Harper's Biochemistry Chapter 31 - Porphyrins & Bile Pigments PDF

C H A P T E R Porphyrins & Bile Pigments Victor W. Rodwell, PhD O B J E C TI V E S 31 Write the structural ormulas o the tw...

C H A P T E R Porphyrins & Bile Pigments Victor W. Rodwell, PhD O B J E C TI V E S 31 Write the structural ormulas o the two amphibolic intermediates whose condensation initiates heme biosynthesis. After studying this chapter, Identiy the enzyme that catalyzes the key regulated step o hepatic heme you should be able to: biosynthesis. Explain why, although porphyrinogens and porphyrins both are tetrapyrroles, porphyrins are colored whereas porphyrinogens are colorless. Speciy the intracellular locations o the enzymes and metabolites o heme biosynthesis. Outline the causes and clinical presentations o various porphyrias. Identiy the roles o heme oxygenase and o UDP-glucosyl transerase in heme catabolism. Defne jaundice, name some o its causes, and suggest how to determine its biochemical basis. Speciy the biochemical basis o the clinical laboratory terms “direct bilirubin” and “indirect bilirubin.” BIOMEDICAL IMPORTANCE pyrrole rings. Examples include iron porphyrins such as the heme o hemoglobin and the magnesium-containing porphy- he biochemistry o the porphyrins and o the bile pig- rin chlorophyll, the photosynthetic pigment o plants. Heme ments are closely related topics. Heme is synthesized rom proteins are ubiquitous in biology and serve diverse unctions porphyrins and iron, and the products o degradation o including, but not limited to, oxygen transport and storage heme include the bile pigments and iron. he biochemistry o (eg, hemoglobin and myoglobin) and electron transport (eg, the porphyrins and o heme is basic to understanding the cytochrome c and cytochrome P450). Hemes are tetrapyr- varied unctions o hemoproteins, and the porphyrias, roles, o which two types, heme b and heme c, predominate a group o diseases caused by abnormalities in the pathway (Figure 31–2). In heme c the vinyl groups o heme b are o porphyrin biosynthesis. A much more common clinical replaced by covalent thioether links to an apoprotein, typically condition is jaundice, a consequence o an elevated level o via cysteinyl residues. Unlike heme b, heme c thus does not plasma bilirubin, due either to overproduction o bilirubin readily dissociate rom its apoprotein. or to ailure o its excretion. Jaundice occurs in numerous Proteins that contain heme are widely distributed in nature diseases ranging rom hemolytic anemias to viral hepatitis (Table 31–1). Vertebrate heme proteins generally bind one and to cancer o the pancreas. mole o heme c per mole, although those o nonvertebrates may bind signiicantly more heme. PORPHYRINS Porphyrins are cyclic compounds ormed by the linkage HEME IS SYNTHESIZED FROM o our pyrrole rings through methyne (—— HC—) bridges (Figure 31–1). Various side chains can replace the eight SUCCINYL-CoA & GLYCINE numbered hydrogen atoms o the pyrrole rings. he biosynthesis o heme involves both cytosolic and mito- Porphyrins can orm complexes with metal ions that orm chondrial reactions and intermediates. Heme biosynthesis coordinate bonds to the nitrogen atom o each o the our occurs in most mammalian cells except mature erythrocytes, 315 316 SECTION VI Metabolism o Proteins & Amino Acids TABLE 31–1 Examples of Important Heme Proteinsa Protein Function Hemoglobin Transport o oxygen in blood Myoglobin Storage o oxygen in muscle Cytochrome c Involvement in the electron transport chain Cytochrome P450 Hydroxylation o xenobiotics Catalase Degradation o hydrogen peroxide Tryptophan pyrrolase Oxidation o tryptophan a The unctions o the above proteins are described in various chapters o this text. Following the exit o δ-aminolevulinate into the cytosol, the reaction catalyzed by cytosolic ALA dehydratase (EC 4.2.1.24; porphobilinogen synthase) condenses two molecules o ALA, orming porphobilinogen: FIGURE 31–1 The porphyrin molecule. Rings are labeled I, II, III, and IV. Substituent positions are labeled 1 through 8. The our (2) 2 δ-Aminolevulinate → porphobilinogen + 2 H2O methyne bridges (=HC—) are labeled α, β, γ, and δ. (Figure 31–4). A zinc metalloprotein, ALA dehydratase is sensitive to inhibition by lead, as can occur in lead poisoning. which lack mitochondria. Approximately 85% o heme syn- he third reaction, catalyzed by cytosolic hydroxymethyl- thesis occurs in erythroid precursor cells in the bone mar- bilane synthase (uroporphyrinogen I synthase, EC 2.5.1.61) row, and the majority o the remainder in hepatocytes. Heme involves head-to-tail condensation o our molecules o por- biosynthesis is initiated by the condensation o succinyl-CoA phobilinogen to orm the linear tetrapyrrole hydroxymethyl- and glycine in a pyridoxal phosphate-dependent reaction cat- bilane (Figure 31–5, top): alyzed by mitochondrial δ-aminolevulinate synthase (ALA (3) 4 Porphobilinogen + H2O → hydroxymethylbilane + 4 NH3 synthase, EC 2.3.1.37). Subsequent cyclization o hydroxymethylbilane, catalyzed (1) Succinyl-CoA + glycine → δ-aminolevulinate by cytosolic uroporphyrinogen III synthase, EC 4.2.1.75: + CoA-SH + CO2 (4) Hydroxymethylbilane → uroporphyrinogen III + H2O Humans express two isozymes o ALA synthase. ALAS1 is ubiquitously expressed throughout the body, whereas ALAS2 orms uroporphyrinogen III (Figure 31–5, bottom right). is expressed in erythrocyte precursor cells. Formation o Hydroxymethylbilane can undergo spontaneous cyclization δ-aminolevulinate is rate-limiting or porphyrin biosynthesis orming uroporphyrinogen I (Figure 31–5, bottom left), in mammalian liver (Figure 31–3). but under normal conditions, the uroporphyrinogen ormed is almost exclusively the type III isomer. he type I isomers o porphyrinogens are, however, ormed in excess in certain porphyrias. Since the pyrrole rings o these uroporphyrino- gens are connected by methylene (—CH2—) rather than by Cys S Cys S N N N N Fe Fe N N N N O OH OH O OH OH O O heme b heme c FIGURE 31–3 Synthesis of δ-aminolevulinate (ALA). This FIGURE 31–2 Structures of heme b and heme c. mitochondrial reaction is catalyzed by ALA synthase.  