Monitoring Antioxidant Enzyme Activity during Exacerbations of Chronic Obstructive Pulmonary Disease

Abstract

Superoxide dismutases (SODs) and catalase (CAT) have been implicated as major antioxidant enzymes of the human lungs. In this study, we investigated whether activities of these enzymes are altered in the airways of patients hospitalized with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). SOD and CAT activities were measured in the sputum, exhaled breath condensate, and serum of 36 COPD patients experiencing a severe exacerbation. Measurements were performed using colorimetric assays in samples collected at the time of hospital admission and at the time of hospital discharge following treatment of AECOPD. For comparison, antioxidants were also assessed in 24 stable COPD patients and 23 healthy control subjects. SOD and CAT activities in sputum were significantly increased in patients with AECOPD compared to those with stable disease (SOD: 0.142 [0.053–0.81] vs. 0.038 [0.002–0.146] U/mL, p < 0.01; CAT: 48.7 [18.7–72.6] vs. 10.2 [2.9–40.6] nmol/min/mL, p < 0.05), while treatment of exacerbation led to a decrease in enzyme activities (SOD: 0.094 [0.046–0.45] U/mL, p < 0.05; CAT: 28.0 [7.3–60.4] nmol/min/mL, p < 0.005). No changes were observed in the serum (p > 0.05). Both SOD and CAT activities significantly correlated with sputum neutrophil and lymphocyte cell counts in patients with AECOPD. Moreover, SOD and CAT values correlated with each other and also with sputum malondialdehyde, an established marker for oxidative stress. Our data demonstrate that sputum antioxidant activity is elevated during COPD exacerbation and suggest that activation of SODs and CAT is an integral part of the human defense mechanism against the increased oxidant production associated with AECOPD.

Keywords:

• Airway
• antioxidants
• biomarker
• COPD
• exacerbation
• sputum

Introduction

Oxidative stress is thought to play a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD), particularly during acute exacerbations of COPD (AECOPD) (1,2). It has been proposed that apart from the burden of inhaled oxidants and reactive oxygen species generated in the airways, depletion of antioxidants can also be partly responsible for the oxidant/antioxidant imbalance characteristic for this condition (3, 4). The role of antioxidants is to counterbalance the deleterious effects of oxidants in the human body.

Enzymes such as superoxide dismutases (SODs) and catalase (CAT) have been implicated as major endogenous antioxidants of the lungs (5, 6). SODs are metalloenzymes that catalyze the dismutation of the highly reactive superoxide anion to molecular oxygen and hydrogen peroxide (H₂O₂), while CAT is involved in converting H₂O₂ into molecular oxygen and water.

Although polymorphisms in genes coding for SODs have been implicated as genetic risk factors for developing COPD (7), studies comparing the amounts of antioxidants between stable COPD patients and healthy subjects yielded conflicting results (8–11). Similarly, in AECOPD both increased (12) and decreased (13) enzyme activities were documented.

The inconsistent findings may be related to the fact that all these measurements were performed in blood that reflects systemic rather than local antioxidant status. Blood levels of antioxidants may or may not be similar to that of the respiratory tract, while direct measurements of SODs and CAT in samples from the respiratory tract should be more representative of antioxidant activity that occurs in the lungs. In agreement with this view, we recently demonstrated that sputum rather than plasma is the sample of choice for estimating the degree of oxidative lipid peroxidation in the airways of pulmonary patients (14, 15).

Various methods of sampling the respiratory tract are known, and among them, sputum and exhaled breath condensate (EBC) collections are simple, noninvasive procedures (16). Both methods yield ideal samples for the identification of pulmonary biomarkers mediating inflammation and oxidative stress in patients with COPD (17, 18).

In this longitudinal study, we aimed to measure the activity of SODs and CAT in sputum and EBC of patients at the onset of AECOPD and following treatment. For comparison, enzyme activities in stable COPD and healthy controls were also determined, and measurements were performed in serum as well. Finally, in order to further explore the relationship between oxidants and antioxidants, malondialdehyde (MDA), an established oxidative stress marker in AECOPD (19), was also assessed.

