Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure
2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês
10.1194/jlr.m300092-jlr200
ISSN1539-7262
AutoresJesús Prades, Sérgio S. Funari, Pablo V. Escribá, Francisca Barceló,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoLipid intake in diet regulates the membrane lipid composition, which in turn controls activities of membrane proteins. There is evidence that fatty acids (FAs) and triacylglycerols (TGs) can alter the phospholipid (PL) mesomorphism. However, the molecular mechanisms involved are not fully understood. This study focuses on the effect of the unsaturation degree of the C-18 FAs, oleic acid (OA), linoleic acid and linolenic acid, and their TGs, triolein (TO), trilinolein, and trilinolenin, on the structural properties of phosphoethanolamine PLs. By means of X-ray diffraction and 31P-NMR spectroscopy, it is shown that both types of molecules stabilize the HII phase in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) liposomes. Several structural factors are considered to explain the correlation between the FA unsaturation degree and the onset temperature of the HII phase.It is proposed that TGs could act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule that would account for the structural parameters of the HII phase. Fluorescence polarization data indicated a fluidification effect of OA on the lamellar phase. TO increased the viscosity of the hydrophobic core with a high effect on the HII phase. Lipid intake in diet regulates the membrane lipid composition, which in turn controls activities of membrane proteins. There is evidence that fatty acids (FAs) and triacylglycerols (TGs) can alter the phospholipid (PL) mesomorphism. However, the molecular mechanisms involved are not fully understood. This study focuses on the effect of the unsaturation degree of the C-18 FAs, oleic acid (OA), linoleic acid and linolenic acid, and their TGs, triolein (TO), trilinolein, and trilinolenin, on the structural properties of phosphoethanolamine PLs. By means of X-ray diffraction and 31P-NMR spectroscopy, it is shown that both types of molecules stabilize the HII phase in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) liposomes. Several structural factors are considered to explain the correlation between the FA unsaturation degree and the onset temperature of the HII phase. It is proposed that TGs could act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule that would account for the structural parameters of the HII phase. Fluorescence polarization data indicated a fluidification effect of OA on the lamellar phase. TO increased the viscosity of the hydrophobic core with a high effect on the HII phase. Dietary fat intake gives the energy storage to the cell and also participates in membrane composition and cell functions (1Denyer G.S. The renaissance of fat: roles in membrane structure, signal transduction and gene expression.Med. J. Aust. 2002; 176: 109-110Google Scholar, 2Maury E. Guerineau N.C. Comminges C. Mollard P. Prevost M.C. Chap H. Potential role for triglycerides in signal transduction.FEBS Lett. 2000; 466: 228-232Crossref PubMed Scopus (7) Google Scholar, 3Alessenko A.V. Burlakova E.B. Functional role of phospholipids in the nuclear events.Bioelectrochemistry. 2002; 58: 13-21Crossref PubMed Scopus (44) Google Scholar). The fat composition of the diet can have an effect on the membrane fatty acid (FA) composition. For example, diets rich in oleic acid (OA), such as the Mediterranean diet, are associated with increases in the levels of this FA in various plasma membrane lipid species in rat and human cells (4Escudero A. Montilla J.C. García J.M. Sánchez-Quevedo M.C. Periago J.L. Hortelano P. Suárez M.D. Effect of dietary (n-9), (n-6) and (n-3) fatty acids on membrane lipid composition and morphology of rat erythrocytes.Biochim. Biophys. Acta. 1998; 1394: 65-73Crossref PubMed Scopus (39) Google Scholar, 5Pagnan A. Corrocher R. Ambrosio G.B. Ferrari S. Guarini P. Piccolo D. Opportuno A. Bassi A. Olivieri O. Baggio G. Effects of an olive-oil-rich diet on erythrocyte membrane lipid composition and cation transport systems.Clin. Sci. 1989; 76: 87-93Crossref PubMed Scopus (60) Google Scholar, 6Vicario I.M. Malkova D. Lund E.K. Johnson I.T. Olive oil supplementation in healthy adults: effects in cell membrane fatty acid composition and platelet function.Ann. Nutr. Metab. 