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The Proceedings of the American Thoracic Society 1:191-199 (2004)
© 2004 The American Thoracic Society

Effects of Corticosteroids on Noninvasive Biomarkers of Inflammation in Asthma and Chronic Obstructive Pulmonary Disease

Sergei A. Kharitonov and Peter J. Barnes

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, Royal Brompton Hospital, London, United Kingdom

Correspondence and requests for reprints should be addressed to Dr. Sergei A. Kharitonov, M.D., Ph.D., Department of Thoracic Medicine, National Heart & Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail: s.kharitonov{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
NONINVASIVE BIOMARKERS OF...
 STANDARDIZATION AND...
 USING BIOMARKERS TO ASSESS...
 NOVEL TECHNOLOGIES FOR BIOMARKER...
 REFERENCES
 
Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in the differential diagnosis of airway diseases, and in the monitoring of therapy. The greatest progress has been made with standardized measurement of exhaled nitric oxide (NO). Bronchial NO is increased in asthma, correlated with other markers of inflammation, and reduced by treatment with corticosteroids and antileukotrienes. Alveolar NO is increased in chronic obstructive pulmonary disease (COPD), reflecting disease severity and progression. Exhaled carbon monoxide and ethane are increased in both asthma and COPD. Increased concentrations of 8-isoprostane, hydrogen peroxide, nitrite, and nitrotyrosine are found in exhaled breath condensate from patients with inflammatory lung diseases. Increased levels of lipid mediators are also found, and the pattern depends on the nature of the disease process. Additional biomarkers are exhaled breath temperature, which is elevated in asthma and reduced in COPD, and bronchial blood flow. It is likely that smaller and more sensitive analyzers will extend the discriminatory value of exhaled breath analysis and that new techniques will become available to diagnose and monitor respiratory diseases in the family practice and home settings.

Key Words: bronchial blood flow • exhaled breath analysis • exhaled breath condensate • exhaled breath temperature • exhaled nitric oxide

The need to monitor inflammation in the lungs has led to the exploration of exhaled gases and condensates that may assist in differential diagnosis of pulmonary diseases, assessment of disease severity, and assessment of response to treatment (1–3). Noninvasive, reliable, biomarkers providing a quick readout are needed to speed up proof-of-concept studies in patients with asthma and chronic obstructive pulmonary disease (COPD). This review examines the practical aspects of the examination of surrogate markers in the assessment of drug efficacy in the treatment of patients with asthma or COPD, with special attention to which indexes are clinically useful.


   NONINVASIVE BIOMARKERS OF INFLAMMATION IN ASTHMA AND COPD
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 NONINVASIVE BIOMARKERS OF...
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Table 1 compiles study results on exhaled gases that have been used as biomarkers of inflammation in asthma and COPD, as well as two alternative quantitative markers, exhaled breath temperature and bronchial blood flow. Tables 2 and 3 review data on biomarkers of exhaled breath condensate (EBC) in asthma and COPD, respectively.


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  		TABLE 1. Exhaled gases, exhaled breath temperature, and bronchial blood flow in asthma and copd

 

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  		TABLE 2. Biomarkers of exhaled breath condensate in asthma

 

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  		TABLE 3. Biomarkers of exhaled breath condensate in copd

 
Exhaled Gases
Nitric oxide.
Nitric oxide (NO) is the most extensively studied exhaled marker of airway inflammation, and abnormalities in exhaled and nasal NO have been documented in several lung diseases, particularly asthma (4). Exhaled NO and nitrite/nitrate levels in EBC can be used to monitor dose-dependent onset and duration of action of corticosteroids (5), and they are valuable parameters to monitor complex NO biochemistry in the clinic. The peripheral airways/alveolar region (6, 7) may be the predominant source of elevated exhaled NO in COPD (8), whereas an increased exhaled NO level in asthma is mainly derived from the larger airways (9).

An interesting method of measuring exhaled NO at several exhalation flow rates has recently been described (10). This novel technique can be used to monitor not only the disease but also the effect of treatment with NO modulators, which may have different mechanisms and sites of action.

