Projets de recherche

Code: 10C19TF

The search for accurate and effective indicators for doping in sport requires the use of many different methods and tools. Traditionally, such tests have relied on the detection of suspected doping agents themselves. However, modern genetic methods have become available that promise not only to improve traditional methods to detect doping substances, but also to provide completely new methods to identify doping by detecting changes in the ways that genes are expressed in doped cells and tissues. WADA has established an extensive research program toward this aim and a number of WADA-supported laboratories have examined the effects of a variety of agents on gene expression such as anabolic steroids, growth and related factors and agents that affect oxygen delivery to tissues. However, detecting such changes is complicated by the fact that the various studies all use different materials and experimental methods.

To identify key changes that identify with scientific and legal certainty the effects of a doping substance and to distinguish useful signals from others caused by training, nutrition, ethnic background, gender, age, etc., it is important to compare the results from many independent studies by using the most powerful modern informatics and computational methods. We have established a WADA-supported informatics facility in this laboratory and have used it to obtain preliminary proof that simultaneous comparative analysis of several independent studies can tentatively identify genes or sets of genes that are altered by exposure to doping agents and that may provide new genetic signatures of doping. We now propose to collaborate with other WADA investigators to expand these initial comparative analyses to a larger set of independent WADA research studies and to published studies in the general biomedical literature. We will include studies related to growth factors and muscle function, oxygen delivery, anabolic steroids and modulators of gene expression.

Main Findings

These two related studies were designed to test the concept that exposure of humans to doping agents disturbs the normal expression of many of the 20,000 genes in human cells and whether those changes can be used as a rigorous proof or a “signature” of exposure to specific doping agents. These kinds of studies are made possible by modern genetic techniques that allow an estimate of the extent of expression of all human genes on a single square inch silicon chip. Our studies also were designed to determine if several studies carried out under different conditions in different laboratories could easily be analyzed even though they used slightly different techniques and methods of analysis. This method of analysis of multiple and slightly different sets of data from different studies is called “meta-analysis” and examples of meta-analysis have been successful in many other kinds of research. We therefore carried out meta-analysis of three independent studies of the effects on gene expression in blood samples from athletes of administration of human growth hormone (HGH) and three separate studies of the effects of erythropoietin (Epo) or hypoxia in the blood cells of human athletes and in mice.

As expected, we found that the genes that are expressed incorrectly after exposure to HGH and Epo are different from each other. However, we also discovered that the separate studies identified only a small number of genes that were disturbed in the same way in all three studies, leading to a conclusion that those genes are probably not specific “signatures” for exposure to HGH or Epo. We have concluded that these results could mean that these drugs do not cause significant changes in gene expression in blood cells. We prefer an explanation that the three separate HGH studies and the diverse Epo/hypoxia differed in many details that introduced too many slight differences in the timing of testing, dosages, methods of tissue preparation and methods of analysis that probably have hidden the gene expressions shared by the different studies. We are impressed that in other very recent studies supported by WADA in which one laboratory has carried out very careful studies of humans exposed to Epo, reproducible changes have been found that consistent with a genetic “signature” for exposure to Epo, demonstrating that extensive and well controlled single studies under some conditions can be an effective approach to identifying the genetic changes in blood samples or other tissues. We propose that a reasonable next step is to carry out a similar study with HGH exposure and compare results from such a study with those obtained in our meta-analysis. We are confident the overall concept of genetic signatures of doping is a correct and robust one and will add important new tools to the prevention and detection of doping.

Code: 10A15MP

As per list of the World Anti-Doping Agency (WADA) 1-Androstenedione is classified as “exogenous anabolic androgenic steroid” and is therefore prohibited in sports. Recently it appears as ingredient of products of the dietary supplement market as so-called prohormone of 1-Testosterone.

Only very limited information is available on its effects and metabolism. It is very likely that the endogenous steroids 17-hydroxy-5-androstane-3-one (5-DHT), 3-hydroxy-5-androstane-17-one (Androsterone), and 5-androstane-3,17-diol occur as metabolites. Altered steroid profiles are therefore expected, especially the ratios of 5/5-androstane-3,17-diol (ADIOL/BDIOL) and androsterone/etiocholanolone (AND/ETIO) may be influenced. A concentration of 5-DHT>21 ng/mL (adjusted for specific gravity) together with ADIOL/BDIOL>1.5, AND/ETIO>2.9, DHT/ETIO>8.2 and DHT/EpiT>0.73 is considered as indicative for the administration of 5-DHT in males. Thus, a potential misinterpretation of the results obtained after 1-Androstenedione administration has to be taken into account.

Thus, the project aims in monitoring the influences on urinary steroid profiles. An identification of other urinary metabolites as well as the evaluation of their time courses will be part of the project as well.

Main Findings

New analogs of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids.

Recently so-called prohormones of 1-testosterone (17β-hydroxy-5α-androst-1-en-3-one), namely 1-androstenedione (5α-androst-1-ene-3,17-dione, 1-AD) and 1-androstenediol (5α-androst-1-ene-3β,17β-diol) are advertised as well. Only very limited information is available on their effects and metabolism.

Within this project the urinary metabolism of 1-androstenedione was investigated and the main metabolites were monitored following administration of a single oral dose of 50 mg to six male volunteers. 1-Testosterone, 3α-hydroxy-5α-androst-1-en-17-one (1-DHA), 3β-hydroxy-5α-androst-1-en-17-one (1-DHEA), 5α-androst-1-ene-3α,17β- and -3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common urinary steroid profile ratios were altered after the administration. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the excretion rate of androsterone were increased for about 2 days post administration. 1-DHA appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to ten days after single administration. It was synthesized within this project and characterized by MS and NMR.

For biological characterization 1-androstenedione, and its metabolites 1-testosterone, 1-DHA, and 1-DHEA were tested in a yeast androgen assay as measure of the androgenic potential. 1-AD displayed androgenic effects itself (1/10 as active as 5α-dihydrotestosterone (DHT)) and thus may not be classified as prohormone but as active hormone. However, its metabolite 1-testosterone is able to transactivate androgen receptor driven reporter genes in the yeast androgen screen with a comparable potency as the reference androgen DHT. The two other tested compounds, 1-DHA and 1-DHEA, were about 1/100 as active as DHT.

Code: 10B12MU

We have developed immunoassays for 20K/22KGH isoforms in serum, and the detectability of GH doping was proven.

This proposal is aiming to make the ELISA kits available for the antidoping laboratories, to extend the methods to detect GH in urine samples, and to maximize the assay performance by multiplexing using flow cytometry that is common to the homologous blood transfusion test.

Main Findings

It is well known that active ingredient of pharmaceutical hGH preparation is 22KDa-hGH isoform (22KGH) and administration of GH increases 22KGH to 20KDa-hGH (20KGH) isoform ratio. Because production of those isoforms is being regulated genetically and formed as a result of splicing mRNA during expression of hGH-N gene, a basal 22KGH/20KGH level in human is very stable. We have established highly sensitive sandwich immunoassays for 22KGH and 20KGH in serum, the assay performance was validated according to WADA ISL and the methods have been implemented on 3 assay platforms, i.e., 96-hole microplate assays, multiplexed 20KGH&22KGH microsphere immunoassay for Luminex® and multiplexed immunoassay by flowcytometry. For all three of the assay platforms, the performance is confirmed to be basically same, and the LOQ for 22KGH and 20KGH are 10 and 20pg/mL serum, respectively. Essentially no age-related decrease, no sex-, ethnical- and sporting type-dependent differences of 22KGH/20KGH are observed. Advantage of the detection of GH doping by monitoring the isoform ratio would be less factors influencing the test results, simplicity of the marker to be monitored, namely, two single molecules that are well characterized in the literatures. Serum isoform ratio immediately goes up from about 10 to several thousands after single GH dose, and returned to the base level in about 32 hours. Our data represented possibility to extend the isoform selective GH test to urine samples.

Code: 10A13CG

Aims

The doping control samples are not protected by environment conditions during their transportation from the collection sites to the WADA Accredited Laboratories. The WADA Technical Document for reporting doping violations concerning endogenous steroids is issuing parameters to check for urinary microbial integrity for certain steroids. Endogenous steroids profiles of urine samples may undergo changes due to the occurrence of microorganisms that can be found in the human body or the surrounding environment, especially during their transportation in the warm periods of the year. Moreover, suspicions of tampering of doping control samples with proteases to mask the administration of peptide hormones were confirmed recently.

The Doping Control Laboratory of Athens, OAKA, was granted in 2005 by a WADA research fund to study different approaches regarding stabilization of urine doping control samples during their transportation to the Laboratories. Evaluation of results showed that the application of the stabilization mixture into urine aliquots had a lethal effect on the populations of the microorganisms tested. Moreover, when the stabilization mixture was included in urine aliquots, enzymatic digestion of rEPO and dissociation of the hCG intact molecule in the presence of proteases were inhibited. However, matrix interferences were recorded in mass spectrometric routine analytical procedures.

The current project focuses on the creation of a specially designed urine sample collection plastic container, coated in its interior surface with a suitable chemical stabilization mixture aiming at improving the quality of sport urine samples. The stabilization mixture should be effective in prohibiting degradation caused by microorganisms and proteolytic enzymes, have immediate efficiency, low cost, lack of toxicity and absence of matrix interferences in the doping control analysis. In the current study, the different components of the stabilization mixture will undergo experimental cycles trying to optimize and compromise between stabilization efficiency and analytical matrix interferences.

Main Findings

The current project focuses on the creation of a specially designed plastic urine collection container, coated in its interior surface with the previously developed in-house stabilization mixture aiming at improving the quality of sport urine samples without posing analytical interferences problems in accredited laboratories. Before implementing the chemical stabilization mixture in an industrial scale, the right application form (liquid, freeze-dried, or spray-coated) should be carefully selected and tested in pilot-scale so that it can be implemented in the doping control sampling protocol. The spray coating application form was selected as the more easily acceptable by the end user. The chemical stabilization mixture was spray coated in the interior walls of plastic urine Collection containers to simulate the doping control urine collection process. Its efficiency against steroid glucuronide degradation caused by five microorganisms (E. coli, N. simplex. E. faecalis, A. flavus, C. albicans) and enzymatic breakdown of intact hCG induced by six proteolytic enzymes (papain, pepsin, trypsin, α-chymotrypsin, bromelain and subtilisin A) was investigated during incubation experiments at 37 ºC. Also, two WADA accredited laboratories, DoCoLab (Ghent) and Laboratorio Antidoping FMSI (Rome) undertook the investigation of the stabilization mixture efficiency against rEPO degradation in the presence of proteolytic enzymes. Moreover, a systematic evaluation of eventual analytical interferences in the presence of the stabilization mixture in spray coated form was conducted by the Athens Doping Control Lab (DCLA), DoCoLab and Laboratorio Antidoping FMSI.

