Projets de recherche

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.

Code: 04D02PD

The classical methodology to detect anabolic steroids or other anabolic substances in doping analytics is GC-MS. However, last year, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances, are sometimes not identified during routine screening by GC-MS if their exact chemical structure is unknown. Obliviously there is the danger that anabolic steroids with unknown chemical structure can be misused without being detected in routine screening. In addition, beside classical anabolic steroids, also substances which interfere with growth factor associated signal transduction pathways may induce anabolic effects. Examples are inhibitors of metalloproteases lie TAPI or BB-3103, which have been demonstrated to modulate myostatin synthesis, and provoke myotube hypertrophy. At present it is completely unknown if such substances are being misused for doping. In addition, problems may arise with nutritional supplements for which purity and efficacy are largely unknown.
To detect anabolic substances, independently of their chemical structure, pharmacological in vitro test systems are promising alternatives and supplement the established systems used in routine doping analytics. In vitro test systems are used routinely in the development of pharmaceuticals and for the detection of poisons or endocrine disruptors in the environment. They are easy to handle, reliable and easy to adapt for high capacity screening. Anabolic steroids and growth factor interfering compounds are known to effect cell proliferation and differentiation of a myoblast cell line. In our project we want on the one hand to establish a test system for anabolic substances based on the analysis of cell cycle distribution and differentiation status of myoblast cells by flow cytometry. This test system would also allow identifying nonsteroidal substances with anabolic properties. In addition, a yeast-based test system has been established for the identification of androgenic compounds in the environment (endocrine disruptor identification) and plant extracts. Because of its sensitivity and robustness, this is a promising tool for the detection of anabolic steroids and metabolites in urine. New designer steroids (synthesized in the Institute of Biochemistry, Deutsche Sporthochschule, Köln) will be used together with well known compounds (DHT, THG etc.) to validate and characterize our bioassays.

Main Findings: 

The classical methodology to detect anabolic steroids or other anabolic substances in anti-doping analytics is GCMS. However, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances, are sometimes not identified during routine screening by GCMS if their exact chemical structure is unknown. Obviously there is the danger that anabolic steroids with unknown chemical structure can be misused without being detected in routine screening. In addition, substances which interfere with growth factor associated signal transduction pathways may induce anabolic effects. At present it is completely unknown if such substances are being misused for doping.
To detect anabolic active substances, independent of their chemical structure, pharmacological in vitro test systems are promising complementary techniques to the established systems used in routine anti-doping analytics. In the current project we have successfully established pharmacological in vitro test systems to detect anabolic substances based on their bio effects. Two strategies were followed. On one hand we tried to establish a test system where anabolic effects of substances could be detected because they affect the biological behaviour of C2C12 myoblast cells. On the other hand we tried to establish a test system for the detection of anabolic steroids in the urine, based on a stable transfected yeast androgen receptor (AR) transactivation system. Whereas the C2C12 system allows to detect all substances with the ability to modulate myotube formation, the yeast AR system will be able to detect all substances with affinity to the AR, but independent of a knowledge of the chemical structure.
The results of our experiments demonstrate that analysing the differentiation of C2C12 cells is a promising strategy to detect the anabolic properties of substances. Treatment of C2C12 cells with anabolic steroids including THG, resulted in an induction of differentiation which could be quantitatively detected by measuring the activity of the creatine kinase. However, the system would not be suitable to detect misuse of such substances in athletes’ urine. Our results obtained with the yeast reporter gene system demonstrate that this test system is a powerful tool to characterize substances with affinity to the AR pharmacologically. Furthermore we could demonstrate in first preliminary experiments that the system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of athletes who have abused steroids and have been identified by GCMS to be positive. Therefore we believe that this system can be developed towards a powerful (pre) screening tool and complement the established anti doping tests. It is characterized by its easy handling without need of high tech equipment, a high robustness, clearness of results and finally by its good cost efficiency. Because the test system is independent of the chemical structure, it is most suitable to be used as a prescreening system to identify the misuse of anabolic steroids including selective androgen receptor modulators (SARMs), especially in training out-of-competition controls. Our future aim is now to develop the yeast assay as a suitable and reliable test system applicable to routine doping analytics. Ultimately we aim to obtain an easy to handle and high sensitive assay with, which can after adoption be used in routine anti-doping analyses.

Code: 04D32PP

Testosterone gel is used for the treatment of testosterone deficiency in males. Testosterone is a hormone with strong anabolic effects and, therefore, belonging to the list of prohibited substances in sport. Testosterone misuse is – in most cases – being found by the analysis of steroid profile parameters and values of testosterone metabolites in urinary samples. In the case of testosterone gel application, however, results of preliminary studies done by the Institute of Biochemistry of the German Sport University suggest that this type of testosterone administration may be more difficult to detect. One possibility to detect the misuse of testosterone gel might be an endocrine test on the integrity of the hypothalamo-pituitary-gonad axis (HPGA) following a routine analysis of the urinary steroid profile. This kind of endocrine testing is routinely used in medical diagnostic investigations including, if indicated, stimulation tests of the HPGA with exogenously injected gonadotropin releasing hormone (GnRH). Under physiological conditions, exogenous GnRH leads to a defined secretion of LH, FSH and, subsequently, testosterone, all of them to be measured in blood samples. In addition, testosterone and its metabolites might also be measured in urine. In the case of chronic suppression of the HPGA, like after a chronic misuse of testosterone gel in sport, a pathological secretion profile of LH, FSH and testosterone is to be expected. However, there is no data available on the exact profiles of the secretion patterns after different time schedules of testosterone gel application and in dependence of physical exercise in athletes. Therefore, the present study will investigate the effects of different time schedules of testosterone gel applications in physically active people on the stimulatory pattern of LH, FSH and testosterone and its metabolites in blood and urinary samples. Concretely, an endocrine test will be developed and validated for the indirect detection of testosterone gel misuse in sport.

Main Findings

The misuse of testosterone gel has not been detectable at the beginning of this study. Therefore, the aim of the present study was to investigate the effects of two different time schedules of testosterone gel applications in physically active male athletes on the stimulatory pattern of LH, FSH and testosterone in blood samples, and thereby developing and validating an endocrine test for the indirect detection of testosterone gel misuse in sports. 17 physically active male subjects agreed to participate and were randomly divided into 2 groups. Group 1 (n=8) received 100 mg of testosterone gel per day over a period of 6 weeks in an intermittent manner with 7 days of application being followed by 7 days of wash-out-phases in between (INT). Group 2 (n=9) constantly received 100 mg of testosterone gel (Tgel) per day over the same period of 6 weeks (CON). Total time of application resulted in 3 weeks in INT and 6 weeks in CON. Serum basal concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone (T), and free testosterone (fT) were determined at 9 a.m. prior to the first Tgel application (W-1), weekly during the 6 week application period (W1 – W6) after each 7-days phase of Tgel application, plus two times after a wash-out period, once after 7 days (W7) and once after 21 days (W10) after the end of Tgel application. Furthermore, we analysed LH, FSH, T and fT 30 min, 75 min, and 120 min after exogenous injection of 100 ug of Gonadotropin Releasing Hormone (GnRH) at each time point of basal hormone measurement. Main results of the study showed a fast reaction of the pituitary gonadotropins after exogenous Tgel application. We observed a suppression of the spontaneous LH and FSH secretion (basal values) after only one week of Tgel, and a fast restitution in the wash-out weeks during intermittent Tgel and after the last week of Tgel during INT and CON. Doping with testosterone, therefore, indirectly is detectable from repetitive measurements of basal LH and FSH values, if 1st blood sample is taken as soon as possible (within a less than one week period) after finishing doping with testosterone gel, and if concentrations are interpreted individually. Although we found a trend for the expected immediate (e.g. after one week of Tgel) increase in exogenous GnRH-induced LH- and FSH-release, and a following decline according to a reduced gonadotropin biosynthesis, exogenous GnRH-induced LH- and FSH increments were not altered significantly beside a significant delayed reduction in GnRH-induced LH and FSH release after 6 weeks of Tgel, which remained reduced for both hormones even after 1 week of wash-out. The missing significance was mainly due to high inter-individual variability in stimulated LH and FSH secretion. For doping analysis of testosterone gel misuse, the combination of (still) reduced GnRH-induced LH and FSH release and already renormalized LH and FSH basal values after a period of testosterone gel application is another indirect indicator. As total and free testosterone concentrations showed a high inter-individual variability and remained in the normal range in many cases, the simple measurement of their blood values is not sufficient for the detection of testosterone gel doping. Serial measurements of blood concentrations in addition to the values of LH and FSH, however, might help in the interpretation of high-normal values.

