Projets de recherche

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.

Code: 05D21FM 

Different substances and methods can be used to increase the oxygen carrying capacity of blood, thereby improving an athlete's ability to perform. Doping control procedures are expensive and the problem always exists of who we should test, by what criteria and when. Research groups have been developing criteria to detect these substances and methods (blood doping, human recombinant erythropoietin, oxygen carriers, the off/on model) International federations, including Biathlon, currently choose athletes based on random selection, standings, high hemoglobin and/or hematocrit and/or reticulocyte counts, off model scores, etc. There is currently no accurate integrated way to combine all variables (individual performance change and laboratory values), to estimate which athletes should be selected at the optimal time for anti-doping tests. This project aims to develop an intelligent system which is able to identify those athletes whose haematological and multiple variables reflect a pattern consistent with the use of banned substances or methods. These athletes could then be chosen at the optimal time for target testing. The focus of this project is the creation of a software program that will consider haematological values abnormal not only on the basis of high values, but also on the basis of raw data considered concurrently (haematological data in relation to the reference population, intraindividual haematological variations including abnormal low data, performance variations, ranking, nation). This system will produce classes of results associated to a diagnostic probability, useful for targeted selection for both in and out of competition controls.The system aims to be fast (analysing multiple data simultaneously), unpredictable and self-learning (the new information will be automatically included to improve the knowledge). The project aims to provide a strong deterrent against doping, reducing the risk of evasion by manipulation, and to be cost-effective, ensuring that anti-doping budgets are spent in an evidence based fashion.

Main Findings: 

Different substances and methods can increase the oxygen carrying capacity of blood and the athlete's performance. Validated detection methods for these ergogenic aids are available but doping control procedures are expensive and the screening phase performed by International Federations remains a critical issue. The wrong selection of both athletes to be tested and samples to be analyzed in a laboratory can be a factor responsible of high costs and unsatisfactory results. This project AR.IE.T.T.A. aimed to develop an intelligent system, able to show athletes’ profile in order to detect those reflecting an abnormal pattern, consistent with the use of banned substances or methods. An intelligent system, with different sections and functions has been developed and tested after the input of haematological and performance data of athletes belonging to the International Biathlon Union, who gave their written informed consent to the study. The AR.I.E.T.T.A. software includes: - Log-in section - Data-Entry section: data are inserted, stored and separated by sports, disciplines, subgroups or by competitions/events; - Analysis section: data can be analysed, validated scores calculated, parameters shown also simultaneously as statistics, table/graphs, individual or population profiles (team, nations, etc); - Screening section: an immediate evaluation of the risk profile can be obtained. A risk score for the present sample can be calculated on the basis of different data considered concurrently (haematological and in future performance data, absolute values and inter-intra-individual variations, etc). An analysis of the athlete’s risk can also be performed by evaluation of all the sample risk scores calculated for the athlete under study. The experience derived by the routine application for a stable target testing program and future studies aiming to evaluate and increase the sensitivity of the diagnostic phase e.g. by inclusion of parameters not altered by haemodilution, will represent a further improvement of the system. AR.I.E.T.T.A. is an efficient database that enables a quick evaluation and interpretation of blood results. It could favour surveillance programs and timely controls on athletes by the International Federations collecting blood samples for targeted testing purposes.

Code: 05D22RG 

Human chorionic gonadotropin (hCG) and human lutropin (hLH) are hormones synthesised or purified by certain pharmaceutical companies for treatment of infertility in women. However, both hormone act through the same receptor in the body and may trigger the production of testosterone. As such these hormones may be attractive molecules for athletes looking for illicit performance enhancement. Both hormones belong to the family of glycoproteins, which means that protein carry a particular decoration known as glycosylation. The glycosylation of both hormones has been found to be of utmost importance for the correct functioning of the molecules. Furthermore, it is known that glycosylation is a phenomenon that is not primarily regulated by particular genes. In stead, glycosylation is the result of the concerted action of many primary gene products, donor and acceptor substrate availability, and additional external factors (temperature, presence of growth factors, etc.). As such, the glycosylation is cell type specific. Recombinant glycoprotein hormones will therefore carry glycans synthesised by the machinery of the host cell and these glycans may be different from those synthesised by the natural producers. Detailed structural analysis of the glycosylation of the endogenous and exogenous hormones will reveal the cell-type specificities. Once established, the cell-type specific structural elements may be used as targets for the development of new, faster, and more reliable screening and confirmation procedures.

Main Findings:

Human chorionic gonadotropin and human lutropin belong to the family of glycoprotein hormones that are used in the treatment of female fertility problems. Under normal circumstances both hormones result in the production of testosterone. This is the main reason for its potential abuse by athletes: increasing the testoterone concentration without affecting the ratio between testosterone and epitestosterone. While both male and female have lutropin (produced in the pituitary), chorionic gonadotropin is produced by the placenta under term conditions, and as such restricted to the female gender. Even though under ordinary circumstances hCG is not produced by men, its production has been described under certain pathological situations. As such, its sole detection in a biological specimen is not sufficient. These hormones, being glycoproteins, carry the particular signature of their origin through the carbohydrate decoration. This post-translational modification depends very much on the environmental settings and could be different in recombinant and endogenous species. As such, the basic principle of this application was the analysis of the glycosylation of exogenous and endogenous CG and LH with the aim of establishing the existence of structural differences that would enable unambiguous discrimination. Both hormones share the α-subunit, glycosylated at Asn52, Asn78, and Thr39 (the latter only described in CG), and have different β-subunits glycosylated at Asn30 in both and at Asn13 only in CG. Furthermore, O-glycosylation has been described for CG at Ser121, Ser127, Ser132, and Ser138. Pure glycoproteins (both prepared from pooled urine of pregnant women, isolated from pituitary material, or recombinant from different sources) were prepared or purchased. Glycoproteins were analysed at the intact level by mass spectrometry and sugar analysis. While the former revealed subtle molecular weight differences mainly in the α-subunit the latter proved too insensitive. A directed analysis of the sialic acid residues that end-cap most of the glycans revealed the presence of the nonhuman residue N-glycolyl neuraminic acid both in proteins expressed in CHO, as well in murine cells. The identification of this residue in hCG and LH, and already previously identified in recombinant erythropoietin, represents a single target to address recombinant glycoprotein pharmaceuticals. Analysis of the N-linked glycans revealed that murine glycosylation is different with the existence of terminal Hex(α1-x)Hex-R epitopes. CHO derived material also displays a slightly different glycosylation for hCG. The endogenous glycosylation machinery results in a significant part of hybrid type structures (predominantly at the α-subunit) that is much less pronounced in the exogenous material. Similar results were obtained for the lutropin preparations. Analysis of the de-N-glycosylated hCG and proteolytic peptides showed that no significant differences exist in terms of other peptide modifications or potential O-glycosylation occupancy although a thorough comparison of the glycans is still ongoing.

Code: 05D11WS

In the fight against doping the laboratories are confronted with an increasing number of substances to screen on. Therefore new methods for the screening of those substances have to be implemented in the laboratories. To keep costs for doping control analysis acceptable, to ensure rapid reporting times and to lower the amount of urine needed to screen for all substances, a comprehensive screening for different classes of substances is desirable. The project aims at combining the screening methods for diuretics, beta-2-agonists, heavy volatile stimulants and narcotics in one LC-MS/MS method. To avoid problems with the stability of diuretics during the hydrolysis, direct analysis of the excreted metabolites (where applicable as intact conjugates) is mandatory. This is also beneficial because relatively clean extracts are obtained and the time consuming hydrolysis is eliminated. Thus the sample preparation has to be adapted, the phase II-metabolism of beta-2- agonists, narcotics and heavy volatile stimulants has to be investigated and reference substances of the metabolites have to be synthesized and characterized by nuclear magnetic resonance (NMR) and mass spectrometric (MS) techniques.

Main Findings:

In the fight against doping in sports the laboratories are confronted with screening an increasing number of substances. Therefore new methods for the screening of those substances have to be implemented. To keep costs for doping control analysis acceptable, to ensure rapid reporting times, and to lower the amount of urine needed to screen for all substances, combining ‘traditional’ screening procedures for different classes of substances is desirable. This project aimed at combining the screening methods for diuretics, beta-2- agonists, heavy volatile stimulants and narcotics in one method. To evade the hydrolysis of the conjugates, which on the one hand is time consuming and on the other hand revealed problems with the stability of diuretics, direct analysis of the excreted metabolites (where applicable as intact conjugates) is desired. Thus, the phase-II metabolism of beta-2 agonists, heavy volatile stimulants and narcotics was investigated and sulfoconjugates of p-Hydroxyamphetamine, pHydroxy¬met¬amphetamine, p-Hydroxyephedrine, p-Hydroxynorephedrine, Etilefrine, and Etamivan were synthesized and characterized by LC-MS/MS. The aglycons were coupled to sulfuric acid by reaction with sulfur trioxide pyridine complex. The structures of the mono-sulfates of p-Hydroxyamphetamine, pHydroxy¬met¬amphetamine, p-Hydroxyephedrine, Etilefrine, and Etamivan were confirmed by nuclear magnetic resonance. Different materials for Solid Phase Extraction (SPE) were investigated and the best results for screening purposes were obtained with PAD I. This also allowed a simple combination with the routine procedure for diuretics used in our laboratory, which also utilizes SPE with PAD I. Validation for the sulfoconjugates and application of the whole method to real urine samples was performed. Due to the very large number of analytes with different ionization behavior, it was necessary to submit the final concentrate to two separate LC-MS/MS runs.

Code: 05C10KH

Current strategies for detecting growth hormone (GH) abuse in sport rely on quantifying GH-responsive proteins or measuring changes in the concentration of pituitary-derived GH isoforms in blood. The aim of this project is to develop a test for GH doping based on the analysis of gene expression in peripheral blood cells. There is strong evidence that white blood cells respond directly to GH and indirectly through many anabolic and metabolic circulating factors. These secondary effects may continue long after the withdrawal of the GH stimulus and their gene profile characterization could provide an extended time window for detecting GH use. We propose to undertake this study by obtaining white blood cells from subjects currently enrolled in a WADA-funded GH administration study being undertaken to identify the most sensitive protein markers of GH in serum and the influence of testosterone on these markers. This proposal will not only enhance the scope and value of the existing study but will also allow comparison of biomarker and gene profiling approaches for diagnostic benchmarking. The gene expression profiles will be measured using microarray technology and analyzed by sophisticated statistical programs to identify a gene set which is a fingerprint of GH use. The gene set will be validated independently by quantitative PCR, a gold standard for measuring gene responses in cells. The gene set will be tested rigorously against placebo and other GH treated samples from the same study, and finally against an independent set with samples from subjects positive and negative for GH excess or administration. It is envisaged that this developmental work will lead to the design of a diagnostic gene chip that can be implemented for large scale diagnostic use.

Main Findings

Initial microarray analysis was undertaken using the Affymetrix platform. We have performed 98 Affymetrix Human U133 Plus 2.0 microarrays spanning four treatment groups and forty individual subjects. To determine the effects of treatment, we have compared an individual's baseline (week 0) and final treatment sample (week 8). Due to the small changes in gene expression, a second analysis was conducted using the Agilent Technologies platform, a more recently developed transcript profiling platform with enhanced sensitivity. The two colour methodology of the Agilent 44K G4112F array allows direct comparison of an individual's week 0 and week 8 sample on the same microarray. We performed 40 Agilent microarrays for 20 GH treated men and women. Validation of the Agilent microarray expression data was performed for seven genes using quantitative real time PCR (qPCR). For both gene expression profiling platforms, the GH-induced alterations were small, and similar in magnitude to variations between individuals. Results from both platforms demonstrate that maximal regulation was of the order of two fold, and that few genes were regulated greater than 1.5 fold. Further, whilst both platforms identify differentially expressed genes, these signatures were different between men and women treated with GH. Using data from the Affymetrix platform at the probe set level, testosterone alone had little effect, whilst the administration of testosterone and GH together resulted in regulation of probe sets that showed little overlap with those regulated by GH alone. We conclude that it is unlikely that gene expression analysis of peripheral blood leukocytes would be a viable approach for the detection of GH doping.