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TO P I C A L R E V I E W

Redox interventions to increase exercise performance Michael B. Reid College of Health and Human Performance, University of Florida, Gainesville, FL 32611, USA

Exercise

The Journal of Physiology

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Abstract Skeletal muscle continually produces reactive oxygen species (ROS) and nitric oxide (NO) derivatives. Both oxidant cascades have complex effects on muscle contraction, metabolic function and tissue perfusion. Strenuous exercise increases oxidant production by muscle, limiting performance during endurance exercise tasks. Conversely, redox interventions that modulate ROS or NO activity have the potential to improve performance. Antioxidants have long been known to buffer ROS activity and lessen oxidative perturbations during exercise. The capacity to enhance human performance varies among antioxidant categories. Vitamins, provitamins and nutriceuticals often blunt oxidative changes at the biochemical level but do not enhance

Mike Reid is professor and dean of the College of Health and Human Performance at the University of Florida. His research focuses on the cellular and molecular mechanisms of muscle weakness with an emphasis on free radical biology and cytokine signalling.

C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

DOI: 10.1113/JP270653

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performance. In contrast, reduced thiol donors have been shown to delay fatigue or increase endurance under a variety of experimental conditions. Dietary nitrate supplementation has recently emerged as a second redox strategy for increasing endurance. Purified nitrate salts and nitrate-rich foods, notably beetroot and beetroot juice, are reported to lessen the oxygen cost of exercise, increase efficiency, and enhance performance during endurance tasks. These findings are exciting but enigmatic since nitrate per se has little bioactivity and cannot be converted to NO by mammalian cells. Overall, the available data suggest exercise endurance can be augmented by redox-active supplements, either reduced thiol donors or dietary nitrates. These findings have clear implications for athletes seeking a competitive edge. More importantly, interventions that increase endurance may benefit individuals whose physical activity is limited by illness, ageing, or frailty. (Received 5 July 2015; accepted after revision 21 October 2015; first published online 20 November 2015) Corresponding author M. B. Reid: College of Health and Human Performance; University of Florida; 1864 Stadium Rd, Suite 200; Gainesville, FL 32611, USA. Email: [email protected] Abstract figure legend Antioxidants, dietary nitrates and exercise performance. Continuous lines: biological actions that are promoted (positive symbol, arrowhead) or opposed (negative symbol, blunt end) by endogenous reactive oxygen species (ROS) and NO derivatives during exercise. Dashed lines: actions by which exogenous antioxidants and dietary nitrate may improve (positive symbol, arrow) or depress (negative symbol, blunt end) exercise performance. NAC, N-acetylcysteine; SOD, superoxide dismutase. Abbreviations cGMP, cyclic guanosine monophosphate; NAC, N-acetylcysteine; NO, nitric oxide; NO3 − , nitrate; NOS, NO synthase; ONOO− , peroxynitrite; PDE5, phosphodiesterase 5; PGC-1, peroxisome proliferator-activated receptor-γ coactivator 1; ROS, reactive oxygen species; SOD, superoxide dismutase.

Introduction

Since the middle of the last century, muscle biologists have recognized that skeletal muscle generates free radicals. Subsequent research has shown that skeletal muscle continually produces reactive oxygen species (ROS) and nitric oxide (NO) derivatives, two families of biologically active molecules that increase dramatically in muscle tissue during exercise. It was easy to imagine that these highly reactive redox species might have deleterious effects on muscle function and exercise performance. The obvious corollary – that antioxidant supplements might oppose oxidant effects and improve performance – was a siren call that lured many scientists into this field of research. The results have been extraordinary. In this review, I shall examine the basics of redox biology in skeletal muscle, the effects of antioxidant supplements in exercise, the emerging story of nitrate supplementation, and other redox strategies that may improve performance. Note that a comprehensive review of this field is beyond the scope of this article, which concisely summarizes key concepts. Similarly, it is not possible to cite a fraction of the original reports that underpin this area of science. Readers who find the topic interesting are encouraged to explore the literature more broadly.

Overview of redox biology in muscle

As reviewed in detail elsewhere (Jackson, 2011), ROS and NO derivatives are essential elements of the

