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CONTENTS |
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TOP
CONTENTS
INTRODUCTION
NITRIC OXIDE
CARBON MONOXIDE
EXHALED HYDROCARBONS
EXHALED BREATH CONDENSATE
OTHER METHODS
FUTURE DIRECTIONS
REFERENCES
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Introduction
Nitric Oxide
Source of NO in exhaled air
Measurement
Asthma
COPD
Cystic fibrosis
Bronchiectasis
Primary ciliary dyskinesia
Rhinitis
Interstitial lung diseases
Pulmonary hypertension
Occupational diseases
Infections
Chronic cough
Lung cancer
Lung transplant rejection
Adult respiratory distress syndrome
Diffuse Panbronchiolitis
Carbon Monoxide
Source of exhaled CO
Measurement
Asthma
COPD
Bronchiectasis
Cystic fibrosis
Interstitial lung disease
Allergic rhinitis
Infections
Other conditions
Exhaled Hydrocarbons
Origin
Measurement
Asthma
COPD
Cystic Fibrosis
Other lung diseases
Exhaled Breath Condensate
Origin
Hydrogen peroxide
Eicosanoids
Products of lipid peroxidation
Vasoactive amines
NO-related products
Ammonia
Electrolytes
Hydrogen ions
Proteins and cytokines
Other Methods
Exhaled temperature
Combined gas chromatography/spectroscopy
The selected ion flow tube (SIFT) technique
Polymer-coated surface-acoustic-wave resonators
Future Directions
Standardization of measurements
Clinical application
Profiles of mediators
Measuring devices
New markers
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INTRODUCTION |
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TOP
CONTENTS
INTRODUCTION
NITRIC OXIDE
CARBON MONOXIDE
EXHALED HYDROCARBONS
EXHALED BREATH CONDENSATE
OTHER METHODS
FUTURE DIRECTIONS
REFERENCES
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There has recently been an explosion of interest in the analysis of breath constituents as a way of monitoring inflammation and oxidative stress in the lungs. Here we review the use of exhaled breath analysis in the diagnosis and monitoring of lung disease. Although most studies have focused on exhaled nitric oxide (NO), recently several other volatile gases (carbon monoxide, ethane, pentane) have also been used. In addition, several endogenous substances (inflammatory mediators, cytokines, oxidants) may be detected in expired breath condensates, opening up new perspectives for exhaled breath analysis.
Many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, and interstitial lung disease, involve chronic inflammation and oxidative stress. Yet these are not measured directly in routine clinical practice because of the difficulties in monitoring inflammation. In asthma fiberoptic bronchial biopsies have become the "gold standard" for measuring inflammation in the airway wall, but this is an invasive procedure that is not suitable for routine clinical practice and cannot be repeated often. It is also unsuitable for use in children and patients with severe disease. Symptoms may not accurately reflect the extent of underlying inflammation because of differences in perception and masking by bronchodilators in airway disease. In asthma measurement of airway hyperresponsiveness by histamine or methacholine challenge has been used as a surrogate marker of inflammation, but interpretation may be confounded by the use of bronchodilator therapy. Furthermore, it is difficult to perform this measurement in children and in patients with severe disease. This has led to the use of induced sputum to detect inflammation. This method is relatively reproducible and allows the quantification of inflammatory cells and mediators (1). However, this technique is somewhat invasive as it involves inhalation of hypertonic saline, which may induce coughing and bronchoconstriction, and it is difficult to use in small children. Furthermore, the technique itself induces an inflammatory response so that it is not possible to repeat measurements in less than 24 h (2). The need to monitor inflammation in the lungs has led to the exploration of exhaled gases and condensates. Noninvasive monitoring may assist in differential diagnosis of pulmonary diseases, assessment of disease severity and response to treatment. Because these techniques are completely noninvasive, they can be used repeatedly to give information about kinetics, they can be used in patients with severe disease, which has been previously difficult to monitor, and they can be used to monitor disease in children, including infants. Breath analysis is currently a research procedure, but there is increasing evidence that it may have an important place in the diagnosis and management of lung diseases in the future (3). This will drive the development of cheaper and more convenient analyzers, which can be used in a hospital and later in a family practice setting, then eventually to the development of personal monitoring devices for use by patients.
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NITRIC OXIDE |
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TOP
CONTENTS
INTRODUCTION
NITRIC OXIDE
CARBON MONOXIDE
EXHALED HYDROCARBONS
EXHALED BREATH CONDENSATE
OTHER METHODS
FUTURE DIRECTIONS
REFERENCES
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NO is the most extensively studied exhaled marker and abnormalities in exhaled NO have been documented in several lung diseases (3), particularly asthma (4).
Source of NO in Exhaled Air
Nitric oxide synthases. Endogenous NO is derived from L-arginine by the enzyme NO synthase (NOS), of which at least three distinct isoforms exist (7) (Figure 1, panel A). Two of these enzymes are constitutively expressed and are activated by small rises in intracellular calcium concentration, secondary to cell activation. Neuronal NOS (NOS1, nNOS) is predominantly expressed in neurones and endothelial NOS (NOS3, eNOS) mainly in endothelial cells, although other cell types also express both of these isoforms. A third enzyme is inducible (NOS2, iNOS), has a much greater level of activity, and is independent of calcium concentration. NOS2 may be induced by inflammatory cytokines, endotoxin, and viral infections and may show increased expression in inflammatory diseases (8). Genetic polymorphisms of all three isoforms of NOS have been detected. Surprisingly, associations have been found between polymorphisms in the NOS1 gene and asthma in Caucasian populations (11, 12). In patients with mild asthma there is a significant association between the length of the AAT repeat polymorphism in intron 20 of the NOS1 gene and exhaled NO levels (13).
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Cellular sources in airways. The cellular source of NO gas
in the lower respiratory tract is not yet certain. Studies with perfused porcine lungs suggest that exhaled NO originates at
the alveolar surface, rather than from the pulmonary circulation (14), and it may be derived from NOS3 expressed in the
alveolar walls of normal lungs. Studies in ventilated perfused
lungs of guinea pigs have shown that exhaled NO is reduced
during perfusion with calcium-free solutions, suggesting that
NO is derived from a constitutive NOS, which is calcium-
dependent (15). Airway epithelial cells may express both
NOS3 and NOS1 and therefore may contribute to NO in the
lower respiratory tract (16, 17). There is some expression of
NOS2 even in airway epithelial cells from normal subjects
(18), and NOS2 appears to be an important isoform contributing to exhaled NO in healthy mice (19). In inflammatory diseases such as asthma it is likely that the increase in exhaled
NO reflects further induction of NOS2 in response to inflammatory signals such as proinflammatory cytokines. Indeed, increased NOS activity has been demonstrated in lung tissue of
patients with asthma, cystic fibrosis, and obliterative bronchiolitis (20). In asthmatic patients there is evidence for increased
expression of NOS2 in airway epithelial cells (21), and this is
likely to be due to increased transcription mediated via the
transcription factors STAT-1 and nuclear factor-
B (NF-B),
and increased availability of L-arginine (22, 23). Proinflammatory cytokines induce the expression of NOS2 in cultured human airway epithelial cells (24, 25), and it is likely that these
same cytokines are released in asthmatic inflammation. NOS2
may be expressed in other cell types such as alveolar macrophages, eosinophils, and other inflammatory cells (26). Further evidence that the increase in exhaled NO is derived from
increased NOS2 expression is the observation that corticosteroids inhibit inflammatory induction of NOS2 in epithelial cells (22, 27), decrease expression in bronchial biopsies of asthmatic patients (26), and also reduce exhaled NO concentrations in asthmatic patients (28) (Figure 1, panel B).
Nonenzymatic sources of NO. NOS is not the only source of NO in exhaled air, and exhaled NO is not therefore a direct measure of NOS activity in the lower respiratory tract. NO reacts with thiol-containing molecules such as cysteine and glutathione to form S-nitrosoproteins and S-nitrosothiols (29). Approximately 70 to 90% of NO is released by S-nitrosothiols, which therefore provide a major source of NO in tissues (30). S-nitrosothiols are potent relaxants of human airways and may play an important role in sequestration, releasing, and transportation of NO to its site of action (29).
NO in exhaled air may also be derived from nitrite protonation to form nitrous acid, which releases NO gas with acidification (31). This pH-related pathway has been implicated in acute asthma, when pH in expired condensate is low (32).
Anatomic origin. NO is produced along the entire length of
human airways. The conducting airways secrete NO into the
lumen, which mixes with alveoli NO during exhalation, resulting in the observed expiratory concentration. The levels of NO
derived from the upper respiratory tract (200 to 1,000 ppb)
(33) and sinuses (1,000 to 30,000 ppb) (36) are a hundred-fold higher than exhaled NO measured in the lower respiratory tract (1 to 9 ppb) (33, 34, 37). Several factors may contribute to high nasal levels. The paranasal sinuses produce a
high level of NO (43). There is a dense innervation with
NOS1-immunoreactive nerve fibers around nasal blood vessels (44). Vasculature-derived NO, however, is not the major
source of NO in nasal mucosa, as neuropeptide Y, a powerful
vasoconstrictor, reduces nasal blood flow by 37%, but NO by
only 7% (45). There appears to be constitutive expression of
NOS2 (46) and the transcription factor NF-B in nasal mucosa (47). Interestingly, the NO outputs from the nostrils are
significantly lower on the operated side (site with the reduced
NO-generating surface) in patients who have undergone unilateral medial maxillectomy (48).
The source of NO in the lower respiratory tract is also of mixed origin and may be derived from airway and alveolar epithelial cells, which express both NOS3 and NOS1. The contribution of endothelial-derived NO is minimal, as inhaled NOS inhibitors are able to reduce exhaled NO by 40 to 70% (49- 51) without any effect on the systemic circulation. By contrast, L-NMMA infusion modulates blood pressure and heart rate but has only a minimal effect on exhaled NO (49).
Simultaneous measurement of expired CO2 and NO demonstrate that exhaled NO precedes the peak value of CO2 (end-tidal), suggesting that NO is derived from airways rather than from alveoli (33, 52). Direct sampling via fiberoptic bronchoscopy in normal subjects shows a similar levels of NO in trachea and main bronchi to that recorded at the mouth, thus indicating that there is NO derived from the lower airways (33, 42). Exhaled NO is therefore most likely to be of epithelial rather than of endothelial origin, and most NO is derived from airways rather than from alveoli.
Measurement
Expiratory flow, soft palate closure, and dead space air may all influence exhaled NO levels. Therefore, exhaled NO is usually determined during single-breath exhalations against a resistance (38) (Figure 2, panel A) (28, 40, 53) to prevent contamination with nasal NO (54, 55), or using reservoir collection with discarding of the dead space (56). However, this method has proven difficult for some children, who may have trouble maintaining a constant flow, and recently a simple flow-driven method for online NO measurements has been developed that does not require active patient cooperation (57). Recently, single breath analysis of exhaled NO has been successfully performed in the newborn when exhaled air was sampled from the tip of a thin nasal catheter placed in the hypopharynx (58). The most commonly used method to measure nasal NO is to sample nasal air directly from one nostril using the intrinsic flow of the chemiluminescence analyzer (36). A novel method of measuring exhaled NO at several exhalation flow rates has recently been described that can be used to approximate alveolar and airway NO production (59). NO is continuously formed in the airways. Mixing during exhalation between the NO produced by the alveoli and the conducting airways, explains its flow dependency (55) and accumulation during a breathhold (33). A relatively simple and robust two-compartment model of NO has been developed that is capable of simulating many important features of NO exchange in the lungs (60). The model assumes that the lung consists of two well-defined, separate regions: a rigid airway compartment and a well-mixed, expansile alveolar compartment. Both compartments seem to contribute to exhaled NO, and the relative contributions of each seems to be a function of minute ventilation (60). Finally, the model suggests that the relationship between exhaled NO at end-exhalation may be a simple, effective, and reproducible technique for determining the relative contribution of the airways and alveoli to exhaled NO.
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It is therefore important to register the flow rate if NO is expressed as a concentration The flow rate recommended in 1997 by a Task Force of the European Respiratory Society is 10 to 15 L/min or 167 to 250 ml/s (53). Most investigators have used about 100 ml/s, but a more recent recommendation from the American Thoracic Society suggests 50 ml/s (61).
Factors Affecting Exhaled NO Measurements
Exhaled and nasal NO in healthy subjects is independent of age, sex, and lung function (34, 62). There is no evidence for significant diurnal variation (63), and exhaled NO measurements are highly reproducible in normal subjects (64, 65). Different phases of the menstrual cycle may influence exhaled NO (66), as estrogen activates NOS3 in airway epithelial cells (67).
There are several major factors, which may change NO levels in normal subjects (Table 1). Either intravenous, or inhaled, or digested L-arginine, the substrate for NOS, increase exhaled NO levels in normal subjects (68). Conversely nebulized L-NMMA and L-NAME, nonspecific inhibitors of NOS, reduce exhaled NO (28, 50) and nasal NO (71, 72). Some routinely used tests can transiently reduce exhaled NO; for example, repeated spirometry (73, 74), physical exercise (75), sputum induction (76). Environmental factors such as NO ozone and chlorine dioxide are known to increase exhaled NO levels (77). Habitual factors such as smoking (80, 81) and alcohol ingestion (82, 83) reduce exhaled NO. Upper respiratory infection significantly increases exhaled NO (84, 85) and nasal NO (86).
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Asthma
Increased levels of exhaled NO have been widely documented in patients with asthma (Figure 2, panel B) (28, 87). The increased levels of exhaled NO in asthma have a predominant lower airway origin (33, 42) and are most likely due to activation of NOS2 in airway epithelial and inflammatory cells (21, 26). However, there may be a small contribution from NOS1 as polymorphisms of NOS1 gene are correlated with exhaled NO (13). Exhaled NO may be further elevated by NO substrate L-arginine (69).
Diagnosis and epidemiology. An elevation of exhaled NO is not specific for asthma, but an increased level may be useful in differentiating asthma from other causes of chronic cough (88). The diagnostic value of exhaled NO measurements to differentiate between healthy subjects with or without respiratory symptoms and patients with confirmed asthma has been recently analyzed by Dupont and colleagues (89) with 90% specificity and 95% positive predictive value when exhaled NO > 15 ppb is used as a cutoff for asthma. The intraindividual coefficient of variation (CoV) of exhaled NO in normal subjects was 15.8% within an interval of 7 d, and 16.8% within 23 d, suggesting that the change of exhaled NO by 30 to 35% or more within the interval of 1 to 3 wk would be abnormal (62). Exhaled and nasal NO may be used to identify subjects with atopy, because nonatopic asthmatics have normal exhaled NO (90). There is a strong association between elevated exhaled and nasal NO and skin prick test scores, total IgE (91), and blood eosinophilia (92) in mild asthma. Elevated nasal NO is also related to the size of skin test reactivity in asymptomatic asthmatic subjects (93). This may denote "subclinical" airway inflammation.
