Projets de recherche

Code: 03C03MB

Research in recent years demonstrated that the analysis of changes in the hGH isoform pattern occurring after administration of recombinant hGH could help to develop a method to detect hGH doping. Basic requirement for such a technique is the availability of appropriate antibodies and the experience in the techniques to utilize them in specific immunoassays. The aim of this research project is to familiarize personnel from 3 WADA accredited laboratories with the use of the specific monoclonal antibodies developed in our group in immunoassays to detect doping with human growth hormone. All steps necessary - ranging from the theoretical background up to practical training with measurement devices - will be performed by experienced members of our group together with the respective delegates from the WADA accredited labs. Each participant will be able to prepare and conduct the tests at the end of the training. In addition, preparation, testing and shipment of reagents necessary to perform a minimum of 5000 analyses is included within this project proposal. Monoclonal antibodies, calibrators and control samples will be shipped on dry ice to the respective WADA accredited laboratories to allow their participation in the international external quality assessment scheme planned for June this year to allow a validated decision about the status of the implementation of the test at the respective sites.

Code: 03B05WS

The 13C/12C-ratio of urinary steroids is an accepted parameter to detect abuse of androgenic anabolic steroids. Significant differences in 13C112C between selected reference and target steroids are regarded as an unequivocal evidence for presence of synthetic material. However theoretically this procedure might result in false positive cases: Significant differences in isotope ratios might result when isotopic composition of diet changes within a short period of time and when physiological carbon fluxes to reference and target compounds are of different order of magnitude. Isotopic composition of diet necessarily changes after transcontinental travelling due to different contributions of isotopically distinct carbon sources of diet. On the other hand, few is known concerning the exchange rates of carbon during steroid biosynthesis. The aim of the study is to elucidate the dynamics of carbon exchange in steroid metabolism. This will help to obtain significantly more insight into the quality of information given by isotope ratio analysis of urinary steroids. The study requires systematic variation of isotopic composition of diet in a longitudinal study with few selected subjects. When possible, we intend to develop explicit models for the carbon fluxes in steroid metabolic pathways.

Main Findings

The exchange rates of the carbon pools for the biosynthesis of steroid hormones may be different for different compounds. As a consequence, the differences in isotopic composition between endogenous urinary steroids (Δ13C-values) temporarily may increase during switch to a diet with largely different isotope signature. Theoretical considerations suggest that in some individuals the increase of the elevated Δ13C-values may remain for comparably long periods (weeks to months). There is a large biological variance concerning these phenomena. Whereas in some individuals the exchange rates are virtually identical, other individuals may exhibit significant differences. The exchange rates vary significantly between individuals in absolute terms. Body mass and physical activity are likely to play a pivotal role. The contribution of cholesterol to the biosynthesis of steroids varies between different individuals. The most robust comparison appears to be A vs. OHA. These two steroids exhibit comparable exchange rates in most of the subjects. However, this is not necessarily the case (see subject 8, 3.2.8). If possible, evaluation of two reference compounds should be considered.

Publications:

Influence of changes in diet on the dynamics of 13C/12C-ratio in selected urinary steroids. Part I: Study design, 13C/12C-ratio of applied foodstuffs and effect on anthropometrical data. U. Flenker, C. von Kuk, U. Güntner, F. Hülsemann, V. Gougoulidis, W. Schänzer. Manfred Donike Workshop 2005

Influence of changes in diet on the dynamics of 13C/12C in delected urinary steroids: diet free from cholesterol. U. Flenker, C. von Kuk, U. Güntner, F. Hülsemann, V. Gougoulidis, W. Schänzer

