Projets de recherche

Code: 08A07LM 

Testosterone is the most commonly reported of all banned substances with 1,124 cases in the WADA statistics for 2006.  As testosterone is a natural hormone, detection requires analysis with highly selective techniques to demonstrate the presence of it and its metabolites outside of normal ranges.  The outcomes of this project will further increase the ability of WADA to identify cheating by the use of testosterone and related compounds. 
A key component in identifying the banned use of the natural hormone testosterone for performance enhancement is the ability of WADA-accredited laboratories to accurately determine the changes that occur in carbon isotope ratios after doping. When an athlete takes synthetic testosterone it has a different 13C/12C ratio to that of the testosterone produced naturally in an athlete’s body. The measurement of these 13C/12C ratios is a complex technical task as the changes in ratio are very small and only specialised mass spectrometers can measure this change.   
A thorough review of the factors affecting accuracy in 13C/12C ratio results will be performed in this project to provide insight into the aspects of the methodology that contribute most to any measurement uncertainty. This will allow the methods to be further optimised to provide even greater accuracy and increase the probability of detection of doping.  

Main Findings: 

The first part of the project investigated opportunities to minimise the uncertainty associated with measurements of stable carbon isotope ratios of steroids related to testosterone in urine. A rigorous investigation of the inputs to a measurement uncertainty budget compliant with the JCGM Guide to Uncertainty in Measurement (GUM) 1 was performed. Optimised analytical method conditions were identified experimentally and a new instrument calibration method was developed that minimises measurement bias and facilitates uncertainty estimation. Using method validation data generated in this project, the combined uncertainty associated with results from the optimised analytical method employing this calibration approach was compared with that obtained when using the calibration method commonly employed in instrument software.
A new approach has been developed for the calculation of carbon isotope ratios when using GC-C-IRMS. The optimised approach uses a single point calibration rather than a linear calibration, and the measurement equation has been re-evaluated to avoid introducing a bias when the sample and internal standard 45R′ ratios do not match. While unmatched sample and internal standard 45R’ ratios do not introduce a bias when using this new approach, matching these ratios does improve the measurement uncertainty.
A number of factors in the sample preparation were identified that influence measurement uncertainty. These sample preparation steps were investigated and optimised to ensure the lowest possible measurement uncertainty: • SPE of conjugated, unconjugated and derivatised steroids
• Hydrolysis of conjugated steroids
• LLE of unconjugated steroids
• HPLC of unconjugated steroids
• Derivatisation
Validation of the optimised method including investigations of ruggedness, reproducibility and accuracy to allow an estimate of measurement uncertainty associated with a typical result. In developing this estimate, the full measurement equation for calculation of the isotope ratio values was expanded to include factors representing any bias in each of the main terms and validation results were used to provide estimates of the potential size of such bias even when it was not detected.
Complete uncertainty budgets were prepared for the carbon isotope ratios of key analytes and their delta differences in line with the GUM1. Detailed calculation of the sensitivity coefficients for each of the terms of the both measurement equations was required. This was relatively simple for the new measurement equation but required the use of mathematical software for the equation used by the IRMS instrument software. Uncertainty budgets were prepared for results from each calibration method for the δ13C values of androsterone, etiocholanolone, 11-oxoetiocholanolone, 11β-hydroxyandrosterone, 5β-androstane-3α,17β-diol, 5α-androstane-3α,17β-diol, 5β-pregnanediol, testosterone and dehydroepiandrosterone. They were also prepared for the differences between δ13C values (Δ13C) for androsterone versus 11-oxoetiocholanolone; etiocholanolone versus 11-oxoetiocholanolone; androsterone versus 11β-hydroxyandrosterone; etiocholanolone versus 11β-hydroxyandrosterone; 5β-androstane-3α,17β-diol versus 5β-pregnanediol; and 5α -androstane-3α,17β-diol versus 5β-pregnanediol.
This allowed a comparison of the measurement uncertainty associated with results from the two calibration approaches. The uncertainties are very similar for the two calculation approaches, although the uncertainty when using the new equation is consistently lower

Code: 08A01ZL

The current strategies used for the detection of EPO in sports are labour-intensive, time-consuming and cost-inefficient. The aim of this project is to develop an innovative strategy towards a fast and cost-efficient EPO assay in sports. This strategy is based on two key steps: 1) nanoparticles-based immunomagnetic extraction (NPIME), and 2) capillary zone electrophoresis (CZE) separation coupled with on-line sample concentration (OLSC) and native fluorescence (NF) detection.  First, a NPIME approach will be developed. Using anti-EPO antibody immobilized magnetic-cored nanoparticles, recombinant and natural EPO in samples is specifically extracted onto the nanoparticles and then desorbed into a much smaller volume. This process effectively concentrates EPO and meanwhile eliminates interference from the sample matrix. Second, a CZE-OLSC-NF method will be established. While the CZE separation provides fast profiling of EPO glycoforms, distinguishing recombinant and natural EPO. The OLSC allows for concentrating EPO from a larger injected sample volume into a much narrower zone before separation, thus significantly reduces the detectable concentration level. The NF detection selectively detects EPO with high sensitivity. Finally, the developed NPIME approach and the CZE-OLSC-NF method will be combined in off-line mode and applied to EPO spiked urine or urine samples of EPO injected patients. The proposed strategy can provide two significant advantages: speed and overall cost-efficiency. Our goal is to reach a total analysis time of less than 4 hours and an overall limit of detection (LOD) of 10-11 M at the end of the project proposed. The overall performance can be further effectively improved by in-depth optimization of the individual techniques. Also, the sensitivity can be further significantly improved by using a deep UV laser as the excitation light source. Thus the strategy proposed can be eventually developed into a reliable and robust analytical method for anti-doping analysis of EPO in the near future. 

Main Findings: 

The current EPO assay is associated with apparent drawbacks: labour-intensive, time-consuming and cost-inefficiency. This assay involves four key component methods: 1) isoelectric focusing (IEF), 2) ultrafiltration, 3) double blotting, and 4) on-gel chemiluminescence detection. The four key component methods are essential for specific and sensitive detection of EPO; however, they are all labour-intensive and time-consuming.  The aim of the project is to explore straightforward and effective substitutes for these sub-methods and then integrate them into a fast and cost-efficient approach. Nanoparticles (or microbeads)-based immunomagnetic extraction (NPIME or MBIME) is used as a replacement of ultrafiltration and double blotting. Capillary zone electrophoresis (CZE) is used as a substitute of IEF and the combination of native fluorescence (NF) and on-line sample concentration (OLSC) is used as an alternative for the on-gel chemiluminescence detection. We have successfully synthesized immunoaffinity magnetic nanoparticles (MNPs) using anti-alpha fetoprotein IgG as a model antibody. However, when the established method was transferred to anti-EPO antibody, the obtained immune-MNPs failed to recognize EPO. The reason for this seems that the antibody used was too susceptible to the reaction conditions for the immobilization. To overcome this issue, we turned to commercial protein A-coated magnetic beads and developed MBIME approach for specific extraction of EPO, which could enrich EPO by one order of magnitude. Besides, we successfully established an in-lab built deep UV laser-induced fluorescence (deep UV-LIF) detection system for detection of the native fluorescence of EPO. It enhanced the detection sensitivity by one order of magnitude as compared with UV absorbance detection. CZE was an effective replacement of IEF, which permitted complete resolution of EPO glycoforms within 25 min. OLSC could enhance the detection sensitivity by 50-100 fold, however, when used for the sample prepared by the MBIME approach, it resulted in notable loss in separation resolution. So it was excluded as a sub-method for the integration. Based on the component approaches established, the finally integrated approach, MBIME-CZE-deep UV LIF, allowed for the extraction, separation and detection of EPO glycoforms within 6.5 hours. However, the detection sensitivity was enhanced only by two orders of magnitude, which is far from the requirement for the analysis of real samples. To further develop the proposed strategy into practical approach for real sample assay, solutions to overcome the experienced issues were found. As human urine exhibited very significant matrix effect, antibody without cross reactivity to other proteins is a critical requirement for specific extraction. Besides,high binding affinity is a critical requirement for more efficient extraction. Furthermore, inertness under immobilization conditions is essential for the synthesis of active immune-affinity MNPs. An anti-EPO antibody that can meet these requirements is the key for the practical use of this approach.

Code: T08A04MS

The aim for the second step is to obtain statistically significant comparison between different delays of transport. The most important point to use for this comparison will be the obtained ratios calculated for both GH detection kits. The aim of the third step is to compare ratios obtained from samples analysed as A-sample, and samples analysed as B-sample after a frozen or cold storage of 10 days.

Main Findings

• For samples separated immediately after clotting, rhGH detection kit appears to have a slightly better sensitivity when samples are stored frozen compared to cooled.

• For samples arriving after 4 days (96h) and centrifuged upon reception, no differences exist for ratio if samples are analyzed immediately or after 7 days if frozen storage is applied.

• For samples separated immediately after clotting, storage must be made frozen. Under cool conditions, ratios is lower after ten days compared to one day. If frozen, ratios do not present any significant difference when analyzed after one day or ten days.

• If delay between end of collection and centrifugation is below 24 hours, no significant differences of ratio is observed between delays.

• Longer delays (up to 3 days; 72h) leads to lower ratio, but difference is close to analytical variation predicted by rhGH kits supplier’s experts.

• Later, ratios get significantly lower and tends to continuously decrease with time.

Code: 08A16CG 

The overall aim of the proposed project is to apply NMR spectroscopy and pattern recognition (PR) methods as innovative method for anti-doping screening and the elucidation of the overall biochemical effects of different doping methods.
This project will test the hypothesis that there are quantifiable variations in the urinary excretion of low molecular weight metabolites, induced by physiological response to doping, and that these changes could be related to different doping methods. By this means doping could be monitored without searching a specific compound but evidenced as a whole, in a fingerprinting approach, through NMR spectra of biofluids and subsequent multivariate analysis. As a first approach, this project will focus the detection of the use of designer steroids, by examining athlete’s positive samples for synthetic steroids like stanozolol.
The project includes the use of the large sample databank of Doping Control Laboratory of Athens, the generation of NMR spectral database and the consecutive analysis as well as the discovery of novel relevant biomarkers. The specific objectives are to apply biofluid NMR spectroscopy and Pattern Recognition in order to build multivariate biochemical models based on the use of prohibited formulations by athletes and to create databases of NMR spectra of characterised biofluids in selected human states. The derived models will be further applied to classify unknown samples according to primary and secondary metabolite variations induced by doping. Data will be analysed using a variety of standard methods to investigate multivariate analysis mapping of endogenous biochemical profiles. The new NMR-based metabonomic approaches will be compared with conventional methods, based on clinical chemistry and mass spectrometry and their relative usefulness in the field of anti-doping control will be evaluated.
To our knowledge the multiparametric approach proposed in this project has not been done before in humans. Such studies could lead to new strategies to complement antidoping control based on the Metabonomic approach maximizing the reliability of screening practices in use.

Main Findings: 

Metabonomics is well established as a powerful method for the evaluation of the metabolic profile of an organism, proffering a holistic view of an organism’s response to different exogenous factors. The aim of the project was to examine whether the NMR based metabonomics approach could be used as complementary tool to the existing methods on the basis of a non-targeted profiling analysis that is able to capture the metabolic signature of athletes who have utilized exogenous steroids. The applicability of the method was tested in a cohort of 263 human urine samples of both men and women athletes targeted to doping controls. Among them, 59 had been reported as positive to the official doping controls for the application of exogenous anabolic steroids.
The study included the NMR measurements of all samples and the chemometric analysis of the resulted spectroscopic data set. The latter consisted of complex 1H NMR spectra containing hundreds of signals from both endogenous and exogenous metabolites and was analyzed by multivariate statistical tools in two ways:
i) using data reduction of the original matrix to a lower dimension, followed by Principal Components Analysis (PCA), Partial Least Square–Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA (OPLS-DA) and ii) using a pre-processed full spectroscopic matrix with peak alignment followed by PCA and interval based Extended Canonical Variate Analysis (iECVA). The method i) resulted in models with partial grouping betweenn the originally classified groups, while method ii) provided a clearer classification that highlighted significant differences of metabolites, such as creatine, creatinine, hippurate, and
acetate among the groups of athletes. It is worth noticing that the samples derived from positive athletes displayed a significant higher variance comparing to the “negative” ones in all developed statistical models. This depicts one limitation of the present study, which arises from the low homogeneity of the sample pool and the lack of sufficient metadata that could be added to the statistical models development.
It is concluded that the NMR based metabonomics approach could be used as an ultra fast and cost effective predictive tool in anti-doping control that could highlight those samples originated from doped athletes among a larger cohort of samples on the basis of their metabolic fingerprint. In order to be fully applicable, the samples collection should be accompanied with extended metadata information that could be utilized in the multivariate statistical analysis.

Code: 08C17PS 

Our proposed project aims at completing the hitherto establishment of our novel direct detection technique for “gene doping” in its primary meaning, i.e. doping by genetically manipulating the human body. In gene therapy trials, aiming at curing diseases, polymerase chain reaction (PCR) is routinely used for monitoring plasma and serum levels of infectious transgenic DNA (tDNA) that often is undetectable within a few hours following gene therapy. In white blood cells gene therapists succeeded to show persistence of non-infectious tDNA for at least 3 months, following many different gene transfer protocols to muscle or other solid tissue of humans (and monkeys). However, this had been achieved under the advantage that the tDNA sequence to be discriminated from the naturally occurring genomic DNA (gDNA) of blood cells has been known exactly.  
Supported by the WADA research grant 06B7PS we establish a novel technology which enables detection of tDNA on a single molecule level even if the distinct “misused” tDNA sequence is not known to us. As a big advantage, our testing procedure only requires conventional blood collection from athletes under ordinary field conditions. As a first step, our patent pending single copy PCR technology (PCT/EP2007/003385) is now established in the laboratory detecting the candidate genes EPO, GDF8 (Myostatin), GH1 (incl. GH2, CSH1, CSH2, CSHL1, CSH1/CSH2), IGF-2, PPARD, PPP3CA, VEGF-A, and VEGF-D. We already succeeded in sensing one single genetically manipulated cell (tDNA containing) among 100.000 normal blood cells even if the probe was stored at room temperature for 3 h following blood collection.  
Our follow-up WADA project now focusses on the following important topics:  
_ further improvement of our “first generation” technical protocol;  
_ extension and updating of our list of gene doping candidates;  
_ verification of specificity and sensitivity for all established gene doping candidates in vivo. 

Main Findings:

In this project we have been able to demonstrate for the first time that direct and long-term detection of gene doping is possible with conventional whole blood samples. Detection protocols using a specifically designed ultra-sensitive detection protocol for transgenic DNA 
(spiPCR) have been developed for EPO, GH1, HIF1a, VEGFA, VEGFD, IGF1 (including MGF), and FST (Beiter et al. 2010). To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions. 
Sensitivity of our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular gene transfer. We employed adenovirus (rAd) and adeno-associated virus (rAAV) as the most likely candidate vector systems to be misused for gene doping. The sensitivity of the methodology was tested in a blinded fashion in mice following prototypic gene transfer with the adenoviral vector rAd-VEGFD and with the adeno-associated viral vector rAAV-VEGFA. Detectability of transgene VEGF elements was tested before and until 8 weeks post- transduction. Both, VEGFA and VEGFD elements could be efficiently detected in all transduced animals for several weeks and for VEGFA as long as 56 days. 
From a practical point of view, our testing procedures for gene transfer with EPO, GH1, HIF1a, VEGFA, VEGFD, IGF1 (including MGF), and FST using our spiPCR are safe and principally applicable for gene doping analysis in our laboratory setting.

Code: 08C15CM 

SPECIFIC AIMS: 1. Detect and identify unique skeletal muscle and blood proteins affected by long-term usage of banned substance.
2. Identify biological markers in muscle and blood that can detect differences between doped and non-doped individuals. SIGNIFICANCE: Detection of anabolic steroids and other banned substances in human blood and urine is only possible for a limited time after intake. However, the effects of such abuse on skeletal muscle morphology can be detected many years after secession of drug administration (Eriksson et al., 2006). The development of a fast and inexpensive method to detect such alterations eliminates the possibility to mask drug abuse, even when the drug is no longer detectable. Abnormal changes in the skeletal muscle proteome may be the only way to detect genetic doping. Supporting a proteomic method development for skeletal muscle can result in a useful tool for future studies regarding genetic doping. BACKGROUND: The use of banned substances among athletes is well-known phenomenon, and the effects on physical performance documented. For example, the myotrophic effects of testosterone have been previosly demonstrated (Sinha-Hikim et al., 2003). These effects are futher enhanced when drug treatment is combined with stength training. However, the mechanisms associated with testosterone and other anabolic steroids on increased muscle fibre area and strength of the muscle fibres has not been clarified. METHODS: A unique group of individuals having used testosterone and anabolic steroids for .5 years is available for muscle and blood sampling. Two dimensional differential gel electrophoresis and mass spectrometry will be used to detect and identify proteins. Multivariate statistical methods can identify discriminating proteins betwen groups. Functional testing can relate performance to protein expression. RESULTS (PRELIMINARY/RELATED) our laboratory have demonstrate changes in the muscle cell after anabolic abuse more than 10 years after secession of intale. Using 2D proteomics we can detect >2300 individual proteins from muscle tissue, >80 of these proteins are changed by using banned substances. Screening for relevant markers  for doping can now be accomplished. 

Main Findings: 

Seventeen trained elite strength athletes were recruited through personal contacts. Ten of the athletes were using AAS (Doped) and seven reported that they had never used AAS (Clean). All subjects were active in sports such as weight lifting, strong men competitions and body building at national and international level. Muscle and blood samples were taken and maximal muscle strength tested. DEXA scanning determined body composition. Blood was analysed for a panel of hormones related to muscle and steroid metabolism. Muscle samples were analysed using fluorescent immunohistochemistry and proteomic screening (2D DIGE/MALDI-TOF).
Doped and Clean athletes had higher bone mineral density at all measured sites compared to Controls, and Doped had more lean body mass compare to Clean and Controls. The use of AAS in combination with strength training results in significantly different skeletal muscle gene expression compared to strength training without AAS. The Doped athletes produced significantly lower results in the strength measurement then the Clean athletes despite more muscle mass and similar muscle fiber size and fiber type composition.. By using cluster analyses, there is a clear separation between Doped and Clean athletes based on morphological, hematological and proteomic data. Major findings were that the number of capillaries/fiber and myonuclei/fiber was higher in Doped compared to Clean athletes. The doped group had approximate one more capillary per muscle fiber compared to the clean group. We conclude that the Doped group has a lower capacity to generate force per muscle mass and fiber cross section area, possible due to the long term use of AAS
Results from the present study should be used as a base for further investigations regarding long term (possible permanent) effect of AAS and may also be used to generate indirect test (hormones, metabolites, proteins) to screen athletes for the use of banned substances in blood, muscle and urine. The advantage of this approach compared to direct measurements of banned substances or its metabolites is that masking agents and clearance rates will be ineffective to avoid detection.
The research approach and analytical methods used in the present study can distinguish a AAS doped athlete from a Clean athlete, regardless of substance used, training regimen, age and duration of AAS supplementation.

Code: 08C05CG 

Training diaries and documents seized during raids associated with ‘Operation Puerto’, together with the discovery of blood transfusion equipment at the Turin 2006 Winter Olympics, has revealed systematic use of autologous blood transfusion by elite athletes. No test currently exists to detect this banned practice.  
Substantial progress has been made by the SIAB research consortium in projects previously funded by the WADA to identify genes that are switched on or off following blood transfusion. Changes in the expression of these genes may thus serve as a diagnostic test to detect the use of autologous transfusion. Further research is required to demonstrate that candidate genes are not perturbed by non-doping activities, such as altitude exposure which may have a similar physiological consequence to blood transfusion. Consequently, this project will collect blood samples from elite athletes at the Australian Institute of Sport to quantify how much the identified gene markers vary from day-to-day during both typical training regimens and altitude exposure. Samples will be collected from both men and women athletes to study the effect of an athlete’s sex on gene markers. 

Main Findings: 

Autologous blood transfusion is one of the most effective methods of illicit performance enhancement for endurance athletes. For instance the USADA report on Lance Armstrong, released on October 10 2012 (http://cyclinginvestigation.usada.org/), indicates widespread systematic blood doping in the professional cycling peloton for at least a decade. Hitherto, no method has been effective in reliably detecting this banned practice. 
This study was based on previous WADA funded research by the Science and Industry Against Blood-doping (SIAB) consortium, which identified via microarray techniques, 16 genes that showed significant alterations in transcript abundance following autologous blood transfusion in healthy volunteers.  
The aim of this study was to assess day-to-day variation of identified gene markers in blood samples from elite male and female athletes at the Australian Institute of Sport (AIS), collected during both typical training regimens and altitude exposure, as a basis for evaluating the usefulness and limitations of using gene expression profiles as indirect markers of autologous transfusion. An additional 25 genes, involved in relevant biological pathways, were added to the initial set of candidate markers to provide a candidate set of 41 genes. Common data-mining tools were used to differentiate between the gene-signatures of transfused subjects (from previous SIAB projects) from those of AIS athletes undergoing different training modalities as well as being sampled at different times of the day. 
We demonstrated that a subset of 16 of the 41 genes (not the same 16 identified by SIAB) were significantly affected by either the between-subject factors of sex and training mode and/or the within-subject factors of being fasted and time of the day of sampling. Other genes proved to be stably expressed and unaffected by either between- and/or within-subject effects in our experimental setting. We also showed that altitude training affected expression levels of some of the markers to the same extent as transfusion, whilst others were robust and not affected by altitude exposure. Finally, we determined that within-subject variation was significantly smaller than between-subject variation for a substantial number of genes, indicating that for diagnostic purposes, individual follow-up and, for instance, inclusion of expression markers into an Athlete Biological Passport approach should be preferred over set population cutoff values. 
Overall, our findings indicated that transfusion led to changes in peripheral blood transcript abundance that are likely to be universal and distinct enough to lead to successful classification of transfused subjects as such, regardless of  sex or training mode. However, there were also some false positives (controls classified as transfused) which suggests that, on their own, the discriminative power of the 16 genes is unlikely to be sufficient in the anti-doping context. 
In regard to increasing robustness of respective gene signatures as a diagnostic tool, we suggest assessment of global gene expression via RNAseq rather than measurement of a single, or small selection, of genes.

Code: 08E12RC 

This project, which constitutes a follow-up from a previous work funded by WADA, aims at widening our understanding of the effects of ?2-agonists on muscle function, cognitive function and exercise performance. The programme is composed of 10 complementary studies and involves 6 Universities of 4 different countries. Three main issues will be addressed:  1- Results of two bronchial provocation tests widely used for asthma detection in athletes (mannitol and eucapnic voluntary hyperpnea) will be confronted;
2- ergogenic and stimulant effects associated with the administration of β2-agonists will be specified;
3- the precise signalling pathways involved in physiological adaptations induced by β2-agonists will be clarified.    The expected outcomes are as follow:   1- Eucapnic Voluntary Hyperpnea should remain the "Gold Standard" for asthma detection in athletes;
2- since β2-receptors are ubiquitous, the psychotropic effects of Terbutaline (i.e., improved cognitive function and increased level of arousal) contribute to its ergogenic effects. At a muscular level, β-agonists facilitate excitation-contraction coupling and protect against fatigue during intermittent high intensity exercise. A synergic effect, characterized by a marked hypertrophy and a proportional increase in maximal tension, is noticed when clenbuterol is associated to strength training;
3- myostatin and interleukin-6 are controlling factors in muscle growth. As such, both those molecules will be proposed as new detection tools for β2-agonists abuse in athletes.

Main Findings: 

We showed that 3 week treatment of high doses of a beta-2 agonist with anabolic and lipolytic properties such as clenbuterol depressed specific isometric tension, particularly in slow twitch muscle. Another potential negative effect for slow twitch muscle lies in the increased ATP cost of shortening, suggesting an altered efficiency in mechano-chemico transduction. However, probably the most marked negative effect is the lesser resistance to fatigue observed with such high doses. These negative effects are of particular importance in sports events where performance depends on endurance and muscle oxidative capacity. In fast-twitch muscles, the slowing of the time course of skeletal muscle contraction and relaxation during force generation induced by clenbuterol treatment could be considered as a negative effect, contrasting the anabolic effect. These last negative impacts had a functional relevance in sport performance since the rate constants of force development and relaxation both control maximal shortening velocity and thus sprint performance. These negative impacts of anabolic beta-2 agonist must be highlighted for prevention strategies against doping. 
We also reported that clenbuterol regulates negatively the expression of myostatin a master regulatory factor of muscle mass. Interestingly, we found that recombinant myostatin was sufficient to antagonize the hypertrophy activities of clenbuterol. Reciprocally, we also found that the genetic deletion of myostatin renders satellite cells refractory to the hypertrophic effect of clenbuterol.  These results suggest that the clenbuterol-induced decrease of myostatin plays a functional role during hypertrophy. Although the beneficial health effects of regular moderate exercise are well established, there is substantial evidence that the heavy training carried out by endurance athletes can cause skeletal muscle damage. This damage is repaired by satellite cells that can undergo a finite number of cell divisions. Our results indicate that clenbuterol-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle. From a sport viewpoint, it predicts that any benefits of clenbuterol treatment are likely to impose any extra stress on the satellite cells and this method could risk the regenerative capacity in the long term. 
In another study, we established the molecular mechanisms of the anabolic action of formoterol. In addition to protein kinase A, cAMP produced by the β2-adrenergic signaling can also activate Epac.  This protein is an exchange factor (guanine nucleotide) for the small G protein Rap. Rap juggles between a GDP inactive form and a GTP active form. Epac protein, activated by cAMP, catalyzes the exchange of GTP in the GDP form. Rap results in the activation of the PI3K/Akt/mTOR pathway and induces protein synthesis. 
In a separate study conducted in trained athletes, we showed that oral administration of terbutaline at supra-therapeutic dose seems to alter the balance that exists between endogenous beta-adrenergic stimulation and exercise-induced stress. Exogenous stimulation of the beta-adrenergic pathway with terbutaline produced the disadvantage of an artificially stimulus and results in negative psychological effects in athletes. In prevention against doping, it is important to highlight the deleterious psychological effects induced by a supra-therapeutic terbutaline administration before a competitive sports event. 
Finally in our last study in athletes we showed that inhalation of terbutaline prevents bronchoconstriction induced by hyperventilation with dry air, without limiting bronchial epithelial damages. Since epithelial damages may be involved in the development of respiratory asthma in many elite athletes, it seems important to identify new pharmacological strategies or nonpharmacological to minimize such damages.  

Code: 08B12YD

Erythropoietin (EPO) is a hormone that stimulates the production of red blood cells in the spine. EPO is predominantly produced in the kidney, and recombinant EPO (rHuEPO) is used therapeutically in the treatment of anaemia seen with chronic kidney disease and certain cancers. Athletes have in addition misused this hormone as a doping agent in order to improve endurance capacity. Endogenous EPO and
rHuEPO have different degree of glycosylation in addition to different combinations of charged groups on the glycan, and detection of misuse today is based on the different isoelectric profiles these charge-differences give rise to. Recently new recombinant EPO analogous (epoetins) have entered the market, some of which display a different and more basic isoform distribution than the traditional rHuEPO. In addition, DynepoTM, the new recombinant EPO that is expressed in a human cell line, has a slightly less basic isoform profile than rHuEPO. The current method for EPO-analysis is based on the profiles of the traditional epoetins, and hence there is a need to further develop this method to meet the new challenges. This project aims to characterize endogenous EPO found in atypical urines and to determine differences in the protein and glycan-groups from the new epoetins, by the use of affinity chromatography, enzymatic deglycosylation and gel electrophoresis.