physiological milieu in skeletal muscle. Both redox cascades are composed of small molecules, each comprising two to four atoms that readily undergo electron exchange reactions. These molecules generally have half-lives in the nanosecond to millisecond range and exist at very low concentrations in biological systems. ROS and NO derivatives undergo complex interactions, both within and between the redox cascades, and function as signalling molecules that frequently regulate the same target proteins. Despite these similarities, the two cascades are distinct in their intracellular origins and biological actions. Superoxide is parent molecule of the ROS cascade (Fig. 1) and may be generated at multiple sites in muscle tissue. These include semiquinones of the mitochondrial electron transport chain, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase and xanthine oxidase. Skeletal muscles of healthy mammals continually produce superoxide, hydrogen peroxide and hydroxyl radicals at low levels under resting conditions. Repetitive contractions increase ROS content in the cytosolic, interstitial and intravascular compartments, a rapid response that is detectable within seconds to minutes. ROS bioavailability is buffered by endogenous antioxidants in muscle. These include two ROS-specific enzymes: superoxide dismutase (SOD), which selectively degrades superoxide anions, and catalase, which selectively inactivates hydrogen peroxide. The major antioxidant pathway in muscle is the glutathione/glutathione peroxidase system which employs thiol cycling to buffer an array of oxidant C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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species. Despite these protective mechanisms, high rates of ROS production can exceed the antioxidant capacity of myofibres, promoting oxidative stress and depressing the force of muscle contraction. NO is produced by NO synthase (NOS), a family of enzymes that are constitutively expressed by skeletal muscle. These include the neuronal-type NOS (nNOS, NOSI) isoform that localizes to the subsarcolemmal region via association with the dystrophin complex, and the endothelial-type NOS isoform (eNOS, NOSIII) that co-localizes with muscle mitochondria. In the presence of oxygen and appropriate co-factors, calcium activation stimulates either nNOS or eNOS to synthesize NO by metabolizing L-arginine to L-citrulline. NO is a potent signalling molecule that regulates target proteins via two general mechanisms: (1) direct redox modification of regulatory thiols or transition metal centres, and (2) indirect cyclic guanosine monophosphate (cGMP)-mediated signalling. Redox derivatives of NO include peroxynitrite (ONOO− ), the product of spontaneous NO reaction with superoxide, and nitrate (NO3 − ), which is an end-product of the NO cascade and is biologically inactive in mammalian systems. Like ROS, NO derivatives are present at low concentrations under resting conditions, at higher levels during contraction, and distribute across all tissue compartments. NO derivatives are buffered by reduced sulfhydryl moieties, notably

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glutathione and protein thiols, and by haem proteins such as myoglobin and haemoglobin. Muscle-derived ROS and NO have complex, multifocal effects on skeletal muscle that may influence exercise performance. First, the force of contraction is depressed by elevated levels of either ROS or NO. These robust responses are largely attributable to changes in myofibrillar calcium sensitivity. Thus, increased production of ROS and NO by exercising muscle has the potential to depress force via redox mechanisms, contributing to fatigue. It is clear that ROS play such a role. Research using selective pharmacological probes has shown that depletion of muscle-derived ROS inhibits fatigue of electrically paced muscle preparations both in vitro and in situ. These findings confirm a causal role for ROS in the fatigue process. The physiological importance of NO is less clear. Animal studies show that muscle is more susceptible to fatigue if nNOS expression or localization is disrupted, either by disease or genetic manipulation. However, muscle structure and function are profoundly abnormal in these animals, obscuring any role that NO may play in the fatigue process. Well-designed studies of healthy animals have yielded conflicting results. Pharmacological blockade of NO synthesis has been shown to either promote or oppose fatigue. Thus, the physiological importance of NO in fatiguing exercise remains a vexed question. Beyond direct effects on contraction, ROS and NO may influence exercise performance by other mechanisms. Mitochondrial respiration is sensitive to both redox cascades and contraction-induced glucose uptake is regulated in part by NO signalling. Vascular regulation during exercise is modulated by NO derivatives that promote vasodilatation in working muscle. ROS are thought to oppose vasodilatation by scavenging NO derivatives, a paracrine yang to the yin of NO. This classical concept is challenged by new evidence that hydrogen peroxide functions as a vasodilator in exercising muscle of trained individuals (Durand et al. 2015). Finally, muscle-derived ROS and NO modulate afferent receptors in working muscle that send afferent traffic to the central nervous system. This can influence somatosensory and cardiovascular reflexes that influence fatigue and can limit the performance of volitional exercise. Antioxidants and exercise performance

GSH

2 H2O + GSSG

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Figure 1. Reactive oxygen cascade Diagram depicts interactions among reactive oxygen species (ROS) detected in skeletal muscle and selected mediators of individual reactions. SQ•− , semiquinone; O2 •− , superoxide anion; • NO, nitric oxide; ONOO− , peroxynitrite; e− , free electron; H2 O2 , hydrogen peroxide; Fe2+ , ferrous iron; • OH, hydroxyl radical; GSH, reduced glutathione; GSSG, oxidized glutathione. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

Since the discovery that strenuous contraction increases free radicals in skeletal muscle (Jackson et al. 1985) there has been widespread interest in the capacity of antioxidant supplements to improve exercise performance. A recent PubMed search using the search terms ‘exercise’ and ‘antioxidants’ identified over 5200 articles over the ensuing 30 years, a 27-fold increase over the previous three decades (Fig. 2). These publications reflect a massive amount of research conducted by thousands of committed scientists.

