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* From the Departments of Pneumonology (Drs. Loukides and Panagou), Clinical Research (Dr. Papatheodorou), and Radiology (Dr. Lachanis), Athens Army General Hospital, Athens, Greece; and the Department of Pneumonology (Drs. Bouros and Siafakas), Medical School, University of Crete, Greece.
Correspondence to: Stelios Loukides, MD, Smolika 2, 16673, Athens, Greece; e-mail ssat@hol.gr
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Design: Cross-sectional study.
Patients: Thirty patients with steady-state bronchiectasis.
Results: Mean (95% confidence interval [CI]) exhaled H2O2 levels were significantly elevated in patients with bronchiectasis compared to normal subjects: 1.1 (0.87 to 1.29) µM vs 0.3 (0.19 to 0.36) µM, respectively (p < 0.0001). Patients treated with ICS had similar values as steroid-naïve patients. The group of patients with P aeruginosa colonization showed a significantly increased concentration of H2O2 compared to the group without P aeruginosa colonization. Patients receiving long-term oral antibiotic treatment had significantly higher values of H2O2 compared to those not receiving antibiotics. There was a significant positive correlation between H2O2 and either the percentage of neutrophils in induced sputum or the extent of the disease as defined by high-resolution CT. A significant negative correlation was found between H2O2 and FEV1 percent predicted. Finally, there was a significant positive correlation between H2O2 and the symptoms score.
Conclusions: Patients with bronchiectasis in stable condition showed increased levels of exhaled H2O2. The above-mentioned levels were not decreased either by ICS or long-term oral antibiotic treatment, but were significantly affected by chronic colonization with P aeruginosa. H2O2 levels could be an indirect index of neutrophilic inflammation, impairment of lung function, and extension and severity of the disease.
Key Words: bronchiectasis • hydrogen peroxide • induced sputum • Pseudomonas aeruginosa • spirometry
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induced sputum is a relatively noninvasive, reliable, valid, and responsive technique to measure airway inflammation.5 It can be applied even in the more severe conditions, and can be used repeatedly to investigate the inflammatory pattern as well as the effects of treatment on various diseases.6
Oxidative stress, defined as an increased exposure to oxidants or decreased antioxidant capacities, is implicated in airway inflammatory diseases including bronchiectasis.7 8 Oxygen-derived free radicals or metabolites, collectively termed reactive oxygen species, are important mediators of cell and tissue injury during inflammation and may be produced by several types of inflammatory cells.9 It has been shown7 that the increased oxidative burden in the lungs of patients with bronchiectasis is being reflected by increased levels of exhaled H2O2.
In testing the hypothesis that reactive oxygen intermediates (ROIs) such as H2O2 are important in bronchial inflammation seen in bronchiectasis as well as in predicting the severity of the disease, it would be necessary and interesting to define which of the inflammatory cells express the main source of H2O2 production in patients with bronchiectasis. Furthermore, it would be interesting to show whether the H2O2 concentration contributes to the development of bronchial inflammation and consequently to the extent of the disease, to disease severity, and to lung function impairment. We therefore measured H2O2 concentrations in the expired breath condensate of patients with steady-state bronchiectasis. We also assessed whether there is an association between H2O2 concentration, airway inflammation as defined by induced sputum cytology, the extent of the disease as defined by a HRCT scoring scale system, the disease activity as defined by a symptoms score, and lung function impairment. To account for possible confounding effects of the use of inhaled corticosteroids (ICS), long-term oral antibiotic treatment, or chronic colonization with P aeruginosa, further analysis was conducted after subclassifying the patients on the basis of treatment with ICS, oral antibiotics, and chronic colonization with P aeruginosa.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12% of predicted
FEV1. All patients had bronchiectasis diagnosed
on the basis of clinical and radiologic features and confirmed by HRCT
of the thorax. Patients were included in the study only if they were in
clinically stable condition and had no evidence of acute infective
exacerbation (lower or upper airways) for at least 4 weeks prior to the
study. Ten patients with chronic colonization with P
aeruginosa were separately studied and compared with the remaining
20 patients who had no evidence of any chronic colonization with
bacteria (Table 1)
. Chronic colonization with P aeruginosa
was defined as more than three isolations of P aeruginosa
from separate samples over 3 months.4
All patients had
negative sweat test results. Patients with cystic fibrosis, allergic
bronchopulmonary aspergillosis, asthma,
1-antitrypsin deficiency, COPD, and atopic
diseases were excluded. Asthma and COPD were excluded by using the
American Thoracic Society criteria.10
None of our patients
were current smokers. Two patients had stopped smoking > 3 years
prior to the study (mean smoking history, < 4 pack-years), and the
remaining 28 patients had a negative smoking history. Twenty-one
patients were receiving regular inhaled
ß2-agonists (short or long acting) and 14
patients were receiving ICS (fluticasone propionate, 500 to 1,000
µg/d, or budesonide, 400 to 800 µg/d). Eleven patients received
long-term oral antibiotics (8 patients in the group with P
aeruginosa colonization). No attempt was made to modify their
treatment with inhaled steroids and long-term oral antibiotics, which
remained unchanged for at least 3 months before sputum induction and
H2O2 collection. None of
the patients were receiving inhaled or oral mucolytics, and none were
receiving oxygen therapy. The study protocol was approved by the ethics
committee of our hospital, and all subjects gave written informed
consent.
