Projets de recherche

Code: 10D5MT

Besides the widely assumed misuse of growth hormone as performance enhancing drug, a new class of compounds has received much interest in doping controls. Recent findings of growth hormone releasing peptides (GHRP) in nutritional supplements have shown the urgency to develop detection methods for these substances. To date the knowledge of the metabolic fate of these agents after oral application are only barely described. In the planned study different GHRPs will be synthesized, chemically characterized by liquid chromatography and mass spectrometry and metabolic products will be identified after in-vitro metabolism and animal feeding experiments. Finally, these metabolites will also be synthesized and a sensitive determination method by means of LC-MS/MS will be established.

Main Findings

The obtained data allow the implementation of metabolic products of a variety of GHRPs into routine doping controls. Although derived from animal in-vivo studies, the model appears valid in a qualitative manner since human in-vitro incubations corroborated the likely presence of the surrogate in-vivo generated compounds in human urine. The obtained results will be published in the near future and all data are available for doping control laboratories to implement a facile LC-MS/MS-based procedure for these prohibited compounds by means of commonly and routinely employed instrumental equipment (triple quadrupole mass spectrometers or equivalent). Furthermore, with the same procedure it is also possible to determine additional prohibited peptides in urine (e.g. desmopressin, gonadorelin) and, thus, the method provides the option to implement a screening for different compound instead of an assay limited to individual compounds only. Up to now the collected blood samples from the in-vivo experiments are not analysed. Here it would be interesting to develop a sophisticated purification procedure to detect the target analytes also in blood. This is of particular interest as doping control tests for hGH are conducted with serum. In case of atypically high amounts of endogenous hGH, the same sample can/should be analysed for the presence of these releasing peptides.

Code: 10C16NL

MicroRNAs (miRNAs) are small (19 to 25-nucleotides) noncoding transcripts involved in many cellular mechanisms, including erythropoiesis and response to hypoxia. MiRNAs have been found in tissues and also in serum and plasma as well as other body fluids, in a remarkably stable form that is protected from endogenous RNase activity and harsh conditions. Moreover, plasmatic miRNAs were shown to be very specific and sensitive biomarkers.

Due to all these aspect miRNAs can serve as potential biomarkers for detection for detection of various cancers, diseases and injuries. Erythropoietin-erythropoietin receptor (EPO-EPOR) signaling plays a master role in the erythtropoiesis. Several studies have reported a major role of miRNAs in erythropoiesis. Specific miRNAs were shown to accumulate to very high levels in red blood cells and were associated with early development and maturation of erythroids.

In this project, we are going to investigate whether circulating microRNAs can serve as biomarkers for erythropoiesis stimulating agent abuse. To this end we will analyze miRNA levels in serum and plasma by miRNA microarrays and quantitative real-time PCR (qRT-PCR). Plasma and serum samples are derived from clinical studies of healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A and Dynepo).

Main Findings

MicroRNAs (miRNAs) are small (19 to 25 nucleotides) non-protein coding transcripts involved in many cellular and physiological mechanisms. The role of miRNAs has been mainly investigated in tissues. Recently, a new class of miRNA was found in cell-free body fluids such as plasma. These new class of miRNAs are called “circulating miRNAs”. Circulating miRNAs have been shown to be very stable, specific and sensitive biomarkers. Therefore, they could be altered in a specific manner by doping interventions.

In this project, we investigated whether circulating microRNAs can serve as biomarkers for erythropoiesis stimulating agent abuse. To this end, we analyzed miRNA levels in plasma by miRNA microarrays and quantitative real-time PCR. Plasma samples are derived from clinical studies of healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A).

Based on microarray results, we observed a highly significant difference in the levels of microRNAs in plasma after C.E.R.A injection. We demonstrated that a specific microRNA, miR-144, exhibit a high increase and that its change can detected significantly in a long-term manner after CERA stimulation. Interestingly, it has been reported that miR-144 is essential in erythropoiesis in different organisms such as zebrafish, mouse and human.

These findings suggest the potential of using specific circulating microRNAs as sensitive and informative biomarkers in anti-doping field.

