Conversion of Amino Acids to Specialized Products

168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 277

Conversion of Amino Acids 21 to Specialized Products

I. OVERVIEW

Dietary In addition to serving as building blocks for proteins, amino acids are protein precursors of many nitrogen-containing compounds that have important 100 g/day typical physiologic functions (Figure 21.1). These molecules include of U.S. diet porphyrins, neurotransmitters, hormones, purines, and pyrimidines. Synthesis of Body nonessential II. PORPHYRIN METABOLISM protein amino acids ~400 g/day varies

Porphyrins are cyclic compounds that readily bind metal ions—usually Fe2+ or Fe3+. The most prevalent metalloporphyrin in humans is heme, 2+ AminoAmino acid acid pool which consists of one ferrous (Fe ) iron ion coordinated in the center of pool (100 g) the tetrapyrrole ring of protoporphyrin IX (see p. 280). Heme is the pros- thetic group for hemoglobin, myoglobin, the cytochromes, catalase, ~30 g/day nitric oxide synthase, and peroxidase. These hemeproteins are rapidly synthesized and degraded. For example, 6–7 g of hemoglobin are synthesized each day to replace heme lost through the Body Synthesis of: normal turnover of erythrocytes. Coordinated with the turnover of hemeprotein • Porphyrins ~400 g/day • Creatine proteins is the simultaneous synthesis and degradation of the associ- • Neuro- ated porphyrins, and recycling of the bound iron ions. transmitters • Purines Varies • Pyrimidines A. Structure of porphyrins • Other nitrogen- containing Porphyrins are cyclic molecules formed by the linkage of four pyrrole compounds rings through methenyl bridges (Figure 21.2). Three structural fea- tures of these molecules are relevant to understanding their medical significance. Glucose, Ketone bodies, glycogen fatty acids, 1. Side chains: Different porphyrins vary in the nature of the side steroids chains that are attached to each of the four pyrrole rings. – Uroporphyrin contains acetate (–CH2–COO ) and prop ionate – CO2 + H2O (–CH2–CH2–COO ) side chains, coproporphyrin contains methyl (–CH3) and propionate groups, and protoporphyrin IX (and heme) contains vinyl (–CH=CH2), methyl, and propionate groups. [Note: Figure 21.1 The methyl and vinyl groups are produced by decarboxylation of Amino acids as precursors of acetate and propionate side chains, respectively.] nitrogen-containing compounds.

277 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 278

278 21. Conversion of Amino Acids to Specialized Products

Porphyrins contain four pyrrole rings (A, B, C, Acetate (A) and and D) joined through methenyl bridges. propionate (P) are P reversed in ring D P A A of uroporphyrin III AB A ABP Porphyrins contain side chains compared with A P N N attached to each of the four uroporphyrin I. Only N N pyrrole rings. In Type I Type III porphyrins are porphyrins, the side chains are physiologically arranged symmetrically, that is, important in humans. N N N N for uroporphyrin I, A (acetate) A DCA P DCA alternates with propionate (P) A P around the tetrapyrrole ring. P P Uroporphyrin I Uroporphyrin III

Figure 21.2 Structures of uroporphyrin I and uroporphyrin III. A = acetate and P = propionate.

2. Distribution of side chains: The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, COO– designated by Roman numerals I to IV. Only Type III porphyrins, which contain an asymmetric substitution on ring D (see Figure CH2 – 21.2), are physiologically important in humans. [Note: CH2 COO CH2 + Protoporphyrin IX is a member of the Type III series.] NH3 O C CoA Glycine Succinyl CoA 3. Porphyrinogens: These porphyrin precursors (for example, uroporphyrinogen) exist in a chemically reduced, colorless form, and ALAS1 Heme serve as intermediates between porphobilinogen and the ALAS2 Iron oxidized, colored protoporphyrins in heme biosynthesis. CoA B. Biosynthesis of heme CO2 COO– The major sites of heme biosynthesis are the liver, which synthe-

CH2 sizes a number of heme proteins (particularly cytochrome P450 pro- CH2 teins), and the erythrocyte-producing cells of the bone marrow, C O which are active in hemoglobin synthesis. [Note: Over 85% of all

CH2 heme synthesis occurs in erythroid tissue.] In the liver, the rate of NH + heme synthesis is highly variable, responding to alterations in the 3 2 δ-Aminolevulinic acid (ALA) cellular heme pool caused by fluctuating demands for heme proteins. In contrast, heme synthesis in erythroid cells is relatively con- δ-Aminolevulinic (Two molecules acid dehydratase condensed) stant, and is matched to the rate of globin synthesis. The initial (cytosolic enzyme) reaction and the last three steps in the formation of porphyrins occur 2 H2O Lead in mitochondria, whereas the intermediate steps of the biosynthetic COO– pathway occur in the cytosol (see Figure 21.8). [Note: Mature red – COO CH2 blood cells lack mitochondria and are unable to synthesize heme.] CH CH 2 2 1. Formation of δ-aminolevulinic acid (ALA): All the carbon and C C nitrogen atoms of the porphyrin molecule are provided by glycine C CH N (a nonessential amino acid) and succinyl coenzyme A (an inter- H CH mediate in the citric acid cycle) that condense to form ALA in a 2 reaction catalyzed by ALA synthase (ALAS) (Figure 21.3) This NH2 Porphobilinogen reaction requires pyridoxal phosphate (PLP) as a coenzyme, and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are two isoforms of ALAS, 1 and 2, each controlled Figure 21.3 by different mechanisms. Erythroid tissue produces only ALAS2, Pathway of porphyrin synthesis: Formation of porphobilinogen. ALAS the gene for which is located on the X-chromosome. Loss of func- = δ-aminolevulinic acid synthase. tion mutations in ALAS2 result in X-linked sideroblastic anemia.] (Continued in Figures 21.4 and 21.5.) a. End-product inhibition of ALAS1 by hemin: When porphyrin production exceeds the availability of the apoproteins that 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 279

