Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of methyltestosterone (17 alpha-methylandrost-4-en-17 beta-ol-3-one) and nandrolone (estr-4-en-17 beta-ol-3-one) metabolites.
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.
Kuuranne
Tiia
T
Viikki Drug Discovery Technology Center, FIN-00014 University of Helsinki, Finland.
Aitio
Olli
O
Vahermo
Mikko
M
Elovaara
Eivor
E
Kostiainen
Risto
R
D000818:Animals; D001711:Biotransformation; D005260:Female; D020719:Glucuronides / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008777:Methyltestosterone / Q000031:analogs & derivatives / Q000378:metabolism*; D008862:Microsomes, Liver; D015394:Molecular Structure; D009277:Nandrolone / Q000031:analogs & derivatives / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D020719:Glucuronides; D009277:Nandrolone; D008777:Methyltestosterone