Effects of Schisandrin B and α-tocopherol on lipid peroxidation, in vitro and in vivo

Effects of Schisandrin B (Sch B) and -tocopherol (-TOC) on ferric chloride (Fe³⁺) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl₄) induced lipid peroxidation in vivo were examined. While -TOC could produce prooxidant and antioxidant effect on Fe³⁺-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with -TOC (3 mmol/kg/day × 3) did not protect against CCl₄-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day × 3) produced a dose-dependent protective effect on the CCl₄-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.Abbreviations ALT alanine aminotransferase - CCl₄ carbon tetrachloride - Fe³⁺ ferric chloride - MDA malondialdehyde - Sch B Schisandrin B - TBA 2-thiobarbituric acid - TBARS thiobarbituric acid reactive substances - -TOC dl--tocopherol     

Clofibric acid or diethylmaleate supplemented diet decrease blood pressure in DOCA-salt treated male Sprague-Dawley rats - relation with liver antioxidant status

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 M) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe³⁺ to Fe²⁺ reduction. High concentration of ascorbate (150 M), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe³⁺/Fe²⁺ ratio. -Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 M concentration. The inhibitory effect of -tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 M -tocopherol and 50 M ascorbate. This strong inhibitory combined effect of -tocopherol and ascorbate was independent of the Fe³⁺/Fe²⁺ ratio, as a-tocopherol is not able to reduce Fe³⁺ to Fe²⁺ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe³⁺ to Fe²⁺. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.     

Effect of melanins on lipid peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe(2+)-induced, Fe(2+)-ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O2). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.     

Nadph-initiated cytochrome P450-dependent free iron-independent microsomal lipid peroxidation: Specific prevention by ascorbic acid

In this paper we demonstrate that ascorbic acid specifically prevents NADPH-initiated cytochrome P450 (P450)-mediated microsomal lipid peroxidation in the absence of free iron. Lipid peroxidation has been evidenced by the formations of conjugated dienes, lipid hydroperoxide and malondialdehyde. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, glutathione, -tocopherol, uric acid, thiourea, mannitol, histidine, -carotene and probucol are ineffective to prevent the NADPH-initiated P450-mediated free iron-independent microsomal lipid peroxidation. Using a reconstituted system comprised of purified NADPH-P450 reductase, P450 and isolated microsomal lipid or pure L--phosphatidylcholine diarachidoyl, a mechanism has been proposed for the iron-independent microsomal lipid peroxidation and its prevention by ascorbic acid. It is proposed that the perferryl moiety P450 Fe³⁺. O₂ initiates lipid peroxidation by abstracting methylene hydrogen from polyunsaturated lipid to form lipid radical, which then combines with oxygen to produce the chain propagating peroxyl radical for subsequent formation of lipid peroxides. Apparently, ascorbic acid prevents initiation of lipid peroxidation by interacting with P450 Fe³⁺. O₂. (Mol Cell Biochem 166: 35-44, 1997)     

Effect ofγ-irradiated carbohydrates on isolated synaptic membranes

The effect of-irradiated solutions of carbohydrates, mainly glucose, upon Na⁺, K⁺-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe²⁺ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe²⁺-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Comparative study of antioxidant properties and cytoprotective activity of flavonoids

Antioxidant properties and cytoprotective activity of flavonoids (rutin, dihydroquercetin, quercetin, epigallocatechin gallate (EGCG), epicatechin gallate (ECG)) were studied. All these compounds inhibited both NADPH- and CCl₄-dependent microsomal lipid peroxidation, and the catechins were the most effective antioxidants. The I ₅₀ values calculated for these compounds by regression analysis were close to the I ₅₀ value of the standard synthetic antioxidant ionol (2,6-di-tert-butyl-4-methylphenol). The antiradical activity of flavonoids to O ₂ was studied in a model photochemical system. Rate constants of the second order reaction obtained by competitive kinetics suggested flavonoids to be more effective scavengers of oxygen anion-radicals than ascorbic acid. By competitive replacement all flavonoids studied were shown to be chelating agents capable of producing stable complexes with transition metal ions (Fe²⁺, Fe³⁺, Cu²⁺). The flavonoids protected macrophages from asbestos-induced damage, and the protective effect increased in the following series: rutin < dihydroquercetin < quercetin < ECG < EGCG. The cytoprotective effect of flavonoids was in strong positive correlation with their antiradical activity to O ₂ .     

