Microsomal fatty acid desaturase activities in vitamin A-deficient rat liver

The activity of microsomal fatty acid delta 9-desaturase was significantly higher in liver microsomes of vitamin A-deficient rats as compared with their controls. Feeding of vitamin A-supplemented control diet to the deficient rats restored the delta 9-desaturase activity to the control values. The activity of delta 6-desaturase was not affected by vitamin A deficiency.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed beta-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of delta 9- and delta 6-desaturases in liver microsomes of rats fed diets supplemented with beta-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg beta-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for delta 9-desaturase and delta 6-desaturase activities. The activity of delta 9-desaturase was lower in liver microsomes of rats fed beta-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal delta 6-desaturase activity was, however, higher in liver of rats fed 13-cis retinoic acid; there was no effect of beta-carotene on delta 6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding beta-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Effects of gamma-linolenic acid supplementation on lipogenesis regulation in pregnant zinc-deficient rat and fetus

In the liver of zinc-deficient pregnant rats fatty acid synthetase and delta 9-desaturase activities decreased and diet supplementation with gamma-linolenic acid potentiated this effect. However, in liver microsomes from the foetuses of zinc-deficient mothers, HMG CoA reductase and delta 9-desaturase activities declined but fatty acid activity rose. The same applied to foetuses from mothers whose diet was supplemented with gamma-linolenic acid and here again, the effect of zinc deficiency was potentiated. The fact that delta 9 activity dropped whereas fatty acid synthetase activity rose implied a defect in the mechanism regulating the functioning of these enzymes. In the non zinc-deficient group of pregnant females, gamma-linolenic acid supplementation had no effect on fatty acid synthetase and delta 9-desaturase activities but significantly increased HMG CoA reductase activity. In foetuses from the same group, the activities of MHG CoA reductase, delta 9-desaturase and fatty acid synthetase all increased.     

Changes in lipid composition and desaturase activities of duodenal mucosa induced by dietary fat

In the present work we have studied the effects of feeding either olive or sunflower oil on lipid composition and desaturase activities of duodenal mucosa microsomes. Duodenal microsomes prepared from dogs fed the sunflower oil diet showed higher percentages of saturated, of linoleic and of n - 3 polyunsaturated fatty acids as well as lower levels of oleic, dihomo-gamma-linolenic and arachidonic acids in phosphatidylcholine and phosphatidylethanolamine than those prepared from animals fed the olive oil diet. In sphingomyelin, the dietary supplementation did not produce significant differences between the two groups. The cholesterol/phospholipid molar ratio was higher in the sunflower oil group than in the olive oil group. The in vitro delta 9-desaturase activity was higher in microsomes from the olive oil dogs. The delta 6-desaturase activity was similar in microsomes from the two groups and lower than that found for delta 9-desaturase activity. Desaturase activities were higher in duodenal microsomes than those previously found for liver microsomes.     

A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver

Expression of antioxidant enzymes (AOE), an important mechanism in the protection against oxidative stress, could be modified by the redox status of the cells. The aim of this project was to evaluate the role of vitamin E deficiency in association with a high-cholesterol diet in the hepatic lipid peroxidation and the expression of AOE. Two groups of 6 male rats were fed with a high-cholesterol or a high-cholesterol vitamin E-deficient diet. All animals were sacrificed at 72 days of treatment. Liver lipid peroxidation index (Malondialdehyde; MDA) and hepatic AOE were evaluated. Total liver RNA was extracted, and the steady state messenger RNA (mRNA) levels of glutathion peroxydase, manganese superoxide dismutase, Cu/Zn superoxide dismutase and catalase were examined by northern blot. After 72 days on the diet, a significant increase in the lipid peroxidation index was observed in the vitamin E deficient group (MDA : 4.45 +/- 0.29 nmol/mg protein versus 3.65 +/- 0.1 nmol/mg protein in vitamin E normal group). Despite this oxidative stress, the activities and mRNA levels of liver AOE were not significantly different in the 2 groups. These preliminary results show that chronic vitamin E deficiency associated with high cholesterol diet is able to increase lipid peroxidation without modulation of AOE expression and activity in the liver. This suggests that beneficial effects of dietary vitamin E are due to a plasma antioxidant effect or a cell mediated action, rather than to a specific modulation of cellular enzymes.     

Effect of long-term feeding olive and sunflower oils on fatty acid composition and desaturation activities of liver microsomes

Changes in microsomal fatty acid composition, delta 9- and delta 6-desaturase activities and cholesterol and phosphorus liver content were studied in dogs fed olive and sunflower oil diets. No changes were observed in the saturated fatty acids between dietary groups. The level of monounsaturated fatty acids was more elevated in animals fed the OO diet, because of its high relative content in this diet although the in vitro delta 9-desaturase activity was similar in microsomes from the two groups. The proportion of arachidonic acid was similar in SO and OO fed animals. This similar level occurred despite a significant increase in the level of linoleic acid in membrane lipids as a result of feeding the SO supplement. The in vitro delta 6-desaturase activity in liver microsomes showed no differences between dogs fed the two diets. Thus, the higher desaturation presented in vivo by microsomes from OO group may be related to the inhibition by linoleic acid of delta 6-desaturase in dogs fed the SO diet. The polyunsaturated fatty acids (PUFA) from the n-3 series were higher in microsomal phosphatidylcholine and phosphatidylethanolamine from animals fed the OO supplemented diet. The cholesterol/phosphorus molar ratio was higher in the SO group in which the unsaturation index was only slightly affected in phospholipids.     

Gamma-linolenic acid dietary supplementation can reverse the aging influence on rat liver microsome delta 6-desaturase activity.

We have recently demonstrated that in rats the process of delta 6-desaturation of linoleic and alpha-linolenic acids slows with aging. One method of counteracting the effect of slowed desaturation of linoleic acid would be to provide the 6-desaturated metabolite, gamma-linolenic acid (18:3(n-6) GLA) directly. We have here investigated the 6-desaturation of both linoleic and alpha-linolenic acids in liver microsomes of young and old rats given GLA in the form of evening primrose oil (EPO) (B diet) in comparison to animals given soy bean oil alone (A diet), monitoring also the fatty acid composition of liver microsomes and relating this to the microviscosity of the membranes. In young rats the different experimental diets did not produce any difference in delta 6-desaturase (D6D) activity on either substrate suggesting that, when D6D activity is at or near its peak, the variations in diet tested are unable to influence it. In the old animals the rate of 6-desaturation of linoleic and particularly of alpha-linolenic acid was significantly greater in the B diet fed animals than in the A diet fed. The effects of the diets on the fatty acid composition of liver microsomes were consistent with the findings with regard to 6-desaturation. Administration of GLA partially corrected the abnormalities of n-6 essential fatty acid (EFA) metabolism by raising the concentration of 20:4(n-6) and other 6-desaturated EFAs. Furthermore, the GLA rich diet also increased the levels of dihomo-gamma-linolenic acid and of 6-desaturated n-3 EFAs in the liver microsomes. The microviscosity of microsomal membranes as indicated by DPH polarization was correlated with the unsaturation index of the same membranes. There was a very strong correlation between the two. In both young and old rats the B diet reduced the microviscosity and increased the unsaturation index. However, the effect was much greater in the old animals.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

The content of lipid peroxidation products and the activity of antioxidant enzymes in the myocardium and liver of rats with various vitamin E allowances

After 2 month of feeding vitamin E-supplemented diet (100.6 and 0 mg/kg; group I-control, II and III, respectively) the concentration of lipid peroxidation products (diene conjugates, malondialdehyde, Schiff's bases) and activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase) was estimated in rat heart and liver. Although the content of alpha-tocopherol in organs of group II was significantly decreased, the concentration of peroxidation products and enzyme activities was unchanged. Moreover, these parameters were constant in rat liver of group III. The heart was more sensitive because in group III to vitamin E deficiency (the alpha-tocopherol level was dropped fourfold) the concentration of diene conjugates and malondialdehyde was increased and superoxide dismutase activity was decreased. Thus insufficiency of vitamin E may result in selective alterations of myocardial functions. In addition, vitamin E may be useful instrument for correction of free radical oxidation and antioxidant system activity in the heart.     

