Projets de recherche

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.

Code: 08D01EE 

The use of prohibited substances is a burden in sport. This is clearly against fair-play and can be  hazardous for health of sp01tspeople. Although the use of doping substances are rare in sports requiring fine tuning motor movements, there are rumours that some archers tend to use such medicines that  diminish anxiety and reduce body sway during shots. This may positively affect shooting performance. 
PITA (International Archery Federation) Medical and Sport Science Committee (MSSC) decided to carry out a project in order to find out whether there is such an effect of anxiolytic substances on performance. FIT A MSSC has proposed to cooperate with WADA Accredited Turkish Doping Control Centre at  Hacettepe University, School of Sports Sciences, Faculty of Medicine Department of Pharmacology,  Ankara University Faculty of Medicine Department of Sports Medicine Department and Turkish Archery Federation. Research group is planning to perform a double blind study on elite archers in order to  evaluate the effects of a benzodiazepine on athletic performance. The analysis method(s) with regard to  the benzodiazepine(s) and their metabolite(s) will be developed and optimized in the laboratory. After the validation of the method(s) in urine due to EUROCHEM and ICH Guidelines, the samples will be  analysed in the laboratory. The results will be submitted to WADA for further studies, if necessary. 

Main Findings: 

It is well-known that athletes may experience some form of stress prior to or during a competition which may reduce or at least affect their athletic performance. Therefore, inhibition or reduction of stress may prove beneficial in athletes which can be easily achieved by utilizing an anxiolytic drug and benzodiazepines are the typical examples of these drugs. The purpose of the present study was to investigate whether the intake of a benzodiazepine would exert positive effects on physical performance capacity, such as an increase in shooting performance in elite archers. The research group has compared the effects of oral diazepam (5 mg) vs placebo. A randomized double-blind trial was used to assess shooting scores, heart rate values, body sway, aiming behaviour, anxiety and clicker reaction time in 24 athletes. The results did not show any difference between the groups, neither in physical performance characteristic nor in other parameters. It is concluded that as regards to the performance capacity, benzodiazepine use does not improve athletic performance in archery. However, the benzodiazepine was applied as a single relatively low dose (5 mg). Benzodiazepines exert calming effects with simultaneous reduction of anxiety at relatively low doses. These effects may be accompanied by some depressant effects on psychomotor and cognitive functions which were not observed in our study. Benzodiazepines, contrary to these depressant effects may also cause disinhibition of previously suppressed behaviour which may be related to their behavioural disinhibitory effects, including euphoria, impaired judgment, and loss of self-control which were also not observed in our study. Single-dose administration and selection of a moderate-low dose of a benzodiazepine derivative may explain why these disinhibitory effects are not observed in our study. It is well known that benzodiazepines also exert dose-dependent anterograde amnesic effects. Since benzodiazepines cause sedation and inhibition of motor activity in higher doses, they are expected to negatively affect motor performance in athletic competitions requiring fine tuning skills. 

Code: 08B02PD

Improvement of a Myostatin Imperacer assay towards a high-sensitive test system for the detection of anabolic manipulations, including gene doping strategies

Main findings

Our recent research has demonstrated that monitoring the fingerprint expression of members of the myostatin signalling pathway is a promising tool to detect manipulations of myostatin. An important result of this project was the development of a sensitive ACT IIB Imperacer. This is a very essential step into the fight against the abuse of myostatin inhibitors because a soluble form of the activin type IIB receptor (ACT IIB), as a strategy for myostatin inhibition, is already examined in clinical trials (Acceleron). In our opinion our assay is applicable to detect the abuse of such myostatin inhibitors. First results demonstrate that it also works in capillary blood. As an alternative to antibody based Imperacers we also have started to develop Imperacer assays based on receptor ligand interactions. For example we could demonstrate that myostatin is detectable in the serum via binding to a recombinant ACT IIB receptor linked to DNA. As a basis for an indirect detection of myostatin inhibition ratios of FOLLI, MYPORO and ACT IIB were determined in long term studies with male volunteers and in females in different phases of the menstrual cycle. The results show notably inter-individual variations, however the distinct individual ratios were stable and not affected by training, menstrual cycle and circadian rhythms.

To determine whether the analysis of FOLLI, MYPORO and ACT IIB ratios are suitable to detect anabolic effects of steroids, their expression was analysed in untrained males, bodybuilders abusing anabolic steroids and “clean” bodybuilders. Our data demonstrate a tendency for a lowered FOLLI/MYOPRO ratio in the serum of natural bodybuilders. However, in our opinion, these variations cannot be used to decide whether somebody has abused anabolic steroids or not.

These data are in agreement to data from matching animal experiments. Fortunately, in these experiments we found that the endocrine profile and IGF-1 expression is strongly affected by anabolic steroid abuse. A WADA funded pilot project based on this observation is ongoing (LIVE) in the moment. Finally we have conducted an animal experiment with myostatin siRNA. The results of this experiment, in agreement to data obtained from Myostatin Knockout (KO) mice, indicate that manipulation of myostatin signalling, even by siRNA, is indeed detectable by comparing ratios of FOLLI, MYOPORO and ACT IIB. Interestingly in these experiments we recognised that fat mass was affected by myostatin siRNA much stronger than muscle mass. This knowledge is very helpful for identification of new biological markers for indirect detection of myostatin inhibition.

