(BQ) Part 2 book Marks’ basic medical biochemistry: A clinical approach presents the following contents: Oxidation of fatty acids and ketone bodies, oxygen toxicity and free radical injury, metabolism of ethanol, basic concepts in the regulation of fuel metabolism by insulin, glucagon, and other hormones, digestion, absorption and transport of carbohydrates,... Invite you to consult.
23 Oxidation of Fatty Acids and Ketone Bodies Fatty acids are a major fuel for humans and supply our energy needs between meals and during periods of increased demand, such as exercise During overnight fasting, fatty acids become the major fuel for cardiac muscle, skeletal muscle, and liver The liver converts fatty acids to ketone bodies (acetoacetate and -hydroxybutyrate), which also serve as major fuels for tissues (e.g., the gut) The brain, which does not have a significant capacity for fatty acid oxidation, can use ketone bodies as a fuel during prolonged fasting The route of metabolism for a fatty acid depends somewhat on its chain length Fatty acids are generally classified as very-long-chain length fatty acids (greater than C20 ), long-chain fatty acids (C12–C20), medium-chain fatty acids (C6–C12), and short-chain fatty acids (C4) ATP is generated from oxidation of fatty acids in the pathway of -oxidation Between meals and during overnight fasting, long-chain fatty acids are released from adipose tissue triacylglycerols They circulate through blood bound to albumin (Fig 23.1) In cells, they are converted to fatty acyl CoA derivatives by acyl CoA synthetases The activated acyl group is transported into the mitochondrial matrix bound to carnitine, where fatty acyl CoA is regenerated In the pathway of -oxidation, the fatty acyl group is sequentially oxidized to yield FAD(2H), NADH, and acetyl CoA Subsequent oxidation of NADH and FAD(2H) in the electron transport chain, and oxidation of acetyl CoA to CO2 in the TCA cycle, generates ATP from oxidative phosphorylation Many fatty acids have structures that require variations of this basic pattern Long-chain fatty acids that are unsaturated fatty acids generally require additional isomerization and oxidation–reduction reactions to rearrange their double bonds during -oxidation Metabolism of water-soluble medium-chain-length fatty acids does not require carnitine and occurs only in liver Odd-chain-length fatty acids undergo -oxidation to the terminal three-carbon propionyl CoA, which enters the TCA cycle as succinyl CoA Fatty acids that not readily undergo mitochondrial -oxidation are oxidized first by alternate routes that convert them to more suitable substrates or to urinary excretion products Excess fatty acids may undergo microsomal -oxidation, which converts them to dicarboxylic acids that appear in urine Very-long-chain fatty acids (both straight chain and branched fatty acids such as phytanic acid) are whittled down to size in peroxisomes Peroxisomal ␣- and -oxidiation generates hydrogen peroxide (H2O2), NADH, acetyl CoA, or propionyl CoA and a short- to medium-chain-length acyl CoA The acyl CoA products are transferred to mitochondria to complete their metabolism In the liver, much of the acetyl CoA generated from fatty acid oxidation is converted to the ketone bodies, acetoacetate and -hydroxybutyrate, which enter the blood (see Fig 23.1) In other tissues, these ketone bodies are converted to acetyl 418 CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES Long-chain Fatty acid-albumin ATP CoA Fatty acid binding proteins Plasma membrane Fatty acyl CoA Outer mitochondrial membrane Carnatine CoA Fatty acyl carnitine Inner mitochondrial membrane Carnatine CoA Fatty acyl CoA β-oxidation spiral FAD (2H) NADH Acetyl CoA (Liver) Ketone bodies TCA cycle 2CO2 NADH, FAD (2H), GTP Fig 23.1 Overview of mitochondrial long-chain fatty acid metabolism (1) Fatty acid binding proteins (FaBP) transport fatty acids across the plasma membrane and bind them in the cytosol (2) Fatty acyl CoA synthetase activates fatty acids to fatty acyl CoAs (3) Carnitine transports the activated fatty acyl group into mitochondria (4) -oxidation generates NADH, FAD(2H), and acetyl CoA (5) In the liver, acetyl CoA is converted to ketone bodies CoA, which is oxidized in the TCA cycle The liver synthesizes ketone bodies but cannot use them as a fuel The rate of fatty acid oxidation is linked to the rate of NADH, FAD(2H), and acetyl CoA oxidation, and, thus, to the rate of oxidative phosphorylation