Vol. 274, Issue 5, R1496-R1499, May 1998
SPECIAL COMMUNICATION
Theta values for
C16O18O
and
C18O2
related to respective pulmonary diffusing capacities
Hartmut
Heller and
Klaus-Dieter
Schuster
Department of Physiology, University of Bonn, 53115 Bonn, Germany
 |
ABSTRACT |
The single-breath diffusing capacities
for singly and doubly 18O-labeled
CO2,
DLC16O18O
and
DLC18O2, as well as for NO, were determined in seven anesthetized rabbits to
investigate whether the theoretically predicted ratio of specific blood
uptake rates of both isotopic CO2
species,
C18O2/C16O18O = 2.0, can be derived from the measured values of
DLC16O18O and
DLC18O2.
Data of DL were obtained by
inflating the lungs with gas mixtures containing 0.35%
C16O18O
or 0.8%
C18O2
or 0.05% NO in nitrogen, with breath-holding periods of 0.05-0.5 s and 2-12 s for the CO2 and
NO tests, respectively.
C18O2/C16O18O was calculated by applying the double-reciprocal Roughton-Forster equation to DL values obtained
in each animal and by assuming that NO diffusing capacity represents
the gas conductance of the alveolar-capillary membrane. The measured
ratio was
C18O2/C16O18O = 1.9 ± 0.2 (mean ± SD), thus comparing reasonably with the
predicted one. Therefore, our findings provide evidence that the
greater value of
DLC18O2
is mainly due to the twofold higher probability (or theta value) for
C18O2
than for
C16O18O
to disappear within red blood cells via isotopic exchange reactions.
artificially ventilated rabbits; oxygen-labeled carbon dioxide; single-breath method
| |
INTRODUCTION |
IN PREVIOUS STUDIES (13, 14), singly and doubly
18O-labeled
CO2,
C16O18O
and
C18O2,
were introduced to determine the pulmonary diffusing capacity for
carbon dioxide in man. The major advantage of this approach is that the
rapid dilution of 18O in the large
water pool (55 M) by isotopic exchange between CO2-bicarbonate and water limits
the development of significant back pressures of indicator gases within
pulmonary capillary blood. The author obtained a ratio of diffusing
capacities of the human lung for
C16O18O
(DLC16O18O)
and
C18O2
(DLC18O2)
of 1.28.
Because the oxygens in bicarbonate are symmetrical
(HC16O18O16O
from
C16O18O
and
HC18O16O18O
from
C18O2),
there is a one in three chance in the case of
C16O18O
but a two in three chance for
C18O2
that the 18O label will be in the
water pool because of the hydration-dehydration reactions of
CO2-bicarbonate interconversion
that is catalyzed by carbonic anhydrase of red blood cells (RBC) and
pulmonary tissue. Because of this twofold higher probability of label
removal for C18O2
than for
C16O18O,
it was concluded (14) that the estimated difference between DLC18O2
and
DLC16O18O is explainable by a higher kinetics of disappearance via isotopic exchange for
C18O2
than for
C16O18O.
In addition, the author stated that, by supposing equal diffusion kinetics of both isotopic species and due to
DLC18O2> DLC16O18O,
the value of
DLC18O2 can be considered to provide a lower limit of the true conductance of
the alveolar-capillary membrane for
CO2.
The present work was undertaken to examine the premises of these
interpretations in an animal study. We used the familiar double-reciprocal Roughton-Forster relationship (12) for data analysis
|
(1)
|
where
DL is the overall pulmonary
diffusing capacity for a test gas and the components Dm and
· Vc represent the true conductance of the
alveolar-capillary membrane and the rate at which the RBC in 1 ml of
pulmonary capillary blood will absorb the test gas () related to the
total pulmonary capillary blood volume (Vc), respectively.
If it is true that singly and doubly
18O-labeled
CO2 diffuse with very similar
facility into pulmonary capillary blood
(DmC16O18O 
DmC18O2)
but that
C18O2
is removed within the
CO2-bicarbonate interconversion
with a twofold higher probability than
C16O18O,
then
C18O2/C16O18O 2 should apply. By use of Eq.
1, the ratio of specific uptake rates
by RBC for both isotopic CO2
species can be derived from
|
(2)
|
We
estimated the membrane diffusing capacity
DmC16O18O
by calculating
DmC16O18O = 12.3 × pulmonary diffusing capacity of NO
(DLNO),
falling back on the theoretical prediction that gases permeate lung
tissue at rates proportional to their solubilities in the lung
[estimated from the solubility constants in water:
CO2/NO = 15.2 (1, 17)] and inversely proportional to the square roots of
molecular weights (
/
= 0.81). Because of the very rapid binding of NO to hemoglobin (4, 11), it is usually expected that alveolar-capillary transfer of NO is mainly
limited by diffusion (2, 7, 10, 11), leading to
1/( · Vc) 
0 and
DLNO DmNO.
We applied the single-breath method to artificially ventilated rabbits
to determine values of
DLC16O18O, DLC18O2,
and DLNO.
If our assumptions are valid, the calculated ratio of specific blood
uptake rates should compare reasonably with the theoretically predicted
value of
C18O2/C16O18O 2.
| |
METHODS |
Single-breath maneuvers were performed on seven adult Chinchilla
cross-breed rabbits weighing 2.8-3.7 kg under pentobarbital sodium
anesthesia (20 mg · kg1 · h1
iv). The animals were paralyzed by alcuronium (0.1 mg · kg1 · h1
iv), endotracheally intubated (2.5-3.5 mm ID), and artificially ventilated with room air by a computerized ventilatory servo system. The animals were instrumented with carotid arterial and ear vein cannulas for blood gas analyses, determinations of the hemoglobin concentration of blood ([Hb]), and the administration of
drugs.
Test gases. To avoid the formation of
oxidation products of NO and to enable the comparison of
DL values of the three test gases, three O2-free gas mixtures,
containing 0.35%
C16O18O
or 0.8%
C18O2
or 0.05% NO in N2, were prepared
and stored in gas-tight flexible aluminum bags. Pure NO was led through
diluted KOH, subsequently collected with a KOH-containing syringe, and
finally injected into the aluminum bags, which had been repeatedly
washed out with N2. To avoid an
artificial isotopic exchange with water, pure C16O18O
or pure
C18O2
was dried within a trap and led into the
N2-containing aluminum bags.
Protocol of experiments. Before the
series of single-breath experiments, pressure-volume curves were
recorded. For this purpose, the lungs were inflated and deflated by
specific volume steps and the airway pressure was measured during short
breath-holds by a differential pressure transducer. The residual volume
(VR) was set at the lung
volume attained at 
20 cmH2O
of airway pressure. It was calculated from the argon (Ar) washout
produced by inflating the rabbit lungs with the Ar-free test gas
mixtures (see Table 1). Anatomic and
apparatus dead spaces were determined in separate expirograms for the
three test gases and were used to calculate the effective inflation and
deflation times (13).
In each animal, the series of
C16O18O,
C18O2,
and NO experiments were performed in random order, starting the
single-breath maneuvers from VR
in each case. The respective times for inflation and deflation were set
at 0.6 s for
C16O18O
and
C18O2
and at 0.8 s for NO. For the two isotopic
CO2 species, experiments with
breath-holding periods of 0.05, 0.10, 0.15, 0.20, and 0.50 s were used
to calculate DL (13, 14). The NO
single-breath maneuvers were performed with breath-holding periods of
2, 4, 6, 8, and 12 s. The lungs of the rabbits were inflated with
30-47 ml of the
C16O18O-
or
C18O2-containing
gas mixtures and with 30-55 ml of the NO-containing test gas.
After breath holding, the total expired gas was sampled by deflating
the lungs via a spiral stainless steel tube (3.5 mm ID, length 5 m).
The gas stored within this tube was dried by freezing and was
continuously sucked into the inlet system of a respiratory magnetic
sector mass spectrometer (M3; Varian MAT, Bremen, Germany). As shown in
Table 1, the ratio of inflation volumes for the
CO2 and NO experiments was varied
between 0.73 and 1.34 to examine the influence of changes in pulmonary
capillary blood volume (and thus of Hb content) on the
C18O2/C16O18O determinations.
Apart from this gas-sampling procedure, continuous recordings of
alveolar partial pressures of O2
and
C16O2
(unlabeled CO2) by mass
spectrometry were used to check ventilatory conditions.
Mass spectrometry. The mass
spectrometer used was modified to measure also isotopic ratios (15).
The relevant gases NO, O2,
C16O2,
Ar,
C16O18O,
and
C18O2
were recorded at two ion collectors and one double collector, which
were set at the following mass-to-charge ratios
(m/e): 30 (NO), 32 (O2), 44 (C16O2),
and 46 (C16O18O).
We determined
C16O2
at the first plate of the double collector,
C16O18O
at the second plate, and
C18O2
at the second plate of the same double collector
(m/e = 48) by repeatedly changing the
accelerating voltage (peak jump). In the same way, Ar
(m/e = 40) was measured at the
C16O2
44-ion collector. To avoid drift errors and cross-talk effects (6, 11,
13), the dry sample gas was repeatedly compared with a reference gas
that only differed in the content of test gases, and by subtracting the
background of the mass peaks. The concentrations of
C16O18O
and
C18O2
were obtained in terms of the difference to natural abundance. The
signal-to-noise ratios were 1,656:1 at 3,500 parts per million (ppm)
C16O18O,
1,905:1 at 8,000 ppm
C18O2,
and 1,351:1 at 500 ppm NO.
Calculations for DL.
We used the partial pressures of the three test gases obtained from
that end-tidal portion of the gas sample where the concentration of
test gases remained unchanged. These values were processed by applying
the DL calculations on the basis
of a coupled system of three differential equations defining gas
transfer during inflation, breath holding, and deflation, as previously
described in detail (13).
Statistics. Averaged data are given as
mean ± SD values. The comparison between the calculated ratios of
specific blood uptake rates and the theoretically predicted value
C18O2/C16O18O 2 was carried out using the Wilcoxon test (one-sample 
test). Multiple regression analysis was performed on
C18O2/C16O18O versus [Hb] and ratios of inflation volume.
| |
RESULTS |
Figure 1 shows the time course of
alveolar-capillary transfer of test gases in a semilogarithmic plot of
ratios of alveolar partial pressures at overall times
t and zero
(PA/PA0)
related to the overall time period of experiments.
PA0 values
were derived by calculating the dilution of inspired test gases within
the alveolar volume (1.5 mmHg < PA0,C16O18O < 2.0 mmHg; 4 mmHg < PA0,C18O2 < 5 mmHg; 0.22 mmHg < PA0,NO < 0.26 mmHg).
C16O18O
as well as
C18O2
were removed according to the following biexponential relationships, where t is time in seconds
|
(3)
|
|
(4)
|
but
NO disappeared monoexponentially from alveolar space
|
(5)
|
During
the initial phase (t < 3 s), the
ratios of PA to
PA0 of
C16O18O
and
C18O2
were reduced to less than 0.01 and 0.001, respectively. The pulmonary
diffusing capacities for both CO2
species were calculated from this fast component by subtracting the
partial pressure of the remaining residues from the respective
PAC16O18O values of the fast phase (13, 14). The smallest
PAC16O18O values, measured during the slow phase
(t > 3 s), came close to the level
of natural abundance of
C16O18O,
whereas the smallest
PA0,C18O2
values determined during the same phase of label removal were 20 times
higher than the natural
C18O2
abundance. By contrast to the biexponential kinetics, >99% of the
inhaled NO disappeared from alveolar gas after a time period of
10 s.
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Fig. 1.
Semilogarithmic plot of removal of
C16O18O
(+),
C18O2
( ), and NO ( ) from alveolar space versus overall time available
for gas transfer (inflation, breath holding, and deflation) obtained
from performing 105 single-breath maneuvers on 7 rabbits.
Ordinate: log of ratios of alveolar
partial pressures at overall times t
and zero
(PA/PA0).
|
|
The DL data of each rabbit are
listed in Table 2. The overall mean ± SD
values are
DLC16O18O = 9.9 ± 1.6 ml · mmHg1 · min1,
DLC18O2 = 13.3 ± 2.1 ml · mmHg1 · min1, and
DLNO = 1.8 ± 0.4 ml · mmHg1 · min1.
During the single-breath maneuvers and due to the inflation of the
rabbit lungs with the O2-free
indicator gas mixtures
[VR/inflation volume = 12.7 ml/(30-55 ml)], the end-tidal
PO2 values averaged 24 ± 4 mmHg.
However, after each maneuver the continuous recording of ventilatory
conditions by mass spectrometry showed a fast recovery of alveolar
PO2 to normal values.
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Table 2.
Single-breath diffusing capacities for
C16O18O2 ,
C18O2, and NO as well as the
corresponding ratio of specific blood uptake rates of both isotopic
CO2 species
|
|
By applying Eq.
2 to the individual mean values of
DL, we calculated the ratios of
specific blood uptake rates of both stable isotopic
CO2 molecules in each rabbit
(see Table 2), averaging C18O2 /C16O18O = 1.9 ± 0.2. This result is not significantly different from the theoretically predicted value of
C18O2/C16O18O 2.0 (Wilcoxon test, = 0.05). Furthermore, there was
no dependence of individual mean values of
C18O2/C16O18O on the ratios of inflation volume for the
CO2 and NO experiments and the
[Hb] of blood (correlation coefficient of multiple
regression analysis: 0.503, n = 7, P < 0.6).
| |
DISCUSSION |
The objective of this study was to investigate whether the pulmonary
diffusing capacities of the
18O-labeled stable isotopic
species of carbon dioxide,
DLC18O2 and
DLC16O18O,
reflect the corresponding ratio of specific blood uptake rates of both
test gases,
C18O2/C16O18O. Our data provide evidence that the higher kinetics of disappearance from alveolar gas found for
C18O2
compared with
C16O18O
is mainly due to the higher removal rate of
C18O2
via the above-mentioned
CO2-bicarbonate interconversion.
This is based on the finding that the calculated ratio
C18O2/C16O18O = 1.9 ± 0.2 compares reasonably with its predicted value (2.0). Because Eq.
2 was applied to data of
DLC16O18O and DLNO,
the calculation
DmC16O18O = 12.3 · DLNO
is also confirmed.
The assumption that the pulmonary diffusing capacity of NO should
provide a very close estimate of the true conductance of the
alveolar-capillary membrane has already been used in recent studies (2,
7, 10, 11). It is mainly based on the observation that there is a very
rapid binding of NO to Hb, the reaction of which is 280 times faster
than that of CO (4). Therefore, it was hypothesized that NO that enters
the RBC is almost entirely bound by Hb (11). By taking this aspect into
account, as well as allowing for the fact that NO solubility is greater
than O2 solubility, we expect
NO values to be >14
ml · ml1 · mmHg1 · min1,
as has recently been determined for
O2 (9). This value is greater than the corresponding value of
NO = 4 ml · ml1 · mmHg1 · min1
that can be derived from the study of Carlsen and Comroe (3), who
measured red cell kinetics by rapid mixing technique. However, this
technique may have been seriously biased by diffusion limitation from
unstirred layers (9). Therefore, we used
NO > 14 ml · ml1 · mmHg1 · min1
to estimate DmNO: on the basis of
the double-reciprocal equation of Roughton and Forster (12), the
overall mean value of
DLNO = 1.8 ml · mmHg1 · min1
found in the present study and a reliable value of pulmonary capillary
blood volume in rabbits, 3 ml, we obtained
DmNO < 1.9 ml · mmHg1 · min1. Thus, by using the
value of
DLNO, the
true NO conductance of the alveolar-capillary membrane is, at worst,
underestimated by 6%.
This evaluation is important to interpreting the deviation between the
measured ratio
DLC16O18O/DLNO = 5.5 and the theoretically predicted value of 12.3 that would apply if
the alveolar-capillary transfer of
C16O18O
were also predominantly diffusion limited
[1/(C16O18O · Vc)0]. From the similarity of
DLNO to
DmNO it can be derived that this
deviation is mainly caused by a significant contribution of the
specific
C16O18O
blood uptake conductance
[1/(C16O18O · Vc)]
to the overall resistance to alveolar-capillary transfer of
C16O18O(1/DLC16O18O). By using the Roughton-Forster equation again and referring to DmC16O18O = 12.3 · DLNO,
one obtains that the overall rate of disappearance of
C16O18O
from alveolar space is limited by 55% because of the label removal within RBC. The corresponding value for
C18O2
amounts to 40%.
The RBC act as a sink to remove
18O-labeled
CO2 via the
CO2-bicarbonate interconversion,
and RBC also act as a sink to very rapidly bind NO to Hb. Therefore,
determinations of the single-breath diffusing capacities of both
isotopic CO2 species and NO might have been biased by using different inflation volumes during the CO2 and NO tests, because
pulmonary capillary Hb content changes because of various inflation
volumes applied to artificially ventilated animals (11). Our finding
that ratios of theta were independent of variations in inflation volume
provides evidence that the specific blood uptake rates of test gases
used are much too high to be influenced by such
variations.
Perspectives
The present animal study revealed that the ratio of pulmonary diffusing
capacities of the two 18O-labeled
stable isotopic CO2 molecules,
C16O18O
and
C18O2,
effectively reflects the difference between the specific blood uptake
rates for both isotopic CO2
species, thus confirming the assumptions previously made (13, 14) with
respect to interpreting the corresponding ratio of diffusing capacities
of
C16O18O
and
C18O2
in man. By using the classical Roughton-Forster equation and an
estimated value of the true conductance of the alveolar-capillary membrane for
C16O18O,
we were able to show that the mean blood uptake resistances of
C16O18O
and
C18O2
contribute to the overall resistance to alveolar-capillary gas transfer
at levels of 55 and 40%, respectively. In reviewing the pertinent
literature, the latter value (of
C18O2)
compares very closely with the corresponding mean value for CO (42%
contribution by blood uptake resistance) that we calculated from
studies in humans using 169 subjects (5, 8, 12, 16). Thus we found that
the blood uptake resistances to
C18O2
and CO equally contribute to the corresponding overall resistances to
alveolar-capillary gas transfer.
| |
ACKNOWLEDGEMENTS |
The authors are very grateful for the valuable technical assistance
provided by Christa Pusch, Barbara Schreiber, and Bernd Eixmann.
| |
FOOTNOTES |
Address for reprint requests: H. Heller, Dept. of Physiology, Univ. of
Bonn, Nussallee 11, D-53115 Bonn, Germany.
