Projets de recherche

Code: 252A09MT

Many substances (also some of those belonging to the WADA Prohibited List) tend to enrich (or deplete) in red blood cells and, thus, will show significantly deviating concentrations in whole blood (such as DBS) and plasma (or serum) samples. For an accurate evaluation and interpretation of analytical results obtained from DBS analyses, the information about the RBC-to-plasma-ratio for each substance is crucial, not only in the context of quantitative result management. The RBC-to-plasma-ratio can be characterized by a simple and straight forward in-vitro approach with incubating the substances of interest in whole blood, followed by the separate analysis of the cells and the plasma fractions. With those experiments, the partitioning of nearly all prohibited substances between erythrocytes and blood plasma can be determined. These data will be of substantial importance for future result management processes where concentrations of MRL- and threshold-substances in DBS are to be evaluated.

Code: 25T03MP

Ecdysterone was demonstrated to exhibit anabolic properties in-vitro and in-vivo (doi10.3390/nu16091382). Thus, ecdysterone supplements are gaining more and more attention in the sports community. In humans, a study funded by WADA (WADA15C18MP, doi10.1007/s00204-019-02490-x) already demonstrated performance enhancement in young man.

To further substantiate the effects and to support an inclusion of ecdysterone in the Prohibited List, the ListEAG suggested to perform additional experiments. A double-blind placebo-controlled intervention trial in humans will be conducted with ecdysterone supplementation in combination with a structured supervised resistance training over 12 weeks in resistance-trained males. Based on previous findings, the study will focus on resistance training and anabolic effects of ecdysterone. A standardized test battery for muscle strength will be used to evaluate potential performance enhancement based on the recommendations of NSCA recommendations with additional on individual muscle thickness etc.

Recovery measures and mechanistic investigations are also added to complement the study. Nutrition will be monitored and all groups are explicitly checked for sufficient protein intake.

Code: 25T242D01CW

The present project will conduct an integrated study on Endogenous Anabolic Androgenic Steroids (EAAS) metabolism in the Chinese population. The population distribution of UGT2B17 gene polymorphism, the long-term comparison of EAAS metabolism among individuals with different UGT2B17 gene deletion polymorphisms, and testosterone intervention experiment are the main focuses of this project. Through population studies, long-term research, and interventional trials, the metabolic profile characteristics of testosterone in the Chinese population will be illustrated. The metabolic characteristics of populations having different UGT2B17 genotypes will be highlighted.

Especially focusing on the long-term study data, which can reflect the environmental influence on urine EAAS and provide a data reference for the optimization of biological passports in the future. By correlating blood steroid profiles, urinary steroid profiles, and stable carbon isotope ratio fingerprint features, basic data and case analyses will be provided for EAAS substance detection in the UGT2B17 del/del population. A comprehensive detection strategy for EAAS, including the combined application of BSP, SP and GC/C/IRMS will be discussed in the project. The influencing factors of diet, region, and other factors on EAAS metabolism in the Chinese population will also be studied. ‌Although DBS analysis is not included in this project, the samples will be collected for subsequent investigation.

Code: 25T04MT

A major concern both for athletes and result management authorities is the possibility of nutritional supplement/dietary product contamination with minute amounts of anabolic agents leading to adverse-analytical findings (AAFs) in doping controls. The analytical sensitivity of anti-doping laboratories has been optimized to allow for utmost retrospectivity in sports drug testing, but the problem arising from such a performance is that also trace amounts introduced into an athlete’s organism by contaminated supplements or food are detected.

To support the process of fair and comprehensive case management, in-depth investigations into the elimination profiles and metabolite ratios of micro-dosed LGD-4033 were conducted in 2020/21. For that purpose, single- and multi-micro-dose excretion studies at 1, 10 and 50 μg LGD-4033 were conducted, and the collected samples analyzed by enzymatic hydrolysis, solid-phase extraction (SPE) and LC-HRMS/MS. A total of 15 phase I metabolites was detected and the ratios of LGD-4033 and the epimer M1 as well as the metabolites M5-a and M5-b were found to shift over time. The combined evaluation of the LGD-4033/metabolite concentrations and ratios can be utilized to estimate whether a minute amount of the drug was recently ingested or a larger, pharmacologically relevant amount of the doping agent was administered several days/weeks ago and thus evaluate the plausibility of potential contamination scenarios.

Within this follow-up research project, the available micro-dose administration study samples will be re-analyzed to quantify the main long-term metabolite of LGD-4033 (M5-a) by using meanwhile available certified reference material.

Code: 25T02AM

Despite suspected high prevalence of Growth Hormone (GH) doping, detection of GH doping is complex. The indirect method based on two biomarkers increased by GH (growth factor IGF-I and procollagen propeptide P-III-NP) has recently been integrated in the endocrine module from the Athlete biological passport (ABP) to better identify doping with GH using a P-III-NP assay for Centaur Analyzer and IGF-I by top-down mass spectrometry.

However Centaur analyzer and its P-III-NP assay are to be ended soon (2027) and need to be replaced. Siemens proposes a new Atellica analyzer with a test for P-III-NP that is described as highly similar to Centaur results in the normal to pathological range, but this need further confirmation in the athletes population range The aims of the project are to evaluate and confirm this new technical option for P-III-NP quantification in the coming years.

1- Comparison of P-III-NP assays performed on two Siemens analyzers and impact of storage : 200 serum samples previously analyzed for endocrine passport will be reanalyzed for P-III-NP in parallel on Advia Centaur and on the new Siemens analyzer Atellica in order to evaluate the similarity of results in an athletes population.

This will also allow a direct comparison to initial values to confirm storage conditions (-80°C) are appropriate for reanalysis;

2- Impact of various degrees of hemolysis on P-III-NP and IGF-I by adding free hemoglobin to clear sample and checking the quantification of both markers.

These data will help to confirm that the new technical options for P-III-NP detection are fit-for-purpose and will increase knowledge on how variations in quality of serum impact endocrine passport.

Code: 25T01RV

Glucocorticoids (GC) are forbidden in sports competitions when used by injectable, oral or rectal routes. They are allowed by other routes for therapeutic purposes. Since most GCs are marketed in different administration forms, the distinction between routes of administration is needed to ensure safe treatments by allowed routes of administration and to detect the use for doping purposes during competitions. Based on scientific data available, compound-specific reporting levels were established for some GC and washout periods after some administration routes.

There is no data in literature regarding concentrations of GCs in urine after intravenous administration. The objective of the present project will be to perform administration studies with dexamethasone (DEX) and methylprednisolone (MP) using intra-venous (IV) administration, to evaluate the suitability of the reporting levels already defined for DEX and MP to detect IV administration and to define the washout periods after IV administration to avoid fase adverse analytical findings due to treatments close to competitions.

Code: 242D03OS

Detecting doping with endogenous substances is challenging due to difficulties in distinguishing between natural production and external sources. Testosterone (T), particularly when administered transdermally, exemplifies this challenge. Recent advances in blood steroid profiling, specifically measuring T and its ratio to androstenedione (A4), have improved detection, particularly in females and individuals with UGT2B17 enzyme deletion. However, current methods and interpretation still require complementary biomarkers to enhance sensitivity and selectivity.

Sulfated steroids, key players in human steroid metabolism, offer potential in this regard. Unlike free steroids, these hydrophilic, conjugated forms are stored in higher concentrations in bodily fluids, providing a reactive reservoir that can be converted back into active steroids to maintain homeostasis. Sulfo-conjugation, a process

mediated by sulfotransferase enzymes (SULT), is a significant metabolic pathway for testosterone and its precursors & metabolites. This makes sulfated steroids, which have a prolonged elimination time compared to free testosterone, ideal long-term biomarkers for targeting exogenous testosterone administration.

In this study, we aim to evaluate the sensitivity of endogenous sulfated steroids in serum and urine for detecting transdermal testosterone use in healthy women. We will analyze samples from two prior clinical trials involving topical testosterone administration. The first trial involved 14 women undergoing 28 days of T gel administration, with serum and urine samples collected every two days for 12 weeks. The second study included 48 women randomized to 10 weeks of daily testosterone cream or placebo treatment with urine and serum samples collected before and after treatment. Urine and serum samples will be analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify steroid sulfates and their conjugated metabolites. Profiles of top candidates will be developed to improve testosterone detection methods.

Code: 242C12EI

The popularity of using plant steroids to improve physical health and performance is on the rise. One such plant steroid is diosgenin (DIO), primarily found in fenugreek (DOI: 10.9755/ejfa.2016-01-086). In vitro studies on adipocytes have demonstrated that glucose and fat metabolism are beneficially influenced by DIO (DOI: 10.1002/mnfr.200900609). These findings are further supported by animal studies (DOI: 10.1155/2019/7213913; DOI: 10.1016/j.jsbmb.2014.02.020). A recent review also supports these observations, indicating that chronic application of fenugreek can significantly reduce fat mass (DOI: 10.1055/a-2048-5925). Additionally, improvement of endurance performance was observed in mice (DOI:10.3177/jnsv.52.287). However, there is a significant lack of data available on the effects of DIO on endurance performance in humans. In a two-arm randomized controlled double-blind crossover trial the effects of 500mg DIO will be tested against a placebo group. A total of 15 endurance-experienced participants will consume a dietary supplement rich in DIO or a placebo product over 14 days. The initial application phase will be followed by a 2-week wash-out period, and then a second application phase. Before and after both application phases, participants' performance will assessed through isometric mid-thigh pull, cycling-ergometer step and ramp test. Thereby, maximal fat oxidation, lactate threshold and maximal oxygen uptake will be analyzed via spiroergometric and lactate data to determine corresponding metabolic profiling & energy metabolism. Training sessions will be monitored throughout the study period to ensure participants maintain their usual training regime. In addition, the metabolism of glucose in C2C12 cells after treatment with DIO will be investigated in our established cell culture system.

Code: 241C01PV

Zeranol (α-ZAL), a prohibited anabolic agent by the World Anti-Doping Agency (WADA), can be present in human urine as a metabolite of zearalenone (ZEN), a mycotoxin occurring in several food commodities. Distinguishing between doping intake and ZEN-contaminated food consumption is challenging. To establish evidence-based criteria, a human intervention trial and toxicokinetic (TK) modeling are fundamental. Up to 74.6% of ZEN is excreted in human urine as reductive metabolites and phase II conjugates. Limited evidence suggests that α-ZAL is metabolized primarily, but not exclusively, into β-ZAL and zearalenone (ZAN) in mammals. Monitoring α-ZAL concentrations relative to other ZEN metabolites helps differentiate illicit intake from food contamination, but WADA LT04 lacks transparent criteria for confident α-ZAL doping identification. This project proposes two independent human intervention trials with 10 healthy adults each to study αZAL and ZEN toxicokinetics. After a wash-out period of two days, the volunteers will ingest a bolus containing α-ZAL or ZEN at the TDI (250 ng/kg bw). From the exposure onwards, the volunteers are requested to collect individual urine and capillary blood samples for 48 hours. ZEN, ZAN, α/β-zearalenol (α/β-ZEL), and α/β-ZAL will be quantified, after extraction and hydrolysis of phase II-conjugates, using GC-MS/MS and GC-HRMS methods validated at ultra-trace levels. We aim to determine the human metabolic excretion profiles over time and use it to build two TK models with Hierarchical Bayesian population structure. The resulting population concentration/time profiles will enable calculating confidence intervals for ZEN/α-ZAL, α-ZEL/α-ZAL, and β-ZEL/β-ZAL ratios, refining WADA report guidelines for Adverse Analytical Finding in urine and capillary blood collected via VAMS Mitra tips. The models will provide population TK parameters of α-ZAL and ZEN and the possibility to estimate intake doses, offering additional proof in legal adjudication.

Code: 241C05MP

Recently, the success of the integration of new long-term markers for anabolic steroids such as metandienone greatly improved the detectability of their misuse in sports. Tetrahydromethyltestosterone-sulfate (THMTS) was proposed as a promising long-term marker for methyltestosterone administration, however, reference material for direct analysis of the intact conjugates is not available for all diastereomers. Reportedly, the longest detectable diastereomer was tentatively assigned as 3α5α17epiTHMTS after solvolysis using GCMS and literature reported Kovac indices for diastereomer allocation. Our preliminary data from intact sulfoconjugate analysis suggest 3α5βTHMTS instead. It remains open until now weather this difference is due to potential epimerization by solvolysis, sample pre-treatment or interindividual variability. Additionally, A-Ring reduced 17β-hydroxymethyl-13-ene (20OHNorTHMT) metabolites have not yet been consequently investigated after methyltestosterone administration. Analogous metabolites resulted in drastic extension of the detection window after Oral Turinabol administration.

Within the project the analysis of tetrahydromethyltestosterone (THMT) diastereomers as classically monitored metabolites of methyltestosterone, A-Ring reduced 17β-hydroxymethyl-13-ene metabolites (20OHNorTHMT), as well as intact sulfoconjugates, will be included in the analysis to evaluate the best suited metabolites after an administration of methyltestosterone.

Therefore, methods will be optimized for sensitive and selective detection using the various diastereomeric sets of target analytes. Furthermore, the suitability of different possibilities for sample preparation will be evaluated specially focusing on the sulfoconjugates. Their diastereomeric structure will unambiguously be confirmed by direct analysis of the intact conjugates and comparison with references of all diastereomers. Full characterization of the relevant THMTS diastereomers with LC-HRMS and NMR will be provided after chemical synthesis and purification. Including postadministration samples from six volunteers will help for investigation of interindividual variabilities of metabolite excretions.