Sergei A. Kharitonov

Increased nitric oxide in exhaled air of asthmatic patients

Nitric oxide (NO) gas is produced by various cells within the lower respiratory tract, including inflammatory and epithelial cells, and is detectable in the exhaled air of normal human subjects. We have measured exhaled NO in patients with asthma, since several cell types that are activated in asthma can produce NO after induction. NO was measured reproducibly by a slow vital capacity manoeuvre and an adapted chemiluminescence analyser. NO was detectable in exhaled air of 67 control subjects (mean peak concentration 80.2 [SE 4.1] ppb) and was significantly reduced by inhalation of the specific NO synthase inhibitor NG-monomethyl-L-arginine. 61 non-steroid-treated asthmatic subjects had significantly higher peak expired NO concentrations than controls (283 [16] ppb, p < 0.001) but 52 asthmatic patients receiving inhaled corticosteroids had levels similar to controls (101 [7] ppb). High exhaled NO concentrations in asthmatic patients may reflect induction of NO synthase, which is known to be inhibited by steroids. Measurement of exhaled NO concentrations may be clinically useful in detection and management of cytokine-mediated inflammatory lung disorders.

Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma

BACKGROUND: Eosinophils in induced sputum and exhaled nitric oxide (NO) are currently used as non-invasive markers in the assessment of airway inflammation in asthma. As both sputum eosinophils (%) and exhaled NO are raised in asthmatic subjects not receiving inhaled steroids and decreased following corticosteroid therapy, a relationship between them is plausible. METHODS: Exhaled NO was measured by chemiluminescence analyser, sputum induction by 3.5% saline inhalation, and bronchial responsiveness was measured as PC20FEV1 methacholine in 35 stable asthmatic patients using beta 2 agonist alone and the correlation between these non-invasive markers of airway inflammation was studied. RESULTS: There were significant correlations between exhaled NO and PC20 (r = -0.64), exhaled NO and sputum eosinophils (%) (r = 0.48), and also between sputum eosinophils (%) and PC20 (r = -0.40). CONCLUSION: The correlation between exhaled NO and PC20 suggests that exhaled NO or the mechanisms leading to its increase may contribute to airway hyperresponsiveness in asthma. Furthermore, the relationship between sputum eosinophils (%), exhaled NO, and PC20 highlight the potential use of eosinophils (%) in induced sputum and exhaled NO to monitor the severity of asthma.

Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections

Viral infection may induce the expression of nitric oxide (NO) synthase, resulting in increased NO formation that has an antiviral effect. NO may be produced by various cells of the upper and lower respiratory tract, and may be detected in the exhaled air. We have studied the levels of exhaled NO in 18 normal subjects during symptomatic upper respiratory tract infections and during recovery 3 weeks later. Exhaled NO was measured using a modified chemiluminescence analyser. At the time of symptoms of upper respiratory tract infection, the peak exhaled NO values were 315 +/- 57 ppb (mean +/- SEM) and decreased to 87 +/- 9 ppb during recovery. Recovery values of exhaled NO were similar to those reported in age-matched normal control subjects (88 +/- 3 ppb, n = 72). These findings suggest that symptomatic upper respiratory tract infections markedly increase the concentration of NO in exhaled air. This may reflect the induction of nitric oxide synthase (NOS) in upper and lower respiratory tract, and may be relevant to viral exacerbations of asthma.

Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress

BACKGROUND Chronic inflammatory diseases are associated with an increased production of oxidants. Induction of a stress protein, heme oxygenase (HO) HO-1, is a cytoprotective mechanism against oxidative cellular injury. HO-1 catabolises heme to bilirubin, free iron, and carbon monoxide (CO). METHODS Exhaled CO and sputum bilirubin levels were measured and HO-1 protein expression in airway macrophages was determined by Western blotting in asthmatic patients as levels of oxidants are raised in asthma and may induce HO-1. RESULTS Exhaled CO was significantly increased in 37 non-steroid treated asthmatic patients compared with 37 healthy subjects (5.8 (95% CI 5.20 to 6.39) ppm vs 2.9 (2.51 to 3.28) ppm; p<0.0001) but was similar to normal in 25 patients who received corticosteroids (3.3 (95% CI 2.92 to 3.67) ppm; p>0.05). In non-treated asthmatic patients more HO-1 protein was expressed in airway macrophages than in normal subjects. Bilirubin levels in induced sputum were also higher than in normal subjects. Inhalation of hemin, a substrate for HO, significantly increased exhaled CO from 3.8 (95% CI 2.80 to 4.87) ppm to 6.7 (95% CI 4.95 to 8.38 CI) ppm (p<0.05) with a concomitant decrease in exhaled nitric oxide levels, suggesting an interaction between the two systems. CONCLUSIONS Increased exhaled CO levels and HO-1 expression may reflect induction of HO-1 which may be inhibited by steroids. Measurement of exhaled CO, an index of HO activity in non-smoking subjects, may therefore be clinically useful in the detection and management of asthma and possibly other chronic inflammatory lung disorders.

Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children

Airway inflammation in asthma is not measured routinely in clinical practice. Fractional exhaled nitric oxide (FENO), a marker of airway inflammation, is increasingly used as an outcome measure in asthma intervention studies and yet the reproducibility of FENO measurements is unknown. The reproducibility, daytoday, diurnal variation and perception of standardised FENO measurements were examined in 59 subjects (40 children aged 7–13 yrs and 19 adults aged 18–60 yrs), both healthy (n=30) and with mild (n=29) asthma. FENO was measured on five consecutive days (four measurements on the same day) for adults and twice on the same day for children. The coefficient of reproducibility expressed as the mean pooled standard deviation (n=59, 675 estimations) was 2.11 parts per billion (ppb) and intraclass correlation coefficient was 0.99 in both children and adults. FENO was significantly higher in asthma subjects (32.3 ppb) than in healthy subjects (16.3 ppb). There was no diurnal or daytoday variation, or a learning effect, as the result of FENO measurements were identical at results of the beginning and at the end of the study. It was concluded that fractional exhaled nitric oxide measurements are simple, reproducible, free from diurnal and daytoday variation, and acceptable by both healthy and asthmatic adults and children, as a part of their routine visit to a physician.

Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma.

BACKGROUND It is desirable to prescribe the minimal effective dose of inhaled steroids to control asthma. To ensure that inflammation is suppressed whilst using the lowest possible dose, a sensitive and specific method for assessing airway inflammation is needed. METHODS The usefulness of exhaled nitric oxide (NO), sputum eosinophils, and methacholine airway responsiveness (PC20) for monitoring airway inflammatory changes following four weeks of treatment with an inhaled corticosteroid (budesonide via Turbohaler) were compared. Mild stable steroid naive asthmatic subjects were randomised into two double blind, placebo controlled studies. The first was a parallel group study involving three groups receiving either 100 μg/day budesonide (n = 8), 400 μg/day budesonide (n = 7), or a matched placebo (n = 6). The second was a crossover study involving 10 subjects randomised to receive 1600 μg budesonide or placebo. The groups were matched with respect to age, PC20, baseline FEV1 (% predicted), exhaled NO, and sputum eosinophilia. RESULTS There were significant improvements in FEV1 following 400 μg and 1600 μg budesonide (11.3% and 6.5%, respectively, p<0.05). This was accompanied by significant reductions in eosinophil numbers in induced sputum (0.7 and 0.9 fold, p<0.05). However, levels of exhaled NO were reduced following each budesonide dose while PC20was improved only with 1600 μg budesonide. These results suggest that exhaled NO and PC20 may not reflect the control of airway inflammation as accurately as the number of eosinophils in sputum. There were dose dependent changes in exhaled NO, sputum eosinophils, and PC20 to inhaled budesonide but a plateau response of exhaled NO was found at a dose of 400 μg daily. CONCLUSION Monitoring the number of eosinophils in induced sputum may be the most accurate guide to establish the minimum dose of inhaled steroids needed to control inflammation. This, however, requires further studies involving a larger number of patients.

Exhaled leukotrienes and prostaglandins in COPD.

Background: The role of eicosanoids, including leukotrienes (LTs) and prostaglandins (PGs), in chronic obstructive pulmonary disease (COPD) is uncertain. The aim of this study was to investigate whether eicosanoids are measurable in exhaled breath condensate (EBC), a non-invasive method of collecting airway secretions, in patients with stable mild to moderate COPD, and to show possible differences in their concentrations compared with control subjects. Methods: LTB4, LTE4, PGE2, PGD2-methoxime, PGF2α, and thromboxane B2 (TxB2) were measured in EBC in 15 healthy ex-smokers, 20 steroid naïve patients with COPD who were ex-smokers, and in 25 patients with COPD who were ex-smokers and who were treated with inhaled corticosteroids. The study was of cross sectional design and all subjects were matched for age and smoking habit. Results: LTB4 and PGE2 concentrations were increased in steroid naïve (LTB4: median 100.6 (range 73.5–145.0) pg/ml, p<0.001; PGE2: 98.0 (range 57.0–128.4) pg/ml, p<0.001) and steroid treated patients with COPD (LTB4: 99.0 (range 57.9–170.5) pg/ml, p<0.001; PGE2: 93.6 (range 52.8–157.0) pg/ml, p<0.001) compared with control subjects (LTB4: 38.1 (range 31.2–53.6) pg/ml; PGE2: 44.3 (range 30.2–52.1) pg/ml). Both groups of patients had similar concentrations of exhaled LTB4 (p=0.43) and PGE2 (p=0.59). When measurable, LTE4 and PGD2-methoxime concentrations were similar in COPD patients and controls, whereas PGF2α concentrations were increased in the former. TxB2-LI was undetectable in any of the subjects. Conclusions: There is a selective increase in exhaled LTB4 and PGE2 in patients with COPD which may be relatively resistant to inhaled corticosteroid therapy.

Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients

An increased concentration of nitric oxide (NO) in the exhaled air of asthmatic patients may reflect inflammation of the airways, and exhaled NO may, therefore, be useful in monitoring asthma control and the optimal use of anti-inflammatory treatment. We have studied the effect of reducing and then increasing the dose of inhaled steroid on exhaled NO, lung function and symptoms in 14 asthmatic patients treated with twice daily budesonide. Baseline measurements were made at the end of a 2 week run-in period, 2 weeks after the daily dose of budesonide was reduced by 200 micrograms daily, and 2 weeks after the dose was then increased by 200 micrograms daily. Exhaled NO increased significantly compared with baseline after the dose was reduced by 200 micrograms daily (from 122 +/- 13 to 246 +/- 52 ppb); whereas, there was no significant decrease in spirometry or change in peak flow variability. There was also a significant increase in symptoms at night, but no change during the day or in the number of rescue doses of inhaled beta 2-agonist. The level of exhaled NO decreased when the dose of inhaled steroids was increased, and this was associated with a reduction in diurnal variability of peak expiratory flow, and in nocturnal symptoms. Our study suggests that exhaled nitric oxide may be a useful means of monitoring control of asthma. Further longitudinal studies in patients of differing asthma severity are now indicated.

Increased leukotriene B4 and 8-isoprostane in exhaled breath condensate of patients with exacerbations of COPD

Background: Exacerbations are an important feature of chronic obstructive pulmonary disease (COPD), accounting for a large proportion of health care costs. They are associated with increased airway inflammation and oxidative stress. Methods: Concentrations of leukotriene B4 (LTB4), a marker of inflammation, and 8-isoprostane, a marker of oxidative stress, were measured in the exhaled breath condensate of 21 patients (11 M) with COPD during an exacerbation and 2 weeks after treatment with antibiotics. In 12 patients who had no further exacerbations these markers were also measured after 2 months. Results: LTB4 concentrations were raised during the COPD exacerbation (mean (SE) 15.8 (1.1) pg/ml and fell after treatment with antibiotics to 9.9 (0.9) pg/ml (p<0.0001). In 12 patients the level of LTB4 fell further from 10.6 (1.1) pg/ml to 8.5 (0.8) pg/ml (p<0.005) after 2 months. In 12 normal age matched subjects the LTB4 levels were 7.7 (0.5) pg/ml. Concentrations of 8-isoprostane were also increased during the exacerbation (13.0 (0.9) pg/ml) and fell after antibiotic treatment to 9.0 (0.6) pg/ml (p<0.0001). In 12 patients there was a further fall from 9.3 (0.7) pg/ml to 6.0 (0.7) pg/ml (p<0.001) after 2 months compared with normal subjects (6.2 (0.4) pg/ml). Conclusions: Non-invasive markers of inflammation and oxidative stress are increased during an infective exacerbation of COPD and only slowly recover after treatment with antibiotics.

Biomarkers of some pulmonary diseases in exhaled breath

Analysis of various biomarkers in exhaled breath allows completely non-invasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchiectasis and interstitial lung diseases. The technique is simple to perform, may be repeated frequently, and can be applied to children, including neonates, and patients with severe disease in whom more invasive procedures are not possible. Several volatile chemicals can be measured in the breath (nitric oxide, carbon monoxide, ammonia), and many non-volatile molecules (mediators, oxidation and nitration products, proteins) may be measured in exhaled breath condensate. Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in differential diagnosis of airway disease and in the monitoring of therapy. Most progress has been made with exhaled nitric oxide (NO), which is increased in atopic asthma, is correlated with other inflammatory indices and is reduced by treatment with corticosteroids and antileukotrienes, but not (β2-agonists. In contrast, exhaled NO is normal in COPD, reduced in CF and diagnostically low in primary ciliary dyskinesia. Exhaled carbon monoxide (CO) is increased in asthma, COPD and CF. Increased concentrations of 8-isoprostane, hydrogen peroxide, nitrite and 3-nitrotyrosine are found in exhaled breath condensate in inflammatory lung diseases. Furthermore, increased levels of lipid mediators are found in these diseases, with a differential pattern depending on the nature of the disease process. In the future it is likely that smaller and more sensitive analysers will extend the discriminatory value of exhaled breath analysis and that these techniques may be available to diagnose and monitor respiratory diseases in the general practice and home setting.