Cholesteryl Linoleate Hydroperoxide Research Articles

Investigation of Lipoproteins Oxidation Mechanisms by the Analysis of Lipid Hydroperoxide Isomers.

The continuous formation and accumulation of oxidized lipids (e.g., lipid hydroperoxides (LOOH)) which are present even in plasma lipoproteins of healthy subjects, are ultimately considered to be linked to various diseases. Because lipid peroxidation mechanisms (i.e., radical, singlet oxygen, and enzymatic oxidation) can be suppressed by certain proper antioxidants (e.g., radical oxidation is efficiently suppressed by tocopherol), in order to suppress lipid peroxidation successfully, the determination of the peroxidation mechanism involved in the formation of LOOH is deemed crucial. In this study, to determine the peroxidation mechanisms of plasma lipoproteins of healthy subjects, we develop novel analytical methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PC 16:0/18:2;OOH) and cholesteryl linoleate hydroperoxide (CE 18:2;OOH) isomers. Using the newly developed methods, these PC 16:0/18:2;OOH and CE 18:2;OOH isomers in the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of healthy subjects are analyzed. Consequently, it is found that predominant PC 16:0/18:2;OOH and CE 18:2;OOH isomers in LDL and HDL are PC 16:0/18:2;9OOH, PC 16:0/18:2;13OOH, CE 18:2;9OOH, and CE 18:2;13OOH, which means that PC and CE in LDL and HDL are mainly oxidized by radical and/or enzymatic oxidation. In conclusion, the insights about the oxidation mechanisms shown in this study would be useful for a more effective suppression of oxidative stress in the human organism.

Effect of vitamin E on low density lipoprotein oxidation at lysosomal pH

Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe3+ or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu2+ or Fe3+ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu2+ and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.

Low density lipoprotein oxidation by ferritin at lysosomal pH

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05–0.2 μM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis.

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography–mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP–TLC blotting) and (ii) gas chromatography–electron ionization–mass spectrometry (GC–EI–MS). CEOOH was applied to DPPP–TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC–EI–MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC–EI–MS analysis combined with DPPP–TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.

Omapatrilat decreased macrophage oxidative status and atherosclerosis progression in atherosclerotic apolipoprotein E-deficient mice.

Oxidative stress is an important risk factor in the pathogenesis of atherosclerosis. Angiotensin-converting enzyme (ACE) inhibitors attenuate atherosclerosis and oxidative stress in animal models. Omapatrilat, a VasoPeptidase-inhibitor, selectively inhibits both Neutral-Endo-Peptidase (NEP) and ACE. In this study, we analyzed the effect of Omapatrilat administration (1, 4, or 20mg/kg/d, for 12 weeks) to atherosclerotic apolipoprotein E-deficient (E0) mice on their blood pressure (BP), serum and macrophage oxidative status, and atherosclerotic lesion area. Following administration of Omapatrilat (4 mg/kg/d and 20 mg/kg/d), the mice systolic and diastolic BP significantly decreased by up to 33% and 25% respectively, compared with placebo-treated mice. However, administration of Omapatrilat at 1mg/kg/d did not affect the mice BP. The Omapatrilat-treated mice serum susceptibility to lipid peroxidation was reduced by up to 21%, and their serum paraoxonase activity was increased by up to 24%, compared with placebo-treated mice. Peritoneal macrophages from Omapatrilat-treated (20 mg/kg/d) mice exhibited a reduced oxidative stress, evidenced by a reduction in macrophage lipid peroxide content (by 45%), cholesteryl-linoleate hydroperoxide content (by 48%), and oxidized glutathione levels (by 40%). Finally, the area of the mice atherosclerotic lesion was dose-dependently reduced, by 50%, 67%, and 82%, following Omapatrilat administration at 1mg/kg/d, 4 mg/kg/d, and 20 mg/kg/d respectively, compared with placebo-treated mice. Omapatrilat has a substantial anti-atherosclerotic effect, which can be related not only to BP reduction but also to its ability to reduce oxidative stress in atherosclerotic E0 mice.

Lipid peroxidation and nitric oxide inactivation in postmenopausal women.

To assess the effect of endogenous estrogens on the bioavailability of nitric oxide (.NO) and in the formation of lipid peroxidation products in pre- and postmenopausal women. NOx and S-nitrosothiols were determined by gaseous phase chemiluminescence, nitrotyrosine was determined by ELISA, COx (cholesterol oxides) by gas chromatography, and cholesteryl linoleate hydroperoxides (CE18:2-OOH), trilinolein (TG18:2-OOH), and phospholipids (PC-OOH) by HPLC in samples of plasma. The concentrations of NOx, nitrotyrosine, COx, CE18:2-OOH, and PC-OOH were higher in the postmenopausal period (33.8+/-22.3 microL; 230+/-130 nM; 55+/-19 ng/microL; 17+/-8.7 nM; 2775+/-460 nM, respectively) as compared with those in the premenopausal period (21.1+/-7.3 microM; 114+/-41 nM; 31+/-13 ng/microL; 6+/-1.4 nM; 1635+/-373 nM). In contrast, the concentration of S-nitrosothiols was lower in the postmenopausal period (91+/-55 nM) as compared with that in the premenopausal p in the premenopausal period (237+/-197 nM). In the postmenopausal period, an increase in nitrotyrosine and a reduction of S-nitrosothiol formation, as well as an increase of COx, CE18:2-OOH and PC-OOH formation occurs. Therefore, NO inactivation and the increase in lipid peroxidation may contribute to endothelial dysfunction and to the greater risk for atherosclerosis in postmenopausal women.

Oxidized Cholesteryl Linoleates Stimulate Endothelial Cells to Bind Monocytes via the Extracellular Signal–Regulated Kinase 1/2 Pathway

Oxidation products of cholesteryl esters have been shown to be present in oxidized low density lipoprotein and in atherosclerotic lesions. Monocyte adhesion to the endothelium is an initiating crucial event in atherogenesis. Here, we show that in vitro oxidized cholesteryl linoleate (oxCL) stimulated human umbilical vein endothelial cells (HUVECs) to bind human peripheral blood mononuclear cells as well as monocyte-like U937 cells but not peripheral blood neutrophils or neutrophil-like HL-60 cells. Among the oxidation products contained in oxCLs, 9-oxononanoyl cholesterol (9-ONC) and cholesteryl linoleate hydroperoxides stimulated U937 cell adhesion. OxCL-induced U937 cell adhesion was inhibited by an antibody against the connecting segment-1 region of fibronectin. Neither oxCL nor 9-ONC induced activation of the classical nuclear factor-kappaB pathway. In contrast, stimulation of HUVECs with oxCL resulted in phosphorylation of the extracellular signal-regulated kinase 1/2. Moreover, U937 cell adhesion induced by 9-ONC and oxCL was blocked by a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor and a protein kinase C inhibitor. Taken together, oxCLs stimulate HUVECs to specifically bind monocytes, involving endothelial connecting segment-1 and the activation of a protein kinase C- and mitogen-activated protein kinase-dependent pathway. Thus, oxidized cholesteryl esters may play an important role as novel mediators in the initiation and progression of atherosclerosis.

Preparation of the addition products of alpha-tocopherol with cholesteryl linoleate-peroxyl radicals.

a-Tocopherol was reacted with cholesteryl linoleate hydroperoxides (Ch18:2-OOH) in the presence of an iron-chelate, Fe(III) acetylacetonate, at 37 degrees C in benzene. The reaction products were isolated and identified as four positional isomers of cholesteryl (8a-dioxy-alpha-tocopherone)-epoxyoctadecenoates and two positional isomers of cholesteryl (8a-dioxy-alpha-tocopherone)-octadecadienoates. The result indicates that the peroxyl radicals from Ch18:2-OOH react with the 8a-carbon radical of alpha-tocopherol to form the addition products.

Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.

