Anti-lipoperoxidation action of food restriction

Chronic food restriction inhibited the age-related increase of malondialdehyde production and lipid hydroperoxides in liver mitochondrial and microsomal membranes of ad libitum fed Fischer 344 rats. The anti-lipoperoxidation action of food restriction could not be attributable to the changes in membrane lipid content nor vitamin E status. Restricting calories modified membrane fatty acid composition by increasing linoleic acid and decreasing docosapentaenoic acid content in both membranes. The significance of the fatty acid modification was discussed in terms of anti-lipoperoxidation and membrane fluidity.     

Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats

The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low.     

Effect of membrane fatty acid substitution and temperature on repair of sublethal damage in mammalian cells

Repair of sublethal radiation damage (SLD) has been investigated as a function of temperature in mouse fibroblast LM cells with different membrane lipid composition. Rigidification or fluidization of the cellular membranes was accomplished by incorporation of myristic acid and arachidonic acid, respectively, in the phospholipids of the membranes. The SLD repair after radiation was essentially the same for the cells with the more rigid (saturated fatty acid) membranes and the cells with the more fluid (polyunsaturated fatty acid) membranes. This observation was made for repair at 37 degrees C as well as for repair at hypothermic temperatures. Incorporation of polyunsaturated fatty acid protected the cells against hypothermic death. These experiments demonstrate that although membranes are likely targets for cell killing by low temperature treatments, membrane lipids are probably not involved in the repair of sublethal radiation damage. It must be concluded that neither the degree of polyunsaturation of the lipids nor the degree of fluidity of the membrane is important for radiation-induced killing of mammalian cells.     

Effect of a hyperlipidic diet on lipid composition, fluidity, and (Na+-K+)ATPase activity of rat erythrocyte membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.     

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.     

Membrane fluidity determines sensitivity of filamentous fungi to chitosan

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan‐resistant but not chitosan‐sensitive fungi. The plasma membranes of chitosan‐sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan‐resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady‐state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.     

Red Blood Cell Membrane Fluidity in the Etiology of Multiple Sclerosis

Organisms adjust the order, or fluidity, of their cellular membranes in response to changes in their physiochemical environment by adjusting the lipid composition of their membranes. We investigated membrane fluidity using the phospholipid, fatty acid and cholesterol content of red blood cells (RBCs) from multiple sclerosis (MS) patients and correlated this with C-reactive protein (CRP) as well as with the severity of neurological outcome as measured by the Kurtzke Expanded Disability Status Scale (EDSS) and its Functional System Scores. The study group consisted of 31 patients with MS and 30 healthy control subjects. Phospholipids were determined using a colorimetric assay, fatty acids by gas chromatography, cholesterol by an enzymatic assay and CRP by a Beckman nephelometer. Cell membrane fluidity was calculated according to previously established formulae. RBC membrane fluidity as measured by the saturated to polyunsaturated fatty acid ratio was higher in patients than in controls (P = 0.04). The phosphatidylethanolamine saturated to polyunsaturated fatty acid ratio showed highly significant positive correlations with the EDSS and CRP < 5 μg/ml. CRP showed significant inverse correlations with the saturated nature but positive correlations with the ordered-crystalline-phase to liquid-crystalline-phase lipid ratio. In this study we show that membrane fluidity as measured by the relationship between membrane fatty acids, phospholipids and cholesterol is closely interrelated with inflammation and disease outcome in patients with MS. In conclusion, our findings suggest that the membrane lipid composition of patients with MS and, consequently, membrane fluidity are altered, which seems to be influenced by the inflammatory status.     

Does membrane fatty acid composition modulate mitochondrial functions and their thermal sensitivities?

We investigated the effect of modifying fatty acid modification of heart mitochondrial membranes by dietary intervention on the functions and thermal sensitivity of electron transport system complexes embedded in the inner mitochondrial membrane. Four groups of rats were fed diets differing in their fat (coconut, olive or fish oil) and antioxidant (fish oil with or without probucol) contents. After 16 weeks of feeding, the coconut and olive oil groups had lower long-chain n-3 polyunsaturated fatty acids contents and a lower unsaturation index compared to both fish oil groups. These differences in fatty acid composition were not related to any differences in the mitochondrial respiration rate induced at Complexes I, II or IV, or to differences in their thermal sensitivity. The coconut oil group showed a lower mitochondrial affinity for pyruvate at 5 degrees C (k(mapp)=6.4+/-1.8) compared to any other groups (k(mapp)=3.8+/-0.5; 4.7+/-0.8; 3.6+/-1.1, for olive, fish oil and fish oil and probucol groups, respectively). At least in rat heart, our results do not support a major impact of the fatty acid composition of the mitochondrial membrane on the function of mitochondrial enzymatic complexes or on their temperature sensitivity.     

Treatment of the enhanced intestinal uptake of glucose in diabetic rats with a polyunsaturated fatty acid diet

Intestinal absorption of most nutrients is enhanced in diabetic rats. We wished to test the hypothesis that manipulation of dietary fatty acids will modify enhanced uptake of glucose in rats with established streptozotocin-diabetes. Chow-fed control rats or animals with one week of streptozotocin-diabetes were continued on chow or were fed ad libitum for three weeks with semisynthetic isocaloric diets containing a high content of either essential polyunsaturated or non-essential saturated fatty acids. The jejunal and ileal in vitro uptake of varying concentrations of glucose was much higher in diabetic than control rats fed chow or the saturated fatty acid diet. In contrast, the enhanced uptake of this sugar was reduced or normalized in diabetic rats fed the polyunsaturated fatty acid diet. Feeding the polyunsaturated fatty acid diet was associated with increased brush-border membrane activity of alkaline phosphatase in diabetic jejunum and ileum, but neither the saturated fatty acid diet nor the polyunsaturated fatty acid diet altered brush-border membrane cholesterol or phospholipids in control or in diabetic rats. Mucosal surface area was similar in diabetic rats fed the saturated fatty acid diet or the polyunsaturated fatty acid diet. Thus, (1) feeding the polyunsaturated fatty acid diet diminishes the enhanced jejunal and ileal uptake of glucose in diabetic rats, and (2) the influence of the polyunsaturated fatty acid diet on uptake in diabetic rats was not explained by alterations in intestinal morphology or brush-border membrane content of cholesterol or phospholipids. This study suggests that manipulation of dietary lipids may play a role in the normalization of the enhanced intestinal glucose uptake in rats with established diabetes.     

Effects of acclimation temperature on enzymatic capacities and mitochondrial membranes from the body wall of the earthworm Lumbricus terrestris

Many ectotherms respond to low temperature by adjusting capacities of enzymes from energy metabolism, restructuring membrane phospholipids and modulating membrane fluidity. Although much is known about the temperature biology of earthworms, it is not known to what extent earthworms employ compensatory changes in enzymatic capacities and membrane physical properties after exposure to low temperature. We examined activities of enzymes from glycolysis and central oxidative pathways as well as fluidity and phospholipid fatty acid composition of mitochondrial membranes prepared from the body wall of the temperate oligochaete Lumbricus terrestris after a one month acclimation to 5 degrees and 15 degrees C. No compensation occurs in central pathways of oxidative metabolism since activities of cytochrome-c oxidase and citrate synthase, when measured at a common temperature, are similar for 5 degrees C and 15 degrees C-acclimated animals. In contrast, activity of pyruvate kinase is elevated 1.3-fold after acclimation to 5 degrees C. Mitochondrial membranes display inverse compensation with respect to temperature (membranes from 5 degrees C animals are more ordered than membranes from 15 degrees C animals). Our results, in combination with earlier reports, indicate that routine metabolism in L. terrestris may be maintained at reduced temperatures with little or no change in enzymatic capacities and inverse compensation of mitochondrial membranes.     

Fish Oil and Oxidative Stress by Inflammatory Leukocytes

We investigated the effects of diets with different fatty acid composition upon the oxidative stress of inflammatory leukocytes of rats. After weaning, two groups of rats were fed isoenergetic semipurified diets for five weeks containing 5% of corn oil or menhaden oil. Polymorphonuclear leukocytes from rats fed menhaden oil diet incorporated n-3 polyunsaturated fatty acids into phospholipid membranes at the expense of arachidonic acid. These cells showed diminished superoxide production and, as a consequence, the total antioxidant status in the inflammatory exudate was increased. However, nitric oxide production was not affected by diet. Free malondialdeyde concentration increased in the exudate because of lower mitochondrial activity. These results add new aspects that help clarifying the anti-inflammatory mechanisms of n-3 polyunsaturated fatty acids.     

Effects of fatty acids on the growth of Caco-2 cells

Epidemiological studies suggest that polyunsaturated fatty acids may protect against colorectal neoplasia. In order to explore this observation, cell proliferation and viability, lipid composition, membrane fluidity, and lipid peroxidation were measured in Caco-2 cells after 48h incubation with various fatty acids. Saturated and monounsaturated fatty acids incorporated less well in the membranes than polyunsaturated fatty acids (PUFAs). All of the PUFAs tested had an inhibitory effect on cell proliferation/viability whereas the saturated and monounsaturated fatty acids did not. Addition of palmitic acid had no significant effect on membrane fluidity whereas unsaturated fatty acids increased membrane fluidity in a dose-dependent manner. PUFAs strongly increased tumor cell lipid peroxidation in a dose-dependent manner. Saturated and monounsaturated fatty acids increased lipid peroxidation in this cell line only at high concentration. Preincubation of Caco-2 cells with vitamin E prevented the inhibition of proliferation/viability, the elevation of the MDA concentration and the increased membrane fluidity induced by PUFAs. Our data indicate that PUFAs are potent inhibitors of the growth of colon cancer cells in vitro.     