Assessment of Disease Severity
Symptoms were assessed as previously described11
using a different analog scale score. Patients were instructed to
record accurately their daily symptoms (wheezing, sputum production,
cough) for a 2-week period. Symptoms were scored by the patient every
day using a subjective 0 to 3 scoring system, where 0 = none,
1 = mild, 2 = moderate, and 3 = severe. Sputum was collected over
a 24-h period and was stored in clear plastic pots (50 mL). Sputum
production was scored as follows: 0 = no sputum, 1 = less than a
half pot, 2 = more than a half pot but less than a full pot,
3 = more than a pot per day. The symptoms were added for each day,
and a mean daily score was calculated for the period of assessment
(score range, 0 to 9).
Lung Function
FEV1 and FVC were measured using a dry
spirometer (Vica-test, model VEP2; Mijnhardt; Rotterdam,
Holland). The best value of three maneuvers was expressed as a
percentage of the predicted value. Airway responsiveness was measured
only in normal subjects by histamine provocation challenge. The
provocative concentration of histamine causing a 20% fall in
FEV1 was calculated by linear interpolation of
the semilogarithmic dose-response curve.
HRCT
A recent HRCT (within a week from entering the study) scan (GE
9800 Highlight Advanced; General Electric Medical Systems; Milwaukee,
WI) of 30 patients was assessed and scored by the same
radiologist, who was blinded to all other details concerning the
patients’ clinical and functional conditions. During the 7-day
interval, none of our patients had an exacerbation of disease. Each
lobe of both lungs was graded for bronchiectatic changes on a scale of
0 to 3 (lingula was scored as a separate lobe), giving a maximum of 18
points (0 = no bronchiectasis, 1 = one bronchopulmonary segment
involved, 2 = more than one bronchopulmonary segment involved, and
3 = gross cystic bronchiectasis). Patients with emphysematous changes
were excluded. This scoring has been used in a previous
study3
with low interobserver variation.
Collection of Expired Breath Condensate and
H2O2 Measurement
A heat-exchanger unit (RHES, model 6V3; Jaeger; Wuerzburg,
Germany) was used to produce cold air of - 15°C to
- 18°C at an air flow of 80 L/min. A double-jacketed glass tube of
30 cm in length was specifically adapted to the cold-air system and
a two-way unidirectional valve (Series 2-200; Hans Rudolph;
Kansas City, MO) was connected to the tube in order to separate
inspiration from expiration. After rinsing their mouths, the subjects
were comfortably seated in a chair wearing nose clips and breathed in a
relaxed manner (tidal breathing) for 10 min. The breath condensate was
collected at the other end of the tube and was immediately stored at
- 70°C for later analysis. According to this design, salivary
contamination was highly unlikely and was easily observed, as the
proximal cold-air connection was 20 cm away from the mouthpiece.
Approximately 1 mL of breath condensate was collected in a 2-mL sterile
plastic tube. H2O2
measurements were performed the same day (minimum of 2 days and maximum
of 20 days from collection time in all samples). To examine the
repeatability of H2O2
measurements within subjects, condensate was collected from 5 normal
subjects and 10 patients on 2 consecutive days. To assess the stability
of H2O2 in the frozen
condensate, 4 mL of condensate was collected from eight subjects (four
patients). The above-mentioned concentration was divided into 1-mL
aliquots, in which H2O2
concentrations were determined after 2 days, 1 week, 2 weeks, and 3
weeks of storage (the maximum time between collection and measurement
in the whole samples). Repeatability of exhaled
H2O2 measurements and
stability of H2O2 frozen
samples were estimated as previously described.12
All condensate samples were tested for salivary contamination by
determination of amylase activity. Amylase activity was carried out
spectrophotometrically (kinetic method) using a commercial reagent kit
(model 981362; KONE Instruments; Espoo, Finland). In this
procedure, -amylase of the sample and the enzyme -glucosidase
hydrolyzes the substrate
p-nitrophenyl--D-maltoheptaoside to glucose and
p-nitrophenol. The liberation of p-nitrophenol is followed at 405 nm
(37°C) for 2 min. Two samples were spiked with saliva to ensure that
it can be detected by our method. In all samples, no amylase was
detected using the method described, suggesting no contamination of
breath condensate with saliva. The samples that were spiked with
saliva, showed levels of > 5,000 IU salivary amylase.
