Linoleic Hydroperoxide Research Articles

α-, γ- and δ- Tocopherols as inhibitors of isomerization and decomposition of cis,trans methyl linoleate hydroperoxides

α-, γ- and δ- Tocopherols as inhibitors of isomerization and decomposition of cis,trans methyl linoleate hydroperoxides

α-, γ- and δ- Tocopherols as inhibitors of isomerization and decomposition ofcis,trans methyl linoleate hydroperoxides

The effects of α-, γ- and δ-tocopherols on the stability and decomposition reactions of lipid hydroperoxides were studied. Isomerization and decomposition of cis,trans methyl linoleate hydroperoxides (cis,trans ML-OOH) in hexadecane at 40 °C were followed by high-performance liquid chromatography. Due to its higher hydrogen donating ability, α-tocopherol was more efficient than γ- and δ-tocopherols in inhibiting the isomerization of cis,trans ML-OOH to trans,trans ML-OOH. α-Tocopherol stabilized hydroperoxides into the cis,trans configuration, whereas γ- and δ-tocopherols allowed hydroperoxides to convert into trans,trans isomers. Thus, the biological importance of α-tocopherol as compared to other tocopherols may be partly due to its better efficacy in protecting the cis,trans configuration of hydroperoxides formed, for example, in the enzymatic oxidation of polyunsaturated fatty acids. The isomeric configuration of hydroperoxides has an impact on biological activities of further oxidation products of polyunsaturated fatty acids. Paradoxically, the order of activity of tocopherols with regard to hydroperoxide decomposition was different from that obtained for hydroperoxide isomerization. γ- and δ-tocopherols were more efficient inhibitors of ML-OOH decomposition when compared to α-tocopherol. A loss of antioxidant efficiency, observed as the tocopherol concentration increased from 2 to 20 mM, was highest for α-tocopherol but was also evident for γ- and δ-tocopherols. Thus, the differences in the relative effects of tocopherols at differing concentrations seem to result from a compromise between their radical scavenging efficiency and participation in side reactions of peroxidizing nature.

Peroxidase-like activity on organic hydroperoxides of ion-exchange resins modified with metal-porphine analogues and analytical application for determination of linoleate hydroperoxide

The ion-exchange resins modified with metal–porphyrins and –phthalocyanines (M–P r) have been found to exhibit a peroxidase (POD)-like activity on organic peroxides in a reaction wherein a quinoid dye is formed from phenol and 4-aminoantipyrine. Among them, Mn– and Co–P r exhibited stronger activity than hemoglobin and Fe–P r, and hence were expected to be practically useful as a solid catalyst for the determination of linoleate hydroperoxide (LOOH). In addition, a resin modified with Co 3+–phthalocyanine tetrasulfonate (Co–PCS r) lacks POD-like activity on hydrogen peroxide in contrast with Mn–P r. We, therefore, concluded that Co–PCS r is superior to both Mn–P r and hemoglobin as a solid catalyst on organic hydroperoxides, and developed a new method for the determination of LOOH.

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.

No effect of esterification with fatty acid on antioxidant activity of lutein

Antioxidant activities of lutein and lutein myristate esters were evaluated by using both the methyl linoleate hydroperoxide method and colorimetry of the quenching activity against 1,1-diphenyl-2-picrylhydrazyl. The antioxidant activities of free lutein (FL), lutein monomyristate ester (LM) and lutein dimyristate ester (LD) were not significantly different in both methods. No synergetic effects among FL, LM and LD were obtained. These results suggest that esterification of OH groups with fatty acids might not affect antioxidant activity of lutein.

Effects of alpha-tocopherol and ascorbyl palmitate on the isomerization and decomposition of methyl linoleate hydroperoxides.

