Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat ...

J Appl Physiol 111: 449–457, 2011. First published June 2, 2011; doi:10.1152/japplphysiol.00766.2010.

Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat surfactant is lung specific and correlates with oral myristic acid supply Wolfgang Bernhard,1 Marco Raith,1 Christopher J. Pynn,1,3 Christian Gille,1 Guido Stichtenoth,2 Dieter Stoll,3 Erwin Schleicher,4 and Christian F. Poets1 Departments of 1Neonatology and 4Internal Medicine IV, Faculty of Medicine, Eberhard-Karls University, Tübingen; 2 Childrens Hospital, University Clinic Schleswig-Holstein, Lübeck; and 3Natural and Medical Sciences Institute, Reutlingen, Germany Submitted 7 July 2010; accepted in final form 1 June 2011

Bernhard W, Raith M, Pynn CJ, Gille C, Stichtenoth G, Stoll D, Schleicher E, Poets CF. Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat surfactant is lung specific and correlates with oral myristic acid supply. J Appl Physiol 111: 449 – 457, 2011. First published June 2, 2011; doi:10.1152/japplphysiol.00766.2010.— Surfactant predominantly comprises phosphatidylcholine (PC) species, together with phosphatidylglycerols, phosphatidylinositols, neutral lipids, and surfactant proteins-A to -D. Together, dipalmitoyl-PC (PC16:0/16:0), palmitoyl-myristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) make up 75– 80% of mammalian surfactant PC, the proportions of which vary during development and in chronic lung diseases. PC16:0/14:0, which exerts specific effects on macrophage differentiation in vitro, increases in surfactant during alveolarization (at the expense of PC16:0/16:0), a prenatal event in humans but postnatal in rats. The mechanisms responsible and the significance of this reversible increase are, however, not understood. We hypothesized that, in rats, myristic acid (C14:0) enriched milk is key to lung-specific PC16:0/14:0 increases in surfactant. We found that surfactant PC16:0/14:0 in suckling rats correlates with C14:0 concentration in plasma chylomicrons and lung tissue triglycerides, and that PC16:0/14:0 fractions reflect exogenous C14:0 supply. Significantly, C14:0 was increased neither in plasma PC, nor in liver triglycerides, free fatty acids, or PC. Lauric acid was also abundant in triglycerides, but was not incorporated into surfactant PC. Comparing a C14:0-rich milk diet with a C14:0-poor carbohydrate diet revealed increased C14:0 and decreased C16:0 in plasma and lung triglycerides, respectively. PC16:0/14:0 enrichment at the expense of PC16: 0/16:0 did not impair surfactant surface tension function. However, the PC profile of the alveolar macrophages from the milk-fed animals changed from PC16:0/16:0 rich to PC16:0/14:0 rich. This was accompanied by reduced reactive oxygen species production. We propose that nutritional supply with C14:0 and its lung-specific enrichment may contribute to decreased reactive oxygen species production during alveolarization. lung development; dipalmitoyl phosphatidylcholine; lipoproteins; myristic acid; neutral lipids LUNGS ARE STABILIZED BY surfactant, a complex containing ⬃80 – 85% phospholipids, 10% neutral lipids, and 5–10% proteins, including the surfactant proteins (SP)-A to -D. The molecular composition of surfactant varies according to differences in pulmonary physiology, anatomy, and development (29). This variability is described not only for its proteins, but also for its major phospholipids (2, 3, 28). Most of the published work describing surfactant composition reports high concentrations of dipalmitoyl-phosphatidylcholine

Address for reprint requests and other correspondence: W. Bernhard, Dept. of Neonatology, Faculty of Medicine, Eberhard-Karls-Univ., Calwer Strasse 7, D-72076 Tübingen, FRG (e-mail: [email protected]). http://www.jap.org

(PC16:0/16:0) (29, 41). However, mammalian surfactant also contains significant amounts of disaturated palmitoyl-myristoylphosphatidylcholine (PC16:0/14:0) and monounsaturated palmitoyl-palmitoleoyl-phosphatidylcholine (PC16:0/16:1), which are preferentially secreted by type II pneumocytes (PN-II) (3). PC16: 0/14:0, for example, ranges from 8 –12% in human surfactant to 25% in some mammals, in contrast to avian lungs, in which it is virtually absent (2, 21). PC16:0/14:0 increases during alveolarization, which takes place in utero in humans and guinea pigs, but occurs postnatally in the rat and mouse (3, 29). In vitro PC16:0/ 14:0 mimics the effects of natural surfactant on macrophage (M⌽) differentiation and M⌽-dependent inhibition of T-lymphocyte proliferation (17), suggesting an important regulatory role in alveolar M⌽ functions. While maternal glucocorticoid treatment administered to trigger fetal PN-II differentiation in premature babies does not alter surfactant phosphatidylcholine (PC) composition (1, 16), maternal lipoprotein loading stimulates fetal surfactant synthesis (33). In keeping with this, triglyceride (TG) stores are increased postnatally in rat lungs (25, 40), suggesting that lipid supply to the lungs may be important for surfactant maturation. Our laboratory recently investigated the impact of nutrition on surfactant PC composition using synthetic TGs, together with stable isotope labeled fatty acids, where we showed that the PC16:0/14:0 concentration was dramatically increased by trimyristin (C14:03), and to a lesser extent by trilaurin (C12: 03), due to lung-specific accumulation of exogenous myristic acid (C14:0) and elongation of lauric acid (C12:0) to C14:0 (27). As rat milk is rich in C12:0 and C14:0, we hypothesized that increased PC16:0/14:0 in postnatal rat surfactant may strongly depend on the physiological variation of these fatty acids. We, therefore, investigated the link between C12:0 and C14:0 supply via rat milk nutrition and the induced changes in surfactant phospholipidomics. Furthermore, we analyzed the lipid fractions of plasma and liver, to further support our hypothesis that C14:0 enrichment in surfactant PC is lung specific, and that such enrichment is variable during normal development. Reasoning that the specific enrichment of myristoylated surfactant PC during mammalian development (5, 28) may play a functional role, we determined the effects of the induced surfactant PC profile changes on surface tension function in vitro. We further investigated the impact of PC16:0/ 14:0 enrichment of surfactant on PC metabolism and homeostasis of alveolar M⌽ in vivo and on their function in terms of the production of reactive oxygen species (ROS). Our results suggest that physiological variations in dietary C14:0 increased PC16:0/14:0 in surfactant at the expense of PC16:0/16:0. This lung-specific effect involves chylomicrons and is limited to

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MILK LIPIDS MODIFY SURFACTANT LIPIDOMICS

C14:0 incorporation, whereas C12:0, also abundant in rat milk, is excluded from incorporation into surfactant PC. Whereas surface tension function of surfactant in vitro, ventilation rate, and animal growth were unchanged, production of ROS by alveolar M⌽ was decreased in cells exposed to high PC16:0/ 14:0 surfactant. We speculate that the surfactant-specific increase in PC16:0/14:0, resulting from the increased C14:0 supply during alveolarization, helps protect against radicalinduced impairment during development. MATERIALS AND METHODS

Phospholipid and fatty acid standards were from Sigma-Aldrich (Deisenhofen, Germany) or Avanti Polar Lipids (Alabaster, AL). Chloroform and methanol [high-performance liquid chromatography (HPLC) grade] were from Baker (Deventer, The Netherlands), whereas n-butanol (analytic grade) was from Merck (Darmstadt, Germany). RPMI medium was from Biochrom (Berlin, Germany), whereas fetal calf serum and phorbol 12-myristate-13-acetate from Sigma-Aldrich (Munich, Germany). Other chemicals (analytical grade) were from various commercial sources. Animal maintenance. Sprague-Dawley rats were kept specific pathogen free and had ad libitum access to carbohydrate-rich standard diet (Provivmi Kliba SA3336, 5.5% fat) and tap water. Spontaneously delivered pups of either sex were removed from their mothers directly before death at postnatal days (d) 1–2 or d14 –15, or at d21 to investigate adult rats. To compare milk (rich in C14:0 and C12:0) with standard diet, animals were either kept with their mothers with no access to standard diet or removed at d19 and fed Provivmi Kliba SA3336 until death at d24; d19 was chosen to ensure animals were mature enough to feed themselves on standard diet. Animal growth was monitored, and gastric content analyzed at death. Experiments were approved by the local government and met the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. For the investigation of PC metabolism of M⌽, rats with surfactant naturally rich in PC16:0/14:0 (d14) or poor in PC16:0/14:0 and rich in PC16:0/16:0 (adult) were injected with 50 mg/kg [D9methyl]choline chloride intraperitoneally 1.5 h before death, based on the finding that it takes ⬃1.5 h before labeled PC is actively secreted, whereas incorporation of [D9-methyl]choline into cell PC starts immediately (4, 27). Harvesting of samples. Rats were killed by intraperitoneal injection of 100 mg ketamine and 20 mg xylazine (WDT, Garbsen, Germany) per kilogram body weight. Blood (0.2–3 ml) was collected into ethylenediaminetetraacetic acid-coated containers by puncturing the right heart ventricle and centrifuged at 1,000 g for 10 min, and the plasma supernatant was harvested. Lungs were perfused with ice-cold 0.9% saline to remove blood and lavaged to harvest 4 –32 ml (depending on age) lung lavage fluid (LLF/surfactant), and LLF cells were separated by centrifugation (200 g, 10 min, 4°C) (3). After decantation of the cell-free supernatant, cell pellets (⬎98% M⌽) of rats exposed in vivo to a surfactant rich in PC16:0/14:0 (d14) or a surfactant poor in PC16:0/14:0 (adult) were washed twice extensively with excess isotonic phosphate-buffered saline without calcium and magnesium (PBS) (14 ml, 4°C). Cells were then resuspended in 2.2 ml PBS, containing 5.6 mmol/l glucose. For mass spectrometric analysis of phospholipids, 0.2-ml aliquots were extracted according to Bligh and Dyer (6), whereas 2 ml were immediately subjected to experiments for the generation of ROS and FACS analysis. Lungs and livers were excised, and gastric contents gently squeezed out. Samples were snap frozen in liquid nitrogen and stored at ⫺80°C until analysis. Lipoprotein preparation. Plasma (0.75 ml) was placed in 5-ml polyethylene tubes, mixed with 0.75-ml 64% (wt/vol) NaBr solution (density: 1.308 g/ml), and sequentially overlaid with 1.5 ml 16% NaBr (1.135 g/ml) and 1.5 ml distilled water (density: 1 g/ml) (11). Samples were centrifuged at 200,000 g for 2.5 h in a model AH-650 rotor using J Appl Physiol • VOL

