Harper’s Illustrated Biochemistry - Part 5 pptx

PORPHYRINS & BILE PIGMENTS / 271 C 2 CCCH C HH 1 N I αδ HC HC 8 HC HC 7 C C NH IV γ CC CC CH 5 H H 6 N III β CH CH 3 4 C C HN II CH HC HC CH N H N Porphin (C 20 H 14 N 4 ) Pyrrole Figure 32–1. The porphin molecule. Rings are la- beled I, II, III, and IV. Substituent positions on the rings are labeled 1, 2, 3, 4, 5, 6, 7, and 8. The methenyl bridges (HC   ) are labeled α, β, γ, and δ. 83 4 7 21 56 I IIIV III AA P P PA AP I IIIV III Figure 32–2. Uroporphyrin III. A (acetate) = CH 2 COOH; P (propionate) = CH 2 CH 2 COOH. droxymethylbilane (HMB). The reaction is catalyzed by uroporphyrinogen I synthase, also named PBG deami- nase or HMB synthase. HMB cyclizes spontaneously to form uroporphyrinogen I (left-hand side of Figure 32–6) or is converted to uroporphyrinogen III by the action of uroporphyrinogen III synthase (right-hand side of Figure 32–6). Under normal conditions, the uropor- phyrinogen formed is almost exclusively the III isomer, but in certain of the porphyrias (discussed below), the type I isomers of porphyrinogens are formed in excess. Note that both of these uroporphyrinogens have the pyrrole rings connected by methylene bridges (CH 2 ), which do not form a conjugated ring sys- tem. Thus, these compounds are colorless (as are all porphyrinogens). However, the porphyrinogens are readily auto-oxidized to their respective colored por- phyrins. These oxidations are catalyzed by light and by the porphyrins that are formed. Uroporphyrinogen III is converted to copropor- phyrinogen III by decarboxylation of all of the acetate (A) groups, which changes them to methyl (M) sub- stituents. The reaction is catalyzed by uroporphyrino- gen decarboxylase, which is also capable of converting uroporphyrinogen I to coproporphyrinogen I (Figure 32–7). Coproporphyrinogen III then enters the mito- chondria, where it is converted to protoporphyrinogen III and then to protoporphyrin III. Several steps are involved in this conversion. The mitochondrial enzyme coproporphyrinogen oxidase catalyzes the decarboxy- lation and oxidation of two propionic side chains to form protoporphyrinogen. This enzyme is able to act only on type III coproporphyrinogen, which would ex- plain why type I protoporphyrins do not generally occur in nature. The oxidation of protoporphyrinogen to pro- toporphyrin is catalyzed by another mitochondrial en- zyme, protoporphyrinogen oxidase. In mammalian liver, the conversion of coproporphyrinogen to proto- porphyrin requires molecular oxygen. Formation of Heme Involves Incorporation of Iron Into Protoporphyrin The final step in heme synthesis involves the incorpora- tion of ferrous iron into protoporphyrin in a reaction catalyzed by ferrochelatase (heme synthase), another mitochondrial enzyme (Figure 32–4). A summary of the steps in the biosynthesis of the porphyrin derivatives from PBG is given in Figure 32–8. The last three enzymes in the pathway and ALA synthase are located in the mitochondrion, whereas the other enzymes are cytosolic. Both erythroid and non- erythroid (“housekeeping”) forms of the first four en- zymes are found. Heme biosynthesis occurs in most mammalian cells with the exception of mature erythro- cytes, which do not contain mitochondria. However, Table 32–1. Examples of some important human and animal hemoproteins. 1 Protein Function Hemoglobin Transport of oxygen in blood Myoglobin Storage of oxygen in muscle Cytochrome c Involvement in electron transport chain Cytochrome P450 Hydroxylation of xenobiotics Catalase Degradation of hydrogen peroxide Tryptophan Oxidation of trypotophan pyrrolase 1 The functions of the above proteins are described in various chapters of this text. ch32.qxd 2/13/2003 3:57 PM Page 271 272 / CHAPTER 32 A P PA AP Uroporphyrin I Uroporphyrin III Coproporphyrin I Coproporphyrin III Uroporphyrins were first found in the urine, but they are not restricted to urine. Coproporphyrins were first isolated from feces, but they are also found in urine. P A M P PM MP P M M P PM MP M P A P PA AP A P Figure 32–3. Uroporphyrins and coproporphyrins. A (acetate); P (propionate); M (methyl) = CH 3 ; V (vinyl) = CHCH 2 . approximately 