Aerobic Fitness Affects Responses to Nitrate Supplementation
Aerobic Fitness Affects Responses to Nitrate Supplementation
Twenty-one young men (mean age, 22.7 ± 1.8 yr; body mass, 62.2 ± 10.5 kg; height, 1.71 ± 0.07 m; body mass index, 21.1 ± 2.3 kg·m) were selected from a larger population, with respect to their fitness levels by means of a questionnaire on physical activity (IPAQ-SF), and volunteered to participate in this study. Six subjects were actively involved in structured training programs and, although endurance training formed a large part of their training routine, they also undertook regular resistance conditioning. Three of these athletes were participants in national and international competitions. Four subjects trained three to four times per week and were participating in regional-level official competitions at the time of data collection. Seven participants were recreationally active individuals who engaged in a variety of activities (e.g., weightlifting, running, and team sports) less than two times per week. Four subjects were young university students sedentary or engaging in exercise for less than 90 min·wk. All participants were nonsmokers, normotensive, and were not assuming any drugs. The procedures used in this study were approved by the local ethics committee. All subjects gave their written informed consent after an explanation of the experimental procedures and before commencement of the study.
Each participant visited the laboratory on five separate occasions. On their first visit, anthropometric measurements and an incremental exercise (pre-examination) test to determine peak oxygen uptake (V̇O2peak) and the gas exchange threshold (GET) were performed. The protocol began with subjects running at 7–10 km·h for 6 min, according to their presumed fitness level estimated by IPAQ-SF; then, the belt speed was increased by 1 km·h·min until volitional exhaustion. The GET was determined as previously described to calculate the treadmill speed that would require 80% of the individual GET (moderate-intensity exercise). The peak values of the main cardiovascular, respiratory, and metabolic parameters were taken as the highest 30-s mean value attained before the subject's volitional exhaustion (Table 1). According to Rowell, subjects were divided into three groups based on their V̇O2peak value (low aerobic fitness (Low, n = 8), V̇O2peak range, 28.2–44.1 mL·kg·min; moderate aerobic fitness (Mod, n = 7), V̇O2peak range, 45.5–57.1 mL·kg·min; high aerobic fitness (High, n = 6), V̇O2peak range, 63.9–81.7 mL·kg·min). Two days after they carried out a 3-km running test on a 400-m outdoor track with no dietary supplementation (preintervention control) to gain familiarity with this test.
Subsequently, each subject was randomly assigned in a double-blind, crossover design to follow 6 d of supplementation with either sodium nitrate (NITR) (approximately 5.5 mmol·d; Sigma, Italy) or the placebo (PLA) (sodium chloride, 8.0 mmol·d; Sigma, Italy) dissolved in water. The daily dose was ingested once before breakfast. The different solutions could not be distinguished by taste or appearance. On day 5 of both supplementation periods, subjects underwent incremental exercise testing and two repetitions of a constant load moderate-intensity exercise. On day 6, the subjects repeated two constant load-moderate-intensity exercises and performed a 3-km time trial (see below for further details). The last ingestion (nitrate or placebo) was 3.5 ± 0.5 h before the exercise tests. Athletes received nutritional guidelines to eat the same amount of moderate- to high-nitrate-content foods (green vegetables, beetroot, strawberries, grapes, and tea). The subjects were also required to abstain from using antibacterial mouthwash and chewing gum, as these are known to destroy the oral bacteria responsible for the reduction of NO3 to NO2. A 14-day washout separated the supplementation periods.
The subjects were instructed to arrive at the laboratory in a rested and fully hydrated state and to avoid strenuous exercise in the 24 h preceding each testing session. In addition, they were told to avoid alcohol and caffeine intake 48 h before the exercise test. All laboratory exercise tests were carried out in a well-ventilated laboratory at 19°C–21°C on a motorized treadmill (Jaeger, Germany) set at a 1% gradient.
The subjects initially performed a ramp incremental exercise test for the determination of V̇O2peak and GET. The protocol began with subjects running at the speed requiring 80% of the individual GET for 6 min; then the belt speed was increased by 1 km·h·min until volitional exhaustion. The peak values of the main cardiovascular, respiratory, and metabolic parameters were taken as the highest 30-s mean value attained before the subject's volitional exhaustion. On days 5 and 6 of both supplementation periods, the subjects completed two 6-min bouts of moderate-intensity running (80% GET) for the determination of pulmonary V̇O2 kinetics.
