Current Perspectives of Oxidative Stress and its Measurement in Chronic Obstructive Pulmonary Disease

ABSTRACT

Cigarette smoking, the principal aetiology of chronic obstructive pulmonary disease (COPD) in the developed countries, delivers and generates oxidative stress within the lungs. This imbalance of oxidant burden and antioxidant capacity has been implicated as an important contributing factor in the pathogenesis of COPD. Oxidative processes and free radical generation orchestrate the inflammation, mucous gland hyperplasia, and apoptosis of the airway lining epithelium which characterises COPD. Pivotal oxidative stress/pro-inflammatory molecules include reactive oxygen species such as the superoxides and hydroxyl radicals, pro-inflammatory cytokines including leukotrienes, interleukins, tumour necrosis factor alpha, and activated transcriptional factors such as nuclear factor kappa-B and activator protein 1. The lung has a large reserve of antioxidant agents such as glutathione and superoxide dismutase to counter oxidants. However, smoking also causes the depletion of antioxidants, which further contributes to oxidative tissue damage. The downregulation of antioxidant pathways has also been associated with acute exacerbations of COPD. The delivery of redox-protective antioxidants may have preventative and therapeutic potential of COPD. Although these observations have yet to translate into common clinical practice, preliminary clinical trials and studies of animal models have shown that interventions to counter this oxidative imbalance may have potential to better manage COPD. There is, thus, a need for the ability to monitor such interventions and exhaled breath condensate is rapidly emerging as a novel and noninvasive approach in the sampling of airway epithelial lining fluid which could be used for repeated analysis of oxidative stress and inflammation in the lungs.

Keywords: :

• COPD
• Oxidants
• Oxidative stress
• Antioxidant capacity
• Inflammation
• Exhaled breath condensate

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation, irreversible airflow limitation, and emphysema (1). COPD is predicted to become the third leading cause of death and the fourth commonest cause of disability in the world by 2020 (2). In the developed countries, cigarette smoking is the principal aetiology of COPD and is believed to account for approximately 90% of the cases (3). There is a strong correlation between oxidative stress from cigarette smoking with the pathogenesis of COPD (4, 5) as smokers have measurably elevated oxidants and depleted antioxidants in their exhaled breath and sputum samples (6, 7) (Figure 1). In the developing countries, a high prevalence of COPD have been documented in nonsmokers and never smokers (8, 9). Risk factors of COPD include indoor air pollution from biomass fuel, pulmonary tuberculosis, chronic asthma, and socioeconomic status (10). Occupational exposure of various compounds such as coal dust and asbestos can severely compromise lung function and may be risk factors for COPD (10). This review will provide a basic overview of oxidative stress and its role in generating an oxidant/antioxidant imbalance in the lungs, contributing to the pathogenesis of COPD and summarize the potential of the exhaled breath analysis, a noninvasive method of airway sampling for assessing airway oxidative stress.

Figure 1.  Schematic representation of oxidative stress. (a) In a healthy individual, there is an abundant of antioxidant capacities in the lungs to counteract inhaled oxidants from air pollution or acute exposures to cigarette smoke. (b) However, chronic smokers experience a reduced antioxidants and increased oxidant burden delivered from the cigarettes. At an early stage, the oxidant burden is still compensated by the antioxidant capacity but eventually (c) the oxidant burden overwhelms the body's antioxidant protection mechanism, leading to oxidative stress and the development of COPD.

OXIDANT GENERATION AND CIGARETTE SMOKING

Oxidative stress is the imbalance between the production of oxidants and the body's ability to detoxify the reactive intermediates or repair the resulting damage (11). Oxidant generation is part of the normal cellular metabolism and is critical for cell homeostasis. Inflammatory cells such as macrophages, neutrophils, and eosinophils are the most important endogenous generators of oxidants (Table 1).

Table 1.  Endogenous and exogenous sources of oxidants

Endogenous oxidant sources	Oxidants	Location
Electron transport system	O2. (oxygen radicals)	Airway epithelial cells
Xanthine oxidase	O2^−	Airway epithelial cells
	H2O2
Inflammatory Process:
1. Oxidative bursts:	Free Oxygen Radicals	Phagocytes:
- Myeloperoxidase enzyme	Superoxides	Monocytes
- Eosinophil peroxidise	Nitric Oxides	Neutrophils
- Heme peroxidase		Eosinophils
2. NADPH oxidases
3. Nitric oxide synthase
Exogenous oxidant sources	Oxidants	Location/mechanism
Cigarette smoking	Superoxides	Induce lung injury and local inflammation in the airways/lungs
H2O2
Peroxy-nitrate
Peroxy-nitrite
Air pollution	SO2, NO2, O3	Polluted air
Infection	Mainly ROS	By inducing lung injury and inflammation.

More than 90% of COPD patients have a long-term cigarette smoking history. Cigarette smoke exposes the lung to extreme levels of oxidative stress. In the gaseous phase, cigarette smoke contains approximately >10¹⁵ reactive molecules per puff, including oxidants such as O₂⁻ and nitric oxides and peroxynitrite. The tar phase of cigarette smoke contains organic radicals such as long-lived semiquinone radicals, hydroxyl radicals, and H₂O₂. Side stream cigarette smoke contains more than 10¹⁷ reactive molecules per puff, including carbon monoxide, nicotine, ammonia, formaldehyde, and other carcinogenic organic molecules (12). Cigarette smoke can induce increased oxidant burden (13) and cause irreversible changes to the antioxidant protective effects in the airways (14). Furthermore, the smoke-derived oxidants damage airway epithelial cells inducing direct injury to membrane lipids, protein, carbohydrates and DNA, leading to chronic inflammation (15) (Figure 2).