Publications

Geyer H, Flenker U, Mareck U, Platen P, Piper T, Schmechel A, Schrader Y, Thevis M, Scha¨nzer W. In Recent Advances in Doping Analysis, vol. 15, Schänzer W, Geyer H, Gotzmann A, Mareck U (eds). Sport & Buch Strauß: Cologne, 2007; 133.

Platen P, Gehlert S, Geyer H, Marek U, Schrader Y, Franke J, Manchado Lopez C, Schänzer W: Endocrinological study on the effects of testosterone gel application in male athletes. Presentation on the Congress of the European College of Sports Sciences (ECSS) 2008, Estoril.

Piper T, Mareck U, Geyer H, Flenker U, Thevis M, Platen P, Schänzer W: Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun Mass Spectrom 22: 2161–2175, 2008.

Code: T04D05RK

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 standardization and validation of specific commercial LH immunoassays, the T/LH ratio remains a useful screening test for hCG doping.

Code: 04D28TK

Anabolic-androgenic steroids (AAS) are synthetic testosterone derivatives, which are designed to maintain the anabolic (beneficial effects) and to minimize the androgenic (side effects) activities of the endogenous prototype. More than 600 testosterone analogues have been synthesized since 1940s and in addition to conventional and bona fide pharmaceutical manufacturers, anabolic steroid products are provided in the market by a wide variety of illicit laboratories. As recently experienced, designer steroids have become a new threat for doping control as the compounds are “tailor made” to avoid the detection in the current analytical procedures. In man, AAS undergo extensive metabolism, in which they are converted into more polar compounds and are better excreted in urine. Therefore, besides new structure modifications, also the metabolism of the “new” AAS is generally not known, which leads to the lack of target compounds in doping control. To ensure the fast response to analytical challenges, the development and set-up of new analytical methods or implementation of new compounds in present procedures should be fast. Despite of its use to some extent, in vivo study of drug metabolism is difficult and time- and resource-consuming because of legislation aspects. Furthermore, designer drugs which are not registered as clinical preparations are not normally allowed to be used in human metabolic studies. An alternative pathway for the examination of metabolism are in vitro applications, which may utilise e.g. mammalian liver slices, cytosolic or microsomal preparations as source of metabolising enzymes. This approach is already well-adapted for in vitro studies in the field of pharmaceutical industry, especially in drug discovery. Utilisation of in vitro preparations is moderately straightforward, fast and amenable for experienced staff, and therefore a method of choice in metabolic studies of new AAS. The aim of the present project is to develop flexible in vitro procedures which can be applied in order to study and predict the metabolic patterns of new AAS with respect to most prominent target compounds for doping control purposes. The correlation between in vitro metabolism of human microsomes and in vitro excretion studies in human will be compared with model compounds and subsequently, the applicability of the in vitro model for prediction of AAS metabolic pathways for new doping agents will be evaluated. Parallel to the synthesis work an analytical aspect is also closely attached to the project. Liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods will be developed for the detection of new AAS metabolites to support the prevailing instrument methodology. Analytical experience gained in metabolic studies will be finally exploited in implementation of potential LC-MS/MS methods in routine doping control.

