Projets de recherche

Code: 03A04RK

Erythropoietin (EPO) improves the oxygen-carrying capacity of blood and the recombinant product has been used by endurance athletes to enhance performance. There is also a strong suspicion that recombinant EPO has also been used to dope racehorses. One of the major requirements of a test for detecting low concentrations of banned substances in blood or urine samples is the ability of the testing procedure to detect the analyte in the presence of many other naturally occurring substances. This can be achieved by a selective extraction of the analyte of interest from the sample matrix or by a selective detection procedure. The current method for detecting recombinant EPO in urine uses a non specific concentration and extraction procedure followed by gel electrophoresis using a highly specific double blotting procedure to detect EPO in the complex of other proteins present. This procedure works well but is slow and very labor intensive. An alternative approach to detecting recombinant EPO would be to use a selective extraction procedure to remove the EPO from the interfering proteins. Immunoaffinity columns are already routinely used by sports drug testing laboratories to extract and purify banned substances such as anabolic steroids from urine. Whilst the initial preparation of the antibodies and evaluation of the columns is relatively difficult, such columns once prepared are robust, simple to use, and capable of regeneration so that they can be reused many times. This collaborative project between Charles Sturt University, the Australian Racing Forensic Laboratory and the Australian Sports Drug Testing Laboratory aims to produce antibodies that have a high binding capacity and selectivity for erythropoietin so that they can used in an immunoaffinity column to extract and concentrate recombinant EPO from urine samples. In this way the isoforms of EPO will be able to be separated by gel electrophoresis and detected by single blotting which should lead to a test for EPO that is not only much simpler to perform, and hence cheaper, but also more sensitive. The ability of the antibodies to selectively extract EPO should enable further developments in the detection of EPO to proceed including the extraction of EPO from serum and the detection of EPO by instrumental techniques such as mass spectrometry.

Main Findings: 

The main aims of this projct were to produce large quantities of polyclonal antibodies to erythropoietin (EPO) by immunising sheep, to extract these antibodies and use them to prepare immuniaffinity columns for selectively extracting EPO from urine, to investigate the composition of such purified extracts with  a viewto imporving the existing double blotting electrophoresis technique for detecting recombinant EPO in urine. The objectives have been achieved. Of the six sheep inoculated with recombinant human EPO all but one produced significant quantites of EPO antibodies in their sera. The antibodies from each sheep were characterised by epitope mapping and some significant differences were observed which may be usefuk in distinguishing various forms of EPO or fragments thereof. Immunoaffinity columns have been prepared using the serum from the sheep with the highest antibody titre. These columns have been found to extraxt EPO from urine typical recoveries ranging from 25 to 40%. The purified extracts have markedly lower levels of other urinary proteins. Gel electrophoresis has shown that the isofowm distribution of the purified EPO is unchanged by the column purification. This applies to both recombinant and urinary EPO although urinary EPO has only hald the recovery of recombinant EPO. the effect of this different recovery behaviour is to make the presence of recombinant EPO in a sample containing both recombinant and urinary EPO more obvious. The unchanged isoform distribution has been confirmed in urine samples which naturally have a greater proportion of more basic isoforms. The unchanged isoform distribution means that further development and simplification of the existing method for detection of doping with recombinant EPO can proceed using the immunoaffinity columns. Over 500 nL of serum is available from each sheep which has the potential to prepare up to 10,000 immunoaffinity columns.

Code: 03A10AC

"MAIIA, which combines a chromatographic separation, based on charge or bio-affinity, with a sensitive and specific immunoassay detection, all integrated in a small microporous sheet has recently been shown to be a sensitive, rapid, simple and inexpensive method for determination of protein isoforms in biological fluids (Lönnberg, M. (2002) Membrane-assisted immunoassay, separation and determination of protein isoforms, Acta Universitatis Upsaliensis, Comprehensive summaries of Uppsala dissertations from Faculty of Science and Technology 691).
Recent findings have indicated that protein isoforms play an important role in healthy and diseased organisms, and their determination should therefore be beneficial in clinical diagnosis (Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct, Glycobiology  3, 97).  In a joint research project the Dept. of Surface Biotechnology at Uppsala university and R&D at Pharmacia Diagnostics AB in Uppsala, Sweden, set out to develop a technique for the quantitative, measurement of protein isoforms, at low concentration, in complex media.  
The MAIIA technique, the result of this research effort, can be used to measure glyco-protein isoforms with different glycosylation-patterns based on their different charges (mainly the sialic acid content) or their varying oligosaccharide structures leading to different behaviour in binding to carbohydrate specific lectin ligands. The concept has been thoroughly demonstrated by measuring carbohydrate-deficient isoforms of transferrin, analytes that e.g. can be used to detect alcohol abuse, in serum (Lönnberg, M., Carlsson, J. (2000) Membrane assisted isoform immunoassay- a rapid method for the separation and determination of protein isoforms in an integrated immunoassay, J. Immunol. Meth. 246, 25 ; Stibler, H., Borg, S., Joustra, M., (1986) Micro anion-exchange chromatography of carbohydrate-deficient transferrin in serum in relation to alcohol consumption, Scand. J. Lab. Inves. 58, 55). 
Preliminary experiments have shown that the MAIIA technique also might be used in a sensitive, rapid and inexpensive test procedure for the discrimination of endogenous EPO from different recombinant EPO-forms in urine. Such a method should be an interesting alternative, as a doping test, to the presently used rather lengthy and expensive IEF-method (Lasne, F. et al (2002) Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones, Anal. Biochem. 311, 119). 
Before starting a project aiming towards a routine EPO doping test, we feel that further research activities on the different test steps (sample treatment, chromatography and immunoassay detection) as well as improvement of reagents and test components are needed.  We also need input about the legal and technical requirements related to the present routine doping testing and access to clinical knowledge and samples for testing. We have therefore established contact with Dr Garle at the doping control laboratory, Huddinge university hospital, Stockholm, Sweden, who has expressed an interest in participating as a coinvestigator to evaluate the MAIIA technique as an EPO doping test.  

