Temperature-dependent molecular motions and phase behavior of cholesteryl ester analogues

The phase behavior and temperature-dependent molecular motions of three cholesteryl ethers (caproyl, myristyl, oleyl) and a cholesteryl carbonate (oleyl) were characterized. The properties of each ether were qualitatively similar to, but quantitatively different from, those of the corresponding cholesteryl ester. For example, cholesteryl oleyl ether exhibited the same phase transitions as cholesteryl oleate, but at much lower temperatures (e.g., the ether isotropic liquid to cholesteric transition is at 29 degrees C). 13C NMR spectra of ethers in the isotropic liquid and liquid crystalline phases were similar to those of the ester analogue. However, near the liquid to liquid crystalline transition, the steroid ring C3 and C6 linewidths, the C3/C6 linewidth ratio, and the steroid ring rotational correlation times tau rx and tau rz calculated from the linewidths were larger for the ether than the ester analogue. The oleyl carbonate had qualitatively different properties from its analogues (e.g., stable vs. metastable cholesteric and smectic phases). Quantitative results (e.g., relatively long tau rx and tau rz in the isotropic liquid phase) for the carbonate were also distinct from those of both the ester and ether analogues. A comparison of analogues in which the polar linkage is the only structural variable yielded insights into the intermolecular interactions which influence phase behavior.     

Physical properties of cholesteryl esters

Cholesteryl esters, the intracellular storage form and intravascular transport form of cholesterol, can exist in crystal, liquid crystal and liquid states. The physical state of cholesteryl esters at physiologic temperatures may be a determinant of their pathogenicity. This review has surveyed saturated aliphatic cholesteryl esters of chain length 1 to 24 carbons and a series of medium-chained unsaturated cholesteryl esters from chain lengths 14 to 24 carbons. A systematic study of transition temperatures by polarizing microscopy and enthalpies by differential scanning calorimetry has provided unifying concepts concerning the phase behavior as a function of chain length and unsaturation. Neat cholesteryl esters show chain-length dependence of transition temperature and enthalpy of both the crystal and liquid crystal transitions. Double bond position along the fatty acyl chain affected stability of the liquid crystal phases; a smectic phase was not observed for any cholesteryl ester with a double bond more proximal than delta 9. 13C NMR spectroscopy in the isotropic liquid phase has provided evidence suggesting a balance of ring-ring vs. chain-chain interactions as a determinant for isotropic liquid----cholesteric vs. isotropic liquid----smectic transitions. Specifically, anisotropic molecular motions of the steroid ring are greater for cholesteryl esters forming a cholesteric phase than a smectic phase from the melt. Chain-chain interactions apparently predominate in smectic phase formation. The X-ray diffraction patterns of cholesteryl esters as a function of chain length reveal several isostructural series and known single crystal data are presented. A chain length depending on the periodicity of the smectic phase is observed which may be different for saturated vs. unsaturated esters. In summary, the phase behavior of cholesteryl ester molecules is complex and cannot be determined a priori from the phase behavior of component cholesterol and fatty acid. The data presented here should provide insight into the biological behavior of this lipid class.     

