Simultaneous measurement of pulmonary diffusing

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Review

Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide Kazuhiro Yamaguchia,n, Takao Tsujib, Kazutetsu Aoshibac, Hiroyuki Nakamurac a Division of Comprehensive Sleep Medicine, Tokyo Women’s Medical University, 8-1 Kawata-cho, Shinjuku-ku, Tokyo 162-8666, Japan b Respiratory Medicine, Institute of Geriatrics Tokyo Women’s Medical University, 2-15-1 Sibuya, Shibuya-ku, 150-0002 Tokyo, Japan c Department of Respiratory Medicine, Tokyo Medical University Ibaraki Medical Center, 3-20-1 Chuou, Ami-machi, Inashiki-gun, 300-0395 Ibaraki, Japan

art i cle i nfo

ab st rac t

Article history:

In Europe and America, the newly-developed, simultaneous measurement of diffusing

Received 1 November 2017

capacity for CO (DLCO ) and NO (DLNO ) has replaced the classic DLCO measurement for

Received in revised form

detecting the pathophysiological abnormalities in the acinar regions. However, simulta-

30 November 2017

neous measurement of DLCO and DLNO is currently not used by Japanese physicians. To

Accepted 8 December 2017

encourage the use of DLNO in Japan, the authors reviewed aspects of simultaneouslyestimated DLCO and DLNO from previously published manuscripts. The simultaneous DLCO -DLNO technique identifies the alveolocapillary membrane-related diffusing capacity

Keywords:

(membrane component, DM ) and the blood volume in pulmonary microcirculation (V C ); V C

Diffusing capacity

is the principal factor constituting the blood component of diffusing capacity (DB ; DB ¼ θ  V C

Membrane component

where θ is the specific gas conductance for CO or NO in the blood). As the association

Blood component

velocity of NO with hemoglobin (Hb) is fast and the affinity of NO with Hb is high in comparison with those of CO, θNO can be taken as an invariable simply determined by

Abbreviations: A,

alveolar gas; BHT,

breath-holding time (sec); CF,

cystic fibrosis; d,

diffusivity of the gas (cm2/s); Dapp,

apparent diffusing capacity for the gas neglecting the effects of functional inhomogeneities (mL/min/mmHg); DB, diffusing capacity for the gas (mL/min/mmHg); DL,

diffusing capacity for the gas (mL/min/mmHg); DL/VA,,

alveolar volume (rate constant of alveolar gas uptake per unit pressure, mL/min/mmHg/L); DM, capacity for the gas (mL/min/mmHg); DPLD, He, PAH,

helium; I,

inspired gas; K,

diffuse parenchymal lung diseases; Hb,

diffusing capacity per unit

membrane component of diffusing

hemoglobin; HbO2, oxyhemoglobin;

Krogh factor for the gas (calculated as S/(PB–PH2O) and equal to DL/VA); MW,

molecular weight (g/moL);

pulmonary arterial hypertension; PAO2, mean alveolar PO2 (surrogate of mean capillary PO2, mmHg); PB,

(mmHg); PC,

partial pressure of the gas in alveolar capillary (mmHg); PH20,

alveolar gas uptake during breath-holding; TL, gas volume (L); VAT, space (mL); VI,

barometric pressure

vapor pressure at body temperature (mmHg); S,

slope of

transfer factor (equal to DL); TL/VA, transfer coefficient (equal to DL/VA); VA,

alveolar

inspired or expired alveolar tidal volume (L); VC,

inspired gas volume (L); α,

overall

blood component of

alveolar capillary blood volume (mL); VD, anatomical dead

Bunsen solubility coefficient of the gas (mL/mL/atm); θ,

specific gas conductance in blood

(mL/min/mmHg/mL); ΥX/Y, relative Krogh diffusion constant of gas X against gas Y ((α  d)X/(α  d)Y); ω, ratio of permeability of erythrocyte membrane to that of erythrocyte interior n Corresponding author. E-mail addresses: [email protected] (K. Yamaguchi), [email protected] (T. Tsuji), [email protected] (K. Aoshiba), [email protected] (H. Nakamura). https://doi.org/10.1016/j.resinv.2017.12.006 2212-5345/& 2018 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

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Specific gas conductance

diffusion limitation inside the erythrocyte. This means that θNO is independent of the

Blood volume

partial pressure of oxygen (PO2 ). However, θCO involves the limitations by diffusion and chemical reaction elicited by the erythrocyte, resulting in θCO to be a PO2 -dependent variable. Furthermore, DLCO is determined primarily by DB (∼77%), while DLNO is determined equally by DM (∼55%) and DB (∼45%). This suggests that DLCO is more sensitive for detecting microvascular diseases, while DLNO can equally identify alveolocapillary membrane and microcirculatory abnormalities. & 2018 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Historical backgrounds on lung diffusing capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Units and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Simultaneous measurement of DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1. Basic parameters of DL/VA and DL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.2. Physical properties of CO and NO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.3. Specific gas conductance in the blood for CO and NO (θCO and θNO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.4. Estimation of DM and VC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.5. Influence of back-pressures of CO and NO on DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.6. Influence of functional inhomogeneities on DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4. Reference equations for DL-associated parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5. Pathophysiological factors affecting DL-related parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.1. Age and height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.2. Alveolar volume (VA) and cardiac output (Q) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.3. DL-related parameters in various lung diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5.4. Differential diagnosis using DLCO- and DLNO-associated parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Appendix A. Supporting information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Historical backgrounds on lung diffusing capacity In Europe and North America, the measurement of the diffusing capacity (DL ) for nitric oxide (NO) is used in a variety of lung diseases to evaluate the impediment of gas transfer in the acinus across the alveolocapillary membrane and microcirculation [1–6]. The use of NO diffusing capacity (DLNO ) required that the Task Force Panel organized by the European Respiratory Society (ERS) create standards for the measurement and interpretation of DLNO [7]. To encourage the use of DLNO in Japan, the authors have comprehensively reviewed the methodological and pathophysiological aspects of clinically using DLNO . It has been over 100 years since Marie Krogh developed the method to measure the single-breath carbon monoxide (CO) uptake through the alveolocapillary membrane [8]. Since then, the single-breath CO diffusing capacity (DLCO ) has become the most clinically useful pulmonary function test after spirometry and the measurement of lung volumes. A practical method to examine the single-breath DLCO was proposed by Ogilvie et al. [9], and at the same time, Roughton and Forster [10] proposed a memorable model describing the

gas transfer in the lung. They assumed that two processes could explain the transfer of CO from the alveolocapillary membrane to hemoglobin (Hb) in erythrocytes: (1) the membrane diffusing capacity for CO (DMCO ) and (2) the blood diffusing capacity for CO (DBCO ). The DMCO reflects the diffusion limitation across the effective alveolocapillary membrane, which consists of gas-phase diffusion (if any), the alveolar wall, and the plasma layer surrounding the erythrocyte. The DBCO is defined as the product of alveolar capillary blood volume (VC ) and specific gas conductance for CO in the blood (θCO ). The θCO signifies the diffusive process across the membrane of the erythrocyte and its interior and incorporates the competitive, replacement reaction of CO with oxyhemoglobin (HbO2 ). Since the reciprocals of DMCO and DBCO are the gas-transfer resistances that are connected in series, the total resistance for CO transfer, 1=DLCO , is expressed as: 1=DLCO ¼ 1=DMCO þ 1=ðθCO  VC Þ

ð1Þ

Roughton and Forster [10] developed a clever method for determining DMCO and V C from DLCO measured at two different alveolar partial pressure of oxygen (alveolar PO2 ) (classic two-step alveolar PO2 technique).

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In the 1980s, Guénard et al. [1] and Borland and Higenbottamet [2] independently proposed a novel method of simultaneously measuring DLCO and DLNO (simultaneous one-step CO-NO technique). Differing from the classic twostep alveolar PO2 technique, to determine DM and VC , the simultaneous one-step CO-NO technique requires the measurement of DLCO and DLNO at a single alveolar PO2 . Therefore, this method is more convenient clinically and reduces the time required for DL measurement.

2.

Units and nomenclature

This review uses traditional units (mL/min/mmHg) to describe DL . The ERS recommends expressing DL in SI units (mmoL/min/kPa), while the American Thoracic Society (ATS) prefers traditional units. Values in traditional units can be divided by 3.0 to convert them to SI units. On behalf of DL , transfer factor, expressed as TL , has been used in Europe and has been suggested to be a more accurate term than DL when considering the mechanism deciding CO transport in the acinus. This is because diffusion is not the sole determinant of CO transport in the acinus, and chemical reactions are involved. However, NO transport in the acinus is almost totally limited by diffusion (see below), which suggests that the term of DL is better for describing NO movement. Furthermore, DL is commonly used in Japan. Based on these facts, the present review will use the term DL . The next important measure is the DL =VA (mL/min/ mmHg/L), in which VA denotes the alveolar volume during a single-breath maneuver. The DL =VA is called the transfer coefficient (TL =V A ) in Europe [11–13]. In the field of respiratory physiology, this parameter expresses the DL per unit alveolar volume, which, in the field of gas kinetics, is equal to the Krogh factor (K: KCO or KNO ), the rate constant of alveolar gas uptake per unit pressure.

3.

Simultaneous measurement of DLCO and DLNO

The 2017 ERS Standards for DLNO [7] recommend that the concentrations of test gases in an inspired gas should be 0.3% of CO, 40-60 ppm of NO, 10% of helium, and 21% of O2 with the balance nitrogen (N2). Following a period of quiet tidal breathing, a bolus of a gas mixture containing known quantities of test gases is rapidly inhaled from residual volume (RV) to total lung capacity (TLC). At full inspiration, the subject holds the breath for a prescribed period near atmospheric intrapulmonary pressure. To obtain the alveolar gas sample, following breath-holding the subject exhales smoothly and rapidly to RV within 4 sec. A sample gas volume of 0.5-1.0 L should be collected. However, if the subject’s vital capacity (VC) is o1.0 L, a sample gas volume o0.5 L can be used [13]. The concentrations of helium and CO in inspired and alveolar gases are measured using a rapid gas analyzer system (90% response time: r150 msec) [13]. Partial pressure of O2 in the alveolar gas sample is measured as a surrogate for mean alveolar PO2 . The gas concentration of NO is measured with a high-sensitivity, high-speed

chemiluminescence NO analyzer, with a detection range from 5 ppb to 500 ppm and a 90% response time of 70 msec. When the chemiluminescence NO analyzer is used, the breath-holding time is prescribed at 10 sec. However, if a low sensitivity, slow-speed electrochemical NO analyzer (detection limit of NO ranging from 0 to 100 ppm with 90% response time of ∼10 sec) is used, a shorter breath-holding time of 4-6 sec is necessary. This is because alveolar NO concentration after a 5-sec period of breath-holding is less than 3-5 ppm [14]. There may be a problem with accuracy to measure NO gas below 3–5 ppm with a low-sensitivity electrochemical NO analyzer. The practical procedures for simultaneous measurement of DLCO and DLNO are summarized in Supplementary Table 1. The equipments for simultaneous measurement of DLCO and DLNO are described in Supplementary Table 2. Since 2008, automated analyzers for simultaneously measuring gas-phase concentrations of CO and NO have been commercially available in Europe. The toxicological and vasodilator effects of inhaled NO should be mentioned. Clutton-Brock [15] demonstrated that severe lung-tissue damage and/or death may result from inhalation of NO over 5000 ppm (0.5%) for 7–50 min in the case of accidental exposure during anesthetic procedures. However, there is no evidence for injurious effects and/or vasodilation of inhaling low concentrations of NO [16–18]. This suggests that the inhalation of low concentrations of NO for short periods of time is safe and induces minimal pulmonary vasodilation. Since the single-breath DL is measured at full inspiration, VA is virtually equal to TLC. The VA is derived from the singlebreath helium dilution after subtracting the estimated anatomic dead space (VD ) from the inspired volume (VI ). The 2017 ERS/ATS Standards for DLCO [13] recommended to use an estimate of 2.2 mL/kg as V D if body mass index (BMI) o30 kg=m2 [19] or H2 /189.4 (H: height in cm) if BMI Z30 kg=m2 . The following formula is used to calculate VA : VA ¼ ðVI − VD Þ  ðHeI =HeAðtÞ Þ

ð2Þ

where HeI denotes the helium concentration in inspired gas and HeAðtÞ the helium concentration in alveolar gas sample at the end of breath-holding. Instead of the estimated value, the VD can be directly measured from the helium washout curve using the Fowler method [20].

3.1.

