J Appl Physiol
91: 2024–2034, 2001.
NO2 interfacial transfer is reduced
by phospholipid monolayers
LYDIA M. CONNOR,1 AKHIL BIDANI,1 JON GOERKE,2,3
JOHN A. CLEMENTS,2,4 AND EDWARD M. POSTLETHWAIT1
1
Pulmonary and Critical Care Medicine, Department of Internal Medicine, University
of Texas Medical Branch, Galveston, Texas 77555; and 2Cardiovascular Research
Institute, Departments of 3Physiology and 4Pediatrics, University of California
at San Francisco, San Francisco, California 94143
Received 27 March 2001; accepted in final form 9 July 2001
(NO2) is a free radical atmospheric
toxicant that initiates a broad range of pulmonary
pathophysiological responses. Inhalation of NO2 induces dose-dependent effects on both conducting airway and alveolar epithelia. Acute low-level exposures
result in oxidative stress, the induction of characteristic epithelial lesions spanning the bronchoalveolar re-
gion, and inflammation (13, 44). Chronic NO2 exposure
produces alterations in lung architecture and obstructive lesions that result in a reduction of both available
alveolar surface area and lung compliance (13, 44).
Although the pathological consequences of both acute
and chronic NO2 intoxication have been extensively
documented, the mechanisms that govern NO2 intrapulmonary dispersion and the induction of tissue
injury have not been fully characterized.
The distribution of acute epithelial injury is related,
in part, to the intrapulmonary airspace concentration
of NO2 and its local rate of absorption. The pulmonary
epithelium is overlain by a continuous but inhomogeneous aqueous layer, epithelial lining fluid (ELF),
which covers all airspace surfaces and protrusions (2).
Absorption of inhaled NO2 is governed by near-interfacial reactions with ELF constituents that serve to
maintain the net driving force for the flux of NO2 from
the gas phase into the ELF, a process termed “reactive
absorption” (33). Because absorption is directly coupled
to reaction, diffusion of NO2 through the ELF is presumed to be limited (3, 35, 52). Products formed as a
consequence of reactive absorption likely initiate the
cascade of events leading to cell injury.
The small-molecular-weight antioxidants glutathione, ascorbic acid, and uric acid have been identified as
primary substrates for NO2 reactive absorption (35,
52). Interestingly, although unsaturated fatty acids as
components of phospholipids appear in abundance in
the lung aqueous surface compartment, under environmentally relevant conditions, NO2 reacts less with unsaturated fatty acids than with other available substrates (14, 35, 37, 38). NO2 reactive absorption
proceeds most rapidly during electron transfer reactions with antioxidant anions, which can exist in significant concentration at lung ELF pH (33, 35, 52).
Under quasi-steady-state, well-mixed exposure conditions (NO2 inflow constant), NO2 absorption rapidly
attains an aqueous substrate concentration-dependent
rate that does not increase with time although the
Address for reprint requests and other correspondence: E. M.
Postlethwait, Environmental Toxicology, Dept. of Preventive Medicine & Community Health, Univ. of Texas Medical Branch, 301 Univ.
Blvd., Galveston, TX 77555-1110 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
nitrogen dioxide; reactive absorption; pulmonary surfactant;
interfacial resistance; lung epithelial lining fluid; dipalmitoyl
phosphatidylcholine
NITROGEN DIOXIDE
2024
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
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Connor, Lydia M., Akhil Bidani, Jon Goerke, John A.
Clements, and Edward M. Postlethwait. NO2 interfacial
transfer is reduced by phospholipid monolayers. J Appl
Physiol 91: 2024–2034, 2001.—Nitrogen dioxide (NO2) is a
ubiquitous, pollutant gas that produces a broad range of
pathological and physiological effects on the lung. Absorption
of inhaled NO2 is coupled to near-interfacial reactions between the solute gas and constituents of the airway and
alveolar epithelial lining fluid. Although alveolar surfactant
imparts limited resistance to respiratory gas exchange compared with that contributed by either the pulmonary membrane or uptake in red blood cells, resistance to NO2 flux
could have a significant effect on NO2 absorption kinetics. To
investigate the effect of interfacial surfactant on NO2 absorption, we designed an apparatus permitting exposure of variably compressed monolayers. Our results suggest that compressed monolayers enriched in 1,2-dipalmitoyl-sn-3-glycerophosphocholine present significant resistance to NO2
absorption even at surface tensions greater than those
achieved in vivo. However, monolayers composed of pure
unsaturated phospholipids failed to alter NO2 absorption
significantly when compressed, in spite of similar reductions
in surface tension. The results demonstrate that phospholipid monolayers appreciably limit NO2 absorption and further that monolayer-induced resistance to NO2 flux is related
to physicochemical properties of the film itself rather than
alterations within the aqueous and gas phases. On the basis
of these findings, we propose that pulmonary surfactant may
influence the intrapulmonary gas phase distribution of inhaled NO2.
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
with 9.0 ml of 0.15 M NaCl. The BALF was pooled, centrifuged at 150 ⫻ g for 10 min to remove cells and debris and
subsequently at 60,000 ⫻ g for 2.5 h to isolate the surfaceactive components (modified from Stevens et al., Ref. 50). The
pellet was extracted by a modified Bligh-Dyer method (5),
with subsequent double reextraction of the upper aqueous
phase. The pooled crude extract was dried under N2 and
redissolved in chloroform for gravimetric estimation of total
extractable material. These weights probably overestimate
the true phospholipid weights because of the presence of
other lipids and hydrophobic proteins. For surface deposition,
solutions (1 mg/ml) were prepared in chloroform, stored under N2 at ⫺20°C, and used within 72 h.
Compressed monolayer studies. An exposure probe-surface
balance with a pantographic barrier was utilized to examine
NO2 absorption across phospholipid monolayers at several
surface tensions (24 ⫾ 1°C) (Fig. 1A). Surface tension was
measured as described below. The glass probe was modeled
after a previously described design (48). Briefly, pollutant gas
entered the probe chamber via a sidearm port and exited
around a disk such that flow proceeded centrally over a
defined surface area (28.3 cm2) (Fig. 1B). The sampling line of
the nitrogen oxides (NOx) analyzer (see below) was connected
to the exit port to actively withdraw gas through the central
tube. Gas flow into the exposure system exceeded the sam-
METHODS
Reagents. DPPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphorac-glycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), and 1,2-dilinoleoyl-sn-glycero-3phosphocholine (DLPC), all in powder form, were purchased
from Avanti Polar Lipids (Alabaster, AL) and used as received. Glutathione (GSH), ascorbic acid (AH2), desferrioxamine, and SeKem agarose were purchased from Sigma
Chemical (St. Louis, MO). Tween 80, HPLC-grade chloroform, methanol, and isopropanol and all other reagents, of
analytical grade, were purchased from Fisher Scientific
(Houston, TX). NO2 and nitric oxide cylinders were purchased from Liquid Carbonic (Pasadena, TX) as certified
standards in N2.
Surfactant isolation. Surfactant was isolated from viral
antigen-free, 250–275 g, male Sprague-Dawley rats (Harlan
Sprague Dawley, Houston, TX). The protocol for harvesting
bronchoalveolar lavage fluid (BALF) was approved by the
Animal Care Use Committee of the University of Texas
Medical Branch, Galveston. Animals were allowed free access to water and food before induction of anesthesia. Animals were anesthetized with 70 mg/kg intraperitoneal pentobarbital sodium and, after tracheal cannulation and
midline thoracotomy, lungs were lavaged in situ five times
J Appl Physiol • VOL
Fig. 1. Schematic representation of the exposure probe-surface balance apparatus. Individual components are identified by letters. A:
Pantograph barrier. Glass exposure probe (a); hinged, deformable
stainless steel frame with Teflon ribbon lining (maximum surface
area ⫽ 387 cm2, minimum surface area ⫽ 157 cm2) (b); platinum
plate (c); and manual leadscrew (d). Plexiglas dish (in which the
compression frame was mounted), adjustable platform, force transducer assembly and enclosure are not shown. B: Vertical crosssection of the probe and aqueous interface: gas inlet port (e); has exit
port to nitrogen oxides (NOx) analyzer (f), central restriction disk (g),
height ⫽ 5 mm (h), and clearance ⫽ 1 mm (i).
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uptake efficiency is ⬍100% (33). This phenomenon can
be observed under transient exposure conditions (NO2
as the limiting reagent) in which the first-order rate
constant for the disappearance of NO2 from the gas
phase plateaus despite further increases in aqueousphase substrate concentration or mixing (34). These in
vitro studies are in agreement with theoretical analyses that suggest that appreciable mass transfer limitations are present at the gas-liquid interface (3, 10).
Pulmonary surfactant functions to prevent alveolar
collapse at end-expiratory lung volumes by lowering
surface tension and, thereby, maintaining high lung
compliance (9, 47). The most abundant surface active
component is 1,2-dipalmitoyl-sn-3-glycero-phosphocholine (DPPC), which may be enriched within the monomolecular film present at the air-liquid interface (30,
51). In general, the flux of respiratory gases across the
serial resistances encountered in transit from the air
space to red blood cells (e.g., surface fluid compartment, tissue, plasma, and red blood cell) is thought to
proceed with limited restriction (53). However, previous experimental evidence suggests that the physicochemical characteristics of the gas-liquid interface may
influence the flux of molecules between the gas and
aqueous phases. For example, interfacial films of oils
and aliphatic alcohols have been demonstrated to limit
significantly the in vitro rate of water evaporation (1,
23), CO2 absorption (4), and O2 transport (21) across
gas-liquid interfaces and to restrict the rate of gasliquid reactions (10). On the basis of these previous
observations, we investigated whether monolayers of
surface active phospholipids alter the interfacial flux
(absorption) of NO2. We utilized an in vitro exposure
apparatus, which facilitated 1) control of phospholipid
monolayer and aqueous phase composition, 2) continuous measurement of surface tension, and 3) determination of steady-state NO2 absorption rates.
2025
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PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
J Appl Physiol • VOL
directly withdrawn into a 50-ml glass syringe containing 10
ml Saltzman reagent (33), and the concentration was determined on the basis of daily standard curves. In both types of
apparatus, NO2 absorption was computed on the basis of the
mass balance across the exposure system and expressed as
either 1) the absorption rate per unit surface area {([NO2]i⫺
[NO2]e) 䡠 outlet flow/surface area}, where [NO2]e is NO2 concentration at the exit port, or 2) the fractional uptake
{([NO2]i ⫺ [NO2]e)/[NO2]i}. Theoretical analyses of the probe
exposure system characteristics and the derivation of a film
resistance term considered in the DISCUSSION are contained in
the APPENDIX.
Surface tension measurements. Surface tension (␥) was
measured by using a flamed 2.5-cm-wide roughened platinum plate. Before phospholipid deposition, the aqueous surface was thoroughly cleaned by aspiration such that ␥ ⫽ 71 ⫾
3 mN/m (25°C) was maintained during several compressionexpansion cycles. Surface tension was measured directly on
all monolayers studied in the probe-surface balance apparatus. Because of geometric limitations, surface tension measurements for the flask studies were determined separately
on films contained in open glass vessels of equivalent interfacial geometry.
