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Departments of 1 Medicine and 2 Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
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ABSTRACT |
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Top
Abstract
Introduction
Appendix
References
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This report describes methods for measuring nitric oxide
production by the lungs' lower alveolar airways
(
NO),
defined as those alveoli and bronchioles well perfused by the pulmonary
circulation. Breath holding or vigorous rebreathing for 15-20 s
minimizes removal of NO from the lower airways and results in a
constant partial pressure of NO in the lower airways
(PL). Then the amount of NO diffusing into the perfusing blood will be the pulmonary diffusing capacity for NO (DNO)
multiplied by PL and by mass
balance equals NO,
or NO = DNO(PL).
To measure PL, 10 normal
subjects breath held for 20 s followed by exhalation at a constant flow
rate of 0.83 ± 0.14 (SD) l/s or rebreathed at 59 ± 15 l/min for 20 s while NO was continuously measured at the mouth.
DNO was estimated to equal five
times the single-breath carbon monoxide diffusing capacity. By using
breath holding, PL equaled 2.9 ± 0.8 mmHg × 10
6
and NO
equaled 0.39 ± 0.12 µl/min. During rebreathing
PL equaled 2.3 ± 0.6 mmHg × 106 and
NO equaled
0.29 ± 0.11 µl/min. Measurements of NO at the mouth during rapid,
constant exhalation after breath holding for 20 s or during rebreathing
provide reproducible methods for measuring NO in humans.
rebreathing; breath holding; nitric oxide in airways; nitric oxide in alveoli; lung nitric oxide
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INTRODUCTION |
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Top
Abstract
Introduction
Appendix
References
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EXHALED NITRIC OXIDE (NO) from the airways of humans has attracted interest as a noninvasive method to detect lung injury (1). Inflammation of the airways in bronchiectasis and acute asthma increases the concentration of exhaled NO (1, 21). Decreased levels of NO in the lungs have been proposed as a cause of the pulmonary vasoconstriction observed in primary pulmonary hypertension (6, 29).
In past studies designed to measure the amount of NO exhaled by the
lungs or the concentration in the lungs, little emphasis has been
placed on the rapid diffusion of NO produced by the lower alveolar
airways, defined as those alveoli and bronchioles well perfused by the
pulmonary circulation, into the perfusing blood. A recent theoretical
analysis (12) showed that ~95% of the NO produced by the lungs'
lower alveolar airways will be taken up by the pulmonary circulation. A
method of calculating the rate of NO production by these airways was
devised on the basis of the prediction that the partial pressure of NO
in the lower alveolar airways
(PL) will reach a constant
value at the end of a period of breath holding or rebreathing for
15-20 s. This constant value is expected because breath holding or
rebreathing minimizes removal or delivery of NO to the lower alveolar
airways from the larger airways and the environment. Then the amount of
NO produced by the lower alveolar airways that enters the air spaces
(NO)
will equal the amount diffusing into the perfusing blood and tissues or
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(1) |
NO is in
milliliters per minute (Fig. 1).
Equation 1 assumes the partial
pressure of NO in the perfusing blood is negligible.
PL can be measured in exhaled
samples after breath holding or during rebreathing (12, 18, 21, 30).
NO can then be
calculated because DNO is readily measured directly or is estimated from the carbon monoxide pulmonary diffusing capacity
(DLCO)
(2, 10, 25). Alternatively, PL
can be interpreted as the production of NO per unit of diffusing capacity of the lower respiratory tract, thereby obviating the need for
measuring DNO or
DLCO.
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In this report, we applied Eq. 1 to
obtain the first measurements of
NO in humans.
In addition to measurements after breath holding and during
rebreathing, we also report values for
NO obtained
during steady-state breathing.
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METHODS |
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Calculation of NO
From Breath Holding Followed by a Single Exhalation and by
Rebreathing Measurements
NO,
PL is multiplied by
DNO (Eq. 1), which is estimated to equal five times the
subject's single-breath
DLCO (2,
10). DLCO
was measured at total lung capacity (TLC) and at 1 liter above
functional residual capacity (FRC) in triplicate with automated
equipment (P. K. Morgan, Haverhill, MA) by using the method described
by Jones and Meade (15). An estimate for
DLCO
present at the middle of the tidal volume
(VT) during rebreathing and
steady-state breathing was obtained by linear extrapolation from the
measurements of
DLCO at TLC
and 1 liter above FRC. This extrapolated value of
DLCO was
multiplied by five to estimate
DNO.
