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. 2006 Nov 24;12(1):51–69. doi: 10.2478/s11658-006-0059-6

Spectroscopic studies of D-α-tocopherol concentration-induced transformation in egg phosphatidylcholne vesicles

Krzysztof Dwiecki 1, Paweł Górnas 1, Agnieszka Wilk 3, Małgorzata Nogala-Kałucka 2,4, Krzysztof Polewski 1,✉
PMCID: PMC6275860  PMID: 17124545

Abstract

The effects of embedding up to 60 mol% of α-tocopherol (α-Toc) on the morphology and structure of the egg phosphatidylcholine (PC) membrane were studied using spectroscopic techniques. The resulting vesicles were subjected to turbidometric and dynamic light scattering measurements to evaluate their size distribution. The α-Toc intrinsic fluorescence and its quenching was used to estimate the tocopherol position in the membrane. Optical microscopy was used to visualize morphological changes in the vesicles during the inclusion of tocopherol into the 2 mg/ml PC membrane. The incorporation of up to 15 mol% of tocopherol molecules into PC vesicles is accompanied by a linear increase in the fluorescence intensity and the simultaneous formation of larger, multilamellar vesicles. Increasing the tocopherol concentration above 20 mol% induced structural and morphological changes leading to the disappearance of micrometer-sized vesicles and the formation of small unilamellar vesicles of size ranging from 30 to 120 nm, mixed micelles and non-lamellar structures.

Key words: Egg phosphatidylcholine, Tocopherol, Fluorescence, Vesicles, Optical microscopy, Dynamic light scattering

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Abbreviations used

α-Toc

D-α-tocopherol

ACR

acrylamide

DLS

dynamic light scattering

DMPC

dimyristoyl phosphatidylcholine

DPPC

dipalmitoyl phosphatidylcholine

GUV

giant unilamellar vesicle

GMV

giant multilamellar vesicle

GLV

giant lamellar vesicle

KI

potassium iodide

KD

dynamic fluorescence quenching constant

KSV

Stern-Volmer quenching constant

LMV

large multilamellar vesicle

NMR

nuclear magnetic resonance

PC

L-α-phosphatydylcholine

SSBV

small single-bilayer vesicle

SUV

small unilamellar vesicle

V

static fluorescence quenching constant

References

  • 1.Ortiz A., Aranda F.J., Gomez-Fernandez J.C. A differential scanning calorimetry study of the interaction of alpha-tocopherol with mixtures of phospholipids. Biochim. Biophys. Acta. 1987;898:214–222. doi: 10.1016/0005-2736(87)90040-X. [DOI] [PubMed] [Google Scholar]
  • 2.Ortiz A., Villalain J., Gomez-Fernandez J.C. Interaction of diacylglycerols with phosphatidylcholine vesicles as studied by differential scanning calorimetry and fluorescence probe depolarization. Biochemistry. 1988;27:9030–9036. doi: 10.1021/bi00425a022. [DOI] [PubMed] [Google Scholar]
  • 3.Walke M., Beckert D., Lasch J. Interaction of UV light-induced alphatocopherol radicals with lipids detected by an electron spin resonance prooxidation effect. Photochem. Photobiol. 1998;68:502–510. doi: 10.1562/0031-8655(1998)068<0502:IOULIT>2.3.CO;2. [DOI] [PubMed] [Google Scholar]
  • 4.Aranda F.J., Sanchez-Migallon M.P., Gomez-Fernandez J.C. Influence of alpha-tocopherol incorporation on Ca(2+)-induced fusion of phosphatidylserine vesicles. Arch. Biochem. Biophys. 1996;333:394–400. doi: 10.1006/abbi.1996.0406. [DOI] [PubMed] [Google Scholar]
  • 5.Grau A., Ortiz A. Dissimilar protection of tocopherol isomers against membrane hydrolysis by phospholipase A2. Chem. Phys. Lipids. 1998;91:109–118. doi: 10.1016/S0009-3084(97)00101-1. [DOI] [PubMed] [Google Scholar]
  • 6.Gutierrez M.E., Garcia A.F., de Africa Madariaga M., Sagrista M.L., Casado F.J., Mora M. Interaction of tocopherols and phenolic compounds with membrane lipid components: evaluation of their antioxidant activity in a liposomal model system. Life Sci. 2003;72:2337–2360. doi: 10.1016/S0024-3205(03)00120-6. [DOI] [PubMed] [Google Scholar]
  • 7.Chen C.S., Patterson M.C., Wheatley C.L., O’Brien J.F., Pagano R.E. Broad screening test for sphingolipid-storage diseases. Lancet. 1999;354:901–905. doi: 10.1016/S0140-6736(98)10034-X. [DOI] [PubMed] [Google Scholar]
  • 8.Nacka F., Cansell M., Meleard P., Combe N. Incorporation of alphatocopherol in marine lipid-based liposomes: in vitro and in vivo studies. Lipids. 2001;36:1313–1320. doi: 10.1007/s11745-001-0846-x. [DOI] [PubMed] [Google Scholar]
  • 9.Stillwell W., Ehringer W., Wassall S.R. Interaction of alpha-tocopherol with fatty acids in membranes and ethanol. Biochim. Biophys. Acta. 1992;1105:237–244. doi: 10.1016/0005-2736(92)90200-6. [DOI] [PubMed] [Google Scholar]
  • 10.Chen H.W., Chiang T., Wang C.Y., Lii C.K. Inhibition of tert-butyl hydroperoxide-induced cell membrane bleb formation by alpha-tocopherol and glutathione. Food Chem. Toxicol. 2000;38:1089–1096. doi: 10.1016/S0278-6915(00)00097-1. [DOI] [PubMed] [Google Scholar]
  • 11.Sezer A.D., Bas A.L., Akbuga J. Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV. J. Liposome Res. 2004;14:77–86. doi: 10.1081/LPR-120039717. [DOI] [PubMed] [Google Scholar]
  • 12.Quinn P.J. Characterisation of clusters of alpha-tocopherol in gel and fluid phases of dipalmitoylglycerophosphocholine. Eur. J. Biochem. 1995;233:916–925. doi: 10.1111/j.1432-1033.1995.916_3.x. [DOI] [PubMed] [Google Scholar]
  • 13.Wang X., Semmler K., Richter W., Quinn P.J. Ripple phases induced by alpha-tocopherol in saturated diacylphosphatidylcholines. Arch. Biochem. Biophys. 2000;377:304–314. doi: 10.1006/abbi.2000.1767. [DOI] [PubMed] [Google Scholar]
  • 14.Massey J.B. Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives. Chem. Phys. Lipids. 2001;109:157–174. doi: 10.1016/S0009-3084(00)00216-4. [DOI] [PubMed] [Google Scholar]
  • 15.Naumowicz M., Figaszewski Z.A. Impedance analysis of phosphatidylcholine/alpha-tocopherol system in bilayer lipid membranes. J. Membr. Biol. 2005;205:29–36. doi: 10.1007/s00232-005-0760-9. [DOI] [PubMed] [Google Scholar]
  • 16.Kaasgaard T., Mouritsen O.G., Jorgensen K. Lipid domain formation and ligand-receptor distribution in lipid bilayer membranes investigated by atomic force microscopy. FEBS Lett. 2002;515:29–34. doi: 10.1016/S0014-5793(02)02391-8. [DOI] [PubMed] [Google Scholar]
  • 17.Leidy C., Mouritsen O.G., Jorgensen K., Peters G.H. Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by timeresolved AFM. Biophys. J. 2004;87:408–418. doi: 10.1529/biophysj.103.036103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pedersen T.B., Kaasgaard T., Jensen M.O., Frokjaer S., Mouritsen O.G., Jorgensen K. Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides. Biophys. J. 2005;89:2494–2503. doi: 10.1529/biophysj.105.060756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Menger F.M., Keiper J.S. Chemistry and physics of giant vesicles as biomembrane models. Curr. Opin. Chem. Biol. 1998;2:726–732. doi: 10.1016/S1367-5931(98)80110-5. [DOI] [PubMed] [Google Scholar]
  • 20.Asai Y. Structural differences in aqueous dispersions of alpha-tocopheryl acetate and phosphatidylcholine upon varying their molar fractions. Pharmazie. 2004;59:849–853. [PubMed] [Google Scholar]
  • 21.Gramlich G., Zhang J., Nau W.M. Diffusion of alpha-tocopherol in membrane models: probing the kinetics of vitamin E antioxidant action by fluorescence in real time. J. Am. Chem. Soc. 2004;126:5482–5492. doi: 10.1021/ja039845b. [DOI] [PubMed] [Google Scholar]
  • 22.Pandey B.N., Mishra K.P. Effect of radiation induced lipid peroxidation on diphenylhexatriene fluorescence in egg phospholipid liposomal membrane. J. Biochem. Mol. Biol. Biophys. 2002;6:267–272. doi: 10.1080/10258140290030870. [DOI] [PubMed] [Google Scholar]
  • 23.Naguib Y.M. A fluorometric method for measurement of peroxyl radical scavenging activities of lipophilic antioxidants. Anal. Biochem. 1998;265:290–298. doi: 10.1006/abio.1998.2931. [DOI] [PubMed] [Google Scholar]
  • 24.Di Giulio A., Saletti A., Oratore A., Bozzi A. Monitoring by cisparinaric fluorescence of free radical induced lipid peroxidation in aqueous liposome suspensions. J. Microencapsul. 1996;13:435–445. doi: 10.3109/02652049609026029. [DOI] [PubMed] [Google Scholar]
  • 25.LaLonde R.T., Xie S. Glutathione and N-acetylcysteine inactivations of mutagenic 2(5H)-furanones from the chlorination of humics in water. Chem. Res. Toxicol. 1993;6:445–451. doi: 10.1021/tx00034a010. [DOI] [PubMed] [Google Scholar]
  • 26.Benezra C., Sigman C.C., Perry L.R., Helmes C.T., Maibach H.I. A systematic search for structure-activity relationships of skin contact sensitizers: methodology. J. Invest. Dermatol. 1985;85:351–356. doi: 10.1111/1523-1747.ep12276961. [DOI] [PubMed] [Google Scholar]
  • 27.Kuksis A. Animal lecithins. J. Am. Oil Chem. Soc. USA. 1985;12:105–162. [Google Scholar]
  • 28.Gennis R.B. Biomembranes: molecular structure and function in Membranes. Springer-Verlag: New York; 1989. [Google Scholar]