CHAPTER 31 Porphyrins & Bile Pigments 317 A P M P I Uroporphyrinogen I A A decarboxylase M M IV II IV II P III P P III P 4CO2 P A P M Uroporphyrinogen III Coproporphyrinogen III FIGURE 31–6 Decarboxylation of uroporphyrinogen III to coproporphyrinogen III. Shown is a representation o the tetrapyr- role to emphasize the conversion o our attached acetyl groups to FIGURE 31–4 Formation of porphobilinogen. Cytosolic por- methyl groups. (A, acetyl; M, methyl; P, propionyl.) phobilinogen synthase converts two molecules o δ-aminolevulinate to porphybilinogen. All our acetate moieties o uroporphyrinogen III next undergo decarboxylation to methyl (M) substituents, orming methyne bridges (— — HC—), the double bonds do not orm a coproporphyrinogen III in a cytosolic reaction catalyzed by conjugated system. Porphyrinogens thus are colorless. hey uroporphyrinogen decarboxylase, EC 4.1.1.37 (Figure 31–6): are, however, readily auto-oxidized to colored porphyrins. (5) Uroporphyrinogen III → coproporphyrinogen III + 4 CO2 his decarboxylase can also convert uroporphyrinogen I, i A HOOC COOH present, to coproporphyrinogen I. P H 2C CH 2 he inal three reactions o heme biosynthesis all occur in CH 2 mitochondria. Coproporphyrinogen III enters the mitochon- C C dria and is converted, successively, to protoporphyrinogen III, C CH and then to protoporphyrin III. hese reactions are catalyzed H 2C N by coproporphyrinogen oxidase (EC 1.3.3.3), which decar- NH 2 H boxylates and oxidizes the two propionic acid side chains to Four molecules of orm protoporphyrinogen III: porphobilinogen (6) Coproporphyrinogen III + O2 + 2 H+ → Uroporphyrinogen I 4 NH 3 synthase protoporphyrinogen III + 2 CO2 + 2 H2O Hydroxymethylbilane his oxidase is speciic or type III coproporphyrinogen, (linear tetrapyrrole) so type I protoporphyrins generally do not occur in humans. Spontaneous Uroporphyrinogen III Protoporphyrinogen III is next oxidized to protoporphyrin cyclization synthase III in a reaction catalyzed by protoporphyrinogen oxidase, EC 1.3.3.4: A P A P A P A P (7) Protoporphyrinogen III + 3 O2 → protoporphyrin III C C H2 C C C C C H2 C C C + 3 H2O2 I II I II C C C C C C C C N N N N he eighth and inal step in heme synthesis involves the CH 2 H H CH 2 CH 2 H H CH 2 incorporation o errous iron into protoporphyrin III in a reac- H H H H tion catalyzed by ferrochelatase (heme synthase, EC 4.99.1.1), N N N N (Figure 31–7): C C C C C C C C IV III IV III C C C C H2 C C C C H2 C C (8) Protoporphyrin III + Fe2+ → heme + 2 H+ P A P A A P P A Figure 31–8 summarizes the stages o the biosynthesis o the Type I Type III uroporphyrinogen uroporphyrinogen porphyrin derivatives rom porphobilinogen. For the above reactions, numbers correspond to those in Figure 31–8 and FIGURE 31–5 Synthesis of hydroxymethylbilane and its in Table 31–2. subsequent cyclization to porphobilinogen III. Cytosolic hydroxy- methylbilane synthase (ALA dehydratase) orms a linear tetrapyrrole, which cytosolic uroporphyrinogen synthase cyclizes to orm uropor- ALA Synthase Is the Key Regulatory phyrinogen III. Notice the asymmetry o the substituents on ring 4, so that the highlighted acetate and propionate substituents are Enzyme in Hepatic Biosynthesis of Heme reversed in uroporphyrinogens I and III. (A, acetate [—CH2COO–]; Unlike ALAS2, which is expressed exclusively in erythrocyte P, propionate [—CH2CH2COO–].) precursor cells, ALAS1 is expressed throughout body tissues. 318 SECTION VI Metabolism o Proteins & Amino Acids Porphobilinogen Uroporphyrinogen I synthase Hydroxymethylbilane Uroporphyrinogen III synthase Spontaneous 6H 6H Uroporphyrin III Uroporphyrinogen III Uroporphyrinogen I Uroporphyrin I Light Light Cytosol Uroporphyrinogen 6H decarboxylase 6H 4CO 2 4CO 2 Coproporphyrin III Coproporphyrinogen III Coproporphyrinogen I Coproporphyrin I Light Light Coproporphyrinogen oxidase Protoporphyrinogen III Mitochondria Protoporphyrinogen Or light in vitro oxidase 6H Protoporphyrin III Fe2+ Ferrochelatase Heme FIGURE 31–7 Biosynthesis from porphobilinogen of the indicated porphyrin derivatives. he reaction catalyzed by ALA synthase 1 (Figure 31–3) is absorption and luorescence spectra. he visible and the rate-limiting or biosynthesis o heme in liver. ypically or an ultraviolet spectra o porphyrins and porphyrin derivatives enzyme that catalyzes a rate-limiting reaction, ALAS1 has a are useul or their identiication (Figure 31–9). he sharp short hal-lie. Heme, acting through the Erg-1 aporepressor absorption band near 400 nm, a distinguishing eature shared and one o its NAB corepressors, acts as a negative regulator by all porphyrins, is termed the Soret band ater its discoverer, o the synthesis o ALAS1 (Figure 31–8). Synthesis o ALAS1 the French physicist Charles Soret. thus increases greatly in the absence o heme, but diminishes Porphyrins dissolved in strong mineral acids or in organic in its presence. Heme also aects translation o ALAS1 and solvents and illuminated by ultraviolet light emit a strong red its translocation rom its cytosolic site o synthesis into the fluorescence, a property oten used to detect small amounts mitochondrion. Many drugs whose metabolism requires the o ree porphyrins. he photodynamic properties o porphy- hemoprotein cytochrome P450 increase cytochrome P450 rins have suggested their possible use in the treatment o cer- biosynthesis. he resulting depletion o the intracellular heme tain types o cancer, a procedure called cancer phototherapy. pool induces synthesis o ALAS1, and the rate o heme syn- Since tumors oten take up more porphyrins than do normal thesis rises to meet metabolic demand. By contrast, since tissues, hematoporphyrin or related compounds are admin- ALAS2 is not eedback regulated by heme, its biosynthesis is istered to a patient with an appropriate tumor. he tumor is not induced by these drugs. then exposed to an argon laser to excite the porphyrins, pro- ducing cytotoxic eects. PORPHYRINS ARE Spectrophotometry Is Used to Detect COLORED & FLUORESCE Porphyrins & Their Precursors While porphyrinogens are colorless, the various porphyrins Coproporphyrins and uroporphyrins are excreted in increased are colored. he conjugated double bonds in the pyrrole amounts in the porphyrias. When present in urine or eces, they rings and linking methylene groups o porphyrins (absent in can be separated by extraction with appropriate solvents, then the porphyrinogens) are responsible or their characteristic identiied and quantiied using spectrophotometric methods.  CHAPTER 31 Porphyrins & Bile Pigments 319 Hemoproteins Proteins Heme Aporepressor 8. Ferrochelatase Fe 2 + Protoporphyrin III 7. Protoporphyrinogen oxidase Protoporphyrinogen III 6. Coproporphyrinogen oxidase Coproporphyrinogen III 5. Uroporphyrinogen decarboxylase Uroporphyrinogen III 4. Uroporphyrinogen III synthase Hydroxymethylbilane 3. Uroporphyrinogen I synthase Porphobilinogen 2. ALA dehydratase ALA 1. ALA synthase – Succinyl-CoA + Glycine FIGURE 31–8 Intermediates, enzymes, and regulation of heme synthesis. The numbers o the enzymes that catalyze the indicated reactions are those used in the accompanying text and in column 1 o Table 31–2. Enzymes 1, 6, 7, and 8 are mitochondrial, but enzymes 2 to 5 are cytosolic. Regulation o hepatic heme synthesis occurs at ALA synthase (ALAS1) by a repression–derepression mechanism mediated by heme and a hypothetical aporepressor (not shown). Mutations in the gene encoding enzyme 1 cause X-linked sideroblastic anemia. Mutations in the genes encoding enzymes 2 to 8 give rise to the porphyrias. DISORDERS OF HEME 1. Similar or identical clinical signs and symptoms can arise rom dierent mutations in genes that encode either a given BIOSYNTHESIS enzyme or an enzyme that catalyzes a successive reaction. Disorders o heme biosynthesis may be genetic or acquired. 2. Rational therapy requires an understanding o the bio- An example o an acquired deect is lead poisoning. Lead can chemistry o the enzyme-catalyzed reactions in both nor- inactivate errochelatase and ALA dehydratase by complexing mal and impaired individuals. with essential thiol groups. Signs include elevated levels o 3. Identiication o the intermediates and side products that protoporphyrin in erythrocytes and elevated urinary levels o accumulate prior to a metabolic block can provide the ALA and coproporphyrin. basis or metabolic screening tests that can implicate the Genetic disorders o heme metabolism and o bilirubin impaired reaction. metabolism (see below) share the ollowing eatures with met- 4. Deinitive diagnosis involves quantitative assay o the activ- abolic disorders o urea biosynthesis (see Chapter 28): ity o the enzyme(s) suspected to be deective. o this might 320 SECTION VI Metabolism o Proteins & Amino Acids TABLE 31–2 Summary of Major Findings in the Porphyriasa Enzyme Involvedb Type, Class, and OMIM Number Major Signs and Symptoms Results of Laboratory Tests c 1. ALA synthase 2 (ALAS2), X-linked sideroblasticanemia Anemia Red cell counts and hemoglobin EC 2.3.1.37 (erythropoietic) (OMIM 301300) decreased 2. ALA dehydratase EC 4.2.1.24 ALA dehydratase defciency Abdominal pain, neuropsychiatric Urinary ALA and coproporphyrin (hepatic) (OMIM 125270) symptoms III increased 3. Uroporphyrinogen I synthased Acute intermittent porphyria Abdominal pain, neuropsychiatric Urinary ALA and PBGe increased EC 2.5.1.61 (hepatic) (OMIM 176000) symptoms 4. Uroporphyrinogen III synthase Congenital erythropoietic Photosensitivity Urinary, ecal, and red cell EC 4.2.1.75 (erythropoietic) (OMIM 263700) uroporphyrin I increased 5. Uroporphyrinogen Porphyria cutaneatarda (hepatic) Photosensitivity Urinary uroporphyrin I increased decarboxylase EC 4.1.1.37 (OMIM 176100) 6. Coproporphyrinogen oxidase Hereditary coproporphyria Photosensitivity, abdominal pain, Urinary ALA, PBG, and EC 1.3.3.3 (hepatic) (OMIM 121300) neuropsychiatric symptoms coproporphyrin III and ecal coproporphyrin III increased 7. Protoporphyrinogen oxidase Variegate porphyria (hepatic) Photosensitivity, abdominal pain, Urinary ALA, PBG, and EC 1.3.3.4 (OMIM 176200) neuropsychiatric symptoms