Calculation of
NO from
Steady-State Measurements
NO during
steady-state breathing is described in the
APPENDIX. In brief, during ventilation
at a steady state, mixed expired partial pressure of NO
(P
) and
VT are measured. From the mixed
expired and end-tidal concentrations of
CO2, dead space volume
(VD) is estimated, and
PL is calculated with the Bohr
equation (4) or
|
(2) |
NO
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(3) |
I is
inspired minute ventilation.
Measurement of NO
A rapidly responding chemoluminescence NO analyzer (Sievers NOA, model 270B, Sievers, Boulder, CO) operating at a sampling rate of 250 ml/min measured NO with a specified response time of <200 ms for a signal 90% of full scale. The analyzer provided 40-400 measurements of the NO concentration per second that could be averaged over any selected time interval. Gas containing 229 parts/billion (ppb) of NO was prepared by diluting gas from a compressed-gas cylinder certified to contain 99,600 ppb of NO in nitrogen (Scott Specialty Gases, Plumsteadville, PA) with room air that was ozonated and charcoal filtered to remove NO and other oxides of nitrogen by using a dynamic calibrator (Monitor Laboratories, Lear Seigler Measurement Controls, Englewood, CA). The NO analyzer was calibrated daily by serial dilutions of the gas containing 229 ppb of NO by mixing with the filtered NO-free air. To obtain gas samples completely free of NO, air from a gas cylinder containing <2 ppb of NO (Scott Specialty Gases) was passed through a filter constructed from a cylinder of 5.8-cm ID and 19 cm in length (Gas Drying Unit, VWR Scientific, Rochester, NY) packed with potassium permanganate (Purafil, Thermoenvironmental Instruments, Franklin, MA) (7). Flow rate through the filter was 300 ml/min. The calibrating gases were stored in 5-liter Tedlar bags (SKC, Eighty Four, PA) that maintained a NO concentration of 229 ppb within 1.1 ± 1.8 (SD) % during multiple measurements for 90 min.Because the NO-free air signal could drift as much as 2 ppb in 10 min, NO-free air was measured within 1 min both before and after each measurement of NO in expired gas samples and averaged to obtain the zero-NO signal. The lag time between the volume signal supplied by the spirometer and the NO signal was 0.84 ± 0.06 (SD) s. To determine the lag time, the subject rapidly inspired 3-4 liters of gas containing 1,000 ppb of NO from a Tedlar bag-in-box system (Fig. 2) and then exhaled at a flow rate of 1.5-2.0 l/s. Lag time was considered equal to the difference in time from the initial rise in the volume signal recorded from the potentiometer attached to the wheel of the spirometer to the initial rise in the NO signal sampled at the mouthpiece.
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Measurements were made to determine whether the NO analyzer provided a
NO signal under dry (ATPD) or wet
conditions (ATPS) during the
analysis of exhaled and rebreathed gases. Multiple gas samples of NO at
concentrations of 1.8-210 ppb that were saturated with water vapor
by storage in a Tissot spirometer were measured directly as well as
after absorption of water vapor by passage through a column 1.5 cm in
diameter by 16 cm in height packed with 6-18 mesh silica gel (J. T. Baker, Phillipsburg, NJ). At 25°C, the dry gas resulted in
samples of NO 2.5 ± 1.3 (SD) % greater than the humidified samples
compared with the predicted increase of 3.2%. [At 25°C,
water vapor pressure
(PH2O)
equals 24 mmHg. At barometric pressure
(PB) = 750 mmHg, drying the
sample should have increased its concentration by 24/750 or
3.2%.] The recorded NO signal from the expired-gas samples was
therefore assumed to provide a measurement at
ATPS. Measurements of NO in ppb
ATPS were converted to partial
pressures of NO in mmHg BTPS by the following relationship: NO (in mmHg
BTPS) = PB (NO in ppb
ATPS)(PB 
47)/(PB PH2O)
(109), where
PH2O = vapor pressure of water at room temperature (4). For example, when
PB = 760 mmHg and
PH2O = 22.4 mmHg, respectively, at a room temperature of 24°, 1 ppb of
NO = 0.735 mmHg × 106
of NO. To ensure that the NO-free air standard remained at
ATPS, the operator exhaled warm
humidified gas from the mouth by the inlet of the NO analyzer
approximately every 5-10 min so that the walls of the unheated
inlet tube were kept moist. The data were processed by using a chart
recorder (MacLab Recording Instrument, AD Instruments, Castle Hill,
Australia) and stored in a Macintosh LC computer (Apple Computer,
Cupertino, CA).
