Marks’ Basic Medical Biochemistry: A Clinical Approach, 2nd Edition • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Chapter 1: Metabolic Fuels and Dietary Components Chapter 2: The Fed or Absorptive State Chapter 3: Fasting Chapter 4: Water, Acids, Bases, and Buffers Chapter 5: Structures of the Major Compounds of the Body Chapter 6: Amino Acids in Proteins Chapter 7: Structure–Function Relationships in Proteins Chapter 8: Enzymes as Catalysts Chapter 9: Regulation of Enzymes Chapter 10: Relationship Between Cell Biology and Biochemistry Chapter 11: Cell Signaling by Chemical Messengers Chapter 12: Structure of the Nucleic Acids Chapter 13: Synthesis of DNA Chapter 14: Transcription: Synthesis of RNA Chapter 15: Translation: Synthesis of Proteins Chapter 16: Regulation of Gene Expression Chapter 17: Use of Recombinant DNA Techniques in Medicine Chapter 18: The Molecular Biology of Cancer Chapter 19: Cellular Bioenergetics: ATP And O2 Chapter 20: Tricarboxylic Acid Cycle Chapter 21: Oxidative Phosphorylation and Mitochondrial Function Chapter 22: Generation of ATP from Glucose: Glycolysis Chapter 23: Oxidation of Fatty Acids and Ketone Bodies Chapter 24: Oxygen Toxicity and Free Radical Injury Chapter 25: Metabolism of Ethanol Chapter 26: Basic Concepts in the Regulation of Fuel Metabolism by Insulin, Glucagon, and Other Hormones Chapter 27: Digestion, Absorption, and Transport of Carbohydrates Chapter 28: Formation and Degradation of Glycogen Chapter 29: Pathways of Sugar Metabolism: Pentose Phosphate Pathway, Fructose, and Galactose Metabolism Chapter 30: Synthesis of Glycosides, Lactose, Glycoproteins and Glycolipids Chapter 31: Gluconeogenesis and Maintenance of Blood Glucose Levels Chapter 32: Digestion and Transport of Dietary Lipids Chapter 33: Synthesis of Fatty Acids, Triacylglycerols, and the Major Membrane Lipids Chapter 34: Cholesterol Absorption, Synthesis, Metabolism, and Fate Chapter 35: Metabolism of the Eicosanoids Chapter 36: Integration of Carbohydrate and Lipid Metabolism Chapter 37: Protein Digestion and Amino Acid Absorption Chapter 38: Fate of Amino Acid Nitrogen: Urea Cycle Chapter 39: Synthesis and Degradation of Amino Acids Chapter 40: Tetrahydrofolate, Vitamin B12, And S-Adenosylmethionine Chapter 41: Purine and Pyrimidine Metabolism Chapter 42: Intertissue Relationships in the Metabolism of Amino Acids Chapter 43: Actions of Hormones That Regulate Fuel Metabolism Chapter 44: The Biochemistry of the Erythrocyte and other Blood Cells Chapter 45: Blood Plasma Proteins, Coagulation and Fibrinolysis Chapter 46: Liver Metabolism Chapter 47: Metabolism of Muscle at Rest and During Exercise Chapter 48: Metabolism of the Nervous System Chapter 49: The Extracellular Matrix and Connective Tissue SECTION ONE Fuel Metabolism n order to survive, humans must meet two basic metabolic requirements: we must be able to synthesize everything our cells need that is not supplied by our diet, and we must be able to protect our internal environment from toxins and changing conditions in our external environment In order to meet these requirements, we metabolize our dietary components through four basic types of pathways: fuel oxidative pathways, fuel storage and mobilization pathways, biosynthetic pathways, and detoxification or waste disposal pathways Cooperation between tissues and responses to changes in our external environment are communicated though transport pathways and intercellular signaling pathways (Fig I.1) The foods in our diet are the fuels that supply us with energy in the form of calories This energy is used for carrying out diverse functions such as moving, thinking, and reproducing Thus, a number of our metabolic pathways are fuel oxidative pathways that convert fuels into energy that can be used for biosynthetic and mechanical work But what is the source of energy when we are not eating— between meals, and while we sleep? How does the hunger striker in the morning headlines survive so long? We have other metabolic pathways that are fuel storage pathways The fuels that we store can be molibized during periods when we are not eating or when we need increased energy for exercise Our diet also must contain the compounds we cannot synthesize, as well as all the basic building blocks for compounds we synthesize in our biosynthetic pathways For example we have dietary requirements for some amino acids, but we can synthesize other amino acids from our fuels and a dietary nitrogen precursor The compounds required in our diet for biosynthetic pathways include certain amino acids, vitamins, and essential fatty acids Detoxification pathways and waste disposal pathways are metabolic pathways devoted to removing toxins that can be present in our diets or in the air we breathe, introduced into our bodies as drugs, or generated internally from the metabolism of dietary components Dietary components that have no value to the body, and must be disposed of, are called xenobiotics In general, biosynthetic pathways (including fuel storage) are referred to as anabolic pathways, that is, pathways that synthesize larger molecules from smaller components The synthesis of proteins from amino acids is an example of an anabolic pathway Catabolic pathways are those pathways that break down larger molecules into smaller components Fuel oxidative pathways are examples of catabolic pathways In the human, the need for different cells to carry out different functions has resulted in cell and tissue specialization in metabolism For example, our adipose tissue is a specialized site for the storage of fat and contains the metabolic pathways that allow it to carry out this function However, adipose tissue is lacking many of the pathways that synthesize required compounds from dietary precursors To enable our cells to cooperate in meeting our metabolic needs during changing conditions of diet, sleep, activity, and health, we need transport pathways into the blood and between tissues and intercellular signaling pathways One means of communication is for hormones to carry signals to tissues about our dietary state For example, a message that we have just had a meal, carried by the hormone insulin, signals adipose tissue to store fat I Dietary components Fuels: Carbohydrate Fat Protein Vitamins Minerals H2O Xenobiotics Digestion absorption, transport Compounds in cells Fuel storage pathways Biosynthetic pathways Body components Fuel stores Detoxification and waste disposal pathways Waste products CO2 H2O O2 Fuel oxidative pathways Energy Fig I.1 General metabolic routes for dietary components in the body The types of Pathways are named in blue In the following section, we will provide an overview of various types of dietary components and examples of the pathways involved in utilizing these components We will describe the fuels in our diet, the compounds produced by their digestion, and the basic patterns of fuel metabolism in the tissues of our bodies We will describe how these patterns change when we eat, when we fast for a short time, and when we starve for prolonged periods Patients with medical problems that involve an inability to deal normally with fuels will be introduced These patients will appear repeatedly throughout the book and will be joined by other patients as we delve deeper into biochemistry Metabolic Fuels and Dietary Components Fuel Metabolism We obtain our fuel primarily from carbohydrates, fats, and proteins in our diet As we eat, our foodstuffs are digested and absorbed The products of digestion circulate in the blood, enter various tissues, and are eventually taken up by cells and oxidized to produce energy To completely convert our fuels to carbon dioxide (CO2) and water (H2O), molecular oxygen (O2) is required We breathe to obtain this oxygen and to eliminate the carbon dioxide (CO2) that is produced by the oxidation of our foodstuffs Fuel Stores Any dietary fuel that exceeds the body’s immediate energy needs is stored, mainly as triacylglycerol (fat) in adipose tissue, as glycogen (a carbohydrate) in muscle, liver, and other cells, and, to some extent, as protein in muscle When we are fasting, between meals and overnight while we sleep, fuel is drawn from these stores and is oxidized to provide energy (Fig 1.1) Fuel Requirements We require enough energy each day to drive the basic functions of our bodies and to support our physical activity If we not consume enough food each day to supply that much energy, the body’s fuel stores supply the remainder, and we lose weight Conversely, if we consume more food than required for the energy we expend, our body’s fuel stores enlarge, and we gain weight Other Dietary Requirements In addition to providing energy, the diet provides precursors for the biosynthesis of compounds necessary for cellular and tissue structure, function, and survival Among these precursors are the essential fatty acids and essential amino acids (those that the body needs but cannot synthesize) The diet must also supply vitamins, minerals, and water Waste Disposal Dietary components that we can utilize are referred to as nutrients However, both the diet and the air we breathe contain xenobiotic compounds, compounds that have no use or value in the human body and may be toxic These compounds are excreted in the urine and feces together with metabolic waste products Essential Nutrients Fuels Carbohydrates Fats Proteins Required Components Essential amino acids Essential fatty acids Vitamins Minerals Water Excess dietary fuel Fed Fuel stores: Fat Glycogen Protein Fasting Oxidation Energy Fig 1.1 Fate of excess dietary fuel in fed and fasting states THE WAITING ROOM Percy Veere is a 59-year-old school teacher who was in good health until his wife died suddenly Since that time, he has experienced an increasing degree of fatigue and has lost interest in many of the activities he previously enjoyed Shortly