The binary phase diagram of lecithin and cholesteryl linolenate

The condensed binary phase diagram of cholesteryl linolenate-egg yolk lecithin has been determined by polarizing light microscopy, differential scanning calorimetry and X-ray diffraction. On increasing the temperature lecithin forms rectangular, cubic and hexagonal liquid-crystalline structures into which varying amounts of cholesteryl linolenate are incorporated. As more cholesteryl linolenate is incorporated, the transition temperatures between different phases are lowered. The rectangular and cubic structures incorporate only small amounts of cholesteryl linolenate; the molar ratios, lecithin to cholesteryl linolenate, being 11:1 and 16:1, respectively. However, the hexagonal phase, in which the phosphorylcholine groups of the lecithin molecules form the core of the rod-like assembly of molecules, incorporates up to approximately 25% cholesteryl linolenate by weight, corresponding to a molar ratio 3:1. At higher concentrations, cholesteryl linolenate forms an excess phase and may be present as crystals, smectic or cholesteric liquid crystals, or as liquid oil, depending on the temperature. At higher temperatures, a large zone of a single isotropic liquid phase exists in which large amounts of lecithin are solubilized by the cholesterol ester. Up to 40% cholesteryl linolenate by weight, the transition temperatures between different phases are influenced by approximately 1% water (by weight) associated with egg lecithin.It is probable that the incorporated apolar cholesterol ester molecules are associated primarily with the apolar hydrocarbon chain region of the different lecithin structures. The resultant decrease in the observed transition temperatures would suggest an overall chain-disordering role for the incorporated cholesteryl linolenate molecules. The influence of cholesteryl linolenate on the thermodynamic stability of the different lecithin structures, together with the models suggested for the molecular orientations of cholesterol esters in the different liquid crystalline structures, may be relevant to the role of these lipids in more complex biological systems, particularly serum lipoproteins.     

Modulation of the physical state of cellular cholesteryl esters by 4,4'-(isopropylidenedithio)bis(2,6-di-t-butylphenol) (probucol).

The effect of 4,4'-(isopropylidenedithio)bis(2,6-di-t-butylphenol) (probucol) on cholesteryl ester physical state was examined in dry mixtures, phospholipid-containing dispersions, and cells. Probucol has little effect on the solid to isotropic transition of cholesteryl oleate, but broadens and decreases the enthalpy of the liquid-crystalline transitions at concentrations as low as 1-2 mol %. A probucol transition is only observed at concentrations greater than 20 mol %. The mesomorphic phases of the cholesteryl oleate/probucol mixtures were identified by visual inspection and polarized light microscopy. Mixtures are liquid at probucol concentrations in excess of 5 mol % at 37 degrees C. Probucol also dramatically reduces the enthalpy of the liquid-crystalline transitions of the cholesteryl oleate core of dispersions of the ester with phospholipids at a concentration of 10 mol %, reducing the enthalpy by greater than 80% and the transition temperatures by approximately 2 degrees C. The phase state of cholesteryl esters in Fu5AH rat hepatoma cells was examined after incubation with cholesterol/phospholipid dispersions that caused the accumulation of anisotropic cholesteryl ester droplets. Differential scanning calorimetry scans of cells incubated with cholesterol-rich phospholipid dispersions indicated a phase transition near 48 degrees C, which was abolished when the cells were co-incubated with 50-100 micrograms/ml of probucol in the loading medium. Subsequent to the formation of isotropic cholesteryl ester droplets in the presence of probucol, the rate of efflux of cholesterol from the cells to phosphatidylcholine-containing acceptors in the medium was increased. These data show that probucol is relatively soluble in cholesteryl esters and that probucol changes the phase state of cholesteryl ester droplets in cells to a more fluid phase in which the cholesteryl esters are more readily mobilized.     

Phase behavior and crystalline structures of cholesteryl ester mixtures: a C-13 MASNMR study.

Cholesteryl esters are a transport and storage form of cholesterol in normal physiology but also a significant lipid in atherosclerotic plaques. To understand better the molecular properties of cholesteryl esters in tissues and plaques, we have studied the polymorphic and mesomorphic features of pure and mixed cholesteryl esters by solid state C-13 NMR with magic angle sample spinning (MASNMR). The temperature-dependent properties of two single components (cholesteryl linoleate (CL, C18:2) and cholesteryl linolenate (CLL, C18:3)), four binary systems (cholesteryl palmitate (CP, C16:0) with CL, CLL or cholesteryl oleate (CO, C18:1), and CO/CL), one ternary system (CO/CP/CL), and one quaternary system (CO/CP/CL/CLL) were studied. The mixing ratios were based on the composition of an atherosclerosis plaque dissected from a cholesterol-fed New Zealand white rabbit. C-13 MASNMR determined the phase transition temperatures, identified the phases present in all systems, and provided novel information about molecular structures. For example, solid CL exhibited a disordered structure with multiple molecular conformations, whereas pure CLL had a crystalline structure different from the three most commonly characterized forms (MLII, MLI, BL). In binary mixtures, the crystalline structure of each cholesteryl ester species was identified by its own characteristic resonances. It was found that CP always existed in its native BL form, but CL and CO were influenced by the composition of the mixture. CL was induced to form MLII crystals by the coexisting CP (55 wt%). When CO was cooled from the isotropic phase, it existed as a mixture of MLII and an amorphous form. The presence of CP significantly accelerated the conversion of the amorphous form to the MLII form. For the ternary mixture co-dried from chloroform, CL cocrystallized with CO in the MLII form and CP existed in BL form. Addition of a small amount of CLL slightly increased the heterogeneity of the solid mixture, but had little effect on the crystal structures or the phase transitions. C-13 MASNMR represents a powerful method for physical characterization of cholesteryl ester mixtures reflecting the composition of biological samples.     

