The Journal of Allergy and Clinical Immunology
Volume 111, Issue 1 , Pages 72-78, January 2003

Increased oxidative stress and altered levels of antioxidants in asthma☆☆☆

Delhi, India

From the Departments of aBiochemistry, bCardiorespiratory Physiology, and cPharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi

Received 15 April 2002; received in revised form 9 September 2002; accepted 26 September 2002.

Article Outline

Abstract 

Background: Reactive oxygen species might play an important role in the modulation of airway inflammation. There is evidence of an oxidant-antioxidant imbalance in asthma. Although several oxidants and antioxidants are likely to be involved, alterations in only limited parameters have been studied in isolation. Objective: We investigated changes in a wide range of oxidants and antioxidants to create a comprehensive picture of oxidant-antioxidant imbalance. Methods: In the peripheral blood of 38 patients with bronchial asthma and 23 control subjects, oxidative stress was measured in terms of superoxide anion generation by leukocytes, lipid peroxidation products, total nitrates and nitrites, total protein carbonyls, and total protein sulfhydrils in plasma. Antioxidant status was evaluated by measuring red blood cell superoxide dismutase and catalase activity, total blood glutathione, and glutathione peroxidase activity in red blood cells and leukocytes and total antioxidant capacity in plasma. Results: Asthmatic patients showed increased superoxide generation from leukocytes, increased total nitrites and nitrates, increased protein carbonyls, and increased lipid peroxidation products and decreased protein sulfhydrils in plasma, indicating increased oxidative stress. They also showed increased superoxide dismutase activity in red blood cells and increased total blood glutathione and decreased glutathione peroxidase activity in red blood cells and leukocytes. Red blood cell catalase activity and the total antioxidant capacity of plasma were not altered. Conclusion: There are alterations in a wide array of oxidants and antioxidants, with balance shifting toward increased oxidative stress in asthma. Therapeutic augmentation of the antioxidant defenses might be beneficial. (J Allergy Clin Immunol 2003;111:72-8.)

Keywords:  Asthma, oxidative stress, superoxide anion, reactive oxygen species, antioxidants, glutathione peroxidase, catalase, superoxide dismutase, glutathione

Abbreviations:  BAL , Bronchoalveolar lavage, GSH-Px , Glutathione peroxidase, NO , Nitric oxide, NOx, Total nitrates and nitrites, O2−, Superoxide anion, ROS , Reactive oxygen species, SOD , Superoxide dismutase, TBARS , Thiobarbituric acid reactive substances

 

Asthma is a chronic inflammatory airway disease.1, 2 The inflammatory cells infiltrating the airways produce several mediators that modulate the inflammatory response. These include a range of toxic reactive oxygen species (ROS), such as superoxide radical, hydrogen peroxide, hypochlorous acid, and hydroxyl radical.3, 4, 5, 6 The ROS likely play a vital role because these have been shown to be associated with many pathophysiologic changes that are relevant in asthma, such as increased lipid peroxidation, increased airway reactivity and secretions, increased production of chemoattractants, and increased vascular permeability.7 The lung and blood are endowed with several antioxidants, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase, glutathione, vitamin E, and vitamin C,8, 9, 10, 11 to counter the oxidant-mediated toxicity. There is increased oxidative stress in asthma, as shown by an increased production of lipid peroxidation products and protein carbonyls in plasma12; increased plasma isoprostanes13; enhanced generation of ROS by blood monocytes, neutrophils, and eosinophils3, 6, 12; increased oxidized glutathione in bronchoalveolar lavage (BAL) fluid14; and increased production of nitric oxide (NO) in exhaled air.15 On the other hand, changes in antioxidant defenses have been reported, including decreased GSH-Px in whole blood, plasma, and platelets; a deficiency of selenium16, 17, 18; decreased protein sulfhydrils and total antioxidant capacity in plasma12; increased SOD activity in BAL cells19; and decreased vitamin C and vitamin E concentration in BAL fluid.14 In the studies carried out thus far, the investigators have studied changes in only a few parameters. Because several oxidants and antioxidants are likely to be involved in the pathogenesis of the inflammatory process in asthmatic airways, a comprehensive study of several parameters of oxidative stress and antioxidant defenses is required to highlight the role of oxidant-antioxidant imbalance in asthma. The present study was carried out with this aim.

