Blood Biomarkers of Elite Athletes

All subjects had a single blood sample taken in the morning. Blood sampling was carried out in one day in August 2021 between 8:00 and 10:00 a.m. from the cubital vein by an experienced technician. All athletes were instructed to rest on the day prior to blood sampling and refrain from alcohol consumption. Analysis of total 25-hydroxyvitamin D (25-OHVITD) in the blood were performed by liquid chromatography-mass spectrometry (LC–MS) on an Agilent 1200 liquid chromatography (Agilent, USA) combined with an AB Sciex 3200 MD mass detector (Sciex, USA). 25 (OH) D concentration was measured using total 25-hydroxyvitamin D levels (25-OHVITD), which is currently considered the most appropriate to reflect vitamin D stores in the body [39 (link), 40 (link)]. Vitamin D bio-chemical marker 25 (OH) D values above 30 ng/ml were considered normal, 20-30 ng/ml were considered insufficient, and below 20 ng/ml were considered deficient [14 (link)]. Analysis of total testosterone in the blood were performed by liquid chromatography-mass spectrometry (LC–MS) on an Agilent 1200 liquid chromatography (Agilent, USA) combined with an AB Sciex 3200 MD mass detector (Sciex, USA). The separation of substances was conducted in a gradient mode, where an acetate buffer was used during the aqueous phase, and methanol was utilized during the organic phase. A reverse-phase column (Phenomenex, USA) was used during the stationary phase. All reagents used were labeled no lower than HPLC-grad, considering the standards for substances produced by TRC (Canada). These procedures have been previously validated and are based on published guidelines for obtaining the most valid, reliable and accurate testosterone values [41 , 42 ].