Harper's Illustrated Biochemistry, 31st Ed. PDF - Porphyrins & Bile Pigments

31 C H A P T E R Porphyrins & Bile Pigments Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD O B J EC T IVES Write the structural formulas of the two amphibolic...

31 C H A P T E R Porphyrins & Bile Pigments Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD O B J EC T IVES Write the structural formulas of the two amphibolic intermediates whose condensation initiates heme biosynthesis. After studying this chapter, Identify the enzyme that catalyzes the key regulated enzyme of hepatic heme you should be able to: biosynthesis. Explain why, although porphyrinogens and porphyrins both are tetrapyrroles, porphyrins are colored whereas porphyrinogens are colorless. Specify the intracellular locations of the enzymes and metabolites of heme biosynthesis. Outline the causes and clinical presentations of various porphyrias. Identify the roles of heme oxygenase and of UDP-glucosyl transferase in heme catabolism. Define jaundice, name some of its causes, and suggest how to determine its biochemical basis. Specify the biochemical basis of the clinical laboratory terms “direct bilirubin” and “indirect bilirubin.” BIOMEDICAL IMPORTANCE >pyrrole rings. Examples include iron porphyrins such as the heme of hemoglobin and the magnesium-containing porphy- & - The biochemistry of the porphyrins and of the bile pig- - rin chlorophyll, the photosynthetic pigment of plants. Heme - - ments are closely related topics. Heme is synthesized from proteins are ubiquitous in biology and serve diverse functions porphyrins and iron, and the products of degradation of including, but not limited to, oxygen transport and storage heme are the bile pigments and iron. The biochemistry of (eg, hemoglobin and myoglobin) and electron transport (eg, the porphyrins and of heme is basic to understanding the - cytochrome c and cytochrome P450). Hemes are tetrapyr- varied functions of hemoproteins, and the porphyrias, a roles, of which two types, heme b and heme c, predominate group of diseases caused by abnormalities in the pathway (Figure 31–2). In heme c the vinyl groups of heme b are of porphyrin biosynthesis. A much more common clinical replaced by covalent thioether links to an apoprotein, typically condition is jaundice, a consequence of an elevated level of via cysteinyl residues. Unlike heme b, heme c thus does not plasma bilirubin, due either to overproduction of bilirubin readily dissociate from its apoprotein. or to failure of its excretion. Jaundice occurs in numerous Proteins that contain heme are widely distributed in nature diseases ranging from hemolytic anemias to viral hepatitis (Table 31–1). Vertebrate heme proteins generally bind one and to cancer of the pancreas. mole of heme c per mole, although those of nonvertebrates may bind significantly more heme. PORPHYRINS Porphyrins are cyclic compounds formed by the linkage of four pyrrole rings through methyne (——O - HC—) bridges HEME IS SYNTHESIZED FROM - - (Figure 31–1). Various side chains can replace the eight - SUCCINYL-CoA & GLYCINE numbered hydrogen atoms of the pyrrole rings. The biosynthesis of heme involves both cytosolic and mito- Porphyrins can form complexes with metal ions that form - chondrial reactions and intermediates. Heme biosynthesis coordinate bonds to the nitrogen atom of each of the four occurs in most mammalian cells except mature erythrocytes, 305 306 SECTION VI Metabolism of Proteins & Amino Acids HC CH TABLE 31–1 Examples of Important Heme Proteinsa HC CH Protein Function N N H Hemoglobin Transport of oxygen in blood Pyrrole Myoglobin Storage of oxygen in muscle 1 2 Cytochrome c Involvement in the electron H H transport chain C C δ I α Cytochrome P450 Hydroxylation of xenobiotics HC C C CH N 3 Catalase Degradation of hydrogen 8 HC C C CH peroxide IV NH HN II Tryptophan pyrrolase Oxidation of tryptophan 7 HC C N C CH 4 HC C C CH The functions of the above proteins are described in various chapters of this text. a γ III β C C H H 6 5 Following the exit of δ-aminolevulinate into the cytosol, the reaction catalyzed by cytosolic ALA dehydratase (EC Porphyrin (C20H14N4) 4.2.1.24; porphobilinogen synthase) condenses two molecules of ALA, forming porphobilinogen: FIGURE 31–1 The porphyrin molecule. Rings are labeled I, (2) 2 δ-Aminolevulinate → porphobilinogen + 2 H2O II, III, and IV. Substituent positions are labeled 1 through 8. The four methyne bridges (=HC—) are labeled α, β, γ, and %. (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% of heme syn- The 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 of the remainder in hepatocytes. Heme