Lipid peroxidation: A mechanism involved in acute ethanol toxicity as demonstrated by in vivo pentane production in the rat

The effect of a single dose of ethanol on lipid peroxidation in three groups of rats fed different amounts of vitamin E was determined by the measurement of pentane in the breath. All rats had increased pentane production above basal levels by 15 min following oral administration of 6 g ethanol/kg body wt. The increase in total pentane production during a 13-hr test period after intragastric administration of ethanol was greater in the rats fed the vitamin E-deficient diet than in the rats, fed vitamin E-supplemented diets (α=2P=0.02). The results support the hypothesis that acute ethanol toxicity involves lipid peroxidation and further demonstrate the usefulness in toxicological studies of monitoring pentane as an index of lipid peroxidation in vivo.     

Volatile hydrocarbon and carbonyl products of lipid peroxidation: A comparison of pentane,ethane, hexanal,and acetone as in vivo indices

A study was undertaken to determine whether respiratory hexanal and acetone as well as pentane and ethane could be measured as potential indices of lipid peroxidation in vivo. The tests of induction of lipid peroxidation in rats included injection of iron-dextran and the vitamin E deficiency status. Injection of 460 mg of iron/100 g body wt over a 28-day period increased pentane and ethane production 4- and 6-fold, respectively. Hexanal production was increased 7-fold after injection of 60 mg of iron/100 g body wt, and then it fell back to the preinjection level in spite of continued injection of iron-dextran. Acetone production was lower in iron-injected rats than in controls, and it was ca. 10-fold higher in fasted vitamin E-deficient rats than in vitamin E-supplemented rats, being ca 48 and 5 nmol/100 g/min, respectively. It was observed that halomethane injection did not increase hexanal production, while acetone and pentane production were increased. Pentane and hexanal, but not acetone, were found to arise from decomposition of linoleic acid hydroperoxide in vitro. It was concluded that hydrocarbon gases are better indices of lipid peroxidation than hexanal, which is enzymatically metabolized, and acetone, the production of which is dominated by factors such as altered carbohydrate metabolism.     

The effect of vitamin C on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production

Measurements of pentane and ethane as indices of in vivo lipid peroxidation were made on samples of breath from vitamin C-sufficient and vitamin C-deficient guinea pigs injected with 23 μl carbon tetrachloride (CCl₄)/100 g body wt. Vitamin C-deficient animals produced significantly more pentane and ethane after CCl₄ treatment than did vitamin C-sufficient guinea pigs. Pretreatment of vitamin C-deficient animals with 75 mg ascorbic acid/100 g body wt significantly lowered both pentane and ethane evolution. Protection against in vivo lipid peroxidation similar to that provided by ascorbic acid was also found when vitamin C-deficient guinea pigs were pretreated with isoascorbic acid, reduced glutathione, α-tocopherol or β-carotene. When animals were pretreated with the radical scavenger mannitol, a protective effect was also observed as measured by pentane evolution.     

Effects of dietary antioxidants on in vivo lipid peroxidation in the rat as measured by pentane production

The hypothesis that pentane is an in vivo product of lipid peroxidation was confirmed by a study of the effects of a nonbiological antioxidant on pentane production in rats fed a diet deficient in vitamin E and supplemented with 0.01% N,N′-diphenyl-p-phenylenediamine (DPPD). Seven rats were fed a vitamin E-deficient diet starting at 3 wk of age. After 5 wk, 0.01% DPPD was added to the diets of three rats (group DPPD) while the diet of the other four rats remained unchanged (group OE). Within 2 wk of the diet change, rats in group DPPD exhaled 65% less pentane than rats of the same age in group OE. After 5 wk of being fed the DPPD-supplemented diet, rats in group DPPD were again fed the basal vitamin E-deficient diet; within 3 wk, these rats produced pentane levels similar to those of rats in group OE. The effects of vitamin E depletion and repletion on in vivo lipid peroxidation in rats were also studied. Three groups of three rats each were initially fed a vitamin E-deficient diet starting at 3 wk of age. After 8, 8, and 5 wk of being fed this diet, the three groups were fed diets supplemented with 3.3 (group 0→3.3E), 11 (group 0→11 E), and 200 (group 200E) i.u. vitamin E acetate/kg diet, respectively. Another group of three rats (group 11 E) was fed a diet supplemented with 11 i.u. vitamin E/kg starting at 3 wk of age for the duration of the study. There were significant decreases in pentane production by rat groups 0→3.3E, 0→11E, and 200E within 2 wk of the change to the vitamin E-supplemented diets. After about 5 wk of being fed their respective vitamin E-supplemented diets, pentane breath levels had stabilized. Breath pentane levels were inversely proportional to the log of dietary vitamin E concentration.     

Effect of dietary vitamin E on expiration of pentane and ethane by the rat

An analytical method for the measurement of hydrocarbon gases in the breath of rats is described. The method was used to follow the expiration in rat breath of in vivo formed scission products of hydroperoxides. The major products are pentane from the linoleic acid family and ethane from the linolenic acid family. Rats were fed 0, 11 or 40 i.u. vitamin E acetate/kg diet for 7 wk starting at age 21 days. Data obtained by gas chromatographic analysis of breath samples were analyzed by the Mann-Whitney nonparametricU-test. This statistical analysis showed that pentane evolved by the group of rats not supplemented with vitamin E was significantly higher during the period 1–7 wk than that evolved by either of the two supplemented groups of rats. Ethane from the nonsupplemented group was significantly higher than that from the group supplemented with 40 i.u. vitamin E/kg of diet by 5 wk, and significantly higher than both supplemented groups by 6 wk. By 7 wk, pentane production was tenfold greater in the nonsupplemented group than in either supplemented group, and ethane was about twofold greater. There was no significant difference between the groups supplemented with 11 and 40 i.u. vitamin E/kg diet for either ethane or pentane. This new technique, which measures scission products from in vivo lipid peroxidation, promises to be useful for application to many experimental areas where lipid peroxidation is expected or known to occur.     

The role of lipid peroxidation during chronic and acute exposure to ethanol as determined by pentane expiration in the rat

Weanling rats were fed one of 3 diets containing 0, 11 or 200 international units (IU) dl-α-tocopherol acetate/kg diet for 4 weeks. Following this period, the drinking water was replaced with an 18% solution of ethanol (v/v). An isocaloric D-glucose solution was substituted for the drinking water of a control group of rats fed the vitamin-E-deficient diet for 4 weeks. The 4 treatment groups were maintained on the diet and drinking regimen for 20 weeks. Basal levels of expired pentane were determined at weeks 0, 1, 3, 5, 7 and 9. Chronic ethanol consumption did not influence basal pentane production during the 9-week treatment. Basal levels of expired pentane were affected by dietary vitamin E. Rats supplemented with vitamin E had basal pentane levels less than one-half of the level of rats fed a vitamin-E-deficient diet (p<0.001). After 14 weeks of treatment, the 2 groups of rats fed a vitamin-E-deficient diet were administered p.o. an acute dose of 6 g of ethanol/kg body wt. Pentane expired above basal levels during the following 4-hr period correlated with the amount of hepatic triglycerides determined at the conclusion of the experiment. The etiology of ethanol toxicity is a complex and multifactorial system made up to many biological variables that influence lipid peroxidation. The appropriate choices of experimental designs and methods are important in examining the role of lipid peroxidation.     

Effect of antioxidants on lipid peroxidation in iron-loaded rats

Indirect evidence has suggested that lipid peroxidation is associated with iron overload in vivo. As a measure of lipid peroxidation, pentane expired in the breath of rats loaded with an accumulated dose of either 100 mg or 186–200 mg of iron injected intraperitoneally as iron dextran was measured over a 7 to 8 week period, and the effect on pentane production of feeding antioxidant-supplemented diets was determined. By the seventh week of feeding the diets, rats fed 0.3% L-ascorbic acid produced 17% less (P=0.03) pentane than did rats fed the basal antioxidant-deficient diet, whereas rats fed 0.004% dl-α-tocopherol acetate produced 92% less (P<0.001). After being fed the basal diet for 7 weeks, iron-loaded rats produced 76±9 pmol pentane/100 g body wt/min. When synthetic antioxidants were added to the diet at a concentration of 0.25%, the order of effectiveness in decreasing pentane production after 1 week was: N,N′-diphenyl-p-phenylenediamine > ethoxyquin > butylated hydroxyanisole > butylated hydroxytoluene > propyl gallate ∼ no antioxidant. After removal of either ethoxyquin or N,N′-diphenyl-p-phenylenediamine from the diets for 1 week, pentane production increased to a high level. The total amount of lipid soluble fluorophores in individual spleens of rats fed N,N′-diphenyl-p-phenylenediamine, ethoxyquin, dl-α-tocopherol acetate, ascorbic acid and no antioxidant were correlated significantly with the corresponding total integrated amount of pentane produced by the individual rats over the 7 to 8 week period. This study has provided some of the most direct evidence to date that lipid peroxidation is associated with iron overload in vivo.     

Effects of dietary vitamin E,selenium, and polyunsaturated fats on in vivo lipid peroxidation in the rat as measured by pentane production

Starting at 21 days of age, groups of six rats each were fed a basal Torula yeast diet supplemented with 0,4% L-methionine and varying amounts of vitamin E as dl-alpha tocopherol acetate, selenium as sodium selenite, and with either 10% stripped corn oil, stripped lard, or coconut oil. By 7 wk, pentane production by rats fed a corn oil diet deficient in both vitamin E and selenium was twice that by rats fed 0.1 or 1 mg of selenium per kg of the same basal diet. Blood glutathione peroxidase activity after 7 wk was proportional to the logarithm of dietary selenium. Groups of rats fed the vitamin E- and selenium-deficient diets with lard or coconut oil had one-half the pentane production of rats fed the vitamin E- and selenium-deficient corn oil diets. The plasma level of linoleic plus arachidonic acid was 1.8 times greater on a wt % basis in rats fed corn oil than in rats fed lard or coconut oil as the fat source. Pentane production by rats fed 40 i.u. dl-alpha tocopherol acetate per kg of the selenium-deficient corn oil diet was one-sixth of that by rats fed the same diet without vitamin E; the plasma of the rats fed the vitamin E-supplemented corn oil diet had a level of vitamin E that was about six times greater than that of the rats fed the vitamin E-deficient corn oil diet.     

Low doses of eicosapentaenoic acid, docosahexaenoic acid, and hypolipidemic eicosapentaenoic acid derivatives have no effect on lipid peroxidation in plasma

It was of interest to investigate the influence of both high doses of eicosapentaenoic acid (EPA) and low doses of 2-or 3-methylated EPA on the antioxidant status, as they all cause hypolipidemia, but the dose required is quite different. We fed low doses (250 mg/d/kg body wt) of different EPA derivatives or high doses (1500 mg/d/kg body wt) of EPA and DHA to rats for 5 and 7 d, respectively. The most potent hypolipidemic EPA derivative, 2,2-dimethyl-EPA, did not change the malondialdehyde content in liver or plasma. Plasma vitamin E decreased only after supplementation of those EPA derivatives that caused the greatest increase in the fatty acyl-CoA oxidase activity. Fatty acyl-CoA oxidase activity increased after administration of both EPA and DHA at high doses. High doses of EPA and DHA decreased plasma vitamin E content, whereas only DHA elevated lipid peroxidation. In liver, however, both EPA and DHA increased lipid peroxidation, but the hepatic level of vitamin E was unchanged. The glutathione-requiring enzymes and the glutathione level were unaffected, and no significant changes in the activities of xanthine oxidase and superoxide dismutase were observed in either low-or high-dose experiments. In conclusion, increased peroxisomal β-oxidation in combination with high amounts of polyunsaturated fatty acids caused elevated lipid peroxidation. At low doses of polyunsaturated fatty acids, lipid peroxidation was unchanged, in spite of increased peroxisomal β-oxidation, indicating that polyunsaturation is the most important factor for lipid peroxidation.     

Effects of dietary oils and methyl ethyl ketone peroxide onin vivo lipid peroxidation and antioxidants in rat heart and liver

Weanling male Sprague-Dawley rats were fed diets for four weeks which differed in their content of n−6 (corn oil; CO) and n−3 fatty acids (fish oil; FO), but were similar in their content of saturated and monounsaturated fatty acids and vitamin E. At the end of the four-week feeding period, each dietary group was subdivided into two groups. One group received a single placebo injection of α-tocopherol-stripped corn oil (TSCO); the other group received a single injection of the free radical generator, methyl ethyl ketone peroxide (MEKP), in TSCO. Twenty-four hours after injection, the effect of dietary oil and MEKP treatment on endogenous lipid peroxide (LPO) production (measured as methylene blue formed by the “Determiner LPO” assay), glutathione (GSH) and vitamin E content, and fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in heart and liver from unfasted animals were measured. FO-fed rats had significantly heavier hearts and livers, increased levels of n−3 fatty acids in membrane phospholipids, and higher liver LPO levels than CO-fed rats. MEKP treatment resulted in significantly lower body weights and liver GSH levels. The data indicate that dietary n−3 fatty acids increase lipid peroxidation in liver somewhat more than in heart. The study also demonstrates that the effect of induced oxidative stress due to a single dose of MEKP on lipid peroxide formation and antioxidant status in tissues from unfasted animals was independent of the dietary oils.     

