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A Sevanian

Publications and source records attributed to A Sevanian.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effect of supplementary antioxidant vitamin intake on carotid arterial wall intima-media thickness in a controlled clinical trial of cholesterol lowering.

BACKGROUND: There is accumulating experimental, epidemiological, and clinical evidence of an association between anti-oxidant vitamin intake and reduced risk of coronary heart disease. Using data from the Cholesterol Lowering Atherosclerosis Study (CLAS), we explored the association of self-selected supplementary antioxidant vitamin intake on the rate of progression of early preintrusive atherosclerosis. METHODS AND RESULTS: CLAS was an arterial imaging trial in which nonsmoking 40- to 59-year-old men with previous coronary artery bypass graft surgery were randomized to colestipol/niacin plus diet or placebo plus diet. The rate of progression of early preintrusive atherosclerosis was determined in 146 subjects using high-resolution B-mode ultrasound quantification of the distal common carotid artery far wall intima-media thickness (IMT). From the nutritional supplement database, 22 subjects had an on-trial average supplementary vitamin E intake of > or = 100 IU per day (high users) and 29 subjects had an average on-trial supplementary vitamin C intake of > or = 250 mg per day (high users). Within the placebo group, less carotid IMT progression was found for high supplementary vitamin E users when compared with low vitamin E users (0.008 versus 0.023 mm/y, P = .03). No effect of vitamin E within the drug group was found. No effect of vitamin C within the drug or placebo group was found. CONCLUSIONS: Supplementary vitamin E intake appears to be effective in reducing the progression of atherosclerosis in subjects not treated with lipid-lowering drugs while the process is still confined to the arterial wall (early preintrusive atherosclerosis).

Adult↗
PubMed1996

Probucol suppresses oxidant stress in hypertensive arteries. Immunohistochemical evidence.

Oxy-free radicals may be involved in the pathogenesis of accelerated atherosclerosis in hypertension. We evaluated the direct antioxidant potential of probucol in hypertensive arteries by studying the spatial immunohistochemical distribution of three primary antioxidant enzymes (AEs). Nineteen normocholesterolemic New Zealand White rabbits were divided into two groups: normotensive controls (NT; n = 6) and 13 animals rendered hypertensive by surgical coarctation of abdominal aorta. The hypertensive group was subdivided into hypertensive alone (HT; n = 8) and hypertensive treated with 1% probucol (PO) for 9 weeks (HT-P; n = 5). Blood pressure rose significantly in both hypertensive groups (P < .005). At autopsy, both hypertensive groups showed similarly significant increases in mean arterial intima-media thickness (IMT) whether or not they were treated with probucol. However, only HT rabbits revealed significant increases in the intima-media depth penetration of glutathione peroxidase, superoxide dismutase, and catalase AEs. By contrast, in HT-P animals probucol produced significant reductions of immunostaining of all three AEs compared to the HT group (P < .05). Additionally, specific macrophage immunostaining revealed that the arterial wall of HT rabbits had numerous (10 to 12 per high power field) subintimal and medial macrophages as compared to the HT-P animals (1 to 2 per high power field). The blood pressure level correlated significantly with IMT in all three groups, but with depth penetration of the three AEs only in the NT and HT groups. Probucol, therefore, appears to act in concert with the native arterial antioxidant enzymes as a potent free radical scavenger to reduce oxidative stress and thus attenuate the macrophage invasive response in hypertensive arteries.

Animals↗
PubMed1996

Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations.

Oxidative modification of LDL is thought to be a radical-mediated process involving lipid peroxides. The small dense LDL subpopulations are particularly susceptible to oxidation, and individuals with high proportions of dense LDL are at a greater risk for atherosclerosis. An oxidatively modified plasma LDL, referred to as LDL-, is found largely among the dense LDL fractions. LDL- and dense LDL particles also contain much greater amounts of lipid peroxides compared with total LDL or the more buoyant LDL fractions. The content of LDL- in dense LDL particles appears to be related to copper- or heme-induced oxidative susceptibility, which may be attributable to peroxide levels. The rate of lipid peroxidation during the antioxidant-protected phase (lag period) and the length of the antioxidant-protected phase (lag time) are correlated with the LDL- content of total LDL. Once LDL oxidation enters the propagation phase, there is no relationship to the initial LDL- content or total LDL lipid peroxide or vitamin E levels. Beyond a threshold LDL- content of approximately 2%, there is a significant increase in the oxidative susceptibility of nLDL particles (ie, purified LDL that is free of LDL-), and this susceptibility becomes more pronounced as the LDL- content increases. nLDL is resistant to copper- or heme-induced oxidation. The oxidative susceptibility is not influenced by vitamin E content in LDL but is strongly inhibited by ascorbic acid in the medium. Involvement of LDL(-)-associated peroxides during the stimulated oxidation of LDL is suggested by the inhibition of nLDL oxidation when LDL- is treated with ebselen prior to its addition to nLDL. Populations of LDL enriched with LDL- appear to contain peroxides at levels approaching the threshold required for progressive radical propagation reactions. We postulate that elevated LDL- may constitute a pro-oxidant state that facilitates oxidative reactions in vascular components.

Adult↗
PubMed1996

Physical effects of biologically formed cholesterol oxidation products on lipid membranes investigated with fluorescence depolarization spectroscopy and electron spin resonance.

Planar oriented membranes of 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) containing cholesterol, 19-hydroxycholesterol, 22S-hydroxycholesterol, or 25-hydroxycholesterol in concentrations up to 5 mol % were investigated with angle-resolved fluorescence depolarization and electron spin resonance measurements. Analyses of the data with the Brownian diffusion model show that the oxysterols have structural effects similar to those of cholesterol: an increase in molecular order and no change in the rotational diffusion coefficients of the probe molecules. Time-resolved fluorescence anisotropy measurements on diphenylhexatriene (DPH) in small unilamellar vesicles of POPC and DOPC were performed using oxysterols commonly found in oxidized low density lipoproteins (LDL) in comparison to membranes containing cholesterol or no sterols. Analyses using the Brownian rotational diffusion model show that most LDL-oxysterols affect the vesicle physical structure in a manner similar to cholesterol, viz. an increase in molecular order and a decrease in the dynamics. Cholesterol-alpha-epoxide has a much smaller ordering effect than cholesterol in POPC-vesicles. A similar effect was found for 7 beta-hydroxycholesterol in DOPC-vesicles. The tendency of the oxysterols to influence the molecular order as compared to pure cholesterol may contribute to cell membrane permeability changes affecting crucial cell functions and events leading to vascular cell injury. Increased LDL oxysterol levels may account for some of the structural changes noted for oxidatively modified LDL as well as its toxicity to vascular cells.

Cholesterol↗
PubMed1995

Effect of linoleic acid hydroperoxide on endothelial cell calcium homeostasis and phospholipid hydrolysis.

The relationship between intracellular free calcium ion concentrations ([Ca2+]i) and release of arachidonic acid from membrane phospholipids following peroxidation was examined in rabbit aortic endothelial cells treated with linoleic acid hydroperoxide (LOOH). LOOH (0.1-0.4) mumol/10(6) cells) caused a rapid and dose-dependent transient increase in [Ca2+]i in the presence of extracellular Ca2+ that remained elevated over baseline for 15 to 30 s. In the absence of extracellular Ca2+, LOOH also evoked a transient increase in [Ca2+]i of lesser magnitude which immediately returned to basal (or below basal) levels. In this regard, the rise in intracellular Ca2+ after LOOH or vasopressin (AVP) treatments involved, at least in part, related intracellular pools that in each case was followed by influx of extracellular Ca2+. The intracellular membrane sources known to be affected by vasopressin were not directly involved. Most notably, the LOOH evoked rise in [Ca2+]i was not associated with release of IP3, suggesting that the source of intracellular Ca2+ is not IP3-sensitive pools. However, pretreatment with LOOH strongly inhibited the rise in [Ca2+]i upon subsequent addition of AVP or LOOH and the extent of such inhibition was dependent on the availability of free intracellular Ca2+ and presence of extracellular Ca2+. These findings suggest that reuptake of Ca2+ into intracellular membrane pools is reduced in the presence of LOOH and/or the availability of Ca2+ from agonist-sensitive sites is inhibited by LOOH. An increase in free 20:4 levels was found after LOOH treatment that was only partly prevented using intracellular Ca2+ chelators which maintained [Ca2+]i at basal levels after LOOH treatment. These findings suggest that LOOH induction of phospholipid hydrolysis proceeds following small transients in [Ca2+]i that are considerably less than that evoked by agents such as AVP, approximating basal Ca2+ concentrations. Inhibition of LOOH-induced lipid peroxidation by vitamin E also prevented the rise in [Ca2+]i and 20:4 release indicating that phospholipid hydrolysis is dependent, at least in part, on membrane lipid peroxidation. Inhibition of protein kinase C (PKC) completely blocked LOOH-induced release of 20:4 but had little effect on the LOOH-induced rise in [Ca2+]i, suggesting an indirect relationship between LOOH-induced membrane Ca2+ signalling events, with intervention via PKC-mediated induction of phospholipid hydrolysis. A rapid and progressive translocation of PKC to the membrane fraction was evident after LOOH addition over the time course corresponding to the maximal release of 20:4 which was also inhibited by vitamin E.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Serial coronary angiographic evidence that antioxidant vitamin intake reduces progression of coronary artery atherosclerosis.

