title: An isocratic high-performance liquid chromatography assay for CYP7A1-catalyzed cholesterol 7alpha-hydroxylation.
abstract: A normal-phase, isocratic high-performance liquid chromatography assay is described for cholesterol 7alpha-hydroxylation catalyzed by CYP7A1, which corresponds to the first and rate-limiting step in the conversion of cholesterol into bile acids. This method is based on the conversion of the primary cytochrome P450 metabolite, 7alpha-hydroxycholesterol, into 7alpha-hydroxy-4-cholesten-3-one in a reaction catalyzed by exogenous cholesterol oxidase, followed by chromatographic separation with monitoring at 254 nm. This technique is applicable to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by cDNA-expressed CYP7A1 and animal or human liver microsomes.
authors.affiliation: Department of Biology, Boston University, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: Thomas K H
authors.initials: TK
authors.lastname: Chang
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006900:Hydroxylation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase