Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 2002 Apr;82(4):2041–2051. doi: 10.1016/S0006-3495(02)75552-5

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers.

Kaushik Nag 1, Jin-Si Pao 1, Robert R Harbottle 1, Fred Possmayer 1, Nils O Petersen 1, Luis A Bagatolli 1
PMCID: PMC1301999  PMID: 11916861

Abstract

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.

Full Text

The Full Text of this article is available as a PDF (842.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bagatolli L. A., Gratton E. A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study. Biophys J. 2000 Jul;79(1):434–447. doi: 10.1016/S0006-3495(00)76305-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bagatolli L. A., Gratton E. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys J. 2000 Jan;78(1):290–305. doi: 10.1016/S0006-3495(00)76592-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bagatolli L. A., Gratton E. Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. Biophys J. 1999 Oct;77(4):2090–2101. doi: 10.1016/S0006-3495(99)77050-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bagatolli L. A., Parasassi T., Gratton E. Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study. Chem Phys Lipids. 2000 Apr;105(2):135–147. doi: 10.1016/s0009-3084(00)00118-3. [DOI] [PubMed] [Google Scholar]
  6. Bagatolli L., Gratton E., Khan T. K., Chong P. L. Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius. Biophys J. 2000 Jul;79(1):416–425. doi: 10.1016/S0006-3495(00)76303-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Batenburg J. J., Haagsman H. P. The lipids of pulmonary surfactant: dynamics and interactions with proteins. Prog Lipid Res. 1998 Sep;37(4):235–276. doi: 10.1016/s0163-7827(98)00011-3. [DOI] [PubMed] [Google Scholar]
  8. Daniels C. B., Orgeig S., Smits A. W., Miller J. D. The influence of temperature, phylogeny, and lung structure on the lipid composition of reptilian pulmonary surfactant. Exp Lung Res. 1996 May-Jun;22(3):267–281. doi: 10.3109/01902149609031775. [DOI] [PubMed] [Google Scholar]
  9. Denicourt N., Tancrède P., Teissié J. The main transition of dipalmitoylphosphatidylcholine monolayers: a liquid expanded to solid condensed high order transformation. Biophys Chem. 1994 Mar;49(2):153–162. doi: 10.1016/0301-4622(93)e0066-e. [DOI] [PubMed] [Google Scholar]
  10. Dietrich C., Bagatolli L. A., Volovyk Z. N., Thompson N. L., Levi M., Jacobson K., Gratton E. Lipid rafts reconstituted in model membranes. Biophys J. 2001 Mar;80(3):1417–1428. doi: 10.1016/S0006-3495(01)76114-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Discher B. M., Maloney K. M., Grainger D. W., Sousa C. A., Hall S. B. Neutral lipids induce critical behavior in interfacial monolayers of pulmonary surfactant. Biochemistry. 1999 Jan 5;38(1):374–383. doi: 10.1021/bi981386h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Discher B. M., Maloney K. M., Schief W. R., Jr, Grainger D. W., Vogel V., Hall S. B. Lateral phase separation in interfacial films of pulmonary surfactant. Biophys J. 1996 Nov;71(5):2583–2590. doi: 10.1016/S0006-3495(96)79450-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dluhy R. A., Reilly K. E., Hunt R. D., Mitchell M. L., Mautone A. J., Mendelsohn R. Infrared spectroscopic investigations of pulmonary surfactant. Surface film transitions at the air-water interface and bulk phase thermotropism. Biophys J. 1989 Dec;56(6):1173–1181. doi: 10.1016/S0006-3495(89)82764-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goerke J. Pulmonary surfactant: functions and molecular composition. Biochim Biophys Acta. 1998 Nov 19;1408(2-3):79–89. doi: 10.1016/s0925-4439(98)00060-x. [DOI] [PubMed] [Google Scholar]
  15. Griese M., Beck J. The interaction of phosphatidylcholine with alveolar type II pneumocytes is dependent on its physical state. Exp Lung Res. 1999 Oct-Nov;25(7):577–594. doi: 10.1080/019021499270033. [DOI] [PubMed] [Google Scholar]
  16. Grunder R., Gehr P., Bachofen H., Schürch S., Siegenthaler H. Structures of surfactant films: a scanning force microscopy study. Eur Respir J. 1999 Dec;14(6):1290–1296. doi: 10.1183/09031936.99.14612909. [DOI] [PubMed] [Google Scholar]
  17. Gustafsson M., Curstedt T., Jörnvall H., Johansson J. Reverse-phase HPLC of the hydrophobic pulmonary surfactant proteins: detection of a surfactant protein C isoform containing Nepsilon-palmitoyl-lysine. Biochem J. 1997 Sep 15;326(Pt 3):799–806. doi: 10.1042/bj3260799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hawco M. W., Davis P. J., Keough K. M. Lipid fluidity in lung surfactant: monolayers of saturated and unsaturated lecithins. J Appl Physiol Respir Environ Exerc Physiol. 1981 Aug;51(2):509–515. doi: 10.1152/jappl.1981.51.2.509. [DOI] [PubMed] [Google Scholar]
  19. Hollars C. W., Dunn R. C. Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy. Biophys J. 1998 Jul;75(1):342–353. doi: 10.1016/S0006-3495(98)77518-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jørgensen K., Sperotto M. M., Mouritsen O. G., Ipsen J. H., Zuckermann M. J. Phase equilibria and local structure in binary lipid bilayers. Biochim Biophys Acta. 1993 Oct 10;1152(1):135–145. doi: 10.1016/0005-2736(93)90240-z. [DOI] [PubMed] [Google Scholar]
  21. Keough K. M., Farrell E., Cox M., Harrell G., Taeusch H. W., Jr Physical, chemical, and physiological characteristics of isolates of pulmonary surfactant from adult rabbits. Can J Physiol Pharmacol. 1985 Sep;63(9):1043–1051. doi: 10.1139/y85-171. [DOI] [PubMed] [Google Scholar]
  22. Korlach J., Schwille P., Webb W. W., Feigenson G. W. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8461–8466. doi: 10.1073/pnas.96.15.8461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mathivet L., Cribier S., Devaux P. F. Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys J. 1996 Mar;70(3):1112–1121. doi: 10.1016/S0006-3495(96)79693-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Menger F. M., Keiper J. S. Chemistry and physics of giant vesicles as biomembrane models. Curr Opin Chem Biol. 1998 Dec;2(6):726–732. doi: 10.1016/s1367-5931(98)80110-5. [DOI] [PubMed] [Google Scholar]
  25. Nag K., Perez-Gil J., Ruano M. L., Worthman L. A., Stewart J., Casals C., Keough K. M. Phase transitions in films of lung surfactant at the air-water interface. Biophys J. 1998 Jun;74(6):2983–2995. doi: 10.1016/S0006-3495(98)78005-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parasassi T., De Stasio G., Ravagnan G., Rusch R. M., Gratton E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys J. 1991 Jul;60(1):179–189. doi: 10.1016/S0006-3495(91)82041-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parasassi T., De Stasio G., d'Ubaldo A., Gratton E. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys J. 1990 Jun;57(6):1179–1186. doi: 10.1016/S0006-3495(90)82637-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parasassi T., Gratton E., Yu W. M., Wilson P., Levi M. Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys J. 1997 Jun;72(6):2413–2429. doi: 10.1016/S0006-3495(97)78887-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Piknova B., Schief W. R., Vogel V., Discher B. M., Hall S. B. Discrepancy between phase behavior of lung surfactant phospholipids and the classical model of surfactant function. Biophys J. 2001 Oct;81(4):2172–2180. doi: 10.1016/S0006-3495(01)75865-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Postle A. D., Mander A., Reid K. B., Wang J. Y., Wright S. M., Moustaki M., Warner J. O. Deficient hydrophilic lung surfactant proteins A and D with normal surfactant phospholipid molecular species in cystic fibrosis. Am J Respir Cell Mol Biol. 1999 Jan;20(1):90–98. doi: 10.1165/ajrcmb.20.1.3253. [DOI] [PubMed] [Google Scholar]
  31. Rouser G., Fkeischer S., Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May;5(5):494–496. doi: 10.1007/BF02531316. [DOI] [PubMed] [Google Scholar]
  32. Schürch S., Green F. H., Bachofen H. Formation and structure of surface films: captive bubble surfactometry. Biochim Biophys Acta. 1998 Nov 19;1408(2-3):180–202. doi: 10.1016/s0925-4439(98)00067-2. [DOI] [PubMed] [Google Scholar]
  33. Veldhuizen R., Nag K., Orgeig S., Possmayer F. The role of lipids in pulmonary surfactant. Biochim Biophys Acta. 1998 Nov 19;1408(2-3):90–108. doi: 10.1016/s0925-4439(98)00061-1. [DOI] [PubMed] [Google Scholar]
  34. Worthman L. A., Nag K., Davis P. J., Keough K. M. Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy. Biophys J. 1997 Jun;72(6):2569–2580. doi: 10.1016/S0006-3495(97)78900-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Worthman L. A., Nag K., Rich N., Ruano M. L., Casals C., Pérez-Gil J., Keough K. M. Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract. Biophys J. 2000 Nov;79(5):2657–2666. doi: 10.1016/S0006-3495(00)76504-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yu S., Harding P. G., Smith N., Possmayer F. Bovine pulmonary surfactant: chemical composition and physical properties. Lipids. 1983 Aug;18(8):522–529. doi: 10.1007/BF02535391. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES