Projets de recherche

Code: 01A06CS 

Recently, we have demonstrated that it is possible to detect the administration of recombinant human growth hormone (hGH) based on a “differential immunoassay approach” (Wu et al., The Lancet, 353 (9156), 1999). The rationale for this approach is that the protein hormone hGH as secreted by the pituitary gland consists of several different isoforms (major hGH isoforms: 22,000 Dalton isoforms, minor isoforms: 20,000 Dalton, 12,000 Dalton and various other chemically modified isoforms and fragments), whereas recombinant growth hormone is a purified form of hUH with a distinct molecular weight of 22,000 Dalton only Screening a large panel of monoclonal antibodies raised against pituitary and recombinant hGH, respectively, we were able to identify specific antibodies and to set up two different immunoassays: One assay, which recognizes all different isoforms of hUH, and another assay, which preferentially recognizes 22,000 DaltonhUH monomers. The administration of recombinant hGH leads to a dramatic proportional increase in the 22,000 Dalton-hGH isoforms in circulation, whereas the other isoforms~ relative abundance is diminished. Measuring a blood sample by both assays, i.e. the permissive assay and the 22,000 Dalton monomer hGH specific assay, allows to calculate the relative abundance of the 22,000 Dalton-hUH isoform. Measuring a series of blinded samples, we were able to identify all those sera which had been drawn after administration of recombinant hUH, because the “22,000 Dalton-hUH specific assay result” in comparison to the “all hUH isoforms assay result” indicated a much higher proportion of 22,000 Dalton-hUH in these samples compared to the placebo group samples. Funded by the German “Bundesinstitut für Sportwissenschaften”, this test method for blood samples is currently being evaluated by a blinded analysis of a large series of samples obtained from the GH2000 double blind study. Proposal for an independent test method, possibly to be used as a confirmatory test The test method described above can be interpreted as a “proof of principle”, that the detection of the administration of recombinant hGH is possible by the analysis of the hUH isoforms composition in a sample. It is most likely that a more detailed analysis of these isoforms will lead to an improved detection method. To make such a method court proof, it is necessary to demonstrate the change in isoforms composition by a test independent from the above described method. This test should include the analysis of other isoforms than the 22,000 Dalton-hUH. Recent experiments in our laboratory indicate that especially the analysis of complexed hUH (dimers and oligomers) and of the 12,000 Dalton isoforms are promising approaches. The most appropriate technology for the identification of protein isoforms in biological fluids is the two-dimensional gel electrophoresis. This technique involves the separation of proteins by two independent physicochemical properties: Jsoelectric point and molecular weight. As we have developed a large panel of monoclonal antibodies against hUH, we are in the unique position to have the opportunity of combining the highly specific recognition of a protein by a specific antibody and the high resolution of proteins provided by gel electrophoresis. Screening many of our antibodies generated against growth hormone, we have been able to detect as little as 1 pie ogramm of recombinant hUH in one dimensional gel electrophoresis. Furthermore, we were able to demonstrate the presence of various hUH isoforms by this technique. This has demonstrated the potential of the combination of gel electrophoresis with specific antibodies against hUH. However, at present the development of a test method based on this technique is hindered by some specific difficulties: - The concentration of hUH in serum (and even more in urine) samples is rather low. Thus, an affinity concentration step is required before the less abundant isoforms of hUH can be analysed by this method. Alternatively, the affinity of the antibodies has to be improved to visualize small amounts of hUH isoforms. - For gel electrophoresis, proteins are diluted in a specific buffer and loaded on a SDS containing gel. This treatment destroys the original three-dimensional structure of the protein and thereby can abolish the recognition by specific antibodies. Therefore, antibodies suitable for analysis of unpretreated serum samples are not neccessarily suitable for gel electrophoresis. Specific antibodies have to be selected which are capable to recognize the “dcnaturalized” protein-hormone molecule on a gel. - Reproducible identification of protein isoforms by gel electrophoresis requires a high degree of standardization of the experimental conditions: Small changes in the quality of the gel, in the pH or in the voltage can lead to major variations in the protein spot pattern detected. This strict criteria of standardization and quality control can not be met by the more simple and variable in-house gels and gel running devices frequently used (and sufficiently precise) for research purposes. - Long term stability and exact documentation of the protein spot pattern derived from gel electrophoresis requires high end detection and documentation systems, especially when a very small amount of protein — as it is the case for hUH isoforms — has to be detected.

Main Findings: 

In this investigation, we have shown that all the 4 immunoassays for GH doping detection have very lower cross-reaction with the most hGH-related homologous proteins or peptides, implicating that the assays are very specific and their performance will not be disturbed by such proteins or peptides in physiological concentration. According to our window-of-opportunity hGH-application study, growth hormone abuse can be detected up to 36 hours with this approach after a single injection of a dose normal for hGH replacement therapy. Doses applied in abuse for performance enhancement are expected to be higher than the doses applied in this study. As an indicator of hGH doping, the ratio calculated from result from the two paired differential immunoassays changes so dramatically after hGH injection that a cut-off can be selected easily to ensure that no false positive result will occur. For the formal establishment of nominative data with regard to the ratios, a study of sera from a large number of hGH-untreated subjects will be performed and analyzed.