involves head-to-tail condensation of four molecules of por- biosynthesis is initiated by the condensation of succinyl-CoA phobilinogen to form 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 of 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 of ALA synthase. ALAS1 is ubiquitously expressed throughout the body, whereas ALAS2 forms uroporphyrinogen III (Figure 31–5, bottom right). is expressed in erythrocyte precursor cells. Formation of Hydroxymethylbilane can undergo spontaneous cyclization δ-aminolevulinate is rate-limiting for porphyrin biosynthesis forming uroporphyrinogen I (Figure 31–5, bottom left), in mammalian liver (Figure 31–3). but under normal conditions, the uroporphyrinogen formed is almost exclusively the type III isomer. The type I isomers of porphyrinogens are, however, formed in excess in certain porphyrias. Since the pyrrole rings of these uroporphyrino- gens are connected by methylene (—CH2—) rather than by Cys S Cys COOH COOH S ALA CH2 synthase CH2 N N N N CH2 CH2 Succinyl-CoA Fe Fe (“active” CoA-SH + CO2 C O C O N N N N succinate) S CoA H C NH2 + H H Glycine H C NH2 δ-Aminolevulinate (ALA) O OH OH O OH OH COOH 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 307 COOH COOH A P M P COOH CH2 COOH CH2 I Uroporphyrinogen I A A decarboxylase M M 2H2O CH2 CH2 CH2 CH2 IV II IV II CH2 O C C C P III P P III P ALA C O H C H C CH 4CO2 dehydratase CH2 H CH2 N P A P M NH H Uroporphyrinogen III Coproporphyrinogen III NH2 NH2 Two molecules of Porphobilinogen FIGURE 31–6 Decarboxylation of uroporphyrinogen III to δ-Aminolevulinate (first precursor pyrrole) coproporphyrinogen III. Shown is a representation of the tetrapyr- role to emphasize the conversion of four 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 of %-aminolevulinate to porphybilinogen. All four acetate moieties of uroporphyrinogen III next undergo decarboxylation to methyl (M) substituents, forming methyne bridges (— — HC—), the double bonds do not form a coproporphyrinogen III in a cytosolic reaction catalyzed by conjugated system. Porphyrinogens thus are colorless. They 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 This decarboxylase can also convert uroporphyrinogen I, if A HOOC COOH present, to coproporphyrinogen I. H 2C CH 2 P The final three reactions of 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. These 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 form protoporphyrinogen III: porphobilinogen (6) Coproporphyrinogen III + O2 + 2 H+ → Uroporphyrinogen I 4 NH 3 synthase protoporphyrinogen III + 2 CO2 + 2 H2O Hydroxymethylbilane This oxidase is specific for 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 The eighth and final step in heme synthesis involves the CH 2 H H CH 2 CH 2 H H CH 2 incorporation of ferrous 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 of the biosynthesis of the Type I Type III uroporphyrinogen uroporphyrinogen porphyrin derivatives from 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) forms a linear tetrapyrrole, which cytosolic uroporphyrinogen synthase cyclizes to form uropor- ALA Synthase Is the Key Regulatory phyrinogen III. Notice the asymmetry of 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. 308 SECTION VI Metabolism of 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. The reaction catalyzed by ALA synthase 1 (Figure 31–3) is absorption and fluorescence spectra. The visible and the rate-limiting for biosynthesis of heme in liver. Typically for an ultraviolet spectra of porphyrins and porphyrin derivatives enzyme that catalyzes a rate-limiting reaction, ALAS1 has a are useful for their identification (Figure 31–9). The sharp short half-life. Heme, probably acting through an aporepres- absorption band near 400 nm, a distinguishing feature shared sor molecule, acts as a negative regulator of the synthesis by all porphyrins, is termed the Soret band after its discoverer, of ALAS1 (Figure 31–8). Synthesis of ALAS1 thus increases the French physicist Charles Soret. greatly in the absence of heme, but diminishes in its presence. Porphyrins dissolved in strong mineral acids or in organic Heme also affects translation of ALAS1 and its translocation solvents and illuminated by ultraviolet light emit a strong red from its cytosolic site of synthesis into the mitochondrion. fluorescence, a property often used to detect small amounts Many drugs whose metabolism requires the hemoprotein of free porphyrins. The photodynamic properties of porphy- cytochrome P450 increase cytochrome P450 biosynthesis. The rins have suggested their possible use in the treatment of cer- resulting depletion of the intracellular heme pool induces syn- tain types of cancer, a procedure called cancer phototherapy. thesis of ALAS1, and the rate of heme synthesis rises to meet Since tumors often take up more porphyrins than do normal metabolic demand. By contrast, since ALAS2 is not feedback tissues, hematoporphyrin or related compounds are admin- regulated by heme, its biosynthesis is not induced by these istered to a patient with an appropriate tumor. The tumor is drugs. then exposed to an argon laser to excite the porphyrins, pro- ducing cytotoxic effects. 