Reaction product analyses. Both probe and flask aqueous
pools were sampled for reaction products. Nitrite (NO2⫺)
concentrations were determined via the Greiss reaction (36)
using standards containing 1.0 mM GSH. Glutathione analyses were performed via HPLC (18). Briefly, immediately
after the termination of an exposure, 1.5 ml of aqueous phase
was withdrawn, diluted 1:2 in HPLC mobile phase, and
stored at 4°C in the dark. Samples (15 ␮l) were injected onto
a 15 ⫻ 150-mm Waters C18 Novapak column and eluted
isocratically at 1.0 ml/min by using 3% methanol in 10 mM
phosphate buffer (pH 3.0) containing 50 ␮M tetrabutyl-ammonium hydrogen sulfate. Peaks were detected at 220 nm
and integrated to compute peak area. GSH was estimated on
the basis of standard curve peak areas. Each sample was
analyzed in triplicate, and a mean peak area was used to
compute concentration.
Data analysis. All values are expressed as means ⫾ SD.
Mean differences between groups were tested by ANOVA and
Dunnett’s test post hoc (49). Significance was defined as P ⬍
0.05.
RESULTS
Effect of DPPC monolayers on NO2 absorption. DPPC
monolayers were deposited onto 1.0 mM GSH solutions
and exposed to NO2 for up to 1 h. During the course of
a single exposure, after the three initial compressionexpansion cycles, monolayers were compressed to four
set areas from an initial molecular area of 121 to 49
Å2/molecule. The presence of the probe prevented further reductions in interfacial surface area or surface
tension. Between 10 and 15 min were allotted at each
stage of compression to permit determination of NO2
absorption as well as measurement of stable surface
tensions. As demonstrated in Fig. 2, when the DPPC
monolayers were cyclically compressed and reexpanded, NO2 absorption rates were coupled to surface
tension. NO2 uptake decreased linearly with surface
tension (Fig. 3; r ⫽ 0.99), attaining a maximal reduction of 47% from the monolayer-free surface value (P ⬍
0.05). On compression, NO2 uptake rates were stable
over ⬎ 45-min periods.
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pling rate by 10–15%. The compression apparatus consisted
of a stainless steel, Teflon ribbon-lined pantograph frame
(17 ⫻ 25 cm) mounted in a Plexiglas dish (20 ⫻ 45 cm), which
contained the aqueous phase. Phospholipids were deposited
within the pantograph frame from chloroform solutions (1
mg/ml) by use of a 25-␮l syringe to achieve an initial area per
molecule of 121 Å2/molecule (⬃0.1 ␮g/cm2). The compression
system was mounted on a moveable platform to permit adjustment of the distance between the aqueous surface and
the bottom edge of the probe. The entire apparatus was
isolated in an enclosure to prevent room air currents from
disturbing the flow pattern under the probe.
NO2 exposure protocols. For exposure of compressed films,
500 ml of buffer (10 mM potassium phosphate, pH 7.0) with
or without GSH (1.0 mM) was placed in the Plexiglas dish,
and the aqueous surface was cleaned by aspiration. The
system was raised to achieve a 1-mm separation between the
bottom surface of the probe and the aqueous surface. The
probe gas inlet and exit lines were connected, and the system
enclosure was closed. After a 10-min control exposure period,
a phospholipid monolayer was deposited from solvent
through a port and allowed to equilibrate for 5 min. Subsequently, three full compression-expansion cycles were performed before compression to the final set area(s) to achieve
a uniform phosphatidylcholine (PC) film devoid of bare areas.
Alternatively, exposures were conducted in a different apparatus utilizing 50-ml flat-bottom Erlenmeyer flasks (33)
when it became necessary either to limit the aqueous phase
volume or to prevent contamination of the compression system. For these exposures, 10 ml of buffer (10 mM or 50 mM
potassium phosphate, pH 7.0) with or without GSH (1.0–50.0
mM) or AH2 (1.0 mM) was introduced into the flask. A
phospholipid film was deposited at a nominal surface density
of 0.5 ␮g/cm2 onto a surface area of 15.9 cm2 and allowed to
equilibrate for 10 min before NO2 delivery. Flasks were
equipped with a Teflon-lined stopper with inlet and exit ports
that extended within 1.0 and 5.0 cm above the interface,
respectively, with the outlet port being sampled by the NOx
analyzer. Films were not compressed after spreading in this
system. Exposures generally lasted 30–45 min. Inlet NO2
concentrations ([NO2]i) were determined before and after
each exposure with the probe or flask out of line.
NO2 exposures. Exposures conducted in both the probe and
flask systems were performed under unstirred, quasi-steadystate conditions (NO2 inflow constant) at 25°C unless stated
otherwise (34). For most studies, [NO2]i was ⬍10 ppm. In this
range, NO2 absorption displays first-order kinetics with respect to [NO2]i (34). GSH (1.0 mM) was used most often as
the aqueous substrate because it effectively drives NO2 absorption (35, 52) and can be maintained in stable concentration with limited autoxidation. Exposure atmospheres were
generated by countercurrent injection of high-concentration
NO2 (in N2) into temperature-equilibrated, humidified air by
using mass flow controllers (Scott Specialty Gas, Houston,
TX) adjusted to achieve the appropriate final NO2 concentrations. Inlet gas flow was controlled to exceed sample flow by
10–15% (128.9 ⫾ 2.4 or 150.9 ⫾ 7.2 ml/min) for both the
probe and flask systems. NO2 exit concentrations were continuously monitored using a model 42 chemiluminescence
NO NO2 NOx analyzer (Thermoenvironmental, Franklin,
MA). The analyzer internal pump sampled at a rate of
112.9 ⫾ 1.6 or 131.8 ⫾ 2.25 ml/min (flow increased after
scheduled pump maintenance) irrespective of the exposure
apparatus used. The analyzer was routinely calibrated by
using a primary standard of 10 ppm NO. Occasionally, analysis of gas phase NO2 was performed via the Saltzman
procedure (17) in which a known volume of exit gas was
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
2027
Fig. 2. Effect of cyclic surface compression on simultaneously recorded surface tension (A) and NO2 concentration ([NO2]) at exit port
([NO2]e) (B). 1,2-Dipalmitoyl-sn-3-glycero-phosphocholine (DPPC)
monolayers were deposited onto 1.0 mM glutathione (GSH) and
exposed to 5.2 ppm NO2. Monolayers were compressed in the surface
balance reducing surface area by 60% at maximum compression
(from an initial phospholipid surface density of 121 Å2/molecule)
through 3 compression and 2 expansion cycles. Plots represent typical compression/expansion cycles that preceded monolayer exposures at selected set area(s). Decreasing the area in the absence of a
surface film did not change either surface tension or [NO2] in the exit
gas. The plot of [NO2] slightly lags the plot of monolayer surface
tension because of delayed NO2 analyzer response.
Similar results were obtained when exposures were
conducted at elevated temperatures (⬃30°C, [NO2] ⫽
7.6 ⫾ 0.2 ppm, 44.3 ⫾ 1.7, and 34.5 ⫾ 2.3 ng 䡠
min⫺1 䡠 cm⫺2 without and with compressed DPPC, respectively, data not shown). In contrast to previous
observations (33), under the experimental conditions
employed we observed only a modest rise in NO2 uptake when the temperature of uncompressed DPPC
systems was increased. Background NO2 loss was approximately twofold greater at the elevated temperature, likely because of condensation of water on system
surfaces, increased NO2 reaction with water, and increased diffusivity of solute NO2 and nitrite within the
J Appl Physiol • VOL
Fig. 3. Relationship between NO2 absorption and surface tension
with graded compression of DPPC monolayers in the surface balance. DPPC was deposited at an initial surface density of 121
Å2/molecule (⬃0.1 ␮g/cm2) onto 1 mM GSH solutions and exposed to
5.2 ppm NO2 for ⬃45 min at 25°C. Monolayers were compressed in
stepwise fashion while [NO2]e was monitored for ⬃10 min at each
compression point. Surface tensions of 71.4 ⫾ 1.5, 60.6 ⫾ 1.3, 53.8 ⫾
2.0, 47.2 ⫾ 4.5, and 26.5 ⫾ 4.4 were recorded at areas of 387, 230,
203, 186, and 157 cm2, respectively. NO2 absorption decreased linearly (r ⫽ 0.99) with surface tension. At the end of each experiment,
[NO2]e returned to baseline levels on surface reexpansion. Values are
means ⫾ SD for n ⱖ 6.
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aqueous phase. The limited temperature effect likely
resulted from an inability to maintain the elevated
temperatures (initial temperature ⬇ 37°C) so that
analyses were conducted under somewhat lower actual
temperatures (gas phase ⬇ 31°C, bulk liquid temperature ⬇ 29°C). However, compression consistently induced significant decreases in the NO2 absorption
rates.
Surface tension dependence. Because NO2 uptake fell
with surface tension, we investigated whether this was
related to surface tension or the nature of the interfacial film. To do this, we used a soluble detergent,
Tween 80, to reduce surface tension, and we conducted
NO2 exposures using GSH ⫹ Tween 80 mixtures. To
avoid possible contamination of the compression system by the Tween 80, we used the exposure flasks. A
0.1% solution of Tween 80 satisfied the criteria of
significantly reducing basal surface tensions (70.3 ⫾
3.6 to 39.5 ⫾ 2.2 mN/m, P ⬍ 0.05) while also permitting
the deposition of stable phospholipid films. Despite
reductions in surface tension, Tween 80 solutions
failed to reduce the rate of GSH-mediated NO2 absorption (Fig. 4). By comparison, when DPPC monolayers
(Fig. 3) were compressed to the same average surface
tension achieved by 0.1% Tween 80 alone (␥ ⫽ 39.5 ⫾
2.2 mN/m), DPPC monolayers reduced NO2 absorption
⬃32%. Similarly, when DPPC films were deposited (0.5
␮g/cm2) onto Tween 80 solutions, NO2 uptake significantly declined by 52% (Fig. 4), suggesting that the
presence of saturated interfacial phospholipids, rather
than surface tension per se, inhibited NO2 flux.
2028
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
significantly decreased NO2 absorption regardless of
substrate concentration (Fig. 6).
Reaction product analysis. To investigate whether
monolayer-induced decrements in NO2 absorption
were related to altered reaction pathways, we measured GSH utilization and the reaction products of
NO2. Under physiological conditions, NO2 undergoes
quantitative univalent reduction to NO2⫺. Both probe
and flask exposures were conducted to determine
whether surface films modulated the stoichiometry between NO2 uptake and NO2⫺ formation (Table 1). For
the probe exposures, a round Teflon dish (66 ⫻ 4 mm
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Fig. 4. Effect of detergent on NO2 absorption. Buffer or 0.1% Tween
80 solutions with or without 1 mM GSH were exposed to 4.6 ppm
NO2 for 15 min in the presence (hatched bar) or absence (open bars)
of a DPPC film in the flask system. DPPC was deposited at an initial
surface density of 0.5 ␮g/cm2 and allowed to equilibrate for 15 min
before exposure initiation. Despite achieving low surface tensions
(listed above bars), 0.1% Tween 80 had no effect on NO2 absorption.
NO2 absorption was significantly reduced, however, when DPPC
monolayers were deposited onto the Tween 80 solutions. Although
surface tension was further reduced by the DPPC (24.3 mN/m), it did
not differ from surface tensions achieved with buffer ⫹ DPPC in the
absence of Tween 80 (25 mN/m). *NO2 uptake was significantly
lower when DPPC was added to the Tween-GSH-containing system
(ANOVA; P ⬍ 0.05). Values are means ⫾ SD for nⱖ 5.
Gas-phase NO2 and aqueous-phase substrate concentration dependence. The gas-phase concentration dependence of monolayer-induced effects was investigated utilizing the probe/surface balance apparatus
with [NO2]i between 4 and 125 ppm. The Saltzman
procedure for NO2 analysis was employed for [NO2]i ⬎
20 ppm. DPPC monolayers were deposited onto GSH
solutions and exposed to 4, 9, 19, or 125 ppm NO2 for 30
min. DPPC-induced reductions in NO2 uptake were
significant (P ⬍ 0.05) at all NO2 concentrations studied
(Fig. 5).
Exposures were also conducted to determine
whether substrate composition and concentration influenced the observed monolayer-induced effect on NO2
absorption. Flask exposures were conducted with
DPPC films deposited onto solutions containing 1.0
mM AH2 and exposed to 5 ppm NO2 for 30 min. Desferrioxamine (50 ␮M) was added to AH2 solutions to
limit autoxidation. As in the experiments with GSH,
AH2-mediated NO2 absorption was significantly reduced from 48.0 ⫾ 0.4 to 16.6 ⫾ 1.2 ng 䡠 min⫺1 䡠 cm⫺2,
suggesting that the monolayer-induced effects were
independent of aqueous substrate composition. In a
separate series of experiments, the effect of interfacial
films was assessed over a broad range of substrate
GSH concentrations. Again DPPC films were deposited
onto solutions containing 1, 10, or 50 mM GSH. Relative to the GSH controls, the addition of DPPC films
J Appl Physiol • VOL
Fig. 5. Relationship between NO2 gas phase concentration at inlet
port ([NO2]i) and monolayer-induced effects on NO2 absorption (A)
and fractional uptake (B). DPPC monolayers, deposited at an initial
surface density of 121 Å2/molecule onto 1 mM GSH in the surface
balance, were exposed to 4, 10, 20, 90, or 125 ppm NO2 (DPPC
uncompressed ⫽ open bars, DPPC compressed ⫽ hatched bars).
Exposures of uncompressed monolayers proceeded for 10 min followed by maximal compression (achieving a 60% reduction in surface
area) and continued exposure for an additional 20 min. At [NO2] ⬎
20 ppm, monolayers were allowed to equilibrate in the absence of
NO2 gas flow, and gas phase analysis was performed by using
Saltzman reagent (17). Values are means ⫾ SD for nⱖ 3. *Uptakes
are significantly lower for compressed DPPC systems compared with
matched uncompressed systems (ANOVA; P ⬍ 0.05).
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PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
internal dimensions) was submerged in the aqueous
phase directly beneath the probe to help restrict reaction product diffusion and subsequent dilution into the
trough bulk phase. Samples were pipetted from the
dish. The results were similar to previous analyses (31,
52) in that we observed an approximate 1:1 ratio of
NO2⫺ formation to NO2 uptake regardless of the exposure system or the presence of a compressed monolayer. For the GSH utilization studies, flask systems
and lower GSH concentrations (200 ␮M) were used.
DPPC films were deposited onto buffer or GSH solutions and exposed for 45 min. HPLC analyses revealed
that the profile and proportions of reaction products,
relative to NO2 uptake, were unaltered by the presence
of a DPPC film (data not shown). In the presence of a
DPPC film, NO2 absorption and GSH consumption
were significantly reduced. NO2 uptake (421 ⫾ 25 or
232 ⫾ 15 nmol without or with PC, respectively) was
less than GSH consumption (709 ⫾ 45 or 514 ⫾ 37
nmol without or with PC, respectively) because of secondary redox cycling reactions (52). Together, the data
suggest that the decline in NO2 uptake was not related
to alterations in the reaction kinetics between NO2 and
the aqueous-phase substrates.
Table 1. Effect of interfacial DPPC on
NO2-derived NO2⫺ production
Exposure
Conditions
NO2⫺ Produced,
nmole
NO2 Uptake,
nmole
Ratio
Nitrite/NO2
Flask ⫺ PC
Flask ⫹ PC
Probe ⫹ PC
1.23 ⫾ 0.03
0.61 ⫾ 0.05
0.63 ⫾ 0.21
0.98 ⫾ 0.04
0.53 ⫾ 0.08
0.65 ⫾ 0.27
1.25 ⫾ 0.03
1.16 ⫾ 0.07
0.99 ⫾ 0.13
Values are means ⫾ SD (n ⱖ 3). After exposures, aqueous-phase
aliquots were withdrawn for immediate nitrite (NO2⫺) analysis as
described in METHODS.
J Appl Physiol • VOL
Effect of monolayer composition on NO2 absorption.
Although it is generally accepted that pulmonary surfactant present at the interface is enriched in DPPC (8,
19), whether unsaturated fatty acyl moieties are retained throughout compression-expansion cycles remains equivocal (24, 39). We investigated the effects of
interfacial unsaturated fatty acids on NO2 uptake utilizing monolayers composed of either DLPC or POPC.
Although not a component of natural surfactant, DLPC
was of particular interest both as a potential direct
reaction substrate for NO2 (because of the abundant
unsaturated bonds) and because of molecular packing
properties induced by the two unsaturated fatty acyl
moieties. On compression, despite surface tension reductions, monolayers of DLPC and POPC did not significantly decrease NO2 absorption rates (Fig. 7).
Alveolar surfactant is a complex mixture containing
both saturated and unsaturated lipids. Consequently,
studies utilizing BALF extracts and phospholipid mixtures were also conducted to examine monolayer systems more representative of natural surfactant. Rat
BALF lipid extract, although a crude preparation, was
used as a surrogate of natural surfactant, and DPPCPOPG (9:1; wt/wt) and DPPC-POPC-DPPG (70:27:3;
wt/wt/wt) mixtures were prepared to simulate published compositions of human surfactant phospholipids
and fatty acids (41). Phospholipid stock solutions (1
mg/ml in chloroform) were prepared by using an averaged molecular weight based on the percent composition of each phospholipid in the solution. All three
compressed monolayers significantly reduced NO2 ab-
Fig. 7. Lack of effect of unsaturated PC on NO2 absorption. Phospholipids were deposited at an initial surface density of 121
Å2/molecule onto 1 mM GSH in the surface balance and exposed to
4.8 ppm NO2. Bars are coded as in Fig. 4 with double-hatched bars
representing unsaturated phosphatidylcholine (PC) films. Exposures of uncompressed films proceeded for 10 min followed by
maximal compression (achieving a 60% reduction in surface area)
with continued exposure for an additional 20 min. Neither
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) nor 1,2dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) reduced NO2 absorption on compression, although surface tensions (listed above
bars) achieved were similar to those with DPPC. Values are
means ⫾ SD for nⱖ 4. *NO2 uptake for the DPPC-covered system
was significantly less than that of the uncompressed control
(ANOVA; P ⬍ 0.05).