Received 11 August 1997; accepted in final form 21 January 1998.
| |
REFERENCES |
1.
Austin, W. H.,
E. Lacombe,
P. W. Rand,
and
M. Chatterjee.
Solubility of carbon dioxide in serum from 15 to 38 C.
J. Appl. Physiol.
18:
301-304,
1963[Abstract/Free Full Text].
2.
Borland, C. D. R.,
and
T. W. Higenbottam.
A simultaneous single breath measurement of pulmonary diffusing capacity with nitric oxide and carbon monoxide.
Eur. Respir. J.
2:
56-63,
1989[Abstract].
3.
Carlsen, E.,
and
J. H. Comroe.
The rate of uptake of carbon monoxide and of nitric oxide by normal human erythrocytes and experimentally produced spherocytes.
J. Gen. Physiol.
42:
83-107,
1958[Abstract/Free Full Text].
4.
Cassoly, R.,
and
Q. H. Gibson.
Conformation, co-operativity and ligand binding in human hemoglobin.
J. Mol. Biol.
91:
301-313,
1975[Medline].
5.
Cotes, J. E.,
and
A. M. Hall.
Normal Values for Respiratory Function in Man. Milan, Italy: Panminerva Medica, 1969, p. 327-343.
6.
Gerster, R.
Cinétique de l'échange des atomes d'oxygène en phase hétérogène entre C18O2 et H2O.
J. Appl. Radiat. Isot.
22:
339-348,
1971.
7.
Guénard, H.,
N. Varène,
and
P. Vaida.
Determination of lung capillary blood volume and membrane diffusing capacity in man by the measurements of NO and CO transfer.
Respir. Physiol.
70:
113-120,
1987[Medline].
8.
Hamer, N. A.
Variations in the components of the diffusing capacity as the lung expands.
Clin. Sci. (Colch.)
24:
275-285,
1963.
9.
Heidelberger, E.,
and
R. B. Reeves.
O2 transfer kinetics in a whole blood unicellular thin layer.
J. Appl. Physiol.
68:
1854-1864,
1990[Abstract/Free Full Text].
10.
Manier, G.,
J. Moinard,
P. Téchoueyres,
N. Varène,
and
H. Guénard.
Pulmonary diffusion limitation after prolonged strenuous exercise.
Respir. Physiol.
83:
143-154,
1991[Medline].
11.
Meyer, M.,
K.-D. Schuster,
H. Schulz,
M. Mohr,
and
J. Piiper.
Pulmonary diffusing capacities for nitric oxide and carbon monoxide determined by rebreathing in dogs.
J. Appl. Physiol.
68:
2344-2357,
1990[Abstract/Free Full Text].
12.
Roughton, F. J. W.,
and
R. E. Forster.
Relative importance of diffusion and chemical reaction rates in determining rate of exchange of gases in the human lung, with special reference to true diffusing capacity of pulmonary membrane and volume of blood in the lung capillaries.
J. Appl. Physiol.
11:
290-302,
1957[Abstract/Free Full Text].
13.
Schuster, K.-D.
Kinetics of pulmonary CO2 transfer studied by using labeled carbon dioxide C16O18O.
Respir. Physiol.
60:
21-37,
1985[Medline].
14.
Schuster, K.-D.
Diffusion limitation and limitation by chemical reactions during alveolar-capillary transfer of oxygen-labeled CO2.
Respir. Physiol.
67:
13-22,
1987[Medline].
15.
Schuster, K.-D.,
K. P. Pflug,
H. Förstel,
and
J. P. Pichotka.
Recent Developments in Mass Spectrometry in Biochemistry and Medicine. New York: Plenum, 1979, p. 451-462.
16.
Werner, F.,
and
H. Kolmer.
The CO single breath transfer factor of the lung.
Pflügers Arch.
393:
269-274,
1982[Medline].
17.
Wilhelm, E.,
R. Baltino,
and
J. Wilcock.
Low-pressure solubility of gases in liquid water.
Chem. Rev.
77:
219-262,
1977.
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Abstract
Introduction
