Background
A balanced and well-chosen nutrient intake is not only crucial for a healthy lifestyle, but also for optimal sport performance. There is a widespread use of dietary antioxidants, including vitamin E, resveratrol, beetroot, quercetin and cocoa flavanols (CF) in the athletic field. The main flavanols in cocoa are epicatechin, catechin (monomers) and procyanidins (oligomers). Their antioxidant (and in some situations perhaps pro-oxidant) effect is determined by the tricyclic structure of the flavanols. In vitro and in vivo studies clearly show that CF have a strong antioxidant capacity [
1,
2]. However, the potential role of chronic or acute CF intake to counteract exercise-induced oxidative stress and its effect on exercise performance is still unclear [
3‐
7]. Oxidative stress refers to the imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and/or molecular damage [
8]. During exhaustive exercise, NADPH oxidase-derived formation of reactive oxygen species (ROS) results in an altered redox state in the muscle [
9]. In contrast to the initial belief that nutritional antioxidant intake would be beneficial for exercise performance by protecting the body against oxidative stress, it is now hypothesized that exercise-induced oxidative stress promotes the expression of antioxidant defence and the adaptive responses to training [
10]. However, when exercise-induced ROS formation exceeds the antioxidant capacity, oxidative stress occurs with detrimental effects on proteins, lipids and DNA, possibly leading to contractile muscle dysfunction, accelerated muscle-fatigue and reduced exercise performance [
11]. Thus, at moments when optimal exercise performance is key and training adaptations are of minor importance (e.g. competition), acute intake of antioxidants may help scavenge free radicals and therefore directly prevent a decline in exercise performance and optimize post-exercise recovery [
9,
12].
In the recent years, CF have also been discovered as potent anti-inflammatory agents [
13]. Exhaustive exercise can cause inflammation [
14], resulting in a potential role for CF as a nutritional intervention to prevent exercise-induced muscle damage. Nevertheless, Allgrove et al. [
6] did not find any beneficial effect of 2 week CF (108 mg) intake on exercise-induced inflammatory markers.
Independent from its antioxidant and anti-inflammatory properties, CF are also known to stimulate vasodilation through increased nitric oxide (NO) availability [
15]. In vitro experiments demonstrated that CF lead to the inhibition of arginase, resulting in a greater L-Arginine availability, the substrate of endothelial NO synthase (eNOS) [
16]. In vitro [
17] and in vivo [
18,
19] experiments showed that CF intake activates eNOS, which converts L-Arginine, in the presence of molecular oxygen, to NO and citrulline. Besides its role in vasodilation, NO can also react at a diffusion-controlled rate with superoxide to form the highly reactive oxidant peroxynitrite. Consequently, by elevating NO production, CF might hypothetically have a pro-oxidant effect. However, because of its antioxidant capacity, CF can reduce ROS formation (e.g. superoxide) and repress the formation of peroxynitrite, thus elevating NO availability [
20]. Based on the fact that NO modulates blood flow and mitochondrial respiration during exercise [
21,
22], it has been suggested that increased NO production may enhance oxygen and nutrient delivery to active muscles, as well as improve mitochondrial efficiency, hence improving tolerance to physical exercise and recovery mechanisms [
21]. Although many athletes use supplements of NO because of their potential ergogenic role, the scientific evidence is very scarce [
21]. eNOS activity and thus NO production are stimulated by exercise-induced shear stress and thus strongly depend on training status [
23]. This suggests a minimal effect for CF and other nutritional supplements on eNOS activity and NO production in healthy trained athletes without vascular restrictions. Whether CF supplementation can alter NO production during exercise, and whether it may improve exercise performance and recovery in well-trained athletes, remains elusive [
5‐
7].
Therefore, the aims of this study were to examine the effects of CF intake on: i) indirect markers of eNOS dependent NO synthesis, L-arginine and citrulline, during exhaustive exercise ii) exercise-induced changes in antioxidant capacity, oxidative stress and inflammation, and iii) exercise performance and recovery in well-trained athletes. We hypothesized that CF intake i) will have little effect on indirect markers of NO production and exercise performance in these healthy, well-trained, subjects, but ii) will decrease exercise-induced oxidative stress and inflammation, leading to an improved recovery.
Discussion
This study aimed to examine the effect of CF intake on: i) indirect markers of eNOS-dependent NO production during exercise, ii) exercise-induced changes in antioxidant capacity, oxidative stress and inflammation, and iii) exercise performance and recovery.
Epicatechin, one of the main components of CFs, is at least partially associated with the observed improvements in NO synthesis and endothelial function [
30]. CF intake increased serum epicatechin concentrations reaching its peak concentration (approximately 450 nM) between 100 and 130 min after intake, exactly during the first exercise bout, i.e. TT1. We hypothesized that CF intake, through the subsequent increase in epicatechin, would increase NO production, which could influence TT performance. In this study, NOS-dependent NO production was indirectly estimated by measuring its substrate L-arginine/ADMA and its by-product citrulline [
31], because of our inability to directly assess NOS activity in our experimental model. L-arginine, a rate limiting factor for NO production, is converted into NO and citrulline by eNOS and arginine supplementation has been shown to affect the release of NO [
32]. As intracellular ADMA and arginine compete for NOS binding, ADMA and arginine levels regulate NO production [
33]. In addition, extracellular ADMA and arginine also compete for cell transport (CAT-2) [
34]. Thus, the higher the arginine/ADMA-ratio, the more arginine may be expected to be available as a substrate for eNOS to produce NO. Although it was shown in vitro that CF inhibits arginase activity, L-arginine/ADMA was not increased by acute CF intake in our study.
Exercise-induced shear stress is the principal physiological trigger for eNOS activation and NO production, promoting vasodilation and blood flow [
35]. Thus, in this population of well-trained cyclists, NO production is already optimized by their regular training [
23]. We examined whether acute CF intake could improve their exercise-induced NO production even more. Citrulline increased and L-arginine/ADMA decreased after both TTs, suggesting an exercise-induced increase in eNOS activity and NO production, but we did not observe an improved eNOS activation by CF as there were no differences in L-arginine/ADMA (eNOS substrate) and citrulline (byproduct) after CF intake, neither in rest, nor in response to exercise. However, recent in vitro [
17] and in vivo [
18,
19] experiments showed that CF intake activates eNOS and improved NO availability. Thus, untrained subjects or a clinical population suffering from reduced NO bioavailability, would probably be more likely to benefit from the CF-induced increase in NO synthesis [
22]. Moreover, a very recent pilot study of Taub et al. [
36] showed that chronic (3 months) intake of epicatechin-rich chocolate increased VO
2max and enhanced mitochondrial efficiency in healthy, but untrained people. Further research measuring NO production (e.g. nitrate, nitrite, nitrosospecies, peroxynitrite, eNOS activity), tissue haemodynamics (e.g. Flow Mediated Dilation, Magnetic Resonance Imaging, Doppler) and mitochondrial efficiency is needed to elucidate the exact role of CF on eNOS activation and NO production in combination with exercise.
In this study, time to complete TT1 was not influenced by CF intake, despite the small significant increase in relative power output at the end of TT1. Increases in RPE, lactate, HR and glucose during TT1 were not affected by CF intake. This suggests that acute CF intake has very limited ergogenic effects in well-trained cyclists. Comparing these results directly with similar studies is difficult because of different exercise protocols, as well as different doses, timing, exact composition of the CF extract and the food matrices of CF intake and placebo used. Davison et al. [
3] investigated the effects of the acute intake of dark chocolate containing 247 mg CF (of which 97 mg epicatechin) on physiological parameters during 2.5 h cycling at 60% of their VO
2max. Similar to our study, heartrate, RPE and respiratory exchange ratio (RER) during the exercise protocol were not affected by CF intake. Exercise performance, however, was not measured or reported in this study. Stellingwerff et al. [
7] similarly examined the effects of the acute intake of dark chocolate containing 240 mg CF (of which 89 mg epicatechin) on exercise performance, but used a different exercise protocol. Two hours after CF intake, subjects performed 2.5 h of steady state exercise at 45% of VO
2max, followed by a 15-min TT. The authors did not find any improvements in the 15-min TT performance. This might be explained by the timing of CF intake and the consequent peak of epicatechin in relation to the exercise protocol: the sum of all epicatechin metabolites reached its maximal concentration (approximately 700 nM) during the steady state exercise (after 190 min) and was already decreased by 65% at the start of the subsequent TT.