Figure 2.  Consequences of airway damage by oxidative stress. Oxidative stress causes airway damages at three levels: airway changes, cellular changes, and biochemical changes. These factors contribute to lung injury and inflammation, which leads to the development of COPD. The ongoing oxidant injuries and inflammation in COPD feeds back to the increased airway damage, restarting the vicious cycle.

Chronic inflammation is a prominent feature in COPD demonstrated by the increased number of activated neutrophils (16), macrophages (17), and inflammatory mediators in the airways (7). The combination of oxidative stress and chronic inflammation accounts for the increased free radicals in the airways and further enhances pro-inflammatory gene expression, inflammatory protein release, and inactivation of anti-proteases, leading to a vicious cycle of oxidative injury and inflammatory cell recruitment (Figure 3) (7, 18, 19).

Figure 3.  Vicious cycle of oxidative stress and inflammation (adapted from Lee 2009) (154). Oxidative stress generated by cigarette smoking increases ROS production in the bronchial mucosa, leading to the recruitment of inflammatory cells, which further increases oxidative stress. These phenomena cause lung injuries, which further enhances lung tissue inflammation and the generation of more ROS.

ROLE OF OXIDATIVE STRESS IN COPD

There is strong evidence to suggest an association between the increased oxidant burden and the development of COPD. Oxidative processes and free radical generation alter gene transcription and orchestrate inflammation, mucous gland hyperplasia, and apoptosis of the airway lining epithelium, all of which characterizes COPD.

Altered gene transcription

Transcription of genes starts with the histone acetyl transferase (HAT) controlled unravelling of DNA from histones to allow access to transcription factors. Oxidative stress activates HAT activity in respiratory epithelial cells, leading to inflammatory gene transcription (20). Reactive oxidant species (ROS) may indirectly cause an upregulation of HAT activity via activating NF-κB, AP-1, and other redox-sensitive transcriptional factors (21).

The oxidant burden within the airway epithelium and alveolar macrophages may generate cytokines which increase recruitment of pro-inflammatory cells (22). The development of COPD may be closely related to the imbalance of pro-inflammatory gene upregulation (23) and depleting the antioxidant protective mechanism in the lungs can induce an increased oxidant burden in the airways.

Mucus gland hypersecretion

Two coexisting mucosal phenomena occur in COPD: impaired mucociliary clearance and increased secretion. Endogenous and exogenous oxidants such as superoxides and H₂O₂ may impair mucociliary clearance by decreasing respiratory epithelial cilia beat frequency as early as 15 minutes after exposure (24). On the other hand, various ROS and inflammatory cytokines have been reported to directly or indirectly stimulate mucus hypersecretion (25, 26). Specifically, TNF-α (27), IL-1β (28), and lipo-polysacchride (29) have been shown to activate mucin synthesis by upregulation and expression of the mucin genes (26).

Apoptosis

COPD is characterized by progressive destruction of lung parenchyma, leading to irreversible air obstruction. Apoptosis, or programmed cell death, is a highly regulated mechanism that allows elimination of unwanted, damaged, or infected cells which may occur prematurely (30) (Table 2), leading to apoptosis of structural cells in the lungs which may possibly be an upstream event in the pathogenesis of COPD (30). Current evidence indicates an increased rate of apoptosis in endothelial cells (31, 32) alveolar epithelial cells (33, 34), lung mesenchymal cells (35), and inflammatory cells (32, 35) in COPD and emphysema patients. Apoptosis within the respiratory tract is also directly related to the decline of lung function in COPD patients (36).

Table 2.  Mechanisms of controlled apoptosis (Recreated from Demedts 2006; Hodge 2005) (30, 35)

	Mechanisms
Extra-cellular apoptotic pathways
Receptor-mediated extrinsic pathway	Mediators of the TNF family can bind to cellular surface “death” receptors resulting in the proliferation of death receptors as well as an activation of caspase pathways.
Cytolytic pathway	Cytotoxic T-cell can release enzymes such as granzyme B and perforin to induce cellular apoptosis.
Growth factor inhibition	Depletion of cellular growth factors leads to apoptosis by the activation of cytochrome C (see below).
Intra-cellular apoptotic pathways
Mitochondrial pathway	In response to stress, mitochondria releases cytochrome C (Cyt C). Together with apoptotic-protease-activating-factor and pro-caspase, Cyt C signals proteolytic activation and cell death.
Endoplasmic reticulum pathway	As a reaction to stress such as hypoxia, the endoplasmic reticulum can directly induce cell death via activating caspase-12, or indirectly induce apoptosis by stimulating the mitochondrial pathway.

Inflammation

Increased airway inflammation is a characteristic for many respiratory diseases including influenza infection, asthma, and COPD; however, the patterns of inflammation differ. Sputum samples of asthmatic patients demonstrate an eosinophil dominated pattern due to the nature of asthma being a hyper-reactivity of the immune system toward allergens (37). The COPD disease process demonstrates a predominantly neutrophilic pattern (16), which is inversely correlated with lung function (FEV₁) (38), indicating that neutrophilic inflammation is closely linked to increased airway obstruction. Oxidative stress may directly recruit inflammatory cells and cytokines to the lungs and set up a positive feedback loop that perpetuates further inflammation and tissue destruction by the infiltration of excess immune cells (39).

Quantification of these inflammatory and oxidative stress markers in COPD has thus become a focus of research. Selective biomarkers may assist in making accurate measurements of disease state/severity and provide potential pharmaceutical targets to better manage COPD.

OXIDATIVE STRESS AND INFLAMMATION MARKERS IN COPD

Oxidative stress markers reflect the level of free oxidants/ROS generating capacity in the airways and lungs. Inflammatory markers reflect the level of inflammation in the airways/lungs. They are generally molecules that directly promote local inflammation or indirectly recruits and attracts inflammatory cells.

Markers of oxidative stress

Hydrogen peroxide (H₂O₂)

H₂O₂ is a reactive oxygen species that can produce the highly reactive oxidants and free radicals including hydrochlorous acid (formed by myeloperoxidase) and hydrobromous acid. An example of which is the transition metal (Fe³⁺, Cu²⁺) catalyzed Haber–Weiss reaction: [1]

H₂O₂ is also a product of many redox-active metal catalyzed reactions, leading to more oxidant generating potential:

• Ascorbic Acid + O₂ → Ascorbyl radical, ·OH, H₂O₂

• NAD(P)H + O₂ → NAD(P)·, ·O₂⁻, ·OH, H₂O₂

• Catecholamines + O₂ → ·O₂⁻, ·OH, H₂O₂

• RSH (thiols) + O₂ → O₂⁻, ·OH, H₂O₂, RS· (thiyl radical).

H₂O₂ is a good indicator of COPD disease state because of the significant difference in exhaled breath/sputum concentration between healthy subjects, COPD patients, and patients with acute exacerbated conditions (40–42).

pH

pH is a robust and reproducible method of sampling airway acidity but it is only an indirect and nonspecific measure for detecting oxidative stress (43). A decreased pH in response to oxidative stress is a result of inflammation and the decline in the action of Na⁺/H⁺ exchanger on the cellular membrane (44). The airway pH is inversely correlated with respiratory disease activity in conditions such as asthma, COPD, and acute exacerbations (45). The stability and bioactivity of many reactive nitrogen and oxygen species are pH dependent. For example, airway acidity contributes to an increased NO concentration since it promotes eosinophil necrosis and causes conversion of endogenous nitrate to NO (21).

8-isoprostane

The detection and measurement of lipid peroxidation products such as prostaglandin also serve as a marker of oxidant burden. In particular, 8-isoprostane, a relatively stable marker at physiological temperature, is a prostaglandin analog produced by free-radical-catalyzed peroxidation of arachidonic acid (46). 8-isoprostane levels were significantly elevated in breath samples of patients with COPD, irrespective of lung function impairment (47). Furthermore, it has been suggested that 8-isoprostane level may reflect the extent of lung emphysema in COPD patients (47).

Malonyldialdehyde (MDA)

Serum MDA, a product of cell membrane lipid peroxidation, is a marker of oxidative stress, and when measured in COPD and healthy control subjects, serum MDA levels were highest in COPD patients with exacerbation, followed by stable COPD patients, followed by healthy control participants (48, 49).

Nitric oxide and reactive nitrogen species

Nitric oxide (NO), nitrite (NO₂⁻), and nitrate (NO₃⁻) have important physiological roles in the respiratory tract including vascular regulation, neurotransmission, host defence, and cytotoxicity. Reactive nitrogen species (RNS) may contribute to oxidative injury via the interaction with reactive oxygen radicals, giving rise to reactive nitrogen intermediates (50), RNS has also been linked to the development of COPD (51). NO is highly unstable and can react with superoxide anions (O₂⁻) to form peroxynitrate, an oxidizing agent that contributes to lipid peroxidation, hydroxylation, and nitration of amino acids. However, despite its ability to induce oxidative stress, the direct involvement of RNS in the development of COPD remains controversial (52–55).

Peroxynitrites 3-nitrotyrosine (3-NT)

Peroxynitrites (PN) are powerful and cytotoxic-reactive intermediates in biologic systems and are able to react with multitude of organic compounds to generate oxidants. PN alter the function of many antioxidant proteins such as superoxide dismutase (SOD), glutathione s-transferase (GST), and metalloproteinase (52, 56). Indirect measurement of PN in COPD EBC indicated elevated PN compared to control samples, with a strong correlation of PN activity and lung function (FEV₁) (52). However, direct quantification of PN is extremely difficult due to their short half-life (<1 s in pH 7.4) (52). RNS-induced oxidative stress is thus quantified by the measurement of NO, nitrite/nitrate, and nitro-tyrosine as a footprint of PN formation.

3-nitrotyrosine (3-NT) is derived from the nitration of the amino acid tyrosine or tyrosine residues within proteins. Using high-performance liquid chromatography (HPLC), 3NT was found to be upregulated in COPD and asthmatic patients (57, 58). A higher number of 3-NT positive cells were also found in the submucosa of severe COPD patients as compared with patients with mild/moderate COPD, healthy smokers, and healthy nonsmokers (59).

