Advertisement

Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure

  • Jesús Prades
    Affiliations
    Molecular and Cellular Biomedicine, Institut Universitari d'Investigacions en Ciencies de la Salut, Associate Unit of Instituto de la Grasa (CSIC), Department of Biology, University of the Balearic Islands, E-07122 Palma de Mallorca, Spain
    Search for articles by this author
  • Sérgio S. Funari
    Affiliations
    Max-Planck Institute for Colloids and Interfaces, HASYLAB, Notkestrasse 85, D-22603 Hamburg, Germany
    Search for articles by this author
  • Pablo V. Escribá
    Affiliations
    Molecular and Cellular Biomedicine, Institut Universitari d'Investigacions en Ciencies de la Salut, Associate Unit of Instituto de la Grasa (CSIC), Department of Biology, University of the Balearic Islands, E-07122 Palma de Mallorca, Spain
    Search for articles by this author
  • Francisca Barceló
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Molecular and Cellular Biomedicine, Institut Universitari d'Investigacions en Ciencies de la Salut, Associate Unit of Instituto de la Grasa (CSIC), Department of Biology, University of the Balearic Islands, E-07122 Palma de Mallorca, Spain
    Search for articles by this author
Open AccessPublished:June 16, 2003DOI:https://doi.org/10.1194/jlr.M300092-JLR200
      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 (
      • Denyer G.S.
      The renaissance of fat: roles in membrane structure, signal transduction and gene expression.
      ,
      • Maury E.
      • Guerineau N.C.
      • Comminges C.
      • Mollard P.
      • Prevost M.C.
      • Chap H.
      Potential role for triglycerides in signal transduction.
      ,
      • Alessenko A.V.
      • Burlakova E.B.
      Functional role of phospholipids in the nuclear events.
      ). 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 (
      • Escudero 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.
      ,
      • Pagnan 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.
      ,
      • Vicario 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.
      ). 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 (
      • Escribá 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.
      ), which are pivotal elements in cell signaling and which control (through these proteins) important physiological functions, such as blood pressure (
      • Escribá 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.
      ).
      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 (
      • Bosner 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.
      ). 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 (
      • Hamilton 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.
      ,
      • Hamilton J.A.
      Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.
      ,
      • Smaby J.M.
      • Brockman H.L.
      Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.
      ,
      • Spooner P.J.
      • Small D.M.
      Effect of free cholesterol on incorporation of triolein in phospholipid bilayers.
      ,
      • Gorrissen H.
      • Tulloch A.P.
      • Cushley R.J.
      Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.
      ,
      • Hamilton J.A.
      • Small D.M.
      Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.
      ). 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 (
      • Smaby J.M.
      • Brockman H.L.
      Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.
      ,
      • Gorrissen H.
      • Tulloch A.P.
      • Cushley R.J.
      Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.
      ). 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 (
      • Engelbrecht 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.
      ) and may be important components in certain membranes, such as the small intestine brush border membrane (
      • Hauser H.
      • Howell K.
      • Dawson R.M.
      • Bowyer D.E.
      Rabbit small intestinal brush border membrane preparation and lipid composition.
      ). Membrane FA composition has a modulating effect on protein activities such as receptors, ion channels, second messengers, and gene expression (
      • Escribá 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.
      ,
      • Ntambi J.M.
      • Bene H.
      Polyunsaturated fatty acid regulation of gene expression.
      ,
      • Sumida C.
      • Graber R.
      • Nunez E.
      Role of fatty acids in signal transduction: modulators and messengers.
      ,
      • Khan 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.
      ,
      • Litman 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.
      ). 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 (
      • Liu 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.
      ,
      • Salem Jr., N.
      • Niebylski C.D.
      The nervous system has an absolute molecular species requirement for proper function.
      ). Also, a recent study has shown strong correlations between membrane PL composition and insulin sensitivity in humans (
      • Storlien 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.
      ).
      Phosphoethanolamine (PE) PLs are the main group of PLs in the inner leaflet of mammalian plasma membranes (
      • Escudero 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.
      ), 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 (
      • Cullis P.R.
      • de Kruijff B.
      Lipid polymorphism and the functional roles of lipids in biological membranes.
      ,
      • Seddon J.M.
      Structure of the inverted hexagonal (HII) phase and non-lamellar phase transitions of lipids.
      ,
      • Turner D.C.
      • Gruner S.M.
      X-ray diffraction reconstruction of the inverted hexagonal (HII) phase in lipid-water systems.
      ,
      • Borovyagin V.L.
      • Sabelnikov A.G.
      Lipid polymorphism of model and cellular membranes as revealed by electron microscopy.
      ,
      • Epand R.
      Lipid Polymorphism and Membrane Properties.
      ). Some special features, such as the control of functions of membrane proteins and the structural organization inside cells, were attributed to HII-prone PLs (
      • Luzzati V.
      Biological significance of lipid polymorphism: the cubic phases.
      ,
      • De Kruijff B.
      Biomembranes. Lipids beyond the bilayer.
      ,
      • Bogdanov M.
      • Sun J.
      • Kaback H.R.
      • Dowhan W.
      A phospholipid acts as a chaperone in assembly of a membrane transport protein.
      ,
      • Boggs J.M.
      Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function.
      ). Under a variety of circumstances, cell membrane lipid composition can be modulated to balance the contents of prolamellar and prononlamellar lipids (
      • Escribá 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.
      ,
      • Gudi S.
      • Nolan J.P.
      • Frangos J.A.
      Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition.
      ,
      • Giorgione J.
      • Epand R.M.
      • Buda C.
      • Farkas T.
      Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.
      ,
      • Rietveld A.G.
      • Koorengevel M.C.
      • de Kruijff B.
      Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.
      ). Therefore, changes in lipid composition may represent a membrane adjustment in order to regulate structural properties to preserve their functions [e.g., (
      • Giorgione J.
      • Epand R.M.
      • Buda C.
      • Farkas T.
      Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.
      ,
      • Rietveld A.G.
      • Koorengevel M.C.
      • de Kruijff B.
      Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.
      )].
      There is evidence that FFAs and TGs can modify the polymorphic properties and fluidity of PLs in model membranes (
      • Hamilton 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.
      ,
      • Hamilton J.A.
      Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.
      ,
      • Smaby J.M.
      • Brockman H.L.
      Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.
      ,
      • Hamilton J.A.
      • Small D.M.
      Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.
      ,
      • Funari 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.
      ,
      • Epand 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.
      ,
      • Langner M.
      • Isac T.
      • Hui S.W.
      Interaction of free fatty acids with phospholipid bilayers.
      ,
      • Ortiz A.
      • Gomez-Fernandez J.C.
      A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes.
      ). 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.

      MATERIALS AND METHODS

      Materials

      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.

      Sample preparation

      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.

      X-ray diffraction

      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 (
      • Boulin C.
      • Kempf R.
      • Koch M.H.J.
      • McLaughlin S.M.
      Data appraisal, evaluation and display for synchrotron radiation experiments: hardware and software.
      ,
      • Boulin C.
      • Kempf R.
      • Gabriel A.
      • Koch M.H.J.
      Data acquisition systems for linear and area X-ray detectors using delay line readout.
      ) 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.

      31P-NMR

      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.

      Fluorescence polarization

      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.

      RESULTS

      X-ray diffraction

      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.
      Figure thumbnail gr1
      Fig. 1Sequence of SAXS (left) and WAXS (right) scattering patterns of (A) 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), (B) DEPE with α-linolenic acid (LN), and (C) DEPE with trilinolenin (TLN) at a molar ratio of 20:1. The heating sequence is shown from 27°C to 72°C. The scan rate was 1°C/min. Successive diffraction patterns were collected for 15 s every min. The thermal sequence of the phases Lβ (up to 39°C), Lα (from 39°C to 57°C for DEPE-LN and from 39°C to 50°C for DEPE-TLN), and HII (up to 72°C) is clearly shown. The phase transition from Lβ to Lα can be identified by the vanishing peak on the WAXS region of the pattern.

      Effects of the FA unsaturation on DEPE membrane structures

      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).
      Figure thumbnail gr2
      Fig. 2Dependence of the interplanar repeat distance d on temperature for DEPE alone (solid squares in A and D) and in the presence of the free fatty acids (A) oleic acid (OA), (B) linoleic acid, and (C) LN (open circles), or the triacylglycerols (D) triolein (TO), (E) trilinolein, and (F) trilinolenin (open diamond). Sample concentration was phospholipid (PL)-fat, 20:1 (mol/mol). Phases represented are: Lβ, Lα, and HII. The coexistence of the Lα + HII two-phase region is indicated by the interval temperature range shown by the vertical lines.

