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(Chest. 2003;124:25-31.)
© 2003 American College of Chest Physicians

Increased Exhaled 8-Isoprostane in Childhood Asthma*

Eugenio Baraldi, MD; Laura Ghiro, MD; Vania Piovan, MD; Silvia Carraro, MD; Giovanni Ciabattoni, MD; Peter J. Barnes, DM and Paolo Montuschi, MD

* From the Department of Pediatrics (Drs. Baraldi, Ghiro, Piovan, and Carraro), School of Medicine, University of Padova, Padova, Italy; Department of Drug Sciences (Dr. Ciabattoni), School of Pharmacy, University "G. d’Annunzio", Chieta, Italy; Department of Thoracic Medicine (Dr. Barnes), Imperial College, School of Medicine, National Heart and Lung Institute, London, UK; and Department of Pharmacology (Dr. Montuschi), School of Medicine, Catholic University of the Sacred Heart, Rome, Italy.

Correspondence to: Paolo Montuschi, MD, Department of Pharmacology, School of Medicine, Catholic University of the Sacred Heart, Largo F. Vito, 1, 00168 Rome, Italy;


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: To quantify lung oxidative stress in asthmatic children by measuring concentrations of 8-isoprostane, a marker of oxidative stress, in exhaled breath condensate (EBC), which is a noninvasive method of sampling airway secretions. Secondary objectives were as follows: (1) to measure levels of exhaled prostaglandin (PG) E2, since impaired PGE2 production has been implicated in the pathogenesis of asthma in adults; and (2) to measure levels of fractional exhaled nitric oxide (FeNO), which is a marker of airway inflammation.

Design: Single-center, cross-sectional study.

Patients: Twelve healthy children, 12 steroid-naïve asthmatic children, and 30 children in stable condition with mild-to-moderate persistent asthma who were being treated with inhaled corticosteroids (ICSs) [average dose, 300 µg per day] were studied.

Interventions: Subjects attended the outpatient clinic on one occasion for the collection of EBC and FeNO measurements.

Measurements and results: 8-Isoprostane and PGE2 concentrations in EBC were measured with specific radioimmunoassays. FeNO was measured online by a chemiluminescence analyzer. 8-Isoprostane was detectable in the EBC of healthy children (mean [± SEM], 34.2 ± 4.5 pg/mL), and its concentrations were increased in both steroid-naïve asthmatic children (mean, 56.4 ± 7.7 pg/mL; p < 0.01) and steroid-treated asthmatic children (mean, 47.2 ± 2.3 pg/mL; p < 0.05). There was no difference in exhaled 8-isoprostane concentrations between the two groups of asthmatic children (p = 0.14). By contrast, exhaled PGE2 concentrations were similar among the three study groups (p = 0.56). FeNO levels were higher in steroid-naïve children with asthma (49.2 ± 9.6 parts per billion [ppb]; p < 0.05) and, to a lesser extent, in steroid-treated asthmatic children (37.8 ± 6.6 ppb; p < 0.05) compared with healthy children (15.2 ± 1.7 ppb).

Conclusions: Lung oxidative stress is increased in children who are in stable condition with asthma, as reflected by increased exhaled 8-isoprostane concentrations. This increase seems to be relatively resistant to treatment with ICSs. Decreased PGE2 lung production is unlikely to play a pathophysiologic role in childhood asthma.

Key Words: airway inflammation • asthma • 8-isoprostane • exhaled breath condensate • nitric oxide • noninvasive markers • oxidative stress • prostaglandin E2