In order to study antioxidant action on lipid hydroperoxide decomposition, the effects of alpha-tocopherol (TOH) and ascorbyl palmitate on the decomposition rate and reaction sequences of 9- and 13-cis,trans methyl linoleate hydroperoxide (cis,trans ML-OOH) decomposition in hexadecane were studied at 40 degrees C. Decomposition of cis,trans ML-OOH as well as the formation and isomeric configuration of methyl linoleate hydroxy and ketodiene compounds were followed by high-performance liquid chromatographic analysis. TOH effectively inhibited the decomposition of ML-OOH. The decomposition rate was two times slower at 0.2 mM and more than 10 times slower at 2 and 20 mM of TOH. Ascorbyl palmitate (0.2, 2, and 20 mM) slightly accelerated the decomposition of ML-OOH. Both compounds had an effect on the reaction sequences of ML-OOH decomposition. At high levels TOH inhibited the isomerization of cis,trans ML-OOH to trans,trans ML-OOH through peroxyl radicals and increased the formation of hydroxy compounds. Further, the majority of the hydroxy and ketodiene compounds formed had a cis,trans configuration, indicating that cis,trans ML-OOH decomposed through alkoxyl radicals without isomerization. These results suggest that when inhibiting the decomposition of hydroperoxides, TOH can act as a hydrogen atom donor to both peroxyl and alkoxyl radicals. In the presence of ascorbyl palmitate, cis,trans ML-OOH decomposed rapidly but without isomerization. In contrast to TOH, the majority of hydroxy compounds were cis,trans, but the ketodiene compounds were trans,trans isomers. This indicates that ascorbyl palmitate reduced cis,trans ML-OOH to the corresponding hydroxy compounds. However, the simultaneous formation of trans,trans ketodiene compounds suggests that ML-OOH decomposition, similar to the control sample, also occurred in these samples. Thus, under these experimental conditions, the reduction of ML-OOH to more stable hydroxy compounds did not occur to an extent significant enough to inhibit the radical chain reactions of ML-OOH decomposition.

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.

A novel fluorescent probe diphenyl-1-pyrenylphosphine to follow lipid peroxidation in cell membranes

Diphenyl-1-pyrenylphosphine (DPPP) was tested whether it could be used as a fluorescent probe to monitor lipid peroxidation in cell membranes. DPPP reacted with organic hydroperoxides and hydrogen peroxide stoichiometrically to give DPPP oxide (DPPPO). DPPP incorporated into phosphatidylcholine liposomal membranes and polymorphonuclear leukocytes (PMNs) reacted with methyl linoleate hydroperoxide rapidly but not with hydrogen peroxide nor with tert-butyl hydroperoxide. This novel method revealed that lipid peroxidation proceeded within membranes of PMNs stimulated with phorbol 12-myristate 13-acetate, which is known to produce several kinds of free radicals. It was found that DPPP is a suitable fluorescent probe to monitor lipid peroxidation within cell membranes specifically.

Peroxidation of linoleate at physiological pH: hemichrome formation by substrate binding protects against metmyoglobin activation by hydrogen peroxide

Peroxidation by metmyoglobin, MbFe(III), by metmyoglobin/hydrogen peroxide, MbFe(III)/H 2O 2 , to yield the myoglobin ferryl radical ( MbFe(IV)=O), or by ferrylmyoglobin, MbFe(IV)=O, was investigated at physiological pH (7.4) in oil-in-water linoleate emulsions. Linoleate peroxidation was followed using second derivative ultraviolet (UV)-spectroscopy for monitoring formation of conjugated dienes and quantitative determination of specific linoleate hydroperoxides by liquid chromatography with photodiode absorption detection. Modifications of myoglobins during lipid peroxidation were followed simultaneously by changes in the Soret absorption band (410 or 424 nm), and in the visible absorption region (from 450 to 700 nm), combined with electron spin resonance (ESR) spectroscopy for direct detection of changes in the spin state of the iron center. In contrast to MbFe(IV)=O, MbFe(III) and MbFe(III)/H 2O 2 were not able to initiate linoleate peroxidation in oil-in-water emulsions, and MbFe(III) was converted, by binding of linoleate (but not methyl linoleate), to a low-spin hemichrome derivate, HMbFe(III), with the distal histidine reversibly bound to the iron center. HMbFe(III) is ineffective in initiating lipid peroxidation and cannot be activated to MbFe(IV)=O or MbFe(IV)=O by addition of moderate amounts of H 2O 2. Addition of MbFe(III) to linoleate emulsions containing H 2O 2 results in the competitive formation of MbFe(IV)=O and HMbFe(III) in favor of HMbFe(III), and little linoleate peroxidation is detected, demonstrating the inherent protection, at physiologic pH, against peroxidation by reversible binding of the substrate to the potential myoglobin catalyst.