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. The conjugated double bonds in the pyrrole amounts in the porphyrias. When present in urine or feces, they rings and linking methylene groups of porphyrins (absent in can be separated by extraction with appropriate solvents, then the porphyrinogens) are responsible for their characteristic identified and quantified using spectrophotometric methods.  CHAPTER 31 Porphyrins & Bile Pigments 309 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 of the enzymes that catalyze the indicated reactions are those used in the accompanying text and in column 1 of Table 31–2. Enzymes 1, 6, 7, and 8 are mitochondrial, but enzymes 2 to 5 are cytosolic. Regulation of 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 from different mutations in genes that encode either a given BIOSYNTHESIS enzyme or an enzyme that catalyzes a successive reaction. Disorders of heme biosynthesis may be genetic or acquired. 2. Rational therapy requires an understanding of the bio- An example of an acquired defect is lead poisoning. Lead can chemistry of the enzyme-catalyzed reactions in both nor- inactivate ferrochelatase and ALA dehydratase by combining mal and impaired individuals. with essential thiol groups. Signs include elevated levels of 3. Identification of the intermediates and side products that protoporphyrin in erythrocytes and elevated urinary levels of accumulate prior to a metabolic block can provide the ALA and coproporphyrin. basis for metabolic screening tests that can implicate the Genetic disorders of heme metabolism and of bilirubin impaired reaction. metabolism (see below) share the following features with met- 4. Definitive diagnosis involves quantitative assay of the activ- abolic disorders of urea biosynthesis (see Chapter 28): ity of the enzyme(s) suspected to be defective. To this might 310 SECTION VI Metabolism of 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 1. ALA synthase 2 (ALAS2), X-linked sideroblasticanemiac 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 deficiency 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, fecal, 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 fecal 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 fecal 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 findings in the active stages of these diseases are listed. Certain biochemical abnormalities are detectable in the latent stages of some of the above condi- tions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias. Condition 2 is rare. b The numbering of 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, %-aminolevulinic acid; PBG, porphobilinogen. be added consideration of the as yet incompletely identified from the accumulation of metabolites prior to the block. factors that facilitate translocation of enzymes and inter- Table 31–2 lists six major types of porphyria that reflect low mediates between cellular compartments. or absent activity of enzymes that catalyze reactions 2 through 5. Comparison of the DNA sequence of the gene that encodes 8 of Figure 31–8. Possibly due to potential lethality, there is no a given mutant enzyme to that of the wild-type gene can known defect of ALAS1. Individuals with low ALAS2 activ- identify the specific mutation(s) that cause the disease. ity develop anemia, not porphyria (Table 31–2). Porphyria consequent to low activity of ALA dehydratase, termed ALA dehydratase-deficient porphyria, is extremely rare. The Porphyrias The signs and symptoms of porphyria result either from a Congenital Erythropoietic Porphyria deficiency of 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. The defective enzyme in congenital erythropoi- etic porphyria is uroporphyrinogen III synthase (Figure 31–5, 5 bottom). The photosensitivity and severe disfigurement exhib- ited by some victims of congenital erythropoietic porphyria has Log absorbency 4 suggested them as prototypes of so-called werewolves. 