The addition of the chemical stabilization mixture in spray-coated form in the interior surface of plastic urine containers inhibited microbial growth and prevented steroid degradation at the end of a 7-day incubation period at 37 °C. The occurrence of the chemical stabilization mixture in spray-coated form prevented rEPO degradation by four proteases (papain, pepsin, trypsin and bromelain) during the 4-day period at room temperature. When α-chymotrypsin or subtilisin A were spiked, IEF signals were detectable at t = 0 in the basic area of the gel only in stabilized aliquots but were eliminated at t = 4 in both the stabilized and unstabilized aliquots. Regarding the degradation of intact hCG induced by proteolytic enzymes, hCG levels were higher in stabilized aliquots at the end of the 4-day incubation period at 37 °C for five out of six proteases tested (bromelain, papain, pepsin, α-chymotrypsin and trypsin). The presence of the chemical stabilization mixture in spray-coated form slowed down the degradation of intact hCG when subtilisin A was spiked but at t = 4, intact hCG levels were undetectable, irrespective of the presence or absence of the stabilization mixture. The evaluation of analytical interferences in the presence of the stabilization mixture in spray coated form, conducted by three WADA accredited labs, showed that some volatile compounds were negatively affected, depending on the urine matrix.

Code: 10A16OP

Testosterone misuse is the most detected doping offence in screening analysis. Nowadays, the ratio between testosterone and epitestosterone (T/E) is measured in the screening method. The exogenous nature of testosterone in those samples showing a T/E higher than 4 is confirmed by GC-C-IRMS. Although this approach gives satisfactory results in most of the testosterone misuse cases, there are some situations in which the value of 4 is not reached even after acknowledged testosterone administration. Those problematic situations include (i) detection of testosterone misuse in population with low basal T/E values, (ii) long term detection of testosterone misuse after oral intake and (iii) detection of testosterone misuse after topical administration.

Recently, four new testosterone metabolites have been reported in our laboratory after treatment of the sample with basic media. The detection of these metabolites in urine samples collected from a volunteer after oral testosterone administration showed that they substantially improved the detection of testosterone misuse in that individual case. Therefore, the addition of these metabolites into screening methods seems to be a promising complement to the commonly used T/E ratio.

Therefore, this project aims to check the usefulness of the quantitative detection of these metabolites for the detection of testosterone misuse in the challenging cases mentioned above: (i) detection of testosterone misuse in population with low basal T/E values, (ii) long term detection of testosterone misuse after oral intake and (iii) detection of testosterone misuse after topical administration. For this purpose, a quantitative method will be developed and validated. Afterwards, the method will be applied to samples collected after different testosterone applications.

Main Findings

Recently, some testosterone metabolites appearing in urine after alkaline treatment have been reported in our laboratory. In this project, the applicability of these metabolites for doping control purposes has been evaluated.

In the first step of the project a quantitative method for the detection of alkaline released metabolites in human urine was developed and validated. Analytical figures (linearity, precision, accuracy and limits of quantification) of the validated method were appropriate for the quantification of both basal levels present in urine of non-treated subjects and concentration levels present after testosterone administration. Besides the satisfactory figures, the method involves an easy and rapid sample treatment which favors its application to the large number of samples required for the development of the project.

In a second step, the validated method was applied to 173 urine samples and reference population concentrations were calculated. Using the software refval a concentration threshold was established for every metabolite and for the ratios calculated between them. The applicability of these thresholds was evaluated. Similarly to T/E, these population based thresholds are useful when large doses of endogenous steroids are administered like in the oral administration of testosterone. However, in small doses, these thresholds are normally not exceeded and, therefore, individual threshold values like the ones used in the Athlete Biological Passport seem to be the option of choice in order to apply these metabolites in the fight against doping.

In the last step, the applicability of these metabolites for the detection of testosterone misuse has been checked. After oral administration, the quantification of these metabolites and several ratios between them allowed for the increase on the retrospectivity. The best marker seems to be 1,4-androstadien-3,17-dione (boldione). The quantification of this marker after alkaline treatment of the urine increased between 3 and 6 times the retrospectivity of the detection of oral administration of testosterone. Ratios involving boldione after alkaline treatment of the sample have been found to be also useful for the detection of administration of single topic dose of testosterone. Although ratios involving 15-AD seem to be useful for this purpose, the absence of the reference material avoid for the quantification of this metabolite.