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Fig. 6. Aqueous substrate concentration dependence of monolayerinduced reduction in NO2 absorption. In flask exposures, excess
DPPC was deposited at a nominal surface density of 0.5 ␮g/cm2 onto
solutions containing 1, 10, and 50 mM GSH concentration ([GSH]).
Exposures were conducted at 6.1 ⫾ 0.1 ppm NO2 for 15 min at 25°C
under unstirred, steady-state exposure conditions. Values are
means ⫾ SD for nⱖ 5.
2029
2030
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
sorption (Fig. 8). The reductions varied with the concentration of unsaturated moieties, resulting in a barrier effectiveness hierarchy of DPPC ⬎ DPPC-POPG ⬎
DPPC-POPC-DPPG. Despite failing to lower surface
tension below 44 mN/m, the BALF extract impeded
NO2 uptake significantly.
DISCUSSION
Investigations into the influences of surface films on
gas-vapor transport processes began in the mid-1920s.
Initial studies were directed at evaluating the effect of
monomolecular oil films on the rate of water evaporation (23, 27, 40). In the early 1940s, Langmuir and
Schaefer (23) designed and utilized an apparatus for
evaluating monolayer effects on the rate of water evaporation. These studies, along with those that followed,
suggested that interfacial monolayers of some fatty
acids and aliphatic alcohols significantly reduced the
rate of water evaporation (1, 42). However, several
years passed before the concept of lung surfactant film
permeability was first introduced: it was initially discussed in the context of a mathematical model (7)
evaluating the stability of bubbles expressed from lung
by Pattle (29). Although studies assessing interfacial
lung surfactant permeability to water vapor have been
conducted (25), monolayer effects on the rate of gas flux
from the gas to the aqueous phase have received limited attention (e.g., 4, 21). This may be due in large
part to the fact that O2 and CO2 partial pressures
J Appl Physiol • VOL
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Fig. 8. Effect of interfacial phospholipid composition on NO2 absorption in the surface balance. Mixed monolayer systems in
which the relative concentration of DPPC varied were exposed to
NO2 (4.6 ⫾ 0.2 ppm) as described in Fig. 7. Stock solutions of the
phospholipid mixtures (1 mg/ml in chloroform) were prepared on
the basis of either a weighted average or estimated molecular
weights for pure chemical and bronchoalveolar lavage fluid
(BALF) systems, respectively. DPPG, 1,2-dipalmitoyl-sn-glycero3-phospho-rac-glycerol; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-rac-glycerol. Double-hatched bars indicate uptakes in the
presence of films. *NO2 absorption was decreased significantly in
all cases (ANOVA; P ⬍ 0.05). Surface tensions achieved on compression (listed above bars) were similar among the pure chemical
systems. Although surface tension remained high for BALF
extract, NO2 uptake was significantly decreased. Values are
means ⫾ SD for nⱖ 3.
normally equilibrate rapidly between alveolar gas and
pulmonary capillary blood (43).
Characterizing the influences of interfacial phospholipids on NO2 absorption required an exposure model
that would enable measurement of changes in NO2 gas
phase concentration over a defined surface during controlled compression of the surface film. We were limited to an in vitro approach because interfacial conditions within the lung cannot be tightly controlled.
Although studies were conducted under quasi-steadystate exposure conditions, intrapulmonary NO2 concentrations vary throughout ventilation. However, previous studies have demonstrated that processes
governing NO2 absorption were equivalent under quasi-steady-state and transient (NO2-limiting) exposure
conditions (32, 34). Despite the design limitations of
the compression-exposure apparatus that prevented
compression of surface films to very low tensions (Fig.
1), the system permitted reproducible control and
study of the effects of interfacial, gas-phase, and aqueous-phase conditions on NO2 absorption.
Using the probe exposure apparatus, we observed
that compression of DPPC monolayers to surface tensions still substantially above those reached in vivo
(e.g., 25 vs. ⬍1 mN/m) significantly reduced the rate of
NO2 absorption (Figs. 3, 6, 7). Increases in monolayer
packing, indicated by lower surface tensions, were rapidly followed by reduced NO2 absorption (Fig. 2). We
therefore felt it important to evaluate whether the
decreased uptake was due to additional resistance imposed directly at the interface or to changes in the gasand/or aqueous-phase conditions. NO2 diffuses through
the gas-phase boundary layer, dissolves in the aqueous-phase boundary layer, and reacts within a nearinterfacial zone. Because of the limited aqueous solubility of NO2, saturation of the aqueous boundary layer
should occur rapidly in the absence of reactive substrates (3). The driving force for the continued net flux
of NO2 is maintained by diffusion of both NO2 and
reactive substrates (e.g., GSH, AH2) into the reaction
zone. Chemical reaction of NO2 serves to limit both its
diffusion into the bulk aqueous phase and the development of backpressures resulting from accumulation of
dissolved NO2. Previous kinetic studies, demonstrating
that NO2 gas-phase disappearance rates were independent of aqueous bulk-phase volume and equivalent for
both wetted filter material and aqueous bulk-phase
systems, support these concepts (34). Studies employing 1% agarose to gel the aqueous phase demonstrated
that impeding aqueous-phase convection did not alter
the steady-state rates of NO2 uptake compared with
unstirred controls (data not shown). In the probe exposure studies, stable uptake rates were observed over
prolonged periods with or without either compressed
monolayers or reaction substrates. It is unlikely that
monolayer compression caused a backpressure-induced decrease in NO2 flux due to aqueous substrate
limitations because this would imply that solute diffusion would be constrained to a new, lower steady-state
level.