Aluminum Enhances Melanin-Induced Lipid Peroxidation

The present study was conducted to characterize the possible interaction of Al³⁺ and Fe²⁺ with synthetic melanin in the potentiation of lipid peroxidation in liposomes and rat caudate-putamen homogenates. Al³⁺ stimulated melanin-initiated lipid peroxidation as measured by the production of 2-thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. The effect of Al³⁺ was dependent on melanin (10–100 g/ml) and Al³⁺ (2.5–250 M) concentrations and no synergism between Fe²⁺ and Al³⁺ was observed. The prooxidant effect of Al³⁺ was partially inhibited by superoxide dismutase indicating the involvement of O ₂ ^- . Ga³⁺ and Be²⁺ which can increase NADH oxidation in the presence of O ₂ ^- , also were shown to stimulate melanin-initiated TBARS production. Based on the effect of Al³⁺ and other non redox metals, we suggest that Al³⁺ does not act through either the induction of melanin free radicals, or the induction of changes in membrane physical properties. Results show that Al³⁺ enhances melanin-initiated lipid peroxidation in part through an interaction with O ₂ ^- generated from the autoxidation of melanin. We speculate that Al³⁺ contributes to neuromelanin-mediated oxidative damage in dopaminergic neurons and subsequent neuronal degeneration and death in Parkinson's disease.     

Cadmium toxicity of rice leaves is mediated through lipid peroxidation

Oxidative stress, in relation to toxicity of detached rice leaves,caused by excess cadmium was investigated. Cd content inCdCl₂-treated detached rice leaves increased with increasingdurationof incubation in the light. Cd toxicity was followed by measuring the decreasein chlorophyll and protein. CdCl₂ was effective in inducing toxicityand increasing lipid peroxidation of detached rice leaves under both light anddark conditions. These effects were also observed in rice leaves treated withCdSO₄, indicating that the toxicity was indeed attributed to cadmiumions. Superoxide dismutase (SOD), ascorbate peroxidase (APOD), and glutathionereductase (GR) activities were reduced by excess CdCl₂ in the light.The changes in catalase and peroxidase activities were observed inCdCl₂-treated rice leaves after the occurrence of toxicity in thelight. Free radical scavengers reduced CdCl₂-induced toxicity and atthe same time reduced CdCl₂-induced lipid peroxidation and restoredCdCl₂-decreased activities of SOD, APOD, and GR in the light. Metalchelators (2,2-bipyridine and 1,10-phenanthroline) reducedCdCl₂ toxicity in rice leaves in the light. The reduction ofCdCl₂ toxicity by 2,2-bipyridine (BP) is closely associatedwith a decrease in lipid peroxidation and an increase in activities ofantioxidative enzymes. Furthermore, BP-reduced toxicity of detached riceleaves,induced by CdCl₂, was reversed by adding Fe²⁺ orCu²⁺, but not by Mn²⁺ or Mg²⁺.Reduction of CdCl₂ toxicity by BP is most likely mediated throughchelation of iron. It seems that toxicity induced by CdCl₂ mayrequire the participation of iron.     

Arsenic‐induced NFκβ transactivation through Erks‐ and JNKs‐dependent pathways in mouse epidermal JB6 cells

Carnosine, a alanylLhistidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe²⁺, H₂O₂)generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fenitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Lignans from the Fruits of Forsythia suspensa (Thunb.) Vahl Protect High-Density Lipoprotein during Oxidative Stress

The objective of the present study was to investigate the beneficial properties lignan compounds obtained from the fruits of Forsythia suspensa (Thunb.) Vahl (Oleaceae) for protecting human high-density lipoprotein (HDL) against lipid peroxidation. The isolated compounds (1–8) inhibited the generation of thiobarbituric acid-reactive substances (TBARS) in a dose-dependent manner with IC₅₀ values from 8.5 to 18.7 μM, since HDL oxidation mediated by catalytic Cu²⁺. They also exerted an inhibitory effect against thermo-labile radical initiator (AAPH)-induced lipid peroxidation of HDL with IC₅₀ values from 12.1 to 51.1 μM. Compounds 1 and 5 exerted inhibitory effects against the Cu²⁺-induced lipid peroxidation of HDL, as shown by an extended lag time prolongation at the concentration of 3.0 μM. These results suggest that the antioxidative effects of F. suspensa are due to its lignans and that these constituents may be useful for preventing the oxidation of HDL.     

Influence of Antioxidants on the Peroxidative Swelling of Mitochondria in vitro

To clarify the role of prooxidative processes during in vitro swelling of freshly isolated rat liver mitochondria, the influence of different antioxidants and free-radical scavengers was tested. Ascorbate below 10 mmol/L without externally added Fe²⁺ acted as a prooxidant and enhanced swelling. Higher concentrations in the presence of Fe²⁺ showed antioxidant properties and a decrease in swelling and lipid peroxidation. Swelling was abolished by -tocopherol and reduced to 50% by butylated hydroxytoluene. Glutathione supplementation decreased both swelling and lipid peroxidation. Oxidized glutathione caused swelling without any effect on peroxidation. Hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide caused progressive decreases in glutathione and reduced niacinamide coenzyme levels, suggesting prooxidative changes. Dithiothreitol was found to abolish this effect. Thus, antioxidants reverse superoxide-induced mito chondrial swelling and lipid peroxidation in vitro.     

Changes in lipid peroxidation, the redox system and ATPase activities in plasma membranes of rice seedling roots caused by lanthanum chloride

Highly purified plasma membranes were isolated by aqueous two-phase partitioning from rice (Oryza sativa) seedling roots. The effects of lanthanum chloride (LaCl₃) on the activities of lipid peroxidation, the redox system and H⁺-ATPase, Ca²⁺-ATPase of plasma membranes were studied. The lipid peroxidation of plasma membranes could be depressed by certain low concentrations of LaCl₃ and enhanced by high concentrations of LaCl₃, while the lipid peroxidation was also dependent on the plasma membrane protein and incubation time. The relative activity of O₂ uptake of plasma membranes was inhibited by all tested LaCl₃ concentrations. In contrast, the reduction rate of Fe(CN)₆ ^3– by plasma membranes was stimulated below 40 M of LaCl₃, but was reduced above 60 M of LaCl₃. The relative activities of both H⁺-ATPase and Ca²⁺-ATPase increased constantly from control to LaCl₃ of concentration 60 M where the activities of both enzymes were the maximum, but decreased remarkably at 80 M LaCl₃ concentrations various LaCl₃ were added to culture solutions. In the other measurement case in which various LaCl₃ concentrations were added directly to reaction medium and the plasma membrane vesicles only came from the control cultured rice seedling roots, the response of H⁺-ATPase activity to La³⁺ was similar to the response in culture solution. However, the La³⁺ concentration was only 20 M when the activity of H⁺-ATPase was the maximum. In contrast to the case of LaCl₃ addition to culture solution, Ca²⁺-ATPase activity was inhibited by all concentrations of La³⁺ which were added directly to the reaction medium. The above results revealed that REEs inhibited electron transfer from NADH to oxygen in plant plasma membranes, depressed the production of active oxygen radicals, and reduced the formation of lipid peroxides through plasma membrane lipid peroxidation. REEs ions also enhanced the H⁺ extrusion by both standard redox system and H⁺-ATPase in plasma membranes at certain concentrations. A possible role for the plant cell wall in REEs effects on plasma membranes was also suggested.     

Antimicrotubular drugs binding to vinca domain of tubulin

The effect of oxidative stress on the Ca²⁺-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO₄/EDTA (0.2 mol Fe²⁺ per mg of protein) at 37°C for 1 h in the presence or absence of antioxidants. FeSO₄/EDTA decreased the maximum velocity of Ca²⁺-ATPase reaction without a change of affinity for Ca²⁺ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca²⁺-ATPase and decrease in tryptophan fluorescence, while the loss of –SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca²⁺-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca²⁺-ATPase activity.     

The multiple effects of ethylenediaminetetraacetate in several model lipid peroxidation systems

Experiments were performed which illustrate the various ways EDTA can influence lipid peroxidation. Either detergent-dispersed linoleate, or liposomes made from extracted microsomal phospholipids were utilized as substrates for peroxidation. Peroxidation was accomplished using Fe²⁺ or Fe³⁺. In systems utilizing Fe²⁺, EDTA chelation facilitated Fe²⁺ autoxidation which in turn caused peroxidation of detergent-dispersed linoleate. Peroxidation was not initiated during EDTA-Fe²⁺ autoxidation when the substrate lipids were in a liposomal configuration. Systems utilizing Fe³⁺ required an enzyme (either xanthine oxidase or NADPH-cytochrome P₄₅₀ reductase) to reduce the iron for peroxidative activity. EDTA chelation of Fe³⁺ enhanced the xanthine oxidase and NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of detergent-dispersed linoleate, presumably by facilitating the reduction of Fe³⁺. Catalase and mannitol inhibited both EDTA-Fe²⁺- and EDTA-Fe³⁺-dependent lipid peroxidation. EDTA-Fe³⁺ was not capable of initiating peroxidation of phospholipid liposomes following enzymatic reduction by either enzyme, but ADP-chelated iron effectively initiated liposomal peroxidation in similar systems. With xanthine oxidase-catalyzed peroxidation of liposomes with ADP-Fe³⁺, the inclusion of EDTA-Fe³⁺ caused a modest enhancement of activity. EDTA-Fe³⁺ greatly stimulated NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of liposomes with ADP-Fe³⁺. In contrast, the addition of EDTA, rather than EDTA-Fe³⁺ inhibited the liposomal peroxidation catalyzed by either enzyme with ADP-Fe³⁺ when the EDTA concentration exceeded the concentration of Fe³⁺.     

Factors affecting lipid peroxidation in guinea-pig adrenal microsomes

Studies were carried out to examine the effects of and interactions between NADPH, Fe²⁺, Fe³⁺ and ascorbate on lipid peroxidation in guinea-pig adrenal microsomes. Fe²⁺, at levels between 10⁻⁶ and 10⁻³ M, produced concentration-dependent increases in lipid peroxidation in adrenal microsomes; Fe²⁺ had a far greater effect than Fe³⁺. In liver microsomes, by contrast, Fe²⁺ and Fe³⁺ had quantitatively similar effects on lipid peroxidation. NADPH alone had no effect on malonaldehyde production by adrenal microsomes. However, in the presence of low Fe²⁺ concentrations (10⁻⁶ M), NADPH stimulated malonaldehyde production; the stimulation was not demonstrable in microsomes which had been heat-treated to inactive microsomal enzymes. In the presence of high Fe²⁺ levels (10⁻³ M), NADPH produced a concentration-dependent inhibition of lipid peroxidation; the inhibition was fully demonstrable in heat-treated microsomes. In the presence of Fe³⁺ (10⁻⁶ to 10⁻³ M), NADPH had little effect on lipid peroxidation, suggesting that NADPH does not significantly promote the reduction of Fe³⁺ to Fe²⁺ in adrenal microsomes. Ascorbate alone increased malonaldehyde production by adrenal microsomes; maximum stimulation occurred at a concentration of 10⁻⁴ M. Ascorbate-induced lipid peroxidation was also inhibited by NADPH. Ascorbate (5 · 10⁻⁶ to 1 · 10⁻⁴ M) synergistically interacted with low levels (10⁻⁶ M) of Fe²⁺ to enhance malonaldehyde production by adrenal microsomes. The synergism was not demonstrable at high concentrations (10⁻³ M) of Fe²⁺ At all concentrations (10⁻⁶ to 10⁻³ M) of Fe³⁺ studied, ascorbate synergistically increased the production of malonaldehyde. The results indicate that interactions between various endogenous substances may be important in the control of adrenal microsomal lipid peroxidation and that there are differences in the regulation of adrenal and hepatic lipid peroxidation.     

Lack of correlation between TBARS production and PUFA degradation during incubation of membrane erythrocytes in an OH⋅ (Fe2+/H2O2) generator system

We investigated the effects of an OH^⋅ (Fe²⁺/H₂O₂) generator system of erythrocyte membrane, particularly the time-course of lipid peroxidation as estimated by measurement of conjugated dienes, thiobarbituric reactive substances (TBARS), lipofuscin-like pigments, and α-tocopherol. Polyunsaturated fatty acids (PUFAs), especially arachidonic acid (20∶4 ω 6) and docosahexenoic acid (22∶6 ω 3), were also measured. Erythrocyte membranes were suspended in phosphate buffer containing Fe²⁺ (200 μM) and H₂O₂ (1.42 mM), and incubated in a shaking water bath at 37°C. Initially, there was an increase in TBARS and lipofuscin-like pigments, two well-known end products of PUFA oxidative degradation, whereas PUFAs remained unchanged (incubation time: 1 h). After two or more hours of incubation, marked lipid peroxidation was noted, with the appearance of conjugated dienes and a decrease of PUFAs, indicating that lipid peroxidation had occurred after a lag phase during which TBARS were not produced from PUFAs. This suggests that another OH^⋅ target was involved.     

