American Journal of Respiratory and Critical Care Medicine

Oxidative stress is implicated in the pathogenesis of asthma, and clinical studies show an imbalance in the level of oxidants to the level of antioxidants in subjects with asthma. Aldehydes and glutathione are examples of biomarkers of oxidant-induced damage and antioxidant status in asthma, respectively. In the study, we applied analytical techniques based on liquid chromatography for the assessment of aldehydes and glutathione in the exhaled breath condensate of children with asthma and in control subjects without asthma. Twelve subjects with asthma were evaluated at exacerbation and after 5 days of therapy with prednisone. At exacerbation, malondialdehyde levels were higher in patients with asthma (30.2 ± 2.4 nM) than in control subjects (19.4 ± 1.9 nM, p = 0.002) and were reduced after steroid therapy (18.5 ± 1.6 nM, p = 0.001). At exacerbation, glutathione levels were lower in subjects with asthma (5.96 ± 0.6 nM) than in control subjects (14.1 ± 0.8 nM, p < 0.0001) and were increased after the therapy (8.44 ± 1.2 nM, p = 0.04). Malondialdehyde and glutathione both in subjects with asthma and control subjects were negatively correlated (r = −0.5, p = 0.001). The study shows that aldehydes and glutathione are detectable in the exhaled breath condensate of children with asthma and healthy children and that their levels are modified during asthma exacerbation and after a 5-day course of therapy with oral prednisone.

Oxidative stress occurs when the production of oxidants exceeds the capacity of the body's antioxidant defenses to detoxify them (1). An imbalance between oxidants and antioxidants, in favor of oxidants leading to oxidative stress, is known to play an important role in the pathogenesis of asthma, especially during exacerbation (2).

There are few published data regarding the evaluation of airway oxidative stress in children with asthma (35), mainly because of the difficulty in performing traditional biologic airway assessments, such as bronchoalveolar lavage and sputum induction, in children.

Exhaled breath condensate (EBC), obtained by cooling exhaled air during spontaneous breathing, is a biologic medium that could be useful for the assessment of airway oxidative stress in vivo (6). EBC is a water solution that is suitable for analytical measurements at the trace level.

EBC collection is totally noninvasive and therefore particularly easy to perform in children. In children with asthma, an increase in EBC levels of oxidants (hydrogen peroxide) (7) and of nitric oxide–related metabolites (nitrite) (8) has been reported. Very recently, Shahid and colleagues showed that interleukin-4 is higher and interferon-γ is lower in the EBC of children with asthma compared with control subjects (9).

However, despite the enthusiasm of a few research groups, much of the skepticism about the validity of EBC as a tool for the assessment of airway oxidative stress derives from analytical problems associated with measurements of trace amounts of biomarkers. These rely on immunochemical or colorimetric assays that lack reference methods and materials, which are affected by poor sensitivity, specificity, and selectivity.

EBC is an aqueous matrix that is ideal for analyses based on liquid chromatography. The high degree of dilution of analytes in EBC requires the use of the most sensitive detection systems available today, namely fluorescence detection and mass spectrometry–related techniques. In the online coupling with liquid chromatography, the latter offers advantages both in terms of identification power of unknown substances and in terms of reliability and accuracy of quantitative results. These techniques are considered the reference techniques for the determination of complex mixtures of organic compounds present at nanomole levels in aqueous matrices.

The aim of the study was to apply reference analytical techniques for the evaluation of selected biomarkers of oxidative stress, namely lipid peroxides (measured as aldehydes) and glutathione, in the EBC of children with asthma, both during exacerbation and after a 5-day course of therapy with oral prednisone.

Design of the Study

The study was performed in children with asthma who were referred to the Department of Pediatrics of Padova for asthma exacerbation. At admission, a pediatrician, who performed spirometry and EBC collection, examined the children. Oral prednisone treatment (1 mg/kg/day) was then started in all patients. After 5 days of therapy, the patients were examined again, and spirometry and EBC collection were repeated.

Population

The study included 12 nonsmoking children with asthma (8 males, age [mean ± SEM] 11.4 ± 0.9 years). The diagnosis of asthma was based on international guidelines (10). Eleven children were atopic, sensitized to common allergens. Ten children were on maintenance therapy at low to medium doses of inhaled corticosteroids (10) at a constant dose for at least 2 months. Asthma exacerbation was defined as an increase in asthma signs and symptoms (coughing, wheezing, shortness of breath) unresponsive to the patients' routine asthma medication and additional β2-agonist therapy.

Ten healthy nonsmoking children (5 males, age 10.1 ± 0.6 years) without a history of asthma formed the control group. They had normal pulmonary function parameters and no history of respiratory infections in the previous 4 weeks.

The Ethics Committee of Padova Hospital reviewed and approved the protocol, and all parents gave their informed consent.

