Projets de recherche

Code: R11B01JS

The research group of N. Leuenberger. J. Saugy, R. B. Mortensen, P. J. Schatz. S. Giraud and M. Saugy has demonstrated the detection or Hema tide™ (OMONTYS.., in anti-doping samples. This approach 1s based on the measurement of pegmesatlde in human serum/plasma samples by an assay and western blot employing specific monoclonal antibodies. This test has been implemented in WADA accredited laboratories as research test system based on ELISA (Enzyme-Linked lmmunoSorbent Assay) technique (screening method) and western blot (confirmation method}.

Actually the coated antibody of the assay is fresh coated directly before the measurement. Between different antibody batches there are variations in the geherated data of the ELISA The aim of this project Is to identfly a method to ensure antibody batches which result m reproducible data. Diflerent methods of antibody production, coating and storage of the antibody and the coated rnicroplate will be tested to establish a safe verilication procedure to detect ?g1nesatide in human blood samples.

The successful development of a stable and functional coated antl-peginesatide antibody is ot

crucial importance for the availability of commercial test kits.

Main findings

The antibodies mAb anti-PEG WADA1A8 and mAb anti-Hematide WADA11F9 of the HematideTM ELISA generate in sandwich with the analyte HematideTM well differentia-ble values. The labs of Paris and Lausanne seem to have problems with the stability of the mAb anti-PEG WADA1A8 antibody. Their results are often not reproducible.

The results of this project cannot trace back these problems to the antibody. The phys-ical QC showed no changing in the structure of the protein. The antibody WADA1A8 shows a stable structure over the time of 12 months. However, the manual preparation showed the same variability in our lab when fresh prepared plates were used. If pa-rameters are fixed (see section 4.3.3) these variability cannot be find. The reason of the problems evolve from the suboptimal conditions for the ELISA and not researched backgrounds for the analyte HematideTM and the both antibodies.

The PEG linker and the anti-PEG antibody are sensitive for detergents because of homologous structures to polyethylene-glycol. In the published assay system [N. Leuenberger et al., Methods for detection and confirmation of Hema-tideTM/peginesatide in anti-doping samples, Forensic Sci. Int.(2011)] Tween 20 is used in several assay steps. Also in cell culture it is common that detergents like Kolli-phor (also known as P188/Pluronic) are added. This could influence the anti-PEG an-tibody already in cell cultivation. This omnipresent application can result in complica-tions of the ELISA. In additional experiments (section 4.3.4) the effect of the replace-ment of Tween 20 against CHAPS shows a significant improvement of the assay sys-tem.

Another possibility to improve the assay is to change the antibodies in the ELISA sandwich. It would be of advantage to use the analyte specific antibody anti-Hematide (WADA11F9) as immobilized captor-antibody. The actual coated antibody anti-PEG antibody (WADA1A8) could be engaged by polyethylene-glycol homologous struc-tures. The assay could be more specific and more stable by this change.

Also critical for the test system is the performance. The microplates should not be coated freshly by the operator. The ELISA should be as easy as possible in practice. A lot of factors are unknown yet. The influence of temperature variation, the endpoints of binding are not determined. The analyte HematideTM has to be analyzed for behav-ior.

After these analysis and optimization of the assay system there are good perspectives to create a well working, reproducible ELISA which is user-friendly and possible to produce commercially.

Code: R10M1MA

There is a consensus in the international community that action must be taken against those behind-the-scene individuals who propagate and facilitate doping in sport. The first step must be to identify those responsible. Law enforcement agencies have used Social Network Analysis (SNA) to tease apart and identify key figures in complex investigations concerning drug trafficking, counter terrorism, money laundering, organised crime and people smuggling. It is proposed to apply SNA to study the social affiliations of convicted blood dopers. This will provide a baseline from which to gain a more sophisticated understanding of the blood doping environment.

Social Network Analysis requires three successive phases of activity. First, information about the links and relationships between the entities under investigation are extracted from existing data (e.g., newspaper reports, internet sites, public databases). Next a structural analysis is conducted to identify central members, subgroups and patterns of interaction between the parties. Finally, those relationships and associations are visualised using a software program that spatially distributes individuals according to their social interactions (i.e., individuals with strong ties are plotted closest together).

Main findings

The use of banned blood transfusions by athletes erodes the integrity of elite sport. Athletes are lured by the substantial performance advantage bestowed by blood doping, together with the realisation that its use cannot be detected by doping controls. Rogue doctors and transfusion technicians have set up extensive and sophisticated doping networks to meet this demand and the covert provision of specialist advice and equipment has proven to be a lucrative trade.

Novel strategies are required to identify participants in blood doping networks. One avenue to gather evidence about networks, and indirectly the athletes who are utilising such facilities, is to obtain eyewitness testimony via investigative interviews. For example, with regard to the transfusion networks that have so far been discovered, invariably some peripheral teammates and support staff of the doped athletes were aware of the existence of the network. However for this interview-based approach to be cost effective, there must be some way to flag persons of interest for interviews amongst the pool of several thousand athletes and support staff.

This study evaluated whether network analysis is an effective method for targeting interviewees for investigations into blood doping networks. Specifically, whether it was possible to identify and rank teammates and staffers in terms of their closeness to riders who had been implicated in blood doping practices.

The study determined that network analysis was capable of prioritising individuals based on the premise that individuals with the greatest exposure to doped athletes would have the highest likelihood of possessing relevant information. These findings have implications for antidoping authorities who seek to rationalise their allocation of scarce investigatory resources. This study makes recommendations for how network analysis can be incorporated into investigative operations.

Code: 10A24XD 

In extremely rare circumstances the presence of 19-norandrosterone (19-NA) in human urine can be explained by the “instability” or “activity” of the urine specimens. This is most likely due to the sample transportation and storage in the laboratory and is based on the 19- demethylation of abundant endogenous steroids in the urine samples.

Tests for the assessment of the activity of the urine samples have been established. The hypothesis is that 19-NA will be produced by 19- demethylation of androsterone, the main androgen metabolite present in the urine. Briefly, an aliquot of sample to be tested for activity will be incubated in the presence of deuterated androsterone. The formation of deuterated 19-NA will be the proof of urine activity.  

From the data collected until now, it appears that the instability of these urine samples is due to enzymatic activity expressed (from exogenous origin) in the sample. It seems unlikely that the 19-demethylation would be the result of a pure, unassisted chemical reaction. If this were the case, this phenomenon would be reproducible at any time, and is not.  The growth of microorganisms in the urine samples is not unlikely. It is reasonable to link this enzymatic activity to the presence of a microorganism growing in the urine.  The expression of aromatase is restricted to the gonads and brain in many vertebrates, from aquatic and avian species to mammals. The removal of the methyl group in the 19 position seems not linked to an aromatization process since no microorganism expresses such an enzyme.  An important demethylase enzyme present in some microorganisms is the 14-demethylase (CYP51A1). The activity of this enzyme is crucial to the life of these microorganisms since it is the responsible for the formation of ergosterol from lanosterol. The hypothesis is that CYP51 uses androsterone as “substrate” consequently producing 19-NA.

Main Findings: 

The present was focused on the hypothesis that the CYP51 (14α-demethylase) of fungal origin may be the cause of the alteration of the metabolic profile found in the so-called active urines. In these cases a concentration of 19-NA beyond the limits allowed, but with a ratio 19-NA/19-NE reversed compared to that A / E (usually the concentration of 19-NA in urine is greater) are observed.
To check if the CYP51 is able to demethylate androsterone and / or etiocholanolone, yeasts such as S. cerevisiae and C. albicans were chosen. As substrates of of the yeasts, androsterone, etiocholanolone and  androstenedione were selected as being the most probable substrates, based on their structure and on the amount present in routine anti-doping samples.
The following experiments were performed:
1. S. cerevisiae and C. albicans were incubated in the presence of androsterone, etiocholanolone, and androstenedione in culture medium and in synthetic urine (in sterile conditions). The products of fungal metabolism were found both in the supernatant and in cell lysate.
2. S. cerevisiae and C. albicans were incubated in human urine in non-sterile conditions. Alterations of the hormonal profile in urine were evaluated according to the protocols of the anti-doping laboratory of Rome, used in the screening of banned substances.
The main conclusions of the present project are:
· The demethylation process of steroids produced in the so-called active urines is not originated chemically, at least under the common laboratory conditions during the sample processing of the urine samples in doping control.
· The aromatization process that is the responsible of the in-vivo formation of 19-norandrosterone is not the responsible for the 19-demethylation ex-vivo during sample storage of the urine samples.
· In cultured C. albicans and S. cerevisiae conducted in SDB medium and in the presence of steroid hormones, any relevant reaction of demethylation was observed. By-products of fungal metabolism on these hormones have been detected.
· In human urine inoculated with the same microorganisms, changes in the hormonal profile were detected, that can be attributed to the activity of C. albicans. These observations were previously detected in culture medium under sterile conditions. Even in these experiments the formation of 19-narandrosterone and 19-noretiocholanolone was not detected.
· Although the formation of 19-norsteroids object of this study was not detected, the changes in urinary steroid profile as a result of contamination with fungi are considered to be relevant for the correct interpretation of the data in doping control.
Under the experimental conditions described, where the functional growth of fungi has been demonstrated, the transformation of androsterone and etiocolanolone to their respective 19-norderivatives, as happens in the case of active urine, was not observed.  With the current obtained knowledge, we can say that the ex-vivo process of demethylation is complex and involving several actors. The proposed test to demonstrate the “activity” of a particular sample provided that deuterated androsterone or etiocholanolone were transformed in their corresponding 19-nor deuterated products. This is not the case with the presence of the tested fungi alone. The process of demethylation implies the presence of an unsaturation in alpha with respect to the methyl group to be removed, absent in androsterone and etiocholanolone. Our experimental evidence supports the hypothesis that the process is a multistep process consisting in a first dehydrogenation of A ring, most probably in the C1 position,
followed by the multistep oxidation or the methyl group

Code: 10A1PV

The project aims to contribute to the development of the steroidal subunit of the Athlete’s Biological Passport (ABP) which is hitherto solely validated on a few markers, such as the T/E ratio.  

Recently, novel biomarkers were found that increase significantly the time detection window after administration of small doses of T, DHT and DHEA using the Adaptive Model of the ABP. These biomarkers consist of steroid ratios including minor metabolites sensitive to steroid administration. More data on intra-individual variation of the involved minor metabolites are nevertheless necessary to validate the proposed biomarkers. Therefore a large-scale investigation of long-term within-subject behaviour of an extended steroid profile will be conducted.

Data obtained on a larger cohort with comprehensive steroid profiling methods will allow the development of a multi-parametric marker of steroid doping that comprises the whole steroid profile. This model statistically classifies abnormal steroid profiles by outputting a single score. Longitudinal evaluation of this ‘Abnormal Steroid Profile Score’ (cfr. Abnormal Blood Profile Score in the Blood Passport) monitors any alteration in the steroid profile regardless to its cause. The goals are twofold. Firstly, when applied at the individual level, this model will allow the general screening of doping with endogenous steroids, food supplements and substances manipulating the steroid profile, such as after ethanol consumption. Secondly, and in contrary to blood doping and doping with growth hormone wherein markers having a detection time long enough to estimate the prevalence of doping already exist, this score might provide accurate estimates of the prevalence of steroid doping in elite sports when applied at the population level.

Moreover, the influence of genetic polymorphism on the new steroid profile parameters and an Abnormal Steroid Profile Score will be studied in order to increase the sensitivity of the model.

Main Findings:

