American Journal of Respiratory and Critical Care Medicine

Free radical injury is believed to be important in diaphragm dysfunction. N-Acetylcysteine (NAC) is a potent free radical scavenger shown in animal models to attenuate diaphragm fatigue; however, its effects on human diaphragm function are unknown. We assessed diaphragm function by electrophrenic twitch stimulation (PdiT) and twitch occlusion (to yield Pdimax) in four healthy subjects 35 ± 3 yr of age (mean ± SD). We intravenously administered NAC (150 mg/kg in 250 ml D5W) or placebo (CON) (250 ml D5W) in a randomized manner after subjects were premedicated with antihistamines. There were no significant side effects with the infusion. After infusion, we measured baseline Pdimax and PdiT at FRC. Diaphragm fatigue was then induced by subjects breathing through an inspiratory resistive load. Pdimax and PdiT were then measured at 15 to 30 min and 1, 2, 3, 4, and 20–25 h after fatigue. Times to fatigue were 13 ± 4 min (CON) and 21 ± 6 min (NAC) (p = 0.04). At 15 min after fatigue, PdiT was reduced to 40% (CON) compared with 30% (NAC) initial PdiT value (p = 0.05). Other twitch characteristics (maximal rate of relaxation and maximal contraction rate) were reduced to a greater degree after placebo compared with NAC. There were no significant differences in the rate of recovery between CON and NAC. Pdimax at 30 min after fatigue was significantly greater with NAC; however, at 1 h after fatigue, Pdimax for CON and NAC were not different, suggesting similar rates of recovery in high-frequency fatigue. These data suggest that NAC may attenuate low-frequency human diaphragm fatigue.

Oxygen-derived free radicals play an important role in the pathogenesis of diaphragm dysfunction (1). N-Acetylcysteine (NAC) is a potent free radical scavenger that has been shown in animal models to attenuate the development of diaphragm fatigue (2-5). NAC also has been shown to attenuate fatigue development in the tibialis anterior muscle of humans subjected to repeated isometric contractions (6). Unlike other skeletal muscle, however, the diaphragm contracts rhythmically throughout life and has a relatively greater proportion of oxidative fibers, making it more fatigue resistant (7). The effects of NAC on the human diaphragm, therefore, may be different than on other skeletal muscle. However, this has not been evaluated.

In this study, we sought to examine the effects of NAC on the induction and rate of recovery of human diaphragm fatigue in normal subjects. Specifically, using bilateral supramaximal electrophrenic diaphragm stimulation, in a randomized, placebo-controlled fashion, we assessed the effect of NAC on the development of low-frequency (PdiT) and high-frequency (twitch occlusion Pdimax) diaphragm fatigue.

Subjects

Four healthy subjects with no prior respiratory illness and a normal physical examination, taking no medications and abstaining from caffeine at least 12 h before each study, were recruited for this study. Approval from our Institutional Review Board for human research was obtained, and subjects gave written informed consent.

Each subject was studied during three sessions, each separated by at least a 1-wk interval. Session 1 involved acquaintance of the subject with the equipment used for testing, measurement of maximal transdiaphragmatic pressure (Pdimax), localization of phrenic nerve motor points, and acquaintance with the fatigue protocol. During Session 2, subjects received either NAC or placebo in a blinded manner. During Session 3, the alternate solution was infused.

Before each infusion, subjects were orally premedicated with diphenhydramine 25 mg and ranitidine 150 mg to decrease the incidence of anaphylactoid side effects from NAC (6).

Measurement of Transdiaphragmatic Pressure

After topical anesthesia (4% xylocaine), two thin-walled balloon-tipped catheters were passed via the nares; one into the lower esophagus (esophageal pressure [Pes]) and the other into the stomach (gastric pressure [Pga]). These catheters were connected to pressure transducers (range ± 100 cm H2O; Validyne, Northridge, CA). Transdiaphragmatic pressure (Pdi) was continuously displayed as the electronic subtraction of Pes from Pga.

A molded plaster cast was placed over the anterior abdomen to prevent diaphragm shortening by minimizing outward abdominal displacement during electrophrenic stimulation. Voluntary Pdimax was measured against an occluded airway using a combined expulsive-Mueller maneuver during visual oscilloscopic feedback (8) while the subject was seated upright in a high-backed chair. The average of three values of Pdimax, all within 5% of each other, are reported as Pdimax.

