Abstract
Viral infection may induce the expression of heme oxygenase, resulting in increased carbon monoxide (CO) formation. CO may be produced by various cells of the upper and lower respiratory tract and may be detected in the exhaled air. Therefore, exhaled CO concentrations were measured on a CO monitor by vital capacity maneuver in subjects with upper respiratory tract infections (URTIs) and in nonsmoking and smoking healthy control subjects. At the time of symptoms of URTI, exhaled CO concentrations were 5.6 ± 0.4 ppm and decreased to 1.0 ± 0.1 ppm during recovery. Recovery values of exhaled CO were similar to those in age-matched nonsmoking healthy control subjects (1.2 ± 0.3 ppm). Smoking healthy control subjects had the highest levels of exhaled CO concentration among the groups (18.5 ± 2.5 ppm). These findings suggest that symptomatic URTIs increase the concentration of CO in exhaled air. This may reflect the induction of heme oxygenase that has an antiviral effect in the airways.
Carbon monoxide (CO) has been reported to have several biologic actions (1-4) and may play a role in pathophysiology of airway diseases (5). CO is made by an enzyme called heme oxygenase, and two forms of heme oxygenase have been characterized (6). Constitutive forms of the enzyme are widely distributed throughout the body with high concentrations in the brain (6), but another isoform is induced in several types of cells after exposure to inflammatory cytokines (7, 8), oxidants (9, 10) and NO (11). CO is detectable in the exhaled air of normal persons (5, 12) and exhaled CO is increased in asthma (5), which may reflect an expression of inducible heme oxygenase in airway epithelial cells (13). This is supported by the fact that inhaled corticosteroids inhibit the increase in exhaled CO in asthmatic patients (5).
Because upper respiratory tract infections (URTIs) are reported to induce increases in interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in nasal lavage fluid (14) as well as increases in exhaled NO (15), it is likely that viral respiratory tract infection increases CO production. We have, therefore, studied whether URTIs increase the concentration of CO in the exhaled air of normal persons.
Twenty subjects with URTIs and 10 control subjects volunteered for this study. None of the subjects in either group were smokers, ex-smokers, or passive smokers. All subjects with URTIs were studied at the time of symptomatic URTIs. Subjects were interviewed regarding the presence and severity of the following 10 symptoms: sneezing, nasal discharge, nasal congestion, malaise, headache, chills, feverishness, sore throat, hoarseness, and cough. Symptoms were rated for severity on a scale from 0 to 3. URTIs were defined if they had a total symptom score more than 5 (16). None of the subjects with URTIs had a history of asthma or sinusitis, and none were receiving medications (antihistamines, nonsteroidal anti-inflammatory drugs) at the time of study. In the 10 control subjects there was no history of URTIs for at least 4 wk prior to the study, and no history of respiratory or cardiovascular disease. None of these 30 nonsmoking subjects were receiving long-term medication. Ten smokers were recruited from volunteers and were studied within 1 to 3 h after the last cigarette. Physical characteristics, symptom score, C-reactive protein (CRP) values, pulmonary function test results, and Brinkman's index (number of cigarettes/d × yr) are shown in Table 1 for these three groups.
| Subjects | Age (yr) | Sex (M/F ) | Symptom Score | CRP (mg/dl ) | FVC (% pred ) | FEV1(% pred ) | Brinkman's Index | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Controls, n = 10 | 44 ± 3 | 6/4 | NO | 0.1 ± 0.1 | 110 ± 3 | 101 ± 3 | 0 | |||||||
| URTI, n = 20 | 45 ± 3 | 11/9 | 10.1 ± 0.7 | 5.7 ± 2.6 | 108 ± 4 | 100 ± 5 | 0 | |||||||
| Smokers, n = 10 | 44 ± 3 | 8/2 | NO | 0.1 ± 0.1 | 102 ± 5 | 96 ± 5 | 518 ± 71 |
Lung function parameters and exhaled CO were measured on two occasions: 1 to 2 d after the onset of URTI symptoms and 3 wk later when they were asymptomatic. The age-matched nonsmoking and smoking control subjects were used as reference groups for comparison regarding the exhaled CO levels. We also measured the amount of CRP in the serum using the method described by Senju and colleagues (17).
To isolate viruses from the subjects with URTIs, we screened 13 kinds of viruses (influenza types A, B, and C, parainfluenza virus, adenovirus, rhinovirus, respiratory syncytial virus, mumps virus, poliovirus, coxsackie B virus, herpes simplex virus, cytomegalovirus, and enteroviruses) in throat swabs from each subject, and viruses were identified using the method described previously (18, 19).
Exhaled CO was measured on a portable Bedfont EC50 analyzer (Bedfont Technical Instruments Ltd, Sittingbourne, UK) using the method described by Jarvis and colleagues (12) 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 (5). Background CO values (0 to 1 ppm) were obtained prior to the subject readings.
To observe the relationship between ambient and exhaled CO, the ambient levels of CO in the small room were varied from 1.0 to 8.1 ppm using a mixture of 50 ppm CO in air. After 5 min of normal breathing at each level of ambient CO (at least 7 points), exhaled CO was measured as described above in five nonsmoking control subjects (mean age, 45 ± 4 yr). Then, the ambient levels of CO and exhaled CO values were plotted on x and y axes, respectively. From linear regression analysis, values of regression slope and regression intercept were obtained from each subject. Mean values were 1.0 ± 0.1 for slope and 1.2 ± 0.1 ppm for intercept. The values of intercept did not differ from those of exhaled CO (1.2 ± 0.2 ppm, p > 0.50, n = 5) measured during air breathing after subtracting the background level. Therefore, the exhaled CO concentration was determined by subtracting the background level from the observed reading in the following experiments as reported previously (12). To avoid analysis with a value of exhaled CO concentration below 1.0 ppm, the background level was subtracted from the average value obtained from three sequential maneuvers in each subject. The exhaled CO concentrations were always the values above 1.0 ppm before subtracting the background level throughout the experiments.
To examine the contribution of the upper airway to the level of exhaled CO, an expiratory resistance was added to maintain a mouth pressure greater than 20 mm Hg during expiration. A continuous pressure in the mouth greater than 20 mm Hg is reported to close the velum during expiration to exclude nasal CO that may leak throughout expiration in the presence of an open velum (20). Mouth pressure was measured by a differential pressure transducer (Model MP45; Validyne, Northridge, CA) and displayed on an oscilloscope (Tektronix 5103 N; Tektronix Corp., Beaverton, OR). Six subjects with URTIs (mean age, 44 ± 4 yr) were recruited from volunteers in this study.
Prior to the start of the study, the analyzer was calibrated with a mixture of 50 ppm CO in air (12). Exhaled 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 nonsmokers (21, 22) as well as the cigarette consumption (22).
Results are reported as means ± SE. 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 measured values were similar among three sequential maneuvers in nonsmoking control subjects (1.2 ± 0.3 versus 1.2 ± 0.2 versus 1.2 ± 0.3 ppm), the subjects during the acute phase of URTIs (5.6 ± 0.4 versus 5.7 ± 0.3 versus 5.6 ± 0.4 ppm), and those after 3 wk of recovery (1.0 ± 0.1 versus 1.0 ± 0.1 versus 1.0 ± 0.1 ppm), respectively. Likewise, measurements of exhaled CO values were reproducible over a 3-wk period (1.2 ± 0.2 ppm before versus 1.2 ± 0.2 ppm after; p > 0.50) in nonsmoking control subjects, and the variation between readings on separate days was small (6.9 ± 2.1%).
Mean exhaled CO concentration was 1.2 ± 0.3 ppm in nonsmoking control subjects. In subjects with URTI, the exhaled CO was significantly higher during the acute phase of URTIs than that in nonsmoking control subjects (Figure 1). As expected, smoking control subjects had the highest CO concentration among the three groups (Figure 1). The exhaled CO during the acute phase of URTIs decreased after 3 wk of recovery and the exhaled CO values in subjects who had recovered from URTIs did not differ significantly from those in nonsmoking control subjects (1.0 ± 0.1 ppm, p > 0.20) (Figure 2). There was a significant relation between changes in the exhaled CO concentration and those in symptom scores from the time of the acute phase to after 3 wk of recovery in subjects with URTIs (r = 0.72, p < 0.01, n = 20).
Fig. 1. Carbon monoxide (CO) concentrations in exhaled air in nonsmoking control subjects (n = 10), smoking control subjects (n = 10), and normal subjects during a symptomatic upper respiratory tract infection (URTI) (n = 20). Mean values ± SE are indicated by closed circles with error bars.
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Fig. 2. Individual values of exhaled carbon monoxide (CO) in 20 normal subjects during a symptomatic upper respiratory tract infection (URTI) and 3 wk afterwards when asymptomatic (Recovery). Mean values ± SE are indicated by closed circles with error bars.
[More] [Minimize]There was a significant difference between the exhaled CO levels measured with and without an expiratory resistance (3.8 ± 0.4 versus 5.4 ± 0.3 ppm, p < 0.05, n = 6).