OBJECTIVE: To explore the association of supplementary and dietary vitamin E and C intake with the progression of coronary artery disease. DESIGN: A subgroup analysis of the on-trial antioxidant vitamin intake database acquired in the Cholesterol Lowering Atherosclerosis Study, a randomized, placebo-controlled, serial angiographic clinical trial evaluating the risk and benefit of colestipol-niacin on coronary artery disease progression. SETTING: Community- and university-based cardiac catheterization laboratories. SUBJECTS: A total of 156 men aged 40 to 59 years with previous coronary artery bypass graft surgery. INTERVENTION: Supplementary and dietary vitamin E and C intake (nonrandomized) in association with cholesterol-lowering diet and either colestipol-niacin or placebo (randomized). OUTCOME: Change per subject in the percentage of vessel diameter obstructed because of stenosis (%S) determined by quantitative coronary angiography after 2 years of randomized therapy on all lesions, mild/moderate lesions (< 50%S), and severe lesions (> or = 50%S). RESULTS: Overall, subjects with supplementary vitamin E intake of 100 IU per day or greater demonstrated less coronary artery lesion progression than did subjects with supplementary vitamin E intake less than 100 IU per day for all lesions (P = .04) and for mild/moderate lesions (P = .01). Within the drug group, benefit of supplementary vitamin E intake was found for all lesions (P = .02) and mild/moderate lesions (P = .01). Within the placebo group, benefit of supplementary vitamin E intake was not found. No benefit was found for use of supplementary vitamin C exclusively or in conjunction with supplementary vitamin E, use of multivitamins, or increased dietary intake of vitamin E or vitamin C. CONCLUSIONS: These results indicate an association between supplementary vitamin E intake and angiographically demonstrated reduction in coronary artery lesion progression. Verification from carefully designed, randomized, serial arterial imaging end point trials is needed.

Adult↗
PubMed1995

Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein.

Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10(-2) U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1995

Characterization of endothelial cell injury by cholesterol oxidation products found in oxidized LDL.

The present study describes the toxicity of oxidized LDL towards rabbit aortic endothelial cells in terms of its lipid components with specific attention to the cholesterol oxidation products (ChOx) found in oxidized LDL isolated from human plasma. Measurements of the major ChOx associated with freshly isolated unmodified LDL, those found in oxidized LDL isolated from human plasma and LDL subjected to oxidation in vitro are described. We have confirmed previous findings that most of the cytotoxicity of freshly isolated human LDL may be attributable to a minor fraction that appears to be oxidatively modified by several criteria. Moreover, this plasma-derived oxidized LDL (referred to as LDL) is highly enriched in ChOx, whereas the content of lipid peroxides or derived products (measured as conjugated dienes and thiobarbituric acid reacting products) are much lower, particularly when compared to copper-induced LDL oxidation. Much of the ChOx found in plasma are associated with LDL, however, the levels and proportions of the various ChOx found in LDL differ from those produced after extensive copper-induced oxidation but resemble those produced after moderate oxidation with copper. The species and concentrations of ChOx found in LDL when applied as a mixture exhibit considerably more toxicity than any individual ChOx alone. At non-toxic levels this ChOx mixture causes an increased influx of several ions, including calcium, an effect not seen with individual ChOx at comparable doses. Perturbations in ionic homeostasis, and particularly the sustained increase in intracellular calcium concentrations, are associated with much of the cytotoxicity, an effect attributable to the membrane disruptive action of ChOx leading to altered ion transporter activity. The effect of the ChOx mixture (but not any individual ChOx) on sodium and potassium flux appears to be due to enhanced Na+/K(+)-ATPase activity based on the complete inhibition produced by ouabain under all treatment conditions. These findings also show that the levels of cholesterol oxidation products found in normal LDL are not cytotoxic whereas those present in oxidized LDL exceed the toxic threshold for endothelial cells and account for most of the cytotoxicity produced by this modified lipoprotein.

