respiratory investigation 56 (2018) 100 –110

Contents lists available at ScienceDirect

Respiratory Investigation

journal homepage: www.elsevier.com/locate/resinv

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

ar t ic l e in f o abs tra ct

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 simultaneously-
Available online 2 February 2018 estimated 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, overall
apparent diffusing capacity for the gas neglecting the effects of functional inhomogeneities (mL/min/mmHg); DB, blood component of
diffusing capacity for the gas (mL/min/mmHg); DL, diffusing capacity for the gas (mL/min/mmHg); DL/VA,, diffusing capacity per unit
alveolar volume (rate constant of alveolar gas uptake per unit pressure, mL/min/mmHg/L); DM, membrane component of diffusing
capacity for the gas (mL/min/mmHg); DPLD, diffuse parenchymal lung diseases; Hb, hemoglobin; HbO2, oxyhemoglobin;
He, helium; I, inspired gas; K, Krogh factor for the gas (calculated as S/(PB–PH2O) and equal to DL/VA); MW, molecular weight (g/moL);
PAH, pulmonary arterial hypertension; PAO2, mean alveolar PO2 (surrogate of mean capillary PO2, mmHg); PB, barometric pressure
(mmHg); PC, partial pressure of the gas in alveolar capillary (mmHg); PH20, vapor pressure at body temperature (mmHg); S, slope of
alveolar gas uptake during breath-holding; TL, transfer factor (equal to DL); TL/VA, transfer coefficient (equal to DL/VA); VA, alveolar
gas volume (L); VAT, inspired or expired alveolar tidal volume (L); VC, alveolar capillary blood volume (mL); VD, anatomical dead
space (mL); VI, inspired gas volume (L); α, 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: yamaguc@sirius.ocn.ne.jp (K. Yamaguchi), tsuji.takao@twmu.ac.jp (T. Tsuji),
kaoshiba@tokyo-med.ac.jp (K. Aoshiba), hiroyuki@tokyo-med.ac.jp (H. Nakamura).

https://doi.org/10.1016/j.resinv.2017.12.006
2212-5345/& 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
 respiratory investigation 56 (2018) 100 –110 101

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.
& 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Contents

1. Historical backgrounds on lung diffusing capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

2. Units and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3. Simultaneous measurement of DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.1. Basic parameters of DL/VA and DL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.2. Physical properties of CO and NO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.3. Specific gas conductance in the blood for CO and NO (θCO and θNO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.4. Estimation of DM and VC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.5. Influence of back-pressures of CO and NO on DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
3.6. Influence of functional inhomogeneities on DLCO and DLNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4. Reference equations for DL-associated parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5. Pathophysiological factors affecting DL-related parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.1. Age and height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.2. Alveolar volume (VA) and cardiac output (Q) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.3. DL-related parameters in various lung diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.4. Differential diagnosis using DLCO- and DLNO-associated parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Appendix A. Supporting information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

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 micro-
circulation [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 measure-
ment and interpretation of DLNO [7]. To encourage the use of
DLNO in Japan, the authors have comprehensively reviewed
the methodological and pathophysiological aspects of clini-
cally 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 mem-
brane diffusing capacity for CO (DMCO ) and (2) the blood
diffusing capacity for CO (DBCO ). The DMCO reflects the diffu-
sion limitation across the effective alveolocapillary mem-
brane, which consists of gas-phase diffusion (if any), the
alveolar wall, and the plasma layer surrounding the erythro-
cyte. 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 incorpo-
rates 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 VC from DLCO measured at two differ-
ent alveolar partial pressure of oxygen (alveolar PO2 ) (classic
two-step alveolar PO2 technique).
102 respiratory investigation 56 (2018) 100 –110
In the 1980s, Guénard et al. [1] and Borland and Higen-
bottamet [2] independently proposed a novel method of
simultaneously measuring DLCO and DLNO (simultaneous
one-step CO-NO technique). Differing from the classic two-
step alveolar PO2 technique, to determine DM and VC , the
simultaneous one-step CO-NO technique requires the mea-
surement 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 =V A (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 =VA ) 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 quan-
tities 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 atmo-
spheric 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 anal-
yzer 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 concentra-
tion 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,
V A is virtually equal to TLC. The VA is derived from the single-
breath helium dilution after subtracting the estimated ana-
tomic 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 VD 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 :
V A ¼ ðVI − V D 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
V D 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 =VA ) 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 breath-
holding (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
 respiratory investigation 56 (2018) 100 –110 103

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  V C ) 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 of the two gases (the Graham’s law). Therefore, the dY =dX
calculated as follows: can be assumed to be the same in any diffusion path. The
FXAð0Þ ¼ ðHeAðtÞ =HeI Þ  FXI ð4Þ relative Krogh diffusion constant of NO against CO (ΥNO=CO Þ is
defined as:
In this equation, FXI is the gas concentration of CO or NO in
ΥNO=CO ¼ ðα  dÞNO =ðα  dÞCO ð7Þ
the inspired gas. From SX , KX and DLX are assumed as:
KX ¼ SX =ðPB − PH2O Þ ð5Þ The ΥNO=CO is the major factor determining the ratio of
membrane diffusing capacity for NO (DMNO ) to CO (DMCO ).
DLX ¼ VA  KX ð6Þ Originally proposed by Wilhelm et al. [26], subsequent studies
agree that the best value of ΥNO=CO in the acinus is 1.97. Thus,
where PB denotes the atmospheric pressure and PH2O the
the DMNO =DMCO can be given by:
vapor pressure.
DMNO =DMCO ¼ 1:97 ð8Þ

3.2. 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 alveolo-
capillary 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 hu-
man biconcave erythrocytes is not infinite but is finite at
104 respiratory investigation 56 (2018) 100 –110

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

1=θCO Conditions References

0.0058  PO2 þ 1.00 ω ¼ 1.5, pH ¼8.0 (rapid-reaction, constant-flow) Roughton (1957) [10]
0.0058  PO2 þ 0.73 ω ¼ 2.5, pH ¼8.0 (rapid-reaction, constant-flow) Roughton (1957) [10]
0.0058  PO2 þ 0.33 ω ¼ 1, pH ¼ 8.0 (rapid-reaction, constant-flow) Roughton (1957) [10]
0.0041  PO2 þ 1.30 ω ¼ 1, pH ¼ 7.4 (rapid-reaction, constant-flow) Forster (1987) [32]
0.0065  PO2 þ 1.08 ω ¼ 1.5 at standard conditions (stopped-flow) Holland (1969) [33]
0.0055  PO2 þ 0.73 Assumed value (ω ¼ 1, pH ¼ 7.4) Stam (1991) [34]
0.0080  PO2 þ 0.0156 Thin blood film exposed to step-change of PCO at varied PO2 Reeves (1992) [35]
0.0062  PO2 þ 1.16 Indirect estimation in vivo (optimal equation preserving DM =VC at the same level under hypoxia Guénard (2016) [29]
and normoxia)

1/θNO Conditions References

1/4.5 Rapid-reaction, constant-flow Carlsen (1958) [27]

0 Assumed value based on infinitely fast reaction of NO with Hb Guénard (1987) [1]
1/3.3 Indirect estimation in vivo Borland (1991) [36]
1/4.0 Indirect estimation in vitro (horse blood) Borland (2006) [28]
1/3.0 Indirect estimation in vivo (optimal value preserving DM =V C at the same level under hypoxia and Guénard (2016) [29]
normoxia)

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 and Ht-dependent change in plasma layer thickness, should
results, they concluded that the NO uptake process by be made using the equations recommended by the 2005
erythrocytes is predominantly limited by diffusion within ATS/ERS Task Force for DLCO [30] as follows:
the erythrocyte but not by a chemical reaction of NO with Hb.

