Funded: 2019

Fellow:
Dr. Federico Ponzetto, University of Turin

Mentor:
Professor Ezio Ghigo; University of Turin

Research Title:

Implementation of a blood steroidal module in the framework of the athlete

Research Summary:

The research project entitled “Implementation of a blood steroidal module in the framework of the Athlete Biological Passport” and carried out at the Department of Medical Sciences of the University of Turin, aimed at providing a robust analytical tool for investigating the variations of Blood Steroid Profile. During the 2 years of the project a novel LC-MS/MS method for the quantification of 27endogenous steroids, including major circulating hormones as well as a wide panel of androgens’ phase II metabolites, was developed and validated according to WADA guidelines. This method was then used for calculating preliminary reference intervals of target analytes, some of which were never reported to date, analyzing serum samples collected from healthy volunteers accessing to the Blood Bank of Turin University Hospital. Furthermore, the potential impact of circadian rhythm and physical exercise on Blood Steroid Profile markers was investigated thanks to the analysis of serum/plasma samples collected during two dedicated clinical studies involving 19 healthy male volunteers and 31professional football players. The outcomes of these studies , beside proving the usefulness of the developed analytical platform for gathering additional information on steroid metabolism, useful for the introduction of blood steroid profiling in anti-doping context, highlighted that serum concentrations of testosterone(T) and dihydrotestosterone (DHT)as well as of androgens phase II metabolites are not significantly perturbated by diurnal variations linked to circadian rhythm and by a moderate physical exercise such as a football training session. These results strongly support the introduction of such markers in the future implementation of Blood Steroid Profile for doping control purposes, remarking the utility of monitoring not only T and DHT but also more concentrated phase II metabolites such as etiocholanolone glucuronide, androsterone glucuronide, epiandrosterone sulphate and androsterone sulphate. During the performed studies it was also observed that the administration of 5α-reductase inhibitors (e.g., Finasteride) could have a strong impact on target markers, stressing the need of dedicated study for better characterizing steroid response to such intake. Finally, with the aim of suggesting an easier sample collection procedure for Blood Steroid Profile, a further analytical method for the measurement of most concentrated steroidal compounds from Volumetric Absorptive Micro-Sampling (VAMS) was developed, validated and applied to assess the medium-term stability of target analytes in blood micro-matrices. The promising results obtained in this proof-of-concept study, underlining the stability of VAMS samples up to 100 days at-80°C,opened the way to the use of such technology for simplifying blood sample collection, storage and transportation in the future studies that are needed to fully characterize the perturbations of endogenous and exogenous potential confounding factors on Blood Steroid Profile. Although the advances obtained thank to the present projects, there is still lack of population studies for the definition of reference intervals in elite athletes’ populations as well as for the assessment of long-term intra-individual variation of most promising serum markers of EAAS doping. These topics could represent the challenge for future research project in the field of steroid analysis in anti-doping context.