Association of oxidative stress markers and C-reactive protein with multidimensional indexes in COPD

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1 Original Paper Association of oxidative stress markers and C-reactive protein with multidimensional indexes in COPD Chronic Respiratory Disease 8(2) ª The Author(s) 2011 Reprints and permission: sagepub.co.uk/journalspermissions.nav DOI: / crd.sagepub.com F Folchini 1, NL Nonato 2, E Feofiloff 2, V D Almeida 3, O Nascimento 2, and JR Jardim 2 Abstract To evaluate the oxidative stress and the C-reactive protein (CRP) in chronic obstructive pulmonary disease (COPD) patients and their correlation between the severity of the disease according to GOLD criteria and multidimensional indexes such as BODE index. A blood sample was collected for thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase, glutathione (GSH), homocysteine (HCY) and CRP analysis from 45 stable COPD patients. Lung function, body nutritional status, dyspnea and 6-min walk test (6MWT) were evaluated. Patients with GOLD stage IV presented a higher value for the TBARS than stage I patients (4.47 þ 1.58 versus 2.27 þ 1.04 nmol/ml, p < 0.05). CRP was higher for GOLD IV (2.46 þ 3.68 mg/dl) than other stages (GOLD I: 0.39 þ 0.25, GOLD II: 0.39 þ 0.18 and GOLD III: 0.48 þ 0.36 mg/dl, p < 0.05). Oxidative stress markers measured as TBARS presented a negative correlation between forced expiratory volume in the first second (FEV 1 ) post bronchodilatador (% predicted; r ¼ 0.39, p ¼ 0.01) and positive correlations with Modified Medical Research Council Scale (MMRC) dyspnea index (r ¼ 0.40, p ¼ 0.01), multidimensional index (r ¼ 0.49, p ¼ 0.001) and BODE index (r ¼ 0.51, p ¼ 0.001). Keywords COPD, oxidative stress, pathophysiology, C-reactive protein, multidimensional index Introduction Chronic obstructive pulmonary disease (COPD) is characterized by a progressive and not completely reversible obstruction of the airways associated with an abnormal inflammatory response of the lungs to particles and noxious gases 1 related to a systemic effect. 2 However, the mechanisms of these effects are not completely known and are likely related to systemic inflammation, oxidative stress, hypoxia and a sedentary lifestyle. 3,4 Oxidative stress plays an important role in lung damage and inflammatory response in COPD. Oxidative stress may affect extra-cellular matrix remodeling, mitochondrial respiration, cell proliferation and lung defense mechanisms. Repair mechanisms and the immune modulation system are directly affected by oxidative stress, which is one of the main events in inflammatory response. 5 One of the consequences of systemic oxidative stress in COPD is muscle dysfunction, comprising of muscle fiber damage, less muscle protein synthesis, protein degradation and increase in muscle apoptosis. Cachexia and loss of non-fat mass may also increase with oxidative stress. 6 Reactive substances to thiobarbituric acid are markers for assessing lipid damage from oxidative stress and consist of the reaction of thiobarbituric acid with malondialdehyde, which is the end product of the peroxidation of the lipid membrane. 7 The antioxidant 1 Federal University of São Paulo (Unifesp/Lesf), Rua Botucatu, São Paulo (SP), Brazil 2 Pulmonary Rehabilitation Center of Federal University of São Paulo, Rua Botucatu, São Paulo (SP), Brazil 3 Institute of Genetics and Inborn Errors of Metabolism (IGEIM) of Federal University of São Paulo (Unifesp), Rua Botucatu, São Paulo (SP), Brazil Corresponding author: José R Jardim, Respiratory Diseases (Pneumologia Unifesp), Rua Botucatu, Andar; São Paulo (SP), Brazil joserjardim@yahoo.com.br

2 102 Chronic Respiratory Disease 8(2) enzymes evaluated are those that catalyze the reactions of reactive oxygen species in water. Glutathione is the main substance of the antioxidant defense system. Correlations between oxidative stress and forced expiratory volume in the first second (FEV 1 ) severity in COPD patients have been described, 8 but there is no study correlating oxidative stress with multisystemic COPD indexes. The multidimensional BODE index is a predictor of mortality in patients with COPD, assessing the pulmonary component as well as the systemic characteristics of the disease, evidence that COPD includes diverse aspects that should be analyzed when severity and prognosis are to be assessed. 9,10 It is possible that oxidative stress is better correlated with multisystemic indexes than to isolated parameters that evaluate COPD severity. Homocystein (HCY) is a non-essential amino acid metabolite of the amino acid methionine. It has been used as a cardiovascular disease marker. The selfoxidation of homocysteine in the presence of molecular oxygen forms reactive oxygen species, evidencing a pro-oxidant role. At high concentrations, HCY can induce oxidative damage. 