and ATP utilization Additional regulation occurs through malonyl CoA, which inhibits formation of the fatty acyl carnitine derivatives Fatty acids and ketone bodies are used as a fuel when their level increases in the blood, which is determined by hormonal regulation of adipose tissue lipolysis THE WAITING ROOM Otto Shape was disappointed that he did not place in his 5-km race and has decided that short-distance running is probably not right for him After careful consideration, he decides to train for the marathon by running 12 miles three times per week He is now 13 pounds over his ideal weight, and he plans on losing this weight while studying for his Pharmacology finals He considers a variety of dietary supplements to increase his endurance and selects one containing carnitine, CoQ, pantothenate, riboflavin, and creatine 419 420 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP The liver transaminases measured in the blood are aspartate aminotransferase (AST), which was formerly called serum glutamate-oxaloacetate transaminase (SGOT), and alanine aminotransferase (ALT), which was formerly called serum glutamate pyruvate transaminase (SGPT) Elevation of liver enzymes reflects damage of the liver plasma membrane Lofata Burne is a 16-year-old girl Since age 14 months she has experienced recurrent episodes of profound fatigue associated with vomiting and increased perspiration, which required hospitalization These episodes occurred only if she fasted for more than hours Because her mother gave her food late at night and woke her early in the morning for breakfast, Lofata’s physical and mental development had progressed normally On the day of admission for this episode, Lofata had missed breakfast, and by noon she was extremely fatigued, nauseated, sweaty, and limp She was unable to hold any food in her stomach and was rushed to the hospital, where an infusion of glucose was started intravenously Her symptoms responded dramatically to this therapy Her initial serum glucose level was low at 38 mg/dL (reference range for fasting serum glucose levels ϭ 70–100) Her blood urea nitrogen (BUN) level was slightly elevated at 26 mg/dL (reference range ϭ 8–25) as a result of vomiting, which led to a degree of dehydration Her blood levels of liver transaminases were slightly elevated, although her liver was not palpably enlarged Despite elevated levels of free fatty acids (4.3 mM) in the blood, blood ketone bodies were below normal Di Abietes, a 27-year-old woman with type diabetes mellitus, had been admitted to the hospital in a ketoacidotic coma a year ago (see Chapter 4) She had been feeling drowsy and had been vomiting for 24 hours before that admission At the time of admission, she was clinically dehydrated, her blood pressure was low, and her breathing was deep and rapid (Kussmaul breathing) Her pulse was rapid, and her breath had the odor of acetone Her arterial blood pH was 7.08 (reference range, 7.36–7.44), and her blood ketone body levels were 15 mM (normal is approximately 0.2 mM for a person on a normal diet) I During Otto’s distance running (a moderate-intensity exercise), decreases in insulin and increases in insulin counterregulatory hormones, such as epinephrine and norepinephrine, increase adipose tissue lipolysis Thus, his muscles are being provided with a supply of fatty acids in the blood that they can use as a fuel Lofata Burne developed symptoms during fasting, when adipose tissue lipolysis was elevated Under these circumstances, muscle tissue, liver, and many other tissues are oxidizing fatty acids as a fuel After overnight fasting, approximately 60 to 70% of our energy supply is derived from the oxidation of fatty acids FATTY ACIDS AS FUELS The fatty acids oxidized as fuels are principally long-chain fatty acids released from adipose tissue triacylglycerol stores between meals, during overnight fasting, and during periods of increased fuel demand (e.g., during exercise) Adipose tissue triacylglycerols are derived from two sources; dietary lipids and triacylglycerols synthesized in the liver The major fatty acids oxidized are the long-chain fatty acids, palmitate, oleate, and stearate, because they are highest in dietary lipids and are also synthesized in the human Between meals, a decreased insulin level and increased levels of insulin counterregulatory hormones (e.g., glucagon) activate lipolysis, and free fatty acids are transported to tissues bound to serum albumin Within tissues, energy