Biol Trace Elem Res DOI 10.1007/s12011-014-0087-4
Increased Oxidative Stress and Altered Levels of Nitric Oxide and Peroxynitrite in Tunisian Patients with Chronic Obstructive Pulmonary Disease: Correlation with Disease Severity and Airflow Obstruction Amel ben Anes & Hamadi Fetoui & Sarra Bchir & Hela ben Nasr & Hassiba Chahdoura & Elyes Chabchoub & Saloua Yacoub & Abdelhamid Garrouch & Mohamed Benzarti & Zouhair Tabka & Karim Chahed
Received: 30 May 2014 / Accepted: 21 July 2014 # Springer Science+Business Media New York 2014
Abstract This study was aimed to evaluate the oxidant–antioxidant imbalance in the pathogenesis of chronic obstructive pulmonary disease (COPD) in Tunisians. We assessed 16 parameters related to the oxidative status that include malondialdehyde (MDA), total protein carbonyls (PCs), and advanced oxidation protein products (AOPP). We also examined the activity of glutathione peroxydase (GSH-Px), catalase, and superoxide dismutase (SOD) in the plasma and erythrocytes. Levels of total thiols, reduced glutathione (GSH), total antioxidant status (TAS), hydrogen peroxide, ascorbic acid, iron, and protein sulfhydryls were determined using spectrophotometry. We also evaluated the level of nitric oxide (NO) and peroxynitrite in plasma from COPD patients and healthy controls. Estimation of DNA damage was
determined using the comet assay. Pulmonary functional tests were performed by body plethysmography. Levels of MDA, PC, DNA damage, and AOPP were significantly increased while total thiols, GSH, and TAS were decreased in COPD patients. GSH-Px activity was higher in COPD patients while no difference was found for catalase and SOD. We also observed a lower level of NO and peroxynitrite in COPD patients. Decreased levels of peroxynitrite were found to correlate with disease progression, as well as with forced expiratory volume in 1 s/forced vital capacity among COPD patients. Multivariate analysis revealed that NO is associated with pathological pathways that help to predict patient outcome independently of the degree of airflow obstruction. These results indicate the presence of a systemic oxidative
Summary at a Glance: This study provides a strong evidence for altered oxidant–antioxidant balance in the plasma from Tunisian patients with COPD. Our findings also point out the relevance of nitric oxide and peroxynitrite as major effectors in COPD development and airflow obstruction. A. ben Anes : S. Bchir : H. ben Nasr : Z. Tabka : K. Chahed (*) Unité de recherche UR12ES06, Physiologie de l’Exercice et Physiopathologie: de l’Intégré au Moléculaire « Biologie, Médecine et Santé », Faculté de Médecine de Sousse, Université de Sousse, Sousse, Tunisia e-mail:
[email protected] H. Fetoui Unité de Recherche de Toxicologie-Microbiologie Environnementale et Santé (UR/11 ES70), Faculté des Sciences de Sfax, Sfax, Tunisia H. Chahdoura Unité de Recherche 03/UR/09-01 «Génome, Diagnostic Immunitaire et valorisation», Institut Supérieur de Biotechnologie de Monastir, Université de Monastir, Monastir, Tunisia
E. Chabchoub Unité de Recherche 04/UR/08-05 Molecular Immunogenetics, Faculté de Médecine, Sousse, Tunisia S. Yacoub Unité de Recherche « UR06SP05 » Centre Régional de Transfusion Sanguine, CHU Farhat Hached, Sousse, Tunisia A. Garrouch : M. Benzarti Service de Pneumo-Allergologie, CHU Farhat Hached, Sousse, Tunisia K. Chahed Faculté des Sciences de Sfax, Sfax, Tunisia
ben Anes et al.
stress and highlight the importance of NO and peroxynitrite as major effectors in COPD development and airflow obstruction. Keywords Airflow obstruction . COPD . Nitric oxide . Oxidative stress . Peroxynitrite Abbreviations AOPP Advanced oxidation protein products BMI Body mass index CAT Catalase COPD Chronic obstructive pulmonary disease FEV1% Forced expiratory volume in 1 s (%predicted) FVC Forced vital capacity GOLD Global Initiative for Chronic Obstructive Lung Disease GSH Glutathione GSH-Px Glutathione peroxydase MDA Malondialdehyde NO Nitric oxide PC Protein carbonyls RNS Reactive nitrogen species ROS Reactive oxygen species SOD Superoxide dismutase TAS Total antioxidant status TBA Thiobarbituric acid
Introduction Chronic obstructive pulmonary disease (COPD) is a major source of morbidity and mortality worldwide that has a large burden on individuals, families, and society [1]. Actually, COPD represents the third most common cause of death in the world and is characterized by airflow limitation and an abnormal inflammatory response of the lungs to noxious particles or gases. It is well established that smoking is the most aetiological factor in the pathogenesis of COPD. However, the contribution of other risk factors is equally important since not all smokers develop COPD. The mechanism of the aetiopathogenesis of COPD implicates several processes, such as inflammation and protease/antiprotease imbalance. Oxidative stress and imbalances in host defense mechanisms may also be among the major causes of COPD development [2]. Under normal conditions, oxidants generated in the biological fluids are efficiently scavenged by antioxidants. Nevertheless, these antioxidants could be dysregulated in response to the increased oxidative burden during COPD development. Actually, oxidative stress has been shown to be a major factor predisposing to the development of COPD. The increased number of neutrophils and macrophages in the lungs of COPD patients can generate large amounts of oxidants that
lead to the activation of several molecular pathways, including kinases, transcription factors, as well as epigenetic events that stimulate the inflammatory response [3]. Oxidants may also affect proliferation and apoptosis and induce mucus hypersecretion, as well as remodeling of extracellular matrix in the lungs. Up to now, increased reactive oxygen species (ROS)/ reactive nitrogen species (RNS) generation has been directly associated with the oxidation of proteins, nucleic acids, and lipids. Free radicals can react with cell membrane fatty acids and produce lipid peroxides which can alter membrane fluidity and affect membrane-bound receptors and enzymes [4]. Reactive species can also attack proteins leading to the formation of protein–protein cross-linkages resulting in fragmentation and generation of protein oxidation products [5]. Protein carbonylation is also an indicator of free radical generation in cells and a useful biomarker of protein oxidation in several human diseases. Assessment of protein carbonyl (PC) levels is also considered as one of the best ways to monitor inflammation [6]. Previous studies have shown that several oxidative markers are altered in the air spaces, fluids, and sputum of patients with COPD. Reports on the relationships between the oxidant– antioxidant imbalance and pulmonary function, as well as disease progression showed, however, contradictory results. As an example, Corradi and colleagues found that exhaled nitric oxide (NO) is increased in patients with stable COPD and correlates positively with lung function as assessed by forced expiratory volume in 1 s (FEV1) [7]. Another study affirmed, however, that NO levels were not different between COPD patients and healthy controls and that an inverse correlation occurs between forced expiratory volume (FEV1) and NO levels in patients with COPD [8]. In a similar context, Rutgers and colleagues found that NO metabolism was not affected in stable COPD patients [9]. Conflicting results were also reported with regard to the involvement of superoxide dismutase and catalase in COPD. As an example, Ahmad and colleagues found decreased activities of superoxide dismutase and catalase whereas others revealed an enhanced activity of these antioxidative proteins in patients with COPD [10, 11]. In a previous study, Kluchova et al. [12] revealed through linear regression analysis a significant inverse relationship between FEV1 and plasma thiobarbituric acid-reactive substances (TBARS) in COPD patients. Other reports unveiled, however, that although TBARs levels were elevated in expired breath condensate from COPD subjects, no significant correlation occurs with the different spirometric parameters [13]. In another context, Nadeem and colleagues found a significant correlation of glutathione with the severity of airways obstruction whereas others revealed no relationships between thiols or total antioxidant status and measurement of airflow limitation in patients with COPD [14, 15]. To the best of our knowledge, no previous studies have investigated the impact of oxidative stress in the development
Nitric Oxide and Peroxynitrite Are Major Effectors in COPD
of COPD in Tunisians. In the current study, we therefore measured simultaneous changes in a wide range of parameters of oxidant–antioxidant balance and relate it to disease progression and the rate of decline of lung function in COPD patients. The extent of oxidative stress was estimated on the basis of its foremost biomarkers: lipid peroxidation products, protein carbonyls, advanced oxidation products, nonenzymatic antioxidants (glutathione, total thiols), and antioxidant defense enzymes (glutathione peroxydase, catalase, superoxide dismutase). Another aim of the study was to compare the levels of NO and peroxynitrite in COPD patients and to evaluate their impact on disease stage and airflow obstruction.
was determined and expressed as weight (kg)/height (m2). Pack years were calculated using the following formula (Number of cigarettes per day×number of years smoked ÷ 20). Blood Sample Collection and Storage Blood samples (10 ml) from both healthy volunteers and COPD patients were obtained by venipuncture and drawn into heparinised tubes. To obtain plasma, tubes were centrifuged at 3,500 rpm for 15 min. Aliquots of erythrocytes and plasma were immediately frozen at −80 °C until used. Washed red cells were lysed using ice-cold distilled water and frozen at −80 °C. Thiobarbituric Acid-Reactive Substances
Materials and Methods Study Subjects Patients with COPD (153) and healthy controls (229) were selected from the same population living in the middle coast of Tunisia. Healthy subjects were recruited from the National Blood Transfusion Center (Farhat Hached Hospital). Patients with COPD were recruited from the Services of Pneumology and Physiology at Farhat Hachad Hospital (Sousse). All participants gave written informed consent, and the study was approved by the local ethical committee. We defined COPD according to the Global Initiative for Chronic Obstructive Lung Disease criteria (GOLD). Airflow limitation in COPD is defined as a postbronchodilator forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC) ratio
