Study of bilirubin metabolism by high-performance liquid chromatography: stability of bilirubin glucuronides

The stabilities of bilirubin (BR) glucuronide, monoglucuronide (BMG), and diglucuronide (BDG) were studied under various conditions by HPLC. In aqueous media, BMG showed a pronounced lability and was easily transformed into equimolar BDG and BR. It was proved by direct analysis of tetrapyrrole isomers that BDG and BR were formed from dipyrrole exchange of BMG molecules. All reducing agents examined (sodium ascorbate, cysteine, GSH, dithiothreitol, NADH, and NADPH) suppressed the transformation of BMG into BDG and BR. Bovine serum albumin and rat liver cytosol fractions also stabilized BMG strongly. BDG was fairly stable in aqueous media as compared with BMG. When BMG was incubated both with and without liver plasma membranes (N2 fraction) from Wistar rats, the formation rates of BDG and BR in both incubation mixtures were exactly the same. The composition of BDG and BR isomers was the same in both mixtures. Also, heat denaturation of the plasma membranes did not affect formation rates. Moreover, the reaction was completely inhibited by sodium ascorbate. These findings indicate that rat liver plasma membranes have no enzyme activity for BDG formation from BMG.