Skip to main content
SpringerLink
Log in

Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6β-Hydroxylation

  • Protocol
Cytochrome P450 Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 320))

  • 4813 Accesses

Abstract

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6β-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6β-hydroxytestosterone formed enzymatically by incubation of [14C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. 14C-labeled enzymatic products are applied to silica gel thin-layer plates, which are developed sequentially with methylene chloride:acetone (80:20) followed by chloroform, ethyl acetate, and absolute ethanol (80:20:14). Metabolite quantification is performed by autoradiography and liquid scintillation counting. This method is applicable to enzymatic studies for the determination of CYP3A-dependent testosterone 6β- hydroxylation activity in both human and animal liver microsomes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nelson, D. R., Zeldin, D. C., Hoffman, S. M. G., Maltais, L. J., Wain, H. M., and Nebert, D. W. (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14, 1–18.

    Article  CAS  PubMed  Google Scholar 

  2. Gibson, G. G., Plant, N. J., Swales, K. E., Ayrton, A., and El-Sankary, W. (2002) Receptor-dependent transcriptional activation of cytochrome P450 3A genes: induction mechanisms, species differences and interindividual variation in man. Xenobiotica 32, 165–206.

    Article  CAS  PubMed  Google Scholar 

  3. Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T., and Kanakubo, Y. (1987) P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16α-hydroxylase of dehydroepiandrosterone 3-sulfate. J. Biol. Chem. 262, 13,534–13,537.

    CAS  PubMed  Google Scholar 

  4. Pichard, L., Fabre, I., Fabre, G., et al. (1990) Cyclosporin A drug interactions: screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes. Drug Metab. Dispos. 18, 595–606.

    CAS  PubMed  Google Scholar 

  5. Schuetz, E. G., Schuetz, J. D., Strom, S. C., et al. (1993) Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 18, 1254–1262.

    Article  CAS  PubMed  Google Scholar 

  6. Chang, T. K. H., Yu, L., Maurel, P., and Waxman, D. J. (1997) Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res. 57, 1946–1954.

    CAS  PubMed  Google Scholar 

  7. Wrighton, S. A., Ring, B. J., Watkins, P. B., and VandenBranden, M. (1989) Identification of a polymorphically expressed member of the human cytochrome P-450III family. Mol. Pharmacol. 36, 97–105.

    CAS  PubMed  Google Scholar 

  8. Westlind-Johnsson, A., Malmebo, S., Johansson, A., et al. (2003) Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab. Dispos. 31, 755–761.

    Article  CAS  PubMed  Google Scholar 

  9. Koch, I., Weil, R., Wolbold, R., et al. (2002) Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab. Dispos. 30, 1108–1114.

    Article  CAS  PubMed  Google Scholar 

  10. Westlind, A., Malmebo, S., Johansson, I., et al. (2001) Cloning and tissue distribution of a novel human cytochrome P450 of the CYP3A subfamily, CYP3A43. Biochem. Biophys. Res. Commun. 281, 1349–1355.

    Article  CAS  PubMed  Google Scholar 

  11. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 270, 414–423.

    CAS  PubMed  Google Scholar 

  12. Chang, T. K. H., Gonzalez, F. J., and Waxman, D. J. (1994) Evaluation of triacetyloleandomycin, α-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Biochem. Biophys. 311, 437–442.

    Article  CAS  PubMed  Google Scholar 

  13. Newton, D. J., Wang, R. W., and Lu, A. Y. H. (1995) Cytochrome P450 inhibitors: evaluation of specificities in the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metab. Dispos. 23, 154–158.

    CAS  PubMed  Google Scholar 

  14. Watkins, P. B., Wrighton, S. A., Maurel, P., et al. (1985) Identification of an inducible form of cytochrome P-450 in human liver. Proc. Natl. Acad. Sci. USA 82, 6310–6314.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff, T., and Waxman, D. J. (1986) 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.

    CAS  PubMed  Google Scholar 

  16. Gorski, J. C., Hall, S. D., Jones, D. R., VandenBranden, M., and Wrighton, S. A. (1994) Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamily. Biochem. Pharmacol. 47, 1643–1653.

    Article  CAS  PubMed  Google Scholar 

  17. Kronbach, T., Mathys, D., Umeno, M., Gonzalez, F. J., and Meyer, U. A. (1989) Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol. Pharmacol. 36, 89–96.

    CAS  PubMed  Google Scholar 

  18. Gonzalez, F. J., Schmid, B., Umeno, M., et al. (1988) Human P450PCN1: sequence, chromosome localization and direct evidence through cDNA expression that P450PCN1 is nifedipine oxidase. DNA 7, 79–86.

    Article  CAS  PubMed  Google Scholar 

  19. Waxman, D. J., Attisano, C., Guengerich, F. P., and Lapenson, D. P. (1988) Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6β-hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys. 263, 424–436.

    Article  CAS  PubMed  Google Scholar 

  20. Gelboin, H. V., Krausz, K. W., Goldfarb, I., et al. (1995) Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3/4. Biochem. Pharmacol. 50, 1841–1850.

