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Code: 12C5YP

a) The use of recombinant human erythropoietin (r-HuEpo) is prohibited by the World Anti-Doping Agency. The purpose of a research project funded by WADA in 2008 entitled “A gene-microarray based approach to the detection of recombination human erythropoietin doping in endurance athletes” was to develop new methods with improved discriminatory power relative to current detection protocols based on gene expression profiles. On the basis of the preliminary data generated by this project, blood gene expression profiles were significantly altered during r- HuEpo administration and for at least 2 weeks after the r-HuEpo administration. These preliminary data are very promising, however, it is well known that athletes are now microdosing with r-HuEpo to minimise the risk of being caught via current detection methods. Therefore, in this project, we will compare blood gene expression profiles altered using a fairly high regimen of r-HuEpo injections of 50 IU/kg body mass every two days for 4 weeks (the previously funded project) with a microdose regimen (< 40 IU/kg body mass twice a week). Blood gene expression profiles following r-HuEpo administration will be assessed using gene-microarray technology. These results will be used to develop more specific and robust blood testing models applicable to the detection of microdose r-HuEpo doping.

b) Our research consortium has previously been funded by WADA as follows: In 2008 “A gene microarray based approach to the detection of recombinant human erythropoietin doping in endurance athletes” and a related project funded in 2010 “Application of a minimally-invasive method for RNA sampling and the addition of miRNA to the detection of recombinant human erythropoietin (r-HuEpo) use by athletes”. These recently funded projects were designed to provide the basis for the development of new detection methods with improved discriminatory capacity relative to current detection protocols using a “system biology” (i.e. gene expression and miRNA profiles assessed by the very latest gene-microarray technology) approach. Current progress with these WADA-funded projects is very encouraging and this success serves to reinforce the feasibility and need for this complex, expensive and technically demanding “systems biology” approach. Here we propose the addition of metabolomics (high resolution mass spectrometry methods) to the established research study aimed at identifying new metabolomic biomarkers that can differentiate between r-HuEpo administration and chronic altitude exposure (using samples already collected from WADA-funded studies). It is envisaged that this vital addition will help formulate new methods with improved discriminatory power relative to current detection protocols and in doing so eliminate the possibility of false-positives due to athletes living and/or training at altitude and false-negatives due to inadequate detection.

Main findings

a) Administration of recombinant human erythropoietin (rHuEpo) improves performance and hence is frequently subject to abuse by athletes. A limited detection window, a lack of sufficiently high sensitivity and specificity limit current testing. Previous WADA-funded research aimed at detecting high doses of rHuEpo doping using gene-based methods has generated promising results. The primary focus of this research is to apply this gene-based approach to the detection of microdoses of rHuEpo. The secondary aim of this research is to assess the performance effects of rHuEpo microdosing. Fourteen healthy endurance trained subjects not involved in competition during the study period participated in a 7 week randomised, double-blind, placebo-controlled crossover microdose rHuEpo regimen previously shown to increase haemoglobin mass. Maximum aerobic capacity and repeated sprint ability was assessed at baseline and during the week after the last rHuEpo/placebo injection. 18 genes were differentially expressed ten days after the first microdoses of rHuEpo. Of these 18 genes, 11 were consistently over-expressed during rHuEpo microdosing and under-expressed post administration thereby further validating the previously identified gene expression signature of rHuEpo. While no clear improvements in measures of anaerobic performance were observed following microdoses of rHuEpo, maximal aerobic capacity was significantly increased pre to post microdose rHuEpo administration by an average of 3.9%. In summary, this research provides strong evidence supporting the idea that gene-based biomarkers have real potential to improve the performance of current anti-doping methods such as the ABP for the detection of doping, not confined only to rHuEpo. In addition, this research highlights the need to improve current detection methods as rHuEpo microdosing has significant performance benefits.

b) Recombinant human erythropoietin (rHuEpo) is prohibited by the World Anti-Doping Agency but rHuEpo remains the drug of choice for many cheating athletes wishing to evade detection using current methods. Currently, the only validated direct test for rHuEpo is isoelectric focusing, but this test is severely limited by the short detection window of approximately 36-48 hours. Recent research funded by WADA has generated exciting new transcriptomics (i.e. gene expression) data to support the proof-of-concept idea of “omics” biomarkers as the preferred next generation anti-doping approach. Metabolomics (a key “omics” technology with widespread application in biomedicine) may further enhance this potential by providing a “snapshot” of the biological state of the organism/cell. The aim of this project was to identify a robust metabolomics signature of rHuEpo using an untargeted approach in blood (plasma and serum) and urine. Plasma, serum and urine samples were analyzed using the hydrophilic interaction liquid chromatography-mass spectrometry in 20 Caucasian males following 4 weeks of rHuEpo administration (i.e. 50 IU∙kg-1 body mass every two days). Three blood (plasma and serum) metabolites associated with red blood cells were identified as potential markers for rHuEpo detection. Although the diagnostic value of the identified metabolites for current rHuEpo detection methods is low, a combination of replicated metabolomic markers will provide a more detailed and thorough understanding of the perturbed system-s, thereby aiding ABP experts identify and differentiate numerous doping substances and methods when reviewing athlete biological passports. The present findings should encourage further metabolomics studies and the integrated reviewing of all “omics” data generated by other WADA anti-doping studies in order to aid the development of new detection models.

Code: 12C4MT

 Metabolomics has become a major tool in systems biology and biomarker discovery studies and possesses great potential also concerning modern anti-doping efforts. Most assays currently applied in doping controls are designed to sensitively and specifically detect the administered drug or its diagnostic metabolite to provide unambiguous evidence for the presence of a banned substance. A few marker-based methods have also been established in the doping control arena such as the steroid profile and the hematological module of the Athlete Biological Passport. 
The influence of ‘external circumstances’ on the metabolome has been recognized decades ago, particularly concerning the diagnosis of diseases. Analogously, the search for biomarkers towards the detection of drug (ab)use in sport will be of great utility for a modern anti-doping fight as demonstrated in pilot studies concerning animal sport and livestock production. Upon drug administration, shifts in metabolomic signatures occur; these are largely unknown but, once identified, potentially easier and/or longer monitored than the administered compound(s). Hence, analysis, quantitation, or profiling of (bio)markers rather than the detection of the drug itself will indicate and possibly provide evidence for a previous illicit intervention on the basis e.g. of growth promoting hormones. This is especially helpful for those therapeutics and designer substances that are difficult to determine (due to short half-life or structures identical with endogenously produced hormones). Modern analytical tools, particularly mass spectrometry-based methods, have proved useful for the identification of parameters that provide information on metabolome alterations. In combination with biostatistics, multivariate statistical analyses are enabled to highlight the value of identified compounds as potential (bio)markers. Upon characterization of marker candidates, these analytes can be implemented in anti-doping methods. Therefore, a comprehensive screening of the urinary metabolome of samples collected from different (administration) studies is planned to provide new insights into new targets for future doping control programs. 

Main Findings:

