Reduced glutathione effects on α-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation

Factors involved in reduced glutathione (GSH) and vitamin E-mediated inhibition of NADPH-dependent rat liver microsomal lipid peroxidation were examined. Lipid peroxidation was monitored over a time-course of 180 min by thiobarbituric acid reactive product formation. The addition of 5 mM GSH to the reaction system containing microsomes from rats fed a diet supplemented with 150 IU/kg of α-tocopherol acetate for eight weeks produced a lag in peroxidation of >30 min. This effect was not observed for microsomes prepared from rats fed a diet deficient in vitamin E. Indeed, a prooxidant effect of 5 mM GSH was observed in assays containing microsomes from rats fed a diet deficient in vitamin E. The inhibition by GSH of lipid peroxidation in microsomes prepared from livers of vitamin E supplemented rats was not restricted by its availability, for it was found that approximately 92% of the GSH remained in the reduced form after 60 min. Additional experiments revealed that the α-tocopherol content of peroxidizing microsomes decreased rapidly in the absence of GSH. The addition of 5 mM GSH to the assay system markedly depressed the loss of microsomal α-tocopherol. The results ofin vivo labeling of liver microsomes with [¹⁴C] α-tocopherol demonstrated that i) GSH addition to thein vitro peroxidizing medium reduced the disappearance of α-tocopherol, and ii) a compound that interfered with the determination of α-tocopherol was separated by HPLC and was not an oxidation product of α-tocopherol. A portion of the microsomal¹⁴C-labeled α-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to α-tocopherol quinone.     

Microsomal lipid peroxidation: Effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase

The role of vitamin E in the protection against iron dependent lipid peroxidation was studied in rat liver microsomes and Triton-dispersed microsomal lipid micelles. In these systems, an antioxidant effect of vitamin E at a physiological ratio to phospholipids could be observed only in the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPX) and glutathione. The rationale of this cooperation is discussed on the basis of the hydroperoxyl radical scavenging capacity of vitamin E and the reduction of membrane hydroperoxides by PHGPX. The scavenging of lipid hydroperoxyl radicals by vitamin E, although inhibiting propagation of the peroxidative chain, produces lipid hydroperoxides from which ferrous iron generates alkoxyl radicals that react with vitamin E almost as fast as with fatty acids. Therefore, only if membrane hydroperoxides are continuously reduced by this specific peroxidase does the scavenging of hydroperoxyl radicals by vitamin E lead to an effective inhibition of lipid peroxidation.     

Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37°C unde 100% O₂. Lipid peroxidation was induced by adding 400 μM adenosine 5′-triphosphate, 2.5 to 20 μM FeCl₃, and 450 μM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992)Arch. Biochem. Biophys. 293, 103–109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes. However, GSH did not protect efficiently against the loss of residual vitamin E in deficient microsomes. These data provide support for the concept that GSH protects against microsomal lipid peroxidation by maintaining protein thiols, and consequently vitamin E, in the reduced state. The lack of protection in vitamin E deficient microsomes may be related to the inability of such low levels of vitamin E to inhibit peroxidation.     

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 μM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 μM) or the combination of ferric ion (10 μM) and ascorbic acid (500 μM), in the presence of reduced glutathione (GSH, 800 μM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 μM). ATG similarly reversed an inhibition of phosphatidyl-choline hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classifical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 μM ATG. ATG, at concentrations up to 250 μM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%). These data demonstrate the potential of ATG to increase toxicity of lipid peroxidative stimuli by inhibition of microsomal and cytosolic defense mechanisms. Although ATG did not inhibit Triton-enhanced PHGPX activity, overall evidence points toward inhibition of this enzyme as the mechanism for ATG-augmented lipid peroxidation and supports the conclusion that PHGPX plays a major role in the cellular defense mechanism.     

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO₂ for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increases were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO₂. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats was comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO₂ exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.     

