Influence of N-acetylcysteine administration on pulmonary O2 uptake kinetics and exercise tolerance in humans

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Abstract

We investigated the influence of the antioxidant N-acetylcysteine (NAC) on plasma nitrite concentration ([NO2]), pulmonary oxygen uptake (V˙O2) kinetics and exercise tolerance. Eight males completed ‘step’ moderate- and severe-intensity cycle exercise tests following infusion of either NAC (125 mg kg−1 h−1 for 15 min followed by 25 mg kg−1 h−1 until the termination of exercise) or Placebo (PLA; saline). Following the initial loading phase, NAC infusion elevated plasma free sulfhydryl groups compared to placebo (PLA: 4 ± 2 vs. NAC: 13 ± 3 μM g−1; P < 0.05) and this elevation was preserved throughout the protocol. The administration of NAC did not significantly influence plasma [NO2] or V˙O2 kinetics during either moderate- or severe-intensity exercise. Although NAC did not significantly alter severe-intensity exercise tolerance at the group mean level (PLA: 776 ± 181 vs. NAC: 878 ± 284 s; P > 0.05), there was appreciable inter-subject variability in the response: four subjects had small reductions in exercise tolerance with NAC compared to PLA (−4%, −8%, −11%, and −14%) while the other four showed substantial improvements (+24%, +24%, +40%, and +69%). The results suggest that exercise-induced redox perturbations may contribute to fatigue development in recreationally-active adults.

Introduction

The initiation of exercise provokes an immediate increase in the ATP turnover rate, but an exponential increase in oxygen consumption within the contracting myocytes. This initial discrepancy between the rates of muscle ATP utilization and ATP supply through oxidative phosphorylation mandates a compensatory energy liberation from both phosphocreatine (PCr) degradation and anaerobic glycolysis (Krogh and Lindhard, 1920, Poole et al., 2008). While pulmonary oxygen uptake (V˙O2), which provides a close approximation of muscle V˙O2 (Grassi et al., 1996, Krustrup et al., 2009), attains a ‘steady-state’ within 2–3 min following the onset of moderate-intensity exercise performed below the gas exchange threshold (GET; Whipp and Wasserman, 1972, Whipp et al., 1982), a supplementary V˙O2 ‘slow component’ emerges during supra-GET exercise. This V˙O2 slow component delays the attainment of the steady-state during heavy-intensity exercise (below the critical power; CP), or prevents the attainment of a steady state during severe-intensity exercise (above CP) (Poole et al., 1988). The development of the V˙O2 slow component is associated with greater muscle PCr (Rossiter et al., 2002) and glycogen (Krustrup et al., 2004) utilization. Therefore, interventions that modulate the dynamic V˙O2 response during exercise, through determining the rate at which the energetic reserves are depleted and fatiguing metabolites accumulated, have important implications for the tolerable duration of exercise (Burnley and Jones, 2007, Bailey et al., 2009a, Bailey et al., 2009b, Bailey et al., 2009c, Jones and Burnley, 2009).

The causes of fatigue during exercise are known to be manifold and complex (Westerblad and Allen, 2003) but the possibility that the production of reactive oxygen species (ROS) during exercise contributes to fatigue development has begun to receive significant attention (Shindoh et al., 1990, Reid et al., 1992a, Moopanar and Allen, 2005, Ferreira and Reid, 2008, Powers and Jackson, 2008, Reid, 2008, Reardon and Allen, 2009). While the administration of nutritional antioxidants fails to reduce muscle fatigue development (Ferreira and Reid, 2008), administration of the pharmacological antioxidant, N-acetylcysteine [NAC], has been shown to delay fatigue in small muscle mass (Reid et al., 1994, Travaline et al., 1997, Koechlin et al., 2004, Matuszczak et al., 2005) and whole body (Medved et al., 2004b, McKenna et al., 2006) exercise in humans. The antioxidant role of NAC is twofold: firstly, NAC has direct antioxidant properties and can scavenge a number of ROS (Aruoma et al., 1989, Benrahmoune et al., 2000); and, secondly, NAC serves as a donor of reduced cysteine (CYS; Cotgreave, 1997) which is subsequently metabolized to reduced glutathione (GSH) by the action of γ-glutamylcysteine synthase (Sen, 1997). Both CYS and GSH have direct antioxidant properties with GSH also serving as the substrate for the enzymatic antioxidant, glutathione peroxidase. How exactly these biological actions of NAC are related to its ability to retard the rate of fatigue development is presently unclear. In a series of experiments (Medved et al., 2003, Medved et al., 2004a, Medved et al., 2004b, McKenna et al., 2006), McKenna and colleagues applied a two-phase intravenous NAC administration procedure in humans that was well-tolerated and demonstrated to increase [NAC], [CYS] and [GSH], to decrease oxidized glutathione concentration ([GSSG]) in blood, and to increase [NAC] and [CYS] in skeletal muscle. While this improved capacity to tolerate exercise-induced redox perturbations has been accompanied by an improved exercise tolerance (Medved et al., 2004b, McKenna et al., 2006), it is presently unclear if this effect is related to changes in V˙O2 dynamics.

Manipulating the bioavailability of the multi-functional physiological signaling molecule, nitric oxide (NO), has important effects on the V˙O2 kinetics during both the fundamental (Jones et al., 2003, Jones et al., 2004, Wilkerson et al., 2004, Bailey et al., 2009a, Bailey et al., 2010) and slow component (Jones et al., 2004, Bailey et al., 2009a, Bailey et al., 2010) phases of the response in humans. Moreover, interventions that increase NO bioavailability have also been demonstrated to improve high-intensity exercise tolerance in conjunction with a reduced V˙O2 slow component (Bailey et al., 2009a, Bailey et al., 2010). Muscle contraction results in an increased production of ROS (Reid et al., 1992a, Reid et al., 1992b, Reid et al., 1993, McArdle et al., 2005), as well as NO (Balon and Nadler, 1994, Kobzik et al., 1994), and it has been reported that ROS are capable of scavenging NO (Huang et al., 2007) and thwarting the enzymatic activity of the nitric oxide synthase (NOS) enzymes (Huang et al., 2001). Accordingly, interventions that scavenge exercise-induced ROS may increase NO synthesis and bioavailability during exercise, resulting in improved V˙O2 dynamics and exercise tolerance. NAC and its derivatives CYS and GSH, not only have non-specific antioxidant properties but are also capable of reacting with reactive nitrogen species (RNS), derived from NO, to generate a reservoir of relatively stable NO that is carried in the form of S-nitrosothiols (Taylor and Winyard, 2007). However, the extent to which these S-nitrosothiols might serve as NO donors during exercise is obscure. The influence of NAC on NO synthesis and bioavailability during exercise is highly complex and in need of clarification.

The purpose of this investigation was therefore to determine the influence of NAC administration on NO synthesis and bioavailability (as reflected by plasma [nitrite]), pulmonary V˙O2 kinetics and exercise tolerance in humans. We reasoned that the potential for NAC to enhance NO synthesis would result in greater NO bioavailability. We therefore hypothesized that the V˙O2 slow component would be reduced and severe-intensity exercise tolerance improved following the administration of NAC.

Section snippets

Subjects

Eight healthy males (mean ± SD, age 27 ± 8 yr, height 180 ± 2 cm, body mass 80 ± 7 kg; V˙O2 max; 51 ± 9 mL kg−1 min−1) volunteered to participate in this study. None of the subjects were tobacco smokers or users of dietary supplements. The subjects were physically trained (engaging in 4–6 h of recreational exercise per week) but were not competitive athletes. All subjects were fully familiar with laboratory exercise testing procedures, having previously participated in studies employing cycle ergometry in our

Results

The NAC administration regime employed in this investigation was well tolerated with no adverse reactions. During the ramp incremental test, subjects attained a peak work rate of 376 ± 61 W and a V˙O2 max of 4.07 ± 0.72 L min−1, while the work rate and V˙O2 values at the GET were 127 ± 20 W and 1.77 ± 0.20 L min−1, respectively.

Discussion

This is the first investigation to assess the influence of NAC administration on indices of NO synthesis, pulmonary V˙O2 kinetics and exercise tolerance in humans. The principal original findings were that intravenous infusion of the potent antioxidant, NAC, which significantly increased total plasma sulfhydryl groups, had no significant influence on plasma [NO2], V˙O2 kinetics or exercise tolerance in healthy adult humans. The unchanged plasma [NO2] suggests that NAC administration did not

Summary

The administration of NAC more than doubled plasma free sulfhydryl (thiol) groups in this investigation, consistent with the observations of an increase in plasma thiols (NAC, CYS and GSH) reported by others using the same NAC administration procedure (Medved et al., 2003, Medved et al., 2004a, Medved et al., 2004b, Merry et al., 2010). While plasma [NO2] increased over the course of the severe-intensity exercise bout, in line with an enhanced NO synthesis during exercise (Balon and Nadler,

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