Projets de recherche

Code: 17C02MP

Increasing numbers of adverse analytical findings were reported in the recent years due to the misuse of the anabolic androgenic steroid dehydrochloromethyltestosterone (DHCMT). Once developed in the former GDR for misuse in sports it regained enormous relevance especially in the samples from Bejing and London Olympic games. Several adverse analytical findings were reported after the samples had been retested for the newly reported long-term metabolite of DHCMT. At present, the use of post administration urines instead of purified reference material has been accepted in confirmatory analyses. Very recently the currently used metabolites have been questioned in the literature. Thus a controlled administration trial in humans will be performed to provide further evidence for tracing back the long term metabolites 20ξOH-NorTHCMT and 20βOH-NorDHCMT to a DHCMT administration. Furthermore, the utilisation of in-vitro experiments will further broaden the scientific insights into metabolic pathways that lead to the generation of these metabolites.

Main Findings

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times. Even if discontinued as pharmaceutical in 1994, there are several adverse analytical findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, a single oral dose of DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. To improve tracing of cheating athletes, it is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Code: 17A24XD

The anabolic androgenic steroids (AAS) are prohibited in sports. They are included in the 2017 list of the World Anti-Doping Agency (WADA) as class S1. In the last years the anabolic agents accounted for most of the adverse analytical findings (AAF) in doping control (e.g. in 2015 50% of all ADAMS reported AAF). AAS undergo extensive metabolization, thus, urinary detection of a prohibited administration is mainly based on the detection of metabolites. As some steroids also occur naturally in the body, their uncoverage generally uses specific ratios, such as testosterone/epitestosterone (T/EpiT), androsterone/etiocholanolone (And/Etio), And/T, and 5α-/5β-androstane-3α,17β-diol (Adiol/Bdiol), that proved to be very stable in humans. Confirmation of the results generally require isotope ratio mass spectrometry. As confirmation is very elaborate and cost intense some minor metabolites came into the focus of anti-doping scientists to increase the efficiency of screening procedures. For the improved detection of an exogenous administration of androstenedione the usefulness of the A- or B-ring hydroxylated metabolites 4-hydroxy-androstenedione, 6z-hydroxy-androstenedione was reported. As already published 2β- and 15β-hydroxylation also occurs in testosterone metabolism with ~10% and 4% of the rate of the most dominant hepatic microsomal 6β-hydroxylation. Furthermore, it was demonstrated that the use of 2- and 4-hydroxyandrostenedione may serve as long term marker of an androstenedione administration. The metabolic generation could be confirmed by in-vitro experiments upon incubation with CYP1A2 and CYP1B1. No reports on the metabolic hydroxylation of androgens by CYP1A2 or by CYP1B1 are found in literature so far. The objective of the project is to further investigate the suitability of A-ring hydroxylation for long-term detection of endogenously occurring androgens and to extent the preliminary investigations to other prohibited steroids.

Main Findings

The hydroxylation pathway in vivo and/or in vitro studies for Testosterone (T), 4-androstenedione (AED), 19-norandrostenediol (NAD) and Methyltestosterone (MT) was investigated. Samples collected after the administration of T showed the presence of 6-OH-T according to an oral administration, but its concentration gave no additional information different to that already known (parameters of the steroid profile). On the other hand, the formation in vivo and in vitro of hydroxylated metabolites of AED, NAD and MT in position 4 allowed the formation of Formestane, Oxabolone and Oxymesterone respectively. Although the formation of these metabolites were significant (in some cases) and easily detectable, any of them showed a detection window longer that those metabolites known for AED (T/E ratio and others steroid profile parameters), NAD (19-norandrosterone, and 19-noretiocholanolone) and MT (5α and 5β reduced metabolites). Nevertheless, a special attention have to be paid when a sample with these compounds is reported, because under these circumstances a concomitant abuse of AED-formestane, NAD-oxabolone or MT-oxymetholone could be misinterpreted reported by the laboratory. Only for AED the 2-hydroxylation demonstrated an added value extending the suspicion of an AED administration based on an extended steroid profile.

Code: ISF17D04SI

The original idea of “athlete`s performance passport” or monitoring
individual performances for better informed decisions on doping
testing has been presented by Schumacher and Pottgiesser. The main objective of an “athlete`s performance passport” in sport is to distinguish between consistent and unexpectedly disproportionate performances. Excellent performance itself is not a proof of any wrongdoing or doping. However, through longitudinal monitoring, inconsistently excellent performance could be a warning sign that need further attention from anti-doping authorities.The purpose of this project is to establish framework for the longitudinal performance monitoring and identification criteria of athletes with outline performance in middle- and long distance
runners population.

Code: 17D09RV 

The detection of endogenous anabolic steroids (EAS) abuse is currently performed using the steroid profile. The steroid profile is composed of concentrations of testosterone and related endogenous metabolites excreted as glucurono-conjugates, and ratios between them, being Testosteone/Epitestosterone ratio the most important one. These parameters are monitored for each individual to define the individual basal ranges, and changes on them reveal the use of EAS. Suspicious samples are confirmed by carbon isotope ratio mass spectrometry to demonstrate the exogenous origin of testosterone and metabolites. The steroid profile is a powerful tool to detect EAS misuse, however improvements are needed to prolong detection windows. Testosterone and metabolites are also excreted as sulfates in urine. The sulfate fraction has not been comprehensively evaluated for the detection of EAS misuse. The objective of the project will be to evaluate the sulfate fraction of testosterone metabolites to look for new biomarkers to prolong the detectability of the misuse of EAS. First, a comprehensive methodology to quantify endogenous steroid sulfates based on their direct analysis by liquid chromatography-tandem mass spectrometry will be optimized and validated. Second, steroid sulfates will be quantified in urines obtained from healthy population to define normal population ranges. Finally, steroid sulfates will be quantified in urines collected after administration of testosterone to healthy volunteers by different routes. Evaluation of sulfate metabolites as markers of EAS administration will be performed by comparison of the excretion profiles of testosterone and metabolites excreted as sulfates with the excretion profiles of metabolites included in the conventional steroid profile. The successful outcome of the project will be directly applicable to sports drug testing by improving the detection of EAS misuse.

Main Findings: 

The objective of the project was a comprehensive evaluation of the sulfate fraction of testosterone (T) metabolites to look for new biomarkers to prolong the detectability of the misuse of endogenous anabolic steroids. First of all, an analytical method was developed and validated to quantify fourteen T related metabolites conjugated with sulfate, based on a mixed-mode solid pahse extraction and the direct measurement of sulfate metabolites by LC-MS/MS. The concentrations of sulfate metabolites in healthy volunteers, including Caucasion and Asian volunteers, were measured.

The usefulness of sulfate metabolites to detect oral T misuse was evaluated after administration of a single oral dose to five Caucasian male volunteers. Using individual threshold limits epiandrosterone sulfate (epiA-S)  improved the detection times (DTs) with respect to T/epitestosterone (E) ration in all five volunteers. Androsterone (A), etiocholanolen (Etio) and two androstanediol sulfates also improved DTs for some volunteers. The most promising results were obtained using ratios between sulfates of epiA, A or androstandediol 1 and E, and also sulfates of epiA or androstanediol 1 and dehydroandrosterone (DHA). These ratios prolonged the DT of oral T administration, in some cases several days after administration, and therefore significantly improving the retorspectivity compared to sulfate concentrations or to the conventionsl T/E ratio.

Sulfate metabolites were also evaluated after a single intramuscular (IM) injection of T to six Caucasian and six Asian helthy male volunteers. Principal component analysis (PCA) was used to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. DTs of each ratio were evaluated using individual thresolds for each volunteer, and the best results were obtained using rations involving T and E sulfates in the denominator. The best marker was the ratio A-S/T-S which prolonged the DTs with respect to T/E ration in all Caucasian volunteers and in two Asian volunteers. Other ratios A-S or Etio-S and E-S, and, sulfates of Etio, DHA or epiA and T-S were also found adequate.

The data obtained in the project provide a comprehensive insight bout the usefulness of endogenous sulfate metabolites as biomarkers for the detection of oral and IM T misuse. They can drastically increase the DTs with respect to the conventional T/E ratio, especially after oral T administration and, according to our results, its inclusion in the steroid profile is strongly recommended.

