Special Feature: Asthma

Immunology and Cell Biology (2001) 79, 178–190; doi:10.1046/j.1440-1711.2001.00990.x

Role of exhaled nitric oxide in asthma

Deborah H Yates1

1 Faculty of Medicine, Sydney University and Department of Respiratory Medicine, Royal North Shore Hospital, St Leonard's, New South Wales, Australia

Correspondence: Dr DH Yates, Suite 706, 26 Ridge Street, North Sydney, NSW 2060, Australia. Email: Deborahy88@hotmail.com

Received 15 September 2000; Accepted 11 October 2000.

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Abstract

Nitric oxide (NO), an evanescent atmospheric gas, has recently been discovered to be an important biological mediator in animals and humans. Nitric oxide plays a key role within the lung in the modulation of a wide variety of functions including pulmonary vascular tone, nonadrenergic non-cholinergic (NANC) transmission and modification of the inflammatory response. Asthma is characterized by chronic airway inflammation and increased synthesis of NO and other highly reactive and toxic substances (reactive oxygen species). Pro- inflammatory cytokines such as TNFalpha and IL-1beta are secreted in asthma and result in inflammatory cell recruitment, but also induce calcium- and calmodulin-independent nitric oxide synthases (iNOS) and perpetuate the inflammatory response within the airways. Nitric oxide is released by several pulmonary cells including epithelial cells, eosinophils and macrophages, and NO has been shown to be increased in conditions associated with airway inflammation, such as asthma and viral infections. Nitric oxide can be measured in the expired air of several species, and exhaled NO can now be rapidly and easily measured by the use of chemiluminescence analysers in humans. Exhaled NO is increased in steroid-naive asthmatic subjects and during an asthma exacerbation, although it returns to baseline levels with appropriate anti-inflammatory treatment, and such measurements have been proposed as a simple non-invasive method of measuring airway inflammation in asthma. Here the chemical and biological properties of NO are briefly discussed, followed by a summary of the methodological considerations relevant to the measurement of exhaled NO and its role in lung diseases including asthma. The origin of exhaled NO is considered, and brief mention made of other potential markers of airway inflammation or oxidant stress in exhaled breath.

Keywords:

airway inflammation, asthma, nitric oxide, nitrosothiols

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Introduction

Nitric oxide (NO), molecule of the year in 1992,1 has recently excited much interest in the scientific community. Ten years ago this simple molecule, one of the smallest biologically active substances, was known primarily for its nuisance value. It was one of the noxious gases produced by car exhausts, destroying the ozone layer, and was implicated in acid rain. Only a short time later, NO was recognized as a key signalling molecule in a wide variety of biological functions, and NO research affects all branches of medicine.2 In the lung, NO acts as a vasodilator, bronchodilator and nonadrenergic non-cholinergic (NANC) neurotransmitter and is an important mediator of the inflammatory response. Nitric oxide is formed in the lungs and the presence of NO has been detected in the exhaled air of several animal species, including humans.3

Although NO is an evanescent gas, it can be measured directly and rapidly by chemiluminescence in vivo. Since such measurements were first reported in 1991 by Gustafsson and colleagues,4 many studies have confirmed the ease and reproducibility of such readings. Levels of exhaled NO are increased in untreated asthma, and decrease with appropriate anti-inflammatory treatment.5, 6 Because exhaled NO is reproducible and totally non-invasive, and levels of NO correlate with some measures of airway inflammation, exhaled NO has been suggested as a simple way of assessing asthma.7

Since the initial studies, exhaled nitric oxide has been reported to be altered in many lung diseases other than asthma, and it has become apparent that other exhaled markers may also be detected.8, 9 This lends promise to the hope that in the future, exhaled gases may be used to provide a 'disease fingerprint', which could lead to better understanding and improved treatments. The rate of advance in this area is rapid. This review can only hope to provide an introduction to this most intriguing mediator, focusing on asthma. Here, the chemical and biological properties of NO will be briefly discussed, followed by a summary of the methodological considerations relevant to the measurement of exhaled NO and its role in lung diseases including asthma. The origin of exhaled NO will be considered, and brief mention made of other potential markers of airway inflammation or oxidant stress in exhaled breath.

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Chemical and biological properties of nitric oxide

Nitric oxide/endothelium-derived relaxing factor

Interest in nitrates is not new. Nitrates were well known in the 19th century, dynamite (or nitroglycerine) having been invented by Alfred Nobel in 1863. Working with dynamite was known to produce a violent headache and hypotension, affecting many workers and also, on occasion, its inventor. The effect was so marked that workers were issued with one legged stools so that if they became faint due to hypotension, they would fall off and thus recover to continue their labours.10 The therapeutic effect of amyl nitrate in angina was first described in 1867, and it is ironic that Alfred Nobel, who later suffered from angina pectoris, was ordered by his doctor to take nitroglycerine internally.11 It was not until 1916 that dietary balance studies first suggested that the body endogenously produced nitrate.12 Initially it was believed that this excess NO3 - was produced by intestinal microorganisms and thus had little relevance to mammalian biology; however, it was subsequently discovered that NO3 - was synthesized outside the intestine and that immune stimulation resulted in increases in urinary excretion.13 The source for these nitrates was found to be the semi-essential amino acid, L-arginine.

It was research within the cardiovascular arena rather than that of the lung that first elucidated the importance of NO. Furchgott and Zadawadzki in 1980 showed that the endothelium was essential for the vasodilator action of acetylcholine in isolated arterial rings, and that removal of the endothelium prevented such relaxation.14 A substance was postulated to be produced by the endothelium after stimulation, which was imaginatively named endothelium-derived relaxing factor, or EDRF. Endothelium-derived relaxing factor was highly unstable, with a half life of only seconds in buffer solution. Factors such as sheer stress, mechanical stretching, hypoxia and abnormal flow could all produce EDRF and result in endothelium-dependent vasodilatation. In addition, a large number of vasoactive substances (such as bradykinin, histamine, adenine nucleotides, thrombin, 5-hydroxytryptamine) also seemed to act through EDRF release.

In 1987, the link was made between EDRF and NO, which were found to be the same substance. Furchgott and Ignarro independently pointed out the similarities between the two substances,15 and Moncada and colleagues, using simultaneous bioassay and chemiluminescence assay, showed that NO accounted for the biological activity of EDRF and that it was formed from L-arginine.16

Chemical properties of nitric oxide

Nitric oxide is one of the smallest known biologically active messenger molecules, and the first example of a completely new class of signalling molecule. At room temperature, it is a colourless gas which, in the absence of oxygen, dissolves in water. At low concentrations NO is fairly stable, even in the presence of oxygen.2 Nitric oxide differs from classical mediators, which have complex structures and a specific receptor, whereas NO is a simple free radical gas that diffuses freely from its site of formation and is not stored. Nitric oxide contains an odd number of electrons and is thus a free radical [NOdot]. This chemical property gives NO paramagnetic properties, prevents its dimerization, and increases its reactivity with a number of atoms and free radicals.17 It rapidly reacts with, and is inactivated by, O2 to form nitrite and nitrates, and with superoxide anion (O2 ) to form an unstable intermediate peroxynitrite anion [ONOO].18 The latter is a potent oxidant and can nitrosate proteins and nucleic acids, and cause lipid peroxidation in vivo. Lipid peroxidation is of importance because this may result in cell membrane dysfunction and lead to cell death, or to damage of DNA.19 Nitric oxide can diazotize primary amines (ArNH2) in conjunction with an oxidant to produce potentially mutagenic substances.20 Nitric oxide reacts with oxyhaemoglobin to form methaemoglobin and nitrate, and also with thiols to form S-nitrosothiols, including S-nitrosocysteine and S-nitrosoglutathione (GSNO). Nitric oxide also exists in the plasma as the S-nitroso adduct of circulating albumin.21

Nitric oxide has a particular affinity for the ferrous (Fe2+) moiety of haemoproteins such as haemoglobin, myoglobin, cytochrome C and soluble guanylate cyclase (sGC), forming nitrosyl products, and will also react with ferric (Fe3+) compounds, although much less readily. The former property can be used for its measurement. The high affinity of the haemoproteins for NO makes it difficult to accurately determine the equilibrium constant directly, but based on a partition constant between NO and carbon monoxide (CO), the affinity of NO for haemoglobin is approximately 1000 fold that for CO, and approximately 3000 fold that of oxygen.2, 22

The chemical reactivity of NO has been used to develop methods for its measurement. Nitric oxide reacts with oxyhaemoglobin in the absence of O2 to form methaemoglobin, and the accompanying shifts in absorption spectra can be used to provide a quantitative spectrophotometric assay of NO. Nitric oxide generates a chemiluminescent product upon reaction with ozone, and catalyses diazotization of sulfanilic acid at acidic pH; both of these reactions are also used to detect NO. The former in particular is used to measure exhaled nitric oxide, while the latter can be used in vitro.

Once produced, NO is freely diffusible, and may enter, for example, vascular smooth muscle cells to act through the stimulation of sGC and subsequently form cyclic GMP. Increased cGMP activates a cGMP sensitive kinase, which phosphorylates a calcium-dependent potassium channel, leading to hyperpolarization and vasodilatation.2 This produces muscle relaxation (Figure 1). Nitric oxide is probably formed on demand in a generator cell such as an endothelial or epithelial cell and acts on nearby target cells such as vascular or bronchial smooth muscle cells. The effects of NO in the lung are not restricted, however, to smooth muscle effects, and the effects on inflammation may be of greater interest with regard to asthma (see later).

Figure 1.
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Constitutive nitric oxide (NO) release. Stimuli such as acetylcholine, bradykinin, ADP or sheer stress increase intracellular calcium, which activates nitric oxide synthase (NOS) and allows the formation of NO.

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It is likely that further mediators similar to NO will be discovered in the future. Carbon monoxide, another activator of guanylate cyclase, acts in a similar manner, is certainly involved in neurotransmission, and may play a role in inflammation within the lung. Carbon monoxide is generated by the enzyme haem oxygenase 1 (HO-1), which catalyses the degradation of haem to biliverdin and CO.23