Another potential use of exhaled NO levels in patient management is the prediction of future asthma. An elevated exhaled NO may be found in patients with "subclinical" forms of asthma (normal lung function, negative bronchodilator tests, and elevated sputum eosinophilic cationic protein concentrations) (94, 95). Elevated levels of NO in patients with "subclinical asthma" are not in conflict with the specificity of exhaled NO as a marker to diagnose asthma, as lack of current asthma symptoms does not exclude the diagnosis of asthma. Perhaps, this subclinical airway inflammation, which is reflected by elevated levels of exhaled NO in adolescent asymptomatic patients with asthma remission (96), should be treated with corticosteroids to prevent the risk of becoming clinically manifest again. This category of patients with "subclinical" forms of asthma, especially children, may be predisposed to develop asthma in the future (97). This may be studied in epidemiologic studies, in which the reservoir collection of exhaled NO has proved to be useful (98, 99). Airway responsiveness measurements (PC20) in this "high risk" group make the combination of exhaled NO and PC20 a more specific test for allergic asthma. This has recently been demonstrated in a study of more than 8,000 adolescents in Norway (100). Because of the noninvasive character and practicality of exhaled and nasal NO measurements they may be used cost effectively for screening of large populations.
Atopy and exposure to proinflammatory stimuli. Exhaled NO is elevated in allergic/atopic adults and children (97, 101, 102). It is further increased as a result of allergen exposure such as during the late phase response to allergen challenge (103, 104), during the grass pollen season (105), or during exposure to indoor allergens (106, 107). In subjects sensitive to house dust mites (HDM) the wheal size for HDM correlates with exhaled and nasal NO levels (93). Both adults (97) and children (102) with atopic asthma have higher levels of exhaled NO than do patients with nonatopic asthma, even without airway hyperresponsiveness (108).
Exhaled NO may represent a useful biomarker of individual exposure to air pollutants, as even healthy subjects may have elevated exhaled NO levels on days with high outdoor air pollution (79, 109). This may reflect an airway inflammatory response to ozone and nitrogen dioxide (110).
Asthma monitoring. It is difficult to monitor the response of different classes of anti-inflammatory drugs in asthma, as there is no single test that can be used to quantify airway inflammation. Peripheral blood markers are unlikely to be adequate as the most important mediator and cellular responses occur locally within airways. Eosinophils in induced sputum originate from more proximal rather than small airway (111). It is clear that different markers of airway inflammation should be considered together to monitor asthma (3).
Exhaled NO has been used to monitor the effect of anti- inflammatory treatment in asthma (6, 112) and asthma exacerbations, both spontaneous (40) and induced by steroid reduction (113, 114). There is a lack of long-term serial studies of exhaled NO, together with other markers of airway inflammation in sputum and exhaled condensate, lung function and symptoms. Exhaled NO behaves as a "rapid response" marker, which is extremely sensitive to steroid treatment, as it may be significantly reduced even after 6 h following a single treatment with a nebulized corticosteroid steroid (115), or within 2 to 3 d after inhaled corticosteroids (112), reaching maximal effect after 2 to 4 wk of treatment (112, 113, 116).
An important issue in asthma management is to prevent overtreatment of patients with steroids. The high sensitivity of exhaled NO to corticosteroid treatment is an advantage, as higher doses of inhaled steroids are not necessary to improve asthma control, e.g., in mild persistent asthma (3). We have demonstrated a dose-dependent reduction in exhaled NO and improvement in asthma symptoms in patients with mild asthmatics after treatment with low doses of inhaled corticosteroids (120), whereas the reduction in sputum eosinophils and similar improvement in symptoms was observed only after the higher dose of steroids (117). This suggests that exhaled NO levels may be too sensitive to determine whether inflammation is adequately controlled (3).
Although exhaled NO levels are normal in patients with moderate asthma treated with corticosteroids (28), increased levels have been observed in patients with severe asthma, despite treatment with oral corticosteroids (98, 121). Individual NO values such as individual peak expiratory flows should be established and monitored, and when the levels are above or below a certain reference level, steroid treatment should be either reduced or increased.
A considerable advantage of exhaled NO is that NO levels may increase before any significant changes in other parameters such as lung function and sputum eosinophils and may therefore serve as an early warning of loss of control (4). Thus, exhaled NO levels increase by 40 and 100% after 2 and 4 wk, respectively, after the reduction in steroid treatment (114). This increase in exhaled NO levels is accompanied by lung function deterioration and asthma symptoms. Although the baseline high number of eosinophils in sputum of patients who eventually develop exacerbations is a good predictor of asthma deterioration, the changes in eosinophils after the steroid reduction are slow (114). Prospective studies, which look at asthma outcomes over a prolonged period of time, where NO is used as a decision point for modifying inhaled corticosteroid treatment will be needed to evaluate the value of exhaled NO as a useful way of monitoring asthma.
Disease severity and control. Treatment with inhaled corticosteroids reduces exhaled NO levels, and therefore exhaled NO cannot be directly related to asthma severity.
Exhaled NO levels are almost three times higher in children with recent symptoms than in symptom-free subjects (122),
and are further elevated during the asthma attack in both adults
(123) and children (124, 125). In fact, the levels of NO in children with acute severe asthma (125) are more than 2-fold higher
than in children with less severe wheezing exacerbations and
almost 4-fold higher than in children with first-time wheeze
(124). A reduction in exhaled NO (by 65% after 5 d of corticosteroid therapy) is accompanied by clinical and FEV1 improvement from asthma exacerbations in children (126), and
NO has been a more sensitive marker of asthma activity than
serum ECP or soluble interleukin-2 receptors (127). Higher
exhaled NO levels are related to asthma symptoms and 
2-agonist use in patients with difficult severe asthma (98). Exhaled
NO is increased in patients who remain symptomatic despite
oral steroids and who have a relative steroid resistance, and
may therefore be useful to quantify steroid resistance in asthma.
It is most likely that exhaled NO is related to asthma control rather than to asthma severity (3), and that serial NO measurements in individual patients over time may be useful to identify patients requiring changes in therapy. In a recent study, Sippel and coworkers (128) have shown that exhaled NO was significantly correlated with markers of asthma control such as asthma symptoms within the previous 2 wk, dyspnea score, daily use of rescue medication, and reversibility of airflow obstruction. However, exhaled NO levels were not correlated with the following markers of asthma severity: history of respiratory failure, health care use, or fixed airflow obstruction.
It is reasonable to believe that subclinical airway inflammation, which is reflected by elevated levels of exhaled NO in adolescent asymptomatic patients with asthma remission (96), should be treated with corticosteroids to prevent this continuous risk of becoming clinically manifest again. However, only longitudinal studies can answer the question whether exhaled NO and bronchial hyperresponsiveness, for example, each reflecting different aspects of the inflammatory process, may guide the anti-inflammatory treatment to prevent asthma relapse later in life.
Although research in asthma has concentrated on complex proinflammatory mechanisms, it is likely that defective expression of cytokines that inhibit allergic inflammation such as interleukin 10 (IL-10), interleukin 12 (IL-12), and interferon gamma might also be important, particularly in determining disease severity and persistence of inflammation in the airways (129). Therapy based on these cytokines might also be useful, with the advantage that it restores the balance of endogenous cytokines. Recently, it has been shown that adenovirus-mediated human IL-10 gene transfer in vivo into lung isografts ameliorates subsequent ischemia-reperfusion injury and results in reduced neutrophil sequestration, and down-regulation of iNOS mRNA expression (130). Potentially, exhaled NO may be useful to monitor this type of treatment.
Relationship to other markers of asthma. The traditional means of monitoring asthma have limitations. Lung function and PC20, measurements are not directly related to airway inflammation, have little room for improvement in mild asthma (FEV1), and are affected by bronchodilators. Both parameters are slow to change and are not able to distinguish the effect of different doses of steroids. There are several areas in which exhaled NO measurements may be advantageous over the traditional means of asthma monitoring: screening for atopy, monitoring the impact of hazardous environmental factors, identification and monitoring of asthma exacerbations, and assessment of the adequacy of anti-inflammatory treatment.
