Plasmalogens effectively reduce the surface tension of surfactant-like phospholipid mixtures

M. Rüdiger, I. Kolleck, G. Putz, R. R. Wauer, P. Stevens, and B. Rüstow

Department of Neonatology, Children's Hospital of the Charité, Medical Faculty of Humboldt University Berlin, 10098 Berlin, Germany; and University Hospital of Anesthesia and Intensive Care, University of Innsbruck, 6020 Innsbruck, Austria

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The alkenyl-acyl subclass of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (plasmalogens) are minor components of alveolar surfactant. Plasmalogens promote and stabilize hexagonal structures of phospholipids. In another study (W. R. Perkins, R. B. Dause, R. A. Parente, S. R. Michey, K. C. Neuman, S. M. Gruner, T. F. Taraschi, and A. S. Janoff. Science 273: 330-332, 1996), it was shown that polymorphic phase behavior may have an important role in the effective functioning of pulmonary surfactant. Therefore, we hypothesized that surface properties of phospholipid mixtures that contain plasmalogens are superior to plasmalogen-free mixtures. The effect of plasmalogens on surface tension of surfactant-like phospholipid mixtures (70 mol% dipalmitoyl phosphatidylcholine, 10 mol% phosphatidylglycerol, and 20 mol% PtdEtn) was measured. Using the pulsating bubble surfactometer, we show that an increasing amount of ethanolamine plasmalogens [plasmenylethanolamine (PlsEtn)] results in reduction of surface tension (0 mol% PlsEtn 44.7 ± 1.7, 2 mol% 33.5 ± 1.7, 4 mol% 36 ± 3.1, 6 mol% 26.2 ± 2.9, and 8 mol% 22.2 ± 0.3 mN/m). By means of the captive bubble surfactometer, minimal surface tension reached with 8 mol% PlsEtn was even lower (3.8 ± 0.7 mN/m). With regard to morphological studies (B. Fringes, K. Gorgas, and A. Reith. Eur. J. Cell Biol. 46: 136-143, 1988), clofibrate treatment of rats might increase the plasmalogen content of alveolar surfactant. However, in the present study, we could not show that synthesis and secretion of plasmalogens are affected by clofibrate treatment.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PLASMALOGENS are the alk-1-enyl-acyl subclass of phosphatidylethanolamine [PtdEtn (plasmenylethanolamine, PlsEtn)] and phosphatidylcholine [PtdCho (plasmenylcholine, PlsCho)]. Together with the alkyl-acyl subclass, plasmalogens belong to the group of ether phospholipids. The biosynthesis of ether phospholipids differs in some aspects from that of the diacyl subclasses of PtdEtn and PtdCho (5, 10). Synthesis of plasmalogens is peculiar because the first two enzymes in the metabolic pathway [dihydroxyacetone phosphate (DHAP) acyltransferase and alkyl-DHAP synthase] are localized in peroxisomes. The following reactions are catalyzed by enzymes of the endoplasmic reticulum (14). Consequently, synthesis of plasmalogens is found to be defective in some genetic disorders that are associated with a significant reduction or loss of peroxisomes.

Plasmalogens are present in most mammalian cells as a minor component. Heart myocytes of several species, however, are an exception because concentration of plasmalogens can be as high as 20-30% of diradyl phospholipids (22). Recently, we have shown that plasmalogens of type II pneumocytes consist predominantly of ethanolamine plasmalogens (PlsEtn) similar to most mammalian cells (19).

Alveolar surfactant is supplemented with plasmalogens during synthesis in type II pneumocytes (17). The content of plasmalogens within the alveolar surfactant has been investigated rarely. We discovered by gas chromatographic analysis ~2 mol% total plasmalogens in rat surfactant (17). Using 31P nuclear magnetic resonance spectroscopy, Rana et al. (16) determined 4 mol% PlsCho in surfactant of dogs. Taking into account that dog surfactant also contains PlsEtn, it can be assumed that the concentration of total plasmalogens is ~5-6 mol%. The different plasmalogen content within the alveolar surfactant of rats and dogs might represent species differences. However, it can not be excluded that the difference is caused by different methods used for the plasmalogen analysis.

