| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance; Centre for Aging, Rehabilitation, Exercise and Sport Science, Victoria University of Technology, Melbourne 8001; 2Department of Anaesthesia, Austin and Repatriation Medical Centre, Melbourne 3084; and 3Department of Anaesthesia and Pain Management, Royal Melbourne Hospital, Melbourne, Victoria 3052, Australia
Submitted 6 April 2004 ; accepted in final form 7 June 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
O2 peak) and then to fatigue at 92% O2 peak. NAC was intravenously infused at 125 mg·kg–1·h–1 for 15 min and then at 25 mg·kg–1·h–1 for 20 min before and throughout exercise. Arterialized venous blood was analyzed for NAC, glutathione status, and cysteine concentration. A vastus lateralis biopsy was taken preinfusion, at 45 min of exercise, and at fatigue and was analyzed for NAC, total glutathione (TGSH), reduced glutathione (GSH), cysteine, and cystine. Time to fatigue at 92% O2 peak was reproducible in preliminary trials (coefficient of variation 5.6 ± 0.6%) and with NAC was enhanced by 26.3 ± 9.1% (NAC 6.4 ± 0.6 min vs. Con 5.3 ± 0.7 min; P < 0.05). NAC increased muscle total and reduced NAC at both 45 min and fatigue (P < 0.005). Muscle cysteine and cystine were unchanged during Con, but were elevated above preinfusion levels with NAC (P < 0.001). Muscle TGSH (P < 0.05) declined and muscle GSH tended to decline (P = 0.06) during exercise. Both were greater with NAC (P < 0.05). Neither exercise nor NAC affected whole blood TGSH. Whereas blood GSH was decreased and calculated oxidized glutathione increased with exercise (P < 0.05), both were unaffected by NAC. In conclusion, NAC improved performance in well-trained individuals, with enhanced muscle cysteine and GSH availability a likely mechanism. reactive oxygen species; antioxidants; muscle fatigue; cysteine; cystine
N-acetylcysteine (NAC), a thiol-containing compound, attenuated fatigue in rabbit and rat diaphragm (12, 42, 44). Furthermore, NAC infusion attenuated fatigue of human muscle, during both low-frequency electrical stimulation of the tibialis anterior muscle (34) and inspiratory resistive loading of the diaphragm (46). Our laboratory developed an NAC infusion model for use during voluntary whole body exercise in humans, finding no effect on intense, intermittent exercise performance (26). However, during prolonged, submaximal exercise, time to fatigue was greater in three well-trained individuals during NAC infusion, with performance change induced by NAC correlated to peak oxygen consumption (O2 peak) (27). This suggested a performance-enhancing effect of NAC, but the limited sample size precluded definitive conclusions. Therefore, the first hypothesis tested in this study was that NAC infusion would increase time to fatigue during prolonged, submaximal exercise performance in a homogenous group of well-trained individuals.
The mechanism(s) underlying this potential ergogenic effect during prolonged exercise was not studied (27) but also deserves investigation. The first problem is that it is not known whether NAC crosses the sarcolemma and thereby directly affects muscle ROS or redox status during exercise. Our laboratory found that NAC infusion increased red blood cell (RBC) NAC concentration ([NAC]) during exercise (26, 27), suggesting that NAC might also enter myocytes, but there are no reports of muscle NAC. Thus the second hypothesis tested was that NAC infusion would increase NAC content in both skeletal muscle and RBCs during prolonged exercise.
NAC potentially reduces the deleterious effects of ROS by direct scavenging of ROS (2) and/or supplying cysteine (Cys) for enhanced glutathione synthesis (9). Our laboratory has demonstrated that NAC maintained blood redox status during high-intensity, intermittent exercise, indicated by an attenuated decline in reduced glutathione (GSH) and rise in oxidized glutathione (GSSG; Ref. 26). It is, therefore, possible that muscle ROS and glutathione status might also be protected by NAC during prolonged exercise, but these effects of NAC in human muscle are unknown.
A complicating factor is that the reported effects of exercise on human skeletal muscle glutathione are scarce and their findings conflicting, with reports of GSH being unchanged (8) or decreased (45) and of GSSG being increased (8) or unchanged (35). A critical role is suggested for endogenous GSH in alleviating exercise-induced oxidative stress and affecting exercise performance (38). In rats, exogenous glutathione administration enhanced glutathione synthesis and increased swim performance by up to 141% (7, 30), whereas glutathione deficiency reduced endurance time by 
50% (38).
