Pulmonary Pharmacology & Therapeutics 18 (2005) 41–47 www.elsevier.com/locate/ypupt
Long-term oral n-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD Fernando De Benedettoa,*, Antonio Acetob, Beatrice Draganib, Antonella Spaconea, Stefano Formisanoc, Riccardo Pelad, Claudio F. Donnere, Claudio M. Sanguinettif a
Department of Pneumology, Ospedale San Camillo De Lellis, Via Carlo Forlanini, 50 66100 Chieti, Italy b Department of Biomedical Sciences, University ‘G.D’Annunzio’, Chieti, Itlay c Rehabilitation Unit, San Francesco d’Assisi Institute, Vasto (CH), Italy d Department of Pneumology, Mazzoni Hospital, Ascoli Piceno, Italy e Department of Pneumology, San Filippo Neri Hospital, Rome, Italy f Department of Pulmonary Disease, Fondazione S. Maugeri IRCCS, Veruno (NO), Italy Received 13 October 2003; revised 5 August 2004; accepted 16 September 2004
Abstract Oxidative stress caused by airway inflammation is increased in chronic obstructive pulmonary disease (COPD) and may account for the progressive deterioration of structure and function of the respiratory tract observed in this disease. Antioxidant defences of the respiratory tract may be overwhelmed by the oxidant burden in COPD and possibly restored with antioxidant therapy. The level of hydrogen peroxide (H2O2) concentration in exhaled air condensate (EAC) is a valuable tool for assessing and monitoring oxidative stress. This study aimed to verify the effect of 2-month oral N-acetylcysteine (NAC) treatment compared to placebo on the H2O2 content in EAC of 55 clinically stable COPD patients (48 males), mean age 65.93G9.3 years. After clinical examination, pulmonary function tests, and collection of EAC for the basal (T0) assay of H2O2, patients were randomly allocated to group A (usual therapy plus oral NAC 600 mg b.i.d. for 2 months) or group B (usual therapy plus placebo b.i.d. for 2 months). H2O2 assay in EAC was repeated at 15 (T15), 30 (T30), and 60 (T60) days after the start of therapy in each group. All patients were non-smokers or ex smokers for at least 5 years and the two groups were comparable in terms of demographic, respiratory function, and EAC data at baseline. The H2O2 level in EAC of group A was significantly decreased at T15 (1.00G0.38 SD mM; pZ0.003), T30 (0.91G0.44 mM; pZ0.007), and T60 (0.83G0.41 mM; pZ0.000) compared to T0 (1.28G0.61 mM). No significant decrease in H2O2 of group B was found at any time point. We conclude that oral NAC 600 mg b.i.d. for 2 months rapidly reduces the oxidant burden in airways of stable COPD patients. q 2004 Elsevier Ltd. All rights reserved. Keywords: COPD; Stable state; Oxidants; Exhaled air condensate; Hydrogen peroxide; Antioxidants; Oral N-acetylcysteine
1. Introduction Oxidative mechanisms are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and other pulmonary disorders where damage of the structure and function of the respiratory apparatus progressively occurs [1–4]. Oxygen reactive species such as hydrogen peroxide
* Corresponding author. Tel./fax: C39 871 358641. E-mail address:
[email protected] (F. De Benedetto). 1094-5539/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2004.09.030
(H2O2) are produced in the lung by activation of inflammatory cells, mainly neutrophils and macrophages [5,6]. Non-invasive methods to measure the oxidant content in exhaled air condensate (EAC) of healthy subjects and of patients affected by COPD and other respiratory diseases have been devised in recent years [7,8]. Dekhuijzen et al. [7] first demonstrated a significant increase in EAC oxidant level of COPD patients, both in stable and exacerbated clinical conditions, compared to healthy control subjects. A successive study confirmed the validity of determining expired H 2O 2 as a biomarker of chronic airway
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inflammation in COPD patients [8]. In healthy subjects the oxidant injury deriving from metabolic processes, food and drug assumption and exposure to environmental pollutants is counterbalanced by enzymatic and non-enzymatic defences. Several enzymatic and non-enzymatic ROS scavenging mechanisms have been implicated and identified in airway epithelial surface liquids. Enzymes that provide such a function include airway lactoperoxidase (LPO), glutathione peroxidase (GPx), superoxide dismutase and catalase. Non-enzymatic components such as mucins and low molecular weight compounds such as vitamin E may also play a role. The balance of these systems protects the airway epithelium from H2O2 [9]. One of the most important defence is the glutathione peroxidase antioxidant system, that catalyzes the reduction of H2O2 by means of reduced glutathione (GSH) as a second substrate [3,6,10]. Thus, a high ratio of reduced to oxidized glutathione (GSH/GSSG) is able to maintain an elevated reducing power, necessary to protect the cells from oxidant injuries. The glutathione antioxidant system has been demonstrated to be heavily challenged in COPD and often overwhelmed by the increased oxidant burden due to chronic airway inflammation [1]. The antioxidant defense of the lung seems incapable of controlling the process of oxidant injury, as is evident from measurement of markers of oxidative stress in COPD patients. To enhance the antioxidant defense could be an attractive strategy for the treatment of COPD. An ideal antioxidant should be very specific and not interfere with the normal oxidative function essential for the inflammatory response and for several metabolic reactions [3]. In this context, an important role seems to be played, both directly and indirectly, by N-acetylcysteine (NAC) in enhancing the antioxidant defences. NAC was selected by Sheffner in the early 1960s as the most suitable sulphydrylcontaining compound for eliciting mucolytic effects in patients with a variety of respiratory disorders characterized by impaired mucokinesis. This action on the respiratory tract is thought to be due to the free sulphydryl group cleaving certain disulphide bounds in the mucus glycoprotein macromolecules through a sulphydryl-disulphide interchanging reaction. But NAC is able to interfere with free radical species and this leads to intermediate formation of NAC thiol radicals, with NAC disulphide as the major end product. It interacts with reactive oxygen intermediates, such as superoxide anions, hydrogen peroxide, hydroxil radicals and hypoclorous acid, all of which are released by inflammatory cells [11–13]. For this reason NAC can exert an effective action to reduce the number and severity of acute COPD exacerbations [14,15]. The aim of the present study was to assay the H2O2 level in EAC of stable COPD patients undergoing long-term treatment with oral NAC to verify whether this drug is able to attenuate the oxidant load in the respiratory tract of these patients.
2. Methods 2.1. Patients Fifty-five patients, 48 males and seven females, mean age 65.93G9.3 years (range: 41–75 years), non-smokers or ex-smokers for at least 5 years, affected by moderate COPD [forced volume in 1 s (FEV1) R50!70 % of predicted] were consecutively recruited among the outpatients attending the pulmonary department of three general hospitals of central Italy. Inclusion criteria were: clinical stability of COPD without episodes of acute exacerbation for at least 3 months, absence of neoplastic or diffuse interstitial pulmonary diseases, and withdrawal of inhaled steroids for at least 15 days.
2.2. Study design After giving informed consent, patients underwent clinical examination and pulmonary function tests, including the measure of forced vital capacity (FVC), FEV1 and the ratio FEV1/FVC (%). Spirometry was performed with a VMAX 22 LV spirometer (Sensormedics Italy srl, Milan). All respiratory function parameters were calculated according to the American Thoracic Society recommendations [16]. Then samples of expired air condensate were taken to determine the basal value of H 2 O2 (T0). Spirometry and EAC sampling were performed in each patient at the same hour in the morning at each time point. After the basal determinations, patients were randomly allocated, in single blind (for the biochemist) fashion, to one of two treatment groups: – group A: treatment with NAC 600 mg b.i.d. for 2 months, plus usual therapy for 2 months. – group B: treatment with placebo, plus usual therapy for 2 months. During the 2 months of the study all patients (in both group A and B) continued their long-term therapy started at least 2 months before the enrollment in the study, and in accordance with the inclusion criteria. The usual therapy was formoterol 50 mg b.i.d. Any significant modification of the usual therapy during the study period had to be declared by patients and constituted grounds for withdrawl from the study. The measurement of H2O2 concentration in expired condensate was repeated after 15 (T15), 30 (T30), and 60 days (T60), and at each time point patients were asked about their clinical history and underwent a clinical examination. In addition, at the end of the study (T60) spirometry was repeated.
F. De Benedetto et al. / Pulmonary Pharmacology & Therapeutics 18 (2005) 41–47
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2.3. Recovery of exhaled air and determination of H2O2 concentration The expired air for H2O2 assessment was collected in all patients always at the same hour in the morning, with the patients sitting in a comfortable position. The exhaled air was directed, via a tube, into a vial kept at 0 8C, and usually 1.5 ml of condensate was recovered in about 20 min (Figs. 1 and 2). The system included a mouthpiece (A), a one-way valve (B), and an air-filter (C). The expired air was directed into a plastic tube (D) connected to a condensation system (E) consisting of an opening connection (E1), a small glass (F) for the sample deposit and an outflow (E2) connected to another tube (G) via a one-way valve (H). The condensation system, opportunely screened, was dipped into a container with ice to facilitate the condensation of acqueous vapor. Patients breathed for 20 min into the collecting device, while wearing a nose clip. The condensate was then immediately added to the reaction mixture according to a method previously reported [8]. The resultant product deriving from the oxidation of the 3.5, 3 0 –5 0 tetramethylbenzidine was stable for 24 h in the dark at 4 8C and it was easily examined spectrophotometrically at 450 nm. Absorbance measurements were directly proportional to H2O2 concentration, as previously determined. Each sample was tested twice and the mean was considered for further analysis. 2.4. Statistical analysis In both groups A and B the values of H2O2 concentration in exhaled air, repeated at various intervals during the study, are given as meanGSD and statistical analysis was carried out using Student’s t-test, p!0.05 being considered significant. In each group the values of H2O2 concentration repeated at various time points (T0 vs. T15; T0 vs. T30; T0 vs. T60; T15 vs. T30; T15 vs. T60; T30 vs. T60) were compared using the paired t-test. We determined the intraindividual variability by calculating the coefficient of variation (CV) at various time points: S/D!100.
Fig. 2. Representation of the system used to sample expired air (details in text).
3. Results There were 32 patients in group A (NAC) and 23 in group B (placebo). No significant difference between groups was present concerning age of patients, respiratory function data, or H2O2 content in EAC at baseline (Table 1). The mean coefficient of variation (CV) of exhaled H2O2 calculated for each time point in groups A and B ranged about 40–50% and the minimum level of H2O2 detection was 0.025 mM with our method. The individual values of H2O2 concentration in each patient in groups A and B are reported in Tables 2 and 3, respectively. The mean(GSD) of the H2O2 values at each time point in group A was: T0Z1.28G0.61 mM; T15Z 1.0G0.38 mM; T30Z0.91G0.44 mM; T60Z0.83G 0.41 mM. Thus, there was a significant decrease in H2O2 value during the treatment with NAC. In group B (placebo) the mean concentration of H2O2 in EAC at the various times of determination was: T0Z1.14G 0.49 mM; T15Z1.28G0.65 mM; T30Z1.40G0.66 mM; T60Z1.60G0.51 mM. Fig. 3 shows the time-course of H2O2 variation in each group with the comparison between groups of the mean Table 1 Demographic, respiratory function, and exhaled air condensate (EAC) data in the two groups of COPD patients at baseline (mean valuesGSD)
Fig. 1. Representation of the system used to sample expired air (details in text).
Parameter
Group A (NAC)
Group B (placebo)
p
Age (years) M/F FVC % FEV1 (% pred) FEV1/ VC % H2O2 in EAC (mM)
66.16G5.31 48 91.62G5.41 60.69G4.84 62.31G3.96 1.28G0.61
66.30G7.39 NS 7 91.39G4.35 60.35G4.91 62.52G3.36 1.14G0.49
NS NS NS NS
NSZnot significant.