Hydrocarbon.
Exhaled ethane levels are higher in steroid-naive patients with mild asthma compared with steroid-treated patients and normal subjects (11). Ethane is increased in normal smokers and patients with COPD (12, 13). Increased levels of volatile organic compounds in exhaled breath could be used as biochemical markers of exposure to cigarette smoke and oxidative damage caused by smoking. In fact, there is a correlation between ethane levels and the degree of airway obstruction in COPD (12).

Exhaled Breath Condensate
Exhaled breath condensate is collected by cooling or freezing exhaled air, a totally noninvasive technique, although the exact source of EBC is unclear. Although the collection procedure has not been standardized, there is strong evidence that abnormalities in EBC composition may reflect biochemical changes of airway lining fluid. Potentially, EBC can be used to measure the targets of modern therapy in clinical trials and to monitor asthma and COPD in the clinic.

Hydrogen peroxide.
Activation of inflammatory cells, including neutrophils, macrophages, and eosinophils, results in increased production of O2– and formation of hydrogen peroxide (H2O2). Because H2O2 is soluble, increased H2O2 in the airway equilibrates with air and can be detected in EBC. Thus, exhaled H2O2 has potential as a marker of oxidative stress in the lungs. H2O2 has been detected in EBC in healthy adults, and increased concentrations have been detected in individuals with asthma (14).

Cigarette smoking causes an influx of neutrophils and other inflammatory cells into the lower airways, and fivefold higher levels of H2O2 have been found in EBC of smokers compared with nonsmokers. In patients with stable COPD, levels of exhaled H2O2 are higher than in normal subjects, and are further increased during exacerbations (15).

Tyrosine, nitrotyrosine, nitrite, nitrate, and reactive nitrogen species.
A significant proportion of NO is consumed by chemical reactions in the lung, leading to formation of nitrite, nitrate, and S-nitrosothiol in the lung epithelial lining fluid. Elevated levels of S-nitrosothiols in EBC have been demonstrated in patients with asthma or COPD (16), increased nitrotyrosine in asthmatic airway epithelium has been inferred from immunostaining of lung biopsies (17), and elevated levels of nitrotyrosine have been observed in EBC from individuals with asthma (18).

Eicosanoids.
Eicosanoids are potent mediators of inflammation responsible for vasodilatation/vasoconstriction, plasma exudation, mucus secretion, bronchoconstriction/bronchodilatation, cough, and inflammatory cell recruitment. Exhaled prostaglandins, for example PGE2 and PGF2{alpha}, are detectable in EBC and are markedly increased in patients with COPD, whereas they are not significantly elevated in asthma (19, 20). In contrast, thromboxane B2 is increased in asthma but is not detectable in normal subjects or in patients with COPD (21).

Detectable levels of the leukotrienes LTB4, LTC4, LTD4, LTE4, and LTF4 have been reported in EBC of subjects with asthma and normal subjects (22, 23). The levels of LTE4, LTC4, and LTD4 in EBC are elevated significantly in patients with moderate and severe asthma (22), and steroid withdrawal in moderate asthma leads to worsening of asthma and further increases in exhaled NO and the concentration of LTB4, LTE4, LTC4, and LTD4 in EBC (24). Concentrations of LTB4 are increased in EBC of patients with stable COPD (21), COPD exacerbations, (25) or moderate or severe asthma (22). This suggests that LTB4 may also be involved in exacerbations of asthma and may contribute to neutrophil recruitment.

Isoprostanes are prostaglandin-like compounds formed by free radical–catalyzed lipid peroxidation of arachidonic acid. They are not simply markers of lipid peroxidation. They also possess biological activity and could be mediators of the cellular effects of oxidant stress and a reflection of complex interactions between reactive nitrogen species and reactive oxygen species. Levels of 8-isoprostane are approximately doubled in patients with mild asthma compared with normal subjects, and they are increased by about threefold in patients with severe asthma, irrespective of treatment with corticosteroids (26).

The relationship to asthma severity is a useful aspect of this marker, in contrast to exhaled NO. A relative lack of effect of corticosteroids on exhaled 8-isoprostane in patients with mild asthma has been confirmed in a placebo-controlled study with two different doses of inhaled corticosteroids (ICSs) (5). This provides evidence that ICSs may not be very effective in reducing oxidative stress.

The concentration of 8-isoprostane in EBC is also increased in normal cigarette smokers and, to a much greater extent, in patients with COPD (27). Interestingly, exhaled 8-isoprostane is increased to a similar extent in patients with COPD who are ex-smokers as in smoking patients with COPD, indicating that the exhaled isoprostanes in patients with COPD are largely derived from oxidative stress from airway inflammation, rather than from cigarette smoking. It has been shown, however, that levels of 8-isoprostane and hydrogen peroxide cannot be reproducibly assessed in exhaled breath condensate from healthy smokers because of their low concentration and/or the lack of sensitivity of the available assays (28).

pH.
Ammonia (NH3), a product of urease hydrolysis of urea to ammonia and carbamate, is one of the key steps in the nitrogen cycle. Ammonia in the respiratory tract may be able neutralize inhaled acid vapors and aerosols, mitigating the pulmonary effects of pollution (29), and has the potential to regulate NOS activity. Exhaled breath ammonia may be an important counteracting agent in a variety of respiratory conditions, as a low pH in exhaled breath condensate has recently been reported in asthma (30, 31). Net production of ammonia by the lungs, however, has not been described and it might be difficult to document because of inhalation of ammonia from the mouth, the arterial–venous pH gradient, and large blood flow (32). Although the pH of the condensate was reduced in asthma, this may be misleading, as CO2 was purged from these samples. Low pH that has been recently reported in patients with COPD (33) may also be related mostly to the CO2 purge, rather than to the disease pathophysiology. The fact that that acidic rinsing results in a considerable (90%), fast, and lasting for 1 hour reduction in exhaled ammonia in normal subjects (29) should be considered when ammonia is measured in exhaled condensate.

Exhaled Breath Temperature and Bronchial Blood Flow
Exhaled breath temperature and bronchial blood flow are other quantitative markers of airway inflammation, as vascular hyperperfusion plays an important role in tissue inflammation and airway remodeling. Exhaled breath temperature and blood flow is increased in asthma because of inflammatory new vessel formation and vasodilatation, but not in COPD, which may reflect changes in bronchial blood flow and tissue remodeling. As discussed in more detail later, we have developed and validated a novel technique for the measurement of exhaled breath temperature and bronchial blood flow (34) in asthma (35) and COPD (36).


   STANDARDIZATION AND REPRODUCIBILITY OF BIOMARKERS
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 NONINVASIVE BIOMARKERS OF...
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Airway inflammation in asthma has been shown to exhibit considerable biological variability, and it is not measured directly and routinely in clinical practice. Several approaches are currently used to measure airway inflammation in COPD, but they are invasive or semiinvasive (bronchoscopy and sputum induction) or indirect (PC20 and lung function), or they may be affected by the patient's perception or the use of bronchodilators. The reproducibility of these approaches is also variable.

Exhaled NO
Serial changes in fractional exhaled NO (FeNO) are more predictive of deterioration of asthma than are single measurements (30). Furthermore, increases in FeNO and asthma symptoms are seen before any significant deterioration in airway hyperresponsiveness, sputum eosinophils, or lung function during asthma exacerbations induced by steroid reduction (37, 38). These data suggest that FeNO may be used as a marker of loss of control in asthma (39).

It can be argued that changes in FeNO may be due to measurement error and/or the natural variability of airway inflammation over time. However, the reproducibility of FeNO measurements within a single day in both adults and children is superior to that of any conventional method of monitoring airway inflammation in asthma (40).

Other major advantages of FeNO measurement include its strong association with airway inflammation (1), even in asymptomatic patients with asthma (41); high sensitivity to steroid treatment; insensitivity to ß2-agonists; and noninvasiveness.

In particular, FeNO measurement has several advantages compared with spirometry. We have shown that the high reproducibility of FeNO measurement in both children and adults may allow the medical practitioner to obtain reliable results with two instead of three exhalations (40).

Secondly, staff training can be minimal, because FeNO measurement with the NO analyzer NIOX (Aerocrine, Solna, Sweden) is fully automated, and incorrect exhalation maneuvers by a patient (shorter than 10 seconds or above the certain limits of the exhaled flow) will not be accepted. Another advantage of FeNO measurement is that it does not require any extra encouragement, as may be the case with peak expiratory flow measurement.

There is no "learning effect" or systematic error with serial FeNO measurements, probably because of its simplicity and high reproducibility. In one of our studies, the mean ± pooled standard deviation of all measurements was 2.1 ± 1.25 ppb (40). These results suggest that if a patient's exhaled FeNO levels change more than 4 ppb between sessions, it is more likely due to the inflammatory process than to inaccuracy of the analyzer. This finding is valuable for potential use of FeNO in routine clinical practice. The ability to perform short-term monitoring of airway inflammation is particularly important in the light of the trend toward use of an ICS in combination with long-acting ß2-agonists (LABAs), such that the antiinflammatory and clinical effect of combination treatment may be seen within days or even hours. Another example is the use of inducible NO synthase (iNOS) inhibitors, a potential additional treatment for severe asthma or COPD, which can have an onset of action within minutes (42, 43) and last for 72 hours (43).

Our data suggest that standardized exhaled NO measurement may be a useful clinical tool to monitor airway inflammation, acceptable to both healthy and asthmatic adults and children as a part of their routine visit to a physician. It may also be useful in drug efficacy studies. Based on the knowledge that FeNO measurements are variable, just as peak expiratory flow measurements are, target FeNO values should be established in clinical trials. When patients exhibit values above or below the reference level, steroid treatment should be reduced or increased, respectively (44).

Exhaled Breath Condensate
Several methods of collecting EBC have been described. The most common approach is to ask the subject to breathe tidally via a mouthpiece through a nonrebreathing valve in which inspiratory and expiratory air is separated. During expiration, the exhaled air flows through a condenser, which is cooled to 0°C by melting ice (14) or to –20°C by a refrigerated circuit (18), and EBC is collected into a cooled vessel. A low temperature may be important for preserving labile markers such as lipid mediators during the collection period, because it usually takes between 10 and 15 minutes to obtain 1 to 3 ml of condensate. Exhaled breath condensate may be stored at –70°C and is subsequently analyzed by gas chromatography and/or extraction spectrophotometry, or by immunoassays (ELISA). Collection of EBC is a simple and well tolerated method that can be safely used in both children (23, 45) and adults (14, 18).

The presence of high concentrations of nitrite/nitrate from the diet may affect NO-related markers in EBC. It is important, therefore, to minimize and monitor salivary contamination. Subjects should rinse the mouth before collection and keep the mouth dry by periodically swallowing saliva.


   USING BIOMARKERS TO ASSESS THE EFFECTS OF TREATMENT
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 NONINVASIVE BIOMARKERS OF...
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Effects of ICS
Acute effect (onset of action).
There have been no direct measurements of acute ICS effects on airway inflammation and microvascular permeability in asthma and COPD, although the newer ICSs are more airway-selective, have greater receptor affinity (i.e., fluticasone), and may have faster onset of action owing to smaller particle size (e.g., beclomethasone in hydrofluoroalkane propellant). A rapid, topical antiinflammatory action may require a rapid means of monitoring the antiinflammatory effect.

Exhaled NO behaves as a "rapid response" marker that is extremely sensitive to steroid treatment, because it may be significantly reduced even 6 hours after a single dose of nebulized budesonide (46), or within 2 to 3 days (47) after regular treatment with ICS. We observed that the onset of action of inhaled budesonide on exhaled NO was dose-dependent, both within the initial phase (first 3 to 5 days of treatment) and during treatment Weeks 1, 2, and 3 (5). The mean difference between the effect of 100 µg and 400 µg budesonide was –1.55 ppb/day. There was no effect of either treatment or placebo on exhaled levels of carbon monoxide (CO) during either the onset or cessation of action (5).