The ternary phase diagram of lecithin, cholesteryl linolenate and water: phase behavior and structure

The ternary phase diagram of cholesteryl linolenate-egg lecithin-water has been determined by polarizing light microscopy, calorimetry and X-ray diffraction at 23 °C. Hydrated lecithin forms a lamellar liquid-crystalline structure into which small amounts of cholesteryl linolenate are incorporated. The maximum incorporation of cholesterol ester into this lamellar structure varies with the degree of hydration. Increasing the water concentration from 10 to 15% (w/w) increased the limiting molar ratio of cholesteryl linolenate to lecithin in the lamellar phase from 1:50 to 1:22. At intermediate concentrations (15 to 30% water) the cholesteryl linolenate:lecithin ratio remains constant at 1:22. When water is increased to 42.5%, the maximum water content in the lamellar phase, the molar ratio decreased to 1:32. At low water concentrations the cholesterol ester appears to be entirely in the apolar region of the lecithin bilayer, while at higher water concentrations the ester groups of cholesteryl linolenate may be located at the lipid-water interface. At high water concentrations the ester appears to disorder the alkyl chains of the lecithin, giving rise to a thinner lipid layer and an increased surface area per lipid molecule when compared to the lecithin-water system in the absence of cholesteryl linolenate.The lamellar phase is the only phase (except at water concentrations less than 5%) in which all three components mutually interact. All mixtures of the three components having compositions outside the one-phase (lamellar) zone produce additional phases of cholesteryl linolenate or water, or both. Between 23 °C and 60 °C only minor changes in the phase diagram are observed.     

13C MAS NMR studies of crystalline cholesterol and lipid mixtures modeling atherosclerotic plaques.

Cholesterol and cholesteryl esters are the predominant lipids of atherosclerotic plaques. To provide fundamental data for the quantitative study of plaque lipids in situ, crystalline cholesterol (CHOL) and CHOL/cholesteryl ester (CE) mixtures with other lipids were studied by solid-state nuclear magnetic resonance with magic-angle-sample spinning. Highly distinctive spectra for three different crystalline structures of CHOL were obtained. When CHOL crystals were mixed with isotropic CE oil, solubilized CHOL (approximately 13 mol % CHOL) was detected by characteristic resonances such as C5, C6, and C3; the excess crystalline CHOL (either anhydrous or monohydrate) remained in its original crystalline structure, without being affected by the coexisting CE. By use of 13C-enriched CHOL, the solubility of CHOL in the CE liquid-crystalline phase (approximately 8 mol %) was measured. When phosphatidylcholine was hydrated in presence of CHOL and CE, magic-angle-sampling nuclear magnetic resonance revealed liquid-crystalline CHOL/phosphatidylcholine multilayers with approximately an equal molar ratio of CHOL/phosphatidylcholine. Excess CHOL existed in the monohydrate crystalline form, and CE in separate oil or crystalline phases, depending on the temperature. The magic-angle-sampling nuclear magnetic resonance protocol for identifying different lipid phases was applied to intact (ex vivo) atherosclerotic plaques of cholesterol-fed rabbits. Liquid, liquid-crystalline, and solid phases of CE were characterized.     

