Projets de recherche

Code: 05C07JR

We are developing a test for exogenous erythropoietin (epo) activity based on transcriptional profiling. This test would work for any method of Epo abuse, including gene doping. Epo, a hormone used to treat anaemia by stimulating red blood cell maturation, is frequently used by endurance athletes to boost their aerobic capacity. Treating anaemia with recombinant Epo is costly and gene therapy (in which the Epo gene is inserted into cells and stimulated to produce the hormone) is a promising alternative. Gene therapy has the potential to revolutionize medicine, but unfortunately many experts believe that illicit application of this technology to performance enhancement is inevitable and that Epo will likely be in the vanguard of this threat. Epo produced by a gene injected into athletes to augment normal production would be identical to the natural product and undetectable by current testing methods; however, the biological pathway of which Epo is part (the response to hypoxia) is complex and involves the coordinate regulation of genes in a variety of distinct, but related pathways. Selectively stimulating only the Epo pathway would not have the same system-wide effects as the natural stimulation of entire hypoxia pathway. Distinguishing between the augmented and the natural response would be possible by comparing transcriptional profiles, which represent the total complement of RNA transcripts (the intermediates that transmit instructions from the genes to the body) in the tissue. In our project, Serial Analysis Gene Expression is being used to compare the transcriptional profile of the hypoxia response to that of foreign Epo activity. We hypothesize that comparing these profiles will identify differentially expressed genes that can be used to develop a diagnostic test. We will identify these genes in a mouse model, locate and characterize their human counterparts, and incorporate the latter into blood-based test for exogenous Epo expression.

Main Findings

qPCR analysis of potential candidate genes (based on differential expression in the mouse in response to hypoxia or EPO) showed that the variation for the most promising candidate genes exceeded the variation between treatments. As the mice are inbred, sex and age matched, and kept in controlled environments, there is too much variation in the candidate genes identified by SAGE analysis of blood for them to be reliable, predictable and consistent markers for Erythropoietin (EPO) use in humans. In the absence of strong candidate genes for follow-up, analysis in humans was not initiated. Given the results of the experiments, differential gene expression in blood cells following red-cell expansion is not a promising method of detecting EPO (or EPO gene) use. As this proposed test was an indirect measure (i.e. measured the effect of EPO rather than EPO itself, it was necessary that the results be: 1) quantitatively and significantly distinguishable from hypoxia induced erythropoiesis and 2) be highly invariant between individuals. I did not find highly differentially expressed genes that could clearly different between EPO and hypoxia and, although promising candidates were found based on un-linking the pathways (i.e. identifying genes expressing unilaterally that that should be co-regulated), none of the genes tested showed sufficient consistency in expression to be reliable biomarkers.

Code: 05A01JC

The purpose of the project is to develop a rapid and easy-to-use erythropoietin (EPO) doping test, which can distinguish recombinant EPO and EPO analogues from the endogenous forms. The technology should also have the potential to reveal new varieties of EPO and its analogues. The aim of this 3-year research project is to provide the basis for the product development phase. The implementation of this test for EPO doping detection will most significantly reduce both hands-on time and total testing time. The easy-to-use set-up will make it possible to perform the testing in laboratories of different degrees of sophistication as well as in the field. This should make the testing more available and much cheaper. The proposed test-procedure is based on the fact that the EPO forms show characteristic differences in their glycosylation structures. The different isoforms are first separated by the use of a chromatographic step (anion- or affinity (lectin)chromatography), performed in certain zones with immobilized anti-EPO, which specifically captures EPO after the chromatographic separation. The EPO captured is then detected by reaction with carbon black labelled antiEPO. The amount of EPO is proportional to the intensity of blackness, as determined by an image scanner. The recently evaluated MAIIA I variety has been shown to efficiently distinguish endogenous EPO from recombinant EPO, although other varieties of the technology has to be evaluated in order to select the best fitted one.

