Projets de recherche

Code: 13D25MT 

The misuse of recombinant human insulin in sport has frequently been mentioned by confessing athletes but is not detectable with currently available methodologies. This is mainly due to the fact that there are no measurable differences to the endogenously produced hormone. Several attempts to establish diagnostic marker ratios to other endogenously produced hormones (C-peptide) failed due to unstable or highly variable conditions in the living organism. With the present study we aim to establish potential marker metabolites, which will uncover a surreptitious insulin application. Pilot studies demonstrated that there are substantial differences in the degradation/metabolism of insulin between the endogenously secreted and the recombinant and injected insulin. It is assumed, that these differences are due to the exposition of insulin to specific enzymes after subcutaneous or intramuscular administration. 
Several urinary metabolites were identified in earlier studies by this research group and an extension of this approach with most modern analytical instruments is planned, potentially revealing new indicative and diagnostic metabolites. Therefore, purification of target compounds from urine followed by the determination of insulin metabolite profiles by LC-MS/MS is planned, which might serve as diagnostic tool to uncover the misuse of recombinant human insulin. 

Main Findings: 

This study was conducted to explore the metabolic fate of subcutaneously administered recombinant human insulin. Due to the exposure to endogenous proteases in the subcutaneous tissue, a minor amount of the bioactive peptide hormone is cleaved to its truncated metabolite DesB30 human insulin, which still owns the full biological activity. In addition to that, DesB30 is a known by-product of recombinant insulin preparations (at ~ 1%) and, thus, it can enter the circulation from the injection site.  Post administration of recombinant human insulin, circulating DesB30 was identified in plasma samples of diabetic patients (type II). No specimen obtained from healthy volunteers with endogenous insulin analyzed in the context of this study contained DesB30, not even when the production of insulin was stimulated by ingestion of a concentrated glucose solution (OGTT).  The analysis of athletes’ routine doping control samples yielded analytical findings of trace amounts (=LOD) of DesB30 HI in 3 out of 64 samples. The additional monitoring of the respective C-peptide levels yielded inconspicuous results for all athlete samples but, conversely, suppressed C-peptide levels in diabetic individuals after HI administration.  Thus, the detection of DesB30 in athlete blood samples in combination with C-peptide levels represents a potential combination of markers for the misuse of recombinant human insulin in sports. Due to degradation processes, urine specimens were not found suitable for this approach. Furthermore, special care regarding the storage and sampling conditions are crucial for the collected plasma samples and degradation processes must be avoided by applying appropriate conditions during the whole post-sampling period until analysis. This is important for both inhibiting the degradation of HI to DesB30 HI on the one hand and stabilizing the C-peptide of the other hand. 

Code: 13D23SS

Several elite athletes were tested positive for clenbuterol and claimed that these findings were caused by the consumption of meat containing clenbuterol. From several studies and EU monitoring programs it is shown that clenbuterol may be present in meat and after consumption of meat concentrations of clenbuterol in urine can be found. To discriminate between clenbuterol administrated via a pharmaceutical preparation or by ingestion of contaminated meat products research was started in 2012 (WADA 11A18SS). The focus of this study was to determine if there was a difference between the ratio of the two enantiomers (left- and right-- hand form of clenbuterol) in meat, and if after consumption of meat there was a difference between the ratio of the two enantiomers in urine when compared to the ratio in urine after illegal oral administration of preparations to humans. 
In 11A18SS the proof is given that the hypothesis is feasible and the analytical methods are in place and capable to detect the differences.  In the proposed project the focus will be on establishing a relation between the consumption of contaminated meat and a change in ratio of clenbuterol enantiomers in urine via a controlled experiment.  The project will result in a decision model that can be used to assess the source of an adverse analytical finding for clenbuterol.  A new technique focusing on untargeted analyses will also be tested on the acquired samples. This will take into account if there are any other changes in metabolic profiles after both ways of ingestion and will try to discriminate on this basis. This technique is known as metabolomics. 