II. Porphyrin Metabolism 279

require it, heme accumulates and is converted to hemin by – 2+ 3+ COO the oxidation of Fe to Fe . Hemin decreases the activity of – COO CH hepatic ALAS1 by causing decreased synthesis of the 2 CH2 CH2 enzyme, through inhibition of mRNA synthesis and use (heme C C decreases stability of the mRNA), and by inhibiting mitochon- C CH drial import of the enzyme. [Note: In erythroid cells, ALAS2 is N controlled by the availability of intracellular iron.] H CH2 b. Effect of drugs on ALA synthase activity: Administration of NH2 4 any of a large number of drugs results in a significant increase Porphobilinogen in hepatic ALAS1 activity. These drugs are metabolized by the Hydroxymethyl- (Four molecules condensed) micro somal cyto chrome P450 monooxygenase system—a bilane synthase hemeprotein oxidase system found in the liver (see p. 149). In 4 NH3 response to these drugs, the synthesis of cytochrome P450 proteins increases, leading to an enhanced consumption of Hydroxymethylbilane heme—a component of cytochrome P450 proteins. This, in turn, causes a decrease in the concentration of heme in liver Uroporphyrinogen III (Ring closure and cells. The lower intracellular heme concentration leads to an synthase isomerization) increase in the synthesis of ALAS1 (derepression), and

prompts a corresponding increase in ALA synthesis. – – OOC CH2 CH2 CH2 COO –OOC CH CH CH COO– 2. Formation of porphobilinogen: The condensation of two 2 N N 2 2 molecules of ALA to form porphobilinogen by Zn-containing ALA H H H H dehydratase (porphobilinogen synthase) is extremely sensitive to – N N – OOC CH2 CH2 COO inhibition by heavy metal ions, for example, lead that replace the – – OOC CH2 CH2 CH2 CH2 COO zinc (see Figure 21.3). This inhibition is, in part, responsible for the Uroporphyrinogen III elevation in ALA and the anemia seen in lead poisoning. Uroporphyrinogen 3. Formation of uroporphyrinogen: The condensation of four decarboxylase (Decarboxylation)

porpho bilinogens produces the linear tetrapyrrole, hydroxymethyl- 4 CO2 bilane, which is isomerized and cyclized by uroporphyrinogen III – synthase to produce the asymmetric uroporphyrinogen III. This OOC CH2 CH2 CH3 CH CH CH COO– cyclic tetrapyrrole undergoes decarboxylation of its acetate 3 N N 2 2 groups, generating coproporphyrinogen III (Figure 21.4). These H H H H reactions occur in the cytosol. N N CH 3 CH3 – – 4. Formation of heme: Coproporphyrinogen III enters the mitochon- OOCCH2 CH2 CH2 CH2 COO drion, and two propionate side chains are decarboxylated to vinyl Coproporphyrinogen III groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe2+) into protoporphyrin IX occurs spontaneously, but the rate is enhanced by ferro - chelatase, an enzyme that, like ALA dehydratase, is inhibited by CH lead (Figure 21.5). H2C CH 3 CH CH CH 3 N N 2 H C. Porphyrias H N N CH Porphyrias are rare, inherited (or occasionally acquired) defects in 3 CH3 –OOC CH CH – heme synthesis, resulting in the accumulation and increased excre- 2 2 CH2 CH2 COO tion of porphyrins or porphyrin precursors (see Figure 21.8). [Note: Protoporphyrin IX With few exceptions, porphyrias are inherited as autosomal dominant disorders.] The mutations that cause the porphyrias are hetero- genous (not all are at the same DNA locus), and nearly every Figure 21.4 affected family has its own mutation. Each porphyria results in the Pathway of porphyrin synthesis: Formation of protoporphyrin IX. accumulation of a unique pattern of intermediates caused by the (Continued from Figure 21.3.) deficiency of an enzyme in the heme synthetic pathway. [Note: “Porphyria” refers to the purple color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis.] 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 280

280 21. Conversion of Amino Acids to Specialized Products

1. Clinical manifestations: The porphyrias are classified as erythro- CH H2C CH 3 poietic or hepatic, depending on whether the enzyme deficiency CH CH CH 3 N N 2 occurs in the erythropoietic cells of the bone marrow or in the H H liver. Hepatic porphyrias can be further classified as chronic or N N CH3 CH3 acute. In general, individuals with an enzyme defect prior to the –OOC CH CH – 2 2 CH2 CH2 COO synthesis of the tetrapyrroles manifest abdominal and neuro- Protoporphyrin IX psychiatric signs, whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensi- Fe2+ Lead tivity—that is, their skin itches and burns (pruritus) when exposed Ferrochelatase to visible light. [Note: Photosenstivity is a result of the oxidation of (mitochondrial enzyme) 2 H+ colorless porphyrinogens to colored porphyrins, which are photo-