Main Findings

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

Code: 05D17BD

The exogenous uptake of nandrolone has been banished by the International Olympic Committee (IOC 2004), due to its anabolic effects. Though maximal authorised concentrations have been published for nandrolone metabolites (19-NE, 19-NA) in urine, some positively controlled athletes claimed that they never used such products and expressed that a natural endogenous secretion is related to their high level of physical training. A previous study from our laboratory (Schmitt et al. 2002) has shown that the urinary concentrations of 19-NE and 19-NA in male athletes (judo and long-distance specialists) following exhaustive exercises (Wingate and limited-time tests on ergocycle) did not exceed 0.008 ± 0.02, and 0.07 ± 0.02 ng.ml-1 , respectively. A maximal concentration of 2ng.ml-1 for 19-NA is, however, authorised in male athletes by the IOC. In females, the endogenous origin of nandrolone is a controversial topic in doping control: firstly, low-dose of norethisterone contained in some birth control pills can produce similar urinary nandrolone-like metabolites and secondly endogenous production of nandrolone by aromatase-rich tissues has been reported in the ovarian follicle and during pregnancy by the placenta. The concentration of nandrolone in blood has been shown to be influenced by estrogens, suggesting that nandrolone may significantly vary during menstrual cycle. It seems possible that high androgens concentrations (e.g. DHEA and testosterone) may influence the production of 19-norsteroids in aromatase-rich tissues. Interestingly, intense exercise has also been associated with raised levels of estrogens in females. Therefore, physical exercise could theoretically influence the urinary excretion of nandrolone metabolites in females. Now, the maximal authorised concentration of 19-NA (IOC 2004) has been set at 2 ng.ml-1 in female athletes without any details concerning the menstrual cycle, the use of oral contraceptive, and physical exercise. Our aim is therefore to determine the influence of oral contraceptive, menstrual cycle, and exhaustive exercises on urinary concentrations of nandrolone metabolites and blood DHEAs and testosterone in sedentary and athlete females. Young voluntary sedentary females (n=16) will be recruited, eight taking similar “low-dosed” monophasic oral contraceptives and eight with regular menstrual cycles. These persons will be investigated at rest, every 30 min during a morning period and at the same time of the day during and after exhaustive exercises. Two kinds of exercises will be studied on separated days: Wingate and limited-time tests on ergocycle. For the subjects with regular menstrual cycles, the sessions (rest and exercises) will be organised during the early follicular phase (5 days after the start of menses) and mid-luteal phase (18-23 days after the start of menses). Each subjects will be investigated during a 3-month period. The chronology of the sessions “rest” and “exercise1”, and “exercise2” will be randomised. For the persons under contraception, the sessions will be organised during the weeks of pill ingestion. Using the same protocol, female judoka (n=16) and long-distance runners (n=16) will be investigated.

Main Findings

• Our results provide strong evidence that the urinary concentration of 2 ng/mL of 19¬NA is fair as the upper acceptable limit in doping control tests for female athletes.

• In young women, neither androsterone nor DHEA and nandrolone metabolite excretions are influenced by menstrual phase and physical training. However, oral contraceptive intake lowered DHEA excretion in urine and androsterone excretion seems to be slightly affected by prolonged exercise.

• Metabolomics is a promising tool in order to gain insight into physiological status and to clarify the changes induced by short-term, intense physical exercise. Further research is needed to determine whether metabolomics could be employed to diagnose specific disorders induced by exercise (such as overreaching and/or overtraining), and if it could be used as a new anti-doping tool).

Publications

1: Enea C, Boisseau N, Diaz V, Dugué B: Biological factors and the determination of androgens in female subjects. Steroids 2008;73:1203-16

2: Enea C, Boisseau N, Dugué B : Facteurs biologiques influençant le profil stéroïdien urinaire lors de contrôles anti-dopage. (Biological factors influencing the urinary steroid profile in doping control analysis –Review article in French-). Science et Sports 2009; 24:119–127

3: Enea C, Boisseau N, Ottavy M, Mulliez J, Millet C, Ingrand I, Diaz V, Dugué B: Effects of menstrual cycle, oral contraception, and training on exercise-induced changes in circulating DHEA-sulfate and testosterone in young women. Eur J Appl Physiology 2009; 106:365-373

4: Bayle ML, Enea C, Goetinck P, Lafay F, Boisseau N, Dugué B, Flament-Waton MM, Grenier-Loustalot MF: Quantitative analysis of DHEA and Androsterone in female urine: application to the evaluation of sport and contraceptive pill intake influences. Analytical and Bioanalytical Chemistry 2009; 393:1315-1325

5: Enea C, Boisseau N, Bayle ML, Flament MM., Denjean A, Diaz V, Dugué B: Nandrolone excretion after exhaustive exercises in females: influence of menstrual cycle, oral contraception and training level. Scand J Med Sci Sports 2010, in press

6: Enea C, Seguin F, Petitpas-Mulliez J, Boildieu N, Boisseau N, Delpech N, Diaz V, Eugène M, Dugué B : (1)H NMR-based metabolomics approach for exploring urinary metabolome modifications after acute and chronic physical exercise. Anal Bioanal Chem, 2010, in press (available on line).

Code: 05C4PD

Great progress has been achieved over the past years by means of innovative molecular techniques which has led to the discovery of new growth factors involved in the regulation of muscle development. These findings provide new starting points to understand the mechanisms involved in the adaptation of skeletal muscle to exercise training. One of these newly identified growth factors is myostatin, a member of the transforming growth factor-β family of proteins that has been demonstrated to play a fundamental role in the regulation of skeletal muscle growth during embryogenesis. Blocking of the myostatin signalling transduction pathway by specific inhibitors and genetic manipulations has been shown to result in a dramatic increase of skeletal muscle mass. Drugs or genetic manipulations with the ability to modulate myostatin signalling may have the potential to enhance physical performance in athletes and therefore probably represent a new class of doping substances. To identify manipulations of myostatin signalling, a promising strategy is the analysis of ratios of factors and molecules associated with the myostatin signal transduction pathway, a so-called molecular fingerprint. Manipulations either by application of a factor or by inhibition of its signalling will change these ratios, resulting in a different fingerprint. A methodological strategy to analyse simultaneously the expression of a variety of these factors and relevant signal transduction molecules with high sensitivity is the detection by Immuno PCR (iPCR). The presented project aims to establish Real time iPCR as a new tool for highly sensitive detection of members of the myostatin signal transduction pathway in skeletal muscle, blood, oral mucosa, and urine. As targets we will choose myostatin itself, myostatin propeptide, follistatins, the follistatin related gene (FLRG), the activin receptors ActRIIA and ActRIIB, the family of activin receptor –interacting proteins (APRIPs) and gasp-1. The technique of iPCR will allow to analyse these parameters simultaneously using multiplex real time PCR. During the initial phase of our project this technique will be established and validated using multiplex Real Time iPCR for the detection of the mentioned factors in tissue, blood, oral mucosa and urine in close cooperation with our partner, who has a wide experience using this technique. In the second phase of the project the technique will be applied to study the expression of myostatin and members of the myostatin signal transduction pathway under training conditions. Such information is very limited but essential to develop test systems to identify manipulations of myostatin signalling either by pharmacological or genetic approaches. Muscle biopsies, blood, oral mucosa, and urine will be investigated. In detail we will determine specific ratios between the different members of the signal transduction pathway (fingerprints) in the different matrices, the effect of physical activity, and inter-individual variations. In a future phase of the project we will try to analyse if the application of myostatin blocking agencies (antibodies, follistatin peptide) will change such fingerprints. For ethical reasons, as long as myostatin inhibitors are not licensed for therapeutical applications, this will be only possible in animal models.

