help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on October 24, 2003, doi:10.1165/rcmb.2003-0351OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0351OCv1
30/5/694    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rau, G. A.
Right arrow Articles by Bernhard, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rau, G. A.
Right arrow Articles by Bernhard, W.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 694-701, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0351OC

Surfactant in Newborn Compared with Adolescent Pigs

Adaptation to Neonatal Respiration

Gunnar A. Rau, Gertrud Vieten, Jack J. Haitsma, Joachim Freihorst, Christian Poets, Benno M. Ure and Wolfgang Bernhard

Departments of Pediatric Pulmonology and Neonatology, and of Pediatric Surgery, Hannover Medical School, Hannover; Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Tübingen, Germany; and Department of Anesthesiology, Erasmus MC-Faculty, Rotterdam, The Netherlands

Address correspondence to: Wolfgang Bernhard, M.D., Ph.D., Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tübingen, Germany. E-mail: wolfgang.bernhard{at}med.uni-tuebingen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant composition and function differ between vertebrates, depending on pulmonary anatomy and respiratory physiology. Because pulmonary development in pigs is similar to that in humans, we investigated surface tension function, composition of phospholipid molecular species, and concentrations of surfactant protein (SP)-A to -D in term newborn pigs (NP) compared with adolescent pigs (AP), using the pulsating bubble surfactometer, mass spectrometry, high-performance liquid chromatography, and immunoblot techniques (IT). NP was more potent than AP surfactant in reaching minimal surface tension values near zero mN/m. Whereas SP-A and SP-D were comparable, SP-B and SP-C were increased 3- to 4-fold in NP surfactant. Moreover, fluidizing phospholipids such as palmitoylmyristoyl-PC (PC16:0/14:0) and palmitoylpalmitoleoyl-PC (PC16:0/16:1) were increased at the expense of PC16:0/16:0 (32.4 ± 0.6 versus 44.5 ± 3.2%, respectively). Whereas concentrations of total anionic phospholipids were similar in NP and AP surfactant (9.9 ± 0.3 and 12.0 ± 0.3%, respectively), phosphatidylinositol was the predominant anionic phospholipid in NP surfactant. We conclude that, compared with AP, NP surfactant displays better surface tension function under dynamic conditions, which is associated with increased concentrations of SP-B and SP-C, as well as fluidizing phospholipids at the expense of PC16:0/16:0.

Abbreviations: adolescent pigs, AP • bronchoalveolar lavage, BAL • BAL fluid, BALF • electrospray ionization mass spectrometry, ESI-MS • high-performance liquid chromatography, HPLC • newborn pigs, NP • phosphatidylcholine, PC • phosphatidylglycerol, PG • phosphatidylinositol, PI • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant is a complex mixture mainly consisting of phospholipids, neutral lipids, and specific proteins. In vertebrates it is essential for normal lung function by reducing surface tension at pulmonary air–liquid interfaces (1, 2), irrespective whether these interfaces are tubular (bronchioles, air capillaries in bird lungs), exclusively saccular (as in newborn rats), alveolar (as in adult mammals and newborn guinea pigs), or in the process of alveolarization as in pigs and humans at term (3–7). Although phospholipids comprise 80% of the mass of surfactants, of which 80–85% are phosphatidylcholines (PC) and ~ 10% anionic phospholipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI) (2, 4), fractions of individual phospholipid molecular species as well as the presence and concentrations of individual surfactant proteins (SP) vary considerably across vertebrate species (3, 4, 8, 9).

Among surfactant phospholipids, dipalmitoyl-PC (PC16:0/16:0) is believed to be the principal component that reduces minimal surface tension ({gamma}min) to near zero mN/m at end-expiration. Due to its straight fatty acid chains and a gel-to-sol phase transition temperature above body temperature (10), lateral compression of PC16:0/16:0 results in tight packing and formation of a metastable liquid condensed PC16:0/16:0 film upon lateral compression during expiration (11). However, because adsorption of PC16:0/16:0 to the air–liquid interface is too slow to cope with the dynamic surface area changes during the respiratory cycle, mammalian surfactant contains SP-B and SP-C together with fluidizing phospholipids to improve adsorption of surfactant to the interface under the conditions of dynamic surface oscillations during the respiratory cycle (11). Recent in vitro studies have questioned the classical concept of a continuous PC16:0/16:0 layer at the air–liquid interface. Instead, the interfacial film contains liquid condensed domains, putatively consisting of PC16:0/16:0, surrounded by a liquid expanded phase of more fluid phospholipids together with SP-B and SP-C (12–15). These and other findings emphasize distinct roles for accessory phospholipids in surfactant function. Adsorption of surfactant from the alveolar hypophase to the surface-associated reservoir apparently depends on surfactant concentration in the hypophase and on its anionic phospholipids. Fusion of adsorbed material with the interfacial surfactant film, however, depends on fluidity of the fatty acyl chains of phospholipids rather than on the charge of their head groups (16).

Similarly, recent studies have shown significant differences in surfactant composition across vertebrate species and effective secretion not only of PC16:0/16:0, PG and SPs, but also of two PC molecular species, namely palmitoylmyristoyl-PC (PC16:0/14:0) and palmitoylpalmitoleoyl-PC (PC16:0/16:1), into the alveolar space (3, 8, 9). These latter two components, being rather unique to mammalian surfactant, directly correlate with the rate of air–liquid interface dynamics, at the expense of PC16:0/16:0, in various animal species. Accordingly, PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 together comprised 75–80% of total surfactant PC, suggesting that surfactant fluidity is adjusted according to physiologic needs by varying these three components (9).

However, differential composition of surfactant may not be restricted to PC molecular species, because interactions of its components are complex. For instance, SP-A mainly interacts with PC16:0/16:0, whereas hydrophobic SP-B and SP-C predominantly interact with fluid phospholipids (17, 18). Consequently, changes in surfactant PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 concentrations may only reflect part of the adaptation to the requirements resulting from differing dynamics of air–liquid interface changes during mammalian respiration. Moreover, such changes may not be restricted to adult organisms with differing pulmonary physiology, but may also apply to newborn compared with older mammals differing in pulmonary development and respiratory rate. Therefore, in surfactant from newborn and adolescent (11 wk) pigs we investigated surface tension function using the pulsating bubble surfactometer technique (19). Moreover, we measured fractional concentrations of the major surfactant phospholipid classes PC, PG, and PI and of their individual molecular species using electrospray ionization mass spectrometry (ESI-MS). Finally, we determined the concentrations of SP-A, -B, -C, and -D in relation to surfactant phospholipids using high-performance liquid chromatography (HPLC) and immunoblotting techniques.

