Projets de recherche

Code: 05D21FM 

Different substances and methods can be used to increase the oxygen carrying capacity of blood, thereby improving an athlete's ability to perform. Doping control procedures are expensive and the problem always exists of who we should test, by what criteria and when. Research groups have been developing criteria to detect these substances and methods (blood doping, human recombinant erythropoietin, oxygen carriers, the off/on model) International federations, including Biathlon, currently choose athletes based on random selection, standings, high hemoglobin and/or hematocrit and/or reticulocyte counts, off model scores, etc. There is currently no accurate integrated way to combine all variables (individual performance change and laboratory values), to estimate which athletes should be selected at the optimal time for anti-doping tests. This project aims to develop an intelligent system which is able to identify those athletes whose haematological and multiple variables reflect a pattern consistent with the use of banned substances or methods. These athletes could then be chosen at the optimal time for target testing. The focus of this project is the creation of a software program that will consider haematological values abnormal not only on the basis of high values, but also on the basis of raw data considered concurrently (haematological data in relation to the reference population, intraindividual haematological variations including abnormal low data, performance variations, ranking, nation). This system will produce classes of results associated to a diagnostic probability, useful for targeted selection for both in and out of competition controls.The system aims to be fast (analysing multiple data simultaneously), unpredictable and self-learning (the new information will be automatically included to improve the knowledge). The project aims to provide a strong deterrent against doping, reducing the risk of evasion by manipulation, and to be cost-effective, ensuring that anti-doping budgets are spent in an evidence based fashion.

Main Findings: 

Different substances and methods can increase the oxygen carrying capacity of blood and the athlete's performance. Validated detection methods for these ergogenic aids are available but doping control procedures are expensive and the screening phase performed by International Federations remains a critical issue. The wrong selection of both athletes to be tested and samples to be analyzed in a laboratory can be a factor responsible of high costs and unsatisfactory results. This project AR.IE.T.T.A. aimed to develop an intelligent system, able to show athletes’ profile in order to detect those reflecting an abnormal pattern, consistent with the use of banned substances or methods. An intelligent system, with different sections and functions has been developed and tested after the input of haematological and performance data of athletes belonging to the International Biathlon Union, who gave their written informed consent to the study. The AR.I.E.T.T.A. software includes: - Log-in section - Data-Entry section: data are inserted, stored and separated by sports, disciplines, subgroups or by competitions/events; - Analysis section: data can be analysed, validated scores calculated, parameters shown also simultaneously as statistics, table/graphs, individual or population profiles (team, nations, etc); - Screening section: an immediate evaluation of the risk profile can be obtained. A risk score for the present sample can be calculated on the basis of different data considered concurrently (haematological and in future performance data, absolute values and inter-intra-individual variations, etc). An analysis of the athlete’s risk can also be performed by evaluation of all the sample risk scores calculated for the athlete under study. The experience derived by the routine application for a stable target testing program and future studies aiming to evaluate and increase the sensitivity of the diagnostic phase e.g. by inclusion of parameters not altered by haemodilution, will represent a further improvement of the system. AR.I.E.T.T.A. is an efficient database that enables a quick evaluation and interpretation of blood results. It could favour surveillance programs and timely controls on athletes by the International Federations collecting blood samples for targeted testing purposes.

Code: 05D22RG 

Human chorionic gonadotropin (hCG) and human lutropin (hLH) are hormones synthesised or purified by certain pharmaceutical companies for treatment of infertility in women. However, both hormone act through the same receptor in the body and may trigger the production of testosterone. As such these hormones may be attractive molecules for athletes looking for illicit performance enhancement. Both hormones belong to the family of glycoproteins, which means that protein carry a particular decoration known as glycosylation. The glycosylation of both hormones has been found to be of utmost importance for the correct functioning of the molecules. Furthermore, it is known that glycosylation is a phenomenon that is not primarily regulated by particular genes. In stead, glycosylation is the result of the concerted action of many primary gene products, donor and acceptor substrate availability, and additional external factors (temperature, presence of growth factors, etc.). As such, the glycosylation is cell type specific. Recombinant glycoprotein hormones will therefore carry glycans synthesised by the machinery of the host cell and these glycans may be different from those synthesised by the natural producers. Detailed structural analysis of the glycosylation of the endogenous and exogenous hormones will reveal the cell-type specificities. Once established, the cell-type specific structural elements may be used as targets for the development of new, faster, and more reliable screening and confirmation procedures.

Main Findings:

Human chorionic gonadotropin and human lutropin belong to the family of glycoprotein hormones that are used in the treatment of female fertility problems. Under normal circumstances both hormones result in the production of testosterone. This is the main reason for its potential abuse by athletes: increasing the testoterone concentration without affecting the ratio between testosterone and epitestosterone. While both male and female have lutropin (produced in the pituitary), chorionic gonadotropin is produced by the placenta under term conditions, and as such restricted to the female gender. Even though under ordinary circumstances hCG is not produced by men, its production has been described under certain pathological situations. As such, its sole detection in a biological specimen is not sufficient. These hormones, being glycoproteins, carry the particular signature of their origin through the carbohydrate decoration. This post-translational modification depends very much on the environmental settings and could be different in recombinant and endogenous species. As such, the basic principle of this application was the analysis of the glycosylation of exogenous and endogenous CG and LH with the aim of establishing the existence of structural differences that would enable unambiguous discrimination. Both hormones share the α-subunit, glycosylated at Asn52, Asn78, and Thr39 (the latter only described in CG), and have different β-subunits glycosylated at Asn30 in both and at Asn13 only in CG. Furthermore, O-glycosylation has been described for CG at Ser121, Ser127, Ser132, and Ser138. Pure glycoproteins (both prepared from pooled urine of pregnant women, isolated from pituitary material, or recombinant from different sources) were prepared or purchased. Glycoproteins were analysed at the intact level by mass spectrometry and sugar analysis. While the former revealed subtle molecular weight differences mainly in the α-subunit the latter proved too insensitive. A directed analysis of the sialic acid residues that end-cap most of the glycans revealed the presence of the nonhuman residue N-glycolyl neuraminic acid both in proteins expressed in CHO, as well in murine cells. The identification of this residue in hCG and LH, and already previously identified in recombinant erythropoietin, represents a single target to address recombinant glycoprotein pharmaceuticals. Analysis of the N-linked glycans revealed that murine glycosylation is different with the existence of terminal Hex(α1-x)Hex-R epitopes. CHO derived material also displays a slightly different glycosylation for hCG. The endogenous glycosylation machinery results in a significant part of hybrid type structures (predominantly at the α-subunit) that is much less pronounced in the exogenous material. Similar results were obtained for the lutropin preparations. Analysis of the de-N-glycosylated hCG and proteolytic peptides showed that no significant differences exist in terms of other peptide modifications or potential O-glycosylation occupancy although a thorough comparison of the glycans is still ongoing.

Code: 05D11WS

In the fight against doping the laboratories are confronted with an increasing number of substances to screen on. Therefore new methods for the screening of those substances have to be implemented in the laboratories. To keep costs for doping control analysis acceptable, to ensure rapid reporting times and to lower the amount of urine needed to screen for all substances, a comprehensive screening for different classes of substances is desirable. The project aims at combining the screening methods for diuretics, beta-2-agonists, heavy volatile stimulants and narcotics in one LC-MS/MS method. To avoid problems with the stability of diuretics during the hydrolysis, direct analysis of the excreted metabolites (where applicable as intact conjugates) is mandatory. This is also beneficial because relatively clean extracts are obtained and the time consuming hydrolysis is eliminated. Thus the sample preparation has to be adapted, the phase II-metabolism of beta-2- agonists, narcotics and heavy volatile stimulants has to be investigated and reference substances of the metabolites have to be synthesized and characterized by nuclear magnetic resonance (NMR) and mass spectrometric (MS) techniques.

Main Findings:

In the fight against doping in sports the laboratories are confronted with screening an increasing number of substances. Therefore new methods for the screening of those substances have to be implemented. To keep costs for doping control analysis acceptable, to ensure rapid reporting times, and to lower the amount of urine needed to screen for all substances, combining ‘traditional’ screening procedures for different classes of substances is desirable. This project aimed at combining the screening methods for diuretics, beta-2- agonists, heavy volatile stimulants and narcotics in one method. To evade the hydrolysis of the conjugates, which on the one hand is time consuming and on the other hand revealed problems with the stability of diuretics, direct analysis of the excreted metabolites (where applicable as intact conjugates) is desired. Thus, the phase-II metabolism of beta-2 agonists, heavy volatile stimulants and narcotics was investigated and sulfoconjugates of p-Hydroxyamphetamine, pHydroxy¬met¬amphetamine, p-Hydroxyephedrine, p-Hydroxynorephedrine, Etilefrine, and Etamivan were synthesized and characterized by LC-MS/MS. The aglycons were coupled to sulfuric acid by reaction with sulfur trioxide pyridine complex. The structures of the mono-sulfates of p-Hydroxyamphetamine, pHydroxy¬met¬amphetamine, p-Hydroxyephedrine, Etilefrine, and Etamivan were confirmed by nuclear magnetic resonance. Different materials for Solid Phase Extraction (SPE) were investigated and the best results for screening purposes were obtained with PAD I. This also allowed a simple combination with the routine procedure for diuretics used in our laboratory, which also utilizes SPE with PAD I. Validation for the sulfoconjugates and application of the whole method to real urine samples was performed. Due to the very large number of analytes with different ionization behavior, it was necessary to submit the final concentrate to two separate LC-MS/MS runs.

Code: 05C10KH

Current strategies for detecting growth hormone (GH) abuse in sport rely on quantifying GH-responsive proteins or measuring changes in the concentration of pituitary-derived GH isoforms in blood. The aim of this project is to develop a test for GH doping based on the analysis of gene expression in peripheral blood cells. There is strong evidence that white blood cells respond directly to GH and indirectly through many anabolic and metabolic circulating factors. These secondary effects may continue long after the withdrawal of the GH stimulus and their gene profile characterization could provide an extended time window for detecting GH use. We propose to undertake this study by obtaining white blood cells from subjects currently enrolled in a WADA-funded GH administration study being undertaken to identify the most sensitive protein markers of GH in serum and the influence of testosterone on these markers. This proposal will not only enhance the scope and value of the existing study but will also allow comparison of biomarker and gene profiling approaches for diagnostic benchmarking. The gene expression profiles will be measured using microarray technology and analyzed by sophisticated statistical programs to identify a gene set which is a fingerprint of GH use. The gene set will be validated independently by quantitative PCR, a gold standard for measuring gene responses in cells. The gene set will be tested rigorously against placebo and other GH treated samples from the same study, and finally against an independent set with samples from subjects positive and negative for GH excess or administration. It is envisaged that this developmental work will lead to the design of a diagnostic gene chip that can be implemented for large scale diagnostic use.

Main Findings

Initial microarray analysis was undertaken using the Affymetrix platform. We have performed 98 Affymetrix Human U133 Plus 2.0 microarrays spanning four treatment groups and forty individual subjects. To determine the effects of treatment, we have compared an individual's baseline (week 0) and final treatment sample (week 8). Due to the small changes in gene expression, a second analysis was conducted using the Agilent Technologies platform, a more recently developed transcript profiling platform with enhanced sensitivity. The two colour methodology of the Agilent 44K G4112F array allows direct comparison of an individual's week 0 and week 8 sample on the same microarray. We performed 40 Agilent microarrays for 20 GH treated men and women. Validation of the Agilent microarray expression data was performed for seven genes using quantitative real time PCR (qPCR). For both gene expression profiling platforms, the GH-induced alterations were small, and similar in magnitude to variations between individuals. Results from both platforms demonstrate that maximal regulation was of the order of two fold, and that few genes were regulated greater than 1.5 fold. Further, whilst both platforms identify differentially expressed genes, these signatures were different between men and women treated with GH. Using data from the Affymetrix platform at the probe set level, testosterone alone had little effect, whilst the administration of testosterone and GH together resulted in regulation of probe sets that showed little overlap with those regulated by GH alone. We conclude that it is unlikely that gene expression analysis of peripheral blood leukocytes would be a viable approach for the detection of GH doping.

Code: 05C07JR

We are developing a test for exogenous erythropoietin (epo) activity based on transcriptional profiling. This test would work for any method of Epo abuse, including gene doping. Epo, a hormone used to treat anaemia by stimulating red blood cell maturation, is frequently used by endurance athletes to boost their aerobic capacity. Treating anaemia with recombinant Epo is costly and gene therapy (in which the Epo gene is inserted into cells and stimulated to produce the hormone) is a promising alternative. Gene therapy has the potential to revolutionize medicine, but unfortunately many experts believe that illicit application of this technology to performance enhancement is inevitable and that Epo will likely be in the vanguard of this threat. Epo produced by a gene injected into athletes to augment normal production would be identical to the natural product and undetectable by current testing methods; however, the biological pathway of which Epo is part (the response to hypoxia) is complex and involves the coordinate regulation of genes in a variety of distinct, but related pathways. Selectively stimulating only the Epo pathway would not have the same system-wide effects as the natural stimulation of entire hypoxia pathway. Distinguishing between the augmented and the natural response would be possible by comparing transcriptional profiles, which represent the total complement of RNA transcripts (the intermediates that transmit instructions from the genes to the body) in the tissue. In our project, Serial Analysis Gene Expression is being used to compare the transcriptional profile of the hypoxia response to that of foreign Epo activity. We hypothesize that comparing these profiles will identify differentially expressed genes that can be used to develop a diagnostic test. We will identify these genes in a mouse model, locate and characterize their human counterparts, and incorporate the latter into blood-based test for exogenous Epo expression.

Main Findings

qPCR analysis of potential candidate genes (based on differential expression in the mouse in response to hypoxia or EPO) showed that the variation for the most promising candidate genes exceeded the variation between treatments. As the mice are inbred, sex and age matched, and kept in controlled environments, there is too much variation in the candidate genes identified by SAGE analysis of blood for them to be reliable, predictable and consistent markers for Erythropoietin (EPO) use in humans. In the absence of strong candidate genes for follow-up, analysis in humans was not initiated. Given the results of the experiments, differential gene expression in blood cells following red-cell expansion is not a promising method of detecting EPO (or EPO gene) use. As this proposed test was an indirect measure (i.e. measured the effect of EPO rather than EPO itself, it was necessary that the results be: 1) quantitatively and significantly distinguishable from hypoxia induced erythropoiesis and 2) be highly invariant between individuals. I did not find highly differentially expressed genes that could clearly different between EPO and hypoxia and, although promising candidates were found based on un-linking the pathways (i.e. identifying genes expressing unilaterally that that should be co-regulated), none of the genes tested showed sufficient consistency in expression to be reliable biomarkers.

Code: 05A01JC

The purpose of the project is to develop a rapid and easy-to-use erythropoietin (EPO) doping test, which can distinguish recombinant EPO and EPO analogues from the endogenous forms. The technology should also have the potential to reveal new varieties of EPO and its analogues. The aim of this 3-year research project is to provide the basis for the product development phase. The implementation of this test for EPO doping detection will most significantly reduce both hands-on time and total testing time. The easy-to-use set-up will make it possible to perform the testing in laboratories of different degrees of sophistication as well as in the field. This should make the testing more available and much cheaper. The proposed test-procedure is based on the fact that the EPO forms show characteristic differences in their glycosylation structures. The different isoforms are first separated by the use of a chromatographic step (anion- or affinity (lectin)chromatography), performed in certain zones with immobilized anti-EPO, which specifically captures EPO after the chromatographic separation. The EPO captured is then detected by reaction with carbon black labelled antiEPO. The amount of EPO is proportional to the intensity of blackness, as determined by an image scanner. The recently evaluated MAIIA I variety has been shown to efficiently distinguish endogenous EPO from recombinant EPO, although other varieties of the technology has to be evaluated in order to select the best fitted one.

