Am J Physiol Regul Integr Comp Physiol 299: R1306–R1316, 2010.
First published September 1, 2010; doi:10.1152/ajpregu.00380.2010.
Myristate is selectively incorporated into surfactant and decreases
dipalmitoylphosphatidylcholine without functional impairment
Christopher J. Pynn,1,2 M. Victoria Picardi,3 Tim Nicholson,4 Dorothee Wistuba,4 Christian F. Poets,1
Erwin Schleicher,2 Jesus Perez-Gil,3 and Wolfgang Bernhard1
Departments of 1Neonatology and 2Internal Medicine IV, Faculty of Medicine, and 4Department of Chemistry,
Eberhard-Karls-University, Tübingen, Germany; and 3Department of Bioquimica y Biologia Molecular, Universidad
Complutense, Madrid, Spain
Submitted 11 June 2010; accepted in final form 18 August 2010
nutrition; lipidomics; lung development; phosphatidylcholine; surfactant
is achieved by the action of
surfactant, a phospholipoprotein complex comprising about
85% phospholipids, 10% neutral lipids and up to 5% specific
proteins SP-A, SP-B, SP-C, and SP-D. The ability of pulmonary surfactant to oppose surface tension forces holds true for
air-liquid interfaces irrespective of their precise anatomy (1,
22). However, its molecular composition varies according to
STABILIZATION OF LUNG PARENCHYMA
Address for reprint requests and other correspondence: W. Bernhard,
Dept. of Neonatology, Faculty of Medicine, Eberhard-Karls-Univ., Calwer
Stra␤e 7, D-72076 Tübingen, Germany (e-mail: wolfgang.bernhard@med.
uni-tuebingen.de).
R1306
differences in pulmonary physiology, anatomy and development (6, 9, 38). Such variability is well described for surfactant
proteins, which define the presence of tubular myelin, opsonization of pathogens, and surface activity under dynamic
conditions, but it is less well understood for the major surfactant phospholipids (6, 36, 38). Most analyses of surfactant
report high concentrations of PC16:0/16:0 as its principal
surface tension-lowering compound (23, 52). However, recent
detailed lipidomic analyses have also revealed important contributions from PC16:0/14:0 and PC16:0/16:1. These components are specifically enriched in the alveolar space, comprising up to 15 and 28%, respectively, of mammalian surfactant
and are fluidic at 37°C in contrast to PC16:0/16:0 (7, 9, 25).
This is in stark contrast to the rigid avian lung, where surfactant nearly exclusively comprises PC16:0/16:0 and SP-B. (6).
In humans and guinea pigs, PC16:0/14:0 increases from
alveolarization in utero onward. In the rat and mouse, this
increase occurs during postnatal alveolarization (7, 9, 21). In
vitro, PC16:0/14:0 mimics the effects of natural lipid extract
surfactant on the differentiation of macrophages and macrophage-dependent inhibition of T-cell proliferation (17), suggesting potential immunoregulatory functions that may be important for parenchyma protection during development. Additionally, the diagnostic and pathophysiological potential of PC16:
0/14:0 has been highlighted by the findings that its concentration
in neonatal surfactant correlates better with lung maturity than
PC16:0/16:0, and that PC16:0/14:0 may be decreased in
chronic lung diseases (7, 9). PC16:0/16:0, PC16:0/14:0 and
PC16:0/16:1 are selectively enriched in surfactant compared
with other lung tissue PC (7). Together, these components
comprise 75– 80% of surfactant PC (7, 9). This is due to the
selective nature of PC accumulation in the lamellar bodies of
mammalian type II pneumocytes (PN-II), where selection occurs according to fatty acyl chain length, possibly involving the
ABC-A3 transporter system of the lamellar body outer membrane (14, 27, 31). When comparing the PC species composition across vertebrates or during development in vivo, PC16:
0/14:0 and PC16:0/16:0 are regulated in an antiparallel fashion
(7). By contrast, hormonal stimulation of lung maturation and
surfactant synthesis in vivo or in isolated PN-II increases
PC16:0/16:0 and PC16:0/14:0 synthesis and pools in a parallel
fashion (11, 19, 35). Furthermore, the pattern of newly synthesized PC in isolated PN-II or lung explants is defined by the
surrounding medium rather than by hormonal stimulation or
lung maturation (11, 12). As lipids from plasma lipoproteins
have been shown to stimulate surfactant formation in rats (42),
developmental changes in surfactant PC composition may
depend on exogenous lipid supply to the lungs.
0363-6119/10 Copyright © 2010 the American Physiological Society
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Pynn CJ, Picardi MV, Nicholson T, Wistuba D, Poets CF,
Schleicher E, Perez-Gil J, Bernhard W. Myristate is selectively
incorporated into surfactant and decreases dipalmitoylphosphatidylcholine without functional impairment. Am J Physiol Regul
Integr Comp Physiol 299: R1306 –R1316, 2010. First published
September 1, 2010; doi:10.1152/ajpregu.00380.2010.—Lung surfactant mainly comprises phosphatidylcholines (PC), together with
phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and
palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75– 80%
of surfactant PC. During alveolarization, which occurs postnatally in
the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/
16:0. As lipoproteins modify surfactant metabolism, we postulated an
extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We,
therefore, fed rats (d19 –26) with trilaurin (C12:03), trimyristin (C14:
03), tripalmitin (C16:03), triolein (C18:13) or trilinolein (C18:23) vs.
carbohydrate diet to assess their effects on surfactant PC composition
and surface tension function using a captive bubble surfactometer.
Metabolism was assessed with deuterated C12:0 (␻-d3-C12:0) and
␻-d3-C14:0. C14:03 increased PC16:0/14:0 in surfactant from 12 ⫾ 1
to 45 ⫾ 3% and decreased PC16:0/16:0 from 47 ⫾ 1 to 29 ⫾ 2%, with
no impairment of surface tension function. Combined phospholipase
A2 assay and mass spectrometry revealed that 50% of the PC16:0/14:0
peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:
0). While C12:03 was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:03, C18:13, and C18:23 had no
significant effect on PC16:0/16:0 or PC16:0/14:0. d3-C14:0 was
enriched in lung PC, either via direct supply or via d3-C12:0 elongation. Enrichment of d3-C14:0 in surfactant PC contrasted its rapid
turnover in plasma and liver PC, where its elongation product d3C16:0 surmounted d3-C14:0. In summary, high surfactant PC16:0/
14:0 during lung development correlates with C14:0 and C12:0 supply
via specific C14:0 enrichment into lung PC. Surfactant that is high in
PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal
respiration and surfactant function in vitro.
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
R1307
MATERIALS AND METHODS
Materials. Phospholipid standards were from Sigma-Aldrich (Deisenhofen, Germany) or Avanti Polar Lipids (Alabaster, AL), while
fatty acid standards were from Sigma. Deuterated fatty acids were
from CDN Isotopes (Pointe Claire, Quebec, Canada), Hydrogen
peroxide (30%, analytical grade) was from Boehringer Ingelheim
(Ingelheim, Germany), whereas perchloric acid (70%, analytical
Table 1. Fatty acid supply in the different feeding groups
Fatty acid
Control
Trilaurin
Trimyristin
Tripalmitin
Triolein
Trilinolein
C12:0
C14:0
C16:0
C18:1
C18:2
0.00
0.01
0.15
0.37
0.75
1.87
0.01
0.15
0.37
0.75
0.00
1.89
0.15
0.37
0.75
0.00
0.01
2.05
0.37
0.75
0.00
0.01
0.15
2.28
0.75
0.00
0.01
0.15
0.37
2.66
Data are given in g fatty acid/100 ml. Data were calculated from the fatty acid concentrations of 20% (wt/vol) dissolved diet (ssniff EF R/M water soluble),
as indicated in the manufacturer’s notes, and the amount of the respective fatty acids of the respective triglycerides added (2%, wt/vol). C12:0, lauric; C14:0,
myristic; C16:0, palmitic; C18:1-N9, oleic; C16:2-N9, linoleic acid. Bolded values indicate the fatty acid added as triglyceride in the respective diet.
AJP-Regul Integr Comp Physiol • VOL
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grade) was from Merck (Darmstadt, Germany). Cholic acid and
pancreas phospholipase A2 were from Sigma. Chloroform and methanol were of HPLC grade and from Baker (Deventer, The Netherlands). All other chemicals (analytical grade) were obtained from
various commercial sources.
Animal maintenance and feeding experiments. Sprague-Dawley
rats of either sex were kept under specific pathogen-free and germfree conditions and had ad libitum access to an irradiated (5 Mrad)
carbohydrate-rich standard rodent diet (Provivmi Kliba SA 3336
containing 5.5% fat) and autoclaved water (134°C, 50 min). All
animals were spontaneously delivered (term: 21–23 days after conception). For feeding experiments, animals were separated from their
mothers at day 19 and put on their respective diets. D19 was chosen
to ensure that the animals were mature enough to feed themselves.
Experiments were approved by the local governmental authorities and
met the National Institutes of Health’s Guidelines for the Care and
Use of Laboratory Animals.