Code: 01C07EA

Recently, a sizeable number of urine samples analysed by official laboratories tested positive (>2ngIml) for the nandrolone metabolites 1 9- nortestosterone (19-NT), 1 9-norandrosterone (19-NA) and 19- noretiocholanolone (19-NE) (1-6). The large number of positive tests in such a short period of time, combined with the fact that some sports involved had not previously been associated with the abuse of anabolic-androgenic steroids (e.g. judo, handball, football and figure skating) have triggered investigations into the possible endogenous production of these metabolites in the adult male (1,3). The JOC and other bodies have concluded that more studies are necessary to fully elucidate the metabolic fate of anabolicandrogenic steroids (AAS) for the parent hormone, its precursors and metabolites. Nandrolone is an AAS that acts in a manner similar to testosterone on many reproductive and non-reproductive target tissues (7). It contributes to the development of male secondary sex characteristics (androgenic) and muscle mass and strength (anabolic). Because most AAS are metabolised extensively, the parent steroids are only detected in the blood for a short period of time following administration. Therefore, detection of the metabolites is of crucial importance in determining over what time period the abuse has occurred (6). The issue of detection is further confused by studies which have indicated nandrolone and its metabolites are produced naturally in men (8). in particular, following intense exercise, there is a relative increase in concentration of nandrolone metabolites in urine (1).

Moreover, the consumption of nutritional supplements containing 19-NA, a precursor of nandrolone which is available over the counter in the USA as a nutritional “supplement” for the enhancement of physical performance (9), produced levels of 19-NA and 19-NE in urine similar to those shown after illicit nandrolone administration and could be detected 7-10 days after a 50 mg single oral dose (2). It has also been argued that too little is known about possible endogenous sources of nandrolone for the testing laboratories to be confident about setting their threshold and that the studies already undertaken have recruited too few subjects.

The methods of measurement for most of these steroids are currently cumbersome, time-consuming and require highly trained personnel. Therefore, our proposal has two strands. In the first instance we propose to develop ELISA-based methods (Enzyme Linked Immuno-Sorbant Assay) for testosterone, nandrolone and some of their rnetabolites to allow routine and rapid screening of urine samples taken from athletes. In conjunction, an HPLC anabolic-androgenic steroids profile will be established for use as a reference for the above immunoassay methods. Secondly, we intend to establish normal ranges for endogenous testosterone, testosterone precursors and metabolites in urine in a number of disparate populations. Development of an HPLCprofilefor anabolic-androgenic steroids: We will optimise the existing HIPLC system -Waters, Cheshire, 13K- in the Department of Dietetics, Nutrition and Biological Sciences, Queen Margaret University College, so that it can detect and identify various AAS. In order to develop reference criteria the system will first be calibrated against authentic androgenic and tritiated-labelled steroids (9).

Following that, urine samples can be diluted appropriately and extracted with dichloromethane. After concentration, the residues will be dissolved in the HPLC mobile phase and aliquots injected on the column (Allure column or C 18 column). The HPLC system will then be utilised to identify various AAS: testosterone, testosterone precursors, testosterone metabolites and other related substances. This procedure will allow us to develop a profile in which any unknown peaks can be identified and investigated. The results of steroid levels (e.g. testosterone and nandrolone) obtained by the HPLC system can then be correlated with established methods such as the GC-MS (10). Development of ELISA methods for the measurement of testosterone and nandrolone in urine samples: The measurement of testosterone (a clinically important steroid) and nandrolone, a potent and widely abused steroid which is detectable in urine samples, has to be quick, simple and routine. Therefore, we are going to develop ELISA (Enzyme Linked Immuno-Sorbant Assay) methods to ascertain the levels of these steroids, in urine, on a routine basis. This will enable us to screen large number of samples within a short time at relatively low cost and without the need for highly skilled personnel. The principle of the ELISA could be either a direct method

Main findings

Highly sensitive and specific Enzyme-Linked Immuno Sorbent Assays (ELISA) have been developed and applied to measure endogenous nandrolone, free testosterone and total testosterone in urine samples. Two clinical studies were performed on normal healthy volunteers who were known not to have taken anabolic steroids. The first one aimed to establish normal ranges for urinary nandrolone and testosterone levels in non-exercising subjects and those who routinely exercise for leisure purposes. The second study investigated the effect of a controlled single bout of exercise on urinary levels of endogenous nandrolone, free and total testosterone in female and male volunteers. An HPLC system that can separate some important anabolic androgenic steroids from glucocorticoids has been developed and established in our laboratories, and work is underway to analyse the urine samples collected from our volunteers under different conditions by this system. Eventually we could produce a correlation between the ELISA results obtained with or without the HPLC separation. Work is still in progress to estimate the low levels of endogenous nandrolone metabolites; 19-norandrosterone and 19- norethiocholanolone, testosterone and testosterone precursors in the urine samples taken from our volunteers in the 2 studies, by a GC-MS system. The results obtained will be compared to those of the immunoassay technique for endogenous nandrolone.