Main Findings

A novel, rapid and easy-to-use method, EPO WGA MAIIA, for determination of aberrant EPO isoform subpopulations in urine or serum, has been tested for its use as an EPO doping control method. The method separates EPO subpopulations due to their different interactions with the lectin wheat germ agglutinin (WGA). The glycosylated structures on recombinant epoetins show stronger interaction with the lectin, probably due to their higher content of polylactose amine. The WGA-based separation of isoforms and the subsequent ultrasensitive EPO determination is rapidly carried out within a few square cm of a thin porous layer formed as a test strip, using an image scanner for quantification. The test takes only 20 min. to perform and is well suited both for determination of single samples and for large series. Before analysis with the EPO WGA MAIIA method, EPO is purified and concentrated from urine or plasma by use of a newly developed disposable EPO affinity purification device (www.maiiadiagnostics.com). With this easy-to-use sample preparation device EPO can rapidly be captured from large sample volumes and be eluted in a final volume of only 55 µL. The high EPO recovery of 65%, and the retained isoform distribution, makes the device a useful pre-step tool also for e.g. IEF, SDS-PAGE and LC/MS. The EPO WGA MAIIA method allows detection of recombinant EPO in urine specimens from patients up to about 7 days after the last injection (p<0.0001). Recombinant epoetin e.g. alpha, beta, omega, delta, zeta and four Chinese types (p<0.0001), and EPO analogues like Aranesp (p<0.0001) and Mircera can be distinguished from endogenous EPO isoforms. Mircera shows less interaction with WGA compared to endogenous EPO, while recombinant EPO:s show stronger and Aranesp shows the strongest interaction. Only 2 pg of EPO is required for isoform detection, which is about 1/10 of the amount of EPO required for the presently used IEF based doping method. When rhEPO beta and endogenous EPO appear in the same sample it is possible to detect rhEPO down to a level where it constitutes only 40% of total EPO. Besides measuring the interaction of the various types of EPO with WGA, it is possible in the same test strip to utilize also their interaction with the anti-EPO immobilized in the detection zone (see J. Immunol. Meth. 339 (2008) 236–244). By interpreting the antibody interaction profile, using the EPO AbQ MAIIA algorithm, it is possible to distinguish EPO and epoetins from EPO analogues like Aranesp and Mircera. The EPO WGA MAIIA test gives also an estimate of the EPO concentration in the eluate, enabling calculation of optimal application volume for the IEF or SDS-PAGE confirmation test. The recommended test set-up for doping control utilizes EPO WGA MAIIA for identification of epoetins and Aranesp, while Mircera preferably is identified by EPO AbQ MAIIA. The EPO WGA MAIIA test classifies all tested epoetin varieties and EPO analogues correctly, shows good resolution between endogenous EPO and epoetins, and can measure very low amounts of EPO. The quality controlled reagents will be supplied worldwide as a complete kit. The hands-on time is reduced compared to presently accredited tests, which significantly decreases the analysis cost. The excellent results and the easyto-use concept seem to fulfil the requirements for a screening EPO doping control test. Such a test makes it possible to considerable increase the number of EPO doping controls performed without increasing the total analysis cost.

Code: 05D17BD

The exogenous uptake of nandrolone has been banished by the International Olympic Committee (IOC 2004), due to its anabolic effects. Though maximal authorised concentrations have been published for nandrolone metabolites (19-NE, 19-NA) in urine, some positively controlled athletes claimed that they never used such products and expressed that a natural endogenous secretion is related to their high level of physical training. A previous study from our laboratory (Schmitt et al. 2002) has shown that the urinary concentrations of 19-NE and 19-NA in male athletes (judo and long-distance specialists) following exhaustive exercises (Wingate and limited-time tests on ergocycle) did not exceed 0.008 ± 0.02, and 0.07 ± 0.02 ng.ml-1 , respectively. A maximal concentration of 2ng.ml-1 for 19-NA is, however, authorised in male athletes by the IOC. In females, the endogenous origin of nandrolone is a controversial topic in doping control: firstly, low-dose of norethisterone contained in some birth control pills can produce similar urinary nandrolone-like metabolites and secondly endogenous production of nandrolone by aromatase-rich tissues has been reported in the ovarian follicle and during pregnancy by the placenta. The concentration of nandrolone in blood has been shown to be influenced by estrogens, suggesting that nandrolone may significantly vary during menstrual cycle. It seems possible that high androgens concentrations (e.g. DHEA and testosterone) may influence the production of 19-norsteroids in aromatase-rich tissues. Interestingly, intense exercise has also been associated with raised levels of estrogens in females. Therefore, physical exercise could theoretically influence the urinary excretion of nandrolone metabolites in females. Now, the maximal authorised concentration of 19-NA (IOC 2004) has been set at 2 ng.ml-1 in female athletes without any details concerning the menstrual cycle, the use of oral contraceptive, and physical exercise. Our aim is therefore to determine the influence of oral contraceptive, menstrual cycle, and exhaustive exercises on urinary concentrations of nandrolone metabolites and blood DHEAs and testosterone in sedentary and athlete females. Young voluntary sedentary females (n=16) will be recruited, eight taking similar “low-dosed” monophasic oral contraceptives and eight with regular menstrual cycles. These persons will be investigated at rest, every 30 min during a morning period and at the same time of the day during and after exhaustive exercises. Two kinds of exercises will be studied on separated days: Wingate and limited-time tests on ergocycle. For the subjects with regular menstrual cycles, the sessions (rest and exercises) will be organised during the early follicular phase (5 days after the start of menses) and mid-luteal phase (18-23 days after the start of menses). Each subjects will be investigated during a 3-month period. The chronology of the sessions “rest” and “exercise1”, and “exercise2” will be randomised. For the persons under contraception, the sessions will be organised during the weeks of pill ingestion. Using the same protocol, female judoka (n=16) and long-distance runners (n=16) will be investigated.