H2C CH CH3 sensitizing molecules that are thought to participate in the forma- CH CH CH 3 N N 2 tion of superoxide radicals from oxygen. These reactive oxygen Fe species can oxidatively damage membranes, and cause the N N release of destructive enzymes from lysosomes.] CH3 CH3 – OOC CH CH CH CH COO– 2 2 2 2 a. Chronic hepatic porphyria: Porphyria cutanea tarda, the most Heme (Fe2+ protoporphyrin IX) common porphyria, is a chronic disease of the liver. The disease is associated with a deficiency in uroporphyrinogen Figure 21.5 decarboxylase, but clinical expression of the enzyme defi- Pathway of porphyrin synthesis: ciency is influenced by various factors, such as hepatic iron Formation of heme. (Continued overload, exposure to sunlight, alcohol ingestion, and the pres- from Figures 21.3 and 21.4) ence of hepatitis B or C, or HIV infections. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms (Figure 21.6), and urine that is red to brown in natural light (Figure 21.7), and pink to red in fluorescent light. b. Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase deficiency, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are characterized by acute attacks of gastro intestinal, neuropsychiatric, and motor symptoms that may be accompanied by photosensitivity. Porphyrias leading to accumulation of ALA and porphobilinogen, such as acute intermittent por phyria, cause abdominal pain and neuro psychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are Figure 21.6 often precipitated by administration of drugs such as barbitu- Skin eruptions in a patient with rates and ethanol, which induce the synthesis of the heme- porphyria cutanea tarda. containing cytochrome P450 microsomal drug oxidation system. This further decreases the amount of available heme, which, in turn, promotes the increased synthesis of ALAS1.

c. Erythropoietic porphyrias: The erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) are characterized by skin rashes and blisters that appear in early childhood. The diseases are complicated by cholestatic liver cirrhosis and progressive hepatic failure. 2. Increased ALA synthase activity: One common feature of the porphyrias is a decreased synthesis of heme. In the liver, heme normally functions as a repressor of the gene for ALAS1. Therefore, Figure 21.7 the absence of this end product results in an increase in the syn- Urine from a patient with porphyria thesis of ALA synthase1 (derepression). This causes an cutanea tarda (right) and from a increased synthesis of intermediates that occur prior to the patient with normal porphyrin excretion (left). genetic block. The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias. 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 281

II. Porphyrin Metabolism 281

LEAD POISONING ERYTHROPOIETIC • Ferrochelatase and ALA dehydratase are PROTOPORPHYRIA particularly sensitive to inhibition by lead. • This disease is caused by to a • Protoporphyrin and ALA accumulate deficiency in ferrochelatase. in urine. • Protoporphyrin accumulates in erythrocytes, bone marrow, and plasma. Heme • Patients are photosensitive.

FeFe2+ ACUTE INTERMITTENT VARIEGATE PORPHYRIA PORPHYRIA • An acute disease caused by a deficiency in • An acute disease caused by a deficiency protoporphyrinogen oxidase. in hydroxymethylbilane synthase1. ProtoporphyrinPrroottoo phyryririn IIXX • Protoporphyrinogen IX and other • Porphobilinogen and δ-amino- intermediates prior to the block levulinic acid accumulate accumulate in the urine. in the urine. • Patients are photosensitive. • Urine darkens on exposure to light and air. • Patients are NOT photosensitive. HEREDITARY COPROPORPHYRIA SuccinylSuccinuccinccincininnnylylyl CoA + GGlycine ly c cine ProtoporphyrinogenProtoopp hyrinogogenen IX • An acute disease caused by a deficiency in coproporphyrinogen oxidase. • Coproporphyrinogen III and other intermediates prior to the block δ-Aminolevulinic-Aminnoolceevulinicc acaacidid accumulate in the urine. • Patients are photosensitive.

CCoproporphyrinogenoprooppo yrinonogogenen III

MITOCHONDRIA

CYTOSOL

Spontaneous δ-Aminolevulinic-AAminolevulinicminolevulinicinolevulinicnolevulinicolevulinicolevulilevulilevulinicevulinicevulinic c aacacidaciid CoproporphyrinogenCopropo y g III Coproporphyrinpppy III

PORPHYRIA CUTANEA TARDA • This disease is caused by a deficiency in uroporphyrinogen KEY: decarboxylase. PorphobilinogenPorphhobilinoobilinoobilinogobilinogen • Uroporphyrin accumulates in the urine. • It is the most common porphyria. • Patients are photosensitive. Hepatic porphyria SpontaneousSpontaneous Hydroxymethylbilane UroporphyrinogenUroporphhyyyrririnnonogogegene III Uroporphyrin III (enzyme bound) CONGENITAL ERYTHROPOIETIC PORPHYRIA Spontaneouss • This disease is caused by a deficiency Uroporphyrinogen I UroporphyrinUroporphyrin I in uroporphyrinogen III synthase. • Uroporphyrinogen I and coproporphyrinogen I Erythro- accumulate in the urine. poietic Spontaneousntaneous • Patients are photosensitive. porphyria Coproporphyrinogen I CCoproporphyrinoproporphyrin I

Figure 21.8 Summary of heme synthesis. 1Also referred to as porphobilinogen deaminase. 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 282

282 21. Conversion of Amino Acids to Specialized Products

3. Treatment: During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of symptoms of the porphyrias can be diminished by MACROPHAGE intravenous injection of hemin and glucose, which decreases the synthesis of ALAS1. Avoidance of sunlight and ingestion of Heme β + -carotene (a free-radical scavenger) are helpful in porphyrias O2, NADPH + H with photosensitivity. Heme oxygenase