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Table 2 Hydrogen peroxide concentration (mM) in the exhaled air condensate (EAC) of group A at different times (days) of observation
Table 3 Hydrogen peroxide concentration (mM) in the exhaled air condensate (EAC) of group B at different times (days) of observation
Group A (32 pts)
Group B (23 pts)
No. of patients
T0
T15
T30
T60
No. of patients
T0
T15
T30
T60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 MeanGSD
1.6 1.6 0.7 0.7 0.7 0.9 1.6 0.8 0.3 0.4 0.9 1.8 2.4 2.0 0.9 1.6 0.8 1.4 1.6 2.0 2.3 2.3 2.0 1.6 2.1 1.0 0.9 0.9 0.7 0.9 0.7 1.0 1.28G0.61
0.7 0.8 1.0 1.3 0.9 0.7 0.9 0.8 0.4 1.0 1.6 2.1 0.9 1.6 1.9 1.9 1.4 1.6 1.0 0.9 1.6 0.8 0.9 0.8 0.6 0.4 0.4 0.5 0.5 0.7 0.6 0.8 1.0G0.38
0.9 0.8 0.6 0.4 0.8 0.7 0.8 0.8 0.4 2.1 1.0 1.6 0.6 1.4 1.9 1.9 0.8 0.7 0.7 0.6 0.7 0.5 0.7 0.9 0.4 0.7 1.4 0.7 0.9 1.0 0.9 0.7 0.91G0.44
0.8 0.8 1.0 0.7 0.8 0.6 0.8 0.8 0.8 2.1 0.8 0.8 0.6 0.8 2.0 1.9 0.9 0.7 0.7 0.4 0.8 0.7 0.7 0.5 0.4 0.6 0.6 0.7 0.7 0.7 0.5 0.9 0.83G0.41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 MeanGSD
2.1 0.9 1.4 1.0 0.8 0.8 0.3 0.7 1 1.6 0.7 0.6 1.6 1.9 1.4 0.6 1 1.9 1 1.6 0.9 1.6 0.8 1.14G0.49
2.1 0.8 1.6 2.2 0.8 0.8 2.5 0.9 1 2.3 1.6 1 2.1 1.8 1.6 1 1.6 1 0.6 0.9 0.4 0.5 0.4 1.28G0.65
2.1 0.3 1.4 2.1 0.7 1 0.9 2.5 2.3 2.3 1.6 1.3 1.9 2.3 0.9 0.8 0.6 0.7 1.6 1.4 0.8 1.9 0.9 1.40G0.66
2.3 1.9 1.4 1.5 0.9 0.8 1.4 0.8 2 1.9 1.6 2 1.8 1.8 1 2.3 2.3 1.4 1.6 2.3 1 1.9 0.9 1.60G0.51
(GSD) values at each time point. At 15 days from the start of treatment a significant difference between the two groups was already evident. There was a trend for the H2O2 value in EAC to increase with time in patients treated with placebo (group B). For each group, a comparison of mean values of H2O2 in EAC between the different time-points and baseline is reported in Table 4. In patients treated with the usual therapy plus NAC significantly lower concentrations of H2O2 were found at 15 (pZ0.03) and 30 (p!0.007) days after the start of treatment, and at the end of the trial (60 days, pZ0.000), compared to the values recorded before the start of treatment. On the contrary, at the end of the study in patients treated with usual therapy plus placebo the H2O2 concentrations in EAC were significantly increased compared to the initial values (p!0.003). Mean spirometry values at the end of the study were not significantly different from those recorded before the beginning of the trial in the two groups of patients (group A: FEV1% at baseline 60.69G4.84 vs. 60.50G4.98 at T60, pZN.S; group B: FEV1% at baseline 60.35G4.91 vs. 59.30.50G3.81 at T60, pZNS).
4. Discussion The increased production of H2O2 in EAC of patients with COPD is a marker of the chronic airways inflammation that persists even during clinical stability and may play an important role in the pathogenesis of the disease. The increased oxidative stress typical of patients with COPD [1]
Fig. 3. Time-course of H2O2 changes in exhaled air condensate (EAC) of Group A (NAC) and of Group B (placebo) during the 2 months of the study. Red and blue solid circles are the mean values and the vertical bars are the SD of the mean. T0Zbaseline, before treatment; T15Z15 days after start of treatment; T30Z30 days after start of treatment; T60Z60 days after start of treatment. Paired-t-test.