Relative susceptibility of microsomes from lung,heart, liver,kidney, brain and testes to lipid peroxidation: Correlation with vitamin E content

Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely among different tissues and species. In rats and rabbits, lung microsomes peroxidized at a 25- to 50-fold lower rate than liver, kidney, testes and brain microsomes. Heart microsomes peroxidized at a rate slightly greater than, but most similar to, lung microsomes. Comparison of tissue homogenates also revealed the unique resistance of lung and heart to lipid peroxidation. The ratio of vitamin E to peroxidizable polyunsaturated fatty acids in lung and heart microsomes was several-fold higher than in microsomes from the other tissues studied, which accounted for the relative resistance of lung and heart to lipid peroxidation. Liposomes of extracted rat lung microsomal lipid were also resistant to peroxidation and the amount of vitamin E contained in the lung lipid extract was sufficient to confer the same degree of resistance when incorported into an equivalent amount of rat liver lipid. Higher rates of peroxidation in mouse lung microsomes relative to rabbit, rat and human lung microsomes were similarly correlated with a lower ratio of vitamin E to peroxidizable fatty acids in mouse lung microsomes. These data provide strong support for the role of vitamin E as the major cellular antioxidant, especially in the highly oxygenated tissues of heart and lung, and demonstrate the utility of the microsomal system in characterizing tissue differences in susceptibility to peroxidative membrane decomposition.     

Promotion of iron-induced rat liver microsomal lipid peroxidation by copper

Although copper has been demonstrated to promote lipid peroxidation in a number of systems, the mechanisms involved have not been fully defined. In this study, the role of copper in modifying lipid peroxidation has been explored in rat hepatic microsomes. In an in vitro system containing reduced glutathione (GSH, 200 μM) and Tris buffer, pH 7,4, cupric sulfate (1–50 μM) potentiated lipid peroxidation induced by ferrous sulfate (10 μM) but was unable to elicit peroxidation in the absence of iron. Higher levels of cupric sulfate (100 μM or greater) were inhibitory. The nature as well as the extent of the peroxidative response of microsomes to cupric sulfate were dependent on glutathione levels in addition to those of iron. Cupric sulfate (100 μM) strongly potentiated ferrous ion-induced lipid peroxidation in the presence of 400–800 μM GSH, while it inhibited peroxidation at lower levels of GSH (0–200 μM) and did not affect ferrous ion-induced peroxidation with glutathione levels of 3–10 mM. The potentiating effect of copper on ferrous ion-induced lipid peroxidation was further explored by investigating: (1) potential GSH-mediated reduction of cupric ions; (2) potential copper/GSH-mediated reduction of ferric ions (formed by oxidation during incubation); and (3) possible promotion of propagation reactions by copper/GSH. Our results indicate that cupric ions are reduced by GSH and thus are converted from an inhibitor to an enhancer of iron-induced lipid peroxidation. Cuprous ions appear to potentiate lipid peroxidation by reduction of ferric ions, rather than by promoting propagation reactions. Iron (in a specific Fe⁺²/Fe⁺³ ratio) is then an effective promoter of initiation reactions.     

Interplay between Vitamin E,Glutathione and Dihydrolipoic Acid in Protection against Lipid Peroxidation