3+ D. Degradation of heme Fe NADP+ CO After approximately 120 days in the circulation, red blood cells are M V MPPMMV taken up and degraded by the reticuloendothelial system, particularly in the liver and spleen (Figure 21.9). Approximately 85% of O N C N C N C N O heme destined for degradation comes from senescent red blood HHH H H cells, and 15% is from turnover of immature red blood cells and Biliverdin cytochromes from nonerythroid tissues. NADPH + H+ 1. Formation of bilirubin: The first step in the degradation of heme is Biliverdin catalyzed by the microsomal heme oxygenase system of the retic- reductase + NADP uloendothelial cells. In the presence of NADPH and O2, the enzyme adds a hydroxyl group to the methenyl bridge between MVMPPMM V two pyrrole rings, with a concomitant oxidation of ferrous iron to Fe3+. A second oxidation by the same enzyme system results in O N C N C N C N O cleavage of the porphyrin ring. The green pigment biliverdin is H H H H H H H 2 produced as ferric iron and CO are released (see Figure 21.9). Bilirubin [Note: The CO has biologic function, acting as a signaling molecule and vasodilator.] Biliverdin 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.] BLOOD Bilirubin–albumin complex Bilirubin, unique to mammals, appears to function as an antioxidant. In this role, it is oxidized to biliverdin, which is then reduced by biliverdin Bilirubin reductase, regenerating bilirubin. LIVER 2 UDP-glucuronic Bilirubin acid glucuronyl- 2. Uptake of bilirubin by the liver: Bilirubin is only slightly soluble in transferase 2 UDP plasma and, therefore, is transported to the liver by binding non- covalently to albumin. [Note: Certain anionic drugs, such as salicyl- Bilirubin diglucuronide ates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system. This causes the potential for neural damage in infants.] Bilirubin dissociates from the carrier albumin molecule, enters a hepatocyte via facilitated dif- Bilirubin diglucuronide fusion, and binds to intracellular proteins, particularly the protein ligandin. 3. Formation of bilirubin diglucuronide: In the hepatocyte, the solu- BILE bility of bilirubin is increased by the addition of two molecules of glucuronic acid. [Note: This process is referred to as conjugation.] The reaction is catalyzed by microsomal bilirubin glucur onyl - Figure 21.9 transferase using uridine diphosphate-glucuronic acid as the glu- Formation of bilirubin from heme. UDP = uridine diphosphate. curonate donor. [Note: Varying degrees of deficiency of this enzyme result in Crigler-Najjar I and II and Gilbert syndrome, with Crigler-Najjar I being the most severe deficiency.] 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 283

II. Porphyrin Metabolism 283

1 Senescent red cells are a major source of hemeproteins.

2 Breakdown of heme to bilirubin BLOOD VESSEL occurs in macrophages of the reticuloendothelial system (tissue macrophages, spleen, and liver).

MACROPHAGE

B U

Bilirubin is taken 4 9 The remainder of the urobilinogen up via facilitated is transported by the blood to the B diffusion by the liver kidney, where it is converted to yellow and conjugated with B urobilin and excreted, giving urine its glucuronic acid. characteristic color. U 3 Unconjugated bilirubin B LIVER is transported through the blood (complexed to albumin) to the liver. A portion of this urobilinogen B B BG 8 participates in the enterohepatic urobilinogen cycle.

BG U GALLBLADDER KIDNEY

5 Conjugated bilirubin is actively secreted INTESTINE into bile and then BG 7 Some of the urobilinogen the intestine. U is reabsorbed from the gut and enters the portal blood. To urine Ub In the intestine, 6 glucuronic acid is removed by bacteria. B U Urobilinogen is oxidized The resulting bilirubin B 10 is converted to U S by intestinal bacteria to urobilinogen. the brown stercobilin. To feces

Figure 21.10 Catabolism of heme B = bilirubin; BG = bilirubin diglucuronide; U = urobilinogen; Ub = urobilin; S = stercobilin.

4. Secretion of bilirubin into bile: Bilirubin diglucuronide (conjugated bilirubin) is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-depen- dent, rate-limiting step is susceptible to impairment in liver disease. [Note: A deficiency in the protein required for transport of conjugated bilirubin out of the liver results in Dubin-Johnson syndrome.] Unconjugated bilirubin is normally not secreted. 5. Formation of urobilins in the intestine: Bilirubin diglucuronide is hydrolyzed and reduced by bacteria in the gut to yield uro - bilinogen, a colorless compound. Most of the urobilinogen is oxidized by intestinal bacteria to ster co bilin, which gives feces the characteristic brown color. However, some of the urobilinogen is reabsorbed from the gut and enters the portal blood. A portion of 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 284

284 21. Conversion of Amino Acids to Specialized Products

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 uro bilin and excreted, giving urine its characteristic color. The metabolism of bilirubin is summarized in Figure 21.10. E. Jaundice Jaundice (also called icterus) refers to the yellow color of skin, nail Figure 21.11 beds, and sclerae (whites of the eyes) caused by deposition of Jaundiced patient, with the sclerae bilirubin, secondary to increased bilirubin levels in the blood (hyper- of his eyes appearing yellow. bili rubinemia, Figure 21.11). Although not a disease, jaundice is usually a symptom of an underlying disorder. Hemolysis Unconjugated bilirubin B 1. Types of jaundice: Jaundice can be classified into three major B B forms described below. However, in clinical practice, jaundice is Hemolytic often more complex than indicated in this simple classification. jaundice For example, the accumulation of bilirubin may be a result of

B LIVER defects at more than one step in its metabolism. a. Hemolytic jaundice: The liver has the capacity to conjugate and B B BG excrete over 3,000 mg of bilirubin per 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 BG a corresponding increase in conjugation and secretion of bili-

INTESTINE rubin diglucuronide. However, massive lysis of red blood cells (for example, in patients with sickle cell anemia, pyruvate BG kinase or glucose 6-phosphate dehydrogenase deficiency) may produce bilirubin faster than it can be conjugated. Unconju- B gated bilirubin levels in the blood become elevated, causing A U U S jaundice (Figure 21.12A). [Note: More conjugated bilirubin is excreted into the bile, the amount of urobilinogen entering the Bilirubin glucuronyl- Unconjugated enterohepatic circulation is increased, and urinary urobilinogen transferase bilirubin is increased.]

