Vol. 86, Issue 1, 159-167, January 1999
Nitric oxide production and absorption in trachea,
bronchi, bronchioles, and respiratory bronchioles of humans
Arthur B.
DuBois1,2,
Patrick M.
Kelley1,
James S.
Douglas1,2, and
Vahid
Mohsenin1,2
1 John B. Pierce Laboratory and
2 Yale University, New Haven,
Connecticut 06519
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ABSTRACT |
Different volumes
of dead-space gas were collected and analyzed for nitric oxide (NO)
content, either immediately after inspiration or after a period of
breath holding on clean air or NO mixtures. This allowed calculation of
NO equilibrium, NO production, and NO absorption. In seven young,
healthy, adult nonsmokers, the mean NO equilibrium values in parts per
billion (ppb) were 56 ± 11 (SE) in the trachea, 37 ± 6 in the
bronchi, 21 ± 3 in the bronchioles, and 16 ± 2 in the
respiratory bronchioles. At any given NO concentration, the NO
absorption rate (in nl/min) equaled the NO concentration (in ppb) times
A (the absorption coefficient in
l/min). A values (in l/min) were 0.11 ± 0.01 in the trachea, 0.17 ± 0.04 in the bronchi, 0.66 ± 0.09 in the bronchioles, and 1.35 ± 0.32 in the respiratory
bronchioles. NO equilibrium concentrations and production rates in one
74-yr-old subject were three to five times as high as those found in
the young subjects. Mouth equilibrium NO concentrations were 3 and 6 parts per million in two subjects who had oral production rates of 6 and 23 nl/min, respectively. In conclusion, production and absorption
of NO occur throughout the first 450 ml of the airways.
expired gas; airway; gas exchange; dead space; mouth
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INTRODUCTION |
THE DISCOVERY of exhaled nitric oxide (NO) in humans
(8) has led to considerable interest in the analysis of exhaled NO as a
possible noninvasive measure of some forms of lung injury. Different
techniques have been employed to collect and analyze respiratory NO.
These have included measurements of peak NO concentrations (18), NO concentration in exhaled fractions (21), the NO
plateau (20), and the NO concentration difference between peak and
end-expiratory samples (10). Researchers have utilized collection of
gas expired from the mouth, nose, conducting airways, and terminal
airways (2, 14, 16, 18). Samples have also been collected via endotracheal tubes (2). Despite the lack of a standardized technique,
exhaled NO has been collected in various pathological conditions
including asthma (1, 13), bronchiectasis (12), liver cirrhosis (17),
Kartagener's syndrome (16), and chronic obstructive pulmonary disease
(10). It is not yet known what either normal or abnormal NO
concentrations actually represent, because the physiological processes
underlying gaseous NO production and absorption in the respiratory
tract have not been characterized.
Earlier studies indicated that a large portion of exhaled NO arose from
the nose (4, 14-16). Studies have shown that the use of a noseclip
does not exclude nasal gas from exhaled samples (3, 20). However, the
nasopharynx and lungs can be studied separately by closing the soft
palate to isolate the nasal cavity from the respiratory tract.
Quantitative studies of NO production, absorption, and equilibrium in
the human nose have been made in this laboratory (5). We postulated
that a similar theoretical analysis could be applied to the conducting
airways of humans to study NO production, absorption, and equilibrium
concentrations within them.
Evidence suggests that, once the nasal component of exhaled breath has
been removed, much of the remaining exhaled NO originates within the
conducting airways (9, 21). Although dead-space gas is discarded during
the single-breath method for measurement of pulmonary diffusing
capacity (7) or for collection of alveolar CO2 samples by the Haldane method,
in the present report, small aliquots of dead-space gas are collected
and analyzed for their NO content either immediately after inspiration
of NO-free air or NO, or after a period of breath holding. This allowed
us to calculate the rates of production and absorption of
NO gas expired from different depths of the conducting airway.
Theoretically, if no change in NO concentration were found during
breath holding after inhalation of a NO mixture, it would indicate that
an equilibrium concentration of NO gas had been reached. At this
concentration, the rates of NO production and absorption would be
equal, and the NO partial pressure within the tissue surface of the
conducting airway would be equal to the partial pressure of NO in the
lumen. A method is presented for collection of exhaled gases from the
various zones of the respiratory tract to examine equilibrium
concentrations, the rates of production, rates of absorption, and
absorption coefficients in each zone.
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METHODS |
Subjects.
Initially, a 74-yr-old subject and a 24-yr-old subject were studied.
The 74-yr-old subject had tracheobronchial NO concentrations which
turned out to be much higher than NO concentrations found in the
younger subject. Data on both subjects were reproducible. Subsequently,
a subject group of adults (ages 20-32) was selected on the basis
of their having no reported respiratory conditions. All were nonsmokers
and had prior experience in respiratory maneuvers. Because nasal gas is
a potential source of NO contamination in the exhaled samples, subjects
were excluded if they were unable to close their soft palate while
breathing against inspiratory and expiratory resistance to exclude
nasal gas (see Procedure for
determination of contamination by nasal NO).
Equipment.
A large rubber mouthpiece (W. E. Collins, Boston, MA) was fitted onto a
Fleisch pneumotachograph (size no. 2), which in turn was connected to
an aluminum three-way stopcock (W. E. Collins). The breathing apparatus
provided enough resistance to aid the subjects in closing their soft
palates naturally. One stopcock arm was attached to a rubber anesthesia
bag. The other arm was attached to 1-in.-diameter tubing connected to a
9-liter Spirometer (W. E. Collins). The spirometer pulley was a
low-torque potentiometer that gave a voltage proportional to expired
gas volume. The anesthesia bag was attached to a Monitor Laboratories
gas-mixing unit which supplied either NO-free air (scrubbed through
Purafil and activated charcoal) or mixtures of NO with NO-free air. Gas
samples were aspirated continuously from the side arm of a T tube on
the proximal pressure tap of the pneumotachograph. The samples were
drawn through an ice bath to condense out water vapor, then through an
infrared CO2 analyzer (Beckman
LB-2), and then a through a needle valve into a chemiluminescence NO
analyzer (Sievers model 270b). The NO,
CO2, expired volume, and
respiratory gas flow led to a signal processor connected to a PC
computer (MacLab 400 with Chart for Windows software; AD Instruments,
Milford, MA). Data read from these charts were tabulated and graphed by
using Cricket Graph software.
Procedure.
After the subject arrived, a brief history was taken. Peak expiratory
flow was obtained by using a peak flowmeter (HealthScan, Cedar Grove,
NJ) to exclude subjects who might have gross evidence of respiratory obstruction.
The subject was then seated in front of the equipment and instructed in
the method. Practice tests were performed until the subject felt
comfortable with the protocol. A thermistor (Yellow Springs
Instruments) was inserted in the nostril to make sure that the soft
palate remained closed during inspiration and expiration. Any leaks
would be indicated by fluctuations of temperature inside the nostril.
The protocol was as follows. The subject twisted the handle of the
three-way stopcock and inhaled a normal tidal volume of either NO-free
air or a mixture of NO and NO-free air from the anesthesia bag. The
subject then twisted the handle of the three-way stopcock back and
expired a partial breath into the spirometer. The subject stopped the
partial breath when the desired exhaled volume was reached. The volume
of gas in the spirometer was maintained by closing the
glottis. The mouth was kept on the mouthpiece, with the glottis closed,
for ~5-10 s to allow time for sampling. The analyzer flow rate
was set by a needle valve at 60 ml/min. This sampling flow was provided
by the vacuum pump of the Sievers NO analyzer. The dead space of the
sampling system caused a small delay in the record; this delay was
taken into account in reading the data. About 10 single expirations,
ranging in volume from 50 to 450 ml, were performed for each inspired
gas concentration. The first 34 ml of this volume was the dead space of
the mouthpiece and pneumotachograph up to the gas-sampling tube. On the
basis of preliminary experiments measuring the volume of the mouth, the
next 40 ml were taken to be the gas that had been held in the mouth.
Preliminary experiments had shown that NO gas became concentrated in
the mouth during breath holding. We report measurements made on the
rate of its accumulation in the mouth, and then we focus on the volumes
of gas that begin after the first 75 ml (from tubing and mouth) have
been expelled.
Initial experiments in the younger subject
(PK) and the older subject
(AD) were done by using several
different inhaled mixtures of NO ranging from 0 to 1,000 parts per
billion (ppb) and several different periods of breath holding ranging
from 0 to 15 s. On the basis of NO analyzed from the samples exhaled as
above, it was decided to collect data on a small group of young,
healthy subjects by using 10 s of breath holding after inhalation of a tidal volume of either NO-free air or NO concentrations above equilibrium values (e.g., inhalation of 396 ppb of NO diluted with
NO-free air).
The actual breath-holding times for the 0- and 10-s intervals were
measured from the pneumotachograph record for each subject. The time of
breath holding was measured from the end of inspiratory flow to the end
of expiratory flow. In the 0-s breath-holding experiment, the time was
called t1 and
averaged ~1-2 s. The time from the end of inspiration to the end
of expiration after the ~10-s breath-holding period was called
t2 and averaged
~11-12 s. When the subject inhaled NO, breath-holding times were
t3 and t4 for
~1-2 and 11-12 s, respectively. The interval value used in
calculations was the difference between these times
(t1 
t2, or
t3 t4)
and equaled ~10 s.
As explained in the APPENDIX, the term
NO
is used to indicate the equilibrium concentration that is
approached during breath holding. The absorption coefficient
A, when it is multiplied by the NO
concentration ([NO]), gives the rate of NO
absorption, and the term
p is the rate
of production of NO gas and could equally well be called the NO output.
The equations utilized in analyzing these data are listed below and are
derived in the
APPENDIX
![([NO]<SUB>1</SUB> + [NO]<SUB>4</SUB> − [NO]<SUB>2</SUB> − [NO]<SUB>3</SUB>)](/content/vol86/issue1/fulltext/159/img002.gif)
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(1)
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![([NO]<SUB>2</SUB> − [NO]∞) × 1 / (<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)](/content/vol86/issue1/fulltext/159/img004.gif)
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(2)
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or the equation can take this alternate form
![<A><AC>Q</AC><AC>˙</AC></A><SUB>p</SUB> = <IT>A</IT> × [NO]∞](/content/vol86/issue1/fulltext/159/img006.gif)
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(3)
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Mouth experiments.
Two sets of experiments were performed in two subjects to measure the
production and absorption of NO in the mouth. In the first set of
experiments, while subjects were breathing through the nose, the gas
was held in the mouth by pressing the back of the tongue against the
roof of the mouth. The mouth was filled and emptied twice with NO-free
air, by using the glossopharyngeal muscles, and then the mouth was
filled with NO-free air, which was then held with positive pressure in
the mouth for periods of 3 s and 2, 5, and 10 min. By using the cheek
muscles, the subject expelled a 10-ml sample into a syringe for NO
analysis. The experiment was repeated with 16.9 parts per million (ppm)
NO instead of NO-free air, and the gas was held in the mouth for 3 s
and 2, 5, and 10 min.
The second set of experiments utilized a constant-flow system. The
subject had a two-holed rubber stopper held firmly between the lips.
One hole of the stopper was attached to a gas-mixing machine which
supplied either NO-free air or a mixture of NO-free air and NO. The
other hole of the stopper led to a syringe, where the effluent gas was
collected and analyzed once a minute. The subject breathed through the
nose and kept the mouth isolated from the rest of the respiratory tract
by pressing the back of the tongue against the roof of the mouth,
maintaining positive pressure in the mouth. The gas was passed into and
out of the mouth for a period of 10 min. Gas flow rates of 19, 40, and
98 ml/min were used. This set of experiments used concentrations of 0, 1.96, 9.43, and 16.9 ppm NO.
Data analysis.
Data were plotted on a Macintosh computer by using Cricket Graph.
Expired NO was plotted against expired volume to give a smooth curve
that was representative of NO levels in each section of the respiratory
dead space. Tracheal, bronchial, bronchiolar, and respiratory
bronchiolar volumes were taken from the data of Weibel (22). For each
inhaled concentration (for example, 0 and 396 ppb), exhaled NO
concentration and volume were graphed at each breath-holding time (for
example, 0 and 10 s) to show the effect of time on an inhaled NO concentration.
The graphs were divided into zones corresponding to the volumes of the
different sections of the respiratory tract. The mean height of the
line was taken for each zone. This was considered the mean NO
concentration for this zone at this time. This yielded four mean NO
concentrations per zone:
[NO]1 at ~1-2 s
of breath holding after inhaling NO-free air,
[NO]2 at ~11-12
s of breath holding after inhaling NO-free air,
[NO]3 at ~1-2 s
of breath holding after inhaling 396 ppb NO, and
[NO]4 at ~11-12
s of breath holding after inhaling 396 ppb NO. The actual time that the
breath was held in the zero breath-holding experiment was
t1, and the actual time that the breath was held in the 10-s breath-holding experiment was t2
(see Fig. 1).
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RESULTS |
Normal subjects.
Based on preliminary experiments and on the curves derived
theoretically (see METHODS), it was
decided to use a 10-s interval of breath holding after the inhalation
of either 0 or 396 ppb NO to test young, healthy subjects, because,
after inhalation of 0 ppb, the NO in the airways increased, whereas,
after 396 ppb, it decreased during breath holding.
Subjects.
Four male and three female subjects, all healthy nonsmokers, were
studied. They ranged in age from 19 to 31 yr, and they had a mean
height of 169 cm and a mean weight of 67 kg. They were all successful
in performing the protocol.
Volumes of respiratory tract.
Volumes for the different areas of the respiratory tract were taken
from Weibel (22). These area volumes were considered to be as follows:
trachea, 30.5 ml; bronchi, 31 ml; bronchioles, 113 ml; and respiratory
bronchioles, 103 ml. The dead space of the mouthpiece and
pneumotachograph until the point of sampling was 34 ml. The volume of
the mouth was 40 ml and was considered dead space in this experiment,
even though some production and absorption of NO occur in the mouth.
This exchange was not the main part of the present investigation. The
total dead space (system + mouth) was 74 ml and was discarded. This
explains why the graphs of NO concentration vs. expired volume (see
Fig. 2, A and
B) begin at a volume of
75 ml on the horizontal axis (to ignore NO collected from the breathing
tube and oropharynx).
Day-to-day variability.
The experiment was repeated for 5 days in one subject
(PK) to assess the day-to-day
variations in NO levels. The young man was 24 yr of age, with a height
of 167.6 cm and a weight of 72.7 kg. Table
1 lists the means, SD, and SE of unpaired
data calculated for NO, A, and
p over these 5 days.
The experiment was also repeated on 5 days in one subject
(AD) aged 74 yr. His height was
184.5 cm, and his weight was 84 kg. The subject's values were
significantly different from those of the young subject
(PK) and other young subjects. The
experiment was repeated on 5 days to determine whether the values were
consistent on a day-to-day basis. Table 2
lists the mean, SD, and SE values of unpaired data for these 5 days.
Rise and fall of NO over time.
When the subject inhaled NO-free air and exhaled without breath
holding, the NO levels were low in all areas of the airways. Expiration
was not immediate, however, and the delay due to turning the stopcock
and the time required to exhale was 1-2 s. During breath-holding
experiments, the delay in onset of expiration was also 1-2 s. The
actual breath-holding times for each subject were obtained from the
pneumotachograph record and were used in the calculations. When breath
holding was performed for 0, 5, 10, or 15 s after inhaling NO-free air,
the NO concentration would build toward an equilibrium concentration in
which the NO would not change if the breath were held longer. Figure
1 shows a model of the behavior of NO in
any zone of the respiratory tract, starting from inhaling either
NO-free air or a concentration above the equilibrium concentration. In
the young adults, this equilibrium concentration agreed reasonably well
with observations made after inhalation of 99 ppb NO. On the other
hand, if the subject inhaled an even higher concentration of NO, such
as 986 ppb, the concentration of NO in the gas samples exhaled from
each zone fell progressively as the breath was held, because of
absorption and reaction of NO in the airway walls. When 396 ppb of NO
were inhaled, the exhaled concentration with no breath holding was
lower than inhaled. After 10 s of breath holding, the concentration was
even lower. The equilibrium concentration reached by inhaling
intermediate concentrations of NO was the same concentration reached
when the rate of rise of NO from zero concentration or rate of decrease
from high concentrations was used to calculate equilibrium. Figure
2 shows exhaled NO
concentrations in a young subject and an older subject after inhaling
various NO concentrations.
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Fig. 1.
Theoretical diagram of rise and fall of nitric oxide (NO) concentration
in a zone of respiratory tract. If a subject were to inhale a NO
concentration greater than the equilibrium concentration
([NO]) (i.e., NO3,
NO4), the concentration would
fall over time until it reached [NO]. If a subject were
to inhale clean air, or a NO concentration less than
[NO] (i.e., NO1,
NO2), the NO concentration would
rise over time until it reached [NO].
t1-t4,
Times in the experiment; see
METHODS.
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Fig. 2.
A: NO concentrations measured
in small volumes of gas quickly exhaled from conducting airways of
subject PK, a young adult man.
Bottom line connects values ( ) that
represent concentrations exhaled from different depths of airways
immediately after inhalation of a tidal breath of air devoid of NO.
Arrow above line points upward in direction taken by NO concentration
when there was a delay between inspiration and the onset of expiration.
 , Values obtained after 10-s breath holding. After subject inhaled
an air mixture that contained 396 parts per billion (ppb) NO, expired
NO was much higher than it had been after inhalation of clean air.
Top line passes through values ( )
representing concentrations found in expired air samples delivered
immediately after inhaling 396 ppm NO. Arrow below this line indicates
decreasing trend of NO concentrations when breath was held between
inspiration and expiration.  , Values obtained after 10 s of breath
holding of 396 ppm NO. Equilibrium values that would have been reached
in each part of the airway if sufficient time had been allowed for the
level in each zone to reach a balance between NO output and uptake were
calculated by using equations given in text. These calculated values
( ) are connected by middle line.
B: NO concentrations measured in older
adult male subject, AD, after
inhalation of air devoid of NO or air containing 980 ppb NO. As in
A, expiration was either immediate or
after 10 s of breath holding; arrows indicate trends. Equilibrium
values (2 sets of ) were obtained after 5 and 10 s of breath holding
after inhalation of 204 ppb NO. Because there was no trend with time,
it was apparent that equilibrium had been reached. Symbols and lines
are same as in A. NO equilibrium
concentrations reached by older subject were higher than those reached
by younger subject.
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Data.
Table 3 lists the means, SD, and SE of
unpaired group data of 7 subjects. Tracheal data for the seven young
subjects had mean values of 56 ppb for the NO, 0.11 l/min for the
A, and 5.7 nl/min for
p. Bronchial
[NO] was 36.9 ppb, A
was 0.17 l/min, and
p was 5.3 nl/min. The bronchioles had values of [NO], 21 ppb;
A, 0.66 l/min; and
p, 13.0 nl/min.
The respective values in the respiratory bronchioles were
[NO], 16 ppb; A, 1.35 l/min; and p,
21.1 nl/min. The SD for [NO] was ~50% of the mean NO concentration in all areas of the respiratory tract. SD for
A and
p also averaged
~50% of the mean values in all areas of the respiratory tract.
The downward trends of [NO] from trachea to bronchi
(P < 0.001), bronchi to bronchioles
(P < 0.001), and bronchioles to
respiratory bronchioles (P < 0.02)
were examined among individuals by calculation of the ratios between
values in adjacent zones, compared with a predicted ratio of 1, by
using Student's two-tailed t-test on paired data. The downward trend was significant on all adjacent zones.
Figure
3A,
which is a graph of each individual, shows this trend.
Similar comparisons on the absorption coefficient
A, by using ratios, showed
P values for each pair of data from
trachea to bronchi (P < 0.4),
bronchi to bronchioles (P < 0.001, upward trend), and bronchioles to respiratory bronchioles
(P < 0.1).
p was not
significantly different between trachea and bronchi or between bronchioles and respiratory bronchioles.
p was greater in
the bronchioles than in the bronchi (P < 0.01). Figure 3, B and
C, shows these trends.
CO2 approached normal alveolar
levels in exhaled volumes of 150 ml or more, and this dead space volume
decreased with time of breath holding, as has been found before.
