Schroepfer, George J., Jr.2009-06-032009-06-031989Needleman, Dolores Heidi. "Studies of the effect of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one on microsomal acyl coenzyme A:cholesterol acyltransferase activity." (1989) Diss., Rice University. <a href="https://hdl.handle.net/1911/16377">https://hdl.handle.net/1911/16377</a>.https://hdl.handle.net/1911/163773$\beta$-Hydroxy-5$\alpha$-cholest-8(14)-en-15-one (15-ketosterol) is a hypocholesterolemic agent which affects cholesterol metabolism at several levels, including sterol biosynthesis and cholesterol absorption. This dissertation describes studies on the effect of 15-ketosterol on intestinal acyl CoA:cholesterol acyltransferase (ACAT) activity, an enzyme involved in the esterification and absorption of dietary cholesterol. Addition of 15-ketosterol to jejunal microsomes decreased the level of cholesterol esterified by ACAT. Incubation of 15-ketosterol with rat jejunal microsomes reduced the (1 -$\sp{14}$C) oleoyl CoA-dependent esterification of microsomal cholesterol (50% inhibition: 3.0 $\mu$M). This reduction in cholesterol esterification was accompanied by the formation of ($\sp{14}$C) labeled material which, upon analysis by thin layer chromatography, comigrated with 15-ketosteryl oleate. The esterification of 15-ketosterol was confirmed by incubating (2,4- 3H) 15-ketosterol with unlabeled oleoyl CoA and jejunal microsomes. Analyses using either normal phase thin layer chromatography or reverse phase high pressure liquid chromatography detected the formation of (3H) material comigrating with 15-ketosteryl oleate. Oral administration of 15-ketosterol to rats lowered ACAT activity relative to pair-fed controls. ACAT activity in rat jejunal microsomes was lowered 82% (P $<$ 0.02) and 77% (p $<$ 0.001) in animals fed a chow diet containing 0.05% and 0.10% 15-ketosterol, respectively. Analysis of the cholesterol and cholesterol ester content of rat jejunal microsomes showed that oral administration of either 0.10% or 0.125% 15-ketosterol did not affect the concentrations of these compounds. Additional studies showed that reduction of rat jejunal ACAT activity was dependent upon both the duration of oral administration of 15-ketosterol and the concentration of the compound in the diet. Reduction of ACAT activity was observed as early as 3 hours after ingestion of the first meal containing 0.10% 15-ketosterol. Significant reductions in microsomal ACAT activity were observed at concentrations as low as 0.05% 15-ketosterol in the diet. Little or no incorporation of (1-$\sp{14}$C) oleoyl CoA into material comigrating with 15-ketosteryl oleate was observed. In additional studies, incubation of 10 $\mu$M (25R)-3$\beta$,26-dihydroxy-5$\alpha$-cholest-8(14)-en-15-one, a metabolite of 15-ketosterol, with rat jejunal microsomes resulted in a 55% reduction in the oleoyl CoA-dependent esterification of microsomal cholesterol.260 p.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.BiochemistryThesisThesis Biochem. 1990 Needleman