Projets de recherche

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.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

 Chan, Hong Kong, Chine

Description à venir.

Ce document n'est actuellement disponible qu'en anglais.

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.