Nutrition with synthetic triglycerides. To compare the effects of
individual fatty acids, rats were fed ad libitum from day 19 until death
at day 26 on a diet based on 20% (wt/vol) of an experimental food for
rat and mouse, according to the American Institute of Nutrition (37)
(EF R/M acc. AIN 93G water soluble, Sniff Spezialdiäten, Soest,
Germany) (EF R/M) ad libitum. Dry EF R/M powder was dissolved
in 60°C tap water, cooled to room temperature, and stored at ⫺20°C
until use. This control diet supplied 61% and 24% of energy as
carbohydrate and protein, respectively, and contained no C12:0,
0.03% C14:0, 0.76% C16:0, 1.86% C18:1, and 3.74% C18:2 (wt/wt).
To assess the effects of individual fatty acids, the control diet was
supplemented with 2% (wt/vol) trilaurin (C12:03), trimyristin (C14:
03), tripalmitin (C16:03), triolein (C18:13), or trilinolein (C18:23) as
follows. An aliquot of the respective triglycerides, together with 25%
wt/wt egg lecithin was dissolved in 5–10 ml chloroform:methanol
(2⫹1) in a glass beaker. The material was distributed on the walls of
the beaker, and the solvent was completely evaporated at 37°C under
a stream of nitrogen. The beaker was then filled with 98 ml 20% EF
R/M per 2 g triglyceride, stirred at 90°C for 1 h, and repeatedly
sonicated using a UW2070 sonicator (Bandelin, Berlin, Germany).
Care was taken that no lipid material remained in the glass beaker.
Suspensions were then aliquotted and stored at ⫺26°C until use. The
resulting fatty acid compositions for the respective feeds are given in
Table 1. Formulations were supplied in drink bottles ad libitum, and
food uptake and growth were monitored twice daily until death.
Labeling with deuterated C12:0 and C14:0. The metabolism of
exogenous C12:0 and C14:0 was assessed at d24 –26, i.e., from 3–5
days after weaning from milk feeding to carbohydrate-based rat chow.
We dissolved 2.44 mmol dodecanoic-12,12,12-d3 acid (d3-C12:0)
(⫽500 mg; mol wt ⫽ 203.34) or tetradecanoic-14,14,14-d3 acid
(d3-C14:0) (⫽563 mg; mol wt ⫽ 231.39) together with 125 mg egg
lecithin in 5 ml chloroform:methanol (2⫹1) in a glass beaker, evaporated the solvent under nitrogen and resuspended, yielding a 2%
suspension with EF R/M as described above (25 ml). Suspensions
were aliquotted and stored at ⫺26°C. Day 24 rats (54.4 ⫾ 1.2 g body
wt) were supplied orally with a bolus dose of 4.92 ␮mol/g body wt (50
There are several factors defining the ability of PN-II to
produce surfactant. First, PN-II have the ability to acquire
substrates such as choline, phosphate, and glucose from the
circulation. Second, PN-II are capable of synthesizing fatty
acids and glycerides from glucose and stored glycogen (40).
Third, PN-II are supplied with fatty acids from triglycerides
stored in lipid interstitial cells of the lungs, as well as from
lipoproteins and free fatty acids from the circulation (30, 42,
51). After birth, when rat pups regularly feed on milk, lipid
interstitial cells and their triglyceride granules increase by
two-fold (43). Also PC16:0/14:0 concentration in surfactant is
increased at the expense of PC16:0/16:0, while shortly after
switching to a carbohydrate-based chow diet, PC16:0/14:0
drops and PC16:0/16:0 increases toward adult values (38).
Since rat milk is rich in fatty acids containing less than 16
carbon units, namely myristic (C14:0) and lauric (C12:0) acid
(7, 9, 47), exogenous fatty acid supply may influence the
surfactant PC molecular profile.
We, therefore, fed rats from d19 to d26 on defined synthetic
triglycerides, comprising either lauric (C12:0), myristic (C14:
0), palmitic (C16:0), oleic (C18:1), or linoleic (C18:2) acid vs.
a carbohydrate-based control. We compared changes in the
surfactant PC profile with triglyceride and fatty acid composition in lung tissue and plasma, using HPLC and gas chromatography. The effects of C12:0 and C14:0 contrasted those of
the other fatty acids and the carbohydrate-based control. Deuterated lauric (␻-d3-C12:0) and myristic (␻-d3-C14:0) acid
were used to address the specificity of C14:0 enrichment in
lung surfactant PC, while hydrolysis with phospholipase A2
together with electrospray ionization tandem mass spectrometry (ESI-MS/MS) of the resulting lyso-PC was used to discriminate PC16:0/14:0 from its isomer myristoyl-palmitoyl-PC
(PC14:0/16:0). Our data suggest that C14:0 enrichment in
surfactant PC is regulated by its supply to the lungs, either
directly or via elongation of C12:0. High C14:0 supply induced
the formation of dimyristoyl-PC (PC14:0/14:0) and increased
that of PC16:0/14:0 and its isomer myristoyl-palmitoyl-PC
(PC14:0/16:0). The C14:0-induced biochemical inversion from
PC16:0/16:0-rich toward PC16:0/16:0-deprived surfactant did
not impair animal growth, respiration, or in vitro surfactant
function.
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MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
AJP-Regul Integr Comp Physiol • VOL
100 ␮l phospholipase A2 (in Tris·HCl, 500 U/ml), the sample was
incubated at 37°C in a shaking water bath, and the resulting lyso-PC
measured via mass spectrometry with minor modifications as described elsewhere (8). Eicosanoyl-lyso-PC (lysoPC20:0) was used as
an internal standard (1 nmol/ml), while electrospray ionization tandem
mass spectrometry (ESI-MS/MS) of lysoPC species was performed on
a TSQ Quantum Discovery Max triple quadrupole mass spectrometer
(Thermo Scientific, Dreieich, Germany) equipped with an electrospray ionization interface. Samples were dissolved in buthanol:methanol:water (75:23:2, vol/vol) at a concentration of 1 ␮mol/l and
introduced into the mass spectrometer by loop injection (25 ␮l) using
a Finnigan Surveyor Autosamples plus and eluted with buthanol:
methanol:water:25% ammonia (75:23:1.7:0.3; vol/vol) as the mobile
phase (0 –2 min: 30 ␮l/min; then 30 to 200 ml/min from 2–3.99 min;
back to 30 ␮l/min at 4 min) using a Finnigan Surveyor MS pump plus
(Thermo Scientific). Following fragmentation with argon gas, a fragment with m/z ⫽ ⫹184, corresponding to the protonated phosphocholine headgroup, was used for precursor scans of the samples from
masses 400 –900 and in the selective reaction monitoring mode for
individual PC and lysoPC species. This allowed for the assessment of
purity of the collected PC16:0/14:0⫹PC14:0/16:0 peak (m/z ⫽ 706)
and its complete hydrolysis to form lysoPC14:0 (m/z⫽468) and
lysoPC16:0 (m/z ⫽ 496) within 1 h. Values were corrected for C13
isotope effects due to increasing carbon content as described elsewhere (8).
Analysis of endogenous fatty acids from free fatty acids, triglycerides, and PC. Analysis of individual free fatty acids and fatty acids
from triglycerides and PC was performed with gas chromatography
after (trans)methylation using 13,16,19-docosatrienoic (C22:3) as an
internal standard (26). In brief, lipids were dried down at room
temperature under a stream of nitrogen. Two milliliters of methanol:
toluol (1:3, vol/vol) containing 10 ␮g/ml C22:3 and 200 ␮l acetylchloride (99% purity; Fluka, Steinheim, Germany) were subsequently
added. Methylation of fatty acids was then performed at 105°C for 60
min in closed Pyrex tubes using a ReactiTherm, model 18971 (Pierce,
Rockford, IL) after which samples were supplemented with 5 ml 6%
potassium carbonate, vortexed, and centrifuged for 5 min at 2800 g.
The upper phase was concentrated to 180 ␮l under a stream of
nitrogen and transferred to a microvial; 1 ␮l was injected for gas
chromatographic analysis using a HP7673A autosampler (Hewlett
Packard, Houston, TX, USA). Analysis was performed on a model HP
5890 gas chromatograph equipped with a split/splitless-injector
(230°C) and flame ionization detector (250°C) with hydrogen and
synthetic air as combustion gases. A 60 m ⫻ 0.25 mm I.D. Rtx 2330
column (Restek, Bellefonte, PA) was used with helium as a carrier
gas. Settings for the column were started at 80°C for 2 min, followed
by linear increases at a rate of 30°C/min up to 120°C, then 2°C/min
increase until a final temperature of 240°C for 10 min. These settings
allowed reproducible separation of fatty acids from lauric (C12:0) to
long-chain saturated and unsaturated (docosaic [C20:0], docosahexaenoic [22:6]) acid in all samples. Quantification was performed using
calibration curves for the respective fatty acids.
Analysis of deuterated fatty acid incorporation. Deuterated fatty
acid methyl esters, prepared from free fatty acids, triglycerides, and
PC (see above), were analyzed via gas chromatography coupled with
mass spectrometry (GC-MS) on an Agilent Technologies (Waldbronn,
Germany) gas chromatograph (HP6890) equipped with a mass selective detector (HP5973) with electron impact (EI) ionization chamber,
a split/splitless injection port, and an autosampler (HP7683). Helium
(99.999%) was used as a carrier gas. Separation of analytes was
performed on a DB23 capillary column (25 m ⫻ 0.25 mm, film
thickness 0.15 ␮m) from J ⫹ W Scientific (Folsom, CA). The oven
temperature program was as follows: 80°C held for 2 min, ramped to
250°C at 4°C/min and held for 4 min. The injection was made in
splitless mode and the injector temperature was 250°C. The ion source
temperature and the transfer line were 230°C and 280°C, respectively.