is derived from oxidation of fatty acids to acetyl CoA in the pathway of -oxidation Most of the enzymes involved in fatty acid oxidation are present as 2-3 isoenzymes, which have different but overlapping specificities for the chain length of the fatty acid Metabolism of unsaturated fatty acids, odd-chain-length fatty acids, and mediumchain-length fatty acids requires variations of this basic pattern The acetyl CoA produced from fatty acid oxidation is principally oxidized in the TCA cycle or converted to ketone bodies in the liver A Characteristics of Fatty Acids Used as Fuels Fat constitutes approximately 38% of the calories in the average North American diet Of this, more than 95% of the calories are present as triacylglycerols (3 fatty acids esterified to a glycerol backbone) During ingestion and absorption, dietary triacylglycerols are broken down into their constituents and then reassembled for transport to adipose tissue in chylomicrons (see Chapter 2) Thus, the fatty acid composition of adipose triacylglycerols varies with the type of food consumed CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES The most common dietary fatty acids are the saturated long-chain fatty acids palmitate (C16) and stearate (C18), the monounsaturated fatty acid oleate (C18:1), and the polyunsaturated essential fatty acid, linoleate (C18:2) (To review fatty acid nomenclature, consult Chapter 5) Animal fat contains principally saturated and monounsaturated long-chain fatty acids, whereas vegetable oils contain linoleate and some longer-chain and polyunsaturated fatty acids They also contain smaller amounts of branched-chain and odd-chain-length fatty acids Medium-chain-length fatty acids are present principally in dairy fat (e.g., milk and butter), maternal milk, and vegetable oils Adipose tissue triacylglycerols also contain fatty acids synthesized in the liver, principally from excess calories ingested as glucose The pathway of fatty acid synthesis generates palmitate, which can be elongated to form stearate, and unsaturated to form oleate These fatty acids are assembled into triacylglycerols and transported to adipose tissue as the lipoprotein VLDL (very-low-density lipoprotein) B Transport and Activation of Long-Chain Fatty Acids Long-chain fatty acids are hydrophobic and water insoluble In addition, they are toxic to cells because they can disrupt the hydrophobic bonding between amino acid side chains in proteins Consequently, they are transported in the blood and in cells bound to proteins CELLULAR UPTAKE OF LONG-CHAIN FATTY ACIDS During fasting and other conditions of metabolic need, long-chain fatty acids are released from adipose tissue triacylglycerols by lipases They travel in the blood bound in the hydrophobic binding pocket of albumin, the major serum protein (see Fig 23.1) Fatty acids enter cells both by a saturable transport process and by diffusion through the lipid plasma membrane A fatty acid binding protein in the plasma membrane facilitates transport An additional fatty acid binding protein binds the fatty acid intracellularly and may facilitate its transport to the mitochondrion The free fatty acid concentration in cells is, therefore, extremely low ACTIVATION OF LONG-CHAIN FATTY ACIDS Fatty acids must be activated to acyl CoA derivatives before they can participate in -oxidation and other metabolic pathways (Fig 23.2) The process of activation involves an acyl CoA synthetase (also called a thiokinase) that uses ATP energy to form the fatty acyl CoA thioester bond In this reaction, the  bond of ATP is cleaved to form a fatty acyl AMP intermediate and pyrophosphate (PPi) Subsequent cleavage of PPi helps to drive the reaction The acyl CoA synthetase that activates long-chain fatty acids, 12 to 20 carbons in length, is present in three locations in the cell: the endoplasmic reticulum, outer mitochondrial membranes, and peroxisomal membranes (Table 23.1) This enzyme has no activity toward C22 or longer fatty acids, and little activity below C12 In contrast, the synthetase for activation of very-long-chain fatty acids is present in peroxisomes, and the medium-chain-length fatty acid activating enzyme is present only in the mitochondrial matrix of liver and kidney cells FATES OF FATTY ACYL COAS Fatty acyl CoA formation, like the phosphorylation of glucose, is a prerequisite to metabolism of the fatty acid in the cell (Fig 23.3) The multiple locations of the longchain acyl CoA synthetase