a Discovery SE ultracentrifuge (Sorvall, Newton, CT). The layer on top of the distilled water was regarded as chylomicrons (density ⬍0.95 g/ml), whereas material from top of the 16% NaBr downwards was regarded as nonchylomicron lipids. Fractions were aspirated with 1-ml syringes and immediately extracted. Lipid analysis. Lipids were extracted from LLF, blood plasma, lipoproteins, and LLF cells (6), as well as from lung, liver, and gastric contents (14). Their phospholipid content was measured as previously described (3), with the exception of the LLF cells due to the minimal phospholipid amounts obtained. For PC and TG isolation (2, 5, 27), material containing 0.5–1.0 ␮mol phospholipid was dried down, dissolved in 1 ml chloroform, applied to 100 mg Strata NH2 cartridges (Phenomenex); and TG and PC were sequentially eluted with 3 ⫻ 1 ml chloroform and 1.2 ml chloroform-methanol (2 ⫹ 1 vol/vol), respectively. Fatty acids from chymus lipids, TG, and PC were analyzed using gas chromatography after transformation to fatty acid methyl esters using 13,16,19-docosatrienoic as an internal standard (22), a HP5890 gas chromatograph (Hewlett Packard) equipped with a flame ionization detector (250°C), hydrogen/synthetic air as combustion gases, and a 60 m ⫻ 0.25 mm Rtx2330 column (Restek) with helium as carrier gas. Column settings were 80°C for 2 min, then 30°C/min increase to 120°C, and 2°C/min increase to 240°C. Quantification was performed using calibration curves for the respective components. Analysis of PC species of all organs, LLF, and plasma was performed with HPLC, as described previously (26). Mass spectrometric analysis of PC in cells of LLF. Lipid extracted M⌽ suspensions were concentrated under nitrogen and resuspended in butanol-methanol-water (75:23:2 vol/vol/vol). Composition of endogenous and [D9-methyl]choline labeled PC species was determined by electrospray ionization tandem mass spectrometry in the positive ionization mode using a Thermo-Finnigan TSQ Quantum Discovery Max (Thermo-Fisher Scientific, Dreieich, Germany), as previously described (27). Quantification of endogenous PC and [methyl-D9]labeled PC was performed in the selected reaction monitoring mode by monitoring transitions from the parent ions of the major PC molecular species (Table 1), which, together, comprise at least 95% of total PC to the diagnostic fragments mass-to-charge ratio (m/z) ⫽ ⫹184 (phosphocholine) and m/z ⫽ ⫹193 (D9-labeled phosphocholine) for the endogenous and newly synthesized PC species, respectively. Water-soluble PC precursors. Choline and phosphocholine were quantified in the water/methanol phase of plasma and lung tissue extracts using D4-choline as an internal standard. Components were separated using an HPLC model 1100 (Agilent, Waldbronn, FRG), an Atlantis HILIC silica column (2.1 ⫻ 30.0 mm) (Waters, MA) at 50°C, and a gradient elution of 500 mM ammonium formate (pH 4.1) and acetonitrile/0.05% formic acid (4). Metabolites were quantified on a Micromass Quattro Micro triple-quadrupole (Waters, Eschborn, Germany) under positive ionization, with multiple-reaction monitoring employing the following transitions: choline ⫽ 103.8 ¡ 59.8 m/z; phosphocholine ⫽ 183.8 ¡ 85.8 m/z. Analysis of surfactant function. Surfactant function was assessed with a pulsating bubble surfactometer (PBS, Electronetics, Amherst, NY), as described previously (2, 28). LLF was centrifuged at 40,000 g for 15 min at 4°C to sediment large aggregates, and the pellets were adjusted with 154 mM NaCl/1.5 mM CaCl2 to give phospholipid concentrations of 3 mg/ml. A sample aliquot was instilled into the chamber of the PBS; a bubble created in the sample and surfactant allowed adsorption to the interface for 10 s. The bubble was then pulsated at a rate of 20/min. The pressure across the bubble was followed for 100 pulsations, and minimum surface tension was calculated using the Laplace equation. Quantification of ROS. LLF cells were counted in an ultraplane Neubauer hemocytometer, adjusted to 1 ⫻ 105 cells/ml in RPMI with 5% fetal calf serum, and placed in a 24-well plate. ROS production was quantified according to Rothe and Valet (32). In brief, cells were incubated with dihydrorhodamine (Sigma, Munich, Germany, 1

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Table 1. Endogenous and [D9-methyl]choline-labeled PC species of alveolar macrophages

PC Species

PC14:0/14:0, PC16:0/14:0, PC16:0/16:0, PC16:0/16:1, PC16:0/18:0, PC16:0/18:1, PC16:0/18:2, PC16:0/18:3, PC16:0/20:4, PC16:0/22:6, PC18:0/18:0, PC18:0/18:1, PC18:0/20:4, PC18:0/22:6, PC18:0/18:2, PC18:1/18:2, PC18:1/20:4, PC20:0/20:0,

dimyristoyl-PC palmitoyl-myristoyl-PC dipalmitoyl-PC palmitoyl-palmitoleoyl-PC palmitoyl-stearoyl-PC palmitoyl-oleoyl-PC palmitoyl-linoleyl-PC palmitoyl-linolenoyl-PC palmitoyl-arachidonoyl-PC palmitoyl-docosahexaenoyl-PC distearoyl-PC stearoyl-oleoyl-PC stearoyl-arachidonoyl-PC stearoyl-docosahexaenoyl-PC stearoyl-linoleyl-PC oleoyl-linoleyl-PC oleoyl-arachidonoyl-PC dieicosanoyl-PC (internal standard)

Mass of Mass of [D9-Methyl] Endogenous Choline-labeled Component Component

678 706 734 732 762 760 758 756 782 806 790 788 810 834 786 784 808 846

687 715 743 741 771 769 767 765 791 815 799 797 819 843 795 793 817

Monoisotopic masses of individual lyso-phosphatidylcholine (PC) and PC species were calculated, and masses analyzed by parent scan of lipid extracts, using endogenous [mass-to-charge ratio (m/z) ⫽ ⫹184], D3- (m/z ⫽ ⫹187), D6- (m/z ⫽ ⫹190), and D9-choline phosphate (m/z ⫽ 193) as diagnostic fragments, as described elsewhere (4, 27). For routine analysis, the endogenous and deuteriated PC components comprising ⬎95% of PC were analyzed in the specific reaction monitoring mode after loop injection, as described in MATERIALS AND METHODS.

␮mol/l) for 5 min at 37°C, followed by induction of ROS production by phorbol 12-myristate 13-acetate (Sigma, 100 nmol/l). After 30 min, cells were placed on ice, stained with phycoerythrin-labeled monoclonal antibody anti-rat-CD45 (BD Biosciences, Heidelberg, Germany) for 15 min, washed with PBS, and immediately analyzed on a FACS Scan cytometer (BD Biosciences). Cells were gated by forward vs. side scatter and CD45 expression. Mean dihydrorhodamine-fluorescence intensities were assessed. Unstimulated cells served as control. Statistics. Data are expressed as means ⫾ SE. Differences were tested by a two-tailed Student’s t-test, or by one-way analysis of

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variance for multiple-group comparisons using Graph Pad Instat 3 (Graph Pad 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