DLCO predicted; male ¼ DLCO  ð10:22 þ HbÞ=ð1:7  HbÞ ð10  1Þ
Although five values for θNO have been reported to date


(Table 1), the θNO reported by Carlsen and Comroe [27] is the DLCO predicted; female ¼ DLCO  ð9:38 þ HbÞ=ð1:7  HbÞ ð10  2Þ
only directly and reliably measured estimate. Therefore, it is
In these equations, DLCO and Hb (g/dL) are the measured
recommended to use 4.5 mL/min/mmHg/mL as the θNO [7].
values.
Further disavowal against reaction limitation during NO
The situation of DLNO differs from that of DLCO . Since the
uptake by erythrocytes was certified by Borland et al. [2,28],
concentration of NO used for DLNO measurement is low (i.e.,
who identified that DLCO is PO2 - and hematocrit (Ht)-depen-
ppm), it is not necessary to correct for Hb absorption of NO.
dent, whereas DLNO is PO2 -independent but Ht-dependent.
Furthermore, due to a large Krogh diffusion constant of NO
The PO2 -independent DLNO implies that NO uptake in the
(Supplementary Table 3), the relative contribution of diffu-
acinus is not limited by chemical reaction between NO and
sion-limited NO transfer, resulting from a Ht-dependent
Hb. The Ht-dependent DLNO indicates that NO uptake is
change in the thickness of the plasma layer, is anticipated
impeded by decreased density of erythrocytes (anemia),
to be small. Indeed, Borland et al. [31] described that the Hb
which results in increased thickness of the plasma layer
adjustment for DLNO is unnecessary until the Hb concentra-
and impairs NO uptake. The PO2 - and Ht-dependent change
tion is approximately 4 g/dL. Therefore, the constant value of
in DLCO indicates that both diffusion limitation and reaction
θNO may be valid under a wide variety of pathophysiological
limitation are involved in CO uptake. As such, although the
conditions, including anemia.
θNO is constant and independent of capillary PO2 , the θCO
changes depending on capillary PO2 . Descriptions of
3.4. Estimation of DM and VC
PO2 -dependent θCO are given in Supplementary Fig. 1. Among
the equations that predict the relationship between PO2 and
The simultaneous equations described below use the mea-
1=θCO (Table 1), the 2017 ERS Standards for DLNO [7] recom-
sured values of DLCO , DLNO , mean alveolar PO2 (PAO2 ), and Hb
mend the equation proposed by Guénard et al. [29]. The
to determine DM and VC :
Guénard et al. equation is:
1=DLCO ¼ 1=DMCO þ 1=ðθCO  VC Þ ð11  1Þ
1=θCO ¼ ð0:0062  PO2 þ 1:16Þ  ðstandard Hb=measured HbÞ ð9Þ
1=DLNO ¼ 1=DMNO þ 1=ðθNO  VC Þ ð11  2Þ
where PO2 is mean capillary PO2 approximated by alveolar PO2
measured at the end of breath-holding. Standard Hb is DMNO ¼ 1:97  DMCO ð11  3Þ
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 1=θCO ¼ ð0:0062  PAO2 þ 1:16Þ  ðstandard Hb=measured HbÞ
measurements. Furthermore, attention should be paid for the ð11  4Þ
Hb term, which aims to correct the Hb capacitance effect on
1=θNO ¼ 1=4:5 ð11  5Þ
absorbing CO, but not the diffusion limitation elicited by
Ht-dependent modification of plasma layer thickness. The Using the previously described simultaneous equations, the
overall correction by Hb, including both Hb capacitance effect relative contribution of 1=DM and 1=DB ð1=ðθ  VC ÞÞ to 1=DLCO or
 respiratory investigation 56 (2018) 100 –110 105

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 limita-
tions caused by diffusion and chemical reaction in the ery-
throcyte, 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 con-
verted to ∼170 of DMO2 , if the relative Krogh diffusion constant
of O2 against NO (ΥO2=NO ) is 0.66. However, the morphometri-
cally 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 ¼ 1, 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).
106 respiratory investigation 56 (2018) 100 –110

