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(Chest. 2004;126:1116-1122.)
© 2004 American College of Chest Physicians

Increased Glutathione Disulfide and Nitrosothiols in Sputum Supernatant of Patients With Stable COPD*

Kai Michael Beeh, MD; Jutta Beier, MD; Nicole Koppenhoefer and Roland Buhl, MD, PhD

* From the Pulmonary Department, Internal Medicine, University Hospital Mainz, Germany.

Correspondence to: Kai Michael Beeh, MD, Pulmonary Department, Internal Medicine, University Hospital, Langenbeckstr. 1, 55131 Mainz, Germany; e-mail: k.beeh{at}3-med.klinik.uni-mainz.de


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Increased oxidant burden is involved in the pathogenesis of COPD. Glutathione (GSH) is a major antioxidant scavenging reactive oxygen and nitrogen species. We studied the concentrations of total and reduced GSH, glutathione disulfide (GSSG), and nitrosothiols in sputum supernatant of patients with COPD.

Design: Twenty-five patients with moderate-to-severe COPD (FEV1 61 ± 12% of predicted) and 25 healthy nonsmoking control subjects underwent sputum induction. Sputum total GSH and GSSG were measured spectrophotometrically, and nitrosothiols were quantified by enzyme assay. Exhaled nitric oxide (eNO) was also measured to correlate eNO with nitrosothiols in induced sputum.

Measurements and results: Compared with healthy subjects, patients with COPD had increased sputum neutrophils (geometric mean, 65%; 95% confidence interval [CI], 57.5 to 71; vs 21%; 95% CI, 13.2 to 31.6; p < 0.001); total GSH (geometric mean, 7.1 µmol/L; 95% CI, 2.95 to 17; vs 5.1 µmol/L; 95% CI, 3.2 to 8.1; p = 0.024); GSSG (geometric mean, 4.1 µmol/L; 95% CI, 1.7 to 10; vs 0.84 µmol/L; 95% CI, 0.35 to 1.99; p = 0.002); and nitrosothiols (geometric mean, 60.4 µmol/L; 95% CI, 40 to 95.5; vs 38 µmol/L; 95% CI, 31.6 to 43.6; p = 0.04). Sputum GSSG was positively correlated with neutrophils ({rho} = 0.47, p = 0.016) and nitrosothiols ({rho} = 0.49, p = 0.024) in patients with COPD, whereas there was no correlation of eNO with nitrosothiols ({rho} = 0.38, p = 0.1).

Conclusions: Sputum concentrations of GSSG and nitrosothiols are increased in patients with COPD and associated with neutrophilic inflammation. These data underline the role of oxidative stress in the pathogenesis of COPD, and suggest that GSH is important to scavenge both reactive oxygen and nitrogen species.

Key Words: antioxidants • COPD • glutathione • induced sputum • neutrophils • nitrosothiols


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxidative stress plays a major role in COPD.1 Due to their ability to rapidly generate reactive oxygen intermediates in response to various stimuli (respiratory burst), neutrophils are a major source of oxidative stress in COPD. The tripeptide glutathione (GSH) [L-{gamma}-glutamyl-L-cysteinyl-glycine] plays a pivotal role in metabolic and cell cycle-related functions in virtually all cells. Its ability to directly scavenge free radicals and to act as cosubstrate in the GSH peroxidase-catalyzed reduction of H2O2 and lipid hydroperoxides makes GSH a central defense mechanisms against intracellular and extracellular oxidative stress.2

Studies34 using BAL and lung epithelial lining fluid have shown that smokers and patients with COPD have increased levels of total and oxidized GSH (GSH disulfide [GSSG]), a finding compatible with the concept of increased pulmonary oxidative stress due to both acute cigarette smoking and chronic inflammation.56 Despite normal levels of exhaled nitric oxide (eNO), increased nitrogen metabolites and protein nitrosylation were found in patients with COPD,7 and this could reflect the rapid reaction of nitric oxide with neutrophil-derived superoxide anion, leading to the formation of reactive nitrogen species, eg, peroxynitrite.8 Peroxynitrite itself again is detoxified by thiol antioxidants like GSH to form nitrosothiols.9

We and others have previously shown that quantification of GSH in induced sputum is valid and reproducible,1011 and reflects known disturbances of oxidant/antioxidant balance in specific disorders.12 Here, we report on sputum concentrations of total GSH, reduced GSH, and GSSG levels in patients with COPD, and their correlation with nitrosothiols and eNO.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
The study population consisted of 25 patients (17 men; mean age, 61 years; age range, 42 to 71 years) with moderate or severe COPD according to criteria of the Global Initiative for Obstructive Lung Disease,13 and 25 healthy nonsmokers (53% men; mean age, 38 years; age range, 25 to 72 years). None of the patients with COPD actively smoked, received oral corticosteroids or antibiotics, or reported an exacerbation for at least 4 weeks prior to sputum induction. Eight of 25 patients with COPD received concomitant inhaled corticosteroids at doses ranging from 400 to 1,200 µg of beclomethasone, or equivalent. The study was approved by the local ethics committee, and each patient gave informed written consent.

Sputum Induction and Processing
Sputum induction was performed according to a method previously described.12 In case sputum plugs were contaminated with saliva, they were immediately separated from salivary secretions, and closely examined by light microscopy to ascertain a least possible contamination of sputum with saliva and squamous cells possibly leading to diffusion of GSH from sputum to saliva. Since dithiothreitol (DTT), which is commonly used for sputum homogenization, interferes with both the GSH and nitrosothiol assays, sputum plugs were dissolved in phosphate-buffered saline solution (Gibco Life; Paisley, Scotland) instead of DTT. Four hundred nonsquamous cells were independently counted by a technician blinded to the sample source. The relative proportion of cell types was counted as percentage of all nonsquamous cells. The sputum supernatant was immediately (within 1 h) processed for GSH quantification.

Measurement of Total and GSSG in Sputum Supernatant
Sputum total GSH was measured using a spectrophotometric method described previously.121415 GSSG was measured with a similar method, but after treatment of samples with 2-vinylpyridine (final concentration, 10 mM; Sigma; Heidelberg, Germany) in ethanol to derivatize reduced GSH. GSSG was quantified only in patients with total GSH levels above the detection limit (0.5 µmol/L). Reduced GSH was calculated as the amount of total GSH minus GSSG.

Nitrosothiols in induced sputum were quantified by a commercially available assay (Alexis Biochemicals; Gruenberg, Germany). The assay uses a standard curve of nitroso-GSH and has a lower detection limit of 0.025 µmol/L.

Measurement of eNO
eNO was quantified according to American Thoracic Society guidelines at a constant flow rate of 50 mL/s using a chemiluminescence analyzer (CLD88sp; ECO MEDICS AG; Duernton, Switzerland). All measurements were performed in triplicate.16

Statistical Analysis
Statistical analysis was performed using the STATA 5.0 intercooled software package (StataCorp; College Station, TX) for personal computer. Unless otherwise stated, data are presented as geometric mean values with 95% geometric confidence intervals (CIs)17 for 25 patients and 25 control subjects. Due to insufficient sputum quantity, nitrosothiol concentrations represent mean data from 21 patients and 23 control subjects. In sputum samples with concentrations below the assay detection limit, a random value between 0 and the lower limit was generated to allow computation of geometric means. However, this was necessary in only two COPD samples for total GSH quantification. eNO levels are geometric mean data from 9 patients with COPD and 16 healthy subjects. Normal distribution was tested by the method of Kolmogorov-Smirnov. If appropriate, log transformation was performed to achieve normal distribution of data. Only for such parameters, group comparisons were done by two-sample t test. For all other comparisons, a nonparametric Mann-Whitney U test was used. Correlations were calculated by a Spearman correlational analysis ({rho}); p < 0.05 was considered statistically significant. Based on a previous study12 on total GSH levels of healthy subjects, the study had approximately 80% power to detect any difference in the primary end point (sputum total GSH) of > 25% on a two-sided {alpha} level of 0.025 (sample power, 2.0; SPSS; Chicago, IL).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sputum Cells
Patients with COPD had increased sputum neutrophils and eosinophils, and lower sputum macrophages in comparison to healthy control subjects (Table 1 ).


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  		Table 1. Characteristics and Sputum Components of Healthy Subjects and Patients With COPD*

 
Sputum Content of Total GSH, Reduced GSH, and GSSG and Nitrosothiols
Compared with healthy subjects, total GSH and GSSG in induced sputum supernatant from patients with COPD was increased. GSSG represented 52.4% (95% CI, 37 to 48) vs 15.4% (95% CI, 6.3 to 37) of total GSH. In contrast, reduced GSH levels were not different in patients with COPD and control subjects. Finally, nitrosothiols were also increased in patients with COPD (Table 1, Fig 1 ). There were no differences with regard to sputum neutrophils, total GSH, GSSG, and nitrosothiols in patients with COPD with or without inhaled corticosteroids (p > 0.1, all comparisons).



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  		Figure 1. Sputum supernatant concentrations of reduced GSH (top, left), GSSG (top, right), and nitrosothiols (bottom, left) in control subjects and patients with COPD. Bars represent geometric means. *p < 0.05 vs control subjects.

 
There was a significant correlation of sputum total GSH and GSSG with neutrophils (Table 2 ). This was also true, when only patients with COPD were analyzed separately (Fig 2 , top, left, and top, right). Nitrosothiols were correlated with neutrophils only in patients with COPD ({rho} = 0.48, p = 0.027). There was also a negative correlation of sputum-reduced GSH and nitrosothiols in patients with COPD (Fig 2, bottom, left). Total GSH and GSSG were inversely correlated with the degree of airway obstruction (FEV1 percentage of predicted) in all patients (Table 2). However, there was no correlation when patients with COPD were analyzed separately (p > 0.1, all correlations). In particular, nine patients with COPD and low or undetectable GSH had FEV1 values comparable to those with GSH > 1 µmol/L (65 ± 10% vs 57 ± 10% [± SD], p = 0.08).


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  		Table 2. Correlation of Sputum Mediators With Lung Function and Cells in All Patients and Control Subjects*

 


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  		Figure 2. Correlation of sputum GSSG with nitrosothiols (n = 21, {rho} = 0.49, p = 0.024) [top, left], GSSG with sputum neutrophils (n = 25, {rho} = 0.47, p = 0.016) [top, right], and reduced GSH with nitrosothiols (n = 21, {rho} = – 0.5, p = 0.02) [bottom, left] in patients with COPD.

 
eNO
eNO levels in patients with COPD (n = 9) were comparable to those of healthy subjects (n = 16). Mean eNO in COPD was 23.6 parts per billion (95% CI, 12.3 to 44.6) vs 20 parts per billion (95% CI, 12.3 to 32.3) in healthy subjects (p = 0.46). Analyzed together or by separate groups, there was no correlation of eNO with sputum nitrosothiols (p > 0.1, all correlations).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
COPD is characterized by an increased oxidative burden in the lower respiratory tract of affected patients in stable disease or exacerbations. Moreover, investigations18 have also linked COPD to increased nitrosative stress in sputum, breath condensate, or airway biopsies, despite normal or mildly increased levels of eNO. Up to now, only few studies have reported direct measurement of antioxidants and markers of oxidative stress in induced sputum of patients with COPD. Kanazawa et al19 recently reported decreased peroxynitrite inhibitory capacity in COPD sputa, and attributed this finding to a general decrease in sputum antioxidants. Our study demonstrates increased levels of total GSH and GSSG in sputum supernatant of patients with COPD, and a close correlation of GSSG with sputum neutrophils, indicating the usefulness of GSSG as a marker of oxidative stress. Moreover, nitrosothiols were elevated in COPD sputa, despite low levels of eNO, and the observed increases were independent of concomitant inhaled corticosteroid use.

Our observation of increased nitrosothiols in COPD is in line with a study18 using breath condensate, although the concentrations observed in our study were a magnitude higher than in breath condensate. There was no correlation of nitrosothiols with eNO, but this negative finding is clearly limited by the small number of patients in whom eNO was measured. In contrast, the correlation of sputum neutrophils with sputum GSSG and nitrosothiols in our patients with COPD is highly remarkable. Neutrophils produce large amounts of superoxide anion, which reacts with nitric oxide to form peroxynitrite. Nitric oxide itself is released by neutrophils under inflammatory conditions.20 Thus, increased production of superoxide anion and nitric oxide by neutrophils would promote the oxidation of GSH, and also result in consumption of reduced GSH to scavenge peroxynitrite, finally leading to increased sputum levels of GSSG and nitrosothiols, as observed in our patients. Although the assay used to quantify nitrosothiols does not exclusively detect nitroso-GSH, the above conclusion still stands as it is, since nitroso-GSH has been shown to be the major nitrosothiol in human airways.21

The observation of increased GSSG in COPD is in contrast to studies in idiopathic pulmonary fibrosis, in which nearly the entire GSH found in BAL fluid22 and sputum supernatant12 is present in the reduced form, but fits well into observations in asthma, where approximately 50% of sputum total GSH was in the oxidized form.11 The finding of increased nitrosothiols in our study clearly indicates that oxidative stress in COPD also leads to the formation of nitroso-GSH; thus, one would relatively underestimate the degree of oxidative stress in COPD by taking into account only GSSG.

