Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC.
Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic 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, liberated after hydrolyzing the GL with beta-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague-Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150 x 4.6 mm, 5 micron). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r2 > 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 days. 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 nmole for TESG, 8HOQG and 4MUG. respectively. The above methods were used to estimate kinetic parameters such as Vmax and Km 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 primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.
Narayanan
R
R
Division of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA 02115, USA.
LeDuc
B
B
Williams
D A
DA
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020719:Glucuronides / Q000032:analysis / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D012739:Gonadal Steroid Hormones; D006923:Hymecromone; D007202:Indicators and Reagents; D007413:Intestinal Mucosa / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D015125:Oxyquinoline; D020033:Protein Isoforms; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013739:Testosterone
D020719:Glucuronides; D012739:Gonadal Steroid Hormones; D007202:Indicators and Reagents; D020033:Protein Isoforms; D006923:Hymecromone; D013739:Testosterone; D015125:Oxyquinoline; D014453:Glucuronosyltransferase