Projets de recherche

Code: 18C18MP

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Part 1 of the project demonstrated the performance enhancement of an ecdysterone supplementation in combination with resistance training in humans. To allow for detection of an ecdysterone administration in doping control, the metabolism of ecdysterone and its urinary excretion will be investigated. For method development additional in vitro experiments will be conducted to help targeting the right potential metabolites. Using the prospected metabolites a targeted method will be used to analyse post-administration urines. This approach will be complemented by non-targeted analyses using high-resolution mass spectrometry. Finally the project aims in providing a method useful for screening of ecdysterone and its metabolites in compliance with existing screening procedures in anti-doping laboratories.

Using these results the prevalence of ecdysterone in samples from human sports anti-doping control from different regions will be monitored.

Main Findings: 

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performanceenhancement in sports was demonstrated recently, and, in 2020, ecdysterone was consequently included in the Monitoring Program of the World Anti-Doping Agency to detect potential patterns of misuse in sport. Dietary supplements containing ecdysterone were analyzed for their quality. Assay revealed to be poor for the majority of the products. Only a few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, a single oral dose of 50 mg of ecdysterone was administered to ten volunteers (five males, five females). After a washout period, two out of the female volunteers were tested a second time. Analysis of serum samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after solid-phase extraction. Kinetic parameters were determined based on this data. Additionally, post-administration urine samples were analyzed using dilute-and-inject LC-MS/MS. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14- deoxy-poststerone, were achieved. Ecdysterone was the most abundant analyte present in postadministration urine samples, detected for more than two days, with a maximum concentration (Cmax) in the 2.8–8.5 h urine (Cmax = 4.4–30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14- deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5–39.5 h (Cmax = 0.1–6.0 µg/mL) and 23.3–41.3 h (Cmax = 0.1–1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14- deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation. Due to the potential generation of metabolites by gut bacteria that may cause significant variations in the metabolic profile, an integration of further isomers and analogues may also be appropriate. Analytical properties of ecdysterone and its metabolites were evaluated to assess the possibility of integrating them into existing procedures currently used for screening in anti-doping laboratories. Ecdysterone and its metabolites may be easily integrated into current initial testing procedures (ITP) for monitoring the prevalence in elite sports. LC-MS/MS showed excellent eligibility for these analytes. Alternatively, GC-MS analysis is possible after TMS derivatization. If extraction is required or desired, SPE was found to be by far superior to LLE. Enzymatic hydrolysis did not provide advantages over the analysis of the unconjugated fraction only. Out of 1100 doping control samples 2% revealed positive findings for ecdysterone. The samples were collected from athletes performing a broad variety of sports. Up to now, no classification of “high prevalence” type of sports is seen. Compared with the concentrations found after oral administration of 50 mg of ecdysterone, their concentrations were rather low.

Code: ISF18D10LL

Fluctuations in plasma volume present a major confounding factor to the analysis of an Athlete's Biological Passport (ABP) profile.  A recently developed model for plasma volume estimation via a simple blood test will be applied to samples collected during a cycling stage race. The model will be used to correct the concentration based hematological markers of the ABP, in order to account for the natural progressive plasma volume expansion induced by the multi day stage racing. The proposed research serves to validate the model in an anti doping context, which if successful presents an exciting step forward for the ABP and anti doping authorities.

Main findings: 

Fluctuations in plasma volume (PV) present potential confounders within the concentration-based markers of the hematological Athlete Biological Passport (ABP). Here, a multi-parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (15 female, 14 male) before, during, and after consecutive days of cycle racing (Tour Down Under, Adelaide, South Australia). Whole blood was analyzed in accordance with the World Anti-Doping Agency (WADA) ABP guidelines at the WADA Accredited Laboratory in Sydney (ASDTL). Serum and plasma were analyzed for hemoglobin concentration ([Hb]), platelets, transferring, albumin, calcium, creatinine, total protein, and low-density lipoprotein. Significant reductions in [Hb] and the OFF-score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. PV was calculated using the CO-rebreathing method. When individual calculations of PV were applied to the adaptive model, the upper and lower reference predictions of [Hb] and the OFF-score were refined such that all outliers consistent with racing induced PV changes were removed. The multi-parametric PV model appears capable of reducing the influence of PV on volumetric markers during competition. This is an important step towards the inclusion of the PV correction in the hematological module of the ABP. Future work should consider intra laboratory and intra analyzer variation as well as adquirig more longitudinal data on elite athletes from different disciplines.  

