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.     

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.     

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.     

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.     

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.     

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.     

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 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 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.     

Edible oil quality as measured by thermal release of pentane

As part of their thermal decomposition products, fatty hydroperoxides produce normal hydrocarbons. The extent of hydrocarbon formation can be measured and associated with the quality and potential stability of an oil. Edible oils containing linoleic acid develop 13-hydroperoxy-9,11-octadecadienoic acid as one product of autoxidation. On thermal decomposition this hydroperoxide yields pentane; the amount released has been correlated with the flavor scores of fresh and aged soybean and cottonseed oils and with the peroxide values of these oils. The quantity of pentane released has an inverse linear relationship to flavor score and a direct linear relationship to peroxide values. Edible oils exposed to light exhibit a different relationship between flavor score and thermally derived pentane than do the same oils when autoxidized in the dark. Presented at AOCS Meeting, New Orleans, May 1967.     

The effect of dihydroergotoxine on lipid peroxidation in vitro

Dihydroergotoxine mesylate (DHET), an ergot alkaloid derivative, is widely used to treat senile cerebral vascular insufficiency. Aspects of this age-related phenomenon may be due to deterioration by lipid oxidation of cellular membranes. DHET stabilizes EEG alpha frequencies, increases cerebral blood flow and oxygen uptake and accumulates in lipid-rich structures of the brain. The effect of DHET was studied on iron-catalyzed peroxidation of liposomes as measured by the thiobarbituric acid assay. It was found that DHET inhibits peroxidation in vitro in a dose-dependent manner. These results suggest that DHET acts in part as a lipid antioxidant when used to treat senile cerebral vascular insufficiency.     

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe⁺³ containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be rquired for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one moth. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85–90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.     

Quantification of lowered cholesterol oxidation in guinea pigs with latent vitamin C deficiency

Fifty-two male guinea pigs fed on a scorbutigenic diet were divided into a control group (10 mg ascorbicacid per animal per day) and a group with latent vitamin C deficiency (2 weeks on the scorbutigenic diet only, followed by a maintaining dose of 0.5 mg ascorbic acid per animal per day). After 13 weeks, 26-¹⁴C-cholesterol was administered intraperitoneally to all the animals, in which the¹⁴C excretion in the expired CO₂ and the urine and cholesterol specific activity in the blood serum and liver were then studied at intervals of 24 hr and 1, 3, 5, 7, 9 and 11 weeks. The ascorbic acid concentration in the liver and spleen of the control animals was five times higher than in vitain C-deficient animals. The total cholesterol concentration in serum and liver was significantly higher in the vitamin C-deficient guinea pigs. A two-pool analysis of the disappearance curves of serum cholesterol specific activity showed that the size of the cholesterol pool A (blood and tissues with rapid cholesterol exchange) was greater in the vitamin C-deficient animals. The rate of the transformation of cholesterol to bile acids was estimated as the ratio of¹⁴CO₂ expired to liver cholesterol specific activity. Latent vitamin C deficiency caused significant slowing down of this process (controls: 11.8±0.6; vitamin C deficiency: 8.3±0.4 mg/24 r/500 g w/w). A significant correlation between the liver ascorbic acid concentration and the rate of cholesterol transformation to bile acids was found. The results demonstrate that ascorbic acid is necessary for a normal course of cholesterol catabolism. In latent vitamin C deficiency, the rate of cholesterol catabolism slows down and cholesterol consequently accumulates in the blood and liver of vitamin C-deficient guinea pigs.     

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.     

The effect of modest vitamin E supplementation on lipid peroxidation products and other cardiovascular risk factors in diabetic patients

Among many factors, elevated lipids and lipid peroxide levels in blood are major risk factors in the development of cardiovascular disease in diabetic patients. This study has examined whether oral supplementation of vitamin E, an antioxidant, has any effect on blood lipid peroxidation products (LP) and lipid profile of diabetic patients. Thirty-five diabetics(D) were supplemented withdl-α-tocopherol (E) capsule (orally, 100 IU/d) or placebo (P) for three months in double-blind clinical trials. Plasma E was analyzed by HPLC and LP by the thiobarbituric acid-reactivity; serum lipids by auto-analyzer. Data were analyzed using pairedt-test and Wilcoxon Signed Rank Test. Vitamin E supplementation significantly lowered LP and lipid levels in diabetic patients; there were no differences in these parameters after P supplementation. There were no differences in the duration of diabetes and ages of D between P-and E-supplemented groups. This study suggests that vitamin E supplementation significantly lowers blood LP and lipid levels in diabetic patients.     

Studies on biochemical effects of nitrogen dioxide: I. Lipid peroxidation as measured by ethane exhalation of rats exposed to nitrogen dioxide

This research was in order to follow the periodic fluctuation of lipid peroxidation by a new method in rats exposed to nitrogen dioxide. Wistar male rats were examined for lipid peroxidation as demonstrated by ethane exhalation. In rats continuously exposed to 10 ppm nitrogen dioxide for 2 weeks, the amount of ethane exhaled fluctuated in a complex manner during the exposure. Ethane exhalation decreased slightly after the first day of exposure and then increased rapidly. The maximal values were observed after the fourth day of exposure and then decreased gradually to the initial level. Furthermore, the activity of glutathione peroxidase in lungs of rats exposed to 10 ppm nitrogen dioxide varied symmetrically against the change of ethane formation. Similar changes in ethane exhalation were observed in rats exposed to the lower levels of nitrogen dioxide (0.4, 1.2 and 4.0 ppm) for 4 months. Compared to 10 ppm nitrogen dioxide exposure for 14 days, the characteristics in rats exposed to the low levels (0.4–4.0 ppm) of nitrogen dioxide were: the decline of ethane formation, the delay in alterations, and the tendency toward gradual increased during the longer period exposure.     

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.     

The effect of beta blocking drugs on lipid peroxidation in rat heartin vitro

Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetolol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetolol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 μM FeSO₄, 5 μMt-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO₄ (liposomes) plus O₂. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetolol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation event at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.     

Influence of dietary soybean and egg lecithins on lipid responses in cholesterol-fed guinea pigs

The comparative influence on plasma and tissue lipids of dietary soybean and egg lecithins, which have contrasting fatty acid compositions, was studied in the hypercholesterolemic guine apig. The polyunsaturated to saturated fatty acid (P/S) ratios of the soybean and egg lecithins were 3.4 and 0.38, respectively. Hypercholesterolemia was induced by feeding guinea pigs a purified diet that contained 15% lard enriched with 0.5% cholesterol. Subsequently, guinea pigs were fed for six wk the same diet supplemented with either soybean or egg lecithin as 7.5% of the diet. A control group continued to be fed the lecithin-free diet. Parameters measured included body weight and relative liver weight; in plasma, total cholesterol, high density lipoprotein cholesterol (HDLC), phospholipid, and nonesterified cholesterol; in liver, total fat, cholesterol, and the specific activity of the catabolic enzyme cholesterol 7α-hydroxylase; (EC 1.14.13.17); and in the aorta, cholesterol. Among the most noteworthy observations were the 49% decrease in total plasma cholesterol of the soybean lecithin group without decreasing HDLC and the 177% increase in HDLC of the egg lecithin group without a significant increase in total cholesterol compared with those values in the control group. These data suggest that dietary lecithin is particularly effective in increasing the HDLC/total cholesterol ratio in plasma. However, the absolute concentrations of those plasma lipids seem to depend upon the fatty acid composition of the lecithin. Presented in part at the annual meeting of the American Oil Chemists' Society, New Orleans LA, May 1987.