Generation of nitric oxide

Nitric oxide is synthesized in mammalian cells by the oxidation of L-arginine to NO and L-citrulline (Figure 2). This reaction is catalysed by enzymes called NO synthases (NOS). Nitric oxide synthases were initially broadly classified into either calcium- and calmodulin-dependent (cNOS), or calcium- and calmodulin-independent (iNOS). Calcium- and calmodulin-dependent NOS was originally thought to be synonymous with constitutive expression of NOS and iNOS with transcriptional regulation (i.e inducibility). Agonists such as sheer stress, bradykinin, acetylcholine and histamine may activate cNOS, resulting in the release of picomolar concentrations of NO within seconds. The synthesis of NO by cNOS appears to be responsible for the vasodilator tone that is essential for the regulation of blood pressure, for neurotransmission, and for regulation of various respiratory, gastrointestinal, and genitourinary functions, as well as playing a role in cardiac contractility and in platelet aggregation. Calcium- and calmodulin-dependent NOS is basally expressed in most cells. In contrast, iNOS is mainly generated in certain pathophysiological conditions such as endotoxic shock, and can be induced by certain cytokines, including TNF-alpha, IFN-gamma and IL1-beta, as well as by endotoxin lipopolysaccharides. Once iNOS is induced, production of NO after several hours rises to much larger levels than with cNOS (nanomolar concentrations) and may continue for days. High NO levels appear more indicative of the level of iNOS expression; this can increase from 0.0005% to almost 1% of the total protein content of cells.24 The resultant high NO levels may produce different pathophysiological effects.2, 3

Figure 2.
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Synthesis of nitric oxide (NO) from L-arginine. ONOO-, peroxynitrite; BH4, tetrahydrobioprotein.

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Nitric oxide synthase

Originally, three distinct isoforms of NOS were identified: neuronal (nNOS), macrophage (iNOS) and endothelial cell (eNOS). These were named after the cells from which they were first isolated, but because they have all since been identified elsewhere, this distinction has been abandoned. For example, nNOS can also be found in skeletal muscle, airway epithelium and neurones. All these isoforms have now been sequenced and cloned, and the more recent consensus classification relies on their molecular identity as well as their calcium- and calmodulin dependence.25 Generally, nNOS is type I NOS, iNOS is type II NOS, and eNOS is type III NOS (Figure 3). All NOS sequences are extremely well conserved between different species, suggesting a key biological function. Additionally, another form of NOS has been recently described in mitochondria, called mtNOS.26 This isoform is calcium-dependent, and plays a role in the regulation of respiration, but its other functions are unknown. The initial classification of NOS types is now recognized to be an oversimplification, because inducibility has been found to be a function of the stimulus and not the isoform, and iNOS expression may be invariant in certain cell types. However, the basic principles with regard to pathophysiology associated with iNOS still apply, and have the advantage of familiarity. Accordingly, such nomenclature will be used for the purposes of discussion about asthma, while acknowledging its deficiencies.

Figure 3.
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(a) Classification of nitric oxide synthase (NOS) isoforms and amino acid sequence identity with cytochrome P450 reductase. Binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), FAD, FMN, calmodulin (CAL) and haem (H) are indicated. Numbers along the bottom refer to amino acid number, with 0 indicating the N-terminus. Adapted from Al-Sa'doni and Ferro with permission.22 (b) Equipment required for measurement of exhaled nitric oxide (eNO) in vivo.

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In human airways, cNOS is expressed in neuronal (NOS1), endothelial (NOS3) and epithelial cells (types 1 and 3 NOS). Calcium- and calmodulin-dependent NOS expression has been described in epithelial cells, macrophages, neutrophils, endothelial cells, and vascular smooth muscle cells. The predominant form in airway epithelia is iNOS.27 Calcium- and calmodulin-dependent NOS can be induced by stimulation of cultured human epithelial cell lines with pro-inflammatory cytokines (TNF-alpha, IFN-gamma, and IL-1beta).28

All NOS isoforms are members of the cytochrome P450 enzyme group, and contain a haem complex at the active site of the enzyme. L-arginine is the usual substrate, with molecular oxygen incorporated to yield NO and L-citrulline. The reaction with L-arginine is stereospecific as D-arginine is ineffective. The cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and tetrahydrobiopterin (BH4) all shunt electrons from the substrate reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the active site, and are essential for NOS action. Radiolabelling studies have shown that NO is generated from one of the terminal guanidino nitrogens of L-arginine, while the remaining oxygen becomes incorporated into a ureido-oxygen.18

Nitric oxide can also be generated from several chemical sources such as S-nitrosothiols, organic nitrates, iron-nitrosyl complexes, sydnonimines, C-nitroso compounds and secondary amine/NO complex ions. S-nitrosothiols occur naturally and may have a role in several physiological and pathophysiological processes.29 The relative importance of NO generation from NOS in the airways versus NO formation from S-nitrosothiols in asthma is currently still being elucidated.

Nitrosothiols

Nitrosothiols (R-SNO) are an important class of NO donors, are present in vivo, and play a role in several physiological and pathophysiological processes. Their decomposition is catalysed by Cu2+ ions, which can themselves be formed by reduction of Cu2+ by thiols. They break down to form NO and the corresponding disulphide (RSRR).

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S-nitrosothiols have been shown to be present in vivo and in human airway lining fluid,30 and are probably produced as natural breakdown products of NO metabolism. They may act as stores of NO that can be released when required. There are several potential mechanisms for the decomposition of thiols to produce NO, including catalysis with copper, transnitrosation, enzymic decomposition with gammaGT, photochemical or thermal decomposition, and reaction with ascorbate (vitamin C). On current evidence, it appears likely that cata-lysis with Cu2+ is the most important mechanism in vivo, but further information is needed in this area.

Inhibitors of nitric oxide synthases

Several stereoselective inhibitors of NOS are now available. L-N methyl arginine (L-NMA) was the first reported inhibitor, but others include NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L- arginine (L-NMMA). NG-nitro-L-arginine methyl ester is a more soluble derivative of L-LNNA and may act as a pro-drug for L-NNA. More recently, selective inhibitors have been developed, such as aminoguanidine, which has a 10–100-fold greater selectivity for iNOS than for cNOS. Inhibition is usually competitive and reversible, but some reports have indicated irreversible inhibition at high concentrations.18 A number of these inhibitors are endogenously produced and have been found in mammalian tissues, including L-NMA, NG and NG dimethylarginine. These inhibitors may accumulate in renal failure and have a therapeutic role in the reversal of septic shock.31

Effect of corticosteroids

Corticosteroids inhibit the expression of the iNOS, but have no effect on other forms of NOS.32, 33 Based on in vitro findings, prednisolone in humans would therefore be expected to inhibit NO formation when iNOS has been induced from a variety of different stimuli, but leave basal cNOS expression unaffected. The neural form (type I NOS) is not sensitive to steroids and therefore neural bronchodilator NO release is not affected.

Actions of nitric oxide in the lung

In the lungs, NO is involved in regulation of vasodilatation, in neurotransmission, and as an agent of inflammation and cell-mediated immunity. Nitric oxide plays a major role in the pulmonary host defence mechanism and has been implicated in bacteriostatic as well as bactericidal processes. It affects ciliary beat frequency, mucus secretion and plasma exudation, and has also been implicated in genotoxicity.3

Because of its role in vascular smooth muscle relaxation, it was initially predicted that NO would prove a bronchodilator within the lung. Nitrovasodilators such as glyceryl trinitrate and other NO donors relax airway smooth muscle in vitro, but the effects in this regard are diminished by the presence of an intact epithelium.34, 35 Nitric oxide will reduce methacholine-induced bronchoconstriction in anaesthetized guinea pigs, as will a NO donor, but the effect is small and requires fairly high levels of inhaled NO.36 Studies in humans have shown that inhalation of NO has only a small effect on resistance and airway calibre, both in normal and asthmatic subjects, so it is unlikely that this will prove a major function of NO in vivo. 37

Smooth muscle exists within the lung in both vascular endothelium and the bronchi. With regard to the pulmonary vasculature, many studies have confirmed that NO plays a key role in regulation of pulmonary arterial vasoconstriction.38, 39 Inhibitors of NO formation reduce the vasodilator response to acetylcholine in pulmonary arteries in vitro and in animals40, 41 and inhaled NO acts as a potent selective pulmonary vasodilator in vivo. 42, 43 Basal release of NO from pulmonary endothelial cells serves to maintain dilatation of the pulmonary vascular bed and release of nitric oxide from patients with pulmonary hypertension is decreased.44 Inhaled NO may have therapeutic potential in the secondary pulmonary hypertension due to chronic obstructive pulmonary disease.45 As this is not the primary topic of this review, interested readers are referred to several excellent summaries of this area.39, 46

Nitric oxide also plays an important role in NANC transmission. Nonadrenergic non-cholinergic transmission nerves are the only neural bronchodilator pathway in human airways. Nitric oxide is released from inhibitory NANC nerves and this is important in several physiological functions in the gut, bladder and reproductive organs.47 It was research about the role of NO in penile erection that resulted in the development of the popular drug sildenafil (Viagra), bringing sustained satisfaction to many a couple. In higher areas such as the lungs, NO accounts for the bronchodilator NANC response in human airways in vitro.48, 49 Such NO probably derives from intrinsic nerves within the airways rather than another transmitter substance releasing NO from candidate cells such as endothelial, epithelial or smooth muscle cells. Nitric oxide may also be involved in the neurogenic vasodilator response in the pulmonary circulation, acting independently from endothelial NO release.50

In keeping with the reputation of NO for ubiquity, the endothelium and nerves are not the only source of NO in the lung. Nitric oxide is produced by a wide variety of structural and inflammatory cells, including those involved in asthma such as eosinophils, macrophages, mast cells, epithelial cells, and smooth muscle cells themselves.51 The highest NO output is from epithelial cells and macrophages, and these origins may be particularly relevant to asthma.52

Epithelial cell stimulation in vitro by cytokines and lipopolysaccharide results in iNOS induction, with the production of large amounts of NO.28 Calcium- and calmodulin-independent nitric oxide synthase immunoreactivity can be demonstrated in lung macrophages and epithelial cells from rats treated with LPS,51, 53 as well as from areas of inflammation in human lungs.51 In asthmatic subjects, iNOS reactivity can be demonstrated in bronchial biopsy specimens, whereas no such staining can be shown in normal volunteers.54 Epithelial-derived NO has been suggested as a physiological defence against infection and such NO could influence airway disease by its antimicrobial activity. Nitric oxide from the airway epithelium would also be expected to have effects on ion transport and, thence, on mucociliary clearance.55 It is interesting that low levels of exhaled NO have been shown in patients with Kartagener's syndrome56 and also with cystic fibrosis (CF).57 Reduced staining for NOS in airways of patients with cystic fibrosis and evidence from CF knockout mice58 have suggested that an inherited inability to increase NO production in CF airways predisposes them to infection.

Alveolar macrophages synthesize NO after exposure to various cytokines and endotoxins, and will express iNOS after such stimulation.59, 60 Nitric oxide is involved in several toxic activities of macrophages such as inhibition of mitochondrial respiration, aconitase activity and DNA synthesis, which are often mediated through iron-containing enzymes, and such activities are inhibited by the use of NO synthase antagonists in laboratory studies.61 The cytotoxic action of NO is important in host defence, and may also be involved in immunosuppression, as alveolar macrophages have a suppressive effect on T lymphocyte proliferation, and this effect may be antagonized by NOS inhibitors.62, 63 Nitric oxide may also promote the development of TH2 responses because NO reduces levels of IFN-gamma, thus allowing proliferation of TH2 cells and perpetuation of the inflammatory response. Eosinophils and mast cells can produce NO, and animal studies have shown that NO plays a key role in eosinophil migration and infiltration in rats,64, 65 as well as the migration of other cells such as neutrophils.66 Activated eosinophils release NO and may also recruit companion eosinophil migration by other mechanisms such as increasing microvascular leakage.67

Role of S-nitrosothiols

S-nitrosothiols have been demonstrated in human airway lining fluid, and high levels of S-nitroso-L-glutathione (GSNO) are found in human bronchial lavage fluid (BALF), approximately 0.3 mmol/L.30 These compounds have bronchodilator activity, and levels have been shown to be reduced in the tracheal aspirates of children with acute asthma exacerbations, thereby implying there may be a defect in nitrosothiol levels in patients with asthma.68 S-nitroso-L- glutathione may also contribute to airway homeostasis by its antimicrobial and anti-inflammatory properties. S-nitroso-L-glutathione also appears to inhibit eosinophil apoptosis, and may serve to protect the airways from inflammatory or chemical insult.29 One study has linked GSNO deficiency with asthmatic respiratory failure, postulating that GSNO is metabolized in the asthmatic airway to NO.68

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Clinical studies on exhaled nitric oxide

Exhaled nitric oxide, or eNO, can easily be measured in the normal subject. The factor which gives this test such an advantage over others is the totally non-invasive character of the measurement. Exhaled nitric oxide has now been measured successfully in normal adults of all sexes and inclinations,69 adolescents, infants, toddlers and small children,70, 71, 72 the elderly,73 and also in many animals.74 It can be measured in various different parts of the respiratory tract from the nose to the distal respiratory tract via a bronchoscopic approach,75 or in patients who are intubated or tracheotomized,76 and has been employed in large epidemiological studies.77 Because of the varied methods that have been used to measure eNO, the European Respiratory Society in 1998 published recommendations for a standardized method of measurement,8 shortly followed by the American Thoracic Society.9 These differ only in minor details, and either can be used for establishment of the technique.

Exhaled nitric oxide is generally measured via chemiluminescence, using the reaction of ozone to generate light, which can be measured photometrically. Several sensitive NO analysers are now commercially available and are generally sensitive to NO levels of <1 p.p.b., with a rapid response time (<3 s). Nitric oxide can be sampled either directly or into a reservoir. Several investigators have used impermeable bags, including Tedlar bags, but metallised balloons are more entertaining for children and wine cask bags have, characteristically, been used in Australia. Preliminary evidence suggests a fairly good correlation between the two techniques.78 Normal levels are between 8 and 14 p.p.b. As the clinical significance of such measurements has not been fully established, the results are currently relevant only to research. Measurement of eNO has, however, been suggested to be a new lung function test, and further research into the clinical relevance of eNO is required.

Exhaled nitric oxide measurement

Nitric oxide is formed both in the upper and lower respiratory tracts, with very high levels in the nose and sinuses (approximately 900 p.p.b.),79 and it is therefore important that techniques are used to exclude nasal contamination if attempting to measure lower respiratory eNO. It is believed NO is formed in the upper and lower respiratory tracts and diffuses into the lumen down a concentration gradient. Alveolar NO is extremely low due to the avidity of the uptake of NO by Hb; studies using isolated porcine lungs have demonstrated that the contribution of the pulmonary vasculature to eNO is small.80 Gastric NO levels may be high and this can be demonstrated by eructating into the analyser; however, this does not usually appear to contaminate eNO. The usual equipment for eNO measurement is shown in Figure 3. Full details are available in the published recommendations.8, 9 The following description relates primarily to direct eNO measurement.

Technical factors

There are three main technical factors that may spuriously affect eNO levels: inspired NO concentration, nasal contamination and exhalation procedure (Table 1). Background ambient NO concentrations may be high, especially in polluted cities such as Paris,81 and although such levels have not been shown to have a large effect on the single breath plateau levels of eNO, it is recommended that subjects should breathe NO-free air.8 This can be achieved either using an NO-free air source, or utilizing an NO scrubber. As saliva contains both nitrite and nitrate, and NO can be formed in the oral cavity by bacteria, it is recommended that a mouthwash containing 10% sodium bicarbonate is used prior to exhalation.82 Nasal contamination can be minimized by making sure that the subject is encouraged to inhale through the mouth to total lung capacity without use of a noseclip, and then exhales immediately against an expiratory resistance of between 5 and 20 cm H2O. The expiratory resistance against a positive mouthpiece pressure closes the posterior nasopharynx and prevents leaks from the velopharyngeal aperture. The immediate exhalation is important as breath holding has been shown to increase exhaled NO. It is common to display the exhalation trace to the subject, who can then be instructed to try to keep the pressure and expiratory flow within a certain range. The exhalation flow rate is important as very low flow rates amplify eNO and too high a rate may decrease eNO.83 Too low a flow rate may be uncomfortable for the subject and may alter NO production and therefore a compromise flow rate of 0.05 L/s (BTPS) has been recommended.


Traces are displayed on a computer monitor as an eNO versus time plot and consist of a washout phase followed by an NO plateau. An early peak before the plateau usually represents nasal contamination and should be ignored. The plateau levels are recorded and are usually reproducible and flat (Figure 4).