Code: 10A8TP

The determination of carbon isotope ratios (CIR) in order to detect the misuse of endogenously occurring steroids is nowadays a routine method in doping control. The comparison of the isotope ratios of androgenic steroids or their metabolites with steroids derived from other metabolic pathways allows for clear discrimination between an endogenous and an exogenous, i.e. administered, androgenic steroid.

Since the 1990´s the microbial degradation of urine samples stored under inappropriate conditions is well investigated. Many microorganisms could be identified and their specific action on steroids and steroid conjugates was ascertained. The main chemical conversions are hydrolysis of gluco- or sulpho-conjugates and dehydrogenation of hydroxyl-groups and the steroid backbone.

The impact of these chemical transformations on the steroid profile with its concentration thresholds and diagnostic ratios is described in literature but no data at all is at hand for possible changes in the CIR of different steroids due to microbial degradation. A change in the 13C/12C ratios of urinary steroids due to degradation might impede the use of this technique in doping control analysis.

Therefore, the stability of CIR for different selected steroids and their degradation products will be investigated in urines stored at 37°C and the impact of these storing conditions on the validity of CIR measurements will be investigated.

Main Findings

The influence on CIR of different urinary steroids and steroid-conjugates during degradation was investigated. Regarding glucuronidated steroids which are used in doping control analysis, no significant influence on CIR during the first weeks of the study could be detected. This changed with emerging of the dehydrogenation products 4DN, ADN and EDN and therefore suggested careful interpretation of CIR results in samples showing these strong indications of degradation. The same applied for steroids excreted as sulfates. Especially at the beginning, unconjugated steroids showed strongly depleted δ13C values and therefore shall not be utilized in doping control analysis. The reasons for this strong fractionation could not be identified unambiguously within this study and further research on the deconjugation of steroid glucuronides seems advisable.

The results obtained for DCM and DHEA supported the theory of a reaction mechanism including an ionic intermediate rather than a concerted reaction. In the context of doping control analysis, the CIR of DCM, DHEA_S and maybe 5EN17b_S should not be taken into consideration due to the strong isotopic fractionation coming along with the cleavage of the sulfate moiety.

Code: 10A20HL

The aim of this project was to provide a method for hCG detection based on immunoaffinity extraction and mass spectrometric detection. Furthermore, a small clinical trial was carried out in order to prove that Hcg that has been administered to male athletes after illicit use of anabolic steroids, can in fact be determined in both serum and urine immuno-MS methodology. Additionally a comparative study with the DELFIA immunoassay also was carried out.

Main Findings

The immuno-MS methodology has been optimized in order to enable the desired LOD and LLOQ, and has furthermore been validated according to existing guideline. Using this method, a clinical study (approved by the regional Committee foe medical Research Ethics) involving 24 healthy voluntary men was carried out. HCG was injected (either Ovitrelle or Pregnyl), and urine and serum samples were collected and analyzed. The method allowed detection and quantitation of administered hCG in urine, from day one until day ten after injection of hCG containing pharmaceutical. On day fourteen after injection, no trace oh hCG can be detected. The method also allowed detection and quantitation of administered hCG in serum, from day one until day seven after injection of hCG containing pharmaceutical. On day fourteen after injection, no trace of hCG can be detected. Furthermore, the presence of different hCG variants in urine and serum samples has been demonstrated. The immuno-MS method has been benchmarked against the DELFIA immunoassay and showed good correlation for the serum samples, For urine samples the DELFIA method showed significant lower hCG values than those obtained using the imuno-MS method. This is ascribed to the hCG instability in urine which has less influence on the immuni-MS method than on the DELFIA immunoassay. Both immuni-ms and DELFIA immunoassay detected hCG in equal long time after administration. The results demonstrate the methods capability of 1) detecting and 2) differentiating between the various hCG variants, thus demonstrating their presence in the biological matrix. Futhermore, the methodology can generate a 3) quantitative measurement of the hCG amount present in the patient as well as in healthy subjects to which hCG was administered. Based on this we conclude that the developed methodology of immunoextraction combined with mass spectrometric detection is capable of revealing the presence of illicit administered hCG in athletes. Additionally, the immuno-MS method gives similar results compared to the conventionally used DELFIA immunoassay.

