Projets de recherche

Code: 04B07RH 

Growth hormone (GH) is a naturally occurring endogenous peptide hormone produced by the pituitary gland. Although banned under the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) list of prohibited substances, the detection of exogenously administered GH poses a formidable challenge.  Two approaches for the detection of GH have been developed in collaboration with WADA and USADA. The first approach is based on the measurement of different GH isoforms. This method is able to detect recombinant human GH (rhGH) within a short ‘window of opportunity’ of <36 hours after the last injection of rhGH but will not detect any injection of pituitary-derived GH (that is readily available).The second approach is based on the measurement of GH-sensitive markers. The GH-2000 group showed that the administration of rhGH caused a significant rise in GH-sensitive markers. The magnitude and duration of the elevation was dependent on the dose of rhGH given, gender and the individual marker. Men were more sensitive to the effects of rhGH than women. Although IGF-I and P-III-P were the best of the markers of GH action and were selected to construct formulae that gave good discrimination between those taking GH and those taking placebo, adjustments have to be made to account for the fall in IGF-I and P-III-P that occurs with age.The results of GH-2000 were reviewed by a panel of international experts (including a representative of the Court of Arbitration in Sport – CAS) at an IOC-organised Workshop in Rome in March 1999. This review produced a number of key issues that needed addressing before the experts felt that there would be a viable test suitable for implementation at an Olympic Games. The main issue raised concerned possible ethnic effects on the GH-sensitive markers proposed, since the large majority of volunteers in GH-2000 were white Caucasians. The GH-2004 project, funded by WADA and USADA, is now currently addressing these issues as well as the effect of injury on GH-sensitive markers. In April 2004, the results of the GH-2000 and preliminary results of the GH-2004 project were presented and reviewed at the “USADA symposium on the detection of hGH abuse in Sport”. Although both WADA and USADA strongly support the marker approach, it was felt that further complementary work remains to be done before such a test can be fully implemented.The major issues relating to the “marker” approach were:
1. The effect of adolescence and puberty on the GH sensitive markers.
2. The need for independent confirmation of the algorithm.
3. Assay calibration
4. The use of other GH sensitive markers
5. Clarification of the collection and storage procedures.
The major issue relating to the “isoform” approach was the need to establish the method in an independent IOC accredited laboratory and validate the method against further sample sets.In this proposal, we plan to address each of these issues.
 

Main Findings: 

• It is believed that growth hormone (GH) is abused by athletes and other professional sports men and women.
• The use of GH is banned under the World Anti-Doping Agency (WADA) list of prohibited substances.
• The detection of abuse with GH by poses several challenges because injectable GH is identical to naturally produced GH.
• The GH-2000 study based at St Thomas’ Hospital, London and led by Professor Peter Sönksen showed that it is possible to detect GH abuse by measuring insulin like growth factor –I (IGF-I) and type 3 procollagen (P-III-P) whose concentrations increase in response to injections of GH and the application of discriminant function analysis.
• The results of GH-2000 were presented to a panel of international experts at an IOC-organised Workshop in Rome in April 1999. This review raised a number of key issues that needed addressing before any test could be implemented. The main issue concerned possible ethnic effects on the proposed markers, since the majority of volunteers in GH-2000 were white European.• The GH-2004 project, which is a 3-year project based at the University of Southampton, is funded by WADA and the US Anti-Doping Agency and aims to address these concerns.
• The first part of the project, which was funded by WADA, measured serum IGF-I and P-III-P in blood samples obtained from 242 male and 62 female elite athletes from different ethnic backgrounds within 2 hours of the end of competition at 9 international sporting events in 13 disciplines.
• The study showed that although there are small differences (<20%) in the concentrations of IGF-I and P-III-P between different ethnic groups, the majority of values for individuals of non-white European background lie within the normal range for the white European subjects.
• Application of the GH-2000 discriminant function analysis formulae to the GH-2004 subjects showed that no individual would have been falsely accused of doping.
• The significance of these results means that the test can be applied across different ethnicities. As it is extraordinarily difficult to define ethnicity, if there had been major differences between ethnic groups, it would have been impossible to apply the test.

