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

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 gluc uronide. A microplate assay has been developed that has improved sensitivit y and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extrac tion method. Absence of extraction and cleanup procedures and the ability t o observe reaction rate directly are also of great advantage to the researc her. Michaelis-Menten kinetic data from one healthy female human liver is p resented. 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 vari ation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, t he mean V-max was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), signifi cantly (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 obs erved in the extraction method. The assay was performed in replicates of si x 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 microplat e 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 nmo l/min/mg of protein, respectively, at 200 mu M 4MU concentration), indicati ng that placental cells may be capable of glucuronidating 4MU.