Vol. 273, Issue 6, R2055-R2058, December 1997
Single-breath diffusing capacity of NO independent of
inspiratory NO concentration in rabbits
Hartmut
Heller and
Klaus-Dieter
Schuster
Department of Physiology, University of Bonn, D-53115 Bonn, Germany
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ABSTRACT |
Pulmonary
diffusing capacity of NO
(DLNO)
was determined by performing single-breath experiments on six
anesthetized paralyzed supine rabbits, applying inspiratory
concentrations of NO
(FINO) within a range of 10 parts per million (ppm) 
FINO 800 ppm. Starting from residual volume, the rabbit lungs were inflated by
50 ml of a NO-nitrogen-containing indicator gas mixture. Breath-holding time was set at 0.1, 1, 3, 5, and 7 s. Alveolar partial pressure of NO
was determined by analyzing the end-tidal portion from expirates, with
the use of respiratory mass spectrometry. In the six animals, pulmonary
diffusing capacity of NO averaged
DLNO = 1.92 ± 0.21 ml · mmHg
1 · min1
(mean ± SD value). Despite extreme variations in
FINO, we
found very similar
DLNO
values, and in three rabbits we found identical values even at such
different
FINO levels
of 80 ppm or 500, 20, or 200 ppm as well as 10 or 800 ppm. There was
also no dependence of
DLNO on the
respective duration of the single-breath maneuvers. In addition, the
time course of NO removal from alveolar space was independent of
applied
FINO
levels. These results suggest that
DLNO
determinations are neither affected by chemical reactions of NO in
alveolar gas phase as well as in lung tissue nor biased by endogenous
release of NO from pulmonary tissue. It is our conclusion that the
single-breath diffusing capacity of NO is able to provide a measure of
alveolar-capillary gas conductance that is not influenced by the
biochemical reactions of NO.
pulmonary diffusing capacity of nitric oxide
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INTRODUCTION |
NITRIC OXIDE (NO) has been successfully introduced as a
new test gas for studying alveolar-capillary diffusion. Because of the
extremely high affinity of NO for hemoglobin and its very fast rate of
association with hemoglobin, it has been suggested that NO uptake in
pulmonary capillary blood is not limited by chemical reaction with
hemoglobin (2, 5, 8, 9). Thus pulmonary diffusing capacity of NO
(DLNO)
has been thought to represent a close estimate of the membrane
component in alveolar-capillary gas transfer. However, chemical
reactions of NO in alveolar gas phase and lung tissue could affect
DLNO
determinations by contributing to the removal of NO from alveolar space
in addition to alveolar-capillary diffusion (9). Furthermore,
endogenous NO generated in lung tissues (6, 14) should also be taken
into account. If significant, pulmonary NO output would increase
alveolar partial pressure of NO, creating an underestimation of the
true diffusive removal rate.
The aim of our work was to check the potency of chemical reactions and
the endogenous production of NO as factors capable of distorting
measurements of pulmonary diffusing capacity. For this purpose, we
performed two series of
DLNO
determinations on each of six mechanically ventilated rabbits, by
applying single-breath maneuvers at two different levels of inspiratory
concentrations of NO
(FINO),
which ranged between 10 and 800 parts per million (ppm). In each
series, breath-holding times of 0.1, 1, 3, 5, and 7 s were executed. It
was hypothesized that 1) if there
were a chemical transformation of NO to
NO2 in alveolar gas phase or a
reversible binding of NO at lung tissue,
DLNO should
increase with increasing breath-holding time;
2) an irreversible binding of NO at
lung tissue should distort the time course of NO removal from alveolar
space; 3) an endogenous NO output of
significant magnitude (compared with
FINO)
should produce detectable traces of NO in alveolar gas.
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METHODS |
Single-breath maneuvers were performed in six anesthetized paralyzed
supine rabbits (Chinchilla cross-breed, mean body weight 4.2 kg, range
3.4-5.6 kg). The experiments were approved by the local animal
ethics committee. The animals were anesthetized with pentobarbital
sodium (11 mg · kg1 · h1), intubated by a
cuffed endotracheal tube (3-4 mm ID) and paralyzed by alcuronium
(0.08 mg · kg1 · h1).
The endotracheal tube was connected to a computerized ventilatory servo-system that we had designed for steady mechanical ventilation of
small laboratory animals as well as to perform lung function testing.
The animals were ventilated with room air. Sufficient conditions of
normoxic ventilation were checked by repeatedly analyzing the partial
pressure of oxygen in arterial blood samples (ABC1, Radiometer,
Copenhagen, Denmark). We replaced fluid loss by intravenously infusing
Ringer solution (5 ml · kg1 · h1).
Preparation of indicator gas mixture.
To avoid a spontaneous transformation of NO to
NO2, we prepared oxygen-free gas
mixtures containing 10-800 ppm NO in nitrogen
(N2). NO (NO 2.5, Messer Griesheim, Cologne, Germany) was led through diluted KOH, subsequently collected with a KOH-containing syringe, and finally injected into
gas-tight flexible aluminium bags (Plastigas-Beutel, Linde Gase,
Cologne, Germany), which had repeatedly been washed out with
N2.
Experimental protocol. After induction
of anaesthesia, we recorded pressure-volume curves in each animal by
inflating and deflating the lungs in predefined volume steps. The
airway pressure was measured with a differential pressure transducer
(Dr. Fenyves and Gut, Basel, Switzerland) stopping the computerized
procedure at airway pressures smaller than 
20
cmH2O or greater than +20 cmH2O (related to atmospheric
pressure). We defined the lung volume attained at 20
cmH2O of airway pressure as
residual volume. In a separate set of single-breath maneuvers, the
rabbit lungs were inflated with a
NO/N2 gas mixture and were
subsequently deflated to determine anatomic and apparatus dead space by
applying Fowler's graphical method (3) to fraction-volume curves for
NO. The results were used to gauge the effective time available for NO disappearence from alveolar space, which was necessary for calculating DLNO.
Starting from the residual volume, we inflated the rabbit lungs using
50 ml of the NO-N2 mixture. We set the time intervals for
both inspiration and expiration at 0.6 s and those of breath-holding at
0.1, 1, 3, 5, and 7 s. After executing the breath-holding periods, alveolar gas was sampled by deflating the lungs via a spiral stainless steel tube (3.5 mm ID, length 5 m), thus storing the entire expirate (~50 ml) within the tube. Then mechanical ventilation of the animals was continued with room air. In each rabbit, the two different levels
of FINO as
well as the various breath-holding times were applied in random order.
The gas stored within the tube was dried by freezing and was
continuously sucked into the inlet system of a respiratory mass
spectrometer. To record NO backpressure, the same experimental
procedure was repeatedly applied by inflating the rabbit lungs with
pure nitrogen.
Mass spectrometry. We used a
variable-collector magnetic sector respiratory mass spectrometer
(modified M3, Varian MAT, Bremen, Germany) to continuously record
partial pressures of oxygen (O2) and carbon dioxide (CO2) during
continued mechanical ventilation as well as to analyze the alveolar
partial pressures of NO and Ar. The relevant gases NO,
O2, Ar, and
CO2 were detected, setting ion
collectors at the following mass-to-charge ratios (m/z):
30 (NO), 32 (O2), and 44 (CO2). We determined Ar (m/z = 40) at the CO2-44-ion collector by repeatedly changing the
accelerating voltage (peak jump). To gain an optimal resolving power
for NO analyses, we put the ion collectors at a maximal distance to the
ion source. As previously introduced (12), we reduced drift errors and
cross-talk effects by repeatedly comparing the dry sample gas with a
reference gas, which only differed in the NO content. We determined the alveolar partial pressure of NO
(PANO)
within the alveolar gas sample by starting at the end-tidal volume
(which was ~10 times greater than dead space) and continuing analysis
as long as
PANO values
remained constant (alveolar plateau). Using a 2-m heated steel inlet
capillary, the gas sampling rate of the mass spectrometer was reduced
to 5 ml/min. The detection limit for NO was 0.07 ppm at
FINO = 10 ppm and 0.5 ppm at
FINO = 800 ppm.
Calculations for
DLNO.
We processed the
PANO values
by performing calculations of the
DLNO on the
basis of differential equations for inspiration, breath-holding, and
expiration, as has been previously introduced (4, 13)
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(1)
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(2)
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(3)
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where
PINO is the
inspiratory partial pressure of NO;
I and
E
are the inspiratory and expiratory flows (ml
BTPS/s), respectively; and
G is the capacitance
coefficient for the gas phase at 37°C (0.00116 ml
STPD · mmHg1 · ml1BTPS)
according to Piiper et al. (11).
VA (ml
BTPS) is the effective alveolar distribution volume
of NO. We determined VA (as well as residual volume) from the Ar washout, induced by inflating the
rabbit lungs with the Ar-free NO-N2 indicator gas mixtures.
We calculated
DLNO by
performing a trial-and-error approximation method (Newton's iteration
procedure) on the coupled system of equations that we obtained from
integrating Eqs. 1-3. First, the
starting value of
DLNO was
set at DLNO = 2 ml · mmHg1 · min1,
and PANO was calculated by using Eqs.
1-3. If the calculated value of
PANO
differed from the measured one,
DLNO was
appropriately adjusted and the procedure was repeated. There was simple
convergence to within a tolerance of 0.01%, usually within five
iterations. The three-equation methodology presented has already been
reported to improve the accuracy and precision in measurements of
pulmonary diffusing capacity significantly when either carbon monoxide
(4) or CO2 (13) was used.
As previously described (13), we considered the variable alveolar
volume to be one compartment. We neglected a backpressure for NO since
we did not detect significant NO traces in alveolar gas samples when
using pure nitrogen. Finally, we also ignored the capability of lung
tissues for taking up NO as it would constitute a further path of NO
removal from alveolar space, which is indistinguishable from
DLNO when
calculated by applying the three-equation methodology.
Data analysis. Values are expressed as
means ± SD. To assess a dependence of the single-breath diffusing
capacity of NO on the inspiratory concentration of NO used, we
performed regression analysis on the
DLNO-to-FINO
relationship. In addition, the
DLNO values
obtained in each animal at the two different inspiratory concentrations
of NO were compared using Student's paired
t-test. At each
FINO level,
regression analysis was applied to the change in
DLNO values
with increasing duration of the single-breath maneuvers (hypothesis 1). Furthermore,
nonlinear regression analyses were used to assess the time course of NO
removal from alveolar space (hypothesis
2). The significance level was set at
P < 0.05.
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RESULTS |
Continuous recordings of alveolar partial pressures of
O2 and
CO2 revealed values around 95 mmHg
(O2) as well as 38 mmHg
(CO2), reflecting normoxic
ventilatory conditions. During the single-breath maneuvers, the
end-tidal partial pressure of O2
ranged between 20 and 25 mmHg. The results of determinations made in
measuring the single-breath diffusing capacity of NO
(DLNO)
are presented in Table 1. The overall mean
value was
DLNO = 1.92 ± 0.21 ml · mmHg1 · min1.
Despite extreme variations in inspiratory concentration of NO (FINO),
we found very similar
DLNO values
(and in 3 rabbits identical values), e.g., even at
FINO = 10 ppm as well as
FINO = 800 ppm (rabbit F). There was no
significant deviation between
DLNO mean values obtained in the same animal and no dependence of
DLNO values on FINO
levels
(DLNO = 1.94 8 · 105 · FINO,
r = 0.12,
n = 12, P > 0.72, power = 0.8).
Table 2 shows the results of regression
analyses. At each
FINO level,
the single-breath diffusing capacity was independent of entire duration
of maneuvers performed, which also applied to the entirety of the 60 DLNO
determinations (see Fig. 1). The time course in
disappearance of NO from alveolar space was also independent of
FINO. At
each level, the ratio of alveolar partial pressures of NO obtained at
the end of gas sampling
(PANO) to initial values
(PAONO, as
calculated from
FINO and Ar
washout) decreased monoexponentially with increasing duration of the
single-breath maneuvers, an aspect that is also valid for the total of
60 PANO/PAONO determinations (see Fig. 2).
Linear transformation yielded that none of the intercept values of
PANO/PAONO
were different from unity at time zero.
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Table 2.
Results of linear regression analysis on
 DLNO vs. t as well as nonlinear
regression analysis on
PANO/PAONO vs. exp(t)
of no. of measurements at various levels of
FINO
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Fig. 1.
Deviation between single pulmonary diffusing capacity of NO
(DLNO)
values and
DLNO mean
values
(DLNO)
obtained at respective levels of inspiratory concentration of NO
against duration of single-breath maneuvers
(t). Linear regression analysis:
correlation coefficient = 0.21
(n = 60, P > 0.2).
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Fig. 2.
Ratio of alveolar partial pressures of NO obtained at the end of
maneuvers
(PANO) to
initial values
(PAONO)
related to various duration of single-breath maneuvers
(t). Nonlinear regression analysis:
correlation coefficient = 0.98
(n = 60, P < 0.001); intercept value at time
zero:
PANO/PAONO = 1.0 ± 0.04.
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DISCUSSION |
The main finding of the present study is that determinations of the
single-breath diffusing capacity of NO are independent of
FINO within
the studied range of 10 ppm FINO 800 ppm. There was also no dependence of
DLNO values
on the respective duration of single-breath maneuvers
(t) over a range of 1.3 s t 8.2 s.
Chemical reaction of NO with alveolar
gas. If there had been a significant decay of NO apart
from diffusive removal, based on a chemical transformation to
NO2,
DLNO should
have been more and more overstated with increasing duration of
single-breath maneuvers as well as increasing values of
FINO, an
aspect that our findings were unable to corroborate. Furthermore, Meyer
et al. (9) showed that a chemical reaction of NO in gas phase (300 ppm
NO in helium by addition of 12%
O2) was much too slow (drop to
100 ppm NO within 1 h) to bias
DLNO data
obtained from their rebreathing experiments on dogs. During the
breath-holding periods of our single-breath study, the
O2 concentration within alveolar
space was ~3% as we added 50 ml of O2-free
NO-N2 gas mixture to residual volume (14.4 ± 1.4 ml),
leading to even lower reaction rates. All in all, we conclude that our
experiments have not been biased by a chemical transformation of NO in
the presence of oxygen.
Chemical reaction of NO in lung
tissue. NO is a highly reactive chemical species.
Therefore, an interaction between NO and lung tissue is also to be
taken into account: if NO is reversibly absorbed by lung tissue, its
effective alveolar distribution volume would exceed that of Ar
[the solubilities of both gases in water are close at 0.04 ml
STPD · ml1 · atm1
(7)]. As alveolar volume is implicit in the calculation of the
single-breath diffusing capacity, we evaluated the role of such an
interaction as might possibly bias our results. We recalculated DLNO in
rabbit E on the basis of our
experimental data
(FINO = 10 ppm), but set alveolar volume at respective values that were 30, 60, and 90% higher. We obtained an underestimation of
DLNO by 2, 37, and 95% at a breath-holding time of 0.1 s, but found an increasing
overstated result with increasing duration of maneuvers, attaining
values of 22, 41, and 56% at a breath-holding time of 7 s. In the same
animal, we calculated even more distinct discrepancies when using
experiments performed on
FINO = 800 ppm. In the face of the time independence of the
DLNO
values, a reversible binding of NO at lung tissue hence appears to be
insignificant, at least when using 10 ppm FINO 800 ppm.
If NO is irreversibly bound to tissue throughout the single-breath
maneuvers, the time course of NO removal from alveolar space should
have been affected in as far as the zero-time intercept of the
PANO/PAONO-to-time
relationship is different than unity. In this connection, it should be
emphasized that we calculated PANO/PAONO = 1 at time zero (see Fig. 2), although we included values obtained
from 10 ppm as well as 800 ppm experiments. From equality in the
zero-time intercept with unity, we inferred that NO could not have been
subjected to an irreversible reaction with lung tissue unless it
developed proportionally to the partial pressure of NO in the gas
phase.
Endogenous production of NO. Recently,
it was shown that NO is also endogenously generated by rabbit lung
tissue (6, 14). Assuming endogenous NO outputs were of significant
magnitude compared with values of
FINO, we
should have obtained significant traces of NO in alveolar gas after
having the animals inhale pure nitrogen or room air. However, we were
unable to detect such traces of NO (detection limit: 0.07 ppm NO).
There is, incidentally, no reason to expect an influence of the
endogenous NO output on the single-breath diffusing capacity as we
determined a lower limit of NO at a removal rate of 30 nmol/min in
rabbit E
(DLNO = 1.68 ml · mmHg1 · min1
at FINO = 10 ppm), although this constitutes a 15-fold higher value compared with
exhalation rates of endogenously produced NO in isolated perfused
rabbit lungs [2 nmol/min (14)]. Evidently, even at 10 ppm
NO its alveolar concentration was much too high to detect an influence
of endogenous NO on measurements of the single-breath diffusing
capacity of NO. However, this observation may be qualified by the
findings of Muramatsu et al. (10), who showed an increased endothelial
NO production in chronic hypoxia-induced hypertensive rat lungs (13 nmol/min), indicating that
DLNO
measurements are possibly limited during conditions of altered
physiology at least when using 10 ppm NO.
Perspectives
The relative roles of chemical reaction and endogenous production of NO
in their ability to influence determinations of pulmonary diffusing
capacity of NO were assessed by performing single-breath experiments on
six mechanically ventilated rabbits. Using very different inspiratory
concentrations of NO as well as a large range of breath-holding times,
we nevertheless obtained unchanged values of
DLNO. This
finding suggests that the single-breath diffusing capacity of NO is
able to provide a measure of alveolar-capillary gas conductance that is
unbiased by biochemical reactions of the indicator gas at least within
the range of 10 ppm FINO 800 ppm. Another aspect of our findings would also seem worthy of discussion, since NO is known to mediate vasodilation in the pulmonary circulation (1). In this respect, our finding that
DLNO values were almost identical at
FINO = 10 ppm as well as at
FINO = 800 ppm may be taken as an indication that within this range of inspiratory concentrations NO did not influence pulmonary diffusion conditions by
changing the effective pulmonary capillary surface area (which, among
other things, depends on pulmonary capillary blood volume). In any
event, to what extent the interpretation of
DLNO data
is limited by functional inhomogeneities of pulmonary gas exchange remains a question requiring further research and investigation.
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ACKNOWLEDGEMENTS |
We gratefully acknowledge the expert technical
assistance of Christa Pusch, Barbara Schreiber, and Bernd Eixmann.
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FOOTNOTES |
Address for reprint requests: H. Heller, Dept. of Physiology, Univ. of
Bonn, Nussallee 11, D-53115 Bonn, Germany.
Received 22 May 1997; accepted in final form 28 August
1997.
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Abstract
Introduction