Main Findings:

The aim of the project was to confirm the proof of principle from a former project with controlled human trials and to build a decision model to be used in sports doping analysis to distinguish AAF for contaminated meat from pharmaceutical preparations. Meat and liver were collected from an animal experiment.  The meat and liver were used for a controlled human trial in which these tissues were consumed by volunteers. Other volunteers ingested a pharmaceutical preparation containing clenbuterol (racemic mixture) or enantio pure clenbuterol. Urine samples of the volunteers were collected for over a week after consumption. All urine samples were analysed using the methods developed in 11A18SS project. The urine samples were also used for untargeted profiling experiments.  
In meat from the animal experiment had S/R clenbuterol ratio of around 1 and 0.9 depending on the animal. This shows that the enrichment of R-clenbuterol in meat of treated animals is not a process that is stable, reproducible and comparable for individual animals and maybe also depends on the concentration administered to the animal. For bovine liver no information on the ratio was available before. Data from bovine liver showed that in liver the S-enantiomer is either enriched or R-clenbuterol very depleted. This is the opposite of the ratio in veal meat. This means that consumption of liver with incurred residues can possibly lead to an opposite ratio in human urine compared to the consumption of veal meat. It was also shown that preparation (cooking and baking) of the incurred meat and liver had little to no effect on the ratio determined prior to preparation.  
In the administration studies a distinction based on the proportion of S-clenbuterol in the human urine samples was possible between those receiving liver (padmin=0.635±0.004) from the other two groups, Spiropent® tablet (padmin=0.499±0.001) respectively meat (padmin=0.509±0.006). A distinction between the volunteers receiving meat and Spiropent® tablets cannot be made based on the enantiomeric composition, due to the reason that the ingested proportions of S-clenbuterol are too close to each other.  
The analysis focusing on potential metabolites of clenbuterol in a targeted non-targeted design predicted a high theoretical number of metabolites/biomarkers. Unfortunately, not many of the predicted metabolites were found in the urine samples of the volunteers. Based on the outcome of the analysis and the statistical processing there is no grouping of the compounds possible at present. There is no underlying mechanism found what could be used to separate the different treatment groups. So it was concluded that it is not possible to use clenbuterol metabolites to discriminate between intentional and unintentional intake of clenbuterol using non-enantiomeric separation.  
Overall it seemed that inter individual differences between elimination kinetics in bovine animals are present. This made setting an absolute threshold or guideline for discrimination not possible. The method developed in the project can be used when there are adverse analytical findings for clenbuterol to obtain additional information. When the S proportion clenbuterol in human urine resembles 0.5 (the S proportion in a pharmaceutical preparation) this is not definite proof of illegal use. However, if the S-proportion is higher than 0.59 this means that in 95% of the cases this is not due to administration of clenbuterol in (racemic) tablet forms.

Code: 13D31PV

Although recent advances of steroid profiling have shown to substantially increase the detection sensitivity, still, the prevalent of misuse is larger than what doping control laboratories can detect. In particular the introduction of slow-release preparations such as T-gels and steroid patches poses anti-doping analysts for greater challenges as the metabolic footprint of misuse with these preparations is very small. 
In 2009, Bayer introduced a long-acting T undecanoate depot Nebido® for therapeutic treatment of hypogonadism in males. Nebido® should be administered only once in two or three months, which is a much longer release period compared to other long-term T-formulations (e.g. Sustanon®). A dose of 1000mg T undecanoate should be injected in the muscle. It is claimed that the insufficient T-levels are restored and do not exceed normal serum levels and likewise cause less unwanted side-effects. 
Also, it has been shown that such high T undecanoate doses can lead to elevated hematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery.  Interesting features for endurance athletes, for whom the biological passport is a well-established tool to screen and record their doping test results. In this light, it is assumed that described effects can be noticed in both the steroidal and blood module of the biological passport and help to find Nebido® misuse in this category of athletes.  This study aims to investigate the direct detectability of T-undecanoate as administered with Nebido® in blood as well as the influence on various blood and steroid parameters as recorded in the biological passport. 