Main Findings: 

The purpose of this eight-months pre-project was to evaluate a new technology as a basis for a rapid and easy-to-use erythropoietin (EPO) doping test. The proposed test-procedure will distinguish recombinant EPO and EPO analogues from the endogenous forms by utilizing the differences in their glycosylation structures. The urine specimen is applied on a small anti-EPO column, which rapidly and efficiently captures EPO in the urine. After a washing step, the bound EPO is released by lowering the pH in the eluent. The novel, chip-based technology, Membrane Assisted Isoform ImmunoAssay (MAIIA), is thereafter used for chromatographic ion-exchange separation of the EPO varieties in the eluate and their on-line detection by a sensitive and quantitative immunoassay in the same small and disposable chip. This procedure will take about one-two hours when processing about 15-20 samples. The obtained results look very promising. Urine specimens from patients (suffering from kidneyrelated deficiency of EPO) receiving recombinant EPO as well as urine specimens from healthy individuals were tested. The difference in results, between the tested patient-urines and the upper normal value (mean +2 stand. dev.), were roughly 3 times larger with the MAIIA technology (average for different test-settings) than for the results obtained with the present IEF EPO doping test, as tested by the Oslo doping laboratory. The implementation of the MAIIA EPO doping test would most significantly reduce the cost of required investments, hands-on time and total test time. The easy-to-use set-up will make it possible to perform the testing in laboratories of different degrees of sophistication as well as in the field. The technology should also have the potential to reveal new varieties of EPO and its analogues, as several interesting selective carbohydrate binders (lectins) can be introduced on the chip as a complement the charge-based separation.

Code: 02A01PS

Growth hormone (GH) is a naturally occurring hormone produced by the pituitary gland, which has strong growth-promoting properties regulating muscle and fat tissue and is widely accepted as being a major drug of abuse in sport. Its use is banned under the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) list of prohibited substances. The detection of doping with GH poses a formidable challenge, as it is identical to that which is produced naturally

in the body. The concentrations of GH vary in the blood during the course of the day as under normal circumstances, it is secreted by the pituitary gland in short bursts, each lasting a few minutes. Exercise and stress are powerful stimuli for GH secretion. The methods for detecting the abuse of androgenic anabolic steroids and related substances measured by mass spectroscopy are highly sophisticated but no such methods have been developed for testing for abuse with hormones such as GH and insulin-like growth factor-I (IGF-I). Immuno-assays and blood sampling are required for the detection of these substances and because the hormones are rapidly degraded in the body, urine analyses are not an option. Although it has been shown that it is possible to detect recombinant human growth hormone (rhGH) in blood, such tests have yet to be properly validated and are only able to detect rhGH within a short window of opportunity’ of <24 hours after the last injection of rhGH. Furthermore they will not detect any injection of pituitaryderived GH that is readily available. GH-2000 was a research project funded jointly by the European Union (EU) and the lOC. Its aim was to develop a method for the detection of GH abuse. It reported its findings to the EU and the IOC in January 1999. The GH-2000 project has shown that a test for GH using GH-dependent markers of GH action is feasible and scientifically valid.

Main findings

• It is believed that growth hormone (GH) is abused by athletes and other professional sports men and women.

• The use of GH is banned under the World Anti-Doping Agency (WADA) list of prohibited substances.

• The detection of abuse with GH by poses several challenges because injectable GH is identical to naturally produced GH.

• The GH-2000 study based at St Thomas’ Hospital, London and led by Professor Peter Sönksen showed that it is possible to detect GH abuse by measuring insulin like growth factor –I (IGF-I) and type 3 procollagen (P-III-P) whose concentrations increase in response to injections of GH and the application of discriminant function analysis.

• The results of GH-2000 were presented to a panel of international experts at an IOC-organised Workshop in Rome in April 1999. This review raised a number of key issues that needed addressing before any test could be implemented. The main issue concerned possible ethnic effects on the proposed markers, since the majority of volunteers in GH-2000 were white European.• The GH-2004 project, which is a 3-year project based at the University of Southampton, is funded by WADA and the US Anti-Doping Agency and aims to address these concerns.

• The first part of the project, which was funded by WADA, measured serum IGF-I and P-III-P in blood samples obtained from 242 male and 62 female elite athletes from different ethnic backgrounds within 2 hours of the end of competition at 9 international sporting events in 13 disciplines.

• The study showed that although there are small differences (<20%) in the concentrations of IGF-I and P-III-P between different ethnic groups, the majority of values for individuals of non-white European background lie within the normal range for the white European subjects.

• Application of the GH-2000 discriminant function analysis formulae to the GH-2004 subjects showed that no individual would have been falsely accused of doping.

• The significance of these results means that the test can be applied across different ethnicities. As it is extraordinarily difficult to define ethnicity, if there had been major differences between ethnic groups, it would have been impossible to apply the test.

Code: 02C05JS

Recombinant human growth hormone (rGH) has found wide-spread appliance in the sport community in the believe that it has ergogenic effect and the understanding that its (mis)use cannot be perceived by current analytical methodologies. Efforts to detect indirect evidences (biochemical markers) of rGH use are encouraging but never will be a definitive proof of the drug. Direct distinction of rGH from endogenously produced GH is difficult based only on the amino acid sequences, which appear to be identical. Additionally, the secretion of the endogenous hormone is a pulsatile event in nature, and that appears to retort differently depending on the kind of exercise performed. An absolute quantification as a single parameter for the detection of misuse is therefore unsustainable. A consequence of the administration of rGH is the down regulation of the endogenous production. As the endogenous hormone consists of an array of molecular-weight “isoforms”, due to splice-variants and other processes, the consumption of rGH, composed usually of only one of the possible “isoforms”, is reflected by variations in the ratio between the different isoforms in the body fluids. An accurate and reliable quantification of this ratio should disclose the origin of the detected GH, as has been suggested by other researchers. This process can be implemented on a chip surface for rapid and high throughput screening by means of surface plasmon resonance (SPR). Additionally, SPR allows unambiguous characterisation of the binding properties of antibodies, both commercially available and generated by the partners in the project, directed against the different known GH “isoforms” . Simultaneously, the proposal includes the scrupulous analysis of the different recombinant human GH preparations, as well as the natural hormone, by state-of-the-art mass spectrometric (MS) equipment, aims at disclosing possible structural differences arising from posttranslational modifications of the amino acids and may result in the production of novel antibodies with conclusive discriminatory capacity. Combinations of different antibodies will be screened, in a single analytical setting, to establish the optimal combination to quantify “isoform” ratios. ~ The project will deliver a test system available for rapid screening rGH administration at the laboratory. MS characterisation of GH “isoforms”. (Expected before summer 2004) Concurrently, generated peptide-fingerprints will serve as reference data for the positive identification of (r)GH. The MS characterisation will be implemented/ applied to samples derived from SPR analyses. The semi-hyphenation of both techniques provides an extremely powerful combination for the detection, quantification and unambiguous structural characterisation of human growth hormone, directly from complex biological matrices such as urine or blood. ~ The project will deliver the specifications for an “on site” screening device and the laboratory mass spectrometric confirmation, directly from the sensor chip. Finally, the approach will be validated using the large number of relevant samples, available from the partners and generated during a clinical study. Additionally, and given the interest in detecting the GH related peptide IGF-1 (potential doping either by recombinant product or gene transfer), preliminary data regarding the feasibility of the same approach for IGF-1 detection will be studied. > The project will deliver the validation of the methodology proposed and the exploration of its use for other doping growth factors.

Main Findings:

Growth hormone comprises a multifarious family of different splice variants, fulllength peptides, proteolytic fragments, homo-, di- and oligomers, and hormonebinding protein complexes. It has been shown that exogenous administration of recombinant 22 kDa GH alters the ratio of the normal isoform balance and that abuse could be detected possibly through isoform-ratio monitoring. The proposal focussed on the 22 and 20 kDa isoforms and originated from the idea that an isoform ratio quantification could be done through either ELISA or Surface Plasmon Resonance (SPR). Whereas the former is a well-established technique that provides information on an “end-point” situation, the latter technique enables both qualitative and quantitative monitoring of multiple antibody-antigen interactions in parallel, real time, under near-physiological conditions and without the need for labelling or a secondary antibody. With this approach several specific and non-specific anti-GH antibodies have been characterised in terms of thermodynamic constants such as association rate and dissociation rate constants, specificity and cross reactivity, surface properties, sensitivity, linear range and so forth. Knowledge of these aspects has proven of utmost importance when different isoforms of the same molecule are to be quantified employing distinct antibodies. As such, it was observed that the anti-20 kDa antibody has inferior surface properties with respect to the anti-22 kDa antibody and that this can be partially compensated for using more surface-bound antibody. Also, another non-specific antibody was shown to display different dissociation rate constants for both isoforms for which a quantification of isoforms based on such an antibody would render different ratios as a function of the number of washes or the total elapsed time between interaction and measurement. In short, the results have shown that it is indeed possible to perform isoform ratio measurements by means of SPR but the limitations in the instrumental design and the low capacity of the surface (approximately 15 ng or 100 fmol), compromises the sensibility and impedes an eminent implementation. Substantial efforts have also been invested, throughout the course of the project, in the sample processing (both for urine and plasma matrices) in order to bring concentrations within the range of the SPR technology as well as in the generation of well defined GH isoforms for the characterisation of the antibodies. Within this context a synthetic 5 kDa has been produced as well as proteolytic 5 kDa and 17 kDa isoforms (recombinant 20 kDa was already produced in Japan and recombinant 22 kDa material is available from different pharmaceutical companies). Furthermore, monoclonal antibodies towards the distinct isoforms have been generated and characterised by means of SPR, or are in the process of being produced. With all information a refined, highly sensitive, differential ELISA system, monitoring the 22 and 20 kDa isoforms, has been validated with limit of detection (LOD) of about 5 pg/ml for which plasma samples can be monitored directly and urine samples with minor processing. This ELISA has been screened with a large number of ordinary samples and is currently in the phase of beta testing employing samples generated through a clinical study with rhGH. Further developments lie in the marketing of the ELISA as well as the advanced transfer of this technology to a higher throughput platform such as Luminex using flowcytometry as basic methodology.

Code: 02B05WS

According to the Dietary Supplement Health and Education Act, prohormones like DHEA, androstenedione and norandrostenedione are sold as nutritional supplements in the USA. Manufacturers are claiming that these hormone precursors are converted to the active hormone after oral ingestion. Because these prohormones are currently marketed legally in the USA, athletes are using these compounds as an alternative to the illicit anabolic steroids to enhance muscle size and strength and to improve performance. Within a clinical study the conversion of 4-norandrostenedione (estr-4-ene-3,17- dione), 4- norandrostenediol (estr-4-ene-3B,1 7B-diol), and 5-norandrostenediol (estr-5-ene-31!,1 7B-diol) to nandrolone shall be found out. Until now there has not been any research dealing with this problem. The purpose of this investigation is to determine the exact plasma levels of physiological active nandrolone after administration of nandrolone prohormones. Six to eight male volunteers are planned to attend the study. Single oral doses of the following products, available on the US nutritional supplement market, will be administered to the subjects: 4-norandrostenedione 100 mg capsule, 4-norandrostenedione 25 mg lozenge, 4- norandrostenediol 100 mg capsule, 4-norandrostenediol 10 mg lozenge, 5-norandrostenediol 100 mg capsule. Pharmacokinetic data will be evaluated for plasma nandrolone and the respective prohormones. Plasma concentrations will be established by venous sampling at baseline and prescribed intervals: 10’, 20’, 30’, 45’, 60’, 90’, 2h, 3h, 4h, 6h, 8h, lOh and 24h from the t=0 point. Analyses will be done by means of gas chromatography/mass spectrometry (GC/MS).

Main Findings:

A new analytical method was developed and validated in order to determine plasma levels of 19-norsteroids, which are linked to the administration of the nandrolone precursors 4-norandrostenedione and 4-norandrostendiol. Compared to urine analysis, blood analysis enables the detection of the applied prohormones themselves and the intermediately formed nandrolone in addition to the main metabolites norandrosterone and noretiocholanolone. Within this project, excretion studies with commercially available nutritional supplements containing 4-norandrostenedione and 4-norandrostenediol, which are advertised as potent nandrolone precursors, were conducted. Both active components were administered to eight volunteers as capsule (100 mg) as well as in form of sublingual formulation (25 mg). Compared to application of capsules sublingual administration provided faster absorption and higher bioavailibility of these prohormones due to circumvention of the gastrointestinal passage and first pass effects of the liver. Our main attention was focused on the generation of unconjugated nandrolone after administration of 4-norandrostenedione and 4-norandrostendiol. These concentrations are an indication for a hormonal activity caused by ingestion of aforementioned nutritional supplements. In fact, relevant plasma levels of unconjugated nandrolone were determined. Within a period of two hours after administration of the different nandrolone precursors, unconjugated nandrolone was detected in plasma samples of most volunteers. After application of only a single dose of both tested formulations containing 4-norandrostenedione, concentrations of generated nandrolone were determined which allow physiological intervention. Administration of sublingual tablets containing 4-norandrostenediol seemed to be particularly suitable for the generation of effective plasma levels of unconjugated nandrolone. While detecting only a marginal concentration after application of 4-norandrostenediol capsules, administration of 4-norandrostenediole sublingual tablets resulted in amounts of unconjugated nandrolone comparable to acute therapeutic treatment with nandrolone pharmaceuticals in all volunteers. Even though these concentration levels were present for only 2-3 hours after administration of a single dose, hormonal action can not be ruled out, especially by considering multiple intake of the respective supplement. In conclusion, administration of 4-norandrostenedione and 4-norandrostendiol gives rise to pharmacologically relevant plasma concnetrations of unconjugated nandrolone. Considering the results of this study, distribution of these particular prohormones as nutritional supplements as done in the past has been irresponsible. In fact, these effective nandrolone 2 precursors should be classified as drugs. Since the commencement of the Anabolic Steroid Control Act of 2004 at the beginning of this year, steroid precursors are banned and classed as Schedule III drugs. Based on this, sales of this products have been officially prohibited, and thus, the development of the black market concerning steroid precursors should be watched carefully in the future.

Code: 02C03PP 

Purified Ghrelin is a 28-amino-acid peptide with a Ser at the third residue. Ghrelin is produced in submucosal cells of the stomach. Small amounts of ghrelin are also produced in the hypothalamic arcuate nucleus. Ghrelin plasma concentrations depend on race and body fat, meal, and time of the day. ghrelin stimulates GH release from pituitary cells in a dose-dependent manner. Intravenous injection of ghrelin induces potent GH release. Endogenous Ghrelin is secreted from the stomach to circulate in the bloodstream and act directly on the pituitary to release GH. Therefore, ghrelin will induce all the effects well known from OH. In skeletal muscle, IGFs are the only known mitogenes that stimulate both the proliferation and differentiation of skeletal muscle cells. It seems very likely that, like GH, ghrelin may also be effective in improving training adaptation processes, especially increasing muscle mass and shortening regeneration processes. Taken together, from the standpoint of anti-doping politics and research in sport, it is important to fully understand the physiological and endocrine effects of endogenous ghrelin in male and female athletes under different conditions of physical activities and eating patterns. It is furthermore important to understand the interactions between exogenous ghrelin, being intravenously injected, and the dependent endocrine parameters like GH and lGFs, in order to be able to differentiate between endogenous and exogenous ghrelin and the respective effects and thereby demonstrating misuse of the peptide. Up to now, no investigations on ghrelin in athletes have been published so far on ghrelin, since this peptide has only very recently be described. The study will be conducted of two steps. In step one we will determine spontaneous action of ghrelin, dependent hormones like GH and lOFs and binding proteins under different conditions in athletes. We will investigate female and male athletes of different sports (especially endurance and power) and different body composition, at rest, during and after exercise (competition and training) and under several nutritional states. We will measure plasma concentrations and will work on establishing a method for measurement of urinary ghrelin concentrations as well. In step two we will investigate effects of exogenous ghrelin administration on endogenous profiles of ghrelin, OH and related peptides. Again, we will investigate female and male athletes of different sports under the above mentioned conditions