Main Findings: 

All the anabolic androgenic steroids (AAS) applied within this study were metabolized using an in vitro model consisting of combined microsomal and S9 fraction of human enzymes. Due to an excessive amount (50 M) of the steroid aglycone, combined fraction of hepatic enzymes and relatively extensive reaction times (4-5 hrs and 16- 17 hrs for phase-I and phase-II reactions, respectively), the number of in vitro formed metabolites was typically higher than those observed from in vivo excretion urine samples of corresponding AAS. The main focus of the analytical part of the project was to examine the applicability of liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method in the detection of phase-I and phase-II metabolites of AAS. As a conclusion it should be drawn the advantage of parallel application of both GC/MS and LC-MS/MS to cover the wide range of AAS metabolites. This is especially taken into account in the case of potential formation of phase-I AAS metabolites with completely saturated A-ring structure (e.g. 5 -estran-3-ol-17-one, the main metabolite of nandolone). Proton affinity of those metabolites is too low to yield [M+H]+ and the analyte is not detectable in LC-MS/MS analysis. However, conjugation with glucuronic acid enhances the ionization efficiency and phase-II metabolites of those same compounds are detected. The situation is completely opposite for the AAS metabolites with extensive conjugated double bond systems among A-, B- and Crings (e.g. allyltrenbolone and its phase-I metabolites), which are extremely problematic in GC/MS analysis. Due to chage delocalization and high proton affinity, LC-MS/MS is a method of choise for analysis of those AAS metabolites. Soft ionization technique, ESI, combined with LC separation also allow the direct detection of intact glucuronide-conjugated AAS metabolites.

Code: 04D25RC 

Beta2-agonist use under medical control is in constant increase in the elite sportsmen, particularly in the endurance sports. However, sensitivity of the exercise-induced bronchoconstriction (EIB) detection test is still under debate, partly due to the fact that several hypothesis exist concerning the underlying action mechanisms leading to EIB. In the first part of our multicentre project, we will re-evaluate the EIB prevalence by a new and more specific test, the mannitol test developed by S. Anderson et al., in order to limit the cases of false positive and thus, both the acute and chronic use of beta2-agonists by athletes. In a second part, we will investigate the potential deleterious effect of chronic use of beta2-agonists in pulmonary function, myocardic contractile function and bone metabolism. If these two hypotesis are confirmed, it would provide a strong argument for prevention against doping. Punishment is not the only way for the anti-doping fight. Prevention and education represent also fundamental aspects. The belief in wonderful effects of substance plays a significant role among the processes leading to doping. The exact knowledge of ergogenic effect and mechanisms involved is necessary to demystify drug effects. There are conflicting results as for the effects of acute or chronic intake of beta2-agonist intake on athletic performance. We propose to contribute to 1°) a more precise evaluation of EIB in elite population of athletes and 2°) a larger knowledge of beta2-agonist action mechanisms on different human systems which both should limit the misuse of beta2- agonists in sports.

Main Findings: 

This project dealing with β2-agonist focused on human performance, skeletal, cardiac, bone tissues and involved several laboratories of different countries. Nine separate studies were conducted.

In the first one, acute therapeutic oral intake of salbutamol appears to induce, an ergogenic effect during supramaximal exercise whatever the subjects' gender. An increase (P<0.05) in peak power and mean power output was observed during sprint exercise. This ergogenic effect could be due a positive impact of salbutamol on excitation-contraction coupling.

A second study was conducted to test this hypothesis in healthy athletes with similar therapeutic acute intake salbutamol (6 mg) under electrical stimulations in isometric condition. The peak torque of stimuli train of 1s duration at a frequency of 20 Hz was significantly modified (P<0.05) in low frequency fatigue condition compared to placebo situation while no change could be evidenced in resting condition suggesting a protective effect of salbutamol in fatigue state. An advantage could be anticipated in sports including maximal intermittent exercise (e.g. tennis, collective sport events). Moreover, in the twitch characteristics, the half time of relaxation was in most cases shortened under salbutamol condition compared to placebo. These protective effects against muscular fatigue elicited by acute salbutamol intake could be due to alterations in Ca2+ release and Ca2+ uptake by the sarcoplasmic reticulum.