Effect of a high cholesterol diet on lipid metabolizing enzymes in spontaneously hypertensive rats

A high cholesterol diet induced a fatty liver and an increase in cholesterol oleate in spontaneously hypertensive rats. The activity of microsomal glycerophosphate acyltransferase in liver increased 2-3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by a 10-fold increase in microsomal delta 9-desaturase activity. Hepatic fatty acid synthetase and diacylglycerol acyltransferase activities were decreased somewhat. These results, together with the fact that the large increases in hepatic cholesterol ester and triacylglycerol were not correspondingly reflected in plasma, indicated that the fatty liver resulted from decreased secretion of lipoprotein rather than increased lipogenesis. Endogenous cholesterol in liver microsomes increased 2-fold and hepatic acyl-CoA:cholesterol acyltransferase activity increased 3-fold, whereas plasma lecithin:cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate seen in spontaneously hypertensive rats fed a high cholesterol diet is due mainly to increases in acyl-CoA:cholesterol acyltransferase and delta 9-desaturase activities.     

Influence of selenomethionine and vitamin E on the antioxidative system in animals with experimentally induced hypercholesterolemia

The aim of this study was to investigate the effect of combined therapy of vitamin E and selenomethionine to pro/ antioxidant status in experimental hypercholesterolemia. Thirty male rabbits were included in the study and randomized into five groups consisting of the control group (standard diet) and four experimental groups staying on a diet rich in cholesterol (0.5 g/100 g diet): cholesterol group and groups supplemented cholesterol with selenomethionine (12.5 μg/kg body mass/24 h) or vitamin E (10 mg of Dl-α-tocopherol/kg body mass/24 h) or combination of the above antioxidants for 3 mo. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) as well as the concentration of malondialdehyde (MDA) was estimated in the blood during every month of experiment. Increased activity of SOD and GPX with a decreased concentration of MDA in comparison to the cholesterol diet was found mainly in the combination of study antioxidants. The supplementation of the cholesterol diet with combined doses of vitamin E and selenomethionine appears to prevent the lesions induced by experimental hypercholesterolemia much more efficiently than single doses of the above.     

Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum.

The ability of the superoxide radical (SOR) generated by xanthine oxidase to activate phospholipase A2 (PLA2) was examined in microsomes prepared from luteinized rat ovaries. Treatment of microsomes with xanthine oxidase resulted in a rapid burst in SOR formation followed by an increase in PLA2 activity. Stimulation of PLA2 activity was dose related and similar in microsomes prepared from control or prostaglandin F2 alpha (PGF2 alpha)-treated rats. Activation was inhibited by the antioxidants, vitamin E and nordihydroguaiaretic acid, and by superoxide dismutase and catalase, which metabolize SOR and H2O2 to remove reactive oxygen species from the cell. The stimulation of PLA2 activity by xanthine oxidase was dependent upon the addition of calcium ions, and it was highest in samples in which cytosol was added to membranes. These results indicate that the SOR and/or H2O2 may mediate PLA2 activation, which may be involved in the luteolytic process.     

Interaction of vitamin E and exercise training on oxidative stress and antioxidant enzyme activities in rat skeletal muscles

It has been shown that free radicals are increased during intensive exercise. We hypothesized that vitamin E (vit E) deficiency, which will increase oxidative stress, would augment the training-induced adaptation of antioxidant enzymes. This study investigated the interaction effect of vit E and exercise training on oxidative stress markers and activities of antioxidant enzymes in red quadriceps and white gastrocnemius of rats in a 2x2 design. Thirty-two male rats were divided into trained vit E-adequate, trained vit E-deficient, untrained vit E-adequate, and untrained vit E-deficient groups. The two trained groups swam 6 h/day, 6 days/week for 8 weeks. The two vit E-deficient groups consumed vit E-free diet for 8 weeks. Vitamin E-training interaction effect was significant on thiobarbituric acid reactive substances (TBARSs), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in both muscles. The trained vit E-deficient group showed the highest TBARS and GPX activity and the lowest SOD activity in both muscles. A significant vit E effect on glutathione reductase and catalase was present in both muscles. Glutathione reductase and catalase activities were significantly lower in the two vit E-adequate groups combined than in the two vit E-deficient groups combined in both muscles. This study shows that vit E status and exercise training have interactive effect on oxidative stress and GPX and SOD activities in rat skeletal muscles. Vitamin E deprivation augmented the exercise-induced elevation in GPX activity while inhibiting exercise-induced SOD activity, possibly through elevated oxidative stress.     