11 Tobacco smoking is one of the factors that contributes to the development of hyper-homocysteinemia and COPD. 12 However, studies on the possible association between HCY and COPD are scarce and inconclusive. The aim of the present study was to assess blood levels of oxidative stress markers through the measurement of substances reactive to thiobarbituric acid (TBARS), superoxide dismutase (SOD), glutathione (GSH), catalase, homocysteine (HCY) and blood inflammation through the measurement of C-reactive protein (CRP) in COPD patients, correlating these variables with the severity of the disease through both FEV 1 according to GOLD criteria and the multidimensional indexes. Materials and methods Study design A cross-sectional sample of 45 stable COPD patients (according to GOLD criteria) 1 who attended the COPD unit at the Pulmonary Rehabilitation Center of the Universidade Federal de São Paulo (Unifesp) Lar Escola São Francisco, Brazil, were chosen for the study. The study was approved by the Ethical Committee of Unifesp and all patients signed an informed consent form. Inclusion criteria were Clinical diagnosis of COPD according to GOLD criteria, 1 clinical stability, no exacerbation during the previous 30 days. Exclusion criteria were as follow orthopedic diseases, rheumatic disease and any other inflammatory systemic diseases that would render testing impossible. Patients were submitted to a clinical evaluation and a blood sample was collected for analysis. Clinical assessment A comprehensive clinical evaluation of the patients was done, including lung symptoms, clinical stability, use of medications, smoking status and dyspnea as perceived by the Modified Medical Research Council Scale (MMRC). This scale measures perceived respiratory disability. Patients were asked about their perceived breathlessness and were then classified into five dyspnea grades (0 minimal to 4 maximum) according to how they perceived their disability. 13 Spirometry was done pre and post-use of bronchodilator (albuterol 400 mcg) and FEV 1, forced volume capacity (FVC), FEV 1 /FVC were measured in accordance with the procedures of the American Thoracic Society on a portable ndd EasyOne 1 spirometer (Switzerland). Predicted values were calculated according to The Third National Health and Nutrition Examination Survey (NHANES III). 14 Body mass index (BMI) was obtained by dividing body weight by height squared (kg/m 2 ). The 6-min walk test (6MWT) was performed according to the American Thoracic Society Statement. 15 Patients were instructed to walk as far as possible for 6 minutes, taking rest periods if necessary. Before each test, the patient s resting heart rate, blood pressure and arterial oxygen saturation level were monitored. The total distance walked was measured to the nearest meter and recorded. BODE index and a multidimensional index were calculated. Blood sample and analysis For blood analysis, 12 ml of venous blood was collected and CRP, oxidative stress markers and homocysteine were analyzed. TBARS were analyzed in reactions with 20% trichloroacetic acid (TCA) and thiobarbituric acid (TBA), following the method described by Ohkawa et al. 16 Homocysteine was analyzed through high performance liquid chromotography (HPLC), following the method described by Pfeiffer et al. 17 Total glutathione was determined by means of reactions with b-nicotinamide adenine dinucleotide phosphate (NADPH) and 5,5-Dithiobis-2-nitrobenzoic acid (DTNB), with the enzyme glutathione reductase

3 Folchini et al. 103 Table 1. Characteristic of 45 patients with chronic obstructive pulmonary disease Variables Values Variables Values Age (years) a MMRC a Smoking (Pack/yr) a FEV 1 /FVC post-bd a Active smokers, n (%) 18 (40) FVC post-bd (L) a Former smokers, n (%) 27 (60) FVC post-bd (%) a Weight (kg) a FEV 1 post-bd (L) a Height (m) a FEV 1 post-bd (%) a BMI (kg/m 2 ) a MWT (m) a Multidimensional index a BODE a Abbreviations: BMI: body mass index, MMRC: Modificated Medical Research Council, FEV 1 \FVC post-bd: forced expiratory volume in the first second\forced vital capacity ratio post bronchodilator, FVC post-bd (L): forced vital capacity (L) post bronchodilator, FVC post-bd (%): forced vital capacity % predicted, FEV 1 post-bd (L): forced expiratory volume in the first second post bronchodilator (L), FEV 1 post BD (%): forced expiratory volume in the first second post bronchodilator % predicted, 6MWT (m): 6-min walk test in meters, Multidimensional index: body mass index, Modificated Medical Research Council and Forced expiratory volume in the first second, BODE index: body mass index, forced expiratory volume in the first second, Modificated Medical Research Council and 6-min walk test in meters. a Mean + SD. and a standard for oxidized glutathione, following the method described by Tietze et al. 18 