An extraordinary degree of structural specificity is required in neural phospholipids for optimal brain function: n-6 docosapentaenoic acid substitution for docosahexaenoic acid leads to a loss in spatial task performance

This study was conducted to determine whether provision of preformed dietary docosapentaenoic acid (DPAn-6) can replace docosahexaenoic acid (DHA) for brain function as assessed by spatial task performance. A newly modified artificial rearing method was employed to generate n-3 fatty acid-deficient rats. Newborn pups were separated from their mothers at 2 days of age and given artificial rat milk containing linoleic acid (LA), or LA supplemented with 1% DHA (DHA), 1% DPAn-6 (DPA) or 1% DHA plus 0.4% DPAn-6 (DHA/DPA). The animals were then weaned onto similar pelleted diets. At adulthood, behavioural tasks were administered and then the brains were collected for fatty acid analysis. The LA and DPA groups showed a lower (63-65%) brain DHA than the dam-reared, DHA and DHA/DPA groups and this loss was largely compensated for by an increase in brain DPAn-6. The brain fatty acid composition in the DPA group was the same as that in the LA group at adulthood. In the Morris water maze, the LA and DPA groups exhibited a longer escape latency than the dam-reared and DHA groups and had a defect in spatial retention. In conclusion, DPAn-6 could not replace DHA for brain function, indicating a highly specific structural requirement for DHA.     

Regulation of rat brain polyunsaturated fatty acid (PUFA) metabolism during graded dietary n-3 PUFA deprivation

Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation may elucidate dietary regulation of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (α-LNA). Compared with control diet (4.6% α-LNA), plasma DHA fell significantly at 1.7% dietary α-LNA while brain DHA remained unchanged down to 0.8% α-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA₂ and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA₂, sPLA₂ and COX-2 activities were increased at or below 0.8% dietary α-LNA, possibly in response to elevated brain DPAn-6. In summary, homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary DHA despite reduced plasma DHA, when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA₂ and COX-1 activities may reduce brain DHA loss.     

Comparison of bloodstream fatty acid composition from African-American women at gestation, delivery, and postpartum

Our aim was to examine the docosahexaenoic acid (DHA; 22:6n-3) status of pregnant African-American women reporting to the antenatal clinic at Wayne State University in a longitudinal study design. Fatty acid compositions of plasma and erythrocyte total lipid extracts were determined and food frequency surveys were administered at 24 weeks of gestation, delivery, and 3 months postpartum for participants (n = 157). DHA (mean +/- SD) in the estimated total circulating plasma was similar at gestation (384 +/- 162 mg) and delivery (372 +/- 155 mg) but was significantly lower at 3 months postpartum (178 +/- 81 mg). The relative weight percentage of DHA and docosapentaenoic acid n-6 (DPAn-6; 22:5n-6) decreased postpartum, whereas their respective metabolic precursors, eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (AA; 20:4n-6), increased. Similar results were found in erythrocytes. Dietary intake of DHA throughout the study was estimated at 68 +/- 75 mg/day. The relative amounts of circulating DHA and DPAn-6 were increased during pregnancy compared with 3 months postpartum, possibly via increased synthesis from EPA and AA. The low dietary intake and blood levels of DHA in this population compared with others may not support optimal fetal DHA accretion and subsequent neural development.     

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.     

The delta 6 desaturase knock out mouse reveals that immunomodulatory effects of essential n-6 and n-3 polyunsaturated fatty acids are both independent of and dependent upon conversion

Typically fatty acids (FA) exert differential immunomodulatory effects with n-3 [α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and n-6 [linoleic acid (LA) and arachidonic acid (AA)] exerting anti- and pro-inflammatory effects, respectively. This over-simplified interpretation is confounded by a failure to account for conversion of the parent FA (LA and ALA) to longer-chain bioactive products (AA and EPA/DHA, respectively), thereby precluding discernment of the immunomodulatory potential of specific FA. Therefore, we utilized the Δ6-desaturase model, wherein knockout mice (D6KO) lack the Fads2 gene encoding for the rate-limiting enzyme that initiates FA metabolism, thereby providing a model to determine specific FA immunomodulatory effects. Wild-type (WT) and D6KO mice were fed one of four isocaloric diets differing in FA source (9 weeks): corn oil (LA-enriched), arachidonic acid single cell oil (AA-enriched), flaxseed oil (ALA-enriched) or menhaden fish oil (EPA/DHA-enriched). Splenic mononuclear cell cytokine production in response to lipopolysaccharide (LPS), T-cell receptor (TCR) and anti-CD40 stimulation was determined. Following LPS stimulation, AA was more bioactive compared to LA, by increasing inflammatory cytokine production of IL-6 (1.2-fold) and TNFα (1.3-fold). Further, LPS-stimulated IFNγ production in LA-fed D6KO mice was reduced 5-fold compared to LA-fed WT mice, indicating that conversion of LA to AA was necessary for cytokine production. Conversely, ALA exerted an independent immunomodulatory effect from EPA/DHA and all n-3 FA increased LPS-stimulated IL-10 production versus LA and AA. These data definitively identify specific immunomodulatory effects of individual FA and challenge the simplified view of the immunomodulatory effects of n-3 and n-6 FA.     

Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-¹³C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10⁻³ min⁻¹, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Gestational age dependent changes of the fetal brain, liver and adipose tissue fatty acid compositions in a population with high fish intakes

IntroductionThere are no data on the intrauterine fatty acid (FA) compositions of brain, liver and adipose tissue of infants born to women with high fish intakes.Subjects and methodsWe analyzed the brain (n=18), liver (n=14) and adipose tissue (n=11) FA compositions of 20 stillborn infants with different gestational ages (range 8–38 weeks) born to Tanzanian women with low linoleic acid (LA) intakes and high intakes of docosahexaenoic (DHA) and arachidonic (AA) acids from local fish.Results and discussionWith advancing gestation, brain saturated-FA (SAFA; in g/100 g FA), polyunsaturated-FA (PUFA), DHA, 20:3ω6, 22:4ω6 and 22:5ω6 increased, while monounsaturated-FA (MUFA), 20:3ω9, 22:3ω9 and AA decreased. Decreasing brain AA might be caused by increasing AA-metabolism to 20:3ω6, 22:4ω6 and 22:5ω6. In the liver, SAFA, PUFA and LA increased, while MUFA decreased with gestation. The steep increase of (mostly de novo synthesized) SAFA in adipose tissue coincided with relative decreases of MUFA, PUFA, DHA, LA and AA with advancing gestation. Compared to Western infants, the currently studied African infants had higher DHA, lower AA, and a higher DHA/AA-ratio in brain and adipose tissue, while the LA content of adipose tissue was lower.ConclusionThe low LA and high DHA and AA intakes by the mothers of these infants might support optimal α-linolenic (ALA) vs. LA competition for Δ5D and Δ6D-activities and DHA vs. AA antagonism. Conversely, the Western diet, characterized by high LA and lower DHA and AA intakes, might disturb these evolutionary conserved mechanisms aiming at an optimal ω3/ω6-balance.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Peroxisome proliferator-activated receptor alpha is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.     

Upregulated mRNA expression of desaturase and elongase,two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

Background  

Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed.     

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).     

Interactions between the APOA5 -1131T>C and the FEN1 10154G>T polymorphisms on ��6 polyunsaturated fatty acids in serum phospholipids and coronary artery disease

We determined the contribution of the combination of FEN1 10154G>T with the most significant association in the analysis of plasma arachidonic acid (AA, 20:4ω6) and the APOA5-1131T>C on phospholipid ω6PUFA and coronary artery disease (CAD). Patients with CAD (n = 807, 27–81 years of age) and healthy controls (n = 1123) were genotyped for FEN1 10154G>T and APOA5-1131T>C. We found a significant interaction between these two genes for CAD risk (P = 0.007) adjusted for confounding factors. APOA5-1131C allele carriers had a higher CAD risk [odds ratio (OR):1.484, 95% confidence interval (CI):1.31–1.96; P = 0.005] compared with APOA5-1131TT individuals in the FEN1 10154GG genotype group but not in the FEN1 10154T allele group (OR:1.096, 95%CI:0.84–1.43; P = 0.504). Significant interactions between these two genes were also observed for the AA proportion (P = 0.04) and the ratio of AA/linoleic acid (LA, 18:2ω6) (P = 0.004) in serum phospholipids of controls. The APOA5-1131C allele was associated with lower AA (P = 0.027) and AA/LA (P = 0.014) only in controls carrying the FEN1 10154T allele. In conclusion, the interaction between these genes suggests that the FEN1 10154T variant allele decreases AA and AA/LA in the serum phospholipids of carriers of the APOA5-1131C allele, but contributes no significant increase in CAD risk for this population subset despite their increased triglylcerides and decreased apoA5.     

Dietary Docosahexaenoic Acid Supplementation Modulates Hippocampal Development in the Pemt?/? Mouse

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt^−/− mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt^−/− mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt^−/− dams fed the control diet had 25–50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt^−/− phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt^−/− mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.     