  • 29.Berne R., Pecora R. Dynamic Light Scattering. New York: Wiley; 1976. [Google Scholar]
  • 30.Eastman S.J., Hope M.J., Cullis P.R. Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients. Biochemistry. 1991;30:1740–1745. doi: 10.1021/bi00221a002. [DOI] [PubMed] [Google Scholar]
  • 31.Moro F., Goni F.M., Urbaneja M.A. Fluorescence quenching at interfaces and permaetion of acrylamide and iodide across phospholipid bilayers. FEBS. 1993;330:129–132. doi: 10.1016/0014-5793(93)80257-U. [DOI] [PubMed] [Google Scholar]
  • 32.Eftink M.R., Ghiron C.A. Fluorescence quenching of indole and model micelle systems. J. Phys. Chem. 1976;80:486–492. doi: 10.1021/j100546a014. [DOI] [Google Scholar]
  • 33.Kalinin S.V., Molotkovsky J.G. Anion binding to lipid bilayers: determination using fluorescent membrane probe by direct quenching or by competitive displacement approaches. J. Biochem. Biophys. Methods. 2000;46:39–51. doi: 10.1016/S0165-022X(00)00125-1. [DOI] [PubMed] [Google Scholar]
  • 34.Zheng B., Wu J.N., Schober W., Lewis D.E., Vida T. Isolation of yeast mutants defective for localization of vacuolar vital dyes. Proc. Natl. Acad. Sci. USA. 1998;95:11721–11726. doi: 10.1073/pnas.95.20.11721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Fukuzawa K., Ikebata W., Sohmi K. Location, antioxidant and recycling dynamics of alpha-tocopherol in liposome membranes. J. Nutr. Sci. Vitaminol. (Tokyo) 1993;39:S9–22. doi: 10.3177/jnsv.39.supplement_s9. [DOI] [PubMed] [Google Scholar]
  • 36.Ferezou J., Nguyen T.L., Leray C., Hajri T., Frey A., Cabaret Y., Courtieu J., Lutton C., Bach A.C. Lipid composition and structure of commercial parenteral emulsions. Biochim. Biophys. Acta. 1994;1213:149–158. doi: 10.1016/0005-2760(94)90021-3. [DOI] [PubMed] [Google Scholar]
  • 37.Chungcharoenwattana S., Ueno M. Size control of mixed egg yolk phosphatidylcholine (EggPC)/oleate vesicles. Chem. Pharm. Bull. (Tokyo) 2004;52:1058–1062. doi: 10.1248/cpb.52.1058. [DOI] [PubMed] [Google Scholar]
  • 38.Wang X., Quinn P.J. Preferential interaction of alpha-tocopherol with phosphatidylcholines in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine. Eur. J. Biochem. 2000;267:6362–6368. doi: 10.1046/j.1432-1327.2000.01720.x. [DOI] [PubMed] [Google Scholar]
  • 39.Wang X., Quinn P.J. The interaction of alpha-tocopherol with bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine. Biochim. Biophys. Acta. 2002;1567:6–12. doi: 10.1016/S0005-2736(02)00636-3. [DOI] [PubMed] [Google Scholar]
  • 40.Nakajima K., Utsumi H., Kazama M., Hamada A. Alpha-tocopherol-induced hexagonal HII phase formation in egg yolk phosphatidylcholine membranes. Chem. Pharm. Bull. (Tokyo) 1990;38:1–4. doi: 10.1248/cpb.38.1. [DOI] [PubMed] [Google Scholar]
  • 41.Chudinova V.V., Zakharova E.I., Alekseev S.M., Chupin V.V., Evstigneeva R.P. Study of the interaction of alpha-tocopherol with phospholipids, fatty acids, and their oxygenated derivatives by (31)P-NMR spectroscopy. Bioorg. Khim. 1993;19:243–249. [PubMed] [Google Scholar]
  • 42.Bagatolli L.A., Gratton E. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys. J. 2000;78:290–305. doi: 10.1016/S0006-3495(00)76592-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Erin A.N., Spirin M.M., Tabidze L.V., Kagan V.E. [. Formation of alpha-tocopherol complexes with fatty acids. Possible mechanism of biomembrane stabilization by vitamin E. Biokhimiia. 1983;48:1855–1861. [PubMed] [Google Scholar]
  • 44.Erin A.N., Spirin M.M., Tabidze L.V., Kagan V.E. Formation of alpha-tocopherol complexes with fatty acids. A hypothetical mechanism of stabilization of biomembranes by vitamin E. Biochim. Biophys. Acta. 1984;774:96–102. doi: 10.1016/0005-2736(84)90279-7. [DOI] [PubMed] [Google Scholar]
