Research projects

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.

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.