reflects the location of different metabolic routes taken by fatty acyl CoA derivatives in the cell (e.g., triacylglycerol and phospholipid synthesis 421 422 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP Fatty acyl CoA Energy β-oxidation ketogenesis O ATP Membrane lipids Phospholipids Sphingolipids – O P O P O – O O – O O Fatty acid P O Adenosine – O– O R C O Storage Triacylglycerols Fig 23.3 Major metabolic routes for longchain fatty acyl CoAs Fatty acids are activated to acyl CoA compounds for degradation in mitochondrial -oxidation, or incorporation into triacylglycerols or membrane lipids When -oxidation is blocked through an inherited enzyme deficiency, or metabolic regulation, excess fatty acids are diverted into triacylglycerol synthesis fatty acyl CoA synthetase O Fatty acyl AMP (enzyme-bound) R C CoASH fatty acyl CoA synthetase Fatty acyl CoA R O P Adenosine O – + O P O P O– O– – O •• O O O O– Pyrophosphate AMP inorganic pyrophosphatase O C ~ SCoA Pi Fig 23.2 Activation of a fatty acid by a fatty acyl CoA synthetase The fatty acid is activated by reacting with ATP to form a high-energy fatty acyl AMP and pyrophosphate The AMP is then exchanged for CoA Pyrophosphate is cleaved by a pyrophosphatase Table 23.1 Chain-Length Specificity of Fatty Acid Activation and Oxidation Enzymes Enzyme Chain Length Comments Very Long Chain 14–26 Only found in peroxisomes Long Chain 12–20 Enzyme present in membranes of ER, mitochondria, and peroxisomes to facilitate different metabolic routes of acyl CoAs Acyl CoA synthetases Medium Chain 6–12 Exists as many variants, present only in mitochondrial matrix of kidney and liver Also involved in xenobiotic metabolism Acetyl 2–4 Present in cytoplasm and possibly mitochondrial matrix Acyltransferases CPTI 12–16 Although maximum activity is for fatty acids 12–16 carbons long, it also acts on many smaller acyl CoA derivatives Medium Chain (Octanoylcarnitine transferase) 6–12 Substrate is medium-chain acyl CoA derivatives generated during peroxisomal oxidation Carnitine:acetyl transferase High level in skeletal muscle and heart to facilitate use of acetate as a fuel Acyl CoA Dehydrogenases VLCAD 14–20 Present in inner mitochondrial membrane LCAD MCAD 12–18 4–12 Members of same enzyme family, which also includes acyl CoA dehydrogenases for carbon skeleton of branched-chain amino acids SCAD 4–6 Other enzymes Enoyl CoA hydratase, Short-chain >4 Also called crotonase Activity decreases with increasing chain length L-3-Hydroxyacyl CoA dehydrogenase, Short-Chain 4–16 Activity decreases with increasing chain length Acetoacetyl CoA thiolase Specific for acetoacetyl CoA Trifunctional Protein 12–16 Complex of long-chain enoyl hydratase, acyl CoA dehydrogenase and a thiolase with broad specificity Most active with longer chains CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES in the endoplasmic reticulum, oxidation and plasmalogen synthesis in the peroxisome, and -oxidation in mitochondria) In the liver and some other tissues, fatty acids that are not being used for energy generation are re-incorporated (re-esterified) into triacylglycerols TRANSPORT OF LONG-CHAIN FATTY ACIDS INTO MITOCHONDRIA Carnitine serves as the carrier that transports activated long chain fatty acyl groups across the inner mitochondrial membrane (Fig 23.4) Carnitine acyl transferases are able to reversibly transfer an activated fatty acyl group from CoA to the hydroxyl group of carnitine to form an acylcarnitine ester The reaction is reversible, so that the fatty acyl CoA derivative can be regenerated from the carnitine ester Carnitine:palmitoyltransferase I (CPTI; also called carnitine acyltransferase I, CATI), the enzyme that transfers long-chain fatty acyl groups from CoA to carnitine, is located on the outer mitochondrial membrane (Fig 23.5) Fatty acylcarnitine crosses the inner mitochondrial membrane with the aid of a translocase The fatty acyl group is transferred back to CoA by a second enzyme, carnitine:palmitoyltransferase II (CPTII or CATII) The carnitine released in this reaction returns to the cytosolic side of the mitochondrial membrane by the same translocase that brings fatty acylcarnitine to the matrix side Long-chain fatty acyl CoA, now located within the mitochondrial matrix, is a substrate for -oxidation Carnitine is obtained from the diet or synthesized from the side chain of lysine by a pathway that begins in skeletal muscle, and is completed in the liver The reactions