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Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.

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

The incorporation of cholesteryl oleate and triolein into phospholipid vesicles was studied in cosonicated mixtures of 94 weight % egg phosphatidylcholine and 6 weight % neutral lipid (0-6% triolein and 6-0% cholesteryl oleate). 13C NMR spectroscopy was used to quantitate both neutral lipids in vesicles containing 90% isotopically substituted [carbonyl-13C]cholesteryl oleate and [carbonyl-13C]triolein. Vesicles were also prepared with radiolabeled cholesteryl oleate and triolein and the composition of ultracentrifugal subfractions determined by chemical and radioisotopic methods. For a given starting composition, the incorporation of neutral lipids into vesicles was similar for vesicles prepared and analyzed by the two methodologies. The maximum solubility in vesicles prepared at 55 degrees C with a single neutral lipid was 3.1 weight % triolein (2.8 mol %) and 2.3 weight % cholesteryl oleate (2.8 mol %). In sonication mixtures with both triolein and cholesteryl oleate, the incorporation of each lipid into vesicles was proportional to the starting concentration; the total incorporation of neutral lipid was less than or equal to 4.0% (weight or mole per cent). The solubility limits were intermediate between the theoretical cases of complete additivity and complete competition. The [13C]carbonyl chemical shifts showed that the carbonyl groups of the vesicle-solubilized neutral lipids were close to the vesicle surface and that excess triolein and cholesteryl oleate partitioned into an oil phase containing both triolein and cholesteryl oleate.

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.

Carbon-13 NMR spectroscopy at 50.3 MHz has been used to study four long-chain cholesterol esters with a double bond in the omega-9 position: cholesteryl oleate, C18:1, omega-9; cholesteryl linoleate, C18:2, omega-6,9; cholesteryl erucate, C22:1, omega-9; cholesteryl nervonate, C24:1, omega-9. The linoleate and oleate esters exhibit two metastable liquid-crystalline phases (cholesteric and smectic), whereas the longer chain esters form a stable smectic phase but no cholesteric phase [Ginsburg, G. S., & Small, D. M. (1981) Biochim. Biophys. Acta 664, 98-107]. Line widths (nu 1/2), spin--lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) were measured for all well-resolved resonances from ring and fatty acyl (FA) carbons at different temperatures in the isotropic liquid of each ester. T1 and NOE values of FA resonances were constant between the FA-2 carbon and olefinic region of each acyl chain and increased markedly for carbons near the chain terminus. FA carbon motions are thus restricted and/or highly correlated in the region between the ring and the olefinic carbons, suggesting that strong interactions occur between cholesterol ester molecules in this region of the FA chain. These results also suggest that the FA chains are approximately extended in the isotropic liquid. Steroid ring methine C-6 and C-3 nu 1/2's increased differentially on cooling to the liquid leads to liquid crystal transition temperature (Tm) of each ester, indicative of increasingly anisotropic ring rotations. The rotational anisotropy was quantitated by using a prolate ellipsoid model for the cholesterol ester molecule for which two correlation times (corresponding to rotations about the long and short molecular axes) were calculated from the C-3 and C-6 nu 1/2 values. The C-3/C-6 nu 1/2 ratio was directly proportional to the anisotropy of the ring motions as measured by the ratio of the two correlation times. At any given temperature relative to Tm, the C-3 and C-6 nu 1/2's and the C-3/C-6 nu 1/2 ratios were larger for cholesterol esters which have a cholesteric phase than for esters which have no cholesteric phase, showing that steroid ring motions were more restricted and more anisotropic prior to the formation of a cholesteric phase. Cholesteryl erucate and cholesteryl nervonate have longer regions of FA chain interactions which result in greater chain cooperativity, apparently preventing the preordering of steroid rings to the degree necessary for formation of a cholesteric phase. Thus, these esters form the smectic phase directly from the isotropic liquid. These results are applied to the cholesterol ester transition in plasma low-density lipoproteins.

