Enzyme-Assisted Synthesis and Structure Characterization of Glucuronide Conjugates of Methyltestosterone (17α-methylandrost-4-en-17β-ol-3-one) and Nandrolone (estr-4-en-17β-ol-3-one) Metabolites

Publisher Website
Google Scholar
Abstract
A new and useful method based on enzyme-assisted synthesis was developed for producing 3α-O-β-d-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17α-methyl-5α-androstane-3α,17β-diol (I), 17α-methyl-5β-androstane-3α,17β-diol (II), 5α-estran-3α-ol-17-one (III), and 5β-estran-3α-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 1H and 13C 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 3α-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.

This publication has 11 references indexed in Scilit: