Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis.

Hepatic biotransformation of bilirubin to the hydrophilic species bilirubin mono- (BMG) and diglucuronide (BDG) by microsomal bilirubin UDP-glucuronosyl-transferase (GT) is a prerequisite for its physiologic excretion into bile. The reaction mechanism of bilirubin-GT and the access of bilirubin and BMG (the intermediate substrate) to the active site of bilirubin-GT are undefined. Highly purified [14C]bilirubin and [3H] BMG were coincubated with rat liver microsomes, and the initial rates of radiolabeled bilirubin glucuronide synthesis were measured. Although these substrates differ markedly in their hydrophilicity, no significant differences were observed in [14C]- and [3H]BDG rates of formation from equimolar [14C]bilirubin and [3H] BMG, in the absence or presence of soluble binding proteins (albumin and hepatic cytosol). In further kinetic studies, [14C]bilirubin and [3H]BMG exhibited mutually competitive inhibition of [3H]- and [14C]BDG synthesis, respectively, and [3H]BMG also inhibited [14C]BMG formation. Finally, unlabeled BMG and BDG inhibited the glucuronidation of [14C]bilirubin, with all three pigments yielding virtual Michaelis-Menten dissociation constants in the 10-20 microM range. These findings indicate that: 1) bilirubin-GT follows Michaelis-Menten kinetics for both bilirubin and BMG glucuronidation over the range of substrate concentrations employed; 2) the findings are consistent with a single active site for the enzymatic synthesis of both BMG and BDG; 3) bilirubin, BMG, and BDG bind competitively to this active site with comparable affinities; and 4) access of both bilirubin and BMG substrates to the enzymatic active site is reduced by soluble binding proteins.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

The formation and distribution of bilirubin monoglucuronide and diglucuronide in rat liver slices.

1. Bilirubin conjugation in rat liver slices was reassessed by using analysis of ethyl anthranilate azopigments to estimate separately the formation of bilirubin mono- and di-glucuronides. 2. Conjugation in slices resembles the situation in vivo more closely than does microsomal conjugation, in that diglucuronide is formed in appreciable quantity. 3. Both bilirubin mono- and di-glucuronides were present in slices in approximately equal amounts, but the monoglucuronide was the major product found in the incubation medium. 4. These results are discussed in relation to recent theories on the relationship between bilirubin mono- and di-glucuronide formation in vivo.     

Carbon monoxide excretion as an index of bilirubin production in rhesus monkeys

The role of increased heme catabolism in neonatal hyperbilirubinemia was investigated in rhesus (Macaca mulatta) neonates through the measurement of carbon monoxide excretion rates (VECO), blood carboxyhemoglobin content (HbCO), and plasma bilirubin concentrations. Neonatal values were compared to those of adult rhesus monkeys. These indices of bilirubin production responded appropriately to administration of NEM-damaged erythrocytes and tin protoporphyrin. Our results indicate that VECO measurements are a valid index of changes in bilirubin production in the newborn rhesus monkey.