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Chromatographic assays of drug oxidation by human cytochrome P450 3A4

Nature Protocols volume 4, pages 12521257 (2009) | Download Citation



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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Cytochrome P450 and NADPH–P450 reductase research in this laboratory is supported by the United States Public Health Service grant R37 CA090426. We thank Dr. M. Wade Calcutt for assistance with MS.

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  1. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

    • Christal D Sohl
    • , Qian Cheng
    •  & F Peter Guengerich


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C.D.S. optimized the assays. C.D.S. wrote most of the paper, with the assistance of F.P.G. F.P.G. and Q.C. checked the protocols, and Q.C. helped with some of the mass spectrometry.

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Correspondence to F Peter Guengerich.

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