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.     

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.     

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 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.     

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.     

Urinary response toin vivo lipid peroxidation induced by vitamin E deficiency

Experiments were carried out to measure the urinary excretion of free and conjugated malonaldehyde (MDA) and other thiobarbituric acid reactive substances (TBARS) in vitamin E deficient and vitamin E supplemented rats. From both dietary groups, six TBA positive fractions were isolated, in addition to that containing free MDA, by high-performance liquid chromatography (HPLC) on a TSK-GEL G-1000PW column. Three of the fractions isolated were found to be significantly increased in vitamin E deficiency. After acid hydrolysis, only one of the above compounds produced free MDA which indicated the presence of derivatized MDA. Only this fraction exhibited fluorescence at excitation 370 nm and emission 450 nm. The five other fractions formed 2,4-dinitrophenylhydrazones (2,4-DNPH), indicating the presence of carbonyl groups, but the derivatized MDA fraction did not. No significant differences were found in free MDA levels between the vitamin E deficient and the vitamin E supplemented groups.     

The influence of vitamin E and selenium on lipid peroxidation and aldehyde dehydrogenase activity in rat liver and tissue

Malondialdehyde (MDA) production and cytosolic aldehyde dehydrogenase (ALDH) response were examined in rat liver tissues after feeding different levels of dietary vitamin E and/or selenium and polyunsaturated fat for 12–38 wk. MDA production was significantly increased by vitamin E deficiency or by high levels of polyunsaturated fat intake, but not by selenium deficiency. The activity of cytosolic ALDH increased upon increased production of MDA after 12–16 wk of feeding the lipid peroxidation-inducing diets. However, ALDH activity was suppressed after 38 wk of feeding the vitamin E-deficient diet. The results indicate that the hepatic cytosolic ALDH may be involved in the metabolism of MDA during a relatively short-term increase inin vivo lipid peroxidation, but that ALDH activity becomes suppressed after more severein vivo lipid peroxidation has been produced. Hepatic and plasma α-tocopherol levels and lipid peroxidation products were measured for the various dietary groups.     

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.     

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.     

Influence of dietary vitamin E,selenium and age on regional distribution of α-tocopherol in the rat brain

Concentrations of α-tocopherol (α-T) in plasma, cerebrum, cerebellum, midbrain and brain stem and activity of selenium (Se)-dependent glutathione peroxidase (GSH-Px) in plasma were measured in 1- and 15-month-old male F344 rats fed diets containing vitamin E (E, IU/kg) and Se (ppm) in the following combinations: 30 E, 0.1 Se (control diet, minimum requirements); 200E, 0.2 Se; 0.0 E, 0.2 Se; 200 E, 0.0 Se; 0.0 E, 0.0 Se for 8 or 20 weeks. α-T and GSH-Px levels in plasma were reflective of dietary treatment in young rats in which an interaction of the two nutrients was noted. A longer period of dietary vitamin E deficiency was necessary to deplete plasma α-T and depress GSP-Px activity significantly in the old rats. Among the brain regions of all ages, cerebrum and midbrain had the highest concentrations of α-T while cerebellum showed the lowest. However, cerebellum of young rats and cerebellum and brain stem of old rats had a greater α-T accumulation with doubly supplemented diets, whereas only cerebellum of young and old rats showed a marked increased of α-T with vitamin E supplementation. In old rats, vitamin E deficiency resulted in greater depletion of α-T in cerebellum and brain stem than cerebrum and midbrain regions. Se deficiency in brain stem of young and old rats significantly decreased α-T accumulation by vitamin E supplementation. Se supplementation marginally alleviates vitamin E depletion in brain. Cerebellum and brain stem of old rats fed the minimum requirements of vitamin E and Se for 20 weeks showed a significant decline in α-T. Therefore, cerebellum and brain stem appear, to have a higher turnover of α-T than cerebrum and midbrain, and older rats may require a higher level, of vitamin E in the diet to maintain steady state levels of α-T in these regions.     

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.     

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.     

Quantitative effects of dietary polyunsaturated fats on the composition of fatty acids in rat tissues

A method combining data on fatty acid composition into subsets is used to illustrate general relative competitive selectivities in the metabolic and transport events that maintain fatty acid compositions in tissue lipids and to minimize differences among tissues or species in the amount of individual fatty acids. Fatty acid compositions of triglycerides and phospholipids in several tissues of the rat were maintained with simple relationships between the exogenous n−3 and n−6 dietary polyunsaturated fatty acids and the endogenous n−7 and n−9 types of fatty acid. The general pattern of fatty acids in triglycerides was similar for liver, plasma and adipose tissue, averaging about 30% as saturated acids, 67% as 16- and 18-carbon unsaturated acids and only about 2% as 20- and 22-carbon highly unsaturated acids. The tissues maintained a linear relationship between the amount of 18-carbon polyunsaturated fatty acids in the diet and in the tissue triglycerides, with the proportionality constant for 18∶3n−3 being 60% of that for 18∶2n−6. The total phospholipids of liver, plasma and red blood cells maintained about 45% of the fatty acids in the form of saturated fatty acids and 20–30% as 20- and 22-carbon highly unsaturated fatty acids irrespective of very different proportions of n−3, n−6 and n−9 types of fatty acids. In all three tissues, the 20-carbon highly unsaturated fatty acids of the n−3, n−6 and n−9 type were maintained in a competitive hyperbolic relationship with apparent EC₅₀ values for dietary 18∶2n−6 and 18∶3n−3 near 0.1% of dietary calories. The consistent quantitative relationships described in this study illustrate an underlying principle of competition among fatty acids for a limited number of esterification sites. This approach may be useful in predicting the influence of diet upon tissue levels of the substrates and antagonists of eicosanoid biosynthesis.     

