Eutectic phase behavior of 1-stearoyl-2-caprylphosphatidylcholine and dimyristoylphosphatidylcholine mixtures

The thermotropic behavior of aqueous dispersions of C(18):C(10)PC/diC(14)PC mixtures with different molar ratios has been investigated by high-resolution differential scanning calorimetry. C(18):C(10)PC is a highly asymmetric lipid molecule, whereas diC(14)PC is a symmetric species with the same molecular weight. Their packing properties in the bilayer are known to be similar at T greater than Tm, but very dissimilar at T less than Tm. Calorimetric results indicate that C(18):C(10)PC and diC(14)PC are completely miscible in the liquid-crystalline state. In the gel state, however, C(18):C(10)PC and diC(14)PC are only partially miscible. The temperature-composition phase diagram for C(18):C(10)PC/diC(14)PC mixtures has the shape characteristic of a typical eutectic system.     

On the peritectic phase behavior of dimyristoyllecithin/distearoyllecithin mixtures. A densitometric study

Multilamellar liposomes of mixtures of dimyristoyllecithin and distearoyllecithin are investigated by mass densitometry. It is shown that the temperature-dependent volumetric properties of this lipid alloy are best described on the basis of a peritectic phase diagram.     

Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures

Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfacial region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylglycerol (DG). Infrared vibrational frequencies of the CH2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L beta-L alpha) and the L alpha to inverted hexagonal (L alpha-HII) phase transitions were observed at approximately 15 degrees and 55 degrees C, respectively. As the DG content increased gradually from 0% to 15%, the L alpha-HII phase transition temperature decreased drastically while the L beta-L alpha phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at approximately 10 degrees C. For the composition study at 23 degrees C, the L alpha-HII transition occurred at approximately 6-10% DG as indicated by abrupt increases in both the CH2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, in these PE/DG mixtures were observed at the L alpha-HII phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientational packing of the lipids were altered at the composition-induced L alpha-HII transition, and agreed with a previous temperature-induced L alpha-HII transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025-1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L alpha-HII phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the L alpha-HII transition.     

Effects of cholesterol and temperature on the permeability of dimyristoylphosphatidylcholine bilayers near the chain melting phase transition

The passive leakage of glucose across bilayers of dimyristoylphosphatidylcholine (DMPC), cholesterol (variable), and dicetyl phosphate (constant 5.9 mol%) has been measured as efflux over 30 min from multilamellar vesicles. Bilayer cholesterol was varied from 20 mol% to 40 mol%. Glucose permeation rates were measured from 10 degrees C to 36 degrees C, and showed a maximum in permeability at 24 degrees C, the DMPC phase transition temperature. Increasing the bilayer cholesterol content above 20 mol% reduced that permeability peak. These results are quite consistent with a large number of similar bilayer permeability studies over the past 25 years. However, they are not consistent with a previous study of these same systems, which reported increased glucose permeability with temperature, without any maximum at or near the lipid chain melting temperature (K. Inoue, Biochim. Biophys. Acta 339 (1974) 390-402).     

Surface rheology and phase transitions of monolayers of phospholipid/cholesterol mixtures

The dynamic surface elasticity and the surface dilational viscosity of three binary phospholipid/cholesterol mixtures were determined with axisymmetric drop shape analysis on a harmonically oscillating pendent drop. Dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine were used to explore the rheological properties and phase transitions of mixtures of saturated and unsaturated phospholipids with cholesterol. The growth rates for surface dilational viscosity and dynamic elasticity are parallel for all film pressures studied. Characteristic breaks and plateaus could be found for these growth rates, indicating phase transitions. For dipalmitoylphosphatidylcholine/cholesterol and dimyristoylphosphatidylcholine/cholesterol mixtures, phase diagrams with six regions separated by phase boundaries were found, which are in good agreement with phase transitions reported in the literature for static measurements of isotherms and isobars on a Langmuir film balance and from fluorescence microscopy. Some phase boundaries were only found by dynamic, but not by static, elasticity measurements. Imaging methods revealed phase separations produced by the formation of condensed stoichiometric complexes leading to micron-sized and mostly circular domains. The effects of these complexes on monolayer rheology in liquid/liquid phases is described. Furthermore, liquid/solid and solid phase transitions are discussed.     

A densitometric study of the effects of free fatty acids on the phase transition of dimyristoylphosphatidylcholine bilayers

The technique of scanning densitometry was applied to a study of the effect of fatty acids on the packing and phase transition properties of dimyristolyphosphaticylcholine (DMPC). It was observed that the three fatty acids studied, i.e. palmitic, palmitoleic and behenic, had the effect of broadening the phase transition and increasing the extent of the change in volume which occurs at the phase transition. Palmitic and behenic acids broaden the phase transition toward higher temperatures while palmitoleic broadens it toward lower temperatures.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and 31P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (approximately 11-15 degrees C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (approximately 23-25 degrees C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing approximately 30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the Lc phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the Lc phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the Lc phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol

The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to approximately 25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base-containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.     

A time-resolved fluorescence anisotropy study of bilayer membranes containing alpha-tocopherol

Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing alpha-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing alpha-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly.     

Steady-state fluorescence anisotropy and multifrequency phase fluorometry on oxidized phosphatidylcholine vesicles

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.     

Ceramide drives cholesterol out of the ordered lipid bilayer phase into the crystal phase in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol/ceramide ternary mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.     

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers.

The ripple structure was studied as a function of temperature in fully hydrated dimyristoylphosphatidylcholine (DMPC)/cholesterol multibilayers using synchrotron x-ray small-angle diffraction and freeze-fracture electron microscopy. In the presence of cholesterol, the ripple structure appears below the pretransition temperature of pure DMPC multibilayers. In this temperature range the ripple periodicity is relatively large (25-30 nm) and rapidly decreases with increasing temperature. In this region, defined as region I, we observed coexistence of the P beta' phase and the L beta' phase. The large ripple periodicity is caused by the formation of the P beta' phase region in which cholesterol is concentrated and the L beta' phase region from which cholesterol is excluded. An increase in ripple periodicity also takes place in the narrow temperature range just below the main transition temperature. We define this temperature region as region III, where the ripple periodicity increases dramatically toward the main transition temperature. In region II, between regions I and III, the ripple periodicity decreases gradually with temperature. This behavior is quite similar to that of pure DMPC. Temperature-versus-ripple periodicity curves are parallel among pure DMPC and DMPCs with various cholesterol contents. We explain this behavior in terms of a model proposed by other workers.     

The phase behavior of hydrated cholesterol.

The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.     

Comparative effects of cholesterol and cholesterol sulfate on hydration and ordering of dimyristoylphosphatidylcholine membranes.

The comparative effect of cholesterol (CH) versus cholesterol sulfate (CS) on dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by optical microscopy, freeze-fracture electron microscopy, x-ray diffraction, and solid state 2H and 31P nuclear magnetic resonance (NMR). The sulfate analogue extends the lamellar phase domain toward high water contents, and substitution of 30 mol % CH by CS in DMPC lamellae induces the trapping of 30 wt % additional water. The greater swelling of the CS-containing systems is evidenced by determination of lamellar repeat distances at maximal hydration: 147 +/- 4 A and 64 +/- 2 A in the presence of CS and CH, respectively. 2H-NMR of heavy water demonstrates that CS binds approximately 12 more water molecules at the interface than CH whereas NMR of deuterium-labeled DMPC chains reveals that 30 mol % CS orders the membrane as 15 mol % CH at high temperature and disorders much more than CH at low temperatures. The various effects of CS versus CH are discussed by taking into account attractive Van der Waals forces and repulsive steric/electrostatic interactions of the negatively charged sulfate group.     

Phase Equilibria in binary mixtures of dimyristoylphosphatidylcholine and cardiolipin

Paramagnetic resonance spectra of the spin-label 2,2,6,6-tetramethylpiperidinyl-l-oxy have been used to study phase separations in binary mixtures of dimyristoyl-phosphatidylcholine and cardiolipin. Two different samples of cardiolipin were used: (i) One sample contained calcium ions at a mole ratio of calcium:cardiolipin = 1:2; the experimental data support the view that cardiolipin is present in the bilayer membrane as calcium ion linked dimers, (CL)2 Ca2+. (ii) A calcium-free sodium cardiolipin sample yielded remarkable spin-label partition data that were quite different from those obtained in the presence of Ca2+. In both cases the spin-label data provide evidence for compound formation and for fluid-fluid immiscibility in the bilayer membrane.