In summary we succeeded in the development of a high sensitive ACT IIB Imperacer, which can be used for the direct detection of specific myostatin inhibitors. This assay seems to be functional in capillary blood samples. Long term studies in males and females indicate that the ratios of FOLLI, MYPORO and ACT IIB are individually very stable and not effected by training, menstrual cycle and anabolic steroids. The use of specific myostatin inhibitors, in our studies specific siRNA to inhibit myostatin in mice, resulted in a significant shift in these patterns. This knowledge is very helpful for indirect detection of myostatin inhibition.

Code: 08A10UF

Stable isotope techniques are successfully employed to detect doping with synthetic steroid hormones such as testosterone or its precursors. The test exploits the fact that synthetic testosterone exhibits a different ratio of the stable carbon isotopes 13C and 12C compared to its natural counterpart. However, the 13C/12C ratio is also influenced by diet and other factors. In some regions the natural 13C/12C-ratio of steroid hormones is close to that of synthetic material. This is due to the 13C/12C-ratios of the diet. Testosterone doping thus can go undetected under these circumstances. The other element present in all steroids is hydrogen. Like carbon it has two stable isotopes, 1H and 2H. The 2H/1H-ratio is probably better suited to discriminate between synthetic and natural testosterone. Especially when the 13C/12C-test fails it can be expected that the 2H/1H-ratio can still betray the presence of synthetic steroids. 

Main Findings: 

Stable carbon isotope analysis of endogenous steroids is a well-established method to demonstrate the illicit administration of synthetic steroids. The latter typically feature lower 13C/12C ratios than their biological congeners. Testosterone, the principal male sex hormone, plays a pivotal role here. Abuse still is frequent but major progress in the fight against doping has been achieved by analysing the 13C/12C ratios of testosterone and of its major metabolites.
The methodology obviously requires that the 13C/12C ratios of the synthetic material sufficiently differ from the biological baseline. Due to variability in the composition of the diet, this not always the case. In addition, black-market testosterone preparations have been found which exhibit inconspicuous carbon isotope signatures.
There is another so-called isotope system which may be useful here. The isotope ratios of hydrogen (2H/1H) typically even exhibit much stronger variation than those of carbon. In fact, it has been demonstrated that synthetic steroids tend to be 2H enriched vs. corresponding biological compounds.
The methodology is, however, much more delicate. This is mostly due to the small abundance of 2H (merely ca. 0.0015 %). Consequently, much more material is required than for 13C/12C analysis. This significantly compromises the applicability of 2H/1H analysis in sports drug testing. Moreover, at low abundances significant and unacceptable bias will be present in the apparent 2H/1H ratios when signal intensities are too low.
Therefore, as a priority, the focus of this research project was to improve the analytical method first in order to render 2H/1H analysis fit for the requirements of sports drug testing.
The conversion of organic material to hydrogen is a mandatory pre-condition for 2H/1H analysis. This is achieved by so-called high temperature conversion at ca 1440°C in reducing environments. The process seems to be sensitive to competing reactions associated by isotope fractionation. This will be less pronounced at higher abundances of the relevant educts.
For these and other reasons, we hypothesized that presence of additional sources of hydrogen would mitigate these problems. A device was designed which allows to evaporate organic solvents and to feed them into the reactor. 
Significant improvements could be achieved by this approach. The required amounts of steroids for valid 2H/1H analysis could roughly be reduced by 50 %. While some refinement still is required, this fundamentally renders 2H/1H analysis of steroids a feasible additional option to counteract abuse of synthetic steroids.

Code: T08C01FD 

Anabolic steroids are amongst the most misused substances in doping control and are intensively metabolized in humans. Adequate screening for misuse of these substances therefore relies on the detection of metabolites in urine samples collected from athletes.
Most of the studies investigating the metabolism of pharmaceutically available steroids were performed in the 1980’s via gas chromatography mass spectrometry (GC-MS). This research resulted in the selection of appropriate metabolites for the detection of steroid misuse. Over the years the selection of metabolites was further elaborated to include several metabolites that can result in prolonged detection times. Over the last decade, liquid chromatography tandem mass spectrometry (LC-MSn) was introduced as a screening technology in doping control laboratories world-wide. As a result several steroids that are difficult to detect via GC-MS became readily detectable. However, it should also be noted that several easily detectable metabolites by GC-MS are virtually undetectable via LC-MS. Indeed, both GC-MS and LC-MS are compatible techniques and the simultaneous application of both technologies is needed to cover the detection of all categories of structures. Recently, it has been shown that precursor ion scanning via LC-MS can be used to detect new steroid metabolites and the use of this technology has resulted in the detection of previously unreported metabolites. These results have also illustrated the limitations of GC-MS for the detection of steroid metabolites. Indeed, some highly abundant metabolites in the LC-MS analysis could hardly be detected by GCMS. This has highlighted the need for a re-investigation of steroid metabolism to allow for their adequate detection by LC-MS. Preliminary results for several steroids indicate that detection times could be prolonged considerably if more appropriate LC-MS metabolites are selected.

Main Findings: 

The use of LC-MS/MS in neutral loss and precursor ion scan modes allows for the detection of steroid metabolites. Several strategies based on this methodology have been applied for metabolic studies of some anabolic steroids. Previously unreported metabolites have been detected. In the case of methyltestosterone one of these metabolites increased the detectability of the steroid misuse. For stanozolol, the screening in negative ionization mode for one of the detected metabolites also improved the detection to stanozolol misuse. Several real samples have been analysed for these metabolites showing their applicability. For other steroids, additional metabolites have been found although more experiments are needed in order to prove their usefulness for doping control analysis.