Although the preliminary results shown in this project seem to indicate that the inclusion of testosterone metabolites released after alkaline treatment in the Athlete Biological Passport can be useful for the detection of endogenous steroid consumption, several parameters have to be evaluated before. Among them, it is remarkable the evaluation of the potential of these metabolites as markers for testosterone administration in those subjects with low basal T/E values.

Code: 10C3JM

In this project, we will develop protein target prediction software to allow the performance-enhancing potential of a molecule to be identified from its chemical structure. This will be made freely available to WADA and to national anti-doping agencies. A Web interface, suitable for intranet deployment, will be underpinned by state-of-the-art predictive software linking molecules to the protein targets against which they are active; both on-target and off-target activities are covered equally well.

We will use machine learning methods to identify substances with potential for use as doping agents. We will primarily employ the Random Forest algorithm, in which we have particular expertise and which has given us excellent results in previous studies. By means of hybrid descriptors combining the geometrical detail of Ultrafast Shape Recognition (UFS) with the chemical information provided by MACCS descriptors, we will encompass both key aspects of molecular recognition. We will use protein target prediction to obtain the on- and off-target bioactivities of molecules with known and unknown doping potential. The profile of activities across a representative panel of protein targets is the molecule’s “bioactivity spectrum”. We will use the bioactivity spectra of known performance-enhancing molecules to predict those of compounds whose performance-enhancing potential is unknown. Thus we will classify molecules, including both licensed pharmaceuticals and other drug-like compounds, as potentially performance-enhancing or otherwise.

While the use of illegal performance-enhancing substances continues to threaten both the integrity of sporting competition and the health of athletes, our software will allow early identification of potential doping molecules. These compounds can then be prioritized for experimental testing, while no further experiments need to be conducted on those with negative in silico predictions. The use of this computational technology will massively reduce the need for animal or human experiments.

Main Findings

In this project, we will develop protein target prediction software to allow the performance-enhancing potential of a molecule to be identified from its chemical structure. This will be made freely available to WADA and to national anti-doping agencies. A Web interface, suitable for intranet deployment, will be underpinned by state-of-the-art predictive software linking molecules to the protein targets against which they are active; both on-target and off-target activities are covered equally well.

We will use machine learning methods to identify substances with potential for use as doping agents. We will primarily employ the Random Forest algorithm, in which we have particular expertise and which has given us excellent results in previous studies. By means of hybrid descriptors combining the geometrical detail of Ultrafast Shape Recognition (UFS) with the chemical information provided by MACCS descriptors, we will encompass both key aspects of molecular recognition. We will use protein target prediction to obtain the on- and off-target bioactivities of molecules with known and unknown doping potential. The profile of activities across a representative panel of protein targets is the molecule’s “bioactivity spectrum”. We will use the bioactivity spectra of known performance-enhancing molecules to predict those of compounds whose performance-enhancing potential is unknown. Thus we will classify molecules, including both licensed pharmaceuticals and other drug-like compounds, as potentially performance-enhancing or otherwise.

While the use of illegal performance-enhancing substances continues to threaten both the integrity of sporting competition and the health of athletes, our software will allow early identification of potential doping molecules. These compounds can then be prioritized for experimental testing, while no further experiments need to be conducted on those with negative in silico predictions. The use of this computational technology will massively reduce the need for animal or human experiments.

Code: 10D4GG

Analytical attempts to monitor the application of testosterone doping -constituting as much as 66% of the all analytical findings for anabolic agents - focus mainly on the profiling of testosterone, its precursors and metabolites in urine samples, especially but not limited to the ratio of testosterone and epitestosterone. The establishment of reference ranges including a large variety of endogenous steroids shall contribute to a better detection of abnormal concentrations or ratios.

Urine as a sample matrix for investigating doping practices collects substances which have already passed the body, being like a waste bin their final destination and a remote collection matrix relating to the application of doping substances. Especially in the case of endogenous substances like steroids the influence of metabolism and excretion is levelling possible markers.

Leveling effects are expected to be smaller in serum samples. Especially in the case of parenteral application, normally having a lower amount of substance to enter the body due to the fact, that the liver passage represents no barrier and first pass metabolism, serum is expected to yield a better detection potential. Testosterone esters, the pharmaceutical form of testosterone applied within the frame of this project, besides unmodified testosterone, can be detected in serum samples, whereas they are undetectable in urine.

Due to the implementation of blood profiling as well as the detection of substances with minimal renal passage the access to blood and serum samples grows to get common practice and increasing importance in doping control. Steroid profiling is part of the concept of the athlete’s passport.

The aim of the proposed project is to target unique markers for parenteral application of testosterone preparations. To reach this goal three different parenteral routes of testosterone application are monitored. In addition a comparison to an oral application of a testosterone ester is done.

Main Findings

The aim of the project was to target endogenous serum markers for application of testosterone preparations. In a clinical study, endogenous hormones in serum were monitored after the application of four different testosterone formulations; gel for transdermal delivery, transdermal patch, intramuscular injection, and oral tablets.

The analytes testosterone (T), epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), dihydrotestosterone (DHT), androstenedione (Adione), 5α-androstane-3α,17β-diol 5β-androstane-3α,17β-diol(5β-diol), dehydroepiandrosterone 17-hydroxyprogesterone(17-OH-Prog),progesterone (Prog), luteinizing hormone (LH), follicle stimulating hormone (FSH) and sex-hormone-binding globulin (SHBG) were included in the project. The samples were assayed for LH, FSH, SHBG, and Prog using immunological methods. For all other analytes, a sensitive method was developed employing liquid-liquid extraction (LLE), derivatization, and liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS).

The study was open and randomized with no blinding procedure. It was divided into two periods during which each participant, randomly assigned, received an oral formulation in one of the periods, and a parenteral application (patch, gel or intramuscular injection) in the other period. All together 12 volunteers (men) participated in the study, and serum samples were collected for 4 weeks after drug application.

Changes in hormone serum concentrations and ratios were assessed using Wilcoxon matched pair test and Friedman ANOVA.

The serum concentrations did not significantly change for any of the hormones measured during the study. Furthermore, no significant changes were observed for the ratios T/LH, T/Prog, T/SHBG and DHT/T. A significant increase was, however, observed for the ratio T/17-OH-Prog in subjects treated with testosterone gel, compared to baseline values. In subjects treated with tablets, patch and intramuscular injections, a trend towards increased T/17-OH-Prog ratios was observed, but the increase was not statistically significant. The findings on elevated T/17-OH-Prog ratios are in accordance with previously published studies [1, 2].

Code: 10A17NB

Since the beginning of the fight against doping, urine is considered to be the preferred biological fluid in which to examine the most commonly used performance enhancing compounds. This was an obvious choice because urine acts as a waste bin where xenobiotics are deposited by the organism. The first analytical techniques used in the fight against doping were thus tailored to detect drugs or drug metabolites at concentrations that can be found in urine. The use of this biological fluid, which could be collected non-invasively during anti-doping tests, became prevalent. Samples could be easily prepared from the urine matrix, and the detection range of performance enhancing drugs was such that it was possible to demonstrate their in-competition use to enhance performance.

Nowadays, analytical techniques have significantly improved in terms of sensitivity and specificity. These improvements allow the scientists to detect forbidden drugs at very low concentration and for a longer time period in multiple different matrices. Even if the blood collection is an invasive procedure, the simplicity of the collection protocol is an important aspect to consider for athletes and doping control officers. Furthermore blood is becoming a biological matrix more and more considered within the antidoping and sport organizations and thus the detection in blood of substances included in the WADA Prohibited list is becoming evident.

The aims of this pilot study are to evaluate the sensitivity and specificity detection, in urine and blood, of 15 different compounds that are the most reported as adverse analytical findings by the WADA laboratories. Samples will be collected after a single dose administration to 3 volunteers for each substance. Finally, we will compare two analytical techniques (LC-MSMS and GC-MSMS) to appraise their use in the fight against doping in urine and blood matrices.

