Clinical Study | Open Access
Additional Effects of Nutritional Antioxidant Supplementation on Peripheral Muscle during Pulmonary Rehabilitation in COPD Patients: A Randomized Controlled Trial
Background. Skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD) is not fully reversed by exercise training. Antioxidants are critical for muscle homeostasis and adaptation to training. However, COPD patients experience antioxidant deficits that worsen after training and might impact their muscle response to training. Nutritional antioxidant supplementation in combination with pulmonary rehabilitation (PR) would further improve muscle function, oxidative stress, and PR outcomes in COPD patients. Methods. Sixty-four COPD patients admitted to inpatient PR were randomized to receive 28 days of oral antioxidant supplementation targeting the previously observed deficits (PR antioxidant group; α-tocopherol: 30 mg/day, ascorbate: 180 mg/day, zinc gluconate: 15 mg/day, selenomethionine: 50 μg/day) or placebo (PR placebo group). PR consisted of 24 sessions of moderate-intensity exercise training. Changes in muscle endurance (primary outcome), oxidative stress, and PR outcomes were assessed. Results. Eighty-one percent of the patients (%pred) showed at least one nutritional antioxidant deficit. Training improved muscle endurance in the PR placebo group (+%, ), without additional increase in the PR antioxidant group (-%; ). Nevertheless, supplementation increased the α-tocopherol/γ-tocopherol ratio and selenium (+%, , and +%, , respectively), muscle strength (+%, ), and serum total proteins (+%, ), and it tended to increase the type I fiber proportion (+%, ). The prevalence of muscle weakness decreased in the PR antioxidant group only, from 30.0 to 10.7% (). Conclusions. While the primary outcome was not significantly improved, COPD patients demonstrate significant improvements of secondary outcomes (muscle strength and other training-refractory outcomes), suggesting a potential “add-on” effect of the nutritional antioxidant supplementation (vitamins C and E, zinc, and selenium) during PR. This trial is registered with NCT01942889.
Chronic obstructive pulmonary disease (COPD) is systematically associated with comorbidities . Peripheral muscle dysfunction, characterized by reduced endurance, atrophy, and weakness, is a common comorbidity that negatively impacts prognosis . Muscle atrophy and weakness are therefore targets of exercise training interventions in pulmonary rehabilitation (PR) . However, the available evidence suggests that the muscle response to training is blunted in COPD patients, with either no or subphysiological responses regarding muscle strength, fiber size, and oxidative fibers [4, 5]. Although still debated , oxidative stress and antioxidant deficits could impair the muscle response to training in COPD.
Oxidative stress is indeed a deleterious factor leading to muscle dysfunction and atrophy in COPD . It results from an imbalance between excessive reactive oxygen species (ROS) production and a deficit in antioxidant defenses. In vitro experiments in COPD atrophic myotubes  match the clinical resting and exercise evidence . Specifically, antioxidants play a key role as they improve muscle endurance  and atrophy in COPD .
Antioxidants also physiologically prevent muscle dysfunction and atrophy by buffering ROS , lowering systemic inflammation , and modulating cell functions . Alpha-tocopherol, zinc, and selenium can protect the muscle against oxidative stress and inflammation in vitro and in vivo [11, 12] and are critical to muscle homeostasis . The combination of antioxidant deficiencies impairs the muscle redox state  and has been implicated in aging sarcopenia  and inflammatory diseases [15, 16]. Conversely, increasing the antioxidant content through dietary vitamin/trace element supplements [14–17] or regular moderate-intensity training  alone or in combination  has improved both systemic oxidative stress or inflammation and muscle function. Significantly, supplementation concomitant with training was efficient only in subjects with preexisting antioxidant deficits .