The MS system was routinely set in selective ion monitoring mode
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␮l/g body wt) d3-C12:0 (n ⫽ 6) or d3-C14:0 (n ⫽ 8) label using a
disposable syringe and were killed 24 h or 48 h after feeding.
Harvesting of blood plasma, bronchoalveolar lavage fluid (BALF)
and tissue samples. Rats were killed by intraperitoneal injection, using
100 mg ketamine (Ketamin 10%, selectavet; Dr. Fischer GmbH,
Weyarn-Holzolling, Germany) and 20 mg xylazine (Sedazylan; WDT,
Garbsen, Germany) per kg body wt, and subsequently bled by puncturing the right ventricle of the beating heart. Blood (0.2–2.7 ml
depending on age) was collected into containers containing EDTA
against coagulation, and the lungs were perfused with ice-cold isotonic saline via the right ventricle to remove remaining blood from
pulmonary blood vessels (7) Blood was centrifuged at 1000 g for 10
min to spin down the cells and blood plasma harvested. BALF was
harvested by repeatedly lavaging the lungs through a tracheal catheter
with ice-cold isotonic saline ( 4 to 32 ml depending on age). Recoveries were 85 to 90% as previously described (9). BALF samples
containing visible signs of blood contamination were discarded. Cells
were removed from BALF by centrifugation at 200 g for 10 min at
4°C. Lungs and livers were excised, snap frozen in liquid nitrogen,
and stored at ⫺80°C.
Lipid extraction. Lipids were extracted from BALF and blood
plasma, according to Bligh and Dyer (5), while lung and liver tissues
were extracted according to Folch et al. (15) and stored at ⫺32°C until
analysis. Total phospholipid was quantified by phospholipid phosphorus determination as described by Bartlett (4) after digestion of the
organic components at 190°C in the presence of 500 ␮l 70% perchloric acid (wt/vol) and 200 ␮l 30% hydrogen peroxide (wt/vol). Triglycerides, free fatty acids, and phosphatidylcholine (PC) molecular
species were isolated from the total lipid extracts as described below.
Analysis of PC molecular species, triglycerides, and free fatty
acids. PC and triglycerides were isolated from total lipid extracts as
described before (34, 36). Briefly, lipid extracts equivalent to 500
(plasma, BALF) or 1,000 (lung and liver tissue) nmol phospholipid
were dried under a stream of nitrogen, dissolved in 1 ml chloroform
and applied to 100 mg Strata NH2 disposable cartridges (Phenomenex,
Torrance, CA). The chloroform solvent was collected and triglycerides were eluted with another 2 ⫻ 1 ml chloroform. The eluate was
then adjusted to 4 ml with chloroform:methanol (2:1), and 2 ml was
transferred to a glass vial for further fatty acid analysis by GC or
GC-MS (see below). PC species were subsequently eluted with 1 ml
chloroform:methanol (2:1, vol/vol) and collected for HPLC analysis.
Free fatty acids were prepared with TLC, according to Stefan et al.
(48). For this, lipid extracts (equivalent to 0.5 to 1.5 ␮mol phospholipid) were applied to 20 ⫻ 20 cm silica TLC plates and developed in
hexan:diethylether:glacial acetic acid (160:40:6, vol/vol) over 50 – 60
min using human plasma extract as a standard.
Analysis of PC molecular species. Eluted PC was dried down under
a stream of nitrogen and dissolved in 125 ␮l trifluoroethanol. Analysis
was carried out twice for each sample, once with 100 nmol dimyristoyl-PC (PC14:0/14:0)/␮mol phospholipid internal standard and once
without the standard. This was necessary because C14:0-nutrition
resulted in the formation of significant amounts of PC14:0/14:0 in the
lungs (see RESULTS). HPLC separation of individual molecular species
was performed with a 4.6 ⫻ 250 mm Sphere Image ODS II column
(Schambeck, Bad Godesberg, Germany) and postcolumn fluorescence
derivative formation in the presence of 1,6-diphenyl-1,3,5-hexatriene
as described before (34). To differentiate between PC16:0/14:0 and
PC14:0/16:0, which cannot be separated by HPLC due to identical
retention times (not shown), PC16:0/14:0⫹PC14:0/16:0 peaks were
collected from HPLC effluents and extracted (5); then, the lipids were
hydrolyzed with porcine pancreas phospholipase A2 with minor variations, as described elsewhere (32). Briefly, to 50 –100 nmol eluted
PC16:0/14:0 ⫹ PC14:0/16:0 50 –100 nmol cholic acid (2 mmol/l in
chloroform:methanol 2:1, vol/vol) was added, and the solvent was
evaporated. Nine-hundred microliters (10 mm) Tris·HCl in 0.9%
saline with 10 mM CaCl2, at pH 8.1, and 8 –10 2-mm glass beads were
added, and the sample was resuspended by vortexing. After adding
R1309
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
the respective time points (24 h, 48 h) for labeling with deuterated
fatty acids that were all included into statistical analysis. Group
differences were tested by a two-tailed Student’s t-test, or by one-way
ANOVA for multiple group comparisons using GraphPad Instat ver.
3 (GraphPad Software, San Diego, CA). Multiple group comparisons
were corrected using the method of Bonferroni-Holm. P values of ⬍0.05
were regarded as significant.
RESULTS
Effects of exogenous triglycerides on overall growth and
neutral lipid parameters. Overall effects of 2% trilaurin (C12:
03), trimyristin (C14:03), tripalmitin (C16:03), triolein (C18:
13), or trilinolein (C18:23) (for total supply, see Table 1) on
growth and neutral and phospholipid parameters are summarized in Table 2. Weight gain in the C18:13 was decreased
compared with other groups. C12:03 and C14:03 slightly increased the amount of surfactant phospholipid in BALF, while
concentrations of total phospholipid and triglyceride fatty acids
were not different compared with controls. The molecular fatty
acid profiles of plasma and lung tissue triglycerides are shown
in Table 3. Control plasma triglycerides were dominated by
C18:2 (34 ⫾ 3%), C18:1 (27 ⫾ 1%), C16:0 (16 ⫾ 2%), and
C20:4 (15 ⫾ 2%), while lung tissue triglyceride contained
more C16:0 (38 ⫾ 1) and C18:1 (33 ⫾ 1%), but less than 5%
C18:2 and C20:4. Also, lung triglyceride contained more
C12:0 (3.1 ⫾ 0.3 vs. 0.4 ⫾ 0.1%, P ⬍ 0.001), C14:0 (and
5.4 ⫾ 0.2 vs. 0.9 ⫾ 0.2%, P ⬍ 0.001) and C16:1 (4.6 ⫾ 0.2%
vs. 2.2 ⫾ 0.3%, P ⬍ 0.001). Other long-chain (C20:0, C22:0)
or polyunsaturated (C20:5, C22:6) fatty acids comprised ⬃10% in
lung tissue triglyceride and less than 5% in plasma triglycerides
(not shown).
Exogenous triglycerides increased the respective fatty acid
concentrations in both plasma and lung tissue triglycerides
(Table 3), except for C18:13 treatment in plasma. Here, the
C18:1 fraction was not significantly increased over control
values, whereas C18:1 was decreased by all other exogenous
triglyceride fatty acids compared with carbohydrate controls.
The increases in C12:0 and C14:0 by their respective exogenous triglycerides were much higher than for other fatty acids.
In particular, C12:0 and C14:0 were increased in plasma 47and 22-fold compared with 1.5–1.8-fold for other fatty acids,
while in lung tissue, the increases in C12:0 and C14:0 were
Table 2. Effects of 6-day triglyceride feeding on growth and lung and plasma lipids
Control
Trilaurin
Trimyristin
Tripalmitin
Triolein
Trilinolein
46.3 ⫾ 1.5
71.6 ⫾ 3.4
25.3 ⫾ 2.3
48.8 ⫾ 2.7
80.1 ⫾ 3.1
31.3 ⫾ 0.6
48.0 ⫾ 0.7
60.1 ⫾ 0.8
12.1 ⫾ 0.2*
48.0 ⫾ 2.8
76.7 ⫾ 2.8
28.7 ⫾ 0.7
15.5 ⫾ 1.1*
191 ⫾ 6.6
60.50 ⫾ 7.28
0.32 ⫾ 0.04
10.0 ⫾ 2.4
165 ⫾ 5.5
36.83 ⫾ 10.81
0.22 ⫾ 0.06
10.10 ⫾ 1.8
179 ⫾ 3.8
48.16 ⫾ 5.60
0.27 ⫾ 0.03
10.5 ⫾ 2.3
176 ⫾ 6.1
39.71 ⫾ 3.34
0.23 ⫾ 0.02
2.42 ⫾ 0.10
0.56 ⫾ 0.05
2.02 ⫾ 0.25
0.39 ⫾ 0.07
2.32 ⫾ 0.04
0.35 ⫾ 0.02
1.83 ⫾ 0.06
0.40 ⫾ 0.07
Body weight, g
day 19
day 26
Weight gain, g
45.6 ⫾ 0.8
77.5 ⫾ 1.3
31.9 ⫾ 0.5
44.3 ⫾ 0.9
63.9 ⫾ 3.1
19.6 ⫾ 2.6
10.8 ⫾ 1.0
186 ⫾ 6.5
36.42 ⫾ 4.32
0.20 ⫾ 0.02
14.4 ⫾ 0.9*
182 ⫾ 3.9
53.83 ⫾ 6.21
0.30 ⫾ 0.04
Lung
BALF phospholipid, nmol/g body wt
Lung phospholipid, nmol/g body wt
Lung-TAG, nmol fatty acid/g body wt
␮mol TAG-fatty acids/␮mol lung phospholipid
Blood Plasma
Phospholipids, ␮mol/ml
TAG, ␮mol fatty acid/ml
2.02 ⫾ 0.02
0.40 ⫾ 0.06
2.27 ⫾ 0.13
0.35 ⫾ 0.04
Rats were fed from days 19 to 26 on diets containing either no added triglyceride (control; n ⫽ 6) or 2 % (wt/vol) trilaurin (n ⫽ 7), trimyristin (n ⫽ 7),
tripalmitin (n ⫽ 4), triolein (n ⫽ 4), or trilinolein (n ⫽ 4). Ssniff EF R/M water soluble served as a carbohydrate control (n ⫽ 3) and was not different from
regular rodent chow (n ⫽ 3; not shown). Data are indicated as means ⫾ SE. BALF, bronchoalveolar lavage fluid; TAG, triglyceride. *P ⬍ 0.05.
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with a solvent delay of 6 min. Agilent Chemstation was used for data
collection and GC-MS control. Methyl esters of fatty acids were
quantified at their respective mass/charge (m/z) values: lauric acid
(m/z ⫽ 214), myristic acid (242), palmitic acid (270), and stearic acid
(298). Deuterated (M⫹3) derivatives of these fatty acids were measured at m/z ⫽ 217, 245, 273, and 301, respectively, and corrected for
the M⫹3 values from C13 effects of the respective endogenous fatty
acids.
Functional characterization of PC16:0/14:0-enriched surfactant.
For functional characterization, surfactant enriched in PC16:0/14:0
(C14:03 feeding) was compared with surfactant of normal composition (PC16:0/16:0 enriched; C18:13 feeding). BALF was first ultracentrifuged at 48,000 g for 1 h at 4°C, and the pellets, consisting of
surfactant large aggregates, were suspended in 0.9% saline solution.
Interfacial adsorption and compression-expansion dynamics of films
formed from these surfactant complexes were assessed using a Captive Bubble Surfactometer, similar to that previously described (45),
and operated as described elsewhere (18). Aliquots of 150 –200 nl of
surfactant at a phospholipid concentration of 8 –10 mg/ml were
deposited at the air-liquid interface of a 50 ␮l air bubble formed inside
the Captive Bubble chamber thermostated at 37°C and filled with 5
mM Tris buffer pH 7, containing 150 mM NaCl and 10% sucrose to
ensure that the small volume of surfactant does not disperse in the full
chamber but instead remains in contact with the bubble. The bubble
was imaged with a video camera (Pulnix TM 7 CN) and recorded for
later analysis. A 5-min adsorption (film formation) period followed
the introduction of the surfactant model suspensions into the chamber
during which the bubble was not manipulated and the change in
surface tension was monitored (initial adsorption). Afterward, the
chamber was sealed and the film-coated bubble rapidly expanded to a
volume of 150 ␮l and imaged for another 5 min to determine
postexpansion adsorption. The process was repeated to generate 4
quasi-static compression/expansion cycles, consisting of stepwise
reductions in chamber volume to compress the film to the minimum
surface tension attainable before the film collapses, followed by
successive stepwise increases back to the initial bubble volume.
Between each compression and expansion step, there was a 4-s delay,
allowing the film to equilibrate, and a 1-min delay between cycles.
After the last quasi-static cycle, dynamic cycling was performed, in
which the bubble was continuously compressed and expanded over
the same volume range determined during the preceding quasi-static
cycles, at a rate of 20 cycles/min. For all imaged bubbles, volume,
interfacial area and surface tension were calculated from the height
and diameter of the bubble, as previously described (44).
Statistical data. were expressed as means ⫾ SE. Three to seven
separate experiments were performed for the triglyceride feeding and
R1310
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
Table 3. Composition of the major triglyceride fatty acids in lavaged lung tissue and blood plasma following C12:03-C18:23
triglyceride-rich diets
Fatty acid
Control
Trilaurin
Trimyristin
Tripalmitin
Triolein
Trilinolein
C12:0
C14:0
C16:0
C16:1-N7
C18:1-N9
C18:2-N6
C20:4-N6
3.06 ⫾ 0.29
5.43 ⫾ 0.21
37.66 ⫾ 0.19
4.55 ⫾ 0.24
32.85 ⫾ 0.08
2.71 ⫾ 0.16
3.70 ⫾ 0.38
16.10 ⫾ 0.83***
8.11 ⫾ 0.62
32.88 ⫾ 0.45
3.99 ⫾ 0.40
26.03 ⫾ 0.29†††
2.25 ⫾ 0.19
2.26 ⫾ 0.49
4.30 ⫾ 1.01
19.21 ⫾ 0.64***
31.79 ⫾ 0.38
4.16 ⫾ 0.40
27.08 ⫾ 0.68†††
2.57 ⫾ 0.14
2.56 ⫾ 0.38
1.50 ⫾ 0.17
3.27 ⫾ 0.23
47.21 ⫾ 1.16***
4.76 ⫾ 0.53
26.80 ⫾ 1.11†††
2.10 ⫾ 0.19
3.73 ⫾ 0.56
6.50 ⫾ 0.32
7.77 ⫾ 0.14
31.29 ⫾ 0.54
3.39 ⫾ 0.14
38.38 ⫾ 1.05***
2.32 ⫾ 0.09
2.31 ⫾ 0.29
2.06 ⫾ 0.34
4.48 ⫾ 0.37
38.28 ⫾ 0.50
4.95 ⫾ 0.20
28.12 ⫾ 0.35††
6.39 ⫾ 0.13***
4.81 ⫾ 0.27
C12:0
C14:0
C16:0
C16:1-N7
C18:1-N9
C18:2-N6
C20:4-N6
0.35 ⫾ 0.08
0.90 ⫾ 0.20
16.27 ⫾ 1.89
2.23 ⫾ 0.25
27.31 ⫾ 0.94
34.50 ⫾ 3.32
14.52 ⫾ 1.61
16.56 ⫾ 2.72***
2.04 ⫾ 0.33
13.03 ⫾ 0.83
1.46 ⫾ 0.22
15.38 ⫾ 0.63†††
27.27 ⫾ 1.03
20.47 ⫾ 1.78
Blood plasma
0.31 ⫾ 0.03
20.14 ⫾ 1.44***
13.37 ⫾ 0.8!
1.49 ⫾ 0.23
16.17 ⫾ 0.66†††
25.95 ⫾ 0.73
18.81 ⫾ 1.80
0.19 ⫾ 0.02
0.87 ⫾ 0.09
29.49 ⫾ 2.66***
2.32 ⫾ 0.19
20.32 ⫾ 0.87†††
24.78 ⫾ 1.53
17.31 ⫾ 2.09
0.46 ⫾ 0.06
0.88 ⫾ 0.04
12.16 ⫾ 0.59
0.80 ⫾ 0.10††,§§
30.40 ⫾ 1.34‡‡‡
24.18 ⫾ 0.57
27.20 ⫾ 1.16†
0.19 ⫾ 0.04
0.55 ⫾ 0.08
9.87 ⫾ 0.47
0.83 ⫾ 0.10††,§§
12.28 ⫾ 1.12†††
52.93 ⫾ 2.56***
20.64 ⫾ 3.37
Lung tissue
5.3- and 3.5-fold vs. 1.2–2.4-fold for other fatty acids after the
respective feedings. Importantly, exogenous C12:0 increased
C14:0 in both plasma and lung tissue triglycerides by 2.3 ⫾
0.4- and 1.5 ⫾ 0.1-fold, respectively, compared with controls
(P ⬍ 0.001).
Specific effects of exogenous triglycerides on surfactant and
lung tissue PC molecular composition. In control rats, surfactant PC comprised 47 ⫾ 1% PC16:0/16:0, 12 ⫾ 1% PC16:0/
14:0, and 23 ⫾ 1% PC16:0/16:1. All other components, palmitoyl-oleoyl-PC (PC16:0/18:1), palmitoyl-linoleyl-PC (PC16:0/
18:2), and palmitoyl-arachidonoyl-PC (PC16:0/20:4) were in
the range of 4 –7% (Fig. 1A). Similarly, lung tissue PC was
enriched in these three components, comprising 32 ⫾ 1%
PC16:0/16:0, 5.5 ⫾ 0.6% PC16:0/14:0, and 9.2 ⫾ 0.6% PC16:
0/16:1. Additionally, lung tissue PC was enriched in PC16:0/
18:1 (17 ⫾ 1%), PC16:0/18:2 (10 ⫾ 0.4%), and PC16:0/20:4
(7.2 ⫾ 0.1%), together with minor PC species containing
stearic (C18:0) acid like stearoyl-linoleyl-PC (PD18:0/18:2)
and stearoyl-arachidonoyl-PC (PC18:0/20:4) (2–3% each)
(Fig. 1B).