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The findings are fascinating, hugely informative, but there is a caveat before we proceed. The term ‘antioxidant’ is a gross simplification that can be misleading. Each of the familiar antioxidant categories – vitamins and provitamins, thiol donors, polyphenols, antioxidant enzymes, etc. – contains families of molecules that vary in their physical and chemical properties. Individual antioxidants differ in their mechanisms of action, redox affinities, bioavailabilities, intracellular distributions and dose dependence. Also, antioxidants administered in combination can interact with one another, complicating the net effect of antioxidant ‘cocktails’ in biological systems. Finally, we have almost no specific data on the uptake, metabolism and mechanism of action of individual antioxidants in skeletal muscle. These limitations constrain the rational design of experiments and must temper our interpretation of results. With these boundary conditions in place, we can explore three basic tenants of antioxidants and exercise. First, many antioxidants do not improve exercise performance. This generalization holds for nutritional antioxidants that include vitamin C (ascorbate, L-ascorbic acid), vitamin E (α-tocopherol), and vitamin A (retinol, retinoic acid, β-carotene), resveratrol, coenzyme Q10, quercitin, and other less-studied compounds. Absent disease or a pre-existing vitamin deficiency, there is little evidence that high-dose supplementation with nutritional antioxidants improves performance of either strength or endurance exercise tasks. These molecules are not ergogenic despite their capacities to increase antioxidant markers in the plasma, circulating cell types, or peripheral tissues. Nutritional antioxidants may also blunt biomarkers for oxidative stress during exercise, e.g. glutathione oxidation or depletion, malondialdehyde or other lipid peroxidation markers, protein carbonyl formation, nitrotyrosine accumulation, etc. However, exercise performance is usually unaffected and may be

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impaired but is almost never improved. Readers who seek more detailed information on nutritional antioxidants and exercise may wish to examine the recent monograph by Wagner (2015). Second, reduced thiol donors can improve exercise endurance. This category of antioxidants is characterized by reduced sulfhydryl moieties that directly quench ROS and NO derivatives and participate in thiol-disulfide exchange reactions. The most fundamental molecules in this category, e.g. cysteine and glutathione, have been shown to limit fatigue but are poorly tolerated upon systemic administration and have limited bioavailability as supplements. Other reduced thiol donors have not been evaluated extensively, e.g. L-ergothioneine, N-acetylcysteine amide, and L-2-oxothiazolidine-4-carboxylate. In contrast, the drug N-acetylcysteine (NAC) effectively opposes oxidative stress when administered systemically and is approved for use in humans. NAC has its limitations in the laboratory. As with many sulfur-rich compounds, the NAC solution smells strongly of rotten eggs and can have mild side effects at pharmacological doses. These traits complicate the design of experiments using NAC, especially in humans. Nevertheless, NAC has proven to be a valuable experimental tool both for basic and translational research. NAC was the first antioxidant shown to inhibit experimental muscle fatigue, a discovery made using auto-perfused neuromuscular preparations in anaesthetized rabbits (Shindoh et al. 1990). Incubation with NAC delayed fatigue of isolated muscle preparations in vitro, independent of blood flow or neural activation, demonstrating that muscle-derived oxidants act within myofibres to promote fatigue at the cellular level (Diaz et al. 1994; Khawli & Reid, 1994). In healthy volunteers, NAC infusion inhibited fatigue of tibialis anterior muscle evoked by repetitive transcutaneous stimulation

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Figure 2. Annual rates of publications on exercise and antioxidants Bars depict numbers of reports published in individual years; arrow highlights the year exercise was shown to increase free radical content in muscle by Jackson et al. (1985); data obtained from PubMed search using terms ‘exercise’ and ‘antioxidants’ performed in June 2015.


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(Reid et al. 1994). Such findings confirm that oxidative stress plays a causal role in fatigue of skeletal muscle, both in quadrupeds and healthy humans. The capacity of NAC to inhibit experimental fatigue depends on the intensity of muscle contraction. NAC is effective at submaximal, quasi-physiological stimulation frequencies but not during near-maximal contractions (Khawli & Reid, 1994; Reid et al. 1994). The cellular mechanisms that depress force differ between these two conditions. This suggests that oxidants contribute to lowbut not high-frequency fatigue. Also, NAC does not alter short-term recovery of force immediately after muscle fatigue. This has been observed in rodent and human studies, in studies of limb and trunk muscles, and in experiments conducted in vitro, in situ and in vivo. This robust finding argues that oxidants do not influence recovery from fatigue. NAC effects on human endurance have been evaluated using a broad array of volitional exercise protocols. In studies of small muscle groups, NAC has lessened markers of oxidative stress and increased endurance during hand-grip exercise (Matuszczak et al. 2005), resistive loading of the diaphragm (Travaline et al. 1997), and respiratory muscle fatigue caused by heavy exercise (Kelly et al. 2009; Smith et al. 2014). NAC pretreatment also increased quadriceps endurance in sedentary men (Leelarungrayub et al. 2011) and individuals with chronic obstructive lung disease (Koechlin et al. 2004). Of broadest importance, NAC has been shown to increase endurance during whole-body exercise. This was first demonstrated during prolonged cycling exercise by endurance athletes; NAC infusion extended the time to task failure (Medved et al. 2004). This finding was replicated in two separate studies of well-trained individuals (McKenna et al. 2006; Slattery et al. 2014). Beyond cycling, NAC has also been shown to delay fatigue during repeated bouts of damaging intermittent exercise by recreationally trained men (Cobley et al. 2011). Interestingly, direct comparisons within subjects suggest that NAC extends time to task failure during exercise at 80% peak power but not at near-maximal levels (Corn & Barstow, 2011), a finding similar to NAC effects on experimental fatigue (above). Studies using NAC also shed light on other aspects of human exercise physiology. During fatiguing exercise, NAC effects suggest that elevated oxidants disrupt potassium regulation, depressing activity of the skeletal muscle Na+ /K+ pump (McKenna et al. 2006) and increasing plasma potassium concentration (Medved et al. 2004). NAC studies also suggest that exercise-associated oxidants limit the bioavailability of circulating nitric oxide, restrict blood flow to the periphery, and depress changes in haematological indices including haemoglobin content, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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and erythropoietin concentration. In aggregate, NAC effects on potassium regulation and perfusion identify two mechanisms by which oxidative stress might lessen exercise endurance. Metabolic regulation during exercise may also be redox sensitive. During high-intensity interval training, NAC effects suggest that elevated oxidants depress fat oxidation and circulating glucose while increasing circulating lactate. In contrast, oxidants do not obviously affect glucose disposal during moderate exercise. Third, antioxidants of all categories can lessen the benefits of exercise. The familiar dichotomy that free radicals are bad and antioxidants are good is pervasive in popular culture. It has fuelled widespread appetite for antioxidant-rich foods and antioxidant supplements. Nowhere has this been more true than at the training table, where athletes have loaded up in the belief that antioxidants improve performance. But a growing body of literature suggests otherwise. Muscle recovery from damaging exercise is mediated in part by oxidant signalling. Antioxidants can blunt these signalling events and the repair mechanisms they regulate. For example, following intense eccentric contractions, NAC administration to healthy men delayed recovery of muscle function and disrupted mechanisms that regulate muscle repair (Michailidis et al. 2013); the latter included an array of signalling events (phosphorylation of protein kinase B, mammalian target of rapamycin, p70 ribosomal S6 kinase, ribosomal protein S6, mitogen-activated protein kinase p38) and the mRNA expression for regulatory proteins (myogenic determination factor, tumor necrosis factor). These findings are consistent with earlier observations that ascorbic acid supplementation inhibited recovery of muscle function after downhill running (Close et al. 2006), a cocktail of nutritional and reduced thiol antioxidants prolonged the elevation of serum creatine kinase levels after kayak competition (Teixeira et al. 2009), and NAC administration prolonged interleukin-10 elevation after eccentric exercise (Silva et al. 2008). Training responses to exercise can also be depressed by antioxidant supplements. For example, administration of high-dose vitamin C during endurance training significantly blunted effects on performance (Gomez-Cabrera et al. 2008). In the same study, muscle analyses showed lower mRNA levels for transcription factors that regulate mitochondrial biogenesis (peroxisome proliferator-activated receptor co-activator 1, nuclear factor 1, mitochondrial transcription factor A), an electron transport chain component (cytochrome c), and antioxidant enzymes (SOD, glutathione peroxidase). In a separate study, healthy young men took vitamins C and E during a programme of physical training; the antioxidants abolished the increase in insulin sensitivity caused by training and

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the upregulation of training-responsive gene products, including peroxisome-proliferator-activated receptor γ coactivator 1α (PGC-1α), PGC-1β, MnSOD, CuZnSOD and glutathione peroxidase (Teixeira et al. 2009). And in older, mildly hypertensive men, consumption of vitamin C, vitamin E and α-lipoic acid abolished the beneficial effects of exercise training on blood pressure and flow-mediated vasodilatation (Silva et al. 2008). Even a single bout of exercise can reveal negative effects of high-dose antioxidants. Pretreatment with antioxidant vitamins have been shown to depress exercise-induced vasodilatation (Richardson et al. 2007) and inhibit post-exercise increases in heat shock protein 70 (Khassaf et al. 2003) and heat shock protein 72 (Fischer et al. 2006). Similarly, NAC pretreatment has blunted the rise in MnSOD mRNA, the phosphorylation of both jun-N-terminal kinase and nuclear factor κB (Petersen et al. 2012), and the upregulation of Na+ /K+ pump mRNA (Murphy et al. 2008). Dietary nitrates and endurance exercise

