Alessandra Vezzoli

Assessment of a Standardized ROS Production Profile in Humans by Electron Paramagnetic Resonance

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.

Effects of Mountain Ultra-Marathon Running on ROS Production and Oxidative Damage by Micro-Invasive Analytic Techniques.

Purpose 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. Methods 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. Results 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. Conclusions 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.

Time-Course Changes of Oxidative Stress Response to High-Intensity Discontinuous Training versus Moderate-Intensity Continuous Training in Masters Runners

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.

Fast reduction of peripheral blood endothelial progenitor cells in healthy humans exposed to acute systemic hypoxia

Non‐technical summary  The endothelium is a thin layer of cells lining the interior surface of the entire circulatory system. Endothelial progenitor cells (EPCs) have a crucial role in maintaining the integrity of the endothelium, as they are recruited from the bone marrow to sites of endothelial injury where they contribute to blood vessel formation and repair. The factors regulating EPC mobilization and trafficking remain incompletely understood. We evaluated the time‐course effects of a single 4 h bout of severe hypoxic breathing (simulating 4100 m altitude) followed by 4 h restoration in room air. We show that hypoxia alone induces a rapid disappearance of EPCs from blood, probably sustained by a prompt cell marginalization followed by a late increase in EPC apoptosis. These observations may broaden our understanding of the mechanisms operated by EPCs to maintain endothelial homeostasis and may help to elucidate the potential role of EPCs in regenerative medicine.

Training Effects on ROS Production Determined by Electron Paramagnetic Resonance in Master Swimmers

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.

Magnetic resonance imaging of molecular transport in living morning glory stems

MRI was applied to investigate the transport pathways in Morning Glory plant stems. The study was carried out on living plants without affecting their integrity. The architecture of a dicotyledonous plant was deeply characterized: the root system structure and the vascular bundle location were identified, the presence of central voids caused by cell maturation and loss were observed in the stem. Molecular transport components were recognized, by observing the concentration profile of a tracer, which changed with time after its absorption by the plant roots. MRI analysis revealed the presence of an axial transport as the progress of the tracer front through the vascular bundles and a radial molecular transport from the vascular bundles toward the surface of the stem. As a result, the tracer molecular transport formed the parabolic tracer front (PTF). A model was built up through the analysis of the PTF that consisted of an axial front at the peak position and a radial front at the width of the parabolic tail. PTF analysis revealed differences between the tracer transport velocities in the axial and the radial directions in the plant stem. The model revealed that the width of the parabolic tail reflected the magnitudes of diffusion and permeation of the tracer in the plant stem.

Effects of temperature and extracellular pH on metabolites: kinetics of anaerobic metabolism in resting muscle by 31P- and 1H-NMR spectroscopy.

SUMMARY Environmental stress, such as low temperature, extracellular acidosis and anoxia, is known to play a key role in metabolic regulation. The aim of the present study was to gain insight into the combined temperature-pH regulation of metabolic rate in frog muscle, i.e. an anoxia-tolerant tissue. The rate of exergonic metabolic processes occurring in resting isolated muscles was determined at 15°C and 25°C as well as at extracellular pH values higher (7.9), similar (7.3) and lower (7.0) than the physiological intracellular pH. 31P and 1H nuclear magnetic resonance spectroscopy high-resolution measurements were carried out at 4.7 T in isolated frog (Rana esculenta) gastrocnemius muscle during anoxia to assess, by means of reference compounds, the concentration of all phosphate metabolites and lactate. Intra- and extracellular pH was also determined. In the range of examined temperatures (15-25°C), the temperature dependence of anaerobic glycolysis was found to be higher than that of PCr depletion (Q10=2.3). High-energy phosphate metabolism was confirmed to be the initial and preferential energy source. The rate of phosphocreatine hydrolysis did not appear to be affected by extracellular pH changes. By contrast, independent of the intracellular pH value, at the higher temperature (25°C) a lowering of the extracellular pH from 7.9 to 7.0 caused a depression in lactate accumulation. This mechanism was ascribed to the transmembrane proton concentration gradient. This parameter was demonstrated to regulate glycolysis, probably through a reduced lactate efflux, depending on the activity of the lactate-H+ co-transporter. The calculated intracellular buffer capacity was related to intra- and extracellular pH and temperature. At the experimental extracellular pH of 7.9 and at a temperature of 15°C and 25°C, calculated intracellular buffering capacity was 29.50 μmol g-1 pH unit-1 and 69.98 μmol g-1 pH unit-1, respectively.

Oxidative stress response to acute hypobaric hypoxia and its association with indirect measurement of increased intracranial pressure: a field study

High altitude is the most intriguing natural laboratory to study human physiological response to hypoxic conditions. In this study, we investigated changes in reactive oxygen species (ROS) and oxidative stress biomarkers during exposure to hypobaric hypoxia in 16 lowlanders. Moreover, we looked at the potential relationship between ROS related cellular damage and optic nerve sheath diameter (ONSD) as an indirect measurement of intracranial pressure. Baseline measurement of clinical signs and symptoms, biological samples and ultrasonography were assessed at 262 m and after passive ascent to 3830 m (9, 24 and 72 h). After 24 h the imbalance between ROS production (+141%) and scavenging (−41%) reflected an increase in oxidative stress related damage of 50–85%. ONSD concurrently increased, but regression analysis did not infer a causal relationship between oxidative stress biomarkers and changes in ONSD. These results provide new insight regarding ROS homeostasis and potential pathophysiological mechanisms of acute exposure to hypobaric hypoxia, plus other disease states associated with oxidative-stress damage as a result of tissue hypoxia.

A novel application for cognitive evaluation in mountain ultramarathons: Olfactory assessment

OBJECTIVE Olfactory function, a cognitive impairment biomarker, was evaluated in mountain ultramarathon (MUM) runners during the Tor des Géants race (332.5 km with an overall altitude gain of 24,000 m; altitude range 330-3296 m above the sea). METHODS An Odor Identification Test was administered before (T0; n = 53), at 148.7 kms (T1; n = 32) and after the race (T2; n = 28). The effect of dehydration and sleep deprivation on olfactory function was assessed. Olfactory function was also assessed in non-MUM athletes and sedentary controls (C) at rest. RESULTS A majority of the athletes completed the olfactory test at all time intervals. Olfactory function decreased throughout the race (T0: 13.8 ± 1.9, T1: 13.7 ± 1.6, T2: 13.1 ± 1.8; T0 vs T2 P = .01). There was no relationship with race time or sleep deprivation on the sense of smell throughout the competition. However, there was a combined effect with decreased olfaction during the second half of the race, while a poor relationship was seen between olfaction and total body water at midterm (T1: rs = -0.427; P = .019), but not at baseline or after the race. MUM athletes had similar olfactory scores to C (13.8 ± 1.9 vs 13.7 ± 1.4) and non-MUM (13.8 ± 1.9 vs 13.9 ± 1.6) athletes. CONCLUSIONS This pilot study showed the feasibility of olfactory evaluation as a minimally invasive cognitive impairment assessment. The test can be used in logistically difficult environments, adding scientific value to this promising method. Although olfaction decreased after prolonged physical activity, further studies are warranted to make the relationship between cognition and external factors (eg, sleep deprivation, dehydration) more clear.

Oxidative Stress Assessment in Response to Ultraendurance Exercise: Thiols Redox Status and ROS Production according to Duration of a Competitive Race

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.