use S-adenosylmethionine to donate methyl groups, and vitamin C (ascorbic acid) is also required for these reactions Skeletal muscles have a ATP + CoA Fatty acid 423 A number of inherited diseases in the metabolism of carnitine or acylcarnitines have been described These include defects in the following enzymes or systems: the transporter for carnitine uptake into muscle; CPT I; carnitineacylcarnitine translocase; and CPTII Classical CPTII deficiency, the most common of these diseases, is characterized by adolescent to adult onset of recurrent episodes of acute myoglobinuria precipitated by prolonged exercise or fasting During these episodes, the patient is weak, and may be somewhat hypoglycemic with diminished ketosis (hypoketosis), but metabolic decompensation is not severe Lipid deposits are found in skeletal muscles CPK levels, and long-chain acylcarnitines are elevated in the blood CPTII levels in fibroblasts are approximately 25% of normal The remaining CPTII activity probably accounts for the mild effect on liver metabolism In contrast, when CPTII deficiency has presented in infants, CPT II levels are below 10% of normal, the hypoglycemia and hypoketosis are severe, hepatomegaly occurs from the triacylglycerol deposits, and cardiomyopathy is also present Cytosol AMP + PPi Fatty acyl CoA Carnitine palmitoyl – transferase I Acyl CoA synthetase (CPT I ) Outer mitochondrial membrane CoA Fatty acyl CoA Fatty acylcarnitine Carnitine Carnitine palmitoyl – transferase II Carnitine acylcar – nitine translocase Matrix (CPT II ) CH3 O CoA CH3 Fatty acylcarnitine Carnitine CH3 Inner mitochondrial membrane Fatty acyl CoA β – oxidation Fig 23.5 Transport of long-chain fatty acids into mitochondria The fatty acyl CoA crosses the outer mitochondrial membrane Carnitine palmitoyl transferase I in the outer mitochondrial membrane transfers the fatty acyl group to carnitine and releases CoASH The fatty acyl carnitine is translocated into the mitochondrial matrix as carnitine moves out Carnitine palmitoyl transferase II on the inner mitochondrial membrane transfers the fatty acyl group back to CoASH, to form fatty acyl CoA in the matrix (CH2)n C + N CH3 CH2 O CH CH2 COO– Fatty acylcarnitine Fig 23.4 Structure of fatty acylcarnitine Carnitine: palmitoyl transferases catalyze the reversible transfer of a long-chain fatty acyl group from the fatty acyl CoA to the hydroxyl group of carnitine The atoms in the dashed box originate from the fatty acyl CoA 424 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP Otto Shape’s power supplement contains carnitine However, his body can synthesize enough carnitine to meet his needs, and his diet contains carnitine Carnitine deficiency has been found only in infants fed a soy-based formula that was not supplemented with carnitine His other supplements likewise probably provide no benefit, but are designed to facilitate fatty acid oxidation during exercise Riboflavin is the vitamin precursor of FAD, which is required for acyl CoA dehydrogenases and ETFs CoQ is synthesized in the body, but it is the recipient in the electron transport chain for electrons passed from complexes I and II and the ETFs Some reports suggest that supplementation with pantothenate, the precursor of CoA, improves performance COASH α O H3C C~ SCoA β Palmitoyl CoA high-affinity uptake system for carnitine, and most of the carnitine in the body is stored in skeletal muscle C -Oxidation of Long-Chain Fatty Acids The oxidation of fatty acids to acetyl CoA in the -oxidation spiral conserves energy as FAD(2H) and NADH FAD(2H) and NADH are oxidized in the electron transport chain, generating ATP from oxidative phosphorylation Acetyl CoA is oxidized in the TCA cycle or converted to ketone bodies THE -OXIDATION SPIRAL The fatty acid -oxidation pathway sequentially cleaves the fatty acyl group into 2carbon acetyl CoA units, beginning with the carboxyl end attached to CoA (Fig 23.6) Before cleavage, the -carbon is oxidized to a keto group in two reactions that generate NADH and FAD(2H); thus, the pathway is called -oxidation As each acetyl group is released, the cycle of -oxidation and cleavage begins again, but each time the fatty acyl group is carbons shorter There are four types of reactions in the -oxidation pathway (Fig 23.7) In the first step, a double bond is formed between the - and ␣-carbons by an acyl CoA dehydrogenase that transfers electrons to FAD The double bond is in the trans Mitochondrial matrix CH3 