Projets de recherche

Code: 13A10PV 

Because of the advances in analytical methodology, it has become possible to Screen for compounds in complex biological matrices at very low concentrations. As a result, during the last decade, the role of minor steroid metabolites have gained importance in steroid detection due to their more specific nature. In this light, our group developed an analytical screening method that encompasses minor androgenic steroid metabolites. Some of these minor steroid metabolites are promising new biomarkers in steroid profiling and can contribute substantially to the evidence of misuse with various endogenous steroids. The endogenous concentrations of these minor metabolites are very low and a strong increase in concentration is obtained when anabolic steroids are administered. This makes these minor metabolites, theoretically, excellent compounds to confirm on IRMS because the endogenous dilution is very small. During this project, a method will be developed that is able to analyze some of these minor metabolites on IRMS. Urinary concentrations beneath a certain concentration threshold are considered as endogenous, urine samples with a higher concentration are suspicious and will be forwarded to IRMS for confirmation. This approach is complementary to the present classic markers (of which the T/E ratio is the most important one) and will reduce the number of false negatives on two levels. It has been shown in the past that the T/E ratio does not always increase with the administration of testosterone. In such cases, minor metabolites might trigger an IRMS confirmation, whereas the classic markers do not. On a second level, elevated values of classic markers might still lead to a negative IRMS result if the endogenous dilution is too big. For minor metabolites this effect can almost be neglected.

Main Findings: 

Screening: 
In most excretion studies, the minor metabolites had shorter detection times than at least one of the traditional screening markers that can trigger on IRMS confirmation (an elevated T/E ratio, elevated concentration of androsterone, etiocholanolone,…). This means that all samples that had elevated concentrations for a minor metabolite, would have been labelled ‘suspicious’ and forwarded to IRMS anyway because at least one other marker had a suspicious value. Nevertheless, when dealing with steroid profiles, one must always keep the large inter-individual differences in mind, meaning that this observation is not necessarily applicable for every individual. In the past, our lab received a sample with perfectly normal values for the traditional steroid profile parameters according to population based reference intervals. However, the 6αOH-ADION concentration was elevated, leading to an IRMS confirmation and consequently an adverse analytical finding was revealed. Based on the traditional parameters alone there would not have been an IRMS confirmation, illustrating the usefulness of monitoring 6αOH-ADION in the steroid profile.  
IRMS analysis 
In general, the IRMS detection time of the minor metabolites does not seem to outperform the traditional IRMS target compounds. There is a sharp concentration increase of minor metabolites after administration, but the concentrations also drop again relatively fast and this neutralizes the advantage of lower endogenous dilution. As a result, the traditional IRMS target compounds have a longer window in which their δ13C values remain in the exogenous range.

Code: T13M06AB

1. We propose to test several sample processing protocols following samples thawing (heating, mixing and digestion with a suitable restriction enzyme) to identify a procedure that ensures full homogeneity of thawed samples and their efficient amplification in PCR.

2. Once such protocol is found, we will use it to test if the presence of background nucleic acid in the RM formulation improves its stability at -20°C and -80°C.

3. In addition to these two main objectives, we propose to continue testing stability of the RM at two additional time points (fifteen and twenty four months).

4. Finally, as the best storage condition identified in our previous study was 4°C, we would like to test if RM ultrafiltration or addition to it of sodium azide as antibacterial measure for prolonged storage at 4°C affect its analysis by real-time PCR or digital PCR.

Main Findings

This study was a continuation of an earlier WADA-funded project on the development of a synthetic DNA reference material (RM) for use in testing for EPO gene doping. The main aim of the study was to investigate the factors that affect stability of the RM during storage at freezing temperatures. Firstly, we confirmed that the previously observed decrease in the concentration of the RM stored frozen was, indeed, the result of the RM degradation and not due to its inadequate reconstitution upon thawing or compromised amplification in polymerase chain reaction. Secondly, we demonstrated that addition to the RM formulation of background nucleic acid improves its stability at freezing temperatures. In the optimal formulation, the material was stable at 4°C, -20°C and -80°C for one year. Finally, we showed that conventional methods to prevent bacterial growth during prolonged storage of the RM at 4°C do not affect its intended application.

As the outcome, we developed optimal protocols for RM formulation, storage and preparation for use. This outcome is important for generating a fit-for-purpose RM for EPO gene doping detection, but also for future development of RMs for detecting other doping genes. Availability of such RMs will facilitate the development of routine tests for direct gene doping detection that are reliable, robust, and able to withstand legal scrutiny.

Code: 13A13MM 

When an athlete dopes the drug is changed by the body and excreted in the
urine. These changed drugs or drug metabolites must be processed by anti-doping laboratories to enable detection using a range of sophisticated techniques. A protein or enzyme called beta-glucuronidase, isolated from Escherichia coli (E. coli) bacteria, is routinely used by anti-doping labs to process samples prior to analysis. It has become an important basic tool used by analysts in the fight against doping. Unfortunately this E. coli beta-glucuronidase enzyme only works on some drug metabolites called glucuronides leaving others such as sulfates unprocessed, and this may mean that the doping goes undetected. Creating a new method to process sulfate metabolites would significantly improve anti-doping analysis.

In this project we will develop a separate bacterial protein isolated from Pseudomonas aeruginosa (P. aeruginosa) called a sulfatase that we have shown is able to process the sulfate metabolites that E. coli beta-glucuronidase cannot. However, the activity for some drug metabolites is low leading to inefficient hydrolysis. We will employ laboratory-based methods of rapid evolution to enhance the activity of the P. aeruginosa sulfatase enzyme for anti-doping applications.

The outcome of the project will be an improved sulfatase enzyme for processing drug metabolites that will complement E. coli beta-glucuronidase.
Including the improved enzyme in the methods used to process drug metabolites will increase the sensitivity of analysis and allow doping to be detected for a longer period after an athlete takes a banned drug. We expect
this improved P. aeruginosa sulfatase will join E. coli beta-glucuronidase and
also become an indispensable tool used by anti-doping laboratories in the fight against doping.

Main Findings: 

Previously, we had identified a bacterial sulfatase, Pseudomonas aeruginosa arylsulfatase (PaS), and found it to compare favourably with commercially available sulfatase preparations (Drug Test. Anal. 2015, 7, 903–911). However, the activity towards β-configured anabolic androgenic steroid sulfates (testosterone sulfate, TS, for example) was several orders of magnitude lower than aryl sulfates such as estrone sulfate. Furthermore, the activity towards α-configured steroid sulfates, such as etiocholanolone 3-sulfate (ECS) or epitestosterone 17sulfate (ETS) and androsterone 3-sulfate (AS) was undetectable. Thus at the outset of our project our goals were to achieve:

• A 10-50-fold improvement for testosterone 17-sulfate and significant activity for other targeted steroid sulfates.
• An improved pH profile with maximum activity closer to neutral pH.

These enhancements in activity and scope will deliver new enzyme preparations suitable for anti-doping applications.

We took two approaches to improve PaS in parallel. The first approach made use of the published structure and our modelling to predict how steroid sulfates bind with the enzyme and identify nearby residues for targeted mutagenesis and screening. The second used directed evolution with random mutagenesis and subsequent selection for improved activity. In both cases we selected for variants with greater affinity and catalytic activity for TS hydrolysis (measured as Vmax/Km or initial rates).

In summary the major aims of the project have been met with a 270-fold improvement (Vmax/Km) for TS hydrolysis, and an improvement in substrate scope with PaS mutants derived from the project capable of hydrolysing ECS and ETS at significant rates where the parent PaS enzyme could not. Further mutants developed by the project show significantly increased absolute and relative activity at pH 7.

Code: 13A25JP 

Detection of rEPO and analogues and their differentiation from endogenous EPO has been hampered by two major reasons: similarity and sensitivity.  Regarding similarity, we already evidenced, through glycan analysis, the presence of minor amounts of N-glycolyl-neuraminic acid (Neu5Gc, MW: 325.27 u) in the recombinant preparations, while in the endogenous hormone, N-acetyl-neuraminic (Neu5Ac, MW: 309.27 u). The direct analysis of sialic acids had the drawback of losing the information of what protein were they attached to. The analysis of glycopeptides where the exogenous moiety is present and the aminoacid sequence unequivocally corresponds to EPO will be a perfect solution. However, sensitivity, particularly for glycopeptides, is greatly reduced given their microheterogeneity. O-glycopeptides, being far less heterogeneous, would the obvious choice. New instrumentation (AB Sciex 6500 QTRAP IonDrive) is able to detect as little as sub-attomol amounts of material. This sensitivity is, at last, compatible with the amounts present in urine of the precious non-human EPO glycopeptides. When combined with advances in immunopurification developed these years, the resulting method for detection of the presence of rEPO or NESP in human urine or serum/plasma will be quite straightforward, as any other doping control screening method and will offer unequivocal evidence. 
Thus, the hypothesis of this project is that MS sensitivity has reached a status in which EPO glycopeptides, particularly O-glycopeptides are detectable in urine or blood samples. The MS differences between endogenous and recombinant EPOs (including NESP) will provide specific detection of the recombinant species as proof of doping. 
The objective of the project are: 
To develop an optimized method of immunopurification and enzymatic digestion of rEPOs. 
To optimize separation and MS conditions for each O-glycopeptide and scale-up sensitivity using the latest state-of-the-art MS instrumentation from AB Sciex.

Main Findings: 

The main objective of the project was to develop an MS‐based analytical procedure for the detection of an EPO O‐glycopeptide containing the non‐human monosaccharide N‐glycolylneuraminic acid (Neu5Gc) using latest generation QTrap‐MS instruments as an unambiguous proof of the exogenous origin of the hormone (i.e. rEPO or analogues). The EPO O‐glycopeptide resulting from a tryptic digest (EAISPPDAAS*AAPLR) was chosen as it is unique for EPO and shows the lowest glycan heterogeneity, thus maximizing signal sensitivity while the peptide backbone will make it unique for EPO. 
A method was developed based on pre‐concentration by immunopurification using MAIIA cartridges followed by tryptic hydrolysis and LC‐MS analysis in MRM mode. The peptide glycoform containing two sialic acids was found to be most abundant; the endogenous‐like glycoform containing two N‐acetyl‐neuraminic acids (Neu5Ac) and its non‐human counterpart containing one Neu5Ac and one N‐glycolyl–neuraminic acid (Neu5Gc). The triply charged species ([M+3H]+3) at m/z 805.0 and 810.3 respectively were therefore selected as the precursor ions for target detection. Scale‐up sensitivity was evaluated comparing standard, micro and nano‐LC‐QTrap6500‐MS systems from AB Sciex with a ca. 10 fold increase in sensitivity between each step. The LOD achieved in nano‐LC‐Qtrap‐MS for the rEPO standard was 80 attomol rEP/ul, quivalent to the final concentration of extracted EPO expected from 20 mL of urine sample of ca. 2 IU/L. However, when urine samples were spiked with low concentrations of rEPO and taken through the procedure, the matrix effect prevented the detection of those low amounts.  For that reason different approaches were tested to circumvent the problem. On one hand, a polyclonal antibody was raised against the EPO peptide backbone (EAISPPDAASAAPLRC), modified with a terminal cysteine residue for synthetic reasons. The polyclonal antibody proved to recognize both the peptide and the EPO‐O‐glycopeptide, making it ideal for further purification of the sample before instrumental analysis. In parallel, magnetic beads coated with titanium dioxide were tested, as they are able to selectively bind acidic residues, as the sialic acids present in the O‐glycopeptide. The approach proved to be successful in quantitatively binding the O‐glycopeptides and elute them under conditions compatible with MS analysis. 
A final sample preparation method needs to be developed taking advantage of those tested methodologies making the unambiguous detection of a non‐human rEPO O‐glycopeptide amenable to MS detection.

Code: 13C28YP 

The main purpose of two research projects funded by the World Anti-Doping Agency in 2008 and 2012 entitled "A gene-microarray based approach to the detection of recombination human erythropoietin (rHuEpo) doping in endurance athletes" and "A systems biology biomarker based approach to the detection of microdose rHuEpo doping" was to develop new improved methods based on gene expression profiles. On the basis of the data generated to date, blood gene expression profiles were profoundly altered during rHuEpo and for at least 4 weeks after administration leading to a "molecular signature" of rHuEpo doping. These most promising data provide the strongest evidence to date that gene biomarkers will add a new dimension to the Athlete Biological Passport in terms of sensitivity and specificity. 
Given the promising results, it is now of paramount importance and of great urgency to evaluate the effects of major confounding factors on this "molecular signature" of rHuEpo doping. These include: 1. The effects of altitude. Altitude training is used by athletes for performance enhancement and has the potential to influence indices of rHuEpo doping. There are concerns about the risk of false positive as well as the misuse of altitude to mask blood doping practices. 2. The effects of prior exercise. Samples are frequently collected at sporting or training venues after intense exercise which could potentially influence the "molecular signature" of rHuEpo. 
In order to provide a set of robust candidate genes that can be used for the detection of rHuEpo doping and a basis for recommendations on the collection of samples, this project will: 1. Compare blood gene expression profiles altered by rHuEpo with altitude exposure and determine genes that can be used to differentiate rHuEpo from altitude training; and
2. Assess the effects of prior exercise on blood gene expression after microdose rHuEpo.

Main Findings: 

All necessary confounder studies were successfully performed (and biobanked for future use) to address the ambitious objectives of this research. Results confirm the enhanced sensitivity of the blood transcriptomic approach over standard haematological markers to detect changes in response to rHuEpo and potentially ABT. Importantly, the confounder analysis clearly demonstrates a large and unique transcriptomic response to rHuEpo involving thousands of genes compared to the major confounders that include altitude and strenuous exercise. On the basis of these results, it can be concluded with greater confidence that “omics” technologies will significantly strengthen current anti-doping strategies. In particular, the use of molecular biomarkers with an improved detection window and high sensitivity and specificity to develop the transcriptionally enhanced ABP model for detecting blood doping. Collectively, the findings of the present research, interpreted in the context of the latest “omics” research in biomedical sciences, are most encouraging and confirm claims that a systems biology approach combining various “omics” signatures from genomics, transcriptomics, proteomics and metabolomics will inevitably provide a deeper understanding of the effects of Epo stimulating agents on erythropoiesis with unparalleled potential to improve current drug detection strategies with particular reference to blood doping.

Code: 13D12VB

Since 2010 the World Anti-Doping Agency (WADA) has changed their restrictions towards certain beta2-agonists on the prohibited list. As of 2013, the beta2-agonists salbutamol, formoterol and salmeterol are allowed by inhalation in therapeutic doses, and no longer require athletes to acquire a therapeutic use exemption. To distinguish allowed therapeutic use from supratherapeutic misuse, urinary thresholds and decision limits have been introduced for salbutamol and formoterol, but due to limited pharmacokinetic studies of salmeterol and terbutaline, no urinary thresholds has yet been established for these beta2-agonists on the prohibited list.

Detection of salmeterol in biological fluids is difficult due to very low concentrations, which only gives a window of approximately 8 to 12 hours of
detection in urine samples. However, the metabolite of salmeterol, Alpha-hydroxysalmeterol, is present in much higher concentrations in the urine than unaltered salmeterol and the metabolite may therefore be a suitable biological marker for excessive use of inhaled salmeterol.

Some athletes with asthma or exercise-induced bronchoconstriction (EIB) use combination therapy of both short and long-acting beta2-agonists. Combined inhalation of various beta2-agonists may affect the excretion of each substance. Lastly, asthmatic athletes usually take their beta2-agonists prior to or during competition and training. Therefore pharmacokinetic studies of beta2-agonists with applicability for the prohibited list should simulate sport-specific situations.