Data from 74-yr-old subject.
NO equilibrium levels in the 74-yr-old man were substantially higher
than in the young subjects. In the trachea, the NO equilibrium was
about five times as high as the young subjects' (266 vs. 56 ppb,
respectively; P < 0.001). The
equilibrated concentration in the bronchi was about five times as high
as the young subjects' (178 vs. 37 ppb, respectively;
P < 0.001). The bronchiolar
equilibrated concentration was 4.4 times as high as the young
subjects' (92 vs. 21 ppb, respectively;
P < 0.001), and the respiratory
bronchiolar concentration was 2.6 times as high as the young subjects'
(41.2 vs. 15.7 ppb, respectively; P < 0.02). The A values were not
statistically different in the older subject, but the rate of NO
production was significantly elevated.
p was 21.0 vs.
5.7 nl/min in the young subjects' tracheas, a 3.7-fold difference
(P < 0.001). Similar differences
were found in the bronchi (19.9 vs. 5.3 nl/min, respectively; P < 0.001), bronchioles (59.7 vs.
13.0 nl/min, respectively; P < 0.001), and respiratory bronchioles (41.2 vs. 21.1 nl/min,
respecticely; P < 0.05).
Mouth data.
When NO-free air was held in the mouth for varying lengths of time, it
was found that NO concentrations increased over time. When 16.9 ppm NO
was held in the mouth for varying lengths of time, it was found that NO
concentration decreased over time. Equations used in the
tracheobronchial tree were utilized to calculate [NO],
A, and
p. In subject
AD, the [NO] was 2.3 ppm, A was 0.0027 l/min, and
p was 6.1 nl/min. In subject PK,
[NO] was 6.8 ppm, A was
0.0034 l/min, and
p was 23.1 nl/min.
When a constant flow of gas was introduced into the mouth, and the
effluent gas was sampled, steady-state NO concentrations were found.
Data from two subjects are listed in Table
4. Steady-state NO was inversely related to
the gas flow rate through the mouth. After NO of 16.9 ppm was
introduced into the mouth at a constant gas flow for 5 min and then
switched to NO-free air, it was found that effluent NO concentrations
were higher than normally found, and a "washout" or desorption
period was observed for up to 13 min.
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DISCUSSION |
The quantitative analysis of NO from all the regions of the respiratory
tract is important, if exhaled NO is to be of clinical utility. The
efficacy of using exhaled NO as a biomarker of inflammation or immune
responses in humans remains undetermined.
This method of NO collection investigates NO from different zones of
the respiratory tract, as determined by expired volume. Forward and
backward mixing of exhaled gas is certain to occur (due to a parabolic
gas front and forward and backward diffusion), and there is some
exchange of NO with airway walls while the gas is passing upward
through the airways. To minimize this exchange, expirations were quick compared with breath-holding times. Because the
bronchioles are longer than they are wide, diffusion within the lung
would favor NO gas exchange with the walls rather than by lengthwise
diffusion. CO2 was found in
exhaled volumes of 150 ml or greater. This indicates that NO gas
exchange is occurring even beyond areas that have classically been
considered dead space or despite diffusion of alveolar
CO2 up the conducting airways. The
reproducibility of results as seen in each subject was reasonably good,
as contrasted to the large difference between one subject who was age
74 yr and all seven of the young subjects.
NO concentration rises in all areas of the respiratory tract with 10 s
of breath holding after inhalation of NO-free air. The smallest
increase in NO level occurs within the respiratory bronchioles. But
local production of NO in the respiratory bronchioles is appreciable,
because this zone has a large volume (22). The NO concentration falls
in all levels of the respiratory tract after inhalation of 396 ppb NO
and after breath holding for 10 s. At the level of the
respiratory bronchioles, the NO concentration approaches an equilibrium
concentration within 10 s of breath holding. The predicted equilibrium
concentration can be found experimentally in all areas of the
respiratory tract by having subjects inhale different concentrations of
NO. An example of this type of approach is shown in Fig.
2B. If the inhaled concentration is
too high, the NO level will drop over time in this zone. If the inhaled
concentration is too low, the NO level will rise over time, as in Fig.
1.
Different equilibrium concentrations apply to the different zones of
the airways. The NO equilibrium concentration drops with depth into the
airways. The rate of production increases with depth, as does the
absorption coefficient. Both of these factors may be related to surface
area, as well as mucosal perfusion, but this is not yet known.
The substantial difference between the 74-yr-old subject's NO
equilibrium concentration and the young subjects' NO
equilibrium concentrations is unexplained. Possible causes may be of
respiratory or circulatory origin or due to differences in ciliary
activity (16) or an age-related effect. The unexplained large
difference between this older subject and the others demonstrates the
complications involved in interpreting exhaled NO.
The data from the mouth show that the mouth is capable of producing
concentrations of NO that are as high or higher than those found in
other areas of the trachea and bronchi. Duncan et al. (6) propose the
following: in addition to production of NO by the family of enzymes NO
synthase, the salivary glands concentrate nitrate (from
diet) in the saliva and excrete it into the mouth. Nitrate is rapidly
reduced to nitrite by facultative anaerobic bacteria found on the rear
of the tongue. NO is generated when the nitrite comes into contact with
acidic conditions, such as those produced by acid-producing bacteria in
the gingival margins (6). Experiments that utilize collection of
expiratory gas from the mouth should take into account the effect of
the mouth in conditioning exhaled gas.
Fractionating an exhaled breath for the study of NO is important. In
pathological conditions, such as asthma, the source of NO may be the
airway tissues that suffer the inflammation. The alveoli and other
portions of the respiratory tract may not be affected. These conditions
are not known, but further studies to characterize exhaled NO need to
incorporate these theoretical considerations. The relationship between
NO concentration (in parts per billion) in the airway and the
underlying nitrate and nitrite concentrations in the adjacent tissues
is unknown and remains to be determined.
Further considerations are acknowledged in the present
experiments. Expiratory transit time, although <1 s,
was not standardized. Conditioning of the gas bolus as it passes
through the conducting airways cannot be discounted because of this
reason. The mouth, which we find in this study to be a site of gas
exchange, will alter exhaled NO concentrations slightly during
expiration. Exhaled NO has been correlated with ciliary action (16),
and ciliary action was not assessed in this experiment. The volumes of
the zones of the respiratory tract are affected by unequal branching and unequal lengths of the bronchi as well as their being dependent on
body size. But the measurement of NO concentration in midvolume of each
segment allows a latitude of ±20% before crossing from one zone to
another. The total test took ~2 h to complete. Some subjects became
fatigued, and controlling the exhaled volume became difficult for them.
Development of a single-breath fractionation device would be beneficial
in future studies. This would enable researchers to perform larger
studies on exhaled NO from all parts of the respiratory tract, and
improvement would be expected in the precision, speed, and
reproducibility of the experiment.
Although investigators have studied exhaled NO and considered this
value to be the NO production value, they have neglected the effect of
absorption. Increased exhaled NO in pathological conditions may be a
result of altered NO absorption as well as an increase in the rate of
NO production.
Studies on NO production and absorption in the nose have shown that the
majority of NO uptake in the nose is due to chemical combination and
not due to simple solubility in blood flow (11). The following
calculation shows that this also applies to the airways. On the basis
of the uptake of chemically inert dimethyl ether, Kimberly et al. (14)
give a value of 6.6 ml/min for mucosal blood flow in the 50 ml of
conducting airway distal to the trachea. Weibel's data (22) for
volumes of the conducting airway give a volume of 31 ml for the
bronchi. Assuming homogenous perfusion for this area, we can calculate
bronchial blood flow by multiplying the blood flow by 0.62 (i.e., 31/50
ml). This gives a mucosal blood flow in the bronchi of 4.1 ml/min. By
neglecting diffusion limitations and basing the uptake on the
solubility of NO in blood (partition coefficient of 0.05 at 37°C),
the rate of NO absorption in the bronchi at the equilibrium
concentration (a quasi-steady-state condition in which the rates of
production and absorption are equal) can be calculated by the equation
|
|
where
a is the rate of NO gas
absorption in the bronchi,
br is bronchial
blood flow, 
NO is the partition
coefficient in blood, and FNO is
the NO gas concentration in the bronchi. On the basis of the mean
values for the seven young subjects, the calculation for the rate of
absorption would be
or
7.6 × 10
9 ml/min. The uptake due to solubility
can be compared with the uptake computed from
equals
6.27 nl/min. Therefore, solubility in blood flow accounts
for (7.6 × 109
ml/min)/(6.27 × 109
l/min) or only ~1/1,000 of the total uptake. A similar comparison, based on the uptake of nitrous oxide, was made in the human nose (11),
and it was found that the uptake of NO was 27 times as much as could be
expected due to solubility. Although Runer and Lindberg (19) found 75%
increased blood flow and 57% increase in ciliary activity after
nebulizing sodium nitroprusside (an NO donor) into the nose of
subjects, no attempt has been made to try to assess the vasodilatory
effects of inhaled NO in the tracheobronchial tree, so the possibility
exists that NO may increase its own uptake by dilating the superficial
mucosal blood vessels. However, it is unlikely that the change in
uptake due to vasodilation would be of the magnitude to explain the
difference in predicted uptake due to solubility in blood and the
experimentally observed uptake. The gas does not desorb on exhalation
(except in the mouth), thus indicating nonreversible reactions. NO
probably forms nitrite and
S-nitrosothiols. The rapid uptake of
NO (in the 1- to 2-s breath-holding period) suggests that, initially, a
majority of the NO reacts at the mucosal surface.
In conclusion, the equilibrium concentrations of NO show a downward
trend with depth into the respiratory tract. Absorption coefficient
values did not show a significant trend from trachea to bronchi or from
bronchioles to respiratory bronchioles, but the values did increase
significantly from the bronchi to the bronchioles. The rate of
production of NO was not significantly different from the trachea to
the bronchi or from the bronchioles to the respiratory bronchioles, but
the rate was greater in the bronchioles than in the bronchi. NO
equilibrium concentrations and rates of production and absorption
showed variability from day to day. A group of young, nonsmoking,
healthy subjects had similar trends of NO rates and concentrations. An
older subject had NO equilibrium concentrations and production rates
that were significantly elevated from the concentrations of the younger subjects. The method of NO collection and analysis demonstrated is
accurate and reproducible.
| |
APPENDIX |
Derivation of the equations for the rate of NO production
(p) and absorption
(a).
During breath holding, the rate at which NO concentration
([NO]) at time t changes
in a volume (V) of the conducting airway is proportional to the
p of NO minus
the a of NO
The
a is
proportional to the [NO] and to an absorption coefficient
(A)
Furthermore,
the [NO] analyzed in small samples of expired air is found
to increase during breath holding until the
a equals the
p. Then, when
t = ,
a = p, and an
equilibrium concentration ([NO]) would be found.
By substitution
Clearing
for [NO]t, where t is
time,
Integrating
or
where
[NO]0 is the concentration of NO when
t = 0.
If we know the values for [NO] and for V, then by
determining the values for
[NO]t at two
different times
(t1 and t2), we can substitute these to calculate
A from the equation
Solution for p.
At equilibrium,
p = a = A[NO]. Substituting
values calculated for A and
[NO] into this, we solve for
p.
Derivation of equations for [NO].
During breath holding, [NO] in the conducting airway
increases with time (due to NO production) or decreases (due to
absorption). Eventually, the [NO] reaches equilibrium at an
asymptote level ([NO]), and the approach of
[NO] toward the asymptote follows a curve such that the
difference of [NO] from the equilibrium point is a
logarithmic function of the time of breath holding
where
A and V are the values of the conducting airway under
consideration. We can assign values to V from the anatomy of the
tracheobronchial tree (22), but values for
A or [NO] are unknown.
To find [NO], we experimentally obtain values of NO
concentration [NO]1
and [NO]2 at two
different times
(t1 and
t2, respectively) from a breath-holding curve after inhalation of clean air
We also determine another pair of [NO] values
([NO]3 and
[NO]4) at times
t3 and
t4, respectively,
from a breath-holding curve after inhalation of a [NO]
After
subtraction of this equation from the previous one to eliminate the
right-hand term (A/V), the
antilogarithms are cross multiplied and solved for [NO]
If
the breath-holding times in the two curves are made experimentally
equal (t4 t3 = t2 t1), the
exponential term becomes 1. Then [NO] is used to find
the value of A.
Calculations.
The process of NO production and absorption in the respiratory tract
will lead to an [NO] in each zone. This equilibrium is
the concentration that will be maintained regardless of the time of
breath holding. To calculate this value, we use the equation derived
above
![([NO]<SUB>1</SUB> + [NO]<SUB>4</SUB> − [NO]<SUB>2</SUB> − [NO]<SUB>3</SUB>)](/content/vol86/issue1/fulltext/159/img023.gif)
|
(A1)
|
The units of [NO] are in parts per billion.
Absorption of NO is occurring in each zone of the respiratory tract
studied. This makes it possible, by using the data from inhaling NO at
396 ppb, to calculate A. The volume of
each zone is represented by V. The formula is
![([NO]<SUB>2</SUB> − [NO]∞)} × 1/(<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)](/content/vol86/issue1/fulltext/159/img025.gif)
|
(A2)
|
or the equation can take this alternate form
The
volume V is converted to liters, and t2 t1 is converted from values measured in seconds (~10 s)
to fractions of minutes (~0.17 min), so
A is in liters per minute. The
p can
now be calculated, since the
p and
a must be
equal at the equilibrated concentration
![<A><AC>Q</AC><AC>˙</AC></A><SUB>p</SUB> = <IT>A</IT> × [NO]∞](/content/vol86/issue1/fulltext/159/img027.gif)
|
(A3)
|
The units of p
are in nanoliters per minute, because the units of
A are in liters per minute and the
units of [NO] are in parts per billion.
Sample calculation.
[NO], A, and
p were
calculated on the bronchial gas obtained from subject
PK by reading the set of curves at 2 and 15 s of breath holding of clean air and after inhalation of 396 ppb of NO in air as follows
where
V = 31 ml (converted to 0.031 liters) and time change
(
t) = 13 s, expressed as 0.22 min, or 4.6 reciprocal min. Substituting to find
[NO]
Substituting
to find the value of A
Substituting
to find the p
| |
ACKNOWLEDGEMENTS |
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-53630.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. B. DuBois, c/o John B. Pierce
Laboratory, 290 Congress Ave., New Haven, CT 06519 (E-mail:
adubois{at}jbpierce.org).
Received 6 July 1998; accepted in final form 28 August 1998.