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Table 4 Comparison of H2O2 mean values in each treatment group at the different times of assessment Comparison
Group A
p
Group B
p
T15 T30 T30 T60 T60 T60
1.00G0.38, 1.28G0.61 0.91G0.44, 1.0G0.38 0.91G0.44, 1.28G0.61 0.83G0.41, 0.91G0.44 0.83G0.41, G0.38 0.83G0.41, 1.28G0.61
0.03 NS 0.007 NS NS 0.000
1.28G0.65, 114G0.49 1.40G0.66, 1.28G0.65 1.40G0.66, 1.14G0.49 1.60G0.51, 1.40G0.66 1.60G0.51, 1.28G0.65 1.60G0.51, 1.14G0.49
NS NS NS NS NS 0.003
vs. T0 vs. T15 vs. T0 vs. T30 vs. T15 vs. T0
may cause various alterations, and not only at the level of the respiratory tract. Lipid peroxidation of the cell membranes, DNA damage, production of transcription factors (Nuclear Factor KB, Activator Protein-1) that regulate the genes to pro-inflammatory mediators (IL-8; TNFa) are only some of the alterations caused by oxidants. Furthermore, reactive oxygen species may inactivate a1-antitrypsin by oxidation of methionine at site 358, with consequent enhancement of neutrophil elastase activity and proteolytic damage to the alveolar structure. Oxidants may also interfere with the production and function of respiratory mucus, so increasing the susceptibility of the respiratory tract to infective agents. As a result of abnormal oxidative stress an exhaustion of the natural antioxidant defences may occur in COPD patients [17]. In the past, several studies have been performed on the role of molecules with antioxidant activity. The study of van Beurden et al. [18] demonstrated that inhaled corticosteroids (ICS) reduce exhaled H2O2 in stable COPD, but there is not general agreement with other authors [19]. The discordance is probably due to technical factors and the duration of treatment in these other studies. Van Beurden et al. confirm the importance of H2O2 in breath condensate as a biomarker of inflammation and as a parameter to compare the effectiveness of different treatments (NAC, ICS, etc.). The evaluation of respiratory oxidant burden and antioxidant capacity has stimulated much interest in recent years, also in relation to possible new therapeutic approaches to apply in a disease like COPD where no drug has been demonstrated capable of influencing the natural course of the disease. Many investigations have pointed out the need for a simple and non-invasive procedure to assess the oxidative status of the lung. We recently found that measurement of the H2O2 content in exhaled air condensate of COPD patients is a useful biomarker of lung oxidative stress [8]. To this end, we devised a new method of H2O2 measurement that minimizes the inaccuracy due to the instability of hydrogen peroxide. In addition, in contrast with previous procedures, our method avoids complex manipulations of the sample and can be routinely used to monitor a large number of patients. Using this modified procedure, we already demonstrated that the H2O2 content in the EAC of stable COPD patients is significantly higher (0.50G0.11 mM) than in healthy nonsmoking subjects (0.12G0.09 mM; pZ0.001) [8]. These
results are in accordance with those of Dekhuijzen et al. [7] and Nowak et al. [20], and the latter also surprisingly observed that current smoking in COPD patients does not influence the expired H2O2 level in comparison to exsmokers or non-smoker COPD patients [17]. In the present study our procedure [8] was utilized to evaluate the efficacy of long-term treatment with NAC compared to placebo and the time-course of the antioxidant effect, in stable COPD patients. For long the therapeutic efficacy of N-acetylcysteine has been attributed to the mucolytic properties of the molecule, which are able to reduce the mucus viscosity and improve the mucociliary clearance [21]. In recent years it has been shown that NAC also has antioxidant properties, which act in both a direct and indirect manner. The direct antioxidant activity consists in the chemical non-enzymatic interaction between its thiolic free groups (–SH) and reactive oxygen species present at the inflammation site. This action is dose-dependent and takes place in the extracellular environment [22]. The indirect antioxidant activity of NAC is that, being a reduced GSH precursor, it provides cysteine for GSH synthesis, and this action occurs at intracellular level [23]. Various clinical findings may confirm the antioxidant properties of NAC. It has been reported that long-term administration of NAC is able to reduce the exacerbation rate in patients with moderate to severe COPD [24], and more recently it has also been demonstrated that NAC can reduce the risk of rehospitalization in patients with COPD [25]. Although the hypothesized antioxidant role of NAC still remains to be confirmed in further studies, these clinical reports draw attention to the increased oxidant production in