Exhaled Nitric Oxide in Asthma

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 groups^20-22 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-arginine^31,43 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

Conclusion
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.

Acknowledgments
The author’s research is supported, in part, by the Fonds de la Recherche en Santé du Québec, the Fonds pour les Chercheurs et l’Aide à la Recherche, the Fondations Bégin et de l’Institut de Cardiologie de Québec, the Asthma Society of Canada, the British Columbia Lung Foundation, MRCC/PMAC (Abbott), and a medical school grant from Merck Frosst.

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