Materials and Methods: The study had a double blind, placebo controlled design. Induced sputum GSH, NO₂ ^-, cell counts and plasma NO₂ ^– contents were evaluated in 11 healthy controls (HCs) and 25 patients with asthma during exacerbation before and after treatment. Fifteen patients with acute asthma attack were treated with inhaled NAC during exacerbation.

Results: Plasma-sputum NO₂ ^- and sputum GSH contents were significantly higher in subjects with asthma subgroups than HCs (p<0.01 for NAC treatment group, p<0.005 for placebo treatment group, for plasma NO₂ ^-; p<0.001 for both groups for sputum NO₂ ^-; p<0.001 for both groups for sputum GSH). NAC treatment group had higher GSH contents in sputum samples after treatment which were, however, not significantly different from those before treatment. There was a significant decrease in sputum (p<0.005 for NAC treatment group, p<0.01 for placebo group) and plasma NO₂ ^- (p<0.005 for NAC treatment group, p<0.01 for placebo group) levels in both NAC treatment group and placebo group after treatment when compared with the pretreatment levels.

Conclusion: These findings indicated that addition of NAC to standard attack therapy does not modify the oxidant-antioxidant status in asthma patients during exacerbation.©2008, Firat University, Medical Faculty.

Despite the abundance of evidence indicating elevated oxidative stress and reduced antioxidant defences in asthma, antioxidant supplementation studies to date have been controversial. N/acetyl-L-cysteine (NAC) is a thiol containing compound that is used to reduce viscosity and elasticity of mucus. It is able to scavenge hydrogen peroxide, hydroxyl radicals and hypochlorous acid⁸. NAC can be deacetylated to cysteine, an important procursor of cellular GSH sythesis, and thus stimulating the cellular GSH system⁹. In view of this, we wondered whether inhaled NAC might also be useful in increasing anti-oxidant capacity during exacerbation. We undertook a double blind, placebo controlled study of inhaled NAC 600 mg bid in addition to standard treatment in patients admitted to hospital with an acute execerbation of asthma.

Patients/Subjects
Acute asthma patients Twenty-five non-smoking atopic asthma patients during exacerbation participated in the study. All subjects were atopic with positive skin prick testing for common aeroallergens from our area (house-dust mites, grass pollen, cat and dog dander, mould mixture). Positive skin prick test was defined when wheal was 3 mm when compared to salin. The diagnosis of asthma was based on international guidelines¹⁰. An exacerbation of asthma was defined by the presence of the dyspnea at rest with wheezing or nocturnal symptoms disturbing sleep. Their current therapy (before the exacerbation) was shown in Table 1.

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Table I: Demographics and lung function datas of all groups, and current medications of asthma subgroups

“Healthy controls” (HCs) Eleven age-matched, nonsmoking healthy subjects were included as control subjects. All subjects were randomly selected from hospital staff. Inclusion criteria for non-smokers were; no history of respiratory or allergic disease, normal baseline spirometric parameters as predicted for age, sex and height, no history of upper respiratory tract infection in the preceeding 6 weeks, and no use of any regular medication. The Ethics Committe of Firat University Faculty of Medicine reviewed and approved the protocol, and all subjects gave informed consent to participate in the study.

Pulmonary Function Test
Pulmonary function parameters (FEV₁, FVC, FEF_25-75) were measured with a spirometer (SuperSpiro, Micromedical Limited, England).

Sputum Induction
Sputum was induced during the acute exacerbation as previously described¹¹. Sputum induction was performed by inhalation of 3% NaCl for 20 minutes from nebuliser [Porta- Neb compressor, Medic-Aid Sidestream nebuliser chamber, mass median diameter 3.18 m (Medic-Aid Limited, UK)]. The standard safety precaution was to premedicate with 200 Ug of inhaled salbutamol¹². Before and after sputum induction lung function measurements were performed. The safety of sputum induction was monitored by measuring peak expiratory flow rates (PEFR). The procedure would have been stopped if PEFR were decreased by 25%. The sputum induction procedure did not cause troublesome symptoms and the PEFR was not decreased by more than 25% in all “acute asthma patients”. Treatment of exacerbation was made according to international guidelines in all acute asthma patients after sputum induction. Expectorated sputum was collected in sterile plastic tubes placed on ice to slow down metabolic processes that might result in loss of GSH.

Sample Processing
Sputum samples were processed within 30 minutes of collection using the method described by Dauletbaev et al¹¹. Samples were diluted with three volumes of chilled phosphate buffered saline (PBS: all reagents were purchased from Sigma- Aldrich Chemie GmbH, Steinheim, Germany). Supernatants were obtained by centrifugation (300 g, 15 minutes, 4ºC) and transferred to another vial by filtering through multiple layers of cotton gauze. Additional centrifugation (800 g, 5 minutes, 4ºC) ensured removal of the remaining cell debris and mucus. Aliquots of the supernatants were placed on ice and assayed immediately for reduced glutathione supernatant was waited at -20 ºC for measuring nitrit (NO₂ ^-) contents.

GSH Measurement
The sputum GSH was measured using an enzymatic recycling assay^13,14. The standard and sample solutions were added to an equal volume of DTNB and 50 µl of this mixture (final concentration of DTNB 0.25 mM) were pipetted into a 1 ml cuvette followed by glutathione reductase and NADPH (final concentrations 1 U/ml and 0.22 µM, respectively). The reaction mixture was equilibrated and the kinetic reaction was followed for two minutes at 412 nm (Techcomp Ltd., UV-VIS 8500 spectrophotometer, Hong Kong).

NO₂ ^– Measurement
The measurement of plasma and supernatant NO was difficult because this radical was poorly soluble in water and had a short half-life in tissue (10-60 s), but its half-life might be as long as 4 min in the presence of oxygen. For these reasons, the determination of NO itself was difficult and required the handling of radioisotopes. In spite of this, the end products, nitrate and nitrite, were preferentially used in clinical biochemistry. Nitrite, a stable end -product of NO, was measured in plasma by using the spectrophotometric Griess reaction¹⁵. One thousand mL experimental samples of deproteinised plasma was reacted with 500 mL N naphthylethylenediamine, 10 g/L sulfanilamide for 45 min at room temperature and analyzed by spectrophotometry at 545 nm. Concentrations were determined by comparison with sodium nitrite. The lower limit of detection was 0.2 mmol/L.

Statistical Analysis
All statistical analyses were done using SPSS v10.0 software. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Kruskal-Wallis test for multiple-group comparisons; Mann-Whitney U test was performed to test any observed differences for significance. Wilcoxon’s rank sum test was performed for comparisons before and after treatment values of asthma subgroups. Chisquare test was performed to compare gender distrubion between groups. A p value of <0.05 was considered as statistically significant.

At exacerbation, sputum GSH contents were higher in both acute asthma subgroups before treatment than HCs (p<0.001 for both groups), and remained higher also after treatment in both acute asthma groups (p<0.001 for both groups). There was no significant difference betweenacute asthma subgroups in sputum GSH levels before and after treatment. There was no significant difference in sputum GSH levels in both acute asthma subgroups after treatment when compared with the pretreatment levels, although there was a trend toward higher levels in patients who received NAC (Table II).

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Table II: Comparison of sputum cell counts, GSH, NO2 - and plasma NO2 - levels from asthma patients during exacerbations and healthy control subjects

Sputum NO₂ ^- contents were higher in both acute asthma subgroups before treatment than HCs (p<0.001 for both groups). After treatment, sputum NO₂ ^- contents were significantly lower in placebo group when compared to HCs (p<0.05) but there was no significant difference between NAC treatment group and HCs. There was no significant difference between acute asthma subgroups in sputum NO₂ ^- contents before and after treatment. We observed a significant decrease in sputum NO₂ ^- levels in both acute asthma subgroups after treatment (p<0.005 for NAC treatment group, p<0.01 for placebo treatment group) when compared with the pretreatment levels (Table II).

Before treatment, plasma NO₂ ^- contents were higher in both acute asthma subgroups than HCs (p<0.01 for NAC treatment group, p<0.005 for placebo treatment group), but there was no significant difference between acute asthma groups. After therapy, there was no significant difference in plasma NO₂ ^- contents between all groups. There was a significant difference in plasma NO₂ ^- contents in both acute asthma subgroups after treatment when compared with the pretreatment levels (p<0.005 for NAC treatment group, p<0.01 for placebo treatment group) (Table II).

FEV₁ was significantly higher in both asthma subgroups after treatment than those before treatment (p<0.005 for NAC treatment group, p<0.01 for placebo treatment group). There was no significant difference in FEV₁ between NAC treatment group and placebo treatment group after treatment and the rate of change in FEV1 was greater with NAC than with placebo but this difference was not statistically significant (Table II).

Sputum GSH, NO₂ ^- and plasma NO₂ ^- contents of both asthma subgroups before and after treatment were shown in figure 1 and 2.

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Figure 1: Sputum GSH, NO2 -, plasma NO2 - contents in patients who received NAC treatment

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Figure 2: Sputum GSH, NO2 -, plasma NO2 - contents in patients who received placebo treatment

