Projets de recherche

Code: 07C10JA

One of the aims of our ongoing (LIVE) study financed by WADA has been to find microbial contaminants of urine samples. We developed an in vitro simulation system for urine to mimic the storage and transportation conditions of urine prior to testing. Our hypothesis was that yeasts and bacteria are the most probable contaminants responsible for the adverse reactions in urine samples. Our preliminary results suggest that a complex microbial community such as that found in human saliva and feaces has a potential to modify the steroid profile of urine. The abovementioned findings have motivated us to accelerate the development in the area of microbial contamination and focus efforts on the real doping control samples. Here we are proposing an one year research project with following objectives;

(i) Identify contaminating microbes from real doping control samples (sent to Helsinki doping control site)

(ii) Analyze the source of contamination for those samples

(iii) Find the most important contamination parameters that affect the reliability of doping control analyzes

(iv) Design routine laboratory test to find seriously contaminated doping control samples

(v) Design procedures to eliminate microbial contamination or reduce its risk to the doping control analyzes

Main Findings

The microbial characterization of urine samples indicates that urogenital and gastrointestinal tract act as most probable contamination sources. All major microbial groups detected can be explained by non-intentional contamination sources. Intentional contamination is an existing possibility, but based on the results of this project we would put the research effort on the bacteria representing natural, unintentional contaminants. Contaminated samples dominated by Lactobacillaceae and Enterococcaceae did not harbor as high microbial numbers as those dominated by Enterobacteriaceae and Pseudomonadaceae. Overall, the detected bacteria are known capable of altering the steroid profiles, emphasizing the importance of high hygiene level at sampling for a reliable doping control. However, in optimal conditions a low bacterial amount can increase exponentially to high levels in a short period of time. Elevated pH is one of those indicators which are used in doping control laboratories to recognize microbial contamination. This screening parameter may be used together with a number of other criteria, e.g. the presence of free steroids in a urine sample, but has very limited selectivity alone. Abnormal smell or turbidity does not correlate with microbial growth. Several studies have been carried out for the stabilization of human urine samples. None of the investigated physiological methods, including sterilization by filtration, ultraviolet radiation, or ultrasonication, have succeeded in preventing microbial growth. Chemical methods have been shown to be more efficient, but the introduction of any chemical substance into athletes’ samples after collection may be difficult to approve legally. Consequently, rapid freezing has proved to be the only feasible method for stabilizing samples and preventing microbial activity.

Publications: Ojanperä S, Leinonen A, Apajalahti J, Lauraeus M, Alaja S, Moisander T, Kettunen A. Characterization of microbial contaminants in urine. Drug Test. Anal. In press.

Presentations: Alaja S, Apajalahti J, Leinonen A, Kettunen A, Ojanperä S, Kuuranne T, Lauraeus M. Characterization of microbial contaminants in urine. Poster in the Manfred Donike Workshop – 28th Cologne Workshop on Dope Analysis, 2010, Köln, Germany.

Code: 07C11AK

In nature, different forms of carbon atoms exist called isotopes. The most common carbon atom has a weight of 12 but a smaller amount naturally exists as a heavier weight of 13. In testosterone, the proportion of carbon13 to carbon-12 present, referred to as the carbon-13/carbon-12 ratio (13C/12C), can be determined by an instrument called a gas chromatograph combustion-isotope ratio mass spectrometer (GC-C-IRMS) The 13C/12C content of testosterone produced in our bodies is ultimately made from carbon broken down from our dietary intake. However, pharmaceutical testosterone is synthesized from soya-plant material that has low 13C/12C ratio. A similarly low 13C/12C ratio of testosterone in urine therefore indicates that the testosterone is of pharmaceutical origin, i.e. a doping offence has occurred. The amount of testosterone in urine is very small, even following its administration, and the separation required to purify and concentrate it for IRMS analysis is time consuming and laborious. A simple and rapid preparatory technique is highly desirable. We will manufacture polymers, which are imprinted with the molecular shape of testosterone, so that testosterone from urine can quickly fit into these imprints, like pieces from a jigsaw. The testosterone can then be easily removed for analysis by GC-C-IRMS. Further development will allow the polymer to be coated onto a small glass bar for stirring in the sample itself prior to the bar being directly transferred into a heated inlet (called a thermal desorption unit) on the GCC-IRMS, where all the testosterone is vaporized for analysis. This ‘on-line process’ is very rapid and all the isolated testosterone is analyzed thus increasing sensitivity. Finally, to prove that an abnormal 13C/12C ratio of the testosterone targeted is not because of impurities present, we intend to simultaneously analyze a portion of the sample with a standard type of spectrometer (quadrupole) attached to the GC-C-IRMS.

Main Findings

Comparison of WADA statistics for adverse findings with those of ten years earlier shows that there has been little change in the ranking of anabolic steroids, with testosterone continuing to account for the most common finding. It is analytically more challenging to prove the administration of testosterone, as it is also naturally produced within the body. An analytical approach that can distinguish administered testosterone from that naturally-produced is by determining the carbon fingerprint of testosterone present in urine. Testosterone has 19 carbon atoms, but collectively the carbon atoms in pharmaceutical testosterone are less heavy than testosterone produced in our body. The difference in heaviness is due to whether or not a carbon atom is present with an extra neutron in its nucleus, that is whether it is the heavier ‘carbon-13’ rather than the ‘carbon-12’. The relative amount of carbon-13 to carbon-12 present, referred to as the carbon-13/carbon-12 ratio (13C/12C), can be determined by an instrument called a gas chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS). For doping control purposes, this approach is used to prove whether an athlete has broken the rules by administering testosterone. The concentration of testosterone in urine is very small, even following its administration (doping), and extensive sample work-up is required to purify and concentrate the testosterone from urine prior to carbon isotope analysis. To reduce the labor and simplify the process, the WADA-accredited at King’s College London (UK) in collaboration with the University of Leeds, manufactured a polymer (a macromolecule) into which the molecular shape of testosterone was imprinted, so that testosterone molecules from urine can quickly fit into these imprints, like jigsaw pieces. The testosterone can then be easily removed for analysis by GC-C-IRMS. The work was challenging, but ultimately successful. Even so, to make the process effective, more R & D work is required, so that an optimized polymer can then be applied by loading into small cartridges through which urine can flow with the testosterone being easily extracted for analysis. In addition to the polymer investigation, the researchers also reconfigured the design of the instrument so that enhanced steroid purification can also be performed within the GC-C-IRMS, a process called multi-dimensional gas chromatography, and they added a different type of mass spectrometer (quadrupole analyzer) to assist with detection. These modifications to the instrument, which are cost-effective and simple to adopt by other laboratories, adds to the certainty that the testosterone measured by isotope analysis is pure, in keeping with the gold standard approach by WADA-accredited laboratories.

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.