Basic parameters of DL/VA and DL

First, the K value (DL =V A ) is measured from the decayed curve of CO or NO (Fig. 1). The slope of CO or NO uptake (SCO , SNO ) during a breath-holding time (BHT) is calculated using the logarithm (Ln) of the gas concentration at the beginning of breath-holding (time: zero) and that at the end of breathholding (time: t). SX ¼ ðLnFXAð0Þ − LnFXAðtÞ Þ=BHT

ð3Þ

In this equation, X is either CO or NO; FXAð0Þ is the gas concentration at the beginning of breath-holding; and FXAðtÞ is that at the end of breath-holding. The gas concentration of

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

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Fig. 1 – Schematic presentation of CO and NO transfer in the lung (A) CO and NO transfer from alveolar gas to erythrocyte. The membrane component (DM ) of diffusing capacity is prescribed by the diffusion through the effective alveolocapillary membrane, including the gas-phase diffusion (if any), the alveolar wall, and the plasma layer. The blood component (DB ¼ θ∙VC ) of diffusing capacity is prescribed by the diffusion in erythrocytes for NO and the diffusion and chemical reactions in the erythrocytes for CO. (B) The alveolar uptake of CO or NO. The slope of the decayed curve of the gas is defined as S (SCO or SNO ), from which K (KCO or KNO ) and DL (DLCO or DLNO ) are calculated. Helium (He) is assumed to be not absorbed by the alveolar capillary blood. CO or NO at the beginning of breath-holding, FXAð0Þ , is calculated as follows: FXAð0Þ ¼ ðHeAðtÞ =HeI Þ  FXI

ð4Þ

In this equation, FXI is the gas concentration of CO or NO in the inspired gas. From SX , KX and DLX are assumed as: KX ¼ SX =ðPB − PH2O Þ

ð5Þ

DLX ¼ VA  KX

ð6Þ

where PB denotes the atmospheric pressure and PH2O the vapor pressure.

3.2.

of the two gases (the Graham’s law). Therefore, the dY =dX can be assumed to be the same in any diffusion path. The relative Krogh diffusion constant of NO against CO (ΥNO=CO Þ is defined as: ΥNO=CO ¼ ðα  dÞNO =ðα  dÞCO

ð7Þ

The ΥNO=CO is the major factor determining the ratio of membrane diffusing capacity for NO (DMNO ) to CO (DMCO ). Originally proposed by Wilhelm et al. [26], subsequent studies agree that the best value of ΥNO=CO in the acinus is 1.97. Thus, the DMNO =DMCO can be given by: DMNO =DMCO ¼ 1:97

ð8Þ

Physical properties of CO and NO

Detailed descriptions of the physical properties of CO and NO are provided in Supplemental Table 3. Briefly, the diffusive process of CO and NO in the acinus is determined by the Krogh diffusion constant, which is defined as (α  d), where α is the Bunsen solubility coefficient of the gas (mL/mL/atm) and d is the gas diffusivity (cm2 =s) in the specific tissue. Since α is almost equal along the diffusion path, including the alveolocapillary membrane, plasma layer, and erythrocyte interior, it can be approximated by the gas solubility in water [21]. The gas diffusivity is distinctly varied along the diffusion path [22–25]. The relative diffusivity of the two gases is defined as dY =dX (X, Y: two indicator gases), which is equal to the reciprocal ratio of square root of the molecular weight (MW)

3.3. Specific gas conductance in the blood for CO and NO (θCO and θNO) The radical difference between CO and NO is recognized in the reactions with Hb. Carlsen and Comroe [27] measured the association velocity constants of CO and NO with Hb using a rapid-reaction, constant-flow apparatus. They identified that the association velocity constant of NO with Hb is ∼400 times that of CO and the affinity of NO with Hb is ∼1800 times that of CO (Supplementary Table 3). This suggests that the chemical reaction of NO with Hb does not affect erythrocyte NO uptake. They also demonstrated that the θNO of human biconcave erythrocytes is not infinite but is finite at

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Table 1 – Equations predicting PO2 -dependent θCO and PO2 -independent θNO in human blood. 1=θCO

Conditions

References

ω¼ 1.5, pH ¼ 8.0 (rapid-reaction, constant-flow) ω¼ 2.5, pH ¼ 8.0 (rapid-reaction, constant-flow) ω¼ 1, pH ¼ 8.0 (rapid-reaction, constant-flow) ω¼ 1, pH ¼ 7.4 (rapid-reaction, constant-flow) ω¼ 1.5 at standard conditions (stopped-flow) Assumed value (ω¼ 1, pH ¼ 7.4) Thin blood film exposed to step-change of PCO at varied PO2 Indirect estimation in vivo (optimal equation preserving DM =V C at the same level under hypoxia and normoxia)

Roughton (1957) [10] Roughton (1957) [10] Roughton (1957) [10] Forster (1987) [32] Holland (1969) [33] Stam (1991) [34] Reeves (1992) [35] Guénard (2016) [29]

1/θNO

Conditions

References

1/4.5 0 1/3.3 1/4.0 1/3.0

Rapid-reaction, constant-flow Assumed value based on infinitely fast reaction of NO with Hb Indirect estimation in vivo Indirect estimation in vitro (horse blood) Indirect estimation in vivo (optimal value preserving DM =VC at the same level under hypoxia and normoxia)

Carlsen (1958) [27] Guénard (1987) [1] Borland (1991) [36] Borland (2006) [28] Guénard (2016) [29]

0.0058  PO2 þ 0.0058  PO2 þ 0.0058  PO2 þ 0.0041  PO2 þ 0.0065  PO2 þ 0.0055  PO2 þ 0.0080  PO2 þ 0.0062  PO2 þ

1.00 0.73 0.33 1.30 1.08 0.73 0.0156 1.16

θ: mL/min/mmHg/mL. ω: ratio of the permeability of erythrocyte membrane to that of erythrocyte interior.

4.5 mL/min/mmHg/(mL  blood) (Table 1). Combining these results, they concluded that the NO uptake process by erythrocytes is predominantly limited by diffusion within the erythrocyte but not by a chemical reaction of NO with Hb. Although five values for θNO have been reported to date (Table 1), the θNO reported by Carlsen and Comroe [27] is the only directly and reliably measured estimate. Therefore, it is recommended to use 4.5 mL/min/mmHg/mL as the θNO [7]. Further disavowal against reaction limitation during NO uptake by erythrocytes was certified by Borland et al. [2,28], who identified that DLCO is PO2 - and hematocrit (Ht)-dependent, whereas DLNO is PO2 -independent but Ht-dependent. The PO2 -independent DLNO implies that NO uptake in the acinus is not limited by chemical reaction between NO and Hb. The Ht-dependent DLNO indicates that NO uptake is impeded by decreased density of erythrocytes (anemia), which results in increased thickness of the plasma layer and impairs NO uptake. The PO2 - and Ht-dependent change in DLCO indicates that both diffusion limitation and reaction limitation are involved in CO uptake. As such, although the θNO is constant and independent of capillary PO2 , the θCO changes depending on capillary PO2 . Descriptions of PO2 -dependent θCO are given in Supplementary Fig. 1. Among the equations that predict the relationship between PO2 and 1=θCO (Table 1), the 2017 ERS Standards for DLNO [7] recommend the equation proposed by Guénard et al. [29]. The Guénard et al. equation is: 1=θCO ¼ ð0:0062  PO2 þ 1:16Þ  ðstandard Hb=measured HbÞ