Fatty acids of lung and plasma TG relative to postnatal surfactant PC. The reversible increase of PC16:0/14:0 in LLF and lung tissue at d14 compared with term and d42, which occurred at the expense of PC16:0/16:0 (Fig. 1, A and C), was accompanied by a concurrent increase in C14:0 and C12:0 of lung tissue and blood plasma TG (Fig. 1, B and D). Other PC species in LLF or lung tissue did not follow this pattern, either continuously decreasing or increasing with age, or maintaining their d14 concentrations. At d14, when PC16:0/14:0 in LLF was doubled relative to term and d42 values, both C14:0 and C12:0 were increased severalfold in lung and plasma TG (P ⬍ 0.001). These reversible increases were mostly at the expense of oleic acid (C18:1) (Fig. 1, B and D). However, changes in C18:1 and palmitic acid (C16:0) were not followed by the equivalent changes of palmitoyloleoyl-PC or PC16:0/16:0 in lung tissue or surfactant (Fig. 1, A–D). Dietary effects on animal growth and overall lipid and fatty acid homeostasis. Fatty acid composition of gastric contents of d14 rats fed on milk, and of d24 rats fed on milk vs. rodent chow, are given in Table 2. Data show that, compared with chow, rat milk comprised 9 –17% C14:0 and 18 –22% C12:0, which was mainly at the expense of C16:0, C18:1, and linoleic acid (C18:2). By contrast, chow contained ⬍2% of C12:0 and C14:0. Animal weight, breathing rate, liver weight, and weight of gastric contents were unaffected by dietary interventions from d19 –24, while the gastric lipid content of milk-fed animals was fivefold higher for milk than for chow-fed animals. Withdrawal of milk feeding at d19 resulted in decreased liver and plasma phospholipids. However, lung tissue phospholipids were unchanged, whereas those in LLF were slightly increased. Diet did not influence the pool sizes of PC precursors, choline, and phosphocholine in lung tissue. Similarly, the plasma concentrations of choline were unchanged (Table 3). In

Fig. 1. Phosphatidylcholine (PC) composition of surfactant [lung lavage fluid (LLF); A] and lung tissue (B), and of fatty acid (FA) composition of lung tissue (C) and blood plasma (D) triglyceride (TG). Values are means ⫾ SE of 3–5 [postnatal day (d) 1, continuous placental feeding until delivery, with less than 24 h free access to milk], 5 (d14 –15, suckling rats), and 4 – 8 (d42, rats that received pure carbohydrate-rich chow from d21 onwards) experiments. TAG, triacylglycerol; PC16:0/14:0, palmitoyl-myristoyl-PC; PC16:0/16:0, dipalmitoyl-PC; PC16: 0/16:1, palmitoyl-palmitoleoyl-PC; PC16:0/18:1, palmitoyloleoyl-PC; PC16:0/18:2, palmitoyl-linoleoyl-PC; PC16:0/20:4, palmitoyl-arachidonoyl-PC; C12:0, lauric; C14:0, myristic; C16.0, palmitic; C16.1, palmitoleic; C18:1 oleic; C18:2, linoleic; C20:4, arachidonic acid. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. newborn; †††P ⬍ 0.001 vs. adult; ‡P ⬍ 0.05 vs. d14 –15.

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Table 2. Fatty acid composition of gastric contents after milk vs. rodent chow nutrition Fatty Acid

Milk Fed Until d24 (N ⫽ 5)

Chow Fed, d19-24 (N ⫽ 3)

Milk Fed, d14 (N ⫽ 4)

Lauric acid (C12:0) Myristic acid (C14:0) Palmitic acid (C16:0) Palmitoleic acid (16:1) (cis-⌬7) Stearic acid (C18:0) Oleic acid (C18:1) (cis-⌬9) Linoleic acid (C18:2) (cis-⌬9,12) Arachic acid (C20:0) Arachidonic acid (C20:4) (cis-⌬5,8,11,14) Eicosapentaenoic acid (C20:5) (cis-⌬5,8,11,14,17) Behenic acid (C22:0) Docosahexaenoic acid (C22:6) (cis-⌬4,7,10,13,16,19)

17.74 ⫾ 1.40 9.21 ⫾ 0.68 29.99 ⫾ 0.49 2.46 ⫾ 0.06 5.66 ⫾ 0.21 29.21 ⫾ 0.92 3.81 ⫾ 0.39 0.24 ⫾ 0.03 1.23 ⫾ 0.04 0.07 ⫾ 0.01 0.13 ⫾ 0.04 0.24 ⫾ 0.01

0.59 ⫾ 0.17*** 1.22 ⫾ 0.18*** 36.02 ⫾ 0.30*** 1.95 ⫾ 0.04* 7.64 ⫾ 0.35*** 40.55 ⫾ 0.71*** 10.01 ⫾ 0.33*** 0.69 ⫾ 0.03 0.59 ⫾ 0.15 0.04 ⫾ 0.01 0.57 ⫾ 0.03** 0.12 ⫾ 0.05

22.10 ⫾ .0.16 16.77 ⫾ 0.24 29.78 ⫾ 0.09 1.35 ⫾ 0.03 4.30 ⫾ 0.06 21.17 ⫾ 0.30 3.03 ⫾ 0.03 0.01 ⫾ 0.01 1.12 ⫾ 0.02 0.12 ⫾ 0.02 0.06 ⫾ 0.01 0.19 ⫾ 0.01

Values are means ⫾ SE; N, no. of rats. Rats were either fed on mother’s milk until death at postnatal day (d) 24 or removed from their mothers at d19 and fed on rodent chow until death at d24. Fatty acid composition (mol%) of total lipids from gastric contents was determined. For comparison, fatty acid composition of d14 gastric contents is shown. * P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. milk fed, d24.

both lung and liver, TG pools and their concentrations relative to phospholipid were decreased in chow controls, whereas differences in plasma were not significant (Table 4). In plasma and lung tissue TG of milk-fed animals, C14:0 and C12:0 were increased severalfold over standard chow (Fig. 2, A and C), where, after 5d, values had dropped to near zero in plasma (P ⬍ 0.001) and by 30 –50% in lung tissue (P ⬍ 0.01). Accordingly, C14:0 withdrawal decreased C14:0 in lung tissue PC from 6.9 ⫾ 0.6 to 2.6 ⫾ 0.3% and in LLF from 11.6 ⫾ 0.3 to 6.3 ⫾ 0.8%. The decrease of C14:0 in PC increased the fraction of C16:0 and palmitoleic acid (Fig. 2, B and D). Interestingly, irrespective of its concentrations in TG (Fig. 2, A and C), C12:0 was virtually absent from lung tissue and surfactant PC (Fig. 2, B and D). Effects of milk diet on surfactant PC molecular species composition and surfactant function. Diet-induced changes in C14:0 concentrations of lung and surfactant PC are reflected by corresponding changes of the intact PC species. While continTable 3. Effects of milk vs. chow diets on growth and phospholipid homoeostasis in lungs, liver, and blood plasma