Main Findings: 

In the 24 months of the funding period of the project we succeeded in the development of functional ELISAs and immuno-pCR assays (Imperacer™) for MYO, MYOPRO, FOLLI, FSTL and the ACT IIA. The Imperacer™ assay detected the respective proteins with significantly higher sensitivity than the corresponding ELISAs. To analyze the effects of training on the ratio of MYOPRO, FOLLI and FSTL in serum, training experiments were performed. A single bout of resistance training did not affect the serum levels of the proteins. To analyze long-term training effects, 30 young male volunteers were divided into a control, a strength and an endurancetraining group and were trained for three months. The serum levels of the respective proteins in serum before and after training were determined and correlated to the tissue expression of the m. vastus lateralis. Skeletal muscle biopsies were taken and the expression of MYO was analyzed by real time PCR. Serum concentrations of all proteins analyzed displayed a moderate inter-individual variability. More important was the finding that the individual ratios of the analyzed proteins were very constant and were not affected by any kind of training. To get an impression whether skeletal muscle mass or intake of anabolic steroids affect the ratios of the chosen proteins, serum concentrations in non-trained, healthy young males, paraplegic patients and body builders abusing anabolic steroids were determined. There were no significant differences in the average serum concentrations off all proteins analyzed between healthy young man and paraplegic patients. Interestingly, the average serum concentration of MYO propeptide was significantly elevated in the body builder group compared to the other groups. Our data provide evidence that bodybuilders abusing anabolic steroids have different MYOPRO/FOLLI or MYOPRO/ACTIIA ratios than untrained controls or paraplegic patients As a step towards a suitable routine test system, we developed a multiplex Imperacer™ assay that is able to simultaneously detect MYOPRO and ACTIIA ratios with high sensitivity in a single well. MYOPRO and ACTIIA ratios were not only successfully determined in venous blood, but also saliva and capillary blood. Summarizing our results, we believe that the data provides evidence that the determination of expression profiles of members of the MYO signaling pathway is a promising strategy to detect potential abuse of MYO inhibitors. We believe that any manipulation of myostatinMYO signaling will be detectable by determination of the ratios of MYO propeptide to other factors involved in MYO signaling. Interestingly this may imply that not only the use of MYO inhibitors but also many other anabolic manipulations (treatment with anabolic steroids including SARMS, GH administration, IGFI and MGF) may be detectable analysing such ratios.

Code: 05D07RK

The WADA lists for 2004 and 2005 have added a large number of new substances that require detection. WADA will require a number of these to be studied for inclusion into the WADA-accredited laboratories routine testing schemes. From the 2004 list the new additions were corticosteroids and a project to study the metabolism and detections of these is currently funded by WADA. The new list 2005 has a variety of new substances and the metabolism and detection of these must also be studied. Before introduction into testing standards, metabolism and funding will be required in order to investigate cost-effective procedures. This is presently undertaken over a lengthy period by a few laboratories that investigate new substances and publish the results for all laboratories to use. The longer the anti=doping community waits for such data the longer athletes can use the substance with impunity. Currently there are over 20 such compounds to be investigated including some new classes such as the 5α-reductase inhibitors eg. Finasteride, the selective estrogen receptor modulators eg. Raloxifene and anti-estrogenic compounds such as fulvestrant. The aim of this project is to study the metabolism of a selected number of these substances and then to determine procedures to allow then to be introduced into the current schemes across all WADA-accredited laboratories.

