Clinical Science (2001) 101, 645–650 (Printed in Great Britain)
Exhaled nitric oxide after inhalation of isotonic
and hypotonic solutions in healthy subjects
Mauro MANISCALCO*, Alessandro VATRELLA*, George CREMONA†, Luigi CARRATU@ *
and Matteo SOFIA*
*Department of Respiratory Medicine, University ‘‘Federico II’’ A. O. Monaldi, Naples, Italy, and †Department of Respiratory
Medicine, San Raffaele Hospital, Milan, Italy
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Airway nitric oxide (NO) homoeostasis is influenced by chemical and mechanical stimuli in
humans ; airway epithelium, which is an important site of NO production, is sensitive to osmotic
challenge. The effect of inhaled hypotonic solutions on exhaled NO (eNO) is not known. In this
study we evaluated the effect of ultrasonically nebulized distilled water (UNDW), a hypotonic
indirect stimulus, on eNO levels. A total of 10 non-smoking healthy subjects were enrolled in the
study. eNO was detected by chemiluminescence, and specific airway conductance (sGaw) was
measured by plethysmography. Bronchial challenges with UNDW and with an isotonic solution
were performed according to a double-blind experimental design. Baseline levels of eNO were
28.1p14.7 p.p.b. UNDW did not cause any significant change in sGaw (from 0.190p0.029 to
0.181p0.036 cmH2O:s−1). With respect to baseline values, the eNO concentration decreased
significantly after inhalation of 8 or 16 ml of UNDW (from 26.0p13.1 to 17.2p8.5 and
16.6p7.7 p.p.b. respectively ; P 0.001, n l 10). After bronchial challenge with UNDW, eNO
was significantly reduced in comparison with after inhalation of the isotonic solution. In five
subjects, pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor NO
synthesis, decreased NO levels from 21.7p8.5 to 10.0p3.3 p.p.b. Subsequent inhalation of 16 ml
of UNDW did not cause any further decrease in NO levels (10.1p3.7 p.p.b. ; not significant
compared with L-NAME). We conclude that inhalation of aqueous solutions decreases eNO
levels in healthy subjects, and that this effect is not associated with any significant change in
airway calibre. The UNDW-induced decrease in eNO is not enhanced by pretreatment with the
NO synthase inhibitor L-NAME, suggesting that inhaled solutions may interfere with the airway
NO pathway in humans.
INTRODUCTION
Endogenous nitric oxide (NO) appears to play an
important role in the airway inflammatory response [1],
and may be involved in the control of airway tone [2].
Exhaled nitric oxide (eNO) provides a useful and noninvasive measure of NO production in vivo in humans
[3,4]. However, the dynamics of NO exchange in the
lung are complex, and the factors affecting NO homoeostasis are still not completely understood. Indeed, recent
studies have shown that both chemical [5] and mechanical
[6] stimuli may be responsible for changes in eNO in
healthy subjects, as well as in asthmatic patients. When
inhaled, NO synthase (NOS) inhibitors and the NOS
substrate L-arginine may respectively decrease and increase eNO levels in humans [7].
Key words: nitric oxide, NG-nitro-L-arginine methyl ester, ultrasonically nebulized distilled water.
Abbreviations: D , NO airway diffusion ; eNO, exhaled NO ; L-NAME, NG-nitro-L-arginine methyl ester ; NOS, nitric oxide
NO
synthase ; sGaw, specific airway conductance ; UDNW, ultrasonically nebulized distilled water.
Correspondence: Dr Mauro Maniscalco, Via Nicolardi 52, 80131 Napoli, Italia (e-mail mauromaniscalco!hotmail.com)
# 2001 The Biochemical Society and the Medical Research Society
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M. Maniscalco and others
The airway epithelium seems to be able to respond to
osmotic stimuli by releasing epithelium-derived relaxing
factors, which could contribute to the regulation of
airway calibre by modulating the responsiveness of the
underlying smooth muscle [8]. Consistent with this
hypothesis is the observation that inhalation of hypertonic solutions induces both bronchoconstriction and
a decrease in eNO levels in asthmatic patients [9].
Ultrasonically nebulized distilled water (UNDW) is an
osmotic\hypotonic indirect stimulus, which elicits
bronchoconstriction in some asthmatic subjects, but not
in healthy individuals [10].
In order to assess the effects of osmolarity on NOdependent airway tone, the effects of UNDW on eNO
levels in healthy subjects were studied.
Phase 2
The subjects were divided randomly into two groups,
receiving either UNDW or isotonic solution (0.9 %
NaCl) ; the stimuli were delivered in a double-blind
manner. Bronchial challenges were performed on two
separate days at the same time ; eNO concentration and
specific airway conductance (sGaw) were determined at
baseline and 1 min after the challenge.
Phase 3
Five subjects also received pretreatment with 15 mg of
NG-nitro-L-arginine methyl ester (L-NAME ; Sigma),
which was followed by inhalant challenge with UNDW.
MATERIALS AND METHODS
Measurement of eNO
Subjects
A total of 10 non-smoking subjects, with no history of
nasal disease, asthma or other chronic airway disorders,
were recruited from hospital personnel and enrolled in
the study (see Table 1). The research was carried out in
accordance with the Declaration of Helsinki (1989) of the
World Medical Association. All participants gave written
informed consent and the study was approved by the
local hospital ethical committee.
Study design
The study included three phases, according to the
following protocol.
Phase 1
eNO levels were measured in all subjects on three
different days. On each day, three measurements were
performed at intervals of 10 min to obtain a baseline
Table 1
estimation of NO production. On the first day, lung
function was also assessed in all subjects 10 min after NO
measurement.
Characteristics of the subjects included in the study
FEV1, forced expiratory volume in 1 s.
Subject
Sex
Age
(years)
FEV1
(% of predicted)
sGaw
(cmH2O:s−1)
NO at baseline
(p.p.b.)
1
2
3
4
5
6
7
8
9
10
m
m
f
m
f
m
f
f
f
m
35
29
30
26
30
31
24
26
25
33
105
114
99
107
102
99
116
121
105
110
0.227
0.176
0.224
0.173
0.160
0.180
0.230
0.156
0.180
0.215
46.0
34.3
28.6
20.3
18.7
37.4
22.0
11.6
8.2
53.5
# 2001 The Biochemical Society and the Medical Research Society
NO was detected with a chemiluminescence analyser
(280 NOA Sievers Instruments, Boulder ; Sensor Medics,
Milan, Italy), characterized by a lower limit of detection
of 1 p.p.b. and a NO sampling rate of 200 ml\min. Daily
two-point calibration was performed with zero gas (Zero
Air Filter, Sievers) and a certified NO gas mixture at
1.01 p.p.m. (SIAD Osio). NO was measured in exhaled
air with the subject performing a single slow vital capacity
manoeuvre against an expiratory resistance according to
ATS guidelines [3]. The breathing circuit consisted of a
mouthpiece connected to a Hans-Rudolph valve, through
which air was inhaled and then exhaled via an expiratory
resistance, while targeting a fixed mouth pressure of
20 mmHg displayed on a pressure gauge. This technique
enables the velum to be closed, thus excluding nasal NO
during expiration. All subjects performed a vital capacity
manoeuvre and then a slow (20 s) exhalation against a
20 cmH O mouth resistance, with a resulting expiratory
#
flow rate of 45 ml\s. The single-breath pattern of eNO
showed an initial washout phase followed by a steady
plateau.
In five subjects eNO was also measured after exhalation against three separate resistances in turn while
maintaining the same expiratory pressure, thus yielding
eNO levels at three different flow rates of 52, 128 and
180 ml\s. NO output was calculated as the product of
flow rate and eNO concentration. The slope of regression
lines through these points reflects NO airway diffusion
(DNO) when NO output values (x axis) are plotted against
eNO values ( y axis) ; the x-axis intercept reflects the
airway wall NO concentration at zero flow [11].
Bronchial challenge
The test was performed as described previously [12].
Briefly, UNDW was generated by a De Vilbiss 65
Exhaled nitric oxide and hypotonic solutions
ultrasonic nebulizer (De Vilbiss Co., Somerset, PA,
U.S.A.), which produces particles with a mass-median
diameter of 4.7 µm. The nebulizer was calibrated to
spontaneously deliver 2 ml:min−". The water container
was weighed before and after each challenge. However,
the output delivered to the patient was likely to be
reduced by the tubing used to connect the patient to the
circuit. Every subject inhaled UNDW by performing
tidal breathing through a mouthpiece while wearing a
nose clip. The subjects inhaled at 4 min intervals for 1, 1,
2 and 4 min (corresponding to 2, 2, 4 and 8 ml of inhaled
water, with cumulative doses of 2, 4, 8 and 16 ml
respectively).
The isotonic solution was delivered using the same
procedure. Every subject inhaled the same cumulative
volume as during the UNDW challenge. L-NAME
administration was also performed using the De Vilbiss
nebulizer.
Lung function measurements
Figure 1 Percentage decrease in eNO concentration in
10 healthy subjects after the inhalation of cumulative doses
of UNDW or isotonic solution
Significance of differences : *P
0.05, **P
0.001.
Forced expiratory volume in 1 s was measured using a
computerized spirometer (FL 2200 ; SensorMedics) ; at
each time point, the best of three consecutive measurements (variability
5 %) was chosen.
Airway conductance, expressed as sGaw, was determined using a compensated whole-body plethysmograph (6200 Autobox ; SensorMedics). Measurements
were carried out in triplicate, with the subjects panting at
2 Hz.
Statistics
The results are expressed as meanspS.D. eNO levels and
sGaw were analysed by ANOVA and Student’s t test for
paired data. A P value of
0.05 was considered to be
significant. The F test was employed for analysis of
regression lines.
Figure 2 eNO concentration under basal conditions and
after pretreatment with L-NAME followed bythe inhalation of
UNDW (16 ml)
NS, not significant.
RESULTS
Baseline levels of eNO are reported in Table 1. eNO
values were comparable on the three different days of
phase 1, showing a coefficient of variation of
10 %.
With respect to baseline values of both sGaw and eNO,
no change was detected after plethysmography.
In healthy subjects, inhalation of 8 and 16 ml of either
UNDW or isotonic solution induced significant
decreases in eNO concentration (from 26.0p13.1 to
17.2p8.5 and 16.6p7.7 p.p.b. after 8 and 16 ml respectively of UNDW, and from 28.1p14.7 to 23.8p11.4
and 19.1p8.1 p.p.b. after 8 and 16 ml respectively of
isotonic solution ; P 0.001, n l 10). The eNO concentration had returned to baseline values 20 min after
UNDW challenge.
No significant change in sGaw was observed after
inhalation of UNDW (from 0.190p0.029 to
0.181p0.036 cmH O:s−").
#
After inhalation of both volumes (8 and 16 ml) of
UNDW, eNO levels were significantly reduced in
comparison with the values obtained after bronchial
challenge with the isotonic solution (P 0.05) (Figure 1).
Pretreatment with L-NAME caused a decrease in eNO
concentration from a baseline value of 21.7p8.5 to
10.0p3.3 p.p.b. (P 0.05, n l 5). After L-NAME
administration, inhalation of 16 ml of UNDW did not
induce any further decrease in eNO (10.1p3.7 p.p.b. ;
not significant) (Figure 2).
eNO levels after inhalation of UNDW or isotonic
solution measured at four points of expiratory flow are
# 2001 The Biochemical Society and the Medical Research Society
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M. Maniscalco and others
Figure 3
Plot of expired NO output against expired NO concentration (from arithmetic mean values) at four points of expiratory flow in five normal subjects at baseline (=)
and after isotonic solution () and UNDW ($).
Table 2 Mean eNO concentration at four expiratory flow rates in five normal subjects at
baseline and after challenge with isotonic solution and UNDW
Values are meanspS.D.
eNO concentration (p.p.b.)
Conditions
Baseline
Isotonic saline
UNDW
Expiratory flow (ml/s) …
45
52
128
180
15.2p4.2
14.1p4.4
11.7p1.1
13.5p3.9
12.2p3.8
10.8p1.4
6.4p2.0
6.0p2.2
5.7p0.4
5.1p2.0
4.0p1.4
4.1p0.1
shown in Table 2. The comparison between the regression
lines obtained under basal conditions and after UNDW
inhalation showed a significant difference (F l 39.09 ;
P l 0.002). The slopes of the regression lines obtained
under basal conditions and after UNDW inhalation were
not different (P l 0.378). However, the x-axis intercept
values obtained under basal conditions and after UNDW
inhalation were significantly different (P l 0.009)
(Figure 3).
DISCUSSION
The present study shows that inhalation of aqueous
solution may significantly reduce eNO levels in healthy
subjects, without affecting bronchial tone. Several factors
reportedly contribute to the decrease in eNO levels,
including the respiratory manoeuvre and the reduction of
# 2001 The Biochemical Society and the Medical Research Society
airway calibre during induced bronchoconstriction, especially when forced spirometry is associated with eNO
measurement [13]. However, no significant variation in
sGaw was observed in any of the subjects during the
inhalation of either UNDW or isotonic solution. Moreover, plethysmography itself was not associated with
changes in eNO, thus corroborating the recent report
showing that panting manoeuvres do not affect eNO
levels in healthy subjects [6]. Furthermore, the slight
modification of bronchial tone that occurs during inhalation is unlikely to have caused a significant decrease
in eNO. These results are in agreement with our previous
findings showing that a decrease in eNO does not modify
airway smooth muscle tone in humans [14].
The observed decreases in eNO levels appear to be
related to the volume of aqueous solution inhaled. This
decrease in eNO may be dependent on the increase in
airway water vapour pressure subsequent to the inhalation of a larger volume of aqueous solution. An
Exhaled nitric oxide and hypotonic solutions
increase in water vapour pressure has been reported to
exert a quenching effect on the chemiluminescence assay
[3]. In the present study, Nafion tubes were used which
allowed equilibration of the samples, and the gas calibration was performed correcting for ambient humidity.
Furthermore, the pressure levels in the reaction chamber
of the analyser are designed to minimize the instrument
tolerance for quenching by CO and water vapour.
#
The observation that UNDW caused a greater fall in
eNO at any volume used may be related to an effect on
the NO signal transduction pathway. Airway epithelium
expresses the inducible isoform of NOS [8] and has also
been reported to be sensitive to osmotic stimuli. In fact,
intraluminal hypo-osmolarity causes papaverine-sensitive contraction of guinea pig trachea, and NO blockers
may modulate the response of pre-constricted trachea to
non-isotonic saline solution [15]. The decrease in eNO
observed in our study was of greater magnitude after
inhalation of hypo-osmolar solutions, indicating a possible effect on NO release from airway epithelium. On the
other hand, inhalation of the NOS inhibitor L-NAME
elicited a significant decrease in eNO levels and also
prevented any further decrease in NO during the
inhalation of UNDW. It is also unlikely that the decrease
in eNO was the consequence of a reduced DNO from the
airway wall to the lumen. In fact, accordingly to the
model proposed recently by Silkoff et al. [11], we
estimated DNO and airway wall NO concentration at zero
flow during the inhalation of UNDW and saline from the
slope and the x-axis intercept respectively of four-point
linear regression of NO output against eNO concentration at four different expiratory flow rates, ranging
from 45 to 180 ml\s. The two regression lines were
significantly different, indicating a change in the relationship between NO airway output and actual eNO.
In particular, calculated DNO values were not significantly
different from baseline during the inhalation of either
saline or UNDW, thus suggesting a limited contribution
of impaired DNO to the mechanisms responsible for the
decrease in eNO induced by aqueous solutions. On the
other hand, the x-axis intercepts were significantly
different, further suggesting an effect of the inhaled
solution on NO release.
The effects on NO release induced by inhaled solutions
on the chemical environment of the airway (i.e. airway
pH and oxygen tension) are not well known. As reported
recently, nitrite in the airway vapour condensate is
converted into NO in a pH-dependent fashion in acidic
(pH 4–5) samples from asthmatic patients, but not in
samples from control subjects [5]. Moreover, Dweik et al.
[16] have demontrated that airway exposure to decreased
oxygen tension could lower eNO levels in humans,
through an effect on NOS II epithelial activity. A
comparable effective decrease in oxygen tension has been
reported in asthmatic patients, but not in healthy subjects,
during UNDW challenge [17].
In conclusion, the results of the present study indicate
that the inhalation of aqueous solutions causes a decrease
in eNO levels in healthy subjects, without inducing any
significant change in airway calibre. The decrease in eNO
was greater after hypo-osmolar challenge, and was not
detectable after treatment with a NOS inhibitor. Since
nebulized solutions are used widely in pharmacological
studies or for treatment, our observations may be of
clinical interest, in that the decrease in eNO was detected
even after inhalation of relatively small volumes of
aqueous solutions. However, the pathophysiological
relevance of the decrease in eNO after solution inhalation
in humans requires further study.
ACKNOWLEDGMENT
We thank Dr Giuseppe Pelaia for his advice.
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Received 28 March 2001/11 June 2001; accepted 22 August 2001
# 2001 The Biochemical Society and the Medical Research Society