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Medicine & Science in Sports & Exercise:
doi: 10.1249/MSS.0b013e3182687e5c
Applied Sciences

Sodium Nitrate Supplementation Does Not Enhance Performance of Endurance Athletes

BESCÓS, RAÚL1; FERRER-ROCA, VENTURA2; GALILEA, PEDRO A.2; ROIG, ANDREU2; DROBNIC, FRANCHEK2; SUREDA, ANTONI3; MARTORELL, MIQUEL3; CORDOVA, ALFREDO4; TUR, JOSEP A.3; PONS, ANTONI3

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Author Information

1Research Group on Sport Sciences, National Institute of Physical Education (INEFC), University of Barcelona, Barcelona, SPAIN; 2Performance and Health Research Group for High Level Sports, High Performance Center of Barcelona, Sant Cugat, SPAIN; 3Laboratory of Physical Activity Science, Research Group on Community Nutrition and Oxidative Stress, University of the Balearic Islands, Palma de Mallorca, SPAIN; and 4Department of Physiology and Biochemistry, School of Physiotherapy, University of Valladolid, Valladolid, SPAIN

Address for correspondence: Raúl Bescós, Ph.D., Physiology Laboratory, National Institute of Physical Education (INEFC), Av., de l’Estadi s/n, 08038 Barcelona, Spain; E-mail: raulbescos@gmail.com.

Submitted for publication April 2012.

Accepted for publication July 2012.

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Abstract

Purpose: Supplementation with inorganic nitrate has been suggested to be an ergogenic aid for athletes as nitric oxide donor. The purpose of this study was to determine whether ingestion of inorganic sodium nitrate benefits well-trained athletes performing a 40-min exercise test in laboratory conditions. In addition, we investigated the effect of this supplement on plasma levels of endothelin-1 (ET-1) and in nitrated proteins.

Methods: Thirteen trained athletes participated in this randomized, double-blind, crossover study. They performed a 40-min cycle ergometer distance-trial test after two 3-d periods of dietary supplementation with sodium nitrate (10 mg·kg−1 of body mass) or placebo.

Results: Concentration of plasma nitrate (256 ± 35 μM) and nitrite (334 ± 86 nM) increased significantly (P < 0.05) after nitrate supplementation compared with placebo (nitrate: 44 ± 11 μM; nitrite: 187 ± 43 nM). In terms of exercise performance, there were no differences in either the mean distance (nitrate: 26.4 ± 1.1 km; placebo: 26.3 ± 1.2 km; P = 0.61) or mean power output (nitrate: 258 ± 28 W; placebo: 257 ± 28 W; P = 0.89) between treatments. Plasma ET-1 increased significantly (P < 0.05) just after exercise in nitrate (4.0 ± 0.8 pg·mL−1) and placebo (2.4 ± 0.4 pg·mL−1) conditions. This increase was significantly greater (P < 0.05) in the nitrate group. Levels of nitrated proteins did not differ between treatments (nitrate: preexercise, 91% ± 23%; postexercise, 81% ± 23%; placebo: preexercise, 95% ± 20%; postexercise, 99% ± 19%).

Conclusion: Sodium nitrate supplementation did not improve a 40-min distance-trial performance in endurance athletes. In addition, concentration of plasma ET-1 increased significantly after exercise after supplementation with sodium nitrate.

Inorganic nitrate has been identified as an important precursor for nitric oxide (NO) syntheses that complements the classical L-arginine NO synthesis pathway. This alternative NO pathway has been suggested to play an important role in the regulation of blood pressure and blood flow, gastric integrity, and tissue protection against ischemic injury (26). Consequently, interest in inorganic dietary nitrate has increased substantially in pharmacology and physiology research during the last decade (26).

Inorganic nitrate supplementation has also been suggested to be a potential ergogenic aid for athletes (7,19). Recent studies have reported that supplementation with beetroot juice rich in nitrate improves exercise performance measured as time to exhaustion during a fixed workload and during an incremental exercise test in healthy humans (2,20,39). Furthermore, three recent studies using supplementation with beetroot have also shown benefits in exercise performance measured in a treadmill (31) and cycle ergometer (7,19) time-trial tests, respectively. The mechanism(s) behind this intriguing effect remains uncertain, although two candidates have been proposed to explain it. The first includes a NO-mediated improvement in muscle contractile efficiency (1), whereas the second relates to an improvement in mitochondrial respiration efficiency (21).

In contrast to previous findings, a recent study demonstrated no increase in exercise performance with well-trained cyclists during a 50-mile time-trial test after an acute supplementation of beetroot juice (41). Three subsequent studies using pharmacological sodium nitrate also showed no increase in exercise capacity during an incremental exercise test (3,22,23). However, it should be noted that, perhaps, the incremental protocols until exhaustion performed in these studies were not the best method in assessing exercise performance. These protocols are commonly shorter and less sensitive to possible improvements in performance compared with endurance events lasting longer than 30 min (10). For this reason, time-trial protocols are used as a better method to assess changes in endurance performance (10). For instance, there is evidence that an enhancement of only 1% of exercise efficiency may improve 40-km cycle time-trial performance by approximately 60 s (30). Therefore, if inorganic nitrate supplementation is able to enhance energy efficiency, it may be expected that this supplement could also promote an increase in exercise performance during a long time-trial test.

On the other hand, a sustained increase in NO synthesis in healthy humans may not always be beneficial. It is known that an excess of NO production may also lead to a reaction with superoxide radical and produce highly reactive peroxynitrite. The latter causes the nitration of tyrosine residues of proteins to form 3-nitrotyrosine (3-NT) and thereby irreversibly alters the biological function of proteins (33). Prolonged, exhaustive endurance exercise may also favor these reactions and increase oxidative stress in athletes (32). In addition, the reduction of nitrite to NO in hypoxic and/or acidic conditions may form nitrogen oxides and raise plasma concentration of 3-NT and nitrated proteins (33). To avoid these harmful effects, an adequate balance between prooxidants and antioxidants is needed. Although exercise training is known to enhance endogenous antioxidant systems (6), there is a lack of studies assessing whether dietary nitrate supplementation affects the level of nitrogen oxides with nitrating properties in athletes after exhaustive exercise. Interestingly, Lundberg et al. (25) have recently launched advice to refrain from the uncontrolled use of nitrate and nitrite supplementation in pharmacological form because there is evidence from internet forums, articles, and discussions within the sports community that the use of these products is spreading rapidly among athletes. However, supplementation with natural sources of nitrate such as whole vegetables or vegetable juices in moderate amounts seems to be safe of any acute risk (16).

It is also known that there are other important factors independent of NO that contribute to the regulation of vascular tone in humans. One of the most important is endothelin-1 (ET-1). This molecule is a potent vasoconstrictor peptide produced by vascular endothelial cells, which interacts with other vascular mediators, most notably NO (36). Very low concentrations of NO (as low as 20 ppm) effectively suppress release and physiological action of ET-1. Moreover, NO can also nitrosylate endothelin receptors and reduce affinity for ET-1 (11). Thus, in addition to its direct vasodilator effects, NO induces vasodilation indirectly by limiting the release of ET-1. Because dietary nitrate is claimed to be a NO donor in humans, its effect on plasma concentration of ET-1 at rest and after exhaustive exercise would extend knowledge of the role of dietary nitrate supplementation on vascular tone.

Accordingly, the first aim of this study was to assess the ergogenic effect of dietary sodium nitrate supplementation in endurance-trained subjects using a 40-min distance-trial test. In addition, we evaluated plasma concentration of nitrated proteins and ET-1 before and after exercise in two experimental groups, one receiving inorganic nitrate supplementation and the other a placebo. We hypothesized that if inorganic nitrate supplementation can enhance energy efficiency of exercise, it may also benefit overall exercise performance during a long distance-trial test. Moreover, we hypothesized that the ingestion of a moderate amount of inorganic nitrate cannot alter plasma concentration of 3-NT after a high-intensity exercise test in well-trained subjects. Because the potential for an increase in free radicals is derived from nitrate–nitrite–NO pathway, these molecules can be effectively reduced by endogenous antioxidant systems of athletes. Lastly, it was anticipated that an increase in NO synthesis after sodium nitrate ingestion may reduce plasma concentration of ET-1 compared with placebo after exercise.

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METHODS

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Subjects

Thirteen nonprofessional male cyclists and triathletes (age, 32.6 ± 5.6 yr; body weight, 72.4 ± 9.7 kg−1; body mass index, 23.4 ± 2.0 kg·m−2; body fat, 9.6% ± 3.3%) volunteered to participate in this study. Athletes were members of competitive cycling or triathlon squads, and none of them reported any medical conditions at the time of the study. They had 8 ± 5 yr of experience in endurance events, and their average weekly training volume was 15.7 ± 5.0 h·wk−1. None of the subjects smoked tobacco. The procedures used in this study were approved by the Ethics Committee of the Catalonian Sports Council. All subjects gave their written informed consent after an explanation of the experimental procedures and before commencement of the study.