85% of heme synthesis occurs in eryth- roid precursor cells in the bone marrow and the major- ity of the remainder in hepatocytes. The porphyrinogens described above are colorless, containing six extra hydrogen atoms as compared with the corresponding colored porphyrins. These reduced porphyrins (the porphyrinogens) and not the corre- sponding porphyrins are the actual intermediates in the biosynthesis of protoporphyrin and of heme. ALA Synthase Is the Key Regulatory Enzyme in Hepatic Biosynthesis of Heme ALA synthase occurs in both hepatic (ALAS1) and ery- throid (ALAS2) forms. The rate-limiting reaction in the synthesis of heme in liver is that catalyzed by ALAS1 (Figure 32–5), a regulatory enzyme. It appears that heme, probably acting through an aporepressor mole- cule, acts as a negative regulator of the synthesis of ALAS1. This repression-derepression mechanism is de- picted diagrammatically in Figure 32–9. Thus, the rate of synthesis of ALAS1 increases greatly in the absence of heme and is diminished in its presence. The turnover rate of ALAS1 in rat liver is normally rapid (half-life about 1 hour), a common feature of an enzyme catalyz- ing a rate-limiting reaction. Heme also affects transla- tion of the enzyme and its transfer from the cytosol to the mitochondrion. Many drugs when administered to humans can re- sult in a marked increase in ALAS1. Most of these drugs are metabolized by a system in the liver that uti- lizes a specific hemoprotein, cytochrome P450 (see Chapter 53). During their metabolism, the utilization of heme by cytochrome P450 is greatly increased, which in turn diminishes the intracellular heme con- centration. This latter event effects a derepression of ALAS1 with a corresponding increased rate of heme synthesis to meet the needs of the cells. MM V P VM MP MM V P VM MP Fe 2 + Protoporphyrin III (IX) (parent porphyrin of heme) Heme (prosthetic group of hemoglobin) FERROCHELATASE Fe 2 + Figure 32–4. Addition of iron to protoporphyrin to form heme. ch32.qxd 2/13/2003 3:57 PM Page 272 PORPHYRINS & BILE PIGMENTS / 273 CH 2 CH 2 CO COOH S CoA H + C COOH NH 2 H CH 2 CH 2 CO COOH C COOH NH 2 H CH 2 CH 2 CO COOH C H NH 2 H Glycine Succinyl-CoA (“active” succinate) ALA SYNTHASE CoA • SH Two molecules of δ-aminolevulinate Porphobilinogen (first precursor pyrrole) CH 2 NH 2 CH 2 CH 2 CO COOH HH CH 2 CH 2 H NH CO C COOH C C C CH CH 2 N H CH 2 CH 2 COOH CH 2 COOH NH 2 ALA DEHYDRATASE 2H 2 O ALA SYNTHASE CO 2 δ-Aminolevulinate (ALA)α-Amino-β-ketoadipate Pyridoxal phosphate Figure 32–5. Biosynthesis of porphobilinogen. ALA synthase occurs in the mitochon- dria, whereas ALA dehydratase is present in the cytosol. Several factors affect drug-mediated derepression of ALAS1 in liver—eg, the administration of glucose can prevent it, as can the administration of hematin (an ox- idized form of heme). The importance of some of these regulatory mecha- nisms is further discussed below when the porphyrias are described. Regulation of the erythroid form of ALAS (ALAS2) differs from that of ALAS1. For instance, it is not in- duced by the drugs that affect ALAS1, and it does not undergo feedback regulation by heme. PORPHYRINS ARE COLORED & FLUORESCE The various porphyrinogens are colorless, whereas the various porphyrins are all colored. In the study of por- phyrins or porphyrin derivatives, the characteristic ab- sorption spectrum that each exhibits—in both the visible and the ultraviolet regions of the spectrum—is of great value. An example is the absorption curve for a solution of porphyrin in 5% hydrochloric acid (Figure 32–10). Note particularly the sharp absorption band near 400 nm. This is a distinguishing feature of the porphin ring and is characteristic of all porphyrins regardless of the side chains present. This band is termed the Soret band after its discoverer, the French physicist Charles Soret. When porphyrins dissolved in strong mineral acids or in organic solvents are illuminated by ultraviolet light, they emit a strong red fluorescence. This fluores- cence is so characteristic that it is often used to detect small amounts of free porphyrins. The double bonds joining the pyrrole rings in the porphyrins are responsi- ble for the characteristic absorption and fluorescence of these compounds; these double bonds are absent in the porphyrinogens. An interesting application of the photodynamic properties of porphyrins is their possible use in the treatment of certain types of cancer, a procedure called cancer phototherapy. Tumors often take up more por- phyrins than do normal tissues. Thus, hematopor- phyrin or other related compounds are administered to a patient with an appropriate tumor. The tumor is then exposed to an argon laser, which excites the porphyrins, producing cytotoxic effects. Spectrophotometry Is Used to Test for Porphyrins & Their Precursors Coproporphyrins and uroporphyrins are of clinical in- terest because they are excreted in increased amounts in ch32.qxd 2/13/2003 3:57 PM Page 273 274 / CHAPTER 32 HOOC COOH CH 2 CH 2 H 2 C CC CHC H N H 2 C NH 2 Four molecules of porphobilinogen NH 3 4 Hydroxymethylbilane (linear tetrapyrrole) CC CC H N H 2 C AP CC CC H N AP CC CC H N H 2 C P CC CC H N APA CH 2 CH 2 IV III CC CC H N H 2 C AP CC CC H N AP CC CC H N H 2 C P CC CC H N APA CH 2 CH 2 IV III Type III uroporphyrinogen Type I uroporphyrinogen III III P A UROPORPHYRINOGEN I SYNTHASE UROPORPHYRINOGEN III SYNTHASE SPONTANEOUS CYCLIZATION Figure 32–6. Conversion of porphobilinogen to uro- porphyrinogens. Uroporphyrinogen synthase I is also called porphobilinogen (PBG) deaminase or hydroxy- methylbilane (HMB) synthase. the porphyrias. These compounds, when present in urine or feces, can be separated from each other by ex- traction with appropriate solvent mixtures. They can then be identified and quantified using spectrophoto- metric methods. ALA and PBG can also be measured in urine by ap- propriate colorimetric tests. THE PORPHYRIAS ARE GENETIC DISORDERS OF HEME METABOLISM The porphyrias are a group of disorders due to abnor- malities in the pathway of biosynthesis of heme; they can be genetic or acquired. They are not prevalent, but it is important to consider them in certain circum- stances (eg, in the differential diagnosis of abdominal pain and of a variety of neuropsychiatric findings); oth- erwise, patients will be subjected to inappropriate treat- ments. It has been speculated that King George III had a type of porphyria, which may account for his periodic confinements in Windsor Castle and perhaps for some of his views regarding American colonists. Also, the photosensitivity (favoring nocturnal activities) and se- vere disfigurement exhibited by some victims of con- genital erythropoietic porphyria have led to the sugges- tion that these individuals may have been the prototypes of so-called werewolves. No evidence to sup- port this notion has been adduced. Biochemistry Underlies the Causes, Diagnoses, & Treatments of the Porphyrias Six major types of porphyria have been described, re- sulting from depressions in the activities of enzymes 3 through 8 shown in Figure 32–9 (see also Table 32–2). Assay of the activity of one or more of these enzymes using an appropriate source (eg, red blood cells) is thus important in making a definitive diagnosis in a sus- pected case of porphyria. Individuals with low activities of enzyme 1 (ALAS2) develop anemia, not porphyria (see Table 32–2). Patients with low activities of enzyme 2 (ALA dehydratase) have been reported, but very rarely; the resulting condition is called ALA dehy- dratase-deficient porphyria. In general, the porphyrias described are inherited in an autosomal dominant manner, with the exception of congenital erythropoietic porphyria, which is inherited in a recessive mode. The precise abnormalities in the genes directing synthesis of the enzymes involved in heme biosynthesis have been determined in some in- stances. Thus, the use of appropriate gene probes has made possible the prenatal diagnosis of some of the porphyrias. As is true of most inborn errors, the signs and symp- toms of porphyria result from either a deficiency of metabolic products beyond the enzymatic block or from an accumulation of metabolites behind the block. If the enzyme lesion occurs early in the pathway prior to the formation of porphyrinogens (eg, enzyme 3 of Figure 32–9, which is affected in acute intermittent porphyria), ALA and PBG will accumulate in body tis- sues and fluids (Figure 32–11). Clinically, patients complain of abdominal pain and neuropsychiatric symptoms. The precise biochemical cause of these symptoms has not been determined but may relate to elevated levels of ALA or PBG or to a deficiency of heme. On the other hand, enzyme blocks later in the path- way result in the accumulation of the porphyrinogens ch32.qxd 2/13/2003 3:57 PM Page 274 PORPHYRINS & BILE PIGMENTS / 275 Uroporphyrinogen I Uroporphyrinogen III P P P P A A A A I III IIIV P P P P A A A A I III IIIV Coproporphyrinogen I Coproporphyrinogen III P P P P M M M M I III IIIV P P P P M M M M I III IIIV UROPORPHYRINOGEN DECARBOXYLASE 4CO 2 4CO 2 Figure 32–7. Decarboxylation of uropor- phyrinogens to coproporphyrinogens in cy- tosol. (A, acetyl; M, methyl; P, propionyl.) Porphobilinogen Hydroxymethylbilane SPONTANEOUS Uroporphyrinogen I 4CO 2 4CO 2 Protoporphyrinogen III 6H Or light in vitro Fe 2 + Heme Light 6H Light 6H Uroporphyrin I Coproporphyrinogen III Light 6H Coproporphyrin I Uroporphyrinogen III Protoporphyrin III Coproporphyrinogen I Coproporphyrin III Light 6H Uroporphyrin III MITOCHONDRIA CYTOSOL PROTOPORPHYRINOGEN OXIDASE FERROCHELATASE UROPORPHYRINOGEN I SYNTHASE UROPORPHYRINOGEN III SYNTHASE UROPORPHYRINOGEN DECARBOXYLASE COPROPORPHYRINOGEN OXIDASE Figure 32–8. Steps in the biosynthesis of the porphyrin derivatives from porphobilinogen. Uropor- phyrinogen I synthase is also called porphobilinogen deaminase or hydroxymethylbilane synthase. ch32.qxd 2/13/2003 3:57 PM Page 275 276 / CHAPTER 32 Protoporphyrinogen III Coproporphyrinogen III Protoporphyrin III Heme Uroporphyrinogen III Hydroxymethylbilane Porphobilinogen ALA Succinyl-CoA + Glycine Fe 2 + Proteins Hemoproteins Aporepressor 6. COPROPORPHYRINOGEN OXIDASE 7. PROTOPORPHYRINOGEN OXIDASE 8. FERROCHELATASE 5. UROPORPHYRINOGEN DECARBOXYLASE 4. UROPORPHYRINOGEN III SYNTHASE 3. UROPORPHYRINOGEN I SYNTHASE 2. ALA DEHYDRATASE 1. ALA SYNTHASE Figure 32–9. Intermediates, enzymes, and regulation of heme syn- thesis. The enzyme numbers are those referred to in column 1 of Table 32–2. Enzymes 1, 6, 7, and 8 are located in mitochondria, the others in the cytosol. Mutations in the gene encoding enzyme 1 causes X-linked sideroblastic anemia. Mutations in the genes encoding enzymes 2–8 cause the porphyrias, though only a few cases due to deficiency of en- zyme 2 have been reported. Regulation of hepatic heme synthesis oc- curs at ALA synthase (ALAS1) by a repression-derepression mecha- nism mediated by heme and its hypothetical aporepressor. The dotted lines indicate the negative ( ᭺ − ) regulation by repression. En- zyme 3 is also called porphobilinogen deaminase or hydroxymethyl- bilane synthase. ch32.qxd 2/13/2003 3:57 PM Page 276 PORPHYRINS & BILE PIGMENTS / 277 300 1 2 3 Log absorbency 4 5 400 500 Wavelength (nm) 600 700 Figure 32–10. Absorption spectrum of hematopor- phyrin (0.01% solution in 5% HCl). Mutations in DNA Photosensitivity Abnormalities of the enzymes of heme synthesis Accumulation of ALA and PBG and/or decrease in heme in cells and body fluids Accumulation of porphyrinogens in skin and tissues Neuropsychiatric signs and symptoms Spontaneous oxidation of porphyrinogens to porphyrins Figure 32–11. Biochemical causes of the major signs and symptoms of the porphyrias. Table 32–2. Summary of major findings in the porphyrias. 1 Enzyme Involved 2 Type, Class, and MIM Number Major Signs and Symptoms Results of Laboratory Tests 1. ALA synthase X-linked sideroblastic anemia 3 Anemia Red cell counts and hemoglobin (erythroid form) (erythropoietic) (MIM decreased 201300) 2. ALA dehydratase ALA dehydratase deficiency Abdominal pain, neuropsychiatric Urinary δ-aminolevulinic acid (hepatic) (MIM 125270) symptoms 3. Uroporphyrinogen I Acute intermittent porphyria Abdominal pain, neuropsychiatric Urinary porphobilinogen positive, synthase 4 (hepatic) (MIM 176000) symptoms uroporphyrin positive 4. Uroporphyrinogen III Congenital erythropoietic No photosensitivity Uroporphyrin positive, porpho- synthase (erythropoietic) (MIM bilinogen negative 263700) 5. Uroporphyrinogen