2.5. Physiological Parameters
SEPARATION, CLOT, BLOOD
We used serum sample separation to analyze the set of physiological parameters (i.e., P1NP and BCTX/1000). After participants fasted for 12 h, a qualified nurse drew 10 mL whole blood samples from an antecubital vein of the participants in a seated position. Blood samples were extracted into dry 10 mL tubes with a silicone gel separator and coagulation activator between 8:00 and 10:00 a.m. (to minimize circadian effects). These samples were kept in a refrigerator at 2–4°C until they were processed, which always occurred within 4 h of extraction. After clot retraction (15–30 min at room temperature), samples were centrifuged with Histopaque (Sigma H-1077) at 3500 rpm for five minutes at 4°C in a Rotina 380R Hettich centrifuge (Tuttlinger, Germany). The professional in charge pipetted and aliquoted the resulting serum supernatant. The aliquots were frozen at −80°C until use. An automated Roche ECLIA system (Cobas 6000, Roche Diagnostics, Mannheim, Germany) measured serum P1NP and BCTX/1000. The person in charge ran the samples in duplicate as per the manufacturer’s instructions to ensure the reliability of the measurements. If the results differed by more than 15%, the analysis was repeated. We used the average of both readings for data analysis.