J Med Biochem 2016; 35 (1) DOI: 10.1515/jomb-2015-0006 UDK 577.1 : 61 ISSN 1452-8258 J Med Biochem 35: 55 –62, 2016 Original paper Originalni nau~ni rad EVALUATION OF SYSTEMIC ANTIOXIDANT LEVEL AND OXIDATIVE STRESS IN RELATION TO LIFESTYLE AND DISEASE PROGRESSION IN ASTHMATIC PATIENTS EVALUACIJA NIVOA ANTIOKSIDANASA I OKSIDATIVNOG STRESA U SISTEMU U ODNOSU NA @IVOTNI STIL I PROGRESIJU BOLESTI KOD OBOLELIH OD ASTME Abhay Singh Yadav, Manisha Saini Department of Zoology, Kurukshetra University, Kurukshetra, Haryana, India Summary Kratak sadr`aj Background: Asthma is a chronic disorder of the airways. Uvod: Astma je hroni~no oboljenje disajnih puteva. Oksi- Oxidative stress is an important part of asthma pathogene- dativni stres ~ini va`an deo u patogenezi astme. Ima pre- sis. It plays a crucial role in exacerbating the disease, as sudnu ulogu u pogor{anju bolesti i predstavlja va`nu posle- well as an important consequence of airways inflammation. dicu upale disajnih puteva. Aim: The present study was undertaken to investigate the Ova studija je preduzeta kako bi se istra`ili lipidna peroksi- lipid peroxidation and catalase activity in serum and antiox- dacija i aktivnost katalaze u serumu i nivo antioksidanasa u idant level in plasma of asthmatic patients and their associ- plazmi kod bolesnika sa astmom i njihova povezanost sa ation with lifestyle and severity of the disease. `ivotnim stilom i te`inom bolesti. Methods: A total of 210 subjects, 120 asthmatics and 90 Metode: Ukupno 210 ispitanika, 120 astmati~ara i 90 healthy controls matched in respect to age, sex, lifestyle zdravih kontrolnih ispitanika odgovaraju}e starosti, pola, `i- and socioeconomic status, were chosen randomly for the votnog stila i socioekonomskog statusa, nasumi~no je iza- present study. The samples were analyzed for MDA con- brano za ovu studiju. Analizom uzoraka odre|ene su kon- centration and catalase activity in serum and ferric reduc- centracija MDA i aktivnost katalaze u serumu i primenjena ing ability of plasma (FRAP). Statistical analysis was done je metoda FRAP (ferric reducing ability of plasma). Stati- using unpaired Student’s t-test, ANOVA with Duncan post sti~ka analiza je izvr{ena pomo}u Studentovog t testa, testa hoc test and Pearson coefficient of correlation. ANOVA sa Duncan post hoc testom i Pearsonove korelacije Results: The serum MDA was found to be significantly koeficijenta. higher in the asthmatics as compared to healthy individu- Rezultati: MDA u serumu bio je zna~ajno vi{i kod astma- als (p<0.01) while catalase activity in serum and antioxi- ti~ara u pore|enju sa zdravim ispitanicima (p<0,01), dok dant level of the plasma were markedly lower in the asth- su aktivnost katalaze u serumu i nivo antioksidanasa u plaz- matics as compared to healthy individuals (p<0.01). A mi bili upadljivo ni`i kod astmati~ara u pore|enju sa zdra- significant difference was observed in serum MDA, cata- vim ispitanicima (p<0,01). Izme|u pacijenata je uo~ena lase activity and plasma antioxidant level among the zna~ajna razlika u nivoima MDA u serumu, aktivnosti kata- patients in relation to the severity of disease. There was a laze i nivoima antioksidanasa u plazmi u odnosu na te`inu marked increase in the serum MDA in the patients with oboljenja. Postojao je upadljiv porast nivoa MDA u serumu longer duration of the disease (p<0.05). kod pacijenata povezan sa du`inom bolesti (p<0,05). Conclusions: The oxidant–antioxidant imbalance occurs in Zaklju~ak: U astmi se javlja poreme}aj oksidantne–antiok- asthma leading to oxidative stress and is an important part sidantne ravnote`e, {to dovodi do oksidativnog stresa i ~ini of the asthma pathogenesis. va`an deo patogeneze astme. Keywords: asthma, antioxidants, oxidants, oxidative Klju~ne re~i: astma, antioksidansi, oksidansi, oksidativni stress, lipid peroxidation stres, lipidna peroksidacija Abhay Singh Yadav, Professor Department of Zoology, Kurukshetra University, Kurukshetra, India e-mail: abyzkukªgmail.com Ph. +91-9416173289 56 Singh Yadav et al.: Systemic oxidative stress in asthmatic patients Introduction version of ferric to ferrous ion at low pH and for- mation of a ferrous-tripyridyltriazine (TPTZ) complex The increasing incidence of asthma is a serious which gives blue color (20). global issue especially in the urban population (1). Asthma is a major cause of chronic morbidity and The present study aimed to compare the malon- mortality with an estimated 300 million people affect- dialdehyde and catalase activity level in serum and ed worldwide and the incidences have increased in the ferric reducing ability of plasma between the asth- the past few years (2). Asthma is characterized by matic patients and healthy individuals. We have also chronic inflammation of the airways which involves studied the effects of lifestyle including smoking, recurrent episodes of wheezing, airflow obstruction drinking and dietary habits and other related factors and airways hyper-responsiveness to a variety of stim- on disease progression. uli (3–5). Oxidative stress is defined as the damage that occurs when the oxidant level in the body over- whelms the antioxidant defense because of the Materials and Methods increased reactive oxygen species such as superoxide Subjects radical (O2*), hydrogen peroxide (H2O2), hypochlor- ous acid (HOCl), and hydroxyl radical (OH*). The A total of 210 subjects, 120 asthmatics and 90 oxidative stress is an important component as well as healthy controls matched with respect to age, sex, consequence of asthma pathogenesis and various lifestyle and socioeconomic status, were chosen ran- factors like age, duration and lifestyle determine the domly for the present study. All the patients were overall impact of disease. Increased production of diagnosed with spirometry tests and clinical symp- ROS in asthma has been reported in many studies (6- toms by a registered medical practitioner and the 9) and also that it is associated with an alteration in severity of disease was determined as per the Global antioxidant activity in the lung and blood (10) and the Initiative for Asthma guidelines (3). The diagnosis was development of airway hyper-responsiveness (11). done on the basis of symptoms, recurrent episodes, The inflammation of the airways leads to overproduc- wheezing and measuring the forced expiratory volu- tion of the reactive oxygen species (ROS) that cause me in 1 second (FEV1) and other spirometric para- oxidative stress by interfering with the tissues in our meters like forced vital capacity (FVC), peak expirato- body like proteins, lipids and DNA and causing dys- ry flow rate (PEF) etc. For control, those individuals function of these molecules. The ROS generate a were selected who had no respiratory symptoms or large number of oxidative modifications in DNA in- any other illness and were not taking any drugs. cluding strand breaks and base oxidations (12–14). In An appropriate written informed consent was addition, the physiological antioxidant system that is taken from each subject prior to taking samples. The equipped to remove the oxidants is also impaired in ethical clearance was obtained from the Institutional asthma due to increased inflammation (15). Further- Ethics Committee of Kurukshetra University, Kuruks- more, poor diet or lifestyle and lesser intake of anti- hetra. A detailed questionnaire was filled in by each oxidants also lead to increased oxidative stress and subject to collect details regarding their sex, age, worsen the symptoms in the patients. The reactive dietary habits, smoking and drinking habits and other oxygen species exaggerate airways inflammation by parameters related to their lifestyle. Each patient’s inducing many proinflammatory mediators including clinical profile, spirometric measurements, history of macrophages, neutrophils and eosinophils (16 –17). allergy and treatment details were also collected. The The enzymatic antioxidants such as catalase present study was carried out keeping in view the Declaration in the pulmonary fluid and interstitial spaces of the of Helsinki ethical guidelines for experiments involv- lungs and also in the blood vessels and airways help ing humans. convert the potent oxidant hydrogen peroxide (H2O2) to H2O thus helping to reduce systemic oxidant level. These enzymatic antioxidants have been reported to Sample collection be decreased in asthmatic patients, which further aids in systemic oxidative stress (7). The increased oxida- Blood samples were drawn from the vein of the tive stress can also be attributed to increased lipid subjects by a registered medical practitioner and peroxidation in asthmatic patients. The malondialde- taken to the laboratory in plastic vials. The blood was hyde (MDA) concentration in serum is an important allowed to clot for 30 minutes and then centrifuged biomarker tool to assess increased oxidative stress at 2500 rpm for 15 minutes. The serum was collect- and lipid peroxidation in asthmatic patients (18 –19). ed and a part of it was analyzed within 24 hours. The Further, the reduced antioxidant level in the diseased rest of it was stored at –20 °C for use at a later stage persons which exacerbates the symptoms is also im- if required. For plasma, blood was collected in portant and should be considered. The Ferric Re- K2EDTA coated vials (Becton Dickinson) and centri- ducing Ability of Plasma (FRAP) assay is a rapid and fuged at 2500 rpm. Analysis was done within 24 novel method to measure the total antioxidant power hours. All the reagents were prepared in the labora- of plasma. The basic principle of this assay is the con- tory conditions. No commercial kit was used. J Med Biochem 2016; 35 (1) 57 Catalase activity in serum assays conducted during the present study and for preparation of graphs. The following tests were The catalase activity was evaluated by the applied: 1) unpaired Student’s t-test, 2) one-way method of Aebi (21) on the basis of oxidation of analysis of variance (ANOVA) with Duncan post hoc H2O2. The activity of catalase was measured by tak- test and 3) Pearson’s correlation coefficient (2-tailed). ing 20 mL serum in 4 mL of 50 mmol/L phosphate A p<0.05 was considered as significant. buffer (HIMEDIA) in a test tube. Then, 0.65 mL of substrate (10 mmol/L H2O2) was added and imme- diately read at 240 nm for 3 minutes by a UV-visible Results nanophotometer (IMPLEN, Germany). The results were expressed in U/mg protein. The general and clinical characteristics of all the subjects are given in Table I. The average age of the subjects was 42.48 years (range 13–80 years). These Lipid peroxidation assay were characterized for gender, age, smoking and drinking habits, dietary habits, daily physical activity For detecting the level of lipid peroxidation in and biomass exposure (use of biomass fuel). Out of serum, MDA concentration was analyzed using the 210 subjects, 97 (46.2%) were males and 113 standard protocol of Buege and Aust (22). For this, (53.8%) were females. A significant difference 0.1 mL sample, 0.1 mL Tris-HCl buffer, 0.1 mL (p<0.01) was observed in the spirometric measure- FeSO4 and 0.1 mL ascorbic acid were added in a test ments (FVC, FEV1, FEV1/FVC, PEF and FEF25 –75% tube, then 0.6 mL dH2O was added to make the vol- predicted) of the asthmatics and the healthy subjects ume 1.0 mL. It was incubated at 37 ˚C for 15 min. (Table I). The serum MDA (0.75±0.08) was found to Then, 1.0 mL tri-chloro acetic acid (TCA) (HIMEDIA) be significantly higher in the asthmatics as compared and 2 mL 2-thiobarbituric acid (TBA) (HIMEDIA) to healthy individuals (0.38±0.02, p<0.01). were added to the reaction mixture. Tubes were plugged and incubated for 15 min in boiling water. Marked differences were observed in the bio- Centrifugation was done at 3000 rpm for 10 min. chemical and spirometric parameters among the Readings were taken with the light pink supernatant patients with different severity of disease except age, at 532 nm against buffer blank. The concentration of BMI and catalase activity of serum (Table II). The MDA was calculated using the extinction coefficient mean serum MDA of asthmatics was found to be sig- of MDA-TBA complex which is 1.56 × 105 mmol/L-1 nificantly increasing with increased level of severity cm-1 and the results were expressed as nmoles/ml of (p<0.05), whereas mean FRAP value was significant- serum. ly decreasing with increased severity level (p<0.01). Markedly higher level of serum MDA was observed in all the categories of asthmatic patients as compared FRAP assay to the similar categories of healthy individuals except Total antioxidant power of plasma was measured for alcohol consumers (Figure 1). The serum catalase by the FRAP (ferric reducing ability of plasma) assay activity was found to be significantly lower in the asth- given by Benzie and Strain (20). One hundred mL of matics as compared to healthy controls (p<0.01) in plasma was mixed with 300 mL distilled water and 3 mL total (Table I) and in all the categories of patients of working FRAP reagent, freshly prepared by adding except smokers and alcohol consumers (Figure 2). 10:1:1 ratio of 300 mmol/L acetate buffer, a 10 mmol/L Total antioxidant status of the plasma was markedly 2,4,6-tripyridyl-S-triazine (HIMEDIA) in 40 mmol/L lower in the asthmatics (367.39±9.95) in compari- HCl and 20 mmol/L FeCl3 × 6H2O (HIMEDIA). son to healthy individuals (466.67±15.52, p<0.01) Absorbance was measured at 593 nm at zero minute in total (Table I) as well as in all the subcategories after vortexing. After that, samples were placed at (Figure 3). Only slight differences were observed in 37 ˚C in a water bath and absorbance was taken after the serum MDA, catalase activity and FRAP values 4 minutes. Ascorbic acid was taken as standard. among asthmatic subjects in relation to various corre- lates like sex, age group, smoking habits, dietary The concentration of serum MDA, catalase acti- habits, drinking habits, drug intake, and family histo- vity and FRAP values were determined in asthmatics ry. However, a significant difference was observed in and controls in relation to various correlates like sex, the plasma antioxidant levels in asthmatic males and age group, smoking habits, dietary habits, drinking females (p<0.05). The comparison of serum MDA, habits, drug intake, family history, duration etc. catalase activity and FRAP values in asthmatics was also done in relation to number of cigarettes smoked in a day, but no significant differences were observed. Statistical analysis Similarly, comparisons were made according to dif- Statistical software, SPSS v16.0 and Microsoft ferent amount of alcohol consumed in a month. Ca- Excel 2007 were used for the analysis of the epidemi- talase activity showed no significant difference while ological data and the experimental results for all the serum MDA was found to be significantly higher in 58 Singh