B B B b. Hepatocellular jaundice: Damage to liver cells (for example, Neonatal in patients with cirrhosis or hepatitis) can cause unconjugated jaundice bilirubin levels in the blood to increase as a result of

LIVER decreased conjugation. Urobilinogen is increased in the urine B because hepatic damage decreases the enterohepatic circu-

B BG lation of this compound, allowing more to enter the blood, from which it is filtered into the urine. The urine thus darkens, whereas stools may be a pale, clay color. Plasma levels of AST and ALT (see p. 251) are elevated. [Note: If conjugated bilirubin is not efficiently secreted from the liver into bile (intra- INTESTINE hepatic cholestasis), it can diffuse (“leak”) into the blood, causing a conjugated hyperbilirubinemia.] BG

B c. Obstructive jaundice: In this instance, jaundice is not caused B B U S by overproduction of bilirubin or decreased conjugation, but S instead results from obstruction of the bile duct (extrahepatic cholestasis). For example, the presence of a tumor or bile Figure 21.12 stones may block the bile ducts, preventing passage of biliru- Alterations in the metabolism of heme. bin into the intestine. Patients with obstructive jaundice experi- A. Hemolytic jaundice. B. Neonatal jaundice. BG = bilirubin glucuronide; B = bilirubin; ence gastrointestinal pain and nausea, and produce stools that U = urobilinogen; S = stercobilin. are a pale, clay color, and urine that dark ens upon standing. The liver “regurgitates” conjugated bilirubin into the blood 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 285

III. Other Nitrogen-Containing Compounds 285

(hyperbilirubinemia). The compound is eventually excreted in the urine. Urinary urobiloinogen is absent. [Note: Prolonged Activity of the enzyme that 1 conjugates bilirubin with obstruction of the bile duct can lead to liver damage and a glucuronic acid, bilirubin subsequent rise in unconjugated bilirubin.] glucuronyltransferase, is low in newborns and especially low in 2. Jaundice in newborns: Newborn infants, particularly if premature, premature babies. often accumulate bilirubin, because the activity of hepatic bilirubin glucuronyltransferase is low at birth—it reaches adult levels in about 4 weeks (Figures 21.12B and 21.13). Elevated bili rubin, in excess of the binding capacity of albumin, can diffuse into the basal ganglia and cause toxic encephalopathy (kernicterus). Thus, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light (Figure 21.14), which converts bilirubin to more polar and, hence, water-soluble isomers. These 0 6 12 18 24 30 photoisomers can be excreted into the bile without conjugation to Postnatal days glucuronic acid.

3. Determination of bilirubin concentration: Bilirubin is most com- Serum levels of bilirubin rise 2 after birth in full-term infants, monly determined by the van den Bergh reaction, in which diazo - although usually not to tized sulfanilic acid reacts with bilirubin to form red azodipyrroles dangerous concentrations. that are measured colorimetrically. In aqueous solution, the water- soluble, conjugated bilirubin reacts rapidly with the reagent (within one minute), and is said to be “direct-reacting.” The unconjugated bilirubin, which is much less soluble in aqueous solution, reacts more slowly. However, when the reaction is carried out in methanol, both conjugated and unconjugated bilirubin are soluble and react with the reagent, providing the total bilirubin value. The “indirect-reacting” bilirubin, which corresponds to the unconjugated bilirubin, is obtained by subtracting the direct-reacting bilirubin from the total bilirubin. [Note: In normal plasma, only about 4% Serum levels of bilirubin in of the total bilirubin is conjugated or direct-reacting, because most 3 premature infants may rise to is secreted into bile.] toxic levels.

III. OTHER NITROGEN-CONTAINING COMPOUNDS Figure 21.13 Neonatal jaundice. GT = glucuronyl- A. Catecholamines transferase. Dopamine, norepinephrine, and epinephrine are biologically active (biogenic) amines that are collectively termed catecholamines. Dopamine and norepinephrine are synthesized in the brain and function as neurotransmitters. Norepinephrine is also synthesized in the adrenal medulla, as is epinephrine.

