Serotoninergic neurotransmission is affected by n-3 polyunsaturated fatty acids in the rat

We explored the effects of chronic alpha-linolenic acid dietary deficiency on serotoninergic neurotransmission. In vivo synaptic serotonin (5-HT) levels were studied in basal and pharmacologically stimulated conditions using intracerebral microdialysis in the hippocampus of awake 2-month-old rats. We also studied the effects of reversion of the deficient diet on fatty acid composition and serotoninergic neurotransmission. A balanced (control) diet was supplied to deficient rats at different stages of development, i.e. from birth, 7, 14 or 21 days of age. We demonstrated that chronic n-3 polyunsaturated fatty acid dietary deficiency induced changes in the synaptic levels of 5-HT both in basal conditions and after pharmacological stimulation with fenfluramine. Higher levels of basal 5-HT release and lower levels of 5-HT-stimulated release were found in deficient than in control rats. These neurochemical modifications were reversed by supply of the balanced diet provided at birth or during the first 2 weeks of life through the maternal milk, whereas they persisted if the balanced diet was given from weaning (at 3 weeks of age). This suggests that provision of essential fatty acids is durably able to affect brain function and that this is related to the developmental stage during which the deficiency occurs.     

Chronic nutritional deprivation of n-3 α-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n-3) were markedly reduced in brains of third-generation (F3) rats nutritionally deprived of alpha-linolenic acid (18 : 3 n-3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n-6), an arachidonic acid (AA, 20 : 4 n-6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3-generation n-3-deficient rats, compared with control concentrations. Brain DPA-CoA could be measured in the deprived but not control rats, and AA-CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA- and DHA-selective enzymes, respectively. Competition among n-3 and n-6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n-3 deprivation), or conversion to bioactive eicosanoids and other metabolites.     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.     

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.     

Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation

Rates of conversion of alpha-linolenic acid (alpha-LNA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3) by the mammalian brain and the brain's ability to upregulate these rates during dietary deprivation of n-3 polyunsaturated fatty acids (PUFAs) are unknown. To answer these questions, we measured conversion coefficients and rates in post-weaning rats fed an n-3 PUFA deficient (0.2% alpha-LNA of total fatty acids, no DHA) or adequate (4.6% alpha-LNA, no DHA) diet for 15 weeks. Unanesthetized rats in each group were infused intravenously with [1-(14)C]alpha-LNA, and their arterial plasma and microwaved brains collected at 5 minutes were analyzed. The deficient compared with adequate diet reduced brain DHA by 37% and increased brain arachidonic (20:4n-6) and docosapentaenoic (22:5n-6) acids. Only 1% of plasma [1-(14)C]alpha-LNA entering brain was converted to DHA with the adequate diet, and conversion coefficients of alpha-LNA to DHA were unchanged by the deficient diet. In summary, the brain's ability to synthesize DHA from alpha-LNA is very low and is not altered by n-3 PUFA deprivation. Because the liver's reported ability is much higher, and can be upregulated by the deficient diet, DHA converted by the liver from circulating alphaLNA is the source of the brain's DHA when DHA is not in the diet.     

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.     

Docosahexaenoic acid promotes neurite growth in hippocampal neurons

Docosahexanoic acid (22:6n-3; DHA) deficiency during development is associated with impairment in learning and memory, suggesting an important role of DHA in neuronal development. Here we provide evidence that DHA promotes neuronal differentiation in rat embryonic hippocampal primary cultures. DHA deficiency in vitro was spontaneously induced by culturing hippocampal cells in chemically defined medium. DHA supplementation improved DHA levels to values observed in freshly isolated hippocampus. We found that DHA supplementation in culture increased the population of neurons with longer neurite length per neuron and with higher number of branches. However, supplementation with arachidonic, oleic or docosapentaenoic acid did not have any effect, indicating specificity of the DHA action on neurite growth. Furthermore, hippocampal cultures obtained from n-3 fatty acid deficient animals contained a lower DHA level and a neuronal population with shorter neurite length per neuron in comparison to those obtained from animals with adequate n-3 fatty acids. DHA supplementation to the deficient group recovered the neurite length to the level similar to n-3 fatty acid adequate cultures. Our data demonstrates that DHA uniquely promotes neurite growth in hippocampal neurons. Inadequate neurite development due to DHA deficiency may contribute to the cognitive impairment associated with n-3 fatty acid deficiency.     

