1Clinic for Neonatology, and 2Institute for Medical Physics and Biophysics, Charité Campus Mitte, Berlin, Germany
Submitted 17 May 2004 ; accepted in final form 13 October 2004
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ABSTRACT |
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plasmalogens; cholesterol; surface-active agents
Plasmalogens and cholesterol, two minor surfactant lipids, promote the formation of hexagonal lipid (HII) structures (26, 33) and thus would support the formation of a surface-associated reservoir. We have shown that addition of only 2 mol% plasmalogens or cholesterol (up to 10 mol%) to a surfactant-like lipid mixture causes a decrease in surface tension and surface viscosity (20, 30, 31). Combination of cholesterol and surfactant protein (SP)-B increases viscosity, an effect that is compensated by addition of plasmalogens (31). Interestingly, plasmalogens do not only improve surface properties, but a high plasmalogen content (about 3.5 mol%) in tracheal aspirates at birth is also associated with a lower incidence in chronic lung disease in preterm infants (23).
Exogenous surfactant substitution represents the standard therapy of respiratory distress syndrome (RDS). Naturally derived surfactants are most often used clinically and consist mainly of lipid extracts from bovine or porcine lungs with a varying amount of lipophilic surfactant proteins. Analysis of molecular species of phosphatidylcholine (PC) revealed significant differences between various commercial surfactant preparations (2). However, no data concerning the content of surfactant minor lipids that promote formation of hexagonal structures are available. Furthermore, it would be of clinical interest to know the surface viscosity of different surfactant preparations, since viscosity represents an important surfactant property.
Thus the present study was performed with the aim to determine surface viscosity and concentrations of the minor lipid components of three different, naturally derived commercial surfactant preparations.
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MATERIALS AND METHODS |
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The chemicals used in the study were of highest purity and obtained from Merck (Darmstadt, Germany). By addition of 14.7 mol DPPC (Sigma, Deisenhofen, Germany), 24.6 mol egg PC used as monoene PC (Sigma), and 17.8 mol 18:0/20:4 PC (Sigma) to 11 mol Survanta-PL, we obtained an artificial surfactant-lipid mixture (Pseudo-Curosurf) that was, in respect to PL pattern and relative proportion of neutral lipids, very similar to Curosurf 120 (see Table 1).
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To analyze lipid composition, lipid extracts were separated by thin-layer chromatography (TLC). For the separation of diacylglycerol (DG), TG, free fatty acids (FFA), and total PL, hexane-ether-acetic acid (80:20:2, vol/vol) was used as solvent. PC and phosphatidylethanolamine (PE) were separated by TLC with chloroform-methanol-water (130:50:8, vol/vol) as solvent. The individual lipids were identified according to the chromatographic properties of authentic lipid standards. After TLC, the areas on the silica gel slides, which contained the individual lipids, were scraped off, and a known concentration of margarinic acid (C17) was added to silica gel as standard.
Thereafter the individual lipids were quantitatively transformed to fatty acid methyl ester (FAME) and dimethyl acetals (DMA), which were separated by gas chromatography as previously described (24). From the FAME pattern of the glycerolipids, the total concentration of PL, DG, and TG was calculated as sum of nmol FAME divided by 2. The mol% of disaturated, monoene, and polyunsaturated PL were calculated from the sum of the appropriate FAME. Plasmalogens were calculated from the sum of the DMA. DPPC (mol%) or disaturated PL (mol%) were calculated from the content of palmitic acid (16:0) or the sum of saturated FA in the FAME patterns of isolated PC or PL, respectively, according to the following formula: the sum of saturated FA minus the sum of unsaturated FA divided by 2.
Analysis of surfactant-associated proteins. Content of surfactant-associated proteins was quantitatively estimated by SDS-electrophoresis in 15% T-; 2.6% C-polyacrylamide gels (11). Proteins were detected by sensitive silver staining (4).
Biophysical methods. Surface tension and surface viscosity of a surface film were simultaneously determined with the method of oscillating drop surfactometer (ODS) as previously described (13, 30).
The ODS uses the harmonic oscillation of a pendant drop. Microdrops of the lipid extracts of each surfactant were applied to the surface of the oscillating drop. The PL concentration in samples was 0.26 µM/ml, and chloroform-methanol (2:1 vol/vol) was used as solvent. After evaporation of the microdrop solvent, the changes of surface viscosity are representative for biophysical properties of the surface film, depending on the appropriate surface tension. All experiments were performed with water in the subphase, with the same amplitude of oscillation and at 20 ± 1°C.
Statistics. All values are shown as means with SD of at least n = 3 experiments.
