Projets de recherche

Code: 07E04KR

Sildenafil (phosphodiesterase-5 inhibitor) is recognized as treatment for pulmonary hypertension and erectile dysfunction. Ingestion at therapeutic dose shows profound improvement in pulmonary artery pressure, cardiac output, VO2peak, and exercise capacity at hypoxic conditions; at sea level breathing 10% O2 and at Mount Everest base camp (>5,000 m) in subjects free of lung disease. Olympic Nordic venues are often at mild altitude; the Salt Lake Olympic Nordic venue is approximately 1,700 m and the Turin venue is at 1,540 m altitude. Although the Vancouver Nordic venue is at 860 m altitude, significant reductions in VO2peak (-5.9%) and maximal 5-min cycle ergometry performance (total kJ, -3.6%) has been documented at simulated 580 m altitude. Therefore, further studies are needed to characterize potential ergogenic effects of sildenafil at mild altitude. Likewise, short-term inhalation of concentrated ambient air particles (PM) promotes vasoconstriction of small pulmonary arteries and produces pathologic features consistent with pulmonary hypertension. We recently found that breathing high levels of combustion-derived PM during exercise caused a significant (~5%) decrease in 6 min cycle ergometry work output. Since the pulmonary vasculature is a target for effects of ambient PM from fossil fuel combustion, oral sildenafil may enhance performance at ice rink venues (resurfaced with fossil-fueled machines) and at the upcoming Beijing and London Olympics venues which are likely to have high levels of PM. The aims of this project are to determine if a therapeutic dose of sildenafil enhances exercise performance and aerobic capacity 1) in high PM pollution and 2) at mild simulated altitude consistent with Olympic venue altitudes, 3) elucidate a potential mechanism, and 4) confirm measurement of sildenafil and metabolites in plasma and urine. These studies will provide evidence detailing performance enhancement from prior-to-competition ingestion of oral sildenafil at 1) high air pollution venues and at 2) mild altitude.

Main Findings

Exercise performance in this study was not significantly decreased in high particulate matter (PM) air for placebo or treatment groups. Although mean performance was 10 kJ lower for high pollution exercise, individual variability did not allow statistical significance. However, when data was analyzed after separating individuals into responders and non-responders as previously done in other studies, significant improvement in performance was noted for those who had a positive response to sildenafil. We further identified increased in mean pulmonary artery pressure (PAP) 1 h after high pollution exercise, but not after exercise in clean air that was associated with decreased performance. Ingestion of 50 mg sildenafil blunted the observed increase in PAP after high PM exposure exercise. The pulmonary artery pressures we recorded were normal for healthy young males, even after the increases recorded from high PM exposure exercise. However, the 18% change in PAP observed from high pollution exercise was significant (p=0.05) and likely affected exercise performance. In conclusion, oral ingestion of sildenafil citrate blunted the increase in pulmonary artery pressure resulting from exercise in high emissions particulate and was likely responsible for a noted improved exercise performance in the majority of subject in this study. Given the likelihood of international competitions being held in high emission environments, sildenafil citrate could provide an unfair advantage for some.

Code: 07C02FD

Diuretics are an indispensable group of therapeutics used to regulate the excretion of water and salts. By definition diuretics are drugs which increase the urinary flow. In sports diuretics are used for two main reasons: to flush previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. Diuretics are banned in sport by WADA. Diuretics cover a wide range of chemical products and one important group of diuretics are the thiazides. Because thiazides cover a whole class of structure related compounds, only a few thiazides are mentioned by name in the prohibited list: i.e. bendroflumethiazide, chlorothiazide and hydrochlorothiazide. Thiazides have an amidophenamide (AP) structure in common. AP is described as an aqueous degradation product for thiazides. Because of the potential hydrolysis of thiazides in urine this latter compound should be included in the screening methods for diuretics. Because the information regarding the degradation is limited a study concerning the degradation parameters can provide new and useful insights for doping analysis. In this project the effect of pH and temperature will be investigated. Three thiazides will be included in this study: altizide, hydrochlorothiazide and chlorothiazide. Altizide and hydrochlorothiazide are commercially available on the Belgian market. Chlorothiazide was also included in this study because it can be detected as metabolite for hydrochlorothiazide and bemetizide. Metabolites or degradation products can often be detected for a longer time in urine than the parent compounds. Therefore, in a second part of this project the detection times of metabolites and degradation products for two commercial available thiazide preparations, namely Docspirochlor (containing hydrochlorothiazide) and Aldactazine (containing Altizide), will be investigated.