The phase behavior of cholesteryl esters in intracellular inclusions.

Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.     

Thermotropic properties and molecular dynamics of cholesteryl ester rich very low density lipoproteins: effect of hydrophobic core on polar surface

Cholesteryl ester rich very low density lipoproteins (CER-VLDL), isolated from the plasma of rabbits fed a hypercholesterolemic diet, have been studied by differential scanning calorimetry (DSC), 13C nuclear magnetic resonance (NMR), and spin-label electron paramagnetic resonance (EPR) to determine the temperature-dependent dynamics of cholesteryl esters in the hydrophobic core and of phospholipids on the polar surface. Intact CER-VLDL exhibit two DSC heating endotherms; these occur at 40-42 and 45-48 degrees C. Cholesteryl esters isolated from CER-VLDL also exhibit two DSC endotherms; these occur at 50.0 and 55.1 degrees C and correspond to the smectic----cholesteric and cholesteric----isotropic liquid-crystalline phase transitions. A model mixture containing cholesteryl linoleate, oleate, and palmitate in a ratio (0.21, 0.51, and 0.28 mol fraction) similar to that in CER-VLDL exhibited comparable DSC endotherms at 45.2 and 51.5 degrees C. CER-VLDL at 37 degrees C gave 13C NMR spectra that contained no resonances assignable to cholesteryl ring carbons but detectable broad resonances for some fatty acyl chain carbons, suggesting the cholesteryl esters were in a liquid-crystalline state. When the mixture was heated to 42 degrees C, broad ring carbon resonances became detectable; at 48 degrees C, they became narrow, indicating the cholesteryl esters were in an isotropic, liquid-like state. With increasing temperature over the range 38-60 degrees C, the resonances for cholesteryl ring carbons C3 and C6 in CER-VLDL narrowed differentially. Similar spectral changes were observed for the synthetic cholesteryl ester mixture, except they occurred at temperatures about 10 degrees C higher. These results indicate that the two DSC transitions in CER-VLDL do not directly correlate with the smectic----cholesteric and cholesteric----isotropic transitions exhibited by pure cholesteryl esters. (5-Doxylpalmitoyl)-phosphatidylcholine (5-DP-PC) and (12-doxylstearoyl)phosphatidylcholine (12-DS-PC) were used to probe the polar surface monolayer of CER-VLDL; the corresponding cholesteryl esters (5-DP-CE and 12-DS-CE) were used to probe the hydrophobic core. None of these probes in CER-VLDL detected an abrupt change in EPR order parameters, S, or maximum splitting, 2T max, over the temperature range 20-58 degrees C even though 12-DS-PC and 5-DP-PC can detect phase transitions in phospholipid bilayers and 12-DS-CE and 5-DP-CE can detect phase transitions in neat cholesteryl esters. However, 12-DS-CE and 5-DP-CE did detect a much greater acyl chain order for the neutral lipids of CER-VLDL than for those of normal triglyceride-rich VLDL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Triolein-cholesteryl oleate-cholesterol-lecithin emulsions: structural models of triglyceride-rich lipoproteins

The organization of lipids within emulsions composed of triolein (TO), cholesteryl oleate (CO), cholesterol (C), and egg yolk phosphatidylcholine (L) was examined. CO was substituted for TO in a series of emulsions to obtain TO:CO ratios comparable to the triglyceride:cholesterol ester ratios observed in subfractions of triglyceride-rich lipoproteins. The weight fraction of TO in the surface phase (0.02-0.05) was independent of the TO content of the emulsions. However, the weight fraction of CO in the surface phase depended upon the percentage of CO in the emulsions and was less than 0.004 even when 13.7% CO was present in the emulsion. When CO was substituted for TO, the percent of the total particle C which was carried in the droplet oil phase was increased. The interparticle equilibration of lipids was studied in subfractions of sonicated emulsions with particle sizes comparable to triglyceride-rich lipoproteins. The TO:CO ratios of the subfractions of a given emulsion were constant and independent of size, but the C:L ratio decreased in particles of smaller diameter. However, the surface C:L ratio was the same in all particles from a given emulsion. The size dependence of the C:L ratios was attributed to the partitioning of C into the oil cores of the emulsions. Because large droplets have the greatest core:surface mass ratios, more of their total particle C is carried in the core.     

