The purpose of this research project is to study the metabolism of cholesterol and bile acids in a group of newly recognized human diseases, i.e., inborn errors of cholesterol and bile acid synthesis. We will investigate the defect in bile acid synthesis in the inherited lipid storage disease, cerebrotendinous xanthomatosis, with the aim of elucidating the enzymatic abnormality and intermediates in the bile acid synthetic path-way. Two new approaches will be tried: (1) the use of a newly developed assay that includes prior removal of all endogenous substrate sterols and will not only overcome the problem of substrate dilution but also eliminate any confounding inhibitory effect of the endogenous sterol pool on key enzymes in the bile acid synthetic pathway, (2) the administration of 24-nor-5beta- cholestane-3alpha, 7alpha,25-triol (a competitive inhibitor of side-chain oxidation) to rats to provide, for the first time, a useful animal model to reproduce the specific enzymatic defect in CTX. In addition, we wish to study the metabolic defect in sitosterolemia with xanthomatosis, a condition in which plant sterols accumulate because of enhanced absorption and reduced removal. Recently, we have discovered reduced cholesterol synthesis in this disease and postulate that the enhanced plant sterol absorption and retention in tissues compensate for decreased cholesterol formation. We will investigate the abnormalities of key enzymes in the cholesterol, 5alpha-stanol and bile acid synthetic pathways. These include: HMG-CoA reductase, cholesterol 7alpha-hydroxylase 7,hydroxy-4-cholesten-3-one 12alpha-hydroxylase, 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26- hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 24S- hydroxylase and the transformation of cholesterol to cholestanol via 3beta-hydroxy-delta4-5-steroid dehydrogenase-isomerase. An important objective of this project will be to isolate in pure form the cytochrome P-450 monooxygenases associated with the 7alpha,12alpha,25- and 26-hydroxylations and study their short-term regulation and biochemical properties. Another objective will be to study in vivo the feedback regulation of bile acid synthesis, specifically the effect of bile acid, sterol and stanol structure on bile acid formation. These experiments will be conducted initially in animal models and the most promising leads will be followed up in humans. Not only will these studies provide important information about the nature of the cholesterol and bile acid synthetic defect in the diseased state but will be useful in elucidating the major pathways and sites of control of cholesterol and bile synthesis in humans.
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