Biochemistry_Lippinco

Biochemistry_Lippinco. Arginine cleavage to ornithine and urea: Arginase-I hydrolyzes arginine to ornithine and urea and is virtually exclusive to the liver. Therefore, only the liver can cleave arginine, thereby synthesizing urea, whereas other tissues, such as the kidney, can synthesize arginine from citrulline. [Note: Arginase-II in kidneys controls arginine availability for nitric oxide synthesis (see p. 150).] 6.


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Biochemistry_Lippinco. Fate of urea: Urea diffuses from the liver and is transported in the blood to the kidneys, where it is filtered and excreted in the urine (see Fig. 19.19). A portion of the urea diffuses from the blood into the intestine and is cleaved to CO2 and ammonia by bacterial urease. The ammonia is partly lost in the feces and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of antibiotics reduces the number of intestinal bacteria responsible for this ammonia production. B. Overall stoichiometry


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Overall stoichiometry Because four high-energy phosphate bonds are consumed in the synthesis of each molecule of urea, the synthesis of urea is irreversible, with a large, negative ∆G (see p. 70). One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST). In effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitrogen from other amino acids (Fig. 19.15). C. Regulation


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Regulation NAG is an essential activator for CPS I, the rate-limiting step in the urea cycle. It increases the affinity of CPS I for ATP. NAG is synthesized from acetyl CoA and glutamate by N-acetylglutamate synthase (NAGS), as shown in Figure 19.16, in a reaction for which arginine is an activator. The cycle is also regulated by substrate availability (short-term regulation) and enzyme induction (long term). activator of carbamoyl phosphate synthetase I. CoA = coenzyme A. VI. AMMONIA METABOLISM


Biochemistry_Lippinco

Biochemistry_Lippinco. activator of carbamoyl phosphate synthetase I. CoA = coenzyme A. VI. AMMONIA METABOLISM Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed of primarily by formation of urea in the liver. However, the blood ammonia level must be kept very low, because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system (CNS). Therefore, a mechanism is required for the transport of nitrogen from the peripheral tissues to the liver for ultimate disposal as urea while keeping circulating levels of free ammonia low. A. Sources


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Sources Amino acids are quantitatively the most important source of ammonia because most Western diets are high in protein and provide excess amino acids, which travel to the liver and undergo transdeamination (that is, the linking of the aminotransferase and GDH reactions), producing ammonia. [Note: The liver catabolizes straight-chain amino acids, primarily.] However, substantial amounts of ammonia can be obtained from other sources. 1. Glutamine: An important source of plasma glutamine is from the catabolism of BCAA in skeletal muscle. This glutamine is taken up by cells of the intestine, the liver, and the kidneys. The liver and kidneys generate ammonia from glutamine by the actions of glutaminase (Fig.


Biochemistry_Lippinco

Biochemistry_Lippinco. 19.17) and GDH. In the kidneys, most of this ammonia is excreted into + the urine as NH4 , which provides an important mechanism for maintaining the body’s acid–base balance through the excretion of protons. In the liver, the ammonia is detoxified to urea and excreted. [Note: α-Ketoglutarate, the second product of GDH, is a glucogenic precursor in the liver and kidneys.] Ammonia is also generated by intestinal glutaminase. Enterocytes obtain glutamine either from the blood or from digestion of dietary protein. [Note: Intestinal glutamine metabolism also produces alanine, which is used by the liver for gluconeogenesis, and citrulline, which is used by the kidneys to synthesize arginine.] 2. Intestinal bacteria: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. This ammonia is absorbed from the intestine by way of the portal vein, and virtually all is removed by the liver via conversion to urea. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Amines: Amines obtained from the diet and monoamines that serve as hormones or neurotransmitters give rise to ammonia by the action of monoamine oxidase (see p. 286). 4. Purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the ring atoms are released as ammonia (see Fig. 22.15 on p. 300). B. Transport in the circulation Although ammonia is constantly produced in the tissues, it is present at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver and to the fact that several tissues, particularly muscle, release amino acid nitrogen in the form of glutamine and alanine, rather than as free ammonia (see Fig. 19.13). 1. Urea: Formation of urea in the liver is quantitatively the most important disposal route for ammonia. Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate. 2.


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Biochemistry_Lippinco. 2. Glutamine: This amide of glutamate provides a nontoxic storage and transport form of ammonia (Fig. 19.18). The ATP-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in skeletal muscle and the liver but is also important in the CNS, where it is the major mechanism for the removal of ammonia in the brain. Glutamine is found in plasma at concentrations higher than other amino acids, a finding consistent with its transport function. [Note: The liver keeps blood ammonia levels low through glutaminase, GDH, and the urea cycle in periportal (close to inflow of blood) hepatocytes and through glutamine synthetase as an ammonia scavenger in the perivenous hepatocytes.] Ammonia metabolism is summarized in Figure 19.19. C. Hyperammonemia


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Hyperammonemia The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of blood ammonia are normally low (5–35 µmol/l). However, when liver function is compromised, due either to genetic defects of the urea cycle or liver disease, blood levels can be >1,000 µmol/l. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS. For example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence (drowsiness), vomiting, cerebral edema, and blurring of vision. At high concentrations, ammonia can cause coma and death. There are two major types of hyperammonemia. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Acquired: Liver disease is a common cause of acquired hyperammonemia in adults and may be due, for example, to viral hepatitis or to hepatotoxins such as alcohol. Cirrhosis of the liver may result in formation of collateral circulation around the liver. As a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. Therefore, the conversion of ammonia to urea is severely impaired, leading to elevated levels of ammonia. 2.


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Biochemistry_Lippinco. Congenital: Genetic deficiencies of each of the five enzymes of the urea cycle (and of NAGS) have been described, with an overall incidence of ~1:25,000 live births. X-linked OTC deficiency is the most common of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal-recessive inheritance pattern. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. [Note: The hyperammonemia seen with arginase deficiency is less severe because arginine contains two waste nitrogens and can be excreted in the urine.] Historically, urea cycle defects had high morbidity (neurologic manifestations) and mortality. Treatment included restriction of dietary protein in the presence of sufficient calories to prevent protein catabolism. Administration of compounds that bind covalently to nonessential amino acids, producing nitrogen-containing molecules that


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Biochemistry_Lippinco. in the presence of sufficient calories to prevent protein catabolism. Administration of compounds that bind covalently to nonessential amino acids, producing nitrogen-containing molecules that are excreted in the urine, has improved survival. For example, phenylbutyrate given orally is converted to phenylacetate. This condenses with glutamine to form phenylacetylglutamine, which is excreted (Fig. 19.20).


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VII. CHAPTER SUMMARY

Biochemistry_Lippinco. VII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (Fig. 19.21). Free amino acids in the body are produced by hydrolysis of dietary protein by proteases activated from their zymogen form in the stomach and intestine, degradation of tissue proteins, and de novo synthesis. This amino acid pool is consumed in the synthesis of body protein, metabolized for energy, or its members used as precursors for other nitrogen-containing compounds. Free amino acids from digestion are taken up by intestinal enterocytes via sodium-dependent secondary active transport. Small peptides are taken up via proton-linked transport. Note that body protein is simultaneously degraded and resynthesized, a process known as protein turnover. The concentration of a cellular protein may be determined by regulation of its synthesis or


Biochemistry_Lippinco

Biochemistry_Lippinco. Note that body protein is simultaneously degraded and resynthesized, a process known as protein turnover. The concentration of a cellular protein may be determined by regulation of its synthesis or degradation. The ATP-dependent, cytosolic, selective ubiquitin–proteasome and ATP-independent, relatively nonselective lysosomal acid hydrolases are the two major enzyme systems that are responsible for degrading proteins. Nitrogen cannot be stored, and amino acids in excess of the biosynthetic needs of the cell are quickly degraded. The first phase of catabolism involves the transfer of the α-amino groups through transamination by pyridoxal phosphate–dependent aminotransferases (transaminases), followed by oxidative deamination of glutamate by glutamate dehydrogenase, forming ammonia and the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine. Some ammonia is used in converting glutamate to glutamine for safe transport, but most is used in the hepatic


Biochemistry_Lippinco

Biochemistry_Lippinco. the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine. Some ammonia is used in converting glutamate to glutamine for safe transport, but most is used in the hepatic synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. Alanine also carries nitrogen to the liver for disposal as urea. The two major causes of hyperammonemia (with its neurologic effects) are acquired liver disease and congenital deficiencies of urea cycle enzymes such as X-linked ornithine transcarbamylase.


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Biochemistry_Lippinco. Choose the ONE best answer. 9.1. In this transamination reaction (right), which of the following are the products X and Y? A. Alanine, α-ketoglutarate B. Aspartate, α-ketoglutarate C. Glutamate, alanine D. Pyruvate, aspartate Correct answer = B. Transamination reactions always have an amino acid and an α-keto acid as substrates. The products of the reaction are also an amino acid (corresponding to the α-keto substrate) and an α-keto acid (corresponding to the amino acid substrate). Three amino acid α-keto acid pairs commonly encountered in metabolism are alanine/pyruvate, aspartate/oxaloacetate, and glutamate/α-ketoglutarate. In this question, glutamate is deaminated to form αketoglutarate, and oxaloacetate is aminated to form aspartate. 9.2. Which one of the following statements about amino acids and their metabolism is correct? A. Free amino acids are taken into the enterocytes by a single proton-linked transport system.


Biochemistry_Lippinco

Biochemistry_Lippinco. 9.2. Which one of the following statements about amino acids and their metabolism is correct? A. Free amino acids are taken into the enterocytes by a single proton-linked transport system. B. In healthy, well-fed individuals, the input to the amino acid pool exceeds the output. C. The liver uses ammonia to buffer protons. D. Muscle-derived glutamine is metabolized in liver and kidney tissue to ammonia + a gluconeogenic precursor. E. The first step in the catabolism of most amino acids is their oxidative deamination. F. The toxic ammonia generated from the amide nitrogen of amino acids is transported through blood as arginine.


