4 hne

Peripheral blood was collected into serum tubes, allowed to stand at room temperature for 30 min, and centrifuged (2000× g RCF, 10 min, room temperature) to separate the serum, which was then aliquoted and frozen at −80 °C. The oxidation status of the serum was determined using the d-ROM assay (measuring ROOH concentration), and the antioxidant potential was determined using the PAT assay (based on the ability to reduce Fe³⁺ to Fe²⁺). We used the REDOX fast kit (Innovatics Laboratories, Philadelphia, PA, USA) and the FRAS5 system (Innovatics Laboratories, Philadelphia, PA, USA). d-ROM: optimal range 250–300, borderline range 301–320, mild oxidative stress 321–340, moderate oxidative stress 341–400, high oxidative stress 401–500, very high oxidative stress > 500 U. Carr. TAC: antioxidant deficiency < 1800, mild deficiency 1800–2000, borderline range 2000–2200, normal range 2200–2800, very high > 2800 U. Cor. Based on the measured ROMs and TAC values, the oxidative stress index (OSI) was automatically determined from both measured values and converted to full numbers for ease of interpretation. Lipid peroxidation was assessed using 4-HNE in serum and DNA damage using 8-OHdG in serum using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions (4-HNE, MyBioSource, San Diego, CA, USA; 8-OHdG, Bio-Connect, Oss, The Netherlands).