Main Findings

During this project, analytical developments for the detection in blood of compounds like the major endogenous and exogenous steroids as well as opioids and clenbuterol were done. The quantification of endogenous steroids linked to testosterone metabolism is well documented and robust in urine matrix. The blood concentrations of these compounds are important also to give another tool to resolve the difficult topics of testosterone and related substances abuse in sports. The future advent of the steroid module of the athlete passport as well as the establishment of reference values for the athlete population was the main motivation of this part of the project. Sample preparation was the main challenge to solve as the extraction of the investigated compound from the blood matrix is totally different than the one used for urine preparation. Different approaches were tested and a simple extraction using a solid support for a liquid extraction (SLE) was selected. Then limit of detection, linearity and first validation data are promising for a near future accomplishment of this part of the project. Collaboration with the London Anti-Doping laboratory is in progress to compare the two different approaches for the quantification of testosterone and epitestosterone in blood. Results are supposed to be available in the following months. The main exogenous steroids were detected in blood by LC-MS/MS in pg/mL ranges. For all the screened substances a LOD of 100 pg/mL was reached and for few specific compounds a 10 pg/mL LOD was obtained. Comparison of the sensitivity and the time for sample preparation bet ween LC-MS/MS and GC-MS/MS revealed that the first one is more suitable and allowed to reach a lower LOD for the exogenous steroids. Opioids quantification in blood was developed and totally validated. Comparison with immunological tests and the GC/MS quantification showed a good correlation indicating that the use of blood for the opioids detection is fit for purpose. Clenbuterol quantification has been developed to evaluate the complementary information that blood could represent in the context of food contamination or doping abuse in case of adverse analytical finding with clenbuterol. Again, first data obtained regarding LOD, linearity and excretion study samples analyses showed that the developed sample preparation and LC-MS/MS analytical tool were adapted to this purpose. This work gave the opportunity to the laboratory to handle with blood matrix for the detection and quantification of forbidden substances. Perspectives are to continue on this way in order to finish the ongoing (LIVE) validation process. Future implementation of blood analyses for the detection of drug of abuse in sports will be of high interest.

Code: 10A21JY

The aim of our project is to prepare metabolites of beta-blockers timolol, bisoprolol, labetalol and carteolol as reference substances to be used in various doping analytical applications. The results of this project will be directly and immediately applicable to the fight against doping in sports. All the above mentioned drugs are included in the World Anti-Doping Agency’s list of prohibited substances.

According to the data available (WADA), there have been many positive doping findings where athletes have been caught using these drugs during recent years. In doping analysis, the analytical data obtained from the sample are compared to the data obtained from a negative and positive reference. According to the WADA’s International Standard for Laboratories and ISO 17025 standard, well-characterized reference materials are recommended to be used as references in the analysis. Availability of well-characterized reference material increases reliability and legal defensibility of confirmation analyses. Furthermore, reference materials could also be used in quality assurance and in the development of new analytical methods.

The work will be carried out in close collaboration with two research organizations: Faculty of Pharmacy of the University of Helsinki and United Medix Laboratories, Helsinki, Finland. The Doping Control Laboratory of the United Medix Laboratories has been IOC accredited laboratory since 1983 and is now accredited by WADA.

Main Findings

Beta blockers are a class of antihypertensive drugs that were originally developed for treatment of angina pectoris. They have multitude of other indications such as coronary artery disease, congestive heart failure, ischemic heart disease and management of cardiac arrhythmias. By using beta blockers athletes can control effects of performance anxiety such as nervousness, hand tremor and high heart rate.

In doping analysis, the analytical data obtained from the sample are compared to the data from a negative and positive reference. According to the WADA’s international laboratory standard and ISO 17025 standard, well-characterized reference materials are recommended to be used as references in the analysis.