1. Function: Outside the nervous system, norepinephrine and its methylated derivative, epinephrine, are hormone regulators of car- bohydrate and lipid metabolism. Norepinephrine 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. Synthesis of catecholamines: The catecholamines are synthe- Figure 21.14 sized from tyrosine, as shown in Figure 21.15. Tyrosine is first Phototherapy in neonatal jaudice hydroxylated by tyrosine hydroxylase to form 3,4-dihydroxyphenyl - 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 286

286 21. Conversion of Amino Acids to Specialized Products

Tyrosine DOPA- – – CH2CHCOO hydroxylase CH2CHCOO decarboxylase CH2CH2NH2 NH NH PLP Ascorbate + 3 + 3 + O2 HO HO HO Tetrahydro- Dihydro- OH CO2 OH biopterin biopterin Tyrosine + O + H O 3,4-Dihydroxy- Dopamine 2 2 phenylalanine Cu+2 (dopa) Dopamine β-hydroxylase

OH H Phenylethanolamine- OH H CH3 C C N N-methyl- C C NH2 Dehydro- transferase H H H H H ascorbate + H2O HO HO OH OH Epinephrine S-Adenosyl- S-Adenosyl- Norepinephrine homocysteine methionine (SAM)

Figure 21.15 Synthesis of catecholamines. PLP = pyridoxal phosphate. alanine (DOPA) in a reaction analogous to that described for the hydroxylation of phenylalanine (see p. 268). The tetrahydro- biopterin (BH4)-requiring enzyme is abundant in the central nervous system, the sympathetic ganglia, and the adrenal medulla, and is the rate-limiting step of the pathway. DOPA is decarboxylated in a reaction requiring pyridoxal phosphate (PLP, see p. 378) to form dopamine, which is hydroxylated by dopamine β-hydroxy- Epinephrine Norepinephrine lase to yield nor epinephrine in a reaction that requires ascorbate (vitamin C) and copper. Epinephrine is formed from nore- MAO MAO pinephrine by an N-methylation reaction using S-adenosylmethionine (SAM) as the methyl donor (see p. 264). COMT COMT Dihydroxymandelic acid

COMT Parkinson disease, a neurodegenerative move- Metanephrine Normetanephrine ment disorder, is due to insufficient dopamine production as a result of the idiopathic loss of MAO MAO dopamine-producing cells in the brain. Administration of L-DOPA (levodopa) is the most Vanillylmandelic acid (VMA) common treatment.

Dopamine 3. Degradation of catecholamines: The catecholamines are inacti- MAO COMT vated by oxidative deamination catalyzed by monoamine oxidase (MAO), and by O-methylation carried out by catechol-O-methyltransferase using SAM as the methyl donor (Figure 21.16). The Dihydroxyphenyl- 3-Methoxytyramine acetic acid two reactions can occur in either order. The aldehyde products of the MAO reaction are oxidized to the corresponding acids. The COMT MAO metabolic products of these reactions are excreted in the urine as vanillylmandelic acid (VMA) from epinephrine and norepinephrine, Homovanillic and homovanillic acid from dopamine. [Note: VMA is increased acid (HVA) with pheochromocytomas, tumors of the adrenal characterized by excessive production of catecholamines.] Figure 21.16 Metabolism of the catecholamines 4. MAO inhibitors: MAO is found in neural and other tissues, such by catechol-O-methyltranferase as the intestine and liver. In the neuron, this enzyme oxidatively (COMT) and monoamine oxidase deaminates and inactivates any excess neurotransmitter (MAO). molecules (norepinephrine, dopamine, or serotonin) that may 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 287

III. Other Nitrogen-Containing Compounds 287

leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors may irreversibly or reversibly inactivate the enzyme, NN permitting neurotransmitter molecules to escape degradation and, therefore, to both accumulate within the presynaptic neuron CH and to leak into the synaptic space. This causes activation of 2 norepinephrine and serotonin receptors, and may be responsible H2 N C COOH H for the antidepressant action of these drugs. Histidine B. Histamine Decarboxylase Histamine is a chemical messenger that mediates a wide range of PLP cellular responses, including allergic and inflammatory reactions, gas- CO2 tric acid secretion, and possibly neurotransmission in parts of the NN brain. A powerful vasodilator, histamine is formed by decarboxylation of histidine in a reaction requiring PLP (Figure 21.17). It is secreted by mast cells as a result of allergic reactions or trauma. Histamine has no CH2 clinical applications, but agents that interfere with the action of his- H2 N CH2 tamine have important therapeutic applications. Histamine C. Serotonin Serotonin, also called 5-hydroxytryptamine (5HT), is synthesized Figure 21.17 and stored at several sites in the body (Figure 21.18). By far the Biosynthesis of histamine. PLP = pyridoxal phosphate. largest amount of serotonin is found in cells of the intestinal mucosa. Smaller amounts occur in the central nervous system, where it functions as a neurotransmitter, and in platelets. Serotonin is synthesized from tryptophan, which is hydroxylated in a BH4- – requiring reaction analogous to that catalyzed by phenylalanine CH2CHCOO hydroxylase. The product, 5-hydroxy tryptophan, is decarboxylated to NH + 3 serotonin, which is also degraded by MAO. Serotonin has multiple N H physiologic roles, including pain perception, regulation of sleep, appetite, temperature, blood pressure, cognitive functions, and Tryptophan mood (causes a feeling of well-being). [Note: Serotonin is converted Tetrahydro- O2 to melatonin in the pineal gland via acetylation and methylation.] biopterin

Dihydro- Hydroxylase D. Creatine biopterin + H O Creatine phosphate (also called phosphocreatine), the phosphory- 2

lated derivative of creatine found in muscle, is a high-energy com- HO – pound that provides a small but rapidly mobilized reserve of CH2CHCOO NH3 + high-energy phosphates that can be reversibly transferred to ADP N (Figure 21.9) to maintain the intracellular level of ATP during the first H 5-Hydroxy- few minutes of intense muscular contraction. [Note: The amount of tryptophan creatine phosphate in the body is proportional to the muscle mass.]