  • 45.Kagan V.E., Quinn P.J. The interaction of alpha-tocopherol and homologues with shorter hydrocarbon chains with phospholipid bilayer dispersions. A fluorescence probe study. Eur. J. Biochem. 1988;171:661–667. doi: 10.1111/j.1432-1033.1988.tb13837.x. [DOI] [PubMed] [Google Scholar]
  • 46.Urano S., Shichita N., Matsuo M. Interaction of vitamin E and its model compounds with unsaturated fatty acids in homogeneous solution. J. Nutr. Sci. Vitaminol. (Tokyo) 1988;34:189–194. doi: 10.3177/jnsv.34.189. [DOI] [PubMed] [Google Scholar]
  • 47.Chudinova V.V., Zakharova E.I., Alekseev S.M., Evstigneeva R.P. [. Interaction of vitamin E (alpha-tocopherol) with oxygenated fatty acid derivatives. Bioorg. Khim. 1993;19:505–511. [PubMed] [Google Scholar]
  • 48.Salgado J., Villalain J., Gomez-Fernandez J.C. Alpha-tocopherol interacts with natural micelle-forming single-chain phospholipids stabilizing the bilayer phase. Arch. Biochem. Biophys. 1993;306:368–376. doi: 10.1006/abbi.1993.1525. [DOI] [PubMed] [Google Scholar]
  • 49.Bourgeois C.F., George P.R., Cronenberger L.A. Automated determination of alpha-tocopherol in food and feed. Part 2. Continuous flow technique. J. Assoc. Anal. Chem. 1984;67:631–634. [PubMed] [Google Scholar]
  • 50.Tiurin V.A., Kagan V.E., Serbinova E.A., Gorbunov N.V., Erin A.N. [. The interaction of alpha-tocopherol with phospholipid liposomes: the absence of transbilayer mobility. Biull. Eksp. Biol. Med. 1986;102:689–692. [PubMed] [Google Scholar]
  • 51.Urano S., Iida M., Otani I., Matsuo M. Membrane stabilization of vitamin E; interactions of alpha-tocopherol with phospholipids in bilayer liposomes. Biochem. Biophys. Res. Commun. 1987;146:1413–1418. doi: 10.1016/0006-291X(87)90807-2. [DOI] [PubMed] [Google Scholar]
  • 52.Urano S., Yano K., Matsuo M. Membrane-stabilizing effect of vitamin E: effect of alpha-tocopherol and its model compounds on fluidity of lecithin liposomes. Biochem. Biophys. Res. Commun. 1988;150:469–475. doi: 10.1016/0006-291X(88)90544-X. [DOI] [PubMed] [Google Scholar]
  • 53.Wassall S.R., Phelps T.M., Wang L.J., Langsford C.A., Stillwell W. Membrane stabilization by vitamin E: magnetic resonance studies of the interaction of alpha-tocopherol with fatty acid acyl chains in phospholipid model membranes. Prog. Clin. Biol. Res. 1989;292:435–444. [PubMed] [Google Scholar]
  • 54.Wang X., Quinn P.J. Vitamin E and its function in membranes. Prog. Lipid. Res. 1999;38:309–336. doi: 10.1016/S0163-7827(99)00008-9. [DOI] [PubMed] [Google Scholar]
  • 55.Srivastava S., Phadke R.S., Govil G. Effect of incorporation of drugs, vitamins and peptides on the structure and dynamics of lipid assemblies. Mol. Cell. Biochem. 1989;91:99–109. doi: 10.1007/BF00228084. [DOI] [PubMed] [Google Scholar]
  • 56.Bellemare F., Fragata M. Transmembrane distribution of alphatocopherol in single-lamellar mixed lipid vesicles. J. Membr. Biol. 1981;58:67–74. doi: 10.1007/BF01871035. [DOI] [PubMed] [Google Scholar]
  • 57.Kagan V.E., Bakalova R.A., Serbinova E.E., Stoytchev T.S. Fluorescence measurements of incorporation and hydrolysis of tocopherol and tocopheryl esters in biomembranes. Methods Enzymol. 1990;186:355–367. doi: 10.1016/0076-6879(90)86129-J. [DOI] [PubMed] [Google Scholar]
  • 58.Bally M.B., Mayer L.D., Loughrey H., Redelmeier T., Madden T.D., Wong K., Harrigan P.R., Hope M.J., Cullis P.R. Dopamineaccumulation in large unilamellar vesicle systems induced by transmembrane ion gradients. Chem. Phys. Lipids. 1988;47:97–107. doi: 10.1016/0009-3084(88)90078-3. [DOI] [PubMed] [Google Scholar]

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