Animals↗
PubMed1994

Linoleic acid hydroperoxide-induced peroxidation of endothelial cell phospholipids and cytotoxicity.

Peroxidation of endothelial cell phospholipids was examined following treatments with linoleic acid hydroperoxide. The treatment effects were analyzed over a range of toxicities and exposure intervals as determined by cell plating efficiencies and survival. Over the concentration ranges where lipid peroxidation was evident (20-40 microM treatments in complete medium), significant cytotoxicity was apparent after 1 h of exposure. The extent of toxicity was dependent on the time interval between the end of peroxide treatment and replating of cells. Maximum toxicity was found when cells were replated 1-3 h after treatment. When cells were replated 4 h after treatment a linear increase in cell survival was found as a function of replating time following peroxide exposure. Analysis of cell phospholipids by HPLC after 1 h of exposure to linoleic acid hydroperoxide revealed that peroxidation (evidenced by conjugated diene content) had taken place among a number of phospholipid species with the most marked increases in phosphatidylcholine. Analysis of the fatty acyl composition of phospholipids also showed that the proportions of polyunsaturated fatty acids were reduced relative to saturated fatty acids, indicating peroxidative damage to phospholipids. Pretreatment of cells with vitamin E prevented the peroxidation of all phospholipids and blocked the cytotoxic action of linoleic acid hydroperoxide. These findings indicate that an immediate cytotoxic action of lipid hydroperoxide is associated with peroxidation of membrane phospholipids. This cytotoxicity is a transient effect, and cells surviving the acute injury display a time-dependent increase in plating efficiency representing a period of repair.

Animals↗
PubMed1994

Lipid hydroperoxide-induced peroxidation and turnover of endothelial cell phospholipids.

The effects of lipid peroxidation on rabbit aortic endothelial cell phospholipid turnover was studied using linoleic acid hydroperoxide (LOOH). Following treatments with 20-40 microM LOOH, cells prelabeled with either arachidonic acid (20:4) or oleic acid (18:1) showed a movement of these fatty acids out of the phospholipids and into neutral lipid and free fatty acid pools. There was also a release of radioactive free fatty acids and phospholipids into the media, which was significantly increased as compared to cells maintained under standard culture conditions. Fatty acid uptake and distribution among phospholipid pools was also affected by LOOH treatment where incorporation of 20:4 and 18:1 into phosphatidylcholine (PC) decreased, while uptake into phosphatidylinositol (PI) increased after 1 h of incubation with 40 microM LOOH. These effects were also inhibited by vitamin E. In cells prelabeled with 20:4 or 18:1 under conditions where approximately 99% of the fatty acids were incorporated into neutral and phospholipid pools, LOOH treatment produced a decrease in radioactivity associated with PC, while the specific activity of PI increased. The extent of these changes was greater for 20:4 than 18:1, but in each case the effects were inhibited by vitamin E. The temporal pattern of uptake for labeled choline and inositol after LOOH treatments paralleled those found for fatty acid incorporation. These cell responses indicate that induction of lipid peroxidation produces rapid fatty acid release and phospholipid turnover involving repair as well as de novo synthesis. The implications of these effects on turnover of specific phospholipids and cell responses to oxidative stress are discussed.

Animals↗
PubMed1994

Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry.

