title: Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate.
abstract: The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.
authors.affiliation: Department of Medicine, Medical College of Virginia-VCU, Richmond 23298.
authors.forename: P B
authors.initials: PB
authors.lastname: Hylemon
authors.forename: E J
authors.initials: EJ
authors.lastname: Studer
authors.forename: W M
authors.initials: WM
authors.lastname: Pandak
authors.forename: D M
authors.initials: DM
authors.lastname: Heuman
authors.forename: Z R
authors.initials: ZR
authors.lastname: Vlahcevic
authors.forename: J Y
authors.initials: JY
authors.lastname: Chiang
mesh_terms: D000818:Animals; D002250:Carbon Radioisotopes; D002784:Cholesterol / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D002784:Cholesterol; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase