This research program concerns selected aspects of the biochemistry of sphingolipid bases and their derivatives and of sterols and other mevalonate-derived products. Of special interest are investigations of interrelationships between regulation of the biosynthesis of sphingolipids and their derivatives and the biosynthesis of cholesterol and other mevalonate-derived products. The results of several studies by others suggest regulation of sphingolipid metabolism by factors of procedures known to affect sterol biosynthesis. Proposed are studies of the effects of two types of potent regulators of sterol biosynthesis, i.e. oxygenated sterols and competitive inhibitors of HMB-CoA reductase, on the synthesis and selected aspects of the metabolism of sphingolipid bases. Also proposes are studies of the effects of sphingolipid bases and their derivatives on sterol biosynthesis and cholesterol esterification and on the levels of HMG-CoA reductase activity in cultured mammalian cells and in in vitro studies with rat liver preparations. These studies are stimulated by our recent demonstration that D-erythro-sphingosine is highly active in lowering HMG-CoA reductase activity in CHO-K1 cells. New and improved methods (involving radioisotopic derivatization and gas chromatography-mass spectrometry) are proposed for determination of the levels of sphingolipid bases and their 1-phosphate derivatives in plasma (and in LDL derived therefrom) and in cells and tissues. Also proposed are studies of the uptake and metabolism of sphingolipid bases and their derivatives by cultured mammalian cells, subjects critical to the interpretation of a large number of studies indicating important changes in cells after their incubation in media containing less compounds. Special concentration will be directed toward two major enzyme activities involved in the control of the levels of 1-phosphate derivatives of the sphingolipid bases, i.e. sphinganine kinase and sphingenine-1-phosphate lyase. Pursuit of these analytical and metabolic studies will involve the chemical syntheses and chromatographic and spectral characterization of a significant number of sphingolipid bases and their derivatives. The proposed studies of the regulation of the biochemistry and cell biology of sterols and sphingolipids will be markedly facilitated by the availability of a recently isolated, temperature-sensitive mutant of CHO- K1 cells in which serine-palmitoyltransferase is affected. Use of this mutant cell line has already permitted the direct demonstration of the requirement of sphingolipids for cellular growth and provides a unique system for studies of the interactions of sphingolipids and sterols in cellular metabolism and function. The potential importance of this work derives from the importance of the concerned compounds in the regulation of the functioning and growth of eukaryotic cells and the generally recognized importance of cholesterol and sphingolipids (especially sphingomyelin) in atherosclerotic lesions in coronary artery disease. Moreover, the results of these studies should provide beneficial for a large number of investigations concerned with the functions of sphingolipids in human cells.
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