On the origin of physiologically high ratios of urinary testosterone to epitestosterone: consequences for reliable detection of testosterone administration by male athletes

Abstract

Testosterone administration to male athletes can be safely detected in the vast majority of cases by the urinary excretion ratio of testosterone to epitestosterone glucuronides (TG/EG), which may not exceed 6. Some rare cases of physiologically high TG/EG ratios (between 6 and 12) are encountered; these may be attributed to a dysregulation of the testicular secretions of epitestosterone which is decreased, and of epitestosterone sulphate (ES) which is normal or increased. Impaired hydrolysis of circulating epitestosterone sulphate by deficiency of a specific sulphatase acting on 17 alpha-sulphates must also be considered as a possible reason for the decreased availability of epitestosterone for hepatic glucuronidation. Urinary excretions of conjugates and metabolites of testosterone and epitestosterone (expressed in nmol/mmol creatinine) have been determined by gas chromatography-mass spectrometry associated with stable isotope dilution, in a reference population of 90 healthy male subjects and in 12 subjects with chronic TG/EG > 4. Urinary excretion ratios such as TG/(EG+ES), EG/ES and TG/5-androstene-3 beta,17 alpha-diol glucuronide are shown to be efficient criteria which allow discrimination between physiologically high and pharmacologically high TG/EG ratios. A simple oral loading test with deuterium-labelled epitestosterone demonstrates the difference between hepatic and total epitestosterone metabolism clearly, particularly in subjects with physiologically high TG/EG in comparison with subjects with normal TG/EG.

UDP-Glucuronosyltransferases (UGTs) 2B7 and UGT2B17 Display Converse Specificity in Testosterone and Epitestosterone Glucuronidation, whereas UGT2A1 Conjugates Both Androgens Similarly

“…2). This observation is in line with a previous report (Dehennin, 1994) and suggestions in the World AntiDoping Agency (WADA Technical Document TD2004EAAS, 2004, http://www.wada-ama.org/rtecontent/document/end_steroids_aug_04. pdf).…”

“…1), and much less is known about its biological role, as well as biosynthesis and elimination (Starka, 2003). Testosterone and epitestosterone are present mainly in the urine as the respective glucuronide conjugates, but sulfate conjugates and even the unconjugated form of these steroids were found (Dehennin, 1994;Borts and Bowers, 2000;Schulze et al, 2008a).…”

“…The interest in testosterone glucuronidation is often linked to studies on anabolic steroid abuse by male athletes (Dehennin 1994;Schulze et al, 2008a,b). The urinary testosterone to epitestosterone concentration ratio, T/E, serves as a marker for possible testosterone abuse among male athletes (Donike et al, 1983;World Anti-Doping Agency, WADA Technical Document-TD2004EAAS, 2004, http:// www.wada-ama.org/rtecontent/document/end_steroids_aug_04.pdf).…”

Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry

“…Under acidic hydrolysis conditions, ES is reported to undergo acid-catalyzed Wagner-Meerwein rearrangement. 18 Such a rearrangement involves a shift of an adjacent group, such as the 18-methyl group, to the carbocation center of the intermediate produced under acidic conditions. Methanolysis, which has been reported to cleave both the glucuronide and sulfate conjugates in a single step, 18,19,24 has resulted in variable cleavage and rearrangements in our hands.…”

“…9,10 Consequently, the aryl sulfatase treatment does not cleave the TS or ES conjugates. 18,19 The GC/MS methods for measuring this ratio are involved, and have a number of potential problems. We report here on the development of an a high-performance liquid chromatographic (HPLC)/electrospray ionization tandem mass spectrometric (ESI-MS/MS) method for the direct measurement of the urinary glucuronide and sulfate conjugates of T and E.…”