Biochemistry_Lippinco

Biochemistry_Lippinco. F. The toxic ammonia generated from the amide nitrogen of amino acids is transported through blood as arginine. Correct answer = D. Glutamine, produced by the catabolism of branched-chain amino acids in muscle, is deaminated by glutaminase to ammonia + glutamate. The glutamate is deaminated by glutamate dehydrogenase to ammonia + αketoglutarate, which can be used for gluconeogenesis. Free amino acids are taken into enterocytes by several different sodium-linked transport systems. Healthy, well-fed individuals are in nitrogen balance, in which nitrogen input equals output. The liver converts ammonia to urea, and the kidneys use ammonia to buffer protons. Amino acid catabolism begins with transamination that generates glutamate. The glutamate undergoes oxidative deamination. Toxic ammonia is transported as glutamine and alanine. Arginine is synthesized and hydrolyzed in the hepatic urea cycle. For Questions 19.3–19.5, use the following scenario.


Biochemistry_Lippinco

Biochemistry_Lippinco. For Questions 19.3–19.5, use the following scenario. A female neonate appeared healthy until age ~24 hours, when she became lethargic. A sepsis workup proved negative. At 56 hours, she started showing focal seizure activity. The plasma ammonia level was found to be 887 µmol/l (normal 5–35 µmol/l). Quantitative plasma amino acid levels revealed a marked elevation of citrulline but not argininosuccinate. 9.3. Which one of the following enzymic activities is most likely to be deficient in this patient? A. Arginase B. Argininosuccinate lyase C. Argininosuccinate synthetase D. Carbamoyl phosphate synthetase I


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Arginase B. Argininosuccinate lyase C. Argininosuccinate synthetase D. Carbamoyl phosphate synthetase I E. Ornithine transcarbamylase Correct answer = C. Genetic deficiencies of each of the five enzymes of the urea cycle, as well as deficiencies in N-acetylglutamate synthase, have been described. The accumulation of citrulline (but not argininosuccinate) in the plasma of this patient means that the enzyme required for the conversion of citrulline to argininosuccinate (argininosuccinate synthetase) is defective, whereas the enzyme that cleaves argininosuccinate (argininosuccinate lyase) is functional. 9.4. Which one of the following would also be elevated in the blood of this patient? A. Asparagine B. Glutamine C. Lysine D. Urea


Biochemistry_Lippinco

Biochemistry_Lippinco. 9.4. Which one of the following would also be elevated in the blood of this patient? A. Asparagine B. Glutamine C. Lysine D. Urea Correct answer = B. Deficiencies of the enzymes of the urea cycle result in the failure to synthesize urea and lead to hyperammonemia in the first few weeks after birth. Glutamine will also be elevated because it acts as a nontoxic storage and transport form of ammonia. Therefore, elevated glutamine accompanies hyperammonemia. Asparagine and lysine do not serve this sequestering role. Urea would be decreased because of impaired activity of the urea cycle. [Note: Alanine would also be elevated in this patient.] 9.5. Why might supplementation with arginine be of benefit to this patient? The arginine will be cleaved by arginase to urea and ornithine. Ornithine will be combined with carbamoyl phosphate by ornithine transcarbamylase to form citrulline. Citrulline, containing one waste nitrogen, will be excreted. Amino Acids: Degradation and Synthesis 20


Biochemistry_Lippinco

Biochemistry_Lippinco. Amino Acids: Degradation and Synthesis 20 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Amino acid degradation involves removal of the α-amino group, followed by the catabolism of the resulting α-keto acids (carbon skeletons). These pathways converge to form seven intermediate products: oxaloacetate, pyruvate, αketoglutarate, fumarate, succinyl coenzyme A (CoA), acetyl CoA, and acetoacetate. The products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose, ketone bodies, or lipids or in the production of energy through their oxidation to carbon dioxide (CO2) by the tricarboxylic acid (TCA) cycle. Figure 20.1 provides an overview of these pathways, with a more detailed summary presented in Figure 20.15 (see p. 269). Nonessential amino acids (Fig. 20.2) can be synthesized in sufficient amounts from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, because the essential amino acids cannot be synthesized (or synthesized in sufficient amounts) by humans, they


Biochemistry_Lippinco

Biochemistry_Lippinco. or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, because the essential amino acids cannot be synthesized (or synthesized in sufficient amounts) by humans, they must be obtained from the diet in order for normal protein synthesis to occur. Genetic defects in the pathways of amino acid metabolism can cause serious disease.


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Biochemistry_Lippinco. postoperative infections, and immunosuppression.] II. GLUCOGENIC AND KETOGENIC AMINO ACIDS Amino acids can be classified as glucogenic, ketogenic, or both, based on which of the seven intermediates are produced during their catabolism (see Fig. 20.2). A. Glucogenic amino acids Amino acids whose catabolism yields pyruvate or one of the intermediates of the TCA cycle are termed glucogenic. Because these intermediates are substrates for gluconeogenesis (see p. 118), they can give rise to the net synthesis of glucose in the liver and kidney. B. Ketogenic amino acids


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Ketogenic amino acids Amino acids whose catabolism yields either acetoacetate or one of its precursors (acetyl CoA or acetoacetyl CoA) are termed ketogenic (see Fig. 20.2). Acetoacetate is one of the ketone bodies, which also include 3hydroxybutyrate and acetone (see p. 195). Leucine and lysine are the only exclusively ketogenic amino acids found in proteins. Their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net synthesis of glucose. III. AMINO ACID CARBON SKELETON The pathways by which amino acids are catabolized are conveniently organized according to which one (or more) of the seven intermediates listed above is produced from a particular amino acid. A. Amino acids that form oxaloacetate


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Amino acids that form oxaloacetate Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate (Fig. 20.3). Aspartate loses its amino group by transamination to form oxaloacetate (see Fig. 20.3). [Note: Some rapidly dividing leukemic cells are unable to synthesize sufficient asparagine to support their growth. This makes asparagine an essential amino acid for these cells, which, therefore, require asparagine from the blood. Asparaginase, which hydrolyzes asparagine to aspartate, can be administered systemically to treat leukemia. Asparaginase lowers the level of asparagine in the plasma, thereby depriving cancer cells of a required nutrient.] B. Amino acids that form α-ketoglutarate via glutamate 1. Glutamine: This amino acid is hydrolyzed to glutamate and ammonia by the enzyme glutaminase (see p. 256). Glutamate is converted to αketoglutarate by transamination or through oxidative deamination by glutamate dehydrogenase (see p. 252). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Proline: This amino acid is oxidized to glutamate. Glutamate is transaminated or oxidatively deaminated to form α-ketoglutarate. 3. Arginine: This amino acid is hydrolyzed by arginase to produce ornithine (and urea). [Note: The reaction occurs primarily in the liver as part of the urea cycle (see p. 255).] Ornithine is subsequently converted to α-ketoglutarate, with glutamate semialdehyde as an intermediate. 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Histidine: This amino acid is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate ([FIGlu], Fig. 20.4). FIGlu donates its formimino group to tetrahydrofolate (THF), leaving glutamate, which is degraded as described above. [Note: Individuals deficient in folic acid excrete increased amounts of FIGlu in the urine, particularly after ingestion of a large dose of histidine. The FIGlu excretion test has been used in diagnosing a deficiency of folic acid. See p. 267 for a discussion of folic acid, THF, and one-carbon metabolism.] C. Amino acids that form pyruvate 1. Alanine: This amino acid loses its amino group by transamination to 2. Serine: This amino acid can be converted to glycine as THF becomes N5,N10-methylenetetrahydrofolate (N5,N10-MTHF), as shown in Figure 20.6A. Serine can also be converted to pyruvate (see Fig. 20.6B). 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Glycine: This amino acid can be converted to serine by the reversible addition of a methylene group from N5,N10-MTHF (see Fig. 20.6A) or oxidized to CO2 and ammonia by the glycine cleavage system. [Note: Glycine can be deaminated to glyoxylate (by a d-amino acid oxidase; see form pyruvate (Fig. 20.5). [Note: Tryptophan catabolism produces alanine and, therefore, pyruvate (see Fig. 20.10 on p. 265).] p. 253), which can be oxidized to oxalate or transaminated to glycine. Deficiency of the transaminase in liver peroxisomes causes overproduction of oxalate, the formation of oxalate stones, and kidney damage (primary oxaluria type 1).] 4. Cysteine: This sulfur-containing amino acid undergoes desulfurization to yield pyruvate. [Note: The sulfate released can be used to synthesize 3′phosphoadenosine-5′-phosphosulfate (PAPS), an activated sulfate donor to a variety of acceptors.] Cysteine can also be oxidized to its disulfide derivative, cystine. 5.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. Threonine: This amino acid is converted to pyruvate in most organisms but is a minor pathway (at best) in humans. D. Amino acids that form fumarate 1. Phenylalanine and tyrosine: Hydroxylation of phenylalanine produces tyrosine (Fig. 20.7). This irreversible reaction, catalyzed by tetrahydrobiopterin-requiring phenylalanine hydroxylase (PAH), initiates the catabolism of phenylalanine. Thus, phenylalanine metabolism and tyrosine metabolism merge, leading ultimately to fumarate and acetoacetate formation. Therefore, phenylalanine and tyrosine are both glucogenic and ketogenic. 2. Inherited deficiencies: Inherited deficiencies in the enzymes of phenylalanine and tyrosine metabolism lead to the diseases phenylketonuria (PKU) (see p. 270), tyrosinemia (see p. 274), and alkaptonuria (see p. 274) as well as the condition of albinism (see p. 273). E. Amino acids that form succinyl CoA: Methionine


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Amino acids that form succinyl CoA: Methionine Methionine is one of four amino acids that form succinyl CoA. This sulfur-containing amino acid deserves special attention because it is converted to S-adenosylmethionine (SAM), the major methyl group donor in one-carbon metabolism (Fig. 20.8). Methionine is also the source of homocysteine (Hcy), a metabolite associated with atherosclerotic vascular disease and thrombosis (see p. 265). the methyl group carrier and donor.] PPi = pyrophosphate; Pi = inorganic phosphate; NH3 = ammonia. 1. S-Adenosylmethionine synthesis: Methionine condenses with ATP, forming SAM, a high-energy compound that is unusual in that it contains no phosphate. The formation of SAM is driven by hydrolysis of all three phosphate bonds in ATP (see Fig. 20.8). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Activated methyl group: The methyl group attached to the sulfur in SAM is activated and can be transferred by methyltransferases to a variety of acceptors such as norepinephrine in the synthesis of epinephrine. The methyl group is usually transferred to nitrogen or oxygen atoms (as with epinephrine synthesis and degradation, respectively; see p. 286) and sometimes to carbon atoms (as with cytosine). The reaction product, Sadenosylhomocysteine (SAH), is a simple thioether, analogous to methionine. The resulting loss of free energy makes methyl transfer essentially irreversible. 3. S-Adenosylhomocysteine hydrolysis: After donation of the methyl group, SAH is hydrolyzed to Hcy and adenosine. Hcy has two fates. If there is a deficiency of methionine, Hcy may be remethylated to methionine (see Fig. 20.8). If methionine stores are adequate, Hcy may enter the transsulfuration pathway, where it is converted to cysteine.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Methionine resynthesis: Hcy accepts a methyl group from N5methyltetrahydrofolate (N5-methyl-THF) in a reaction requiring methylcobalamin, a coenzyme derived from vitamin B12 (see p. 379). [Note: The methyl group is transferred by methionine synthase from the B12 derivative to Hcy, regenerating methionine. Cobalamin is remethylated from N5-methyl-THF.] b. Cysteine synthesis: Hcy condenses with serine, forming cystathionine, which is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). This vitamin B6–requiring sequence has the net effect of converting serine to cysteine and Hcy to α-ketobutyrate, which is oxidatively decarboxylated to form propionyl CoA. Propionyl CoA is converted to succinyl CoA (see Fig. 16.20 on p. 195). Because Hcy is synthesized from the essential amino acid methionine, cysteine is not an essential amino acid as long as sufficient methionine is available.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Relationship of homocysteine to vascular disease: Elevations in plasma Hcy levels promote oxidative damage, inflammation, and endothelial dysfunction and are an independent risk factor for occlusive vascular diseases such as cardiovascular disease (CVD) and stroke (Fig. 20.9). Mild elevations (hyperhomocysteinemia) are seen in ~7% of the population. Epidemiologic studies have shown that plasma Hcy levels are inversely related to plasma levels of folate, B12, and B6, the three vitamins involved in the conversion of Hcy to methionine and cysteine. Supplementation with these vitamins has been shown to reduce circulating levels of Hcy. However, in patients with established CVD, vitamin therapy does not decrease cardiovascular events or death. This raises the question as to whether Hcy is a cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma Hcy as a result of rare deficiencies in cystathionine β-synthase of the transsulfuration pathway are


Biochemistry_Lippinco

Biochemistry_Lippinco. cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma Hcy as a result of rare deficiencies in cystathionine β-synthase of the transsulfuration pathway are seen in patients with classic homocystinuria (resulting from severe hyperhomocysteinemia [>100 µmol/l], see p. 273).] Deficiencies in the remethylation reaction also result in a rise in Hcy.


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Biochemistry_Lippinco. Elevated homocysteine and decreased folic acid levels in pregnant women are associated with increased incidence of neural tube defects (improper closure, as in spina bifida) in the fetus. Periconceptual supplementation with folate reduces the risk of such defects. F. Other amino acids that form succinyl CoA Degradation of valine, isoleucine, and threonine also results in the production of succinyl CoA, a TCA cycle intermediate and gluconeogenic compound. [Note: It is metabolized to pyruvate.] 1. Valine and isoleucine: These amino acids are branched-chain amino acids (BCAA) that generate propionyl CoA, which is converted to methylmalonyl CoA and then succinyl CoA by biotin-and vitamin B12 – requiring reactions. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Threonine: This amino acid is dehydrated to α-ketobutyrate, which is converted to propionyl CoA and then to succinyl CoA. Propionyl CoA, then, is generated by the catabolism of the amino acids methionine, valine, isoleucine, and threonine. [Note: Propionyl CoA also is generated by the oxidation of odd-numbered fatty acids (see p. 193).] G. Amino acids that form acetyl CoA or acetoacetyl CoA Tryptophan, leucine, isoleucine, and lysine form acetyl CoA or acetoacetyl CoA directly, without pyruvate serving as an intermediate. As noted earlier, phenylalanine and tyrosine also give rise to acetoacetate during their catabolism (see Fig. 20.7). Therefore, there are a total of six partly or wholly ketogenic amino acids. 1. Tryptophan: This amino acid is both glucogenic and ketogenic, because its catabolism yields alanine and acetoacetyl CoA (Fig. 20.10). [Note: Quinolinate from tryptophan catabolism is used in the synthesis of nicotinamide adenine dinucleotide ([NAD], see p. 383).] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Leucine: This amino acid is exclusively ketogenic, because its catabolism yields acetyl CoA and acetoacetate (Fig. 20.11). The first two reactions in the catabolism of leucine and the other BCAA, isoleucine and valine, are catalyzed by enzymes that use all three BCAA (or their derivatives) as substrates (see H. below). 3. Isoleucine: This amino acid is both ketogenic and glucogenic, because its metabolism yields acetyl CoA and propionyl CoA. 4. Lysine: This amino acid is exclusively ketogenic and is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. Lysine is ultimately converted to acetoacetyl CoA. H. Branched-chain amino acid degradation


Biochemistry_Lippinco

Biochemistry_Lippinco. H. Branched-chain amino acid degradation The BCAA isoleucine, leucine, and valine are essential amino acids. In contrast to other amino acids, they are catabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. Because these three amino acids have a similar route of degradation, it is convenient to describe them as a group (see Fig. 20.11). 1. Transamination: Transfer of the amino groups of all three BCAA to α ketoglutarate is catalyzed by a single, vitamin B6–requiring enzyme, branched-chain amino acid aminotransferase, that is expressed primarily in skeletal muscle. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Oxidative decarboxylation: Removal of the carboxyl group of the α-keto acids derived from leucine, valine, and isoleucine is catalyzed by a single multienzyme complex, branched-chain α-keto acid dehydrogenase (BCKD) complex. This complex uses thiamine pyrophosphate, lipoic acid, oxidized flavin adenine dinucleotide (FAD), NAD+, and CoA as its coenzymes and produces NADH. [Note: This reaction is similar to the conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase (PDH) complex (see p. 109) and α-ketoglutarate to succinyl CoA by the α-ketoglutarate dehydrogenase complex (see p. 112). The dihydrolipoyl dehydrogenase (Enzyme 3, or E3) component is identical in all three complexes.] 3.


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Biochemistry_Lippinco. Dehydrogenations: Oxidation of the products formed in the BCKD reaction produces α-β-unsaturated acyl CoA derivatives and FADH2. These reactions are analogous to the FAD-linked dehydrogenation in the β-oxidation of fatty acids (see p. 192). [Note: Deficiency in the dehydrogenase specific for isovaleryl CoA causes neurologic problems and is associated with a “sweaty feet” odor in body fluids.] 4. End products: The catabolism of isoleucine ultimately yields acetyl CoA and succinyl CoA, rendering it both ketogenic and glucogenic. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetoacetate and acetyl CoA. In addition, NADH and FADH2 are produced in the decarboxylation and dehydrogenation reactions, respectively. [Note: BCAA catabolism also results in glutamine and alanine being synthesized and sent out into the blood from muscle (see p. 253).] IV. FOLIC ACID AND AMINO ACID METABOLISM


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. FOLIC ACID AND AMINO ACID METABOLISM Some synthetic pathways require the addition of single-carbon groups that exist in a variety of oxidation states, including formyl, methenyl, methylene, and methyl. These single-carbon groups can be transferred from carrier compounds such as THF and SAM to specific structures that are being synthesized or modified. The “one-carbon pool” refers to the single-carbon units attached to this group of carriers. [Note: CO2, coming from bicarbonate (HCO3–), is carried by the vitamin biotin (see p. 385), which is a prosthetic group for most carboxylation reactions but is not considered a member of the one-carbon pool. Defects in the ability to add or remove biotin from carboxylases result in multiple carboxylase deficiency. Treatment is supplementation with biotin.] A. Folic acid and one-carbon metabolism


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Folic acid and one-carbon metabolism The active form of folic acid, THF, is produced from folate by dihydrofolate reductase in a two-step reaction requiring two nicotinamide adenine dinucleotide phosphate (NADPH). The one-carbon unit carried by THF is bound to N5 or N10 or to both N5 and N10 . Figure 20.12 shows the structures of the various members of the THF family and their interconversions and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. [Note: Folate deficiency presents as a megaloblastic anemia because of decreased availability of the purines and of the thymidine monophosphate needed for DNA synthesis (see p. 303).] V. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS


Biochemistry_Lippinco

Biochemistry_Lippinco. V. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS Nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from the essential amino acids phenylalanine and methionine, respectively. The synthetic reactions for the nonessential amino acids are described below and are summarized in Figure 20.15. [Note: Some amino acids found in proteins, such as hydroxyproline and hydroxylysine (see p. 45), are produced by posttranslational modification (after incorporation into a protein) of their precursor (parent) amino acids.] A. Synthesis from α-keto acids


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Synthesis from α-keto acids Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the α-keto acids pyruvate, oxaloacetate, and α-ketoglutarate, respectively. These transamination reactions (Fig. 20.13; also see p. 250) are the most direct of the biosynthetic pathways. Glutamate is unusual in that it can also be synthesized by reversal of oxidative deamination, catalyzed by glutamate dehydrogenase, when ammonia levels are high (see p. 252). B. Synthesis by amidation 1. Glutamine: This amino acid, which contains an amide linkage with ammonia at the γ-carboxyl, is formed from glutamate by glutamine synthetase (see Fig. 19.18, p. 256). The reaction is driven by the hydrolysis of ATP. In addition to producing glutamine for protein synthesis, the reaction also serves as a major mechanism for the transport of ammonia in a nontoxic form. (See p. 256 for a discussion of ammonia metabolism.) 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Asparagine: This amino acid, which contains an amide linkage with ammonia at the β-carboxyl, is formed from aspartate by asparagine synthetase, using glutamine as the amide donor. Like the synthesis of glutamine, the reaction requires ATP and has an equilibrium far in the direction of amide synthesis. C. Proline Glutamate via glutamate semialdehyde is converted to proline by cyclization and reduction reactions. [Note: The semialdehyde can also be transaminated to ornithine.] D. Serine, glycine, and cysteineThe pathways of synthesis for these amino acids are interconnected. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Serine, glycine, and cysteineThe pathways of synthesis for these amino acids are interconnected. 1. Serine: This amino acid arises from 3-phosphoglycerate, a glycolytic intermediate (see Fig. 8.18, p. 101), which is first oxidized to 3phosphopyruvate and then transaminated to 3-phosphoserine. Serine is formed by hydrolysis of the phosphate ester. Serine can also be formed from glycine through transfer of a hydroxymethyl group by serine hydroxymethyltransferase using N5,N10-MTHF as the one-carbon donor (see Fig. 20.6A). [Note: Selenocysteine (Sec), the 21st genetically encoded amino acid, is synthesized from serine and selenium (see p. 407), while serine is attached to transfer RNA. Sec is found in ~25 human proteins including glutathione peroxidase (see p. 148) and thioredoxin reductase (see p. 297).] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycine: This amino acid is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyltransferase (see Fig. 20.6A). THF is the one-carbon acceptor. 3. Cysteine: This amino acid is synthesized by two consecutive reactions in which Hcy combines with serine, forming cystathionine, which, in turn, is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). [Note: Hcy is derived from methionine, as described on p. 264. Because methionine is an essential amino acid, cysteine synthesis requires adequate dietary intake of methionine.] E. Tyrosine


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Tyrosine Tyrosine is formed from phenylalanine by PAH (see p. 263). The reaction requires molecular oxygen and the coenzyme tetrahydrobiopterin (BH4), which is synthesized from guanosine triphosphate. One atom of molecular oxygen becomes the hydroxyl group of tyrosine, and the other atom is reduced to water. During the reaction, BH4 is oxidized to dihydrobiopterin (BH2). BH4 is regenerated from BH2 by NADH-requiring dihydropteridine reductase. Tyrosine, like cysteine, is formed from an essential amino acid and is, therefore, nonessential only in the presence of adequate dietary phenylalanine. VI. AMINO ACID METABOLISM DISORDERS


Biochemistry_Lippinco

Biochemistry_Lippinco. VI. AMINO ACID METABOLISM DISORDERS These single gene disorders, a subset of the inborn errors of metabolism, are caused by mutations that generally result in abnormal proteins, most often enzymes. The inherited defects may be expressed as a total loss of enzyme activity or, more frequently, as a partial deficiency in catalytic activity. Without treatment, the amino acid disorders almost invariably result in intellectual disability or other developmental abnormalities as a consequence of harmful accumulation of metabolites. Although >50 of these disorders have been described, many are rare, occurring in <1 per 250,000 in most populations (Fig. 20.14). Collectively, however, they constitute a very significant portion of pediatric genetic diseases (Fig. 20.15).


Biochemistry_Lippinco

Biochemistry_Lippinco. boxes. Classification of amino acids is color coded: Red = glucogenic; brown = glucogenic and ketogenic; green = ketogenic. Compounds in BLUE ALL CAPS are the seven metabolites to which all amino acid metabolism converges. CoA = coenzyme A; NAD(H) = nicotinamide adenine dinucleotide. A. Phenylketonuria


Biochemistry_Lippinco

Biochemistry_Lippinco. PKU is the most common clinically encountered inborn error of amino acid metabolism (incidence 1:15,000). It is caused by a deficiency of PAH (Fig. 20.16). Biochemically, PKU is characterized by hyperphenylalaninemia. Phenylalanine is present in high concentrations (ten times normal) not only in plasma but also in urine and body tissues. Tyrosine, which normally is formed from phenylalanine by PAH, is deficient. Treatment includes dietary restriction of phenylalanine and supplementation with tyrosine. [Note: Hyperphenylalaninemia may also be caused by rare deficiencies in any of the several enzymes required to synthesize BH4 or in dihydropteridine reductase, which regenerates BH4 from BH2 (Fig. 20.17). Such deficiencies indirectly raise phenylalanine concentrations, because PAH requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the


Biochemistry_Lippinco

Biochemistry_Lippinco. requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the catecholamines. Simply restricting dietary phenylalanine does not reverse the central nervous system effects due to deficiencies in neurotransmitters. Supplementation with BH4 and replacement therapy with L-3,4 dihydroxyphenylalanine and 5-hydroxytryptophan (products of the affected tyrosine hydroxylase– and tryptophan hydroxylase–catalyzed reactions) improves the clinical outcome in these variant forms of hyperphenylalaninemia, although the response is unpredictable.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Screening of newborns for a number of treatable disorders, including inborn errors of amino acid metabolism, is done by tandem mass spectrometry of blood obtained from a heel prick. By law, all states must screen for >20 disorders, with some screening for >50. All states screen for PKU. 1. Additional characteristics: As the name suggests, PKU is also characterized by elevated levels of a phenylketone in the urine. a. Elevated phenylalanine metabolites: Phenylpyruvate (a phenylketone), phenylacetate, and phenyllactate, which are not normally produced in significant amounts in the presence of functional PAH, are elevated in PKU (Fig. 20.18). These metabolites give urine a characteristic musty (“mousy”) odor. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Central nervous system effects: Severe intellectual disability, developmental delay, microcephaly, and seizures are characteristic findings in untreated PKU. The affected individual typically shows symptoms of intellectual disability by age 1 year and rarely achieves an intelligence quotient (IQ) >50 (Fig. 20.19). [Note: These clinical manifestations are now rarely seen as a result of newborn screening programs, which allow early diagnosis and treatment.] c. Hypopigmentation: Patients with untreated PKU may show a deficiency of pigmentation (fair hair, light skin color, and blue eyes). The hydroxylation of tyrosine by copper-requiring tyrosinase, which is the first step in the formation of the pigment melanin, is decreased in PKU because tyrosine is decreased. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Newborn screening and diagnosis: Early diagnosis of PKU is important because the disease is treatable by dietary means. Because of the lack of neonatal symptoms, laboratory testing for elevated blood levels of phenylalanine is mandatory for detection. However, the infant with PKU frequently has normal blood levels of phenylalanine at birth because the mother clears increased blood phenylalanine in her affected fetus through the placenta. Normal levels of phenylalanine may persist until the newborn is exposed to 24–48 hours of protein feeding. Thus, screening tests are typically done after this time to avoid false negatives. For newborns with a positive screening test, diagnosis is confirmed through quantitative determination of phenylalanine levels. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Prenatal diagnosis: Classic PKU is caused by any of 100 or more different mutations in the gene that encodes PAH. The frequency of any given mutation varies among populations, and the disease is often doubly heterozygous (that is, the PAH gene has a different mutation in each allele). Despite this complexity, prenatal diagnosis is possible (see p. 493). 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Treatment: Because most natural protein contains phenylalanine, an essential amino acid, it is impossible to satisfy the body’s protein requirement without exceeding the phenylalanine limit when ingesting a normal diet. Therefore, in PKU, blood phenylalanine level is maintained close to the normal range by feeding synthetic amino acid preparations free of phenylalanine, supplemented with some natural foods (such as fruits, vegetables, and certain cereals) selected for their low phenylalanine content. The amount is adjusted according to the tolerance of the individual as measured by blood phenylalanine levels. The earlier treatment is started, the more completely neurologic damage can be prevented. Individuals who are appropriately treated can have normal intelligence. [Note: Treatment must begin during the first 7–10 days of life to prevent cognitive impairment.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below


Biochemistry_Lippinco

Biochemistry_Lippinco. begin during the first 7–10 days of life to prevent cognitive impairment.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below normal is avoided. In patients with PKU, tyrosine cannot be synthesized from phenylalanine, and, therefore, it becomes an essential amino acid and must be supplied in the diet. Discontinuance of the phenylalanine-restricted diet in early childhood is associated with poor performance on IQ tests. Adult PKU patients show deterioration of IQ scores after discontinuation of the diet (Fig. 20.20). Therefore, lifelong restriction of dietary phenylalanine is recommended. [Note: Individuals with PKU are advised to avoid aspartame, an artificial sweetener that contains phenylalanine.] 5. Maternal phenylketonuria: If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring can be affected with maternal PKU syndrome. High blood phenylalanine in the mother has a teratogenic


