COPD, 11:539–545, 2014 ISSN: 1541-2555 print / 1541-2563 online Copyright © Informa Healthcare USA, Inc. DOI: 10.3109/15412555.2014.898028
ORIGINAL RESEARCH
Paraoxonase 1 Activity in Patients with Chronic Obstructive Pulmonary Disease
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
Lada Rumora,1 Marija Grdic´ Rajkovic´,1 Lara Milevoj Kopcˇinovic´,2 Dolores Pancirov,3 Ivana Cˇepelak,1 and Tihana Žanic´ Grubišic´1 1
Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
2
University Department of Chemistry, Clinical Unit of Medical Biochemistry in Traumatology and Orthopedics, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia
3
Department of Biochemistry and Hematology Diagnosis, Dr. Ivo Pedišic´ general Hospital, Sisak, Croatia
Abstract Introduction: Paraoxonase 1 (PON1) is an antioxidative enzyme manly associated with high density lipoproteins (HDL) in the peripheral blood. The aim of this study was to determine the PON1 paraoxonase and arylesterase activities in patients with chronic obstructive pulmonary disease (COPD). We also aimed to determine the concentration of reduced thiol groups as a marker of protein oxidation. Materials and methods: The study included 105 patients with stable COPD and 44 healthy controls. PON1 activities and thiols concentration were assayed in sera by spectrophotometry. Results: PON1 basal (POX) and salt-stimulated paraoxonase activity (POX1) as well as arylesterase activity (ARE) were significantly reduced in COPD patients. In addition, concentration of reduced thiol groups was significantly decreased in COPD group. PON1 activities were similar in patients with different disease severity (GOLD stages). However, a significant reduction in POX, POX1 and ARE was observed already in GOLD II stage when compared to controls. POX and POX1 showed modest while ARE yielded very good power for discrimination between healthy subjects and COPD patients. Univariate and multivariate logistic regression analysis indicated that ARE is a good COPD predictor. Conclusion: Reduction of PON1 activity observed in COPD patients could be partly caused by oxidative environment. Lower concentrations of reduced thiol groups in COPD patients suggest that a decrease in PON1 activity could reflect oxidative changes of enzyme free cysteine residues. Furthermore, decreased PON1 arylesterase activity might indicate a down-regulation of PON1 concentration. Our results suggest that ARE could be considered as potential biomarker for COPD diagnosis.
Introduction
Keywords: paraoxonase 1, chronic obstructive pulmonary disease, thiols, oxidative stress, paraoxonase activity, arylesterase activity Correspondence to: Lada Rumora, Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Domagojeva 2, 10000 Zagreb, Croatia, phone: +38516394782, fax: +38514612716, email: [email protected]
Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory disease with increasing prevalence, morbidity and mortality (1,2). Cigarette smoking, air pollution and increase of free radicals in respiratory tract (by inflammation and infections) are the leading causes of oxidative stress in COPD patients (3,4). Smoking may enhance oxidative stress directly through the release of reactive oxygen and nitrogen radicals, and indirectly by weakening the antioxidant defence systems. Paraoxonase 1 (PON1) is an enzyme localized in Clara cells, endothelial cells, and type 1 cells of the alveolar epithelium with supposed protective role against oxidative stress (3, 5–7). After being synthesized in the liver, PON1 is secreted into plasma where it is mainly bound to the high density lipoproteins (HDL) (8). PON1 hydrolyzes 539
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
540
Rumora et al.
different substrates by its organophosphatase, arylesterase and lactonase activities (8–14). PON1 acts as antioxidative and antiatherogenic enzyme by hydrolyzing lipid peroxides in oxidized lipoproteins, thus protecting both HDL and low density lipoproteins (LDL) from oxidation (8,15,16,17). It was shown that three cysteine residues at positions 42, 284 and 353 (C42, C284, C353) are important for PON1 activity. C42 and C353 form a disulfide bond and these residues are important for secretion and catalytic activity of the enzyme (14,18). C284 is free and is assumed to be located closely to the active centre of the enzyme, thus possibly participating in orientation or binding of the substrate (14,19). Furthermore, it is not specifically required for paraoxonase/arylesterase activity, but is required for prevention of copper-induced LDL oxidation (14,19). Decreased, but not abolished, paraoxonase and arylesterase activities were observed when mutating the C284 residue to alanine or serine (8,19). Large inter-individual variability of up to 13 times for PON1 concentration and up to 40 times for PON1 activity was demonstrated. Both genetic and non-genetic factors affect PON1 concentration and activity. Genetic factors include polymorphisms in promoter and coding regions of the pon1 gene, while non-genetic factors comprise diet, alcohol consumption, exposure to environmental toxins, and different physiological and pathological conditions (8,20–25). Cigarette smoke is considered as one of the non-genetic factors associated with down-regulation of PON1 concentration and activity (3,5,6,20,22,23). Furthermore, reduced PON1 activity is reported in different diseases where aethiology is associated with increased oxidative stress (8,20,22). In the present study, we aimed to investigate PON1 paraoxonase and arylesterase activity in COPD patients. Furthermore, we explored the effect of disease severity and smoking history on PON1 activity. As PON1 antioxidative role depends on reduced sulfhydryl groups, and oxidation of those groups is indicator of oxidative stress in general, we measured thiol concentration in healthy and COPD individuals.