1998; 42: 160-169Crossref PubMed Scopus (40) Google Scholar). Also, many of the unsaturated FAs, such as linoleic acid (LL) and α-linolenic acid (LN), are components of the membrane phospholipids (PLs) and are not produced by human cells. Moreover, the FA composition of membranes influences the localization and activity of G proteins and protein kinase C (7Escribá P.V. Ozaita A. Ribas C. Miralles A. Fodor E. Farkas T. García-Sevilla J.A. Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes.Proc. Natl. Acad. Sci. USA. 1997; 94: 11375-11380Crossref PubMed Scopus (100) Google Scholar), which are pivotal elements in cell signaling and which control (through these proteins) important physiological functions, such as blood pressure (8Escribá P.V. Sánchez-Dominguez J.M. Alemany R. Perona J.S. Ruíz-Gutiérrez V. Alteration of lipids, G proteins, and PKC in cell membranes of elderly hypertensives.Hypertension. 2003; 41: 176-182Crossref PubMed Scopus (65) Google Scholar). Triacylglycerols (TGs) constitute the largest source of dietary FAs. It is well known that TG cleavage at the brush border membrane is a prerequisite for efficient FA absorption (9Bosner M.S. Gulick T. Riley D.J. Spilburg C.A. Lange L.G. Heparin-modulated binding of pancreatic lipase and uptake of hydrolyzed triglycerides in the intestine.J. Biol. Chem. 1989; 264: 20261-20264Abstract Full Text PDF PubMed Google Scholar). On the other hand, it has been demonstrated that TGs can partition with a preferred orientation in phosphocholine (PC) lipids, although they are neutral lipids (10Hamilton J.A. Vural J.M. Carpentier Y.A. Deckelbaum R.J. Incorporation of medium chain triacylglycerols into phospholipid bilayers: effect of long chain triacylglycerols, cholesterol, and cholesteryl esters.J. Lipid Res. 1996; 37: 773-782Abstract Full Text PDF PubMed Google Scholar, 11Hamilton J.A. Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.Biochemistry. 1989; 28: 2514-2520Crossref PubMed Scopus (88) Google Scholar, 12Smaby J.M. Brockman H.L. Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.J. Biol. Chem. 1987; 262: 8206-8212Abstract Full Text PDF PubMed Google Scholar, 13Spooner P.J. Small D.M. Effect of free cholesterol on incorporation of triolein in phospholipid bilayers.Biochemistry. 1987; 26: 5820-5825Crossref PubMed Scopus (53) Google Scholar, 14Gorrissen H. Tulloch A.P. Cushley R.J. Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.Chem. Phys. Lipids. 1982; 31: 245-255Crossref PubMed Scopus (25) Google Scholar, 15Hamilton J.A. Small D.M. Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.Proc. Natl. Acad. Sci. USA. 1981; 78: 6878-6882Crossref PubMed Scopus (137) Google Scholar). These data suggest the possibility that TGs could also be present in small proportions inserted in PL bilayers of biological membranes. In fact, it has been suggested that TGs, as interfacial molecules, could be important in the interaction between the membrane and some lipolytic enzymes and carrier proteins (12Smaby J.M. Brockman H.L. Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.J. Biol. Chem. 1987; 262: 8206-8212Abstract Full Text PDF PubMed Google Scholar, 14Gorrissen H. Tulloch A.P. Cushley R.J. Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.Chem. Phys. Lipids. 1982; 31: 245-255Crossref PubMed Scopus (25) Google Scholar). In this case, the intracellular pool of TGs could participate in several cellular events, such as the formation of TG-rich lipoproteins and (transient) functional bilayer structures rich in TG and FA storage. FAs are major components of membranes, mainly bound to PLs and cholesterol esters, although low levels of free fatty acids (FFAs) can also be found in natural membranes (16Engelbrecht A.M. Louw L. Cloete F. Comparison of the fatty acid compositions in intraepithelial and infiltrating lesions of the cervix: part II, free fatty acid profiles.Prostaglandins Leukot. Essent. Fatty Acids. 1998; 59: 253-257Abstract Full Text PDF PubMed Scopus (7) Google Scholar) and may be important components in certain membranes, such as the small intestine brush border membrane (17Hauser H. Howell K. Dawson R.M. Bowyer D.E. Rabbit small intestinal brush border membrane preparation and lipid composition.Biochim. Biophys. Acta. 1980; 602: 567-577Crossref PubMed Scopus (246) Google Scholar). Membrane FA composition has a modulating effect on protein activities such as receptors, ion channels, second messengers, and gene expression (7Escribá P.V. Ozaita A. Ribas C. Miralles A. Fodor E. Farkas T. García-Sevilla J.A. Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes.Proc. Natl. Acad. Sci. USA. 1997; 94: 11375-11380Crossref PubMed Scopus (100) Google Scholar, 18Ntambi J.M. Bene H. Polyunsaturated fatty acid regulation of gene expression.J. Mol. Neurosci. 2001; 16: 273-278Crossref PubMed Scopus (98) Google Scholar, 19Sumida C. Graber R. Nunez E. Role of fatty acids in signal transduction: modulators and messengers.Prostaglandins Leukot. Essent. Fatty Acids. 1993; 48: 117-122Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Khan W.A. Blobe G.C. Hannun Y.A. Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.J. Biol. Chem. 1992; 267: 3605-3612Abstract Full Text PDF PubMed Google Scholar, 21Litman B.J. Niu S.L. Polozova A. Mitchell D.C. The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways: visual transduction.J. Mol. Neurosci. 2001; 16: 237-242Crossref PubMed Scopus (179) Google Scholar). In addition, there is evidence suggesting the existence of a close correlation between some functions of the cells and the various degrees of unsaturation in the sn-2-acyl chains of membrane PLs (22Liu S. Baracos V.E. Quinney H.A. Clandinin M.T. Dietary omega-3 and polyunsaturated fatty acids modify fatty acyl composition and insulin binding in skeletal-muscle sarcolemma.Biochem. J. 1994; 299: 831-837Crossref PubMed Scopus (94) Google Scholar, 23Salem Jr., N. Niebylski C.D. The nervous system has an absolute molecular species requirement for proper function.Mol. Membr. Biol. 1995; 12: 131-134Crossref PubMed Scopus (126) Google Scholar). Also, a recent study has shown strong correlations between membrane PL composition and insulin sensitivity in humans (24Storlien L.H. Tapsell L.C. Fraser A. Leslie E. Ball K. Higgins J.A. Helge J.W. Owen N. Insulin resistance. Influence of diet and physical activity.World Rev. Nutr. Diet. 2001; 90: 26-43Crossref PubMed Google Scholar). Phosphoethanolamine (PE) PLs are the main group of PLs in the inner leaflet of mammalian plasma membranes (4Escudero A. Montilla J.C. García J.M. Sánchez-Quevedo M.C. Periago J.L. Hortelano P. Suárez M.D. Effect of dietary (n-9), (n-6) and (n-3) fatty acids on membrane lipid composition and morphology of rat erythrocytes.Biochim. Biophys. Acta. 1998; 1394: 65-73Crossref PubMed Scopus (39) Google Scholar), where they are mainly organized into lamellar structures. However, PE PLs are also prone to form nonlamellar structures, such as the inverted hexagonal HII phase (25Cullis P.R. de Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes.Biochim. Biophys. Acta. 1979; 559: 399-420Crossref PubMed Scopus (1522) Google Scholar, 26Seddon J.M. Structure of the inverted hexagonal (HII) phase and non-lamellar phase transitions of lipids.Biochim. Biophys. Acta. 1990; 1031: 1-69Crossref PubMed Scopus (1012) Google Scholar, 27Turner D.C. Gruner S.M. X-ray diffraction reconstruction of the inverted hexagonal (HII) phase in lipid-water systems.Biochemistry. 1992; 31: 1340-1355Crossref PubMed Scopus (129) Google Scholar, 28Borovyagin V.L. Sabelnikov A.G. Lipid polymorphism of model and cellular membranes as revealed by electron microscopy.Electron Microsc. Rev. 1989; 2: 75-115Crossref PubMed Scopus (22) Google Scholar, 29Epand R. Lipid Polymorphism and Membrane Properties. Academic Press, San Diego, CA1997: 25-102Google Scholar). Some special features, such as the control of functions of membrane proteins and the structural organization inside cells, were attributed to HII-prone PLs (30Luzzati V. Biological significance of lipid polymorphism: the cubic phases.Curr. Opin. Struct. Biol. 1997; 7: 661-668Crossref PubMed Scopus (221) Google Scholar, 31De Kruijff B. Biomembranes. Lipids beyond the bilayer.Nature. 1997; 386: 129-130Crossref PubMed Scopus (184) Google Scholar, 32Bogdanov M. Sun J. Kaback H.R. Dowhan W. A phospholipid acts as a chaperone in assembly of a membrane transport protein.J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 33Boggs J.M. Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function.Biochim. Biophys. Acta. 1987; 906: 353-404Crossref PubMed Scopus (693) Google Scholar). Under a variety of circumstances, cell membrane lipid composition can be modulated to balance the contents of prolamellar and prononlamellar lipids (7Escribá P.V. Ozaita A. Ribas C. Miralles A. Fodor E. Farkas T. García-Sevilla J.A. Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes.Proc. Natl. Acad. Sci. USA. 1997; 94: 11375-11380Crossref PubMed Scopus (100) Google Scholar, 34Gudi S. Nolan J.P. Frangos J.A. Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition.Proc. Natl. Acad. Sci. USA. 1998; 95: 2515-2519Crossref PubMed Scopus (285) Google Scholar, 35Giorgione J. Epand R.M. Buda C. Farkas T. Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.Proc. Natl. Acad. Sci. USA. 1995; 92: 9767-9770Crossref PubMed Scopus (72) Google Scholar, 36Rietveld A.G. Koorengevel M.C. de Kruijff B. Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.EMBO J. 1995; 14: 5506-5513Crossref PubMed Scopus (141) Google Scholar). Therefore, changes in lipid composition may represent a membrane adjustment in order to regulate structural properties to preserve their functions [e.g., (35Giorgione J. Epand R.M. Buda C. Farkas T. Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.Proc. Natl. Acad. Sci. USA. 1995; 92: 9767-9770Crossref PubMed Scopus (72) Google Scholar, 36Rietveld A.G. Koorengevel M.C. de Kruijff B. Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.EMBO J. 1995; 14: 5506-5513Crossref PubMed Scopus (141) Google Scholar)]. There is evidence that FFAs and TGs can modify the polymorphic properties and fluidity of PLs in model membranes (10Hamilton J.A. Vural J.M. Carpentier Y.A. Deckelbaum R.J. Incorporation of medium chain triacylglycerols into phospholipid bilayers: effect of long chain triacylglycerols, cholesterol, and cholesteryl esters.J. Lipid Res. 1996; 37: 773-782Abstract Full Text PDF PubMed Google Scholar, 11Hamilton J.A. Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.Biochemistry. 1989; 28: 2514-2520Crossref PubMed Scopus (88) Google Scholar, 12Smaby J.M. Brockman H.L. Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.J. Biol. Chem. 1987; 262: 8206-8212Abstract Full Text PDF PubMed Google Scholar, 15Hamilton J.A. Small D.M. Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.Proc. Natl. Acad. Sci. USA. 1981; 78: 6878-6882Crossref PubMed Scopus (137) Google Scholar, 37Funari S.S. Barceló F. Escribá P.V. Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membrane.J. Lipid Res. 2003; 44: 567-575Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 38Epand R.M. Epand R.F. Ahmed N. Chen R. Promotion of hexagonal phase formation and lipid mixing by fatty acids with varying degrees of unsaturation.Chem. Phys. Lipids. 1991; 57: 75-80Crossref PubMed Scopus (68) Google Scholar, 39Langner M. Isac T. Hui S.W. Interaction of free fatty acids with phospholipid bilayers.Biochim. Biophys. Acta. 1995; 1236: 73-80Crossref PubMed Scopus (41) Google Scholar, 40Ortiz A. Gomez-Fernandez J.C. A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes.Chem. Phys. Lipids. 