Projets de recherche

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.

Code: 02A03RK

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

Main Findings: 

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

Code: 01C15CA

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

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

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

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

Main findings

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

Code: 01C10DM

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

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

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

Main Findings

(The project was not finished)

Code: 01A07CH

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

Main Findings: 

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