Main Findings

• Our results provide strong evidence that the urinary concentration of 2 ng/mL of 19¬NA is fair as the upper acceptable limit in doping control tests for female athletes.

• In young women, neither androsterone nor DHEA and nandrolone metabolite excretions are influenced by menstrual phase and physical training. However, oral contraceptive intake lowered DHEA excretion in urine and androsterone excretion seems to be slightly affected by prolonged exercise.

• Metabolomics is a promising tool in order to gain insight into physiological status and to clarify the changes induced by short-term, intense physical exercise. Further research is needed to determine whether metabolomics could be employed to diagnose specific disorders induced by exercise (such as overreaching and/or overtraining), and if it could be used as a new anti-doping tool).

Publications

1: Enea C, Boisseau N, Diaz V, Dugué B: Biological factors and the determination of androgens in female subjects. Steroids 2008;73:1203-16

2: Enea C, Boisseau N, Dugué B : Facteurs biologiques influençant le profil stéroïdien urinaire lors de contrôles anti-dopage. (Biological factors influencing the urinary steroid profile in doping control analysis –Review article in French-). Science et Sports 2009; 24:119–127

3: Enea C, Boisseau N, Ottavy M, Mulliez J, Millet C, Ingrand I, Diaz V, Dugué B: Effects of menstrual cycle, oral contraception, and training on exercise-induced changes in circulating DHEA-sulfate and testosterone in young women. Eur J Appl Physiology 2009; 106:365-373

4: Bayle ML, Enea C, Goetinck P, Lafay F, Boisseau N, Dugué B, Flament-Waton MM, Grenier-Loustalot MF: Quantitative analysis of DHEA and Androsterone in female urine: application to the evaluation of sport and contraceptive pill intake influences. Analytical and Bioanalytical Chemistry 2009; 393:1315-1325

5: Enea C, Boisseau N, Bayle ML, Flament MM., Denjean A, Diaz V, Dugué B: Nandrolone excretion after exhaustive exercises in females: influence of menstrual cycle, oral contraception and training level. Scand J Med Sci Sports 2010, in press

6: Enea C, Seguin F, Petitpas-Mulliez J, Boildieu N, Boisseau N, Delpech N, Diaz V, Eugène M, Dugué B : (1)H NMR-based metabolomics approach for exploring urinary metabolome modifications after acute and chronic physical exercise. Anal Bioanal Chem, 2010, in press (available on line).

Code: 05C4PD

Great progress has been achieved over the past years by means of innovative molecular techniques which has led to the discovery of new growth factors involved in the regulation of muscle development. These findings provide new starting points to understand the mechanisms involved in the adaptation of skeletal muscle to exercise training. One of these newly identified growth factors is myostatin, a member of the transforming growth factor-β family of proteins that has been demonstrated to play a fundamental role in the regulation of skeletal muscle growth during embryogenesis. Blocking of the myostatin signalling transduction pathway by specific inhibitors and genetic manipulations has been shown to result in a dramatic increase of skeletal muscle mass. Drugs or genetic manipulations with the ability to modulate myostatin signalling may have the potential to enhance physical performance in athletes and therefore probably represent a new class of doping substances. To identify manipulations of myostatin signalling, a promising strategy is the analysis of ratios of factors and molecules associated with the myostatin signal transduction pathway, a so-called molecular fingerprint. Manipulations either by application of a factor or by inhibition of its signalling will change these ratios, resulting in a different fingerprint. A methodological strategy to analyse simultaneously the expression of a variety of these factors and relevant signal transduction molecules with high sensitivity is the detection by Immuno PCR (iPCR). The presented project aims to establish Real time iPCR as a new tool for highly sensitive detection of members of the myostatin signal transduction pathway in skeletal muscle, blood, oral mucosa, and urine. As targets we will choose myostatin itself, myostatin propeptide, follistatins, the follistatin related gene (FLRG), the activin receptors ActRIIA and ActRIIB, the family of activin receptor –interacting proteins (APRIPs) and gasp-1. The technique of iPCR will allow to analyse these parameters simultaneously using multiplex real time PCR. During the initial phase of our project this technique will be established and validated using multiplex Real Time iPCR for the detection of the mentioned factors in tissue, blood, oral mucosa and urine in close cooperation with our partner, who has a wide experience using this technique. In the second phase of the project the technique will be applied to study the expression of myostatin and members of the myostatin signal transduction pathway under training conditions. Such information is very limited but essential to develop test systems to identify manipulations of myostatin signalling either by pharmacological or genetic approaches. Muscle biopsies, blood, oral mucosa, and urine will be investigated. In detail we will determine specific ratios between the different members of the signal transduction pathway (fingerprints) in the different matrices, the effect of physical activity, and inter-individual variations. In a future phase of the project we will try to analyse if the application of myostatin blocking agencies (antibodies, follistatin peptide) will change such fingerprints. For ethical reasons, as long as myostatin inhibitors are not licensed for therapeutical applications, this will be only possible in animal models.