The application of gas chromatography and high-pressure liquid chromatography/mass spectrometry techniques for analysis of plasma cholesterol oxidation products is described. Cholesterol oxides that are widely identified in biological samples were subjected to gas (GC) and high-pressure liquid chromatographic (HPLC) separations, and their detection and characterization by mass spectrometry (MS) were compared. Analysis of cholesterol oxides from plasma samples revealed distinct advantages for each method according to the specific cholesterol oxide in question. Whereas HPLC/MS analysis of cholesterol oxides provided less resolution and lower sensitivity as compared to GC/MS, a distinct advantage was evident for direct measurements of cholesterol-7-hydroperoxides and 7-ketocholesterol. These two cholesterol oxides are particularly sensitive to storage in solvents, derivatization procedures, and analytical conditions used for GC analysis, which are minimized or avoided using the HPLC/MS conditions described. Analysis of human and rabbit plasma samples identified cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol); 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol (cholesterol-5 alpha, 6 alpha-epoxide); 5 alpha-cholestane-3 beta, 5,6 beta-triol (cholestanetriol); 3 beta-hydroxycholest-5-ene-7-one (7-ketocholesterol); and 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide) as commonly occurring components (trivial names indicated in parentheses). The latter two compounds were dramatically increased in hypercholesterolemic samples and were found in approximately equal amounts in the free cholesterol and cholesteryl ester fractions. Although most of the plasma cholesterol oxides are found in the dietary cholesterol, others are not, particularly cholesterol-5 beta,6 beta-epoxide, suggesting that at least some of these compounds are formed by in vivo oxidation of cholesterol. Despite the readily measurable levels of the above cholesterol oxides, as well as other less prominent oxides, there was no evidence of cholesterol-7-hydroperoxides associated with plasma free cholesterol. Although several of the plasma cholesterol oxides may derive from cholesterol-7-hydroperoxides, it appears that the latter are either unstable and decompose in plasma, are metabolized to other cholesterol oxidation products, or break down during their isolation.

Animals↗
PubMed1994

Phospholipase A2 stimulation by methyl mercury in neuron culture.

In primary prelabeled cultures of cerebellar granule cells, methyl mercury (MeHg) induced a concentration- and time-dependent release of [3H]arachidonic acid. MeHg-induced [3H]arachidonate release was partially dependent on the extracellular Ca2+ concentration. MeHg at 10-20 microM also stimulated basal 45Ca2+ uptake after 20 min of incubation at 37 degrees C, and at 10 microM inhibited K+ depolarization-stimulated uptake. MeHg stimulated [3H]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A2 (PLA2) activation preceded cytotoxicity, but at higher concentrations of MeHg such dissociation was not evident. Inhibition of MeHg-induced PLA2 activation by 100 microM mepacrine failed to modify cytotoxicity. MeHg-induced lipoperoxidation, measured as the production of thiobarbituric acid-reacting products, was inhibited by alpha-tocopherol without inhibition of [3H]arachidonate release. The absence of alpha-tocopherol inhibition of MeHg-induced arachidonate release precludes a causal role for lipoperoxide-induced PLA2 activation in this system. Moreover, MeHg induced an increased susceptibility of unilamellar vesicles to exogenous PLA2 in the presence of low Ca2+ concentrations without evidence of lipid peroxidation. [3H]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relatively high proportional incorporation was found in the combined phosphatidylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the phosphatidylinositol fraction, indicating a preferential turnover in this phospholipid species in the presence of MeHg.

Acylation↗
PubMed1994

Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-).

Using ion exchange high pressure liquid chromatography, total plasma low density lipoprotein (LDL) from 30 hypercholesterolemic and 10 normocholesterolemic cynomolgus monkeys was subfractionated into unmodified LDL (n-LDL) and more negatively charged LDL (LDL-). In hypercholesterolemic monkeys, the absolute LDL-cholesterol level was 16.54 +/- 2.82 mg/dl (mean +/- SE) whereas in normocholesterolemic monkeys it was 2.39 +/- 0.12 mg/dl (P < 0.0001); the percentage of LDL- was 5.2 +/- 0.71% and 4.9 +/- 0.19% of the total LDL for hypercholesterolemic versus normocholesterolemic monkeys, respectively. LDL- averaged 5% and n-LDL 95% of the total plasma LDL cholesterol. To confirm and further elucidate the oxidative nature of LDL-, cholesterol and cholesterol oxide contents of LDL- and n-LDL were determined by capillary gas chromatography; 53.98 +/- 2.24% (mean +/- SE) of the LDL- cholesterol was oxidized whereas in n-LDL only 10.70 +/- 1.06% of the cholesterol was oxidized (P < 0.00001). The spectrum of oxysterols identified, which was similar for LDL- and n-LDL, suggested a free radical-mediated process for cholesterol oxidation. The principal oxysterols identified were: cholest-5-ene-3 beta, 7 alpha-diol, cholesta-3,5-diene-7-one, cholest-5-ene-3 beta, 7 beta-diol, 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol, 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, 5 alpha-cholestan-3 beta,5,6 beta-triol, 3 beta-hydroxycholest-5-ene-7-one, and cholest-5-ene-3 beta,25-diol. To model one of the steps in the possible mechanism of atherogenesis, the cytotoxicity of LDL- was demonstrated to be greater against subconfluent than confluent aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Enhanced interfacial catalysis and hydrolytic specificity of phospholipase A2 toward peroxidized phosphatidylcholine vesicles.