The urinary metabolome, i.e the entire set of low molecular mass substances of the human organism eliminated into urine, is composed of the endogenous and the exogenous metabolome. This reflects the metabolic products naturally produced by the individual and metabolites resulting from xenobiotics (e.g. drugs, toxins, food additives, etc.), respectively. It is consequently an extraordinary complex and dynamic object, which is significantly influenced (amongst others) by drug (mis)use. In order to assess whether –omics strategies such as urine metabolomics can indicate the misuse of drugs relevant in a doping control context, a pilot study was conducted, probing for the potential of this analytical strategy of identifying samples collected during and after applications of anabolic-androgenic steroids. Three scenarios were pursued with controlled administration studies including (1) oral, and (2) transdermal testosterone, where both intra- and interindividual variations of the metabolome were measured. In addition, the metabolome of a reference population of healthy and active individuals was compared to the urinary metabolome of athletes tested positive for the misuse of anabolic-androgenic steroids in routine doping controls (3). The urinary metabolomic ‘signatures’ were recorded using high resolution/high accuracy mass spectrometry with both gas chromatographic and liquid Chromatographic separation of analytes to provite utmost comprehensiveness. Followwing smaples analyses, non-targeted data evaluation was conducted to enable pattern recognition as supported by statistical methods such as principal component analysis (PCA) and orthogonal projection to latent structures (OPLS). 
Comparing the samples of the reference population with those resulting from adverse analytical findings (i.e. scenario 3), extreme interindividual variations were observed. Nevertheless, distribution patterns of typical non-suspicious and atypical urine samples were also recognized that potentially contribute information supporting the identification of unusual anti-doping analyses. Within both testosterone administration studies (1 and 2) it was readily possible to distinguish between pre- and post-administration samples on an individual basis. By comparing the undisturbed metabolome of each volunteer with the effects induced by testosterone, many entities or metabolites could be detected that were either up- or down-regulated. Even for the application of low doses of testosterone via transdermal testosterone gel, the metabolome was significantly influenced in all 4 volunteers on an individual basis. However, the interindividual variability was extensive and the effects of testosterone administrations on the metabolome became more or less negligible compared to the large biological differences. These individual variations are composed of many factors such as the different regulation of enzymes and metabolic pathways due to physical activity or simply the individual´s diet. Consequently, metabolome analyses seem to have potential to indicate drug (mis)use based on pattern positioning when compared to a reference population. Moreover, supporting information on anabolic-androgenic steroid misuse (here: testosterone) is obtained when intraindividual metabolomics data are available.  
On a sideline, another unexpected result was observed. The UDP-glucuronosyltransferase 2B17 genotype obviously has a large impact not only on selected glucuronidated (steroid) metabolites but seems to significantly influence the entire urinary metabolome. Since only 2 participants with this polymorphism were included in this study it remains speculative if identifying the genotype can be accomplished by a distinctive urinary metabolome pattern; however, the preliminary dataset suggests this possibility.

Code: 12A2MT

Within the last few years a new class of substances emerged in the focus of sports drug testing, so called gene doping agents. One of these called AICAR (aminoimidazolecarboxyamide-ribonucleoside) has proved to enhance physical performance, even without training, in animal experiments. 
In the course of clinical trials AICAR has been administered to human beings to investigate its health care properties related to type-2 diabetes. As during these studies no major side effects were reported it cannot be excluded that AICAR will be of interest for cheating athletes.  Therefore, the World Anti-Doping Agency included AICAR on their list of prohibited substances in 2009 and doping control laboratories started to establish methods for detecting AICAR-abuse. Until now, urinary concentrations of AICAR have been determined and reference-based thresholds were suggested due to the fact that AICAR is an endogenously produced substance and occurs in every urine specimen. As the observed biological variability in urinary concentration was high – as also reported for other classes of substances such as steroids – these thresholds are quite high and endogenously produced AICAR can potentially be found beyond the reference limit in rare cases. In analogy to steroids, the carbon isotope ratio determination would be the method of choice to distinguish endogenous from exogenous/administered AICAR. Hence, the aims of this study are to develop and validate a method for the determination of carbon isotope ratio values of AICAR and to elucidate the endogenous 13C/12C ratios of this compound. Additionally, the 13C/12C ratios of synthesized AICAR will be determined to enable the calculation of a clear cut-off criterion between the endogenous and the exogenous AICAR. 

Main Findings: 

AICAR (5-Aminoimidazole-4-carboxamide 1β-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples suspicious of AICAR abuse by measuring its urinary concentration and comparing the observed level to naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore a method for the determination of carbon isotope ratios (CIR) of urinary AICAR has been developed and validated. Concentrated urine samples were fractionated by means of liquid chromatography for analyte clean up. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography-combustion-isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study.    
The developed method fulfils all requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in CIR of AICAR as for endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforward to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h.  
Determination of CIR is the method of choice to distinguish between an endogenous or exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR. 

Code: 12C14NL

Autologous blood transfusions are a prohibited doping intervention in anti-doping field. The most promising attempt to detect autologous transfusions are the longitudinal measurement of different biomarkers such the Athlete Biological Passport (ABP).

Previously, the implementation of the detection of DEHP metabolite in urine showed to be an efficient method to detect autologous blood transfusion several hours after re-infusion. However, cheated athletes could easily use DEHP-free blood bags to prevent the detection of DEHP-metabolites in urine. For this reason, other biomarkers based on physiological response to transfusion of stored blood have to be discovered to detect autologous transfusion in anti-doping field.

In this project, we plan to perform clinical study in which volunteers will perform autologous DEHP-free blood transfusion. DEHP-free blood bags will be supplied thank to a laboratory-industry collaboration. Collection of different matrix such as plasma, serum and fingertip prick test samples will be carried out at different time points. In these samples multiple biomarkers such as blood cells, non- transferrin-bound iron (NTBI), total bilirubin, interleukin and circulating microRNAs will be measured and compared in order to detect autologous DEHP-free blood transfusion in recipient.

Main Findings

BACKGROUND: Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin and new urinary metabolites of plasticizers may be a alternative to detect ABT.

STUDY DESIGN AND METHODS: clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. The impact of ABT on hepcidin and plasticizers metabolites as well as haematological parameters, iron metabolism, and inflammation markers was investigated. RESULTS: Blood transfusion had a particularly marked effect on hepcidin concentrations and long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl)phthalate compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hours and 1 day after blood re-infusion, these concentrations rose by 7- and 4-fold, respectively. No significant change was observed in the control phase. Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the leukocyte filter (22.3%).

CONCLUSION: Hepcidin quantification and urinary DEHP metabolite measurement is a cost-effective way to detect ABT in anti-doping field even when BTHC bags are used for blood storage.

Code: 12A17VB 

Growth hormone (GH) is included in the WADA list of prohibited doping substances. The detection of GH misuse is currently achieved using immunological assays to determine the abundance ratio between the 22 kDa and the 20 kDa GH isoforms. Another strategy, which profiles indirect markers of GH administration (mainly blood IGF-1 increase), has also attracted some interest. The aim of the present project is to develop an innovative liquid chromatography - mass spectrometry-based assay to detect and quantify simultaneously the 22 kDa GH isoform and IGF-1 in serum. We anticipate that this assay will also provide information on the GH isoform ratio. Serum sample treatment will be designed: (i) to circumvent the use of antibodies to overcome the current dependence on antibody manufacturers and (ii) to be compatible with highthroughput sample analysis. Mass spectrometry analysis will be performed using the Selected Reaction Monitoring (SRM) mode renowned for its high specificity and sensitivity. Quantification will be achieved using the PSAQ method (Protein Standard Absolute Quantification) which uses stable isotope-labeled versions of the targeted proteins as quantification standards. We expect that this PSAQ-SRM assay, which targets both direct and indirect biomarkers of GH misuse, will improve the reliability of the biological information delivered. This should be an important contribution to efficient sports drug testing. 