Methods

Study subjects

Patients hospitalized with AECOPD were recruited for the study between January 2016 and June 2017. Inclusion and exclusion criteria are summarized in Figure 1. AECOPD was defined as increased dyspnea, cough, or sputum expectoration that led the subject to seek medical attention, as specified in international guidelines (20).

Figure 1. Flowchart showing the study profile in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). EBC: exhaled breath condensate.

Additionally, 24 clinically stable, ex-smoker COPD patients and 23 healthy, ex-smoker controls were enrolled in the study (Table 1). All patients were >40 years of age, had a smoking history of >10 pack-years, and documented airway obstruction with forced expiratory volume in one second (FEV₁) < 80% of predicted and post-bronchodilator FEV₁/forced vital capacity (FVC) < 0.7. Control subjects had normal lung function values and no history of acute or chronic respiratory diseases in the previous 4 weeks. The research protocol was approved by local Ethics Committee, and all subjects gave written informed consent to participate in the study.

Table 1. Demographic and clinical characteristics of study subjects.

	Healthy controls	Stable COPD	AECOPD
Subjects (n)	23	24	36
Sex (male/female, n)	13/10	14/10	19/17
Age (years)	59.3 ± 2.8	64.5 ± 2.6	68.5 ± 3.1
Smoking (pack-years)	40.1 ± 2.5	46.5 ± 4.3	44.2 ± 2.3
GOLD stages (n, %)	–
I–II.		14 (58)	16 (44)
III–IV.		10 (42)	20 (56)
			At admission	After treatment
Pulmonary function
FVC (L)	3.96 ± 0.17	3.66 ± 0.20	1.99 ± 0.12	2.25 ± 0.13*
FVC (% predicted)	101.6 ± 3.3	79.3 ± 3.1	69.9 ± 3.34	74.8 ± 3.24*
FEV1 (L)	3.1 ± 0.12	1.87 ± 0.16	0.93 ± 0.08	1.09 ± 0.09***
FEV1 (% predicted)	107.3 ± 2.88	65.3 ± 4.92	41.9 ± 3.78	46.6 ± 3.4***
FEV1/FVC	0.78 ± 0.01	0.51 ± 0.03	0.47 ± 0.03	0.48 ± 0.02*
Blood gases
PaCO2 (kPa)	5.23 ± 0.09	5.09 ± 0.12	5.69 ± 0.16	6.03 ± 0.18
PaO2 (kPa)	9.9 ± 0.51	8.51 ± 0.21	6.87 ± 0.19	7.77 ± 0.21*
FENO (ppb)^†	14.0 (10.4–20.3)	16.2 (11.0–25.8)	19.2 (9.78–33.1)	10.9 (7.13–16.1)**

Data are presented as mean ± SEM unless stated otherwise. FVC: forced vital capacity, FEV₁: forced expiratory volume in one second, FENO: fractional exhaled nitric oxide, ppb: parts per billion, PaCO₂: arterial carbon dioxide tension, PaO₂: arterial oxygen tension, GOLD: Global Initiative for Chronic Obstructive Pulmonary Disease. *p < 0.05, **p < 0.01, and ***p < 0.005 vs. admission, ^†median (interquartile ranges).

Study design

In patients with AECOPD, EBC, spontaneously expectorated sputum, and serum were collected and fractional exhaled nitric oxide (FENO), blood gases, and lung function parameters were measured at hospital admission and at the day of discharge. In stable COPD patients and healthy controls, EBC, induced sputum and serum were collected during routine clinical visits. The ex-smoker status of all participants was confirmed by measuring exhaled carbon monoxide (eCO) levels. All subjects had eCO levels <4 ppm indicating that they truly refrained from smoking prior to the study.

Sputum induction, EBC collection with an EcoScreen condenser (Jaeger, Hoechberg, Germany), FENO measurement using a chemiluminescence analyzer (Model LR2500, Logan Research, Rochester, UK), eCO measurement with a portable CO monitor (Smokerlyzer Micro, Bedfont Scientific, Kent, UK), and all other laboratory tests were performed, as previously described (21, 22). All samples were stored frozen at –80 °C before analysis.