Free radicals can disturb the intracellular homeostasis by either modification of essential free sulfhydryl groups or by inducing lipid peroxidation. The damage provoked by oxidation of sulfhydryl groups might be reversible but the damage induced by the process of lipid peroxidation is probably not reversible. The main protective constituents of the cell are thiols and vitamin E. Thiols, especially glutathione, protect the cytosol while vitamin E protects the lipid membranes against free radicals. In the scavenging of free radicals in the lipid membrane, vitamin E becomes oxidized. However continuous recycling of vitamin E by a reductase, with the cytosolic thiol glutathione as cofactor, will keep the vitamin E levels high enough to protect against lipid peroxidation. In the recycling glutathione is oxidized. Dihydrolipoic acid cannot provide directly reducing equivalents for the recycling of vitamin E by the free radical reductase. However indirectly, via the reduction of oxidized glutathione, dihydrolipoic acid can mediate the regeneration of vitamin E. One of the secondary mechanisms that mediates free radical induced damage is the rise in intracellular free Ca²⁺-concentration caused by inactivation of the endoplasmic reticulum ca²⁺-ATPase. The Ca²⁺-ATPase can be inactivated either by sulfhydryl alkylation or by lipid peroxidation. The authors used the thiol-alkylating agent N-ethylmaleimide, cystamine and ebselen. Dithiothreitol reversed the inhibition caused by all the three agents, while dihydrolipoic acid reversed the inhibition caused by ebselen. Glutathione was not able to reverse the effects of the sulfhydryl reactive agents. The reactivation of the microsomal Ca²⁺-ATPase by dihydrolipoic acid, may – besides the reduction of oxidized glutathione – contribute to the protective effect of dihydrolipoic acid on lipid peroxidation.     

Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation

The effect of dietary vitamin E and/or selenium (Se) supplementation (200 IU and/or 0.2 ppm, respectively) or deficiency for two months on lipid peroxidation in cerebrum, cerebellum, mid-brain, and brain stem of one-month-old male F344 rats was investigated. Dietary treatment had a minimal effect on weight gain of rats for the period tested. Plasma α-tocopherol (α-T) concentration and glutathione peroxidase (GSH-Px) activity were reflective of dietary treatments. Supplementation of diets with vitamin E and/or Se increased plasma α-T and/or GSH-Px activity, while diets devoid of these nutrients reduced them significantly. Increased GSH-Px activity in Sesupplemented rats was further enhanced by vitamin E supplementation. Differential concentrations of α-T among brain regions were affected by dietary vitamin E but not by Se. In vitro lipid peroxidation of brain homogenates was inhibited by dietary vitamin E supplementation and increased by deficiency. Addition of 0.25 mM ascorbic acid or 0.1 mM of Fe²⁺ to brain homogenates markedly increased in vitro lipid peroxidation. Ascorbic acid-induced lipid peroxidation was inversely correlated with dietary vitamin E and Se in cerebrum. In vitro Fe²⁺-addition induced the greatest stimulation of lipid peroxidation, with cerebellum and brain stem of vitamin E-deficient rats showing the highest response to Fe²⁺ challenge. These findings indicate that concentrations of α-T among the brain regions are different and can be altered by dietary vitamin E treatments, cerebellum and brain stem are more susceptible to in vitro challenge by peroxidative agents than other regions, and the degree of lipid peroxidation of brain regions is partially affected by dietary vitamin E but not by Se in the levels tested.     

Effect of dietary vitamin E levels on fatty acid profiles and nonenzymatic lipid peroxidation in the guinea pig liver

Guinea pigs were fed for five weeks with three diets containing different levels of vitamin E: LOW (but nondeficient, 15 mg of vitamin E/kg diet), MEDIUM (150 mg/kg diet), and HIGH (1,500 mg/kg diet). Dietary vitamin E supplementation did not change oxidative stress indicators in the hydrophilic compartment but increased liver α-tocopherol in a dose-dependent way and strongly decreased sensitivity to nonenzymaticin vitro liver lipid peroxidation. This last effect was already observed in group MEDIUM, and no further decrease inin vitro lipid peroxidation occurred from group MEDIUM to group HIGH. The protective effect of vitamin E againstin vitro lipid peroxidation was observed even though an optimum dietary concentration of vitamin C for this animal model was present in the three different vitamin E diets. Both HIGH and LOW vitamin E decreased percentage fatty acid unsaturation in all phospholipid fractions from membrane origin in relation to group MEDIUM. The results, together with previous information, show that both vitamin E and vitamin C at intermediate concentrations are needed for optimal protection against lipid peroxidation and loss of fatty acid unsaturation even in normal nonstressful conditions. These protective concentrations are higher than those needed to avoid deficiency syndromes.     

Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation

The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 μM iron as ferric sulfate and 50 μM ascorbate, ALDH, glucose-6-phosphate (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathioneS-transferase and nicotinamide adenine dinucleotide phosphate-cytochromec reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected, andN,N′-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 μM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid, peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.     

Enrichment of eggs with n-3 polyunsaturated fatty acids: effects of vitamin E supplementation

Eggs enriched with n−3 polyunsaturated fatty acids (PUFA) could contribute to dietary intake of these healthful fatty acids (FA). Because n−3 PUFA are highly susceptible to peroxidation, a first part of the study with Leghorn laying hens was carried out to investigate the influence of different levels of fish oil (0, 0.7, 1.4, 2.8, or 5.6%, respectively) in the diet on n−3 PUFA, cholesterol, vitamin E, and lipid peroxidation product contents in eggs. Addition of fish oil to a complete diet based on wheat, rye, tapioca, and soybean constituents containing 11 IU vitamin E/kg resulted in increased n−3 PUFA content in egg yolk, mainly due to accumulation of docosahexaenoic acid. Cholesterol was not altered up to 2.8% fish oil in the diet. The vitamin E content of the yolk was insufficient for the protection of PUFA from peroxidation. Addition of up to 2.8% fish oil to laying hen diets increased the n−3 PUFA content of yolks with a concomitant imbalance between vitamin E and PUFA, leading to increased levels of cytotoxic aldehydic lipid peroxidation products such as malondialdehyde (MDA). In a second part of the studies, the balance between vitamin E, PUFA, and lipid peroxidation was analyzed during the period of storage of n−3 PUFA-enriched eggs produced after feeding the laying hens with 1.5% fish oil diets with different concentrations of vitamin E (0, 5, 10, 20, 40, 80, 160 IU/kg). Storage of eggs resulted in a marked loss of vitamin E in yolk. In stored eggs, the cytotoxic lipid peroxidation products MDA, 4-hydroxynonenal, and 4-hydroxyhexenal were reduced in response to vitamin E supplementation. To prevent the increase of cytotoxic aldehydic lipid peroxidation during production and storage of n−3 PUFA-enriched eggs, a high vitamin E supplementation with at least 80 IU vitamin E/kg is needed.     

A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5′-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPHvia NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.     

Lipid peroxide catalyzed chemical carcinogenesis

Peroxides, including lipid peroxides, with heme catalysts cause the binding of C¹⁴-acetylaminofluorene to DNA if microsomes are present. This binding was 96% inhibited by paraoxon, a deacetylase inhibitor. It is concluded that peroxide-peroxidase systems rapidly oxidize acetylated arylamines to proximate carcinogens following deacetylation by microsomal deacetylases. The DNA binding observed was greater than that observed with the liver microsomal mixed function oxidase catalyzed activation to N-OH-acetylaminofluorene, which binds to DNA following deacetylation by microsomal deacetylase. Lipid peroxidation or prostaglandin synthesis should therefore enhance carcinogenesis induced by arylamides.     

The effect of a moderately thermoxidized dietary fat on the vitamin E status,the fatty acid composition of tissue lipids,and the susceptibility of low-density lipoproteins to lipid peroxidation in rats

