cholesteryl-palmitate and cholesteryl-oleate

Changes in cholesterol ester (CE) content regulate the progression of atherosclerosis. However, the spatial dynamics of CE subsets and their quantitative changes during lesion progression are not well understood due to a lack of appropriate imaging techniques. In this study, we developed an imaging-based analysis method to map the distribution of CE subsets using time-of-flight secondary ion mass spectrometry (ToF-SIMS).. Serial sections of atherosclerotic aortic sinuses from apolipoprotein E knock-out mice (n = 15) fed a 0.15% high-fat diet for 12-20 weeks were examined by ToF-SIMS.. We found that the ratio of cholesteryl palmitate (Ch-PA) to cholesteryl oleate (Ch-OA) increased by approximately 99% (p = 0.02) as atherosclerosis progressed, whereas the ratios of cholesteryl linoleate (p = 0.09) and cholesteryl stearate (p = 0.22) to Ch-OA did not change significantly. In advanced atherosclerotic plaques, in situ and in-vitro cell death assays showed that local Ch-PA densities were highly correlated with an increase in the number of apoptotic cells. These results suggest that increased Ch-PA may contribute to the formation of a necrotic core by increasing cell death. Our results indicate that the regional ratio of CEs as measured by ToF-SIMS might be a valuable new marker of atherosclerotic progression.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Cholesterol Esters; Diet, High-Fat; Disease Progression; Male; Mice; Mice, Knockout; Spectrometry, Mass, Secondary Ion

The reaction products of three major cholesteryl esters, cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE), and cholesteryl linoleate (C18:2-CE), present in human low-density lipoprotein (LDL) treated with ozone were isolated and characterized. In vitro ozonization of C16:0-CE was found to form the palmitoyl ester of secosterol-A (3β-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product secosterol-B (3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde). On the other hand, when C18:1-CE and C18:2-CE were oxidized by ozone, the aldehyde 9-oxononanoyl cholesterol (9-ONC) was formed as a primary product, which was then further oxidized to form 9-oxononanoyl secosterol-A (9-ON-secoA) and 9-oxononanoyl secosterol-B (9-ON-secoB). The compounds 9-ON-secoA and -B, but not 9-ONC, were found to exhibit strong cytotoxicity against human leukemia HL-60 cells. An LC-ESI-MS/MS method was developed for the detection of these cholesteryl ester ozonolysis products by derivatizing them with dansyl hydrazine. Using this method, we found for the first time that low concentrations of 9-ON-secoA and -B, but not palmitoyl secosterols, were present in human LDL. These novel oxidized cholesterol esters, 9-ON-secoA and -B, probably play important roles in the pathogenesis of several inflammatory disorders such as cancer, diabetes, atherosclerosis, and neurodegenerative diseases.

Topics: Aldehydes; Cell Proliferation; Cholestanones; Cholesterol; Cholesterol Esters; HL-60 Cells; Humans; Leukemia; Lipoproteins, LDL; Oxidation-Reduction; Ozone; Secosteroids

Raman spectroscopy is a non-destructive analytical technique and previous results have shown that qualitative analysis of the lipid component of human atheromatous arteries is feasible. In this paper, we describe a quantitative analytical method for cholesterol and cholesteryl esters in human atherosclerotic plaques, combined with Raman spectroscopic results, using partial least-squares (PLS) regression, a statistical multivariate method based on factorial analysis. Twenty-nine human atherosclerotic pooled samples were studied and the results of Raman spectroscopy coupled with the PLS method were compared to biochemical results. The standard error of prediction was 16.1, 13.6, 1.9, 3.3 and 3.4 mg/g for total cholesterol, free cholesterol, palmitate cholesteryl, oleate cholesteryl and linoleate cholesteryl, respectively. The repeatability of Raman spectroscopy was found to be excellent. Our results show that Raman spectroscopy is a promising technique to obtain a consistent and non-destructive quantitative analysis of cholesterol and cholesteryl esters in human atherosclerotic lesions. In situ and in vivo analysis is a possibility in the near future.

Topics: Aged; Aged, 80 and over; Arteriosclerosis; Cholesterol; Cholesterol Esters; Female; Humans; Least-Squares Analysis; Male; Middle Aged; Multivariate Analysis; Spectrum Analysis, Raman; Sterol Esterase

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.

Topics: alpha-Linolenic Acid; Animals; Arteriosclerosis; Carbon Isotopes; Cholesterol Esters; Cholesterol, Dietary; Crystallization; Diet, Atherogenic; Magnetic Resonance Spectroscopy; Rabbits

A reversed phase high performance liquid chromatographic (HPLC) method was developed for direct profiling and determination of adrenal cholesteryl ester composition. Cholesteryl adrenate and cholesteryl cervonate, which are not commercially available, were synthesized as markers. Lipid extracts of rat adrenal homogenates or lipid droplets were individually applied to a conditioned silica gel-60 column which separated cholesteryl esters from other native lipids. The eluted cholesteryl ester fraction was then analyzed by HPLC. With cholesteryl heptadecanoate as internal standard, seven adrenal cholesteryl esters were detected and quantified: cholesteryl cervonate, cholesteryl arachidonate, cholesteryl adrenate, cholesteryl myristate, cholesteryl oleate, cholesteryl palmitate, and cholesteryl stearate. Among them, cholesteryl adrenate appeared to be the major sterol ester stored in the rat adrenal.

Topics: Adrenal Glands; Animals; Arachidonic Acids; Cholesterol Esters; Chromatography, High Pressure Liquid; Male; Rats; Rats, Sprague-Dawley

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.

Topics: Calorimetry, Differential Scanning; Cholesterol Esters; Crystallization; Thermodynamics; X-Ray Diffraction

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)

Topics: Animals; Calorimetry, Differential Scanning; Cattle; Cholesterol Esters; Foam Cells; Kinetics; Macrophages; Models, Biological; Particle Size; Thermodynamics