Wednesday, March 24, 2010

Exhaled Nitric Oxide in Asthma

by Bruno Battistini, PhD

Exhaled nitric oxide may allow clinicians to improve diagnosis, determine proper initial treatment, and monitor the progression of various pulmonary diseases including asthma.

Nitric oxide, an atmospheric gas and free radical, was found in the 1970s to activate guanylate cyclase and increase guanosine 3':5'-cyclic monophosphate (cGMP) levels in various tissue preparations.1 Soon after (in 1981), a relationship between cGMP formation and the relaxation of coronary arterial smooth muscle in response to glyceryl trinitrate, nitroprusside, nitrite, and nitric oxide was established.2 It is reported that activation of endothelial cells by acetylcholine and other agents lead to arterial vasodilatation, from which emerged the new concept of endothelium-derived relaxing factor (EDRF) and the subsequent endothelium-related increase in smooth muscle cGMP.3 Nitric oxide emerged as the molecule responsible for the biological activity of EDRF.4 Of the more than 40,000 publications that can be found on nitric oxide today, about 5,000 (13%) focus on the biochemistry, pharmacology, and molecular biology of this molecule in the lung and pulmonary system. The first description of the presence and measurement of endogenous nitric oxide in the exhaled air of various species, including humans, was reported by Gustafsson et al.5 As of March 2001, 502 publications can be found on exhaled nitric oxide, but close to 2,000 can be found on the the inhalation of nitric oxide as a mean of therapeutic intervention, such as reversing pulmonary vasoconstriction.6

Biotransformation function
Nitric oxide is endogenously produced in the lung and many other organs, tissues, and cells by three subtypes of nitric oxide synthase (NOS I, II, and III).7 Once released, nitric oxide (having a half-life of 0.05 to <1 second) is almost immediately transformed into nitrite and nitrate. It may also react with superoxide to form peroxynitrite, as well as reacting with many bioreactants in circulating blood.8 Nitric oxide, as a vasodilator released by the normal endothelium, counterbalances, with atrial natriuretic peptide and bradykinin, the effects of vasoconstrictors (angiotensin II and endothelins) on blood flow and pressure.9 Thus, nitric oxide, under normal pulmonary-circulation and respiratory conditions, dilates human pulmonary arteries.10 Under hypoxic conditions, the release and action of nitric oxide are reduced.10 In hypoxia and other disease states such as acute lung injury and pneumonia, the loss or attenuation of endogenous nitric oxide inevitably leads to pulmonary hypertension.

Nitric oxide is produced not only by endothelial cells, but also by many other types of cells, such as epithelial cells, macrophages, eosinophils, neutrophils, and neurons11 that contribute to the roles of nitric oxide in respiration and as a bronchodilator.12-14 Nitric oxide is also involved as a neurotransmitter for the nonadrenergic noncholinergic nerves,15 and it shows antimicrobial activity.16 Conversely, nitric oxide, reacting with superoxide and forming peroxynitrite, can induce membrane lipid peroxidation that leads to cell-membrane damage, thus revealing the cytotoxic potential of superoxide and nitric oxide.17 Nitric oxide can also cause DNA breaks, apoptosis, and cytostasis, and it can be involved in angiogenesis and tumor progression.18 In short, nitric oxide is not merely a marker; it plays significant vascular and nonvascular regulatory and host-defense roles in pulmonary physiology and pathophysiology.

Measuring exhaled nitric oxide
Considering the close anatomical proximity of blood capillaries to membranous airways (alveolar space), pulmonary endothelial nitric oxide was expected to enter the airspace and, therefore, to be measured in the exhaled air. The first measurement of exhaled nitric oxide in normal human subjects5 used chemiluminescence (based on a photochemical reaction between nitric oxide and ozone), diazotization, and mass spectrometry, and was later confirmed using gas chromatography/mass spectrometry.19 Several other groups20-22 have since measured basal levels of exhaled nitric oxide in normal human subjects. Some noticeable variations in exhaled–nitric-oxide values were reported. A number of recommendations have been made to help ensure the reproducibility of the technique.23 At first, the exact origin of nitric oxide in exhaled air was not known. Most variations in exhaled nitric oxide can be explained by contamination from the upper respiratory tract.24 Such contamination was not fully eliminated by the use of nasal blockage (encouraging only oral airflow). Rather, the problem was solved by the selection of an online single constant expiratory flow-controlled rate.25 This later observation suggested that exhaled nitric oxide is released within the conducting airways, whereas alveolar nitric oxide levels are negligible. Thus, under normal conditions, levels of exhaled nitric oxide are less than 10 parts per billion (109), reflecting the minor contribution of alveolar nitric oxide and the total absence of nasal nitric oxide.