ð9Þ

where PO2 is mean capillary PO2 approximated by alveolar PO2 measured at the end of breath-holding. Standard Hb is 14.6 g/dL for men and 13.4 g/dL for women. However, it should be noted that Eq. 9 is not directly derived from the kinetic measurements. Furthermore, attention should be paid for the Hb term, which aims to correct the Hb capacitance effect on absorbing CO, but not the diffusion limitation elicited by Ht-dependent modification of plasma layer thickness. The overall correction by Hb, including both Hb capacitance effect

and Ht-dependent change in plasma layer thickness, should be made using the equations recommended by the 2005 ATS/ERS Task Force for DLCO [30] as follows:
ð10  1Þ DLCO predicted; male ¼ DLCO  ð10:22 þ HbÞ=ð1:7  HbÞ
DLCO predicted; female ¼ DLCO  ð9:38 þ HbÞ=ð1:7  HbÞ

ð10  2Þ

In these equations, DLCO and Hb (g/dL) are the measured values. The situation of DLNO differs from that of DLCO . Since the concentration of NO used for DLNO measurement is low (i.e., ppm), it is not necessary to correct for Hb absorption of NO. Furthermore, due to a large Krogh diffusion constant of NO (Supplementary Table 3), the relative contribution of diffusion-limited NO transfer, resulting from a Ht-dependent change in the thickness of the plasma layer, is anticipated to be small. Indeed, Borland et al. [31] described that the Hb adjustment for DLNO is unnecessary until the Hb concentration is approximately 4 g/dL. Therefore, the constant value of θNO may be valid under a wide variety of pathophysiological conditions, including anemia.

3.4.

Estimation of DM and VC

The simultaneous equations described below use the measured values of DLCO , DLNO , mean alveolar PO2 (PAO2 ), and Hb to determine DM and V C : 1=DLCO ¼ 1=DMCO þ 1=ðθCO  VC Þ

ð11  1Þ

1=DLNO ¼ 1=DMNO þ 1=ðθNO  VC Þ

ð11  2Þ

DMNO ¼ 1:97  DMCO

ð11  3Þ

1=θCO ¼ ð0:0062  PAO2 þ 1:16Þ  ðstandard Hb=measured HbÞ ð11  4Þ 1=θNO ¼ 1=4:5

ð11  5Þ

Using the previously described simultaneous equations, the relative contribution of 1=DM and 1=DB ð1=ðθ  VC ÞÞ to 1=DLCO or

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to 1=DLNO was estimated (Fig. 2). The contribution to 1=DLCO of 1=DMCO is 23% and that of 1=DBCO is 77%. The contribution to 1=DLNO of 1=DMNO is 55% and that of 1=DBNO is 45%. This suggests that the DLCO is primarily determined by the limitations caused by diffusion and chemical reaction in the erythrocyte, whereas the DLNO is almost equally limited by the diffusion through the effective alveolocapillary membrane and

Fig. 2 – Relative contribution of 1=DM and 1=DB to overall resistance of 1=DL Calculated using previously reported DL -related values [7], θCO value [29], and θNO value [27].

the interior of the erythrocyte. Guénard et al. [1] assumed that θNO must be infinite, because the chemical reaction of NO with Hb is extremely rapid, leading them to postulate that DLNO is equal to DMNO . However, based on the previously described analysis, the assumption of Guénard et al. [1] is incorrect. The important pathophysiological message from this analysis is that DLNO is equally sensitive to morphological abnormalities of the effective alveolocapillary membrane and the pulmonary microcirculation. In contrast, DLCO is more sensitive to the microcirculatory abnormalities. Among the values physiologically measured for normal subjects, the DMNO (∼260 mL/min/mmHg adopted from ref. [7]) should provide the best estimate of the alveolocapillary membrane-associated diffusive processes. This value is converted to ∼170 of DMO2 , if the relative Krogh diffusion constant of O2 against NO (ΥO2=NO ) is 0.66. However, the morphometrically determined DMO2 is between ∼350 and ∼550 [37,38], which is much larger than the physiologically determined DMO2 . The difference may be attributed to the fact that morphological and functional inhomogeneities in the acinar regions have been eliminated in the morphometric DMO2 measurements.

Fig. 3 – Functional inhomogeneities in a lung model with two acinar regions PA : alveolar gas pressure in each region (i ¼1, 2), PC : partial pressure of gas in each alveolar capillary, D: diffusing capacity (DL ) in each region, VA : alveolar gas volume in each region, VAT : inspired or expired alveolar tidal volume in each region, Dapp : overall apparent diffusing capacity in an assumed lung with no inhomogeneities, which is inevitably lower than the true D defined as (D1 þ D2 ). (A) CO or NO uptake with time in region 1. Alveolar CO and NO decay linearly. (B) CO and NO uptake rates differ between regions 1 and 2. (C) CO or NO uptake in a lung with inhomogeneities expressed by a curved line. (D) CO or NO uptake in a lung assumed to have no inhomogeneity (see Supplement 5 for further details).

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]

3.5. Influence of back-pressures of CO and NO on DLCO and DLNO

7

4. Reference equations for DL-associated parameters

Detailed descriptions of the back-pressure of CO and NO in the pulmonary microcirculation are presented in Supplement 4. Since the chemical reaction of NO with Hb is very rapid and the affinity between them is very high, the NO back-pressure in pulmonary microcirculation is negligible during NO inhalation [28]. The CO back-pressure during DLCO measurements is estimated at ∼3% of the alveolar PCO in a nonsmoking subject (Supplementary Fig. 2), indicating that the effect of CO back-

The 2017 ERS Standards for DLNO [7] generated comprehensive regression equations that predict a variety of DL -associated parameters in a normal lung. The partial regression coefficients for these equations are presented in Supplementary Table 4. However, these equations were developed based on data primarily from Caucasian populations. Therefore, it is unclear if these equations are applicable to non-Caucasian populations, including Asians, such as Japanese.

pressure on DLCO can also be ignored in subjects who do not smoke. However, in subjects who are heavy smokers, and repeated measurements of DLCO, result in CO back-pressure

5. Pathophysiological factors affecting DL-related parameters

that should not be disregarded (Supplement 4).

5.1. 3.6.

Influence of functional inhomogeneities on DLCO and DLNO

The effect of functional inhomogeneities on single-breath DLCO and DLNO is schematically depicted in Fig. 3. Detailed descriptions for Fig. 3 are provided in Supplement 5. The analysis identifies that the transfer of CO and NO in the acini during breath-holding is influenced by the three inhomogeneities. These inhomogeneities include: (1) the distribution of inspired/expired alveolar tidal volume to alveolar volume
VATi =VAi ; (2) the ratio of alveolar tidal volume in each region
to total alveolar tidal volume VATi =VAT ; and (3) the ratio of

diffusing capacity to alveolar volume DLi =VAi . The DLi =VAi determines the gas uptake rate KCO (or KNO ) in each acinar region (i). The functional inhomogeneities have a negative impact on DLCO and DLNO , particularly in diseased lungs with

Age and height

The regression equations constructed by the 2017 ERS Standards for DLNO [7] identified that all DL -associated parameters decrease with aging irrespective of sex (Supplementary Table 4). As height increases, DLCO , DLNO , and V C increase, which is explained by the height-dependent increase in lung volume. Although the absolute values differ, the DL -associated parameters normalized by their maximal values suggest the following (Supplementary Fig. 5): (1) the negative impact of aging on DLCO and DLNO is analogous for both sexes; (2) for both sexes, the decline in DMCO with aging is larger than the decrease in V C , suggesting that the age-dependent distortion of alveolocapillary membrane structures differs from that of microcirculatory structures; and (3) the aging-related decline in DLCO , DLNO , or DMCO is greater in females than males, suggesting that as to the diffusing capacity, females have a higher sensitivity to aging.

destruction of alveolocapillary membrane and/or pulmonary microcirculation. The effect of the gas-phase diffusion limitation on overall

5.2.

gas transfer of CO and NO in the acinar regions is discussed

The modified Roughton-Foster formula is useful to understand the complicated VA -elicited changes in DLCO - and DLNO -related parameters. The modified Roughton-Foster formula is shown below:

in Supplement 6, which concludes that the gas-phase diffusion-related interference with gas transfer of CO and NO is small [39].

Alveolar volume (VA) and cardiac output (Q)

Fig. 4 – Effect of reduced VA without loss of acini on DL -related parameters The values are normalized against those obtained at the maximal VA . DM indicates DMCO . Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

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respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]

Table 2 – Pathophysiological factors affecting DLCO - and DLNO -related parameters. DM =V A

V C =VA

DL

DL =V A

Age

↓ (DM ↓)

↓ (V C ↓)

↓

↓

Height

↓(1) (DM ∼)

∼ (V C ↑)

↑

∼

Reduced VA Without loss of acini Respiratory muscle weakness

↓ (DM ↓)

↑↑ (V C ∼)

↓

↑↑

Chest wall/pleural restriction With loss of acini Pneumonectomy Increased Q or redistribution of Q Left-to-right shunt

↑ (DM ↓) ↑ (DM ↑)

↑ (V C ↓) ↑ (V C ↑)

↓↓(2)

↑(3)

↑

↑

↓ (DM ↓) ∼↓ (DM ∼↓)

↓ (V C ↓) ↓ (V C ↓)

↓

↓

↓

↓

↑ (DM ↑) ↓ (DM ↓)

∼ (V C ∼) ∼ (V C ∼)

↑

↑

↓

↓

↓ (DM ↓)

↓ (V C ↓)

↓

↓

↓ (DM ↓)

∼↓ (V C ↓)

↓

∼↓

Exercise Asthma (redistribution of Q) Reduced Q Heart failure Microvascular damage Pulmonary arterial hypertension Angiitis Alveolar hemorrhage Without change in V A Reduced density of erythrocytes Anemia Destruction of acinar structures COPD (destruction of alveoli and microcirculation)

Diffuse interstitial lung diseases (destruction of alveoli and microcirculation þ reduced V A with loss of acini)

(1): decrease in DMCO =V A but no change in DMNO =V A . (2): effects of reduced VA on DL are larger in lungs with loss of acini. (3): effects of reduced V A on DL =VA are larger in lungs without loss of acini.


1= DLX =VA ¼ 1=ðDMX =V A Þ þ

1=ðθX  VC =VA Þ

ð12Þ

where X is CO or NO. The analysis based on the regression equations proposed by the 2017 ERS Standards for DLNO [7] demonstrates that a decline in V A without a loss of acini accompanies: (1) a minimal decrease in DM =VA with a greater increase in VC =VA ; (2) a greater decrease in DLNO than DLCO ; and (3) a greater increase in DLCO =VA (KCO ) than DLNO =VA (KNO ) (Fig. 4, Table 2). Although DM =V A has been considered to remain constant with decreasing V A [13,34], the analysis indicates that in lungs without a loss of acini, the reduction in DM and VA is not isotropic. Overall VC remains constant due to the stability of Q, which reinforces the redistribution of pulmonary perfusion during lung volume changes, thus VC =VA increases with decreasing VA . Reduced VA decreases DM , but does not change VC , resulting in decreased DLCO and DLNO , and the effect of VA is larger on DLNO . This is because DLNO is more sensitive to DM than DLCO . As DLCO =VA is primarily determined by DB =VA (∝ VC =VA ), and DLNO =VA is equally determined by DM =VA and DB =V A (Fig. 2), the reduced VA -elicited augmentation in VC =VA is more pronounced on DLCO =VA than DLNO =VA . This suggests that a decline in VA produces a greater improvement of gas uptake for CO than NO.

The situation is different where reduced expansion is the result of a loss of acini, such as postpneumonectomy. When comparing a decreased V A without loss of acini to a decreased VA with loss of acini, the analysis for DLCO -associated parameters based on the proposal of Michael et al. [11] suggests the following (Fig. 5, Table 2): (1) a decrease in both DM and VC is associated with a larger decrease in DLCO (due to pneumonectomy); (2) a smaller increase in VC =VA is accompanied by an increase in DM =VA (attributed to the redistribution of Q); and (3) a smaller increase in DLCO =VA . The trend of DM =VA is opposite to that without loss of acini. The extent of increased VC =VA in lungs with loss of acini is less than that without loss of acini, leading to a restricted rise in DLCO =VA (i.e., a lesser improvement of CO uptake) in lungs with loss of acini. The increased Q results in capillary distension and/or recruitment, leading to an increased alveolocapillary membrane surface area (an increase in DM and DM =VA ) and pulmonary blood volume (an increase in V C and VC =V A ), which

increases

DL -associated

parameters

(Table

2).

