Hepatic glucuronidation is essential for the biliary excretion of bilirubin-IXAlpha, and numerous other hydrophobic endogenous compounds, xenobiotics and toxins. In human, monkey and rat liver, the synthesis of bilirubin mono- and diglucuronides (C8 and C12 BMG isomers and BDG) is catalyzed by microsomal bilirubin UDP-glucuronyltransferase (BGT). Bilirubin and its polar glucuronides exhibit a spectrum of physicochemical properties, and as such they represent a unique series of physiologic probes to investigate the UDP-glucuronyltransferase system and the pathways of organic anion transport in the hepatocyte. Specifically, we propose to characterize the catalytic properties of BGT, and to determine the manner in which the enzyme is regulated by its membrane environment and the cosubstrate UDP-glucuronic acid. Monkey (M, fascicularis) and rat liver microsomes will be used in these studies, with a new BGT radioassay and sensitive methods for glucuronide analysis. The findings should not only clarify the mechanisms of bilirubin glucuronide formation and the factors regulating BGT activity, but also provide a basis for understanding the patterns of BMG isomers and BDG excreted in different species and the pathogenesis of various forms of jaundice (e.g. Crigler-Najjar disease, Gilbert's syndrome and neonatal hyperbiliribinemia). This information also will have implications with regard to the behavior of other membrane-bound enzymes and the hepatic biotransformation of toxic lipophilic compounds. Bulirubin incorporated into the lipid phase of liposomes will be used as a model to investigate whether lipophilic substrates undergo membrane-membrane transfer and lateral membrane diffusion to reach the sites of glucuronidation. The intracellular pathways by which bilirubin glucuronides and other polar organic anions are transported to the canalicular membrane for biliary excretion, and the role of a microtubule-dependent bile salt/lipid vesicle system will be explored in intact rats and isolated perfused liver. Finally, we will optimize conditions for the selective targeting of unilamellar liposomes to hepatocytes, and thereby provide a basis for an attempt to correct the genetic deficiency of BGT activity in Gunn rats by infusing liposomes containing purified BGT. These experiments may provide a model for the therapeutic application of this approach in patients with life-threatening Crigler Najjar disease.
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