Several studies demonstrated that dietary oxidized oils markedly affect the vitamin E status and alter the fatty acid composition of tissue lipids in animals. It must however be emphasized that highly oxidized oils reduce the feed intake of animals, which makes it difficult to interpret the results. Therefore, the present study used a moderately thermoxidized soybean oil (peroxide value: 75 mEq O₂/kg), having a similar fatty acid composition as fresh soybean oil (peroxide value: 9.5 mEq O₂/kg) which was used as control. Moreover, according to a bifactorial design, two different vitamin E supplementary levels (11 vs. 511 mg α-to-copherol equivalents per kg diet) were used. The experiment was conducted with male Sprague-Dawley rats. The feeding period lasted for 40 days. In order to assess the vitamin E status, the vitamin E concentrations in plasma, liver, heart, kidney, and adipose tissue were determined. The vitamin E supply had a pronounced effect on the vitamin E concentrations of those tissues whereas the type of fat had only a slight effect. The fatty acid composition of total lipids from liver, erythrocytes, and low-density lipoproteins was also only slightly influenced by the oxidized fat. The osmotic fragility of erythrocytes was even reduced by feeding the oxidized oil. With a low vitamin E supply, the in vitro susceptibility of low-density lipoproteins to lipid peroxidation was slightly increased by feeding the oxidized oil. In contrast, with a high vitamin E supply, there was no adverse effect of the dietary oxidized oil on the susceptibility of low-density lipoproteins to lipid peroxidation. Feeding the oxidized oil, however, increased the concentrations of malondialdehyde in low-density lipoproteins suggesting an increased in vivo lipid peroxidation. Therefore, it cannot be ruled out that moderately oxidized dietary fats increase the atherogenicity of low-density lipoproteins. In contrast, a moderately oxidized oil scarcely affected the vitamin E status and the fatty acid composition of tissue lipids.     

Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate

The purpose of this study was to test the hypothesis that multiple antioxygenic nutrients provide increased protection against lipid peroxidative damage to rat liver. Rats were fed diets (i) deficient in vitamin E and selenium (Diet 1), (ii) supplemented with vitamin E and selenium (Diet 2), (iii) supplemented with (ii) and in addition trolox C,N-acetylcysteine, coenzyme Q₀, and (+)-catechin (Diet 3), or (iv) supplemented with (iii) and in addition β-carotene, ascorbic acid palmitate, canthaxanthin, and coenzyme Q₁₀ (Diet 4). Liver homogenates were obtained from three rats fed each of the diets for six weeks and were incubated at 37°C up to two hours with and without exogenous tertiary-butyl hydroperoxide (TBHP) or Cu²⁺. Lipid peroxidation was determined by measurement of thiobarbituric acid substances. Diets 2 and 3 significantly protected againstin vivo hepatic lipid peroxidation, and this protection was augmented by Diet 4. Diets 2, 3, and 4 were protective against mild oxidation induced by TBHP or Cu²⁺. During incubations with exogenous TBHP and Cu²⁺, there were only small differences between diets supplemented with antioxidants in inhibition of lipid peroxidation, indicating that diets supplemented with vitamin E and selenium (Diet 2) may have provided the maximal protection for liver. The possible mechanisms of protection provided by multiple antioxidants in diets were discussed. Protection by multiple antioxidants against lipid peroxidation may translate to prevention of peroxidative damage to human tissue, a factor in human disease.     

Protective effect of estrogens and catecholestrogens against peroxidative membrane damagein vitro

The antioxidant effects of natural estrogens (estrone E₁; 17β-estradiol), synthetic estrogens (17α-ethynylestradiol, EE₂; mestranol, MES; diethylstilbestrol, DES) and catechle-strogens (2-hydroxyestradiol; 4-hydroxyestradiol 4-OHE₂) on lipid peroxidation induced by different means in rat liver microsomes were investigated. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances. Prooxidants included Fe³⁺/ADP/reduced NADPH, Fe²⁺/ascorbate,tert-butyl hydroperoxide (t-BOOH) and 2,2′-azobis (2-amidinopropane) (AAPH). Estrogens and catecholestrogens decreased lipid peroxidation in all four systems tested. In the iron/ascorbate model it was shown that (i)-OHE₂ and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsómes, and (ii) the antioxidant activities of E₁, EE₂ and MES were dependent on the assay conditions with the activity being markedley higher when estrogen metabolism was favored. When peroxidation was initiated by the peroxyl radical generator AAPH, the inhibitory effects observed were least pronounced. Our data also showed that, in each of the systems, all inhibitors displayed the same order of inhibitory potency with DES and catecholestrogens being the most potent antioxidants under all experimental conditions used. The present results confirm earlier findings and point toward a link between estrogen metabolism and estrogen antioxidant activity. The data also indicate that estrogens and catecholestrogens interact with the peroxidative process at different levels with their interactions with iron or the metal-derived species being the most important modes of inhibition.     

