Eutectic interactions between saturated and unsaturated chain cholesteryl esters: comparison of calculated and observed phase diagrams

Binary phase behavior of saturated chain with unsaturated chain cholesteryl esters is evaluated by analysis of the phase diagrams in terms of ideal solution theory. Cholesteryl palmitate, which crystallizes in the bilayer structure, forms a eutectic with either cholesteryl oleate or cholesteryl linoleate and, as indicated by low angle X-ray data, the components are nearly totally fractionated in the solid state. The fit of the two experimental liquidus curves by a calculation of freezing point depression for an ideal solution indicates that the molecular interactions are nonspecific in the binary liquid state. Cholesteryl caprylate and cholesteryl oleate, both of which crystallize as the monolayer II form, also form a eutectic. X-ray data again indicate nearly total fractionation. The liquidus curve is reasonably well matched by calculation of ideal freezing point depression. However, dissimilar molecular volumes can cause the melt-cholesteric transition line to deviate from an ideal concentration dependence. Possible fractionation mechanisms for cholesteryl esters in arterial lesions are thereby indicated. For example, when the molecules have greatly different volumes, clustering can occur in the liquid crystalline state. Even when the molecular volumes are similar, the saturated component can solidify in regions where it is relatively abundant, because of the incompatibility of two crystal structures with greatly different layer structures.     

Cholesteryl esters of saturated fatty acids: cosolubility and fractionation of binary mixtures

Factors affecting the solid state miscibility of saturated chain cholesteryl esters were determined from electron diffraction and differential scanning calorimetric measurements on a homologous series which included two types of crystal packing. Electron diffraction patterns from solution- and epitaxially crystallized microcrystals gave measured unit cell constants consistent with the bilayer crystal form for myristate, pentadecanoate, palmitate, and stearate esters. Cholesteryl undecanoate crystallized as the monolayer I structure and cholesteryl laurate was polymorphic, packing in either monolayer I or bilayer forms. No evidence was found for the monolayer II form of the laurate claimed in earlier work. It is clear that solid solution formation follows general rules formulated earlier by Kitaigorodskii for molecular crystals. A symmetry criterion must be satisfied first of all, i.e., two compounds that solidify in greatly different crystal structures will not form continuous solid solutions (e.g., cholesteryl undecanoate/cholesteryl myristate). Within a given crystal structure type, solid solution is permitted when the molecular volumes are similar. (For example, cholesteryl myristate forms an ideal solid solution with cholesteryl pentadecanoate, a nonideal solution with cholesteryl palmitate, and a eutectic of solid solutions with cholesteryl stearate.) For the polymorphic cholesteryl laurate, solid solutions of either the monolayer I structure (e.g., with cholesteryl undecanoate) or bilayer structure (e.g., with cholesteryl myristate) are permitted.     

Polymorphic forms of 1,2-dipalmitoyl-sn-glycerol: a combined X-ray and electron diffraction study

Quantitative crystallographic structure analyses are carried out for two polymorphic forms of 1,2-dipalmitoyl-sn-glycerol. A single crystal X-ray determination on the higher melting beta'L-form reveals that the hairpin conformer structure is essentially identical to that of the dilauroyl homolog reported earlier (I. Pascher, S. Sundell and H. Hauser (1981) J. Mol. Biol. 153, 791-806) with inclined acyl chain packing in the O perpendicular methylene subcell. Lamellar electron diffraction intensity data from epitaxially crystallized samples were used to determine the structure of the lower melting alpha L-form. The chains pack in the hexagonal subcell and are perpendicular to the lamellar surface. An appropriately oriented molecular model based on the beta'L-polymorph does not lead to a satisfactory structure solution but models based on the conformationally different 1,2-diglyceride moiety of several phospholipid structures does lead to a closer match to the observed diffraction data. In this proposed packing model for the alpha L-form, the hydroxyl oxygens are somewhat farther away from the unit cell origin than in the beta'L-form crystal structure, and, in combination with the different molecular conformation, this might explain the observed stability of this crystal polymorph against acyl shifts.     

Computer-based modeling of the conformation and packing properties of docosahexaenoic acid

We used a molecular modeling approach to search for a conformation of docosahexaenoic acid (DHA) that might uniquely influence acyl chain packing in cell membranes. Studies of DHA models containing six cis double bonds and five intervening methylene groups identified two conformations of special interest. Both had nearly straight chain axes formed by methylene carbon alignment. In one, the carbons of the six double bonds projected outward from the methylene axis in two nearly perpendicular planes to form an angle iron-shaped molecule. In the other, the double-bond carbons projected outward from the axis at nearly 90 degree-intervals to form a helix. Studies of packed arrays of these hexaenes with or without saturated hydrocarbons showed that tight intermolecular packing arrangements were possible, particularly in the case of the angle iron-shaped molecules. The planar surfaces of two or more such molecules could be brought into contact "back to back," while the interplanar "V groove" of each molecule could come into close apposition with a saturated chain. Because a similar mixed chain packing arrangement was found also for 1,2 diacylglycerols, these results raise the possibility that DHA may, in certain circumstances, promote tight, regular acyl chain packing arrays in DHA-rich membranes.     

Solid-liquid phase behavior of binary fatty acid mixtures 3. Mixtures of oleic acid with capric acid (decanoic acid) and caprylic acid (octanoic acid)

Solid-liquid phase behavior of binary mixtures of oleic acid (OA)/capric acid (C10A) and OA/caprylic acid (C8A) were investigated by means of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction. The phase diagram of OA/C10A mixture constructed from the DSC results suggested that a molecular compound with the composition of OA:C10A = 3:2 is formed in a solid phase, and OA and the molecular compound are miscible, while C10A and the molecular compound are completely immiscible. The formation of the molecular compound was supported by the IR spectroscopic observation, and a possible model of the structure was proposed on the basis of X-ray diffraction spectrum in small angle region. This compound formation is characteristic of the OA/C10A mixture, and may be attributed to the similarity of the acyl chain length of C10A to the lengths of Delta- and omega-chains of OA (i.e., the chain segments divided by cis-double bond). The mixture of OA and C8A, whose chain length is close to but shorter than the two chain segments of OA, provided a eutectic-type phase diagram showing a partial mixing of the two components in OA-rich region. Thermodynamic analysis of the liquidus line in the phase diagram exhibits a systematic trend for the non-ideality parameter of mixing with the variation of the chain length difference between OA and saturated fatty acid species.     

Co-solubility in binary phospholipid crystals

Crystalline binary solid solutions of phosphatidylethanolamines are obtained when various fractions of compounds with different chain lengths are dissolved in chloroform and allowed to evaporate to dryness. Phase diagrams and electron diffraction measurements on chain mixtures with a difference of two or four methylene groups indicate that solubility is continuous, although non-ideal. Average molecular volume appears to increase according to Vegard's rule although deviations are noted. These deviations are similar to those observed for binary paraffin solids. Substitution of ether-links for ester-links in one component does not alter solubility behavior. In general the rules of solid solution formation appear to conform to those originally proposed by Kitaigorodskii [1961) Organic Chemical Crystallography, pp. 231-240, Consultants Bureau, New York).     

Gas--liquid chromatography of silylated mixtures from lipolysis of triglycerides containing unusual fatty acyl groups

A convenient and rapid procedure involving methylation, silylation, and temperature-programmed gas-liquid chromatography (GLC) is described for analyzing unfractionated products from the hydrolysis of triglycerides with pancreatic lipase. The conditions employed for GLC were selected to provide maximum and rapid separation of silylated monoglycerides in which the acyl moieties differ in chain length or degree of substitution with oxygen-containing functional groups. Derivatives differing only in the number of double bonds present were not separated. In the GLC curves obtained, peaks representing methyl esters are generally readily distinguishable from those of other components. Therefore, the extent of lipolysis and the composition (with respect to chain length and substitution of acyl groups) of monoglycerides formed are determined simultaneously. The accuracy of the new method was demonstrated with standard mixtures and by comparison of results for several lipolysis products with data obtained by conventional procedures.     

Calorimetric studies on the influence of N-methylated headgroups on the mixing behavior of diheptadecanoyl phosphatidylcholine with 1-behenoyl-2-lauroylphosphatidylcholine.

Recent studies of five different phosphatidylcholine/phosphatidylcholine (PC/PC) systems indicate that binary mixtures of phosphatidylcholines in which one component has a normalized chain length difference (delta C/CL) in the range of 0.09-0.40 and the other a delta C/CL in the range of 0.42-0.57 exhibit the phase behavior of a eutectic system. Here, delta C is the effective chain-length difference between the two acyl chains, and CL is the effective length of the longer of the two acyl chains for the same lipid molecule in the gel state. In each mixture, gel phase immiscibility occurs over a wide compositional range due to the difference in the gel phase acyl chain packing properties of each component. Although the mixtures differ in the location of their eutectic horizontal, with respect to temperature, all have a similar eutectic point that occurs at a composition of approximately 40 mol percent of the component with the delta C/CL value in the range of 0.42-0.57. Here, we extend these studies by systematically modifying the headgroup of C(17):C(17)PC and then analyzing the mixing behavior of the modified lipid with C(22):C(12)PC using DSC. Progressive demethylation of the C(17):C(17)PC headgroup leads to an increase in gel phase immiscibility and a decrease in the amount of C(22):C(12)PC that comprises the eutectic composition. The temperature defining the location of the eutectic horizontal, however, remains virtually unchanged in all three phase diagrams. Our results suggest that the eutectic composition is influenced by changes in gel phase acyl chain packing that are dependent on headgroup-headgroup interactions. In contrast, the eutectic nature of the phase diagram and the location of its solidus line are properties of acyl chain interactions that are independent of phospholipid headgroup-headgroup interactions.     

Synthesis and characterization of monosaccharide lipids as novel hydrogelators

Self-assembly of small molecules is a useful strategy for forming functional supramolecular structures. Three new series of methyl alpha-D-glucopyranoside derivatives, including esters and carbamates, have been synthesized and characterized. Several of these compounds are excellent hydrogelators and formed interesting self-assembled network structures, including birefringent fibers and tubules. The gelation properties depend on the acyl chain length and the headgroup structures. Small molecule sugar-based hydrogelators have potential applications in drug delivery and enzyme immobilization.     

Smoothed acyl chain orientational order parameter profiles in dimyristoylphosphatidylcholine-distearoylphosphatidylcholine mixtures: a 2H-NMR study.

The accommodation of chain-length mismatch in liquid crystal phase bilayers was examined by using deuterium nuclear magnetic resonance to obtain smoothed orientational order parameter profiles for acyl chains of both components in binary lipid mixture bilayers. Mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) covering a range of compositions were prepared with either DSPC acyl chains or DMPC acyl chains perdeuterated. Orientational order parameters in the plateau regions of the smoothed profiles for both components were found to increase smoothly with increasing DSPC concentration. The orientational order parameters in the DSPC-smoothed profile were found to be slightly higher than corresponding values for DMPC over a wide range of bilayer composition. The shapes of the smoothed profiles for both components were found to be sensitive to bilayer composition. At low DSPC concentration, DSPC methylene deuterons near the bilayer center display a secondary plateau at low orientational order. At high DSPC concentration, the plateau of the DMPC-smoothed profile is stretched slightly. The concentration dependence of the smoothed profiles at low DSPC concentration appears to be consistent with a picture in which the last few segments of the DSPC chain cross the bilayer midplane, on average, but remain very disordered.     

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.     

Investigation into the acyl chain packing of endotoxins and phospholipids under near physiological conditions by WAXS and FTIR spectroscopy

The acyl chain packing of various endotoxins and phospholipids was monitored via the main wide-angle reflection between 0.410 and 0.460 nm by wide-angle X-ray scattering (WAXS) and via the absorption band of the symmetric stretching vibration of the methylene groups v(s)(CH(2)) around 2849 to 2853 cm(-1) by Fourier-transform infrared spectroscopy. The lipids investigated included various rough mutant (R) and smooth form (S) lipopolysaccharides (LPS) differing in the length of the sugar portion, lipid A, the "endotoxic principle" of LPS, and various saturated and unsaturated phospholipids with different head groups under a near physiological (>/=85%) water content. The packing density of the saturated endotoxin acyl chains is lower than those of saturated phospholipids but similar to those of monounsaturated phospholipids, each in the gel phase. The hydrophobic moiety of endotoxins thus exhibits significant conformational disorder already in the gel phase. The acyl chain packing of the endotoxins decreases with increasing length of the sugar chain lengths, which seems to be relevant to the observed differences in biological activity. For Re-LPS with different counterions (salt forms), in the presence of externally added cations or at reduced water content (50%), no change of the acyl chain packing density is deduced in the X-ray data, although the FT-IR data indicate its increase. The position of the v(s)(CH(2)) vibration is, thus, only a relative measure of lipid order, in particular when lipids with different head groups and in the presence of external agents are compared.     

Influence of chain length and unsaturation on sphingomyelin bilayers

Sphingomyelins (SMs) are among the most common phospholipid components of plasma membranes, usually constituting a mixture of several molecular species with various fatty acyl chain moieties. In this work, we utilize atomistic molecular dynamics simulations to study the differences in structural and dynamical properties of bilayers comprised of the most common natural SM species. Keeping the sphingosine moiety unchanged, we vary the amide bonded acyl chain from 16 to 24 carbons in length and examine the effect of unsaturation by comparing lipids with saturated and monounsaturated chains. As for structural properties, we find a slight decrease in average area per lipid and a clear linear increase in bilayer thickness with increasing acyl chain length both in saturated and unsaturated systems. Increasing the acyl chain length is found to further the interdigitation across the bilayer center. This is related to the dynamics of SM molecules, as the lateral diffusion rates decrease slightly for an increasing acyl chain length. Interdigitation also plays a role in interleaflet friction, which is stronger for unsaturated chains. The effect of the cis double bond is most significant on the local order parameters and rotation rates of the chains, though unsaturation shows global effects on overall lipid packing and dynamics as well. Regarding hydrogen bonding or properties related to the lipid/water interface region, no significant effects were observed due to varying chain length or unsaturation. The significance of the findings presented is discussed.     