Code: 01C16CA

Extremely low levels of 19-norandrosterone can be naturally excreted in human urine. Sensitive methods being applied for the detection and identification of anabolic agents, the International Olympic Committee safely recommended in 1998, a threshold in males and females for reporting positive results. However, as it is the case with the other androgens, which could be endogenous in human, natural factors are systematically invoked to challenge the positive test results. The inadvertent intake of 1 9- nortestosterone by eating contaminated meat of animals treated with growth promoters or from animals in which I 9-nortestosterone has been suspected to be endogenously formed, is a recurrent argument. This project is aimed at applying the combination of GO/MS and GC/C/IRMS (isotope ratio mass spectrometry) to the detection, identification and quantification of 19-norsteroids in human urine samples collected following the ingestion of pork meat and offal. Complement of the existing GO/MS methods, the GO/O/IRMS permits the differentiation of the exogenous (from synthetic standards) or endogenous origin of urinary metabolites, by measuring the isotopic content of their carbon atoms. The 130 contents of 1 9-nortestosterone reported to be present naturally in boar offal and of 19- norandrosterone, expected to be excreted following its consumption, could be measured and compared to those of the other urinary reference steroids. There are reports from the laboratories involved in the analysis of drug residues in animal tissues, of the presence of endogenous l7cL- or 17pnortestosterone, in a few animal species. One paper described last year the excretion of 19-norandrosterone in the urine of male volunteers having eaten offal and meat of an uncastrated pig. This study should provide the means to estimate and document objectively, the link between the detection of 19-norandrosterone excreted in human urine and the consumption of meat from animals in which 1 9- nortestosterone has been reported to be endogenous. The importance of this study goes beyond the scope of athletic drug testing. If androgens such as 19-nortestosterone are present in edible tissues of animals, in sufficient amount to detect its metabolites in T i.e. urine of humans having eaten that meat, it should be known and the risks for the health of the population properly evaluated.

Main Findings

Administration of 19-nortestosterone, a well known anabolic steroid, leads mainly to the excretion of 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone. Nortestosterone and the precursors, norandrostenedione and norandrostenediol are listed as prohibited substances by the International Olympic Committee and the presence of 19- norandrosterone in an amount greater than 2 ng/mL in athletes’ samples constitutes a doping offence. Excreted in very low amounts in human urine samples, endogenous 19- norandrosterone is not detected by the methods routinely employed in drug testing laboratories. A more sensitive instrumentation, larger volumes of urine and extensive sample clean-up are needed to detect, identify and quantify endogenous 19- norandrosterone. The physiological levels of 19-norandrosterone measured in samples collected from males and females are 0.6 ng/mL and 1 ng/mL, respectively. Recently, the results of the only truly controlled study involving athletes demonstrated that exercise does not influence the excretion of 19-norandrosterone. Again, very low levels ranging from undetectable to a maximum of 0.25 ng/mL were measured. This work aimed at studying the phase II metabolites originating physiologically during pregnancy or after intake of norsteroids in the three typical following conditions: 1) intake of a “dietary supplement” of 19-norandrost-4-en-3,17-dione; 2) ingestion of edible parts of non-castrated and castrated pig; 3) in several athlete’s samples found to be positive during routine doping controls. We have estimated by GC/HRMS the excreted levels of 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone when possible after selective hydrolysis of the glucuro and sulfoconjugated metabolites. The 13C content of the metabolites present in sufficient amounts after the ingestion of edible parts of non-castrated pig was measured by isotope ratio mass spectrometry. Our results indicate that when the norsteroids conjugates are properly measured, 19- norandrosterone and 19-noretiocholanolone glucuronides and sulfates are present in relative amounts which do not enable a distinction between either exogenous or endogenous origin. The use of isotope ratio mass spectrometry is the only way, when the norsteroids are present in a sufficient amount, to prove the origin of the metabolites.

Code: 01A09JJ

Growth hormone secretagogues (GHS) are small synthetic molecules that stimulate the release of growth hormone (GH) from the pituitary. In 1996 the GHS-receptor was cloned’, and in 1999 Kojima et al. discovered Ghrelin, the endogenous ligand of the GHS-receptor. Ghrelin is a peptide hormone that is synthesized in the stomach. It strongly stimulates the production and release of GH from the pituitary, with only minor effects on other pituitary hormones. The regulation and physiological significance of ghrelin is only gradually being revealed. It appears, that the hormone plays a major role in the regulation of the growth hormone axis and in the central regulation of appetite, but so far data on plasma levels of ghrelin have only been reported in the original paper by Kojima2. The International Olympic Committee bans administration of GH by athletes to enhance performance, but there is currently no approved method of detection. Measurement of serum GH itself is of limited use because recombinant GH and endogenous GH have identical amino acid sequences, and therefore markers of GH action are being investigated as potential tests for GH abuse. The rationale for the abuse of GH by athletes to enhance performance is that GH, at least in studies of GH-deficient adults, has been shown to reduce fat mass and to increase lean body mass, skeletal muscle mass, muscle force and aerobic performance. The abuse is limited by the high cost of recombinant GH and by the need of parenteral administration. However, in the near future relatively cheap growth hormone secretagogues for oral administration might become widely available, leading to a new type of indirect GH doping. The assessment of circulating ghrelin is difficult due to the fragility of the molecule as well as to a high level of protein binding, and so far no generally accepted assay has been developed. However, we have recently established collaboration with Dr. Kojima and his group on the measurements of ghrelin, and we have preliminary data indicating that circulating levels of ghrelin are strongly suppressed by GH. Thus measurements of plasma ghrelin may by a useful tool in the detection of both direct and indirect GH doping. Our department has a longstanding tradition in development and optimization of hormone assays, and from our involvement in the IOC sponsored GH2000 project we have thorough know-how in conducting exercise related clinical trials.

