Standardisation and application of the single-breath determination of nitric oxide uptake in the lung

Gerald S. Zavorsky; Connie C.W. Hsia; J. Michael B. Hughes; Colin D.R. Borland; Hervé Guénard; Ivo van der Lee; Irene Steenbruggen; Robert Naeije; Jiguo Cao; Anh Tuan Dinh-Xuan

doi:10.1183/13993003.00962-2016

Figures

Tables
Supplementary Materials
Additional Files

Download figure
Open in new tab
Download powerpoint
FIGURE 1
Diagram of the uptake pathways for inhaled nitric oxide (NO) and carbon monoxide (CO) from the alveolar membrane to their combination with haemoglobin (Hb) within the red blood cell, in terms of the Roughton–Forster equation, 1/D_L=1/D_M+1/(θ·V_C), where 1/D_L is the total resistance to NO or CO uptake, 1/D_M is the resistance from the alveolar membrane to the red cell membrane (membrane resistance) and 1/(θ·V_C) is the diffusion and chemical combination resistance (red cell resistance) within the erythrocyte (1). The chief barrier to CO uptake is within the red cell (∼70–80%); the ∼25% remaining resistance to CO diffusion is located in the alveolar membrane (2). The main resistance barrier for NO lies between the alveolar and red blood cell membranes (∼60%; 3), with the red cell resistance (4) comprising ∼40% of the resistance to NO diffusion, as observed by Borland et al. [13]. Specifically, the red cell interior is the determinant part of the membrane resistance to NO [14]. Reproduced and adapted from [20] with permission from the publisher.
Download figure
Open in new tab
Download powerpoint
FIGURE 2
a) The association between diffusing capacities of the lung for nitric oxide (D_LNO) and carbon monoxide (D_LCO) measured at rest (single-breath; average breath-hold time was ∼6 s). Several published studies were used [57, 106, 107]. D_LNO=4.65·(D_LCO)+3.8, R² 0.90, standard error of the estimate (see) 11.8, p<0.001, 95% CI of the slope 4.51–4.79; n=493 healthy subjects. b) The association between pulmonary diffusing capacity and alveolar volume (V_A) measured at rest (single-breath; average breath-hold time was ∼6 s). Several published studies were used [57, 106, 107]. D_LNO=23.0·(V_A)+2.4, R² 0.64, see 21.9, p<0.001, 95% CI of the slope 21.4–24.5; n=493. D_LCO=4.63·(V_A)+1.55, R² 0.62, see 4.5, p<0.001, 95% CI of the slope 4.31–4.94; n=493. All healthy subjects. c) The association between pulmonary diffusing capacity and cardiac output (Q) measured at rest and during exercise by rebreathing. Data from two published studies [35, 109], including ∼45% of previously unpublished data. D_LNO=6.3·(Q)+58.2, R² 0.42, see 31.3, p<0.001, 95% CI of the slope 5.5–7.2; n=76, four data points per subject. D_LCO=2.0·(Q)+9.0, R² 0.71, see 5.3, p<0.001, 95% CI of the slope 1.8–2.1; n=76, four data points per subject. All healthy subjects. When using rebreathing manouvres, D_LCO is more tightly associated with cardiac output than D_LNO (comparison of correlation coefficients z-statistic 5.52, p<0.01); however, D_LNO is more tightly related to alveolar volume compared to D_LCO (comparison of correlation coefficients z-statistic 2.27, p=0.023). The association between D_LNO and D_LCO in relation to V_A during rebreathing manouvres (r=0.73 between D_LNO versus V_A, and r=0.63 between D_LCO versus V_A) is not shown here.
Download figure
Open in new tab
Download powerpoint
FIGURE 3
Plots of a) pulmonary diffusing capacity for nitric oxide (D_LNO) and pulmonary diffusing capacity for carbon monoxide (D_LCO), their ratio (D_LNO/D_LCO), alveolar–capillary membrane diffusing capacity for carbon monoxide (D_MCO) and the D_MCO to pulmonary capillary blood volume (V_C) ratio (D_MCO/V_C), as they relate to the percentage of maximal alveolar volume (V_A) (x-axis) compared to their percentage value at maximal V_A (y-axis); and b) rates of alveolar uptake for NO and CO per unit time and pressure, K_NO and K_CO (mathematically equivalent to D_LNO/V_A and D_LCO/V_A, respectively), and the membrane diffusing capacity (D_M) and pulmonary capillary volume (V_C), both per unit alveolar volume (V_A) (D_M/V_A and V_C/V_A), as the expansion of the lung is changed voluntarily in normal subjects (100% of maximal V_A, which is approximately total lung capacity, and 50% of maximal V_A, which is approximately functional residual capacity). Note in a) that with diminishing lung expansion (ΔV_A), ΔD_LNO is better related to membrane diffusing capacity (ΔD_MCO) and ΔD_MCO/V_C change than the D_LCO change. In b), ΔK_CO is a better reflection of changes in the pulmonary microcirculation (capillary volume per unit alveolar volume, V_C/V_A) than the K_NO; decrease of D_M/V_A with V_A change suggests isotropic change as alveolar dimensions reduce with concomitant thickening of the alveolar–capillary membranes. Interrupted line (in b) signifies no change with change of V_A. Data from [21, 57].
Download figure
Open in new tab
Download powerpoint
FIGURE 4
a) Pulmonary capillary blood volume (V_C) and b) alveolar–capillary membrane diffusing capacity for carbon monoxide (D_MCO), measured using four different formulas for specific conductance in the blood for carbon monoxide (θCO). Based on the subject data from table 1, D_MCO and V_C were calculated from the four formulas/constants listed in table 5. The mean values for both V_C and D_MCO were statistically significant between all four formulas (p<0.01). Each box represents the 25th (bottom border), 50th (middle) and 75th (top border) percentiles. The error bars above and below each box represent the 90th and 10th percentiles, respectively. The 5th and 95th percentiles are represented by solid black circles below and above the error bars, respectively. The formula from Guénard et al. [16] provided the highest V_C and lowest overall D_MCO, while the formula from Roughton and Forster [11] provided the highest D_MCO and lowest V_C. The three formulas developed by Guénard et al. [16], Forster [4] and Holland [28] provided the closest mean values with one another. Taken overall, these formulas show reasonable agreement with one another.