We studied the pig because the developmental pattern of alveolarization and respiratory rates closely resembles that of humans, in contrast to rat, mouse, and guinea pig (7, 20). Our study was aimed to investigate functional and biochemical intraspecies variability of surfactant after delivery. We clearly demonstrate a better surface tension function of newborn compared with adult lung surfactant and the biochemical basis of this superiority.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Phospholipid standards were from Sigma-Aldrich (Deisenhofen, Germany). Hydrogen peroxide (30%, analytical grade) was from Boehringer Ingelheim (Ingelheim, Germany). Perchloric acid (70%, analytical grade) and dichloromethane (Suprasolv) were from Merck (Darmstadt, Germany). Chloroform and methanol were of HPLC grade and from Baker (Deventer, The Netherlands). All other chemicals were of analytical grade and obtained from various commercial sources.

Harvesting of Surfactant from Newborn and Adult Pigs
Mixed-breed York-Pyatrain-Landrace newborn piglets (23 ± 15 h old, 1.4 ± 0.3 kg) and adolescent pigs of the same race (11 ± 1 wk old, 34–36 kg) were used to obtain porcine surfactant material. Porcine surfactant was harvested by bronchoalveolar lavage (BAL) of the lungs with 154 mmol/liter saline as previously described (9). Original BAL fluid (BALF) was centrifuged for 15 min at 200 x g to remove cells.

Analysis of Surfactant Function
Surfactant function was assessed with the pulsating bubble surfactometer (PBS; Electronetics, Amherst, NY) (19) as described previously (4). Briefly, cell-free BALF was centrifuged at 40,000 x g for 15 min at 4°C to sediment large aggregates and the pellets adjusted with 154 mmol/liter NaCl supplemented with 1.5 mmol/liter CaCl2 to give phospholipid concentrations of 1.33 or 0.33 mmol/liter (1 or 0.25 mg/ml). Suspensions were vortexed for 30 s to ensure complete homogenization. Aliquots were instilled into the sample chamber of the PBS and their surface tension function measured at 37°C. A bubble communicating with ambient air was created in the surfactant suspension and surfactant allowed to adsorb to the air–liquid interface for 10 s. After this time, the bubble was pulsated at a rate of 20 oscillations per minute between a minimum radius of 0.4 mm and a maximum radius of 0.55 mm. The pressure across the bubble was measured by a pressure transducer and the surface tension calculated using the Laplace equation. Minimum ({gamma}min) and maximum ({gamma}max) surface tensions were determined for 100 pulsations.

Analysis of Phospholipids
Phospholipids from BALF were extracted according to the method of Bligh and Dyer (21). Phospholipid phosphorus was quantified as described by Bartlett and coworkers (22) after digestion of the organic components at 190°C for 35 min in the presence of 500 µl 70% perchloric acid (wt/vol) and 200 µl 30% hydrogen peroxide (wt/vol). Molecular species of PC, PG, and PI, the major phospholipids of lung surfactant, were analyzed with ESI-MS as described elsewhere (23). Briefly, 50 nmol dimyristoyl-PC (PC14:0/14:0) and 20 nmol dimyristoyl-PG were added as standards to 50 nmol phospholipid extract. ESI-MS was performed using a single-quadrupole liquid chromatography mass spectrometer (API I; Perkin Elmer–Sciex, Toronto, ON, Canada) equipped with an electrospray ionization interface. Samples were dissolved in 200 µl chloroform: methanol (1:2, vol/vol), supplemented with 500 µmol/liter sodium acetate. Samples were infused into the mass spectrometer via a syringe pump (Model 22; Harvard Apparatus, South Natick, MA) at a flow rate of 3 µl/min. Dry heated nitrogen gas was used as both the cone gas (60 liters/h) and the dissolvation gas (60 liters/h with a pressure of 40 PSI). For PC analysis samples were measured in the positive ionization mode and data recorded at atomic resolution as their sodium adducts (M+22), with a signal average of 20 scans/collection. Similarly, PG and PI molecular species were measured in the negative ionization mode (M-1). Data were processed using BioMultiview software (Perkin Elmer–Sciex). After correction for 13C isotope effects, molecular species of PC, PG, and PI were expressed as percentages of the respective phospholipid class in the sample. Concentrations of the three major phospholipid classes of surfactant, PC, PG, and PI, were calculated from the sums of their respective molecular species as previously described (23).

Analysis of SP-A and SP-D
Concentrations of SP-A and SP-D were determined in BALF samples by immunoblot analysis after electrophoresis as described before (3, 4), using sample aliquots containing 10 or 2 nmol phospholipid for determination of SP-A and SP-D, respectively. Purified porcine SP-A and human SP-D isolated from BALF samples served as standards. After heating the samples (10 min, 70°C) under reducing conditions with NuPAGE LDL sample buffer (Invitrogen, Groningen, The Netherlands), proteins were separated on a pre-cast 10% neutral polyacrylamide Bis/Tris gel using MOPS running puffer pH 7.7 (NuPAGE; Invitrogen). Prestained multicolored molecular weight standard (SeeBlue Plus2; Invitrogen) was used as a control. After electrophoresis, the proteins were blotted onto a nitrocellulose membrane (Protran BA85; Schleicher and Schuell, Dassel, Germany) and efficiency of transfer was assessed using Ponceau S red dye. For SP-A determination the primary antibody was a polyclonal rabbit anti-human SP-A antibody (kindly provided by Byk Gulden, Konstanz, Germany). This antibody cross-reacted with porcine, rat, and bovine SP-A and was used at 1:2,000 dilution in phosphate-buffered saline with 0.04% bovine serum albumin. A porcine horseradish peroxidase–labeled anti-rabbit IgG antibody (1:2,000 in 10 mM Tris, pH7.4, with 1% bovine serum albumin; DAKO, Hamburg, Germany) served as the secondary antibody. For the determination of SP-D a polyclonal rabbit anti-mouse IgG antibody, cross-reacting with porcine and rat SP-D (dilution 1:2,000 in 10 mM Tris, pH7.4, with 1% bovine serum albumin; Chemicon International, Hofheim, Germany), and porcine horseradish peroxidase–labeled anti-rabbit IgG were used as the first and second antibodies, respectively. The blots were visualized using a chemiluminescence kit (Amersham Pharmacia Biotech, Braunschweig, Germany) according to the manufacturer's instructions. Densitometric scanning was performed with the Phoretix 1D software (Biostep, Jahnsdorf, Germany).