Main Findings

Ghrelin is the newly discovered endogenous ligand for the GH secretagogue receptor. Exogenous Ghrelin and its synthetic analogues are powerful stimulators of GH release from the pituitary gland. In addition ghrelin also stimulates appetite and food intake. We hypothesized that administration of exogenous GH would suppress endogenous ghrelin levels in serum and that this could be used as a means to detect GH abuse in the future. Before such a paradigm translates into a feasible test several issues need to be scrutinized. We have reported that ghrelin levels depend on energy balance and is increased by weight loss (1). Surprisingly, ghrelin levels are unaltered during an acute exercise bout despite a concomitant increase in GH (2). Administration of GH in healthy subjects induces a moderate decline in ghrelin concentrations (3). In addition, our studies have uncovered new and complex associations between energy status and ghrelin secretion (3, 4, 5, 7, 8). More recently, a large study in fit non-elite athletes have documented that exercise is associated with a subsequent suppression of ghrelin secretion and the study also showed that GH administration lowered ghrelin in serum (presented as an abstract at the European Congress of Endocrinology 2005). Our studies are ongoing (LIVE), but it remains a possible option that ghrelin measurements can be factored into a GH doping test. Our studies are extremely well publicized; the first paper (1) has been cited 110 times in less than 3 years (effective impact factor 37 !), and the most recent publication has been selected as a hot topic by the journal in its forthcoming issue (“What’s hot in European Journal of Endocrinology?”) (8).

Code: 01C18MG 

Administration of testosterone (T) or testosterone derivatives is one of the mainstays in human sports doping. Our knowledge about potential risks and the alleged advantage is limited as most doping is concealed. Very often the structure and dose of the used agents is unknown. In addition, doping tests are generally designed for qualitative identification of the agents with use of predetermined cut-off levels. There are many pitfalls with the current routines for interpretation of analytical results. As an example, the interindividual variations in turnover and metabolic patterns of endogenous and exogenous androgens are not considered in the antidoping laboratory tests, In addition, dose — sample collection interval is generally unknown which confers an uncertainty about the interpretation of the results. Genetic and constitutional factors are the major causes of variations in androgen metabolism and effects. Research carried out over the past decades has revealed important genetic differences in the capacity of enzymes in drug metabolism, particularly the members of the cytochrome P450 (CYP) family. Some of these enzymes, albeit relatively few, are inherited in a polymorphic way which may confer a 100 — 1000-fold interindividual metabolic variation in the population (1). The function of the majority of these enzymes is however governed by many genes polygenic inheritance. Nevertheless, their interindividual variation is often 10- 50-fold and it has been estimated that 50-60 % of the variation may be ascribed to genetic variation. As many androgens and androgen precursors are metabolised by the same or related enzyme members of the CYP family, there are reasons to believe that a similar genetic variability exists in the metabolism of anabolic androgenic doping agents. This would have conspicuous influence on turnover and excretion of such agents, which should be considered in the anti-doping test. The ethnic differences in the epimerisation of testosterone is only one example (2) which is probably based on genetic grounds. The mapping of the human genome (3, 4) has now paved the way to identify important sites of variation such as single nucleotide polymorphisms (SNPs) in genes of relevance for the synthesis, metabolism, and receptors of androgens (5). These genes encode several enzymes in the androgen and estrogen metabolism (see Appendix 2), as well as androgen and estrogen receptors. Many of the problems in anti-doping tests are associated with the identification of testosterone doping: assessment of the T/epitestosterone (TIE) ratio is probably affected by interindividual and ethnic genetic differences and variation, as well as interaction with concomitantly used agents or drugs. The various methods to check the validity of the TIE ratio includes repeated measurement of 17 OHprogesterone in serum after presumed doping. (6, 7). Another method is to determine ‘ 2C-T/’3C-T ratio which is changed in testosterone doping due to different relative concentrations of ‘ 2C and 13C in natural sterol precursors in the testosterone synthesis. Naturally elevated TIE ratios may also be due to inherently low epitestosterone concentration. In identification of testosterone doping one must consider the following: 1. The possibility of a naturally high T/E ratio must be excluded. 2.In males: monitoring of TIE ratios over three months is necessary in order to ascertain that the ratio is stable in absence of doping. 3.In females: low absolute TIE values may be influenced by ovulation. There is no information about the effect of contraceptives on the TIE ratio.

Main Findings: 

