Miller Grover P, Jones Drew R, Sullivan Shane Z, Mazur Anna, Owen Suzanne N, Mitchell Neil C, Radominska-Pandya Anna, Moran Jeffery H
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, USA.
Chem Res Toxicol. 2009 Jul;22(7):1239-45. doi: 10.1021/tx900031z.
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.
作为探索靶向代谢组学方法用于华法林（香豆素）个性化治疗的第一步，我们开发了一种液相色谱 - 串联质谱（LC-MS/MS）方法，该方法能够定量华法林（WAR）的特定对映体（R和S）贡献以及分别由细胞色素P450（CYP）和UDP - 葡萄糖醛酸转移酶（UGT）产生的相应羟基华法林（OH-WAR）和葡萄糖醛酸苷（-GLUC）。对质量控制样品和三种市售人样品的评估表明，我们的分析方法能够测量人尿中24种独特的WAR代谢物。对人体数据的评估还为评估WAR毒性提供了新的见解，并开始表征负责WAR解毒的重要UGT代谢途径。数据揭示了患者中特定代谢物的重要性以及产生这些化合物的相应酶活性，包括直接WAR葡萄糖醛酸化的首次报道。基于总OH-WAR水平，（S）-7-OH-WAR是主要代谢物，表明CYP2C9在WAR代谢中的重要性，尽管其他CYP2C酶也参与了该异构体的清除。患者中（R）-WAR羟基化生成OH-WAR的情况更为多样，这反映在CYP1A2和多种CYP2C酶的不同贡献上。WAR和OH-WAR的葡萄糖醛酸化在化合物和患者方面存在很大差异。与4'-OH-WAR和8-OH-WAR不同，6-和7-OH-WAR主要（>70%）以葡萄糖醛酸苷形式排泄。对于所有患者，UGT1A1可能负责6-O-GLUC的产生，尽管UGT1A10在一名患者中也可能有贡献。7-O-GLUC水平反映了可能五种不同UGT1A酶的贡献。在所有情况下，WAR、4'-OH-WAR、8-OH-WAR以及相应的葡萄糖醛酸苷相对于其他代谢物而言都是次要代谢物。综上所述，这些数据表明P450和UGT反应都有助于人尿中可排泄产物的生成，从而形成复杂的代谢网络。
