Dual Test Gas Pulmonary Diffusing Capacity Measurement During Exercise in Humans Using the Single-Breath Method

Summary

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (D_L,CO) and nitric oxide (D_L,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

Abstract

The combined single-breath measurement of the diffusing capacity of carbon monoxide (D_L,CO) and nitric oxide (D_L,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant's ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in D_L,NO and D_L,CO from rest to exercise at increasing intensities of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung's ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

Introduction

Exercise leads to a considerable increase in energy demand compared to the resting state. The heart and lungs respond by increasing cardiac output and ventilation resulting in an expansion of the alveolar-capillary bed, mainly the recruitment and distention of pulmonary capillaries¹. This ensures a sufficient pulmonary gas exchange, which can be measured by an increase in pulmonary diffusing capacity (D_L)^2,3,4. The first attempts to measure D_L during exercise date back more than a century^5,6,7. The ability to increase D_L from the resting state is often referred to as the alveolar-capillary reserve^{8, 9}.

Experimentally, the relative contributions of alveolar-capillary membrane diffusing capacity (D_M) and pulmonary capillary blood volume (V_C) to alveolar-capillary reserve can be assessed by different methods, including the classical multiple fractions of inspired oxygen () method¹⁰. An alternative technique that may be useful in this context is the dual-test gas method, in which the D_L to carbon monoxide (CO) and nitric oxide (NO) (D_L,CO/NO) are concurrently measured¹¹. This technique was developed in the 1980s, and takes advantage of the fact that the reaction rate of NO with hemoglobin (Hb) is substantially greater than that of CO, such that the pulmonary diffusion of CO depends more on V_C than does NO. Hence, the main site of resistance (~75%) to CO diffusion is located within the red blood cell, while the main resistance (~60%) to NO diffusion is at the alveolar-capillary membrane and pulmonary plasma¹². The concurrent measurement of D_L,CO and D_L,NO thus permits the assessment of the relative contributions of D_M and V_C to D_L¹², where the change in D_L,NO observed during exercise thus largely reflects the expansion of the alveolar-capillary membrane. An additional advantage of this method when obtaining measurements during exercise is that it involves a relatively short breath-hold time (~5 s) and fewer maneuvers compared to the classical technique, where multiple repeated maneuvers with a standardized 10 s breath-hold are performed at different oxygen levels. Although has recently been applied with a shorter breath-hold time and fewer maneuvers at each intensity¹³. Nevertheless, only permits a total of six D_L,CO maneuvers per session, whereas up to 12 repeated D_L,CO/NO maneuvers can be performed without any measurable effect on the resultant estimates¹⁴. These are important considerations when obtaining measurements during exercise since both a long breath-hold and multiple maneuvers may be difficult to perform at very high intensities or in patient populations who experience dyspnea.

The present paper provides a detailed protocol, including theoretical considerations and practical recommendations on the measurement of D_L,CO/NO during exercise and its use as an index of the alveolar-capillary reserve. This method is easily applicable in the experimental setting and permits the assessment of how diffusion limitation in the lungs may affect oxygen uptake in different populations.

Theory and measurement principles
The D_L,CO/NO method involves a single breath of a gas mixture with the assumption that the gases distribute equally in the ventilated alveolar space after inhalation. The gas mixture consists of several gases including an inert tracer gas. The dilution of the tracer gas in the ventilated alveolar space, as based on its fraction in end-expiratory air, can be used to calculate the alveolar volume (V_A)¹⁵. The gas mixture also includes the test gas CO and NO, both of which are diluted in the ventilated alveolar space and diffuse across the alveolar-capillary membrane. Based on their alveolar fractions, their individual rates of disappearance (k), also termed the diffusing constant, from the alveolar space can be calculated. By convention, the D_L for a test gas measured during a single-breath maneuver, is derived by the following equation¹⁶:

