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Lung Diffusing Capacities (DL) for Nitric Oxide (NO) and Carbon Monoxide (CO): The Evolving Story

Colin D.R. Borland, J. Mike B. Hughes

10.1002/cphy.c190001

Source: Volume 10, Issue 1, January 2020

Published online: December 2019

Full Article on Wiley Online Library



Abstract

Nitric oxide and carbon monoxide diffusing capacities (DLNO and DLCO) obey Fick's First Law of Diffusion and the basic principles of chemical kinetic theory. NO gas transfer is dominated by membrane diffusion (DM), whereas CO transfer is limited by diffusion plus chemical reaction within the red cell. Marie Krogh, who pioneered the single‐breath measurement of DLCO in 1915, believed that the combination of CO with red cell hemoglobin (Hb) was instantaneous. Roughton and colleagues subsequently showed, in vitro, that the reaction rate was finite, and prolonged in the presence of high . Roughton and Forster (R‐F) proposed that the resistance to transfer (1/DL) was the sum of the membrane resistance (1/DM) and (1/θVc), the red cell resistance (θ being the CO or NO conductance for blood uptake and Vc the capillary volume). From this R‐F equation, DM for CO and Vc can be solved with simultaneous NO and CO inhalation. At near maximum exercise, DMCO and Vc for normal subjects were 88% and 79%, respectively, of morphometric values. The validity of these calculations depends on the values chosen for θ for CO and NO, and on the diffusivity of NO versus CO. Recent mathematical modeling suggests that θ for NO is “effectively” infinite because NO reacts only with Hb in the outer 0.1 μM of the red cell. An “infinite θNO” recalculation reduced DMCO to 53% and increased Vc to 95% of morphometric values. © 2020 American Physiological Society. Compr Physiol 10:73‐97, 2020.

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Figure 1. Figure 1. Outline of apparatus for studying O2 kinetics in Hb solutions or red cells. Reagents were driven from pressure bottles by compressed N2 through the mixing chamber at a fixed flow and constant velocity (0.8 cm/ms) so that 1 cm = 1.25 ms along the observation tube. The reversion spectroscope was set up at intervals along the tube (multiple measurements being required), and the HbO2 ± HbCO composition, in a particular time frame, was determined from the position of the α band on two identical spectra mounted one above the other (with one reversed). Reused, with permission, from Hughes JMB and Bates DV, 2003 45.
Figure 2. Figure 2. Replotting by Kang and Sapoval 54 of the relationship between ΔLCO (∼DLCO,sb) from data of Forster et al. 27 and Borland and Higenbottam 15, showing a 55% to 60% decrease in ΔLCO from alveolar PO2 100 to 600 mmHg. Reused, with permission, from Kang M‐Y and Sapoval B, 2016 54.
Figure 3. Figure 3. The first published figure of the uptake of nitric oxide (NO) in relation to the simultaneous uptake of carbon monoxide (CO). Data collected from a series of breath holds (from 4 to 11 s) in one subject. Log alveolar concentrations plotted as % initial concentration at breath hold onset (t = 0) against breath hold times. From the slopes of gas disappearance, a rate constant (k) proportional to DLCO/VA and DLNO/VA has been calculated. Since DLCO (or DLNO) = k × VA (and VA is common to both), the ratio kNO/kCO is identical to the ratio DLNO/DLCO. Reused, with permission, from Borland CD and Higenbottam TW, 1989 15.
Figure 4. Figure 4. Diffusing capacity for NO (DLNO) in individual animals (closed symbols, dashed lines) rose significantly after successive exchange transfusion with Oxyglobin solution compared to baseline at 0 mL/kg (p < 0.05 by repeated measures ANOVA), while DLCO in each animal (open symbols, solid lines) remained unchanged. Reused, with permission, from Borland CD, et al., 2010 14.
Figure 5. Figure 5. Circuit for measurement of nitric oxide diffusing capacity (DNO) and carbon monoxide diffusing capacity (DCO) under standard conditions using double membrane oxygenators (above) and deoxygenators (below). Closure of the forceps (X) stops perfusion and ventilation to half the oxygenator. Reused, with permission, from Borland CD, et al., 2006 13.
Figure 6. Figure 6. Membrane oxygenator (see text). Resistance to CO transfer (1/DCO) increases as PO2 increases, but there is no change in NO transfer resistance (1/DNO). Reused, with permission, from Borland CD, et al., 2006 13.
Figure 7. Figure 7. Membrane oxygenator. Change in DNO and DCO with added nitrite, converting Hb to methemoglobin. See text for explanation. Reused, with permission, from Borland C, et al., 2014 4.
Figure 8. Figure 8. Cross‐sectional diagram of diffusion cell for measuring NO permeability and diffusivity in blood elements. The liquid cell contained plasma or nitrited red cells (to block uptake by hemoglobin itself) enclosed by silicone (silastic) membranes, maintained in position by a porous stainless steel scaffolding. Reused, with permission, from Borland C, et al., 2018 10.
Figure 9. Figure 9. (A) The estimated fall in NO concentration in the continuous flow rapid reaction apparatus (CFRRA) from the surface of the red cell to the interior as measured by its half thickness [0.8 μM 14]. (B) Similar for CO during a single‐breath DLCO. (C) Similar for NO during a single‐breath DLNO. The [Hb]/[NO] molar concentration ratio was 1.0 for (A), but 3.5 × 105 for (C). Thus, the excess in Hb molecules in (C) restricts incoming NO to the outer red cell layers. See text for further explanation. Reused, with permission, from Borland C, et al., 2017 11.


