pulmonary capillary blood volume and alveolar-membrane diffusing capacity are components to overall pulmonary diffusing capacity. The equation deriving pulmonary diffusing capacity into these components was first published in 1957 by Francis Roughton and Robert Forster (19) in which the total resistance to pulmonary diffusion is the addition of alveolar-membrane resistance and red blood cell resistance placed in series, such that where, DL is the overall diffusing capacity of the lung, DM is the true alveolar-membrane diffusing capacity separating the alveolar air from the blood, Vc is the total volume of blood in the pulmonary capillaries exposed to alveolar air, and Θ is the number of milliliters of gas taken up by red blood cells in 1 ml of blood per 1 mmHg gradient of partial pressure of dissolved gas between the plasma and the interior of the red blood cell. While technically difficult to measure pulmonary diffusing capacity for oxygen, carbon monoxide (CO) is most commonly used as a surrogate of oxygen transfer. Therefore, DL and DM are reported as DMCO (alveolar-membrane diffusing capacity for CO) and DLCO (pulmonary diffusing capacity for CO) while Θ is reported as ΘCO (the blood transfer conductance for CO). According to the Roughton and Forster equation (19), the resistance of the red blood cell to the uptake of CO is about equal to the resistance of the alveolar-capillary membrane to the diffusion of gas across it.
To obtain DMCO and Vc, DLCO has been traditionally measured at two different levels of alveolar Po2 (PaO2), e.g., at ∼100–120 mmHg and ∼600 mmHg. For each PaO2 level, 1/DLCO is plotted on the y-axis and 1/ΘCO is plotted on the x-axis. A line is drawn through the two points and the y-intercept (1/DMCO) and slope (1/Vc) can be solved. The formula for 1/ΘCO varies across studies, but the most predominant formula used in studies today are from the original Roughton and Forster paper or a modification of this formula (12, 19).
However, there are at least three main technical issues with this traditional two-step method in obtaining DMCO and Vc. First, at least four tests are needed (2 at low and 2 at high PaO2) to obtain a reliable measure of DMCO and Vc. This places considerable time restraint in a clinical setting and increases patient effort. Second, with the traditional method, the CO gas distribution in the lungs may be different at two inspirations at two different oxygen tensions, affecting DMCO and Vc. Third, with the standard method, cardiac output may vary between measurements of DLCO at different oxygen tensions, which then have to be interpolated to obtain DLCO at the two oxygen tensions at the same cardiac output. This would also affect DMCO and Vc.
In 1983, Colin Borland and his colleagues (3) examined the fate of inhaled NO, as it was an important component of cigarette smoke. They determined that nitric oxide (NO) uptake behaved similarly to CO uptake such that the ratio of pulmonary diffusing capacity for NO (DLNO) to DLCO was ∼4.6, and concluded inhaled NO does not readily convert to nitrogen dioxide (NO2), a toxic gas (11, 21).1 Later, in 1987, Hervé Guénard et al. (13) made the assumption that since the reaction on NO with hemoglobin is effectively infinite, the blood transfer conductance for NO (ΘNO) must also be infinite (13). Therefore, as the diffusivity of NO is about twice that of CO (DLNO = DMNO ≈ 2 DMCO), Guénard assumed that DMCO and Vc could be calculated using a one-step maneuver in which both CO and NO are inhaled together. This was an ingenious idea. Hence, all the technical issues with the traditional Roughton and Forster two-step method are avoided with the new modified, one-step DLNO-DLCO technique. Since 1987, most studies that use this modified technique have assumed that ΘNO approaches infinity.
Nonetheless, a disagreement soon arose whether DLNO = DMNO (2, 6). Colin Borland and colleagues believed that ΘNO was less than finite, at ∼4.5 ml NO·(ml blood·min·mmHg)−1. This was derived from a value obtained in vitro with human red blood cells in 1958 in unphysiological conditions (8). In 2006, Borland and colleagues used a membrane oxygenator as a model for NO and CO transfer (5). Their membrane oxygenator tests various factors that affect NO and CO in physiological conditions that would be impossible to do in vivo or in an isolated lung preparation. The authors postulated that if DLNO = DMNO then hemolysis (which eliminates red blood cell resistance) would not alter DLNO. However, hemolysis actually increased DNO, providing evidence that there is significant red blood cell diffusive resistance to NO (i.e., DNO<DMNO). A limitation of this study was that the results were neither obtained in human blood nor were they obtained in vivo.
In this issue of the Journal of Applied Physiology, Colin Borland and colleagues (7) go one step further in that they use both in vitro (membrane oxygenator perfused with whole blood) and in vivo (foxhounds) data directly to examine whether ΘNO was infinite. They postulated if that there was significant red blood cell resistance to NO, both in vitro DNO and in vivo DLNO would increase progressively as red blood cells were replaced with cell-free heme-based blood substitute (cell-free hemoglobin-based oxygen carrier). Both in vitro and in vivo replacement of whole blood with hemolyzed blood and a cell-free hemoglobin solution (see Fig. 1) caused DLNO to progressively increase, implying that DLNO<DMNO. The in vitro and in vivo data demonstrate a finite ΘNO of ∼4.5 ml NO·(ml blood·min·mmHg)−1, similar to data from 1958 (8). Borland and colleagues further demonstrate that the overall resistance to pulmonary NO uptake (1/DLNO) is 63% alveolar-capillary membrane resistance (1/DMNO) and 37% red blood cell resistance (1/ΘNO·Vc) (7).
What are the clinical implications of this study? First, although ΘNO is finite, DLNO does not need to be adjusted unless [Hb] is <8 g/dl (7). Therefore, in routine clinical practice and in research studies involving humans, ΘNO can still be assumed to be infinite and DLNO can be “clinically” equal to DMNO. In fact, altering [Hb] in humans does not alter DLNO (23). Second, to ensure internal consistency, it is recommended that future studies using combined DLNO and DLCO report the absolute values for alveolar oxygen pressure, hemoglobin concentration, and the formula for ΘCO2 so that, if need be, DM and Vc can be recalculated.
The use of DLNO may have possible health implications. Both DLCO and DLNO at rest are related to aerobic capacity (26), a strong independent predictor of death in women (14) and men (16). Thus a measurement of DLNO could be a prognostic marker for mortality in several patient populations. DLCO and DLNO are also sensitive indicators of the morphological changes assessed with computed tomography to detect emphysema and cystic fibrosis (10, 22). But as with aerobic capacity (26), DLNO seems to be better compared with DLCO to detect emphysema and cystic fibrosis (10, 22). Furthermore, individuals who have a high DM-to-Vc ratio (which is proportional to the DLNO-to-DLCO ratio) may indicate high pulmonary artery pressure compatible with pulmonary hypertension/occlusion (1, 4, 15, 24). However, in a wide spectrum of pulmonary vascular diseases, such as sarcoidosis (18) or systemic microangiopathy brought on by type 2 diabetes (9), there would be a disproportionate reduction in DM relative to Vc decreasing the DM-to-Vc ratio (and thus decreasing the DLNO-to-DLCO ratio). Roughton and Forster indicate that there is an interdependence of DM and Vc in diseases of the pulmonary vascular bed (19). Any change in the vascular bed that decreases Vc will have an obligate reduction in surface area and therefore DM. When pulmonary vascular disease results in a reduction of the radius of all capillaries throughout the lung, Vc will decrease more than DM, because capillary volume is a function of radius squared while surface area is a function of the radius (17). Therefore, a future research direction would be to determine which patient population(s) would benefit by assessing DLNO and DLCO together such that diagnosis, treatment, and outcome would be better than just obtaining DLCO itself. For example, spirometry is related to perinatal outcomes (20), thus DLNO and/or DLCO may also be related too (25), a possibility that has not yet been tested. Individuals such as Colin Borland, Connie Hsia, Hervé Guénard, Ivo van der Lee, and I have already made great strides in the clinical and basic science research of DLNO. Let's press on.
↵1 Other studies have determined that NO2 is formed at a rate of ∼0.02 ppm/s (∼1.2 ppm NO2/min) in a gas mixture containing 21% oxygen and 60 ppm NO. Therefore, the production of NO2 is negligible provided care is taken to prevent the mixture of NO with oxygen until immediately prior to inhalation.
↵2 Currently there are about eight different equations that describe the reaction rate of CO with hemoglobin (ΘCO) for humans, so reporting the equation for ΘCO in research studies would aid in between-study comparison.
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