Yadav et al.: Systemic oxidative stress in asthmatic patients Table I General, clinical and demographic characteristics of the subjects studied. Characteristics Asthmatics Controls p value N 120 90 – Age (years) 45.22±1.48 38.84±1.32 – Age range (years) 13–80 18–67 – BMI (Kg/m2) 23.84±0.47 23.61±0.39 0.707 FVC (% Predicted)** 71.66±1.79 95.26±1.18 0.000 FEV1 (% Predicted)** 66.97±1.94 94.33±0.98 0.000 FEV1/FVC (% Predicted)** 95.40±1.37 104.79±0.78 0.000 PEF (% Predicted)** 54.27±2.11 80.59±1.82 0.000 FEF 25–75 (% Predicted)** 46.35±2.01 74.53±2.06 0.000 Sex (Males/Females) 44/76 53/37 – Smokers/Nonsmokers 19/101 29/61 – Alcohol consumers/Abstainers 17/103 25/65 – Vegetarians/Non-vegetarians 81/39 53/37 – Mild to moderately active/Sedentary 24/96 38/52 – Biomass smoke exposure (Exp/Non-exp) 37/83 29/61 – Serum MDA (nmol/mL)** 0.75±0.08 0.38±0.02 0.000 FRAP (mmol/L)** 367.39±9.95 466.67±15.52 0.000 Catalase activity (U/mg protein)** 0.20±0.02 0.30±0.02 0.000 **Significant (p<0.01), unpaired Student’s t-test. BMI – Body Mass Index, FVC – Forced Vital Capacity, FEV1 – Forced Expiratory Volume in 1 Second, PEF – Peak Expiratory Flow Rate, FEF25-75% – Forced Expiratory Flow at 25–75% in % predicted, Exp – Exposed. The values of age, BMI, FVC, FEV1, FEV1/FVC, PEF and FEF 25-75 are expressed as Mean ± S.E of mean. Table II Comparison of various parameters among patients with different severity of asthma. Level of asthma severity Variables p-value Intermittent Mild Moderate Severe N 18 34 30 38 – Age 36.39±3.15A 41.56±2.32AB 46.86±2.60BC 51.37±3.02C – BMI# 24.10±1.23A 24.16±0.86A 24.04±0.88A 23.26±0.90A 0.874 FVC** 90.39±3.42A 85.32±2.39B 68.50±2.08C 53.05±2.05C 0.000 FEV1** 87.55±3.15A 77.59±1.46B 63.73±2.30C 50.29±3.9D 0.000 FEV1/FVC** 101.44±2.13A 96.03±1.94B 98.17±3.20AB 89.79±2.71B 0.024 PEF** 76.78±3.85A 69.18±2.69B 47.30±2.52C 35.76±3.20C 0.000 FEF25-75%** 67.89±4.84A 57.79±2.72B 43.47±3.44C 28.18±2.15D 0.000 Serum MDA* 0.36±0.05A 0.66±0.09AB 0.88±0.15B 0.91±0.12B 0.018 FRAP** 397.99±20.31A 405.20±17.82A 363.34±19.99AB 322.25±17.7B 0.006 Catalase activity# 0.25±0.06A 0.21±0.03A 0.21±0.04A 0.16±0.03A 0.487 The values are expressed as Mean± S.E. *The values are significantly different at 0.05 level (p<0.05), ANOVA. **The values are significantly different at 0.01 level (p<0.01), ANOVA. #The values are not significantly different. The values with different alphabets in superscript along a row are significantly different from each other (Duncan multiple range test, 0.05 level). The alphabets in superscript denote the significant difference, along a row; the highest value being denoted by A, lower by B and so on. J Med Biochem 2016; 35 (1) 59 1 ** ** 0.9 ** ** ** ** ** 0.8 ** Serum MDA (nmol/mL) 0.7 Asthmatics 0.6 Control 0.5 ** ** ** 0.4 ** ** ** ** 0.3 0.2 0.1 0 Figure 1 MDA concentration expressed as nmol of MDA/ml of serum in asthmatic patients and controls. **Significant (p<0.01), unpaired Student’s t-test. 0.4 ** ** * ** 0.35 ** ** * 0.3 Catalase activity (Unit/mg) * * 0.25 ** ** ** ** 0.2 ** Asthmatics 0.15 Control 0.1 0.05 0 Figure 2 Catalase activity in serum expressed as units/mg protein in asthmatic patients and controls. *Significant (p<0.05), **Significant (p<0.01), unpaired Student’s t-test. 60 Singh Yadav et al.: Systemic oxidative stress in asthmatic patients 600 ** ** ** ** ** ** 500 ** ** ** ** ** ** ** ** ** FRAP value (μmol/L) 400 ** ** ** 300 Asthmatics 200 Control 100 0 Figure 3 Ferric reducing antioxidant power (FRAP) of plasma expressed as mmol/L in asthmatic patients and controls. **Significant (p<0.01), unpaired Student’s t-test. Table III Comparison of serum MDA, catalase activity and plasma antioxidant status among asthmatic subjects in relation to con- sumption of alcohol (all kinds) in a month. Specific activity FRAP value Consumption of alcohol N of catalase Serum MDA (nmol/mL) (mmol/L) (U/mg protein) <1 L/month 5 0.17±0.08# 0.41±0.13B 511.80±40.33A 1–7 L/month 6 0.22±0.09# 0.53±0.06B 359.47±35.61B >7 L/month 6 0.26±0.10# 0.10±0.27A 306.70±41.28B The values are expressed as Mean± S. E. Values with different alphabets in superscript along a column show significant difference (p<0.05), ANOVA with Duncan post hoc test. #The values show nonsignificant difference (p>0.05, ANOVA). persons consuming more than 7 L in a month. The FVC (r=–0.171, p<0.05) and FEV1 (r=–0.200, FRAP value was found to be markedly lower in the p<0.05) and a positive correlation with the age asthmatic subjects who were taking 1–7 L and more (r=0.212, p<0.05) of the patients and duration of than that in a month as compared to those who were the disease (r=0.237, p<0.05). The catalase activity taking less than 1 L/month (Table III). No significant of serum was slightly correlated with FVC (r=0.091) differences were observed in biochemical parameters and FEV1 (r=0.083), but a markedly negative corre- with different age groups (class interval for 13–50 lation was observed with duration of disease and 50–80 years were compared with each other) (r=–0.167, p<0.05). The total antioxidant status of and family history (subjects with or without a family the plasma exhibited a significantly positive correla- history of asthma were compared) among the asth- tion with the FVC (r=0.196, p<0.05) and FEV1 matic patients. (r=0.227, p<0.01) of the patients and a significant negative correlation with duration (r=–0.210, There was a remarkable negative correlation of p<0.05). the serum MDA with the spirometric observations, J Med Biochem 2016; 35 (1) 61 Discussion lower in asthmatic smokers, which indicates that smo- king also affects disease pathogenesis. The increased The oxidative stress is an important constituent MDA concentration and decreased FRAP value with of asthma, where chronic inflammation leads to gen- increasing consumption of alcohol among asthmatics eration of reactive oxygen species and also exacer- also imply a dose response relationship of alcohol bates the disease. Many studies have reported elevat- consumption and oxidative stress among asthmatic ed levels of lipid peroxidation in asthmatics especially patients. MDA was found to be noticeably increasing during an acute asthmatic attack (18, 23–24). The while plasma antioxidant was observed to be decreas- data is inconsistent because of the varying lifestyle, ing with duration and also with severity of asthma. A dietary factors, techniques used for analysis and other significant positive correlation was obser ved between factors, but overall it has been reported that the lipid the serum MDA and age of the asthmatic patients, peroxidation level increases with severity of disease. which implies that age plays a critical role in oxidative Rahman et al. (23) reported that the plasma MDA stress. The age-related increase in oxidative stress has level was significantly higher in asthmatic patients as also been reported in several earlier studies (28–30). compared to controls and it was found to be increas- Serum MDA was also found to be positively correlat- ing with the severity of disease. Increase in mean ed with the duration of the disease, which signifies serum MDA level with increasing severity among that lipid peroxidation increases with disease progres- asthmatic children has been reported by Al-Abdulla et sion. There was a negative correlation of serum MDA al. (24). Ozaras et al. (18) studied lipid peroxidation with the FVC and FEV1 of the patients which indi- in BAL fluid and reported