Anabolic steroid glucuronides : Enzyme-assisted synthesis and liquid chromatographic-mass spectrometric analysis
Year: 2003
Type: dissertation
Abstract: Anabolic–androgenic steroids (AAS) are testosterone derivatives, widely misused by athletes because of their potential enhancing effect on physical performance. Within the human body, AAS are transformed by phase-I and phase-II metabolic reactions and most often they are excreted in urine as glucuronide-conjugates. Electron impact (EI) and GC–MS based methods have conventionally been applied in the analysis of AAS metabolites in urine. The methods rely on the detection of hydrolyzed and derivatized steroid aglycons and, for that, laborious sample preparation must be carried out. Soft ionization methods, such as electrospray (ESI), enable the connection of liquid chromatographic (LC) separation to mass spectrometric (MS) detection, and thereby, the direct analysis of non-volatile, bulky, and polar compounds, such as the AAS glucuronides. Glucuronide-conjugated AAS standards are required for LC–MS method development, but only a few are commercially available. An enzyme-assisted synthesis was optimized to produce milligrams of glucuronideconjugated metabolites of the most widely misused AAS. The uridine diphosphoglucuronosyl-transferase (UGT) enzymes that catalyze the glucuronidation reaction were obtained from the hepatic microsomal fraction of induced Wistar rats. To allow characterization of regioand stereoselectivity and substrate specificity of UGTs, the glucuronidation reaction was additionally examined in vitro with recombinant human UGT isoenzymes, as well as with human liver microsomes. After the structural characterization of the synthesized substances, the conjugates were utilized in the development of a liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method designed for the determination of intact AAS glucuronides in enzyme-kinetic assays and in human urine. Enzyme-assisted synthesis was successfully applicable to the production of stereochemically pure AAS glucuronides in amounts sufficient for LC–MS/MS method development. The glucuronide-conjugated AAS were recovered in milligram amounts (1.3–6.5 mg), with yields (13-78%) highest for steroid substrates with 4-ene-3-one structure. The only recombinant human isoenzymes showing evidence of regioselectivity were UGT1A8, 1A9, and 2B15, which appeared preferentially to catalyze 17β-hydroxyl glucuronidation. Most recombinant human UGTs did not exhibit a clear preference for conjugation to either the 3α-hydroxyl or the 17β-hydroxyl group. Apparent stereoselectivity was detected in the formation of nandrolone metabolites 5α-NG and 5β-NG with most of the UGT isoenzymes, but the corresponding isomeric pair of methyltestosterone metabolites, 5αMTG and 5β-MTG, did not show the similar behavior, however. The substrate specificities were closely similar among the groups of structurally analogous UGTs, although interindividual differences were observed in their relative activities. In a comparison of rat and human liver preparations and recombinant UGT isoenzymes, the main difference was found in the conjugation of methyltestosterone, which was glucuronidated only with the human and rat liver microsomal UGTs, from which the induced rat liver UGTs were clearly more active.
Source: Työväentutkimus Vuosikirja
Author: Tiia Kuuranne
Language: English
Topic: Doping in Sports
Subfield: Sociology and Political Science
Field: Social Sciences
Domain: Social Sciences
Open Access status: green
OpenAlex ID: W2242404639