An isocratic high-performance liquid chromatography assay for CYP7A1-catalyzed cholesterol 7alpha-hydroxylation.
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.
Waxman
David J
DJ
Department of Biology, Boston University, MA, USA.
Chang
Thomas K H
TK
D002784:Cholesterol / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006900:Hydroxylation; D013056:Spectrophotometry, Ultraviolet
D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase