Projets de recherche

Code: 02A03RK

The purpose of this project is to determine the variability of the natural isoform pattern of EPO in a group of subjects and determine the extent to which the isoform distribution can be influenced by external factors such as vigorous exercise and disease. Such information is needed to support the urinary test for human recombinant EPO. At present the detection of doping with human recombinant erythropoietin (EPO) relies on the detection of abnormal blood parameters such as those reported by Parisotto (Parisotto et al 2001) coupled with the presence of recombinant human EPO in a corresponding urine sample. The urine test developed by LNDD uses isoelectric focussing and a patented double blotting technique (Lasne 2001) to separate the EPO isoforms into a series of bands. A positive cannot be declared unless the urine has bands which correspond to those found in human recombinant EPO. Data collected so far indicate that normal urinary EPQ has isoforms which are more acidic than those found in human recombinant EPO although there is some overlap (Lasne and De Ceaurriz 2000). A positive is declared if the percentage of basic isoforms is greater than 80%. This value was statistically determined on the basis of the range of values found in a relatively small number (a few hundred) of normal subjects. Whilst some data has been obtained showing that the distribution of urinary EPO isoforms is not significantly affected by external factors such as altitude there has been no large systematic study on factors such as acute, extreme and long term exercise, and disease. It is only a matter of time before the technical aspects of the urinary EPO test are legally challenged and hence it is essential that data be collected and published in advance to establish whether extreme exercise or disease can alter the pattern of isoforms present so that they more closely resemble those found in recombinant EPO. Such data will be essential to support the urinary EPO test if it is ever to be used without a blood sample to confirm doping with recombinant EPQ.

Main Findings: 

This project was undertaken to determine if there were any significant changes on the distribution of urinary erythropoietin (EPO) isoforms induced by exercise or disease. It is important to know if such changes do occur as the current test for detecting doping with recombinant EPO depends on the fact that the EPO isoform distribution in the urine of those who have been administered EPO is more basic than the distribution found naturally. In an attempt to determine whether exercise can induce a change to more basic isoforms, subjects were studied who underwent a range of exercise regimes ranging from a short duration (10 minute) exercise test to exhaustion, through a full marathon of approximately three hours, to a 100 km cross country run with a typical duration of over 24 hours. In all cases urine samples were collected in order to measure both the concentration of urinary EPO and the distribution of isoforms. The results show that the concentration of EPO in urine is not affected by any of the levels of exercise. It was also found that the concentration of EPO in urine is highly variable and for some individuals can vary by more than a factor of four from one collection to the next. For most levels of exercise up to including a full marathon the variation in distribution of urinary EPO isoforms was small and within the range of normal variability found for individual subjects. However it appears that the extreme long duration exercise can produce a small but significant increase in the percent basic isoforms found in the urine. It is not known whether this increase relates to changes in EPO production or changes in EPO excretion. The magnitude of the change was not sufficient to require changes in the criteria currently used to assess whether a urine sample contains recombinant EPO. The samples from the subjects who were part of the disease study were all suffering from anaemia resulting from severe kidney disease. It was hoped to determine if the EPO excreted from such subjects was different in isoform distribution possibly due to a greater contribution from the liver. Unfortunately it was not possible to draw any conclusions from this aspect of the project because the current method was found to be unsuitable for such urine samples owing to their high protein content.

Code: 01C15CA

Detecting the use of androgenic anabolic steroids, potentially endogenous in humans, and which are prohibited substances in sport doping control programmes, still represents a major challenge to the analysts. These steroids include testosterone, its precursors androstenedione and dehydroepiandrosterone, and the 19-norsteroids equivalents, some of which are commercially available for oral selfadministration. This project is aimed at applying the IRMS (isotope ratio mass spectrometry) to the detection of the “natural” testosterone and 19-nortestosterone anabolic agents. Complement of the existing GC/MS methods, the GCIC/IRMS permits the differentiation of the exogenous or endogenous origin of urinary androgens metabolites, by measuring the isotopic content of their carbon atoms. That novel applcation of an otherwise well-known technique, now requires its validation in different experienced laboratories, the determination of international reference ranges of the metabolites of endogenous origin and the documentation of the changes commonly observed following the administration of these steroids. Ultimately, one of its most direct outcomes will be to verify that although different methodological approaches and different equipment are used, the same determination is made from the analysis of common specimens. Testosterone and 19-nortestosterone (nandrolone) are potent androgenic anabolic steroids of known abuse in sport. Anabolic steroids are banned in Olympic sports for more than 20 years and since 1986, the highest number of positive cases reported are due to these two steroids. The administration of testosterone and its precursors, androstenedione and DHEA has been described to significantly alter the parameters of the urinary androgens steroid profile measured by GC/MS. The administration of testosterone is first detected in human urine by the GC/MS measurement of a testosterone/epitestosterone (TIE) value higher than 6, which is caused by the relative increase of excreted testosterone glucuronide (Donike, (1983)). The oral intake of androstenedione and DHEA was shown to transiently increase the excreted T/E value in females and in some male volunteers, from whom T/E values slightly higher than one were measured (Uralets (1999); Lévesque (2000); Bowers (1999); Garle (1998)). Other alterations of the urinary steroid profile, such as abnormally high concentration of androsterone and etiocholanolone and the presence of the characteristic hydroxylated metabolites

glucuro- and sulfoconjugated, 6c~-androstenedione, 6P-epiandrosterone, had permit to report positive findings (Lévesque, (1999)). Disruption of the normal urinary profiles of androgens metabolites can be demonstrated by comparison to the described population reference ranges and to the individual’s norm (Donike (1993); Ayotte (1997) and reference cited therein). That requires the investigation of the athlete’s previous or subsequent tests results in order to exclude the few individuals who naturally produce urine samples in which elevated TIE values are systematically measured. Although successfully applied in many cases, this method is time-consuming and complex. Considering only the T/E values above 6 also leads to false negative results since it is known that the limit will not be exceeded following the administration of testosterone and precursors, when the basal values are lower than one, which is a characteristic but not exclusively, of the Asian population (Shackleton, (1997)). The level of androgens in female samples is generally very low and the uncertainty of the

measurements may represent a problem to which must be add reports of alteration of the normal values attributed to other sources than the administration of

androgens. The administration of 19-nortestosterone and of its precursors, 19- norandrostenedione and 19- norandrostenediol, which are available for oral selfadministration, results in the excretion of 19- norandrosterone and 19- noretiocholanolone, mostly found in the glucuroconjugated form. The period, during which the metabolites can be detected, is drastically reduced when the oral preparations are taken (Engel (1958); Masse (1985); Ayotte (1996); Schanzer (1996); Kintz (1999)). In the last years, many positive cases were reported with low levels of the urinary metabolites. Extremely low levels of 19-norandrosterone can be endogenously excreted in human urine, and that has prompted the IOC to safely recommend a threshold in males and females. However, as it is the case with the androgens, natural factors are systematically invoked to challenge the positive test results.

Main findings

The three laboratories participating to the project have years of experience in testing urinary steroids by GC/MS and by GC/C/IRMS. For the latter, they have developed and validated techniques which make use of different instruments, different sample preparation and analytical methods. The internal urinary reference steroids also differ. Nothing has been changed to the laboratory validated protocols. However, having observed in the early phases of the project that one laboratory had values differing significantly from the two others when authentic standards were analysed, the project was halted until the necessary verifications and adjustments were made. The results indicate that similar conclusions are reached by the three laboratories when sharing urine samples. It further confirms the need to consider and compare the difference of delta values between the intact and altered urinary metabolites for each sample and not the absolute individual values which were found to vary. This does not limit in any way the applicability of the technique since already only the difference of 13C/12C values (delta values per mil) is significant in individual samples, allowing for individual variations that could be due to external environmental factors. As an example, absolute mean 13C/12C values of alcanes of know and certified isotopic content and authentic standards of different steroids were found to vary in the three laboratories by less than 0,53 and up to 1,6 0 /00 respectively. When urine samples were shared, we observed that the absolute delta values of some steroids could vary up to 2,3 0 /00. However, when the differences of the values between the metabolites and the reference steroids were compared, before and after the administration of the testosterone precursor, coefficients of variation of less than 21% were obtained for the main urinary metabolites. In all three laboratories, significant alterations of the 13C/12C values of androsterone and etiocholanolone (after the hydrolysis of the glucuronide) were recorded in samples collected following the administration of testosterone, androstenedione and DHEA. The values measured in reference steroids e.g., pregnandiol, pregnantriol and cholesterol remained stable, as expected.

Code: 01C10DM

The use of performance-enhancing substances has historic foundations in man’s desire to create a body-building “wonder drug”. In the United States, there has been a rapid rise in the availability and use of unregulated herbal products. The extent of use of these products within the sports community is largely unknown. However, it has been demonstrated that many of these products contain steroids, steroid precursors, or steroid-like compounds (1).

The goal of this proposal is to develop a comprehensive laboratory and clinical model by which new anabolic steroid compounds of interest, particularly those contained in herbal products, can be quickly evaluated to elucidate their metabolism and improve detection. We propose that a dual system of in vitro human liver metabolism studies, coupled with clinical dose-response studies in human subjects, can offer distinct advantages in determining both the profile of metabolites expected after use of a specific compound (or product) as well as the optimum biological specimen(s) for detecting use. Emphasis in this proposal is placed on developing the model with a focus on studying the metabolism and distribution of androstenedione. If successful, this model could be used to study future compounds of interest to the World Anti-Doping Agency and other sports testing bodies, such as 19-norsteroids.

Two general objectives will guide us in reaching our goals. First, we will investigate whether an in vitro human liver assay system can be used to predict the qualitative and quantitative profile of androstenedione and metabolites as compared to in vivo studies. Second, we will evaluate the pharmacokinetics and in vivo metabolism of this steroid precursor in healthy human volunteers. The disposition of parent compound and relevant metabolites into various biological matrices, including plasma, urine, sweat, oral fluids and hair will be investigated. To accomplish our specific aims, we will also develop and rigorously validate sensitive and specific laboratory methods for the analysis of androstenedione and its relevant metabolites in several biological matrices.