Code: 03C01GG

The present project aims to introduce microarray technology as a new analytical tool into the field of doping control analysis to gain insight in specific effects of recombinant human growth hormone (hgh) on blood cells (leukocytes). Microarray technology will be used to search for changes in leukocyte gene expression which are directly related to the application of human growth hormone (hgh). Knowledge generated along this research project and the introduction of microarray technology into doping analysis should open novel approaches and strategies for future detection of doping substances. In the first phase of the project cell lines of specific leukocyte subsets (THP-1/monocyte, IM-9/T lymphocyte, H9/B lymphocyte), PBMCs (peripheral blood mononuclear cells) from healthy donors, respectively will be treated in vitro with hgh to identify candidate genes influenced by hghapplication. Whole genome cDNA microarrays as well as oligonucleotide microarrays including leukocyte-relevant genes will be used to characterize potential hgh-specific genes by comparing gene expression profiles of treated and untreated cells. To find additional hgh-candidate genes, suppression subtractive hybridization (SSH) technology will be applied on hgh-treated PBMCs and on those cultured leukocyte subsets which showed a response to hgh before during in vitro studies. After confirming SSH-genes to be differentially expressed in a series of microarray experiments, oligonucleotide probes will be designed for both SSH-genes and the candidate genes identified along gene expression profiling using whole genome/leukocyte relevant arrays. To establish a selective “hgh-chip” the newly designed oligonucleotide probes for the hghcandidate genes will be printed on a microarray. The “hgh-chip” will then be thoroughly evaluated by performing numerous gene expression experiments on hgh-treated and untreated leukocyte subsets, cultured PBMCs as well as PBMCs obtained from hgh-patients. During the proposed project new, nucleic acid-based analytical techniques will be used to gain insight into specific effects of hgh on gene expression of leukocytes. Experience and expertise gained along this project should be of great importance for novel strategies esp. in fields of doping analysis, where so far no satisfying analytical test exists.

Main Findings: 

The aim was to establish a selective human Growth Hormone (hgh) microarray which can be used in hgh doping control by detecting the specific effects of hgh administration rather than the presence of the hormone itself as its very short half-life complicates direct detection. To this purpose cell lines of specific leukocyte subsets (THP-1/ monocyte, IM-9/ B- lymphocyte, H9/ T-lymphocyte) and PBMCs (peripheral blood mononuclear cells) from healthy donors, respectively were treated in vitro with hgh to identify candidate hgh-sensitive genes. Results obtained from gene expression profiling utilising whole genome arrays were in accordance with published data. Additional a range of novel hgh sensitive genes could be discovered. Hgh treatment caused anabolic effects mainly by diversion of energy to protein synthesis. More genes were found to be up-regulated than down-regulated after hgh administration. Responses of the cell subtypes were highly different. The T lymphocyte model cell line H9 was the most responsive. Genes clustering in the categories fatty acid beta oxidation, cell adhesion, DNA replication and polyamine biosynthesis were up-regulated indicating increased lipolysis, cell attachment, proliferation and growth. Genes for non-apoptotic cell death and regulation of osmotic pressure were down-regulated. The B lymphocyte cell line RA-1 showed gene upregulation in the categories opioid receptor, oxidoreductase and GMP-reductase activity. Genes of groups similar to those detected as repressed in H9 cells were also down-regulated in the RA-1 cell line along with genes responsible for muscle development. THP-1 cells, a model system for monocytes, were least sensitive to the application of hgh due to the lack of insulin-like growth factor 1 (IGF-1) production. At doping relevant hgh concentrations effects on THP-1 cells were observed only after a 30 min incubation when hgh was still present. Several collagen types, fatty acid metabolism genes and superoxide dismutase 1 expression were upregulated. After 180 min hgh incubation, only a high hgh dosage led to over-expression of genes indicating increased cell proliferation, hormone and androgen catabolism and cell cycle checkpoint control. In PBMCs hgh administration led to increased cell maintenance and anti-apoptotic activity. Cell proliferation, C21-steroid hormone biosynthesis, insulin receptor signalling pathways and protein amino acid phosphorylation and glycosylation were activated.