| |
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J APPL PHYSIOL 86(1):159-167
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Copyright © 1999 the American Physiological Society
Top
Abstract
Introduction
![[NO]∞ = ([NO]<SUB>1</SUB>[NO]<SUB>4</SUB> − [NO]<SUB>2</SUB>[NO]<SUB>3</SUB>) /](/content/vol86/issue1/fulltext/159/img001.gif)
![<IT>A</IT> = V × ln ([NO]<SUB>1</SUB> − [NO]∞)/](/content/vol86/issue1/fulltext/159/img003.gif)
![<IT>A</IT> = V × ln ([NO]<SUB>3</SUB> − [NO]∞)/([NO]<SUB>4</SUB> − [NO]∞) × 1/(<IT>t</IT><SUB>4</SUB> − <IT>t</IT><SUB>3</SUB>)](/content/vol86/issue1/fulltext/159/img005.gif)
![<A><AC>Q</AC><AC>˙</AC></A><SUB>a</SUB> = <IT>A</IT>[NO]∞ = 0.17 × 36.9 × 10<SUP>−9</SUP> l/min](/content/vol86/issue1/fulltext/159/img009.gif)
![d[NO]<SUB><IT>t</IT></SUB> /d<IT>t</IT> = (<A><AC>Q</AC><AC>˙</AC></A><SUB>p</SUB> − <A><AC>Q</AC><AC>˙</AC></A><SUB>a</SUB>)/V](/content/vol86/issue1/fulltext/159/img010.gif)
![<A><AC>Q</AC><AC>˙</AC></A><SUB>a</SUB> = <IT>A</IT>[NO]<SUB><IT>t</IT></SUB>](/content/vol86/issue1/fulltext/159/img011.gif)
![d[NO]<SUB><IT>t</IT></SUB> /d<IT>t</IT> = (<IT>A</IT>[NO]∞ − <IT>A</IT>[NO]<SUB><IT>t</IT></SUB>)/V](/content/vol86/issue1/fulltext/159/img012.gif)
![d[NO]<SUB><IT>t</IT></SUB> /([NO]∞ − [NO]<SUB><IT>t</IT></SUB> ) = (<IT>A</IT>/V)d<IT>t</IT>](/content/vol86/issue1/fulltext/159/img013.gif)
![ln([NO]∞ − [NO]<SUB><IT>t</IT></SUB> ) = (<IT>A</IT>/V)<IT>t</IT> − ln([NO]∞ −[NO]<SUB>0</SUB>)](/content/vol86/issue1/fulltext/159/img014.gif)
![([NO]∞ − [NO]<SUB><IT>t</IT></SUB> )/([NO]∞ − [NO]<SUB>0</SUB>) = 1/<IT>e</IT><SUP>(<IT>A</IT> / V)<IT>t</IT></SUP>](/content/vol86/issue1/fulltext/159/img015.gif)
![<IT>A</IT> = ln{([NO]∞ − [NO]<SUB>2</SUB>)/([NO]∞ − [NO]<SUB>1</SUB>)} ⋅ {V /(<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)}](/content/vol86/issue1/fulltext/159/img016.gif)
![ln{([NO]∞ − [NO]<SUB><IT>t</IT><SUB>2</SUB></SUB>)/([NO]∞ − [NO]<SUB><IT>t</IT><SUB>1</SUB></SUB>)} {1/(<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)} = −(<IT>A</IT>/V)](/content/vol86/issue1/fulltext/159/img017.gif)
![ln{([NO]∞ − [NO]<SUB>2</SUB>)/([NO]∞ − [NO]<SUB>1</SUB>)} {1/<IT>t</IT><SUB>2</SUB> − <IT>t</IT><SUB>1</SUB>)} = −<IT>A</IT>/V](/content/vol86/issue1/fulltext/159/img018.gif)
![ln{([NO]∞ − [NO]<SUB>4</SUB>)/([NO]∞ − [NO]<SUB>3</SUB>)} {1/(<IT>t</IT><SUB>4</SUB> − <IT>t</IT><SUB>3</SUB>)} = −<IT>A</IT>/V](/content/vol86/issue1/fulltext/159/img019.gif)
![[NO]∞ = {([NO<SUB>1</SUB>][NO]<SUB>4</SUB> − [NO]<SUB>2</SUB>[NO]<SUB>3</SUB>)/([NO]<SUB>1</SUB> + [NO]<SUB>4</SUB> − [NO]<SUB>2</SUB> − [NO]<SUB>3</SUB>)}](/content/vol86/issue1/fulltext/159/img020.gif)
![[NO]∞ = ([NO]<SUB>1</SUB>[NO]<SUB>4</SUB> − [NO]<SUB>2</SUB>[NO]<SUB>3</SUB>/](/content/vol86/issue1/fulltext/159/img022.gif)
![<IT>A</IT> = V × {ln([NO]<SUB>1</SUB> − [NO]∞)/](/content/vol86/issue1/fulltext/159/img024.gif)
![<IT>A</IT> = V × {ln([NO]<SUB>3</SUB> − [NO]∞)/([NO]<SUB>4</SUB> − [NO]∞)} × 1/(<IT>t</IT><SUB>4</SUB> − <IT>t</IT><SUB>3</SUB>)](/content/vol86/issue1/fulltext/159/img026.gif)
![[NO]<SUB>1</SUB> = 17.5, [NO]<SUB>2</SUB> = 45, [NO]<SUB>3</SUB> = 272, [NO]<SUB>4</SUB> = 148 ppb](/content/vol86/issue1/fulltext/159/img028.gif)
![[NO]∞ = (17.5 ppb × 148 ppb − 45 ppb × 272 ppb)](/content/vol86/issue1/fulltext/159/img029.gif)
![<IT>A</IT> = 0.031 × 4.6 × ln[(272 − 63.7)/(148 − 63.7)] = 0.129 l/min](/content/vol86/issue1/fulltext/159/img031.gif)
