Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Chromatographic assays of drug oxidation by human cytochrome P450 3A4

Nature Protocols volume 4, pages 1252–1257 (2009)Cite this article

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3: UV–HPLC chromatogram of nifedipine oxidation by human liver microsomes, monitoring at A254.
Figure 4: UV–HPLC chromatogram of testosterone oxidation by human liver microsomes, monitoring at A244.
Figure 5: HPLC–MS chromatogram of testosterone oxidation by human liver microsomes.

Similar content being viewed by others

References

  1. Guengerich, F.P. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol. 14, 611–650 (2001).

    Article  CAS  Google Scholar 

  2. Guengerich, F.P. Cytochrome P450s, drugs, and diseases. Mol. Interventions 3, 8–18 (2003).

    Article  Google Scholar 

  3. Wienkers, L.C. & Heath, T.G. Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov. 4, 825–833 (2005).

    Article  CAS  Google Scholar 

  4. Obach, R.S., Zhang, Q.Y., Dunbar, D. & Kaminsky, L.S. Metabolic characterization of the major human small intestinal cytochrome p450s. Drug Metab. Dispos. 29, 347–352 (2001).

    CAS  PubMed  Google Scholar 

  5. Lamba, J.K., Lin, Y.S., Schuetz, E.G. & Thummel, K.E. Genetic contribution to variable human CYP3A-mediated metabolism. Adv. Drug Deliv. Rev. 54, 1271–1294 (2002).

    Article  CAS  Google Scholar 

  6. Lin, Y.S. et al. Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol. Pharmacol. 62, 162–172 (2002).

    Article  CAS  Google Scholar 

  7. Yano, J.K. et al. The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A resolution. J. Biol. Chem. 279, 38091–38094 (2004).

    Article  CAS  Google Scholar 

  8. Ekroos, M. & Sjogren, T. Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc. Natl. Acad. Sci. USA 103, 13682–13687 (2006).

    Article  CAS  Google Scholar 

  9. Williams, P.A. et al. Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305, 683–686 (2004).

    Article  CAS  Google Scholar 

  10. Shou, M. et al. Activation of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site. Biochemistry 33, 6450–6455 (1994).

    Article  CAS  Google Scholar 

  11. Ueng, Y.-F., Kuwabara, T., Chun, Y.-J. & Guengerich, F.P. Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36, 370–381 (1997).

    Article  CAS  Google Scholar 

  12. Korzekwa, K.R. et al. Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. Biochemistry 37, 4137–4147 (1998).

    Article  CAS  Google Scholar 

  13. Harlow, G.R. & Halpert, J.R. Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. Proc. Natl. Acad. Sci. USA 95, 6636–6641 (1998).

    Article  CAS  Google Scholar 

  14. Chen, Y., Monshouwer, M. & Fitch, W.L. Analytical tools and approaches for metabolite identification in early drug discovery. Pharm. Res. 24, 248–257 (2007).

    Article  CAS  Google Scholar 

  15. Liesener, A. & Karst, U. Monitoring enzymatic conversions by mass spectrometry: a critical review. Anal. Bioanal. Chem. 382, 1451–1464 (2005).

    Article  CAS  Google Scholar 

  16. Guengerich, F.P. et al. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem. 261, 5051–5060 (1986).

    CAS  PubMed  Google Scholar 

  17. Guengerich, F.P. & Bartleson, C.J. in Principles and Methods of Toxicology 5th edn. (ed. Hayes, A.W.) 1981–2048 (CRC Press, Boca Raton, FL, 2007).

    Google Scholar 

  18. Krauser, J.A. et al. 1β-Hydroxylation of testosterone by human cytochrome P450 3A4. Eur. J. Biochem. 271, 3962–3969 (2004).

    Article  CAS  Google Scholar 

  19. Krauser, J.A. & Guengerich, F.P. Cytochrome P450 3A4-catalyzed testosterone 6β-hydroxylation: stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps. J. Biol. Chem. 280, 19496–19506 (2005).

    Article  CAS  Google Scholar 

  20. Wang, D.D. & Zhang, M.L. Rapid quantitation of testosterone hydroxyl metabolites by ultra-performance liquid chromatography and mass spectrometry. J. Chromatogr. B 855, 290–294 (2007).

    Article  CAS  Google Scholar 

  21. Arlotto, M.P., Trant, J.M. & Estabrook, R.W. Measurement of steroid hydroxylation reactions by high-performance liquid-chromatography as indicator of P450 identity and function. Methods Enzymol. 206, 454–462 (1991).

    Article  CAS  Google Scholar 

  22. Shaw, P.M., Hosea, N.A., Thompson, D.V., Lenius, J.M. & Guengerich, F.P. Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b 5 . Arch. Biochem. Biophys. 348, 107–115 (1997).

    Article  CAS  Google Scholar 

  23. Wang, P.P. et al. Purification and characterization of six cytochrome P-450 isozymes from human liver microsomes. Biochemistry 22, 5375–5383 (1983).

    Article  CAS  Google Scholar 

  24. Guengerich, F.P., Martin, M.V., Sohl, C.D. & Cheng, Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat. Protoc., 4, 1245–1251.

  25. Miyazaki, M. et al. Defective activity of recombinant cytochromes P450 3A4.2 and 3A4.16 in oxidation of midazolam, nifedipine, and testosterone. Drug Metab. Dispos. 36, 2287–2291 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  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

Authors
  1. Christal D Sohl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qian Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F Peter Guengerich
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to F Peter Guengerich.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sohl, C., Cheng, Q. & Guengerich, F. Chromatographic assays of drug oxidation by human cytochrome P450 3A4. Nat Protoc 4, 1252–1257 (2009). https://doi.org/10.1038/nprot.2009.122

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.122

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing