Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • Log out
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • Log out
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Development of a Non-High Pressure Liquid Chromatography Assay to Determine Testosterone Hydroxylase (CYP3A) Activity in Human Liver Microsomes

Alison J. Draper, Ajay Madan, Kevin Smith and Andrew Parkinson
Drug Metabolism and Disposition April 1998, 26 (4) 299-304;
Alison J. Draper
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ajay Madan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kevin Smith
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew Parkinson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Abstract

The major pathway of testosterone oxidation by human liver microsomes is the formation of 6β-hydroxytestosterone, which is catalyzed by CYP3A4/5 and which accounts for 75–80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-3H]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in no extraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6,7-3H]testosterone paralleled rates of testosterone 6β-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent Km andVmax values). The sample-to-sample variation in tritium release from [1,2,6,7-3H]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6β-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-3H]testosterone, and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-3H]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-3H]testosterone.

Footnotes

  • Send reprint requests to: Dr. Andrew Parkinson, Ph.D., Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417.

  • This work was supported by Grant ES03765 from the National Institutes of Health. A.J.D. was supported by NIH Training Grant ES07079.

  • Abbreviations used are::
    CYP
    cytochrome P450
    P450
    cytochrome P450
    4MA
    17β-N,N-dimethylcarbamoyl-4-methyl-4-aza-5α-androstan-3-one
    testosterone
    17β-hydroxy-4-androsten-3-one
    HPLC
    high pressure liquid chromatography
    • Received June 3, 1997.
    • Accepted November 26, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
View Full Text

 

DMD articles become freely available 12 months after publication, and remain freely available for 5 years. 

Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page. 

 

  • Click here for information on institutional subscriptions.
  • Click here for information on individual ASPET membership.

 

Log in using your username and password

Forgot your user name or password?

Purchase access

You may purchase access to this article. This will require you to create an account if you don't already have one.
PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition
Vol. 26, Issue 4
1 Apr 1998
  • Table of Contents
  • Index by author
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Development of a Non-High Pressure Liquid Chromatography Assay to Determine Testosterone Hydroxylase (CYP3A) Activity in Human Liver Microsomes
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

Development of a Non-High Pressure Liquid Chromatography Assay to Determine Testosterone Hydroxylase (CYP3A) Activity in Human Liver Microsomes

Alison J. Draper, Ajay Madan, Kevin Smith and Andrew Parkinson
Drug Metabolism and Disposition April 1, 1998, 26 (4) 299-304;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

Development of a Non-High Pressure Liquid Chromatography Assay to Determine Testosterone Hydroxylase (CYP3A) Activity in Human Liver Microsomes

Alison J. Draper, Ajay Madan, Kevin Smith and Andrew Parkinson
Drug Metabolism and Disposition April 1, 1998, 26 (4) 299-304;
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results and Discussion
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • TMDD Affects PK of IL-10 Fc-fusion Proteins
  • Uptake as the RDS in Pevonedistat Hepatic Clearance
  • In vitro downregulation of OATP1B1 by retinoids
Show more Article

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2022 by the American Society for Pharmacology and Experimental Therapeutics