Physical state of cholesteryl esters deposited in cultured macrophages

J774 macrophages load with cholesteryl ester (CE) when incubated with acetylated low-density lipoprotein and cholesterol-rich liposomes; the CE accumulates as cytoplasmic inclusions 1-2 micron in diameter. The CE core of the droplet comprises about 90% of its mass; the predominant CE species present are cholesteryl palmitate (CP, 41%) and cholesteryl oleate (CO, 37%). The thermotropic properties of the inclusions, both in intact cells and after isolation, have been characterized by differential scanning calorimetry. On heating, the inclusions exhibit two endothermic transitions at about 41 and 53 degrees C with a total enthalpy of 7.7 +/- 1.2 cal/g of CE. Very similar thermal behavior is exhibited by a binary mixture containing equal weights of CO and CP; this indicates that these two species dominate the phase behavior of CE in J774 inclusions. A phase diagram for the CO/CP system has been generated, and this reflects simple eutectic behavior. The eutectic is 83% w/w CO, and it melts at 49-50 degrees C. Below this temperature, CO and CP form two immiscible crystalline phases due to the very limited ability of the unsaturated oleate and saturated palmitate acyl chains to mix in the crystal phase. On heating a 1/1 w/w CO/CP mixture, an isotropic liquid of eutectic composition forms at 49 degrees C, and the remaining crystalline cholesteryl palmitate melts over the temperature range 50-69 degrees C. The phase diagram indicates that bulk mixtures of CE molecules in J774 inclusions should be crystalline at 37 degrees C, the growth temperature of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermal transitions and structural properties of 5alpha-cholestan-3beta-ol esters of aliphatic acids.

5 alpha-Cholestan-3 beta-ol esters of aliphatic acids undergo both enantiotropic and monotropic changes of state. Ten saturated and three unsaturated esters have been examined by differential scanning calorimetry and polarizing microscopy to determine transition temperatures, enthalpies, and entropies. The results are compared with an analogous series of cholesterol esters. All esters of even-numbered n-alkanoic acids from C2 to C20 melt from a crystalline state to an isotropic liquid. The crystalline state has been studied by X-ray powder diffraction. The C8 to C20 esters have progressively increasing crystalline melting transition temperatures from 76 to 99 degrees C and possess similar X-ray powder diffraction patterns, suggesting that these compounds form an isostructural series. Esters of C2, C4, and C6 acids exhibit polymorphism. Crystalline cholestanol oleate melts to an isotropic liquid, whereas cholestanol linoleate and linolenate fail to crystallize, even after several months at -20 degrees C. Esters of the even-numbered saturated acids from C4 to C14 form monotropic cholesteric liquid crystalline phases. Esters C10, C12, and C14 form smectic liquid crystalline phases. Cholestanol oleate, linoleate, and linolenate form both cholesteric and smectic mesophases. The lower smectic to cholesteric and cholesteric to isotropic transition temperatures of the cholestanol esters compared to the corresponding transition temperatures of the analogous cholesterol esters suggest that the delta 5 double bond in cholesterol increases the thermal stability of the mesophases of cholesterol esters.     

