Identification of human UGT2B7 as the major isoform involved in the O-glucuronidation of chloramphenicol

Drug Metab Dispos. 2010 Mar;38(3):368-75. doi: 10.1124/dmd.109.029900. Epub 2009 Dec 11.

Abstract

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.

MeSH terms

  • Anti-Bacterial Agents / metabolism*
  • Chloramphenicol / metabolism*
  • Glucuronides / analysis
  • Glucuronides / biosynthesis*
  • Glucuronosyltransferase / antagonists & inhibitors
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism*
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Metabolic Detoxication, Phase II
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Recombinant Proteins / metabolism
  • Statistics, Nonparametric
  • Substrate Specificity
  • Zidovudine / metabolism

Substances

  • Anti-Bacterial Agents
  • Glucuronides
  • Isoenzymes
  • Recombinant Proteins
  • Zidovudine
  • Chloramphenicol
  • UDP-glucuronosyltransferase, UGT1A6
  • UGT2B7 protein, human
  • Glucuronosyltransferase
  • UDP-glucuronosyltransferase 1A9