Projets de recherche

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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Description à venir.

Code: 07A08JS

Recently, autologous blood transfusion is one of biggest challenges for abuse detection. For reasons of convenience and safety, autologous transfusions, in which the cells are the athlete's own, are reportedly far more common than homologous transfusions, in which the cells belong to someone else with compatible blood. So far there is no adequate and unequivocal method to confirm illicit boosting of an athlete's red blood-cell via transfusion of one’s own blood. Presently our institution is running the WADA sponsored project “Overall approach for blood transfusion detection (autologous/homologous)”. As a complementary approach the present project aims to add another alternative to the possible ways to demonstrate autologous blood transfusion developing tests, based on comparison of red blood cell membrane proteomes of fresh red blood cells and stored red blood cells for different times, at standard storage conditions. Several studies employing different strategies describe in details normal human erythrocyte proteome, while some studies have postulated changes in erythrocyte proteome after red blood cell storage (Anniss et al.; Manojkumar et al.) This project is presented with an idea to identify detectable changes in erythrocyte membrane proteome before and after standard storage procedures in order they can be used as a methods to identify autologous blood transfusion. For such purpose we will apply proteomics methods such as 2D electrophoresis, 2D-DIGE, mass spectrometry of identified proteins. The project is presented as a preliminary step of one year in order to test the hypothesis. If, as expected, differential proteome appears as an actual possibility next step would be to develop adequate tests which would employ flow cytometry and proper markers in order to demonstrate existence of different populations of erythrocytes in the blood of the same subject, which would confirm anterior autologous transfusion.

Main Findings

In the present project we employed two complementary proteomic strategies in order to get an insight to the changes occurring at the membranes of the RBC over prolonged storage time. 2D gel electrophoresis as a strategy of protein separation has its drawbacks such a difficulties to separate efficiently extremely basic, acidic, or hydrophobic proteins. So, it was necessary to complement it with isotope tagging for relative and absolute quantitation (iTRAQ) methodology, which is peptide oriented and helps to achieve satisfactory resolution of the changes occurred. Not a single method is sufficient to follow all the changes happening. From the application of each one of the methodologies, several important proteins appear as serious candidates for further targeting. However, within the same 2D spot it is possible to identify several proteins with different degree of certainty, even though that experimentally some of them may be discarded taking into account molecular mass, sequence coverage, % or score number and other indicators. When it was possible we compared also graphs of corresponding spots (increase or decrease of spots volume ratio) and ratios of iTRAQ results when corresponding identified proteins appeared in both of them. According to the results from our studies and also available results of other authors we propose to group observed findings such as:

• Transmembrane proteins

• Cytoskeletal proteins

• Other proteins

• Other investigator’s findings related to our studies

Code: T07B01JS 

The aim of this proposal is the characterisation of distinct antibodies that specifically recognise growth hormone isoforms. Some of them are to be employed in an ELISA-based test to detect growth hormone abuse by athletes. In addition, other antibodies with different specificities and different origin will be studied. For the characterisation of the antibodies the technique of surface plasmon resonance will be employed. In a straight-forward strategy the antibodies will be immobilised onto the carboxymethylated surface through standard amide-bond ligation chemistry. Different chips (each one with four channels, including controls) will be required to respond to different aspects of the characterisation. Available GH isoforms structurally related to the pituitary GH-N form will be flown across for the determination of specificity, cross-reactivity and to determine kinetic parameters. Also the effect of other growth-hormone related substances, both structurally and functionally, may be included in the study.

Main Findings

A rigorous study on the binding characteristics of thirteen different monoclonal antibodies, that address growth hormone isoforms, has been successfully conducted. In this study, antibodies were compared using the Surface Plasmon Resonance method and this comparison helped in the selection of hGH antibodies for the purpose of anti-doping testing.