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УДК 615.40:54
THE RELATIONSHIP BETWEEN OXIDANT-ANTIOXIDANT STATUS
AND BRONCHIAL OBSTRUCTIVE PARAMETERS IN PATIENTS WITH COPD
© Khurts Solongo
PhD in Medicine, RespiratoryDivision, Department of Internal Medicine, Mongolian National University of Medical Sciences E-mail: [email protected] © Budbazar Gombosuren
PhD in Medicine, Professor, Respiratory Division, Department of Internal Medicine, Mongolian National University of Medical Sciences E-mail: [email protected] © Jambalsuren Narantsetseg
Postgraduate Student, Lecturer of Medical Institute "New Medicine" E-mail: [email protected] © Miegombo Ambaga
MD, Professor, Rector of Medical Institute "New Medicine" E-mail: [email protected]
Air pollution has major health impacts on people living in Ulaanbaatar. As written in the WORLD BANK report:"Ambient annual average particulate matter concentrations in the capital of Mongolia are 10-25 times greater than Mongolian air quality standards and are among the highest recorded measurements in any world capital."Chronic obstructive pulmonary disease (COPD) induced by air pollution wasfound to be a major cause of illness in Mongolia. We measured a wide range of parameters of the oxidant-antioxidant status in erythrocyte membrane, cytosol, plasma and urine of 188 patients with COPD and 48 healthy controls (HC). All data used in this paper is gathered since 2008 at the First National Central Hospital of Mongolia. Using the data mining methodology we selected highly effective features among them. Correlations between features were determined by SPSS 20. Also the results were analyzed using SPSS 20 for Windows; data are reported as mean, standard deviations and standard errors. The statistical significance was given by a p value<0.05.
An oxidant-antioxidant imbalance is thought to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). We hypothesized that antioxidant capacity reflected by cytochrome c oxidase (COX), free radical scavenging substances (FRSC), and levels of the lipid peroxidation product malondialdehyde (MDA) in erythrocyte, plasma and urine may be related to the bronchial obstructive parameters in patients with COPD.
The findings of the present study suggest that antioxidant capacity reflected by COX and the lipid peroxidation products MDA in erythrocyte's membrane are linked to the severity of COPD. Keywords: chronic obstructive pulmonary disease, free radical scavenging activity, cytochrome c oxidase, Lipid peroxides products, Malondialdehyde.
Introduction
COPD represents a major health problem, and its prevalence and mortality rates are increasing worldwide. COPD mainly caused by cigarette smoking and also a number of studies have shown a link between COPD and air pollution. Air pollution has major health impacts on people living in Ulaanbaatar. Ambient annual average particulate matter concentrations in the capital of Mongolia are 10-25 times greater than Mongolian air quality standards and are among the highest recorded measurements in any world capital.The excessively high particulate matter concentrations, especially in the winter and in the ger areas, increase the incidence of heart and lung diseases, and lead to premature deaths [1]. Oxidative stress, defined as an imbalance between increased exposure to oxidant and/or decreased anti-oxidative capacities, represents one of the key pathogenic mechanisms in the development of COPD [2]. A number of antioxidant disturbances have been observed in patients with COPD. Lipid peroxidation products, one of the key indicators of oxidative stress [3], are elevated in sputum and exhaled breath condensate of patients with COPD [4]. At the same time, the antioxidant mechanisms are attenuated in these patients, as indicated by reducing glutathione levels in the lungs [5], reduced glutathione peroxidase activity in erythrocytes [6] and lower antioxidant capacity in plasma [7] during exacerbations of COPD. Nevertheless, studies on the relationships between the oxidant-antioxidant imbalance and pulmonary functions showed inconsistent results. On the one hand, airway obstruction, reflected by reductions in forced expiratory volume in one second (FEV1), was shown to correlate with antioxidant substances such glutathione and myeloperoxidase levels [8]. Furthermore, lipid peroxidation products as measured as malondialdehyde (MDA) content
Kh. Solongo, B. Gombosuren, J. Narantsetseg, M. Ambaga. The relationship between oxidant-antioxidant status and bronchial ob-strnctive parameters in patients with copd_
correlated inversely with the degree of small airway obstruction [9]. On the other hand, however, more recent studies failed to find a significant relationship between plasma antioxidant capacity and pulmonary function in patients with COPD [7]. The aim of the present study was to assess the relationships between the COPD and antioxidant activity reflected by free radical scavenging capacity, cytochrome c oxidase and MDA levels in erythrocyte's membrane and cytosol, plasma and urine.
Methods
Patients with COPD were consecutively recruited to the study in 2008, 2010 and 2012, at the Pulmonology Department of First National Central Hospital (Ulaanbaatar, Mongolia). All patients classified into four stages according to the American Thoracic Society/European Respiratory Society guidelines [10].
