Projets de recherche

Code: R04A12PS

The aim of the project is to evaluate a procedure to detect the use of any substance (i.e. erythropoietin and analogs) or method (i.e. homologous or autologous transfusions) that induce an increase in total haemoglobin.

The proof of principle is based in monitoring variations in the oxygen saturation curve by comparing measurements under normoxia or hypoxia in the resting state. It is proposed that the results will show that individuals who resorted to any of the aforementioned prohibited substances or methods will show a better adaptation under hypoxic conditions, evidenced by a significantly increased oxygen saturation value.

In all, the overall objective is to develop an indirect field test to instantly measure this type of doping in addition to urinary and/or blood tests used to that effect. It is envisioned to use such method on the same grounds as the currently utilized hematocrit determination.

Main findings

The aim of this study was to investigate an indirect method based on variations in haematological markers that could be used to identify haemoglobin-enhancing substances or methods that improve oxygen transfer.

The selection of statistical markers has been realised from experimental variations obtained during different phases including an increase in training volume at sea level, high altitude training, blood withdrawal and autologous blood transfusion. Blood arterial oxygen saturation under hypoxia conditions at resting state, like other markers, could not be taken into account due to the absence of statistical uniformity. The markers selected were hematocrit (Hct), haemoglobin concentration ([Hb]) and stimulation index (Off-hr). An absolute norm of variation (normΔ) for each selected markers allowing the distinction between normal and abnormal variations was established at the time. The absolute norms of variation obtained are:

normΔHct>6%

normΔ[Hb]>4%

normΔOff-hr>20%

Application of this method for one day competitive trials would ideally consist in taking the first blood sample 15 days before the competition and the second one upon its completion. For competitive trials of several days or weeks or during championships lasting several months blood samples should be taken without warning at a maximum interval of 15 days. In the future, this maximal interval could be increased with use of localisation data collected with the ADAMS system, and specifically those linked with presence at high altitude or not of an athlete.

Code: 04D22CG

The aims of the proposed research study are the following:

• Unification of the WADA Accredited Doping Control Laboratories screening procedures of classes of prohibited substances including classical small molecules like stimulants, narcotics, steroid agents, diuretics, etc, in order to free laboratory resources to new classes of prohibited substances: e.g. proteins. Modern sensitive high mass resolution, full mass range fast scanning mass spectrometer technology Time-Of-Flight, coupled with GC and LC chromatographic systems, capable to cover all classes of small molecules prohibited substances in minimum analytical runs per samples will be used. Sample preparation techniques using the minimum of steps but compatible with all classes of small molecules/prohibited substances will also be used. Incorporation of new drugs or metabolites, belonging to similar classes of molecules, with minimum or no changes to the laboratory preparative/analytical methods will be facilitated.

• Development of two new analytical tools, based on the instrumentation referred to in the previous paragraph, in order to widen the spectrum of the detection of prohibited substances or metabolites that do not exist as reference materials in the laboratory: a) creation of predictive analytical data, for molecules that are computer designed in the laboratory, b) bioassay-directed identification of unknown molecules

Main findings

A novel, rapid and easy-to-use method, EPO WGA MAIIA, for determination of aberrant EPO isoform subpopulations in urine or serum, has been tested for its use as an EPO doping control method. The method separates EPO subpopulations due to their different interactions with the lectin wheat germ agglutinin (WGA). The glycosylated structures on recombinant epoetins show stronger interaction with the lectin, probably due to their higher content of polylactose amine. The WGA-based separation of isoforms and the subsequent ultrasensitive EPO determination is rapidly carried out within a few square cm of a thin porous layer formed as a test strip, using an image scanner for quantification. The test takes only 20 min. to perform and is well suited both for determination of single samples and for large series. Before analysis with the EPO WGA MAIIA method, EPO is purified and concentrated from urine or plasma by use of a newly developed disposable EPO affinity purification device (www.maiiadiagnostics.com). With this easy-to-use sample preparation device EPO can rapidly be captured from large sample volumes and be eluted in a final volume of only 55 µL. The high EPO recovery of 65%, and the retained isoform distribution, makes the device a useful pre-step tool also for e.g. IEF, SDS-PAGE and LC/MS. The EPO WGA MAIIA method allows detection of recombinant EPO in urine specimens from patients up to about 7 days after the last injection (p<0.0001). Recombinant epoetin e.g. alpha, beta, omega, delta, zeta and four Chinese types (p<0.0001), and EPO analogues like Aranesp (p<0.0001) and Mircera can be distinguished from endogenous EPO isoforms. Mircera shows less interaction with WGA compared to endogenous EPO, while recombinant EPO:s show stronger and Aranesp shows the strongest interaction. Only 2 pg of EPO is required for isoform detection, which is about 1/10 of the amount of EPO required for the presently used IEF based doping method. When rhEPO beta and endogenous EPO appear in the same sample it is possible to detect rhEPO down to a level where it constitutes only 40% of total EPO. Besides measuring the interaction of the various types of EPO with WGA, it is possible in the same test strip to utilize also their interaction with the anti-EPO immobilized in the detection zone (see J. Immunol. Meth. 339 (2008) 236–244). By interpreting the antibody interaction profile, using the EPO AbQ MAIIA algorithm, it is possible to distinguish EPO and epoetins from EPO analogues like Aranesp and Mircera. The EPO WGA MAIIA test gives also an estimate of the EPO concentration in the eluate, enabling calculation of optimal application volume for the IEF or SDS-PAGE confirmation test. The recommended test set-up for doping control utilizes EPO WGA MAIIA for identification of epoetins and Aranesp, while Mircera preferably is identified by EPO AbQ MAIIA. The EPO WGA MAIIA test classifies all tested epoetin varieties and EPO analogues correctly, shows good resolution between endogenous EPO and epoetins, and can measure very low amounts of EPO. The quality controlled reagents will be supplied worldwide as a complete kit. The hands-on time is reduced compared to presently accredited tests, which significantly decreases the analysis cost. The excellent results and the easyto-use concept seem to fulfil the requirements for a screening EPO doping control test. Such a test makes it possible to considerable increase the number of EPO doping controls performed without increasing the total analysis cost.