Materials and methods Study design The study included 105 patients with clinically stable COPD (32 smokers, 27 ex-smokers, 46 non-smokers) and control group of 44 healthy subjects (16 smokers, 13 ex-smokers, 15 non-smokers). Smokers were defined as current smokers who smoke more that 2 cigarettes daily and those who quit smoking up to 6 months before study enrolment; ex-smokers were defined as subjects who had smoking history during their lifetime but quit smoking more than 6 months before study enrolment; non-smokers were defined as subjects who had never smoked. Inclusion criterion for the COPD group was clinical diagnosis of COPD according to GOLD (Global Initiative for Chronic Obstructive Lung Disease) report (26).
COPD was diagnosed by a pulmonary specialist according to clinical examination (chronic and progressive dyspnoea, cough and sputum production) and spirometry results, measured on the first admission at the Department for Pulmology in Dr. Ivo Pedišić General Hospital (Sisak, Croatia). Fixed forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio FEV1/ FVC < 0.70 and percentage of FEV1 predicted were used to diagnose and classify patients into GOLD subgroups, according to disease severity (stage GOLD II: 50% ≤ FEV1 < 80% predicted; stage GOLD III: 30% ≤ FEV1 < 50% predicted; stage GOLD IV: FEV1 < 30% predicted or FEV1 < 50% predicted with the presence of chronic respiratory failure). Healthy subjects were recruited from the same geographic area by a general practitioner and all were apparently healthy according to the exclusion criteria with normal spirometry results. Exclusion criteria, for both COPD patients and healthy subjects, included the presence of pulmonary diseases other than COPD, infective and inflammatory diseases, neoplastic pathologies, renal, gastrointestinal, endocrine and hepatic diseases, and excessive alcohol consumption (≥40 g/day). All patients were in the stable phase of the disease for at least 3 months without need for hospitalization. Their medication therapy consisted of bronchodilators, anticholinergic agents, theophylline and inhaled corticosteroids. No therapy modification was applied. The study was approved by the Medical Ethics Committee for Human Studies of Dr. Ivo Pedišić General Hospital, and informed consent was signed by all subjects enrolled in the study. The study was designed according to the Declaration of Helsinki. Serum samples were collected from all subjects after an overnight fast and they were stored at –20°C until further analysis.
Methods Lipid parameters Concentrations of triglycerides and total cholesterol were determined on Alcyon 300 (Abbott Diagnostics, IL, USA) with commercially available reagents (Herbos Diagnostic d.o.o, Sisak, Hrvatska). Concentration of HDL-cholesterol was measured on Dimension Xpand Plus (Siemens Healthcare Diagnostics, Deerfield, Illinois USA) with commercial reagent (Siemens Healthcare Diagnostics, Deerfield, Illinois USA). Concentration of LDL-cholesterol was calculated using Friedwald equation [LDL-cholesterol (mmol/L) = total cholesterol – HDL-cholesterol - (triglycerides/2.2)]. PON1 paraoxonase and arylesterase activities PON1 activity in serum was assessed by two different substrates: paraoxon (PON1 paraoxonase activity) and phenylacetate (PON1 arylesterase activity). PON1 paraoxonase activity was measured in the absence and in the presence of NaCl (basal and Copyright © 2014 Informa Healthcare USA, Inc
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
Paraoxonase 1 and chronic obstructive pulmonary disease
salt-stimulated paraoxonase activity), using the Olympus 540 analyzer (Beckman Coulter, Brea, CA, USA) at 37°C, as described previously (27). Briefly, reaction mixture contained 15 μL of serum and 300 μL of reagent (2.5 mmol/L paraoxon of ~ 90% purity, 2.2 mmol/L CaCl2 in 0.1 mol/l Tris-HCl buffer, pH 8.0). For salt-stimulated paraoxonase activity buffer contained 1.0 mol/L NaCl. The release of p-nitrophenol was measured at 410/480 nm (ε = 17900 L/mol cm) and the PON1 enzyme activity was calculated. PON1 arylesterase activity was determined by previously described methods (28,29), with some modifications. Briefly, 200 μL of freshly prepared substrate (3.26 mmol/L phenylacetate in 20 mmol/L Tris-HCL buffer with 1 mmol/L CaCl2, pH 8.0) was added to 20 μL of serum samples (diluted 1:80 with buffer). Reaction mixture for determination of spontaneous substrate hydrolysis contained 20 μL of buffer and 200 μL of substrate. Distilled water was used as blank. The release of phenol was measured at room temperature continuously (every 30 seconds) during 4 minutes at 260 nm (ε = 1310 l/mol cm) on a microplate reader (1420 Victor3, PerkinElmer, USA). Calculated PON1 arylesterase activities in serum samples were corrected for spontaneous substrate hydrolysis.