Treatment

Body weight, g Breathing rates, breaths/min Gastric content, g Lipid concentration in gastric content, %wet wt Lung tissue phospholipids, ␮mol/ lavaged lung LLF phospholipids, nmol/total LLF Lung tissue choline, nmol/total lung Lung tissue phosphocholine, nmol/lung Liver weight, g Liver phospholipid pool, ␮mol/total liver Liver phospholipid concentration, ␮mol/g wet wt Plasma phospholipids, mmol/l Plasma choline, ␮mol/l

Milk Fed Until d24 (N ⫽ 11)

No Milk From d19 to d24 (N ⫽ 9)

59.27 ⫾ 2.14 122 ⫾ 6 0.729 ⫾ 0.108 10.22 ⫾ 0.24*

55.74 ⫾ 1.06 130 ⫾ 6 0.859 ⫾ 0.578 2.07 ⫾ 0.24

14.06 ⫾ 0.44

14.32 ⫾ 0.51

887.7 ⫾ 49.1* 35.5 ⫾ 6.0 82.0 ⫾ 8.7

1,209.4 ⫾ 123.9 34.7 ⫾ 5.9 88.1 ⫾ 1.0

2.59 ⫾ 0.09 105.09 ⫾ 3.57*

2.35 ⫾ 0.09 92.35 ⫾ 2.73

40.71 ⫾ 0.57

39.36 ⫾ 0.56

2.91 ⫾ 0.15** 22.1 ⫾ 4.2

2.21 ⫾ 0.14 18.4 ⫾ 0.6

Values are means ⫾ SE; N, no. of rats. Rats fed on mother’s milk throughout or on rodent chow from d19 onwards were killed at d24. LLF, lung lavage fluid. *P ⬍ 0.05; **P ⬍ 0.01. J Appl Physiol • VOL

uous milk feeding resulted in pulmonary PC profiles identical to those expected for d14 animals, 5d milk/C14:0 withdrawal reduced PC16:0/14:0 significantly, while PC16:0/16:0 was increased, i.e., composition was directed toward that of adult rat surfactant (Fig. 1, A and C, Fig. 3, A and B). We investigated the potential physiological role of such lipidomic changes by measuring surfactant surface tension and alveolar M⌽ function. In vitro, surface tension function was only minimally altered (Fig. 3C). However, the basal production of ROS of freshly isolated alveolar M⌽ from 14d milk-fed rats, naturally exposed to a surfactant rich in PC16:0/14:0 (for comparison, see Fig. 1A), was reduced by 50 – 65% compared with M⌽ from adult rats, exposed to a surfactant low in PC16:0/14:0. Furthermore, phorbol 12-myristate-13-acetate did not stimulate ROS production in the PC16:0/14:0-enriched d14 rat alveolar M⌽ at all (Fig. 4A). Addressing PC metabolism and composition of these cells (Fig. 4, B and C) by [D9-methyl]choline labeling and electrospray ionization tandem mass spectrometry showed that both d14 and adult M⌽ Table 4. Effects of milk feeding on postnatal triglyceride fatty acids in lungs, liver, and blood plasma Treatment

Milk Fed Until d24

Lavaged lung tissue ␮mol fatty acid/total 8.642 ⫾ 0.838*** lavaged lung ␮mol fatty acid/␮mol 0.617 ⫾ 0.051*** phospholipid Liver ␮mol fatty acid/total liver 34.926 ⫾ 3.770** ␮mol fatty acid/␮mol 0.323 ⫾ 0.028** phospholipid Plasma ␮mol fatty acid/ml plasma 2.279 ⫾ 0.602 ␮mol fatty acid/␮mol 0.811 ⫾ 0.123 phospholipid

No Milk From d19 to d24

4.505 ⫾ 0.493 (⫺48%) 0.315 ⫾ 0.034 15.927 ⫾ 2.019 (⫺54%) 0.189 ⫾ 0.017 1.227 ⫾ 0.084 (⫺46%) 0.673 ⫾ 0.056

Values are means ⫾ SE of 3–11 experiments. Rats were either milk fed from d1 until death at d24, or removed from their mothers at d19 and fed on rodent chow for 5 days until sacrifice. Triglycerides of lavaged lung tissue, liver, and blood plasma were analyzed and expressed relative to total phospholipid pools as ␮mol triglyceride fatty acids. Values in parentheses are percentage changes relative to controls (⫽100%). P values were calculated according to two-sided t-test, as described in MATERIALS AND METHODS. **P ⬍ 0.01; ***P ⬍ 0.001.

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Fig. 2. FA composition of plasma (A) and lung tissue (B) TG and of lung tissue (C) and LLF (D) PC of 24d rats. Rats were either fed on mother’s milk from birth until death at d24 or were removed from their mothers at d19 and fed chow for 5 days before death (d19 – d24). Values are means ⫾ SE of 5 vs. 3 (plasma TG), 9 vs. 11 (lung TG), 6 vs. 6 (lung PC), and 9 vs. 11 (LLF PC) experiments, respectively. C22:6, docosahexaenoic acid. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

primarily synthesized PC species containing arachidonic acid, together with components containing C18:1 and C18:2 (Fig. 4C, 1.5 h labeling with [D9-methyl]choline, narrow striped bars), whereas surfactant-specific PC16:0/16:0, PC16:0/14:0,