Code: 17C09DL

The present proposal builds up on a previous project funded by the WADA entitled: Tramadol and sport: Effects on physical and sustained attention performance during cycling exercise. The aim of this project is to investigate further the effect of tramadol on cycling performance at the physical, cognitive and brain levels by: 1) addressing the potential moderator effect of physical fatigue; 2) testing the effect of the pain-killer on intense short anaerobic efforts (as final sprint); 3) studying the effect of the combination of tramadol and caffeine. This is particularly relevant as it appears from anecdotal reports that pro-cyclists are consuming a combination of the two drugs in order to improve performance.
The project consists of a placebo-controlled, double blind experiment. A single dose of 100mg of tramadol, 6mg/kg of caffeine, and the combination of both, versus placebo, will be administered to participants (in separate sessions). They will then complete a 40-min cycling submaximal exercise (at 60% VO2max). A 20-min indoor Time-Trial (TT) will follow this. Participants will complete a visual discrimination task while cycling both during the submaximal and TT tests. After that, participants will undertake three 30-sec Wingate tests. The Psychomotor Vigilance Task will be performed prior to and
after the exercise phase. Electroencephalography will be continuously recorded throughout the cycling exercise and at rest. We will also obtain measures of subjective performance such as perceived effort and mental fatigue. Subjects will complete the Profile of mood state questionnaire before and after every session.
We will investigate the effect of tramadol, paracetamol and the combination of both vs. placebo on physiological and subjective parameters related to cycling performance (at the physical, cognitive and brain levels) at constant load, during a self-paced indoor time trial and during short maximal intensity efforts.

Main Findings:

The use of tramadol, a weak opioid, is a controversial current topic of debate in cycling. However, little is known about is potnetial ergogonic and cognitive (harmful) effects. In this project, we aimed at providing novel empirical evidence on this issue. As control conditions we included placebo and paracetamol, the other mild analgesic commonly used in cylcing. A randomized, double-blind, placedbo-controlled trial (EudraCT number: 2018-000388-10) design study was performed. 20 participants completed a 40-min constant work-rate at 60% of the VO2max followed by a 20-min time trial (TT) on a cycle-ergometer after ingesting 100 mg tramadol, 1.5 g paracemtamol, or placebo (in seprate days/sessions). Participants completed the Psychomotor Vigilance Task (PVT) before substance administration and ten minutes before starting the warm-up, and the Sustained Attnetion to Response Task (SART) during the 60 min of exercise. Electroencephalography (EEG), heart rate (HR), and power output were recorded throughout the sessions. The rating of perceived effort (RPE; by means of he Borg scale) was measured at the end of the 40-min constant work-rate and the 20-min TT.

The PVT (baseline-corrected) results showed that participants were faster in the tramadol condition than in the paracetamol and placebo conditions. The average power output during the 20-min TT only revealed greater performance in the tramadol than in the paracetamol condition. Higher HR values were reported in the tramadol condition than in the paracetamol and placebo conditions. RPE findings evidenced lower values in hte tramadol condition than the other conditions in the 40-min constant work while no differences were observed in the 20-min TT. Cognitive performance in the SART did not differ between substances in the 20-min TT. During the 40-min constant work-rate, better accuracy was reported in the tramadol condition than in the placebo condition, accompanied by slower RT. The speed-accuracy trade off could be a sign of improved congitive control (i.e., better ability to inhibit unwanted responses at the expense of being slower). The analysis of EEG tonic spectral power revearled increased power in the tramadol condition than in the other conditions at rest, while no differences in EEG oscillatory activity (either tonic or event-related) were found during the exercise.

In sum, the results of this clinical trial sugest that neither tramadol nor paracetamol (vs. placebo) seem to improve exercise performance or impair the ability to stay focused during a pre-loaded 20-min TT.

Code: ISF17B03BS 

Recombinant erythropoietins (EPOs) have long been one of the most frequently abused drugs for increasing blood oxygen capacity. However, numerous compounds with EPO-like activity but completely different chemical structures (either short peptides or even nonpeptidic synthetic molecules) have been developed so far and due to such chemical diversity, detection of their potential abuse is notoriously difficult. To our knowledge, no universal and reliable analytical method is available to confirm the presence of all EPO mimetics in biological sample regardless of their chemical structure. Here, we describe development of a rapid and universal assay for detection of any ligand binding to the erythropoietin receptor (EpoR) which could be used as a low-cost screening test in doping control to identify individuals for further detailed examination. Our innovative platform comprises combination of immunoprecipitation steps and two modified ELISA tests where recombinant filamentous phage particles displaying EpoR are used instead of primary antibodies. As evaluated with spiked artificial urine, such assay can detect the presence of EpoR ligands chemically unrelated to EPO in specimens. Site-specific proteolytic cleavage and isolation of EpoR:ligand complex from phage particles enables analysis of the complex by mass spectrometry and potential identification of previously unknown EpoR ligand. The described platform may also be further developed into the lateral flow immunoassay format similar to common over-the-counter pregnancy test which would greatly increase its applicability out of the laboratory. To emphasize, such concept can also be applied to detection and identification of other growth factor or hormone mimetics by implementation of appropriate combination of receptor, ligands and antibodies. Moreover, based on our platform a multiplex assay capable of simultaneous detection of substances with distinct activity can be developed by utilizing Luminex® technology and appropriate combination of recombinant phage particles displaying different hormone and growth factor receptors. 