      Lamellar phases

      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 (
      • Funari 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.
      ). 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) properties
      Compositionδd /δT (Lα)
      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.
      δd /δT (HII)
      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.
      ΔT
      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.
      d
      dLα at 40°C and dHII at 72°C.
      d HII
      dLα at 40°C and dHII at 72°C.
      nm/°Cnm/°C°Cnmnm
      DEPE−0.012−0.02438–61(66)5.436.27
      DEPE-OA
      No single Lα phase was observed.
      −0.014−0.020(38) 39 (53)5.445.90
      DEPE-LL−0.012−0.027(33)37–50(60)5.406.15
      DEPE-LN−0.017−0.021(36)39–57(64)5.406.20
      DEPE-TO−0.017−0.03638–43(51)5.436.72
      DEPE-TLL−0.012−0.039(39)40–49(62)5.436.58
      DEPE-TLN−0.013−0.03739–50(56)5.446.45
      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.
      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 ΔT.
      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 d at 40°C and dHII at 72°C.
      DEPE-OA mixtures were characterized by X-ray diffraction measurements (
      • Funari 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.
      ). 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).

      Hexagonal HII phases

      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.

      Effects of TGs on DEPE membrane structures

      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°C to 13°C) (Figs. 2D–F). The spacing of the hexagonal phase started at a high value (∼9 nm), showed a strong decay during the coexistence of the Lα and HII phases, and then followed the pattern of the DEPE-FFA systems. iii) TGs increased the intercylinder distance of the DEPE HII phases. The lattice spacing was in the range of 6.7–6.4 nm for DEPE-TG (20:1, molar ratio) and 6.27 nm for DEPE liposomes (data taken at 72°C; Table 1). In addition, the negative value of the compressibility factor decreased (∂d/∂T ≈ −0.037 nm/°C) compared with DEPE alone (∂d/∂T ≈ −0.024 nm/°C) (Table 1). Also, notice that for DEPE-TG, the hexagonal (HII) phase contraction factor values are about three times greater than that of the lamellar Lα phase (∂d/∂T ≈ −0.013 nm/°C) (Table 1).

      31P-NMR data

      The phase behavior of DEPE in the presence of OA or TO was also analyzed by 31P-NMR, working under quasiequilibrium conditions, with a 15 min rest at each temperature. The spectra showed line shapes characteristic of Lα and HII phases (Fig. 3). OA and TO lowered the onset transition temperature of the HII phase with different degrees of cooperation, in agreement with the X-ray diffraction data. DEPE underwent a lamellar-to-inverted hexagonal phase transition between 45°C and 50°C, and above 52°C, a single HII phase was observed. The systems DEPE-OA and DEPE-TO (molar ratio 20:1) had no pure Lα phase. OA showed the highest degree of cooperativity and the HII structure appeared at 42°C. TO presented a lamellar-hexagonal transition in the range of 37–42°C, and from 47°C upward, a single HII phase was observed.
      Figure thumbnail gr3
      Fig. 331P-NMR spectra of DEPE aqueous dispersions (15%, w/w) in D2O, as a function of temperature. A: DEPE alone and in the presence of (B) OA or (C) TO PL-fat, 20:1; mol/mol). Samples were equilibrated for 15 min at each temperature before data acquisition. Scans were done from lower to upper temperature. The temperatures at which the systems had Lα, Lα + HII, and pure HII phases are indicated. Note that DEPE showed the HII phase at 52°C, DEPE-OA at 42°C, and DEPE-TO at 47°C.

