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Am. J. Respir. Cell Mol. Biol., Volume 25, Number 6, December 2001 725-731

Phosphatidylcholine Molecular Species in Lung Surfactant
Composition in Relation to Respiratory Rate and Lung Development

Wolfgang Bernhard, Simone Hoffmann, Heike Dombrowsky, Gunnar A. Rau, Andrea Kamlage, Martin Kappler, Jack J. Haitsma, Joachim Freihorst, Horst von der Hardt, and Christian F. Poets

Departments of Pediatric Pulmonology and Neonatology and of Biometrics, Hannover Medical School, Hannover, Germany; and Department of Anesthesiology, Erasmus University, Rotterdam, The Netherlands


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

Surfactant reduces surface tension at the air-liquid interface of lung alveoli. While dipalmitoylphosphatidylcholine (PC16:0/ 16:0) is its main component, proteins and other phospholipids contribute to the dynamic properties and homeostasis of alveolar surfactant. Among these components are significant amounts of palmitoylmyristoylphosphatidylcholine (PC16:0/ 14:0) and palmitoylpalmitoleoylphosphatidylcholine (PC16:0/ 16:1), whereas in surfactant from the rigid tubular bird lung, PC16:0/14:0 is absent and PC16:0/16:1 strongly diminished. We therefore hypothesized that the concentrations of PC16:0/14:0 and PC16:0/16:1 in surfactants correlate with differences in the respiratory physiology of mammalian species. In surfactants from newborn and adult mice, rats, and pigs, molar fractions of PC16:0/14:0 and PC16:0/16:1 correlated with respiratory rate. Labeling experiments with [methyl-3H]choline in mice and perfused rat lungs demonstrated identical alveolar proportions of total and newly synthesized PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0, which were much higher than those of other phosphatidylcholine species. In surfactant from human term and preterm neonates, fractional concentrations not only of PC16:0/16:0 but also of PC16:0/14:0 and PC16:0/ 16:1 increased with maturation. Our data emphasize that PC16:0/14:0 and PC16:0/16:1 may be important surfactant components in alveolar lungs, and that their concentrations are adapted to respiratory physiology.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

Pulmonary surfactant is a complex mixture consisting of phospholipids, neutral lipids, and specific proteins. It is essential for normal lung function because it reduces surface tension at the air-liquid interface of alveolar spaces (1, 2, 3). Phospholipids comprise 80% of the mass of surfactant, of which 80-85% are phosphatidylcholines (PC) (1, 4). Among the PC molecular species, dipalmitoyl-PC (PC16:0/16:0) is the principle surface tension-lowering molecule, ranging from 40 to 60 mol % in adult mammals, whereas disaturated palmitoylmyristoyl-PC (PC16:0/14:0) together with the monounsaturated palmitoylpalmitoleoyl-PC (PC16:0/16:1) and palmitoyloleoyl-PC (PC16:0/18:1) comprise up to 38% of total PC (4, 5, 6). Interestingly, PC16:0/ 14:0 is virtually absent and PC16:0/16:1 strongly reduced in the surfactant of bird lungs (7). These lungs principally contain air capillaries instead of alveoli. Bird lungs are rigid organs and are not ventilated via expansion and deflation, but by a flow-through mechanism involving a highly differentiated system of air sacs, mesobronchi, and parabronchi. The volume of the pulmonary air spaces and the area of the gas-exchanging surface do not change during the respiratory cycle in bird lungs (8, 9). Consequently, dynamic surface properties are far less important for avian than for mammalian lung surfactant and less well developed, which has previously been demonstrated in vitro in the pulsating bubble surfactometer (7). We therefore postulated that concentrations of PC16:0/14:0 and PC16:0/16:1 in surfactants may correlate with parameters of respiratory physiology. To assess the impact of respiratory physiology on the molecular composition of surfactant, we analyzed the fractional concentrations of PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0 in surfactants from the rigid lungs of duck and chicken and from mammals with highly different respiratory rates, namely 8-d-old and adult mice and rats, and newborn as well as adult pigs. These data were correlated with the respective respiratory rates. Moreover, we expected functionally important PC species to be secreted with comparable kinetics to PC16:0/16:0. We therefore investigated in mice and in isolated perfused rat lungs the fractions of newly synthesized PC species in the alveolar space, using [methyl-3H]choline labeling techniques. Furthermore, we expected that functionally important PC species might increase in human surfactant during pulmonary maturation, similarly to the increase of PC16:0/16:0 at late gestation. We therfore analyzed the composition of PC molecular species in surfactant preparations isolated from pharyngeal suctionings of term and preterm newborn infants, and correlated the fractional concentrations with the gestational age.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

Organic solvents were of high-performance liquid chromatography (HPLC) grade and from Baker (Deventer, The Netherlands). All other chemicals were of analytical grade and from various commercial sources.

Animal and Human Samples

Specific pathogen-free Ztm:MF1 mice (8 d old, 3.5-4.8 g, and 5-7 wk old, 25-30 g) as well as male Sprague Dawley rats (8 d old, 16-19 g, and 6 wk old, 170-180 g) were bred at our local animal facilities. Animals were kept and the hygienic status tested monthly according to the methods of Kunstyr (10). Adult animals were fed an irradiated (5 Mrad) standard diet (Altromin 1314; Altromin GmbH, Lage, Germany), and autoclaved water (134°C, 50 min) was given ad libitum, whereas mouse and rat pups were removed from their mothers directly before being killed. Mixed-breed York-Pyatrain-Landrace newborn piglets (23 ± 15 h old, 1.4 ± 0.3 kg) and pigs of the same race (11 ± 1 wk old, 34-36 kg) were used to obtain porcine surfactant material. Adult ducks and chickens were purchased from our local animal facilities. Pharyngeal aspirates from 29 neonates (mean gestational age: 36.7 ± 4.9 [SD] wk; range, 26-42 wk) were harvested by routine suctioning after delivery as decribed previously (11). Birth weight ranged from 520 to 4,219 g (2,869 ± 1,160 g). Six infants were delivered by Cesarean section, of which two developed respiratory distress syndrome (RDS). The other infants were delivered spontaneously, of which one developed RDS. Sample volumes of the aspirates ranged from 0.5 to 2 ml.

Determination of Respiratory Rates

Respiratory rates were either taken from the literature (adult pig, newborn piglet [12]) or were determined by manual counting. For this purpose unanesthetized resting mice and rats were filmed for 3 min using a digital camera (model MV 30i; Canon, Krefeld, Germany) and respiratory rates counted in the slow motion mode (1/5 of normal velocity) at a model PIII 500 MHz personal computer (Medion, Müeheim, Germany) using Adobe Premiere 5.1 software (Adobe Systems Inc., San Jose, CA). Periods of active sniffing were excluded from counting. Because our aim was to correlate the biochemical composition of surfactants with dynamic parameters of pulmonary ventilation, and because mouse pups, contrarily to newborn rats, display periods of apnea during physiologic ventilation (up to 35% of time) (13 and authors' personal observations), we excluded periods of apnea in these animals for the assessment of respiratory rates.

Analysis of Surfactant from Lung Lavage Fluids and from Pharyngeal Aspirates

Mice and rats were anesthetized with intraperitoneal injection of 100 mg ketamine hydrochloride (Ketanest; WDT Corp., Garbsen, Germany) and 4 mg xylacaine hydrochloride (Rompun; Bayer AG, Leverkusen, Germany)/kg body weight, and the lungs perfused with saline via the right ventricle to remove blood as described previously. Six-week-old mice were then lavaged with 5× 1.0 ml 154 mmol/liter saline and rats with 4× 8 ml of saline. Eight-day-old mice and rats were gently lavaged with 10× 0.15 ml and 0.5 ml saline, respectively. Total recovery of bronchoalveolar lavage fluid (BALF) was 4.4-4.6 ml and 28-30 ml for the older mice and rats, respectively (> 85%), whereas this could not be assured with the newborn animals due to the fragility and the poor retraction of their lung tissue (53-81% recovery). Porcine surfactant was harvested by bronchoalveolar lavage of the lungs with 154 mmol/liter saline as described before (4). Surfactant from duck and chicken was harvested after cerebral disintegration and bleeding of the animals by lavaging the lungs in situ with 154 mmol/liter saline via a tracheal catheter as described elsewhere (7). Because BALF was not available from healthy newborn infants for ethical reasons, surfactant was isolated from their pharyngeal aspirates by sodium bromide density gradient centrifugation (14). This technique resulted in a surfactant fraction with phospholipid compositions similar to those of BALF and low in tissue components (4, 11). Samples containing visible signs of blood contamination were discarded. Cells were removed from BALF by centrifugation at 200 × g for 15 min at 4°C as described before (5), and samples stored at -80°C until further analyses.

Labeling Experiments with [methyl-3H]choline and Investigation of PC Metabolism in Mouse and Rat Lungs

Six-week-old MF-1 mice were intraperitoneally injected with 111 kBq [methyl-3H]choline/g body weight from a stock solution of 37 MBq/ml in saline as described elsewhere (15). After 3 h the animals were anesthetized, and BALF (5 × 1 ml ice cold saline) as well as lung tissue were harvested. Rat lungs were isolated and perfused as decribed before, which included constant ATP levels of lung tissue for at least 3.15 h (16). Lungs were ventilated with water-saturated 95% air/5% CO2 at a rate of 12 strokes/min, a peak inspiratory pressure of 10 cm H2O and an end-expiratory pressure of 3 cm H2O. Lungs were perfused with 7-8 ml/min of 100 ml recycling Krebs-Ringer-bicarbonate buffer (37°C, pH 7.35-7.40), which was equilibrated with water-saturated 95% O2/ 5% CO2, and supplemented with 5% bovine serum albumin (fraction V, > 96% purity; Sigma, Steinheim, Germany), 5.6 mmol/liter glucose, 2 mmol/liter glycerol, 1 mmol/liter sodium acetate, 0.1 mmol/liter sodium palmitate and 0.1 mmol/liter sodium oleate. Fifteen minutes after start of perfusion, 370 kBq [methyl-3H]- choline in 2.5 µmol total choline chloride were added to the perfusate. Lungs showed no visible signs of atelectasis or edema (16) throughout the entire experiment. Time course of [methyl-3H]- choline label in the perfusate was monitored, and after 3.15 h the lungs were lavaged with 4 × 8 ml ice cold 154 mmol/liter saline.

Analysis of Composition and [methyl-3H]choline Incorporation of Phosphatidylcholine Molecular Species

Phospholipids (PL) from BALF and pharyngeal aspirates were extracted according to Bligh and Dyer (17), and from lung tissue according to the method of Folch and colleagues (18). PL phosphorus was quantified as described by Bartlett and coworkers (19) 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). PC molecular species were analyzed as described before (20). Briefly, 50 nmol dimyristoyl-PC (PC14:0/14:0) were added as a standard to 200-1,000 nmol PL extract. Total PC was then isolated with 100 mg Varian Bondelut NH2 disposable cartridges (Varian, Hamburg, Germany), followed by HPLC analysis of individual PC molecular species with a 4.6 × 250-mm Sphere Image ODS II column (Schambeck, Bad Godesberg, Germany) and postcolumn fluorescence derivative formation in the presence of 1,6-diphenyl-1,3,5-hexatriene. For measurement of [methyl-3H]choline incorporation, HPLC peaks of individual PC species were collected and measured in a model 2000 CA TriCarb liquid scintillation analyzer (Packard, Groningen, The Netherlands). Solvents were evaporated before the addition of scintillation cocktail (LumaSafe Plus; Lumac*LCS, Groningen, The Netherlands).