Biochemistry_Lippinco

Biochemistry_Lippinco. If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring can be affected with maternal PKU syndrome. High blood phenylalanine in the mother has a teratogenic effect, causing microcephaly and congenital heart abnormalities in the fetus. Because these developmental responses to high phenylalanine occur during the first months of pregnancy, dietary control of blood phenylalanine must begin prior to conception and be maintained throughout the pregnancy.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Maple syrup urine disease Maple syrup urine disease (MSUD) is a rare (1:185,000), autosomalrecessive disorder in which there is a partial or complete deficiency in BCKD, the mitochondrial enzyme complex that oxidatively decarboxylates leucine, isoleucine, and valine (see Fig. 20.11). These BCAA and their corresponding α-keto acids accumulate in the blood, causing a toxic effect that interferes with brain functions. The disease is characterized by feeding problems, vomiting, ketoacidosis, changes in muscle tone, neurologic problems that can result in coma (primarily because of the rise in leucine), and a characteristic maple syrup–like odor of the urine because of the rise in isoleucine. If untreated, the disease is fatal. If treatment is delayed, intellectual disability results. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Classification: MSUD includes a classic type and several variant forms. The classic, neonatal-onset form is the most common type of MSUD. Leukocytes or cultured skin fibroblasts from these patients show little or no BCKD activity. Infants with classic MSUD show symptoms within the first several days of life. If not diagnosed and treated, classic MSUD is lethal in the first weeks of life. Patients with intermediate forms have a higher level of enzyme activity (up to 30% of normal). The symptoms are milder and show an onset from infancy to adolescence. Patients with the rare thiamine-dependent variant of MSUD respond to large doses of this vitamin. 2. Screening and diagnosis: As with PKU, prenatal diagnosis and newborn screening are available, and most affected individuals are compound heterozygotes. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Screening and diagnosis: As with PKU, prenatal diagnosis and newborn screening are available, and most affected individuals are compound heterozygotes. 3. Treatment: MSUD is treated with a synthetic formula that is free of BCAA, supplemented with limited amounts of leucine, isoleucine, and valine to allow for normal growth and development without producing toxic levels. [Note: Elevated leucine is the cause of the neurologic damage in MSUD, and its level is carefully monitored.] Early diagnosis and lifelong dietary treatment are essential if the child with MSUD is to develop normally. [Note: BCAA are an important energy source in times of metabolic need, and individuals with MSUD are at risk of decompensation during periods of increased protein catabolism.] C. Albinism


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Albinism Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a deficiency in the production of melanin. These defects result in the partial or full absence of pigment from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of several modes: autosomal recessive (primary mode), autosomal dominant, or X linked. Total absence of pigment from the hair, eyes, and skin (Fig. 20.21), tyrosinase-negative oculocutaneous albinism (type 1 albinism), results from an absent or defective copper-requiring tyrosinase. It is the most severe form of the condition. In addition to hypopigmentation, affected individuals have vision defects and photophobia (sunlight hurts their eyes). They are at increased risk for skin cancer. D. Homocystinuria


Biochemistry_Lippinco

Biochemistry_Lippinco. The homocystinurias are a group of disorders involving defects in the metabolism of Hcy. These autosomal-recessive diseases are characterized by high urinary levels of Hcy, high plasma levels of Hcy and methionine, and low plasma levels of cysteine. The most common cause of homocystinuria is a defect in the enzyme cystathionine β-synthase, which converts Hcy to cystathionine (Fig. 20.22). Individuals homozygous for cystathionine β-synthase deficiency exhibit dislocation of the lens (ectopia lentis), skeletal anomalies (long limbs and fingers), intellectual disability, and an increased risk for developing thrombi (blood clots). Thrombosis is the major cause of early death in these individuals. Treatment includes restriction of methionine and supplementation with vitamin B12 and folate. Additionally, some patients are responsive to oral administration of pyridoxine (vitamin B6), which is converted to pyridoxal phosphate, the coenzyme of cystathionine β-synthase. These patients usually


Biochemistry_Lippinco

Biochemistry_Lippinco. some patients are responsive to oral administration of pyridoxine (vitamin B6), which is converted to pyridoxal phosphate, the coenzyme of cystathionine β-synthase. These patients usually have a milder and later onset of clinical symptoms compared with B6nonresponsive patients. [Note: Deficiencies in methylcobalamin (see Fig.


Biochemistry_Lippinco

Biochemistry_Lippinco. 20.8) or N5,N10-MTHF reductase ([MTHFR]; see Fig. 20.12) also result in elevated Hcy.] E. Alkaptonuria Alkaptonuria is a rare organic aciduria involving a deficiency in homogentisic acid oxidase, resulting in the accumulation of homogentisic acid (HA), an intermediate in the degradative pathway of tyrosine (see Fig.


Biochemistry_Lippinco

Biochemistry_Lippinco. 20.15 on p. 269). The condition has three characteristic symptoms: homogentisic aciduria (the urine contains elevated levels of HA, which is oxidized to a dark pigment on standing, as shown in Fig. 20.23A), early onset of arthritis in the large joints, and deposition of black pigment (ochronosis) in cartilage and collagenous tissue (see Fig. 20.23B). Dark staining of diapers can indicate the disease in infants, but usually no symptoms are present until about age 40 years. Treatment includes dietary restriction of phenylalanine and tyrosine to reduce HA levels. Although alkaptonuria is not life threatening, the associated arthritis may be severely crippling. [Note: Deficiencies in fumarylacetoacetate hydrolase, the terminal enzyme of tyrosine metabolism, result in tyrosinemia type I (see Fig. 20.15) and a characteristic cabbage-like odor to urine.] VII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Amino acids whose catabolism yields pyruvate or an intermediate of the tricarboxylic acid cycle are termed glucogenic (Fig. 20.24). They can give rise to the net formation of glucose in the liver and kidneys. The solely glucogenic amino acids are glutamine, glutamate, proline, arginine, histidine, alanine, serine, glycine, cysteine, methionine, valine, threonine, aspartate, and asparagine. Amino acids whose catabolism yields either acetoacetate or one of its precursors, acetyl coenzyme A (CoA) or acetoacetyl CoA, are termed ketogenic. Leucine and lysine are solely ketogenic. Tyrosine, phenylalanine, tryptophan, and isoleucine are both ketogenic and glucogenic. Nonessential amino acids can be synthesized from metabolic intermediates or from the carbon skeletons of essential amino acids. Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine, and lysine. Phenylketonuria (PKU) is


Biochemistry_Lippinco

Biochemistry_Lippinco. Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine, and lysine. Phenylketonuria (PKU) is caused by a deficiency of phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine. Hyperphenylalaninemia may also be caused by deficiencies in the enzymes that synthesize or regenerate the coenzyme for PAH, tetrahydrobiopterin. Untreated individuals with PKU suffer from severe intellectual disability, developmental delay, microcephaly, seizures, and a characteristic musty (mousy) smell of the urine. Treatment involves controlling dietary phenylalanine. Tyrosine becomes an essential dietary component for people with PKU. Maple syrup urine disease (MSUD) is caused by a partial or complete deficiency in branched-chain a-keto acid dehydrogenase, the enzyme that decarboxylates the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Symptoms include feeding


Biochemistry_Lippinco

Biochemistry_Lippinco. or complete deficiency in branched-chain a-keto acid dehydrogenase, the enzyme that decarboxylates the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Symptoms include feeding problems, vomiting, ketoacidosis, changes in muscle tone, and a characteristic sweet smell of the urine. If untreated, the disease leads to neurologic problems that result in death. Treatment involves controlling BCAA intake. Other important genetic diseases associated with amino acid metabolism include albinism, homocystinuria, methylmalonic acidemia, alkaptonuria, histidinemia, tyrosinemia, and cystathioninuria.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. For Questions 20.1–20.3, match the deficient enzyme with the associated clinical sign or laboratory finding in urine. 0.1. Cystathionine β-synthase 0.2. Homogentisic acid oxidase 0.3. Tyrosinase


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.1. Cystathionine β-synthase 0.2. Homogentisic acid oxidase 0.3. Tyrosinase Correct answers = F, A, D. A deficiency in cystathionine β-synthase of methionine degradation results in a rise in homocysteine. A deficiency in homogentisic acid oxidase of tyrosine degradation results in a rise in homogentisic acid, which forms a black pigment that is deposited in connective tissue (ochronosis). A deficiency in tyrosinase results in decreased formation of melanin from tyrosine in the skin, hair, and eyes. A sweaty feet– like odor is characteristic of isovaleryl coenzyme A dehydrogenase deficiency. Cystine crystals in urine are seen with cystinuria, a defect in intestinal and renal cystine absorption. Increased branched-chain amino acids are seen in maple syrup urine disease, increased methionine is seen in defects in homocysteine metabolism, and increased phenylalanine is seen in phenylketonuria.


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.4. A 1-week-old infant, who was born at home in a rural, medicallyunderserved area, has undetected classic phenylketonuria. Which statement about this baby and/or her treatment is correct? A. A diet devoid of phenylalanine should be initiated immediately. B. Dietary treatment will be discontinued in adulthood. C. Supplementation with vitamin B6 is required. D. Tyrosine is an essential amino acid. Correct answer = D. In patients with phenylketonuria, tyrosine cannot be synthesized from phenylalanine and, hence, becomes essential and must be supplied in the diet. Phenylalanine in the diet must be controlled but cannot be eliminated entirely because it is an essential amino acid. Dietary treatment must begin during the first 7–10 days of life to prevent intellectual disability, and lifelong restriction of phenylalanine is recommended to prevent cognitive decline. Additionally, elevated levels of phenylalanine are teratogenic to a developing fetus.


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.5. Which one of the following statements concerning amino acids is correct? A. Alanine is ketogenic. B. Amino acids that are catabolized to acetyl coenzyme A are glucogenic. C. Branched-chain amino acids are catabolized primarily in the liver. D. Cysteine is essential for individuals consuming a diet severely limited in methionine. Correct answer = D. Methionine is the precursor of cysteine, which becomes essential if methionine is severely restricted. Alanine is a key glucogenic amino acid. Acetyl coenzyme A (CoA) cannot be used for the net synthesis of glucose. Amino acids catabolized to acetyl CoA are ketogenic. Branched-chain amino acids are catabolized primarily in skeletal muscle. 0.6. In an individual with the dihydrolipoyl dehydrogenase (E3)-deficient form of maple syrup urine disease, why would lactic acidosis be an expected finding?


