Bilirubin diglucuronide formation by rat liver microsomes: demonstration by affinity and thin layer chromatography of bile pigment tetrapyrroles

The conjugates formed in vitro by bilirubin UDP-glucuronyl transferase were studied by examining reaction products as intact tetrapyrroles, rather than as dipyrrolic azoderivatives. Bile pigments were extracted from conventional microsomal enzyme reaction mixtures by affinity chromatography over albumin-agarose, eluted with 50% ethanol, and separated by a silica gel thin layer chromatographic system. In the presence of UDPGA, native and activated microsomal preparations all formed both bilirubin mono- and diglucuronides from unconjugated bilirubin, and bilirubin diglucuronide from bilirubin monoglucuronide. No significant non-enzymatic conversion of mono- to diglucuronide occurred without UDPGA, or in the presence of denatured enzyme. Hence, bilirubin diglucuronide is a major product of bilirubin-UDP-glucuronyl transferase.