and PC16:0/16:1 only comprised a small portion of newly synthesized M⌽ PC (Fig. 4B). However, enrichment of newly synthesized M⌽ PC in [D9-methyl]choline after 1.5 h was only 1.72 ⫾ 0.08 and 1.44 ⫾ 0.65% of total PC in d14 and adult M⌽, respectively. Whole endogenous (nonlabeled) PC of freshly isolated d14 and adult alveolar M⌽, by contrast, comprised 54 ⫾ 2 and 60 ⫾ 4% “surfactant-PC,” i.e., the sum of PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1, with a molecular distribution similar to that of the age-matched surfactant. In adult M⌽, PC16:0/16:0 was the predominant component (43 ⫾ 3% of total M⌽ PC), whereas PC16:0/14:0 was not different from endogenous values (6 ⫾ 1%; P ⬎ 0.05). By contrast, in d14 rats, PC16:0/14:0 was increased to 19 ⫾ 1%, while PC16:0/16:0 only reached 21 ⫾ 2% of total M⌽ PC. Six hours after [D9-methyl]choline administration, D9 enrichment was 10.7 ⫾ 0.84 and 5.6 ⫾ 1.2% of PC in d14 and adult M⌽. At this time point (where substantial amounts of [D9-methyl]choline-labeled surfactant PC is already secreted into the alveoli, data not shown), the molecular composition of newly synthesized PC in the isolated M⌽ had approximated the surfactantlike composition of whole nonlabeled M⌽-PC (P ⬎ 0.05) (Fig. 4, B and C, wide striped bars). Lung specificity of C14:0 incorporation into PC. Analysis of plasma fractions from 14d suckling rats showed that C14:0 and C12:0 were highly enriched in the chylomicron fraction (Fig. 5A). Neither liver TG or PC (Fig. 5, B and C), nor total plasma PC (not shown) comprised significant amounts of C14:0 or PC16: 0/14:0, nor were their concentrations increased by C14:0/milk diet. However, there was a direct correlation between total plasma C14:0 and lung surfactant PC16:0/14:0, which inversely correlated PC16:0/16:0 (P ⬍ 0.001). No such correlation was found for C16:0 supply (Fig. 6). DISCUSSION

Fig. 3. Composition of lung tissue (A) and surfactant (LLF; B) PC and surfactant function in vitro (C) of d24 rats receiving mother’s milk from birth until death (d24) or removed from their mothers at d19 and fed chow for 5 days (d19–d24). Values are means ⫾ SE of 9 vs. 11 experiments. ␥min, Minimal surface tension; ns, nonsignificant. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001; ****P ⬍ 0.0001. J Appl Physiol • VOL

Surfactant comprises approximately two-thirds PC, together with other phospholipids, neutral lipids, and proteins SP-A to -D. Together PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 comprise 75– 80% of the PC, the proportions of which change

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nary glycogen stores (20, 35, 38). Our different nutrition regimens did not change choline and phosphocholine concentrations in plasma and lungs, suggesting no role for altered choline/phosphocholine supply in the surfactant changes observed. Moreover, PN-II comprise all the enzymes to synthesize, desaturate, and elongate fatty acids (for review, see Ref. 30), suggesting a high degree of metabolic self supply for the generation and storage of the PC species characteristic of surfactant. In utero, the 2-carbon units for C16:0 synthesis, the principle fatty acid of the main surfactant component PC16:0/ 16:0, originate from circulating glucose or from fetal pulmonary glycogen (31). Such fatty acid synthesis is essential during fetal development, where 80% of synthesized fatty acids are found in lung phospholipids (7). However, while the end product of endogenous fatty acid synthesis in the lungs is predominantly C16:0, fatty acid synthesis decreases shortly before term, following exhaustion of the glycogen stores (d20 – d21 in rats), whereas choline incorporation as a parameter of surfactant synthesis further increases (31). From these findings, it must be assumed that, from end gestation onwards, exogenous supply of the lungs with fatty acid may significantly modify the surfactant PC profile, if its chain length fits into the incorporation and sorting program of PN-II. Role of exogenous fatty acid supply for surfactant synthesis. Our laboratory previously demonstrated the dependence of

Fig. 4. Production of reactive oxygen species (ROS) by PC16:0/14:0-rich (d14) and -poor (adult) macrophage (M⌽; A), and composition of newly synthesized and endogenous PC (B and C). A: ROS production of such different M⌽ was quantified after 30 min, with and without 100 nmol/l phorbol myristate acetate (PMA), as indicated in MATERIALS AND METHODS. B and C: rats were labeled for 1.5 h or 6 h with [D9-methyl]choline chloride, and composition of D9-choline labeled and endogenous PC of isolated M⌽ was measured by electrospray ionization tandem mass spectrometry, as indicated in MATERIALS AND METHODS. A: the virtual absence of synthesis of PC16:0/16:0 and PC16:0/14:0 (1.5 h; narrow striped bars), whereas endogenous PC composition (shaded bars, d14; open bars, adult) shows a surfactant-like composition. After 6 h, M⌽ are already enriched with a D9-choline labeled surfactant-like PC profile. B: the predominantly synthesized mono-, di-, and polyunsaturated PC (1.5 h) is surmounted by surfactant-PC, resulting in a low C20:4-PC (6 h, endogenous) fraction. Values are means ⫾ SE of 4 –9 (PC16:0/14:0-poor, adult) and 5–13 (PC16:0/14:0-rich, 14d) experiments. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.

during development, while PC16:0/14:0 is decreased in chronic lung diseases (3, 29). In contrast, hormonal stimulation induces parallel increases (1, 16), whereas the PC synthesis pattern of human fetal lung explants is defined by incubation conditions, rather than hormones or tissue maturity (8, 9). The preferential enrichment of PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 in surfactant is explained by intracellular transport mechanisms favoring PC components containing fatty acids with 14 –16 carbon (3, 13, 23). Consequently, the processes resulting in variable PC16:0/16:0-to-PC16:0/14:0 ratios of surfactant appear to occur before surfactant assembly into lamellar bodies. Relevance of precursors for surfactant PC synthesis. A large body of evidence suggests that PC synthesis in PN-II depends on exogenous choline for head group formation, whereas the glycerol moiety originates from circulating glucose or pulmoJ Appl Physiol • VOL

Fig. 5. FA composition of plasma lipoproteins from 14d succling rats (A) and of liver TG (B) and PC (C) following milk or chow diets. Values are means ⫾ SE of 3–11 experiments. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.