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.6. In an individual with the dihydrolipoyl dehydrogenase (E3)-deficient form of maple syrup urine disease, why would lactic acidosis be an expected finding? The three α-keto acid dehydrogenase complexes (pyruvate dehydrogenase [PDH], α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase [BCKD]) have dihydrolipoyl dehydrogenase (Enzyme 3, or E3) in common. In E3-deficient maple syrup urine disease, in addition to the branched-chain amino acids and their α-keto acid derivatives accumulating as a result of decreased activity of BCKD, lactate will also be increased because of decreased activity of PDH. 0.7. In contrast to the vitamin B6–derived pyridoxal phosphate required in most enzymic reactions involving amino acids, what coenzyme is required by the aromatic amino acid hydroxylases? Tetrahydrobiopterin, made from guanosine triphosphate, is the required coenzyme. Amino Acids: Conversion to Specialized Products 21


Biochemistry_Lippinco

Biochemistry_Lippinco. Tetrahydrobiopterin, made from guanosine triphosphate, is the required coenzyme. Amino Acids: Conversion to Specialized Products 21 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW In addition to serving as building blocks for proteins, amino acids are precursors of many nitrogen-containing compounds that have important physiologic functions (Fig. 21.1). These molecules include porphyrins, neurotransmitters, hormones, purines, and pyrimidines. [Note: See p. 151 for the synthesis of nitric oxide from arginine.] II. PORPHYRIN METABOLISM


Biochemistry_Lippinco

Biochemistry_Lippinco. II. PORPHYRIN METABOLISM Porphyrins are cyclic compounds that readily bind metal ions, usually ferrous (Fe2+) or ferric (Fe3+) iron. The most prevalent metalloporphyrin in humans is heme, which consists of one Fe2+ coordinated in the center of the tetrapyrrole ring of protoporphyrin IX (see p. 279). Heme is the prosthetic group for hemoglobin (Hb), myoglobin, the cytochromes, the cytochrome P450 (CYP) monooxygenase system, catalase, nitric oxide synthase, and peroxidase. These hemeproteins are rapidly synthesized and degraded. For example, 6–7 g of Hb is synthesized each day to replace heme lost through the normal turnover of erythrocytes. The synthesis and degradation of the associated porphyrins and recycling of the iron are coordinated with the turnover of hemeproteins. A. Structure


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Structure Porphyrins are cyclic planar molecules formed by the linkage of four pyrrole rings through methenyl bridges (Fig. 21.2). Three structural features of these molecules are relevant to understanding their medical significance. 1. Side chains: Different porphyrins vary in the nature of the side chains attached to each of the four pyrrole rings. Uroporphyrin contains acetate (−CH2–COO–) and propionate (−CH2–CH2–COO–) side chains; coproporphyrin contains methyl (−CH3) and propionate groups; and protoporphyrin IX (and heme b, the most common heme) contains vinyl (−CH=CH2), methyl, and propionate groups. [Note: The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side chains, respectively.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Side chain distribution: The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, designated by Roman numerals I to IV. Only type III porphyrins, which contain an asymmetric substitution on ring D (see Fig. 21.2), are physiologically important in humans. [Note: Protoporphyrin IX is a member of the type III series.] 3. Porphyrinogens: These porphyrin precursors (for example, uroporphyrinogen) exist in a chemically reduced, colorless form and serve as intermediates between porphobilinogen (PBG) and the oxidized, colored protoporphyrins in heme biosynthesis. B. Heme biosynthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. The major sites of heme biosynthesis are the liver, which synthesizes a number of heme proteins (particularly the CYP proteins), and the erythrocyte-producing cells of the bone marrow, which are active in Hb synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. [Note: Over 85% of all heme synthesis occurs in erythroid tissue. Mature red blood cells (RBC) lack mitochondria and are unable to synthesize heme.] The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. [Note: Fig. 21.8 summarizes heme synthesis.] 1. δ-Aminolevulinic acid formation: All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a


Biochemistry_Lippinco

Biochemistry_Lippinco. pathway occur in the cytosol. [Note: Fig. 21.8 summarizes heme synthesis.] 1. δ-Aminolevulinic acid formation: All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl coenzyme A (a tricarboxylic acid cycle intermediate) that condense to form δ-aminolevulinic acid (ALA) in a reaction catalyzed by ALA synthase ([ALAS], Fig. 21.3). This reaction requires pyridoxal phosphate ([PLP] see p. 382) as a coenzyme and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are two ALAS isoforms, each produced by different genes and controlled by different mechanisms. ALAS1 is found in all tissues, whereas ALAS2 is erythroid specific. Loss-of-function mutations in ALAS2 result in X-linked sideroblastic anemia and iron overload.] (Continued in Figs. 21.4 and 21.5.) a.


Biochemistry_Lippinco

Biochemistry_Lippinco. Heme (hemin) effects: When porphyrin production exceeds the availability of the apoproteins that require it, heme accumulates and is converted to hemin by the oxidation of Fe2+ to Fe3+ . Hemin decreases the amount (and, thus, the activity) of ALAS1 by repressing transcription of its gene, increasing degradation of its messenger RNA, and decreasing import of the enzyme into mitochondria. [Note: In erythroid cells, ALAS2 is controlled by the availability of intracellular iron (see p. 475).] b.


Biochemistry_Lippinco

Biochemistry_Lippinco. Drug effects: Administration of any of a large number of drugs results in a significant increase in hepatic ALAS1 activity. These drugs are metabolized by the microsomal CYP monooxygenase system, a hemeprotein oxidase system found in the liver (see p. 149). In response to these drugs, the synthesis of CYP proteins increases, leading to an enhanced consumption of heme, a component of these proteins. This, in turn, causes a decrease in the concentration of heme in liver cells. The lower intracellular heme concentration leads to an increase in the synthesis of ALAS1 and prompts a corresponding increase in the synthesis of ALA. 2. Porphobilinogen formation: The cytosolic condensation of two ALA to form PBG by zinc-containing ALA dehydratase (PBG synthase) is extremely sensitive to inhibition by heavy metal ions (for example, lead) that replace the zinc (see Fig. 21.3). This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Uroporphyrinogen formation: The condensation of four PBG produces the linear tetrapyrrole hydroxymethylbilane, which is cyclized and isomerized by uroporphyrinogen III synthase to produce the asymmetric uroporphyrinogen III. This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups by uroporphyrinogen III decarboxylase (UROD), generating coproporphyrinogen III (Fig. 21.4). The reactions occur in the cytosol. 4. Heme formation: Coproporphyrinogen III enters the mitochondrion, and two propionate side chains are decarboxylated by coproporphyrinogen III oxidase to vinyl groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe2+) into protoporphyrin IX produces heme. This step can occur spontaneously, but the rate is enhanced by ferrochelatase, an enzyme that, like ALA dehydratase, is inhibited by lead (Fig. 21.5). C. Porphyrias


Biochemistry_Lippinco

Biochemistry_Lippinco. Porphyrias are rare, inherited (or sometimes acquired) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors (see Fig. 21.8). [Note: Inherited porphyrias are autosominal-dominant (AD) or autosomal-recessive (AR) disorders.] Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway. [Note: Porphyria, derived from the Greek for purple, refers to the red-blue color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis.] 1. Clinical manifestations: The porphyrias are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and


Biochemistry_Lippinco

Biochemistry_Lippinco. or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs, whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensitivity (that is, their skin itches and burns [pruritus] when exposed to sunlight). [Note: Photosensitivity is a result of the oxidation of colorless porphyrinogens to colored porphyrins, which are photosensitizing molecules thought to participate in the formation of superoxide radicals from oxygen. These radicals can oxidatively damage membranes and cause the release of destructive enzymes from lysosomes.] a. Chronic hepatic porphyria: Porphyria cutanea tarda, the most common porphyria, is a chronic disease of the liver. The disease is associated with severe deficiency of UROD, but clinical expression of the deficiency is influenced by various factors, such as


Biochemistry_Lippinco

Biochemistry_Lippinco. most common porphyria, is a chronic disease of the liver. The disease is associated with severe deficiency of UROD, but clinical expression of the deficiency is influenced by various factors, such as hepatic iron overload, exposure to sunlight, alcohol ingestion, estrogen therapy, and the presence of hepatitis B or C or HIV infections. [Note: Mutations to UROD are found in only 20% of affected individuals. Inheritance is AD.] Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms (Fig. 21.6) as well as urine that is red to brown in natural light (Fig.


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Biochemistry_Lippinco. 21.7) and pink to red in fluorescent light. b. Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase–deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are characterized by acute attacks of gastrointestinal (GI), neuropsychiatric, and motor symptoms that may be accompanied by photosensitivity (Fig. 21.8). Porphyrias leading to accumulation of ALA and PBG, such as acute intermittent porphyria, cause abdominal pain and neuropsychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are often precipitated by use of drugs, such as barbiturates and ethanol, which induce the synthesis of the heme-containing CYP microsomal drug-oxidation system. This further decreases the amount of available heme, which, in turn, promotes increased synthesis of ALAS1. c.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Erythropoietic porphyrias: The chronic erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) cause photosensitivity characterized by skin rashes and blisters that appear in early childhood (see Fig. 21.8). 2. Increased δ-aminolevulinic acid synthase activity: One common feature of the hepatic porphyrias is decreased synthesis of heme. In the liver, heme normally functions as a repressor of the ALAS1 gene. Therefore, the absence of this end product results in an increase in the synthesis of ALAS1 (derepression). This causes an increased synthesis of intermediates that occur prior to the genetic block. The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias. 3.