coproporphyrin III and ecal protoporphyrin IX increased 8. Ferrochelatase EC 4.99.1.1 Protoporphyria (erythropoietic) Photosensitivity Fecal and red cell protoporphyrin (OMIM 177000) IX increased a Only the biochemical indings in the active stages o these diseases are listed. Certain biochemical abnormalities are detectable in the latent stages o some o the above condi- tions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias. Condition 2 is rare. b The numbering o the enzymes in this Table corresponds to that used in Figure 31–8. c X-linkedsideroblastic anemia is not a porphyria but is included here because ALA synthase is involved. d This enzyme is also called PBG deaminase or hydroxymethylbilane synthase. e PBG = porphyrobilinogen III. Abbreviations: ALA, δ-aminolevulinic acid; PBG, porphobilinogen. be added consideration o the as yet incompletely identiied rom the accumulation o metabolites prior to the block. actors that acilitate translocation o enzymes and inter- able 31–2 lists six major types o porphyria that relect low mediates between cellular compartments. or absent activity o enzymes that catalyze reactions 2 through 5. Comparison o the DNA sequence o the gene that encodes 8 o Figure 31–8. Possibly due to potential lethality, there is no a given mutant enzyme to that o the wild-type gene can known deect o ALAS1. Individuals with low ALAS2 activ- identiy the speciic mutation(s) that cause the disease. ity develop anemia, not porphyria (able 31–2). Porphyria consequent to low activity o ALA dehydratase, termed ALA dehydratase-deicient porphyria, is extremely rare. The Porphyrias he signs and symptoms o porphyria result either rom a Congenital Erythropoietic Porphyria deficiency o intermediates beyond the enzymatic block, or While most porphyrias are inherited in an autosomal dominant manner, congenital erythropoietic porphyria is inherited in a recessive mode. he deective enzyme in congenital erythropoi- etic porphyria is uroporphyrinogen III synthase (Figure 31–5, 5 bottom). he photosensitivity and severe disigurement exhib- ited by some victims o congenital erythropoietic porphyria has Log absorbency 4 suggested them as prototypes o so-called werewolves. 3 Acute Intermittent Porphyria 2 he deective enzyme in acute intermittent porphyria is 1 hydroxymethylbilane synthase (Figure 31–5, bottom). ALA and porphobilinogen accumulate in body tissues and luids (Figure 31–10). 300 400 500 600 700 Wavelength (nm) Subsequent Metabolic Blocks FIGURE 31–9 Absorption spectrum of hematoporphyrin. The Blocks later in the pathway result in the accumulation of spectrum is o a dilute (0.01%) solution o hematoporphyrin in 5% HCl. the porphyrinogens indicated in Figures 31–8 and 31–10.  CHAPTER 31 Porphyrins & Bile Pigments 321 o hematin to repress ALAS1 synthesis to diminish production Mutations in various genes o harmul heme precursors. Patients exhibiting photosensi- tivity beneit rom sunscreens and possibly rom administered Abnormalities of the β-carotene, which appears to lessen production o ree radi- enzymes of heme synthesis cals, decreasing photosensitivity. Accumulation of ALA and PBG and/or Accumulation of CATABOLISM OF HEME porphyrinogens in skin decrease in heme in cells and body fluids and tissues PRODUCES BILIRUBIN Human adults normally destroy about 200 billion erythrocytes per day. A 70-kg human thereore turns over approximately Spontaneous oxidation Neuropsychiatric signs of porphyrinogens to 6 g of hemoglobin daily. All products are reused. he globin and symptoms porphyrins is degraded to its constituent amino acids, and the released iron enters the iron pool. he iron-ree porphyrin portion o heme is also degraded, mainly in the reticuloendothelial cells Photosensitivity o the liver, spleen, and bone marrow. he catabolism o heme rom all heme proteins takes FIGURE 31–10 Biochemical basis of the major signs and place in the microsomal fraction o cells by heme oxygenase, symptoms of the porphyrias. EC 1.14.18.18. Heme oxygenase synthesis is substrate-inducible, and heme also serves both as a substrate and as a coactor or heir oxidation to the corresponding porphyrin derivatives the reaction. he iron o the heme that reaches heme oxy- cause photosensitivity to visible light o about 400-nm genase has usually been oxidized to its ferric form (hemin). wavelength. Possibly as a result o their excitation and reaction Conversion o one mole o heme-Fe3+ to biliverdin, carbon with molecular oxygen, the resulting oxygen radicals injure monoxide, and Fe3+ consumes three moles o O2, plus seven lysosomes and other subcellular organelles, releasing proteolytic electrons provided by NADH and NADPH–cytochrome P450 enzymes that cause variable degrees o skin damage, including reductase: scarring. Fe3+-Heme + 3 O2 + 7 e– → biliverdin + CO + Fe3+ CLASSIFICATION OF THE Despite its high ainity or heme-Fe2+ (see Chapter 6), the carbon monoxide produced does not severely inhibit heme PORPHYRIAS oxygenase. Birds and amphibians excrete the green-colored bili- Porphyrias may be termed erythropoietic or hepatic based verdin directly. In humans, biliverdin reductase (EC 1.3.1.24) on the organs most aected, typically bone marrow and the reduces the central methylene bridge o biliverdin to a methyl liver (able 31–2). Dierent and variable levels o heme, toxic group, producing the yellow-pigment bilirubin (Figure 31–11): precursors, or metabolites