GSH is the key antioxidant in the respiratory tract lining fluid (RTLF). Disturbed GSH status is reported in asthma patients. Kelly and Smith obtained an increased GSH (total and oxidised) status in BAL fluid in asthma patients ^16,17. Doulatbaev et al also demonstrated that stable asthma patients tended to have higher GSH concentrations in sputum samples¹¹. There are several clinical studies of the oxidantantioxidant status in asthma. Acute exacerbations of these conditions have received much less attention. We hypothesised that increased numbers of inflammatory leukocytes in the bronchoalveolar space and/or those sequestered in the pulmonary vasculature in acute exacerbations of asthma, may create an increased oxidant burden. Rahman et al showed that decreased antioxidant and increased oxidant capacity in plasma of patients presenting with acute exacerbations of asthma, and antioxidant capacity tended to improve by the time of discharge⁶. We observed increased amount of sputum GSH in patients with asthma during exacerbation. This result suggests that GSH synthesis and/or transport were increased in response to the presence of excess oxidants during acute exacerbation. A compensatory but probably inadequate increase in GSH has been reported in other conditions of oxidative stress^18,19. If ROS and RNS are important in asthma especially during acute exacerbation, enhancement of the antioxidant defences would be expected to have beneficial effects in the disaese. On the contrary, some studies evaluated GSH of exhaled breath condensate in children with asthma during acute exacerbations were found decreased GSH levels compared with control subjects and stable asthma patients^20,21. At present, it is not clear whether altered RTLF GSH status is the cause or a consequence in respiratory disease. Studies carried out with normal volunteers showed that GSH and other antioxidants present in RTLF play an important role in protecting the lung surface from oxidative attack²².

Sputum concentrations of NO₂ ^- and nitrate, which are stable metabolites of NO have been shown to be elevated in asthma^23,24. NO can be measured in the exhaled air of asthmatic patients^25,26, and it is thought to reflect the inflammatory state of the airways. In the current study, we demonstrated that sputum NO₂ ^- content was higher in acute asthma exacerbation when compared with the healthy subjects and their levels were significantly decreased after treatment. To our knowledge, this is the first study in which sputum NO₂ ^- contents in acute asthma exacerbation were evaluated, therefore the data cannot be compared with previous findings. Recently, Hunt et al showed that the pH in the airways drops dramatically during acute asthma attack, which facilitates the conversion of nitrite to NO. Hence, increased NO concentrations in the exhaled air of asthmatic patients may reflect nitrite conversion rather than NOS activity. Acidosis at levels seen in subjects with acute asthma causes both necrosis of human eosinophils and conversion of airway NO₂ ^- to NO effects relevant to asthma pathophysiology²⁷. In addition, increased sputum NO₂ ^- contents may develop due to increased numbers of inflammatory leukocytes in the bronchoalveolar space during acute exacerbations of asthma. High concentrations of NO released in an inflammatory context, it has putatively detrimental effects via the formation of ROS²⁸.

Despite the abundance of evidence indicating elevated oxidative stress and reduced antioxidant defences in asthma, antioxidant supplementation studies to date have been limited. NAC is one of the most widely investigated antioxidants having shown beneficial effects in disease states. In contrast with the activity found different pulmonary disease, the influence of NAC in clinical asthma remains uncertain^29,30 but has not been recently evaluated. We found that short-term administration of inhaled 600 mg/day NAC increased sputum GSH contents in acute asthma patients, but this increase was not significant according to before treatment. When compared with placebo group, NAC group had increased sputum GSH levels after treatment but it was not significant. Administration of NAC had no significant effect on FEV₁, serum and plasma NO₂ ^-, and sputum GSH levels in asthma patients during exacerbations. Improvement in postreatment levels of FEV₁ could be attributed to the use of standard attack therapy (Â2 agonists and/or inhaled steroids). NAC can easily be deacetylated to cysteine, an important precursor of cellular GSH synthesis, and thus stimulate the cellular GSH system⁹. The upper level of cellular GSH is regulated by feedback inhibition, but cellular levels also depend upon availability of the substrates, especially cysteine³¹. In several studies, it was shown that intracellular and plasma cysteine levels were increased following NAC administration^32,33. Meyer et al found that GSH levels increased in BAL fluid after the oral NAC (3x600 mg NAC per day for 5 days) augmentation compared with pretherapy³⁴. On the contrary, Cotgreave et al demonstrated that the cysteine and GSH content in the lavage fluid were unaltered during NAC (600 mg, daily for 2 weeks) administration in healthy subjects³⁵. NAC in a daily dose up to 1800 mg administered for 5 days did not significantly increase the concentration of GSH or cysteine in BAL fluid³⁶. Oral administration of NAC for several days did not elevate the concentration of GSH in the airways. There has been many studies about oral administration of NAC but a few studies is present with nebulized NAC administration. In these studies it is shown that inhaled NAC has anti- or prooxidant action³⁷ and it is reported that nebulized NAC transiently increases exhaled H₂O₂ level, whereas it has no effect on other oxidative parameters³⁸. In our study, sputum GSH contents were not increased significantly in patients with acute asthma attacks. This may be due to both short-term and nebulized administration of NAC during exacerbation.

In conclusion, the results of this study demonstrate that short-term inhaled treatment with NAC produced insufficient increase in sputum GSH contents in the airways in patients with asthma during exacerbation. In addition, this therapy had no effect on sputum NO₂ ^- content. For this reason, our study does not suggest that NAC supplementation therapy is effective during asthma exacerbations. Further studies can be made by using higher doses of NAC to mild to moderate asthma patients.

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