An Isocratic High-Performance Liquid Chromatography Assay for CYP7A1-Catalyzed Cholesterol 7α-Hydroxylation

A normal-phase, isocratic high-performance liquid chromatography assay is described for cholesterol 7α-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, 7α-hydroxycholesterol, into 7α-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 7α- hydroxylation activity catalyzed by cDNA-expressed CYP7A1 and animal or human liver microsomes.