On-line drug metabolism in capillary electrophoresis. 1. Glucuronidation using rat liver microsomes.
A rat liver microsome pseudostationary phase has been used for the on-line capillary electrophoresis monitoring of glucuronidation. Uridine diphosphate glucuronosyltransferase (EC 2.4.1.17) containing microsomes was isolated from rat liver and directly injected onto neutrally coated capillary containing polymeric replaceable gels followed by injection of the substrate mixture. On-line glucuronidation was observed within 15 min without any sample preparation. The factors affecting the separation of glucuronides and parent compounds were investigated by varying the applied electric fields and the size (length and internal diameter) of capillary. The Michaelis-Menten parameters (Km and Vmax) for the glucuronidation of 4-methyl-7-hydroxy coumarin and 4-nitrophenol were determined using the CE method and by off-line microsomal incubation. No significant differences were observed for Km and Vmax values for 4-methyl-7-hydroxycoumarin and 4-nitrophenol between on-line and off-line glucuronidation of these two compounds. This method was also used to determine the inhibition constant (IC50 value) for the competitive inhibition of morphine glucuronidation by codeine, IC50 (on-line) = 170 vs 580 microM (off-line). The results demonstrate that this method can be used to screen for the glucuronidation of test compounds and should reduce the time required for this screening process.
Kim
Hee Seung
HS
Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Wainer
Irving W
IW
D000818:Animals; D003061:Codeine / Q000378:metabolism / Q000494:pharmacology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005965:Glucuronates / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009020:Morphine / Q000378:metabolism / Q000494:pharmacology; D009862:Online Systems; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D051381:Rats
D005965:Glucuronates; D004364:Pharmaceutical Preparations; D009020:Morphine; D014453:Glucuronosyltransferase; D003061:Codeine