Projets de recherche

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.

Code. 12A21RV 

Anabolic androgenic steroids (AAS) are the most important group of forbidden substances detected in antidoping analyses, and improvement of the detection capabilities of their misuse is a priority in antidoping control. Studies on steroid metabolism have been traditionally performed using GC-MS methods. Phase II metabolic reactions of AAS have been normally studied by using hydrolysis with -glucuronidase enzymes, thus mainly conjugates with glucuronic acid have been systematically studied using both GC-MS and LC-MS/MS approaches. The study of sulfates has been limited by the difficulties of their efficient hydrolysis to phase I metabolites. LC-MS/MS offers the possibility to directly detect sulfate conjugates without previous hydrolysis to phase I metabolites. The application of LC-MS/MS has enabled the detection and characterization of unreported sulfate metabolites for boldenone and methyltestosterone, useful to improve detection capabilities of these AAS. Sulfated metabolites of boldenone can be used as markers of exogenous administration of boldenone. For methyltestosterone, long-term sulfated metabolites have been identified that allow for an increase between two and three times the retrospectivity of the detection. These results indicate that sulfatation is an important metabolic pathway for AAS and deserves to be comprehensively studied for all AAS using adequate technology (LC-MS/MS).
The objective of the present proposal is to continue the systematic study of sulfate conjugated metabolites of other AAS that could be used to improve detection capabilities of their misuse. A methodology based on the direct analysis of the sulfates by LC-MS/MS will be applied. Detected sulfate metabolites will be characterized and their excretion profiles will be compared with those of the steroid metabolites targeted in conventional screening procedures, in order to evaluate their interest as long-term markers of steroid misuse. Finally, a methodology addressed to the reliable detection and confirmation of new sulfate metabolites in routine antidoping analysis will be developed.

Main Findings: 

The objective of the study was to evaluate sulfate conjugated metabolites of different anabolic androgenic steroids (AAS). Based on the common ionization and fragmentation behaviour of steroid sulfates, precursor and neutral loss scan methods, and selected reaction monitoring methods including theoretical transitions of potential sulfate metabolites were used to detect new sulfate metabolites in samples obtained after administration of different AAS, including 4-chloro-metnadienone, stanozolol and clostebol.
For 4-chloro-metandienone, four conjugates with sulfate were detected, and two of them (identified as isomers of 4-chloro-18-nor-17€-hydroxymethyl-17€-methylandrost-1,4,13- triene-3-one conjugated with sulfate) were detected in urine up to the last samples collected in the excretion studies available, showing for these studies the same detection window as the recently reported long-term metabolites excreted in the glucuronide fraction.
For stanozolol, eleven sulfate metabolites were detected, including 16Å-hydroxy-stanozolol 16Å-sulfate and other metabolites characterized based on mass spectrometric data: two isomers of 16Å-hydroxy-stanozolol 16Å-sulfate; two isomers of stanozolol N-sulfate; three isomers of hydroxy-stanozolol N-sulfate; 16-oxo-stanozolol N-sulfate; and 4Å-hydroxy-16- oxo-stanozolol N-sulfate. Detection times up to 10 days were obtained after oral administration and, therefore, the sulfate metabolites did not improve the retrospectivity obtained with the recently described epistanozolol N-glucuronide, resulting from the initial formation of a sulfate conjugate.
For clostebol, sixteen sulfate metabolites were detected. One of them, characterized as 4€- chloro-5‚-androst-3Å-ol-17-one 3Å-sulfate, was detected up to the last sample collected in the excretion studies evaluated (31 days) and, therefore, significantly improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction.
The results of the project demonstrate the importance of sulfatation as a phase II metabolic pathway of AAS and the interest to study this metabolic fraction to look for new long-term metabolites, and the need to incorporate these metabolites in the initial testing procedures for AAS in all antidoping laboratories.

Code: 12C17MM 

The metabolic steps that lead to drug clearance in the human body are divided into two distinct parts: chemical modifications of the parent compound (phase I); and conjugation of either the parent drug and/or the phase I metabolite(s) with endogenous molecules (phase II).  Generally, cytochrome P450 systems are most important for phase I, while UDP glycosyltransferases (UGTs) for phase II reactions. UGTs catalyze the conjugation of the glucuronic acid moiety from UDP-glucuronic acid to many xenobiotics, including drugs/metabolites banned by the WADA. Conjugation facilitates the excretion of the compounds into urine. The kinetics of elimination of a compound could be influenced by genetic variability and/or by the co-administration of drugs that interact with UGTs. In this context, previous studies demonstrated that non steroidal anti-inflammatory drugs, like diclofenac and ibuprofen, decrease the enzymatic activity of the most important UGTs involved in the phase II metabolism of testosterone, whereas benzodiazepines and antifungals alter the glucuronidation kinetics of morphine and codeine.