Although plasmalogens have been known as constituents of mammalian cells for some decades, our knowledge about their physiological function is very limited (14), and nothing is known about their function in the alveolar surfactant. Two properties of plasmalogens might be closely related to their physiological function in the alveolar surfactant. 1) Plasmalogen can act as an antioxidant. Recently, Zoeller et al. (24) showed that cellular plasmalogens act as antioxidants against ultraviolet light-induced lipid peroxidation. In addition, plasmalogens seem to be able to act as antioxidants in low-density lipoproteins (6). 2) Plasmalogens stabilize and promote hexagonal lipid structures (9, 14). In the hypophase of the alveoli, phospholipids are stored mainly as bilayers in tubular myelin before entering the monolayer at the air-water interface. Metcalfe et al. (12) and Yu et al. (23) noted that nonbilayer structures of the tubular myelin seem to be involved in the process of film formation. Recently, Perkins et al. (15) showed that a phospholipid mixture containing dioleoylphosphatidylethanolamine, dipalmitoyl phosphatidylcholine (DPPC), and cholesterol in a molar ratio of 7:3:7 exists above 35°C as a mixture of lamellar and hexagonal phases. Although this phospholipid mixture differs strongly from the phospholipid composition of natural surfactant, testing this phospholipid mixture in vivo in a neonatal rabbit model shows that the phospholipid mixture elicited an onset of action equal to that of native human surfactant. From these results, the authors assume that lipid polymorphic phase behavior may have an important role in the effective functioning of pulmonary surfactant. Because plasmalogens affect the rearrangement from bilayer to hexagonal phase (9), we hypothesize that plasmalogens within the surfactant system support the transition of phospholipid from bilayer structures (such as tubular myelin) to the monolayer at the air-water interface. By influencing the transition of phospholipids, plasmalogens would have an effect on surface properties of phospholipid mixtures. That can be measured as changes in surface tension.

To analyze the effect of plasmalogens on surface properties, surfactant mixtures with different concentrations of plasmalogens are needed. However, to measure only an effect of plasmalogens, the original phospholipid composition must remain unchanged. The best way would be an enrichment in vivo during synthesis of surfactant. Fringes et al. (4) showed by morphometric methods that clofibrate treatment of rats caused an increase in the number of peroxisomes and lamellar bodies of type II pneumocytes. From their morphological study, the authors hypothesized that clofibrate influences the metabolism of the pulmonary surfactant and that peroxisomes may be involved in the processing of surfactant. Because the first steps of plasmalogen biosynthesis take place in peroxisomes, we investigated whether clofibrate treatment of rats will increase synthesis and secretion of plasmalogens by type II pneumocytes. Although we were able to reproduce the known clofibrate effects, no effect on the incorporation of hexadecanol into plasmalogens or secretion of hexadecanol-labeled plasmalogens and palmitic acid-labeled phospholipids by type II pneumocytes could be shown.

Using an artificial, PlsEtn-enriched phospholipid mixture, we showed in the present study that PlsEtn affects surface properties of the mixture. By measurement of surface tension, using the pulsating bubble surfactometer and the captive bubble surfactometer, we can show that plasmalogens reduce effectively the surface tension of a surfactant-like synthetic phospholipid mixture containing 70 mol% DPPC, 20% PtdEtn, and 10 mol% phosphatidylglycerol (PtdGro).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

DPPC, PtdGro, and clofibrate [2-(p-chlorophenoxy)-2-methylpropionic acid] were obtained from Sigma. [14C]hexadecanol (specific activity: 10.4 mCi/mmol) was from Sigma, and [3H]palmitic acid (specific activity: 54 Ci/mmol) was from Amersham. All other chemicals used were of analytical grade and were purchased from different suppliers.

PtdEtn containing ~40% plasmalogens was isolated from the lipid extract of pig heart by thin-layer chromatography (TLC). Plasmalogen-free PtdEtn was isolated by TLC after HCl treatment of the isolated plasmalogen-containing PtdEtn. Because treatment with HCl does not remove alkyl-acyl species, this subclass is present in the same quantity and quality in the plasmalogen-containing and plasmalogen-free PtdEtn that was used.

All of the phospholipid mixtures used for measuring surface tension contained 70% DPPC, 10% PtdGro, and 20% PtdEtn. The amount of PlsEtn in the PtdEtn fraction was varied between almost 0 and 40%, resulting in phospholipid mixtures that contained 0, 2, 4, 6, or 8% PlsEtn of total phospholipids.

Methods

Measurement of the surface tension. Phospholipids of all mixtures were suspended in 150 mM saline/3 mM CaCl2 at a concentration of 2 mg phospholipid/ml and were frozen at -80°C. Before surface tension was measured, the frozen mixtures were thawed at 37°C, sonicated with a sonifier equipped with a 0.3-cm-diameter microtip for 1 min at 70 W, and incubated at 37°C. After 30 min, the mixtures were agitated on a vortex mixer. A 25-µl (pulsating bubble surfactometer) or 1-ml (captive bubble surfactometer) aliquot of the phospholipid mixtures was used for measurement.

Surface tension-lowering properties of mixtures were investigated with a pulsating bubble surfactometer (Electronics) as described by Enhorning (3). After automatic formation of the bubble, the suspension was allowed to adsorb at minimum bubble size for 10 s. Thereafter, the bubble was cycled automatically (20 cycles/min). Once the radius is known, measurement of pressure difference across the bubble allows the calculation of surface tension by using the Laplace equation. Each phospholipid mixture was prepared three times and tested consecutively in triplicate, resulting in nine different measurements.

The measurements with the captive bubble surfactometer were carried out as described earlier (20). After the stirred (100 revolutions/min) phospholipid mixtures had reached 37°C, a 30-µl air bubble was injected at 1.0 atmosphere (ATA). Each phospholipid mixture was measured two times. To measure adsorption properties of the mixtures, we abruptly increased the bubble area by a sudden lowering of chamber pressure from 1.0 to 0.5 ATA. The pressure was kept constant at 0.5 ATA for 10 s. Thereafter, the bubble area was automatically cycled by changing the pressure between 2.8 and 0.5 ATA (10 cycles/min).

Determination of phospholipids. The lipids were extracted according to Bligh and Dyer (1) and analyzed as previously described (17, 19). After methanolysis, the fatty acid methyl esters and the dimethyl acetals were separated by gas chromatography using margarinic acid (C-17) as an internal standard for quantification. The sum of the dimethyl acetals represents the concentration of plasmalogens.

Treatment of animals. As described by Fringes et al. (4), Wistar rats (body mass: 120 ± 15 g) received clofibrate (300 mg/kg body mass) or 0.9% NaCl (control animals) by daily intraperitoneal injection for 7 days. The animals were killed by intraperitoneal injection of 30 mg pentobarbital sodium, and the lungs were perfused free of blood, removed, and used for the isolation of type II pneumocytes. Type II pneumocytes of each animal were isolated by digestion of lung tissue with elastase (porcine pancreas from Boehringer) and purified by panning on immunoglobulin G-coated plates according to Dobbs et al. (2).

Labeling and secretion of phospholipids. As described previously (19), all experiments were carried out as double-labeling experiments. Type II cells were cultured for 18-20 h in the presence of [3H]palmitate (0.3 µCi/106 cells) and [14C]hexadecanol (0.2 µCi/106 cells) solubilized in fetal calf serum used in the culture medium (17). Thereafter, the cells were washed, and the relative secretion of hexadecanol-labeled PlsCho and PlsEtn and of palmitic acid-labeled phospholipids was determined. For the stimulation of surfactant secretion, we used terbutaline (Sigma; final concentration 10-6 M; see Ref. 4). The incorporation of label into plasmalogens and phospholipids was determined as described previously (19).

Isolation and subfractionation of lamellar bodies of type II pneumocytes. Type II cells prelabeled with [3H]palmitate and [14C]hexadecanol for 18 h were suspended in 1 M sucrose and disrupted in an N2 bomb at 2,300 psi N2 pressure for 15 min at 4°C. The 1 M sucrose containing the disrupted cells was overlayered consecutively with 0.8-0.2 M sucrose in 0.1 M steps. After centrifugation at 80,000 g at 4°C for 3 h in a Beckman ultracentrifuge using an SW-41 swinging bucket rotor, the lamellar bodies were located between densities 1.3472 and 1.3568 (13). This fraction of the gradient was collected, diluted 1:1 (vol/vol) with water, and centrifuged according to Oosterlaken-Dijksterhuis et al. (13) first at 8,000 g for 30 min at 4°C (heavy lamellar bodies) and thereafter at 80,000 g for 60 min at 4°C (light lamellar bodies). In the subfractions of the lamellar bodies, we determined [3H]DPPC, [14C]PlsCho, and [14C]PlsEtn as relative proportions of total labeled lipids present in the nonfractionated lamellar body fractions.

Other methods. Catalase activity was determined using H2O2 as the substrate. DPPC was determined by OsO4 oxidation of the PtdCho fraction according to Mason et al. (11). The separation of DPPC from the oxidation products was carried out by TLC as previously described (18).

Statistics. The statistical analyses of our data were performed using STATGRAPHICS (Manugistics). The results are expressed as medians and ranges. Differences were considered significant with P < 0.05 using the rank test for independent unpaired samples (Mann-Whitney test).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Measurement of surface tension of mixtures that contain either no or 8% PlsEtn reveals significant differences. As shown in Figs. 1 and 2, surface tension is always lower in samples that contain PlsEtn. Differences between the two methods of surface tension measurement (captive bubble surfactometer vs. pulsating bubble surfactometer) have been found with regard to absolute values that were reached. Using the captive bubble surfactometer, minimal surface tensions came close to zero. However, both methods show lower surface tension for samples that contain PlsEtn and similar time courses in reaching low surface tension during pulsation. Therefore, pulsating bubble surfactometer and captive bubble surfactometer can be used equally to show the effect of PlsEtn on surface tension.


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Fig. 1.   Surface tension of lipid mixtures that were either free of plasmenylethanolamine (PlsEtn; X) or that contained 8% PlsEtn (black-lozenge ) measured in a captive bubble surfactometer. After 10 compressions, surface tension is lower in the PlsEtn-containing samples (P < 0.001).


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Fig. 2.   Surface tension of lipid mixtures that were either free of PlsEtn (X) or that contained 8% PlsEtn (black-lozenge ) measured in a pulsating bubble surfactometer. Surface tension is always lower in the PlsEtn-containing samples (P < 0.01).

Using the pulsating bubble surfactometer for measurement of surface tension of mixtures that contain different percentages of PlsEtn (0, 2, 4, 6, and 8%), we were able to show that even the smallest concentrations of PlsEtn (2% of total phospholipids) have an effect on surface properties (Table 1). With increasing amounts of total PlsEtn, the difference in surface tension between the PlsEtn-free mixtures increases linearly (coefficient of linearity r = -0.86).

                              
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Table 1.   Surface tension of lipid mixtures that contain increasing amounts of PlsEtn (ranging between 0 and 8% of total phospholipids) measured in the pulsating bubble surfactometer

Table 2 shows the fatty acid and the dimethyl acetal patterns of plasmalogen-containing and plasmalogen-free PtdEtn, which were used for the phospholipid mixtures. The degree of unsaturation and the patterns of unsaturated fatty acid showed no significant differences. Comparison of the fatty acid and dimethyl acetal patterns before and after surface tension measurement showed no differences (results not shown).

                              
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Table 2.   FA and DMA patterns of plasmalogen-free and plasmalogen-containing PtdEtn used for the PL mixtures

In our study, treatment of rats with clofibrate had an effect on the weight (increase per week 36.5 ± 4.7 g in control animals vs. 26.6 ± 4.9 g in clofibrate treated), serum concentration of triglycerides (0.34 ± 0.13 vs. 0.19 ± 0.11 mmol/l), or cholesterol (1.42 ± 0.3 vs. 1.08 ± 0.1 mmol/l). Analyzing the effect of clofibrate on catalase activity in type II cells, we found increased activity of catalase in clofibrate-treated rats (Table 3). No changes were found regarding the incorporation of [3H]palmitic acid into phospholipids and the incorporation of [14C]hexadecanol into plasmalogens. Clofibrate treatment also had no effect on the spontaneous or terbutaline-stimulated secretion of labeled phospholipids and plasmalogens (Table 3). Spontaneous and terbutaline-stimulated secretion of radiolabeled PlsCho is greater than PlsEtn. As discussed previously (17), the reason could be that PlsCho is secreted as a component of surfactant phospholipids, whereas PlsEtn might be secreted independent from surfactant secretion. Analysis of the distribution of labeled lipids among the lamellar body fractions revealed no significant differences between the label of DPPC, PlsEtn, and PlsCho in either 8,000- or 80,000-g pellets (Table 4). The lamellar body fraction contained 14.1 ± 3.7% (n = 3) of the total cellular [3H]palmitic acid-labeled phospholipids, 11.4 ± 4.8% (n = 3) of the total cellular [14C]hexadecanol-labeled phospholipids, and 3.1 ± 0.8% (n = 3) of total cellular [14C]hexadecanol-labeled plasmalogens.

                              
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Table 3.   Effect of clofibrate treatment of rats on parameters measured in isolated type II pneumocytes

                              
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Table 4.   Relative proportion of palmitic acid-labeled DPPC and hexadecanol-labeled PlsCho and PlsEtn from total in subfractions of LBs isolated from type II cells of clofibrate-treated and control animals

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using the captive bubble surfactometer and pulsating bubble surfactometer in the present study, we were able to show that PlsEtn, a minor component in the pulmonary surfactant system, improves surface tension-lowering properties of a surfactant-like phospholipid mixture (Figs. 1 and 2).