that the MDA level was cates that lipid peroxidation increases with the level of higher in asthmatic patients. Kanazawa et al. (25) severity. On the other hand, the catalase activity and also observed variations related to severity with acute plasma antioxidant level were found to be negatively exacerbations. correlated with duration of the disease, and positively There is some evidence of lower plasma antiox- correlated with the measured FVC and FEV1 of the idant levels in asthmatics as compared to healthy indi- patients, which implies that the lower antioxidant sta- viduals. Rahman et al. (17) have reported lower anti- tus causes airway obstruction. Similar findings have oxidant capacity of plasma in asthmatics with been reported by Ahmed et al. (26). increased oxidative load. Ahmad et al. (26) studied In conclusion, the present study depicted that MDA level, catalase activity in erythrocytes and the the oxidative stress is an important part of asthma antioxidant level in plasma in asthmatic subjects. The pathogenesis. The oxidant – antioxidant imbalance study showed that the MDA level was higher while which occurs in asthma plays a crucial role in disease catalase activity in erythrocytes and antioxidant ca- progression and severity. This study fortifies the evi- pacity of plasma were lower in asthmatic patients with dence shown by the abovementioned studies. increased severity of disease and also in comparison to healthy controls. The lower activity of catalase in the red blood cells of asthmatic patients has also Acknowledgement been reported by Rai and Phadke (27). The present study entails that oxidant – antioxidant imbalance The present study was financially supported by occurs in asthma and plays a role in disease progres- Haryana State Council for Science and Technology, sion. Our results are in accordance with the above- Panchkula (India). The authors duly acknowledge the mentioned studies. In the present study it was found authorities of Kurukshetra University, Kurukshetra for that the MDA level was significantly higher, while ca- providing the laboratory and equipment facility and talase activity in serum and plasma antioxidant status HSCST for providing funds for the present study. were decreased in the asthmatic patients as compa- Sincere thanks are due to Dr. Subhash Garg and Dr. red to healthy individuals. Noticeable differences were Madan Khanna for their help in collection of blood observed for almost all the subcategories, viz males, samples. females, smokers, nonsmokers, alcohol consumers and non-consumers, vegetarians and non-vegetarians. For Conflict of interest statement example, in asthmatic smokers a significantly higher level of MDA was observed as compared to healthy The authors stated that they have no conflicts of smokers while the value of FRAP was significantly interest regarding the publication of this article. 62 Singh Yadav et al.: Systemic oxidative stress in asthmatic patients References 1. Masoli M, Fabian D, Holt S, Besley R. The global burden mediators in asthma. Pulm Pharmacol Ther 2001; 14: of asthma: Executive summary of the GINA Disse- 409–20. mination Committee report. Allergy 2004; 59: 469–78. 17. Rahman I, Morrison D, Donaldson K. Systemic oxidative 2. Safwat T. Towards a deep understanding of Bronchial stress in asthma, COPD, and smokers. Am J Respir Crit Asthma. Egypt J Bronch 2007; 1: 120. Care Med 1996; 154: 1055–60. 3. Global Strategy for Asthma Management and Prevention 18. Ozaras R, Tahan V, Turkmen S, Talay F, Besirli K, Aydin S, (revised 2006): Global Initiative for Asthma (GINA). et al. Changes in malondialdehyde levels in bronchoalve- Available at: http://www.ginasthma.org/local/uploads/ olar fluid and serum by the treatment of asthma with files/GINA_Report_2010_1.pdf. inhaled steroid and beta2-agonist. Respirology 2001; 5(3): 289–92. 