We screened 30 individuals for inclusion in the study. Seventeen healthy subjects participated (age 30 ± 2 years, body weight 86 ± 5 kg). The study followed a double-blinded, placebo-controlled, crossover design. Subjects received NAC in capsules (Arm 1) or solution (Arm 2). Ten individuals completed each arm of the study, with 3 subjects involved in both arms. The inclusion criteria were age 21–55 years, absence of acute illness (e.g., common cold) and chronic diseases, and systolic/diastolic blood pressure ≤130/90 mm Hg. We excluded individuals taking any medication other than aspirin on a regular basis and those who could not follow our exercise protocol. Volunteers read and signed a consent form agreeing to participate in the study. All experiments and procedures were approved by the institutional review board of the University of Kentucky and performed at the institution’s General Clinical Research Center.
For each trial, the subjects received two capsules containing either 0 mg (placebo), 300 mg, or 600 mg of NAC yielding total doses of 0, ~9, or ~18 mg/kg per trial. The subjects ingested one capsule while in the hospital between 11 a.m. and 2 p.m. and another capsule on the same day between 9 and 11 p.m. We contacted them by phone in the evening of the trial to remind them to take the capsule and to keep track of the time of capsule intake. The subjects returned to the hospital the following morning (8–9 a.m.) for the experiments. Trials were separated by 1 week. On the day of the experiment blood was drawn from the antecubital vein and each subject performed a bout of fatiguing handgrip exercise. Five to ten minutes after finishing the exercise bout subjects answered a questionnaire about adverse reactions experienced during the trial.
For each trial, the subjects drank a placebo (0.9% saline) or NAC solution containing doses of 35, 70, and 140 mg/kg. Because of the characteristic sulfur smell of NAC the subjects wore a nose clip during exposure to and intake of the experimental solution. Moreover, they drank the experimental solution in a room with gauze soaked in full-strength NAC solution. Experimental visits were between 8 and 11 a.m. and separated by 1–2 weeks. As an attempt to mask the salty taste of NAC, we used saline (0.9%) to adjust the drug concentration to the desired dose. The volume of all solutions matched the highest dose of NAC. A venous blood sample was drawn 60 min after the subject drank the solution. Approximately 75–90 min after ingestion of the experimental solution, and within 5–10 min of finishing a bout of fatiguing exercise (see below), subjects answered a questionnaire about adverse reactions to NAC.
The questionnaire on adverse reactions was derived from responses reported by subjects who received NAC in previous studies (Matuszczak et al., 2005
; Reid et al., 1994
). Reactions on the questionnaire included upset stomach; nausea; stomach or intestinal gas; sleepiness; metallic taste; lightheadedness; redness of eye, face, or hand; cough; welts; and wheezing. Subjects were asked to grade the intensity of each reaction and sensation as none, mild, moderate, or severe. They were asked to describe and grade any reactions that were not included in the questionnaire. Although our focus was on responses immediately after ingestion of NAC, we also contacted the subjects after their visits to inquire about adverse reactions experienced during the 24–48 hr after the experiment. We also asked the subjects if they thought they had received NAC or placebo.
Blood Sampling and Analysis
Venous blood samples were collected from the antecubital vein using a 22-gauge intravenous catheter (Jelco, Medex Medical Ltd., Rossendale, UK) connected to polyethylene tubing and a syringe. Approximately 2 ml of blood per sample were drawn slowly to prevent hemolysis. Hemolyzed samples were discarded because of plasma cysteine and glutathione contamination by lysed red blood cells. The blood was placed in a microcentrifuge tube containing a preservative solution (100 mM serine-borate [pH 8.5] containing [per ml] 0.5 mg sodium heparin, 1 mg bathophenanthroline disulfonate, and 2 mg iodoacetic acid] and gently inverted to ensure adequate mixing. After centrifugation (12,000 rpm, 60 s), 200 μl of plasma were placed in a tube containing 200 μl perchloric acid solution and stored at −80 °C until later analysis. Plasma samples were analyzed for glutathione (GSH), oxidized glutathione (GSSG), cysteine (CySH), cystine (CySS), and cysteine-glutathione disulfide (CySSG) using high-performance liquid chromatography (Clinical Biomarkers Laboratory, Emory University, Atlanta, GA). For further details on processing and analysis of blood samples see Jones (2002)
. Total glutathione (TGSH) was calculated as GSH + 2•GSSG + CySSG. Total cysteine (TCyS) was calculated as CySH + 2•CySS + CySSG.
Our focus was the perception of adverse reactions in the context of fatiguing exercise. Thus, subjects performed a handgrip exercise test. This test was conducted using a custom-made handgrip dynamometer connected to a personal computer (Matuszczak et al., 2005
). The exercise protocol consisted of three maximal voluntary contractions (MVC) of ~5 s duration separated by 1 min rest followed by a sequence of repetitive isometric contractions (3 s on, 3 s off). Exercise was stopped when the subject failed to reach the target force (70% MVC) in three consecutive efforts (task failure). Subjects were familiarized with the exercise protocol before entering the study. We asked all volunteers to avoid caffeinated beverages for a minimum of 12 hr before each experimental visit.
To assess handgrip performance we determined the number of efforts in which the subject reached the target force and calculated the force-time integral and maximal rate of force development (dF/dtMAX) of each effort. All efforts before task failure were added to estimate the amount of work performed during the exercise bout (total force-time integral). The decline in dF/dtMAX during the fatigue trial was analyzed by linear regression. The slope of this relationship was used as an indicator of muscle fatigue, wherein negative slopes suggest faster fatigue.
NAC Capsules and Solution
NAC capsules and solution were obtained from PhysioLogics (Northglenn, CO) and American Regent Laboratories Inc. (Shirley, NY), respectively. Investigational Drug Services of the University of Kentucky College of Medicine prepared the solutions, capsules, and randomization sequence for each arm of the study. Investigators and subjects were blinded regarding the content of the capsules or solutions being administered during data collection and analysis. We limited our study to oral administration because in the United States, the Food and Drug Administration approves the clinical use of the intravenous route only for treatment of acetaminophen overdose.
Comparison of plasma thiol data among doses in each experimental arm was performed using one-way repeated-measures ANOVA. Post hoc analyses were conducted using Tukey’s test. Side effects were ranked from 0 (none) to 3 (severe), categorized as GI and non-GI, and compared by Page’s test with post hoc analysis based on Hollander’s multiple comparison test. The relationship between variables was determined using Pearson’s correlation and linear regression. Values of p less than .05 were considered statistically significant. All statistics were calculated using commercially available software (Prism 5.0b for Mac OS X, GraphPad Software Inc., La Jolla, CA). Data are shown as M ± SE.