Fig. 1. BALF (A) and lavaged lung tissue (B) phosphatidylcholine (PC) molecular composition following C12:03C18:23 triacylglycerol (TAG)-enriched diets vs. carbohydrate (CH) control. Rats were fed for 6 days on a carbohydrate-based diet (CH-control) or on the same diet
supplemented with 2% trilaurin (C12:0), trimyristin (C14:
0), tripalmitin (C16:0), triolein (C18:1) or trilinolein (C18:
2). Data are expressed as means ⫾ SE of 4 to 7 experiments. Individual PC species were determined by HPLC as
described in MATERIALS AND METHODS. PC14:0/14:0,
dimyristoyl-PC; PC16:0/14:0, palmitoyl-myristoyl-PC;
PC14:0/16:0, myristoyl-palmitoyl-PC; PC16:0/16:0, dipalmitoyl-PC; PC16:0/16:1, palmitoyl-palmitoleoyl-PC;
PC16:0/18:1, palmitoyl-oleoyl-PC; PC16:0/18:2, palmitoyl-linoleoyl-PC; PC16:0/20:4, palmitoyl-arachidonoyl-PC;
PC18:0/18:2, stearoyl-linoleoyl-PC; PC18:0/20:4, stearoylarachidonoyl-PC. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001
vs. CH-control.
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Data are expressed as means ⫾ SE of the major fatty acids, comprising 89 –92% of total TAG in lung tissue and 95-97% in plasma; n ⫽ 4 to 7 experiments.
Rats were fed for 6 days on diets containing either no TAG supplementation (control) or with 2% trilaurin, trimyristin, tripalmitin, triolein, or trilinolein. Fatty
acids of triglycerides were determined by gas chromatography as described in MATERIALS AND METHODS. C20:4-N6, arachidonic acid.***P ⬍ 0.001 vs. all other
groups; ††P ⬍ 0.01, †††P ⬍ 0.001 vs. control; ‡‡‡P ⬍ 0.001 vs. C12:0, C14:0, C16:0 and C18:2-N6; §§P ⬍ 0.01 vs. C16:0.
R1311
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
vs. 115 ⫾ 5 breaths/min for C18:13 controls; n ⫽ 6; P ⬎ 0.05),
nor did the animals with such an “inverted” low PC16:0/16:0
surfactant show cyanosis or decreased growth (Table 2). Moreover, in vitro function of inverted surfactant was not different
from high-PC16:0/16:0 surfactant, neither initially nor after
expansion (Fig. 2A). Both surfactants reached equilibrium
surface tensions of 22–24 mN/m within 15 s. Also, minimal
surface tension values of below 5 mN/m were readily achieved
by both surfactants (Fig. 2B). Under quasi-static conditions,
inverted surfactant induced by trimyristin (C14:03; Fig. 2B,
bottom left) reached near-zero surface tension during the first
compression-expansion cycle with slightly less compression
(35% vs. 40%) than normal high PC16:0/16:0 surfactant after
triolein feeding (C18:13; Fig. 2B, top left). Under dynamic
conditions repeated compression-decompression resulted in
nearly identical surface tension behavior of inverted and normal surfactant (Fig. 2B, right).
Metabolism of exogenous d3-C14:0 and d3-C12:0 in lung,
plasma, and liver. Of the 4.92 ␮mol/g body wt d3-C14:0
applied to the rats, 31.4 ⫾ 5.7 nmol and 233 ⫾ 76 nmol were
detected in PC from BALF and lavaged lung tissue after 24 h.
These values were maintained for 48 h in BALF and lung
tissue PC with no significant decreases (Fig. 3, A and B).
Whereas the amounts of deuterated palmitic acid (d3-C16:0)
tended to increase continuously, deuterated stearic acid (d3C18:0) was below the limit of detection (Fig. 3, A and B). By
contrast, in plasma and liver, PC concentrations of d3-C16:0
surmounted those of d3-C14:0, and d3-C18:0 was present in
measurable amounts (Fig. 3, C and D). Moreover, d3-C16:0
and d3-C14:0 in plasma and liver PC peaked at 24 h and then
rapidly decreased in contrast to BALF and lung tissue PC (Fig.
3, C and D). Regarding the fatty acid substrates for lung PC
synthesis, d3-C14:0 surmounted d3-C16:0 in both lung and
plasma free fatty acids and triglycerides, while d3-C18:0 was
below the detection level during the 48-h period of investigation (Fig. 4, A–D). Again, for lung free fatty acids and triglyceride d3-C14:0 and d3-C16:0, concentrations were maintained
for at least 48 h (Fig. 4, A and C), while in plasma, their
concentrations peaked at 24 h and were nearly absent at 48 h
(Fig. 4, B and D).
In light of the virtual absence of C12:0 from surfactant PC,
but its effect on increasing myristic acid of PC (Table 4, Fig.
1), we investigated the metabolism of its deuterated analog
(d3-C12:0) and elongation products (d3-C14:0 and d3-C16:0) in
lungs and plasma. After ingestion of 4.92 ␮mol/g body wt
d3-C12:0, this deuterated fatty acid was below detection level
in PC of BALF. In contrast, however, lung tissue PC contained
Table 4. Fatty acid composition of lung tissue PC following C12:03-C18:23 TAG-rich diets vs. carbohydrate control
Fatty acid
Control
Trilaurin
Trimyristin
Tripalmitin
Triolein
Trilinolein
C12:0
C14:0
C16:0
C16:1-N7
C18:1-N9
C18:2-N6
C20:4-N6
0.07 ⫾ 0.01
3.31 ⫾ 0.13
52.51 ⫾ 0.63
3.21 ⫾ 0.18
13.94 ⫾ 0.19
8.56 ⫾ 0.26
8.27 ⫾ 0.14
0.59 ⫾ 0.05***
5.33 ⫾ 0.08***
51.07 ⫾ 0.37
2.28 ⫾ 0.12***
12.82 ⫾ 0.26
9.21 ⫾ 0.22
8.22 ⫾ 0.11
0.10 ⫾ 0.01
19.12 ⫾ 1.74***
40.58 ⫾ 1.16***
1.66 ⫾ 0.06***
11.96 ⫾ 0.37***
8.77 ⫾ 0.10
7.81 ⫾ 0.15††
0.07 ⫾ 0.003
3.28 ⫾ 0.13
54.23 ⫾ 0.52***
2.71 ⫾ 0.07
12.51 ⫾ 0.63***
9.06 ⫾ 0.15
8.66 ⫾ 0.19
0.09 ⫾ 0.01
3.85 ⫾ 0.11
52.56 ⫾ 0.15
1.50 ⫾ 0.06***
15.71 ⫾ 0.23***
7.66 ⫾ 0.22***
7.65 ⫾ 0.12††
0.07 ⫾ 0.01
3.00 ⫾ 0.13
52.13 ⫾ 0.33
1.77 ⫾ 0.14***
9.81 ⫾ 0.59***
16.34 ⫾ 0.33***
7.21 ⫾ 0.12†††
Data are expressed as means ⫾ SE of the major fatty acids, comprising 89 –91% of fatty acids in lung tissue PC; n ⫽ 4 to 7 experiments. Rats were fed for
6 days on diet containing either no TAG supplementation (control) or with 2% trilaurin, trimyristin, tripalmitin, triolein, or trilinolein. Phosphatidylcholines (PC)
fatty acids were determined by gas chromatography as described in MATERIALS AND METHODS. ***P ⬍ 0.001 vs. control; ††P ⬍ 0.01, †††P ⬍ 0.001 vs.
tripalmitin.
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In surfactant (BALF) and lung tissue, C14:03 increased
PC16:0/14:0 by three-fold at the expense of PC16:0/16:0 and
PC16:0/16:1, while only in lung tissue, PC16:0/18:1 was decreased (Fig. 1, A and B). Additionally, C14:03 induced the
formation of PC14:0/14:0 (7.0 ⫾ 0.3%), which in controls was
virtually absent. C12:0 also increased PC16:0/14:0 from
11.6 ⫾ 1.05 to 15.1 ⫾ 0.28% (P ⬍ 0.05) and PC14:0/14:0
from 0.14 ⫾ 0.01 to 0.62 ⫾ 0.02% (P ⬍ 0.05). C16:03 and
C18:13 exerted no significant effects on surfactant PC16:0/
14:0, PC16:0/16:0, or PC16:0/16:1 distribution, while C18:23
increased PC16:0/18:2 from 6.5 ⫾ 0.7% to 13.0 ⫾ 0.5%,
which was at the expense of PC16:0/16:1 (P ⬍ 0.05) and with
a small increase in PC16:0/16:0 (55 ⫾ 2% compared with 47 ⫾
1% in controls; P ⬍ 0.05) (Fig. 1A). Similar changes were
induced by exogenous triglycerides in lung tissue PC. Additionally, PC18:0/18:2 was increased here by C18:23 (Fig. 1B).
Consistent with these findings, analysis of the fatty acid composition of PC revealed that exogenous C12:03 and C14:03
increased C14:0 in both lung tissue (Table 4) and BALF (not
shown). Importantly, while the relative increases in C12:0 and
C14:0 in lung tissue PC were in the same range (8.4- and
5.8-fold by the respective diets) as in its triglycerides (Table 3),
total incorporation of C12:0 remained low so that in lung tissue
PC, it was always below 1% and did not impact on other PC
fatty acids, while C14:0 increased up to 19.2 ⫾ 0.6% and
decreased C16:0, C16:1, and C18:1 in lung PC (Table 4).
ESI-MS/MS analysis of the PC16:0/14:0 HPLC peaks collected from different samples revealed a mass by charge
(m/z) ⫽ ⫹706 as their single component, which is equivalent
to the masses of both PC16:0/14:0 and PC14:0/16:0, with
m/z ⫽ ⫹184 (phosphocholine) as a diagnostic fragment (not
shown). Complete hydrolysis of this peak with phospholipase A2 generated 1-myristoyl-glycero-3-phosphocholine
(lysoPC14:0; m/z ⫽ ⫹468) and 1-palmitoyl-glycero-3-phosphocholine (lysoPC16:0; m/z ⫽ ⫹496) as the only reaction
products, indicating that the PC16:0/14:0 peak from HPLC the
analysis was a mixture of 1-palmitoyl-2-myristoyl-PC (PC16:
0/14:0) and 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). In
C14:03-fed animals, 44.4 ⫾ 2.0% of the m/z ⫽ ⫹706 peak was
PC14:0/16:0. This was not different from PC16:0/16:0-enriched surfactant after triolein feeding (46.6 ⫾ 0.9% PC14:0/
16:0) or later development (adult, n ⫽ 5: 41.7 ⫾ 0.8%) (P ⬎
0.05).
Effects of PC composition on respiratory and surfactant
function. Resting respiratory rate of the animals with increased
PC16:0/14:0, PC14:0/16:0, and PC14:0/14:0 after C14:03 feeding was not different from PC16:0/16:0-rich animals (126 ⫾ 5
R1312
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
450 ⫾ 149 pmol/g body wt after 24 h, although by 48 h values
were below the detection limit (not shown). By contrast,
d3-C14:0, as well as d3-C16:0, increased in lung tissue and
surfactant PC (Fig. 5A). Interestingly and in contrast to the
d3-C14:0 experiments, absolute values of d3-C16:0 were higher
than those of d3-C14:0 (Fig. 5B). d3-C12:0 and its elongation
products d3-C14:0 and d3-C16:0, were detected in all potential
fatty acid sources for lung PC synthesis, free fatty acids and
triglycerides from both plasma and lung tissue (Fig. 5, C and
D). Significantly, concentrations of d3-C14:0 and d3-C16:0 in
lung tissue free fatty acids were much greater than those of
d3-C12:0 (Fig. 5C), while in lung tissue triglycerides, as well
as in plasma free fatty acids and triglycerides, d3-C12:0 values
were much higher than those of d3-C14:0 and d3-C16:0. d3C12:0 in lung triglycerides peaked at 24 h and then decreased,
while its elongation products further increased (Fig. 5D) in
contrast to exogenous d3-C14:0 experiments, where the d3C14:0 concentration was maintained for 48 h in lung tissue
triglycerides (Fig. 4C).
AJP-Regul Integr Comp Physiol • VOL
DISCUSSION
Phosphatidylcholine (PC) synthesis in type II pneumocytes
(PN-II) depends on exogenous choline for the formation of the
head group, while the glycerol moiety originates from exogenous glucose or endogenous glycogen stores (24, 46, 49). The
lung contains all of the enzymes to synthesize, desaturate or
elongate fatty acids (for review, see Ref. 39), suggesting that
PN-II may possess considerable “autarchy ” for generating a
PC molecular profile characteristic for surfactant. In utero, the
C2-units for palmitic acid (C16:0) synthesis are derived from
exogenous glucose or from pulmonary glycogen (40). Such
fatty acid synthesis of the lung is essential during fetal development, where 80% thereof are found in PC and other lung
phospholipids (10). In most vertebrates surfactant is highly
enriched in dipalmitoyl-PC (PC16:0/16:0), the end product of
de novo synthesis, as well as acyl remodeling. However, in all
mammals myristic acid (C14:0) containing PC species, like
PC16:0/14:0 or its isomers and alkyl-acyl-analogs are present,
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Fig. 2. Adsorption and expansion-compression curves for
surfactant prepared from 2% trimyristin (C14:03) vs. 2%
triolein (C18:13)-fed rats. Interfacial adsorption (a) of
C18:13 (Œ) and C14:03 () surfactants spread onto the
surface of a captive bubble formed on a 5 mM Tris subphase (pH 7) containing 150 mM NaCl and 10% sucrose.
Left: surface tension reduction kinetics upon spreading 150
nl of surfactant (10 mg/ml phospholipid) at the surface of a
clean 50-␮l air bubble. Right: surfactant adsorption upon
expansion of the bubble to a volume of 150 ␮l. Insets: rescaled
adsorption curves showing the fastest adsorption rates. Data
are expressed as means ⫾ SE of at least 4 independent
experiments for each surfactant sample. B: representative
compression-expansion surface tension-area isotherms of
C18:13 (top) and C14:03 (bottom) adsorbed surfactant films,
under quasi-static (left) or dynamic (right) cycling conditions as described in MATERIALS AND METHODS. Shown here
are the isotherms corresponding to the first (circles), second
(triangles), third (squares), and fourth (diamonds) quasistatic compression-expansion cycles, and first (circles), 10th
(triangles) and 20th (diamonds) dynamic cycles.
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
R1313
and sometimes highly enriched, either transiently during alveolar development as in humans, rats, and mice or continuously
as in pygmy shrews or some marsupials (7, 9, 25, 38). Although the regulation of surfactant-specific PC components has
hitherto been attributed mostly to ontological development and
hormonal regulation, our data suggest that exogenous supply of
the lungs with saturated fatty acids shorter than C16:0, particularly C14:0, may be a major determinant of surfactant PC
composition.
Role of exogenous fatty acid supply to the PN-II for surfactant PC homoeostasis. PN-II utilize exogenous fatty acids,
triglycerides and very low-density lipoproteins to synthesize
surfactant PC (13, 16, 42), suggesting that neutral lipids and
lipoproteins are intimately linked to surfactant metabolism. In
this context, the data presented here show that the fatty acid
composition of exogenous triglycerides differentially influence
the PC profile of lung surfactant. While all fatty acids supplied
change the fatty acid composition in plasma and lung tissue
triglycerides to similar extents, the effects of C12:0 and C14:0
were most significant compared with their concentrations in
carbohydrate controls or rats fed on longer fatty acids. Furthermore, supply with exogenous C14:0 increased the myristoylated PC components of surfactant at the expense of PC16:0/
16:0, while exogenous supply with C16:0, C18:1, and C18:2
Fig. 4. Incorporation of deuterated myristic acid and its
elongation products into free fatty acids and triglycerides
of lung tissue and blood plasma. Rats were fed 4.92
␮mol/g body wt d3-C14:0 and its presence and those of
its elongation products palmitic (d3-C16:0) and stearic
(d3-C18:0) acid determined in the free fatty acid (A, B)
and triglyceride (C, D) fractions of lung tissue (A, C) and
blood plasma (B, D) Deuterated fatty acids were determined after methylation by GC-MS as described in
MATERIALS AND METHODS. Data are expressed as means ⫾
SE of 4 experiments per data point. A, D: **P ⬍ 0.01,
***P ⬍ 0.001 vs. d3-C16:0 and d3-C18:0 of the corresponding time points; B, D: ‡‡P ⬍ 0.01, ‡‡‡P ⬍ 0.001
vs. 24 h of the respective fatty acid (d3-C14:0, d3-C16:0).
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Fig. 3. Incorporation of deuterated myristic acid (d3C14:0) and its elongation products palmitic (d3-C16:0)
and stearic (d3-C18:0) acid into lung, liver, and plasma
PC. Rats were fed 4.92 ␮mol/g body wt d3-C14:0 and its
incorporation and those of its elongation products d3C16:0 and d3-C18:0 acid into the PC fractions of surfactant (BALF) (A), lavaged lung tissue (B), blood plasma
(C), and liver (D) determined. Deuterated fatty acids were
determined by GC-MS as described in MATERIALS AND
METHODS. Data are expressed as means ⫾ SE of 4 experiments per data point. A, B: *P ⬍ 0.05, ***P ⬍ 0.001 vs.
d3-C16:0 and d3-C18:0 of corresponding time points; C,
D: †††P ⬍ 0.001 vs. d3-C14:0 and d3-C18:0 of corresponding time point; ‡‡P ⬍ 0.01, ‡‡‡P ⬍ 0.001 vs. 24 h
of respective fatty acid.
R1314
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
had no major effect on PC composition, which is consistent
with previous findings showing minor effects on PC16:0/16:0
concentrations when mainly feeding fatty acids longer than 16
carbon units, although supply with linoleic or long-chain polyunsaturated fatty acids may exert increased inflammatory reactions of the lung (2, 53). These data are also in line with
Longmuir and Rossi (28), showing that the lung does not
shorten fatty acids to a significant extent to generate C14:0.
Taken together, our data suggest that C14:0 is preferentially
enriched in the lungs, and C14:0 supply to the lungs is the most
significant exogenous modulator of surfactant PC profile.
In the rat and mouse, PC16:0/14:0 increases postnatally,
correlating with the high supply of milk containing C12:0 and
C14:0 (47), while such changes occur antenatally in other
species, such as humans and guinea pigs (7, 9, 21, 38). These
differences imply different mechanisms of regulation across
mammalian species. Fatty acids from very low density lipoproteins have been shown to be incorporated into PC (42), and
data from other groups (2) exclude a major hormonal control of
surfactant PC profile. Hence, we postulate that the fractions of
PC16:0/16:0 and PC16:0/14:0 in surfactant are determined by
the supply of the lungs with C14:0 from neutral lipids. This
metabolic principle may be true for both, direct supply from
plasma lipoproteins, as well as triglycerides stored in pulmonary lipid interstitial cells (50, 51).
