Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.     

Copper-induced peroxidation of phosphatidylserine-containing liposomes is inhibited by nanomolar concentrations of specific antioxidants

Copper-induced peroxidation of liposomal palmitoyllinoleoyl-phosphatidylcholine (PLPC) is inhibited by -tocopherol at micromolar concentrations. In our previous study we found that when the liposomes contain phosphatidylserine (PS), nanomolar concentrations of Toc were sufficient to inhibit peroxidation. In an attempt to gain understanding of the origin of this extreme antioxidative potency, we tested the antioxidative potency of 36 additional antioxidants and the dependence of their potency on the presence of PS in the liposomes. The results of these studies reveal that only 11 of the tested antioxidants possess similar antioxidative potency to that of Toc. These include trolox, butylated hydroxytoluene (BHT), curcumin, nordihydroguaiaretic acid (NDGA), diethylstilbestrol (DES), 2 of the 13 tested flavonoids (luteolin and 7,3′,4′-trihydroxyflavone; T-414), -naphthol, 1,5-, 1,6- and 1,7-dihydroxynaphthalenes (DHNs). Propyl gallate (PG), methyl syringate, rosmarinic acid, resveratrol, other flavonoids, as well as β-naphthol, 1,2-, 1,3-, 1,4-, 2,3-, 2,6-, and 2,7-DHNs were either moderately antioxidative or pro-oxidative. For liposomes made of PLPC (250 μM) and PS (25 μM) the “lag” preceding copper-induced peroxidation (5 μM copper) was doubled upon addition of 30–130 nM of the “super-active” antioxidants.

We propose that the mechanism responsible for the extreme antioxidative potency against copper-induced peroxidation in PS-containing liposomes involves replenishment of the antioxidant in a ternary PS–copper-antioxidant complex. Based on structure–activity relationship of the 37 tested antioxidants, the “super-antioxidative potency” is attributed to the recycling of relatively stable semiquinone or semiquinone-like radicals.     

Oxidation of liposomal cholesterol and its effect on phospholipid peroxidation

Lipid peroxidation is believed to play an important role in the pathogenesis of many diseases. Much research has therefore been devoted to peroxidation of different lipids in biomembranes and in model systems (liposomes) of different compositions. Yet, in spite of the relative simplicity of the liposomes, the existing literature is insufficient to reach definite conclusions regarding basic questions including the susceptibility of cholesterol to oxidation, its effect on the peroxidation of polyunsaturated phospholipids such as palmitoyllinoleoylphosphatidylcholine (PLPC) and how cholesterol influences the effect of water-soluble antioxidants such as urate on the peroxidation. The aim of the present study was to clarify these issues. Its major findings are that: (i) AAPH-induced peroxidation of cholesterol is slow and independent of the peroxidation of PLPC. In turn, AAPH-induced peroxidation of PLPC is not affected by cholesterol, independent of the presence of urate in the system. (ii) Cholesterol is not susceptible to copper-induced oxidation, but its inclusion in PLPC liposomes affects the peroxidation of PLPC, slowing down the initial stage of oxidation but promoting later stages. (iii) Addition of urate accelerates copper-induced peroxidation of PLPC in the absence of cholesterol, whereas in cholesterol-containing liposomes it inhibits PLPC oxidation. We attribute the complexity of the observed kinetics to the known cholesterol-induced rigidization of liquid crystalline bilayers.     

Lipid peroxidation in the presence of albumin, inhibitory and prooxidative effects

Oxidative modifications of LDL are involved in atherogenesis. Previously we have developed a simple assay to evaluate the susceptibility of lipids to copper-induced peroxidation in the relatively natural milieu of unfractionated serum in the presence of excess citrate. Based on our previous results we have proposed that the inducer of peroxidation in our optimized assay is a copper-citrate complex. Recent investigations indicate that under certain conditions a copper-albumin complex may induce peroxidation of ascorbate. Two different complexes may be formed in albumin-containing systems (e.g. serum) namely 1:1 and 2:1 copper-albumin complexes. The aim of the present work was to evaluate the possibility that at least one of these complexes may be responsible for the induction of peroxidation of lipids in lipidic systems containing copper and albumin, including our optimized assay. Towards this end, we have investigated the dependence of copper-induced peroxidation on the concentration of added albumin in lipidic systems in the absence and presence of citrate. In all the systems investigated in this study (PLPC liposomes, LDL, HDL and mixtures of HDL and LDL) we found that at low concentrations of free copper (e.g. in the presence of excess citrate) the 2:1 copper-albumin complex is redox-active and that this complex is the major contributor to the initiation of lipid peroxidation in these systems and in our optimized assay. The possible relevance of the induction of peroxidation in vivo by the latter complex has yet to be studied. *This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Dorit Samocha-Bonet, Sackler Faculty of Medicine, Tel-Aviv University, Israel.     

Lipid Peroxidation in the Presence of Albumin,Inhibitory and Prooxidative Effects

Oxidative modifications of LDL are involved in atherogenesis. Previously we have developed a simple assay to evaluate the susceptibility of lipids to copper-induced peroxidation in the relatively natural milieu of unfractionated serum in the presence of excess citrate. Based on our previous results we have proposed that the inducer of peroxidation in our optimized assay is a copper-citrate complex. Recent investigations indicate that under certain conditions a copper-albumin complex may induce peroxidation of ascorbate. Two different complexes may be formed in albumin-containing systems (e.g. serum) namely 1:1 and 2:1 copper-albumin complexes. The aim of the present work was to evaluate the possibility that at least one of these complexes may be responsible for the induction of peroxidation of lipids in lipidic systems containing copper and albumin, including our optimized assay. Towards this end, we have investigated the dependence of copper-induced peroxidation on the concentration of added albumin in lipidic systems in the absence and presence of citrate. In all the systems investigated in this study (PLPC liposomes, LDL, HDL and mixtures of HDL and LDL) we found that at low concentrations of free copper (e.g. in the presence of excess citrate) the 2:1 copper-albumin complex is redox-active and that this complex is the major contributor to the initiation of lipid peroxidation in these systems and in our optimized assay. The possible relevance of the induction of peroxidation in vivo by the latter complex has yet to be studied. <footnote id="fn1"><no>*</no>This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Dorit Samocha-Bonet, Sackler Faculty of Medicine, Tel-Aviv University, Israel. </footnote>     

In vitro lipid peroxidation of human serum catalyzed by cupric ion: antioxidant rather than prooxidant role of ascorbate.

The dual role of ascorbate as an antioxidant and a prooxidant has been clearly documented in the literature. Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, we observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protective effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar alpha-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with alpha-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by plpha-tocopherol after serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanism is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH. Our results also indicate an outstanding anti-oxidant property of human serum due to the chelation of transition metal ions (even at high concentrations) by various serum proteins.     

Kinetic studies of copper-induced oxidation of urate, ascorbate and their mixtures

Urate and ascorbate are the major water-soluble low molecular weight antioxidants in serum. Much attention has been devoted to the effect of these antioxidants on lipoprotein peroxidation in vivo and on their effect on copper-induced peroxidation ex vivo. These studies revealed that urate inhibits ascorbate oxidation in vitro, whereas the effect of ascorbate on urate oxidation has not been systematically studied thus far. The present study addresses mechanistic aspects of the kinetics of copper-induced oxidation of both these antioxidants and their mutual effects in aqueous solutions. We found that: (i) ascorbate becomes oxidized much faster than urate. (ii) Urate inhibits the oxidation of ascorbate but, even in the presence of excess urate, ascorbate becomes oxidized much faster than urate. (iii) Ascorbate, as well as the products of its oxidation (and/or hydrolysis) inhibit the copper-induced oxidation of urate. All these results are consistent with the hypothesis that the rate of ascorbate oxidation is determined by the rate of reoxidation of reduced copper (Cu(I)) to Cu(II) by molecular oxygen, whereas the rate of urate oxidation is governed by the rate of oxidation of urate within a 2:1 urate/copper complex. We think that the mutual effects of urate and ascorbate on each other's oxidation are likely to enhance their inhibitory effect on lipid peroxidation in biologically relevant systems including membranes and lipoproteins.     

Kinetic analysis of copper-induced peroxidation of HDL, autoaccelerated and tocopherol-mediated peroxidation

Comparison of the kinetic profiles of copper-induced peroxidation of HDL and LDL at different copper concentrations reveals that under all the studied experimental conditions HDL is more susceptible to oxidation than LDL. The mechanism responsible for HDL oxidation is a complex function of the copper/HDL ratio and of the tocopherol content of the HDL. At high copper concentrations, the kinetic profiles were similar to those observed for LDL oxidation, namely, relatively rapid accumulation of oxidation products, via an autoaccelerated, noninhibited mechanism, was preceded by an initial "lag phase." Under these conditions, the maximal peroxidation rate (V(max)) of HDL and LDL depended similarly on the molar ratio of bound copper/lipoprotein. Analysis of this dependency in terms of the binding characteristics of copper to lipoprotein, yielded similar dissociation constant (K = 10(-6) M) but different maximal binding capacities for the two lipoproteins (8 Cu(+2)/HDL as compared to 17 Cu(+2)/LDL). Given the size difference between HDL and LDL, these results imply that the maximal surface density of bound copper is at least 2-fold higher for HDL than for LDL. This difference may be responsible for the higher susceptibility of HDL to copper-induced oxidation in the presence of high copper concentrations. At relatively low copper concentrations, the kinetic profile of HDL oxidation was biphasic, similar to but more pronounced than the biphasic kinetics observed for the oxidation of LDL lipids at the same concentration of copper. Our results are consistent with the hypothesis that the first phase of rapid oxidation occurs via a tocopherol-mediated-peroxidation (TMP) mechanism. Accordingly, enrichment of HDL with tocopherol resulted in enhanced accumulation of hydroperoxides during the first phase of copper-induced oxidation. Notably, the maximal accumulation during the first phase decreased upon increasing the ratio of bound copper/HDL. This behavior can be predicted theoretically for peroxidation via a TMP mechanism, in opposition to autoaccelerated peroxidation. The possible pathophysiological significance of these findings is discussed.     

