Projets de recherche

Code: 19A04MP 

In 2018, the current GC-QQQMS routine screening method (i.e., initial testing procedure) for human doping control was successfully converted into an equivalent and complete GC high resolution acquisition screening method for routine purposes by using low energy electron ionization (EI) GCQTOF. This GC-QTOF screening method is compliant with the WADA requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min). Because of the full scan high resolution data acquisition ability of TOF technology (and the corresponding retrospective capabilities), this proved to be a big step forward in comparison with the current GC-QQQMS routine screening methods. Taking into account that anti-doping samples can be stored and reanalyzed for up to ten years, the retrospectivity and sensitivity offered by GC-QTOF opened the door to a cleaner sport. Sensitivity is obviously compound depended, but in general the sensitivity of the low energy EI GCQTOF is situated between EI GC-QQQMS and CI GC-QQQMS.

Chemical ionization (CI) is a softer ionization than low energy EI and has the potential to further increase the sensitivity, in parallel with our previous EI/CI work on GC-QQQMS. Combining GC-QTOF with CI is the next logical step and this project aims at exploring, testing and exploiting the potential of CI GC-QTOF in all its aspects. This will result in the development of a high-resolution acquisition screening method with higher sensitivities. Higher sensitivities lead to more flexibility, longer detection times and a more extended list of compounds that can be monitored.

Main Findings

The main objective of this project was to improve the sensitivity of the GC-QTOF by using CI instead of EI, in order to maximize the capabilities of the GC-QTOF. Our experiments show that at high concentrations, CI is indeed more sensitive than EI, as we expected. Unfortunately, at lower concentrations, in general, the signal generated by CI ionization drops substantially faster than with EI ionization, making CI less sensitive than EI at those crucial low concentrations. To make It worthwhile to shift from EI to CI ionization on the GC-QTOF, the sensitivity at the low levels should be at least 5 times higher. Only then, it becomes worthwhile shifting from EI to CI ionization as CI also has some important inherent disadvantages such as the requirement for laborious and frequent instrument maintenance. The performance of CI GC-QTOF is insufficient and was found unsuitable for ITP purposes. At high concentrations, CI indeed outperforms EI, as we expected. Unfortunately, at lower concentrations, in general, the signal generated by CI ionization drops substantially faster than with EI ionization, making CI less suitable than EI at those low concentrations. The reason for this phenomena is up to this day unclear to us and we are at this stage cooperating with the R&D department of Agilent the share our experiences and to examine possible causes and solutions. From a theoretically point of view CI should outperform EI. However, in practice, this is only the case at high concentrations. That is the main cause and issue, preventing the use of CI for an ITP. Due to the gain/loss of sensitivity/specificity when comparing CI GC-QTOF versus EI GC-QTOF, CI might be useful to use as a CP. However, this needs to be checked on a substance to substance basis and depends on sample prep, GC parameters, etc. For example, for some AAS, CI will be a better option, for others it will not.

Code: 19D06RF

The goal of our first study is to investigate the correlation between the Athlete Biological Passport (ABP) variables and performance data from competitive elite athletes analyzed from training and racing power outputs (elite cyclists), apneic times, depth or distance (trained apneic divers) and endurance capacity (sport students). In this way, our study would challenge the complexity of confounding factors affecting the interpretation of the ABP. Testing the hypothesis that variations in performances are related to variations in the ABP in different sport disciplines would allow the ABP model to be strengthened. Finally, our project will evaluate the range of variability of identified confounding factors altering the blood formula in a population of trained apnea divers. It is hypothesized that breath-holding methods and specific apnea training techniques may significanlty alter blood parameters from the ABP. Indeed, it has already been shown that 3 repeated breath-holdings increase hemoglobin concentration ([Hb]) in divers, skiers and untrained humans acutely while an extension of such findings in the context of the ABP is still needed. Overall, out study aims at improving the ABP by challenging the individual whithin-subject variance in light of known confounding factors. The objectives of this study are threefold: firstly, this study will allow to further investigate the relationship between Hbmass and aerobic performances in three distinct populations (elite cyclists, apnea divers, sport students). Second, blood variables data collected monthly will allow us to discriminate between the influence of specific confounding factors thanks to the multi-sourcing data and heterogeneous groups (cyclists, apnea divers, sport students). Overall, this study will enable us to strengthen the ABP after looking at the within-subject variations to propose a robust evaluation of known underlying confoundng factors.

Second, we will conduct a study investigating the influence of the menstrual cycle on hematological varaibles to identify potential variations factors specific to blood losses and hormonal variations.

Thirdly, we will conduct a study on elite race walkers to describe hematological variations observed before, during and after a prolonged exposure to i) altitude and ii) heat in their final preparation block before a major competition.

Main Findings

In the supported project, we first conducted a study to investigate the relationship between Hbmass and aerobic performances in three distinct populations (elite cyclists, apnea divers, sport students) with blood variables data collected monthly to discriminate between the influence of specific confounding factors thanks to the multi-sourcing data and heterogeneous groups (cyclists, apnea divers, sport students). This resulted in a peer-reviewed publication (1). Although individual hematological variations were observed, all ABP variables remained within the individually calculated limits. We showed that acute training load variations in elite cyclists significantly affect (Hb), likely due to short-term PV fluctuations, underlining the importance of considering training load when interpreting individual ABP variations for antidoping purposes.

Second, we conducted a study investigating the influence of the menstrual cycle on hematological variables (total Hbmass, ABP variables & indicators of plasma volume changes) to identify potential variations factors specific to blood losses and hormonal variations. This resulted in another peer-reviewed publication (2). A multi-parametric model previously validated in elite cyclists was applied to compare inferred and actual PV variations. Some significant intra-individual PV variations were observed, in good agreement with the estimated PV changes; and it can be concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.

Thirdly, we had planned to conduct a study on elite race walkers to describe hematological variations observed before, during and after a prolonged exposure to i) altitude and ii) heat in their final preparation block before a major competition. However, this could not be completed yet, due to 2020/2021 travel restrictions with the context of the pandemic. We worked however instead on the monitoring data from the apnea divers cohort. This resulted in a peer reviewed publication (3). Whilst we hypothesized that regular breath-hold training could affect or alter ABP variables, the results of our study do not outline any particular significant effect of regular breath-hold training on ABP variables. Overall, this project will enable us to strengthen the ABP after looking at the within-subject variations to propose a robust evaluation of known underlying confounding factors.

PUBLICATIONS

(1) Astolfi T, Crettaz von Roten F, Kayser B, Saugy M, Faiss R. The Influence of Training Load on Hematological Athlete Biological Passport Variables in Elite Cyclists. Front Sports Act Living. 2021 Mar 18;3:618285. doi:10.3389/fspor.2021.618285. PMID: 33817634

(2) Basile Moreillon, Tristan Equey, Tiffany Astolfi, Olivier Salamin 1 3, Raphael Faiss. Removal of the influence of plasma volume fluctuations for the athlete biological passport and stability of haematological variables in active women taking oral contraception. Drug Test Anal 2022 Jun;14(6):1004-1016.doi: 10.1002/dta.3218.

(3) Astolfi, T., Crettaz Von Roten, F., Kayser, B., Saugy, M., & Faiss, R. Hematological variables in recreational breath-hold divers: a longitudinal study. J Sports Med Phys Fitness. 2021 Sep 9. doi: 10.23736/S00224707.21.12918-4.

Code: 19C02MT

The anti-estrogen clomiphene is prohibited at all times in sport and since 2011 a continuous trend in increasing numbers of adverse analytical findings is noted. Recent studies have outlines a particularly long detection window for clomiphene in human urine; further, few studies have demonstrated a significantly enhanced egg production if layig hens are treated with clomiphene. Hence, concerns have ben raised whether trace amounts of clomiphene are present in eggs or poultry due to a potential use of clomiphene in the farming industry, and whether such trace amounts could lead to adverse analytical findings in doping controls. In order to protect the athletic community, a controlled administration study is planeed, where clomiphene is administered to laying hens, and both the produced aggs as well as the edible tissue will be tested for residues of clomiphene. Fruther, eggs and edible tissue will be consumed by study volunteers, and urine sample will be subjected to routine doping control analytical assays to prove for th presence of the prohibited substance. The information gained from this study is vital to fair result managment and decision-making processes in case of clomiphene findings in sports drug testing programs. If the results prove the possibility of clomiphene contamination in dietary products such as eggs of poultry, athletes and anti-doping organizations must be warned and informed.

Main Findings: 

Athletes are permanently at risk of inadvertent ingestion of prohibited substances, as shown so prominently across countries by the clenbuterol scandal. In consideration of the growing number of AAFs related to clomiphene, it cannot be excluded that food contamination is one reason of this phenomenon. In this study, the possible contamination of eggs and chicken meat with clomiphene was investigated since it had been shown that clomiphene administration can lead to an increased egg production rate in hens.

When a drug is administed to a chicken, it can accumulate in certain parts of the body or even in the eggs. This distribution depends on physical and chemical properties such as the diffusivity of trhe substances, the molecular mass, lipophilicity and on other properties such as the ability to bind to plasma proteins. All this has an influence on if clomiphene accumulates in the meat and eggs of hens fed with the drug and if yes, how fast, how long and how much of it can be found.

The results of this study reinforce the concern that clomiphene given orally to laying hens migrates to and is stored in eggs. The determined clomiphene levels result in absolute amounts of 10 to 20 µg per egg. These amounts are more than 1000-fold below the daily therapeutic dose, so no effect would be expected with the consumption of a contaminated egg. However, many athletes have a high-protein diet and may consume clomiphene-contaminated eggs in larger quantities. This could subsequently lead to clomiphene residues in urine, leading to adverse analytical findings. This was simulated in the second part of the study where healthy male volunteers consumed the eggs produced in the animal administration study. A single consumption of two clomiphene-containing eggs led to maximum hydroxy-clomiphene concentrations between 82 and 266 pg/mL in the tested urine. Discrimination of the isomers already took place in eggs. Therefore, it was not surprising that almost exclusively (Z)-clomiphene and its metabolites were detected in urine. The (Z)-form of clomiphene has a longer half-life and is mostly found as the only isomer in urine long after clomiphene therapy. Hence, the findings indicated that it is possible to generate an adverse analytical finding by consuming clomiphene-containing eggs. Consequently, a more detailed study of the metabolites was conducted.

The intake of the substance differed between the both studies: While the microdose study volunteers took a preparation containing 42 % (Z)- and 58 % (E)-clomiphene, the egg consumers took almost only (Z)-clomiphene. Additionally, the egg consumers took 10-20 % of the clomiphene amount as hydroxy clomiphene. As (Z) 4 HC was already present in eggs, it was suggested that this had an influence on the formation of the metabolites. By analyzing the phase-II metabolites, it was found that the preferably formed hydroxy metabolite must differ between egg consumers and the comparator group, since the glucuronide peaks differed in retention time and product ion mass spectra. This was confirmed when analyzing the samples with the method for differentiation of the hydroxy metabolites of clomiphene by derivatization with dansyl chloride to achieve chromatographic separation. After consumption of clomiphene-containing eggs, over 90 % of the hydroxy metabolites was (Z)-4-HC, which was found neither in samples from the microdose study nor in the re-analyzed samples with clomiphene AAFs. 

In conclusion, a method was developed to assist in distinguishing between the consumption of eggs containing clomiphene and ingestion of the drug.

Code: ISF19C05MT

Due to the erythropoiesis-stimulating effects, the misuse of cobalt and cobalt salts in sports is prohibited both in- and out-of-competition. While total urinary cobalt levels can be determined by means of inductively coupled plasma-mass spectrometry (ICP-MS), there are currently no assays for the detection of inorganic cobalt which exclude cobalt-containing molecules such as Vitamin-B12. But especially in cases of atypical findings with elevated cobalt concentrations, the analysis of Vitamin-B12-depleted urine is required to provide accurate information on the ionic cobalt content of the sample. Therefore, a quantitative test method for inorganic urinary cobalt will be developed within this study by using different depletion approaches such as solid phase extraction (SPE) or liquid chromatography (LC) in combination with ICP-MS. In particular during prolonged exposure to high concentrations, cobalt was found to be irreversibly incorporated into red blood cells. As the determination of the cobalt content in erythrocytes could be highly relevant to uncover long-term cobalt exposure in a doping control context, an assay for the quantitative determination of cobalt from a defined amount of erythrocytes will be additionally set up. Both assays will eventually be used to analyze blood and urine samples collected within two administration studies with cobalt chloride and Vitamin-B12 (dose: 1 mg/day over a period of 14 days). The Vitamin B12 administration study will provide important insights into the influence of Vitamin-B12 supplementation – which is legitimately used by many athletes – on urinary cobalt levels.

Main Findings

The manipulation of blood and blood components, commonly referred to as “blood doping” is one of the continuing challenges in the field of sports drug testing. For that purpose, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. As a cheap an easy available alternative to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport. To illegally improve the athlete's aerobic capacity and endurance performance, the administration of ionic cobalt (Co2+, e.g. CoCl2) can be used to stimulate the endogenous erythropoietin (EPO) biosynthesis. By contrast, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, the need of analytical differentiation of urinary Co-concentrations is desirable. To this end, an excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days where urine, plasma, and concentrated red blood cells were analyzed. The samples were collected starting 7 days before the administration until 7 days after. For total cobalt analyses, inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake, was utilized. Moreover, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. Especially for illegal Co2+ supplementation in sports this approach can be complemented to a prospective detection method.

Finally, for adequate method characterization and quantitative analyses, one or more internal standards need to be implemented and the chromatographic separation of additional cobalt-containing organic species as well as the stability of different variants of cyanocobalamin, especially with regard to photolytic degradation and possible conversions, need to be clarified. Nevertheless, despite the fast and preparative chromatographic run, inorganic cobalt is clearly separated and Co2+ concentrations attributed to unbound cobalt and exceeding future threshold levels will be regarded as antidoping rule violations. With regard to routine doping controls the presented approach offers an initial testing tool in order to identify those doping control samples that justify subsequent accurate cobalt quantification.

Code: 19B05CR

Class S4 of WADA’s Prohibited List 2019 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents preventing activin receptor IIB activation”). Like follistatin, myostatin-propeptide suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. In serum, >70% of myostatin is bound to myostatin-propeptide and thus myostatin-propeptide regulates skeletal muscle mass, i.e. if myostatin-propeptide is administered, more myostatin will be inhibited and then more muscle mass will be developed. Myostatin-propeptide is a glycoprotein containing one N-glycosylation site and 243 amino acids. Typical concentrations in serum and plasma are in the range of ng/mL.

So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes”. They are labelled either “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”. All of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous myostatin-propeptide. This fact will be exploited in order to detect doping with myostatin-propeptide. After immunoaffinity purification (serum, urine), myostatin-propeptide will be separated by electrophoresis (SDS- or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” products will not only differ in molecular mass but also isoelectric point (pI) from endogenous myostatin-propeptide.

Main Findings

Myostatin propeptide is prohibited according to chapter S4 of the “WADA 2022 List of Prohibited Substances and Methods.” So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes.” A study on black market myostatin propeptide products was performed and electrophoretic detection methods for serum and urine were develeoped. Out of the 12 tested products, only nine actually contained the protein. Separation by SDS-PAGE revealed that the nine products were relatively impure and that the main compound had a much higher mass (approximately 54–55 kDa) than expected (approximately 33 kDa). Further analyses by mass spectrometry showed that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three antimyostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 μL serum, the detection limit of the method is approximately 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with a limit of detection (LOD) of approximately 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible. Publication: Reichel C, Gmeiner G, Thevis M. Electrophoretic detection of black market myostatin propeptide. Drug Test Anal. 2022;14(11-12):1812-1824.

Code: 19A14MT 

Several new performance enhancing peptides have necessitated particular attention of doping control laboratories. These peptides own significant energy modulating properties by acting as insulin receptor modulators (S507, S519). Athletes will benefit from these modulations and availability is given via internet-based sources for the non-approved candidates or via the pharmacy for approved compounds. For this study, peptides will be purchased, in-vitro metabolized and characterized by mass spectrometric methods. Afterwards, simple and fast detection methods will be developed by means of solid phase extraction and mass spectrometry. Ideally, it is aimed that these simplified methods will be combined with the already established assays for large peptides (insulin, GRFs etc.) to obtain one single multiplexed initial testing procedure for a large number of different prohibited peptides.

Main Findings: 

Due to their insulin mimetic properties, the two bioactive peptide-based drugs S519 and S597 represent prohibited compounds in sports. They act as selective insulin receptor modulators and can potentially trigger performance enhancing effects comparable to insulin or its analogs. So far, no analytical method exists to uncover the misuse of these peptides in sports. Within this study, a detection assay was developed to determine S519 and S597 in human plasma by means of liquid chromatography – mass spectrometry (LC-MS) after solid-phase extraction (SPE). The peptides together with their stable isotope labelled internal standards were custom synthesized and characterized by mass spectrometry. Moreover, the method was comprehensively characterized and found to show excellent specificity and sufficient limits of detection (< 0.5 ng/mL). In addition, different in-vitro experiments were conducted with both peptides and 15 different metabolic products were identified by means of high-resolution mass spectrometry (HRMS).

Code: 19D08OP

Screening for EAAS misuse remains one of the main challenges in doping control. Currently, relevant EAAS are quantified by enzymatic hydrolysis, TMS-derivatization and GC-MS determination. However, several markers might be either lost or underestimated by this approach.

In a previous WADA funded project (15A29OP, BICEPS), we obtained promising results with the detection of two steroid bis-sulfates, which substantially improved the retrospectivity of the T/E marker for oral testosterone misuse. Based on their MS behaviour, we hypothesize that these markers are two isomeric forms of the compund 3,16-dihydroxy-5-androstane-17-one bis-sulfate (16PHAnd_EtioSS1 and 16OHAnd_EtioSS2). Synthess of reference materials is required to confirm these results. In BICEPS, we also evaluated the occurence of steroid glucoronide-sulfates in human urine. We found that one of them (5a-androstane-3β,17β-diol 3-sulfate 17-glucoronide) clearly increased after oral administration of testosterone supporting its potential usefulness for doping control.

This follow-up project (BICEPS II) aims to continue with the evaluation of the potential of steroid bis-conjugates for the detection of EAAS misuse. The project will be divided in three parts: Part I will be focused on the elucidation of the exact structure of 16OHAnd_EtioSS1 and 16OHAnd_EtioSS2. Reference materials for a range of isomers will be sythesized and the confirmation of the marker identity will be performed by comparison with excretion urines already available at IMIM. Part II will be focused on the evaluation of the potential of mixed steroid glucuronide-sulfate conjugates for the detection of testosterone misuse by developing an untareted screening method. Part III will evaluate the actual potential of these conjugates for the detection of testosterone misuse. For that purpose, a quantative analytical methodology will be validated and applied to samples from excretion studies already available at IMIM.

Code: ISF19D02JP 

Ethanol affects the steroid profile in a way that may mask testosterone administration. Our group has shown that urinary ratios 6OHAndrosterone3G/Epitestosterone17G and 6OHEtiocholanolone3G/Epitestosterone17G increase after testosterone administration while preliminary results show they decrease after ethanol consumption. This behavious suggests that those two glucoronides may be useful to distinguish between changes in T/E due to ethanol consumption and those due to the combined administration of testosterone and ethanol.

A project to investogate those markers was approved in 2018 (ISF18D13OP). The clinical trial includes the administration of placebo, testosterone, alcohol and the combination of testosterone plus alcohol. Samples of urine, blood and saliva are collected. However, a budget reduction in the approved grant prevented investigating the new biomarkers not just in saliva, but even in blood.

The steroid profile in blood is very relevant. Previous attempts to develop a blood steroid profile lost the focus including a mixture of a few androgens, plus estrogens and corticoids. However, the key analytes in blood will also be steroid conjugates. The ratio of free to conjugated testosterone is known to change greatly after oral testosterone administration. Our primary results show how the new glucurono-conjugated biomarkers 6OH-A3G and 6OH-Etio-3G and others can be monitored in blood. The administration of ethanol affects phase II metabolism and therefore this specific blood steroid profile needs to be studied.

This project aims at studying the blood samples collected in project ISF18D13OP to study the behaviour of the new biomarkers 6OH-A-3G and 6OH-Etio-3G in blood as part of a more selective steroid profile, and the usefulness of the combination of phase I plus phase II metabolites in blood to differentiate between the consumption of alcohol alone and its consumption during testosterone administration.

Main Findings

The steroid profile is subject to several confounding factors. The use of alcohol is probably the most prevalent one. This projects aimed at finding new markers to tell the difference between the administration of testosterone and the administration of alcohol or the combination of both substances.

In the present study, urine and whlole blood samples (as for ABP hematological module) were collected from four volunteers after the administration of placebo, alcohol, testosterone transdermal or the combination alcohol plus testosterone.

The analysis of the plasma from blood samples collected as per ABP hematological module purposes, showed that from the many markers studied, androsterone glucuronide was the one showing a clearly different behaviour when administering alcohol (decrease) or testosterone (increase). This has been the key factor in the development of a new marker calculated as the product of the concentrations of testosterone glucuronide and androsterone glucuronide (T-G x AN-G) in plasma samples. This new marker has shown to be:

· sensitive to the administration of testosterone (transdermal)

· insensitive to the administration of alcohol

· still sensitive to testosterone when administered jointly with alcohol

Other markers, i.e. the recently proposed ratio testosterone over androstenedione (T/A4) have shown unable to discriminate between both substances.

The new marker (T-G x AN-G) has a great potential for the detection of testosterone use even when co-administered with alcohol. Furthermore, it can be readily implemented by accredited laboratories as these analytes are commercially available and laboratories have experience in the analysis of those substances. In order to confirm and validate this new marker, further studies wil be necessary increasing the cohort of subjects and to test its behaviour for other dosages and routes administration of testosterone, the impact of genetic/ethnic differences and other potential confounding factors.

Considering the latest publication of the WADA guideline Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport (July 2023), analysis of these new markers in serum instead of plasma would be advisable for consistency with the approach followed by WADA.

Code: 19C09CR 

Class S4 of WADA's Prohibited List 2019 ("Hormone and Metabolic Modulators") lists follistatin under sub-class 4 ("Agents preventing activin receptor IIB activation, Myostatin inhibitors") as prohibited substances.

So far, no approved follistatin pharmaceutricals are available. On the other hand, there are two groups of follistatins sold on the black market (FST-344 and FST-315). But the administratio of black market follistatins to human test persons will be ethically not justifiable. For that reason, we plan a study with rats. The test animals will receive black maket FST-344 (group 1) and FST-315 (group 2) at a dosage, which can be clearly detected after 48 hours in serum (1 mg/rat, weight-adjusted). Subsequently, serum and urine will be collected and tested for follistatin with eletrophoresis and Western blotting.

The study will help to clarify (1) if both FSTs (FST-344, FST-315) are still observable after 48 hours of circulation in blood, and (2) if these FSTs can also be detected in urine. We have already shown that black market FSTs can be clearly differentiated from endogenous FSTs by electrophoresis (SDS-PAGE) and Western blotting.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

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