About 125 publications have presented data on the levels of exhaled nitric oxide in children. The first of these was by Lundberg et al.24 Further relationships between exhaled nitric oxide and childhood asthma have also been reported.26-28 Compared to induced sputum, exhaled nitric oxide presents undeniable advantages29 as a novel, noninvasive way to assess the degree of airway inflammation in this particular population.

Exhaled Nitric Oxide in Children
In mildly atopic asthma patients, the levels of exhaled nitric oxide seen during oral breathing were two to three times those seen in normal human subjects.30 In many other groups of asthma patients, the level of nitric oxide was also shown to be elevated.10-22,25,31 Thus, since increased production of nitric oxide in the lower airways may involve activated macrophages or neutrophils, exhaled nitric oxide was proposed for use to monitor underlying bronchial inflammation. Exhaled nitric oxide was not shown to be increased in patients with stable chronic obstructive pulmonary disease (COPD), but it was higher in unstable COPD,32 in bronchiectasis,33 following lung transplantation, and in association with obliterative bronchiolitis.34 Patients with cystic fibrosis had lower levels of exhaled nitric oxide35 as did patients with primary ciliary dyskinesia.36

The lungs of patients with primary pulmonary hypertension produced low levels of exhaled nitric oxide that may reflect the reduced blood capillary volume in these patients, rather than a decreased basal production of nitric oxide.37 The levels of exhaled nitric oxide are also reduced in patients with hypertension.38 The decrease is more pronounced in males than in females. Other patients with renal failure presented no differences, but sepsis was associated with a significant increase.38 Upon major surgery, the levels of exhaled nitric oxide dropped 79% , rising toward normal postoperatively (but still remaining 30% below baseline values).38 Furthermore, the absence of nasal nitric oxide in children with Kartagener syndrome was proposed as a simple noninvasive test supporting the diagnosis.

Stimulation and Inhibition
In addition to the elevation of exhaled nitric oxide associated with various pulmonary and other disorders, upregulation of the endogenous basal levels of exhaled nitric oxide can be induced by other factors. Nitric oxide concentrations in exhaled air have been reported to increase during physical exercise.39,40 Exercise on a stationary bicycle also produces rapid and reversible increases in pulmonary nitric oxide excretion rates that are well correlated with observed changes in heart rate.41 Conversely, graded dynamic (treadmill) exercise does not affect exhaled nitric oxide, which is maintained at the same level as work rates increase.42

The generation of nitric oxide in the human lung is endogenous, and it has been shown to be inhibited by L-NAME and NG-monomethyl-L-arginine (L-NMMA), inhibitors of cNOS.5 In human study subjects, exhaled–nitric oxide levels were significantly reduced by the inhalation of the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine31,43 or intravenous administration of L-NMMA.44 Voluntary twofold hyperventilation for 1 minute decreased expired nitric oxide by 50% (from 9.5±2.5 parts per billion) in six subjects.42 Smoking attenuated the levels of exhaled nitric oxide by 21% in men and by 41% in women.38 This effect is also supported by other studies.45 Ethanol (given at 0.25 g/kg and 1 g/kg in four times its volume of orange juice) produced dose-dependent reductions of exhaled nitric oxide in humans.46 Thus, drinking prior to analysis may affect the levels of exhaled nitric oxide in human subjects. Prednisolone, a glucocorticosteroid, did not inhibit exhaled nitric oxide in normal subjects, suggesting that the increased exhaled nitric oxide seen in asthma patients is likely to be caused by the induction of inducible NOS.43