Code: T04D27JD

The aim of this project is to set criteria for the misuse of glucocorticosteroids including synthetic glucocorticosteroids, natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen as stimulators of the adrenal cortex. For synthetic glucocorticosteroids, a general methodology has to be established including oral and intra-muscular administration as systemic routes and nasal and/or inhalation administration as local routes, with a high probability of systemic effect. The other local routes are to be considered as having a low probability of systemic action. According to this scheme, a two step study was built. First, for each test substance, time-related studies will be carried out under well controlled clinical conditions (ie clinical trials) for the systemic routes (oral and IM), as well as for the local routes with a high probability of systemic recovery (nasal and/or inhalation). These results will be used to set preliminary criteria for positivity (part I). Secondly, for some substances, time-related studies will be performed under ambulatory treatments (uncontrolled conditions) for the other local routes (dermal, intra-articular). These results will be used to confirm the preliminary criteria or to modulate them if necessary (part II). Moreover, urine indicators of native cortisol metabolism breakdown will be monitored by GC-MS in parallel to the measure of synthetic glucocorticosteroids urinary concentration by LC-MS/MS, whatever the mode of administration (part I and part II of this study). It is expected that these additional results will offer a way to confirm the general authorization for local routes or to modulate their initial status, especially if there is any evidence of a substance-related health risk due to cortico-adrenal gland suppression. For natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen, the determination of endogenous glucocorticosteroid profiles by GC-MS analysis is also regarded as a key step in this study. Consequently, complementary analyses of the main cortisol metabolites by GC-C-IRMS and of other native steroid compounds by CC-MS will be performed.

Main Findings:

AFLD Laboratory: Via deux études de population distinctes nous avons établi des seuils de suspicion correspondant à la moyenne + 2 écarts types, puis des seuils de positivité correspondant à une déplétion isotopique de 3 ‰ sur la base de la moyenne +é- 3 écarts types. Ces seuils sont d’accord avec les études d’excrétion au cortisol et à la cortisone. Ces études contrôles ont démontrée que l’interprétation des rapport Y+⁄THF et THS et F⁄THS lorsqu’ils sont pris ensemble sont globalement en concordance avec la technique. Nos études étant limitées a des sous population d’origine caucasienne, l’universalité des résultats devrait être examinée a travers des études de sus population multiethnique. Une variante de la méthode directe d’oxydation utilisée pour IRMS avec séparation des métabolites du cortisol et de la cortisone de la désoxycortocosterone avant l’oxydation améliorait la spécificité de l’analyse isotopique. Lausanne laboratory : Les résultats de dosage obtenus après investigations des divers glucocorticostéroïdes montrent une grande variabilité en fonction des modes d’administration, des concentrations maximales éliminées par les individus, du rapport entre les fractions libres et conjuguées, de la durée d’élimination. Néanmoins, au vu des résultats, il est déjà envisageable de distinguer les voies systémique (orale, intramusculaire) des autres voies locales d’administration (pulmonaire, nasale) en fonction des concentrations urinaires éliminées des divers glucocorticostéroïdes investigués et de leur rémanence dans l’urine. Néanmoins, les voie intra-articulaire et péridurale devront être re-évaluées car ne peuvent être différenciées des voies systémiques. Après synthèse des résultats obtenus par les laboratoires de Paris, Lausanne et Sydney, il sera probablement possible d’affiner le seuil de positivité, permettant de réglementer l’administration des glucocorticostéroïdes. D’autre part, il serait intéressant de poursuivre et d’approfondir les investigations au niveau des injections intra-articulaires et péridurales, et éventuellement des autres modes d’administration (péri-articulaire, péri-tendineux, mais aussi les pommades, collyres et autres solutions). Au vu des résultats préliminaires, il serait possible en dosant le Cortisol de distinguer la voie systémique (orale et surtout intramusculaire) des autres voies locales d’administration, comme l’inhalation pulmonaire. Un affaissement du Cortisol urinaire avec des concentrations proches de zéro pourrait indiquer la nature de l’administration du glucocorticostéroïde. Néanmoins, des analyses complémentaires devraient encore être réalisées dans le but de confirmer et de généraliser cette observation. En particulier, sur un plus grand nombre de volontaires et surtout sur les échantillons provenant des IA et des autres applications locales.