Code: 17A28PA 

Detection of the use of the anabolic steroid nandrolone is based primarily upon the identification of the main urinary metabolite, 19-norandrosterone. Laboratories use the sophisticated technique of gas chromatography mass spectrometry to detect 19-norandrosterone in athletes' urine. WADA have recently taken steps to harmonize the analysis of nandrolone, issuing WADA Technical Document TD2016NA in 2016.

Reference materials are a vital tool for validating analytical testing methods and for ongoing laboratory quality assurance. International best practice specifies that CRMs beused as part of routine laboratory quality control. However CRMs for the measurement of steroid metabolites in human urine are not readily available. The project involves certification of the stable carbon isotope delta value of 19-norandrosterone (19-NA) in CRM MX017, currently being prepared for WADA project 15J04JM. Laboratories will reconstitute this material with water, and then analyse it alongside samples of urine taken from athletes for doping control. The testing laboratory can the compare the measured values for the CRM to those certified, providing assurance of the validity of the testing procedure.

Main Findings:

A new freeze-dried human urine NMIA MX017 certified reference material (CRM) has been prepared by NMIA, under the funding rom WADA, to replace the depleted MX005 CRM. Approximately 2400 units had been produced and made available to WADA laboratories since April 2019. This material will assist WADA laboratories to demonstrate comparability of measurements for the Athlete Biological Passport introduced in 2014. The replacement material will continue to support the longitudinal profiling measurements for detection of testosterone abuse and to provide SI-traceable values for the mass fraction and mass concentration of the glucuronides of six steroids specified in the WADA technical document TD2018EAAS: testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5a-androstane-3a,17b-diol (5a-Adiol) and 5b-androstane-3a,17b-diol (5b-Adiol).

In February 2020, the carbon isotope delta value of (δ13CVPDB) 19-norandrosterone (19-NA) has been added to the MX017 certificate. The new property value (δ13CVPDB of 19-NA) is traceable to the VPDB reference via the NMIA MX018 steroid carbon isotopic reference materials normalised by two secondary isotopic reference materials IAEA-CH-6 (sucrose) and IAEA-CH-7 (polyethylene). The reference value, δ13CVPDB -29.82 ± 0.41 , was determined using 36 bottles of MX017 analysed in five batches of experiments. The combined standard uncertainties of the reference value was expanded with a coverage factor of 2.0 to provide a 95% level of confidence.

Analysis was performed on 20 mL of reconstituted urine CRM (MX017) after hydrolysis with β-glucuronidase (E.colie). Free steroids were extracted into hexane and purified by two-dimensional HPLC. In-house quality control samples including water spikes, matrix spikes and solvent standards were included to monitor fractionation due to sample transformation procedures. The purified fractions were dried and reconstituted in cyclohexan/2-propanol (4:1) for anaylis by GC-C-IRMS. The identical treatment priniciples was followed to ensure samples and calibration soltions were combusted and transferred the same way into the IRMS. Each samples was injected twice in a randomised order bracketed by calibration standards. Frequent bracketing of the test samples with calibration solutions allowed any drift in the instrument to be fully captured. An approximate δ-value of the internal working gas was used to calculate all raw δ-values for samples and standards. A multi-point isotopic bracketing calibration approach was adopted to normalise all the measured δ-values of samples using steroid isotopic CRM mixtures NMIA MX018-1 and MX018-3. A linear regression line (R2> 0.99) fitted through the measured and the reference delta values from the eight steroid compounds, in the two calibration mixtures, allowed the measured δ-values of 19-norandrosterone in the MX017 to be normalised to δ13CVPDB the VPDB scale (Figure 1). The multi-point steroid isotopic calibration approach has allowed the property value to be assigned with ower uncertainty than the measurement approach employing calibrated reference gas.