3 Acute Intermittent Porphyria 2 The defective enzyme in acute intermittent porphyria is 1 hydroxymethylbilane synthase (Figure 31–5, bottom). ALA and porphobilinogen accumulate in body tissues and fluids (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 the spectrum is of a dilute (0.01%) solution of hematoporphyrin in 5% HCl. porphyrinogens indicated in Figures 31–8 and 31–10. Their  CHAPTER 31 Porphyrins & Bile Pigments 311 of hematin to repress ALAS1 synthesis to diminish production Mutations in various genes of harmful heme precursors. Patients exhibiting photosensi- tivity benefit from sunscreens and possibly from administered Abnormalities of the β-carotene, which appears to lessen production of free radi- enzymes of heme synthesis cals, decreasing photosensitivity. Accumulation of ALA and PBG and/or Accumulation of CATABOLISM OF HEME decrease in heme in cells and body fluids porphyrinogens in skin and tissues PRODUCES BILIRUBIN Human adults normally destroy about 200 billion erythrocytes per day. A 70-kg human therefore turns over approximately Spontaneous oxidation Neuropsychiatric signs of porphyrinogens to 6 g of hemoglobin daily. All products are reused. The globin and symptoms porphyrins is degraded to its constituent amino acids, and the released iron enters the iron pool. The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells Photosensitivity of the liver, spleen, and bone marrow. The catabolism of heme from all heme proteins takes FIGURE 31–10 Biochemical basis of the major signs and place in the microsomal fraction of 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 cofactor for oxidation products, the corresponding porphyrin derivatives, the reaction. The iron of the heme that reaches heme oxy- cause photosensitivity to visible light of about 400-nm wave- genase has usually been oxidized to its ferric form (hemin). length. Possibly as a result of their excitation and reaction with Conversion of one mole of heme-Fe3+ to biliverdin, carbon molecular oxygen, the resulting oxygen radicals injure lyso- monoxide, and Fe3+ consumes three moles of O2, plus seven somes and other subcellular organelles, releasing proteolytic electrons provided by NADH and NADPH–cytochrome P450 enzymes that cause variable degrees of skin damage, including reductase: scarring. Fe3+-Heme + 3 O2 + 7 e– → biliverdin + CO + Fe3+ CLASSIFICATION OF THE Despite its high affinity for 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 affected, typically bone marrow and the reduces the central methylene bridge of biliverdin to a methyl liver (Table 31–2). Different and variable levels of heme, toxic group, producing the yellow-pigment bilirubin (Figure 31–11): precursors, or metabolites probably account for why spe- Biliverdin + NADPH + H+ → bilirubin + NADP+ cific porphyrias differentially affect some cell types and organs. Alternatively, porphyrias may be classified as acute Since 1 g of hemoglobin yields about 35 mg of bilirubin, or cutaneous based on their clinical features. The diagnosis of human adults form 250 to 350 mg of bilirubin per day. This a specific type of porphyria involves consideration of the clini- is derived principally from hemoglobin, and also from ineffec- cal and family history, physical examination, and appropriate tive erythropoiesis and from catabolism of other heme proteins. laboratory tests. Table 31–2 lists the major signs, symptoms, Conversion of heme to bilirubin by reticuloendothe- and relevant laboratory findings in the six principal types of lial cells can be observed visually as the purple color of the porphyria. heme in a hematoma slowly converts to the yellow pigment of bilirubin. Drug-Induced Porphyria Certain drugs (eg, barbiturates, griseofulvin) induce the produc- Bilirubin Is Transported to the Liver tion of cytochrome P450. In patients with porphyria, this can Bound to Serum Albumin precipitate an attack of porphyria by depleting heme levels. The Unlike bilirubin, which is only sparingly water soluble, biliru- compensating derepression of synthesis of ALAS1 then results bin bound to serum albumin is readily transported to the liver. in increased levels of potentially harmful heme precursors. Albumin appears to have both high-affinity and low-affinity sites for bilirubin. The high-affinity site can bind approxi- Possible Treatments for Porphyrias mately 25 mg of bilirubin/100 mL of plasma. More loosely Present treatment of porphyrias is essentially symptomatic: bound bilirubin can readily be detached and diffused into tis- avoiding drugs that induce production of cytochrome P450, sues, and antibiotics and certain other drugs can compete with ingestion of large amounts of carbohydrate, and administration and displace bilirubin from albumin’s high-affinity site. 312 SECTION VI Metabolism of