Blood Collection and Plasma Extraction

Blood was collected on an empty stomach on the same day as, but prior to, the patient's examination and saliva collection. 7.5 mL of venous blood was collected in a S-Monovette® EDTA K3 tube (Sarstedt, Nümbrecht, Germany). Under laboratory conditions, the samples were centrifuged in order to separate the plasma from erythrocytes (1500 × g; 4°C, 10 minutes). Erythrocytes were rinsed three times with 0.9% cold NaCl (v/v) and then haemolysed by adding 50 mM cold phosphate buffer (pH 7.4) 1 : 9 (v/v) [28 (link)]. To prevent sample oxidation, 0.5 M BHT (Sigma-Aldrich, Saint Louis, MO, USA; 10 μL/mL blood) was added to the blood [28 (link)]. The samples with signs of undesired haemolysis were excluded from the study. All samples were stored at -82°C (but not longer than six months).