Main Findings

(The project was not finished)

Code: 01A07CH

Androgen replacement therapy is usually life-long, and should only be started after androgen deficiency has been proven by hormone assays. The therapeutic goal is to maintain physiological testosterone levels (lc). However, the normal range for serum-testosterone for 20-30 yr. old men has not been established. The existing studies have been on limited number of subjects using recruited healthy subjects, who have not been evaluated for hypogonadism, or blood donors (la, 3,4, 4a). The study design of earlier studies may not have taken into account the circadian rhytm in testosterone levels. The acrophase (time of maximum value) of the rhytm occurs several hours prior to the time of awakening and the nadir is in the late afternoon or early evening (lb). These problems with physiological levels of testosterone may be especially important when looking at use, misuse and abuse of androgens. Therefore when defining “hypogonadism” as serum total testosterone levels consistently below the lower limit of normal, it is impossible from the data available in the literature to extract an estimated prevalence for hypogonadism. The best estimation in otherwise healthy, non-obese male subjects aged 20-30 yrs. is between 2 and 4% (4a). Testosterone circulates in the blood in a free form and in a protein bound form. Only approximately 2% of the circulating testosterone is free, 30% is tied to albumin and the remaining part is tied to the sexual hormone-binding-protein (SHBG), which is a glucoprotein with special affinity to androgens and oestrogens. The concentration of SHBG is increased by hypogonadism, cirrhosis of the liver and decreased by obesity and treatment with androgens in supraphysiological doses. Muscle mass, strength and exercise Androgens are known for the anabolic effects, especially in high doses. Patients with hypogonadism can increase muscle mass during androgen substitution therapy (5, 6, 7, 8, 11). In cell cultures, androgens will stimulate mitosis of mioblasts and initiate a cascade of biochemical changes. In studies using eugonadal young males, pharmacological doses of testosterone did only influence muscle mass if they were administrated during increased physical activity or weight lifting training (5, 7). Large population-based studies on the relationship between muscle mass, exercise, strength, and testosterone levels are not avaiable. Neither do we know of any publication, which in a controlled study show long-term effects of testosterone substitution therapy on these parameters in hypogonadal men. Cardiovascular risk factors (fat mass, lipids, and glucose metabolism) Patients with hypogonadism have increased total cholestrole, increased LDL and increased HDL (5-7, 12). These patients have increased fat mass and decreased lean body mass (LBM) (6,7, 11, 13, 14). The increased fat mass is accompanied by increased s-leptin and decreased s-IGF- 1 and growth hormone concentration in blood (7). It has been shown that the increased fat mass is mainly located as abdominal fat. This leads to increased glucose concentration, insulin concentration and later probably to increased blood pressure. One of the hypotheses for these changes is that the decreased testosterone leads to increased frequency of syndrome X. The primary trigger mechanism should be increased stress and thus increased activity of the hypothalamus pituitary adrenal access (10, 14, 16- 18). Bone metabolism Long-term treatment of hypogonal men with testosterone decreases bone resorption and increases bone formation markers (8) and increase bone mineral density (9). Behre et al. (9) found that 36 months of T substitution therapy of hypogonadal men restored BMD to the age-dependent reference range. A larger increase was seen in patients with initial low BMD during the first year of treatment. There was no significant changes in BMD after 18 to 24 months of treatment. There are no clinical control studies available in this area.

Main Findings: 

The Odense Androgen study is a population-based cohort study of hypogonadism and growth factor (IGF-1) deficiency in male subjects, 20-30 years of age. 783 males aged 20-30 years were included in the study. The 783 participants included responded to a detailed questionnaire and underwent: full physical examination, blood tests (including androgens/estrogens, bone metabolism, general metabolism, thyroid function, liver parameters, hemoglobin, endocrine parameters), urine tests, DNA analysis of the androgen and estrogen receptor, diverse physiological measurements, DEXA scan, MRI scan, muscle strength tests. The objectives of these studies are:
a) To establish a reference interval for the serum concentration of total-T, free-T,
total-E2, free-E2
b) To evaluate the impact of BMI, fat parameters, chronic disease (gynecomastia,
microtestis etc.) and medication on testosterone, free-testosterone and estradiol
levels.
using probit evaluation.
c) To evaluate a positive role of high, medium, and low testosterone levels on:
a) muscle mass, muscle strength, muscle power, and oxygen uptake
b) bone mineralization (BMD) and bone metabolism
c) cardiovascular risk factors/metabolic syndrome: fat mass, serum
lipids, glucose, metabolism, blood pressure
d) hematocrit, growth factors
e) signs of hypogonadism by medical history as well as clinical signs.
f) sexual function
g) quality of life

