The interrelationship of the processes of lipid peroxidation and lipid phospholipase hydrolysis in the synaptosomes

The influence of Fe2+, alpha-tocopherol, phospholipase A2 and mepacrine on the activity of lipid peroxidation (LPO) and phospholipid hydrolysis (PLH) was studied in synaptosomes. It was established that there is the tight direct interconnection between LPO and PLH in synaptosomes. It is assumed that activation of endogenous phospholipases in neurons is one of the causes of uncompensated LPO-activation during epileptogenesis.     

Carnosine protection of Ca2+ transport against damage induced by lipid peroxidation

The authors studied the protective action of carnosine on sarcoplasmic reticulum (SR) membranes from frog skeletal muscles destroyed by ascorbic acid-dependent lipid peroxidation (LPO). It was demonstrated that addition of carnosine to the incubation medium at a concentration of 25 mM sharply decelerated inactivation of Ca-ATPase of SR membranes, maintaining at the same time the coupling of hydrolysing and transport functions of the Ca-pump. When given at the same concentration carnosine inhibited the accumulation of LPO products reacting with 2-thiobarbituric acid. This effect of carnosine was followed by its utilization.     

Mechanism of the stabilization of synaptosomes with alpha-tocopherol during activation of lipid peroxidation

Using the fluorescent probe technique, it was shown that activation of lipid peroxidation decreases the value of transmembrane potential of rat brain synaptosomes. Depolarization of synaptosomes may be due to the impairment of the "barrier" properties of synaptosomal membranes and the decrease in Na,K-ATPase activity. alpha-Tocopherol and its model derivative devoid of the phytol chain--2,2,5,7,8-pentamethyl-6-oxychromanol--stabilize the transmembrane potential value during inhibition of lipid peroxidation. alpha-Tocopherol acetate causes no stabilizing or inhibiting effects. Unlike 2,2,5,7,8-pentamethyl-6-oxychromanol, alpha-tocopherol exerts a structuralizing action which manifests itself in the stabilization of the synaptosomal membrane potential during incomplete inhibition of lipid peroxidation. The previously established ability of alpha-tocopherol to protect synaptosomes from the damaging action of phospholipases and the experimental results of this work permit to regard vitamin E as a universal stabilizer of brain synaptosomal membranes.     

The role of phospholipase A2 in microsomal lipid peroxidation induced with t-butyl hydroperoxide

The role of phospholipase A2 (PlA2) in lipid peroxidation induced with t-butyl hydroperoxide was examined in rat liver microsomes. Exposure of microsomes to t-butyl hydroperoxide was associated with activation of endogenous PlA2. When PlA2 was inhibited with chlorpromazine, mepacrine, or p-bromphenacyl bromide, the accumulation of thiobarbituric acid reactive substances (TBARS) was reduced in a dose dependent manner. In contrast, the accumulation of conjugated dienes was not affected by chlorpromazine, and was slightly increased by mepacrine. When endogenous PlA2 was activated with mellitin prior to induction of peroxidation, accumulation of both TBARS and dienes was reduced. Analogously, pretreatment with exogenous PlA2 reduced both dienes and TBARS. In contrast, addition of mellitin following the induction of peroxidation did not alter either TBARS or dienes.     

Role of isoprenoid chain of lateral mobility of alpha-tocopherol in lipid bilayer]

Using the quenching effect of the fluorescence by nitroxyl radicals the lateral mobility of chromanols in the lipid bilayer was studied. The lateral mobility of the chromanols was shown to increase when the length of phytol chain was diminished. The result is consistent with the idea that antioxidant affect of the chromanols depends on their lateral mobility.     

The role of phospholipase A activity in rat liver microsomal lipid peroxidation

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.     