Code: 04D06DH

The recent identification of two designer androgens, norbolethone and tetrahydrogestrinone (THG), has raised new concerns regarding risks from novel designer androgens custom-produced as undetectable sports doping agents. These two synthetic androgens were produced by modification of existing steroids to produce previously unknown (THG) or non-marketed (norbolethone) potent androgens. THG was produced from gestrinone, a progestin known to have androgenic activity and banned by WADA. Yet, among 36 commercially marketed progestins many have similar or greater androgenic potential than gestrinone and the capability to be chemically modified, potentially forming potent and currently undetectable designer androgens. Banning all synthetic progestins is not feasible given their importance in female reproductive health. Therefore it is essential to characterize the available progestins according to their risk profile and to develop detection methods for those with most androgenic activity and most capable of being developed into so-far undetectable doping agents. This project involves 3 stages, the first involves an in vitro yeast based androgen bioassay that we used to provide the first proof that THG was a potent androgen. This in vitro androgen bioassay is a sensitive and efficient screening method that can be applied to all available progestins and their analogs or metabolites as compared with a reference panel of synthetic androgens. The second stage involves confirming the androgenic bioactivity identified as positive in the in vitro screening bioassay by an in vivo mouse-based androgen bioassay. The third stage involves developing urinary Mass Spectrometric detection methods for progestins with significant in vitro and in vivo androgenic bioactivity.

Main Findings

A comprehensive review of commercially marketed synthetic progestins was completed using the yeast-based in vitro androgen bioassay. This led to a detailed description of steroid androgen receptor (SAR) for androgenic activity of synthetic progestins. The practical implications of this analysis for the WADA antidoping program were that the risk from newer synthetic progestins was relatively low as they lacked significant intrinsic androgenic potency. The major risk was from the older, first generation synthetic progestins, especially those derived from 19-nortestosterone, which had significant intrinsic androgenic potency that could be enhanced by simple chemical modifications. Nevertheless existing GC/MS profiling was already available for these compounds. These conclusions are supported by the rarity of novel progestin-derived illicit designer androgens subsequent to the discovery of THG. The simplified and modified mouse in vivo androgen bioassays gave quite consistent findings with rank order of potency of the in vitro androgen bioassays. Specific class-based comparisons confirmed that the newer third generation synthetic progestin, as exemplified by nesterone, had negligible androgenic bioactivity whereas the older early generation progestin, norgestrel, had significant intrinsic androgenic potency in vivo. These findings confirmed that no further detailed metabolic analysis of excretory products of modern progestins in humans is warranted as the significant risks are already covered by the known commercially marketed progestins.

Publications

L. McRobba, D.J. Handelsman, R. Kazlauskas, S. Wilkinson, M.D. McLeode, A.K. Heather. Structure–activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay. Journal of Steroid Biochemistry & Molecular Biology, 110 (2008) 39–47.

Code: R04D35CA

This project is aimed at providing rapid answers to testing authorities following the seizure and/or the discovery of use of a new steroid. As a matter of fact little can be done at the analytical level until the substance has bee fully characterized, its urinary metabolites identified, the reference material made available and its potential performance-enhancing properties investigated. Since 2000, the testing authorities have been alerted to the use of new steroids by certain athletes. Public comments made by individuals involved in the Balco scandal are adding to the information obtained from other sources, mainly informants, to the effect that other “designer” potent and undetectable steroids have been prepared and would be available to some athletes. That has become a certainty in December 2003, following seizure at the Canadian border of hGH and two steroid products one of which being THG. The identification of the second one, DMT (17α-methyl-5α-androst-2-en-17β-ol) has been done within the scope of this grant application. Lastly, urine samples of athletes at “high risks” have been found to contain metabolites of two isomers of a steroid which are not currently tested for. Steroids or urinary steroid metabolites will be identified employing analytical techniques such as mass spectrometry and NMR; structures will be proposed. The synthesis of reference standards will permit the full characterization of the compounds (parent and metabolites)> The hormonal properties of the steroids, androgenic/anabolic, estrogenic and progestagenic will be assayed. Studies will be conducted to enable the identification of the metabolites (phase I and II) which will permit ultimately their inclusion to testing methods.

Main Findings

We consider having fully met the objectives contained in the original application. New steroids were characterised: DMT1 ‐ 17α‐methyl‐5α‐androst‐2‐en‐17β‐ol major isomer along with the 3‐en isomer, methyldrostanolone ‐ 2α,17α‐dimethyl‐5α‐androstan‐17β‐ol‐3‐one (sold under the names of Superdrol, Methasterone; and Guggulsterone ‐4,17(20)‐pregnadiene‐3,16‐dione. Anticipating what could be the new molecules introduced clandestinely we have synthesized 17‐methylated derivatives of popular steroids such as methenolone and its isomer stenbolone. The phase I metabolites were successfully produced from incubations with cryopreserved human hepatocytes for several steroids particularly drostanolone and its 17‐methylated derivative, “Superdrol”. Structures were proposed for a novel metabolite hydroxylated in C‐2 and confirmed by chemical synthesis. NMR and mass spectrometry were utilised to characterise the metabolites. The model failed however to produce conjugated phase II metabolites and significant amounts of phase 1 metabolites from desoxymethyltestoterone, DMT. In spite of these limitations, the identification of metabolites was much easier in incubation medium when compared to the heavily interfered urine matrix. The utilisation of human hepatocytes significantly reduces to the minimum the administration to human volunteers. We have extended by the work in order to finish the characterisation of the metabolites of methyldrostanolone and drostanolone including new ones that were not reported previously.

