beta-Glucuronidase-resistant bilirubin glucuronide isomers in cholestatic liver disease--determination of bilirubin metabolites in serum by means of high-pressure liquid chromatography.

"Direct reacting bilirubin" in serum of patients with cholestatic liver disease and in serum of bile duct-ligated rats consists of a complex mixture of bilirubin metabolites. These metabolites were studied by means of high-pressure liquid chromatography. Bilirubin glucuronides in normal bile are beta-glycosidic 1-O-acyl conjugates which are completely hydrolyzed on incubation with beta-glucuronidase. Cholestatic serum contains glucuronide and non-glucuronide bilirubin metabolites. The glucuronides were only partially hydrolyzable with beta-glucuronidase. Compernolle et al. [11] showed that the 1-O-acyl bond of bilirubin glucuronides is labile and prone to migrate from the C1 position at the glucuronosyl residue to positions C2, C3 and C4. The isomerisation products are non-beta-glycosidic, beta-glucuronidase-resistant conjugates. The main beta-glucuronidase-resistant conjugates in cholestatic serum were characterized as: non-beta-glycosidic bilirubin monoglucuronide, non-beta-glycosidic diglucuronide and a diglucuronide isomer with beta-glycosidic and non-beta-glycosidic glucuronosyl groups. Moreover, a substantial amount of bilirubin monoglucoside monoglucuronide was detected in cholestatic human serum.