American Journal of Respiratory and Critical Care Medicine

Exhaled carbon monoxide (CO) concentrations were measured on a CO monitor by vital capacity maneuvers in asthmatic patients receiving or not receiving inhaled corticosteroids and in nonsmoking and smoking healthy control subjects. CO was detectable and measured reproducibly in the exhaled air of all subjects. The exhaled CO concentrations were higher in asthmatic patients not receiving inhaled corticosteroids (5.6 ± 0.6 ppm, p < 0.001) and similar in asthmatic patients receiving inhaled corticosteroids (1.7 ± 0.1 ppm) compared with those in nonsmoking healthy control subjects (1.5 ± 0.1 ppm). Smoking healthy control subjects had the highest levels of exhaled CO concentration among the groups (21.6 ± 2.8 ppm, p < 0.001). To examine whether inhaling corticosteroids reduce exhaled CO concentration in a given asthmatic patient, 12 patients with symptomatic asthma who were being treated by inhaled β2-agonists alone underwent measurements of exhaled CO concentration before and 4 wk after the initiation of inhaled corticosteroid treatment. All patients had reductions in exhaled CO concentration (p < 0.001) and eosinophil cell counts in sputum (p < 0.01) that were accompanied by an improvement in airway obstruction. Changes in exhaled CO concentration were significantly related to those in the eosinophil cell counts in sputum (p < 0.001). The present study shows an elevation of exhaled CO in asthmatic patients that decreases with corticosteroid therapy. Increases in the exhaled CO levels therefore may reflect inflammation in the asthmatic lung.

Carbon monoxide (CO), like nitric oxide (NO), has been reported to have biologic actions such as smooth muscle relaxation (1) or inhibition of platelet aggregation (2), and to act as a neural messenger in the brain (3, 4). CO is made in many tissues of the body by an enzyme called heme oxygenase, the only system that produces CO as a product of heme degradation (5). Two forms of heme oxygenase have been characterized (5). Heme oxygenase-1 is induced by heme and expressed at high concentrations in the spleen and liver, where it is responsible for the destruction of heme from red blood cells. Heme oxygenase-2 is not inducible and is widely distributed throughout the body, with high concentrations in the brain (5).

So far, measurements of exhaled CO in humans have been used as an indicator of smoking habit (6) and CO poisoning (7). However, heme oxygenase is present in the pulmonary vascular endothelium (8) and alveolar macrophages (9) and is upregulated by oxidative stress (8, 10), inflammatory cytokines (11, 12), and NO (13). These findings imply a role of endogenous CO in airway inflammatory diseases. We therefore examined whether asthmatic patients produce more CO than do healthy control subjects and if the levels of the exhaled CO concentration are reduced in asthmatic patients receiving regular inhaled corticosteroids, which control inflammation in the asthmatic airways (14).

Asthmatic patients and control subjects were recruited from volunteers and patients attending the Miyagi National Hospital. None of the 30 nonsmoking control subjects had a history of respiratory or cardiovascular disease or were receiving long-term medication. Asthma was defined as a clinical history of intermittent wheeze, cough, chest tightness, or dyspnea, and documented reversible airflow limitation either spontaneously or with treatment during the preceding year (15). All the asthmatic subjects were nonsmokers and their airway obstruction was stable for at least 2 wk before the study. One group received inhaled β2-agonists only and the others received regular inhaled corticosteroids (beclomethasone dipropionate 400 to 1,200 μg daily). Smokers were recruited from volunteers and were studied at least 1 h after the last cigarette. Physical characteristics, pulmonary function test results, and Brinkman's index (number of cigarettes/day × year) are shown in Table 1.


Subjects (n)Age (yr)SexFVC (% pred  )FEV1(% pred  )PaO2 (mm Hg)PaCO2 (mm Hg)Brinkman's Index
Controls3043 ± 311M/19F112 ± 3103 ± 386 ± 339 ± 10
 Untreated 3041 ± 315M/15F 98 ± 5 92 ± 578 ± 537 ± 20
 Treated 3041 ± 317M/13F101 ± 5 98 ± 580 ± 538 ± 30
Smokers2041 ± 316M/4F102 ± 4 93 ± 580 ± 341 ± 2623 ± 84

*Values are mean ± SEM.

No inhaled corticosteroids.

Regular inhaled corticosteroids.

In order to further investigate the effect of inhaled corticosteroids on exhaled CO concentration, 12 patients with symptomatic asthma, which was being treated by inhaled β2-agonists alone and which was considered severe enough to require prophylactic treatment for disease control, were followed before and 4 wk after the initiation of inhaled corticosteroid treatment (beclomethasone dipropionate 400 μg daily). Their baseline physical characteristics and pulmonary function test results are shown in Table 2. We also examined eosinophil cell counts in sputum. Before inhaled corticosteroid therapy, spontaneous sputum was collected in the morning. After corticosteroid therapy, sputum was induced by inhalation of hypertonic saline as previously described (16). Sputum plugs arising from the lower respiratory tract were selected and incubated with dithiothreitol 0.1% (Sigma Chemical, St. Louis, MO) at 37° C for 20 min and washed with phosphate-buffered saline. The cell suspension was centrifuged in a cytocentrifuge (Shandon Cytospin 2; Shandon, Oakland, CA), and slides were kept frozen at −20° C until analyzed. Two slides were fixed in acetone/methanol (1:1) and stained with May-Grünwald-Giemsa for differential cell counts of leukocytes and squamous epithelial cells. The slides were coded, and 400 cells were counted blind for differential leukocyte count. A sample was considered adequate when the percentage of squamous cells was lower than 20% (17). To correct for variable salivary contamination, the results of eosinophil counts were expressed as a percentage of nucleated cells excluding squamous cells (average counts of two slides for each case) (17). The study was approved by the Tohoku University Ethics Committee, and informed consent was obtained from each subject.


Subjects (n)Age (yr)SexFVC1(% pred  )FEV1(% pred  )PaO2 (mm Hg)PaCO2 (mm Hg)Brinkman's Index
Asthmatics1242 ± 45M/7F90 ± 567 ± 370 ± 439 ± 20

*Values are mean ± SEM,

Exhaled CO was measured on a portable Bedfont EC50 analyzer (Bedfont Technical Instruments Ltd., Sittingbourne, UK) using the method described by Jarvis and coworkers (6) in which subjects are asked to exhale fully, inhale deeply, and hold their breath for 20 s before exhaling rapidly into a disposable mouthpiece. This procedure was repeated three times, with 1 min of normal breathing between each repetition, and the mean value was used for analysis. Background CO values (0 to 1 ppm) were obtained prior to the subject readings. The subject readings were determined by subtracting the background level from the observed reading (6). Prior to the start of the study, the analyzer was calibrated with a mixture of 50 ppm CO in air (6). Expired CO concentration measured by the Bedfont EC50 analyzer is reported to correlate closely with blood carboxyhemoglobin concentration over the range of values encountered in smokers and in nonsmokers (18, 19).

Results are reported as mean ± SEM. Statistical analysis was performed by one-way analysis of variance and followed by the Newman-Keuls test. Significance was accepted at p < 0.05.

Exhaled CO was reproducible in all subjects and the subject readings on the EC50 analyzer were similar among three sequential maneuvers in nonsmoking control subjects (1.5 ± 0.1 ppm versus 1.5 ± 0.1 ppm versus 1.5 ± 0.1 ppm), asthmatic patients not receiving corticosteroids (5.7 ± 0.6 ppm versus 5.6 ± 0.6 ppm versus 5.5 ± 0.5 ppm), and asthmatic patients receiving inhaled corticosteroids (1.7 ± 0.1 ppm versus 1.7 ± 0.1 ppm versus 1.7 ± 0.1 ppm), respectively. Likewise, measurements in individual subjects were reproducible on separate days. In 30 normal subjects the variation between readings on separate days was small (5.1 ± 1.9%).

The mean exhaled CO concentration was 1.5 ± 0.1 ppm in nonsmoking control subjects. In asthmatic patients not receiving corticosteroids the exhaled CO concentration was significantly higher (5.6 ± 0.6 ppm, p < 0.001), whereas in asthmatic patients receiving inhaled corticosteroids the exhaled CO concentration did not differ significantly from that in nonsmoking control subjects (1.7 ± 0.1 ppm, p > 0.20) (Figure 1).

As expected, smoking control subjects had a higher CO concentration in exhaled air than did asthmatic patients not receiving corticosteroids (21.6 ± 2.8 ppm; range, 4.3 to 50.0 ppm; p < 0.01).

The exhaled CO concentration before and after the initiation of inhaled corticosteroid treatment is shown in Figure 2. Inhaled corticosteroids decreased the exhaled CO concentration from 8.4 ± 0.6 to 1.8 ± 0.3 ppm (p < 0.001) (Figure 2) and eosinophil cell counts in sputum from 35.0 ± 3.0 to 9.8 ± 1.2% (p < 0.01) in association with increases in FEV1 (percent predicted value) from 67 ± 3 to 92 ± 2% (p < 0.01). There was a significant relation between changes in the exhaled CO concentration and those in eosinophil cell counts in sputum (p < 0.001) (Figure 3). Likewise, changes in the exhaled CO concentration significantly correlated with those in FEV1 (r = 0.71, p < 0.01).

The present study has shown that exhaled CO can be reliably measured in healthy control subjects and asthmatic patients; the latter has an elevated exhaled CO concentration. The values of exhaled CO in nonsmoking and smoking control subjects were similar to those of previous studies (6, 18, 19). Furthermore, we found that improved lung function was accompanied by concomitant decreases in exhaled CO concentration and eosinophil cell counts in sputum in patients who needed inhaled corticosteroid therapy. Unlike that in smokers, exhaled CO seems to be derived from an endogenous source since none of the asthmatic patients were smokers, ex-smokers, or passive smokers, and background CO values were subtracted prior to studies. The source of CO within the lung is unknown, but heme oxygenase-1, the inducible form of heme oxygenase, is likely expressed in endothelial cells (8) and alveolar macrophages (9).

In the present study, eosinophil cell counts were performed in spontaneous sputum before corticosteroid treatment and in induced sputum thereafter. However, in inducing sputum, it has been reported that pretreatment with β2-agonists and hypertonic saline inhalation do not affect cell counts (20, 21). Furthermore, it is unlikely that the salivary contamination of sputum affected the results: first, because it was relatively low and the counts were corrected for the number of squamous cells; second, it has been proved that saliva does not contribute to the leukocytes in expectorated samples, as 99% of the nucleated cells in saliva are squamous cells (16). Finally, it has recently been reported that there are no significant differences in the differential leukocyte counts between selected plugs and residual portions of expectorate (22).

The present study has shown the first evidence that exhaled CO concentrations in asthmatic patients not receiving inhaled corticosteroids are significantly higher than those in nonsmoking healthy control subjects. High levels of exhaled CO concentration may reflect inflammation of the asthmatic lung. Many cytokines are involved in asthmatic inflammation, including interleukin-1, interleukin-6, and tumor necrosis factor, which can upregulate heme oxygenase-1 activity in animal and human tissues (11, 12). Furthermore, asthmatic airways produce high levels of NO (23-25) and NO is shown to decrease cytochrome P450 and microsomal heme through increases in the activity of heme oxygenase-1 (13). The normal exhaled CO levels in corticosteroid-treated patients suggest that inhaled corticosteroids downregulate heme oxygenase-1 activities, probably through direct action on the heme oxygenase promoter (12), and reduction of inflammatory cytokines and NO (23). In fact, exhaled CO levels and eosinophil cell counts in sputum in a given individual patient with asthma decreased after inhaled corticosteroid therapy. Thus, it is tempting to speculate that the anti-inflammatory effects of corticosteroids result in the down regulation of heme oxygenase-1, but this hypothesis has not yet been tested.

Although we have shown an elevation of exhaled CO in asthmatic patients that decreases with corticosteroid therapy, we do not know whether the level of CO in exhaled air is merely an indicator of asthma activity or a causative link in the biology of asthma. Furthermore, the present study did not refer to the contribution of the upper airway to the level of CO in exhaled air. However, our demonstration that corticosteroid treatment resulted in a decrease of exhaled CO levels concomitant with improved lung function and reduced eosinophil cell counts in sputum in individual asthmatic patients raises the possibility that an increase in exhaled CO concentration may reflect inflammation of the asthmatic lung.

The writers thank the Chest Institute of Technology for technical assistance and Mr. G. Crittenden for reading the manuscript.

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Correspondence and requests for reprints should be addressed to Hidetada Sasaki, M.D., Professor and Chairman, Department of Geriatric Medicine, Tohoku University School of Medicine, Aoba-ku Seiryo-machi 1-1 Sendai 980, Japan.


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