Genotype‐dependent metabolism of exogenous testosterone – new biomarkers result in prolonged detectability

Abstract

Testosterone (T) misuse still represents a major problem in sports drug testing. Many strategies have been developed and applied to routine doping controls in recent years to enable both to identify suspicious samples in initial testing procedures and to confirm the exogenous origin of urinary T by means of carbon isotope ratio (CIR) determinations. Depending on the tested individual's genotype of UGT2B17, significantly different amounts of T are glucuronidated and excreted, which results in unaffected T/epitestosterone ratios after T misuse in those subjects with the deletion/deletion polymorphism (del/del). The aim of this study was to investigate differences in metabolic pathways of orally administered T between persons of del/del and insertion/insertion (ins/ins) genetic polymorphism. Therefore, a recently established method using hydrogen isotope ratios together with high-resolution and high-accuracy mass spectrometry was applied after administration of deuterated T to n = 4 subjects including both genotypes. Participants collected urine specimens directly before and for up to 8 days after the application. Urine aliquots were prepared to yield unconjugated, glucuronidated, and sulphoconjugated fractions of urinary steroids. Besides the significant difference in the excretion of T-glucuronide, all measured metabolites varied rather on an individual basis than due to a genotype difference. New T metabolites (both methylated and de-methylated) were detected and investigated regarding their potential to enhance the screening for T misuse. Sulphoconjugated epiandrosterone was further identified as the biomarker allowing for a prolonged retrospective detection of T misuse by means of CIR determinations for up to 5 days compared to 1 day if currently applied sports drug testing procedures were used. Copyright © 2016 John Wiley & Sons, Ltd.

Sulfate metabolites improve retrospectivity after oral testosterone administration

“…24 As an example, concentrations of sulfate metabolites for volunteers 2, 3, and 5 are plotted in Figure S1. 24 As an example, concentrations of sulfate metabolites for volunteers 2, 3, and 5 are plotted in Figure S1.…”

“…The endogenous sulfate fraction is also affected by the administration of EAAS, as demonstrated previously. 24 Excretion studies after oral administration of DHEA, 4androstenedione, dihydrotestosterone (DHT), and epiA and, after application of T gel also demonstrated that epiA-S improved the detection capabilities of EAAS misuse. After administration of dehydroepiandrosterone (DHEA) to seven healthy volunteers, an increase in the concentrations of epiandrosterone sulfate (epiA-S), and some androstanediol and androstenediol sulfates was observed.…”

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

“…Consequently, an important research activity was developed in the recent years to look for additional markers, [5][6][7][8][9][10][11][12][13][14] including other endogenous metabolites excreted in the glucuronide fraction, 5,6 metabolites excreted as conjugates with cysteine, 7,8 or glucuronide metabolites resistant to enzymatic hydrolysis. [15][16][17] However, few studies have been performed dealing with the quantitation of endogenous steroid sulfates, 13,14,[18][19][20][21][22][23][24][25] mainly because of the challenges in the analysis of these conjugated metabolites. [15][16][17] However, few studies have been performed dealing with the quantitation of endogenous steroid sulfates, 13,14,[18][19][20][21][22][23][24][25] mainly because of the challenges in the analysis of these conjugated metabolites.…”

“…27 As a result, in recent years, some methods have been described to directly analyze sulfate metabolites in urine using LC-MS systems. [15][16][17] These results suggest that additional studies need to be performed to evaluate the role of the sulfate fraction on the detection of EAAS administration, including as much as T related metabolites conjugated with sulfate as possible (eg, epiA, androstanediols). After administration of DHEA to 7 healthy volunteers, an increase in concentrations of epiandrosterone (epiA) sulfate, and some androstanediol and androstenediol sulfates was observed, 19 In recent studies with a lower number of volunteers and using GC-IRMS, epiA sulfate was also suggested as the biomarker allowing for a prolonged retrospective detection of oral and gel T administration, and DHEA, DHT, and 4androstenedione oral administrations.…”

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate

“…Other advantages can also arise from the study of phase II metabolites. Monitoring of phase II sulfate conjugates can increase detection windows for some analytes, and has been used to distinguish between the endogenous or exogenously administered steroids . Further, the formation and subsequent decomposition of phase II sulfate conjugates has also been implicated in epimerization of 17α‐alkyl‐17β‐hydroxy steroids, leading to the formation of 17β‐alkyl‐17α‐hydroxy compounds and other minor but significant metabolites .…”