Identification of human UGT2B7 as the major isoform involved in the O-glucuronidation of chloramphenicol.
Chloramphenicol (CP), a broad spectrum antibiotic, is eliminated in humans by glucuronidation. The primary UGT enzymes responsible for CP O-glucuronidation remain unidentified. We have previously identified the 3-O-CP (major) and 1-O-CP (minor) glucuronides by beta-glucuronidase hydrolysis, liquid chromatography-tandem mass spectrometry, and 1D/2D H NMR. Reaction phenotyping for the glucuronidation of CP with 12 expressed human liver UGT isoforms has identified UGT2B7 as having the highest activity for 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. The kinetics of CP 3-O-glucuronidation by pooled human liver microsomes (HLMs) exhibited biphasic Michaelis-Menten kinetics with the apparent high-affinity K(m1) and low-affinity K(m2) values of 46.0 and 1027 microM, whereas expressed UGT2B7 exhibited Michaelis-Menten kinetics with the apparent K(m) value of 109.1 microM. The formation of 1-O-CP glucuronide by pooled HLM and expressed UGT2B7 exhibited substrate inhibition kinetics with apparent K(m) values of 408.2 and 115.0 microM, respectively. Azidothymidine (AZT) and hyodeoxycholic acid (substrates of UGT2B7) inhibited 3-O- and 1-O-CP glucuronidation in pooled HLMs. In 10 donor HLM preparations, both CP 3-O- and CP 1-O-glucuronidation showed a significant correlation with AZT glucuronidation (UGT2B7) (r(s) = 0.85 and r(s) = 0.83, respectively) at 30 microM CP, whereas no significant correlation was observed between CP 3-O-glucuronidation and serotonin glucuronidation (UGT1A6) or propofol glucuronidation (UGT1A9) at this CP concentration. These results suggest that UGT2B7 is the primary human hepatic UDP-glucuronosyltransferase isoform catalyzing 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9.
Chen
Mei
M
Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts 02115, USA.
LeDuc
Barbara
B
Kerr
Stephen
S
Howe
David
D
Williams
David A
DA
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002701:Chloramphenicol / Q000378:metabolism*; D020719:Glucuronides / Q000032:analysis / Q000096:biosynthesis*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D050217:Metabolic Detoxication, Phase II; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018709:Statistics, Nonparametric; D013379:Substrate Specificity; D000090264:UDP-Glucuronosyltransferase 1A9; D015215:Zidovudine / Q000378:metabolism
D000900:Anti-Bacterial Agents; D020719:Glucuronides; D007527:Isoenzymes; D011994:Recombinant Proteins; C000715591:UGT1A9 protein, human; D015215:Zidovudine; D002701:Chloramphenicol; C110655:UDP-glucuronosyltransferase, UGT1A6; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9