Projets de recherche

Code: 06A14MA

The long term goal of this research is to develop a test or tests to detect the presence of autologous transfused red cells in peripheral blood samples. Autologous transfusion is the reinfusion of the donor's blood that has been withdrawn and stored for an indefinite period, with the intent to increase total red cell mass and thereby endurance performance. There is unequivocal evidence that autotransfusion has been used, and is currently used, by elite endurance athletes. Prior research has found that autotransfusion alters red cell membrane structure, haematological parameters, and antigen expression. It is self-evident that autotransfusion also alters total red cell mass (which represents the goal of this practice). Our pilot research has also found that the expression levels of multiple genes remains altered for several weeks after reinfusion of autologous blood. Each of these parameters can be measured using appropriate technology, consequently they offer potential avenues for the detection of autologous transfusion for use by antidoping agencies. We hypothesize that a combination of these methods supplemented with additional biologic information, will yield a diagnostic with superior sensitivity and specificity.

We will conduct autologous transfusion trials at the Copenhagen Muscle Research Centre (CMRC) in Denmark which has successfully performed such trials in the past. All samples and measurements required for each candidate parameter will be collected simultaneously, which will enable us to retrospectively scrutinize the sensitivity and specificity of various permutations of test data. We will evaluate cumulative sensitivities when the different approaches are applied to a single blood sample, and thereby determine whether one, or a combination of several, methodologies have potential to be applied in an antidoping setting.

Main Findings

In the absence of a test to detect autologous blood transfusion, this project was an exploration and comparison of several different avenues of research. The initial 2-year project was extended to permit investigation of promising early findings that autologous transfusion altered gene expression levels. Our follow up studies have demonstrated that the gene signal was most evident 7 days after reinfusion of three bags of blood, remained strong at 14 days and persisted through 28 days. There was an indication that a change does occur following transfusion with only one bag of blood, but this was of much smaller magnitude. We found that haematological parameters were relatively insensitive to the reinfusion of blood, but were disturbed to a greater extent in the days after blood had been withdrawn. A novel marker was found to have greater sensitivity than conventional variables such as haemoglobin concentration or OFF-score. An in-depth evaluation of total haemoglobin mass estimation via the CO rebreathing method showed that this variable was the only one able to detect transfusion within the first few days after blood had been reinfused. However from one week onwards its sensitivity was no better than the OFF-score model. Following initial promising results exploring red cell lesions as a means to reveal transfused cells, basic problems in the development of that assay led us to conclude that a test based on this premise was most unlikely to be developed at the moment.

Code: 06C12FD

Anabolic steroids are widely misused in sports. Because anabolic steroids are extensively metabolised in the human body, methods for the detection of these steroids need to focus on metabolites rather than on the parent drug. The liver is the principal organ where anabolic steroids are metabolised. Recently, several anabolic steroids have appeared on the market either as so-called prohormones, available as “nutritional” supplement, or underground as designer steroid (e.g. THG). Regular anabolic steroids –like all other drugs- from pharmaceutical companies undergo a vast amount of toxicological tests before they are administered to test subjects in the final phase before their release on the market. This is not the case for these new steroids. Hence, essential toxicological data is missing and administration of these steroids to human subjects for the identification of marker metabolites for their detection is medically and ethically questionable. This study will evaluate an animal model in which mice harbouring a functional human liver will be used for investigation of the metabolism of steroids. In contrast to, in-vitro models using single cells or cell cultures, this animal uses a functional human liver and resembles administration studies better than any other model available so far.

Main Findings

The aim of this project was to investigate whether the uPA+/+ -SCID mice transplanted with human hepatocytes can be used as an alternative for the human excretion studies and in vitro hepatocyte cultures. This small animal model would be used to investigate drug metabolism and to find urinary markers that allow for the detection of steroid abuse.