Catalase was analyzed through the reaction with Tris HCl 1 M EDTA 5 mm ph 8.0, following the method described by Adamo et al. 19 SOD was analyzed through reactions in the presence of the enzyme xanthine oxidase, following the method described by McCord et al. 20 Hemoglobin was measured by means of a spectrophotometric reading at a 540 nm, reacting with Drabkin s solution. Statistical analysis Data are expressed as mean and standard deviation. The Kolmogorov-Smirnov test was used to determine the distribution of the sample. Pearson s correlation was performed to assess possible associations between oxidative stress, homocysteine, severity of COPD, multidimensional indexes, the 6MWT and nutritional state. Analysis of variance (ANOVA) was used for the comparisons between groups with regard to COPD severity, with the Bonferroni post-test. The sample size was calculated using the lowest correlation coefficient obtained for a ¼ 0.01 and b ¼ 0.20, which determined a minimum of 37 individuals. 21 Results Anthropometric and baseline pulmonary function data of the 45 patients (31 men and 14 women) are presented in Table 1. A total of 21% had co-morbidities, the most common being systemic arterial hypertension. Fifty-one percent of the patients used no medication, 41% used b2 agonist and anticholinergic agents, 8% used inhaled corticosteroids. Assessment of oxidative stress markers and C-reactive protein A few samples were lost due to conditioning problems and/or blood coagulation. The actual number of analyzed samples are shown in Table 2. The mean TBARS was nmol/ml. The mean catalase, SOD, GSH were respectively U/mg of Hb; U/mg of Hb; mmol/g Hb. The mean HCY was mm andthemeancrp was mg/dl. TBARS exhibited a correlation with CRP (r ¼ 0.54; p < 0.01), but not with antioxidants and homocystein. CRP showed a negative correlation with 6MWT (r ¼ 0.48; p < 0.003), but not with HCY (r ¼ 0.21; NS), and weak and negative correlation with BMI (r ¼ 0.27; p < 0.01) (Table 3). Distribution of oxidative stress and CRP markers with COPD severity Table 4 displays the oxidative stress markers, CRP and HCY mean values according to the severity of COPD based on FEV 1. TBARS showed higher values for GOLD IV than GOLD I (p < 0.05). No differences between GOLD stages and antioxidant enzymes, glutathione or homocystein were observed. CRP values were significantly higher for GOLD IV in comparison with the other stages (p < 0.05). TBARS showed a negative correlation with post-bronchodilator FEV 1

4 104 Chronic Respiratory Disease 8(2) Table 2. Values of thiobarbituric acid reactive substances, antioxidant enzymes, glutathione, homocysteine and C-reactive protein. Variables Mean + SD Thiobarbituric acid reactive substances (nmol/ml; n ¼ 38) Catalase (U/mg of Hb; n ¼ 45) SOD (U/mg of Hb; n ¼ 45) GSH (mmol/ghb; n ¼ 41) HCY (mm; n ¼ 43) CRP (mg/dl; n ¼ 38) r = 0.51 p = Abbreviations: SOD: superoxide dismutase, GSH: glutathione, HCY: homocysteine, CRP: C-reactive protein. L r = 0.39 p = 0.01 Figure 2. Correlation of thiobarbituric acid reactive substances and BODE index. and oxidative stress markers measured by TBARS (r ¼ 0.49; p ¼ 0.001) was observed (Table 3). Assessment of exercise capacity The only observed correlation was between CRP and the distance traveled during the 6MWT (r ¼ 0.48; p < 0.003; Table 3). Figure 1. Correlation between thiobarbituric acid reactive substances and forced expiratory volume in the first second (FEV 1 ) post-bronchodilatador (%). (% predicted; r ¼ 0.39; p ¼ 0.01; Figure 1). The other markers were not correlated to FEV 1. MMRC dypsnea scale and oxidative stress parameters A positive correlation between TBARS and the MMRC dyspnea scale was found (r ¼ 0.40; p ¼ 0.01; Table 3). Assessment of oxidative stress markers and inflammation with a multidimensional index A multidimensional index comprising FEV 1, BMI and the MMRC dyspnea scale was evaluated. 10 Index values ranged from 1 to 7, with a mean of 2.9 þ 1.4 (Table 1). A positive correlation between this index L BODE index and correlations with oxidative stress, homocysteine and CRP The BODE index score was (Table 1) and correlated with TBARS (r ¼ 0.51; p ¼ 0.001; Figure 2) as with CRP (r ¼ 0.46; p < 0.004). No correlations between the BODE index and others oxidative stress markers were observed (Table 3). Discussion The principal finding of our study was the observation that oxidative stress and CRP correlated better with multidimensional indexes of patients with COPD than with the isolated factors or GOLD stage in a sample of patients. The main result that oxidative stress and inflammatory markers including CRP correlate better with multidimensional indexes confirms the systemic characteristics of the disease. We analyzed oxidative stress markers through validated methods that have been well described in the literature. 22 The assessment of TBARS is one of the most widely used and simplest marker methods for assessing lipid damage from oxidative stress. It consists of the reaction of TBA with

