Oxidized Alpha-1-Antitrypsin as a Potential Biomarker Associated with Onset and Severity of Chronic Obstructive Pulmonary Disease in Adult Population

Abstract

Oxidative stress could reduce inhibitor activity of the alpha-1-antitrypsin (A1AT). Oxidative-modified A1AT (oxidized alpha-1-antitrypsin, OxyA1AT) significantly loses ability to protect the lungs from neutrophil elastase. We aimed to investigate OxyA1AT as a potential biomarker associated with onset and severity of chronic obstructive pulmonary disease (COPD) in adult population. The study included 65 patients with COPD (33 smokers and 32 no-smokers) and 46 healthy participants (17 smokers and 29 no-smokers). Determination of OxyA1AT in serum was based on the difference between the inhibitory activities of normal and oxidized A1AT against trypsin and elastase. The level of OxyA1AT was significantly increased in the group of COPD smokers compared to healthy no-smokers (p = 0.030) and COPD no-smokers (p = 0.009). The highest level of OxyA1AT was found in group of smokers with severe and very severe COPD in comparison to the following: no-smokers with the same stage of disease (p = 0.038), smokers with moderate COPD (p = 0.022), and the healthy control group, regardless of the smoking status (control no-smokers p = 0.001 and control smokers p = 0.034). In conclusion, serum level of OxyA1AT would be potentially good biomarker for the assessment of harmful effect of smoking to the onset and severity of COPD. Also, clinical significance of OxyA1AT as prognostic biomarker could be useful in assessing the effectiveness of antioxidant therapy for COPD and emphysema. Suitable and inexpensive laboratory method for determination of OxyA1AT is additional benefit for the introduction of OxyA1AT into routine clinical practice for diagnosis and monitoring of COPD.

Keywords:

• Chronic obstructive pulmonary disease
• oxidized alpha-1-antitrypsin
• specific inhibitor activity to elastase
• specific inhibitor activity to trypsin

Introduction

World Health Organization report states that the main risk factors for chronic obstructive pulmonary disease (COPD) are tobacco smoke (including passive exposure), indoor air pollution (such as biomass fuel used for cooking and heating), outdoor air pollution, occupational dust, and exposure to chemicals (vapors, irritants, and fumes) (1). Tobacco smoke contains harmful toxins, which inhaled directly into the lungs over prolonged periods of time can lead to severe lung irritation, triggering the onset of COPD. The earliest pathological changes that include hyperplasia of the basal cells of airway epithelium induced by smoking are critical to the pathogenesis of COPD (2). At least three interrelated pathophysiological mechanisms affect the lung function due to the influence of tobacco smoke: inflammation, oxidative stress, and protease–antiprotease imbalance (3). Prolonged exposure to the tobacco smoke causes the recruitment of inflammatory cells to the lung, which presents a risk for tissue damage through the release of toxic mediators, including proteolytic enzymes and reactive oxygen species (4). The protease–antiprotease imbalance in the pathogenesis of COPD is closely related to tobacco smoke via oxidative stress and inflammatory response (5). Tobacco smoke promotes inflammatory cells influx into lungs, which under certain conditions excessively releases a variety of proteases, including neutrophil elastase (NE), proteinase 3, matrix metalloproteinases, and cathepsins, which inhibited their endogenous inhibitors. Furthermore, pro-oxidants originating from tobacco smoke could promote oxidative modification and inactivate the protease inhibitors. Consequently, over-activated proteases could degrade components of the extracellular matrix, elastin fibers, and collagen, and therefore can destroy lung tissue.

Clinical studies indicated that imbalance between NE and their main circulating inhibitor alpha-1-antitrypsin (A1AT) causes the development of COPD and even panlobular emphysema with characteristic widespread destruction of alveolar tissue and dilation of small airspaces throughout the lungs (6).

A1AT is an acute-phase glycoprotein, mainly synthesized in hepatocytes and subsequently secreted into the plasma. A1AT is able to inhibit a variety of serine proteinases, but its major physiological role is to inhibit NE in lower respiratory tract, and protect pulmonary connective tissue from NE released from triggered neutrophiles. Human NE (EC 3.4.21.20) stored in the primary (azurophil) granules of polymorphonuclear neutrophils (PMNs) is able to degrade most of the components of the pulmonary extracellular matrix (ECM), including elastin, collagens, proteoglycans, and laminin (7).

The active sites of A1AT at residue Met³⁵⁸-Ser³⁵⁹, as well as at the residue Met³⁵¹ are susceptible to oxidation by several oxidants from tobacco smoke, and form stimulated inflammatory cells (8). Oxidation of both Met³⁵⁸ and Met³⁵¹ to methionine sulfoxide significantly reduces the ability of A1AT to inhibit NE (9).

Hereditary alpha-1-antitrypsin deficiency (A1ATD) is associated with retention of mutant A1AT polymers in hepatocytes which leads to decrease of circulating A1AT with less than 15% of normal level in A1ATD homozygotes. Since the integrity of lung alveoli is maintained by proper circulating level of A1AT, severe deficiency of this protein was identified as genetic risk factor for COPD and emphysema. Clinical manifestation of emphysema in patients with A1ATD occurs in 3rd decade in smokers, in the 5th decade in nonsmokers, and requires replacement therapy with purified A1AT pooled from plasma of blood donors (10). The cut-off level of A1AT which protects the lung tissue from proteases is 0.49 g/L (11 μM), and this value is therapeutic goal of the so-called “augmentation therapy” for patients with severe A1ATD. Many epidemiological studies investigated the significance of hereditary A1ATD on lung function decline, but much less attention is devoted to the study of non-inherited functional deficiency of A1AT. Acquired functional deficiency of A1AT, caused by pro-oxidants released from tobacco smoke or activated phagocytes, such as peroxide, hydroxyl radicals, hypochloride, chloramines, and peroxynitrite may reduce antiprotease activity of A1AT, although the level of A1AT in serum may stay normal (11).

Thitherto, it seems that utility of circulating level of A1AT as a biomarker of susceptibility to onset of COPD is limited due to the complex interrelationship among tobacco smoke exposure, circulating level of A1AT, lung function, and systemic inflammatory status.

Therefore, we aimed to investigate the utility of oxidized alpha-1-antitrypsin (OxyA1AT) as a potential diagnostic and prognostic biomarker associated with COPD onset and severity in adult population.

Materials and methods

Subjects and samples

This case–control study included 65 patients with COPD (42 males and 23 females), who were recruited from the Clinic for Pulmonary Diseases, Clinical Centre of Kragujevac, Serbia. Patients included in this study were hospitalized for COPD. Among the patients, 33 were current smokers, and 32 current no-smokers. Diagnosis of COPD and severity of disease was established based on medical history, physical examination, and pulmonary function tests (spirometry parameters: forced expiratory volume in 1 second, % of predicted value, FEV1%; forced vital capacity, % of predicted value, FVC%; and FEV1/FVC ratio). Severity of COPD was classified according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification (12): Moderate COPD (GOLD 2; 50% ≤ FEV1 < 80% predicted), Severe COPD (GOLD 3; 30% ≤ FEV1 < 50% predicted) and very severe COPD (GOLD4; FEV1 < 30% predicted). Exclusion criteria were mild and severe hereditary A1AT deficiency, and COPD-patients with less than 60% of normal serum level of A1AT (<0.9 g/L) were not included in the study. The second exclusion criteria were the presence of other pulmonary diseases, such as bronchial asthma, bronchiectasis, tuberculosis, lung cancer, and other.

The control group included 46 healthy subjects (41 males and 5 females), with normal laboratory test results and normal pulmonary status assessed by physical examination, carried out in Health Centre, Belgrade, Serbia. In the control group, 29 subjects were current no-smokers, and 17 were current smokers.

Venous blood samples for laboratory testing were collected, and after centrifugation the sera were isolated and stored at –80 °C until the start of the analysis.

Basic data about age and current smoking status (smokers or no-smokers) were collected through questionnaires completed by all participants. All participants in study provided written informed consent. The study was approved by the local Ethics Committee and was carried out according to the principles of the Declaration of Helsinki.