Markers of inflammation

Pro-inflammatory transcription precursors

Nuclear factor κB (NF-κB) is a protein complex that controls transcription of DNA as well as playing a key role in cellular immune responses to potentially damaging stimuli such as stress, cytokines, free radicals, oxidized LDL, and bacterial or viral antigens. Activator protein-1 (AP-1) is also a transcription factor protein that regulates gene expression in response to a variety of stimuli including cytokines, stress, oxidants, and infection. AP-1 controls cellular processes including the differentiation, proliferation, and apoptosis of inflammatory cells. NF-κB and AP-1 are upregulated by oxidants, leading to an increase in airway inflammatory cytokines, direct recruitment of inflammatory cells, resulting in lung tissue injuring and causing a further inflammatory response (7, 18, 41, 45, 60).

Leukotrienes

Leukotrienes are lipid-derived mediators of the immune system produced endogenously from arachidonic acid by the enzyme 5-lipoxygenase, which plays a key role in the inflammation of asthma and COPD (21). Leukotriene B₄ (LTB₄) is chemotactic to neutrophils and is found in elevated concentrations in exhaled breath (61) and induced sputum (21) in COPD patients and smokers as compared with nonsmokers. Furthermore, it has been shown that LTB₄ concentrations are significantly correlated with interleukin-6 (61).

The cysteinyl (Cys)-leukotrienes—LTC₄, LTD₄, and LTE₄—are responsible for airway smooth muscle contraction, increasing vascular permeability and stimulate mucus secretion, all of which would induce an acute asthmatic attack (62). The use of leukotriene antagonists in the treatment of asthma is commonplace; however, there has not been sufficient data to suggest a benefit of leukotriene antagonism in the treatment of COPD.

Interleukins

Interleukins (ILs) are a group of pro-inflammatory cytokines (signalling molecules) that are expressed by leukocytes. Several ILs have been associated with COPD, including IL-1β (63), IL-6 (61), IL-8 (64), IL-10 (65), among others. Their primary functions are to stimulate inflammatory cell chemotaxis and to induce the maturation and proliferation of immune cells including neutrophils, macrophages, T-helper cells, B-cells, and natural killer cells. IL-6 levels are significantly higher in smokers than control subjects (61) and IL-8 levels are significantly higher in induced sputum of COPD patients (66), suggesting a direct relationship between airway IL activity and local inflammation and tissue damage.

Tumor necrosis factor-α

Tumor necrosis factor-α (TNF-α) is a cytokine that stimulates acute phase reaction and is involved in systemic inflammation. The primary function of TNF is the regulation of immune cells but it is also capable of inducing apoptosis. TNF-α is produced mainly by macrophages, which are increased in smokers and COPD patients. Exhaled breath condensate and induced sputum studies have shown an elevated TNF-α concentration in smokers as compared to nonsmokers (66, 67). Correlations between TNF-α have been found in COPD patients with other mediators of inflammation such as IL-1β, LTB4, sputum neutrophils, and sputum macrophages (68). However, there was considerable variation in the strength of the correlations.

Transforming growth factor-β

Transforming growth factor-β (TGF-β) is a fibrogenic and immune-modulatory cytokine that controls a range of cellular functions including proliferation, differentiation, apoptosis, and recruitment of inflammatory cells when activated (69). TGF-β has been shown to perpetuate the pathogenesis of asthma by airway remodelling (70) and COPD through its induction of inflammation (69).

Volatile organic compounds

Volatile organic compounds (VOCs) refer to chemical compounds that have significant vapor pressure and low water solubility. VOCs can include both naturally occurring and man-made chemical compounds that can affect the environment and human health. These compounds are generally not acutely toxic but have chronic effects in which the mechanism have yet to be fully elucidated.

In the area of respiratory medicine and COPD, exhaled breath analysis has identified six potential VOCs (isoprene, C16 hydrocarbon, 4,7-dimethy undecane, 2,6-dimethy heptane, 4-methyloctane, and hexadecane) having early diagnostic potential for COPD (71). Additionally, VOC “fingerprints” from exhaled breath have been documented to be able to discriminate between COPD and healthy control participants (72). This finding furthers the potential for exhaled VOC as a tool for early detection and diagnosis of COPD.

Other inflammatory markers

Vascular endothelial growth factor (VEGF) has been implicated in the pulmonary remodelling of chronic progressive airflow limitation of COPD. Although VEGF is most potent as a regulator of angiogenesis, VEGF expression is increased in chronic inflammation and has been implicated in the pathogenesis of emphysema through apoptotic and oxidative stress mechanisms (73–75). The median VEGF concentration is also significantly elevated in induced sputum of COPD patients compared to nonsmokers and asymptomatic smokers (66).

ANTIOXIDANTS OF THE LUNGS

Antioxidants are agents that decrease steady-state ROS and protect cellular macromolecules from oxidative modification (76). A classic antioxidant rapidly reacts with ROS, producing less-reactive species. Broadly speaking, there are two categories of endogenous antioxidants: enzymatic and nonenzymatic antioxidants (77). Antioxidant enzymes include SOD, catalase, glutathione (GSH) peroxidases, GSH S-transferase, peroxiredoxin, and the heme-oxygenase-I system. Nonenzymatic antioxidants include GSH, ascorbate, urate, alpha-tocopherol, bilirubin, carbon monoxide, ferritin, and lipoic acid.

