Lipid peroxide formation in microsomes. The role of non-haem iron

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe²⁺ or Fe³⁺) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe²⁺ is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe(2+) or Fe(3+)) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe(2+) is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

Time-course of oxidation of lipids in human cerebrospinal fluid in vitro

Oxidative mechanisms play an important role in the pathogenesis of Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. To assess whether the oxidation of brain lipoproteins plays a role in the development of these pathologies, we investigated whether the lipoproteins of human cerebrospinal fluid (CSF) are susceptible to oxidative modification in vitro. We studied oxidation time-course for up to 100 h of human CSF in the absence (autooxidation) or presence of exogenous oxidants. Autooxidation of diluted CSF was found to result in a slow accumulation of lipid peroxidation products. The time-course of lipid hydroperoxide accumulation revealed three consecutive phases, lag-phase, propagation phase and plateau phase. Qualitatively similar time-course has been typically found in human plasma and plasma lipoproteins. Autooxidation of CSF was accelerated by adding exogenous oxidants, delayed by adding antioxidants and completely inhibited by adding a chelator of transition metal ions. Autooxidation of CSF also resulted in the consumption of endogenous ascorbate, α-tocopherol, urate and linoleic and arachidonic acids. Taking into account that (i) lipid peroxidation products measured in our study are known to be derived from fatty acids, and (ii) lipophilic antioxidants and fatty acids present in CSF are likely to be located in CSF lipoproteins, we conclude that lipoproteins of human CSF are modified in vitro during its autooxidation. This autooxidation appears to be catalyzed by transition metal ions, such as Cu(II) and Fe(III), which are present in native CSF. These data suggest that the oxidation of CSF lipoproteins might occur in vivo and play a role in the pathogenesis of neurodegenerative diseases.     

The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes.

Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.     

Ascorbate- and hemoglobin-dependent brain chemiluminescence

It has been indicated recently that ascorbic acid is responsible for the hemoglobin-mediated oxidative damage to the central nervous system (Sadrzadeh & Eaton, J. Clin. Invest. 82:1510-1515, 1988). In this paper we describe the changes in chemiluminescence accompanying hemoglobin- and ascorbate-dependent oxidative injury to brain tissue. Addition of either hemoglobin (15 microM) or ascorbate (1 or 2 mM) to rat brain homogenates stimulated spontaneous chemiluminescence in a synergistic manner. This increase in chemiluminescence was inhibited by desferrioxamine indicating that free iron was involved in the reactions leading to lipid peroxidation. Preincubation with ascorbate oxidase inhibited both spontaneous and hemoglobin-dependent chemiluminescence, suggesting that ascorbate was required for the reactions leading to lipid peroxidation. Supplementation with aminotriazole (an irreversible inhibitor of the catalase-H2O2 complex) increased chemiluminescence in a time-dependent manner, as catalase reacted with accumulated H2O2, suggesting that ascorbic acid has a dual action being involved in the production of H2O2 and also maintaining Fe in the reduced state to catalyze a Fenton-like reaction. The excited species responsible for the chemiluminescence were partially characterized by adding specific fluorescent energy acceptors: dibromoanthracene (DBA) and diphenylanthracene (DPA). Both DBA and DPA stimulated chemiluminescence several-fold indicating that triplet and singlet species are responsible for the observed chemiluminescence. Excited singlet carbonyls (identified with DPA) may be produced during the collision of two ROO.. Singlet oxygen may also be generated during the same reaction. It decays to the triplet state (emitting chemiluminescence at 634 nm) and reacts with double bonds producing dioxetanes, which may breakdown generating triplet carbonyls (identified with DBA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.     

In vitro Antioxidant Activity of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> Against Different Neurotoxic Agents

Valeriana officinalis L. (Valerian) is widely used as a traditional medicine to improve the quality of sleep. Although V. officinalis have been well documented as promising pharmacological agent; the exact mechanisms by which this plant act is still unknown. Limited literature data have indicated that V. officinalis extracts can exhibit antioxidant properties against iron in hippocampal neurons in vitro. However, there is no data available about the possible antioxidant effect of V. officinalis against other pro-oxidants in brain. In the present study, the protective effect of V. officinalis on lipid peroxidation (LPO) induced by different pro-oxidant agents with neuropathological importance was examined. Ethanolic extract of valerian (0–60 μg/ml) was tested against quinolinic acid (QA); 3-nitropropionic acid; sodium nitroprusside; iron sulfate (FeSO₄) and Fe²⁺/EDTA induced LPO in rat brain homogenates. The effect of V. officinalis in deoxyribose degradation and reactive oxygen species (ROS) production was also investigated. In brain homogenates, V. officinalis inhibited thiobarbituric acid reactive substances induced by all pro-oxidants tested in a concentration dependent manner. Similarly, V. officinalis caused a significant decrease on the LPO in cerebral cortex and in deoxyribose degradation. QA-induced ROS production in cortical slices was also significantly reduced by V. officinalis. Our results suggest that V. officinalis extract was effective in modulating LPO induced by different pro-oxidant agents. These data may imply that V. officinalis extract, functioning as antioxidant agent, can be beneficial for reducing insomnia complications linked to oxidative stress.     