Main Findings: 

Growth hormone (GH) is included in the WADA list of prohibited doping substances. The recombinant form of GH and its endogenous counterpart (22 kDa) are structurally analogous, making detection of GH misuse challenging. A strategy to overcome this issue is based on the determination of the abundance ratio between the 22 kDa and the 20 kDa GH isoforms in athlete’s serum samples. This is effective because exogenously administered recombinant GH exerts negative feedback on endogenous expression of the 20 kDa isoform, resulting in a modified 20 to 22 kDa abundance ratio (Wu et al., 1999). 
In this context, this project aimed at developing an innovative liquid chromatography - mass spectrometry -based assay to detect and quantify both the 22 kDa and 20 kDa GH isoforms in serum. Mass spectrometry analysis was performed using Selected Reaction Monitoring (SRM) acquisition (Lange et al., 2008). Quantification was performed using the PSAQ method (Protein Standard Absolute Quantification) (Brun et al., 2007). The first task of the project consisted in the synthesis of PSAQ standards, i.e. isotope labeled versions of the 22 kDa and 20 kDa GH isoforms. Both standards were successfully produced with a final purity over 95% and an isotope incorporation greater than 99%. These standards are now available as validated products for the anti-doping community. Then, using these standards as model proteins, we implemented a LC-SRM method on a 6500QTRAP mass spectrometer to simultaneously detect and quantify GH isoforms. This method was specifically optimized to allow the detection of peptides discriminating the 22 kDa from the 20 kDa isoform. In a third step, we assessed different biochemical methods to extract GH isoforms from serum matrix. Although immunocapture was effective to capture GH isoforms, it did not allow the discrimination of the 2 GH isoforms. Detection sensitivity was determined at 50 ng/ml of serum. 
Biochemical optimizations are still ongoing to improve the detection limit of the method and investigate the abundance ratio between the 2 GH isoforms. 

Code: 12D10RD 

The imminent introduction of the so called ‘endocrine module’ of the athlete’s biological passport needs to consider the numerous reports showing the effect of ethanol ingestion on the steroid profile. A steroid profile would only be useful for longitudinal monitoring and statistical evaluation if it has not been altered by any uncontrolled circumstance, very particularly alcohol consumption. Ethylglucuronide (EtG) and in a lesser extent Ethylsulfate (EtS) are well established ethanol biomarkers. Recent studies have shown the correlation between their urinary concentrations and changes in T/E ratios and other parameters. Studies are needed to develop cut-off values for biomarkers able to ensure that no alteration of the steroid profile can be claimed so that data can be incorporated in a longitudinal intra-individual passport. We have already performed a number of placebo controlled cross-over studies in which male volunteers have received different doses of alcohol and for which urine samples have been collected. This is a precious material that can be used to monitor the steroid profile before and after the ethanol ingestion and determine the intra-individual effect and correlation between the dose, the variation of the parameters monitored as well as the biomarkers chosen (EtG and EtS). 
The main objectives of the project are: 
1.- study the intra-individual variation of steroid profile parameters as a result of the ingestion of different doses of ethanol using urine samples already available from previous ethanol administration studies performed by our group. 
2.- Develop cut-off values for the selected biomarkers (EtG and EtS) granting that the steroid profile in a particular urine sample could not have been altered by ethanol consumption. 
3.- Perform additional clinical studies incorporating Asian volunteers in order to extend the validity of those markers to other populations, particularly those with the well known UGT2B17 gene deletion (e.g. Asians). 

Main Findings: 

Background: the introduction of the so called ‘endocrine module’ of the athlete’s biological passport needs to consider the numerous reports showing the effect of ethanol ingestion on the steroid profile. A steroid profile would only be useful for longitudinal monitoring and statistical evaluation if it has not been altered by any uncontrolled circumstance, very particularly alcohol consumption. Ethylglucuronide (EtG) and Ethylsulfate (EtS) are well established ethanol biomarkers and have been proposed in doping control for the detection of its consumption. Large intakes of alcohol produce increases of the T/E ratio, probably because alcohol, excreted in large amounts as glucuronide, compete and disturb the glucuronidation of steroids. Studies had been performed showing those variations and how EtG determination in urine, grossly correlated with the variation in T/E ratios. Those studies led to the impression that a cut-off value for EtG could be developed to justify changes in T/E ratios. Values below EtG would show a consumption of alcohol sufficiently low to not justify variations of T/E. 
Study aims included: the evaluation of intra-individual variations of steroid profile parameters as a result of the ingestion of moderate alcohol consumption (15g, 18g, 30g and 42 g of ethanol, n=6-12 per dose). Also the potential modifications of those variations depending on the kind of alcohol source (i.e. vodka or wine, 30g of ethanol, n=6) were also considered.    Results: Doses as low as 15 g ethanol (equivalent to ‘one drink’) and up to 42 g (the starting dose where subjects display symptoms of acute intoxication) were studied here. T/E values were found to be altered very fast, in parallel with the absorption of alcohol, in the period 0-2h after ingestion. Variations, even with low doses, accounted for more than 70% change in T/E value that would have triggered a confirmation and IRMS analysis. EtG concentrations in urine had a delayed profile, reaching their peak in the period 2-4h. The implication is that while EtG concentrations were quite low in the period 0-2h, T/E values had already varied significantly or even reached their maximum variation. However, when EtG concentrations were maximal, T/E values had already begun to decline or had already returned to normal. Before 12h all T/E values had returned to normal. A linear correlation was found between the alcohol dose and the T/E variation, clearly showing their linkage. Both EtG and EtS have shown a very good correlation. But both have shown to be badly correlated kinetically with the variations produced in T/E ratios with the consequent risk of leading to wrong conclusions regarding the reasons for T/E changes. The ratio T/A (expressed as 100T/A for numerical reasons) kinetically fits much better with the EtG or EtS concentrations. Alterations in the steroid profile observed were independent of the kind of alcohol source. 
Future developments: (i) studies need to be performed in women (milder first pass metabolism and lower T and E concentrations), (ii) the effect of repeated doses of alcohol along several hours and studying particularly the terminal elimination phase with multiple sampling after 8h of the last dose) resembling usual alcohol consumption behavior, and ‘next morning’ doping control, need to be evaluated and (iii) the performance of studies in ethnicities other than Caucasian and the evaluation of the impact of the UGT2B17 polymorphism (low T/E ratios) and its influence on alcohol effects.

Code: 12D5EE 

According to the consultation process by WADA on 2012 Prohibited List, World Archery reevaluated the removal of beta blockers, as it is always a major concern regarding TUEs over decades. A study has been carried out many years ago for pistol shooting which has shown the effect to be existing, which is why the substance ended on the prohibited list. It was taken granted that the use of beta blockers would affect archery performance and was added to the list. 
However, no scientific research was carried out on archers concerning the effects of beta blockers. On the other hand, there has been an enormous development especially in the field of Beta-blockers. The so called cardioselective Beta-blockers (means there is only effect on heart activity) are widely used and may keep archery open to a great number of archers throughout the world. This would be one of the issues to test during such Beta Blockers-project and also illustrates the challenge since how do we differentiate between these and more traditional betablockers. 
With this perspective on mind, WA is willing to carry out a Project to search the effect of betablockers on the effect of archery shooting performance, more specifically fine motor tuning during shooing. In this Project, archers will be given beta blockers and their performances will be evaluated in a laboratory setting. The main parameters that will be measured are postural sway, aiming behaviour, mechanical clicker reaction time and actual shooting scores. 

Main Findings: 

It is well-known that athletes may experience some form of stress prior to or during a competition which may reduce or at least negatively affect their athletic performance. Therefore, inhibition or reduction of stress related behaviour and responses, like tremor or increased body sway, in sports requiring fine motor tuning may prove beneficial by utilizing beta blockers. The purpose of the present study was to investigate whether the intake of a beta blocker would exert positive effects on physical performance capacity, such as an increase in shooting performance in elite archers. The reserach group has compated the effects of a single dose oral selective (nebivolol) and non-selective (propranolol) beta blockers vs placebo and control shooting. A randomized double-blind cross over trial was used to assess shooting scores, heart rate values, body sway and aiming behaviour on 15 archers. The results, despite the lower heat rate after taking beta blockers, did not show any difference between the groups in archery performance characteristic. In addition, beta blockers did not effect aiming behaviour and body sway during shooting. It was concluded that as regards to the performance capacity, neither selective nor non-selective beta blocker use do not improve athletic performance in archery.

Code: 12C12PD

Drug abuse and physical activity result in a variety of adaptation processes of the organism down to the molecular level. Because direct detection of drugs in doping analysis is getting more and more complex, long term monitoring of physiological parameters is a promising supportive strategy in the fight against doping. A new field for the identification of biomarkers is epigenetics. Epigenetics is defined as the heritable change in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence such as DNA methylation and histone deacetylation. Evidence is accumulating that drugs, nutrition, but also physical activity result in the modulation of the epigenome. Identification of relevant mechanisms has resulted in pharmaceutical strategies. It’s very likely that such drugs will be abused in doping. 
Knowledge of specific epigenetic modulations as the result of abusing drugs can serve as biomarkers for an indirect doping detection. In the proposed project experts from the center of preventative doping research of the german sports university and from the Division Epigenomics and Cancer Risk Factors of the German Cancer Research Center want to combine their skills to identify a signature of genes differentially methylated by doping abuse. Therefore data from animal experiments, training studies in humans and a field study with bodybuilders abusing anabolic steroids will be combined. In white blood cells (WBCs) and muscle tissue from the animal study and WBCs and urine from the human studies genome wide methylation patterns will be monitored by methyl-CpG immunoprecipitation (MCIp) of methylated DNA followed by next generation sequencing (NGS). In a second step methylation of identified differentially methylated regions will then quantified in a high-throughput manner by Maldi-TOF Mass spectroscopy.

Main Findings: 

Endocrine active agents have been shown to influence the epigenome. The kinetics, extent and persistence of such changes are however not thoroughly established and need to be investigated in well controlled studies both in animal models as well as in human pilot studies. 
The aim of our research project was to identify a methylation profile specific for doping by genome-wide methylation profiling. Based on this profile we intended to develop a methylation signature that might be useful as high throughput biomarker for drug abuse when measured by mass spectrometry-based quantitative methylation analyses in blood cells. 
In a human pilot study performed with healthy control subjects (C), natural (non-doping) body builders (BB), and body builders abusing anabolic substances (ABB) DNA of 31 subjects participating in the pilot study was available for 450k analyses. When combining all the information gathered on DNA methylation in blood derived DNA, we conclude that its potential use as a biomarker for the detection of drug abuse is hampered by several limitations and confounding factors. First we could only detect few and small significant methylation differences. The detected methylation differences between groups are small, in the range of 5-10%. We analyzed >450.000 CpG sites, but only about 0.1% of the sites (around 500) passed our selection cutoff criteria: Significant differences between groups with p<0.05, methylation differences >5%, standard deviation below 15%. Of these, most were significant only between two of the analyzed groups, but not between all three groups. Second the genome of individuals differs. Although we removed all sites affected by SNPs during our bioinformatic analysis, some sites might still overlap with less frequent or not annotated SNPs. Even one of our most discriminating site cg21365902 carries a SNP with a very low minor allele frequency of 0.06. Since the frequency of SNPs differs between races, the ethnic background and the variability of the study group also contributes to whether a site might be a good biomarker or not. Third, DNA methylation is cell type specific. Since the blood cell composition of the study subjects differed, we cannot exclude that the methylation differences between the groups are merely due to differences in blood cell composition. The fact that we had genome wide methylation data allowed us to correct for the blood cell composition. However, these genome-wide analyses are time and cost-intensive. Overall, detection of drug abuse by measuring DNA methylation from blood samples appears to be prone to artifacts and is affected by several confounding factors. In order to be able to understand the relevance of DNA methylation changes identified in blood cells, we also performed an animal study with male Wistar rats (rat training study). Half of the animals were treated with methandienone (5 mg/kg/bw/d) once at the age of 91 days to mimick “doping”. In addition, half or the rats underwent a training program for 6 weeks. The animals were allocated to four groups, a control group (C), a training-only group (T), a “doping”-only group (D) and a group with combined training and doping (TD). DNA from muscle tissue and blood cells were available for methylation profiling. Methylation analyses in the muscle tissue provide interesting new insights into potential epigenetic gene regulation related to doping. However, as outlined in the human study, ideally methylation levels should be measured in all cell types composing a tissue, as changes in cell type composition cannot be excluded. So far, the rat genome is less characterized than the human or mouse genome, and further epigenomic information not available. To confirm the results, it might be preferable to perform a study in mice instead of rats, or to analyze human biopsies. In PBMCs obtained from the rat training study we identified only few significant methylation changes that passed our selection criteria. This demonstrates that in comparison to muscle tissue, the number of training or MD induced methylation changes is even smaller.  
We conclude from these evaluations that the analysis of blood cells only provides very limited information, and methylation profiling of target tissues such as muscle should be preferred.

Code: 12B7CP

Reliable, sensitive and specific measurement of the hormone EPO + related analogues, used to illegally enhance sports performance, is a continuing problem for WADA. To date, immunoassay tests for EPO suffer sensitivity and specificity problems due to the multiple forms of EPO normally present in blood and also from newer synthetic short forms that can be used by competitive athletes. This project will validate a novel immunoassay technology for the detection of EPO in human blood through the measurement of the protein's signal peptide. Signal peptides are short, discrete components of hormones that were previously thought to be degraded by cells once the hormone has been produced. However, we have shown that the signal peptide of EPO is not destroyed in human blood and can present as a novel, measurable factor to determine the presence or absence of abnormal EPO administration. The ratio of EPO to its signal peptide in human blood and urine samples will be validated in normal healthy people before and after exercise. We will also characterise the molecular forms of EPO signal peptide in blood and urine and monitor its normal daily circadian rhythm. This project has the potential to offer a novel, simple, robust and specific method to detect illegal EPO, via measurement of the ratio of EPO to EPO signal peptide, for use in competitive sports. We envisage the ratio forming part of individual athletes 'Athlete Biological Passport' or ABP.

Main Findings

Erythropoietin (EPO) and its illegal use to boost performance represents a challenge to sporting regulatory bodies. Further, micro-dosing regimens mean EPO in blood and urine samples can be difficult to detect. The WADA athlete biological passport goes some way to improving EPO detection, but there is room for improvement. We previously made the novel discovery that signal peptide fragments from proteins and peptides are present in human blood and urine and have applied this concept to testing for the signal peptide of EPO in healthy human blood and urine samples. We developed a robust and reliable immunoassay for the detection of human EPO signal peptide (EPOsp). This assay does not cross react with EPO or related peptides and proteins and is not subject to interference from common drugs and medications. Immunoreactive EPOsp in human blood and urine samples eluted on high performance chromatography consistent with a nonapeptide fragment. EPOsp in the blood of 109 healthy control subjects did not correlate with EPO and there was no difference between levels in men and woman. EPOsp levels in blood showed modest but significant correlations with body mass index and systolic blood pressure. The ratio of EPOsp:EPO was 8.2:1. Blood levels of EPO displayed a diurnal rhythm in which levels rose approximately 50% during the day: EPOsp showed no such change, which meant the EPOsp:EPO ratio was higher in the AM than in the PM. EPOsp was measurable in reasonable amounts in human urine whereas EPO was undetectable. Finally, based on regional venous and arterial sampling in 11 subjects, EPOsp is secreted in greatest amounts from the kidney and heart, consistent with other renal and cardiac data. Thus, we have established a robust working assay for measurement of EPOsp in human samples. We confirm that EPOsp is present in human samples as a distinct entity, separate from EPO, and may potentially be used as a method for the detection of EPO dosing.