Unlike GSSG, reduced GSH was not increased in our patients with COPD. This is in contrast to other authors,23 who reported an adoptive increase of airway GSH in long-term smokers. A potential explanation of this finding is that the principally healthy lung of asymptomatic cigarette smokers is initially able to augment and maintain increased levels of reduced GSH. However, increasing GSSG concentrations in the long term may indicate that this capacity is progressively lost in COPD. Nine of our patients with COPD had low or undetectable levels of GSH, although these patients were not those with more severe COPD (FEV1 percentage of predicted). Nevertheless, given the observed increase of GSSG in patients with COPD, this would also support the rationale of antioxidant supplementation as a therapeutic concept in COPD. In fact, several studies24 have confirmed that oral antioxidants, eg, N-acetylcysteine (NAC), a GSH precursor, may reduce the frequency of exacerbations in patients with chronic bronchitis. The ongoing Bronchitis Randomized on NAC Cost Utility Study25 to assess the effectiveness of the antioxidant NAC in patients with moderate-to-severe COPD may shed further light on this question. Besides clinical outcome parameters like exacerbation rates or decline of lung function requiring prospective long-term trials with antioxidant supplementation, it would also be desirable to evaluate the effect of antioxidant supplementation in the target compartment. Unfortunately, a study by Bridgeman and colleagues26 failed to demonstrate increased BAL GSH levels following high-dose oral (600 mg/d) administration of NAC. However, the relatively noninvasive and safe method of sputum induction would allow to repeatedly follow larger study populations, thus increasing study power and chances to detect minor increases in sputum GSH content.

In fact, our study also demonstrates the possibility to directly measure GSH in induced sputum from patients with COPD. Several considerations need to be taken into account when quantifying sputum GSH. A GSH gradient with low or undetectable concentrations in the nose, and high concentrations in BAL has been described previously.27 The concentrations of GSH in sputum both from patients and control subjects were a magnitude lower than those in BAL or epithelial lining fluid, as reported by others.2328 Given the degree of oxygen exposure in the alveolar space, this finding is not surprising. We have previously shown that GSH levels in induced sputum increase with the duration of sputum induction, indicating that different lung compartments were sampled at different time points during sequential sputum induction.10 This observation also fits into a study by Smith et al,29 who demonstrated significantly higher GSH levels in BAL than in bronchial washings of asthmatics. However, strict adherence to standardized protocols, with particular regard to induction length, therefore appears crucial to achieve valid results.

There are some apparent limitations of our study. First, patients and control subjects were not perfectly age matched. It has been shown that oxidative stress in general increases with age.30 This phenomenon may theoretically in part account for our results, although several authors1831 failed to describe a correlation of airway GSH or nitrosothiols with age. Adding to this, there was no correlation of GSH or nitrosothiols with age in our group of healthy subjects. Second, we chose not to include "healthy" smokers as additional control group, despite the fact that active smoking per se may increase lung antioxidants like GSH. However, all patients with COPD were ex-smokers for at least 6 months prior to our study; therefore, the observed changes truly reflect underlying COPD and are not confounded by short-term consequences of active cigarette smoking. Moreover, we found it hardly possible to find a group of age-matched (ie, elderly) "healthy" smokers with comparable amounts of pack-years, in particular since most of these persons at least displayed clinical signs of chronic bronchitis, ie, COPD stage 0. Hence, we believe the basic finding of our study, ie, increased GSSG in sputa of former smokers with COPD, stands as it is. Finally, the quantification of thiols requires strict avoidance of reducing agents such as DTT, which is commonly used during sputum processing to release cells from mucus. However, the use of DTT also increases cell viability,32 and the avoidance of DTT in our investigation might have influenced our cell populations to a certain extent. Efthimiadis et al32 were able to demonstrate that cell differentials of sputum samples treated with DTT or phosphate-buffered saline solution were comparable. Moreover, the reported total cells and differentials of patients with COPD and control subjects in our study compare well with published data3334 from investigations using standard protocols including DTT, supporting the conclusion that the effect of DTT avoidance on the basic results from our study is negligible.

In summary, this study describes increased levels of GSSG and nitrosothiols in airway secretions of patients with COPD, and their correlation with neutrophilic inflammation assessed by induced sputum. Our study supports the concept of increased oxidative and nitrosative stress in COPD and highlight the role of GSH as scavenging molecule in both pathways. Sputum analysis of oxidative and nitrogen metabolism might offer a future tool to further elucidate their role in the pathogenesis of COPD.


   Footnotes
 
Abbreviations: CI = confidence interval; DTT = dithiothreitol; eNO = exhaled nitric oxide; GSH = glutathione; GSSG = glutathione disulfide; NAC = N-acetylcysteine

The presented work was a part of a doctoral thesis by Ms. Koppenhoefer.

Received for publication October 13, 2003. Accepted for publication May 7, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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