Blood Collection and Sample Handling

Blood was collected on an empty stomach, during routine examinations in the case of adolescents from the control group, and for the study groups: during admission to the Pediatric and Nephrology Clinic of the Medical University of Bialystok. Blood was collected in the amount of 10 mL using an S-Monovette^® EDTA K3 tube (Sarstedt, Nümbrecht, Germany). After collection, the blood was centrifuged (3000 g, 10 min, 4 °C). No hemolysis was observed in any of the obtained plasma samples. Blood cell mass was rinsed 3 times with 0.9% NaCl, and then underwent osmotic lysis using 50 mM cold phosphate buffer (pH 7.4) 1:9 (v/v) [17 (link)]. To prevent sample oxidation, 0.5 M BHT (Sigma-Aldrich, Saint Louis, MO, USA; 10 µl/ml blood) was added to the plasma and red blood cell lysate [17 (link)]. Plasma and blood lysate were frozen (–82 °C). The samples were stored deep-frozen for no longer than 6 months.