Projets de recherche

Code: 15A31JP 

A project (acronym GOpep) was approved by WADA with the objective of detecting a Neu5Gc containing EPO O-glycopeptide using latest generation MS instruments (i.e. AB Sciex Qtrap 6500).

The EPO O-glycopeptide shows the lowest glycan heterogeneity, thus being the best choice to reach the necessary MS sensitivity. Results obtained showed that the glycopeptide isoform containing Neu5AcNeu5Gc was found to be the most abundant form with its triply charge species at m/z 810.3 giving the best signal. A limit of detection of around 2 IU EPO/L, from a standard preparation was achieved, compatible with the expected concentrations in human urine. An antibody against the peptide was also developed and initial results show that it also recognizes the glycopeptide. However, matrix effect when spiking real samples at very low concentrations,
as well instrumental conditions to speed-up the analysis are still to be solved.
The hypothesis of this project is that MS sensitivity has reached a status in which EPO glycopeptides, particularly O-glycopeptides as they present lower heterogeneity, are detectable in urine or blood samples. Using a proper combination of specific peptide immunopurification with other desalting techniques, matrix effects can be avoided and the required sensitivity for the EPO O-glycopeptide containing the non-human tag (Neu5Gc) reached.

Objectives:
1.- To improve sample clean-up by using the already developed polyclonal antibody against the peptide backbone and other desalting techniques.
2.-. To improve the nanoLC-MS/MS set-up using monolithic columns for high throughput and on-line sample clean-up.
3.- To develop monoclonal antibody for future use of the methodology if the use of the polyclonal proves successful.
4.- To validate the procedure in urine and serum samples

Main Findings:

The main objective of the project was to develop an MS-based analytical procedure for the detection of an EPO O-glycopeptide containing the non-human monosaccharide N-glycolyl-neuraminic acid (Neu5Gc) as an unambiguous proof of the exogenous origin of the hormone (i.e. rEPO or analogues). The trypsin released EPO O-glycopeptide T13: (E117-R131) shows the lowest glycan heterogeneity, thus maximizing signal sensitivity while the peptide backbone will make it unique for EPO.
Through the method development, it was found that the formation of ubiquitous ammonium adducts was unavoidable, even under conditions where no ammonium salts were used.

The ammonium adducts of the doubly and triply charged species of the “endogenous” species (T13 O-2Neu5Ac) have masses identical (within 0.5 m/z) to the non-adduct forms of the “exogenous” species (T13 O-1Neu5Ac-1Neu5Gc) containing a 13C atom. The potential risk of having false positives forced the development of a method in which there was a complete chromatographic separation between these two very similar species.

The overall results demonstrated that the “exogenous” glycopeptide (T13 O-1Neu5Ac-1Neu5Gc) could be detected in rEPO using both a QTRAP6500 (low resolution) and an Orbitrap Fusion Lumos (high resolution). This approach includes the development of an MRM and PRM method respectively and the enrichment of the target EPO T13 O-1Neu5Ac1Neu1Gc using the anti-EPO T13 antibodies.

The method developed in this project is relatively straightforward, however the LOD using the most sensitive instrument in the market is still far from its applicability for real biological samples.

Still, as opposed to SAR-PAGE method, the proposed strategy may have the potential to unequivocally identify rEPO by detecting the 1-2% of T13 O-1NeuAc1NeuGc present in the sample using the signal of T13 O2Neu5Ac, which is present in all forms of EPO (exogenous and endogenous), as its own internal standard and quality control. The peptide chosen (T13) is unique for EPO, so it might allow the development of specific immunopurification techniques.

Code: ISF15D14ZG

The ability to increase oxygen carrying capacity to exercising skeletal muscles is a highly effective method for improving athletic performance. Unfortunately, some athletes seeking to gain an edge over their competition, have turned to artificially enhanced performance gains through blood transfusions, despite these methods being banned by the World Anti-Doping Agency. While methods such as flow cytometry can reveal heterogeneity in red blood cell (RBC) surface antigens and thereby detect homologous transfusions – or blood doping from a different person, there is currently no method available to detect autologous blood transfusions (ABT) with an athletes own blood. The lack of a direct detection method represents a significant problem for endurance sports, and the absence of a test means that this performance enhancing method is still widely utilized. There is evidence that biochemical changes occur in RBCs stored ex-vivo, including changes in their cell membrane that do not occur in a normal RBC population. One major obstacle to the development of a specific and reliable method for detecting ABT is then the lack of an available technique for detecting these age-related changes in circulation and at low concentration.  
The goal of this proposal is to develop the ability to quantify these modifications in red blood cell storage age using a combination of dielectrophoretic spectroscopy and a storage sensitive membrane cross-linking reaction, and to employ this approach to develop a simple and specific test for the detection of autologous blood transfusions in endurance athletes. The successful outcome of this project will lead to the development of an entirely new electrical approach to monitoring an athlete’s blood sample, and will lead to a new ABT indicator that is simple, rapid, require only a small droplet of blood, and capable of being integrated into an athlete's Biological Passport. 

