Despite the growing interest in the role of reactive oxygen species (ROS) in health and disease, reliable quantitative noninvasive methods for the assessment of oxidative stress in humans are still lacking. EPR technique, coupled to a specific spin probe (CMH: 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) is here presented as the method of choice to gain a direct measurement of ROS in biological fluids and tissues. The study aimed at demonstrating that, differently from currently available “a posteriori” assays of ROS-induced damage by means of biomolecules (e.g., proteins and lipids) spin-trapping EPR provides direct evidence of the “instantaneous” presence of radical species in the sample and, as signal areas are proportional to the number of excited electron spins, lead to absolute concentration levels. Using a recently developed bench top continuous wave system (e-scan EPR scanner, Bruker) dealing with very low ROS concentration levels in small (50 μL) samples, we successfully monitored rapid ROS production changes in peripheral blood of athletes after controlled exercise and sedentary subjects after antioxidant supplementation. The correlation between EPR results and data obtained by various enzymatic assays (e.g., protein carbonyls and thiobarbituric acid reactive substances) was determined too. Synthetically, our method allows reliable, quick, noninvasive quantitative determination of ROS in human peripheral blood.

The growing interest in the role of Reactive Oxygen Species (ROS) and in the assessment of oxidative stress in health and disease clashes with the lack of consensus on reliable quantitative noninvasive methods applicable. The study aimed at demonstrating that a recently developed Electron Paramagnetic Resonance microinvasive method provides direct evidence of the “instantaneous” presence of ROS returning absolute concentration levels that correlate with “a posteriori” assays of ROS-induced damage by means of biomarkers. The reliability of the choice to measure ROS production rate in human capillary blood rather than in plasma was tested (step I). A significant (P < 0.01) linear relationship between EPR data collected on capillary blood versus venous blood (R 2 = 0.95), plasma (R 2 = 0.82), and erythrocytes (R 2 = 0.73) was found. Then (step II) ROS production changes of various subjects' categories, young versus old and healthy versus pathological at rest condition, were found significantly different (range 0.0001–0.05 P level). The comparison of the results with antioxidant capacity and oxidative damage biomarkers concentrations showed that all changes indicating increased oxidative stress are directly related to ROS production increase. Therefore, the adopted method may be an automated technique for a lot of routine in clinical trials.

Beneficial systemic effects of regular physical exercise have been demonstrated to reduce risks of a number of age-related disorders. Antioxidant capacity adaptations are amongst these fundamental changes in response to exercise training. However, it has been claimed that acute physical exercise performed at high intensity (>60% of maximal oxygen uptake) may result in oxidative stress, due to reactive oxygen species being generated excessively by enhanced oxygen consumption. The aim of this study was to evaluate the effect of high-intensity discontinuous training (HIDT), characterized by repeated variations of intensity and changes of redox potential, on oxidative damage. Twenty long-distance masters runners (age 47.8±7.8 yr) on the basis of the individual values of gas exchange threshold were assigned to a different 8-weeks training program: continuous moderate-intensity training (MOD, n = 10) or HIDT (n = 10). In both groups before (PRE) and after (POST) training we examined the following oxidative damage markers: thiobarbituric acid reactive substances (TBARS) as marker of lipid peroxidation; protein carbonyls (PC) as marker of protein oxidation; 8-hydroxy-2-deoxy-guanosine (8-OH-dG) as a biomarker of DNA base modifications; and total antioxidant capacity (TAC) as indicator of the overall antioxidant system. Training induced a significant (p<0.05) decrease in resting plasma TBARS concentration in both MOD (7.53±0.30 and 6.46±0.27 µM, PRE and POST respectively) and HIDT (7.21±0.32 and 5.85±0.46 µM, PRE and POST respectively). Resting urinary 8-OH-dG levels were significantly decreased in both MOD (5.50±0.66 and 4.16±0.40 ng mg−1creatinine, PRE and POST respectively) and HIDT (4.52±0.50 and 3.18±0.34 ng mg−1creatinine, PRE and POST respectively). Training both in MOD and HIDT did not significantly modify plasma levels of PC. Resting plasma TAC was reduced in MOD while no significant changes were observed in HIDT. In conclusion, these results suggest that in masters runners high-intensity discontinuous does not cause higher level of exercise-induced oxidative stress than continuous moderate-intensity training, inducing similar beneficial effects on redox homeostasis.

Acute exercise induces an increase in Reactive Oxygen Species (ROS) production dependent on exercise intensity with highest ROS amount generated by strenuous exercise. However, chronic repetition of exercise, that is, exercise training, may reduce exercise-induced oxidative stress. Aim of this study was to evaluate the effects of 6-weeks high-intensity discontinuous training (HIDT), characterized by repeated variations of intensity and changes of redox potential, on ROS production and antioxidant capacity in sixteen master swimmers. Time course changes of ROS generation were assessed by Electron Paramagnetic Resonance in capillary blood by a microinvasive approach. An incremental arm-ergometer exercise (IE) until exhaustion was carried out at both before (PRE) and after (POST) training (Trg) period. A significant (P < 0.01) increase of ROS production from REST to the END of IE in PRE Trg (2.82 ± 0.66 versus 3.28 ± 0.66 µmol·min−1) was observed. HIDT increased peak oxygen consumption (36.1 ± 4.3 versus 40.6 ± 5.7 mL·kg−1·min−1 PRE and POST Trg, resp.) and the antioxidant capacity (+13%) while it significantly decreased the ROS production both at REST (−20%) and after IE (−25%). The observed link between ROS production, adaptive antioxidant defense mechanisms, and peak oxygen consumption provides new insight into the correlation between ROS response pathways and muscle metabolic function.

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