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

DLCO and DLNO
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 inhala-
tion [28].
The CO back-pressure during DLCO measurements is esti-
mated at ∼3% of the alveolar PCO in a nonsmoking subject
(Supplementary Fig. 2), indicating that the effect of CO back-
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
that should not be disregarded (Supplement 4).
4. Reference equations for DL-associated
parameters
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 coeffi-
cients 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.
5. Pathophysiological factors affecting
DL-related parameters
5.1. Age and height

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 inhomo-
geneities. These inhomogeneities include: (1) the distribution
of inspired/expired alveolar tidal volume to alveolar volume

(2) for both sexes, the decline in DMCO with aging is larger than
V ATi =VAi ; (2) the ratio of alveolar tidal volume in each region

the decrease in VC , suggesting that the age-dependent dis-
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
destruction of alveolocapillary membrane and/or pulmonary
microcirculation.
The effect of the gas-phase diffusion limitation on overall
gas transfer of CO and NO in the acinar regions is discussed
in Supplement 6, which concludes that the gas-phase diffu-
sion-related interference with gas transfer of CO and NO is
small [39].
The regression equations constructed by the 2017 ERS Stan-
dards for DLNO [7] identified that all DL -associated parameters
decrease with aging irrespective of sex (Supplementary
Table 4). As height increases, DLCO , DLNO , and VC increase,
which is explained by the height-dependent increase in lung
volume. Although the absolute values differ, the DL -asso-
ciated parameters normalized by their maximal values sug-
gest the following (Supplementary Fig. 5): (1) the negative
impact of aging on DLCO and DLNO is analogous for both sexes;
tortion 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.
5.2. Alveolar volume (VA) and cardiac output (Q)
The modified Roughton-Foster formula is useful to under-
stand the complicated VA -elicited changes in DLCO - and
DLNO -related parameters. The modified Roughton-Foster for-
mula is shown below:

Fig. 4 – Effect of reduced V A without loss of acini on DL -related parameters. The values are normalized against those obtained
at the maximal VA . DM indicates DMCO .
 respiratory investigation 56 (2018) 100 –110 107

Table 2 – Pathophysiological factors affecting DLCO - and DLNO -related parameters.

DM =VA V C =V A DL DL =VA

Age ↓ ↓ ↓ ↓
(DM ↓) (VC ↓)

Height ↓(1) ∼ ↑ ∼
(DM ∼) (VC ↑)

Reduced V A
Without loss of acini ↓ ↑↑ ↓ ↑↑
Respiratory muscle weakness (DM ↓) (VC ∼)

Chest wall/pleural restriction

With loss of acini ↑ ↑ ↓↓(2) ↑(3)
Pneumonectomy (DM ↓) (VC ↓)
Increased Q or redistribution of Q ↑ ↑ ↑ ↑
Left-to-right shunt (DM ↑) (VC ↑)

Exercise

Asthma (redistribution of Q)
Reduced Q ↓ ↓ ↓ ↓
Heart failure (DM ↓) (VC ↓)
Microvascular damage ∼↓ ↓ ↓ ↓
Pulmonary arterial hypertension (DM ∼↓) (VC ↓)

Angiitis
Alveolar hemorrhage ↑ ∼ ↑ ↑
Without change in VA (DM ↑) (VC ∼)
Reduced density of erythrocytes ↓ ∼ ↓ ↓
Anemia (DM ↓) (VC ∼)
Destruction of acinar structures
COPD (destruction of alveoli and microcirculation) ↓ ↓ ↓ ↓
(DM ↓) (VC ↓)

Diffuse interstitial lung diseases (destruction of alveoli ↓ ∼↓ ↓ ∼↓

and microcirculation þ reduced V A with loss of acini) (DM ↓) (VC ↓)

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

1= DLX =V A ¼ 1=ðDMX =VA Þ þ 1=ðθX  VC =VA Þ
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 VA 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 =V A (KCO ) than DLNO =VA (KNO )
(Fig. 4, Table 2). Although DM =VA has been considered to
remain constant with decreasing VA [13,34], the analysis
indicates that in lungs without a loss of acini, the reduction
in DM and V A 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 =V A increases with decreasing V A . Reduced VA decreases
DM , but does not change VC , resulting in decreased DLCO and
DLNO , and the effect of V A is larger on DLNO . This is because
DLNO is more sensitive to DM than DLCO . As DLCO =VA is
primarily determined by DB =V A (∝ VC =VA ), and DLNO =VA is
equally determined by DM =VA and DB =VA (Fig. 2), the reduced
VA -elicited augmentation in V C =VA is more pronounced
on DLCO =V A than DLNO =V A . This suggests that a decline in VA
produces a greater improvement of gas uptake for CO
than NO.
ð12Þ The situation is different where reduced expansion is the
result of a loss of acini, such as postpneumonectomy. When
comparing a decreased VA without loss of acini to a
decreased VA with loss of acini, the analysis for DLCO -asso-
ciated 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 accom-
panied by an increase in DM =VA (attributed to the redistribu-
tion of Q); and (3) a smaller increase in DLCO =VA . The trend of
DM =V A 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 =V A (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 VC and
VC =VA ), 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 decrea-
sed [40,41].
108 respiratory investigation 56 (2018) 100 –110
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 VA .
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 =VA , DMCO , and VC were significantly lower
than in normal controls. Furthermore, the same investigators
[48] showed that in patients with pulmonary arterial hyper-
tension (PAH), DLCO , DLCO =VA , 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 VC is not greatly reduced, other parameters, includ-
ing DLCO , DLCO =VA , DMCO , DLNO , and DLNO =V A , 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 =V A is not correlated with
the CT scores of CF.
Using a rebreathing maneuver, Phansalkar et al. [50]
identified that the DM and VC in patients with pulmonary
sarcoidosis without overt fibrosis (stages II-III) were much
lower than normal controls. The reduction of V C was sig-
nificantly 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 pulmon-
ary sarcoidosis with little fibrosis, patchy alveolar deposition
of noncaseating granulomas selectively obliterates pulmon-
ary capillaries, which is accompanied by inflammation that
may damage the alveolar membrane. However, the hypoth-
esis 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 condi-
tions (Table 2). As described in the section of 5.2, decreased
DL with increased DL =VA is characteristic of decreased V A
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 =V A is more obvious in conditions without the
loss of acini.
Increase in both DL and DL =V A can result from increased
cardiac output (left-to-right shunt or exercise loading); redis-
tribution 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 reduc-
tion 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 alveoloca-
pillary 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
 respiratory investigation 56 (2018) 100 –110
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 V C (Fig. 2). Similarly,
DLCO and/or DLCO =V A may be sensitive to microvascular
damage associated with PAH or angiitis. However, DLNO and/
or DLNO =V A may be useful to detect progressive microvascular
damage if it is associated with destruction of alveolar struc-
tures. The morphological changes in ILD are complicated, i.e.,
there is destruction of alveolar and microvascular structures
in association with a reduction in VA due to the loss of acini.
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
the reduction in DLNO =VA , leading, in some cases, to a
reduction in DLNO but relative maintenance of DLNO =VA .
If the reduction of DLCO -associated parameters is greater
than the reduction of DLNO -associated parameters, it suggests
that the primary pathophysiologic factor involved is pulmon-
ary 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 func-
tional inhomogeneities, in addition to alveolocapillary mem-
brane 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 com-
pared 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 techni-
que requires only a single sample for the measurement of
both DLCO and DLNO . The convenient simultaneous measure-
ment of DLCO and DLNO can be used to determine DM and VC .
Since DLCO is determined by DB and DLNO is equally deter-
mined by DM and DB , DLCO -associated parameters have a high
sensitivity for detecting pulmonary microvascular damage,
while DLNO -associated parameters equally detect alveoloca-
pillary 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.
109
Acknowledgements
There is no financial support to be declared regarding the
publication of this paper.
Conflict of interest
The authors have no conflicts of interest.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at https://doi.org/10.1016/j.resinv.2017.12.006.
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