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.     

Lipid peroxidation in rat tissue slices: Effect of dietary vitamin E,corn oil-lard and menhaden oil

Rats were fed for 5 weeks either 10% (w/w) menhaden oil (MO) or a 10% corn oil-lard (COL) mixture (1∶1) in diets with ≤5 IU or ≤2 IU/kg vitamin E, respectively, or the same diets supplemented with d-α-tocopheryl succinate to a total of 35 and 180 IU vitamin E/kg, respectively. Slices of liver and heart from these rats were used to study lipid peroxidationin vitro. Thiobarbituric acid-reactive substances (TBARS) were measured in the medium after incubation of the slices at 37°C for 1 hr in the absence (uninduced) and presence of 0.5 mM tert-butyl hydroperoxide (induced). The release of TBARS from slices of heart and liver from rats fed either lipid decreased with increasing levels of dietary vitamin E. At the same level of dietary vitamin E, TBARS release was greater for slices of liver and heart from the MO-fed rats than from the COL-fed rats. Application of the TBARS data to a model simulating the experimental conditions showed a good correlation (r=0.95, p<0.001) between experimental and simulated values. Of the 16∶0–22∶6 fatty acids measured in liver from MO-fed rats, 15.4% was n−6 fatty acids and 29.9% was n−3 fatty acids; in liver from COL-fed rats, the respective values were 37.4% and 3.7%. Liver and kidney vitamin E levels were unaffected by the dietary lipid. This study demonstrated that a dietary fish oil increased the susceptibility of rat liver and heart toin vitro lipid peroxidation, and that vitamin E decreased TBARS in tissues from rats fed COL to lower levels than for tissues from rats fed MO. The results suggest that there might also be an increased requirement for dietary antioxidants by humans using fish oil supplements.     

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.     

Effects of dietary linseed oil and marine oil on lipid peroxidation in monkey liverin vivo andin vitro

Diets rich in linoleic acid (CO) from corn oil, or in linoleic acid and either α-linolenic acid (LO) based on linseed oil or n−3 fatty acids (MO) from menhaden oil were fed to male and female Cynomolgus monkeys for 15 wk. In the liver a 40% reduction of α-tocopherol occurred in the MO group relative to the CO and LO groups followed by increased formation of lipofuscinin vivo. A four-fold increase of α-tocopherol in the MO diet (MO+E) brought the level in the liver to that found with CO and LO. The increased peroxidation in the MO group in the liver phospholipids was associated with the replacement of 60% of the n−6 fatty acids by n−3 fatty acids from menhaden oil. Similar fatty acid profiles were found in groups fed MO and MO+E, respectively. Compared to the CO fed group, feeding α-linolenic acid only resulted in a slight incorporation of n−3 fatty acids in the liver membranes mainly due to a direct incorporation of α-linolenic acid. However, in monkeys fed menhaden oil more than 30% of the total fatty acids in the liver phospholipids were n−3 fatty acids. The various diets did not influence the activity of liver catalase (EC 1.11.1.6) nor superoxide dismutase (EC 1.15.1.1), but glutathione-peroxidase activity (EC 1.11.1.9) was higher in monkeys fed the MO diet. The catalase activity in females was 20% higher than in males. In anin vitro assay, liver microsomes from monkeys fed the MO diet or the MO diet supplemented with tocopherol produced similar amounts of thiobarbituric acid reactive substances and at a much higher rate than microsomes from the CO and LO groups. It appeared that α-tocopherol did not protect long-chain n−3 C₂₀ and C₂₂ fatty acids as well as n−6 fattya acids against peroxidation. The present data showed that monkeys were not fully able to compensate for increased peroxidative stress but a four-fold supplement of vitamin E to the diets reduced the oxidation.     

Unsaturated lipid peroxidation catalyzed by hematin compounds and its inhibition by vitamin E1

Evidence favoring hematin catalysis over autoxidation as the dominant mechanism of lipid peroxidation in animal tissues is presented. Lipid peroxidation in Erlich ascites tumor cells and isolated electron transport particles has been studied. Random destruction of the cytochromes and a loss of catalytic activity correlate with peroxidation of the electron transport particle. Mixtures of α-, β-, and γ-tocopherols show no synergistic effect. Synergism with ascorbate and citrate greatly enhance the antioxidant activity of α-tocopherol. A tocopherol-ascorbate-glutathione-triphosphopyridine nucleotide couple could act synergistically to inhibit lipid peroxidation in animal tissues. Supported in part by the Bureau of Commercial Fisheries.     

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.     

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.