Cys is a precursor to glutathione synthesis, and increased intracellular Cys availability enhanced intracellular glutathione (9, 16, 41). Supplementation with a Cys donor increased performance during sprint cycling (22), suggesting that enhanced glutathione synthesis can augment performance in humans. No studies have comprehensively investigated the effects of prolonged exercise or NAC infusion on muscle total glutathione (TGSH), GSH, Cys, and cystine. We therefore also tested the hypotheses that fatiguing exercise would decrease muscle GSH and increase GSSG and that each of TGSH, GSH, and Cys would be augmented by NAC infusion.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Eight healthy men (age, 27.1 ± 5.6 yr; body mass, 76.7 ± 10.9 kg; height, 180.3 ± 5.4 cm; means ± SD) volunteered for the study after being informed of all risks and giving written, informed consent. The subjects were endurance trained, completing either running or cycling activity, four to five times per week for 1–2 h, for a minimum of 2 yr. Subjects refrained from vigorous activity and avoided ingesting caffeine, alcohol, or other drugs and also consumed standard food packages for 24 h before their two experimental trials. Ethical approval was obtained from the Victoria University of Technology Human Research Ethics Committee.
Exercise Trials
Overview. Subjects attended the laboratory on six separate occasions, separated by a 7-day period. All exercise trials were completed on an electronically braked cycle ergometer (Lode Excalibur, Groningen, The Netherlands).
O2 peak.
Subjects first completed an incremental exercise test to determine their O2 peak, with all equipment, calibration, and procedures as previously detailed (24, 26).
Prolonged, submaximal exercise.
An identical exercise protocol was used as previously described (27), with subjects cycling at 70% O2 peak for 45 min and then to volitional fatigue at 90% O2 peak (27).
Experimental trials. The two experimental prolonged, submaximal exercise trials were conducted in a double-blind, randomized, crossover design, to determine the effects of NAC (Parvolex, Faulding Pharmaceuticals) or saline [control (Con)] infusion on exercise performance and on muscle and blood thiols (26, 27). For ethical reasons, the attending medical practitioner was nonblinded. To prevent possible unblinding of experimenters due to the NAC odor, all ampules and syringes containing NAC and saline were handled and sealed in a room separate to the laboratory. The medical practitioner also removed the cannulas after the experiment.
NAC Infusion
The NAC intravenous infusion protocol comprised an initial loading dose of 125 mg·kg–1·h–1 for 15 min to increase plasma [NAC], followed by a constant infusion of 25 mg·kg–1·h–1 to achieve a plateau in [NAC], with exercise commencing after 20 min of constant infusion (26, 27). NAC infusion was continued throughout exercise until fatigue, and any adverse reactions were assessed according to the scale previously detailed (27).
Blood Processing and Analyses
A 20-gauge catheter was inserted into a dorsal hand vein for arterialized venous blood sampling, and a 22-gauge catheter was inserted into a superficial median forearm vein for infusion of either NAC or saline. Arterialized venous blood was sampled from a dorsal hand vein (13) at 0, 1, 2, 5, 10, 15, 25, and 35 min during the preexercise infusion period. Further samples were taken at 15, 30, and 45 min and at fatigue during exercise and during recovery at 1, 2, 5, 10, and 30 min. A 5-ml sample was used for measurement of reduced and total thiols in blood and plasma, with all processing and analyses as previously detailed (26, 27). Blood and plasma thiol concentrations, including NAC, total glutathione (TGSH), GSH, and Cys, were analyzed by high-pressure liquid chromatography (HPLC; Waters Associates, Milford, MA), with fluorescence detection (Hitachi, Tokyo, Japan). Because of laboratory freezer failure, for three subjects whole blood total and reduced cysteine could not be measured and consequently cystine and RBC concentrations could not be calculated.
Muscle Biopsy Sampling and Analyses
Muscle biopsy sampling. After injection of a local anesthetic (1% Xylocaine) into the skin and fascia, three small incisions were made in the midportion of the vastus lateralis muscle. Muscle samples were taken preinfusion, after 45 min of exercise, and at fatigue and analyzed for muscle thiols, including NAC, glutathione, and Cys. The subject's contralateral leg was biopsied on their second experimental trial. The time taken for the subject to stop pedaling at 45 min, undergo a muscle biopsy and recommence cycling did not differ between trials (Con 55 ± 7 s vs. NAC 53 ± 7 s).
Muscle thiol analyses. The muscle sample was immediately blotted on filter paper, frozen in liquid nitrogen, and stored at –80°C for later analysis of muscle thiols. Approximately 20 mg of frozen muscle were homogenized for 20 s in 200 ml of 20 mM monobromobimane by using a handheld homogenizer (Omni 1000, Omni International, Gainesville, VA). For analysis of total thiols, 100 ml of homogenate were added to 200 ml of 4 mM dithiothreitol in an Eppendorf tube. The sample was immediately vortexed and left at room temperature for 10 min to reduce oxidized thiols. Free thiols were derivatized with 50 ml of 20 mM monobromobimane, vortexed, and incubated at room temperature in darkness for 10 min. Proteins were precipitated with 25 ml of sulfosalicylic acid (50% wt/vol) and immediately vortexed and centrifuged at 1,000 g for 5 min. The supernatant (50 ml) was injected into the HPLC for analysis. For reduced thiols, 100 ml of muscle homogenate was mixed with 250 ml of H2O and 25 ml of sulfosalicylic acid (50% wt/vol) in an Eppendorf tube, immediately vortexed, and centrifuged at 1,000 g for 5 min, before injection of 50 ml of the supernatant into the HPLC for analysis. The HPLC method and conditions were identical to those used for plasma and blood thiol analyses (26).
The HPLC mobile phase was methanol (18:82 vol/vol) and 20 mM KH2PO4 at pH 2.9 and 5 mM octanesulfonic acid running through a 150 by 3.9-mm Novapak C18 column (Waters Associates) at 1 ml/min, with fluorescence detection at 400-nm excitation and 475-nm emission. This gives baseline separation of thiol compounds from each other and the reagent peaks, with a quantitation limit of 100 nM and coefficient of determination of <5% for each. Total concentrations of the thiols were determined in a similar manner except that the oxidized thiols were reduced with dithiothreitol before a second derivitization with monobromobimane was performed. The supernatant was then extracted and injected for HPLC analysis.
Calculations
Thiol concentrations in RBCs were calculated as previously described (26, 27). Hct could not be measured in one subject because of technical difficulties, and consequently RBC NAC data are reported for seven subjects. Individual coefficients of variation (CV) for time to fatigue were calculated for all subjects within the exercise protocol and averaged to obtain an overall CV (18). This allowed clearer delineation of NAC effects from typical test variation.
Statistical Analyses
All data are presented as means ± SE, except anthropometric data. Single comparisons (e.g., time to fatigue) were analyzed by using a paired Student's t-test. A one-way ANOVA with repeated measures was used for blood and plasma [NAC]. All other blood and muscle analyses were analyzed by using a two-way (treatment, time) ANOVA with repeated measures on both factors. Post hoc analyses were conducted by using the Student-Newman-Kuels test. Significance was accepted at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
The subjects' O2 peak was 65.6 ± 2.2 ml·kg–1·min–1, and their submaximal work rates were 239 ± 20 and 336 ± 25 W, corresponding to 71 ± 1.3 and 92 ± 1.9% O2 peak, respectively. Time to fatigue at 92% O2 peak was reproducible during the two variability trials (CV 5.6 ± 0.6%, Table 1), and no trial order effect was observed (data not shown). NAC increased, by 26.3 ± 9.1% (P < 0.05), time to fatigue at 92% O2 peak (Con 5.3 ± 0.7 vs. NAC 6.4 ± 0.6 min) and thus also work done (Con 104.9 ± 15.3 KJ vs. NAC 126.5 ± 11.6 kJ).
|
Reactions. No moderate or severe adverse reactions to NAC were observed during the preinfusion, exercise, or recovery periods (Table 2).
|
|
|
Cysteine and cystine. Muscle total and reduced Cys as well as cystine were unchanged from preinfusion levels during Con and were not different between NAC and Con before infusion (Fig. 3). However, NAC markedly increased both total and reduced Cys and cystine in muscle compared with preinfusion levels (P < 0.001). Muscle total and reduced Cys and cystine were greater at 45 min and fatigue with NAC than in Con (P < 0.005; Fig. 3).
|
|
Cysteine and cystine. No change in total and reduced Cys concentration ([Cys]) or cystine from preinfusion levels occurred in Con at any time (Tables 3 and 4). Before infusion, no differences were found between NAC and Con in whole blood, plasma, or RBC for total and reduced cysteine ([Cys], Table 3) or cystine (Table 4). However, NAC increased total and reduced [Cys] and cystine in whole blood, plasma, and RBCs by up to threefold above preinfusion levels (P < 0.005). Consequently, NAC increased total and reduced [Cys] and cystine in whole blood, plasma and RBC immediately before and throughout the exercise and recovery periods (P < 0.005).
|
|
|
Plasma Glutathione
Plasma glutathione levels were too low to be reliably detected consistent with other studies (14, 26).
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Increased Muscle NAC and Increased Muscle Performance
Our data clearly demonstrate that NAC infusion substantially enhanced performance in well-trained individuals, with a 26% increase in time to fatigue during prolonged exercise. This finding confirms our earlier preliminary observation based on only a few athletes (27) and provides the first evidence of attenuated fatigue by NAC during voluntary, whole body exercise in humans. We show, for the first time, that NAC infusion increased NAC content in skeletal muscle. It is probable that a small portion of this NAC detected in muscle was due to contamination by blood. However, given the magnitude of increase in muscle NAC, it is highly likely that muscle intracellular [NAC] was elevated. Furthermore, we demonstrate here and in previous studies (26, 27) that NAC infusion increases RBC [NAC]. Together these indicate that an elevation in muscle intracellular NAC is highly likely.
Muscle Cys, Glutathione, and Exercise
Because the proportion of oxidized to total muscle glutathione was 15%, it is possible that some ex vivo oxidation may have occurred, perhaps due to the small delay in freezing muscle samples. However, this is an inevitable limitation when obtaining muscle samples via needle biopsy, and our proportion of muscle GSSG to TGSSG is consistent with others (45). We show that muscle TGSH content decreased during submaximal cycling exercise, which differs from other studies (35, 45). The reason for this discrepancy is unclear, but it may be related to the fact that our subjects were more highly trained than subjects used in previous studies (35, 45). However, our findings of a tendency for a decrease in muscle GSH (P = 0.06) and no change in muscle GSSG during submaximal exercise are in agreement with others (35, 45). More importantly, we demonstrate for the first time that NAC was able to increase both muscle TGSH and GSH during whole body exercise. The maintenance of GSH is dependent on regeneration from GSSG by glutathione reductase (40). However, this seems unlikely as a mechanism responsible for the elevation in GSH, because there was no change in GSSG with exercise or NAC, therefore implicating another mechanism. This was probably due to increased availability of the glutathione precursor, Cys, as evidenced by increases in each of muscle, RBC, and plasma.
Cys can be actively transported into cells (4) and increased intracellular Cys availability enhances intracellular GSH (40). Therefore, the increased extracellular Cys consequent to NAC infusion increases the intracellular availability of this amino acid to regenerate GSH (40, 41). In addition, we also report that NAC infusion increased muscle NAC. NAC is rapidly deacylated to produce free Cys (11), and this may have also contributed to the threefold increase in muscle cysteine. Although the increase in muscle Cys and cystine with NAC could also be due to contamination with blood, this is unlikely because there was no increase in Con trials. Thus our results strongly suggest that the greater muscle GSH with NAC may be consequent to increased intra- and extracellular Cys availability. Furthermore, NAC attenuated the decline in muscle TGSH, which, being predominantly in the reduced form (11), would have also increased muscle GSH availability.
Our results are consistent with conclusions from other studies that increasing glutathione availability improved exercise performance in rats (7, 30, 38) and in humans (22). We further demonstrate that the greater muscle GSH and TGSH during exercise with NAC was associated with increased time to fatigue. However, this effect was already evident by 45 min of exercise and suggests that factors additional to increased Cys and GSH availability may have also contributed to muscle fatigue.
Muscle ROS Scavenging
Numerous studies implicate ROS production as an important factor in muscle fatigue (5, 12, 23, 33, 43). Although we did not measure ROS concentration, NAC is known to scavenge a number of ROS, including hypochlorous acid, hydroxyl radical, and hydrogen peroxide (H2O2) (2). Furthermore, NAC is rapidly deacetylated to Cys, which itself is a known free radical scavenger (9). Thus, in addition to supporting glutathione synthesis, NAC can scavenge ROS within skeletal muscle, which may be another possible mechanism explaining enhanced performance with NAC. Further research is required to determine whether NAC scavenges ROS in vivo and whether this is sufficient to improve exercise performance.
Increased GSH availability with NAC could have indirect effects on antioxidant enzyme activity. GSH acts as a substrate for glutathione peroxidase activity, which decreases H2O2 and also leads to the formation of the deleterious hydroxyl radical. We speculate that the increased GSH availability with NAC may also have supported the function of glutathione peroxidase, thus facilitating the removal of H2O2. However, we did not measure glutathione peroxidase activity, and the concentration of H2O2 in human muscle is unknown.
Other Potential Mechanisms of NAC on Exercise Performance
Although enhanced glutathione status and NAC scavenging of ROS may have contributed to increased exercise performance, other potential mechanisms may also be responsible. NAC blunts unpleasant sensations produced during exhaustive exercise (46). Whether this occurred in this study cannot be determined. However, this seems unlikely because of the large effect of NAC on muscle TGSH, GSH, and Cys, which were already evident at 45 min, where work was matched to Con trials.
It is possible that elevated muscle antioxidant capacity may have exerted a protective effect on key ion transporting or ion channel proteins in muscle, including the Na+-K+-ATPase enzyme, the sarcoplasmic reticulum Ca2+-release channel (ryanodine receptor), and Ca2+-ATPase enzyme, each of which is deleteriously affected by increased ROS production (20). Our laboratory has found that the activities of Na+-K+-ATPase and Ca2+-ATPase and also the rate of Ca2+ release are depressed at fatigue in human muscles (6, 13, 24), with increased ROS a proposed mechanism. Animal models demonstrate that ROS deleteriously affects Na+-K+-ATPase activity (21, 39), ryanodine-receptor function (31, 32), and Ca2+-ATPase activity (1, 47). Whether NAC blunts these effects in human skeletal muscle remains to be elucidated.
Blood and Plasma Thiols
The decreased whole blood [GSH] and increased [cGSSG] with prolonged exercise is consistent with other human studies (14, 15, 38). However, the lack of modification with NAC contrasts our laboratory's previous findings during high-intensity, intermittent exercise (26), where our group found a clear effect of NAC on blood thiol status during exercise. This may reflect differences in the training status of subjects and the exercise intensity and duration. Our laboatory's previous study utilized untrained individuals (26), whereas this study utilized endurance-trained individuals. Endurance-trained individuals have increased blood antioxidant activity compared with sedentary subjects (25) and training increases blood antioxidant enzyme capacity (28), which may reduce the overall oxidative stress in RBCs (19). Increasing exercise intensity results in increased blood glutathione oxidation (36) and ROS production (3), which might also explain an effect of NAC on blood thiol status during intense (26) but not in submaximal exercise. More profound changes in blood glutathione may have occurred in blood draining the exercising muscles, and hence NAC effects on GSH and cGSSG may be greater at these sites.
In conclusion, we demonstrate for the first time that NAC infusion during prolonged, submaximal exercise increased muscle NAC and skeletal muscle cysteine, cystine, and glutathione availability during exercise and that it substantially enhanced performance in well trained individuals.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. F. Ferreira and M. B. Reid Muscle-derived ROS and thiol regulation in muscle fatigue J Appl Physiol, March 1, 2008; 104(3): 853 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. McKenna, I. Medved, C. A. Goodman, M. J. Brown, A. R. Bjorksten, K. T. Murphy, A. C. Petersen, S. Sostaric, and X. Gong N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans J. Physiol., October 1, 2006; 576(1): 279 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sahlin, J. S. Nielsen, M. Mogensen, and M. Tonkonogi Repeated static contractions increase mitochondrial vulnerability toward oxidative stress in human skeletal muscle J Appl Physiol, September 1, 2006; 101(3): 833 - 839. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
Significant interaction effect; no difference at preinfusion but NAC > Con at 45 min and fatigue (P < 0.05).
Significant treatment main effect, NAC > Con (P < 0.05). 
Time main effect; less than preinfusion (P = 0.06).
, NAC; 
, Con. Shaded bar denotes exercise comprising 45 min at 71% peak oxygen consumption and then to fatigue (F) at 92% peak oxygen consumption. Values are means ± SE; n = 8 subjects. *Significant time effect; less than preinfusion (P < 0.05). **Significant time effect; greater than preinfusion (P < 0.05).