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
  • 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
  • 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

Variables in Human Liver Microsome Preparation: Impact on the Kinetics of L-α-Acetylmethadol (LAAM)N-Demethylation and DextromethorphanO-Demethylation

Ann C. Nelson, Wei Huang and David E. Moody
Drug Metabolism and Disposition March 2001, 29 (3) 319-325;
Ann C. Nelson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wei Huang
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David E. Moody
  • 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

We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochromeb5 reductase, cytochromeb5, absorbance at 420,p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage ofp-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphanO-demethylation to dextrorphan and theN-demethylation of l-α-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.

Footnotes

  • Send reprint requests to: David E. Moody, Ph.D., University of Utah, Center for Human Toxicology, 20 S. 2030 E. RM 490, Salt Lake City, UT 84112-9457. E-mail: dmoody{at}alanine.pharm.utah.edu

  • ↵1 Current Address: Secor International, Inc., Salt Lake City, UT 84107.

  • This research was supported by U.S. Public Health Service Grant R01 DA10100. A preliminary report of these results was presented at the Experimental Biology 1999 meeting in Washington, D.C.

  • Abbreviations used are::
    HLM
    human liver microsomes
    P450
    cytochrome P450
    LAAM
    l-α-acetylmethadol
    HPLC
    high-performance liquid chromatography
    • Received November 9, 2000.
    • Accepted December 5, 2000.
  • 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: 29 (3)
Drug Metabolism and Disposition
Vol. 29, Issue 3
1 Mar 2001
  • Table of Contents
  • About the Cover
  • 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.
Variables in Human Liver Microsome Preparation: Impact on the Kinetics of L-α-Acetylmethadol (LAAM)N-Demethylation and DextromethorphanO-Demethylation
(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

Variables in Human Liver Microsome Preparation: Impact on the Kinetics of L-α-Acetylmethadol (LAAM)N-Demethylation and DextromethorphanO-Demethylation

Ann C. Nelson, Wei Huang and David E. Moody
Drug Metabolism and Disposition March 1, 2001, 29 (3) 319-325;

Citation Manager Formats

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

Share
Research ArticleArticle

Variables in Human Liver Microsome Preparation: Impact on the Kinetics of L-α-Acetylmethadol (LAAM)N-Demethylation and DextromethorphanO-Demethylation

Ann C. Nelson, Wei Huang and David E. Moody
Drug Metabolism and Disposition March 1, 2001, 29 (3) 319-325;
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

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

Related Articles

Cited By...

More in this TOC Section

  • Ontogeny of CPPGL
  • Expression of AKR and SDR Isoforms in the Human Intestine
  • Metabolism of Lufotrelvir in Humans
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 © 2023 by the American Society for Pharmacology and Experimental Therapeutics