Physical and chemical modulation of lipid rafts by a dietary n-3 polyunsaturated fatty acid increases ethanol-induced oxidative stress

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to modulate lipid raft-dependent signaling, but not yet lipid raft-dependent oxidative stress. Previously, we have shown that ethanol-induced membrane remodeling, i.e., an increase in membrane fluidity and alterations in physical and biochemical properties of lipid rafts, participated in the development of oxidative stress. Thus, we decided to study n-3 PUFA effects in this context, by pretreating hepatocytes with eicosapentaenoic acid (EPA), a long-chain n-3 PUFA, before addition of ethanol. EPA was found to increase ethanol-induced oxidative stress through membrane remodeling. Addition of EPA resulted in a marked increase in lipid raft aggregation compared to ethanol alone. In addition, membrane fluidity of lipid rafts was markedly enhanced. Interestingly, EPA was found to preferentially incorporate into nonraft membrane regions, leading to raft cholesterol increase. Lipid raft aggregation by EPA enhanced phospholipase Cγ translocation into these microdomains. Finally, phospholipase Cγ was shown to participate in the potentiation of oxidative stress by promoting lysosome accumulation, a major source of low-molecular-weight iron. To conclude, the ability of EPA to modify lipid raft physical and chemical properties plays a key role in the enhancement, by this dietary n-3 PUFA, of ethanol-induced oxidative stress.     

One generation of n-3 polyunsaturated fatty acid deprivation increases depression and aggression test scores in rats

Male rat pups at weaning (21 days of age) were subjected to a diet deficient or adequate in n-3 polyunsaturated fatty acids (n-3 PUFAs) for 15 weeks. Performance on tests of locomotor activity, depression, and aggression was measured in that order during the ensuing 3 weeks, after which brain lipid composition was determined. In the n-3 PUFA-deprived rats, compared with n-3 PUFA-adequate rats, docosahexaenoic acid (22:6n-3) in brain phospholipid was reduced by 36% and docosapentaenoic acid (22:5n-6) was elevated by 90%, whereas brain phospholipid concentrations were unchanged. N-3 PUFA-deprived rats had a significantly increased (P = 0.03) score on the Porsolt forced-swim test for depression, and increased blocking time (P = 0.03) and blocking number (P = 0.04) scores (uncorrected for multiple comparisons) on the isolation-induced resident-intruder test for aggression. Large effect sizes (d > 0.8) were found on the depression score and on the blocking time score of the aggression test. Scores on the open-field test for locomotor activity did not differ significantly between groups, and had only small to medium effect sizes. This single-generational n-3 PUFA-deprived rat model, which demonstrated significant changes in brain lipid composition and in test scores for depression and aggression, may be useful for elucidating the contribution of disturbed brain PUFA metabolism to human depression, aggression, and bipolar disorder.     

Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes

DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.     

The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae

Livestock meat is generally low in n-3 polyunsaturated fatty acids (PUFAs), which are beneficial to human health. An alternative approach to increasing the levels of n-3 PUFAs in meat is to generate transgenic livestock animals. In this study, we describe the generation of cloned pigs that express the cbr-fat-1 gene from Caenorhabditis briggsae, encoding an n-3 fatty acid desaturase. Analysis of fatty acids demonstrated that the cbr-fat-1 transgenic pigs produced high levels of n-3 fatty acids from n-6 analogs; consequently, a significantly reduced ratio of n-6/n-3 fatty acids was observed. We demonstrated that the n-3 desaturase gene from C. briggsae was functionally expressed, and had a significant effect on the fatty acid composition of the transgenic pigs, which may allow the production of pork enriched in n-3 PUFAs.     