Effects of β-carotene and retinal on the formation of methyl linoleate hydroperoxides

In order to clarify the prooxidative role of carotenoids on the oxidation of unsaturated lipids this study examined the effects of β-carotene and its oxidative breakdown product, retinal, on primary oxidation products of linoleic acid methyl ester. Formation as well as isomer distribution of methyl linoleate hydroperoxides were followed by highperformance liquid chromatography. Oxidation of methyl linoleate without or with added β-carotene (5, 20, 200 μg/g) or retinal (7, 18, 180, 360 μg/g) was carried out in the dark under air at 40 °C. Both β-carotene and retinal promoted the formation of hydroperoxides and thus acted as prooxidants in a concentration-dependent way. Moreover, carotenoids also had an effect on the isomeric distribution of primary oxidation products as high contents of retinal increased the portion (%) of trans,trans-hydroperoxides. Being thermodynamically more stable isomers than cis,trans-isomers of hydroperoxides they are known to accumulate during later phases of oxidation or during hydroperoxide decomposition. The results showed that β-carotene and retinal were not effective hydrogen donors. These findings raise the question that carotenoids and their oxidative breakdown products enhance the decomposition of lipid hydroperoxides and this effect partially explains the prooxidative effect of carotenoids.

Effects of β-carotene and retinal on the formation of methyl linoleate hydroperoxides

In order to clarify the prooxidative role of carotenoids on the oxidation of unsaturated lipids this study examined the effects of β-carotene and its oxidative breakdown product, retinal, on primary oxidation products of linoleic acid methyl ester. Formation as well as isomer distribution of methyl linoleate hydroperoxides were followed by high-performance liquid chromatography. Oxidation of methyl linoleate without or with added β-carotene (5, 20, 200 μg/g) or retinal (7, 18, 180, 360 μg/g) was carried out in the dark under air at 40 °C. Both β-carotene and retinal promoted the formation of hydroperoxides and thus acted as prooxidants in a concentration-dependent way. Moreover, carotenoids also had an effect on the isomeric distribution of primary oxidation products as high contents of retinal increased the portion (%) of trans,trans-hydroperoxides. Being thermodynamically more stable isomers than cis,trans-isomers of hydroperoxides they are known to accumulate during later phases of oxidation or during hydroperoxide decomposition. The results showed that β-carotene and retinal were not effective hydrogen donors. These findings raise the question that carotenoids and their oxidative breakdown products enhance the decomposition of lipid hydroperoxides and this effect partially explains the prooxidative effect of carotenoids.

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.

Identification of new geometric isomers of methyl linoleate hydroperoxide and their chromatographic behavior.

New geometric isomers, methyl (9Z,11Z)-13-hydroperoxy-9,11-octadecadienoate and methyl (10Z,12Z)-9-hydroperoxy-10,12-octadecadienoate, were proved to be present in methyl linoleate hydroperoxide produced by autoxidation. They were identified from their UV, MS, and 1H-NMR spectra after conversion to the corresponding oxo derivatives: methyl (9Z,11Z)-13-oxo-9,11-octadecadienoate and methyl (10Z,12Z)-9-oxo-10,12-octadecadienoate. Their chromatographic behavior is described.

Chemiluminescence properties of soybean protein fraction in the hydroperoxide and hydrogen donor system

Whey fraction, a constituent of soybean protein, produced a photon emission in the presence of gallic acid and hydrogen peroxide. Identification of the chemiluminescence agent from the whey fraction indicated the participation of lipoxygenase in the emission. The reactivity of lipoxygenase with peroxides in the gallic acid solidus hydroperoxide system was in the order of methylethyl hydroperoxide (MEK-OOH, 4800 cps) > tert-butyl hydroperoxide (tert-BuOOH, 607 cps) > hydrogen peroxide (H(2)O(2), 455 cps) > cumene hydroperoxide (cumene-OOH, 261 cps). Emission maxima for H(2)O(2) and cumene-OOH were 670 nm, and emission maxima for MEK-OOH and tert-BuOOH were at 510 nm. The photon intensity from the gallic acid lipoxygenase system corresponded to the linoleic acid hydroperoxide value. A high correlation of photon intensity with hydroperoxide, including linoleic hydroperoxide was useful as a simple and sensitive method for the direct detection of hydroperoxides in biomaterials.

Hydroperoxide Assay with the Ferric–Xylenol Orange Complex

A range of hydroperoxides were reduced by ferrous ions in acid solutions and the amount of ferric product was measured as a xylenol orange complex at 560 nm. Dilute sulfuric acid, 50% acetic acid, and acidified 90% methanol proved to be suitable solvents. The color developed within 15 min and was stable for several hours in most solvents. The apparent molar absorption coefficients (εapp) of H2O2 and of the t-butyl, cumene, bovine serum albumin, and linoleate hydroperoxides were measured, using known hydroperoxide concentrations determined independently by an iodometric assay. The εapp values differed significantly and depended on the hydroperoxide, the solvent, and the source of the xylenol orange. The numbers of Fe3+ ions formed by a range of hydroperoxides in different solvents showed that H2O2 gave 2.5, t-butyl and cumene hydroperoxides 5, and the other hydroperoxides 2 Fe3+ ions per –OOH group. This general finding allows the determination of approximate hydroperoxide concentrations even in chemically complex systems. Accurate measurements require knowledge of the nature of the hydroperoxide and its εapp and careful control of the assay conditions. However, the convenience of the assay makes it potentially useful in a variety of applications.

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.

Effect of pH and temperature on comparative nonenzymatic browning of proteins produced by oxidized lipids and carbohydrates.

Bovine serum albumin (BSA) was incubated for 24 h in the presence of 10 mM ribose (RI), methyl linoleate hydroperoxides, or the secondary products of methyl linoleate oxidation (SP), at five temperatures (25, 37, 50, 80, and 120 degrees C) and different pHs (4, 7, and 10), to study the influence of these variables in the browning, fluorescence, amino acid losses, and pyrrolization of the modified proteins. All treated proteins exhibited similar colors and fluorescence spectra, and the spectra of their Ehrlich adducts were also analogous. However, at 25-50 degrees C the proteins treated with oxidized lipids exhibited higher color changes, amino acid losses, and pyrrolization than the BSA treated with RI, and these effects were much higher in proteins treated with RI at 80-120 degrees C. The effect of pH was similar in proteins treated with RI or SP. These results suggested a similarity for browned proteins obtained from both carbohydrates and oxidized lipids. In addition, both reactions seem to be complementary, because melanoidin formation derived from oxidized lipids can be produced under conditions different from those carbohydrate/protein reactions.

New Geometric Isomers of Oxooctadecadienoate in Copper-catalyzed Decomposition Products of Linoleate Hydroperoxide.

Methyl linoleate hydroperoxide produced by autoxidation was refluxed with 10(-4) M Cu-naphthenate in benzene. Two new geometrical isomers of oxooctadecadienoate (compounds I and II) were found in addition to the four known isomers. They were isolated by a Sephadex LH-20 column chromatography with chloroform-hexane (2:1) and purified by HPLC on Nucleosil ®100-5 and Zorbax ODS columns. UV, IR, MS, and (1)H-NMR spectra were measured. The geometry of conjugated dienes were assigned from the coupling constants of the olefinic protons. Compounds I and II were identified as 13-oxo-trans-9, cis-11- and 9-oxo-cis-10, trans-12-octadecadienoate, respectively. Each of them had a cis double bond adjacent to the oxo group. The hydroperoxides of the same geometry as compounds I and II were also detected in autoxidation products.