Particles closely resembling rat high density lipoproteins (HDL) in terms of equilibrium density profile and particle size were prepared by sonication of apoA-I with a microemulsion made with egg lecithin and cholesterol oleate. These particles, like authentic HDL, allowed selective uptake of their cholesterol ester moieties by cultured cells without parallel uptake of the particle itself. That uptake was saturable and competed by HDL. In rats, the plasma decay kinetics and sites of uptake of a cholesteryl ether tracer were similar whether that tracer was incorporated into synthetic or authentic HDL. Synthetic particles containing other apoproteins were made by generally the same method, but using in place of apoA-I either a mixture of rat apoCs or apoE that was either competent or reductively methylated to prevent interaction with the B/E receptor. These particles, of lower density and larger Stokes radius than those made with apoA-I, also allowed selective uptake of cholesterol esters, albeit with a lower degree of selectivity than in the case of apoA-I. Thus a specific apoprotein component in the subject lipoprotein particle is not required for selective uptake. However, selective uptake was shown to be a function of particle density or size, and part of the difference in rates of selective uptake from the particles made with various apoproteins was explained by their differences in density or size.

Reassembled low density lipoprotein (LDL) complexes have been prepared by the interaction of lipid-free sodium deoxycholate-solubilized apoprotein B (apoB) of native human LDL with preformed, 200 A in diameter, microemulsions of cholesteryl oleate (CO), surface-stabilized by either egg yolk phosphatidylcholine ( EYPC ) or dimyristoyl phosphatidylcholine (DMPC). Gel chromatography of PC/CO/apoB complexes shows co-elution of the complex at 43% PC, 43% CO, and 14% apoB. Negative stain electron microscopy shows the particles to be circular, homogeneous, and approximately 200 A in diameter. PC/CO/apoB complexes exhibit beta-migration on agarose gels and show one high molecular weight protein band on 3.0% sodium dodecyl sulfate-polyacrylamide gels. Differential scanning calorimetry and x-ray scattering show the lipids in the complexes to undergo at least two specific thermal transitions depending on lipid composition, one associated with the core-located cholesterol esters similar to LDL and the protein-free microemulsions and the other from the phospholipid forming the surface monolayer. In addition, particle disruption-protein unfolding/denaturation occur irreversibly at 80-85 degrees C. At 4 degrees C, the secondary structure of apoB on complexes of EYPC /CO/apoB is similar to that of native LDL. For complexes of DMPC/CO/apoB, the secondary structure shows less alpha-helix which correlates with the difference in surface lipid environment. The reassembled complexes of PC/CO/apoB provide a defined system in which the components may be varied systematically in order to study the molecular organization, molecular interactions, and metabolism of LDL.

The phase equilibria of human very low density lipoprotein (VLDL) and monkey chylomicron lipids was examined. Triglyceride (TG), cholesterol ester (CE), and cholesterol (C) partitioned into both surface monolayer and oil "core" phases of emulsions of lipoprotein lipids, whereas phospholipid was found exclusively in the surface. In addition to phospholipid, the surface lipids consisted of 2-4% (by weight) TG, less than 1% CE, and 22% C (VLDL), 5-8% C (chylomicrons). The oil lipids consisted mainly of TG, but also 13-16% CE (VLDL), 3% CE (chylomicrons), and 1-2% C (VLDL), 0.3-0.4% C (chylomicrons). The equilibrium state of lipids within size subfractions of native lipoproteins was defined using phase diagram analysis. Subfractions were in equilibrium with respect to surface-to-core and interparticle distributions of C molecules. In contrast, subfracitions were not in a state of interparticle TG and CE equilibrium. By using the phase diagrams, the percentages of the total particle lipids carried in the phases of lipoproteins of varying size were calculated. For Sf greater than 400 particles, greater than or equal to 30% of the total particle C molecules are carried in the core, and less than 3% of the TG and CE molecules are located in the surface. Nascent, plasma, remnant, and beta-migrating TG-rich lipoprotein compositions taken from the literature were compared using phase diagrams. Although the total compositions of nascent liver VLDL and lymph chylomicrons vary substantially, they have identical concentrations of C in their respective phases. Upon equilibration with plasma, the surface and core of nascent TG-rich lipoproteins become enriched (2-4-fold) with C. Remnants and beta-VLDL have the most C-rich phases of the TG-rich lipoproteins examined. The analysis indicates that the C concentrations of the phases of a lipoprotein are related to its metabolic status.

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.

Human plasma low density lipoprotein (LDL) exhibits a thermal transition over the temperature range 20-40 degrees. This transition is associated with a structural change within the lipoprotein particle and is reflected in the small-angle x-ray scattering profiles from LDL. The scattering profile of the quasispherical LDL particle at 10 degrees shows a relatively intense maximum at 1/36 A-1 which is absent from the scattering of LDL at 45 degrees. Theoretical calculations, using model electron density distributions, have been carried out to describe the packing of arrangement of the cholesterol esters, based on perturbations of the molecular packing of crystalline cholesteryl myristate, adequately reproduces the high relative intensity of the x-ray scattering maximum at 1/36 A-1. The perturbations of the packing in the crystal structure of cholesteryl myristate involve "melting" of the hydrocarbon chains of the esters together with translations of pairs of molecules parallel to the molecular long axis. The interaction of opposing steroid moieties, with C18 and C19 angular methyl groups interlocked, exhibited in the crystal structure is retained in the perturbed arrangement. At 45 degrees, thermally induced disorder of this arrangement averages the electron density of the central core. The x-ray scattering profiles of particles with a homogeneous electron density in the core region do not show a high relative intensity of the subsidiary maxima in the 1/36 A-1 region, in agreement with experimental observation. The results of these calculations support the concept that the thermal transition observed for LDL is due to a smectic leads to disordered transition of the cholesterol esters in the core of the LDL particle.

The accumulation of cholesterol esters in foam cells of the arterial intima is an important characteristic of fatty streak lesions of atherosclerosis. We wished to know if cholesterol ester accumulations in cells could be mobilized by altering their external milieu. Thus, phospholipid dispersions were used to remove cholesterol from a cholesterol ester-enriched cell line. Rat hepatoma cells, Fu5AH, were loaded with cholesterol esters by incubation in medium supplemented with hyperlipemic rabbit serum. After removing the loading medium, we incubated the cells in serum-free medium containing egg phosphatidylcholine dispersions. Unesterified cellular cholesterol level decreased in the first 4 h and then remained at a constant level. The cholesterol esters decreased after a lag time of about 2 h and the triacylglycerol level increased after 3 h. The decrease in cellular cholesterol ester depended on the amount of phospholipid in the medium. Cellular cholesterol ester decreased with increasing concentration of medium phospholipid to 2 mumols/ml and then plateaued. The removed cellular sterols appeared in the medium as free cholesterol. Since there was no measurable cholesterol esterase activity in the medium, the cholesterol ester in the cells was hydrolyzed before it appeared in the medium. The fatty acyl composition of the cellular cholesterol esters remained unchanged after significant reduction, suggesting that the hydrolysis of cholesterol esters was not specific for the acyl chain. Sphingomyelin and dimyristoyl phosphatidylcholine dispersions, though cytotoxic, were also effective in reducing cellular cholesterol esters. These experiments demonstrate that cholesterol ester accumulations in these cells can be reduced when phospholipid dispersions are used as cholesterol acceptors in the extracellular medium.

A wide variety of cholesteryl ester-rich apoB-containing lipoproteins undergo an order-disorder transition in the cholesteryl ester core at approximately normal body temperature. The transition occurs over several degrees C with the mid-point being as high as 57 degrees C in some cholesterol-fed animals. The transition mid-point of normal human low density lipoprotein (LDL) appears to vary from as low as 26 degrees C to about body temperature. However, to screen a large population of patients at risk for atheroscerlotic cardiovascular disease (ACD), a rapid method for determining the transition temperature of LDL is needed. Since apoB-containing lipoproteins (VLDL and LDL) are readily precipitated from plasma by dextran sulfate and magnesium sulfate, we have studied the thermal properties of this precipitate using differential scanning calorimetry (DSC). The VLDL-LDL precipitate undergoes a reversible thermal transition similar in transition temperature and enthalpy to the cholesteryl ester transition of isolated pure LDL. The transition is seen with the precipitate from VLDL-free plasma, but no transition is seen when VLDL and LDL have been removed. Cholesteryl ester-rich apoB containing lipoproteins were isolated from a variety of sources (man, cholesterol-fed monkeys, and rabbits) and their transition temperatures compared with the apoB-containing lipoprotein precipitates from the same source. The mid-point of individual transitions varied over a wide range (17-57 degrees C) and the correlation between the pure lipoprotein and the plasma precipitate was strong (r = 0.98, P < 0.001. Thus, DSC of the plasma apoB precipitate may be used as a rapid method of determining the cholesteryl ester transition of LDL and other apoB-containing lipoproteins.-Waugh, D. A., and D. M. Small. Rapid method for determining cholesteryl ester transitions of apoB-containing lipoproteins.