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
J Appl Physiol • VOL
Fig. 9. Relationship between interfacial resistance to NO2 flux and
DPPC surface pressure. As discussed in the APPENDIX, film resistance
is defined as the difference between total resistance and apparatus
resistance.
air in water is taken into account). A plot of film
resistance vs. surface pressure (Fig. 9) clearly demonstrates that film resistance is related to the degree of
DPPC monolayer compression. Under the exposure
conditions employed in these studies, monolayer resistance to NO2 absorption increased from 0.3 to 12.3 s/cm
when the film was compressed from 121 to 49 Å2/
molecule. Because the surface tensions achieved in
these studies were still higher than those that occur in
vivo, and film molecular packing was lower, it is reasonable to speculate that physiological resistance to
NO2 transfer within the pulmonary gas exchange regions could exceed those observed in this study. Differences in estimates of film resistance between previous
studies and those reported herein likely reflect variations in experimental materials and methods and in
the definition of film resistance.
We cannot be sure that NO2 absorption would have
continued to decrease during surface compression to
lower, physiological levels of surface tension; however,
the relation is reasonably linear in the range of surface
tension measured (Fig. 3). Nevertheless, it is likely
that the low resistance of mixed monolayers would be
raised by further compression, which produces a more
DPPC-enriched film. Furthermore, alveolar surface
tension varies throughout the ventilation cycle even
during tidal breathing: large increases in lung volume
increase alveolar surface tension above equilibrium (25
mN/m) (46), at which point renewal of the surfactant
monolayer begins. Interfacial lipids are probably also
responsible for the low surface tension of conducting
airway lining fluid (30 mN/m) (45). As demonstrated by
the Tween 80 (Fig. 4) and unsaturated fatty acid (Fig.
7) studies, mere reduction in surface tension is not
sufficient to restrict NO2 transfer. Consequently, because airway interfacial properties have not been fully
characterized, it is difficult to predict whether significant film resistance to NO2 transfer might occur in
these anatomic sites.
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Although previous NO2 studies have shown limitations to mass transfer in the gas phase (3), we concluded that the addition of a monolayer would not
affect gas-phase NO2 transport. Nevertheless, appreciable mass transfer resistance is associated with the
gas phase as is shown by the saturation of NO2 absorption rates in the presence of vigorous interfacial stirring and aqueous substrates in great excess (e.g., [NO2]
⬇ 10 ppm, GSH concentration ⬇ 10 mM) (33, 34). NO2
absorption in the presence of an uncompressed DPPC
film did reach maximal values (Fig. 6) similar to those
reported previously under steady-state, well-stirred
conditions (33). We attributed the reduction in NO2
absorption observed with DPPC to resistance introduced by the interfacial films themselves rather than
effects of the films on the adjacent bulk phases. We
based this conclusion on the following considerations:
1) the constant background loss of NO2 in buffer systems despite the absence or presence of DPPC, 2) the
theoretical lack of monolayer-induced diffusion effects
within the aqueous reaction zone, 3) the lack of direct
surface tension effects on absorption (Fig. 4), 4) a
film-induced reduction in NO2 absorption at various
aqueous substrate concentrations and compositions,
and NO2 gas-phase concentrations (Fig. 5), and 5) the
consistent stoichiometry of NO2⫺ formation from NO2
(Table 1). It is noteworthy that DPPC films reduced
NO2 uptake significantly even at 90 and 125 ppm
(⬃60%, Fig. 5). These data clearly demonstrate that
interfacial monolayers limit NO2 transfer.
Despite their lowering of surface tension to ⬃25
mN/m, unsaturated PC moieties did not restrict NO2
transfer (Fig. 7). The area per molecule of unsaturated
PC in a fully compressed monolayer remains quite high
(e.g., POPC ⬎ 70 Å2/molecule) (11) compared with
disaturated PC (⬍49 Å2/molecule). Moreover, had exposure-induced oxidation of the unsaturated fatty acid
occurred, the products would have further disordered
molecular packing. NO2 reaction with unsaturated
fatty acids proceeds via either allylic hydrogen abstraction or double-bond addition (15, 16). For polyunsaturated fatty acids, the molecule isomerizes and adds O2
to produce a cis-trans conjugated diene fatty acid hydroperoxide. For both mono- and polyunsaturates, NO2
addition products may potentially result (22, 38). The
conformational changes induced by these molecular
modifications could have introduced further disorder
into the films (20).
Others have reported monolayer-induced resistance
to transport processes ranging from ⬍1 to 103 s/cm (4,
23, 48). Estimates of film resistance, which we calculated from the data of Sebba and Briscoe (48) (for
C16–C20 fatty acid and aliphatic alcohols), ranged
from ⬍0.1 to 25 s/cm, with the exception of n-docosanol,
which achieved a resistance of 180 s/cm when maximally compressed. Blank and Roughton (4) reported
resistance to CO2 transport across (C16–C18 fatty acid
and aliphatic alcohol) films of 80–392 s/cm. Clements
(7) estimated the film resistance of Pattle’s bubbles
(surfactant bubbles extruded from whole lung) to be
⬃104 s/cm (180 s/cm when the solubility coefficient of
2031
2032
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
When GSH solutions were exposed to NO2 under the
probe, the exit concentration remained constant for ⬎45 min,
indicating steady-state uptake over the time intervals used
in our experiments and suggesting that the thickness of the
NO2 reaction zone remained constant, allowing a calculation
of the NO2 gradient across the monolayer.