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Nitrate supplementation

Subjects were randomly assigned in a double-blind, crossover design to follow 3 d of supplementation with either sodium nitrate (10 mg·kg−1 of body mass, code 18211; Acofarma, Spain) or the placebo (sodium chloride, 10 mg·kg−1 of body mass) dissolved in water. Supplementation was ingested each morning before breakfast. On the last day, subjects ingested the supplement or placebo 3 h before the exercise test. A diet with low levels of moderate or high nitrate content foods (green vegetables, beetroot, strawberries, grapes, and tea) was followed 2 d before the tests. During this time, athletes received nutritional guidelines and were encouraged to follow a high-carbohydrate diet to optimize glycogen deposition. In addition, they were told to avoid alcohol, caffeine products, and dietary supplements 24 h before the exercise test. A 4-d washout separated the supplementation periods.

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Ergometry test

Subjects were required to report to the laboratory on four occasions, each separated by 1 wk. During the first week, subjects performed an anthropometric assessment and an incremental exercise test under laboratory-controlled conditions to determine maximal oxygen uptake (V˙O2max), maximal power output (Wmax), ventilatory threshold (VT), and respiratory compensation point (RCP). The exercise protocol started at 50 W and increased 25 W every minute until voluntary exhaustion. An electronically braked cycle ergometer (Schoberer Rad Messtechnik (SRM), Germany) was used for all tests. The configuration of the ergometer—crank length, pedals, saddle, and handlebar position—was adapted to the measurements of subjects’ own road bicycles. Before each test, the cycle ergometer was calibrated following the manufacturer’s instructions. Pedaling cadence was individually chosen within the range of 70–100 rpm. In the next 3 wk, subjects performed three distance trials in the laboratory with controlled environmental conditions (23.8°C ± 1.0°C). The first was carried out to familiarize subjects with the bicycle ergometer, the gas analyzer, and the testing procedure. The following two distance trials were carried out in both conditions (placebo and nitrate) at the same time of day (±1 h). They were asked to cover as much distance as possible during the 40 min. We chose this duration because it was related to the distance (22 km) and time (approximately 35–40 min) of the regional and national time-trial championships. Some athletes in the current study were training for these championships and accepted to participate in this study as part of their training for these events. Before the test, athletes performed 15 min of warm-up at 60% of V˙O2max followed by 5–10 min of passive recovery before starting the distance trial. The ergometer (SRM) was programmed in the mode “open end test.” Subjects started the test in “gear 9” and were allowed to change gear. In this mode, power output varies with pedal rate and/or a gear change. For each distance trial, time, distance, power, and torque was recorded every second by SRM software. To avoid any experimental bias, the only feedback available to cyclists during the distance trial was time elapsed. In addition, they were strongly encouraged verbally during both distance trials. Food and fluid ingestion was forbidden during the test.

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Gas analysis

During the incremental exercise test, oxygen uptake (V˙O2), minute ventilation (V˙E), carbon dioxide production (V˙CO2), and RER were measured continuously breath by breath by a computerized gas analyzer (Jaeger Oxycon Mobile, Germany). V˙O2peak was determined as the mean V˙O2 measured during the final 60 s of exercise. The criteria for a true V˙O2peak were the attainment of a plateau in oxygen uptake (increase less than 150 mL·min−1 during the last minute of exercise) despite an increase in workload. Wmax and HRmax were defined as the HR and W at the point of exhaustion during the test. To determine VT and RCP, data were averaged for 30-s intervals and analyzed by two independent reviewers as described previously (3). During the 40-min distance trials, the respiratory response was not measured continuously. Three samples of respiratory gas exchange were taken during the test: 1) between 12 and 15 min, 2) between 22 and 25 min, and 3) between 32 and 35 min. Data regarding V˙O2, V˙E, V˙CO2, and RER were recorded breath by breath, and values of the last minute were averaged and used to assess the respiratory response during exercise (Jaeger Oxycon Mobile). In addition, HR was continuously recorded (beat by beat) with a portable heart rate monitor (RS800 SD; Polar, Finland).

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Blood sampling

Two blood samples were collected from the antecubital vein to analyze nitrate and nitrite: 1) after 3 d of nitrate supplementation or placebo in resting conditions before the exercise test, 2) during the first 3 min after distance trials (placebo and nitrate). Venous blood was drawn with a 5-mL syringe ethylenediaminetetraacetic acid and was immediately centrifuged at 1000 g for 20 min to separate plasma from blood cells. Plasma samples were then centrifuged for 30 min at 14,000 g in 10 K filters (Amicon Ultra; Millipore, EMD Millipore Corporation, Billerica, MA) to remove proteins. The supernatant was recovered and used to measure nitrite and nitrate concentration by detecting liberated NO in a gas-phase chemiluminescence reaction with ozone using a NO analyzer (NOA 280i; Sievers, GE Power & Water, Boulder, CO) as described previously (3).

In addition, from the same blood samples, a determination of plasma ET-1 concentration was made using commercially available immunoassay kits (Assay Designs, Inc., Ann Arbor, MI) following the manufacturer’s instructions. Assays were performed in duplicate, and optical density was determined using a microplate reader set to 450 nm. In addition, plasma 3-NT concentration in proteins was analyzed using commercially available immunoassay kits (OxiSelect™ Nitrotyrosine Immunoblot; Cell Biolabs, Inc., San Diego, CA).

Lastly, during the distance trials, four samples of capillary blood (10 μL) were collected from the ear lobe to analyze lactate ([Hla]) using a Lange Miniphotometer LP2 (Germany) system: 1–3) in minute 10, 20, and 30 of the test, and 4) 3 min after the maximal test.

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Urine sampling

To analyze nitrate–nitrite urine concentration, two samples were collected: 1) in resting conditions before the exercise test after 3 d of nitrate supplementation or placebo ingestion, 2) during the first hour postexercise (placebo and nitrate). The same method used for blood samples was applied to analyze nitrate and nitrite concentration in urine. Percentage urinary nitrate excretion with respect to supplemented dietary nitrate was estimated based on nitrate excreted in the urine before and after the exercise test (approximately 45 min each) for each treatment using the following formula:

Equation (Uncited)
Equation (Uncited)
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where USNO3− represents urinary nitrate excretion (mg) after dietary nitrate supplementation, UPNO3− represents urinary nitrate excretion (mg) after placebo treatment, and DSNO3− represents the amount of dietary nitrate supplementation per day (mg).

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Statistics

Results are expressed as means ± SD of the mean. The coefficient of variation (CV) for distance and power output between the third and fourth distance trial was calculated by dividing each subject’s SD by his mean. A spreadsheet proposed by Hopkins (14) that analyzes validity by linear regression was used for calculations. In addition, an intraclass correlation coefficient was also computed for the same variables. To investigate the influence of treatment (S) and time (T) and interaction between both these variables (S × T), the data were treated with two-way repeated-measures ANOVA. The sets of data in which there was significant S × T interaction were tested by one-way ANOVA test. When significant effects of S or T were found, a Student’s t-test for paired data was used to determine differences between the groups (nitrate and placebo) involved. In addition, Pearson correlation coefficient was used to assess the relation between variables. Differences between two independent groups (low responders and high responders) were assessed using a Student’s t-test for unpaired data. All data were analyzed to determine the normal distribution, and post hoc analyses were performed using Tukey HSD. Significance level was set at P < 0.05, whereas a trend was noted when P < 0.10.

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RESULTS

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Performance during the V˙O2max test

Mean results of V˙O2max, HRmax, and Wmax during the incremental exercise test were 4.3 ± 0.3 L·min−1 (60 ± 7 mL·kg−1·min−1), 180 ± 11 beats·min−1, and 378 ± 30 W (5.3 ± 0.8 W·kg−1), respectively. VT was determined at a mean intensity of 215 ± 38 W, 144 ± 12 beats·min−1 (80% ± 4% of HRmax), and 38.6 ± 7.7 mL·kg−1·min−1 of V˙O2 (64.3% ± 8.3% of V˙O2max), whereas RCP was estimated at an average intensity of 301 ± 37 W, 166 ± 12 beats·min−1 (92% ± 3% of HRmax), and 50.8 ± 7.8 mL·kg−1·min−1 of V˙O2 (85.1% ± 8.1% of V˙O2max).

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Performance during 40-min distance trial

The mean CV (CV %) for distance and power output between the third and fourth distance trial independent of treatment was 1.1% (95% confidence interval (CI) = 0.9–1.8) and 2.2% (95% CI = 1.7–3.5), respectively. These differences were not statistically significant (P > 0.05). In addition, the intraclass correlation coefficient for distance was 0.98 (95% CI = 0.95–1.00) and 0.99 (95% CI = 0.96–1.00) for power output.

Average distance and power output profiles are shown in Figure 1. There were no significant differences between nitrate and placebo groups in overall distance (nitrate: 26.4 ± 1.1 km; placebo: 26.3 ± 1.2 km; P = 0.61) or mean power output (nitrate: 258 ± 28 W and 3.6 ± 0.6 W·kg−1; placebo: 257 ± 28 W and 3.6 ± 0.6 W·kg−1; P = 0.89) achieved during the 40-min distance trial. Athletes performed the distance trial at a mean intensity of 91.1% ± 3.3% (85.1% ± 5.0% V˙O2max) and 91.3% ± 3.0% of HRmax (85.1% ± 6.3% V˙O2max) in placebo and nitrate conditions, respectively. Mean cadence during distance trials was 93 ± 7 and 93 ± 6 rpm for placebo and nitrate.

Figure 1
Figure 1
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Cardiorespiratory and metabolic response during the 40-min distance trial

Main respiratory variables (V˙O2, V˙CO2, V˙E, and RER) were unaffected after nitrate supplementation compared with placebo (Table 1). HR increased significantly during the test in both treatments (P < 0.001) (Table 1). There were no differences in average blood lactate concentration at any point of the test between placebo and nitrate groups (Table 1).

Table 1
Table 1
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Plasma and urinary concentration of nitrate and nitrite

Concentration of plasma nitrate (256 ± 35 μM, P < 0.001) and nitrite (334 ± 86 nM, P = 0.008) increased significantly after sodium nitrate supplementation compared with placebo (nitrate: 44 ± 11 μM; nitrite: 187 ± 43 nM) (Fig. 2). After exercise, plasma nitrate concentration were higher in nitrate condition compared with placebo (nitrate: 272 ± 54 μM; placebo: 52 ± 8 μM; P < 0.001). Otherwise, physical exercise did not significantly alter plasma nitrate just after a 40-min distance trial in either group (placebo or nitrate). Although concentration of plasma nitrite showed a reduction after the exercise test (plasma: 248 ± 72 nM) compared with values in resting conditions (plasma: 334 ± 86 nM), these differences were not statistically significant (plasma: P = 0.107) (Fig. 2).

Figure 2
Figure 2
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We performed analyses of correlations between data of the incremental test (V˙O2max and Wmax), without supplementation, with basal plasma concentration of nitrate and nitrite, as well as with plasma values of both anions after dietary nitrate supplementation. We did the same analysis of correlation between distance, power, and speed performed during the 40-min distance-trial tests in both conditions (placebo and nitrate) and plasma concentration of nitrate and nitrite. These analyses were performed to assess whether subjects with high-performance parameters were more or less sensitive to changes in plasma concentration of nitrate and nitrite after dietary nitrate supplementation. We found that subjects with high V˙O2max (r = 0.56, P = 0.048) and Wmax (r = 0.59, P = 0.032) in the incremental test (without supplementation) showed a greater increase in plasma nitrate concentration after nitrate treatment. In addition, there was found to be a positive trend between the increase in plasma nitrate concentration after dietary consumption of nitrate and increase in distance (r = 0.53, P = 0.062), speed (r = 0.52, P = 0.066), and power (r = 0.54, P = 0.055) in the 40-min distance time trial. However, no effect was found between performance parameters and changes in plasma nitrite (P > 0.05).

Furthermore, it was found that seven subjects showed a small increase (<30%) in plasma nitrite after dietary supplementation with nitrate compared with placebo. For this reason, these subjects were classified as low responders (Table 2). The remaining six subjects were classified as high responders because of a greater increase in plasma nitrite concentration (>50%) after nitrate treatment. However, despite this fact, no statistical differences were found in exercise performance (distance and power output) parameters between both groups after nitrate and placebo treatments (Table 2). A small reduction, but not statistically significant, was found in V˙O2 (approximately 1.7%), V˙CO2 (approximately 3.4%), and ratio between oxygen consumption and power (approximately 1.1%) in the high-responder group during the 40-min distance-trial test after nitrate supplementation compared with placebo. In addition, blood lactate concentration showed a trend to decrease at 3 min postexercise after nitrate treatment in high responders (P = 0.076) (Table 2).

Table 2
Table 2
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Urinary nitrate (7624 ± 795 μM, P = 0.004) and nitrite (283 ± 122 nM, P = 0.004) excretion rose significantly after dietary nitrate supplementation compared with placebo in resting conditions (nitrate: 1299 ± 121 μM; nitrite: 111± 40 nM) (Fig. 2). After exercise, urinary nitrate excretion was also higher in nitrate condition (7046 ± 1354 μmol) compared with placebo (705 ± 165 μmol, P < 0.001). Percentage urinary nitrate losses with respect to supplemented dietary nitrate were 4.8% ± 1.9% and 3.7% ± 1.5% before and after exercise. Like plasma concentration, urinary nitrite showed a decrease after exercise test (188 ± 85 nM) compared with peak values at resting conditions (283 ± 102 nM) in nitrate treatment (Fig. 2). However, this decrease was not statistically significant (P = 0.174).

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Plasma concentration of ET-1 and 3-NT

In resting conditions, plasma ET-1 concentration did not differ between nitrate and placebo conditions (Fig. 3). However, a significant increase was shown just after exercise in placebo (P = 0.030) and nitrate (P < 0.001) groups compared with resting values. In addition, this effect was significantly (P = 0.010) greater in the nitrate group compared with placebo. On the other hand, plasma concentration of nitrated proteins was not affected by dietary nitrate supplementation in resting or postexercise conditions (Fig. 4). There were no differences in plasma concentration of ET-1 and 3-NT between high- and low-responder groups (Table 2).

Figure 3
Figure 3
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Figure 4
Figure 4
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DISCUSSION

The main finding of this study is that supplementation with sodium nitrate did not enhance the performance of endurance-trained athletes during a 40-min test. These results are contrary to the first hypothesis of this study. However, we confirmed the second hypothesis by showing that the ingestion of a moderate amount of sodium nitrate did not induce an increase of plasma concentration of nitrated protein just after exhaustive exercise in well-trained athletes. In addition, and in contrast to the third hypothesis, we found that the concentration of plasma ET-1 was significantly higher just after the exercise test in the nitrate condition compared with placebo.

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Effects of dietary inorganic nitrate supplementation on endurance performance

Several recent studies have reported that supplementation with beetroot can enhance exercise performance in healthy subjects (2,5,7,19,20,31,39,40) or in patients with cardiovascular disease (17) throughout different form of protocols such as time to exhaustion or time trials. However, the ergogenic effect of nitrate supplementation in well-trained endurance athletes remains uncertain (4). For instance, several studies supplementing endurance athletes for at least 4 d with pharmacological sodium nitrate have not reported an increase in exercise performance during incremental protocols until exhaustion (3,22,23). Furthermore, a recent study by Wilkerson et al. (41) showed that an acute dose of beetroot juice was not effective to enhance exercise performance during a 50-mile time-trial test in well-trained cyclists. The present results are in agreement with this study, indicating that the ingestion of sodium nitrate for 3 d did not show an ergogenic effect in a 40-min distance-trial test. However, in contrast with these results, another recent study by Cermak et al. (7) showed that 6 d of supplementation with beetroot juice was able to reduce the time required for cyclists to complete a time-trial test of 10 km.

In an attempt to explain the above discrepancies between studies, there are at least three factors that need to be discussed. The first is related to the duration of the nitrate supplementation. Although Cermak et al. (7) analyzed 6 d of supplementation with beetroot juice and found a significant increase in exercise performance, Wilkerson et al. (41) and the present study did not find such benefits after an acute dose of beetroot juice rich in nitrate and 3 d of sodium nitrate ingestion, respectively. Therefore, it could be suggested that trained subjects may need at least 1 wk of nitrate supplementation to induce some benefit in exercise performance.

The second factor is associated with the duration and intensity of the exercise test. These parameters are also an important key point because it is suggested that the nitrate–nitrite–NO pathway is mainly activated under anaerobic and acidic conditions (26). A recent study in mice has shown that dietary nitrate supplementation increased force production and calcium handling in fast-twitch muscles with a larger capacity to use anaerobic metabolism (13). In contrast, no effects were found in slow-twitch muscles with high aerobic capacity. Consistent with this fact, we found previously that in well-trained athletes, inorganic nitrate supplementation significantly decreased the ratio between oxygen consumption and power output only at maximal loads of exercise above their RCP (3). From this viewpoint, Cermak et al. (7) and Lansley et al. (19) found that a supplementation of beetroot juice rich in nitrate improved performance in a time-trial tests shorter than 30 min. In addition, another recent study by Bond et al. (5) has indicated that beetroot juice supplementation improved performance of rowers during a high-intensity intermittent test. On the contrary, the study by Wilkerson et al. (41) and the current study evaluated the effect of nitrate supplementation over a longer duration (>30 min). Accordingly, perhaps one confounding variable of these last studies might be the longer duration of the test. At exercise intensities below the RCP, the energy to sustain exercise is mainly provided by the aerobic system, and therefore, the nitrate–nitrite–NO pathway might not have been fully elicited. In addition, this fact could be more pronounced in well-trained endurance subjects compared with normal population because the increase in capillary density in skeletal muscle resulting from endurance training is likely to reduce the likelihood of developing hypoxic and/or acidic environment in the active muscle (15).

The third factor is related to the response to increase plasma nitrite after nitrate ingestion. This is an important issue because nitrite is a more sensitive marker of NO synthesis and provides a nitric oxide synthase (NOS)-independent source for NO generation (26). A recent study by Totzeck et al. (38) has shown that plasma nitrite concentration is related to exercise capacity in moderate trained subjects. This finding could also be related to an increase of nitrite throughout NOS activity because there is evidence that endurance exercise stimulates skeletal muscle NOS function in humans during exercise (24). Regarding the above studies with nitrate supplementation, study by Cermak et al. (7) did not analyze plasma levels of nitrite, and consequently, it is not possible to know whether all subjects responded equally to nitrate treatment. Interestingly, in this study, we found that seven subjects showed a low response (<30%) to increase plasma nitrite after nitrate load. Wilkerson et al. (41) also found that three of their eight subjects had a low response to increase plasma nitrite after nitrate supplementation. Perhaps this lower capacity to increase plasma nitrite concentration after nitrate ingestion could be related to some modification in the microflora of the oral cavity (12). Although the use of antibacterial mouthwash was discarded in this study because no subjects reported a regular use of these products, we found that all low responders were triathletes performing a considerable amount of training in the pool, whereas all high responders, except one, were cyclists who did not swim regularly. This fact suggests to us that a possible explanation for the low response showed by triathletes could be related to the presence of disinfectants such as chlorine in the water pool. Because water commonly enters the mouth during swimming, there is the possibility that the chlorine may interfere with the oral bacteria in a similar manner shown with antibacterial mouthwash (12). However, this is only a speculation and further studies are needed to analyze if regular training in chlorinated swimming pools can or cannot attenuate the conversion from nitrate to nitrite after the ingestion of supplement or food rich in nitrate.

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Effects of dietary inorganic nitrate supplementation on oxygen consumption during exercise

Different studies have reported that inorganic nitrate supplementation in the form of beetroot juice, as well as sodium nitrate supplementation, reduces the oxygen demands of moderately trained subjects during exercise at several intensities (2,20,22,23,39). Although the mechanism(s) behind this intriguing effect remains to be elucidated, two possible explanations have been reported recently, linking the dietary nitrate consumption with an improvement in muscle contractile efficiency (1) and in mitochondrial respiration (21). In well-trained athletes, we found previously that sodium nitrate supplementation significantly reduced oxygen consumption only at maximal loads of exercise (V˙O2peak) without significant changes at intensities below the RCP. Interestingly, the results of the current study were in agreement with our previous data because athletes performed exercise at an average intensity equivalent to RCP (approximately 85.1% of V˙O2peak), and no differences in oxygen consumption were found between treatments. In addition, although there was a trend, not statistically significant, toward a decrease in V˙O2, V˙CO2, and ratio between V˙O2 and power output after nitrate supplementation in the high-responder group, this effect was not associated with an increase in exercise performance (Table 2). These results were in agreement with the recent study by Wilkerson et al. (41), indicating no substantial change in the respiratory response of well-trained endurance athletes after nitrate treatment during a long time-trial test.

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Effects of dietary inorganic nitrate ingestion on blood concentration of 3-NT

In accordance with the second hypothesis of this study, a modest amount of dietary nitrate ingestion, which could also be achieved by ingestion of several nitrate-rich foods such as green leafy vegetables and beetroot juice, did not raise plasma levels of nitrated proteins at rest or after exhaustive exercise. Similar results were reported by two previous studies assessing the effect of dietary nitrate consumption on plasma nitrated protein levels in resting conditions (32,34). In addition, a recent study in mice has reported that an increase in nitrite bioavailability was associated with lower production of nitrotyrosine, superoxide production, and expression of nicotinamide adenine dinucleotide phosphate oxidase (37). However, no previous studies analyzed the effect of dietary nitrate ingestion on the formation of reactive nitrogen species (RNS) after exhaustive endurance exercise. This fact could be important because high-intensity endurance exercise, which induces an increase in muscle acidosis and nitrite, has been demonstrated to be involved in the formation of RNS under acidic conditions in vitro (33). In addition, there is also evidence that exhaustive exercise itself may increase the formation of RNS in athletes (35). Nevertheless, although we could not corroborate data in mice (37) because no differences were found between the low- and high-responder groups in both conditions, pre- and postexercise (Table 2), we found that the ingestion of moderate amounts of dietary inorganic nitrate did not alter plasma concentration of nitrated proteins in resting conditions and immediately after an intense exercise.

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Effects of dietary inorganic nitrate ingestion on blood concentration of ET-1

In contrast to our third hypothesis, plasma ET-1 levels rose significantly after exercise in the group supplemented with nitrate compared with the placebo group. There is evidence that acute exercise causes a tissue-specific change in the release of ET-1 (28). Studies by Maeda et al. (27,29) in humans exercising one leg showed that the concentration of ET-1 increased in the venous blood of the nonexercising leg, whereas it remained unchanged in the exercising one. Two endogenous mechanisms have been put forward to explain this response. The first is associated with the stimulus of shear stress induced by physical exercise. The release of ET-1 in cultured vascular endothelial cells is linked to low levels of shear stress, whereas higher levels of stress depress the release of this molecule (18,29). In the present study, athletes performed a cycle ergometer test exercising leg muscles; however, blood samples were taken from the antecubital vein. Thus, on the basis of studies by Maeda et al. (27,29), an increase in plasma ET-1 would be expected in the forearms just after exercise in both treatments (nitrate and placebo).

The second mechanism of ET-1 release is associated to neurohumoral factors, such as NO, prostacyclin, and arginine vasopressin, which may be released during exercise (36). From this viewpoint, there is evidence that nitrite induces vasodilation and increases the blood flow in humans (8). This response is mediated by hemoglobin in red blood cells, which sense hypoxia (9). Interestingly, using near-infrared spectroscopy device, Bailey et al. (2) showed that nitrate supplementation with beetroot juice was able to increase blood volume of active tissues during exercise, linking this fact with an increase of vasodilation via nitrite reduction to NO. Considering these data and the present results, we hypothesize that an increase in plasma ET-1 in nonactive tissues, derived from dietary nitrate ingestion, causes enhanced vascular tone and the consequent decrease in blood flow in these tissues, which could contribute to increasing blood flow in exercising muscles or in the lungs (8). Future studies are needed to corroborate or discard this hypothesis analyzing the arterial–venous gradient of ET-1 between the main exercising and the nonexercising muscles after ingestion of inorganic nitrate.

In conclusion, 3 d of dietary inorganic nitrate supplementation (10 mg·kg−1 of body mass) did not enhance performance during a 40-min distance-trial test in well-trained endurance athletes. Perhaps the duration of supplementation, as well as the duration of the exercise test performed in this study, might partially explain the more limited effect of sodium nitrate. Interestingly, this study confirmed that a moderate amount of inorganic nitrate ingestion did not stimulate an increase in plasma concentration of nitrated proteins after exhaustive exercise, indicating that this dose is safe. Furthermore, we found that nitrate supplementation induced a significant increase in plasma ET-1 concentration in forearms just after exercise. However, the mechanism behind this intriguing response remains to be elucidated.

This study was funded by the High Performance Center of Barcelona (CAR-Sant Cugat), the National Institute of Physical Education (INEFC), the Laboratory of Physical Activity Science of the University of the Balearic Islands, the Spanish Ministry of Health (DPS2008-07033-C03-03 and DPS2008-07033-C03-02), and the FEDER funds. Raúl Bescós was a PhD candidate supported by the University Department, Research and Information Society (AGAUR) of the Generalitat of Catalunya.

We are indebted to Dr. Joan Riera and Montse Banquells for their technical support during the study.

There is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

The results of the present study do not constitute an endorsement by the American College of Sports Medicine.

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Keywords:

NITRIC OXIDE; NITRATE; NITRITE; EXERCISE PERFORMANCE; ENDOTHELIN-1

©2012The American College of Sports Medicine

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