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Research ArticleArticle

A Highly Sensitive Fluorescent Microplate Method for the Determination of UDP-Glucuronosyl Transferase Activity in Tissues and Placental Cell Lines

Abby C. Collier, Malcolm D. Tingle, Jeffrey A. Keelan, James W. Paxton and Murray D. Mitchell
Drug Metabolism and Disposition October 2000, 28 (10) 1184-1186;
Abby C. Collier
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Malcolm D. Tingle
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Jeffrey A. Keelan
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James W. Paxton
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Murray D. Mitchell
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Abstract

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a meanVmax of 19.9 nmol/min/mg of protein and a mean Km of 652.5 μM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the meanVmax was 36.21 ± 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P < .0001) higher than for the extraction method. The meanKm, 175.4 ± 24.2 μM (CV = 14.5%), was significantly lower (P < .0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% forVmax and 8 and 35% forKm, respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2.6 nmol/min/mg of protein, respectively, at 200 μM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.

Footnotes

  • Send reprint requests to: Abby Collier, C/- Department of Pharmacology and Clinical Pharmacology, University of Auckland Medical School, 85 Park Rd., Grafton, Auckland, New Zealand. E-mail:a.collier{at}auckland.ac.nz

  • This work was generously supported by the Maurice and Phyllis Paykel Trust and Uniservices (Auckland) LTD.

  • Abbreviations used are::
    UDPGT
    5′-uridine diphosphate glucuronosyl transferase
    UDPGA
    uridine 5′-diphosphate glucuronic acid
    4MU
    4-methylumbelliferone
    CV
    coefficient of variation
    BSA
    bovine serum albumin
    sacchrolactone
    d-saccharic 1,4-lactone
    Vmax
    maximum reaction velocities
    Km
    Michaelis-Menten constant
    • Received April 27, 2000.
    • Accepted July 5, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 28 (10)
Drug Metabolism and Disposition
Vol. 28, Issue 10
1 Oct 2000
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Research ArticleArticle

A Highly Sensitive Fluorescent Microplate Method for the Determination of UDP-Glucuronosyl Transferase Activity in Tissues and Placental Cell Lines

Abby C. Collier, Malcolm D. Tingle, Jeffrey A. Keelan, James W. Paxton and Murray D. Mitchell
Drug Metabolism and Disposition October 1, 2000, 28 (10) 1184-1186;

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Research ArticleArticle

A Highly Sensitive Fluorescent Microplate Method for the Determination of UDP-Glucuronosyl Transferase Activity in Tissues and Placental Cell Lines

Abby C. Collier, Malcolm D. Tingle, Jeffrey A. Keelan, James W. Paxton and Murray D. Mitchell
Drug Metabolism and Disposition October 1, 2000, 28 (10) 1184-1186;
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