Statistics

Data are expressed as mean ± SD. To compare groups, two-tailed t tests were used and in the case of three or more groups ANOVA was performed using GraphPad Instat Version 1.11a (GraphPad Software, San Diego, CA). To determine in pharyngeal aspirate preparations if the variables of interest depend on the gestational age in linear or quadratic form, the change of the global F-statistic was calculated and used as criterion to add the corresponding term to the model. Regression analyses were performed with the statistics software package SPSS Release 10.0.7 (SPSS Inc., Chicago, IL).

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

Surfactant PC Composition in Relation to Respiratory Rates

Values of respiratory rates measured in adult and 8-d-old mice were 254 ± 28 (n = 43) and 372 ± 75 (n = 18) per minute, respectively, whereas in adult and 8-d-old rats they were 112 ± 11 (n = 6) and 302 ± 24 (n = 51), respectively. Values were set as zero for birds, because in their rigid, tubular lungs the respiratory cycle is not associated with volume and surface area changes (8, 9). Figure 1 demonstrates the molar fraction of PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 of surfactant PC in relation to respiratory rates. In all animal species, irrespective of respiratory rate or age, the sum of PC16:0/16:0, PC16:0/14:0, and PC16:0/ 16:1 was ~ 80% of total PC (Figures 1 and 2). For surfactant from duck and chicken, molar fractions of PC16:0/16:0 in relation to total PC were highest (71.5 ± 2.5% [n = 4] and 72.5 ± 3.2% [n = 5], respectively), whereas they were low for PC16:0/14:0 (1.1 ± 0.4% and 1.9 ± 0.8%) and PC16:0/16:1 (3.5 ± 1.2% and 3.5 ± 0.6%, respectively). PC16:0/18:1 were 15.0 ± 1.1 and 12.5 ± 2.4, respectively. With increasing respiratory rates of mammals, the concentrations of PC16:0/14:0 and PC16:0/16:1 increased in relation to total PC, whereas that of PC16:0/16:0 decreased. Including the periods of apnea of the 8-d-old mice into the calculation of respiratory rates did not affect these correlations (data not shown).


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  		Figure 1.   Concentrations of phosphatidylcholine (PC) molecular species in surfactant in relation to respiratory rates. Molar fractions (%) of PC16:0/16:0, PC16:0/14:0, PC16:0/16:1 as well as of PC16:0/14:0 + PC16:0/16:1 in relation to total PC were plotted against the respiratory rates of 8-d-old and adult mice and rats, of newborn and adult pigs, and of chicken and ducks. Rates in birds were set as zero, because these animals possess rigid lungs without surface area changes during the respiratory cycle (see Introduction). Symbols represent means ± SD of 3 to 20 experiments. Circles: PC16:0/14:0; diamonds: PC16:0/16:1; triangles: PC16:0/16:0; squares: PC16:0/14:0 + PC16:0/16:1. *P < 0.05; dagger P < 0.01.


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  		Figure 2.   PC composition of surfactant from 8-d-old versus adult mice and rats, and newborn versus adult pigs. Surfactant from lungs of 8-d- and 6-wk-old mice (A) and rats (B) and from 1-d-old and adult pigs (C) was extracted with chloroform:methanol. PC species were analyzed with HPLC as described in MATERIALS AND METHODS. Data are means ± SD of 3 (mouse, 8 d, pooled from 2-3 BALF), 20 (mouse, 6 wk), 7 (rat, 8 d), 10 (rat, 6 wk), 7 (newborn piglet) and 10 (adult pig) experiments. *P < 0.05; dagger P < 0.01; ddager P < 0.001; §P < 0.0001 versus adult.

Surfactant PC Species in 8-d-Old Mice and Rats and Newborn Piglets Compared with Adult Animals

As demonstrated in Figures 2A-2C, surfactant from 8-d-old mice and rats and from newborn pigs was significantly different from that of their adult counterparts. Surfactant PC from adult animals contained ~ 60% PC16:0/16:0, whereas that from 8-d-old mice and rats and newborn piglets contained only 38.9 ± 2.2%, 39.6 ± 2.6%, and 51.4 ± 1.9%, respectively. Instead, compared with adult mice and rats (5.9 ± 0.8% and 8.6 ± 1.1%, respectively), PC16:0/14:0 was increased in 8-d-old animals (22.9 ± 4.0% and 24.1 ± 1.2%, respectively). These results were consistent with the PC composition of lung tissue, where increased concentrations of PC16:0/14:0 and reduced concentrations of PC16: 0/16:0 were found in 8-d-old mice and rats compared with adult animals (data not shown). In newborn piglets both PC16:0/14:0 and, particularly, PC16:0/16:1 (9.3 ± 0.9% and 14.6 ± 0.8%, respectively) were increased compared with adult animals (6.2 ± 0.7% and 6.7 ± 2.5%, respectively) (Figure 2C).

Alveolar Fractions of Individual PC Molecular Species in Mice and Rats

Lung tissue and BALF from both mouse (Figure 3A) and rat (Figure 4A) contained significant amounts not only of PC16:0/16:0, but also of PC16:0/14:0, PC16:0/16:1, and other unsaturated PC species like palmitoyloleoyl-PC (PC16:0/ 18:1) and palmitoyllinoleoyl-PC (PC16:0/18:2). The mass fractions of PC16:0/14:0 and PC16:0/16:1 in BALF, compared with whole lung, were identical to those of PC16:0/16:0 (29-31% for the mouse and 13-17% for the rat, respectively), and were significantly higher than those of other PC species of mouse and rat BALF (12-16% and 3-7%, respectively (Figures 3B and 4B). Comparable results were obtained in the radiolabeling experiments. Most [methyl- 3H]choline label was found in PC16:0/16:0, whereas PC16:0/ 14:0 and the unsaturated PC species contained lower amounts of [methyl-3H]choline label (Figures 3C and 4C). However, the [methyl-3H]choline-labeled fractions in BALF, compared with whole lung, were identical for PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0 (1.9-2.7% and 3.2-3.7% of total lung label in mouse and rat lungs, respectively) (Figures 3D and 4D). In contrast, BALF fractions of [methyl- 3H]choline-labeled PC species were much lower for other PC species like PC16:0/18:1 and PC16:0/18:2 in both mice and rats (Figures 3D and 4D).


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  		Figure 3.   Pool sizes of and [methyl-3H]choline incorporation into PC molecular species of mouse lung tissue and bronchoalveolar lavage fluid (BALF). Mice were intraperitoneally injected with 111 kBq [methyl-3H]choline/g body weight and lavaged 3 h after radiolabeling as described in MATERIALS AND METHODS. Pool sizes of PC molecular species (A) as well as their [methyl-3H]choline incorporation after 3 h (C) are indicated for BALF and total lungs (lavaged lung + BALF). The fractions of masses (B) and of [methyl- 3H]choline label of PC molecular species (D) were determined in BALF in relation to total lung. Data are means ± SD of 10 (A and B) and 4 (C and D) experiments. dagger P < 0.01; ddager P < 0.001 versus PC16:0/16:0, PC16:0/14:0 and PC16:0/16:1; n.s., not significantly different from PC16:0/16:0 and PC16:0/ 16:1 or PC16:0/14:0.


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  		Figure 4.   Pool sizes of and [methyl-3H]choline incorporation into PC molecular species of rat lung tissue and BALF. Rat lungs were perfused for 3 h in the presence of 370 kBq [methyl-3H]choline in 100 ml perfusate and then lavaged as described in MATERIALS AND METHODS. Pool sizes of PC molecular species (A) as well as their [methyl-3H]choline incorporation (C) are indicated for BALF and total lungs (lavaged lung + BALF). Fractions of masses (B) and of [methyl-3H]choline-labeled PC molecular species (D) were determined in BALF in relation to total lung. Data are means ± SD of five separate experiments. *P < 0.05; ddager P < 0.001 versus PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1; n.s., not significantly different from PC16:0/16:0 and PC16:0/16:1 or PC16:0/14:0.

