Kuuranne Tiia, Aitio Olli, Vahermo Mikko, Elovaara Eivor, Kostiainen Risto
Viikki Drug Discovery Technology Center, FIN-00014 University of Helsinki, Finland.
Bioconjug Chem. 2002 Mar-Apr;13(2):194-9. doi: 10.1021/bc010038g.
A new and useful method based on enzyme-assisted synthesis was developed for producing 3 alpha-O-beta-D-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (I), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (II), 5 alpha-estran-3 alpha-ol-17-one (III), and 5 beta-estran-3 alpha-ol-17-one (IV) are urinary metabolites, indicating the human misuse of the above-mentioned anabolic androgenic steroids (AAS). The common lack of reference material precludes the use and validation of these biomarkers in human doping control. Liver microsomes from Aroclor 1254-induced rats were used as a highly active source of mammalian UDP-glucuronosyltransferases (UGT, EC 2.4.1.17). After purification by protein precipitation, liquid-liquid extraction (dichloromethane), C-18 solid-phase extraction, and lyophilization, the steroid glucuronide structures were characterized by (1)H and (13)C NMR spectroscopy and tandem mass spectrometry. The enzymatic method was highly stereoselective, producing a single major conjugate from the parent steroids I-IV. The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (1.0-2.8 mg, yield 12-29%), which is sufficient for veterinary and human doping control analyses; for pharmaco-, toxico-, and enzyme kinetic studies in the pharmaceutical industry; for clinical laboratories; and for forensic medicine. A new sensitive LC-MS method was developed for controlling the product purity in syntheses, as well as for enzyme kinetic characterization of AAS-metabolizing UGT activities in rat liver toward the aglycones I-IV. In this study, the UGT enzymes responsible for the formation of 3 alpha-O-linked glucuronides from the substrates I, II, III, and IV exhibited the specific enzyme activity values: 25, 124, 48, and 212 nmol/mg microsomal protein in a 2-h incubation, respectively.
开发了一种基于酶辅助合成的新的实用方法，用于从甲基睾酮和诺龙的合成I相代谢物中制备3α-O-β-D-葡萄糖醛酸苷缀合物。17α-甲基-5α-雄甾烷-3α,17β-二醇（I）、17α-甲基-5β-雄甾烷-3α,17β-二醇（II）、5α-雌甾-3α-醇-17-酮（III）和5β-雌甾-3α-醇-17-酮（IV）形成的葡萄糖醛酸苷缀合物是尿液代谢物，表明人类滥用了上述合成代谢雄激素类固醇（AAS）。由于普遍缺乏参考物质，这些生物标志物无法在人体兴奋剂检测中使用和验证。来自Aroclor 1254诱导大鼠的肝微粒体被用作哺乳动物UDP-葡萄糖醛酸基转移酶（UGT，EC 2.4.1.17）的高活性来源。通过蛋白质沉淀、液-液萃取（二氯甲烷）、C-18固相萃取和冻干进行纯化后，通过（1）H和（13）C NMR光谱以及串联质谱对类固醇葡萄糖醛酸苷结构进行了表征。该酶法具有高度的立体选择性，从母体类固醇I-IV中产生单一的主要缀合物。立体化学纯的类固醇葡萄糖醛酸苷缀合物以毫克量回收（1.0-2.8 mg，产率12-29%），这足以用于兽医和人体兴奋剂检测分析；用于制药行业的药物、毒理和酶动力学研究；用于临床实验室；以及用于法医学。开发了一种新的灵敏LC-MS方法，用于控制合成中的产物纯度，以及对大鼠肝脏中AAS代谢UGT活性对苷元I-IV的酶动力学表征。在本研究中，负责从底物I、II、III和IV形成3α-O-连接葡萄糖醛酸苷的UGT酶在2小时孵育中的比酶活性值分别为：25、124、48和212 nmol/mg微粒体蛋白。