Main Findings

The WADA list since 2004 has had a large number of new substances added to the various classes that require detection by the WADA-accredited laboratories. WADA will eventually require most of these new substances to be studied and included in the WADA-accredited laboratories routine testing schemes. The 2004 list saw new additions such as corticosteroids and a project to study the metabolism and detection of these in several laboratories that is currently being funded by WADA. The recent lists introduced in 2005 and 2006 had a variety of new substances added. Before the introduction of detection methods for all of these substances in the laboratory programmes for routine testing, both research and ongoing (LIVE) testing will be required in order to initiate costeffective testing procedures. Such studies are presently undertaken over a lengthy period by a few laboratories that investigate new substances and publish the results for all laboratories to use. However, the longer the anti-doping community waits for such data the longer athletes can use the substances with impunity. The aim of this project was to obtain as many of the compounds added to the list as possible, to study the metabolism of a select number of these substances and then to determine procedures to allow them to be introduced into current detection schemes across all WADA-accredited laboratories. All substances were obtained but not all had metabolic studies undertaken. The substances that can be considered as “new“ additions are: Aromatase inhibitors (Formestane and testolactone); Selective estrogen receptor modulators (Raloxifine and toremifine); Anti-estrogenic substances (Cyclofenil and fulvestrant); 5 α -reductase inhibitors (Finasteride and dutasteride); Anabolic agents (18α-homo-17-hydroxyestr-4-ene-3-one, boldione, 4-hydroxy-19-nortestosterone, methyldienolone, methyltrienolone, methasterone, methyl-1-testosterone, methylnortestosterone, prostanozol, tibolone, zilpaterol and norclostebol); Stimulants (Famprofazone, benzylpiperazine, cyclazodone, fenbutrazate, fencamine, isometheptene, p-methylamphetamine, norfenefrine, octopamine, ortetamine, oxilofrine, phenpromethamine, sibutramine and tuaminoheptane). One of the biggest challenges of the project was to obtain the substance suitable for the studies. These were either purchased, often with difficulty in finding a supplier, or synthesised at NMIA’s Chemical Reference Materials group. Most of the substances on the WADA list could be analysed by one of two analytical techniques, both used within NMIA and most other WADA-accredited laboratories. These techniques use either solidphase extraction or solvent-solvent extraction followed by measurement using either Gas Chromatography – Mass Spectrometry (GCMS) or Liquid Chromatography – Mass Spectrometry (LCMS). Many of the materials or excretion studies have been provided to laboratories through cooperation with WAADS.

Code: 05D3KC 

Glucocorticoids are widely administered for acute and chronic musculoskeletal pain as well as for several other pain syndromes. Increasing concern in sport has recently been raised due to the common use of glucocorticoids amongst elite athletes. WADA has therefore placed certain restrictions on the use of corticosteroids and presently lists them as a legal medication permitted in the local form only when prescribed by a physician with a simplified therapeutic use exemption, while systemic administration is currently banned. Indeed, the influence of corticosteroids on physiological and psychological variables could theoretically enhance performance and therefore explain their overuse by athletes. They have been shown at rest to increase blood glucose and energy store mobilization and to induce euphoria and erythropoiesis but surprisingly little work has been done to determine whether systemic glucocorticoid administration has a direct or indirect beneficial effect on physical performance in healthy subjects. Controversies remain therefore as to whether elite athletes who use glucocorticoids may gain a competitive advantage after acute or chronic intake. We propose to contribute to a wider knowledge of glucocorticoid action mechanisms during exercise thanks to a multi-centre project with, in particular, an investigation of the impact of acute/chronic glucocorticoid administration on: 1) energy cost and substrate utilization during submaximal exercise; 2) eating behaviour; 3) psychological aspects and mood change; 4) erythropoiesis; 5) performance during submaximal exercise with regard to the fatigue status of the subjects.

Main Findings: 

We showed that an acute therapeutic administration of oral prednisolone (20 mg) does not improve the time of cycling until exhaustion during submaximal exercise (70-75% VO2 max) in healthy moderately trained male volunteers, despite a probable increase in lipid oxidation and decrease in CHO oxidation. However, our results demonstrated that short-term therapeutic prednisolone intake (60 mg per day for 7 days) does not significantly modify erythropoiesis or eating behavior but improves performance in healthy recreationally men during submaximal exercise (70-75% VO2 max) both with or without combined intense training. The concomitant alterations in the psychological, hormonal and metabolic parameters analyzed show that short-term administration of this drug had both central and peripheral effects but the complexity of these responses makes dissociation of the possible causal effects on performance difficult to distinguish. Subsequent research are therefore needed to elucidate the mechanisms of these hormonal and metabolic changes in particular after short-term intake in order to determine which changes may be associated with the marked performance improvement obtained only after this mode of administration. Moreover, to our knowledge, no study has focused on women and a specific gender response to glucocorticoid can be questioned. We therefore propose to contribute to a wider knowledge of glucocorticoid action mechanisms during exercise with in particular investigation of: 1) the ergogenic impact of this drug in women, after both acute and short-term administration with, in parallel, investigation of potential eating behavior change and psychological effects; 2) the metabolic and endocrine responses after glucocorticoid administration during longer exercise (3 hours), with regard to the gender status of the subjects in order to elucidate the mechanism(s) of action involved in the improvement in performance occurring after shortterm intake.