Main Findings

The goal of this project was to investigate the stability of the thiazide diuretics altizide, hydrochlorothiazide and chlorothiazide both in vitro and in vivo. Not only the degradation of the parent drug was investigated also the formation of the degradation compound 4-amino-6-chloro-1,3-benzenedisulponamide was monitored. The results of the in vitro studies show that the thiazides are degradated faster at higher pH and higher temperature. In particular the lower pH improves the stability. When altizide and hydrochlorothiazide were exposed to UV-light, they photodegradate to chlorothiazide. When the degradation rate between the different compounds was compared for a given temperature and pH, altizide is the most unstable compound. Concentrations ranged between 41-239 ng/mL and 60-287 ng/mL after altizide and hydrochlorothiazide administration, respectively.

Publications

Deventer K, Baele G, Van Eenoo P, Pozo OJ, Delbeke FT. Stability of selected chlorinated thiazide diuretics. J Pharm Biomed Anal. (2009); 49(2):519-24.

Deventer K, Pozo OJ, Van Eenoo P, Delbeke FT. Detection of urinary markers for thiazide diuretics after oral administration of hydrochlorothiazide and altizide-relevance to doping control analysis. J Chromatogr A. (2009); 1216(12):2466-73.

Code: 07C03FD

Recently, a few methods for urinalysis of threshold substances via LC-MS have been developed and published. These methods allow for the direct analysis of urine samples without the need for extraction. The minimal sample preparation of these methods offers several advantages besides cost effectiveness and speed. Indeed, lower sample preparation leads to a reduction in the factors contributing to the measurement uncertainty of a result. Moreover, the required volume of urine for the quantification is limited and the methodology allows for the direct quantification of Phase 2 metabolites. Direct quantification of conjugates is preferred above hydrolysis followed by a quantification of the deconjugated substances since incomplete hydrolysis and or degradation effects (e.g. endogenous steroids) can lead to quantification errors. These errors are an important factor in the bias of current quantitative methods and of the uncertainty estimates. The current project would develop direct LC-MS quantification methods for all WADA threshold substances with a minimal sample preparation procedure. The developed methods would then be implemented and validated in all participating laboratories and a common measurement uncertainty estimate would be made to harmonize methodologies and decision limits (concentration above which a sample can be regarded as exceeding the threshold taking into consideration MU). As such the project would not only harmonize methodologies but also decision criteria leading to a more uniform interpretation of results. Moreover, taking into account the simplified sample preparation and the extensive harmonization of these methods among the participating laboratories it can be expected that the inter-laboratory variation will be lower than currently. Such a reduction in inter-lab variability is a primary objective for an adequate use of individual athlete passports with biometrical data. This hypothesis will be tested in the last phase of the project during which PTsamples will be distributed for every threshold substance to all laboratories. These samples will be quantified using the procedures currently applied in these laboratories as well as with the new unified methodologies. This approach will allow for a direct comparison and evaluation of the effectiveness of the harmonization of methods.

Main Findings

One of the goals of this project was to develop simple and robust quantification methods to quantify all WADA threshold substances using asimplea dilute-and-shoot approach. These methods were developed and validated and MU was estimated for each of them. All were compliant with WADA’s TD’s. Every laboratory implemented the developed methods. A proficiency test was set-up and the values obtained via these methods and the laboratories own methods revealed that there were no clear differences in the quality of the data. No real benefit was obtained and no clear cut reasons could be identified when smaller differences were noticed. Perhaps, this is caused by the high quality standard WADA already demands from accredited methods/laboratories.

Publications/Presentations

Deventer K et al. :Direct quantification of morphine-glucuronides and free morphine in urine using LC-MS/MS. Cologne, 13-02-2011, 29st Cologne Workshop on Dope Analysis.

Deventer, K., Pozo Mendoza, O. J., Verstraete, A., Van Eenoo, P. (2014). Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis in doping control and analytical toxicology. TRAC-TRENDS IN ANALYTICAL CHEMISTRY, 55, 1–13.

Deventer, K., Pozo, O., Delbeke, F., Van Eenoo, P. (2012). Direct quantification of morphine glucuronides and free morphine in urine by liquid chromatography-tandem mass spectrometry. FORENSIC TOXICOLOGY, 30(2), 106–113.

Sardela, V., Deventer, K., Pereira, H., Neto, F., Van Eenoo, P. (2012). Development and validation of a ultra high performance liquid chromatography-tandem mass spectrometric method for the direct detection of formoterol in human urine. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 70, 471–475.