Analysis of SP-B and SP-C
Porcine SP-B and SP-C standards were kindly provided by Dr. M. van Eijk (Dept. of Biochemistry, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands). Concentrations of SP-B and SP-C were determined in BALF as well as 60,000 x g pellets of BALF (1 h at 4°C) by HPLC on a VYDAC C4 analytical column (4.6 x 500 mm, 30 nm pores, 5 µm particle size) at room temperature as previously described by Bünger and colleagues (24), using an HPLC device equipped with a Merck L-4000 ultraviolet detector and a Merck-Hitachi F-1050 fluorescence detector as used previously (3). To improve ultraviolet and fluorescence detection of the proteins, chloroform in the mobile phase was replaced by dichloromethane (dichloromethane:methanol:0.1 mol/liter trifluoroacetic acid 47.5:47.5:5, vol/vol) as previously described by van Eijk and associates (25). As demonstrated, this resulted in good separation of the hydrophobic proteins (Figure 1A) as well as linear ultraviolet detection of either protein at 228 nm and of SP-B with fluorescence detection (exc.: 280 nm; em.: 365 nm) (Figure 1B).




View larger version (53K):
[in this window]
[in a new window]
  		Figure 1. (A) Separation of SP-B and SP-C with HPLC. Purified porcine SP-B (10 µg; A, B) and SP-C (10 µg; C, D) as well as lipid extracts of porcine surfactant (500 nmol PL; E, F) were separated on a 500 x 4.6 mm Vydac C4 column with dichloromethane: methanol: 0.1 N trifluoroacetic acid (47.5:47.5:5, vol/vol) as a mobile phase and detected with ultraviolet absorbance at 228 nm (A, C, E) as well as fluorescence detection (excitation: 280 nm; emission: 365 nm) (B, D, F) as described in MATERIALS AND METHODS. (B) Calibration curves of SP-B and SP-C with HPLC. Purified SP-B (1–23 µg) and SP-C (5–112 µg) were separated on a 500 x 4.6 mm Vydac C4 column with dichloromethane: methanol: 0.1 N trifluoroacetic acid (47.5:47.5:5, vol/vol) as a mobile phase as described in MATERIALS AND METHODS.

 
Statistics
Data are expressed as means ± SE. One-way analyses of variance were calculated using Graph Pad Instat Version 3 (Graph Pad Software, San Diego, CA). Group differences were tested by a two-tailed Student's t test. Multiple group comparison was performed using the Bonferroni correction, with P < 0.05 being considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surfactant Function in Newborn Compared with Adolescent Pigs
Under the dynamic conditions of the PBS, surfactant from newborn piglets displayed a significantly better surface tension function than that from adolescent pigs, as it reached {gamma}min values after fewer pulsations. This was evident for a concentration of 1.33 mmol/liter phospholipid (Figure 2A) and even more for 0.33 mmol/liter phospholipid (Figure 2B), where {gamma}min values near zero mN/m were only achieved by surfactant from newborn but not from adolescent pigs. Similarly, {gamma}max started with higher values for surfactant from 12-wk-old pigs, and for the lower concentration (0.33 mmol/liter phospholipid) {gamma}max was lower for surfactant from the newborn piglets throughout the study.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Minimal surface tension of surfactant from newborn and adolescent pigs. Surfactant samples from newborn (diamonds) and adolescent (squares) pigs were investigated for minimal (A, B) and maximal (C, D) surface tension in the pulsating bubble surfactometer for 1–100 cycles at a pulsation rate of 20 cycles/min, and at 37°C. Investigation was performed at 1.33 (A, C) and 0.33 (B, D) mmol/liter phospholipid (PL) concentrations. Data points are means ± SE of three to five experiments.

 
Composition of Phospholipid Classes
Table 1 shows that the sum of the anionic phospholipids was lower in newborn compared with adolescent pigs (9.9 ± 0.3% versus 12.0 ± 0.3%; P < 0.01), causing a small but significantly higher fraction of PC in relation to all major surfactant phospholipids. Sphingomyelin and dihydrosphingomyelin, being detected in the positive ionization mode of ESI-MS analysis were negligible components of surfactant from either age (not shown). However, PI was the dominant anionic phospholipid in newborn piglet surfactant (7.2 ± 0.3%), whereas PG comprised only 2.7 ± 0.2%. By contrast, in surfactant from adolescent pigs, PG was the dominant anionic phospholipid (9.2 ± 0.2%, P < 0.0001) over PI (2.8 ± 0.2%, P < 0.0001). Moreover, PC, PG, and PI showed substantial differences in molecular species composition in newborn piglets compared with adolescent pigs (Figures 3 and 4).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Concentration of major phospholipid classes in surfactant of newborn compared with adolescent pigs

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Composition of PC molecular species in surfactant from newborn compared with adolescent pigs. Data are molar fractions (%) of PC species in relation to total PC. Data are means ± SE from n = 8 (newborn; solid bars) and n = 5 (adolescent; shaded bars). 16:0/14:0, palmitoylmyristoyl-PC; 16:0/16:0, dipalmitoyl-PC; 16:0/16:1, palmitoylpalmitoleoyl-PC; 16:0/18:0, palmitoylstearoyl-PC; 16:0/18:1, palmitoyloleoyl-PC; 16:0/18:2, palmitoyllinoleoyl-PC; 16:0/20:4, palmitoylarachidonoyl-PC; 16:0/22:6, palmitoyldocosahexaenoyl-PC. The prefix aa indicates the 1-alkyl-2-acyl derivatives of PC, whereas the other molecular species are 1,2-diacyl PC. *P < 0.05; {dagger}P < 0.01; {ddagger}P < 0.001; §P < 0.0001.

