Exercise-Related Oxidative Stress and Inflammatory Response in COPD are Modulated by Dichloroacetate

Dichloroacetate Modulates the Oxidative Stress and Inflammatory Response to Exercise in COPD 1. Evi M. Mercken, MSc, 2. Lori D. Calvert, MD, 3. Sally J. Singh, PhD, 4. Geja J. Hageman, PhD, 5. Annemie M. Schols, PhD and 6. Michael C. Steiner, MD + Author Affiliations 1. Affiliations: From the Departments of Respiratory Medicine (Ms. Mercken and Dr. Schols), and Health Risk Analysis and Toxicology (Dr. Hageman), School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, the Netherlands; and the Department of Respiratory Medicine (Drs. Calvert, Singh, and Steiner), Institute for Lung Health, University Hospitals of Leicester National Health Service Trust, Leicester, UK. 1. Correspondence to: Evi M. Mercken, MSc, Department of Respiratory Medicine, Maastricht University, PO Box 5800, 6202 AZ Maastricht, the Netherlands; e-mail: Next Section Abstract Background: Impaired skeletal muscle function contributes to exercise intolerance in patients with COPD. Exercise-induced oxidative stress may initiate or accelerate impaired muscle function. Dichloroacetate (DCA) activates muscle pyruvate dehydrogenase complex (PDC) at rest, reducing inertia in mitochondrial energy delivery at the onset of exercise and thereby diminishing anaerobic energy production. This study aimed to determine whether DCA infusion also may reduce exercise-induced systemic oxidative stress and inflammatory response in patients with COPD. Methods: A randomized, double-blind crossover design was used in which 13 patients with COPD performed maximal cycle exercise after an IV infusion of DCA (50 mg/kg body mass) or saline solution (placebo). Venous blood was sampled before exercise, and immediately, 30 min, and 2 h after exercise. Urine samples were obtained before and 2 h after exercise. Results: Peak workload improved significantly after DCA infusion compared to placebo (10%; p < 0.01). Urinary uric acid levels after exercise were significantly lower in the DCA condition than in the placebo condition, whereas no significant difference was observed for urinary malondialdehyde levels. Oxidized glutathione (GSSG) levels were significantly increased 2 h after exercise in the placebo condition (p < 0.02) but not after DCA infusion. No changes in reduced glutathione (GSH), GSSG/GSH ratio, and superoxide dismutase activity were observed. Plasma interleukin (IL)-6 levels significantly increased 2 h after exercise only in the DCA condition (p < 0.01). Conclusions: This study shows that improved performance after a pharmacologic intervention known to activate PDC was accompanied by an enhanced IL-6 response and a limited reduction in exercise-induced systemic oxidative stress. Peripheral skeletal muscle dysfunction is a common feature in patients with COPD, and contributes to exercise intolerance, disability, and poor quality of life and survival.1 Chronic inflammation and oxidative stress have been implicated in the etiology of peripheral muscle dysfunction.2,3 Increased systemic oxidative stress has been demonstrated in patients with COPD at rest, which is further intensified after exercise.4,5 A reduction in skeletal muscle mitochondrial oxidative capacity, which also may contribute to exercise intolerance, has been observed in COPD patients1,6–8 and may result in significant muscle adenine nucleotide loss despite the low exercise absolute workload in COPD patients because adenosine triphosphate (ATP) resynthesis is unable to meet the energy demands of exercise.9 This situation has been described as metabolic stress because the reduction in adenine nucleotides available for phosphorylation is not sustainable. Metabolic stress has the potential to increase oxidative stress because of increased metabolism of purine nucleotide derivatives, leading to increased activity of xanthine oxidase, which is a known source of free radicals.10,11 However, the relationship between metabolic and oxidative stress induced by exercise has not been explored in COPD patients. Skeletal muscle mitochondrial ATP production is delayed at the onset of exercise, which results in a reliance on nonoxidative sources of energy production to meet the shortfall in ATP supply in the early stages of exercise. Some evidence12 has suggested that this metabolic inertia resides at the level of the pyruvate dehydrogenase complex (PDC), a mitochondrial, multienzyme complex that regulates carbohydrate entry into the tricarboxylic acid cycle and catalyzes the irreversible oxidation of pyruvate to mitochondrial acetyl-coenzyme A. Dichloroacetate (DCA), a pharmacologic agent, increases mitochondrial aerobic energy production through the activation of PDC. The mechanism of action takes place by the inhibition of PDC kinase.13 DCA has been used in humans for the treatment of mitochondrial diseases and lactic acidosis.14 Recently, Calvert et al15 demonstrated that by activating PDC, blood lactate accumulation decreases during exercise and maximal exercise performance increased in subjects with COPD. Moreover, this study suggested that DCA reduces