EBC Collection and Assays

EBC samples were collected in a condensing device formed by two glass chambers, as described previously (11). For further details see online supplement.

Exhaled breath condensate collection at different exhaled flow rates.

In four healthy subjects, EBC was collected at four constant expiratory flows (200, 150, 100, and 50 ml · s−1). Different expiratory resistances were created using a restrictor set (HTF 5019X; Sievers Instruments Inc., Boulder, CO). An ultrasonic flow sensor (N.D.D. Medizintechnik AG, Zürich, Switzerland) was inserted between the mouth of the subject and the condenser. The flow meter was connected to a computer display provided with a special software (Ecomedics, Duernten, Switzerland) for the visual control of the expiratory flow. The ultrasonic flow meter is particularly suited for this purpose, as the exhaled air is not trapped or filtered.

Exhaled breath condensate lipid peroxides.

Lipid peroxides were evaluated as aldehydic products, namely malondialdehyde, α,β-unsaturated aldehydes (4-hydroxyhexenal and 4-hydroxynonenal), and saturated aldehydes (hexanal, heptanal, and nonanal). Aldehydes were determined, after derivatization with 2,4-dinitrophenylhydrazine, by liquid chromatography–tandem mass spectrometry (API 365; Perkin Elmer Sciex, Thornhill, Canada). Ionization of the analytes was obtained by atmospheric pressure chemical ionization in positive-ion mode for malondialdehyde, and in negative ion mode for 4-hydroxyhexenal, 4-hydroxynonenal and saturated aldehydes. Dinitrophenylhydrazone derivatives were separated on a Supelcosil C18 DB column, 75 × 4.6 mm inside diameter, 3 μm (Supelco, Milan, Italy) using variable proportions of 20 mM of aqueous acetic acid and methanol.

Exhaled breath condensate glutathione.

Reduced glutathione was measured by high-performance liquid chromatography with fluorescence detection using a recent method, validated for the evaluation of glutathione in biologic samples at the femtomole level (12). The method was adapted to glutathione analysis in EBC. Briefly, 100 μl of EBC sample was derivatized with 100 μl of an ortho-phthalaldeyde solution and 800 μl of a 500 mM of sodium phosphate (pH 7.00). As it has been reported that the glutathione-ortho-pthalaldeyde derivatives are stable for no longer than 24 hours when stored at 4°C in darkness (12), EBC samples were derivatized before the assay. In addition, we did not find any differences in EBC glutathione levels between samples derivatized and assayed immediately after the collection compared with samples stored at −80°C and derivatized before the assay (data not shown). Chromatography of the glutathione-ortho-phthalaldeyde adduct was achieved with an isocratic elution on a Superchrom LC18 column, 250 × 4.6 mm inside diameter, 5 μm (Varian, Milan, Italy), kept at 30°C, with a mobile phase composed of 50 mM of sodium acetate buffer (pH 6.20)/acetonitrile (88/12). Fluorimetric detection was performed at 420 nm after excitation at 340 nm.

Preliminarily, we verified that the levels of oxidized glutathione (GSSG) were undetectable in EBC samples.

Pulmonary Function Test

Pulmonary function parameters (FVC, FEV1, FEF25–75) were measured by means of a 10-L bell spirometer (Biomedin, Padova, Italy).

Statistical Analysis

Statistical analysis was performed using Graph Pad Prism version 3.0 for Window NT (Graph Pad, San Diego, CA). Data were expressed as mean ± SEM. Comparisons between groups and correlation between variables were based on parametric tests (t test and Pearson, respectively). A p value of less than 0.05 indicated statistical significance.

Aldehyde, glutathione levels in EBC are shown in Table 1

TABLE 1. Comparison of aldehydes and glutathione in exhaled breath condensate from 12 subjects with asthma and 10 healthy control subjects




Subjects with Asthma
 (n = 12)


LOD*
Exacerbation
After Treatment
Control Subjects
 (n = 10)
Malondialdehyde1.0730.2 ± 2.4,18.5 ± 1.619.4 ± 1.9
Hexanal1.0740.5 ± 4.751.5 ± 4.336.1 ± 6.3
Heptanal0.3429.8 ± 3.123.7 ± 1.729.6 ± 5.2
Nonanal0.3126.9 ± 4.628.9 ± 3.048.1 ± 3.6
Glutathione
2.0
5.96 ± 0.6,
8.44 ± 1.2
14.1 ± 0.8

*Limit of detection (S/N = 3).

p < 0.05 versus after treatment.

p < 0.05 versus control subjects.

Definition of abbreviation: LOD = limit of detection.

Aldehyde and glutathione levels are expressed in nM. Data are shown as mean ± SEM. Subjects with asthma were evaluated at exacerbation and after 5 days of therapy with oral prednisone (1 mg/kg/day).