Exhaled NO in patients with asthma is correlated with sputum eosinophils (117, 131, 132) and methacholine reactivity (133, 134), as well as peak flow variability (113, 116). However, the
relationship between exhaled NO and airway inflammation is still uncertain, and in smaller studies no significant relationship is seen between exhaled NO and eosinophils in bronchial
biopsies or bronchoalveolar lavage (116), and the induction of
sputum eosinophils by inhaled LTE4 is not associated with increased exhaled NO (135, 136). This may indicate that increased exhaled NO reflects some, but not all, aspects of airway inflammation, and further work is needed to determine
how it relates to some other markers of airway inflammation.
On the other hand, a more comprehensive spectrum of inflammatory markers (for example, IL-4, IL-5, IL-6, IL-8, IL-10,
and TNF-
) can be measured in induced sputum, and in the
future these should be correlated with changes in exhaled NO.
Corticosteroids. Systemic corticosteroids have no effect on
exhaled NO in normal subjects, but they decease its levels in patients with asthma (40, 50). Oral dexamethasone (4 mg/d for
2 d) similarly has no effect on exhaled NO or on serum concentrations of interferon-
and IL-1 in normal subjects (137).
A large dose (1 mg/kg/d for 5 d) of oral prednisolone normalized exhaled NO in infants and young children with wheezing exacerbations (124), whereas the same dose in children with more severe asthma only shifted their exhaled NO down to the levels of mild-to-moderate asthma, in spite of the improvement in lung function (125). A cumulative dose of methylprednisolone (180 to 500 mg) causes 36% reduction within 50 h in the majority of severe adult patients with severe, acute asthma (40), and a combination of oral prednisolone and inhaled steroids reduces exhaled NO by 65% in children with acute asthma (126).
Recently, it has been shown that NO levels correlate with
the percentage improvement in FEV1 from baseline to the
poststeroid (30 mg prednisolone/d for 14 d) postbronchodilator value. A NO level of > 10 ppb at baseline has a positive predictive value of 83% for an improvement in FEV1 of 
15%,
and therefore may be useful in predicting the response to a
trial of oral steroid in asthma (138).
A key question is why has it been so difficult to show a
dose-dependent effect of inhaled corticosteroids in the treatment of asthma? First, it is possible that the small change in
doses makes it difficult to detect changes in asthma symptoms
and lung function (FEV1). Secondly, the currently recommended doses may be at the upper end of the dose-response
curve, making it difficult to detect a relatively small change in
dose. In view of concerns about systemic effects and the better
effects of adding an inhaled long-acting 2-agonist compared
with doubling the dose of inhaled steroid, there is now a trend
towards use of lower doses of inhaled corticosteroids. Exhaled
NO as an inflammatory marker sensitive to corticosteroids
may be the ideal tool to demonstrate a dose-response effect
and to adjust the dose in clinical practice. It may also be useful
in patients using a fixed combination inhalers (corticosteroids and long acting 2-agonist) to ensure that inflammation is controlled, as this may be difficult to assess from symptoms when
a long-acting bronchodilator is taken. On the other hand, caution should be exercised as once-daily combination therapy
In fact, inhaled corticosteroids reduce exhaled NO in asthmatic patients (112) and this effect is dose-related (117). However, a plateau effect on exhaled NO measured after 6 to 12 h since the last treatment may be seen at a dose of 400 µg budesonide and higher (117, 139) in contrast to dose-related improvements in adenosine monophosphate and methacholine reactivity up to 1,600 µg in patients with mild-to-moderate asthma (120, 140). The effect of inhaled steroids on exhaled NO is very rapid and may occur within 6 h after a single high-dose (8 mg) of budesonide (Pulmicort Respules) in symptomatic moderate asthma (115). Therefore, chronic and acute reduction in exhaled NO may be of a different magnitude. Recently, it has been shown that the onset of action of inhaled BUD on exhaled NO and the time to reach the maximal reduction were also dose-dependent (120). A gradual reduction in exhaled NO is seen during the first week of regular treatment (112, 119, 120) with maximal effect between 3 wk (112, 118) or 4 wk (116, 117).
It is still uncertain whether exhaled NO is useful to direct changes in asthma therapy. Recently, it has been shown that exhaled NO values above 13 ppb had a sensitivity of 0.67 and a specificity of 0.65 to predict a step up in therapy (141), but clearly more studies are needed using exhaled NO to direct therapy.
Corticosteroids may reduce exhaled NO by directly inhibiting the induction of NOS2 (22) or by suppressing the proinflammatory cytokines that induce NOS2. There is inhibition of NOS2 immunoreactivity with inhaled corticosteroid treatment in asthmatic patients and a parallel reduction in immunoreactivity for nitrotyrosine, which may reflect local production of peroxynitrite from an interaction of NO and superoxide anions (26).
2-agonists. Neither short-acting (112, 125, 142) nor long-
acting (125, 139, 142, 144, 146) 2-agonists reduce exhaled NO.
This is consistent with the fact that they do not have any anti-inflammatory effects in asthma, although it has been shown that regular treatment with inhaled formoterol reduces inflammatory cells in the mucosa of asthmatic patients (147).
There may even be a short-term increase in exhaled NO after
2-agonists, which may be due to opening up of airways with
higher local NO concentrations (148).
Antileukotrienes. The leukotriene receptor antagonist pranlukast blocks the increase in exhaled NO when inhaled corticosteroids are withdrawn (149), and montelukast rapidly reduces exhaled NO by 15 to 30% in children with asthma (150). Antileukotrienes have a moderate effect in patients with asthma and seasonal allergic rhinitis (151, 152). Both formoterol and zafirlukast were equally effective in maintaining asthma control, and zafirlukast caused a significant reduction in exhaled NO (143).
NOS inhibitors. Nebulized L-NMMA and L-NAME, which are nonselective inhibitors of NOS, both reduce exhaled NO in asthmatic patients, although this is not accompanied by any changes in lung function (50, 153). Aminoguanidine, a more selective inhibitor of NOS2, reduces exhaled NO in asthmatic patients, but it has little effect in normal subjects, indicating that NOS2 is an important source of the increased exhaled NO in asthma (51).
Prostaglandins. Prostaglandin (PG)E2 down-regulates NOS2
expression (154) and inhaled PGE2 and PGF2
decrease exhaled NO in normal and in asthmatic subjects (155).
Other drugs. The immunosuppressive drugs cyclosporin and rapamycin inhibit NOS2 expression (156), suggesting that exhaled NO can be used to monitor their effect. Ibuprofen, a cyclooxygenase inhibitor, reduces the elevated levels of exhaled NO in normal subjects after intravenous administration of endotoxin (157), and indomethacin partially prevents an increase in exhaled NO and asthma symptoms in patients whose dose of steroids was reduced (158). A low dose of theophylline has no effect on exhaled NO levels in asthmatic patients (159). Nebulized IL-4 receptor (altrakincept) reduces exhaled NO in patients with moderate asthma (160).
COPD
Exhaled NO levels in patients with stable COPD (80, 81, 161) and chronic bronchitis (162) are lower than in either smoking or nonsmoking asthmatics (163) and are not different from those in normal subjects. This reduction in exhaled NO is due to the effect of tobacco smoking, which down-regulates eNOS (164) and reduces exhaled NO (80), suggesting that this may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers. In addition to the effects of cigarette smoking, a relatively low value of exhaled NO in COPD may reflect more peripheral inflammation than in asthma, low NOS2 expression (161), and increased oxidative stress that may consume NO in the formation of peroxynitrite (165).