Chebbah C1, Pozo OJ, Deventer K, Van Eenoo P, Delbeke FT. Direct quantification of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis. Rapid Commun Mass Spectrom. 2010 Apr 30;24(8):1133-41

Code: 07C01FD

Beta-agonists are substances frequently used for the treatment of asthma. These drugs are most frequently administered by inhalation of aerosol, powder or nebulised solution. Besides the main pharmacological effect, at higher doses, side effects of the use of these products result in anabolic action. Hence, b2-agonists might be misused for their stimulating effect on respiration and growth promoting action when administered in higher doses. As a result, WADA has prohibited the use of these drugs in sports. Exceptions are formoterol, salbutamol, fenoterol, salmeterol and terbutaline which can be used by athletes if a proper medical justification (TUE) is issued. Although this group of medication is frequently declared by athletes on the doping control forms these substances are not always detected. In addition, no minimum required performance levels (MRPL) for laboratories have been presently issued by WADA. Information about the excretion of b2-agonists in urine after the use by different administration routes could result in the establishment of MRPL levels based upon scientific evidence. Therefore this project will focus on the administration of different b2-agonists by different administration routes. In this project 5 b2-agonists will be administered (i.e. salbutamol, formoterol, fenoterol, salmeterol and terbutaline) of which 2 of them will be administered both by inhalation and orally. All these studies will be conducted following a strict research protocol as approved by an ethical committee. As the supposed outcome of this research is a MRPL level, the first step of this project is to develop and validate quantitative analytical methods for all administered b2-agonists (except salbutamol for which these methods already exist).

Main Findings

The goal of this project was to investigate which concentrations can be detected in urine after therapeutical inhalation of β2-agonists. For this purpose a quantitative GC-MS method was developed for the detection of salbutamol. For salmeterol, formoterol and terbutaline LC-MS/MS methods were developed and validated. Since no isotopic labeled internal standard was available for fenoterol, only a semi quantitative LC-MS/MS method could be developed. After the validation, the developed methods were used to investigate the urinary excretion of the β2-agonist after oral administration of 4 mg of salbutamol (Ventolin®) and by inhalation of: Ventolin® (800 μg salbutamol), Serevent® (100 μg salmeterol), Oxis® (18 μg formoterol), Bricanyl® (500 μg terbutaline) and DuoventHFA® (100 μg fenoterol). After oral administration of salbutamol urinary concentrations reached 2626 ng/mL. After inhalation of a high therapeutic dose of salbutamol, the threshold of 1000 ng/mL was almost exceeded for one volunteer (994 ng/mL). The results show that the concentration of salmeterol and formoterol reach a maximum after 1 and 3 hours after ingestion. The highest salmeterol concentration detected was 1.27 ng/mL. For formoterol highest concentration was 11.4 ng/mL. After inhalation of terbutaline and fenoterol highest concentration detected were 197 ng/mL and 58.3 ng/mL, respectively. These concentrations were then compared with routine samples in which an above mentioned β2-agonist was found. In this way samples containing salmeterol (n=45). Formoterol (n=82), terbutaline (n=8) and fenoterol (n=3) were collected and quantified using the developed methods. Concentrations in these samples could not be related to the use of abnormal high doses of β2-agonists. Publications/Presentations

Presentations:

• Presenting “Excretion Studies with β2-Agonists” at the VI Latin American Workshop in Doping Analysis, November 8 to 11, Asunción- Paraguay, 2009.

• Presenting “Excretion Studies with β2-Agonists” at the 28th Workshop on dope analysis, 2010, Cologne, Germany.

Publications:

• Excretion Studies with β2-Agonists, K. Deventer, W. Van Thuyne, O.J. Pozo, P. Van Eenoo, F.T. Delbeke, Proceedings of the 28th Workshop on dope analysis, 2011, Cologne, Germany.

• Quantitative detection of inhaled salmeterol in human urine and relevance to doping control analysis, Submitted to Drug Testing and Analysis.

• Quantitative detection of inhaled formoterol in human urine and relevance to doping control analysis, in preparation.

• Quantitative detection of inhaled terbutaline in human urine and relevance to doping control analysis, in preparation.