      Steady-state fluorescence polarization measurements

      DEPE and DOPE were used to assess the effect of the presence of OA and TO on the properties of the lamellar and hexagonal (HII) phases. The fluorescent probe HPE is localized in the hydrophobic core of the membrane and provides structural information on this region. The probes pyS DHPE and TMA-DPH allow monitoring of the lipid environments close to the bilayer surface; pyS DHPE is anchored in close proximity to the bilayer surface, and TMA-DPH anchors its nonfluorescent polar moiety in the lipid/water interface and interacts with both the PL polar headgroups and the first part (C8–C10) of the fatty acyl chain region (
      • Andrich M.P.
      • Vanderkooi J.M.
      Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phospholipid artificial membranes.
      ,
      • Kaiser R.D.
      • London E.
      Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth.
      ). The fluorescence polarization of HPE-labeled DEPE liposomes in the presence and absence of OA and TO showed the same trend (Fig. 4A), with an apparent gel-to-liquid crystalline transition temperature of ∼30–35°C, which was not affected significantly by the presence of fats. OA induced a decrease and TO an increase in the HPE-probe polarization in the lamellar phase, Lβ and Lα. However, both types of molecules produced an increase of the fluorescence polarization of HPE-labeled DOPE liposomes, although TO had a significantly higher effect (Fig. 4C). DOPE has an HII structure under the experimental conditions; thus, one expects that increasing the temperature will increase the molecular Brownian movement and therefore will decrease the polarization of the probe. Nevertheless, fluorescence polarization data showed an opposite trend, more acute in the DOPE-OA sample. This peculiar property could be explained considering the X-ray diffraction data of DOPE-OA (20:1; mol/mol) reported previously (
      • Funari 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.
      ), which showed a value for the thermal compression factor (−0.014 nm/°C) significantly lower than for DOPE alone (−0.021 nm/°C), indicating a tighter packing of the lipid molecules with the temperature, which would hinder the rotational movement of the probe. In the other region of the membrane, OA and TO showed a similar effect. The fluorescence polarization of TMA-DPH-labeled DEPE (Fig. 4B) and pyS DHPE-labeled DOPE (Fig. 4D) membranes increased with a higher value for the probe pyS DHPE.
      Figure thumbnail gr4
      Fig. 4Fluorescence polarization versus temperature of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (HPE)-labeled DEPE (A), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate-labeled DEPE (B), HPE-labeled 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (C), and N-(1-pyrenesulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (pyS DHPE)-labeled DOPE (D) liposomes, in the absence or presence of OA or TO. Sample composition was: probe-phospholipid (PL), 1:1000 (mol/mol); PL-OA and PL-TO, 20:1 (mol/mol).

      DISCUSSION

      It has been demonstrated that the composition of some biological membranes is influenced by dietary fats (4–6, 22). Among the biologically unsaturated FAs, the C-18s are important in the fat composition of the Mediterranean diets. Thus, considering the importance and influence of diet on membrane properties, we focused our interest on the effect of unsaturated fats, which are an essential component of biological membranes, on the structural properties of PE membranes.
      From the experimental data reported, it seems quite plausible that membranes can incorporate small amounts of FFA and TG into the bilayer structure, in agreement with previous reports (
      • Hamilton 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.
      ,
      • Hamilton J.A.
      Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.
      ,
      • Smaby J.M.
      • Brockman H.L.
      Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.
      ,
      • Spooner P.J.
      • Small D.M.
      Effect of free cholesterol on incorporation of triolein in phospholipid bilayers.
      ,
      • Gorrissen H.
      • Tulloch A.P.
      • Cushley R.J.
      Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.
      ,
      • Hamilton J.A.
      • Small D.M.
      Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.
      ,
      • Funari 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.
      ,
      • Epand 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.
      ,
      • Langner M.
      • Isac T.
      • Hui S.W.
      Interaction of free fatty acids with phospholipid bilayers.
      ,
      • Ortiz A.
      • Gomez-Fernandez J.C.
      A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes.
      ). But, to our knowledge, this is the first detailed study of the thermotropic phase behavior of PE PLs and TGs. Interestingly, the unsaturated FAs (OA, LL, and LN) and their respective TGs (TO, TLL, and TLN) induced a marked decrease of the Lα-to-HII phase transition temperature in DEPE lipids (Figs. 1, 2). In addition, 31P-NMR measurements done under quasiequilibrium conditions showed that TO, like OA, favored the formation of the HII nonlamellar phase (Fig. 3). Thus, the main effect of the unsaturated FFAs and their TGs would be to stabilize the HII phase, although they showed a differential relative efficiency in the perturbation of the lipid phase properties in the lamellar and inverted hexagonal structures.

