alamethicin and estradiol-3-glucuronide

Traditionally, the Michaelis-Menten equation has been used to determine kinetic parameters for in vitro glucuronidation assays. Recently, estradiol-3-glucuronide formation was shown to exhibit non-Michaelis-Menten kinetics consistent with autoactivation. A concern with the observation of nontraditional kinetics is that they may result as an artifact of the incubation conditions. To examine this concern, the formation of estradiol-3-glucuronide was investigated using human liver microsomes prepared by two different methods, a range of assay conditions, and activation by alamethecin, sonication, or Brij 58 (polyoxyethylene monocetyl ether). Interestingly, holding the other assay components constant, estradiol-3-glucuronide formation was up to 2.5-fold greater using microsomes prepared in phosphate buffer compared with those prepared in sucrose. Incubations activated by alamethecin consistently exhibited the highest rates of estradiol glucuronidation versus the other activators. Furthermore, estradiol-3-glucuronidation exhibited autoactivation kinetics in all of the conditions investigated (n = 1.2-1.7). Nontraditional kinetics were also observed when other UGT1A1 substrates such as ethinylestradiol, buprenorphine, and anthraflavic acid were studied with both human liver microsomes and recombinant UGT1A1. Naphthol, propofol, morphine, and androstanediol were used as probe UGT substrates selective for UGT1A6, UGT1A9, UGT2B7, and UGT2B15, respectively. Of these substrates, only androstanediol exhibited nontraditional kinetics using human liver microsomes. In conclusion, the Hill and/or Michaelis-Menten equations should be used to fit kinetic data to obtain an accurate assessment of in vitro glucuronidation.

Topics: Alamethicin; Cetomacrogol; Chromatography, Liquid; Enzyme Activation; Estradiol; Glucuronosyltransferase; Humans; In Vitro Techniques; Kinetics; Mass Spectrometry; Microsomes, Liver; Phosphates; Recombinant Proteins; Sonication; Substrate Specificity; Sucrose

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

Topics: Acetaminophen; Alamethicin; Alleles; Estradiol; Genetic Variation; Genotype; Glucuronosyltransferase; Homozygote; Humans; Intestines; Kidney; Kinetics; Liver; Lung; Metabolic Clearance Rate; Microsomes, Liver; Morphine; Morphine Derivatives; Mutation; Promoter Regions, Genetic; Tissue Banks; Tissue Distribution