As recently reviewed by Jones (2014), dietary nitrate supplements can increase exercise efficiency and improve endurance performance. Typically, nitrate supplements are consumed orally as nitrate-rich beetroot, beetroot juice, or as sodium nitrate per se. The surprising capacity of nitrate supplements to lower oxygen consumption in exercising humans was first observed by Larsen et al. (2007) and has since been confirmed by numerous studies. The mechanism of this action, which increases exercise efficiency, remains the focus of intense research. Possible mechanisms include greater ATP supply via altered mitochondrial respiration and more efficient ATP utilization during muscle contraction. The latter concept is consistent with the discovery of Hernandez et al. (2012) that nitrate supplementation for 7 days increases calcium release during contraction of fast-type murine muscle fibres. This enhances the force developed at activation frequencies ࣘ50 Hz and shifts the force–frequency L-Arginine

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relationship leftward: changes that are predicted to lessen motor unit recruitment for a given exercise task. In parallel, Bailey et al. (2009) showed that nitrate supplementation increases exercise endurance during high-intensity cycling. This triggered a flurry of reports that nitrate loading can increase endurance during cycling, rowing, running, kayaking and team sport-specific exercise. Nitrate efficacy appears to be dose- and time-dependent and can vary according to exercise intensity, fitness level and age. Accordingly, nitrate supplementation does not improve exercise performance under all conditions. Larsen et al. (2007) originally proposed that nitrate supplements influence exercise by increasing the bioavailability of NO derivatives. Nitrate consumption is thought to promote NO signalling via the ‘nitrate–nitrite–NO pathway’ (Fig. 3). The first step in this pathway poses a redox enigma. Nitrate conversion to nitrite is catalysed by nitrate reductase, an enzyme that is not expressed by mammalian cells. If so, how might humans metabolize dietary nitrate? The dominant hypothesis is that buccal flora convert nitrate to nitrite via bacterial nitrate reductase. The nitrite is then absorbed in the gastrointestinal tract, accounting for the rapid rise in plasma nitrite levels seen in subjects following nitrate consumption. Circulating nitrite is readily converted to NO, a reaction facilitated by deoxymyoglobin, deoxyhaemoglobin, xanthine oxidase and an acidic environment. These factors are typical of the microvascular milieu in exercising muscle. Thus, increased nitrite delivery creates a source of bioactive NO that may be generated in response to local metabolic demand. This is proposed to increase exercise performance by augmenting oxygen delivery (Masschelein et al. 2012), increasing energetic efficiency (Larsen et al. 2011), and increasing the force of contraction (Haider & Folland, 2014). Beyond the working muscle, nitrate supplements may also influence performance via effects on the central nervous system. Nitrates appear to modulate aspects of cortical function that affect motor control and can limit

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Figure 3. Nitric oxide and dietary nitrate Left to right: canonical pathway for enzymatic synthesis and redox degradation of nitric oxide (• NO) to nitrite (NO2 − ) and nitrate (NO3 − ). Right to left: proposed pathway for conversion of dietary NO3 − to • NO. NOS, NO synthase; Hgb, haemoglobin; Mgb, myoglobin. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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exercise endurance. These include cognitive function and perceived exertion (Thompson et al. 2014) as well as apnoeic duration (Engan et al. 2012). Other redox interventions