Enzymatic antioxidants

Superoxide dismutases family

SODs are a class of enzymatic antioxidants that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. The chemical reaction can be written as follows:

• (Transitional metal⁽ⁿ⁺¹⁾⁺ − SOD) + O₂⁻ → (transitional metalⁿ⁺ − SOD) + O₂

• (Transitional metalⁿ⁺ − SOD) + O₂⁻ + 2H⁺ → (transitional metal⁽ⁿ⁺¹⁾⁺ − SOD) ⁺ H₂O₂.

The are three forms of superoxide dismutases in humans, Cu/ZnSOD and MnSOD are primarily localized in lung cells and extracellular SOD (EcSOD) is mainly in the extracellular space of the lung (78).

Cu/ZnSOD is the most abundant superoxide dismutase in lung tissue, and is especially concentrated in the ciliated epithelial cells to combat inhaled oxidants (77). An overexpression of transgenic CuZnSOD can resist allergen-induced lung toxicity and pulmonaryoxygen toxicity (79, 80). MnSOD is predominantly located in cells in airway walls and mitochondria of the lungs (81). An over-expression of transgenic MnSOD in lungs of mice had protective effects toward hyperoxia (82). MnSOD expression is sensitive to oxidative stress. The concentration of both bronchial/alveolar epithelium MnSOD is higher in COPD than in nonsmokers and MnSOD activity is increased by 50% in smokers as compared to nonsmokers (83).

EcSOD is the major SOD in the extracellular fluid that exists in abundance in the lungs, the fluid lining the lung and in the vasculature (78, 84, 85). EcSOD is capable of protecting the lung from free radical damage and in controlling inflammation (86). There is strong evidence documenting the role of EcSOD in controlling inflammation and fibrosis of the lungs via reducing the expression of inflammatory cytokines TNF-α and MIP-2 (39). Additionally, functional polymorphisms of the SOD gene correlate well with lung function and disease status (87–91) (Table 3).

Table 3.  Summary of genetic association studies of COPD with EcSOD (Reproduced from Oberley-Deegan et al. 2009) (78)

Reference	Study population	Subjects	Findings
Juul et al. (2006) (91)	Danish Population	n = 9258	EcSOD R213G mutation protects smokers from COPD.
Young et al. (2006) (90)	European Decent Population	n = 440
Wilk et al. (2007) (89)	Framingham Heart Study	n = 1758	SNPs near EcSOD gene exhibited association with lung function.
Dahl et al. (2008) (155)	Copenhagen City Heart Study	n = 9093	An EcSOD polymorphism (E1/l1) was associated with COPD morbidity.
	Copenhagen General Population Study	n = 35,635	No association of EcSOD (E1/l1) with COPD morbidity was found.
Arcaroli et al (2009) (156)	In vitro study of EcSOD genetic variant	In vitro study	EcSOD haplotypes are associated with acute lung injury and mortality.

Glutathione (GSH) redox system

GSH is the most abundant intracellular thiol-based antioxidant that reacts with peroxides forming less-toxic disulfide products and is recycled by gluthathione-reductases (GR). GSH is more concentrated in the epithelial lining fluids and plays an important role in the maintenance of the redox balance in the lungs. In addition, GSH, under catalysis of GSH-S-transferase, is capable of rapidly detoxifying harmful components of tobacco smoke (92). Glutathione peroxidases (GPx) also catalyze a variety of GSH reactions including the breakdown of H₂O₂ (Figure 4):

Figure 4.  Diagrammatic representation of the GSH redox system. The oxidant hydrogen peroxide (H₂O₂) generated from oxidative stress is neutralized by the glutathione redox system with the assistance of GSH-reductase and glutathione peroxidase.

• 2GSH + H₂O₂ → GS–SG + 2H₂O.

The GSH recycle reaction catalyzed by GR occurs as follows:

• GS – SG + NADPH + H⁺ → 2 GSH + NADP⁺.

GSH is sensitive to oxidative stress. Acute cigarette smoke exposure of alveolar epithelial cells in culture or to lung in animal models depleted GSH (93, 94). However, GSH levels increased in the epithelial lining fluids of chronic cigarette smokers (95), suggesting a transient upregulation of GSH in acute exposure to counteract the increased oxidants, but it is eventually overwhelmed by ongoing oxidative stress. Cigarette smoke also affects GPx, GR, and GSH-S-transferase, all of which are detoxifying enzymes involved in the GSH redox system in the lungs. These observations suggest that smokers are predisposed to oxidative injuries.

Peroxiredoxins and thioredoxins

Peroxiredoxins are a family of antioxidant enzymes that control cytokine-induced peroxide levels by using cysteine as the catalytic center rather than selenocysteine in the GPx enzymes (96). Thioredoxins are proteins that act as antioxidants by facilitating (as a cofactor) the reduction of H₂O₂ and organic hydroperoxides. The antioxidant capacity of the peroxiredoxins (mainly the peroxiredoxin-6) has been assessed by attenuating the gene expression. A decreased expression of peroxiredoxins in rat lung epithelial cell line resulted in increased oxidant sensitivity and apoptosis (97). Conversely, an increased expression resulted in decreased oxidative stress and increased survival in hyperoxia (98, 99). Peroxiredoxins have increased expression in chronic oxidative diseases such as COPD (96).