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxidative stress, a major component of inflammation, plays an important role in the pathophysiology of asthma in adults,1 as indicated by elevated levels of exhaled carbon monoxide, a marker of oxidant stress, and ethane, a marker of lipid peroxidation, in patients with asthma.2 3 Exhaled breath condensate (EBC), the liquid phase of exhaled breath, contains aerosol particles in which nonvolatile substances have been identified.4 5 Markers of free radical production, such as hydrogen peroxide,6 S-nitrosothiols,7 and 3-nitrotyrosine,8 have been detected in the EBC of healthy adults, and its levels have been found to be increased in adults with asthma. EBC pH is over two log orders lower than normal in patients with acute asthma, normalizes with corticosteroid therapy,9 and is probably regulated by glutaminase activity.10 The analysis of EBC is a novel, noninvasive method with which to monitor lung inflammation, which is likely to reflect the local production of free radicals rather than their systemic production.11 In fact, using freely diffusible molecules (eg, urea or electrolytes) as markers, it has been shown that a measurable fraction of the EBC in healthy subjects is derived from aerosolized airway lining fluid.12 13 We have previously shown that isoprostanes, prostaglandin (PG) analogs that are formed by the peroxidation of arachidonic acid due to reactive oxygen species (ROS),14 are elevated in the EBC of adults with asthma.15 Measurements of these compounds in EBC have several advantages over other markers of oxidative stress14 since isoprostanes are chemically stable, are formed in vivo, are specific for lipid peroxidation, and are used to define the clinical pharmacology of antioxidants.14 Moreover, 8-isoprostane exerts potent biological activity such as the contraction of human bronchial smooth muscle in vitro16 that may be relevant to the pathophysiology of asthma. Most of the studies demonstrating a pathogenetic role of increased ROS production in asthma have been performed in adults, but less is known about the role of oxidative stress in childhood asthma. In this disease, elevated levels of exhaled carbon monoxide and hydrogen peroxide have been reported,17 18 but the latter is less stable than other markers of oxidant stress such as isoprostanes.

PGE2 has bronchoprotective and inhibitory effects on inflammatory cells at the concentrations known to occur in the airways, and the impaired production of PGE2 has been proposed to contribute to the pathogenesis of asthma.19 The findings of a recent study20 do not support this hypothesis, since the PGE2 concentrations in the EBC of adults with asthma were found to be similar to those measured in healthy subjects. The aim of this study was to quantify lung oxidative stress in children with asthma who were steroid-naïve or had been treated with inhaled corticosteroids (ICSs) by measuring 8-isoprostane concentrations in EBC. A secondary objective was to measure PGE2 concentrations in EBC to ascertain whether a decreased production of this eicosanoid could contribute to the pathophysiology of childhood asthma. As part of the assessment of airway inflammation and oxidative stress, we also measured the levels of fractional exhaled nitric oxide (FeNO) in healthy and asthmatic children.21


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Subjects
The following three groups of white subjects were studied: 12 healthy children; 12 steroid-naïve asthmatic children; and 30 children in stable condition with mild-to-moderate persistent asthma who were treated with ICSs (Table 1 ). The asthmatic children (age range, 5 to 16 years) and healthy children (age range, 6 to 13 years) were matched for age.


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  		Table 1.. Subject Characteristics*

 
The diagnosis and classification of asthma was based on clinical history, physical examination, and pulmonary function parameters, which were measured according to international guidelines.22 Children with asthma who were treated with steroids received maintenance therapy with low to medium doses of ICSs (budesonide fluticasone propionate) at a constant dose for at least 2 months. The overall average daily dose of ICS was 300 µg/d. Thirteen steroid-treated asthmatic children also received treatment with long-acting ß2-agonists (salmeterol, 100 µg/d), whereas steroid-naïve asthmatic children received no drugs. Atopy was assessed by means of skin-prick tests for common allergens (eg, house dust mite, grass pollen, Parietaria, Artemisia, Alternaria, and cat). Patients had experienced no upper respiratory tract infections in the previous 3 weeks. Patients were excluded from the study if they had used oral steroids in the last 4 weeks or nonsteroidal anti-inflammatory drugs in the last 2 weeks.

Twelve healthy children without a history of asthma and atopy were recruited. They had negative results of skin-prick tests, normal pulmonary function parameters, and no history of respiratory infections in the previous 4 weeks.

Study Design
The type of study was cross-sectional. Children attended the Pulmonology/Allergy Outpatient Clinic of the Department of Pediatrics of the University of Padova on one occasion for clinical examination, FeNO measurement, EBC collection, and spirometry. Informed consent was obtained from parents, and the study was approved by the Ethics Committee of the University of Padova.

Pulmonary Function
FEV1, FVC, and midexpiratory phase of forced expiratory flow (FEF25–75) were measured by means of a 10-L bell spirometer (Biomedin; Padova, Italy), and the best value of three maneuvers, expressed as the percentage of predicted values, was chosen.