Methods

Assay for trypsin inhibitory capacity

Trypsin inhibitory capacity (TIC) was determined using modified methods by Schwert and Takenaka (13). In brief, N-α-benzoyl-l-arginine ethyl ester (BAEE) purchased from SERVA was used as a substrate for determining trypsin activity. Five microliters of porcine trypsin (Trypsin from porcine pancreas ca. 60 U/mg, SERVA), previously dissolved in 0.001 M HCl and 3.0 mL of Tris-HCl (pH 8.0) were added to the serum samples and to control analysis (40 g/L albumin). After incubation period of 10 minutes at 37 °C, 20 µL BAEE was added, and the excess of trypsin activity which was not inhibited by A1AT from serum sample was measured. Trypsin activity was measured spectrophotometrically (UV-1800 Shimadzu UV/VIS spectrophotometer) as the change in absorbance at 253 nm per minute (ΔA/min) for 5 minutes. TIC in serum samples were calculated by equitation: TIC (kU or mM/L/min) = (ΔA/min_control – ΔA/min_serum) × f; f = (1/1.15 mM) × (3045/5) = 529, where 1.15 mM is molar absorptivity of BAEE at 253 nm, and 3045/5 is dilution factor of serum samples.

Assay for elastase inhibitor capacity

Elastase inhibitory capacity (EIC) was assayed by modified methods by Bieth et al. (14). The method was optimized for automatic analyzer, Instrumentation Laboratory® ILab 650. The elastase activity was determined using substrate N-succinyl-l-Alanyl-l-Alanyl-l-Alanyl-p-nitroanilide (STAPNA) purchased from SERVA. Stock elastase was prepared by dissolving elastase (Elastase from porcine pancreas min. 200 U/mg, SERVA) in a buffer 0.05 M TRIS/0.05 NaCl, pH 8.0. The three microliters of stock elastase and 0.05 M Tris-HCl pH 8.0 were added to the diluted serum samples (1:5) and control analysis (40 g/L albumin), respectively. The mixture was incubated for 10 minutes at 37 °C. The assay started by adding of 12 microliters of STAPNA that hydrolyze the remaining elastase, which was not inhibited by A1AT from sample, and the increase in absorbance at 405 nm was monitored for 5 minutes. EIC in serum samples were calculated by equation: EIC (kU or mM/L/min) = (ΔA/min_control – ΔA/min_serum) × f; f = (1/9.9 mM) × (357/3) × 5 = 60, where 9.9 mM is the molar absorptivity of p-nitroaniline at 405 nm, 357/3 is dilution factor of serum, and the factor value 5 is initial serum dilution factor.

The serum level of A1AT (g/L) was determined by immunoturbidimetric method using Beckman Coulter AU480 analyzer and Randox reagent for A1AT RX series (Cat. No AA 2471). Specific inhibitory activity of A1AT toward trypsin (SIA-Trypsin) and elastase (SIA-Elastase) was calculated as a ratio between functional activity of A1AT (TIC or EIC) and immunoreactive level of A1AT (g/L).

Determination of oxidized A1AT

Determination of oxidized A1AT in serum is based on difference between the inhibitory activities of normal and oxidized A1AT against a trypsin-like enzyme and elastase, without influences of other proteins in serum (15). The functionally active A1AT inhibits both porcine trypsin and porcine pancreatic elastase, while oxidized A1AT lost its inhibitory activity toward porcine pancreatic elastase, and its net trypsin inhibitory capacity was partially preserved.

According to the proposed method, percent of oxidized A1AT (OxyAAT, %) was calculated by equation: OxyA1AT (%) = [1 – (1.27/(TIC_sample/EIC_sample)] × 100; the value of 1.27 in equation represents the TIC_reduced/EIC_reduced ratio of the fully mercaptoethanol-reduced purified A1AT. Thus, the ratio of values TIC_samle/EIC_sample in serum sample determines the oxidized ratio. Finally, for the certain concentration of A1AT (g/L), the level of OxyA1AT (g/L) was calculated by equation: OxyA1AT (g/L) = [OxyA1AT (%) × A1AT (g/L)]/100.

Statistical analysis

Normality of variables was assessed by the one-sample Kolmogorov–Smirnov test (K-S test). Student t-test for independent samples, one-way analysis of variance (one-way ANOVA), and post hoc comparison (Least Significant Difference, LSD test) were used for comparison of continuous variables among the groups. The two-way ANOVA was used for comparison of differences in the level of OxyA1AT between patients according to the smoking status and severity of COPD. The linear relationship between functional activity of A1AT (SIA-Trypsin, SIA-Elastase) and OxyA1AT was evaluated using the Spearman’s correlation analysis. Statistical analysis was performed using SPSS 20.0 software. In this study, a p value <0.05 was considered statistically significant.

Results

Demographic and clinical characteristics of healthy participants and patients with COPD are presented in Table 1. Patients with diagnosed COPD were older than participants in control group. Also, COPD-patients had significantly higher serum level of A1AT than in control group. As expected, the values of spirometry parameters of lung function in patients with severe and very severe COPD (GOLD 3 and 4) were significantly worse than those in patients with moderate COPD (GOLD 2). Frequency of current smokers was not different among investigated groups.

Table 1. Characteristics of investigated control group, all patients with COPD and patients divided into two groups according to COPD severity.

Characteristic	Control (n = 46)	COPD patients
		All (n = 65)	GOLD 2 (n = 18)	GOLD 3 and 4 (n = 47)
Age, years	56 ± 5*	64 ± 9	65 ± 7	63 ± 10
Males, n (%)	41 (89)	42 (65)	11 (61)	31 (66)
Smokers, n (%)	17 (37)	33 (51)	7 (39)	26 (55)
A1AT, g/L	1.5 ± 0.34*	1.7 ± 0.40	1.6 ± 0.35	1.8 ± 0.41
FEV1%	–	39 ± 22	68 ± 14**	28 ± 13
FVC%	–	62 ± 25	89 ± 16**	51 ± 19
FEV1/FVC	–	48 ± 11	56 ± 9**	45 ± 11

Continuous data are presented as mean ± standard deviation.

* Difference from COPD patient group (p < 0.05).

**Differences from patients with COPD stage GOLD 3 and 4 (p < 0.001).

The effects of tobacco smoke on the functional activity of A1AT (SIA-Trypsin and SIA-Elastase), serum level of A1AT, and level of OxyA1AT, which were obtained in control group and in patients with COPD are shown in Figure 1. One-way ANOVA and post hoc tests revealed that: SIA-Trypsin was decreased in COPD patients than in control, regardless of their smoking status (Figure 1a) (COPD smokers vs. control no-smokers p = 0.002, COPD no-smokers vs. control smokers p = 0.017, COPD no-smokers vs. control nonsmokers p < 0.001, difference between COPD smokers and control group smokers was close to significance p = 0.063); SIA-Elastase was decreased in COPD smokers than in control no-smokers (p = 0.032) and COPD no-smokers (differences were close to significance p = 0.069) (Figure 1b); serum level of A1AT was increased in COPD smokers than in control no-smokers (p = 0.011) (Figure 1c); OxyA1AT level was increased in COPD smokers than in control no-smokers (p = 0.030) and COPD no-smokers (p = 0.009) (Figure 1d).

Figure 1 Smoking-related mean values (95% confidence interval) obtained in control group (29 no-smokers and 17 smokers) and patients with COPD (32 no-smokers and 33 smokers) for: (a) SIA-Trypsin (*significant difference to COPD smokers and COPD no-smokers), (b) SIA-Elastase (#significant difference to COPD smokers), (c) levels of A1AT (& significant difference to COPD smokers), (d) OxyA1AT (Δ significant difference to COPD smokers.

The main results of Spearman’s correlation analysis are presented in Table 2. Significant inverse relation was found between SIA-Elastase and serum level of OxyA1AT in all investigated groups. However, the stronger inverse relation was found in groups of COPD no-smokers and COPD smokers (Spearman’s R: –0.927 and –0.890, respectively), than it was found in no-smokers and smokers in control group (Spearman’s R: –0.671 and –0.640, respectively).

Table 2 Spearman’s correlation analysis between parameters of functional activity of A1AT (SIA-Trypsin and SIA-Elastase) and OxyA1AT.

Group	Correlated parameters
	OxyA1AT and SIA-Trypsin	OxyA1AT and SIA-Elastase
Control-all	–0.247 (0.188)	–0.640 (<0.001)
Control no-smokers	–0.342 (0.179)	–0.671 (<0.001)
Control smokers	–0.077 (0.802)	–0.640 (<0.001)
COPD all	–0.386 (0.024)	–0.919 (<0.001)
COPD no-smokers	–0.442 (0.066)	–0.927 (<0.001)
COPD smokers	–0.284 (0.286)	–0.890 (<0.001)

* Data presented as Spearman's correlation coefficient (p value)

Figure 2 presents smoking-related levels of OxyA1AT and SIA-Elastase in the control group and in two patient groups divided according to the severity of the disease (GOLD 2 group: patients with moderate COPD, and GOLD 3 + 4 group: patients with severe and very severe COPD). Mean values of SIA-Elastase in GOLD 3 + 4 group who were no-smokers was lower than in healthy no-smokers (p = 0.037). The lowest value of SIA-Elastase was found in group of COPD-smokers with severe and very severe stages of disease, and it was significantly lower than in both healthy smokers and no-smokers (p = 0.034, p = 0.001, respectively). Contrary to that, COPD-smokers with end-stage of disease had the highest level of OxyA1AT, in comparison to all other investigated groups (vs. control no-smokers, p = 0.009; vs. control smokers, p = 0.033; vs. GOLD 2 smokers, p = 0.022, and vs. GOLD 3 + 4 no-smokers, p = 0.038).

Figure 2. Smoking-related mean values of SIA-Elastase (full line, label for mean values ●) and OxyA1AT (dashed line, label for mean values ▴) in control group (29 no-smokers, 17 smokers) and patients divided into two groups according to COPD severity (11 no-smokers with GOLD 2; 7 smokers with GOLD 2; 21 no-smokers with GOLD 3 + 4; 26 smokers with GOLD 3 + 4); *difference of SIA-Elastase in relations to control no-smokers; **difference of SIA-Elastase in relations to control no-smokers and control smokers; # difference of OxyA1AT in relations to control no-smokers and smokers; smokers with GOLD 2 and no-smokers with GOLD 3 + 4.

Discussion

In the past fifty years, since the discovery of the hereditary A1ATD, many epidemiological studies have investigated the association between this genetic abnormality and the onset of the COPD. However, in clinical practice of the diagnosis, prevention and monitoring of COPD, non-hereditary functional deficiency of A1AT caused by its oxidative modification is overlooked. Mostly due to the lack of a suitable and inexpensive method that would be used for assessment of the oxidative modified A1AT in routine laboratory practice.

In this study, COPD patients who were smokers had significantly higher levels of serum A1AT than healthy no-smokers (Figure 1c). A1AT as an acute phase protein increases rapidly (3- to 4-fold) in response to inflammation or infection (16). The obtained raised level of A1AT in COPD patients was expected, considering that the COPD is characterized by lung inflammation. Our results agree with the data obtained by Serapinas et al. (17), who found an elevated level of A1AT in COPD smokers, with the continued elevation of A1AT, even after cessation of smoking. The similar influence of smoking on serum level of A1AT was found in general population (18) that is dose-dependent, with highest A1AT levels in active smokers who consume at least 15 cigarettes per day. Moreover, the twenty-five year follow-up study revealed that the raised plasma level of A1AT was associated with an increased incidence of COPD exacerbations which require hospitalization (19). An association between elevated level of A1AT in exhaled breath condensate and COPD exacerbations was revealed too (20).

Contrary to the increased immunoreactive serum level of A1AT, the SIA-Elastase was significantly decreased in COPD-smokers in comparison with COPD no-smoker and no-smokers in control group (Figure 1b). Similarly, data from literature showed the significant decreased inhibitory activity of A1AT to elastase in lavage fluid from the epithelial surface of the lower respiratory tract in asymptomatic smokers and smokers with idiopathic pulmonary fibrosis (21). In vitro kinetics study (22) conducted with A1AT isolated from lungs has shown that cigarette smoking is associated with the significant reduction of association rate constant of A1AT for NE. All of these findings indicate that the serum level of A1AT has limited diagnostic accuracy in evaluation of functional capacity of A1AT to protect the respiratory tract from the proteolytic activity of NE.