Iron binding to alpha-tocopherol-containing phospholipid liposomes

Tocopherols (vitamin E) located in the hydrophobic domains of biological membranes act as chain breaking antioxidants preventing the propagation of free radical reactions of lipid peroxidation. The naturally occurring form, d-alpha tocopherol is an exquisite molecule in that it is intercalated in the membrane in such a way that the hydrophobic tail anchors the molecule positioning the chromanol ring containing the hydroxyl group, which is the essence of its antioxidant function, at the polar hydrocarbon interface of phospholipid membranes. The interaction of this group with water soluble substances is not very well understood. In the present study, an investigation was made of the interaction of ascorbate and ferrous ions (Fe+2) initiators of lipid peroxidation with alpha tocopherol. The results show that tocopherol increases membrane associated iron. The formation of a tocopherol iron complex in the presence of phospholipid liposomes and ascorbate in its reduced form is indicated. These results suggest a new way in which tocopherols act to inhibit lipid peroxidation.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Kinetics of lipid peroxidation in mixtures of HDL and LDL, mutual effects.

In view of the proposed central role of LDL oxidation in atherogenesis and the established role of HDL in reducing the risk of atherosclerosis, several studies were undertaken to investigate the possible effect of HDL on LDL peroxidation. Since these investigations yielded contradictory results, we have conducted systematic kinetic studies on the oxidation in mixtures of HDL and LDL induced by different concentrations of copper, 2, 2'-azo bis (2-amidinopropane) hydrochloride (AAPH) and myeloperoxidase (MPO). These studies revealed that oxidation of LDL induced either by AAPH or MPO is inhibited by HDL under all the studied conditions, whereas copper-induced oxidation of LDL is inhibited by HDL at low copper/lipoprotein ratio but accelerated by HDL at high copper/lipoprotein ratio. The antioxidative effects of HDL are only partially due to HDL-associated enzymes, as indicated by the finding that reconstituted HDL, containing no such enzymes, inhibits peroxidation induced by low copper concentration. Reduction of the binding of copper to LDL by competitive binding to the HDL also contributes to the antioxidative effect of HDL. The acceleration of copper-induced oxidation of LDL by HDL may be attributed to the hydroperoxides formed in the "more oxidizable" HDL, which migrate to the "less oxidizable" LDL and enhance the oxidation of the LDL lipids induced by bound copper. This hypothesis is supported by the results of experiments in which native LDL was added to oxidizing lipoprotein at different time points. When the native LDL was added prior to decomposition of the hydroperoxides in the oxidizing lipoprotein, the lag preceding oxidation of the LDL was much shorter than the lag observed when the native LDL was added at latter stages, after the level of hydroperoxides became reduced due to their copper-catalyzed decomposition. The observed dependence of the interrelationship between the oxidation of HDL and LDL on the oxidative stress should be considered in future investigations regarding the oxidation of lipoprotein mixtures.     

Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.     

The effect of albumin on copper-induced LDL oxidation

In an attempt to gain deeper understanding of the mechanism or mechanisms responsible for the protective effect of serum albumin against Cu²⁺-induced peroxidation of low density lipoprotein (LDL), we have examined the influence of the concentrations of bovine serum albumin (BSA), Cu²⁺ and LDL on the kinetics of peroxidation. Since the common method of monitoring the oxidation by continuous recording of the absorbance of conjugated dienes at 234 nm cannot be used at high BSA-concentrations because of the intensive absorption of BSA, we have monitored the time-dependent increase of absorbance at 245 nm. At this wavelength, conjugated dienes absorb intensely, whereas the background absorbance of BSA is low. Using this method, as well as the TBARS assay for determination of malondialdehyde, over a large range of BSA concentrations, we show that in many cases the influence of BSA on the kinetics of oxidation can be compensated for by increasing the concentration of copper. This reconciles the apparent contradiction between previously published data. Detailed studies of the kinetic profiles obtained under different conditions indicate that binding of Cu²⁺ to albumin plays the major role in its protective effect while other mechanisms contribute much less than copper binding. This conclusion is consistent with the less pronounced effect of BSA on the oxidation induced by the free radical generator AAPH. It is also shown that the copper-albumin complex is capable of inducing LDL oxidation, although the kinetics of the latter process is very different from that of copper-induced oxidation. Nevertheless, when compared to copper induced oxidation at similar concentration of the oxidation-promotor, the kinetics of oxidation induced by copper-albumin complex is very different and is consistent with a tocopherol mediated peroxidation, characteristic under low radical flux. Similar kinetics was observed for copper-induced oxidation only at much lower copper concentrations.     

Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes

Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes.     

Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.     

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.     

Differences in the susceptibility of plant membrane lipids to peroxidation

Peroxidation of three membrane lipid preparations from plants was initiated using Fe-EDTA and ascorbate and quantified as the production of aldehydes and loss of esterified fatty acids. Using liposomes prepared from commercial soybean asolecithin, the degree of peroxidation was shown to be dependent on: the free radical dose, which was varied by the ascorbate concentration; the presence of tocopherol in the liposome; the configuration, of the liposome, multilamellar or unilamellar; and time after initiation. There were dramatic interactions among these factors which led to the conclusion that in comparing the susceptibility of different membrane preparations it is essential to examine the kinetics of the peroxidation reactions. The composition of the liposome was a major determinant of the degree of peroxidation and of the type of degradative reactions initiated by the oxygen free radicals. A fresh polar lipid extract from Typha pollen had very similar fatty acid composition to the soybean asolecithin, but was more resistant to peroxidation as shown by less aldehyde production and increased retention of unsaturated fatty acids after treatment. Similarly, microsomal membranes from the crowns of non-acclimated and cold acclimated winter wheat (Triticum aestivum L.) seedlings had a much higher linolenic acid content than soybean asolecithin but was much more resistant to peroxidation. In the winter wheat microsomes, the loss of esterified fatty acids was not selective for the unsaturated fatty acids; consequently, even with 40% degradation, the degree of unsaturation in the membrane did not decrease. These different reaction mechanisms which occur in plant membranes may explain why measurements of fatty acid unsaturation fail to detect peroxidative reactions during processes such as senescence, aging and environmental stress.     

Chelating and antiradical effect of rutin during peroxidation of lipids from microsomes and liposomes

The antioxidative effect of rutin (vitamin P) on Fe2+-induced lipid peroxidation (LPO) in bovine heart microsomes and lecithin liposomes was studied. It was shown that the LPO-induced inhibition of microsomes and liposomes in the presence of rutin occurs via two mechanisms, i.e., association of Fe2+ ions to form an inactive complex and a direct interaction between rutin and free radicals. The contribution of these mechanisms depends on the composition of the reaction mixture. In bovine heart microsomes and liposomes, ascorbic acid has a dual activity towards LPO. At high concentrations of Fe2+ necessary for LPO induction (approximately 1 x 10(-3) M), ascorbic acid blocks LPO, whereas at low Fe2+ concentrations (less than 1 x 10(-4) M) it has a prooxidative effect. A combined use of ascorbic acid and rutin results in an additive antioxidative effect at high Fe2+ concentrations (approximately 1.10(-3) M). However, at low Fe2+ concentrations rutin acts as an antagonist of the prooxidative effect of ascorbic acid.     

Hg2+ and Cu+ are ionophores, mediating Cl-/OH- exchange in liposomes and rabbit renal brush border membranes.

Organometals, including organomercurials, are capable of mediating Cl-/OH- exchange across lipid membranes by forming neutral ion pairs. In this study, the ability of inorganic metals to catalyze Cl-/OH- exchange was examined. In the presence of an inwardly directed chloride gradient, HgCl2 at concentrations as low as 30 nM resulted in quenching of acridine orange fluorescence in liposomes, indicating liposomal acidification. In the presence of the reducing agent, ascorbate, CuSO4 at concentrations as low as 0.6 microM also mediated chloride-dependent liposomal acidification. Copper in the absence of ascorbate, iron (with or without ascorbate), cobalt, cadmium, zinc, nickel, and lead were without an effect. 36Cl efflux from rabbit renal brush border membrane vesicles was also markedly stimulated by micromolar concentrations of mercury or copper plus ascorbate. Vesicle integrity was not altered by the concentrations of mercury or copper employed in these studies. In the absence of ascorbate, CuCl stimulated chloride efflux only under anaerobic conditions, confirming that it is the reduced form of copper (Cu+) that mediates chloride transport across the membrane. In the presence of mercury or reduced copper, an inside alkaline pH gradient stimulated the uphill accumulation of 36Cl and 82Br, respectively, confirming Cl-/OH- exchange. Studies in liposomes and brush border membranes demonstrate that this is an electroneutral process. These results show that Hg2+ and Cu+ are capable of acting as ionophores, mediating electroneutral Cl-/OH- exchange in liposomes and brush border membrane vesicles. This effect could contribute to the toxicity of these two metals.