Concentration of reduced thiol groups Concentration of reduced thiol groups in serum was measured by the method described by Hu et al. (30). Statistical analysis Statistical analysis was performed using SigmaStat for Windows, version 3.0, and MedCalc for Windows version 12.4.0. Normality of distribution was tested by the Kolmogorov-Smirnov test. Depending on the distribution, the difference between two groups was tested using Mann–Whitney Rank Sum Test or t-test for nonparametric and parametric data, respectively. The difference between more than two groups was tested using the Kruskal–Wallis Analysis of Variance on Ranks or by One Way Analysis of Variance, for nonparametric and parametric data, respectively. Post hoc testing was performed using pairwise comparisons by the Holm–Sidak or Dunn methods. Linear regression analysis was used to assess the relationship of gender, age, BMI, smoking status, lung function parameters, lipid parameters and thiol concentration with PON1 activities. Diagnostic accuracy for PON1 activities was assessed using receiver operating characteristic (ROC) curve analysis. Univariate and multivariate logistic regression were used to analyze the suitability of PON1 activities in predicting COPD. Chi-square test was used for comparison of proportions. Quantitative data are presented as mean and standard deviation or as median and interquartile range, while qualitative data are presented as absolute numbers and percentages. All p values lower than 0.05 were considered statistically significant. www.copdjournal.com
Table 1. Demographic characteristics, lung function, lipid and oxidative stress parameters of healthy and COPD individuals Controls n = 44 Age
COPD n = 105
p
52 (46–56)
71 (65–76)
ORIGINAL RESEARCH
Paraoxonase 1 Activity in Patients with Chronic Obstructive Pulmonary Disease
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
Lada Rumora,1 Marija Grdic´ Rajkovic´,1 Lara Milevoj Kopcˇinovic´,2 Dolores Pancirov,3 Ivana Cˇepelak,1 and Tihana Žanic´ Grubišic´1 1
Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
2
University Department of Chemistry, Clinical Unit of Medical Biochemistry in Traumatology and Orthopedics, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia
3
Department of Biochemistry and Hematology Diagnosis, Dr. Ivo Pedišic´ general Hospital, Sisak, Croatia
Abstract Introduction: Paraoxonase 1 (PON1) is an antioxidative enzyme manly associated with high density lipoproteins (HDL) in the peripheral blood. The aim of this study was to determine the PON1 paraoxonase and arylesterase activities in patients with chronic obstructive pulmonary disease (COPD). We also aimed to determine the concentration of reduced thiol groups as a marker of protein oxidation. Materials and methods: The study included 105 patients with stable COPD and 44 healthy controls. PON1 activities and thiols concentration were assayed in sera by spectrophotometry. Results: PON1 basal (POX) and salt-stimulated paraoxonase activity (POX1) as well as arylesterase activity (ARE) were significantly reduced in COPD patients. In addition, concentration of reduced thiol groups was significantly decreased in COPD group. PON1 activities were similar in patients with different disease severity (GOLD stages). However, a significant reduction in POX, POX1 and ARE was observed already in GOLD II stage when compared to controls. POX and POX1 showed modest while ARE yielded very good power for discrimination between healthy subjects and COPD patients. Univariate and multivariate logistic regression analysis indicated that ARE is a good COPD predictor. Conclusion: Reduction of PON1 activity observed in COPD patients could be partly caused by oxidative environment. Lower concentrations of reduced thiol groups in COPD patients suggest that a decrease in PON1 activity could reflect oxidative changes of enzyme free cysteine residues. Furthermore, decreased PON1 arylesterase activity might indicate a down-regulation of PON1 concentration. Our results suggest that ARE could be considered as potential biomarker for COPD diagnosis.