A combination of the Support Vector Machine (SVM) algorithm with a comprehensive approach of steroid profiling resulted in a steroidomic model that enables to differentiate normal steroid profiles from abnormal ones. Theoretically, the SVM tool plots all monitored\steroids in a multi-dimensional hyperspace which makes the use of steroid ratios redundant to obtain a strategy with optimal detection sensitivity. Hence, the whole set of steroid profile values can be evaluated at once. In our model, however, the degree of abnormality was quantified by an Abnormal Steroid Profile Score (ASPS) for which values greater than 0.79 could be considered as deviating from normal.  
Since the introduction of the Athlete Biological Passport, the results of steroid profiling tests can be systematically stored in a central database enabling the estimation of the individual reference ranges. From such databases, longitudinal steroid profiling data can be made readily available to elaborate longitudinal strategies, thereby omitting a large contribution of the inter-individual variance. Similarly, the raw SVM model was improved by standardizing the training set using individual mean and standard deviation obtained with the adaptive model. The combination of the adaptive model and the SVM enhances the general performance accuracy of the raw SVM model from 62% to 84%, disregarding the kind of endogenous steroid administered. The diagnostic sensitivity of the resulting ASPS was 55% in a post-administration period of 7 days. Altered steroid profiles can be found until 5 days after ingesting a small single doses of T or DHEA or after topical application of T or DHT in therapeutically recommended doses. This drastic increase in sensitivity can be explained by the ability of the model to sensitively distinguish a prolonged recovery state of the steroid metabolism which is restoring the homeostasis of steroid profile to known basal levels.  
Since the model was trained on data obtained after T, DHT and DHEA administration, the model risked to be overfitted i.e. a specific detection tool for these steroids. This problem was addressed by leave-one-subject-out cross-validation and testing of the model on another volunteer, with another dose of DHEA and with other steroids. Testing of the excretion data from a 100mg dose of DHEA, 50mg Adion and 7-keto-DHEA ingested by another volunteer showed a clear response of the ASPSs. This indicates the polyvalent nature of the SVM model to detect any small disturbance of the steroid profile. Moreover, the high sensitivity of 97% obtained for this new test set illustrates the potential of the ASPS as a powerful biomarker for the general detection of misuse with endogenous steroids. Although, this single model shows excellent sensitivity for a wide range of administered steroids, it cannot specify which cause resulted in an aberrant steroid profile. For this information, specific metabolites should be evaluated separately. 
Despite the excellent preliminary results on low dose administration studies conducted on a limited study population - including subjects with atypical T/E’s that challenge the classification -, the applicability of this strategy will require further work and large scale validation procedure. In order to implement the ASPS in routine testing as a sensitive marker for of any misuse with endogenous steroids, the model should be tested on larger cohorts of data and external influences on the steroid profile that can alter the ASPS should be scrutinized in the future.  
In conclusion, a new strategy was developed that returns a single value ASPS as a denotation of the degree of abnormality of a steroid profile containing 24 steroid metabolites. With this strategy, the alteration of the steroid profile, caused by a variety of endogenous steroids, can be detected very sensitively. The longitudinal SVM model was shown to be a general model which can result in long detection of small doses of oral and topical steroid formulations up to 5 days. The overall model performance was very good, particularly when coupled with the longitudinal results from the adaptive Bayesian model. The combination of computer aided techniques as the Bayesian adaptive model and SVM algorithm provide a valuable steroidomic strategy for the long term detection of misuse with endogenous steroids in complement with current steroid profiling methods.

Code: 10D3AR

Anabolic androgenic steroids (AAS) behave differently in the human body. The human organism deals with these compounds differently in respect of uptake, distribution into different organs, metabolism and excretion. We recently demonstrated that ¾ of Oriental people have a severely compromised capacity to excrete testosterone in the urine compared to only 10 % in people from the west. This is a confounder in the doping test program. In the way towards personalised test programmes, Bayesian inference techniques are known to suit particularly well. To further improve the new individualised steroid profile passport, we will conduct several human studies with different routes of administration, preparations, and doses of testosterone in order to assess the sensitivity and specificity of the test program. Our research program encompasses projects designed to investigate variation in AAS disposition including inter-ethnic and gender differences. We will study both testosterone and synthesized agents with anabolic androgenic effects (nandrolone, stanozolone). We also plan to study interactions between AAS turnover and common drugs used by sportsmen, e.g. non steroidal antiinflammatory drugs. Theoretically such drugs may mask the use and enhance the effect of AAS.

Main Findings: 

The overall aim of the project has been to identify and quantitate genetic and other mechanisms of variation in the bioavailability, metabolism, and excretion of androgens (endogenous as well as exogenous). We have also focussed on the serum concentration profiles of testosterone in genetic panels of healthy volunteers. Serum concentrations are relevant comparators to effects of, and adverse reactions to androgens. Therefore, they are highly interesting. 
Serum concentrations and bioavailability have been studied and related to bioactivating enzymes and transporters. Variation in these parameters are likely determinants of the effects of androgens and of interest not only for the capacity of doping tests but also for the user profile, risk exposure etc.
a) Validation of putative biomarkers that could be used to increase the positive and the negative predictive values of testosterone doping (PPV, NPV) was addressed in the following publication: Schulze JJ, Thörngren JO, Garle M, Ekström L, Rane A. “Androgen sulfation in healthy UDP-glucuronosyl transferase 2B17 enzyme-deficient men” J Clin Endocrinol Metab. 2011 Nov;96(11):3440-7. Epub 2011 Aug 17.
b) Other endpoints related to adverse effects of testosterone were also studied in healthy volunteers, e.g. the serum lipid profile. Garevik N, Skogastierna C, Rane A, Ekstrom L. “Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA Reductase” Subst Abuse Treat Prev Policy. 2012 Mar 20;7(1):12. [Epub ahead of print]                                     c) The effect of androgen abuse on endocrine pituitary-gonadal axis was studied in Gårevik N, Strahm E, Garle M, Lundmark J, Ståhle L, Ekström L, Rane A. “Long term perturbation of endocrine parameters and cholesterol metabolism after discontinued abuse of anabolic androgenic steroids” J Steroid Biochem Mol Biol. 2011 Nov;127(3-5):295-300. Epub 2011 Aug 22
d) We have shown that the main nandrolone metabolite (19 norandrosterone glucuronide) could be detected in urine up to one year in AAS abusers.
e) The androgen profile in urine in different female populations was studied. It is of great importance that the athlete’s steroid passport program, will be able to correct for and consider all possible variability in longitudinal steroid profiles in women.  Ekström L, Gök E, Johansson M, Garle M, Rane A and Schulze JJ. Doping and Genetic Testing: Sex Difference in UGT2B15 expression, Testosterone Glucuronidation Activity and Urinary Testosterone/Epitestosterone ratio. Current Pharmacogenomics and Personalized medicine, 2012, June 10:125-131
f) We have observed conspicuous inter-individual differences in serum concentrations of testosterone after administration of the same testosterone dose to healthy volunteers.  Ekström, L., Schulze, J., Guillemette, C., Belanger, A., Rane A. “Bioavailability of testosterone enanthate dependent on genetic variation in the phosphodiesterase 7B (PDE7B) but not on the UDP-glucuronosyltransferase (UGT2B17) gene” Pharmacogenetics and genomics 2011 Jun;21(6):325-32.            
g)  Determinants of androgen access to androgen receptors also include transporters, along with metabolising enzymes form various families. We have investigated organic anion transporting polypeptides (OATP). Schulze JJ, Johansson M, Rane A, Ekström L. “Genetic variation in SLCO2B1 is associated with serum levels of testosterone and its metabolites prior to and two days after testosterone administration” Current Pharmacogenomics and Personalized Medicine” to be published Vol. 10, No. 3, 2012.      h) In one publication; Sten, T., Finel, M., Ask, B., Rane, A., Ekström, L. “Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation”, Steroids 2009 Nov;74(12):971-7, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. 
i)  Since many of the individuals devoid of the UGT2B17 gene would not reach a T/E ratio of 4.0 after testosterone intake future test programs will most likely shift from the population based- to an individual-based T/E cut-off ratios using Bayesian inference.   Schulze, JJ., Lundmark, J., Garle., Ekström, L., Sottas, PE., Rane, A. ” Substantial advantage of a combined bayesian and genotyping approach in testosterone doping tests.” Steroids. 2009 Mar;74(3):365-8

Code: 10C23YP

A World Anti Doping Agency (WADA) funded research project in 2008 entitled “A Gene Microarray Based approach to the Detection of recombinant Human Erythropoietin Doping in Endurance Athletes” was designed to formulate new methods with improved discriminatory power relative to current available 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. The ongoing (LIVE) funded project, albeit of great scientific and diagnostic potential given recent data, focuses solely on mRNA as mRNA transcribed from DNA is translated into protein.

However, current opinion would suggest that micro RNA (miRNA), a class of recently discovered small RNA molecules, play a major role in post-transcriptional regulation, thought to regulate approximately 30% of all human protein coding genes. Therefore, it would be prudent for the purposes of the ongoing (LIVE) research project funded by WADA to assess the miRNA target interactions that correlate with effects on target mRNA levels in order to provide the necessary insight into the interaction between mRNA and miRNA and consequently the impact of gene expression on protein synthesis. This elaborate study provides a unique opportunity to gather all the data necessary to develop a robust diagnostic test in the shortest timescale possible. The additional funds requested will also allow the piloting of a simple, minimally invasive, safe and cost effective method of sampling, storing and extracting RNA from saliva.

Main Findings

Recombinant human erythropoietin (rHuEPO) improves performance and is frequently subject to abuse by athletes. The use of rHuEPO is prohibited by the World Anti-Doping Agency. To improve the sensitivity of current detection methods, new “omics”-based methods such as gene expression have generated promising results using whole blood (08C19YP). Current research focused on saliva gene expression and on blood-derived microRNAs (key post-transcriptional regulators of gene expression). 9 genes were commonly expressed in saliva after 2 weeks of rHuEPO administration, at the end of administration and 4 weeks after the end of rHuEPO administration Furthermore, a handful of blood-derived miRNAs were identified using different techniques, i.e. microarray, qPCR and sequencing. Further thorough validation experiments are required before any solid conclusion can be drawn. Nevertheless, “omics” signatures of rHuEPO administration from saliva gene expression and blood-derived miRNA provide further support for the idea that “omics” biomarkers have the greatest known potential to improve the performance of current anti-doping methods such as the Athlete Biological Passport for rHuEPO detection.

Code: 10B9US

Human chorionic gonadotropin (hCG) is used to restore gonadal function after use of anabolic hormones. These suppress gonadal function by inhibiting pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone. hCG stimulates steroid production in the gonads after use of anabolic steroids. hCG is available as pharmaceutical product and is used for doping.

Use of gonadotropins in sports is prohibited in males. hCG can be detected by immunological techniques in urine 7-10 days after administration. A concentration exceeding 5 IU/L is considered positive. Various hCG assays are used in anti-doping laboratories although results obtained by these differ. Urine contains degradation products of hCG, which different assays detect differently. Presently available hCG assays are clinically approved for use on serum but not for urine. The excretion rate of urine varies causing up to 10-fold variation in hCG concentration. Measuring urinary creatinine and correcting the hCG result accordingly can compensate for this, but these methods have not been validated. Furthermore, hCG immunoreactivity may be lost when urine is stored at -20 C and also by adsorption of hCG to the collection tubes.

We propose to develop standardized methods for determination of hCG in urine. The project comprises the following parts. 1. Characterization of the forms of hCG in urine after parenteral administration of hCG.

2. Development of a reference method for determination of hCG in urine including correction for variation in urine excretion rate.

3. Establishment of procedures for collection and storage of urine before assay.

4. Establishment of reference values for various forms of hCG in urine from males.

5. Comparison of the ability of selected commercial assays to identify the various forms of hCG occurring in urine.

6. Development of quality control procedures for assay of hCG in urine.

Main Findings

In order to evaluate methods to be used in doping control for human chorionic gonadotropin (hCG), we have determined the urine concentrations of intact hCG and its subunits, hCGβ, hCGα and the core fragment of hCGβ (hCGβcf ) in about 1000 doping control urine samples from male athletes, who agreed to the use of their samples for research purposes. In addition, hCG and hCGβwere determined in samples obtained during the first 9 days after injection of urinary or recombinant hCG to 12 male volunteers.

The results obtained by various hCG assays varied considerably, but results above 5 IU/L were observed in only 3 samples with two assays. After correction for urine density, no sample had a result above 3 IU/L in two different assays. With the present decision limit of 5 IU/L, no falsely elevated results were obtained if a positive test required elevated results with two different assays. The problem caused by adsorption of hCG to the precipitate forming when urine is frozen needs to be taken into account when selecting methods and sample handling protocols to be used for doping control. In the present study, the samples were not frozen before analysis and thus the results for these are valid for establishment of cut-off values for doping control provided that samples are not frozen before assay. Methods to avoid formation of precipitates in frozen urine need to be developed.

Of the methods studied, the AutoDelfia and Delfia Express assays are best suited for doping control.

Code: 10C12PS

Project Aims

Many of the different substances and procedures that have the potential to be abused for doping, finally act in the same way within the athlete’s body by activating just a few key pathways relevant for mediating performance enhancing effects. This common feature may eventually lead to common and more or less permanent alterations on the molecular level within the athlete’s blood. Finding just a single specific signature for doping would therefore serve as an indirect prove for many different procedures and substances that could have been abused.

In this project we will exploit the possibility to detect molecular signatures of gene doping based on the level of proteins (proteome), RNAs (transcriptome) or the non-coding, regulatory microRNA that are modified upon modifications of the pathways induced by IGF1 and HIF1a, the former improving muscle properties and the latter increasing oxygen delivery and aerobic capacity. For this purpose we will in first place assess data on the variability of gene expression signatures found in the blood cells of athletes under various physiological conditions.

In second place we will then look for molecular signatures of doping in two mouse models using gene transfer with HIF1a and with IGF1 as a procedure that is currently undetectable. Along with our search for signatures specific for these doping procedures we will try to show long-term, direct detection using a novel PCR procedure which we have recently developed for WADA.

All data obtained in this project will be supplied to the WADA Informatics Facility for extensive cross study comparison in order to assess the specificity of potential molecular signatures for the detection of doping.

