色谱法
细胞色素P450
化学
高效液相色谱法
CYP3A4型
药物代谢
基质(水族馆)
质谱法
微粒体
血红素
酶
生物化学
生物
生态学
作者
Christal D. Sohl,Qian Cheng,F. Peter Guengerich
出处
期刊:Nature Protocols
[Springer Nature]
日期:2009-08-06
卷期号:4 (9): 1252-1257
被引量:34
标识
DOI:10.1038/nprot.2009.122
摘要
Cytochrome P450 enzymes (P450s) are heme-thiolate mono-oxygenases involved in the oxidation of many endogenous and exogenous substrates. Herein, we describe two protocols for measuring the activity of a key enzyme of drug metabolism, P450 3A4. In this protocol, the substrate is incubated with human liver microsomes, the reaction is quenched, and the substrates and products are extracted and subjected to liquid chromatography (LC) separation and detection. Oxidation of the calcium-channel blocker nifedipine is measured using UV–Vis spectroscopy in-line with high performance liquid chromatography (HPLC). 6β-Hydroxytestosterone formation from testosterone is measured by HPLC coupled to mass spectrometry (MS). Both of these procedures are rapid, requiring 2 h or less, and can be used to confirm and measure P450 3A4 activity and can also be used as a guide for developing other assays for measuring P450 catalysis. The separation strategy described here is more rapid than many available methods, except when ultra-performance liquid chromatography (UPLC) is used.
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