. At exacerbation, malondialdehyde was higher in subjects with asthma than in control subjects (p = 0.002) and was decreased after the therapy (p = 0.001) to values no longer different from those of control subjects (Figure 1) . α,β-Unsaturated aldehydes were detectable in only a few (in two subjects with asthma and two control subjects) EBC samples. Hexanal and heptanal levels in subjects with asthma did not differ from those of control subjects at both times and were unaffected by the therapy with prednisone. Nonanal levels in subjects with asthma, at both times, were lower than those in control subjects (p = 0.001).

Glutathione levels in subjects with asthma at exacerbation were lower than those of control subjects (p < 0.001) and remained lower also after 5 days of therapy with oral prednisone (p = 0.001); 5 days of therapy with oral prednisone slightly increased glutathione levels compared with pretreatment levels (p = 0.04) (Figure 2)

.

Malondialdehyde and glutathione levels were negatively correlated, considering either subjects with asthma (at both times) and control subjects together (r = −0.5, p = 0.001) (Figure 3)

or subjects with asthma only (r= −0.5, p = 0.01).

The concentrations of malondialdehyde and of glutathione (mean ± SEM) in EBC collected at flow rates of 200, 150, 100, and 50 ml · s−1 were as follows: for malondialdehyde, 18.4 ± 4.2, 18.6 ± 3.2, 16.4 ± 0.1, and 15.8 ± 1.6 nM, respectively; for glutathione, 10.2 ± 2.3, 6.6 ± 3.1, 7.2 ± 3.0, and 7.4 ± 2.9 nM, respectively. No differences were observed among malondialdehyde values and among glutathione levels obtained at the different flow rates (one-way analysis of variance, p = 0.5 and 0.1, respectively). No correlation was found between exhaled flow rates and malondialdehyde levels (p = 0.3) and glutathione levels (p = 0.9).

After the prednisone course, there was a significant improvement in spirometric parameters (FVC before 83.2 ± 5.3% of predicted versus after 97.2 ± 4.4% of predicted, p = 0.0001; FEV1 69.2 ± 5.2% of predicted versus 87.7 ± 4.1% of predicted, p = 0.0005; FEF25–75 52.2 ± 6.7% of predicted versus 77.1 ± 5.6% of predicted, p = 0.003). No correlation was found between spirometric values and EBC biomarker levels.

The study shows that aldehydes and glutathione are detectable in the EBC of children with asthma and in healthy children, and that their levels are modified during acute asthma attack and after a 5-day course of therapy with oral prednisone.

Among different aldehydes, malondialdehyde in EBC seems to mirror better the clinical status of children with asthma, as it is higher in subjects with asthma at acute phase than in control subjects and is decreased after 5 days of therapy with oral prednisone. The levels of EBC malondialdehyde that we observed in subjects with asthma were of the same order as those reported by Larstad and colleagues in EBC (13) and by Ozaras and colleagues in bronchoalveolar lavage (BAL) (14).

The increased amount of malondialdehyde that we observed in children with asthma during acute exacerbation could be reasonably explained by an enhanced oxidant formation during asthma exacerbation (2), therefore leading to an increased oxidative-induced damage. This accords with previous findings in blood reported by Shanmugasundaram and colleagues, who showed increased levels of blood malondialdehyde during severe episodes of wheeze in children suffering from asthma (15).

Malondialdehyde measurement is usually used to evaluate lipid peroxides in biological fluids, and it is most of the time quantified as thiobarbituric-reactive substances (16). However, the colorimetric thiobarbituric reactive substances assay has been criticized because of its lack of specificity and because thiobarbituric-reactive material usually forms during heating of the sample rather than being present from the outset (16). Very recently, malondialdehyde was measured in EBC from subjects with and without asthma by high-performance liquid chromatography (13), but no statistically significant difference in malondialdehyde levels between patients with and without asthma was found.

Besides malondialdehyde, which is generated mainly by arachidonic acid and docosahexenoic acid, other classes of aldehydes are produced during lipid peroxidation (17): α,β-unsaturated aldehydes, namely 4-hydroxynonenal and 4-hydroxyhexenal, are formed by peroxidation of ω-6 (arachidonic and linoleic acid) and ω-3 polyunsaturated fatty acids (oleic acid). Saturated aldehydes (hexanal, heptanal, and nonanal) are known to be break down products of oxidized linoleic acid and arachidonic acid, palmitoleic acid, and oleic (16).

α,β-Unsaturated aldehydes were detectable only in a few EBC samples. This could probably be explained by the fact that these aldehydes are highly reactive and, therefore, may be scavenged rapidly by thiol groups (16). In contrast, saturated aldehydes, which are chemically stable, were detectable in all EBC samples.