Alternative redox strategies for enhancing exercise performance have largely focused on NO signalling via cGMP. The canonical NOS-regulated pathway has been selectively targeted by L-arginine supplementation. This approach provides exogenous substrate for NO synthesis with the goal of stimulating NO production. The rationale is that greater bioactivity will enhance NO-sensitive processes that can limit exercise performance, e.g. blood flow, mitochondrial metabolism and muscle protein synthesis. An early report by Maxwell et al. (2001) indicated that L-arginine supplementation increases NO production and aerobic exercise capacity in mice. This set the stage for human studies that have yielded conflicting results. For example, Bailey et al. (2010) found that L-arginine supplementation increased performance of high-intensity exercise while decreasing the oxygen cost of exercise. A more recent study by the same group (Bailey et al. 2015) showed that L-citrulline, an L-arginine derivative, improved tolerance to severe intensity exercise and increased total work performance whereas L-arginine had no ergogenic effect. Nor did L-arginine improve performance in a different study by the same research team (Vanhatalo et al. 2013) or in tests of endurance and sprint performance of elite athletes by other investigators (Sandbakk et al. 2014). During resistance exercise, L-arginine does not appear to alter muscle blood flow or protein synthesis and does not increase muscle strength. Such inconsistencies suggest the ergogenic benefits of systemic L-arginine administration are not robust. Downstream interventions may promote NO signalling more effectively. Activity of the NO/cGMP pathway can be enhanced by pharmacological blockade of phosphodiesterase 5 (PDE5), the enzyme that degrades cGMP. Two commercial PDE5 inhibitors are approved for human use (sildenafil/Viagra; tadalafil/Cialis) and have been tested in healthy subjects for exercise-related actions. The strongest evidence that PDE5 inhibition might improve performance is from Sheffield-Moore et al. (2013) who showed that short-term, daily administration of sildenafil increases muscle protein synthesis and reduces muscle fatigue. Consistent with this finding, tadalafil has been shown to increase time-to-peak power during anaerobic testing (Guidetti et al. 2008) and sildenafil improved time-trial performance during cycling in normobaric hypoxia (Hsu et al. 2006). However, cycling performance was not altered by PDE5 inhibitors in at least one study of healthy subjects under normoxic conditions (Di Luigi et al. 2008). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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Conclusion

Thiol antioxidants and dietary nitrates appear to improve performance during endurance exercise. Both are safe for human use at laboratory doses. Both are effective as a single dose taken shortly before exercise. And both are thought to work via distinct redox mechanisms. Thiol antioxidants appear to preserve contractile function of myofibres by buffering exercise-induced ROS. Dietary nitrates increase exercise efficiency by augmenting NO actions on mitochondrial metabolism, blood flow distribution, or both. However, important issues remain unresolved. Thiol antioxidants are not practical for widespread use. The archetype NAC stinks of rotten eggs and is only effective at pharmacological doses, limiting use to research laboratories and clinical settings. Dietary nitrates such as beetroot and beetroot juice are freely available but poorly understood. Their purported mechanism of action is largely speculative and virtually nothing is known about long-term use. If taken together, NAC and dietary nitrates have the potential to interact with each other. Reduced thiols react directly with nitric oxide, altering bioactivity in a complex manner that has not been studied in exercise and cannot be predicted. Combining NAC plus nitrates might yield additive effects, improving exercise performance more than either supplement alone. Or the two types of compounds might inactivate one another and abolish the performance benefit altogether. This field is at a critical crossroads. Robust proofs-of-concept exist for thiol antioxidants and dietary nitrates as ergogenic aids that might benefit athletes, the frail and the ill. However, well-defined questions limit practical use of such supplements. A growing community of scientists is pursuing new research to solve these problems so stay tuned. The next few years should be exciting. References Bailey SJ, Varnham RL, DiMenna FJ, Breese BC, Wylie LJ & Jones AM (2015). Inorganic nitrate supplementation improves muscle oxygenation, O2 uptake kinetics, and exercise tolerance at high but not low pedal rates. J Appl Physiol (1985) 118, 1396–1405. Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N & Jones AM (2009). Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol (1985) 107, 1144–1155. Bailey SJ, Winyard PG, Vanhatalo A, Blackwell JR, DiMenna FJ, Wilkerson DP & Jones AM (2010). Acute L-arginine supplementation reduces the O2 cost of moderate-intensity exercise and enhances high-intensity exercise tolerance. J Appl Physiol (1985) 109, 1394–1403.

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Close GL, Ashton T, Cable T, Doran D, Holloway C, McArdle F & MacLaren DP (2006). Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process. Br J Nutr 95, 976–981. Cobley JN, McGlory C, Morton JP & Close GL (2011). N-Acetylcysteine’s attenuation of fatigue after repeated bouts of intermittent exercise: practical implications for tournament situations. Int J Sport Nutr Exerc Metab 21, 451–461. Corn SD & Barstow TJ (2011). Effects of oral N-acetylcysteine on fatigue, critical power, and W’ in exercising humans. Respir Physiol Neurobiol 178, 261–268. Di Luigi L, Baldari C, Pigozzi F, Emerenziani GP, Gallotta MC, Iellamo F, Ciminelli E, Sgro P, Romanelli F, Lenzi A & Guidetti L (2008). The long-acting phosphodiesterase inhibitor tadalafil does not influence athletes’ VO2max , aerobic, and anaerobic thresholds in normoxia. Int J Sports Med 29, 110–115. Diaz PT, Brownstein E & Clanton TL (1994). Effects of N-acetylcysteine on in vitro diaphragm function are temperature dependent. J Appl Physiol (1985) 77, 2434–2439. Durand MJ, Dharmashankar K, Bian JT, Das E, Vidovich M, Gutterman DD & Phillips SA (2015). Acute exertion elicits a H2 O2 -dependent vasodilator mechanism in the microvasculature of exercise-trained but not sedentary adults. Hypertension 65, 140–145. Engan HK, Jones AM, Ehrenberg F & Schagatay E (2012). Acute dietary nitrate supplementation improves dry static apnea performance. Respir Physiol Neurobiol 182, 53–59. Fischer CP, Hiscock NJ, Basu S, Vessby B, Kallner A, Sjoberg LB, Febbraio MA & Pedersen BK (2006). Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans. J Appl Physiol (1985) 100, 1679–1687. Gomez-Cabrera MC, Domenech E, Romagnoli M, Arduini A, Borras C, Pallardo FV, Sastre J & Vina J (2008). Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance. Am J Clin Nutr 87, 142–149. Guidetti L, Emerenziani GP, Gallotta MC, Pigozzi F, Di Luigi L & Baldari C (2008). Effect of tadalafil on anaerobic performance indices in healthy athletes. Br J Sports Med 42, 130–133. Haider G & Folland JP (2014). Nitrate supplementation enhances the contractile properties of human skeletal muscle. Med Sci Sports 46, 2234–2243. Hernandez A, Schiffer TA, Ivarsson N, Cheng AJ, Bruton JD, Lundberg JO, Weitzberg E & Westerblad H (2012). Dietary nitrate increases tetanic [Ca2+ ]i and contractile force in mouse fast-twitch muscle. J Physiol 590, 3575–3583. Hsu AR, Barnholt KE, Grundmann NK, Lin JH, McCallum SW & Friedlander AL (2006). Sildenafil improves cardiac output and exercise performance during acute hypoxia, but not normoxia. J Appl Physiol (1985) 100, 2031–2040. Jackson MJ (2011). Control of reactive oxygen species production in contracting skeletal muscle. Antioxid Redox Signal 15, 2477–2486. Jackson MJ, Edwards RH & Symons MC (1985). Electron spin resonance studies of intact mammalian skeletal muscle. Biochim Biophys Acta 847, 185–190.

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Jones AM (2014). Dietary nitrate supplementation and exercise performance. Sports Med 44, Suppl. 1, S35–45. Kelly MK, Wicker RJ, Barstow TJ & Harms CA (2009). Effects of N-acetylcysteine on respiratory muscle fatigue during heavy exercise. Respir Physiol Neurobiol 165, 67–72. Khassaf M, McArdle A, Esanu C, Vasilaki A, McArdle F, Griffiths RD, Brodie DA & Jackson MJ (2003). Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J Physiol 549, 645–652. Khawli FA & Reid MB (1994). N-acetylcysteine depresses contractile function and inhibits fatigue of diaphragm in vitro. J Appl Physiol (1985) 77, 317–324. Koechlin C, Couillard A, Simar D, Cristol JP, Bellet H, Hayot M & Prefaut C (2004). Does oxidative stress alter quadriceps endurance in chronic obstructive pulmonary disease? Am J Respir Crit Care Med 169, 1022–1027. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO & Weitzberg E (2011). Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab 13, 149–159. Larsen FJ, Weitzberg E, Lundberg JO & Ekblom B (2007). Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol (Oxf) 191, 59–66. Leelarungrayub D, Khansuwan R, Pothongsunun P & Klaphajone J (2011). N-Acetylcysteine supplementation controls total antioxidant capacity, creatine kinase, lactate, and tumor necrotic factor-alpha against oxidative stress induced by graded exercise in sedentary men. Oxid Med Cell Longev 2011, 329643. McKenna MJ, Medved I, Goodman CA, Brown MJ, Bjorksten AR, Murphy KT, Petersen AC, Sostaric S & Gong X (2006). N-Acetylcysteine attenuates the decline in muscle Na+ ,K+ -pump activity and delays fatigue during prolonged exercise in humans. J Physiol 576, 279–288. Masschelein E, Van Thienen R, Wang X, Van Schepdael A, Thomis M & Hespel P (2012). Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in hypoxia. J Appl Physiol (1985) 113, 736–745. Matuszczak Y, Farid M, Jones J, Lansdowne S, Smith MA, Taylor AA & Reid MB (2005). Effects of N-acetylcysteine on glutathione oxidation and fatigue during handgrip exercise. Muscle Nerve 32, 633–638. Maxwell AJ, Ho HV, Le CQ, Lin PS, Bernstein D & Cooke JP (2001). L-Arginine enhances aerobic exercise capacity in association with augmented nitric oxide production. J Appl Physiol (1985) 90, 933–938. Medved I, Brown MJ, Bjorksten AR, Murphy KT, Petersen AC, Sostaric S, Gong X & McKenna MJ (2004). N-acetylcysteine enhances muscle cysteine and glutathione availability and attenuates fatigue during prolonged exercise in endurance-trained individuals. J Appl Physiol (1985) 97, 1477–1485. Michailidis Y, Karagounis LG, Terzis G, Jamurtas AZ, Spengos K, Tsoukas D, Chatzinikolaou A, Mandalidis D, Stefanetti RJ, Papassotiriou I, Athanasopoulos S, Hawley JA, Russell AP & Fatouros IG (2013). Thiol-based antioxidant supplementation alters human skeletal muscle signaling and attenuates its inflammatory response and recovery after intense eccentric exercise. Am J Clin Nutr 98, 233–245. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

J Physiol 594.18


Murphy KT, Medved I, Brown MJ, Cameron-Smith D & McKenna MJ (2008). Antioxidant treatment with N-acetylcysteine regulates mammalian skeletal muscle Na+ -K+ -ATPase α gene expression during repeated contractions. Exp Physiol 93, 1239–1248. Petersen AC, McKenna MJ, Medved I, Murphy KT, Brown MJ, Della Gatta P & Cameron-Smith D (2012). Infusion with the antioxidant N-acetylcysteine attenuates early adaptive responses to exercise in human skeletal muscle. Acta Physiol (Oxf) 204, 382–392. Reid MB, Stokic DS, Koch SM, Khawli FA & Leis AA (1994). N-acetylcysteine inhibits muscle fatigue in humans. J Clin Invest 94, 2468–2474. Richardson RS, Donato AJ, Uberoi A, Wray DW, Lawrenson L, Nishiyama S & Bailey DM (2007). Exercise-induced brachial artery vasodilation: Role of free radicals. Am J Physiol Heart Circ Physiol 292, H1516–H1522. Sandbakk SB, Sandbakk O, Peacock O, James P, Welde B, Stokes K, Bohlke N & Tjonna AE (2014). Effects of acute supplementation of L-arginine and nitrate on endurance and sprint performance in elite athletes. Nitric Oxide 48, 10–15. Sheffield-Moore M, Wiktorowicz JE, Soman KV, Danesi CP, Kinsky MP, Dillon EL, Randolph KM, Casperson SL, Gore DC, Horstman AM, Lynch JP, Doucet BM, Mettler JA, Ryder JW, Ploutz-Snyder LL, Hsu JW, Jahoor F, Jennings K, White GR, McCammon SD & Durham WJ (2013). Sildenafil increases muscle protein synthesis and reduces muscle fatigue. Clin Transl Sci 6, 463–468. Shindoh C, DiMarco A, Thomas A, Manubay P & Supinski G (1990). Effect of N-acetylcysteine on diaphragm fatigue. J Appl Physiol (1985) 68, 2107–2113. Silva LA, Silveira PC, Pinho CA, Tuon T, Dal Pizzol F & Pinho RA (2008). N-acetylcysteine supplementation and oxidative damage and inflammatory response after eccentric exercise. Int J Sport Nutr Exerc Metab 18, 379–388. Slattery KM, Dascombe B, Wallace LK, Bentley DJ & Coutts AJ (2014). Effect of N-acetylcysteine on cycling performance after intensified training. Med Sci Sports 46, 1114–1123.


5133

Smith JR, Ade CJ, Broxterman RM, Skutnik BC, Barstow TJ, Wong BJ & Harms CA (2014). Influence of exercise intensity on respiratory muscle fatigue and brachial artery blood flow during cycling exercise. Eur J Appl Physiol 114, 1767–1777. Teixeira VH, Valente HF, Casal SI, Marques AF & Moreira PA (2009). Antioxidants do not prevent postexercise peroxidation and may delay muscle recovery. Med Sci Sports 41, 1752–1760. Thompson KG, Turner L, Prichard J, Dodd F, Kennedy DO, Haskell C, Blackwell JR & Jones AM (2014). Influence of dietary nitrate supplementation on physiological and cognitive responses to incremental cycle exercise. Respir Physiol Neurobiol 193, 11–20. Travaline JM, Sudarshan S, Roy BG, Cordova F, Leyenson V & Criner GJ (1997). Effect of N-acetylcysteine on human diaphragm strength and fatigability. Am J Respir Crit Care Med 156, 1567–1571. Vanhatalo A, Bailey SJ, DiMenna FJ, Blackwell JR, Wallis GA & Jones AM (2013). No effect of acute L-arginine supplementation on O2 cost or exercise tolerance. Eur J Appl Physiol 113, 1805–1819. Wagner KH (2015). Antioxidants in sport nutrition: All the same effectiveness? In Antioxidants in Sport Nutrition, ed. Lamprecht M. CRC Press/Taylor & Francis, Boca Raton, FL, USA.

Additional information Competing interests None declared. Funding This publication was funded by the University of Florida. Acknowledgements The author was assisted by Christine Coombes (graphics) and Delainie McNeil (manuscript preparation).