β CH2 CH2 O α C ~ SCoA CH2 Fatty acyl CoA [total C = n] H3C FAD O acyl CoA dehydrogenase C ~ SCoA + O CH3 C~ SCoA Repetitions of the β–oxidation spiral CH3 CH2 ~ 1.5 ATP FAD (2H) β O CH CH C ~ SCoA trans ∆2 Fatty enoyl CoA Acetyl CoA Acetyl CoA Fig 23.6 Overview of -oxidation Oxidation at the -carbon is followed by cleavage of the ␣— bond, releasing acetyl CoA and a fatty acyl CoA that is two carbons shorter than the original The carbons cleaved to form acetyl CoA are shown in blue Successive spirals of -oxidation completely cleave an evenchain fatty acyl CoA to acetyl CoA H2O enoyl CoA hydratase β–Oxidation Spiral CH2 CH3 β OH CH CH2 C ~ SCoA L – β – Hydroxy acyl CoA NAD+ β-hydroxy acyl CoA dehydrogenase CH3 O CH2 β NADH + H+ O C ~ 2.5 ATP O CH2 C ~ SCoA β – Keto acyl CoA CoASH β-keto thiolase O CH3 [total C =(n – 2)] CH2 C SCoA + CH3 Fatty acyl CoA O C ~ SCoA Acetyl CoA Fig 23.7 Steps of -oxidation The four steps are repeated until an even-chain fatty acid is completely converted to acetyl CoA The FAD(2H) and NADH are reoxidized by the electron transport chain, producing ATP 425 CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES configuration (a ⌬2-trans double bond) In the next step, an OH from water is added to the -carbon, and an H from water is added to the -carbon The enzyme is called an enoyl hydratase (hydratases add the elements of water, and “ene” in a name denotes a double bond) In the third step of -oxidation, the hydroxyl group on the -carbon is oxidized to a ketone by a hydroxyacyl CoA dehydrogenase In this reaction, as in the conversion of most alcohols to ketones, the electrons are transferred to NADϩ to form NADH In the last reaction of the sequence, the bond between the - and ␣-carbons is cleaved by a reaction that attaches CoASH to the -carbon, and acetyl CoA is released This is a thiolytic reaction (lysis refers to breakage of the bond, and thio refers to the sulfur), catalyzed by enzymes called -ketothiolases The release of two carbons from the carboxyl end of the original fatty acyl CoA produces acetyl CoA and a fatty acyl CoA that is two carbons shorter than the original The shortened fatty acyl CoA repeats these four steps until all of its carbons are converted to acetyl CoA -Oxidation is, thus, a spiral rather than a cycle In the last spiral, cleavage of the four-carbon fatty acyl CoA (butyryl CoA) produces two acetyl CoA Thus, an even chain fatty acid such as palmitoyl CoA, which has 16 carbons, is cleaved seven times, producing FAD(2H), NADH, and acetyl CoA ENERGY YIELD OF -OXIDATION Like the FAD in all flavoproteins, FAD(2H) bound to the acyl CoA dehydrogenases is oxidized back to FAD without dissociating from the protein (Fig 23.8) Electron transfer flavoproteins (ETF) in the mitochondrial matrix accept electrons from the enzyme-bound FAD(2H) and transfer these electrons to ETF-QO (electron transfer flavoprotein -CoQ oxidoreductase) in the inner mitochondrial membrane ETF-QO, also a flavoprotein, transfers the electrons to CoQ in the electron transport chain Oxidative phosphorylation thus generates approximately 1.5 ATP for each FAD(2H) produced in the -oxidation spiral The total energy yield from the oxidation of mole of palmityl CoA to moles of acetyl CoA is therefore 28 moles of ATP: 1.5 for each of the FAD(2H), and 2.5 for each of the NADH To calculate the energy yield from oxidation of mole of palmitate, two ATP need to be subtracted from the total because two high-energy phosphate bonds are cleaved when palmitate is activated to palmityl CoA The -oxidation spiral uses the same reaction types seen in the TCA cycle when succinate is converted to oxaloacetate CH2 CH2 H C H C Palmitoyl CoA Palmitoloyl CoA FAD Acyl CoA DH FAD (2H) Acyl CoA DH FAD (2H) ETF FAD ETF FAD ETF • QO FAD (2H) ETF • QO CoQH2 CoQ Electron transport chain Fig 23.8 Transfer of electrons from acyl CoA dehydrogenase to the electron transport chain Abbreviations: ETF, electron-transferring flavoprotein; ETF-QO, electron-transferring flavoprotein–Coenzyme Q oxidoreductase What is the total ATP yield for the oxidation of mole of palmitic acid to carbon dioxide and water? CHAIN LENGTH SPECIFITY IN -OXIDATION The four reactions of -oxidation are catalyzed by sets of enzymes that are each specific for fatty acids with different chain lengths (see Table 23.1) The acyl dehydrogenases, which catalyze the first step of the pathway, are part of an enzyme family that have four different ranges of specificity