Code: 01B03PM 

After regular injections of rhEPO, there is a feedback effect depleting the endogenous production of rhEPO (figure 1, lane f, almost no endogenous EPO can be detected). The detection of a “positive case” has been based on the ratio between the sum of the areas of all bands appearing in the P1 range of an rhEPO standard analysed in parallel and the sum of the areas of all bands. The determination of criteria for a positive sample has not yet been appreciated. It is proposed that independent experts produce a report of evaluation on the already existing data obtained in the Laboratory of Paris. Some validation process has already been set up in this laboratory in order to produce a publication including the whole description of the method and the results analyses of samples from double blind rhEPO treatment protocols. The aim of this project is to have three IOC accredited laboratories capable of measuring the haematocrit and reficulocyte count before major competitions and also capable of detecting the presence of rhEPO in urine. The analysis of blood parameters has to be done, because it is a test sensitive, cheep, fast and selective enough to screen a maximum of athletes in a very short time. The doubtful samples will be selected and will be confirmed with the urinary test. As mentioned, this latter test is for the time being the only way to determine the presence or absence of rhEPO in urine, It is efficient, but it is expensive and time consuming. Some sport federation (UCI, ISU, FIS, UIPMB) have introduced unannounced blood testing in order to evaluate the haematocrit and/or haemoglobin level before competitions. These tests have been going on for a few years and they have proved to be feasible at the beginning of any race/competition around the world. The possibility of measuring at the same time, in the same conditions, the reticulocyte count as well as the haematocrit level would be a very simple way to screen most of the athletes taking part to sport’s events. In these conditions, both parameters can be measured immediately. Will be considered as suspicious the blood samples with a haematocrit level > 47 % or with a reticulocyte count> 2.4 %. In such a case a urinary sample will have to be taken under the same regulation as an antidoping control and will have to be sent for analysis to one of the three lOG accredited laboratory. In cycling, UCI regulations will be applied every time the haematocrit is above 50 % (47 % for women). The urinary test is still under evaluation by the lOG experts, but it was performed during the Olympic Games in Sydney. Two major sport federation, the UCI and the IAAF have accepted to introduce this test specially for the cycling season 2001 and the Athletics World Championship in Edmonton, as a pilot scheme. Due to technical and financial reasons it is compulsory to combine it with blood tests as mentioned above to avoid too many analysis (in case of acceptance, the detailed protocol will have to be set up, but at this time, one can presume that it will be inspired by the experience acquired in cycling). This project has the advantage of being feasible in a short time period, because most of the analytical and pre analytical procedures have been tested for more than four years and they have proved to be robust. Moreover, the analysis of the reticulocyte count is nowadays something realisable thanks to entirely automatic analysers. These apparatus have become smaller and they can easily be transported and installed at the beginning of a stage of a major cycling event or an athletics’ competition.

Main Findings:

Part 1: In conclusion, we strongly recommend to the federations to have the blood samples analysed during or prior to the competitions with their own equipment to avoid any inter-technological variations, and especially to have control of the entire process from blood sampling to blood analysis. Therefore, the confidentiality and the return of the results are optimal. concerning the medical follow-up, parameters stable over time and temperature should be preferred to unstable parameters such as the haematocrit. Indeed, most of the time, the blood samples  are not analysede right after venipuncture and travel from the medical surgery to the laboratory. The use of a SpyT during the sending off of biological samples (blood, urine) should be strongly required in order to identify strange results which notably come from an interruption of the refrigeration procedure. Futhermore, the follow-up of temperature is compulsory when using a transportable refrigerator, because it is necessary to cool down the temperature, but above all to avoid freezing of the samples (especially blood samples).                                                                                           

Part 2: In conclusion, the ideal way of performing blood analysis prior to competitions is to have first excellent pre-analytical conditions and have the analysis done as soon as possible. Then it is strongly recommended to have a unique type of technology for the determination of the blood parameters. In such a case the haematological/biological passport for example. Otherrwise, the lackof data does not enable the federations to focus on the athletes potentially manipulating their blood precautions, to introduce the OFF-model in order to reduce the number of athletes abusing of rhEPO and/or blood transfusions. The health of the athletes and sport will certainly be the winner of such a strategy as it was the case with the haematocrit limitation introduced in 1997.

Code: 01A03KH

Tests for detecting abuse of peptide hormones such as growth hormone (GH) in sport have proved elusive. However, there has recently been considerable progress in this field. Following the work of the GH 2000 project, two approaches were proposed for detecting GH doping; one using indirect markers of GH action and one based on the quantilation of GH isoforms secreted from the pituitary gland [1-3]. The difficulties associated with development of tests for doping are compounded by the fact that athletes frequently take performance-enhancing agents (PHAs) from more than one class. A PHA acting on one system can influence the activity of a different system. How physiological interactions between biological systems may alter the result of a doping test has not received detailed attention. For example, anabolic steroids stimulate the GH system [4, 5], and both anabolic steroids and GH stimulate erythropoiesis [6, 7]. Physiologic and pharmacologic interactions of this nature will have an impact on the predictive value of a test. Therefore, the validity of a test is dependent on basic information on how one P1-IA interferes with tests for another group. An alternate approach for detecting exogenous GH use is based on the difference between recombinant human GH (r-hGH) and pituitary GH, which consists of different molecular isoforms including l7kD (17K) and 20 kD GH (20K). Initial work using subtraction assays indicates that GH doping can be reliably detected using this method [3]; however, isoform specific assays are preferable. We have developed a highly sensitive and specific immunoassay for 20K-hGH [8] and have also developed an immunoassay for 17K-hGH [9]. Use of 20K- and 17K-hGH together is likely to provide a more robust measure of GH abuse than use of one isoform alone. Our Consortium is uniquely placed to conduct these investigations. The Australian Sports Drug Testing Laboratory (ASDTh) is IOC accredited, and has extensive experience of drug testing in the Olympic setting. Members of the Consortium are internationally recognised leaders in their field and each provides complementary expertise within this project. These areas include GH physiology, sex steroid interactions and clinical research facilities to undertake interventional studies (Prof Ho and co-workers), 20K GH assay development (Prof Irie and co-workers), 17K GH assay development (Prof Ho and co-workers), insulin-like growth factor-I (IGFI) and IGF-binding proteins measurement (Prof Baxter and co-workers), anabolic steroid and androgen pharmacology (Prof Handelsman) and EPO detection (Dr. Kazlauskas and co-workers). Several members have already established a network for large scale collection of samples and links with National sporting bodies (Prof Irie and Dr.Kazlauskas). We have already collected more than 4000 serum samples from elite athletes as part of the EPO 2000 project involving the ASDTh. Multiple samples were obtained from more than 1100 athletes over a two week period. These athletes represented a wide variety of nationalities and ethnic groups. This archival collection is a powerful resource for the ultimate validation of tests which will be selected for their potential from studies of pharmacologic interactions, and will define the reference data for the application of these tests.

Main Findings:

This summarizes the results obtained in our project to develop a robust test for doping in sport with growth hormone (GH), based on the detection of (i) GH-responsive proteins and (ii) pituitary GH isoforms in blood. This involved the conduct of two major studies: (a) a cross-sectional study of over 1000 elite athletes to determine the potential influence of demographic factors (Demographic Study) (b) a prospective intervention study in recreational athletes to identify the most sensitive GH-responsive markers and the potential influence of co-administration of androgens (Intervention Study) The main achievements are:                                                                                                           - Demographic Study : (i) Publication of the study characterising the influence of demographic factors and sport type on GH-responsive markers in the leading scientific journal in the field. (ii) Analysis of the influence of demographic factors on pituitary GH isoforms (20K and 22K GH). (iii) Addition to (i) and (ii) above, by recruitment of young elite athletes and Japanese elite athletes GH/testosterone

-Intervention Study: (i) Completion of a major placebo-controlled GH and testosterone administration study in over 90 recreational athletes. (ii) Measurement of IGF markers, collagen markers and GH isoforms, and a preliminary analysis of completed markers. The key findings are:                                                                                                             Demographic) Study : (i) For GH-responsive markers, age and gender were the major determinants of variability, except for IGFBP-3 and ALS. Tests for GH doping based on IGF-I and on collagen markers must take age into account and ethnicity need not be considered for these markers. (ii) For pituitary GH isoforms, the relative concentrations of 20K and 22K GH, are minimally influenced by demographic factors. The stability of the isoform ratio to the effects of these factors renders it a promising measure of exogenous 22K GH abuse. (iii) Additional data from young athletes and Japanese athletes have further consolidated the age-related changes in the GH-responsive markers, particularly in early adolescence.                                                                                                                                Intervention Study: From a preliminary analysis of the IGF axis markers (IGF-I, IGFBP-3 and ALS) and PIIINP, the responses were greater in men than in women for all markers, and testosterone enhanced the response of PIIINP to GH , but not the responses of the IGF axis markers to GH.

Code: 01B09WS

A comprehensive method of sugar analysis which should include the plasma volume expanders HES (already published) and dextran and additionally the diuretic mannitol should extend and improve the possibilities of analysis in doping control urine samples. The scandals of the world championships in Lahti showed the necessity of the detection of this class of substances and, due to the close chemical similarity of some plasma volume expanders and mannitol, methods for the determination of abuse of these compounds should be established. Therefore, the development of sensitive and specific identification procedures are planned based on chemical analysis of excretion study urine specimen, reference compounds, consideration of earlier metabolism studies and routine analysis of doping control samples. Here, the suitability of gas chromatography and mass spectrometry was already proven with HES. The applicability of liquid chromatography coupled to mass spectrometry will also be tested with different ionization techniques. Concerning dextran the identification of the pattern of glucose and its polymers with 1,6-linkage should be elucidated and the origin of possibly occurring 1,6-linked oligo- or polysaccharides in human urine determined. A validation of a method for identification and quantification of the 1,6-linked glucose as 1,5,6- triacetyl-2,3,4-trimethyl-glucitol will be performed. Normal values for this PMAA in urine of athletes in endurance sports will be established based on routine samples in the Cologne laboratory for doping analysis. Urine samples collected after application of mannitol and the different forms of dextran will be obtained from patients who are therapeutically treated with the medicaments.

Main Findings:

The intravenously administered plasma expander dextran and the diuretic agent mannitol are prohibited substances according to the “Prohibited list of substances” of the WorldAnti-Doping-Agency. Plasma expanders are colloidal solutions that increase the blood volume by an influx of interstitial fluid. The plasma expander dextran is administered in cases of loss of blood, e.g. treatment of burns or hypovolaemic shock and for the stabilisation of the circulation of blood during narcosis. This “diluting effect” is of great interest in sports in order to control haematological parameters and masking of an EPO misuse. Mannitol is used as an osmotic diuretic by intravenous infusion to preserve renal function in acute renal failure and to reduce raised intracranial and intra-ocular pressure. In sports it may be administered to impair the excretion of prohibited substances. Both dextran and mannitol are highly polar saccharide-based compounds. The related chemical properties of dextran, HES and the diuretic mannitol allow an implementation of the two compounds into the existing HES screening method. The screening method enables a semi-quantitative estimation of mannitol and glucose levels resulting from an entire hydrolysis of dextran. For mannitol, a method based on GC-MS, which enables the detection and quantification of the analyte, was developed. The procedure allows distinguishing between the six stereoisomers of mannitol such as allitol, altritol, dulcitol, iditol, mannitol and sorbitol. Urinary mannitol concentrations could be determined following oral application of mannitol originating from commercially available sources (i.e. sweeteners). However, the method does not enable to distinguish between orally and intravenously administered mannitol. A novel method, enabling the identification and quantification of the plasma volume expander dextran in human urine by LC-APCI-MS/MS was successfully developed. The concentration of dextran after intravenous application is 100-250 times higher than “normal” concentration levels of dextran (polymeric a-1,6-glucose) in human urine. Based on these results, a misuse of dextran in sports can be revealed by establishing a threshold level for dextran. Furthermore, an additional qualitative evidence for the presence of dextran can be accomplished by means of partially methylated alditol acetates (PMAAs) analysis, which provides precise information about the linkage positions of glucose monomers.