Code: 05D08LM

Violations for the steroid testosterone are typically the second most common (after Cannabis) of all banned substances. To detect testosterone abuse an initial screening of athletes’ urine occurs and the testosterone/epitestosterone ratio is measured. Enhanced levels of testosterone with respect to its very close analogue epitestosterone are indicative of steroid abuse. The new 2005 WADA Prohibited Substances List took effect on January 2005 and changed the level of the testosterone/epitestosterone ratio at which further investigation for potential testosterone abuse must be conducted from 6/1 to the lower level of 4/1. The measurement of T/E ratios is technically not a trivial issue ant his reduction in ratio makes it even more essential that WADA-accredited laboratories can accurately determine T/E ratios. A main aim of this project is the production and certification of such a urine matrix certified reference material (CRM) certified to a very accurate level for the concentrations of both testosterone and epitestosterone (and their ratio) will also be produced to provide laboratories with a suite of reference materials to ensure that as many parts of their analytical process as possible can be controlled. These certified reference materials will be produced by Australia’s National Measurement Institute (NMI) using techniques which are considered to be primary methods to ensure the highest accuracy and lowest uncertainty. Once they are produced these reference materials can be used by all WADA accredited laboratories to test their specific methods and ensure accurate and internationally comparable results are produced. These materials will provide an unequivocal international benchmark for all measurements of these steroids around the world.

Main Findings

The preparation and characterisation of two matrix reference materials to underpin the accuracy of measurements of testosterone to epitestosterone (T/E) ratios has been successfully completed. A methanol solution of testosterone and epitestosterone was prepared as reference material NMIA MX006 and distributed into 2386 glass ampoules, each containing 1 mL. Composite human urine containing natural levels of epitestosterone glucuronide and testosterone glucuronide, the key excreted metabolites of testosterone and epitestosterone, was fortified with additional testosterone glucuronide to an approximate T/E ratio of 4. This material (designated NMIA MX005) was distributed into 1282 bottles in 20 mL aliquots and freeze-dried to maximise stability in storage. During the first two years of the project, high accuracy methods of analysis for testosterone, epitestosterone and their glucuronides were developed, optimised and validated. Pilot batches of the two proposed reference materials were produced to demonstrate the feasibility of preparing homogenous and stable products with the required property values. This enabled successful preparation of bulk quantities of the candidate certified reference materials (CRM), which were then apportioned into individual units. These were then extensively evaluated to provide initial reference values and associated uncertainties during the second year of the project. Assessment of homogeneity and stability of the CRMs during storage, transport and use was performed and alternate high accuracy methods of analysis developed to investigate possible measurement biases. Tasks performed during the third and final year of the project involved completing the estimates of the uncertainty associated with the reference values for the measurands to be certified. These measurands are the mass fractions and concentrations of testosterone and epitestosterone in the methanol solution (NMIA MX006), the mass fractions and concentrations of the glucuronide conjugates of testosterone (as testosterone) and epitestosterone (as epitestosterone) following reconstitution of the freeze dried urine (NMIA MX005), and the T/E ratios in both materials. The long term stability trials on both reference materials at their normal storage temperature (-20oC) continued and no significant changes were observed in the measurands.

Code: 05B2MT 

Insulin-like growth factors (IGFs) are circulating peptides that are involved in the regulation of cell proliferation, differentiation and apoptosis. In particular IGF-I has been found to be a critical modulator of skeletal muscle growth when administered locally rather than systemically. The infusion of IGF-I into target tissues such as selected skeletal muscles results in a significant increase in total protein and DNA content, an effect that is highly desirable for athletic performance in various sports disciplines. Owing to the growth promoting properties of IGF-I it belongs to the prohibited list of the World Anti-Doping Agency, and the determinations of its abuse or corresponding synthetic derivatives is of paramount importance. The amino acid composition of recombinant IGF-I is identical to that of endogenously produced IGF-I, and preliminary approaches to reveal IGF-I misuse are based on its quantification. By means of mass spectrometry, a precise determination of IGF-I amounts in human plasma is aspired in addition to an unambiguous identification of this peptide. Moreover, synthetic analogues such as R3 -IGF-I and long IGF-I are commercially available, and strategies enabling the detection of three structurally related compounds are required. A selective isolation of IHG-I and analogues from human plasma is possible by means of immunoaffinity extraction and subsequent liquid chromatography coupled to mass spectrometry provides substantial data allowing a qualitative and quantitative determination of target analyses.