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Biochemistry_Lippinco. 3. Treatment: During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of acute symptoms of the porphyrias can be diminished by intravenous injection of hemin and glucose, which decreases the synthesis of ALAS1. Protection from sunlight, ingestion of β-carotene (provitamin A; see p. 386) that scavenges free radicals, and phlebotomy (removes porphyrins) are helpful in porphyrias with photosensitivity. D. Heme degradation After ~120 days in the circulation, RBC are taken up and degraded by the mononuclear phagocyte system (MPS), particularly in the liver and spleen (Fig. 21.9). Approximately 85% of heme destined for degradation comes from senescent RBC. The remainder is from the degradation of hemeproteins other than Hb.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Bilirubin formation: The first step in the degradation of heme is catalyzed by microsomal heme oxygenase in macrophages of the MPS. In the presence of nicotinamide adenine dinucleotide phosphate and oxygen, the enzyme catalyzes three successive oxygenations that result in opening of the porphyrin ring (converting cyclic heme to linear biliverdin), production of carbon monoxide (CO), and release of Fe2+ (see Fig. 21.9). [Note: The CO has biologic function, acting as a signaling molecule and anti-inflammatory. Iron is discussed in Chapter 29.] Biliverdin, a green pigment, is reduced, forming the red-orange bilirubin. Bilirubin and its derivatives are collectively termed bile pigments. [Note: The changing colors of a bruise reflect the varying pattern of intermediates that occurs during heme degradation.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Bilirubin, unique to mammals, appears to function at low levels as an antioxidant. In this role, it is oxidized to biliverdin, which is then reduced by biliverdin reductase, regenerating bilirubin. 2. Bilirubin uptake by the liver: Because bilirubin is only slightly soluble in plasma, it is transported through blood to the liver by binding noncovalently to albumin. [Note: Certain anionic drugs, such as salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system (CNS). This causes the potential for neural damage in infants (see p. 285).] Bilirubin dissociates from the carrier albumin molecule, enters a hepatocyte via facilitated diffusion, and binds to intracellular proteins, particularly the protein ligandin. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Bilirubin diglucuronide formation: In the hepatocyte, bilirubin solubility is increased by the sequential addition of two molecules of glucuronic acid in a process called conjugation. The reactions are catalyzed by microsomal bilirubin UDP-glucuronosyltransferase (bilirubin UGT) using uridine diphosphate (UDP)-glucuronic acid as the glucuronate donor. The bilirubin diglucuronide product is referred to as conjugated bilirubin (CB). [Note: Varying degrees of deficiency of bilirubin UGT result in Crigler-Najjar I and II and Gilbert syndrome, with Crigler-Najjar I being the most severe.] 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Bilirubin secretion into bile: CB is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease. [Note: A rare deficiency in the protein required for transport of CB out of the liver results in Dubin-Johnson syndrome.] Unconjugated bilirubin (UCB) is normally not secreted into bile. 5.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. Urobilin formation in the intestine: CB is hydrolyzed and reduced by gut bacteria to yield urobilinogen, a colorless compound. Most of the urobilinogen is further oxidized by bacteria to stercobilin, which gives feces the characteristic brown color. However, some is reabsorbed from the gut and enters the portal blood. A portion of this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is taken up by the liver and then resecreted into the bile. The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted, giving urine its characteristic color. The metabolism of bilirubin is summarized in Figure 21.10. E. Jaundice


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Jaundice Jaundice (or, icterus) refers to the yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by bilirubin deposition, secondary to increased bilirubin levels in the blood (hyperbilirubinemia) as shown in Figure 21.11. Although not a disease, jaundice is usually a symptom of an underlying disorder. [Note: Blood bilirubin levels are normally ≤1 mg/dl. Jaundice is seen at 2–3 mg/dl.] 1. Types: Jaundice can be classified into three major types described below. However, in clinical practice, jaundice is often more complex than indicated in this simple classification. For example, the accumulation of bilirubin may be a result of defects at more than one step in its metabolism.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Hemolytic (prehepatic): The liver has the capacity to conjugate and excrete >3,000 mg of bilirubin/day, whereas the normal production of bilirubin is only 300 mg/day. This excess capacity allows the liver to respond to increased heme degradation with a corresponding increase in conjugation and secretion of CB. However, extensive hemolysis (for example, in patients with sickle cell anemia or deficiency of pyruvate kinase or glucose 6-phosphate dehydrogenase) may produce bilirubin faster than it can be conjugated. UCB levels in the blood become elevated (unconjugated hyperbilirubinemia), causing jaundice (Fig. 21.12A). [Note: With hemolysis, more CB is made and excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased.] b.


Biochemistry_Lippinco

Biochemistry_Lippinco. Hepatocellular (hepatic): Damage to liver cells (for example, in patients with cirrhosis or hepatitis) can cause unconjugated hyperbilirubinemia as a result of decreased conjugation. Urobilinogen is increased in the urine because hepatic damage decreases the enterohepatic circulation of this compound, allowing more to enter the blood, from which it is filtered into the urine. The urine consequently darkens, whereas stools may be a pale, clay color. Plasma levels of alanine and aspartate transaminases (ALT and AST, respectively; see p. 251) are elevated. If CB is made but is not efficiently secreted from the liver into bile (intrahepatic cholestasis), it can leak into the blood (regurgitation), causing a conjugated hyperbilirubinemia. c.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Obstructive (posthepatic): In this instance, jaundice is not caused by overproduction of bilirubin or decreased conjugation but, instead, results from obstruction of the common bile duct (extrahepatic cholestasis). For example, the presence of a tumor or bile stones may block the duct, preventing passage of CB into the intestine. Patients with obstructive jaundice experience GI pain and nausea and produce stools that are a pale, clay color. The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is eventually excreted in the urine (which darkens over time) and is referred to as urinary bilirubin. Urinary urobilinogen is absent.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Jaundice in newborns: Most newborn infants (60% of full term and 80% of preterm) show a rise in UCB in the first postnatal week (and a transient, physiologic jaundice) because the activity of hepatic bilirubin UGT is low at birth (it reaches adult levels in about 4 weeks), as shown in Figures 21.12B and 21.13. Elevated UCB, in excess of the binding capacity of albumin (20–25 mg/dl), can diffuse into the basal ganglia, causing toxic encephalopathy (kernicterus) and a pathologic jaundice. Therefore, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light (phototherapy), as shown in Figure 21.14, which converts bilirubin to more polar and, therefore, water-soluble isomers. These photoisomers can be excreted into the bile without conjugation to glucuronic acid. [Note: Because of solubility differences, only UCB crosses the blood–brain barrier, and only CB appears in urine.] 3. Bilirubin measurement: Bilirubin is commonly measured by the van den


Biochemistry_Lippinco

Biochemistry_Lippinco. acid. [Note: Because of solubility differences, only UCB crosses the blood–brain barrier, and only CB appears in urine.] 3. Bilirubin measurement: Bilirubin is commonly measured by the van den Bergh reaction, in which diazotized sulfanilic acid reacts with bilirubin to form red azodipyrroles that are measured colorimetrically. In aqueous solution, the water-soluble CB reacts rapidly with the reagent (within 1 minute) and is said to be direct reacting. The UCB, which is much less soluble in aqueous solution, reacts more slowly. However, when the reaction is carried out in methanol, both CB and UCB are soluble and react with the reagent, providing the total bilirubin value. The indirect-reacting bilirubin, which corresponds to the UCB, is obtained by subtracting the direct-reacting bilirubin from the total bilirubin. [Note: In normal plasma, only ~4% of the total bilirubin is conjugated, or direct reacting, because most is secreted into bile.]


Biochemistry_Lippinco

Biochemistry_Lippinco. III. A. Dopamine, norepinephrine (NE), and epinephrine (or, adrenaline) are biologically active (biogenic) amines that are collectively termed catecholamines. Dopamine and NE are synthesized in the brain and function as neurotransmitters. Epinephrine is synthesized from NE in the adrenal medulla. 1. Function: Outside the CNS, NE and its methylated derivative, epinephrine, are hormone regulators of carbohydrate and lipid metabolism. NE and epinephrine are released from storage vesicles in the adrenal medulla in response to fright, exercise, cold, and low levels of blood glucose. They increase the degradation of glycogen and triacylglycerol as well as increase blood pressure and the output of the heart. These effects are part of a coordinated response to prepare the individual for stress and are often called the “fight-or-flight” reactions. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Synthesis: The catecholamines are synthesized from tyrosine, as shown in Figure 21.15. Tyrosine is first hydroxylated by tyrosine hydroxylase to form L-3,4-dihydroxyphenylalanine (DOPA) in a reaction analogous to that described for the hydroxylation of phenylalanine (see p. 263). The tetrahydrobiopterin (BH4)-requiring enzyme is abundant in the CNS, the sympathetic ganglia, and the adrenal medulla, and it catalyzes the rate-limiting step of the pathway. DOPA is decarboxylated in a reaction requiring PLP to form dopamine, which is hydroxylated by dopamine βhydroxylase to yield NE in a reaction that requires ascorbic acid (vitamin C) and copper. Epinephrine is formed from NE by an N-methylation reaction using S-adenosylmethionine (SAM) as the methyl donor (see p. 264).


Biochemistry_Lippinco

Biochemistry_Lippinco. Parkinson disease, a neurodegenerative movement disorder, is due to insufficient dopamine production as a result of the idiopathic loss of dopamine-producing cells in the brain. Administration of L-DOPA (levodopa) is the most common treatment, because dopamine cannot cross the blood–brain barrier.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Degradation: The catecholamines are inactivated by oxidative deamination catalyzed by monoamine oxidase (MAO) and by Omethylation catalyzed by catechol-O-methyltransferase (COMT) using SAM as the methyl donor (Fig. 21.16). The reactions can occur in either order. The aldehyde products of the MAO reaction are oxidized to the corresponding acids. The products of these reactions are excreted in the urine as vanillylmandelic acid (VMA) from epinephrine and NE and homovanillic acid (HVA) from dopamine. [Note: VMA and the metanephrines are increased with pheochromocytomas, rare tumors of the adrenal gland characterized by excessive production of catecholamines.] 4. Monoamine oxidase inhibitors: MAO is found in neural and other tissues, such as the intestine and liver. In the neuron, this enzyme oxidatively deaminates and inactivates any excess neurotransmitter molecules (NE, dopamine, or serotonin) that may leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors (MAOI)


Biochemistry_Lippinco

Biochemistry_Lippinco. oxidatively deaminates and inactivates any excess neurotransmitter molecules (NE, dopamine, or serotonin) that may leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors (MAOI) may irreversibly or reversibly inactivate the enzyme, permitting neurotransmitter molecules to escape degradation and, therefore, both to accumulate within the presynaptic neuron and to leak into the synaptic space. This causes activation of NE and serotonin receptors and may be responsible for the antidepressant action of MAOI. [Note: The interaction of MAOI with tyraminecontaining foods is discussed on p. 373.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Histamine Histamine is a chemical messenger that mediates a wide range of cellular responses, including allergic and inflammatory reactions and gastric acid secretion. A powerful vasodilator, histamine is formed by decarboxylation of histidine in a reaction requiring PLP (Fig. 21.17). It is secreted by mast cells as a result of allergic reactions or trauma. Histamine has no clinical applications, but agents that interfere with the action of histamine have important therapeutic applications. C. Serotonin


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Serotonin Serotonin, also called 5-hydroxytryptamine (5-HT), is synthesized and/or stored at several sites in the body (Fig. 21.18). The largest amount by far is found in the intestinal mucosa. Smaller amounts occur in the CNS, where it functions as a neurotransmitter, and in platelets (see online Chapter 35). Serotonin is synthesized from tryptophan, which is hydroxylated in a BH4 requiring reaction analogous to that catalyzed by phenylalanine hydroxylase. The product, 5-hydroxytryptophan, is decarboxylated to 5HT. Serotonin has multiple physiologic roles including pain perception and regulation of sleep, appetite, temperature, blood pressure, cognitive functions, and mood (causes a feeling of well-being). [Note: Selective serotonin reuptake inhibitors (SSRI) maintain serotonin levels, thereby functioning as antidepressants.] Serotonin is degraded by MAO to 5hydroxy-3-indoleacetic acid (5-HIAA). D. Creatine


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Creatine Creatine phosphate (also called phosphocreatine), the phosphorylated derivative of creatine found in muscle, is a high-energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to adenosine diphosphate (Fig. 21.19) to maintain the intracellular level of ATP during the first few minutes of intense muscular contraction. [Note: The amount of creatine phosphate in the body is proportional to the muscle mass.] 1. Synthesis: Creatine is synthesized in the liver and kidneys from glycine and the guanidino group of arginine, plus a methyl group from SAM (see Fig. 21.19). Animal products are dietary sources. Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP as the phosphate donor. [Note: The presence of creatine kinase (MB isozyme) in the plasma is indicative of heart damage and is used in the diagnosis of myocardial infarction (see p. 65).] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Degradation: Creatine and creatine phosphate spontaneously cyclize at a slow but constant rate to form creatinine, which is excreted in the urine. The amount excreted is proportional to the total creatine phosphate content of the body and, therefore, can be used to estimate muscle mass. When muscle mass decreases for any reason (for example, from paralysis or muscular dystrophy), the creatinine content of the urine falls. In addition, a rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly cleared from the blood and excreted. A typical adult male excretes ~1–2 g of creatinine/day. E. Melanin


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Melanin Melanin is a pigment that occurs in several tissues, particularly the eye, hair, and skin. It is synthesized from tyrosine in melanocytes (pigmentforming cells) of the epidermis. It functions to protect underlying cells from the harmful effects of sunlight. [Note: A defect in melanin production results in oculocutaneous albinism, the most common type being due to defects in copper-containing tyrosinase (see p. 273).] IV. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Amino acids are precursors of many nitrogen (N)-containing compounds including porphyrins, which, in combination with ferrous (Fe2+) iron, form heme (Fig. 21.20). The major sites of heme biosynthesis are the liver, which synthesizes a number of hemeproteins (particularly cytochrome P450 enzymes), and the erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. Heme synthesis starts with glycine and succinyl coenzyme A. The committed step is the formation of δ-aminolevulinic acid (ALA). This mitochondrial reaction is catalyzed by ALA synthase-1 (ALAS1) in the liver (inhibited by hemin, the oxidized form of heme that accumulates when heme is being underutilized) and ALAS2 in


Biochemistry_Lippinco

Biochemistry_Lippinco. (ALA). This mitochondrial reaction is catalyzed by ALA synthase-1 (ALAS1) in the liver (inhibited by hemin, the oxidized form of heme that accumulates when heme is being underutilized) and ALAS2 in erythroid tissues (regulated by iron). Porphyrias are caused by inherited or acquired (lead poisoning) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. Enzymic defects early in the pathway cause abdominal pain and neuropsychiatric symptoms, whereas later defects cause photosensitivity. Degradation of heme occurs in the mononuclear phagocyte system, particularly in the liver and spleen. The first step is the production by heme oxygenase of biliverdin, which is subsequently reduced to bilirubin. Bilirubin is transported by albumin to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase (bilirubin UGT). Bilirubin


Biochemistry_Lippinco

Biochemistry_Lippinco. by albumin to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase (bilirubin UGT). Bilirubin diglucuronide (conjugated bilirubin) is transported into the bile canaliculi, where it is first hydrolyzed and reduced by gut bacteria to yield urobilinogen, which is further oxidized by bacteria to stercobilin. Jaundice (icterus) refers to the yellow color of the skin and sclerae that is caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Three commonly encountered types of jaundice are hemolytic (prehepatic), obstructive (posthepatic), and hepatocellular (hepatic) (see Fig. 21.20). Other important N-containing compounds derived from amino acids include the catecholamines (dopamine, norepinephrine, and epinephrine), creatine, histamine, serotonin, melanin, and nitric oxide.


Biochemistry_Lippinco

Biochemistry_Lippinco. bilirubin or decreased secretion of conjugated bilirubin from the liver into bile.] CoA = coenzyme A; CO = carbon monoxide; Fe = iron. Choose the ONE best answer. 1.1. δ-Aminolevulinic acid synthase activity: A. catalyzes the committed step in porphyrin biosynthesis. B. is decreased by iron in erythrocytes. C. is decreased in the liver in individuals treated with certain drugs such as the barbiturate phenobarbital. D. occurs in the cytosol. E. requires tetrahydrobiopterin as a coenzyme. Correct answer = A. δ-Aminolevulinic acid synthase is mitochondrial and catalyzes the rate-limiting and regulated step of porphyrin synthesis. It requires pyridoxal phosphate as a coenzyme. Iron increases production of the erythroid isozyme. The hepatic isozyme is increased in patients treated with certain drugs.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1.2. A 50-year-old man presented with painful blisters on the backs of his hands. He was a golf instructor and indicated that the blisters had erupted shortly after the golfing season began. He did not have recent exposure to common skin irritants. He had partial complex seizure disorder that had begun ~3 years earlier after a head injury. The patient had been taking phenytoin (his only medication) since the onset of the seizure disorder. He admitted to an average weekly ethanol intake of ~18 12-oz cans of beer. The patient’s urine was reddish orange. Cultures obtained from skin lesions failed to grow organisms. A 24-hour urine collection showed elevated uroporphyrin (1,000 mg; normal, <27 mg). The most likely diagnosis is: A. acute intermittent porphyria. B. congenital erythropoietic porphyria. C. erythropoietic protoporphyria. D. hereditary coproporphyria. E. porphyria cutanea tarda.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. acute intermittent porphyria. B. congenital erythropoietic porphyria. C. erythropoietic protoporphyria. D. hereditary coproporphyria. E. porphyria cutanea tarda. Correct answer = E. The disease is associated with a deficiency in uroporphyrinogen III decarboxylase (UROD), but clinical expression of the enzyme deficiency is influenced by hepatic injury caused by environmental (for example, ethanol) and infectious (for example, hepatitis B virus) agents. Exposure to sunlight can also be a precipitating factor. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms and urine that is red to brown. Treatment of the patient’s seizure disorder with phenytoin caused increased synthesis of δaminolevulinic acid synthase and, therefore, of uroporphyrinogen, the substrate of the deficient UROD. The laboratory and clinical findings are inconsistent with other porphyrias.


Biochemistry_Lippinco

Biochemistry_Lippinco. 21.3. A patient presents with jaundice, abdominal pain, and nausea. Clinical laboratory results are shown below. What is the most likely cause of the jaundice? A. Decreased hepatic conjugation of bilirubin B. Decreased hepatic uptake of bilirubin C. Decreased secretion of bile into the intestine D. Increased hemolysis Correct answer = C. The data are consistent with an obstructive jaundice in which a block in the common bile duct decreases the secretion of bile containing conjugated bilirubin (CB) into the intestine (stool will be pale in color). The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is excreted in the urine (which darkens) and is referred to as urinary bilirubin. Urinary urobilinogen is not present because its source is intestinal urobilinogen, which is low. The other choices do not match the data.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1.4. A 2-year-old child was brought to his pediatrician for evaluation of gastrointestinal problems. The parents report that the boy has been listless for the last few weeks. Lab tests reveal a microcytic, hypochromic anemia. Blood lead levels are elevated. Which of the enzymes listed below is most likely to have higher-than-normal activity in the liver of this child? A. δ-Aminolevulinic acid synthase B. Bilirubin UDP glucuronosyltransferase C. Ferrochelatase D. Heme oxygenase E. Porphobilinogen synthase


Biochemistry_Lippinco

Biochemistry_Lippinco. A. δ-Aminolevulinic acid synthase B. Bilirubin UDP glucuronosyltransferase C. Ferrochelatase D. Heme oxygenase E. Porphobilinogen synthase Correct answer = A. This child has the acquired porphyria of lead poisoning. Lead inhibits both δ-aminolevulinic acid dehydratase and ferrochelatase and, consequently, heme synthesis. The decrease in heme derepresses δaminolevulinic acid synthase-1 (the hepatic isozyme), resulting in an increase in its activity. The decrease in heme also results in decreased hemoglobin synthesis, and anemia is seen. Ferrochelatase is directly inhibited by lead. The other choices are enzymes of heme degradation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells. Without them, neither ribonucleic acid (RNA) nor deoxyribonucleic acid (DNA) can be produced, and, therefore, proteins cannot be synthesized or cells proliferate. Nucleotides also serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids, and conjugated proteins (for example, uridine diphosphate [UDP]-glucose and cytidine diphosphate [CDP]choline) and are structural components of several essential coenzymes, such as coenzyme A, flavin adenine dinucleotide (FAD[H2]), nicotinamide adenine dinucleotide (NAD[H]), and nicotinamide adenine dinucleotide phosphate (NADP[H]). Nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), serve as second messengers in signal transduction pathways. In addition, nucleotides play an important role as energy sources in the cell. Finally, nucleotides are important regulatory compounds