Naturally derived commercial surfactants differ in composition of surfactant lipids and in surface viscosity

Mario Rüdiger,1,* Angelika Tölle,1,* Wolfgang Meier,2 and Bernd Rüstow1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary surfactant biophysical properties are best described by surface tension and surface viscosity. Besides lecithin, surfactant contains a variety of minor lipids, such as plasmalogens, polyunsaturated fatty acid-containing phospholipids (PUFA-PL), and cholesterol. Plasmalogens and cholesterol improve surface properties of lipid mixtures significantly. High PUFA-PL and plasmalogen content in tracheal aspirate of preterm infants reduces the risk of developing chronic lung disease. Different preparations are available for exogenous surfactant substitution; however, little is known about lipid composition and surface viscosity. Thus lipid composition and surface properties (measured by oscillating drop surfactometer) of three commercial surfactant preparations (Alveofact, Curosurf, Survanta) were compared. Lipid composition exhibited strong differences: Survanta had the highest proportion of disaturated PL and total neutral lipids and the lowest proportion of PUFA-PL. Highest plasmalogen and PUFA-PL concentrations were found in Curosurf (3.8 ± 0.1 vs. 26 ± 1 mol%) compared with Alveofact (0.9 ± 0.3 vs. 11 ± 1) and Survanta (1.5 ± 0.2 vs. 6 ± 1). In Survanta samples, viscosity increased >8 x 10–6 kg/s at surface tension of 30 mN/m. Curosurf showed only slightly increased surface viscosity below surface tensions of 25 mN/m, and viscosity did not reach 5 x 10–6 kg/s. By adding defined PL to Survanta, we obtained a Curosurf-like lipid mixture (without plasmalogens) that exhibited biophysical properties like Curosurf. Different lipid compositions could explain some of the differences in surface viscosity. Therefore, PL pattern and minor surfactant lipids are important for biophysical activity and should be considered when designing synthetic surfactant preparations.

plasmalogens; cholesterol; surface-active agents


THE ALVEOLAR AIR-LIQUID INTERFACE is covered by pulmonary surfactant (6). The size of the surfactant layer changes during the respiratory cycle. Surfactant-phospholipids (PL) enter the layer during inspiration. Upon compression (expiration), predominantly nonlecithin PLs are squeezed out. The "squeeze out" is thought to cause an enrichment of the layer with lecithin. Subsequently, surface tension is reduced and end-expiratory alveolar collapse is prevented (6). This explanation of surfactant activity represents a helpful model; however, it is most likely an oversimplification, since physicochemical properties of the major surfactant-PL fraction are not in accordance with the predicted requirements of the squeeze-out model (22). Recently, the concept of a "surface-associated reservoir" was suggested as an improved model of surfactant function (18, 25). According to that concept, large areas of the surfactant layer are folded into the subphase during expiration, however, remaining adherent with the monolayer. Lipids that promote and stabilize the formation of nonbilayer structures are required to connect the surfactant layers of the subphase with the monolayer at the air-liquid interface and to serve as an initiation site for folding and re-integration (22). The squeeze-out model predicts a rigid lipid surfactant film at end-expiration with high surface viscosity (1). In contrast, the film would be less rigid (e.g., low surface viscosity) if the monolayer remained connected with a surface-associated reservoir. Thus not only surface tension but also surface viscosity represents an important parameter of surfactant activity (22).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Three different, commercially available surfactant preparations that are used for therapy of RDS were analyzed: Curosurf 120 (Nycomed Pharma, Ismaning, Germany) is a lipid extract from whole minced porcine lung tissue. Survanta (Abbott, Wiesbaden, Germany) is prepared from minced bovine lung extract with added dipalmitoyl phosphatidylcholine (DPPC), triacylglyerol (TG), and palmitic acid. Alveofact (Boehringer Ingelheim Pharma, Ingelheim, Germany) is produced by lipid extraction from bovine lung lavage.

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).


View this table:
[in this window]
[in a new window]
 
Table 1. Composition of glycerolipids isolated from Survanta, Curosurf, and Alveofact

 
Analysis of lipids. Lipids were analyzed as described previously (24). In short, aliquots of the individual surfactant suspensions were extracted according to Bligh and Dyer (3). The quantity of lipids was determined by phosphate determination (5). To measure the cholesterol content, we used a commercially available test kit from Merck (Darmstadt, Germany).

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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid patterns of surfactant preparations. The lipid composition of the commercial surfactant preparation is shown in Table 1. The relative proportion of total neutral lipids (NL) differs by a factor of 4; Survanta (44 mol%) contained the highest and Curosurf (11 mol%) contained the lowest proportion of NL. The major fraction in all surfactant NL was FFA, with the highest level in Survanta (80 mol% of total NL) and the lowest level in Curosurf (54 mol% of total NL).

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).