4. Chung KF. Role of inflammation in the hyperreactivity of the airways in asthma. Thorax 1986; 41: 657–62. 19. Nadeem A, Chhabra S, Masood A. Increased oxidative stress and altered levels of antioxidants in asthma. J 5. Barnes PJ. Reactive oxygen species and airway inflam- Allergy Clin Immunol 2003; 111: 72–8. mation. Free Radic Biol Med 1990; 9: 235–43. 20. Benzie IF, Strain JJ. The ferric reducing ability of plasma 6. Wood LJ, Gibson PG, Garg ML. Biomarkers of lipid per- (FRAP) as a measure of »antioxidant power«: the FRAP oxidation, airway inflammation and asthma. Eur Respir J assay. Anal Biochem 1996; 239(1): 70–6. 2003; 21: 177–86. 21. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 7. Bowler RP. Oxidative stress in the pathogenesis of asth- 121–6. ma. Curr Allergy Asthma Rep 2004; 4: 116–22. 22. Buege JA, Aust SD. Microsomal lipid peroxidation. 8. Caramori G, Papi A. Oxidants and asthma. Thorax 2004; Methods Enzymol 1978; 52: 302–10. 59: 170–3. 23. Rahman I, Biswas SK, Kode A. Oxidant and antioxidant 9. Riedl MA, Nel AE. Importance of oxidative stress in the balance in the airways and airways diseases. Eur J pathogenesis and treatment of asthma. Curr Opin Allegy Pharmacol 2006; 533: 222–39. Clin Immunol 2008; 8: 49–56. 24. Al-Abdulla NO, Al Namma LM, Hassan MK. Antioxidant 10. Sackesen C, Ercan H, Dizdar E, Soyer O, Gumus P, Tosun status in acute asthmatic attack in children. J Pak Med BN, et al. A comprehensive evaluation of the enzymatic Assoc 2010; 60(12): 1023–7. and nonenzymatic antioxidant systems in childhood asth- ma. J Allergy Clin Immunol 2008; 122: 78–85. 25. Kanazawa H, Kurihara N, Hirata K, Takeda T. The role of free radicals in airway obstruction in asthmatic patients. 11. Saleh D, Ernst P, Lim S, Barnes PJ, Giaid A. Increased for- Chest 1991; 100: 1319–22. mation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of NO 26. Ahmad A, Shameem M, Husain Q. Relation of oxidant – synthase: effect of inhaled glucocorticoid. FASEB J 1998; antioxidant imbalance with disease progression in asth- 12: 929–37. ma. Ann Thoracic Med 2012; 7(4): 226–32. 12. Cadet J, Bellon S, Berger M, Bourdat AG, Douki T, 27. Rai RR, Phadke MS. Plasma oxidant – antioxidant status Duarte V, et al. Recent aspects of oxidative DNA dam- in different respiratory disorders. Ind J Clin Biochem age: guanine lesions, measurement and substrate speci- 2006; 21: 161–4. ficity of DNA repair glycosylases. Bio Chem 2002; 383: 28. Radovi} J, Vojinovi} J, Bojani} V, Jevtovi}-Stoimenov T, 933–43. Koci} G, Milojkovi} M, Veljkovi} A, Markovi} I, Stojanovi} 13. Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H. Free S, Pavlovi} D. Lipid peroxidation and oxidative protein radical induced damage to DNA: mechanisms and products in children with episodic fever of unknown ori- measurement. Free Radic, Bio Med 2002; 32: 1102–15. gin. J Med Biochem 2014; 33: 197–202. 14. Bjelland S, Seeberg E. Mutagenicity, toxicity and repair of 29. Yildirism Z, Uegren NI, Yildirim F. The Role of Oxidative DNA base damage induced by oxidation. Mut Res 2003; Stress and Anti-oxidants in the Pathogenesis of Age-relat- 53: 137–80. ed Macular Degeneration. Clinics 2011; 66(5): 743–6. 15. Fujisawa T. Role of oxygen radicals on bronchial asthma. 30. Cencioni C, Spallotta F, Martelli F, Vanete S, Mai A, Curr Drug Targets Inflamm Allergy 2005; 4(4): 505–9. Zeiher A, et al. Oxidative Stress and Epigenetic Regu- lation in Ageing and Age-related Diseases. Int J Mol Sci 16. Henricks PA, Nijkamp FP. Reactive oxygen species as 2013; 14: 17643–63. Received: January 31, 2015 Accepted: March 12, 2015