A small amount of inhaled nitric oxide does not increase lung diffusing capacity

The equation of the diffusing capacity of the lung for carbon monoxide (D_L,CO) has been classically described as: where D_m,CO represents pulmonary membrane diffusing capacity for carbon monoxide (CO) and ΘCO is the specific blood transfer conductance for CO. V_c represents pulmonary capillary blood volume 1. Membrane resistance (1/D_m,CO) and red cell resistance (1/ΘCO·V_c) usually contribute equally to the overall diffusive resistance across the lung 2, although this has been debated 3. To obtain D_m,CO and V_c, D_L,CO has been traditionally measured at two different levels of alveolar oxygen tension (P_A,O2), ∼13.3–16.0 kPa (∼100–120 mmHg) and ∼79.8 kPa (∼600 mmHg). For each level of P_A,O2, 1/D_L,CO is then plotted on the y-axis and 1/ΘCO is plotted on the x-axis. A line is then drawn through two points and the x-intercept (1/D_m,CO) and slope (1/V_c) can be solved. This two-step method can be time consuming and uncomfortable to perform, especially during exercise.

However, over the past 15 yrs, the measurement of diffusing capacity of the lung using the transfer gases nitric oxide (NO) and CO together permits one to obtain D_m,CO and V_c in a single-breath manoeuvre, thus allowing a similar distribution of the two gases and reducing the number of measurements and testing time by half 4, 5. The velocity constant of the combination of NO with haemoglobin is about 280 times faster than that of CO 6, and thus the specific blood transfer conductance for NO (ΘNO) is so large that the red cell resistance to NO (1/ΘNO) approaches zero 7. Therefore, diffusing capacity of the lung for nitric oxide (D_L,NO) equals the membrane diffusing capacity for NO (D_m,NO), and is independent of V_c and haemoglobin concentration 8. Others have made the same assumption that (1/ΘNO) is negligible 9–14, and it was recently determined that a nonzero 1/ΘNO would not be able to explain their experimental data 12. Therefore, these data suggest that D_L,NO is a good measure of D_m,NO. Given that the molecular weight of CO and NO are 28 and 30 g·M⁻¹, respectively, and solubilities (Bunsen coefficients) of CO and NO in plasma at 37°C are 0.0215 and 0.0439 15, respectively, the diffusivity of NO, which is the solubility of NO divided by the square root of the molecular weight (MW) of NO (), is ∼1.97 times greater than that of CO and, thus, the theoretical factor between membrane diffusing capacities for NO and CO is: Indeed, the solubility of either gas will depend upon the composition of the fluid that the gas has to diffuse through. If the fluid changes composition, the relative solubilities may well be different pre- and post-exercise. Nevertheless, data obtained from sick and healthy patients performing rebreathing manoeuvres suggest that the actual ratio of D_L,NO to D_m,CO is ∼2.42 11, 12. The larger ratio may be due to a higher than assumed NO solubility in plasma as well as NO facilitated diffusion 12.

Several researchers have previously obtained D_L,NO from single-breath 4, 5, 9, 10, 13, 14, 16–19 or rebreathing manoeuvres 11, 12, along with the simultaneous measurement of D_L,CO to obtain D_m,CO and V_c. Since brief exposure to NO does not interfere with physiological function 11, 12, 20, 21, it seems pertinent to use NO as a test gas to assess lung diffusion capacity. However, there is still debate as to whether inhalations of high NO concentrations (∼40–50 ppm) during sequential single-breath manoeuvres can affect the pulmonary microcirculation. At those NO concentrations, pulmonary vasodilation may occur, increasing D_L,CO, V_c and perhaps D_L,NO (D_m,CO) 22. Therefore, the first objective of the present study was to determine whether five sequential single breath-hold manoeuvres inhaling ∼40 ppm of NO increase D_L,CO, D_L,NO (and thus D_m,CO) and V_c. The current hypothesis was that five repeated inhalations of 40–50 ppm of NO would not increase D_L,CO, D_L,NO (D_m,CO) or V_c.

Furthermore, the recent 2005 American Thoracic Society (ATS)/European Respiratory Society (ERS) task force guidelines of standardisation of the single-breath determination of CO uptake in the lung 23 mentioned that more research is needed to determine the actual number of tests required to provide a reasonable estimate of the average D_L,CO. Therefore, the second objective of the current study was to determine the actual number of tests required to provide a reasonable estimate of not only D_L,CO, but D_L,NO (D_m,CO) and V_c for a given person. The present authors' hypothesis was that an average of three measurements would be needed to obtain a reasonable estimate of all those parameters.

The third objective was to determine the repeatability of lung diffusing capacity and its components over five trials in a given patient using the newer single-breath method of CO and NO. This would help to decide whether a change in an observation of D_L,NO, D_L,CO, D_m,CO and V_c represents a real clinical change in the pulmonary system or just measurement error. The current authors' hypothesis was that the repeatability for D_L,NO, D_L,CO and D_m,CO would be 10, 2 and 6 mL·min⁻¹·mmHg⁻¹, respectively, and 10 mL for V_c.

METHODS

RESULTS

Of the 31 subjects, 29 had normal spirometry function (table 1⇓) based on the fact that FEV₁ was >80% pred for all subjects, and the FEV₁/FVC was >0.7 for all but two subjects (FEV₁/FVC 0.67 and 0.68, respectively). The single breath-hold manoeuvres, on the whole, were performed adequately (table 2⇓). Breath-hold time was consistent within 0.3 s for all five trials, and the average inspired volume was always >90% of the FVC. The average alveolar volume was also maintained within 0.2 L for all trials.

The data show that D_L,NO and, thus, D_m,CO remained unaltered from trial one to trial five (table 3⇓). However, compared with the first trial, D_L,CO and V_c significantly decreased by the fourth (−4±5%; 95% confidence interval (CI) −5– −2%; p<0.05) and third trial (−5±7%; 95% CI −7– −2%; p<0.05), respectively. When D_m,CO = D_L,NO/2.42, the per cent predicted based on age, height and sex was 111±31% (range 62–164%; p<0.05), but when D_m,CO = D_L,NO/1.97, the per cent predicted was 136±39% (72–202%; p<0.05). For V_c, when D_m,CO = D_L,NO/2.42 the per cent predicted based on height and sex was 116±19% (76–155%; p<0.05), but when D_m,CO = D_L,NO/1.97 the per cent predicted for V_c was 100±16% (66–131%; p>0.05). Therefore, when D_m,CO = D_L,NO/2.42, the D_m,CO is reduced closer to normative values and the V_c is increased above normative values. The opposite occurs when D_m,CO = D_L,NO/1.97 as the D_m,CO is largely increased above the predicted value, but V_c is reduced to 100% pred.

The measurement error and repeatability of five trials are presented in table 4⇓. The repeatability represents the critical value in which a clinically measurable change in a given patient occurs between sessions. From the repeated measures ANOVA, a real clinical change in a patient's pulmonary diffusion capacity and its components was 17.2 and 3.2 mL·min⁻¹·mmHg⁻¹ for D_L,NO and D_L,CO, respectively. When D_m,CO = D_L,NO/2.42, a real clinical change for D_m,CO and V_c were 7.1 mm·min⁻¹·Hg⁻¹ and 13 mL, respectively. When D_m,CO = D_L,NO/1.97, a real clinical change for D_m,CO and V_c was any change >8.7 mL·min⁻¹·mmHg⁻¹ and 9.8 mL, respectively.

The variables age, weight and height were examined by forward stepwise multiple regression to predict D_L,NO values at rest. The only variable that appreciably affected D_L,NO was height. Predicted D_L,NO in mL·min⁻¹·mmHg⁻¹ = 2.0164×height in cm–175.63 (r² = 0.39; see = 23.3). Adding the other two variables to the equation did not increase the coefficient of determination significantly. Therefore, D_L,NO at rest was best predicted by height.

DISCUSSION

The present study showed that repeated inhalations of 40–50 ppm NO during single breath-hold manoeuvres does not increase D_L,NO, D_m,CO, D_L,CO or V_c. In fact, five sequential breath-hold manoeuvres did not change D_L,NO and, thus, D_m,CO. However, D_L,CO significantly decreased by the fourth trial, and V_c significantly decreased by the third trial. The drop in D_L,CO by the fourth trial due to progressive increase in carboxyhaemoglobin was similar to that reported elsewhere 33. As such, the data show that the actual number of tests required to provide a reasonable estimate of D_L,NO, D_L,CO, D_m,CO and V_c during properly performed manoeuvres is two trials. The average of the two trials should then be reported. Any more than two properly performed manoeuvres will lower V_c, and more than three will lower D_L,CO. Therefore, two properly performed manoeuvres are sufficient to obtain all the components of lung diffusion capacity using the D_L,NO–D_L,CO method.

There has been some concern that NO may affect the pulmonary microcirculation due to its vasoactive properties 22. One study showed that there is a small nonsignificant increase in D_L,CO in the presence of NO 17. However, the current authors have shown that brief exposure to NO does not interfere with physiological function. Other human 11, 12, 20, 21 and animal studies 34 are in agreement with the present study. Rebreathing 20–40 ppm NO over 16 s to 10 min resulted in no significant changes to oxygen uptake, arterial oxygen tension, alveolar–arterial oxygen tension difference, D_L,CO, D_m or V_c in sick and healthy subjects at rest or during exercise 11, 12, 21. In mechanically ventilated rabbits, D_L,NO values remained unchanged despite inspiratory NO concentrations varying from 10–800 ppm 34. Furthermore, the pulmonary toxicity of inhaled NO at concentrations of ∼40 ppm over a prolonged period of time is minimal 35.