Changes of PC Molecular Species Composition of Human Surfactant during Gestational Maturation

In surfactant isolated from pharyngeal aspirates of human neonates, the mean value for PC16:0/16:0 in relation to total PC was increased in term (37-42 wk gestational age [GA]) over preterm infants (< 37 wk GA) (Table 1). However, due to interindividual variations this did not reach statistical significance (P > 0.05). In contrast, PC16:0/14:0 and PC16:0/16:1 were significantly increased (P < 0.0001 and P < 0.05, respectively) in term compared with preterm neonates. Fractional concentrations of other mono- or polyunsaturated PC species like PC16:0/18:1, palmitoylarachidonoyl-PC (PC16:0/20:4), and stearoylarachidonoyl-PC (PC18:0/20:4) were lower, whereas palmitoyllinoleyl-PC (PC16:0/18:2) and palmitoyldocosahexaenoyl-PC (PC16:0/ 22:6) were similar in both groups. More detailed analyses of the time course of PC molecular species composition from 26 to 42 wk GA revealed that PC16:0/16:0 followed a polynomial curve, increasing from mean values of 30% at 26 wk GA to 57% at 38 wk, and then falling back to 52% at 42 wk (P < 0.01, Figure 5A). In contrast, PC16:0/14:0 and PC16:0/16:1 increased linearily (P < 0.001 and P < 0.01, respectively) during the entire period investigated (Figures 5B and 5C). Consequently, the ratios of PC16:0/ 14:0 to PC16:0/16:0 and of PC16:0/16:1 to PC16:0/16:0 increased after 38 wk GA (P < 0.05 and P < 0.001, respectively; data not shown). The increases in PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 occurred at the expense of other unsaturated PC molecular species, namely PC16:0/ 18:1, PC18:0/18:2, and PC16:0/20:4, which declined linearily from 26 to 42 wk GA (Figures 5D-5F).

                              
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TABLE 1
Composition of phosphatidylcholine molecular species in pharyngeal aspirates of term versus preterm newborn infants


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  		Figure 5.   Phosphatidylcholine molecular species in surfactant isolated from pharyngeal aspirates in relation to gestational age. (A) Dipalmitoyl-PC. (B) Palmitoylmyristoyl-PC. (C ) Palmitoylpalmitoleoyl-PC. (D) Palmitoyloleoyl-PC. (E ) Stearoyllinoleoyl-PC. (F ) Palmitoylarachidonoyl-PC. Surfactant was isolated by sodium bromide density gradient centrifugation from routine pharyngeal suctions (n = 29) of newborn infants (26-42 wk gestational age). Individual PC molecular species were resolved by HPLC and the fraction of PC species determined in relation to total PC as described in MATERIALS AND METHODS. dagger P < 0.01; ddager P < 0.001.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

Differences of Surfactant Phosphatidylcholine Composition in Relation to Lung Function

Biochemical characterization of lung surfactant usually focuses on PC16:0/16:0, phosphatidylglycerol (PG) and apoproteins surfactant protein (SP)-A, SP-B, SP-C and SP-D as characteristic components (1, 2). In this study we demonstrate for the first time the major differences in the molar fractions of PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 of lung surfactants in relation to respiratory rates of animals, and in human neonates in relation to pulmonary development. It is well known that both the anionic PG and the monounsaturated PC16:0/18:1 contribute to dynamic surfactant functions of alveolar surfactant in vitro (21, 22). However, most surfactants contain only 5-12% PC16:0/ 18:1, whereas lung tissue and secretions from other organs are highly enriched in this molecule (4, 23, 24). In contrast, all mammalian surfactants investigated so far contain significant amounts of PC16:0/14:0 and PC16:0/16:1, which are not prominent in other organs (4, 5, 6, 25, 26). The data of this study clearly demonstrate that the fractional concentrations of PC16:0/14:0 and PC16:0/16:1 are directly correlated to the respiratory rates of mammals, whereas that of PC16:0/16:0 is inversely correlated. The possible impact of PC16:0/14:0 and PC16:0/16:1 on dynamic functions of alveolar surfactant is supported by the finding that surfactant from the rigid and essentially nonalveolar bird lung contains virtually no PC16:0/14:0 and only low concentrations of PC16:0/16:1 (7). Such avian surfactants display poor surface tension function under the dynamic cycling conditions of the pulsating bubble surfactometer (7). In contrast, surfactant from newborn piglets reaches values of minimal surface tension below 5 mN/m more rapidly than that from adult pigs, particularly at high oscillation rates of 100/min (authors' unpublished data). Consequently, the data of this study support the concept of a functional role not only of PC16:0/16:0, but also of PC16:0/14:0 and PC16:0/16:1 in lung surfactant and the adjustment of their concentrations to the physiologic needs of the respective lungs.