Code: 07C28CA

Insulin issued from the recombinant technology present the same amino acid sequence as the human endogenous hormone and a molecular weight of 5807 Da. Different analogues have been commercialized with slight amino acid modifications affecting their molecular weight, except for Humalog (Eli Lily) that presents only two interchanged amino acids resulting in a polypeptide chain of same molecular weight than human insulin. A LCMS/MS detection method for rapid-acting analogues in plasma and urine was recently published (Thevis et al., 2005; Thevis et al., 2006). The urine method proposes an immunoaffinity purification from a 25 mL urine aliquot and a top-down sequencing based mass spectrometric approach on a triple quadrupole instrument. The study of the lowest molecular weight ions after fragmentation in the collision cell allows differentiation of the analogues, even if their precursor mass is identical. This approach is innovative but can not be adapted on regular 3D ion trap as fragmentation of large molecule is difficult. As a result, no low molecular weight ions can be observed. Preliminary works have shown that a bottom-up approach using peptide hydrolysis can release smaller fragments that can easily fragment to the desired diagnostic ions. It is proposed that hydrolysis can be performed prior to analysis on an ion trap to monitor the smaller peptide containing the susceptible amino acid interchange, and then fragmentation pattern can qualify the analogues. The goal of this project is to develop a detection method for hydrolysis products of insulin and rapid-acting analogues using an ion trap mass spectrometer. Complementarily, a simpler purification method using single-use immunoprecipitation will be tentatively developed and compared with the 3steps approach.

Main Findings

Insulin testing is not widespread; as a matter of fact, in 2012 it is available in only a handful of laboratories. This method consuming a quite important volume of the urine sample and requiring cumbersome sample preparation must be applied aside from those routinely employed, the testing organizations have been reluctant as well to request insulin testing considering the costs involved. Whilst research is still ongoing (LIVE), this relatively pragmatic approach was put in place for the analysis of the 2010 Olympic Games samples. The simplified purification with immunoprecipitation allowed the analysis of a greater number of specimens. As a result, for the first time during Olympic Games, over 200 samples, i.e. 10% of samples could be analyzed. With current advancements in LC-MS/MS technologies (nanoflow, nanospray ionization, selexION, Qtrap 6500 etc), this method could be further improved as extraction volumes could be reduced to a more practical volume in the low mL range and as well, lower injection volumes would reduce ion suppression.

Code: 07D02SL

Numerous pharmacological and/or genetic strategies exist that simulate the effects of hypoxia at the cellular level and increase expression of hypoxia-induced genes such as hypoxia-inducible factor (HIF), its downstream targets such as erythropoietin (EPO) and consequently increase red blood cell production. Many of these strategies / molecules were developed to treat diseases such as the anemia associated with chronic renal failure and indeed are mainstays in the medical management of these conditions. However, these strategies have great potential to be abused by elite athletes seeking to gain a (unfair) competitive advantage, since they are, 1) currently available and 2) not efficiently detectable by current anti-doping protocols (particularly the newer molecular / gene-based strategies that are subject of this proposal). Prime amongst the pharmacological and / or genetic strategies that have great doping potential are those aimed at modulating hypoxia-induced genes by inhibiting HIF-prolyl hydroxylase (HIF-PHD), a key regulatory enzyme that regulates HIF. Both small molecule inhibitors against HIF-PHD (such as iron chelators or small molecules are able to efficiently inhibit HIF-PHD) and direct gene transfer (i.e. RNAi-based) strategies knocking down HIF have been shown be effective. Of particular concern regarding 'doping' by athletes is the availability drugs/small molecules such as clinically used iron chelators (desferrioxamine-DFO, ciclopirox olamine-CPX) and small molecules (e.g. FG-2216, FG-4519) developed by FibroGen, that are currently in human Phase II/III trials. Currently, tests do not exist to detect hypoxia induced gene 'doping' with pharmacological and/or genetic strategies such as those listed above. The fact that a number of web-based resources and pharmaceutical firms currently offer 'HIF-PHD inhibitory reagents, underscores the need for rapidly developing a test to detect hypoxia induced-gene doping not just the currently described reagents but also those that would be used in the near future, such as gene transfer using RNA-based inhibitors (such as RNAi molecules against HIF). Thus, the challenge is to develop standardized detection tests that would be specific, sensitive and standardized enough to hold up to legal challenges that anti-doping agencies would almost certainly face by athletes caught using these tests. These challenges are not insurmountable; we propose to develop a robust and standardized, blood-based test using 'molecular signatures' of hypoxia that will detect all the different forms (of small molecule and gene-based) hypoxia-induced gene doping that are currently in use (or likely to be developed in the near future) with great sensitivity and specificity to serve this need.

Main Findings

Numerous physical, pharmacological and/or genetic strategies exist that simulate the effects of hypoxia at the molecular and cellular level and increase expression of hypoxia-induced genes such as hypoxia-inducible factor (HIF), its downstream targets such as erythropoietin (EPO) and consequently increase red blood cell production. While hypoxia was classically achieved by exposure to high altitude (hypobaric hypoxic exposure), there are currently numerous methodologies for achieving hypoxia-induced gene doping including chambers (normobaric hypoxia), chemicals and genetic manipulation. Our basic hypothesis is that exposure to different types of hypoxia lead to both a unique 'molecular signature' specific to the type of hypoxia as well as core 'molecular signature' irrespective of the type of hypoxia. Testing the 'molecular signatures of hypoxia' using blood samples from athletes will detect all the different forms (of physical, small molecule and gene-based) hypoxia-induced gene doping that are currently in use (or likely to be developed in the near future) with great sensitivity and specificity. Identification and definition of these molecular signatures would allow detection of hypoxia-inducible gene doping and preclude abuse by elite athletes seeking to gain a (unfair) competitive advantage using this strategy. The project was funded to provide proof of concept that the 'molecular signatures' (in peripheral blood) are distinct for different types of hypoxiainduced doping. We have been able to demonstrate that different hypoxic conditions tested experimentally (hypobaric/high-altitude, normobaric/ chambers or tents and chemical) have both a unique 'molecular signature' specific to the type of hypoxia as well as core 'molecular signature' irrespective of the type of hypoxia. To be comprehensive and rigorous experiments were conducted both in laboratory conditions and in field studies on Mt. Everest at altitudes up to c. 8400 mts. The 'molecular signature' generated during the pilot studies have been deposited into the WADA Informatics website and GEO databases (GEO Accession Numbers GSE 15894, 15901 and 15902). Further we have been able to validate the data by Taqman based qPCR assays. These findings are of great relevance for development of anti-doping efforts as it provide the first evidence that sensitive and specific anti-doping tests to detect hypoxia-inducible gene doping can indeed be developed using the bioinformatically generated 'molecular signatures' we have identified. Based on the progress and tangible resources that we have generated, we are confident of achieving our overall goals to develop sensitive and specific tests to comprehensively and rigorously detect hypoxia induced gene doping and preclude abuse by elite athletes seeking to gain a (unfair) competitive advantage using these strategies.

Code: 07E06KC

It is generally accepted in the sporting world that glucocorticoid use enhance maximal performance, and, as a consequence, this pharmacological class is banned by the World Antidoping Agency (WADA) after systemic administration. Literature on the ergogenic effects of glucocorticoid intake appears, however, very scarce. Indeed, as a matter of fact, research is limited to a few studies for both acute and short-term administration. Using an animal model, Gorostiaga et al. (1988) showed that a single injection of glucocorticoids (cortisol acetate, 100 mg.kg body wt-1) is capable of improving endurance in female rats. In contrast, Soetens et al. (1995) did not find any significant increase of maximal performance with 1 mg ACTH injection in professional cyclists. Similarly, we showed previously (2005, submitted) that an acute therapeutic administration of oral prednisolone (20 mg) does not improve the time of cycling until exhaustion during submaximal exercise (70-75% VO2 max and 80-85%VO2 max) in healthy moderately trained male volunteers, despite a probable increase in lipid oxidation and decrease in CHO oxidation (in press). Regarding published studies following short-term administration of glucosteroids, only one study has focused on the effects of short-term dexamethasone intake (0,5 and 1,5 mg per day for 4.5 days) (Marquet et al., 1999) during maximal exercise without demonstrating any ergogenic effect of the treatment. We recently demonstrated (in press), however, that short-term therapeutic prednisolone intake (60 mg per day for 7 days), contrarily to acute intake, significantly improved performance in healthy men during submaximal exercise (70-75% VO2 max). The concomitant alterations in the hormonal and metabolic exercise parameters analyzed showed that shortterm administration of this drug had both central and peripheral effects. Further studies will, however, be necessary to elucidate the mechanisms of these hormonal and metabolic changes in particular after short-term intake in order to determine which changes may be associated with the marked performance improvement obtained only after this mode of administration. Moreover, to our knowledge, no study has focused on women and a specific gender response to glucocorticoid can be questioned. We therefore propose to contribute to a wider knowledge of glucocorticoid action mechanisms during exercise with in particular investigation of: 1) the ergogenic impact of these drugs in women; 2) the endocrine and metabolic responses after short-term glucocorticoid administration during longer exercise (3 hours), with regard to the gender status of the subjects in order to elucidate the mechanism(s) of action involved in the improvement in performance.

Main Findings

We showed in the first study that short-term therapeutic prednisolone (Cor) intake (50 mg per day for 7 days) improves significantly performance in healthy recreationally women during submaximal exercise (70-75% VO2 max). ACTH and DHEA remained completely blunted throughout the experiment with Cor vs. Placebo (Pla), whereas growth hormone and prolactin were significantly decreased with Cor during exercise. No significant difference in insulin or blood glucose values was found between the two treatments, suggesting that women may be less sensitive than men to glucocorticoid-induced insulin-resistance. These data indicate that short-term glucocorticoid intake improved endurance performance in women, but further investigation is needed to determine whether these results are applicable to elite female athletes. In parallel, during a long-lasting exercise (2 hrs), no significant difference in glucagon, insulin or free fatty acid values was found between the treatments. However, essential amino acids (in particular BCAA) and blood glucose were significantly higher after Cor vs Pla during the second part of exercise. It appears therefore that proteolysis probably increases with glucocorticoid during long-lasting exercise and that the related higher plasma EAA concentrations may contribute as energy substrates. Regarding saliva DHEA and cortisol concentrations, they decreased strongly immediately after the start of prednisone treatment, demonstrating a rapid suppression of the HPA axis. However, it is only a short-lasting suppression, as 3 days after concluding prednisone administration, both saliva DHEA and cortisol had returned to pretreatment levels. We can therefore conclude that: 1) this is no high risk of adrenal insufficiency after such treatment (i.e., 1 week at high therapeutic dosage). 2) non invasive saliva samples may offer a practical approach to assessing pituitary-adrenal function continuously during and after short-term corticosteroid therapy. No significant change was found in body weight, body composition or food intake after 7 days of glucocorticoid treatment, which induces however a significant leptin increase. In parallel, no significant physiological repercussions were noted. Such glucocorticoid treatment does not promote obesity in recreationally trained women. However, further studies are necessary to understand its stimulating effects on the metabolically active hormone leptin.

Publications

Short-term glucocorticoid intake improves exercise endurance in healthy recreationally trained women. Le Panse B, Thomasson R, Jollin L, Lecoq AM, Amiot V, Rieth N, De Ceaurriz J, Collomp K. Eur J Appl Physiol 107(4): 437-443, 2009.

Saliva DHEA and cortisol responses following short-term corticosteroid intake. Jollin L, Thomasson R, Le Panse B, Baillot A, Vibarel-Rebot N, Lecoq AM, Amiot V, De Ceaurriz J, Collomp K. Eur J Clin Invest 40(2): 183-186, 2010.

Short-term glucocorticoid and metabolic responses during long-lasting exercise Thomasson R, Rieth N, Jollin L, Lecoq AM, Amiot V, Lasne F, Collomp K. Eur J Appl Physiol

Congress Presentations

Short-term glucocorticoid intake improves exercise endurance in healthy recreationally trained women. Collomp K, Le Panse B, Thomasson R, Jollin L, Lecoq AM, Amiot V, Rieth N, De Ceaurriz J. Abstract accepted (April 2010) in European Congress of Sport Science (ECSS) congress, Antalya, July 2010

Saliva DHEA and cortisol responses following short-term corticosteroid intake. Jollin L, Thomasson R, Le Panse B, Baillot A, Vibarel-Rebot N, Lecoq AM, Amiot V, De Ceaurriz J, Collomp K. Abstract accepted (April 2010) in European Congress of Sport Science (ECSS) congress, Antalya, July 2010

Code: 07C26CG

Uncertainty is an important analytical and legal issue for reporting WADA Prohibited List threshold substances. Threshold substances require quantitative analysis to compare analytical signal and concentration of the substance of interest in the athlete’ sample to the threshold value. The importance of the uncertainty parameter in reporting an adverse analytical finding of a threshold substance is the fact that the uncertainty value is used as an, among others, additional protection for the athlete from a highly improbable false positive report. On the other hand, a high value of uncertainty can guide to false negative results. For that reason, uncertainty has been found in the middle of court evidence arguments. Consequently, a correct and harmonized calculation of uncertainty is required not only for legal reasons but also to reveal probable analytical drawbacks. More specifically, the current project proposes:

1. To evaluate uncertainty estimation for isotope ratio mass spectrometry and hematological analyzers technologies, where pour literature has been published so far.

2. To survey within the WADA Accredited Doping Control Laboratories various ways for uncertainty estimation of the quantitative procedures.

3. To incorporate uncertainty specifications from the under preparation WADA technical document on uncertainty

4. To develop a software tool for uncertainty evaluation, specific for the WADA Accredited Doping Control Laboratories based on the 1-3 above paragraphs

5. To distribute a first version of the software tool to WADA Accredited Doping Control Laboratories to perform a first evaluation.