Effect of vitamin E- and selenium-deficiency on rat liver chemiluminescence.

The role of vitamin E and selenium as protective agents against oxidative stress was evaluated by measuring liver chemiluminescence in situ. Weanling rats fed a vitamin E- and selenium-deficient diet showed liver chemiluminescence that was increased 60 and 100% over control values at 16 and 18 days respectively after weaning. At day 21, the double deficiency led to hepatic necrosis, as observed by optical and electron microscopy, and increased serum levels of lactate dehydrogenase and alanine aminotransferase. Single deficiencies, in either vitamin E or selenium, did not produce liver necrosis but increased liver chemiluminescence. Vitamin E deficiency led to a 23 and 50% increase in liver emission at days 18 and 20 respectively; selenium deficiency produced a 64% increase at day 16. The activity of liver selenium-glutathione peroxidase diminished to 13% of the control value in the rats fed doubly deficient and selenium-deficient diets. Activities of superoxide dismutase, catalase and non-selenium-glutathione peroxidase were not modified by the different diets. These results suggest that oxy-radical generation may play a major role in hepatic necrosis in vitamin E- and selenium-deficiency.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Lipoperoxides, vitamin E, and activities of superoxide dismutase, glutathione peroxidase, and catalase in regenerating rat liver

Lipoperoxides in homogenates of regenerating rat liver increased from 6 hours after the operation and reached a peak (about 7 times the control level) 18-24 hours after the operation. The concentration of blood lipoperoxides rapidly decreased after the operation. The enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase, and vitamin E content in regenerating livers were also determined. Among these antioxidant factors, the catalase level changed markedly.     

Essential fatty acid interconversion during gestation in the rat

The synthesis of arachidonic acid has been investigated in fetal and pregnant rat liver microsomes in the course of the gestation. The delta 5-desaturase activity decreased 2-3 times in rat liver between the 19th and 22nd day of the pregnancy. During this period the delta 5-desaturate activity increased 3-fold in the fetal liver, exceeding the activity of the maternal liver. In contrast, the activity of the fetal delta 6-desaturase was in the same range as in pregnant rat liver and the liver of control animals and did not change between these two stages of the gestation. The elongation rate of linoleic acid in fetal liver was 2-3 times lower than in maternal liver but this increased during the pregnancy. The fatty acid activate rate was always higher than the activity of the desaturases. At the 19th day, the activity of the delta 5-desaturase was apparently the rate limiting step of arachidonic acid synthesis in fetal liver. We did not find any delta 5- and delta 6-desaturase activities or linoleic acid elongation in the placenta microsomes.     

Activities of liver microsomal fatty acid desaturases in zinc-deficient rats force-fed diets with a coconut oil/safflower oil mixture of linseed oil

