Cholesteryl oleate

Lipemic activity. Cigarette smoke extract, in macrophages with LDL culture at a dose of 100 Xg/mL, stimulated cholesteryl oleate synthesis approximately equal to 12.5-fold. Enhancement in cholesteryl ester synthesis was dependent on the concentration of smoke-modified LDL and exhibited saturation kinetics. There was extensive fragmentation of apo B. This LDL modification depended on the incubation time and concentration of the smoke extract. Superoxide dismutase inhibited LDL modification by 52%, suggesting that superoxide anion is involved. The results indicated that smoke extract alters LDL into a form recognized and incorporated by macrophages . En-... 

Add 200 pi water to the cell pellet and sonicate briefly at low power. Use 50 pi homogenate for a protein determination and 100 pi for lipid extraction. To 100 pi homogenate add 4 ml chloroform/methanol 2/1 (v/v). Leave for 1 h at room temperature and add 0.8 ml of 0.73% NaCl. Vortex and centrifuge for 5 min at 1000xg to separate the phases. Remove the lower chloroform phase and add 10 nmol cho-lesteryl oleate and 10 nmol triolein as a cold carrier. Dry the sample under nitrogen. Take the sample up in 25 pi chloroform/methanol 2/1 (v/v) and spot the sample on a silica gel plate (0.5 cm streak). Rinse the tube with 25 pi chloroform/methanol 2/1 (v/v) and spot the sample on a silica gel plate. Develop the plate with hexane/diethyl-ether/acetic acid, 70/30/1 (v/v/v). Remove the plate from the tank when the solvent front almost reaches the top, dry the plate, and expose briefly to iodine vapor to localize the cholesteryl oleate. Scrape the spots into scintillation vials, mix thoroughly, and count in a scintillation counter. 

Egg yolk phosphatidylcholine. Cholesterol. Cholesteryl oleate. [3H]-cholesteryl oleate. Chloroform. Ethanol. Sodium phosphate. EDTA. NaN3. Sodium cholate. NaCl. 

To prepare proteoliposomes, 7 mg egg yolk phosphatidylcholine, 1.16 pg cholesterol, 77.5 pg cholesteryl oleate and 10 pCi [3H]-cholesteryl oleate, all dissolved in chloroform are mixed. After evaporation of chloroform with nitrogen, the lipids are resolved in 400 pi ethanol. The ethanolic solution is injected into 5 ml of a vortex-ing buffer with 39mmol/l sodium phosphate, 0.01% EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) 3 mg apoA-I is added. The solution is subsequently dialyzed against a buffer with 39 mmol/1 sodium phosphate, 0.01 % EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) at 4°C. At the end the solution is filled up with analysis buffer containing 39 mmol/1 sodium phosphate,... 

Figure 7. The ternary system cholesteryl oleate-cholesterol-triolein (CO-C-TO) at different temperatures (10). The darkened region corresponds to one isotropic phase whereas the remainder consists of two or three phases.

Cholesteryl oleate (CO Aldrich, 97%) contained impurities which absorb strongly below 320 nm. Even after purification (see below) it developed extraneous absorptions unless stored and handled in the absence of air and below room temperature. The purification procedure has been described (10). The Isolated material exhibited an enantlotropic phase (T +c 41-42°C and Tc+i 55.0°C [lit (24) mp 50.5°C]) and no discernible absorption above 300 nm. Cholesteryl chloride (CC1) from Aldrich was recrystallized from 95% ethanol, mp 96.5-99°C (lit (9) 95-96°C). 

Handa, T., Eguchi, Y., and Miyajima, K. (1994) Effects of cholesterol and cholesteryl oleate on lipolysis and liver uptake oftriglycerides/phosphatidylcholine emulsions in iBIsarm. Res., 11 1283-1287. 

Method B. Bisgaier et al. [78] reported a CETP assay using fluorescent cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenedodecanoate (BODIPY-CE, Fig. 4) in microemulsions. Microemulsions for donor/acceptor contain triolein, BODIPY-CE/cholesteryl oleate, and l-hexadecanoyl-2-[cw-9-octadecenoyl]-s -glycero-3-phosphocholine. The assay mixtures consist of acceptor microemulsions, donor microemulsions, and a test sample in each well of flat-bottom 96-well plates. After preincubation at 37°C for 10 min, the reaction is initiated by addition of CETP solution. The fluorescent intensity in each well of the plates is periodically (every 10 sec) detected at 37°C with a fluorescent 96-well plate reader equipped with 485- and 538-nm bandpass filters in the excitation and emission paths, respectively. 

Atherosclerosic aortic sinuses from ApoE knockout mice Cholesterol esters (cholesteryl palmitate and cholesteryl oleate) markers of atherosclerosis progression ... 

Choleterol in tissues is sometimes esterified by fatty acids, (a) Draw the structure of cholesteryl oleate. (b) Would this ester give a positive Lieberman-Burchard test ... 

Heikela M, Vattulainen I, Hyvonen MT. Atomistic simulation studies of cholesteryl oleates model for the core of lipoprotein particles. Biophys. I. 2006 90 2247-2257. 