1987; 45: 75-91Crossref PubMed Scopus (64) Google Scholar). However, the molecular mechanisms involved in the modulation of the membrane structure and function are not fully understood. Therefore, the study of structural properties of PL membranes in which FFAs or TGs are present is important for a better understanding of their crucial biological function. The present work was planned to analyze the effect of the unsaturation in the C-18 acyl chain of the FAs, (OA, LL, and LN) and their sterified derivatives, such as TGs [triolein (TO), trilinolein (TLL), and trilinolenin TLN)], on the structural properties of lamellar and nonlamellar PE PLs, as model membranes. 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, U.S.A.). 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (HPE), N-(1-pyrenesulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (pyS DHPE), and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) were from Molecular Probes (Leiden, The Netherlands). OA (18:1cΔ9), LL (18:2c,cΔ9,12), α-LN (18:3c,c,cΔ9,12,15), TO, TLL, TLN, and N-(2-hydroxy ethyl) piperazine-N′-(2-ethanesulfonic acid) sodium salt (HEPES) were obtained from Sigma Chemical Co. (Madrid, Spain). Lipids, FAs, and TGs were stored under argon at −80°C until use. Differential scanning calorimetry (DSC) of multilamellar liposomes from these phosphatidylethanolamine derivatives was used to evaluate the PL purity. DSC calorimetric scans showed highly cooperative phase transitions. Multilamellar lipid vesicles 15% (w/w) were prepared in 10 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.4 (HEPES buffer) for X-ray studies, or in D2O for NMR experiments. PLs with FAs or TGs were prepared at a molar ratio of 20:1 (PL-fat). Lipid mixtures were thoroughly homogenized with a pestle-type minihomogenizer (Sigma) and vortexed until a homogeneous mixture was obtained. Then the suspensions were submitted to five temperature cycles (heated up to 70°C and cooled down to 4°C). Samples for X-ray scattering experiments were stored at −80°C under argon and allowed to equilibrate at 4°C for 48 h before measurements. Samples for NMR experiments were equilibrated at 4°C for at least 24 h prior to data acquisition. For fluorescence spectroscopy experiments, PL and fluorophore (PL-probe; 1,000:1; mol/mol), in the presence or absence of OA or TO (PL-fat; 20:1; mol/mol), were dissolved in chloroform-methanol (2:1; v/v), evaporated under argon, and vacuum-dried for at least 3 h. The lipid film was resuspended in HEPES buffer by vortex shaking at ∼45°C. The lipid suspension (120 μM) was subjected to five freeze/thaw cycles to ensure the complete hydration of the lipid vesicles. To obtain large unilamellar vesicles (LUVs), the resulting multilamellar suspension was passed 11 times through polycarbonate membranes (0.1 μm) in an extruder (Avanti Polar Lipids, Inc.). LUVs were used immediately. Small- and wide-angle (SAXS and WAXS) synchrotron radiation X-ray scattering data were collected simultaneously, using standard procedures on the Soft Condensed Matter beamline A2 (41Boulin C. Kempf R. Koch M.H.J. McLaughlin S.M. Data appraisal, evaluation and display for synchrotron radiation experiments: hardware and software.Nucl. Instrum. Meth. Phys. Res. 1986; A249: 399-407Crossref Scopus (296) Google Scholar, 42Boulin C. Kempf R. Gabriel A. Koch M.H.J. Data acquisition systems for linear and area X-ray detectors using delay line readout.Nucl. Instrum. Meth. Phys. Res. 1988; A269: 312-320Crossref Scopus (232) Google Scholar) of Hasylab at the storage ring DORIS III of the Deutsches Elektronen Synchrotron. Data were acquired continuously for 15 s at each temperature, followed by a waiting time of 45 s with a local shutter closed. During data collection, samples were heated from 27°C to 75°C at a scan rate of 1°C/min. Then they were kept at the highest temperature for 5 min and finally cooled down to the lowest temperature at the same scan rate. The experimental conditions did not affect the phase sequence structures or their parameters. Positions of the observed peaks were converted into distances, d, after calibration with the standards rat tendon tail and poly-(ethylene therephtalate) for the SAXS and WAXS regions, respectively. Interplanar distances, dhkl, were calculated according to equation 1: where s is the scattering vector, 2θ is the scattering angle, λ (0.154 nm) is the X-ray wavelength, and hkls are the Miller indexes of the scattering planes. Measurements were conducted on a model AMX-300 multinuclear NMR spectrometer (Bruker Instruments) in 4 mm tubes. Samples were equilibrated at the working temperatures for 15 min before data acquisition. 