The main objective of the present study is to help establish urinary thresholds for salmeterol and terbutaline on the prohibited list as done with salbutamol and formoterol. A secondary objective is to address how combined inhalation of beta2-agonists influences the excretion of each substance in the urine. Lastly, an objective is to investigate the relationship between the specific gravity of urine samples and the concentrations of each beta2-agonist.

Main Findings: 

In Northern Europe, terbutaline is a commonly prescribed beta2-adrenoceptor agonist in treatment of asthma and exercise induced bronchoconstriction. We investigated the pharmacokinetics of single-dose inhalation of 2 or 4 mg terbutaline, oral ingestion of 10 mg terbutaline, and daily inhalation of 2 mg terbutaline for seven days in urine samples collected 0-24 h after administration in trained male subjects that had performed 90 min of cycling exercise. Terbutaline accumulated in urine after seven days of daily inhalation compared to single-dose inhalation during the first day, revealing incomplete elimination after 24 h. Urine concentrations of terbutaline were higher after inhalation of 4 mg than after oral ingestion of 10 mg during the first 4 h after administration, whereas concentrations of terbutaline were lower after inhalation of 4 mg than after oral ingestion of 10 mg 12 h after administration. It is concluded that inhalation of 2-4 mg terbutaline cannot be discriminated from 10 mg oral ingestion on the basis of urine concentrations for doping control purposes.

Salmeterol is another beta2-agonist widely used in treatment of asthma and exercise-induced bronchoconstriction among athletes. We investigated urine concentrations of salmeterol 0-24 h after therapeutic (200 µg) and supratherapeutic (400 µg) inhalation, as well as after seven days of daily therapeutic inhalation (200 µg/d) in endurance athletes who performed cycling exercise to mimic real-life doping control settings. Mean maximum urine concentrations of salmeterol unadjusted for specific gravity reached 4.0, 1.6, and 2.0 ng/mL after single-dose inhalation of 400 and 200 µg, and seven consecutive days of inhalation of 200 µg, respectively. Corresponding individual maximum urine concentrations of salmeterol were 6.9, 5.0, and 4.8 ng/mL. Salmeterol was incompletely eliminated 24 h after inhalation, which should be taken into consideration if a urine threshold is introduced for this beta2-agonist.3

We further investigated urine concentrations 1½, 4 and 6 h of beta2-agonists salbutamol, terbutaline, salmeterol and formoterol after combined inhalation of all of them in endurance athletes who also performed cycling exercise. In this study, subjects completed two trials consisting of inhalation of short-acting beta2-agonists salbutamol (8 × 200 µg) and terbutaline (8 × 500 µg), or combined inhalation of short- and long-acting beta2-agonists salbutamol (8 × 200 µg), terbutaline (8 × 500 µg), salmeterol (4 × 50 µg), and formoterol (4 × 9 µg).Concentrations of salbutamol and terbutaline 1½-6 h after inhalation ranged from 124 to 2198 ng/mL and 82 to 2543 ng/mL, respectively, with 16% of samples above the decision limit for salbutamol when unadjusted for specific gravity, and 11% when adjusted for specific gravity. 
When all four beta2-agonists were inhaled, concentrations of salbutamol and terbutaline 1½-6 h after inhalation ranged from 66 to 2958 ng/mL and 44 to 3806 ng/mL, respectively, while concentrations of salmeterol and formoterol 1½-6 h after inhalation ranged from 0.0 to 5.7 ng/mL and 0.4 to 25.7 ng/mL, respectively. At no point did concentrations of formoterol exceed the decision limit. The most important finding from this study was that single-dose inhalation of 1600 µg salbutamol resulted in several samples that exceeded the current threshold and decision limit.

Code: 13C31JC

With the increased capacity to detect the drugs and chemicals used for doping to enhancing performance, more instances of blood transfusion (allogeneic or autologous), termed "blood doping", have been found in different athletic competition. While blood doping methods was prohibited by the International Olympic Committee in the 1980s, there is a lack of direct and reliable methods to detect blood doping, especially from their own blood (autologous blood transfusion (ABT)). Therefore, there is an urgent need for the development of better and novel methods to detect ABT and other blood doping. These blood doping efforts are to increase the number of red blood cells (RBC) to better the oxygen-carrying capacity and athletic performance. We have discovered and characterized the large amount of genetic information in the red blood cells (RBC) that are usually used for the blood doping. We plan to apply genomic technology and advanced bioinformatics to global analyze the changes of the genetic materials of RBC during in vitro storage to identify "gene signatures" of blood storages. These gene signatures will be validated and used to identify individuals who may have received their own stored blood to enhance their athletic performance.

Main Findings:

With the increased capacity to detect the drugs and chemicals used for doping to enhance performance, more instances of blood transfusion (allogeneic or autologous), termed “blood doping”, have been found in different athletic competitions. While blood doping methods were prohibited by the International Olympic Committee in the 1980s, there is a lack of direct and reliable methods to detect autologous blood transfusions (ABT). Therefore, there is an urgent need for the development of better and novel methods to detect ABT and other forms of blood doping. We have previously discovered the presence of large amount of microRNAs in the human mature RBC. These genetic materials offer an unique window into the development history and environmental exposure of the RBC, the main cell types used for blood doping. In our previous works, we have found that the genomic analysis of the RBC microRNAs in sickle cell diseases offer important insights into the heterogeneity of the anemia severity and malaria resistance that are caused by the elevated miR-144 and miR-451, respectively. In this project, we will perform global microRNA analysis of the RBC during storage to identify a unique gene signature that is found only in the stored RBC. This signature can then be used to analyze the blood of athletes to detect the possibility of blood doping based on the presence of these signatures of stored RBC. To achieve this scientific goal, we first used the high throughput sequencing to comprehensively analyze all the long and short-sized RNAs in the fresh RBCs before placed under storage. In addition, we used a state-of-the-art profiling procedures to analyze the changes in the RBC microRNA during storages. We have identified and validated several microRNAs that are specifically found in the stored red cells that can be used for detecting blood doping. We will continue to seek support to further investigate the basis of this “storage gene signature” and validate their ability to detect blood doping in additional samples and real volunteers who have received ABT.

Code: 13D24GJ

Oral dosing of salbutamol is known to lead to beneficial performance effects in athletes and is banned, however, it is allowed to be delivered by inhalation for use in athletes with asthma. The drug is usually used as the racemic mixture consisting of active R- and inactive S-enantiomers (non-superimposable mirror image molecules) which have a different time course in the blood and urine. 
These differences are further amplified by whether the drug is taken orally or by inhalation. To date, anti-doping strategies have not capitalised on this difference in how enantiomers are eliminated from the body. Furthermore, studies have not adequately investigated the effects of repeated dosing of both inhaled and oral salbutamol over several days on urine levels in a doping control context. We will apply our advanced analytical technique that can measure both R- and S-salbutamol in urine, to samples from patients treated with either oral or inhaled therapy over the course of a week. The project will validate the benefits, namely increased sensitivity and reduced false positives and false negatives, of measuring R- and S-salbutamol enantiomers. This will allow to better discriminate between oral and inhaled salbutamol.

Main Findings: 

Background: Salbutamol is a chiral drug, consisting of non-superimposable mirror image molecules called enantiomers (R- and S- salbutamol). Salbutamol is usually administered as a 1:1 racemic mixture of these enantiomers. The body metabolises and eliminates R- and S- salbutamol differently over time (called enantioselective pharmacokinetics).  

Objectives: The primary objective of this project was to improve discrimination between prohibited oral and permitted inhaled dosing by using ratios of salbutamol enantiomers in spot urine samples collected over one week of treatment duration with high doses. 

Results: As predicted, the S:R ratio was greater in oral versus inhaled dosing but the difference was not a reliable overall determinant of route of salbutamol administration, with significant intra- and interpatient variability. Enantiomer ratios offered modest improvement over the current urine threshold in diagnostic capability. During the trial, several subjects exceeded the current urine threshold of 1000 ng/ml after administering salbutamol via inhalation in a permitted manner, both in one week of treatment (800 microgram daily), as well as after an acute dosing regimen within 24 hours (2x 800 micrograms 12 hours apart). Some individuals appeared to have a higher likelihood of exceeding the threshold and more work is required to understand these determinants. A secondary finding of the study was that use of urine specific gravity (USG) normalisation to 1.020 for all samples to account for hydration improved diagnostic performance (mainly sensitivity), compared to both correction for concentrated samples only (as per TD2018DL) and uncorrected concentration. 

Conclusions: Salbutamol S:R ratio is different between oral and inhaled delivery, but the ratio only provided a modest increase in diagnostic performance over existing approaches. The 1000 ng/ml urine threshold is an imperfect classifier of prohibited dosing with potential for some individuals to exceed the threshold using permitted inhalation high and repetitive dosing. USG normalisation for all samples improves the performance of the salbutamol urine threshold 

Code: 13D21DS 

Among the misuse of erythropoietic stimulating agents, the application of substances that induce EPO gene expression by stabilizing HIF 1 as cobalt (II) ions or carbon monoxide inhalation seems to be widely distributed. For athletes, the advantages of the misuse of both substances are low costs, high erythropoietic effectiveness, and no risk of detection, because they are not yet included into the prohibited list of WADA.
The aim of this project is to demonstrate significant effects of cobalt and CO-inhalation on the erythropoietic system which can be detected by the Athlete Biological Bloodpass and direct methods. Special aims are: 1. to monitor the physiological effects of different doses of cobalt and carbon monoxide on plasma-EPO, Hb-mass, and aerobic performance, 2. to prove if the effects of cobalt and CO can be detected by the recently developed Bayesian probabilistic inference techniques, which are the basis for the athletes biological passport, 3. to develop and evaluate direct detection methods for cobalt-misuse, and 4. to evaluate whether the endogenously produced EPO after stimulation by cobalt and CO differs from normal native EPO. 
In a first study the optimal dosage of cobalt and carbon monoxide on endogenous EPO production will be determined. In a second step, application of either cobalt or CO for 3 weeks will provide data on (1.) performance effects of both drugs, (2.) stimulation of endogenous EPO, (3.) changes of hemoglobin mass, and (4) changes of hematological parameters which will be analyzed by the statistics of the athlete biological passport. Cobalt concentration and EPO isoforms will be analyzed in urine and/or blood samples. We expect to develop methods which can be used for the detection of endogenous stimulation of erythropoietin by cobalt and carbon monoxide.

Main Findings: 

Introduction: From the mid 40ies until the late 70ies of the last century cobaltous ions (Co++) were used as an effective drug to treat anemia of different origins. Due to severe side effects this kind of treatment was replaced by other erythropoietic stimulating agents like recombinant human erythropoietin. Co++ acts similarly as a hypoxic stimulus; it stabilizes HIF-1α and HIF-2α and thereby increases the endogenous erythropoietin production. Because of its possible performance enhancing effect Co++ has been added to WADA’s list of prohibited substances and methods. Co++ can be easily purchased and it is recommended from suppliers of nutritional supplements to athletes to boost their performance. The aim of this project was to investigate the effects of low dose Co++ ingestion and to determine the minimum oral dosage which is necessary to increase erythropoietic processes.
Methods: Three sub-studies were carried out: I. Single Co++ dosages between 1mg and 10mg; II. Co++ administration for 5 days with the lowest dosage found to be effective in study I; III. 3-week Co++ administration with the lowest effective dosage (5 mg/day). We determined plasma [EPO], all parameters used for the athlete’s biological passport (ABP) as well as in study III hemoglobin mass and performance. In total, 63 male recreational athletes participated at the studies which were conducted in a double blind design.
Results and discussion: Study I: Single dosages until 2mg Co++ had no erythropoietic effect. Plasma [EPO] increased 5h after the 5mg administration by 18 ±14% (p<0.05) and until 7h following the 10mg Co++ administration by 41 ±15% (p<0.001). Study II: Following the 5-day Co++ application 5mg showed no significant effects on any parameter while 10mg increased [EPO] by 28 ±26% (p<0.05) and the immature reticulocyte fraction by 50 ±21% (p<0.001). [Ferritin] decreased in the 10mg group by 22 ±17ng/ml, p<0.01). Study III: During the 3-week application period plasma [EPO] increased by 30 ±39% (p<0.001) and total hemoglobin mass by 17.1 ±16.8g (p<0.001) while Ferritin decreased by 17 ±18 ng/ml (p<0.01). Although VO2max was not affected, submaximal performance (+11 ±xy Watts at 2 mmol/l lactate, p<0.05) and time until exhaustion (35 ±xy sec., p<0.05) was slightly improved after the 3-week administration period.
Discussion: Co++ administration using dosages 2-5-times above the recommendations of suppliers of nutritional supplements and ~ 2-times above the amount which is considered to be safe and without any side effects in case of life-long daily administration show measurable erythropoietic and performance-enhancing effects. We therefore recommend to install threshold limits for Co++ concentrations in urine and in blood to deter and detect blood manipulation by Co++ administration.

Code: 13D22RV 

Glucocorticosteroids are widely used in sports medicine for the treatment of conditions such as asthma and acute injuries. The use of glucocorticosteroids by oral, intravenous, intramuscular and rectal routes is forbidden, and their use by other routes is allowed. Since some glucocorticosteroids are marketed in different administration forms, the distinction between different routes of administration through the analysis of urine samples is needed. A reporting level of 30 ng/mL for glucocorticosteroids and their metabolites has been established by WADA to detect corticosteroid misuse. Unfortunately, this reporting level is not suitable to distinguish therapeutic use from forbidden administration for all corticosteroids and the investigation of more effective criteria of discrimination is needed. 
The aim of the project is to elaborate analytical strategies to be used in the effective differentiation between legal and forbidden administration routes for synthetic glucocorticosteroids. A comprehensive study of the metabolic pathways of corticosteroids will be performed using liquid chromatography coupled to tandem mass spectrometry. Both phase I (including saturated A-ring and acidic metabolites) and phase II (including conjugates with sulphate and cysteine) metabolism will be studied in several excretion studies already available in our lab for triamcinolone acetonide, betamethasone, prednisolone, methylprednisolone and budesonide. 
Differences between responses of every metabolite detected in the different administration routes will be evaluated. Potential markers will be selected based on these results. Selectivity and sensitivity of these potential markers will be compared with current reporting level. 

Main Findings: 

The objective of the project was to evaluate the current WADA criterion to detect glucocorticoid (GC) misuse.  The urinary profiles of budesonide (BUD), triamcinolone acetonide (TA) and betamethasone (BET) were studied in clinical studies were they were administered by allowed and forbidden administration routes in order to look for the best discrimination markers for each GC. 

For BUD, the metabolite 6β-hydroxy-BUD was confirmed to be the best the marker to detect BUD misuse. However, high 6β-hydroxy-BUD concentrations were found after high-dose inhaled treatments and, for this reason, a reporting level of 40 ng/mL is proposed to discriminate allowed from forbidden administrations. 
For TA, a reporting level of 30 ng/mL of TA did not allow the detection of intramuscular administration. A reporting level of 5 ng/mL for TA was proposed to detect intramuscular use, and to discriminate topical and intranasal from intramuscular administrations. Concentrations obtained after intraarticular and periarticular administrations, which are currently regarded as allowed administration routes, need be taken into account before proposing a definitive reporting level to discriminate allowed from forbidden administrations of TA. 

For BET, a reporting level of 40 ng/mL for BET is proposed to discriminate topical and intranasal from oral and intramuscular administrations. However, concentration values after intraarticular and periarticular administrations are similar to those obtained after intramuscular administrations. 

The results of the present project demonstrate the need of studying the metabolism of each GC after allowed and prohibited administration routes in order to establish the best discrimination criterion for each GC.