The objectives of this application are to use biophysical and pathophysiologic rationale and physical- chemical, biochemical and molecular biologic techniques to advance fundamental understanding of bile and its two major dysfunctions, namely cholestasis and gallstones of both pigment and cholesterol types, i) The first aim will determine whether enterohepatic infection with Heficobacter rodentium and hepaticus, both common human and murine enterobiliary pathogens, is the final common trigger in cholesterol gallstone pathogenesis via nucleation of cholesterol supersaturated bile. Studies will be carried out on genetically gallstone-susceptible mice coupled with in vitro analysis of cholesterol nucleation from model and native biles as well as quantifying evidence of Heficobacter infection in human biliary tissues, ii) Aim two will quantify the activity of the pathway responsible for trimethylation of phosphatidylethanolamine to phosphatidylcholine on the canalicular membrane in the lithogenic state, which may be coupled with biliary cholesterol hypersecretion. Elevation of plasma and bile homocysteine, a by-product of this reaction, may serve as a lithogenic marker and shed light on vicinal and distal organ dysfunction, iii) The third aim will investigate, and unambiguously prove, enterohepatic cycling of bilirubin and 'black'pigment gallstones in the setting of severe bile salt malabsorption in the SlclOa2 null mouse and determine its prevention by non- absorbed hydrogels containing covalently linked cholic acid, a strong bilirubin binder, or 13-glucaro,l-4 lactone, a potent inhibitor of bacterial 6-glucuronidase. iv) Aim four will work out, both experimentally and by molecular dynamic simulations, the physical states of conjugated and unconjugated bilirubins in native biles and define the thermodynamic conditions required for """"""""black"""""""" pigment gallstone formation in human biles, v) The fifth aim will explore cholestasis and pigment """"""""sludge"""""""" and gallstone formation associated with total parenteral nutrition, focusing on the lack of CCK and secretin from enteral starvation. The hormonal deficits cause enterohepatic cycling of unconjugated bilirubin which engenders hyperbilirubinbilia and also results in less alkaline hepatic bile, where endogenous 13-glucuronidase hydrolysis of bilirubin glucuronides and calcium bilirubinate precipitation occurs. The results of these hypothesis-driven basic research aims will provide insights that correlate native and model systems and should be readily translatable to prevent and treat these common heDatobiliarv diseases in humans.
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