There is concern that different P_A,O2 may slightly alter D_L,NO 36. However, more recent data has shown that varying the P_A,O2 in those with or without pulmonary disease does not affect D_L,NO 11, 12, implying that the combined D_L,NO–D_L,CO method can be used in hypoxaemic patients. Therefore, taken together, there is no reason to refrain from using NO and CO concurrently as a test gas to assess lung diffusion capacity.

The clinical implication of using both NO and CO concurrently in research and medical practice is that scientists and clinicians can immediately partition and quantify the components of lung diffusing capacity in a subject from a single 4-s breath-hold manoeuvre that requires minimal effort on the part of the patient, while simultaneously being able to pinpoint which component (V_c, D_m) is causing low (or high) total lung diffusion capacities. The ability to estimate D_m,CO and V_c from one-step simultaneous measurement of D_L,NO and D_L,CO represents significant conceptual advantages. One conceptual advantage of the D_L,NO technique is that with the standard Roughton–Forster method, cardiac output can vary between measurements of D_L,CO at different O₂ tensions, which then have to be interpolated to obtain D_L,CO at two O₂ tensions, but at the same cardiac output 12. With the D_L,NO method, all measurements are obtained at the same cardiac output and O₂ tension; no interpolation is necessary. Another conceptual advantage is that with the traditional method, the distribution of CO gas in the lungs may be different at two different O₂ tensions, but with the D_L,NO method only one inspiration is required, which results in a similar distribution of NO and CO gases. A third conceptual advantage is that with the traditional method, there is systematic underestimation of V_c and an overestimation of D_m since the inspiration at two different O₂ tensions affects alveolar–capillary membrane diffusion 37. The D_L,NO–D_L,CO method avoids this error altogether and should improve the accuracy of estimated D_m,CO and V_c.

The ratio D_m,CO = D_L,NO/2.42 gives a better estimate of Dm. When the present measured values were compared against the published norms, using the traditional two-step Roughton–Forster method 25, the predicted D_m,CO was 136% pred when D_m,CO = D_L,NO/1.97, but only 111% pred when D_m,CO = D_L,NO/2.42. However, the better per cent predicted values for V_c occurred when D_m,CO = D_L,NO/1.97. Nonetheless, since the traditional Roughton–Forster method underestimates V_c, the current authors feel that the predicted Vc values by Zanen et al. 25 slightly underestimate V_c. Therefore, the best compromise is to estimate D_m and V_c from a single breath using the D_L,NO–D_L,CO method and the formula D_m,CO = D_L,NO/2.42.

It was also important to clarify the repeatability of lung diffusion capacity and its components over five trials from the single-breath D_L,NO–D_L,CO method. The repeatability allows clinicians to identify a true clinically meaningful change from measurement error. The difference between any two measurements for the same subject is expected to be <2.77 multiplied by the within-subject standard deviation; therefore, the repeatability was defined as 2.77 multiplied by the within-subject standard deviation obtained from the ANOVA 30. Table 4⇑ shows a true measurable clinical change in D_L,NO and D_L,CO as an absolute change of >17 and 3 mL·min⁻¹·mmHg⁻¹, respectively. In addition, a true clinical change in D_m,CO and V_c is an absolute change of >7 mL·min⁻¹·mmHg⁻¹ and 13 mL, respectively. Any change that is less than these values is considered a true measurement error. Indeed, D_m,CO equals D_L,NO divided by a fixed value, and V_c is derived from the D_L,NO and D_L,CO; therefore the repeatability of D_m and V_c can be calculated from (or explained by) the repeatability of the D_L,NO.

Based on ATS and ERS criteria, the average value of two trials whose difference in diffusing capacity is within 10% of each other is considered acceptable. The present authors looked at the average D_L,NO and D_L,CO for all 31 subjects over five trials, and the repeatability data of this study are in agreement with the ATS/ERS guidelines as D_L,NO (and thus D_m,CO) and D_L,CO are found to be 10%. However, since the variability of the parameters D_L,NO and D_L,CO were independent of the magnitude of the measurement, the results invalidate the use of percentage value to describe the repeatability. Using a percentage will lead to underestimation of variability in low values and overestimation for high values. Others studies have also suggested using an absolute value rather than a percentage, since the diffusing capacity was also independent of the magnitude of the measurement 38, 39. Therefore, the current authors recommend using an absolute difference rather than a percentage as alternative criteria for repeatability. It is also recommended to report the average of two trials when the absolute difference between the two measurements is within 17 mm·min⁻¹·mmHg⁻¹ for D_L,NO, 3 mm·min⁻¹·mmHg⁻¹ for D_L,CO, and 13 mL for V_c.

It is believed that the present paper is the first to actually quantify important measurable clinical changes in the parameters obtained from the single-breath D_L,NO–D_L,CO method.