      FAs

      The X-ray diffraction data of the FFAs in DEPE liposomes showed that the presence of one, two, or three cis double bonds in the acyl chain, such as in OA, LL, and LN, drastically affected the temperature range of the Lα phase presence, as a unique phase or in coexistence with the nonlamellar HII phase (Table 1). One interesting observation is that the increase in the degree of unsaturation of the FA contributes to increases in the temperature range of existence of the lamellar Lα phase. Also, the onset temperature of a single hexagonal phase was directly related to the degree of unsaturation of the FA. Thus, the interval of temperatures where lamellar and nonlamellar phases coexisted depended on the FA. In addition, the C-18 unsaturated FAs did not alter the lamellar bilayer thickness and had a relatively minor effect on the HII lattice spacing (d = 6.27 nm for DEPE alone, 5.90 nm in the presence of OA, and ≅ 6.2 nm in presence of LL and LN).
      From the structural point of view, several factors could influence the ability of these molecules to determine the lipid phase propensity. A DSC study of FAs with varying degrees of unsaturation demonstrated the importance of the pH in controlling the degree of carboxyl ionization of the FA and the electrostatic repulsions among the bilayers (
      • Epand 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.
      ). It is reported that the pK of FAs is quite insensitive to the unsaturation degree [LL had a pK value 0.1 units higher than stearic acid (SA)]; thus, the ionization state of the C-18 FAs would be about the same, independent of the FA structure. However, the DSC data obtained for the series C-18 and C-22 at pH 7.4 point to the fact that structural factors such us unsaturation and length affect the hexagonal phase-forming ability of the FFAs. Our X-ray diffraction data are in agreement with these previous DSC results with the series C-18; the increase in the unsaturation degree of FAs or their TGs correlates with an increase in the Lα-HII transition temperature (Table 1 and Fig. 2).
      A comparative X-ray diffraction analysis at pH 7.4 of the effect of the C-18 FAs, OA, elaidic acid (EA), and SA showed the importance of the steric disturbance induced by the FA molecular shape on the acyl-chain moiety of the PE membrane (
      • Funari 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.
      ). From these results, a cis double bond (such as OA) would bring the greatest disruption of the lamellar phase, while the presence of a trans double bond (EA) or the absence (SA) affected slightly the HII propensity of PE membranes. These data could be explained by the lipid-packing theory (
      • Cavagnetto F.
      • Relini A.
      • Mirghani Z.
      • Gliozzi A.
      • Bertoia D.
      • Gambacorta A.
      Molecular packing parameters of bipolar lipids.
      ). Another parameter could be the molecular location of FAs within the membrane, which could be influenced by structural properties. Some works have provided evidence;. e.g., spin label electron paramagnetic resonance experiments on 1,2-dimyristoyl-sn-glycero-3-phosphocholine/myristic acid (1:2 molar ratio) mixtures showed that the FA acyl chain was located approximately one methylene group deeper than the sn-2 chain of the PC in the HII phase (
      • Rama Krishna Y.V.
      • Marsh D.
      Spin label ESR and 31P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures.
      ). FAs labeled with spectroscopic probes showed differences between the depth of FAs and the PC fatty acyl chains (
      • Kaiser R.D.
      • London E.
      Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth.
      ). In addition, the carboxyl ionization decreased FA depth in membranes (
      • Abrams F.S.
      • Chattopadhyay A.
      • London E.
      Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.
      ). In our system, the degree of unsaturation could affect the depth of the C-18 FAs in the membrane. Differences in depth could modify the hydrophobic/hydrophilic balance in DEPE-FA membranes, reducing the DEPE headgroup area relative to its volume and increasing the intrinsic monolayer curvature. In this case, the HII phase would be induced at lower temperatures.

      TGs

      The X-ray diffraction data showed that TG, TO, TLL, and TLN were incorporated into DEPE liposomes. Their gel and liquid-crystalline lamellar phases had similar properties, and the structural parameters, such as the lattice spacing and the compressibility factor, were not affected by the nature of the TG molecule (Figs. 2D–F and Table 1). Therefore, there should be little discrimination between the different TG species incorporated into a lamellar phase of a biological membrane. It was proposed that TO incorporated into PC bilayers had the carbonyl groups oriented toward the aqueous interface, with the 1- and 3-carbonyls slightly closer to the aqueous interface than the 2-carbonyl (
      • Hamilton J.A.
      Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.
      ,
      • Gorrissen H.
      • Tulloch A.P.
      • Cushley R.J.
      Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.
      ). Extrapolating this possible model to our PE systems, TO would be integrated in the membrane with the polar headgroup and the acyl chains oriented parallel to the PE molecules. We propose that the presence of the bulky TG molecules in the lamellar DEPE liposomes increases the monolayer bending to force the kinetic formation of an HII structure with a high intercylinder distance (Figs. 2D–F). As the temperature increases, the interface bending provides for an approach of lipid headgroups and forces the slightly polar TG molecule depth into the hydrocarbon matrix. TG homogenization in the membrane will reduce the acyl chain stress up to the final HII structure. TGs could be considered to act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule and in the diameters of the water cylinders. Also, differences in the decrease in the cross-sectional area of DEPE and TG molecules upon dehydration, as the temperature increases, could be responsible for the high negative value of the compressibility factor (Table 1). The structural differences among the different molecules, TO, TLL, and TLN, mainly due to the number of double bonds per acyl chain, would result in differences in the intercylinder distances (Table 1).

      Fluorescence polarization data

      The effect of the FAs or the TGs on the properties of the lamellar and HII phases was analyzed using OA or TO as references. Both types of molecules had a reduced effect on the fluorescence polarization of the probes located in the upper region of the membrane. Their differences came from the hydrophobic acyl region. OA notably increased the fluidity of the acyl chain region in the gel and in the liquid-crystalline lamellar phases (Fig. 4A). An increase in the fluidity would favor a diffusion process across the membrane. Consequently, an important role for the unsaturated FFAs in the biological membranes could be to create a dynamic lipid environment to allow a rapid lateral diffusion of transient molecules within the hydrophobic region of the lipid bilayer. However, OA had a slight effect on the fluorescence polarization of the HPE probe in the DOPE HII structure (∼10%) (Fig. 4C). Because the polarization of the probe depends on the rotational mobility and the degree of restriction of its motion, these results suggest that the increase in fluidity induced by OA is probably counteracted by an increase in the acyl chain packing. Note that DOPE samples showed an increase in the polarization value with the temperature, probably due to a higher packing of the lipid molecules, as was reported in X-ray diffraction studies with DOPE (
      • Tate M.W.
      • Gruner S.M.
      Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes.
      ) and DOPE-OA (
      • Funari 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.
      ). Therefore, the rotational movement of the HPE probe would be hindered as the temperature increased. On the other hand, TO slightly increased the HPE fluorescent polarization in the lamellar phase (∼20% at 37°C) and had a main effect in DOPE HII structures (∼85% at 20°C), probably due to an increase in the microviscosity of the acyl surface. Considering the model proposed from the X-ray diffraction data, if the HII phase stabilization by TG molecule is accompanied by an increase in the depth position of the TG molecule in the lipid matrix, one would expect to get an increase in the HPE polarization, as we observed.
      In summary, the incorporation of an unsaturated FA or a TG into a PE membrane would facilitate the Lα-HII phase transition. However, the temperature range of the presence of the Lα phase was related to the degree of unsaturation of the FA. Although it is rather improbable that TGs are incorporated into cell membranes, it is important to note that their presence in PE liposomes would favor the kinetic process of the transition Lα to HII. The reported data indicate the possibility that the membrane lipid composition could be modulated, e.g., by the FFA composition and/or appropriate metabolic adjustments, to counteract a necessity, such as to preserve the lamellar liquid crystalline phase essential to membrane stability and functions, or facilitate the formation of a transient microdomain prone to an HII structure.

      Acknowledgments

      This work was supported in part by grants FIS 00/1029 and SAF2001-0839 from the Ministerio of Sanidad y Consumo and Ministerio de Ciencia y Tecnología (Spain), by project I-02-066EC from Deutsches Elektronen-Synchrotron DESY, Hasylab (Hamburg, Germany), and by project CAO001-002 of Junta de Andalucía. The authors are grateful to Conselleria d'Innovació i Tecnologia and Sanitat for their financial support.