Sputum processing

Sputum samples were processed in PBS containing dithiothreitol (DTT), as previously described (23). At least 400 inflammatory cells were counted for each cytospin slide. The number of inflammatory cells in sputum was recorded as a percentage of total nonsquamous cells.

Measurement of SODs and CAT

Activity of SODs and CAT was determined in sputum supernatant, EBC, and serum by colorimetric assay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol except when measuring sputum DTT was added to the standards at a concentration corresponding to that in sputum supernatant (0.04%), in agreement with other studies (24). The dynamic ranges of the SOD and CAT assays were 0.025–0.25 U/mL and 2–35 nmol/min/mL, respectively. The intra- and inter-assay coefficients of variation (CV) were 3.2% and 3.7% for the SOD assay, and 3.8% and 9.9% for the CAT assay, respectively.

The repeatability of SOD and CAT measurements was determined in a pilot study. Sputum and serum samples collected from a subset of COPD patients (n = 8) were divided into two aliquots, stored frozen, and analyzed separately.

Additionally, in order to compare antioxidant levels in induced and spontaneous sputum, from a subgroup of patients (n = 6) capable of expectorating sputum spontaneously, both induced and spontaneous sputum samples were collected.

Measurement of MDA

MDA concentrations in sputum supernatant of patients with stable disease and AECOPD were measured with high-performance liquid chromatography, according to the method established previously in our laboratory (19). The limit of quantification was 0.01 μmol/L.

Statistical analysis

Data are presented as mean ± SEM or median with interquartile range, as appropriate. SOD and CAT activities were compared using the Kruskal–Wallis test followed by the Dunn’s test for multiple comparisons. Paired Student’s t-test (parametric data) and the Wilcoxon signed-rank test (nonparametric data) were used to compare variables measured at the time of hospital admission and discharge. Baseline parameters between stable patients and controls were analyzed by unpaired t-test or the Mann–Whitney U-test. Correlation coefficients were calculated by Spearman’s method. The repeatability of the SOD and CAT measurements was estimated by the CV and the limits of agreement (Bland–Altman test). Calculations were performed by GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). A p value <0.05 was considered significant.

Results

From the AECOPD patients admitted to our hospital during the study period, 45 fulfilled inclusion criteria and agreed to participate (Figure 1). During hospital treatment, 9 patients were withdrawn due to unforeseen complications or their inability producing sputum samples. Demographic and clinical data of the 36 patients who completed the study are presented in Table 1.

Clinical variables during treatment of AECOPD

Exacerbations were treated with systemic glucocorticoids, bronchodilators (short- or long-acting anticholinergics and/or β₂-agonists) and oxygen with a flow rate of 2 L/min through a nasal cannula in all cases. Antibiotics were given to 32 patients, while 5 patients received nonsteroidal anti-inflammatory drugs. The mean length of hospitalization was 9.4 ± 1.6 days. During the course of recovery, lung function variables and arterial oxygen tension increased, while FENO, sputum total cell counts, and the number of sputum neutrophils and lymphocytes decreased (Table 1 and Supplementary Table 1).

Repeatability of SOD and CAT measurements

SOD and CAT activities in the two aliquots of the same sputum and serum samples, and the mean CV for the repeated measurements were similar (p > 0.05, Supplementary Table 2). The corresponding Bland–Altman graphs are provided in Supplementary Figure 1.

Comparison of antioxidants between induced and spontaneous sputum

SOD and CAT activities in spontaneous and induced sputum samples were similar (SOD: 0.88 [0.23–4.31] vs. 0.97 [0.16–5.27] U/mL, p > 0.05, CV: 16.4 ± 4.5%; CAT: 60.2 [20.2–70.3] vs. 61.1 [20.1–100.0] nmol/min/mL, p > 0.05, CV: 10.9 ± 4.8%) indicating that the induction by itself had no effect on antioxidant readings.