Porphyria cutanea tarda (he- Photosensitivity Uroporphyrin positive, porpho- decarboxylase patic) (MIM 176100) bilinogen negative 6. Coproporphyrinogen Hereditary coproporphyria Photosensitivity, abdominal pain, Urinary porphobilinogen posi- oxidase (hepatic) (MIM 121300) neuropsychiatric symptoms tive, urinary uroporphyrin positive, fecal protopor- phyrin positive 7. Protoporphyrinogen Variegate porphyria (hepatic) Photosensitivity, abdominal pain, Urinary porphobilinogen posi- oxidase (MIM 176200) neuropsychiatric symptoms tive, fecal protoporphyrin positive 8. Ferrochelatase Protoporphyria (erythropoietic) Photosensitivity Fecal protoporphyrin posi- ` (MIM 177000) tive, red cell protoporphyrin positive 1 Only the biochemical findings in the active stages of these diseases are listed. Certain biochemical abnormalities are detectable in the la- tent stages of some of the above conditions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias. 2 The numbering of the enzymes in this table corresponds to that used in Figure 32-9. 3 X-linked sideroblastic anemia is not a porphyria but is included here because δ−aminolevulinic acid synthase is involved. 4 This enzyme is also called porphobilinogen deaminase or hydroxymethylbilane synthase. ch32.qxd 2/13/2003 3:57 PM Page 277 278 / CHAPTER 32 indicated in Figures 32–9 and 32–11. Their oxidation products, the corresponding porphyrin derivatives, cause photosensitivity, a reaction to visible light of about 400 nm. The porphyrins, when exposed to light of this wavelength, are thought to become “excited” and then react with molecular oxygen to form oxygen radicals. These latter species injure lysosomes and other organelles. Damaged lysosomes release their degradative enzymes, causing variable degrees of skin damage, in- cluding scarring. The porphyrias can be classified on the basis of the organs or cells that are most affected. These are gener- ally organs or cells in which synthesis of heme is partic- ularly active. The bone marrow synthesizes considerable hemoglobin, and the liver is active in the synthesis of another hemoprotein, cytochrome P450. Thus, one classification of the porphyrias is to designate them as predominantly either erythropoietic or hepatic; the types of porphyrias that fall into these two classes are so characterized in Table 32–2. Porphyrias can also be classified as acute or cutaneous on the basis of their clinical features. Why do specific types of porphyria af- fect certain organs more markedly than others? A par- tial answer is that the levels of metabolites that cause damage (eg, ALA, PBG, specific porphyrins, or lack of heme) can vary markedly in different organs or cells de- pending upon the differing activities of their heme- forming enzymes. As described above, ALAS1 is the key regulatory en- zyme of the heme biosynthetic pathway in liver. A large number of drugs (eg, barbiturates, griseofulvin) induce the enzyme. Most of these drugs do so by inducing cy- tochrome P450 (see Chapter 53), which uses up heme and thus derepresses (induces) ALAS1. In patients with porphyria, increased activities of ALAS1 result in in- creased levels of potentially harmful heme precursors prior to the metabolic block. Thus, taking drugs that cause induction of cytochrome P450 (so-called micro- somal inducers) can precipitate attacks of porphyria. The diagnosis of a specific type of porphyria can generally be established by consideration of the clinical and family history, the physical examination, and ap- propriate laboratory tests. The major findings in the six principal types of porphyria are listed in Table 32–2. High levels of lead can affect heme metabolism by combining with SH groups in enzymes such as fer- rochelatase and ALA dehydratase. This affects por- phyrin metabolism. Elevated levels of protoporphyrin are found in red blood cells, and elevated levels of ALA and of coproporphyrin are found in urine. It is hoped that treatment of the porphyrias at the gene level will become possible. In the meantime, treat- ment is essentially symptomatic. It is important for pa- tients to avoid drugs that cause induction of cyto- chrome P450. Ingestion of large amounts