1. Synthesis: Creatine is synthesized from glycine and the guanidino CO Decarboxylase 2 PLP group of arginine, plus a methyl group from SAM (see Figure 21.19). Creatine is reversibly phosphorylated to creatine phos- HO CH2CH2NH2 phate by creatine kinase, using ATP as the phosphate donor. [Note: The presence of creatine kinase (MB isozyme) in the N plasma is indicative of heart damage, and is used in the diagnosis H of myocardial infarction (see p. 65).] Serotonin

2. Degradation: Creatine and creatine phosphate spontaneously Figure 21.18 cyclize at a slow but constant rate to form creatinine, which is Synthesis of serotonin. PLP = excreted in the urine. The amount of creatinine excreted is propor- pyridoxal phosphate. 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 288

288 21. Conversion of Amino Acids to Specialized Products

tional to the total creatine phosphate content of the body, and thus can be used to estimate muscle mass. When muscle mass NH2 + decreases for any reason (for example, from paralysis or muscular C NH2 NH dystrophy), the creatinine content of the urine falls. In addition, any rise in blood creatinine is a sensitive indicator of kidney mal- CH2 function, because creatinine normally is rapidly removed from the CH2 blood and excreted. A typical adult male excretes about 15 mmol CH2 H + + of creatinine per day. HCNH3 + HCNH3 COO– COO– E. Melanin Arginine Glycine Melanin is a pigment that occurs in several tissues, particularly the eye, hair, and skin. It is synthesized from tyrosine in the epidermis by Amidino- pigment-forming cells called melanocytes. Its function is to protect transferase Ornithine underlying cells from the harmful effects of sunlight. [Note: A defect in melanin production results in albinism, the most common form being due to defects in copper-containing tyrosinase (see p. 273).] NH2 + C NH2 NH IV. CHAPTER SUMMARY CH2 COO– Guanidinoacetate Amino acids are precursors of many nitrogen-containing compounds including porphyrins, which, in combination with ferrous (Fe2+) iron, S-Adenosylmethionine form heme (Figure 21.20). The major sites of heme biosynthesis are Methyltransferase the liver, which synthesizes a number of heme proteins (particularly cytochrome P450), and the erythrocyte-producing cells of the bone S-Adenosylhomocysteine marrow, which are active in hemoglobin synthesis. In the liver, the rate NH2 + of heme synthesis is highly variable, responding to alterations in the C NH 2 cellular heme pool caused by fluctuating demands for hemeproteins. In NCH 3 contrast, heme synthesis in erythroid cells is relatively constant, and is CH 2 matched to the rate of globin synthesis. Porphyrin synthesis start with COO– Creatine glycine and succinyl CoA. The committed step in heme synthesis is the formation of δ-aminolevulinic acid (ALA). This reaction is catalyzed ATP ATP H20 by ALA synthase-1 in liver (inhibited by hemin, the oxidized form of Creatine heme that accumulates in the cell when heme is being underutilized) kinase and ALA synthase-2 in erythroid tissues (regulated by iron). C NH ADP ADP + H+ Porphyrias are caused by inherited or acquired defects in heme syn- NCH3 O NH – – thesis, resulting in the accumulation and increased excretion of por- CH2 OOP + phyrins or porphyrin precursors. With few exceptions, porphyrias are CO NH2 Creatinine C NH inherited as autosomal dominant disorders. Degradation of hemepro- Pi teins occurs in the reticuloendothelial system, particularly in the liver NCH3 and spleen. The first step in the degradation of heme is the production CH2 COO– of the green pigment biliverdin, which is subsequently reduced to Creatine phosphate bilirubin. Bilirubin is transported to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid. Bilirubin Figure 21.19 diglucuronide is transported into the bile canaliculi, where it is first Synthesis of creatine. hydrolyzed and reduced by bacteria in the gut to yield urobilinogen , then oxidized by intestinal bacteria to sterco bilin . Jaundice refers to the yellow color of the skin and sclera that is caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Three commonly encountered type of jaundice are hemolytic jaundice, obstructive jaundice, and hepatocellular jaundice. Other important N- containing compounds derived from amino acids include the catecholamines (dopamine, norepinephrine, and epinephrine), creatine , histamine, serotonin, and melanin. 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 289

IV. Chapter Summary 289

Heme synthesis Heme degradation occurs in occurs in SPLEEN, LIVER Erythrocytes, heptocytes LIVER BONE MARROW Glycine Iron Glycine Hemoglobin, + + Cytochromes Succinyl CoA δ-Amino- δ-Amino- Succinyl CoA catalyzed by Amino 1 catalyzed by levulinate levulinate 1 acids δ-Aminolevulinic acid synthase-1 synthase-2 δ-Aminolevulinic acid Heme 2 Lead Lead catalyzes catalyzes 2 Porphobilinogen Porphobilinogen Biliverdin, CO, Fe2+ 3 3 Rate-limiting step Hydroxymethylbilane Hydroxymethylbilane Bilirubin 4 4 BLOOD Uroporphyrinogen III Uroporphyrinogen III Bilirubin-albumin 5 5 LIVER Coproporphyrinogen III Coproporphyrinogen III 6 6 Bilirubin Protoporphyrinogen IX Protoporphyrinogen IX 7 7 Bilirubin glucuronide Protoporphyrin IX Protoporphyrin IX INTESTINE 8 Lead 8 Lead Heme Heme Bilirubin glucuronide

Bilirubin Inherited enzyme Inherited enzyme deficiencies in deficiencies in liver bone marrow Urobilinogen lead to lead to 3 Acute intermittent porphyria 4 Congenital erythropoietic porphyria Stercobilin Hepatic 5 Porphyria cutanea tarda Erythropoietic (brown pigment in stools) porphyrias 6 Hereditary coproporphyria porphyrias 7 Variegate porphyria 8 Erythropoietic protoporphyria Urobilin (yellow pigment in urine)

