Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bo und proteins which catalyze the transfer of glucuronic acid from UDP-glucur onic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberat ed after hydrolyzing the GL with P-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site su bstrates. Therefore the purpose of this study was to develop simple and sen sitive HPLC methods for the direct and simultaneous analysis of the GL(s) a nd their aglycones without the drawbacks of the conventional methods. The t hree classical substrates we chose were 4-methylumbelliferone (4MU), testos terone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isofoms 1A6, 2B 1 and 2B12 of the rat family, respectively. Here we report the validated HP LC conditions, for the detection and separation of 4-methylumbeliferone glu curonide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glu curonide (8HOQG) and their aglycones in incubation media containing male Sp rague-Dawley rat liver and intestinal microsomal preparations. The separati ons were achieved on a Zorbax SB-CN column (150 x 4.6 mm, 5 mu). The analys is time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r (2) > 0.99) over the concentration ranges tested (0.25-5.0 nmoles of TESG; 0.125-18.75 nmoles of 8HOQG and 0.125-12.5 nmoles of 4MUG), good precision and accuracy (RSD < 2.5%). Inter-day variability studies (n = 3) showed no significant difference between the regression lines obtained on the three d ays. Recoveries were good (> 90%) at all three points (low, mid-point, high ) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nm ole for TESG, 8HOQG and 4MUG, respectively. The above methods were used to estimate kinetic parameters such as V-max and K-m for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primaril y in the liver while UGTs 1A6 and 2B12 were present in comparable amounts i n both tissues. (C) 2000 Elsevier Science B.V. All rights reserved.