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Methods 

Subjects 

Thirty-eight patients with bronchial asthma, 28 male and 10 female patients 15 to 40 years of age, were included in the study from the outpatient department of the institute. The diagnosis was established on the basis of recurrent symptoms of breathlessness and wheezing and an improvement of greater than 200 mL and 12% in FEV1 after inhalation of 200 μg of salbutamol from a metered-dose inhaler. All were nonsmokers. Only symptomatic ambulatory patients with mild-to-moderate asthma symptoms were included. The patients were classified according to disease severity into mild persistent, moderate persistent, and severe persistent groups, as recommended by the National Institutes of Health Expert Panel Report.20 All the patients were already receiving inhaled budesonide or fluticasone, with salbutamol as the rescue bronchodilator. None of the patients were taking any antioxidant vitamins. Evidence of other concurrent pulmonary or systemic disease or of any upper or lower respiratory tract infection was an exclusion criterion. Also excluded were patients who had an acute exacerbation of asthma within the past 4 weeks. No drug was allowed on the day of testing. Twenty-three age-matched, nonsmoking healthy subjects were included as control subjects. Informed consent was obtained. The study was approved by the institutional ethics committee.

Leukocyte harvesting 

Twenty milliliters of blood was withdrawn and mixed with dextran (molecular weight, 509,000 d; Sigma) and allowed to stand for 45 minutes at room temperature. The resulting leukocyte-rich plasma was centrifuged at 250g (4°C) for 12 minutes to obtain leukocytes. Contaminating red blood cells were lysed with ice-cold 0.2% NaCl for 30 seconds, followed by restoration of molarity with the addition of 1.6% NaCl. After centrifugation, the leukocytes were washed twice in Krebs-Ringer phosphate buffer (pH 7.35) containing 2 mg/mL dextrose and finally suspended in this buffer at a concentration of 5 million cells per milliliter. The viability of leukocytes harvested with this technique was greater than 95%, as determined by means of trypan blue exclusion.

Superoxide anion assay 

Superoxide anion (O2−) generation by leukocytes (2.5 million cells) was measured as the SOD-inhibitable reduction of cytochrome C (Sigma).21 Release of O2− was stimulated by the addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (final concentration, 0.1 μmol/L) for 15 minutes at 37°C. The difference in absorbance of supernatant fluids in the presence or absence of SOD was determined with a spectrophotometer at 550 nm. The amount of reduced cytochrome C was calculated on the basis of an extinction coefficient of 21 per millimole per centimeter for cytochrome C. Results were expressed in nanomoles of O2− produced per 15 minutes per 2.5 million leukocytes.

Leukocyte gsh-px assay 

This assay was carried out at 340 nm according to the method of Little et al22 by using cumene hydroperoxide (Sigma) as the substrate. A half million washed leukocytes were used for the assay. Results were expressed in nanomoles of the reduced form of nicotinamide adenine dinucleotide phosphate oxidized per minute per 0.5 million cells.

Additionally, 5 mL of blood was centrifuged for 5 minutes at 1000g and 4°C, and plasma was stored at −70°C until the following analyses.

Total antioxidant capacity (ferric reducing ability of plasma assay) 

The ferric reducing ability of plasma as a measure of total antioxidant power was measured at 593 nm by using the method of Benzie and Strain.23 The components of plasma that contributed to total antioxidant capacity in percentage terms were ascorbate at 15%, α-tocopherol at 5%, uric acid at 60%, bilirubin at 5%, and the remaining antioxidants at 15%. FeSO4•7H2O, and L-ascorbic acid were used as standards. Results were expressed in micromoles per liter.

Total nitrites and nitrates 

The assay for total nitrites and nitrates (NOx) was carried out according to the method of Tracey et al24 by using nitrate reductase (Sigma) to convert all the nitrates present in plasma into nitrites. The incubation was carried at 37°C for 1 hour. Griess reagent (1:1 mixture of 1% sulfanilamide in 5% orthophosphoric acid and 0.1% N-[1-naphthyl] ethylenediamine, Sigma) was added for the development of color, and readings were taken after 10 minutes in a 96-well assay plate at 540 nm on Microscan-5405 A. A standard curve was generated by using known concentrations of potassium nitrate. Results were expressed as micromoles of NOx per liter.

Lipid peroxides assay 

Plasma lipid peroxides were precipitated by using the phosphotungstic acid-sulfuric acid system and were measured at 532 nm with the method of Dousset et al.25 Results were expressed as micromoles of thiobarbituric acid reactive substances (TBARS) per liter.

Plasma protein sulfhydrils and carbonyls 

Plasma protein sulfhydrils were measured at 412 nm by using Ellman's reagent according to the method of Hu et al.26 The content of protein-bound carbonyls in plasma, an indicator of protein oxidation, was measured at 380 nm by using 2,4-dinitrophenylhdrazine according to the method of Levine et al.27

After removing the plasma, sedimented red blood cells were washed twice in normal saline and used for the following assays.

Red blood cell gsh-px 

Washed red blood cells were lysed with ice-cold distilled water and frozen at −20°C. Before the assay, Drabkin reagent was added in hemolysate, and further dilutions were made in potassium phosphate buffer (50 mmol/L, pH 7). The assay was carried out by using the method of Little et al,22 with cumene hydroperoxide as the substrate. Results were expressed as micromoles of the reduced form of nicotinamide adenine dinucleotide phosphate oxidized per minute per gram of hemoglobin.

Red blood cell catalase 

Washed red blood cells were lysed with ice-cold distilled water and frozen at −20°C. Before the assay, further dilution of the hemolysate was made in potassium phosphate buffer (50 mmol/L, pH 7). The assay of this enzyme was carried out by using the method of Aebi28 at 240 nm. The results were expressed in units per gram of hemoglobin.

Red blood cell sod 

Washed red blood cells were hemolysed by means of the addition of ice-cold distilled water, and SOD was extracted in a chloroform/ethanol mixture in the ratio of 1.5:2.5. After centrifugation at 4°C, the supernatant was kept at −70°C until analysis. The assay was carried out according to the method of Abbe,29 with a slight modification. Xanthine-xanthine oxidase (Sigma) was used as a generating system. A standard curve was made from known concentrations of purified bovine SOD (Sigma). Results were expressed in units per gram of hemoglobin.

Total glutathione (oxidized glutathione plus reduced glutathione; whole blood) 

Whole blood was lysed by means of the addition of 6% acetic acid, and total glutathione was immediately precipitated by means of the addition of 10% 5-sulfosalicylic acid. After centrifugation at 4°C, the supernatant was kept at −70°C and analyzed within 15 days. The assay was carried out according to the method of Griffith,30 in which the rate of reduction of 5,5′-dithio-bis (2-nitrobenzoic acid; Ellman's reagent) is measured spectrophotometrically at 412 nm. Results were expressed as millimoles per liter.

Statistical analysis 

The unpaired t test was used to compare the healthy control subjects and patients with asthma. The biochemical parameters were compared among patients with different severities of asthma by using ANOVA. The relationship between different study parameters and the degree of airways obstruction was evaluated by computing the Pearson correlation coefficient. The differences were considered significant at a P value of less than .05.

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Results 

Measures of oxidative stress 

Superoxide generation from leukocytes 

Superoxide generation from leukocytes was significantly increased in asthmatic patients (mean ± SEM, 11.9 ± 0.62 nM O2− produced/15 min/2.5 million cells; 5th-95th percentile, 6.08-20.71 nM O2− produced/15 min/2.5 million cells; n = 35) compared with that seen in control subjects (7.45 ± 0.59 nM O2− produced/15 min/2.5 million cells; 5th-95th percentile, 3.29-12.72 nM O2− produced/15 min/2.5 million cells; n = 21; P < .001; Fig 1).

Lipid peroxides, NOx, total protein carbonyls, and total protein sulfhydrils in plasma 

Plasma levels of lipid peroxidation products, measured as TBARS, NOx, and total protein carbonyls, were increased in asthmatic patients compared with that seen in control subjects. The levels of these products in asthmatic patients and control subjects were as follows: (1) TBARS, 3.67 ± 0.27 μmol/L (5th-95th percentile, 2.04-6.98 μmol/L; n = 32) and 2.31 ± 0.2 μmol/L (5th-95th percentile, 1.33-4.82 μmol/L; n = 22; P < .001; Fig 2); (2) NOx, 49.02 ± 5.39 μmol/L (5th-95th percentile, 11.46-92.16 μmol/L; n = 24) and 26.51 ± 2.88 μmol/L (5th-95th percentile, 7.75-52 μmol/L; n = 18; P < .001; Fig 3); and (3) total protein carbonyls, 1.28 ± 0.05 nmol/mg protein (5th-95th percentile, 0.9-1.77 nmol/mg protein; n = 32) and 0.8 ± 0.03 nmol/mg protein (5th-95th percentile, 0.52-1.03 nmol/mg protein; n = 22; P < .001; Fig 2).

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  • Fig. 2. 

    Lipid peroxides and total protein carbonyls in plasma expressed as micromoles of TBARS per liter and nanomoles per milligram of protein, respectively. ***P < .001.

Total protein sulfhydrils were decreased in asthmatic patients compared with that seen in control subjects, with the levels being 0.539 ± 0.05 mmol/L (5th-95th percentile, 0.45-0.65 mmol/L; n = 31) and 0.6 ± 0.09 mmol/L (5th-95th percentile, 0.5-1.0 mmol/L; n = 22; P < .01; Fig 4), respectively.

Measures of antioxidant status 

Red blood cell SOD and catalase activity, total blood glutathione, and GSH-Px activity in red blood cells and leukocytes 

SOD activity in red blood cells was significantly increased in asthmatic patients, with values of 2434 ± 128.39 U/g hemoglobin (5th-95th percentile, 1348.05-4235.4 U/g hemoglobin; n = 31) compared with 1931 ± 113.7 U/g hemoglobin (5th-95th percentile, 931.2-2824.8 U/g hemoglobin; n = 23) in control subjects (P < .01, Fig 5).

There was no difference in the activity of red blood cell catalase between asthmatic patients and control subjects, with the values being 4951 ± 184.7 U/g hemoglobin (5th-95th percentile, 3063.9-7100.2 U/g hemoglobin; n = 38) and 5166 ± 223.6 U/g hemoglobin (5th-95th percentile, 3607-7333.6 U/g hemoglobin; n = 21), respectively (P > .05, Fig 5). Total blood glutathione was increased in asthmatic patients compared with that seen in control subjects (0. 83 ± 0.05 mmol/L [5th-95th percentile, 0.32-1.46 mmol/L; n = 29] and 0.61 ± 0.09 mmol/L [5th-95th percentile, 0.12-1.41 mmol/L, n = 20], respectively, P < .05; Fig 4). Mean red blood cell GSH-Px activity was significantly reduced in asthmatic patients (48.32 ± 2.319 μM NADPH oxidized/min/g hemoglobin; 5th-95th percentile, 21.14-72.55 μM NADPH oxidized/min/g hemoglobin; n = 38) compared with that seen in control subjects (64.42 ± 4.1 μM NADPH oxidized/min/g hemoglobin; 5th-95th percentile, 32.08-102.55; n = 23; P < .001). Similarly, the leukocyte GSH-Px activity was significantly reduced in asthmatic patients (55.1 ± 2.93 nM NADPH oxidized/min/0.5 million cells; 5th-95th percentile, 34.72-100.32 nM NADPH oxidized/min/0.5 million cells; n = 33) compared with that seen in control subjects (68 ± 4.89 nM NADPH oxidized/min/0.5 million cells; 5th-95th percentile, 38.64-119.36 nM NADPH oxidized/min/0.5 million cells; n = 20; P < .05; Fig 6).
  • View full-size image.
  • Fig. 6. 