EBC Collection
EBC samples were collected in a specially designed condenser consisting of a glass chamber cooled by ice that was suspended in a larger glass chamber, as described previously.15 EBC was collected between the two glass surfaces. Children were instructed to breath tidally through a mouthpiece connected to the condenser for 15 min without noseclip. To minimize salivary contamination, the two-way valve served as a saliva trap, with a 12-cm banded tube vertically positioned between the mouthpiece and the condensing device while the mouth of the subject remained at a lower position with respect to the inlet of the device. In addition, children were asked periodically to swallow their saliva. Approximately 1 to 1.2 mL EBC was collected and stored at -70°C in a 2-mL sterile plastic tube. Saliva contamination of EBC was excluded by measuring amylase concentrations, as previously described.23

Measurement of Exhaled 8-Isoprostane and PGE2
8-Isoprostane and PGE2 concentrations in EBC were measured by specific radioimmunoassays (RIAs) that were developed in our laboratory. The specificity of eicosanoid RIAs was confirmed by the following two different criteria: (1) qualitative reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of the EBC 8-isoprostane-like immunoreactivity (LI) and PGE2-LI; and (2) simultaneous measurement of a set of EBC samples with two antisera with different cross-reactivities.

EBC samples were obtained from 16 subjects. Samples were pooled together (23 mL), extracted (Sep-Pak C18 cartridges; Waters Associates; Milford, MA), and vacuum-dried, as described previously.24 25 Recoveries for 8-isoprostane and PGE2 were evaluated by adding 220,000 disintegrations per minute [3H]-8-isoprostane or 140,000 disintegrations per minute [3H]PGE2 to an equal volume of distilled water that was subjected to the same process of extraction and RP-HPLC purification as the EBC sample pool. The individual fractions eluted by RP-HPLC were assayed for radioactivity. Retention times for 8-isoprostane and PGE2 standards were 13 min and 21 min, respectively. The extracted EBC pool was recovered with 100 µL methanol and was subjected to RP-HPLC (C18, 125 x 4.6 mm, 5 µm; LiChrospher column; Merck; Darmstadt, Germany) with the solvent system acetonitrile-water-acetic acid (27:73:0.18 [vol/vol/vol]). One-minute samples were collected for 30 min at a flow rate of 1 mL/min for RIA analysis of eicosanoid immunoreactivity. All RP-HPLC purifications were carried out isocratically. Each RP-HPLC fraction was vacuum-dried and recovered with 1 mL buffer. Aliquots of the fractions eluted then were analyzed for 8-isoprostane-LI and PGE2-LI by RIAs24 26 or for radioactivity. We used specific antisera anti-8-isoprostane (Rab 1)24 25 and anti-PGE2 (GP 356).26 The detection limit for both the 8-isoprostane and PGE2 RIAs was 10 pg/mL. The intraassay (n = 6) and interassay (n = 8) coefficients of variation for 8-isoprostane were ± 2.0% and ± 2.9%, respectively, at the lowest concentration of the standard (2 pg/mL) and ± 3.7% and ± 10.8%, respectively, at the highest concentration of the standard (250 pg/mL). The intraassay (n = 8) and interassay (n = 4) coefficients of variation for PGE2 were < 4% and < 5%, respectively, across the range of values measured (10 to 250 pg/mL).

RP-HPLC separation and RIA analysis of the eluted fractions showed a single peak of 8-isoprostane-LI and PGE2-LI that coeluted with authentic standards of 8-isoprostane and PGE2, respectively, indicating that the unknown 8-isoprostane-LI and PGE2-LI in EBC have identical chromatographic behavior with the authentic respective standards. The recoveries for 8-isoprostane and PGE2 were 66.1% and 65.5%, respectively.

To confirm the specificity of 8-isoprostane and PGE2 RIA measurements, 10 unextracted EBC samples were tested simultaneously with two different anti-8-isoprostane sera and two different anti-PGE2 sera, since every antiserum has a unique immunologic profile of cross-reactivity. For this purpose, a second anti-8-isoprostane serum (L9)24 25 and a second anti-PGE2 serum (FG-2) were used. Similar concentrations for both 8-isoprostane (limits of agreement, -4.8 and 3.6 pg/mL; SEM, 1.15) and PGE2 (limits of agreement, -4.2 and 6.5 pg/mL; SEM, 1.46) were obtained (data not shown). Taken together, these data strongly suggest that immunoreactive material measured in EBC is represented by authentic 8-isoprostane and PGE2.

