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Bilirubin metabolism

Author Section Editor Namita Roy Chowdhury, PhD Sanjiv Chopra, MD Jayanta Roy Chowdhury, MD, Elizabeth B Rand, MD MRCP Deputy Editor Peter A L Bonis, MD

Last literature review for version 16.1: January 31, 2008 | This topic last updated: November 20, 2006 INTRODUCTION Bilirubin is the potentially toxic catabolic product of heme metabolism. Fortunately, there are elaborate physiologic mechanisms for its detoxification and disposition. Understanding these mechanisms is necessary for interpretation of the clinical significance of high serum bilirubin concentrations. Furthermore, because bilirubin shares its metabolic pathway with various other sparingly water soluble substances that are excreted in bile, understanding bilirubin metabolism also provides insight into the mechanisms of transport, detoxification, and elimination of many other organic anions. An overview of the major aspects of bilirubin formation and disposition will be reviewed here. The settings in which bilirubin disposition is impaired will also be discussed briefly. Clinical aspects of serum bilirubin determination, the evaluation of patients with hyperbilirubinemia, and the classification of causes of jaundice are presented separately. (See "Clinical aspects of serum bilirubin determination" and see "Diagnostic approach to the patient with jaundice or asymptomatic hyperbilirubinemia" and see "Classification and causes of jaundice or asymptomatic hyperbilirubinemia"). FORMATION OF BILIRUBIN Bilirubin is formed by breakdown of heme present in hemoglobin, myoglobin, cytochromes, catalase, peroxidase and tryptophan pyrrolase. Eighty percent of the daily bilirubin production (250 to 400 mg in adults) is derived from hemoglobin [1] ; the remaining 20 percent being contributed by other hemoproteins and a rapidly turningover small pool of free heme. Enhanced bilirubin formation is found in all conditions associated with increased red cell turnover such as intramedullary or intravascular hemolysis (eg, hemolytic, dyserythropoietic, and megaloblastic anemias). Heme consists of a ring of four pyrroles joined by carbon bridges and a central iron atom (ferroprotoporphyrin IX). Bilirubin is generated by sequential catalytic degradation of heme mediated by two groups of enzymes:

Heme oxygenase Biliverdin reductase

Heme oxygenase initiates the opening of the porphyrin ring of heme by catalyzing the oxidation of the alpha-carbon bridge (show figure 1). This leads to formation of the green pigment, biliverdin, which is then reduced by the biliverdin reductase to the orange-yellow pigment

bilirubin IXa. Iron is released in this process, and the oxidized alpha-bridge carbon is eliminated as carbon monoxide (CO). Measurement of intrinsic CO production has been used to quantify bilirubin production [2] . Heme oxygenase is present in the high concentrations in reticuloendothelial cells of the spleen, the principal site of red cell breakdown, and in the Kupffer cells in the liver [3] . It is also induced by heme, eg, in hemolytic states. Heme oxygenase is thought to be rate-limiting in bilirubin production [4] . As an example, pharmacologic inhibition of heme oxygenase by tin-protoporphyrin or tin-mesoporphyrin reduces bilirubin production [5] . Conjugation Bilirubin is very poorly soluble in water at physiologic pH because of internal hydrogen bonding that engages all polar groups and gives the molecule a contorted "ridge-tile" structure [6] . The fully hydrogen-bonded structure of bilirubin is designated bilirubin IXalphaZZ. Water-insoluble unconjugated bilirubin is associated with all known toxic effects of bilirubin. Thus, the internal hydrogen bonding is critical in producing bilirubin toxicity and also prevents its elimination. Conversion of bilirubin-IXa to a water-soluble form, by disruption of the hydrogen bonds, is essential for the elimination by the liver and kidney. This is achieved by glucuronic acid conjugation of the propionic acid side chains of bilirubin. Bilirubin glucuronides are watersoluble and are readily excreted in bile. Bilirubin is primarily excreted in normal human bile as the diglucuronide; unconjugated bilirubin accounts for only 1 to 4 percent of pigments in normal bile. During phototherapy, which is used to reduce serum bilirubin concentrations in babies with severe neonatal hyperbilirubinemia, configurational and structural photoisomers of bilirubin are produced [7] . These isomers lack hydrogen bonding and are excreted into bile without further metabolism. In the bile, the configurational isomers may revert back to the hydrogen-bonded bilirubin-IXa-ZZ. Measurement of serum bilirubin Reaction of bilirubin with diazo reagents causes breakdown of the tetrapyrrole to two azodipyrroles (the van den Bergh reaction) [8] which can be readily measured spectrophotometrically. Diazo assays are most commonly used for quantification of bile pigments for clinical purposes via the following sequence.

