Sulfanilamide

Study objectives: We recently found that peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients than in normal control subjects. Current guidelines recommend inhaled corticosteroids as first-line control therapy in asthma. Therefore, this study was designed to examine the effect of inhaled beclomethasone dipropionate (BDP) on peroxynitrite inhibitory activity in induced sputum from asthmatic patients.

Design: Interventional study.

Setting: University hospital.

Patients: Twenty-one asthmatic patients and 10 age-matched, normal control subjects.

Interventions: Inflammatory indexes in induced sputum were examined in all study subjects, and peroxynitrite inhibitory activity was also assayed by monitoring rhodamine formation. For 8 weeks after the first sputum induction, BDP 400 [micro]g bid, was administered to all asthmatic patients and sputum induction was repeated.

Measurements and results: Nitrite and nitrate levels in induced sputum were significantly higher in asthmatic patients (1,121 [micro]mol/L [SD, 205 [micro]mol/L], p < 0.0001) than in normal control subjects (642 [micro]mol/L [SD, 137 [micro]mol/L]). In contrast, peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients (50.0% [SD, 25.7%], p < 0.0001) than in normal control subjects (93.0% [SD, 3.6%]). After 8 weeks of BDP therapy, nitrite and nitrate levels were significantly decreased (847 [micro]mol/L [SD, 143 [micro]mol/L], p < 0.0001) and peroxynitrite inhibitory activity was increased (73.9% [SD, 19.2%], p = 0.0005). Moreover, the increase in peroxynitrite inhibitory activity from before to after BDP therapy was significantly correlated with decrease in nitrite and nitrate levels (r = 0.79, p = 0.0004). We also found the significant relationship between increase in peroxynitrite inhibitory activity in induced sputum and increase in FE[V.sub.1] percentage of predicted after BDP therapy (r = 0.68, p = 0.0023).

Conclusions: Large amounts of peroxynitrite, which are exaggerated in acute asthma attacks, might overwhelm endogenous antioxidant defenses. However, inhaled corticosteroid therapy enhanced antioxidant activity against peroxynitrite, and therefore might reduce the susceptibility to peroxynitrite-induced injury in asthmatic airways.

Key words: antioxidant; bronchial asthma; corticosteroids; epithelial lining fluid

Abbreviations: BDP = beclomethasone dipropionate; ECP = eosinophil cationic protein; ELF = epithelial lining fluid; NO = nitric oxide; PBS = phosphate buffer solution

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Peroxynitrite is a potent oxidant formed by the rapid reaction of nitric oxide (NO) with superoxide anion. (1) In airway inflammation, simultaneous cellular production of superoxide anion and NO may occur, potentially leading to the formation of peroxynitrite. It has been previously reported (2,3) that increased production of superoxide anion and NO have been implicated in the pathogenesis of asthma. In fact, high levels of superoxide anion have been found in BAL fluid from asthmatic patients, and its levels were inversely correlated with airway obstruction. (2) Moreover, levels of NO were also elevated in exhaled air (3) and induced sputum (4) from asthmatic patients, but most cytotoxic effects of high levels of NO were found to be mediated by peroxynitrite. (5) From the existing evidence, it is likely that high levels of peroxynitrite are formed in asthmatic airways.

The thin layer of epithelial lining fluid (ELF) may provide antioxidant protection and serve as a frontline defense for airway epithelial cells. The major antioxidants in ELF are based on thiols such as glutathione, (6) and various types of thiols may also function as antioxidants against peroxynitrite in the respiratory tract. Peroxynitrite can either react with thiols or become protonated to form peroxynitrous acid, which decays rapidly and give rise to nitrite and nitrate. (7) Peroxynitrite adds a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine to produce the stable product nitrotyrosine, resulting in alterations of protein function. (8) It also enhances airway hyperresponsiveness, airway epithelial damage, and inflammatory cell recruitment in asthmatic airways. (9) In our earlier studies, we determined that peroxynitrite altered [[beta].sub.2]-aderenoceptor function (10) and inactivated neutral endopeptidase in the airways. (11) On this basis, it is important to evaluate antioxidant activity against peroxynitrite in ELF to examine the physiologic effects of peroxynitrite in asthmatic airways. We recently found that peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients than in normal control subjects. (12) Current guidelines recommend inhaled corticosteroids as first-line control therapy in asthma. However, little is known concerning the effects of inhaled corticosteroids on antioxidant activity against peroxynitrite in asthmatic airways. Therefore, this study was designed to examine the effect of inhaled beclomethasone dipropionate (BDP) on peroxynitrite inhibitory activity in induced sputum from asthmatic patients.

MATERIALS AND METHODS

Subjects

The normal control subjects consisted of 10 subjects (mean age, 33.0 years; mean FE[V.sub.1], 111.5%). All normal control subjects were healthy, life-long nonsmoking volunteers who had no history of lung disease. All asthmatic patients satisfied the American Thoracic Society criteria for asthma. (13) The clinical characteristics of the 21 asthmatic patients are shown in Table 1. All asthmatic patients were nonsmokers (mean age, 39.6 years; mean FE[V.sub.1], 90.2%). Methacholine inhalation challenge testing was performed for all study subjects as previously described. (14) All challenge tests were performed at 1 PM to eliminate the effects of diurnal variation. Following baseline spirometry and inhalation of diluent to establish the stability of FE[V.sub.1], the subjects were instructed to take slow inspirations in each set of inhalations. All asthmatics in this study demonstrated bronchial hyperreactivity to methacholine, but all normal control subjects did not. Their regular medication consisted of [[beta].sub.2]-agonists and theophylline, and none were receiving oral or inhaled corticosteroids. Medications were not changed for a 1-month period preceding the study and were withdrawn for at least 12 h before the methacholine challenge test and sputum induction. All patients were in clinically stable condition, and none had a history of respiratory infection for at least the 4-week period preceding the study. All subjects gave their written informed consent for participation in the study, which was approved by the Ethics Committee of Osaka City University.

Sputum Induction and Processing

The sputum induction was performed 3 days after the methacholine challenge test. Spirometry was performed prior to inhalation of salbutamol, 200 [micro]g, via a metered-dose inhaler. All subjects were instructed to wash their mouths thoroughly with water. They then inhaled 3% saline solution at room temperature, nebulized by an ultrasonic nebulizer (NE-U12; Omron; Tokyo, Japan) at maximum output. They were encouraged to cough deeply after 3-min intervals thereafter. After sputum induction, spirometry was repeated. If the FE[V.sub.1] fell, the subjects were required to wait until it returned to the baseline value. The sputum sample diluted with phosphate buffer solution (PBS) containing dithiothreitol (final concentration, 1 mmol/L) was then centrifuged at 400g for 10 min, and the cell pellet was resuspended. Total cell counts were performed with a hemocytometer, and slides were made by using a cytospin (Cytospin 3; Shandon; Tokyo, Japan) and stained with May-Grunwald-Giemsa stain for differential cell counts. The supernatant was stored at -70[degrees]C for subsequent assay of eosinophil cationic protein (ECP). ECP concentration was measured by using a radioimmunoassay kit (Pharmacia Diagnostics; Uppsala, Sweden). Nitrite and nitrate in induced sputum were assayed colorimetrically after the Griess reaction. (15) Two hundred microliters of sputum sample or standard were deproteinated by adding 20 [micro]L of NaOH (1.0 mol/L, 4[degrees]C; Wako Chemical; Osaka, Japan) and 30 [micro]L of ZnS[O.sub.4] (1.3 mol/L, 4[degrees]C; Wako Chemical). Samples were mixed and allowed to stand on ice for 15 min. After centrifugation (5 min at 4[degrees]C at 2,600g), 100 [micro]L of supernatant was mixed with 5 x [10.sup.-2] units of nitrate reductase (Sigma Chemical; St. Louis, MO), 20 [micro]L of 0.2 mol/L N-Tris (hydroxymethyl) methylamino enthanesulphonic acid (pH 7.0, Sigma Chemical), and 20 [micro]L of 0.5 mol/L sodium formate (Wako Chemical). After anaerobic incubation at room temperature for 20 min, 1.0 mL of water was added to the samples, and nitrite was assayed in supernatants obtained by centrifugation (5 min at 260g). Deproteinated samples of standards (200 [micro]L) were mixed with 20 [micro]L of 1% sulfanilamide (Sigma Chemical) in 15% phosphoric acid (Wako Chemical). After 10 min, 20 [micro]L of 0.1% N-(1-naphtyl) ethylenediamine (Sigma Chemical) was added, and the absorption at 540 nm was determined. All subjects produced an adequate specimen of sputum; a sample was considered adequate if the patient was able to expectorate at least 2 mL of sputum and, if on differential cell counting, the slides contained < 10% squamous cells. For 8 weeks after the first sputum induction, BDP 400 [micro]g bid was administered to all asthmatic patients. During this 8-week period, all subjects continued their previous treatment with [[beta].sub.2]-agonists and theophylline. All of the above sputum induction protocol was repeated following treatment with BDP.

Measurement of Peroxynitrite Inhibitory Activity

Peroxynitrite inhibitory activity in induced sputum was measured as previously described. (12) We chose to use the sol phase of sputum for measurement of peroxynitrite inhibitory activity, avoiding the potential confounding influence of dithiothreitol. (16) The sol phase was obtained by ultracentrifuging the remaining portion of the sputum sample at 60,000g for 60 min at 4[degrees]C. This was allocated and stored at -70[degrees]C for subsequent assay for peroxynitrite inhibitory activity. Working solutions of peroxynitrite (Wako Pure Chemical Industries; Osaka, Japan) were prepared by dilution in 0.1 normal NaOH just before use as [10.sup.-2] mol/L solutions, and further dilutions were made in PBS. Peroxynitrite concentration was determined spectrophotometrically by measuring the absorption at 302 nm ([member of]mol/L = 1.670 mol/ L x cm). Peroxynitrite readily oxidizes dihydrorhodamine 123, whereas superoxide anion, [H.sub.2][O.sub.2], and NO alone do not. (17) A standard curve of oxidizing activity of dihydrorhodamine 123 to rhodamine was constructed by employing peroxynitrite. Peroxynitrite inhibitory activity was assayed by monitoring rhodamine formation at 500 nm in reaction mixtures containing 200 [micro]L sputum sample, 1.3 mL dihydrorhodamine 123 diluted with PBS (pH 7.4), and 500 [micro]L peroxynitrite for 30 min at 37[degrees]C. Peroxynitrite inhibitory activity was assayed at least in triplicate. Our previous findings supported the specificity of this assay system for peroxynitrite. (18)

Statistical Analysis

All values are presented as mean (SD). The Wilcoxon signed-rank test was used to compare paired values. Multiple comparisons among groups were analyzed by one-way analysis of variance followed by the Bonferroni correction. The significance of correlations was evaluated by determining Spearman rank correlation coefficients; p < 0.05 was considered significant.

RESULTS

Baseline pulmonary function and bronchial hyperreactivity to methacholine in the 21 asthmatic patients are shown in Table 1. The percentage of eosinophils and concentration of ECP in induced sputum were significantly higher in asthmatic patients (percentage of eosinophils, 17.5% [SD, 8.6%], p < 0.0001; ECP, 760 ng/mL [SD, 401 ng/mL], p < 0.0001) than in normal control subjects (percentage of eosinophils, 0.8% [SD, 0.5%]; ECP, 119 ng/mL [SD, 44 ng/mL]) [Fig 1]. Moreover, the concentration of nitrite and nitrate in induced sputum was also significantly higher in asthmatic patients (1,121 [micro]mol/L [SD, 205 [micro]mol/L], p < 0.0001) than in normal control subjects (642 [micro]mol/L [SD, 137 [micro]mol/L]) [Fig 2]. In contrast, peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients (50.0% [SD, 25.7%], p < 0.0001) than in normal control subjects (93.0% [SD, 3.6%]). We also found the significant relationships between biochemical markers in induced sputum and FE[V.sub.1] (percentage of predicted) before BDP therapy (nitrite and nitrate levels, p = 0.014; peroxynitrite inhibitory activity, p = 0.014) [Table 2].

[FIGURES 1-2 OMITTED]

After 8-weeks of BDP therapy, the percentage of eosinophils and concentration of ECP in induced sputum were significantly decreased to the levels in normal control subjects (percentage of eosinophils, 0.9% [SD, 0.6%], p < 0.0001; ECP, 169 ng/mL [SD, 92 ng/mL], p < 0.0001). Moreover, the concentrations of nitrite and nitrate in induced sputum were also significantly decreased after BDP therapy (847 [micro]mol/L [SD, 143 [micro]mol/L], p < 0.0001). In contrast, peroxynitrite inhibitory activity was increased after BDP therapy (73.9% [SD, 19.2%], p = 0.0005). However, the concentrations of nitrite and nitrate after BDP therapy were still higher in asthmatic patients than in normal control subjects (p = 0.0029). In addition, peroxynitrite inhibitory activity in asthmatic patients after BDP therapy was lower than in normal control subjects (p = 0.019). Moreover, increase in peroxynitrite inhibitory activity from before to after BDP therapy was significantly correlated with decrease in nitrite and nitrate levels (r = 0.79, p = 0.0004) [Fig 3]. We also found the significant relationship between increase in peroxynitrite inhibitory activity in induced sputum and increase in FE[V.sub.1] percentage of predicted after BDP therapy (r = 0.68, p = 0.0023) [Fig 4].