Co-sonicated mixtures of egg phosphatidylcholine and small amounts (less than or equal to 4%, w/w) of [carbonyl-13C]cholesteryl oleate have been studied by 13C NMR spectroscopy at 50.3 MHz. The carbonyl chemical shift from cholesteryl oleate solubilized in vesicles was 1 ppm downfield from the carbonyl chemical shift of cholesteryl oleate present in a separate oil phase. The maximum solubility of the steroid in vesicles determined by chemical analysis of purified vesicles was 1.6 weight % (approximately 2 mol %), in close agreement with the maximum solubility estimated from NMR speak intensity measurements (1.4 weight %). The downfield shift indicates hydrogen bonding of solvent (H2O) molecules with the cholesteryl oleate carbonyl group, suggesting that vesicle-solubilized cholesteryl oleate molecules are located in the phospholipid bilayer with the carbonyl group close to the aqueous interface and the sterol ring and fatty acyl chain approximately parallel to the fatty acyl chains of the phospholipid. Such a folded conformation and localization of the carbonyl group at the aqueous interface may facilitate interactions of sterol esters with cholesterol ester transfer proteins and cholesterol esterases.

The properties of human plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and their extracted lipids were compared using calorimetric, X-ray scattering, and polarizing microscopy techniques. Intact LDL, and cholesterol esters isolated from LDL and VLDL each undergo reversible changes in their physical state around body temperature. These transitions are associated with ordered liquid crystalline to liquid phase changes of the cholesterol esters. In contrast to LDL, VLDL has no reversible transitions and shows no evidence of ordered liquid crystalline structures between 10 and 45 degrees C. Therefore, unlike LDL, VLDL does not contain a separate cholesterol ester region capable of undergoing cooperative melting. Solubility studies at 37 degrees C of cholesterol esters and triglyceride isolated from VLDL show that even at a weight ratio of 1:1, which greatly exceeds the relative amount of cholesterol esters in VLDL, cholesterol ester is completely soluble in triglyceride. Thus, the cholesterol ester in VLDL is not sequestered in a separate domain within VLDL, but is dissolved in the liquid core of the particle.

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

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [carbonyl-13C]cholesteryl oleate. The carbonyl-13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger (520 +/- 152 A diameter) than those with no cholesterol (291 +/- 97 A diameter), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.

By polarizing microscopy and differential scanning calorimetry we observed that the relative stability of the smectic and cholesteric mesophases of cholesteryl esters of acyl chain length of 20 carbons or more depends on the length of the acyl chain and its degree of unsaturation. Significantly, the addition of a single double bond to the acyl chain of a fully saturated cholesteryl ester which exhibits no mesophases (e.g., cholesteryl behenate (C22:0) and cholesteryl lignocerate (C24:0) yields an ester which displays an unusually stable smectic mesophase, bot no cholesteric mesophase. In fact, increasing unsaturation was found to have a destabilizing effect on the cholesteric phase. Similarly, a decrease in thermal stability of the cholesteric mesophase was observed with increasing thermal stability of the smectic mesophase increased in the same series. X-ray scattering data are presented on the smectic mesophase of cholesteryl erucate (C22:1) and cholesteryl nervonate (C24:1). Significant differences in molecular packing of these two monounsaturated omega = 9 cholesteryl esters in the crystalline state are demonstrated by preliminary X-ray scattering experiments.