Our research program was aimed to study the large inter-individual variation in the disposition of exogenous and endogenous doping agents, such as anabolic androgenic steroids. There are reasons to believe that genetic variation is the single most important cause of variation in disposition of many androgenic compounds as it is for drugs. The conventional way to detect testosterone abuse is to measure the urinary ratio between testosterone glucuronide and epitestosterone glucuronide, (T/E). However, the large differences in this ratio observed within and between ethnic populations make it difficult to disclose all testosterone abusers. We have shown that the distribution of urinary testosterone excretion showed a distinct bimodal pattern, both in a Caucasian and a Korean population. However, the distribution into the low and high excretion mode differed markedly in that 74 % of Koreans, but only 7 % of the Caucasians belonged to the low excretion group. Our research group has identified the genetic explanation for this inter-individual and inter-ethnic discrepancy. Individuals avoid of the uridine diphospho (UDP) glucuronosyl transferase 2B17 (UGT2B17) gene all belong to the low excretion group, and hence also the low T/E-ratio group [1] [2]. We have continued our work to evaluate the sensitivity and specificity of the T/E test in different UGT2B17 genotype panels of 55 healthy volunteers. Our results show that the excretion of administered testosterone is highly dependent on the UGT2B17 genotype. In fact, 40 % of the subjects devoid of the UGT2B17 gene never reached the cut-off T/E ratio on any of the days investigated [3]. Our results strongly suggest that urine analysis should be combined with a genetic test of the UGT2B17 deletion polymorphism in order to refine and improve the testosterone doping test. Additionally we have identified polymorphisms in other androgen metabolising enzyme genes, e.g. in the aldo-keto reductase (AKR) 1C3 gene [4] and the P450 cytochrome (CYP) 7B1 gene [5]. An AKR1C3 single nucleotide polymorphism (SNP) was located in the promoter region and was 50 times more common in Caucasians compared to Oriental subjects. Another SNP, which conferred a Glu77Gly exchange in the protein was completely absent in the Oriental population, but occurred in 4.8 % of the Caucasians. Interestingly, subjects with this polymorphism had significantly lower levels of testosterone in serum and in urine. The results will pave the way towards personalised test strategies where the genetic factor will be considered in the assessment of the individual’s androgen excretion profile in the current test program. A better sensitivity and specificity of the test is mandatory for the fairness in sports and to the concerned individuals.

Code: 01A04GG 

With the increasing availability of recombinant human growth hormone (rhGH) and insulin- like growth factor I (IGF-I) these two peptides are becoming increasingly used to improve performance in strength and power events in track and field athletics and other sports. Both can now be purchased over the internet and are relatively inexpensive. GH is hyper- and JGFI hypo-glycaemic and instructions are provided on how these substances should be taken so that glucose homeostasis is not unduly disturbed, yet a marked growth effect may be
produced. As both are human peptides that are naturally upregulated during athletic training it is difficult to detect exogenous from endogenous, GH and JGF-I peptides. Methods have been developed that enable us to distinguish between the different isoforms and determine the ratios of the different IGF-I splice variants so that it is now possible to distinguish between the introduced and the endogenous peptides. At the present time the abuse includes injection as well as the ingestion of the peptides. The latter is a grey area as far as banned substances is concerned as colostrum which can be purchased from health stores is also known to have high levels of IGF-I that survive pasteurization and digestion in the gastrointestinal (GJ) system. It can do this because it is stabilized by binding to specific binding proteins. In the newborn, which derives many of its growth factors in this way, there is an uptake mechanism in the GI system and it is believed that may persist to some extent into adulthood. This would explain the apparent “beneficial” effects of ingesting colostrum or freeze dried IGF-I. Certainly it is known that the age-related loss of muscle mass is associated with a marked decrease in GH and IGF-I levels which decline by about 60% between the teenage years and old age (Rudman et al., 1981). An anticipated problem in future years is gene doping using the cDNA of different forms of IGF-I. Several laboratories in the world including Professor Goldspink’s lab are developing gene therapy methods for maintaining muscle mass in medical conditions such as muscular dystrophy, prolonged space flight and in ageing. The experiments on animals have been very successful (Skarli et al, 1998; Goldspink et al, 1999; MacCoil et al, 1998; MacCoil et al, 2000; BartonDavis et aT, 1998)) and once the engineered gene becomes more widely available it is predicted that there will be an increasing misuse. It is anticipate that this will happen within 5 to 10 years. Further work is required to characterise
these growth factors in relation to the development and maintenance of muscle mass and in relation to the misuse of these potent growth factors. Previous work on the GH/IGF-I system The growth hormone /insuiin growth factor 1 (GH/IGF-l) axis is the main regulator of tissue mass during early life and JGF 1 is one of the main growth factors that stimulates protein synthesis in muscle tissue. In adult muscles, increasing evidence suggests that IGF-l can act in an autocrine/paracrine fashion. As described below (Yang et al, 1997; Goldspink, 2000) showed that exercised muscle produces an autocrine splice variant of IGF-I that appears to be an important liilk between mechanical activity and the local cellular effects that result in muscle hypertrophy. Although the liver is usually thought of as the source of circulating IGFs, it has been shown that during exercise skeletal muscle not only produces much of the circulating IGF-l but the active musculature also utilises most of the JGF-l produced (Brahm et al, 1997). It has long been appreciated that there is local as well as systemic regulation of muscle growth. Using Differential Display (RT-PCR) Professor Goldspink’s group have identified and cloned the cDNA of two growth factors which are expressed by muscle when it is subjected to activity which are derived from the IGF-I gene by alternative splicing (Yang et al 1997). One (L.IGF-I) is very similar to the liver endocrine type of IGF-J. The other is a new growth factor that can only be detected in exercised/overloaded muscle and has been.