The next studies focused on skeletal muscle adaptations. Obviously, the invasive investigations required were performed on animal. Three weeks of high doses of clenbuterol treatment induced anabolic effects (P<0.05) especially in EDL muscle and a greater isometric force was observed in both fast (P<0.001) and slow (P<0.001) twitch muscles (EDL and soleus, respectively) compared to control groups. Nevertheless, when maximal tetanic force was corrected for muscle cross-sectional area, specific tension was unchanged and even depressed in soleus (P<0.001) muscle. The increase in myofibrilar ATPase activities observed both in relaxed (P<0.001) and activated conditions (P<0.001) in soleus muscle suggests that the depressed specific tension is not due the contractile machinery it self. Another step in the muscle shortening process must be involved (see Ca2+ transient study below). The unchanged specific tension observed during clenbuterol treatment in EDL was associated with an unchanged myosin ATPase activity in fast twitch muscle. In the same line, the shortening duration of myofibrils in unloading condition was not significantly modified suggesting a lack of effect on maximal shortening velocity. Finally, significant reduction of fatigue resistance with clenbuterol treatment was observed in EDL suggesting changes within the muscle: MHC shift to fast and more fatigable isoforms, alteration in muscle calcium-handling ability and/or alteration in muscle energetic pathways. Concerning practical implications in doping prevention, one could argue that high dosage of clenbuterol would induce depressed specific isometric force especially in slow twitch muscle. This negative effect is of particular importance in sport events where performance depends on body mass. Another potential negative effect for slow twitch muscle lies in the increased ATP cost of shortening suggesting an altered efficiency in mechanochemico transduction and energy cost of locomotion. The most marked adverse effect concerning sport performance is probably the lesser resistance to fatigue observed with such high doses of clenbuterol especially for sport events with exercise durations around 1 min. Since these depressive effects could be linked with alteration in Ca2+ homeostasis the goal of next study was to characterise clenbuterol effects on calcium transients in fast twitch muscle. A decreased amplitude of global Ca2+ transients (P<0.01), associated with a reduced sarcoplasmic reticulum Ca2+ load (P<0.01) were observed after 14 days of clenbuterol administration. Local Ca2+ release events were also impaired (P<0.01) after only 9 days of clenbuterol administration. Ca2+ sparks in EDL muscle from clenbuterol-treated animals exhibit a reduced frequency, a smaller amplitude and a shorter spatial spread (P<0.01) compared to controls. The Ca2+ disturbances observed in the present study could explain firstly the negative effect observed in specific tension and secondly the skeletal muscle remodelling usually evidenced in the preceding study.

In this context, the two next works focused on hypertrophy and Ca2+ dependent of protein expression than its activity level. In our study, hypertrophy was significant (P<0.05) after 14 days of treatment in EDL muscle but calcineurin expression was increased (P<0.05) only after 21 days. The myogenic factor MyoD is usually associated with a fast phenotype of muscle fibers. Although in our study, both soleus and EDL underwent a phenotypic shift toward a greater proportion of fast fibers, MyoD protein expression was increased (P<0.05) only in soleus. In EDL, the proportion of type IIb fibers increased whereas type IIa decreased. In contrast, in soleus, the main alteration lies in the increased proportion of type IIa fibers. One can expect that MyoD protein expression correlates with the proportion of type IIb fibers. The mechanisms underlying the enhancement in cross sectional area seems to implicate both the calcineurin/NFAT pathway as well as the PI3/Akt/mTOR/p70SK6 pathway. The increase in soleus MyoD expression suggests that hypertrophy may come from the stimulation of satellite cells. However in this muscle, a necro/apoptotic phase appeared in the early stage signalling pathways. At doping doses, clenbuterol provoked an hypertrophy (P<0.05) more marked in fast twitch muscles (EDL) compared to slow twitch muscle (soleus). In EDL muscle, phenotypic conversion became significant earlier (9 days of treatment) than hypertrophy (14 days) suggesting that the phenotype fiber shift may be a prerequisite for hypertrophy. The rate constants that described phenotypic shift during treatment were similar between soleus and EDL suggesting a similar kinetics of phenotypic remodelling between slow and fast twitch fibers. Calcineurin protein expression is often used in the literature as a marker of muscle hypertrophy. Nevertheless, the implication of this protein in muscle anabolism seems less of the treatment, quickly followed by regeneration. Here we show that the ubiquitous calcium dependant proteolysis system, the calpains 1 & 2, are differentially activated (P<0.05). Soleus, characterized by a high density of !2-adreno-receptors, responded to doping doses of clenbuterol by a transient decrease (p<0.05) in calpain 1 activation and by a rapid calpain 2 activation. EDL, a rapid muscle responds by a progressive increase (p<0.05) in both calpain 1 & 2. The main difference between these two muscles is that soleus muscle underwent a rapid necro/apoptotic transient phase followed by regeneration, EDL did not. Thus, rapid muscles, with low β2-adreno-receptors density, seem to be protected against a transient necro/apoptotic phase. Calpain 1 may participate to the hypertrophy/ phenotypic process by cleaving the protein of cytoskeleton to insure the addition/replacement of the protein of the myofilaments. The precise role of calpain 2 is less clear. The calpain 2-induced partial proteolysis and activation of intermediates in the signalling cascades could participate to hypertrophy and/or the phenotypic shift, like protein kinase C. Here, the main practical implications in doping prevention lie in the two following findings: 1°) high doses of clenbuterol determine a transient deleterious impact on slow twitch muscle, rich in !2-adrenoreceptors, 2°) the ubiquitous calcium dependent proteolysis system is differentially activated in oxidative and glycolytic muscles and their activation may explain the disorganization/organization of the cytoskeleton for the addition/replacement of myofilaments required in hypertrophy. It is not excluded that cardiac muscle, very rich in !2-adrenoreceptors, could display a similar transient deleterious impact to soleus.