Main Findings:

Today the steroid passport is a very performant tool to detect abnormal variations in steroid profile markers. Although recent advances of current methods for endogenous steroids have shown to substantially increase the detection sensitivity, the prevalent of misuse is far larger than what doping control laboratories can detect. Particularly, slow-release preparations pose anti-doping analysts for great challenges as the metabolic footprint of misuse with these preparations can be very small. Nebido (Bayer) is one of such long-acting slow release T preparations, containing T undecanoate, that is used for therapeutic treatment of hypogonadism in males. In sports, slow releasing T formulation are applied to rapid repair of micro damage to muscles after exercise for faster recovery. In addition, it has been shown that such high T doses can lead to elevated haematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery. Both features are interesting for endurance athletes, whereas T used to be related to cheating power athletes for its anabolic effects.  
The T/E ratio was clearly altered by the use of Nebido as slow release T formulation and remain detectable using longitudinal following during 3 months. Androstanediol  ratio and androsterone over etiocholanolone remain mostly unaltered, despite elevated concentrations. Minor steroid metabolites did not contribute to longer or better detection as likely the major pathways were not saturated. As a result the steroidomic model showed similar sensitivities as those observed for longitudinal evaluation of the T/E ratio.  
A very sensitive method for the detection of T in saliva was developed and applied to the oral fluid samples collected after Nebido. Although, salivary T concentrations increased to 7 fold and detectable using population based reference ranges, the basal values were restored after 14 days. Oral fluid analysis of T did not provide any added value for doping control. Hence, the pharmacokinetics of salivary T are much different compared with the observations for T-gel, also a slow-release formulation. 
Blood passports were created after the Nebido T supplementation and results demonstrated that the blood markers were significantly altered but did not results in atypical passport findings. The reticulocyte production was stimulated in the month after Nebido injection, after which a suppressive effect was observed in the second month. By this time haemoglobin was formed to give rise to an elevated plateau in the blood passport that remained below the upper individual limit. Also the off-score showed the alterations that was typical for bone marrow stimulation. 

Code: 13A08MT

The necessities generated by the high standards and quality requirements of modern sports drug testing entails numerous challenges for doping control authorities as well as accredited laboratories. Among these challenges, several prominent aspects can be approached and solved by collecting and, when indicated, testing an additional/ a complementary specimen referred to as dried blood spot (DBS). 1. An important element of efficient doping controls is the frequent and unpredictable sampling of athletes, especially out-of-competition and concerning e.g. anabolic agents, erythropoiesis-stimulating agents, etc. Here, costs associated with sample collection and analysis are particularly limiting factors.
2. With regard to in-competition controls, the differentiation whether a substance such as a stimulant or cannabinoid (i.e. not prohibited at all times) was present in the athlete's blood at pharmacologically relevant concentrations or not is required. From urine analyses, conclusions as to the blood concentration of a drug is a complex issue and has been subject of numerous studies and discussions in the past.2-5
3. The issue of instable compounds can be solved as e.g. in case of Synacthen, a substance that was confiscated in the Spanish Fuentes scandal and admittedly misused by several athletes. Due to its limited stability in blood and urine, its analysis was hampered despite the availability of analytical methods. DBS collection and analysis is considered as a valuable means to improve currently conducted doping control strategies as it addresses all of the above mentioned aspects. DBS are substantially cheaper than conventional doping control samples and thus larger collectives can be processed; drug concentrations at the time of competition can be determined; and instable analytes can be conserved. As a result, both relevant aspects of doping controls, i.e. deterrence from doping and protecion of honest athletes will be considerably strengthened. 