Main Findings: 

The group at the Royal Free and UCL medical have presented results in relation to hGH doping that show:
1. Two weeks after hGH administration of young untrained and trained adult male subjects results in markedly increased serum levels of IGF-I
2. Two weeks after hGH administration PIIIP levels remain elevated longer after cessation hGH administration longer than IGF-I.
3. A single bout of exercise intensive raining does not affect IGF-I expression in thigh muscles. Hence it cannot be claimed that the elevated levels in 1 and 2 are due to exercise.
4. In addition to validating the use of IGF-I and PIIIR as markers, two further approaches to detecting hGh and IGF-I abuse have been pursued.
A. These include a rapid screening method using “cell biosensors”. Serum from the athlete suspected of cheating following rapid screening is placed on muscle cells maintained in culture. Non physiological levels containing exogenous hGH result in the expression of MGF (human IGF-IEc) RNA which is a involved in the muscle hypertrophy process.
This is a normal physiological process but in excess and therefore should stand up in a Court of Law.
B. In addition a new approach using computerised mass spectrometry method involving neural net work analyses has been studied as an extension of the present project. Human biopsy samples have been studied to determine if those receiving hGH had a different pattern of molecules in the serum. This work is being carried out with the HFL in NewMarket that carries out much of the equine doping tests and Nottingham Trent University who have experts in mass spectrometry analysis. Good repeatability is being obtained with murine as well as human serum samples following hGH administration and this approach would be very suitable for routine screening of athletes as only very small serum samples are required and hundreds of these can be analyses within a few days as the process is robotically controlled. The work is refined to identifying the marker molecules that differ in hGh doping so that it might also act as a confirmatory test.

Code: 01C02DC

Over 98 percent of the carbon atoms in nature are ‘ 2C but about 1.1% are ‘ 3C, an isotope of carbon. If molecules of testosterone (T) or any other steroid are completely burned in a furnace at 800 0C, the molecules are combusted to carbon dioxide and water. The gas chromatography- combustion-isotope ratio mass spectrometer (GC-C-IRMS) instrument measures the ‘ 3C/’2C ratio of the combusted steroid, relative to a standard. The steroids are first extracted from urine, then separated in the GC, then combusted in the furnace, and finally the ~‘3C value is determined. There is a measurable difference in V3C values between pharmaceutical T (- 30 0/) manufactured from soy compounds and T biosynthesized by the human body (- 25 0/)~ This arises from differential ‘fixing’ of ‘in soy and related plants.’ Thus by determining the ö’3C value of urinary T one can determine its origin. This brief overview greatly oversimplifies an exceedingly complex method which is highly dependent on the underlying analytical methodology. If the extraction and chromatography steps are not perfectly executed to provide complete baseline separation of the steroids, the results will be unintelligible.2 Correct interpretation of the ~‘3C values requires an understanding of factors that influence them. This proposal focuses on clinical studies that probe the underlying physiology and pharmacology of the method. This study is concerned with endocrinology, steroid metabolism and pharmacokinetics. In the past seven years we have developed and optimized the IRMS method,25 determined ö’3C values on various urinary steroids, validated our screening method which determines ö’3C values on androsterone and etiocholanolone,4 and validated a confirmation method which determines ~‘3C values of three urinary diolsi’5 We have also presented evidence that the IRMS method is useful for distinguishing cases of naturally elevated TIE5 from T users. Others have confirmed6 and extended our studies8 (Page limitations prevent citing all the relevant publications.) Our results show, as expected, that T administration lowers ~‘3C values of urine T metabolites. All the previous studies delve into the method, its efficacy, and the ~‘3C values of healthy subjects, whereas the study proposed herein probes the underlying physiology, endocrinology, and pharmacology of the application of the IRMS method. It is reported 9,11 that some individuals do not experience an increase in TIE when T or related steroids are administered. This lack of response is often attributed to the Asian ethnic group, although we have found that some nonAsians also do not respond to T. We believe, but have yet to unequivocally prove,9 that the population we study (U.S. students) is bimodal with respect to baseline T/E and that this is one determinant of the response to T. The high mode (HM-T/E) group is characterized by relatively high baseline T and E excretion rates, whereas the opposite is the case for the low mode (LM-T/E) group.

Main Findings: 

We investigated the hypothesis that a population of males might exist that coul dope with testosterone and yet not be detected by the conventional TE that is used in doping control to detext testosterone users. Twelve healthy male subjects were stratified as high mode TE (HM) or low mode (LM) based on their resting baseline urine TE. All received a three-day continous intravenous infusion of testosterone. Serum and urine were collected for one day prior the trstosterone infusion, during the infusion, and for two days dollowing the infusion. The main hypotheses were that subjects with low TEs would be difficult to detect by the conventional TE screen used in doping conytol and that te carbon isotope ratio method would be a superior method for the conventional screening method. The hypothesis is proved to be correct. The excretion rates of all steroids increased during the first day of the infusion when the dose was equivalent to the normal production rate of testosterone. When the dose was increased to double and three times the normal production rate, the excretion rates increased still more. While the rates increased for both the HM and the LM groups, in the case of the HM subjects the increases occurred earlier and were higher. In fact 5 of the 6 LM subjects never archieved a TE greater than 4 while the TEs exceeded 4 in all the HM subjects. This study provides convincing evidence that all males do not metabolize testosterone in the same fashion and that at least two subgroups exist. Those with low TEs have low testosterone excretion ratees and un general are not able to increase the rate above 5μg/h. These subjects are, in effect, protected from detection by the conventional doping control TE method. Futher studies are needed to define these groups. The carbon isotope ratio method detects all testosterone users in both the HM and LM groups and is therefore superior to the conventional TE method. In addition the carbon isotope ratio method is known to detect the use of other doping agents that are difficult to detect by the conventional TE method.