Intensity of free radical processes and regulation of cytoplasmic NADP-isocitrate dehydrogenase in rat cardiomyocytes under normal and ischemic conditions

The intensity of free radical processes and the regulation of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) activity have been studied in the cytoplasmic fraction of normal and ischemized rat myocardium. Chemiluminescence parameters, such as the light sum (S) of slow flash and the tangent of the kinetic curve slope angle (tan₁), which characterize the intensity of free radical processes, were increased in ischemia 2.1- and 20.0-fold, respectively. The slow flash intensity (I_max) was increased 22-fold. The contents of lipid peroxidation products–diene conjugates and malonic dialdehyde–were increased 11.9- and 4.7-fold, respectively, suggesting pronounced oxidative stress. Using homogenous enzyme preparations of NADP-IDH isolated from the normal and experimentally ischemized rat myocardium, a number of catalytic properties of the enzyme were characterized for normal and pathologic conditions. NADP-IDH from the normal and ischemized myocardium had the same electrophoretic mobility and was regulated similarly by Fe²⁺, Cu²⁺, Zn²⁺, and also with succinate and fumarate. However, under normal and pathologic conditions NADP-IDH was different in the affinity for substrates and in the sensitivity to inhibitory effects of hydrogen peroxide, reduced glutathione, and of Ca²⁺. The degree of synergy in the enzyme inhibition with Fe²⁺ and H₂O₂ was less pronounced in ischemia. The inhibitory effect of the reaction product 2-oxoglutarate was higher under normal conditions than in ischemia (the K _i values were 0.22 and 0.75 mM, respectively). The specific features of the NADP-IDH regulation in ischemia are suggested to promote the stimulation of the enzyme functioning during increased level of free radical processes, and this seems to be important for NADPH supplying for the glutathione reductase/glutathione peroxidase antioxidant system of cardiomyocytes.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Direct Measurement of Lipid Hydroperoxides in Iron-Dependent Spinal Neuronal Injury

Abstract: The relationship between iron-dependent fetal mouse spinal cord neuron injury and the generation of endogenous lipid hydroperoxides (LOOHs) has been investigated. Cultured spinal cord neurons were incubated with ferrous iron (3–200 µM). Cell viability was measured in terms of the uptake of α-[methyl-³H]aminoisobutyric acid ([³H]AIB). Both endogenously and iron-generated LOOH, i.e., free fatty acid hydroperoxide (FFAOOH), phosphatidylethanolamine hydroperoxide (PEOOH), and phosphatidylcholine hydroperoxide (PCOOH), were measured directly by an HPLC-chemiluminescence (HPLC-CL) assay. The FFAOOH, PEOOH, and PCOOH levels in neurons incubated with 200 µM Fe²⁺ for 40 min were, respectively, 22-, 158-, and sevenfold higher than those in non-iron-exposed cultures, demonstrating that phosphatidylethanolamine (PE) was most sensitive to peroxidation. The dose-response and time course of Fe²⁺-induced generation of these LOOHs were also established. In both experiments, the LOOH levels were correlated directly with loss of neuronal viability, suggesting strongly a direct relationship between lipid peroxidation and cell injury. On examination of the time course of the LOOH generation, an immediate increase in PEOOH and PCOOH levels with only 30 s of Fe²⁺ incubation was observed. In contrast, a lag phase in the increase in FFAOOH level (2 min after Fe²⁺ addition) suggested a delay in the activation of phospholipase A₂ (PLA₂) required for the hydrolysis and generation of FFAOOH. This culture system provides an excellent model for screening antioxidant neuroprotective compounds with regard to their ability to protect against iron-dependent peroxidative injury and the relationship of the neuroprotection to inhibition of lipid peroxidation and/or PLA₂.