Catalase

Catalase is a common enzymatic antioxidant found in living organisms. It functions to catalyze the decomposition of H₂O₂ to water and oxygen:

• 2H₂O₂ → 2H₂O + O₂.

Catalase is an extremely well-researched antioxidant whose imbalance has been linked to aging (100), atherosclerosis (101), among others. In terms of respiratory health, catalase has been demonstrated to be significantly downregulated in former smokers with COPD compared to never-smokers and former smokers without COPD (102). Additionally, polymorphisms of the catalyse gene have been documented to be related to an imbalanced oxidant/antioxidant system in patients of COPD (103).

Heme-oxygenase-1 (HO-1)

Heme-oxygenase (HO) is an enzyme that catalyzes the degradation of heme, an oxidant, and generates biliverdin-IXα, iron, and carbon monoxide (CO) (Figure 3). These downstream products of heme catabolism mediate the antioxidant properties of HO. In the airways, HO-1 is expressed in the epithelium, smooth muscle, type II pneumocytes, and alveolar macrophages. HO-1 is induced by heme, hypoxia, hyperoxia, NO, endotoxin, and pro-inflammatory cytokines (104). HO-1 expression is extremely sensitive to agents that cause oxidative stress and exhibits, in turn, powerful antioxidant and anti-inflammatory properties. HO-1 expression is upregulated in several pulmonary diseases, including COPD and asthma. The over-expression of HO-1, demonstrated in animal models, can be beneficial in several lung diseases including COPD (105) (Figure 5).

Figure 5.  Integrated diagram of oxidative stress and the heme breakdown pathway by HO-1. Heme is a product of haemoglobin metabolism and is further broken down into carbon monoxide, iron, and biliverdin, which is then converted into bilirubin. Each of these downstream products of heme metabolism has antioxidant capacities that function at different levels. CO acts as anti-inflammatory, anti-apoptotic, anti-proliferative agent. The iron-storage protein ferritin has antioxidant actions that counteract oxidative stress, and bilirubin is also an antioxidant and anti-inflammatory molecule.

Nonenzymatic antioxidants

HO-1 downstream products (bilirubin, carbon monoxide, ferritin)

HO-1 generates biliverdin, which is then converted to bilirubin, a potent endogenous antioxidant (106). CO has numerous biological functions including anti-inflammatory, anti-apoptotic, anti-coagulation, and anti-proliferative effects (53). CO can also inhibit smooth muscle proliferation, platelet aggregation, and modulate vascular tone by increasing cGMP levels (104, 107, 108). Free iron produced by HO-1 is taken up by ferritin, a major intracellular iron storage protein that removes excess free iron, thus minimizing the generation of iron-catalyzed reactive oxygen species.

α-Tocopherol and ascorbate

Vitamins E (α-tocopherol) and C (ascorbate) are two of the dominant naturally occurring antioxidants in human. They function by binding with ROS to produce less reactive radicals that can be recycled by cellular reductases (76). Oral vitamin A, C, E supplementations have protective effects upon oxidant-induced airway damage (109, 110). A water soluble derivative of α-tocopherol is 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox®). Trolox® is used in biological/biomedical applications to reduce oxidative stress or damage. Because of the difficulties in measuring individual antioxidant components of a complex mixture, Trolox® equivalency antioxidant capacity (TEAC) is used as a benchmark for antioxidant capacity.

N-acetyl-L-cysteine

N-acetyl-L-cysteine (NAC) is a powerful antioxidant capable of removing several ROS such as hypochlorous acid, peroxides, hydroxyl radicals, and ONOO⁻ (76, 111). Additionally, NAC stimulates GSH synthesis, enhances glutathione-S-transferase activity, and promotes liver detoxification. The therapeutic use of NAC in COPD has yield conflicting results, and while some studies have shown beneficial effects of NAC with regard to lung function (112) and COPD exacerbations (113–115), some clinical trials concluded that NAC was ineffective at preventing deterioration of lung function or preventing exacerbations (116, 117).

Role of antioxidants in COPD

A relationship between dietary antioxidants, pulmonary function, and the development of COPD has been shown in several studies. Population surveys showed the inverse relationship between both dietary and serum vitamin C to FEV₁ and chronic respiratory symptoms (118, 119). A positive relationship was found between the levels of dietary vitamin C, vitamin E, selenium, and beta-carotene and FEV₁ in healthy and COPD subjects (120–123).

As a response to oxidative stress, there is a transient upregulation of protective antioxidant generation within the lungs including MnSOD, metallothionein, and GSH peroxidase (124). However, in COPD patients, serum/airway antioxidant levels are depleted (125, 126), suggesting that the increase of airway antioxidants in response to oxidative stress is a temporary phenomenon and this protective mechanism can be overwhelmed by chronic exposure of airway and lungs to oxidative stress, for example in chronic smokers.

EXACERBATIONS OF COPD

Exacerbations of COPD are characterized by an acute increase in symptoms, associated with a deterioration in lung function, resulting in poor health and high mortality. Causes of exacerbations include bacterial/viral infection, continued oxidant damage, air pollution, comorbidities of COPD, pulmonary hypertension, and cor pulmonale (127). Advanced diagnostic techniques have demonstrated rhinovirus (128) and seasonal influenza (129) as a common cause of COPD exacerbation by directly infecting the upper-respiratory and lower-respiratory tract. Bacterial colonization is often reported in patients with COPD and associated with frequency of exacerbations (130). Exacerbations of COPD reflect a worsening of airway and lung oxidative stress caused by acute-on-chronic inflammation that overwhelms the protective oxidant-scavenging activity. The increased oxidative state can be caused by intrinsic factors due to worsening of the oxidant burden and depleted antioxidant capacity or can be triggered by extrinsic factors such as air pollution and acute bacterial/viral infections (7, 126).

