Nasal nitric oxide in man

J O N Lundberg, E Weitzberg

The past decade has witnessed an explosion inthe interest of biologists in the gas nitric oxide(NO). This highly reactive free radical, firstconsidered only a noxious air pollutant, is pro-duced in mammalian cells by specific enzymesand is believed to play a vital role in many bio-logical events including regulation of bloodflow, platelet function, immunity, andneurotransmission.1 2 Direct measurement ofNO in biological tissues is diYcult to performbecause this gas reacts rapidly with, forexample, haemoglobin or other Fe2+-containing proteins. It is therefore often neces-sary to rely on indirect measurements in orderto detect NO synthesis in vivo. Unlike the situ-ation in most biological tissues where NO israpidly destroyed, in the gas phase NO is fairlystable at low concentrations.3 NO produced insuperficial structures of hollow organs will dif-fuse into the lumen and thus be detectable ingas collected from such organs.

The presence of NO in exhaled breath ofhumans was first demonstrated by Gustafssonet al in 1991.4 Later studies clearly showed that,in healthy controls at rest, almost all NO foundin exhaled air originates from the upper airwayswith only a minor contribution from the lowerrespiratory tract and the lungs.5–7

Origin of nasal NOThe exact origin of the NO found in nasal airand the relative contribution from diVerentsources within the nasal airways are not known.There are, however, some indications thatfavour the paranasal sinuses rather than themucosa of the nasal cavity as a major source ofnasal NO in adult healthy humans.8 Firstly, NOrelease in the sinuses is markedly reduced byintrasinus instillation of an NO synthaseinhibitor (L-NAME) whereas nasal NO con-centrations are only slightly reduced followingintranasal administration of this inhibitor.9

Secondly, immunohistochemical and in situhybridisation studies showed dense staining fornitric oxide synthase (NOS) and its mRNA insinus epithelium whereas only weak stainingwas found in the nasal epithelium.9 Thirdly, atransient decrease in nasal NO (measured fromone nostril) was observed in a study when airwas continuously removed from one maxillarysinus during nasal sampling and the opposite(transient elevation) occurred when air wasinjected into the same sinus.10 Fourthly, nasalNO levels are markedly reduced in patientswho generally have mucus filled paranasalsinuses and obstructed sinus ostia (Karta-gener’s syndrome,6 cystic fibrosis,11 acutesinusitis12). Finally, nasal NO levels are high inhumans and other primates13 but very low inbaboons,14 the only mammal lacking paranasalsinuses.

Both immunohistochemical and in situhybridisation studies indicate that the NOSfound in healthy sinus epithelium9 is identicalor very closely related to the inducible NOS(iNOS) that has been cloned from activatedhuman hepatocytes.15 Moreover, NOS activityin sinus mucosa is predominantly calciumindependent, a characteristic associated withiNOS.16 However, the regulation of the expres-sion and the activity of sinus NOS seems todiVer from that previously described for iNOS.Thus, sinus NOS is constantly expressed andseems to be resistant to steroids,9 16 propertiesnormally associated with the low rate NO pro-ducing endothelial and neuronal NOS.Immunostaining for NOS in the nasal mucosahas shown only weak staining for iNOS.9

Moreover, Ramis et al found only calciumdependent NOS activity in nasal mucosa fromhealthy subjects.17 Human nasal epitheliumfrom patients with nasal polyp disease expressincreased iNOS mRNA compared with normalepithelium.18

Measurement of nasal NOAlmost all studies in the literature on themeasurement of nasal NO have used thechemiluminescence method. This method usesan excess of ozone (O3) which reacts with NOto produce nitric dioxide (NO2) with anelectron in an excited stage (NO2*). NO2*changes back to the ground state whileemitting electromagnetic radiation in the wave-length range 600–3000 nm. A photomultipli-cator tube that proportionally converts theintensity of luminescence into an electrical sig-nal detects this chemiluminescence. Thechemiluminescence technique is highly sensi-tive and NO can be detected in levels down toparts per trillion.3 19 It is rapid and easy to usewhich allows for on line measurements ofexhaled NO. Other techniques that have beenused to establish that NO is present in theexhaled breath of humans include massspectroscopy and gas chromatography-massspectroscopy.4 20

Several techniques have been used forcollecting nasal air, the most commonly usedmethod being to sample nasal air directly fromone nostril.21 22 Using the intrinsic flow of thechemiluminescence analyser or an externalpump, air is aspirated from one nostril. Thecontralateral nostril is left open and the subjecteither holds his/her breath or breathes throughthe mouth. Air is thereby forced from one sideof the nose to the other via the nasopharynx. Toavoid contamination with NO from the lowerairway the subject can be asked to exhale orallyagainst a resistance in order to close the softpalate.23 24

Another suggested method of measuringnasal NO is to exhale through the nose after a

Thorax 1999;54:947–952 947

Department ofPhysiology andPharmacology,Karolinska Institute,171 77 Stockholm,SwedenJ O N Lundberg

Department ofAnesthesiology andIntensive Care,Karolinska Hospital171 76 Stockholm,SwedenE Weitzberg

Correspondence to:Dr J O N Lundberg.

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full inhalation to vital capacity. The exhalationshould be made at a constant flow rate.Following this procedure the subject thenexhales through the mouth from full vitalcapacity at the same flow rate and the NO valuefrom oral exhalation is subtracted from thatmeasured during nasal exhalation (unpub-lished observation).

The optimal measurement technique fornasal NO is yet to be determined. A reliableand standardised method will allow for bettercomparisons of results between diVerent labo-ratories. Attempts to standardise nasal NOmeasurements are in progress and recommen-dations for suitable sampling techniques will begiven in the near future.

Factors that influence nasal NO levelsMEASUREMENT TECHNIQUE

Since NO is continuously released into thenasal airways the concentration will be depend-ent on the flow rate by which the sample isaspirated.21 Thus, nasal NO concentrations arehigher at lower flow rates. It may therefore bepreferable to express nasal NO as the outputper time unit (e.g. nl/min) which has beenshown to be relatively independent of flowrate.25

PHYSIOLOGICAL FACTORS

In an early study nasal NO was measured inhealthy subjects at diVerent ages ranging from0 to 70 years.9 Interestingly, nasal NO wasalready present at birth. In a later study Sche-din et al found significant nasal NO levels innewborn infants including those delivered bycaesarian section.26 In a study by Lundberg et althe same flow rate was used in all age groupsand nasal NO was found to be much lower innewborn babies and in infants than in adults.However, if the nasal NO concentration fromthese data is calculated in relation to bodyweight, the concentration in children around10 years of age is found to be approximatelytwice as high as that in newborn infants andadults. There is no evidence of sex diVerencesin nasal NO, but variation in NO levels has notyet been studied in relation to the menstrualcycle. NO output in the nasal airways is acutelydecreased by physical exercise.27–30 In the studyby Lundberg et al27 direct measurement fromone maxillary sinus showed an 83% reductionin sinus NO concentrations after five minutesof exercise. The decrease in nasal NO levelscannot be explained merely by dilution of nasalair due to changes in nasal cavity volume orincreased ventilation. Instead, these changes innasal NO have been attributed to a reduction

in the blood flow in the mucosa of the nasalairways with a concomitant decrease in sub-strate supply to the high rate producing NOS inthe paranasal sinuses.27

DISEASES

The eVects of various diseases on nasal NOlevels are summarised in table 1. In childrenwith Kartagener’s syndrome—a triad consist-ing of sinusitis, bronchiectasis, and situsinversus—nasal NO levels are extremely lowcompared with healthy, age matched controls.6

Nasal NO levels are also very low in patientswith cystic fibrosis (fig 1).11 31 32 Baraldi et almeasured nasal NO in a group of children withacute sinusitis and found low nasal NO levels.12

In these patients nasal NO levels increasedwhen the patients improved following treat-ment with antibiotics. Chronic sinusitis is asso-ciated with more than a 50% reduction in nasalNO levels.33 In general, the above studies indi-cate that nasal NO levels are lower in subjectswith sinus disorders. It has not been estab-lished whether the low nasal NO levels inpatients with sinus disorders are purely a resultof altered passage of NO gas from the sinusesto the nasal cavity or whether the actualproduction of NO in the sinuses is decreased inthese patients.

Whether or not nasal NO levels are altered inpatients with rhinitis of diVerent aetiology hasnot been fully established. Some groups havereported higher nasal NO levels in patientswith allergic rhinitis.34–38 In one study nasal NOdecreased following treatment with nasal topi-cal steroids.35 Kirsten et al reported increasednasal NO levels after nasal challenge withendotoxin-containing swine confinementdust.39 In contrast, we found no alterations innasal NO levels in a group of children withperennial rhinitis.11 The reasons for the dis-crepancies between the studies are not clear.One might speculate that iNOS is upregulatedin the nose during rhinitis,40as is the case in thelower airways in asthma.41 This would explainthe higher levels of NO reported in some stud-ies. On the other hand, the swelling of the nasalmucosa present during rhinitis might also leadto partial blockage of the sinus ostia whichwould result in reduced passage of sinus NO tothe nasal cavity where it is measured.

Eccles et al measured nasal NO levels inpatients with a common cold but failed to see

Table 1 Nasal nitric oxide (NO): eVects of disease

Disease Age Nasal NO Reference

Kartagener’s syndrome Children 2.5–12 yrs Extremely low 6Primary ciliary dyskinesia Adults Extremely low 69Cystic fibrosis Children 5–15 yrs Decreased 11,31,32Allergic rhinitis Children 5–15 yrs Unchanged 11

Adults Increased 34–37Acute sinusitis Children Decreased 12Chronic sinusitis Adults Decreased 33Common cold Adults Unchanged 42Preeclampsia Adults Unchanged 44

Figure 1 Steady state NO levels in air sampled directlyfrom one nostril of healthy children, children with cysticfibrosis, and children with Kartagener’s syndrome. Datafrom Lundberg.6 11

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any diVerences compared with healthycontrols.42 In another study on experimentalhuman influenza nasal NO levels decreasedslightly during the symptomatic period.43

A general NO deficiency has been implicatedin the pathogenesis of pre-eclampsia, a diseaseof pregnancy characterised by oedema, hyper-tension, and proteinuria. However, in a recentstudy we found similar nasal levels of NO inpatients and healthy pregnant controls.44

DRUGS

The eVects of various drugs on nasal NO aresummarised in table 2. Early reports on theeVect of topical glucocorticoids showed noeVect of these drugs on nasal NO levels.6 Infact, not even systemic steroids in high dosesseemed to alter nasal NO levels.16 Gerlach et alsuggested that bacteria in the nasopharynxinduced nasal NO release.7 However, we foundno eVects of systemic antibiotic treatment onnasal NO levels.6 Moreover, as already men-tioned, newborn babies delivered by caesariansection had high nasal NO levels.26 Nasal NOrelease therefore seems to be independent ofbacteria normally present in the nasopharynx.

Topical nasal decongestants such asoxymetazoline have been shown to decreasenasal NO levels.37 42 45 The reason for this is notclear but it has been suggested that thedecrease in nasal/sinus blood flow induced bythese drugs leads to a reduction of substratesupply to the high producing iNOS in thesinuses.45 Indeed, substrate supply seems to beof importance for nasal/sinus NO release sinceintravenous administration of L-arginine re-sulted in an increase in nasal NO levels.16

Rinder et al looked at the eVects of histamineand capsaicin on nasal NO levels but found noeVects of these drugs.45

The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) has onlyminor eVects on nasal NO levels when admin-istered locally in the nose.9 45 In contrast, Albertet al found a substantial decrease in nasal NOlevels following intravenous administration ofNG-monomethyl-L-arginine (L-NMMA), an-other NOS inhibitor.46 This fits well with thetheory that the paranasal sinuses are the mainsource of NO in the nasal airways. Thus, NOSinhibitors probably penetrate poorly into thesinuses if administered locally in the nosewhereas, if given intravenously, these drugs willreach the sinus mucosa.

Nasal NO levels have been reported to belower in cigarette smokers7 47 but increase oneweek after smoking cessation.47

Animal modelsSchedin et al have measured nasal NO in alarge number of species and found high levelsonly in primates and in elephants.13 Lewand-owski et al recently measured nasal NO inbaboons and found only low levels.14 Interest-ingly, baboons are the only mammal known tolack paranasal sinuses. Because of the largevariation in nasal NO output between differentspecies, caution should be taken when compar-ing data from animal models with results fromstudies in humans.

Role of nasal NOAlthough the exact role of nasal NO is far fromclear, it is reasonable to believe that thispluripotent gas is involved in a variety of physi-ological as well as pathophysiological events inthe airways.

HOST DEFENCE

Among the various biological properties of NOare its eVects on the growth of variouspathogens including bacteria, fungi, andviruses.2 48 NO produced by white blood cells isthought to be important in the killing of certainmicro-organisms by these cells. Furthermore,some bacteria are sensitive to authentic NO gasin concentrations as low as 100 parts per billion(ppb).49 The fact that local NO concentrationsin the nasal airways may be several hundredtimes higher than this supports the notion thatNO is involved in local host defence in theupper airways. Local concentrations in theparanasal sinuses can reach 30 000 ppb.9 Wehave suggested that this NO may help to keepthe sinuses sterile under normal conditions.One might speculate that very low nasal NOlevels in patients with Kartagener’s syndromeor cystic fibrosis8 contribute to the increasedsusceptibility to airway infections in thesepatients. If this is correct, stimulation ofendogenous NO production could increase theresistance to airway infections in patients withlow nasal NO levels as suggested earlier.6 It isnot clear whether NO itself acts directly onmicro-organisms or whether it combines withother components to yield other reactive nitro-gen intermediates that are toxic.50

Besides acting directly on micro-organisms,NO may also contribute to local host defenceby stimulating ciliary motility.51 A study byRuner et al showed that application of an NOdonor in the nasal mucosa of humans did,indeed, cause an increase in ciliary beatfrequency.52 Furthermore, the same group hasshown that low levels of nasal NO correlatewith impaired mucociliary function in thehuman upper airways.53

INFLAMMATION

NO synthesis is clearly enhanced locally at sitesof inflammation.54 This has been described notonly in asthma5 but also in, for example,inflammatory bowel disease55 56 and cystitis.57

However, the role of NO in inflammation is far

Table 2 Nasal nitric oxide (NO): eVects of drugs, cigarette smoke and physical exercise

Drug Administration Nasal NO Reference

GlucocorticoidsHealthy subjects Nasal instillation Unchanged 6

Systemic Unchanged 16Allergic rhinitis Nasal instillation Decreased 35

NO synthase inhibitors Nasal instillation Minor decrease 9,45Systemic Decreased 46

L-arginine Nasal spray Unchanged 45Nasal spray Increased 69Systemic Increased 16,44

Histamine, capcaisin Nasal spray Unchanged 45Alpha-2 agonists Nasal spray Decreased 37, 42, 45Cigarette smoke Decreased 7,47Antibiotics Systemic Unchanged 6Physical exercise Decreased 27–30

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from certain. Some studies indicate a harmfulrole for NO during inflammation, whereasothers indicate the opposite. Possible pro-inflammatory actions of NO include activationof enzymes such as cyclo-oxygenase or metallo-proteases. Moreover, peroxynitrite, which isformed from the reaction of NO with super-oxide, can exert toxic eVects to tissues.36 58 Thepossible harmful eVects of NO have beenattributed to the large amounts of this gas pro-duced by iNOS during inflammation. How-ever, the recent finding of a constantlyexpressed iNOS in the nasal airways compli-cates this picture since it clearly demonstratesthat the sole expression of iNOS and thesubsequent increased production of NO is notassociated with tissue damage. On the con-trary, iNOS in the upper airways may serveimportant protective functions as discussedabove. Furthermore, in an animal model ofcolon inflammation McCaVerty et al haveshown that mice lacking the iNOS genedevelop a much more severe inflammation thanwild type mice.59

NASAL NO AS AN AIRBORNE MESSENGER

The eVect of inhaled exogenous NO iscurrently being investigated in large scale clini-cal trials in patients with pulmonary hyper-tension and/or adult respiratory distress syn-drome and its clinical use in newborn childrenwith persistent pulmonary hypertension hasbeen reported.60 Several investigators havefound clear eVects on arterial oxygenation andpulmonary arterial pressure using concentra-tions of inhaled NO as low as 10–100 ppb.61 62

Interestingly, during normal breathing endo-genous NO is inhaled at concentrations(approximately 100 ppb) which are known tohave vasodilating eVects in the pulmonarycirculation.6 7 We have shown that nasalbreathing reduces pulmonary vascular resist-ance and improves arterial oxygenation com-pared with oral breathing in subjects withoutlung disease (fig 2).8 63 64 The addition of 100ppb NO during oral breathing mimicked theeVect of nasal breathing while moistened airduring oral breathing had no eVect.63 Intubatedpatients are deprived of the natural inhalationof endogenous upper airway NO. Supplemen-tation of nasal air to intubated patients treated

with a ventilator also improves arterial oxy-genation and reduces pulmonary vascularresistance.65 Moreover, Pinsky et al have shownthat the hospital pressurised air may containNO levels similar to those described above(6–500 ppb) which may consequently haveeVects on arterial oxygenation and pulmonaryarterial pressure in mechanically ventilatedpatients.66 67 In a recent study nostril wideningwith a nasal tape improved arterial oxygenationin spontaneously breathing patients, probablyby enhancing ventilation through the nasal air-ways thereby increasing the delivery of NOfrom the nasal airways to the lungs.68

These results show that NO derived from theupper airways is capable of improving oxygenuptake and reducing pulmonary vascularresistance (fig 3). It is tempting to speculatethat the production of NO in the paranasalsinuses has the purpose of modulating lungfunction in humans. Since NO is producedabove the bifurcation of the large airways thisvasodilating gas will only aVect pulmonary ves-sels in contact with ventilated alveoli, therebyimproving ventilation/perfusion matching.This new physiological principle of NO as anairborne mediator may extend beyond vasodi-lation. Further studies will reveal whetherinhalation of endogenous NO also has antimi-crobial eVects, upregulates ciliary motility, andinhibits platelet aggregation. If this is true thewell known complications associated with longterm intubation and ventilation, such as ciliarydysfunction and bacterial infections, maypartly be explained by the lack of NO due todisruption of the natural low dose flushing ofthe lower airways by self-inhalation of endo-genous NO from the upper airways.69

Clinical value of nasal NO measurementsMeasurements of nasal NO levels are attractivesince they are completely non-invasive and caneasily be performed even in small children. The

Figure 2 Representative tracing showing transcutaneousoxygen tension in a healthy subject during periods of oralbreathing (empty bars), nasal breathing (filled bar), or oralbreathing of air containing exogenous NO at aconcentration of 100 ppb (hatched bar). Reprinted fromLundberg et al63 with permission.

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Figure 3 Nitric oxide (NO) is released in the nasalairways in humans. During inspiration through the nosethis NO will follow the airstream to the lower airways andthe lungs. Nasally derived NO has been shown to increasearterial oxygen tension and reduce pulmonary vascularresistance, thereby acting as an airborne messenger.

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finding of low nasal NO levels in patients withchronic sinus disorders such as Kartagener’ssyndrome and cystic fibrosis are interesting.8 Itis tempting to speculate that nasal NOmeasurements may be used to facilitate earlydiagnosis of these two respiratory disorders.

The possible usefulness of nasal NO meas-urements in the diagnosis and treatment moni-toring of allergic rhinitis clearly needs to befurther evaluated. If nasal NO is a reliablemarker of local inflammation, this simple testmay be used to monitor patients with rhinitis ofdiVerent aetiology.

As mentioned above, intubated patients aredeprived of inhaling the NO produced in thenasal airways. Considering the possible benefi-cial eVects of nasal NO on pulmonaryfunction, it is possible that replacement of nasalNO would be of benefit to patients being ven-tilated with an endotracheal tube.

It is reasonable to believe that drugs thataVect endogenous NO synthesis will be usedclinically in the future. These drugs may eitherfacilitate or decrease NO production by, forexample, interfering with diVerent NOS.Measurement of nasal NO could be aninteresting non-invasive means of monitoringthe eVects of such drugs and of optimising thedosage. The non-selective NOS inhibitorL-NMMA is currently being investigated foruse in patients with sepsis. Interestingly, thisinhibitor, when administered intravenously,leads to a dose dependent decrease in nasal NOlevels.46 Conversely, systemic administration ofL-arginine increases nasal NO levels.16

Summary and future directionsLarge amounts of NO are constantly beingreleased in the nasal airways of humans and theparanasal sinuses seem to be the major sourceof this NO. Nasal NO may have important localas well as distal eVects in, for example, hostdefence and regulation of pulmonary function.

The measurement of nasal NO levels may behelpful in further exploring the physiologicaland pathophysiological roles of this gas in theairways. Moreover, this simple test may beclinically useful in the diagnosis and treatmentmonitoring of certain respiratory disorders. Toevaluate fully the potential of nasal NOmeasurements in the clinical setting it isimportant that we develop a standardised andreliable technique. We also need to know moreabout the diVerent factors that aVect nasal NOlevels.

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