Use of mass spectrometry omic strategies to find markers of hydrocortisone misuse

Code: 18E08CB

Glucocorticoids are prohibited in sport in competition and only when administered by oral, intravenous, intramuscular or rectal routes. The World Anti Doping Agency (WADA) proposed a criteria of 30 ng/mL above which a result should be reported for this class of substances. However such criteria cannot be applied to cortisone and hydrocortisone which are naturally present in urine samples. Regarding these two substances confirmatory method by GC-C-IRMS is applicable for the determination of endogenous or exogenous origin. However, no efficient screening criterion is available and applicable in Antidoping laboratories to trigger GC-C-IRMS analysis. Our project aims to use omics strategies to highlight biomarkers of effect and pattern changes in urine and blood samples able to give evidence of hydrocortisone administration. For this project, a clinical study will be conducted with two routes of administration: one prohibited such as oral route in order to find biomarkers of effects of hydrocortisone abuse and one authorized route such as topic application with a cream in order to confront the potential biomarkers found with prohibited administration. The impact of topical application on carbon isotopic ratio of cortisol and its metabolites will also be explored in a second step.

Main findings

In the present project, two administration studies with hydrocortisone were performed, one with a prohibited route (oral) and one with an authorized route (topic). The effects of such treatments on urinary concentrations and 13C-delta values of a set of endogenous corticosteroids were studied. The clinical trial was conducted with 20 healthy recreational athletes. For the prohibited route, 50 mg hydrocortisone was taken orally by 10 subjects every day for 5 days. For the authorized route study, 10 other subjects used a cream with 1% hydrocortisone at the therapeutic dose of 25 mg twice a day also for 5 days. The overall goal of this study was twofold, to find biomarkers of the hydrocortisone treatment but also to investigate if the two routes tested could be differentiated.

In the first part of this project, blood and saliva samples collected in this study were analyzed using ELISA kits. It was found that therapeutic oral administration of hydrocortisone once daily for 5 days modified adrenal DHEA secretion by inhibiting pituitary ACTH. However, this effect was only transient, with no significant alteration in basal adrenal or pituitary function 24 hours after administration. Given the high correlations between plasma and saliva, noninvasive saliva sampling may therefore be a sensitive alternative to invasive venous blood sampling for estimating pituitary and adrenal function after hydrocortisone treatment.

Urine samples were analyzed by LC-MS/MS to determine the concentration and by GC-C-IRMS to determine carbon isotopic ratios focusing on hydrocortisone and its metabolites: cortisone, tetrahydrocortisol (THF), allo-THF, tetrahydro-11-desoxycortisol (THS), 11-keto-Etiocholanolone, 11β- OH-Androsterone, 11β-OH-Etiocholanolone, β-cortol and 6β-hydrocortisol. As a main result, the topical application of 1% hydrocortisone cream on safe skin (25 mg twice daily for 5 days) had no impact on cortisone, hydrocortisone or metabolite concentrations. On the contrary, as expected, a significant increase in concentrations could be observed after oral treatment for all compounds studied except 11bOH-etiocholanolone. This compound being a minor metabolite of cortisone, a significant impact on this substance was expected. On the basis of the results obtained with oral treatment, we were also able to observe that the detection windows in the context of anti-doping controls were dependent on the metabolite considered and on the subject. A clear impact on concentrations could be observed on all the target compounds during less than 24 hours except for b-cortol which seems to be impacted for a longer period of time (return to basal values in 48 hours). The multivariate statistical analysis carried out on all the data obtained made it possible to highlight new markers of detection of unauthorized hydrocortisone intake. Sixteen markers based on specific target metabolites or ratios of these metabolites were selected to create a statistical model for screening samples from doping controls. The steroid profile was also measured in all samples collected. No clear trend could be confirmed regarding the impact of hydrocortisone intake on these steroids.

The GC-C-IRMS analysis was then performed on the urine samples from this study. These analyses concluded that 1% hydrocortisone cream does not exhibit transdermal activity when used under therapeutic conditions and apply on safe skin (25 mg twice daily for 5 days): no changes in 13C/12C isotope ratios were observed for all subjects. The results of the IRMS analysis showed that in addition to the metabolites already studied in the literature such as allo-THF or allo-THE or 11keto-Etiocholanolone. The compound 11bOH-etiocholanolone could also be a good target for this application but with a detection window observed up to 36h, the interpretation of results in the context of out-of-competition treatment could be more complex.