Code: ISF18D13OP 

The screening of the misuse of endogenous androgenic anabolic steroids (EAAS) is currently performed by the quantification of the urinary steroid profile. One of the main problems related with this approach is the great impact of alcohol consumption on the profile. The fact that ethanol affects and invalidates the steroid profile opens the door to the use of ethanol as masking agent for testosterone administration. 

Our group has shown that urinary ratios 6OH-Etio3G/EG and 6OH-Andros3G /EG increase after EAAS administration. Conversely, preliminary results show that those ratios decrease after ethanol consumption. This behavior suggests that those two glucuronides 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 (used as masking agent). Additionally, the determination of phase II metabolites in alternative matrices is a promising tool for the screening of EAAS misuse and might provide new insights in differentiating testosterone administration from (simultaneous) ethanol consumption. 

This project aims to (i) evaluate the usefulness of phase II metabolites to differentiate between the consumption of alcohol alone and its consumption during testosterone administration, (ii) explore the potential of the determination of phase II metabolites in alternative matrices for the screening of testosterone misuse and (iii) look for the differences in a comprehensive steroid profile (in urine, plasma and saliva) between samples collected after testosterone administration and after the combination of testosterone and ethanol. For this purpose, we plan to perform a pilot study with 4 male volunteers. Each volunteer will be treated with placebo, testosterone gel, ethanol and the combination of ethanol and testosterone. Urine, plasma and saliva will be analyzed to look for differences between the different conditions.

Main findings:

Considering that the most common situation is the consumption of moderate amounts of alcohol and that the transdermal application of testosterone is the most challenging to detect, we aimed at testing those new markers for those difficult conditions. Following our clinical trial scheme, 4 volunteers received (using a randomized protocol) either placebo, 100 mg/day for 3 days transdermal testosterone, 30 g/day for 3 days ethanol, or the combination of the same dosages of testosterone plus ethanol.

Code: T18M04NB 

The International Standard for Testing and Investigations requires that blood samples for the Athlete Biological Passport (ABP) are collected at least two hours after training or competition, in order to harmonize the passport data by reducing the impact of variation in volume of blood plasma that is induced by intensive exercise. Removal of this requirement would facilitate more frequent, convenient, cost-effective sample collection for athletes and testing authorities right after competition. A set of common clinical chemistry markers have recently been demonstrated to correct for fluctuations in plasma volume (PV) in experiments with healthy volunteers, indicating that they have potential to refine the ability of the ABP to detect blood manipulations by improving the sensitivity of the primary ABP markers. This project will evaluate the feasibility and validity of these markers in routine anti-doping settings by analyzing the markers in four WADA-accredited anti-doping laboratories. Successful validation of the PV correction method in this study will support the future implementation of this approach as a pertinent component of the ABP hematological module and enable the collection of ABP blood samples immediately after training or competition.

Main findings

Contrary to the prevailing perception, an interesting observation within this study population was that none of the hemoglobin (HGB) values exceeded the predicted ABP reference intervals 1 h post-exercise, providing evidence for a low probability of concentration-based hematological markers being affected by an exercise-induced PV shift 1 h post-physical activity in football. Regarding the PV markers examined, calculated confidence levels (based on the agreement in the magnitude and directionality of change in the PV markers values) across the entire study population were generally very low. Preliminary data processing revealed significant differences in the mean values of the results between the four laboratories, which used differing immunoassays for the analysis of PV markers. Therefore, a round of inter-laboratory comparison was added to the study design to further understand this inter-lab bias. Investigations through the ring-test confirmed, indeed, that the absolute concentrations of PV markers were dependent on the assay used, which might be one of the root causes of the observed heterogeneity in measurement. Laboratory-dependent pre-analytical conditions of samples have also been identified as a potential source of variability of PV markers measured in four different laboratories.

Code: 18A07XD

Prednisolone and prednisone are two glucorticosteroids included in the section S9 of the WADA Prohibited List of substances and methods. Glucocorticosteroids are prohibited in competition when administered by oral, intravenous or rectal routes. A reporting level of 30 ng/mL has been established by the WADA in the attempt to disclose permitted from illicit administration of GC.

Urine samples used for Antidoping purposes are not collected under sterile conditions. This implies that depending on the healthy conditions of the athlete, quality of collection material used and transport conditions to the laboratory, microorganisms’ growth may occur. The metabolic activity of the microorganisms has several impacts on the validity of the sample including the interpretation of the results. Antidoping laboratories have been dealing since many years with “active” urines where atypical findings have been ex-vivo in the urine sample by the microorganisms’ activity. For a correct identification of the origin of such findings into, the application of isotope ratio mass spectrometry (IRMS) has become mandatory.

