Ethyl docosahexaenoate-associated decrease in fetal brain lipid peroxide production is mediated by activation of prostanoid and nitric oxide pathways

Previously we have shown that intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to pregnant rats resulted in decreased lipid peroxidation in the fetal brain, under a variety of conditions (S. Glozman, P. Green, E. Yavin, J. Neurochem. 70 (1998) 2482-2491). In the present study we examine the potential mechanisms to explain this effect. This was done by a pharmacological approach, utilizing brain slice preparations from Et-DHA treated or control rats in the presence of various agents and examining the formation of products in the tissue slices or incubation medium. Et-DHA treated brains produced 2-3-fold more prostanoids (PN) than control brains, indicating cyclooxygenase (COX) activation. Indomethacin at 50 microM inhibited PN formation and also abolished Et-DHA induced decrease in lipid peroxides, as evident by the levels of thiobarbituric acid reactive substances (TBARS) released in the medium. The phospholipase A2 inhibitors quinacrine and p-bromophenacyl bromide added at 0.1 mM concentration each to either slices from controls or Et-DHA treated fetal brains, decreased TBARS production. Et-DHA treated brains released 2.2-fold more nitric oxide (NO) than control brains and NO synthase (NOS) inhibitors abolished this effect. Increasing the concentration of NO by the addition of an NO donor greatly decreased the concentration of the TBARS in the medium. These results suggest that at least some of the effect of Et-DHA on decreased lipid peroxidation may be explained by a shift of oxygen species utilization via enzymatically regulated, therefore metabolically controlled, COX and NOS activities.     

Modulation of Fetal Rat Brain and Liver Phospholipid Content by Intraamniotic Ethyl Docosahexaenoate Administration

Abstract: In an attempt to elucidate the role of docosahexaenoic acid (DHA; 22:6n-3) in the developing brain, a method was devised whereby rapid enrichment of fetal brain and liver lipid with DHA was achieved. Fetal rats at 17 days of gestation were injected intraamniotically with ethyl docosahexaenoate (EtDHA). Control fetuses were administered ethyl oleate (EtOle). Brain lipid DHA content increased by almost 21% (p = 0.02) 3 days after EtDHA administration as compared with EtOle-injected fetuses, whereas liver lipid DHA content increased by almost 60% (p = 0.0002). At this time brain phosphatidylinositol content doubled, whereas phosphatidylserine (PS) content increased by >50% (p = 0.03). Increases in liver PS (+25.8%; p = 0.015) and sphingomyelin (+43.6%; p = 0.01) content were observed. A redistribution of total brain phospholipid (PL) DHA was observed following Et-DHA administration, resulting in a 56.4% increase in PS-DHA abundance (p < 0.05) and an 8.8% decrease in phosphatidylethanolamine-DHA abundance (p = 0.05). These results suggest modulation of fetal brain and liver PL and provide a method for enrichment of DHA content in discrete PLs during intrauterine life.     

Enhanced free radical scavenging and decreased lipid peroxidation in the rat fetal brain after treatment with ethyl docosahexaenoate

In order to explore possible mechanisms to explain previously observed decreases in fetal brain lipid peroxidation (LPO) following intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to near term fetuses, the hydroxyl radical trapping capacity of Et-DHA treated fetal brain preparations was compared to control ethyl oleate injected fetuses by electron spin resonance using 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) probe. Lipid extracts from control brains showed little hydroxyl radical scavenging activity, whereas those from the Et-DHA injected animals exhibited an almost 70% decrease in the amount of DMPO-OH adducts. A marked decrease (58%) in LPO formation was noticed in the Et-DHA treated animals compared to controls. The Et-DHA treatment related trapping capacity resided in the phospholipid fraction of the lipid extract, which was enriched in both docosahexaenoic acid and aminophospholipid contents. The decreased LPO production, as well as increased production of prostaglandin E(2) and nitric oxide by the fetal brain following Et-DHA administration, could be mimicked by a synthetic quinone possessing both hydroxyl radical producing and LPO propagation inhibiting properties. The data are consistent with the possibility that the neuroprotective effect of Et-DHA might be due to possible free radical scavenging ability of the brain tissue and interference with LPO propagation.     

Docosahexaenoic acid-deficient phosphatidyl serine and high alpha-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain.     

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.     