FeNO Measurement
FeNO was measured (NIOX system; Aerocrine; Stockholm, Sweden) using a single-breath on-line method according to American Thoracic Society guidelines.27 Children inhaled nitric oxide (NO)-free air and exhaled through a dynamic flow restrictor. Subjects were asked to exhale and inhale to total lung capacity through an NO filter (Gasmask breathing filter according to EN141; Dräger; Lübeck, Germany) and then to exhale into the device through the same mouthpiece. A negative pressure tracing on the screen was used to confirm that subjects were inhaling NO-free air from the system. A visual feedback on a computer screen was used to maintain a constant flow of 50 mL/s for at least 7 s.28

Statistical Analysis
One-way analysis of variance with the Newman-Keuls test for multiple comparisons was used to compare groups. Linear regression analysis was used to assess the relationship among the exhaled markers (ie, 8-isoprostane, PGE2, and FeNO) and between the exhaled markers and lung function tests. Relationships of 8-isoprostane and PGE2 concentrations in EBC measured with different antisera were assessed by limits of agreement according to Bland and Altman analysis.29 The precision of the estimated limits of agreement was expressed as the SEM. All data were expressed as the mean ± SEM, and significance was defined as a value of p < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
8-Isoprostane concentrations were detectable in EBC in healthy children (34.2 ± 4.5 pg/mL; 95% confidence interval [CI], 24.3 to 44.0) and were increased in both steroid-naïve children with asthma (56.4 ± 7.7 pg/mL; p < 0.01; 95% CI, 39.4 to 73.4) and steroid-treated children with asthma (47.2 ± 2.3 pg/mL; p < 0.05; 95% CI, 42.4 to 52.0) [Fig 1 , top]. There was no difference in exhaled 8-isoprostane concentrations between the two groups of asthmatic children (p = 0.14). In contrast, exhaled PGE2 concentrations were similar (p = 0.052) in healthy children (46.8 ± 5.3 pg/mL, 95% CI, 35.1 to 58.5), in steroid-naïve children with asthma, (44.6 ± 5.0 pg/mL; 95% CI, 35.5 to 55.7), and steroid-treated children with asthma (51.5 ± 4.0 pg/mL; 95% CI, 43.4 to 59.6) [Fig 1 , bottom]. FeNO levels were higher in steroid-naïve children with asthma (mean, 49.2 ± 9.6 parts per billion [ppb]; p < 0.05; 95% CI, 27.6 to 70.8.) and, to a lesser extent, in asthmatic children who were treated with ICSs (37.8 ± 6.6 ppb; p < 0.05; 95% CI, 24.3 to 51.4) compared with healthy children (15.2 ± 1.7 ppb; 95% CI, 11.4 to 18.9) [Fig 2 ]. There was no correlation between the level of exhaled eicosanoids or FeNO and age, sex, and lung function in any study group. There was no correlation between the different exhaled markers (ie, 8-isoprostane, PGE2, and FeNO) in any of the study groups. There was no correlation between exhaled 8-isoprostane concentrations and ICS dose in those children who were treated with these drugs.



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  		Figure 1.. Top: 8-isoprostane levels in EBC of healthy children ({square}), steroid-naïve asthmatic children ({blacksquare}), and children with asthma who were treated with ICSs ({blacktriangleup}). Bottom: PGE2 levels in EBC in healthy children ({square}), steroid-naïve asthmatic children ({blacksquare}), and children with asthma who had been treated with ICSs ({blacktriangleup}). The mean values are shown by horizontal bars.

 


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  		Figure 2.. FeNO levels in healthy children ({square}), steroid-naïve asthmatic children ({blacksquare}), and children with asthma who were treated with ICSs ({blacktriangleup}). In one child, FeNO was not measured. Mean values are shown by horizontal bars.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, attention has focused on analyzing EBC for studying the composition of the fluid that lines the respiratory tract.4 As the measurement of EBC is completely noninvasive, this method is potentially useful for the long-term monitoring of lung inflammation in patients with inflammatory airway diseases, including children.30

In this study, we have shown that 8-isoprostane, a marker of free radical-induced lipid peroxidation, is detectable in the EBC of healthy children, indicating that ROS production occurs under physiologic conditions even when there is no evidence of airflow obstruction. Children with asthma who were steroid-naïve and children who had been treated with ICSs had higher exhaled 8-isoprostane concentrations than did healthy children, indicating that lipid peroxidation, which is an important component of oxidant stress, is increased in the lungs of children with asthma. Similar results were reported previously in adults with mild-to-moderate asthma who had increased lung oxidative stress, as indicated by 8-isoprostane levels in EBC.15 The concentrations of exhaled 8-isoprostane that we measured in healthy and asthmatic children were higher than those reported in their respective adult counterparts.15 This discrepancy may be explained partly by the different analytical methods used to measure 8-isoprostane levels in EBC in the two studies (ie, RIA vs enzyme immunoassay).15 There was a certain degree of overlapping in 8-isoprostane concentrations between healthy children and children with asthma. This could reflect a different relevance of lipid peroxidation and oxidative stress to the airway inflammatory process in the single child with asthma. For this reason, the measurement of 8-isoprostane in EBC should be considered as part of an integrated assessment of airway inflammation in the single child with asthma. However, larger and longitudinal studies are required to establish the clinical utility of exhaled 8-isoprostane measurement in the diagnosis and management of children with asthma. Similar to asthmatic adults,15 there was no correlation between 8-isoprostane concentrations in EBC and the results of lung function tests in asthmatic children. These findings may be explained by interindividual differences in susceptibility to the same levels of oxidative stress and/or mechanisms different from oxidant stress contributing to lung inflammation. Moreover, the relationship between 8-isoprostane and lung function in children with asthma who were treated with ICSs could have been affected by these drugs. ICS treatment was effective in controlling bronchial obstruction (mean FEV1, 89.9 ± 2.6% predicted) but not lung oxidative stress, as indicated by elevated levels of 8-isoprostane in the EBC of these patients.