after his wife’s death, one of his married children moved far from home Since then, Mr Veere has had little appetite for food When a Percy Veere has a strong will He is enduring a severe reactive depression after the loss of his wife In addition, he must put up with the sometimes life-threatening antics of his hyperactive grandson, Dennis (the Menace) Veere Yet through all of this, he will “persevere.” SECTION ONE / FUEL METABOLISM neighbor found Mr Veere sleeping in his clothes, unkempt, and somewhat confused, she called an ambulance Mr Veere was admitted to the hospital psychiatry unit with a diagnosis of mental depression associated with dehydration and malnutrition Otto Shape is a 25-year-old medical student who was very athletic during high school and college, and is now “out-of-shape.” Since he started medical school, he has been gaining weight (at feet 10 inches tall, he currently weighs 187 lb) He has decided to consult a physician at the student health service before the problem gets worse Heat ATP CO2 Energy production Carbohydrate Lipid Protein O2 Energy utilization Biosynthesis Detoxification Muscle contraction Active ion transport Thermogenesis ADP + Pi Fig 1.2 The ATP–ADP cycle Oxidative pathways are catabolic; that is, they break molecules down In contrast, anabolic pathways build molecules up from component pieces Amino acids e– e– e– Acetyl CoA TCA cycle CO2 CO2 e– electron transport chain H2O Ann O’Rexia is a 23-year-old buyer for a woman’s clothing store Despite the fact that she is feet inches tall and weighs 99 lb, she is convinced she is overweight Two months ago, she started a daily exercise program that consists of hour of jogging every morning and hour of walking every evening She also decided to consult a physician about a weight reduction diet I Fatty acids Glucose ATP Ivan Applebod is a 56-year-old accountant who has been morbidly obese for a number of years He exhibits a pattern of central obesity, called an “apple shape,” which is caused by excess adipose tissue deposited in the abdominal area His major recreational activities are watching TV while drinking scotch and soda and doing occasional gardening At a company picnic, he became very “winded” while playing baseball and decided it was time for a general physical examination At the examination, he weighed 264 lb at feet 10 inches tall His blood pressure was slightly elevated, 155 mm Hg systolic (normal ϭ 140 mm Hg or less) and 95 mm Hg diastolic (normal ϭ 90 mm Hg or less) O2 Fig 1.3 Generation of ATP from fuel components during respiration Glucose, fatty acids, and amino acids are oxidized to acetyl CoA, a substrate for the TCA cycle In the TCA cycle, they are completely oxidized to CO2 As fuels are oxidized, electrons (eϪ) are transferred to O2 by the electron transport chain, and the energy is used to generate ATP DIETARY FUELS The major fuels we obtain from our diet are carbohydrates, proteins, and fats When these fuels are oxidized to CO2 and H2O in our cells, energy is released by the transfer of electrons to O2 The energy from this oxidation process generates heat and adenosine triphosphate (ATP) (Fig 1.2) Carbon dioxide travels in the blood to the lungs, where it is expired, and water is excreted in urine, sweat, and other secretions Although the heat that is generated by fuel oxidation is used to maintain body temperature, the main purpose of fuel oxidation is to generate ATP ATP provides the energy that drives most of the energy-consuming processes in the cell, including biosynthetic reactions, muscle contraction, and active transport across membranes As these processes use energy, ATP is converted back to adenosine diphosphate (ADP) and inorganic phosphate (Pi) The generation and utilization of ATP is referred to as the ATP–ADP cycle The oxidation of fuels to generate ATP is called respiration (Fig 1.3) Before oxidation, carbohydrates are converted principally to glucose, fat to fatty acids, and protein to amino acids The pathways for oxidizing glucose, fatty acids, and amino acids have many features in common They first oxidize the fuels to acetyl CoA, a precursor of the tricarboxylic acid (TCA) cycle The TCA cycle is a series of reactions that completes the oxidation of fuels to CO2 (see Chapter 19) Electrons lost from the fuels during oxidative reactions are transferred to O2 by a series of proteins in the electron transport chain (see Chapter 20) The energy of electron transfer is used to convert ADP and Pi to ATP by a process known as oxidative phosphorylation CHAPTER / METABOLIC FUELS AND DIETARY COMPONENTS In discussions of metabolism and nutrition, energy is often expressed in units of calories “Calorie” in this context really means kilocalorie (kcal) Energy is also expressed in joules One kilocalorie equals 4.18 kilojoules (kJ) Physicians tend to use units of calories, in part because that is what their patients use and understand A Carbohydrates The major carbohydrates in the human diet are starch, sucrose, lactose, fructose, and glucose The polysaccharide starch is the storage form of carbohydrates in plants Sucrose (table sugar) and lactose (milk sugar) are disaccharides, and fructose and glucose are monosaccharides Digestion converts the larger carbohydrates to monosaccharides, which can be absorbed into the bloodstream Glucose, a monosaccharide, is the predominant sugar in human blood (Fig 1.4) Oxidation of carbohydrates to CO2 and H2O in the body produces approximately kcal/g (Table 1.1) In other words, every gram of carbohydrate we eat yields approximately kcal of energy Note that carbohydrate molecules contain a significant amount of oxygen and are already partially oxidized before they enter our bodies (see Fig 1.4) B Proteins The food “calories” used in everyday speech are really “Calories,” which ϭ kilocalories “Calorie,” meaning kilocalorie, was originally spelled with a capital C, but the capitalization was dropped as the term became popular Thus, a 1-calorie soft drink actually has Cal (1 kcal) of energy Table 1.1 Caloric Content of Fuels kcal/g Carbohydrate Fat Protein Alcohol Proteins are composed of amino acids that are joined to form linear chains (Fig 1.5) In addition to carbon, hydrogen, and oxygen, proteins contain approximately 16% nitrogen by weight The digestive process breaks down proteins to their constituent amino acids, which enter the blood The complete oxidation of proteins to CO2, H2O, and NH4ϩ in the body yields approximately kcal/g C Fats Fats are lipids composed of triacylglycerols (also called triglycerides) A triacylglycerol molecule contains fatty acids esterified to one glycerol moiety (Fig 1.6) Fats contain much less oxygen than is contained in carbohydrates or proteins Therefore, fats are more reduced and yield more energy when oxidized The complete oxidation of triacylglycerols to CO2 and H2O in the body releases approximately kcal/g, more than twice the energy yield from an equivalent amount of carbohydrate or protein CH2 OH O CH2 OH O O OH OH HO CH2 OH O O HO CH2 OH O O OH CH2 O O OH HO or O OH HO Starch (Diet) An analysis of Ann O’Rexia’s diet showed she ate 100 g carbohydrate, 20 g protein, and 15 g fat each day Approximately how many calories did she consume per day? Glycogen (Body stores) HO CH2 OH O C H H H C C H OH HO OH C C H OH Glucose Fig 1.4 Structure of starch and glycogen Starch, our major dietary carbohydrate, and glycogen, the body’s storage form of glucose, have similar structures They are polysaccharides (many sugar units) composed of glucose, which is a monosaccharide (one sugar unit) Dietary disaccharides are composed of two sugar units SECTION ONE / FUEL METABOLISM Miss O’Rexia consumed 100 ϫ ϭ 400 kcal as carbohydrate 20 ϫ ϭ 80 kcal as protein 15 ϫ ϭ 135 kcal as fat NH R1 O CH C O NH CH C NH R3 O CH C R + H3N CH COO– R2 for a total of 615 kcal/day Protein Amino acid Fig 1.5 General structure of proteins and amino acids R ϭ side chain Different amino acids have different side chains For example, R1 might be –CH3; R2, ; R3, –CH2 –COOϪ O O CH3 (CH2)7 CH CH (CH2)7 C CH2 O O (CH2)14 CH3 O CH CH2 C O C (CH2)16 CH3 Triacylglycerol CH2 OH HO C H O CH3 CH2OH (CH2)14 C O– Palmitate Glycerol O CH3 (CH2)7 CH CH (CH2)7 C O– Oleate O CH3 (CH2)16 C O– Stearate Fig 1.6 Structure of a triacylglycerol Palmitate and stearate are saturated fatty acids, i.e., they have no double bonds Oleate is monounsaturated (one double bond) Polyunsaturated fatty acids have more than one double bond Ivan Applebod ate 585 g carbohydrate, 150 g protein, and 95 g fat each day In addition, he drank 45 g alcohol How many calories did he consume per day? D Alcohol Many people used to believe that alcohol (ethanol, in the context of the diet) has no caloric content In fact, ethanol (CH3CH2OH) is oxidized to CO2 and H2O in the body and yields approximately kcal/g—that is, more than carbohydrate but less than fat II BODY FUEL STORES It is not surprising that our body fuel stores consist of the same kinds of compounds found in our diet, because the plants and animals we eat also store fuels in the form of starch or glycogen, triacylglycerols, and proteins Although some of us may try, it is virtually impossible to eat constantly Fortunately, we carry supplies of fuel within our bodies (Fig 1.7) These fuel stores are light in weight, large in quantity, and readily converted into oxidizable substances Most of us are familiar with fat, our major fuel store, which is located in adipose tissue Although fat is distributed throughout our bodies, it tends to increase in quantity in our hips and thighs and in our abdomens as we advance into middle age In addition to our fat stores, we also have important, although much smaller, stores of carbohydrate in the form of glycogen located primarily in our liver and muscles Glycogen CHAPTER / METABOLIC FUELS AND DIETARY COMPONENTS Muscle glycogen 0.15 kg (0.4%) Liver glycogen 