The subsequent steps of the spiral use enzymes specific for long- or short-chain enoyl CoAs Although these enzymes are structurally distinct, their specificity overlaps to some extent After reviewing Lofata Burne’s previous hospital records, a specialist suspected that Lofata’s medical problems were caused by a disorder in fatty acid metabolism A battery of tests showed that Lofata’s blood contained elevated levels of several partially oxidized medium-chain fatty acids, such as octanoic acid (8:0) and 4-decenoic acid (10:1, ⌬4) A urine specimen showed an increase in organic acid metabolites of medium-chain fatty acids containing to 10 carbons, including medium-chain acylcarnitine derivatives The profile of acylcarnitine species in the urine was characteristic of a genetically determined medium-chain acyl CoA dehydrogenase (MCAD) deficiency In this disease, long-chain fatty acids are metabolized by -oxidation to a medium-chain-length acyl CoA, such as octanoyl CoA Because further oxidation of this compound is blocked in MCAD deficiency, the medium chain acyl group is transferred back to carnitine These acylcarnitines are water soluble and appear in blood and urine The specific enzyme deficiency was demonstrated in cultured fibroblasts from Lofata’s skin as well as in her circulating monocytic leukocytes In LCAD deficiency, fatty acylcarnitines accumulate in the blood Those containing 14 carbons predominate However, these not appear in the urine 426 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP Palmitic acid is 16 carbons long, with no double bonds, so it requires oxidation spirals to be completely converted to acetyl-CoA After spirals, there are FAD(2H), NADH, and acetyl-CoA Each NADH yields 2.5 ATP, each FAD(2H) yields 1.5 ATP, and each acetyl-CoA yields 10 ATP as it is processed around the TCA cycle This then yields 17.5 ϩ 10.5 ϩ 80.5 ϭ 108 ATP However, activation of palmitic acid to palmityl-CoA requires two high-energy bonds, so the net yield is 108 – 2, or 106 moles of ATP Linoleate, although high in the diet, cannot be synthesized in the human and is an essential fatty acid It is required for formation of arachidonate, which is present in plasma lipids, and is used for eicosanoid synthesis Therefore, only a portion of the linoleate pool is rapidly oxidized As the fatty acyl chains are shortened by consecutive cleavage of two acetyl units, they are transferred from enzymes that act on longer chains to those that act on shorter chains Medium- or short-chain fatty acyl CoAs that may be formed from dietary fatty acids, or transferred from peroxisomes, enter the spiral at the enzyme most active for fatty acids of their chain length Approximately one half of the fatty acids in the human diet are unsaturated, containing cis double bonds, with oleate (C18:1, ⌬9) and linoleate (18:2,⌬9,12) being the most common In -oxidation of saturated fatty acids, a trans double bond is created between the 2nd and 3rd (␣ and ) carbons For unsaturated fatty acids to undergo the -oxidation spiral, their cis double bonds must be isomerized to trans double bonds that will end up between the 2nd and 3rd carbons during -oxidation, or the double bond must be reduced The process is illustrated for the polyunsaturated fatty acid linoleate in Fig 23.9 Linoleate undergoes -oxidation until one double bond is between carbons and near the carboxyl end of the fatty acyl chain, and the other is between carbons and An isomerase moves the double bond from the 3,4 position so that it is trans and in the 2,3 position, and -oxidation continues When a conjugated pair of double bonds is formed (two double bonds separated by one single bond) at positions and 4, an NADPH-dependent reductase reduces the pair to one trans double bond at position Then isomerization and -oxidation resume In oleate (C18:1, ⌬9), there is only one double bond between carbons and 10 It is handled by an isomerization reaction similar to that shown for the double bond at position of linoleate The medium-chain-length acyl CoA synthetase has a broad range of specificity for compounds of approximately the same size that contain a carboxyl group, such as drugs (salicylate, from aspirin metabolism, and valproate, which is used to treat epileptic seizures), or benzoate, a common component of plants Once the drug acyl CoA is formed, the acyl group is conjugated with glycine to form a urinary excretion product With certain disorders of fatty acid oxidation, medium- and