Mongolian Journal of Health Sciences, 2013, 1(10)

Oxidant-antioxidant status in chronic obstructive pulmonary disease: relationship with disease severity

( Судалгааны өгүүлэл )

Solongo Kh1*, Narantsetseg J2, Narantsetseg B2, Gombosuren B3, AmbagaM2

¹ Department of Pulmonology, First National Central Hospital, Ulaanbaatar, Mongolia

² Department of Newly Coded Medicine, New Medicine Institute, Ulaanbaatar, Mongolia  ³Department of Pulmonology, Health Sciences University of Mongolia, Ulaanbaatar

 

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Абстракт

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 (FRSS), and levels of the lipid peroxidation product malondialdehyde (MDA) in erythrocyte, plasma and urine may be related with disease severity in COPD patients. We measured several parameters of oxidant-antioxidant status in erythrocyte membrane and cytosol, plasma and urine in 188 patients with COPD and 48 healthy controls (HC). Lung function was measured by spirometry. Partial oxygen pressure in blood (PaO2) was determined in correlation with an oxy-hemoglobin saturation (SaO2) estimate measured by finger pulse oxy-meter. Free radical scavenging substances in erythrocyte cytosol (FRSSc) and membrane (FRSSm) were significantly lower, but greater in urine (FRSSu) in patients with COPD as compared to HC (FRSSc: 0.331±0.025 vs. 0.311±0.015 mcg/ml, p<0.05, FRSSm: 0.526±0.024 vs. 0.481±0.011, mcg/ml, p<0.05, FRSSu: 0.045±0.008 vs. 0.052±0.008 mcg/ml, p<0.05). In contrast, no differences were seen between the two groups in the plasma FRSS (0.320±0.013 vs. 0.321±0.011 mcg/ml). COX in plasma (COXp), urine (COXu) and erythrocyte membrane (COXm) were significantly lower, but greater in erythrocyte cytosol (COXc) in patients with COPD as compared to those in HC (COXp: 3.40±0.21 vs. 2.92±0.11, COXu: 24.39±1.34 vs. 21.92±1.27, COXm: 37.01±1.21 vs. 33.67±1.11, Oc: 14.19±0.71 vs. 19±1.2, p<0.05). Plasma, urinary and membrane MDA levels were significantly higher in study group as compared to HC (MDAp: 0.080±0.007 vs. 0.057±0.004, MDAu: 0.101±0.02 vs. 0.054±0.005, MDAm: 0.145±0.01 vs. 0.109±0.007, p<0.05). Linear regression analysis revealed a significant direct relationship between FVC, FEV1 and COXm (r=0.299, p<0.01, r=0.260, p<0.01), and a significant inverse relationship between FVC, FEV1 and MDAm levels (r=-0.277, p<0.01, r=-0.235, p<0.05). Findings of the present study suggest that antioxidant capacity reflected by COX and the lipid peroxidation products MDA in erythrocyte membrane are linked to the severity of COPD.

INTRODUCTION

COPD represents a major health problem, and its prevalence and mortality rates are increasing worldwide¹. Oxidative stress, defined as an imbalance between increased exposure to oxidant and/or decreased anti-oxidative capacities, represents one of the key pathogenetic mechanisms in the development of COPD². A number of antioxidant disturbances have been observed in patients with COPD. Lipid peroxidation products, one of the key indicators of oxidative stress³, are elevated in sputum and exhaled breath condensate of patients with COPD⁴. At the same time, the antioxidant mechanisms are attenuated in these patients, as indicated by reduced glutathione levels in the lungs⁵, reduced glutathione peroxidase activity in erythrocytes^6, and lower antioxidant capacity in plasma⁷ 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 levels⁸. Furthermore, lipid peroxidation products measured as malondialdehyde (MDA) content correlated inversely with the degree of small airway obstruction⁹. On the other hand, more recent studies showed that there was no significant relationship between plasma antioxidant capacity and pulmonary function in patients with COPD⁹. The aim of the present study was to assess the relationships between the COPD stages and antioxidant activity reflected by free radical scavenging capacity, cytochrome c oxidase and MDA levels in erythrocyte membrane and cytosol, plasma and urine.

MATERIALS AND METHODS

Subjects: 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 had COPD according to the American Thoracic Society /European Respiratory Society guidelines¹⁰. Patients with other respiratory disorders than COPD, malignancy, overt cardiac failure, recent surgery, severe endocrine, hepatic or renal diseases were not included. The control group included 48 healthy persons with similar ages, having normal pulmonary function tests.

Ethical considerations: The study was conducted in accordance with national policies on ethics for surveys involving human subjects. The study protocol was approved by Ethical Review Committee of Health Science University of Mongolia. In accordance with their approval, all participants signed an informed consent form before participating in the study.

Pulmonary function testing: Pulmonary functional tests were evaluated with using of spirometer ST-320 (Mitsubishi, Japan). All pulmonary function tests were performed at the 10-15 minute after inhaling short-term β2-agonist salbutamol in dosage 200 mcg. Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were expressed as a percentage of the predicted values for age, sex, and height. Three technically acceptable measurements were performed in each patient, and the best value was included in the analyses. Partial oxygen pressure in blood (PaO2) was determined in correlation with an oxy-hemoglobin saturation (SaO2) estimate measured by finger pulse oxy-meter (504-US; Criticare Systems, Waukesha, WI) and expressed in a percentage.

Parameters related to oxidant-antioxidant status: Fasting venous blood samples were collected in EDTA vial and in plain vials (without anticoagulant). Samples were used for the estimations of cytochrome c oxidase, free radical scavenging substances, lipid peroxidation products in plasma, erythrocyte cytosol and membrane suspension. Assessment of similar parameters were performed in urine, taken under standardized condition.

Free radical scavenging substences, measured as protonized products in plasma, urine, erythrocyte cytosol and membrane suspension were determined with method, using of the stable free radical 2,2-diphenyl-picryl-hydrazyl (DPPH), described by Brand-Williams (1995) and expressed as microgramm per mililiter.

Cytochrome c oxidase (COX) in plasma, urine, erythrocyte’s cytosol and membrane was estimated with using of 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 membrane, plasma and urine were assessed by measuring concentration of thiobarbituric acid reactive substances (MDA-TBA) in spectrophotometry at 535 nm. MDA levels are expressed as nanomoles of thiobarbituric acid reactive substances formed per liter of erythrocyte membrane suspension, plasma and urine.

Statistical analyses: Statistical analysis was carried out using SPSS 20. Continuous variables are shown as means ± S.E.M. To assess the relationship between selected variables, linear regression analyses was used. P-value less than 0.05 (P<0.05) was considered as significant.

RESULTS

One hundred and eighty eight patients, 127 men and 61 women, were included 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. COPD severity in patients was classified as Stage I, II, III, or IV, depending on the FEV1% predicted, as described in the GOLD guidelines⁸. Stages III and IV were combined into one group designed “Stage III+IV”, because the number of subjects in Stage IV was too small for separate examination. The control group included 48 healthy persons with similar ages, smoking history of 4.5±1.2 pack-years and having normal pulmonary function tests. No differences were found in the demographic data between the 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 values in COPD group compared to HC (p<0.001, p=0.08, respectively) (Table 1). Erythrocyte cytosol FRSS were significantly lower in Stage II and Stages III+IV (p<0.05), but greater in Stage I. Erythrocyte membrane FRSS were lower in COPD group as compared to HC (p<0.05), although not all differences were statistically significant. Urinary FRSS were lower in Stage I and greater in the other Stages as compared to HC. In contrast, no differences of plasma FRSS were seen between the control and COPD groups. COX activity in plasma, urine and erythrocyte’s membrane are significantly lower, but having greater in erythrocyte cytosol in patients with COPD compared to HC (p<0.05). Plasma and urine COX in Stage III+IV were significantly lower than that in both Stage II and Stage I. Erythrocyte membrane COX also tended to decrease with COPD progression, although not all differences were statistically significant. Plasma, urinary and membrane lipid peroxides measured as MDA-TBA products were greater in COPD group significantly, compared to HC (p<0.05) (Table 2). Erythrocyte membrane MDA in Stages III+IV was significantly greater than that in both Stage II and Stage I. This suggests that MDAm increased with the progression of COPD. Linear regression analysis revealed a significant direct relationship of FVC and FEV1 with COX activity of erythrocyte 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). Findings of the present study suggest that oxidant-antioxidant capacity reflected by erythrocyte’s membrane cytochrome c oxidase and membrane levels of the lipid peroxidation product MDA are linked to the severity of COPD.

DISCUSSION

By studying patients with different stages of COPD we have demonstrated that the erythrocyte’s membrane COX activity, plasma COX activity and membrane MDA levels correlate with disease severity as assessed by FVC and FEV1. Our present study suggests a significant negative relationship between COX activity of erythrocyte’s membrane and the stage of COPD. These findings are extend investigations of imbalance between oxidant and antioxidant capacities, represents one of the key pathogenetic mechanisms in the development of COPD^3,5,11.

The present study yields several interesting and novel findings. First, the antioxidant activity measured as FRSS in erythrocyte’s cytosol were increased in Stage I and decreased in progressing of COPD. This finding may be related to the activation of compensate mechanism in the early stage of disease. The previous investigation by Halliwell В. also suggested the increase of antioxidant activity in smokers¹². Numerous studies have shown depletion of antioxidant activity in plasma and erythrocyte^3,5,6,12. Indeed, several¹³ but not all¹⁴ studies documented that certain markers of oxidative stress may be related to the severity of obstructive lung impairment in patients with COPD. Relationships between anti-oxidative enzymatic systems and lung function impairment were also reported in previous studies, where the anti-oxidative enzymes were measured in erythrocytes¹⁵ but not in plasma⁷. Similarly, we didn’t observe any relationship between plasma antioxidant capacity and spirometric variables in present study. One reason for failing to find a significant relationship between FRS activity 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 stress².