Cellular cholesteryl ester clearance. Relationship to the physical state of cholesteryl ester inclusions

The hypothesis that clearance of cellular cholesteryl ester deposits may be a function of the physical state of the stored lipid has been investigated. Cultured rat hepatoma cells were induced to store cholesteryl ester in either anisotropic inclusions by exposure to free cholesterol-rich phospholipid dispersions or isotropic inclusions by exposure to identical dispersions supplemented with oleic acid. Differential scanning calorimetry demonstrated an order/disorder transition at 43 degrees C for cholesteryl esters stored in anisotropic inclusions; the enthalpy of this transition was consistent with a smectic liquid crystalline to liquid transition. Lipids in cells with isotropic inclusions displayed no order/disorder transitions over the range 20-80 degrees C, indicating that the lipids are in a liquid state. The presence of oleic acid did not influence the mass of cholesteryl ester stored but increased the amount of stored triglyceride. Fatty acyl compositions of the cholesteryl esters were different under the two loading conditions; in particular, there was 38% cholesteryl oleate in anisotropic inclusions and 65% cholesteryl oleate in isotropic inclusions. Kinetics of cholesteryl ester clearance from cells with either anisotropic or isotropic inclusions were studied during a 12-h exposure to acceptors of free cholesterol. In both cases, cholesteryl ester clearance is essentially linear over 12 h and is directly proportional to the initial content of cholesteryl ester. However, the fraction of initial content of cholesteryl ester cleared in 12 h is 0.17 +/- 0.05 for cells with anisotropic inclusions and 0.34 +/- 0.09 for cells with isotropic inclusions. Our data demonstrate that the more rapid clearance of cholesteryl ester by cells with isotropic inclusions can be correlated with the physical state of the cholesteryl ester.     

Enantioselective separations using chiral supported liquid crystalline membranes

Porous and nonporous supported liquid crystalline membranes were produced by impregnating porous cellulose nitrate supports with cholesteric liquid crystal (LC) materials consisting of 4-cyano-4'-pentylbiphenyl (5CB) mixed with a cholesterol-based dopant (cholesteryl oleyl carbonate [COC], cholesteryl nonanoate [CN], or cholesteryl chloride [CC]). The membranes exhibit selectivity for R-phenylglycine and R-1-phenylethanol because of increased interactions between the S enantiomers and the left-handed cholesteric phase. The selectivity of both phenylglycine and 1-phenylethanol in 5CB/CN membranes decreases with effective pore diameter while the permeabilities increase, as expected. Phenylglycine, which is insoluble in the LC phase, exhibits no transport in the nonporous (completely filled) membranes; however, 1-phenylethanol, which is soluble in the LC phase, exhibits transport but negligible enantioselectivity. The enantioselectivity for 1-phenylethanol was higher (1.20 in 5CB/COC and 5CB/CN membranes) and the permeability was lower in the cholesteric phase than in the isotropic phase. Enantioselectivity was also higher in the 5CB/COC cholesteric phase than in the nematic phase of undoped 5CB (1.03). Enantioselectivity in the cholesteric phase of 5CB doped with CC (1.1), a dopant lacking hydrogen bonding groups, was lower than in the 5CB/COC phases. Finally, enantioselectivity increases with the dopant concentration up to a plateau value at approximately 17 mol%.     

Lipid structure and the behavior of cholesteryl esters in monolayer and bulk phases.

The behavior of cholesteryl esters at the air-buffer interface was studied as a function of molecular area and the presence of noncholesterol-containing lipids (colipids). The data obtained indicate that cholesteryl esters with other than long, saturated acyl groups can be present in surface phases up to packing densities approximately those in natural membranes. Their apparent molecular areas in such phases, which are largely determined by colipid structure, suggest their orientation with the ester function toward the interface. The extent of miscibility in the surface phase is also a strong function of colipid structure. Reversibility of the monolayer to bulk phase transition is determined exclusively by the acyl structure of the cholesteryl ester. Of the esters examined, only those with cis unsaturation collapsed reversibly. Our data predict that cholesteryl esters should be present in small, but finite amounts on the surface of arterial lipid deposits and that a prerequisite for the removal of such deposits is that the bulk lipid phase be in a liquid or liquid crystalline state.     

