The formation of bilirubin diglucuronide by rat liver microsomal preparations

Abstract

Bilirubin transformation in vitro to bilirubin conjugates in the presence of activated rat liver microsomal preparations and UDP-glucuronate was assessed with a method involving isolation of the products as tetrapyrroles. The proportions of bilirubin monoglucuronide and diglucuronide formed by the microsomal bilirubin UDP-glucuronosyltransferase (EC 2.4.1.17) were governed by the concentration of bilirubin present and the nature of the activation of the microsomal membrane. Activation of the microsomal preparations with the nonionic detergents Triton X-100 or Emulgen 911, or with digitonin for 24 h, produced bilirubin monoglucuronide as the only product at all concentrations of bilirubin investigated. In contrast, bilirubin diglucuronide was the only conjugate formed when hepatic microsomal preparations were activated with digitonin for periods of less than 2 h, and the concentration of bilirubin was 20 .mu.M. Increasing the concentration of bilirubin utilized in this assay system changed the relative amounts of bilirubin monoglucuronide and diglucuronide formed. As the level of bilirubin was increased from 20-166 .mu.M, the proportion of bilirubin diglucuronide decreased and that of bilirubin monoglucuronide increased, until at levels of 108 and 166 .mu.M bilirubin only bilirubin monoglucuronide was formed. No evidence was found with liver plasma membranes that transglucuronidation plays a major role in the formation of bilirubin diglucuronide from bilirubin monoglucuronide.