Effect of n-3 and n-6 polyunsaturated fatty acids on hare reproductive performances

The study was carried out on 42 breeder couples (42 males and 42 females) of European brown hare (Lepus europaeus), divided into three groups fed three different experimental diets (14 couples/treatment). Two diets were supplemented with n-3 and n-6 polyunsaturated fatty acids (PUFAs; 2% of linseed oil and soybean oil, respectively) and were compared with a control diet supplemented with a monounsaturated fatty acids (2% of olive oil). During the experimental period (from 15 April to 30 September), the following parameters were recorded: days from the beginning of trial to the first parturition, parturition interval, number of parturitions, number of leverets born (alive and dead), dead during suckling, the total number of leverets weaned and feed intake per cage (of males, females and leverets until weaning). Feed intake was not influenced by treatments. In hares fed n-3 and n-6 diets, the days from the beginning of the trial to the first parturition and the parturition interval were similar and were lower compared with control group (63.1 v. 70.6 days, and 37.8 v. 40.9 days, respectively; P < 0.05). Hares from n-6 group had a higher (P < 0.05) number of parturitions per cage during the experimental period than the n-3 and control group that showed a similar value (3.00 v. 2.36, respectively). The total number of leverets born per cage and parturition in n-6 and n-3 groups increased with respect to those fed control diet (P < 0.05). The leverets' mortality rate at birth was higher in n-6 than in n-3 and control group (3.50 v. 2.17, respectively; P < 0.05). In control group, leverets' mortality rate during suckling was lower with respect to n-3 (P < 0.05) and n-6 (P < 0.05), showing the highest value for the latter (P < 0.05). In spite of this higher mortality, the number of leverets weaned per cage and parturition was higher (P < 0.05) in n-6 compared with n-3 group, being the latter higher than the control group (3.12, 2.79 and 2.43, respectively). Our results show that the dietary PUFAs, particularly n-6 supplementation, have a positive influence on the reproductive performances of the European brown hare.     

Effect of n-6 and n-3 dietary fatty acids on phospholipid composition of plasma, platelets and aorta in the rat

Female Wistar rats were fed with diets containing as dietary lipids 10% of hydrogenated coconut oil, grape-seed oil, olive oil, linseed oil and fish oil, respectively, for a period of 60 days. At the end of dietary treatment plasma, platelets and aorta phospholipids were extracted and fatty acid spectra determined. Plasma and platelet phospholipids showed the largest diet dependent changes. Anyway in aorta samples too, phospholipids showed marked increase in oleic (olive oil group) linoleic (grape-seed oil group) and alpha linoleic (linseed oil group) acids percentage. Conversely decreased amounts of arachidonic acid were detected in rats fed with diets containing linseed and fish oils. In these samples eicosapentenoic acid partly replaced arachidonic one.     

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.     

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.     

Effect of n-3 and n-6 polyunsaturated fatty acids and their ethylesters on stimuli-dependent superoxide generation in neutrophils

Polymorphonuclear leukocytes from healthy volunteers (HPMN) generated superoxide (O2*-) following treatment with various stimuli, such as phorbol myristate acetate (PMA), opsonized zymozan (OZ) and arachidonic acid (AA). Other types of n-3 polyunsaturated fatty acids (PUFAS), such as docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), also stimulated O2*- generation. The free form of DHA enhanced the generation of O2*- induced by PMA but inhibited that induced by OZ. In contrast, the ethylester of DHA (DHA-E) inhibited O2*- generation induced by PMA but stimulated that induced by OZ. Similar effects were also observed with ethylesters of EPA (EPA-E), DPA (DPA-E) and AA (AA-E). High concentrations of DHA-E reduced the PMA-induced formation of superoxide without affecting the cellular activity of protein kinase C (PKC). Similar phenomena were also observed with oral neutrophils from healthy volunteers (OPMN). These results indicate that PUFAS and their esters affect 02*- generation in human PMN via different pathways, thereby modulating inflammatory reactions.     

The effects of n-3 and n-6 polyunsaturated fatty acids on plasma lipids and fatty acids of treated phenylketonuric children

Dietary-treated phenylketonuric patients (PKUs) display low levels of long-chain polyunsaturated fatty acids (PUFA) in plasma lipids. In a 6-month clinical trial we observed a decrease of triglycerides and an increase of n-3 long-chain PUFA in plasma of PKUs supplemented with fish oil, while no major differences in respect to the baseline values were found in a group supplemented with blackcurrant oil. A more complete source of long-chain PUFA of both the n-6 and n-3 series should be investigated for dietary supplementation of PKU patients.     

n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.     

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.     

Differential induction of electrophile-responsive element-regulated genes by n-3 and n-6 polyunsaturated fatty acids

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.     