Lipid peroxidation in chronic ethanol treated rats: In vitro uncoupling of peroxidation from reduced nicotine adenosine dinucleotide phosphate oxidation

Chronic ethanol treated rats were found to have enhanced ethanol metabolism and to metabolize ethanol in vivo in the presence of an inhibitor of alcohol dehydrogenase. In vitro studies of the hepatic microsomal system thought to the responsible for this activity showed it to be markedly induced. Lipid peroxidation also was enhanced in the ethanol treated animals. The lipid peroxidation was shown to be uncoupled from the microsomal nicotinamide adenine dinucleotide phosphate, reduced form, oxidase activity by a low concentration of azide.     

Einfluß der Vitamin E-Supplementierung auf den antioxidativen Stoffwechsel des Ferkels bei unterschiedlicher Linolsäureversorgung

Influence of Vitamin E Supplementation on the Antioxidative Metabolism of Young Pigs at Different Intakes of Linoleic Acid To characterize the antioxidative metabolism of young pigs influenced by different vitamin E and linoleic acid concentrations in the diet, the in vitro pentane production of the liver microsomes, the α-tocopherol concentration in total liver and liver microsomes, and the fatty acid content of the liver microsomes were investigated. Increased vitamin E concentrations in the diet resulted in decreased pentane production, increased α-tocopherol concentration of total liver and microsomes and reduced absolute content of total fatty acids of the microsomes. However, the n-6-fatty acids decreased to a smaller degree than the saturated fatty acids. Increased supplementation with linoleic acid resulted in an elevated pentane production when the vitamin E concentration of the diet was low. The α-tocopherol concentration of total liver and microsomes were not influenced by increased supplementation of linoleic acid, however, the monoenoic fatty acid content of the microsomes was reduced.     

The effects of vitamin E and selenium intake on oxidative stress and plasma lipids in hamsters fed fish oil

The aim of the present work was to test the effects of large-dose supplementation of vitamin E (Vit E) and selenium (Se), either singly or in combination, on fish oil (FO)-induced tissue lipid peroxidation and hyperlipidemia. The supplementation of Se has been shown to lower blood cholesterol and increase tissue concentrations of the antioxidant glutathione (GSH); however, the effects of Se supplementation, either alone or in combination with supplemental Vit E, on FO-induced oxidative stress and hyperlipidemia have not been studied. Male Syrian hamsters received FO-based diets that contained 14.3 wt% fat and 0.46 wt% cholesterol supplemented with Vit E (129 IU d-α-tocopheryl acetate/kg diet) and/or Se (3.4 ppm as sodium selenate) or that contained basal requirements of both nutrients. The cardiac tissue of hamsters fed supplemental Se showed increased concentrations of lipid hydroperoxides (LPO) but decreased oxidized glutathione (GSSG) concentrations. The higher concentrations of LPO in the hearts of Se-supplemented hamsters were not lowered with concurrent Vit E supplementation. In the liver, Se supplementation was associated with higher Se-dependent glutathione peroxidase activity and an increase in the GSH/GSSG ratio, whereas a lower hepatic non-Se-dependent glutathione peroxidase activity was seen with Vit E supplementation. Supplemental intake of Se was associated with lower plasma concentrations of total cholesterol and low density lipoprotein cholesterol plus very low density lipoprotein cholesterol. In view of the pro-oxidative effects of Se supplementation on cardiac tissue, a cautionary approach needs to be taken regarding the plasma lipid-lowering properties of supplemental Se.