Last, in contrast to healthy individuals, COPD patients experience baseline antioxidant (vitamin C, vitamin C/E ratio, zinc, and selenium) deficits [7, 20], which worsen after training . Thus, training alone did not reduce the oxidative damage and inflammation in patients . Given the role of antioxidants in muscle dysfunction and the muscles’ lack of improvement with training, antioxidants constitute a candidate for the blunted muscle response to training in COPD. Currently, only one study has tested the combination of training and antioxidant supplementation in COPD patients and showed no additional effect of the combination versus training alone. However, the single supplemented antioxidant did not increase its cellular target (i.e., the GSH/GSSG ratio)  or improve the other antioxidant deficits observed in COPD patients [7, 20].
In order to assess the potential of combining exercise training and antioxidant supplementation to target antioxidant deficits in stable COPD patients , we therefore conducted a randomized double-blind controlled trial during a PR program. We specifically tested the effects of oral antioxidant supplementation with vitamins and trace elements (i.e., vitamins C and E, zinc, and selenium) versus placebo on muscle endurance (primary outcome) and muscle strength, oxidative stress, inflammation, and PR outcomes (secondary outcomes).
2. Material and Methods
2.1. Study Design, Randomization, and Ethics
In this 28-day monocentric trial, COPD patients were randomly allocated (1 : 1 ratio) with permuted blocks of four patients, ensuring an equal number of patients receiving antioxidant supplementation (PR antioxidant group) and placebo (PR placebo group) during a PR program. The program included exercise training and followed the recommendations for chronic respiratory patients . All patients received a detailed information letter about the study before providing written informed consent. Randomization was carried out after consent was obtained and centralized by computer software at the medical information department of Montpellier University Hospital. Investigators, PR caregivers, the statistician, and COPD patients were blinded to the nature of supplementation until trial completion. The study was conducted in accordance with good clinical practice and the Declaration of Helsinki. It was approved by the ethics committee Montpellier Sud-Mediterranée IV (no. 2011-A00842-39). The trial was preregistered in https://www.clinicaltrials.gov (ClinicalTrials.gov identifier: NCT01942889).
Stable COPD patients (40 to 78 years old) referred to the La Solane Pulmonary Rehabilitation Center (5 Santé /Fontalvie Corporation, F-66340, Osséja, France) were recruited. COPD was defined by the occurrence of dyspnea, chronic cough or sputum production, and/or a history of exposure to risk factors for the disease and postbronchodilatator %, evaluated by plethysmography (Body Box 5500, Medisoft, Belgium) according to the validated methodology . The exclusion criteria were as follows: exacerbations within the last month, unstable disease incompatible with a PR program, antioxidant supplementation (vitamins, trace elements, etc.) or use of drugs such as allopurinol and N-acetylcysteine within the last month, and use of oral corticosteroids over the last six months. The BODE index was determined to assess global disease severity . It was determined from the body mass index (B), the degree of airflow obstruction with (O), dyspnea (D) as assessed by the Medical Research Council (MRC) scale, and exercise capacity (E) measured by the 6-minute walk test (6MWT).
In line with the current guidelines for pulmonary rehabilitation, 24 sessions of endurance exercise (stationary cycling, walking) over 28 days were proposed . Endurance training intensity was individualized for each patient and corresponded to the target heart rate at the ventilatory threshold , as assessed during a maximal cardiopulmonary exercise test. For each patient, the ventilatory threshold was assessed blindly by two investigators. This intensity was continuously monitored with a heart rate monitor, and the workload was progressively increased. Each session lasted 1 h and 30 min: 45 minutes of endurance training completed by 30 minutes of strength exercise (8-10 exercises, with sets of 10-15 repetitions) progressively increased using a perceived exertion scale (with a target of 5-6 on a 10-point scale) . All sessions were supervised blindly by an experienced clinician. This exercise training was part of a multicomponent PR program. In order to promote long-term health behavior changes and long-term adherence, the PR also included dietary counseling, smoking cessation help, and educational interventions when needed .