To further investigate the mechanisms involved in clenbuterol induced hypertrophy, another study was conducted with primary human muscle cell culture. To this end we compared the action of clenbuterol with the action of DAPT, a pharmacological inhibitor of Notch signalling and a prohibited substance in athletes. The results obtained here showed that clenbuterol and DAPT act as hypertrophic agents in vitro. They also indicate that these “anabolic” agents possess the ability to down-regulate the myostatin pathway. Indeed, myostatin could be a major player in the anabolic action of these compounds. These results raise the possibility that myostatin level might be a predictor of the use of anabolic agents. Therefore screening manipulations of myostatin signalling could be in the future a way in the fight against doping. Based on significant positive effects of chronic β-2 treatment on both muscle mass and force, we further investigate bone micro architecture because bone tissue is in principle closely related to mechanical loading and obviously to muscle force. For the first time, we observed deleterious effects of salbutamol on bone in trained rats, despite an increase in muscle mass. The alteration of bone tissue due to increased bone resorption occurred mainly in trabecular bone micro architecture (P<0.05) and was also observed in biomechanical measurements of cortical and trabecular bone (P<0.05). The doses used in our study are not so different to those delivered during the chronic administration of such drugs, particularly by young athletes under 20 years of age. This population is using more and more of these substances because of their lypolytic and anabolic effects on skeletal muscles. Unfortunately, the effects of these substances at doping doses have never been tested in sedentary humans or in athletes. We believe that testing salbutamol and clenbuterol on the rat bones has provided a valuable initial assessment of the skeletal phenotype of some athletes who take chronic salbutamol or clenbuterol for doping purposes. Of course an increased risk of fracture and especially risk of osteoporosis in senior female athlete could be anticipated.

Finally, we studied the effect of clenbuterol administration on cardiac performance and calcium transients in cardiomyocyte. Chronic treatment of animals with clenbuterol, for three weeks induced a positive inotropism at the cellular cardiac level (P<0.05). This is characterized by an increase (P<0.05) in cellular shortening mostly due to an increase (P<0.05) in calcium release from the reticulum at each contraction. These modifications increase with time of treatment (P<0.05) suggesting a lack of β-receptors desensitization between 9 and 21 days of treatment. The clenbuterol treatment was insufficient to modify the contractile properties of the myofilaments while at least of its component, titin, is phosphorylated by the clenbuterol treatment. Three weeks of treatment in our conditions were insufficient to induce deleterious effects on cellular contractility. However considering the large increase in the calcium transient (by 30%, P<0.05), it is possible that a maintained hyper stimulation of the myocytes at this level or to higher levels of calcium transient will have potential arythmogenic effects.