Figure 4. Normal phase high-pressure liquid chromatography of cholesterol esters isolated from atherosclerotic lesions of human aorta (I), free cholesterol (II), oxygenated cholesterol esters (III), cholesteryl arachidonate (IV), cholesteryl linoleate (V), cholesteryl oleate and cholesteryl palmitate.

The relationship between crystal packing and liquid crystalline ordering has been discussed for cholesteryl n-alkanoates with between two and eighteen carbon atoms in the acyl group.159 The mesomorphic transition temperatures of cholesteryl oleate, linoleate, and linolenate are pressure-dependent.160 The thermal stabilities... 

The activation parameters from the PnP in H, 70/30 (w/w) CH/CCl and cholesteryl oleate ( ,) are presented in Table 3. On first inspection, it is obvious that the ability of the pyrenyl groups to interact is influenced by at least one aspect of solvent structure. Additionally, the data in 11 clearly show... 

Acid CEH has an optimum pH of 4.5 and is located within lysosomes (Fig. 1). Like many lipolytic enzymes, it is water soluble whereas its substrate is not. For this reason, devising a reliable assay method is difficult, and results should be viewed with caution [26,35]. Recently developed conditions foimd to yield satisfactory linearity with both time of incubation and enzyme concentration are 12.7 jitM cholesteryl oleate, dispersed in 1.27 mM egg PC 50 mM acetate buffer 2.0 mM sodium taurocholate 0.005% digitonin pH 3.9 [36]. In aortic cells these conditions gave an apparent of 1.5 /iM for cholesteryl oleate. It should be noted, however, that the nature, physical form, and molecular organization of the CE and associated molecules can have a critical effect on the activity of any preparation of CEH [37]. Rat liver acid CEH, for example, shows K s of 15.3, 14.3, and 7.3 jaM for cholesteryl oleate when the latter is present in vesicles, micelles, and emulsions, respectively [35]. 

The hydrolytic activity of acid CEH toward CE appears to have no specificity as to the nature of the fatty acyl ester [39]. Furthermore, the enzyme appears to hydrolyze emulsions of TG, and the ratio of the enzyme s activity toward cholesteryl oleate and TG remains the same during enzyme purification [38], suggesting that acid CEH may be a single polypeptide with a fairly broad spectrum of activity. Consistent with this possibility, the activities toward CE and TG show identical inhibition patterns, fractionation and anion-exchange behavior, and thermal inactivation patterns [38], It should be noted that a separate, highly specific TG hydrolase (EC 3.1.1.3) is known to be present in rat liver [38]. 

Neutral CEH activity (optimum pH 7.5) is found in the cytoplasm of most cells (Fig. 1). It has been assayed in vitro at 37°C, pH 6.6, using mixed miceUes containing radioactive cholesteryl oleate, PC, and sodium taurocholate [43]. Other research has demonstrated that cholesteryl oleate, presented in an acetone dispersion. yields submaximum rates of hydrolysis [44,45]. 

The formation of plasma lipoprotein CE begins in cells of the intestinal mucosa and liver (Fig. 2B). ACAT activity in these cells leads to the formation of cholesteryl palmitate and cholesteryl oleate, which can be included in nascent chylomicrons, VLDL, and apparently also small spherical HDL [15,80-82], These esters presumably accumulate when more than enough UC is present in the cells to provide for the requirements of cell membranes and lipoprotein surfaces. (For a more detailed discussion of ACAT activity in intestinal epithelial cells, see Chapter 5.)... 

Studies of cells in culture have provided considerable information about the control of this pathway [97]. In early studies, cultured skin fibroblasts were maintained for 24 h in a medium containing lipoprotein-deficient serum. These cells showed increased binding of LDL to cell surface receptors and increased HMG-CoA reductase activity. When LDL were added to the medium, binding, uptake, and degradation of the LDL followed. LDL CE were hydrolyzed, and decreased levels of both HMG-CoA reductase and the apo B/E receptor were seen. Furthermore, increased formation of cholesteryl oleate could be demonstrated. In subsequent studies, modified LDL with a net positive charge were used. These cationized LDL were internalized by a mechanism that did not depend on the apo B/E receptor, and that led to a substantial increase in cell cholesterol [103]. Under these conditions there was again increased synthesis of cholesteryl oleate. These findings support two principal conclusions (1) plasma lipoprotein CE is an important source of cholesterol for fibroblasts and similar cells and (2) the formation of intracellular... 

The levels of palmitic acid, palmitoleic acid, stearic acid and oleic acid increased in both groups, after 4 h of copper-oxidation. While concentrations of cholesteryl oleate, cholesteryl linoleate, cholesteryl arachidonate and cholesteryl docosahexanoate were reduced, following copper stimulated oxidation, in both groups [85]. 

Cholesteryl oleate was probably a component of the mixture of steryl esters described in fine-cured tobacco by Rowland and Latimer (3358) and in tobacco smoke by Rodgman et al. (3296). The steryl esters included sterols esterified with a series of saturated (palmitic, stearic, etc) and unsaturated (oleic, linoleic, etc.) acids. 

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