0.08 kg (0.2%) Mr Applebod consumed 585 ϫ ϭ 2,340 kcal as carbohydrate 150 ϫ ϭ 600 kcal as protein 95 ϫ ϭ 855 kcal as fat 45 ϫ ϭ 315 kcal as alcohol for a total of 4,110 kcal/day Fat 15 kg (85%) Protein kg (14.5%) Fig 1.7 Fuel composition of the average 70-kg man after an overnight fast (in kilograms and as percentage of total stored calories) consists of glucose residues joined together to form a large, branched polysaccharide (see Fig 1.4) Body protein, particularly the protein of our large muscle masses, also serves to some extent as a fuel store, and we draw on it for energy when we fast A Fat Our major fuel store is adipose triacylglycerol (triglyceride), a lipid more commonly known as fat The average 70-kg man has approximately 15 kg stored triacylglycerol, which accounts for approximately 85% of his total stored calories (see Fig 1.7) Two characteristics make adipose triacylglycerol a very efficient fuel store: the fact that triacylglycerol contains more calories per gram than carbohydrate or protein (9 kcal/g versus kcal/g) and the fact that adipose tissue does not contain much water Adipose tissue contains only about 15% water, compared to tissues such as muscle that contain about 80% Thus, the 70-kg man with 15 kg stored triacylglycerol has only about 18 kg adipose tissue B Glycogen Our stores of glycogen in liver, muscle, and other cells are relatively small in quantity but are nevertheless important Liver glycogen is used to maintain blood glucose levels between meals Thus, the size of this glycogen store fluctuates during the day; an average 70-kg man might have 200 g or more of liver glycogen after a meal but only 80 g after an overnight fast Muscle glycogen supplies energy for muscle contraction during exercise At rest, the 70-kg man has approximately 150 g of muscle glycogen Almost all cells, including neurons, maintain a small emergency supply of glucose as glycogen C Protein In biochemistry and nutrition, the standard reference is often the 70-kg (154-lb) man This standard probably was chosen because in the first half of the 20th century, when many nutritional studies were performed, young healthy medical and graduate students (who were mostly men) volunteered to serve as subjects for these experiments What would happen to a 70-kg man if the 135,000 kcal stored as triacylglycerols in his 18 kg of adipose tissue were stored instead as skeletal muscle glycogen? It would take approximately 34 kg glycogen to store as many calories Glycogen, because it is a polar molecule with –OH groups, binds approximately times its weight in water, or 136 kg Thus, his fuel stores would weigh 170 kg Protein serves many important roles in the body; unlike fat and glycogen, it is not solely a fuel store Muscle protein is essential for body movement Other proteins serve as enzymes (catalysts of biochemical reactions) or as structural components of cells and tissues Only a limited amount of body protein can be degraded, approximately kg in the average 70-kg man, before our body functions are compromised III DAILY ENERGY EXPENDITURE If we want to stay in energy balance, neither gaining nor losing weight, we must, on average, consume an amount of food equal to our daily energy expenditure The daily energy expenditure (DEE) includes the energy to support our basal metabolism (basal metabolic rate or resting metabolic rate) and our physical activity, plus the energy required to process the food we eat (diet-induced thermogenesis) Daily energy expenditure ϭ RMR ϩ Physical Activity ϩ DIT where RMR is the resting metabolic rate and DIT is diet-induced thermogenesis BMR (basal metabolic rate) is used interchangeably with RMR in this equation SECTION ONE / FUEL METABOLISM A Resting Metabolic Rate Table 1.2 Factors Affecting BMR Expressed per kg Body Weight Gender (males higher than females) Body temperature (increased with fever) Environmental temperature (increased in cold) Thyroid status (increased in hyperthyroidism) Pregnancy and lactation (increased) Age (decreases with age) What are Ivan Applebod’s and Ann O’Rexia’s RMR? (Compare the method for a rough estimate to values obtained with equations in Table 1.3.) Registered dieticians use extensive tables for calculating energy requirements, based on height, weight, age, and activity level A more accurate calculation is based on the fat-free mass (FFM), which is equal to the total body mass minus the mass of the person’s adipose tissue With FFM, the BMR is calculated using the equation BMR ϭ 186 ϩ FFM ϫ 23.6 kcal/ kg per day This formula eliminates differences between sexes and between aged versus young individuals that are attributable to differences in relative adiposity However, determining FFM is relatively cumbersome— it requires weighing the patient underwater and measuring the residual lung volume Indirect calorimetry, a technique that measures O2 consumption and CO2 production, can be used when more accurate determinations are required for hospitalized patients A portable indirect calorimeter is used to measure oxygen consumption and the respiratory quotient (RQ), which is the ratio of O2 consumed to CO2 produced The RQ is 1.00 for individuals oxidizing carbohydrates, 0.83 for protein, and 0.71 for fat From these values, the daily energy expenditure (DEE) can be determined The resting metabolic rate (RMR) is a measure of the energy required to maintain life: the functioning of the lungs, kidneys and brain, the pumping of the heart, the maintenance of ionic gradients across membranes, the reactions of biochemical pathways, and so forth Another term used to describe basal metabolism is the basal metabolic rate (BMR) The BMR was originally defined as the energy expenditure of a person mentally and bodily at rest in a thermoneutral environment 12 to18 hours after a meal However, when a person is awakened and their heat production or oxygen consumption is measured, they are no longer sleeping or totally at mental rest, and their metabolic rate is called the resting metabolic rate (RMR) It is also sometimes called the resting energy expenditure (REE) The RMR and BMR differ very little in value The BMR, which is usually expressed in kcal/day, is affected by body size, age, sex, and other factors (Table 1.2) It is proportional to the amount of metabolically active tissue (including the major organs) and to the lean (or fat-free) body mass Obviously, the amount of energy required for basal functions in a large person is greater than the amount required in a small person However, the BMR is usually lower for women than for men of the same weight because women usually have more metabolically inactive adipose tissue Body temperature also affects the BMR, which increases by 12% with each degree centigrade increase in body temperature (i.e., “feed a fever; starve a cold”) The ambient temperature affects the BMR, which increases slightly in colder climates as thermogenesis is activated Excessive secretion of thyroid hormone (hyperthyroidism) causes the BMR to increase, whereas diminished secretion (hypothyroidism) causes it to decrease The BMR increases during pregnancy and lactation Growing children have a higher BMR per kilogram body weight than adults, because a greater proportion of their bodies is composed of brain, muscle, and other more metabolically active tissues The BMR declines in aging individuals because their metabolically active tissue is shrinking and body fat is increasing In addition, large variations exist in BMR from one adult to another, determined by genetic factors A rough estimate of the BMR may be obtained by assuming it is 24 kcal/day/kg body weight and multiplying by the body weight An easy way to remember this is kcal/kg/hr This estimate works best for young individuals who are near their ideal weight More accurate methods for calculating the BMR use empirically derived equations for different gender and age groups (Table 1.3) Even these calculations not take into account variation among individuals B Physical Activity In addition to the RMR, the energy required for physical activity contributes to the DEE The difference in physical activity between a student and a lumberjack is enormous, and a student who is relatively sedentary during the week may be much Table 1.3 Equation for Predicting BMR from Body Weight (W) in kg Males Age Range (years) 0–3 3–10 10–18 18–30 30–60 >60 Females BMR kcal/day 60.9W 22.7W 17.5W 15.3W 11.6W 13.5W Ϫ ϩ ϩ ϩ ϩ ϩ 54 495 651 679 879 487 Age Range (years) 0–3 3–10 10–18 18–30 30–60 Ͼ60 BMR kcal/day 61.0W 22.5W 12.2W 14.7W 8.7W 10.5W Ϫ ϩ ϩ ϩ ϩ ϩ 51 499 746 496 829 596 From Energy and protein requirements: report of a Joint FAO/WHO/UNU Expert Consultation Technical report series no 724 Geneva World Health Organization, 1987:71 See also Schofield et al Hum Nutr Clin Nutr 1985;39 (suppl) 906 SECTION EIGHT / TISSUE METABOLISM Cell movement within the extracellular matrix requires remodeling of the various components of the matrix This is accomplished by a variety of matrix metalloproteinases (MMPs) and regulators of the MMPs, tissue inhibitors of matrix metalloproteinases (TIMPs) Dysregulation of this delicate balance of the regulators of cell movement allows cancer cells to travel to other parts of the body (metastasize) as well as to spread locally to contiguous tissues THE WAITING ROOM Sis Lupus (first introduced in Chapter 14) noted a moderate reduction in pain and swelling in the joints of her fingers when she was taking a 6-week course of high-dose prednisone, an anti-inflammatory steroid As the dose of this drug was tapered to minimize its long-term side effects, however, the pain in the joints of her fingers returned, and, for the first time, her left knee became painful, swollen, and warm to the touch Her rheumatologist described to her the underlying inflammatory tissue changes that her systemic lupus erythematosus (SLE) was causing in the joint tissues Ann Sulin complained of a declining appetite for food as well as severe weakness and fatigue The reduction in her kidneys’ ability to maintain normal daily total urinary net acid excretion contributed to her worsening metabolic acidosis This plus her declining ability to excrete nitrogenous waste products, such as creatinine and urea, into her urine (“azotemia”) are responsible for many of her symptoms Her serum creatinine level was rising steadily As it approached a level of mg/dL, she developed a litany of complaints caused by the multisystem dysfunction associated with her worsening metabolic acidosis, retention of nitrogenous waste products, and so forth (“uremia”) Her physicians discussed with Ann the need to consider peritoneal dialysis or hemodialysis I COMPOSITION OF THE EXTRACELLULAR MATRIX A Fibrous Proteins Fig 49.2 The triple helix of collagen COLLAGEN Collagen, a family of fibrous proteins, is produced by a variety of cell types but principally by fibroblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells Type I collagen [collagen(I)], the most abundant protein in mammals, is a fibrous protein that is the major component of connective tissue It is found in the extracellular matrix (ECM) of loose connective tissue, bone, tendons, skin, blood vessels, and the cornea of the eye Collagen(I) contains approximately 33% glycine and 21% proline and hydroxyproline Hydroxyproline is an amino acid produced by posttranslational modification of peptidyl proline residues (see Chapter 7, section V.C., for an earlier introduction to collagen) Procollagen(I), the precursor of collagen(I), is a triple helix composed of three polypeptide (pro-␣) chains that are twisted around each other, forming a rope-like structure Polymerization of collagen(I) molecules forms collagen fibrils, which provide great tensile strength to connective tissues (Fig 49.2) The individual polypeptide chains each contain approximately 1,000 amino acid residues The three polypeptide chains of the triple helix are linked by interchain hydrogen bonds CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Each turn of the triple helix contains three amino acid residues, such that every third amino acid is in close contact with the other two strands in the center of the structure Only glycine, which lacks a side chain, can fit in this position, and indeed, every third amino acid residue of collagen is glycine Thus, collagen is a polymer of (Gly-X-Y) repeats, where Y is frequently proline or hydroxyproline, and X is any other amino acid found in collagen Procollagen(I) is an example of a protein that undergoes extensive posttranslational modifications Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues These reactions occur after the protein has been synthesized (Fig 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes, for example, prolyl hydroxylases and lysyl hydroxylase Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose) The side chains of lysine residues also may be oxidized to form the aldehyde, allysine These aldehyde residues produce covalent cross-links between collagen molecules (Fig 49.4) An allysine residue on one collagen molecule reacts with the amino group of a lysine residue on another molecule, forming a covalent Schiff base that is converted to more stable covalent cross-links Aldol condensation also may occur between two allysine residues, which forms the structure lysinonorleucine 907 The role of carbohydrates in collagen structure is still controversial The hydroxyproline residues in collagen are required for stabilization of the triple helix by hydrogen bond formation In the absence of vitamin C (scurvy), the melting temperature of collagen can drop from 42oC to 24oC, because of the loss of interstrand hydrogen bond formation from the lack of hydroxyproline residues i Types of Collagen At least 19 different types of collagen have been characterized (Table 49.1) Although each type of collagen is found only in particular locations in the body, more than one type may be present in the ECM at a given location The various types of collagen can be classified as fibril-forming (types I, II, III, V, and XI), network-forming (types IV, VIII and X), those that associate with fibril surfaces (types IX, XII, and XIV), those that are transmembrane proteins (types XIII and XVII), endostatin-forming (types XV and XVIII), and those that form periodic beaded filaments (type VI) O N CH H 2C O C N + α -Ketoglutarate CH2 prolyl hydroxylase H H2C Ascorbate C O2 H CH CH2 CO2 H OH - Hydroxyproline residue O H CH CH2 CH2 CH2 O C + α -Ketoglutarate lysyl hydroxylase Ascorbate O2 CO2 N CH H CH2 C + Succinate CH2 CH CH2 CH2 + NH + NH Lysine residue + Succinate C Proline residue N C OH - Hydroxylysine residue Fig 49.3 Hydroxylation of proline and lysine residues in collagen Proline and lysine residues within the collagen chains are hydroxylated by reactions that require vitamin C Protein 908 SECTION EIGHT / TISSUE METABOLISM δ ε Table 49.1 Types of Collagen + CH2 CH2 NH3 Collagen Type Lysine residue A Gene Structural Details Localization I II III Col1A1-Col1A2 Col2A1 Col3A1 Fibrils Fibrils Fibrils IV Col4A1–Col4A6 Nonfibrillar, mesh collagen V Col5A1-Col5A3 Small fibers, N-terminal globular domains VI Col6A1-Col6A3 VII Col7A1 Microfibrils, with both N and C-terminal globular domains An anchoring collagen VIII Col8A1-Col8A2 Nonfibrillar, mesh collagen IX Col9A1-Col9A3 X Col10A1 XI XII Col11A1-Col11A3 Col12A1 Fibril-associated collagens with interrupted triple helices (FACIT); N-terminal globular domain Nonfibrillar, mesh collagen, with C-terminal globular domain Small fibers FACIT Skin, tendon, bone, cornea Cartilage, vitreous humour Skin, muscle, associates with type I collagen All basal laminae (basement membranes) Associates with type I collagen in most interstitial tissues Associates with type I collagen in most interstitial tissues Epithelial cells; dermal– epidermal junction Cornea, some endothelial cells Associates with type II collagen in cartilage and vitreous humour XIII XIV XV XVI XVII XVIII XIX Col13A1 Col14A1 Col15A1 Col16A1 Col17A1 Col18A1 Col19A1 Transmembrane collagen FACIT Endostatin-forming collagen Other Transmembrane collagen Endostatin-forming Other O2 lysyl oxidase NH3 + OH – ε O C H δ CH2 + ε H2N δ CH2 CH2 Second lysine residue Allysine residue B H2O δ CH2 ε CH ε N CH2 δ CH2 Schiff base δ CH2 ε O C H HO ε + C H Allysine (aldo form) C δ CH Allysine (enol form) Aldol condensation δ CH2 δ Cartilage, vitreous humor Interacts with types I and II collagen in soft tissues Cell surfaces, epithelial cells Soft tissue Endothelial cells Ubiquitous Epidermal cell surface Endothelial cells Ubiquitous See the text for descriptions of the differences in types of collagen HO ε H ε O C CH δ CH H2O CH2 Growth plate, hypertrophic and mineralizing cartilage ε H ε O C CH C δ Lysinonorleucine Fig 49.4 Formation of cross-links in collagen A Lysine residues are oxidized to allysine (an aldehyde) Allysine may react with an unmodified lysine residue to form a Schiff base (B), or two allysine residues may undergo an aldol condensation (C) All collagens contain three polypeptide chains with at least one stretch of triple helix The non–triple helical domains can be short (such as in the fibril-forming collagens) or can be rather large, such that the triple helix is actually a minor component of the overall structure (examples are collagen types XII and XIV) The FACIT (fibril-associated collagens with interrupted triple helices, collagen types IX, XII, and XIV) collagen types associate with fibrillar collagens, without themselves forming fibers The endostatin-forming collagens are cleaved at their C-terminus to form endostatin, an inhibitor of angiogenesis The network-forming collagens (type IV) form a mesh-like structure, because of large (approximately 230 amino acids) noncollagenous domains at the carboxy-terminal (Fig 49.5) And finally, a number of collagen types are actually transmembrane proteins (XIII and XVII) found on epithelial or epidermal cell surfaces, which play a role in a number of cellular processes, including adhesion of components of the ECM to cells embedded within it Types I, II, and III collagens form fibrils that assemble into large insoluble fibers The fibrils (see below) are strengthened through covalent cross-links between lysine residues on adjacent fibrils The arrangement of the fibrils gives individual tissues their distinct characteristics Tendons, which attach muscles to bones, contain collagen Endostatins block angiogenesis (new blood vessel formation) by inhibiting endothelial cell migration Because endothelial cell migration and proliferation are required to form new blood vessels, inhibiting this action blocks angiogenesis Tumor growth is dependent on a blood supply; inhibiting angiogenesis can reduce tumor cell proliferation CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE 909 A Protomer Carboxy terminal Amino terminal B Dimer Carboxy terminal hexamer (NC1 domain) C Type IV collagen tetramer Aggregation at amino termini (7S domain) D Suprastructure NC1 hexamer 7S domain Fig 49.5 Type IV collagen contains a globular carboxy-terminal domain (A), which forms tropocollagen dimers (hexamers of collagen, B) Four dimers associate at the amino-terminal domains to form a 7S domain (C), and the tetramers form a lattice (D), which provides structural support to the basal lamina fibrils aligned parallel to the long axis of the tendon, thus giving the tendon tremendous tensile strength The types of collagen that not form fibrils perform a series of distinct roles Fibril-associated collagens bind to the surface of collagen fibrils and link them to other matrix- forming components The transmembrane collagens form anchoring fibrils that link components of the extracellular matrix to underlying connective tissue The network- forming collagens (type IV) form a flexible collagen that is part of the basement membrane and basal lamina that surround many cells ii Synthesis and Secretion of Collagen Collagen is synthesized within the endoplasmic reticulum as a precursor known as preprocollagen The presequence acts as the signal sequence for the protein and is cleaved, forming procollagen within the endoplasmic reticulum From there it is transported to the Golgi apparatus (Table 49.2) Three procollagen molecules associate through formation of intrastrand disulfide bonds at the carboxy-terminus; once One type of osteogenesis imperfecta (OI) is caused by a mutation in a gene that codes for collagen The phenotype of affected individuals varies greatly, depending on the location and type of mutation See the Biochemical Comments for more information concerning this type of OI 910 SECTION EIGHT / TISSUE METABOLISM Table 49.2 Steps Involved in Collagen Biosynthesis Location Rough endoplasmic reticulum Lumen of the ER Lumen of ER and Golgi apparatus Secretory vesicle Extracellular Process Synthesis of preprocollagen; insertion of the procollagen molecule into the lumen of the ER Hyroxylation of proline and lysine residues; glycosylation of selected hydroxylysine residues Self-assembly of the tropocollagen molecule, initiated by disulfide bond formation in the carboxy-terminal extensions; triple helix formation Procollagen prepared for secretion from cell Cleavage of the propeptides, removing the amino- and carboxy-terminal extensions, and self-assembly of the collagen molecules into fibrils, and then fibers these disulfides are formed, the three molecules can align properly to initiate formation of the triple helix The triple helix forms from the carboxy-end toward the amino-end, forming tropocollagen The tropocollagen contains a triple helical segment between two globular ends, the amino- and carboxy-terminal extensions The tropocollagen is secreted from the cell, the extensions are removed using extracellular proteases, and the mature collagen takes its place within the ECM The individual fibrils of collagen line up in a highly ordered fashion to form the collagen fiber ELASTIN Elastin is the major protein found in elastic fibers, which are located in the ECM of connective tissue of smooth muscle cells, endothelial and microvascular cells, chondrocytes, and fibroblasts Elastic fibers allow tissues to expand and contract; this is of particular importance to blood vessels, which must deform and reform repeatedly in response to the changes in intravascular pressure that occur with the contraction of the left ventricle of the heart It is also important for the lungs, which stretch each time a breath is inhaled and return to their original shape with each exhalation In addition to elastin, the elastic fibers contain microfibrils, which are composed of a number of acidic glycoproteins, the major ones being fibrillin-1 and fibrillin-2 i Tropoelastin Elastin has a highly cross-linked, insoluble, amorphous structure Its precursor, tropoelastin, is a molecule of high solubility, which is synthesized on the rough endoplasmic reticulum (RER) for eventual secretion Tropoelastin contains two types of alternating domains The first domain consists of a hydrophilic sequence rich in lysine and alanine residues The second domain consists of a hydrophobic sequence rich in valine, proline, and glycine, which frequently occur in repeats of VPGVG or VGGVG The protein contains approximately 16 regions of each domain, alternating throughout the protein (Fig 49.6) Hydrophilic C-terminal Signal peptide Hydrophilic cross-linking exons Hydrophilic exon 26a (for alternative splicing) 3'-untranslated region Hydrophobic exons Fig 49.6 The cDNA structure of elastin, indicating the repeating cross-linking and hydrophobic domains CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE On secretion from the cell, the tropoelastin is aligned with the microfibrils, and lysyl oxidase initiates the reactions that cross-link elastin molecules, using lysine residues within the hydrophilic alternating domains in the proteins This cross-linking reaction is the same as that which occurs in collagen In this reaction, 2, 3, or lysine residues are cross- linked to form a stable structure The net result of the cross-linking is the generation of a fibrous mesh that encircles the cells ii Elastic Properties of Elastin Elastic fibers have the ability to stretch and then to reform without requiring an obvious energy source to so The mechanism by which this stretching and relaxing actively occurs is still controversial but does relate to the basic principles of protein folding described in Chapter When the elastic fibers are stretched (such as when a breath is taken in and the lung fills up with air), the amorphous elastin structure is stretched This stretching exposes the repeating hydrophobic regions of the molecule to the aqueous environment This, in turn, leads to a decrease in the entropy of water, because the water molecules need to rearrange to form cages about each hydrophobic domain When this stretching force within the lung is removed (e.g., when the subject exhales), the elastin takes on its original structure because of the increase in entropy that occurs because the water no longer needs to form cages about hydrophobic domains Thus, the hydrophobic effect is the primary force that allows this stretched structure to reform Elastin is inherently stable, with a half-life of up to 70 years LAMININ After type IV collagen, laminin is the most abundant protein in basal laminae Laminin provides additional structural support for the tissues through its ability to bind to type IV collagen, to other molecules present in the ECM, and to cell surface–associated proteins (the integrins, see section D) 911 Supravalvular aortic stenosis (SVAS) results from an insufficiency of elastin in the vessel wall, leading to a narrowing of the large elastic arteries Current theory suggests that the levels of elastin in the vessel walls may regulate the number of smooth muscle cell rings that encircle the vessel If the levels of elastin are low, smooth muscle hypertrophy results, leading to a narrowing and stenosis of the artery α-chain HNH Gobular domains H HN β-chain H NH γ-chain Disulfide bonds HOOC Coiled-coil; rigid rods COOH i Laminin Structure Laminin is a heterotrimeric protein shaped, for the most part, like a cross (Fig 49.7) The trimer is composed of ␣, ␤, and ␥ subunits There are five possible ␣ proteins (designated ␣1–␣5), three different versions of the ␤ subunit (␤1–␤3), and three different ␥ forms (␥1 – ␥3) Thus, there is a potential for the formation of as many as 45 different combinations of these three subunits However, only 12 have been discovered (designated laminins 1–12) Laminin 1, composed of ␣1␤1␥1, is typical of this class of proteins The major feature of the laminin structure is a coiled ␣-helix, which joins the three subunits together and forms a rigid rod All three chains have extensions at the amino-terminal end Only the ␣ chain has a significant carboxy-terminal extension past the rod-like structure It is the laminin extensions that allow laminin to bind to other components within the ECM and to provide stability for the structure Components of the ECM that are bound by laminin include collagen, sulfated lipids, and proteoglycans ii Laminin Biosynthesis Like other secreted proteins, laminin is synthesized with a leader sequence targeting the three chains to the endoplasmic reticulum Chain association occurs within the Golgi apparatus before secretion from the cell After laminin is secreted by the cell, the amino terminal extensions promote self-association, as well as the binding to other ECM components Disulfide linkages are formed to stabilize the trimer, but there is much less posttranslational processing of laminin than there is of collagen and elastin COOH Fig 49.7 The structure of laminin Defects in the structures of laminin or laminin (proteins that contribute to the cohesion of the dermis and epidermis) lead to the disorder referred to as junctional epidermolysis bullosa (JEB) In this disorder, there can be severe spontaneous blistering of the skin and mucous membranes A severe form of the disease, JEB gravis, is often fatal early in life Death occurs as a result of epithelial blistering of the respiratory, digestive, and genitourinary systems Congenital muscular dystrophy (CMD) results from a defect in laminin 2, which is a component of the bridge that links the muscle cell cytoskeleton to the extracellular matrix Lack of this bridge triggers muscle cell apoptosis, which results in weakened muscles 912 SECTION EIGHT / TISSUE METABOLISM The ECM is not simply a glue that holds cells together; it also serves to keep cells from moving to other locations and to prevent large molecules and other particles, such as microorganisms, from reaching contiguous and distant cells This confining property of the matrix is medically important For example, infections spread, in part, because the infectious agent alters the “containing” capacity of the ECM Cancer cells that metastasize (migrate to other tissues) can so only by altering the integrity of the matrix Diseases such as rheumatoid arthritis (an autoimmune destruction of articular and periarticular tissues) and osteoarthritis (degenerative joint disease often associated with aging) involve damage to the functional capacity of the matrix Alterations in the structural characteristics of the matrix of the renal glomerulus may allow proteins to be excreted into the urine, an indication of inexorable decline in renal function Genetic defects may cause components of the matrix to be structurally and functionally abnormal, resulting in connective tissue disorders such as the EhlersDanlos syndrome (caused by a number of mutations that affect specific collagen genes) and Marfan’s syndrome (a defect in the protein, fibrillin, in which over 330 different mutations, many of which give rise to different phenotypes, have been identified) Deficiencies of lysosomal enzymes involved in normal degradation of molecules of the matrix result in diseases such as the mucopolysaccharidoses The principal components of the matrix of cartilage are collagen and proteoglycans, both of which are produced and degraded by the chondrocytes that are embedded in this matrix An autoimmune attack on articular proteins alters the balance between cartilage degradation and formation The resulting loss of cartilage organization accompanied by an inflammatory response is responsible for the symptoms experienced by Sis Lupus The collagen component forms a network of fine fibrils that give shape to the cartilage The proteoglycans embedded in the cartilage are responsible for its compressibility and its deformability B Proteoglycans The fibrous structural proteins of the ECM are embedded in gels formed from proteoglycans Proteoglycans consist of polysaccharides called glycosaminoglycans (GAG) linked to a core protein The GAGs are composed of repeating units of disaccharides One sugar of the disaccharide is either N-acetylglucosamine or Nacetylgalactosamine, and the second is usually acidic (either glucuronic acid or iduronic acid) These sugars are modified by the addition of sulfate groups to the parent sugar A proteoglycan may contain more than 100 GAG chains and consist of up to 95% oligosaccharide by weight The negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules, thereby creating a hydrated gel The gel provides a flexible mechanical support to the ECM The gel also acts as a filter that allows the diffusion of ions (e.g., Ca2ϩ ), H2O, and other small molecules, but slows diffusion of proteins and movement of cells Hyaluronan is the only GAG that occurs as a single long polysaccharide chain and is the only GAG that is not sulfated STRUCTURE AND FUNCTION OF THE PROTEOGLYCANS Proteoglycans are found in interstitial connective tissues, for example, the synovial fluid of joints, the vitreous humor of the eye, arterial walls, bone, cartilage, and cornea They are major components of the ECM in these tissues The proteoglycans interact with a variety of proteins in the matrix, such as collagen and elastin, fibronectin (which is involved in cell adhesion and migration), and laminin Proteoglycans are proteins that contain many chains of GAGs (formerly called mucopolysaccharides) Glycosaminoglycans are long, unbranched polysaccharides composed of repeating disaccharide units (Fig 49.8) The repeating disaccharides usually contain an iduronic or uronic acid and a hexosamine and are frequently sulfated Consequently, they carry a negative charge, are hydrated, and act as lubricants After synthesis, proteoglycans are secreted from cells; thus, they function extracellularly Because the long, negatively charged glycosaminoglycan chains repel each other, the proteoglycans occupy a very large space and act as “molecular sieves,” determining which substances enter or leave cells (Table 49.3) Their properties also give resilience and a degree of flexibility to substances such as cartilage, permitting compression and reexpansion of the molecule to occur At least seven types of glycosaminoglycans exist, which differ in the monosaccharides present in their repeating disaccharide units—chondroitin sulfate, dermatan sulfate, heparin, heparin sulfate, hyaluronic acid, and keratan sulfates I and II Except for hyaluronic acid, the glycosaminoglycans are linked to proteins, usually attached covalently to serine or threonine residues (Fig 49.9) Keratan sulfate I is attached to asparagine SYNTHESIS OF THE PROTEOGLYCANS The protein component of the proteoglycans is synthesized on the ER It enters the lumen of this organelle, where the initial glycosylations occur UDP-sugars serve as The long polysaccharide side chains of the proteoglycans in cartilage contain many anionic groups This high concentration of negative charges attracts cations that create a high osmotic pressure within cartilage, drawing water into this specialized connective tissue and placing the collagen network under tension At equilibrium, the resulting tension balances the swelling pressure caused by the proteoglycans The complementary roles of this macromolecular organization give cartilage its resilience Cartilage can thus withstand the compressive load of weight bearing and then reexpand to its previous dimensions when that load is relieved 913 CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Table 49.3 Some Specific Functions of the Glycosaminoglycans and Proteoglycans Glycosaminoglycan Hyaluronic acid Chondroitin sulfate proteoglycans Keratan sulfate proteoglycans Dermatan sulfate proteoglycans Heparin Heparan sulfate (syndecan) Hyaluronate – COO Function CH2OH O H H O Cell migration in: Embryogenesis Morphogenesis Wound healing Formation of bone, cartilage, cornea Transparency of cornea Transparency of cornea Binds LDL to plasma walls Anticoagulant (binds antithrombin III) Causes release of lipoprotein lipase from capillary walls Component of skin fibroblasts and aortic wall; commonly found on cell surfaces H H OH H H O H OH Glucuronic acid H – COO H H CH2OSO3 O HO H O OH H H H OH Glucuronic acid H Synovial lining Cartilage Calcified cartilage Synovial cavity Capsule NHCOCH3 N – Acetylgalactosamine – H O H COO – O CH2OSO3 O H H H O OH H H OH H H OSO3– H NHSO3– Glucuronic acid α (1 4) Glucosamine Keratan sulfate CH2OH O HO H H – CH2OSO3 O H O O H H OH H H OH H NHCOCH3 Galactose β (1 4) N – Acetylglucosamine Dermatan sulfate – H Bone H H H β (1 3) O Heparin H The functional properties of a normal joint depend, in part, on the presence of a soft, well-lubricated, deformable, and compressible layer of cartilaginous tissue covering the ends of the long bones that constitute the joint In Sis Lupus’ case, the pathologic process that characterizes SLE disrupted the structural and functional integrity of her articular (joint) cartilage NHCOCH3 N – Acetylglucosamine Chondroitin 6– sulfate – O the precursors that add sugar units, one at a time, first to the protein and then to the nonreducing end of the growing carbohydrate chain (Fig 49.10) Glycosylation occurs initially in the lumen of the ER and subsequently in the Golgi complex Glycosyltransferases, the enzymes that add sugars to the chain, are specific for the sugar being added, the type of linkage that is formed, and the sugars already present in the chain Once the initial sugars are attached to the protein, the alternating action of two glycosyltransferases adds the sugars of the repeating disaccharide to the growing glycosaminoglycan chain Sulfation occurs after addition of the sugar 3Ј-Phosphoadenosine 5Ј-phosphosulfate (PAPS), also called active sulfate, provides the sulfate groups (see Fig 33.34) An epimerase converts glucuronic acid residues to iduronic acid residues After synthesis, the proteoglycan is secreted from the cell Its structure resembles a bottle brush, with many glycosaminoglycan chains extending from the core protein (Fig 49.11) The proteoglycans may form large aggregates, noncovalently attached by a “link” protein to hyaluronic acid (Fig 49.12) The proteoglycans interact with the adhesion protein, fibronectin, which is attached to the cell membrane protein integrin Cross-linked fibers of collagen also associate with this complex, forming the ECM (Fig 49.13) H H HO β (1 3) O O H COO– O OH H H H OH Iduronic acid O3S CH2OH O O H H H H H β (1 3) O NHCOCH3 N – Acetylgalactosamine Fig 49.8 Repeating disaccharides of some glycosaminoglycans These repeating disaccharides usually contain an N-acetylated sugar and a uronic acid, which usually is glucuronic acid or iduronic acid Sulfate groups are often present but are not included in the sugar names in this figure 914 SECTION EIGHT / TISSUE METABOLISM Core protein Glycosaminoglycan B Link trisaccharide Galactose A Galactose O Xylose N H CH2 C H Serine O C n Uronic acid N –Acetylated sugar Fig 49.9 Attachment of glycosaminoglycans to proteins The sugars are linked to a serine or threonine residue of the protein A and B represent the sugars of the repeating disaccharide n B A PAP PAPS B A UDP UDP UDP UDP UDP A UDP UDP B A 6 A UDP Xyl–transferase Gal– transferase I Gal– transferase II GlcUA–transferase I GalNAc– transferase I GlcUA–transferase II GalNAc– transferase II Sulfotransferase UDP UDP UDP A UDP UDP A B B B UDP Protein core Xylose Galactose N – Acetylgalactosamine Glucuronic acid Sulfate Fig 49.10 Synthesis of chondroitin sulfate Sugars are added to the protein one at a time, with UDP-sugars serving as the precursors Initially a xylose residue is added to a serine in the protein Then two galactose residues are added, followed by a glucuronic acid (GlcUA) and an Nacetylglucosamine (GalNAc) Subsequent additions occur by the alternating action of two enzymes that produce the repeating disaccharide units One enzyme (6) adds GlcUA residues, and the other (7) adds GalNAc As the chain grows, sulfate groups are added by phosphoadenosine phosphosulfate (PAPS) Modified from Roden L In: Fishman WH, ed Metabolic Conjugation and Metabolic Hydrolysis, vol II Orlando, FL: Academic Press, 1970:401 915 CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Table 49.4 Defective Enzymes in the Mucopolysaccharidoses Disease Hunter Hurler ϩ Scheie Maroteaux-Lamy Mucolipidosis VII Sanfilippo A Sanfilippo B Sanfilippo D Enzyme Deficiency – Accumulated Products Iduronate sulfatase ␣-L-Iduronidase N-Acetylgalactosamine sulfatase ␤-Glucuronidase Heparan sulfamidase N-Acetylglucosaminidase N-Acetylglucosamine 6-sulfatase Heparan sulfate, Dermatan sulfate Heparan sulfate, Dermatan sulfate Dermatan sulfate Heparan sulfate, Dermatan sulfate Heparan sulfate Heparan sulfate Heparin