Code: 01B02PM 

The SLAB project will generate a comprehensive blood screening procedure capable of providing same-day feedback on the use of recognized methods of blood doping. These detection methodologies are designed to complement the ‘SAFE mobile’ project presented to the WADA Health and Medical Committee. Implementation of the strategies will deter athletes from experimenting with blood substitutes and other substances that enhance oxygen delivery. Researchers who were instrumental in the success of the EPO2000 project will collaborate with pharmaceutical companies to develop tests for yet-to-be-released pharmaceutical products. This pro-active approach will dissuade athletes who in the past have sought to ‘stay ahead’ of doping authorities by progressing to novel drugs before appropriate tests can be implemented. Features of the SLAB proposal: • Internationally-renowned scientists with a proven ability to undertake and successfully complete anti- doping research (incorporating both blood and urine matrices) • Collaboration with the pharmaceutical companies who develop blood substitutes, as well as with medical experts and manufacturers of analytical instruments • Substantial in-kind contributions from industry partners subsequent to appropriate negotiation and confidentiality agreements • A Steering Committee (including invitees from the WADA and the IOC/Australian Olympic Committee Medical Commissions) and a Probity Officer to ensure appropriate transparency and propriety.

Main Findings:

The SIAB research consortium seeks to deter blood doping in sport by combining the expertise of sports scientists, haematologists and analytical chemists with the knowledge and experience of industry partners, to proactively develop tests for novel pharmaceutical products that may be prone to abuse by athletes. One important aspect of research focused upon haemoglobin-based oxygen carriers (HBOCs). The first requirement was to enter collaborative arrangements with each of the five pharmaceutical companies developing products, in order to obtain a sample for analytical chemists at the University of Montpellier and the Laboratoire Nationale Depistage du Dopage to develop analytical techniques to detect the different substances. Two manuscripts were drafted and subsequently accepted. These manuscripts describe an electrophoretic technique that was subsequently adopted by WADA for use as a screening method (Lasne et al. Clin Chem 50(2):410-5, 2004), and a high performance liquid chromatography technique that was adopted by WADA for use as a confirmation technique (Varlet-Marie et al. Clin Chem. 2004 Apr;50(4):723-31). Both methods were shown to be valid for each of the different HBOC products currently under development. A second aspect of research was the Haematological Passport. A preliminary goal was to overcome the practical limitations inherent in the different proprietary technologies used to measure reticulocytes by different instrument manufacturers. This leads inevitably to interinstrument bias which makes results collected on different instruments difficult to compare. We conceived and published an approach that allows reticulocyte percentage results derived on different platforms, at different times, and in different locations, to be compared directly. In keeping with SIAB’s goal to provide strategies compatible with sport federations, it was recognised that the disparate approaches utilised by some international sport federations to blood testing of their athletes was detrimental to the widespread acceptance of blood testing as a valid strategy to fight blood doping in sport. Efforts were undertaken to liaise with key federations, to better understand their rationale and stance, and to seek to find a compromise approach that was both inclusive and an effective deterrent against doping. Having addressed these practical concerns, we focussed upon the statistical interpretation of longitudinal data collected from international athletes in order to understand the degree of biological variation inherent in key blood parameters. We proposed a third-generation approach to blood testing to assist authorities to detect EPO doping, in particular focussing on the need to detect cynical athletes who manipulated EPO injections in order to escape sanction with the existing urine test. The research consortium also completed a pilot trial utilising a low-dose EPO regimen, which demonstrated that it is likely that an athlete could only be sanctioned for rHuEPO use if a urine test was conducted within 24 hours of the last injection if the athlete had resorted to ‘maintenance’ doses of the drug.