Oral capsules of the antioxidant supplements or placebo were delivered to the patients daily by the nurses of the PR center. In accordance with the deficits observed previously in COPD patients , the antioxidant supplementation was associated with vitamin E (α-tocopherol: 30 mg/day), vitamin C (ascorbate: 180 mg/day), zinc gluconate (15 mg/day), and selenium (selenomethionine: 50 μg/day). Although no vitamin E deficit was observed in the COPD patients, it was administered because of its synergistic effects with ascorbate . Vitamin E was given as α-tocopherol because this is the form preferentially absorbed by humans, and it has more antioxidant effects than other tocopherols or tocotrienols . The nutritional supplementation doses were in line with (selenium) or above (vitamins C and E and zinc) the Recommended Dietary Allowances and Adequate Intakes (Food and Nutrition Board) .
2.4.1. Primary Outcome
The quadriceps endurance time (Qend, in seconds) may be more sensitive to interventions targeting the limb muscles, and the international guidelines therefore recommend its assessment in research studies . The quadriceps endurance time was determined only for the dominant leg, as previously described by our group . The patients performed knee extensions (6 movements per minute) with a workload to 30% of quadriceps maximal voluntary isometric contraction () until exhaustion. Immediately after this test, they performed a to evaluate quadriceps fatigue, and a reduction in % was necessary to validate the test, as previously published .
2.4.2. Secondary Outcomes
Secondary outcomes included changes in (1) quadriceps maximal voluntary isometric contraction () and (2) oxidative stress (plasma vitamin and trace elements, lipid peroxidation, and GSH/GSSG ratio), as previously described . was compared with the reference values. The predicted in kg was given by the following regression equation:
The residual standard deviation from the analysis was 8.58 kg, and patients with (observed-predicted )/ were considered weak .
In addition, we assessed validated parameters of inflammation, muscle mass, exercise capacity, and nutritional status, as previously described . Briefly, the muscle mass was assessed by bioelectrical multifrequency segmental impedance (Biacorpus RX Spectral; Medical HealthCare GmbH, Karlsruhe, Germany), as previously described . Muscle biopsies were performed in the vastus lateralis of the quadriceps before and after the interventions as previously described . Muscle fiber type and mean cross-sectional area (CSA) were assessed after immunohistochemistry on frozen sections, using anti-MHCI monoclonal antibodies . The nutritional status was assessed by the evaluations of the body mass index (BMI) and body composition by bioelectrical multifrequency impedance. Total serum protide, albumin, and prealbumin were determined on venous blood collected in dry tubes. Samples were immediately centrifuged and frozen (-80°C) until tested later. Total serum protide, albumin, and prealbumin were determined using an immune electrochemiluminescence assay on the Cobas 8000/e602® immunochemistry system (Roche Diagnostics, Meylan, France). Last, the inflammation was assessed by a dosage of highly sensitive C-reactive protein (hsCRP) on venous blood collected in dry tubes. Samples were immediately centrifuged and frozen (-80°C). Determination of CRP was run on the Cobas 8000/e502® analyzer (Roche Diagnostics, Meylan, France) using the immunoturbidimetric method .
Treatment compliance was recorded daily by the nurses. Adverse events, the number of patients experiencing acute exacerbations of COPD (defined as an acute worsening of respiratory symptoms that results in additional therapy) , and additional medications (indication, cumulative systemic corticosteroid dose, and treatment length) were recorded and examined as covariables. Last, physical training (training session attendance, intensity) and micronutrient intake are influenced by pulmonary rehabilitation interventions, and therefore, these confounding variables were also recorded before and during the study.
2.5. Sample Size and Statistical Analysis
Sample size was calculated based on the change in Qend in seconds . We estimated that with a sample of 28 patients per group evaluated for the primary outcome, the study had at least 80% power to determine the superiority of the PR antioxidant group compared with the PR placebo group. For the intention-to-treat analysis, the following assumption was made: Qend change of s in the PR antioxidant group versus s in the PR placebo group. As we assumed a dropout rate of 10%, we included 32 stable COPD patients per group. Quantitative data are presented as deviation (SD), and qualitative data are described using proportions. To compare all the parameters between the PR placebo and PR antioxidant groups at baseline and to examine within-group relative variation, we used a Student -test (or a Mann-Whitney for nonnormally distributed variables) for quantitative data and Pearson’s chi-squared test for qualitative data. To assess the group effect on the relative variation, linear regressions were performed and covariable effects and their interactions were tested. If a confounder was identified, adjusted outcome analyses were performed by adding the confounder factor as a covariable of the test. For all analyses, the level of significance was set at . Statistical analyses were performed on an intention-to-treat basis using R version 3.3.1 and SAS version 9.3 (SAS Institute, Cary, NC).
3.1. Study Flow Chart and Population
Patients we enrolled between 3 June 2012 and 17 December 2014. As shown in the CONSORT flow diagram (Figure 1), 64 of the 389 screened COPD patients were randomized to receive placebo (PR placebo group, ) or antioxidant supplementation (PR antioxidant, ), 57 COPD patients completed the study and were included in the analysis, and 43 patients underwent muscle biopsy.
Postbronchodilatation. Results are expressed as . Definition of abbreviations: COPD: chronic obstructive pulmonary disease; PR: pulmonary rehabilitation; W/M: women/men; BMI: body mass index (kg/m2); (%pred): forced expiratory volume in 1 second; BODE index: body mass index, airway obstruction, dyspnea, exercise capacity index (); MRC: Medical Research Council; (m): six-minute walking distance; : symptom-limited power output; : symptom-limited oxygen uptake; VT: ventilatory threshold; (N·m-1): quadriceps maximal voluntary contraction expressed in Newtons; Qend (s): quadriceps endurance time expressed in seconds; CSA: cross-sectional area expressed in μm2.
Results are expressed as . In the first column, we specify the lower and upper limits of reference values obtained in a large healthy subject cohort, as previously described . Definition of abbreviations: PR: pulmonary rehabilitation; M: male; W: women; GSH: reduced glutathione; GSSG: oxidized glutathione; SOD: superoxide dismutase; GPx: peroxidase glutathione; LDL: oxidized low-density lipoprotein.
When measured values were compared with predicted values, the COPD patients (49% males, %pred) were exercise-intolerant (symptom-limited oxygen uptake %pred and 6-minute walking distance %pred) and had muscle weakness (%pred). Based on the reference values, 81% had at least one nutritional antioxidant deficit, with 26%, 24%, and 67%, respectively, deficient in vitamin C, zinc, and selenium. As shown in Table 1, no significant differences were noted between the two groups for age, sex ratio, disease severity, body composition, or functional and skeletal muscle parameters. The GSH/GSSH ratio in the PR antioxidant group tended to be higher than that in the PR placebo group (454 ± 281 vs. 334 ± 241, respectively; , Table 2). The characteristics of the COPD patients who underwent muscle biopsy did not differ significantly from those of the whole group (Table 3).
Results are expressed in . Definition of abbreviations: W/M: women/men; BMI: body mass index (kg/m2); (%pred): forced expiratory volume in 1 second; (m): six-minute walking distance; : symptom-limited oxygen uptake; (N·m-1): quadriceps maximal voluntary contraction expressed in Newtons; Qend (s): quadriceps endurance time expressed in seconds; CSA: cross-sectional area expressed in μm2.
As shown in Table 4, PR did not differ between groups in terms of training, treatment compliance, or micronutrient intake. However, despite a similar number of acute exacerbations, the PR placebo patients received more short-term systemic corticosteroid treatment than the PR antioxidant patients (31% vs. 6%, respectively; ).
Results are expressed as . Definition of abbreviations: PR: pulmonary rehabilitation; : symptom-limited oxygen uptake; VT: ventilatory threshold; RDA: Recommended Dietary Allowances and Adequate Intakes.
3.2. Effect of the PR Program
The significant improvements in quadriceps endurance and exercise capacity (symptom-limited workload: and ) in the PR placebo patients show that the 4-week moderate-intensity training program was efficient. In addition, the PR placebo group showed improved outcomes that were driven not only by training (Tables 5 and 6), as we observed improvements in vitamin C ( to ; ) and GPx ( to ; ).