Code: T04D27JD

The aim of this project is to set criteria for the misuse of glucocorticosteroids including synthetic glucocorticosteroids, natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen as stimulators of the adrenal cortex. For synthetic glucocorticosteroids, a general methodology has to be established including oral and intra-muscular administration as systemic routes and nasal and/or inhalation administration as local routes, with a high probability of systemic effect. The other local routes are to be considered as having a low probability of systemic action. According to this scheme, a two step study was built. First, for each test substance, time-related studies will be carried out under well controlled clinical conditions (ie clinical trials) for the systemic routes (oral and IM), as well as for the local routes with a high probability of systemic recovery (nasal and/or inhalation). These results will be used to set preliminary criteria for positivity (part I). Secondly, for some substances, time-related studies will be performed under ambulatory treatments (uncontrolled conditions) for the other local routes (dermal, intra-articular). These results will be used to confirm the preliminary criteria or to modulate them if necessary (part II). Moreover, urine indicators of native cortisol metabolism breakdown will be monitored by GC-MS in parallel to the measure of synthetic glucocorticosteroids urinary concentration by LC-MS/MS, whatever the mode of administration (part I and part II of this study). It is expected that these additional results will offer a way to confirm the general authorization for local routes or to modulate their initial status, especially if there is any evidence of a substance-related health risk due to cortico-adrenal gland suppression. For natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen, the determination of endogenous glucocorticosteroid profiles by GC-MS analysis is also regarded as a key step in this study. Consequently, complementary analyses of the main cortisol metabolites by GC-C-IRMS and of other native steroid compounds by CC-MS will be performed.

Main Findings:

AFLD Laboratory: Via deux études de population distinctes nous avons établi des seuils de suspicion correspondant à la moyenne + 2 écarts types, puis des seuils de positivité correspondant à une déplétion isotopique de 3 ‰ sur la base de la moyenne +é- 3 écarts types. Ces seuils sont d’accord avec les études d’excrétion au cortisol et à la cortisone. Ces études contrôles ont démontrée que l’interprétation des rapport Y+⁄THF et THS et F⁄THS lorsqu’ils sont pris ensemble sont globalement en concordance avec la technique. Nos études étant limitées a des sous population d’origine caucasienne, l’universalité des résultats devrait être examinée a travers des études de sus population multiethnique. Une variante de la méthode directe d’oxydation utilisée pour IRMS avec séparation des métabolites du cortisol et de la cortisone de la désoxycortocosterone avant l’oxydation améliorait la spécificité de l’analyse isotopique. Lausanne laboratory : Les résultats de dosage obtenus après investigations des divers glucocorticostéroïdes montrent une grande variabilité en fonction des modes d’administration, des concentrations maximales éliminées par les individus, du rapport entre les fractions libres et conjuguées, de la durée d’élimination. Néanmoins, au vu des résultats, il est déjà envisageable de distinguer les voies systémique (orale, intramusculaire) des autres voies locales d’administration (pulmonaire, nasale) en fonction des concentrations urinaires éliminées des divers glucocorticostéroïdes investigués et de leur rémanence dans l’urine. Néanmoins, les voie intra-articulaire et péridurale devront être re-évaluées car ne peuvent être différenciées des voies systémiques. Après synthèse des résultats obtenus par les laboratoires de Paris, Lausanne et Sydney, il sera probablement possible d’affiner le seuil de positivité, permettant de réglementer l’administration des glucocorticostéroïdes. D’autre part, il serait intéressant de poursuivre et d’approfondir les investigations au niveau des injections intra-articulaires et péridurales, et éventuellement des autres modes d’administration (péri-articulaire, péri-tendineux, mais aussi les pommades, collyres et autres solutions). Au vu des résultats préliminaires, il serait possible en dosant le Cortisol de distinguer la voie systémique (orale et surtout intramusculaire) des autres voies locales d’administration, comme l’inhalation pulmonaire. Un affaissement du Cortisol urinaire avec des concentrations proches de zéro pourrait indiquer la nature de l’administration du glucocorticostéroïde. Néanmoins, des analyses complémentaires devraient encore être réalisées dans le but de confirmer et de généraliser cette observation. En particulier, sur un plus grand nombre de volontaires et surtout sur les échantillons provenant des IA et des autres applications locales.

Code: 04D04DH

The project aims are to establish parameters to allow the detection of the use of recombinant human Luteinizing Hormone (LH) as a sports doping agent. LH is a natural pituitary hormone which, although on the banned list, has never been available previously but has become available as a commercial recombinant hormone marketed in Europe (Nov 2000). Clinically, LH is intended for use to trigger ovulation in anovulatory infertile women as well as potentially in in vitro fertilization hyperstimulation regimens. In men it is the natural stimulus to testicular Leydig cells to increase synthesis and secretion of testosterone. The availability of the recombinant LH as a potential doping agent for male athletes requires the development of a reliable and valid detection test. Like hCG, LH is most likely to be used mainly by athletes who have reduced testicular size and suppressed endogenous testosterone production due to use of synthetic androgens, but (in contrast to hCG) without risk of detection. As LH and hCG act upon the same LH/CG receptor, their biological effects are likely to be very similar. However, as hCG has a much longer circulating half-life than LH due to the C-terminal sialic acids residues in the CG β subunit, the effects of LH are likely to be of shorter duration making it easier to continue use closer to competition events which are subject to doping tests. Furthermore, in contrast to hCG, LH occurs normally in easily detectable concentrations in blood and urine so it also becomes necessary to distinguish between exogenous recombinant LH from endogenous LH.