TABLE 1

Subject characteristics from previously published studies from which prediction equations were made [57, 106, 107]

	Males	Females	Combined
Subjects	248	242	490
Age years	44±17 (18–93)	45±18 (18–87)	44±17 (18–93)
Weight kg	76.7±9.4 (55.0–105.0)	61.6±8.8 (44.0–95.0)	69.3±11.8 (44.0–105.0)
Height cm	176±8 (154–196)	164±7 (147–182)	170±10 (147–196)
Body mass index kg·m^-2	24.7±2.5 (18.9–29.9)	23.0±3.0 (17.2–29.8)	23.8±2.9 (17.2–29.9)
D_LNO mL·min⁻¹·mmHg⁻¹	164±31 (67–235)	119±25 (47–186)	142±36 (47–235)
D_LCO mL·min⁻¹·mmHg⁻¹	34.1±6.3 (11.9–49.9)	25.1±5.3 (11.3–38.6)	29.6±7.4 (11.3–49.9)
D_MCO mL·min⁻¹·mmHg⁻¹	161±39 (72–250)	104±26 (33–182)	133±44 (33–250)
V_C mL	78±16 (25–121)	65±15 (30–105)	72±17 (25–121)
D_MCO/V_C ratio min⁻¹·mmHg⁻¹	2.11±0.57 (1.01–4.03)	1.63±0.40 (0.88–2.96)	1.90±0.57 (0.88–4.03)
K_CO mL·min⁻¹·mmHg⁻¹·L⁻¹	4.9±0.8 (2.7–7.1)	4.8±0.7 (3.0–6.8)	4.9±0.8 (2.7–7.1)
K_NO mL·min⁻¹·mmHg⁻¹·L⁻¹	23.8±3.9 (13.7–34.2)	22.8±3.2 (13.5–31.5)	23.3±3.6 (13.5–34.2)
D_LNO/D_LCO ratio	4.83±0.40 (3.83–5.82)	4.74±0.39 (3.85–5.78)	4.79±0.40 (3.83–5.82)

Data are presented as n or mean±sd (range). The alveolar–capillary membrane diffusing capacity for carbon monoxide (D_MCO) and total volume of blood in the lung capillaries exposed to alveolar air (V_C) values reported in these studies [57, 106, 107] have been recalculated according to the parameters listed in table 4. D_LNO: diffusing capacity of the lung for nitric oxide; D_LCO: diffusing capacity of the lung for carbon monoxide; K_CO: rate of uptake of carbon monoxide from alveolar gas; K_NO: rate of uptake of nitric oxide from alveolar gas.

TABLE 2

Predictive equations for white adults at a breath-hold time of ∼6 s, inspired nitric oxide (NO) of ∼35 ppm and inspired oxygen of ∼19.5%, from three studies [57, 106, 107]

	Height cm	Age²	Sex	Constant	Adjusted R²	see	LLN and ULN
D_LCO mL·min⁻¹·mmHg⁻¹	0.23	−0.002	6.0	−8.5	0.68	4.2	±8.2
D_LNO mL·min⁻¹·mmHg⁻¹	0.81	−0.010	34.4	9.7	0.69	20.0	±39.2
D_MCO mL·min⁻¹·mmHg⁻¹		−0.011	56.4	129.6	0.61	27.3	±53.5
V_C mL	0.84	−0.004		−59.9	0.49	12.0	±23.5
V_A L	0.079		0.73	−7.7	0.67	0.72	±1.4
V_C/V_A mL·L⁻¹		−0.0006	−1.25	13.9	0.27	1.89	±3.70
D_MCO/V_A mL·min⁻¹·mmHg⁻¹·L⁻¹	−0.200	−0.002	5.9	56.6	0.41	3.81	±7.47
K_CO mL·min⁻¹·mmHg⁻¹·L⁻¹		−0.00027		5.5	0.34	0.6	±1.2
K_NO mL·min⁻¹·mmHg⁻¹·L⁻¹		−0.00137		26.4	0.39	2.8	±5.5

Alveolar–capillary membrane diffusing capacity for carbon monoxide (D_MCO) and total volume of blood in the lung capillaries exposed to alveolar air (V_C) values in these studies [57, 106, 107] have been recalculated according to the formulas and constants in table 4 and then re-analysed for the regression. A predictive model was not found for the ratio of diffusing capacities of the lung for nitric oxide and carbon monoxide (D_LNO/D_LCO). Sex: 1 for male, 0 for female; see: standard error of the estimate. To convert D_LNO, D_LCO and D_MCO to mmol·min⁻¹·kPa⁻¹, divide by 3. Lower limit of normal (LLN)=2.5th percentile; upper limit of normal (ULN)=97.5th percentile. n=490. V_A: alveolar lung volume; K_CO: rate of uptake of carbon monoxide from alveolar gas; K_NO: rate of uptake of nitric oxide from alveolar gas. Supplementary appendix H allows the patient's individual values to be inserted in relation to the predicted values.

TABLE 3

Intra-session and inter-session variability of single-breath measurements of the diffusing capacities of the lung for nitric oxide (D_LNO) and carbon monoxide (D_LCO) (5 s breath hold) at rest

	Test-to-test measurement error (within the same testing session)	Repeatability^# (within the same testing session)	Reproducibility^¶ (week-to-week or month-to-month change) More stringent	Smallest measurable change⁺ (week-to-week or month-to-month change) Less stringent
D_LNO
mL·min⁻¹·mmHg⁻¹	6.2 (4)	17 (10)	20 (13)	10 (7)
mmol·min⁻¹·kPa⁻¹	2.1 (4)	5.8 (10)	6.5 (13)	3.3 (7)
D_LCO
mL·min⁻¹·mmHg⁻¹	1.2 (4)	3.2 (10)	4.9 (16)	2.5 (8)
mmol·min⁻¹·kPa⁻¹	0.4 (4)	1.1 (10)	1.6 (16)	0.8 (8)
D_LNO/D_LCO ratio	0.12 (2)	0.36 (7)	0.23 (5)	0.13 (3)
D_MCO
mL·min⁻¹·mmHg⁻¹	12 (7)	34 (19)	47 (28)	24 (28)
mmol·min⁻¹·kPa⁻¹	4.1 (7)	11.2 (19)	15.8 (28)	8 (14)
V_C mL	4 (5)	10 (13)	16 (24)	8 (12)

Numbers are presented as the value with the percentages in parentheses. Within-session data [49] and reproducibility data (between sessions) [88] were obtained from healthy subjects. The diffusing capacity of the membrane for carbon monoxide (D_MCO), nitric oxide (D_MNO) and total volume of blood in the lung capillaries exposed to alveolar air (V_C) values are recalculations from the original dataset using the formulas and constants in table 4 as well as the supplementary appendices. The D_LCO repeatability and reproducibility in subjects with pulmonary pathophysiology (mean D_LCO 11–18 mL·min⁻¹·mmHg⁻¹) are 2.7 and 4 mL·min⁻¹·mmHg⁻¹, respectively [113, 114]. The repeatability was calculated as follows: the mean within-subject standard deviation (which is the average standard deviation between several diffusing capacity tests performed in one session) multiplied by 2.77. The reproducibility is performed the same way, except the mean week-to-week standard deviation is used (which is the average standard deviation between diffusing capacity measured over several weeks multiplied by 2.77). Refer to supplementary appendix G for in-depth statistical methodology of the calculation. ^#: the difference between two trials for D_LNO, D_LCO, D_MCO and V_C measured on the same subject in the same testing session is expected to be <17, <3.2 and <34 mL·min⁻¹·mmHg⁻¹ and <10 mL, respectively, for 95% of observations; ^¶: the difference in D_LNO, D_LCO, D_MCO and V_C measured on the same subject over two different weeks is expected to be less than 20, 4.9, and 47 mL·min⁻¹·mmHg⁻¹ and 16 mL, respectively, 95% of the time. Any diffusing capacity parameter that has a week-to-week or month-to-month change that is equal to or greater than the reproducibility has only a 5% chance that it is not a real change; ⁺: half the reproducibility and thus less stringent than the reproducibility. Any week-to-week or month-to-month change that is equal to the smallest meaningful change has a 20% chance that it is not a real change. The reproducibility and smallest meaningful change are most correct when using the same equipment for the duration of the assessment.

TABLE 4

Summary consensus statement for simultaneous single-breath measurement of diffusing capacities of the lung for nitric oxide (D_LNO) and carbon monoxide (D_LCO) in healthy adults

Issue	Agreement
Breath-hold time	10 s is desired for better gas mixing 4–6 s is acceptable if using a single electrochemical NO cell that measures in the ppm range
Measured inspired NO concentration	40–60 ppm, placed in the inspiratory bag ≤2 min before use
Measured inspired O₂ concentration	Close to 21%
Measured expired O₂ concentration	Used to calculate P_AO₂ and θCO
1/θCO [16]	(0.0062·P_AO₂+1.16)·(ideal Hb÷measured Hb)
θNO [14, 29]	4.5 mL NO·(mL blood·min·mmHg)⁻¹ (1/θNO=0.222)^#
θNO/θCO ratio	Average 8.01 (male Hb 14.6 g·dL⁻¹), 8.73 (female Hb 13.4 g·dL⁻¹) at P_AO₂ of 100 mmHg^#
D_MCO	D_MNO divided by 1.97^#
Presentation of results	Report D_LNO, D_LCO, K_NO and K_CO in absolute numbers and as % predicted from regression equations (at the appropriate breath-hold time), with the corresponding LLN, ULN and z-score (standardised residuals: number of standard deviations above or below the reference value) Report alveolar volume in L BTPS and as TLC % pred

NO: nitric oxide; O₂: oxygen; θCO (NO): specific conductance in the blood for carbon monoxide (NO) in mL·(mL blood·min·mmHg)⁻¹; D_MCO: alveolar–capillary membrane diffusing capacity for CO; P_O₂: oxygen tension; P_AO₂: alveolar oxygen tension; Hb: haemoglobin; D_MNO: alveolar–capillary membrane diffusing capacity for NO; K_NO: rate of change of NO from alveolar gas; K_CO: rate of change of CO from alveolar gas; LLN: lower limit of normal; ULN: upper limit of normal; BTPS: body temperature and pressure, saturated (760 mmHg, 37°C, 100% humidity); TLC: total lung capacity. ^#: used in tables 1, 2 and 3 and the supplementary appendices.

TABLE 5

1/θCO equations that show reasonable agreement

	Formula for 1/θCO	1/θCO		θNO/θCO
		Ideal Hb (14.6 g·dL⁻¹)	Ideal Hb (13.4 g·dL⁻¹)	Ideal Hb (14.6 g·dL⁻¹)	Ideal Hb (13.4 g·dL⁻¹)
Derived in vivo
Guénard et al. [16]	(0.0062·P_AO₂+1.16)·(ideal Hb÷measured Hb)	1.780	1.939	8.010	8.727
Derived in vitro
Forster [4] α=∞, pH=7.4	(0.0041·P_AO₂+1.3)·(ideal Hb÷measured Hb)	1.710	1.863	7.695	8.384
Roughton and Forster [11] α=1.5, pH=8.0	(0.0058·P_AO₂+1.0)·(ideal Hb÷measured Hb)	1.580	1.721	7.110	7.747
Holland [28] α=1.5	(0.0065·P_AO₂+1.08)·(ideal Hb÷measured Hb)	1.730	1.885	7.785	8.482

Numbers given are for the following standards: alveolar oxygen tension (P_AO₂) 100 mmHg and specific conductance in the blood for nitric oxide (θNO) 4.5 mL NO· (mL blood·min·mmHg)⁻¹ and thus 1/θNO=0.222, from [14, 29]. θCO: specific conductance in the blood for carbon monoxide; Hb: haemoglobin; α: the ratio of permeability of the red cell membrane to that of the red cell interior.

Supplementary Materials

Figures
Tables
Additional Files

Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material: Appendix H ERJ-00962-2016_Appendix_H
Supplementary material: Appendices A to G ERJ-00962-2016_Appendices_A-G

Additional Files

Figures
Tables
Supplementary Materials

ERJ Podcast

Duration: 13:34 min

Presenter: Marc Humbert, Chief Editor, ERJ

Interviewee: Gerald S. Zavorsky, Dept of Respiratory Therapy, Georgia State University, Atlanta, GA, USA

Articles discussed:

Zavorsky GS, Hsia CCW, Hughes JMB, et al. Standardisation and application of the single-breath determination of nitric oxide uptake in the lung. Eur Respir J 2017; 49: 1600962. Full Text

Subscribe to the ERJ Podcast via iTunes, SoundCloud or RSS

Files in this Data Supplement:

Download the podcast - in MP3 format