Second, COX activity in erythrocyte cytosol was increased in all stages of COPD, in contrast to erythrocyte’s membrane, plasma and urine COX, where they decreased respectively. This finding may be related to migration of Fe⁺⁺ from hemoglobin to erythrocyte’s cytosol due to damage of their membrane. A previous investigation by Sauleda et al.¹⁶ also suggested that activity of COX is increased in circulating lymphocytes, although numerous studies have shown depletion of COX in lungs, plasma and erythrocytes of patient with COPD^17,18.

Finally, a significant inverse relationship between erythrocyte membrane MDA levels and the degree of obstructive lung impairment reflected by FEV1 and FVC was observed in the present study. Previously, lipid peroxidation products measured as MDA content in plasma correlated inversely with the degree of small airway obstruction reflected by low maximal expiratory flow rates in smokers⁹. Our findings support these original reports by suggesting that high level of MDA in erythrocyte membrane as well as in plasma might be associated with lung function in patients with COPD. The numerous of studies showed elevated levels of other markers of lipid peroxidation such as urinary and plasma concentrations of 8-isoprostane¹⁹ and exhaled ethane²⁰ in patients with COPD. Lipid peroxidation products were elevated in sputum, exhaled breath condensate⁴ and plasma²¹ of patients with stable COPD. Moreover, exacerbations of COPD lead to even further elevations in various markers of oxidative stress²².  In contrast, we didn’t found any significant correlation of MDA levels, measured in plasma and urine with COPD stages in present study.

In conclusion, our results indicate that the COX activity is increased in erythrocyte’s cytosol, but decreased in membrane. Moreover, FRSS are reduced and lipid peroxidation activity is increased with progressing of COPD. Further studies are needed to analyze the pathophysiological mechanisms involved in lung injury related to oxidant-antioxidant imbalance. 

Table 1. Demographic data and pulmonary functional tests in control and study groups.

Variable	Control group n=48	COPD group
		Total n=188	Stage I n=45	Stage II n=90	Stage III+IV n=53
Age (years)	59.9±5.6	59.32±5.5	58.18±11.23	59.66±12.61	60.8±10.74
Men/women	32/16	127/61	25/20	57/33	45/8
Pack-years	4.5±1.2	31.3±7.3*	27.9±14.8	30.6±15.5	35.4±12.8
Smoke index	11.3±2.2	21.7±16.5*	18.6±10.8	20.99±16.9	27.6±17.4
Body mass index (kg/m2)	27.2±4.4	26.9±6.1*	28.28±6.10	26.92±5.71	25.7±6.8**
FVC (%)	96.04±3.40	90.544±21.6**	114.6±12.3	92.9±12.4	66.1±12.7**
FEV1 (%)	83.98±11.77	57.82±17.07**	78.1±6.2	59.97±9.1	36.9±8.02**
FEV1/FVC (%)	0.83±0.11	0.62±0.08**	0.68±0.03	0.64±0.06	0.55±0.09**
SaO2 (%)	97.6±0.77	92.6±4.63**	95.6±1.8	92.97±3.5	89.55±5.9**
PaO2 (%)	89.9±3.4	69.4±14.2**	79.02±10	69.3±12	61.2±14**

                               *p<0.05; **p<0.01 Data are means ± S.E.M. 

 

Table 2. Parameters of oxidant-antioxidant status in control and study groups.

Parameters	Control group n=48	COPD group
		Total n=188	Stage I n=45	Stage II n=90	Stage III+IV n=53
Plasma FRSS (mcg/ml)	0.321±0.01	0.320±0.013	0.310±0.013	0.311±0.015	0.350±0.014
Urinary FRSS  (mcg/ml)	0.052±0.01	0.045±0.008	0.061±0.012	0.039±0.005	0.042±0.07
Cytosol FRSS (mcg/ml)	0.311±0.02	0.331±0.025*	0.28±0.02	0.37±0.03	0.38±0.03*
Membrane FRSS (mcg/ml)	0.481±0.01	0.526±0.024*	0.57±0.03*	0.50±0.02	0.54±0.02*
Plasma COX (min)	2.92±0.11	3.40±0.21*	3.18±0.23	3.39±1.79	3.58±0.24
Urinary COX (min)	21.92±1.27	24.39±1.34*	24.3±1.42	24.2±1.34	24.9±1.31
Cytosol COX (min)	19±1.2	14.19±0.71*	13.7±0,5.	14.8±0,7	14.1±0.7
Membrane COX (min)	33.67±1.11	37.01±1.21*	33.7±1.2	36.6±1.3	42.5±1.3*
Plasma MDA (mmol/L)	0.057±0.01	0.080±0.007*	0.075±0.006	0.089±0.009*	0.071±0.005
Urinary MDA  (mmol/L)	0.054±0.01	0.101±0.02*	0.054±0.004	0.122±0.002*	0.106±0.012*
Membrane MDA (mmol/L)	0.109±0.01	0.145±0.01*	0.096±0.01	0.143±0.03	0.170±0.01*

                              *p<0.05 Data are means ± S.E.M. 

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