31P-NMR free induction decays were accumulated for up to 160 transients by employing a 6.75 μs 90° radio frequency pulse, 12.2 kHz sweep width, and 32 K data points. The delay between transients was 2 s. The spectra were obtained by scanning from lower to higher temperatures. Experiments were done with a MPF-66 fluorescence spectrophotometer (Perkin-Elmer). The cuvette was thermostated during the experiment. LUV suspensions, containing the fluorophores HPE or pyS DHPE, were excited at 340 or 350 nm, and fluorescence polarization was registered at 378 or 379 nm, respectively. TMA-DPH was excited at 360 nm, and the fluorescence emission was recorded at 427 nm. The bandpass was 4 nm for excitation and for emission. Samples were equilibrated for 5 min at each temperature, prior to measurement. In our experimental conditions, the inner filter effect became critical when sample absorption was ∼0.1. Thus, lipid samples were diluted until the fluorescence samples had an absorption at the excitation wavelength of ∼0.04. Light scatter was checked by a control experiment with unlabeled liposomes. A residual fluorescence (∼5% for DOPE and 4% for DEPE) was observed in the control experiments with unlabeled liposomes, and its contribution to the fluorescence polarization was insignificant. Fluorescence polarization was calculated according to equation 2: where IVV and IVH are fluorescence intensity values measured with the excitation and emission polarizers in parallel and perpendicular, respectively. G is an instrumental factor. Figure 1illustrates the SAXS and WAXS X-ray scattering patterns of DEPE alone or in the presence of the FA, LN, or the TG, TLN, both at a molar ratio of 20:1 (DEPE-fat). The sequence of diffraction patterns collected showed defined phase transitions that allowed unequivocal characterization of the structures and their respective lattice parameters. The Lβ phase was identified by a sharp and intense SAXS reflection (s ≈ 0.15 nm−1), accompanied by a reflection in the WAXS region. The Lα phase was identified by a single reflection peak at s ≈ 0.19 nm−1, with a very good signal-to-noise ratio. The appearance of three diffraction orders in the SAXS region with a d spacing ratio of 1:1/√3:1/√4 indicated the formation of the HII phase. All the samples showed similar X-ray scattering patterns. DEPE alone and in the presence of the unsaturated FAs OA, LL, and LN showed a phase sequence from gel Lβ to liquid crystalline Lα and to hexagonal HII phases with increasing temperature (Fig. 2A–C). DEPE-FA (20:1; mol/mol) samples showed an Lβ phase up to 39°C with a constant repeat distance of ∼5.4 nm (Table 1), similar to the value shown for DEPE alone and previously reported (37Funari S.S. Barceló F. Escribá P.V. Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membrane.J. Lipid Res. 2003; 44: 567-575Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Nevertheless, the presence of the FAs OA, LL, and LN strongly destabilized the DEPE Lα phase (Fig. 2A–C). Although the temperature range of the Lα phase presence was dependent upon the number of unsaturations of the acyl chain (Table 1), the repeat distance decreased linearly with temperature and showed a constant value (∼−0.013 nm/°C) for the compression coefficient (Table 1).TABLE 1Summary of the physical (structural) propertiesCompositionδd /δT (Lα)aThe compressibility of Lα is linear in the single or two-phase regions. Single HII phases have linear compressibility factor. The temperature range in which the Lα phase is observed is shown in ΔTLα.