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
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 exhalednitric-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, exhalednitric 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 patients 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.
The authors research is supported, in part, by the Fonds de la Recherche en Santé du Québec, the Fonds pour les Chercheurs et lAide à la Recherche, the Fondations Bégin et de lInstitut 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.
1. Arnold WP, Mittal CK, Katsuki S, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci USA. 1977;74:3203-3207.
2. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3′:5′-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin. J Pharmacol Exp Ther. 1981;219:181-186.
3. Furchgott RF, Cherry PD, Zawadzki JV, Jothianandan D. Endothelial cells as mediators of vasodilation of arteries. J Cardiovasc Pharmacol. 1984;6:S336-S343.
4. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526.
5. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun. 1991;181:852-857.
6. Frostell C, Fratacci MD, Wain JC, Jones R, Zapol WM. Inhaled nitric oxide. A selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation. 1991;83:2038-2047.
7. Schmidt HH, Pollock JS, Nakane M, Forstermann U, Murad F. Ca2+/calmodulin-regulated nitric oxide synthases. Cell Calcium. 1992;13:427-434.
8. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA. 1990;87:1620-1624.
9. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J. 1989;3:2007-2018.
10. McCormack D. Endothelium-derived relaxing factors and the human pulmonary circulation. Lung. 1990;168:35-42.
11. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med. 1994;120:227-237.
12. Adnot S, Raffestin B, Eddahibi S. NO in the lung. Respir Physiol. 1995;101:109-120.
13. Matera MG. Nitric oxide and airways. Pulm Pharmacol Ther. 1998;11:341-348.
14. Di Maria GU, Spicuzza L, Mistretta A, Mazzarella G. Role of endogenous nitric oxide in asthma. Allergy. 2000;55:31-35.
15. Kiss JP. Role of nitric oxide in the regulation of monoaminergic neurotransmission. Brain Res Bull. 2000;52:459-466.
16. Fang FC. Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest. 1997;99:2818-2825.
17. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996;271:1424-1437.
18. Brune B, von Knethen A, Sandau KB. Nitric oxide (NO): an effector of apoptosis. Cell Death Differ. 1999;6:969-975.
19. Leone AM, Gustafsson LE, Francis PL, Persson MG, Wiklund NP, Moncada S. Nitric oxide is present in exhaled breath in humans: direct GC-MS confirmation. Biochem Biophys Res Commun. 1994;201:883-887.
20. van Amsterdam JG, Nierkens S, Vos SG, Opperhuizen A, van Loveren H, Steerenberg PA. Exhaled nitric oxide: a novel biomarker of adverse respiratory health effects in epidemiological studies. Arch Environ Health. 2000;55:418-423.
21. Kharitonov SA, Barnes PJ. Clinical aspects of exhaled nitric oxide. Eur Respir J. 2000;16:781-792.
22. Silkoff PE. Noninvasive measurement of airway inflammation using exhaled nitric oxide and induced sputum. Current status and future use. Clin Chest Med. 2000;21:345-360.
23. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: recommendations. Eur Respir J. 1997;10:1683-1693.
24. Lundberg JO, Weitzberg E, Nordvall SL, Kuylenstierna R, Lundberg JM, Alving K. Primarily nasal origin of exhaled nitric oxide and absence in Kartageners syndrome. Eur Respir J. 1994;7:1501-1504.
25. Silkoff PE, McClean PA, Slutsky AS, et al. Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med. 1997;155:260-267.
26. Frank TL, Adisesh A, Pickering AC, et al. Relationship between exhaled nitric oxide and childhood asthma. Am J Respir Crit Care Med. 1998;158:1032-1036.
27. Chedevergne F, Le Bourgeois M, de Blic J, Scheinmann P. The role of inflammation in childhood asthma. Arch Dis Child. 2000;82:II6-II9.
28. Kissoon N, Duckworth L, Blake K, Murphy S, Silkoff PE. Exhaled nitric oxide measurements in childhood asthma: techniques and interpretation. Pediatr Pulmonol. 1999;28:282-296.
29. Gibson PG, Henry RL, Thomas P. Noninvasive assessment of airway inflammation in children: induced sputum, exhaled nitric oxide, and breath condensate. Eur Respir J. 2000;16:1008-1015.
30. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J. 1993;6:1368-1370.
31. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet. 1994;343:133-135.
32. Corradi M, Majori M, Cacciani GC, Consigli GF, deMunari E, Pesci A. Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease. Thorax. 1999;54:572-575.
33. Kharitonov SA, Wells AU, OConnor BJ, et al. Elevated levels of exhaled nitric oxide in bronchiectasis. Am J Respir Crit Care Med. 1995;151:1889-1893.
34. Boehler A, Estenne M. Obliterative bronchiolitis after lung transplantation. Curr Opin Pulm Med. 2000;6:133-139.
35. Lundberg JO, Nordvall SL, Weitzberg E, Kollberg H, Alving K. Exhaled nitric oxide in paediatric asthma and cystic fibrosis. Arch Dis Child. 1996;75:323-326.
36. Loukides S, Kharitonov S, Wodehouse T, Cole PJ, Barnes PJ. Effect of arginine on mucociliary function in primary ciliary dyskinesia. Lancet. 1998;352:371-372.
37. Cremona G, Higenbottam T, Borland C, Mist B. Mixed expired nitric oxide in primary pulmonary hypertension in relation to lung diffusion capacity. QJM. 1994;87:547-551.
38. Schilling J, Holzer P, Guggenbach M, Gyurech D, Marathia K, Geroulanos S. Reduced endogenous nitric oxide in the exhaled air of smokers and hypertensives. Eur Respir J. 1994;7:467-471.
39. Persson MG, Wiklund NP, Gustafsson LE. Endogenous nitric oxide in single exhalations and the change during exercise. Am Rev Respir Dis. 1993;148:1210-1214.
40. Sheel AW, Road J, McKenzie DC. Exhaled nitric oxide during exercise. Sports Med. 1999;28:83-90.
41. Bauer JA, Wald JA, Doran S, Soda D. Endogenous nitric oxide in expired air: effects of acute exercise in humans. Life Sci. 1994;55:1903-1909.
42. Iwamoto J, Pendergast DR, Suzuki H, Krasney JA. Effect of graded exercise on nitric oxide in expired air in humans. Respir Physiol. 1994;97:333-345.
43. Yates DH, Kharitonov SA, Robbins RA, Thomas PS, Barnes PJ. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am J Respir Crit Care Med. 1995;152:892-896.
44. Krejcy K, Schmetterer L, Kastner J, et al. Role of nitric oxide in hemostatic system activation in vivo in humans. Arterioscler Thromb Vasc Biol. 1995;15:2063-2067.
45. Persson MG, Zetterstrom O, Agrenius V, Ihre E, Gustafsson LE. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet. 1994;343:146-147.
46. Persson MG, Cederqvist B, Wiklund CU, Gustafsson LE. Ethanol causes decrements in airway excretion of endogenous nitric oxide in humans. Eur J Pharmacol. 1994;270:273-278.
47. Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med. 1996;153:454-457.
48. Bratton DL, Lanz MJ, Miyazawa N, White CW, Silkoff PE. Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study. Pediatr Pulmonol. 1999;28:402-407.
49. Yates DH, Kharitonov SA, Barnes PJ. Effect of short- and long-acting inhaled beta2-agonists on exhaled nitric oxide in asthmatic patients. Eur Respir J. 1997;10:1483-1488.
50. Fuglsang G, Vikre-Jorgensen J, Agertoft L, Pedersen S. Effect of salmeterol treatment on nitric oxide level in exhaled air and dose-response to terbutaline in children with mild asthma. Pediatr Pulmonol. 1998;25:314-321.
51. Steudel W, Hurford WE, Zapol WM. Inhaled nitric oxide: basic biology and clinical applications. Anesthesiology. 1999;91:1090-1121.
52. Massaro AF, Gaston B, Kita D, Fanta C, Stamler JS, Drazen JM. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med. 1995;152:800-803.