Effect of dietary menhaden oil and vitamin E onin vivo lipid peroxidation induced by iron

Weanling rats were fed diets containing 10% menhaden oil (MO) or 10% corn oil-lard (1∶1, COL) with low (≤5 IU/kg) or supplementary (35 IU/kg) vitamin E for six weeks. The rats were killed 30 min after injection with 24 mg iron/kg as ferrous chloride because thiobarbituric acid-reactive substances (TBARS) in liver homogenates were highest at 30 min after injection of iron into rats fed a standard diet. Tissue homogenates were used either without incubation (zero-time) or after incubation at 37°C for 1 hr. In addition to TBARS and conjugated dienes, headspace hexanal and total volatiles (TOV) determined by capillary gas chromatography were useful indices of lipid peroxidation since they were decreased by vitamin E supplementation and were increased with increasing iron dose. Regardless of the dietary lipid used, vitamin E supplementation decreased headspace hexanal, TOV, TBARS and conjugated dienes in both zero-time and incubated homogenates of liver and kidney. Dietary MO increased TBARS in both zero-time and incubated homogenates of tissue from rats injected with iron. In contrast, dietary MO decreased hexanal and TOV in incubated tissue homogenates. The study demonstrated the usefulness and limitations of using hexanal and TOV as indices of lipid peroxidation.     

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.     

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.     

Lipid peroxidation and antioxidant status is affected by different vitamin E levels when feeding fish oil

The protective role of vitamin E and changes in the status of several physiological antioxidants after feeding rats a fish oil diet were investigated. Six-week-old male Sprague-Dawley rats were divided into four groups and fed experimental diets for 8 wk. Three fish oil (FO) groups were fed a menhaden fish oil and soybean oil (SO) (9∶1) mixture as 10% (w/w) of the diet. These groups were provided with ≤3, 45 or 209 IU of vitamin E/kg diet. One SO group was used as control and was fed ≤45 IU of vitamin E/kg diet. Plasma vitamin E levels, when expressed as vitamin E per mL plasma, were extremely low in the group fed FO and ≤3 IU of vitamin E, and were lower in the groups fed FO than in the group fed SO. However, plasma vitamin E levels when expressed per mg plasma lipid were higher in the FO groups provided with ≤45 and 209 IU of vitamin E than in the SO group. Compared with the SO group, plasma levels of thiobarbituric acid reactive substances (TBARS), when expressed per mg lipid, were higher in the three FO groups, plasma retinol levels were lower in the FO groups provided with ≤3 and 45 IU of vitamin E, and ascorbic acid levels were lower only in the FO group provided with ≤3 IU of vitamin E. Blood glutathione (GSH) levels were lower in all three FO groups than in the SO group. Liver vitamin E levels increased as the dietary level of vitamin E increased, but all FO groups had higher liver levels of TBARS than the SO group. The dietary vitamin E levels were correlated positively with plasma vitamin E (r=0.71) and negatively with TBARS in both the plasma and liver of rats fed FO. Among the antioxidants measured, correlations were found between plasma retinol and vitamin C (r=0.64), and plasma vitamin C, uric acid (r=0.72) and blood GSH (r=0.60). Weaker correlations were found between plasma retinol, uric acid and blood GSH. It is concluded that vitamin E requirements are higher when feeding fish oil. Vitamin E seems necessary to prevent enhanced lipid peroxidation and to maintain appropriate levels of other physiological antioxidants.     