Code: 03E08WS 

As notified by the 100 and wada on june 1, 2001 the use of aromatase inhibitors is prohibited for male athletes beginning september 1, 2001. Aromatase inhibitors are listed in the class of peptid hormones, mimetics and analogues. Three classes of aromatase inhibitors are differentiated: 1. aminoglutethimide 2. non steroidal aromatase inhibitors (anastrozole. letrozole, vorozole) 3. steroidal aromatase inhibitors (exemestane, formestane) Actually there exists only a mass spectrometric method for the detection of the misuse of aminoglutethimide in urine The aim of the study is to develop mass spectrometric methods for the detection of the misuse of the non steroidal aromatase inhibitors anastrozole, letrozole and vorozole in urine. If possible the methods should be comprehensive methods for the simultaneous detection of several aromatase inhibitors and should be added to existing screening procedures of doping analysis. The project includes investigations of the metabolism of the aromatase inhibitors, development of methods for suitable sample preparation and derivatisation, choice of the suitable mass spectrometric method (gc/ms; lc/ms) and validation of the methods. For the investigation urines of female patients treated with non steroidal aromatase inhibitors will be used.

Main Findings: 

The methodology used for the detection of the misuse of the aromatase inhibitors anastrozole and letrozole could be incorporated into the existing screening procedures for doping substances. An inclosure of anastrozole parent compound into the screening procedure for selected anabolic androgenic steroids [1] via LC-MS/MS is recommended. The fragmentation pattern shows suitable ion transitions at m/z 294/225, 294/210, 294/142 and 294/130. The characterization of the method shows a linear and homoskedastic calibration curve with a detection limit of 0.02 ng anastrozole per milliliter as well as high accuracy and precision. The standard operating procedure for selected anabolic androgenic steroids via LC-MS/MS is a robust screening method with a recovery of 97% for anastrozole. [2] * For the detection of letrozole misuse screening for the letrozole metabolite bis-(4- cyanophenyl)-methanol by GC-MS is an excellent tool. The EI-MS fragmentation pattern of the TMS derivative shows suitable ions with high intensity [m/z 217 (M+ - 89 / base peak), m/z 291 (M+ -15) and m/z 306 (• M+ )]. The validation of the method shows a linear and homoskedastic calibration curve with an estimated lower limit of detection of 4.4 ng/ml. The standard operating procedure for anabolic steroids [1,3] is a robust screening method with a recovery of 90% for bis-(4-cyanophenyl)-methanol. [4]

Code: 03A02GG 

Since the development of an analytical method for the direct detection of the application of recombinant erythropoietin in urine samples by Lasne et. al. (Nature, 2000, 405, 635), great efforts have been made to implement the method in different laboratories and to standardize and harmonize the interpretation of the data obtained by isoelectric focussing, double blotting and chemiluminiscence detection. Up to now no final decision has been made by the labs accredited for the cited method, which sets of criteria for positivity are to be applied for the interpretation of the gels. The use of different software products with different tools of data interpretation interferes the search for a common basis of gel interpretation. The aim of this project proposal is to develop a reliable and easy-to-use software package called GASEPO2, specially designed for quantification of recombinant erythropoietin in urine samples as a standardized and unified tool for the use across doping-control laboratories worldwide thus providing for international harmonization of EPO analysis. This software is an advancement of the precursor software GASEPO1, designed as research prototype in 2003. The development includes research in design of decision criteria based on image information as well as in mathematical methods of image analysis including image segmentation and 3D visualisation. By using the concept of band segmentation for the differentiation between band and background, the isolated single band serves as basis for further calculation rather than a 1 D transformation of the entire lane. GASEPO2 will equally well handle images taken by a dedicated digital camera for chemiluminescence or scanned from a photographic film. The software will come with a validation package that allows auditing. The implementation of alternative evaluation methods in the software will contribute to and facilitate the process of finding and harmonizing the decision criteria.

Main Findings: 

The Project fulfilled the original goals, namely: • A software tool has been created that has been generally accepted by the doping control community and is now being routinely used in 75% of all laboratories doing epo-analytics. • Thus, the product of the Project contributed directly to the superordinate aim of standardization and harmonization of the epo analytics. • An effective epo-analytics expert network has been created. • Scientific know-how has been extended and disseminated via a number of scientific publications. • WADA-funded research in doping control has been promoted by a number of project-related presentations for broader public.