Code: 01B04MM

In order to improve their performance, some athletes utilize methods which optimize the physiological characteristics needed for their sport. The considerable availability of recombinant human erythropoietin (rEPO) has allowed the widespread use of this drug in aerobic sports to increase oxygen transfer capacity. Like endogenous EPO the recombinant hormone interacts with the precursor erythroid cells causing proliferation and differentation of these cells in mature erythrocytes (1). Although rEPO has banned by the medical commission of the International Olympic Committee, the anti-doping tests currently available cannot detect it with confidence. A direct detection of rEPO in urine has recently been suggested (2). However, while the plasma half-life of rEPO varies between 4 and 13 hours (3) its biological effects occur several days after treatments and thus the erythropoietic effect becomes evident when rEPO is no longer detectable in circulation. Moreover, EPO concentrations are not only very low but also vary considerably from one person to another and are influenced by environmental factors such as fatigue, stress and body hydration. In order to overcome these limitations and make the abuse of rEPO detectable , Gareau et a! (4) and subsequently Bressoll et a! (5) suggested that, besides the hematocrit value, the ratio between the concentration of the soluble transfenmn receptor (a marker of erythroid activity) and the concentration of serum ferritin (a measure of the iron stored in the body) should also be evaluated. In our previous study (7) we evaluated the concentration of the soluble tansferrin receptor (sTfr) and the concentration of serum ferritin (fr), to detect the abuse of rEPO as suggested by Gareau et a!. (4). In groups of athletes treated utilizing different protocols for rEPO, iron, fohic acid and vitamin B 12 administration, we demonstrated that the sTfr/fr ratio depends on the administration schedule and can vary upon iron supplementation. Our results confirm the conclusion of Gareau only when rEPO is administered at high doses and without iron supplementation. In contrast, when rEPO is administered at low doses and associated with an iron supplement, as is common in clinical practice to obtain a significant and lasting hematocrit increase (8), the sTfr/fr ratio cannot be considered a reliable marker of rEPO abuse (7). The main indirect method currently available for the detection of rEPO abuse (9) utilizes simultaneously multiple indirect hematohogical and biochemical markers. This method is potentially effective to identify users of rEPO but it doesn’t exclude the possibility of registering false positives. Moreover, in this method there are no appropriate internal standards for inter-assay calibration and quality control procedures. In our laboratory we evaluated the effects of different rIEPO administration protocols on the levels of ~3-globin mRNA, a selective marker of erythroid activity expressed during erythropoiesis stimulation, and Tfr mRNA, detected by RT-PCR. We found that the amounts of j3-ghobin and Tfr mRNAs in whole blood significantly increase in all treatments investigated. Prompted by these results, we developed a mathematical equation that takes into account hematological, biochemical and molecular values that changed most significantly during treatment. The values considered were hematocrit (Ht), reticulocyte count (Ret), sTfr, Tfr mRNA and f3-globin mRNA and allowed the detection of rEPQ abuse regardless of the administration schedule and iron supplement. in our multiparametric equation the values that play a critical role are f3-ghobin and Tfr mRNAs. For J3-globin a quantitative competitive RT-PCR assay was developed; in fact accurate quantitation of nucleic acids by RT-PCR needs a valid internal standard and an adequate mathematical model for data analysis. To address these points we first generated an internal standard, referred to as the “competitor”, with a sequence very similar to that of the target amplified fragment, natural ~3-globin cDNA, but with a different size due to a 29-bp deletion. We also evaluated that the competitor and natural target f3-globin cDNA have a similar amplification efficiency, that is a crucial point for an adequate quantitative competitive RT-PCR assay. The present proposal aims at: a. validating the method developed in a great number of athletes; these goals will be reached by means of further population studies. In particular the selected biological markers will be evaluated in non-pro athletes with regard to the sport practiced, gender, race, intake of iron, vitamins and other supplements; b. possibly reducing the number of determinations to be performed on blood samples in order to simplify the procedure, reduce the cost and time required, and provide definitive conclusions without decreasing statistical significance; c. generating an internal standard for Tfr mRNA in order to assure accurate quantitation by competitive RT-PCR and inter-laboratory calibration, as just performed for f3-ghobin; d. automating as much as possible the molecular procedures employed in order to reduce the time required; e. identifying blood denaturing solutions that allow stability and delivery of RNAs at room temperature; f developing computer softwares allowing a rapid evaluation of the results obtained and thus a rapid detection of rEPO abuse. Finally, the acceptability of this procedure for athletes will be favoured by the very small amount of blood needed to perform all determinations. We expect to validate a procedure able to detect rEPO abuse with a probability higher than 0.999999 that can be applied in different laboratories around the world.

Main Findings:

The considerable availability of substance such us EPO has allowed the widespread use of this drug in aerobic sports to increase oxygen transfer capacity. Like endogenous EPO the recombinant hormone interacts with the precursor erythroid cells causing proliferation and differentiation of these cells in mature erythrocytes. Although rEPO has been banned by the medical commission of the International Olympic Committee, the anti-doping tests currently used cannot detect it with confidence because of the short half-life of rEPO in plasma (4 and 13 hours), while its biological effects occur several days after treatments. We have previously developed a new indirect method based on the use of a multiparametric formula which utilizes simultaneously multiple hematological, biochemical and molecular markers changing significantly after rEPO administration. The values considered are hematocrit (Ht), reticulocyte count (Ret), soluble Transferrin receptor (sTfr-R) and β-globin mRNA (Magnani et al., Identification of blood erythroid markers useful in revealing erythropoietin abuse in athletes, Blood Cells Mol Dis. 2001 27:559-71. In our formula the beta-globin mRNA parameter plays a very important role since it is heavily influenced by Epo administration. During these two years, we have optimized a procedure to collect and store the blood samples in order to assure beta-globin mRNA stability for a long term before analyses. Moreover, standardized conditions were established for determination of beta-globin mRNA in a blood sample. Subsequently the method was validated in non pro athletes receiving different forms of recombinant-Epo (Eprex® and Aranesp®) and with different protocols. Based on our previous experience, the amount of beta-globin mRNA was considered together with several other parameters including the hematocrit, reticulocyte percentage and plasma soluble transferrin receptor. These parameters were included in a multiparametric formula that provides a value for the detection for Epo abuse. The results obtained suggest that the method we have developed can be conveniently used in a large interval of days, but is also dependent upon Epo administration regime, sex and Epo molecular form.