COPD subjects with exacerbations exhibit an increased oxidative stress associated with elevated IL-8 and neutrophil activity in the distal airspaces (7), in combination with depleted antioxidants such as decreased plasma TEAC and GSH levels in patients with acute exacerbations of COPD (7, 126, 131). Worsening of oxidative stress also induces the transcription of various pro-inflammatory factors such as NF-κB (132), AP-1 (7), which translates to inflammation and a decline in lung function. A compensatory mechanism in response is the transient upregulation of antioxidant genes, such as the HO-1 pathway, but fails to restore oxidant/antioxidant balance completely (124–126).

A rational approach to the treatment of COPD exacerbations should thus focus on its three most common causes—oxidative stress, viruses, and bacterial infections. These factors amplify the innate inflammatory response, triggering increased oxidant production and might inhibit anti-inflammatory responses. Therefore, antioxidants may have an important role in the treatment of COPD exacerbations.

THERAPEUTIC POTENTIAL OF OXIDANT INHIBITION AND ANTIOXIDANT IN COPD

Various approaches have been taken to redress the oxidant/antioxidant imbalance with the intervention of ameliorating lung function decline and to better manage COPD. Two potential therapeutic options are the reduction of oxidant burden and the delivery of antioxidants, with their therapeutic effects indicated in countering oxidative stress in the airways (115, 133, 134) (Table 4).

Table 4.  Recent clinical trials for the efficacy of antioxidants in pulmonary disease and in the general population

References	Antioxidant used	Disease condition	Aim of the study	Result/outcome
Stav D (2009) (112)	NAC	COPD	Effect of NAC on FVC and exercise tolerance.	NAC has beneficial effect on physical performance in 24 patients.
Sagiura et al. (2009) (157)	NAC	COPD	Effect of NAC on TGF-β1 mediated tissue remodelling/production of VEGF, fibronectin.	NAC significantly decreased TGF-β1 augmented fibronectin and VEGF production in vitro.
Schermer et al. (2009) (116)	NAC	COPD/chronic bronchitis	Effect of NAC in primary care patients.	No beneficial treatment were observed in the study population (n = 286).
Zuin et al. (2005) (137)	NAC	COPD exacerbation	Effect of high dose NAC (1200mg/day) in treatment of COPD exacerbation.	Improved biological markers and clinical outcomes in exacerbated COPD.
Decramer et al. (2001, 2005) (158, 117)	NAC	COPD	Effect of NAC on FEV1.	30% reduction in COPD hospitalization obtained without change on decline in FEV1.
Kirkil et al. (2008) (159)	Zinc picolinate	COPD	Effect of zinc supplementation on oxidant stress and pulmonary function test (PFT).	May have a favorable effect on oxidant–antioxidant balance. PFT showed no significant improvement probably due to the short duration of supplementation.
Hirayama et al. (2009) (135)	Diet high in vegetables and fruits	COPD	Investigate relationship between vegetable and fruit consumption and risk of COPD.	Inverse association between vegetable consumption and risk of COPD.
Nadeem et al. (2009) (160)	Vitamin E	COPD	Effect of Vitamin E supplementation on oxidative stress.	No significant benefits.
Conklin et al. (2010) (161)	Vitamins C and E, alpha-lipoic acid	COPD	Effect of antioxidants on skeletal muscle oxygen delivery and blood flow.	Oxidative stress determines skeletal muscle blood flow and muscle metabolism in COPD.
Wu et al. (2007) (110)	Vitamins C and E	COPD	Effect of vitamins C and E on white blood cell DNA damage.	Vitamin E or C may improve resistance of DNA in WBC against oxidative challenge.
Guenegou et al. (2006) (109)	Vitamin E β-carotene	Lung function	Effect of low level serum β-carotene and vitamins A and E on 8-year lung function.	β-carotene protects against decline in FEV1. Both β-carotene and vitamin E are protective of lung function decline in heavy smokers.
Griese et al. (2004) (139)	Inhaled GSH	Cystic fibrosis	Effect of GSH on lung function and oxidative stress.	Improvement in lung function but no change in oxidative stress state of the lungs.
Bishop et al. (2005) (140)	Inhaled GSH	Cystic Fibrosis	Effect of GSH on clinical parameter of CF patients.	Improvement in clinical parameters.

Direct antioxidants

Dietary antioxidant supplementation is one of the simplest approaches in delivering antioxidants to the body. The most common dietary antioxidant supplementations are vitamins A, C, E including β-carotene, through tablets or vegetables and fruits. A diet high in vegetables and fruits has an inverse relationship with COPD risk factors (135), and supplementation with vitamins C and E is associated with an improvement of oxidant/antioxidant imbalance in COPD (110). Supplementation of β-Carotene may also protect against FEV₁ decline in the general population (109). Oral administration of NAC in the management of COPD has yielded some positive results as NAC may improve physical performance and exercise tolerance (112), reduce odds of exacerbation (136), lower oxidant markers, and improve clinical outcome in managing acute exacerbation (137). Some studies have also shown that the use of NAC could reduce rate of hospitalization of COPD patients by 30% with no decline of FEV₁ (117), although not all studies have yielded beneficial results. The use of intra-tracheal injection of MnSOD in an animal model revealed protective effect in otherwise healthy lungs (138).