Figure 1. Outline of apparatus for studying O2 kinetics in Hb solutions or red cells. Reagents were driven from pressure bottles by compressed N2 through the mixing chamber at a fixed flow and constant velocity (0.8 cm/ms) so that 1 cm = 1.25 ms along the observation tube. The reversion spectroscope was set up at intervals along the tube (multiple measurements being required), and the HbO2 ± HbCO composition, in a particular time frame, was determined from the position of the α band on two identical spectra mounted one above the other (with one reversed). Reused, with permission, from Hughes JMB and Bates DV, 2003 45.


Figure 2. Replotting by Kang and Sapoval 54 of the relationship between ΔLCO (∼DLCO,sb) from data of Forster et al. 27 and Borland and Higenbottam 15, showing a 55% to 60% decrease in ΔLCO from alveolar PO2 100 to 600 mmHg. Reused, with permission, from Kang M‐Y and Sapoval B, 2016 54.


Figure 3. The first published figure of the uptake of nitric oxide (NO) in relation to the simultaneous uptake of carbon monoxide (CO). Data collected from a series of breath holds (from 4 to 11 s) in one subject. Log alveolar concentrations plotted as % initial concentration at breath hold onset (t = 0) against breath hold times. From the slopes of gas disappearance, a rate constant (k) proportional to DLCO/VA and DLNO/VA has been calculated. Since DLCO (or DLNO) = k × VA (and VA is common to both), the ratio kNO/kCO is identical to the ratio DLNO/DLCO. Reused, with permission, from Borland CD and Higenbottam TW, 1989 15.


Figure 4. Diffusing capacity for NO (DLNO) in individual animals (closed symbols, dashed lines) rose significantly after successive exchange transfusion with Oxyglobin solution compared to baseline at 0 mL/kg (p < 0.05 by repeated measures ANOVA), while DLCO in each animal (open symbols, solid lines) remained unchanged. Reused, with permission, from Borland CD, et al., 2010 14.


Figure 5. Circuit for measurement of nitric oxide diffusing capacity (DNO) and carbon monoxide diffusing capacity (DCO) under standard conditions using double membrane oxygenators (above) and deoxygenators (below). Closure of the forceps (X) stops perfusion and ventilation to half the oxygenator. Reused, with permission, from Borland CD, et al., 2006 13.


Figure 6. Membrane oxygenator (see text). Resistance to CO transfer (1/DCO) increases as PO2 increases, but there is no change in NO transfer resistance (1/DNO). Reused, with permission, from Borland CD, et al., 2006 13.


Figure 7. Membrane oxygenator. Change in DNO and DCO with added nitrite, converting Hb to methemoglobin. See text for explanation. Reused, with permission, from Borland C, et al., 2014 4.


Figure 8. Cross‐sectional diagram of diffusion cell for measuring NO permeability and diffusivity in blood elements. The liquid cell contained plasma or nitrited red cells (to block uptake by hemoglobin itself) enclosed by silicone (silastic) membranes, maintained in position by a porous stainless steel scaffolding. Reused, with permission, from Borland C, et al., 2018 10.


Figure 9. (A) The estimated fall in NO concentration in the continuous flow rapid reaction apparatus (CFRRA) from the surface of the red cell to the interior as measured by its half thickness [0.8 μM 14]. (B) Similar for CO during a single‐breath DLCO. (C) Similar for NO during a single‐breath DLNO. The [Hb]/[NO] molar concentration ratio was 1.0 for (A), but 3.5 × 105 for (C). Thus, the excess in Hb molecules in (C) restricts incoming NO to the outer red cell layers. See text for further explanation. Reused, with permission, from Borland C, et al., 2017 11.
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Teaching Material

Colin D.R. Borland, J. Mike B. Hughes, Lung Diffusing Capacities (DL) for Nitric Oxide (NO) and Carbon Monoxide (CO): The Evolving Story. Compr Physiol 10 : 2020, 73-97.

Didactic Synopsis

Major Teaching Points:

  1. In the lung, the nitric oxide (NO) and carbon monoxide (CO) diffusing capacities (DLNO and DLCO) are measured with a single inhalation of NO and CO gases, followed by a 5s or 10s breath hold, exhalation and alveolar gas sampling.
  2. DLNO is 4.5–5.0 times DLCO. The main reason is that NO reacts x 280 times faster with red cell hemoglobin (Hb) than does CO. NO also diffuses through tissues and plasma approximately twice as fast as CO.
  3. As resistances (reciprocal of conductance) the Roughton–Forster equation states 1/DL = 1/DM + 1/?Vc, where 1/DM is the membrane resistance and 1/?Vc the specific resistance of blood combining with CO or NO; ?is the rate of uptake of a specified gas by hemoglobin per mL of blood per mmHg gas partial pressure (a specific conductance) and Vc is capillary blood volume.
  4. The ratio DMNO/DMCO (~ tissue-plasma diffusivity) is 1.97, and for ?NO/?CO in normoxia 8.01. These ratios are not set in stone, and are currently being reassessed.
  5. DLNO is weighted towards the membrane conductance (DM), the purely diffusive pathways from the alveolar lining layer to the interior of the red cell: the DLCO, on the other hand, is more weighted towards the number of red cells (and their Hb content) in the pulmonary capillary bed.
  6. Thus, the DLNO/DLCO ratio is related to the DM/Vc ratio.
  7. A higher than normal DLNO/DLCO ratio, in relation to local controls, is associated with pulmonary vasculopathies, and a lower than normal DLNO/DLCO with alveolar destruction, either interstitial fibrosis or emphysema. There is overlap; more studies are awaited.

Didactic Legends

The following legends to the figures that appear throughout the article are written to be useful for teaching.

Figure 1. Teaching points. Diagram of the method (continuous flow rapid reaction apparatus, CFRRA) used, almost 100 years ago, to show, for the first time, that the combination of hemoglobin [Hb] (either free in solution or packaged within a red cell) with oxygen or carbon monoxide was not "instantaneous", but took a finite time which could be measured. Briefly, in the case of oxygen (O2) uptake velocity, a red cell or Hb solution, oxygen–free and very dilute (flask K), and O2 dissolved in water (flask J) were forced together in a rotating mixing chamber (design based on a car carburetor!), and then down a narrow tube of fixed dimensions at a known rapid rate, such that each cm of travel corresponded to a finite time (in ms) from the moment of combination. The reversion spectroscope measured, in a complex way, the fraction of O2 (or CO) combined with Hb. By moving the spectroscope, for successive "runs" or "mixes", to different parts of the observation tube, a plot of HbO2% or HbCO% against time (in ms) could be drawn. The reversion spectroscope measured HbO2 or HbCO spectra (the ? band) in duplicate, one spectra being reversed, so the observer could, with a micrometer scale, superimpose them — the sum of the shifts doubling the accuracy of the measurement.

Figure 2. Teaching points. The diffusing capacity for carbon monoxide (?LCO = DLCO) has units of conductance (= "ease of transfer"). This conductance is "hindered" (?LCO falls) when the oxygen pressure in the lung air sacs (alveoli) — normally 100 mmHg — is increased experimentally. Individual subject data shown for the first published study (1959) and group data for a later study (1989). The reason for the O2 "hindrance" is competition between O2 and CO for combination sites on the Hb molecules within red cells. Molecular diffusion from alveoli to the Hb molecule is not affected by O2 partial pressure.

Figure 3. Teaching points. When a rapid inhalation brings carbon monoxide (CO) or nitric oxide (NO) gas, in tracer quantities, into contact with the blood–gas barrier in the alveoli, their uptake as a function of time (during a breath hold) is monoexponential (note log scale on the y–axis). Each point represents the alveolar concentration (as % its concentration at breath hold = zero) expired after a defined period of breath holding. Note the NO disappearance rate is x 5 that of CO.

Figure 4. Teaching points. These experiments, in anesthetized dogs, show that replacing red cells (with their Hb molecules) with a cell-free artificial hemoglobin (oxyglobin) in solution, by means of progressive exchange transfusions (total blood hemoglobin remaining about the same), leads to an increase in NO uptake from the lung (DLNO), but no increase in DLCO. This shows that the packaging of hemoglobin molecules within red cells increases the resistance or "hindrance" to NO uptake. The uptake of CO by red cells is much slower (about eightfold) than for NO, so easier "access" makes no difference to the DLCO.

Figure 5. Teaching points. Circuit and apparatus for measuring the uptake of NO and CO (for the derivation of DNO and DCO) in vitro under "controlled" conditions, by substituting two pairs of membrane oxygenators (as used in heart–lung machines), and deoxygenators — one pair acting as a "lung" and the other pair as "systemic tissue" oxygen consumers. Closure of the forceps (X) stops airflow and blood flow to half the oxygenator. Figures 6 and 7 show different applications of this in vitro gas exchange system.

Figure 6. Teaching points. The membrane oxygenator (Figure 5) shows, under strictly controlled conditions, that resistance to CO uptake (1/DCO has units of resistance) increases as PO2 increases, just as in Figure 2 its reciprocal (DLCO, a conductance) decreases as PO2 increases. There is no PO2 effect on NO transfer because NO affinity for Hb binding far outweighs that of oxygen.

Figure 7. Teaching points. Converting Hb (as oxyhemoglobin) to methemoglobin (metHb) by adding sodium nitrite to the blood in the membrane oxygenator (Figure 5) reduces DNO and DCO because NO and CO cannot combine with the metHb molecule. But, for NO, the explanation may be more subtle. After metHb formation and fall of DNO, induction of hemolysis with water (not shown in the graph) caused DNO to rise again, although metHb concentration in the blood had not changed. This suggests that metHb forms predominantly in the outer layers of the red cell, hindering NO access to Hb in the interior. This "access" was restored by releasing HbO2 from the interior of the red cells by hemolysing them.

Figure 8. Teaching points. Apparatus for measuring the permeability and diffusivity of nitric oxide in plasma or red cells, under controlled conditions, in a liquid cell or chamber, enclosed by silicone (silastic) membrane. The liquid cell contained plasma or nitrited red cells — nitrited to prevent NO uptake by Hb, since the aim of the study was to measure molecular diffusion, i.e. the membrane conductance for NO (~ DMNO).

Figure 9. Teaching points. Calculations in panel A, from in vitro kinetics based on CFRRA experiments (see Figure 1), of concentration profiles from surface to centre of a red cell rapidly exposed to NO. In panel C, the profile is calculated for a simulated single breath test, as in vivo. In C, the uptake is more rapid than A, and entirely in the outer layers of the cell; the reason is that the NO concentration (in moles) is more than a thousand fold less in C versus A with a Hb concentration in C greatly in excess of NO concentration. Thus, all the NO in C is rapidly consumed in the outermost layers of the cell. This does not happen in the case of CO in the single breath simulation (B) because CO reacts with Hb molecules very much more slowly, or in A because NO molecules are as abundant as Hb molecules.

 


Related Articles:

Diffusion of Gases Across the Alveolar Membrane
Diffusing‐Capacity Heterogeneity
Carbon Monoxide Transport and Actions in Blood and Tissues
History of Respiratory Gas Exchange
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Article Title: Lung Diffusing Capacities (DL) for Nitric Oxide (NO) and Carbon Monoxide (CO): The Evolving Story
 

How to Cite

Colin D.R. Borland, J. Mike B. Hughes. Lung Diffusing Capacities (DL) for Nitric Oxide (NO) and Carbon Monoxide (CO): The Evolving Story. Compr Physiol 2019, 10: 73-97. doi: 10.1002/cphy.c190001