Pieter Van Renterghem

Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes.

The detection of misuse with naturally occurring steroids is a great challenge for doping control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous use of these steroids can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference ranges. Elevated values of the steroid profile constitute an atypical finding after which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous origin of a natural steroid. However, the large inter-individual differences in urinary steroid concentrations and the recent availability of a whole range of natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a different effect on the monitored parameters in doping control complicate the interpretation of the current steroid profile. The screening of an extended steroid profile can provide additional parameters to support the atypical findings and can give specific information upon the steroids which have been administered. The natural concentrations of 29 endogenous steroids and 11 ratios in a predominantly Caucasian population of athletes were determined. The upper reference values at 97.5%, 99% and 99.9% levels were assessed for male (n=2027) and female (n=1004) populations. Monitoring minor metabolites and evaluation of concentration ratios with respect to their natural abundances could improve the interpretation of the steroid profile in doping analysis.

A pilot study on subject-based comprehensive steroid profiling: novel biomarkers to detect testosterone misuse in sports

Context  Until now, the testosterone/epitestosterone (T/E) ratio is the main marker for the detection of testosterone (T) misuse in athletes. As this marker can be influenced by a number of confounding factors, additional steroid profile parameters indicating T misuse can provide substantiating evidence of doping with endogenous steroids. The evaluation of a steroid profile is currently based upon population statistics. As large inter‐individual variations exist, a paradigm shift towards subject‐based references is ongoing in doping analysis.

Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports

Doping with natural steroids can be detected by evaluating the urinary concentrations and ratios of several endogenous steroids. Since these biomarkers of steroid doping are known to present large inter-individual variations, monitoring of individual steroid profiles over time allows switching from population-based towards subject-based reference ranges for improved detection. In an Athlete Biological Passport (ABP), biomarkers data are collated throughout the athletes sporting career and individual thresholds defined adaptively. For now, this approach has been validated on a limited number of markers of steroid doping, such as the testosterone (T) over epitestosterone (E) ratio to detect T misuse in athletes. Additional markers are required for other endogenous steroids like dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA). By combining comprehensive steroid profiles composed of 24 steroid concentrations with Bayesian inference techniques for longitudinal profiling, a selection was made for the detection of DHT and DHEA misuse. The biomarkers found were rated according to relative response, parameter stability, discriminative power, and maximal detection time. This analysis revealed DHT/E, DHT/5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol as best biomarkers for DHT administration and DHEA/E, 16α-hydroxydehydroepiandrosterone/E, 7β-hydroxydehydroepiandrosterone/E and 5β-androstane-3α,17β-diol/5α-androstane-3α,17β-diol for DHEA. The selected biomarkers were found suitable for individual referencing. A drastic overall increase in sensitivity was obtained. The use of multiple markers as formalized in an Athlete Steroidal Passport (ASP) can provide firm evidence of doping with endogenous steroids.

Population based evaluation of a multi-parametric steroid profiling on administered endogenous steroids in single low dose.

Steroid profiling provides valuable information to detect doping with endogenous steroids. Apart from the traditionally monitored steroids, minor metabolites can play an important role to increase the specificity and efficiency of current detection methods. The applicability of several minor steroid metabolites was tested on administration studies with low doses of oral testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone (DHEA). The collected data for all monitored parameters were evaluated with the respective population based reference ranges. Besides the traditional markers T/E, T and DHT, minor metabolites 4-OH-Adion and 6α-OH-Adion were found as most sensitive metabolites to detect oral T administration. The most sensitive metabolites for the detection of DHEA were identified as 16α-OH-DHEA and 7β-OH-DHEA but longest detection up to three days (after oral administration of 50 mg) was obtained with non-specific 5β-steroids and its ratios. Steroids applied as a gel had longer effects on the metabolism but were generally not detectable with universal decision criteria. It can be concluded that population based reference ranges show limited overall performance in detecting misuse of small doses of natural androgens. Although some minor metabolites provide additional information for the oral testosterone and DHEA formulations, the topical administered steroids could not be detected for all volunteers using universal reference limits. Application of other population based threshold limits did not lead to longer detection times.

Development of a GC/C/IRMS method – Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

Detection of dihydrotestosterone gel, oral dehydroepiandrosterone, and testosterone gel misuse through the quantification of testosterone metabolites released after alkaline treatment.