Biogenic Synthesis, Purification, and Chemical Characterization of Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)

Enzymatically oxygenated derivatives of the ω-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197–1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677–14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S™ oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.Enzymatically formed oxygenation products of C-20 and C-22 long chain polyunsaturated fatty acids (LC-PUFAs),⁴ have important biological roles in inflammation, allergies, and blood clotting and are thus believed to have therapeutic potential in several chronic immune diseases (1–10) Several biologically important products of cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA), and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) have been described (4, 11, 12). Proinflammatory oxylipins, such as leukotrienes and some prostaglandins, are derived from ARA, an ω-6 fatty acid. Interestingly, the same fatty acid also serves as a precursor to anti-inflammatory or proresolution molecules like lipoxins (13, 14). Stable analogues of lipoxins are being developed as drugs for asthma and other inflammatory airway diseases (15, 16). Oxylipins derived from ω-3 fatty acids, such as DHA and EPA, known as resolvins, are primarily anti-inflammatory in nature (17). EPA acts as a precursor to the E-series resolvins that have shown potential in the treatment of colitis, arthritis, and periodontitis (18–20). The resolvins of the D-series derived from DHA are useful as neuroprotective agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or neuroprotectin D1 is a resolvin that is formed endogenously in the human brain and eye and is believed to exert its protective effect against cell injury-induced oxidative stress (21–23).The main enzymes responsible for the production of these oxygenated LC-PUFA products are primarily lipoxygenases and, in addition, cyclo-oxygenases and cytochromes P450. These enzymes produce oxylipins via transcellular activity, often involving multiple cell types (24). This activity mainly results in mono-, di-, and tri-hydroxylation products of fatty acids that have varying potencies, depending on the exact structure of the compound. Lipoxygenases are non-heme, iron-containing dioxygenases that catalyze the regioselective and enantioselective oxidation of polyunsaturated fatty acids containing one or more cis,cis-1,4-pentadienoic moieties to give the corresponding hydroperoxy derivatives (25, 26). We thus considered that, in addition to DHA and EPA, other C-22 PUFAs containing such methylene interrupted double bonds may also be substrates for lipoxygenases and that resulting products may have anti-inflammatory activity similar to DHA-derived resolvins. DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present in algal oils, and recent studies have demonstrated that this fatty acid has anti-inflammatory activities in vitro and, in conjunction with DHA, also has anti-inflammatory activity in vivo.⁵ Also, it has been suggested that a combination of DHA and DPAn-6 could be a beneficial natural therapy in neuroinflammatory conditions like Alzheimer disease. Specifically, in a 3×Tg-AD mouse model of Alzheimer disease, DPAn-6 was shown to reduce levels of early stage phospho-Tau epitopes, which in turn correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a putative Tau kinase (27). Although the precise mechanism of action of DPAn-6 in these inflammatory milieus is not known, it suggests a possible role for oxylipin products of DPAn-6 in resolution of inflammation. Also, another LC-PUFA, DPAn-3 (cis-7,10,13,16,19-docosapentaenoic acid) usually present along with DHA and EPA in marine oils is known to be a potent inhibitor of platelet aggregation (28–30). In addition, this LC-PUFA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 (vascular endothelial-cell growth factor receptor 2) expression. Angiogenesis is known to contribute to tumor growth, inflammation, and microangiopathy, again pointing to the possibility that anti-inflammatory activity of DPAn-3 might be mediated through resolvin-like products as in the case of DHA and EPA (31).The purpose of this research was to determine whether oxylipins are formed from the C-22 LC-PUFAs, DPAn-6 and DPAn-3, by lipoxygenase activity; to compare them to products formed from DHA; to chemically characterize products; to purify key oxylipin products from the DPAn-6/15-lipoxygenase reaction; and to test whether these compounds have resolvin-like anti-inflammatory activity. This research also sets the stage for preparation and isolation of a wide range of other C-22 oxylipins that could be evaluated as potential anti-inflammatory compounds.     

Long-chain conversion of [13C]linoleic acid and alpha-linolenic acid in response to marked changes in their dietary intake in men

We studied the long-chain conversion of [U-13C]alpha-linolenic acid (ALA) and linoleic acid (LA) and responses of erythrocyte phospholipid composition to variation in the dietary ratios of 18:3n-3 (ALA) and 18:2n-6 (LA) for 12 weeks in 38 moderately hyperlipidemic men. Diets were enriched with either flaxseed oil (FXO; 17 g/day ALA, n=21) or sunflower oil (SO; 17 g/day LA, n=17). The FXO diet induced increases in phospholipid ALA (>3-fold), 20:5n-3 [eicosapentaenoic acid (EPA), >2-fold], and 22:5n-3 [docosapentaenoic acid (DPA), 50%] but no change in 22:6n-3 [docosahexanoic acid (DHA)], LA, or 20:4n-6 [arachidonic acid (AA)]. The increases in EPA and DPA but not DHA were similar to those in subjects given the SO diet enriched with 3 g of EPA plus DHA from fish oil (n=19). The SO diet induced a small increase in LA but no change in AA. Long-chain conversion of [U-13C]ALA and [U-13C]LA, calculated from peak plasma 13C concentrations after simple modeling for tracer dilution in subsets from the FXO (n=6) and SO (n=5) diets, was similar but low for the two tracers (i.e., AA, 0.2%; EPA, 0.3%; and DPA, 0.02%) and varied directly with precursor concentrations and inversely with concentrations of fatty acids of the alternative series. [13C]DHA formation was very low (<0.01%) with no dietary influences.