Because the central carbon bridge of bilirubin is buried within the molecule by hydrogen bonds, unconjugated bilirubin reacts slowly with the diazo reagent. In comparison, conjugated bilirubin, which lacks hydrogen bonds, reacts rapidly even in the absence of accelerators ("direct" van den Bergh reaction). On disruption of hydrogen bonds by addition of "accelerators" such as methanol or caffeine, the reaction is completed rapidly (total bilirubin).

Unconjugated bilirubin ("indirect" bilirubin) concentration is calculated by subtracting the direct-reacting fraction from total bilirubin.

Direct reacting bilirubin slightly overestimates the conjugated bilirubin concentration because a fraction of unconjugated bilirubin (about 10 to 15 percent) also gives a direct van den Bergh reaction. There are several other potential sources of error. As an example, endogenous substances, such as plasma lipids, and drugs, such as propranolol, interfere with the diazo assay. This can produce unreliable results, but only when the bilirubin concentration is normal or slightly elevated. Albumin-bilirubin complexes (delta-bilirubin) also may give a direct reaction. More accurate and sensitive quantification of bilirubin requires chromatographic analysis, such as high performance liquid chromatography and reflectance fluorimetry [9-11] . DISPOSITION OF BILIRUBIN A number of steps are provided in the disposition of bilirubin (show figure 2). Albumin binding of bilirubin in plasma Binding to albumin and, to a much lesser degree, high density lipoprotein, keeps bilirubin in solution in plasma; only a small fraction of bilirubin circulates in the unbound state. Binding to high density lipoprotein may become significant in states of severe hypoalbuminemia. Albumin binding keeps bilirubin in the vascular space, thereby preventing its deposition into extrahepatic tissues, including sensitive tissues such as the brain, and minimizing glomerular filtration. It also transports bilirubin to the sinusoidal surface of the hepatocyte, where the pigment dissociates from albumin and enters the hepatocyte (see below). Free bilirubin can cause cerebral toxicity when the molar concentration of bilirubin exceeds that of albumin (see "Bilirubin toxicity" below). On the other hand, the plasma bilirubin content increases after albumin infusion because of transfer of the pigment from tissue stores to the intravascular space. Several other ligands bind to albumin at the same site as bilirubin, including sulfonamides, warfarin, antiinflammatory drugs, and cholecystographic contrast media. These agents can displace bilirubin from albumin, thereby precipitating bilirubin encephalopathy in newborns without an alteration in the total serum bilirubin concentration [12] . Many other compounds such as fatty acids bind at a different albumin site but may, in some cases, reduce the binding constant of albumin for bilirubin [13] . Albumin binding of bilirubin is usually reversible. However, irreversible binding can occur in the presence of prolonged conjugated hyperbilirubinemia, eg, during biliary obstruction. The bilirubin fraction irreversibly bound to albumin (delta-bilirubin) is not cleared by the liver or the kidney and, because of the long half-life of albumin, lingers in the plasma [14] . This may result in prolonged hyperbilirubinemia after endoscopic or surgical relief of biliary obstruction. Because delta-bilirubin gives a "direct" diazo reaction, this may give a false impression of a persistent blockage of the bile ducts [15] . The presence of delta-bilirubin can be inferred by the absence of bilirubin excretion in the urine despite the apparent presence of direct

hyperbilirubinemia and can be identified by high performance liquid chromatography of serum (show figure 3). Uptake and storage of bilirubin by hepatocytes In the liver sinusoids, the albumin-bilirubin complex dissociates, and the bilirubin is taken up efficiently by the hepatocyte while the albumin remains in the circulation. Bilirubin is taken up by hepatocytes by a process of facilitated diffusion, which is not energy-consuming; as a result, transport cannot occur against a concentration gradient, and is bidirectional. Defects in the specific transporters that mediate each of the steps in bilirubin transport can lead to hyperbilirubinemia. (See "Inherited disorders associated with conjugated hyperbilirubinemia" and see "Gilbert's syndrome and unconjugated hyperbilirubinemia due to bilirubin overproduction"). Sinusoidal bilirubin uptake requires inorganic anions, such as chloride, and is thought to be mediated by carrier proteins which have not been fully characterized [16,17] . Passage of bilirubin across the hepatocyte sinusoidal surface membrane is bidirectional. Within the hepatocyte, bilirubin and other organic anions bind to glutathione S-transferases (GSTs). GSTbinding reduces the efflux of the internalized bilirubin, thereby increasing net uptake (show figure 2). This process of bilirubin uptake is impaired in certain disease states:

Bilirubin uptake is reduced in some, but not all, patients with Gilbert syndrome (see "Gilbert's syndrome and unconjugated hyperbilirubinemia due to bilirubin overproduction"), and is inhibited by certain drugs, eg, rifampin, flavaspidic acid, and cholecystographic dyes. In patients with cirrhosis, a portion of the bilirubin produced in the spleen may bypass the liver via portosystemic collaterals. Furthermore, the sinusoidal endothelium, which is normally fenestrated, may lose the fenestrae (capillarization), thereby creating a barrier between the plasma and the hepatocytes. As a result, serum unconjugated bilirubin concentrations often increase in this condition.

Conjugation of bilirubin Glucuronidation of bilirubin, a large variety of endogenous compounds (eg, steroid hormones, thyroid hormones, catecholamines), and a wide array of exogenous substrates (eg, drugs, toxins, carcinogens and laboratory xenobiotics) is mediated by a family of enzymes, termed uridine-diphosphoglucuronate glucuronosyltransferase (UGT) (show figure 2) [18] . As noted above, glucuronides are more water soluble, and are readily excreted in bile and urine. Enzyme-catalyzed glucuronidation is one of the most important detoxification mechanisms of the body. Of the various isoforms of the UGT family of enzymes, only one isoform, UGT1A1 is physiologically important in bilirubin glucuronidation (show figure 3) [19] . UGT1A1 is an intrinsic protein of the endoplasmic reticulum (ER) and its catalytic site is located within the ER lumen. Therefore, the sugar donor substrate uridinediphosphoglucuronate (UDPGA) must enter the ER lumenal space for conjugation. UDP-N-acetylglucosamine is the natural stimulator of the

transport of the polar substrate, UDPGA into the ER lumen. It has been suggested that four nucleotide sugar transporter proteins mediate UDP-N-acetylglucosamine stimulation of [20] . Bilirubin diglucuronide is the predominant pigment in normal adult human bile, representing over 80 percent of the pigment. However, in subjects with reduced bilirubin-UGT activity, the proportion of bilirubin diglucuronide decreases, and bilirubin monoglucuronide may constitute more than 30 percent of the conjugates excreted in bile. Reduction of conjugating enzyme activity to approximately 30 percent of normal results in a mild but discernible increase in serum bilirubin concentrations. (See "Gilbert's syndrome and unconjugated hyperbilirubinemia due to bilirubin overproduction"). Inhibitory factor(s) for hepatic UGT1A1 is secreted in the milk of some mothers (maternal milk jaundice). In other cases, an inhibitory factor present in maternal plasma may be transplacentally transferred to the fetus (the Lucey Driscoll syndrome). UGT1A1 deficiency also may be seen in neonates, chronic hepatitis, and in certain inherited disorders (Gilbert's syndrome and CriglerNajjar syndrome I and II). Excretion of conjugated bilirubin Conjugated bilirubin and other substances destined to be excreted in bile are secreted across the bile canalicular membrane of the hepatocyte against a concentration gradient that may reach 1:1000, indicating the presence of active transport [21] . Among the four types of canalicular transporters, the multispecific organic anion transporter (cMOAT) appears to be the most important for the canalicular secretion of bilirubin and many other organic anions, with the exception of bile acids (show figure 2) [22] . Enhanced bile flow (eg, by infusion of bile salts) or phenobarbital treatment increases the maximal bilirubin excretory capacity. On the other hand, the excretion of conjugated bilirubin is impaired in a number of acquired conditions (eg, alcoholic or viral hepatitis, cholestasis of pregnancy) and inherited disorders (eg, Dubin-Johnson syndrome, Rotor syndrome, benign recurrent intrahepatic cholestasis). It can also be caused by a variety of drugs (eg, alkylated steroids, chlorpromazine). (See "Classification and causes of jaundice or asymptomatic hyperbilirubinemia" and see "Gilbert's syndrome and unconjugated hyperbilirubinemia due to bilirubin overproduction"). Degradation of bilirubin in the digestive tract Bile pigment appearing in bile is mostly (more than 98 percent) conjugated. Conjugated bilirubin is water soluble and is not absorbed across the lipid membrane of the small intestinal epithelium; in comparison, the unconjugated bilirubin fraction is partially reabsorbed and undergoes enterohepatic circulation (show figure 4) [23] . This fraction increases during phototherapy because of the excretion of photoisomers of bilirubin. Oral administration of charcoal, agar, or cholestyramine may interfere with the absorption of unconjugated bilirubin, thereby increasing the efficacy of phototherapy. In contrast, excessive amounts of bilirubin are available for reabsorption in neonates with obstruction of the upper intestinal tract, delayed passage of meconium, or fasting; this may increase the intensity and duration of neonatal jaundice. Bilirubin is reduced by bacterial enzymes in the colon to a series of molecules, termed urobilinogens [24] . The two major urobilinoids found in stool, urobilinogen and stercobilinogen,

