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

DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY

Annalise Di Marco, Michelle D'Antoni, Silvia Attaccalite, Pietro Carotenuto and Ralph Laufer
Drug Metabolism and Disposition June 2005, 33 (6) 812-819; DOI: https://doi.org/10.1124/dmd.105.004333
Annalise Di Marco
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michelle D'Antoni
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Silvia Attaccalite
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Pietro Carotenuto
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ralph Laufer
  • 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

A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDP-glucuronic acid, and 14C-labeled glucuronidation products are separated from the unreacted nucleotide sugar by solid-phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic, and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics were studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone; UGT1A6 and UGT1A8 for 1-naphthol; UGT2B7 for naloxone; UGT1A3 and UGT2B7 for ketoprofen; and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies.

Footnotes

  • This work was supported in part by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.105.004333.

  • ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; SPE, solid-phase extraction; HLM, human liver microsomes; RLM, rat liver microsomes; DLM, dog liver microsomes; MS, mass spectometry; MS/MS, tandem mass spectometry; 1NP, 1-naphthol; 4MU, 4-methylumbelliferone; HFC, 7-hydroxy-4-trifluoromethylcoumarin; LC, liquid chromatography.

  • ↵1 Current address: Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

  • ↵2 Current address: CEINGE Biotecnologie Avanzate, Napoli, Italy.

    • Received February 17, 2005.
    • Accepted March 18, 2005.
  • 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: 33 (6)
Drug Metabolism and Disposition
Vol. 33, Issue 6
1 Jun 2005
  • 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.
DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY
(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

DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY

Annalise Di Marco, Michelle D'Antoni, Silvia Attaccalite, Pietro Carotenuto and Ralph Laufer
Drug Metabolism and Disposition June 1, 2005, 33 (6) 812-819; DOI: https://doi.org/10.1124/dmd.105.004333

Citation Manager Formats

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

Share
Research ArticleArticle

DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY

Annalise Di Marco, Michelle D'Antoni, Silvia Attaccalite, Pietro Carotenuto and Ralph Laufer
Drug Metabolism and Disposition June 1, 2005, 33 (6) 812-819; DOI: https://doi.org/10.1124/dmd.105.004333
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

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

Related Articles

Cited By...

More in this TOC Section

  • CYP3A-mediated oxidation of DABE and BIBR0951
  • Adipocyte PXR does not play an essential role in obesity.
  • Biodistribution of Lipid in Rats
Show more Articles

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