Bilirubin formation

The 1 mL reaction mixture contained heme oxygenase, 5 mM deferroxa-mine, 25 piM hemin, 15 fiM bovine serum albumin, 1 mM NADPH, 0.1 M potassium phosphate (pH 7.4), and 0.05 mL of 105,000g supernate from 20% (w/v) perfused rat liver homogenate as a source of biliverdin reductase. To stop the reaction, 0.1 mL of the reaction mixture was added to 0.1 mL of ethanol-DMSO (95 5, v/v). After 2 minutes incubation on ice, the mixture was centrifuged and 0.1 mL of the supernate was injected on HPLC. The rate of bilirubin formation was linear up to 0.63 mg protein/0.237 mL assay, and up to 9 minutes incubation at 37°C. 

The use of high doses of naturally occurring vitamin K (Kj and K2) appears to have no untoward effect however, menadione (K3) treatment can lead to the formation of erythrocyte cytoplasmic inclusions known as Heinz bodies and hemolytic anemia. With severe hemolysis, increased bilirubin formation and undeveloped capacity for its conjugation may produce kernicterus in the newborn. 

Studies of bilirubin formation with tracers, using bile fistula animals, or isolated whole liver, have revealed that there are several sources of bilirubin (Table 1). One is a very early labeled bilirubin (ELB) with a half-life of a few hours derived from the conversion by the liver of exogenous tracer ALA via heme to bilirubin. The labeled bilirubin appeared [Israels et al., 94] 15 minutes after injection of tracer ALA into the rat the bilirubin increased to a maximum in 60 minutes. Both the liver and the kidney converted ALA to bilirubin, but the liver was the main contributor. When protein synthesis of the liver was blocked by cycloheximide to prevent new hemoprotein synthesis, ELB still continued to be formed. This experiment suggests that heme does not have to be attached to a hemoprotein other than heme oxygenase to be rapidly oxidized. Thus, the earliest fraction of labeled bilirubin may be... 

Hemoglobin Breakdown and Bilirubin Formation 385 Bilirubin Transport 385 Bilirubin Excretion 387 Urobilinogens 388 Jaundice 389 Neonatal Jaundice... 

Type I Crigler-Najjar syndrome is a rare autosomal recessive disorder. It is characterized by severe congenital jaundice (serum bilirubin usually exceeds 20 mg/dL) due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease is often fatal within the first 15 months of life. Children with this condition have been treated with phototherapy, resulting in some reduction in plasma bilirubin levels. Phenobarbital has no effect on the formation of bilirubin glucuronides in patients with type I Crigler-Najjar syndrome. A liver transplant may be curative. 

Biliary sludge A deposit of tiny stones or crystals made up of cholesterol, calcium bilirubinate, and other calcium salts. The cholesterol and calcium bilirubinate crystals in biliary sludge can lead to gallstone formation. 

G10. Gydell, K., Transient effect of nicotinic acid on bilirubin metabolism and formation of carbon monoxide. Acta Med. Scand. 167, 1 (1960). 

It should be noted that charged micelles create medium polarity, which is a very important factor for the ion-radical formation. The reaction of bilirubin with peroxy radicals is a prominent example. In biological systems, bilirnbin acts as an antioxidant. It has also been claimed that it is a... 

Glucuronide synthesis is the rate-determining step in hepatic bilirubin metabolism. Drugs such as phenobarbital, for example, can induce both conjugate formation and the transport process. 

Phase II reactions (conjugate formation). Type II reactions couple their substrates (bilirubin, steroid hormones, drugs, and products of phase I reactions) via ester or amide bonds to highly polar negatively charged molecules. The enzymes involved are transferases, and their products are known as conjugates. 

Studies of UDP-glucuronyltransferase with bilirubin as the acceptor substrate are technically difficult. This is indicated by frequent modification of the initial assay systems (A8, G9, L4, S4) and by the wide range of reported enzyme activities (Table 1). Possible causes of these discrepancies, which are manyfold, will be discussed in some detail below. The conclusions drawn should be helpful in the design of assays of conjugate formation of bilirubin and of the synthesis of mono- and diconjugates. 

Most frequently, binding protein is added to the incubation mixtures as either serum or purified serum albumin. With human serum albumin, at equilibrium, the acceptor substrate will largely be protein-bound, when the bilirubin albumin molecular ratio is smaller than one (the dissociation constant of the first binding site of purified human serum albumin is approximately 7 X 10 M with 2 X 10 M for two additional binding sites) (J2). The first binding site of albumin, measured with rat serum, has a dissociation constant of about 5 X 10" M (M8). The unbound fraction will normally not exceed the very low solubility of the pigment. Addition of albumin to an alkaline solution of bilirubin, or its addition immediately after neutralization, prevents colloid formation, if the bilirubin albumin molecular ratio is smaller than one (B25). However, colloidal bilirubin, once formed, cannot be redissolved by the addition of albumin (B26). 

At a bilirubin albumin molecular ratio below one the added binding protein will thus act as a kind of buffer, keeping the concentration of unbound substrate sufficiently low to inhibit colloid formation (B25) or precipitation onto bound bilirubin (B26), and will prevent aspecific binding to cell particulates. The binding protein can also be thought of as a reservoir providing a continuous stream of molecularly dispersed sub-... 

Some authors have used carrier-free enzymatic incubation mixtures at pH 8.0-8.3 (J5, P3, W12). In general, the final concentrations used (incubation at 37°C) were 5-10-fold higher than the solubility of bilirubin at 25°C (B25). Although solubility data at 37°C are not available, it is likely that in most instances the solubility was exceeded. It is not known whether, and to what extent, bilirubin is solubilized in an aspecific way, e.g., by dissolution in lipid membrane regions. Formation of colloidal bilirubin is possible (B25). Aging of the initial, supersaturated (B25) bilirubinate solution is expected to depend (B26) on the procedure of initial solubilization, the time elapsed between lowering the alkaline... 

With fresh rat liver homogenates that were not fortified with UDP-sugar, appreciable conjugate formation of bilirubin occurred (HIO, M5). The process occurred in the absence of added Mg-+ and was inhibited by digitonin (HIO). The conjugation rates at 37°C were constant for the first 3- to 5-minute period, then decreased gradually to zero (M5). 

A8. Arias, I. M., and London, I. M., Bilirubin glucuronide formation in vitro demonstration of a defect in Gilbert s disease. Science 126, 563-564 (1957). 

Brodersen, R., and Theilgaard, J., Bilirubin colloid formation in neutral aqueous solution. Scand. J. Clin. Invest. 24, 396-398 (1969). 

H4. Halac, E., DiPlazza, M., and Detwiler, P., The formation of bilirubin mono- and diglucuronide by rat liver microsomal fractions. Biockim. Biophys. Acta 279, 544-553 (1972). 

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