Another possible approach of antioxidant therapy is to deliver antioxidants to the lungs. GSH is a potential antioxidant in the management of COPD. A range of studies have suggested that inhaled GSH may be beneficial in other lung diseases such as cystic fibrosis (139, 140) and idiopathic pulmonary fibrosis (141, 142).

Indirect antioxidants

Indirect antioxidants include the use of antioxidant analogs that are converted into antioxidants in vivo or the use of medications to modulate the oxidant/antioxidant level in the body. N-acystelyn (NAL), a salt of NAC, is a mucolytic and antioxidant compound. It can be aerosolized into the lungs to enhance intracellular GSH in alveolar epithelial cells and inhibit the formation of ROS (143). All-trans retinoic acid, an acidic form of vitamin A, can modulate protease/anti-protease balance in the lungs (144).

Oxidant inhibitors

Inhibition of superoxide generation via phosphodiesterase 4 (PDE4) inhibitors is a novel method to control oxidative stress. PDE4 inhibitors promote intracellular levels of cyclic adenosine monophosphate (cAMP), which has a broad anti-inflammatory effect. Oral intake of PDE4 inhibition produced significant improvement in lung function of COPD patients (145); however, an inhaled PDE4 inhibitor showed no improvement in lung function of COPD symptoms (146). Other potential pharmaceutical targets include redox sensitive molecules such as NF-κB, AP-1, or inflammatory cytokines such as TNF-α and monocyte inflammatory protein (MIP)-1β.

QUANTIFYING OXIDATIVE STRESS AND ANTIOXIDANTS IN THE AIRWAY AND LUNGS

The accepted method of investigating the respiratory system has depended upon invasive approaches such as induced sputum, broncho-alveolar lavage, or lung biopsy. However, the invasive nature of these procedures made it sub-optimal for as tools of disease diagnosis or long-term monitoring. Thus, there has been an increasing interest in using exhaled breath as a simple, noninvasive means to repeatedly sample and monitor the lower respiratory tract (63).

Exhaled breath condensate

Exhaled breath contains aerosol particles and proteins that originate in the lower respiratory tract (147). Proteomic comparisons between frozen aerosol particles and saliva samples demonstrate the presence of extra protein in aerosol particles that are not found in saliva, suggesting that nonvolatile substances such as proteins from the lower respiratory tract can be transported in the form of aerosols in exhaled breath (63).

Exhaled breath can be collected in the gaseous phase or the liquid phase. The gaseous phase comprises products of oxidative stress and antioxidants such as CO, NO, and volatile organic compounds (148). The liquid phase, or the exhaled breath condensate (EBC), comprises nonvolatile mediators and inflammatory markers such as hydrogen peroxide, nitrites/nitrates, nitrotyrosine, adenosine, vasoactive amines, ammonia, eicosanoids, peptides, cytokines, and other larger molecules (149, 150).

EBC collection method

EBC is collected by the condensation of expired air through a cooling system. Successful collection has been reported with many devices, but different designs may contribute to biomarker variability (151). There is intersubject variability of EBC volume generated and minute ventilation remains the major determinant of the amount of condensate obtained over time (147, 149).

EBC samples airway lining fluid (65, 152) from the upper respiratory tract (nasopharynx, trachea), lower respiratory tract (bronchi and alveoli), and the ambient environment and the oral cavity (149). Therefore, prevention of contamination is an important step in designing and sampling of EBC. Saliva is a major source of contamination, and salivary amylase can be tested in EBC to rule out salivary contamination (147). In summary, the EBC apparatus should be designed to maximize condensate collection and minimize the risk of sample contamination.

Many oxidative stress or inflammatory markers have been measured in EBC including H₂O₂ (42), nitrates (149), leukotrienes (60), among others. However, the potential weaknesses of EBC are the low concentration of detectable biomarkers as compared with induced sputum or bronchoalveolar lavage and the poor reproducibility between studies (151, 153). Several recommendations to improve and standardize the collection of EBC are in place, including the use of argon gas to promote biomarker stability and adopting methods to concentrate EBC for easier detection of biomarkers.

CONCLUDING REMARKS

Current research demonstrates a link between oxidative stress and the development of COPD. The oxidative process can be triggered by endogenous factors such as cytokines, pro-inflammatory markers, and immune complexes or by exogenous factors such as pollution and cigarette smoke. Antioxidants may neutralize the induced oxidant burden and have been suggested to play an important role in the prevention of oxidative tissue injury. Recent studies have documented correlations between depleted antioxidant levels and the exacerbation of COPD. Yet, the evidence of different antioxidants and their therapeutic potentials remains limited partly due to the difficulty in the specific sampling of the pulmonary pathway. The exhaled breath analysis thus stands out as a promising tool for the accurate and noninvasive sampling and monitoring of oxidant burden and antioxidant capacity. Ultimately, more research will be needed on EBC of COPD patients to further explore the role of oxidant burden and antioxidant pathways and their potential therapeutic manipulations in the treatment of COPD.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.