Projets de recherche

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.

Code: 08A13UF 

The analysis of 13C/12C is a well proven and powerful method to detect abuse of endogenous steroids. However, the method is rather complicated as compared to more classical analytical techniques. Successful application requires skilled and
experienced technicians and scientists. However some possible problems appear to be exaggerated. This has led partly to the impression that only measurements obtained under absolutely perfect conditions can be trusted. Self evidently this has been exploited to dishonestly charge the method. The project aims to find out to which degree 13C/12C analyses are impaired when the conditions are not ideal.

Main Findings: 

• Chromatographic resolution in GC-C-IRMS is significantly impaired by ineffective combustion.
• Lack of oxygen and the state of the furnace themselves are more significant than e. g. combustion temperatures in this respect.
• By contrast, the effects of dead volumes, cold spots etc. seem to be less important.
• Observed peak overlap which is induced by cold spots (and possibly also by dead volumes) does not necessarily result in invalid data.
• This even applies when the (true) δ13C-values of incompletely resolved signals differ largely.
• Nonetheless, poor peak shapes and insufficient resolutions require careful investigation of the corresponding causes.
• In order to still benefit from analyses obtained from poorly resolved chromatograms, incomplete combustion must definitely be excluded.
• The peak shapes of highly oxidized compounds are a sensitive indicator for the effectiveness of combustion, increasing peak tailing corresponding inefficient combustion. This however might confound with the effects of higher retention times.
• The δ13C-values of lowly oxidized compounds are more sensitive to incomplete combustion than those of highly oxidized compounds.  However, these conclusions must be confined to steroids currently.

Code: 07E03HG

In some sports about 100% of athletes take nutritional supplements (NS) without any reflection of the risk/benefit relation. One of the risks connected with the use of NS is the risk of inadvertent doping originating either from contaminated or faked NS. An international IOC study performed in 2002 has shown that about 15% of nonhormonal nutritional supplements contained anabolic androgenic steroids (mainly prohormones) not declared on the label. Since that time athletes have been warned by their federations, information systems have been established, legislation towards anabolic-androgenic steroids as nutritional supplements has been changed e.g. by the anabolic steroid act 2004 in the USA and some companies have improves their quality control systems. The question is: has the situation on nutritional supplement market improved or got worse? To answer this question on an international level an extended follow-up of the 2002 IOC study should be performed. NS from different countries and from internet sources should be purchased and analysed for prohormones, classic anabolic steroids, new designer steroids, β2-agonists and stimulants. The results should be used to educate athletes to reduce the non-reflected use of NS, to force the industry for the improvement of quality control systems for NS and to motivate governmental institutions to regulate and restrict the market for NS.

Main findings

Previous studies have shown that supplements may be contaminated with anabolic androgenic steroids so that these supplements present a considerable risk of inadvertent doping for athletes involved in a doping-control system. The purpose of the present study was to investigate the international supplement market for an update on the contamination rate of nutritional supplements. A total of 597 supplement samples were obtained from 17 countries and from the Internet. Samples were analyzed for presence of 43 different anabolic-androgenic steroids by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. For most substances, the limit of detection was in the range of 10-50 ng/g or lower. In four samples, the presence of a prohibited substance was confirmed (Androsta-1,4-diene-3,17dione (twice), DHEA, and Androsta-1,4,6-triene-3,17-dione). A total of 561 samples were found to be negative of the selected anabolic agents and in 32 samples, no analytically acceptable result was obtained. The present results indicate that the prevalence of supplement contaminations has decreased in recent years. Nevertheless, supplement contaminations and adulterations still present a risk of inadvertent doping to athletes who are involved in a doping-control system. Further studies are necessary to identify the risk of contamination for further prohibited substances.

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