sulfate – – – – n – – – – – – – – – – – – n – n – – – – – – n Core protein These disorders share many clinical features, although there are significant variations between disorders, and even within a single disorder, based on the amount of residual activity remaining In most cases, multiple organ systems are affected (with bone and cartilage being a primary target) For some disorders, there is significant neuronal involvement, leading to mental retardation – n – – – – Repeating disaccharide DEGRADATION OF PROTEOGLYCANS Lysosomal enzymes degrade proteoglycans, glycoproteins, and glycolipids, which are brought into the cell by the process of endocytosis Lysosomes fuse with the endocytic vesicles, and lysosomal proteases digest the protein component The carbohydrate component is degraded by lysosomal glycosidases Lysosomes contain both endoglycosidases and exoglycosidases The endoglycosidases cleave the chains into shorter oligosaccharides Then exoglycosidases, specific for each type of linkage, remove the sugar residues, one at a time, from the nonreducing ends Deficiencies of lysosomal glycosidases cause partially degraded carbohydrates from proteoglycans, glycoproteins, and glycolipids to accumulate within membrane-enclosed vesicles inside cells These “residual bodies” can cause marked enlargement of the organ with impairment of its function In the clinical disorder known as the mucopolysaccharidoses (caused by accumulation of partially degraded glycosaminoglycans), deformities of the skeleton may occur (Table 49.4) Mental retardation often accompanies these skeletal changes Fig 49.11 “Bottle-brush” structure of a proteoglycan, with a magnified segment Chondroitin sulfate Protein II INTEGRINS Integrins are the major cellular receptors for ECM proteins and provide a link between the internal cytoskeleton of cells (primarily the actin microfilament system) and extracellular proteins, such as fibronectin, collagen, and laminin Integrins Proteoglycan Fibronectin Collagen Link proteins Keratan sulfate Hyaluronic acid Fig 49.12 Proteoglycan aggregate Cell membrane Integrin Fig 49.13 Interactions between the cell membrane and the components of the extracellular matrix 916 SECTION EIGHT / TISSUE METABOLISM consist of an ␣ and a ␤ subunit There are 18 distinct ␣ and eight distinct ␤ gene products Twenty-four unique ␣/␤ dimers have been discovered Mice have been genetically engineered to be unable to express many of the integrin genes (one gene at a time), and the phenotypes of these knockout mice vary from embryonic lethality (the ␣5 gene is an example) to virtually no observable defects (as exemplified by the ␣1 gene) In addition to anchoring the cell’s cytoskeleton to the ECM, thereby providing a stable environment in which the cell can reside, the integrins are also involved in a wide variety of cell signaling options Certain integrins, such as those associated with white blood cells, are normally inactive because the white cell must circulate freely in the bloodstream However, if an infection occurs, cells located in the area of the infection release cytokines, which activate the integrins on the white blood cells, allowing them to bind to vascular endothelial cells (leukocyte adhesion) at the site of infection Leukocyte adhesion deficiency (LAD) is a genetic disorder that results from mutations in the ␤2 integrin such that leukocytes cannot be recruited to the sites of infection Conversely, drugs are now being developed to block either the ␤2 or ␣4 integrins (on lymphocytes) to treat inflammatory and autoimmune disorders by interfering with the normal white cell response to cytokines Integrins can be activated by “inside-out” mechanisms, whereby intracellular signaling events activate the molecule, or “outside-in” mechanisms, in which a binding event with the extracellular portion of the molecule initiates intracellular signaling events For those integrins that bind cells to ECM components, activation of specific integrins can result in migration of the affected cell through the ECM This mechanism is operative during growth, during cellular differentiation, and in the process of metastasis of malignant cells to neighboring tissues III ADHESION PROTEINS Fibronectin was first discovered as a large, external transformationsensitive protein (LETS), which was lost when fibroblasts were transformed into tumor cells Many tumor cells secrete less than normal amounts of adhesion protein material, which allows for more movement within the extracellular milieu This, in turn, increases the potential for the tumor cells to leave their original location and take root at another location within the body (metastasis) Because MMPs degrade extracellular matrix (ECM) components, their expression is important to allow cell migration and tissue remodeling during growth and differentiation In addition, many growth factors bind to ECM components and, as a bound component, not exhibit their normal growth-promoting activity Destruction of the ECM by the MMPs releases these growth factors, thereby allowing them to bind to cell surface receptors to initiate growth of tissues Thus, coordinated expression of the MMPs is required for appropriate cell movement and growth Cancer cells that metastasize require extensive ECM remodeling and usually use MMP activity to spread throughout the body Adhesion proteins are found in the ECM and link integrins to ECM components Adhesion proteins, of which fibronectin is a prime example, are large multidomain proteins that allow binding to many different components simultaneously In addition to integrin binding sites, fibronectin contains binding sites for collagen and glycosaminoglycans As the integrin molecule is bound to intracellular cytoskeletal proteins, the adhesion proteins provide a bridge between the actin cytoskeleton of the cell and the cells’ position within the ECM Loss of adhesion protein capability can lead to either physiologic or abnormal cell movement Alternative splicing of fibronectin allows many different forms of this adhesion protein to be expressed, including a soluble form (versus cell-associated forms), which is found in the plasma The metabolic significance of these products remains to be determined IV MATRIX METALLOPROTEINASES The ECM contains a series of proteases known as the matrix metalloproteinases, or MMPs These are zinc-containing proteases that use the zinc to appropriately position water to participate in the proteolytic reaction At least 23 different types of human MMPs exist, and they cleave all proteins found in the ECM, including collagen and laminin A propeptide is present in newly synthesized MMPs that contains a critical cysteine residue The cysteine residue in the propeptide binds to the zinc atom at the active site of the protease and prevents the propeptide from exhibiting proteolytic activity Removal of the propeptide is required to activate the MMPs Once activated, certain MMPs can activate other forms of MMP Regulation of MMP activity is quite complex These regulatory processes include transcriptional regulation, proteolytic activation, inhibition by the circulating protein ␣2-macroglobulin, and regulation by a class of inhibitors known as CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE 917 tissue inhibitors of metalloproteinases, or TIMPs It is important that the synthesis of TIMPs and MMPs be coordinately regulated, because dissociation of their expression can facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis CLINICAL COMMENTS Articular cartilage is a living tissue with a turnover time determined by a balance between the rate of its synthesis and that of its degradation (Fig 49.14) The chondrocytes that are embedded in the matrix of intraarticular cartilage participate in both its synthesis and its enzymatic degradation The latter occurs as a result of cleavage of proteoglycan aggregates by enzymes produced and secreted by the chondrocytes In SLE, the condition that affects Sis Lupus, this delicate balance is disrupted in favor of enzymatic degradation, leading to dissolution of articular cartilage and, with it, the loss of its critical cushioning functions The underlying mechanisms responsible for this process in SLE include the production of antibodies directed against specific cellular proteins in cartilage as well as in other intra-articular tissues The cellular proteins thus serve as the “antigens” to which these antibodies react In this sense, SLE is an “autoimmune” disease because antibodies are produced by the host that attack “self” proteins This process excites the local release of cytokines such as interleukin-1 (IL-1), which increases the proteolytic activity of the chondrocytes, causing further loss of articular proteins such as the proteoglycans The associated inflammatory cascade is responsible for Sis Lupus’ joint pain Pericellular matrix Intercellular matrix Synovial fluid Chondrocyte Biosynthesis t1 = 100– 800 d t = – 30 d Degradation products Lysosomal 0degradation Fig 49.14 Synthesis and degradation of proteoglycans by chondrocytes From Cohen RD, et al The Metabolic Basis of Acquired Disease, vol London: Bailliere Tindall, 1990:1859 918 SECTION EIGHT / TISSUE METABOLISM The microvascular complications of both type and type diabetes mellitus involve the small vessels of the retina (diabetic retinopathy), the renal glomerular capillaries (diabetic nephropathy), and the vessels supplying blood to the peripheral nerves (autonomic neuropathy) The lack of adequate control of Ann Sulin’s diabetic state over many years caused a progressive loss of the filtering function of the approximately one-and-one-half million glomerular capillary–mesangial units that are present in her kidneys Chronic hyperglycemia is postulated to be a major metabolic initiator or inducer of diabetic microvascular disease, including those renal glomerular changes that often