Mechanisms of Hepatic Bilirubin Metabolism

Gordon, Ellen

Abstract

To facilitate secretion, bilirubin is converted to polar compounds by glucuronidation in the hepatic mictosomal uridine diphosphate glucuronyl transferase system (UDPG). Yet, the metabolic steps forming bilirubin diglucuronide, the major polar compound in bile, have not yet been resolved. Two important aspects of hepatic bilirubin metabolism will be examined: 1) the mechanism of glucuronidation of bilirubin in the liver (Section A), and 2) the characterization and metabolism of an unique bilirubin species covalently bound to albumin, which has recently been detected in the serum of subjects with hepatobiliary disease (Section B). Our investigations of the glucuronidation of bilirubin will focus on defining the enzyme systems involved. We will determine in reconstituted systems: a) if more than one microsomal uridine diphosphate glucuronyl transferase is involved in the formation of bilirubin diglucuronide by examining the specificity of the various purified forms of UDPG transferases for bilirubin and bilirubin monoglucuronide. b) the role of phospholipids and c) the specificity of the uridine diphosphate glycoside for the transferases and the metabolic source of bilirubin (hemoglobin or hepatic heme proteins) in the formation of the conjugates of bilirubin. In another set of experiments(d) the role of transglucuronidation in the formation of bilirubin diglucuronide will also be studied by determining if a newly developed highly purified canalicular membrane fraction is capable of forming bilirubin diglucuronide from bilirubin monoglucuronidate enzymatically. In the second section (part B) of this grant, it is proposed to investigate an unique species of bilirubin covalently linked to albumin found in the serum of subjects with hepatobiliary disease. In the first set of experiments, the chemical identity of the compound will be determined, utilizing colulmn and high performance liquid chromatography enzymic and chemical treatments, gel isoelectric focusing and electrophoresis and NMR. Both in vivo and in vitro studies are described to deremine the metabolic site of formation of the bilirubin species, as well as determining its tissue distribution, half-life amd rate of secretion. We will also determine if other organic anions which normally are glucuronidated to facilitate secretion also form complex with albumin during cholestasis.