Pharmacological inhibition of exhaled nitric oxide by inhaled L-NMMA was also reported43 in asthma patients and those with other pulmonary diseases, just like in normal subjects. Existing drugs such as inhaled or oral corticosteroids were also shown to attenuate the increased levels of exhaled nitric oxide found in asthma significantly.31,43,47 Anti-leukotrienes also inhibit the rise in exhaled nitric oxide moderately.48 Conversely, bronchodilators, such as albuterol or salmeterol, did not influence exhaled nitric oxide.49,50

The levels of exhaled nitric oxide in normal and ill subjects have been established for different age categories, and significant relationships with more direct measurements of inflammation in the airways (induced sputum, bronchoalveolar lavage, bronchial biopsy, and clinical signs and symptoms of asthma, especially during acute exacerbations) have been described. While exhaled nitric oxide has attracted much interest, the inhalation of nitric oxide as a therapy in patients with pulmonary hypertension and other conditions has attracted even more interest.51

Combining rapid, noninvasive, standardized analysis of exhaled nitric oxide with traditional techniques for assessing pulmonary function, airway reactivity, and inflammation may allow the clinician to assess airway inflammation and oxidative stress more accurately in patients with pulmonary diseases that include asthma, COPD, and interstitial lung disease. It may also allow the clinician to improve diagnosis, determine the proper initial treatment, monitor the progression of disease, and assess the efficacy of treatment and subsequent adjustments over time.52 Knowing the level of nitric oxide (and, by association, airway inflammation) may help clinicians keep the patient’s medication to a minimum while maintaining therapeutic efficacy. Thus, more longitudinal studies are needed to confirm that the analysis of exhaled nitric oxide may be used for the short-term and long-term management and treatment of diseases. Then, nitric oxide analysis may take the place that it deserves in pulmonology. Technological advances will make it possible to miniaturize nitric oxide analyzers (and will make them less expensive) so they will become portable and may even be used, in the not-so-distant future, at home in conjunction with peak flow meters.

Bruno Battistini, PhD, is assistant professor of medicine and research scientist, Department of Medicine, Laval University, Laval Hospital Research Center, Institut de Cardiologie et de Pneumologie, Ste-Foy, Quebec, Canada.


Sunday, March 14, 2010

The Use of Exhaled Nitric Oxide to Guide Asthma Management

Rationale: Current asthma guidelines recommend adjusting antiinflammatory treatment on the basis of the results of lung function tests and symptom assessment, neither of which are closely associated with airway inflammation.

Objectives: We tested the hypothesis that titrating corticosteroid dose using the concentration of exhaled nitric oxide in exhaled breath (FENO) results in fewer asthma exacerbations and more efficient use of corticosteroids, when compared with traditional management.

Methods: One hundred eighteen participants with a primary care diagnosis of asthma were randomized to a single-blind trial of corticosteroid therapy based on either FENO measurements (n = 58) or British Thoracic Society guidelines (n = 60). Participants were assessed monthly for 4 months and then every 2 months for a further 8 months. The primary outcome was the number of severe asthma exacerbations. Analyses were by intention to treat.

Measurements and Main Results: The estimated mean (SD) exacerbation frequency was 0.33 per patient per year (0.69) in the FENO group and 0.42 (0.79) in the control group (mean difference, –21%; 95% confidence interval [CI], –57 to 43%; p = 0.43). Overall the FENO group used 11% more inhaled corticosteroid (95% CI, –17 to 42%; p = 0.40), although the final daily dose of inhaled corticosteroid was lower in the FENO group (557 vs. 895 µg; mean difference, 338 µg; 95% CI, –640 to –37; p = 0.028).

Conclusions: An asthma treatment strategy based on the measurement of exhaled nitric oxide did not result in a large reduction in asthma exacerbations or in the total amount of inhaled corticosteroid therapy used over 12 mo, when compared with current asthma guidelines.


Wednesday, March 10, 2010

Exhaled nitric oxide and asthma control: a longitudinal study in unselected patients

Controlled studies have shown that monitoring of the exhaled nitric oxide fraction (FeNO) improves asthma management. However, the studies seldom consider the full range of patients seen in clinical practise. In the present study, the ability of FeNO to reflect asthma control over time is investigated in a regular clinical setting, and meaningful FeNO cut-off points and changes are identified.