Recent studies on interactions between n-3 and n-6 polyunsaturated fatty acids in brain and other tissues

Recent literature provides a basis for understanding the behavioral, functional, and structural consequences of nutritional deprivation or disease-related abnormalities of n-3 polyunsaturated fatty acids. The literature suggests that these effects are mediated through competition between n-3 and n-6 polyunsaturated fatty acids at certain enzymatic steps, particularly those involving polyunsaturated fatty acid elongation and desaturation. One critical enzymatic site is a delta6-desaturase. On the other hand, an in-vivo method in rats, applied following chronic n-3 nutritional deprivation or chronic administration of lithium, indicates that the cycles of de-esterification/re-esterification of docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6) within brain phospholipids operate independently of each other, and thus that the enzymes regulating each of these cycles are not likely sites of n-3/n-6 competition.     

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.     

Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein

Synthesis of n-3 and n-6 very long chain-PUFAs (VLC-PUFAs) from 18-carbon essential fatty acids is differentially regulated. The predominant product arising from n-3 fatty acids is docosahexaenoic acid (22:6n-3), with the liver serving as the main site of production. The synthetic pathway requires movement of a 24-carbon intermediate from the endoplasmic reticulum to peroxisomes for retroconversion to 22:6n-3. The mechanism of this intra-organelle flux is unknown, but could be binding-protein facilitated. We thus investigated binding of a series of previously untested VLC-PUFAs to liver fatty acid-binding protein (L-FABP). Three fluorometric assays were employed, all of which showed strong binding (K(d)' approximately 10(-8) to 10(-7) M) of 20-, 22-, and 24-carbon n-3 PUFAs to L-FABP. In contrast, synthesis of the predominant n-6 PUFA product, arachidonic acid, does not require intra-organelle transport. However, we found that n-6 VLC-PUFAs bound to L-FABP with affinities (K(d)' approximately 10(-8) to 10(-7) M) comparable to their n-3 counterparts.Although these results raise the possibility that L-FABP may participate in the cytoplasmic processing of n-3 and n-6 VLC-PUFAs, there is no evidence on the basis of binding affinities that L-FABP accounts for differences in the predominant products formed by the n-3 and n-6 PUFA metabolic pathways.     

Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.     

Onset of changes in phospholipid fatty acid composition and prostaglandin synthesis following dietary manipulation with n-6 and n-3 fatty acids in the rat

A synthetic diet preparation supplemented with 10% by weight of either safflower oil, hydrogenated coconut oil containing 3% safflower oil, or 'max EPA' fish oil was fed to rats over a 8-week period. Serial measurements of serum fatty acids, serum thromboxane B2 and urinary prostaglandin excretion were taken during the treatment period to assess the rate of change in fatty acid composition and prostaglandin synthesis following dietary manipulation. There was no significant change in weight gain between the dietary groups during the treatment period. Significant changes in serum fatty acids occurred within 48 h of treatment, with the 'max EPA' oil group having arachidonic acid levels reduced by 23% (P less than 0.01) compared to the coconut oil group. Conversely, rats fed safflower oil had an 18% enhancement of arachidonic acid during the same time period. Whole blood synthesis of thromboxane B2 was significantly depressed (P less than 0.01) after 48 h in rats fed 'max EPA' oil compared to the safflower oil or coconut oil groups. This suppression reached a maximum of 65% (P less than 0.001) after 7 days of dietary 'max EPA' oil treatment. The safflower oil and coconut oil-fed groups showed the same levels of serum thromboxane B2 production over the treatment period. Urinary excretion of both 6-ketoprostaglandin F1 alpha and prostaglandin E2 varied significantly (P less than 0.01) between the groups after 7 days of dietary treatment. Rats fed 'max EPA' oil had depressed urinary prostanoid excretion compared to the safflower and coconut oil groups which remained very similar to each other. After the 8-week treatment period rats were killed and the phospholipid fatty acid composition and prostaglandin-generating capacity of platelets, aorta and renal tissue was examined. Prostanoid production by kidney cortex and medulla and segments of aorta was consistently suppressed in rats fed 'max EPA' oil. These observations correlated well with changes in the phospholipid fatty acid profiles in these tissues. This study shows rapid changes in serum fatty acids and thromboxane B2 generation following dietary manipulation, while changes in urinary excretion or prostanoid metabolites occur only after a longer time period.     

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.     

Effect of dietary n-3 and n-6 fatty acids on tobramycin induced nephrotoxicity

Post fish oil(n-3 fatty acids) treatment (5mg/kg/day for 12 days) was effective in bringing the reversal of tobramycin (160mg/kg/day,ip for 12 days) induced nephrotoxicity in albino rats as was evident by normal urea, creatinine, cholesterol and inorganic phosphate levels in the serum of the treatment group compared with group receiving tobramycin only. The return of normal levels of alkaline and acid phosphatase in kidney homogenates of post fish oil treatment group also indicated the beneficial effect of dietary n-3 fatty acids(fish oil) more than n-6 fatty acids(olive oil).The results suggest that oral supplements of dietary n-3 fatty acids (fish oil) for nearly two weeks after tobramycin exposure is more beneficial than n-6 fatty acids (olive oil) as it results in reversal of nephrotoxicity induced by tobramycin.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.