A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines

AC Collier, MD Tingle, Jeffrey Keelan, JW Paxton, MD 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 mean V-max of 19.9 nmol/min/mg of protein and a mean K-m of 652.5 mu M on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V-max was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K-m, 175.4 +/- 24.2 mu M (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% for V-max and 8 and 35% for K-m, 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 mu M 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.
    Original languageEnglish
    Pages (from-to)1184-1186
    JournalDrug Metabolism and Disposition
    Volume28
    Publication statusPublished - 2000

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