Exclusion criteria were respiratory disorders other than the COPD, malignancy, overt cardiac failure, recent surgery, severe endocrine, hepatic or renal diseases. The control group included 48 healthy persons with similar ages, having normal pulmonary function tests. Pulmonary functional tests were evaluated by using of spirometer ST-320 (Mitsubishi, Japan). All pulmonary function tests were performed at the 10-15 minute after inhaling short-term p2-agonist Salbutamol in dosage 250 mcg. Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were expressed as a percentage of the predicted value for age, sex, and height. Three technically acceptable measurements were performed in each patient, and the highest value was included in the analyses. PaO2 was correlated with an oxygen saturation (SaO2), what was measured by finger pulse-oxymeter and expressed as a percentage.
Fasting venous blood samples were collected for the study of various parameters and taken in EDTA vial and in plain vials (without anticoagulant). Samples were used for the estimations of cytochrome c oxidases, free radical scavenging activity, lipid peroxidation products in plasma, erythrocyte's cytosol and membrane suspension. Assessment of similar parameters was performed on urine, taken under standardized conditions.
Free radical scavenging activity measured as protonized products in plasma, urine, erythrocyte's cytosol and membrane suspension were determined by method, using of the stable free radical 2,2-dipheny l-2-picrylhydrazyl (DPPH), described by Brand-Williams (1995) and expressed asmicrogramm per milliliter.
Cytochrome c oxidases (COX) in plasma, urine, erythrocyte's cytosol and membrane were estimated by using the HIMEDIA oxidase disks, based on the method described by Kovacs, developed by Gaby and Hadley (1957) and expressed as a minute.
Lipid peroxidation in erythrocyte membranes, plasma and urine were assessed by measuring the concentration of thiobarbituric acid reactive substances (MDA-TBA) by spectrophotometry at 535 NM [11]. MDA levels are expressed as nanomoles of thiobarbituric acid reactive substances formed per liter of erythrocyte membrane suspension, plasma and urine.
Statistical analysis was carried out using SPSS 20. Continuous variables are shown as means ± S.E.M. To assess the relationships between selected variables, linear regression analyses was used. A p — value less than 0.05 (P<0.05) was considered as significant.
Results
Hundred and eighty eight patients, 127 men and 61 women, were enrolled in this study. They were generally late middle-aged (mean age 59.3±5.5 years), with the average smoking history of 31.3±7.3 pack-years. The control group included 48 healthy persons with similar ages, smoking history of 4.5±1.2 pack-years, having normal pulmonary function tests. No differences were found in the demographic data between the two groups (Table 1). FVC, FEV1, and the ratio of FEV1/FVC were all significantly lower in patients with COPD compared to HC (p<0.001 for all spirometric variables). Examination of SaO2 and PaO2 revealed significantly lower in the study group compared to HC (p<0.001, p=0. 08, respectively) (Table 1).
Erythrocyte's cytosol and membrane FRSA were significantly lower, urinary FRSA is significantly greater in the study group compared to HC (p<0.05). In contrast, no differences of plasma FRSA were seen between the two groups. COX activity in plasma, urine and erythrocyte's membrane are lower, but having greater in erythrocyte's cytosol in patients with COPD compared to HC (p<0.05). Plasma, urinary and membrane lipid peroxides measured as MDA-TBA products were greater in study group significantly, compared to HC (p<0.05) (Table 2). Linear regression analysis revealed a significant direct relationship of FVC and FEV1 with COX activity of erythrocyte's membrane (r=0.260, p<0.01), and a significant inverse relationship of FVC and FEV1 with membrane MDA levels (r=-0.235, p<0.05). The findings of the present study suggest that oxidant-antioxidant capacity reflected by erythrocyte's membrane cytochrome c oxidases and membrane levels of the lipid peroxidation product MDA are linked to the severity of COPD.
Table 1
Demographic data and pulmonary functional tests in control and study groups
Variable Control group COPD group
n=48 n=188
Age (years) 59.9±5.6 59.32±5.5
Men/women 32/16 124/64
Pack-years 4.5±1.2 31.3±7.3*
Smoke index 11.3±2.2 21.7±16.5*
Body mass index (kg/m2) 27.2±4.4 26.9±6.1*
FVC ( %) 96.04±3.403 90.544±21.6**
FEV1 ( %) 83.98±11.774 57.82±17.07**
FEV1/FVC ( %) 0.83±0.11 0.62±0.08**
SaÜ2 ( %) 97.6±0.77 92.6±4.63**
PaÜ2 ( %) 89.9±3.4 69.4±14.2**
*p<0.05; **p<0.01Data are means ± S.E.M.