Main Findings: 

In the 24 months of the funding period of the project we succeeded in the development of functional ELISAs and immuno-pCR assays (Imperacer™) for MYO, MYOPRO, FOLLI, FSTL and the ACT IIA. The Imperacer™ assay detected the respective proteins with significantly higher sensitivity than the corresponding ELISAs. To analyze the effects of training on the ratio of MYOPRO, FOLLI and FSTL in serum, training experiments were performed. A single bout of resistance training did not affect the serum levels of the proteins. To analyze long-term training effects, 30 young male volunteers were divided into a control, a strength and an endurancetraining group and were trained for three months. The serum levels of the respective proteins in serum before and after training were determined and correlated to the tissue expression of the m. vastus lateralis. Skeletal muscle biopsies were taken and the expression of MYO was analyzed by real time PCR. Serum concentrations of all proteins analyzed displayed a moderate inter-individual variability. More important was the finding that the individual ratios of the analyzed proteins were very constant and were not affected by any kind of training. To get an impression whether skeletal muscle mass or intake of anabolic steroids affect the ratios of the chosen proteins, serum concentrations in non-trained, healthy young males, paraplegic patients and body builders abusing anabolic steroids were determined. There were no significant differences in the average serum concentrations off all proteins analyzed between healthy young man and paraplegic patients. Interestingly, the average serum concentration of MYO propeptide was significantly elevated in the body builder group compared to the other groups. Our data provide evidence that bodybuilders abusing anabolic steroids have different MYOPRO/FOLLI or MYOPRO/ACTIIA ratios than untrained controls or paraplegic patients As a step towards a suitable routine test system, we developed a multiplex Imperacer™ assay that is able to simultaneously detect MYOPRO and ACTIIA ratios with high sensitivity in a single well. MYOPRO and ACTIIA ratios were not only successfully determined in venous blood, but also saliva and capillary blood. Summarizing our results, we believe that the data provides evidence that the determination of expression profiles of members of the MYO signaling pathway is a promising strategy to detect potential abuse of MYO inhibitors. We believe that any manipulation of myostatinMYO signaling will be detectable by determination of the ratios of MYO propeptide to other factors involved in MYO signaling. Interestingly this may imply that not only the use of MYO inhibitors but also many other anabolic manipulations (treatment with anabolic steroids including SARMS, GH administration, IGFI and MGF) may be detectable analysing such ratios.

Code: 05D07RK

The WADA lists for 2004 and 2005 have added a large number of new substances that require detection. WADA will require a number of these to be studied for inclusion into the WADA-accredited laboratories routine testing schemes. From the 2004 list the new additions were corticosteroids and a project to study the metabolism and detections of these is currently funded by WADA. The new list 2005 has a variety of new substances and the metabolism and detection of these must also be studied. Before introduction into testing standards, metabolism and funding will be required in order to investigate cost-effective procedures. This is presently undertaken over a lengthy period by a few laboratories that investigate new substances and publish the results for all laboratories to use. The longer the anti=doping community waits for such data the longer athletes can use the substance with impunity. Currently there are over 20 such compounds to be investigated including some new classes such as the 5α-reductase inhibitors eg. Finasteride, the selective estrogen receptor modulators eg. Raloxifene and anti-estrogenic compounds such as fulvestrant. The aim of this project is to study the metabolism of a selected number of these substances and then to determine procedures to allow then to be introduced into the current schemes across all WADA-accredited laboratories.