AJP-Regul Integr Comp Physiol • VOL
Effects of molecular inversion of surfactant PC profile on
surface tension function. The view that mammalian surfactant
is highly enriched in PC16:0/16:0, a critical component
deemed necessary for lowering alveolar surface tension (1, 22),
has recently been challenged since surfactant contains very low
PC16:0/16:0 concentrations in several mammals (7, 25). Consistent with this, in vitro observations have shown that the
biophysical properties of the overall surfactant phospholipid
mixture are markedly different from those of the individual
components (33). In this context, it is not surprising that a
surfactant with an “inverted composition,” containing only
29% PC16:0/16:0, 45% PC16:0/14:0 and its isomer PC14:0/
16:0, as well as even 7% PC14:0/14:0 relative to total PC,
shows good surface tension function in vitro and, judging by
the normal development and breathing rates of the C14:03-fed
rats, in vivo. PC16:0/14:0 is possibly important for the immune
homoeostasis of developing lungs due to its effects on macrophages (17). Interestingly, PC16:0/14:0 is not present in surfactant of healthy bird lungs, which only contain interstitial
macrophages not exposed to the surfactant-containing air
spaces (29). On the other hand, phase transition temperatures
of PC14:0/14:0, PC16:0/14:0 and PC14:0/16:0 are below body
temperature (transition temperatures: 23°C, 27°C, 35°C, respectively) compared with PC16:0/16:0 (41.5°C) (20). Although this may well decrease the viscosity and possibly
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Fig. 5. Incorporation of deuterated lauric acid (d3-C12:0)
and its elongation products myristic (d3-C14:0) and
palmitic acid (d3-C16:0) into free fatty acids, PCs and
TAGs of lung tissue and plasma. Rats were fed 4.92
␮mol/g body wt d3-C12:0 and its presence and those of its
elongation products d3-C14:0 and d3-C16:0 determined in
BALF and lung tissue PC (A, B), lung tissue, and plasma
free fatty acids (C, E) and lung tissue and plasma TAG (D,
F). Deuterated fatty acids were determined as methyl esters
by GC-MS as described in MATERIALS AND METHODS. Data
are means ⫾ SE of 3 experiments per data point. A: **P ⬍
0.01 vs. 24 h; ††P ⬍ 0.01 vs. lung tissue d3-C14:0 and
d3-C16:0; B: *P ⬍ 0.05 vs. BALF d3-C14:0; **P ⬍ 0.01
vs. BALF d3-C16:0; C: ‡‡P ⬍ 0.01 vs. d3-C12:0 and
d3-C16:0; D, E: ¶¶P ⬍ 0.01 vs. d3C14:0 and d3-C16:0.
MYRISTIC ACID DECREASES SURFACTANT DIPALMITOYLPHOSPHATIDYLCHOLINE
Perspectives and Significance
The molecular profile of surfactant PC is related to lung
physiology, and to the supply and metabolism of neutral lipids,
where myristic acid is specifically enriched in the lung, and has
the most direct effect on components preferentially secreted
into the alveolar space. A significant contribution not only of
PC16:0/16:0, but also of PC16:0/14:0 and PC16:0/16:1 to lung
surfactant is characteristic of all mammals, including humans.
Respiratory function in vivo and surface tension function in
vitro of organisms possessing “inverted” surfactants with high
PC16:0/14:0, considerable PC16:0/16:1 and low PC16:0/16:0
suggest that inclusion of all these PC components, rather than
only of PC16:0/16:0, should be considered when constructing
synthetic therapeutic surfactants. Nevertheless, several issues
have yet to be addressed: do the actions of PC16:0/14:0 on
macrophage differentiation and function in vitro (17) hold also
true for alveolar immune homeostasis in vivo, where PC16:0/
14:0 is particularly increased during alveolar formation (7)?
AJP-Regul Integr Comp Physiol • VOL
Moreover, are these in vitro actions of surfactant PC16:0/14:0,
eventually together with the general accumulation of C14:0 in
other lung lipids, important for inflammatory reactions, where
unsaturated fatty acid supply appears to be harmful rather than
protective to the lung (53)? Finally, it will be essential to
address the safety and efficacy of PC16:0/14:0-enriched surfactants in vivo under pathological conditions. These include
maintenance of alveolar stability during hypothermia or fever,
as frequently found in preterm neonates and older patients in
the intensive care setting.
ACKNOWLEDGMENTS
The authors thank Verena Müller, Marco Raith, and Jamila Borchert for
excellent technical assistance.
GRANTS
This work was supported by an institutional grant of the medical faculty of
the University of Tübingen (F.1275127) and by the German Research Council
(BE2223/2-1). V. P. and J. P. -G. were supported by grants from the Spanish
Ministry of Science (BIO2009-09694, CSD2007-00010) and the Community
of Madrid (P-MAT-000283-0505).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
REFERENCES
1. Adams FH, Fujiwara T, Emmanouilides GC, Räihä N. Lung phospholipids of human fetuses and infants with and without hyaline membrane
disease. J Pediatr 77: 833–841, 1970.
2. Alam SQ, Alam BS. Lung surfactant and fatty acid composition of lung
tissue and lavage of rats fed diets containing different lipids. Lipids 19:
38 –43, 1984.
3. Ashton MR, Postle AD, Smith DE, Hall MA. Surfactant phosphatidylcholine composition during dexamethasone treatment in chronic lung
disease. Arch Dis Child Fetal Neonatal Ed 71: F114 –F117, 1994.
4. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem
234: 466 –468, 1959.
5. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and
purification. Can J Biochem Physiol 37: 911–917, 1959.
6. Bernhard W, Gebert A, Vieten G, Rau GA, Hohlfeld JM, Postle AD,
Freihorst J. Pulmonary surfactant in birds: coping with surface tension in
a tubular lung. Am J Physiol Regul Integr Comp Physiol 281: R327–R337,
2001.
7. Bernhard W, Hoffmann S, Dombrowsky H, Rau GA, Kamlage A,
Kappler M, Haitsma JJ, Freihorst J, von der Hardt H, Poets CF.Phosphatidylcholine molecular species in lung surfactant: composition in
relation to respiratory rate and lung development. Am J Respir Cell Mol
Biol 25: 725–731, 2001.
8. Bernhard W, Pynn CJ, Jaworski A, Rau GA, Hohlfeld JM, Freihorst
J, Poets CF, Stoll D, Postle AD. Mass spectrometric analysis of surfactant
metabolism in human volunteers using deuteriated choline. Am J Respir
Crit Care Med 170: 54 –58, 2004.
9. Bernhard W, Schmiedl A, Koster G, Orgeig S, Acevedo C, Poets CF,
Postle AD. Developmental changes in rat surfactant lipidomics in the
context of species variability. Pediatr Pulmonol 42: 794 –804, 2007.
10. Buechler KF, Rhoades RA. Fatty acid synthesis in the perfused rat lung.
Biochim Biophys Acta 619: 186 –195, 1980.
11. Caesar PA, McElroy MC, Kelly FJ, Normand IC, Postle AD. Mechanisms of phosphatidylcholine acyl remodeling by human fetal lung. Am
J Respir Cell Mol Biol 5: 363–370, 1991.
12. Caesar PA, Wilson SJ, Normand CS, Postle AD. A comparison of the
specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture. Biochem J 253: 451–457,
1988.
13. Darrah HK, Hedley-Whyte J. Rapid incorporation of palmitate into
lung: site and metabolic fate. J Appl Physiol 34: 205–213, 1973.
14. Fitzgerald ML, Xavier R, Haley KJ, Welti R, Goss JL, Brown CE,
Zhuang DZ, Bell SA, Lu N, Mckee M, Seed B, Freeman MW. ABCA3
inactivation in mice causes respiratory failure, loss of pulmonary surfac299 • NOVEMBER 2010 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
improve therapeutic applicability of surfactant (41), further
research will be necessary to evaluate its function in vivo at
variable body temperatures. However, since artificial PC16:0/
16:0-based surfactants appear to be less effective than natural
surfactants, inclusion of physiological surfactant components,
such as PC16:0/14:0 should be considered in the construction
of future artificial lung surfactants.
Metabolism of C14:0 in the lungs relative to plasma and
liver. The preferential enrichment of exogenously labeled d3C14:0 in lung tissue and surfactant PC is in clear contrast to the
liver, where preferentially, its elongation products d3-C16:0 &
d3-C18:0 were incorporated into PC and where turnover was
much faster. Moreover, while d3-labeled C12:0 was effectively
excluded from incorporation, its elongation products, d3-C14:0
& d3-C16:0 were enriched in lung PC, highlighting the specificity of pulmonary fatty acid incorporation into surfactant.
This is consistent with the preference of the lungs to incorporate exogenous C14:0, and with data on human volunteers
showing a smaller turnover of surfactant compared with
plasma PC (8). The results presented here extend those findings
to the neutral lipids, since in lung tissue triglycerides and free
fatty acids, the accumulation of d3-C14:0 and d3-C16:0 persisted, resembling the overall low lipid turnover of the lungs,
while in plasma (see Fig. 5, B and D) and liver (not shown)
d3-labeled free fatty acids and triglycerides had declined to
near zero values after 48 h.
Another specific indicator of the lung’s selectivity of d3C14:0 over d3-C12:0 metabolism is their turn over time in the
neutral lipid fraction of the lungs compared with plasma.