Thermotropic mesomorphism of cholesteryl myristate. An electron diffraction study

Electron diffraction measurements on heated or cooled microcrystals of cholesteryl myristate, which are grown from solution or epitaxially, on benzoic acid, provide further structural information about its mesomorphic behavior. At subambient temperatures (less than -65 degrees C), a new crystal form is observed which doubles the unit cell axes in the (001) plane. At the major crystalline in equilibrium with smectic endotherm at 70 degrees C, evidence is found for the existence of a pretransition crystal packing. The smectic phase, which coexists with this pretransition crystal form, is composed of relatively well-ordered layers, probably with a monolayer-type packing. Cooling the cholesteric phase to the crystalline form causes a rotational disorder which is not yet understood.     

Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microemulsions of cholesteryl oleate and dimyristoylphosphatidylcholine: a model for cholesteryl ester rich very low density lipoproteins

This study describes the preparation, purification, and characterization of a cholesteryl oleate/dimyristoylphosphatidylcholine microemulsion as a model for the interaction of lipid domains in cholesteryl ester rich very low density lipoproteins. These lipids were chosen specifically because their thermal transitions were distinct from each other, and their differences in chemical structure permitted the motion(s) of each lipid component to be monitored independently by 13C nuclear magnetic resonance (NMR). The model particles were formed by cosonication of cholesteryl oleate and dimyristoylphosphatidylcholine in a 4:1 molar ratio for 45 min at 55-60 degrees C (above both lipid phase transition temperatures). The crude microemulsion was fractionated by low-speed centrifugation and Sepharose CL-2B chromatography. Microemulsion particles which eluted from the column at a volume similar to that of cholesteryl ester rich very low density lipoproteins had high cholesteryl ester:phospholipid ratios (2.5:1----6:1). Electron micrographs of negatively stained particles showed them to be large spheres devoid of multilamellar or unilamellar vesicle structures. Particle size calculated from a simple compositional model correlated well with sizes determined by electron microscopy (500-1000 A) for various column fractions. Differential scanning calorimetry studies of the microemulsion revealed two thermal transitions for the model particles, at 31.0 and 46.6 degrees C, which were tentatively assigned to the surface phospholipid and core cholesteryl ester domains, respectively. These assignments were confirmed by 13C NMR which demonstrated that, at temperatures near the lower thermotropic transition, only resonances derived from carbon atoms of dimyristoylphosphatidylcholine (DMPC) were observable. As the temperature was raised to 38.6 degrees C, resonances from the olefinic carbons in the cholesteryl ester acyl chain appeared in the spectrum. At 46.6 degrees C, the center of the higher temperature endotherm, resonances from both the steroid ring and remaining acyl chain carbons of cholesteryl oleate became observable in the spectrum. Further increases in temperature did not result in the appearance of new resonances; however, those that were present narrowed and increased in intensity. The elevation in transition temperature for DMPC in these particles (31 degrees C) as compared to that for DMPC in small unilamellar (18 degrees C) and large multilamellar (23 degrees C) vesicles suggested a stabilization of the phospholipid monolayer, possibly by interaction with the nonpolar core lipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fourier-transform infrared spectroscopy study of dioleoylphosphatidylcholine and monooleoylglycerol in lamellar and cubic liquid crystals

The liquid-crystalline phases of the systems monooleoylglycerol (MO)/water, dioleoylphosphatidylcholine (DOPC)/water, and MO/DOPC/water have been studied by Fourier-transform infrared (FTIR) spectroscopy. In the latter ternary system, the sn-3 OH group of MO competes with water to interact with the polar head group of DOPC, and an intramolecular hydrogen bonding of MO is broken up. The hydration of the ester carbonyl groups in the lamellar phases of both the MO/water and DOPC/water systems increases with increasing water content. Similarly, the addition of small amounts either of MO to a DOPC/water system or of DOPC to an MO/water system also results in an increase in the hydration of the ester carbonyl groups. This leads to an unfavorable hydrocarbon-water contact which is counteracted by the formation of a cubic phase, except for the DOPC/water system, where the lamellar phase is stable also at the highest water concentrations. The phase behavior of the different systems can be described in terms of lipid monolayer curvature and molecular packing in the lipid aggregates. Finally, it is shown by the water association band in the FTIR spectrum that the water hydrogen bonding is considerably different in the liquid-crystalline phases than in bulk water.     

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.