Derivation of film resistance. We derived a mathematical
expression for film resistance under the probe on the basis of
the following assumptions: 1) a steady state existed during
the measurements of uptake, 2) the profile of changing stagnant layer thickness in the gas phase was not affected by the
surface films, 3) an edge-to-center NO2 gradient existed, 4)
vertical gradients in gas-phase [NO2] were negligible, 5) the
effective depth of the reaction zone was constant, and 6)
specific film resistance was equal to the difference between
total resistance (probe-flask system with PC deposited onto
GSH solution, Rtotal) and probe resistance (probe-flask system with GSH solution alone, Rapparatus).
We used a washer-shaped control volume, of thickness h
with inner radius r and outer radius r ⫹ ⌬r, to perform a
steady-state mole balance on NO2 (Eq. 1).
关Q̇C兴r ⫽ 关Q̇C兴r⫹⌬r ⫹ 2␲r⌬rN
where Q̇ is the outlet gas flow (ml/min), C is [NO2] in the gas
phase (mol/ml), and N is NO2 flux from gas to liquid
(mol 䡠 cm⫺2 䡠 min⫺1). By assuming that Q̇ is constant, Eq. 1 can
be rearranged to
关C兴r⫹⌬r ⫺ 关C兴r 2␲rN
⫽
⌬r
Q̇
APPENDIX
J Appl Physiol • VOL
(2)
Taking the limit as ⌬r 3 0, this equation becomes
dC 2␲rN
⫽
dr
Q̇
Validation of probe exposure apparatus. Because of the
design characteristics of the probe apparatus, some NO2 loss
to the atmosphere was inevitable (Fig. 1). To evaluate NO2
flow characteristics under the probe and to determine optimal probe positioning, round filters wetted with Saltzman
reagent (17) were placed on a solid support and positioned
directly beneath the probe, and the pattern of color development was examined. When 1) the central restriction disc was
recessed 4 mm from the probe lip and 2) the probe lip was
positioned 1 mm above the wetted filter, a circular pattern of
homogeneous staining (34.1 ⫾ 1 cm2) formed on the wetted
filters. The surface area of the colored circle was similar to
that of the probe itself (28.3 cm2) and was not altered when
exposures were conducted with the pantograph barrier set to
the smallest allowable surface area.
The background (buffer without GSH) rate of NO2 gas
phase disappearance {([NO2]i ⫺ [NO2]e) 䡠 exit flow 䡠 surface
area⫺1} represents the combined contributions of aqueous
solubility and reaction with water (which is relatively slow;
Ref. 12). The background rates among buffer only, buffer plus
DPPC (121 Å2/molecule), and buffer plus compressed DPPC
(49 Å2/molecule) systems were comparable (data not shown),
indicating that monolayer-induced effects on transfer were
small under these conditions. In addition, the rates of NO2
gas-phase disappearance were comparable for GSH solutions
with or without uncompressed DPPC or POPC (121 Å2/
molecule) (23.0 ⫾ 0.4, 23.1 ⫾ 0.3, and 23.1 ⫾ 0.4 ng 䡠 min⫺1 䡠
cm⫺2 for GSH, DPPC, and POPC, respectively). Collectively,
the data suggest that the gas phase distribution under the
probe was not appreciably affected by the presence of a
monolayer.
(1)
(3)
We then defined the rate equation for interfacial flux as
N⫽
1
共C ⫺ ␭Cliquid兲
R
(4)
where R is the interfacial resistance (s/cm), Cliquid is [NO2] at
the aqueous surface, and ␭ is the solubility of NO2 in the
aqueous phase. Assuming that the backpressure from the
liquid is negligible (N ⬇ C/R) and
冉 冊
(5)
ci
␲ 2
⫽
共rf ⫺ ri2兲
cf
Q̇R
(6)
dC
2␲
rC
⫽
dr
Q̇R
integrating Eq. 5 yields
ln
冉冊
where subscripts i and f refer to initial and final parameters,
(i.e., radii or concentration) so that
R ⫽ 60 A
冒 冋 冉 冊册
Q̇ ln
ci
cf
(7)
We then defined specific film resistance as the difference
between Rtotal, the system resistance (GSH with PC deposited) and Rapparatus, probe resistance (GSH alone). R, therefore, represents a composite coefficient that includes the
effects of surface adsorption, aqueous solubility, diffusion,
and chemical reaction of NO2.
Assistance in the design and construction of specialized equipment by Frederick Schuster and Gordon Ansell and technical assis-
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The implications of these results are intriguing. If
NO2 absorption in proximal air spaces is limited by
interfacial surfactant, then NO2 movement into more
distal regions would occur. In contrast, the absence of
exposure-induced injury has been accepted as prima
facie evidence for the lack of NO2 distribution into
distal alveoli. The intrapulmonary distribution of inhaled reactive gases is related to the balance between
removal at the airway walls and longitudinal transport
(3, 6). Dosimetry models predict that the gas-phase
concentration of inhaled NO2 rapidly declines beyond
the proximal alveolar spaces (26, 28). Most models,
which attempt to characterize the tissue dose of inhaled oxidants, predict a rapid equilibration between
gas and aqueous (ELF) phases on the basis of Henry’s
law solubility coefficients (3). Data from our studies
lead us to speculate that the resistance of surfactant
films to NO2 diffusion could result in increased NO2
transport to more distal airspaces. If uptake were sufficiently restricted, cell injury would not directly correlate with the local gas-phase [NO2] concentration. Furthermore, because in vitro exposure models (e.g., cell
cultures) generally do not include an interfacial film of
surfactant, dose-effect relationships may differ appreciably from those occurring in vivo. Consequently, differing relationships between gas-phase concentration
and cell injury may confound direct extrapolations
between in vitro exposure models and from models to
animals.
PHOSPHOLIPID MONOLAYERS AND NO2 ABSORPTION
tance of Leon Katigbak are gratefully acknowledged. We are also
indebted to a referee for an alternative derivation of the equation for
film resistance.
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-54696 and HL-24075 and by National
Institute of Environmental Health Sciences Grants 5-F31-ES05749
and T32-ES-07254 (to L. M. Connor).
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