Although in lung and plasma triglycerides, as well as in plasma
free fatty acids, d3-C12:0 concentrations far exceeded those of
d3-C14:0 (and d3-C16:0), in lung tissue free fatty acids d3C14:0 as a fatty acid source of PC assembly exceeded d3-C12:0
(Fig. 5, C and D). This rules out that lung tissue free fatty acids
are merely an overflow of plasma or that they simply resemble
the hydrolysis products of lung tissue triglycerides. Instead,
d3-C14:0 (and d3-C16:0) must either be preferentially absorbed
from the circulation as free fatty acids or effectively elongated
from pulmonary and extrapulmonary triglyceride stores. Hence,
C14:0 is specifically enriched in surfactant PC by either direct
supply or via elongation of shorter fatty acids (e.g., C12:0).
R1315
R1316
15.
16.
17.
18.
19.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
tant, and depletion of lung phosphatidylglycerol. J Lipid Res 48: 621–632,
2007.
Folch J, Lees M, Stanley GHS. A simple method for the isolation and
purification of total lipids from animal tissues. J Biol Chem 226: 497–509,
1956.
Gal S, Bassett DJ, Hamosh M, Hamosh P. Triacylglycerol hydrolysis in
the isolated, perfused rat lung. Biochim Biophys Acta 713: 222–229, 1982.
Gille C, Spring B, Bernhard W, Gebhard C, Basile D, Lauber K, Poets
CF. Differential effect of surfactant and its saturated phosphatidylcholines
on human blood macrophages. J Lipid Res 48: 307–317, 2007.
Gomez-Gil L, Goormaghtigh E, Perez-Gil J. Pulmonary surfactant
protein SP-C counteracts the deleterious effects of cholesterol on the
activity of surfactant films under physiologically relevant compressionexpansion dynamics. Biophys J 97: 2736 –2745, 2009.
Gonzales LW, Guttentag SH, Wade KC, Postle AD, Ballard PL.
Differentiation of human pulmonary type II cells in vitro by glucocorticoid
plus cAMP. Am J Physiol Lung Cell Mol Physiol 283: L940 –L951, 2002.
Huang CH. Mixed-chain phospholipids: structures and chain-melting
behavior. Lipids 36: 1077–1097, 2001.
Hunt AN, Kelly FJ, Postle AD. Developmental variation in whole human
lung phosphatidylcholine molecular species: a comparison with guinea pig
and rat. Early Hum Dev 25: 157–171, 1991.
Ikegami M, Jobe A. Surfactant function in respiratory distress syndrome.
J Pediatr 102: 443–447, 1983.
Ingenito EP, Mora R, Mark L. Pivotal role of anionic phospholipids in
determining dynamic behaviour of lung surfactant. Am J Respir Crit Care
Med 161: 831–838, 2000.
Ishiguro N, Oyabu M, Sato T, Maeda T, Minami H, Tamai I.
Decreased biosynthesis of lung surfactant constituent phosphatidylcholine
due to inhibition of choline transporter by gefitinib in lung alveolar cells.
Pharm Res 25: 417–427, 2008.
Lang C, Postle A, Orgeig S, Possmayer F, Bernhard W, Panda A,
Jurgens K, Milsom W, Nag K, Daniels C. Dipalmitoylphosphatidylcholine is not the major surfactant phospholipid species in all mammals. Am
J Physiol Regul Integr Comp Physiol 289: R1426 –R1439, 2005.
Lepage G, Roy CC. Direct transesterification of all classes of lipids in a
one-step reaction. J Lipid Res 27: 114 –120, 1986.
Longmuir KJ, Haynes S. Evidence that fatty acid chain length is a type
II cell lipid-sorting signal. Am J Physiol Lung Cell Mol Physiol 260:
L44 –L51, 1991.
Longmuir KJ, Rossi ME. Fatty acid chain-shortening activity in the
pulmonary type II cell. Biochim Biophys Acta 961: 144 –147, 1988.
Lorz C, Lopez J. Incidence of air pollution in the pulmonary surfactant
system of the pigeon (Columba livia). Anat Rec 249: 206 –212, 1997.
McGowan SE, Torday JS. The pulmonary lipofibroblast (lipid interstitial
cell) and its contributions to alveolar development. Annu Rev Physiol 59:
43–62, 1997.
Mulugeta S, Gray JM, Notarfrancesco KL, Gonzales LW, Koval M,
Feinstein SI, Ballard PL, Fisher AB, Shuman H. Identification of
LBM180, a lamellar body limiting membrane protein of alveolar type II
cells, as the ABC transporter protein ABCA3. J Biol Chem 277: 22147–
22155, 2002.
Petkovic M, Julia Müller J, Matthias Müller M, Jürgen Schiller J,
Klaus Arnold K, Arnhold J. Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A2. Anal Biochem 308: 61–70, 2002.
Piknova B, Schief WR, Vogel V, Discher BM, Hall SB. Discrepancy
between phase behaviour of lung surfactant phospholipids and the classical model of surfactant function. Biophys J 81: 2172–2180, 2001.
Postle AD. Method for the sensitive analysis of individual molecular
species of phosphatidylcholine by high-performance liquid chromatogra-
AJP-Regul Integr Comp Physiol • VOL
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
phy using post-column fluorescence detection. J Chromatogr 415: 241–
251, 1987.
Postle AD, Gonzales LW, Bernhard W, Clark GT, Godinez MH,
Godinez RI, Ballard PL. Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells. J
Lipid Res 47: 1322–1331, 2006.
Rau GA, Vieten G, Haitsma JJ, Freihorst J, Poets CF, Ure BM,
Bernhard W. Surfactant in newborn compared to adolescent pigs: adaptation to neonatal respiration. Am J Respir Cell Mol Biol 30: 694 –701,
2004.
Reeves PG, Nielsen FH, Fahey Jr GC. AIN-93 purified diets for
laboratory rodents: final report of the american institute of nutrition ad hoc
writing committee on the reformulation of the AIN-76A rodent diet. J Nutr
123: 1939 –1951, 1993.
Ridsdale R, Roth-Kleiner M, D’Ovidio F, Unger S, Yi M, Keshavjee S,
Tanswell AK, Post M. Surfactant palmitoylmyristoylphosphatidylcholine
is a marker for alveolar size during disease. Am J Respir Crit Care Med
172: 225–232, 2005.
Rooney SA. Fatty acid biosynthesis in developing fetal lung. Am J Physiol
Lung Cell Mol Physiol 257: L195–L201, 1989.
Rooney SA, Gobran LI, Chu AJ. Thyroid hormone opposes some
glucocorticoid effects on glycogen content and lipid synthesis in developing fetal rat lung. Pediatr Res 20: 545–550, 1986.
Rüdiger M, Tölle A, Meier W, Rüstow B. Naturally derived commercial
surfactants differ in composition of surfactant lipids and in surface
viscosity. Am J Physiol Lung Cell Mol Physiol 288: L379 –L383, 2005.
Ryan AJ, Medh JD, McCoy DM, Salome RG, Mallampally RK.
Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis. Am J Physiol Lung Cell Mol Physiol 283: L310 –L318,
2002.
Schmiedl A, Vieten G, Mühlfeld C, Bernhard W. Distribution of
intracellular and secreted surfactant during postnatal rat lung development.
Pediatr Pulmonol 42: 548 –562, 2007.
Schoel WM, Schurch S, Goerke J. The captive bubble method for the
evaluation of pulmonary surfactant: surface tension, area, and volume
calculations. Biochim Biophys Acta 1200: 281–290, 1994.
Schurch S, Bachofen H, Goerke J, Green F. Surface properties of rat
pulmonary surfactant studied with the captive bubble method: adsorption,
hysteresis, stability. Biochim Biophys Acta 1103: 127–136, 1992.
Sheehan PM, Yeh YY. Lung lipid synthesis from acetoacetate and
glucose in developing rats in vitro. Lipids 18: 295–301, 1983.
Skottová N, Palkovic M, Macho L. Effect of premature weaning on the
fatty acid composition of rat adipose tissue. Physiol Bohemoslov 2:
97–101, 1977.
Stefan N, Peter A, Cegan A, Staiger H, Machann J, Schick F, Claussen
CD, Fritsche A, Häring HU, Schleicher E. Low hepatic stearoyl-CoA
desaturase 1 activity is associated with fatty liver and insulin resistance in
obese humans. Diabetologia 51: 648 –656, 2008.
Tian Y, Zhou R, Rehg JE, Jackowski S. Role of phosphocholine
cytidylyltransferase alpha in lung development. Mol Cell Biol 27: 975–
982, 2007.
Torday J, Hua J, Slavin R. Metabolism and fate of neutral lipids of fetal
lung fibroblast origin. Biochim Biophys Acta 1254: 198 –206, 1995.
Tordet C, Marin L, Dameron F. Pulmonary di-and-triacylglycerols
during the perinatal development of the rat. Experientia 37: 333–334,
1981.
Veldhuizen EJA, Batenburg JJ, van Golde LMG, Haagsman HP. The
role of surfactant proteins in DPPC enrichment of surface films. Biophys
J 79: 3164 –3171, 2000.
Wolfe RR, Martini WZ, Irtun O, Hawkins HK, Barrow RE. Dietary
fat composition alters pulmonary function in pigs. Nutrition 18: 647–653,
2002.
299 • NOVEMBER 2010 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
20.
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