Main Findings:

We included 44 endurance (E, n = 23) and power (P, n = 21) athletes of both gender (m: n = 24 , f: n = 20). All 24 subjects participated in the main part 1 of the study (analysis of physiologic profile of Ghrelin and corresponding parameters), 2 subjects additionally participated in part 2 (injection of exogenous Ghrelin). The main results are: 1. Total Ghrelin values significantly declined over time in both active groups (endurance and strength), while values remained nearly constant in the inactive groups, independent from gender. Furthermore, values remained constant without carbohydrate intake but declined significantly over time under carbohydrate loading. This trend was also independent from gender and from kind of activity. 2. Active Ghrelin values significantly declined over time under carbohydrate loading in the active but not in the inactive groups, which was independent from kind of sport or gender. The differences against the first value did not show any clear time effect, but showed significantly lower values in the strength versus the endurance groups, and in the female athletes under carbohydrate loading compared to the low-carbohydrate condition. 3. Exogenous Ghrelin injection led to extremely high increases in total and active Ghrelin plasma concentrations with a maximum 15 min after the injection. Baseline values of total Ghrelin were not yet reached 180 min after the injection, while active Ghrelin baseline concentration was reached already after about 90 min. 4. HGH values declined significantly over time in the female group but remained constant in the male athletes. This effect was independent from type of sport and from carbohydrate intake. 5. Exogenous Ghrelin injection led to high increases in HGH serum concentration (15 – 30 ng/ml over baseline) 60 min after the injection. Values declined to baseline after 120 min. This effect was independent from carbohydrate intake, but was more pronounced during run exercise. 6. IGF1 declined significantly over time in the active but not in the inactive groups. This effect was independent from gender, type of sport, and carbohydrate intake. 7. In spite of high exogenous Ghrelin injection-induced HGH increments, we could not find any effect on IGF1 concentration 60 – 180 min after Ghrelin injection. In the active group, IGF 1 declined over time after Ghrelin, while values remained constant
in the inactive situation. 8. Urinary total Ghrelin concentrations were measured 1.4 - 1.6 times higher after stabilizing with HCL or HCL plus PMSF as compared to untreated urine. 8 – 9 hrs overnight urinary total Ghrelin values were 1.3 – 1.6 time higher as compared to 3 hrs intervention-period daytime values. Neither carboloading, nor gender or physical activity did affect urinary total Ghrelin concentrations. 9. Urinary active Ghrelin concentrations were 20 – 30 times lower as compared to total Ghrelin concentrations. Urinary active Ghrelin concentrations were measured 1.1 - 1.4 times higher after stabilizing with HCL or HCL plus PMSF as compared to untreated urine. 8 – 9 hrs overnight urinary active Ghrelin values were not different from 3 hrs intervention-period daytime values. Neither carboloading, nor gender or physical activity did affect urinary active Ghrelin concentrations. 10. In spite of extremely high plasma total and active Ghrelin concentrations 60 min after exogenous Ghrlein injection, urinary values did not show any increase during the sampling period until 3 hrs after the injection. To summarize, Ghrelin seems to be mainly independent from acute or chronic physical activity. This makes it unlikely that Ghrelin plays a major role in any physiological anabolic adaptation processes according to training. As the effect of exogenous Ghrelin on serum Ghrelin and hGH concentration is very short lasting, it seems to be unlikely that this substance might play a major role as a doping agent. However, it could be demonstrated, that the effect on hGH secretion was very high, so that Ghrelin should stay on the list of forbidden substances.

Code: 02A06NP

As the use of protein drugs for enhancing athletic ability becomes more prevalent it is essential to have a general detection technology able to be used to detect the protein drugs available now and any new protein variants that will be used in the future. This proposal is for the development of such a generic protein analysis platform based on protein concentration procedures, gel electrophoresis separations and in the first instance, antibody detection. Specifically, methods for the improved detection of the endurance drug, erythropoietin in urine will be developed, as well as testing the feasability of a new detection process for human growth hormone in plasma. When proteins are produced for medical uses they almost invariably change their properties of isoelectric point (p1) and/or molecular mass (MW) or are metabolized differently from the native protein in the body. The pharmaceutical drug companies usually have no need to make proteins identical in structure and/ or composition to the native protein if the efficacy of the protein drug is not affected. Synthetic proteins are difficult and costly to “humanize” and are sometimes actually deliberately modified in the production process in order to affect their delivery and/or their time in the body. These characteristics can be exploited experimentally to enable the drug forms to be differentiated from the native human forms of the protein. Proteome Systems has a proven track record in proteomic analysis and in the development of technology to characterize proteins. Their scientists have a proven research track record in innovative methods for protein analysis, including protein separation, identification and characterization of their modifications. The company has developed and manufactured several instruments to automate the electrophoretic and mass spectrometric analysis and have produced informatics programs to track samples and data. They have also engineered diagnostic antibody/antigen point-of-care new technology that will facilitate the introduction of any developed antibody based drug screening test. Proteome Systems is thus well positioned to take on the task of solving the varied challenges of detecting the abuse of clinical proteins by athletes who hope that the similarity of these drugs to the human proteins will make their detection difficult. The close collaboration with the Australian Sports Drug Testing Laboratories and the Penang Doping Control Centre will enable athlete urine and blood samples that they have collected for other tests to be used for method development, as well as providing a highly experienced testing laboratory for validation of the assays developed. As the protocols and instrumentation become available they will be transferred to these laboratories for accreditation. It is expected that substantial improvements and provision of instrumentation and quality controlled consumables to enable a more reliable and automated EPO test will become available to IOC laboratories by the end of the grant.