adenine nucleotide loss, as evidenced by a reduction in exercise-induced ammonia accumulation following DCA infusion. In the present study, we aimed to explore the effect of DCA on exercise-induced oxidative stress in patients with COPD. We hypothesized that the DCA-mediated reduction in metabolic stress would result in a reduction in exercise-induced oxidative stress. Exercise is recognized to result in immediate effects on circulating levels of inflammatory cytokines. A rise in blood interleukin (IL)-6 levels is a normal physiologic response to exercise.16 In this context, IL-6 is thought to originate from skeletal muscle and may have important antiinflammatory effects, including an inhibitory effect on proinflammatory cytokines such as tumor necrosis factor (TNF)-α.17 Evidence18 also shows that exercise-induced IL-6 production is sensitive to muscle carbohydrate utilization, and it has been suggested that IL-6 may be an “energy-sensing” myokine that has an important regulatory role in the longer term antiinflammatory effects of exercise and training. An additional aim of the present study, therefore, was to explore the effect of the DCA-mediated improvements in skeletal muscle energy delivery on the exercise-induced IL-6 and TNF-α response in patients with COPD. Previous SectionNext Section Materials and Methods Study Population The decision to undertake the current investigation was made after the start of the original trial; therefore, 13 of the 18 patients with stable COPD examined in the study of Calvert et al15 were included. Patients met the clinical and spirometric Global Initiative for Chronic Obstructive Lung Disease criteria for COPD.19,20 Patients were excluded if they were receiving maintenance therapy with oral corticosteroids, were unable to perform exercise tests, or had experienced exercise desaturation (arterial oxygen saturation, < 80%), cardiac dysfunction, exacerbation of their condition within the previous 6 weeks, or received pulmonary rehabilitation within the past year. Full approval was obtained from the Leicestershire Research Ethics Committee, and all participants provided informed written consent. Assessment of Body Composition Body mass index was calculated from height measured by wall-mounted stadiometer to the nearest 0.1 cm, and weight was measured with the patient in light clothing to the nearest 0.1 kg. Fat-free mass was estimated with a lightweight bioimpedance analyzer (Bodystat 1500; Bodystat Ltd; Douglas, UK) and was calculated using disease-specific regression equations.21 Pulmonary Function Tests Spirometry (Model R; Vitalograph; Buckingham, UK) was performed to European Respiratory Society standards. Predicted values were calculated from European Respiratory Society20 regression equations. Study Design Details on the study design have been reported previously.15 Briefly, patients performed a maximal incremental exercise test on an electrically braked cycle ergometer. After a 2-week washout period, they repeated the exercise challenge. Prior to exercise, patients received either DCA (25 mg/mL sodium salt), 50 mg/kg body mass, or an equivalent volume of normal saline solution as an IV infusion into a forearm vein for > 45 min. Following the infusion, patients rested for 30 min before undergoing exercise to ensure that PDC activation was achieved. Peripheral venous blood samples were obtained with the patient at rest, and immediately, 30 min, and 2 h after performing the maximal incremental exercise test. Urine samples were obtained at rest and 2 h after the exercise test. Moreover, arterialized venous blood samples for lactate and ammonia analyses were taken at different time points during and after exercise, as described previously.15 Sample Preparation Venous blood samples were drawn into ethylenediaminetetraacetic acid-containing tubes. Plasma was obtained by centrifugation (800g for 10 min at 4°C) and stored at −80°C until analysis. Urine samples were collected in sterile containers and stored at −20°C until further analysis. Markers of Systemic Oxidative Stress Urinary malondialdehyde (MDA) and urinary uric acid were assessed by high-performance liquid chromatography as described by Lepage et al22 and Lux et al,23 respectively. Both urinary MDA and uric acid concentrations were corrected for creatinine content.24 Erythrocyte superoxide dismutase (SOD) activity was determined according to the method of Sun et al.25 Hemoglobin concentration was determined.26 Erythrocyte glutathione was measured in its reduced form27 and its oxidized form.28 Markers of Systemic Inflammation Plasma concentrations of IL-6 and TNF-α were determined with a quantitative, high-sensitivity, enzyme-linked immunosorbent assay kit (R&D Systems; Minneapolis, MN). Statistical Analysis Data are expressed as the mean ± SD or as the mean ± SEM. A paired t test was used to test the effect of treatment on exercise-induced biological markers. Putative correlations between variables were evaluated with Pearson product moment correlations. A difference of p < 0.05 was considered statistically significant. Statistical analyses were conducted with a statistical