RT Journal Article SR Electronic T1 A Highly Sensitive Fluorescent Microplate Method for the Determination of UDP-Glucuronosyl Transferase Activity in Tissues and Placental Cell Lines JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP 1184 OP 1186 VO 28 IS 10 A1 Abby C. Collier A1 Malcolm D. Tingle A1 Jeffrey A. Keelan A1 James W. Paxton A1 Murray D. Mitchell YR 2000 UL http://dmd.aspetjournals.org/content/28/10/1184.abstract AB 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. The American Society for Pharmacology and Experimental Therapeutics