Of this study was to evaluate the effects of short-term nitrate supplementation on endurance performance in subjects with different levels of aerobic fitness. Methods Twenty-one subjects (mean age, 22.7 ± 1.8 yr) with different aerobic fitness level (V˙O2peak value ranging from 28.2 to 81.7 mL·kg−1·min−1) participated in a crossover double-blind placebo-controlled study. Subjects were tested after 6 d of supplementation with either 0.5 l per day of nitrate (5.5 mmol)-containing water (NITR) or nitrate-free water (PLA). Participants performed an incremental running test until exhaustion and four repetitions of 6-min submaximal (approximately 80% of gas exchange threshold) constant load exercise on a motorized treadmill. Moreover, subjects performed a 3-km running time trial on the field. Results After NITR, a negative correlation between reduction of O2 cost of submaximal exercise and individual aerobic fitness level was observed (r2 = 0.80; P < 0.0001). A significant inverse correlation was also found between aerobic fitness level and improvement in performance for 3-km time trial after NITR (r2 = 0.76; P < 0.0001). Additionally, subjects responded differently to dietary nitrate supplementation according to aerobic fitness level with higher-fit subjects showing a lower increase in plasma [NO3−] (r2 = 0.86; P < 0.0001) and [NO2−] (r2 = 0.75; P < 0.0001). Conclusions 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....

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.

Aiming 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. Forty-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. Twenty-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. The 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.

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 (R2=0.95), plasma (R2=0.82), and erythrocytes (R2=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 0.0001–0.05Plevel). 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.

Amino acid (AA) sequences of 807 bioactive peptides from foods of animal origin were examined in order to correlate peptide structure with activity (antihypertensive, antioxidative, immunomodulatory, antimicrobial, hypolipidemic, antithrombotic, and opioid) and stability in vivo . Food sources, such as milk, meat, eggs, and marine products, show different frequencies of bioactive peptides exhibiting specific effects. There is a correlation of peptide structure and effect, depending on type and position of AA. Opioid peptides contain a high percentage of aromatic AA residues, while antimicrobial peptides show an excess of positively charged AAs. AA residue position is significant, with those in the first and penultimate positions having the biggest effects on peptide activity. Peptides that have activity in vivo contain a high percentage (67%) of proline residues, but the positions of proline in the sequence depend on the length of the peptide. We also discuss the influence of processing on activity of these peptides, as well as methods for predicting release from the source protein and activity of peptides.

Purpose. Response to an ultraendurance competitive race on thiols redox status, reactive oxygen species (ROS) production, and oxidative stress (OxS) was investigated according to duration. Methods. Twenty‐four elite runners were examined: six completed 50 km and eighteen 100 km. Blood and urine samples were collected before and immediately after the race. Erythrocytes and plasma aminothiols by high‐performance liquid chromatography, total antioxidant capacity (TAC), and OxS biomarkers (protein carbonyl (PC), thiobarbituric acid‐reactive substances (TBARS), 8‐isoprostane (8‐iso‐PGF2α), and 8‐OH‐2‐deoxyguanosine (8‐OH‐dG)) by immunoenzymatic assays and ROS production by Electron Paramagnetic Resonance were assessed. Results. Significant increases (P between <0.05 and <0.0001) were recorded in plasma total and oxidized aminothiols concentration and TAC (P < 0.0001) only after 100 km: plasmatic (ROS production (+12 versus +29%), PC (+54 versus +115%), and TBARS (+28 versus +55%)) and urinary (8‐OH‐dG.creatinine⁻¹ (+71 versus +158%) and 8‐iso‐PGF2α.creatinine⁻¹ (+43 versus +135%)) concentrations for 50 and 100 km (duration 4 h 3′ versus 8 h 42′), respectively. Conclusion. Very prolonged ultraendurance exercise causes an increase in ROS production and OxS depending on specific biomarker examined but always linearly and directly related to exercise duration. Redox status of erythrocytes was preserved. A relationship between running performance and both prerace ROS production and antioxidant‐redox status was found in 100 km race.