Improving the Athlete Biological Passport: Inclusion of specific and long term biomarkers
The detection of the exogenous administration of synthetic androgens having the same chemical structure of the compounds produced endogenously (i.e the so called “pseudoendogenous” steroids, like testosterone, 5α-dihydrotestosterone and androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles.
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories are being collected since 1st January 2014 in a global database integrated in the endocrinological module of the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges for every athlete. Once the ABP detects an atypical profile, an isotope ratio mass spectrometric confirmation must be applied.
The ABP will be effective once a sufficient number of data of a given individual will be collected. In normal conditions, almost two years are needed to collect such information. This will delay in any case the investigations and the time to take the appropriate decisions. New and long term specific endogenous steroids markers have been detected by our research group and others. The data collected up to now suggest that by monitoring such metabolites, the detection window of the abuse of pseudoendogenous steroids can be enlarged. The use of such markers will permit to suspect of a steroid misuse and to carry out a confirmation process by IRMS, even in such in cases where the use of the current criteria will not be effective.
The main goal of this project is to define and include in the ABP the more relevant and specific pseudoendogenous steroids metabolites permitting to suspect from a steroid abuse and to proceed with an IRMS confirmation. This should reduce the gap between the suspicion and confirmation capacities of laboratories and antidoping authorities.
The detection of pseudoendogenous steroids (testosterone or its precursors) is based on the monitoring of selected markers of the steroid profile after a longitudinal evaluation using the Bayesian adaptive model set up in ADAMS. If an individual profile is found atypical, then the application of isotope ratio mass spectrometry (GC-C-IRMS) is mandatory according to WADA Technical Documents in order to disclose the endogenous or synthetic origin of the target compounds found in urine.
GC-C-IRMS requires a comlex sample preparation in order to guarantee the purity of the extracts and subsequent reliability of the measurements performed. The intrinsic charactertistic of the technique makes that is use is limited to those cases that really deserve its application.
In order to better select the samples for IRMS analysis, we have investigated the potential incorporation of additional markers of the steroid profile into the steroid module of the ABP. We have investigated the modifications of the steroid profile including the current parameters of the ABP and the specific hydroxylated metabolites (positions 2, 4, 6, 7 and 16), after controlled administrations of testosterone, androstenedione and dehydroepiandrosterone (DHEA). The results have been compared to the modifications of 13C (%0) delta values obtained by IRMS.
There is no doubt that the specificity of the hydroxylation of pseudo-endogenous steroids is an additional value to get a better insight of the alterations of the steroid profile provoked after the exogenous administrations of testosterone or its precursors (androstenedione or DHEA). Compared to the current markers of the steroid profile included in the steroid module of the ABP, the hydroxylated compounds allow in some cases a better retrospectivity after the administrations (i.e. 2 hydroxylation for AED and T, 4 and 7 hydroxylation for DHEA). These depends on the compound selected, the settings of the detection method used and to some extent on the inter-individual variability.
Hydroxylated steroids being endogenous compounds are present in all urine samples although this can depend on the detection capacity of the method used. In our Laboratory, when working MS/MS and trying to differentiate by MS some isobars (i.e. 2 and 4 OH-androstenedione by selecting minor but specific ion transitions) some compounds can only be detected above some concentration. This indirectly helps to detect at a glance the exogenous administration by monitoring of such compounds. In other cases (i.e. 6OH metabolites, usually present in larger concentrations), it would be necessary to establish reference ranges and to harmonize their quantification among WADA accredited laboratories (to increase their robustness as biomarkers) before thinking in their incorporation to the ABP. The monitoring of hydroxylated metabolites of pseudo-endogenous steroids is less trivial that what one may a priori think. Most of the current GC methods used to screen synthetic and pseudo-endogenous steroids have been designed for the correct separation and quantification of the compounds currently in the steroid module of the ABP. Hydroxylated compounds are late eluding, in an area of the chromatogram of "less interest", where frequent co-elutions may happen, also with some endogenous corticosteroids and pregnanes. To concentrate in a single GC run the capacity of monitoring all the compounds simultaneously is challenging. In our case we have evaluated as much as possible the data obtained under routine conditions and applied an alternative GC method with a different selectivity in those cases where the routine conditions were not adequate.
Although some hydroxylated compounds, specifically in C2, increased for some administrations the detection capacity compared to the current markers of the ABP, their analysis is challenging and in absolute terms, the analysis by IRMS showed the better retrospectivity. The detection of DHEA doping is the most challenging situation. The more significant metabolites of this compound follow sulfo-conjugation before the excretion into urine. This makes the detection of such compoun challenging by the usual and unique monitoring the metabolites excreted in the free + glucuronated fraction. A better knowledge of the sulfate fraction is needed.
A method based on IRMS analysis at the ITP level, considering also the sulfate conjugates may be an option to be investigated in the future to cope with this situation.