The results of the present study suggest that the individual aerobic fitness level affects the ergogenic benefits induced by dietary nitrate supplementation. The optimal nitrate loading regimen required to elevate plasma [NO2] and to enhance performance in elite athletes is different from that of low-fit subjects and requires further studies.

PurposeAiming to gain a detailed insight into the physiological mechanisms involved under extreme conditions, a group of experienced ultra-marathon runners, performing the mountain Tor des Géants® ultra-marathon: 330 km trail-run in Valle d’Aosta, 24000 m of positive and negative elevation changes, was monitored. ROS production rate, antioxidant capacity, oxidative damage and inflammation markers were assessed, adopting micro-invasive analytic techniques.MethodsForty-six male athletes (45.04±8.75 yr, 72.6±8.4 kg, 1.76±0.05 m) were tested. Capillary blood and urine were collected before (Pre-), in the middle (Middle-) and immediately after (Post-) Race. Samples were analyzed for: Reactive Oxygen Species (ROS) production by Electron Paramagnetic Resonance; Antioxidant Capacity by Electrochemistry; oxidative damage (8-hydroxy-2-deoxy Guanosine: 8-OH-dG; 8-isoprostane: 8-isoPGF2α) and nitric oxide metabolites by enzymatic assays; inflammatory biomarkers (plasma and urine interleukin-6: IL-6-P and IL-6-U) by enzyme-linked immunosorbent assays (ELISA); Creatinine and Neopterin by HPLC, hematologic (lactate, glucose and hematocrit) and urine parameters by standard analyses.ResultsTwenty-five athletes finished the race, while twenty-one dropped out of it. A significant increase (Post-Race vs Pre) of the ROS production rate (2.20±0.27 vs 1.65±0.22 μmol.min-1), oxidative damage biomarkers (8-OH-dG: 6.32±2.38 vs 4.16±1.25 ng.mg-1 Creatinine and 8-isoPGF2α: 1404.0±518.30 vs 822.51±448.91 pg.mg-1Creatinine), inflammatory state (IL-6-P: 66.42±36.92 vs 1.29±0.54 pg.mL-1 and IL-6-U: 1.33±0.56 vs 0.71±0.17 pg.mL1) and lactate production (+190%), associated with a decrease of both antioxidant capacity (-7%) and renal function (i.e. Creatinine level +76%) was found.ConclusionsThe used micro-invasive analytic methods allowed us to perform most of them before, during and immediately after the race directly in the field, by passing the need of storing and transporting samples for further analysis. Considered altogether the investigated variables showed up that exhaustive and prolonged exercise not only promotes the generation of ROS but also induces oxidative stress, transient renal impairment and inflammation.

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.

It has been reported that nitrate supplementation can improve exercise performance. Most of the studies have used either beetroot juice or sodium nitrate as a supplement; there is lack of data on the potential ergogenic benefits of an increased dietary nitrate intake from a diet based on fruits and vegetables. Our aim was to assess whether a high-nitrate diet increases nitric oxide bioavailability and to evaluate the effects of this nutritional intervention on exercise performance. Seven healthy male subjects participated in a randomized cross-over study. They were tested before and after 6 days of a high (HND) or control (CD) nitrate diet (~8.2 mmol∙day−1 or ~2.9 mmol∙day−1, respectively). Plasma nitrate and nitrite concentrations were significantly higher in HND (127 ± 64 µM and 350 ± 120 nM, respectively) compared to CD (23 ± 10 µM and 240 ± 100 nM, respectively). In HND (vs. CD) were observed: (a) a significant reduction of oxygen consumption during moderate-intensity constant work-rate cycling exercise (1.178 ± 0.141 vs. 1.269 ± 0.136 L·min−1); (b) a significantly higher total muscle work during fatiguing, intermittent sub-maximal isometric knee extension (357.3 ± 176.1 vs. 253.6 ± 149.0 Nm·s·kg−1); (c) an improved performance in Repeated Sprint Ability test. These findings suggest that a high-nitrate diet could be a feasible and effective strategy to improve exercise performance.