To evaluate sensitivity of the NO measurements, 10 repetitive
measurements were made on a gas sample initially containing 2.92 mmHg × 106 of NO. The
analyzer recorded 2.83 ± 0.08 (SD) mmHg × 106 of NO. A sample
initially containing 8.20 mmHg × 106 of NO resulted in 8.22 ± 0.09 mmHg × 106
of NO. If the detection limit is assumed to equal twice the SD of the
measurements, our analytic technique provides a 95% confidence in
detecting a signal change of 0.2 mmHg × 106 of NO.
Subjects
Measurements of PL andNO were
performed in 10 healthy, nonsmoking subjects, ranging in age from 30 to
72 yr (mean 47 yr). Their mean height was 172 ± 9 cm, and weight
was 70 ± 8 kg. Seven were men, and three were women.
Spirometry showed values >80% of predicted for the forced expiratory
volume in 1 s, with a mean value of 108% (5). Residual volume (RV) was
calculated from measurements of FRC performed with plethysmography (8),
and values of expiratory reserve volume were measured with spirometry. Subjects did not use noseclips during the maneuvers for measuring NO.
This study was approved by the University of Rochester's Research
Subjects Review Board.
Protocols for Breathing Maneuvers and Gas Samples
Protocol for breath holding followed by a single exhalation. The subject exhaled to RV through the mouthpiece of the apparatus (Fig. 2) into the room and then turned the four-way valve (Hans Rudolph, Kansas City, MO) to connect the mouthpiece to the bag-in-box system previously filled with room air. The subject then rapidly inhaled to TLC and breath held for 15-20 s to obtain a constant value for PL (12). At the end of the breath hold, the mouthpiece valve was turned 90° to the spirometry circuit, and the subject exhaled to RV at a constant rate that was varied from 80 to 2,600 ml/s BTPS (Fig. 3). Marks on the spirometer bell at 500-ml intervals allowed the subject to gauge the expiratory flow rate. The subject repeated the maneuver if the measured flow rate was not within the targeted range.
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Protocol for rebreathing. After the bag-in-box apparatus was filled with a volume of air equal to ~90% of the subject's vital capacity, the subject exhaled to RV. The mouthpiece valve was then turned to the bag-in-box, and the subject rapidly inhaled until the bag was empty. The subject then rebreathed a volume of 1.4-3.8 liters at a rate of 8-60 breaths/min for 20-30 s, emptying the bag with each inspiration (Fig. 4). By watching the volume marks on the spirometer bell, the subject maintained the selected rebreathing volume. The value for PL during rebreathing was measured from the last 3 s of the NO signal after the final exhalation during rebreathing was discarded.
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Protocol for steady-state breathing.
A mouthpiece attached to a one-way valve assembly (valve no. 2600, Hans
Rudolph) permitted the subject to inspire from a 120-liter Tissot
spirometer and exhale into a 60-liter Douglas bag (Warren E. Collins,
Braintree, MA). After three 2-min runs of tidal breathing to
equilibrate the tubing, dead space, and the Douglas bag with expired
air, the Douglas bag was emptied, and the subject performed tidal
breathing for 4 min while expired air was collected in the Douglas bag.
To determine the dead
space-to-VT ratio
(VD/VT), end-tidal PCO2
(PETCO2) was recorded at
30-s intervals by using a CO2
analyzer (Capnograph IV, Gould Medical BV, Bilthoven, The Netherlands).
The NO concentration in the Tissot spirometer at the beginning and end
of the maneuver and the NO concentration in the Douglas bag were
measured with the NO analyzer. Mixed expired
PCO2
(P
)
and mixed expired PO2
(P
)
in the Douglas bag and the inspired PCO2 and
PO2 in the Tissot spirometer were
measured by using a blood-gas analyzer (Radiometer ABL 520, Copenhagen, Denmark). Partial pressure of inspired and expired nitrogen in these
gas samples was estimated by subtracting
PCO2, PO2, and
PH2O
from the PB.
VD/VT
was assumed equal to (PETCO2 P
)/PETCO2
(4). The kymograph on the Tissot spirometer provided
measurements of
I. Expired
minute ventilation was measured by multiplying
I by the
ratio of the partial pressure of inspired to expired nitrogen (24).
Volume of NO inspired per minute
(INO)
was calculated as the product of
I and the NO
concentration in the Tissot spirometer. Volume of NO expired per minute
(ENO)
was calculated as expired minute ventilation multiplied by the NO
concentration in the Douglas bag. NO production by the nose during the
period of steady-state breathing was measured as described below.