Determination of ethane and pentane in free oxygen radical-induced lipid peroxidation

It has been proposed that ethane and pentane reflect free oxygen radical-induced lipid peroxidation. However, methodological difficulties limit the use of these gases for assessment of free oxygen radical activity. In the present report we describe an improved method for the accurate analysis of picomole quantities (≥1 pmol) of ethane and pentane. They are first quantitatively trapped into an adsorbent and then heat-desorbed directly into a capillary column for gas chromatographic quantitation. During oxidation of linolenic (n−3) and linoleic (n−6) acid, ethane and pentane were formed, respectively. Nonstimulated granulocytes formed pentane. Upon addition of phorbol 13-myristate 12-acetate, the generation of pentane was increased by 540%. Addition of superoxide dismutase plus catalase inhibited lipid peroxidation in both a cell-free system and in isolated cells. The present method is useful in the evaluation of free oxygen radical induced damage.     

The effect of dietary vitamin E supply and a moderately oxidized oil on activities of hepatic lipogenic enzymes in rats

Diets rich in polyunsaturated fatty acids (PUFA) are well known to suppress hepatic lipogenic enzymes compared to fat-free diets or diets rich in saturated fatty acids. However, the mechanism underlying suppression of lipogenic enzymes is not quite clear. The present study was undertaken to investigate whether lipid peroxidation products are involved in suppression of lipogenic enzymes. Therefore, an experiment with growing male rats assigned to six groups over a period of 40 d was carried out. Rats received semisynthetic diets containing 9.5% coconut oil and 0.5% fresh soybean oil (coconut oil diet, peroxide value 5.1 meq O₂/kg oil), 10% fresh soybean oil (fresh soybean oil diet, peroxide value 0.5 meq O₂/kg oil), or 10% thermally treated soybean oil (oxidized soybean oil diet, peroxide value 74 meq O₂/kg oil). To modify the antioxidant state of the rats, we varied the vitamin E supply (11 and 511 mg α-tocopherol equivalents per kg of diet) according to a bi-factorial design. Food intake and body weight gain were not influenced by dietary fat and vitamin E supply. Activities of hepatic lipogenic enzymes were markedly influenced by the dietary fat. Feeding either fresh or oxidized soybean oil diets markedly reduced activities of fatty acid synthase, (FAS), acetyl CoA-carboxylase, (AcCX), glucose-6-phosphate dehydrogenase, (G6PDH), 6-phosphogluconate dehydrogenase, and ATP citrate lyase (ACL) relative to feeding the coconut oil diet. Moreover, feeding oxidized soybean oil slightly, but significantly, lowered activities of FAS, AcCX, and ACL compared to feeding fresh soybean oil. Activities of hepatic lipogenic enzymes were reflected by concentrations of triglycerides in liver and plasma. Rats fed the coconut oil diet had markedly higher triglyceride concentrations in liver and plasma than rats consuming fresh or oxidized soybean oil diets, and rats fed oxidized soybean oil had lower concentrations than rats fed fresh soybean oil. The vitamin E supply of the rats markedly influenced concentrations of thiobarbituric acid-reactive substances in liver, but it did not influence activities of hepatic lipogenic enzymes. Because the vitamin E supply had no effect, and ingestion of an oxidized oil had only a minor effect, on activities of hepatic lipogenic enzymes, it is strongly suggested that neither exogenous nor endogenous lipid peroxidation products play a significant role in the suppression of hepatic lipogenic enzymes by diets rich in PUFA. Therefore, we assumed that dietary PUFA themselves are involved in regulatio of hepatic lipogenic enzymes. Nevertheless, the study shows that ingestion of oxidized oils, regardless of the vitamin E supply, also affects hepatic lipogenesis, and hence influences triglyceride levels in liver and plasma.     

Rat vitamin E status and heart lipid peroxidation: Effect of dietary α-Linolenic acid and marine n−3 fatty acids