Main Findings:

Background. The ability to increase oxygen carrying capacity to exercising skeletal muscles is an effective method for improving athletic performance. Unfortunately, some athletes have turned to artificially enhanced performance gains using blood transfusions, despite these methods being banned by the World Anti-Doping Agency (WADA). One main limitation in detecting autologous blood transfusions (ABT) is that there is no direct method capable of performing specific detection across a large transfusion regime, including detecting re-infusion with a small volume of blood under conditions when re-infusion has occurred multiple weeks prior to a competition. The significance of this project is based on this effort to develop a new method to overcome this problem using a combination of electrokinetics and microfluidics.

Results. We used electrodes to measure the electrical behavior of RBCs. To perform our experiments, RBCs were collected from healthy human volunteers and stored in storage buffer. We discovered that the electrical properties of RBCs change when cells are stored. We also developed an ABT assay that can quantify differences in mechanical elasticity of RBCs based on the ability for RBCs to deform in a microfluidic channel.

Conclusions. We believe that these two ABT assays complement each other and speculate that the microfluidic deformability assay can be used as a rapid screen for athletes to detect potential doped subpopulations of RBCs. The electrokinetic assay could then be used as a secondary detection method to verify the presence of aged RBCs. We are looking forward to evaluating the performance of these assays on in future work from samples collected from doping volunteers. 

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Code: 15C18MP 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported. 
Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study. However, scientific studies in humans are very rarely accessible.  
Thus, our project aims at investigating the effects of ecdysterone containing products on human athletic performance. A 12-week intervention study in young man will be conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements will be administered during the study to evaluate the performance enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement will be conducted. 
To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents regular screening for prohibited compounds is included in the project. Furthermore, the administered supplements will be tested for the absence of anabolic steroid contaminations. 

Main Findings: 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported.

Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible.

Thus, our project aimed at investigating the effects of ecdysterone containing products on human athletic performance. A ten-week intervention study in young man has been conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements have been administered during the study to evaluate the performance enhancing effect. Analyses of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement have been conducted.

To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents screening for prohibited compounds was also performed. Furthermore, the administered supplements have been tested for the absence of anabolic steroid contaminations prior to administration.

The ecdysterone administration led to increased serum IGF1 concentrations in comparison to the control group while thyroxin (T4) concentrations decreased.

Significantly higher increases in muscle mass were observed in those volunteers that were dosed with the ecdysterone supplements. Even more relevant with respect to sports performance, also significantly more pronounced increases in one-repetition bench press performance were observed.
These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. We therefore strongly recommend to include ecdysterone in the list of prohibited substances and methods in sports to improve clean competition in the future. As the exact mechanism of action is not yet fully understood, we suggest to include it in class S1.2 “other anabolic agents”.

Code: 15A29OP 

Screening for endogenous androgenic anabolic steroid (EAAS) misuse remains one of the main challenges in doping control.  Currently, this challenge is addressed by quantification of the seven markers forming the steroid profile included in the steroid module of the Athlete Biological Passport (ABP). The common approach for this quantification involves enzymatic hydrolysis, TMS-derivatization and GC-MS(/MS) analysis. However, several markers might be either lost or underestimated by this approach. 
Preliminary experiments performed by the project team show the occurrence of EAAS excreted as bis-sulfates and highlight their potential usefulness for the detection of testosterone misuse.  
The BIs-Conjugates in the Endogenous Profile of Steroids (BICEPS) project aims to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse. For this purpose, the project will be divided in two parts. 
In Part I, the usefulness of bis-sulfates for the detection of EAAS misuse will be evaluated. Bis-sulfate reference materials will be quantitatively synthesized and characterized, and an analytical approach for the quantification of urinary EAAS bis-sulfates will be developed and validated. The validated method will be applied to several samples from available drug administration excretion studies. 
In Part II, the occurrence and usefulness of other EAAS bis-conjugates will be evaluated. Libraries of steroid bis-glucuronides and glucuronide-sulfates will be synthesized. From these available reference materials an open screening strategy will be developed based on MS behavior of the synthesized compounds enabling the detection of EAAS bis-conjugates. The detection of these bis-conjugates in selected postadministration samples will be evaluated. 
Taken together, the BICEPS project will reveal which bis-conjugate metabolites are useful for screening of EAAS misuse. Further, the project will deliver a range of characterised reference materials derived by chemical synthesis for their further study and quantification. 