    GSH-Px activity in red blood cells (RBC) and leukocytes expressed as micromoles of the reduced form of nicotinamide adenine dinucleotide phosphate oxidized per minute per gram of hemoglobin and nanomoles of the reduced form of nicotinamide adenine dinucleotide phosphate oxidized per minute per 0.5 million cells, respectively. *P < .05; ***P < .001.

Total antioxidant power of plasma (ferric reducing ability of plasma) 

The total antioxidant power of plasma, measured as its ferric reducing ability, was reduced in asthmatic patients (733.68 ± 59.2 μM/L; 5th-95th percentile, 249.5-1418.5 μM/L; n = 29) compared with that seen in control subjects (856.4 ± 39.39 μM/L; 5th-95th percentile, 607.8-1321.75 μM/L; n = 20). However, the difference was statistically not significant (P > .05, Fig 7).

The biochemical parameters were compared among patients with different severities of asthma. No significant differences were observed for any of the parameters studied (Table I).

Table I. Comparison of indices of oxidant-antioxidant status between patients with different severities of asthma
Study parameterMild persistent asthmaModerate persistent asthmaSevere persistent asthma
Leukocyte superoxide generation* (nM O2− produced/15 min/2.5 million cells)11.19 ± 0.6711.61 ± 1.5913.21 ± 1.28
TBARS (μmol/L)*3.87 ± 0.553.76 ± 0.523.51 ± 0.35
NOx, plasma (μmol/L)*53.02 ± 7.6451.04 ± 15.7141.34 ± 6.83
Total protein carbonyls, plasma (nmol/mg protein)*1.2 ± 0.041.22 ± 0.081.43 ± 0.09
Total protein sulfhydrils, plasma (mmol/L)*0.54 ± 0.180.55 ± 0.160.53 ± 0.12
Red blood cell SOD (U/g hemoglobin)*2336.28 ± 175.182159.78 ± 218.722590.92 ± 251.51
Red blood cell catalase (U/g hemoglobin)*4845.7 ± 255.345118.96 ± 616.615021.2 ± 206.11
Total blood glutathione (mmol/L)*0.83 ± 0.090.82 ± 0.070.87 ± 0.101
Red blood cell GSH-Px* (μM NADPH oxidized/min/g hemoglobin)49.30 ± 3.6149.75 ± 5.3145.62 ± 4.25
Leukocyte GSH-Px* (nM NADPH oxidized/min/0.5 million cells)48.01 ± 3.1356.00 ± 5.0161.38 ± 6.75
Total antioxidant power of plasma* (μ143M/L)861.31 ± 101.18706.29 ± 102.59569.75 ± 79.46
*P > .05 (ANOVA), not significant.
Similarly, the correlation between the different study parameters and the degree of airways obstruction, expressed as FEV1 percent predicted, was not significant, except for that between total antioxidant capacity and FEV1 percent predicted (r = 0.47, P < .05; Table II).
Table II. Coefficients of correlation between study parameters and FEV1 percent predicted
Study parameterCoefficients of correlation (r)
Leukocyte superoxide generation*−0.32
TBARS* plasma0.02
NOx, plasma*0.18
Total protein carbonyls, plasma*−0.33
Total protein sulfhydrils, plasma*−0.05
Red blood cell SOD*−0.18
Red blood cell catalase*0.03
Total blood glutathione*−0.16
Red blood cell GSH-Px*0.14
Leukocyte GSH-Px*−0.23
Total antioxidant power of plasma†0.47
*P > .05 (not significant). †P < .05.

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Discussion 

In the present study we have shown that patients with asthma have increased oxidative stress, as shown by increased superoxide generation from leukocytes, increased NOx, increased protein carbonyls, and increased lipid peroxidation products and decreased protein sulfhydrils in plasma compared with that seen in healthy subjects. This is accompanied by alterations in several antioxidants in blood, including decreased GSH-Px activity in red blood cells and leukocytes and increased glutathione and SOD activity in red blood cells. No significant change was observed in the red blood cell catalase activity and total antioxidant power of plasma in asthmatic patients.