Code: 03C03MB

Research in recent years demonstrated that the analysis of changes in the hGH isoform pattern occurring after administration of recombinant hGH could help to develop a method to detect hGH doping. Basic requirement for such a technique is the availability of appropriate antibodies and the experience in the techniques to utilize them in specific immunoassays. The aim of this research project is to familiarize personnel from 3 WADA accredited laboratories with the use of the specific monoclonal antibodies developed in our group in immunoassays to detect doping with human growth hormone. All steps necessary - ranging from the theoretical background up to practical training with measurement devices - will be performed by experienced members of our group together with the respective delegates from the WADA accredited labs. Each participant will be able to prepare and conduct the tests at the end of the training. In addition, preparation, testing and shipment of reagents necessary to perform a minimum of 5000 analyses is included within this project proposal. Monoclonal antibodies, calibrators and control samples will be shipped on dry ice to the respective WADA accredited laboratories to allow their participation in the international external quality assessment scheme planned for June this year to allow a validated decision about the status of the implementation of the test at the respective sites.

Code: 03B05WS

The 13C/12C-ratio of urinary steroids is an accepted parameter to detect abuse of androgenic anabolic steroids. Significant differences in 13C112C between selected reference and target steroids are regarded as an unequivocal evidence for presence of synthetic material. However theoretically this procedure might result in false positive cases: Significant differences in isotope ratios might result when isotopic composition of diet changes within a short period of time and when physiological carbon fluxes to reference and target compounds are of different order of magnitude. Isotopic composition of diet necessarily changes after transcontinental travelling due to different contributions of isotopically distinct carbon sources of diet. On the other hand, few is known concerning the exchange rates of carbon during steroid biosynthesis. The aim of the study is to elucidate the dynamics of carbon exchange in steroid metabolism. This will help to obtain significantly more insight into the quality of information given by isotope ratio analysis of urinary steroids. The study requires systematic variation of isotopic composition of diet in a longitudinal study with few selected subjects. When possible, we intend to develop explicit models for the carbon fluxes in steroid metabolic pathways.

Main Findings

The exchange rates of the carbon pools for the biosynthesis of steroid hormones may be different for different compounds. As a consequence, the differences in isotopic composition between endogenous urinary steroids (Δ13C-values) temporarily may increase during switch to a diet with largely different isotope signature. Theoretical considerations suggest that in some individuals the increase of the elevated Δ13C-values may remain for comparably long periods (weeks to months). There is a large biological variance concerning these phenomena. Whereas in some individuals the exchange rates are virtually identical, other individuals may exhibit significant differences. The exchange rates vary significantly between individuals in absolute terms. Body mass and physical activity are likely to play a pivotal role. The contribution of cholesterol to the biosynthesis of steroids varies between different individuals. The most robust comparison appears to be A vs. OHA. These two steroids exhibit comparable exchange rates in most of the subjects. However, this is not necessarily the case (see subject 8, 3.2.8). If possible, evaluation of two reference compounds should be considered.

Publications:

Influence of changes in diet on the dynamics of 13C/12C-ratio in selected urinary steroids. Part I: Study design, 13C/12C-ratio of applied foodstuffs and effect on anthropometrical data. U. Flenker, C. von Kuk, U. Güntner, F. Hülsemann, V. Gougoulidis, W. Schänzer. Manfred Donike Workshop 2005

Influence of changes in diet on the dynamics of 13C/12C in delected urinary steroids: diet free from cholesterol. U. Flenker, C. von Kuk, U. Güntner, F. Hülsemann, V. Gougoulidis, W. Schänzer