The present study was conducted to investigate the effect of zinc deficiency on fatty acid desaturation in rats fed two different types of dietary fat, a mixture of coconut oil and safflower oil (7∶1, w/w, “coconut oil diet”) or linseed oil (“linseed oil diet”). In order to ensure an adequate food intake, all rats were force-fed by gastric tube. Zinc deficiency caused statistical significant reducion of Δ9-desaturase activity in liver microsomes of rats fed coconut oil diet and tendencial reduction (p<0.15) in rats fed linseed oil diet compared with control rats fed diets with the same type of fat. In agreement with this effect, zinc deficiency in the rats fed both types of dietary fat increased the ratio between total saturated and total monounsaturated fatty in liver phospholipids and liver microsomes. Zinc deficient rats on the coconut oil diet had unchanged Δ6-desaturase activity with linoleic acid as substrate and lowered activity with α-linolenic acid as substrate. In contrast, zinc deficient rats on the linseed oil diet had increased Δ6-desaturase activity with linoleic acid as substrate and unchanged activity with α-linolenic acid. Because linoleic acid is the main substrate for Δ6-desaturase in the rats fed coconut oil diet, and α-linolenic acid is the main substrate in the rats fed linseed oil diet, it is concluded that in vivo Δ6-desaturation was not changed by zinc deficiency in the rats fed both types of dietary fat. Activity of Δ5-desaturase was also not changed by zinc deficiency in the rats fed both dietary fats. Levels of fatty acids in liver phospholipids and microsomes derived by Δ4-, Δ5-, and Δ6-desaturation were not consistently changed by zinc deficiency in the rats fed both types of dietary fat. Thus, the enzyme studies and also fatty acid composition data of liver phospholipids and microsomes indicate that zinc deficiency does not considerably disturb desaturation of linoleic and α-linolenic acid. Therefore, it is suggested that similarities between deficiencies of zinc and essential fatty acids described in literature are not due to disturbed desaturation of linoleic acid in zinc deficiency. The present study also indicates that zinc deficiency enhances incorporation of eicosapentaenoic acid into phosphatidylcholine of rats fed diets with large amounts ofn-3 polyunsaturated fatty acids.     

Effects of dietary vitamin E and selenium on antioxidative defense mechanisms in the liver of rats treated with high doses of glucocorticoid

The aim of this work was to determine the effects of dietary intake vitamin E and selenium (Se) on lipid peroxidation as thiobarbituric acid reactive substances (TBARS) and on the antioxidative defense mechanisms in the liver of rats treated with high doses of prednisolone. Two hundred fifty adult male Wistar rats were randomly divided into five groups. The rats were fed a normal diet, but groups 3, 4, and 5 received a daily supplement in their drinking water of 20 mg vitamin E, 0.3 mg Se, and a combination of vitamin E and Se, respectively, for 30 d. For 3 d subsequently, the control group (group 1) was treated with a placebo, and the remaining four groups were injected intramuscularly with 100 mg/kg body weight (BW) prednisolone. After the last administration of prednisolone, 10 rats from each group were killed at 4, 8, 12, 24, and 48 h and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) enzymes and the levels of glutathione (GSH) and TBARS in their livers were measured. GSH-Px, SOD, and CAT enzyme activities and GSH levels in prednisolone-treatment group (group 2) began to decrease gradually at 4 h, falling respectively to 38%, 55%, and 40% of the control levels by 24 h, and recovering to the control levels at 48 h. In contrast, prednisolone administration caused an increase in the hepatic TBARS, reaching up to four times the levels of the control at 24 h. However, supplementation with vitamin E and Se had a preventive effect on the elevation of the hepatic TBARS and improved the diminished activities of the antioxidative enzymes and the levels of GSH. Therefore, the present study demonstrates the effectiveness of vitamin E and Se in reducing hepatic damage in glucocorticoid-treated rats and suggests that reductions in increased TBARS as a result of prednisolone may be an important factor in the action of vitamin E and Se.     

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia).

Hepatic microsomes prepared from vitamin E deficient and supplemented rats were analyzed for cytochrome P-450 content and drug metabolizing activity. Reduced levels of benzo[α]pyrene hydroxylase and ethylmorphine N-demethylase activities were observed in microsomes derived from rats fed a diet deficient in vitamin E compared to those of control rats. NADPH-mediated destruction of P-450, and pentobarbital and zoxazolamine sleeping times were similar in the two groups. Induction with 3-methylcholanthrene raised the levels of benzo[α]pyrene hydroxylase activity of both supplemented and deficient rats to the same absolute levels. No differences were noted in cytochrome P-450 or P-448 content between control and tocopherol deficient rats, nor did the activity of liver catalase differ between the two dietary groups. Thus, these studies did not demonstrate any impairment of heme protein synthesis in vitamin E deficient rats.