The metabolic profile of the chimeric mouse model was first validated with 3 selected steroids. The results of the administration studies with androt-4-ene-3,17-dione (AD), methandienone (MTD) and 19-norandrost-4-ene-3,17-dione (19-norAD) to the chimeric mice showed a good correlation with the previously described metabolism in humans. Morever the major metabolites described in humans were all confirmed in the chimeric mice. To further illustrate the applicability of the chimeric mice, new marker metabolites for 17α-methyltestosterone (17α-MT) and stanozolol were found and implemented in the routine doping control screening since they are longer detectable (e.g. 4,16-dihydroxystanozolol).

All these results including the analytical data were presented at conferences (6 presentations/posters) and/or via 7 scientific publications.

The model combines the ethical advantages of in vitro studies with the reality of in vivo studies. In the future this promising mouse model will be used to further encourage the fight against doping by evalutaing some prohormones and food supplement based on the urinary results of the chimeric mice.

Code: 06C08FD

Anabolic steroids are widely misused in sports. Recently, several new anabolic steroids have appeared on the “nutritional supplement” market. Some of these steroids can be classified as designer steroids (e.g. madol, THG) that were specifically designed to circumvent detection in doping control laboratories, while others are structural analogues of steroids previously produced as pharmaceuticals (e.g. superdrol as an analogue of drostanolone). Via the internet these steroids are rapidly distributed world-wide. Unlike for pharmaceutical preparations, no clinical studies need to be performed for these substances and they can be introduced onto the market without delay. Hence, it is of key importance to the anti-doping community that the introduction of such new steroids is detected as early as possible. Moreover, the chemical names attributed to the active ingredient of such supplements is usually incorrect. Hence, the supplements need to be tested to identify exactly their content. This project would aim at identifying new anabolic steroids introduced on the supplement market and disseminate this information as well distribute reference solutions of these compounds to all WADA-accredited doping control laboratories, to accelerate their incorporation into detection methods.

Main Findings

For the project over 40 different supplements were purchased and analyzed using different techniques (LC-MS and GC-MS) for the presence of known and unknown steroids. Several new steroids were identified. In many cases, it was shown that the labelled steroids/substances were not present or that other steroids were present. However, for several preparations “new” steroids were detected. These included 3bhydroxy-androst-5-ene-17-one, 1,4,6-androstene-3,17-dione, 11-oxo-androstenedione, 17a-methyl-5a-androstan-17b-ol, 6a-bromo-androstenedione, 6b-bromoandrostenedione, 2a,3a-epithio-17a-methyl-17b-hydroxy-5a-androstane, 4-chloro-17amethyl-androst-4-ene-3,17b-diol, 3a-hydroxy-androstane ethyl ester and 17a-methyl5a-androstane-3a,17b-diol. Moreover, the survey also revealed that steroids that were officially removed from the market were still present (e.g. madol, superdrol). All of the newly detected steroids were distributed to the WADA-accredited laboratories and information on their existence (including analytical data) was disseminated via different forms of communication (presentations at conferences (3), scientific publications (3) and multiple personal communications). During these communications the importance for the incorporation of several of these substances was stressed. In several cases besides the parent compound, useful metabolites were identified and reported to the community of accredited laboratories. Several of the newly reported steroids have since then been incorporated into methods used in doping control laboratories and a few adverse analytical findings have been reported for some of these steroids (e.g. 1,4,6-androstratiene-3,17-dione). For several others, it was shown that the regular screening procedures would be satisfactory to detect their misuse, but that the new substances needed to be taken into consideration when the results are interpreted.

Publications

Van Eenoo P, Lootens L, Van Thuyne W, Deventer K, Pozo-Mendoza O, Delbeke FT. Results of several (small) research projects at DoCoLab in 2006. Manfred Donike Workshop, Cologne, 2007.

Van Eenoo P, Van Thuyne W, Pozo-Mendoza O, Deventer K, Lootens L, Van Renterghem P, Delbeke FT. Results of several (small) research projects at DoCoLab in 2007. Manfred Donike Workshop, Cologne, 2008.

Van Eenoo P, Van Thuyne W, Pozo-Mendoza O, Deventer K, Lootens L, Van Renterghem P, Delbeke FT. Results of several (small) research projects at DoCoLab in 2008. Manfred Donike Workshop, Cologne, 2009.

Code: 06B11JJ

Growth hormone (GH) is an anabolic hormone produced throughout life from the pituitary gland. The actions in adult subjects include promotion of muscle and bone growth and stimulation of lipid consumption. Numerous evidence from official bodies as well as the lay press and underground literature strongly indicate that GH is being used as a doping agent. Excess exposure to GH is associated with hypermetabolism, irreversible bone deformations, a risk of type 2 diabetes mellitus, and probably also cardiovascular morbidity. Growth hormone is therefore on the prohibited list of drugs and related compounds. No approved or licensed method is so far available for the detection of GH doping. The GH preparations used for doping are not structurally different from naturally produced GH and injected GH is rapidly eliminated by the organism. Detection of GH in urine is also not a viable method. The strategies pursued thus far relates to the detection in the blood of growth factors known to be stimulated by long-term GH abuse. The specificity of these methods remain a matter of debate. Proteomics or protein profiling by means of so-called two dimensional gel electrophoresis is a powerful and unique method to demonstrate the expression of proteins in human tissues. We have recently refined this method for the use of human serum, which can be obtained by a single blood sample. The protein profile, or proteome, in serum can thus be characterised and we have preliminary data to indicate that the proteome change significantly when the individual has been exposed to GH. Moreover, proteomics also allows the detection of novel GH-related proteins. The project aims to further characterise the impact of GH exposure on serum proteomics. Serum obtained from individuals with known GH disturbances will be evaluated together with serum obtained after high dose GH administration to healthy adults. In addition, the impact of physical exercise on serum proteomics will be investigated.

Main Findings

The background for the project was that doping with GH is a significant problem in the world of sports and that viable novel methods for its detection are still needed. Our hypothesis was that serum proteomics may provide the basis for future GH doping detection strategies based on either global changes in the protein pattern or identification of new GH-dependent candidate proteins. To this end a collaboration was established between Aarhus University Hospital and Edison Biotechnology Institute, Ohio University in order to provide pertinent serum samples from clinical protocols involving patients and healthy subjects exposed to conditions with alterations in GH status, and to perform proteomic analysis on serum samples from these protocols. Our project has included several modes of GH exposure ranging from patients with overproduction of GH (acromegaly) before and after treatment with either surgery or medication (a GH antagonist = pegvisomant) to patients with GHdeficiency before and after substitution with GH. More importantly, we also included a study with high dose exogenous GH administration to young and healthy male subjects for 8 days (i.e. “doping”). We successfully completed serum proteomics in these protocols and identified several new biomarkers of GH activity that could serve as future targets for an anti doping strategy. Our study also revealed that many biomarkers exist as isoforms and that these isoforms may respond to GH in a reciprocal manner such that the total level of the protein remains unchanged. Several isoforms of apo A-1, which is a lipoprotein in serum, changed in all the studies and may as such serve as a sensitive (but perhaps unspecific) GH biomarker. By contrast, one isoform of α-1 antitrypsin only changed in the study involving exogenous GH administration. Thus, this biomarker seems specific to exogenous GH. Exercise alone was also studied and this was associated with changes in two of the GH-dependent biomarkers. In conclusion, our study has identified new GH biomarkers that merit further investigations from an anti doping perspective. We suggest a study focusing on high dose GH administration (to mimic GH doping) to a larger number of subjects of both sexes and including a wash-out period. The candidate proteins should include isoforms of apo A-1, α-1 antitrypsin and should be assayed by means of proteomics as well as with more conventional assays.

Publications

Ding J, List EO, Okada S, Kopchick JJ. Perspective: Proteomic approach to detect human growth hormone doping. Growth Hormone & IGF Research 2009;19:399-407

Diana Cruz-Topete, Britt Christensen, Lucila Sackmann-Sala, Shigeru Okada, Jens Otto L. Jorgensen , John J. Kopchick. Serum Proteome Changes in Acromegalic Patients following Transsphenoidal Surgery: Novel Biomarkers of Disease Activity. (Submitted)

Juan Ding, Shigeru Okada, Jens Otto L. Jorgensen, John J. Kopchick. Biomarkers of GH doping in healthy human subjects (in preparation) Diana Cruz-Topete, Christensen B, Jorgensen JOL, Kopchick. Serum proteomic profiles in GH-deficient patients before and after GH substitution (in preparation)

Christensen B, Jorgensen JOL, Kopchick. Serum proteomic profiles before and after pegvisomant administration in patients with acromegaly as well as in healthy subjects (in preparation)

Code: 06A08CA

Erythropoietin (EPO) is detected in urine by isoelectric focusing followed by immunoblotting procedures. The sample preparation involves mainly a concentration step by ultrafiltration on MW-selective filters. The retenate loaded on gel is therefore containing all other retained urinary proteins and the resulting impact is the frequent generation of distorted patterns of isoforms. It is suggested that a more selective purification step before loading the urine retenate on the IEF gel may improve the resolution of the isoform profiles, facilitate the interpretation and provide better quantification. Different chromatographic and affinity techniques of purification were evaluated in preliminary works. It was shown that chromatographic separation by anion-exchange chromatography improved significantly the resolution of the bands of urine retentates that would otherwise present smeared, diffused or arc-shaped bands. Immunoaffinity was also tested by purification through in-tube immunoprecipitation. This method allowed the total recovery of urinary EPO and combined with the elimination of other interfering proteins on gel, could also improve significantly the quality of the profiles.

It is suggested that adding sequentially both of these purification steps before loading the urine samples on IEF gels would increase the resolution and the clarity of the profiles of isoforms, and faciliate greatly the densitometric evaluation. This would contribute to standardize the interpretation of the results and limit its subjectivity. It can also perhaps represent an alternative to the second immunoblotting that is necessary to avoid non-specific binding of secondary anti-body, with the dual advantage of increasing both the resolution and the sensistivity of the test.

In perspective, developing a low-scale purification method is the starting point to explore new approaches for the detection of urinary EPO such as analysis of biomarkers by mass spectrometry and 1D or 2D gel electrophoresis that were up to now, limited by the complexity of the urinary matrix.

Main Findings

The objectives of this study were to explore purification processes to improve the resolution and quality of the EPO isoform profiles on IEF gel, but also to set the basis for the exploration of new detection approaches by 1D gel electrophoresis or mass spectrometry, that were limited by the complexity of the urinary retentates. The resolution of urinary profiles of EPO isoforms was improved following purification, either by anion exchange chromatography or by immunoprecipitation of the retentates obtained after concentration of the specimens. The anion exchange resin approach was found to be of impossible routine application since rapid and irreversible deterioration of the column occurred. However, the immunopurification of urinary EPO isoforms was successfully developed and implemented in routine testing, which allows their analysis on SDS-PAGE gels.

Code: 06C24CS 

Nandrolone and other 19-norsteroids, potent anabolic steroids are prohibited in sports for 30 years. The detection of its main urinary metabolite, 19- norandrosterone in an amount greater than 2 ng/mL constitutes an adverse analytical finding. The presence in nutritional sport supplements of steroids not listed on the label has undoubtedly caused positive tests. The project deals with detection and quantification in urine and blood samples of free and conjugated forms of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). This analytical method appears appropriate to study the phase II metabolism of nandrolone as it does not require deconjugation and derivatization steps prior to analysis. In this context, urine and blood samples collected during excretion studies of oral administration of nandrolone and also to samples containing 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) of endogenous origin will be analyzed to find specific biomarkers of a doping with this norsteroid. Our results for low concentrations of analyte (< 10 ng/mL) will be validated using GC/HRMS and GC/C/IRMS methods to determine 19-norandrosterone glucuronide concentrations and 13C/12C ratio, respectively.

Main findings:

The aim of the study was to developan analytical tool using liquid chromatography ion trap mass spectrometry LC/MS/MS to determine if the presence of nandrolone metabolites originates from administration of nandrolone prohormones or are produced at the endogenous level. In the first part of the project, a LC/MS/MS assay was validated for the quantification of the four major phase 2 nandrolone metabolites in human urine, 19-norandrosterone and 19-noretiocholanolone in their glucuronide and sulfate forms. This method was subsequently appplied to samples collected after oral administration of 100 mg 19-norandrostenedione and samples containing nandrolone matabolites from endogenous origin. 19-Norandrosterone sulfate was detected over 200 hours after precursor absorption. Based on concentrations or ratios of 19-norsteroids conjugates in urine specimens, it was not possible to disminate between endogenous and exogenous production at the ng/mL conventration levels. However, our study showed that the concentration of 19-norandrosterone sulfate often exceeds 19-norandrosterone glucuronide. Therefore, il might be conceivable in the futur to quantify 19-norandrosterone by LC/MS/MS as a routine procedure to support the positive resultats obtained for 19-norandrosterone glucuronide at a concentration above the 2 ng/mL threshold. 

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Code: 05C11GG

The present project aims to establish 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). The obtained information shall be finally used for the development of a selective “hGHmicroarray”, applicable for hGH-doping control in the near future. Experience and expertise gained along this project will be of general importance and interest for developing similar assays for other doping substances, where so far also no satisfying analytical test exists. Preceding the current project a feasibility study was already done on cell lines of specific leukocyte subsets (THP-1/monocyte, H9/T lymphocyte, RA1/B lymphocyte), PBMCs (peripheral blood mononuclear cells) from healthy donors, respectively which had been treated in vitro with hGH. Comparing gene expression profiles of treated and untreated cells on genome wide microarrays, numerous genes could be identified that showed a specific response to hGH treatment. In the current project time series gene expression studies will be performed on PBMCs obtained from athletes receiving a 3 weeks’ hGH-treatment in comparison to healthy controls. To find additional hGH-doping candidate genes, suppression subtractive hybridization (SSH) technology will be applied on PBMCs from hGH-doped athletes and non-doped individuals. 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 arrays. To establish a selective “hGH-microarray” the newly designed oligonucleotide probes for the hGH-candidate genes will be printed on a microarray. The “hGH-microarray” will then be thoroughly evaluated by performing numerous gene expression experiments on PBMCS from hghtreated and untreated individuals. Results from microarray studies on the hGH-microarray will be spot check-like validated with microarrayindependent methods such as quantitative PCR (Taqman).

Main Findings

Testing for capture antibody specificity using various recombinant dopingrelevant antigens we found that for the same capture antibodies cross reactivity with other antigens and for some antibodies antigen affinity was rather modest leading to detection limits which were not suited for detection of doping relevant substances in serum/plasma or urine. In the case of hGH detection, on which we were focusing in this project, we were not able to detect any reliable changes in hGH levels in samples which had been treated with hGH compared to placebo-treated controls even when testing with 5 different types of hGH antibodies on the microarray. The inability of detecting changes in hGH is most likely related to the detection limit (caused by rather low affinity of the capture antibodies used) and to the fact that matrices such as serum and plasma contain interfering substances which obviously hinder antigen detection. The latter could be clearly seen in experiments where hGH antigen was spiked in PBS compared to undiluted serum or plasma or in 1:10 diluted serum or plasma samples. Whereas spiked hGH antigen could be detected in PBS and the diluted serum/plasma samples, it was not possible to detect an increase in hGH in undiluted plasma or serum samples.