The overall research plan applies physical-chemical rationale as well as biochemical and biophysical techniques to define the pathophysiology of biologically important alimentary tract lipids and lipid-protein systems. As phase behavior, fine structures and properties of model systems are elucidated, this information is correlated with actual pathophysiological phenomena. The overall goals aim at understanding how the delicate physical-chemical balance of lipids and lipid-protein systems in multicomponent lipid-rich tissues is perturbed hepatobiliary diseases so that strategies for their prevention and treatment can be developed. In studies of normal and abnormal bile formation, fluorescence spectroscopy will be used to define putative roles of hepatic phosphatidylcholine transfer protein in combination with sterol carrier protein-2 and submicellar bile salts in promoting cytosolic transport of biliary lecithins. 13C nuclear magnetic resonance spectroscopy will be applied to detection of canalicular membrane translocators (""""""""flippases"""""""") and defining their roles in lecithin and cholesterol secretion into bile. With hepatocyte couplets, the physical-chemical state and phase transitions of canalicular bile will be defined using the complementary approaches of microscope quasielastic light scattering spectroscopy and electron spectroscopic imaging. The origin and fate of plasma lipoprotein X, an abnormal lipoprotein causing hypercholesterolemia in cholestasis, will be studied. The hypothesis that lipoprotein X is a misdirected """"""""nascent"""""""" biliary vesicle will be explored by showing that biliary lipid """"""""flippases"""""""" and anion transporters are misplaced in basolateral membranes during cholestasis but are correctly inserted into canalicular membranes following ursodeoxycholic acid therapy. The physical-chemical state of bile and gallstone formation will be studied by combining physical- chemical, ultrastructural, and pathophysiological approaches with particular reference to critical nuclei and vesicle fusion in mucin gel. Cholesterol nucleation, crystallization and growth from bile will be investigated employing model bile systems as well as biles from humans and inbred mice where the lithogenic genes are being defined. Altered hydrophilic-hydrophobic balance of biliary proteins as a secondary response to lithogenic bile will be investigated by hydrophobic interaction chromatography and the effects of purified human as well as prairie-dog biliary proteins on the nucleation maps from model systems will be tested. Phase equilibria and micellar properties of the muricholate epimers, biomedically-relevant hydrophilic bile salts resistant to bacterial catabolism, will be investigated to establish molecular mechanisms required for inhibiting crystalline phase transitions in human bile. Elucidation of unconjugated bilirubin absorption from the colon and its enterohepatic cycling in ileal dysfunction induced by chemical and dietary means will be investigated using the intact rat; the pathophysiologic paradigm developed should serve as a model for investigating the etiology of non-hemolytic pigment gallstone formation in humans.
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