We chose to study the oxidant-antioxidant balance in the various components of blood (ie, red blood cells, leukocytes, and plasma) for several reasons. First, blood is an important pool of antioxidant defenses in the body.9, 10, 11 Second, asthma is an inflammatory disease in which cells are recruited from the peripheral blood into the airways. The activated leukocytes are likely to release increased amounts of ROS that can produce many of the pathologic abnormalities observed in asthma.1, 2, 3, 4, 5, 6 Third, blood is an easily available source to study the oxidant-antioxidant imbalance. BAL fluid can provide direct samples of airway cells. However, the technique is invasive and, moreover, can only be performed in patients with stable asthma who are likely to show less marked alterations in the oxidant-antioxidant balance than could be observed in a more unstable state.

Increased generation of superoxide radical has been shown to correlate with the severity of airway obstruction.31 Generation of oxygen radicals in the airways at night has been proposed as a possible mechanism for nocturnal asthma.32 Superoxide anions, although directly promoting inflammation, also have other interactions that might perpetuate the inflammatory process. These have been shown to interact with GSH-Px, reducing its activity.33, 34 Furthermore, reaction with NO can lead to the formation of peroxynitrite and hydroxyl radicals,35 which would amplify the inflammatory process.

The role of NO in the development of airway inflammation is increasingly being recognized. NOx in plasma are the end products of NO metabolism and provide one of the most useful methods to quantify systemic NO production.36 We have found a significant increase in NOx in asthmatic patients compared with that seen in control subjects, which reflects increased production of NO from inflammatory cells. Kharitinov et al15 have also reported an increase in exhaled NO in asthmatic patients. Peroxy-nitrite anion is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydrils.37 It also induces lipid peroxidation. The increased superoxide generation, NOx, TBARS, and protein carbonyls and decreased protein sulfhydrils observed in the present study shows increased oxidative stress in asthmatic patients.

Oxidative stress is believed to play an important role in the pathophysiology of asthma because several of the characteristic changes in the airways can be produced by the actions of ROS.7 Earlier documentation of increased oxidative stress in asthma includes the presence of increased lipid peroxidation products and protein carbonyls in plasma12; increased plasma isoprostanes13; enhanced production of ROS in blood monocytes, neutrophils, and eosinophils3, 6, 12; increased oxidized glutathione in BAL fluid14; and increased production of NO in exhaled air.15 The present study confirms the earlier reports.

Several authors have reported alterations in different antioxidants in asthma. The alterations in antioxidant defenses might be an increase or a decrease depending on whether the changes are due to a defense response (increase) or neutralization by oxidants (decrease), whereas if the reserves are sufficient, there might be no change. Decreased activity of GSH-Px has been well documented.16, 17, 38 This might arise as a result of deficiency of selenium, but inactivation caused by •OH and O2− radicals might play a role.16, 17, 18, 33, 34 GSH-Px is essential for removing toxic lipid oxidation products and hydrogen peroxide, which are continuously generated as a result of sequestration and infiltration of inflammatory leukocytes in the lung. An important mechanism by which leukocyte GSH-Px might be involved in the inflammatory process is as a regulator of the lipoxygenase pathway of arachidonic acid metabolism. It has been known to reduce 12-hydroperoxyeicosatetraenoic acid to 12-hydroxyeicosatetraenoic acid, a precursor of leukotriene B4 and many other sulfidopeptide leukotrienes, which are considered to be important inflammatory mediators in asthma.39, 40 A decreased GSH-Px activity in leukocytes will lead to accumulation of 12-hydroperoxyeicosatetraenoic acid in the airways that might stimulate lipoxygenase and inhibit the cyclooxygenase pathways, thus leading to an increased flow of arachidonic acid metabolites along the former pathway. Thus inactivation of this enzyme by oxidants might be one of the prerequisites for inflammation in asthmatic airways.

Red blood cells are thought to be active scavengers of ROS by virtue of their large antioxidant reservoirs.9, 10, 11 We found increased concentrations of glutathione and SOD in red blood cells that might be a compensatory response to the increased oxidative stress.41, 42, 43, 44 It has been reported that superoxide anion can traverse red blood cell membranes easily.45 Another mechanism to remove the superoxide anion is an interaction with NO to form peroxynitrite, a reaction that is 2 to 3 times faster than enzymatic dismutation of O2− catalyzed by SOD.46 However, Powell et al38 did not find increased activity of red blood cell SOD in their study.