[FIGURES 3-4 OMITTED]

DISCUSSION

In the present study, we found that peroxynitrite inhibitory activity in induced sputum from asthmatic patients was significantly increased after BDP therapy. We previously found that peroxynitrite inhibitory activity in induced sputum was related to the degree of airway obstruction, bronchial hyperreactivity to methacholine, and degree of eosinophilic airway inflammation in asthmatic patients. (12) Moreover, we also found the significant relationship between increase in peroxynitrite inhibitory activity in induced sputum and increase in FE[V.sub.1] percentage of predicted after BDP therapy. These findings suggest that inhaled BDP therapy is effective for the treatment of asthma at least in part by increasing peroxynitrite inhibitory activity in asthmatic airways. However, the precise mechanism by which inhaled BDP therapy enhances peroxynitrite inhibitory activity in asthmatic airways is unclear. Since corticosteroids prevent the induction of NO synthase, (19) inhaled corticosteroids decrease NO production in asthmatic airways. (20) In this study, nitrogen oxides levels in induced sputum were significantly decreased after BDP therapy. Moreover, we found that an increase in peroxynitrite inhibitory activity in induced sputum was correlated with decrease in levels of nitrogen oxides before to after BDP therapy. It is possible that an increase in peroxynitrite inhibitory activity reflects decrease in levels of nitrogen oxides due to inhaled BDP therapy.

The reaction of peroxynitrite with airway thiols is associated with oxidation of airway thiols to the corresponding disulfide (dimethyl disulfide). In this study, we evaluated the reduced-form antioxidant activity against peroxynitrite. In fact, local antioxidant defenses in asthmatic airways are mediated by the reduced-form antioxidants alone. It seems likely that a balance exists between peroxynitrite generation and antioxidant defenses to maintain normal airway function, and that increase in peroxynitrite generation during exacerbation of asthma might overwhelm endogenous antioxidant defenses. Thus, when this balance is shifted toward increased peroxynitrite generation, the reduced-form antioxidants are diminished and therefore asthmatic airways have markedly increased the susceptibility to peroxynitrite. The high statistical significance observed in this study may reflect well on the importance of peroxynitrite inhibitory activity in the pathogenesis of asthma. However, asthma is a very complicated disease and corticosteroids are multifunctional compounds. Therefore, inhaled BDP therapy would also improve clinical status of asthma via a decrease in levels of other biochemical markers. Thus, possibilities of the overall effects of inhaled BDP therapy should be considered. Moreover, though we have an adequate basis about the analytical methodology, there are also some methodologic limitations in this study. For example, in the assay we used the sputum sample instead of ELF, which has a complicated nature.

Although eosinophilic inflammation was completely inhibited by inhaled BDP therapy in asthmatic patients, nitrogen oxides levels were still higher and peroxynitrite inhibitory activity was lower in BDP-treated asthmatics than in normal control subjects. Asthmatic airway inflammation may therefore be a heterogeneous process of which sputum eosinophilia comprises only one part, and it may be that sputum eosinophilia and peroxynitrite stress reflect different components of the inflammatory process. In this study, the range of levels of nitrogen oxides and peroxynitrite inhibitory activity was large, with some having normal values and others having significant differences from normal control subjects despite treatment with inhaled BDP. Little et al (21) also found that the range of exhaled NO levels did not return to normal in all asthmatic patients despite treatment with oral corticosteroids.

In conclusion, since even in stable asthmatics ELF lacks peroxynitrite inhibitory activity, large amounts of peroxynitrite, which are exaggerated in acute asthma attacks, would not be completely inactivated. Inhaled corticosteroid therapy enhanced antioxidant activity against peroxynitrite, and therefore might reduce the susceptibility to peroxynitrite-induced injury in asthmatic airways.

ACKNOWLEDGMENT: The authors thank Miss Yukari Matsuyama for her help in the preparation and editing of the manuscript.

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* From the Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan.

This work was supported by a grant-in-aid for Scientific Research (13670611) from the Ministry of Education, Science and Culture, Japan.

Manuscript received November 19, 2002; revision accepted June 3, 2003.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Hiroshi Kanazawa, MD, Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, 1-4-3, Asahi-machi, Abenoku, Osaka, 545-8585, Japan; e-mail address: kanazawa-h@med.osaka-cu.ac.jp

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