Projets de recherche

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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