Nasal suction and gas-collection system.
Air exhaled through the mouth could be contaminated by the much higher
concentrations of NO in the nasopharynx (1, 13, 18, 20). To determine
whether negative pressure in the nasal passages (produced by sucking a
continuous gas flow through the nasal passages) could decrease this
possible contamination and result in lower values of
PL, the steady-state
measurements and some of the breath-holding and rebreathing experiments
were performed while a continuous flow of room air passed from one
nostril to the other by the application of 10 cmH2O to one nostril. A rubber cork surrounding copper tubing 1/4 in. in OD and 5 cm in length was
fitted snugly to one nostril (Fig. 2). Tygon tubing (3/8-in. OD, Norton
Plastics, Pittsburgh, PA) connected the cork to a T-shaped stopcock
valve (no. 0211045, Warren E. Collins) that could connect the cork
directly to a source of 10
cmH2O pressure (Chest tube drainage set-up, Atrium Medical, Hudson, NH) or a 19-liter bag-in-box with 10 cmH2O applied to
the box. The negative pressure was applied 2 min before as well as
during the measurements of PL.
For all of the steady-state measurements, the amount of NO expired by the nose
(nose) was
measured by connecting the tubing from the nostril to the bag-in-box
during steady-state breathing. By measuring the concentration of NO in
room air and in the collecting bag, as well as the volume and length of
time the gas was collected in the bag, the rate of
nose could be calculated.
Specific Experiments
Selection of expired samples after breath holding. During breath holding, high concentrations of NO accumulate in the upper airways (30) and appear as an initial peak during subsequent exhalation (Fig. 3). This peak is followed by a constant value that is assumed to represent the concentration of NO in the expirate from the lower alveolar airways. To select this value without contamination from the peak level, we discarded from analysis an initial expired volume equal to three or four times the subject's anatomic dead space estimated to equal (in cm) the ideal body weight (in lb.) (4), plus the instrument's dead space of 100 ml. We also discarded the final 10% of the volume of the expired breath because the NO concentration usually increased with the slowing of expiratory flow near the end of the breath. The exact increments of volume analyzed to select a part of the exhalation not contaminated by the initial NO peak are shown in Fig. 3.
Effect of continuous nasal suction with 10
cmH2O.
To determine whether nasal suction reduced contamination of
PL from NO in the nasopharynx,
we compared breath-holding and rebreathing measurements performed on
the same day with and without nasal suction.
Comparison of breath-holding and rebreathing maneuvers to determine PL. The subjects performed the breath-holding and rebreathing maneuvers in triplicate on the same day. For breath holding, PL was determined after a volume four times the sum of the subject's dead space and the instrument's dead space (PL4 in Fig. 3) was discarded.
Effect of expiratory flow rates on breath-holding measurements of PL. Exhaled NO levels measured at the mouth progressively increase with slower expiratory flow rates (30). To determine the rapidity of expiratory flow rates required to obtain a stable, constant value for PL not influenced by changes in the expiratory flow rate, five subjects performed breath-holding maneuvers followed by exhalations at constant flow rates that varied between 80 and 2,600 ml/s BTPS. PL was measured after an initial volume equal to four times the dead space (PL4 in Fig. 3) was discarded. However, at expiratory flow rates >900 ml/s, the initial expiratory peak was not separated from the subsequent plateau after a volume four times the dead space was discarded. For these measurements at flow rates >900 ml/s, PL was calculated from data recorded after the peak had disappeared.
Effect of different ventilatory rates on PL measured during rebreathing. In five subjects, the ventilatory rate during rebreathing was varied by changing the breathing rate from 8 to 60 breaths/min while keeping the volume rebreathed constant at 1.4-3.8 liters BTPS. To obtain ventilatory rates as high as 145 l/min, subjects also rebreathed as deep and fast as possible (maximum voluntary ventilation maneuver). Measurements were performed in triplicate.
Effect of varying alveolar lung volume on rebreathing measurements of PL. Five subjects rebreathed 1.6 liters at 25 breaths/min and changed their alveolar volume by starting the rebreathing maneuver at either 1 liter above RV or two liters below TLC.
Intraday and interday variation of PL in five subjects during breath holding and rebreathing. Intraday variations in PL were determined from three measurements made on the same day. Interday variation was calculated from the mean of three or more measurements made on three to five different days.
Statistical Methods
In all experiments, subjects served as their own control so that results were compared with control measurements by using a two-tailed paired t-test. For each set of control measurements and interventions, measurements of PL were made in triplicate and the mean values were compared. A level of P < 0.05 was required for statistical significance.| |
RESULTS |
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Selection of Expired Gas Samples After Breath Holding
Table 1 shows the mean values for PL in five subjects obtained from various segments of the expiratory curve for NO described in Fig. 3. The various expired samples showed little difference from each other with the exception of PL1, the first of three segments analyzed after a volume equal to three times the dead space was discarded. PL1 was significantly greater than the later segments (P < 0.05 with nasal suction, P < 0.02 nonsuction). PL4, data collected after the discarding of a volume equal to four times the dead space, was slightly lower than PL1-3, the data collected after the discarding of a volume three times the dead space, but this difference was only significant without nasal suction (P < 0.02). However, differences were small between PL4 vs. PL1-3, being only 0.2 mmHg × 106 with nasal suction and
0.4 mmHg × 106
without nasal suction. In the subsequent measurements of
PL by breath holding,
PL4 was
chosen because of the theoretical advantage of less influence from
contamination by the NO peak at the start of exhalation and the
inclusion of a larger number of data points than were obtained with
either PL2
or PL3.
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Effects of Continuous Nasal Suction with 10
cmH2O
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Comparison of Measurements of PL by Breath Holding, Rebreathing, and Steady-State Breathing
Table 3 shows the mean values obtained for PL by breath holding and rebreathing performed without nasal suction in 10 subjects. Individual values are illustrated in Fig. 5. The expiratory flow rate after breath holding equaled 830 ± 141 ml/min (range: 640-1,160 ml/min), and minute ventilation with rebreathing equaled 59 ± 15 l/min (range: 41-86 l/min). The steady-state values were measured on different days in five of the subjects while using nasal suction. Rebreathing values for PL were 0.6 ± 0.6 mmHg × 106 less than
observed during breath holding (P < 0.001). PL of 7.1 ± 2.5 mmHg × 106 measured during
steady-state breathing in five subjects significantly exceeded the
rebreathing measurements of 2.3 ± 0.6 mmHg × 106
(P < 0.02) and the breath-holding
measurements of 2.9 ± 0.8 mmHg × 106
(P < 0.04), suggesting the more
leisurely flow rates with steady-state breathing were not effective in
minimizing contamination of the expired samples by NO generated in the
upper airways.
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Effects of Expiratory Flow Rates on PL Obtained After Breath Holding in Five Subjects
Figure 6 shows PL obtained after breath holding by using different constant expiratory flow rates that varied between 80 and 2,600 ml/s. As previously reported by Silkoff and co-workers (30), PL progressively increased as expiratory flow rates were reduced. These data show that PL remained constant once flow rates exceeded 550 ml/s.
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Effect of Changes in Minute Ventilation on PL Measured by Rebreathing in Five Subjects
By varying the rebreathing rate while keeping VT at 1.4-3.8 liters, the effects of minute ventilation on PL were determined. In addition, subjects performed the maximum voluntary ventilation (MVV) maneuver to obtain values of PL at ventilatory rates from 75 to 145 l/min. Figure 7 shows that a minute ventilation greater than ~35 l/min results in stable values for PL. Data obtained during MVV did not significantly differ from data obtained by rebreathing 1.4-3.8 liters at minute ventilations >35 l/min. PL equaled 2.1 ± 0.5 mmHg × 106 of NO during
MVV of 112 ± 14 l/min compared with 2.3 ± 0.6 mmHg × 106 of NO at the lower rate
of 66 ± 5 l/min (P = 0.7).
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Effects of Varying Alveolar Lung Volume on Rebreathing Measurements of PL
To see whether increases in alveolar lung volume altered PL during rebreathing, five subjects rebreathed 1.4 to 1.9 liters at 25 breaths/min, starting at either 1 liter above RV or 2 liters below TLC. The resultant thoracic lung volume at the midpoint of rebreathing was 3.1 ± 1.0 liters BTPS at the lower lung volume and 4.4 ± 1.6 liters BTPS at the higher lung volume. Values for PL at the lower lung volume were 2.9 ± 0.7 mmHg × 106 and 2.8 ± 0.6 mmHg × 106 of NO at the
higher volumes. They did not differ significantly (P = 0.8).
Rate of NO by
the Lower Alveolar Respiratory Tract in Humans
NO by the
lower alveolar respiratory tract of humans, 10 healthy subjects had
breath-holding and rebreathing measurements of
PL without nasal suction as well
as measurements of
DLCO. Five
of the subjects had measurements during steady-state breathing. For the
breath-holding measurements, subjects exhaled at 640-1,156 ml/s.
For the rebreathing measurements, they rebreathed 40-85 l/min.
Table 3 shows that the breath-holding measurements of
NO of 0.39 ± 0.2 µl/min were 29% greater than the rebreathing measurements
of 0.29 ± 0.12 µl/min (P < 0.001). Steady-state measurements of
NO of 0.74 ± 0.25 µl/min were approximately twice as great as the
rebreathing measurements (P < 0.01)
and breath-holding measurements (P = 0.07).
Resting Steady-State Measurements of Respiratory Tract NO Exchange
Table 4 shows values for lower alveolar respiratory tract NO production (NO), net NO
exchange with the environment, and nasal NO removal
(nose) in five
subjects during resting tidal breathing. Subjects
1, 2, and
5 were studied on days with low levels
of environmental NO (1.4, 1.2, and 4.6 mmHg × 106 of NO). They exhaled
more NO than they inspired. Subjects 3 and 4 inhaled 13.2 and 9.6 mmHg × 106 of NO,
respectively, and absorbed NO from the environment.
nose of 0.44 ± 0.20 µl/min was less than
NO of 0.74 ± 0.20 µl/min, but the difference was not significant
(P = 0.14). Ninety-five percent or
more of the calculated value of
NO
was absorbed by the respiratory tract, and only 5% or less of
NO was
exhaled.
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Intraday and Interday Variability of PL
Table 5 shows the intraday and interday variability of PL measured during breath holding and rebreathing in five subjects expressed as the coefficient of variation (CV). Breath-holding measurements had an intraday CV of 9 ± 2% compared with 8 ± 3% with rebreathing and did not differ significantly (P = 0.6). The interday CV was 24 ± 7% with breath holding and 26 ± 9% with rebreathing, respectively. This difference was not significant (P = 0.8).
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DISCUSSION |
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These experiments show that the lower alveolar airways, defined as the
alveoli and respiratory bronchioles well perfused by the pulmonary
circulation, have a
NO of 0.39 ± 0.12 µl/min, determined from rapidly expired samples collected
after 15-20 s of breath holding and 0.29 ± 0.11 µl/min
measured during rebreathing. The steady-state measurements shown in
Table 4 and theoretical analyses (12) show that only ~5% of
NO is expired
and 95% is taken up by the tissues of the airways and perfusing blood flow. As a result,
NO is
substantially greater than the amount of NO in mixed expired samples of
resting subjects. For example, the five subjects in Table 4 expired
0.054 ± 0.024 µl/min of NO during steady-state breathing but had
values for NO
of 0.42 ± 0.09 µl/min after breath holding and 0.30 ± 0.04 µl/min during rebreathing.
Despite the large surface area of the lower alveolar airways, on the
order of 70 m2 (33), these values
for NO are
similar to the net amount of nose of 0.44 ± 0.20 µl/min in five of our subjects, 0.32 ± 0.09 µl/min
reported by Imada and co-workers (13), and 0.22 ± 0.02 µl · min1 · m2
by Kimberly and co-workers (18) in eight normal subjects. However, NO is more
than fivefold greater than the amount of NO produced by the larger
conducting airways extending from above the respiratory bronchioles to
the mouth [0.05 ± 0.02 (SD) µl/min]. This value was calculated
from measurements of expired NO after breath holding. These were made
by using multiple exhalations at different expiratory flow rates in
five normal subjects, as described in a preliminary report by Perillo
and co-workers (26).
Comparison With Measurements by Others
There are no reported previous measurements ofNO to compare
with our values, but several groups have measured the concentration of
NO in expired gas samples collected in a manner that suggest the
NO came primarily from the lower alveolar airways. In 10 subjects, after a 30-s breath hold that should result in a constant
value for PL, Silkoff and
co-workers (30) reported 2.3 ± 0.1 mmHg × 106 of NO in samples
exhaled at 1,550 ml/s. They did not scrub their reference gas of NO
through potassium permanganate, which we find decreases the zero
reference signal by ~0.5 mmHg × 106. Therefore, the
concentration of NO their subjects actually exhaled was likely ~2.8 ± 1.1 mmHg × 106.
This value is similar to our post-breath-holding
PL values of 2.9 ± 0.8 mmHg × 106 (Table 3). NO
in gas samples suctioned from the bronchial lumen after tracheal
intubation should supply gases not contaminated with the high
concentration of NO in the nasopharynx. Tsujino and co-workers (32)
observed 3.5 ± 0.7 mmHg × 106 of NO in expired
samples collected from the lumen of a cuffed endotracheal tube after
the first 200 ml of the expired gas in six normal subjects were
discarded. Lundberg and co-workers (20) observed 2.2 ± 0.7 mmHg × 106 of NO in
exhaled air collected from the stoma of four adults with chronic
tracheostomies. Collectively, the above data indicate that the
PL in normal humans is on the
order of 3 mmHg × 106.
Minimizing Errors in Measurements of
NO from
Contamination by NO in the Nasopharynx
NO depend on
collection of alveolar gas samples free of contamination from the high
concentrations of NO in the nasopharynx. To evaluate possible
nasopharyngeal contamination of expired and rebreathed gas samples, we
compared measurements of PL made
with and without continuous application of 10 cmH2O
pressure to one nostril. This nostril (Table 4) and would be expected
to lower the concentration of NO in the nasopharynx. Also, the nasal
suction should result in negative pressure in the posterior
nasopharynx, thereby closing the velum to the oropharynx and making any
leak across the velum move gases into the nasopharynx. However, this
negative pressure did not significantly change values of
PL. These results indicate that nasopharyngeal contamination was minimal during the breath-holding and
rebreathing maneuvers. The similarity of our values of
PL to measurements of NO
collected from the airways of subjects breathing through cuffed
endotracheal tubes that should eliminate nasopharyngeal contamination
(21, 32) also suggests that the breath-holding and rebreathing
maneuvers used in this study prevented significant nasopharyngeal
contamination. Other investigators have employed positive airway
pressure during breath holding and exhalation to prevent nasopharyngeal
contamination (30), and as little as +5
cmH2O of positive pressure is
considered sufficient to prevent contamination (16). Recently,
experiments have shown that breath holding even without positive
pressure prevents contamination of the oropharynx by the high
concentrations of NO in the nasopharynx (17).
Potential Errors in Measurements of
NO From NO
Produced in the Conducting Airways
Cause of Higher Values for
NO Measured After
Breath Holding Compared With Rebreathing Measurements
Steady-State Estimates of PL and
NO
NO from mixed
expired samples collected during steady-state breathing resulted in
values of PL and NO that were
twice those obtained with rebreathing at a rate >41 l/min or after
breath holding at expiratory flow rates in excess of 640 ml/min shown
in Table 3 and Fig. 5. A logical explanation is that the dead space
fraction of ~200 ml/breath will contain NO being produced by the
conducting airways. During the slower exhalation during steady-state
breathing, a larger amount of NO will accumulate in the dead space
fraction and contribute more NO to the mixed expired gas and increase
the size of PL calculated with
Eq. 2. The equations for measuring
PL during steady-state breathing
do not take into account the NO being produced by the conducting
airways and thereby lead to an overestimation of
NO and
PL. Data obtained during
steady-state ventilation are therefore not suitable for
determining NO
or PL with Eqs.
2 and 3.
Potential Errors in
NO From Assuming
Partial Pressure of NO in the Perfused Blood (Pc) is Negligible
NO with
Eq. 1 stating
NO = PL(DNO)
is correct. However, if Pc is not negligible, Eq. 1 becomes (12)
|
(4) |
NO
calculated with Eq. 1, we have
initiated in vitro estimates of Pc (28). Fifty milliliters of blood are
incubated in a Tedlar bag at 37°C containing 2,500 ml of gas at a
PCO2 of 40 Torr,
PO2 of 100 Torr, and a partial
pressure of NO
(PNObag)
of 40 mmHg × 106.
PNObag is
measured every 10 min. In four sets of measurements of
PNObag
containing blood from four healthy subjects,
PNObag initially fell exponentially, until reaching a constant value of 0.4 ± 0.2 (SD) mmHg × 106 at 50 min. At the
present time we know of no other estimates of Pc. Because
PL is on the order of 3 mmHg × 106, these
preliminary data suggest
NO should be
calculated with Eq. 4, multiplying
DLNO by
3 0.4 or 2.6 mmHg × 106 in place of 3.0 mmHg × 106. These
observations suggest the
NO calculated
with Eq. 1, which assumes Pc is zero,
overestimates its true value by ~15% in normal subjects. If
NO is measured
in diseased subjects, measurements of Pc will be required to determine
whether Pc is large enough to cause significant overestimates of
NO calculated
with Eq. 1 that assumes Pc is negligible.