In the present study the red blood cell catalase activity was not found to be changed. This might be because the hydrogen peroxide formed after dismutation of O2− by SOD can be actively scavenged by normal levels of catalase and increased levels of glutathione, with the latter being the main defender against hydrogen peroxide toxicity.9 Rahman et al12 reported a decreased total antioxidant capacity of plasma in asthmatic patients. In the present study, although the total antioxidant capacity in asthmatic patients was lower than that in control subjects, the difference was not significant. One possible reason for our observation might be the small contribution of protein albumin to the total antioxidant capacity and low pH (3.6) because sulfhydrils are bound to the albumins that are not reactive at such a low pH.23 Assayed separately, protein sulfhydrils were found to be decreased in asthmatic patients, which is in agreement with the findings of Rahman et al,12 who have attributed the decreased antioxidant capacity to oxidized sulfhydril groups associated with albumin. Moreover, peroxynitrite is reported to be 1000 times more active in oxidizing sulfhydril groups than other ROS. Thus it might be expected that protein sulfhydrils are the main targets of ROS-mediated attack,37 reducing their levels.

In the present study, the measures of oxidative stress and antioxidant status did not differ significantly between patients grouped according to severity of asthma. Similarly, none of the measured indices had a significant correlation with the degree of airways obstruction, except total antioxidant capacity. This suggests that the lower the antioxidant defenses, the greater the degree of airway obstruction (decreased FEV1). In studies that have found differences related to severity,31 patients with acute exacerbations have been included, thus giving a wider spectrum of severity. We did not include such patients.

We did not attempt to separate the constituents of leukocytes into separate fractions. This was done because the inflammatory response in asthma involves several types of cells, and changes in measures of oxidant-antioxidant imbalance have been reported in many of these by other workers. Derangements of oxidant-antioxidant balance are thus not restricted to any specific fraction of leukocytes. However, the method used by us to separate leukocytes from whole blood provides more than 90% of cells as neutrophils.

All the patients included in the present study were receiving treatment with inhaled corticosteroids. However, they were symptomatic and had airways obstruction, indicating that airway inflammation was not under control and that asthma was active. The biochemical alterations observed corroborate this. The effect of treatment with antiasthma drugs on parameters reflecting oxidative stress and on antioxidant levels has not been evaluated systematically, although it has been suggested that the changes tend to revert toward normal values with treatment.32 Thus the changes observed in the present study might have been even more marked, had the patients not been receiving treatment. Among other possible confounders could be food intake. However, we did not attempt to control this factor. This might be considered as a limitation of the study.

The present study is the first to examine the changes in several measures of oxidative stress and antioxidant status. Unlike previous studies that focused on only a few parameters, we have investigated alterations in a wide variety of antioxidants and carried out measurements on several parameters of oxidative stress. Increased oxidative stress is likely to contribute to perpetuation and amplification of the inflammatory response. Whether exposure to oxidative stress in the airways can induce airway inflammation is not certain. It might be pointed out that exposure to ozone has been shown to induce airway inflammation and hyperreactivity,47 and in vivo generation of oxygen radicals by means of inhalation of xanthine-xanthine oxidase has been shown to induce airway hyperreactivity.48 The observations in the present study also have clinical implications. Demonstration of a shift in oxidant-antioxidant balance in favor of the former suggests that augmentation of the antioxidant defenses by means of therapeutic interventions might be beneficial.

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☆ Ahmed Nadeem is the recipient of a Junior Research Fellowship from the Council of Scientific and Industrial Research (CSIR).

☆☆ Reprint requests: Sunil K. Chhabra, MD, Department of Cardiorespiratory Physiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi - 110 007, India.

PII: S0091-6749(02)91279-0

doi:10.1067/mai.2003.17

The Journal of Allergy and Clinical Immunology
Volume 111, Issue 1 , Pages 72-78, January 2003

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