Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.     

Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.     

Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the simultaneous determination of phosphatidylcholine hydroperoxides (PCOOH) and cholesteryl ester hydroperoxides (CEOOH). Lipid hydroperoxides (LOOH) were quantitatively extracted from human plasma with a mixture of n-hexane and ethyl acetate, and separated by column-switching high-performance liquid chromatography using one aminopropyl column and two octyl columns followed by chemiluminescence detection. LOOHs could be completely separated from each other and detected at picomole levels. The results of method validation tests were satisfactory. This method was then applied to determine LOOH in normal human plasma; the levels of PCOOH and CEOOH found were 36.0±4.0 nM (mean±S.D., n=6) and 12.3±3.1 nM (mean±S.D., n=6), respectively.     

Reduced effect on apoptosis of 4-hydroxyhexenal and oxidized LDL enriched with n-3 fatty acids from postmenopausal women

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in atherosclerotic plaques in the vascular wall, a process mediated through its oxidized lipids. 4-Hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), derived from oxidation of n-6 and n-3 fatty acids, respectively, are among the major oxidized products in oxLDL. HYPOTHESIS: This study hypothesized that eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA)-rich versus linoleic acid-rich oxLDL obtained from postmenopausal women and HNE versus HHE differentially influence apoptosis in U937 cells. EXPERIMENTAL DESIGN: Thirty healthy postmenopausal women were supplemented with 14 g/day safflower oil (SO), 7 g/day of both fish oil and SO (low dose LFO) or 14 g/day fish oil (high dose HFO) for 5 weeks. Low-density lipoprotein, obtained after supplementation, was oxidized with 5 microM CuSO(4) at 37 degrees C for 6 h. The concentration of cholesteryl ester hydroperoxides (CEOOH) and conjugated dienes was measured in the oxidized LDL (oxLDL). U937 cells were incubated with the oxLDL, 10 microM of HHE, 7 muM of HHE plus 3 microM of HNE, 5 microM of both HHE and HNE or 10 microM of HNE and the extent of apoptosis measured three ways. RESULTS: The concentration of CEOOH and conjugated dienes in oxLDL did not differ among the three treatment groups. The percent of apoptotic cells was approximately 40% lower when incubated with oxLDL obtained from the HFO-supplemented group than the SO-supplemented group measured by both the Annexin V and the DNA fragmentation assays (P = .04 and .004, respectively). Apoptosis of U937 cells was significantly lower in cells incubated with 10 microM of HHE, and mixtures of HHE and HNE than the 10 microM HNE when measured by the Annexin V, DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) staining. CONCLUSIONS: These data suggest that the cardioprotective properties of n-3 fatty acids may derive in part from their less reactive oxidized lipid metabolites.     

Low density lipoproteins develop resistance to oxidative modification due to inhibition of cholesteryl ester transfer protein by a monoclonal antibody

Although numerous studies have investigated the relationship between cholesteryl ester transfer protein (CETP) and high density lipoprotein (HDL) remodeling, the relationship between CETP and low density lipoproteins (LDL) is still not fully understood. In the present study, we examined the effect of the inhibition of CETP on both LDL oxidation and the uptake of the oxidized LDL, which were made from LDL under condition of CETP inhibition, by macrophages using a monoclonal antibody (mAb) to CETP in incubated plasma. The 6-h incubation of plasma derived from healthy, fasting human subjects led to the transfer of cholesteryl ester (CE) from HDL to VLDL and LDL, and of triglycerides (TG) from VLDL to HDL and LDL. These net mass transfers of neutral lipids among the lipoproteins were eliminated by the mAb. The incubation of plasma either with or without the mAb did not affect the phospholipid compositions in any lipoproteins. As a result, the LDL fractionated from the plasma incubated with the mAb contained significantly less CE and TG in comparison to the LDL fractionated from the plasma incubated without the mAb. The percentage of fatty acid composition of LDL did not differ among the unincubated plasma, the plasma incubated with the mAb, and that incubated without the mAb. When LDL were oxidized with CuSO4, the LDL fractionated from the plasma incubated with the mAb were significantly resistant to the oxidative modification determined by measuring the amount of TBARS and by continuously monitoring the formation of the conjugated dienes, in comparison to the LDL fractionated from the plasma incubated without the mAb. The accumulation of cholesteryl ester of oxidized LDL, which had been oxidized for 2 h with CuSO4, in J774.1 cells also decreased significantly in the LDL fractionated from the plasma incubated with mAb in comparison to the LDL fractionated from the plasma incubated without the mAb. These results indicate that CETP inhibition reduces the composition of CE and TG in LDL and makes the LDL resistant to oxidation. In addition, the uptake of the oxidized LDL, which was made from the LDL under condition of CETP inhibition, by macrophages also decreased.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Cholesteryl ester enrichment of plasma low-density lipoproteins in hamsters fed cereal-based diets containing cholesterol

