Immunology and Cell Biology (2001) 79, 178–190
Special Feature
Role of exhaled nitric oxide in asthma
D E B O R A H H YAT E S
Faculty of Medicine, Sydney University and Department of Respiratory Medicine, Royal North Shore Hospital,
St Leonard’s, New South Wales, Australia
Summary 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). Proinflammatory cytokines such as TNFα and IL-1β 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.
Key words: airway inflammation, asthma, nitric oxide, nitrosothiols.
Introduction
1
Nitric oxide (NO), molecule of the year in 1992, 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.
Chemical and biological properties of nitric oxide
Correspondence: Dr DH Yates, Suite 706, 26 Ridge Street, North
Sydney, NSW 2060, Australia. Email: [email protected]
Received 15 September 2000; accepted 11 October 2000.
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
Role of exhaled nitric oxide in asthma
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
[NO.]. 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
179
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 (Fig. 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).
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 (Fig. 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
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DH Yates
Figure 1 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.
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-α, IFN-γ and IL1-β, 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
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
(Fig. 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
Figure 2 Synthesis of nitric oxide (NO) from
ONOO-, peroxynitrite; BH4, tetrahydrobioprotein.
L -arginine.
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.
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-α, IFN-γ, and IL-1β).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).
2RSNO → RSSR + 2NO
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
Role of exhaled nitric oxide in asthma
181
Figure 3 (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.
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 γGT, photochemical or thermal decomposition, and reaction with ascorbate
(vitamin C). On current evidence, it appears likely that catalysis with Cu2+ is the most important mechanism in vivo, but
further information is needed in this area.
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.
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-Larginine methyl ester (L-NAME) and NG-monomethyl-Larginine (L-NMMA). NG-nitro-L-arginine methyl ester is a
more soluble derivative of L-LNNA and may act as a prodrug 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
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
182
DH Yates
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 calmodulinindependent 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-γ, 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-Lglutathione may also contribute to airway homeostasis by its
antimicrobial and anti-inflammatory properties. S-nitroso-Lglutathione 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
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,70–72
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
Role of exhaled nitric oxide in asthma
Table 1
183
Technical factors altering exhaled nitric oxide (NO)
Increased NO
Decreased NO
Low exhalation rate
Breathholding
High background
concentrations
of NO (>20 p.p.b.)
Contamination by
nasal NO
High exhalation flow rate
Water vapour
Use of plastic tubing
Suboptimal storage or leakage of reservoir
Poor response time/sensitivity
Repeated spirometry
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
Figure 4 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.).
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 (Fig. 4).
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.
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DH Yates
Table 2
Physiological and pharmacological factors affecting exhaled NO in humans
Increase exhaled NO
Pharmacological factors
Ingested and inhaled L-arginine
in normal and asthmatic subjects
Isocapnic cold air hyperventilation
Hyperoxia
?IV nitrates
Physiological factors
Menstrual cycle variations
Breathholding
Physical exercise
Diseases
Asthma
Allergen challenge
(late response)
Viral respiratory tract infection
Active tuberculosis
?Ozone
Formaldehyde in children
Decrease exhaled NO
Leave NO unchanged
Inhaled or IV NOS inhibitors
(L-NMMA, L-arginine, aminoguanidine, etc.)
Inhaled or oral glucocorticosteroids
Oral leukotriene antagonists
Bradykinin
Smoking
Passive smoking
Acute alcohol ingestion in asthmatics
Capsaicin
Caffeine
Heart failure
Prostaglandin E2 and F2α
Inhaled aspirin
Menstrual cycle variation
Repeated forced expiratory manoeuvres
Circadian rhythm (nocturnal asthmatics only)
Systemic sclerosis with pulmonary hypertension
Cystic fibrosis
Sarcoidosis
Acute respiratory distress syndrome
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 nitritecontaining 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
Role of exhaled nitric oxide in asthma
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 nonasthmatic 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
185
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 doserelated, 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.116–118
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-α and
IL-1β 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
186
DH Yates
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-α and IL-1β 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 byproduct 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
β-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
Role of exhaled nitric oxide in asthma
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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