Patients with unstable COPD, however, have high NO levels compared with stable smokers or ex-smokers with COPD (166), which may be explained by increased neutrophilic inflammation and oxidant/antioxidant imbalance. Eosinophils that are capable of expressing NOS2 and producing NO are present in exacerbations of COPD (167). Acidosis, which is frequently associated with exacerbations of COPD, may increase the release of NO (32). Pulmonary hypertension has the opposite effect, as COPD patients with cor pulmonale have low exhaled NO levels (168), which may reflect their impaired endothelial NO release.
A small proportion of patients with COPD appear to response to corticosteroids, and these patients, who are likely to have coexistent asthma, have an increased proportion of eosinophils in induced sputum (169). These patients also have an increased in exhaled NO (170). This suggests that exhaled NO may be useful in predicting which patients with COPD will respond to long-term inhaled corticosteroid treatment.
Cystic Fibrosis
Surprisingly, exhaled and nasal NO levels are significantly
lower in patients with cystic fibrosis (CF) than in normal subjects, despite the intense neutrophilic inflammation in the airways (35) (Figure 3) (171) leading to the release of superoxide
anions, which convert NO to nitrate and may result in the formation of peroxynitrite (172). Increased oxidative stress in CF
is likely to be a consequence of this neutrophilic inflammation,
malnutrition, and IL-10 deficiency (173, 174). Although there
is a trend toward both exhaled and nasal NO being higher in
patients who were not homozygous for the 
F508 CF transmembrane regulator mutation (175, 176), there is no strong
association between exhaled NO and disease severity in CF
(176) or infection with Pseudomonas (35).
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There are several possible reasons for the low levels of NO in patients with CF. First, there is a deficiency of NOS2 in patients with CF (177). Constitutive expression of NOS2, which has been demonstrated in normal human airway epithelium, and of non-CF mouse is essentially absent in the epithelium of CF airways (178). Neutrophils enhance expression of NOS2 in normal human bronchial epithelial cells but not in CF epithelial cells (179). The low expression of NOS2 would account for the low levels of NO in nasal as well as exhaled air. Secondly, an association between the length of a repeat polymorphism in the NOS1 gene and exhaled NO in patients with CF has recently been demonstrated, and exhaled NO is significantly lower in patients with CF with two alleles with a high number of repeats than in those alleles with fewer repeats at this locus (180). Interestingly, Pseudomonas aeruginosa colonization is more common in patients with CF with high numbers of repeats in the NOS1 gene, hence with lower exhaled NO.
Sexual hormones have impact on cystic fibrosis transmembrane mRNA expression (181) and it is not unusual that female patients with CF have reported worsening of lung symptoms prior to menstruation. Changes in exhaled NO during the menstrual cycle with the lowest NO levels during menstruation have been observed (66). Although NO is a weak bronchodilator and the physiologic significance of this finding is still not known, it has been shown that FEV1 was significantly lower during menstruation in female patients with CF (182).
Bronchiectasis
An increase in exhaled NO is found in bronchiectasis and the increase in NO is related to the extent of disease as measured by a computerized tomography score (183). As in asthma, the elevation of exhaled NO is not seen in patients treated with inhaled corticosteroids. This suggests that exhaled NO in bronchiectasis may reflect active inflammation in the lower airways and may be used to monitor disease activity. This is supported by increased NOS2 expression in lungs from patients with bronchiectasis (184). However, in another study, exhaled NO levels were not elevated compared with normal subjects in clinically stable patients with bronchiectasis, and it was suggested that NO is either trapped in viscous airway secretions or removed by reaction with reactive oxygen species (185).
Primary Ciliary Dyskinesia
Primary ciliary dyskinesia (PCD), including Kartagener's syndrome, is a genetic disease characterized by defective motility of cilia, in which the levels of exhaled NO are very low compared with normal subjects (186) (Figure 3) (187, 188). Such low values of exhaled and nasal NO are not seen in any other condition and are therefore of diagnostic value. Measurement of exhaled NO might be used as a screening procedure to detect PCD among patients with recurrent chest infections or male infertility caused by immotile spermatozoa, and the diagnosis of PCD is then confirmed by the saccharine test, nasal nitric oxide, ciliary beat frequency, and electron microscopy (189). NO plays an important role in bactericidal activity in the lungs, sodium and chloride transport in nasal epithelium, and ciliary beating (190), so that a lack of endogenous NO production might contribute to the characteristic recurrent chest infections in patients with PCD. Low levels of exhaled and nasal NO in patients with PCD are related to mucociliary dysfunction (186, 191), and treatment with NO donor L-arginine increases nasal NO and also improves mucociliary transport in patients with PCD (3, 186). The mechanism for such a low NO production by nasal and airway epithelia in PCD is unknown, but it might be linked to genetic abnormalities in NOS2 gene expression as in CF.
Rhinitis
The levels of NO derived from the upper respiratory tract are more than 100-fold higher than those from lower airways. This fact is mostly due to its high production in human paranasal sinuses (43), which is due to high basal activity of constitutively expressed form of NOS2 (192), and nasal NO may be significantly reduced by L-NAME in normal subjects (72), and this inhibition of NOS may induce hyperresponsiveness of the nasal airway (193). Strong NOS3- and weak NOS2-immunoreactivity are found in nasal epithelium and submucosal glands of normal subjects, but NOS2 reactivity is increased in patients with allergic rhinitis (46). There is increased immunoreactivity of nitrotyrosine in the nasal mucosa of patients with perennial rhinitis and is related to the severity of the nasal symptoms (194). However, an increased expression of iNOS is not necessarily associated with a higher 3-nitrotyrosine-labeling intensity (195), suggesting that iNOS-derived NO may have a role in the pathophysiology of rhinitis, but the production of peroxynitrite in patients with rhinitis is not dependent on the level of iNOS alone. Eotaxin causes chemotaxis of eosinophils, an increase of nitrotyrosine-immunoreactivity in nasal mucosa and increased levels of nasal NO in clinically symptomatic patients with allergic rhinitis (196). Instillation of LTB4 into the nasal segment caused a time-dependent increase in the volume of airway fluid and in the recruitment of neutrophils in dogs, and was prevented by L-NAME (197). Recently, it has been shown that the nasal decongestants oxymetazoline and xylometazoline, frequently used in the topical treatment of rhinitis and sinusitis, may have a dose dependent inhibitory effect on total iNOS activity (198).
Elevated nasal NO has been reported in allergic and perennial rhinitis (199, 200), which is reduced by treatment with nasal corticosteroids (200). Similar results are seen in children with allergic rhinitis (201). In addition, exhaled NO is also significantly elevated in allergic rhinitis in the nonpollen season and is increased further in the pollen season (202). However, the differences between the levels of nasal NO in rhinitis compared with those in normal subjects and much less marked than the differences between exhaled NO between patients with asthma and normal subjects because of the very high baseline values. This makes nasal NO less useful for diagnosis and monitoring treatment in rhinitis than exhaled NO in asthma.
Interstitial Lung Diseases
Systemic sclerosis. In patients with systemic sclerosis who have developed pulmonary hypertension, there is a reduction in exhaled NO compared with that in normal subjects and with that in patients with interstitial lung disease without pulmonary hypertension (203, 204). This may be due to reduced expression of NOS3 in pulmonary vessels, or a reduction in the pulmonary vascular endothelial surface. However, the presence of NOS3 in pulmonary vessels is variable, and it has been found to be either reduced (205), increased (208), variable (209), or unaltered (210).
Fibrosing alveolitis. There is strong expression of nitrotyrosine and NOS2 in macrophages, neutrophils, and alveolar epithelium in lungs of patients with idiopathic pulmonary fibrosis with active inflammation during the early to intermediate stage of the disease (211). This is consistent with elevated levels of exhaled NO in patients with fibrosing alveolitis. Increased exhaled NO levels are associated with disease activity, as assessed by BAL lymphocyte counts, and are reduced in patients treated with corticosteroids (212).