Introduction
Keywords: paraoxonase 1, chronic obstructive pulmonary disease, thiols, oxidative stress, paraoxonase activity, arylesterase activity Correspondence to: Lada Rumora, Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Domagojeva 2, 10000 Zagreb, Croatia, phone: +38516394782, fax: +38514612716, email: [email protected]
Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory disease with increasing prevalence, morbidity and mortality (1,2). Cigarette smoking, air pollution and increase of free radicals in respiratory tract (by inflammation and infections) are the leading causes of oxidative stress in COPD patients (3,4). Smoking may enhance oxidative stress directly through the release of reactive oxygen and nitrogen radicals, and indirectly by weakening the antioxidant defence systems. Paraoxonase 1 (PON1) is an enzyme localized in Clara cells, endothelial cells, and type 1 cells of the alveolar epithelium with supposed protective role against oxidative stress (3, 5–7). After being synthesized in the liver, PON1 is secreted into plasma where it is mainly bound to the high density lipoproteins (HDL) (8). PON1 hydrolyzes 539
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
540
Rumora et al.
different substrates by its organophosphatase, arylesterase and lactonase activities (8–14). PON1 acts as antioxidative and antiatherogenic enzyme by hydrolyzing lipid peroxides in oxidized lipoproteins, thus protecting both HDL and low density lipoproteins (LDL) from oxidation (8,15,16,17). It was shown that three cysteine residues at positions 42, 284 and 353 (C42, C284, C353) are important for PON1 activity. C42 and C353 form a disulfide bond and these residues are important for secretion and catalytic activity of the enzyme (14,18). C284 is free and is assumed to be located closely to the active centre of the enzyme, thus possibly participating in orientation or binding of the substrate (14,19). Furthermore, it is not specifically required for paraoxonase/arylesterase activity, but is required for prevention of copper-induced LDL oxidation (14,19). Decreased, but not abolished, paraoxonase and arylesterase activities were observed when mutating the C284 residue to alanine or serine (8,19). Large inter-individual variability of up to 13 times for PON1 concentration and up to 40 times for PON1 activity was demonstrated. Both genetic and non-genetic factors affect PON1 concentration and activity. Genetic factors include polymorphisms in promoter and coding regions of the pon1 gene, while non-genetic factors comprise diet, alcohol consumption, exposure to environmental toxins, and different physiological and pathological conditions (8,20–25). Cigarette smoke is considered as one of the non-genetic factors associated with down-regulation of PON1 concentration and activity (3,5,6,20,22,23). Furthermore, reduced PON1 activity is reported in different diseases where aethiology is associated with increased oxidative stress (8,20,22). In the present study, we aimed to investigate PON1 paraoxonase and arylesterase activity in COPD patients. Furthermore, we explored the effect of disease severity and smoking history on PON1 activity. As PON1 antioxidative role depends on reduced sulfhydryl groups, and oxidation of those groups is indicator of oxidative stress in general, we measured thiol concentration in healthy and COPD individuals.
Materials and methods Study design The study included 105 patients with clinically stable COPD (32 smokers, 27 ex-smokers, 46 non-smokers) and control group of 44 healthy subjects (16 smokers, 13 ex-smokers, 15 non-smokers). Smokers were defined as current smokers who smoke more that 2 cigarettes daily and those who quit smoking up to 6 months before study enrolment; ex-smokers were defined as subjects who had smoking history during their lifetime but quit smoking more than 6 months before study enrolment; non-smokers were defined as subjects who had never smoked. Inclusion criterion for the COPD group was clinical diagnosis of COPD according to GOLD (Global Initiative for Chronic Obstructive Lung Disease) report (26).
COPD was diagnosed by a pulmonary specialist according to clinical examination (chronic and progressive dyspnoea, cough and sputum production) and spirometry results, measured on the first admission at the Department for Pulmology in Dr. Ivo Pedišić General Hospital (Sisak, Croatia). Fixed forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio FEV1/ FVC < 0.70 and percentage of FEV1 predicted were used to diagnose and classify patients into GOLD subgroups, according to disease severity (stage GOLD II: 50% ≤ FEV1 < 80% predicted; stage GOLD III: 30% ≤ FEV1 < 50% predicted; stage GOLD IV: FEV1 < 30% predicted or FEV1 < 50% predicted with the presence of chronic respiratory failure). Healthy subjects were recruited from the same geographic area by a general practitioner and all were apparently healthy according to the exclusion criteria with normal spirometry results. Exclusion criteria, for both COPD patients and healthy subjects, included the presence of pulmonary diseases other than COPD, infective and inflammatory diseases, neoplastic pathologies, renal, gastrointestinal, endocrine and hepatic diseases, and excessive alcohol consumption (≥40 g/day). All patients were in the stable phase of the disease for at least 3 months without need for hospitalization. Their medication therapy consisted of bronchodilators, anticholinergic agents, theophylline and inhaled corticosteroids. No therapy modification was applied. The study was approved by the Medical Ethics Committee for Human Studies of Dr. Ivo Pedišić General Hospital, and informed consent was signed by all subjects enrolled in the study. The study was designed according to the Declaration of Helsinki. Serum samples were collected from all subjects after an overnight fast and they were stored at –20°C until further analysis.