A reduction in airway responsiveness to adenosine monophosphate (AMP) was seen within 2 hours of a single inhalation (100 µg) of fluticasone (48). A dose-dependent reduction in exhaled NO has been reported in patients with mild asthma within 3 to 5 days from the beginning of treatment (5). However, no dose-dependent effect (single dose of 100, 250, or 1,000 µg fluticasone) or cumulative effect (3 vs. 7 inhalations of 1,000 µg fluticasone) on airway responsiveness to AMP has been shown (48).

The above effects were evident without concomitant improvements in lung function. This disconnection between lung function and inflammatory markers may reflect the short-term positive effects of ICSs, which may occur within even 3 to 5 days (5), whereas improvements in lung function may take longer to occur. It remains unclear whether "rapid improvement" in inflammatory markers translates into a commensurate improvement in airway remodeling or exacerbations over the long term.

The reduction in nitrite/nitrate levels in EBC during the onset and cessation of action of ICS may also be fast, but it is not dose-dependent (5). This may be a reflection of the rather complex process of nitrite/nitrate formation and metabolism, as well as greater variability in the measurements compared with exhaled NO.

Exhaled CO has been suggested as a marker of airway inflammation in asthma (49). However, we did not find any changes in either exhaled NO or CO levels 3 and 6 hours after a single dose of 100 µg or 400 µg budesonide (5).

An acute vasoconstrictive effect of ICS can be demonstrated on skin by the skin blanching test, and this effect can now be shown in the lung using a new method of assessment of bronchial blood flow—perhaps best called the "lung blanching test." Measured by a novel method that we have recently developed (35, 50), a single dose of inhaled budesonide (800 µg) caused a rapid (within 30 minutes), significant, but transient (recovery at 60 minutes) reduction in bronchial blood flow (P. Paredi and colleagues, unpublished observations) (Figure 1) (34). Although the maximal fluticasone-induced (880 µg) reduction in airway mucosal blood flow was seen 30 minutes after drug inhalation in subjects with and without asthma (51), the recovery was slower (90 minutes). Both in absolute and relative terms, the maximum decrease in bronchial blood flow was greater in individuals with asthma than in normal subjects, which may be explained by the vasoconstrictive effect and/or reduction of airway microvascular permeability seen after a single dose of ICS.



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  		Figure 1. The effect of ICSs on airway mucosal blood flow. Bud indicates budesonide; FP, fluticasone propionate; NS, not significant.

 
Cessation of action (recovery).
An important question is how quickly exhaled NO levels recover when steroid treatment is stopped. We have shown that exhaled NO levels recovered rapidly during the first 3 to 5 days in all patients who stopped inhaled budesonide, and recovery was complete by the end of the first week off treatment. Interestingly, recovery of exhaled NO levels during the first 3 to 5 days was faster in patients who had stopped 400 µg/day of budesonide than in those who had been receiving 100 µg, independent of the degree of reduction in exhaled NO by the end of week 3 of treatment (5). The assumption that the effect of 400 µg/day of budesonide lasts longer when treatment is stopped does not, therefore, appear to be true.

Long-term effect.
In patients with COPD, treatment with steroids resulted in a significant reduction in both nitrotyrosine and iNOS immunoreactivity in sputum cells and correlated with improvement in FEV1 (6). Reactive nitrogen species may be involved in the reversible component of inflammation and the long-term progression of COPD and asthma that is suppressed by steroids or by the combination of iNOS inhibitor and an ICS (52). Patients with COPD with a partial bronchodilator response to inhaled salbutamol show a better response to ICS treatment, in reduction of elevated levels of exhaled NO, than do those without reversible airflow limitation (53).