6. To collect evaluation points, apply a second software version and distribute the software to the WADA Accredited Doping Control Laboratories.

Main Findings

Uncertainty is an important analytical gauge for the quality of the analytical data that are produced from a quantitative procedure. Uncertainty estimation is based mainly on validation data for the ISO 17025 accredited analytical methodologies. Moreover, uncertainty is added or subtracted to the concentration of the, according to the WADA List, threshold substances found in doping control urine or blood samples in order to decide to report for an adverse analytical finding or not, i.e. uncertainty is a major decision parameter. According to the International Standard for Laboratories, measurement uncertainty reporting for the prohibited substances required quantification is mandatory and WADA Accredited Laboratories are obliged to comply with this specification. WADA will bring into force by September 1st, 2010, the technical document TD2010DL entitled “DECISION LIMITS FOR THE CONFIRMATORY QUANTIFICATION OF THRESHOLD SUBSTANCES”, where a full description of the uncertainty estimation scheme is included. Facilitating and harmonising the way that Accredited Doping Control Laboratories estimate uncertainty to their quantitative methodologies will add one more ring in the WADA lab network chain. The project has concentrated information for the various ways of uncertainty estimation within the WADA Accredited Laboratories, the WADA Technical Document and from literature sources and it has provided with a friendly software tool called MUADS for this purpose.

Code: 07A01JR

As autologous blood doping augments the athlete’s blood with their own cells, detecting the procedure is challenging. Blood cell shape and distribution are being explored as potential markers for; however, changes in the transcript (the mRNA intermediate in the DNA-to-protein pathway) complement of the blood may be a more informative indicator of reinfused blood cells in an athlete. The transcript complement of a tissue is extremely sensitive to the environment, so blood cells that have been removed, processed and stored may show changes in transcript frequencies that could be diagnostic for autologous blood doping. RNA is an excellent candidate for doping tests as it is stable when stored correctly, quantifiable using commercially supported techniques, and measurable in very small blood samples. This proposal describes a one year pilot project using high-resolution transcription profiling of blood to identify transcripts with the potential to be used as a diagnostic test for autologous blood doping. The project has three phases: 1) identification of transcripts in whole and leuko-depleted blood that represent genes that are activated or up-regulated in blood cells in response to post-withdrawal processing and/or short-term storage, 2) use of simulated autologous blood doping to determine if these transcripts could be detected in a “doped” athlete; and 3) characterization of the inter- and intra-individual variation of the transcripts shown to have potential test utility under a variety of conditions experienced by athletes (e.g. physical exertion, hypoxia/altitude, time of day, and time in menstrual cycle). The ultimate goal of the project is to identify transcripts that are characteristic of stored blood, that are of sufficient quantity as to be identifiable in a recipient’s circulation following reinfusion, and have patterns of expression that are consistent, predictable, and informative in athletes, which believe could form the basis for a blood-based, gene-expression test for autologous blood doping.

Main Findings

The aim of this study was to determine if there were genes overexpressed in blood in response to refrigerated storage that could be used as markers for the presence of stored blood in an athlete’s circulation. To be considered a potential transcript for follow-up, there had to be a minimum of a 10 fold expression difference (as this represented he the typical dilution that would occur in an autologous reintroduction of a unit of blood into the ‘doper’s’ circulation. We also felt that the overexpressed transcripts needed to be part of a unique pathway and not a variant of other commonly expressed pathways, as those would be unlikely sufficiently robust to serve as a diagnostic tool. Outcome: Transcriptional analysis identified only a single non-hemoglobin gene that met the criteria for being a candidate for a test for blood storage. The gene was GADD45G (encoding: Growth arrest and DNA-damage-inducible protein GADD45 gamma). This gene is a stress response gene that has been shown to be up-regulated in circulating cells in response to ‘behavioral stress’ in animal models (e.g. restraint, Flint at al., J Neuroimmunol. 2005; 167:34-44). This response would likely make it an unpromising candidate for a doping control assay as expression levels would likely fluctuate between athletes due to differential responses to perceived stress (e.g. doing control, imminent or recent competition). The only other genes that fit the candidacy profile were ‘rare’ hemoglobins (delta and zeta). As the adult blood transcriptome is overwhelmingly made up of hemoglobin alpha and beta transcripts, it is unlikely that an assay sufficiently specific and sensitive could be developed based on differentiating between the highly homologous hemoglobin transcripts against this background. Overall, the results of the project do not support the use of transcriptomic analysis of athletes' blood as a method for detecting the inclusion of stored blood (i.e. autologous blood doping).