Code: 03E09WS

Insulin, a peptide hormone consisting of two disulfide-linked chains composed by 51 amino acids, is part of the endogenous system regulating blood glucose levels by inducing glycogen synthesis. Owing to inconvenient properties of insulin preparations, rapid- as well as long-acting insulins have been developed, which overcome the drawbacks of remedies consisting of recombinant insulin only. Both classes of synthetic insulins, rapid and long acting analogues, as well as human insulin itself are possibly misused in sports, some reasons of which were discussed in the literature. The testimony of an athlete of having used insulin as well as findings of injection solutions in luggage of cyclists substantiate this suspect, and sensitive, selective and comprehensive procedures are necessary to determine these compounds in doping control samples. So far, analyses of insulins are mainly performed by means of immunoassays, but also mass spectrometric approaches were demonstrated in the past, which indicate advantages of this technique over immunochemical determination of insulins such as elimination of cross-reaction. The improvements and innovations in soft ionization of large biomolecules by electrospray (ESI) or matrix-assisted laser desorption (MALDI), as well as the production of highly sensitive mass selective detectors enable the development of procedures capable of screening and confirmation of insulin and its new biotechnologically synthesized analogues for doping control purposes. In order to identify rapid- and long-acting insulins, their mass spectrometric properties after electrospray ionization have to be elucidated. As differences of endogenous and synthetic insulins are limited to exchanges of few amino acids, and one compound is identical with human insulin regarding molecular weight, product ion experiments have to be optimized to efficiently analyze the target compounds in low concentrations in biological fluids. Furthermore, isolation of insulin and its synthetic counterparts from plasma will be performed by different methods, e.g. solidphase extraction (SPE) and immunoaffinity chromatography (IAC). CommerciaUy available plasma will be fortified with the analytes of interest, extraction recoveries, reproducibility and detection limits will be determined.

Main Findings: 

Synthetic insulin derivatives with modified amino acid sequences such as Humalog Lispro, Novolog Aspart or Lantus Glargine have been introduced to the pharmaceutical market as they provide better controllability, faster or prolonged bioactivity and improved convenience compared to conventional recombinant human insulin preparations. However, the misuse of insulin in sports has been reported manifold, and the international doping control system has required a reliable and robust assay to determine the presence or absence of related prohibited drugs. A qualitative evidence of administered substances is of utmost importance, which is preferably obtained by mass spectrometry. Hence, a top-down sequencing-based assay was developed that allows the detection of synthetic insulin derivatives in human plasma. Therefore, specimens of 2 mL were fortified with three synthetic insulin analogues to simulate plasma levels after subcutaneous administration. A subsequent purification by immunoaffinity chromatography and solid-phase extraction was performed, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This approach has allowed the assignment of individual fragment ions, in particular of those comprising modifications that are originating from C-termini of Bchains, and thus, enabled the unambiguous detection of synthetic derivatives of human insulin in plasma samples. Humalog Lispro contains the same set of amino acid residues as endogenous insulin with one pair having switched positions (B28 and B29, lysine and proline). Here, MS/MS data are essential to allow mass spectrometric separation and identification of target analytes. Other synthetic drugs (Novolog Aspart or Lantus Glargine) possess different amino acid compositions and deviate in molecular mass from human insulin, which facilitates their mass spectrometric and chromatographic differentiation. Recoveries of synthetic insulins from plasma aliquots ranged from 91-98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes, which represents plasma levels of insulin for non-fasting normal subjects.

Code: 03B06FD 

3,6,17-androstenetrione is an anabolic steroid and is also referred to as 6-oxo. This product is sold as an over the counter product in the United States where it is regarded as a nutritional supplement. It is widely available on the internet and it is marketed as an antiestrogenic agent. Athletes might be tempted to threat the adverse effects of an extensive abuse of anabolic steroids (e.g. suppression of androgens and gynaecomastia) by using this type of drugs. Although at present no results from clinical studies supporting the claims of the manufacturer are available, it seems likely that athletes will use this product. This is especially true because the manufacturer of this product is the same company that introduced 1 -androstenediol, 4- androstenedione, 4-androstenediol and 1 9-nor-4-androstenediol on the prohormone market. All of these products have rapidly become a commercial succes. Although the manufacturer claims that 6-oxo is the natural first antiestrogenic product, it is clear that based upon its structure this product can be regarded as an anabolic steroid as well. The producer claims that 6-oxo works as an aromatase inhibitor, blocking the aromatisation of endogenous as well as exogenously administered drugs resulting in higher levels of endogenous steroids as well as synthetic co-administered steroids. Based upon its structure, it is likely that 6-oxo is not metabolised into endogenous steroids commonly screened for during doping control, misuse of this substance remains undetected. It seems therefore necessary to determine whether or not the use of this supplement does indeed result in higher levels of endogenous steroids (testosterone). Moreover elucidation of the metabolism and identification of 6-oxo metabolites is necessary to allow for the detection of this supplement. This research would therefore investigate the metabolism and excretion of 6-oxo to allow for its detection in the urine of athletes.

Main Findings: 

The analysis of the 6-OXO supplement resulted in the detection of 6-oxo-androstenedione but also of 6b-OH-androstenedione. No other contaminants were detected. Excretion studies resulted in the detection of the parent drug 6-oxo-androstenedione and 6a-OH-androstenedione and 6a-OH-testosterone as metaboites. A GC-MS-SIM method was developed and validated to fulfil requirements of WADA for doping control laboratories. Using this method, the ingestion of the supplement, accordin the manufacturers recommendations resulted in the detection of a 6a-OH-androstenedione up to 37 h after the administration, while 6-oxo-androstenedione and 6a-OH-testosterone could be detected up to 24h post administration. Because of the longer detection time and its presence in urine after administration of androstenedione, it is recmmended that beides 6-oxo-androstenedione, 6a-OH-androstenedione is also included in screening methods for doping control purposes. The presence of 6-oxo-androstenedione or 6a-OH-testosterone can be used to disciminate between the administration of 6-oxo-androstenedione and androstenedione

Code: 03C02TF 

The use of gene transfer methods for athletic enhancement is inevitable. To prepare for such eventuality, it will be necessary to develop more efficient and more effective methods for detection of the foreign genetic information and/or the vector used to deliver the transgene. Current screening methods rely partly on the existence of detectable differences between the functional gene products of endogenous genes and the foreign or exogenously administered proteins. For instance, the detection of erythropoietin administration relies on structural differences between endogenous EPO and the administered drug. However, those committed to either drug- or gene-based doping will certainly eventually have available those forms of drugs that are indistinguishable from the endogenous human form or gene transfer vectors that express gene products completely identical to the endogenous function. In that case, current approaches to detection of doping will become ineffective. We propose to take advantage of powerful and broad new methods for gene expression screening to detect secondary changes in gene expression that result from systemic or local administration of genes most likely to be subjects for gene-based doping attempts – erythropoietin (EPO), growth hormone (GH) and insulin-like growth factor (IGF- 1). The hypothesis underlying this proposal is that administration of these extremely and widely bioactive agents or the genes expressing them will be associated with reproducible and detectable secondary changes in gene expression in many affected tissues, including peripheral blood. Erythropoietin is very likely to produce changes in gene expression of nucleated cell lineages in the peripheral blood and therefore detectable in simple blood samples. In addition, exposure to the powerful growth effects of GH or IGF-1 administered locally may also produce effects on the pituitary GH-GHRH-IGF-1 axis and also be detectable in peripheral blood. It is the purpose of this proposal to examine the patterns of gene expression in cells from peripheral blood of mice exposed to GH, IGF-1 and EPO and to gene transfer vectors expressing them and to identify associated changes in gene expression through global microarray techniques.

Main Findings: 

The goal of these studies has been the use of molecular genetic tools of transcriptional profiling and proteomic analysis to identify molecular “signatures” of exposure to doping substances. The underlying principle is the hypothesis that exposure to growth factors and to many other kinds of doping agents will be accompanied by systemic changes in gene expression and in the proteomes if tissues readily accessible for testing. As an introduction to that concept, we have concentrated on the demonstration of genetic and proteomic changes in a number of tissues both in vulture and in vivo after exposure to one of the important potential doping agents – IGF-1. In our in in vitro studies of muscle IGF-treated stem cells, we have identified acute changes in gene expression of several hundred genes, with far more genes being up-regulated than down-regulated. Most impressively, we have found uniform reproducible up-regulation of the genes that regulate cholesterol and steroid biogenesis and of genes fatty acid biosynthesis. If any of these changes come to be documented in vivo, combinations of these changes are candidates for assays for exposure to IGF-1. Under the same conditions, we have used 2-dimensional gel/mass spectroscopic proteomic methods to establish a large data set of proteins that show altered levels acutely after IGF-exposure. The in vivo studies in skeletal muscle in IGF-treated mice have also identified altered expression of several hundred genes, but, in contrast to the in vitro results with muscle stem cells, more genes are down-regulated than upregulated in vivo in skeletal muscle, including a number of genes that specify several muscle growth factors. The most important conclusion that we come to as a result of these studies is that the identification of methods useful for doping detection and screening will require a much higher degree of data sharing and comparison among the WADA-sponsored studies undertaking such genetic studies than is currently occurring. These disparate studies are using many different experimental designs, research platforms and analytical tools to identify genes and proteins perturbed by drug exposure, and it seems highly likely that these disparate studies have identified many common or inter-related functions but that those results are not being identified by current analytical methods. Toward that end, our laboratory has proposed a pilot study with WADA to test the feasibility of establishing a core bioinformatics facility to collect, collate and analyze data from all relevant WADA-supported laboratories to greatly enhance the likelihood of identifying authentic and diagnostic molecular and metabolic changes that can be used in the fight against sport doping.