Code: 04C06JR 

The development of molecular biological technology over the last decade has resulted in dramatic advances in our ability to monitor, control and manipulate cellular systems. As a result the genetic modification of athletes (gene doping) considered a distant threat no more than five years ago is now considered a real threat to the integrity of sport. Recently the Netherlands Centre for Doping Affairs published a monograph on the issue of Gene doping. Genes suitable for use as doping agents were identified, as were suitable vectors for their introduction. The threat to sport of gene doping is made more pressing by a number of factors: • By its nature Gene doping cannot be controlled by the application of technologies currently in use in sports testing laboratories. • Vectors and genes that have been identified as potential doping agents reflect current knowledge and will change with our understanding of molecular biology. • The possible side effects and potential for serious impact upon the subjects undergoing gene doping has not yet been quantified but are likely to be profound. The introduction of a gene for production of IGF-1 is considered a high probability target for potential gene doping. The potential to increase muscle mass has already been demonstrated in model systems and its use in gene therapy is under investigation. For these reasons IGF-1 has been chosen as the model for gene doping for this research project. Modern biochemical techniques such as those employed for research into gene expression are immensely powerful, and provide tools for investigating subtle changes in the genetic makeup of organisms or the presence of foreign DNA. A radically different approach to detection, possibly more applicable as a screening methodology, could be developed through the application of proteomic or transcriptomic techniques. Following gene doping (or the application of an anabolic agent) the expression of one or more proteins will be altered, this expression will also be reflected in the translated RNA. Proteomic and transcriptomic approaches are targeted at identifying such changes in expression/translation. The application of multiple techniques to identify gene expression using cellular chemistry (London group), advanced mass spectrometry with ESI (Newmarket group) and MALDI (Nottingham group) for circulating proteins significantly improves the possibility of success.

Main Findings: 

The administration of GH and IGF-I gene therapy to a murine population, and the generation of mass spectral profiles produced suitable ANNs models for discriminating between doped and control populations. The GH gene therapy batches showed inconsistent results between batches, however the initial batch showed very promising results. The IGF-I gene therapy administration generated models that were not as accurate as the first GH batch, but were significantly better than the large GH gene therapy batch. The application of ANNs to the human GH administrations also generated a number of highly accurate models, and in the case of the LC-MS analysis, an important peptide biomarker ion was characterised as being derived from the protein leucine-rich alpha-2-glycoprotein. The subsequent development of a 5 minute UPLC-MS/MS assay enabled a partial validation of the protein and comparison with IGF-I, an existing biomarker to GH administration. It has been demonstrated that the combination of quantitative data for the A2GL and IGF-I proteins increases the separation of the placebo and treated states compared to IGF-I alone. However, the data from both GH administrations show that the combined values still cannot be used in isolation to discriminate doped from normal populations. This is the first demonstration that quantitative data from an established biomarker related to GH may be combined with data from an unrelated protein to enhance predictive discrimination between treated and untreated samples. The A2GL protein could therefore be used in combination with existing biomarkers of GH such as PIIINP, IGF-BP3 and IGF-I , and with further method development, the application of UPLCMS/MS could give quantitative information on all four proteins in serum or plasma in a single analysis. We have demonstrated that the use of ANNs is a valid and potentially useful approach to the detection of gene and protein doping, and that it could possibly be used to study the effects of other protein targets, for example erythropoietin. The project has also resulted in the generation of a high throughput and sensitive assay for the quantitation of IGF-I in human serum and, following further method development, in human plasma. Recent publications on the analysis of IGF-I by LC-MS/MS require the use of antibody coated magnetic beads and subsequent analysis using 35 minute runtime. The IGF-I assay we have developed requires only acetonitrile for the extraction process in a 96 well plate format, and a 5 minute UPLC-MS/MS analysis. This approach gave similar sensitivity levels to the recent paper, with a significantly higher throughput LCMS/MS analysis.