software package (SPSS for Windows, version 13.0; SPSS, Inc; Chicago, IL). Previous SectionNext Section Results Patients’ Personalities Table 1 displays anthropometric and spirometric data. Airflow blockage that was moderate in severity was present in the COPD group. Patients (n = 8) or current smokers (n = 5) were both smokers.). View this table: In this window In a new window Table 1 Characteristics of the Study Population Activity Capacity Table 2 displays the outcomes of the incremental exercise test carried out following infusions of DCA and normal saline solution. Peak effort considerably increased (10%; p 0.01) following DCA therapy. Additionally, after DCA, both peak oxygen consumption and peak ventilation were significantly higher. infusion compared with placebo (9% [p < 0.01%] and 12% [p < 0.02], respectively). Respiratory exchange ratio at peak exercise was significantly greater following DCA infusion (p < 0.001) than placebo. DCA infusion also significantly reduced blood lactate and ammonia accumulation compared to placebo (p < 0.01 and p < 0.001, respectively).15 View this table: In this window In a new window Table 2 Exercise Data From Maximal Cycle Exercise in Patients With COPD After DCA and Saline Solution Infusion Markers of Systemic Oxidative Stress and Inflammation The results of the systemic oxidative stress and inflammatory markers at rest and 2 h after exercise following placebo and DCA infusion are shown in Table 3. At other time points, we observed no significant differences in our outcome variables. Baseline values of systemic oxidative stress and inflammation were not significantly different between both conditions. Urinary uric acid at 2 h after exercise was significantly lower in the DCA condition than in the placebo condition (p < 0.05) [Fig 1A]. For urinary MDA, no significant differences between the groups were observed 2 h after exercise (Fig 1B). No significant effect of exercise was observed for erythrocyte reduced glutathione (GSH) levels after both conditions, whereas erythrocyte oxidized glutathione (GSSG) levels were significantly elevated 2 h after exercise in the placebo condition (p < 0.02) [Fig 2]. However, GSSG/GSH ratio values were unchanged after both conditions. Furthermore, no significant exercise-induced change in erythrocyte SOD activity was observed after both conditions. View this table: In this window In a new window Table 3 Markers of Systemic Oxidative Stress and Inflammation in Patients With COPD at Rest and 2 h After Exercise Following DCA and Saline Solution Infusion View larger version: In this page In a new window Download as PowerPoint Slide Figure 1 A: Concentration of urinary uric acid 2 h after exercise in placebo and DCA conditions. B: urinary MDA level 2 h after exercise in placebo and DCA conditions. * = p < 0.05 (significantly different 2 h after exercise between patients in the placebo and DCA groups). View larger version: In this page In a new window Download as PowerPoint Slide Figure 2 Effect of exercise on erythrocyte GSSG levels in placebo and DCA conditions. * = p < 0.02 (significantly different from baseline values). As shown in Figure 3, the exercise-induced IL-6 response was significantly higher in the DCA condition than in the placebo condition (p < 0.001), and IL-6 levels were significantly increased 2 h after exercise following DCA treatment (p < 0.01). No significant exercise-induced effects were observed for TNF-α after both conditions. However, a negative correlation was observed between exercise-induced changes in IL-6 and TNF-α levels (r = −0.80; p < 0.01) [Fig 4] after DCA treatment. No correlation was found between exercise-induced changes in IL-6 and TNF-α levels after the placebo condition, although there were two outlying values for TNF-α that may have affected this association (see Fig 4). No significant correlations were found between changes in the levels of blood markers of the metabolic response and markers of oxidative stress and inflammation. View larger version: In this page In a new window Download as PowerPoint Slide Figure 3 Effect of exercise on plasma IL-6 levels in placebo and DCA conditions. * = p < 0.01 (significantly different from baseline values). # = p < 0.001 (significantly different between placebo and DCA groups). View larger version: In this page In a new window Download as PowerPoint Slide Figure 4 There was an inverse and significant correlation between exercise-induced changes in IL-6 and TNF-α after DCA infusion (r = −0.80; p < 0.01). Previous SectionNext Section Discussion This study shows that a pharmacologic intervention with DCA, which has been previously reported15 to improve exercise performance and reduce metabolic stress in patients with COPD, resulted in a reduction in the levels of markers of exercise-induced oxidative stress (erythrocyte GSSG and urinary uric acid levels) and led to an enhanced exercise IL-6 response in patients with COPD. We also observed an inverse relationship between exercise-induced changes in IL-6 and TNF-α levels after DCA treatment. We15 previously reported that DCA improves the supply of ATP from oxidative