Short-term n-3 fatty acid supplementation but not aspirin increases plasma proresolving mediators of inflammation

Resolution of inflammation is an active process involving specialized proresolving mediators (SPM) formed from the n-3 fatty acids. This study examined the effect of n-3 fatty acid supplementation and aspirin on plasma SPMs in healthy humans. Healthy volunteers (n = 21) were supplemented with n-3 fatty acids (2.4g/day) for 7 days with random assignment to take aspirin (300 mg/day) or placebo from day 5 to day 7. Blood was collected at baseline (day 0), day 5, and day 7. Plasma 18R/S-HEPE, E-series resolvins, 17R/S-HDHA, D-series resolvins, 14R/S-HDHA, and MaR-1 were measured by LC/MS/MS. At baseline concentrations of E- and D- series resolvins and the upstream precursors 18R/S-HEPE, 17R/S-HDHA ranged from 0.1nM to 0.2nM. 14R/S-HDHA was 3-fold higher than the other SPMs at baseline but MaR-1 was below the limit of detection. Supplementation with n-3 fatty acids significantly increased RvE1, 18R/S-HEPE, 17R/S-HDHA, and 14R/S-HDHA but not other SPMs. The addition of aspirin after 5 days of n-3 fatty acids did not affect concentrations of any SPM. N-3 fatty acid supplementation for 5 days results in concentrations of SPMs that are biologically active in healthy humans. Aspirin administered after n-3 fatty acids did not offer any additional benefit in elevating the levels of SPMs.     

Chemical and immunochemical identification of propanoyllysine derived from oxidized n-3 polyunsaturated fatty acid

It is known that n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and eicosapentaenoic acid, are rapidly oxidized in vitro. N?-(propanoyl)lysine (propionyllysine, or PRL) is formed from the reaction of the oxidized products of n-3 PUFAs and lysine. To evaluate the oxidized n-3 PUFA-derived protein modifications in vivo, we have developed detection methods using a novel monoclonal antibody against PRL as well as liquid chromatography–mass spectrometry (LC/MS/MS). The antibody obtained specifically recognized PRL. A strong positive staining in atherosclerotic lesions of hypercholesterolemic rabbits was observed. We have also simultaneously identified and quantified both urinary PRL and urinary N?-(hexanoyl)lysine, using LC/MS/MS using isotope dilution methods. The level of urinary PRL (21.6 ± 10.6 μmol/mol of creatinine) significantly correlated with the other oxidative stress markers, 8-oxo-deoxyguanosine, dityrosine, and isoprostanes. The increase in the excretion of amide adducts into the urine of diabetic patients was also confirmed compared to healthy subjects. These results suggest that PRL may be good marker for n-3 PUFA-derived oxidative stress in vivo.     

Interactions of saturated, n-6 and n-3 polyunsaturated fatty acids to modulate arachidonic acid metabolism

Anti-thrombotic effects of omega-3 (n-3) fatty acids are believed to be due to their ability to reduce arachidonic acid levels. Therefore, weanling rats were fed n-3 acids in the form of linseed oil (18:3n-3) or fish oil (containing 20:5n-3 and 22:6n-3) in diets containing high levels of either saturated fatty acids (hydrogenated beef tallow) or high levels of linoleic acid (safflower oil) for 4 weeks. The effect of diet on the rate-limiting enzyme of arachidonic acid biosynthesis (delta 6-desaturase) and on the lipid composition of hepatic microsomal membrane was determined. Both linseed oil- or fish oil-containing diets inhibited conversion of linoleic acid to gamma-linolenic acid. Inhibition was greater with fish oil than with linseed oil, only when fed with saturated fat. delta 6-Desaturase activity was not affected when n-3 fatty acids were fed with high levels of n-6 fatty acids. Arachidonic acid content of serum lipids and hepatic microsomal phospholipids was lower when n-3 fatty acids were fed in combination with beef tallow but not when fed with safflower oil. Similarly, n-3 fatty acids (18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3) accumulated to a greater extent when n-3 fatty acids were fed with beef tallow than with safflower oil. These observations indicate that the efficacy of n-3 fatty acids in reducing arachidonic acid level is dependent on the linoleic acid to saturated fatty acid ratio of the diet consumed.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites.

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.     

Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increase turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-(14)C]DPAn-6 was infused intravenously for 5min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2-7.7 fold, while turnover rates were increased 2.1-4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. (196 words).     

