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.     

Crystal structure of cholestanyl caprylate and binary phase behavior with cholesteryl caprylate

The crystal structure of cholestanyl n-octanoate (caprylate) (C35H62O2) is monoclinic with space group A2 and cell dimensions a = 10.103(7), b = 7.646(7), c = 87.63(7) A, beta = 90.51(6) degrees; Z = 8 [two molecules (A, B) in asymmetric unit], V = 6769 A3, Dc = 1.010 g cm-3. Integrated X-ray intensities for 3798 reflections with I greater than 2 sigma (I) were measured with a rotating anode diffractometer at room temperature. The structure was determined using direct methods. Block diagonal least squares refinement gave R = 0.111. Molecules A and B have almost fully extended conformations, but differ significantly in the rotation about the ester bond and in the C17 chains. The molecular packing in the crystal structure of cholestanyl caprylate consists of stacked bilayers each having d002 = 43.8 A in thickness and within each bilayer, cholestanols pack with cholestanols and caprylate chains pack with caprylate chains. The crystal structure is very similar to that of cholesteryl myristate but is quite different from that of cholesteryl caprylate. The phase equilibria of the cholestanyl caprylate/cholesteryl caprylate binary system have been shown to involve limited mutual solubility of the two components and to have a eutectic point at 73% cholestanyl caprylate. The cholesteric mesophase is monotropic at all compositions except for a narrow range near the eutectic point where it is enantiotropic.     

The temperature-composition phase diagram and mesophase structure characterization of monopentadecenoin in water.

The temperature-composition phase diagram of monopentadecenoin, a monoacylglycerol with a cis monounsaturated fatty acid 15 carbon atoms long (C15:1c10) in water was constructed using x-ray diffraction. Low- and wide-angle diffraction patterns were collected from samples of fixed hydration as a function of temperature in the heating direction on x-ray-sensitive film. The temperature and hydration ranges investigated were 0-104 degrees C and 0-60% (w/w) water, respectively. The phases identified in the system include the lamellar crystalline phase, the lamellar liquid crystalline phase, the fluid isotropic phase, and two inverted cubic phases belonging to space groups la3d (Q230) and Pn3m (Q244). Particular attention has been devoted to the issues of phase equilibrium, phase boundary verification, and structure characterization. The phase diagrams of monopentadecenoin, monomyristolein (C14:1c9), and monoolein (C18:1c9) are compared, and the impact of molecular structure on mesophase stability and structure is discussed.     

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.     

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.     

Molecular motions and thermotropic phase behavior of cholesteryl esters with triolein

The phase behavior of cholesteryl esters with triglyceride has been characterized by differential scanning calorimetry (DSC), light microscopy, and polarizing light microscopy (PLM). Temperature-dependent molecular motions determined by 13C NMR spectroscopy were correlated with thermotropic phase behavior. Two systems, cholesteryl oleate (CO) and a 3/1 w/w mixture of cholesteryl linoleate (CL) and CO, were examined in the presence of small amounts of triolein (TO). Both systems exhibited metastable cholesteric and smectic (or only smectic) phases. Increasing amounts of TO progressively lowered the liquid-crystalline phase transition temperatures and eventually abolished the cholesteric phase, but at differing amounts of TO for the two systems (between 4% and 5% with CL/CO and between 7% and 10% with CO). DSC and PLM showed a progressive broadening of the phase transitions as well as an overlapping of the temperature ranges of the cholesteric and smectic phases. At greater than or equal to 4% TO, a separate isotropic liquid phase coexisted with liquid-crystalline phases. 13C NMR spectroscopy was used to monitor the molecular motions of the cholesteryl ester steroid ring and acyl chain in liquid and liquid-crystalline phases. In the liquid phase, no significant changes in fatty acyl motions, as reflected in spin-lattice relaxation time (T1) and nuclear Overhauser enhancement (NOE) values, were found on addition of TO. The line width (v 1/2) of the steroid ring resonances increased markedly near (1-5 degrees C above) the isotropic liquid----liquid-crystal phase transition temperature (TLC). However, the C3/C6 v 1/2 ratio at 1 degree C above TLC was greater for mixtures exhibiting an isotropic----cholesteric transition than for mixtures exhibiting an isotropic----smectic transition. Rotational correlation times calculated for motions about the long molecular axis and the nonunique axis showed (i) that the ring motions became more anisotropic as TLC was approached and (ii) that the motions were more anisotropic at TLC + 1 degree C for systems exhibiting a cholesteric phase than for systems exhibiting only a smectic phase. 13C line widths in spectra of the cholesteryl ester liquid-crystalline phases suggested that TO perturbed the cholesteryl ester intermolecular interactions and increased the rates of cholesteryl ester molecular motions relative to neat esters.     

Formation,isolation and structure determination of methyl linolenate diperoxides

Autoxidation of methyl linolenate gives rise to isomeric mono-hydroperoxides by reaction with one mole of oxygen but further reaction with a second mole of oxygen readily occurs to produce an isomeric mixture of diperoxides. Autoxidation of individual pure methyl hydroperoxylinolenate isomers has been used as a method of obtaining less complex diperoxide mixtures which can be separated into their pure components by preparative high-pressure liquid chromatography (HPLC). The major diperoxide isomers arising from the autoxidation of pure 9R- and 13S- hydroperoxides of methyl linolenate have been isolated and characterised as isomeric epidioxyhydroperoxides of methyl linolenate. These same compounds have been identified as components of the more complex mixture of diperoxides produced during methyl linolenate autoxidation. The structures of the isolated diperoxides have been determined by physico-chemical methods and a mechanism for their formation is proposed.     

Interactions of cholesterol esters with phospholipids: cholesteryl myristate and dimyristoyl lecithin.

The ternary phase diagram of cholesteryl myristate--dimyristoyl lecithin--water has been determined by polarizing light microscopy, scanning calorimetry, and x-ray diffraction. Hydrated dimyristoyl lecithin forms a lamellar liquid--crystalline phase (L alpha) at temperatures greater than 23 degrees C into which limited amounts of cholesteryl myristate (less than 5 wt. %) can be incorporated. The amount of cholesterol ester incorporated is dependent upon the degree of hydration of the L alpha phase. Below 23 degrees C dimyristoyl lecithin forms ordered hydrocarbon chain structures (L beta' and P beta') which do not incorporate cholesterol ester. Comparison with other phospholipid--cholesterol ester--water phase diagrams suggests the following general principles: i) the incorporation of cholesterol ester occurs only into liquid crystalling phospholipid bilayers, ii) the extent of incorporation is temperature-dependent, with increasing amounts of cholesterol ester being incorporated at higher temperatures, and iii) unsaturated cholesterol esters induce increased disordering of the phospholipid bilayers.     

The crystal structure of cholesteryl octanoate

Crystals of cholesteryl octanoate (C₃₅H₆₀O₂) are monoclinic, space group P2₁, with $\text{a = 12.80(3), b = 9.20(2), c = 14.12(3)}\text{A}\text{?}$, β = 93.81(3)° and 2 molecules per unit cell. The structure has been determined by Patterson rotation and translation methods from the X-ray intensities (Mo-Kα radiation) of 1320 reflections ($\text{sin}\text{θ/λ < 0.59}\text{A}\text{?}{}^{\mathrm{?1}}$) measured with a diffractometer. Refinement by block diagonal least squares and Fourier methods gave R = 0.096. The molecules are arranged in monolayers with their long axes antiparallel and severely tilted (28°). There is a close packing of cholesteryls within the monolayers, but the octanoate chains which form the layer interface regions are conformationally and thermally disordered. The crystal structure is quite different from that of cholesteryl nonanoate, as expected from the discontinuity in thermodynamic properties and phase behaviour which occurs at this point in the homologous series.     

The ionic structure of lecithin monolayers

Surface potentials of mixed monolayers of dicetyl phosphate and eicosanyl trimethylammonium bromide (1:1) were the same on subsolutions of 0.02 M NaCl or 0.01 M CaCl(2), which indicated that ionic phosphate does not interact with Ca(++) in the presence of a neighboring trimethylammonium group. Surface potential-pH plots of dicetyl phosphate, and of dipalmitoyl, egg, and dioleoyl lecithins showed that as the pH of the subsolution is decreased the phosphate groups in the monolayer are neutralized in the order: dicetyl phosphate > dipalmitoyl lecithin > egg lecithin > dioleoyl lecithin. The binding of cations (Na(+), Ca(++)) to the phosphate group of lecithin also showed the same order. The binding of Ca(++)) to egg phosphatidic acid monolayers, as measured by the increase in surface potential, is considerably greater than that to egg lecithin. These results suggest that there is an internal salt linkage between the phosphate and trimethylammonium groups on the same lecithin molecule. An increase in unsaturation of fatty acyl chains increases the intermolecular spacing, which reduces the ionic repulsion between polar groups, and hence strengthens the internal salt linkage. The results support the concept of a vertical rather than coplanar orientation of the phosphoryl choline group with respect to the interface. A position has been proposed for Ca(++) in the dipole lattice of lecithin from a consideration of the surface potential measurements.     

