Effect of dietary nitrate supplementation on tolerance to supramaximal intensity intermittent exercise

Highlights

• Dietary nitrate increased tolerance to supramaximal intensity intermittent exercise.

• Red blood cells muscle concentration was increased in the working muscle.

• This occurred without changes in resting blood pressure and femoral artery blood flow.

• In contrast to lower intensity exercise, oxygen uptake was not reduced.

Abstract

Dietary nitrate $\left({\text{NO}}_3^{-}\right)$ supplementation has been shown to increase exercise tolerance and improve oxidative efficiency during aerobic exercise in healthy subjects. We tested the hypothesis that a 3-day supplementation in beetroot juice (BJ) rich in ${\text{NO}}_3^{-}$ would improve the tolerance to supramaximal intensity intermittent exercise consisting of 15-s exercise periods at 170% of the maximal aerobic power interspersed with 30-s passive recovery periods. The number of repetitions completed before reaching volitional exhaustion was significantly higher in the BJ than in the placebo condition (26.1 ± 10.7 versus 21.8 ± 8.0 respectively, P < 0.05). In contrast to previous findings during exercise performed at intensity below the peak oxygen uptake (VO_2peak), oxygen uptake (VO₂) was unaffected (BJ: 2735 ± 345 mL kg⁻¹ min⁻¹ vs. placebo: 2787 ± 346 mL kg⁻¹ min⁻¹, NS). However, the Area Under the Curve for microvascular total hemoglobin (AUC-THb) in the vastus lateralis muscle assessed by near infrared spectroscopy during 3 time-matched repetitions was significantly increased with ${\text{NO}}_3^{-}$ supplementation (BJ: 9662 ± 1228 a.u. vs. placebo:8178 ± 1589a.u.; P < 0.05). Thus, increased ${\text{NO}}_3^{-}$ (BJ: 421.5 ± 107.4 μM vs placebo:39.4 ± 18.0 μM) and ${\text{NO}}_2^{-}$ (BJ: 441 ± 184 nM vs placebo: 212 ± 119 nM) plasma levels (P < 0.001 for both) are associated with improved muscle microvascular Red Blood Cell (RBC) concentration and O₂ delivery during intense exercise, despite no effect on resting femoral artery blood flow, and vascular conductance. Maximal voluntary force during an isometric leg extensor exercise, and blood lactate levels were also unaffected by ${\text{NO}}_3^{-}$ supplementation.

To conclude, dietary ${\text{NO}}_3^{-}$ supplementation enhances tolerance to exercise at supramaximal intensity, with increased microvascular total RBC concentration in the working muscle, in the absence of effect on contractile function and resting hemodynamic parameters.

Introduction

Nitric oxide (NO) is a gaseous signaling molecule with a wide range of physiological effects, that include the regulation of skeletal muscle oxidative efficiency, contractile properties and blood flow [1], [2], [3]. Circulating NO is short lived and rapidly converted to nitrite $\left({\text{NO}}_2^{-}\right)$ and nitrate $\left({\text{NO}}_3^{-}\right)$, which is considered as the main body pool of NO [4], [5]. Besides NO synthesis from l-arginine and oxygen by the endothelial, neural and inducible isoforms of NO synthases, the ingestion of dietary inorganic nitrate $\left({\text{NO}}_3^{-}\right)$ is also able to significantly increase ${\text{NO}}_3^{-}$ and ${\text{NO}}_2^{-}$ plasma levels [3], [6], [7], [8]. Once ingested and absorbed from the gastrointestinal tract into the blood, approximately 25% of dietary ${\text{NO}}_3^{-}$ are taken up by the salivary gland, concentrated into the saliva, and converted to ${\text{NO}}_2^{-}$ by oral nitrate reductase bacteria [3]. Once swallowed, salivary ${\text{NO}}_2^{-}$ is either being reduced to NO and other nitrogen species in the acidic stomach or absorbed from the intestine [3], [9]. During exercise in the aerobic domain, supplementation with inorganic nitrate translates to effects such as increased time to exhaustion [6], higher work rate achievement [10] and remarkable decrease in whole body VO₂ for a given work rate [6], [11], [12]. Larsen et al. also clearly showed a tight relationship between the reduction of the oxygen cost of cycling exercise and the increase of the ratio of ATP produced to oxygen consumed in skeletal muscle mitochondria from subjects supplemented with sodium nitrate [2]. There are evidences supporting the inhibition of enzyme cytochrome c oxidase activity by NO and inhibition of uncoupled respiration as candidate factors responsible for the increased mitochondrial respiratory efficiency that prevent low O₂ availability from becoming a limiting factor to respiration [13]. In contrast, it is less evident to date that enhanced exercise performances can be mediated by the vasodilator effect of NO. Nitrite can be reduced to NO by various mechanisms involving deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, complexes of the mitochondrial transport chain, ascorbate, polyphenols and protons [3]. Among these mechanisms, vasodilation has been shown to particularly increase under hypoxic condition in response to enhance reduction of ${\text{NO}}_2^{-}$ to NO and other nitric oxide species by deoxyhemoglobin [5]. Similarly when intracellular pO₂ decreases in muscle cells, the enhanced conversion of ${\text{NO}}_2^{-}$ to NO by deoxymyoglobin increases the inhibition of mitochondrial respiration [4].

Athletes are frequently engaged in exercise of intermittent character, where the power output during working periods is above their power output at VO_max. During recovery periods, local muscle blood flow and O₂ delivery to skeletal muscle determine the rate of PCr resynthesis by oxidative phosphorylation and therefore the ability to repeat exercise bouts at high work rates [14], [15]. Considering the pronounced increase of deoxyhemoglobin [5] in regions of poor oxygenation [16], skeletal muscle could provide a enabling environment to the generation of NO generation from ${\text{NO}}_2^{-}$ during intense exercise. A facilitated NO generation from ${\text{NO}}_2^{-}$ in acidic condition at pH typically encountered in heavy working skeletal muscle is another factor that could increase NO availability [17]. Considering that the formation of NO occurs in a nitrite concentration-dependent manner at low pH levels, increasing circulating nitrite levels by a dietary nitrate supplementation may delay fatigue development by facilitating local O₂ supply to active skeletal muscle.

Altogether, these data suggest that tolerance to supramaximal intermittent exercise may be particularly sensible to NO bioavailability due to specific physicochemical conditions occurring with its achievement: important type II fibers recruitment (greater force production), ischemia and hypoxia (imbalance between O₂ demand and delivery), and intracellular acidosis (low muscle pH). However, previous studies that examined the effects of ${\text{NO}}_3^{-}$ supplementation during supramaximal exercise have yielded conflicting results, ranging from improved [18], to impaired performances without effects on oxygen uptake [19] during repeated sprints.

The aim of the present study was therefore to determine in young men the effects of a 3-day supplementation in beetroot juice rich in ${\text{NO}}_3^{-}$ on the tolerance to supramaximal intermittent exercise, oxygen uptake, the change of muscle microvascular THb concentration and oxygen extraction. Our hypotheses after dietary ${\text{NO}}_3^{-}$ supplementation were as follow: 1) tolerance to exercise would be significantly increased, 2) VO₂ during exercise would be significantly decreased, 3) and muscular O₂ delivery (characterized by microvascular RBC concentration) would be significantly increased during intermittent exercise.