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RESULTS |
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Disaturated PL contain DPPC as major component and disaturated species of phosphatidylglycerol and phosphatidylinositol as minor components. The content of disaturated PL differs strongly among the commercial surfactant preparations. Disaturated PL was the major PL fraction only in Survanta (66 mol% of total PL); in Curosurf and Alveofact the amount of monoene PL exceeds that of disaturated PL significantly.
Accordingly, the proportion of DPPC in total PC was highest in Survanta (39 mol%) and significantly lower in Curosurf (25 mol%) and in Alveofact (17 mol%) (Table 2).
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The content of plasmalogen was highest in Curosurf, where it was 2.5 times higher than in Survanta and four times higher than in Alveofact (Table 1).
The cholesterol content of the commercial surfactant preparations is extremely different. The cholesterol-total PL ratio was highest in Alveofact (100 x 103) followed by Survanta (15 x 103); almost no cholesterol was found in Curosurf (0.8 x 103).
Curosurf and Survanta differed strongly in the relative proportion of NL and in the content and composition of PL (Table 1). By adding different PL to Survanta, we obtained an artificial surfactant lipid mixture (Pseudo-Curosurf) similar to Curosurf in respect to PL composition and relative proportion of NL. The plasmalogen content of Pseudo-Curosurf was <0.2%; the composition of the neutral lipid fraction was identical to the Survanta NL fraction.
Content of surfactant-associated proteins. Recently, Bernhard et al. (2) published quantitative data of the concentration of surfactant-specific proteins in commercial surfactant preparations.
To validate the presence of SP-B and SP-C in surfactant preparations qualitatively, surfactant proteins were silver stained after electrophoretic separation of lipid extracts. The highest amount of SP-B was found in Curosurf followed by Alveofact, whereas the band of SP-B was missing in Survanta. For SP-C the highest amount was found in lipid extract of Survanta. The amount of SP-C was lower in lipid extracts of Curosurf and Alveofact. These qualitative data are in accordance with the quantitative data (2).
Biophysical properties of commercial surfactant preparations. Lipid extracts of all surfactant preparations showed a decreasing surface tension with an increasing amount of lipids on the surface of the oscillating drop. However, we found great differences in biophysical properties when comparing the different commercial surfactant preparations.
The surface viscosity data are presented as a function of surface tension (Fig. 1).
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For Alveofact (high concentration of cholesterol, small amount of plasmalogens), the increase in surface viscosity started at a surface tension of 25 mN/m, surface viscosity remained <10 x 106 kg/s (Fig. 1).
Curosurf (highest plasmalogen level, lowest cholesterol content) showed only a small increase of surface viscosity below surface tensions of 25 mN/m; viscosity did not reach 5 x 106 kg/s (Fig. 1).
The biophysical properties of Pseudo-Curosurf compared with Curosurf and Survanta are shown in Fig. 2. Similar to Curosurf, low surface tensions without a rise in viscosity were achieved with Pseudo-Curosurf.
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DISCUSSION |
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Surface viscosity, an important biophysical property of surfactant. Surfactant has been considered to prevent end-expiratory alveolar collapse by forming a monolayer at the air-liquid interface. For end-expiratory stability of alveoli, a low surface tension is required, and thus quality of surfactant was mainly judged according to its surface tension-lowering properties. However, a more complex model that describes surfactant activity better has been suggested recently (25). According to the concept of a surface-associated reservoir, a low surface viscosity is required at low surface tensions (22).
Comparison of the three different surfactant preparations revealed significant differences with regard to surface viscosity. Whereas surface viscosity significantly rose with decreasing surface tension in Survanta, lower surface tensions without an increase in viscosity were achieved with Curosurf.
We recently showed that lipid mixtures that contain nonbilayer structure-promoting lipids achieve low surface tensions without a rise in viscosity (30, 31). The addition of only 2 mol% plasmalogens, a very potent promoter and stabilizer of HII structures (12), to a surfactant-like lipid mixture is sufficient to reduce surface tension and viscosity (20). In the present study, Curosurf had the highest concentration of plasmalogens; 3.8 mol% of all PL are plasmalogens. The high plasmalogen concentration could explain low surface viscosity of Curosurf. By adding different PL to the lipid mixture of Survanta we obtained a nearly plasmalogen-free Curosurf-like mixture containing a high amount of PUFA-PL. The surface properties were similar to Curosurf. From the results, it can be concluded that either plasmalogens or PUFA-PL are required for low surface viscosity. However, plasmalogens are more effective than PUFA-PL, since 3 mol% of plasmalogens or 26 mol% of PUFA-PL, respectively, are sufficient to achieve a low surface viscosity (30).