SOD and CAT in sputum

In the healthy control group, SOD and CAT activities were only detectable in sputum supernatants of 13 and 19 subjects, respectively (Figure 2). In the stable COPD group, SOD activity was below the detection limit only in 4, while CAT in 2 patients. Among patients with AECOPD, both antioxidants were detectable in all cases.

Figure 2. Superoxide dismutase (SOD) (a) and catalase (CAT) (b) activities in sputum supernatant of healthy controls (controls), stable COPD patients (stable), and AECOPD patients at the time of acute exacerbation (ex) and after hospital treatment (treat). Horizontal bars represent median values. *p < 0.05 and **p < 0.01 vs. stable COPD patients, ^§p < 0.001 vs. healthy controls, ^#p < 0.05 and ^##p < 0.005 vs. acute exacerbation.

In stable COPD patients, sputum SOD and CAT activities were higher compared to healthy controls, although the differences have not reached statistical significance Table 2 (p > 0.05 for both). In patients with AECOPD, both SOD and CAT activities further increased (p < 0.01 and p < 0.05, respectively). Treatment of AECOPD resulted in a significant decrease in the activities of sputum antioxidants (p < 0.05 and p < 0.005, respectively).

SOD and CAT in EBC

Using EBC samples, both SOD and CAT activities were found to fall below the detection limit of the assay in nearly all (>90%) samples irrespective of the patient group. Therefore, EBC data were not further analyzed in the study.

SOD and CAT in serum

Both antioxidants were detectable in all serum samples. However, SOD and CAT readings were similar among the groups, and treatment of AECOPD had no major effect on those values either (Table 2, p > 0.05).

Table 2. Superoxide dismutase (SOD) and catalase (CAT) activities in sputum and serum of healthy controls, stable COPD patients, and AECOPD patients at the time of acute exacerbation and after hospital treatment.

			AECOPD
Antioxidants	Healthy controls	Stable COPD	At admission	After treatment
Sputum
SOD (U/mL)	0.01 (0–0.043)	0.038 (0.002–0.146)	0.142 (0.053–0.81)^§**	0.094 (0.046–0.45)^#
CAT (nmol/min/mL)	5.2 (0.44–16.6)	10.2 (2.9–40.6)	48.7 (18.7–72.6)^§*	28.0 (7.3–60.4)^##
Serum
SOD (U/mL)	7.69 (6.31–12.7)	9.94 (6.93–13.4)	6.14 (3.53–12.8)	8.28 (6.04–11.8)
CAT (nmol/min/mL)	39.4 (5.94–57.7)	42.5 (5.15–58.3)	36.6 (4.65–58.9)	31.2 (4.77–50.0)

Data are presented as median (interquartile ranges). *p < 0.05 and **p < 0.01 vs. stable COPD patients, ^§p < 0.001 vs. healthy controls, ^#p < 0.05 and ^##p < 0.005 vs. at admission.

MDA in sputum

Sputum MDA levels were increased in patients with AECOPD compared to stable COPD patients (179.3 ± 17.6 vs. 126.3 ± 12.5 nmol/L, p < 0.05), while treatment of exacerbation led to a significant decrease in MDA concentrations (151.2 ± 15.2 nmol/L, p < 0.05). In healthy controls MDA levels were 88.8 ± 13.6 nmol/L.

Correlations

Major correlation data are summarized in Table 3. Briefly, in both the stable and the AECOPD groups, significant correlations were observed between the number of neutrophils and SOD or CAT activities in the sputum. Additionally, in patients with AECOPD, the percentage of sputum neutrophils, the number of lymphocytes, and the total sputum cell count showed an association with SOD and CAT values. Moreover, sputum MDA level was shown to positively correlate with SOD and CAT activities in AECOPD. Finally, SOD and CAT values correlated with each other in both stable COPD patients (r = 0.582, p < 0.05) and in patients with AECOPD (r = 0.412, p < 0.05).

Table 3. Correlations between sputum superoxide dismutase (SOD)/catalase (CAT) activities and clinical variables, sputum inflammatory cells, and sputum MDA levels in stable COPD patients and in those with AECOPD.