Antioxidant Defense Systems in the Brains of Type II Diabetic Mice

Abstract: The specific activities of superoxide dismutase, catalase, and glutathione S -transferase (μ subtype) were significantly lower in the brains of mice with type II diabetes than in the brains of control mice. On the other hand, the specific activity of glutathione peroxidase was unaltered. The concentration of vitamin E, but not that of total glutathione and ascorbate, was increased in the brains of the type II diabetic mice. The relative amount of polyunsaturated fatty acids (as determined with soybean lipoxygenase) was increased in whole brains and crude synaptosomal membranes of the type II diabetic mice. Endogenous levels of thiobarbituric acid-positive material were decreased in both whole brain homogenates and crude synaptosomal membranes of the db/db mice. Susceptibility of lipids within whole brain homogenates and crude synaptosomal membranes of mice with type II diabetes to peroxidation with iron/ascorbate was also markedly decreased compared with that of controls. Vitamin E is known to quench lipid peroxidation. Therefore, decreased lipid peroxidation in the type II mouse brain may be due to increased vitamin E content.     

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.     

In the presence of ferritin,visible light induces lipid peroxidation of the porcine photoreceptor outer segment

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.     

Lipid Peroxidation in the Cortex and Medulla of Rabbit Kidneys Subjected to Cold Ischaemia and the Value of Protective Agents

The storage of rabbit kidneys for 24hr at 0 C in isotonic saline resulted in significantly increased rates of lipid peroxidation, as measured by the formation of thiobarbituric acid-reactive material and Schiff bases during in vitro incubation of homogenates prepared from the cortex and medulla. In addition, the content of thiobarbituric acid-reactive material in the medulla was also significantly elevated as a result of cold storage for 24 hr.

The effects of antioxidants (vitamin E), iron-chelation (desferoxamine) and inhibitors of arachidonic acid oxidation (indomethacin and dazmegrell on the rate of lipid peroxidation in homogenates prepared from ischaemic kidneys were studied. This demonstrated that lipid peroxidation in the cortex was predominantly non-specific and iron-catalysed whereas in the medulla approximately 50% of the TBA-reactive material was formed enzymically from arachidonic acid by cyclooxygenase.     

Induction of Betalain Pigmentation in Hairy Roots of Red Beet under Different Radiation Sources

The effects of aluminum on lipid peroxidation and activities of antioxidative enzymes were investigated in detached rice leaves treated with 0 to 5 mM AlCl₃ at pH 4.0 in the light. AlCl₃ enhanced the content of malondialdehyde but not the content of H₂O₂. Superoxide dismutase activity was reduced by AlCl₃, while catalase and glutathione reductase activities were increased. Peroxidase and ascorbate peroxidase activities were increased only after prolonged treatment, when toxicity occurred. The results give evidence that Al treatment caused oxidative stress and in turn, it caused lipid peroxidation.     

Carboxyfullerene prevents iron-induced oxidative stress in rat brain

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron-induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+-evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co-infusion of carboxyfullerene prevented iron-induced oxidative injury. Furthermore, tyrosine hydroxylase-immunoreactive staining showed that carboxyfullerene inhibited the iron-induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron-induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose-dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron-induced oxidative injury.     

Novel 21-amino steroids as potent inhibitors of iron-dependent lipid peroxidation

Two representative compounds from a novel chemical series of potent inhibitors of lipid peroxidation are described. The compounds 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione monomethane sulfonate (U74006F) and 21-[4-(3,6-bis(diethylamino)-2-pyridinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)triene-3,20-dione hydrochloride (U74500A) inhibited lipid peroxidation in brain homogenates and purified brain synaptosomes under a variety of conditions involving iron. With IC50 values ranging from 2 to 60 microM, U74006F and U74500A were comparable in potency to alpha-tocopherol or butylated hydroxytoluene and were nearly 100 times as potent as desferrioxamine. Some specificity for intact phospholipid membranes is suggested since the ability of U74006F or U74500A to inhibit lipid peroxidation was greatly reduced in methanol solutions of arachidonic acid. Despite close similarities in their structures, their response to increasing concentrations of Fe2+ in lipid peroxidation assays differed qualitatively. One of the compounds, U74500A, may act as a membrane localized chelator of iron.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.     

Effects of zinc on copper-induced and spontaneous lipid peroxidation

Zinc (Zn) is an essential nonredox metal that has been regarded as having antioxidant properties. Some epidemiological indications and therapeutic results point to a role of Zn in restricting the development and the progression of some diseases. Redox-active metals like iron and copper are involved in oxidative injury mechanisms, and a decrease in the Zn∶Cu ratio may be associated with certain pathologies. We studied the effect of Zn on the copper-induced lipid peroxidation in diluted human plasma. Lipid peroxidation was evaluated by measuring the formation of conjugated dienes and of thiobarbituric acid reactive products. We found that 20 μM Zn reduced the 125-μM copper-dependent formation of conjugated dienes by 27% and of thiobarbituric acid reactive products by 49%, during a 3-h incubation period. The inhibition of lipid peroxidation by 125 μM Zn is almost total in the same conditions. The time-course study of the inhibitory effect of 125 μM Zn showed that it lasted for 7 h, which was the maximum incubation period tested. We also found that Zn had an inhibitory effect on the spontaneous lipid peroxidation in rat brain whole homogenates. Our results support the antioxidant properties of Zn, which may be potentially relevant to the protection of human plasma constituents, competing with the transition metals for redox reactions.     