Main Findings

The WADA list since 2004 has had a large number of new substances added to the various classes that require detection by the WADA-accredited laboratories. WADA will eventually require most of these new substances to be studied and included in the WADA-accredited laboratories routine testing schemes. The 2004 list saw new additions such as corticosteroids and a project to study the metabolism and detection of these in several laboratories that is currently being funded by WADA. The recent lists introduced in 2005 and 2006 had a variety of new substances added. Before the introduction of detection methods for all of these substances in the laboratory programmes for routine testing, both research and ongoing (LIVE) testing will be required in order to initiate costeffective testing procedures. Such studies are presently undertaken over a lengthy period by a few laboratories that investigate new substances and publish the results for all laboratories to use. However, the longer the anti-doping community waits for such data the longer athletes can use the substances with impunity. The aim of this project was to obtain as many of the compounds added to the list as possible, to study the metabolism of a select number of these substances and then to determine procedures to allow them to be introduced into current detection schemes across all WADA-accredited laboratories. All substances were obtained but not all had metabolic studies undertaken. The substances that can be considered as “new“ additions are: Aromatase inhibitors (Formestane and testolactone); Selective estrogen receptor modulators (Raloxifine and toremifine); Anti-estrogenic substances (Cyclofenil and fulvestrant); 5 α -reductase inhibitors (Finasteride and dutasteride); Anabolic agents (18α-homo-17-hydroxyestr-4-ene-3-one, boldione, 4-hydroxy-19-nortestosterone, methyldienolone, methyltrienolone, methasterone, methyl-1-testosterone, methylnortestosterone, prostanozol, tibolone, zilpaterol and norclostebol); Stimulants (Famprofazone, benzylpiperazine, cyclazodone, fenbutrazate, fencamine, isometheptene, p-methylamphetamine, norfenefrine, octopamine, ortetamine, oxilofrine, phenpromethamine, sibutramine and tuaminoheptane). One of the biggest challenges of the project was to obtain the substance suitable for the studies. These were either purchased, often with difficulty in finding a supplier, or synthesised at NMIA’s Chemical Reference Materials group. Most of the substances on the WADA list could be analysed by one of two analytical techniques, both used within NMIA and most other WADA-accredited laboratories. These techniques use either solidphase extraction or solvent-solvent extraction followed by measurement using either Gas Chromatography – Mass Spectrometry (GCMS) or Liquid Chromatography – Mass Spectrometry (LCMS). Many of the materials or excretion studies have been provided to laboratories through cooperation with WAADS.

Code: 05D3KC 

Glucocorticoids are widely administered for acute and chronic musculoskeletal pain as well as for several other pain syndromes. Increasing concern in sport has recently been raised due to the common use of glucocorticoids amongst elite athletes. WADA has therefore placed certain restrictions on the use of corticosteroids and presently lists them as a legal medication permitted in the local form only when prescribed by a physician with a simplified therapeutic use exemption, while systemic administration is currently banned. Indeed, the influence of corticosteroids on physiological and psychological variables could theoretically enhance performance and therefore explain their overuse by athletes. They have been shown at rest to increase blood glucose and energy store mobilization and to induce euphoria and erythropoiesis but surprisingly little work has been done to determine whether systemic glucocorticoid administration has a direct or indirect beneficial effect on physical performance in healthy subjects. Controversies remain therefore as to whether elite athletes who use glucocorticoids may gain a competitive advantage after acute or chronic intake. We propose to contribute to a wider knowledge of glucocorticoid action mechanisms during exercise thanks to a multi-centre project with, in particular, an investigation of the impact of acute/chronic glucocorticoid administration on: 1) energy cost and substrate utilization during submaximal exercise; 2) eating behaviour; 3) psychological aspects and mood change; 4) erythropoiesis; 5) performance during submaximal exercise with regard to the fatigue status of the subjects.

Main Findings: 

We showed 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) in healthy moderately trained male volunteers, despite a probable increase in lipid oxidation and decrease in CHO oxidation. However, our results demonstrated that short-term therapeutic prednisolone intake (60 mg per day for 7 days) does not significantly modify erythropoiesis or eating behavior but improves performance in healthy recreationally men during submaximal exercise (70-75% VO2 max) both with or without combined intense training. The concomitant alterations in the psychological, hormonal and metabolic parameters analyzed show that short-term administration of this drug had both central and peripheral effects but the complexity of these responses makes dissociation of the possible causal effects on performance difficult to distinguish. Subsequent research are therefore needed 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 this drug in women, after both acute and short-term administration with, in parallel, investigation of potential eating behavior change and psychological effects; 2) the metabolic and endocrine responses after 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 occurring after shortterm intake.