Supplementation of postmenopausal women with fish oil does not increase overall oxidation of LDL ex vivo compared to dietary oils rich in oleate and linoleate

Although replacement of dietary saturated fat with monounsaturated and polyunsaturated fatty acids (MUFA and PUFA) has been advocated for the reduction of cardiovascular disease risk, diets high in PUFA could increase low density lipoprotein (LDL) susceptibility to oxidation, potentially contributing to the pathology of atherosclerosis. To investigate this possibility, 15 postmenopausal women in a blinded crossover trial consumed 15 g of sunflower oil (SU) providing 12.3 g/day of oleate, safflower oil (SA) providing 10.5 g/day of linoleate, and fish oil (FO) providing 2.0 g/day of eicosapentaenoate (EPA) and 1.4 g/day of docosahexaenoate (DHA). During CuSO4-mediated oxidation, LDL was depleted of α-tocopherol more rapidly after FO supplementation than after supplementation with SU (P = 0.0001) and SA (P = 0.05). In LDL phospholipid and cholesteryl ester fractions, loss of n-3 PUFA was greater and loss of n-6 PUFA less after FO supplementation than after SU and SA supplementation (P < 0.05 for all), but loss of total PUFA did not differ. The lag phase for phosphatidylcholine hydroperoxide (PCOOH) formation was shorter after FO supplementation than after supplementation with SU (P = 0.0001) and SA (P = 0.006), whereas the lag phase for cholesteryl linoleate hydroperoxide (CE18:2OOH) formation was shorter after FO supplementation than after SU (P = 0.03) but not SA. In contrast, maximal rates of PCOOH and CE18:2OOH formation were lower after FO supplementation than after SA (P = 0.02 and 0.0001, respectively) and maximal concentrations of PCOOH and CE18:2OOH were lower after FO supplementation than after SA (P = 0.03 and 0.0006, respectively). Taken together, our results suggest that FO supplementation does not increase the overall oxidation of LDL ex vivo, especially when compared with SA supplementation. Consequently, health benefits related to increased fish consumption may not be offset by increased LDL oxidative susceptibility. —Higdon, J. V., S. H. Du, Y. S. Lee, T. Wu, and R. C. Wander. Supplementation of postmenopausal women with fish oil does not increase overall oxidation of LDL ex vivo compared to dietary oils rich in oleate and linoleate.

Measurement of free cholesterol, cholesteryl esters and cholesteryl linoleate hydroperoxide in copper-oxidised low density lipoprotein in healthy volunteers supplemented with a low dose of n-3 polyunsaturated fatty acids

A study was designed to evaluate the effect of supplementation with a low dose (0.9 g/d) of n-3 polyunsaturated fatty acids ( n-3 PUFA) in fish oil on the oxidative modification of low-density lipoprotein (LDL) in a group of healthy volunteers. Eight volunteers were randomly selected from a larger fish oil supplementation study, and were required to take either 0.9g n-3 PUFA as fish oil (FO group) or 0.9g olive oil [control oil (CO group)] for 16 weeks. Oxidative modification of LDL was assessed by measuring concentrations of free cholesterol (FC), cholesteryl esters (CE) and cholesteryl linoleate hydroperoxide (Ch18:2-OOH) in LDL following copper-induced lipid peroxidation for 0, 2, 3 and 4 h. The composition of LDL fatty acids over 4 h of copper-induced oxidation was also investigated. LDL eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3) compositions were significantly ( P < 0.05) higher in the FO compared with the CO group, following supplementation. Linoleic acid (C18:2 n-6), arachidonic acid (C20:4 n-6), C20:5 n-3 and C22:6 n-3 were significantly ( P < 0.05) oxidised in LDL following 4 h copper-oxidation. The proportions of palmitic acid (C16:0) ( P < 0.05), palmitoleic acid (C16:1) ( P < 0.05), stearic acid (C18:0), and oleic acid (C18:1) increased in the FO and CO groups, after 4 h of copper-oxidation. Concentrations of cholesteryl oleate (Ch18:1), cholesteryl linoleate (Ch18:2 n-6), cholesteryl arachidonate (Ch20:4 n-6) and cholesteryl docosahexanoate (Ch22:6 n-3) were significantly ( P < 0.05) reduced following copper-stimulated oxidation, in both groups. Ch18:2-OOH concentrations were significantly increased ( P < 0.05) following 3 h oxidation in both groups compared with 0 h copper-oxidation, but decreased after 4 h. There was no significant difference in concentrations of Ch18:2-OOH between the groups over the time-course of copper-mediated oxidation. The results of this study suggest that moderate dietary intakes of n-3 PUFA do not significantly influence the susceptibility of LDL to copper-induced oxidation in vitro.

Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities.

Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions. We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.

Regioisomeric distribution of cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans provides evidence for free radical-mediated lipid peroxidation in vivo

We have previously reported the detection of cholesteryl ester hydroperoxides, consisting mainly of cholesteryl linoleate hydroperoxides (Ch18:2-OOH), at nm levels in plasma from healthy humans (Y. Yamamoto and E. Niki, 1989. Biochem. Biophys. Res. Commun. 165: 988–993). To elucidate their production mechanism in vivo, we examined the distribution of Ch18:2-O(O)H regioisomers in blood plasma from nine healthy young subjects using a sequential method consisting of methanol/hexane extraction in the presence of antioxidant, reductant, and internal standard, solid phase extraction to remove unoxidized cholesteryl linoleate, purification by reversed-phase high-performance liquid chromatography (HPLC), and detection by normal phase HPLC. Furthermore, we confirm that little artifactual oxidation of cholesteryl linoleate occurred during analytical procedures indicated by the absence of oxidation products of cholesteryl 11Z,14Z-eicosadienoate (Ch20:2) when provided as an exogenous substrate to the experimental procedure. We detected nm levels of all free radical-mediated oxidation products, 13ZE-, 13EE-, 9-EZ-, and 9-EE-forms of Ch18:2-O(O)H, in blood plasma, whereas the 13ZE-isomer resulting from enzymatic 15-lipoxygenase oxidation was not evident as a major product. These results indicate that free radical chain oxidation of lipids occurs even in healthy young individuals. —Mashima, R., K. Onodera, and Y. Yamamoto. Regioisomeric distribution of cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans provides evidence for free radical-mediated lipid peroxidation in vivo.

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.

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%–61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme’s only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON’s free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON’s ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation

The effect of the consumption of glabridin, an isoflavan isolated from Glycyrrhiza glabra (licorice) root, on the susceptibility of low density lipoprotein (LDL) to oxidation was studied in atherosclerotic apolipoprotein E deficient (E° mice) and was compared with that of the known flavonoids, quercetin and catechin. Glabridin inhibitory activity on in vitro oxidation of human LDL was also investigated by determining the formation of lipid peroxides and oxysterols and the consumption of LDL-associated lipophilic antioxidants. Determination of the extent of LDL oxidation by measuring the formation of thiobabituric acid reactive substances (TBARS) after 2 h of LDL incubation with CuSO 4 (10 μM) or 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH) (5 mM), revealed that glabridin or quercetin consumption resulted in a 53 and 54% reduction in copper ion induced oxidation, respectively, and a 95 and 83% reduction in AAPH induced LDL oxidation, respectively. No inhibition was obtained with consumption of catechin. About 80% of glabridin was found to bind to the LDL human particle. In the in vitro oxidation of LDL induced by AAPH (5 mM), glabridin inhibited the formation of TBARS, lipid peroxides and cholesteryl linoleate hydroperoxide (CLOOH) at all the concentrations tested (5–60 μM), while in oxidation induced by copper ions (10 μM), glabridin exhibited a pro-oxidant activity at concentrations lower than 20 μM, and a clear antioxidant activity at concentrations greater than 20 μM. Glabridin (30 μM) inhibited the formation of cholest-5-ene-3,7-diol (7-hydroxycholesterol), cholest-5-ene-3-ol-7-one (7-ketocholesterol) and cholestan-5,6-epoxy-3-ol (5,6-epoxycholesterol) after 6 h of AAPH induced LDL oxidation, by 55, 80 and 40%, respectively, and after 6 h of copper ion induced LDL oxidation, by 73, 94 and 52%, respectively. Glabridin also inhibited the consumption of β-carotene and lycopene by 38 and 52%, respectively, after 0.5 h of LDL oxidation with AAPH, but failed to protect vitamin E. The in vivo and in vitro reduction of the susceptibility of LDL to oxidation obtained with glabridin, may be related to the absorption or binding of glabridin to the LDL particle and subsequent protection of LDL from oxidation by inhibiting the formation of lipid peroxides and oxysterols, and by protecting LDL associated carotenoids.