Main Findings

The project consisted out of three main parts. Part 1 was an in vivo study in cyclists looking for changes in the transcriptome in response to training in normoxia and in normobaric hypoxia simulating an altitude training session. Part 2 was investigating in an in vitro study which candidate genes could be assessed on mRNA or miRNA level in order to detect gene expression changes in response to hypoxia or Insulin like growth Factor 1 (IGF1). Part 3 was an in vivo study in mice that underwent gene transfer with IGF1 as a potential gene doping scenario and the feasibility of both indirect detection and direct detection were investigated. Results and general conclusions

In study one we revealed that a typical endurance training session at 75 % of the individual anaerobic threshold under hypoxic and under normoxic conditions only induced some minor alterations in gene expression. 16 candidate genes showed a more than two-fold alteration in gene expression compared to resting conditions. The changes in gene expression between training in normoxic or in hypoxic settings were for most parts non-significant. Gene expression analysis revealed ankyrin repeat domain 37 (Ankrd37) as the only candidate. Subsequent in vitro testing and bioinformatics analysis in study part 2 confirmed Ankrd37 as a hypoxia sensitive candidate gene. Additionally, we revealed a list of miRNAs that are potential candidates for indirect detection of hypoxia or proliferative effects induced by IGF1. However, critical factors are a striking cell type specificity of these effects and a limited magnitude of gene expression alteration of candidates that show response in vivo and in vitro. High inter- and intra-individual differences that can be assumed to exist in typical settings in elite sports may therefore limit the applicability of our findings for indirect detection of the alteration of the HIF1 pathway.

With this regards the results of our third study following AAV1, AAV2 and AAV9 mediated gene transfer of IGF1 to mouse muscle in vivo were much more promising. Gene transfer of IGF1 to muscle cells evoked a strong proliferative effect and along with this the miRNA profile of all investigated candidates was severely down regulated. We then established a digital droplet PCR (ddPCR) based transgene detection approach using a priming strategy previously described by our working group that specifically amplifies sequences devoid of intronic DNA. We showed that ddPCR was able to directly detect the transgene following AAV9 mediated IGF1 gene transfer in the peripheral blood taken from the living animals for as much as 33 days following gene transfer.

Two of our findings are primarily of interest for the development of a doping test. First the highly proliferative effect of IGF1 that was highly associated with a general down regulation of miRNAs and second the ability to directly detect gene transfer on the level of the transgenic DNA.

Code: 10D21CG

Some athletes consume high volumes of drinks before the anti-doping sample collection in order either to recover from dehydration body conditions, which is normal after competition in high temperature and humid environmental conditions, or to dilute the urine and try to manipulate and mask detection of doping agents, where dilution as practice cannot be prohibited. Currently, for the International Standard of Testing, the suitable specific gravity (sg) of the doping control samples is 1.005 or higher measured with a refractometer, or 1.010 or higher with lab sticks.

Inappropriate sg for urine during sample collection procedure is faced by the collection of multiple samples until the Code requirement of the sg is met. Even if, the current specification of the appropriateness of the sg is scientifically valid, extensive knowledge on pharmacokinetics of hyperhydrated urine samples and subsequent influence on the detection of doping agents is lacking.

The goal of the current project is the performance of a series of pharmacokinetic studies of three different doping agents under various controlled hydration conditions to compare and examine the hydration influence in the urine excretion profile. Relating the hydration status to the pharmacokinetics of the three doping agents will allow drawing conclusions for the additional measures, if necessary, that can be taken during sample collection against the delivery of diluted urine that probably can mask or disturb the detection of the use of prohibited substances.

Main Findings

Hyperhydration effect on the endogenous androgenic anabolic steroids (EAAS) concentration levels was clearly demonstrated in the present study based on the individual and study volunteers population data. No significant difference was observed between the two hyperhydration agents with the water and Gatorade to act similarly on the urinary ‘steroid profile’ markers. The conventional WADA applied SG-adjustment method can eliminate the dilution induced effect and correct the EAAS concentrations by adjusting approximately to the baseline values. All the steroid ratios included to the urinary ‘steroid profile’ were remained unaffected by the hyperhydration due to the homeostasis of the steroid biosynthesis with the variability to be within 30% for the majority of data. No interference on the detectability of the selected transitions was observed due to the dilution of the samples after hyperhydration. The evaluation of the collected data using the steroid module of the ABP would be performed in order to examine in which extent the individually calculated thresholds of the ABP software are exceeded under hyperhydration conditions.

As final statement: the use of hyperhydration as a masking procedure by altering the urinary ‘steroid profile’ is not effective. Although, excessive fluid intake can alter the urinary ‘steroid profile’ markers, the steroid ratios remain unaffected and the SG-adjustment method officially used by WADA can fully eliminate any effect caused by dilution.

Hyperhydration induced decrease in LH urine concentration levels can be eliminated by adjusting the measured concentrations. Since reduced LH concentration levels may serve as an indicator for steroid abuse, adjustment of LH concentrations would be useful for the urinary ‘steroid profile’ evaluation. The WADA applied SG adjustment method can compensate the dilution induced effect and correct the LH concentrations by adjusting them close to the baseline values. Based on the results of the present study, SG-adjustment of LH concentrations in all the samples, as it is applied on the endogenous ‘steroid profile’ markers, could be a useful practice even if the low LH threshold is not applicable in the WADA TD2017.

In the present study, hyperhydration induced by two different agents (water and Gatorade) showed no significant changes on the hematological module of the ABP at any time during the study. The magnitude of difference between pre- and post-hydration values was too small and not statistically different 30 min after the ingestion independently of the hyperhydration agent. The evaluation of the collected data using the hematological module of the ABP has been requested from WADA in order to examine the extent that the individually calculated thresholds of the ABP software are exceeded under hyperhydration conditions.

Based on the serum PK profiles as well as on SAR-PAGE analysis and analyte mobility of both urine and serum samples no effect of hyperhydration was observed under the conditions examined on the present study in the anti-doping urine and blood analysis of rHuEPO.

Urine concentrations of budesonide and its metabolites were affected due to dilution after hyperhydration leading to increased percentage of samples below the MRPL of 30 ng/mL. However, WADA SG-adjustment based on the equation of Levine-Fahy was able to compensate the dilution effect by adjusting the concentration values completed to the baseline and therefore, decreasing the % of samples below the MRPL.

Code: 10D16RM

Changes in the rate of urine formation will affect the concentration of doping agents in the urine, but may not do so in a uniform manner. This raises the possibility that athletes may manipulate urine flow rates to evade detection of prohibited substances. It also offers a potential basis for appeal by an athlete who has failed a test. A tested athlete may be acutely dehydrated after training or competition, resulting in an extremely high total solute concentration in the urine sample obtained. If athletes are unable to produce a sample, and are allowed water or other drinks to stimulate urine flow the solute concentration of the urine sample obtained may then be extremely low due to an acute increase in urine flow rate. This is likely to be followed by a refractory period during which lipid-soluble metabolites will be excreted in lower than normal amounts as these compounds will be released from lipid depots at a relatively constant rate. Any sudden change in urine flow rate is likely to disturb this equilibrium and result in a temporary mis-match between concentrations in the tissues and the urine. It is therefore important to quantify the effects of changes in urine flow rate on the excretion patterns of prohibited substances so that allowance may be made for these effects. Urine composition may be affected by acute changes in the rate of urine formation, due to differences in the size, polarity, lipid solubility and other physio-chemical properties of components excreted. Changes in the measured concentration and/or time-course of excretion of diagnostic markers for prohibited substances will be of interest to anti-doping. A fundamental understanding of the effect of acute changes in hydration status will be particularly important in establishing acceptable parameters for metabolite excretion profiles with the application of the athlete passport.

Main Findings

This project examined changes in the time-course of excretion of diagnostic markers for prohibited substances in response to acute fluid loads and dehydrating exercise. Fundamentally this work aimed to answer the following questions; could an athlete cheat a doping test by drinking large volume of water or inadvertently test positive when dehydrated and is the current urine specific gravity correction fit for purpose? The present data demonstrate that ingestion of a large bolus of fluid not only results in the dilution of all steroids present in the urine, but also alters the excretion profile of these compounds to varying degrees; as evidenced by varying changes in T:E and the other ratios. While these effects display somewhat high inter-individual variability, the overall responses are consistent across both studies described in this report and highlight a need to further consider hydration status when interpreting steroid profile data collected as part of the Steroid Module of the Athlete Biological Passport. To limit opportunities for athletes to manipulate the outcome of the test, firm guidelines should be established to limit drinking to a fixed volume known to simulate sufficient diuresis in the vast majority of cases.