The crystal structure of cholesteryl dihydrogen phosphate

Crystals of cholesteryl dihydrogen phosphate grown from 1,4-dioxane solution are monoclinic, space group C2 with $\text{a = 24.40, b = 6.27, c = 40.86}\text{A}\text{?}\text{and}\text{β = 102.7°}$. The asymmetric unit contains two molecules of cholesteryl phosphate CP and one dioxane molecule of the solvent. The CP molecules pack tail to tail in a bilayer structure. Within the layer they are arranged in double rows with their phosphate groups linked to ribbons by hydrogen bonds. Laterally the double strands of phosphate groups are separated by rows of dioxane molecules. The dioxane serves as hydrogen bond acceptor and as a spacer molecule that compensates the differences in cross-sectional area of the cholesteryl residue (38.4 Ų and the phosphate group (24 Ų). In the cholesterol matrix the CP molecules joined to double rows have packing contact with the smooth side of their skeleta and interdigitate with their annular methyl groups with those of molecules of the adjacent double rows. The branched cholesteryl side chains facing the bilayer center are loosely packed and show considerable disorder and/or thermal motion.     

The crystal structure of cholesteryl 17-bromoheptadecanoate

Crystals of cholesteryl-17-bromoheptadecanoate (C₄₄H₇₇BrO₂) are monoclinic (P2₁) with a = 7.663(2), b = 10.311(5), c = 55.96(2) A and β = 103.10(3°). These are two molecules in the asymmetric unit which have different conformations of the cholesterol side chain and about the ester bond. The molecules pack with regions of only steroid skeleta alternating with regions of hydrocarbon chains. Due to the packing requirements of the skeleta the carbon chains are forced into a hybrid type packing which contains features of the earlier known O⊥ and T∥ subcells. The subcell (HS1) is orthorhombic with a_s = 10.3, b_s = 7.5 and c_s = 2.54A. The molecular packing is such that the ω-bromine atoms do not continue the trans-carbon chains but adopt a gauche conformation.     

Distribution and localization of lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity in A-I-containing lipoproteins

Two types of A-I-containing lipoproteins are found in human high density lipoproteins (HDL): particles with A-II (Lp(A-I with A-II] and particles without A-II (Lp(A-I without A-II]. We have studied the distribution of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer (CET) activities in these particles. Lp(A-I with A-II) and Lp(A-I without A-II) particles were isolated from ten normolipidemic subjects by anti-A-I and anti-A-II immunosorbents. Most plasma LCAT mass (70 +/- 15%), LCAT (69 +/- 16%), and CET (81 +/- 15%) activities were detected in Lp(A-I without A-II). Some LCAT (mass: 16 +/- 7%, activity: 17 +/- 8%) and CET activities (7 +/- 8%) were detected in Lp(A-I with A-II). To determine the size subspecies that contain LCAT and CET activities, isolated Lp(A-I with A-II) and Lp(A-I without A-II) particles of six subjects were further fractionated by gel filtration column chromatography. In Lp(A-I without A-II), most LCAT and CET activities were associated with different size particles, with the majority of the LCAT and CET activities located in particles with hydrated Stokes diameters of 11.6 +/- 0.4 nm and 10.0 +/- 0.6 nm, respectively. In Lp(A-I with A-II), most of the LCAT and CET activities were located in particles similar in size: 11.1 +/- 0.4 nm and 10.6 +/- 0.3 nm, respectively. Ultracentrifugation of A-I-containing lipoproteins resulted in dissociation of both LCAT and CET activities from the particles. Furthermore, essentially all CET and LCAT activities were recovered in the non-B-containing plasma obtained by anti-LDL immunoaffinity chromatography. This report, therefore, provides direct evidence for the association of LCAT and CET protein with A-I-containing lipoproteins. Our conclusions pertain to fasting normolipidemic subjects and may not be applicable to hyperlipidemic or nonfasting subjects.     

The behavior of proteases in lecithin reverse micelles

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and alpha-chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co-surfactant. The C8-lecithin system, i.e. 1,2-dioctanoyl-sn-glycero-3-phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to wo 60, where wo = [H2O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 1H-NMR; the proton chemical shift as a function of wo was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2-ethylhexyl sulfosuccinate). 31P-NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and alpha-chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (kcat and Km have been determined for a few systems). The bell-shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell-shaped curves are also obtained when studying the influence of wo on the enzyme activity, with an optimal wo which is in the range 7-10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.     

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.     

The calcium- and iron bound phosphate phase diagram

A phase diagram for Ca- and Fe-bound phosphate has been calculated based on two criteria:

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.