H2O2 concentration was determined by an enzymatic assay as previously described.13 14 Briefly, 250 µL of 420 µM 3', 3,5,5' tetramethylbenzidine (dissolved in 0.42 mol/L citrate buffer, pH 3.8) and 10 µL of 52.5 U/mL of horseradish peroxidase (HRP; Sigma Chemicals; St Louis, MO) were reacted with 250 µL of the condensate for 20 min at room temperature. Subsequently, the mixture was acidified to a pH of 1 with 10 µL of 18 N sulfuric acid. The reaction product was quantitated at the absorbency of 450 nm using a double-beam spectrophotometer (Uvicon 940; Kontron Instruments; Zurich, Switzerland). A standard curve was performed for the assay, with limit of determination of 0.1 µM H2O2. The investigator who performed the H2O2 measurements was blinded to the clinical and functional status of the subjects as well as to the results of sputum inflammatory cells.
Sputum Induction and Processing
Sputum was induced by the inhalation of an aerosol 3.5%
hypertonic saline solution generated by an ultrasonic nebulizer (model
2696; DeVilbiss; Somerset, PA) with modifications to improve its
safety.6
At least 2 mL of sputum was collected into a
sterile container. The sample from the first cough was discarded, as
this is heavily contaminated with squamous epithelial
cells.15
An adequate sample was defined if the number of
squamous epithelial cells was < 30% of the total number of
inflammatory cells.16
Cytospin slides were prepared and
stained with May-Grunwald-Giemsa. The person who performed the
differential cell counts was not aware of the clinical and functional
status of the patients, as well as of the expired breath condensate
measurements. Two slides were used for counting, and at least 300
inflammatory cells were counted for each slide.
Statistical Analysis
Data concerning characteristics of the subjects are shown as
mean (SD) and range. Data concerning the comparisons between the
various parameters in the study groups are given as mean with 95%
confidence intervals (CIs) for the differences. Parameters from
patients and control subjects, as well as from all the subgroups, were
compared using the Student’s t test. Spearman’s rank
correlation coefficient was used to investigate the relation between
the various parameters. A p value < 0.05 was considered significant.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mean H2O2 levels were significantly increased in patients with bronchiectasis compared to normal subjects: 1.1 µM (95% CI, 0.87 to 1.29 µM) vs 0.3 µM (95% CI, 0.19 to 0.36 µM), respectively (p < 0.0001; Fig 1 , top). Patients treated with ICS had similar values than those not receiving ICS: 1.15 µM (95% CI, 0.81 to 1.5 µM) vs 1.02 µM (95% CI, 0.73 to 1.3 µM), respectively (p = 0.55; Fig 1 , top). Patients with P aeruginosa colonization had significantly higher levels of H2O2 compared to patients without P aeruginosa colonization: 1.6 µM (95% CI, 1.25 to 1.92 µM) vs 0.8 µM (95% CI, 0.64 to 1.01 µM), respectively (p < 0.05; Fig 1 , top). H2O2 levels were significantly increased in patients receiving long-term oral antibiotic treatment compared to those not receiving any antibiotics: 1.3 µM (95% CI, 0.95 to 1.7 µM) vs 0.9 µM (95% CI, 0.67 to 1.16 µM), respectively (p < 0.05). The total cell count was significantly higher in patients with bronchiectasis compared to normal subjects: 3.7 x 106/mL (95% CI, 1.6 to 3.9 x 106/mL) vs 2.1 x 106/mL (95% CI, 1.4 to 2.3 x 106/mL) respectively (p < 0.001). The percentage of neutrophils obtained from induced sputum was significantly higher in patients with bronchiectasis than in normal subjects: 59% (95% CI, 54 to 63%) vs 27% (95% CI, 24 to 36%), respectively (p < 0.0001; Fig 1 , bottom). Sputum macrophage levels were significantly lower in patients than in normal subjects: 40% (95% CI, 32 to 53%) vs 72% (95% CI, 65 to 84%), respectively (p < 0.0001; Fig 1 , bottom).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concentration of H2O2 measured in exhaled-breath condensate may be considered as the net result of production vs scavenging. Compared with the cellular antioxidant scavenging systems, the extracellular space and airway have significantly less ability to scavenge reactive oxygen species.17 Thus, the airway inflammation that occurs in bronchiectasis and other acute or chronic inflammatory processes involving the respiratory tract may be particularly likely to induce increased oxidant production that is detectable by increased H2O2 in expired breath. Activation of inflammatory cells, including neutrophils, eosinophils, and macrophages, induces a respiratory burst, resulting in marked production of reactive oxygen species, including H2O2.18 However, the precise role of ROIs in the pathophysiology of inflammatory process remains unclear due to the difficulty in identifying which cells produce ROIs in the inflamed lung and bronchi in each disease, as well as to specify the activated state of the above-mentioned cells. Therefore, the major problem is to determine a strong relationship between oxidant production and the development of an inflammatory reaction. Airway assessments performed by bronchoscopy biopsy and lung lavage have suggested that inflammation contributes to the development