As for running performance, participants completed a 3-km running time trial on a 400-m outdoor track. To minimize the variability in pacing strategy (i.e., suboptimal or inconsistent pacing strategies) reported to occur at the start of a self-paced time trial independent of any intervention, the participants were encouraged to begin time trials based on their experiences in preliminary trials. For this reason, the participants were provided with feedback on their lap times for the first three laps but were blinded to exercise time thereafter. Performance times were recorded by using two synchronized stopwatches.
Pulmonary ventilation (V̇E, in BTPS), O2 consumption (V̇O2), and CO2 output (V̇CO2), both in STPD, were determined breath by breath by a metabolic cart (Vmax29c; SensorMedics, Bilthoven, The Netherlands). Expiratory flow was determined by a mass flow sensor (hot wire anemometer). V̇O2 and V̇CO2 were determined by continuously monitoring PO2 and PCO2 at the mouth throughout the respiratory cycle and from established mass balance equations. Gas exchange ratio (RQ) was calculated as V̇CO2/V̇O2. Heart rate (HR) was determined from the ECG signal. At rest and at various times (1, 3, and 5 min) during recovery, 20 μL of capillary blood was obtained from a preheated earlobe for the determination of blood lactate concentration ([La]b) by an enzymatic method (Biosen 5030; EKF, Cosmed, Italy) both for laboratory and field tests.
V̇O2 kinetics were evaluated during transitions from rest to constant load exercise. Breath-by-breath V̇O2 values obtained during the four repetitions of the exercises were time aligned and then superimposed for each subject. Mean V̇O2 values every 10 s were calculated. Data obtained during the first 20 s of the transition ("cardiodynamic" phase) were excluded from analysis. Thus, V̇O2 kinetics analysis focused on "phase 2" (or "fundamental" component) of the response, which more closely reflects gas exchange kinetics occurring in skeletal muscles.
To mathematically evaluate the V̇O2 kinetics, data were first fitted by a monoexponential function of the type:
where yBAS indicates the V̇O2 value at baseline; Af the amplitude of the V̇O2 response calculated between the baseline value and the steady-state value for the fundamental component; TDf is the time delay, and τf is the time constant of the function for the fundamental component.
To check the presence of a slow component of the kinetics, data were also fit by a double exponential function of the type:
where As, TDs, and τs indicate, the amplitude, the time delay, and the time constant of the slow component of the kinetics, respectively. No slow component was observed in all the constant load exercises both for NITR and PLA.
Resting blood sample was collected to determine plasma levels of nitrate and nitrite both on days 5 and 6. Venous blood was drawn from the antecubital vein into a 5-mL EDTA vacutainer tube (Vacutainer, Becton Dickinson, USA). Plasma was immediately separated by centrifuge (5702R, Eppendorf, Germany) at 1000g for 10 min at 4°C. Plasma samples were then ultrafiltered through a 10-kd molecular weight cutoff (AmiconUltra; Millipore, EMD Millipore Corporation, Billerica, MA) using a ultracentrifuge (4237R, ALC, Italy) at 14,000g for 60 min at 4°C to reduce background absorbance due to the presence of hemoglobin. The ultrafiltered plasma was recovered and used to measure nitrite and nitrate concentration. We used a commercial colorimetric assay kit (Cayman Chemical, USA), which provides an accurate and convenient method for measurement of nitrate and nitrite concentration. Samples were read by the addition of Griess reagents at 545 nm by a microplate reader spectrophotometer (Infinite M200, Tecan, Austria). A linear calibration curve was computed from pure nitrite and nitrate standard. All samples were determined in duplicate, and the interassay coefficient of variation was in the range indicated by the manufacturer.
Results are expressed as mean ± SD. A two-way mixed-design ANOVA with repeated measures (independent measures on aerobic fitness level and repeated measures on supplementation) has been used to examine the effects of intervention, aerobic fitness level, and the interaction between the two. Post hoc analysis was completed using Bonferroni multiple comparisons. Significance level was set at P < 0.05. When significant effects of intervention were found, a Student t-test for paired data was used to determine differences between PLA and NITR conditions. Pearson statistical test and the square of Pearson correlation coefficient (r) have been used to examine the relationship between variables. All statistical procedures were completed using Prism 6.0 (GraphPad Software, CA, USA).
Methods
Subjects
Twenty-one young men (mean age, 22.7 ± 1.8 yr; body mass, 62.2 ± 10.5 kg; height, 1.71 ± 0.07 m; body mass index, 21.1 ± 2.3 kg·m) were selected from a larger population, with respect to their fitness levels by means of a questionnaire on physical activity (IPAQ-SF), and volunteered to participate in this study. Six subjects were actively involved in structured training programs and, although endurance training formed a large part of their training routine, they also undertook regular resistance conditioning. Three of these athletes were participants in national and international competitions. Four subjects trained three to four times per week and were participating in regional-level official competitions at the time of data collection. Seven participants were recreationally active individuals who engaged in a variety of activities (e.g., weightlifting, running, and team sports) less than two times per week. Four subjects were young university students sedentary or engaging in exercise for less than 90 min·wk. All participants were nonsmokers, normotensive, and were not assuming any drugs. The procedures used in this study were approved by the local ethics committee. All subjects gave their written informed consent after an explanation of the experimental procedures and before commencement of the study.
Experimental Design
Each participant visited the laboratory on five separate occasions. On their first visit, anthropometric measurements and an incremental exercise (pre-examination) test to determine peak oxygen uptake (V̇O2peak) and the gas exchange threshold (GET) were performed. The protocol began with subjects running at 7–10 km·h for 6 min, according to their presumed fitness level estimated by IPAQ-SF; then, the belt speed was increased by 1 km·h·min until volitional exhaustion. The GET was determined as previously described to calculate the treadmill speed that would require 80% of the individual GET (moderate-intensity exercise). The peak values of the main cardiovascular, respiratory, and metabolic parameters were taken as the highest 30-s mean value attained before the subject's volitional exhaustion (Table 1). According to Rowell, subjects were divided into three groups based on their V̇O2peak value (low aerobic fitness (Low, n = 8), V̇O2peak range, 28.2–44.1 mL·kg·min; moderate aerobic fitness (Mod, n = 7), V̇O2peak range, 45.5–57.1 mL·kg·min; high aerobic fitness (High, n = 6), V̇O2peak range, 63.9–81.7 mL·kg·min). Two days after they carried out a 3-km running test on a 400-m outdoor track with no dietary supplementation (preintervention control) to gain familiarity with this test.
Subsequently, each subject was randomly assigned in a double-blind, crossover design to follow 6 d of supplementation with either sodium nitrate (NITR) (approximately 5.5 mmol·d; Sigma, Italy) or the placebo (PLA) (sodium chloride, 8.0 mmol·d; Sigma, Italy) dissolved in water. The daily dose was ingested once before breakfast. The different solutions could not be distinguished by taste or appearance. On day 5 of both supplementation periods, subjects underwent incremental exercise testing and two repetitions of a constant load moderate-intensity exercise. On day 6, the subjects repeated two constant load-moderate-intensity exercises and performed a 3-km time trial (see below for further details). The last ingestion (nitrate or placebo) was 3.5 ± 0.5 h before the exercise tests. Athletes received nutritional guidelines to eat the same amount of moderate- to high-nitrate-content foods (green vegetables, beetroot, strawberries, grapes, and tea). The subjects were also required to abstain from using antibacterial mouthwash and chewing gum, as these are known to destroy the oral bacteria responsible for the reduction of NO3 to NO2. A 14-day washout separated the supplementation periods.
Exercise Tests
The subjects were instructed to arrive at the laboratory in a rested and fully hydrated state and to avoid strenuous exercise in the 24 h preceding each testing session. In addition, they were told to avoid alcohol and caffeine intake 48 h before the exercise test. All laboratory exercise tests were carried out in a well-ventilated laboratory at 19°C–21°C on a motorized treadmill (Jaeger, Germany) set at a 1% gradient.
The subjects initially performed a ramp incremental exercise test for the determination of V̇O2peak and GET. The protocol began with subjects running at the speed requiring 80% of the individual GET for 6 min; then the belt speed was increased by 1 km·h·min until volitional exhaustion. The peak values of the main cardiovascular, respiratory, and metabolic parameters were taken as the highest 30-s mean value attained before the subject's volitional exhaustion. On days 5 and 6 of both supplementation periods, the subjects completed two 6-min bouts of moderate-intensity running (80% GET) for the determination of pulmonary V̇O2 kinetics.
As for running performance, participants completed a 3-km running time trial on a 400-m outdoor track. To minimize the variability in pacing strategy (i.e., suboptimal or inconsistent pacing strategies) reported to occur at the start of a self-paced time trial independent of any intervention, the participants were encouraged to begin time trials based on their experiences in preliminary trials. For this reason, the participants were provided with feedback on their lap times for the first three laps but were blinded to exercise time thereafter. Performance times were recorded by using two synchronized stopwatches.
Measurements
Pulmonary ventilation (V̇E, in BTPS), O2 consumption (V̇O2), and CO2 output (V̇CO2), both in STPD, were determined breath by breath by a metabolic cart (Vmax29c; SensorMedics, Bilthoven, The Netherlands). Expiratory flow was determined by a mass flow sensor (hot wire anemometer). V̇O2 and V̇CO2 were determined by continuously monitoring PO2 and PCO2 at the mouth throughout the respiratory cycle and from established mass balance equations. Gas exchange ratio (RQ) was calculated as V̇CO2/V̇O2. Heart rate (HR) was determined from the ECG signal. At rest and at various times (1, 3, and 5 min) during recovery, 20 μL of capillary blood was obtained from a preheated earlobe for the determination of blood lactate concentration ([La]b) by an enzymatic method (Biosen 5030; EKF, Cosmed, Italy) both for laboratory and field tests.
Kinetics Analysis
V̇O2 kinetics were evaluated during transitions from rest to constant load exercise. Breath-by-breath V̇O2 values obtained during the four repetitions of the exercises were time aligned and then superimposed for each subject. Mean V̇O2 values every 10 s were calculated. Data obtained during the first 20 s of the transition ("cardiodynamic" phase) were excluded from analysis. Thus, V̇O2 kinetics analysis focused on "phase 2" (or "fundamental" component) of the response, which more closely reflects gas exchange kinetics occurring in skeletal muscles.
To mathematically evaluate the V̇O2 kinetics, data were first fitted by a monoexponential function of the type:
where yBAS indicates the V̇O2 value at baseline; Af the amplitude of the V̇O2 response calculated between the baseline value and the steady-state value for the fundamental component; TDf is the time delay, and τf is the time constant of the function for the fundamental component.
To check the presence of a slow component of the kinetics, data were also fit by a double exponential function of the type:
where As, TDs, and τs indicate, the amplitude, the time delay, and the time constant of the slow component of the kinetics, respectively. No slow component was observed in all the constant load exercises both for NITR and PLA.
Blood Sampling
Resting blood sample was collected to determine plasma levels of nitrate and nitrite both on days 5 and 6. Venous blood was drawn from the antecubital vein into a 5-mL EDTA vacutainer tube (Vacutainer, Becton Dickinson, USA). Plasma was immediately separated by centrifuge (5702R, Eppendorf, Germany) at 1000g for 10 min at 4°C. Plasma samples were then ultrafiltered through a 10-kd molecular weight cutoff (AmiconUltra; Millipore, EMD Millipore Corporation, Billerica, MA) using a ultracentrifuge (4237R, ALC, Italy) at 14,000g for 60 min at 4°C to reduce background absorbance due to the presence of hemoglobin. The ultrafiltered plasma was recovered and used to measure nitrite and nitrate concentration. We used a commercial colorimetric assay kit (Cayman Chemical, USA), which provides an accurate and convenient method for measurement of nitrate and nitrite concentration. Samples were read by the addition of Griess reagents at 545 nm by a microplate reader spectrophotometer (Infinite M200, Tecan, Austria). A linear calibration curve was computed from pure nitrite and nitrate standard. All samples were determined in duplicate, and the interassay coefficient of variation was in the range indicated by the manufacturer.
Statistical Analysis
Results are expressed as mean ± SD. A two-way mixed-design ANOVA with repeated measures (independent measures on aerobic fitness level and repeated measures on supplementation) has been used to examine the effects of intervention, aerobic fitness level, and the interaction between the two. Post hoc analysis was completed using Bonferroni multiple comparisons. Significance level was set at P < 0.05. When significant effects of intervention were found, a Student t-test for paired data was used to determine differences between PLA and NITR conditions. Pearson statistical test and the square of Pearson correlation coefficient (r) have been used to examine the relationship between variables. All statistical procedures were completed using Prism 6.0 (GraphPad Software, CA, USA).
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