Code: 07A04CR

Non-specific binding of the monoclonal anti-human EPO antibody (clone AE7A5; R&D Systems, Inc.) used for the detection of doping with recombinant human erythropoietin by the worldwide practised isoelectric focusing (IEF) and Western double blotting method has been in discussion for several years. Publications in peer-reviewed journals addressed this subject in 2005 and 2006 and led to some scientific argumentation on the specificity of the employed detection antibody. However, under experienced users of the Epo-method several additional bands (usually two to four) in the basic region of the pH 2-6 IEF-gel caused scientific interest, because theses bands could only be occasionally detected among the tested urinary samples. Their relationship to Epo remained unclear for years. Nevertheless, it was obvious that these bands did not interfere with the endogenous urinary and recombinant Epo-IEF profiles and thus the evaluation of the profiles was not disturbed or questioned at all. We were able to identify the protein causing these basic IEF-bands by means of carrier ampholyte IEF-PAGE, SDS-PAGE, Western blotting, and nano-ESI high resolution mass spectrometry and presented the results at the 2007 Cologne Workshop on Dope Analysis. The name of the protein is Zinc-alpha-2-glycoprotein. It is a high abundant urinary protein. The purpose of this project is a detailed study on the specificity of the clone AE7A5 antibody. According to the WADA technical document on the “Harmonization of the method for the identification of epoetin alfa and beta (Epo) and darbepoetin alfa (NESP) by IEF-double blotting and chemiluminescent detection” the IEF-method “relies on the particular specificity of the monoclonal antibody with which it was developed (clone AE7A5)” and “this antibody is considered a critical reagent and shall not be changed”. However, during SDS-PAGE experiments we were able to show that AE7A5 also binds to urinary proteins other than erythropoietin and Zinc-alpha-2-glycoprotein. These proteins might interfere with the interpretation of the Epo-IEF profiles.

Main Findings

A comprehensive study on the non-specific binding behaviour of monoclonal anti-EPO antibody clone AE7A5, which has to be used according to the technical document on EPO-analysis of WADA (TD2009EPO) for the detection of EPO-doping, was performed. Human urine and serum were used as matrices, since both are also used for EPO-testing. Additionally, E. coli and S. cerevisiae lysates were investigated. Aside from zinc-alpha2-glycoprotein (ZAG) a weak interaction with transferrin was detected (urine, serum). However, the non-specific interaction of the four proteins, which were mentioned by Khan et al. (2005) [1] in the context of 2D-PAGE (Tamm Horsfall glycoprotein, alpha-1antichymotrypsin, alpha-2-thiol proteinase inhibitor, alpha-2-HS-glycoprotein precursor), could not be confirmed – neither on carrier ampholyte IEF- nor SDS-PAGE. Strong binding was observed for thioredoxin reductase of E. coli, but no interaction occured with human thioredoxin reductases 1 and 2. Another strong non-specific binding was found for enolase from S. cerevisiae. Contrary to E. coli thioredoxin reductase, human enolases (tested were two enolases from human brain) were also non-specifically detected by the antibody. E. coli thioredoxin reductase as well as human and S. cerevisiae enolases are also non-specifically bound on IEF-PAGE. However, due to their higher isoelectric points (pI) these proteins focus outside the region of the gel used for EPO evaluation and therefore have no influence on the analysis results. Due to the observed non-specific interactions of the clone AE7A5 anti-EPO antibody with proteins other than EPO, immunoaffinity purification is mandatory for SDS-PAGE analyses. This pre-cleaning step has already been performed in the past, since the high protein content of urinary retentates and serum samples impedes the accurate detection of EPO by SDS-PAGE. This step is mandatory due to the non-specific binding of the antibody to thioredoxin reductase of E. coli. The molecular mass of this protein is within the range of rhEPOs on SDS-PAGE. An interference with the detection of rhEPO-doping is excluded by the combined usage of (1) two monoclonal antibodies directed against different epitopes for the immunoaffinity purification step, (2) the Western double blotting procedure, and (3) the usage of IEF- and SDS-PAGE for the interpretation of analysis results. This strategy has already been implemented in TD2009EPO. Reference: [1] Khan A, Grinyer J, Truong ST, Breen EJ, Packer NH (2005) New urinary EPO drug testing method using two-dimensional gel electrophoresis. Clin Chim Acta 358:119-30.