Answers to the Asthma Control Questionnaire and FeNO were recorded at least once in 341 unselected asthma patients. The whole population and subgroups were considered, i.e. both inhaled corticosteroid (ICS)-naïve and low or high-to-medium (≤ or >500 µg beclomethasone dipropionate equivalents·day–1) ICS-dose groups.

An FeNO decrease <40% or increase <30% precludes asthma control optimisation or deterioration, respectively (negative predictive value 79 and 82%, respectively). In the present study’s low-dose group, a decrease >40% indicated asthma control optimisation (positive predictive value (PPV) 83%). In ICS-naïve patients, FeNO >35 ppb predicted asthma control improvement in response to ICS (PPV 68%). In most cases, forced expiratory volume in one second assessments were not useful.

In conclusion, in a given patient, exhaled nitric oxide fraction was found to be significantly related to asthma control over time. The overall ability of exhaled nitric oxide fraction to reflect asthma control was reduced in patients using high doses of inhaled corticosteroids. Forced expiratory volume in one second had little additional value in assessing asthma control.


Sunday, March 7, 2010

Exhaled Nitric Oxide and its role in the treatment of Asthma Part I

In medicine, exhaled nitric oxide (eNO) can be measured in a breath test for asthma which is characterized by airway inflammation. Nitric oxide (NO) is a gaseous molecule produced by certain cell types in an inflammatory response. Exhaled NO (also referred to as FENO) is a promising biomarker as a guide to therapy in adults and children with asthma. The breath test has recently become available in many well-equipped hospitals in developed countries. Clinical trials have looked at whether tailoring asthma therapy based on eNO values is better than conventional care, in which therapy is gauged by symptoms and the results of lung function tests.
An excerpt from the New England Journal of Medicine in a study by Andrew D. Smith, M.B., Ch.B., Jan O. Cowan, Karen P. Brassett, G. Peter Herbison, M.Sc., and D. Robin Taylor, M.D. details the trial to establish the use of eNO in the measurements to guide treatment in chronic asthma.

The findings of the study are detailed below.


International guidelines for the treatment of asthma recommend adjusting the dose of inhaled corticosteroids on the basis of symptoms, bronchodilator requirements, and the results of pulmonary-function tests. Measurements of the fraction of exhaled nitric oxide (FeNO) constitute a noninvasive marker that may be a useful alternative for the adjustment of inhaled-corticosteroid treatment.


In a single-blind, placebo-controlled trial, the investigators randomly assigned 97 patients with asthma who had been regularly receiving treatment with inhaled corticosteroids to have their corticosteroid dose adjusted, in a stepwise fashion, on the basis of either FeNO measurements or an algorithm based on conventional guidelines. After the optimal dose was determined (phase 1), patients were followed up for 12 months (phase 2). The primary outcome was the frequency of exacerbations of asthma; the secondary outcome was the mean daily dose of inhaled corticosteroid.


Forty-six patients in the FeNO group and 48 in the group whose asthma was treated according to conventional guidelines (the control group) completed the study. The final mean daily doses of fluticasone, the inhaled corticosteroid that was used, were 370 μg per day for the FeNO group (95 percent confidence interval, 263 to 477) and 641 μg per day for the control group (95 percent confidence interval, 526 to 756; P=0.003), a difference of 270 μg per day (95 percent confidence interval, 112 to 430). The rates of exacerbation were 0.49 episode per patient per year in the FeNO group (95 percent confidence interval, 0.20 to 0.78) and 0.90 in the control group (95 percent confidence interval, 0.31 to 1.49), representing a non-significant reduction of 45.6 percent (95 percent confidence interval for mean difference, ¡78.6 percent to 54.5 percent) in the FeNO group. There were no significant differences in other markers of asthma control, use of oral prednisone, pulmonary function, or levels of airway inflammation (sputum eosinophils).


With the use of FeNO measurements, maintenance doses of inhaled corticosteroids may be significantly reduced without compromising asthma control.