The role of lipid peroxidation in liver damage

The consequences of the peroxidative breakdown of membrane lipids have been considered in relation to both the subcellular and tissue aspects of liver injury. Mitochondrial functions can be impaired by lipid peroxidation probably through the oxidation of pyridine nucleotides and the consequent alteration in the uptake of calcium. Several enzymatic functions of the endoplasmic reticulum are also affected as a consequence of peroxidative events and among these are the activities of glucose 6-phosphatase, cytochrome P-450 and the calcium sequestration capacity. Moreover, a release of hydrolytic enzymes from lysosomes and a decrease in the fluidity of plasma membranes can contribute to the liver damage consequent to the stimulation of lipid peroxidation. Extensive studies carried out in vivo and integrated with the use of isolated hepatocytes have shown that lipid peroxidation impairs lipoprotein secretion mainly at the level of the dismission from the Golgi apparatus, rather than during their assembly. However, such an alteration appears to give a late and not essential contribution to the fat accumulation. A more critical role is played by peroxidative reactions in the pathogenesis of acute liver necrosis induced by several pro-oxidant compounds as indicated by the protective effects against hepatocyte damage exerted by antioxidants. In addition, even in the cases where lipid peroxidation has been shown not to be essential in causing cell death there is evidence that it can still act synergistically with other damaging mechanisms in the amplification of liver injury.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

Species differences in adrenal lipid peroxidation: role of alpha-tocopherol

Previous reports have noted high levels of lipid peroxidation (LP) in vitro in a variety of adrenocortical preparations. However, we have observed that susceptibility to adrenal LP seems to vary considerably from species to species. The current study was done to confirm these apparent species differences in adrenal LP in vitro and to determine if they were attributable to differences in alpha-tocopherol content. Incubation of mitochondrial or microsomal preparations from guinea pig or rabbit adrenal glands with ferrous ion (Fe2+) caused a time-dependent increase in the formation of thiobarbituric acid reactive substances (TBARS) accompanied by depletion of alpha-tocopherol. By contrast, incubation of adrenal mitochondria or microsomes from rats or monkeys with Fe2+ had little or no detectable effect on TBARS and basal adrenal alpha-tocopherol levels were five to ten-fold greater than those in guinea pigs or rabbits. In addition, there was little change in alpha-tocopherol concentrations during incubation of rat or monkey adrenal tissue. Dietary alpha-tocopherol deficiency in rats reduced adrenal alpha-tocopherol to concentrations approximating those in guinea pigs. Incubation with Fe2+ induced high levels of TBARS in adrenal mitochondria and microsomes from the alpha-tocopherol deficient rats. Conversely, dietary alpha-tocopherol supplementation in rabbits increased adrenal alpha-tocopherol levels and prevented Fe2+ induced TBARS formation in mitochondria and microsomes. The results indicate that there are large species differences in adrenal susceptibility to LP in vitro and that these differences are at least partly attributable to species differences in adrenal alpha-tocopherol concentrations.     

Nickel-mediated inhibition in the glutathione-dependent protection against lipid peroxidation

Glutathione protects liver microsomes against the rapid onset of lipid peroxidation via a sulfhydryl dependent heat labile factor known as free radical reductase. The administration of nickel to mice resulted in an inhibition in the activity of free radical reductase, and enhanced lipid peroxidation and the activity of glutathione S-transferase in a dose dependent manner. The pretreatment of cyclam, a known specific chelator of nickel restored free radical reductase and glutathione S-transferase activities and alleviated nickel mediated enhancement of lipid peroxidation. Our results indicate that nickel-mediated inhibition in free radical reductase activity and activation of glutathione S-transferase may be due to the interaction of nickel with sensitive-SH groups located on these proteins.     

The influence of alpha-tocopherol and pyritinol on oxidative DNA damage and lipid peroxidation in human lymphocytes

Unscheduled DNA synthesis (UDS) and lipid peroxidation (LPO) were measured in human peripheral lymphocytes from healthy volunteers. These processes were induced by the catalytic system Fe2+-sodium ascorbate. The degree of induced LPO was measured spectrophotometrically by the thiobarbituric acid assay. UDS was detected by scintillometric measurement of the incorporation of 3H-thymidine into DNA. The protective action by fat-soluble vitamin E (D,L-alpha-tocopherol) and the artificial antioxidant pyritinol on UDS and LPO was also investigated. The system Fe2+ (2 mumole/l)-sodium ascorbate (30 mumole/l) increased the LPO level in healthy volunteers approximately 2.5 times and the incorporation of 3H-thymidine by 60-70%. alpha-Tocopherol (0.2 mmole/l) very efficiently suppressed LPO processes (p less than 0.01) and the oxidative damage of DNA measured as UDS was also significantly diminished (p less than 0.05). Pyritinol had no effect on LPO and UDS under our experimental conditions.     