Steroid treatment may also reduce lipid peroxidation in COPD, because patients receiving steroid treatment have lower levels of exhaled ethane than untreated patients (12). Exhaled 8-isoprostane levels are increased in asthma and COPD irrespective of treatment with corticosteroids (26). The lack of effect of corticosteroids on exhaled 8-isoprostane either suggests that ICSs may not be very effective in reducing oxidative stress or could be a function of the dose.

It is well established that exhaled NO is gradually reduced during the first week of regular treatment with an ICS, with maximal reduction at 3 or 4 weeks (1). The reduction in airway mucosal blood flow after a 2-week course of intermediate-dose fluticasone (440 µg daily) was modest (11%) but consistent in patients with asthma (50). It may be that a longer duration of ICS treatment would have resulted in a further decrease of airway mucosal blood flow in the patients with asthma.

Dose-dependent effect.
We have shown that the acute reduction in exhaled NO (within the first 3 to 5 days of treatment) and the chronic reduction (Days 7 to 21) are dose dependent in patients with mild asthma who are treated with low doses of budesonide (5). Serial exhaled NO measurements, as we recently suggested (1), may therefore be useful in studying the onset and duration of action of ICS, as well as in monitoring patient compliance. It is still unclear whether exhaled NO levels can be used to study the effect of higher doses of ICSs. We did not find any further reduction in NO levels in patients with mild asthma who were treated with 1,600 µg/day of budesonide for 3 weeks (54).

Single-dose effect.
We did not find any changes in either exhaled NO or CO levels 3 and 6 hours after a single dose of 100 µg or 400 µg budesonide (5). As discussed above, a single dose of inhaled budesonide (800 µg) caused a rapid (within 30 minutes), significant, but transient (recovery at 60 minutes) reduction in bronchial blood flow (P. Paredi and coworkers, unpublished observation) (34).

Effects of Combination Treatment
Combination inhalers (ICS and LABA) are currently in use as the first-line treatment in asthma. Recently, it has been shown that combination treatment produces a clinically significant improvement in health status and reduces daily symptoms in COPD. It is important, however, to monitor the underlying airway and alveolar inflammation in both diseases, independently of patients' lung function and symptoms, which are affected by LABAs. Surrogate markers may help us to see whether there is an additional antiinflammatory effect of combination treatment in these patients in the clinic.

In patients with mild to moderate persistent asthma, double the dose of fluticasone alone was superior to fluticasone plus salmeterol in exhaled NO and PC20 AMP, but not lung function (55). This shows that exhaled NO and PC20 AMP are more direct markers of underlying airway inflammation than is lung function and that they are not affected by salmeterol.

There is evidence that symptom-driven dosing with combination inhalers may be useful in the future, as long as the dose of the steroid can be determined by the degree of symptoms at a particular time. We suggest that the high sensitivity of exhaled NO may be used to adjust doses of combination therapy based on control of inflammation in asthma. This is important because a LABA may control symptoms and, therefore, mask underlying inflammation that is not adequately suppressed by corticosteroids. Portable, simple, and inexpensive exhaled NO analyzers (based on measurements other than the chemiluminescence principle of NO detection) could be available in the next few years.

Effects of Other Treatments
Inhaled ß2-agonist.
Neither short-acting ß2-agonists nor LABAs reduce exhaled NO (49). This is consistent with the fact that they do not have any antiinflammatory effects in asthma.

Leukotriene antagonist.
It is difficult to assess the antiinflammatory action of compounds that have no bronchodilator action and none of the profound immunomodulatory effects of corticosteroids. Nevertheless, some noninvasive inflammatory markers may be used in clinical studies to test the efficacy of leukotriene antagonists. Pranlukast blocks the increase in exhaled NO when ICSs are withdrawn, and montelukast rapidly reduces exhaled NO by 15 to 30% in children with asthma (56). Zafirlukast, which is as effective as formoterol in maintaining asthma control, causes a significant reduction in exhaled NO (57).