where F_A0 is the alveolar fraction of the test gas (CO or NO) at the onset of the breath-hold of the individual D_L maneuver, while F_A is the alveolar fraction of the test gas at the end of the breath-hold, and tBH is the breath-hold time. D_L is mechanically equivalent to the conductance of the test gas across the alveolar-capillary membrane, through plasma and the red blood cell interior to hemoglobin. It thus depends both on the conductance of D_M and the so-called specific conductance of pulmonary capillary blood (θ), of which the latter depends both on the conductance of the test gas in blood and on its reaction rate with hemoglobin¹⁰. Given that the reciprocal of conductance is resistance, the total resistance to the transfer of a test gas depends on the following resistances in series¹⁰:

These components may be distinguished by concurrently measuring the D_L to CO and NO, because these have different θ-values, and their respective D_L values thus depend differently on V_C. The pulmonary diffusion of CO depends more heavily on V_C than does NO, with the main site of resistance (~75%) to CO diffusion being located within the red blood cell¹². In contrast, the main resistance (~60%) to NO diffusion is at the alveolar-capillary membrane and pulmonary plasma, because the reaction rate of NO with hemoglobin is substantially greater than that of CO. Hence, by concurrently measuring D_L,CO and D_L,NO, changes in both D_M and V_C will markedly affect the former, while the latter will depend much less on V_C, thus permitting an integrative assessment of the factors that determine D_L.

The reporting of D_L,CO/NO metrics may be done using different units. Hence, the European respiratory society (ERS) uses mmol/min/kPa, whereas the American Thoracic society (ATS) uses mL/min/mmHg. The conversion factor between the units is 2.987 mmol/min/kPa = mL/min/mmHg.

Protocol

The Scientific Ethical Committee for the Capital Region of Denmark has previously approved the measurement of DL,CO/NO at rest, during exercise, and in the supine position in both healthy volunteers and patients with chronic obstructive pulmonary disease (COPD) at our institution (protocols H-20052659, H-21021723, and H-21060230). NOTE: Before DL,CO/NO is measured during exercise, a dynamic spirometry, and a cardiopulmonary exercise test (CPET) must be performed. The dyna…

Representative Results

The protocol was implemented in 2021 and at the time of writing a total of 124 measurements during exercise (i.e., 51 in healthy volunteers and 73 in patients with COPD of various severities) had been performed. The maneuvers, as well as data on fulfilled acceptability and repeatability criteria, and the failure rate are all provided in Table 3. Calculations As an example, calculations from a single DL,CO/NO maneuver are …

Discussion

The protocol provides a standardized approach to the measurement of D_L,CO/NO during exercise using the dual test gas single-breath technique. Since the obtained D_L,CO/NO-metrics increase due to pulmonary capillary recruitment and distension, the method provides a physiologically meaningful measure of the alveolar-capillary reserve.

Critical steps in the protocol
The method requires an exhalation to residual volume followed by an inspiration to total…

Materials

HemoCue Hb 201+	HemoCue, Brønshøj, Denmark	Unkown	For measurements of hemoglobin
Jaeger MasterScreen PFT pro (Lung Function Equipment)	CareFusion, Höchberg, Germany	Unkown	For measurements of DLCO/NO
Mouthpiece	SpiroBac, Henrotech, Aartselaar, Belgium	Unkown	Used together with the Lung Fuction Equipment. (dead space 56 ml, resistance to flow at 12 L s−1 0.9 cmH2O)
Nose-clip	IntraMedic, Gentofte, Denmark	JAE-892895
Phenumotach	IntraMedic, Gentofte, Denmark	JAE-705048	Used together with the Lung Fuction Equipment
SentrySuite Software Solution	Vyaire's Medical GmbH, Leibnizstr. 7, D-97204 Hoechberg Germany	Unkown
Test gasses	IntraMedic, Gentofte, Denmark	Unkown	Concentrations: 0.28% CO, 20.9% O2, 69.52% N2 and 9.3% He