and ferritin (an iron-containing protein) are made in the liver with inflammation. Consequently, hepatic zinc and iron concen- trations increase, while plasma zinc and iron concentrations decrease. Such changes diminish the likelihood of microor- ganisms utilizing the body’s zinc and iron for their own prolif- eration. Another nutrient affected by inflammation is vitamin A; plasma retinol concentrations typically decrease with both infection and trauma.

Restoration of homeostasis following an inflammatory response involves a group of anti-inflammatory compounds including endogenous lipid mediators like resolvins, protec- tins, lipoxins, and maresins, as well as anti-inflammatory

cytokines like IL-10, transforming growth factor β, and cytokine antagonist IL1Ra. While a discussion of the resolu- tion to inflammation and stress is beyond the scope of this Perspective, it is important to note that inadequate energy and nutrient intakes impair the ability to rebuild lost muscle mass as well as diminish the immune and antioxidant defense responses. Research is focused on determining optimal nutrition support required for the treatment of stress and inflammatory conditions, including the identification of nutrients that may serve as immunomodulators. (However, the benefits of physical activity [early ambulation] should not be ignored.) Supplementation of the diet with essential amino acids, including sufficient amounts of leucine, has been shown to offset the catabolic effects associated with bedrest and acute hypercortisolemia [3]. However, the absolute quan- tities of protein as well as other nutrients needed to reverse the catabolic state associated with stress and inflammation have not yet been determined.

References Cited

1. Biolo G, Ciocchi b, Lebenstedt M, Barazzoni R, Zanetti M, Platen P, Heer M, Guarnieri G. Short-term bed rest impairs amino acid-induced protein anabolism. J Physiol. 2004;558:331–8.

2. Biolo G. Protein metabolism and requirements. World Rev Nutr Diet. 2013;105:12–20.

3. Paddon-Jones D, Sheffiled-Moore M, Urban RJ, Aarsland A, Wolfe RR, Ferrando AA. The catabolic effects of prolonged inactivity and acute hypercortisolemia are offset by dietary supplementation. J Clin Endocrinol Metab. 2005;90:1453–9.

Suggested Readings

Finnerty CC, Mabvuure NT, Ali A, Kozar RA, Herndon DN. The surgically induced stress response. JPEN. 2013;37:21S–29S.

Parlato M. Host response biomarkers in the diagnosis of sepsis: a general overview. Methods Molec Biol. 2015;1237:149–211.

Watt DG, Horgan PG, McMillan DC. Routine clinical markers of the magnitude of the systemic inflammatory response after elective operation: a systemic review. Surgery. 2015;157:362–80.

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245

M ETABOLISM IS OFTEN DEFINED as all chemical reactions and pathways that occur in a living organism to maintain life. Such a broad definition may seem overwhelming, although a closer look at metab- olism reveals a highly coordinated set of events that occur around the central theme of energy homeostasis. Humans require frequent input of energy to perform mechanical work, including cardiac and skeletal muscle contractions; active transport of molecules and ions; and synthesis of complex molecules from simple precursors. The demand for energy by cells and the intake of energy from food are rarely synchronized, so the body is constantly adjusting metabolic pathways to maintain energy homeostasis. Despite the complexity, metabolic integration is achieved by the cells’ ability to use a common energy currency (i.e., ATP) and surprisingly few intermediates (e.g., pyruvate and acetyl-CoA) that tie together metabolic pathways.

Chapters 3, 5, and 6 featured carbohydrate, lipid, and protein metabolism. Those chapters discussed how the pathways are regulated at the level of regulatory enzymes by substrate availability, allosteric mechanisms, and covalent modifications such as phosphorylation. This chapter focuses on the integration of carbohydrate, lipid, and protein metabolism as it occurs in different organs and tissues—and the interconnections among them. The important topics discussed are: (1) the homeostasis of cellular energy and the control between catabolism and anabolism; (2) how the major organs and tissues interact through integration of their metabolic pathways to redistribute energy, (3) hormonal regulation of these metabolic processes; and (4) examples of the body’s ability to maintain homeostasis under the daily events of fasting, refeeding, and physical activity. This chapter also discusses exercise—planned, structured physical activity to enhance physical fitness—and sports nutrition.

EnErgy HomEostasis in tHE CEll

Metabolic pathways generally belong to two broad categories: those that yield energy (degradative; catabolic) and those that require energy (synthetic; anabolic). The main purpose of catabolic reactions is to break down macronutrients so their inherent energy can be released and transformed into ATP. To a lesser extent other high-energy molecules such as GTP and UTP are formed, although ATP is ubiquitous and the primary cellular energy carrier. Anabolic reactions, on the other hand, synthesize complex molecules from simple precursors by utilizing the energy from ATP (and the other nucleoside triphosphates in a few key reactions).

Despite the thousands of reactions that occur in the body, there are only a few common pathways in the metabolic roadmap that control whether a

IntegratIon and regulatIon of MetabolIsM and the IMpact of exercIse

7

ENERGY HOMEOSTASIS IN THE CELL Regulatory Enzymes

INTEGRATION OF CARBOHYDRATE, LIPID, AND PROTEIN METABOLISM Interconversion of Fuel Molecules Energy Distribution among Tissues

THE FED-FAST CYCLE The Fed State The Postabsorptive State The Fasting State The Starvation State

HORMONAL REGULATION OF METABOLISM Insulin Glucagon Epinephrine Cortisol Growth Hormone

EXERCISE AND NUTRITION Muscle Function Energy Sources in Resting Muscle Muscle ATP Production during Exercise Fuel Sources during Exercise

SUMMARY

P E R S P E C T I V E

THE ROLE OF DIETARY SUPPLEMENTS IN SPORTS NUTRITION

BY KARSTEN KOEHLER, PhD

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246 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

the TCA cycle and oxidative phosphorylation. And when carbohydrates and lipids are in short supply, amino acids are converted to pyruvate and acetyl-CoA, thus providing needed energy for ATP production. Amino acids are also used to replenish many of the TCA cycle intermediates to ensure the cycle’s continued operation.

Pyruvate and acetyl-CoA may be used to produce more complex molecules when the cellular energy status favors anabolic reactions (Figure 7.1). Pyruvate is converted to glucose via gluconeogenesis, whereas acetyl-CoA is mostly used for fatty acid synthesis. Note that the conversion

cell or organ is engaged in catabolism or anabolism. As depicted in Figure 7.1, pyruvate and acetyl-CoA are critical junctions in the roadmap that connect the metabolism of carbohydrate, lipid, and protein. The energy status of the cell largely determines the direction in which molecules flow. If cellular energy (ATP) is needed, the pyruvate from glycolysis is sent to the mitochondria, decarboxylated to acetyl-CoA, and oxidized via the TCA cycle to produce ATP through oxidative phosphorylation (see Chapter 3). Similarly, fatty acids may be catabolized to acetyl-CoA in mitochondria, resulting in the production of ATP via

Figure 7.1 Metabolic pathways involved in the maintenance of energy homeostasis. Bidirectional pathways with separate arrows indicate separate regulatory enzymes controlling each direction. Not all pathway intermediates are shown.

TCA cycle

Succinyl-CoA

Oxaloacetate

Acetyl-CoA

Pyruvate

Citrate

α-ketoglutarate

Fumarate

Glyceraldehyde 3-phosphate

Phosphoenolpyruvate

Glycogen Glucose

Fructose

Carbohydrate

Propionyl-CoA

Propionate

Isoleucine Methionine Threonine

Tyrosine Phenylalanine

Aspartate

Alanine

Serine

Cysteine

Acetoacetate

Tyrosine Phenylalanine

Leucine

Fat

Triacylglycerol

Glycerol Fatty acids

Tryptophan

Glycine

Methionine +

Serine

Lactate

Threonine Isoleucine

Phenylalanine Tryptophan

Tyrosine Lysine

Leucine

Valine

Protein

Amino acids

Arginine Glutamine Histidine Proline

Glutamate

Galactose

Asparagine

Aspartate

Threonine

β-hydroxybutyrate

Cholesterol

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 247

of the needed NADPH is supplied by the pentose phosphate pathway, which also produces ribose-5-phosphate used in the synthesis of nucleotides (see Chapter 3).

Regulatory Enzymes Maintaining the balance between catabolism and anabolism is achieved by the regulation of distinct enzymes that are highly sensitive to changes in energy status within the cell. Regulatory enzymes are located at strategic points in metabolic pathways and are mostly unidirectional (irreversible). The majority of regulatory enzymes is controlled allosterically and respond immediately to cellular signals, although some are controlled by covalent modification, usually phosphorylation, in response to hormonal signals. Glycogen synthase and phosphorylase are examples of covalently modified regulatory enzymes (see Chapter 3). Table 7.1 summarizes key enzymes that are controlled allosterically in response to immediate changes in cellular signals.

As the energy status of the cell declines, so does the concentration of acetyl-CoA, citrate, and ATP due to decreased glycolysis, lipolysis (β-oxidation), and TCA cycle reactions. At the same time, increased amounts of ADP and AMP indicate that ATP has been used up in anabolic reactions and more ATP is needed. Furthermore, decreased concentration of malonyl-CoA reflects little or no fatty acid synthesis occurring during low energy status. Each of these cellular signals will trigger the allosteric stimulation of regulatory enzymes to increase glycolysis, b-oxidation, and the TCA cycle to replenish ATP.

When cellular ATP is abundant, the concentration of ATP, acetyl-CoA, and citrate are relatively high and act to allosterically inhibit regulatory enzymes that

of pyruvate to acetyl-CoA is an irreversible reaction, preventing appreciable amounts of acetyl-CoA from being used for gluconeogenesis. Other metabolic intermediates, particularly those of the TCA cycle, can be diverted into anabolic pathways as needed. TCA cycle intermediates used for anabolic reactions are largely replenished by the conversion of pyruvate to oxaloacetate, although a variety of molecules are available to ensure the TCA cycle continues to function. Examples of TCA cycle intermediates entering anabolic pathways include the following:

● Citrate can move from the mitochondria into the cyto- sol, where citrate lyase cleaves it into oxaloacetate and acetyl-CoA, the latter being used for fatty acid synthesis.

● Malate, in the presence of NADP1-linked malic enzyme, may provide a portion of the NADPH required for reduction reactions in fatty acid synthesis.

● Succinyl-CoA can combine with glycine in the mito- chondria to form Δ-aminolevulinic in initial step in heme synthesis (see Figure 13.7).

● Oxaloacetate may be used for conversion to amino acids or it may enter the gluconeogenesis pathway.

● CO2 produced by the TCA cycle is a source of cellular carbon dioxide for carboxylation reactions that initiate fatty acid synthesis and gluconeogenesis. This CO2 also supplies the carbon of urea and certain portions of the purine and pyrimidine rings (see Figures 6.7, 6.27, and 6.31).

It is important to remember that anabolic reactions generally require NADPH to provide reductive power for synthesis of complex molecules. NADPH is the major electron donor in cells that drives anabolic reactions. Most

Low Energy Status

Metabolic Response Cellular Signals Regulated Enzyme

↑ Glycolysis ↑ AMP, ↓ ATP, ↓ citrate, ↓ acetyl-CoA ↑ Phosphofructokinase ↓ ATP, ↓ acetyl-CoA ↑ Pyruvate kinase

↑ TCA cycle ↑ ADP, ↑ pyruvate ↑ Pyruvate dehydrogenase ↑ ADP ↑ Isocitrate dehydrogenase

↓ Gluconeogenesis ↑ AMP ↓ Fructose-1,6-bisphosphatase ↑ Fatty acid b-oxidation ↓ Malonyl-CoA ↑ Carnitine acyltransferase I

Abundant Energy Status

Metabolic Response Cellular Signals Regulated Enzyme

↓ Glycolysis ↑ ATP, ↑ citrate ↓ Phosphofructokinase ↓ TCA cycle ↑ ATP ↓ Isocitrate dehydrogenase

↑ ATP ↓ a-Ketoglutarate dehydrogenase ↑ Gluconeogenesis ↑ Acetyl-CoA ↑ Pyruvate carboxylase

↑ Citrate ↑ Fructose-1,6-bisphosphatase ↑ Fatty acid synthesis ↑ Citrate ↑ Acetyl-CoA carboxylase

↑ Malonyl-CoA ↓ Carnitine acyltransferase I

Table 7.1 Cellular Energy Status, Metabolic Response, and Allosteric Control Sites

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248 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

Note that malonyl-CoA and acetyl-CoA are part of a rapid cycle in the cytosol that is mediated by two opposing enzymes, acetyl-CoA carboxylase (ACC) and malonyl- CoA decarboxylase (MCD), as depicted in the shaded area in Figure 7.2. If energy is not needed by the cell, the acetyl- CoA will be carboxylated to form malonyl-CoA, which is the first step of fatty acid synthesis (see Figure 5.27). Malonyl-CoA levels are highest in the fed state and decline with fasting (the fed-fast cycle is discussed in detail later in this chapter). The amount of malonyl-CoA in skeletal muscle is increased by glucose and insulin, resulting in a decrease in b-oxidation of fatty acids due to the inactivation of AMPK. In lipogenic tissues such as the liver, adipose tissue, and lactating mammary glands, malonyl-CoA is a cosubstrate for the cytosolic fatty acid synthase system for the de novo synthesis of palmitic acid (see Chapter 5). Malonyl-CoA is also involved in the elongation reactions of fatty acids in the ER.

When the energy status of the cell is low, recruitment of fatty acids from the circulation increases the cytosolic

govern glycolysis and the TCA cycle while stimulating gluconeogenesis and fatty acid synthesis as a way of capturing and storing the energy for later use. Each of the regulatory enzymes highlighted in Table 7.1 have been discussed in more detail in Chapters 3 and 5.

