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
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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
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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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