Proteins & Amino Acids COOH COOH facilitated transport system. Even under pathologic condi- tions, transport does not appear to be rate-limiting for the metabolism of bilirubin. The net uptake of bilirubin depends on its removal by subsequent metabolism. Once internal- ized, bilirubin binds to cytosolic proteins such as glutathione N N S-transferase, previously known as a ligandin, to prevent bili- Fe3+ rubin from reentering the bloodstream. N N Conjugation of Bilirubin With Glucuronate Ferric heme Bilirubin is nonpolar, and would persist in cells (eg, bound to lipids) if not converted to a more water-soluble form. Bili- 3O2 + 7e– rubin is converted to a more polar molecule by conjugation with glucuronic acid (Figure 31–12). A bilirubin-specific CO + Fe3+ UDP-glucuronosyltransferase (EC 2.4.1.17) of the endoplas- mic reticulum catalyzes stepwise transfer to bilirubin of two HOOC COOH glucosyl moieties from 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 of conjugated bilirubin into the bile occurs by an NADPH Biliverdin active transport mechanism, which probably is rate-limiting for the entire process of hepatic bilirubin metabolism. The Reductase NADP + protein involved is a multispecific organic anion trans- porter (MOAT) located in the plasma membrane of the bile canaliculi. A member of the family of ATP-binding cassette HOOC COOH transporters, MOAT transports a number of organic anions. The hepatic transport of conjugated bilirubin into the bile is inducible by the same drugs that can induce the conjugation of bilirubin. Conjugation and excretion of bilirubin thus con- O N N N N O stitute a coordinated functional unit. H H H H Most of the bilirubin excreted in the bile of mammals is Bilirubin bilirubin diglucuronide. Bilirubin UDP-glucuronosyltransfer- 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 of ferric 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 of bilirubin takes place in three stages: H2C CH2 uptake by the liver, conjugation with glucuronic acid, and H2C 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 of Bilirubin is removed from albumin and taken up at the sinu- bilirubin. Clinically, the diglucuronide is also termed “direct reacting” soidal surface of hepatocytes by a large capacity, saturable bilirubin.  CHAPTER 31 Porphyrins & Bile Pigments 313 Blood presence of added methanol measures total bilirubin. The 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 of biliru- Neonatal jaundice bin per dL (17 μmol/L), may result from production of more UDP-GlcUA “Toxic” jaundice UDP-GlcUA Crigler-Najjar syndrome bilirubin than the normal liver can excrete, or from the failure Gilbert syndrome of a damaged liver to excrete normal amounts of bilirubin. In the absence of hepatic damage, obstruction of the excretory Bilirubin diglucuronide ducts of the liver prevents the excretion of bilirubin, and will also cause hyperbilirubinemia. In all these situations, when 3. SECRETION Dubin-Johnson syndrome the blood concentration of bilirubin reaches 2 to 2.5 mg/dL, it diffuses 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 of hyperbilirubinemia include retention hyperbiliru- major processes (uptake, conjugation, and secretion) involved in the transfer of bilirubin from blood to bile. Certain proteins of binemia due to overproduction of bilirubin, and regurgita- hepatocytes bind intracellular bilirubin and may prevent its efflux tion hyperbilirubinemia due to reflux into the bloodstream into the bloodstream. The processes affected in certain conditions because of biliary obstruction. that cause jaundice are also shown. Because of its hydrophobicity, only unconjugated bilirubin can cross the blood–brain barrier into the central nervous sys- the three major processes involved in the transfer of bilirubin tem. Encephalopathy due to hyperbilirubinemia (kernicterus) from blood to bile. Sites that are affected in a number of condi- thus occurs only with unconjugated bilirubin, as in retention tions causing jaundice are also indicated. hyperbilirubinemia. Alternatively, because of its water solubil- ity, only conjugated bilirubin can appear in urine. Accordingly, Intestinal Bacteria Reduce Conjugated choluric jaundice (choluria is the presence of 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 of an the large intestine, the glucuronosyl moieties are removed by excess of unconjugated bilirubin. Table 31–3 lists some causes specific bacterial β-glucuronidases (EC 3.2.1.31). Subsequent of unconjugated and conjugated hyperbilirubinemia. A mod- reduction by the fecal flora forms a group of colorless tetra- erate hyperbilirubinemia accompanies hemolytic