Effect of Docosahexaenoic Acid on the Synthesis of Phosphatidylserine in Rat Brain Microsomes and C6 Glioma Cells

Abstract: Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 ± 0.1%) when compared with control animals (15.0 ± 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [³H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10–40 µ M ) showed a significant increase in the synthesis of [³H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.     

Lipid Peroxides are Generated by the Fetal Rat Brain After Episodes of Global Ischemia in Utero

Complete arrest of maternal-fetal blood supply for up to 30 min caused a time-dependent increase in the endogenous levels of lipid peroxides (LPO) in fetal brain and liver extracts and fetal blood and amniotic fluids as indicated by the appearance of thiobarbituric acid reactive substances (TBARS), A steady increase of TBARS from 48.0 ± 2.2 pmol/g wet weight to 75.0 ± 5.6 pmol/g wet weight up to 30 min restriction was noticed in the fetal brain. The fetal liver TBARS values increased by approximately 69% after 5 min restriction and remained high, above the control level, for 30 min. After two days reperfusion following 30 min restriction, the TBARS levels in the fetal brain were 1.8 fold higher above the control, while those of the liver returned to control values. The levels of the lipid-soluble antioxidant -tocopherol were reduced by about 40% and 50% in the placenta and brain tissues after 5 min restriction, respectively. Slices of fetal brain incubated at 37°C in DMEM under oxygen in the presence of 50 M Fe²⁺ were able to generate LPO in a time- and tissue concentration-dependent manner. After 15 min incubation, about 6.3 fold increase in total TBARS levels could be measured in the presence of 50 M Fe²⁺, most of which was released in the medium. The iron chelator desferrioxamine (25 M) and the antioxidant -tocopherol (10 M) added to the incubation medium, each inhibited by about 88% TBARS production. After 20 min episode of ischemia, fetal brain slices released into the medium 138.5 ± 9.8 nmol/15 min/mg DNA compared to 75.9 ± 4.5 nmol/15 min/mg DNA released by the sham preparations. After 3 h reperfusion, brain slices from fetuses exposed to 20 min ischemia continued to produce TBARS above control levels, whereas those of brief ischemia (5 min) returned to control levels. The data indicate that the limited resistance of the fetal brain to brief, rather than prolonged, periods of ischemia, is likely due to a lack of free FA for LPO generation, rather than the levels of tissue lipid antioxidants.     

Chronic daily administration of ethyl docosahexaenoate protects against gerbil brain ischemic damage through reduction of arachidonic acid liberation and accumulation

Recently, we reported that dietary ethyl docosahexaenoate (Et-DHA) intake decreases the level of membrane arachidonic acid (AA), which reduces the generation of AA metabolites in ischemic gerbil brain. As an extended study, we further investigated the influence of the chronic administration of Et-DHA on free AA levels after ischemia. In addition, Na,K-ATPase activity, cation content, cerebral edema and brain damage were also evaluated. Weanling male gerbils were orally pretreated with either Et-DHA (200 mg/kg) or vehicle, once a day for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. Time-course analyses revealed that pretreatment with Et-DHA, compared with pretreatment with the vehicle, significantly decreased the brain's free AA levels during ischemia (5, 15 and 30 min) and after reperfusion (5, 10, 15 and 30 min), and attenuated the decline of Na,K-ATPase activity at examined time points. Pretreatment with Et-DHA significantly prevented an increase in Na(+) concentration and a decrease in K(+) concentration after 24 h of reperfusion, which resulted in lower cerebral water content. Reduced brain infarct volume and low animal mortality were also observed in Et-DHA-treated animals. These data suggest that the reduction of ischemia-induced AA liberation and accumulation by Et-DHA pretreatment may be attributable to (a) protection against the decline of Na,K-ATPase activity, (b) postischemic cerebral edema and brain damage and (c) animal mortality.     

n-3 Deficient and docosahexaenoic acid-enriched diets during critical periods of the developing prenatal rat brain.

The last period of the intrauterine life in the rat (embryonic day 17 to 21, ED17-ED21) is demarcated by an increase in brain and body weight and active neuronogenesis. During this period, a rapid accumulation of DHA (22:6 n-3), unparalleled to other fatty acids, takes place. The details of DHA rapid acquisition in the fetal brain were investigated after imposing a diet deficient in n-3 fatty acids (FA) as of ED1 and subsequently examining the distribution of DHA in major brain phospholipid (PL) classes on ED20, having added on ED15 a triglyceride (TG) mixture enriched up to 43% with DHA. The n-3 deficiency maintained for 19 days resulted at ED20 in more than 30% reduction of DHA in PL, which was counterbalanced by an increase of docosapentaenoic acid (DPA, 22:5 n-6). No effect on body weight, nor major changes in PL composition or other FA in fetal brain PL were observed. Feeding dams a DHA-TG diet on ED15 induced an immediate increase of DHA in maternal liver PL, followed by a subsequent increase of DHA in fetal liver PL, as well as in fetal brain PL.Thus the content of fetal brain DHA in n-3 deficient embryos could be restored within 48 hours. Dietary manipulation of fetal tissues is a rapid phenomenon and can be used to enrich DHA at critical periods of development in utero.     

Recovery of brain docosahexaenoate leads to recovery of spatial task performance

Infants fed vegetable oil-based formulas may have poorer visual function, lower cognitive scores and acquire learning tasks more slowly in comparison with those breast fed or those fed formulas supplemented with docosahexaenoate. The aim of the present study was to determine the reversibility of losses in brain function associated with the loss of brain DHA. Rats were fed very low or adequate levels of n-3 fatty acids through three generations. The n-3 fatty acid deficient animals of the F3 generation were then given an n-3 adequate diet containing alpha-linolenic and docosahexaenoic acids (DHA) at birth, weaning (3 weeks) or young adulthood (7 weeks). The spatial task performance of these animals returned to the n-3 adequate diet was then compared using the Morris water at two different ages, at 9 or 13 weeks. Our results indicate that animals repleted since birth or at weaning were able to achieve nearly the same level of brain DHA and spatial task performance as animals maintained for three generations on an n-3 adequate diet. In the case of young adult animals, the degree of DHA and behavioral performance recovery depended upon the duration of dietary repletion with substantial recovery in animals after 6 weeks but little recovery of function after two weeks. The significance of these findings is that they indicate that at least some of the adverse effects of DHA deficiency during neurodevelopment may be reversible with an n-3 fatty acid supplemented diet.     

Dietary α-Linolenic Acid Increases the Biosynthesis of the Choline Glycerophospholipids from [14C]CDPcholine in Rat Liver and Kidney But Not in Brain

The effect of feeding rats for 30 days with diets containing high levels of linoleic acid (sunflower oil, SO) or -linolenic acid (perilla oil, PO) was studied in the liver, kidney and brain. The PO group showed a higher labeling of choline glycerophospholipids (CGP) in liver and kidney but no difference with the SO group in ethanolamine glycerophospholipids (EGP) labeling. The brain displayed the lowest incorporation of both precursors and no difference between the two diets. Analyses of brain CGP and EGP fatty acid compositions showed that in the PO group the ratio n-6/n-3 was lower than in the SO group, mainly as a consequence of lower levels of n-6 fatty acids. The mole % of docosahexaenoate (DHA) in these lipids was the same for both groups and only triacylglycerols (TAG) displayed a higher DHA. Therefore, at least in the brain, the magnitude of fatty acid changes observed in CGP and EGP for the PO group does not affect the uptake/incorporation of the precursors into phospholipids.     

Versatile roles of docosahexaenoic acid in the prenatal brain: from pro- and anti-oxidant features to regulation of gene expression