5 Folchini et al. 105 Table 3. Pearson s correlation coefficients between the oxidative stress, C-reactive protein values, homocystein and antioxidants variables. Variables Pearson (r) p Value TBARS versus CRP <0.001 TBARS versus catalase NS TBARS versus GSH NS TBARS versus SOD NS TBARS versus HCY 0.05 NS CRP versus BMI <0.01 TBARS versus BMI NS TBARS versus MMRC Multidimensional index versus TBARS CRP versus 6MWT <0.003 BODE index versus CRP <0.004 CRP versus HCY NS Abbreviations: TBARS: thiobarbituric acid reactive substances, CRP: C reactive protein, GSH: glutathione, SOD: superoxide dismutase, HCY: homocysteine, 6MWT (m): 6-min walk test in meters, multidimensional index: body mass index, Modificated Medical Research Council and forced expiratory volume in the first second, BODE index: body mass index, forced expiratory volume in the first second, Modificated Medical Research Council and 6-min walk test in meters. malondialdehyde, the main end product of the peroxidation of the lipid membrane. 7 The other antioxidant enzymes assessed are those that act as catalysts in reactions of reactive oxygen species in water. We also evaluated glutathione, which is the main component of the antioxidant defense system. Most of the patients in the present study were classified as GOLD stages I and II, reflecting the prevalence of the disease in Latin America according to the PLATINO study. 23 Therefore, comparisons of our results with the literature are limited, as other studies used patients with more severe COPD The values of the oxidative stress markers from the severe and very severe groups in our study were always higher than those of the patients with mild or moderate COPD, which is in keeping with what has been published. 28 Studies assessing other oxidative stress markers, such as 4-hydroxy-2-nonenal (4-HNE) in airways and alveolar epithelial cells, 28 and an analysis of condensed air 25 also found a negative correlation between these markers and the severity of the disease assessed through FEV 1 as we found in our study (r ¼ 0.39; p ¼ 0.01; Figure 1). Despite the difference between the response of the more severe patients and the moderate ones, 70% of our patients exhibited increased values for TBARS according to reference values of our laboratory. 22 Recently, Barreiro et al. 29 demonstrated that in severe COPD patients the chronic endurance exercise induced a substantial increase in quadriceps nitrosative stress. This was accompanied by an inefficient adaptation of the muscle antioxidant systems to the changes generated by chronic exercise in these patients exhibiting systemic effects of the disease. A multidimensional index comprising postbronchodilator FEV 1 values (predicted %), the MMRC dypsnea scale and body mass index 10 showed a better correlation with lipid peroxidation (r ¼ 0.49; p ¼ 0.001; Table 3) than with its isolated variables, thereby confirming that the use of multidimensional indexes reflect more accurately the COPD systemic process. We also noted an association between lipid peroxidation and the BODE index (r ¼ 0.51; p ¼ 0.001; Figure 2), which adds the 6MWT in its calculation to the three other previously mentioned parameters. Reactive C protein has been reported as a good risk predictor for cardiovascular disease 30 and is considered an important marker of inflammation in COPD and could be useful in monitoring the disease during treatment. 31 We found a significantly higher value among patients in GOLD stage IV than in the other stages (Table 4), reaffirming the role of inflammation in the pathogenesis of COPD. We also found that CRP correlated with the multidimensional index BODE. Other studies have shown that the BODE index is associated with nitrotyrosine, a protein degradation marker, 26 as well as with CRP. 24 Our results and the ones from these studies confirm the systemic nature of COPD and its better assessment through multidimensional parameters than through an isolated pulmonary parameter. High levels of CRP in COPD have been shown to be associated to poorer pulmonary function, 24 bronchial hyperreactivity 32 a rapid decline in FEV 33 1 ; hospitalization and death. 31 We showed that CRP was moderately related to TBARS. Block et al. 30 also demonstrated an association between CRP and MDA, 30 which confirms that inflammation is associated to oxidative stress in the pathogenesis of COPD. The negative correlation between CRP and the distance walked during the 6MWT found by us is similar to the findings of Pinto-Plata et al. 27 and Torres et al. 24 The fact that the correlation was stronger between CRP and the BODE index than a multidimensional index that does not take into account the 6MWT 10 seems to demonstrate that CRP is actually well associated with physical capacity. It was recently shown that physical activity reduces the risk of having high levels of circulating TNF-a and CRP. 34