Antioxidant and Neuroprotective Properties of Sour Tea (Hibiscus sabdariffa, calyx) and Green Tea (Camellia sinensis) on some Pro-oxidant-induced Lipid Peroxidation in Brain in vitro

Oxidative stress is the cause of neurodegenerative disorders such as Lou Gehrig’s disease, Parkinson’s disease, and Huntington’s disease; one practical way to prevent and manage neurodegenerative diseases is through the eating of food rich in antioxidants (dietary means). This present study sought to compare the ability of aqueous extract of sour tea (Hibiscus sabdariffa, calyx) and green tea (Camellia sinensis) to prevent some pro-oxidant [Fe (II), sodium nitroprusside, quinolinic acid]-induced lipid peroxidation in rat’s brain in vitro. Aqueous extracts of both teas were prepared (1 g tea in 100 ml of hot water). Thereafter, the ability of the extracts to prevent 25 μM FeSO₄, 7 μM sodium nitroprusside, and 1 mM quinolinic acid-induced lipid peroxidation in isolated rat’s brain tissue preparation was determined in vitro. Subsequently, the total phenol content, reducing power, Fe (II) chelating and OH radical scavenging ability were determined. The results of the study revealed that both teas significantly (P < 0.05) inhibit lipid peroxidation in basal and pro-oxidant-induced lipid peroxidation in the rat’s brain homogenates in a dose-dependent manner. Also, the teas had high total phenol content [sour (13.3 mg/g); green (24.5 mg/g)], reducing power, and Fe (II) chelating and OH radical scavenging ability (except sour tea). However, green tea had a significantly higher (P < 0.05) ability to inhibit lipid peroxidation in both the basal and pro-oxidant-induced lipid peroxidation in rat’s brain homogenates in vitro. Therefore, it is obvious from the study that both teas had high antioxidant properties and could inhibit Fe²⁺, sodium nitroprusside, and quinolinic acid-induced lipid peroxidation in brain. However, green tea had a higher inhibitory effect, which may probably be due to its high total phenol content, reducing power, Fe (II) chelating ability, and OH radical scavenging ability.     

Glutathione enzymes,glutathione content and t-butyl hydroperoxide induced lipid peroxidation in the gill and digestive gland of the estuarine clam,Rangia cuneata

1. Reduced glutathione (GSH), glutathione reductase (GSSG-reductase) and glutathione peroxidase (GSH-peroxidase) activities were measured in the gill and digestive gland of Rangia cuneata.2. Substantial GSH concentrations were found in both gill (820 ± 80 nmole/g tissue) and digestive gland (930 ± 130 nmole/g tissue). The digestive gland exhibited 2.5-fold greater GSSG-reductase activities and 0.5-fold lower GSH-peroxidase activities relative to the gill.3. In vivo exposure to t-butyl hydroperoxide (BHP) elicited a dose-dependent increase (P < 0.05) in lipid peroxidation in both tissues. Lipid peroxidation occurred earlier and to a greater extent in the digestive gland versus the gill. GSH concentrations in both tissues were unaffected by BHP exposure.4. The study results indicate that gill and digestive gland differ in susceptibility to BHP induced oxidative damage, and the difference is accounted for by differences in tissue GSH metabolism.     

Metalloporphyrins are potent inhibitors of lipid peroxidation

The objectives of these studies were to determine whether metalloporphyrins could inhibit lipid peroxidation, characterize factors that influence their potency and compare their potency to prototypical antioxidants. Lipid peroxidation was initiated with iron and ascorbate in rat brain homogenates and the formation of thiobarbituric acid reactive species was used as an index of lipid peroxidation. Metalloporphyrins were found to be a novel and potent class of lipid peroxidation inhibitors. Inhibition of lipid peroxidation by metalloporphyrins was dependent on the transition metal ligated to the porphyrin, indicating that metal centered redox chemistry was important to the mechanism of their antioxidant activities. Manganese porphyrins with the highest superoxide dismutase (SOD) activities, MnOBTM-4-PyP and MnTM-2-PyP (charges are omitted throughout text for clarity), were the most potent inhibitors of lipid peroxidation with calculated IC50s of 1.3 and 1.0 microM, respectively. These manganese porphyrins were 2 orders of magnitude more potent than either trolox (IC50 = 204 microM) or rutin (IC50 = 112 microM). The potencies of the manganese porphyrins were related not only to their redox potentials and SOD activities, but also to other factors that may contribute to their ability to act as electron acceptors. The broad array of antioxidant activities possessed by metalloporphyrins make them attractive therapeutic agents in disease states that involve the overproduction of reactive oxygen species.