Sarcoidosis. Cytokines, including TNF- and interferon-,
are increased in the pulmonary inflammation of sarcoidosis
and there is an up-regulation of NOS2 in respiratory epithelium and granulomata in patients with sarcoidosis (213). The
magnitude of the rise in exhaled NO in sarcoidosis may be related to the activity of the disease and is reduced by steroid
therapy. This is, perhaps, the reason behind two conflicting
observations reporting either elevated (213) or normal (214)
exhaled NO in patients with active pulmonary sarcoidosis.
Pulmonary Hypertension
The pathogenesis of pulmonary hypertension remains poorly understood. Vasoconstriction is likely to be a major factor in the initial stages of the disease, and a reduction in endogenous NO may contribute to the development of pulmonary hypertension. In fact, nebulized epoprostenol increased exhaled NO in patients with pulmonary hypertension, but not in normal control subjects, suggesting that this effect on the hypertensive circulation has a NO-related mechanism (215). In contrast, the angiotensin-converting enzyme (ACE) inhibitor enalapril, used to treat pulmonary hypertension, increases exhaled NO levels in normotensive subjects, but not in patients with systemic hypertension (216).
Biochemical reaction products of NO are inversely correlated with pulmonary artery pressures in patients with primary pulmonary hypertension and with years since the diagnosis (217). This may reflect reduced expression of NOS3 in patients with pulmonary hypertension, as reduced NOS3 expression has been reported in patients with primary pulmonary hypertension (205). In fact, aerosolized NOS2 gene transfer increases pulmonary NO production and reduces hypoxic pulmonary hypertension in rats (218) and may be a promising future strategy to target pulmonary vascular disorders.
However, interpretation of these low NO levels should be made cautiously and in the context of potential influence of Hb on NO. Although stimulation of NO production by pulmonary vascular endothelial cells in response to shear stress has been described, it is not an important determinant of NO production. Low exhaled NO in patients with pulmonary hypertension may be consistent with flow redistribution from alveolar septal capillaries to extra-alveolar vessels and decreased surface area or a direct, stretch-mediated depression of lung epithelial NO production (219), or increased Hb NO scavenging. It may be difficult to use exhaled NO changes as an accurate measure of lung tissue NO production.
Occupational Diseases
Allergens from rats, mice, guinea pigs, or rabbits cause as much as 30% of exposed persons to develop specific immunoglobulin E (IgE) responses. Laboratory animal allergy (LAA) is among the highest occupational risks for asthma. Exhaled NO is raised in subjects with LAA symptoms and correlates with symptom severity (97). The progressive increase in exhaled NO from asymptomatic to early LAA to symptomatic asthma suggests that exhaled NO measurements may be useful in monitoring occupational asthmas, and of environmental health effects of air pollution (220) in epidemiologic surveys. Recently, measurement of exhaled NO and induced sputum were evaluated in occupational asthma. Aluminum potroom workers (exposure to dust and fluorides) with asthmalike symptoms had higher concentrations of exhaled NO than did those with no symptoms (221), suggesting that exhaled NO may be an early marker of airway inflammation in potroom workers. High levels of exhaled NO and asthmalike symptoms in subjects with occupational exposure to high levels of ozone and chlorine dioxide (78), or in swine confinement workers (162), may indicate the presence of chronic airway inflammation. Latex sensitivity is an increasing problem among healthcare workers. Although allergen challenge with natural rubber latex increased exhaled NO levels after 22 h in some subjects with suspected occupational asthma (222), further studies are needed to demonstrate a clear relationship between exhaled NO and routine latex workplace exposure.
Infections
NO may play an important role in nonspecific host defenses against bacterial, viral and fungal infections. One of the general mechanisms of antimicrobial defenses involving NO is S-nitrosylation by NO of cysteine proteases, which are critical for virulence, or replication of many viruses, bacteria, and parasites. The reduced endogenous NO production, resulting in low exhaled and nasal NO levels, may contribute to recurrent chest infections in patients with PCD or CF, as discussed above. Low nasal NO is associated with colonization of the upper respiratory tract with Staphylococcus aureus in active Wegener's granulomatosis (223).
Viral infections. Exhaled, but nasal, NO is elevated during
viral infections in adults and in children (84, 86). Exhaled NO
is also increased in experimental human influenza (224) and rhinovirus infection (225). The increase in NO production
during viral infection is likely to be protective, as NO inhibits
virus replication either by inhibiting viral RNA synthesis, or/
and by S-nitrosylation of the cysteine proteases that are critical for virulence and replication of viruses (226). Viral infection may also induce the expression of NOS2 via activation of
NF-B and other transcription factors (227). Exhaled (228)
and nasal NO (229) in HIV positive patients is less than in
control subjects, and NO synthesis is further depressed in terminally ill patients with HIV (230), suggesting that low NO
may indicate a mechanism of impaired host defense in HIV infection. This may be explained by an inhibitory role of the
HIV type 1 regulatory protein Tat on NOS2 activity in a murine macrophage cell line (231).
Tuberculosis. NO plays an important role in resistance to Mycobacterium tuberculosis infection, and exposure of extra-cellular M. tuberculosis to < 100 ppm of NO for a short period (< 24 h) results in microbial killing (232). Elevated exhaled NO and NOS2 expression in alveolar macrophages is found in patients with active tuberculosis and is reduced with antituberculosis therapy (233).
Bacterial infections. Nitrate concentrations are significantly higher in BAL in immunosuppressed children with pneumonia than in normal control subjects (234), and elevated exhaled NO levels are found in patients with lower respiratory tract inflammation and chronic bronchitis (162).
Chronic Cough
Increased levels of exhaled NO do not accompany all forms of airway inflammation. Patients with chronic cough that is not attributable to asthma have lower NO values than do healthy volunteers and patients with asthma (88, 134), including those with cough caused by gastroesophageal reflux (235). Measurement of exhaled NO may therefore be a useful screening procedure for patients with chronic cough and would readily identify those patients with cough caused by asthma (88).
Lung Cancer
The levels of nitrite in epithelial lining fluid and exhaled NO are significantly higher in patients with lung cancer than in control subjects, and they are correlated with the intensity of NOS2 expression in alveolar macrophages (236). The level of nitrite was also significantly higher in epithelial lining fluid from patients with cancer, but the increased NO production is not specific to the tumor side and might be attributed to a tumor-associated nonspecific immunologic and inflammatory mechanism.
Lung Transplant Rejection
Monitoring endogenous NO release may be useful in lung transplantation. Loss of endogenous production of NO by cadaver lung allografts in the perioperative period (237), and the fact that reduced exhaled NO after hypoxia-reoxygenation might reflect bronchial epithelial dysfunction (238), may provide a rationale for interventions to restore NO production and, therefore, to improve the outcome of the surgery. The development of postlung transplant obliterative bronchiolitis is the commonest cause of late graft failure and is characterized by intense airway inflammation and high exhaled NO, which are higher than in either control subjects or stable lung transplant recipients (239). In stable lung transplant recipients, exhaled NO concentrations are highly dependent upon the severity of BAL neutrophilia and the intensity and extent of expression of NOS2 in the bronchial epithelium, but not in the subepithelial area (240). This suggests that serial exhaled NO measurements may have a role in the early detection of obliterative bronchiolitis (240) or of acute rejection (241).
Adult Respiratory Distress Syndrome
Adult respiratory distress syndrome (ARDS) is associated with a neutrophilic alveolar inflammation. In animal models of ARDS induced by endotoxin there is increased production of NO (242). Exhaled NO values are low, presumably because of the concomitant oxidative stress and consumption of NO by superoxide anions to form peroxynitrite (243). Association of reduced exhaled NO levels with the increases in pulmonary artery pressure and alveolar-arterial oxygen pressure and the decrease in lung compliance (244) suggests that exhaled NO may be an indicator of lung injury in adult patients after cardiopulmonary bypass.