Code: 01B01RK 

Blood substitutes based on stabilised or polymerised haemoglobins are already approved for use in animals eg Oxyglobin is approved for use in dogs in the USA. Due to increasing demand for blood during surgical procedures and the perceived risk from blood borne diseases there is demand for such products to be made available for human use. At least two companies Biopure and Hemosol have products in stage three clinical trials. The haemoglobin based blood substitutes have several advantages over whole blood which makes their use by athletes highly probable. At present athletes who seek to increase their performance in endurance sports can do so by blood transfusions or by the injection of recombinant EPO. Both procedures increase the red blood cell concentration in the blood but with blood transfusions there is always the risk of mismatching unless homologous blood is used. Also blood has a relatively short storage life and must be kept refrigerated. With EPO there are no particular health risks but the rate of increase of red blood cells is quite slow requiring a doping regime over several weeks prior to an event. Also since the Sydney 2000 Olympic Games there is a risk of detection by the combined blood and urine test (Parisotto et al, 2001 and Lasne and de Ceaurriz, 2000). The haemoglobin based blood substitutes at present have none of the disadvantages of either blood or EPO. They have shelf lives of up to two years at room temperature, their effect on increasing the oxygen carrying capacity is almost immediate, and there is no risk of mismatching. Thus unless detection methods are developed and put in place soon then it is likely that haemoglobin based blood substitutes will be keenly sought after by athletes who are willing to go beyond ethical means to improve performance (Birkeland and Hemmersbach, 1999). Three sources of haemoglobin have been considered for use in HBOCs namely human haemoglobin, bovine haemoglobin, and recombinant haemoglobin (Chapman, 1998). Each has advantages and disadvantages but one problem that must be overcome with any type is the kidney toxicity problem caused by the rapid dissociation of cell-free haemoglobin into its dimer and monomer sub-units (Bunn, 1995). To overcome this the haemoglobin molecule must be stabilised and four approaches have been investigated (Chang, 1999). The four types of stabilised haemoglobin are intramolecular cross-linked haemoglobin, polymerised haemoglobin, conjugated haemoglobin, and recombinant haemoglobin. Problems with vasoconstriction leading to increased blood pressure (Alayash, 1999) have limited the development of the Intramolecular cross-linked haemoglobins, and products based on polymerised human or bovine haemoglobin are the furthest advanced in clinical trials.

Main Findings: 

Blood substitutes are oxygen-carrying therapeutics developed for use in operations and emergencies in place of donated blood. Increased oxygen-carrying capacity through the use of blood substitutes can help elite athletes to improve their performance and lengthen endurance capacity. Only one product, Hemopure™ (Biopure Corporation), a glutaraldehyde-polymerised bovine haemoglobin solution with a high molecular weight range (130-500 kDa), has limited approval for human use but there are others in phase III clinical trials. As blood substitutes become more readily available, it is essential that a detection method, for their abuse in sport, is available. The aim of this study was to investigate methods that could be used as screening procedures for polymerised haemoglobin in plasma and to identify tests that can unequivocally confirm their presence. Direct visual screening of plasma discolouration was the most appropriate screening method with detection limits lower than 1% haemoglobin-based oxygen carrier (HBOC) in plasma. Three methods have been shown to confirm the presence of exogenous haemoglobin in plasma samples: size-exclusion chromatography with photodiode array detection (SEC-PDA), native-polyacrylamide gel electrophoresis (native-PAGE) with luminol exposure and enzymatic digestion with detection by mass spectrometry. SEC separates the plasma proteins according to size with the largest molecules eluting first. Size exclusion separation coupled with the spectral analysis of the HBOCs compared to human and bovine haemoglobin standards uniquely identified the polymerised haemoglobin molecules by their retention time and spectral match to haemoglobin standards. The SEC-PDA method was able to determine the presence of HBOCs at a 1% spiking level in plasma. Native-PAGE separates the plasma proteins by both molecular weight and conformation. Haemoglobin and modified haemoglobin were identified on gels by their production of chemiluminesence after exposure to luminol and their migration point relative to each other. Hemopure™ and Oxyglobin™ were clearly detectable down to a 1% level in plasma samples. The LC/MS and LC/MS/MS analysis of tryptic digests of Hemopure™ and Oxyglobin™ identifies peaks which are from polymerised bovine haemoglobin and not from human haemoglobin origin. The methods have been validated by demonstrating their ability to detect and confirm the presence of Hemopure™ in incurred plasma samples. Furthermore, the methods which have been developed are not specific to Hemopure™ and should be capable of detecting and confirming the presence of any HBOC which is chemically cross-linked and has an average molecular weight significantly higher than human haemoglobin. All HBOCs which are in advanced stages of development meet these criteria.