Main Findings: 

A drop of whole blood dried on filter paper (Dried Blood Spots, DBS) represents an aspiring technique for minimal-invasive sample collection in a multitude of analytical disciplines, e.g., therapeutic drug monitoring, preclinical drug development and diagnostic analysis of metabolic disorders in newborns. DBS sampling is characterized by cost-effectiveness, straightforwardness, robustness and facilitated storage and shipment conditions. 
The present investigation was conducted to highlight the opportunities arising from the implementation of DBS as complementary matrix in doping control programs. Being frequently abused, three model compounds were chosen to represent the classes of anabolic agents (stanozolol and dehydrochloromethyltestosterone) and stimulants (pseudoephedrine). A quantitative method was developed and validated for the detection of the target analytes from DBS using liquid chromatography coupled to high resolution/ high accuracy tandem mass spectrometry. The imprecision of the assay amounted to < 8% for intraday and < 18% for day-to-day measurements. Highly purified DBS sample extracts exhibited no ion suppression effects due to interfering matrix components and provided limits of detection of 20 pg/mL for stanozolol and 0.8 ng/mL for DHCMT and pseudoephedrine, respectively, notwithstanding an overall recovery of 26%. Deuterium-labeled internal standards were used to yield reliable quantitative results (accuracy 84-125%). Stability of the analytes was shown for at least 28 days at room temperature.  
Proof-of-principle for the method presented was substantiated by means of the analysis of authentic specimens obtained from administration studies with stanozolol, DHCMT and pseudoephedrine. The results provided, to our best knowledge, unprecedented detection windows for the tested anabolic agents accomplished by DBS sampling to support out-of-competition control efforts for the tested anabolic agents. Furthermore, the unambiguous proof of pharmacologically relevant blood concentrations at given urinary analyte levels are noteworthy for the improvement of in-competition controls, e.g., with regard to stimulant analysis.

Code: 13C35CC 

Stimulants are banned in competition by the World Anti-Doping Agency, except for nicotine. Psychoactive substance as nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance.
In healthy humans nicotine administration increases heart rate and blood pressure. Plasma nicotine concentrations improve anaerobic energy production and suggest a nicotine-induced sympathetic stimulation of the heart. Nicotine has been placed on WADA's 2013 Monitoring Program in order
to detect potential patterns of abuse. Smokeless tobacco is a blanket term that refers to a number of tobacco products that are used by means other than smoking. Snus is a smokeless tobacco consumed orally, which has a long tradition of use that, is widespread in North America and Scandinavian countries. It is typically placed between the upper lip and the gum, toward the front of the mouth, for approximately 30min before it is discarded. Recently, some studies on snus in competitive events showed a high use among athletes mostly in a winter sporting environment.  The aim of this study is to investigate the snus effects on sport performance. In the first year we will investigate the effects of snus in an aerobic performance by an exercise intensity nearly 80% VO2max until exhaustion correlated with the perception of effort and time to exhaustion along with a range of physiological parameters. In the second and third year the aim is to investigate the effect of snus on high-intensity exercise by on a, short-term high-intensity cycling test, (Wingate) using trained athletes. If results will show a snus effect of increasing time to exhaustion, reduced perception of effort and increase anaerobic performance, an informed decision on the potential doping of nicotine will be done. WADA could consider to include nicotine in Prohibited List of Substances and Methods, prohibited in-competition

Main Findings: 

Snus is a smokeless, orally consumed tobacco, traditionally produced and used in Sweden and in Scandinavian countries. Analytical chemistry studies showed an increase of nicotine use in athletes practicing different sports. Nicotine has also several physiological effects on heart and on the vascular system. This effect influence sport performances and exercise capacity.

The purpose of this study was to investigate the snus effects on aerobic performance (80% VO2max) during exercise until exhaustion and the perception of fatigue during abstinence (AC) and satiety condition (SC). We recruited 18 healthy male athletes daily snus users. The study was a double-blind cross-over design. Subjects were tested during three sessions on cycle-ergometer.  Experimental session 1 (EXP1) consisted in maximal incremental exercise test (MIT) to determine the VO2max. EXP2 and EXP3 consisted in exercise, after 25 min from snus administration, at 80% VO2max until exhaustion (TTE) under AC and SC conditions. During the tests, muscle and cerebral oxygenation was recorded by means of NIRS (near-infrared spectroscopy) and the global rating of perceived exertion (RPE). We measured, non-invasively the dynamic response of tissue O2 saturation (Ox%) on tight muscle by a vascular occlusion test (VOT).

