title: Assessing cytochrome P450 and UDP-glucuronosyltransferase contributions to warfarin metabolism in humans.
abstract: As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, USA.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
authors.forename: Drew R
authors.initials: DR
authors.lastname: Jones
authors.forename: Shane Z
authors.initials: SZ
authors.lastname: Sullivan
authors.forename: Anna
authors.initials: A
authors.lastname: Mazur
authors.forename: Suzanne N
authors.initials: SN
authors.lastname: Owen
authors.forename: Neil C
authors.initials: NC
authors.lastname: Mitchell
authors.forename: Anna
authors.initials: A
authors.lastname: Radominska-Pandya
authors.forename: Jeffery H
authors.initials: JH
authors.lastname: Moran
mesh_terms: D000925:Anticoagulants / Q000378:metabolism* / Q000633:toxicity / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D055432:Metabolomics; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry; D014859:Warfarin / Q000378:metabolism* / Q000633:toxicity / Q000652:urine
chemical_list: D000925:Anticoagulants; D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase