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.     

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 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.     

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.     

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.     

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.     

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.     

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)     

幼虫信息素中三种酯类对中华蜜蜂工蜂发育和采集行为的影响

为研究中华蜜蜂Apis cerana cerana幼虫信息素中的3种酯类成分(甲基棕榈酸酯、乙基棕榈酸酯和乙基油酸酯)对其工蜂发育和采集行为的影响, 取1日龄工蜂喂食不同食物, 把喂食食物(炼糖)中添加0.1%或1%(w/w)某种酯类的工蜂组作为处理组, 以喂食食物(炼糖)中不添加酯类(0%)的工蜂组作为对照组, 然后测定7日龄、14日龄工蜂的卵巢发育和3, 5, 7, 12, 18和21日龄工蜂王浆腺的宽度, 以及工蜂首次参加采集的日龄。结果表明: (1)在无王群中, 甲基棕榈酸酯两处理组(1%, 0.1%)的7日龄和14日龄工蜂卵巢发育率都显著低于对照组; 在无王群中, 0.1%乙基棕榈酸酯处理组的14日龄工蜂卵巢发育率极显著低于对照组; 在有王群中, 1%甲基棕榈酸酯处理组和乙基油酸酯两处理组的14日龄工蜂卵巢发育都显著低于对照组;(2)在有王群中, 甲基棕榈酸酯两处理组的5日龄和7日龄工蜂王浆腺宽度显著大于对照组, 12, 18, 21日龄工蜂王浆腺宽度极显著小于对照组; 乙基油酸酯两处理组的7~21日龄工蜂王浆腺宽度显著小于对照组;(3)3种酯类, 只有甲基棕榈酸酯处理组的工蜂首次采集日龄显著大于对照组。这些结果说明, 不同蜜蜂幼虫信息素对中华蜜蜂工蜂发育和采集行为具有不同的生物学效应。     

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.     

Specificity of lipid transfer protein for molecular species of cholesteryl ester

The capacity of the plasma-derived lipid transfer protein to facilitate the transfer of various cholesteryl ester species has been investigated. Four different molecular species of cholesteryl ester were incorporated into either reconstituted high density lipoproteins or phosphatidylcholine liposomes, and the resulting particles were used as donors in standardized lipid transfer assays. With reconstituted high density lipoproteins as substrate, the rate of transfer of cholesteryl esters was cholesteryl oleate greater than cholesteryl linoleate greater than cholesteryl arachidonate greater than cholesteryl palmitate. The transfer rate for cholesteryl oleate was 154% of that for cholesteryl palmitate. Liposome substrates gave similar results. It is concluded that lipid transfer protein transfers all major species of cholesteryl ester found in plasma; however, the relative rates of transfer were significantly affected by acyl chain composition. The transfer rates appeared to reflect substrate specificity rather than substrate availability within the donor particle.     

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)     

Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate

The pancreatic enzyme carboxyl ester lipase (CEL) has been shown to hydrolyse a large number of different esters, including triacylglycerols, cholesteryl esters and retinyl esters with an absolute requirement for bile salts. Some of the lipids that are substrates for CEL can also be hydrolysed by pancreatic lipase. In order to investigate the relative roles of human CEL and pancreatic lipase, the two enzymes were incubated on a pH-stat with isotope-labelled lipid substrate mixtures in physicochemical forms resembling the state of the dietary lipids in human intestinal contents. In the first set of experiments, cholesteryl oleate (CO) and retinyl palmitate (RP) were solubilised in an emulsion of triolein (TO) stabilised by egg phosphatidylcholine and bile salts. Lipase (always added together with its cofactor, colipase) hydrolysed TO, with monoolein and oleic acid as end-products, whereas CEL alone could not hydrolyse TO in the presence of phosphatidylcholine (PC). Lipase alone did not hydrolyse CO or RP, but CEL did hydrolyse these esters if lipase was present. Release of [3H]glycerol from labelled TO increased only slightly if CEL was added compared to lipase alone, suggesting that monoolein hydrolysis was slow under these conditions. In the second set of experiments, CO and RP were dissolved in bile salt/monoolein/oleic acid dispersions with varying bile salt concentrations. CEL hydrolysed CO and RP more rapidly in a system with a high bile salt concentration containing mixed micelles than in a system with a low bile salt concentration, where the lipids were dispersed in the form of mixed micellar and non-micellar aggregates; both types of aggregate have been reported to exist in human intestinal contents. In conclusion, these data suggest that the main function of CEL under physiological conditions is to hydrolyse cholesteryl and retinyl esters, provided that the triacylglycerol oil phase is hydrolysed by pancreatic lipase, which probably causes a transfer of the substrate lipids of CEL from the oil emulsion phase to an aqueous bile salt/lipolytic product phase. Depending on the bile salt/lipolytic product ratio, the substrate will reside in either micellar or non-micellar lipid aggregates, of which the micellar state is preferred by CEL.     

The sterol esters of maize seedlings

1. The composition of the sterol ester fraction of the shoot, root, scutellum and endosperm of 10-day-old maize seedlings was investigated. 2. The scutellum and endosperm together contain 80% of the sterol ester of the seedling. 3. beta-Sitosteryl linoleate is the major sterol ester of the scutellum and endosperm. 4. beta-Sitosteryl and stigmasteryl palmitate, palmitoleate, oleate and linoleate are the major sterol esters of the root. 5. In the shoot phytosterol linoleate is less abundant than phytosterol myristate, palmitate, palmitoleate and oleate. 6. There is a greater proportion of cholesteryl ester in the shoot than in the other tissues of the seedling.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

The study of nonspecific internalization of cholesteryl esters by the Hep G2 hepatoma cells

The cholesteryl oleate-POPC dispersions (1:3, mol/mol, mean particle size 110+/-20 nm) were taken up by the human hepatoma line Hep G2 cells via endocytosis. Internalization of the cholesteryl oleate-POPC dispersions by Hep G2 cells was dependent on the incubation time and dispersion concentration. At the cholesteryl oleate concentration 100 microM, its total uptake and internalization were found to be 1.5 nmol and 0.8 nmol per 1 mg of cell protein/24 h, respectively. Intracellular cleavage of the cholesteryl oleate incorporated in dispersions resulted in accumulation of free cholesterol capable of being released into the medium and metabolized to water-soluble polar products, presumably bile acids; oleic acid released is, apparently, involved in biosynthesis of triacylglycerides. The low-density lipoprotein receptor is not involved in internalization of lipid dispersions, and the presence of the cholesteryl oleate-POPC dispersions has no effect on the receptor-dependent internalization of cholesteryl esters of the low-density lipoproteins. The obtained data allow us to consider nonspecific internalization of cholesteryl esters by hepatocytes as a substantial part of the nonpolar lipid clearance.     

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.     

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.     

Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity.

Cholesterol ester hydrolase activity of pig aorta has been examined under optimum experimental conditions for hydrolysis of different cholesterol esters. The enzyme specific activity values were in the numerical order of substrates hydrolyzed: cholesteryl linoleate larger than or equal to linolenate greater than palmitate larger than or equal to stearate greater than oleate. The results are discussed in relation to the arterial accumulation of cholesterol esters.     

Effect of fish oil versus lard diets on the chemical and physical properties of low density lipoproteins of nonhuman primates

Twenty-four adult male African green grivet monkeys were fed diets containing 42% of calories as lard or menhaden oil and 0.76 mg of cholesterol/kcal for a period of 8 months. Plasma samples from fasting animals were then taken and low density lipoproteins (LDL) were isolated by ultracentrifugation and agarose column chromatography. The LDL were analyzed chemically, and physical properties of the particles were studied by differential scanning calorimetry. The fish oil group had significantly smaller LDL (2.91 vs. 3.43 g/mumol), which contained fewer molecules per particle of all lipid constituents, except triglyceride, compared to the lard-fed animals. The fish oil-fed group had 15% of the total cholesteryl esters as n-3 fatty acyl species and the number of n-3, but not n-6, cholesteryl esters per LDL particle was proportional to LDL size. The numbers of saturated and monounsaturated cholesteryl ester species per LDL particle were highly correlated with LDL size for both diet groups. The LDL of the fish oil group had broad reversible thermotropic transitions that were 12-13 degrees C lower than those of the lard group. These transitions were indicative of order-disorder transitions of the LDL core cholesteryl esters. The peak transition temperature of LDL of the lard group was proportional to the ratio of saturated and monounsaturated to polyunsaturated cholesteryl ester species (CEFA ratio). However, the much lower peak transition temperature of the LDL of the fish oil group was not related to the CEFA ratio nor to the triglyceride content of the particles, but rather, to the n-3 cholesteryl ester content of the particles. Studies of cholesteryl ester model systems demonstrated that relatively small amounts of n-3 cholesteryl esters (less than 15% of total cholesteryl ester) could result in a lowering of the peak transition temperature of cholesteryl linoleate similar to that seen for intact LDL. We conclude that n-3 cholesteryl esters in small quantities have a marked disordering effect on the core cholesteryl esters of LDL, resulting in a striking depression of LDL transition temperature. In addition, we conclude that n-3 cholesteryl esters are preferentially utilized relative to n-6 cholesteryl esters to increase the number of cholesteryl esters per LDL particle with LDL enlargement in fish oil-fed animals.