Figure 4.
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Schematic diagram demonstrating traces during measurement of exhaled nitric oxide (eNO) in a normal subject. The trace on the left represents an early peak followed by a plateau after the subject has inhaled through the nose. The trace on the right is a mouth inhalation after mouthwash. FENO, fractional exhaled NO (p.p.b.).

Full figure and legend (8K)

Usually at least 6 s are needed to produce a plateau, and three readings are performed, or sufficient readings to produce at least three readings that agree within 10% of the mean value. Specific criteria have been published to accurately define a plateau.9 Subjects should rest for at least 30 s between readings, as repeated forced expiration can change NO output.

Exhaled nitric oxide in normal subjects

Patient factors may also affect eNO, and are summarized in Table 2. In adults, there have been no consistent relationships described between age or sex and eNO levels; possible racial factors are uncharacterized. One study showed a relationship with the menstrual cycle in women, and another a change in eNO with age in children, but these have yet to be confirmed. A circadian rhythm of NO production has been suggested, but more data are needed.84 Vigorous exercise has been shown to increase eNO,85 so it is recommended that subjects rest before the readings. Similarly, spirometric measurements have been reported to transiently reduce NO, so it is suggested that these should be performed after eNO.86 Studies on the effect of airway calibre on eNO have been conflicting, with some describing no effect of bronchodilators, and others reporting a reduction in eNO.87, 88 Food and beverages may alter eNO, but there are few data on this topic. Nitrate or nitrite- containing foods such as lettuce may alter eNO82 (although this effect may be reduced by a mouthwash), and alcohol and caffeine-containing drinks may similarly change eNO,89, 90 but only if ingested recently. Several drugs may affect eNO, including inhaled and oral glucocorticosteroids, especially in asthmatics, where eNO is exquisitively sensitive to such treatment.6 Normal subjects are more resistant. Oral L-arginine in high dosage increases eNO in normal subjects, as does nebulized L-arginine in normals91 and patients with cystic fibrosis. Smoking, both active and passive, can reduce eNO, and a close correlation is observed between eNO levels and the number of cigarettes smoked.92, 93 Upper and lower respiratory tract infections increase eNO, although interestingly sinusitis does not appear to increase nasal NO.79


Exhaled nitric oxide in diseases other than asthma

Because eNO is raised in asthma, it might be expected that levels of eNO would also be elevated in patients with chronic obstructive pulmonary disease (COPD), where airway inflammation also exists. However, the inflammation in COPD is primarily neutrophilic rather than eosinophilic. The situation regarding eNO in COPD is unclear; eNO levels have been reported to be increased in two studies,94, 95 and unchanged in another.96 The absolute NO values appear to be much lower than those of asthma, possibly reflecting the differences in inflammatory cell involvement in these different diseases. In patients with COPD, concentrations of NO derivatives were significantly correlated with the percentage of neutrophils and sputum IL-8,94 whereas in patients with asthma the correlations were with sputum eosinophils.97

In other diseases associated with airway inflammation, such as bronchiectasis, eNO may be raised, and the level of NO has been reported to be correlated to the extent of disease as measured by a computed tomography score.98 However, patients with CF do not have raised eNO, even during an infective exacerbation.57 This is unexpected, because in CF, iNOS staining is prominent in inflammatory cells that surround the airway. Calcium- and calmodulin-independent NOS staining is however, absent in airway epithelium, and the low eNO levels may reflect defective NO production, or else local metabolism or trapping.

Active pulmonary tuberculosis (TB) has also been reported to result in raised eNO, to levels approximately twice that of control subjects; levels decrease after appropriate antituberculous treatment for 3 months. Calcium- and calmodulin-independent NOS is upregulated in alveolar macrophages from patients with TB and correlates with NO values, and BALF macrophage nitrite generation is also increased and correlates with radiographic disease extent.60 Interestingly, however, active pulmonary sarcoidosis, where the macrophage has also been implicated as the dominant cell mediating the inflammatory response, does not result in raised eNO levels, nor in raised BALF nitrates/nitrites.99 Neither are NO levels raised in acute respiratory distress syndrome, where a neutrophilic infiltrate is predominant within the lung, and peroxynitrite is found in elevated concentrations in BALF. In fact, measured eNO values have paradoxically been shown to be low. Nitric oxide levels are also low in systemic sclerosis with associated pulmonary hypertension.100

Exhaled nitric oxide in asthmatic subjects

Many studies have now confirmed that exhaled NO is higher in steroid-naive asthmatic subjects compared with non- asthmatic subjects, both in adults and in children. In normal subjects, levels are higher in atopic compared with non-atopic subjects,101 but do not reach eNO levels seen in the patient with uncontrolled asthma not using steroids, or during an acute asthma exacerbation. Exhaled NO rises during a deterioration in asthma control6 and is also high during an acute asthma exacerbation,102 rapidly returning to normal levels soon after initiation of glucocorticosteroid (GCS) treatment.

