The long term objectives are to identify the metabolic pathways of bile acid synthesis particularly as they exist in humans and to characterize the interrelationships that exist between cholesterol and bile acid metabolism. We will focus on two metabolic pathways that relate to: (1) the pathogenesis of cholestatic syndromes particularly during the newborn period and (2) the regulation of cholesterol and bile acid synthesis. To investigate further the metabolic defect that has been found in a child with a familial form of cheolestasis, we have now prepared the naturally occurring bile acid intermediates before and after the metabolic block in 7Alpha-hydroxylation. A combination of 3Beta-hydroxy-5-cholenoic acid(14C) and 3Beta,7Alpha-dihydroxy(7Beta 3H)-5-cholenoic acid will be given intravenously and their metabolites in urine identified using HPLC, GLC-MS and reverse isotope dilution. These substrates will also be used for the isolation and purification of the putative microsomal cytochrome P-450 7Alpha-hydroxylase. Depending on the success of the purification procedure, an antibody will be prepared which can then be used to obtain cDNA clones for this enzyme. The hypothesis that an extrinsic pathway of bile acid synthesis exists, beginning with the production of hydroxycholesterol(s) in extrahepatic tissues that regulate cholesterol synthesis and ending with metabolism to predominantly chenodeoxycholic acid will be explored using both in vivo and in vitro studies. A new tritium labeled cholesterol(3H27) has been synthesized and with reconstitution methods incorporated into LDL and other lipoproteins as the fatty acid ester. Intravenous administration of these cholesterol labeled lipoproteins to hamsters that are functionally hepatectomized will permit the quantitation of extrahepatic cholesterol metabolism to hydroxycholesterol(s). In vitro, rates of hydroxycholesterol synthesis will be determined in normal human fibroblasts and those from individuals with cerebrotendinous xanthomatosis. In another series of studies we will analyze the changes in the hydroxycholesterol and bile alcohol content of the liver at short intervals during the induction of enhanced bile acid synthesis by feeding cholestyramine. For this purpose we will utilize a newly developed method which quantitates these compounds as anthracene-9-carboxylic acid esters at the picogram level using HPLC and a fluorescent detector.
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