Bilirubin is an important metabolite of heme (ferroprotoporphyrin IX), a coordination complex that serves to coordinate iron in various proteins. It is a potentially toxic substance. However, the body has developed mechanisms for its safe detoxification and disposition. Bilirubin and its metabolites also provide the distinctive yellow color to bile and stool and a lesser degree, urine. This article will summarize the mechanism of heme metabolism and bilirubin synthesis.
Formation of Bilirubin
Bilirubin is derived from two main sources, and about 4 mg/kg body weight of bilirubin is produced daily. Roughly, 80% of bilirubin is made from the breakdown of hemoglobin in senescent red blood cells and prematurely destroyed erythroid cells in the bone marrow. The remainder originates from the turnover of various heme-containing proteins found in other tissues, primarily the liver and muscles. These proteins include myoglobin, cytochromes, catalase, peroxidase, and tryptophan pyrrolase.
Cellular Heme Metabolism
Heme is a ring of four pyrroles joined by carbon bridges and a central iron atom. Bilirubin is generated by a by a two-stage sequential catalytic degradation reaction that primarily takes place in the cells of the reticuloendothelial system, notably the spleen. Other cells include phagocytes and the Kupffer cells of the liver. Heme is taken up into These cells take up the heme, and enzyme heme oxygenase acts on them. The enzyme liberates the chelated iron by catalyzing the oxidation of the alpha carbon bridge. This reaction produces an equimolar amount of carbon monoxide which is excreted by the lungs and leads to the formation of the green pigment, biliverdin. This green pigment is acted upon further by the nicotinamide adenine dinucleotide phosphate (NADPH) dependent enzyme, biliverdin reductase. This process releases an orange-yellow pigment known as bilirubin. Heme oxygenase as mentioned above is present in high concentrations in the Kupffer cells of the liver and the cells of the reticuloendothelial system. Heme oxygenase is the rate-limiting factor in bilirubin production.
The final structure is highly compacted by hydrogen bonding rendering the molecule essentially insoluble in aqueous solutions at neutral pH. The fully bonded structure of bilirubin is designated as bilirubin IX-alpha-ZZ. Bilirubin, being insoluble in aqueous solution, is carried in circulation bound to albumin which is a reversible and covalent type of bonding.
Metabolism of Bilirubin
Conjugation is mandatory to render bilirubin aqueous soluble and facilitate its secretion across the canalicular membrane and excretion into bile. Bilirubin is conjugated within the hepatocyte to glucuronic acid by a family of enzymes, termed uridine-diphosphoglucuronic glucuronosyltransferase (UDPGT). The process of glucuronidation is one of the many crucial detoxification mechanisms of the human body. Many different isoforms of UDPGT exist, but the physiologically important isoform in bilirubin glucuronidation is UDPGT1A1. The enzyme esterifies two glucuronide moieties to the propionic acid side chains of bilirubin. Under normal conditions, bilirubin diglucuronide is the predominant molecule synthesized. However, if the conjugation system is overwhelmed under conditions of excessive bilirubin synthesis, the majority of bilirubin may be conjugated as bilirubin monoglucuronide. The ratio of mono-conjugated to the dis-conjugated pigment in bile is 1:4. Conjugation of bilirubin to the water-soluble form involves the disruption of the hydrogen bonds, an essential process for its elimination by the liver and kidney. This is achieved by glucuronic acid conjugation of the propionic acid side chains of bilirubin.
The process of conjugation alters the physiochemical properties of bilirubin giving it many special properties. Most importantly, it makes the molecule water soluble which allows it to be transported in bile without a protein carrier. Conjugation also increases the size of the molecule. Conjugation prevents bilirubin from passively being reabsorbed by the intestinal mucosa due to its hydrophilicity and large molecular size. Thus, conjugation works to promote the elimination of potentially toxic metabolic waste products. Furthermore, conjugation modestly decreases the affinity of bilirubin for albumin.
Measurement of Serum Bilirubin
Serum bilirubin is measured spectrophotometrically when the molecule undergoes a reaction with diazo reagents causing the breakdown of the tetrapyrrole to two azodipyrroles. This reaction is termed as the “Van den Bergh.” Unconjugated bilirubin reacts slowly with the diazo reagent as the central carbon bridge of bilirubin is buried within the hydrogen bonds. In contrast, conjugated bilirubin lacks these hydrogen bonds, and the reaction occurs rapidly even in the absence of accelerators. Addition of accelerators such as caffeine or methanol disrupts the hydrogen bonds, and the reaction is quickly completed yielding the value of total bilirubin. Unconjugated bilirubin is measured by subtracting the direct-reacting fraction from total bilirubin. Potential sources of error include plasma lipids, drugs such as propranolol and several other endogenous substances. These interfere with the diazo assay and can potentially produce an unreliable result.
As unconjugated bilirubin is always bound to albumin in serum, it cannot be filtered by the glomeruli (in the absence of glomerular disease). Thus, unconjugated bilirubin is never found in urine even when there is an elevated level of unconjugated bilirubin in circulation. Jaundice that occurs with unconjugated hyperbilirubinemia is termed acholuric because the urine is not darkened. Dark urine, however, occurs when there is excretion of an excess of water-soluble, conjugated bilirubin. This is seen in conjugated hyperbilirubinemia and signifies the presence of either liver or biliary disease. Thus the presence of bilirubin in urine will help identify subtle hepatobiliary dysfunction leading to conjugated hyperbilirubinemia, even when the measured concentration of conjugated bilirubin is serum is only slightly elevated. An exception to this rule is when bilirubinuria is not detected in a patient with prolonged cholestasis and marked jaundice. This is due to the formation of delta bilirubin or conjugated bilirubin that is tightly bound to serum albumin. The absence of bilirubinuria in such patients should not cause any difficulty in diagnosing conjugated hyperbilirubinemia, as the patient is clearly jaundiced and serum conjugated bilirubin is markedly elevated in such cases.
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