Abstract
Melatonin, a key regulator of the circadian rhythm, exerts strong antioxidant effects by scavenging reactive oxygen species (ROS) and modulating enzymatic redox balance. Xanthine oxidoreductase (XOR), a molybdenum- and iron-sulfur-containing enzyme, catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid-the final steps of purine catabolism-serving as an important enzymatic source of ROS under physiological conditions. XOR exists in three interconvertible isoforms: xanthine dehydrogenase (XDH), which uses NAD(+) as an electron acceptor; xanthine oxidase (XO), which transfers electrons to oxygen, producing superoxide and hydrogen peroxide; and an intermediate form (XDO) that reflects the redox-dependent interconversion between the two. This study aimed to evaluate temporal and sex-dependent variations in XOR isoforms and their relationship with melatonin levels in healthy individuals. Sixty-six volunteers (33 women aged 24-38 and 33 men aged 24-44) were examined. Blood samples were collected at 02:00, 08:00, 14:00, and 20:00. Serum melatonin was measured using ELISA, and XOR isoform activities were determined spectrophotometrically. Melatonin exhibited a precise 24 h rhythm with a nocturnal peak at 02:00 (~98 pg/mL) and a daytime nadir at 14:00 (~9 pg/mL). XO activity varied significantly (p < 0.01), showing an inverse correlation with melatonin in men (ρ = -0.52, p = 0.006), while XDO activity correlated positively with melatonin in women at 14:00 (ρ = 0.48, p = 0.01). These findings indicate sex-specific and time-dependent regulation of XOR isoforms, suggesting that redox homeostasis is modulated differently in men and women throughout the day. Understanding these dynamics may refine the interpretation of oxidative stress biomarkers and help optimize diagnostic and chronotherapeutic approaches in redox-related disorders.