   NOVEL TECHNOLOGIES FOR BIOMARKER ASSESSMENT
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 NONINVASIVE BIOMARKERS OF...
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Genomics, proteomics, and metabonomics, which respectively can characterize the response of living systems to chemical exposure regarding gene expression, protein expression, or metabolic regulation, may help speed up drug development by the pharmaceutical industry. These noninvasive technologies offer rapid, mechanistic information and are, to some degree, quantitative. They may facilitate incorporation of toxicologic and clinical data at earlier stages of drug development and particularly aid biomarker discovery and characterization.

Proteomics
Measurement and identification of proteins in EBC is controversial (4), although higher concentrations of total protein in EBC have been found in young smokers compared with nonsmokers. Various proteins derived from airways and unlikely to be contaminated with saliva have been detected in EBC by two-dimensional electrophoresis (58). Although their range and source are still unclear, the proteins recovered in EBC might be used to monitor respiratory diseases noninvasively in the future.

Many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence (genomics). Proteomics has the potential to identify novel targets of tyrosine nitration in cells and tissues and depict the nitro- and phosphoproteome, or to identify proteins undergoing S-nitrosylation in vivo. Recently, a proteomic approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response (58). These targets included proteins involved in oxidative stress, apoptosis, adenosine triphosphate production, and other metabolic functions.

We have identified low levels of proteins in EBC that are distinct from saliva proteins (S.A.K. and P.J.B., unpublished data). Proteomics analysis has allowed us to detect proteins that undergo endogenous nitration and, therefore, to eliminate artifacts that may arise when EBC is exposed to exogenous NO or nitrating agents. The use of proteomics to detect proteins in exhaled breath promises to be a sensitive means to study the mechanisms, selectivity, and consequences of biological tyrosine nitration in asthma and COPD.

Metabonomics
Metabonomics is an emerging technology that enables rapid in vivo screening for toxicity, disease state, or drug efficacy (59). It combines the power of high-resolution nuclear magnetic resonance imaging with statistical data analysis methods to rapidly evaluate the metabolic "status" of a patient or healthy volunteer. Complementary to other profiling technologies such as proteomics and genomics, metabonomics provides a fingerprint of the small molecules contained in a given biofluid, such as EBC, and captures the whole metabolic profile of the cell through the time course of a study.

CONCLUSIONS
Exhaled breath analysis allows completely noninvasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including asthma and COPD. The techniques are simple to perform, may be repeated frequently, and may be applied to children and to patients with severe disease. They are useful in making a differential diagnosis, quantifying inflammation and oxidative stress, and assessing drug efficacy. Measurement of bronchial and alveolar NO is particularly promising, but other biomarkers worth pursuing include CO, ethane, 8-isoprostane, hydrogen peroxide, nitrite, nitrotyrosine, and lipid mediators.

Asthma is characterized by elevated exhaled temperature and blood flow, which may be an index of airway inflammation, in contrast to low exhaled temperature in COPD. This may further indicate the use of exhaled temperature of NO as a simple method for monitoring of pulmonary circulation, as well as the effect of ICS and NO modulators in asthma and COPD.


   ACKNOWLEDGMENTS
 
S.A.K. is a member of scientific Advisory Boards for Aerocrine and has received lecture fees from Aerocrine, AstraZeneca, MSD and research grants from GlaxoSmithKline, AstraZeneca, Duska, MSD and Aerocrine; P.J.B. has previously served as a consultant to GlaxoSmithKline and is a member of scientific Advisory Boards for GSK, Boehringer Ingelheim, Altana, Pfizer and has received lecture fees from GSK, AstraZeneca, Boehringer Ingelheim and unrestricted grants from GSK, AstraZeneca, Boehringer Ingelheim, Novartis, Millenium, and Scios.

(Received in original form February 17, 2004; accepted in final form May 4, 2004)


   REFERENCES
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  1. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 2001;163:1693–1722.[Free Full Text]
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  5. Kharitonov SA, Donnelly LE, Montuschi P, Corradi M, Collins JV, Barnes PJ. Dose-dependent onset and cessation of action of inhaled budesonide on exhaled nitric oxide and symptoms in mild asthma. Thorax 2002;57:889–896.[Abstract/Free Full Text]
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