The natural occurrence of endogenous anabolic steroids together with their availability in different administration forms makes the detection of their misuse a great challenge for doping control laboratories. Nowadays, the detection of endogenous steroids abuse is performed by the analysis of the steroid profile. Recently, androst-1,4-dien-3,17-dione (1,4-AD), androst-4,6-dien-3,17-dione (4,6-AD), 17β-hydroxy-androst-4,6-dien-3-one (6-T), and androst-15-en-3,17-dione (15-AD) have been described as testosterone (T) metabolites released after basic treatment of the urine. In the present work, the usefulness of these metabolites has been evaluated detecting the use of three different forms of endogenous steroids in a single dose: dihydrotestosterone gel (DHT), oral dehydroepiandrosterone (DHEA), and T gel. After the independent administration of these endogenous steroids, a rise in the value of several of the ratios calculated between the tested metabolites was noticed. For DHT, a small increase was observed for the ratios 1,4-AD/15-AD, 6-T/15-AD and 4,6-AD/15-AD although only for one volunteer. Better results were obtained for oral DHEA and T gel where an increase was observed in all volunteers for several of the tested ratios. The detection time in which the misuse can be detected (DT) has been evaluated using two different approaches: (1) comparison with population based reference limits, and (2) comparison with individual threshold levels. The obtained DTs were compared with the results of previously published markers for the misuse of such substances. When using basic released metabolites, shorter DTs were obtained for DHT, similar DTs for DHEA, and the detectability was substantially improved for T gel.

Statistical discrimination of steroid profiles in doping control with support vector machines.

Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society. To screen for misuse with these compounds, drug testing laboratories monitor the urinary concentrations of endogenous steroid metabolites and their ratios, which constitute the steroid profile and compare them with reference ranges to detect unnaturally high values. However, the interpretation of the steroid profile is difficult due to large inter-individual variances, various confounding factors and different endogenous steroids marketed that influence the steroid profile in various ways. A support vector machine (SVM) algorithm was developed to statistically evaluate urinary steroid profiles composed of an extended range of steroid profile metabolites. This model makes the interpretation of the analytical data in the quest for deviating steroid profiles feasible and shows its versatility towards different kinds of misused endogenous steroids. The SVM model outperforms the current biomarkers with respect to detection sensitivity and accuracy, particularly when it is coupled to individual data as stored in the Athlete Biological Passport.

Steroid metabolism in chimeric mice with humanized liver.

Anabolic androgenic steroids are considered to be doping agents and are prohibited in sports. Their metabolism needs to be elucidated to allow for urinary detection by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Steroid metabolism was assessed using uPA(+/+) SCID mice with humanized livers (chimeric mice). This study presents the results of 19-norandrost-4-ene-3,17-dione (19-norAD) administration to these in vivo mice. As in humans, 19-norandrosterone and 19-noretiocholanolone are the major detectable metabolites of 19-norAD in the urine of chimeric mice.A summary is given of the metabolic pathways found in chimeric mice after administration of three model steroid compounds (methandienone, androst-4-ene-3,17-dione and 19-norandrost-4-ene-3,17-dione). From these studies we can conclude that all major metabolic pathways for anabolic steroids in humans are present in the chimeric mouse. It is hoped that, in future, this promising chimeric mouse model might assist the discovery of new and possible longer detectable metabolites of (designer) steroids.

Profiling of urinary formestane and confirmation by isotope ratio mass spectrometry.

Formestane (F, androst-4-en-4-ol-3,17-dione) is an irreversible aromatase inhibitor with the ability to suppress the estrogen production from anabolic steroids. Consequently, F is mentioned on the World Anti-Doping Agency (WADA) prohibited list and because studies have shown that F is produced endogenously in small amounts, a threshold for urinary excreted F of 150 ng/mL was introduced. Lower concentrations could be due to endogenous production and need further investigation to prove the exact origin through determination of the carbon isotope ratio. However, because the current screening methods are a lot more sensitive, F is detected in practically every urine sample. A strict implementation of this WADA rule would imply that almost every urine sample needs additional investigation to verify an exogenous or endogenous origin. The main aim of this study was to propose and introduce a lower concentration limit of 25 ng/mL beneath which the detected F is considered as being endogenous and no further investigation is needed. The data presented in this paper suggests that this threshold provides a good balance between a sufficiently large detection window and not having to perform isotope ratio mass spectrometry (IRMS) analyses on negative urine samples.

Studies on the minor metabolite 6a-hydroxy-androstenedione for doping control purposes and its contribution to the steroid profile.

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a-hydroxy-androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC-MS) because of the low concentrations; this is especially the case for 6aOH-ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH-ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a-OH-ADION. For this purpose, the more sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH-ADION. If a urine sample has an elevated 6aOH-ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH-ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH-ADION.