Code: ISF17D05FM & ISF17E06RG

In a funded WADA study recently completed (“Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs”) we tested the hypothesis that novel biomarkers as fetal hemoglobin (HbF) and related microRNAs might show changes of interest in a group of trained healthy volunteers exposed to ABT respect to controls. The first result of this study is the production of a validated WADA-UNIFE-Biobank constituted of around 2000 plasma samples from control and ABT trained subjects. It is established that hypoxic and hyperoxic stimuli and blood manipulation during procedures of withdrawal and reinfusion (distress, ageing, apoptosis/degradation of circulating blood cells, effects of preservative substances, etc.) might induce a predictable and an unpredictable series of changes of miRNAs expression. Therefore, we hypothesized that after significant blood collection and autologous reinfusion, the miRNA network in the athlete’s plasma is changed, allowing to generate integrated molecular profiles permitting to predict ABT. Therefore, the hypothesis is that a miRNA pathway might be much more informative than a single miRNA, even if demonstrated to be associated with ABT in a sub-set of athletes. To this end, a small sample of subjects enrolled for the cited study was recently spontaneously tested in our laboratory for a full genome analysis. Therefore, the aim of this study is the validation of the first release of ABT-miRNA list and the identification of novel miRNAs of putative interest in predicting ABT following global miRNA analyses.

Main Findings

This project was aimed at identifying novel molecular markers useful in the anti-doping field to detect autologous blood transfusion (ABT). The hypothesis was that both the phases of ABT, blood withdrawal and reinfusion, might be accompanied by changes in different parameters to be identified by OMICS approaches in the level of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression. In a previous funded study “Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs” twenty-four healthy trained male subjects were enrolled and randomized into Transfusion (T) and a Control (C) groups. The T subjects underwent nine seriated blood samples before and after the procedures of withdrawal and reinfusion. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects at 12 days time-point after re-infusion.

In the present study, using samples stored in the biobank, we aimed to validate the first release of ABT-miRNA list at other time-points (i.e. D-25 and D+3) to confirm the interest of this miRNA list in predicting ABT. ABT prediction was also demonstrated after global miRNA analyses, thus identifying novel ABT-informative miRNAs. The levels of selected informative microRNAs in relation to sex was determined and protocols/assays for ABT detection were developed. of specific interest. To this aim, microarray analysis and droplet digital RT-ddPCR were also performed for subjects of the T group and C groups for selected time-points.

The data obtained studying the global miRNome pattern demonstrated clustering of ABT-treated plasma samples at D+3 and D+15. These results support the concept that identification of ABT can be performed in one-step using the developed microarrays procedure for miRNA global analysis. The blood withdrawal was not found to be a “confounding factor”. Secondarily, the data obtained by RT-ddPCR performed in plasma samples from ABT trained subjects at D-25, D+3 and D+15 time points were first aimed at validation the “erythroid-associated miRNA list” (let-7a-5p, miR-126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p and miR-92a-3p) representing ABT associated miRNAs involved in hypoxia, erythroid differentiation and fetal hemoglobin production. This set of 8 erythroid associated miRNAs were informative in detecting ABT in most (5/6) of the ABT-treated subjects at all the time-points considered. We then considered miRNA found specifically dysregulated at D-25, D+3 and D+15 time points according with the miRNA microarray analyses. This additional list was proposed in consideration of the miRNA fold changes, the availability of the assays for miRNA amplification and the necessity to have both up- and down-regulated miRNAs to test as possible ABT-associated biomarkers. A final list of six miRNAs was identified and selected for further experimental activity. While the data obtained for down-regulated miRNAs were not informative (no major differences with the control set), differential expression of miR-766-3p (D-25), miR-636 (D-25), miR-425-3p (D+3), miR-4284 (D+3), miR-3151-3p (D+15) and miR-5787 (D+15) was found. When the two miRNA lists (erythroid and global) were used, only one ABT subject was not identified as abnormal or potentially positive for ABT (score 11/12 = 92%). Despite a high heterogeneity within the male and female groups, minimal sex-biased miRNAs differences were observed. Interestingly several miRNAs found up-regulated at the D-25 time point, which is 10 days after the blood withdrawal, may represent “follow-me” markers to identify athletes at risk of ABT to be tested in the proximity of major agonistic events. The results of the present project allow to validate a list of 14 miRNA useful, by RT-ddPCR based analysis, to detect ABT in the majority (over 90%) of the ABT-treated subjects.