Main Findings: 

This one year project aimed to undertake clinical administration studies using single doses of recombinant human LH (rhLH) and recombinant hCG (rhCG) to determine their effects on (a) conventional urine steroid profiles, (b) blood hormone analyses and (c) to provide samples for development of novel tests for rhLH and rhCG as sports doping agents in men. The clinical studies recruited healthy young men to have one of two rhLH doses (75 IU, 225 IU) with or without prior suppression of endogenous testosterone (T) by a single dose of 200 mg nandrolone decanoate. The original target of 32 men in a balanced design (2 LH doses [75 I, 225IU], with or without 200 mg nandrolone decanoate pre-treatment and 8 per group) was modified in the light of the interim analysis. This showed no consistent or significant effects on urinary LH or T at either LH dose. We therefore decided to incorporate a higher rhLH dose. A protocol amendment was approved and we completed the 4 original groups with 5 (rather than 8) men per dose but added 3 more men studied at a higher LH dose (750 IU, in two evenly divided doses 4 hr apart) without nandrolone pre-treatment. The LH component of this project was completed with 23 men having rhLH without adverse effects. Analysis of the time-course of serum LH and testosterone as well as urinary LH, testosterone and testosterone/epitestosterone ratio are consistent in showing no significant effects of any LH dose on blood or urinary LH or testosterone. Artefacts influencing urinary LH measurements were identified and shown to be rectified by redissolving the urinary sediment (to correct for non-specific adsorption of LH onto urinary sediment) and correcting for urinary creatinine or specific gravity (to correct for time of sampling and urine dilution effects). The hCG component of the study was completed with 24 men having one of two rhCG doses. The pharmacokinetics and pharmacodynamics of rhCG was defined with dose-proportionality of peak serum and urinary hCG levels but no effect of concurrent gonadal suppression by nandrolone. By contrast neither serum nor urinary LH or testosterone were influenced by rhCG dose and serum but not urinary T was lowered by concurrent nandrolone treatment. The T/LH ratio was highly sensitive to rhCG administration with a progressive and steep rise lasting well over a week after a single rhCG dose but without influence by rhCG dose or nandrolone treatment. rhCG and nandrolone had modest effects of increasing the T/E ratio. Subject to standardisation and validation of specific commercial LH immunoassays, the T/LH ratio remains a useful screening test for hCG doping.

Code: T04B8AK

Human growth hormone (hGH) is assumed to be abused as an anabolic hormone among athletes to enhance their physical performance. Several methods to detect hGH doping are under development, among others the “Marker approach”. Application of hGH effectuates a number of processes in the organism, which can lead to changes in the concentration of peptides and proteins. Some of these parameters measurable in serum are insulin-like growth factor I (IGF-I), acid labile subunit (ALS), IGF-binding protein-3, N-terminal propeptide of the type III procollagen (PIIINP), crosslinks (ICTP) and osteocalcin. The concentration of these markers vary inter-individually. Therefore it is impossible to discriminate between treated and untreated athletes using only one of these markers. As a consequence, it is necessary to calculate a discriminant function combining some of these parameters. As a result of our hGH application study with 15 athletes, we published recently a discriminant function which separated hGH-treated and placebo-treated subjects clearly. On the other hand there are indications that some markers are influenced by physical activities. As shown in some studies, acute physical stress influences the levels of these markers not very strong and only temporarily, but there are no data concerning the effects on long-term variation in physical stress (e.g. changes in intensity, category of physical activity, intermission and restart of training) on the parameters until now. The proposed study will give information about the long-term intra-individual variations of hGH markers and about the effects of long-term changes in physical activities on the hGH marker level.

Main Findings

For more than ten years intensive efforts have been made to develop the “marker approach” for detecting hGH doping. Due to the fact that exogenous and endogenous hGH are nearly identical and the metabolic half live of hGH is only 20 – 30 min, the search for a marker based method is reasonable.

From the vast number of GH-dependent peptides the insulin-like growth factor I (IGF-I) and the N-terminal pro-peptide of type III procollagen (PIIINP) seem to be the most promising markers for usage in doping analysis at the moment. Both markers are mainly GH-regulated and less sensitive to the acute effects of exercise. On the other hand in both markers the magnitude of the response to exogenous hGH and the concentration at baseline vary widely inter-individually.

Factors influencing these markers have been investigated in extensive studies (e.g. acute physical stress, differences between normal subjects and athletes, illnesses, injuries, different sports, medication, ethnicity).

Several studies tried to define age-dependent cut-off levels for the marker concentrations in athletes; however the ranges of these limits are very wide because of the enormous inter-individual variation.

Congruously, it seems to be reasonable to establish profiles of athletes including the marker concentrations of each subject to reveal possible manipulations. However for the evaluation of such profiles a thorough knowledge concerning the actual intra-individual fluctuation ranges and possible influences to these markers are indispensable.

It is known that both IGF-I and PIIINP show no diurnal variation and have a good long-term stability of their concentrations in normal subjects. However, only few investigations exist concerning the effects of deep changes in the intensity or category of heavy physical stress.

In this context the aim of the present study was to evaluate the long-term stability of IGF-I and PIIINP concentrations in high level athletes with special respect to changes in the intensity of the training workload.