Allergen challenge, the closest laboratory simulation of the asthmatic exacerbation in vivo, results in a rise in eNO, but only with the late response to allergen, and only in those asthmatics who experience a late asthmatic reaction (LAR).103, 104 It seems likely that this reflects the inflammatory cell infiltration that accompanies the LAR. The increase in eNO correlates with the magnitude of the LAR.103 Exhaled nitric oxide also rises with exposure to inhaled allergen such as grass pollens.105 Exhaled nitric oxide is increased during upper respiratory tract infections, presumably viral, in normal subjects,106 and also rises with influenza vaccination.107 In asthmatics, experimental infection with rhinovirus (RV16) led to increased levels of eNO, but only 2–3 days later; the level of NO was inversely correlated with histamine bronchial responsiveness.108

Corticosteroids alter eNO in asthma, but their effects are different in asthma from when administered to normal subjects. Oral prednisolone (30 mg for 3 days) reduces elevated eNO in mild asthmatic patients, but has no significant effect in subjects without asthma.109 Oral dexamethasone similarly produces no change in eNO in normals.110 Presumably, this is because iNOS is the major source of eNO in asthmatics, but in normals the source is more likely to be cNOS, which is not affected by steroids. Controlled studies using inhaled budesonide and also beclomethasone have shown that eNO is reduced by inhaled steroid treatment;6, 111 also, eNO rises if GCS therapy is withdrawn.6 The time course of the response is such that there appears to be a rapid onset of reduction in eNO, followed by a more slowly progressive reduction that does not plateau before 3 weeks. A single dose of nebulized budesonide produces a rapid reduction in eNO,112 presumably due to a rapid inhibitory effect on iNOS expression. Such rapid effects have also been seen in animal studies. In a recent bronchial biopsy study, 4 weeks treatment with inhaled budesonide reduced expression of iNOS in both airway epithelial cells and inflammatory cells as well as decreasing nitrotyrosine immunoreactivity (a marker of peroxynitrite formation).113 The effect of inhaled corticosteroids is dose-related, but appears to plateau at a relatively low dose of 400 mcg daily.114 Patients with severe asthma and continuing symptoms have persistently raised eNO despite treatment, possibly reflecting continuing inflammation in the respiratory tract.115

In asthma, airway hyperresponsiveness (AHR) is often used as a surrogate marker of airway inflammation, and therefore it might be expected that eNO would correlate with AHR. Several recent studies have shown a relationship between AHR to methacholine and eNO, as well as a correlation between eNO and sputum eosinophils.98, 114 Despite this, no relationship was seen between direct indices of airway inflammation in airway biopsies and eNO in one recent study.113

If endogenously produced NO serves to maintain bronchodilatation in asthma, analagous to NO regulating vascular tone, it would be expected that NOS inhibitors would precipitate bronchoconstriction. Raised eNO in asthma might be a compensatory response towards a marked tendency towards bronchoconstriction. However, current information seems to indicate that this is not an important function of NO in humans. Nitric oxide synthase inhibitors do not appear to have a large effect either on bronchoconstriction or on airway responsiveness in humans,5, 20, 109, 116 either when used in low or high doses. Although infusion of L-NMMA causes an increase in blood pressure in normal volunteers, neither L-NAME or L-NMMA, when nebulized, had any significant effect on heart rate or blood pressure, suggesting that NOS inhibitors, when given by this route, are confined to the respiratory tract.5, 109 Inhalation of aminioguanidine, a more selective inhibitor of iNOS than L-NAME, caused a decrease in eNO in asthmatic, but not in normal subjects, adding support to the view that the increased eNO in asthma derives from iNOS within the respiratory tract.116, 117, 118 Similarly, both a high and a low dose of L-NAME, inhaled by asthmatics prior to bronchial provocation challenge, had no effect on forced expiratory volume in 1 s (FEV1) and only the high dose had a minor effect on AHR to histamine and AMP.119

Source of origin of exhaled nitric oxide in asthma

What then is the reason for the raised eNO levels in asthma? It seems likely that the high eNO reflects a pathological state produced by the chronic inflammation that is characteristic of asthma. Pro-inflammatory cytokines such as TNF-alpha and IL-1beta are secreted in asthma and result in inflammatory cell recruitment, but also induce iNOS and perpetuate the inflammatory response within the airways. Nitric oxide produced in small quantities locally by cNOS activation may have a beneficial role, in neural transmission and in bacterial defence, but have deleterious effects when produced in much higher concentrations by iNOS. Nitric oxide could contribute to the hyperaemia of asthmatic airways through its vasodilatory effect, and this in turn could increase plasma exudation and inflammation. Nitric oxide is chemotactic for a variety of inflammatory cells, and may also have cytotoxic effects that could contribute to the epithelial shedding that is seen in asthma. Nitric oxide, through its reaction with superoxide, produces peroxynitrite, which is a powerful oxidant that can react with several proteins and lipids. Recently, a marker of peroxynitrate (nitrotyrosine) has been demonstrated in bronchial biopsy specimens in asthmatics after allergen challenge120, 121 and both nitrotyrosine and iNOS are reduced by the use of inhaled corticosteroids.113 Nitric oxide also plays a key role in the modulation of eotaxin, a chemokine that has selective chemotactic activity for eosinophils.122

What is the cellular origin of the NO detected in exhaled air? There are several possible sources. Nitric oxide could derive from the endothelium, airway nerves, or from the large amounts of NO produced by cytokine-generated iNOS induction either in airway inflammatory cells, in the airway epithelium, or both. Another alternative is that NO is generated in the airway lining fluid due to a drop in airway pH.123

Several lines of evidence suggest that the increased NO measurable in asthma reflects inflammation in the lower respiratory tract. As mentioned above, levels of eNO are increased in conditions associated with airway inflammation such as asthma, pollen inhalation, viral respiratory tract infections, experimental virus infections, and increases in eNO also accompany rejection in lung transplant recipients.124 Exhaled nitric oxide rises after allergen challenge, and is increased when measured during acute asthma exacerbations.5, 102 Raised NO is found in several other diseases where inflammation occurs (e.g. rheumatoid arthritis). It seems likely that the increase in exhaled NO is due to induction of iNOS, as increased NOS activity has been found in the lung tissue of patients with asthma, CF and obliterative bronchiolitis. In asthmatic patients there is evidence for the expression of iNOS in airway epithelial cells, whereas in normal subjects cNOS immunostaining alone is found.54 Pro-inflammatory cytokines such as TNF-alpha and IL-1beta induce iNOS expression in vitro and these same cytokines are also released in asthma.3 Nitric oxide synthase inhibitors reduce eNO in asthmatic subjects, and a NOS inhibitor more selective for iNOS reduces eNO in asthmatics, but not in normal volunteers.116 The differential effect of corticosteroids, inhibiting iNOS, but not cNOS, is also seen in vivo. 109

An alternative hypothesis is that NO is a metabolic by-product that reflects a metabolic deficiency in the asthmatic airway.68, 123 S-nitrosothiols such as GSNO are found in normal human airway lining fluid, and levels have been shown to be reduced during an acute asthma exacerbation.68 S-nitrosoglutathione is a more potent bronchodilator and antimicrobial agent than NO,30, 125 has anti-apoptotic properties,125 and may serve to stabilize NO and modify its cytotoxic potential.123 S-nitrosoglutathione may normally protect the epithelial lining of the airway from inflammatory or chemical insult. In conditions of stress, however, levels of GNSO may be depleted by metabolism to NO, thereby exposing the airway to further damage. Levels of GNSO have been shown to be reduced in the airway lining fluid of asthmatics in exacerbation compared to normal subjects, although asthmatics generally have slightly higher levels of nitrites than in the normal lung. A fall in airway pH, which has recently been shown to occur in acute asthma,125 could provide the ideal milieu for such a reaction to occur. Low airway pH would also allow release of NO from nitrous acid which in its turn has originated from the nitrite reserve of the airway lining fluid. It is currently unclear, however, whether the changes in airway pH are a consequence or the cause of the inflammatory process, and why any such metabolic defect should occur in asthma. Changes in pH are well recognized to occur in conditions of stress (e.g. septic shock), and these may simply be a reflection of the severity of the inflammatory process. Also, such an explanation for the generation of NO could not account for the marked fall in eNO observed by the use of NOS inhibitors.

Given the ubiquity of NO and the importance of NO in pathophysiology, it seems unlikely that eNO is derived from a single cell type or part of the airway. Exhaled nitric oxide may be generated by a number of different mechanisms, with contributions made by various sources in different diseases. Currently, the likeliest source for the majority of eNO in asthma appears to be iNOS, derived from airway inflammatory cells, epithelium or both, but other sources are indeed possible.

Clinical relevance of exhaled nitric oxide in asthma

Exhaled nitric oxide has been proposed as a sensitive marker of airway inflammation, a simple and non-invasive measurement that may predict the efficacy of anti-inflammatory treatment in asthma. It has also been proposed as a method for assessing compliance with anti-inflammatory therapy. Despite its apparent promise in this regard, there are few studies currently available that have addressed this issue, and those that have been performed have generally studied mild asthmatics whose response to therapy is usually not a problem.

It is widely assumed that a surrogate marker of airway inflammation will lead to better asthma control, in particular in the prevention of the airway wall remodelling that can lead to fixed airflow obstruction. The importance of treating airway inflammation rather than simply treating the symptoms is not, however, definitively established, although a recent study by Sont and colleagues would suggest that using AHR in addition to usual measures of asthma control may improve outcomes.126 With regard to eNO, two studies have assessed the effect of differing doses of budesonide on inflammatory markers after initiation of treatment in mild asthma,113, 114 but only one115 has included more severe asthmatics. The latter was a cross-sectional study of a selected group at a tertiary referral hospital and demonstrated a correlation between eNO, symptom frequency and rescue beta-agonist use, but no correlation with lung function. Exhaled nitric oxide levels were higher in subjects with difficult asthma already established on inhaled steroids and also in patients requiring oral corticosteroids. When eNO was assessed in comparison to other methods of assessment of airway inflammation in a placebo controlled cross-over manner using inhaled budesonide, a correlation was observed between eNO and PC20 prior to initiation of inhaled steroid, but not afterwards. In addition, no relationship was found between change in eNO and biopsy improvement.113 This can perhaps be accounted for by the apparent extreme sensitivity of eNO for the effect of inhaled steroid, which plateaus at 400 mcg.114 These data suggest that eNO may not be as useful as a way of monitoring asthma treatment or adherence as had previously been suggested. However, it is not yet possible to be certain with current information. The appropriate prospective studies in moderate or severe asthmatic patients have simply not yet been performed. It is possible that in the future eNO may provide further insights into different types of asthma and that different exhaled markers, along with other measures of asthma control, will be useful to assess differing categories of disease.

Other exhaled markers

Several other markers have been identified in exhaled air or breath condensate that may also prove useful for furthering understanding of lung disease, or for improving diagnosis or treatment in respiratory medicine in the future. These include exhaled carbon monoxide,127 hydrogen peroxide, 8-isoprostane, volatile organic compounds and ethane.128 All have the same advantage as NO in being completely non-invasive, and there is early evidence for their different expression in different types of lung disease. Information on these markers is rapidly emerging, and it is possible that the next 20 years may allow the development of breath 'fingerprints' that will cause our understanding of pulmonary pathophysiology to explode, just as it has with NO.

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