probably account or why spe- Biliverdin + NADPH + H+ → bilirubin + NADP+ ciic porphyrias dierentially aect some cell types and organs. Alternatively, porphyrias may be classiied as acute Since 1 g o hemoglobin yields about 35 mg o bilirubin, or cutaneous based on their clinical eatures. he diagnosis o human adults form 250 to 350 mg of bilirubin per day. his a speciic type o porphyria involves consideration o the clini- is derived principally rom hemoglobin, and also rom ineec- cal and amily history, physical examination, and appropriate tive erythropoiesis and rom catabolism o other heme proteins. laboratory tests. able 31–2 lists the major signs, symptoms, Conversion o heme to bilirubin by reticuloendothe- and relevant laboratory indings in the six principal types o lial cells can be observed visually as the purple color o the porphyria. heme in a hematoma slowly converts to the yellow pigment o bilirubin. Drug-Induced Porphyria Certain drugs (eg, barbiturates, griseoulvin) induce the produc- Bilirubin Is Transported to the Liver tion o cytochrome P450. In patients with porphyria, this can Bound to Serum Albumin precipitate an attack o porphyria by depleting heme levels. he Bilirubin is only sparingly soluble in water. Consequently, it compensating derepression o synthesis o ALAS1 then results must be bound to serum albumin or transport to the liver. in increased levels o potentially harmul heme precursors. Albumin appears to have both high-ainity and low-ainity sites or bilirubin. he high-ainity site can bind approxi- Possible Treatments for Porphyrias mately 25 mg o bilirubin/100 mL o plasma. More loosely Present treatment o porphyrias is essentially symptomatic: bound bilirubin can readily be detached and diused into avoiding drugs that induce production o cytochrome P450, tissues, and antibiotics and certain other drugs can compete ingestion o large amounts o carbohydrate, and administration with and displace bilirubin rom albumin’s high-ainity site. 322 SECTION VI Metabolism o Proteins & Amino Acids COOH COOH facilitated transport system. Even under pathologic condi- tions, transport does not appear to be rate-limiting or the metabolism o bilirubin. he net uptake o bilirubin depends on its removal by subsequent metabolism. Once internal- ized, bilirubin binds to cytosolic proteins such as glutathione N N S-transerase, previously known as a ligandin, to prevent bili- Fe3+ rubin rom reentering the bloodstream. N N Conjugation of Bilirubin With Glucuronate Ferric heme Bilirubin is nonpolar, and would persist in cells (eg, bound to lipids) i not converted to a more water-soluble orm. Bili- 3O2 + 7e– rubin is converted to a more polar molecule by conjugation with glucuronic acid (Figure 31–12). A bilirubin-speciic CO + Fe3+ UDP-glucuronosyltransferase (EC 2.4.1.17) o the endoplas- mic reticulum catalyzes stepwise transer to bilirubin o two HOOC COOH glucosyl moieties rom UDP-glucuronate: Bilirubin + UDP-glucuronate → bilirubin monoglucuronide + UDP Bilirubin monoglucuronide + UDP-glucuronate → bilirubin diglucuronide + UDP O N N N N O H H H Biliverdin Secretion of Bilirubin Into the Bile Secretion o conjugated bilirubin into the bile occurs by an NADPH Biliverdin active transport mechanism, which probably is rate-limiting or the entire process o hepatic bilirubin metabolism. he Reductase NADP + protein involved is a multispecific organic anion trans- porter (MOAT) located in the plasma membrane o the bile canaliculi. A member o the amily o AP-binding cassette HOOC COOH transporters, MOA transports a number o organic anions. he hepatic transport o conjugated bilirubin into the bile is inducible by the same drugs that can induce the conjugation o bilirubin. Conjugation and excretion o bilirubin thus con- O N N N N O stitute a coordinated unctional unit. H H H H Most o the bilirubin excreted in the bile o mammals is Bilirubin bilirubin diglucuronide. Bilirubin UDP-glucuronosyltranser- ase activity can be induced by several drugs, including pheno- FIGURE 31–11 Conversion of ferric heme to biliverdin, and then to bilirubin. (1) Conversion o erric heme to biliverdin is cata- barbital. However, when bilirubin conjugates exist abnormally lyzed by the heme oxygenase system. (2) Subsequently, biliverdin in human plasma (eg, in obstructive jaundice), they are pre- reductase reduces bilirubin to bilirubin. dominantly monoglucuronides. Figure 31–13 summarizes Further Metabolism of Bilirubin Occurs O O Primarily in the Liver – OOC(CH2O)4C O C C O C(CH2O)4COO– Hepatic catabolism o bilirubin takes place in three stages: H2C CH2 uptake by the liver, conjugation with glucuronic acid, and H2 C CH2 M V M M M V secretion in the bile. II III IV I O C C C O Uptake of Bilirubin by Liver Parenchymal Cells FIGURE 31–12 Bilirubin diglucuronide. Glucuronate moi- eties are attached via ester bonds to the two propionate groups o Bilirubin is removed rom albumin and taken up at the sinu- bilirubin. Clinically, the diglucuronide is also termed “direct reacting” soidal surace o hepatocytes by a large capacity, saturable bilirubin.  CHAPTER 31 Porphyrins & Bile Pigments 323 Blood presence o added methanol measures total bilirubin. he Bilirubin Albumin difference between total bilirubin and direct bilirubin is known as “indirect bilirubin,” and is unconjugated bilirubin. 