Variables	Sputum SOD		Sputum CAT
	r value	p value	r value	p value
Stable COPD
PaO2 (kPA)	–0.123	0.559	−0.228	0.308
FEV1 (% predicted)	–0.114	0.587	−0.32	0.166
Sputum
Total cell counts (×10^6/g)	0.402	0.052	0.642	0.003*
Neutrophils (×10^6/g)	0.435	0.033*	0.543	0.016*
MDA (nmol/L)	–0.004	0.986	0.009	0.973
AECOPD (at admission)
PaO2 (kPA)	–0.02	0.242	–0.047	0.829
FEV1 (% predicted)	–0.116	0.536	–0.271	0.223
Sputum
Total cell counts (×10^6/g)	0.432	0.005*	0.447	0.017*
Neutrophils (×10^6/g)	0.557	<0.001*	0.514	0.007*
Neutrophils (%)	0.376	0.024*	0.493	0.009*
Lymphocytes (×10^6/g)	0.417	0.012*	0.392	0.048*
MDA (nmol/L)	0.402	0.023*	0.48	0.015*
AECOPD (at discharge)
PaO2 (kPA)	–0.102	0.626	–0.121	0.622
FEV1 (% predicted)	–0.167	0.361	–0.289	0.192
Sputum
Total cell counts (×10^6/g)	0.054	0.746	0.491	0.007*
Neutrophils (×10^6/g)	0.249	0.156	0.468	0.014*
Neutrophils (%)	0.140	0.407	0.442	0.018*
Lymphocytes (×10^6/g)	–0.183	0.454	0.079	0.696
MDA (nmol/L)	0.424	0.019*	0.498	0.011*

FEV₁: forced expiratory volume in one second, PaO₂: arterial oxygen tension, MDA: malondialdehyde, *significant correlations.

Spirometric and other clinical variables showed no correlations with sputum SOD or CAT values in stable COPD patients or in patients with AECOPD. Similarly, serum SOD and CAT exhibited no significant associations with sputum inflammatory cell counts or other clinical parameters recorded (data not shown).

Discussion

In this study, we investigated the activities of the major antioxidants SODs and CAT in the airways of patients with severe AECOPD requiring hospitalization. The main finding of the study was that activity of these enzymes in sputum is increased in AECOPD compared to stable disease, while recovery from exacerbation leads to a decrease in enzyme activities. In contrast, activity of SODs and CAT in serum was not altered during the course of AECOPD indicating that sputum but not blood could be the sample of choice for monitoring exacerbation-associated changes in antioxidant status of patients with COPD.

Lung tissue is protected against free radical-mediated injury by a variety of defense mechanisms. Among these are SODs that enzymatically convert superoxide radicals to H₂O₂ (5, 6). Since superoxide anion is one of the most powerful oxidants produced from a variety of sources, its dismutation by SODs is of primary importance for the survival of cells. In humans, three types of SODs (cytosolic, mitochondrial, and extracellular) have been characterized, and all of them are expressed in the lung tissue (6). Notably, the assay used in our study measures all three types of SODs. Another ubiquitous antioxidant enzyme CAT, expressed preferentially in bronchial epithelial cells and alveolar macrophages (25, 26), acts as one of the most important H₂O₂ scavenging enzymes in the airways.

To the best of our knowledge, no previous study has investigated changes in the activities of SODs or CAT in the airways of patients with COPD recovering from an exacerbation. Among the few studies exploring antioxidants in respiratory samples, Zeng et al. (27) have reported that sputum SOD level is decreased in stable and exacerbated patients compared to healthy controls. Our findings are in contrast to their results, but this is likely due to the differences in study populations: In our study, all AECOPD patients received systemic corticosteroids and were ex-smokers, while in the trial of Zeng et al. (27), only milder cases (patients without systemic corticosteroid treatment), and both smokers and nonsmokers were included. Additionally, there are differences between the two studies regarding the methods used for measuring SODs. Using an assay similar to the one employed here, Regan and co-workers showed that the activity of extracellular SOD is higher in sputum of stable COPD patients compared to controls (28). In our study, a similar tendency was noted in stable patients.

It is well known that AECOPD is characterized by the increased production of reactive oxygen and nitrogen species that induce lipid peroxidation, protein/DNA damage, and aggravate airway inflammation via multiple mechanisms (1, 2). We speculate that the increase in sputum SOD and CAT activity in AECOPD represents a protective regulatory response to counteract the harmful effects of oxidants during exacerbation. In support of this concept, both the current and our previous studies (19) demonstrated that the level of MDA, a by-product of polyunsaturated fatty acid peroxidation, is increased in the sputum of patients with AECOPD. Moreover, in this study, the level of MDA and the number of neutrophils and lymphocytes present in sputum samples at the time of exacerbation showed a positive correlation with SOD and CAT activity suggesting a direct relationship between the degree of inflammation/lipid peroxidation and antioxidant activity in the airways. The correlation between SOD and CAT activities favors our concept as well.

In contrast to findings in sputum, SOD and CAT activity in serum remained unchanged in patients with AECOPD indicating that blood may not be suitable sample for estimating antioxidant activity present locally in the airways. Such lack of agreement between plasma and airway samples is not uncommon; for example, there is evidence that the severity of COPD (based on GOLD stages) influences SOD levels in sputum, while no corresponding changes can be detected in plasma (28). It appears that a local imbalance of the oxidant/antioxidant status in the airways does not necessarily lead to systemic imbalance. This may be due to differences in the regulation of redox state between circulation and the airways or the confounding effect of various factors such as co-morbidities and nutrition that influence the systemic redox state.

Longitudinal studies investigating changes in oxidative markers in the airways of patients with COPD experiencing an exacerbation are scarce. Our data showing that airway MDA levels are elevated in AECOPD and decrease upon treatment are in accordance with the findings of Footitt et al. (29), who reported similar kinetics for various oxidative and nitrosative stress markers in sputum of patients with rhinovirus-induced exacerbations.

Sputum oxidant levels and antioxidant activities at the time of exacerbation often show considerable variability that may reflect either heterogeneous inflammatory responses present in patients or the uneven delay between the first-onset of symptoms and hospital admission.

Although SOD and CAT levels significantly decreased with treatment of AECOPD, at discharge they were still higher compared to those of stable COPD patients. This can be explained by the attenuated but not completely eliminated acute inflammation present in the lungs at the time of discharge from the hospital. Indeed, delayed resolution of inflammatory response during recovery from exacerbation has been observed by others as well (30). Further studies with longer follow-up periods are needed to investigate this issue.

As mentioned before, antioxidant activity could be detected only sporadically in EBC samples. Mediator level in EBC often remains below the detection limit of the assay due to the fact that EBC is an extremely diluted type of respiratory sample (17). Our data suggest that EBC is of limited value for assessing enzyme activity in the airways.

Several lines of evidence indicate that cigarette smoking is a confounding factor in the measurement of antioxidants (8, 28). Since our recruitment strategy called for only ex-smokers, smoking status had no influence on our results.

Conclusions

Our data suggest that, in contrast to some previous assumptions, AECOPD is accompanied by increased antioxidant activity in the airways, which could represent a defense mechanism against free radical-mediated injury during the course of AECOPD. Since antioxidants have been suggested to play an important role in the prevention of oxidative tissue injury, it is tempting to speculate that addition of these agents may be clinically beneficial for patients with AECOPD, particularly for those who have impaired defense capacity. Nonetheless, further studies are needed to explore the role of antioxidant pathways and their potential as therapeutic targets in the management of patients with COPD.

Declaration of interest

No potential conflict of interest was reported by the authors.

Acknowledgments

We thank M. Mikoss and O. Drozdovszky (National Koranyi Institute for TB and Pulmonology) for technical assistance in FENO measurements, sputum and EBC collections. The study was supported by the Hungarian Respiratory Society and the Hungarian National Scientific Foundation (OTKA 124343).