 


View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. Composition of phosphatidylglycerol (PG) (A) and phosphatidylinositol (PI) (B) molecular species in surfactant from newborn (solid bars) compared with adolescent (shaded bars) pigs. Data are molar fractions (%) of PG and PI species in relation to total PG and PI, respectively. Data are means ± SE from n = 5–11 experiments. 16:0/14:0, palmitoylmyristoyl-; 16:0/16:0, dipalmitoyl-; 16:0/16:1, palmitoylpalmitoleoyl-; 16:0/18:0, palmitoylstearoyl-; 16:0/18:1, palmitoyloleoyl-; 16:0/18:2, palmitoyllinoleoyl-; 16:0/20:4, palmitoylarachidonoyl-; 16:0/16:1, 16:0/22:6, palmitoyldocosahexaenoyl- 18:0/18:0, distearoyl-; 18:0/18:1, stearoyloleoyl-; 18:0/18:2, stearoyllinoleoyl-; 18:0/18:3, stearoyllinolenoyl-; 18:0/20:4, stearoylarachidonoyl-; 18:0/22:6, stearoyldocosahexaenoyl-; 18:1/18:2, oleoyllinoleoyl-; 18:1/20:4, oleoylarachidonoyl-. *P < 0.05; {dagger}P < 0.01; {ddagger}P < 0.001; §P < 0.0001.

 
Composition of PC Molecular Species
As ESI-MS allowed more detailed analyses of individual PC molecular species than rpHPLC (8), alkyl-acyl derivatives of PC (aaPC) species as well as minor long-chain saturated and unsaturated diacyl PC species were determined (Figure 3). In newborn pigs PC16:0/16:1 and PC16:0/14:0 were increased at the expense of long-chain saturated PC16:0/16:0 and PC16:0/18:0, whereas no age-related differences were observed for palmitoyloleyl-PC (PC16:0/18:1) and palmitoyllinoleoyl-PC (PC16:0/18:2). All other PC species were minor components with individual concentrations of < 5%. However, in newborn piglets palmitoylarachidonoyl-PC (PC16:0/20:4) and the alkyl-acyl derivatives of dipalmitoyl-PC (aaPC16:0/16:0) and of palmitoylpalmitoleoyl-PC (aaPC16:0/16:1) were increased, whereas that of palmitoyldocosahexaenoyl-PC (aaPC16:0/22:6) was higher in adolescent pigs. Although these minor components only comprised up to 15% of total lung surfactant PC at either age, their inclusion into compositional analysis resulted in lower fractions of the major components (diacyl)PC16:0/16:0, (diacyl)PC16:0/14:0, and (diacyl)PC16:0/16:1 compared with previous studies with rpHPLC (3, 9). To exclude the possibility that lower PC16:0/16:0 concentrations were compensated for by other PC species with high phase transition temperatures, we determined the sum of all saturated long-chain PC molecular species of the surfactant, namely the diacyl species PC16:0/16:0 and PC16:0/18:0, alkyl-acyl PC16:0/16:0 (aaPC16:0/16:0), and alkyl-acyl PC16:0/18:0 (aaPC16:0/18:0). These calculations confirmed that surfactant from newborn piglets contained less disaturated longchain PC species than that from adolescent pigs (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Concentration of saturated long chain PC species in surfactant of newborn compared to adolescent pigs

 
Composition of PG and PI Molecular Species
ESI-MS analysis of anionic phospholipids revealed substantial differences in molecular species composition of both PG and PI between differently aged pigs (Figures 4A and 4B). In adolescent pigs, dipalmitoyl-PG (PG16:0/16:0) and palmitoylstearoyl-PG (PG16:0/18:0) comprised 18 ± 3% and 1.7 ± 0.2% of total PG, respectively, whereas the majority of components were the mono- or polyunsaturated PG species palmitoylpalmitoleoyl-PG (PG16:0/16:1; 10.9 ± 2.4%), palmitoyloleoyl-PG (PG16:0/18:1; 21.8 ± 1.0%), palmitoyllinoleoyl-PG (PG16:0/18:2; 11.5 ± 2.2%), stearoyloleoyl-PG (PG18:0/18:1; 5.0 ± 0.5%), and stearoyllinoleoyl-PG (PG18:0/18:2; 6.9 ± 1.4%) (Figure 4A). By contrast, in newborn pig surfactant PG16:0/16:0 was virtually absent, whereas it contained higher amounts of PG16:0/18:0 (12.2 ± 1.9%; P < 0.01) and of PG16:0/16:1 (34.1 ± 2.2%; P < 0.0001) compared with adolescent pigs. Taken together, mono- and diunsaturated PG species were lower in surfactant from newborn than in adolescent pigs (58.1 ± 3.1 and 69.3 ± 2.9, respectively; P < 0.05). In contrast, polyunsaturated PG species like palmitoylarachidonoyl-PG (PG16:0/20:4), stearoylarachidonoyl-PG (PG18:0/20:4), and palmitoyldocosahexaenoyl-PG (PG16:0/22:6) were higher in surfactant from newborn than in adolescent pigs, comprising 17.0 ± 1.7% versus 7.7 ± 1.1% (P < 0.01), 7.3 ± 1.7% versus 1.4 ± 0.6% (P < 0.05), and 4.9 ± 1.2% versus 0.9 ± 0.6% (P < 0.05) of total PG, respectively.

The pattern of PI composition differed substantially from that of PG, with disaturated species being virtually absent (Figure 4B). Although differences in molecular species composition were evident, in both newborn and adolescent pigs the majority of PI was composed of the mono- and diunsaturated species palmitoylpalmitoleoyl-PI (PI16:0/16:1), palmitoyloleoyl-PI (PI16:0/18:1), palmitoyllinoleoyl-PI (PI16:0/18:2), stearoyloleoyl-PI (PI18:0/18:1), and stearoyllinoleoyl-PI (PI18:0/18:2). Taken together, these PI species comprised 72.4 ± 1.2% and 73.5 ± 2.2% (P > 0.05) at either age. However, similar to PG (see Figure 4A), surfactant from 1-d-old piglets contained significantly more PI species with arachidonic acid residues than that from adolescent pigs (Figure 4B).

Fractional Concentrations of PG and PI Molecular Species in Surfactant
Whereas the concentration of total PC was almost identical in newborn compared with adolescent surfactant, fractions of total PG and PI were substantially different. We therefore determined the fractions of their molecular species in relation to total phospholipid. Figures 5A and 5B show that concentrations of individual anionic phospholipid species comprised 2% of total phospholipid or less. With the exception of PG16:0/16:0, which comprised 1.6 ± 0.3% of phospholipid in adolescent pigs, disaturated PG or PI species were negligible components. The majority of anionic surfactant phospholipids were mono- and diunsaturated components, possessing oleic or linoleic acid residues. However, whereas in adolescent pigs these components were PG species, in newborn piglets these were negligible. By contrast, newborn piglet surfactant contained mono- and diunsaturated PI species, together with increased amounts of polyunsaturated PI species possessing an arachidonic acid residue (Figure 5B).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 5. Fractional concentrations of phosphatidylglycerol (PG) (A) and phosphatidylinositol (PI) (B) molecular species in surfactant from newborn (solid bars) compared with adolescent (shaded bars) pigs. Data are molar fractions (%) of PG and PI species in relation to the sum of all major surfactant phospholipids (PC+PG+PI). Data are means ± SE from n = 5–11 experiments. For PG and PI molecular species see legend to Figure 4 for abbreviations. *P < 0.05; {dagger}P < 0.01; {ddagger}P < 0.001; §P < 0.0001.

 
Surfactant Proteins SP-A, SP-B, SP-C, and SP-D
To further explain the superior surface activity of surfactant from newborn compared with older pigs and to rule out an immaturity of the surfactant system from newborn pigs compared with their older counterparts, we investigated the concentrations of SP-A to -D in the surfactant preparations. Table 3 shows that BALF from newborn piglets contained amounts of both SP-A and SP-D similar to that of their older counterparts. In contrast, SP-B and SP-C were significantly increased in surfactant from newborn compared with adolescent pigs. This was evident for both cell-free BALF and surfactant large aggregates enriched by differential centrifugation (60,000 x g pellets).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Concentrations of SP-A to -D in surfactant from newborn compared with adolescent pigs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Biochemical characterization of lung surfactant usually focuses on PC16:0/16:0 as its major surface tension lowering component, and anionic phosphatidylglycerol (PG) as well as apoproteins SP-A, SP-B, SP-C, and SP-D for their functions in assisting surfactant adsorption to, and enrichment at, the air–liquid interface as well as their role in alveolar surfactant homoeostasis (1, 2, 26, 27). However, there is increasing evidence that fluidic phospholipid components other than PG contribute to the dynamic properties of surfactant (12, 16), and that the complex composition of surfactant is adapted to fulfill the demands of respiratory physiology and pulmonary anatomy differing across animal species (3, 9). Although initial adsorption of surfactant to the interface depends on the charge of head groups from anionic phospholipids rather than their fatty acid chains, subsequent fusion with the interfacial layer depends on the mixed acyl chains of phospholipids irrespective of the head group (16). Functional relevance of surfactant phospholipids other than PC16:0/16:0 was further suggested by surface pressure–area isotherms together with fluorescence and Brewster angle microscopy, revealing that even at near zero surface tension, liquid condensed clusters of PC16:0/16:0 remain surrounded by a liquid expanded phase of fluidic phospholipids together with hydrophobic proteins SP-B and SP-C (12, 14–16).

These considerations question the classical model of a purified interfacial PC16:0/16:0 layer at the air–liquid interface as necessary for near zero surface tensions (high surface pressures near 70 mN/m) (12). These data, together with the observation that fluidic phospholipids in the presence of SP-B and SP-C improve surfactant function under dynamic conditions, and the idea that molecular adaptation to physiologic needs is a general feature of surfactant biochemistry (for review see Ref. 30), imply that molecular adaptation of surfactant occurs not only across vertebrate species but also within a species to optimize surface tension function according to the differences in respiratory physiology and lung development.

The data presented here agree with this concept, as surfactant from newborn piglets, breathing at higher rates than older pigs (52 versus 8–18 breaths per min at rest) (28), displayed a higher potency as defined by the minimal concentration necessary to achieve surface tensions near zero mN/m upon interface lateral compression (29). However, such improved function was not associated with increased concentrations of PC16:0/16:0 or the sum of all long-chain saturated PC species with phase transition temperatures above body temperature (see Figure 3 and Table 2). Instead, it was associated with a decrease in PC16:0/16:0 and largely increased concentrations of more fluidic PC species, namely PC16:0/14:0 and PC16:0/16:1, together with increased SP-B and SP-C. SP-B and -C concentrations from adolescent porcine lung surfactant as analyzed by HPLC proved to be identical to those previously shown with Sephadex LH-60 gel filtration, demonstrating comparability of these two techniques (25, 29). Notably, therapeutic surfactants from adult pig or cattle being used for treatment of neonatal respiratory distress syndrome contain only 33–49% of SP-B and -C concentrations compared with those of native surfactant from these animals (29), whereas in term newborn pigs SP-B and -C are 3- to 4-fold increased compared with their older counterparts.

We have previously demonstrated that PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1, but not PC16:0/18:1 and PC16:0/18:2, are effectively secreted into the alveolar spaces of mammalian lungs (9). Moreover, fractional concentrations of PC16:0/16:0 correlated inversely, whereas those of PC16:0/14:0 and PC16:0/16:1 correlated directly with the rate of dynamic changes at the air–liquid interface during respiration across animal species (3, 9). Moreover, surfactant from rigid bird lungs, which contains mammalian concentrations of SP-B and is particularly enriched in PC16:0/16:0 at the expense of PC16:0/14:0 and PC16:0/16:1, did not reach minimal surface tension values near zero mN/m under dynamic conditions (3). Similar to those studies, no adaptive changes were found for PC16:0/18:1 or PC16:0/18:2 in neonatal compared with older pigs. Consequently, not only across vertebrate species (for review see Ref. 30), but also during postnatal development within mammalian species, surfactant composition may generally be adapted to respiratory physiology by varying fractional concentrations of PC16:0/16:0, PC16:0/14:0, PC16:0/16:1, SP-B, and SP-C. This was shown previously for PC16:0/14:0 and PC16:0/16:1 in the mouse, rat, and guinea pig (9, 31). In contrast to these animal models, where alveolarization has either not started (mouse, rat) or is already complete at birth (guinea pig), pre- and postnatal development of pig lung is more similar to that of human lungs, where alveolarization starts antenatally and continues after birth (6, 7). Consequently, our findings on neonatal compared with older pigs may be important for our understanding of human lung development and compositional adjustment. This is further emphasized by data on human surfactant composition at birth, where PC16:0/14:0 and PC16:0/16:1 are better indicators of pulmonary maturation than PC16:0/16:0 (9).

It remains unclear, however, why increases in fluidic phospholipids in surfactant are achieved by PC16:0/14:0 and PC16:0/16:1, but not by the abundant PC16:0/18:1, which displays phase transition temperatures below body temperature (10). Although the sorting of PC species within the type II alveolar epithelial cell and their integration into lamellar bodies is generally determined by fatty acyl chain length and not by fatty acid saturation (32), information on specific functions of PC16:0/14:0 and PC16:0/16:1 is sparse, but may include inhibition of the respiratory burst and of oxygen radical formation (33, 34; for review see Ref. 30). From our data, however, it is unlikely that decreased concentrations of PC16:0/16:0 and increases in PC16:0/14:0 as well as in PC16:0/16:1 are due to pulmonary immaturity. First, differences in the fractional concentrations of these components in correlation with air–liquid interface dynamics are not restricted to newborn compared with adult animals, but are also present across adult vertebrates (3, 9). Moreover, pulmonary immaturity is generally associated with increased PC16:0/18:1 and not with PC16:0/14:0 or PC16:0/16:1, the latter being characteristic surfactant components (3, 8, 35). Furthermore, the concentrations of surfactant proteins presented in this study being equal to or even superior to adult levels, do not suggest a functional immaturity of type II alveolar epithelial cells in newborn piglets. By contrast, high concentrations of fluidizing phospholipids together with increased SP-B and SP-C, which interact with these components (12, 14–16), suggest that newborn pig surfactant is optimally designed for breathing at a high rate in ambient air.

In addition to the compositional differences in PC species and hydrophobic surfactant proteins, anionic phospholipids were different in newborn piglets, with PI largely exceeding PG, whereas PG was the predominant anionic phospholipid in adolescent pigs. Whereas in humans PG is a marker of pulmonary maturity, and increased PI indicates pulmonary immaturity or trauma (36, 37), PI species as the predominant anionic surfactant phospholipids are not only characteristic for neonatal pigs but also for newborn sheep and rhesus monkeys (38, 39). Temporary differences of PI versus PG are well described and are likely due to higher plasma concentrations of myoinositol during development (40). Increased PI at the expense of PG does not impair surface tension function of surfactant, as PI can compensate for PG (41, 42). However, PI may have an impact on pulmonary alveoli aside from its contribution to surface tension function, as it specifically binds to SP-D (43, 44), which displayed concentrations in newborn pigs comparable to those of older pigs. PI inhibits macrophage activating factor induced priming of alveolar macrophages and the SP-D–mediated synthesis of matrix metalloproteinases by alveolar macrophages, which may be important for the protection of the developing lung from oxidative stress (45, 46). Why PI is increased in neonatal pigs and lambs, but not in mature human infants, remains unclear. Our data, however, emphasize that the pig model does not consistently reflect all aspects of the human lung surfactant system.

In both newborn and adolescent pigs the majority of anionic phospholipids is composed of fluidic unsaturated molecular species. This is consistent with their role in supporting rapid surface adsorption and (possibly) refinement of the interfacial layer (12, 16), and with data on human surfactant, where most anionic phospholipids are mono- and diunsaturated as well (23). However, composition of anionic phospholipids is distinct from that of guinea pigs, rats, and rabbits, where up to one-third of anionic phospholipids are of the 16:0/16:0 series. Although PG16:0/16:0, similar to PC16:0/16:0, may well form liquid condensed clusters at air–liquid interfaces (12), this appears to be of minor relevance for the pig and human, where 16:0/16:0 species of PG and PI are low. Particularly, in newborn pigs long chain saturated PG and PI species are virtually absent and replaced by monounsaturated PI species (Figure 5).

A characteristic feature of neonatal surfactant is the high concentration in polyunsaturated phospholipids (see Figures 3– 5). Although arachidonic acid containing PC, PG, and PI species may contribute to surfactant fluidity due to their low phase transition temperature (10), they provide the terminal airspaces with arachidonic acid as a substrate for eicosanoid formation. Increased concentrations of arachidonic acid containing phospholipids in neonatal lungs is common not only in pigs but also in infants, and may be an important source of eicosanoid precursors in amniotic fluid around term (47). However, its role within the alveolar space in vivo is unclear. It provides the alveolar spaces and their cells with arachidonic acid, which in turn may contribute to surfactant secretion as well as to protection from hyperoxia (48, 49), whereas during inflammatory processes it may contribute to inhibition of surfactant synthesis (50).

Conclusion
Surfactant from newborn piglets is significantly different from that of older pigs with respect to both functional and biochemical parameters. It is of superior surface tension function, which is associated with 3- to 4-fold increases in hydrophobic surfactant proteins SP-B and SP-C. Consistent with general concepts of adaptation of phospholipid molecular species composition to differences in respiratory physiology, the fluidizing components PC16:0/14:0 and PC16:0/16:1 are increased at the expense of PC16:0/16:0. Unlike human infants, PI instead of PG is the predominant anionic phospholipid in newborn piglets. However, similar to humans and in contrast to rodents, anionic phospholipids of porcine surfactant generally consist of unsaturated fluidic molecular species. Compared with adolescent pigs, surfactant from newborn piglets contains more arachidonic acid–containing phospholipids, which may influence alveolar eicosanoid metabolism. We suggest a concept of molecular adaptation of surfactant function and biochemistry to the age-dependent requirements in pulmonary development and respiratory physiology.


   Acknowledgments
 
The authors thank Mrs. Christa Acevedo, Mrs. Son-Eun Pautsch, and Mrs. Birgit Teichmann for excellent technical assistance, and Larainne Visser-Isles (Erasmus MC-Faculty) for English language editing. This work was supported by an institutional grant of the medical faculty of the University of Tübingen (F.1275089).