Effect of a zinc-deficient diet on mitochondrial and microsomal lipid composition in TEPC-183 plasmacytoma

A zinc-deficient diet caused an increase in microsomal membrane phospholipid levels compared to ad libitum controls. Cholesterol levels were found to be decreased 50% compared to either pair-fed or ad libitum controls, resulting in a sharp decline in the cholesterol/phospholipid ratio. No differences were observed in the distribution of phospholipid classes among all three groups, either in mitochondrial or microsomal membrane fractions. Fatty acid analysis of PC and PE revealed a rise in the 18:2 fraction from zinc-deficient mitochondrial and microsomal membrane fractions. Mitochondrial PE and PC from zinc-deficient animals revealed a rise in the 22:6 fatty acid fraction while microsomal PC also revealed a corresponding decrease in 20:4. None of the zinc-deficient preparations differed significantly from either ad libitum or pair-fed controls in the content of long-chain alk-l-enyl ethers. The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in tumor subcellular membranes which may account for the decreased rate of tumor growth observed in zinc-deficient animals.     

Sensitivity of 5'-nucleotidase and phospholipase A2 towards liver plasma membranes modifications

The changes in the phospholipid and fatty acid composition of liver plasma membranes isolated from rats, fed two different diets, containing either saturated or unsaturated fatty acids, were investigated. We established that dietary treatment can considerably modify the fatty acid as well as the phospholipid composition of liver plasma membranes. Lipid transfer proteins were used for enrichment of liver plasma membranes with sphingomyelin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and phosphatidylinositol. A marked sphingomyelin and membrane fluidity dependence of the membrane-bound 5'-nucleotidase and phospholipase A2 was observed.     

Effect of n-6 and n-3 polyunsaturated fatty acids ingestion on rat liver membrane-associated enzymes and fluidity

The influences of diets having different fatty acid compositions on the fatty-acid content, desaturase activities, and membrane fluidity of rat liver microsomes have been analyzed. Weanling male rats (35–45 g) were fed a fat-free semisynthetic diet supplemented with 10% (by weight) marine fish oil (FO, 12.7% docosahexaenoic acid and 13.8% eicosapentaenoic acid), evening primrose oil (EPO, 7.8% γ-linolenic acid and 70.8% linoleic acid) or a mixture of 5% FO-5% EPO. After 12 weeks on the respective diets, animals fed higher proportions of (n-3) polyunsaturated fatty acids (FO group) consistently contained higher levels of 20:3(n-6), 20:5(n-3), 22:5(n-3), and 22:6(n-3), and lower levels of 18:2(n-6) and 20:4(n-6), than those of the EPO (a rich source of (n-6) polyunsaturated fatty acids) or the FO + EPO groups. Membrane fluidity, as estimated by the reciprocal of the order parameter S_DPH, was higher in the FO than in the EPO or the FO + EPO groups, and the n-6 fatty-acid desaturation system was markedly affected.     

Changes in cerebral membrane lipid composition and fluidity during thioacetamide-induced hepatic encephalopathy

Lipids are an essential structural and functional component of cellular membranes. Changes in membrane lipid composition are known to affect the activities of many membrane-associated enzymes, endocytosis, exocytosis, membrane fusion and neurotransmitter uptake, and have been implicated in the pathophysiology of many neurodegenerative disorders. In the present study, we investigated changes in the lipid composition of membranes isolated from the cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin that induces fulminant hepatic failure (FHF) and thereon hepatic encephalopathy (HE). HE refers to acute neuropsychiatric changes accompanying FHF. The estimation of membrane phospholipids, cholesterol and fatty acid content in cerebral cortex membranes from TAA-treated rats revealed a decrease in cholesterol, phosphatidylserine, sphingomyelin, a monounsaturated fatty acid, namely oleic acid, and the polyunsaturated fatty acids gamma-linolenic acid, decosa hexanoic acid and arachidonic acid compared with controls. Assessment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in the annular membrane fluidity, whereas the global fluidity was unaffected. The level of the thiobarbituric acid reactive species marker for lipid peroxidation also increased in membranes from TAA-treated rats, thereby indicating the prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane lipid composition and fluidity during TAA-induced HE, and their possible implications in the pathogenesis of this condition are also discussed.     

Effect of hyperthermia on the repair of sublethal radiation damage in normal and membrane fatty acid substituted fibroblasts

The interaction of heat and X irradiation was studied in normal and polyunsaturated fatty acid (PUFA) substituted mouse fibroblast LM cells. As a result of the substitution the membranes of the PUFA cells were more fluid than the membranes of the normal cells. Three different heat doses were applied (60 min 42 degrees C, 20 min 43 degrees C, and 10 min 44 degrees C) in combination with single or split doses of X rays. Heat radiosensitization was the largest for the 60 min 42 degrees C treatment. Heat radiosensitization and the heat-induced inhibition of the rate of sublethal damage repair were the same for the normal and the PUFA cells. It is concluded from the experiments reported that the processes of hyperthermic inhibition of SLD repair and hyperthermic radiosensitization are independent of membrane fluidity and membrane fatty acid composition.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.