Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.     

Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE,and 4-HNE to ethanolamine phospholipid subclasses

Lipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl. We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes.     

Aldehydes from n-6 fatty acid peroxidation. Effects on aminophospholipids

4-Hydroxy-nonenal (4-HNE) is a major by-product of n-6 fatty acid peroxidation. It has been described to covalently bind biomolecules expressing primary amine, especially the Lys residues in proteins. Low-density lipoproteins (LDL) are well-described macromolecules to be modified by 4-HNE, making them available to scavenger receptors on macrophages. Those macrophages then become foam cells and play an active role in atherogenesis. This paper reports on the covalent binding of 4-HNE to phosphatidylethanolamine (PE), a major aminophospholipid in biological membranes. In contrast, phosphatidylserine (PS) is virtually not modified by 4-HNE. One stable adduct, the Michael adduct PE/4-HNE is a poor substrate of secreted phospholipase A(2) and is not cleaved by phospholipase D. Plasmalogen PE, an important subclass of PE, is covalently modified by 4-HNE as well, but appears to be further degraded on its sn-1 position, the alkenyl chain, which might alter the antioxidant potential of the molecule. An aldehyde homologous to 4-HNE has been characterized as a breakdown product of 12-hydroperoxyeicosatetraenoic acid (12-HpETE) and named 4-hydroxy-2E,6Z-dodecadienal (4-HDDE). This compound as well as 4-HNE was detected in human plasma. Finally, 4-HDDE appears almost 3-fold more active than 4-HNE to make covalent adducts with PE. We conclude that 4-HNE and 4-HDDE are two biologically relevant markers of n-6 fatty acid peroxidation that may alter the phospholipid-dependent cell signaling.     

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.     

Maternal dietary fat alters amniotic fluid and fetal intestinal membrane essential n-6 and n-3 fatty acids in the rat

We investigated whether maternal fat intake alters amniotic fluid and fetal intestine phospholipid n-6 and n-3 fatty acids. Female rats were fed a 20% by weight diet from fat with 20% linoleic acid (LA; 18:2n-6) and 8% alpha-linolenic acid (ALA; 18:3n-3) (control diet, n = 8) or 72% LA and 0.2% ALA (n-3 deficient diet, n = 7) from 2 wk before and then throughout gestation. Amniotic fluid and fetal intestine phospholipid fatty acids were analyzed at day 19 gestation using HPLC and gas-liquid chromotography. Amniotic fluid had significantly lower docosahexaenoic acid (DHA; 22:6n-3) and higher docosapentaenoic acid (DPA; 22:5n-6) levels in the n-3-deficient group than in the control group (DHA: 1.29 +/- 0.10 and 6.29 +/- 0.33 g/100 g fatty acid; DPA: 4.01 +/- 0.35 and 0.73 +/- 0.15 g/100 g fatty acid, respectively); these differences in DHA and DPA were present in amniotic fluid cholesterol esters and phosphatidylcholine (PC). Fetal intestines in the n-3-deficient group had significantly higher LA, arachidonic acid (20:4n-6), and DPA levels; lower eicosapentaenoic acid (EPA; 20:5n-3) and DHA levels in PC; and significantly higher DPA and lower EPA and DHA levels in phosphatidylethanolamine (PE) than in the control group; the n-6-to-n-3 fatty acid ratio was 4.9 +/- 0.2 and 32.2 +/- 2.1 in PC and 2.4 +/- 0.03 and 17.1 +/- 0.21 in PE in n-3-deficient and control group intestines, respectively. We demonstrate that maternal dietary fat influences amniotic fluid and fetal intestinal membrane structural lipid essential fatty acids. Maternal dietary fat can influence tissue composition by manipulation of amniotic fluid that is swallowed by the fetus or by transport across the placenta.     

Age-related changes of n-3 and n-6 polyunsaturated fatty acids in the anterior cingulate cortex of individuals with major depressive disorder

Accumulating evidence finds a relative deficiency of peripheral membrane fatty acids in persons with affective disorders such as unipolar and bipolar depression. Here we sought to investigate whether postmortem brain fatty acids within the anterior cingulate cortex (BA-24) varied according to the presence of major depression at the time of death. Using capillary gas chromatography we measured fatty acids in a depressed group (n=12), and in a control group without lifetime history of psychiatric diagnosis (n=14). Compared to the control group, the depressed group showed significantly lower concentrations of numerous saturated and polyunsaturated fatty acids including both the n-3 and n-6 fatty acids. Additionally, significant correlations between age at death and precursor (or metabolites) in the n-3 fatty acid pathway were demonstrated in the depressed group but not in control subjects. In the n-6 fatty acid family, the ratio of 20:3(n-6)/18:2(n-6) was higher in patients than in control groups, whereas the ratio of 20:4(n-6)/20:3(n-6) was relatively decreased in patients. Lastly, a significant negative correlation between age and the ratio of 20:4(n-6) to 22:6(n-3) was found in patients, but not in controls. Taken together, decreases in 22:6(n-3) may be caused, at least in part, by the diminished formation of 20:5(n-3), which is derived from 20:4(n-3) through a Δ5 desaturase reaction. The present findings from postmortem brain tissue raise the possibility that an increased ratio of 20:4(n-6) to 22:6(n-3) may provide us with a biomarker for depression. Future research should further investigate these relationships.     

Liver triacylglycerols and free fatty acids in streptozotocin-induced diabetic rats have atypical n-6 and n-3 pattern

In diabetes there is a decrease in membrane arachidonic (AA) and docosahexaenoic (DHA) acids and a concomitant increase in linoleic (LA) and alpha-linolenic (ALA) acids. This metabolic perturbation is thought to be due to impaired activity of Delta(6)- and Delta(5)-desaturases. Triacylglycerols are the major lipid pool in plasma and liver tissue and have a significant influence on fatty acid composition of membrane and circulating phospholipids. Data on the distribution of n-6 and n-3 polyunsaturated fatty acids of triacylglycerols in diabetes are sparse. We investigated whether streptozotocin-induced diabetes in Sprague-Dawley rats alters fatty acid composition of triacylglycerols and free fatty acids of liver tissue. The animals were fed a breeding diet prior to mating, during pregnancy and lactation. On days 1-2 of pregnancy, diabetes was induced in 10 of the 25 rats. Liver was obtained at post partum day 16 for analysis. Relative levels of LA (P=0.03), dihomo-gamma-linolenic acid (DHGLA) (P=0.02), AA (P=0.049), total n-6 (P=0.02), ALA (P=0.013), eicosapentaenoic acid (EPA) (P=0.004), docosapentaenoic acid (22:5n-3, DPA) (P=0.013), DHA (P=0.033), n-3 metabolites (P=0.015) and total n-3 (P=0.011) were significantly higher in the triacylglycerols of the diabetics compared with the controls. Similarly, liver free fatty acids of the diabetics had higher levels of LA (P=0.0001), DHGLA (P=0.001), AA (P=0.001), n-6 metabolites (P=0.002), total n-6 (P=0.0001), ALA (P=0.003), EPA (P=0.015), docosapentaenoic (22:5n-3, P=0.003), DHA (P=0.002), n-3 metabolites (P=0.005) and total n-3 (P=0.001). We conclude that impaired activity of desaturases and/or long chain acyl-CoA synthetase could not explain the higher levels of AA, DHA and n-6 and n-3 metabolites in the diabetics. This seems to be consistent with an alteration in the regulatory mechanism, which directs incorporation of polyunsaturated fatty acids either into triacylglycerols or phospholipids.     

Alterations of the fatty acid composition and lipid metabolome of breast muscle in chickens exposed to dietary mixed edible oils

The fatty acid composition of chicken’s meat is largely influenced by dietary lipids, which are often used as supplements to increase dietary caloric density. The underlying key metabolites and pathways influenced by dietary oils remain poorly known in chickens. The objective of this study was to explore the underlying metabolic mechanisms of how diets supplemented with mixed or a single oil with distinct fatty acid composition influence the fatty acid profile in breast muscle of Qingyuan chickens. Birds were fed a corn-soybean meal diet supplemented with either soybean oil (control, CON) or equal amounts of mixed edible oils (MEO; soybean oil : lard : fish oil : coconut oil = 1 : 1 : 0.5 : 0.5) from 1 to 120 days of age. Growth performance and fatty acid composition of muscle lipids were analysed. LC-MS was applied to investigate the effects of CON v. MEO diets on lipid-related metabolites in the muscle of chickens at day 120. Compared with the CON diet, chickens fed the MEO diet had a lower feed conversion ratio (P < 0.05), higher proportions of lauric acid (C12:0), myristic acid (C14:0), palmitoleic acid (C16:1n-7), oleic acid (C18:1n-9), EPA (C20:5n-3) and DHA (C22:6n-3), and a lower linoleic acid (C18:2n-6) content in breast muscle (P < 0.05). Muscle metabolome profiling showed that the most differentially abundant metabolites are phospholipids, including phosphatidylcholines (PC) and phosphatidylethanolamines (PE), which enriched the glycerophospholipid metabolism (P < 0.05). These key differentially abundant metabolites – PC (14:0/20:4), PC (18:1/14:1), PC (18:0/14:1), PC (18:0/18:4), PC (20:0/18:4), PE (22:0/P-16:0), PE (24:0/20:5), PE (22:2/P-18:1), PE (24:0/18:4) – were closely associated with the contents of C12:0, C14:0, DHA and C18:2n-6 in muscle lipids (P < 0.05). The content of glutathione metabolite was higher with MEO than CON diet (P < 0.05). Based on these results, it can be concluded that the diet supplemented with MEO reduced the feed conversion ratio, enriched the content of n-3 fatty acids and modified the related metabolites (including PC, PE and glutathione) in breast muscle of chickens.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.     

Modification of liver fatty acid metabolism in mice by n-3 and n-6 delta 6-desaturase substrates and products

The effects of dietary supplementation of either alpha-linolenic acid (18:3(n-3)) or stearidonic acid (18:4(n-3)) in combination with either linoleic acid (18:2(n-6)) or gamma-linolenic acid (18:3(n-6)) on liver fatty acid composition in mice were examined. Essential fatty acid deficient male C57BL/6 mice were separated into four groups of seven each and were fed a fat-free semi-purified diet supplemented with 1% (w/w) fatty acid methyl ester mixture (1:1), 18:2(n-6)/18:3(n-3), 18:2(n-6)/18:4(n-3), 18:3(n-6)/18:3(n-3), or 18:3(n-6)/18:4(n-3). After 7 days on the diets, fatty acid compositions in liver phosphatidylcholine and phosphatidylethanolamine fractions were analyzed. In groups fed 18:4(n-3) (18:2(n-6)/18:4(n-3) or 18:3(n-6)/18:4(n-3)) as compared to those fed 18:3(n-3) (18:2(n-6)/18:3(n-3) or 18:3(n-6)/18:3(n-3)), the levels of 20:4(n-3), 20:5(n-3) and 22:5(n-3) were increased, whereas those of 20:3(n-6) and 20:4(n-6) were decreased. When 18:3(n-6) replaced 18:2(n-6) as the source of n-6 acids, the levels of 18:3(n-6), 20:3(n-6), 20:4(n-6) and 22:5(n-6) were increased, whereas those of 20:4(n-3) and 20:5(n-3) were reduced. Replacing 18:3(n-3) by 18:4(n-3) reduced the (n-6)/(n-3) ratio by approx. 30%, whereas replacing 18:2(n-6) by 18:3(n-6) increased the (n-6)/(n-3) ratio by approx. 2-fold. These findings indicated that delta 6-desaturase products were metabolized more readily than their precursors. Both products also competed for the subsequent metabolic enzymes. However, the n-6 fatty acids derived from 18:3(n-6) were incorporated more favourably into liver phospholipids than n-3 fatty acids derived from 18:4(n-3).     

Sex differences in n-3 and n-6 fatty acid metabolism in EFA-depleted rats

We studied the effect of sex on the distribution of long-chain n-3 and n-6 fatty acids in essential fatty acid-deficient rats fed gamma-linolenate (GLA) concentrate and/or eicosapentaenoate and docosahexaenoate-rich fish oil (FO). Male and female weanling rats were rendered essential fatty acid deficient by maintaining them on a fat-free semisynthetic diet for 8 weeks. Thereafter, animals of each sex were separated into three groups (n = 6) and given, for 2 consecutive days by gastric intubation, 4 g/kg body wt per day of GLA concentrate (containing 84% 18:2n-6), n-3 fatty acid-rich FO (containing 18% 20:5n-3 and 52% 22:6n-3), or an equal mixture of the two oil preparations (GLA + FO). The fatty acid distributions in plasma and liver lipids were then examined. GLA treatment increased the levels of C-20 and C-22 n-6 fatty acids in all lipid fractions indicating that GLA was rapidly metabolized. However, the increases in 20:3n-6 were less in females than those in males, while those in 20:4n-6 were greater, suggesting that the conversion of 20:3n-6 to 20:4n-6 was more active in female than in male rats. FO treatment increased the levels of 20:5n-3 and 22:6n-3 and reduced those of 20:4n-6. The increase in n-3 fatty acids was greater in females than that in males and the reduction in 20:4n-6 was smaller. Consequently, the sum of total long-chain EFAs incorporated was greater in females than that in males. The administration of n-3 fatty acids also reduced the ratio of 20:4n-6 to 20:3n-6 in GLA + FO-treated rats indicating that n-3 fatty acids inhibited the activity of delta-5-desaturase. However, this effect was not affected by the sex difference.     

