Oxidative Stress and Antioxidant Enzyme Activities in Elite Female Turkish Cross-Country Skiers

Overview

This study is designed to investigate oxidative stress and antioxidant defense biomarkers in elite female cross-country skiers compared with sedentary women. Oxidative stress plays a central role in exercise adaptation, and the balance between reactive oxygen species production and antioxidant capacity can influence performance and recovery. Despite growing interest in redox biology, evidence from elite female endurance athletes is limited. This cross-sectional observational study enrolled 17 elite female cross-country skiers, who were members of the Turkish national junior and senior teams, and 17 age- and BMI-matched sedentary women. All participants reported to the laboratory between 07:00 and 09:00 a.m. after an overnight fast. Anthropometric characteristics, including height, weight, and BMI, were measured under standardized conditions. Venous blood samples were collected from the antecubital vein using aseptic technique. Serum was separated, stored at -80 °C, and analyzed for key redox biomarkers. Catalase (CAT) activity was measured spectrophotometrically using the method of Aebi, reduced glutathione (GSH) concentration was determined using Ellman's reagent, and malondialdehyde (MDA), a marker of lipid peroxidation, was assessed using the thiobarbituric acid reactive substances (TBARS) assay. All analyses were performed in triplicate in a certified clinical biochemistry laboratory. The primary objective of this study is to characterize and compare systemic antioxidant capacity and oxidative stress levels between trained athletes and sedentary controls. The data generated will provide insight into how chronic endurance training in cold outdoor environments affects enzymatic and non-enzymatic antioxidant systems in young women.

This study was designed as a cross-sectional observational investigation to examine systemic oxidative stress and antioxidant defenses in elite female cross-country skiers compared with sedentary women of similar age and body mass index. The study followed the STROBE guidelines for reporting observational research and complied with the principles of the Declaration of Helsinki. Ethical approval was granted by the Muş Alparslan University Scientific Research and Publication Ethics Committee (Approval No: 27754; Date: 27 October 2021). Participants A total of 34 women participated in the study, including 17 elite female cross-country skiers who were current members of the Turkish national junior and senior teams (2021-2025 competitive seasons) and 17 sedentary women matched for age and BMI. Inclusion criteria were: (a) female sex; (b) age between 15-20 years; (c) absence of chronic metabolic, cardiovascular, or inflammatory diseases; (d) no regular medication use; (e) no vitamin or antioxidant supplementation within the preceding three months; and (f) non-smoker and non-alcohol consumer. Exclusion criteria included acute infection, recent musculoskeletal injury, or failure to comply with pre-test requirements (e.g., overnight fasting, rest). Written informed consent was obtained from all participants; for those under 18 years of age, parental or guardian consent was also collected. Anthropometric and Pre-Analytical Procedures All participants attended the laboratory between 07:00 and 09:00 a.m. following a 10-12-hour overnight fast to minimize the effect of circadian variation and dietary intake on oxidative stress markers. Upon arrival, participants were asked to remain seated in a quiet, temperature-controlled room (22-24 °C) for at least 30 minutes to achieve resting physiological conditions. Anthropometric measurements were taken barefoot and in light clothing using standardized procedures: standing height was measured to the nearest 0.1 cm using a stadiometer, and body weight was measured to the nearest 0.1 kg with a calibrated digital scale. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m²). Blood Sampling and Processing Venous blood samples (approximately 3 mL) were collected from the antecubital vein using sterile vacutainer tubes. Samples were allowed to clot for 20-30 minutes at room temperature and subsequently centrifuged at 5000 rpm for 10 minutes to separate serum. Serum aliquots were transferred into labeled polypropylene tubes and stored at -80 °C until biochemical analysis. Previous studies have confirmed the stability of oxidative stress biomarkers, including reactive oxygen metabolites and antioxidant potential, in serum stored under these conditions for up to five years. Biochemical Analyses All analyses were performed in the Central Biochemistry Laboratory of Van Yüzüncü Yıl University Faculty of Medicine by the same experienced laboratory technician to minimize inter-operator variability. Catalase (CAT) activity was determined according to the spectrophotometric method of Aebi, which measures the rate of hydrogen peroxide decomposition at 240 nm over a two-minute interval at 25 °C. Results were expressed as units per milliliter (U/mL). Reduced Glutathione (GSH) concentration was quantified using the colorimetric method of Beutler et al., employing Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB) to produce a yellow-colored compound measurable at 412 nm. Malondialdehyde (MDA) concentration, a marker of lipid peroxidation, was measured by the thiobarbituric acid reactive substances (TBARS) assay, based on the formation of a pink MDA-TBA complex read at 532 nm. Each sample was analyzed in triplicate, and mean values were used for subsequent statistical processing to increase measurement reliability. Data Management and Planned Analyses Data were entered into a secure electronic database and cross-checked for accuracy by two independent researchers. Descriptive statistics (mean ± standard deviation, minimum-maximum) were calculated for all continuous variables. The normality of data distributions was assessed using the Shapiro-Wilk test. For variables not meeting the assumption of normality, non-parametric tests (Mann-Whitney U test) were pre-specified to compare group differences. Effect sizes were to be calculated using Cohen's d with 95% confidence intervals to quantify the magnitude of between-group differences. Correlations between CAT, GSH, and MDA were planned using Spearman rank-order correlation coefficients. Statistical significance was set at p \< 0.05.