Fraction of Total
NO Measured with
Eq. 1
NO assumes NO
produced in the tissues of the lower airways enters the air spaces and
then diffuses into the perfusing blood and tissues. Some NO produced in
these tissues will react with substances in the tissues and never enter the air spaces. NO produced in bronchioles and small bronchi may be
taken up by blood in the bronchial circulation or by other tissues
without entering the air spaces (19). Because these forms of NO
produced by the lower alveolar airways do not enter the air spaces and
contribute to PL, they will not
be measured with Eq. 1. Therefore,
total NO may
be considerably higher than 0.3 to 0.4 µl/min calculated with
Eq. 1. We are unaware of methods that
can measure both the airway-exchangeable and -nonexchangeable components of NO produced by the lower airways.
Importance of Measurements of DNO
According to Eq. 1, PL can be interpreted asNO per unit of
DNO. If the process or disease
being studied does not alter DNO,
PL is proportional to
NO, and direct
measurements or estimates of DNO
add little information. However, if the intervention or disease has the
potential of changing DNO, such
as a decrease secondary to acute lung injury or passive lung
hyperinflation (12), or an increase with heavy exercise,
PL can change its value
secondary to the change in DNO
with no alteration in
NO. Therefore,
direct measurements of DNO at
the time PL is measured will be
required to make accurate measurements of
NO if the process or disease being studied can significantly alter
DNO.
In summary, these experiments show that the NO concentration measured
at the mouth during moderately vigorous rebreathing or forceful
exhalation can provide data to calculate NO produced by the lower
alveolar airways that enters the air spaces. This technique is
sufficiently reproducible to determine whether factors that increase
expired NO, such as positive end-expiratory pressure (3, 7, 27) and
elevated pH (3), or that decrease expired NO, such as hypoxia (3, 7,
9), are associated with a similar change in
NO. Other
potential applications include studies in patients with diseases
suspected of causing overproduction of NO, such as asthma (1, 21) and
cirrhosis of the liver (22, 31), and underproduction of NO described in
primary pulmonary hypertension (6, 29). Some drugs designed to reduce
lung inflammation lower NO production (11) so that measurements of NO or
PL may have application in
monitoring responses to therapeutic interventions designed to reduce
lung inflammation.
| |
APPENDIX |
|---|
|
Top
Abstract
Introduction
Appendix
References
|
|---|
Detailed Derivation of Method to Calculate
NO From
Steady-State Measurements of Expired NO
NO) will
equal the sum of the amount being removed by diffusion
(PL × DNO) and removed by alveolar
ventilation
(A),
A[PL/(PB 47)] or
|
(A1) |
A is
alveolar ventilation (in ml STPD/min),
PB is barometric pressure (in
mmHg), and 47 is the
PH2O
at body temperature (in mmHg).
Equation A1 can be used if inspired NO
concentration is negligible. However, if the partial pressure of NO in
inspired air (PINO) is
appreciable, the term
A[PL/(PB 47)] must be expanded to include the NO inspired from the
environment and airways. The NO inspired into the lower alveolar
airways from the environment is expressed as
I (1 VD/VT)
[PI/(PB 47)], where
I is the inspired minute ventilation and
VD/VT
is the dead space-to-tidal volume ratio. NO inspired from the dead
space is expressed as I
(VD/VT)
PL/(PB 47). The amount of NO exhaled from the lower alveolar airways
is expressed as (E)
[PL/(PB 47)], where E is expired
minute ventilation or
|
(A2) |
E nearly equals
I,
Eq. A2 can be simplified
|
(A3) |
|
(A4) |
) and
VT can be measured and
PL can be calculated from
P and
PI by the Bohr equation (4)
|
(A5) |
(VT)
is proportional to total NO in an exhaled breath, and the right-hand
terms represent the contributions from the dead space
(VD) and the lower airways (VT VD), respectively. Solving for
PL gives
|
(A6) |
NO during
steady-state breathing. The extrapolated volume for
DLCO
present at the middle of the VT
during steady-state breathing was multiplied by five to provide a value
for DNO used in
Eq. A4.
VD was calculated from
measurements of inspired, end-tidal, and mixed expired
CO2 concentrations (4).
E was determined from the product of I measured
from the Tissot spirometer multiplied by the inspired nitrogen
concentration and divided by the expired nitrogen concentration (24).
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by National Institutes of Health Grants HL-07216, HL-51701, ES-02679, and ES-07026.
| |
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: R. W. Hyde, Pulmonary and Critical Care Unit, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 692, Rochester, NY 14642-8692.
Received 19 March 1998; accepted in final form 16 September 1998.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Appendix
References
|
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