Besides previously mentioned decreased functional activity of A1AT which was estimated as SIA-Elastase in COPD smokers (Figure 1b), another important finding in this study is highest level of OxyA1AT in COPD smokers in relation to no-smokers in both COPD and control groups (Figure 1d). Interestingly, the OxyA1AT was in strong inverse relation with SIA-Elastase in COPD-patients (Spearman’s R was –0.919), however in control group the inverse relation was moderate (Spearman’s R was –0.640) (Table 2). This data qualifies the SIA-Elastase and level of OxyA1AT as specific biomarkers for the assessment of functional inactivation of A1AT associated with COPD in smokers. Moreover, the highest serum level of OxyA1AT was found in group of smokers with severe and very severe stage of COPD, while the SIA-Elastase was the lowest in the same group of patients (Figure 2). Similarly, Fujita et al. (23) found that inhibitory capacity of A1AT, measured in bronchoalveolar lavage fluid (BAL), correlated inversely with emphysema severity.

To our best knowledge, this study has shown, for the first time that increased serum level of OxyA1AT, which was induced by smoking, is associated with a worse prognosis of COPD. These data contribute to highlighting the significant role of smoking-related oxidative stress in pathogenesis of COPD, as well as the importance of the application of OxyA1AT as a biomarker in clinical practice. The pro-oxidative species which are delivered to lungs by the tobacco smoke and/or by stimulated inflammatory cells are involved in several mechanisms responsible for oxidative damage of A1AT (11). The main target of oxidative modification is active center of A1AT at Met³⁵⁸ to methionine sulfoxide_, which consequently decreases the second order rate constant for association with NE (9, 24). In support of this, Carp et al (25) identified methionine sulfoxide in purified A1AT from healthy smokers’ BAL fluids, while in the nonsmokers’ BAL fluid it was not found. In the same study was shown decrease of the elastase inhibitory capacity of A1AT for 40% in smokers’ BAL fluid compared to no-smokers, which suggest that methionine oxidation may be the cause of decreased functional activity of A1AT in the lungs of the smokers.

It is well known that tobacco smoke abounds with pro-oxidants such as hydrogen peroxide, nitrogen dioxide, and transition metals. In case of the reduced antioxidant defense, these pro-oxidants can cause the oxidative stress-related inactivation of A1AT. Additionally, inflammatory cells at sites of acute or chronic inflammation liberate a number of reactive species and create the microenvironment which contributes to the oxidative inactivation of A1AT. Smoking, by itself provokes infiltration of neutrophils and alveolar macrophage in the lower respiratory tract which induces the release of a spectrum of oxidants and pro-oxidative enzymes that may inactivate A1AT in their local environment. As already mentioned, the oxidants released by inflammatory cells could oxidize A1AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD. Both risk factors, released proteases from triggered phagocytes and the reduced activity of anti-proteases, could be involved in lung tissue damage (26). Besides the loss of the anti-elastase activity, the OxyA1AT takes on the other biological properties which are involved in the pathogenesis of COPD. Activated myeloperoxidase-hydrogen peroxide system (MPO-H₂O₂) could oxidize the A1AT. The oxidized A1AT has a tendency to form complexes with IgA, and to lose inhibitor activity to proteases (27). A few studies revealed immunomodulatory role of OxyA1AT. One of them found that OxyA1AT could activate the monocytes (28). Other study revealed that OxyA1AT-generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1), which in turn attracts macrophages and neutrophils into the airways (29).

Conclusions

The level of A1AT is commonly used in clinical practice as an initial diagnostic biomarker of hereditary A1ATD associated with the premature onset of pulmonary emphysema. However, we have shown that serum biomarkers which are relatively simple for determination and inexpensive, SIA-Elastase and OxyA1AT may improve diagnosis, management, and prevention of COPD in clinical practice. The potential utility of OxyA1AT in clinical practice could be in the prevention of COPD progression, both in emphysema caused by inherited A1ATD, as well as in acquired functional deficiency of A1AT caused by smoking-induced oxidative stress. In many studies were evaluated various biomarkers of oxidative stress associated with COPD onset and severity (reviewed in Ref. 30). However, the advantage of OxyA1AT as potential biomarker of oxidative stress specific for COPD is its direct involvement in the pathogenesis of COPD. On the other hand, clinical significance of OxyA1AT as a prognostic biomarker could be in assessing the effectiveness of antioxidant therapy for COPD and emphysema.

Declaration of interest

The authors declare that there are no conflicts of interest.

Additional information

Funding

This work was supported by grants 173008 from the Ministry of Education and Science, Republic of Serbia.