Main Findings: 

Several important EAAS might remain undetectable with the current approach used for the determination of the steroid profile i.e. gas chromatography-mass spectrometry (GC-MS) analysis after an enzymatic hydrolysis with E. coli β-glucuronidase, and the silylation of the steroids. Among them steroid bis-conjugates remain unexplored. The main goal of BICEPS is to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse.

Firstly, we have characterized the MS behavior of steroid bis-sulfates and we have developed an open screening method for their detection in urine. We have quantitatively synthesized 12 steroid bis-sulfates and we have developed and validated a quantitative method for their determination in urine. The method has been applied to samples collected after oral administration of testosterone undecanoate. We have evaluated several ratios between the validated analytes and we found that they have limited applicability for doping control purposes. However, we found two additional steroid bis-sulfates which allowed for the screening of the misuse with promising results. We hypothesize that these markers are two isomeric forms of the compound 3,16-dihydroxy-5-androstane-17-one bis sulfate. Synthesis of reference material is required to confirm the identity. Using these two markers, we obtained results comparable with those obtained with the best retrospective markers for oral misuse (resistant glucuronides and cysteinyl conjugates).

In a second part of the project we have synthesized some steroid bis-glucuronides and steroid glucuronide-sulfates. We have demonstrated the occurrence of some of them in human urine samples. Preliminary results showed that one of them (5α-androstane-3β,17β-diol 3-sulfate 17-glucuronide) clearly increased after oral testosterone administration supporting its potential usefulness for doping control.

Taken together, the results of BICEPS provide the first evidence about the potential usefulness of bis-conjugates in the doping control field.

The main results of BICEPS have been published at :
1.- McLeod MD, Waller CC, Esquivel A, Balcells G, Ventura R, Segura J, Pozo OJ*. A constant ion loss method for the untargeted detection of bissulfate metabolites. Anal Chem 2017; 89(3): 1602-1609.
2.- Pranata A, Fitzgerald CC, Khymenets O, Westley E, Anderson NJ, Ma P, Pozo O J, McLeod MD*. Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide Reference Materials: Unearthing Neglected Treasures of Steroid Metabolism. Steroids 2019: doi 10.1016/j.steroids.2018.11.017.
Some results have been presented at: 1.- “Steroid bis-sulfates: a forgotten minority” Oral presentation at SUPA2017, Targeting Steroid Sulfation Pathways, Birmingham April 2017

Code: 15E05AB 

We will develop a new approach to gene doping detection based on the leading edge technology, targeted massively parallel sequencing (MPS). Similarly to PCR-based methodology, doping genes will be detected by identifying sequences that do not feature introns present in natural genes. However, the MPS approach will be more reliable since multiple splice sites will be analyzed simultaneously. 
Its sensitivity is likely to be superior due to target enrichment during library preparation and to greater flexibility in choosing targeted regions. The MPS multiplexing capability will allow simultaneous analysis of many samples and genes, reducing test’s cost and turnaround time. We will develop a reference material for the MPS detection of five genes, most likely candidates for doping, and validate the test using blood samples. 

Main Findings:

In this project we developed and tested the protocol for a targeted MPS-based method for simultaneous detection of five transgenes in solutions of doping genes and genomic DNA that we used to mimic DNA extracts from athletes’ blood samples. Enrichment is achieved by PCR that amplifies a large portion of each transgene covering several exon-exon junctions, which should increase the likelihood of transgene detection. This also allows greater flexibility in selecting regions targeted by primers for enrichment PCR and provides a potential advantage of MPS over real-time PCR methods, where assay design is confined to a small area around the exon-exon border. Enrichment PCR for each transgene is performed separately in simplex, before the materials from five PCR, each for one transgene, are mixed prior to being subjected to library preparation and sequencing. With this experimental workflow, it will be relatively easy to add more genes to the detection panel; this will require designing primers and optimising enrichment PCR for new target genes.