Received in original form September 26, 2003

Received in final form October 21, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ikegami, M., and A. Jobe. 1983. Surfactant function in respiratory distress syndrome. J. Pediatr. 102:443–447.[CrossRef][Medline]
  2. Adams, F. H., T. Fujiwara, G. C. Emmanouilides, and N. Räihä. 1970. Lung phospholipids of human fetuses and infants with and without hyaline membrane disease. J. Pediatr. 77:833–841.[CrossRef][Medline]
  3. Bernhard, W., A. Gebert, G. Vieten, G. A. Rau, J. M. Hohlfeld, A. D. Postle, and J. Freihorst. 2001. Pulmonary surfactant in birds: coping with surface tension in a tubular lung. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 281:R327–R337.[Abstract/Free Full Text]
  4. Bernhard, W., H. P. Haagsman, T. Tschernig, C. F. Poets, A. D. Postle, M. E. van Eijk, and H. von der Hardt. 1997. Conductive airway surfactant: surface tension function, biochemical composition and possible alveolar origin. Am. J. Respir. Cell Mol. Biol. 17:171–180.
  5. Burri, P. 1974. The postnatal growth of the rat lung: III. Morphology. Anat. Rec. 180:77–98.[CrossRef][Medline]
  6. Winkler, G. C., and N. F. Cheville. 1985. Morphometry of postnatal development in the porcine lung. Anat. Rec. 211:427–433.[CrossRef][Medline]
  7. Tschanz, S. A., and P. H. Burri. 1999. Prä- und postnatale Entwicklung und Wachstum der Lunge. In Pädiatrische Pneumologie. C. Rieger, H. von der Hardt, F. H. Sennhauser, U. Wahn, and M. Zach, editors. Springer, Heidelberg. 3–16.
  8. Postle, A. D., E. L. Heeley, and D. C. Wilton. 2001. A comparison of the molecular species compositions of mammalian lung surfactant phospholipids. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129:65–73.[CrossRef][Medline]
  9. Bernhard, W., S. Hoffmann, H. Dombrowsky, G. A. Rau, A. Kamlage, M. Kappler, J. J. Haitsma, J. Freihorst, H. von der Hardt, and C. F. Poets. 2001. Phosphatidylcholine molecular species in lung surfactant: composition in relation to respiratory rate and lung development. Am. J. Respir. Cell Mol. Biol. 25:725–731.[Abstract/Free Full Text]
  10. Small, D. M. 1988. Appendix VI-Phospholipids, Major endothermic phase-transition parameters for phospholipids in excess water. In Handbook of Lipid Research, Vol. 4. The Physical Chemistry of Lipids: from Alkanes to Phospholipids. D. M. Small, editor. Plenum Press, New York. 629–635.
  11. Possmayer, F., K. Nag, K. Rodriguez, R. Qanbar, and S. Schürch. 2001. Surface activity in vitro: role of surfactant proteins. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129:209–220.[CrossRef][Medline]
  12. Piknova, B., W. R. Schief, V. Vogel, B. M. Discher, and S. B. Hall. 2001. Discrepancy between phase behaviour of lung surfactant phospholipids and the classical model of surfactant function. Biophys. J. 81:2172–2180.[Abstract/Free Full Text]
  13. Takamoto, D. Y., M. M. Lipp, A. von Nahmen, K. Y. Lee, A. J. Waring, and J. A. Zasadzinski. 2001. Interaction of lung surfactant proteins with anionic phospholipids. Biophys. J. 81:153–169.[Abstract/Free Full Text]
  14. Schief, W. R., M. Anita, B. M. Discher, S. B. Hall, and V. Vogel. 2003. Liquid-chrystalline collapse of pulmonary surfactant monolayers. Biophys. J. 84:3792–3806.[Abstract/Free Full Text]
  15. Wang, Z., S. B. Hall, and R. H. Notter. 1995. Dynamic surface activity of lung surfactant phospholipids, hydrophobic proteins, and neutral lipids. J. Lipid Res. 36:1283–1293.[Abstract]
  16. Walters, R. W., R. R. Jenq, and S. B. Hall. 2000. Distinct steps in the adsorption of pulmonary surfactant to an air-liquid interface. Biophys. J. 78:257–266.[Abstract/Free Full Text]
  17. Casals, C., E. Miguel, and J. Perez-Gil. 1993. Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles. Biochem. J. 296:585–593.
  18. Pérez-Gil, J. 2002. Molecular interactions in pulmonary surfactant films. Biol. Neonate 81:6–15.
  19. Enhorning, G. 1977. Pulsating bubble technique for evaluating pulmonary surfactant. J. Appl. Physiol. 43:198–203.[Abstract/Free Full Text]
  20. Lechner, A. J., and N. Banchero. 1982. Advanced pulmonary development in newborn guinea pigs (Cavia porcellus). Am. J. Anat. 163:235–246.[CrossRef][Medline]
  21. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Med. Sci. 37:911–917.
  22. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466–468.[Free Full Text]
  23. Postle, A. D., A. Mander, K. B. M. Reid, J.-Y. Wang, S. M. Wright, M. Moustaki, and J. O. Warner. 1999. Deficient hydrophilic lung surfactant proteins A and D with normal surfactant phospholipid molecular species in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 20:90–98.[Abstract/Free Full Text]
  24. Bünger, H., L. Kaufner, and U. Pison. 2000. Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection. J. Chromatogr. A 870:363–369.[CrossRef][Medline]
  25. Van Eijk, M., C. G. De-Haas, and H. P. Haagsman. 1995. Quantitative analysis of pulmonary surfactant proteins B and C. Anal. Biochem. 232:231–237.[CrossRef][Medline]
  26. Veldhuizen, E. J. A., J. J. Batenburg, L. M. G. van Golde, and H. P. Haagsman. 2000. The role of surfactant proteins in DPPC enrichment of surface films. Biophys. J. 79:3164–3171.[Abstract/Free Full Text]
  27. Ingenito, E. P., R. Mora, and L. Mark. 2000. Pivotal role of anionic phospholipids in determining dynamic behaviour of lung surfactant. Am. J. Respir. Crit. Care Med. 161:831–838.[Abstract/Free Full Text]
  28. Plonait, H. 1997. Fieberhafte Allgemeinerkrankungen. In Lehrbuch der Schweinekrankheiten. H. Plonait and K. Bickhardt, editors. Blackwell Science, Berlin. 93–110.
  29. Bernhard, W., J. Mottaghian, A. Gebert, G. A. Rau, H. von der Hardt, and C. F. Poets. 2000. Commercial versus native surfactants: surface activity, molecular components, and the effect of calcium. Am. J. Respir. Crit. Care Med. 162:1524–1533.[Abstract/Free Full Text]
  30. Bernhard, W., P. L. Haslam, and J. Floros. 2004. From birds to humans: new concepts on airways relative to alveolar surfactant. Am. J. Respir. Cell Mol. Biol. 30:6–11.[Abstract/Free Full Text]
  31. Burdge, G. C., F. J. Kelly, and A. D. Postle. 1993. Synthesis of phosphatidylcholine in guinea-pig fetal lung involves acyl remodelling and differential turnover of individual molecular species. Biochim. Biophys. Acta 1166:251–257.[Medline]
  32. Longmuir K. J., and S. Haynes. 1991. Evidence that chain length is a type II cell lipid-sorting signal. Am. J. Physiol. (Lung Cell Mol. Physiol.) 260:L44–L51.[Abstract/Free Full Text]
  33. Chao, W., R. G. Spragg, and R. M. Smith. 1995. Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils: attenuation of p47phox and p67phox membrane translocation as the mechanism. J. Clin. Invest. 96:2654–2660.
  34. Smith, R. M., R. Spragg, and J. Connor. 1999. Surfactant phospholipids inhibit neutrophil NADPH oxidase activity in a cell free system. Am. J. Respir. Crit. Care Med. 159:A894. (Abstr.)
  35. Ashton, M. R., A. D. Postle, M. A. Hall, S. L. Smith, F. J. Kelly, and I. C. Normand. 1992. Phosphatidylcholine composition of endotracheal tube aspirates of neonates and subsequent respiratory disease. Arch. Dis. Child. 67:378–382.[Abstract]
  36. James, D. K., M. L. Chiswick, A. Harkes, M. Williams, and V. R. Tindall. 1984. Maternal diabetes and neonatal respiratory distress: I. Maturation of fetal surfactant. Br. J. Obstet. Gynaecol. 91:316–324.[Medline]
  37. Pison, U., W. Seeger, R. Buchhorn, T. Joka, M. Brand, U. Obertacke, H. Neuhof, and K. P. Schmit-Neuerburg. 1989. Surfactant abnormalities in patients with respiratory failure after multiple trauma. Am. Rev. Respir. Dis. 140:1033–1039.[Medline]
  38. Egberts, J., A. Beintema-Dubbledam, and A. de-Boers. 1987. Phosphatidylinositol and not phosphatidylglycerol is the important minor phospholipid in rhesus-monkey surfactant. Biochim. Biophys. Acta 919:90–92.[Medline]
  39. Benson, B. J., J. A. Kitterman, J. A. Clements, E. J. Mescher, and W. H. Tooley. 1983. Changes in phospholipid composition of lung surfactant during development in the fetal lamb. Biochim. Biophys. Acta 753:83–88.[Medline]
  40. Egberts, J., and W. A. Noort. 1986. Gestational age-dependent changes in plasma inositol levels and surfactant composition in the fetal rat. Pediatr. Res. 20:24–27.[Medline]
  41. Beppu, O. S., J. A. Clements, and J. Goerke. 1983. Phosphatidylglycerol-deficient lung surfactant has normal properties. J. Appl. Physiol. 55:496–502.[Abstract/Free Full Text]
  42. Perez-Gil, J., J. Tucker, G. Simatos, and K. M. Keough. 1992. Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung. Biochem. Cell Biol. 70:332–338.[Medline]
  43. Ogasawara, Y., Y. Kuroki, and T. Akino. 1992. Pulmonary surfactant protein D specifically binds to phosphatidylinositol. J. Biol. Chem. 267:21244–21249.[Abstract/Free Full Text]
  44. Persson, A. V., B. J. Gibbons, J. D. Shoemaker, M. A. Moxley, and W. J. Longmore. 1992. The major glycolipid recognized by SP-D in surfactant is phosphatidylinositol. Biochemistry 31:12183–12189.[CrossRef][Medline]
  45. Hayakawa, H., G. Giridhar, Q. N. Myrvik, and L. Kucera. 1992. Pulmonary surfactant phospholipids modulate priming of rabbit alveolar macrophages for oxidative responses. J. Leukoc. Biol. 51:379–385.[Abstract]
  46. Trask, B. C., M. J. Malone, E. H. Lum, H. G. Welgus, E. C. Crouch, and S. D. Shapiro. 2001. Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D. J. Biol. Chem. 276:37846–37852.[Abstract/Free Full Text]
  47. Bernal, A. L., and P. J. Phizackerley. 2000. Fetal surfactant as a source of arachidonate in human amniotic fluid. Prostaglandins Other Lipid Mediat. 60:59–70.[CrossRef][Medline]
  48. Baybutt, R. C., J. E. Smith, M. N. Gillespie, T. G. Newcomb, and Y. Y. Yeh. 1994. Arachidonic acid and eicosapentaenoic acid stimulate type II pneumocyte surfactant secretion. Lipids 29:535–539.[Medline]
  49. Sosenko, I. R., S. M. Innis, and L. Frank. 1988. Polyunsaturated fatty acids and protection of newborn rats from oxygen toxicity. J. Pediatr. 112:630–637.[CrossRef][Medline]
  50. Arias-Diaz, J., E. Vara, C. Garcia, and J. L. Balibrea. 1994. Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins. J. Clin. Invest. 94:244–250.



This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
C. Gille, B. Spring, W. Bernhard, C. Gebhard, D. Basile, K. Lauber, C. F. Poets, and T. W. Orlikowsky
Differential effect of surfactant and its saturated phosphatidylcholines on human blood macrophages
J. Lipid Res., February 1, 2007; 48(2): 307 - 317.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. D. Postle, L. W. Gonzales, W. Bernhard, G. T. Clark, M. H. Godinez, R. I. Godinez, and P. L. Ballard
Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells
J. Lipid Res., June 1, 2006; 47(6): 1322 - 1331.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. J. Lang, A. D. Postle, S. Orgeig, F. Possmayer, W. Bernhard, A. K. Panda, K. D. Jurgens, W. K. Milsom, K. Nag, and C. B. Daniels
Dipalmitoylphosphatidylcholine is not the major surfactant phospholipid species in all mammals
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1426 - R1439.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
R. Ridsdale, M. Roth-Kleiner, F. D'Ovidio, S. Unger, M. Yi, S. Keshavjee, A. K. Tanswell, and M. Post
Surfactant Palmitoylmyristoylphosphatidylcholine Is a Marker for Alveolar Size during Disease
Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 225 - 232.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0351OCv1
30/5/694    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rau, G. A.
Right arrow Articles by Bernhard, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rau, G. A.
Right arrow Articles by Bernhard, W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.