lead to end-stage renal disease (“glucose toxicity”) For a comprehensive review of the four postulated molecular mechanisms by which chronic hyperglycemia causes these vascular derangements, the reader is referred to an excellent review by Sheetz and King (see suggested references) Regardless of which of the postulated mechanisms (increased flux through the aldose reductase or polyol pathway [see Chapter 30], the generation of advanced glycosylation end products [AGEs], the generation of reactive oxygen intermediates [see Chapter 24], or excessive activation of protein kinase C [see Chapter 18]) will eventually be shown to be the predominant causative mechanism, each can lead to the production of critical intracellular and extracellular signaling molecules (e.g., cytokines) These, in turn, can cause pathologic changes within the glomerular filtration apparatus that reduce renal function These changes include: (1) increased synthesis of collagen, type IV, fibronectin, and some of the proteoglycans, causing the glomerular basement membrane (GBM; Fig 49.15) to become diffusely thickened throughout the glomerular capillary network This membrane thickening alters certain specific filtration properties of the GBM, preventing some of the metabolites that normally enter the urine from the glomerular capillary blood (via the fenestrated capillary endothelium) from doing so (a decline in glomerular filtration rate or GFR) As a result, these potentially toxic substances accumulate in the blood and contribute to the overall clinical presentation of advancing uremia In spite of the Glomerulus Capillary loops Fenestrated capillary endothelium Urinary space Capillary lumen Capillary lumen Parietal epithelium (Bowmans capsule) Proximal tubule Bowmans space (urinary space) Urine Mesangial cells Mesangial matrix Glomerular basement membrane Bowmans space (urinary space) (proximal-most part of a nephron) Capillary lumen Capillary lumen Fig 49.15 A cross-section of a normal renal glomerulus showing four capillary tufts delivering blood to the glomerulus for filtration across the fenestrated capillary endothelium then through the glomerular basement membrane into the Bowman’s space to form urine The urine then enters the proximal tubule of the nephron This filtration removes potentially toxic metabolic end products from the blood The mesangium, by contracting and expanding, controls the efficiency of these filtering and excretory functions by regulating the hydraulic filtration pressures within the glomerulus An intact basement membrane must be present to maintain the integrity of the filtering process CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE thickening of the GBM, this membrane becomes “leaky” for some macromolecules (e.g., albumin) that normally not enter the urine from the glomerular capillaries (microalbuminuria) Suggested mechanisms for this increased permeability or leakiness include reduced synthesis of the specific proteoglycan, heparan sulphate, as well as increased basement membrane production of vascular endothelium growth factor (VEGF), a known angiogenic and permeability factor; and expansion of the extracellular matrix in the mesangium The mesangium consists of specialized tissue containing collagen, proteoglycans, and other macromolecules that surround the glomerular capillaries and that, through its gel-like and sieving properties, determine, in part, the glomerular capillary hydraulic filtration pressure as well as the functional status of the capillary endothelium–mesangial glomerular basement membrane filtration apparatus (see Fig 49.15) As the mesangial tissue expands, the efficiency of glomerular filtration diminishes proportionately The cause of these mesangial changes is, in part, the consequence of increased expression of certain growth factors, especially transforming growth factor ␤ (TGF-␤) and connective tissue growth factor (CTGF) Current therapeutic approaches in patients with early diabetic nephropathy include the use of antibodies that neutralize TGF-␤ BIOCHEMICAL COMMENTS Osteogenesis imperfecta (OI) is a heterogenous group of diseases that have in common a defect in collagen production This defect can be either of two types: The first type is associated with a reduction in the synthesis of normal collagen (due to a gene deletion or splice-site mutation) The second type is associated with the synthesis of a mutated form of collagen Most of the mutations have a dominant-negative effect, leading to an autosomal dominant mode of transmission In the second type of OI, many of the known mutations involve substitutions of another amino acid for glycine This results in an unstable collagen molecule, because glycine is the only amino acid that can fit between the other two chains within the triple helix of collagen If the mutation is near the carboxy-terminal of the molecule, the phenotype of the disease is usually more severe than if the mutation is near the aminoterminal end (recall that triple helix formation proceeds from the carboxy- to the aminoterminal end of the molecule) Of interest are mutations that replace glycine with either serine or cysteine Such mutations are more stable than expected, because of the hydrogen-bonding capabilities of serine and the ability of cysteine to form disulfide bonds Both would aid in preventing the strands of the triple helix from unwinding Children with OI can be treated with a class of compounds known as bisphosphonates, which consist of two phosphates linked by a carbon or nitrogen bridge (thus, they are analogs of pyrophosphate, in which the two phosphates are linked by oxygen) Normal bone remodeling is the result of a coordinated “coupling” between osteoclast activity (cells that resorb bone) and osteoblast activity (cells that form bone) In OI, bone resorption outpaces bone formation because osteoclast activity is enhanced (perhaps because of the reduced levels of normal collagen present to act as nucleating sites for bone formation) This leads to a net loss of bone mass and fragility of the skeleton Bisphosphonates inhibit osteoclast action with the potential to increase bone mass and its tensile strength Suggested References Bosman FT, Stamenhovic I Functional structure and composition of the extracellular matrix J Pathol 2003;200:423–428 Byers PH Disorders of collagen biosynthesis and structure In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds The Metabolic and Molecular Bases of Inherited Disease, vol IV, 8th Ed New York: McGraw-Hill, 2001:5241–5286 919 920 SECTION EIGHT / TISSUE METABOLISM Hynes RO Integrins: biodirectional allosteric signalling machines Cell 2002;110:673–687 Neufeld EF, Muenzer J The mucopolysaccharidoses In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds The Metabolic and Molecular Bases of Inherited Disease, vol IV, 8th Ed New York: McGraw-Hill, 2001:3421–3452 Sheetz MJ, King GL Molecular understanding of hyperglycemia’s adverse effects for diabetic complications JAMA 2002;288(20):2579–2588 REVIEW QUESTIONS—CHAPTER 49 Individuals who develop scurvy suffer from sore and bleeding gums and loss of teeth This is due, in part, to the synthesis of a defective collagen molecule The step that is affected in collagen biosynthesis attributable to scurvy is which of the following? (A) The formation of disulfide bonds, which initiates tropocollagen formation (B) The formation of lysyl cross-links between collagen molecules (C) Secretion of tropocollagen into the extracellular matrix (D) The formation of collagen fibrils (E) The hydroxylation of proline residues, which stabilizes the collagen structure The underlying mechanism that allows elastin to exhibit elastic properties (expand and contract) is which of the following? (A) Proteolysis during expansion, and resynthesis during contraction (B) Breaking of disulfide bonds during expansion, reformation of these bonds during contraction (C) A decrease in entropy during expansion, and an increase in entropy during contraction (D) The breaking of salt bridges during expansion, and reformation of the salt bridges during contraction (E) Hydroxylation of elastin during expansion, and decarboxylation of elastin during contraction The underlying mechanism by which glycosaminoglycans allow for the formation of a gel-like substance in the extracellular matrix in which of the following? (A) Charge attraction between glycosaminoglycan chains (B) Charge repulsion between glycosaminoglycan chains (C) Hydrogen bonding between glycosaminoglycan chains (D) Covalent cross-linking between glycosaminoglycan chains (E) Hydroxylation of adjacent glycosaminoglycan chains The movement of tumor cells from their site of origin to other locations within the body requires the activity of which of the following proteins? (A) Collagen (B) Laminin (C) Proteoglycans (D) Elastin (E) Matrix metalloproteinases Fibronectin is frequently absent in malignant fibroblast cells One of the major functions of fibronectin is which of the following? (A) To inhibit the action of matrix metalloproteinases (B) To coordinate collagen deposition within the extracellular matrix (C) To fix the position of cells within the extracellular matrix (D) To regulate glycosaminoglycan production (E) To extend glycosaminoglycan chains using nucleotide sugars ... from amino acids is an example of an anabolic pathway Catabolic pathways are those pathways that break down larger molecules into smaller components Fuel oxidative pathways are examples of catabolic... serve as a goal for intake by individuals The AI is a recommended intake value that is used when not enough data are available to establish an RDA 11 Are Ivan Applebod and Ann O’Rexia gaining... biosynthetic pathways include certain amino acids, vitamins, and essential fatty acids Detoxification pathways and waste disposal pathways are metabolic pathways devoted to removing toxins that can be