Metabolism and Developmental Changes of Surfactant PC16:0/14:0 and PC16:0/16:1

We postulated that a PC species which is important for surfactant functions should have a synthesis and secretion pattern similar to that of PC16:0/16:0. This concept is supported by our labeling experiments in mice in vivo as well as in perfused rat lungs ex vivo. Total amounts and newly synthesized PC16:0/14:0 and PC16:0/16:1 are enriched in BALF compared with lung tissue to a similar extent as PC16:0/16:0. In contrast, other [methyl-3H]choline-labeled PC species, like PC16:0/18:1 and PC16:0/18:2, were predominantly retained in lung tissue. The concept of selective release of PC16:0/14:0 and PC16:0/16:1 into the alveolar spaces was further supported by our analyses of human surfactant in relation to pulmonary maturation. For ethical reasons BALF samples from term and preterm newborn infants could not be collected. However, our data on surfactant from nasopharyngeal suctionings clearly demonstrate that during human pulmonary maturation the fractional concentrations not only of PC16:0/16:0 but also of PC16:0/14:0 and PC16:0/16:1 increased. Similar to the selective incorporation of these PC species into surfactant of the mouse and rat, these increases occurred at the expense of other unsaturated PC species, predominantly PC16:0/ 18:1, PC18:0/18:2, and PC16:0/20:4. Particularly at term, when a fetus faces respiration in ambient air for the first time and when respiratory rate is highest during the entire human life, the molar fractions of PC16:0/14:0 and PC16:0/ 16:1 are highest in relation to PC16:0/16:0. Our data support and extend those from Hunt and associates, who demonstrated an increase not only of PC16:0/16:0, but also PC16:0/14:0 during end-gestation in human, guinea pig, and rat fetal lung tissue (26). Hence, both in mammalian animals and in humans PC16:0/14:0 and PC16:0/16:1 are primarily synthesized to serve as alveolar surfactant components. The metabolic pathways for the formation of PC16:0/14:0 and PC16:0/16:1, i.e., acyl remodeling together with de novo synthesis, may be similar to those of PC16:0/ 16:0, and a possible mechanism for selective accumulation during end-gestation is their increasing turnover time (27).

PC16:0/14:0 and PC16:0/16:1 Function in Relation to Other Surfactant Components

In mammalian surfactant the concentrations of PC16:0/ 14:0 and PC16:0/16:1 are in the same range or even higher than that of the functionally important PG (8-10% of total phospholipid) (21). It is unclear, however, what the specific functions of PC16:0/14:0 and PC16:0/16:1 and, therefore, their importance for exogenous therapeutic surfactants are. It is well described that not only PG but also PC16:0/18:1 improve surfactant functions. The latter increases adsorption properties of surfactant in vitro and the fusogenic action of SP-B on PC16:0/16:0 containing liposomes (5, 21, 28). However, PC16:0/18:1 is only increased in surfactant from cattle (5, 12). In contrast, surfactant from other mammals, particularly the mouse and rat, is low in PC16:0/18:1 but enriched in PC16:0/14:0 and PC16:0/ 16:1. Principally PC16:0/14:0 and PC16:0/16:1 are not good candidates for lowering surface tension, as collapse of the monolayer would occur at temperatures far below body temperature (29). Such assumptions, however, must be reconsidered for the complex molecular interactions in native surfactants, particularly at different respiratory rates. It must, moreover, be considered that there are additional differences in alveolar morphology and respiratory physiology across different species and ages. Such differences include alveolar diameter and radius of curvature, the latter ranging from 23.8 ± 4.6 and 52 ± 1.5 µm in mice and rats to over 100 µm in larger mammals, as well as the degree of surface area oscillation during a respiratory cycle, which may be low at very high respiratory rates (30).

