Projets de recherche

Code: 10A26AK

Cheaters in sport commonly use testosterone for anabolic purposes, not least because it is more difficult to detect such doping with a steroid that is also produced naturally in the body. It is suspected that lower doses of testosterone are often administered, particularly formulations designed for daily use, such as hydroalcoholic gels, so that users can rapidly clear the drug from their bodies in anticipation of a drug test. An increasing number of blood samples are now being taken to detect transfusion and growth hormone administration and this also permits the analysis of testosterone for profiling. Serum analysis has an advantage over urine in that the concentration of testosterone should be relatively constant in ‘clean’ athletes. By contrast, administration of low doses of testosterone does raise serum testosterone but this may not exceed the upper limit of normal for men in every case. However, proving that an increase in serum testosterone is uncharacteristic in men and women should enable detection of low-dose administration.

Methods will be developed to enable the accurate measurement of testosterone in a small volume of blood serum using a gold-standard approach of liquid chromatography-mass spectrometry. The natural variation in the profile of serum testosterone in male and female athletes will be monitored over a year to assess whether the concentration of serum testosterone within each subject is sufficiently stable to be worthy as a biomarker for evidential analysis (athlete passport Bayesian approach). Once established, further funding will be sought to validate the effectiveness of the approach by administering small doses of testosterone to volunteers that are not competing in sports. The approach should provide a more sensitive method for the detection of doping with testosterone than that provided by urine analysis alone.

Main Findings

This study attempts to reveal whether within individual serum testosterone concentrations might be sufficiently stable biologically in the normal individual to provide a useful marker to add to the ABP to evidence anabolic steroid administration. The study required the design and validation of a suitable analytical method based on LC-MS/MS instrumentation initially to be able to measure serum testosterone from blood samples collected from male sports competitors. Blood and urine samples were collected from the competitors by UK anti-doping agency ltd (UKAD) trained doping control officers in accordance with WADA protocols. Generally the samples were collected out of competition on multiple occasions from the same competitors in order to be able to assess any change of the values over the time. The competitors were selected by UKAD as part of their normal process but, in particular, because these competitors had previously been shown to have more variable urinary testosterone to epitestosterone ratios (T/E) than the more normal 30% limit for males. The laboratory was blinded to the identity of the competitors and were not provided with any further information about the extent of any exercise undertaken by the competitors immediately prior to sample collection. Data was obtained from 15 different competitors. We found that the urinary T/E in the cohort studied had a variation over time (CV%) greater than 30% in 7 out of the 15 cases investigated. We consider that it is reasonable to assume that these individuals may also have a greater variability in their serum testosterone concentrations. Indeed we found that these athletes had an equivalent CV% for their within subject serum testosterone concentrations greater 30% in 11 out of 15 cases investigated. However, these values were not clearly correlated between serum and urine although five exceeded 30% for both serum testosterone concentrations and urinary T/E. The ABP software provided a useful tool for evaluating the data enabling us readily to assess the sensitivity of the method, i.e. at what point would an athlete be shown to be "abnormal". No sample exceeded the calculated limits nor did any sample exceed the normal male reference interval. It is likely that a cheating male athlete would need to administer sufficient testosterone to overcome their normal endocrine homeostasis that would simply reduce endogenous production when the serum testosterone is greater than the norm for the individual. The fact that we have evaluated a cohort selected on their larger than normal variability in urinary T/E gives us reason to believe that the majority of the population would fit well within the model that we have used, Thus the results indicated to us that the majority of the population would fit well within the model that we have used. Thus the results indicated to us that serum testosterone measurements may be a useful part of a steroid passport scheme. An administration study would be required to confirm this assertion.

Code: 10B11RG

Growth hormone secretagogues (GHS) are molecules that stimulate the secretion of human growth hormone from the pituitary. They have proven to be potent agonist and as they have been developed by pharma even before the natural ligand or receptor were known, GHS display large structural heterogeneity. In order to address the entire family of molecules one can only target what all GHS have in common: the interaction with the receptor. Based on this premise we developed a competition assay and have demonstrated already its functionality.

Within the context of this project the assay validation shall be performed as well as further development of improvements to facilitate implementation in other anti-doping laboratories.

Main Findings

Growth hormone secretagogues (GHS) are a large group of, structurally very diverse, chemical compounds that all share the interaction with the growth hormone secretagogue receptor 1a (GHSR-1a). Following the interaction of GHS with GHSR-1a, located in the pituitary gland, the release of growth hormone (GH) is stimulated through the intracellular Ca2+-release mechanism that is completely independent from the cAMP mechanism of the growth hormone releasing hormone. Both the receptor GHSR-1a as the natural ligand (ghrelin) were discovered less than 20 years ago whereas the development of GHS was initiated more than 30 years ago giving rise to large structural heterogeneity in the pharmacophores. Within the framework of this project we have developed a universal screening method for all GHS, irrespective of the chemical nature and structural characteristics, by using the single feature that all share: the interaction with GHSR-1a. We have established a stable and recombinant expression of this receptor and use this in combination with 125I-ghrelin to establish a 100% binding situation. The co-incubation with a processed urine extract (from 2.5 ml urine for a triplicate measurement) indicates the presence of a secretagogue if binding falls below an established threshold.

Following the development of the protocol we established the threshold value and limit of detection, the functional assay sensitivity, analytical stability, and intra- and inter-assay accuracy. We have assessed the influence of ethnicity, gender, age, and exercise, finding no significant influence of any of these factors on the assay readouts. Finally, we evaluated specimens from a pralmorelin administration study and compared the results to those obtained from analysis by LC-MS. We found that for the intact compound both protocols provided very similar results.

Code: 10A1PV

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

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

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

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

Main Findings

A combination of the Support Vector Machine (SVM) algorithm with a comprehensive approach of steroid profiling resulted in a steroidomic model that enables to differentiate normal steroid profiles from abnormal ones. Theoretically, the SVM tool plots all monitored\steroids in a multi-dimensional hyperspace which makes the use of steroid ratios redundant to obtain a strategy with optimal detection sensitivity. Hence, the whole set of steroid profile values can be evaluated at once. In our model, however, the degree of abnormality was quantified by an Abnormal Steroid Profile Score (ASPS) for which values greater than 0.79 could be considered as deviating from normal.

Since the introduction of the Athlete Biological Passport, the results of steroid profiling tests can be systematically stored in a central database enabling the estimation of the individual reference ranges. From such databases, longitudinal steroid profiling data can be made readily available to elaborate longitudinal strategies, thereby omitting a large contribution of the inter-individual variance. Similarly, the raw SVM model was improved by standardizing the training set using individual mean and standard deviation obtained with the adaptive model. The combination of the adaptive model and the SVM enhances the general performance accuracy of the raw SVM model from 62% to 84%, disregarding the kind of endogenous steroid administered. The diagnostic sensitivity of the resulting ASPS was 55% in a post-administration period of 7 days. Altered steroid profiles can be found until 5 days after ingesting a small single doses of T or DHEA or after topical application of T or DHT in therapeutically recommended doses. This drastic increase in sensitivity can be explained by the ability of the model to sensitively distinguish a prolonged recovery state of the steroid metabolism which is restoring the homeostasis of steroid profile to known basal levels.

Since the model was trained on data obtained after T, DHT and DHEA administration, the model risked to be overfitted i.e. a specific detection tool for these steroids. This problem was addressed by leave-one-subject-out cross-validation and testing of the model on another volunteer, with another dose of DHEA and with other steroids. Testing of the excretion data from a 100mg dose of DHEA, 50mg Adion and 7-keto-DHEA ingested by another volunteer showed a clear response of the ASPSs. This indicates the polyvalent nature of the SVM model to detect any small disturbance of the steroid profile. Moreover, the high sensitivity of 97% obtained for this new test set illustrates the potential of the ASPS as a powerful biomarker for the general detection of misuse with endogenous steroids. Although, this single model shows excellent sensitivity for a wide range of administered steroids, it cannot specify which cause resulted in an aberrant steroid profile. For this information, specific metabolites should be evaluated separately.

Despite the excellent preliminary results on low dose administration studies conducted on a limited study population - including subjects with atypical T/E’s that challenge the classification -, the applicability of this strategy will require further work and large scale validation procedure. In order to implement the ASPS in routine testing as a sensitive marker for of any misuse with endogenous steroids, the model should be tested on larger cohorts of data and external influences on the steroid profile that can alter the ASPS should be scrutinized in the future.

In conclusion, a new strategy was developed that returns a single value ASPS as a denotation of the degree of abnormality of a steroid profile containing 24 steroid metabolites. With this strategy, the alteration of the steroid profile, caused by a variety of endogenous steroids, can be detected very sensitively. The longitudinal SVM model was shown to be a general model which can result in long detection of small doses of oral and topical steroid formulations up to 5 days. The overall model performance was very good, particularly when coupled with the longitudinal results from the adaptive Bayesian model. The combination of computer aided techniques as the Bayesian adaptive model and SVM algorithm provide a valuable steroidomic strategy for the long term detection of misuse with endogenous steroids in complement with current steroid profiling methods.

Code: 10B1GC

The reliable determination of human recombinant erythropoietin (rhEpo) abuse to enhance the athletic performance is an important problem for sport and represents a continuous challenge for investigators involved in anti-doping. The analytical tests used to detect rhEpo in urines are often inadequate and suffer from many interfering factors. Hepcidin, a liver-derived peptide which is the major regulator of body iron metabolism, is an accurate indicator of changes in blood levels of Epo. Indeed, Epo administration in humans caused a marked reduction in urinary and circulating hepcidin.

Therefore, the aim of the present project is to verify whether the determination of hepcidin may represent a valid alternative method to detect an inappropriate use of rhEpo for doping purposes. Such an indirect approach could be also useful for the detection of the use of last generation pharmacological agents such as continuous erythropoietin receptor activators (CERA). The detection of hepcidin in urines and serum has been performed so far by means of immunological assays or SELDI-TOF-MS techniques, which present problems related to quantitative determination and requirement for expensive equipment and skilled personnel, respectively.

Our challenge is to provide an innovative analytical process for the evaluation of the presence of erythropoiesis stimulators abuse. The heart of the project is a new multi-screening affinity sensing platform and innovative low cost devices, for the detection of biomarkers such as hepcidin and for the doping substance itself (erythropoietin).

In this context, a flexible platform, consisting of a biochip coupled to a label free technology for simultaneous measurements in short time could represent an innovative approach for selectively detecting biomarkers of erythropoiesis stimulators abuse. The project will also consider the possibility of developing an innovative, electrochemical biosensor which can be also used in a low density array format for the simultaneous detection of several doping markers.

Main Findings

The aim of this project, which was funded as one year pilot project by WADA, was to evaluate the role of hepcidin, the major regulator of iron homeostasis, as an alternative or complementary marker to detect the abuse of rHuEpo for doping. During this period of time, we used a multidisciplinary approach combining biochemistry, molecular biology, analytical chemistry, etc., in different experimental models, and we obtained several interesting and promising results.

These results indicate that affinity-based biosensing can be an innovative approach to hepcidin detection, although the assays have to be improved in order to compete with more established analytical methods. A preliminary evaluation indicates that the developed systems, based on an antibody and a biomimetic receptor specific for hepcidin-25, have several advantages, i.e. they allow the direct determination of hepcidin, are reproducible, label-free, easy to achieve, quite cheap, and fast. In fact, both systems allowed hepcidin quantification in very short time (15 min), if compared to previous methods requiring several hours.

Anti-hepcidin resulted more performing than HBD: it was sensitive and reproducible in the range of physiological hepcidin levels. On the other hand, the antibody showed, compared with HBD, a short life time once immobilized on the biochip. However, this problem could be prevented by conceiving different immobilization chemistries (e.g. through Protein A/G immobilization). Finally, a general consideration should be done on the necessity to handle hepcidin (particularly standard solutions) in a way that minimizes variation and under conditions that assure reliable analytical results. ongoing (LIVE) work is now devoted to exploring new antibodies and synthetic receptors which could be successfully applied to SPR for hepcidin determination, i.e. aptamers.

To our knowledge, the results described in this report are the first attempt to detect hepcidin, a relevant but “tricky” peptide, by affinity-based biosensors (ABBs). We are aware that there still are limitations in terms of performing features, but are currently exploring several strategies to overcome these problems. Possible approaches to enhance the analytical response by improving the detection limit could be: changing the immobilization procedures, and/or in adding suitable tails to HBD to confer more freedom to the peptide once immobilized on the biochip. Once identified a pool of suitable bioreceptors displaying low-cost, stability, easy availability, and high sensitivity to the analytical target, we believe that affinity biosensors can be successfully applied in the near future to real samples, in both urine and serum matrix (initially flanking conventional and profiling methodologies). When validated, the biosensing approach will contribute to the development of an an innovative methods for a fast, low cost, and easy to use methods for hepcidin detection.

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.