Code: 01C08SW 

The aim of the project is to produce Certified Reference Materials (CRM5) of marker metabolites for testosterone, testosterone precursors and 1 9- norsteroids. These materials are needed by laboratories undertaking testing and research in this area. Their availability will contribute to improving the quality assurance procedures, the intercomparability and the assessment of the measurement uncertainty in such analyses, and to fundamental research into the qualitative and quantitative detection of abuse of these compounds. Project Output: Ten Certified Reference Materials, with appropriate analysis certificates, for laboratories testing for doping involving androstendione, DHEA, testosterone or 1 9-norsteroids. The aim will be the preparation of sufficient material for the anticipated worldwide requirements for five years. Background & Discussion: The availability of the appropriate certified reference materials (CRM5) is a fundamental necessity for harmonizing methodology for the detection of anabolic steroid abuse. It is a condition of accreditation to ISO Guide 17025 that testing laboratories use the appropriate reference materials. The availability of CRMs produced under a rigorous quality assurance protocol is thus a key requirement for improving the confidence in results obtained by worldwide programmes of doping analysis and detection. CRMs are necessary as analytical benchmarks for method development and validation, for verification of the application of standardised procedures, for establishing the intercomparability and traceability of measurements, and for assessment of the measurement uncertainty associated with test results. They can assist in the evaluation of the technical performance of a laboratory and in ensuring the comparability of and confidence in analytical measurements obtained in different locations.

Main Findings: 

The objective of the project is the preparation, certification and provision for WADA-accredited laboratories of pure substance reference materials for use in detecting doping with precursors ("prohormones") for testosterone and for 19- nor steroids. The followong activities, in line with the approved project plan, have been undertaken:                                        - consultation with IOC/WADA accredited doping laboratories                                                                                                                                                                    - Development of a target CRM  list for production.                                                                                                                                                                                - external tendering for candidate material preparation.                                                                                                                                                                             - Production by the CRM team of four candidate CRMs (19-norandrosterone sulfate, 19-noretiocholanolone sulfate, 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3, 17-dione).                                                                                                                                                                                                                                                       - Production throught sub-contracting four candidate CRMs (7-keto DHEA, 16β-hydroxy DHEA, 4β-hydroxy DHEA and 16α-hydroxyandrosterone.)                                         -Production throught sub-contracting five additional candidate CRMs (7α-hydroxy DHEA, 7β-DHEA, 6β-hydroxyandrosterone, 6β-hydroxyetiocholanolone, 3α-hydroxy-4-estren-17-one).                                                                                                                                                                                                                                     - Certification by NMI of thirteen materials (6β-hydroxyetiocholanolone,6β--hydroxyandrosterone, 7-keto DHEA, 7α-hydroxy DHEA, 7β-hydroxy DHEA, 4β-hydroxy DHEA, 16β-hydroxy DHEA, 16 α-hydroxy androsterone, 19- norandrosterone sulfate, 3α-hydroxy-5α-androst-1-ene-17-one, 5α-androst-1-en-3, 17-dione, 3α-hydroxy-4-estren-17-one and 19-noretiocholanolone sulfate) for issue to doping control laboratories as NMI CRMs                                                                                                                              - Review and approval of all thirteen CRMs by an external panel of experts.

Code: 01C06PH 

Problems related to the detection of 1 9-norsteroid misuse in analytical doping control have been of outstanding importance during the last decade. Both the endogenous production of I 9-nortestosterone (nandrolone) and its metabolites as well as the administration and metabolism of 1 9-nortestosterone precursors have been issues of an ongoing scientific discussion. In this respect our project should contribute with essential new and supplementary information on the establishment of normal urinary levels as well as the mechanism of endogenous production. Investigations of 19-norsteroid metabolites by means of IRMS should contribute to methodological evaluation of IRMS and interpretation of metabolic effects. Based on studies on reference steroids, interfering analytical parameters will be considered and standardisation will be proposed. This will permit the investigation of influences of metabolic steps on carbon isotope ratios. Following up previous publications1 ,2 and recommendations3 we would like to investigate further the excretion of nandrolone metabolites as norandrosterone and noretiocholanolone from individuals who have not ingested any nandrolone or nandrolone precursor. The reporting threshold of 2 ng/ml and 5 ng/ml for the concentration of norandrosterone in urine of male and female athletes, respectively, has been challenged in many doping cases. Although no clear evidence has yet been shown that the excretion of norandrosterone may occur in higher concentrations, it would strenghten the fight against doping to extend the scientific data in this respect. Our investigations shall therefore include: a) the monitoring of norandrosterone levels in males (at least 100 persons) not having ingested any nandrolone or its precursors. b) the influence of physical exercise on the excretion of nandrolone metabolites. c) the influence of alcohol consumption on the excretion of nandrolone metabolites. In animals the natural production of 19-norsteroids is well documented and their synthesis related to the aromatisation of testosterone/androstenedione to estradiol/estrone4’ 5. Additionally to the observation of 1 9-norsteroids in females during pregnancy 6,7 metabolites of nandrolone can also be detected in small amounts during the menstrual cycle8. The concentrations follow the blood levels of estrogen, supporting that both estrogens and nandrolone are synthesised in the maturing follicle9. Because estradiol is mainly synthesised in the dominant follicle’s granulosa cells during the time period close to the ovulation, we choose granulosa cells from in vitro fertilizing experiments as model to investigate the mechanism of endogenous nandrolone production. The project intends to study a) the nandrolone excretion of human granulosa cells, b) the aromatisation of different amounts of testosterone in this aromatase rich medium with respect to nandrolone or norandrostenedione synthesis, c) aromatase independent production of nandrolone, d) the influence of aromatase inhibitors on these reactions, e) nandrolone as possible substrate for in vitro aromatisation.