      REFERENCES

        • Denyer G.S.
        The renaissance of fat: roles in membrane structure, signal transduction and gene expression.
        Med. J. Aust. 2002; 176: 109-110
        • Maury E.
        • Guerineau N.C.
        • Comminges C.
        • Mollard P.
        • Prevost M.C.
        • Chap H.
        Potential role for triglycerides in signal transduction.
        FEBS Lett. 2000; 466: 228-232
        • Alessenko A.V.
        • Burlakova E.B.
        Functional role of phospholipids in the nuclear events.
        Bioelectrochemistry. 2002; 58: 13-21
        • Escudero 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: 65-73
        • Pagnan 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: 87-93
        • Vicario 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: 160-169
        • Escribá 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: 11375-11380
        • Escribá 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: 176-182
        • Bosner 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: 20261-20264
        • Hamilton 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: 773-782
        • Hamilton J.A.
        Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions.
        Biochemistry. 1989; 28: 2514-2520
        • Smaby J.M.
        • Brockman H.L.
        Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.
        J. Biol. Chem. 1987; 262: 8206-8212
        • Spooner P.J.
        • Small D.M.
        Effect of free cholesterol on incorporation of triolein in phospholipid bilayers.
        Biochemistry. 1987; 26: 5820-5825
        • Gorrissen H.
        • Tulloch A.P.
        • Cushley R.J.
        Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.
        Chem. Phys. Lipids. 1982; 31: 245-255
        • Hamilton J.A.
        • Small D.M.
        Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.
        Proc. Natl. Acad. Sci. USA. 1981; 78: 6878-6882
        • Engelbrecht 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: 253-257
        • Hauser H.
        • Howell K.
        • Dawson R.M.
        • Bowyer D.E.
        Rabbit small intestinal brush border membrane preparation and lipid composition.
        Biochim. Biophys. Acta. 1980; 602: 567-577
        • Ntambi J.M.
        • Bene H.
        Polyunsaturated fatty acid regulation of gene expression.
        J. Mol. Neurosci. 2001; 16: 273-278
        • Sumida C.
        • Graber R.
        • Nunez E.
        Role of fatty acids in signal transduction: modulators and messengers.
        Prostaglandins Leukot. Essent. Fatty Acids. 1993; 48: 117-122
        • Khan 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: 3605-3612
        • Litman 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: 237-242
        • Liu 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: 831-837
        • Salem Jr., N.
        • Niebylski C.D.
        The nervous system has an absolute molecular species requirement for proper function.
        Mol. Membr. Biol. 1995; 12: 131-134
        • Storlien 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: 26-43
        • Cullis P.R.
        • de Kruijff B.
        Lipid polymorphism and the functional roles of lipids in biological membranes.
        Biochim. Biophys. Acta. 1979; 559: 399-420
        • Seddon J.M.
        Structure of the inverted hexagonal (HII) phase and non-lamellar phase transitions of lipids.
        Biochim. Biophys. Acta. 1990; 1031: 1-69
        • Turner D.C.
        • Gruner S.M.
        X-ray diffraction reconstruction of the inverted hexagonal (HII) phase in lipid-water systems.
        Biochemistry. 1992; 31: 1340-1355
        • Borovyagin V.L.
        • Sabelnikov A.G.
        Lipid polymorphism of model and cellular membranes as revealed by electron microscopy.
        Electron Microsc. Rev. 1989; 2: 75-115
        • Epand R.
        Lipid Polymorphism and Membrane Properties.
        Academic Press, San Diego, CA1997: 25-102 (167–189.)
        • Luzzati V.
        Biological significance of lipid polymorphism: the cubic phases.
        Curr. Opin. Struct. Biol. 1997; 7: 661-668
        • De Kruijff B.
        Biomembranes. Lipids beyond the bilayer.
        Nature. 1997; 386: 129-130
        • Bogdanov 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: 11615-11618
        • Boggs J.M.
        Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function.
        Biochim. Biophys. Acta. 1987; 906: 353-404
        • Gudi 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: 2515-2519
        • Giorgione 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: 9767-9770
        • Rietveld 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: 5506-5513
        • Funari 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: 567-575
        • Epand 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: 75-80
        • Langner M.
        • Isac T.
        • Hui S.W.
        Interaction of free fatty acids with phospholipid bilayers.
        Biochim. Biophys. Acta. 1995; 1236: 73-80
        • Ortiz 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: 75-91
        • Boulin 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: 399-407
        • Boulin 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: 312-320
        • Andrich M.P.
        • Vanderkooi J.M.
        Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phospholipid artificial membranes.
        Biochemistry. 1976; 15: 1257-1261
        • Kaiser R.D.
        • London E.
        Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth.
        Biochemistry. 1998; 37: 8180-8190
        • Cavagnetto F.
        • Relini A.
        • Mirghani Z.
        • Gliozzi A.
        • Bertoia D.
        • Gambacorta A.
        Molecular packing parameters of bipolar lipids.
        Biochim. Biophys. Acta. 1992; 1106: 273-281
        • Rama Krishna Y.V.
        • Marsh D.
        Spin label ESR and 31P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures.
        Biochim. Biophys. Acta. 1990; 1024: 89-94
        • Abrams F.S.
        • Chattopadhyay A.
        • London E.
        Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.
        Biochemistry. 1992; 31: 5322-5327
        • Tate M.W.
        • Gruner S.M.
        Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes.
        Biochemistry. 1989; 28: 4245-4253