Decreased Q results in the opposite effects. In patients with heart failure and reduced Q, DLCO and DLNO are decreased [40,41].

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]

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Fig. 5 – Effects of reduced VA with and without loss of acini on DLCO -related parameters The values are normalized against those obtained at the maximal V A .

5.3.

DL-related parameters in various lung diseases

There are conflicting reports on the effect of overweight or obesity on DL -related parameters. Saydain et al. [42] and Jones et al. [43] reported that DLCO increases with increasing body weight. However, Oppenheimer et al. [44] demonstrated no effect of body weight on DLCO . Furthermore, Biring et al. [45] reported that DLCO decreases as body weight increases. Zavorsky et al. [46] using simultaneous measurements of DLCO and DLNO , identified no increase in DL -related parameters in morbidly obese subjects. In heavy smokers and patients with chronic obstructive pulmonary disease (COPD), van der Lee et al. [47] reported that although DLCO , DLCO =VA , DLNO , and DLNO =VA decreased, DLNO =VA has the highest sensitivity for detecting the emphysematous lesions determined by the computed tomography (CT). In patients with diffuse parenchymal lung diseases (DPLD), van der Lee et al. [48] demonstrated that DLCO , DLCO =VA , DLNO , DLNO =V A , DMCO , and VC were significantly lower than in normal controls. Furthermore, the same investigators [48] showed that in patients with pulmonary arterial hypertension (PAH), DLCO , DLCO =V A , DLNO , DLNO =VA , DMCO , and VC showed decreases similar to the decreases observed in patients with DPLD. Although microvascular damage is a distinctive feature of PAH, these findings do not support the trait of microvascular damage, i.e., a relative maintenance of DM with a large reduction in VC (Table 2). Measuring DLCO - and DLNO -associated parameters in patients with cystic fibrosis (CF), Dressel et al. [49] found that although V C is not greatly reduced, other parameters, including DLCO , DLCO =VA , DMCO , DLNO , and DLNO =VA , are significantly decreased. The severity of CF as scored by CT, including bronchiectasis, mucus plugging, peribronchial thickening, and parenchymal diseases, is more sensitively detected by DLNO than DLCO . Furthermore, DLCO =VA is not correlated with the CT scores of CF. Using a rebreathing maneuver, Phansalkar et al. [50] identified that the DM and V C in patients with pulmonary sarcoidosis without overt fibrosis (stages II-III) were much lower than normal controls. The reduction of VC was significantly greater than the reduction of DM . These findings

suggest that microvascular damage plays a primary role in the pathology of pulmonary sarcoidosis. Based on these findings, Phansalkar et al. [50] hypothesized that in pulmonary sarcoidosis with little fibrosis, patchy alveolar deposition of noncaseating granulomas selectively obliterates pulmonary capillaries, which is accompanied by inflammation that may damage the alveolar membrane. However, the hypothesis proposed by Phansalkar et al. [50] has not been verified.

5.4. Differential diagnosis using DLCO- and DLNO-associated parameters The combination of DLCO - and DLNO -associated parameters can differentiate various acinar pathophysiological conditions (Table 2). As described in the section of 5.2, decreased DL with increased DL =VA is characteristic of decreased VA without the loss of acini (respiratory muscle dysfunction) or with the loss acini (pneumonectomy). The decrease in DL is more obvious in conditions with the loss of acini, while the increase in DL =VA is more obvious in conditions without the loss of acini. Increase in both DL and DL =VA can result from increased cardiac output (left-to-right shunt or exercise loading); redistribution of cardiac output (bronchial asthma); or alveolar hemorrhage with no change in VA . It should be noted that alveolar hemorrhage may be accompanied by a reduction in VA due filling alveoli with blood. In this case, the increase in DL =VA may be more obvious than when there is no change in VA . Decrease in both DL and DL =VA suggests the following: (1) the destruction of acinar structures, including alveolocapillary membrane and/or microvasculature (COPD, diffuse interstitial lung diseases (ILD), or microvascular diseases); (2) decreased density of erythrocytes (anemia); or (3) decreased cardiac output (heart failure). Moinard and Guenard [51] suggested that a reduction in VC is a specific feature of early COPD, while there is a concurrent reduction in DM and VC in advanced COPD. This suggests that DLCO and/ or DLCO =VA may act as a useful measure to sensitively detect the patchy destruction of microvasculature in early COPD, while DLNO and/or DLNO =VA may be valuable for detecting the

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

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respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]

pathological changes in alveolocapillary membrane and microvasculature in advanced COPD. This is because DLCO -associated parameters are primarily associated with changes in VC while the DLNO -associated parameters are equally influenced by changes in DM and VC (Fig. 2). Similarly,

Acknowledgements There is no financial support to be declared regarding the publication of this paper.

DLCO and/or DLCO =VA may be sensitive to microvascular damage associated with PAH or angiitis. However, DLNO and/ or DLNO =VA may be useful to detect progressive microvascular damage if it is associated with destruction of alveolar structures. The morphological changes in ILD are complicated, i.e.,

Conflict of interest The authors have no conflicts of interest.

there is destruction of alveolar and microvascular structures in association with a reduction in VA due to the loss of acini.

Appendix A.

Supporting information

The DLNO -related parameters, which detect abnormal DM and VC equally, are reduced in parallel to advancing ILD. However, in ILD, the decreased VA due to the loss of acini suppresses

Supplementary data associated with this article can be found in the online version at https://doi.org/10.1016/j.resinv.2017.12.006.

the reduction in DLNO =VA , leading, in some cases, to a reduction in DLNO but relative maintenance of DLNO =V A . If the reduction of DLCO -associated parameters is greater

r e f e r e n c e s

than the reduction of DLNO -associated parameters, it suggests that the primary pathophysiologic factor involved is pulmonary microvascular damage. In contrast, if the reduction in DLNO -associated parameters is greater than the reduction in DLCO -associated parameters, it suggests that the primary pathophysiologic factor involved is the destruction of the alveolocapillary membrane. When the reduction of DLCO - and DLNO -associated parameters is similar, the effect of the functional inhomogeneities, in addition to alveolocapillary membrane and/or microvasculature damage, is involved. The combination of DLCO - and DLNO -associated parameters enables us to detect the pathological changes and the functional inhomogeneities in the acinar regions more precisely compared to the single measure of DLCO or DLNO . The DLNO =DLCO ratio, first described by Guénard et al. [1], has been used by many investigators. However, this ratio has significant limitations in the diagnosis of acinar pathology [6]. A critique on the use of this ratio is provided in Supplement 8.

6.

Conclusions

The newly developed simultaneous one-step CO-NO technique requires only a single sample for the measurement of both DLCO and DLNO . The convenient simultaneous measurement of DLCO and DLNO can be used to determine DM and VC . Since DLCO is determined by DB and DLNO is equally determined by DM and DB , DLCO -associated parameters have a high sensitivity for detecting pulmonary microvascular damage, while DLNO -associated parameters equally detect alveolocapillary membrane damage and microvascular damage. When compared to measuring DLCO or DLNO , measuring both DLCO and DLNO improves the diagnostic precision of identifying pathological changes and functional inhomogeneities in the acinar regions. The authors suggest that the simultaneous measurement of DLCO and DLNO should be used clinically in the diagnosis of lung diseases in Japan.

[1] Gue´nard H, Varene N, Vaida P. Determination of lung capillary blood volume and membrane diffusing capacity in man by the measurements of NO and CO transfer. Respir Physiol 1987;70:113–20. [2] Borland CD, Higenbottam TW. A simultaneous single breath measurement of pulmonary diffusing capacity with nitric oxide and carbon monoxide. Eur Respir J 1989;2:56–63. [3] van der Lee I, Zanen P, Stigter N, van den Bosch JM, Lammers JW. Diffusing capacity for nitric oxide: reference values and dependence on alveolar volume. Respir Med 2007;101:1579–84. [4] Aguilaniu B, Maitre J, Gle´net S, Gegout-Petit A, Gue´nard H. European reference equations for CO and NO lung transfer. Eur Respir J 2008;31:1091–7. [5] Zavorsky GS, Cao J, Murias JM. Reference values of pulmonary diffusing capacity for nitric oxide in an adult population. Nitric Oxide 2008;18:70–9. [6] Martinot JB, Gue´nard H, Dinh-Xuan AT, Gin H, Dromer C. Nitrogen monoxide and carbon monoxide transfer interpretation: state of the art. Clin Physiol Funct Imaging 2017;37:357–65. [7] Zavorsky GS, Hsia CC, Hughes JM, Borland CD, Gue´nard H, van der Lee I, et al. Standardisation and application of the singlebreath determination of nitric oxide uptake in the lung. Eur Respir J 2017. http://dx.doi.org/10.1183/13993003.00962-2016 1600962. [8] Krogh M. The diffusion of gases through the lungs of man. J Physiol 1914;49:271–300. [9] Ogilvie C, Forster R, Blakemore W, Morton JW. A standardized breath-holding technique for the clinical measurement of the diffusing capacity of the lung for carbon monoxide. J Clin Investig 1957;36:1–17. [10] Roughton F, Forster R. Relative importance of diffusion and chemical reaction rates in determining rate of exchange of gases in the human lung, with special reference to true diffusing capacity of pulmonary membrane and issue of blood in the lung capillaries. J Appl Physiol 1957;11:290–302. [11] Hughes JM, Pride NB. Examination of the carbon monoxide diffusing capacity (DLCO) in relation to its KCO and VA components. Am J Respir Crit Care Med 2012;186:132–9. [12] Cotes JE, Chinn DJ, Quanjer PH, Roca J, Yernault JC. Standardization of the measurement of transfer factor (diffusing capacity). Eur Respir J 1993;16:41–52. http://dx.doi.org/ 10.1183/09041950.041s1693.

Please cite this article as: Yamaguchi K, et al. Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide. Respiratory Investigation (2018), https://doi.org/10.1016/j.resinv.2017.12.006

respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]

[13] Graham BL, Brusasco V, Burgos F, Cooper BG, Jensen R, Kendrick A, et al. 2017 ERS/ATS standards for single-breath carbon monoxide uptake in the lung. Eur Respir J 2017. http: //dx.doi.org/10.1183/13993003.00016-2016 1600016. [14] Murias JM, Zavorsky GS. Short-term variability of nitric oxide diffusing capacity and its components. Respir Physiol Neurobiol 2007;157:316–25. [15] Clutton-Brock J. Two cases of poisoning by contamination of nitrous oxide with higher oxides of nitrogen during anaesthesia. Br J Anaesth 1967;39:388–92. [16] Asadi AK, Sa´ RC, Kim NH, Theilmann RJ, Hopkins SR, Buxton RB, et al. Inhaled nitric oxide alters the distribution of blood flow in the healthy human lung, suggesting active hypoxic pulmonary vasoconstriction in normoxia. J Appl Physiol 2015;118:331–43. http://dx.doi.org/10.1152/japplphysiol.01354.2013. [17] Sheel AW, Edwards MR, Hunte GS, McKenzie DC. Influence of inhaled nitric oxide on gas exchange during normoxic and hypoxic exercise in highly trained cyclists. J Appl Physiol 2001;90:926–32. [18] Tamhane RM, Johnson Jr RL, Hsia CC. Pulmonary membrane diffusing capacity and capillary blood volume measured during exercise from nitric oxide uptake. Chest 2001;120:1850–6. [19] Crapo R, Morris A. Standardized single breath normal values for carbon monoxide diffusing capacity. Am Rev Respir Dis 1981;123:185–9. [20] Fowler WS. Lung function studies. II. The respiratory dead space. Am J Physiol 1948;154:405–16. [21] Hook C, Yamaguchi K, Scheid P, Piiper J. Oxygen transfer of red blood cells: experimental data and model analysis. Respir Physiol 1988;72:65–82. [22] Chick H, Lubrzynska E. The viscosity of some protein solutions. Biochem J 1914;8:59–69. [23] Goldstick TK, Ciuryla VT, Zuckerman L. Diffusion of oxygen in plasma and blood. Adv Exp Med Biol 1976;75:183–90. [24] Kreuzer F, Hoofd LJ. Facilitated diffusion of oxygen in the presence of hemoglobin. Respir Physiol 1970;8:280–302. [25] Stein TR, Martin JC, Keller KH. Steady-state oxygen transport through red blood cell suspensions. J Appl Physiol 1971;31:397–402. [26] Wilhelm E, Battino R, Wilcock RJ. Low-pressure solubility of gases in liquid water. Chem Rev 1977;77:219–62. [27] Carlsen E, Comroe Jr. JH. The rate of uptake of carbon monoxide and nitric oxide by normal human erythrocytes and experimentally produced spherocytes. J Gen Physiol 1958;4:83–107. [28] Borland CD, Dunningham H, Bottrill F, Vuylsteke A. Can a membrane oxygenator be a model for lung NO and CO transfer? J Appl Physiol 2006;100:1527–38. [29] Gue´nard HJ, Martinot JB, Martin S, Maury B, Lalande S, Kays C. In vivo estimates of NO and CO conductance for haemoglobin and for lung transfer in humans. Respir Physiol Neurobiol 2016;228:1–8. http://dx.doi.org/10.1016/j.resp.2016.03.003. [30] MacIntyre N, Crapo RO, Viegi G, Johnson DC, van der Grinten CP, Brusasco V, et al. Standardisation of the single-breath determination of carbon monoxide uptake in the lung. Eur Respir J 2005;26:720–35. http://dx.doi.org/10.1183/09031936.05.00034905. [31] Borland CD, Dunningham H, Bottrill F, Vuylsteke A, Yilmaz C, Dane DM, et al. Significant blood resistance to nitric oxide transfer in the lung. J Appl Physiol 2010;108:1052–60. http: //dx.doi.org/10.1152/japplphysiol.00904.2009. [32] Forster RE. Diffusion of gases across the alveolar membrane. In: Fishman AP, Farhi LE, Tenney SM, Geiger SR, editors, Handbook of Physiology, The Respiratory System, Gas Exchange, Bethesda MD: Am Physiol Soc, sect. 3, vol. IV, chap. 5; 1987, p. 71-88.

11

[33] Holland RAB. Rate at which CO replaces O2 from O2Hb in red cells of different species. Respir Physiol 1969;7:43–63. [34] Stam H, Kreuzer FJ, Versprille A. Effect of lung volume and positional changes on pulmonary diffusing capacity and its components. J Appl Physiol 1991;71:1477–88. [35] Reeves RB, Park HK. CO uptake kinetics of red cells and CO diffusing capacity. Respir Physiol 1992;88:1–21. [36] Borland CD, Cox Y. Effect of varying alveolar oxygen partial pressure on diffusing capacity for nitric oxide and carbon monoxide, membrane diffusing capacity and lung capillary blood volume. Clin Sci 1991;81:759–65. [37] Gehr P, Bachofen M, Weibel ER. The normal human lung: ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol 1978;32:121–40. [38] Weibel ER, Sapoval B, Filoche M. Design of peripheral airways for efficient gas exchange. Respir Physiol Neurobiol 2005;148:3–21. [39] Yamaguchi K, Mori M, Kawai A, Takasugi T, Asano K, Oyamada Y, et al. Ventilation-perfusion inequality and diffusion impairment in acutely injured lungs. Respir Physiol 1994;98:165–77. [40] Magini A, Apostolo A, Salvioni E, Italiano G, Veglia F, Agostino P. Alveolar-capillary membrane diffusion measurement by nitric oxide inhalation in heart failure. Eur J Prev Cardiol 2013. 〈http://www.ncbi.nlm.nih.gov/pubmed/24165475〉. [41] Zavorsky GS, Borland CD. Confusion in reporting pulmonary diffusing capacity for nitric oxide and the alveolar-capillary membrane conductance for nitric oxide. Eur J Prev Cardiol 2014. http://dx.doi.org/10.1177/2047487314528872. [42] Saydain G, Beck KC, Decker PA, Cowl CT, Scanlon PD. Clinical significance of elevated diffusing capacity. Chest 2004;125:446–52. [43] Jones RL, Nzekwu MM. The effects of body mass index on lung volumes. Chest 2006;130:827–33. [44] Oppenheimer BW, Berger KI, Rennert DA, Pierson RN, Norman RG, Rapoport DM, et al. Effect of circulatory congestion on the components of pulmonary diffusing capacity in morbid obesity. Obesity (Silver Spring) 2006;14:1172–80. [45] Biring MS, Lewis MI, Liu JT, Mohsenifar Z. Pulmonary physiologic changes of morbid obesity. Am J Med Sci 1999;318:293–7. [46] Zavorsky GS, Kim DJ, McGregor ER, Starling JM, Gavard JA. Pulmonary diffusing capacity for nitric oxide during exercise in morbid obesity. Obesity (Silver Spring) 2008;16:2431–8. http://dx.doi.org/10.1038/oby.2008.402. [47] van der Lee I, Gietema HA, Zanen P, van Klaveren RJ, Prokop M, Lammers JW, et al. Nitric oxide diffusing capacity versus spirometry in the early diagnosis of emphysema in smokers. Respir Med 2009;103:1892–7. http://dx.doi.org/10.1016/j. rmed.2009.06.005. [48] van der Lee I, Zanen P, Grutters JC, Snijder RJ, van den Bosch JMM. Diffusing capacity for nitric oxide and carbon monoxide in patients with diffuse parenchymal lung disease and pulmonary arterial hypertension. Chest 2006;129:378–83. [49] Dressel H, Filser L, Fischer R, Marten K, Mu¨ller-Lisse U, de la Motte D, et al. Lung diffusing capacity for nitric oxide and carbon monoxide in relation to morphological changes as assessed by tomography in patients with cystic fibrosis. BMC Pulm Med 2009;9:30. http://dx.doi.org/10.1186/1471-2466-9. [50] Phansalkar AR, Hanson CM, Shakir AR, Johnson RL, Hsia CC. Nitric oxide diffusing capacity and alveolar microvascular recruitment in sarcoidosis. Am J Respir Crit Care Med 2004;169:1034–40. [51] Moinard J, Guenard H. Determination of lung capillary blood volume and membrane diffusing capacity in patients with COLD using the NO-CO method. Eur Respir J 1990;3:318–22.

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Supplemental Materials Simultaneous measurement of pulmonary diffusing capacity for carbon monoxide and nitric oxide

S.1 practical procedures and equipments for simultaneous measurement of DLCO and DLNO The practical procedures of how to measure DLCO and DLNO simultaneously are described in S-Table 1 and the equipments allowing simultaneous measurements of DLCO and DLNO are shown in S-Table-2. S-Table 1. Practical procedures for simultaneous measurements of single-breath DLCO and DLNO Inspired CO concentration Inspired NO concentration Inspired He concentration

0.3% (for DLCO) 40-60 ppm (for DLNO) 10% (alternatively, 0.3% of methane or neon) (for VA) 21% (for mean alveolar PO2) 10 sec

Inspired O2 concentration Breath-holding time for high-sensitivity chemiluminescent NO analyzer (90% response time: 70 msec) Breath-holding time for low-sensitivity electrochemical NO analyzer (90%

4-6 sec

response time: 10 sec) 90% response time of CO analyzer

150 msec