Fifty male and female athletes (all Caucasians; 31 f; 19 m, 19,3 +/- 3,2y) were included in the study performing at least six intensive training sessions per week in different sports. Eight blood samples were taken from each subject over a period of 18 months with an interval of at least 4 weeks between each sampling, with special respect to changes in the training intensity and overall physical strain of the athletes.

IGF-I and PIIINP concentrations were determined in all serum samples using immunoassays.

About fifteen percent of the samples had IGF-I and PIIINP concentrations above the upper limits of the 95 % age dependent reference ranges specified by the manufacturer. The IGF-I concentrations did not differ significantly between male and female athletes; PIIINP levels in male subjects were significantly higher than in females.

As expected, there was a wide variation of the IGF-I and PIIINP levels between the athletes independent from age, reflecting the inter-individual variability of the marker.

The intra-individual variability of the IGF-I and PIIINP concentration was in female considerably higher than in male subjects. Partly, the fluctuations exceeded significantly the expected level. In single cases changes in the marker concentrations could be associated with changes in the workload level. The influence of the performed sport discipline couldn’t be proven, as the sports were not uniformly distributed among the gender.

Summarizing the study revealed unexpected high fluctuations in the hGH marker concentrations in high level athletes. The adaptation of the body to heavy physical stress caused by strong changes in intensity and category of the training workload can be observed in alterations of the marker levels.

Code: 04D18ES

Currently, chemical-analytical methods are the only methods used to detect specific compounds, compound profiles and metabolites in urine and serum samples from athletes as well as in natural- and synthetic ingredients in sport supplements and in food. Such methods have the advantage of being sensitive and highly selective, being able to fingerprint the use of particular drugs. They have the disadvantage in that the compounds being looked for are defined single chemical substances. Substances with slightly different chemical structure but with similar biological activity can be missed as their specific analytical detection parameters are not defined. In contrast, bioassays can directly probe the biological pathway of drug action rather than individual compound detection. Such methods have the advantage that bypassing detection is hardly possible since they will detect all compounds interacting with the endpoint of choice. BioDetection Systems’ CALUX® (Chemical Activated LUciferase eXpression) bioassays, being mechanism based, highly selective and extremely sensitive, have potential to be employed as broad screening tools, being able to identify the presence of hormone levels outside of established population normal ranges. These stable cell lines have incorporated the firefly luciferase gene coupled to specific hormone responsive elements together with selective receptors for the hormonal class of interest. Exposure of the designed CALUX® cells to their respective class of hormones induces the production of luciferase and the consequent emission of light. The amount of light produced is proportional to the amount of specific hormonal activity present. We have recently developed a panel of highly sensitive and selective CALUX® cell lines allowing sensitive, rapid, cost-effective and straightforward measurement of not only androgen-, but also estrogen-, progesterone-, and glucocorticoid receptor interacting compounds. As CALUX® bioassays also respond to endogenous hormones, we envisage determination of normal values within a specific population or sub-population followed by screening for deviations from these norms. A system consisting of screening for known compounds using sensitive chemical-analytical methods in conjunction with broader, effect-based CALUX® bioassays is expected to establish a robust system able to detect the use of almost any chemical compound that interferes with normal steroid hormone action. In this project suitable CALUX® bioassays will be established to detect relevant anabolic agents (with androgenic, estrogenic, progestagenic or glucocorticoid activity) as well as agents with anti-estrogenic activity capturing all steroidal compounds from WADA prohibited list.