Hexanal and heptanal levels in subjects with asthma did not differ from those of control subjects, and nonanal levels were higher in control subjects than in subjects with asthma. We speculate that these different changes could be related to either different membrane cell lipid targets of the reactive oxygen specie attack or a different composition in membrane cell phospholipids occurring in lung disease (18). Further studies are warranted and are being performed in this regard.

Five days of therapy with oral prednisone reduced malondialdehyde levels in children with asthma compared with pretreatment levels, being then no longer different from those of control subjects. Similar findings, but in blood, were obtained by Shanmugasundaram and colleagues (15). α,β-Unsaturated and saturated aldehydes seem to be unaffected by oral steroid therapy in our patients.

EBC glutathione levels in children with asthma during acute asthma exacerbation were lower than those of control subjects. This could be related to a depletion of this antioxidant in response to an increased load of oxidants and is consistent with the study of Bibi and colleagues (19), which shows that children having acute asthma attacks exhibit lower levels of blood glutathione peroxidase compared with stable subjects with asthma and with control subjects. In addition, it has been reported (8) that during acute asthma the drop in EBC pH levels would favor the conversion ex vivo of glutathione to S-nitrosoglutathione through protonation of nitrite.

Glutathione levels in subjects with asthma rose after oral steroid treatment compared with pretreatment levels, and this could be explained by the fact that corticosteroids have been demonstrated to increase glutathione synthase in both animal models and in vitro systems (20, 21). In four children with asthma, glutathione levels did not increase after therapy with prednisone, although there were no differences in how these subjects did clinically. Further studies are needed to evaluate the effect of prednisone on EBC malondialdehyde and glutathione levels in the absence of an asthma exacerbation.

To our knowledge, this is the first report dealing with reduced glutathione in EBC, and therefore, the data cannot be compared with previous findings. The concentration of reduced glutathione that we observed (nM) is much lower than those (μM range) observed in BAL (22) and in sputum (23). This could probably be explained by the fact that glutathione, because of its high molecular weight, is poorly volatile. Therefore, it looks like there is a 1:1,000 dilution with the lavage fluid.

Oxidized glatathione was undetectable in EBC samples, and this was probably due to its EBC very low concentration. Data reported by other authors (12) showed that the oxidized fraction (oxidized glatathione, expressed as glutathione equivalents) in red blood cells and in cultured fibroblasts was 8.5 and 5%, respectively, being the corresponding glutathione concentrations in the order of mM and μM. These concentrations are considerably higher than those found in EBC, which are in the low nanomole range. Assuming that oxidized glatathione could represent no more than 10% of glutathione also in EBC, we expect oxidized glatathione concentrations at the pmol level, definitely too low and undetectable with the method we used.

Malondialdehyde and glutathione levels in EBC of both subjects with asthma and control subjects are negatively correlated. As it is known that glutathione and malondialdehyde chemically react together very slowly (16), it is plausible that the observed negative correlation levels could be explained by assuming that both malondialdehyde and α,β-unsaturated aldehydes are formed proportionally, but mainly α,β-unsaturated aldehydes have particular chemical properties to react with glutathione (forming glutathionyl adducts), leading to its depletion. Studies dealing with the formation of glutathionyl adducts are currently in progress.

Both malondialdehyde and glutathione concentrations were not exhaled flow rate–dependent, and no correlation was found between spirometric values and EBC biomarker levels.

We acknowledge that an independent method of determining the dilution of the EBC by water vapor would be necessary to calculate properly the biomarker concentrations in the airway fluid lining the respiratory tract based on those measured in EBC (24). However, the observed bidirectional changes in biomarker levels in asthma (increased malondialdehyde and decreased glutathione values) argue against EBC analysis simply representing relatively alterations in the number of aerosolized droplets recovered in the condensate from the airway lining fluid. If this were the explanation for increased concentrations of the solutes in EBC (as we have found for malondialdehyde), then the concentration of glutathione would also be expected to be increased.

We do believe that the biomarkers that we measured are not sufficiently volatile to reach the condensate as a gas. This is due to the fact that they have such particular physical (i.e., boiling point) and chemical (i.e., hydrophilic) properties, which leads us to believe that they remain in water solution in the fluid lining the airways, and therefore, they are poorly volatile as gas.

To summarize, the study shows that aldehydes and glutathione are measurable in EBC of children with asthma and are modified during acute asthma exacerbation and after a 5-day course of therapy with oral prednisone. The possibility of using reference analytical techniques for measuring biomarkers of oxidative stress in EBC could open the way for using these biomarkers to assess the oxidative stress status in clinical practice and to predict the usefulness of antioxidant drugs.

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Correspondence and requests for reprints should be addressed to Dr. Massimo Corradi, M.D., Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Via Gramsci 14, 43100 Parma, Italy. E-mail:

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