Diffuse Panbronchiolitis
Diffuse panbronchiolitis (DPB), a pulmonary disease of unknown origin with chronic inflammation in the respiratory bronchioles leading to chronic chest infections resulting from mucociliary dysfunction, is the third disease (after primary ciliary dyskinesia, and cystic fibrosis) with diagnostically low nasal NO levels (245). Airway impaired NOS activity may be involved in its pathogenesis, and NO measurements may serve as a noninvasive test in the diagnosis of DPB.
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CARBON MONOXIDE |
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CONTENTS
INTRODUCTION
NITRIC OXIDE
CARBON MONOXIDE
EXHALED HYDROCARBONS
EXHALED BREATH CONDENSATE
OTHER METHODS
FUTURE DIRECTIONS
REFERENCES
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Carbon monoxide (CO) is a gas that may be formed endogenously and is detectable in exhaled air.
Source of Exhaled CO
There are three major sources of CO in exhaled air: enzymatic degradation of heme, non-heme-related release (lipid peroxidation, xenobiotics, bacteria) and exogenous CO. The predominant endogenous source of CO (~ 85%) in the body is from the degradation of hemoglobin by the enzyme heme oxygenase (HO), and approximately 15% arises from degradation of myoglobin, catalase, NO synthases, guanylyl cyclase and cytochromes (246). Several bacteria produce CO (247), but this does not play an appreciable role in the turnover of CO that is inhaled or endogenously produced. Approximately 85% of the CO in the body is bound to hemoglobin in circulating erythrocytes and the remaining 15% is bound to other compounds (such as myoglobin) or in tissues, and less than 1% is unbound and dissolved in body fluid (248). Approximately 80% of the CO formed from heme degradation is exhaled (249). CO uptake or excretion across the skin is minimal, except in premature infants, and the amount of CO consumed by the tissues is very small (3% of the rate of endogenous CO production) (250).
There are several reasons to consider that the alveoli are the predominant site of exhaled CO in normal subjects. First, levels of exhaled CO measured at the end of exhalation are similar to those measured via a bronchoscope at the level of main bronchus (251). Second, exhaled CO levels are less flow- or breathhold-dependent than exhaled NO (252), suggesting less airway contribution. Third, maximal CO levels are seen close to the end of exhalation, as for CO2. There is also a small proportion of CO derived from the airways, which is higher after allergen challenge measured either via bronchoscope (251), or at the mouth (104). The fact that breathing through the nose increases the CO levels obtained in the exhaled air (253) suggests that nose and paranasal sinuses may also contribute to the CO production of the human airways. Indeed, HO-like immunoreactivity is seen in the respiratory epithelium, in connection with seromucous glands and in the vascular smooth muscle of the nose (253).
Heme oxygenase. CO is a by-product of rate-limited oxidative cleavage of hemoglobin by HO, which exists in three isoforms, i.e., HO-1, HO-2, and HO-3. HO-2 is constitutively expressed in most tissues, whereas HO-3 is, so far, only described in rats (254). HO-1 has been identified as the major 32 kD heat shock (stress) protein (255). Like other stress proteins HO-1 can be induced by a variety of stimuli, such as proinflammatory cytokines, bacterial toxins, heme, ozone, hyperoxia, hypoxia, reactive oxygen species, and reactive nitrogen species. Both HO-1 and HO-2 are expressed in human airways and are found in most cell types, with particularly strong immunfluorescence in airway epithelial cells (256). Heme is converted by HO to biliverdin and thence to bilirubin, with the formation of CO and ferritin (Figure 4).
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Interactions with NO. Like NO, CO is also capable of up-regulating cyclic guanine monophosphate (cGMP) via activation of guanylyl cyclase causing vasodilatation, smooth-muscle relaxation, and platelet disaggregation. The vasodilatory effect of CO may be important in maintaining adequate tissue oxygenation and perfusion in the lung during normal physiology and in hypoxic conditions that result from pulmonary vascular diseases and acute lung injury. It has been suggested that the HO pathway exerts important counter-regulatory effects on the NOS pathway and, when blocked, the underlying NOS pathway is unmasked leading to increased and prolonged release of NO (257). In contrast, exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells resulting in a higher resistance to oxidant damage (258). This effect of NO is related to the release of free heme from heme proteins, which are able to transcriptionally up-regulate HO-1 and lead to their own degradation. CO also directly inhibits NOS2 activity by binding to the heme moiety of the enzyme (259). The effect of hemoglobin scavenging, as a function of the extent of bronchial arterial neovascularization (e.g. bronchiectasis, thromboembolic disease) may play an important role in the reaction between erythrocytic hemoglobin and NO. This interaction has been generally considered in the context of mechanisms that safely detoxify NO. More recently, hemoglobin-dependent mechanisms that preserve, not destroy, NO bioactivity in vivo have also been proposed (260). The emerging picture suggests that the interplay between NO and erythrocytic hemoglobin is important in regulating the functions of both these molecules in vivo. Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has been successfully studied in Phase II clinical trials, and may be used for the treatment of shock associated with the systemic inflammatory response syndrome (261). The redox activity of modified hemoglobins can be attenuated, so that modified hemoglobins containing endogenous antioxidants such as PHP may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including severe asthma, CF, COPD, and bronchiectasis.
Effect of oxidative stress. There is a close link between the reactive oxygen and nitrogen species and CO. Thus, a dose-dependent increase in exhaled CO has been shown after a 1-h exposure to different concentrations of O2 (262). HO-1 activation can be diminished by N-acetylcysteine, a precursor of glutathione with antioxidant properties (263). Both, superoxide anions and peroxynitrite can stimulate HO-1 activation (264), and subsequent release of CO is an important negative-feedback regulatory mechanism limiting the release of these cytotoxic substances (265). Animals exposed to a low concentration of CO exhibit a marked tolerance of the lungs to lethal concentrations of hyperoxia in vivo (266).
The precise mechanisms for this protection are not fully
understood, but both the degradation of heme (with removal
of iron and induction of ferritin) and the generation of bilirubin (an antioxidant) may be involved. There is evidence that
the deleterious effects of ROS, such as superoxide and H2O2,
are dependent on the presence of iron. The intracellular pool
of free iron can react with both H2O2 and superoxide, giving
rise to the OH
radical via the Fenton reaction. The free iron
that is not metabolized intracellularly sequestered in cells as
ferritin. Thus, ferritin serves as a reservoir to restrict iron from
participating in the Fenton reaction. It has been shown that
free iron released from heme by HO may induce ferritin synthesis, and heme-induced HO-1 protein also activates ferritin
via mRNA expression (267). Furthermore, the metabolite of
heme degradation, bilirubin, is itself an effective antioxidant
of peroxynitrite-mediated protein oxidation and may be even
more effective than vitamin E in preventing lipid peroxidation (268). Moderate overexpression of HO-1 improves the resistance of cells to oxygen toxicity (269). However, there is cytotoxicity associated with HO-1 overexpression.
HO-2 may also protect against oxidative stress. HO-2 knockout mice are sensitized to hyperoxia-induced oxidative injury, have a higher mortality, and increased lung iron content without increased ferritin, suggesting accumulation of available redox-active iron (270).
Measurement
Exhaled CO as a marker to assess different diseases (cardiovascular, diabetes, and nephritis) was first described in Russia 1972 (271). Over the last 20 yr exhaled CO has been measured to identify current and passive smokers, to monitor bilirubin production, including hyperbilirubinemia in newborns, and in the assessment of the lung diffusion capacity.
