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    Home > Biochemistry News > Biotechnology News > An Isocratic High-Performance Liquid Chromatographic Assay for CYP7Al-Catalyzed Cholesterol 7-Hyciroxylation

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

    • Last Update: 2021-03-02
    • Source: Internet
    • Author: User
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    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.
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