Code: 10A10MT

Small interfering RNA (siRNA) is a tool to influence and manipulate gene expression, which might be misused in sports and has therefore been prohibited according to established anti-doping rules. siRNA molecules bind to messenger molecules (so-called mRNA) to downregulate the synthesis of selected proteins. In general, any target gene could be downregulated on the mRNA level by applying the corresponding, complementary siRNA. Therefore, the fields to use these molecules for performance enhancement are manifold.

Due to a very short plasma half life, siRNA molecules are modified and protected from degradation by RNAses, which complicates the prediction of a future pharmaceutical product but identifies the xenobiotic nature of such molecules and is considered a starting point for method development for doping control procedures.

The planned project includes the development of a confirmation method targeting modified siRNA and shall further provide the proof-of-principle by oral application or injection of siRNA to laboratory rodents with subsequent blood and/or urine analysis. A screening method based on the isolation of the intact siRNA strands from plasma samples followed by high resolution/ high accuracy mass spectrometry measurement was recently developed and validated and serves as basis for a confirmatory assay. For a confirmation method, tandem-mass spectra may be recorded and evaluated after optimization of the sensitivity to identify a sequence and modified nucleotides. Alternatively, modified nucleotides may be detected after degradation of the strands in plasma or urine samples. For that purpose, administration studies are of particular importance and, due to the clinical status of the substances, only preclinical studies are aimed. For the modified nucleotides, analysis is planned to be performed by LC/MS procedures.

Main Findings

The issue of gene doping with modified genetic information that is introduced into the athletes’ organism has been an emerging field in sports drug testing. Within the present project several strategies were developed in order to uncover the misuse of small interfering (si) RNA for performance enhancement. By means of siRNA as doping agent, literally every gene of interest can be temporarily silenced (knocked-down). In the present study the muscle regulator myostatin was chosen as target gene. It was shown that specific model siRNAs (designed to knock-down the myostatin messenger RNA) are detectable in rat urine after single intravenous administration at arguably therapeutic dosing for up to 24 hours. The unambiguous identification of the metabolites in urine was realized by a combination of liquid chromatography-mass spectrometry approaches and gel electrophoretic-based assays under consideration of intact metabolites as well as their hydrolysis products. The assay’s performance was characterized and validated for the designed model siRNA substances, and a generic protocol and approach to uncover the misuse with to-date unknown target molecules was suggested. Here, strategies comparable to proteomics methodologies allowing for de novo sequencing were tested, which were successfully applied as long as the artificially modified nucleotides were included in the available data evaluation software. Further work will be required to expand the test to the virtually unlimited options of sequence modifications; however, the presence of a xenobiotic nucleotide or sequence of nucleotides in doping control specimens is a substantial hint towards the misuse of RNA-interfering substances.

Code: 10B10MT

The misuse of recombinant growth hormone in elite sports is well known from confiscations and confessions and additionally, the first two positive samples were reported in 2009 using the luminescence immunoassay developed by Bidlingmaier and Strasburger. The luminescence immunoassay (LIA) provides a powerful screening tool but a complementary method for confirmation providing more detailed information would be desirable. Therefore, a method based on immunoaffinity purification, 2D-PAGE and immunoblotting was developed which detects discrete endogenous variants of growth hormone. After successful development and validation, the methods´ sensitivity and robustness need to be optimized.

The project is planned to improve the sensitivity to be similar to that reached by the LIA to yield a powerful confirmation method. This can be done by optimizing a) the immunoaffinity purification e.g. by coupling specific antibodies directly to magnetic beads, b) the blotting conditions or the immunodetection, e.g. by using different secondary antibodies for the visualization and detection. Furthermore, the robustness of the method should be improved by providing another primary antibody which could replace the currently used one to ensure continuous availability.

After optimization, another follow-up project could include the measurement of a reference population to allow the calculation of reference values.