Main Findings: 

The contractors from the WADA-accredited laboratories in Kreischa/Germany and Oslo/Norway account in this report for their results of the WADA supported project ”Studies related to the metabolism of endogenously produced nandrolone and exogenously administered nandrolone precursors”. The results are laid down in 9 presentations/publications. As their main outcome it was shown that: 1. In very few cases certain matrix and storage conditions in urine could lead to a formation of low amounts of 19-norsteroids from androsterone and etiocholanolone. A WADA Technical Note already describes the necessary procedures to take into account this phenomenon. 2. The natural production of nandrolone metabolites can be explained as a side reaction during the aromatisation of testosterone. This explains plausible the occurrence of norandrosterone of natural origin in females during pregnancy and mid-cyclic, while the production in males is considerably less. The threshold for norandrosterone should reflect these circumstances. 3. Investigations of the glucuronide and sulphate fraction of nandrolone metabolites of natural origin and after administration of norandrostenedione show that the distribution between the glucuronide fraction and the sulphate fraction of norandrosterone and noretiocholanolone can not be used as a factual evidence to distinguish between endogenous production and application. The results also show that excretion in the free fraction is of insignificant value. 4. After oral and transdermal administration of the nandrolone precursor 4- estrene-3,17-dione no significant discrimination effect could be observed in relation to a possible influence of the biotransformation reactions on the carbon isotope ratio values of the terminal metabolites norandrosterone and noretiocholanolone.

Code: 01B06JP 

The abuse of recombinant hormones, as erythropoietin (EPO), in the world of sport is of special relevance. For long time, the absence of a methodology able to differentiate the recombinant pharmaceutical hormone (rhEPO) from its natural engonenic counterpart neither in blood nor in urine has created the suspicion of its widespread use in some sports. Recently, two methods with completely different approaches have been proposed. - The first one, using blood, is based on monitoring some ‘indirect haematological parameters” affected by the administration of EPO (soluble transferrin receptors, hematocrit, reticulocite, etc.). It is an indirect method that allows suspecting the misuse of EPO, but that needs a confirmation procedure to draw a definitive case. - The second one, using urine is based on the ability of isoelectric focusing (IEF) in gel to separate between the different glycoforms of the protein based on their charge. Ultimately, the method uses a chemiluminescent detection system that allows visualising the spots corresponding to the different separated glycoforms. Up to now, however, there is no definitive confirmation showing structural differences in the more basic isoforms characteristic of the rhEPO. The aim of the present study is to identify the precise carbohydrate structure that is unique for the recombinant hormone (rhEPO) so being able to, in an absolute way, differentiate between endogenous and exogenous EPO. The study will be based, primarily on the more promising glycoforms already separated by the existing IEF method to do: - Analysis of the N-glycans using HPLC and fluorescent detection. - Analysis of the peptide fingerprint using last generation mass spectrometric techniques. (MALDI-TOF, nanoLC-ESI-Q-TOF). Once the differences have been identified and characterized, research will be conducted for the use of those techniques on the direct urine analysis making the whole method applicable for high throughput analysis, as required by incompetition doping control.

Main Findings: 

The project “Unequivocal confirmation of recombinant erythropoietin (rhEPO) in human urine through structural evidences of specific glycosylation” (acronym:RHEPOSE) had as its central objective the structural elucidation of the glycans present in rEPO and NESP. The ultimate goal was obtaining the information that will lead to the establishment of the differences between those recombinantly produced materials and the endogenous urinary EPO. We developed and applied all necessary tools for the structural analysis of the Nglycans present in each single IEF band and characterise the microheterogeneity of rEPO and NESP (E. Llop et al. Proteomics, 2007; 7: 4278-4291). The results showed that EPO alfa and beta as well as NESP, although very heterogeneous, have been purified to contain a great proportion of tri and tetraantenary structures most of them fully sialylated. No charges other than sialic acids were found in the carbohydrates of those recombinant species while there were present in endogenous urinary EPO. Some enzymatic activities potentially present in urine would justify the behaviour described for EPO in some samples. The addition of inhibitors or competitive substrates of such enzymes was shown to be a potential solution to those situations (V. Belalcazar et al. Electrophoresis, 2006; 27(22): 4387- 4395). The presence of N-glycolyl-neuraminic acid, a sialic acid not produced in humans was found in NESP, and confirmed in rEPO (BRP standard), in both cases in amounts of ca. 1% of the total sialic acid content. The presence of such monosaccharide in EPO may be used as an unequivocal proof of its exogenous origin. The lack of a pure urinary EPO standard prevented us from achieving the same structural analysis with the endogenous protein. An appropriate purification procedure is still being developed, although the trace amounts of EPO in urine and the need to isolate its glycans from other glycoproteins, make it particularly challenging. In a complementary approach, polyclonal antibodies were developed using short peptides containing the differing aminoacid sequences of rEPO and NESP. Those antibodies were able to differentiate between both species, although the sensitivity achieved prevented its use in routine analysis (E. Gimenez et al. Anal Bioanal Chem 2007; 388: 1531-1538)