    Article  CAS  PubMed  Google Scholar 

  21. Mei, Q., Tang, C., Assang, C., et al. (1999) Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 in assessment of human drug metabolism. J. Pharmacol. Exp. Ther. 291, 749–759.

    CAS  PubMed  Google Scholar 

  22. Shou, M., Lu, T., Krausz, K. W., et al. (2000) Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism. Eur. J. Pharmacol. 394, 199–209.

    Article  CAS  PubMed  Google Scholar 

  23. Wang, R. W. and Lu, A. Y. H. (1997) Inhibitory anti-peptide antibody against human CYP3A4. Drug Metab. Dispos. 25, 762–767.

    CAS  PubMed  Google Scholar 

  24. Wrighton, S. A., Brian, W. R., Sari, M. A., et al. (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA3 (HLp3). Mol. Pharmacol. 38, 207–213.

    CAS  PubMed  Google Scholar 

  25. Aoyama, T., Yamano, S., Waxman, D. J., et al. (1989) Cytochrome P-450 hPCN3, a novel cytochrome P-450IIIA gene product that is differentially expressed in adult human liver: cDNA and deduced amino acid sequence and distinct specificites of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine. J. Biol. Chem. 264, 10,388–10,395.

    CAS  PubMed  Google Scholar 

  26. Waxman, D. J., Lapenson, D. P., Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Korzekwa, K. (1991) Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s. Arch. Biochem. Biophys. 290, 160–166.

    Article  CAS  PubMed  Google Scholar 

  27. Sanwald, P., Blankson, E. A., Dulery, B. D., Schoun, J., Huebert, N. D., and Dow, J. (1995) Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites. J. Chromatogr. 672, 207–215.

    Article  CAS  Google Scholar 

  28. Wood, A. W., Ryan, D. E., Thomas, P. E., and Levin, W. (1983) Regio-and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes. J. Biol. Chem. 258, 8839–8847.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Walsky, R. L. and Obach, R. S. (2004) Validated assays for human cytochrome P450 activities. Drug Metab. Dispos. 32, 647–660.

    Article  CAS  PubMed  Google Scholar 

  31. Pearce, R., Greenway, D., and Parkinson, A. (1992) Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation. Arch. Biochem. Biophys. 298, 211–225.

    Article  CAS  PubMed  Google Scholar 

  32. Pearce, R. E., McIntyre, C. J., Madan, A., et al. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch. Biochem. Biophys. 331, 145–169.

    Article  CAS  PubMed  Google Scholar 

  33. Brian, W. R., Sari, M. A., Iwasaki, M., Shimada, T., Kaminsky, L. S., and Guengerich, F. P. (1990) Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae. Biochemistry 29, 11,280–11,292.

    Article  CAS  PubMed  Google Scholar 

  34. Buters, J. T. M., Korzekwa, K. R., Kunze, K. L., Omata, Y., Hardwick, J. P., and Gonzalez, F. J. (1994) cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus. Drug Metab. Dispos. 22, 688–692.

    CAS  PubMed  Google Scholar 

  35. Peyronneau, M. A., Renaud, J. P., Truan, G., Urban, P., Pompon, D., and Mansuy, D. (1992) Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5. Eur. J. Biochem. 207, 109–116.

    Article  CAS  PubMed  Google Scholar 

  36. Lee, C. A., Kadwell, S. H., Kost, T. A., and Serabjit-Singh, C. J. (1995) CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. Arch. Biochem. Biophys. 319, 157–167.

    Article  CAS  PubMed  Google Scholar 

  37. Waxman, D. J. (1991) P450-catalyzed steroid hydroxylation: assay and product identification by thin-layer chromatography. Methods Enzymol. 206, 462–476.

    Article  CAS  PubMed  Google Scholar 

  38. Busby W. F. Jr., Ackermann, J. M., and Crespi, C. L. (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab. Dispos. 27, 246–249.

    CAS  PubMed  Google Scholar 

  39. Chauret, N., Gauthier, A., and Nicoll-Griffith, D. A. (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab. Dispos. 26, 1–4.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the National Institutes of Health (grant DK33765) and the Canadian Institutes of Health Research (grant MOP-42385).

Author information

Authors and Affiliations

  1. Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA, USA

    David J. Waxman

  2. Faculty of Pharmaceutical Sciences, University of British Columbia, Vaqncouver, British Columbia, Canada

    Thomas K. H. Chang

Authors
  1. David J. Waxman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas K. H. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. School of Biological Sciences, Queen Mary University of London, London, UK

    Ian R. Phillips

  2. Department of Biochemistry and Molecular Biology, University College London, London, UK

    Elizabeth A. Shephard

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Waxman, D.J., Chang, T.K.H. (2006). Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6β-Hydroxylation. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:133

Download citation

  • DOI: https://doi.org/10.1385/1-59259-998-2:133

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-441-8

  • Online ISBN: 978-1-59259-998-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation