Human airways produce nitric oxide (NO), and
exhaled NO increases as expiratory flow rates fall. We show that mixing
during exhalation between the NO produced by the lower, alveolar
airways (
LNO)
and the upper conducting airways
(UNO)
explains this phenomenon and permits measurement of
LNO,
UNO,
and the NO diffusing capacity of the conducting airways
(DUNO).
After breath holding for 10-15 s the partial pressure of alveolar
NO (PA) becomes constant, and
during a subsequent exhalation at a constant expiratory flow rate the
alveoli will deliver a stable amount of NO to the conducting airways.
The conducting airways secrete NO into the lumen
(UNO),
which mixes with PA during
exhalation, resulting in the observed expiratory concentration of NO
(PE). At fast exhalations, PA makes a large contribution to
PE, and, at slow exhalations, NO
from the conducting airways predominates. Simple equations describing
this mixing, combined with measurements of
PE at several different
expiratory flow rates, permit calculation of
PA,
UNO, and DUNO.
LNO
is the product of PA and the
alveolar airway diffusion capacity for NO. In seven normal subjects,
PA = 1.6 ± 0.7 × 10
6 (SD) Torr,
LNO = 0.19 ± 0.07 µl/min,
UNO = 0.08 ± 0.05 µl/min, and
DUNO = 0.4 ± 0.4 ml · min1 · Torr1.
These quantitative measurements of
LNO
and
UNO
are suitable for exploring alterations in NO production at these sites
by diseases and physiological stresses.
nitric oxide diffusing capacity of airways; nitric oxide production
by airways; lung nitric oxide; breath holding
 |
INTRODUCTION |
INCREASED EXHALED nitric oxide (NO) concentrations have
attracted interest as a means for detecting inflammation of the airways in asthma (17). NO production by the lungs may be abnormal in diseases
such as sepsis, cirrhosis, primary pulmonary hypertension, and
interstitial lung diseases (18, 21, 26, 27). The exhaled concentration
of NO (PE) increases as
expiratory flow rates
(
E) fall
(24), so E must be kept constant to
obtain reproducible measurements of
PE (Fig.
1). The reason for this flow dependence has
recently been elucidated by Tsoukias and co-workers (28, 29). They show
that during exhalation the mixing between NO from the lower alveolar
airways perfused by the pulmonary circulation (LNO)
with NO produced in the upper conducting airways
(UNO) perfused by the bronchial circulation explains this phenomenon. Simple
equations can describe this mixing. When combined with multiple
measurements of PE at different
E, these
equations permit calculation of
UNO
and the partial pressure of NO in the lower alveolar airways
(PA). In this report, we
describe an analysis of expired NO at different
E that also
permits calculation of the diffusing capacity of the upper airways
(DUNO)
and
LNO. LNO
is determined from the product of
PA and measurements of the
pulmonary diffusing capacity of the lower airways
(DLNO)
(12). Because diseases and physiological stress may cause changes in NO
production and diffusing capacity by the alveoli different from those
by the conducting airways, measurement of
LNO,
UNO, DLNO, and
DUNO may
provide new information about factors that alter NO production by the
lungs.
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Fig. 1.
Exhaled nitric oxide (NO) (PE)
vs. flow rate
(E) in
normal subject AP after 15 s of breath
holding. Note marked decrease in
PE as flow rates increase.
|
|
Glossary
| DLNO |
Diffusing capacity of the lower, alveolar airways recorded as
milliliters of NO STPD moving from the
air spaces into the tissues and blood per minute per Torr of NO in the
air spaces
|
| DUNO |
Diffusing capacity of the upper, conducting airways recorded as
milliliters of NO STPD moving from the
air spaces into the tissues and blood per minute per Torr of NO in the
air spaces
|
| f |
Small fraction of
DUNO,
PU, or
UNO
|
| FVC |
Forced vital capacity
|
| NO |
Nitric oxide
|
| PA |
Partial pressure of NO in the alveoli
|
| PB |
Barometric pressure
|
| PE |
Partial pressure of NO in exhaled gas
|
| PU |
Partial pressure on NO in all or a segment of the upper conducting
airway
|
| E |
Expiratory flow rate
|
| RV |
Residual volume of gases in the lungs
|
| TLC |
Total capacity of gases in the lungs
|
| LNO |
Rate of production of NO by the lower alveolar airways that enters the
airways
|
| UNO |
Rate of production of NO by the conducting airways that enters the
airways
|
| |
METHODS |
NO Exchange in the Alveolar Airways
The alveolar airways are defined as those tissues and air spaces well
perfused by the pulmonary circulation, such as the alveoli, alveolar
ducts, and respiratory bronchioles. In this zone, some of the NO
produced by these lower airways diffuses into the air spaces. The
fraction of the total NO produced in this alveolar compartment that
enters the air spaces is called
LNO.
The NO in the alveoli can react with the surrounding tissues (16) or diffuse rapidly through the alveolar capillary membrane into the perfusing blood. After elimination of ventilation by breath holding for
10-15 s, a steady state will develop, and the amount of NO entering the alveoli
(LNO)
equals the amount of NO diffusing into the perfusing blood and
surrounding tissues (12) or
|
(1)
|
where
DLNO is the
alveolar airway NO diffusing capacity, which is considered equivalent
to the NO pulmonary diffusing capacity. Therefore, determination of
PA multiplied by an independent
measurement of
DLNO
permits calculation of
LNO.
DLNO was
determined by a modification of the constant single exhalation
method for measuring the pulmonary carbon monoxide diffusing capacity
(DLCO)
described by Newth and co-workers (19) and Perillo and co-workers (20). Multiple values of
DLNO are
calculated during the exhalation and averaged.
NO Exchange in the Conducting Airways
The conducting airways are defined as those airways extending from the
alveolar airways to the mouth. Strategies such as continuous positive
pressure in the mouth (13, 24) or constant suction of gases from one
nostril (9, 28, 29) can be used to avoid contamination of expired NO
from the conducting airways by the much higher concentration in the
nasopharynx (14). NO gas exchange in the conducting airways can be
analyzed in the same manner as in the alveolar airways. Namely, a
fraction of NO production by the conducting airways
(UNO)
enters the lumen. Some of this NO can diffuse back into the tissues of
the conducting airways and enter the bronchial circulation in
proportion to the partial pressure of NO in the lumen of the conducting
airways (PU). If the bronchial
blood flow maintains the partial pressure of NO in the blood perfusing
the tissues of the conducting airways at a negligible level, the amount
of NO in the lumen that diffuses back into these tissues will equal
PU · DUNO.
With exhalation at a constant flow rate,
PU will reach a constant value,
and during this steady state
UNO
will equal the amount of NO diffusing back into the tissues or
|
(2)
|
We
describe two models of the conducting airways based on the above
assumptions that allow the simultaneous calculation of PA,
UNO,
and DUNO
from multiple measurements of PE
performed at different constant
E.
Model 1.
Model 1 assumes a uniform
concentration of NO throughout the conducting airways (Fig.
2), so
PU = PE. After breath holding for
10-15 s, a constant PA is
achieved (12), and subsequent exhalation at a steady flow rate
(E)
delivers a constant amount of NO to the conducting airways equal to
E[PA
/ (PB 
47)], where PB is the
barometric pressure, 47 is the partial pressure of water at body
temperature in Torr,
E is
expressed in milliliters per minute
STPD, and
PA is expressed in Torr. This NO
from the alveolar airways instantaneously mixes with NO in the
conducting airways, resulting in a uniform partial pressure of NO in
the conducting airways and the expired breath
(PE). The amount of NO exhaled
at any instant (STPD) equals
E[PE
/ (PB 47)].
This equals the contribution from the alveolar airways
{E[PA
/ (PB 47)]} plus
UNO
less the NO diffusing from the lumen of the conducting airways back
into the tissues and bronchial circulation of the conducting airways
(PE · DUNO)
or
|
(3)
|
Rearranging
gives
|
(4)
|
Multiple
sets of measurements of PE at
different E
provide the data needed to determine
PA,
UNO,
and DUNO in
Eqs. 3 and 4. First,
PA is determined graphically by
taking advantage of the following observation: At higher values of
E (i.e.,
>200 ml/s), PE is relatively
small and results in the term
PE · DUNO decreasing to <3% of
UNO.
If
PE · DUNO
is considered insignificant at such flow rates, Eq. 4 becomes
![P<SC>e</SC> = <FR><NU>1</NU><DE><A><AC>Q</AC><AC>˙</AC></A><SC>e</SC></DE></FR> [<A><AC>V</AC><AC>˙</AC></A><SC>u</SC><SUB>NO</SUB> (P<SC>b</SC> − 47)] + P<SC>a</SC>](/content/vol87/issue4/fulltext/1532/img005.gif)
|
(5)
|
Equation 5 has the following form:
y = mx + b. A plot of
PE vs.
1/E results
in PA at the
y-intercept when
1/E = 0, which is also the point where
E = 
. The
slope equals
UNO(PB 47). We therefore calculated
PA from the linear regression of
PE plotted vs.
1/E when
E > 200 ml/s (Fig. 3). If these data failed to result in a doubling of PE, data
at the next slower flow rate <200 ml/s were added until
PE doubled its lowest value.
This value of PA was combined
with all the measurements of PE
and E
collected at different constant
E to
calculate the remaining two variables, UNO
and DUNO,
with use of Eq. 4 with the assistance
of a curve-fitting program utilizing a quasi-Newton regression (8)
(Fig. 4). The program forced the fit
through the calculated value of
PA. To determine whether the
quasi-Newton regression-fitting algorithm supplied a unique solution
for
UNO
and DUNO,
we also calculated their values using the Newton and the steepest
descent-fitting algorithms for a representative subject. The three
algorithms yielded the same values for
UNO
and DUNO.
Therefore, the choice of curve-fitting algorithm does not influence
identification of the unique solutions from these data. The
curve-fitting program requires assumed starting values for
UNO
and DUNO.
These were arbitrarily chosen to be 0.1 µl/min and 0.3 ml · min1 · Torr1,
respectively. In a representative subject, these starting values could
be systematically varied 4- to 10-fold before deterioration of the
fitted curve became apparent. If a poor fit is obtained, starting
values would need to be changed to allow the program to identify a
reasonable fit to the data.
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Fig. 2.
Two-compartment model consisting of lower alveolar airways and upper
conducting airways that assumes a constant concentration of NO within
each compartment (model 1). After
breath holding for 10-20 s, concentration of NO in alveolar
airways (PA) becomes constant,
because alveolar airway NO production
(LNO)
then equals amount of NO diffusing out of airways, or
LNO = DLNO · PA,
where DLNO
is diffusing capacity of NO in alveolar airways. During subsequent
exhalation at a constant
E, alveolar
airways deliver a constant amount of NO to conducting airways, which
equals PA /
(PB 47), where
PB is barometric pressure.
LNO
mixes with NO production by conducting airways
(UNO),
resulting in an NO concentration of
PE. Some of
PE diffuses back into tissues of
conducting airways
(DUNO · PE,
where DUNO
is diffusing capacity for NO of conducting airways).
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Fig. 3.
PE vs. reciprocal of
E
(1/E) for 4 fastest exhalations by subject AP.
Note linear relationship between different pairs of values of
PE and
1/E.
Extrapolation of line of least mean squares to
1/E = 0 (i.e.,
E = )
results in PA = 1.8 × 106 Torr.
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Fig. 4.
PE vs.
1/E with use
of same data from subject AP in Fig.
1. y-Intercept, where
1/E = 0 (i.e.,
E = )
equals PA and was determined
from a linear extrapolation with a linear regression through 4 fastest
values for E
in Fig. 3. Computer then used this value for
PE and all 7 data points with
Eq. 4 (model
1) or Eq. 7
(model 2) to determine
UNO
and DUNO.
Solid curved line is computer solution that uses Eq. 5. Both models resulted in similar close fits to data,
with r2 > 0.998 in all subjects. As slope of curve becomes steeper,
UNO
increases. As curvature increases,
DUNO
increases.
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|
To determine whether a reliable measurement of
UNO
was possible from just the faster values of
E used to
calculate PA, UNO
was also calculated from these data with Eq. 5 and compared with
UNO
determined with all values of
E by use of
Eq. 4.
This method for measuring PA,
UNO,
and DUNO
with model 1 assumes rapid arrival at
a new steady state when the NO coming from the alveolar airways mixes
with the NO in the conducting airways during exhalation.
APPENDIX A describes an equation for
calculating the changes in PE
during mixing and shows the amount of gas needed to be exhaled to reach
a steady state. The equation shows that once ~30% of the expiratory
vital capacity has been exhaled after the initial breath-holding
period, PE is within 99% of the
constant equilibrated value, so Eqs. 4 and 5 are valid for measuring
PA,
UNO,
and DUNO.
Model 2.
Model 2 assumes stratification of the
NO concentration in the conducting airways so the concentration
of NO can gradually increase as the expired gas moves through
the conducting airway (Fig. 5). In contrast
to model 1, the conducting airway is
considered to be a cylinder with a total volume K and an infinite
number of uniform segments. Each segment has an equal
fraction (f) of K, UNO,
and
DUNO, so
that the dimensions of any segment are fV,
fUNO,
and fDUNO.
At the start of exhalation at a constant E,
PA enters the first segment,
where
fUNO
adds NO and
fDUNO removes NO at a rate proportional to the partial pressure of NO in the
segment. The bronchial blood flow in the wall of the upper airway is
assumed to keep its partial pressure of NO at a negligible level. The
resultant partial pressure of NO in the lumen of the segment equals
PU1.
PU1 then
enters the next segment, and its fraction of
UNO
and DUNO
results in
PU2, and so
forth. At the proximal end of the conducting airway,
PU = PE.
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Fig. 5.
Model of lower alveolar airways and upper conducting airways that
assumes a progressive change in partial pressure of NO in conducting
airways (PU) during exhalation
(model 2). In contrast to
model 1 in Fig. 2, conducting airways
are divided into an infinite number of segments, each with same
fraction (f) of conducting airway volume, conducting airway NO
production
(fU), and
conducting airway diffusing capacity
(fDU). In 1st segment,
fU adds NO to
lumen and fDuNO removes NO,
resulting in a partial pressure of NO equal to
PU1.