The second goal of this study was to examine the effects of acute CF intake on exercise-induced changes in antioxidant capacity, oxidative stress and inflammation and its possible implications for exercise recovery. Post-exercise recovery can be optimized by acute intake of antioxidants by scavenging free radicals [
9,
12]. Antioxidant capacity refers to the cumulative action of all antioxidants present in the plasma and is modulated by either radical overload or by exogenous antioxidant intake [
37]. UA, a strong scavenger of free radicals, contributes to more than 60% of the total plasma antioxidant capacity. However, UA is also upregulated by exercise as a result of an elevated energy-rich purine phosphate catabolism [
38]. Therefore, next to assessing the total antioxidant capacity of plasma as TEAC values, UA plasma concentrations were determined and TEAC/UA was assessed [
39]. Consistent with previous research which showed that acute exercise induces oxidative stress and antioxidant capacity [
8], TT1 and TT2 increased both TEAC/UA and UA plasma concentrations. In line with our hypothesis that CF intake enhances the antioxidant capacity, TEAC/UA and UA were significantly increased by CF intake, before and after exercise. In the study of Davison [
3], resting antioxidant capacity was also slightly increased by acute dark chocolate intake. Their exercise protocol (2.5 h cycling at 60% VO
2max) was longer and less intense than ours, but also increased TEAC values. However, in contrast to our results, they found that dark chocolate intake blunted the exercise-induced rise in antioxidant capacity. In the study of Allgrove [
6], a 2-week dark chocolate supplementation tended to increase TEAC at each time point, independently of exercise, which is consistent with our results. MDA, a by-product of lipid peroxidation, is the most frequently studied marker of oxidative stress during exercise [
39]. Although TT1 increased MDA plasma concentrations, this temporal change was not affected by CF intake. Davison [
3] and Allgrove [
6] used F2-isoprostanes as a marker of lipid peroxidation and found that acute and chronic chocolate intake lowered post-exercise plasma free F2-isoprostane levels. In the study of Wiswedel [
1], plasma F2-isoprostane concentrations increased after acute CF (187 mg) intake in rest, whereas plasma TEAC and MDA remained unaffected by CF intake. Thus, we cannot rule out that other biomarkers (such as F2-isoprostane) of oxidative stress might have been affected by CF intake in the present study. Although acute CFs intake upregulated the exercise-induced antioxidant capacity, the resultant oxidative stress (lipid peroxidation) was not decreased, suggesting that the small CF-induced increase in antioxidant capacity is not sufficient to counteract the increase in ROS formation during exhaustive exercise.
The TTs induced an increase in inflammatory cytokines IL-1 and IL-6, but not in TNF-α. Although anti-inflammatory properties of CF are well documented by in vitro and ex vivo studies [
15], acute intake of CF did not modify the exercise-induced inflammatory response. Similarly, Davison [
3] and Allgrove [
6] could not find any beneficial effects of acute and chronic dark chocolate intake on exercise-induced inflammatory markers. IL-6, next to its role as pro-inflammatory cytokine, also serves as a myokine produced by skeletal muscle in response to acute exercise to regulate muscle metabolism [
40]. Therefore, the inability of acute CF to blunt exercise-induced IL-6 elevations is not detrimental in this context. However, future research should focus on the effect of chronic CF intake on chronic diseases involving inflammation. Gastrointestinal, nervous, and cardiovascular diseases might benefit from chronic anti-inflammatory properties of CF [
41], as evidenced by a lowered TNF-α and IL-6 in type 2 diabetes patients following 6 weeks of CF supplementation [
42].
Given that CF intake did not reduce time to complete a second TT, exercise-induced inflammation and lipid peroxidation, a biomarker of oxidative stress, we failed to support the hypothesis that CF intake could enhance post-exercise recovery. However, the finding that acute CF intake increased total antioxidant capacity in response to exercise is promising. Future research should focus on its implications on a broader range of biomarkers of oxidative stress, metabolic and vascular parameters in healthy and diseased populations.