CO can be quantified by a number of different techniques. Most of the measurements in humans have been made using electrochemical CO sensors. The sensor is selective, gives reproducible results (272), and is inexpensive. However, these instruments are susceptible to interference from a large number of substances, for example, hydrogen, which is present in exhaled breath and may be increased after glucose ingestion. H2-insensitive CO sensors, which are now available, are therefore recommended.
Exhaled CO can also be measured (at ppb level) by adjustable laser spectrophotometer (262, 273), or by a near-infrared CO analyzer (274). Near-infrared instruments, are used for continuous monitoring of atmospheric CO, and are fairly sensitive and stable. However, they are larger than electrochemical CO sensors, sensitive to water and CO2 concentrations, and require large sample volumes (275). This may explain the low CO levels detected by these instruments even after a prolonged breathhold time of 20s (274). Gas chromatography is a reference method for CO measurements, but its use is limited to specialized laboratories.
End-tidal exhaled CO measurements can be made during a single exhalation and is a routine in cooperative adults. It can also be easily performed in children older than 5 yr of age (276). A method for measuring CO in nasally sampled exhaled air in noncooperative neonates has been developed that involves the relatively noninvasive placement of a small catheter into the posterior of the nasopharynx and the collection of breath samples either manually or automatically (249).
Factors Affecting Exhaled CO Measurements
CO exists in the atmosphere as a by-product of incomplete combustion and oxidation of hydrocarbons, and is oxidized to CO2 by hydroxyl radicals, or eliminated either by soil microorganisms or by stratospheric diffusion. Regional and local levels of CO in ambient air can vary significantly depending on time of the day and season, on wind velocity, industrialization, traffic, and altitude. Although some exposure to CO may occur in normal day-to-day life because of environmental pollution, active or passive smoking are the most likely reason for high levels of exhaled CO. After inhalation, CO displaces oxygen in the erythrocyte to form carboxyhemoglobin (COHb), which has a half-life of about 5 to 6 h in this form. A cutoff level of 6 ppm (277) effectively separates nonsmokers from smokers, and the previously used cutoff 8 ppm (278) or 10 ppm (279) may be too high. Other individual factors, which can markedly affect the amount of CO that a person may inhale, are type and location of home and occupation, cooking/ heating appliances, and mode of transportation.
Many pathologic conditions and factors can increase the rate of hemoprotein breakdown and potentially increase the levels of exhaled CO, including anemias, hematomas, and preeclampsia. Nonpathologic factors may also increase endogenous CO production, including fasting, dehydration, some drugs (phenobarbitone), and xenobiotic compounds (paint remover) (280) (Table 2).
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Asthma
Elevated levels of exhaled CO have been reported in stable asthma (281, 282) with normal levels in patients treated with inhaled corticosteroids (282). The difference in exhaled CO between normal and asthmatic subjects, however, is much less than in exhaled NO (283), and the effect of inhaled steroids on exhaled CO in patients with mild asthma, as it has been reported recently, is negligible (256). Both HO-1 and HO-2 are extensively distributed in airways of normal and asthmatic subjects (256). The increased levels in stable asthma are likely to be due to preferential increase of HO-1 expression, which is seen in alveolar macrophages in induced sputum of patients with asthma (263). There is also an increase in the concentration of bilirubin in induced sputum, indicating increased HO-1 activity (263). Further evidence that exhaled CO increases may reflect HO activity is the demonstration that inhaled hemin, which is a substrate for HO, results in a significant increase in exhaled CO concentration in normal and asthmatic subjects (263). Increased levels of exhaled CO are seen in acute exacerbations of asthma, and are reduced after treatment with oral corticosteroids (284). Significantly elevated CO levels are found in patients with severe asthma (285), including patients treated with 30 mg of prednisolone for 2 wk (286). In view of the simplicity of CO measurements and the portability of CO analyzers, exhaled CO may be useful in noninvasive monitoring of pediatric asthma. For example, children with persistent asthma despite treatment with steroids, which reduce their NO levels, have significantly higher exhaled CO than do those with infrequent episodic asthma (276).
COPD
A major limitation of exhaled CO in COPD is the marked effects of cigarette smoking, which masks any increase that may occur because of the disease process. There is no difference in exhaled CO in patients with chronic bronchitis (without airflow obstruction) when compared with normal subjects (287). However, exhaled CO levels are elevated in ex-smoking patients with COPD (288), suggesting ongoing oxidative stress or inflammation. HO is induced in fibroblasts exposed to cigarette smoke (289). There is an increase in exhaled CO during acute exacerbations of COPD, with a decline after recovery (290).
Bronchiectasis
Exhaled CO levels are elevated in patients with bronchiectasis, irrespective of whether they are treated with inhaled corticosteroids (291).
Cystic Fibrosis
In contrast to NO, exhaled CO levels were markedly elevated
in patients with stable CF (292) and increased further
during exacerbations and reduced with antibacterial treatment (Figure 5) (176). This suggests that exhaled CO is not
only a marker of oxidative stress/inflammation in CF, but is
also a marker of disease severity. This is further confirmed by
the finding of lower CO levels in patients receiving oral corticosteroid treatment (292). In fact, by reducing airway inflammation and the release of oxidants by inflammatory cells
steroids may attenuate HO-1 expression and the synthesis of
CO. We have shown that patients homozygous for the CF
transmembrane regulator F508 mutation have higher exhaled CO levels than do heterozygous patients (292). Considering the growing interest in gene therapy in cystic fibrosis, further studies are needed to investigate the role of CO levels in the assessment of effective therapeutic gene delivery or to confirm the diagnosis in patients with borderline sweat tests where more extensive genetic analysis is not available.
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Interstitial Lung Disease
Elevation of exhaled CO is related to lung function deterioration (295) and impaired gas transfer in patients with cryptogenic fibrosing alveolitis and scleroderma (296). Elevated levels of exhaled CO in patients with fibrosing alveolitis are also associated with disease activity as assessed by BAL cell counts (212). This suggests that exhaled CO may be used to monitor disease progression and response to therapy in interstitial lung diseases.
Allergic Rhinitis
Stable levels of CO are recorded during continuous sampling from one nostril during normal breathing through the mouth in normal subjects (253). Sampling through a drainage tube inserted into the maxillary sinus reveals CO levels comparable to the levels obtained by sampling through the nose. In patients with allergic rhinitis exhaled CO is increased during the pollen season and returns to normal values after the season (297). The levels of exhaled CO are significantly higher in patients with symptoms than in those without. However, there is no correlation between nasal and exhaled samples, suggesting that the increase is derived from the lower respiratory tract. We did not measure any direct nasal CO production in either normal or asthmatic subjects (283).
Infections
HO-1 is induced by many infectious agents, and HO-1 may provide protection to cells against attack by infectious agents. Upper respiratory tract viral infections may induce the expression of HO-1, resulting in increased exhaled CO in adults (298) and in children (276). Elevated exhaled CO levels might provide an early warning signal for an acute infective episode, which may lead to exacerbation of asthma and COPD. Elevated levels of CO have been measured in patients in general practice with lower respiratory tract infection, which were significantly reduced after 5 d of treatment with antibiotics (290).
Other Conditions
Critically ill patients have a significantly higher CO concentration in exhaled air as well as total CO production than do healthy control subjects (299), but inspired oxygen concentration has to be measured, as it can influence CO excretion in mechanically ventilated patients (300). Interestingly, the levels of exhaled CO in these patients are similar to the levels seen in severe asthma and may be a reflection of systemic rather than the local oxidative stress.
Exhaled CO levels are also increased in diabetes, and the level is significantly related to the level of hyperglycemia (301). The mechanism is unclear, but hyperglycemia and oxidative stress in uncontrolled diabetes may activate HO-1.
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