Main Findings

Currently, chemical-analytical methods are used to detect specific compounds, compound profiles and metabolites in urine and serum samples from athletes, as well as natural- and synthetic ingredients in sport supplements and in food. Such methods have the advantage of being sensitive and highly selective, being able to specifically detect the use of particular compounds. The disadvantage of these chemical-analytical methods is that they can only detect a pre-defined list of chemical substances, or their metabolites. Substances with slightly different chemical structure but with similar biological activity can be missed as their specific analytical detection parameters are not defined. In contrast, bioassays can directly probe the biological mechanism of drug action rather than individual compound detection. Such methods have the advantage that bypassing detection is hardly possible since bioassays will detect all compounds interacting with the endpoint of consideration. BDS’ CALUX® (Chemical Activated LUciferase eXpression) bioassays, being highly selective and extremely sensitive, have potential to be employed as broad screening tools, are able to identify hormonally active compounds irrespective of their mode of action. These stable cell lines have incorporated the firefly luciferase gene coupled to specific hormone responsive elements together with selective receptors for the hormonal class of interest. Exposure of CALUX cells to their respective class of hormones induces the production of luciferase and the consequent emission of light. The amount of light produced is proportional to the amount of specific hormonal activity present. We have recently developed a panel of highly sensitive and selective CALUX cell lines allowing sensitive, rapid, cost-effective and straightforward measurement of not only androgen-, but also estrogen-, progesterone-, and glucocorticoid receptor interacting compounds. As BDS’ CALUX bioassays also respond to endogenous hormones, we envisage determination of normal values within a specific population or sub-population followed by screening for deviations from these normal values. A system consisting of screening for known compounds using effect-based CALUX bioassays in conjunction with more specific sensitive and identifying chemical-analytical methods such as GC-MS is expected to establish a robust system able to detect the use of almost any chemical compound that interferes with normal steroid hormone action. Almost all anabolic androgenic steroids (AAS) from the 2005 List of Prohibited Compounds were tested for bioactivity using CALUX bioassays. Also other potential anabolic designer steroids were included. Testing of AAS from the prohibited list demonstrated that anabolic androgenic steroids relevant in the field of doping control can be measured with CALUX bioassays. 98% of the tested compounds were active in one or more of the bioassays, and most compounds showed androgenic activity (88%). Only certain urinary metabolites of androgenic steroids on the list of the prohibited compounds, did not generate a measurable response in the AR CALUX bioassay. However, also estrogenic and progestagenic activities were demonstrated for many compounds. The AR CALUX bioassay can be used for determining the presence of bioactive compounds with known chemical structures as well as bioactive compounds with unknown chemical structures, the latter being especially helpful for the analysis of new designer steroids. Various potential AAS as yet not present on the WADA prohibited list, showed strong androgenic activities, as well as progestagenic activities. Methods were developed for the sample pre-treatment of urine and serum for analysis with CALUX bioassays. Steroidal activities in serum and urine samples could be analyzed both in extracts and un-extracted samples. Human urine samples had to be deconjugated prior to analysis. Extremely low limits of detection were obtained for the developed methods (for androgenic activity 0.02-0.04 ng DHT-equivalents/ml urine). Shake solvent extraction and solid phase extraction both proved to be suitable methods for the clean-up and concentration of urine and plasma samples for AAS analysis using CALUX bioassays. Analysis was also possible after direct exposure of CALUX cells to intact urine and plasma samples. All methods gave comparably high and reproducible recoveries of androgenic, estrogenic and progestagenic activity in spiked samples. Mixtures of AAS behaved additively in the AR CALUX bioassay and the total sum of androgenic effects could be correctly described with the concept of concentration addition. Comparison between the sum activity (total equivalent) of mixtures and chemical analysis of individual compounds can be facilitated by multiplying the concentration times the relative potency in the bioassay and summing them all up. This can be used in the development of an integrated system for doping analysis. One of the consequences of additive behaviour of mixtures of androgens is that low concentrations of compounds that are undetectable on an individual basis, together may generate a detectable response. This will e.g. be extremely helpful in the detection of multi drug abuse. CALUX bioassays were successfully applied to determine endogenous levels of steroidal activities in human urine samples collected from volunteers and from athletes competing at high level sports. Almost all samples showed activities several orders higher than the limit of detection. Androgenic, progestagenic and glucocorticoidal activities appeared to be significantly related with gender. Correlations between e.g. ethnical background and androgenic activity, and between contraception pill use and progestagenic activity were observed as well. By analyzing steroidal activities in this large set of samples normal values for endogenous levels of steroidal activities in urine could be determined. Comparison of AR CALUX bioassay results with GC-MS analysis of these human urine samples revealed that the endogenous androgens testosterone and DHT are the main contributors to normal endogenous steroidal activities measured by the AR CALUX bioassay. The comparison of AR CALUX bioassay activities with the main endogenous steroids detected with GC-MS in human urine samples showed an excellent correlation, providing proof of principle of the compatibility of both techniques. Since in the current project positive AAS doping samples were not available, the WADA proficiency test (PT) containing human urine samples of unknown composition was used to re-create a doping control setting. Very high androgenic activities were observed in some of the WADA PT samples, deviating from the “normal range”. To determine if the high androgenic (and progestagenic) activities of certain WADA PT samples are due to exogenous bioactive AAS or to high (supra-physiological) endogenous androgen levels, also GC-MS analysis for the main endogenous androgens in urine was applied on these samples, revealing a clear difference between endogenous androgenic activities (GC-MS derived) and AR CALUX derived androgenic activities. This suggests the presence of exogenous bioactive AAS in these samples. Parallel, human urine samples spiked with different concentrations of two AAS (THG and nandrolone) were analysed to obtain an impression of the capability of the AR CALUX bioassay to discriminate between AAS “positive” and “negative” samples. Indeed the AR CALUX bioassay could detect the presence of these active exogenous compounds above endogenous androgen levels and therefore was able to discriminate between doping negative and positive urine samples. In conclusion, in the strategy of an integrated system design, all experiments performed in the current project indicate that CALUX bioassays and chemical analytical analysis are complementary methods. These first experiments indicate that GC-MS is the method of choice for identification purposes and CALUX is the method of choice for screening unknown active compounds and mixture effects.

Publications

Besselink, H., Van de Heijning, M.P.M., Sonneveld, E., Brouwer, B. (2007) Validation of the ERα CALUX bioanalysis for the determination of estrogenic activity in human plasma. Organohal. Comp. 69, 3006-3009.

Brouwer, A., Sonneveld, E., Sterk, S.S., Stephany, R., van der Burg, B. (2006) A05 Development of CALUX bioassay-based systems as instruments to detect hormones and contaminants. J. Vet. Pharmacol. Ther. 29, Suppl 1:55-56.

Brouwer, A., Sonneveld, E., Sterk, S.S., Stephany, R., van der Burg, B. (2006) WS10 (workshop) Development of CALUX bioassay-based systems as instruments to detect hormones and contaminants. J. Vet. Pharmacol. Ther. 29, Suppl 1:35.