Breathing Circuit and Recording Apparatus

During the study, subjects breathed through a pneumotachograph (Hans Rudolph, Inc., Kansas City, MO) connected to an in-line three-way valve enabling subjects to breathe spontaneously or against an occluded airway. The airflow signal, inspired and expired volumes derived via integration of the airflow signal, and airway pressure measured at the mouth were continuously displayed on a strip chart recorder (ES 1000; Gould, Dayton, OH).

Electrophrenic Stimulation

Compound diaphragm action potentials (CDAP) were measured bilaterally by two 3-mm EMG surface electrodes placed 2 mm apart in the seventh intercostal space in the anterior axillary line.

In each subject, the area for optimal phrenic nerve stimulation was located by using well-determined neck anatomic landmarks (9). Once identified, an electrical stimulus was applied and the CDAP was displayed on a recording oscilloscope to confirm phrenic nerve stimulation. Both the right- and left-sided EMG signals were amplified and, after each twitch, both signals were recorded on a storage oscilloscope (Model 1604; Gould). The stimulus voltage was incrementally increased until there was no further increase in CDAP amplitude. Once maximal stimulus voltage was achieved, it was increased by 20% to ensure supramaximal diaphragm activation.

A modified neck brace, which housed the phrenic nerve stimulus probes, was used throughout the study to ensure consistency in stimulation. The phrenic nerves were then stimulated transcutaneously (S88 Stimulator; Grass, Quincy, MA) with 100 to 140 V (approximately 30 mA), 0.1 ms in duration, to produce diaphragm twitch pressures.

Pdi Twitch (PdiT) at FRC

With the subject seated in an upright position with the anterior abdomen casted, bilateral phrenic nerve stimulation was applied at FRC after closure of the three-way valve at end-expiration. Pes was continuously monitored to ensure that end-expiratory lung volume had returned to a consistent baseline prior to valve closure. Six to 10 consecutive twitches at FRC were applied at each study point.

Twitch Occlusion

In addition to PdiT at FRC, the twitch occlusion technique was performed in each subject. This was done by having the subject voluntarily contract his diaphragm by making an inspiratory effort against an occluded airway to the level of Pdimax that was displayed as the visual oscilloscopic target. Once the subject achieved the targeted PdiT (20%, 40%, 60%, 80%, and 100% of Pdimax), bilateral electrophrenic stimuli (five to six twitches) were applied, producing a superimposed PdiT.

Pdi Contractile Characteristics

Twitch contractile characteristics were measured in each of the twitches, and all were included in the analysis. Their definitions are as follows: Twitch amplitude: maximal height of the twitch from baseline pressure to its peak. Time to peak tension: time from twitch onset to the generation of peak pressure. Half relaxation time: time from the peak pressure to half of the peak pressure during relaxation. Maximal rate of relaxation (MRR): slope of a line drawn between two points during the initial third of the relaxation portion of the twitch curve. Maximal contraction rate (MCR): slope of the line drawn between two points during the middle third of the ascending limb of the twitch curve where the rate of rise is maximal.

Induction of Diaphragm Fatigue

Subjects breathed against a resistive load using oscilloscopic feedback to achieve a targeted Pdi 80% of Pdimax. Inability to maintain the visual Pdi target for five consecutive breaths despite coaching by one of the investigators determined the end of the fatigue run. To ensure fatigue, subjects underwent four consecutive fatigue runs each separated by a 1-min pause (10). Endurance was calculated as the total time of all four fatigue runs.

Recovery of PdiT

After fatigue induction, PdiT at FRC and twitch occlusion Pdimax were measured once between 15 and 30 min and then 1, 2, 3, and 4 h after fatigue. If total recovery to baseline values did not occur by 4 h, the subjects were studied the following day (20 to 25 h after fatigue) until the return of values to baseline. The rate of recovery in contractile characteristics was calculated as the change between the post-fatigue value and the subsequent value divided by the time interval between those values.

Protocol

Day 1. Acquaintance of the subjects to the apparatus and techniques.

Days 2 and 3. Measurement of voluntary Pdimax and PdiT at FRC. Intravenous NAC (Apothecon, Princeton, NJ) 150 mg/kg in 250 ml D5W or placebo (250 ml D5W) via an infusion pump over 1 h was administered in a random fashion. Repeat measurements of PdiT at FRC and twitch occlusion were performed. Fatigue induction was done with inspiratory resistive loading and then, after fatigue, measurement of PdiT at FRC and twitch occlusion were performed until return to baseline.

Days 4 and 5. Repeat measurements of the above protocol were performed with the alternate infusion (NAC or placebo).

Data Analysis

Determination of sample size was based on accepting a test power of 0.8 and considering a p value < 0.05 as statistically significant. Analysis of variance was used for repeated measures of PdiT characteristics over time. Student's paired t test was used to compare values between NAC and placebo after a normality test of the data was passed. All values are expressed as mean ± SE unless otherwise specified.

Four subjects 35 ± 3 yr of age (mean ± SD) were infused with both placebo and NAC. There were no serious adverse reactions during drug infusion.

Although subjects were premedicated with ranitidine and diphenhydramine, two subjects experienced mild transient skin flushing and pruritus. Two other subjects experienced a mild degree of nausea occurring approximately 20 to 35 min after the end of NAC infusion and lasting only a few minutes. There were no hemodynamic or other physical exam abnormalities detected during the infusions or during the immediate postinfusion period.

Table 1 shows the individual subject characteristics and group data, which include voluntary Pdimax and baseline PdiT expressed as percent of Pdimax determined by twitch occlusion. There were no significant differences in either of these parameters during the control or NAC studies.

Table 1. BASELINE SUBJECT CHARACTERISTICS

Subject No.Age (yr)InfusionVoluntary Pdimax(cm H2O)Baseline PdiT/Pdimax
132CON1490.21
NAC1540.16
233CON1890.21
NAC1860.19
339CON1730.14
NAC1710.18
434CON1600.18
NAC1440.17
Mean ± SD35 ± 3CON168 ± 170.19 ± 0.03
NAC164 ± 190.18 ± 0.01

Definition of abbreviations: CON = control (placebo infusion) study; NAC = N-acetylcysteine infusion study; Pdimax = maximal transdiaphragmatic pressure; PdiT/Pdimax = transdiaphragmatic twitch pressure at FRC divided by maximal transdiaphragmatic pressure determined by twitch occlusion.

Endurance was increased in each subject after NAC preadministration, with a mean increase of approximately 50% when compared with placebo (p = 0.04; Figure 1).

The percent of PdiT reduction at 15 min after fatigue was significantly lower following NAC preadministration. There was an approximately 40% reduction in PdiT after placebo administration compared with an approximately 30% reduction after NAC administration (p = 0.05; Figure 2). Also, at all time points during recovery from fatigue, the values of PdiT expressed as percent of initial value were significantly higher after NAC preadministration when compared with placebo (Figure 2).

The MCR and MRR determined for each twitch expressed as percent of initial value are also shown in Figure 2. The reductions in MRR and MCR after fatigue were similar to the reduction in PdiT, with a trend towards less reduction after NAC preadministration than placebo. There were no significant differences, however, in the rate of recovery of diaphragm contractile properties.

Pdimax expressed as a percent of the initial Pdimax derived from twitch occlusion at 30 and 60 min after fatigue is shown in Figure 3. There was significantly (p = 0.03) less reduction in Pdimax 30 min after fatigue with NAC pretreatment. At 1 h into recovery, however, there were no significant differences in Pdimax between NAC and placebo pretreatment (p = 0.25).

The major findings of this study are (1) in healthy subjects breathing against an inspiratory resistive load, task endurance is significantly greater after NAC administration; (2) NAC administered before the induction of diaphragm fatigue attenuates the fall in PdiT after fatigue; (3) NAC lessens the immediate fall in Pdimax after fatigue; (4) NAC has no significant effect on the rate of recovery of diaphragm fatigue; and (5) intravenously administered NAC appears to be tolerated with minimal side effects in normal subjects, when administered after premedication with antihistamines.

Oxidants or free radicals may contribute to the pathogenesis of skeletal muscle fatigue or respiratory failure (1). Antioxidant therapy in the form of free radical scavengers in various models has been shown to inhibit skeletal muscle fatigue (2, 3, 6, 11-13). Shindoh and associates (3), for example, evaluated the effects of NAC on in situ diaphragm contractility in anesthetized rabbits and found during rhythmic diaphragm stimulation that the rate of fatigue development was greater in the saline-treated animals than in the NAC-treated animals. They concluded that NAC pretreatment reduced the rate of diaphragm fatigue and that these effects were not related to alterations in blood flow or gas exchange.

Investigating human skeletal muscle, Reid and coworkers (6) showed that intravenously administered NAC inhibited fatigue of the tibialis anterior in normal subjects and that the inhibition of muscle fatigue occurred only at low-frequency stimulation. These investigators also demonstrated that intravenous administration of NAC was well tolerated by the subjects, given antihistamine premedication.

Our study adds information specifically about NAC effects on human diaphragm fatigue. We have shown that human diaphragm fatigue induced by loaded breathing can be attenuated by pretreatment with NAC. We found that while there may have been a small effect on high-frequency diaphragm fatigue that was lessened by NAC, the predominant effect of NAC was in reducing the magnitude of low-frequency fatigue. We found an approximate 17% increase in postfatigue PdiT after NAC preadministration. This is similar to the effect of the same dose of NAC in nonrespiratory skeletal muscle reported by Reid and coworkers (6). They showed an approximately 15% increase in sustainable forces during peripheral fatigue in the tibialis anterior after subjects were pretreated with NAC. These increases in muscle strength, however, contrast with the nearly 50% increase in rabbit diaphragm tension after fatigue in NAC-pretreated animals demonstrated by Shindoh and associates (3). Because the dose and route of NAC administration in each of these studies was the same (150 mg/kg), the differences between the human and animal data probably reflect interspecies variations in response to NAC or differences in the methodology of measuring diaphragm strength.

Furthermore, in our study, while endurance was greater after NAC preadministration, there appeared to be no effect on the rate of recovery of either high- or low-frequency diaphragm fatigue. This finding is also consistent with previous data in both nonrespiratory skeletal muscle (6) and the diaphragm in animal studies (3).

Interestingly, while NAC has been shown to have effects in inhibiting skeletal muscle fatigue, Khawli and Reid (14) showed that NAC depressed peak twitch tension and altered time to peak tension in a dose-dependent fashion in an unfatigued diaphragm strip from a rat. In three of our four subjects in the unfatigued state, PdiT expressed as percent of Pdimax tended to be lower after NAC preadministration when compared with placebo. Voluntary Pdimax after NAC preadministration at baseline also tended to be lower than after placebo. These differences, however, were not statistically significant and may be more consistent with the results of other investigators who have shown no effect of NAC in the unfatigued skeletal muscle (6).

The mechanism of NAC's ability to attenuate low-frequency fatigue we believe is secondary to its effects on inactivating oxygen-derived free radicals. We cannot discount the possible effect that NAC may have had on blood flow to the diaphragm since no parameter of this was specifically measured. We do know, however, that NAC had no significant effects on blood pressure or heart rate during its infusion to suggest an alteration in hemodynamics. Moreover, based on invasive studies in animals, NAC has not been found to produce significant hemodynamic effects (3).

NAC is known, however, to have effects on the central nervous system (6). It is conceivable that the greater endurance seen in our subjects after NAC may have been related to an effect of NAC blunting unpleasant sensations produced by doing an exhaustive fatigue run. While we cannot absolutely discount this possibility, it seems unlikely for two reasons. One, the subjects experienced some unpleasant side effects from the NAC, indicating that unpleasant stimuli were perceived. Moreover, given the presence of unpleasant side effects, if there was any effect related to a subject's comfort level, one would expect the effect to decrease endurance. Secondly, NAC's effect in attenuating the fall in PdiT after fatigue is consistent with a greater endurance, again suggesting an effect of NAC on the muscle itself, not related to the central nervous system.

In summary, we have shown that preadministration of intravenous NAC may attenuate the development of diaphragm fatigue in normal subjects breathing against high inspiratory loads. These data suggest that antioxidant therapy may have a potential clinical benefit for patients with ventilatory failure secondary to respiratory muscle fatigue.

The writers wish to thank Apothecon for their donation of intravenous N-acetylcysteine solution, the subjects who participated in this study, and Mrs. Kim Williams for her secretarial assistance.

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Correspondence and requests for reprints should be addressed to John M. Travaline, M.D., Assistant Professor of Medicine, Division of Pulmonary and Critical Care Medicine, Temple University School of Medicine, Parkinson Pavilion, 9th Floor, 3401 N. Broad St., Philadelphia, PA 19140.

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