1. UPTAKE HYPERBILIRUBINEMIA Hepatocyte Bilirubin CAUSES JAUNDICE 2. CONJUGATION Hyperbilirubinemia, a blood level that exceeds 1 mg o biliru- Neonatal jaundice bin per dL (17 μmol/L), may result rom production o more UDP-GlcUA “Toxic” jaundice UDP-GlcUA Crigler-Najjar syndrome bilirubin than the normal liver can excrete, or rom the ailure Gilbert syndrome o a damaged liver to excrete normal amounts o bilirubin. In the absence o hepatic damage, obstruction o the excretory Bilirubin diglucuronide ducts o the liver prevents the excretion o bilirubin, and will also cause hyperbilirubinemia. In all these situations, when 3. SECRETION Dubin-Johnson syndrome the blood concentration o bilirubin reaches 2 to 2.5 mg/dL, it diuses into the tissues, which turn yellow, a condition termed jaundice or icterus. Bile ductule Bilirubin diglucuronide Occurrence of Unconjugated Bilirubin in Blood FIGURE 31–13 Diagrammatic representation of the three Forms o hyperbilirubinemia include retention hyperbiliru- major processes (uptake, conjugation, and secretion) involved in the transfer of bilirubin from blood to bile. Certain proteins o binemia due to overproduction o bilirubin, and regurgita- hepatocytes bind intracellular bilirubin and may prevent its elux tion hyperbilirubinemia due to relux into the bloodstream into the bloodstream. The processes aected in certain conditions because o biliary obstruction. that cause jaundice are also shown. Because o its hydrophobicity, only unconjugated bilirubin can cross the blood–brain barrier into the central nervous sys- the three major processes involved in the transer o bilirubin tem. Encephalopathy due to hyperbilirubinemia (kernicterus) rom blood to bile. Sites that are aected in a number o condi- thus occurs only with unconjugated bilirubin, as in retention tions causing jaundice are also indicated. hyperbilirubinemia. Alternatively, because o its water solubil- ity, only conjugated bilirubin can appear in urine. Accordingly, Intestinal Bacteria Reduce Conjugated choluric jaundice (choluria is the presence o bile pigments Bilirubin to Urobilinogen in the urine) occurs only in regurgitation hyperbilirubine- When conjugated bilirubin reaches the terminal ileum and mia, and acholuric jaundice occurs only in the presence o an the large intestine, the glucuronosyl moieties are removed by excess o unconjugated bilirubin. Table 31–3 lists some causes speciic bacterial β-glucuronidases (EC 3.2.1.31). Subsequent o unconjugated and conjugated hyperbilirubinemia. A mod- reduction by the ecal lora orms a group o colorless tetra- erate hyperbilirubinemia accompanies hemolytic anemias. pyrroles called urobilinogens. Small portions o urobilino- gens are reabsorbed in the terminal ileum and large intestine TABLE 31–3 Some Causes of Unconjugated and and subsequently are reexcreted via the enterohepatic urobi- Conjugated Hyperbilirubinemia linogen cycle. Under abnormal conditions, particularly when excessive bile pigment is ormed or when liver disease disrupts Unconjugated Conjugated this intrahepatic cycle, urobilinogen may also be excreted in Hemolytic anemias Obstruction o the biliary tree the urine. Most o the colorless urobilinogens ormed in the Neonatal “physiological Dubin–Johnson syndrome colon are oxidized there to colored urobilins and excreted in jaundice” the eces. Fecal darkening upon standing in air results rom Crigler-Najjar syndromes types I Rotor syndrome the oxidation o residual urobilinogens to urobilins. and II Gilbert syndrome Liver diseases such as the Measurement of Bilirubin in Serum various types o hepatitis Quantitation o bilirubin employs a colorimetric method Toxic hyperbilirubinemia based on the reddish-purple color ormed when bilirubin reacts with diazotized sulanilic acid. An assay conducted in These causes are discussed briely in the text. Common causes o obstruction o the biliary tree are a stone in the common bile duct and cancer o the head o the pan- the absence o added methanol measures “direct bilirubin,” creas. Various liver diseases (eg, the various types o hepatitis) are requent causes which is bilirubin glucuronide. An assay conducted in the o predominantly conjugated hyperbilirubinemia. 324 SECTION VI Metabolism o Proteins & Amino Acids Hyperbilirubinemia is usually modest (< 4 mg bilirubin per dL; tends not to exceed 20 mg/dL o serum, and patients respond < 68 μmol/L) despite extensive hemolysis, due to the high to treatment with large doses o phenobarbital. capacity o a healthy liver to metabolize bilirubin. Toxic Hyperbilirubinemia DISORDERS OF BILIRUBIN Unconjugated hyperbilirubinemia can result rom toxin- induced liver dysfunction caused by, or example, chloro- METABOLISM orm, arsphenamines, carbon tetrachloride, acetaminophen, Neonatal “Physiologic Jaundice” hepatitis virus, cirrhosis, or Amanita mushroom poisoning. hese acquired disorders involve hepatic parenchymal cell he unconjugated hyperbilirubinemia o neonatal “physi- damage, which impairs bilirubin conjugation. ologic jaundice” results rom accelerated hemolysis and an immature hepatic system or the uptake, conjugation, and secretion o bilirubin. In this transient condition, bilirubin- Obstruction in the Biliary Tree Is the glucuronosyltranserase activity, and probably also synthesis Most Common Cause of Conjugated o UDP-glucuronate, are reduced. When the plasma concen- Hyperbilirubinemia tration o unconjugated bilirubin exceeds that which can be tightly bound by albumin (20–25 mg/dL), bilirubin can pen- Conjugated