Influenza type A viruses were identified from all subjects and other viruses were not detected in any of the 20 subjects with URTIs.
The present study has shown that exhaled CO can be reliably measured in healthy control subjects and subjects during the acute phase of URTIs. The values of exhaled CO in nonsmoking and smoking control subjects were similar to those of previous studies (12, 21, 22). Because the carboxyhemoglobin level in the blood declines exponentially and becomes normal 24 h after the cessation of smoking (23), the present study should underestimate the exhaled CO concentration in smoking control subjects. However, we have demonstrated that URTIs are associated with an increase in exhaled CO in normal persons in the acute phase when symptoms are present, and that there is a reduction in exhaled CO after recovery to values that are similar to those in age-matched normal subjects. This suggests that URTI, presumably caused by influenza viral infection, increases the production of CO in the respiratory tract.
However, several factors might influence the exhaled CO concentration in the present study. First, some variations in the exhaled CO concentration among three sequential maneuvers were observed. Although the reason for observed variations is uncertain, the mean exhaled CO concentrations were similar among three sequential maneuvers in nonsmoking control subjects and the subjects during and after URTIs. Furthermore, the variation between the exhaled CO concentrations on separate days in nonsmoking control subjects was small. Second, the subjects exposed to cigarette smoke as passive smokers might have the same range as the subjects with URTIs. However, it seems unlikely because we selected the subjects with URTIs and none of them were smokers, ex-smokers, or passive smokers. Finally, the subjects might encounter much higher ambient CO levels than those at the place of the CO measurement on their way to the hospital. However, the route and means of transportation to the hospital were the same in the subjects with URTIs between the time of the acute phase and after 3 wk of recovery, and they visited the hospital at a similar time of day. Therefore, the ambient CO levels on the way to the hospital could little influence the difference in the exhaled CO concentration between the time of the acute phase and after 3 wk of recovery in the subjects with URTIs.
The exhaled CO concentration was higher without an expiratory resistance used to close the velum during expiration to exclude nasal CO that may leak throughout expiration in the presence of an open velum (20). Therefore, the increase in CO may be derived from both nasal tissues and the lower respiratory tract. It is of interest that a large proportion of subjects with URTIs in this study complained of lower respiratory tract symptoms, including cough and chest pain accentuated by coughing, indicating that the lower respiratory tract was likely to have been involved.
Heme oxygenase plays an important role in the resolution of inflammation in animals (24, 25) and pulmonary epithelial cells in vitro (26). Likewise, heme oxygenase protects against viral infection and replication in cultured human airway epithelial cells (13). Viral infections may induce heme oxygenase in a variety of cell types, including airway epithelial cells and macrophages (27) via the induction of proinflammatory cytokines (7, 8) and NO (11). The increased heme oxygenase activity may then serve to limit the virus infection by inhibiting viral replication (13) and airway inflammation by anti-inflammatory actions (24-26). Because inhaled corticosteroids inhibit the increase in exhaled CO in asthmatic patients (5), corticosteroids may impair host defenses against the viral infection through downregulation of heme oxygenase activity. However, Farr and colleagues (28) reported that the trend toward less increase in nasal obstruction, middle ear pressure, mucus production, and nasal mucus kinin and albumin concentrations during the first 2 d after rhinovirus inoculation was temporally related to the simultaneous administration of oral prednisone and intranasal beclomethasone. Furthermore, dexamethasone inhibits rhinovirus-induced production of cytokines and intercellular adhesion molecule-1 productions and replication of rhinovirus in the cultured human airway epithelial cells in vitro (29). Therefore, corticosteroids may have effects on upper respiratory tract infections through several mechanisms, and a further study is needed to clarify this issue. Furthermore, the administration of NO synthease inhibitors by nebulization to normal subjects and patients with asthma produces a fall in exhaled NO levels (30). Therefore, NO synthase inhibitors may downregulate heme oxygenase activity, thereby decreasing the exhaled CO.
Although we have shown an elevation of exhaled CO in URTIs that decreases after recovery, it is uncertain whether the level of CO in exhaled air is merely an indicator of airway inflammation or a causative link in the biology of airway viral infection. However, the demonstration that URTIs are associated with a high level of exhaled CO suggests that respiratory viral infections may induce the expression of heme oxygenase in the airway. Heme oxygenase therefore may act as a host defense against URTIs.
The writers thank the Chest Institute of Technology for technical assistance and Mr. G. Crittenden for correcting the English.
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