The preliminary results of an ongoing project, also funded by the WADA, focused on the study of CYP450s inhibitors as potential “masking agents”, proved that in the presence of CYP450s inhibitors the in vitro phase I metabolic profiles of several classes of banned substances were extensively modified. In this project we plan to follow an analogous approach to preliminarily investigate whether the co-administration of doping agents with other xenobiotics that are substrate of the UGTs could be used by athletes as a strategy to evade anti-doping testing.  Specifically, the in vitro phase II metabolic profile of selected banned compounds, with especially emphasis on threshold substances (i.e 19-norandrosterone, morphine and tetrahydrocannabinol) will be assessed individually and in the presence of selected medicaments commonly used by athletes (non steroidal anti-inflammatory agents, antifungal agents and benzodiazepines).

Main Findings: 

This research project focused on the evaluation of, the inhibitory effect of  on-prohibited drugs on the phase II metabolism of prohibited drugs. More specifically, we have considered the effect, on the kinetics of the glucuronation reaction catalyzed by uridinediphosphateglucuronosyltransferases (UGT) of 19-norandrosterone, testosterone, epitestosterone and morphine, of non-prohibited drugs belonging to the classes of antifungals (fluconazole, itraconazole, ketoconazole and miconazole), benzodiazepines (alprazolam, bromazepam, diazepam and triazolam) and non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen and nimesulide).

In the first part of this project, the in vitro metabolism protocol using either pooled human liver microsomes or recombinant human UGT isoenzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) was optimized and validated in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro protocol was subsequently used to perform the inhibition studies in the presence of non banned medicaments commonly used by athletes (primarily among them antifungals, benzodiazepines and non-steroidal anti-inflammatory drugs).

19-norandrosterone, testosterone, epitestosterone and morphine undergo extensive glucuronidation confirming that the in vitro model developed and optimized in this study provides a good representation of the metabolic glucuronidation reactions in humans, being these data in accordance with information reported by other investigators.  The UGT isoforms principally involved in the phase II metabolism are the UGT2B7 and UGT2B17 for 19-
norandrosterone, the UGT2B15 and UGT2B17 for testosterone, the UGT2B4 and UGT2B7 for epitestosterone and UGT2B7 for morphine.

Concerning the inhibition study, the results obtained showed that the enzymatic activity of the UGT isoforms involved in the glucuronidation of 19-norandrosterone, testosterone, epitestosterone and morphine are extensively altered in the presence of the antifungals ketoconazole and miconazole. Moderate variations were, instead, registered in the presence of itraconazole, triazolam, diclofenac and ibuprofen and no significant modifications were measured in the presence of the others agents studies. The inhibitory effect of diclofenac, ketoconazole, ibuprofen, itraconazole, miconazole and triazolam on the 19-norandrosterone, epitestosterone, testosterone and morphine glucuronide formation is not an irreversible process, but involves competitive and miхed type mechanisms.

These evidences impose that drug-drug interaction has to be taken into account in doping control analysis, to reduce the risk of reporting uncorrected results.

Code: 12D18TM

In 2011 a study reported that of 2185 urine samples from professional athletes (spanning 43 different sport disciplines), ‘active’ consumption (not passive environmental exposure) of nicotine and/or tobacco-related alkaloids were detected in 15% immediately prior to and during sport practice. That same year WADA added nicotine to its Monitoring Program. Taken together, this highlights the extensive and alarming use of nicotine in professional sport.

Inclusion on the Monitoring Program implies nicotine satisfied three criteria of potentially i) enhancing performance, ii) representing a health threat for athletes and, iii) altering the spirit of sport. However, whilst evidence for the latter two criteria features in abundance the former criterion suffers from a lack of evidence or examination. To date, only two studies have investigated the effects of nicotine on sporting/exercise performance, with one showing an endurance improvement of 17% whilst the other found no effect.

However, evidence from similarly acting drugs such as bupropion and methylphenidate suggests that any performance improvements only occur at warmer ambient temperatures, when their action as central stimulants becomes more obvious. These substances also cause body temperature and heart rate to be pushed closer to the limit of safety.

Many competitive sporting events, especially endurance, take place during the summer period or in warm climates. Nicotine is known to reduce skin blood flow. When combined with exercise this raises a serious concern over the safety of its use during exercise/sport in a warm environment, where increasing skin blood flow and sweating are the primary routes of heat loss, possibly placing an athlete at greater risk of developing a heat illness.

This project will extend the limited data on whether nicotine does improve endurance performance, and determine whether at higher ambient temperatures nicotine may lead to developing heat illness, increasing an athlete’s health risk.

Main Findings

Nicotine was added to the 2012 WADA Monitoring Program after it was found that 15% of professional athletes actively consume it. Previous research has demonstrated that transdermal nicotine administration improves cycling time to exhaustion but does not influence 1h cycling time-trial performance in a moderate (~20°C) ambient temperature. However, evidence from similarly acting drugs suggests that any performance improvements only occur at warmer ambient temperatures, when they also cause body temperature and cardiovascular function to be pushed closer to the limit of safety.

Many competitive sporting events take place during the summer period or in warm climates. Nicotine is known to reduce skin blood flow and when combined with exercise this raises a serious concern over the safety of its use during exercise in a warm environment, where increasing skin blood flow and sweating are the primary routes of heat loss, possibly placing an athlete at greater risk of developing a heat illness.

Therefore, this project extended the limited data on whether nicotine does improve endurance performance, and whether at higher ambient temperatures it may lead to developing heat illness, increasing an athlete’s health risk. A final aim was to determine whether measurement of nicotine’s major metabolite, cotinine, should be performed using blood (serum) or urine. Ten trained, male cyclists cycled for 60 min at 55% of their maximum power output followed by a work-dependent time-trial at 75% of their maximum power output (~30 min) on four occasions. Twice trials were conducted in 18°C and twice in 30°C and in each environment they were administered placebo and nicotine via transdermal patch (7 mg·24hr-1) the evening before.

Ambient temperature affected time-trial performance, such that both time to complete the set work was faster (4±1min) and mean power output was higher (24±8W) in 18°C than 30°C. Forearm blood flow (venous occlusion plethysmography) values increased from 18°C to 30°C (6±1ml·100ml·min-1) and decreased from placebo to nicotine (3±1ml·100ml·min-1). Mean weighted (12-site) skin temperature values were lower in 18°C than 30°C (3.7±0.3°C) and higher with nicotine than placebo (0.5±0.2°C). Core (gastro-intestinal) temperature values were lower in 18°C than 30°C (0.4 ± 0.1°C) and higher with nicotine than placebo (0.2±0.1°C). Total sweat loss and % body weight loss were lower in 18°C than 30°C (0.6±0.1L and 0.7±0.2%). Heart rate was lower in 18°C than 30°C (8±1 beats·min-1). Cotinine concentrations immediately before exercise were higher in urine (140 ± 127 ng/ml-1) than serum (31 ± 29 ng/ml-1).

On the basis of these results, i) under these conditions, nicotine is neither ergogenic nor ergolytic, ii) nicotine does increase the risk for heat illness when administered during exercise coupled with heat stress, and iii) anti-doping efforts should concentrate on athlete urine sampling.

Code: 12C18PS

The detectability of erythropoiesis stimulating agent abuse is a major challenge on the way to a doping free sport. Despite existing tests, the detection of ESA abuse has proven to be very difficult. According to the large number of novel ESAs that are marketed around the world, or are in preclinical or clinical trials it seems to be virtually impossible to develop direct tests for each substance. To face this problem, indirect detection strategies have been suggested. During the last decade, molecular biological techniques enabled screening for the human transcriptome.

However, the ´transcriptomic´ approach is still facing a number of problems before robust biomarker can be detected. The problems include biological- and technical bias that are accompanied with the choice of target RNA, sample type and sample processing. Here, we suggest for the first time to utilize micro RNA (miRNA) extracted from mature red blood cells.

Since erythrocytes do not contain nuclei, gene – environment interactions that typically hamper establishing gene signatures for doping are excluded and will not affect miRNA levels. This stability against physiological bias or other factors along with their long life-span makes the erythrocytes the ultimate cell type for addressing the question of whether gene signatures for ESA doping can be found. With this pilot study we want to evaluate the feasibility of erythropoiesis specific miRNA profiling for ESA doping detection in red blood cells.

Main findings

This study intended to examine recombinant human EPO related effects on microRNA (miRNA) profile of red blood cells (RBCs), to discover markers which are indicative for altered erythropoiesis in induced by rhEPO. RBCs, as a homogenous sample material, were chosen to avoid typical bias that can hamper reliable miRNA marker detection, such as shifts in blood cell composition and cell lysis.

A double blind placebo controlled cross over study, including 14 healthy male participants, who received microdoses of recombinant human erythropoietin was conducted at the University of Glasgow, supervised by Prof. Pitsiladis. To discover potential novel biomarkers, which are indicative for rhEPO doping with microdoses, highly purified red blood cell samples from 7 subjects before and after 6 weeks of rhEPO administration were analyzed using Next Generation Sequencing. As a result of 6 weeks rhEPO administration mean haematocrit and haemoglobin concentration values increased significantly (p<0.01) from baseline values (42,57 ± 1,71 to 47,41 ± 2,97 (%) and 14.77 ± 0.61 to 16.36 ± 1,12 (g/dl), respectively). Concomitantly, the percentage of reticulocyte remained unchanged.

Using the Illumina NextSeq500 sequencing system -Illumina, San Diego, CA, USA- for small RNAs, RBC miRNA profiles of 7 subjects were discovered before and after 6 weeks of microdoses rhEPO administration. 13 out of 1,202 discovered mature miRNAs were significantly dysregulated (P<0.05, FDR<0.05). The most dysregulated miRNA, has-miR-150-5p is known to be down-regulated in RBCs and erythropoietic progenitors cells of polycythemia vera patients who suffer from disordered erythropoiesis. This information is concordant with our findings that has-miR-150-5p is down-regulated in healthy subjects who received rhEPO. Presumably, microdoses of rhEPO dysregulate the tightly controlled process of erythropoiesis and can be reflected by screening the RBCs miRNA transcriptome. This makes RBC specific miRNAs valuable biomarker candidates, which could be used in an additive way to generate a predictive marker for rhEPO doping, independent of changes in reticulocyte counts.