sources and thereby attenuates adenine nucleotide loss, as measured by a reduction in ammonia accumulation. Consequently, this enhanced mitochondrial (oxidative or aerobic) energy production could play a role in decreasing exercise-induced systemic oxidative stress and inflammatory response in patients with exercise limitation due to COPD. During strenuous exercise, ATP is consumed at a greater rate than it can be replenished, leading to adenine nucleotide degradation followed by increases in the plasma concentrations of hypoxanthine, xanthine, and uric acid. Uric acid is an indicator of xanthine oxidase activity, which is seen as an important source of reactive oxygen species.10,11 In the present study, urinary uric acid levels were significantly lower after exercise in the DCA condition than in the placebo condition, which could point to the greater production of free radicals due to xanthine oxidase activity in patients in the placebo group. In addition, the higher exercise-induced urinary uric acid levels after placebo condition also might reflect greater muscle adenine nucleotide loss during exercise because the formation of uric acid is irreversible. This finding is supported by the previously published data of Calvert et al,15 who observed greater blood ammonia accumulation after placebo infusion compared with DCA infusion. This finding also is in keeping with previous data9,29 showing that adenine nucleotide loss, which is associated with a rise in blood ammonia levels, occurs during exercise in COPD patients. These observations indicate that ATP resynthesis is unable to meet the energy demands of exercise after placebo treatment, suggesting that metabolic stress occurs in patients with COPD during exercise, despite the low absolute exercise work rates these individuals can achieve. This aberrant energy production can be countered by DCA, which activates PDC at the onset of exercise, leading to an increase in the provision of mitochondrial acetyl-coenzyme A and resulting in an exponential rise in mitochondrial ATP production.27,30,31 We observed increased blood GSSG levels after placebo treatment, also indicating increased oxidative stress; however, no between-group differences were observed for GSH, for the GSSG/GSH ratio, or for the activity of the antioxidant enzyme SOD between the two conditions. Similarly, no significant difference in urinary MDA levels between patients under both conditions was found after exercise. The reduction in exercise-induced erythrocyte GSSG levels coupled by a reduction in urinary uric acid accumulation following DCA suggests a potential link between skeletal muscle metabolic and oxidative stress, which warrants further study. However, taken together, these results suggest that in COPD, the DCA-mediated reduction in metabolic stress only had a limited effect on wider markers of exercise-induced oxidative stress. Furthermore, we were unable to find significant correlations between changes in markers of oxidative and metabolic stress. Differences may exist in the kinetics of formation and disposal of ammonia, lactate, and oxidation products, and we recognize that further research involving larger sample sizes will be required to address these questions. A marked increase in plasma IL-6 levels after exercise has been a consistent finding in healthy subjects.32 It has been suggested,16 therefore, that contraction-induced IL-6 production in skeletal muscle may mediate several diverse metabolic and physiologic effects. IL-6 has been proposed to mediate glucose homeostasis during exercise by stimulating hepatic glucose production. In the present study, there was a significant difference in the exercise-induced IL-6 response between patients receiving DCA and placebo infusions such that plasma IL-6 levels were significantly increased after exercise following DCA infusion only. Moreover, a negative correlation between changes in IL-6 and TNF-α levels was observed after DCA treatment. This finding is in line with previous studies17,33,34 showing that increased IL-6 levels inhibit the production of the proinflammatory cytokine TNF-α. Our data are consistent with previous studies35 supporting the hypothesis that IL-6 has important antiinflammatory effects in relation to exercise. Following exercise, the high circulating levels of IL-6 are followed by increased circulating levels of well-known antiinflammatory cytokines, such as IL-1ra and IL-10.36,37 Therefore, IL-6 induces an antiinflammatory environment by inducing the production of IL-1ra and IL-10 on the one hand and suppressing TNF-α production on the other. The mechanism by which the DCA reduction in metabolic stress increased exercise-induced IL-6 production cannot be determined from our data. Possibilities include a direct effect of increased flux through mitochondrial oxidative phosphorylation (with the concomitant reduction in the accumulation of products of anaerobic metabolism, such as lactate) or a reduction in traffic through the purine nucleotide degradation pathway through an improvement in ATP resynthesis. Our observation of reduced urinary uric acid accumulation following DCA infusion suggests that the adenine nucleotide metabolism through the purine nucleotide pathway was indeed reduced, although we cannot determine from our data whether this was responsible for the alteration in the inflammatory response. In addition, DCA is known to result in the preferential oxidation of carbohydrate during exercise. The purported role of IL-6 in exercise-related glucose homeostasis,18 therefore, could be relevant to our observations. Clinical Implications The intriguing finding of the present study is that treatment with DCA appeared to restore the expected IL-6 response to exercise that was absent following treatment with placebo, suggesting that increasing the supply of ATP from oxidative sources may have an influence on the inflammatory response to exercise in COPD patients. This finding could have implications for the systemic effects of regular physical activity for patients with COPD, which may be characterized by excess anaerobic energy production. The wider clinical implications of these observations and the impact of interventions such as aerobic training remain to be determined, but our observation of an inverse relationship between IL-6 and TNF-α is tantalizing because TNF-α is thought to be a key intermediary in the development of insulin resistance and skeletal muscle wasting in patients with COPD and other chronic diseases.38,39 Limitations of the Study We acknowledge several limitations to the interpretation of our data. The study involved a relatively small number of patients and, therefore, may have lacked the statistical power to detect significant between-group differences in the exercise response. The correlation between the IL-6 and TNF-α responses was seen only in the DCA group. Given the small sample size, the two outlying values for TNF-α in the placebo group may have affected the correlation calculation (Fig 4). We found a difference in postexercise urinary uric acid levels between patients in the two conditions. The change from baseline urinary uric acid values is difficult to interpret because dietary intake (known to affect urine uric acid excretion) was not controlled at baseline. Moreover, the postexercise measurement represents accumulation for the specific duration of the exercise test, which would be different from the baseline test. We recognize that our observations were made during a standardized, maximal cycle exercise test. The reproducibility of these findings following submaximal exercise and with other exercise modalities, such as walking, will be needed to determine the wider clinical implications of observations. Finally, we did not directly measure the activity of PDC, but the data on activation by DCA in this dose are fairly robust.40–42 In summary, we have shown that the modulation by DCA of the skeletal muscle energy response to exercise in COPD patients may have implications for the oxidative stress and inflammatory response. Targeting the PDC may be a useful means of studying the wider consequences of metabolic stress during exercise and aerobic training in patients with COPD. Previous SectionNext Section Acknowledgments Author contributions: Dr. Calvert was responsible for data collection. Ms. Mercken analyzed the data and wrote the manuscript. Drs. Singh, Hageman, and Schols were responsible for reviewing the manuscript. Dr. Steiner was responsible for the design of the study, data analyses, and review of the manuscript. All authors read, commented on, and contributed to the submitted manuscript. Financial/nonfinancial disclosures: The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Other contributions: We thank Jeff Graham in the Pharmacy Department at Nottingham University Hospitals NHS Trust, University Hospital Campus (UK), and Eoin Barrett in the Pharmacy Department at Leicester Royal Infirmary, University Hospitals of Leicester NHS Trust (UK), for supervising the preparation of DCA and placebo and allocation of randomization. Previous SectionNext Section Footnotes This research was supported by a University Hospitals of Leicester NHS Trust award of a personal research fellowship to Dr. Calvert to conduct this study. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( Abbreviations:: ATP adenosine triphosphate DCA dichloroacetate GSH reduced glutathione GSSG oxidized glutathione IL interleukin MDA malondialdehyde PDC pyruvate dehydrogenase complex SOD superoxide dismutase TNF tumor necrosis factor Received December 11, 2008. Accepted March 30, 2009. © 2009 American College of Chest Physicians Previous Section References 1. ↵ (1999) Skeletal muscle dysfunction in chronic obstructive pulmonary disease: a statement of the American Thoracic Society and European Respiratory Society. Am J Respir Crit Care Med 159:S1–S40. FREE Full Text 2. ↵ 1. Agusti AG (2005) Systemic effects of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2:367–370. Abstract/FREE Full Text 3. ↵ 1. Berton E, 2. Antonucci R, 3. Palange P (2001) Skeletal muscle dysfunction in chronic obstructive pulmonary disease. Monaldi Arch Chest Dis 56:418–422. 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