Docosahexaenoic acid (DHA) is the most ubiquitous polyunsaturated fatty acid (FA) in brain tissue. It is selectively esterified to amino phospholipids (PL) and therefore it is highly prevalent at the cytofacial site of the plasma membrane where it may specifically participate in intracellular events. A highly selective DHA accumulation prior to birth is the result of maternal supply via the placenta through a bio-magnification process. Supplements of DHA via the intra-amniotic route to the fetal rat increase brain DHA levels and also confer neuroprotection to fetuses subjected to global ischemic stress. The protective effect has been attributed to an enhanced free radical scavenging capacity of DHA. Dietary deprivation of linolenic acid (LNA) during the perinatal life on the other hand, resulted in losses of DHA from cerebral PLs [M. Schiefermeier, E. Yavin, n-3 deficient and DHA-enriched diets during critical periods of the developing prenatal rat brain, J. Lipid Res. 43 (2002) 124-131]. LNA deprivation also caused changes in a number of gene markers the identification of which was attained by a labor-intensive suppression subtractive hybridization protocol using mRNA from 2-week-old postnatal brains [E. Yakubov, P. Dinerman, F. Kuperstein, S. Saban, E. Yavin, Improved representation of gene markers on microarray by PCR-select subtracted cDNA targets, Mol. Brain Res. 137 (2005) 110-118]. Most notable was a remarkable elevation of dopamine (DA) receptor (D1 and D2) genes as evaluated by quantitative RT-PCR, SDS-PAGE gel electrophoresis and immunochemical staining [F. Kuperstein, E. Yakubov, P. Dinerman, S. Gil, R. Eylam, N. Salem Jr., E. Yavin, Overexpression of dopamine receptor genes and their products in the postnatal rat brain following maternal n-3 FA dietary deficiency, J. Neurochem. 95 (2005) 1550-1562]. Over-expression of DA receptors has been attributed to a compensatory mechanism resulting from impairment in DA neurotransmitter production, storage and processing. In conclusion, DHA is a versatile molecule with a wide range of actions spanning from participation in cellular oxidative processes and intracellular signaling to modulatory roles in gene expression and growth regulation.     

Metabolic conversion of intra-amniotically-injected deuterium-labeled essential fatty acids by fetal rats following maternal n-3 fatty acid deficiency

Accumulation of polyunsaturated fatty acids (PUFA) in the fetal brain is accomplished predominantly via a highly selective flow of docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, AA) through the placenta. Little is known regarding the endogenous capability of the fetus to generate its own DHA and AA from lower homologues such as linolenic (18:3n-3, ALA) and linoleic (18:2n-6, LA) acids, respectively. Deuterium-labeled d5-ALA and d5-LA at millimolar concentrations were injected directly into the amniotic fluid in order to investigate maternal-independent metabolic conversion of the stable isotopes in brain and liver of the fetus near delivery. After 48 h under adequate maternal diet, the levels of d5-ALA metabolites in the fetal brain and fetal liver were 45 ± 2.2 pmol/mg and 86 ± 4 pmol/mg of which 79% and 63.6% were comprised of d5-DHA. At this time point, incorporation of d5-LA metabolites was 103 ± 5 pmol/mg and 772 ± 46 pmol/mg for brain and liver, of which 50% and 30% were comprised of d5-AA. Following sustained maternal dietary ALA deficiency, the levels of total d5-ALA derived metabolites in the fetal brain and fetal liver were increased to 231 pmol/mg and 696 pmol/mg of which 71% and 26% were comprised of d5-DHA. From the time course and relative rates of d5-ALA precursor displacement by d5-DHA in cellular phosphoglycerides, it is concluded that the fetal rat brain can generate its own DHA from its d5-ALA precursors particularly under dietary stress.     

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.     

Behavioral deficits associated with dietary induction of decreased brain docosahexaenoic acid concentration

Docosahexaenoic acid (DHA), an n-3 fatty acid, is rapidly deposited during the period of rapid brain development. The influence of n-3 fatty acid deficiency on learning performance in adult rats over two generations was investigated. Rats were fed either an n-3 fatty acid-adequate (n-3 Adq) or -deficient (n-3 Def) diet for three generations (F1-F3). Levels of total brain n-3 fatty acids were reduced in the n-3 Def group by 83 and 87% in the F2 and F3 generations, respectively. In the Morris water maze, the n-3 Def group showed a longer escape latency and delayed acquisition of this task compared with the n-3 Adq group in both generations. The acquisition and memory levels of the n-3 Def group in the F3 generation seemed to be lower than that of the F2 generation. The 22:5n-6/22:6n-3 ratio in the frontal cortex and dams' milk was markedly increased in the n-3 Def group, and this ratio was significantly higher in the F3 generation compared with the F2 generation. These results suggest that learning and cognitive behavior are related to brain DHA status, which, in turn, is related to the levels of the milk/dietary n-3 fatty acids.     