The aim of this study was to synthesize phase I metabolites of bisoprolol and timolol, however the synthesis of a timolol metabolite could not be completed. The synthesized bisoprolol metabolite was characterized by NMR and mass spectrometry and compared to the metabolites extracted from urine samples to confirm the structure. Stability studies of the metabolite were also performed. Samples of O-desisopropylbisoprolol were provided to the accredited doping laboratories at the Cologne Workshop for Dope Analytics 2013.

Code: 10C24ZN

Detection of autologous blood transfusion is probably currently the greatest challenge in the doping control. For the reasons of convenience, for its considerable security and the difficulty of detecting it, autologous transfusion has become the method chosen by many athletes to illegally increase their oxygen delivery capacity. This project aims is to study the proteome changes of erythrocytes during storage, prior to (re) infusion of the blood to the same athlete.

For the project were selected proteins associated with the erythrocyte membrane, because they are easily available and have been well characterized in previous studies with methodologies such as 2D electrophoresis, DIGE, liquid chromatography coupled with mass spectrometry, and other combinations of the above.

The studies made so far can be classified broadly as protein-centered or peptide-centered. In the first case, the proteomics approach is based on the separation of membrane proteins from the sample by 2D electrophoresis; in the second case, the most powerful among those described is the isobaric tagging (iTRAQ), which allows quantitative change detection in multiple samples.

The results of our preliminary studies, as well as those available from other authors, identified proteins which can be grouped as follows:

• cytoskeletal proteins,

• transmembrane proteins

• other proteins related to our research findings by other researchers. To continue our study, we consider efforts in two directions:

First, to validate the results obtained, confirm the same with the largest number of subjects,

Second, to verify by independent technical changes detected in the membrane by proteomic methods, we use Western blot and/or flow cytometry. Ultimately, the goal is to develop a reliable test for the analysis of doping by autologous transfusion, using appropriate markers from membrane proteins or the cytoskeleton of erythrocytes, whose levels vary due to storage of blood in standard transfusion conditions.

Main Findings

Autologous blood transfusion doping (ABT) is defined as the transfusion of stored red blood cells (RBCs) from the same individual. The World Anti-Doping Agency (WADA) includes ABT in the List of Prohibited Substances but no official method exists yet to directly detect it.

In this project the focus was directed towards quantification of erythrocyte membrane protein changes after transfusion. Previously described data showed that some proteins translocate from cytosol to the membrane during erythrocyte storage under standard blood banking conditions. Based on this data, we have selected a panel of proteins to be further characterized. The list included peroxiredoxin-2, glyceraldehyde-3-phosphate dehydrogenase, catalase, tropomodulin-1, sorcin and annexin-7. Using immunoblotting we have observed that in general these proteins were already present in fresh blood samples and their levels increased on the membrane of the erythrocyte during in storage. Additionally, when stored RBC concentrates were mixed in vitro with fresh blood simulating a transfusion procedure, an increase of the marker proteins in the mixtures was observed. However, when blood from recently transfused patients was analyzed, there was no a clear trend in the increment or decrement of the marker proteins. On the other hand, we have observed that the training for endurance disciplines as compared to non-endurance disciplines (both in aquatic sports) seem to influence the basal concentrations of some of the marker proteins, especially those linked with the oxidation protection of RBC.

Based on all the above considerations, it is doubtful whether the changes in marker RBC membrane proteins by translocation from cytosol to membrane may afford useful methodology for detecting blood transfusion in antidoping control by analyzing blood collected from a transfused athlete. However, the analysis of a transmembrane protein, glycophorin A, unraveled modifications that modify protein molecular weight during blood bag preparation or storage conditions, suggesting that characteristic changes on erythrocyte surface are occurring. Therefore, the characterization of the time sequence line of these modifications produced on integral membrane proteins could represent a useful approach to detect autologous blood transfusion.