The long-term goals of this project are to determine the mechanisms of biliary cholesterol and phosphatidylcholine (PC) secretion in man. As a step in this direction the aims of the current project are to identify the major pathway(s) by which cholesterol and PC are transported from plasma and/or hepatocyte into bile. These goals are germain to the pathogenesis of atherosclerosis and cholesterol cholelithiasis. Pilot studies in subjects without HDL showed a decrease in 1) the normally high rate of free cholesterol (FC) transport between liver and plasma and 2) biliary cholesterol secretion. Additional experiments will be conducted in subjects who are totally deficient in HDL, VLDL and LDL (abetalipoproteinemia), and ApoB receptors. By studying a variety of molecular defects it may be possible to pin-point the mechanisms of HDL-hepatic FC exchange and biliary cholesterol secretion. Methodology includes simultaneous administration of 14C mevalonic acid and 3H-FC- HDL (or LDL); blood and bile are collected for 1-3 days; cholesterol and bile acid mass and radioactivity data are analyzed by compartmental analysis (SAAM-27 computer program). The in vivo effects of cholesterol enriched chylomicrons and of heparin- releasable hepatic lipase on hepatic FC transport will also be examined. The origin of biliary PC in man will be determined by compartmental analysis of data from IV administration of 3H- choline and HDL (or LDL) labeled with palmityl-14C linoleic-PC (PLPC). To critically determine if 14C-PLPC found in bile is from recycled 14C-linoleic acid or from C-PLPC transported intact from plasma, 3H linoleic acid and 14C-PLPC-HDL will be administered. Compartmental analysis of mass and radioactivity in bile and blood of linoleic acid, triglyceride and PLPC will be carried out. Small pigs will be administered PLPC labelled with 3H and 14C in combinations of the glycerol, choline, linoleate or palmitate moieties (one with 3H, one with 14C); the ratio of 3H to 14C in bile PLPC will be examined for intact plasma-liver-bile transport. Isotopic precursors of PC will be administered to rats and for 2 hr the appearance of labeled PC will be quantitated in microsomes, bile, canalicular membrane (cLPM) cytosolic surface and cLPM canalicular surface. In vitro experiments will examine potential synthesis file on by several pathways, lipid asymmetry in cLPM vesicles, and transport of PC and FC to/from cLPM vesicles and various acceptors. Elucidation of bile PC secretion is crucial for understanding cholesterol secretion and is therefore a major priority.
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