Hemolytic jaundice Obstructive jaundice Hepatocellar jaundice Neonatal jaundice

Lysed erythrocytes Erythrocytes, heptocytes Erythrocytes, heptocytes Erythrocytes, heptocytes

Hemoglobin Hemoglobin, Hemoglobin, Hemoglobin, Cytochromes Cytochromes Cytochromes Amino acids Amino Amino Amino Heme acids acids acids Heme Heme Heme

Biliverdin, CO, Fe2+ Biliverdin, CO, Fe2+ Biliverdin, CO, Fe2+ Biliverdin, CO, Fe2+

Bilirubin Bilirubin Bilirubin Bilirubin

Bilirubin glucuronide Bilirubin glucuronide Bilirubin glucuronide Bilirubin glucuronide Gallstone INTESTINE Bilirubin Bilirubin Bilirubin Bilirubin

Urobilinogen Urobilinogen Urobilinogen Urobilinogen

Stercobilin Stercobilin Stercobilin Stercobilin Urobilin Urobilin Urobilin Urobilin

Figure 21.20 Key concept map for heme metabolism. = Block in the pathway. 168397_P277-290.qxd7.0:21 Specialized products5-27-04 2010.4.4 6:17 PM Page 290

290 21. Conversion of Amino Acids to Specialized Products

Study Questions

Choose the ONE best answer. Correct answer = B. The activity of δ-aminolevulinic acid synthase controls the rate of por- 21.1 δ-Aminolevulinic acid synthase activity: phyrin synthesis. The hepatic form of the A. in liver is frequently decreased in individuals enzyme is increased in patients treated with cer- treated with drugs, such as the barbiturate pheno- tain drugs, and requires pyridoxal phosphate as a coenzyme. Another enzyme in the pathway (δ- barbital. aminolevulinic acid dehydrase) is extremely sen- B. catalyzes a rate-limiting reaction in porphyrin sitive to the presence of heavy metals. biosynthesis. C. requires the coenzyme biotin. D. is strongly inhibited by heavy metal ions such as lead. E. occurs in the cytosol. Correct answer = B. The cyclic heme molecule is oxidatively cleaved to form biliverdin. The 21.2 The catabolism of hemoglobin: catabolism occurs in the cells of the reticulo - endothelial system, particularly the spleen, and A. occurs in red blood cells. results in the liberation of carbon monoxide. B. involves the oxidative cleavage of the porphyrin ring. Protoporphyrinogen is an intermediate in the C. results in the liberation of carbon dioxide. synthesis, not degradation, of heme. D. results in the formation of protoporphyrinogen. Cytochromes and other non-hemoglobin hemeproteins are also precursors of bilirubin. E. is the sole source of bilirubin.

21.3 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 poison ivy or sumac, new soaps or Correct answer = A. The disease is associated detergents, or new medications. He denied having with a deficiency in uroporphyrinogen decar- previous episodes of blistering. He had partial com- boxylase, but clinical expression of the enzyme plex seizure disorder that had begun about three deficiency is influenced by hepatic injury caused years earlier after a head injury. The patient had by iron overload, chronic ethanol consumption, and the presence of hepatitis B or C and HIV been taking phenytoin—his only medication—since infections. Exposure to sunlight can also be a the onset of the seizure disorder. He admitted to an precipitating factor. Clinical onset is typically average weekly ethanol intake of about eighteen 12- during the fourth or fifth decade of life. Porphyrin oz cans of beer. The patient’s urine was reddish accumulation leads to cutaneous symptoms and orange. Cultures obtained from skin lesions failed to urine that is red to brown. Treatment of the grow organisms. A 24-hour urine collection showed patient's seizure disorder with phenytoin caused elevated uroporphyrin (1,000 mg; normal, <27mg). increased synthesis of ALA synthase, and, The most likely presumptive diagnosis is: therefore, of uroporphyrinogen, the substrate of the deficient enzyme. The laboratory and clinical A. porphyria cutanea tarda. findings are inconsistent with other porphyrias. B. acute intermittent porphyria. C. hereditary coproporphyria. D. congenital erythropoietic porphyria. E. erythropoietic protoporphyria. Correct answer = C. A deficiency of uropor- 21.4 A 10-year-old boy is referred to a specialist because phyrinogen III synthase results in accumulation of skin that blisters easily, urine that darkens on of hydroxymethylbilane and the spontaneous standing, and stained teeth. Lab studies are remark- conversion of this substrate to porphyrins of the able for high levels of uroporphyrin I and copropor- Type I series. A deficiency of ALA synthase or phyrin I in plasma, with uroporphyrin I being present inhibition of ALA dehydratase by lead would not in the urine. The most likely biochemical pathology in allow the synthesis of porphobilinogen, the first this case is: pyrrole product in the heme biosynthetic pathway, and thus would not result in uro- or copro- A. deficiency of ALA synthase. porphyrin synthesis. Deficiency of the B. deficiency of bilirubin glucuronyltransferase. glucuronyltransferase would not present with the C. deficiency of uroporphyrinogen III synthase. systems described, and lab studies would be remarkable for an elevation of unconjugated D. down-regulation of tyrosinase. bilirubin. Down-regulation of tyrosinase would E. inhibition of ALA dehydratase by lead. result in decreased pigmentation.