The minimal surface tension obtained by the captive bubble surfactometer (Fig. 1) is lower than that measured by the pulsating bubble surfactometer (Fig. 2). This effect might be caused by the leakage phenomenon that is known for the pulsating bubble surfactometer. However, both methods show a reduction in surface tension in PlsEtn-containing mixtures and can therefore be used to measure the effect of PlsEtn on surface properties of phospholipid mixtures. The advantage of the pulsating bubble surfactometer is the much smaller amount of phospholipid mixture that is needed for measurement (25 vs. 1,000 µl). For this reason, we preferred the pulsating bubble surfactometer after having shown that both methods can be used equally.

Surface properties of natural surfactant or surfactant-like phospholipid mixtures are mainly the result of their ability to form an elastic monolayer at the air-water interface. In the alveoli, the size of the surfactant monolayer is changing during a single breath. In expiration, unsaturated surfactant phospholipids are squeezed out from the monolayer, resulting in an enrichment of DPPC and a further decrease in surface tension. During inspiration, phospholipids from bilayer structures (mainly tubular myelin) in the hypophase enter the monolayer. This transfer requires discontinuities in otherwise stable bilayers (8). It has been assumed that nonbilayer structures, such as hexagonal lipid structures (HII), of tubular myelin might act as the initiation sides of the phospholipid transfer (12, 23). If a sufficient amount of HII promoter phospholipids is present, even phospholipid mixtures with a pattern far from that of natural surfactant show surfactant-like properties (15).

Because the degree of unsaturation of the fatty acid/dimethyl acetal pattern of plasmalogen-free and plasmalogen-containing PtdEtn used for our phospholipid mixtures is well comparable (Table 2) and the handling of the phospholipid mixtures during surface tension measurements did not change the fatty acid pattern, we assume that only the vinyl ether moiety in the sn-1 position of glycerol is responsible for the measured effect of PlsEtn on surface tension.

It has been assumed that the transport of surfactant phospholipids from bilayer structures to the monolayer is unlikely to be a transfer of single phospholipid molecules but rather a cooperative movement of large units of surfactant phospholipids (7, 21). This process requires only few initiation sides in the bilayer structures. Therefore, low concentrations of lipids that promote or stabilize nonbilayer structures, such as PlsEtn, will have a strong effect on surface tension despite a high concentration of DPPC and PtdGro. In accordance with this idea, we were able to show an almost linear decrease (r = -0.86) in surface tension in our phospholipid mixture, with increasing PlsEtn concentrations ranging between 2 and 8 mol% (Table 3). Due to the relatively high standard deviations of the measurement of the surface tension, we assume that the increase in the surface tension from 2 to 4 mol% is accidental.

Because such low plasmalogen concentrations have big effects on surface tension-lowering properties even in the presence of 70 mol% DPPC and 10 mol% PtdGro, we assume that small variations of the plasmalogen concentration affect the surface properties of natural surfactant to a much higher extent than variations of the DPPC or PtdGro concentration. This speculation could be verified by measurement of natural surfactant with different plasmalogen content. Unfortunately, there is no method available to change the plasmalogen concentration in vitro without changing the entire lipid composition. Therefore, we thought that clofibrate treatment might be sufficient to manipulate the plasmalogen content in vivo. Fringes et al. (4) showed by electronic microscopy that clofibrate treatment increases the number of peroxisomes and lamellar bodies in type II pneumocytes of rats. Even though in our experiments clofibrate treatment of rats caused the known clofibrate effects, we did not find any change in incorporation of hexadecanol or secretion of hexadecanol-labeled plasmalogens by type II pneumocytes (Table 3). Fringes et al. (4) showed that the size of the lamellar bodies seems to be affected by clofibrate. Therefore, we measured the content of hexadecanol-labeled plasmalogens in light and heavy lamellar bodies. However, we did not find any clofibrate-induced differences in this lamellar body fraction (Table 4). Although morphological investigations give the indication that clofibrate treatment of rats might increase the plasmalogen concentration of the alveolar surfactant, we did not find any biochemical evidence for this hypothesis. Therefore, up to now, no method is available to increase the amount of plasmalogens in pulmonary surfactant.

Summarizing our results, we were able to show for the first time that a phospholipid, which can be found as a minor component in natural surfactant, reduces significantly the surface tension of a phospholipid mixture containing high concentrations of DPPC and PtdGro. From these results, we assume that plasmalogens are a functionally very important constituent of the alveolar surfactant.

    ACKNOWLEDGEMENTS

This work was supported by Deutsche Forschungsgemeinschaft 517 Ru 1-2 and BMBF Projekt Perinatale Lunge.

    FOOTNOTES

Address for reprint requests: B. Rüstow, Charité Hospital, Dept. of Neonatology, Schumannstr. 20/21, 10098 Berlin, Germany.

Received 10 June 1997; accepted in final form 14 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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