FeNO, a marker of airway inflammation and oxidant stress, was elevated in steroid-naïve asthmatic children and, to a lesser extent, in children with asthma who were treated with low-to-medium doses of ICSs compared to healthy children. These results provide further evidence for a role of ROS in childhood asthma. Considering the wide variety of biological actions that isoprostanes exert within the lungs, including contraction of human bronchial smooth muscle in vitro,16 isoprostanes may be considered not just markers of oxidative stress but also mediators of lung injury.31 However, the role of isoprostanes in the pathophysiology of asthma, if any, is currently unknown.

PGE2 was detectable in EBC in all the children studied, and there was no difference in its concentrations among the three study groups. In view of its bronchodilator and anti-inflammatory effects in the lung, a decreased production of PGE2 has been proposed to contribute to the pathophysiology of asthma.19 Our data do not support this hypothesis and indicate similar PGE2 levels in the EBC of children with asthma, as we have previously demonstrated in adults with asthma.20

8-Isoprostane seems to be relatively resistant to ICS therapy, as indicated by the lack of difference in exhaled 8-isoprostane concentrations between steroid-naïve asthmatic children and children with asthma who were treated with these drugs. This hypothesis is supported further by a study32 that showed no effect of pretreatment with high-dose inhaled budesonide (1,600 µg/d for 14 days) on exhaled 8-isoprostane increase following short-term ozone exposure (ie, 400 ppb for 2 h) in healthy adults. However, controlled studies are required to establish the effects of ICS therapy on 8-isoprostane levels in the EBC of children with asthma.

EBC analysis cannot provide any information on the cellular origin of mediators. For this reason, we were unable to ascertain the cellular source of 8-isoprostane and PGE2 in EBC for which invasive studies, such as bronchial biopsies, are required. In view of the fact that 8-isoprostane is produced by free radical-induced peroxidation of arachidonic acid on plasma membrane phospholipids,14 all cells in the airways may release this eicosanoid. PGE2 is largely produced by epithelial and airway smooth muscle cells.33

In conclusion, we have shown that oxidative stress is increased in children who are in stable condition with mild-to-moderate persistent asthma who were steroid-naïve or had been treated with ICSs, as reflected by increased 8-isoprostane concentrations in EBC. These findings may justify clinical trials with antioxidants in childhood asthma for which the measurement of 8-isoprostane in EBC may provide a useful noninvasive biological marker.


   Footnotes
 
Abbreviations: CI = confidence interval; EBC = exhaled breath condensate; FEF25–75 = midexpiratory phase of forced expiratory flow; FeNO = fractional exhaled nitric oxide; ICS = inhaled corticosteroid; LI = like immunoreactivity; NO = nitric oxide; PG = prostaglandin; ppb = parts per billion; RIA = radioimmunoassay; ROS = reactive oxygen species; RP-HPLC = reverse-phase high-performance liquid chromatography

This work was supported by the Catholic University of the Sacred Heart, Rome, Italy, and by the University of Padova, Padova, Italy.

Received for publication June 26, 2002. Accepted for publication January 22, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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 Am. J. Respir. Crit. Care Med.Home page
R. M. Effros, J. Biller, B. Foss, K. Hoagland, M. B. Dunning, D. Castillo, M. Bosbous, F. Sun, and R. Shaker
A Simple Method for Estimating Respiratory Solute Dilution in Exhaled Breath Condensates
Am. J. Respir. Crit. Care Med., December 15, 2003; 168(12): 1500 - 1505.
[Abstract] [Full Text] [PDF]

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