Main Findings

Human endogenous growth hormone (hGH) is one of the most important growth promoting hormones in the human body. It regulates bone growth in childhood and has an important impact on many metabolic processes such as muscle growth and increased fat consumption. Recombinant growth hormone (rGH) is supposedly misused by various athletes as performance enhancing agent because of its lipolytic and anabolic effects. It is a protein composed by 191 amino acids with a molecular weight of 22 kDa and is produced in the pituitary gland. Alternative splicing results in a smaller isoform of 20 kDa missing the amino acids 32-46, and different posttranslational modifications such as phosphorylation, acylation, glycosylation as well as proteolytic cleavage and dimerization lead to a large heterogeneity. The detection of discrete variants of hGH by immunoaffinity purification, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunoblotting could serve as a complementary detection assay to uncover the misuse of hGH in sport. The aim of this project was to enhance the sensitivity of an existing 2D-PAGE detection assay for hGH doping by optimizing different steps of the sample preparation protocol plus tests for alternative primary antibodies. Out of four tested antibodies, one proved to be an adequate alternative as it was not only able to detect rGH amounts down to 0.25 ng but also to bind all endogenous variants of hGH. The antibody was subjected to protein A purification and the sample preparation protocol was optimized by modifying antibody concentrations, incubation times, secondary antibodies, amplification systems and fluorescence detection. Finally, an optimized protocol was composed comprising immunoaffinity purification, 2D-PAGE and immunoblotting (with secondary antibody amplification); however, due to these new aspects and requirements of the methodology, further evaluation of the performance and applicability to authentic administration study samples might be required. In the absence of technical alternatives (e.g. MS-based methodologies) to immunologically driven assays, the use of monoclonal antibodies (as employed in the currently routinely used LIA test system) might be preferable to ensure constant quality and comparability of analytical results.

Code: 10D20VB

The higher prevalence of asthma and shortness of breath during exercise among elite athletes has resulted in increases use of anti-asthmatic medication among elite athletes. Studies of therapeutic doses of beta-agonists have in general not shown effect on the cardio-respiratory capacity. But theses doses are probably not the case in prohibited use of medication among athletes. The last international doping cases have shown significantly high intake of beta2-agonist, indicating either above therapeutic level of inhaled use, or systemic use of ordinary used anti-asthma medication. If intake of high doses of beta-agonist leave the aerobic capacity, muscular power or recovery is unchanged, ordinary used beta-agonist probably should be removed entirely from the prohibited list. Before this substantial change of the WADA code, more research is needed.

The cardiovascular effect of high doses of beta-agonists, with better overall performance, oxygen uptake and oxygen kinetics, has never been thoroughly studied, which is of importance as doping doses seldom are within therapeutic level.

Purpose: To examine whether high doses of salbutamol and Terbutaline is a class effect of beta2-agonists, different from therapeutic doses. Animal models have shown that salbutamol, in higher doses than normal therapeutic asthma doses, increased the contraction of skeletal muscles and keep a persistent effort. Furthermore, another animal model has recently shown increased force and recovery of the skeletal muscles after Salbutamol. Based on the knowledge of therapeutic doses beta2-agonists seems to be unimportant, but illegal use of beta2- agonist probably would be in higher doses which could be of substantial importance.

Purpose: To examine whether Salbutamol and Terbutaline in a human in vivo model similar to the Rat model can show the same positive effect on peripheral leg muscles given as a single high dose prior to exercise and continuously over 2 weeks.

Main Findings

Our purpose was to investigate effects of high dose beta2-agonists on aerobic and anaerobic exercise performance along with muscular effects on contractile force, metabolism, and ion handling in healthy trained men. To investigate these purposes, we conducted seven experiments.

First four experiments investigated the effects of high dose inhaled terbutaline (15-20 mg) on exercise performance and sprinting peak and mean power, as well as on muscle metabolism, contractile properties, and ion handling. Our data showed that terbutaline increased contractile force and enhanced sprinting peak and mean power, but had no effect on time-trial performance or on endurance performance. In skeletal muscles, terbutaline improved Ca2+ handling and counteracted exercise-induced reductions in Na+/K+-ATPase Vmax. Furthermore, terbutaline elevated rate of glycolysis. Thus, performance-enhancing effects of high dose terbutaline may be attributed to effects on skeletal muscles through enhanced Ca2+ handling and elevated glycolytic activity.

In the latter three experiments, we investigated the effects of acute and short-term administration of oral salbutamol (8 mg) and terbutaline (20-30 mg) on exercise performance, muscle contractile properties, and sprinting peak and mean power. Our data showed an acute enhancing effect of terbutaline on contractile properties of m. quadriceps. Four-week administration of oral terbutaline (2∙10-15 mg/d) elicited muscle hypertrophy, reduced fat mass and increased contractile force. Acute and two-week administration of oral salbutamol enhanced sprinting peak power, but had no effect on endurance performance.

In conclusion, our data supports the restriction of high dose inhaled terbutaline and oral beta2-agonists in competitive sports in and out of competition.

Code: 10D12GF

In doping controls, knowledge of steroid metabolism is a key issue to ensure adequate detection of steroids in a urine sample. Metabolism studies are usually performed in vivo by collecting urine samples over a certain period of time after administration of the steroid. However, with new designer steroids on the marked lacking a toxicological profile, this approach has become difficult. Moreover, metabolites may be present at low concentrations in urine, making detection and characterization difficult.

In vitro metabolism studies is an alternative to excretion studies in humans, and human hepatocytes are recognized to be a very close model to the human liver with intact cell membranes and the full compliment of enzymes and cofactors. The limited availability of fresh human hepatocytes has long been the major disadvantage using hepatocytes as a model. The progress in cryopreservation techniques, however, has resulted in an increased accessibility and human cryopreserved hepatocytes are now commercially available.

In the proposed project, the use of cryopreserved human hepatocytes to investigate metabolism of doping agents will be evaluated. Emphasis will be put on anabolic steroids, and the developed model will be tested on two steroids with a well known metabolic profile and one designer steroid with an unknown profile. After hepatocyte incubation and a clean-up procedure, analysis will be performed with liquid chromatography and gas chromatography, combined with mass spectrometry. The in vitro biotransformations and metabolic profiles will be studied in detailed, including the correlation to known in vivo urinary profiles, if available.

Main Findings

The present study deals with the usefulness of cryopreserved hepatocytes to access metabolic mixtures of doping substances. The described in vitro methods are simple systems which are fairly easy and fast to perform. The in vitro hepatocyte metabolism has proven to be close to the human metabolism. It does, however, not reflect the whole complexity of human metabolism, particularly in the light of long term metabolites and phase 2 metabolism.

Despite this, incubations with hepatocytes represent a very useful model for identifying and predicting potential metabolites. Major advantages are amongst others:

- A fast access to a metabolic mixture, which can directly be used for detection strategies of doping substances.

- New non-approved substances with unknown toxicological profiles can be used as substates resulting in metabolites as markers for screening and confirmation purposes.

- No ethical approval is necessary

- Easy detection of possible metabolites due to limited matrix interference of the incubation mixture. Compared to microsomes, the in vivo situation is better reflected with hepatocytes, taking into account drug transport. Furthermore, cryopreserved hepatocytes have several advantages over fresh hepatocytes, first of all a better availability. In addition, cells from several donors can easily be pooled for experiments. Drugs that are metabolized to a high extent in the liver tend to show higher interindividual differences and sometimes genetic polymorphism. Hence, a pooled experiment can better reflect the metabolic picture of the average population.

In this study, human hepatocyte incubations have led to biotransformations generating major metabolites of androstendione and metandienone reported in vivo in humans. Additionally, several metabolites of the synthetic steroid norbolethone were detected, which of only two have been previously described.

Code: 10D1PD 

The classical methodology to detect anabolic steroids or other anabolic substances in doping analytics is GCMS and LCMS. However, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not identified during routine screening if their exact chemical structure is unknown. Moreover pharmaceutical companies are developing non steroidal androgen receptor modulators (SARMs) which have a complete different chemical structure and metabolism than classical anabolic steroids. Therefore indirect detection techniques may be very helpful in the future to supplement the direct detection of anabolic substance abuse. From the classical anabolic steroids, including Testosterone, it is known that exogenous administration of these substances directly affect the production of endogenous hormones via feedback mechanisms on the hypothalamic pituitary gland axis (Mahabadi et al. 2009). This concept is so efficient that treatment with low testosterone doses is a well established pharmaceutical concept for male contraception (Gu et al. 2009, Misro et al. 2009). The simultaneous detection of ACTH, TSH, PRL, FSH, and LH in comparison to inhibin, estradiol, thyroxin and testosterone levels and indicators for anabolic activity, such as IGF-1 and myostatin, could be a supportive strategy to detect anabolic substance misuse, including SARMs. 

Main Findings: 

The classical methodology to detect anabolic steroids or other anabolic substances in doping analytics is GCMS and LCMS. However, the example of THG has demonstrated that even substances with a chemical structure typical for this class of substances are sometimes not identified during routine screening if their exact chemical structure is unknown. Moreover pharmaceutical companies are developing non steroidal androgen receptor modulators (SARMs) which have a complete different chemical structure and metabolism than classical anabolic steroids. Therefore indirect detection techniques may be very helpful in the future to supplement the direct detection of anabolic substance abuse. From the classical anabolic steroids, including Testosterone, it is known that exogenous administration of these substances directly affect the production of endogenous hormones via feedback mechanisms on the hypothalamic pituitary gland axis (Mahabadi et al. 2009). This concept is so efficient that treatment with low testosterone doses is a well established pharmaceutical concept for male contraception (Gu et al. 2009, Misro et al. 2009). The simultaneous detection of ACTH, TSH, PRL, FSH, and LH in comparison to inhibin, estradiol, thyroxin and testosterone levels and indicators for anabolic activity, such as IGF-1 and myostatin, could be a supportive strategy to detect anabolic substance misuse, including SARMs

Code: 10C2BC

Already in 1990, rHuEpo was banned by the American Medical Association and the International Olympic Committee (IOC). However, the abuse of rHuEpo continues and a sensitive and robust detection test is needed. The current method directly measures urinary rHuEpo, and is based on differences in glycosylation between rHuEpo and endogenous Epo. However, this method is expensive and has low sensitivity.

Protein profiling, or proteomics, provides a novel and powerful method for characterizing the complete catalogue of proteins expressed in a biological system. The aim of the current project is to study the impact of more prolonged rHuEpo exposure on serum proteomics in healthy human subjects, in order to detect novel biomarkers for Epo abuse.

The study will have a randomized double-blinded design. A total of 4 experimental groups will be included in the study; 1. rHuEpo treatment, 2. rHuEpo + exercise, 3. Placebo, 4. Placebo + exercise. A total of 12 healthy inactive male subjects will be included in each group. The treatment period will be 12 weeks followed by a 3 week washout period. rHuEpo will be administrated s.c. at a dose of 5000 IU (~60IU/kg) every second day for the first 2 weeks, on three consecutive days during week 3, and once weekly during weeks 4-12. All subjects will be supplemented by 100 mg iron orally/week. The exercise will consist of 1½h biking at 60-75% of VO2max 3 times per week.

Proteomics will be performed on serum samples collected before treatment and on days 16, 84 (end of treatment) and 105 (end of washout period). It is the hope that the current study will identify novel biomarkers that are specific for rHuEpo treatment, and not effected by exercise itself, that can be used in future anti doping test.

Main Findings

RESULTS: a total 125 spots were identified in the serum 2DE gels, here of was 80 observed in all gels in the current study, isoforms of 6 proteins changed significant during the intervention and washout period in response to ESA treatment. When comparing all 4 experimental groups, isoforms of 2 proteins (sertransferrin and haptoglobin, respectively) showed a significant response to ESA treatment. Hereof, one spot (g), containing haptoglobin, showed a significant lower intensity in all subjects in the training-Epo group during the ESA treatment period, and an increase in intensity during the washout period. Thus, this isoform of haptoglobin could be a potential new anti-doping marker.

CONCLUSION: It is possible by a proteomic approach to identify serum protein isoforms that change significantly in response to Epo abuse without being affected by endurance training. Especially spot G seems promising as a novel biomarker for EPO abuse, since the level of this haptoglobin isoform was significantly decreased in all subjects during the treatment period, and increased again in all subject during the washout period.