Three groups of sixteen male rats each were fed semipurified diets containing 15% by weight of lipid for a period of 4 wk. The diets contained the same amount of polyunsaturated fatty acids (PUFA) (20% of total fatty acids) and saturated fatty acids (19% of total fatty acids). Dietary PUFA were represented exclusively by linoleic acid (18∶2 diet), or 10% linoleic acid and 10% linolenic acid (18∶3 diet), or 10% linoleic acid and 10% long-chain n−3 fatty acids (LCn−3 diet). The overall amount of vitamin E was similar in the three diets,i.e, 140, 133 and 129 mg/kg diet, respectively. Following appropriate extraction, tocopherol levels in heart, liver, brain, adipose tissue (AT) and plasma were measured by high-performance liquid chromatography. The level of vitamin E in the heart decreased with n−3 PUFA diets, most markedly with LCn−3 PUFA. Liver and AT vitamin E contents also decreased with n−3 PUFA diets when expressed as μg/mg total lipids and μg/mg phospholipids, respectively. Total plasma vitamin E was lower in rats fed the LCn−3 diet, but there was no significant difference when expressed as μg/mg total lipids. Brain vitamin E was not affected by the various diets.In vitro cardiac lipid peroxidation was quantified by the thiobarbituric acid reactive substances (TBARS) test. Heart homogenates were incubated at 37°C for 15 and 30 min in both the absence (uninduced) or presence (induced) of a free radical generating system (1 mM xanthine, 0.1 IU per mL xanthine oxidase, 0.2 mM/0.4 mM Fe/ethylenediaminetetraacetic acid). TBARS release was time-independent but significantly higher when LCn−3 fatty acids were fed to rats in either the uninduced or induced system. The study demonstrated that n−3 PUFA diets can influence vitamin E status of rats even in short-term experiments and can change the susceptibility of the heart toin vitro lipid peroxidation.     

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.     

Effect of n−3 fatty acid supplementation on lipid peroxidation and protein aggregation in rat erythrocyte membranes

Human erythrocytes in the circulation undergo dynamic oxidative damage involving membrane lipid peroxidation and protein aggregation during aging. The present study was undertaken to determine the effect of n−3 fatty acid supplementation on lipid peroxidation and protein aggregation in the circulation and also the in vitro susceptibility of rat erythrocyte membranes to oxidative damage. Wistar male rats were fed a diet containing n−6 fatty acid-rich safflower oil or n−3 fatty acid-rich fish oil with an equal amount of vitamin E for 6 wk. n−3 Fatty acid content in erythrocyte membranes of rats fed fish oil was significantly higher than that of rats fed safflower oil. The degree of membrane lipid peroxidation and protein aggregation of rats fed fish oil was not significantly higher than that of rats fed safflower oil when the amounts of phospholipid hydroper-oxides, thiobarbituric acid-reactive substances, and detergent-insoluble protein aggregates were measured. When isolated erythrocytes were oxidized under aerobic conditions in the presence of Fe(III), the degree of membrane lipid peroxidation of erythrocytes from rats fed fish oil was increased to a greater extent than that of rats fed safflower oil, whereas the degree of membrane protein aggregation of both groups was increased in a similar extent. Hence, n−3 fatty acid supplementation did not affect lipid peroxidation and protein aggregation in membranes of circulating rat erythrocytes, and the supplementation increased the susceptibility of isolated erythrocytes to lipid peroxidation, but not to protein aggregation, under the aerobic conditions. If a sufficient amount of vitamin E is supplied, n−3 fatty acid supplementation may give no undesirable oxidative effects on rat erythrocytes in the circulation.     

Effect of n−3 polyunsaturated fatty acid supplementation on lipid peroxidation of rat organs

The present study was undertaken in order to reexamine the effect of n−3 polyunsaturated fatty acid (PUFA)-rich diet supplementation on lipid peroxidation and vitamin E status of rat organs. Male Wistar rats were fed a diet containing safflower or fish oil at 50 g/kg diet and an equal amount of vitamin E at 59 mg/kg diet (1.18 g/kg oil; and 1.5 g/kg PUFA in safflower oil diet, and 4.3 g/kg PUFA in fish oil diet) for 6 wk. Fatty acid composition of total lipids of brain, liver, heart, and lung of rats fed fish oil was rich in n−3 PUFA, whereas that of each organ of rats fed safflower oil was rich in n−6 PUFA. The vitamin E levels in liver, stomach, and testis of the fish oil diet group were slightly lower than those of the safflower oil diet group, but the levels in brain, heart, lung, kidney, and spleen were not different between the two diet groups. The levels of phospholipid hydroperoxides were determined by the high-performance liquid chromatography-chemiluminescence method and the levels of thiobarbituric acid-reactive substances (TBARS) were determined at pH 3.5 in the presence of butylated hydroxytoluene with or without EDTA. Levels of phospholipid hydroperoxides and TBARS in the brain, liver, heart, lung, kidney, spleen, stomach and testis of the fish oil diet group were similar to those of the safflower oil diet group. The results indicate that high fish oil intake does not induce increased levels of phospholipid hydroperoxides and TBARS in rat organs.