Male Syrian hamsters were fed 0.02, 0.03, or 0.05% cholesterol to test the hypothesis that moderate cholesterol intake increases the cholesteryl ester content of the plasma low-density lipoproteins (LDL). Dietary cholesterol levels of 0.02%-0.05% were chosen to reflect typical human intakes of cholesterol. Hamsters were fed ad libitum a cereal-based diet (modified NIH-07 open formula) for 15 weeks. Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased plasma LDL and high-density lipoprotein cholesterol concentration, increased liver cholesterol concentration, and increased total aorta cholesterol content. The cholesteryl ester content of plasma LDL was determined as the molar ratio of cholesteryl ester to apolipoprotein B and to surface lipid (i.e., phospholipid + free cholesterol). Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased cholesteryl ester content of LDL particles. Furthermore, cholesteryl ester content of LDL was directly associated with increased total aorta cholesterol, whereas a linear relationship between plasma LDL cholesterol concentration and aorta cholesterol was not observed. Thus, the data suggest that LDL cholesteryl ester content may be an important atherogenic feature of plasma LDL.     

Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine

Oxidative modification of low-density lipoproteins (LDLs) is an important feature in the initiation and progression of atherosclerosis. Aminoguanidine (AMG), classically described as an inhibitor of advanced glycation end products, turned out to be also efficient in animal models as an antioxidant against lipid peroxidation. The originality of the present study was based on the simultaneous assessment of the oxidation of LDL lipid and protein moieties in order to characterize the molecular sites of AMG protection. Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes (CD) and hydroperoxide molecular species from cholesteryl esters (CEOOH) and phosphatidylcholines (PCOOH). LDL protein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. The LDL oxidation was mediated by water gamma radiolysis, which has the advantage of being quantitative and highly selective with regard to the free radicals produced. Here, we reported that AMG resulted in a protection of LDLs against lipid peroxidation (both in the lag phase and in the propagation phase) and against apoB fragmentation in a concentration-dependent manner, due to the scavenging effect of AMG toward lipid peroxyl radicals. Paradoxically, AMG was poorly efficient against apoB carbonylation that began during the lag phase. We hypothesize that, even in the presence of AMG, a nonnegligible proportion of (*)OH radicals remained able to initiate oxidation of the LDL protein moiety, leading to apoB carbonylation.     

Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages

Oxidative modification of low density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway in cultured macrophages, and it has been proposed that this process might lead to cholesterol accumulation in macrophages in the arterial wall in vivo. However, oxidation of LDL is accompanied by a substantial reduction in LDL total cholesterol content and hence the amount of cholesterol delivered by oxidatively modified LDL may be less than that delivered by scavenger receptor ligands such as acetyl LDL which results in massive cholesterol accumulation in cultured macrophages. The present studies were done to determine whether the decrease in total cholesterol content during LDL oxidation was due to oxidation of cholesterol and cholesteryl ester, and to determine whether the resulting oxidized sterols could affect cholesterol esterification in cultured macrophages. It was found that when LDL prelabeled with [3H]cholesteryl linoleate was oxidized, there was a decrease in cholesterol mass but no change in radioactivity. The radioactive substances derived from cholesteryl linoleate appeared more polar than the parent compound when analyzed by reverse-phase liquid chromatography, but were not identical with free cholesterol. Thin-layer chromatography of oxidized LDL lipids confirmed the loss of esterified cholesterol, and revealed multiple new bands, some of which matched reference oxysterols including 7-ketocholesterol, 5,6-epoxycholesterol, and 7-hydroxycholesterol. In addition to oxysterols, oxidized cholesteryl esters were also present. Quantitation by gas chromatography indicated that 7-ketocholesterol was the major oxysterol present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

Differential effects of cis and trans fatty acid isomers, oleic and elaidic acids, on the cholesteryl ester transfer protein activity.

In a previous publication (Lagrost, L. and Barter, P.J. (1991) Biochim. Biophys. Acta 1085, 209-216), saturated and cis unsaturated non-esterified fatty acids have been shown to modulate the rate at which cholesteryl esters are transferred from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the presence of the human cholesteryl ester transfer protein (CETP). In the present report, the effects of cis (oleic acid) and trans (elaidic acid) monounsaturated isomers on the CETP-mediated transfer of cholesteryl esters between HDL and LDL were compared. Mixtures of human LDL and HDL3, containing or not radiolabelled cholesteryl esters, were incubated at 37 degrees C with CETP in the presence or in the absence of either stearic (18:0), oleic (18:1 cis) or elaidic (18:1 trans) acids. It was observed that oleic acid and elaidic acid had different effects on the CETP-mediated redistribution of radiolabelled cholesteryl esters as well as on the net mass transfer of cholesterol from HDL3 to LDL. In particular, at high non-esterified fatty acid/lipoprotein ratio, the transfer of cholesteryl esters was significantly inhibited by the cis isomer and increased by the trans isomer.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.     

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.     

Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E.

Two microbial metabolites, bafilomycin B1 and destruxin E, have been found to inhibit significantly the oxidized low density lipoprotein (LDL)-induced accumulation of lipid droplets at 3 nM and 0.5 microM, respectively, in macrophage J774. The incorporation of [14C]oleate into cholesteryl esters in the cells incubated with oxidized LDL was inhibited to the same extent by the two compounds. Both compounds had no effect on the cell surface binding at 4 degrees C and the internalization of oxidized 125I-LDL as well as on the activity of acyl-CoA:cholesterol acyltransferase. However, when incubated with these compounds at 37 degrees C, receptors for oxidized LDL were partially trapped within the cell. In accordance with receptor accumulation, ATP-dependent acidification of endosomes and lysosomes was significantly inhibited by 50 nM bafilomycin B1 and 1 microM destruxin E, respectively. From these results it was concluded that the inhibition of ATP-dependent acidification of endosomes and lysosomes by bafilomycin B1 and destruxin E resulted in the reduction of oxidized LDL-induced synthesis of cholesteryl ester and thereby caused a reduced accumulation of lipid droplets in macrophage J774.     

Lipid transfer protein-catalyzed exchange of cholesteryl ester between high density lipoproteins and apoB-containing lipoproteins

Low density lipoproteins (LDL), lipoprotein (a)(Lp(a)), and lipoprotein(a) after removal of the a-protein (Lp(a-)) were compared with respect to their ability to accept cholesteryl ester from high density lipoproteins (HDL). The incubations were performed at constant concentrations of HDL and various concentrations of either LDL, Lp(a), or Lp(a-). Lp(a) exchanged cholesteryl ester with HDL, but at a rate that was only 48.5 +/- 3.8% of the exchange rate found in the presence of autologous LDL. Cleavage of the apo(a) from Lp(a) resulted in Lp(a-), an LDL-like particle, with characteristics of cholesteryl ester exchange very similar to LDL.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.