anemias. pyrroles called urobilinogens. Small portions of 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 formed or when liver disease disrupts Unconjugated Conjugated this intrahepatic cycle, urobilinogen may also be excreted in Hemolytic anemias Obstruction of the biliary tree the urine. Most of the colorless urobilinogens formed in the Neonatal “physiological Dubin–Johnson syndrome colon are oxidized there to colored urobilins and excreted in jaundice” the feces. Fecal darkening upon standing in air results from Crigler-Najjar syndromes types I Rotor syndrome the oxidation of residual urobilinogens to urobilins. and II Measurement of Bilirubin in Serum Gilbert syndrome Liver diseases such as the various types of hepatitis Quantitation of bilirubin employs a colorimetric method Toxic hyperbilirubinemia based on the reddish-purple color formed when bilirubin reacts with diazotized sulfanilic acid. An assay conducted in These causes are discussed briefly in the text. Common causes of obstruction of the biliary tree are a stone in the common bile duct and cancer of the head of the pan- the absence of added methanol measures “direct bilirubin,” creas. Various liver diseases (eg, the various types of hepatitis) are frequent causes which is bilirubin glucuronide. An assay conducted in the of predominantly conjugated hyperbilirubinemia. 314 SECTION VI Metabolism of Proteins & Amino Acids Hyperbilirubinemia is usually modest (< 4 mg bilirubin per dL; tends not to exceed 20 mg/dL of serum, and patients respond < 68 μmol/L) despite extensive hemolysis, due to the high to treatment with large doses of phenobarbital. capacity of a healthy liver to metabolize bilirubin. Toxic Hyperbilirubinemia DISORDERS OF BILIRUBIN Unconjugated hyperbilirubinemia can result from toxin- induced liver dysfunction caused by, for example, chloro- METABOLISM form, arsphenamines, carbon tetrachloride, acetaminophen, Neonatal “Physiologic Jaundice” hepatitis virus, cirrhosis, or Amanita mushroom poisoning. These acquired disorders involve hepatic parenchymal cell The unconjugated hyperbilirubinemia of neonatal “physi- damage, which impairs bilirubin conjugation. ologic jaundice” results from accelerated hemolysis and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin. In this transient condition, bilirubin- Obstruction in the Biliary Tree Is the glucuronosyltransferase activity, and probably also synthesis Most Common Cause of Conjugated of UDP-glucuronate, are reduced. When the plasma concen- Hyperbilirubinemia tration of unconjugated bilirubin exceeds that which can be tightly bound by albumin (20–25 mg/dL), bilirubin can pen- Conjugated hyperbilirubinemia commonly results from etrate the blood–brain barrier. If left untreated, the resulting blockage of the hepatic or common bile ducts, most often hyperbilirubinemic toxic encephalopathy, or kernicterus, due to a gallstone or to cancer of the head of the pancreas can result in mental retardation. Exposure of 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, of 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 of uric jaundice), and the stools typically are a pale color. bilirubin metabolism, may be administered. The term cholestatic jaundice includes both all cases of extrahepatic obstructive jaundice and also conjugated hyper- bilirubinemia due to micro-obstruction of intrahepatic biliary Defects of Bilirubin ductules by damaged hepatocytes, such as may occur in infec- UDP-Glucuronosyltransferase tious hepatitis. Glucuronosyltransferases (EC 2.4.1.17), a family of enzymes with differing substrate specificities, increase the polarity of various drugs and drug metabolites, thereby facilitating 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 reflects the severity of the impairment include Gilbert syndrome and two types of Crigler-Najjar syndrome. HEPATIC Liver diseases (Liver) (eg, hepatitis, cancer) Gilbert Syndrome Providing that about 30% of the bilirubin UDP-glucuronosyl- transferase activity is retained in Gilbert syndrome, the condi- tion is harmless. Gallstone Type I Crigler-Najjar Syndrome POST-HEPATIC Pancreatic The severe congenital jaundice (over 20 mg bilirubin per dL (Biliary system & cancer serum) and accompanying brain damage of type I Crigler- pancreas) Najjar syndrome reflect the complete absence of hepatic UDP-glucuronosyltransferase activity. Phototherapy reduces plasma bilirubin levels somewhat, but phenobarbital has no beneficial effect. The disease is often fatal within the first 15 months of life. 