Houtman, C.J. (2007) Oral presentation at BioDetectors 2007, Amsterdam, the Netherlands Houtman, C. J., Sterk, S.S., Brouwer, A., Stephany, R., van der Burg, B., Sonneveld, E. (2008) Development of a tight bioassay-based control system for steroids in sport doping. Euroresidue VI ,Egmond aan Zee, the Netherlands. poster presentation.

Houtman, C. J., Sterk, S.S., Brouwer, A., Stephany, R., van der Burg, B., Sonneveld, E. (2008) Development of a tight bioassay-based control system for steroids in sport doping. Donike Workshop, Cologne, Germany . poster presentation.

Houtman, C. J., van de Heijning, M.P.M., Sterk, S.S., Brouwer, A., Stephany, R., van der Burg, B., Sonneveld, E. (2008) Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassay. Anal. Chim. Acta, in press.

Sonneveld, E., Jansen, H.J., Riteco, J.A., Brouwer, A., Van der Burg, B. (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid responsive bioassays. Toxicol. Sci., 83, 136-148.

Sonneveld, E., Riteco, J.A., Jansen, H.J., Pieterse, B., Brouwer, A., Schoonen, W.G., Van der Burg, B. (2006) Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities. Toxicol. Sci., 89, 173-187.

Sonneveld, E., Sterk, S.S., Pieterse, B., Brouwer, A., Stephany, R., van der Burg, B. (2006) Development of a tight bioassay-based control system for steroids in sport doping. Donike Workshop, Cologne, Germany. Poster presentation.

Code: 04D29AL 

The appearance of new abused molecules (e.g. HES, THG, adrafinil, modafinil) has increased the number of substances on the list and has given to the doping control laboratories a big challenge to keep their analytical procedures updated. Occasionally totally new analytical methods should be designed which is time consuming and may also require additional, expensive instrumentation or reagents. Furthermore, the increase in the number of separate analytical procedures renders the laboratory analysis more complex, delays reporting, increases the workload, and raises the cost of one test. Inclusion of new drugs and their metabolites in screening procedures is sometimes slow or impossible due to the lack of reference substances. For a long time, the analytics has mainly been based on different gas chromatography – mass spectrometric techniques However, recently, the excellent suitability of liquid chromatography – mass spectrometry (LC/MS) has been demonstrated for multi-analyte screening of many classes of prohibited substances (e.g. anabolic steroids, betaadrenergic drugs, diuretics and glucocorticosteroids). Actually, many new compounds added to the list of banned substances can be effectively screened only by LC/MS. In theory, LC/MS would have capabilities “almost for an all-in-one screening procedure”. Unfortunately, at the moment, this approach is greatly restricted by the use of non-universal sample preparation procedures and by the use of scanning-type of mass spectrometers. Recently, a novel toxicological screening method for urine samples based on liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) has been established. In the method, acidic, neutral, and basic drugs are extracted in urine and analyzed by LC/TOFMS with positive-ion ionspray and continuous accurate mass measurement. The method has been used effectively for screening of several different drugs, metabolites, and pesticides. In this project a general LC/TOFMS-based screening method will be developed and its effectiveness will be evaluated for several chemically and pharmacologically different doping agents. Compounds will be identified based on their monoisotopic masses and retention times. A substance database containing accurate masses and retention behaviour of the prohibited drugs will be built up and will be accessible without charge for all WADA/IOC accredited doping control laboratories. The method will be validated with respect to limit of detection, repeatability, extraction recovery and specificity. To investigate the efficiency of the method for formula-based metabolite identification, excretion studies with different drugs will be carried out.

Main Findings: 

The demanding task of the anti-doping laboratories is to detect substances on the prohibited list of World Anti-Doping Agency (WADA).The constant appearance of new prohibited substances challenges the laboratories to update their methods. Today, doping analysis requires the use of several different chromatographic and mass spectrometric methods. Consequently, a large number of separate analytical methods results in a more complex, time-consuming and laborious screening strategy. The aim of our research was to develop and validate a general liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) –based screening method for several chemically and pharmacologically different doping agents, in order to reduce the number of separate screening procedures used at present in antidoping laboratories. Included in the study were the following classes of prohibited subtances: agents with antiestrogenic activity, aromatase inhibitors, cannabinoids, beta-blockers, beta- 2-adrenergic agonists, diuretics, narcotics, and stimulants. The project began on March 2005 and terminated in September 2007. The method consisted of enzymatic hydrolysis of urine samples, solid phase extraction, separation of compounds on a reversed phase column and continuous accurate mass measurement by positive-ion ionspray TOFMS. Compounds were identified based on their monoisotopic masses, isotopic patterns and retention times. The method was validated with 124 different substances. The minimum required performance limit (MRPL) established by WADA was attained to 97 substance. The maximum mass error of the method was 0.7 mDa. Extraction recoveries varied between 33 and 98 %. Repeatability of the method for spiked urine samples (median of relative standard deviations at concentrations of MRPL and 10 times MRPL) were 14% and 9%, respectively. The method exhibited high specificity and proved to be suitable also for formula-based metabolite identification enabling preliminary identification of new drugs and their metabolites even without reference substances. A substance database containing chromatographic and mass spectrometric behaviour of all investigated substances was built up and will be accessible for all anti-doping laboratories for their method development. The developed LC/TOFMS method allowed identification of chemically and pharmacologically different drugs in urine in the same run and has great potential in doping analysis and simplifies analytical screening strategies in anti-doping laboratories.