An Isocratic High-Performance Liquid Chromatographic Assay for CYP7Al-Catalyzed Cholesterol 7-Hyciroxylation

CYP7A1, which is the only CYP7A subfamily P450 tdentified to date (1 ), catalyzes cholesterol 7α-hydroxylation, the first, and rate-limiting, step in the converston of cholesterol to bile acids. CYP7Al has been Isolated from human liver and purified to apparent homogeneity (2 ). Rodent-model studies have established that CYP7A1 is highly regulated by physiological factors that influence hepatic-bile-acid biosynthesis, including cholesterol feedmg, dmrnal factors, and bile acids, whtch feedback-inhibit the overall btosynthetlc pathway in large part at the level of CYP7A1 gene expression (3 ). Relatrvely little is known about the regulation of human CYP7A1, although liver biopsy analyses have shown that hepatic CYP7A1 protem content is elevated in patients treated with the bile-acid sequesterant cholestyramme (4 ). Purified CYP7A1 is catalytically active in cholesterol 7α-hydroxylation (2 ) and immunoinhibition experiments with antihuman CYP7A1 antibodies have shown that CYP7A1 accounts for most of the cholesterol 7a-hydroxylase acttvtty in human liver microsomes (4 ). Thus, mlcrosomal cholesterol 7α-hydroxylase activity can be used as a marker for human liver CYP7A1. Several analytical methods have been developed to quantify hepatrc mrcrosomal cholesterol 7α-hydroxylation, including reversed-phase high-performance hquid chromatography (RPHPLC) (5 ), isotope dilution-mass spectrometry (6 ) and thin-layer chromatography (TLC) (7 ). This chapter describes a normal-phase, isocratic HPLC assay for the determination of cholesterol 7α-hydroxylase acttvity based on the conversion by cholesterol oxidase of the primary P450 metabohte 7α-hydroxycholesterol to 7α-hydroxy-4-cholesten-3-one, which can be detected at 254 nm.