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 from hepatitis or other liver diseases (eg, cancer). Posthepatic jaundice refers to events in In type II Crigler-Najjar syndrome, some bilirubin UDP- the biliary tree, for which the major causes are obstruction of the glucuronosyltransferase activity is retained. This condition common bile duct by a gallstone (biliary calculus) or by cancer of the thus is more benign than the type I syndrome. Serum bilirubin head of the pancreas.  CHAPTER 31 Porphyrins & Bile Pigments 315 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 if micro-obstruction Present if micro-obstruction Decreased is present occurs Obstructive jaundicea ↑Direct Absent Present Trace to absent a The most common causes of obstructive (posthepatic) jaundice are cancer of the head of the pancreas and a gallstone lodged in the common bile duct. The presence of bili- rubin in the urine is sometimes referred to as choluria—therefore, hepatitis and obstruction of the common bile duct cause choluric jaundice, whereas the jaundice of hemolytic anemia is referred to as acholuric. The laboratory results in patients with hepatitis are variable, depending on the extent of damage to parenchymal cells and the extent of micro-obstruction to bile ductules. Serum levels of alanine aminotransferase and aspartate aminotransferase are usually markedly elevated in hepatitis, whereas serum levels of alkaline phosphatase are elevated in obstructive liver disease. Dubin-Johnson Syndrome of prothrombin time) and on serum (eg, electrophoresis of This benign autosomal recessive disorder consists of conju- proteins; alkaline phosphatase and alanine aminotransferase gated hyperbilirubinemia in childhood or during adult life. and aspartate aminotransferase activities) also help to distin- The hyperbilirubinemia is caused by mutations in the gene guish between prehepatic, hepatic, and posthepatic causes of encoding the protein involved in the secretion of conjugated jaundice. bilirubin into bile. SUMMARY Some Conjugated Bilirubin Can Bind The heme of hemoproteins such as hemoglobin and the Covalently to Albumin cytochromes is an iron-containing porphyrin consisting of four pyrrole rings joined by methyne bridges. When levels of conjugated bilirubin remain high in plasma, a The eight methyl, vinyl, and propionyl substituents on the four fraction can bind covalently to albumin. This fraction, termed pyrrole rings of heme are arranged in a speci%c sequence. The 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 four nitrogen atoms of the pyrrole rings. recovery from obstructive jaundice. Some patients therefore Biosynthesis of the heme ring involves eight enzyme-catalyzed continue to appear jaundiced even after the circulating conju- reactions, some of which occur in mitochondria, others in the gated bilirubin level has returned to normal. cytosol. Synthesis of heme commences with the condensation of Urinary Urobilinogen & Bilirubin Are succinyl-CoA and glycine to form ALA. This reaction Clinical Indicators is catalyzed by ALAS1, the regulatory enzyme of heme In complete obstruction of the bile duct, bilirubin has no biosynthesis. access to the intestine for conversion to urobilinogen, so no Synthesis of ALAS1 increases in response to a low level of urobilinogen is present in the urine. The presence of conju- available heme. For example, certain drugs (eg, phenobarbital) gated bilirubin in the urine without urobilinogen suggests indirectly trigger enhanced synthesis of ALAS1 by promoting synthesis of 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 of bilirubin leads to increased production of urobilin- synthesis of cytochrome P450. ogen, which appears in the urine in large amounts. Bilirubin Genetic abnormalities of seven of the eight enzymes of heme is not usually found in the urine in hemolytic jaundice, so biosynthesis result in inherited porphyrias. Erythrocytes the combination of increased urobilinogen and absence of and liver are the major sites of expression of the porphyrias. bilirubin is suggestive of hemolytic jaundice. Increased blood Photosensitivity and neurologic problems are common destruction from any cause brings about an increase in urine complaints. Intake of certain toxins (eg, lead) can cause urobilinogen. acquired porphyrias. Increased amounts of porphyrins or their Table 31–4 summarizes laboratory results obtained in precursors can be detected in blood and urine, facilitating patients with jaundice due to prehepatic, hepatic, or pos- diagnosis. thepatic causes: hemolytic anemia (prehepatic), hepatitis Catabolism of 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 of biliverdin in the cytosol tion of the possibility of a hemolytic anemia and measurement forms bilirubin.