of carbohy- drates (glucose loading) or administration of hematin (a hydroxide of heme) may repress ALAS1, resulting in di- minished production of harmful heme precursors. Pa- tients exhibiting photosensitivity may benefit from ad- ministration of β-carotene; this compound appears to lessen production of free radicals, thus diminishing photosensitivity. Sunscreens that filter out visible light can also be helpful to such patients. CATABOLISM OF HEME PRODUCES BILIRUBIN Under physiologic conditions in the human adult, 1–2 × 10 8 erythrocytes are destroyed per hour. Thus, in 1 day, a 70-kg human turns over approximately 6 g of he- moglobin. When hemoglobin is destroyed in the body, globin is degraded to its constituent amino acids, which are reused, and the iron of heme enters the iron pool, also for reuse. The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells of the liver, spleen, and bone marrow. The catabolism of heme from all of the heme pro- teins appears to be carried out in the microsomal frac- tions of cells by a complex enzyme system called heme oxygenase. By the time the heme derived from heme proteins reaches the oxygenase system, the iron has usu- ally been oxidized to the ferric form, constituting hemin. The heme oxygenase system is substrate-in- ducible. As depicted in Figure 32–12, the hemin is re- duced to heme with NADPH, and, with the aid of more NADPH, oxygen is added to the α-methenyl bridge between pyrroles I and II of the porphyrin. The ferrous iron is again oxidized to the ferric form. With the further addition of oxygen, ferric ion is released, carbon monoxide is produced, and an equimolar quantity of biliverdin results from the splitting of the tetrapyrrole ring. In birds and amphibia, the green biliverdin IX is ex- creted; in mammals, a soluble enzyme called biliverdin reductase reduces the methenyl bridge between pyrrole III and pyrrole IV to a methylene group to produce bilirubin, a yellow pigment (Figure 32–12). It is estimated that 1 g of hemoglobin yields 35 mg of bilirubin. The daily bilirubin formation in human adults is approximately 250–350 mg, deriving mainly from hemoglobin but also from ineffective erythro- poiesis and from various other heme proteins such as cytochrome P450. The chemical conversion of heme to bilirubin by reticuloendothelial cells can be observed in vivo as the purple color of the heme in a hematoma is slowly con- verted to the yellow pigment of bilirubin. ch32.qxd 2/13/2003 3:57 PM Page 278 PORPHYRINS & BILE PIGMENTS / 279 HN HN H H HN HN P P O O Bilirubin HN HN N HN P P O O Biliverdin II III IV I NADP NADPH N II N N I III N IV Fe 3 + P P N II N N I III N IV Fe 2 + P P N II N N I III N IV Fe 3 + P P NADPH NADP NADPH NADP O 2 OH Hemin Heme α Heme O 2 Fe 3 + (reutilized) CO (exhaled) α Microsomal heme oxygenase system Figure 32–12. Schematic representation of the microsomal heme oxygenase system. (Modified from Schmid R, McDonough AF in: The Porphyrins. Dolphin D [editor]. Academic Press, 1978.) ch32.qxd 2/13/2003 3:57 PM Page 279 280 / CHAPTER 32 Bilirubin formed in peripheral tissues is transported to the liver by plasma albumin. The further metabolism of bilirubin occurs primarily in the liver. It can be di- vided into three processes: (1) uptake of bilirubin by liver parenchymal cells, (2) conjugation of bilirubin with glucuronate in the endoplasmic reticulum, and (3) secretion of conjugated bilirubin into the bile. Each of these processes will be considered separately. THE LIVER TAKES UP BILIRUBIN Bilirubin is only sparingly soluble in water, but its solu- bility in plasma is increased by noncovalent binding to albumin. Each molecule of albumin appears to have one high-affinity site and one low-affinity site for bilirubin. In 100 mL of plasma, approximately 25 mg of bilirubin can be tightly bound to albumin at its high- affinity site. Bilirubin in excess of this quantity can be bound only loosely and thus can easily be detached and diffuse into tissues. A number of compounds such as antibiotics and other drugs compete with bilirubin for the high-affinity binding site on albumin. Thus, these compounds can displace bilirubin from albumin and have significant