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Fig. 6. Correlation between C14:0 and C16:0 in plasma TG and PC16:0/14:0 (A) and PC16:0/16:0 (B) in LLF/surfactant. Fractions of PC16:0/14:0 and CP16:0/16:0 in LLF were plotted against the concentrations of C14:0 and C16:0 in plasma TG. Values are means ⫾ SE of 3 (plasma d1) to 11 (milk fed, d24) experiments, respectively. 1, Newborn; 2, d14 – d15; 3, d24, milk fed; 4, d24, no milk; 5, d42– d44. r, Correlation coefficient. ***P ⬍ 0.001.

PC16:/14:0 on the dietary intake of C14:0. Using C14:03 as the exclusive exogenous fatty acid source resulted in the generation of a surfactant where PC16:0/14:0 became the major PC species at the expense of PC16:0/16:0. The same, but less pronounced, effect was induced by C12:03, although C12:0 was only incorporated into PC following lung-specific elongation to C14:0 (27). The occurrence of the same processes under normal physiological conditions was demonstrated in these experiments, where newborn rat surfactant transiently switched from being a high-PC16:0/16:0 to a high-PC16:0/14:0, containing surfactant during postnatal milk feeding, which also coincides with alveolar formation, and where prolonged milk feeding maintains a surfactant with increased PC16:0/14:0. We found a positive correlation between C14:0 enrichment in lung tissue and surfactant PC (PC16:0/14:0), and C14:0 concentrations in plasma and lung TG. Since the lungs express lipoprotein lipase (18), exogenous TG from very low density lipoproteins (12, 15, 25, 33, 40), as well as from chylomicrons (see RESULTS), are potent fatty acid sources of the lungs. Our data demonstrate that chylomicron-mediated effects play a major role in the profile changes of disaturated PC of surfactant. Importantly, they are restricted to the incorporation of C14:0 into PC, resulting in an enrichment of surfactant with PC16:0/14:0. Although milk nutrition also provides C12:0, this fatty acid was not found in PC. This is in accordance with our laboratory’s previous finding that C12:0 is elongated to C14:0 before incorporation, and that any traces of C12:0 directly incorporated into lung PC are rapidly removed (27). In essence, enrichment of C14:0 in PC is specific to mammalian lung surfactant, based on preferential accumulaJ Appl Physiol • VOL

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tion of C14:0 in pulmonary lipid stores, but not in other organs like the liver, where C14:0 is readily metabolized, or in birds in which PC16:0/16:0 comprises 75% of surfactant PC and in which PC16:0/14:0 is virtually absent (2, 3, 23, 27, 29). Lung specificity of PC16:0/14:0 synthesis. The enrichment in C12:0 and C14:0 of lung TG correlates well with their values in plasma TG. In accordance with previous experiments, PN-II use C14:0 from lung, as well as from plasma TG (27, 39). Also, in suckling rats, C14:0 is increased in the plasma free fatty acid fraction (34). However, while C14:0 rapidly vanishes from plasma TG after cessation of exogenous supply, its decrease in lung TG and PC is slower, highlighting its slow pulmonary turnover. By contrast, exogenous C14:0 did not increase C14:0 in liver and plasma PC or liver TG, implying that neither PC nor TG secreted by the liver contribute to increased PC16:0/14:0 in lung surfactant of rats, although the liver may well contribute to overall surfactant homoeostasis (12), and C14:0 metabolism may be different in humans and guinea pigs (3, 5). However, our data not only suggest lungspecific accumulation of this fatty acid under physiological conditions, but also raise the question of the underlying physiological importance. Function of PC16:0/14:0 in mammalian surfactant. Surface tension function of surfactant enriched in PC16:0/14:0 was not impaired, which is consistent with the finding that even “inverted” PC profiles with high PC16:0/14:0 and PC16:0/16:0 down to 2–20% are compatible with normal respiration (3, 21, 27). On the other hand, low PC16:0/14:0 concentrations are a better indicator of lung immaturity than PC16:0/16:0 and correlate with bronchopulmonary dysplasia and lung emphysema (3, 29). Hence, it is tempting to assume a role of PC16:0/14:0 in alveolar maintenance. This is supported by previous findings that bird lungs do not possess alveoli and that the alveolar M⌽-like cells in their lungs do not have PC16:0/ 14:0 in their surfactant (2, 24). Furthermore, in vitro data show that PC16:0/14:0 specifically mimics several of those effects on M⌽ differentiation characteristic of natural lipid extract surfactant containing this component, like increased HLA-DR and CD80 expression, and decreased M⌽-triggered T-lymphocyte proliferation (17). In keeping with this, we show here that, in freshly isolated alveolar M⌽, the production of ROS, another pivotal proinflammatory function of M⌽, is attenuated in rats with elevated pulmonary PC16:0/14:0 levels. ROS are generated by alveolar M⌽ upon activation and are essential for local host defense (19), whereas uncontrolled ROS release may lead to severe tissue damage (36). Previous age-related investigations on rat alveolar M⌽ showed that conventionally fed young rats exhibit elevated ROS production (37). Conversely, our experiments show diminished ROS production in 14d rats fed on a C14:0-rich diet. Therefore, we speculate that an elevated PC16:0/14:0 proportion in surfactant may attenuate ROS production. Surfactant and PC metabolism of alveolar M⌽. Comparing the PC metabolism of alveolar M⌽ after 1.5 h [D9-methyl]choline incorporation, when only minute amounts of newly synthesized D9-labeled surfactant PC have entered the alveolar space (not shown), with that of nonlabeled PC suggests that PC homeostasis of alveolar M⌽ is dictated by the lipid composition of the surrounding surfactant. If recycling of endocytosed surfactant fatty acids were to play a significant role in M⌽ PC homeostasis, one would expect higher concentrations of D9-

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choline-labeled PC16:0/14:0, PC16:0/16:0, and PC16:0/16:1 from M⌽ synthesis after 1.5 h, rather than the predominant synthesis of polyunsaturated PC. Rather, it is dictated by the presence and lipidomic composition of the surfactant-rich surrounding, which may be important for the exposure to synthetic surfactant lacking PC16:0/14:0, and the function of these cells. However, the PC metabolism of alveolar M⌽ appears independent of surfactant fatty acid supply, although it cannot be excluded that some C14:0 released from absorbed PC16:0/14:0 is used for protein myristoylation, e.g., of the inhibitory ␣-subunit of G proteins regulating M⌽ function (10). Instead, these cells predominantly ad- and absorb surfactant PC, as defined by their alveolar environment and the metabolic characteristics of surfactant producing PN-II. While the latter clearly depends on C14:0 nutrition and development, direct examination of the effect of diets high in C14:0 fatty acids compared with those low in C14:0 in age-matched rats (27) will be necessary to investigate in more detail the specificity of C14:0 and PC16: 0/14:0-enriched surfactant on M⌽ function for pulmonary homeostasis. In conclusion, the concentrations of saturated PC components in surfactant, PC16:0/14:0, and PC16:0/16:0 are modified by differences in the exogenous supply of C14:0. Whereas shorter fatty acids, such as C12:0, are excluded from PC synthesis, C14:0 enrichment in PC is restricted to the lungs. In rats, this metabolic process is mediated by chylomicrons, whereas, in other mammals, transplacental secretion may contribute. Physiologically, C16:0/14:0 enrichment may be more important for alveolar M⌽ homoeostasis than for surface tension function, where the development-dependent surfactant PC metabolism influences the PC homeostasis of alveolar M⌽. Taken together, these findings strongly support further investigations into the association between PC16:0/14:0, alveolar immune-homeostasis, and pulmonary disease. GRANTS This work was supported by an institutional grant of the medical faculty of the University of Tübingen (F.1275127) and by the Deutsche Forschungsgemeinschaft (BE2223/2-1). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Ashton MR, Postle AD, Smith DE, Hall MA. Surfactant phosphatidylcholine composition during dexamethasone treatment in chronic lung disease. Arch Dis Child 71: F114 –F117, 1994. 2. 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. 3. 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. 4. 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. 5. 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. 6. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959. J Appl Physiol • VOL

7. Buechler KF, Rhoades RA. Fatty acid synthesis in the perfused rat lung. Biochim Biophys Acta 619: 186 –195, 1980. 8. 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. 9. 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. 10. Chen CA, Manning DR. Regulation of G proteins by covalent modification. Oncogene 20: 1643–1652, 2001. 11. Chung BH, Wilkinson H, Geer JC, Segrest JP. Preparative and quantitative isolation plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 21: 284 –291, 1980. 12. Darrah HK, Hedley-Whyte J. Rapid incorporation of palmitate into lung: site and metabolic fate. J Appl Physiol 34: 205–213, 1973. 13. 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 surfactant, and depletion of lung phosphatidylglycerol. J Lipid Res 48: 621–632, 2007. 14. 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. 15. Gal S, Bassett DJ, Hamosh M, Hamosh P. Triacylglycerol hyrolysis in the isolated, perfused rat lung. Biochim Biophys Acta 713: 222–229, 1982. 16. Gesche J, Fehrenbach H, Koslowski R, Ohler FM, Pynn CJ, Griese M, Poets CF, Bernhard W. rhKGF stimulates lung surfactant production in neonatal rats in vivo. Pediatr Pulmonol. DOI:10.1002/ppul.21443. 17. 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. 18. Hamosh M, Yeager M Jr, Shechter Y, Hamosh P. Lipoprotein lipase in rat lung. Effect of dexamethasone. Biochim Biophys Acta 431: 519 –525, 1976. 19. Henriet SS, Hermans PW, Verweij PE, Simonetti E, Holland SM, Sugui JA, Kwon-Chung KJ, Warris A. Human leukocytes kill Aspergillus nidulans by reactive oxygen species-independent mechanisms. Infect Immun 79: 767–773, 2011. 20. 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. 21. 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. 22. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27: 114 –120, 1986. 23. 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. 24. Lorz C, Lopez J. Incidence of air pollution in the pulmonary surfactant system of the pigeon (Columba livia). Anat Rec 249: 206 –212, 1997. 25. McGowan SE, Torday JS. The pulmonary lipofibroblast (lipid interstitial cell) and its contributions to alveolar development. Annu Rev Physiol 59: 43–62, 1997. 26. Postle AD. Method for the sensitive analysis of individual molecular species of phosphatidylcholine by high-performance liquid chromatography using post-column fluorescence detection. J Chromatogr 415: 241– 251, 1987. 27. 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. 28. Rau GA, Vieten G, Haitsma JJ, Freihorst J, Poets CF, Ure BM, Bernhard W. Surfactant in newborn compared with adolescent pigs: adaptation to neonatal respiration. Am J Respir Cell Mol Biol 30: 694 – 701, 2004. 29. Ridsdale R, Roth-Kleiner M, D’Ovidio F, Unger S, Yi M, Keshavjee S, Tanswell AK, Post M. Surfactant palmitoylmyristoylphosphatidylcholine

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30. 31. 32. 33. 34. 35.

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. Rothe G, Valet G. Flow cytometric assay of oxidative burst activity in phagocytes. Methods Enzymol 23: 539 –548, 2010. 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. Sámel M, Pullmann R. The development of lipid metabolism. Changes in the level of fatty acids in serum during postnatal ontogenesis in the rat. Bratisl Lek Listy 94: 361–365, 1993. Sheehan PM, Yeh YY. Lung lipid synthesis from acetoacetate and glucose in developing rats in vitro. Lipids 18: 295–301, 1983.

J Appl Physiol • VOL

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36. Tasaka S, Amaya F, Hashimoto S, Ishizaka A. Roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome. Antioxid Redox Signal 10: 739 –753, 2008. 37. Tasat DR, Mancuso R, O’Connor S, Molinari B. Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages. Aging Cell 2: 159 –164, 2003. 38. Tian Y, Zhou R, Rehg JE, Jackowski S. Role of phosphocholine cytidylyltransferase alpha in lung development. Mol Cell Biol 27: 975– 982, 2007. 39. Torday J, Hua J, Slavin R. Metabolism and fate of neutral lipids of fetal lung fibroblast origin. Biochim Biophys Acta 1254: 198 –206, 1995. 40. Tordet C, Marin L, Dameron F. Pulmonary di-and-triacylglycerols during the perinatal development of the rat. Experientia 37: 333–334, 1981. 41. 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.

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