Main Findings: 

Erythropoietin (EPO) is a hormone that stimulates the production of red blood cells in the spine. EPO is predominantly produced in the kidney, and recombinant EPO and analogues (epoetins) are used therapeutically in the treatment of anaemia seen with chronic kidney disease and certain cancers. Athletes misuse epoetins to improve endurance capacity. Endogenous EPO and the epoetins have different degree of glycosylation and different combinations of charged groups on the glycan, and detection of misuse is based on the different isoelectric profiles these charge-differences give rise to. New epoetins which display a more basic isoform distribution than the traditional epoetins, and the deviating (more basic) isoelectric EPO-profiles seen in active urines and in effort-urines, pose a challenge to the current EPO-method. The aim of this project was to characterize endogenous EPO found in atypical urines and to determine differences in the protein and glycan-groups from the new epoetins, by the use of affinity chromatography, enzymatic deglycosylation and gel electrophoresis. 
We found that EPO isolated from umbilical cord blood (predominantly produced in the liver) has more basic isoelectric profile than EPO from adult blood, and similar to that seen in some effort urines. Unlike the recombinant epoetins with hyper basic profiles, umbilical cord EPO, as well as EPO from effort urines, display the same electrophoretic mobility on SDS/SAR-PAGE as normal urinary and blood EPO.  
EPO produced from a human liver cell line, HepG2, has a hyper basic isoelectric profile, and similar WGA-affinity as EPO from umbilical cord blood and effort urines. However, HepG2 EPO migrates differently from endogenously produced EPO when analysed with SDS-PAGE. HepG2 EPO and also EPO from adult blood, was differently (less) affected by enzymatic deglycosylation than urinary and recombinant EPO, indicating structural differences in the glycan groups.  EPO was isolated from human liver tissue, to our knowledge for the first time. The results show that EPO produced in the liver has a different (more basic) isoelectric profile than EPO produced in the kidney, and the same electrophoretic mobility when analysed with SDS/SAR-PAGE.  

Code: 08E02VB

Asthma is the most common respiratory disorder among adolescents and young adults and the majority (70%) of ATUE certificates in Denmark concerns beta2-agonist.

Asthmatic athletes who request permission to use inhaled beta2-agonist (salbutamol, terbutaline, salmeterol, and formoterol) will be given permission, whereas systemically taken beta2-agonist will never be allowed. Therefore high urine level of beta2-agonist will be considered as doping.

Some cross-sectional studies of blood and urine levels of inhaled beta2-agonists exist, but follow-up studies are needed, taking blood and urine samples concurrently over several hours. Such studies will show the relationship between intakes and out-put of beta2-agonist.

Furthermore, asthmatics using beta2-agonist daily might be better in breakdown beta2-agonist, and therefore have different level in their urine compared with their blood. Moreover elite athletes have not participated in those studies and lastly systemic intake of the drug has not been tested.

Finally, the existence of salbutamol glucuronide in human urine has not been proven or reported in the scientific literature yet. Consequently, an accurate and rapid confirmation procedure will be developed based on direct injection of the urine specimens into the analytical instrument and subsequent determination of concentrations of unconjugated (i.e., free) salbutamol, salbutamol glucuronide and salbutamol sulfate. This is of significant importance for the future doping analysis of beta-agonists.

Main Findings

Results: No differences were demonstrated between normal, asthmatics and elite athletes with asthma in urine or serum levels of beta2-agonist. Urine concentrations peaked in the collecting period 0–4 h after administration of inhaled medication as well as oral salbutamol in all groups. The mean (SD) urine concentration of salbutamol was 356 (162), terbutaline 553 (299), formoterol 7.7 (4.7) and salmeterol 0.37 (0.14) after inhalation and after systemic administration salbutamol values of 2724 (1810) and of terbutaline values of 549 (424) was found. The maximum concentration after inhalation of salbutamol was 742, terbutaline was 1370, formoterol 18.30 and salmeterol 0.62. (ng mL-1). Two samples of salbutamol exceeded the threshold value of 1000 ng x mL-1 (1082 and 1057 ng x mL-1) uncorrected for urine specific gravity, but when corrected values of 746 and 661 ng x mL-1, respectively was found. While salbutamol glucuronide was not detected in excretion urine samples after inhalation, 26 out of 82 specimens collected after oral ingestion showed salbutamol glucuronide with a peak value of 63 ng/mL. The percentage of salbutamol glucuronide compared to unconjugated salbutamol was less than 3 %.

In conclusion: Mean values have been established for four beta2-agonists. Uncorrected urine values are higher than values correction for gravity. The excretion of salbutamol glucuronide in urine after administration of salbutamol has been proven and is reported for the first time.