δd /δT (HII)aThe compressibility of Lα is linear in the single or two-phase regions. Single HII phases have linear compressibility factor. The temperature range in which the Lα phase is observed is shown in ΔTLα.ΔT LαbThe parentheses indicate the temperature limit of the Lα phase in a two-phase region. Values on the left correspond to Lβ + Lα and on the right to Lα + HII temperature range coexistence.d LαddLα at 40°C and dHII at 72°C.d HIIddLα at 40°C and dHII at 72°C.nm/°Cnm/°C°CnmnmDEPE−0.012−0.02438–61(66)5.436.27DEPE-OAcNo single Lα phase was observed.−0.014−0.020(38) 39 (53)5.445.90DEPE-LL−0.012−0.027(33)37–50(60)5.406.15DEPE-LN−0.017−0.021(36)39–57(64)5.406.20DEPE-TO−0.017−0.03638–43(51)5.436.72DEPE-TLL−0.012−0.039(39)40–49(62)5.436.58DEPE-TLN−0.013−0.03739–50(56)5.446.45DEPE, 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine; OA, oleic acid; LL, linoleic acid; LN, linolenic acid; TO, triolein; TLL, trilinolein; TLN, trilinolenin. Sample DEPE-FFA and DEPE-TG composition was 20:1 (molar ratio). The angular coefficient of the dependence of the interplanar distance d10 on the temperature ∂d/∂T <0 indicates a compression process.a The compressibility of Lα is linear in the single or two-phase regions. Single HII phases have linear compressibility factor. The temperature range in which the Lα phase is observed is shown in ΔTLα.b The parentheses indicate the temperature limit of the Lα phase in a two-phase region. Values on the left correspond to Lβ + Lα and on the right to Lα + HII temperature range coexistence.c No single Lα phase was observed.d dLα at 40°C and dHII at 72°C. Open table in a new tab DEPE, 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine; OA, oleic acid; LL, linoleic acid; LN, linolenic acid; TO, triolein; TLL, trilinolein; TLN, trilinolenin. Sample DEPE-FFA and DEPE-TG composition was 20:1 (molar ratio). The angular coefficient of the dependence of the interplanar distance d10 on the temperature ∂d/∂T <0 indicates a compression process. DEPE-OA mixtures were characterized by X-ray diffraction measurements (37Funari S.S. Barceló F. Escribá P.V. Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membrane.J. Lipid Res. 2003; 44: 567-575Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). It was observed that the temperature range in which the lamellar and nonlamellar phases coexisted depended on the DEPE-OA molar ratio. For example, in the mixture DEPE-OA (20:1; mol/mol), the Lα phase was observed from 38°C to 53°C and in coexistence with either Lβ or HII phases (Table 1). DEPE in the presence of LL or LN (20:1; mol/mol) showed a single Lα phase in the range of 37–50°C or 39–57°C, respectively, with a repeat distance value of 5.4 nm at 40°C. Above these temperatures, lamellar (Lα) and hexagonal (HII) phases coexisted in a range that depended also on the FA (Fig. 2B, C and Table 1). The threshold temperature for DEPE-based HII phases formed by the binary system (DEPE-FFA; 20:1; mol/mol) depended on the unsaturation degree of the FA (Fig. 2A–C). The temperature at which a single inverted hexagonal phase appeared was directly related to the degree of unsaturation of the FA. It is important to note that the repeat distance of the DEPE HII phase (6.27 nm at 72°C) was reduced by the FA OA (5.90 nm at 72°C), while LL and LN produced a smaller effect (6.15–6.20 nm at 72°C). The compressibility factor (∼−0.024 nm/°C) did not change in a significant mode in the presence of an unsaturated FFA at a molar ratio of 20:1, suggesting a similar packing of the PL molecules in the DEPE and DEPE-FFA systems. DEPE in the presence of the TGs TO, TLN, and TLL had the same phase sequence pattern, Lβ to Lα to HII, as DEPE in presence of FFAs (Fig. 1). The diffraction patterns of the DEPE-TG samples (Figs. 2D–F) showed several important general trends: i) TGs, like FFAs, did not alter the interplanar distance of the Lα phase (5.4 nm at 40°C), or the compressibility factor (∂d/∂T ≈ −0.012) (Table 1). ii) TO, TLL, and TLN facilitated formation of the HII phase, and there was a long range of temperatures where Lα and HII phases coexisted (∼6°
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