More recently, the unusual presence of prednisone/prednisolone has been observed due to steroid 1-dehydrogenase activity of the microorganisms over naturally occurring compounds present in urine (cortisol or cortisone).  For prednisone/prednisolone findings between the reporting level and 60 ng/mL, in addition to verify markers of microbial activity and the presence of specific metabolites (20b-hydroxy metabolite), WADA recommends to use GC/C/IRMS analysis to confirm the exogenous origin. In order to close the debate, the aim of this study is to develop and validate a GC/C/IRMS method to confirm the origin of prednisone/prednisolone in human urine in samples collected during Antidoping sessions and showing prednisone/prednisolone concentrations between 30 and 60 ng/mL, without modifying the structure of the compounds of interest, fulfilling WADA requirements.

Code: T18M02TK

The aim of the study is to re-assess the sensitivity of the hGH biomarkers test, in its current assay format, to detect doping with hGH. This study includes the analysis of multiple samples already collected before, during and after administration of low, medium or high doses of hGH (and placebo as control).

Samples will be analyzed for P-III-NP (Siemens Centaur assay). These measurements will be combined with IGF-I determinations (to be performed in another lab, using LC-MS/MS) to calculate the GH-2000 scores and re-assess the sensitivity of the current hGH Biomarkers Test.

Code: 18C08MH

Salbutamol is a short-acting beta2-agonist used for asthma and exercise induced bronchoconstriction and is permitted in inhaled doses up to 1600 μg in 24 h, not exceeding 800 μg in 12 h. A corresponding urine threshold of 1000 ng/mL and decision limit of 1200 ng/mL has been established to discriminate permitted inhalation from prohibited misuse. If an athlete exceeds the decision limit in doping control, this is recorded as an Adverse Analytical Finding (AAF) that may result in the athlete being charged with an anti-doping violation.

While salbutamol has been available for half a century, knowledge about its pharmacokinetics in relation to athletes and anti-doping control is incomplete. A handful of clinical trials underpin that high urine concentrations of salbutamol, relative to the dose inhaled, can occur, indicating that the risk of AAFs reported for salbutamol is possibly greater than previously assumed. These observations are further substantiated by the fact that several supplements and medications may interfere with the metabolism and subsequent excretion of salbutamol, which to our knowledge, are unexplored with respect to the salbutamol decision limit. Thus, athletes are exposed to several factors that may affect the pharmacokinetics of inhaled salbutamol, including, but not limited to strenuous exercise, dehydration, diet and supplements, and other medication.

This project will provide a comprehensive investigation into the factors that affect the intra- and inter-individual pharmacokinetics of salbutamol in female and male athletes. The primary objective is to model the peak urine excretion rate and peak urine concentrations of unchanged salbutamol and its major 4'O sulfate metabolite after permitted inhalation and prohibited oral ingestion to possibly improve the predictive value and accuracy of the decision limit for salbutamol.

Main Findings

The 2021 Prohibited List allows athletes to administer salbutamol at inhaled doses up to 1600 µg in 24 hours, not exceeding 800 µg in 12 hours. A urine threshold of 1000 ng/mL with a corresponding decision limit of 1200 ng/mL has been established to discriminate permitted therapeutic use from prohibited supratherapeutic use during doping control. Despite many studies investigating the pharmacokinetics of salbutamol, limited data are available on factors that may confound its pharmacokinetics. In this project, we investigated the effect of dietary flavonoid intake, dehydration, combined flavonoid intake and dehydration, and consecutive days of salbutamol inhalation on urine pharmacokinetics in highly trained females and males who were subjected to prolonged strenuous exercise. Additionally, we evaluated the diagnostic performance of the current threshold and decision limit in discriminating permitted inhaled doses (1600 µg in 24 hours) from prohibited oral doses of 8 mg salbutamol. In this comprehensive repeated measures study, we collected and analyzed more than 750 urine samples and demonstrated that the current decision limit approach, whereby concentrated urine samples are adjusted to a specific gravity (SG) of 1.020, has robust sensitivity and specificity for application in doping control. Only 1 sample, collected from a female participant following consecutive days of inhalation, exceeded the decision limit after SG-adjustment (reaching 1303 ng/mL), and only 4 samples (from 3 different participants; also all from consecutive days of inhalation) exceeded the threshold with SG adjustment. As such, employing SG-adjusted data from 0-6 h at all trials, which is the most relevant window of detection, sensitivity at the decision limit was 40% with 99% specificity, while at the threshold, sensitivity was 47% with 98% specificity. Females generally presented with higher urine concentrations of salbutamol than males, and 3 of the 4 samples exceeding the threshold were from female participants. Consecutive days of inhalation resulted in higher urine concentrations of salbutamol, indicating that 24 hours are insufficient for complete urine 2 elimination of salbutamol for most individuals. Prior bioflavonoid intake lowered urine concentrations of salbutamol after inhalation, while dehydration, as expected, led to higher urine concentrations of salbutamol which could be accounted for with SG adjustment. In conclusion, the current threshold approach with SG adjustment displays robust sensitivity and specificity, even in the face of dehydration, bioflavonoid intake, and consecutive days of treatment. Female athletes generally present with higher urine concentrations than male athletes.

Code: 18D06XD 

Among the substances declared by the athletes during the sample collection sessions and reported in the doping control forms (DCF), it is quite frequent to observe supplements or medication based on thyroid hormones. This supplements or medications contain not only levothyroxine (T4) or triiodotironine (T3) but some of their derivatives as Triacana (3,5,3’-triiodothyroacetic acid) or Tetrac (3,5,3’,5’-tetraidothyroacetic acid). In 2017, at the WADA accredited antidoping laboratory of Rome, the athletes declaring to consume this kind of substances, was ten times higher compared to the prevalence of hypothyroidism in Italy. This high incidence of their consumption among the athletes in conjunction with their metabolic actions and the consequences of their intake over the health, impose to investigate which is the real use of these compounds and to start to investigate a new potential doping practice.

We plan to investigate the real analytical possibilities for determining the prevalence of use in sports using the available capabilities in the antidoping laboratories, ideally in already existing methods starting from urine or serum samples collected for other antidoping analyses.  This would allow, if considered convenient, to include an additional section in the athletes biological passport (ABP) endocrinological module for these hormones.

The main goal is to monitor thyroxine hormones (TSH, freeT3, freeT4 and freeT4/freeT3 ratio) in athlete’s serum and investigate the best biomarkers in urine focusing the attention in their potential inclusion in the ABP endocrinological module. Once the method developed and biomarkers chosen, the proposed approach will be applied to different thyroid conditions and the profiles under some administrations evaluated. 

Main findings

A method to detect thyroid hormones (TH) and some related metabolites in serum was validated using the LCMS/MS technique. The correlation of the validated method with RIA showed adequate results and one of the advantages of the LCMS technique with respect to RIA is that it does not show cross-interferences as is the case of T3 with triiodo-thyronacetic acid, which could happen with other metabolites. One of the disadvantages is the matrix effect, so it must be treated with particular care in the implementation process. Analysis of serum samples from athletes who reported levothyroxine consumption showed elevated levels of thyronine and tetraiodo-thyronacetic acid. At least preliminarily, these two compounds and the relationships that involve them (with T3 and T4) could be used as markers for the detection of levothyroxine consumption in serum.

Also, a method was validated for urine that allows the detection of THs and their metabolites. The method was applied to the total urine fraction (free + glucuronide conjugates + sulfate conjugates) but can be applied to the fractions separately. It was based on extraction at neutral pH to have a global idea of the excretion in urine of acidic (thyronacetic acids) and basic (thyronamines) metabolites.

Applying the validated method in LCMS to the total fraction of serum and urine, it was possible to verify that the levels of T3 and T4 of athletes are lower than those of a euthyroid population even when they are under the administration of TH supplements, coinciding with episodes of hypothyroidism described in reports focused

on high-performance sports. However, the high efficiency of the HTP axis (which proved to be, throughout the study, the most strict, refined, and exquisite) does not allow us to observe differences between athletes who do not declare and those who declare having used TH supplementation.

Direct measurements of T3 and T4 do not seem to be useful in these cases since everything seems to indicate that external supplementation aims to cover the needs of thyroid hormones to maintain homeostasis. Therefore, it could be assumed that the metabolic pathways observed in a euthyroid individual after the administration of TH are not the same as those observed in athletes. The detection of the administration of TH, at least triiodothyronine (T3) and levothyroxine (T4) in urine could work when dealing with euthyroid individuals, either by applying cut-off values of ratios (for T3) or the presence of T1 (for T4). In individuals with hypothyroidism where the tendency is towards the maintenance of homeostasis (as in the case of athletes), it was not possible to clearly detect the consumption.

Regarding to Endogenous Steroid Profile in urine, differences in the markers of the ABP steroidal module were found considering samples of athletes declaring and not declaring the supplementation with TH in the Doping Control Form. Although not all assessed parameters showed significant differences, there was observed a tendency to decrease steroid concentrations in the group of athletes who declared the consumption of levothyroxine. Female groups showed higher differences between declaring and not-declaring athletes' respect to males. The results for pregnanediol were a remarkable point, especially in the case of males. The information on TH therapy by athletes may be helpful for the correct interpretation of the ABP as happens for other markers considered confounding factors of the urinary steroid profile in the current WADA documents.

Code: ISF18E01JC 

The blood doping is a significant challenge in our fight to ensure fair athletic competition. The blood doping typically involved the transfusion of red blood cells which have been stored outside of human body. We have found that red blood cells have significant amount of RNAs which are not well characterized. Therefore, we will employ cutting edge single cell technology to identify whether subsets of RBCs and/or RNA will undergo storage-associated changes to distinguish these stored red cell from fresh red cells. We will develop single cell RNA-FISH methods identify individual stored red cells as a novel way to detect blood doping.

Main Findings: 

1. During blood storage, we have extensively validated the induction of miR-720 during storage. The miR-720 induction are consistent in all 15 tested samples and likely to comprise the stroage signature that can be used to detect ABT.

2. miR-720 is a cleave product from threonine-tRNA. During storage, there is concordant increase in miR-720 and remaining tRNA fragments based on Northern blots.

3. However, we noted that increased tRNA cleavage is not a general phenomenon. When we probed the small RNA Northern with differenet tRNA probes, we did not see the increased tRNA fragments seen for the miR-720. In addition, when we perform small RNA-Seq of fresh vs. stored RBC, we did not observe a general increase in tRNA fragments.

4. Small RNA-Seq of the fresh vs. stored RBC samples further reprodcued the induction of miR-720 by 16 folds. In addition, we identified additional transcripts which were increased by at least 10-fold during storage. We have reproduced the RNA-Seq in 4 additional paired samples and in the process of analyzing the data. Therefore, these storage-enriched transcripts can serve as robust storage signatures.

5. The stored RBC lysates contain the cleavage activities that include angiogenin, a stress-responsive nuclease. Angiogenin in the storage solution is increased during in vitro cleavage. The immune-depletion of angiogenin significantly reduced the miR0720 cleavage and addition of the recombinant angiogenin enhance the miR-720 induction during storage.

6. We have also optimized the single cell RNA-FISH methods to validate the increased miR-720 positive population during storages. This detection method can be used to detect small portion of RBC cells which have been stored and mixed with fresh RBCs.

7. It is also possible to separate fresh vs stored RBC cells based on opitcal volume changes stressed in microfluidic devices using quantitative phase imaging. Such methods can detect single stored red cells without labeling.

Code: 18C07KD 

Insulins are therapeutically used for the treatment of diabetes mellitus. There are rumors that insulins are used by athletes for anabolic properties. Consequently their use is prohibited by WADA. To control their abuse several sensitive methods for the detection of insulins in blood and urine have been described for doping control purposes. Generally, the applicability of these methods is illustrated with blood and urine samples from diabetic-patients. Indeed, blood and urine samples can be easily collected from diabetic patients without major ethical concern. Unfortunately, it can not be excluded that these samples are not representative for a healthy athlete population because of the diabetic status of the patients. Additionally, these spot-samples don’t give information on detection times. In general, no administration studies, from which the results are readily applicable to doping-control, have been performed. Therefore the aim of this project is to administer a single dose of insulins to healthy volunteers and to investigate detection times in blood and urine.

Because administration of a high dose of insulins can result in a life threatening situation only a low dose will be administered (0.05IU/kg). Three short acting insulins Lispro, Aspart and Glulisine will be investigated. Blood and urine samples will be collected from 1 week before administration, until 3 days after administration. The result of this project will be useful for doping organizations to set testing windows and for doping laboratories to evaluate their detection methods.

Main Findings: 

A simplified immunoaffinity purification LC-HRMS method was presented for the identification of the synthetic insulin analogue Lispro, Aspart and Glulisine in serum and urine samples. LODs obtained in serum for all 3 compounds was 500 pg/ml. In post administration serum samples, the insulins were detectable up to 6 hours.

More importantly, urinary detection resulted in much better results in terms of method development, validation (LODs 5 pg/ml, LOIs 10 pg/ml) and detection windows of these three rapid-acting insulins. Following the single injection (0.05 IU/kg), the administered analogues could be detected up to 12 hours and identified according to the TDICR2015 document up to about 9 hours using the presented analytical strategy.