hyperbilirubinemia commonly results rom etrate the blood–brain barrier. I let untreated, the resulting blockage o the hepatic or common bile ducts, most oten hyperbilirubinemic toxic encephalopathy, or kernicterus, due to a gallstone or to cancer of the head of the pancreas can result in mental retardation. Exposure o jaundiced neo- (Figure 31–14). Bilirubin diglucuronide that cannot be nates to blue light (phototherapy) promotes hepatic excretion excreted regurgitates into the hepatic veins and lymphatics, o unconjugated bilirubin by converting some to derivatives conjugated bilirubin appears in the blood and urine (chol- that are excreted in the bile, and phenobarbital, a promoter o uric jaundice), and the stools typically are a pale color. bilirubin metabolism, may be administered. he term cholestatic jaundice includes both all cases o extrahepatic obstructive jaundice and also conjugated hyper- bilirubinemia due to micro-obstruction o intrahepatic biliary Defects of Bilirubin ductules by damaged hepatocytes, such as may occur in inec- UDP-Glucuronosyltransferase tious hepatitis. Glucuronosyltranserases (EC 2.4.1.17), a amily o enzymes with diering substrate speciicities, increase the polarity o various drugs and drug metabolites, thereby acilitating their PRE-HEPATIC Hemolytic anemias excretion. Mutations in the gene that encodes bilirubin UDP- (Vascular) glucuronosyltransferase can result in the encoded enzyme having reduced or absent activity. Syndromes whose clinical presentation relects the severity o the impairment include Gilbert syndrome and two types o Crigler-Najjar syndrome. HEPATIC Liver diseases (Liver) (eg, hepatitis, cancer) Gilbert Syndrome Providing that about 30% o the bilirubin UDP-glucuronosyl- transerase activity is retained in Gilbert syndrome, the condi- tion is harmless. Gallstone Type I Crigler-Najjar Syndrome POST-HEPATIC Pancreatic he severe congenital jaundice (over 20 mg bilirubin per dL (Biliary system & cancer serum) and accompanying brain damage o type I Crigler- pancreas) Najjar syndrome relect the complete absence o hepatic UDP-glucuronosyltranserase activity. Phototherapy reduces plasma bilirubin levels somewhat, but phenobarbital has no beneicial eect. he disease is oten atal within the irst 15 months o lie. FIGURE 31–14 Major causes of jaundice. Prehepatic jaun- dice indicates events in the bloodstream, major causes being various Type II Crigler-Najjar Syndrome hemolytic anemias. Hepatic jaundice arises rom hepatitis or other liver diseases (eg, cancer). Posthepatic jaundice reers to events in In type II Crigler-Najjar syndrome, some bilirubin UDP- the biliary tree, or which the major causes are obstruction o the glucuronosyltranserase activity is retained. his condition common bile duct by a gallstone (biliary calculus) or by cancer o the thus is more benign than the type I syndrome. Serum bilirubin head o the pancreas.  CHAPTER 31 Porphyrins & Bile Pigments 325 TABLE 31–4 Laboratory Results in Normal Patients and Patients With Three Different Causes of Jaundice Condition Serum Bilirubin Urine Urobilinogen Urine Bilirubin Fecal Urobilinogen Normal Direct: 0.1-0.4 mg/dL 0-4 mg/24 h Absent 40-280 mg/24 h Indirect: 0.2-0.7 mg/dL Hemolytic anemia ↑Indirect Increased Absent Increased Hepatitis ↑Direct and indirect Decreased i micro-obstruction Present i micro-obstruction Decreased is present occurs Obstructive jaundicea ↑Direct Absent Present Trace to absent a The most common causes o obstructive (posthepatic) jaundice are cancer o the head o the pancreas and a gallstone lodged in the common bile duct. The presence o bili- rubin in the urine is sometimes reerred to as choluria—thereore, hepatitis and obstruction o the common bile duct cause choluric jaundice, whereas the jaundice o hemolytic anemia is reerred to as acholuric. The laboratory results in patients with hepatitis are variable, depending on the extent o damage to parenchymal cells and the extent o micro-obstruction to bile ductules. Serum levels o alanine aminotransferase and aspartate aminotransferase are usually markedly elevated in hepatitis, whereas serum levels o alkaline phosphatase are elevated in obstructive liver disease. Dubin-Johnson Syndrome o prothrombin time) and on serum (eg, electrophoresis o his benign autosomal recessive disorder consists o conju- proteins; alkaline phosphatase and alanine aminotranserase gated hyperbilirubinemia in childhood or during adult lie. and aspartate aminotranserase activities) also help to distin- he hyperbilirubinemia is caused by mutations in the gene guish between prehepatic, hepatic, and posthepatic causes o encoding the protein involved in the secretion o conjugated jaundice. bilirubin into bile. SUMMARY Some Conjugated Bilirubin Can Bind Te heme o hemoproteins such as hemoglobin and the Covalently to Albumin cytochromes is an iron-porphyrin complex. Porphyrin consists o our pyrrole rings joined by methyne bridges. When levels o conjugated bilirubin remain high in plasma, a Te eight methyl, vinyl, and propionyl substituents on the our raction can bind covalently to albumin. his raction, termed pyrrole rings o heme are arranged in a specic sequence. Te δ-bilirubin, has a longer half-life in plasma than does con- metal ion (Fe2+ in hemoglobin; Mg2+ in chlorophyll) is linked to ventional conjugated bilirubin, and remains elevated during the our nitrogen atoms o the pyrrole rings. recovery rom obstructive jaundice. Some patients thereore Biosynthesis o the heme ring involves eight enzyme-catalyzed continue to appear jaundiced even ater the circulating conju- reactions, some o which occur in mitochondria, others in the gated bilirubin level has returned to normal. cytosol. Synthesis o heme commences with the condensation o Urinary Urobilinogen & Bilirubin Are succinyl-CoA and glycine to orm ALA. Tis reaction Clinical Indicators is catalyzed by ALAS1, the regulatory enzyme o heme In complete obstruction of the bile duct, bilirubin has no biosynthesis. access to the intestine or conversion to urobilinogen, so no Synthesis o ALAS1 increases in response to a low level o urobilinogen is present in the urine. he presence o conju- available heme. For example, certain drugs (eg, phenobarbital) gated bilirubin in the urine without urobilinogen suggests indirectly trigger enhanced synthesis o ALAS1 by promoting synthesis o the heme protein cytochrome P450, which thereby intrahepatic or posthepatic obstructive jaundice. depletes the heme pool. By contrast, ALAS2 is not regulated In jaundice secondary to hemolysis, the increased pro- by heme levels, and consequently not by drugs that promote duction o bilirubin leads to increased production o urobilin- synthesis o cytochrome P450. ogen, which appears in the urine in large amounts. Bilirubin Genetic abnormalities o seven o the eight enzymes o heme is not usually ound in the urine in hemolytic jaundice, so biosynthesis result in inherited porphyrias. Erythrocytes the combination o increased urobilinogen and absence o and liver are the major sites o expression o the porphyrias. bilirubin is suggestive o hemolytic jaundice. Increased blood Photosensitivity and neurologic problems are common destruction rom any cause brings about an increase in urine complaints. Intake o certain toxins (eg, lead) can cause urobilinogen. acquired porphyrias. Increased amounts o porphyrins or their Table 31–4 summarizes laboratory results obtained in precursors can be detected in blood and urine, acilitating patients with jaundice due to prehepatic, hepatic, or pos- diagnosis. thepatic causes: hemolytic anemia (prehepatic), hepatitis Catabolism o the heme ring, initiated by the mitochondrial (hepatic), and obstruction of the common bile duct (posthe- enzyme heme oxygenase, produces the linear tetrapyrrole, patic); see Figure 31–14. Laboratory tests on blood (evalua- biliverdin. Subsequent reduction o biliverdin in the cytosol tion o the possibility o a hemolytic anemia and measurement orms bilirubin. 326 SECTION VI Metabolism o Proteins & Amino Acids Bilirubin binds to albumin or transport rom peripheral Jaundice results rom an elevated level o plasma bilirubin. tissues to the liver, where it is taken up by hepatocytes. Te iron Te causes o jaundice can be distinguished as prehepatic o heme is released and reutilized. (eg, hemolytic anemias), hepatic (eg, hepatitis), or posthepatic Te water solubility o bilirubin is increased by the addition (eg, obstruction o the common bile duct). Measurements o two moles o the highly polar glucuronosyl moiety, derived o plasma total and nonconjugated bilirubin, o urinary rom UDP-glucuronate, per mole o bilirubin. Attachment urobilinogen and bilirubin, o the activity o certain serum o the glucuronosyl moieties is catalyzed by bilirubin UDP- enzymes, and the analysis o stool samples help distinguish glucuronosyltranserase, one o a large amily o enzymes o between the causes o jaundice. diering substrate specicities that increase the polarity o various drugs and drug metabolites, thereby acilitating their excretion. REFERENCES Mutations in the encoding gene may result in reduced or Ajioka RS, Phillips JD, Kushner JP: Biosynthesis o heme in absent bilirubin UDP-glucuronosyltranserase activity. Clinical mammals. Biochim Biophys Acta 2006;1763:723. presentations that refect the severity o the mutation(s) include Desnick RJ, Astrin KH: Te porphyrias. In Harrison’s Principles Gilbert syndrome and two types o Crigler-Najjar syndrome, of Internal Medicine, 17th ed. Fauci AS (editor). McGraw-Hill, conditions whose severity depend on the extent o remaining 2008. glucuronosyltranserase activity. Duour DR: Liver disease. In Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. Burtis CA, Ashwood ER, Following secretion o bilirubin rom the bile into the gut, Bruns DE (editors). Elsevier Saunders, 2006. bacterial enzymes convert bilirubin to urobilinogen and Higgins , Beutler E, Doumas B: Hemoglobin, iron and urobilin, which are excreted in the eces and urine. bilirubin. In Tietz Textbook of Clinical Chemistry and Molecular Colorimetric measurement o bilirubin employs the color Diagnostics, 4th ed. Burtis CA, Ashwood ER, Bruns DE (editors). ormed when bilirubin reacts with diazotized sulanilic acid. Elsevier Saunders, 2006. Assays conducted in the absence o added methanol measure Pratt DS, Kaplan MM: Evaluation o liver unction. In Harrison’s “direct bilirubin” (ie, bilirubin glucuronide). Assays conducted Principles of Internal Medicine, 17th ed. Fauci AS (editor). in the presence o added methanol measure total bilirubin. Te McGraw-Hill, 2008. dierence between total bilirubin and direct bilirubin, termed Wolko AW: Te hyperbilirubinemias. In Harrison’s Principles of “indirect bilirubin,” is unconjugated bilirubin. Internal Medicine, 17th ed. Fauci AS (editor). McGraw-Hill, 2008.