The hydrolytic action of phospholipase A2 was examined with unilamellar vesicles composed of soybean phosphatidylcholine in terms of the calcium dependency of the enzyme and substrate specificity following lipid peroxidation. Experiments were performed under conditions where enzyme:substrate ratios were low, specifically in the range of one to five enzyme molecules for every 10 vesicle particles. Accordingly, low hydrolytic activities were found where less than 15% of the phospholipids were hydrolyzed under the various conditions of time, enzyme:substrate ratios, calcium concentrations, and extent of peroxidation utilized. Vesicle peroxidation increased the Ca2+ binding potential to a degree comparable to addition of the anionic phospholipid, dioleoylphosphatidic acid (DOPA). A remarkable similarity was found between the binding profiles for Ca2+ and phospholipase A2 activity; however, enzyme activity toward oxidized vesicles was beyond the increases observed for Ca2+ binding. Under conditions where approximately 5% of the phospholipids were peroxidized the effective Ca2+ concentration required for half-maximal activity was less than one-half that required for unoxidized vesicles. Peroxidation of vesicle phospholipids markedly increased the rate and extent of hydrolysis, even in the presence of DOPA or deoxycholate. Deoxycholate is known to induce vesicle fusion such that a larger proportion of enzyme is associated with a fewer number of enlarged vesicles. Using a dual isotope technique to measure hydrolysis of oxidized vs unoxidized phospholipids and covesicle preparations to study enzyme binding and activity, a significantly greater apparent intervesicle exchange of enzyme was found after peroxidation of vesicles with more than a twofold hydrolytic specificity toward the oxidized phospholipids. We postulate that a combination of structural and Ca2+ binding affinity changes are produced in membranes following lipid peroxidation which evoke an additive effect on PLA2 activity. Although oxidized phospholipids may serve as activators of phospholipase A2 by presenting the interface in a form where Ca2+ and enzyme binding and/or specific activity are increased, an additional and important factor appears to involve membrane fusion or vesicle-vesicle interactions. This process facilitates enzyme activity through the replenishment of substrates wherein the otherwise limited interaction of enzyme and substrate is overcome by more rapid or extensive vesicle fusion which increases access to the phospholipids available in the preparation.

Calcium↗
PubMed1993

The genotoxic effects of arachidonic acid in V79 cells are mediated by peroxidation products.

Chinese hamster lung fibroblasts (V79 cells) were exposed to 2, 20, 160, and 200 microM arachidonic acid (AA). At 20 microM, a stimulatory effect on cell growth was observed, while at 200 microM, an inhibitory effect was found. AA at 160 and 200 microM induced a 10- to 12-fold increase of 6-thioguanine-resistant mutants in V79 cells, whereas increased alkaline labile sites on DNA were consistently produced only with 200 microM treatment doses. The induction of alkaline labile lesions was partially inhibited in the presence of an antioxidant NDGA. [3H]AA metabolism in cells was examined by HPLC analysis following reductive transmethylation of the cell lipid extracts. Two fractions corresponding to [3H]AA-derived peroxidation products were identified by HPLC-radiochromatography and analyzed by mass spectrometry. Quantitative evaluation of the radioactivity associated with these peaks revealed an increase in [3H]AA peroxidation products when cells were exposed to increasing amounts of AA. The AA-dependent stimulation of lipid peroxidation product formation was partially inhibited when cells were treated in the presence of NDGA. The results provide evidence that substantially enlarged AA pools in cells may induce genotoxic effects mediated through increased endogenous lipid peroxidation products.

Animals↗
PubMed1992

Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.

Diphenylhexatriene↗
PubMed1992

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.

Animals↗