Oxidation of High Density Lipoproteins: II. EVIDENCE FOR DIRECT REDUCTION OF LIPID HYDROPEROXIDES BY METHIONINE RESIDUES OF APOLIPOPROTEINS AI AND AII

Human high density lipoproteins (HDL) can reduce cholesteryl ester hydroperoxides to the corresponding hydroxides (Sattler W., Christison J. K., and Stocker, R. (1995) Free Radical Biol. & Med. 18, 421-429). Here we demonstrate that this reducing activity extended to hydroperoxides of phosphatidylcholine, was similar in HDL2 and HDL3, was independent of arylesterase and lecithin:cholesteryl acyltransferase activity, was unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing apoAI or apoAII only, as well as isolated human apoAI. Concomitant with the reduction of lipid hydroperoxides specific oxidized forms of apoAI and apoAII formed in blood-derived and reconstituted HDL. Similarly, specific oxidized forms of apoAI accumulated upon treatment of isolated apoAI with authentic cholesteryl linoleate hydroperoxide. These specific oxidized forms of apoAI and apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction and the associated formation of specific oxidized forms of apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was dose-dependent and accompanied by the conversion of specific Met residues of apoAI and apoAII into Met(O). Canine HDL, which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker lipid hydroperoxide reducing activity and lower extents of formation of oxidized forms of apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and phosphatidylcholine associated with human HDL.

Cholesteryl Ester Hydroperoxide Formation in Myoglobin-Catalyzed Low Density Lipoprotein Oxidation: Concerted Antioxidant Activity of Caffeic and p-Coumaric Acids with Ascorbate

Two diet-derived phenolic acids, caffeic and p-coumaric acids, interplayed with ascorbate in the protection of low density lipoproteins (LDL) from oxidation promoted by ferrylmyoglobin. Ferrylmyoglobin, a two-electron oxidation product from the reaction of metmyoglobin and H 2O 2, was able to oxidize LDL, degrading free cholesterol and cholesteryl esters. Upon exposure to ferrylmyoglobin, LDL became rapidly depleted of cholesteryl arachidonate and linoleate, which turn into the corresponding hydroperoxides. Cholesteryl oleate and cholesterol were, comparatively, more resistant to oxidation. Caffeic (2 μM) and p-coumaric (12 μM) acids efficiently delayed oxidations, as reflected by an increase in the lag times required for linoleate hydroperoxide and 7-ketocholesterol formation as well as for cholesteryl linoleate consumption. At the same concentration, ascorbate, a standard water-soluble antioxidant, was less efficient than the phenolic acids. Additionally, phenolic acids afforded a protection to LDL that, conversely to ascorbate, extends along the time, as inferred from the high levels of cholesteryl linoleate and cholesteryl arachidonate left after 22 hr of oxidation challenging. Significantly, the coincubation of LDL with ascorbate and each of the phenolic acids resulted in a synergistic protection from oxidation. This was inferred from the lag phases of cholesteryl linoleate hydroperoxide (the major peroxide found in LDL) formation in the presence of mixtures of ascorbate with phenolic acids longer than the sum of individual lag phases of ascorbate and the phenolic acids. A similar description could be drawn for the accumulation of a late product of oxidation, 7-ketocholesterol. It is concluded that ferrylmyoglobin induces a typical pattern of LDL lipid peroxidation, the oxidation rate of cholesteryl esters being a function of unsaturation; furthermore, there is a synergistic antioxidant activity of diet-derived phenolic acids with ascorbate in the protection of LDL from oxidation, a finding of putative physiological relevance.