Code: 17D15XD 

The detection of pseudoendogenous steroids is based on the alterations of the parameters of the steroid profile included in the steroid module of the Athletes’s Biological Passport (ABP). A Bayesian statistical inference approach allows fixing the boundaries for the ABP parameters of a given individual based on the previous data collected. In the case of a suspicious sequence of data or data outside the population based ranges when previous values are not available, a specific confirmatory method based on isotope ratio mass spectrometry (GC-C-IRMS) is mandatorily applied to the suspicious sample to disclose the synthetic from endogenous origin of the steroids detected. The current approach, although scientifically valid, has some limitations: (1) delay in taking appropriate decisions by the results management authorities (RMA) if the first value of the sequence is abnormal since the model needs the collection of additional samples to detect it; (2) ineffective for those athletes not tested with some frequency; (3) unable to detect low dosages with physiological effect.

In addition, new pharmaceutical formulations of synthetic steroids are not easily detectable by GC-C-IRMS if isotopic analyses of additional samples of the athlete are not performed. The aim of this study is to expand the use of IRMS at a screening level for those disciplines at higher risk of pseudoendogenous steroids abuse, simplifying the current confirmatory procedure. This would allow reducing the time of evaluating an abuse of pseudoendogenous steroids including those athletes less frequently tested, detecting lower dosages of administrations and for a longer period of time and finally being able to detect those preparations not currently detectable by the longitudinal evaluation of the IRMS data since these data would be now available.

Code: ISF17R02NN

This project is aimed at identifying markers of rHuEPO misuse and separate these from markers of natural hypoxic exposure by metabolomics. Male (n=20) and female (n=20) non-competing athletes are exposed to 4 weeks sea-level investigation and after a full washout period, participants are exposed to hypobaric hypoxia at 2.320 m for four weeks. Four groups are formed in a blinded randomized and gender balanced fashion: One group (n=20) receives EPO during the sea-level period and placebo during the stay
at altitude. Another group (n=8) receives EPO both at sea-level and altitude. A third group (n=8) receives placebo both at sea-level and altitude and the fourth group (n=4) receives placebo at sea-level and EPO at altitude. The design facilitates identification of markers of EPO misuse at sea-level (n=28) as well as markers of natural altitude exposure (n=28). Each participant’s treatment response is analyzed in relation to a 4 week baseline period. Moreover, the design includes an intra-individual possibility to verify identified targets expected to be unique to rHuEPO misuse, since 8 participants are treated with rHuEPO both at sea-level and altitude. Runners are enrolled due to the good possibility of recruitment and conduction of training at altitude. The project results in a highly valuable biobank for identification of markers sensitive to initiation and termination of rHuEPO injections. Metabolomics discovery is carried out in collaboration with a world-leading research group as well as the experts at University of Cologne, Germany. Importantly, the project is a joint effort between world-leading University research, Anti-doping organizations and WADA laboratories.

Main Findings

With the present study we were able to demonstrate that recombinant human erythropoietin (rhEpo) treatment combined with altitude exposure provide an additive erythropoietic response compared to rhEpo treatment or altitude exposure alone. Accordingly, analysing the results in the Athlete

Biological Passport revealed that the passport is more sensitive to rhEpo treatment at altitude than at sea level, but a concurrent compromised specificity exist with altitude exposure.

We also investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rhEpo treatment. With this study we demonstrate that a low-dose rhEpo treatment alters IRF and IR/RBC compared with placebo, which also exceeds the fluctuations induced by altitude exposure in the post-treatment period. Similarly, during rhEpo treatment at altitude, both biomarkers exceeded the fluctuations induced by placebo injections at altitude. When individual thresholds were calculated, we were able to identify ~90% of the rhEpo treated participants at sea level with each variable, whereas we were able to identify ~30% and ~65% of the rhEpo treated participants at altitude for IRF and IR/RBC, respectively. Importantly, these sensitivities were obtained with a specificity >99%.

Plasma samples have been subjected to untagerted metabolomics followed and bioinformatic analyses. No biomarkers with sufficient sensitivity and specificity to discriminate between sea-level or altitude exposure could be identified. Likewise, no biomarkers able to discriminate rhEpo and placebo treatment could be identified.

Finally, the present study demonstrated that the novel markers of iron homeostasis, hepcidin and erythroferrone, are more sensitive markers of altered iron homeostasis within one week of changes in iron demands than routine iron markers. When individual thresholds were calculated, 30-40% of the rhEpo treated participants exceeded the thresholds. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise as novel biomarkers for detecting rhEpo misuse at altitude, but intra-individual variability is of concern and additional studies are required