clinical effects. In the liver, the bilirubin is removed from albumin and taken up at the sinusoidal surface of the hepato- cytes by a carrier-mediated saturable system. This facil- itated transport system has a very large capacity, so that even under pathologic conditions the system does not appear to be rate-limiting in the metabolism of bilirubin. Since this facilitated transport system allows the equilibrium of bilirubin across the sinusoidal mem- brane of the hepatocyte, the net uptake of bilirubin will be dependent upon the removal of bilirubin via subse- quent metabolic pathways. Once bilirubin enters the hepatocytes, it can bind to certain cytosolic proteins, which help to keep it solubi- lized prior to conjugation. Ligandin (a family of glu- tathione S-transferases) and protein Y are the involved proteins. They may also help to prevent efflux of biliru- bin back into the blood stream. Conjugation of Bilirubin With Glucuronic Acid Occurs in the Liver Bilirubin is nonpolar and would persist in cells (eg, bound to lipids) if not rendered water-soluble. Hepato- cytes convert bilirubin to a polar form, which is readily excreted in the bile, by adding glucuronic acid mole- cules to it. This process is called conjugation and can employ polar molecules other than glucuronic acid (eg, sulfate). Many steroid hormones and drugs are also converted to water-soluble derivatives by conjugation in preparation for excretion (see Chapter 53). The conjugation of bilirubin is catalyzed by a spe- cific glucuronosyltransferase. The enzyme is mainly located in the endoplasmic reticulum, uses UDP- glucuronic acid as the glucuronosyl donor, and is re- ferred to as bilirubin-UGT. Bilirubin monoglucuronide is an intermediate and is subsequently converted to the diglucuronide (Figures 32–13 and 32–14). Most of the bilirubin excreted in the bile of mammals is in the form of bilirubin diglucuronide. However, when bilirubin conjugates exist abnormally in human plasma (eg, in obstructive jaundice), they are predominantly mono- glucuronides. Bilirubin-UGT activity can be induced by a number of clinically useful drugs, including phe- nobarbital. More information about glucuronosylation is presented below in the discussion of inherited disor- ders of bilirubin conjugation. Bilirubin Is Secreted Into Bile Secretion of conjugated bilirubin into the bile occurs by an active transport mechanism, which is probably rate- limiting for the entire process of hepatic bilirubin me- tabolism. The protein involved is MRP-2 (multidrug resistance-like protein 2), also called multispecific or- ganic anion transporter (MOAT). It is located in the plasma membrane of the bile canalicular membrane and handles a number of organic anions. It is a member of the family of ATP-binding cassette (ABC) trans- porters. The hepatic transport of conjugated bilirubin into the bile is inducible by those same drugs that are capable of inducing the conjugation of bilirubin. Thus, the conjugation and excretion systems for bilirubin be- have as a coordinated functional unit. Figure 32–15 summarizes the three major processes involved in the transfer of bilirubin from blood to bile. Sites that are affected in a number of conditions caus- ing jaundice (see below) are also indicated. OCC OC VMMMVM H 2 C H 2 C C O – OOC(CH 2 O) 4 C CH 2 CH 2 C O C(CH 2 O) 4 COO – O O II III IV I Figure 32–13. Structure of bilirubin diglucuronide (conjugated, “direct-reacting” bilirubin). Glucuronic acid is attached via ester linkage to the two propionic acid groups of bilirubin to form an acylglucuronide. ch32.qxd 2/13/2003 3:57 PM Page 280 [...]