Role of Malonyl-CoA Malonyl-CoA deserves special mention because of its role in both fatty acid synthesis and b-oxidation. Its regulatory role is expressed through its allosteric control of carni- tine acyltransferase I (CAT I), although the cellular lev- els of malonyl-CoA are mediated by the phosphorylation of AMP-activated protein kinase (AMPK). Recall from Chapter 5 that CAT I is required to transport activated fatty acids (as fatty acyl-CoA) into the mitochondria for oxidation. Increased cellular concentration of malonyl- CoA blocks CAT I and prevents transport and subsequent oxidation, whereas the absence of malonyl-CoA allows CAT I to function. Figure 7.2 illustrates the cellular signals that control the concentration of malonyl-CoA.

Figure 7.2 Role of malonyl-CoA in fatty acid synthesis and oxidation. Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; CAT, carnitine acyltransferase (I and II); and MCD, malonyl-CoA decarboxylase.

+

AcylcarnitineCarnitine

Mitochondrial outer membrane

Mitochondrial inner membrane

Fatty acyl-CoA

CoA

CoA

Fatty acyl-CoA

Acetyl -CoA

Malonyl -CoA

ACC MCD

AMPK-P (active)

AMPK (inactive)

Fatty acid

Fatty acid

Fatty acyl-CoA

CoA

+

Glucagon Plasma

membrane

Acetyl -CoA

Malonyl -CoA

ACC MCD

Abundant Cellular Energy Low Cellular Energy

Fatty acid synthesis

Fatty acid oxidation

Glucose

Glucose

– CAT ICAT I

CAT II CAT II

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 249

activator of phosphofructokinase (reaction 3 in Figure 3.17). Furthermore, activated AMPK prevents cellular energy from being diverted into anabolic pathways by inhibiting glycogen synthase (see Figure 3.13) and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose- 6-phosphatase (see Figure 3.30).

Another role of AMPK in lipid metabolism when the cellular AMP:ATP ratio is high includes the promotion of fatty acid uptake into cardiac muscle by increasing the translocation of the fatty acid transporter CD36 to the plasma membrane. Activated AMPK also inhibits acyltransferases involved in triacylglycerol and phospholipid synthesis (see Figure 5.31), and HMG-CoA reductase, the rate-limiting step in cholesterol synthesis (see Figure 5.32). With regard to protein metabolism, AMPK appears to inhibit protein synthesis by phosphorylating at least two enzymes involved in the translation of mTOR, itself a protein kinase that controls many cellular processes including protein synthesis [1].

AMPK also plays a role in energy metabolism through its action in the hypothalamus, the primary appetite control center of the body. Details of the role of AMPK and hormones in appetite regulation are discussed in Chapter 8.

intEgration of CarboHydratE, lipid, and protEin mEtabolism

The chapter thus far has focused mainly on individual pathways and regulation of those pathways by specific enzymes. We now shift our attention to a broader view of metabolism in which many pathways are coordinated simultaneously through common cellular and extracellular signals. In fact, the entire human body must be continually synchronized for normal metabolism to occur. This expanded view of metabolism reveals a type of “communication” within cells, between cells, and even among tissues and organs. The constantly changing metabolic status of various tissues throughout the body requires communication across great distances, facilitated by the nervous, endocrine, and vascular systems. Described here is an overview of how fuel molecules can be interconverted and redistributed among the body’s tissues to maintain energy homeostasis. The impact of the fed-fast cycle and skeletal muscle activity (exercise) on these events are discussed later in this chapter.

Interconversion of Fuel Molecules The body needs a constant supply of energy from macronutrients to function optimally. The amount of food consumed and the frequency of intake have profound effects on metabolic pathways that maintain the balance

fatty acyl-CoA concentration (Figure 7.2). The need for ATP production is accelerated by fasting and muscle contraction. Under these conditions, increased levels of fatty acyl-CoA and glucagon activate AMPK by phosphorylation, which in turn activates MCD and inactivates ACC by phosphorylation. In cardiac muscle 50–80% of the energy is derived from fatty acids. Fatty acids provide less energy following consumption of a high-carbohydrate meal and more following a high-fat meal. Malonyl-CoA is also thought to function as one of the signals for b-cells of the pancreas to secrete insulin in response to elevated blood glucose levels. The elevated malonyl-CoA levels in b-cells inhibit the transfer of fatty acids into the mitochondria, and the increased fatty acid levels in the cytosol act as a coupling factor for insulin secretion.

Malonyl-CoA is also associated with the restraint of food intake. It acts through the hormone leptin released by adipose tissue to signal that triacylglycerol storage in adipose is adequate. This is discussed in Chapter 8 under the section on the control of food intake.

Role of AMP-Activated Protein Kinase The previous section described how AMPK participates in fatty acid metabolism. AMPK appears to play a much larger role in metabolism and can be viewed as a master energy sensor, controlling both catabolic and anabolic pathways involving all the macronutrients [1]. Regulatory systems that are responsive to AMPK represent another common mechanism of regulation that links carbohy- drate, lipid, and protein metabolism. AMPK is activated by an increasing cellular AMP and declining ATP (high AMP:ATP ratio), indicating low energy status of the cell. This can be caused by declining cellular glucose that occurs during fasting, increased ATP utilization that occurs with muscle contraction, and metabolic stresses that interfere with ATP production such as hypoxia. Several dietary phytochemicals can activate AMPK, including capsaicin in peppers, resveratrol in red wine and grapes, curcumin in turmeric, and epigallocatechin gallate in green tea.

AMPK influences glucose metabolism in several ways. In response to increasing AMP:ATP ratio, activated AMPK in turn activates a transporter protein in adipocytes and muscle cells involved in the translocation of GLUT4 to the plasma membrane, thus increasing the uptake of glucose into the cell. A common drug used to treat type 2 diabetes, metformin, exerts part of its hypoglycemic effect by activating AMPK and increasing the translocation of GLUT4 to the cell surface by mechanisms independent of insulin. AMPK also promotes glucose uptake in cells that express only GLUT1 by activating the transporter that is already located in the plasma membrane (see Table 3.2). AMPK stimulates glycolysis mainly in cardiac muscle by phosphorylating phosphofructokinase-2, which produces 2,6-bisphosphate, a potent allosteric

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250 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

Glucose is the precursor for the glycerol moiety of triacylglycerol in adipose tissue. It can be formed from dihydroxyacetone phosphate (DHAP), a three- carbon intermediate in glycolysis (see Figure 3.17). Reduction of DHAP by glycerol-3-phosphate dehydrogenase and NADH produces glycerol-3- phosphate (see Figure 5.35). The fatty acid components of triacylglycerols in adipose tissue can come from the diet, from adipose tissue (via lipolysis), or from the liver, where they are synthesized and packaged for delivery to adipocytes by VLDL. Triacylglycerols are synthesized by the reaction of the glycerol-3-phosphate with CoA- activated fatty acids (Figure 5.29). Recall that muscle and adipose tissue lack the glycerol kinase that can phosphorylate glycerol directly and must obtain the glycerol-3-phosphate through glycolysis.

While carbohydrate can be converted into both the glycerol and the fatty acid components of triacylglycerols, only the glycerol portion of triacylglycerols can be converted into carbohydrate. The conversion of fatty acids into carbohydrate is not possible because the pyruvate dehydrogenase reaction is not reversible. This fact prevents the direct conversion of acetyl-CoA, the sole catabolic product of even-numbered-carbon fatty acids, into pyruvate for gluconeogenesis. In addition, gluconeogenesis from acetyl-CoA as a TCA cycle intermediate cannot occur because for every two carbons in the form of acetyl-CoA entering the TCA cycle, two carbons are lost by decarboxylation in early reactions of the cycle (see Figure 3.18). Consequently, there can be no net conversion of acetyl-CoA to pyruvate or to the gluconeogenic intermediates of the cycle. Acetyl- CoA produced from any source must be used for ATP production, lipogenesis, cholesterol synthesis, or ketogenesis (Figure 7.1).

Although fatty acids that have an even number of carbons are degraded exclusively to acetyl-CoA and therefore are not glucogenic (resulting in glucose production), fatty acids that possess an odd number of carbon atoms are partially glucogenic. Fatty acids with an odd number of carbons can be partially converted to glucose because propionyl-CoA (CH CH COSCoA)3 22 2 , ultimately formed by b-oxidation, is carboxylated and rearranged to succinyl-CoA, a glucogenic TCA cycle intermediate (see Figure 5.25). Fatty acids with an odd number of carbon atoms are not abundant in the diet, although ruminant milk fat and some fish are known sources.

Metabolism of the amino acids gives rise to a variety of amphibolic intermediates, some of which produce glucose (glucogenic), while others produce ketone bodies (ketogenic) by their conversion to acetyl-CoA or acetoacetyl-CoA. Only the amino acids leucine and lysine are purely ketogenic. The dispensable (nonessential) glucogenic amino acids can be converted to carbohydrate,

between catabolism and anabolism both short term (minutes, hours) and long term (days, weeks, months), as discussed later in this chapter. Dietary carbohydrate and lipid provide the majority of energy for ATP production. Amino acids are also used for energy, although the high demand for body proteins generally diverts most food-derived amino acids into protein synthesis. Amino acids may be called upon for energy when carbohydrate and lipid are insufficient.

Figure 7.1 illustrates how macronutrients are catabolized to common intermediates (pyruvate and acetyl-CoA), which can be resynthesized into glucose, triacylglycerol, and amino acids as warranted by the metabolic status of the cell. Pyruvate and acetyl-CoA represent key intersections in the metabolic roadmap where interconversion among the nutrients can occur. For example, as explained in Chapter 6, certain amino acids can be synthesized in the body from carbohydrates or fatty acids; conversely, most amino acids can serve as precursors for glucose or fatty acid/triacylglycerol synthesis. Carbohydrates can be used to synthesis fatty acids and triacylglycerols. Fatty acids, in contrast, cannot be used to make glucose because humans lack the necessary enzymes to convert acetyl-CoA to pyruvate or any intermediate along the gluconeogenic pathway. Not evident from the figure, but important to recall, is that the TCA cycle is an amphibolic pathway, meaning that it not only functions in the oxidative catabolism of carbohydrates, fatty acids, and amino acids but also provides precursors for many biosynthetic pathways, particularly gluconeogenesis (see Figure 3.31). Amino acids can be converted into several TCA cycle intermediates. When needed, a-ketoglutarate, succinate, fumarate, and oxaloacetate can be used as gluconeogenic precursors.

When ATP is needed, glucose and amino acids may be catabolized to pyruvate, which is translocated from the cytosol into the mitochondria while simultaneously decarboxylating it to acetyl-CoA. The acetyl-CoA can be oxidized to CO2 and H O2 to produce ATP by the TCA cycle and oxidative phosphorylation. Another fate of pyruvate is its reduction in the cytosol to lactic acid (Figure 7.1). The lactate can be transported to other tissues, converted back to pyruvate and oxidized in the muscle, or used for gluconeogenesis in the liver. Most of the acetyl-CoA is produced in the mitochondria through the b-oxidation of fatty acids. When acetyl-CoA is involved in anabolic reactions, it has to be translocated back to the cytosol across the mitochondrial membrane, which is not permeable to it. Therefore, the acetyl-CoA in the mitochondria combines with oxaloacetate to form citrate (as in the TCA cycle), to which the mitochondrial membrane is freely permeable. The citrate moves into the cytosol and can break down again to oxaloacetate and acetyl-CoA. The acetyl-CoA may undergo a carboxylation reaction catalyzed by acetyl-CoA carboxylase to form malonyl-CoA (see Figure 5.27), the first step of fatty acid synthesis.

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 251

and transported among tissues via the bloodstream under all metabolic conditions to provide energy where needed. The following discussion highlights the unique metabolic features and primary differences of tissues that participate in energy distribution.

Liver The liver plays a central role in metabolism. Most nutrients absorbed by the small intestine first pass through the liver, and many fuel molecules released by extrahepatic tissues travel to the liver for additional processing. Figures 7.3, 7.4, and 7.5 illustrate the fate of glucose-6-phosphate, amino acids, and fatty acids in the liver. In these figures, anabolic pathways are shown pointing up; catabolic pathways are pointing down; and distribution to other tissues is running horizontally. The pathways indicated are described in detail in Chapters 3, 5, and 6, which deal with carbohydrate, lipid, and protein metabolism, respectively.

Glucose entering hepatocytes from the hepatic portal vein and, to a lesser extent, the systemic circulation is phosphorylated by glucokinase to glucose-6-phosphate. Dietary galactose is also phosphorylated and rearranged to glucose-6-phosphate. Figure 7.3 shows the possible metabolic routes available to glucose-6-phosphate. The liver uses relatively little glucose-6-phosphate for its own energy needs and, instead, stores a significant amount

but like the ketogenic amino acids, they can also be converted indirectly into fatty acids by undergoing oxidation to acetyl-CoA. Fatty acids cannot be converted into the glucogenic amino acids for the same reason that fatty acids cannot be converted into glucose—namely, the irreversibility of the pyruvate dehydrogenase reaction. Although metabolically possible, the conversion of the glucogenic amino acids into fatty acids is rather uncommon. Only when protein is supplying a high percentage of calories would glucogenic amino acids be expected to be used in fatty acid synthesis. All the amino acids producing acetyl-CoA directly—isoleucine, threonine, phenylalanine, tryptophan, tyrosine, lysine, and leucine—are indispensable. Tyrosine is conditionally indispensable because it is formed by hydroxylation of phenylalanine. The catabolism of the individual amino acids is covered in Chapter 6.

Energy Distribution among Tissues The ability to interconvert fuel molecules is crucial for maintaining energy homeostasis in the body, where metabolism in every tissue is unique and markedly dif- ferent. The metabolic events that occur in one tissue will significantly affect metabolism in other tissues. In this way, fuel molecules are constantly being interconverted

Figure 7.3 Pathways of glucose-6-phosphate metabolism in the liver.

Liver glycogen

Pyruvate Triacylglycerols, phospholipids

Glycolysis

Acetyl-CoA

Cholesterol Fatty acids

TCA cycle

Blood glucose

NADPH

Nucleotides

Ribose-5-phosphate

CO2 O2 H2O

e–

ADP + Pi ATP

Glucose- 6-phosphate

in the liver

Pentose phosphate

pathway

Oxidative phosphorylation

Glucose

Arrows of catabolic pathways point downward.

Arrows of reactions to distribute products to other tissue are horizontal.

Arrows of anabolic

reactions point upward.

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252 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

These substances in turn can be oxidized for energy or converted to glucose or fatty acids. Glucose formed from gluconeogenesis can be transported to muscle, brain, nerve cells, red blood cells, and other tissues for energy utilization. Newly synthesized fatty acids can be transported to adipose tissue for storage or used as fuel primarily by cardiac and skeletal muscle. Hepatocytes are the exclusive site for the formation of urea, the major excretory form of amino acid nitrogen.

The fate of fatty acids in the liver is outlined in Figure 7.5. Hepatic fatty acids are derived from chylomicron remnants and from de novo synthesis. In humans, most fatty acid synthesis takes place in the liver rather than in adipose tissue. Fatty acids can be assembled into liver triacylglycerols and released into the circulation as plasma VLDL. Circulating VLDL interact with tissues expressing lipoprotein lipase, namely muscle and adipose tissue, where the triacylglycerols are delivered. Adipocytes store the triacylglycerols, whereas muscle will mostly hydrolyze the triacylglycerols and oxidize the resulting fatty acids for ATP production. Under most circumstances, fatty acids

as glycogen for times when glucose is in short supply. Glycogen synthesis occurs when blood glucose levels are high and the liver takes in more glucose, especially after a meal. About two-thirds of the glucose-6-phosphate entering glycogenesis is derived from glucose absorbed by the small intestine. The remaining glucose-6-phosphate entering glycogenesis is derived, paradoxically, from newly synthesized glucose because gluconeogenesis continues to function under all metabolic conditions. This is due to the constant flow of lactate into systemic circulation that the liver must convert to glucose-6-phosphate to keep blood lactate levels in check. The lactate comes from extrahepatic tissues, notably skeletal muscle and red blood cells.

Figure 7.4 reviews the particularly active role of the liver in amino acid metabolism. The liver is the site of synthesis of many different proteins, both structural and plasma- borne, from amino acids. The liver can also convert amino acids into nonprotein products such as nucleotides and porphyrins. Catabolism of amino acids can take place in the liver, where most are transaminated and degraded to acetyl-CoA and other TCA cycle intermediates.

Figure 7.4 Pathways of amino acid metabolism in the liver.

Liver proteins

Plasma proteins

Tissue proteins

Pyruvate

Fatty acids Cholesterol

TCA cycle

Amino acids

in blood

Alanine

Urea cycle

Nucleotides, hormones and

porphyrins

Glycogen

Glucose

Glucose

Gluconeogenesis

Triacylglycerols and phospholipids

NH3 Urea

CO2 O2 H2O

e–

ADP + Pi ATP

Amino acids in the liver

Oxidative phosphorylation

Amino acids in muscle

and TCA cycle intermediates

Arrows of catabolic pathways point downward.

Arrows of reactions to distribute products to other tissue are horizontal.Arrows of

anabolic reactions

point upward. Acetyl-CoA

Glycogen

Blood glucose Glycolysis

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 253

glucose-6-phosphate by the enzyme hexokinase. Cardiac muscle has a very limited capacity to synthesize glycogen and therefore uses the glucose-6-phosphate for immediate energy needs. Skeletal muscle, on the other hand, can store large amounts of glycogen for use at a later time. Skeletal and cardiac muscle express other GLUT proteins and can take up glucose from the circulation during fasting or when energy demands exceed incoming dietary sources of carbohydrate. Under these conditions, the blood glucose originates from the liver via glycogenolysis or gluconeogenesis. The liver releases glucose into the circulation after dephosphorylation by the enzyme glucose-6-phosphatase. Muscle cells lack this enzyme and cannot export glucose; therefore, glucose stored in muscle as glycogen is used for glycolysis.

Fatty acids are a primary fuel source for cardiac muscle and resting skeletal muscle. b-oxidation of fatty acids is entirely aerobic, so it is not surprising that cardiac muscle has a high concentration of mitochondria. Cardiac muscle will increase its use of glucose when glucose is abundant, but still favors fatty acids as the main fuel source. Similarly, resting skeletal muscle can increase its use of glucose in the fed state when there is ample glucose, insulin, and GLUT4. The use of fatty acids by active skeletal muscle can be augmented by the ability of muscle to store moderate

are a major fuel supplying energy to the liver via the TCA cycle and oxidative phosphorylation. The acetyl-CoA that cannot be used for energy may be converted to ketone bodies, which are important fuels for certain peripheral tissues such as the brain and heart muscle, particularly during periods of prolonged fasting.

Muscle Fatty acids and glucose are the major fuels for both skel- etal and cardiac muscle. Muscle can also use ketone bodies for energy when the availability of fatty acids and glucose is insufficient. Cardiac muscle requires a continuous sup- ply of energy, whereas skeletal muscle’s demand for fuel molecules is quite low when at rest, but will increase as muscle contractions increase. The relative contribution of fatty acids and glucose use in skeletal muscle can change dramatically depending on the duration and intensity of physical activity (discussed later in this chapter).

Skeletal and cardiac muscle expresses GLUT4 on the cell surface when the blood concentration of glucose and insulin are elevated such as following a carbohydrate- rich meal. Recall that GLUT4 is the only GLUT protein whose function is dependent on insulin (see Table 3.2). Consequently, muscle cells can take up large amounts of glucose and will quickly phosphorylate it to

Figure 7.5 Pathways of fatty acid metabolism in the liver.

Liver lipids

β-oxidation

Acetyl-CoA Ketone bodies in blood

Cholesterol

Steroid hormones Bile salts

TCA cycle

Free fatty acids in blood

NADH FADH2

CO2 O2 H2O

e–

ADP + Pi ATP

Oxidative phosphorylation

Arrows of catabolic pathways point downward.

Arrows of reactions to distribute products to other tissue are horizontal.

Arrows of anabolic

reactions point upward.

Delivery to muscle and adipose tissue

Fatty acids in the liver

Plasma VLDL

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254 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

Brain Glucose is the primary fuel used by the brain and nerve cells. Under normal conditions, glucose is the sole energy source, but the brain can adapt to using ketone bodies during prolonged energy deficit as occurs when consuming energy-restricted diets and in starvation. The brain cannot use fatty acids because these large molecules do not transport across the blood–brain barrier. The brain requires a constant and relatively large supply of glucose. Unlike skeletal muscle whose energy requirements are highly variable, mental activity does not increase energy utilization by the brain. The brain accounts for about 20–25% of total energy used in the body and the majority of glucose removed from the blood when at rest.

Maintaining adequate blood glucose levels is imperative for normal brain function. In the short term when the diet is unable to supply adequate amounts of glucose, the liver releases glucose into the circulation. Liver glucose is derived from the breakdown of glycogen and from gluconeogenesis using noncarbohydrate precursors (lactate, glycerol, and certain amino acids). Prolonged energy deficit leads to accelerated breakdown of triacylglycerols in adipose tissue, resulting in an overabundance of fatty acids that the liver oxidizes to acetyl-CoA. The acetyl-CoA is converted to ketone bodies that the brain and other tissues can convert back to acetyl-CoA and use for ATP production via the TCA cycle and oxidative phosphorylation.

Red Blood Cells Red blood cells rely exclusively on glucose as their only energy source under all metabolic conditions. During their development, red blood cells lose their organelles, including mitochondria. Without mitochondria, anaerobic glycolysis is the only means of producing ATP. The metabolic advantage is that red blood cells are unable to consume any of the oxygen they transport. The disadvantage is that glycolysis is an inefficient means of producing ATP from glucose. However, the end product of glycolysis (pyruvate) is quickly converted to lactate, released into the blood plasma, and taken up by the liver. The lactate is then used to synthesize glucose via gluconeogenesis and released back into the circulation.

Kidneys The kidneys require about 10% of the total energy used by the body. Their main function is to produce urine and, in the process, remove metabolic waste products from the blood plasma. The kidneys filter the plasma approximately 60 times per day. Most of the constituents of plasma filtered by the kidney are desirable, such as glucose and water, and need to be retained. Reabsorbing these constituents in the kidney tubules requires substantial amounts of energy.

The role of the kidneys in helping maintain energy homeostasis has not been studied as thoroughly as other major organs, although their contribution appears to have

amounts of triacylglycerol adjacent to mitochondria. However, skeletal muscle relies increasingly on glucose for energy as physical activity increases. Most fatty acids used for energy by muscle are derived from the circulation. Cardiac and skeletal muscle express lipoprotein lipase on the cell surface that will bind to circulating chylomicrons (derived from the intestine following a meal) and VLDL (derived from the liver). The triacylglycerols transported by chylomicrons and VLDL are hydrolyzed by lipoprotein lipase and the fatty acids are transferred into the cell. Muscle cells can also utilize free fatty acids released by adipose tissue and transported by serum albumin.

Adipose Tissue Adipose tissue has the ability to store huge amounts of triacylglycerols and thus serves as an energy reservoir in the body. Triacylglycerols derived from the diet are transported by chylomicrons to adipose tissue where lipo- protein lipase hydrolyzes the triacylglycerols, facilitating the transfer of fatty acids into the cell. In a similar manner, triacylglycerols derived from the liver are transported by VLDL to adipose tissue. Triacylglycerols secreted by the liver come from the catabolism of chylomicron remnants as well as hepatic synthesis of fatty acids from nonlipid precursors, including “excess” glucose and fructose. Imme- diately following the uptake of fatty acids into adipocytes, the fatty acids are esterified with glycerol-3-phosphate to form triacylglycerols. The action of lipoprotein lipase does not facilitate the transfer of glycerol into the cell, so adipocytes rely on the presence of glucose as the source of glycerol-3-phosphate. The free glycerol may be transported to the liver and used as a precursor for gluconeogenesis.

Adipocytes express GLUT4 on the cell surface, which promotes glucose uptake when blood glucose levels are elevated. Glucose is converted to glucose-6-phosphate and rapidly enters glycolysis. The glycolytic pathway provides glycerol-3-phosphate for triacylglycerol assembly (as mentioned above) and pyruvate that can be converted to acetyl-CoA. Some acetyl-CoA may be oxidized via the TCA cycle to address the energy needs of the cell, while the remaining is used for fatty acid synthesis. Recall that lipogenesis requires the reducing power of NADPH. Some of the glucose-6-phosphate is directed into the pentose phosphate pathway to supply the necessary NADPH. The rate of lipogenesis is higher in the liver than in adipose tissue when measured on a gram-per-gram basis. However, the mass of adipose tissue can be many times greater than liver, especially in obese individuals, demonstrating that an overabundance of carbohydrate can contribute significantly to adiposity.

When dietary energy is in short supply, the triacylglycerols in adipose tissue are hydrolyzed and released as free fatty acids into the circulation where they bind to albumin for transport to other tissues. Many tissues in the body use fatty acids as a fuel, most notably cardiac and skeletal muscle.

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 255

The time frames assigned to each phase are only approximate and are strongly influenced by factors such as activity level, the caloric value and nutrient composition of the meal, and the person’s metabolic rate. While somewhat variable, the established time frames represent distinctive metabolic events that characterize each phase. The following discussion highlights these hallmark events that occur as time extends beyond a person’s last meal. In a normal eating routine, only the fed and postabsorptive (early fasting) states will apply, although prolonged energy deprivation will occur in extreme dieting, involuntary starvation, and certain metabolic diseases.

The Fed State Figure 7.6 illustrates the disposition of glucose, fat, and amino acids among the major tissues during the fed state. A primary indicator of the fed state is the release of insulin by the β-cells of the pancreas in response to increased blood glucose levels (discussed in detail later in this chapter). The liver is the first tissue to have the opportunity to use dietary glucose. Only some glucose is retained in the hepatocyte on first pass, while about two-thirds passes into the systemic circulation. Glucose that is retained may enter glycolysis or, to a lesser extent, be converted into glycogen. Liver glycogen is preferentially made from newly synthesized glucose. Even in the fed state, when ample dietary glucose is present, gluconeogenesis continues to function as a result of lactate returning to the liver mainly from glycolysis occurring in red blood cells and, under certain conditions, skeletal muscle (discussed later in this chapter). Red blood cells do not have mitochondria and therefore cannot oxidize fatty acids or glucose aerobically; they can oxidize glucose only anaerobically and produce lactate. The preferential use of gluconeogenic precursors for glycogen synthesis is further encouraged by the low phosphorylating activity (high Km) of hepatic glucokinase at physiological concentrations of glucose.

Most dietary glucose enters the systemic circulation and is delivered to red blood cells, skeletal muscle, the brain and nervous tissue, adipose tissue, and other tissues of the body. Red blood cells and the brain rely on glucose for energy and have no metabolic mechanisms by which glucose or fatty acids can be converted to energy stores. These tissues cannot make glycogen or store triacylglycerols. Glucose available to these tissues is oxidized immediately to produce ATP. On the other hand, skeletal muscle can store glucose as glycogen and a moderate amount of fatty acids and triacylglycerols in the fed state. With the exception of red blood cells, all of the tissues included in Figure 7.6 actively catabolize glucose for energy by glycolysis and the TCA cycle.

When available glucose or its gluconeogenic precursors exceed the glycogen storage capacity of the liver, the excess glucose can be converted to fatty acids (and triacylglycerols), as shown in Figure 7.3. The conversion

been underappreciated. It is useful to think of the kidneys as two separate organs, with glucose utilization occurring mostly in the renal medulla and glucose synthesis and secretion occurring in the renal cortex [2]. These separate activities are the result of different enzymes located within each region of the kidney. The renal medulla is similar to the brain in that it requires glucose for energy, whereas the renal cortex uses fatty acids as a primary fuel source under normal conditions. Cells of the renal medulla are able to convert glucose to glucose-6-phosphate for entry into glycolysis; however, they lack glucose-6-phosphatase and are unable to release glucose into the circulation. In contrast, cells of the renal cortex possess gluconeogenic enzymes—they lack phosphorylating capacity and cannot synthesize glycogen—and therefore can make and release glucose. The renal cortex increases glucose synthesis during prolonged starvation and may contribute up to half of the circulating glucose. Interestingly, any lactate produced as a result of glycolysis in the renal medulla can be used by the renal cortex for gluconeogenesis.

tHE fEd-fast CyClE

The best way to appreciate the integration of metabolic pathways and the involvement of different organs and tissues in metabolism is to understand the fed-fast cycle. Humans are “meal eaters” and typically consume food at routine times, followed by periods of not eating. Most meals provide significantly more energy than is needed at that moment, which triggers regulatory hormones and enzymes designed to capture and store the excess energy largely as glycogen and triacylglycerols in tissues equipped to handle such molecules. Because glucose is a major fuel for tissues, it is important that glucose homeostasis be maintained, whether the person has just consumed food or is in a fasting state. If the period since the last meal is short (less than 18 hours), the mechanisms used to main- tain glucose homeostasis are different from those used if the fasting state is prolonged. The extent to which differ- ent organs are involved in carbohydrate, fatty acid, and amino acid metabolism varies within the fed-fast cycles that underlie the eating habits of humans. A fed-fast cycle can be divided into four states, or phases:

● the fed state, lasting about 3 hours after a meal is ingested and characterized by insulin secretion

● the postabsorptive state, occurring from about 3 to 18 hours following the meal and accompanied by a rise in glucagon secretion

● the fasting state, lasting from 18 hours to about 2 days without additional intake of food and accompanied by further increases in glucagon

● the starvation state or long-term fast, a fully adapted state of food deprivation lasting longer than about 2 days.

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256 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

largely by glucagon secreted by the pancreas in response to declining blood glucose.

Lactate, formed in and released by red blood cells, is a constant noncarbohydrate carbon source for hepatic gluconeogenesis. Skeletal muscle may also contribute lactate resulting from anaerobic glycolysis. The glucose–alanine cycle, in which alanine returns to the liver from muscle cells, also becomes important (see Figure 6.35). The alanine is then converted to pyruvate by the transfer of the amino group to a-ketoglutarate as the first step in the gluconeogenic conversion of alanine in the liver. Alanine cannot be converted to glucose in skeletal muscle. In the postabsorptive state, glucose provided to the muscle by the liver comes primarily from the recycling of lactate and alanine and, to a lesser extent, from hepatic glycogenolysis. Muscle glycogenolysis provides glucose as fuel only for muscle cells in which the glycogen is stored because muscle lacks the enzyme glucose-6-phosphatase, which converts glucose-6-phosphate to free glucose. Once phosphorylated in the muscle, glucose is trapped there and cannot leave except as lactate or alanine.

The brain is an extravagant consumer of glucose, oxidizing it for energy and releasing no gluconeogenic precursors in return. At rest, the brain uses about 20–25% of the available energy even though it is only about 2% of the body by weight. Mental activity does not increase energy utilization by the brain. The rate of

of glucose to fatty acids appears to occur only in the fed state when energy intake exceeds energy expenditure. Chronic overconsumption of carbohydrate can therefore lead to triacylglycerol accumulation in the liver as well as increased secretion of triacylglycerol-rich VLDL into the circulation. The VLDL deliver their lipid cargo to adipose tissue for storage and may contribute to increased body fat. Adipose tissue itself uses glucose as a precursor for both the glycerol and fatty acid components of triacylglycerols, although most triacylglycerols are delivered to adipocytes from circulating lipoproteins.

The Postabsorptive State With the onset of the postabsorptive state, tissues can no longer derive energy directly from ingested macronutri- ents, but instead must begin to depend on fuel sources already in the body (Figure 7.7). During the short period of time marking this phase (3–18 hours after eating), hepatic glycogenolysis is the major provider of glucose to the blood, which transports it to other tissues for use as fuel. When glycogenolysis is occurring, the synthesis of glycogen and triacylglycerols in the liver is diminished, and the de novo synthesis of glucose (gluconeogenesis) becomes a more important contributor in maintaining blood glucose levels. Each of these events is controlled

Figure 7.6 Disposition of dietary glucose, amino acids, and triacylglycerols in the fed state. Abbreviations: RBC, red blood cells; TAG, triacylglycerols.

Glucose

VLDL Chylomicrons

Fatty acids Amino acids

Glycogen

Protein Amino acids Pyruvate Glucose

CO2, H2O Protein

TAG

GlucoseAmino acids TAG

Lactate

TAG

Protein

Gut

Liver

RBC

Muscle

Glucose

Protein

Glycogen TAGCO2, H2O

Lactate

Adipose tissue

Glucose

Lactate

Brain

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 257

red blood cells are unable to use fatty acids, but both cardiac and skeletal muscle are well adapted to using fatty acids. The liver can also oxidize fatty acids for energy in the absence of insulin, which promotes fatty acid synthesis rather than oxidation. The glycerol that results from triacylglycerol hydrolysis in the adipose tissue is released into the circulation and used as a gluconeogenic precursor by the liver. Free glycerol—that which is not

glucose use in the postabsorptive state is greater than the rate of glucose production by gluconeogenesis, and thus the stores of liver glycogen begin to diminish rapidly. In the course of an overnight fast, nearly all reserves of liver glycogen are depleted.

Fatty acids released from adipose tissues are another valuable source of energy for tissues that can oxidize fatty acids via the TCA cycle. The brain, nerve cells, and

Figure 7.7 Distribution of fuel molecules in the postabsorptive state. Abbreviations: RBC, red blood cells; TAG, triacylglycerols. Source: Modified from Zakim D, Boyer T. eds., Hepatology: A Textbook of Liver Disease, 4th ed., Philadelphia: WB Saunders. Copyright Elsevier, 2003.

Liver

RBC

Muscle

Adipose tissue

Brain

Glucose

Lactate +

ATP

Pentoses +

NADPH

Alanine Glucose

Glycogen

Lactate

Glycerol

Fatty acids

CO2, H2O, ATP

Triacylglycerols

Fatty acids +

Glycerol

CO2, H2O, ATP

Alanine

Glucose

Glycogen

Fatty acids

Lactate +

ATP CO2, H2O, ATP

Glucose CO2, H2O, ATP

Glycogenolysis is the main provider of glucose to the blood in the postabsorptive state.

Lipolysis supplies fatty acids to liver, muscle, and other tissues for energy.

Lactate from red blood cells is a constant gluconeogenic precursor under all metabolic conditions.

Lactate from muscle occurs during anaerobic conditions when muscle activity exceeds oxygen supply.

ATP, CO2, H2O

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258 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

Amino acids from muscle protein breakdown provide the chief substrates for gluconeogenesis during this time, although the glycerol from lipolysis and the lactate from red blood cells (and skeletal muscle if exercising anaerobically) continue to provide gluconeogenic precursors. The release of fatty acids from adipose tis- sues continues to occur during the early fasting state at about the same or slightly higher rate as during the postabsorptive state. This supplies many tissues with fatty acids for ATP production, while the glycerol is converted to glucose in the liver.

phosphorylated or attached to fatty acids—is not used in the adipocyte and is released to the blood. Plasma free glycerol levels have thus been used as an indication of triacylglycerol turnover in adipose tissue.

The Fasting State The postabsorptive state evolves into the fasting state after 18–48 hours of no food intake. Particularly notable in the liver is the increase in gluconeogenesis that occurs in the wake of hepatic glycogen depletion (Figure 7.8).

Figure 7.8 Distribution of fuel molecules in the fasting state.

Liver

RBC

Muscle

Adipose tissue

Brain

Glucose

Lactate +

ATP

Pentoses +

NADPH

Amino acids

Glucose

Lactate

Glycerol

Fatty acids

CO2, H2O and ATP

Triacylglycerols

Fatty acids +

Glycerol

CO2, H2O and ATP

Protein

Amino acids

Glucose Fatty acids

Lactate

CO2, H2O and ATP

Glucose CO2, H2O, ATP

A constant supply of glucose from the liver is necessary for certain tissues, including the brain and red blood cells, when dietary sources of glucose are absent.

Hydrolysis of muscle proteins is a major source of carbon atoms for gluconeogenesis during the fasting state.

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 259

for the transport of oxygen to tissues. Several important changes in metabolism characteristic of the starvation state occur in order to spare protein: (1) accelerated lipolysis, (2) increased use of fatty acids as fuel in certain tissues, (3) increased use of glycerol for gluconeogenesis, and (4) increased ketone body synthesis and utilization (Figure 7.10).

The protein-sparing shift to lipolysis makes use of the ample triacylglycerol stores in most people. Free fatty acids are released by adipose tissue, becoming the primary fuel for the kidneys, liver, heart, and skeletal muscle. Hydrolysis of triacylglycerols also provides glycerol, now the primary source of carbon atoms for gluconeogenesis in the liver. Lactate is still a gluconeogenic precursor because red blood cells continually produce lactate from glycolysis under all metabolic conditions. And while muscle protein breakdown is significantly diminished, muscle cells still release some amino acids (notably alanine and glutamine), which can be used for gluconeogenesis. During this time the kidneys become a major supplier of glucose through gluconeogenesis, apparently using glutamine and possibly glycerol as substrates, although research on the role of the kidney is somewhat sparse [2]. Total production of glucose in humans during starvation is about 80 g/day.

Eventually, the use of TCA cycle intermediates for gluconeogenesis depletes the supply of oxaloacetate (see Figure 3.31). Low levels of oxaloacetate, coupled with rapid production of acetyl-CoA from fatty acid catabolism, cause acetyl-CoA to accumulate, favoring formation of acetoacetyl-CoA and ketone bodies in the liver. The ketone bodies are then released into the blood for delivery to tissues. Skeletal muscle, heart, and brain preferentially oxidize the ketone bodies instead of glucose via the TCA cycle. In fact, after several weeks of starvation, about two-thirds of the biological fuel for the brain comes from b-hydroxybutyrate and acetoacetate [3]. Table 7.2 shows the dramatic increase in ketone body utilization that occurs during many days of starvation. The kidneys also produce ammonia (NH )3 that helps neutralize the acidity associated with ketone bodies. Furthermore, although cardiac and skeletal muscle can use fatty acids, they also shift to using ketone bodies when available. These shifts away from using glucose help to conserve the blood glucose for tissues that depend solely on glucose as a fuel source. As long as ketone bodies are maintained at a high concentration by increased lipolysis and hepatic fatty acid oxidation, the need for glucose and gluconeogenesis is reduced, thus sparing valuable protein.

Survival time in starvation depends mostly on the quantity of triacylglycerols stored before starvation. Stored triacylglycerols in the adipose tissue of a person of normal weight and adiposity can provide enough fuel to sustain basal metabolism for about 3 months. A very obese adult probably has enough fat calories stored to endure a fast of

The shift to gluconeogenesis using amino acids during the fasting state is mediated by the increased secretion of glucagon and cortisol. Proteins are hydrolyzed in muscle cells at an accelerated rate, providing amino acids for gluconeogenesis. The high rate of breakdown of muscle protein is accompanied by large daily losses of nitrogen through the urine. Of all the amino acids, only leucine and lysine cannot directly contribute to gluconeogenesis because they are ketogenic. However, these two amino acids can nonetheless be used for energy due to their conversion to acetyl-CoA and ketone bodies (acetoacetate and b-hydroxybutyrate), thus providing a source of energy for the brain, heart, and skeletal muscle.

An amino acid of particular significance during the fasting state is alanine, which is involved in the alanine– glucose cycle (see Figure 6.35). During fasting, as muscle protein breaks down to amino acids, the nitrogen from the amino acids is transaminated to a-ketoglutarate (formed in the TCA cycle) to make glutamate. The a-amino group from glutamate is then transaminated to pyruvate (formed from glycolysis) to make alanine. The alanine enters the bloodstream and is transported to the liver, where it again transaminates its amino group to a-ketoglutarate. Alanine is converted to pyruvate, and a-ketoglutarate is converted to glutamate. This cycle serves several functions. It removes the nitrogen from muscle during a period of high proteolysis and transports it to the liver in the form of alanine. This process also transfers the carbon structure of pyruvate to the liver, where it can be made into glucose through gluconeogenesis. The synthesized glucose can be transported back to the muscle and used for energy by that tissue.

Glutamine also plays a central role in transporting and excreting amino acid nitrogen, which is greatly increased during the fasting state. Many tissues, including the brain, form glutamine from glutamate and generate ammonia. In the form of glutamine, the ammonia can then be released from the tissues and carried through the blood to the liver or kidneys for excretion as urea or ammonium ion, respectively. Figure 7.9 gives an overview of organ cooperation and other aspects of amino acid metabolism. See also Chapter 6, “Interorgan ‘Flow’ of Amino Acids and Organ-Specific Metabolism,” for a more detailed discussion of amino acid metabolism, particularly Figure 6.37.

The Starvation State If the fasting state persists and progresses into a starvation state (often referred to as a long-term fast), a more dramatic metabolic fuel shift occurs, this time in an effort to spare body protein. This new priority is justified by the vital physiological importance of many body proteins such as hemoglobin, which is necessary

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260 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

inhibits insulin secretion by the pancreas. In normal eating patterns, the postabsorptive phase would be reversed by food consumption and thus prevent the more dramatic changes that occur with prolonged food deprivation. Continued declines in glucose during fasting and starvation states cause greater secretion of glucagon. Increased lipolysis in adipose tissue and subsequent rise in free fatty acids cause the liver to produce significant amounts of ketone bodies.

more than a year, but physiological damage and even death could result from the accompanying extreme ketoacidosis. When triacylglycerol reserves are gone, the body begins to use essential protein, leading to the loss of liver and muscle function.

To summarize, Figure 7.11 illustrates the changes that occur in plasma concentration of fuel molecules following a single meal. The gradual decrease in glucose during the postabsorptive state stimulates glucagon and

Figure 7.9 Interchanges of selected amino acids and their metabolites among body organs and tissue. Source: Modified from Munro, H.N., “Metabolic Integration of Organs in Health and Disease,” Journal of Parenteral and Enteral Nutrition 1982, 6; 4:271–279. Copyright © 1982 by Sage Publications. Reprinted by permission of SAGE Publications.

Branched-chain amino acids

Tryptophan Serotonin

Glutamate, α-ketoglutarate, and NH3

Glucose

Glucose

Gluconeogenesis

Glutamine

Alanine

Actomyosin

Pyruvate

NH2

NH3 Urea

3-methylhistidine

Alanine

Gut

Urea

Liver

Brain

Fat depot

Muscle

Kidney

Branched-chain amino acids

Aromatic amino acids

Glutamate and glutamine

Gluconeogenesis

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 261

transduction) leading to a metabolic response. Refer to Figure 3.10 for a brief overview of the insulin signaling pathway and the metabolic events resulting from insulin binding to its receptor. Hormones that control metabolism may be generally categorized as those promoting anabolic reactions and energy storage (insulin) versus those that promote catabolic reactions and energy utilization (glucagon, epinephrine, and cortisol). Some hormones elicit both anabolic and catabolic responses depending on the target tissue (growth hormone). Table 7.3 summarizes

Hormonal rEgulation of mEtabolism

The organs of the endocrine system secrete hormones that play a major role in regulating metabolism. Tissues and cells that respond to hormones are called target tis- sues and cells because they express membrane receptors to which the hormones bind. The act of binding triggers a series of intracellular reactions (referred to as signal

Figure 7.10 Distribution of fuel molecules in the starvation state.

Kidney

Glucose

Lactate

ATP +

Glutamine

CO2, H2O and ATP

Fatty acids

Liver

RBC

Muscle

Adipose tissue

Brain

Glucose

Lactate +

ATP

Pentoses +

NADPH

Alanine and Glutamine

Glucose

Lactate

Glycerol

Fatty acids

CO2, H2O, Ketones ATP

Triacylglycerols

Fatty acids +

Glycerol

CO2, H2O, ATP

Alanine and Glutamine Fatty

acids

CO2, H2O, ATP

Glucose

Ketones

Ketones

CO2, H2O and ATP

Muscle uses no glucose during starvation, only fatty acids and ketone bodies.Protein hydrolysis is

significantly decreased, although these amino acids continue to be released by muscle.

Accelerated lipolysis provides fatty acids for direct energy and for ketone body production. Glycerol becomes the main gluconeogenic precursor in the liver.

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262 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

glycogen synthesis in the liver and skeletal muscle and inhibits gluconeogenesis in the liver. With regard to controlling blood glucose levels, insulin is a master hor- mone without peer. Failure of insulin to function properly causes chronic hyperglycemia and increases the risk of cardiovascular diseases (discussed in detail in Chapter 8).

Insulin also stimulates fatty acid synthesis—using excess glucose and fructose as precursors—that leads to increased triacylglycerol assembly for energy storage. Newly synthesized triacylglycerols in the liver are packaged in VLDL and shipped out to adipose tissue. As an anabolic hormone, insulin inhibits lipolysis in adipose tissue and proteolysis in muscle, while promoting protein synthesis in muscle, liver, and many other tissues expressing insulin receptors.

the role of key regulatory hormones and the target tissues most affected. Regulatory hormones involved in nutri- ent digestion and absorption were discussed in Chapter 2 and are not elaborated on further here.

Insulin Insulin is the major anabolic hormone that impacts glu- cose, fatty acids, and protein synthesis and storage [4]. It is a protein secreted by the b-cells of the pancreas in response to rising blood glucose and has a half-life in the circulation of 4–6 minutes. The impact of insulin is critical in the fed state when large amounts of blood glucose must be removed to prevent hyperglycemia. Insulin promotes the uptake of glucose into muscle and adipose tissue by stimulating the translocation of GLUT4 from storage vesicles to the cell surface (see Figure 3.10). It also increases

Figure 7.11 Changes in plasma concentration of fuel molecules following a single meal.

8

7

6

5

4

3

2

1

0

Pl as

m a

co nc

en tr

a� on

(m M

)

0 1 2 4 8 12 16 Time following single meal (days)

Starva�on stateFas�ng state

Postabsorp�ve state

Ketone bodies

Fa�y acids

Glucose

• •

Fed state

Insulin Glucagon Epinephrine Cortisol Growth hormone

Gluconeogenesis ↓ Liver ↑ Liver ↑ Liver Glycogenolysis ↑ Liver ↑ Liver, skeletal muscle ↑ Liver, skeletal muscle ↑ Liver Glycogenesis ↑ Liver, skeletal muscle ↓ Liver ↓ Liver, skeletal muscle Glycolysis ↑ Liver, skeletal muscle ↓ Liver ↑ Skeletal muscle Glucose uptake by GLUT4 ↑ Liver, heart, skeletal muscle Fatty acid oxidation ↑ Liver Fatty acid synthesis ↑ Adipose tissue, skeletal

muscle, liver ↓ Adipose tissue, liver

↓ Adipose tissue, skeletal muscle, liver

Fatty acid uptake from plasma lipoproteins

↓ Skeletal muscle; ↑ Adipose tissue

↑ Skeletal muscle ↑ Skeletal muscle, liver

Triacylglycerol synthesis ↑ Adipose tissue ↓ Liver Triacylglycerol breakdown (lipolysis) ↓ Adipose tissue ↑ Adipose tissue ↑ Adipose tissue, skeletal

muscle ↑ Adipose tissue ↑ Adipose tissue

Protein synthesis (translation) ↑ Skeletal muscle, liver ↑ Skeletal muscle, liver

Protein breakdown (proteolysis) ↓ Skeletal muscle ↑ Skeletal muscle ↓ Skeletal muscle Ketone body production ↑ Liver

Table 7.3 Hormonal Regulation of Energy Metabolism

Fuel Exchanges and Consumption Amount Formed or Consumed in 24 Hours (g)

Day 3 Day 40

Fuel Use by the Brain

Glucose 100 40

Ketone bodies 50 100

All other use of glucose 50 40

Fuel Mobilization

Adipose tissue lipolysis 180 180

Muscle protein degradation 75 20

Fuel Output of the Liver

Glucose 150 80

Ketone bodies 150 150

Source: Adapted from Berg JM, Tymoczko JL, Stryer L. Biochemistry. 6th ed. New York: Freeman. 2007 p. 773.

Table 7.2 Fuel Metabolism in Starvation

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 263

Cortisol Cortisol is a corticosteroid hormone produced in the adrenal cortex from cholesterol (see Figure 5.9). It is released from the adrenal cortex in response to low blood glucose levels and has a half-life of about 1 hour in the circulation. Cortisol travels in the circulation bound to albumin and corticosteroid-binding globulin (also called transcortin). After being delivered to target cells, cortisol passes freely through plasma membranes, then binds to intracellular cortisol receptors residing in the cytosol.

In the liver, cortisol stimulates gluconeogenesis and glycogenolysis. It also increases the activity of glucose-6- phosphatase, thus promoting the release of free glucose into the circulation. In skeletal muscle, cortisol stimulates glycogenolysis and inhibits the translocation of GLUT4 to the cell membrane. Cortisol also stimulates lipolysis in adipose tissue, thus providing free fatty acids for energy use in the liver, kidneys, and cardiac and skeletal muscle. Persistently high levels of cortisol, as seen in prolonged fasting (starvation) and vigorous exercise, stimulate protein breakdown in skeletal muscle so amino acids may be used for gluconeogenesis.

Growth Hormone Growth hormone (GH), also known as somatotropin, is a protein hormone produced by the anterior pituitary gland. It is transported in the circulation bound to GH-binding protein and has a half-life of 12–16 minutes. GH is secreted in response to a variety of stimuli, including fasting and strenuous exercise. GH receptors are present in the liver, adipose tissue, heart, skeletal muscle, kidney, brain, and pancreas.

In adipose tissue, GH stimulates lipolysis and the release of fatty acids into circulation. This lipolytic action occurs predominantly in the visceral adipose tissue and to a lesser extent in the subcutaneous adipose tissue. It also stimulates lipoprotein lipase in skeletal muscle, thus promoting triacylglycerol uptake from circulating VLDL. This may seem paradoxical in view of increased lipolysis in adipocytes, but GH is tissue- specific and has no effect on lipoprotein lipase in adipose tissue.

In the liver, GH increases triacylglycerol uptake from VLDL by inducing the expression of lipoprotein lipase and hepatic lipase. Once again, the action of GH seems paradoxical since the liver produces VLDL. Apparently increased lipase activity in the liver occurs mainly in starvation as a way to recapture energy from the circulation. GH also functions to conserve protein by inhibiting protein breakdown while stimulating protein synthesis [6].

Glucagon All metabolic effects of glucagon reflect the need to liberate stored energy for ATP production while maintaining blood glucose levels in the absence of dietary carbohydrate. The metabolic responses elicited by glucagon oppose those of insulin. Therefore, it is a prominent hormone in nonfed states and its concentration in the blood increases as starvation approaches. Glucagon is a protein secreted by the a-cells of the pancreas when blood glucose levels decline and has a half-life in the circulation of 3–6 minutes. The main tissues expressing glucagon receptors are the liver and adipose tissue. In the liver, glucagon causes an increase in gluconeogenesis and glycogenolysis, while inhibiting glycogen synthesis, so that more glucose can be released into the circulation and thus reverse the effects of insulin (Table 7.3). Additional effects include increased lipolysis in adipose tissue (for release of free fatty acids into the circulation) and increased fatty acid oxidation and ketone body production in the liver as starvation progresses. Glucagon also stimulates thermogenesis in brown adipose tissues, presumably to maintain body heat during periods of low or no food intake [5].

Skeletal muscle does not make glucagon receptors and is unresponsive to the hormone. The kidneys do have receptors, although the effect of glucagon in the kidney is not well studied. It is possible that glucagon contributes to the increased gluconeogenesis known to occur in the kidney during starvation.

Epinephrine Epinephrine is a catecholamine produced in the adrenal medulla from the amino acids phenylalanine and tyrosine (see Figure 6.10). It functions both as a neurotransmitter in the nervous system and as a stress hormone in the circula- tion. Epinephrine has a half-life in the circulation of 1–2 minutes. It binds to two classes of adrenergic receptors on cell membranes, a and b receptors. The receptors function as part of a cAMP signal transduction cascade, an exam- ple of which is shown in Figure 1.9. As a stress hormone, epinephrine can increase cardiac muscle contractions and increase vasodilation and blood flow to skeletal muscle and the liver. Epinephrine levels are known to increase during exercise.

The binding of epinephrine to a receptors in the pancreas inhibits insulin secretion; in the liver and skeletal muscle it stimulates glycogen breakdown and inhibits glycogen synthesis; and in skeletal muscle it stimulates glycolysis. Epinephrine binding to b receptors in the pancreas stimulates glucagon secretion; and in adipose tissue and skeletal muscle it stimulates lipolysis and inhibits fatty acid synthesis. Each of these responses leads to increased blood glucose and free fatty acids, allowing stored fuels to be used when dietary sources are insufficient.

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264 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

The combination of fibers is an important property of skeletal muscle that allows the human body to respond to a variety of physical demands. The presence of fast-twitch fibers allows for a rapid and intense muscle contraction, whereas the presence of slow-twitch fatigue-resistant fibers allows for muscular endurance. When a low amount of force is required, muscle contraction involves predominantly type I fibers. Increasing force requirements will recruit progressively more type IIa fibers and lastly type IIx fibers when the greatest force is required. Each muscle in the body exhibits different proportions of fiber types depending on its function. For example, muscles involved in maintaining posture engage in prolonged but relatively low-force contractions and thus have a high proportion of type I fibers. Muscles that engage in rapid or high-force contractions, such as jaw muscles, have a high proportion of type IIa and IIx fibers. The proportion (relative number) of each type of muscle fiber a person has is genetically determined; however, with appropriate exercise training, the metabolic potential of muscle can be influenced by effecting changes in fiber size and its components.

Exercising muscle causes several changes in hormones and other regulatory molecules. Such changes are required to support the increasing energy demands of muscle while simultaneously maintaining blood glucose levels for other tissues that rely on glucose for energy. It is well known that exercise increases the circulating levels of epinephrine, cortisol, and growth hormone. Collectively these regulatory molecules increase gluconeogenesis, glycogenolysis, and lipolysis, thus promoting the release of more glucose and fatty acids into the bloodstream. Most of the fatty acids and some glucose can be taken up by muscle and used for energy, whereas the remaining glucose is intended for the brain and red blood cells. Exercise of higher intensity also activates AMPK, resulting in increased lipolysis and fatty acid oxidation, as well as increased GLUT4 translocation to the muscle cell surface for glucose uptake independent of insulin (discussed earlier in this chapter). Furthermore, physical inactivity is associated with chronic inflammation and related conditions, including atherosclerosis and insulin resistance. Recent studies have suggested that exercise, even a single bout of exercise in untrained adults, stimulates the secretion of cytokines from skeletal muscle that promotes the clearance of glucose and lipoproteins from the circulation and may improve insulin sensitivity [7].

An important tool used to measure exercise capacity is the concept of maximum oxygen consumption ( 2VO2 max). As physical work increases, the volume of oxygen taken up by the body also increases. The VO2 max is defined as the point at which a further increase in the intensity of the exercise no longer results in an increase in the volume of oxygen uptake. VO2 max is unique for each person and is generally expressed in milliliters of oxygen consumed per kilogram of body mass per minute (mL kg min )1 13 32 2 . As a person goes from an untrained state to a trained state,

ExErCisE and nutrition

Movement of the human body requires the contraction of skeletal muscle. Significant amounts of energy may be needed to support muscle function, especially in people who are physically active. Some people may be physically active due to the nature of their jobs, while others engage in exercise—defined as planned, structured physical activ- ity to enhance physical fitness. Whether it be an average person wishing to stay physically fit or an elite athlete, the amount and type (and timing) of nutrient intake can influ- ence health and performance outcomes. Only in recent years has the connection between exercise and nutrition been fully appreciated, which has led to an increase of research on the topic. The following sections address the energy demands of skeletal muscle, the fuel sources avail- able to muscle under different types of exercise, and the special application of sports nutrition.

Muscle Function Skeletal muscle is composed of striated cells (called myo- cytes or muscle fibers) that generally extend the length of the muscle. The main proteins in muscle are actin and myosin; upon stimulation, myosin ATPase hydrolyzes ATP that provides the energy for muscle contraction. Muscle fibers also contain myoglobin that can store oxygen to be quickly used when needed. The typical red color of muscle is due to the presence of myoglobin. On the basis of their metabolic characteristics, muscle fibers are classified as type I, type IIa, and type IIx. Type I muscle fibers are also called oxidative, slow-twitch fibers. Type I fibers contain a large number of mitochondria and a relatively high con- centration of myoglobin, both features designed to sup- port aerobic metabolism. These fibers are surrounded by more capillaries than other fiber types in order to facilitate oxygen transport. Type I fibers are capable of oxidizing fatty acids and glucose to CO2 and H O2 via the TCA cycle and oxidative phosphorylation. Because of their reliance on aerobic metabolism, the speed of contraction of type I fibers is considered slow but resistant to fatigue. In contrast are type IIx fibers, also called glycolytic, fast-twitch fibers. Type IIx fibers have significantly fewer mitochondria and less myoglobin, giving these fibers a white appearance. This type of muscle fiber has increased myosin ATPase and an active glycolytic pathway for rapid ATP replenishment in the absence of oxygen. Type IIx fibers have an increased ability to store glycogen and higher phosphofructokinase activity to support glycolysis. The metabolic characteristics of type IIa fibers lie between those of types I and IIx fibers, having both glycolytic (fast) and oxidative (slow) function. Type IIa fibers are red in appearance and contain interme- diate levels of both mitochondria and myoglobin. They are resistant to fatigue, but have relatively high myosin ATPase activity and can contract rapidly when necessary.

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE 265

The ATP-Phosphocreatine System

The ATP-phosphocreatine system is a cooperative system in muscle cells using the high-energy phosphate bond of phosphocreatine to quickly regenerate ATP (see Figure 3.22 and Figure 6.24). When the body is at rest, energy needs of skeletal muscle are fulfilled by glucose and fatty acid oxidation because the low demand for oxygen can easily be met by oxygen exchange in the lungs and by the oxy- gen carried to the muscle by the cardiovascular system. At the onset of physical activity, the energy requirement of contracting muscle is initially met by existing ATP. However, stores of ATP in muscle fibers are limited, pro- viding enough energy for only a few seconds of maximal exercise. As ATP levels diminish, they are replenished rapidly by the transfer of high-energy phosphate from phosphocreatine to ADP to regenerate ATP. The muscle fiber concentration of phosphocreatine is only four to five times greater than that of ATP, and therefore most energy furnished by this system is diminished after the first 15–25 seconds of strenuous exercise. As the ATP-phosphocreatine system is exhausted, the lactic acid system (anaerobic glycolysis) picks up to produce more ATP. Performance demands of high intensity and short duration such as weightlifting, 100-m sprinting, gymnastics, and various short-duration field events benefit most from the ATP-phosphocreatine and lactic acid systems. Lower-intensity activity may allow skeletal muscle to use the combined ATP-phosphocreatine and lactic acid systems for several minutes.

The Lactic Acid System

This system involves the glycolytic pathway, which anaerobically produces ATP through substrate phosphorylation by the incomplete breakdown of one molecule of glucose into two molecules of lactate in skeletal muscle (see Figure 3.17). The sources of glucose are primarily muscle glycogen and, to a lesser extent, circu- lating glucose. The system can generate ATP quickly for high-intensity exercise. The rapid rise in cellular AMP resulting from ATP hydrolysis is a strong allosteric stimulator of phosphofructokinase, the most prominent regulatory enzyme in glycolysis (Table 7.1). As pointed out in Chapter 3, the lactic acid system is not efficient from the standpoint of the quantity of ATP produced. However, because the process is so rapid, the relatively small amount of ATP is produced quickly and supplies important energy for a short duration.

The lactate produced by this system rapidly crosses the muscle cell membrane into the bloodstream, from which it can be cleared by other tissues (including the liver) for aerobic production of ATP or gluconeogenesis. If the rate of production of lactate exceeds its rate of clearance, blood lactate accumulates. The quantity of lactate released at the onset of strenuous exercise is low, but when lactate

the VO2 max increases. A sedentary (untrained) person may have a VO2 max of 30 or 40, whereas a trained runner may have a VO2 max of 80 or 90. Consequently, VO2 max can be used as a measure of cardiovascular fitness. Another application of VO2 max is in quantifying the intensity of exercise. If the VO2 max for an individual is known, then the intensity of exercise can be expressed as a percentage of VO2 max. When at rest, the relative intensity is 90%) and rest. The event would be performed on day 7 of the regimen. The classical method yielded muscle glycogen levels approaching 220 mmol/kg wet weight, more than double the athlete’s resting level. However, because of various undesirable side effects of the classical regimen, such as irritability, dizziness, and a diminished exercise capacity, a less stringent regimen of diet and exercise has evolved that produces comparably high muscle glycogen levels. In the modified regimen, runners perform tapered-down exercise sessions over the course of 5 days, followed by 1 day of rest. During this time, 3 days of a 50% carbohydrate diet are followed by 3 days of a 70% carbohydrate diet, generally achieved by consuming large quantities of pasta, rice, or bread. The modified regimen, which can increase muscle glycogen stores 20–40% above normal, has been shown to be as effective as the classical approach, with fewer adverse side effects [12].

In contrast to carbohydrate loading, emerging research suggests that a low-glycogen approach to endurance training may also enhance performance. The strategy is to deliberately train in conditions of low carbohydrate intake to limit glycogen storage. This promotes adaptations in skeletal muscle that increases mitochondria and improves oxidative capacity, particularly fatty acid oxidation. Then a high-carbohydrate meal is consumed immediately prior to an important competition. Some athletes have claimed success using a “train low, compete high” approach, although strategies that create optimal conditions are unknown and a common protocol has not been established [13].

Figure 7.15 Schematic representation of the “classical” and modified regimens of muscle glycogen supercompensation. Source: From Sherman, W.M., Carbohydrate, muscle glycogen, and muscle glycogen supercompensation, In Ergogenic Aids in Sport by Williams, M.H., Champaign, IL: Human Kinetics Publishers, 1983, p. 14. Reprinted by permission.

10

20

30

220

165

110

55

40

0 1 2 3 4 5 6

Days

Modif ied

50% CHO

CHO = carbohydrate

10% CHO

70% CHO

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Classical

G ly

co ge

n (g

/k g

w et

w ei

gh t)

G ly

co ge

n (m

m ol

es /k

g w

et w

ei gh

t)

Classical (arrows indicate exhaustive exercise)

10% CHO

90% CHO

Modif ied (tapering exercise, 90, 40, 40, 20, 20 min; rest)

50% CHO 73% VO2 max

70% CHO

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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270 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

summary

Metabolic pathways are constantly adjusting in response to the energy status of cells, tissues and organs, and the whole body. Humans require frequent input of energy from dietary sources to perform mechanical work, including active transport at the cell level, synthesis of complex molecules, and muscle contractions. Dietary carbohydrates and fats (triacylglycerols) are the primary fuel molecules, although amino acids from dietary protein can also be used for energy when necessary.

All cells of the body require energy to function. The liver, cardiac and skeletal muscle, kidneys, and adipose tissue can use both glucose and fatty acids for energy. The brain and nerve cells cannot use fatty acids and rely on glucose, but can adapt to ketone bodies made from fatty acids during long-term fasting. Red blood cells lack mitochondria and have no oxidative capacity, so they depend solely on glucose and anaerobic glycolysis for energy.

In the fed state, ample energy is consumed in excess of immediate needs, resulting in energy storage as triacylglycerols in adipose tissue and muscle, and as glycogen in liver and muscle. The fed state is characterized by high insulin levels that stimulate anabolic reactions by allosteric regulation of key enzymes. In the postabsorptive

state, insulin diminishes and glucagon increases, which causes the release of stored molecules to provide the energy for cellular function between meals or sleeping through the night. Long-term energy deprivation that occurs in starva- tion can result in severe loss of body fat and muscle mass as the body sacrifices protein to meet critical energy needs.

Energy demands during exercise are strongly influenced by the intensity and duration of exercise. Contracting mus- cles that require an immediate burst of energy depend on ATP and phosphocreatine inherently present in the muscle fibers, then shift to anaerobic glycolysis as required in the first several seconds of maximal activity. Aerobic oxida- tion of fatty acids and glucose becomes the major source of energy as muscle contractions continue beyond 2 or 3 minutes.

Skeletal muscle engaged in normal daily activities at low intensity uses primarily fatty acids derived from adi- pose tissue for energy. As exercise intensity increases, the muscle uses glycogen stores until it is depleted. Skeletal muscle engaged in long-duration, high-intensity exercise becomes increasingly dependent on plasma glucose for energy and continues to use free fatty acids released from adipose tissue.

1. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sen- sor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012; 13:251–62.

2. Triplitt CL. Understanding the kidney’s role in blood glucose regula- tion. Am J Manag Care. 2012; 18:S11–S16.

3. Cahill, GF Jr. Fuel metabolism in starvation. Annu Rev Nutr. 2006; 26:1–22.

4. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001; 414:799–806.

5. Marroqui L, Alonso-Magdalena P, Merino B, et al. Nutrient regulation of glucagon secretion: involvement in metabolism and diabetes. Nutr Res Rev 2014; 27:48–62.

6. Vijayakumar A, Novosyadlyy R, Wu YJ, et al. Biological effects of growth hormone on carbohydrate and lipid metabolism. Growth Horm IGF Res. 2010; 1–14.

7. Brown WMC, Davison GW, McClean CM, Murphy MH. A systematic review of the acute effects of exercise on immune and inflammatory indices in untrained adults. Sports Med. 2015; 1:35.

8. Gastin PB. Energy system interaction and relative contribution during maximal exercise. Sports Med. 2001; 31:725–41.

9. Coyle EF. Substrate utilization during exercise in active people. Am J Clin Nutr. 1995; 61:S968–79.

10. Spriet LL, Watt MJ. Regulatory mechanism in the interaction between carbohydrate and lipid oxidation during exercise. Acta Physiol Scand. 2003; 178:443–52.

Suggested Readings

Brown WMC, Davison GW, McClean CM, Murphy MH. A systematic review of the acute effects of exercise on immune and inflammatory indices in untrained adults. Sports Med. 2015; 1:35.

The article reviews the transient changes in immune and inflammatory markers evoked by a single bout of exercise and the resulting health benefits, including reduced risk of cardiovascular disease.

McArdle WD, Katch FL, Katch VL. Exercise Physiology: Nutrition, Energy, and Human Performance. 8th ed. Baltimore: Wolters Kluwer, 2015.

A textbook that provides in-depth coverage of the metabolic principles con- necting nutrition, muscle function, energy metabolism, and exercise performance.

Schnyder S, Handschin C. Skeletal muscle as an endocrine organ: PGC-1a, myokines and exercise. Bone. 2015; 80:115–25.

A review article that describes the regulatory molecules secreted by exercis- ing muscle and their metabolic impact on health.

11. Bergstrom J, Hultman E. A study of the glycogen metabolism during exercise in man. Scand J Clin Lab Invest. 1967; 19:218–28.

12. Ivy JL. Dietary strategies to promote glycogen synthesis after exercise. Can J Appl Physiol. 2001; 26 (suppl):S236–45.

13. Bartlett JD, Hawley JA, Morton JP. Carbohydrate availability and exer- cise training adaptation: too much of a good thing? Eur J Sport Sci 2015; 15:3–12.

References Cited

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271

P E R S P E C T I V E

the role of dIetarY suppleMents In sports nutrItIon bY Karsten Koehler, phd

At least since the Ancient Olympics, athletes have been exploring possible ways to gain a competitive edge. While the diet provides ample opportunities to maximize performance and recovery during training and competi- tion, it has become an appealing option for many athletes to supplement their diet with isolated nutrients in highly concentrated form. Supplements, particularly those with claimed ergogenic effects, appear as a legal and healthy alternative to performance-enhancing drugs, which are prohibited by the strict antidoping regulations in competi- tive sports. Who would not be intrigued by “miracle pills” that promise one to run faster, jump higher, and be stronger, all while being safe and legal?

Considering this appeal, it is not surprising that ath- letes are much more prone to using supplements than the average population. In fact, depending on the sport and the level of competition, it may be hard to find a single athlete who does not use supplements on a regular basis [1]. Many athletes use supplements for the obvious motive of improving their performance and health, but supple- ments use is also often done in an attempt to emulate the behavior of opponents and peers [2]. These trends can be observed not only in competitive athletes but also in the world of recreational sports, which is eminent by the ever- increasing market of “sport supplements.” This market has been expanding both in revenue as well as in the number of products on the market. Due to varying product definitions and categorizations, it is difficult to estimate the true size of the market, but it is safe to assume that annual revenue from sport supplements is in the multibillion-dollar range.

However, contrary to popular beliefs about supple- ments among competitive and recreational athletes, scientific evidence for potential ergogenic effects is rather scarce. In fact, only a few selected substances are unanimously considered as performance enhancing, and their ergogenic properties are limited to certain sports and activities. Furthermore, for most substances available on the market, research has failed to demonstrate the claimed ergogenic effects or, more importantly, scientifically valid studies on these effects are completely lacking. Despite the lack of concrete evidence, many supplements are heavily advertised using anecdotal reports from athletes or pseu- doscientific publications, which makes it difficult for the layperson to tell fact from fiction. Considering that many athletes obtain their supplement knowledge from coaches, athletic trainers, physical therapists, or team physicians (and not from peer-reviewed scientific journals), it is not

surprising that most athletes are inadequately educated about the true effects of supplements [3].

SUPPLEMENTS WITH CONFIRMED ERGOGENIC EFFECTS

Only for a handful of substances available as supplements is there sufficient scientific evidence available to confirm performance-enhancing effects: caffeine, creatine, buff- ering agents, and nitrate-containing, carbohydrate, and protein and amino acid supplements.

Caffeine Caffeine is probably the most accepted dietary compound used to enhance performance, even in the nonathletic popula- tion. Caffeine is available in various forms, including food and beverages as well as supplements and drugs. As an adenosine receptor antagonist, caffeine has numerous central, neuronal, and metabolic effects. The ergogenic properties of caffeine are most likely modulated through neuromuscular effects that include improved neuromuscular coupling, increased recruitment of motor units, and reduced fatigue. To a lesser extent, caffeine may also influence metabolism by increas- ing the rate of lipolysis. At doses of approximately 3 mg/kg body weight and higher, caffeine improves endurance exercise performance. Ergogenic effects are likely for other modes of exercise, such as team and racquet sports as well as sports involving prolonged high-intensity exercise, even though scientific data is limited [4]. Despite the well-documented performance-enhancing effects, caffeine is currently not banned by the World Anti-Doping Agency [5]. Possible side effects of caffeine include insomnia, gastrointestinal bleed- ing, muscle tremor, and coordinative impairments. The diuretic properties of caffeine are minimal during exercise.

Creatine Creatine, a nonessential nutrient and a component of phos- phocreatine involved in intracellular energy storage, is a very popular supplement among strength athletes. In doses of 3 g/d and greater, creatine supplementation is associated with improved contractile performance during high-intensity exercise as well as muscle hypertrophy. Furthermore, creatine may also improve intramuscular glycogen storage [6]. For healthy individuals, creatine supplementation is considered safe, even though larger doses of 20–30 g/d, which are frequently endorsed for initial “charging” or “ loading” phases, are not recommended [7].

Buffering Agents During high-intensity exercise, buffering agents may improve performance by increasing fatigue resistance. Extra- cellular buffers with demonstrated ergogenic properties include sodium bicarbonate, which improves performance during repeated high-intensity interval exercise at doses of 0.3 g/kg and greater [8]. However, sodium bicarbonate supplementation is frequently associated with severe gastrointestinal distress, including nausea and vomiting. Beta-alanine, a precursor of the intracellular buffer carnosine, has been shown to increase performance and attenuate fatigue during short, high-intensity interval exercise at doses of 4–6 g/day. Potential side effects of beta-alanine supplementation include paresthesia [9].

Nitrate-Containing Supplements The ingestion of nitrate, either from supplementation or through nitrate-rich foods such as beetroot juice, is associated with increased nitric oxide generation and an increase in metabolic efficiency during submaximal exer- cise. However, it remains to be determined whether these effects translate into meaningful improvements in athletic performance, particularly in highly trained individuals [10].

Carbohydrate Supplements The ingestion of carbohydrates during prolonged aerobic exercise can improve endurance performance, and per- formance benefits are maximized at intakes of 60–80 g/h when multiple absorbable carbohydrates such as glucose and fructose are utilized. Carbohydrates are obviously not limited to supplements, as many sport-specific products such as beverages, bars, or gels as well as conventional foods can provide similar amounts and types of carbohy- drates, and the ergogenic effects appear to be independent of their form of presentation. However, the ingestion of car- bohydrates in highly concentrated form has been linked to increased gastrointestinal distress [11].

Protein and Amino Acid Supplements Protein and amino acids supplements are extremely popular among athletes who wish to increase their muscle mass or strength. It is well established that the ingestion of protein or essential amino acids in association with resistance training can support anabolic adaptations to training. How- ever, there is currently no scientific evidence to suggest that protein or amino acid supplementation is superior to protein from conventional food sources [12].

Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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272 C H a p t E r 7 • INTEgrATION AND rEguLATION OF METABOLISM AND ThE IMpAcT OF ExErcISE

POPULAR SUPPLEMENTS WITHOUT SCIENTIFIC EVIDENCE OF ERGOGENIC EFFECTS

For products such as ribose or b-hydroxy-b-methylbutyrate (HMB), which are popular among strength athletes, as well as L-carnitine or medium-chain triglycerides (MCT), which are claimed to improve fatty acid oxidation and promote weight loss, scientific data are mixed. Whereas preliminary studies and early publications acknowledged ergogenic effects, subsequent research failed to confirm these effects [1]. Other popular supplements with mixed or limited findings include quercetin, exotic berries, glutamine, and glucosamine. For many other supplements, sound scientific data is mostly lack- ing. As new products enter the supplement market almost on a daily basis, a list of supplements without sufficient evidence will always remain incomplete.

CHALLENGES IN THE EVALUATION OF ERGOGENIC EFFECTS

Research addressing the effects of supplements on performance is often complicated by the fact that most studies are exclusively laboratory based and typically employ standardized tasks such as treadmill running, bicycle ergometry, or isometric strength tests to assess physical and physiological measures of performance. While these tests are mostly validated and well accepted, they may not adequately reflect true sports performance in a competitive setting. It has further been questioned whether these laboratory- based tests as well as the statistical approaches employed in laboratory-based research are sufficiently sensitive to detect differences in performance that decide between victory and defeat, which can be as minute as hundreds of a second or millimeters [13]. Another caveat is that most research is con- ducted in moderately trained or untrained subjects; whereas scientific studies explicitly conducted in elite athletes are scarce. For several supplements, including buffering agents and nitrate-containing supplements, the ergogenic properties appear to be more pronounced in untrained or moderately trained individuals. As such, study results may not be transfer- able across the whole fitness spectrum.

POTENTIAL NEGATIVE EFFECTS OF SUPPLEMENTATION

It has further been questioned whether the use of certain supplements could potentially impair athletic performance. For example, antioxidant supplementation has been shown to attenuate beneficial effects of exercise training in untrained or moderately trained individuals. However, it remains to be determined whether antioxidant supplementation is similarly detrimental in trained athletes [14].

The consumption of supplements further bears the risk of ingesting substances that are not adequately declared on the label. Despite their form of presentation (i.e., pills, tablets, capsules, or powders), dietary supplements are regulated as food in the United States as well as in many other countries. As such, they are controlled less tightly than pharmaceuticals. There have been numerous cases in which supplements were found to contain substances that were harmful and/or that represented doping agents. For example, numerous rapid weight loss or muscle gain supplements were found to con- tain large amounts of prohibited stimulants (e.g., ephedrine, sibutramine) or anabolic steroids [15]. In addition, there have also been findings of supplements containing trace amounts of doping agents, most likely due to cross-contamination dur- ing the production process. Even though these minute doses were rarely pharmacologically relevant, they were sufficient to trigger a positive doping test [15]. Consequently, athletes enrolled in antidoping programs must be particularly cautious when using supplements. Several countries have fortunately adopted programs in recent years to better protect athletes from adulterated and contaminated supplements.

SUMMARY

Based on current scientific evidence, there is only a handful of dietary supplements, including caffeine, creatine, buffering agents, and nitrate-containing products, that can improve performance during certain sporting events. In addition, dietary supplements may also serve to improve nutrient intake in situations of special needs, such as during travel or weight loss, as well as in athletes with dietary insensitivities or severe dietary restrictions. Furthermore, supplements may also improve athletic performance through placebo effects. However, the use of supplements may also be associated with adverse events that include physical side effects as well as the unintentional uptake of prohibited substances. There- fore, supplements should only be used following a careful benefit–risk analysis, and athletes are encouraged to limit their use of supplements to specific situations [16,17].

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2. Braun H, et al. Dietary supplement use among elite young German athletes. Int J Sport Nutr Exerc Metab. 2009; 19(1):97–109.

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7. European Commission. Scientific Committee on Food. Opinion of the Scientific Committee on Food on safety aspects of creatine supplementation. 2000; Available from: http://ec.europa.eu/food/safety/ docs/labelling_nutrition-special_group_foods – portspeople-out70_en.pdf. Accessed March 20, 2016.

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14. Gomez-Cabrera MC, et al. Redox modulation of mitochondriogenesis in exercise. Does antioxidant supplementation blunt the benefits of exercise training? Free Radic Biol Med. 2015; 86:37–46.

15. Geyer H, et al. Nutritional supplements cross- contaminated and faked with doping substances. J Mass Spectrom. 2008; 43(7):892–902.

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17. IOC consensus statement on sports nutrition 2010. J Sports Sci. 2011; 29(Suppl 1):S3–4.

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273

ENERGY IS CONSTANTLY BEING USED by every cell in the body. Consequently, humans must consume food on a regular basis to meet energy demands. When the amount of food energy matches energy expenditure over time, a person is in energy balance. A person who habitually consumes energy in excess of energy needs is said to be in positive energy balance and will convert the unused energy into triacylglycerols for storage as body fat. The previous chapter discussed how the body automatically adjusts to the daily inconsistencies in energy intake and energy expenditure by redistributing fuel molecules among tissues during the fed-fast cycle and during exercise. Over lon- ger periods of time, however, maintaining whole-body energy balance is largely under external control, influenced by how much we eat and how much we exer- cise. These controllable factors inevitably form the basis of recommendations and interventions aimed at reducing the prevalence of obesity in the United States and other developed countries. This chapter addresses the common meth- ods used to measure energy expenditure and body composition. The chapter also discusses energy balance, the concept of healthy weight, and the genetic and hormonal factors that regulate appetite and body composition.

MEASURING ENERGY EXPENDITURE

Techniques for measuring energy expenditure have been important tools for health professionals in developing dietary and exercise strategies for maintain- ing healthy weight and improving athletic performance. Energy expenditure can be assessed through direct or indirect calorimetry. Another method uti- lizes doubly labeled water that compares well with calorimetric methods and is considered by many to be a “gold standard” for determining total energy expenditure. These methods have provided data that have been used to develop formulas by which energy expenditure can be quickly calculated based on body weight, height, gender, and age. Each of these methods of assessment is explained in the following sections.

Direct Calorimetry Recall from Chapter 1 that metabolic processes in the body result in the pro- duction of heat. Figure 1.12 illustrates how the metabolic oxidation of a typical fatty acid releases more energy as heat than is captured in ATP molecules. Consequently, energy expenditure can be quantified by measuring heat dis- sipated by the body. The technique of direct calorimetry is highly accurate and includes both sensible heat loss and heat of water vaporization. Although the concept of direct calorimetry is relatively simple, direct measurement of

ENERGY EXPENDITURE, BODY COMPOSITION, AND HEALTHY WEIGHT

8

MEASURING ENERGY EXPENDITURE

Direct Calorimetry Indirect Calorimetry Doubly Labeled Water

COMPONENTS OF ENERGY EXPENDITURE

Basal and Resting Metabolic Rate Energy Expenditure of Physical Activity Thermic Effect of Food Thermoregulation

BODY WEIGHT: WHAT SHOULD WE WEIGH?

Ideal Body Weight Formulas Body Mass Index

MEASURING BODY COMPOSITION

Field Methods Laboratory Methods

REGULATION OF ENERGY BALANCE AND BODY WEIGHT

Hormonal Influences Intestinal Microbiota Environmental Chemicals Lifestyle Influences

HEALTH IMPLICATIONS OF ALTERED BODY WEIGHT

Metabolic Syndrome Insulin Resistance Weight Loss Methods

SUMMARY

P E R S P E C T I V E

EATING DISORDERS

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274 C H A P T E R 8 • ENERGY EXPENDITURE, BODY COMPOSITION, AND HEALTHY WEIGHT

The Respiratory Quotient Measuring gas exchange in indirect calorimetry provides additional information about the fuel sources used in the body. Carbohydrates, fats, and proteins each required dif- ferent amounts of O2 to completely oxidize to CO2 and water because of primary differences in their chemical structures. Thus, the ratio of CO2 produced relative to O2 consumed, called the respiratory quotient (RQ), is characteristic for each fuel source. The RQ for carbohydrate, fat, and protein is 1.0, 0.70, and 0.82, respectively. An RQ value that falls somewhere between the lowest (0.70) and highest (1.0) value indicates a mixture of fuels were used for energy. Measuring gas exchange over a known period of time provides the necessary data to calculate not only total energy expenditure, but also the relative contribution of fuel sources. It is assumed that no proteins are oxidized for energy during short-duration activity. Over longer periods, the amount of protein being oxidized can be estimated from the amount of urinary nitrogen excreted, and the remainder of the metabolic energy must be made up of a combination of carbohydrate and fat. Should the principal fuel source shift from mainly fat to carbohy- drate, the RQ correspondingly increases, and a shift from carbohydrate to fat lowers the RQ. Table 8.1 includes the thermal (caloric) equivalents of oxygen consumed at RQ values between 0.70 and 1.0, assuming no contribution of proteins to energy expenditure. The use of RQ to calculate energy expenditure also assumes that gas exchange in the lungs reflects the ratio of oxygen consumption and carbon dioxide production at the cell level.

body heat loss is expensive, impractical, cumbersome, and usually rather unpleasant for the subject or subjects involved. Direct calorimetry is seldom used and has been replaced by the indirect methods discussed in the follow- ing sections.

Indirect Calorimetry In addition to heat production, metabolic processes also consume oxygen in a quantifiable manner. Therefore, the heat released by metabolic oxidation can be calculated indirectly by measuring the consumption of oxygen. Indirect calorimetry is used most often to assess energy expenditure because the required instrumentation can be portable and, under most conditions, does not interfere with physical activities. The expiration of carbon dioxide is also measured so that the ratio of carbon dioxide produced relative to oxygen consumed (termed the respiratory quo- tient) can be determined. While carbohydrate and fat are the major fuels used in the body, it is recommended that urinary nitrogen excretion also be measured to account for the contribution of protein oxidation to energy expendi- ture. Oxygen consumption and carbon dioxide production are measured using either portable equipment (Figure 8.1) that can be placed on a person, enabling collection and analysis of gases while mobile, or stationary equipment often referred to as a metabolic cart (Figure 8.2). The rela- tive ease of indirect calorimetry makes it a widely used method in research settings when measured data is desired rather than calculated estimates based on body weight.

Figure 8.1 A portable device to measure oxygen consumption and carbon dioxide production. The headgear contains oxygen and carbon dioxide sensors on the left side and the flow sensor on the right side, all tethered to an electronics box (to the left of the headgear) that fits into a small wearable pack.Ph

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C H A P T E R 8 • ENERGY EXPENDITURE, BODY COMPOSITION, AND HEALTHY WEIGHT 275

Nonprotein RQ

Caloric Value of O2 (kcal/L)

Caloric Value of CO2 (kcal/L)

Source of Calories

Carbohydrate (%) Fat (%)

0.707 4.686 6.629 0 100

0.71 4.690 6.606 1.10 98.9

0.72 4.702 6.531 4.76 95.2

0.73 4.714 6.458 8.40 91.6

0.74 4.727 6.388 12.0 88.0

0.75 4.739 6.319 15.6 84.4

0.76 4.751 6.253 19.2 80.8

0.77 4.764 6.187 22.8 77.2

0.78 4.776 6.123 26.3 73.7

0.79 4.788 6.062 29.9 70.1

0.80 4.801 6.001 33.4 66.6

0.81 4.813 5.942 36.9 63.1

0.82 4.825 5.884 40.3 59.7

0.83 4.838 5.829 43.8 56.2

0.84 4.850 5.774 47.2 52.8

0.85 4.862 5.721 50.7 49.3

0.86 4.875 5.669 54.1 45.9

0.87 4.887 5.617 57.5 42.5

0.88 4.899 5.568 60.8 39.2

0.89 4.911 5.519 64.2 35.8

0.90 4.924 5.471 67.5 32.5

0.91 4.936 5.424 70.8 29.2

0.92 4.948 5.378 74.1 25.9

0.93 4.961 5.333 77.4 22.6

0.94 4.973 5.290 80.7 19.3

0.95 4.985 5.247 84.0 16.0

0.96 4.998 5.205 87.2 12.8

0.97 5.010 5.165 90.4 9.58

0.98 5.022 5.124 93.6 6.37

0.99 5.035 5.085 96.8 3.18

1.00 5.047 5.047 100 0

Source: Adapted from McArdle, W.D., Katch, F.I., Katch, V.L., Exercise Physiology, 2nd ed., p. 127 (Philadelphia: Lea & Febiger, 1986). Adapted by permission.

Table 8.1 Thermal Equivalent of O2 and CO2 for Nonprotein RQ

RQ and Substrate Oxidation An RQ equal to 1.0 suggests that carbohydrate is being oxidized because the amount of oxygen required for the combustion of glucose equals the amount of carbon dioxide produced, as shown here:

C6H12O6 + 6 O2 6 CO2 + 6 H2O RQ = 6 CO2/6 O2 = 1.0

The RQ for fat is 95th percentile is considered obese [16]. Body weight

Men Women

Broca formula (1871) Weight(kg) height(cm) 100 10%5 2 6 Weight(kg) = height(cm) Weight(kg) height(cm) 100 10%5 2 6 100 Weight(kg) height(cm) 100 10%5 2 6 15%

Hamwi formula (1964) 48.1 kg 1 2.7 kg/inch over 60 inches 45.4 kg 1 2.3 kg/inch over 60 inches

Devine formula (1974) 50.0 kg 1 2.3 kg/inch over 60 inches 45.5 kg 1 2.3 kg/inch over 60 inches

Miller formula (1983) 56.2 kg 1 1.41 kg/inch over 60 inches 53.1 kg 1 1.36 kg/inch over 60 inches

Robinson formula (1983) 51.7 kg 1 1.85 kg/inch over 60 inches 48.7 kg 1 1.65 kg/inch over 60 inches

Deitel-Greenstein formula* (2003) 61.3 kg 1 1.36 kg/inch over 63 inches 54.0 kg 1 1.36 kg/inch over 60 inches

Kammerer formula* (2015) 64.5 kg 1 1.36 kg/inch over 63 inches 1 0.45 kg/inch over 71 inches 54.0 kg 1 1.36 kg/inch over 60 inches

* IBW formulas were developed in bariatric surgical patients.

Table 8.4 Ideal Body Weight (IBW) Formulas

Figure 8.4 BMI values used to categorize weight. Source: U.S. Department of Agriculture and Human Services, Nutrition and Your Health: Dietary Guidelines for Americans. Washington, DC, 2000, p. 7.

50 75 100 125 150

Underweight Healthy

BMI (kg/m2)

18.5 25 30

Overweight Obese

175 200 225 250 275 4’10”

4’11”

5’0″

5’1″

5’2″

5’3″

5’4″

5’5″

5’6″

5’7″

5’8″

5’9″

5’10”

5’11”

6’0″

6’1″

6’2″

6’3″

6’4″

6’5″

6’6″

Pounds (without clothes)

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Key: BMI 40 in (102 cm) Women > 35 in (88 cm)

Underweight ( 18.5 kg/m )2, — — Normal (18.5–24.9 kg/m2) — — Overweight (25.0–29.9 kg/m2) Increased High

Obese, class I (30.0–34.9 kg/m2) High Very high

Obese, class II (35.0–39.9 kg/m2) Very high Very high

Obese, class III ( 40 kg/m )2$ Extremely high Extremely high

Source: National Institutes of Health. Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults. Publication No. 98-4083. Bethesda, MD: National Institutes of Health; National Health, Lung, and Blood Institute. 1998.

Table 8.5 Disease Risk* Relative to Normal BMI and Waist Circumference

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284 C H A P T E R 8 • ENERGY EXPENDITURE, BODY COMPOSITION, AND HEALTHY WEIGHT

Bioelectrical Impedance Bioelectrical impedance analysis is another commonly used field technique that assesses the two-compartment model. The method is based on the principle that the flow of electricity (conductivity) is facilitated in fat-free tissue high in water and electrolyte content, but is impeded by fat tissue low in water and electrolytes. Electrical conduc- tivity is measured by various techniques. For example, an instrument generates a painless current or multiple electri- cal current frequencies that are passed through the body by means of the electrodes. Other devices are available in which the subject stands barefoot on a scale or grasps a hand-held device that measures electrical conductivity (Figure 8.8). In each case, opposition to the electric cur- rent, called impedance, is detected and measured by the instrument. Impedance is the inverse of conductance. The lowest resistance value of a person is used to calcu- late conductance and predict lean body mass or fat-free mass. For example, muscle, organs, and blood, which have high water and electrolyte contents, are good conductors. Tissues containing little water and electrolytes (such as adipose tissue) are poor conductors and have a high resis- tance to the passage of electrical current. When multiple frequencies are used, the higher frequencies can estimate both intracellular and extracellular water because the higher-frequency current can penetrate cell membranes. At lower frequencies, the flow of the current is blocked, and the measured resistance indicates extracellular water.

Bioelectrical impedance is a safe, noninvasive, and rapid means to assess body composition. The equipment is relatively inexpensive, portable, and fairly easy to operate. Bioelectrical impedance readings are affected by hydration and electrolyte imbalances. Thus, the technique is more useful for healthy subjects. Several bioelectrical impedance analysis prediction equations have been developed for various populations. The use of multifrequency techniques provides results that are in good agreement with other body composition methods because this technique estimates both total body water and extracellular water.

Laboratory Methods Densitometry: Underwater Weighing The density of body fat is about 0.9 g/mL, whereas the den- sity of fat-free mass is about 1.1 g/mL. Percent body fat of an individual can therefore be calculated if whole-body density is known. The Greek mathematician Archimedes discovered that the volume of an object submerged in water is equal to the volume of water displaced by the object. The density of an object can then be calculated by dividing the object’s weight (wt) in air by its loss of weight in water. For example, for a person who weighs 47 kg in air and 2 kg underwater, 45 kg represents the loss of body weight and the weight of the water displaced. After an adjustment for the change in density of water at different temperatures is made, the vol- ume of the person can be calculated. Figure 8.9 illustrates an apparatus for weighing under water. Correction for residual air volume in the lungs (RLV) and gas in the gastrointestinal tract (GIGV) must be made.

Body density is calculated using the following formula:

Wt of body in air Body density =

( Wt of body in air − Wt of body underwater ) − RLV − GIGV Density of water

Residual lung volume is thought to be about 24% of vital lung capacity. The volume of gas in the gastrointestinal tract is estimated to range from 50 to 300 mL. This volume typically is neglected, or a value of 100 mL may be used in calculations. The density or the weight of water is known for a wide range of temperatures and must be obtained for the calculation. Once density of the human body is known, an estimation of body fat can be determined. At any known body density, estimating the percentage of body fat is possible using established equations.

Underwater weighing is considered a noninvasive and relatively precise method for assessment of percent body fat. The standard error of body fat measurements

Figure 8.7 Measuring triceps skinfold with a Lange caliper to estimate body fat.

Figure 8.8 Hand-held device used to measure bioelectrical impedance.

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C H A P T E R 8 • ENERGY EXPENDITURE, BODY COMPOSITION, AND HEALTHY WEIGHT 285

suit and wears a bathing cap (to displace pockets of air in the hair). The measurement takes only a few minutes to complete. The apparatus has an advantage in that it can measure the body composition in age groups that are not suitable for underwater weighing, such as older adults or the very young. A similar instrument called the PEA POD is designed for infants and small children. Once the density of the body is obtained, the calculation of percent body fat is the same as with underwater weighing using established equations.

Dual-Energy X-ray Absorptiometry Dual-energy X-ray absorptiometry (abbreviated DXA or DEXA) involves scanning subjects with X-rays at two dif- ferent energy levels, illustrated in Figure 8.11. The subject lies on a table while an X-ray source beneath the table and the detector above the table pass across the subject’s body. Attenuation of the beam of X-rays as it passes through the body is calculated by computer. Percentage of fat mass, bone- free fat-free mass, and bone mineral (total body or specific sites) can be calculated based on the restriction in the flux of the X-rays across the fat and the fat-free masses [19].

DEXA is considered to be the gold standard technique for diagnosing osteoporosis and osteopenia and is a commonly used method for body composition measurements. It is widely available and entails relatively low X-ray exposure: 1–10% that of a chest X-ray [19]. Limitations to the use of absorptiometry include the expense of the equipment and the exposure of subjects to radiation. In addition, trained personnel are required to run the instrument and analyze the scans. DEXA

using densitometry has been estimated at 2.7% for adults and about 4.5% for children and adolescents [18]. Measurements obtained by underwater weighing correlate well in broad populations with those obtained by other techniques. Limitations of underwater weighing include its relatively high equipment cost, the inability to measure gas volume in the gastrointestinal tract, its impracticality for large numbers of subjects, and the high level of cooperation and time required of subjects, who must be submerged and remain motionless for an extended time. Thus, the technique is not suitable for young children, older adults, or subjects in poor health.

Densitometry: Air Displacement Another way to determine the volume—and thus density—of the body is with air displacement plethysmog- raphy. In the commercially available apparatus shown in Figure 8.10 (BOD POD, Cosmed Inc.), the subject is seated in a sealed chamber of known volume, separated from a second chamber by a membrane. The instrument measures the change in pressure caused by the volume occupied by the person. The person is dressed in a tight-fitting bathing

Figure 8.9 Apparatus for