PU1 then
moves to next segment, where
fU and
fDU result in
PU2. At end
of conducting airway, final value for
PU equals
PE. Other symbols are identified
in Fig. 2. See text and APPENDIX B for
more details.
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|
For any segment of the conducting airways, the amount of NO in the
segment of volume fK equals
fK[PU /
(PB 47)]. It is
changed by the NO production
(fUNO)
entering the segment less the amount diffusing out
(PU · fDUNO)
or
|
(6)
|
The
solution of Eq. 6 given in detail in
APPENDIX B is
|
(7)
|
PA
is obtained from data obtained at the faster
E, as
described above. With this value of
PA and all the measured pairs of
PE and
E,
UNO
and DUNO
are calculated using Eq. 7 with the
assistance of a curve-fitting program utilizing a quasi-Newton regression (8).
Measurement of NO
Details of methods for measuring NO have been recently published (9).
Briefly, a rapidly responding chemiluminescence NO analyzer (Sievers
NOA, model 270B, Sievers, Boulder, CO) operating at a sample rate of
250 ml/min measured exhaled levels of NO at the mouthpiece with a
150-cm-long, 1.6-mm-ID, 3.2-mm-OD Tygon inlet tube. Response time of
the analyzer was <200 ms for a signal 90% of full scale. The
analyzer was adjusted to provide 40 measurements of the NO
concentration per second that could be averaged over any time interval.
The NO analyzer was calibrated daily by serial dilutions of a gas
containing 229 parts per billion (ppb) of NO. To obtain gas samples
free of NO, air from a gas cylinder containing <2 ppb of NO (Scott
Specialty Gases, Plumsteadville, PA) was passed through a filter
constructed from a 5.8-cm-ID, 19-cm-long cylinder (Gas Drying Unit, VWR
Scientific, Rochester, NY) packed with potassium permanganate (Purafil,
Thermoenvironmental Instruments, Franklin, MA) (4).
Because the air signal free of NO could drift as much as 2 ppb in 10 min, measurements of NO-free air were performed within 1 min before and
after each NO measurement from expired gas samples, and these values
were averaged to obtain the zero NO signal. The lag time between the
volume signal obtained from a potentiometer attached to the spirometer
and the change in the NO signal was determined daily and equaled 0.8 ± 0.1 (SD) s. Multiple repetitive measurements of gas mixtures of
2.8 and 8.2 × 106
Torr of NO showed a standard deviation of 0.09 × 106 Torr. We assumed that
the detection limit of our analyzer was two times the standard
deviation of these multiple measurements or 0.2 × 106 Torr. During gas
sampling the operator exhaled warm humidified gas from the mouth by the
inlet of the NO analyzer approximately every 5-10 min, so the
walls of the unheated inlet tubing were kept moist. This resulted in
all gases being considered measured at
ATPS. Measurements of NO in parts per
billion ATPS were converted to partial
pressure of NO in Torr BTPS as
follows: NO in Torr = (NO in ppb
ATPS)(PB)(PB 47) / (PB PH2O)(109),
where PH2O is
partial pressure of water at room temperature. For example, at
PB of 760 Torr and room temperature of 24°C where PH2O = 22.4 Torr, 1 ppb NO = 0.735 × 106 Torr of NO. The chart
recorder (MacLab Recording Instrument, AD Instruments, Castle Hill,
Australia) stored the volume signal and NO signal in a Macintosh LC
computer (Apple Computer, Cupertino, CA). To obtain a stable constant
value for the measurement of PE
after breath holding, we discarded an initial portion of the exhalate
equal to four times the sum of the subject's estimated anatomic dead
space and the instrument dead space of 100 ml, as well as the final
10% of the exhalate (Fig. 6). At flow
rates <45 ml/s, a constant value for
PE was obtained earlier during exhalation (APPENDIX A). At flow
rates >1,000 ml/s, a constant value for
PE was frequently not present
until 40-50% of the breath had been expired. In these cases, the
NO plateau level was determined by visual assessment of the NO signal
displayed on the computer.
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Fig. 6.
Record of NO concentration at mouth
(PE) and lung gas volume
signal during 20 s of breath holding followed by exhalation at 600 ml/s. PE is obtained from NO
plateau that follows NO peak seen at start of exhalation. Peak is
attributed to accumulation of NO in conducting airways during breath
holding that is then flushed through mouthpiece at beginning of
exhalation. Data for calculating
PE was obtained after an initial
expired volume equal to 4 times subject's and instrument's dead space
(DS), as well as final 10% of forced vital capacity (FVC), was
discarded. E
was calculated from volume signal after initial and final 10% of FVC
was discarded. NO signal was moved to left by 0.8 s to correct for lag
between NO and volume signals.
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Maneuvers Used to Measure PE and
E
Subjects exhaled to residual volume (RV) through the mouthpiece of the
apparatus into the room and then rapidly inhaled room air from a
bag-in-box device to total lung capacity (TLC) (Fig. 7). The subject then held this breath
for 10-20 s, so that
PA reached a constant
concentration irrespective of the inhaled ambient NO
concentration (12). At the end of the breath hold, the mouthpiece valve
was turned 90° into the spirometry circuit, and the subject exhaled, maintaining mouth pressure at +5
cmH2O by watching a water
manometer. Corks with various-sized holes bored through their
centers were placed in the expiratory tubing and resulted in different
expiratory resistances and
E. Each
subject performed measurements at seven different flow rates that were
as low as 6 ml/s and as high as 1,355 ml/s. Exhalations at each flow
rate were performed in triplicate, and the values for
PE and
E were averaged. PE was measured as
described above, and
E was
obtained from the spirometer's volume signal after the initial and
final 10% of the expired volume were discarded (Fig. 6). The entire experiment for each subject was completed within 4 h on the same day.
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Fig. 7.
Apparatus for measuring NO production of conducting and alveolar
airways. After exhalation to residual volume, subject is connected via
valve at mouthpiece to bag-in-box apparatus filled with room air and
inspires to total lung capacity. After breath is held for 10-20 s,
valve is turned 90° into spirometer circuit, and subject exhales,
maintaining a mouth pressure of +5
cmH2O by watching water manometer.
Corks with different-sized apertures were placed in expiratory tubing
and provided variable resistance to exhalation, resulting in different
constant E. NO
concentration is continuously measured via side port on mouthpiece.
Changes in gas volume are measured with a spirometer attached to a
potentiometer. NO and volume signals are displayed on a chart recorder
and stored in a computer.
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Measurement of
DLNO
DLNO for
each subject was calculated from the expired NO concentration measured
after inspiring 10 parts/million of NO in air placed in the bag in Fig.
7 from RV to TLC, breath holding for 5 s, and then exhaling to RV at a
constant flow rate of 500 ml/s with a modification of the single-breath
exhalation method for continuously measuring
DLCO during
exhalation described by Newth and co-workers (19) and Perillo and
co-workers (20). Lung volume at any instant during exhalation used in
the calculation of the multiple values of
DLNO was
obtained by adding the amount of exhaled gas remaining above RV
recorded by the spirometer (Fig. 7) to the subject's RV. RV was
obtained from the subject's functional residual capacity (FRC)
measured with body plethysmography (5) by subtracting the expiratory
reserve volume obtained from a spirometer (P. K. Morgan, Haverhill, MA)
from FRC. The multiple measurements of
DLNO during
the exhalation were averaged and performed in triplicate, and the mean
value was recorded.
Subjects
PA,
UNO,
and DUNO
were measured in seven healthy, nonsmoking, 31- to 72-yr-old (mean 46 ± 18 yr) subjects. Five were men and two were women. All subjects
were free of cardiopulmonary disease. Spirometry showed values >90%
of predicted for the forced expiratory volume in 1 s, with a mean value
of 104 ± 16 (SD)% (2). This study was approved by the University
of Rochester's committee for investigations involving human subjects.
Statistical Methods
Values are means ± SD. In experiments where subjects served as
their own control, results were compared using a two-tailed paired
t-test. Groups of subjects were
compared with an unpaired t-test.
P < 0.05 was required for
statistical significance. Regression lines and curves were fitted to
the experimental data by the line of least mean squares referenced to
PE.
| |
RESULTS |
PA,
LNO,
and DLNO
Figure 8 shows the values for
PE and the reciprocal of
E
(1/E) used
to determine PA from the faster
exhalations in the seven subjects. The linear regression of these
points extrapolated to infinite flow, where
1/E = 0, equals PA. The regression line fitted the data closely, with
r2 = 0.965-0.999. PA was 1.6 ± 0.7 × 106 (SD)
Torr. DLNO
was 123 ± 19 ml · min1 · Torr1.
LNO
(i.e.,
PA · DLNO)
was 0.19 ± 0.07 µl/min.
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Fig. 8.
PE vs. reciprocal of faster
values of E in
7 healthy subjects. Extrapolation of linear regression for each subject
to infinite flow
(1/E = 0) provided PA on vertical
axis. Slope equals
UNO(PB 47). See Eq. 5. Seven subjects
represented by different symbols.
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|
UNO
and DUNO
Figure 9 shows the paired values for
PE and
1/E for all
exhalations by the seven subjects used to determine
UNO
and DUNO. E ranged
from 6 to 1,355 ml/s. For model 1,
UNO
was 0.077 ± 0.053 µl/min and
DUNO was
0.4 ± 0.4 ml · min1 · Torr1;
for model 2 the values were similar:
0.074 ± 0.052 µl/min and 0.5 ± 0.4 ml · min1 · Torr1, respectively. The
regression lines for both models fit the data closely, with
r2 > 0.998 in
all subjects. The value of
r2 for the two
models did not differ significantly: 0.9996 ± 0.0003 for
model 1 and 0.9994 ± 0.0006 for model 2 (P = 0.30).
UNO calculated with just the faster values of
E shown in
Fig. 8 with use of Eq. 4 was 0.070 ± 0.048 µl/min. Although this value is slightly lower than 0.077 ± 0.053 µl/min with model 1 and
0.074 ± 0.052 µl/min with model
2, the difference was not significant (P = 0.2).
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Fig. 9.
PE vs.
1/E in 7 healthy subjects. Note linear relationship between
PE and
1/E at faster
E seen more
clearly in Fig. 8. Straight lines connecting data points become less
steep as 1/E
increases (or
E decreases),
because higher values for PE
at slower E
result in more NO diffusing into walls of conducting airways. Different
symbols identify each subject (see Fig. 8).
|
|
Comparison of
LNO
and
UNO
Figure 10 shows that
LNO
of 0.19 ± 0.07 µl/min was consistently greater than
UNO
of 0.077 ± 0.053 µl/min with use of model 1 (P < 0.01).
Calculating with model 2 gave similar
results.
LNO was 0.19 ± 0.07 µl/min compared with
UNO
of 0.074 ± 0.052 µl/min (P < 0.01).
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Fig. 10.
UNO
(calculated using model 1) vs.
LNO
in 7 healthy subjects. In all subjects,
LNO
exceeded
UNO,
and difference was significant (P < 0.01). Model 2 gave similar results
(P < 0.01).
|
|
Comparison of
DLNO and
DUNO
Table 1 shows that
DLNO is
>100-fold greater than
DUNO
calculated with model 1 or
model 2.
| |
DISCUSSION |
These data show that a model of the human airways where exhaled NO from
the alveoli mixes with the NO produced by the conducting airways
precisely predicts the PE
observed at different
E. Simple equations describing this mixing combined with values for
PE at different values of
E result in
measurements of
UNO,
DUNO, and
PA.
PA multiplied by a separate
measurement of
DLNO gives a measurement of
LNO.
Besides these separate quantitative measurements of
LNO
and
UNO,
this model provides a reasonable physiological explanation for the rise
in expired NO with slower
E and helps define the physiological basis for observed values of expired NO
reported by many investigators (6, 13, 17, 24, 29).
Common practice is to measure expired NO at a single relatively slow
E on the order
of 100-250 ml/s (13). The resultant observed values of
PE are three to five times
PA and, therefore, predominantly
represent
UNO.
Although these measurements at single relatively slow
E values
provide a useful index of
UNO, they are at a disadvantage for detecting changes in
PA and
LNO.
A number of studies suggest that the mechanisms altering
UNO
and
LNO
may be different. The large increases in
PE seen in bronchial asthma
likely come from upregulation of inducible NO synthase in the
conducting airways (11, 30). Endothelial-derived NO synthase is
reported to be located in the alveolar capillary membrane (10) and is
upregulated in a rat model of the hepatopulmonary syndrome (7). This
upregulation could explain the high levels of exhaled NO observed in
some patients with cirrhosis and the hepatopulmonary syndrome (18).
Downregulation of endothelial-derived NO synthase may account for the
low levels of expired NO reported in primary pulmonary hypertension (3,
21). The technique described in this report for measuring
UNO
and
LNO
should provide a quantitative method to localize alteration in NO
production to the alveoli or the conducting airways. Such measurements
may result in more precision in the use of exhaled NO to assess lung injury or alterations in regulation of NO production by the lungs than
that obtained with observations at a single
E.
Choice of Lung Models to Explain the Change in
PE With Different Values of
E
The simpler model (model 1) of the
airways, where the conducting airways are considered one single uniform
compartment, precisely described the observed data obtained at
different values of
E, with
r2 > 0.998 in
all subjects. The multicompartment model of the conducting airways
(model 2), with the more realistic
assumption that NO concentration in the conducting airways gradually
approaches PE during exhalation,
does not provide a better fit to the observed data. We also performed
theoretical calculations to see if measurements of
PE at
E in humans as
low as the practical limit of ~5 ml/s can be used to distinguish
between the two models. These models generate different values for
PE shown in Fig.
11 for the same assumed values of
PA,
UNO,
and DUNO.
Fitting the equation of one model to the data generated by the
other model results in a very tight fit, with
r2 > 0.999 (Fig. 12). Therefore, observed values of
PE measured over a wide spectrum
of E values
cannot be expected to distinguish which model provides a more realistic
prediction of the observed data.
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Fig. 11.
Theoretical values for PE at
different constant
E vs.
1/E for
models 1 and
2 (dashed curves). Assumed values were
2.0 × 106 Torr for
PA, 0.075 µl/min for
UNO,
and 0.5 ml · min1 · Torr1
for DUNO.
Solid curves represent realistically obtainable values for
PE in human subjects, where
E > 5 ml/s.
Note similarity of shape of solid curves generated by
models 1 and
2. This similarity makes it difficult
to distinguish models 1 and
2 by use of measurements of exhaled NO
and E.
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Fig. 12.
Theoretical values of PE vs.
1/E generated
by models 1 and
2 by use of assumed values for
PA,
UNO,
and DLNO in
Fig. 11 legend.
E = 1,000, 540, 300, 80, 75, 40, and 6 ml/s. Fitting model
1 to data generated by model
2 and vice versa results in very tight fits, with
r2 > 0.999. Because fits are so precise, observed data cannot be used to
distinguish which model most closely predicts observed changes in
PE at different values of
E.
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Models 1 and
2 have limitations in their assumed
dimensions, because the conducting airways must contain multiple
compartments where the ratio of the surface area of the conducting
airways that secretes NO into the gas volume in the lumen decreases as exhaled gas moves from the alveoli through the trachea (6, 23, 31).
This anatomy results in uneven distribution between UNO,
DUNO, and
conducting airway gas volume. Because the simple one-compartment model
of the conducting airways so accurately predicts
PE at different values of
E, use of more
realistic models of the conducting airways is not likely to result in a better measurable prediction of the experimental data.
Lung Model Where NO Production Is Uniformly Distributed Throughout
the Walls of the Conducting Airways
Tsoukias and George (28) reported what may be a more realistic model of
the dynamics of pulmonary NO exchange in the conducting and alveolar
airways. They define NO production as taking place uniformly throughout
the walls of the lungs' tissues. From the differential mass balance of
NO in the tissue, they derive a second-order partial differential
equation (Eq. 1 in Ref. 28) that
allows determination of the changes in
PE by interventions such as
varying breath-holding time before exhalation, accelerating or slowing flow rates during exhalation, and varying the inspired NO
concentration. Their experimental data obtained by measuring expired NO
concentrations in seven normal subjects at different constant
E levels
result in a fit close to their model, similar to that obtained using models 1 and
2 described above. Therefore, expired
NO concentrations collected at different
E in normal
subjects unfortunately do not provide a means to determine which of
these various models most closely accounts for the observed profiles of
expired NO concentration.
Potential Errors in
LNO
Calculated With Eq. 2 With the Assumption That
DLNO Is Constant
If the decrease with lung volume observed for
DLCO is the
same as that observed for
DLNO,
LNO
might be falsely high when values for
DLNO
obtained at high lung volumes are used and falsely low when
measurements of
DLNO
measured at low lung volumes are used. In the calculation of
LNO
with Eq. 2, we used a mean value of
DLNO
obtained from
DLNO
continuously calculated from the expired NO concentration recorded
during expiration. The calculation started at a maximum volume equal to
the subject's TLC less four times the subject's estimated anatomic
dead space and ended when the subject reached a volume equal to the RV
plus 15% of the forced vital capacity (19, 20). Newth and co-workers
(19) reported that
DLCO
measured with this method was unchanged as lung volume decreased.
Preliminary measurements in nine subjects (20) showed that
DLNO
decreased 9% over this volume interval, but this change did not reach
statistical significance (P = 0.3).
Therefore, the change in
DLNO with
different lung volumes with use of the continuously calculated values
during exhalation appears modest and would not be expected to result in
large errors in
LNO.
However, use of single-breath measurements of
DLNO
obtained at TLC could result in overestimation of
LNO.
Fraction of Total
LNO
and
UNO
Measured From Analyses of PE
This method of measuring
LNO
and
UNO
assumes that NO produced in the tissues enters the air
spaces and then diffuses into the surrounding tissues and
perfusing blood. Some of the NO produced in the alveoli and the
conducting airways will react with the tissues and blood and never
enter the air spaces (16). This NO will not be measured by analyses of
NO in the airways; therefore, LNO
and
UNO
are likely underestimates of the true amount of NO produced by the
alveoli and conducting airways. We are unaware of methods that can
measure the fraction of NO that does not communicate with
airways, and its size may be increased by diseases that impair diffusion of NO from the tissues into the air spaces.
Comparison to Estimates of
LNO
and PA From Data of Others
Because determination of PA
requires breath holding or rebreathing for 10-15 s to achieve a
constant value as well as rapid exhalations, most published values of
PE do not permit calculations of
PA. However, Silkoff and
co-workers (24) measured PE in
10 subjects at
E of 1,550 ml/s preceded by a 30-s breath hold and obtained a
PE of 2.4 ± 1.0 × 106 Torr. With use of their
mean data for PE at slower
E,
extrapolation of their data to an infinite value for
E gives
PA of 1.9 ± 0.8 × 106 Torr, which is in close
agreement with our value of 1.6 ± 0.7 × 106 Torr observed in our
seven subjects.
Recently, Tsoukias and co-workers (28, 29) published a similar
two-compartment model consisting of a nonexpansile compartment representing the conducting airways and an expansile compartment representing the alveolar region of the lungs. In their seven normal
subjects, they determined PA
from 8-12 measurements of PE and
E performed at
constant values of
E that varied
from 175 to 600 ml/s. With an equation equivalent to
Eq. 3, they calculated PA and the flux of NO from the
tissues of the conducting airways to the lumen. For
model 1, flux equals
UNO (PE · DUNO).
By plotting
E[PE
/ (PB 47)]
on the vertical axis vs.
E on the horizontal axis, the intercept on the vertical axis equals flux and the
slope equals PA /
(PB 47). Their values of
PA of 4.1 ± 2.3 × 106 Torr were significantly
greater than 1.6 ± 0.7 × 106 Torr obtained in our
seven normal subjects (P = 0.025). We
have no explanation for the higher values of
PA obtained by Tsoukias and
co-workers. However, their flow rates ranged from only 175 to 600 ml/s,
whereas E for
the subjects of Silkoff et al. (24) and our subjects varied from 4 ml/s
to as high as 1,550 ml/s. This greater range in
E may provide
more precision in determining PA.
Comparison to Estimates of
UNO
and DUNO From Data of
Others
Only a few investigators have measured
PE at a number of different
constant E
that permit calculation of
UNO
or DUNO. Silkoff and co-workers (24) reported
PE at nine different values of
E between 4.2 and 1,550 ml/s in 10 subjects. Their data shown in Fig.
13 permit calculation of
UNO
and DUNO by
use of Eq. 4 or
7. Note the similarity of their data
to the findings in our subjects shown in Fig. 9. Model
1 closely fit the data of Silkoff and co-workers, with
a mean r2 of
0.996 for their 10 subjects.
UNO
from their data was 0.061 ± 0.056 µl/min compared with 0.076 ± 0.053 µl/min in our subjects and did not differ significantly
(P = 0.22).
DUNO in
their subjects was 0.4 ± 0.3 ml · min1 · Torr1
compared with 0.4 ± 0.4 ml · min1 · Torr1
in our subjects (P = 0.61).
Model 2 gave similar results with a
close fit to the data
(r2 = 0.995).
UNO
was 0.053 ± 0.039 µl/min compared with 0.074 ± 0.052 µl/min
in our subjects (P = 0.20),
and DUNO
was 0.5 ± 0.3 ml · min1 · Torr1
vs. 0.5 ± 0.4 ml · min1 · Torr1
in our subjects (P = 0.46). The data
of Silkoff and co-workers and our data show a wide scatter for the
values of
UNO
and DUNO in
normal subjects, with coefficients of variation (CV) ranging from 60 to
90%. PA and
LNO
show less scatter, with a CV on the order of 40%.
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Fig. 13.
PE vs.
1/E in 10 subjects reported by Silkoff and co-workers (24).
E = 4.2-1,550 ml/s. Note similarity to data in Fig. 9 for our 7 subjects. Values for
UNO
and DUNO
determined from these data are given in text.
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Tsoukias and co-workers (28, 29) calculated flux from the data in their
seven subjects, as described above. With use of representative values
of PE in our subjects at
E of
175-600 ml/s used by Tsoukias and co-workers, their values of flux
would only be ~1-3% smaller than
UNO.
Flux in their subjects was 0.043 ± 0.015 µl/min and did not
significantly differ from the values of
UNO
of 0.070 ± 0.048 µl/min in our subjects with use of the faster
E shown in
Fig. 8 (P = 0.20) or 0.077 ± 0.053 µl/min with model 1 (P = 0.16) or 0.074 ± 0.52 µl/min with model 2 (P = 0.18) with use of faster and
slower
E.
Evaluation of a Simplified Method to Measure
UNO
by Use of Only Faster
E
Measurement of
UNO
with E > 80-100 ml/s would have the advantage of fewer measurements of
PE and elimination of the slow exhalations that are more difficult to perform because expiration must
be continued for 25-150 s. The disadvantage is that
DUNO cannot
be measured with any precision, because its accuracy requires the
higher concentrations of NO in the conducting airways achieved with
low values for
E. In our
subjects,
UNO
calculated with only the faster
E shown in
Fig. 8 with use of Eq. 4 was 0.070 ± 0.048 µl/min compared with 0.077 ± 0.053 µl/min for
model 1 and 0.074 ± 0.052 µl/min
for model 2 by use of all the values of PE and
E shown in
Fig. 9. The three values did not differ significantly
(P = 0.2) and have similar CVs of
~70%. Measuring UNO
with the useful expediency of using only faster
E provides acceptable values for
UNO
but at the expense of measurements of
DUNO.
Choice of Analytic Method to Determine
PA,
UNO,
and DUNO From
Measurements of PE and
E Performed at
Different Constant
E
Tsoukias and co-workers (28, 29) measured
PA and flux by plotting the
quantity of NO exhaled, which is the product of E and
PE /
(PB 47) vs.
TOP
ABSTRACT
INTRODUCTION