... H OH OH PRPP (II) N 5, N10MethenylH 4 folate H 4 folate NH 3 Mg 2+ ATP ADP + Pi H H OH OH α-D-Ribose 5- phosphate (I) C4 O O 1 H PRPP SYNTHASE Glutamine H 2O H O 1 P O Glutamate PPi NH 3+ C4 O 5 CH 2 H NH 3+ FORMYLTRANSFERASE C4 O OH OH Formylglycinamide ribosyl -5 - phosphate (V) – OOC + HC VI SYNTHETASE NH3 Aspartate O 9 NH R- 5- P Glycinamide ribosyl -5 - phosphate (IV) 5- Phospho-β-D-ribosylamine (III) 7... ATP Mg 2 + Glu 5 CH2 – OOC COO – O – OOC CH HC HC H 2C – OOC N1 H C 6 3 H2 N – OOC O C 5 4 C H 2O N –O 8 C 6 CH N IX SYNTHETASE 3 H2 N R- 5- P CO2 N 5 7 HC N CH 4 N ATP, Mg 2 + H2O Ring closure 6 CH C VII CARBOXYLASE H2N N R- 5- P R- 5- P Fumarate Aminoimidazole succinyl carboxamide ribosyl -5 - phosphate (IX) 9 Aminoimidazole ribosyl -5 - phosphate (VII) Aminoimidazole carboxylate ribosyl -5 - phosphate (VIII)... N C H2N C N 10 CH C O N 10 -FormylH 4 folate H 4 folate N FORMYLTRANSFERASE R- 5- P Aminoimidazole carboxamide ribosyl -5 - phosphate (X) H2N O C O C C C H N H H 2O N 11 HN Ring closure C HC C N CH CH N R- 5- P IMP CYCLOHYDROLASE Formimidoimidazole carboxamide ribosyl -5 - phosphate (XI) N C N R- 5- P Inosine monophosphate (IMP) (XII) Figure 34–2 Purine biosynthesis from ribose 5- phosphate and ATP See text... H2C5 3 HN 7 8 CH C4 9 O NH R- 5- P Formylglycinamidine ribosyl -5 - phosphate (VI) ch34.qxd 2/13/2003 4:04 PM Page 2 95 Glycine ch34.qxd 2/13/2003 4:04 PM Page 296 296 / CHAPTER 34 – OOC O N – H – OOC C C COO H2 + H 2O NH 3 12 HN N GTP, Mg 2 N R- 5- P Inosine monophosphate (IMP) NAD + – OOC H C H C 13 COO – NH 2 N N + ADENYLOSUCCINATE SYNTHASE N N ADENYLOSUCCINASE R- 5- P Adenylosuccinate (AMPS) N N R- 5- P... inhibition of enzymes essential for nucleic acid synthesis or their incorporation into nucleic acids with resulting disruption of base-pairing Oncologists employ 5- fluoro- or 5iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mercaptopurine, 5- or 6-azauridine, 5- or 6-azacytidine, and 8-azaguanine (Figure 33–12), which are incorporated into DNA prior to cell division The purine analog allopurinol, used in treatment... is catabolized to 6-mercaptopurine, is employed during organ transplantation to suppress immunologic rejection ch33.qxd 3/16/04 11:00 AM Page 291 NUCLEOTIDES I HN 5 6 O O N HO O O F HN HO H 5- Iodo-2′-deoxyuridine 6 8 N H2N N H HO 5- Fluorouracil SH N 6 N H 6-Mercaptopurine H2N N N 8-Azaguanine OH N N1 6 5 2 N N H OH 6-Azauridine SH N N HN 5 O 2′ N N HO O O N 291 O O HN / N H N H N 6-Thioguanine N 4 3... G, C, T, and U, traces of 5- methylcytosine, 5- hydroxymethylcytosine, pseudouridine (Ψ), or N-methylated bases • Most nucleosides contain D-ribose or 2-deoxy-Dribose linked to N-1 of a pyrimidine or to N-9 of a purine by a β-glycosidic bond whose syn conformers predominate • A primed numeral locates the position of the phosphate on the sugars of mononucleotides (eg, 3′GMP, 5 -dCMP) Additional phosphoryl... N 3 3 O C O 5 CH 2 + 2 6 1 P + H3 N Carbamoyl phosphate (CAP) C O –O C 4 H2 N 3 ASPARTATE TRANSCARBAMOYLASE H COO – 2 O DIHYDROOROTASE 5 CH 2 6 CH 3 1 – N COO H Carbamoyl aspartic acid (CAA) C 2 C O O Pi Aspartic acid CH 2 HN C H2O N H Dihydroorotic acid (DHOA) NAD + DIHYDROOROTATE DEHYDROGENASE + 4 NADH + H O HN 3 O CO 2 4 6 5 PP i O N OROTIDYLIC ACID DECARBOXYLASE R- 5- P O COO – N R- 5- P UMP OROTATE... detected even in fossils RNAs are far less stable than DNA since the 2′-hydroxyl group of RNA Phosphodiester bonds link the 3 - and 5 -carbons of adjacent monomers Each end of a nucleotide polymer thus is distinct We therefore refer to the 5 - end” or the “3 - end” of polynucleotides, the 5 - end being the one with a free or phosphorylated 5 -hydroxyl Polynucleotides Have Primary Structure The base sequence... dUDP (deoxyuridine diphosphate) H2O UDP ATP 8 RIBONUCLEOTIDE REDUCTASE 11 ADP Pi dUMP UTP ATP N 5, N10 -Methylene H4 folate Glutamine THYMIDYLATE SYNTHASE CTP SYNTHASE 9 H2 folate NH 2 N O 12 O CH 3 HN N R- 5- P - P - P CTP O N dR- 5- P TMP Figure 34–7 The biosynthetic pathway for pyrimidine nucleotides O PRPP 5 HN 2 1 6 CH COO – HN O N H COO – Orotic acid (OA) ch34.qxd 2/13/2003 4:04 PM Page 299 METABOLISM . their in- corporation into nucleic acids with resulting disruption of base-pairing. Oncologists employ 5- fluoro- or 5- iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mer- captopurine, 5- or 6-azauridine,. is D -ribose, and in deoxyribonucleo- sides it is 2-deoxy- D -ribose. The sugar is linked to the heterocyclic base via a ␤-N-glycosidic bond, almost al- ways to N-1 of a pyrimidine or to N-9 of. 5 -nucleotides are nucleosides with a phospho- ryl group on the 3 - or 5 -hydroxyl group of the sugar, respectively. Since most nucleotides are 5 -, the prefix 5 - is usually omitted when naming them.