Main Findings: 

Human IGF-1 is a promising part of strategy to reveal the abuse of human growth hormone by serving as a biomarker. Therefore, it is of paramount importance to quantify the plasma level of IGF-1 in order to be able to compare the results with valid reference ranges and thus to distinguish between 'normal' and 'abnormal' concentrations. The definition of such reference values is still ongoing because there are many parameters to consider. Regarding the IGF-1 analogues as a possible threat in sports and doping control analysis, there is a lack of information because pharmacological data in human are currently not avaible. Recent investigations in the internet have, however, shown that LONG(tm)R(3)IGF-1 in particular is already in use. Althought it is not approved for the use in humans, it is probably favoured due to its increased potency (e.g. prolonged half-life). Based on its higher biological activity the administered doses and, hence the costs, are much lower than excepted for an application of recombinant human IGF-1. Thus, an assay enabling the determination in athletes' plasma specimens is required. The described procedure allows the validquantification of human IGF-1 as well as the unequivocal identification of its analogues LONG(TM)R(3)IGF-1 and des(1-3) IGF-1. The combination of immunoaffinity isolation and purification with LC-ESI-MS/MS provides utmost specifity for the analysis of banned peptides as recently demonstrated. It enables absolute quantification without distortion caused by, e.g., cross-reactions.

Code: 05D4JY

Mesocarb (Sydnocarb®) is an amphetamine-related psychostimulant that have been used for doping purposes due to their central nervous system stimulating effects. Moreover, it is very likely that mesocarb is being used by drug addicts. Mesocarb is included in the World Anti-Doping Agency’s list of substances and methods that are prohibited in sports. Mesocarb is chemically a cyclic and mesoionic N-phenylcarbamoyl-3-(betaphenylisopropyl) sydnone imine. The dominant urinary metabolites of mesocarb are identified to be its mono-, di- and trihydroxylated isomers. The exact chemical structures of regioisomeric metabolites of mesocarb have not been solved yet. Furthermore, the metabolites are not commercially available. According to the WADA’s international laboratory standard and ISO 17025 standard, well characterized pure reference materials are recommended to be used as references in the analysis. In this project a high-yielding and selective synthesis methods for the preparation of mesocarb metabolites as reference substances will be developed. The synthesized metabolites will be purified and characterized and their chemical stability will be tested. The synthesized substances will be used to solve so far unknown regiochemical structures of the main urinary metabolites of mesocarb. Using the synthesized substances, the feasibility of various screening methods used in doping laboratories for the abuse of mesocarb will be tested, and a liquid chromatographic – tandem mass spectrometric confirmation method will be developed and validated. Finally, the synthesized mesocarb metabolites would be available free of charge as reference substances to the world’s antidoping community. The pure and fully characterized reference substances would enable the reliable and legally defensible analysis of mesocarb and could also be used in quality assurance and in development of new analytical methods.

Main Findings: 