Table 2
Parameters of oxidant-antioxidant status in control and study groups
Parameters Control group COPD group
n=48 n=188
Plasma FRSA (mcg/ml) 0.321±0.011 0.320±0.013
Urinary FRSA (mcg/ml) 0.052±0.008 0.045±0.008
Cytosol FRSA (mcg/ml) 0.311±0.015 0.331±0.025*
Membrane FRSA (mcg/ml) 0.481±0.011 0.526±0.024*
Plasma COX (min) 2.92±0.11 3.40±0.21*
Urinary COX (min) 21.92±1.27 24.39±1.34*
Cytosol COX (min) 19±1.2 14.19±0.71*
Membrane COX (min) 33.67±1.11 37.01±1.21*
Plasma MDA (|amol/L) 0.057±0.004 0.080±0.007*
Urinary MDA (^mol/L) 0.054±0.005 0.101±0.02*
Membrane MDA (|amol/L) 0.109±0.007 0.145±0.01*
*p<0.05 Data are means ± S.E.M.
Discussion
In the present study, we have demonstrated by studying patients with all stages of COPD, that the erythrocyte's membrane COX activity and membrane MDA levels correlate with disease severity as assessed by FVC and FEV1. In agreement, our present study suggests a significant relationship between COX activity in erythrocyte membrane and pulmonary functions in patients with COPD. These findings extend those of Yang. M et al. 2010 [12] by indicating that the COX expression and activity, which were often associated with cigarette smoking, were present in COPD patients. Antioxidant activity measured as FRSA in plasma, erythrocyte membrane and urine are decreased in patients with COPD, but increased in erythrocyte's cytosol. We suggest that increasing of FRSA in cytosol may be related with increasing of F++ in cytosol due to damage of hemoglobin's membrane. No relationship was observed between FRSA and pulmonary functions in the present study. Numerous studies have shown depletion of antioxidant capacity in patients with COPD compared to healthy subjects, but also compared to smokers without COPD [13]. Indeed, several [17] but not all [18] studies documented that certain markers of oxidative stress may be related to smoking and to the severity of obstructive lung impairment in patients with COPD. However, Rahman et al. (2000) failed to document any relationship between plasma antioxidant capacity and spirometric variables. A similar result was shown in recent study. One reason for failing to find a significant relationship between FRSA and pulmonary function parameters may be related to the earlier described phenomenon that various enzymatic systems differ substantially in their responses to smoking-induced increases in oxidative stress [20].
Lipid peroxidation products are elevated in sputum, exhaled breath condensate [21] and plasma [2] of patients with stable COPD. Moreover, exacerbations of COPD lead to even further elevations in various markers of oxidative stress [3]. In addition, the oxidant-antioxidant balance is deteriorating further by the
Kh. Solongo, B. Gombosuren, J. Narantsetseg, M. Ambaga. The relationship between oxidant-antioxidant status and bronchial obstructive parameters in patients with copd_
depletion of antioxidant mechanisms. Indeed, deficiencies in both enzymatic and non-enzymatic anti-oxidative systems were described in patients with COPD [6]. Relationships between anti-oxidative enzymatic systems and lung function impairment were found in previous reports studying the anti-oxidative enzymes in erythrocytes [6] but not in plasma [19]. One of the mechanisms by which oxidants can cause lung injury, is lipid peroxidation. Malondialdehydeis the principal and most studied product of polyunsaturated fatty acid production [4]. In the present study, a significant inverse relationship between erythrocyte's membrane MDA levels and the degree of obstructive lung impairment reflected by FEV1 and FVC was observed. Previously, lipid peroxidation products measured as MDA content correlated inversely with the degree of small airway obstruction reflected in low maximal expiratory flow rates in smokers [17]. Our findings extend these original reports by suggesting that high levels of MDA may be associated with lung function not only in plasma, but also in erythrocyte membrane in patients with COPD. These observations indicate that lipid peroxidation in erythrocyte membrane is markedly increased in patients with COPD, in agreement with previous findings showing elevated levels of other markers of lipid peroxidation such as urinary and plasma concentrations of 8-isoprostane [18] and exhaled ethane [15] in patients with COPD.
In conclusion, our results indicate that the activity COX and FRSA are reduced, and that lipid peroxidation is more active in patients with COPD suggesting that reductions in the capacity of anti-oxidative enzymes and increases in toxic lipid peroxidation products might be related to the progression of the disease. Further studies are needed to analyze the pathophysiological mechanisms involved in lung injury related to an oxidant / antioxidant imbalance. Therefore more computational approaches needed to analyze the correlation between air pollution and lung disease.
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