Nicotine and cotinine analysis confirmed the different initial conditions (AC vs. SC). We found a significant increase (+13.1%) in TTE (24.1 ± 10.7 min; means ± SD during AC session; 20.9 ± 8.0 min during SC session; means ± SD, P = 0.0131). RPE values did not significantly change during exercise. We found significant differences in the oxygenation of the cerebral and muscular tissues at rest (T0) and over VOT. Interestingly, our data showed significant differences in metabolic responses (VO2; VCO2; HR; VO2/HR) and in cardiovascular responses (SV; EJT) at baseline (T0). Our sample of athletes consumed 8.1 ± 4.0 (means ± SD) daily snus portions with a value of 5.9 ± 1.9 (means ± SD) of the Fagerstrom Test for Nicotine Dependence-Smokeless Tobacco (FTNDST).

The results of this project research showed that the increase of performance (+13.1% TTE) due to snus administration could be associated to the nicotine effects in order to keep the allostatic state.  In conclusion, our study highlighted that athletes addicted to nicotine would use snus to maintain their nicotine level to “feel normal” in order to improve sport performance.

Code: 13C36AB 

Over the last decade, significant efforts by us and other researchers in the anti-doping community have led to the development of methodology for direct detection of gene doping, a new threat to the world of sport.  The methodology involves polymerase chain reaction (PCR) assays targeting intronless complementary DNA (cDNA) sequences that are present in doping genes but absent in intron-containing endogenous genes. To ensure the method for detection of erythropoietin (EPO) gene doping performs as intended when implemented in routine test laboratories, an EPO DNA reference material (RM) has been prepared and characterized in our laboratory for use as a control. This RM has been carefully designed to ensure that inadvertent cross-contamination of a tested sample with the RM will not lead to a false positive test result. We are currently optimizing and validating protocols for blood sample collection, processing and storage. This will then provide a complete protocol from sample collection through to PCR detection together with an appropriate RM for ongoing monitoring of the method performance. 
This project aims to translate the method for direct detection of gene doping into a routine test method which is implemented in three WADA-accredited laboratories. The method will be initially implemented in the Australian Sports Drug Testing Laboratory (ASDTL). The close association of the applicants' laboratory and ASDTL (both laboratories within the National Measurement Institute, Australia (NMI)) will allow efficient review of the method when conducted in a routine testing environment and enable rapid finalization of the routine test method protocol. The method will then be implemented in two additional WADA-accredited laboratories and the competency of the three laboratories demonstrated through interlaboratory analysis of test samples. 
The project is scheduled for completion early 2016 which is several months prior to the Rio de Janeiro 2016 Olympic Games.

Main Findings: 

Over the last decade, significant efforts have led to the development and validation of methodology for detecting erythropoietin gene doping together with preparation of a DNA reference material to monitor test method performance. This project aimed to translate the method into a routine test and to implement it in one WADA-accredited laboratory, the Australian Sports Drug Testing Laboratory, which is part of the National Measurement Institute of Australia. We documented the complete test for detecting erythropoietin gene doping, provided staff training and assisted the laboratory during method implementation. Following test implementation in a routine laboratory environment, we assessed staff competence in analyzing blind samples and validated the test using blood samples which were prepared to model positive and negative gene doping samples from athletes

