Molecular organization and motions of crystalline monoacylglycerols and diacylglycerols: a C-13 MASNMR study.

Six saturated acylglycerols (1-myristoyl-sn-glycerol, 1-palmitoyl-sn-glycerol, 1,2-dimyristoyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycerol, 1,2-dipalmitoyl-rac-glycerol, and 1,3-dimyristoylglycerol) were studied in their various polymorphic forms (sub-alpha, alpha, beta') by natural abundance C-13 nuclear magnetic resonance (NMR) with magic angle spinning (MASNMR). C-13 MASNMR does not require single crystals and can observe relatively disordered crystals, distinct advantages over crystallographic diffraction methods. Well resolved spectra were obtained for each acylglycerol, and the chemical shifts of corresponding carbons were different for each crystalline phase and the isotropic liquid phase; moreover, in the case of monoacylglycerols, the symmetrically nonequivalent molecules in the same crystalline structure gave distinct C-13 resonances for the same carbon. The C-13 chemical shifts corresponding to each polymorphic phase were interpreted in terms of differences in intramolecular bond distances, intermolecular interactions (such as H bonding), and molecular motions. Mobilities of the glycerol backbone and acyl chains were assessed by the C-13 linewidths and the C-H dipolar relaxation rates. The chemical shift anisotropy(ies) (delta sigma) of the carbonyl group(s) of each acylglycerol was determined from slow-spinning MAS spectra, and was discussed in terms of the conformational and/or motional changes for the carbonyl carbon(s).     

Phase behavior of cholesteryl ester dispersions which model the inclusions of foam cells

In order to understand the phase behavior of the approximately 1-micron-diameter droplets which occur in the cytoplasm of cholesterol-enriched cells, differential scanning calorimetry has been utilized to elucidate the factors controlling the rate of crystallization of cholesteryl esters. The kinetics of the thermotropic transitions between liquid, liquid-crystal, and crystal states which occur in mixtures of cholesteryl oleate and cholesteryl palmitate present in monodisperse, phospholipid-stabilized, emulsion droplets have been determined and are compared to the characteristics of these transitions in bulk mixtures. Cholesteryl palmitate is observed to crystallize in undercooled phospholipid-stabilized dispersions of cholesteryl palmitate/cholesteryl oleate (50/50 w/w) at temperatures up to 50 degrees C lower than it does in bulk mixtures of the same cholesteryl ester composition. It is postulated that this difference between crystallization temperatures is due primarily to the presence of impurities present in bulk mixtures which act as catalysts that promote crystallization. It is suggested that phospholipid-stabilized dispersions of cholesteryl palmitate/cholesteryl oleate are more appropriate models than bulk mixtures of these cholesteryl esters for studying the kinetic and thermodynamic basis of the phase behavior in cholesteryl ester rich inclusions characteristic of foam cells and atherosclerotic plaque. The thermotropic phase behavior of these dispersions can be satisfactorily analyzed by using the equations of homogeneous nucleation theory. The interfacial tension between the crystal nucleus and the surrounding fluid cholesteryl ester is about 10 erg/cm2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.     

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.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

Phase behavior and dynamic heterogeneities in lipids: a coarse-grained simulation study of DPPC-DPPE mixtures

The coarse-grained Marrink-model for biomembrane simulation is used to study mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) at various concentrations and temperatures. At high temperatures close to ideal mixing is observed. In the low temperature ordered phase dynamic heterogeneities are identified under some conditions. These are correlated with heterogeneities in the local order and define local neighborhoods.     

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.     

Phase behavior of lipid mixtures

Biological membranes are two-dimensional mixtures of an enormous number of different components. Modeling cell membranes as simple bilayer mixtures reveals rich phase behavior, but how can we use the observed phase behavior to understand the real membranes?     

Effect of estrogens on the acitivities of cholesteryl ester synthetase and cholesteryl ester hydrolases in pigeon aorta.

This study is the first to report the effect of conjugated equine estrogens on the acitivity of cholesteryl ester synthetase and cholesteryl ester hydrolases in the aorta. In spontaneously atherosclerosis-susceptible White Carneau pigeons, estrogens significantly decreased (P less 0.01) the activity of cholesteryl ester synthetase and increased (P less than 0.01) the cholesteryl ester hydrolase activity in the microsomal fraction of the aorta. There was no effect on the cholesteryl ester hydrolase activity in the supernatant fraction. The inhibition of cholesteryl ester synthesis and the stimulation of cholesteryl ester hydrolase might be responsible for the decreased content of cholesteryl esters noted in pigeon aorta after estrogen treatment.     