COPD, particularly during exacerbations, and to the possibility that an antioxidant treatment might reduce the consequences of inflammation at the respiratory level, so preventing further exacerbations of the disease. The results of the present study show that COPD patients treated with usual therapy plus NAC (group A) already have a significantly (p!0.03) decreased concentration of H2O2 in the EAC at 15 days after start of treatment. Still lower levels of H2O2 in the EAC were measured at 30 days (p!0.002) and at the end of the treatment (60 days pZ0.000). On the contrary, patients treated with usual therapy plus placebo (group B) were characterized by levels of H2O2 in the exhaled air condensate that remained stable or tended
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to increase at all time points of the study (T30: 0.91G0.44 vs. 1.40G0.66; T60: Z0.83G0.41vs. 1.60G0.51). Furthermore, at the end of the trial H2O2 mean concentration in EAC was significantly higher (p!0.003) compared to the baseline value in this group of patients. Such an event is not easy to explain but it is probably related to the fact that all the studied patients, in both group A and B, had suspended the treatment with inhaled corticosteroids at the beginning of the trial. This could have determined, in patients treated with placebo, a decrease in the control of chronic inflammation exerted by inhaled steroids, with resulting increased production of oxygen reactive species, while such effect would have been blunted by the antioxidant action of N-acetylcysteine in patients treated with this compound. The coefficient of variation ranged between 40 and 50% for the patient and control groups, respectively. These CV values were similar to or lower than those found in previous investigations [7]. The mean CV estimated by Van Beurden et al. [26] in a study where the samples of exhaled air were obtained early in the morning was 45%. When the intraindividual variability was tested at different expiratory flow rates, the corresponding CV values ranged between 60 and 80% [27]. In general the variability appears to be higher for exhaled H2O2 than for exhaled NO [28]. The cause of this high variability is still unclear, and further investigations are likely necessary to establish the importance that other factors such as age, history of smoking, and diet may play in determining the observed high intra-individual variability. Since it has been recently reported that the H2O 2 concentration has a circadian variability, increasing during the day from 9 a.m. to 3 p.m. [29], we examined all our patients at the same hour in the morning to minimize the possible circadian effect. There were no significant changes in respiratory function values at the end of the study compared to baseline. Since in a previous investigation [24] we observed a mild but significant increase in respiratory function values after six months of administration of NAC, we can suppose that the duration of the present study is likely not long enough to reveal possible effects, if any, in terms of respiratory function improvement. The results obtained in the present study support the effective antioxidant properties of NAC and demonstrate that 2 weeks of 1200 mg/day administration of this antioxidant are already sufficient to decrease the respiratory oxidant burden. Our results are different from those reported by Kasielski and Novak [30], who observed no reducing effect of NAC on H2O2 in the first six months of administration of the drug compared to placebo. The reason for this delay in the effect of NAC could be that the dosage of the drug administered by these authors was 600 mg once a day, instead of 600 mg b.i.d. as in our patients. However, at 9 and 12 months of treatment patients given NAC 600 mg u.i.d. exhaled 2.3–2.6-fold less H2O2 than patients treated with placebo [30].
Thus, it seems that if one wants to reach a fast antioxidant action, an oral dose of NAC as high as 1200 mg should be used and this effect further increases continuing the administration with this dosage. The problem of drug tolerability does not appear important, at least in our experience, because all patients tolerated well this dosage of NAC. Our results also point out the importance of the assessment of H2O2 content in the airways of COPD patients, as a marker of inflammation and, possibly, of the anatomical and functional progression of the disease. In this context, the technique used in our study is easy, noninvasive and sufficiently stable. Furthermore, H2O2 assessment may be a valuable tool to verify the efficacy of drugs such as N-acetylcysteine in reducing the oxidant stimuli in the lung, in view of their possible inclusion in the therapeutic options for this disease.
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