Main Findings: 

In 2003, the Peltre –Thormann report commissioned by the World Anti-Doping Agency identified specific issues to address in order to improve the one-dimensional isoelectric focusing electrophoresis method currently being used for identifying drug doping with recombinant erythropoietin (rHuEPO). The normal human form (HuEPO) and the drug (recombinant) form of EPO (rHuEPO) have exactly the same protein component. The reason they can be differentiated in tests is that the number and type of sugar groups attached to the protein, which account for about 40 per cent of the mass of EPO, differ between the two proteins and change the charge and mass of the different forms. The First Year report described a new method for the detection of recombinant drug erythropoietin (EPO) in urine using two dimensional gel electrophoresis (2D Method). The new protocol covered changes to urine sample preparation, 2D electrophoretic separation of the endogenous from the exogenous protein isoforms, single blotting, Western detection and a software algorithm for analysis of the 2D image data. The 2DE method separates HuEPO and rHuEPO by both iso-electric point and molecular mass. This method was published in Clinical Chemica Acta Vol. 238 (2005) p.119-130, and was presented at the Manfred Donike workshop in Cologne in 2004. A favourable comparison of this method with that of the current testing protocol (1D Method) was carried out on spiked samples in conjunction with the WADA accredited doping control laboratory in Sydney, Australia (National Measurement Institute) and Penang, Malaysia (Doping Control Center), and was presented at the Manfred Donike workshop in Cologne in 2005 and published in the proceedings. The 2DE method presented dealt with most of the WADA recommendations for an improved EPO test and provides a sensitive and accurate detection of the EPO drug in urine. The Second Year of the project largely concentrated on optimising this EPO 2D method protocol for transfer to the anti-doping laboratories for validation and for comparison with the current IPG separation method (1D Method). We had identified major urinary proteins, and removed some but not all, non-specific binding by addition of an acidic wash of the blot in the revised protocol. An optimized 2DE method Standard Operating Procedure for EPO testing in urine was developed with the support of some WADA accredited laboratories. Figure left: separation of normal human EPO (HuEPO) from the recombinant drug form (rHuEPO) in urinary proteins separated by the 2D method HuEPO rHuEPO Conclusions: It is our conclusion that once fully validated in an anti-doping environment the 2D Method for EPO testing could become an attractive complementary test of the current 1D Method until such time as it becomes proven in reproducibility and reliability in accredited WADA laboratories. The 2D Method is still laboratory based and requires some skill level and time but likely offers improvements in separation of the isoforms from each other and from other urinary proteins which may react with either the primary and/or secondary antibodies, removes the need for double blotting, and has a horizontal as well as vertical separation of the drug form from the endogenous EPO protein. The two dimensional gel separation in the 2D Method also offers the advantage of being able to add a migration reference standard to the 2D gel electrophoresis for accurate measurement of the migration of the detected EPO, and the opportunity to use image analysis of this result for automated identification of the drug form. Preliminary studies were also carried out on the feasibility of using two dimensional electrophoresis as a method for the detection of human growth hormone (hGH). There was question as to whether the high turnover and variability of the endogenous hormone make the detection of the actual administered protein a desirable drug doping test method. In addition, the primary differences between the endogenous form and the exogenous drug appears to be low level, variable proteolysis as well as possible single amino acid substitutions, phosphorylation, oxidation and other modifications. These low abundance, small mass and pI differences are difficult to detect by the method of 2D PAGE so this approach is not recommended to pursue further for the development of a hGH drug detection protocol.

Code: 02B06LM 

The aim of the project is to produce solution and urine matrix Certified Reference Materials (CRM5) certified for the presence of 19-norandrosterone, the main metabolite of the anabolic steroid nandrolone, at the level of 2 nanograms/ millilitre (ng/ml). This is the level above which a doping violation has occurred for a male athlete, as specified in the current ICC Medical Commission prohibited substance list. The production and inclusion of CRMs of these types into routine testing procedures will serve the twin purposes of assisting laboratories in establishing the traceability of their measurement results and helping them to make more accurate estimation of the measurement uncertainty associated with their results. The matrix CRMs would specifically be useful for benchmarking the capabilities of laboratories and would also allow them to more readily detect and address bias in their analytical methods. Ultimately their production and use will result in greater confidence, particularly under potentially aggressive legal scrutiny, in these critical results. They would also assist research into new or improved methods for the qualitative and quantitative detection of doping with nandrolone.

Main Findings: 

The World Anti-Doping Agency (WADA) statistics show that nandrolone was the second most commonly abused steroid of those detected in 2004. A urine matrix CRM has thus been produced in conjunction with WADA for the major nandrolone metabolite, 19- norandrosterone. The material was prepared at the allowed cut-off level for 19- norandrosterone of 2 ng/ mL. The exact measurand for the CRM was defined to link in with the requirements of the WADA technical document TD2004 NA “ Reporting Norandrosterone Findings” and the measurand was thus defined as the total of the free and glucuronide forms of 19-norandrosterone. A freeze dried human urine, fortified with 19-norandrosterone glucuronide, was produced following ISO guides 34 and 35 and a high-accuracy isotope dilution mass spectrometry (IDMS) method was developed and used to certify the concentration of 19-norandrosterone in the reference material. Certification of the material included homogeneity testing of the 1,200 units produced and stability testing over the temperature ranges of –20 to 40°C. Results for 19-norandrosterone concentration from the within-bottle homogeneity testing of 30 units of the CRM, selected in a stratified random manner, had an RSD of 1.3%, indicating excellent agreement over the batch of 1,200 units. Stability testing of the material at its storage temperature of -20°C showed excellent stability over the 12-months of testing to date. The accelerated stability trial carried out at 4°C, 22°C and 40°C showed a change in level of the analyte only at the 40°C; at this elevated temperature the level had dropped by 25% after 12 months. Stability testing of the reconstituted urine, kept in its liquid form at 4°C for 4 weeks, showed no change in analyte level and therefore the freeze-dried material may be reconstituted and then refrigerated for later use. The rigorous application of a primary ratio method such as IDMS should ensure that the value assigned to the CRM will be traceable to the SI and have a very well-defined uncertainty. A high-accuracy exact-matching isotope dilution mass spectrometry (IDMS) method for 19-norandrosterone (NNA) in human urine was developed. The developed IDMS method was based on a published GC/ HRMS procedure [ 1] . However the various components of the method were specifically optimised for analysis of the specific urine matrix of the CRM which was being produced and certified. This included: • optimisation of the hydrolysis step and measurement of the hydrolysis efficiency • optimisation of clean-up of the hexane extract with HPLC fractionation employed • optimisation of GC/ HRMS conditions. In addition, the calibration standards used for this project were rigorously investigated and standards of both the free and glucuronide forms of the steroid were used and compared. A confirmatory LC/ MS/ MS method was also developed to monitor the level of the glucuronide. Exact-matching IDMS involves a one-point calibration procedure whereby the isotopicallylabelled d4 Sample Blend m/z Calibration Blend m/z Equal ratios in the two blends Equal intensities in the two blends NNA d4-NNA NNA d4-NNA 405 409 405 409 -19-norandrosterone internal standard is added at the very beginning of the process to both the sample and calibration standard solution to create two blends. The ratios of analyte to internal standard in each of the sample and calibration solution blends are matched to be equal and the instrumental intensities of all of the analytes are also matched. This technique minimises many of the systematic biases involved in high-accuracy MS measurements. The uncertainty of the assigned value was thoroughly assessed with all analytical biases investigated and factors covering sample homogeneity and stability incorporated. The overall expanded relative uncertainty at the 95% confidence level was estimated as 8%, which should meet the needs of the WADA-accredited user community. The certified level of 19 norandrosterone (as the sum of the free and glucuronide forms of the steroid) in the CRM is certified as 2.13 ± 0.17 ng/ g or 2.15 ± 0.17 ng/ mL as mass fraction or mass concentration units. 

Code: 02E04WS 

Finasteride is an inhibitor of 5-alpha reductase, the enzyme responsible for conversion of testosterone to dihydrotestosterone. It is administered orally in a dose of 5 mg daily for the treatment of benign prostatic hypertrophy. Since 1999 it is also admitted in several countries for the treatment of men with hair loss (androgenetic alopecia) and it seems to become a so called ,,life style drug”. The recommended dose for the treatment of hair loss is 1 mglday. Recent studies with finasteride have shown, that this substance can be misused as a potential masking agent. The application of finasteride may prevent the detection of misuse of anabolic-androgenic steroids like nandrolone, norandrostendione, norandrostenediols, dihydrotestosterone and testosterone. These preliminary results should be confirmed by more extensive studies with several volunteers. If the preliminary results can be confirmed, it should be discussed, if finasteride is added to the prohibited class of masking agents. The second aim of the study is to develop and validate a sensitive and specific method for the detection of finasteride misuse.

Main Findings: 

Finasteride is an inhibitor of 5-alpha reductase and used for the treatment of benign prostatic hypertrophy and androgenetic alopecia. Investigations with finsteride with only one volunteer have shown, that the use of finasteride complicates the detection of the misuse of several anabolic steroids in doping control. To confirm this result and to study the influence of finasteride on the urinary steroidprofile and on the metabolism of anabolic androgenic steroids excretion studies with single oral administrations of 5 mg and 1 mg finasteride were performed with 5 volunteers. Urine samples were collected before and till 8 days after the application and the profiles of endogenous urinary steroids were analysed by GC/MS. It could be shown, that finasteride led to obvious changes of several steroidprofile parameters. The excretion of 5-alpha-steroids like androsterone, 5α-androstane-3α, 17ß-diol, allo-tetrahydrocortisol, 11ß-hydroxy-androsterone, and dihydrotestosterone decreased, whereas the excretion of the 5ß-steroids increased or didn’t change. The results were obvious decreases of the ratios between epimeric 5α-and 5ß steroids like e.g. androsterone/ etiocholanolone, 5α-androstane-3α, 17ß-diol/5ß-androstane-3α, 17ß- diol and allo-tetrahydrocortisol/ tetrahydrocortisol. These changes could be detected for more than 8 days both with 5 mg and 1 mg finasteride. The suppression of the excretion of the 5-alpha-steroids showed the same extent for 5 mg and 1 mg finasteride, whereas the increase of the excretion of the 5ß-steroids was weaker with 1 mg finasteride compared to 5 mg finasteride. The ratio testosterone/ epitestosterone showed no changes after the application of finasteride and varied within the normal variation. Further excretion studies with 5 mg finasteride were performed with volunteers, who administered additionally 20 µg norandrostendione. It could be shown that under the influence of finasteride the excretion of the 5α-steroid norandrosterone, the main metabolite of norandrostendione, is suppressed to 20-40% of values without finasteride, whereas the excretion of the 5ß-metabolite noretiocholanolone increased under the influence of finasteride up to 400% of the values without finasteride. Based on these results the ratios of norandrosterone/noretiocholanolone changed from values between 1.7-8.4 to values between 0.3-0.7. The results of the present study show, that the use of finasteride may cause serious problems for the interpretation of steroidprofiles which play an important role in doping control (detection of the misuse of endogenous steroids, longitudinal studies, individualisation of samples, etc.). Furthermore finasteride can complicate or even prevent the detection of 19-norsteroids, which is mainly based on the detection of the their 5-alpha metabolite norandrosterone. These results show that finasteride can be misused as masking agent. Within this research project a method for the detection of the use of finasteride was developed. As main urinary metabolite a carboxy metabolite of finasteride was identified by LC/MS. This metabolite could be included in an existing screening procedure for doping substances. After a single oral application of 5 mg of finasteride the carboxy metabolite could be detected for 90 hours.

Code: 02A08GT

To establish a facility within ASDTL which is capable of meeting the increasing demands for confirmation of peptide hormones and other large biologically active molecules using liquid chromatography mass spectrometry (LC/MS). There is an urgent need to develop the skill and resource base needed to carry out the mass spectral analysis of bio-molecules used for doping. Whilst at present the use of immunoassays and other immuno-reactive techniques is accepted as proof of doping this is unlikely to continue once it has been demonstrated that mass spectral confirmation is possible. Recently published work has shown that it is now possible to detect and identify proteins in biological matrices at the extremely low concentrations found naturally. At present the confirmation of the presence of peptide hormones and other large bioactive molecules is done using techniques that rely on specific antibody reactions to large molecules. Unfortunately, such reactions are not completely specific and the current IOCIWADA anti-doping code includes the need for two separate antibodies to confirm doping with HCG. All drugs that are detected, other than peptide hormones, must be confirmed by the use of mass spectrometry using gas chromatography mass spectrometry. The reason the peptide hormones were excluded from this requirement was that it was not practicable to attempt mass spectrometric analysis of large bio-molecules both because of their high molecular weight and because of the very low concentrations found in blood and urine. However with the ever increasing demands of proteomics research the use and capabilities of mass spectrometry using LC/MS for the analysis of bio-molecules has increased dramatically in the last few years and will continue to do so. The aim is to research processes to allow:

• A validated method to confirm cases of HCG doping using LC/MS.

• A LC/MS method to identify and confirm the presence of haemoglobin based blood substitutes.

• A mass spectral method to distinguish between recombinant EPO and urinary EPO • Mass spectral methods to detect and identify other significant biologically active molecules such as NESP, EPO mimetics, growth hormone isomers, IGF1 etc. at the low levels found in blood and urine samples.

Main Findings

In the first year of this three year project we completed a validated method for confirming the presence of haemoglobin based oxygen carriers in serum and began to investigate methods that could be used for confirmation of the abuse of peptide hormones. The first has been achieved and a paper describing the work has been published (Goebel, 2005). The latter activity was focused on two peptide hormones, human chorionic gonadotropin (hCG) and erythropoietin (EPO). Mass spectrometric analysis of peptides and proteins provides structural information which can uniquely identify the compound being examined. WADA rules mass spectrometry as the definitive method for confirmation except in the case of peptide hormones. It has been seen with the development of new techniques such as carbon isotope ratio mass spectrometry that, once a mass spectral technique can be shown to replace or supplement a less rigorous procedure, its use becomes essential both to confirm guilt and to demonstrate innocence. The same will apply to the peptide hormones in the near future and positive cases will require mass spectral confirmation once it is possible to do so. It was intended that in the second stage of this project that we would establish capillary chromatography coupled with high resolution mass spectrometry (HRMS) as a routine procedure for the analysis of peptide digests and proteins. We have completed the development and validation of a precise MS method for the reliable identification and quantitation of hCG in urine at physiological levels. In addition we have extended this procedure to assist in developing a method with the potential to distinguish between recombinant and urinary hCG. We have developed of methods for the concentration and purification of hormones such as hCG, EPO, insulins and IGF-I from urine and serum to assist in their subsequent analysis by LC/MS/MS. A method has been developed and validated for the detection of synthetic insulins in both serum and urine (Goebel et al 2008). The method will soon be an ISO17025 method for routine use in our laboratory. Work has also proceeded on methodology for the detection and quantitation of IGF-I and related analogues such as long R3 IGF-I in serum. Our ability to detect and identify peptide hormones at physiological levels has recently been enhanced by our purchase of a Thermo LTQ-Orbitrap XL with an Eksigent 2D Nano LC and a Michrom Advance Nanospray source. Using this instrument we are capable of detecting natural gonadotrophin releasing hormone (GnRH) in urine at levels significantly below those previously reported (Thomas et al 2008)

Publications:

Trout G.J. and Kazlauskas R. Sports drug testing – an analyst’s perspective. Chemical Society Reviews 2004, 33, 1-13. Goebel C., Alma C., Trout G.J., Kazlauskas R. HBOC detection – progress since 2000.

Schanzer W., Geyer H., Gotzmann A., Mareck U. (eds) Recent Advances in Doping Analysis (13). Sport und Buch Strauss, Koln, 2005, 235-242. Goebel C., Alma C., Howe C., Kazlauskas R. and Trout G.J. Methodologies for detection of haemoglobin-based oxygen carriers. Journal of Chromatographic Science 2005, 43, 39-46.