View this table:
[in this window]
[in a new window]
 
Table 2. Fatty acid patterns of PC and PE isolated from Survanta, Curosurf, and Alveofact

 
Unsaturated lipids, in particular polyunsaturated FA (PUFA)-containing PL, serve as a liquidifier of disaturated PL at body temperature and increase the fluidity of lipid layers. As Table 1 shows, Curosurf contains by far the highest concentration of PUFA-PL, whereas Alveofact contains the highest proportion of monoene PL (PL containing fatty acids with one double bond). These results are corroborated by the FA patterns of PC (most abundant PL in surfactant) and PE (most unsaturated PL in surfactant). Both PC and PE of Curosurf showed the highest level of PUFA-PL (8.0 and 41.8 mol%), whereas the PUFA-PL concentration was clearly lower in Survanta (1.7 and 17.8 mol%) and Alveofact (4.2 and 18.3 mol%).

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 10–3) followed by Survanta (15 x 10–3); almost no cholesterol was found in Curosurf (0.8 x 10–3).

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).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Relationship between surface tension ({gamma}) and damping constant (b) of natural surfactant preparations measured with an oscillating drop surfactometer. The drop was covered with Survanta ({blacktriangleup}), with Alveofact ({triangledown}), or with Curosurf ({bullet}). The bars show SD of at least 3 independent experiments (n = 3).

 
Samples obtained from Survanta (with a low content of cholesterol, plasmalogen, and SP-B) showed a dramatic increase of surface viscosity at surface tension of 30 mN/m (Fig. 1). The surface viscosity increased above 18 x 10–6 kg/s.

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 10–6 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 10–6 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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Relationship between {gamma} and b of Survanta ({blacktriangleup}), Curosurf ({bullet}), and Pseudo-Curosurf ({triangledown}) measured with the oscillating drop surfactometer. The bars show SD of at least 3 independent experiments (n = 3).

 
Comparison of viscosity and proportion of saturated PL in relation to plasmalogen or PUFA-PL, respectively, shows that viscosity was highest when saturated PL were high and plasmalogens or PUFA-PL were low (Survanta). In contrast, Curosurf contains a low proportion of saturated PL but a high proportion of plasmalogens and PUFA-PL and exhibited low viscosity. Similar low viscosity was found in Pseudo-Curosurf with a high proportion of PUFA-PL. The values measured in Alveofact were between Survanta and Curosurf.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, the concentration of plasmalogens, PUFA-PL, and cholesterol was determined in three naturally derived, commercial surfactant preparations. Furthermore, an important biophysical property, the surface viscosity, was measured. Significant differences regarding lipid composition and surface viscosity were found between the studied surfactant preparations.

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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Rüstow, Clinic for Neonatology, Charité-Mitte; Schumannstr. 21, 10098 Berlin, Germany (E-mail: bernd.ruestow{at}charite.de)

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. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bangham AD, Morley C, and Phillips MC. The physical properties of an effective lung surfactant. Biochim Biophys Acta 573: 552–556, 1979.[ISI][Medline]
  2. Bernhard W, Mottaghian J, Gebert A, Rau GA, von der Hardt H, and Poets C. Commercial versus native surfactants Surface activity, molecular components, and the effect of calcium. Am J Respir Crit Care Med 162: 1524–1533, 2000.[Abstract/Free Full Text]
  3. Bligh ER and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem 37: 911–917, 1959.[ISI]
  4. Blum H, Beier H, and Gross HJ. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99, 1987.[ISI]
  5. Chen PS, Tonibara TY, and Warner H. Microdetermination of phosphorus. Anal Chem 28: 1758–1762, 1956.
  6. Clements JA, Hustead RF, Johnson RP, and Gribetz I. Pulmonary surface tension and alveolar stability. J Appl Physiol 16: 444–450, 1961.[ISI]
  7. Daniels CB, Barr HA, Power JH, and Nicholas TE. Body temperature alters the lipid composition of pulmonary surfactant in the lizard Ctenophorus nichalis. Exp Lung Res 16: 435–449, 1990.[ISI][Medline]
  8. Doyle IR, Jones ME, Barr HA, Orgeig S, Crockett AJ, McDonald CF, and Nicholas TE. Composition of human pulmonary surfactant varies with exercise and level of fitness. Crit Care Med 149: 1619–1627, 1994.
  9. Herting E, Gefeller O, Land M, van Sonderen L, Harms K, and Robertson B. Surfactant treatment of neonates with respiratory failure and group B streptococcal infection. Pediatrics 106: 957–964, 2000.[Abstract/Free Full Text]
  10. Herting E, Rauprich P, Stichtenoth G, Walter G, Johansson J, and Robertson B. Resistance of different surfactant preparations to inactivation by meconium. Pediatr Res 50: 44–49, 2001.[Abstract/Free Full Text]
  11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[ISI][Medline]
  12. Lohner K, Balgavy P, Hermetter A, Paltauf F, and Laggner P. Stabilization of non-bilayer structures by the etherlipid ethanolamine plasmalogen. Biochim Biophys Acta 1061: 132–140, 1991.[ISI][Medline]
  13. Meier W, Greune G, Meyboom A, and Hofmann KP. Surface tension and viscosity of surfactant from the resonance of an oscillating drop. Eur Biophys J 29: 113–124, 2000.[CrossRef][ISI][Medline]
  14. Neumeister B, Woerndle S, and Bartmann P. Effects of different surfactant preparations on bacterial growth in vitro. Biol Neonate 70: 128–134, 1996.[ISI][Medline]
  15. Notter RH, Tabak SA, and Mavis RD. Surface properties of binary mixtures of some pulmonary surfactant components. J Lipid Res 21: 10–22, 1980.[Abstract]
  16. Orgeig S, Barr HA, and Nicholas TE. Effect of hyperpnea on the cholesterol to disaturated phospholipid ratio in alveolar surfactant of rats. Exp Lung Res 21: 157–174, 1995.[ISI][Medline]
  17. Perkins WR, Dause RB, Parente RA, Minchey SR, Neuman KC, Gruner SM, Taraschi TF, and Janoff AS. Role of lipid polymorphism in pulmonary surfactant. Science 273: 330–332, 1996.[Abstract]
  18. Post A, Nahmen AV, Schmitt M, Ruths J, Riegler H, Sieber M, and Galla HJ. Pulmonary surfactant protein C containing lipid films at the air-water interface as a model for the surface of lung alveoli. Mol Membr Biol 12: 93–99, 1995.[ISI][Medline]
  19. Rauprich P, Moller O, Walter G, Herting E, and Robertson B. Influence of modified natural or synthetic surfactant preparations on growth of bacteria causing infections in the neonatal period. Clin Diagn Lab Immunol 7: 817–822, 2000.[Abstract/Free Full Text]
  20. Rüdiger M, Kolleck I, Putz G, Stevens P, Wauer RR, and Rüstow B. Plasmalogens effectively reduce the surface tension of surfactant-like phospholipid mixtures. Am J Physiol Lung Cell Mol Physiol 274: L143–L148, 1998.[Abstract/Free Full Text]
  21. Rüdiger M, Proquitté H, Dushe T, Meier W, Tölle A, Schmalisch G, Rüstow B, and Wauer RR. Two commercial surfactant preparations-biochemical & clinical differences (Abstract). Pediatr Res 54: 570, 2003.
  22. Rüdiger M, Tölle A, Meier W, and Rüstow B. Effect of minor components of alveolar surfactant lipids on surface properties. In: Recent Research Developments in Chemistry and Physics of Lipids, Trivandrum, India: Transworld Research Network, 2003, p. 1–14.
  23. Rüdiger M, von Baehr A, Haupt R, Wauer RR, and Rüstow B. Preterm infants with high polyunsaturated fatty acid and plasmalogen content in tracheal aspirates do develop bronchopulmonary dysplasia less often. Crit Care Med 28: 1572–1577, 2000.[CrossRef][ISI][Medline]
  24. Rüstow B, Kolleck I, Guthmann F, Haupt R, and Stevens P. Synthesis and secretion of plasmalogens by type-II pneumocytes. Biochem J 302: 665–668, 1994.[ISI][Medline]
  25. Schürch S, Qanbar R, Bachofen H, and Possmayer F. The surface-associated surfactant reservoir in the alveolar lining. Biol Neonate 67: 61–76, 1995.[ISI][Medline]
  26. Seddon JM, Cevc G, Kaye RD, and Marsh D. X-ray diffraction study of the polymorphism of hydrated diacyl- and dialkylphosphatidylethanolamines. Biochemistry 23: 2634–2644, 1984.[ISI][Medline]
  27. Seeger W, Grube C, Gunther A, and Schmidt R. Surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations. Eur Respir J 6: 971–977, 1993.[Abstract]
  28. Speer CP, Gefeller O, Groneck P, Laufkötter E, Roll C, Hanssler L, Harms K, Herting E, Boenisch H, Windeler J, and Robertson B. Randomised clinical trial of two treatment regimes of natural surfactant preparations in neonatal respiratory distress syndrome. Arch Dis Child 72: F8–F13, 1995.[ISI]
  29. Tegtmeyer FK, Gortner L, Ludwig A, and Brandt E. In vitro modulation of induced neutrophil activation by different surfactant preparations. Eur Respir J 9: 752–757, 1996.[Abstract/Free Full Text]
  30. Tölle A, Meier W, Greune G, Rüdiger M, Hofmann KP, and Rüstow B. Plasmalogens reduce the viscosity of surfactant-like phospholipid monolayer. Chem Phys Lipids 100: 81–87, 1999.[CrossRef][ISI]
  31. Tölle A, Meier W, Rüdiger M, Hofmann KP, and Rüstow B. Effect of cholesterol and surfactant protein B on the viscosity of phospholipid mixtures. Chem Phys Lipids 114: 159–168, 2002.[CrossRef][ISI][Medline]
  32. Woerndle S and Bartmann P. The effect of three surfactant preparations on in vitro lymphocyte functions. J Perinat Med 22: 119–128, 1994.[ISI][Medline]
  33. Yeagle PL. Hydration and the lamellar to hexagonal II phase transition of phosphatidylethanolamine. Biochemistry 25: 7518–7522, 1986.[ISI][Medline]