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* From the Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX.
Correspondence to: Connie C. W. Hsia, MD, FCCP, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9034; e-mail: Connie.Hsia{at}utsouthwestern.edu
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Setting and
participants: The study was performed at a university medical
center involving 12 nonsmoking healthy volunteers (age range, 23 to 79
years). DLCO, DLNO, cardiac output (
c),
and lung volume were measured simultaneously at rest and during graded
ergometer exercise by a rebreathing technique. Pulmonary membrane
diffusing capacity and Vc were compared by (1) the classic technique of
Roughton and Forster from DLCO measured at two alveolar
oxygen tension (PAO2) levels, and (2) from
DLNO and DLCO assuming negligible erythrocyte
resistance to nitric oxide (NO) uptake, ie,
DLNO approximately equal to pulmonary membrane diffusing
capacity for nitric oxide.
Results: In all
subjects, DLNO increased linearly from rest to exercise;
age, c, and lung volume were the major determinants of
DLNO by stepwise regression analysis. The
DLNO/DLCO ratio averaged 3.98 ± 0.38
(± SD) and the DLNO/DMCO ratio averaged
2.49 ± 0.28 irrespective of exercise intensity. Changing
PAO2 did not alter DLNO. Brief
exposure to 40 ppm of inhaled NO during 16 s of rebreathing did
not alter either DLCO or c. Estimates of pulmonary
membrane diffusing capacity and Vc by the two methods showed a strong
correlation.
Conclusion: Results support DLNO as a direct measure of pulmonary membrane diffusing capacity, allowing the estimation of Vc in a single rebreathing maneuver during exercise. The DLNO-DLCO rebreathing technique can be applied clinically in the investigation of pulmonary microvascular regulation.
Key Words: carbon monoxide • cardiac output • lung diffusing capacity • pulmonary capillary blood volume • rebreathing technique
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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CO is the rate of carbon monoxide uptake by whole blood
and combination with hemoglobin (Hb) measured in vitro; and
Vc is the pulmonary capillary blood volume. In normal subjects,
resistances of the membrane
(1/DMCO) and erythrocyte
(1/[CO · Vc]) contribute almost equally to
the overall diffusive resistance across the lung. In order to estimate
DMCO and Vc from carbon monoxide uptake,
DLCO must be measured at two alveolar oxygen
tension (PAO2) levels, with
the assumption that PAO2 does not
alter DMCO.
Lung diffusing capacity estimated using nitric oxide (NO) as the tracer
gas (DLNO) has been proposed2
as a direct
measure of the conductance of alveolar membrane (pulmonary membrane
diffusing capacity for NO [DMNO]). Because the
reaction rate of NO binding to Hb is some 280 times faster than that of
carbon monoxide, the rate of NO uptake by blood (NO) is
extremely large and 1/(NO · Vc) becomes
negligible, ie, DLNO is approximately
equal to DMNO.
Based on the molecular weights (MWs) of carbon monoxide
(MW = 28) and NO (MW = 30) and the solubility factors (
) of
carbon monoxide (0.0183 mL-1 · atm-1) and
NO (0.0364 mL-1 · atm-1) in water at
37.5°C,3
the theoretical relationship between membrane
diffusing capacities for NO and carbon monoxide (CO) is as follows:
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Previous studies have measured
DLNO and DLCO simultaneously at rest in
humans2
4
and in animals using a
single-breath5
or rebreathing3
technique.
However, DLNO and DLCO have not been compared
during exercise. There has been no direct comparison of
DLNO and DMCO at rest or during exercise, and
the relationship between DLNO and cardiac output (c)
has not been established.
Our objective is to validate the measurement of
DLNO during exercise as an index of pulmonary membrane
diffusing capacity by determining (1) the relationships between
DLNO and c and between DLNO and
DMCO, and (2) the effect of a brief exposure to inhaled NO
during testing on c and DLCO. We hypothesized that
there is a close correlation between DLNO and
DMCO from rest to exercise, and that the
DLNO/DMCO ratio should be approximately 2
irrespective of exercise intensity. We utilized a rebreathing technique
to measure DLCO, DLNO, and c in normal
subjects at rest and during exercise at two levels of inspired oxygen
tension. In addition, we compared Vc estimated from DLCO
measured at two inspired oxygen tensions with that estimated from
simultaneous measurements of DLNO and DLCO. If
our hypothesis is true, it should be possible to simultaneously
estimate DLNO, DMCO, Vc, and c at rest
or exercise without having to repeat the measurement at more
than one level of inspired oxygen tension.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Rebreathing Apparatus and Technique
The experimental apparatus has been described.6
7
Subjects exercised on a bicycle ergometer, breathing through two
three-way pneumatically controlled balloon valves (model 8500; Hans
Rudolph; Kansas City, MO). Airflow was measured by a turbine flowmeter
(VMM 2; Interface Associates; Aliso Viejo, CA). Oxygen uptake, carbon
dioxide production, ventilation, and heart rate were measured
continuously by a metabolic cart (Vmax 229; Sensormedics; Yorba Linda,
CA). At the end of a selected expiration, the pneumatic valves switched
to allow the subject to inspire to total lung capacity one breath of
test gas mixture from a reservoir bag containing 0.3% carbon monoxide,
0.3% methane, 0.8% acetylene, and either 30% oxygen in a balance of
nitrogen or 99% oxygen. When needed, medical-grade NO at a
concentration of 40 ppm was added to the test gas mixture just prior to
each measurement. After inspiring the test gas to total lung capacity,
a second valve switch allowed the subject to rebreathe in and out of an
anesthetic bag for 16 s while gas concentrations were continuously
monitored at the mouth. At rest, the rebreathing rate was set at 30
breaths/min synchronized by a metronome. During exercise, subjects were
allowed to breathe at their spontaneously chosen rate.
Concentrations of carbon monoxide, methane, and acetylene were measured by rapid-response infrared gas analyzers (Sensors; Saline MI) via a recirculating sampling system. The concentration of NO was measured by a chemiluminescence analyzer (model NOA280; Sievers Instruments; Boulder, CO); linearity and reproducibility of the analyzer were verified in the range from 0 to 400 ppm. The sampling flow rate was 200 mL/min. The response time of the analyzer was < 500 ms. The NO analyzer was calibrated daily using a zero NO filter and a test gas containing 26 ppm NO. Analog output of the NO analyzers was passed through a medium-gain, direct-current amplifier and digitized by a computer along with signals from the infrared analyzer, flowmeter, and the metabolic cart.
Calculation of Diffusing Capacities and c
Diffusing capacities for carbon monoxide or NO were calculated
from the slope of the exponential disappearance rate of each gas with
respect to methane during rebreathing. The first three end-tidal points
and points after approximately 12 s were routinely discarded to
avoid incomplete mixing and recirculation, respectively. The rate of
carbon monoxide uptake by erythrocytes (CO) was
calculated from the mean PAO2 during
rebreathing and Hb concentration1
8
:
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DMCO and Vc were calculated from DLCO
measured at the two levels of PAO2;
results were used to calculate DLCO at a standard Hb
concentration of 14.6 g/dL and a PAO2
of 120 mm Hg. All results of DLCO are expressed under these
standard conditions. Vc was also calculated from DLCO and
DLNO measured during a single rebreathing maneuver.
c was measured from the slope of the end-tidal exponential
disappearance of acetylene with respect to methane.9
Protocol
Measurements were conducted on two separate days. On day 1,
spirometry was measured. Maximal oxygen uptake was determined by an
incremental exercise protocol; workload was increased by 30 W every 2
min until volitional termination or until a plateau in oxygen uptake
with respect to workload was obtained. On day 2, the subject exercised
at workloads equivalent to 25%, 50%, and 80% of their predetermined
maximal oxygen uptake. Each workload was sustained for 3 min to reach a
quasisteady state in heart rate, respiratory rate, and oxygen uptake;
the rebreathing maneuver was then performed. Baseline measurements were
obtained at rest sitting on the bicycle. Between measurements, the
subject rested for 10 to 20 min or until heart rate and respiratory
rate returned to baseline. Measurements were repeated with 30% or 99%
oxygen in the test gas mixture corresponding to a mean
PAO2 during rebreathing of about 150
mm Hg and 600 mm Hg, respectively. Prior to rebreathing the test
mixture containing 99% oxygen, the subject prebreathed 100% oxygen
for 4 min at rest and 1 min during heavy exercise. All measurements
were performed first without NO in the test gas mixture, and then
repeated with 40 ppm of NO added to the reservoir bag. A venous blood
sample was drawn to measure Hb concentration.
Data Analysis
Diffusing capacities were normalized by body surface area and
analyzed with respect to cardiac index. The slope and correlation
coefficient of the DLNO vs cardiac index plot were
calculated for each subject and averaged for all subjects. The
dependence of DLNO on cardiac index, lung volume, age, body
surface area, height, and weight was examined by stepwise linear
regression analysis. The correlation between DLNO and
DLCO, between DLNO and DMCO, and
between DLNO measured at two levels of
PAO2 was determined. The
DLNO/DMCO and DLNO/DMCO
standard ratios at different workloads were compared by
repeated-measures analysis of variance. Statistical analysis was
performed using commercial software (Statview, version 4.5; SAS
Institute; Cary, NC).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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c, end-inspiratory lung volume, and age. Using age as an
independent variable accounts for more of the variance than height,
weight, or body surface area. The relationship between observed
DLNO and DLNO fitted to the
multivariate regression equation is shown in Figure 1
,
bottom.
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c (Fig 2
, top and bottom), closely matching previously
published reference values from our laboratory based on c, age,
and body surface area.6
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Effect of NO on DLCO and c
The brief exposure to 40 ppm NO during 16 s of rebreathing
does not significantly affect the measurement of DLCO or
c. Pooled data from all subjects at all workloads performed in
the presence or absence of NO in the rebreathing mixture show the
following correlations: DLCO (with NO) = 0.968 x
DLCO (without NO) [r2 =
0.93]. c (with NO) = 0.977 x c (without NO)
[r2 = 0.92].
Vc
Similarly, there is a close correlation between Vc (in
milliliters) estimated using the technique of Roughton and
Forster1 and that estimated from simultaneous measurements
of DLNO and DLCO: Vc
(DLNO-DLCO technique) = 1.04 x Vc
(Roughton-Forster technique)
[r2 = 0.74].
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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c; the slope of this relationship is remarkably
constant among subjects, while the variability in elevation can be
largely attributed to differences in lung volume and age. The
dependence of DLNO on c and lung volume is similar
to that of DLCO.6
10
The volume dependence of
DLNO is also consistent with previous reports at rest by
the single-breath technique.4
11
Our estimates of
DLNO by the rebreathing method correlate highly with
simultaneously measured DMCO at rest and during exercise;
these data support the use of DLNO as a direct measure of
membrane diffusing capacity. Although the average
DLNO/DMCO ratio is 29% higher than that
expected based on physical constants of NO and carbon monoxide alone,
the difference is in the wrong direction to be explained by a
significant erythrocyte resistance to NO uptake. Changing
PAO2 has no significant effect on
DLNO. Brief exposure to NO during rebreathing does not
alter DLCO or c.
Critique of Methods
The acetylene rebreathing method for estimating c has been
validated not only at rest but also during high-intensity exercise
against both dye dilution and direct Fick techniques in normal
subjects12
13
as well as exercising
animals.14
Different inspired NO concentrations have been
used to measure DLNO, ranging from 8 ppm in humans to 600
ppm in animals.3
Long-term exposure to high concentrations
of NO, such as for the treatment of respiratory failure, can
potentially reduce oxygen-carrying capacity of the blood through the
formation of methemoglobin, and the oxidation products of NO may cause
lung injury.15
Also, NO-mediated pulmonary vasodilatation
during physiologic increases in blood flow16
17
may alter
the physiologic parameters under investigation. Our data show that
brief exposures to NO (16 s) at an initial concentration of 40 ppm have
no toxic effects, do not alter c or DLCO, and allow
an adequate number of end-tidal points to be sampled for calculating
the slope of NO disappearance.
Comparison With Previous Measurements of DLNO
Previous measurements of DLNO in human subjects have
been conducted using the single-breath technique at rest. Guénard
et al4
reported a resting DLNO of 136 ± 32
mL/min/mm Hg (mean ± SD) with a breath-hold of 3 s and an
inspired NO concentration of 8 ppm. Borland and
Higenbottam2
reported a resting DLNO of
147 ± 31 mL/min/mm Hg (mean ± SD) with a breath-hold of 10 s
and an inspired NO concentration of 40 ppm. Moinard and
Guénard18
reported a resting DLNO of
124 ± 13 mL/min/mm Hg (mean ± SE) with a breath-hold of 3 s
and an inspired NO concentration of 8 ppm. Because of the faster
NO-uptake kinetics, the breath-hold time had to be reduced. In
comparison, our measurement of DLNO at rest by the
rebreathing technique (117 ± 7 mL/min/mm Hg, mean ± SE; range, 69
to 178 mL/min/mm Hg) is somewhat lower. The
DLNO/DLCO ratio of 4.0 in this study is similar
to the DLNO/DLCO ratio of 4.3 reported by
others.4
18
However, in previous studies,
DLCO was not expressed at a standard
PAO2 or Hb concentration.
Correlation Between DLNO and DMNO
If there was a significant erythrocyte resistance to NO uptake,
then the measured DLNO would underestimate true
DMNO, ie, the observed
DLNO/DMCO ratio would be
lower than the expected ratio of 1.93. We observed a
DLNO/DMCO ratio of 2.49,
29% higher than expected. A number of factors that could potentially
contribute to this difference are discussed below:
1. NO undergoes spontaneous transformation to nitrogen dioxide in the presence of oxygen. This chemical reaction could lead to an overestimation of DLNO, as the NO lost during conversion to nitrogen dioxide is measured as uptake by the pulmonary capillary blood. The reaction rate of NO to nitrogen dioxide is directly proportional to the square of NO concentration and to the concentration of oxygen.3 However, the rate of gas-phase oxidation of NO is slower by several magnitudes compared to the kinetics of NO uptake in the lungs.3 11 We added NO to the test gas mixture just before each measurement; thus, it seems unlikely that NO oxidation should be a significant factor affecting the measurement of DLNO.
2. Chemical transformation of NO can occur in the superficial lining fluid, bronchial mucosa, and other tissues of the respiratory system. This reaction is difficult to quantify, but both in vivo and in vitro studies using radiolabeled NO have shown that NO passes rapidly into the blood before reacting significantly with lung tissue.19 20 Spriestersbach et al,21 using isolated rabbit lungs perfused with buffer equilibrated with NO, also showed that diffusive uptake of NO is not affected by its reaction with lung tissue; the rate of reaction is much slower than the rate of diffusion across the alveolar-capillary membrane and hence should not affect estimates of DLNO.
3. Reversible gas exchange of NO occurs in the conducting airways.22 23 24 The NO diffusing capacity of the conducting airways calculated by Pietropaoli et al24 is in the range of 0.4 to 1.2 mL/min/mm Hg; these values are too small in magnitude (< 1%) to cause a major error in our measured DLNO.
4. NO is produced within the respiratory tract.22 23 24 Endogenous NO from the lung, airway, and nasal tissue could potentially cause an underestimation of DLNO, as it is not accounted for in the inspired NO concentration. The rate of endogenous NO production measured in spontaneously exhaled air is in the parts per billion (10-9 L/min) range.22 23 24 It is unlikely that such minute concentrations of NO would significantly affect our measurements, which were carried out in the (parts per million) range.
5. The expected DMNO/DMCO ratio of 1.93 is based on the solubility of NO in water with the implicit assumption that NO solubility in alveolar tissue and plasma is similar. The accuracy of this assumption is untested; thus, the expected ratio is only a rough approximation.
6. The accuracy of DMCO calculated using the
Roughton-Forster technique depends on the accuracy of the relationship
of 1/CO to intraerythrocyte oxygen tension. This
relationship varies among the studies in which it has been
measured,25
and could easily account for a 25%
underestimation of true DMCO. In addition, the calculation
of 1/CO assumes that mean
PAO2 within capillary erythrocytes is
the same as that in alveolar air; this assumption may also be in error.
Thus, errors in the assumed value of CO may well explain
the observed difference in DLNO/DMCO ratios by
the two techniques. Since the observed difference between
experimental and theoretical DLNO/DMCO ratios
is in the wrong direction to be caused by a significant erythrocyte
resistance to NO uptake, these data do not contradict the major tenet
underlying the combined DLCO-DLNO method,
ie, DLNO approximates the true
diffusing capacity of the alveolar-capillary membrane.
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Conclusion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Acknowledgements |
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Footnotes |
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c = cardiac output; Vc = pulmonary capillary blood
volume; CO = rate of carbon monoxide uptake This study was supported in part by American Heart Association grant No. 00–50274N.
Received for publication January 24, 2001. Accepted for publication June 4, 2001.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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where EILV = end-inspiratory lung volume at full
inspiration in liters, and age is in years. Solid line = identity;
dashed line = regression through data points.