Metabolism of polyunsaturated (n-3) fatty acids by monkey seminal vesicles: isolation and biosynthesis of omega-3 epoxides.

Monooxygenases of monkey seminal vesicles can metabolize arachidonic acid (20:4(n-6)) by w3-hydroxylation to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and eicosapentaenoic acid (20:5(n-3)) to 17,18-dihydroxyeicosatetraenoic acid (Oliw, E.H. (1989) J. Biol. Chem. 264, 17845-17853). The present study aimed to further characterize the oxygenation of (n-3) polyunsaturated fatty acids. 14C-Labelled 22:6(n-3), 20:5(n-3), 20:4-(n-3) and 18:3(n-3) were incubated with microsomes of seminal vesicles of the cynomolgus monkey, NADPH and a cyclooxygenase inhibitor, diclofenac, and the main metabolites were identified by capillary gas chromatography-mass spectrometry. 22:6(n-3) was slowly metabolized to 19,20-dihydroxy-4,7,10,13,16-docosapentaenoic acid, while 20:5(n-3), 20:4(n-3) and 18:3(n-3) were metabolized more efficiently to the corresponding w4,w3-diols. The w3 epoxides, which were obtained from 20:5(n-3) and 18:3(n-3), were isolated in the presence of an epoxide hydrolase inhibitor, 1(2)epoxy-3,3,3-trichloropropane, and the geometry of the epoxides was determined to be 17S, 18R and 15S, 16R, respectively. While 20:5(n-3) was metabolized almost exclusively to the epoxide and diol pair of metabolites, 18:3(n-3) was metabolized not only to the w3 epoxide and the corresponding diol, but also to the w2 alcohol, 17(R)-hydroxy-9,12,15-octadecatrienoic acid. 22:6(n-3) and 5,8,11,14-eicosatetraynoic acid inhibited the biosynthesis of 18(R)-HETE from arachidonic acid (IC50 0.16 and 0.14 mM, respectively). In comparison with 20:4 or 18:3(n-3), 18:1(n-9) and 22:5(n-6) appeared to be slowly metabolized by seminal monooxygenases, while 18:2(n-6) was converted to the w3 alcohol and to smaller amounts of the w2 alcohol (4:1). Together, the results indicate that the w3-hydroxylase and w3-epoxygenase enzyme(s) metabolize 20:4(n-6) and 20:5(n-3) almost exclusively to the w3(R) alcohol and the w3(R, S) epoxide, respectively, while longer and shorter fatty acids either are poor substrates or metabolized with a lesser degree of position specificity.     

Statin treatment alters serum n-3 and n-6 fatty acids in hypercholesterolemic patients

Statins are highly effective cholesterol-lowering drugs but may have broader effects on metabolism. This investigation examined effects of simvastatin on serum levels of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Subjects were 106 healthy adults with hypercholesterolemia randomly assigned to receive placebo or 40 mg simvastatin daily for 24 weeks. Serum fatty acids were analyzed by gas chromatography. Total fatty acid concentration fell 22% in subjects receiving simvastatin (P<.001), with similar declines across most fatty acids. However, concentrations of arachidonic acid (AA, 20:4n-6), eicosapentanoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were unchanged. Relative percentages of linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (LNA, 18:3n-3), decreased while AA and DHA increased (P's < or = .007). In addition, simvastatin increased the AA:EPA ratio from 15.5 to 18.8 (P<.01), and tended to increase the AA:DHA ratio (P=.053). Thus, simvastatin lowered serum fatty acid concentrations while also altering the relative percentages of important PUFAs.     

The role of n-3 fatty acids in gestation and parturition

Preterm birth is the most common cause of low infant birth weight and infant morbidity and mortality. Evidence from human and animal studies indicates that essential fatty acids of both the n-3 and n-6 series, and their eicosanoid metabolites, play important and modifiable roles in gestational duration and parturition, and n-3 fatty acid intake during pregnancy may be inadequate. Prostaglandins (PG) of the 2-series are involved in parturition and connective tissue remodeling associated with cervical maturation and rupture of membranes. In the absence of infections, preterm birth is characterized by lower reproductive tissue PG production and decreased inducible cyclooxygenase expression. Women who deliver prematurely have increased pools of n-6 fatty acid and decreased n-3 fatty acids, despite the lower PG production. Several human pregnancy supplementation trials with n-3 fatty acids have shown a significant reduction in the incidence of premature deliver and increased birth weight associated with increased gestational duration. Supplementation with long chain n-3 fatty acids such as docosahexaenoic acid may be useful in prolonging the duration of gestation in some high-risk pregnancies. Evidence presented in this review is discussed in terms of the roles of dietary n-3 and n-6 fatty acids in gestation and parturition, mechanisms by which they may influence gestational duration and the human trials suggesting that increased dietary long-chain n-3 fatty acids decrease the incidence of premature delivery.     

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.     

Cellular interactions between n-6 and n-3 fatty acids: a mass analysis of fatty acid elongation/desaturation, distribution among complex lipids, and conversion to eicosanoids.

The biologic effect of eicosanoids depends in large measure upon the relative masses in tissues of eicosanoids derived from the n-6 fatty acids, dihomogammalinolenic acid and arachidonic acid, and the n-3 fatty acid, eicosapentaenoic acid. Generation of this tissue balance is related to the relative cellular masses of these precursor fatty acids, the competition between them for entry into and release from cellular phospholipids, and their competition for the enzymes that catalyze their conversion to eicosanoids. In order to better understand these processes, we studied the cellular interactions of n-6 and n-3 fatty acids using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Unlike studies using cells with endogenous pools of n-6 and n-3 fatty acids, the use of EFD-1 cells enabled us to examine the metabolic fate of each family of fatty acids both in the presence and in the absence of the second family of fatty acids. Thus, the specific effects of one fatty acid family on the other could be directly assessed. In addition, we were able to replete the cells with dihomogammalinolenic acid (DHLA), arachidonic acid (AA), and eicosapentaenoic acid (EPA) of known specific activities; thus the masses of cellular DHLA, AA, and EPA, and their metabolites, PGE1, PGE2, and PGE3, respectively, could be accurately quantitated. The major findings of this study were: 1) n-6 fatty acids markedly stimulated the elongation of EPA to 22:5 whereas n-3 fatty acids inhibited the delta 5 desaturation of DHLA to AA and the elongation of AA to 22:4; 2) n-6 fatty acids caused a specific redistribution of cellular EPA from phospholipid to triacylglycerol; 3) n-3 fatty acids reduced the mass of DHLA and AA only in phosphatidylinositol whereas n-6 fatty acids reduced the mass of EPA to a similar extent in all cellular phospholipids; and 4) n-3 fatty acids caused an identical (33%) reduction in the bradykinin-induced release of PGE1 and PGE2, whereas n-6 fatty acids stimulated PGE3 release 2.3-fold. Together, these highly quantitative metabolic data increase our understanding of the regulation of both the cellular levels of DHLA, AA, and EPA, and their availability for eicosanoid synthesis. In addition, these findings provide a context for the effective use of these fatty acids in dietary therapies directed at modulation of eicosanoid production.     

The metabolism of (n-3) and (n-6) fatty acids and their oxygenation by platelet cyclooxygenase and lipoxygenase

Arachidonic acid is the principal unsaturated acid in most membrane lipids. Membrane lipids also contain a variety of other (n-6) and (n-3) fatty acids. The amounts of (n-6) and (n-3) fatty acids in membrane lipids can be modified by dietary fat change. Our studies show that long chain (n-6) and (n-3) acids are metabolized by platelet lipoxygenase and cyclooxygenase. When cells are exposed to various agonists, a variety of unsaturated fatty acids may be released. Our studies show that they have the potential of modifying physiological function both by mediating arachidonic acid metabolism and as direct precursors for oxygenated metabolites which themselves may interact with specific receptors to regulate biological processes.