Code: 03E09WS

Insulin, a peptide hormone consisting of two disulfide-linked chains composed by 51 amino acids, is part of the endogenous system regulating blood glucose levels by inducing glycogen synthesis. Owing to inconvenient properties of insulin preparations, rapid- as well as long-acting insulins have been developed, which overcome the drawbacks of remedies consisting of recombinant insulin only. Both classes of synthetic insulins, rapid and long acting analogues, as well as human insulin itself are possibly misused in sports, some reasons of which were discussed in the literature. The testimony of an athlete of having used insulin as well as findings of injection solutions in luggage of cyclists substantiate this suspect, and sensitive, selective and comprehensive procedures are necessary to determine these compounds in doping control samples. So far, analyses of insulins are mainly performed by means of immunoassays, but also mass spectrometric approaches were demonstrated in the past, which indicate advantages of this technique over immunochemical determination of insulins such as elimination of cross-reaction. The improvements and innovations in soft ionization of large biomolecules by electrospray (ESI) or matrix-assisted laser desorption (MALDI), as well as the production of highly sensitive mass selective detectors enable the development of procedures capable of screening and confirmation of insulin and its new biotechnologically synthesized analogues for doping control purposes. In order to identify rapid- and long-acting insulins, their mass spectrometric properties after electrospray ionization have to be elucidated. As differences of endogenous and synthetic insulins are limited to exchanges of few amino acids, and one compound is identical with human insulin regarding molecular weight, product ion experiments have to be optimized to efficiently analyze the target compounds in low concentrations in biological fluids. Furthermore, isolation of insulin and its synthetic counterparts from plasma will be performed by different methods, e.g. solidphase extraction (SPE) and immunoaffinity chromatography (IAC). CommerciaUy available plasma will be fortified with the analytes of interest, extraction recoveries, reproducibility and detection limits will be determined.

Main Findings: 

Synthetic insulin derivatives with modified amino acid sequences such as Humalog Lispro, Novolog Aspart or Lantus Glargine have been introduced to the pharmaceutical market as they provide better controllability, faster or prolonged bioactivity and improved convenience compared to conventional recombinant human insulin preparations. However, the misuse of insulin in sports has been reported manifold, and the international doping control system has required a reliable and robust assay to determine the presence or absence of related prohibited drugs. A qualitative evidence of administered substances is of utmost importance, which is preferably obtained by mass spectrometry. Hence, a top-down sequencing-based assay was developed that allows the detection of synthetic insulin derivatives in human plasma. Therefore, specimens of 2 mL were fortified with three synthetic insulin analogues to simulate plasma levels after subcutaneous administration. A subsequent purification by immunoaffinity chromatography and solid-phase extraction was performed, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This approach has allowed the assignment of individual fragment ions, in particular of those comprising modifications that are originating from C-termini of Bchains, and thus, enabled the unambiguous detection of synthetic derivatives of human insulin in plasma samples. Humalog Lispro contains the same set of amino acid residues as endogenous insulin with one pair having switched positions (B28 and B29, lysine and proline). Here, MS/MS data are essential to allow mass spectrometric separation and identification of target analytes. Other synthetic drugs (Novolog Aspart or Lantus Glargine) possess different amino acid compositions and deviate in molecular mass from human insulin, which facilitates their mass spectrometric and chromatographic differentiation. Recoveries of synthetic insulins from plasma aliquots ranged from 91-98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes, which represents plasma levels of insulin for non-fasting normal subjects.

Code: 03B06FD 

3,6,17-androstenetrione is an anabolic steroid and is also referred to as 6-oxo. This product is sold as an over the counter product in the United States where it is regarded as a nutritional supplement. It is widely available on the internet and it is marketed as an antiestrogenic agent. Athletes might be tempted to threat the adverse effects of an extensive abuse of anabolic steroids (e.g. suppression of androgens and gynaecomastia) by using this type of drugs. Although at present no results from clinical studies supporting the claims of the manufacturer are available, it seems likely that athletes will use this product. This is especially true because the manufacturer of this product is the same company that introduced 1 -androstenediol, 4- androstenedione, 4-androstenediol and 1 9-nor-4-androstenediol on the prohormone market. All of these products have rapidly become a commercial succes. Although the manufacturer claims that 6-oxo is the natural first antiestrogenic product, it is clear that based upon its structure this product can be regarded as an anabolic steroid as well. The producer claims that 6-oxo works as an aromatase inhibitor, blocking the aromatisation of endogenous as well as exogenously administered drugs resulting in higher levels of endogenous steroids as well as synthetic co-administered steroids. Based upon its structure, it is likely that 6-oxo is not metabolised into endogenous steroids commonly screened for during doping control, misuse of this substance remains undetected. It seems therefore necessary to determine whether or not the use of this supplement does indeed result in higher levels of endogenous steroids (testosterone). Moreover elucidation of the metabolism and identification of 6-oxo metabolites is necessary to allow for the detection of this supplement. This research would therefore investigate the metabolism and excretion of 6-oxo to allow for its detection in the urine of athletes.

Main Findings: 

The analysis of the 6-OXO supplement resulted in the detection of 6-oxo-androstenedione but also of 6b-OH-androstenedione. No other contaminants were detected. Excretion studies resulted in the detection of the parent drug 6-oxo-androstenedione and 6a-OH-androstenedione and 6a-OH-testosterone as metaboites. A GC-MS-SIM method was developed and validated to fulfil requirements of WADA for doping control laboratories. Using this method, the ingestion of the supplement, accordin the manufacturers recommendations resulted in the detection of a 6a-OH-androstenedione up to 37 h after the administration, while 6-oxo-androstenedione and 6a-OH-testosterone could be detected up to 24h post administration. Because of the longer detection time and its presence in urine after administration of androstenedione, it is recmmended that beides 6-oxo-androstenedione, 6a-OH-androstenedione is also included in screening methods for doping control purposes. The presence of 6-oxo-androstenedione or 6a-OH-testosterone can be used to disciminate between the administration of 6-oxo-androstenedione and androstenedione

Code: 03C02TF 

The use of gene transfer methods for athletic enhancement is inevitable. To prepare for such eventuality, it will be necessary to develop more efficient and more effective methods for detection of the foreign genetic information and/or the vector used to deliver the transgene. Current screening methods rely partly on the existence of detectable differences between the functional gene products of endogenous genes and the foreign or exogenously administered proteins. For instance, the detection of erythropoietin administration relies on structural differences between endogenous EPO and the administered drug. However, those committed to either drug- or gene-based doping will certainly eventually have available those forms of drugs that are indistinguishable from the endogenous human form or gene transfer vectors that express gene products completely identical to the endogenous function. In that case, current approaches to detection of doping will become ineffective. We propose to take advantage of powerful and broad new methods for gene expression screening to detect secondary changes in gene expression that result from systemic or local administration of genes most likely to be subjects for gene-based doping attempts – erythropoietin (EPO), growth hormone (GH) and insulin-like growth factor (IGF- 1). The hypothesis underlying this proposal is that administration of these extremely and widely bioactive agents or the genes expressing them will be associated with reproducible and detectable secondary changes in gene expression in many affected tissues, including peripheral blood. Erythropoietin is very likely to produce changes in gene expression of nucleated cell lineages in the peripheral blood and therefore detectable in simple blood samples. In addition, exposure to the powerful growth effects of GH or IGF-1 administered locally may also produce effects on the pituitary GH-GHRH-IGF-1 axis and also be detectable in peripheral blood. It is the purpose of this proposal to examine the patterns of gene expression in cells from peripheral blood of mice exposed to GH, IGF-1 and EPO and to gene transfer vectors expressing them and to identify associated changes in gene expression through global microarray techniques.

Main Findings: 

The goal of these studies has been the use of molecular genetic tools of transcriptional profiling and proteomic analysis to identify molecular “signatures” of exposure to doping substances. The underlying principle is the hypothesis that exposure to growth factors and to many other kinds of doping agents will be accompanied by systemic changes in gene expression and in the proteomes if tissues readily accessible for testing. As an introduction to that concept, we have concentrated on the demonstration of genetic and proteomic changes in a number of tissues both in vulture and in vivo after exposure to one of the important potential doping agents – IGF-1. In our in in vitro studies of muscle IGF-treated stem cells, we have identified acute changes in gene expression of several hundred genes, with far more genes being up-regulated than down-regulated. Most impressively, we have found uniform reproducible up-regulation of the genes that regulate cholesterol and steroid biogenesis and of genes fatty acid biosynthesis. If any of these changes come to be documented in vivo, combinations of these changes are candidates for assays for exposure to IGF-1. Under the same conditions, we have used 2-dimensional gel/mass spectroscopic proteomic methods to establish a large data set of proteins that show altered levels acutely after IGF-exposure. The in vivo studies in skeletal muscle in IGF-treated mice have also identified altered expression of several hundred genes, but, in contrast to the in vitro results with muscle stem cells, more genes are down-regulated than upregulated in vivo in skeletal muscle, including a number of genes that specify several muscle growth factors. The most important conclusion that we come to as a result of these studies is that the identification of methods useful for doping detection and screening will require a much higher degree of data sharing and comparison among the WADA-sponsored studies undertaking such genetic studies than is currently occurring. These disparate studies are using many different experimental designs, research platforms and analytical tools to identify genes and proteins perturbed by drug exposure, and it seems highly likely that these disparate studies have identified many common or inter-related functions but that those results are not being identified by current analytical methods. Toward that end, our laboratory has proposed a pilot study with WADA to test the feasibility of establishing a core bioinformatics facility to collect, collate and analyze data from all relevant WADA-supported laboratories to greatly enhance the likelihood of identifying authentic and diagnostic molecular and metabolic changes that can be used in the fight against sport doping.

Code: 03E05DM 

The purpose of this study is to determine the dose-response effects of inhaled B2-agonists on exercise performance in elite non-asthmatic athletes using a sport specific test of performance. To investigate the dose-response effect of inhaled B2-agonists (IBA) on athletic performance elite, male, non-asthmatic cyclists will volunteer to participate in this randomized, double-blind repeated measures study. The sample size will be determined following a pilot study to determine the within-subject variability in a simulated 20km time trial. The minimum worthwhile difference in performance will be equivalent to 0.5-0.7 of the within-subject coefficient of variance. Subjects will be screened for asthma using a eucapneic voluntary hyperpnea test with positive responders excluded from the study. Following baseline measurements, height, weight, V02, average performance velocity, average performance in watts, and 20km time trial performance will be measured in the laboratory during each of 4 testing sessions. The order of treatment will be randomized among placebo, 200~.tg, 400~.ig, and 800~.tg of salbutamol administered by inhaler. The results of this study will determine whether the administration of BA influences performance in elite cyclists from competitive and statistically significant perspectives. Additionally, this study will determine if there is a dose response relationship between IBA, exercise performance and the appearance of salbutamol in the urine.

Main Findings: 

The intention of this project was to address two questions: what are the relationships between SAL dose and exercise performance in a simulated cycling time-trail, and what are the effects of dose on cSAL as used in doping control? Two studies were used to demonstrate that inhaled SAL does not enhance endurance performance in non-asthmatic athletes when using a highly reproducible and sport-specific test. This is the first examination of the dose-response effect of inhaled salbutamol using a sport-specific performance evaluation and used a substantially larger sample size (n=27) compared to most previous work. The lack of a dose-response relationship further supports previous findings that acute SAL inhalation does not enhance exercise performance in non-asthmatics. Furthermore, the short duration of the time trial may not have provided sufficient stimulus for changes in hydration status that can accompany longer duration exercise. Even though most urine samples generally fell well below the WADA limit of 1000 ng/mL, the possibility exists for individuals to exceed this value following inhaled administration. A significant relationship between cSAL and urine SG was observed at higher doses, signifying the potential impact of hydration on values observed in doping control. As with exercise, the role of hydration and individual differences in absorption, metabolism, and excretion on cSAL require further investigation. It is also noted that the finding of SAL to be non-ergogenic cannot preclude the possibility that continued, short-term (2-3 weeks) use of inhaled SAL would not be performance enhancing. Regular use of SAL during both training and competition would be expected and I is possible that continued elevated plasma levels following inhalation may increase ergogenic properties of SAL. Future research needs to be conducted to eliminate this possibility. Lastly, it was observed that a large portion (~19%) of the cyclists/triathletes tested were susceptible to airway hyperresponsiveness. Although a small number of cyclists and triathletes were recruited for these studies, the possibility exists that there is a significant portion of this athlete population competing with impaired airway function unbeknownst to them. Although potential mechanisms for increased airway hyperresponsiveness in certain athletes have been postulated, longitudinal research is required to track changes in airway function with length of time in specific sports. In conclusion, this project demonstrated a lack of dose-response in relationship with SAL and exercise performance in non-asthmatic athletes and that urine cSAL following exercise are highly variable and dosedependent.