Code: 13D18GF 

Synthetic cannabinoids mimic the psychoactive effects of tetrahydrocannabinol (THC) and are found in herbal smoking mixtures, commonly sold as "spice".  Like THC, these compounds are cannabinoid receptor agonists. Today, there is an immense variety of synthetic cannabinoids and spice blends available on the market and new substances constantly appear. Slight chemical modifications of existing compounds make many new cannabimimetics possible. For doping analysis, a fast elucidation of the metabolic pathways of new synthetic cannabinoids is of high importance in order to be able to implement them in doping control procedures. Due to a lack of a toxicological profile, in vivo excretion studies are however difficult to perform. Hence, the current study will employ in vitro metabolism studies on selected cannabimimetics using human cryopreserved hepatocytes. Human cryopreserved hepatocytes are recognized to be a close model to the human liver, showing excellent metabolizing and transporting activities. After hepatocyte incubation and a clean-up procedure, analysis will be performed with liquid chromatography and gas chromatography, combined with mass spectrometry. The selection of the target substances for the in vitro experiments will be based on the frequency of their detection in forensic samples. This is due to a rapidly changing market. The proposed metabolism is verified through the analysis of samples from traffic controls.

Main Findings: 

For doping analysis, the elucidation of the metabolic pathways of new synthetic cannabinoids is of high importance in order to be able to implement them in doping control procedures. Because of the high variability of synthetic cannabinoids and new herbal mixtures in a rapidly changing marked, a fast characterisation of metabolites is required. In terms of non-approved substances like this, though, human excretion studies are difficult to perform due to ethical considerations. In vitro studies, however, can identify human metabolites, and human primary hepatocytes are recognized to be a very close model to the human liver. Furthermore, the improvement of cryopreservation techniques has facilitated the use of cryopreserved hepatocytes which are now commercial available. 
The aim of the current project was to use human cryopreserved hepatocytes to study the metabolism and characterize metabolites of selected synthetic cannabinoids predominating on the marked today. Two frequently reported cannabinoids in herbal smoking mixtures are JWH-307 and AM-694. Thus, these two compounds were selected for the study. 
The incubation mixtures were analysed for substrate and metabolites using liquid chromatography-mass spectrometry (LC/MS/MS) with electrospray ionisation (ESI). Free, glucuronide and sulphate products were separated during sample preparation. Although varied conjugation rates were observed, the majority of the main metabolites were to a high extent excreted as glucuronide conjugates. For all metabolites, the structures are proposed, not confirmed. 
After incubation of AM-694 with human hepatocytes, 10 metabolites were recovered in the extracted culture medium. The most abundant generated products were a defluoroniated monohydroxylated metabolite and a carboxylated metabolite. Furthermore, the results from the hepatocyte assay were compared to analytical results from a forensic AM-694 urine sample. The in vitro data correlated well with observed metabolites in the urine sample. 
After incubation of JWH-307 with human hepatocytes, 22 metabolites were identified. A monohydroxylated compound, a dihydroxylated compound, and three dihydrodiol metabolites were the prevailing metabolites generated in the incubation mixture. 
In conclusion, incubations with human hepatocytes seems to represent a useful model for identifying and predicting major urinary metabolites. 

Code: 13A17MT

Most substances relevant to doping controls undergo considerable metabolism following administration. The generated metabolites are commonly less toxic and more polar which allows for renal clearance. Consequently, in urine the analysis of metabolites is more promising than attempts to detect the ingested or injected compound, particularly when these metabolites are excreted for a longer period of time than the drug. These so-called long-term metabolites are of great interest especially for those doping agents that are predominantly used during out-of-competition periods as for instance in case of anabolic androgenic steroids. 
In order to identify drug metabolites in biological matrices, stable isotope labeling of compounds has proven to be a helpful means within the last 3 decades. Usually, hydrogen is replaced by its heavier isotope deuterium at 3 to 5 locations within the steroid nucleus and the resulting mass shift was detected by means of conventional mass spectrometers. The sensitivity of this approach has recently been considerably improved by using hydrogen isotope ratio (HIR) mass spectrometry instead of conventional mass spectrometers as these HIR-dedicated systems are built to determine the deuterium/hydrogen ratio at natural abundance, i.e. with approximately 1 out of 10000 hydrogen atoms representing a deuterium atom. For deuterium-labeled compounds commonly up to 10 % of hydrogen is substituted and thus enables a highly sensitive detection of any metabolite of the administered compound as long as it carries the deuterium-label.  This feature will be exploited in a novel approach to investigate the metabolism of clenbuterol and nandrolone. Both substances are in the scope of sports drug testing since several years and the systematic study of their metabolism will be conducted to support their long-term detection and determination of respective sources (i.e. endogenous/natural or artificial/contamination). 

Main Findings: 

Employing stable isotope-labeled drugs and GC/IRMS for metabolism studies significantly facilitates the detection of relevant analytes also at lowest abundance in complex matrices such as human urine. In the present research project, the metabolism of two critical anabolic agents, 19-nortestosterone and clenbuterol, was revisited utilizing the recently established approach. Triply deuterated 19-nortestosterone and nine-fold deuterated clenbuterol was used to characterize the drugs’ metabolic profile and to complement and/or corroborate the picture of long-term metabolites as well as the possibilities to differentiate the origin of analytes. In case of 19-nortestosterone, metabolites supporting the classification of low abundant 19-norandrosterone findings as of natural or xenobiotic nature is desirable; in case of clenbuterol, information facilitating the differentiation of contaminated food ingestion from deliberate drug misuse is needed.
The results obtained for both drugs were manifold and a substantial number of known and several yet unknown metabolites as well as sample preparation-derived artefacts were identified. A new 19-nortestosterone metabolite was observed, which was traceable as long as 19-norandrosterone in human urine in the conducted elimination study. The structure of this compound is not entirely clarified yet, and since it appeared to exhibit significant interindividual variability (as suggested by means of routine doping control samples and confirmed adverse analytical findings), its utility for sports drug testing purposes might unfortunately be rather limited. Also for clenbuterol, metabolites were detected and attributed to oxygenation products of the drug. As these are expected to possess different elimination kinetics than clenbuterol itself, a means to differentiate the recent intake of a minute amount (i.e. via food contamination) from remnants of clenbuterol being eliminated from drug abuse weeks ago could be obtained. Here, urinary clenbuterol / metabolite ratios need to be determined in future studies.

Code: 13B10MU 

Prohibited glycoprotein hormones such as ESA, hCG, FSH etc. are currently tested by immunological methods, and the decision is made by immuno-blotting electrophoresis or by comparing concentration with the applicable threshold. However, heterogeneity of the markers or multiple sources of the target compound, e.g. pregnancy, hormone producing tumor, doping etc. can sometime cause in difficulty of decision making. We confirmed that all recombinant glycoproteins from CHO cell line studied were lacking certain human type glycanes, which are not always identifiable by mass spectrometry. Our first year results represented that recombinant and human glycoprotein hormone can be isolated by stereo specific lectin-glycane interactions, and the recombinant fraction does not show any human type glycane specific lectin interactions. Thus, findings of our 1st year project supported the possibility of origin specific detection of glycoproteins by lectin specific extraction coupled to a normal immunoassay instruments that commonly used by all WADA laboratories.

Main Findings: 

Genetically manufactured glycoprotein has the unique isoform profile and the heterogeneity can sometimes cause in interpretation difficulties. Prohibited glycoprotein hormones are currently tested by immunological methods with the result evaluation using the applicable threshold or by comparing the isoform profile with that of the reference standard as no procedure yet available to identify their origins. Glycoproteins from human show a strong lectin-glycan interaction with certain lectins whereas those expressed in CHO-cell lines do not show any interaction with the selected lectins. Immobilized SSA lectin was suspended with a sample aliquot and non-reactive glycoprotein was collected by an exclusion chromatography.  Rest of captured hormone was then eluted with Lactose solution and each fraction was measured by immunoassays. SSA captures LH and FSH from human, and Lutropin (rLH) and Follitropin (rFSH) are collected in the Filtrate. IEF gel electrophoresis western blotting analysis and well-evaluated immunoassays confirmed that FSH in the Filtrate and the Eluate were identical to those of rFSH and uFSH respectively. Pre-analysis lectin fractionation coupled with immunoassay has enabled an origin identification of LH and FSH. Some other possible markers to indicate origin of glycoprotein were evaluated.

Code: 13D13JD 

This research project will contribute to the understanding of inhaling high therapeutic doses of asthma medication (long acting Beta-2-agonists) over a sustained period of time on athletic performance. The majority of research undertaken with inhaled Beta-2-agonists involves acute doses of short acting Beta-2-agonists in male athletes. Investigating acute doses of Beta-2-agonists does not necessarily replicate real life situations, where athletes prescribed Beta-2-agonists will use them on a daily basis for a prolonged period of the time.

The potential performance enhancement from inhaled Beta-2-agonists focuses on increases in skeletal muscle protein, leading to increased muscle mass.  Increased muscle mass is associated with increased muscle force production.  In addition the long term use of Beta-2-agonists may also decrease body fat.  No study has investigated the potential for these performance gains to occur when athletes inhale long acting Beta-2-agonists over a prolonged period of time.

There is limited ecologically valid data available that demonstrates whether long acting Beta-2-agonists, taken over a prolonged period of time is performance enhancing. Further to this there is no data available that details the movement of Beta-2-agonists through the body following long-term use of high therapeutic doses of inhaled long-acting Beta-2-agonists.

This research project will investigate the impact of inhaling the long acting Beta-2-agonists Salmeterol or Formoterol twice daily over a 12 week period on sprint performance, strength, power, recovery and body composition in male and female athletes. In addition the movement of the inhaled long acting Beta-2-agonists through the body will be analysed at 6 weeks and 12 weeks.  The results of this study will provide data to inform the use of inhaled Beta-2-agonists by elite athletes. In particular they will provide data to assist in the implementation of regulations on the use of inhaled Salmeterol and Formoterol and assist in the resolution of contested doping violations.
 

Main Findings: 

The purpose of this study was to contribute to the understanding of inhaling high therapeutic doses of long acting β2-agonists over a sustained period of time on athletic performance. In particular this study investigated the impact of inhaling Salmeterol or Formoterol twice daily over a ten week period on strength, power, recovery and body composition in male and female athletes.
To carry out the investigation into the potential ergogenic effect of long term use of long acting β2-agonists we recruited 38 participants (23 male and 15 female) who had no history of asthma. All participants underwent a eucapnic voluntary hyperpnoea challenge to confirm absence of asthma. Participants completed baseline assessments which included measuring strength and power performance as well as assessing body composition. Participants were randomly assigned to one of three groups: placebo inhaler, salmeterol inhaler (100 µg twice daily) or formoterol inhaler (12 µg twice daily). The participants undertook a 10 week training protocol with a repeat of the baseline assessments taking place at five and ten weeks. Over the course of the training periods participants took their assigned inhaled dose of β2-agonists twice daily.  During the training weeks participants attended three training sessions a week that focused specifically on developing strength, power and sprint performance. Assessments of recovery and mood were recorded via questionnaires during the training period. 
All 38 participants completed all data collection between weeks 0 and 5, however only the male participants completed all 10 weeks. Between week zero and week five 30 m sprint time improved in both the formoterol (–0.29 ± 0.11 s; P<0.049) and salmeterol (– 0.35 ± 0.05 s; P<0.04) groups when compared to the placebo group (+0.01 ± 0.11 s).  However, there were no changes in other strength and power measurements, recovery, mood or sum of 4 skin folds between groups over the course of the 5 weeks between groups. Between weeks 5 and 10 decreases in sprint time in salmeterol and formoterol groups were similar to placebo group in the male participants. 
Our results suggest that large daily doses of salmeterol and formoterol may lead to improvements in sprint performance over a 5 week period. However the improvements in sprint performance are not sustained over a longer period of time.  Future work investigating the mechanisms will provide an understanding how daily use of long acting β2-agonists may influence sprint performance over a sustained period of time.