Determination of n-3 HUFA content in Atlantic salmon flesh based on the lipid content, morphometric measurements and blood fatty acid composition: A modeling approach

Fish are the most important source of n-3 highly unsaturated fatty acids (HUFA) in the human diet and, with declining wild stocks, an increasing proportion is being provided by aquaculture. Paradoxically, aquaculture fish diets have traditionally incorporated fish oil and meal derived from wild fisheries. Continued aquaculture development requires fish oil to be replaced with vegetable oils, the only sustainable alternative. However, vegetable oils lack n-3 HUFA and so flesh from fish reared on these diets can also have reduced n-3 HUFA and thus reduced nutritional quality. This accepted, the flesh n-3 HUFA content should be an economically important trait, however to be included in the breeding goal the trait must be measurable. In the present study, we investigated whether the flesh n-3 HUFA content of salmon can be estimated in a non-fatal way. We showed that a general regression model based on flesh lipid content, morphometric and blood fatty acid measurements could estimate and predict flesh n-3 HUFA content. This would allow a choice from a range of selection methods, including mass selection or within-family selection, if this important flesh quality trait is to be included in future salmon breeding programmes.     

Specific phospholipid fatty acid composition of brain regions in mice. Effects of n-3 polyunsaturated fatty acid deficiency and phospholipid supplementation

This study examined the effects of dietary alpha-linolenic acid deficiency followed or not by supplementation with phospholipids rich in n;-3 polyunsaturated fatty acid (PUFA) on the fatty acid composition of total phospholipids in 11 brain regions. Three weeks before mating, mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group were supplemented with n;-3 polyunsaturated fatty acids (PUFA) from either egg yolk or pig brain phospholipids for 2 months. Saturated and monounsaturated fatty acid levels varied among brain regions and were not significantly affected by the diet. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all regions. alpha-Linolenic acid deficiency decreased the level of 22:6 n-3 and was compensated by an increase in 22:5 n-6 in all regions. However, the brain regions were affected differently. After the pituitary gland, the frontal cortex, and the striatum were the most markedly affected with 40% reduction of 22:6 n-3. Supplementation with egg yolk or cerebral phospholipids in deficient mice restored a normal fatty acid composition in brain regions except for the frontal cortex. There was a regional distribution of the fatty acids in the brain and the impact of deficiency in alpha-linolenic acid was region-specific. Dietary egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for the recovery of altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Assessment of blood measures of n-3 polyunsaturated fatty acids with acute fish oil supplementation and washout in men and women

Changes in n-3 highly unsaturated fatty acids (HUFA, ≥20 carbons and ≥3 carbon–carbon double bonds) at baseline, during fish oil supplementation (4 weeks) and during washout (8 weeks) were compared in venous plasma, erythrocytes, whole blood and fingertip prick blood (weeks 0, 4, 8 and 12) with additional weekly fingertip prick samples. Correlations between the various blood fractions were slightly stronger when n-3 HUFA status was expressed as the percentage of n-3 HUFA in total HUFA as compared with the sum of EPA and DHA. Increases and decreases in n-3 HUFA were more dramatic in plasma, and EPA responded rapidly (within 1 week) with fish oil supplementation and cessation. Sex differences in the proportions of n-3 HUFA in blood were also apparent at baseline with females (n=7) having a tendency for higher docosahexaenoic acid (DHA, 22:6n-3) relative to eicosapentaenoic acid (EPA, 20:5n-3) and n-3 docosapentaenoic acid (DPAn-3, 22:5n-3) as compared with males (n=9). Further n-3 biomarker research in larger populations is required.     

Black tea increases the resistance of human plasma to lipid peroxidation in vitro, but not ex vivo.

A number of in vitro studies have shown that polyphenols and flavonoids in tea exert significant antioxidant activity. However, epidemiologic and experimental studies have produced conflicting results. The purpose of the present study was to compare the antioxidant activity of black tea in vitro with that ex vivo. Black tea polyphenols (BTP), black tea extract (BTE), or their major polyphenolic antioxidant constituent, epigallocatechin gallate (EGCG), were added to human plasma and lipid peroxidation was induced by the water-soluble radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Following a lag phase, lipid peroxidation was initiated and occurred at a rate that was lowered in a dose-dependent manner by BTP. Similarly, EGCG and BTE added to plasma in vitro strongly inhibited AAPH-induced lipid peroxidation. The lag phase preceding detectable lipid peroxidation was due to the antioxidant activity of endogenous ascorbate, which was more effective at inhibiting lipid peroxidation than the tea polyphenols and was not spared by these compounds. In contrast, when eight healthy volunteers consumed the equivalent of six cups of black tea, the resistance of their plasma to lipid peroxidation ex vivo did not increase over the next 3 h. These data suggest that, despite antioxidant efficacy in vitro, black tea does not protect plasma from lipid peroxidation in vivo. The striking discrepancy between the in vitro and ex vivo data is most likely explained by the insufficient bioavailability of tea polyphenols in humans.     

Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells

The antioxidant effects of dipyridamol (DIP), a coronary vasodilator, and its derivative RA-25 were compared in intact red blood cells (RBC) and in isolated ghost membranes. Both compounds are quite effective antioxidants in cumene hydroperoxide-induced lipid peroxidation of RBC, showing a much smaller effect for hydrogen peroxide oxidation. The antioxidant effect of DIP was considerably higher than that of RA25. For isolated ghost membranes, the apparent IC50 (the drug concentration that produces 50% inhibition of lipid peroxidation) in cumene hydroperoxide-induced peroxidation was 25 microM, while the maximum protective effect of RA-25 was around 30% in the drug concentration range of 50-100 microM. The drugs can protect the oxidative hemolysis induced by cumene hydroperoxide with a lower effect when the hemolysis is induced by H2O2. The significant antioxidant effect against damages induced by cumene hydroperoxide suggests that DIP, due to its lipophilic character, can interact with RBC membranes, and the protective effect is associated with the binding of the drug to the membrane. On the other hand, RA-25 is more hydrophilic than DIP, binds to the membrane to a smaller extent, and, for this reason, has a lower antioxidant effect.     

Dietary n-3 polyunsaturated fatty acid deprivation together with early maternal separation increases anxiety and vulnerability to stress in adult rats

Low concentrations of n-3 polyunsaturated fatty acid (PUFA) and chronic stress are implicated in susceptibility to mood disorders. We have investigated the combined effects of chronic n-3 PUFA dietary deficiency and early maternal separation (MS) stress on the reactivity to stressful situations of rats as adults. Pups fed a control or an n-3 PUFA deficient diet were daily separated for two weeks before weaning They were all tested at 3 month-old to determine their anxiety, and their ability to learn two aversive tasks differing in the control they could exert on the situation: auditory fear conditioning and brightness avoidance discrimination. Neither the n-3 PUFA-deficient diet nor MS alone significantly affected behavior. But n-3 PUFA-deficient rats that had been separated were more anxious and fearful in inescapable situations, while their ability to cope with an aversive avoidance task remained unaffected. These results support the notion that PUFA-unbalanced diet, together with stress, may be a determinant risk factor in emotional disorders.     

Mechanisms of enhanced apoptosis in HL-60 cells by UV-irradiated n-3 and n-6 polyunsaturated fatty acids

We examined the effects of arachidonic acid (AA), eicosapentaenoic acid (EPA), and their ultraviolet (UV)-irradiated products on HL-60 cells and isolated mitochondria to explore the following four obscure points in the mechanism of polyunsaturated fatty acids (PUFAs)-induced apoptosis: (i). the role of reactive oxygen species, (ii). the interaction of PUFAs and their metabolites with mitochondria in situ, (iii). the cyclosporine A (CsA)-sensitivity in PUFA-induced membrane permeability transition, (iv). the specificity of oxidized n-3 PUFAs in the induction of apoptosis in cancer cells. UV-oxidized PUFAs contained conjugated dienes and thiobarbituric acid reactive substances (TBARS). The apoptotic effects of PUFAs on HL-60 cells were increased by UV-irradiation whereas the swelling effect of PUFAs on isolated mitochondria was decreased. Both oxidized n-3 and n-6 PUFAs induced increased depolarization, ferricytochrome c release, the activation of various caspases, and DNA-fragmentation in a CsA-insensitive mechanism concomitant with a slight increase in the value of TBARS in cells. Furthermore, there were no significant differences in the mechanism of apoptosis induced by either oxidized AA or oxidized EPA. On the basis of these results, it was concluded that both oxidized n-3 or n-6 PUFAs induced apoptosis in HL-60 cells by a similar mechanism in a CsA-insensitive manner and also that oxidized products of PUFAs, but not the cellular oxidation process itself, play an important role in the mechanism of apoptosis in HL-60 cells.