Code: 03E05DM 

The purpose of this study is to determine the dose-response effects of inhaled B2-agonists on exercise performance in elite non-asthmatic athletes using a sport specific test of performance. To investigate the dose-response effect of inhaled B2-agonists (IBA) on athletic performance elite, male, non-asthmatic cyclists will volunteer to participate in this randomized, double-blind repeated measures study. The sample size will be determined following a pilot study to determine the within-subject variability in a simulated 20km time trial. The minimum worthwhile difference in performance will be equivalent to 0.5-0.7 of the within-subject coefficient of variance. Subjects will be screened for asthma using a eucapneic voluntary hyperpnea test with positive responders excluded from the study. Following baseline measurements, height, weight, V02, average performance velocity, average performance in watts, and 20km time trial performance will be measured in the laboratory during each of 4 testing sessions. The order of treatment will be randomized among placebo, 200~.tg, 400~.ig, and 800~.tg of salbutamol administered by inhaler. The results of this study will determine whether the administration of BA influences performance in elite cyclists from competitive and statistically significant perspectives. Additionally, this study will determine if there is a dose response relationship between IBA, exercise performance and the appearance of salbutamol in the urine.

Main Findings: 

The intention of this project was to address two questions: what are the relationships between SAL dose and exercise performance in a simulated cycling time-trail, and what are the effects of dose on cSAL as used in doping control? Two studies were used to demonstrate that inhaled SAL does not enhance endurance performance in non-asthmatic athletes when using a highly reproducible and sport-specific test. This is the first examination of the dose-response effect of inhaled salbutamol using a sport-specific performance evaluation and used a substantially larger sample size (n=27) compared to most previous work. The lack of a dose-response relationship further supports previous findings that acute SAL inhalation does not enhance exercise performance in non-asthmatics. Furthermore, the short duration of the time trial may not have provided sufficient stimulus for changes in hydration status that can accompany longer duration exercise. Even though most urine samples generally fell well below the WADA limit of 1000 ng/mL, the possibility exists for individuals to exceed this value following inhaled administration. A significant relationship between cSAL and urine SG was observed at higher doses, signifying the potential impact of hydration on values observed in doping control. As with exercise, the role of hydration and individual differences in absorption, metabolism, and excretion on cSAL require further investigation. It is also noted that the finding of SAL to be non-ergogenic cannot preclude the possibility that continued, short-term (2-3 weeks) use of inhaled SAL would not be performance enhancing. Regular use of SAL during both training and competition would be expected and I is possible that continued elevated plasma levels following inhalation may increase ergogenic properties of SAL. Future research needs to be conducted to eliminate this possibility. Lastly, it was observed that a large portion (~19%) of the cyclists/triathletes tested were susceptible to airway hyperresponsiveness. Although a small number of cyclists and triathletes were recruited for these studies, the possibility exists that there is a significant portion of this athlete population competing with impaired airway function unbeknownst to them. Although potential mechanisms for increased airway hyperresponsiveness in certain athletes have been postulated, longitudinal research is required to track changes in airway function with length of time in specific sports. In conclusion, this project demonstrated a lack of dose-response in relationship with SAL and exercise performance in non-asthmatic athletes and that urine cSAL following exercise are highly variable and dosedependent.

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.