Mesocarb (Sydnocarb®) is an N-alkylated amphetamine derivative, and has been used for doping purposes due to its central nervous system stimulating effects. It belongs to the list of prohibited substances and methods in sports of World Anti-Doping Agency (WADA). According to the WADA’s international standard for laboratories and ISO 17025 standard, well-characterized reference compounds should be used for the identification of a prohibited substance, if available. Presently, the metabolites of mesocarb are not commercially available, and additionally, the exact chemical structures of several mesocarb metabolites have not been solved yet. In this WADA-funded research project we synthesized six potential mono-, di-, and trihydroxylated regioisomeric metabolites of mesocarb for the doping analytical purposes. The synthesized metabolites were purified, characterized and compared with the in vitro synthesized mesocarb metabolites using human liver enzymes and with the in vivo formed metabolites extracted from human urine after oral administration of mesocarb. An LC-MS/MS-method for the characterization of mesocarb and its metabolites in urine was developed. p-Hydroxymesocarb was found to be the main (conjugated and non-conjugated) metabolite in human urine, emphasizing the importance of its synthesis and availability as a reference material with respect to doping analysis. The synthesized and fully characterized p hydroxymesocarb enables the reliable and legally defensible confirmation analysis of mesocarb, and could also be used in quality assurance and in development of new analytical methods. The metabolite synthesized within this project will be available without charge to all WADAaccredited anti-doping laboratories.

Code: 05B9RK 

It is known that anabolic steroids are very effective in promoting muscle growth. The detection of synthetic anabolic steroids is now very effectively carried out by WADA laboratories but the effective detection of the abuse of naturally occurring anabolic steroids is still a major challenge. Doping control laboratories need criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. For some endogenous steroids such as testosterone the criteria have been established and measurements of the T/E ratio coupled with isotope ratio measurements can effectively detect such doping. There are however several other endogenous androgenic steroids which can potentially increase active androgen levels and boost performance. 4-androstenediol and 5-androstenediol are two such prohormones which are banned but for which there is incomplete understanding of their metabolism. Recent evidence indicates that at least in some individuals the administration of 4-androstenediol does not lead to a significant change in the T/E ratio. Thus there is a need for improved detection capabilities which can not only detect the abuse of endogenous androgens but also specify which steroid has been administered. It is proposed to use the same methodology which has been developed in this laboratory for the detection of dehydoepiandrosterone (DHEA) abuse. Specific markers arising from 4-androstenediol and 5- androstenediol abuse will be looked for in both the glucuronide and sulfate steroid fractions. Once identified these compounds will be used both for GCMS screening and for GC-C-IRMS confirmation procedures.

Main Findings: 

Compound specific detection (CSD) of endogenous steroid abuse using complementary Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) techniques is an important requirement for effective doping control in sport. This project was undertaken to determine if 4-androstenediol (4-ADIOL; androst-4-ene- 3β,17β-diol) and 5-androstenediol (5-ADIOL; androst-5-ene-3β,17β-diol) administration could be selectively identified in urine. Single and multiple oral doses of 4- and 5-ADIOL were performed on two volunteers with human ethics approval and informed consent to monitor changes in urinary endogenous steroid excretion and 13C content. The results showed significant increases in the excretion of androsterone glucuronide and etiocholanolone glucuronide from 4- and 5-ADIOL administration. This behaviour, which is similar to that observed for dehydroepiandrosterone and androstenedione in previous studies, is an effective screening indicator of endogenous steroid administration. 4-ADIOL was found to produce the diagnostic urinary dehydrogenation products: androst-2,4-diene-17-one and androst-3,5-diene- 17-one. These can be used as screening markers in the GC-MS steroid profile to identify suspicious samples that require confirmation by carbon isotope ratio (δ13C) analysis using GC-C-IRMS. Differences in post-administration detection times based on Δδ13C values, relative to δ13C measurement of an endogenous reference compound, were found between glucuronide and sulfoconjugated steroids that were dependent on the individual’s metabolism. Preferential glucuronide excretors provided a detection period of up to 72 hours, while sulfoconjugate excretors may provide up to 68 hours. The administration of 5-ADIOL did not result in the urinary excretion of the diagnostic diene marker compounds, thereby enabling a distinction to be made between the 4- and 5-ADIOLs. Additionally, CSD between 4- and 5-ADIOL may be performed using δ13C analysis of the etiocholanolone sulfoconjugate relative to the sulfoconjugates of androsterone, epiandrosterone and dehydroepiandrosterone. The detection of 5-ADIOL doping based on Δδ13C values, derived from sulfoconjugates greater than 4.0‰ is capable of achieving a 60 hour post-administration time.