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)     

Phase behavior and dynamic heterogeneities in lipids: A coarse-grained simulation study of DPPC-DPPE mixtures

The coarse-grained Marrink-model for biomembrane simulation is used to study mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) at various concentrations and temperatures. At high temperatures close to ideal mixing is observed. In the low temperature ordered phase dynamic heterogeneities are identified under some conditions. These are correlated with heterogeneities in the local order and define local neighborhoods.     

Low density lipoprotein uptake: holoparticle and cholesteryl ester selective uptake.

Low density lipoproteins (LDL) contain apolipoprotein B-100 and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the lipolysis of very low density lipoproteins. This review will describe the receptors responsible for uptake of whole LDL particles (holoparticle uptake), and the selective uptake of LDL cholesteryl ester. The LDL-receptor mediates the internalization of whole LDL through an endosomal-lysosomal pathway, leading to complete degradation of LDL. Increasing LDL-receptor expression by pharmacological intervention efficiently reduces blood LDL concentrations. The lipolysis stimulated receptor and LDL-receptor related protein may also lead to complete degradation of LDL in presence of free fatty acids and apolipoprotein E- or lipase-LDL complexes, respectively. Selective uptake of LDL cholesteryl ester has been demonstrated in the liver, especially in rodents and humans. This activity brings five times more LDL cholesteryl ester than the LDL-receptor to human hepatoma cells, suggesting that it is a physiologically significant pathway. The lipoprotein binding site of HepG2 cells mediates this process and recognizes all lipoprotein classes. Scavenger receptor class B type I and CD36, which mediate the selective uptake of high density lipoprotein cholesteryl ester, are potentially involved in LDL cholesteryl ester selective uptake, since they both bind LDL with high affinity. It is not known whether they are identical to the uncloned lipoprotein binding site and if the selective uptake of LDL cholesteryl ester produces a less atherogenic particle. If this is verified, pharmacological up-regulation of LDL cholesteryl ester selective uptake may become another therapeutic approach for reducing blood LDL-cholesterol levels and the risk of atherosclerosis.     

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.     

Acyl unsaturation and cholesteryl ester miscibility in surfaces. Formation of lecithin-cholesteryl ester complexes

The surface behavior of a series of cholesteryl esters was studied in mixtures with a model phospholipid, 1-palmitoyl-2-oleoyl phosphatidylcholine. The cholesteryl esters were representative of the predominant forms occurring naturally and qualitative similarities in their phase behavior permits generalization of their surface properties. Quantitative differences, however, show that the availability of cholesteryl esters in all surface states is dependent on the structure of the acyl moiety. All except cholesteryl stearate were surface-active and formed preferred packing arrays, i.e., complexes, with the lecithin at compositions grouped around cholesteryl ester mol fractions of 0.015. Exceptions were cholesteryl arachidonate and docosahexaenoate, with complex stoichiometries of 0.034 and 0.032, respectively. Lecithin had the same apparent area in all complexes, 56.5 +/- 1.04 A2, which was larger than that of uncomplexed lecithin, 53.3 +/- 0.7 A2. This implies that the conformation or orientation of the two polyunsaturated species in complexes is markedly different than the others studied. The areas and hydrations of all uncomplexed cholesteryl esters were similar. Because mixing of complex with uncomplexed cholesteryl ester deviated positively from ideality, the apparent molecular areas of the uncomplexed cholesteryl esters ranged from 161 +/- 22 (complex-rich) to 107 +/- 15 A2 (cholesteryl ester-rich). The similarity of the monolayer phase complex stoichiometries and the bilayer miscibilities of cholesteryl oleate suggests a correspondence between states. If so, the availability of cholesteryl arachidonate or docosahexaenoate in bilayers should be approximately twice that of other naturally occurring cholesteryl esters.     

Structural features of cholesteryl ester transfer protein: A molecular dynamics simulation study

Cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl esters (CEs) from atheroprotective high‐density lipoproteins (HDLs) to atherogenic low‐density lipoproteins (LDLs) or very‐low‐density lipoproteins (VLDLs). Inhibition of CETP raises HDL cholesterol (good cholesterol) levels and reduces LDL cholesterol (bad cholesterol) levels, making it a promising drug target for the prevention and treatment of coronary heart disease. Although the crystal structure of CETP has been determined, the molecular mechanism mediating CEs transfer is still unknown, even the structural features of CETP in a physiological environment remain elusive. We performed molecular dynamics simulations to explore the structural features of CETP in an aqueous solution. Results show that the distal portion flexibility of N‐terminal β‐barrel domain is considerably greater in solution than in crystal; conversely, the flexibility of helix X is slightly less. During the simulations the distal end of C‐terminal β‐barrel domain expanded while the hydrophilic surface increasing more than the hydrophobic surface. In addition, a new surface pore was generated in this domain. This surface pore and all cavities in CETP are stable. These results suggest that the formation of a continuous tunnel within CETP by connecting cavities is permitted in solution. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Phase behavior and molecular interactions in mixtures of ceramide with dipalmitoylphosphatidylcholine.

In mixtures with dipalmitoylphosphatidylcholine, ceramide induces broadening of the calorimetric main phase transition that could be deconvoluted into at least three components: the first represents isothermal melting of a phosphatidylcholine-enriched phase; the second and third represent phases with increasing proportions of ceramide melting at progressively higher temperatures. The partial phase diagram (up to 40 mole % ceramide) indicates complete or partial gel-phase immiscibility, and complete gel- and liquid-phase miscibility depending on the ceramide content. Cluster distribution function analysis of each individual transition reveals decreased cooperativity and domain size with increased amounts of ceramide. Compared to individual lipids, mixed monolayers with dipalmitoylphosphatidylcholine show unchanged mean molecular areas or slight expansions at 24 degrees C with dipole potentials exhibiting hyperpolarization; by contrast, already at 27 degrees C the mean molecular areas become condensed and dipole potentials show little changes or are slightly depolarized. This suggests that favorable ceramide;-phosphatidylcholine dipolar matching in the liquid state can be one of the local determinants for close molecular interactions while unfavorable matching may explain lateral domain segregation of ceramide-enriched gel phases. The changes are detected at relatively low proportions of Cer (1;-12 mole %) which are comparable to variations of Cer levels in membranes of cultured cells undergoing functional responses mediated by the sphingomyelin signaling pathway.     

Transfer of cholesteryl ester into high density lipoprotein by cholesteryl ester transfer protein: effect of HDL lipid and apoprotein content

Recombinant high density lipoprotein (rHDL) particles were prepared by cosonication of purified lipids and human apoproteins and incubated with partly purified cholesteryl ester transfer protein (CETP) and low density lipoprotein (LDL) containing [3H]cholesteryl ester. Increasing the triglyceride content relative to cholesteryl ester in rHDL significantly decreased the ability of the particles to accept cholesteryl esters transferred by CETP. Kinetic analysis of the data was performed to numerically define the maximum velocity of lipid transfer, Tmax, and the HDL concentration required for half maximal velocity, KH. Increases in rHDL-triglyceride content were shown to result in a significant reduction in the Tmax without a major change in KH. When the free cholesterol content was increased relative to phospholipid, the ability of the particles to accept cholesteryl esters was also decreased in a similar manner. Conversely, rHDL prepared from purified apoprotein A-I, A-II, or mixtures of both, had significantly elevated Tmax and KH values for their interaction with CETP. The results suggest that increases in triglyceride or free cholesterol content of an rHDL particle decrease the catalytic ability of CETP by noncompetitive inhibition. In addition, some component(s) of HDL apoproteins, other than A-I or A-II, were shown to uncompetitively inhibit the activity of CETP, by modifying both Tmax and the KH for the reaction. This study has shown that altered HDL composition may have marked effects on the transfer and equilibration of cholesteryl esters within the HDL pool.     

Establishment of anti-human cholesteryl ester transfer protein monoclonal antibodies and radioimmunoassaying of the level of cholesteryl ester transfer protein in human plasma.

Plasma cholesteryl ester transfer protein (CETP) facilitates the net transfer and exchange of cholesteryl ester (CE), triglyceride (TG), and phospholipids between lipoproteins. A series of monoclonal antibodies (mAbs) against human CETP was obtained, comprising mAbs either inhibiting or not inhibiting these transfer activities. One mAb (LT-J1) inhibited the transfer activity of TG almost completely, but not that of CE, indicating that CE and TG binding sites on the CETP molecule may be distinct from each other, and that this mAb may specifically recognize the TG binding site. A radioimmunoassay system for determining the level of CETP was also established using these mAbs, and the plasma CETP levels in 20 normolipemic Japanese adults were found to range from 2.1 to 2.7 mg/liter.