Cholesterol also supports formation of nonbilayer structures (15) and reduces surface viscosity (31); however, higher concentrations are required. Furthermore, the presence of SP-B also lowers surface viscosity of lipid mixtures (31). In accordance with these in vitro data, Survanta, the surfactant preparation with the lowest content of plasmalogen, cholesterol, PUFA-PL, and SP-B, showed an increase in surface viscosity at high surface tension.
However, the impact of a single component on surface viscosity of a complex lipid mixture has to be interpreted with care. Addition of each of the three minor components (plasmalogen, cholesterol, and SP-B) to a lipid mixture causes low surface viscosity at low surface tensions (30, 31). The combination of SP-B and plasmalogen further reduced viscosity, whereas SP-B increased the viscosity in the presence of cholesterol, an effect we reversed by adding plasmalogens (31).
Lipid minor components and PUFA-PL of surfactant. Lecithin has been considered as most important for surface properties of surfactant. However, addition of small amounts of plasmalogens or cholesterol to lipid mixtures will have a greater impact on surface properties than large amounts of DPPC (1). Even in artificial lipid mixtures that contain DPPC as a minor component, low surface tensions are achieved if HII structure-supporting lipids are present (17). When tested in vivo in a neonatal rabbit model, such lipid mixtures elicited the same effect as native human surfactant, indicating that lipid polymorph phase behavior is very important for the biological function of surfactant (17). An endogenous variation in the composition of minor lipids could be a mechanism that supports an adaptation to physiological requirements (7, 8, 16).
Clinical data further support the importance of minor surfactant lipids and PUFA-PL. Our group has shown that the concentration of plasmalogens and PUFA-PL in tracheal aspirates at birth correlates with the subsequent development of bronchopulmonary dysplasia (BPD) (23). Preterm infants who did not develop BPD had a significantly higher PUFA-PL (26 ± 9 mol%) and plasmalogen (3.5 ± 1.2 mol%) content at birth than term infants or preterm infants who subsequently developed BPD (14 ± 4 vs. 1.8 ± 0.9 mol%). In the present study the highest plasmalogen and PUFA-PL content (3.8 mol% and 26 mol% of total PL) was found in Curosurf.
Clinical relevance. Good surface properties of surfactants are considered as a prerequisite for clinical efficacy. Differences in surface viscosity could affect the clinical response to surfactant therapy. But preliminary data of a retrospective study comparing Curosurf and Alveofact do not show a difference in short-term effects such as oxygenation, duration of ventilation, etc. (21). Comparison of Curosurf and Survanta revealed a greater arterial-alveolar oxygen tension ratio and lower ventilatory requirements in the Curosurf group (28). The data, however, have to be interpreted with care, since the recommended dosage of Curosurf is 200 mg/kg, whereas only 100 mg/kg of Survanta was administered. Retrospective analysis of infants with group B Streptococci pneumonia did not reveal any differences in short-term outcome between Alveofact, Survanta, or Curosurf treatment (9).
The limited clinical data seem to suggest that differences in surface properties of surfactant preparation are less important for short-term in vivo efficacy. Further prospective clinical trials are required to decide whether a low surface viscosity of exogenous surfactant preparations will improve clinical outcome of treated patients.
Other surfactant properties. During recent years, properties of surfactant other than the biophysical are being considered as clinically important. Research was aimed to develop surfactant preparations that are less sensitive to exogenous inhibition of surface activity (10). For natural surfactant preparations, differences were found with regard to the sensitivity to inhibition (27). Animal and clinical studies have suggested an immunomodulatory effect of surfactant (32), showing significant differences between the effect of Curosurf, Survanta, and Alveofact (29). In association with immunomodulatory effects, bactericidal properties of surfactant preparations were discussed. Survanta seemed to accelerate the growth of Escherichia coli in vitro (14, 19). Furthermore, in the presence of Alveofact, bacterial numbers declined, and Curosurf clearly exhibited bactericidal properties in a dose-dependent manner (19).
These effects could be explained by differences in surfactant protein content but also by different lipid composition. Further research will be necessary to understand the effects of single surfactant component and, more importantly, the complex interaction in the surfactant mixture.
In summary, a great variation concerning the in vivo and in vitro effects of surfactant preparations were found; however, no sufficient explanation has been given yet. In the present study we describe striking differences in composition of lipid minor components and surface viscosity for the first time. The different lipid composition could explain some of the differences in function. To further understand the impact of each component of the complex surfactant system, further studies are required. Nevertheless, the importance of some minor components has to be considered in the design of new synthetic surfactant preparations. Not only surfactant-associated proteins, but also lipids are of importance. Furthermore, investigators should consider viscosity as well as surface tension when evaluating properties of surfactant preparations.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. Rüdiger and A. Tölle contributed equally to this work.
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REFERENCES |
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