of bronchiectasis.2 Most observations dealing with inflammatory cells in bronchiectasis have focused on neutrophils that undoubtedly impact on the inflammatory process and may alter the oxidant-antioxidant balance.19 9 Similar results were observed in our study using a noninvasive method, such as sputum induction. The high percentage of neutrophils observed in this study, the strong correlation with H2O2 concentration, and the lack of correlation between macrophages and H2O2 concentration lead to the plausible explanation that in patients with bronchiectasis, neutrophils express the main source of H2O2 production, while macrophages seem not to be the cells that generate H2O2. These findings are partially supported by previous observations,1 9 where accumulation of neutrophils in bronchiectatic airways is associated with airways damage. Polymorphonuclear leukocytes release several molecules that mediate this damage, particularly proteases and oxidants.9 20 This means that neutrophilic inflammation in the bronchiectatic airways is an endogenous source of oxidants leading to proteolytic and cellular damage and thereby contributes to oxidant stress.
In patients with bronchiectasis, a correlation between the extent of the disease and the degree of lung function impairment has already been shown.21 Furthermore, the degree of lung function impairment and the number of neutrophils recovered from lung lavage appeared to be strongly correlated in most patients.19 These earlier observations lead to the plausible explanation that lung function impairment expresses both the degree of neutrophilic inflammation in the airway lumen and the extent of the disease. These data are partially confirmed by our study, which also showed a strong positive correlation between neutrophil differential counts in induced sputum and disease severity. The last observation is the first, to our knowledge, to give evidence that the inflammatory process in the airway of patients with bronchiectasis is strongly associated with both the severity and extent of the disease. Taking all these points into consideration and based on previous observations where early knowledge of these parameters could be beneficial for the monitoring and treatment of the disease,3 22 H2O2 concentration seems to be an appropriate way to detect and follow up the course of these features. However, our study was designed as cross-sectional and could not strongly support the above-mentioned statements, which would require prospective controlled longitudinal data.
The present cross-sectional study cannot demonstrate a causal relationship between ICS usage, long-term oral antibiotic treatment, and H2O2 concentration. In fact, patients receiving ICS had similar values with steroid-naïve patients, while patients receiving long-term oral antibiotics consistently showed the highest values. Prospective, controlled studies are needed for that purpose. However, our findings are similar to those previously reported, where steroids have failed to alter H2O2 production from polymorphonuclear leukocytes,23 and antibiotics reduced effectively the amount of inflammation during an exacerbation but they are inadequate in dealing with persistent airway inflammation.24 The finding that patients receiving long-term oral antibiotic treatment had the highest H2O2 values may be due to the fact that patients with the most severe infections were the only patients receiving antibiotic therapy. The results of our study and the findings of others4 25 suggest that P aeruginosa colonization in patients with bronchiectasis is associated with more extensive disease, more severe disease, and greater lung function impairment. However, it remains unclear whether P aeruginosa is the cause or the result of advanced disease. We additionally showed that chronic colonization with P aeruginosa may have a causal role in the oxidant-mediated lung injury. This is partially supported by previous observations where bacterial products released from P aeruginosa impair ciliary activity and lead to persistent inflammation by a pathway that involves neutrophils and is mediated by ROIs, which are not inhibited by antioxidants.26
In summary, exhaled H2O2, an index of oxidative stress, may be an indirect index of neutrophilic inflammation, lung function impairment, and severity and extension of disease. As collection of breath condensate is simple and noninvasive, it may be useful in patients with severe disease who may not tolerate more invasive investigations. If certain oxygen species contribute to airway inflammation in patients with bronchiectasis, then oxygen radical scavengers or antioxidants could play a critical role in treating the disease. However, no justification exists for this treatment, and further studies are needed to confirm this hypothesis.
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Footnotes |
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Received for publication October 25, 2000. Accepted for publication June 12, 2001.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-5 in induced sputum. J Allergy Clin Immunol 99,693-698[CrossRef][ISI][Medline]
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