are colorless and turn orange-yellow only after oxidation to urobilins. In complete biliary obstruction or severe intrahepatic cholestasis (eg, in the early phase of acute viral hepatitis), feces may take the appearance of china clay. Thus, the absence of urobilinogen in stool and urine in a jaundiced patient indicates complete biliary obstruction. Urobilinogens and their derivatives are partly absorbed from the bowel, undergo enterohepatic recycling, and are eventually excreted in urine and feces (show figure 4). In states of enhanced bilirubin production and in liver disease, the amount of urinary urobilinogen increases. However, urinary urobilinogen excretion is an unreliable marker of bilirubin metabolism because of tubular reabsorption and instability of the pigment in acid urine. The intestinal microflora influence serum levels of bilirubin. In a study in Gunn rats (which have a congenital deficiency of bilirubin UDB-glucuronsyltransferase), treatment with oral clindamycin and neomycin resulted in the disappearance of fecal urobilinoids while serum bilirubin increased dramatically (from 10.8 mg/dL {186 micromol/L} to 17 mg/dL {289 micromol/L}) [25] . Intestinal colonization with C. perfringens led to reappearance of fecal urobilinoid production accompanied by a partial decrease in serum bilirubin levels. The authors speculated that prolonged use of certain antibiotics may lead to an increase in serum bilirubin levels in humans. Patients with abnormal bilirubin conjugation may be at particular risk. Alternative pathways of bilirubin elimination Alternative pathways of bilirubin elimination may be important in certain clinical settings. One such pathway is oxidation of bilirubin by mixed function oxidases in liver and other organs. In addition, induction of cytochrome P-450c by chlorpromazine reduced the serum bilirubin concentration in one patient with Crigler-Najjar syndrome type I (enzyme catalyzed oxidation) [26] . In conditions associated with elevated conjugated plasma bilirubin concentrations (eg, intrahepatic or extrahepatic cholestasis), the kidney is responsible for 50 to 90 percent of conjugated bilirubin excretion [27,28] . However, bile remains the main excretion route for unconjugated hyperbilirubinemia. BILIRUBIN IN DISEASE STATES In normal plasma, about 4 percent of bilirubin is conjugated. This relationship may vary in disease states:

In inherited disorders of bilirubin conjugation, the proportion of conjugated bilirubin is reduced. In biliary obstruction or hepatocellular diseases, both conjugated and unconjugated bilirubin accumulate in plasma [14] . In hemolytic jaundice, total plasma bilirubin increases, but the proportion of the unconjugated and conjugated fractions remains unchanged.

Bilirubin binds to the elastic tissue of skin and sclera, and is also found in all tissue fluids with a high albumin content. The usual tight but reversible binding to albumin precludes glomerular filtration of unconjugated bilirubin; similar principles apply to irreversible covalently bound

bilirubin (delta-bilirubin). In contrast, conjugated bilirubin is less strongly bound to albumin and can be excreted in the urine. Thus, the finding of bilirubin in the urine, in the absence of albuminuria, indicates the presence of an increased amount of conjugated bilirubin in the plasma. Potential beneficial effects of bilirubin Although clinicians are generally concerned with serum bilirubin levels as a marker of liver disease and the direct toxicity of bilirubin (see below), within a near-physiological range of serum bilirubin concentrations, the antioxidative action of bilirubin may provide beneficial effects. An inverse relationship between serum bilirubin levels and risk of ischemic coronary artery disease has been reported in middle-aged British men [29] . A population study in Belgium showed an inverse relationship between serum bilirubin levels and cancer mortality [30] . More recently, the study of a very large number of subjects in the US revealed that the odds ratio for the history of colorectal cancer is reduced to 0.295 in men and 0.186 in women per 1 mg/dl increment in serum bilirubin levels [31] . However, such statistical associations do not conclusively prove a causative role of bilirubin. Bilirubin toxicity Unconjugated bilirubin is toxic to many cells and organelles. Physiologic mechanisms that protect against bilirubin toxicity include, as described above, binding to plasma albumin, and rapid uptake, conjugation, and clearance by the liver. Because of these efficient protective mechanisms, the harmful effects of unconjugated bilirubin is limited to neonates with a high degree of unconjugated hyperbilirubinemia and subjects with inherited disorders of bilirubin conjugation. Markedly elevated serum unconjugated bilirubin concentrations (usually over 20 mg/dL) in the newborn may result in clinical evidence of brain damage, ranging from subtle neurologic abnormalities to severe encephalopathy or permanent neurologic damage (kernicterus) [32] . The serum concentration at which clinical signs of neurotoxicity occur is variable. (See "Clinical manifestations of unconjugated hyperbilirubinemia in term and near-term infants" section on Kernicterus).

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