Vol. 28, Issue 10, 1184-1186, October 2000

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

Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand

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 µM 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 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% 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 µM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.