6 106 Chronic Respiratory Disease 8(2) Table 4. Values of oxidative stress markers and CRP with GOLD severity of patients with chronic obstructive pulmonary disease Variables GOLD I GOLD II GOLD III GOLD IV TBARS (nmol/ml) n ¼ 14; n ¼ 10; n ¼ 8; n ¼ 6; a Catalase (U/mg of Hb) n ¼ 16; n ¼ 11; n ¼ 12; n ¼ 6; SOD (U/mg of Hb) n ¼ 16; n ¼ 11; n ¼ 12; n ¼ 6; GSH (mmol/ghb) n ¼ 14; n ¼ 11; n ¼ 11; n ¼ 5; HCY (mm) n ¼ 16; n ¼ 10; n ¼ 11; n ¼ 6; PCR (mg/dl) n ¼ 11; n ¼ 10; n ¼ 11; n ¼ 6; b Abbreviations: CRP: C-reactive protein, GSH: glutathione, HCY: homocysteine, SOD: superoxide dismutase, TBARS: thiobarbituric acid reactive substances. a Substances reactive to thiobarbituric acid higher in GOLD IV than in GOLD I (p < 0.05). b CRP higher in GOLD IV than in others severity stages(p < 0.05). The main non-enzymatic human antioxidant in the human organism is glutathione (GSH). We found mean GSH values slightly increased, which may express an attempt a compensation by the antioxidant system in response to the increase in oxidative stress. However, we observed no correlation with any of the parameters studied. Homocysteine is an important marker for cardiovascular disease 11 and its association to CRP is an efficient cardiovascular risk marker. 35 Homocysteine has been assessed in smokers, non-smokers and patients with COPD; higher values have been observed for the group with COPD than for smokers or non-smokers, thereby correlating it to FEV 1 and its annual rate of decline. 12 To our knowledge, this is the only study that to date has assessed the role of homocysteine in COPD. However, contrary to these findings, we did not find abnormal levels of homocysteine; also there was no association to physiological parameters, multidimensional indexes or tobacco consumption. Unlike patients in the study by Kai et al., 12 who had more severe COPD and hypoxemia, our sample of patients was made up predominantly of patients with mild and moderate COPD, which probably may explain why we did not find high levels of homocysteine. Cardiovascular complications are higher in patients with more severe COPD and hypoxemia. 12 Further studies are needed to assess the role of homocysteine in COPD: might homocysteine be an isolated marker for COPD or is it only increased when there is an associated cardiovascular co-morbidity? One partial limitation of this study was the relatively low number of severe and very severe patients; however, the literature is already quite clear with regard to the high degree of inflammation and high indices of oxidative stress in patients with severe COPD and muscle depletion, but the profile of oxidative stress in patients with mild COPD is not yet well clarified. The majority of our patients were classified in stages I and II (GOLD classification). Our aim was to analyze the situation of an outpatient clinic at a pulmonary lung rehabilitation center in accordance with the prevalence of COPD in Latin America based on the PLATINO study. This is a reflection of what is seen daily in the COPD outpatient clinics. It has been found that oxidative stress and CRP are better correlated to the multidimensional index and this is a novel finding and this confirms the systemic characteristics of the disease. Second limitation, the sample size was sufficient to prove the power of the study but was not sufficient to carry out multivariate regression analysis that control for multiple comparisons. With a sample of 45 patients, we could have Type I and II errors, which would reduce the accuracy of the results. A lower number of events per independent variable means greater variability in the regression coefficient and greater bias. Finally, the measurement of systemic markers of oxidative stress in blood is highly non-specific as biopsy to the target sites of systemic dysfunction in COPD, but the analysis of markers of oxidative stress in the peripheral blood is a simpler, practical, minimally invasive method and is well described in the literature for an overall evaluation of patients. We also agree that more specific methods, such as biopsy of the quadriceps, although more invasive, can determine the degree of muscle impairment in these patients, which could be the object of subsequent studies. In conclusion, oxidative stress and inflammation are associated with COPD and their measurements may be important tools in monitoring the evolution of the disease. The understanding of the mechanism of their actions may indicate possible alternative therapies for reducing the progression of the disease. In addition, in view of the correlation between

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