Effects of phospholipase A2 and alpha-tocopherol on the activity of adenylate cyclase of rat brain synaptosomes

The action of phospholipase A2 and alpha-tocopherol on adenylate cyclase system functioning and on the lipid bilayer microviscosity of the rat brain synaptosome membranes was investigated. It was shown that the exposure of the synaptosomes to phospholipase A2 increases the adenylate cyclase activity stimulated by guanylyl imidotriphosphate (GITP), decreases the adenylate cyclase activity stimulated both by isoproterenol and by isoproterenol with GITP. The preincubation of synaptosomes in medium containing alpha-tocopherol does not change the character of the phospholipase action on the adenylate cyclase activity stimulated by isoproterenol but normalizes the adenylate cyclase activity stimulated both by GITP and by GITP with isoproterenol. In the last case the normalizing action of alpha-tocopherol is not caused by alteration of the microviscosity of the lipid bilayer. It appears to be due to the modification of the lipid-protein interactions of annular lipids with activated complex of catalytic subunit and guanyl nucleotide-binding protein.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity

Treatment of non-induced or phenobarbital-induced, glutathione-depleted mice with 400 mg/kg paracetamol led to a marked ethane exhalation as an index of in vivo lipid peroxidation (LPO) and to a significant elevation of liver-specific serum enzyme activities. Similar effects were seen with rats treated with 0.5 ml/kg CCl4. Pretreatment with the iron-chelating agent desferrioxamine (DFO) clearly suppressed lipid peroxidation in all cases, but inhibited only the CCl4-induced hepatotoxicity. Treatment of mice with desferrioxamine alone showed no hepatotoxicity at all, nor did it influence liver GSH-levels. In addition, DFO had no effect on hepatic microsomal enzyme activities responsible for the bioactivation of both paracetamol and CCl4. These findings are consistent with the theories which indicate that lipid peroxidation requires the presence of Fe2+-ions, regardless of the initiating agent, and that LPO is involved in CCl4-toxicity, but most probably not in paracetamol-induced liver damage. Furthermore, Fe2+-ions might play a role as mediators of CCl4-hepatotoxicity.     

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.     

Inhibition of lipid peroxidation in muscle homogenates by phospholipase A2 inhibitors

Chlorpromazine, mepacrine, tetracaine, dibucaine, chloroquine, and procaine have been shown to inhibit the iron- and ascorbate-induced lipid peroxidation of skeletal-muscle hornogenates in vitro. These compounds are known to be inhibitors of phospholipase activity, but they were also found to be effective in blocking free-radical-mediated damage to lipids in denatured homogenates, to linoleate suspensions, and to glutamic acid solutions where phospholipase activity was not a relevant factor. The inhibitory action did not appear to be related to any iron-binding activity of the compounds.     

Antioxidant protection of phospholipid bilayers by alpha-tocopherol. Control of alpha-tocopherol status and lipid peroxidation by ascorbic acid and glutathione

Factors affecting the balance between pro- and antioxidant effects of ascorbic acid and glutathione were studied in soybean phosphatidylcholine liposomes challenged with Fe2+/H2O2. Effective antioxidant protection by alpha-tocopherol appeared to be due to efficient reaction with lipid oxy-radicals in the bilayer rather than to interception of initiating oxygen radicals. At concentrations above a threshold level of approximately 0.2 mol % (based on phospholipid content), alpha-tocopherol completely suppressed lipid oxy-radical propagation, which was measured as malondialdehyde production. Both ascorbic acid and glutathione, alone or in combination, enhanced lipid oxy-radical propagation. Alpha-Tocopherol, incorporated into liposomes at concentrations above its threshold protective level, reversed the pro-oxidant effects of 0.1-1.0 mM ascorbic acid but not those of glutathione. Ascorbic acid also prevented alpha-tocopherol depletion. The combination of ascorbic acid and subthreshold levels of alpha-tocopherol only temporarily suppressed lipid oxy-radical propagation and did not maintain the alpha-tocopherol level. Glutathione antagonized the antioxidant action of the alpha-tocopherol/ascorbic acid combination regardless of alpha-tocopherol concentration. These observations indicate that membrane alpha-tocopherol status can control the balance between pro- and antioxidant effects of ascorbic acid. The data also provide the most direct evidence to date that ascorbic acid interacts directly with components of the phospholipid bilayer.     

Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate

The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.