short-chain fatty acylglycines may appear in the urine, together with acylcarnitines or dicarboxylic acids OXIDATION OF UNSATURATED FATTY ACIDS ODD-CHAIN-LENGTH FATTY ACIDS Fatty acids containing an odd number of carbon atoms undergo -oxidation, producing acetyl CoA, until the last spiral, when five carbons remain in the fatty acyl CoA In this case, cleavage by thiolase produces acetyl CoA and a three-carbon fatty acyl CoA, propionyl CoA (Fig 23.10) Carboxylation of propionyl CoA yields methylmalonyl CoA, which is ultimately converted to succinyl CoA in a vitamin B12–dependent reaction (Fig 23.11) Propionyl CoA also arises from the oxidation of branched chain amino acids The propionyl CoA to succinyl CoA pathway is a major anaplerotic route for the TCA cycle and is used in the degradation of valine, isoleucine, and a number of other compounds In the liver, this route provides precursors of oxaloacetate, which is converted to glucose Thus, this small proportion of the odd-carbonnumber fatty acid chain can be converted to glucose In contrast, the acetyl CoA formed from -oxidation of even-chain-number fatty acids in the liver either enters the TCA cycle, where it is principally oxidized to CO2, or is converted to ketone bodies D Oxidation of Medium-Chain-Length Fatty Acids Dietary medium-chain-length fatty acids are more water soluble than long-chain fatty acids and are not stored in adipose triacylglyce After a meal, they enter the blood and pass into the portal vein to the liver In the liver, they enter the mitochondrial matrix by the monocarboxylate transporter and are activated to acyl CoA derivatives in the mitochondrial matrix (see Fig 23.1) Medium-chain-length acyl CoAs, like long-chain acyl CoAs, are oxidized to acetyl CoA via the -oxidation spiral Medium-chain acyl CoAs also can arise from the peroxisomal oxidation pathway CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES 12 18 O C SCoA β oxidation (three spirals) 427 Linoleolyl CoA cis – ∆9, cis – ∆12 Acetyl CoA O C cis – ∆3, cis – ∆6 SCoA enoyl CoA isomerase C SCoA trans – ∆2, cis – ∆6 O One spiral of β oxidation and the first step of the second spiral Acetyl CoA SCoA C trans – ∆2, cis – ∆4 O NADPH + H+ 2,4-dienoyl CoA reductase NADP+ O C trans – ∆3 SCoA enoyl CoA isomerase O C trans – ∆2 SCoA β oxidation (four spirals) Acetyl CoA Fig 23.9 Oxidation of linoleate After three spirals of -oxidation (dashed lines), there is now a 3,4 cis double bond and a 6,7 cis double bond The 3,4 cis double bond is isomerized to a 2,3-trans double bond, which is in the proper configuration for the normal enzymes to act One spiral of -oxidation occurs, plus the first step of a second spiral A reductase that uses NADPH now converts these two double bonds (between carbons and and carbons and 5) to one double bond between carbons and in a trans configuration The isomerase (which can act on double bonds that are in either the cis or the trans configuration) moves this double bond to the 2,3-trans position, and -oxidation can resume O ω O CH3 CH2 E Regulation of -Oxidation Fatty acids are used as fuels principally when they are released from adipose tissue triacylglycerols in response to hormones that signal fasting or increased demand Many tissues, such as muscle and kidney, oxidize fatty acids completely to CO2 and H2O In these tissues, the acetyl CoA produced by -oxidation enters the TCA cycle The FAD(2H) and the NADH from -oxidation and the TCA cycle are C ~ SCoA C ~ SCoA Propionyl CoA O CH3 C ~ SCoA Acetyl CoA Fig 23.10 Formation of propionyl CoA from odd-chain fatty acids Successive spirals of -oxidation cleave each of the bonds marked with dashed lines, producing acetyl CoA except for the three carbons at the -end, which produce propionyl CoA 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 ␣11␥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 ... blood are aspartate aminotransferase (AST), which was formerly called serum glutamate-oxaloacetate transaminase (SGOT), and alanine aminotransferase (ALT), which was formerly called serum glutamate... MCFA CoA CAT Acetylcarnitine Acetyl CoA TCA cycle Acetylcarnitine NADH CO2, H2O CAC MCFA CoA SCFA CoA COT SCFA-carnitine MCFA-carnitine SCFA-carnitine MCFA-carnitine n turns of β-oxidation Further... and ␣-oxidation pathways, which are essentially chain-shortening pathways O R CH2 CH2 C SCoA FAD FADH2 H R C VERY-LONG-CHAIN FATTY ACIDS Very-long-chain fatty acids of 24 to 26 carbons are oxidized