Binary mixtures of asymmetric phosphatidylcholines with one acyl chain twice as long as the other

High-resolution differential scanning calorimetry and 31P NMR spectroscopy have been used to study aqueous phosphatidylcholine (PC) dispersions prepared from colyophilized mixtures of C(10):C(22)PC/C(22):C(12)PC of various molar ratios. These two lipid species are highly asymmetric but have a common structural feature; namely, one acyl chain in the fully extended conformation is about twice as long as the other. Our experimental results support two conclusions: (1) These two component lipids are miscible in all proportions in both gel and liquid-crystalline states. This type of system behaves as a nearly ideal mixture. Its calorimetric parameters are those expected on the basis of the mole fraction weighted average of the corresponding parameters for the pure components. (2) The component lipids appear to self-assemble, at T less than Tm, into a mixed interdigitated bilayer in excess water. In a mixed interdigitated bilayer, the short acyl chain of one asymmetric phosphatidylcholine on one side of the bilayer leaflet is apposed with the short acyl chain of another lipid molecule on the other side of the bilayer leaflet, while the longer acyl chain from each of the two leaflets crosses the entire hydrocarbon width of the bilayer. The fundamental packing unit, as well as the dynamic unit describing the axial rotator motion about the bilayer normal for this mixed interdigitated bilayer, is thus a dimer, whereas the packing unit assigned for the noninterdigitated bilayer such as C(16):C(16)PC lamellae is a monomer.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

Molecular packing of a crystalline ether-linked phosphatidylcholine: an electron diffraction study

Electron diffraction data from solution- and epitaxially-crystallized samples of 1,2-dihexadecyl-sn-glycerophosphocholine are used in an analysis of its molecular packing in the minimally hydrated crystal form. The molecular chain axes are found to be perpendicular to the bilayer plane and the chains pack in the hexagonal methylene subcell. Translational search of a model based on a known diacyl phosphatidylcholine crystal structure indicates that a crystallographic residual minimum corresponds to a headgroup packing distance similar to values found for the dipalmitoyl analog at low relative humidity. The bilayer packing for the ether-linked phosphatidylcholine is therefore similar to the one reported for a sphingomyelin.     

Molecular packing in steroid-lecithin monolayers,part II: Mixed films of cholesterol with dipalmitoylphosphatidylcholine and tetradecanoic acid

Mixed film studies of the systems cholesterol/tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine have been carried out over the entire compositional range at 21°C. When compared on an acyl chain basis the condensing effects were found to be essentially independent of which host-lipid was utilized. The phase change of the host lipid was shifted to higher pressures, then broadened and eliminated. Maximal condensation occurred at just above 42 mol% for the cholesterol/DPPC system. In both systems the two components were initially found to be miscible at all proportions.The results are interpreted in terms of the molecular packing of cholesterol with acyl boundary layers, one significantly, one weakly affected. Maximum condensation is a result of packing that provides maximum cholesterol/acyl chain contact. Consideration is given to both long term stability of such mixed monolayers and the behaviour of the corresponding bilayer states.     

Waxes of the social spider Anelosimus eximius (Araneae,Theridiidae): Abundance of novel n-propyl esters of long-chain methyl-branched fatty acids

The pentane extract of the social spider, Anelosimus eximius (Araneae, Theridiidae), contains hydrocarbons, fatty acids and their methyl esters, and a series of novel propyl esters of long-chain methyl-branched fatty acids. The propyl esters comprise almost three-fourths of the extract and consist predominantly of odd-numbered carbon chain components. Mass spectrometric analyses of the propyl esters, their methyl esters and cyanide derivatives showed that mono-, di- and trimethyl branched components with methyl branches on even numbered carbons predominate. The major components are propyl 4,20- and 4,30-dimethylhentriacontanoate and propyl 6,20- and 6,30-trimethylhentriacontanoate. The hydrocarbon fraction consists of n-, monomethyl- and dimethylalkanes, containing a relatively high proportion of even-numbered carbon chain components. The abundance of even-numbered carbon chain length alkanes and odd-numbered carbon chain length fatty acyl groups, along with abundant methyl-branches suggest that the propionyl-CoA and its carboxylated product, methylmalonyl-CoA, play important roles in the biosynthesis of these unique waxes. Arch. Insect Biochem. Physiol. 36:295–314, 1997. © 1997 Wiley-Liss, Inc.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.