Wednesday, March 3, 2010

Exhaled Nitric Oxide Measurements Can Be Used to Both Diagnose and Rule Out Asthma: Presented at AAAAI

Doctors have been using fractional exhaled nitric oxide (FeNO) measurements to indicate airway inflammation and aid in asthma diagnosis for some time, leading many to believe that these measurements could play a role in actually diagnosing the problem. As it turns out, this noninvasive, inflammatory marker may not only send up a red flag for a positive diagnosis but also aid in confirming a negative one, according to a study presented here at the 2010 American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting.

In the study, a group of 115 patients who were reporting asthma symptoms were assembled. The patients had "perennial symptoms consistent with asthma," with normal spirometry and a negative bronchodilator test. They also met medication withdrawal requirements before testing and completed an Asthma Control Test and Asthma Control Questionnaire before the research protocol began. After undergoing a methacholine challenge test following the standard 5-breath dosimeter protocol, FeNO measurements were taken with a portable device at a 50-mL/s exhalation flow rate.

Thirty-five of the 115 patients had a positive methacholine test and were diagnosed with asthma, making the prevalence of asthma in the test population 30.43%. FeNO levels in asthmatic patients averaged 58 parts per billion (ppb) (interquartile range [IQR] 36.00-112.00), whereas they averaged 29.5 ppb in patients not diagnosed with asthma (IQR 21.00-42.75; P = .0001). Maria Pedrosa, MD, University Hospital La Paz, Madrid, Spain, stated in a presentation on February 27 that these results showed that "FeNO levels are significantly higher for asthmatics…than non-asthmatics."


Tuesday, March 2, 2010

Relationship of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children

Exhaled nitric oxide (eNO) is a noninvasive test that measures airway inflammation.eNO provides information about the asthmatic state consistent with information from other markers of inflammation. It is a noninvasive technique that could be used in decisional management of children with asthma.

Asthma control is monitored by means of symptoms using pulmonary function tests, particularly FEV1 and FEV1/forced vital capacity (FVC), to supplement the clinical information. There is growing interest in determining the role of biomarkers in asthma care. Most of these studies have been conducted in adults. Peripheral blood eosinophils1,and sputum eosinophils correlate with response to oral corticosteroids. Studies in adults have shown that improved asthma control can be attained by use of airway responsiveness to methacholine and sputum eosinophils to modulate asthma therapy rather than waiting for clinical symptoms to appear or pulmonary functions to deteriorate. Because the application of biomarkers to assessment of asthma management has become more widespread, exhaled nitric oxide (eNO) has been studied to predict the clinical course with reductions in or response to medications. eNO levels correlate with response to inhaled corticosteroids (ICSs), in that patients with higher levels have better responses.Jones et al demonstrated that increases of eNO have a positive predictive value for loss of control of asthma as ICSs are withdrawn, providing information about control of asthma equal to induced sputum eosinophils and methacholine responsiveness. eNO is a particularly attractive biomarker, because the test requires little effort from the patient, can be measured even in young children,and the results of the test can be immediately available.


Thursday, February 25, 2010

Comparisons between Exhaled Nitric Oxide Measurements and Conventional Tests

International guidelines recommend a range of clinical tests to confirm the diagnosis of asthma. These focus largely on identifying variable airflow obstruction and responses to bronchodilator or corticosteroid. More recently, exhaled nitric oxide (FENO) measurements and induced sputum analysis to assess airway inflammation have been highlighted. However, to date, no systematic comparisons to confirm the diagnostic utility of each of these methods have been performed. To do so, we investigated 47 consecutive patients with symptoms suggestive of asthma, using a comprehensive fixed-sequence series of diagnostic tests. Sensitivities and specificities were obtained for peak flow measurements, spirometry, and changes in these parameters after a trial of steroid. Comparisons were made against FENO and sputum cell counts. Sensitivities for each of the conventional tests (0–47%) were lower than for FENO (88%) and sputum eosinophils (86%). Overall, the diagnostic accuracy when using FENO and sputum eosinophils was significantly greater. Results for conventional tests were not improved, using a trial of steroid. We conclude that FENO measurements and induced sputum analysis are superior to conventional approaches, with exhaled nitric oxide being most advantageous because the test is quick and easy to perform.c asthma, do not display elevated FENO.