We optimised all wet lab steps and designed, produced and tested a prototype reference material that is suitable for use in positive controls in the test.

We developed a tailored bioinformatics pipeline that reliably distinguishes doping genes from the corresponding endogenous genes and from the reference material. The pipeline enabled step-wise analysis of global distribution of alignments for sample and the RM, checking whether the sample is positive for the RM and doping genes, followed by analysis of distribution of alignments across the individual doping genes and their corresponding fragments in the RM and, lastly, making a final positive/negative call for individual doping genes. The process allows optimum elimination of false positives due to accidental contamination of sample with the RM.

Using ‘mock’ samples, we showed that MPS-based approach can detect down to five copies of doping genes in a background of genomic DNA similar in quantity to that in one mL of blood. We demonstrated that the amount of genomic DNA in a sample affects target enrichment for some transgenes more than others and this can affect the sensitivity of their detection.

As part of the evaluation of the long-term potential of the MPS for transgene detection, we performed preliminary assessment of the method's cost, sensitivity, reliability, turn-around time and required infrastructure and expertise, and compared it with the real-time PCR-base transgene detection.

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

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

Elite athletes can be persuaded not to take banned substances – either by appealing to their sense of morality or educating them about the risks of using performance-enhancing drugs, according to previous research.

The aim of this research project was to develop, implement, and evaluate an evidence-based moral intervention, and determine whether it is more effective than a standard educational (i.e., knowledge-based) intervention, in reducing doping likelihood in young athletes, in the UK and Greece.

Researchers developed two separate intervention programmes – one targeting moral factors associated with doping likelihood, the other introducing doping and providing information about the health consequences of banned substances and the risks of sport supplements.

Methodology

Phase 1 - Intervention Development and Screening Survey:

In this phase, the researchers developed a moral intervention consisting of six one-hour sessions aimed at changing predictors of doping intentions: moral identity, moral disengagement, and moral atmosphere. A screening survey was conducted involving over 1000 athletes across Greece and the UK to identify eligible participants with some likelihood of doping. The interventions were pilot-tested with athlete feedback guiding revisions.

Phase 2 - Intervention Delivery and Evaluation:

A total of 280 athletes from 24 clubs in Greece and the UK were selected for the study. Clubs were randomly assigned to receive either the moral or educational intervention. Participants were aged 16-22, participating in individual and team sports. The interventions were delivered over six-to-eight weeks, with measures collected at four time points. Data analysis employed a repeated measures multivariate approach, assessing outcomes for intervention type, gender, and time.

Phase 3 - Focus Groups:

After the interventions, focus groups were conducted with participants (32 in Greece, 35 in the UK) to gather qualitative insights.

Main finding

Both programs were tested on young elite athletes from the UK and Greece, finding that both approaches were effective at deterring the athletes from taking banned substances over a six-month period.

Impact for Clean Sport

1. Program Evaluation: The project highlights the importance of evaluating the effectiveness and outcomes of antidoping education programs. Understanding the impact of interventions is crucial for refining strategies and ensuring that efforts are yielding the desired results.
2. Promoting Values-Based Education: The findings advocate for the promotion of values-based education programs for doping prevention. By emphasizing values such as honesty, fair play, and ethical behavior, these programs can effectively reduce athletes' likelihood of engaging in doping.
3. Fostering Clean Sport Ideals: Values-based education can contribute to fostering clean sport ideals. By emphasizing the significance of ethical behavior and integrity, athletes are more likely to align with the principles of fair competition and uphold the spirit of clean sport.
4. Long-Term Effectiveness: The research demonstrated that the moral intervention had lasting effects on athletes' attitudes towards doping even six months after completion. This highlights the potential for creating interventions that have a sustained impact over time.
5. Policy and Advocacy: The study underscores the importance of advocating for values-based anti-doping education programs at policy levels. Such programs can contribute to a cultural shift within the sports community towards integrity, ethics, and honesty.

Overall, this research project contributes to the advancement of antidoping efforts by providing evidence-based insights into effective education strategies. By targeting athletes' moral identity and values, the project aligns with the overarching goal of maintaining a level playing field, upholding fair competition, and promoting clean sport principles.

Related Publications:

• A Moral Intervention Reduces Doping Likelihood in British and Greek Athletes: Evidence From a Cluster Randomized Control Trial
• Teaching athletes about morality in sport can help reduce doping

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