Methods Lipid parameters Concentrations of triglycerides and total cholesterol were determined on Alcyon 300 (Abbott Diagnostics, IL, USA) with commercially available reagents (Herbos Diagnostic d.o.o, Sisak, Hrvatska). Concentration of HDL-cholesterol was measured on Dimension Xpand Plus (Siemens Healthcare Diagnostics, Deerfield, Illinois USA) with commercial reagent (Siemens Healthcare Diagnostics, Deerfield, Illinois USA). Concentration of LDL-cholesterol was calculated using Friedwald equation [LDL-cholesterol (mmol/L) = total cholesterol – HDL-cholesterol - (triglycerides/2.2)]. PON1 paraoxonase and arylesterase activities PON1 activity in serum was assessed by two different substrates: paraoxon (PON1 paraoxonase activity) and phenylacetate (PON1 arylesterase activity). PON1 paraoxonase activity was measured in the absence and in the presence of NaCl (basal and Copyright © 2014 Informa Healthcare USA, Inc
COPD Downloaded from informahealthcare.com by University of Liverpool on 10/08/14 For personal use only.
Paraoxonase 1 and chronic obstructive pulmonary disease
salt-stimulated paraoxonase activity), using the Olympus 540 analyzer (Beckman Coulter, Brea, CA, USA) at 37°C, as described previously (27). Briefly, reaction mixture contained 15 μL of serum and 300 μL of reagent (2.5 mmol/L paraoxon of ~ 90% purity, 2.2 mmol/L CaCl2 in 0.1 mol/l Tris-HCl buffer, pH 8.0). For salt-stimulated paraoxonase activity buffer contained 1.0 mol/L NaCl. The release of p-nitrophenol was measured at 410/480 nm (ε = 17900 L/mol cm) and the PON1 enzyme activity was calculated. PON1 arylesterase activity was determined by previously described methods (28,29), with some modifications. Briefly, 200 μL of freshly prepared substrate (3.26 mmol/L phenylacetate in 20 mmol/L Tris-HCL buffer with 1 mmol/L CaCl2, pH 8.0) was added to 20 μL of serum samples (diluted 1:80 with buffer). Reaction mixture for determination of spontaneous substrate hydrolysis contained 20 μL of buffer and 200 μL of substrate. Distilled water was used as blank. The release of phenol was measured at room temperature continuously (every 30 seconds) during 4 minutes at 260 nm (ε = 1310 l/mol cm) on a microplate reader (1420 Victor3, PerkinElmer, USA). Calculated PON1 arylesterase activities in serum samples were corrected for spontaneous substrate hydrolysis.
Concentration of reduced thiol groups Concentration of reduced thiol groups in serum was measured by the method described by Hu et al. (30). Statistical analysis Statistical analysis was performed using SigmaStat for Windows, version 3.0, and MedCalc for Windows version 12.4.0. Normality of distribution was tested by the Kolmogorov-Smirnov test. Depending on the distribution, the difference between two groups was tested using Mann–Whitney Rank Sum Test or t-test for nonparametric and parametric data, respectively. The difference between more than two groups was tested using the Kruskal–Wallis Analysis of Variance on Ranks or by One Way Analysis of Variance, for nonparametric and parametric data, respectively. Post hoc testing was performed using pairwise comparisons by the Holm–Sidak or Dunn methods. Linear regression analysis was used to assess the relationship of gender, age, BMI, smoking status, lung function parameters, lipid parameters and thiol concentration with PON1 activities. Diagnostic accuracy for PON1 activities was assessed using receiver operating characteristic (ROC) curve analysis. Univariate and multivariate logistic regression were used to analyze the suitability of PON1 activities in predicting COPD. Chi-square test was used for comparison of proportions. Quantitative data are presented as mean and standard deviation or as median and interquartile range, while qualitative data are presented as absolute numbers and percentages. All p values lower than 0.05 were considered statistically significant. www.copdjournal.com
Table 1. Demographic characteristics, lung function, lipid and oxidative stress parameters of healthy and COPD individuals Controls n = 44 Age
COPD n = 105
p
52 (46–56)
71 (65–76)