Whereas the contribution of PC16:0/14:0 and PC16:0/ 16:1 to dynamic surface tension functions of surfactant is only supported by circumstantial evidence, their actions on phagocytic cells is better evaluated. PC molecular species with short or unsaturated acyl chains in position 2, like PC16:0/12:0 and PC16:0/16:1, suppress oxygen superoxide production by phagocytic cells more efficiently than other components, like PC16:0/18:1 and PC16:0/16:0 (31, 32). The underlying mechanism possibly is inhibition of the translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane (33). As analyzed with immunoblotting, surfactant from newborn compared with adult rats contains less SP-A in relation to total phospholipid (authors' unpublished data). Because SP-A stimulates the respiratory burst of alveolar macrophages (34), our findings are consistent with a biological concept of suppression of inflammatory processes in developing lungs.

    Conclusion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

The molecular design of pulmonary surfactant displays significant differences among vertebrate species and between newborn compared with adult organisms. Differences in the fractional concentrations of PC16:0/16:0, PC16:0/14:0, and PC16:0/16:1 correlate with the respiratory rates of animals, with PC16:0/14:0 and PC16:0/16:1 increasing at the expense of PC16:0/16:0. This suggests that the fractional concentrations of PC16:0/14:0 and PC16:0/16:1 in alveolar surfactants are adjusted to the physiologic needs of mammalian lungs. Our theory is supported by the increase in the molar fractions of PC16:0/14:0 and PC16:0/16:1 during fetal lung maturation and by selective secretion of newly synthesized PC16:0/14:0 and PC16:0/16:1 along with PC16:0/ 16:0 into the alveolar space. Further investigations are necessary to reveal mechanisms of action of PC16:0/14:0 and PC16:0/16:1 in the alveolus, and their possible clinical relevance in respiratory failure.

    Footnotes

Address correspondence to: Wolfgang Bernhard, M.D., Ph.D., Department of Pediatric Pulmonology & Neonatology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. E-mail: bernhard.wolfgang{at}mh-hannover.de

(Received in original form April 25, 2001 and in revised form August 7, 2001).

Abbreviations: bronchoalveolar lavage fluid, BALF; gestational age, GA; high-performance liquid chromatography, HPLC; phosphatidylcholine(s), PC; dipalmitoyl-PC, PC16:0/16:0; palmitoylmyristoyl-PC, PC16:0/14:0; palmitoyllinoleoyl-PC, PC16:0/18:2; palmitoyloleoyl-PC, PC16:0/18:1; palmitoylpalmitoleoyl-PC, PC16:0/16:1; palmitoylarachidonoyl-PC, PC16:0/20:4; stearoylarachidonoyl-PC, PC18:0/20:4; palmitoyldocosahexaenoyl-PC, PC16:0/22:6; phosphatidylglycerol, PG; phospholipid, PL; respiratory distress syndrome, RDS; surfactant protein, SP.

Acknowledgments: The authors thankfully acknowledge the excellent technical assistance of Mrs. Christa Acevedo and Ms. Ivonne Strenger. This work was supported by the Deutsche Forschungsgemeinschaft (Grant Ha1959/2), the Appenrodt Stiftung, and the Forschungsgemeinschaft Mukoviszidose.
    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References

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