Omega-3 fatty acid deficiency increases constitutive pro-inflammatory cytokine production in rats: Relationship with central serotonin turnover

Omega-3 (n-3) fatty acid deficiency, elevated inflammatory signaling, and central serotonin (5-HT) turnover have separately been implicated in the pathophysiology of major depressive disorder (MDD). In the present study we investigated the interrelationship between n-3 fatty acid status, pro-inflammatory signaling activity, and central 5-HT turnover in vivo. Rats were fed diets with or without the n-3 fatty acid precursor α-linolenic acid (ALA) during perinatal development (E0-P100), and a subset of rats fed the ALA− diet were switched to the ALA+ diet post-weaning (P21-P100, repletion). In adulthood (P100), plasma interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), and C-reactive protein (CRP) levels were measured. Additionally, indices of liver n-6 fatty acid biosynthesis, erythrocyte fatty acid composition, and regional brain monoamine turnover were determined. Indices of liver delta-6 desaturase activity were up-regulated in n-3-deficient rats, and were associated with greater erythrocyte membrane arachidonic acid (AA, 20:4 n-6) composition. Plasma IL-6 (p=0.001), TNFα (p=0.02), and CRP (p=0.001) concentrations were significantly greater in n-3-deficient rats relative to controls. The 5-HIAA/5-HT ratio was significantly greater in frontal cortex, hypothalamus, and ventral striatum of n-3-deficient rats relative to controls. Changes in membrane n-3 and n-6 fatty acid composition, elevations in plasma IL-6 and TNFα, and increased central 5-HT turnover were all prevented by normalization of n-3 fatty acid status. Erythrocyte docosahexaenoic acid (DHA, 22:6 n-3) was inversely correlated, and AA and the AA/DHA and AA/eicosapentaenoic acid ratios were positively correlated, with plasma IL-6, TNFα, and CRP levels. Plasma IL-6 levels were positively correlated with 5-HIAA/5-HT ratios in all brain regions. These preclinical data provide evidence for a functional link between n-3 fatty acid deficiency, elevated peripheral inflammatory signaling, and increased central 5-HT turnover.     

Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats

Dietary docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) are considered important for maintaining normal heart and brain function, but little EPA is found in brain, and EPA cannot be elongated to DHA in rat heart due to the absence of elongase-2. Ingested EPA may have to be converted in the liver to DHA for it to be fully effective in brain and heart, but the rate of conversion is not agreed on. This rate was determined in male adult rats fed a standard n-3 PUFA, containing diet by infusing unesterified albumin-bound [U-¹³C]EPA intravenously for 2 h and measuring esterified [¹³C]labeled PUFAs in arterial plasma lipoproteins, as well as the specific activity of unesterified plasma EPA. Whole-body (presumably hepatic) synthesis secretion rates from circulating unesterified EPA, calculated from peak first derivatives of plasma esterified concentration × volume curves, equaled 2.61 μmol/day for docosapentaenoic acid (22:5n-3) and 5.46 μmol/day for DHA. The DHA synthesis rate was 24-fold greater than the reported brain DHA consumption rate in rats. Thus, dietary EPA could help to maintain brain and heart DHA homeostasis because it is converted at a relatively high rate in the liver to circulating DHA.     

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.     

Omega-3 fatty acid deficiency during brain maturation reduces neuronal and behavioral plasticity in adulthood

Omega-3-fatty acid DHA is a structural component of brain plasma membranes, thereby crucial for neuronal signaling; however, the brain is inefficient at synthesizing DHA. We have asked how levels of dietary n-3 fatty acids during brain growth would affect brain function and plasticity during adult life. Pregnant rats and their male offspring were fed an n-3 adequate diet or n-3 deficient diets for 15 weeks. Results showed that the n-3 deficiency increased parameters of anxiety-like behavior using open field and elevated plus maze tests in the male offspring. Behavioral changes were accompanied by a level reduction in the anxiolytic-related neuropeptide Y-1 receptor, and an increase in the anxiogenic-related glucocorticoid receptor in the cognitive related frontal cortex, hypothalamus and hippocampus. The n-3 deficiency reduced brain levels of docosahexaenoic acid (DHA) and increased the ratio n-6/n-3 assessed by gas chromatography. The n-3 deficiency reduced the levels of BDNF and signaling through the BDNF receptor TrkB, in proportion to brain DHA levels, and reduced the activation of the BDNF-related signaling molecule CREB in selected brain regions. The n-3 deficiency also disrupted the insulin signaling pathways as evidenced by changes in insulin receptor (IR) and insulin receptor substrate (IRS). DHA deficiency during brain maturation reduces plasticity and compromises brain function in adulthood. Adequate levels of dietary DHA seem crucial for building long-term neuronal resilience for optimal brain performance and aiding in the battle against neurological disorders.