Thematic Review Series: Living History of Lipids: Discovery of essential fatty acids

Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research.     

In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

Dietary n-6 PUFA deprivation for 15 weeks reduces arachidonic acid concentrations while increasing n-3 PUFA concentrations in organs of post-weaning male rats

Few studies have examined effects of feeding animals a diet deficient in n-6 polyunsaturated fatty acids (PUFAs) but with an adequate amount of n-3 PUFAs. To do this, we fed post-weaning male rats a control n-6 and n-3 PUFA adequate diet and an n-6 deficient diet for 15 weeks, and measured stable lipid and fatty acid concentrations in different organs. The deficient diet contained nutritionally essential linoleic acid (LA,18:2n-6) as 2.3% of total fatty acids (10% of the recommended minimum LA requirement for rodents) but no arachidonic acid (AA, 20:4n-6), and an adequate amount (4.8% of total fatty acids) of α-linolenic acid (18:3n-3). The deficient compared with adequate diet did not significantly affect body weight, but decreased testis weight by 10%. AA concentration was decreased significantly in serum (− 86%), brain (− 27%), liver (− 68%), heart (− 39%), testis (− 25%), and epididymal adipose tissue (− 77%). Eicosapentaenoic (20:5n-3) and docosahexaenoic acid (22:6n-3) concentrations were increased in all but adipose tissue, and the total monounsaturated fatty acid concentration was increased in all organs. The concentration of 20:3n-9, a marker of LA deficiency, was increased by the deficient diet, and serum concentrations of triacylglycerol, total cholesterol and total phospholipid were reduced. In summary, 15 weeks of dietary n-6 PUFA deficiency with n-3 PUFA adequacy significantly reduced n-6 PUFA concentrations in different organs of male rats, while increasing n-3 PUFA and monounsaturated fatty acid concentrations. This rat model could be used to study metabolic, functional and behavioral effects of dietary n-6 PUFA deficiency.     

Abnormal n-6 fatty acid metabolism in cystic fibrosis is caused by activation of AMP-activated protein kinase

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.     

Metabolism of polyunsaturated fatty acids by larvae of the waxmoth,Galleria mellonella

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio-high-pressure liquid chromatography and radio-gas-liquid chromatography. In addition to undergoing β-oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.     

Influence of phospholipid long chain polyunsaturated fatty acid composition on neonatal rat cardiomyocyte function in physiological conditions and during glucose-free hypoxia-reoxygenation

There is evidence that dietary polyunsaturated fatty acids (PUFA) may protect against cardiovascular diseases, but the involvement of the cardiac muscle cell in this beneficial action remain largely unknown. The present study compared the respective influence of n-3 and n-6 PUFA on the function of cultured neonatal rat cardiomyocytes (CM). Cells were grown for 4 days in media enriched either n-3 (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) or n-6 (arachidonic acid, AA) PUFA. The PUFA n-6/n-3 ratio in the phospholipids was close to 1 and 20 in the n-3 and n-6 cells, respectively. The transmembrane potentials were recorded using microelectrodes and the contractions were monitored with a photoelectric device. In physiological conditions, the increase of n-6 PUFA level in the phospholipids resulted in a significant decrease in the maximal rate of initial depolarization (–16%). In opposition, the action potential amplitude and duration were not altered, and the cell contractio n outline was not affected. Ischemia was simulated in vitro using a substrate-free, hypoxia-reoxygenation procedure in a specially designed gas-flow chamber. The progressive loss of electrical activity induced by the substrate-free, hypoxic treatment was affected by the n-6/n-3 ratio, since the n-6 rich CM displayed a slower depression of the AP amplitude and duration parameters. Conversely, the recovery of the resting potential (MDP) during reoxygenation was faster in n-3 CM, whereas the recovery of the contraction parameters was unaffected by the fatty acid composition of the cells. These results suggested that, in physiological conditions, the modification of long chain PUFA balance in the phospholipids of cardiac muscle cells may modulate the initial AP upstroke, which is governed by sodium channels. Moreover, the presence of n-3 PUFA appeared to accelerate the electrical depression during substrate-free hypoxia but in turn to allow a faster recovery upon reoxygenation. (Mol Cell Biochem 175: 253–262, 1997)     

Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids

Male rat pups (21 days old) were placed on a diet deficient in n-3 polyunsaturated fatty acids (PUFAs) or on an n-3 PUFA adequate diet containing alpha-linolenic acid (alpha-LNA; 18 : 3n-3). After 15 weeks on a diet, [4,5-3H]docosahexaenoic acid (DHA; 22 : 6n-3) was injected into the right lateral cerebral ventricle, and the rats were killed at fixed times over a period of 60 days. Compared with the adequate diet, 15 weeks of n-3 PUFA deprivation reduced plasma DHA by 89% and brain DHA by 37%; these DHA concentrations did not change thereafter. In the n-3 PUFA adequate rats, DHA loss half-lives, calculated by plotting log10 (DHA radioactivity) against time after tracer injection, equaled 33 days in total brain phospholipid, 23 days in phosphatidylcholine, 32 days in phosphatidylethanolamine, 24 days in phosphatidylinositol and 58 days in phosphatidylserine; all had a decay slope significantly greater than 0 (p < 0.05). In the n-3 PUFA deprived rats, these half-lives were prolonged twofold or greater, and calculated rates of DHA loss from brain, Jout, were reduced. Mechanisms must exist in the adult rat brain to minimize DHA metabolic loss, and to do so even more effectively in the face of reduced n-3 PUFA availability for only 15 weeks.     

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

The conversion of the plant-derived omega-3 (n-3) α-linolenic acid (ALA, 18:3n-3) to the long-chain eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) can be increased by ALA sufficient diets compared to ALA deficient diets. Diets containing ALA above an optimal level result in no further increase in DHA levels in animals and humans. The present study evaluates means of maximizing plasma DHA accumulation by systematically varying both linoleic acid (LA, 18:2n-6) and ALA dietary level. Weanling rats were fed one of 54 diets for three weeks. The diets varied in the percentage of energy (en%) of LA (0.07–17.1 en%) and ALA (0.02–12.1 en%) by manipulating both the fat content and the balance of vegetable oils. The peak of plasma phospholipid DHA (>8% total fatty acids) was attained as a result of feeding a narrow dietary range of 1–3 en% ALA and 1–2 en% LA but was suppressed to basal levels (～2% total fatty acids) at dietary intakes of total polyunsaturated fatty acids (PUFA) above 3 en%. We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA is low (<3 en%).     

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 M isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 M, compared with no fatty acid addition (from 99.0 ±: 1.0% to 30.7 ± 5.2% (p < 0.05) for DHA and 23.8 ± 2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7 ± 1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r_ss) of atrial myocytes compared with no addition of fatty acid (control) (from r_ss = 0.203 ±0.004 to 0.159 ± 0.004 (p < 0.01) for DHA, 0.166 ± 0.001 (p < 0.01) for EPA and 0.186 ±0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.     

alpha-Linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid

Adult male unanesthetized rats, reared on a diet enriched in both alpha-linolenic acid (alpha-LNA) and docosahexaenoic acid (DHA), were infused intravenously for 5 min with [1-(14)C]alpha-LNA. Timed arterial samples were collected until the animals were killed at 5 min and the brain was removed after microwaving. Plasma and brain lipid concentrations and radioactivities were measured. Within plasma lipids, > 99% of radioactivity was in the form of unchanged [1-(14)C]alpha-LNA. Eighty-six per cent of brain radioactivity at 5 min was present as beta-oxidation products, whereas the remainder was mainly in 'stable' phospholipid or triglyceride as alpha-LNA or DHA. Equations derived from kinetic modeling demonstrated that unesterified unlabeled alpha-LNA rapidly enters brain from plasma, but that its incorporation into brain phospholipid and triglyceride, as in the form of synthesized DHA, is < or = 0.2% of the amount that enters the brain. Thus, in rats fed a diet containing large amounts of both alpha-LNA and DHA, the alpha-LNA that enters brain from plasma largely undergoes beta-oxidation, and is not an appreciable source of DHA within brain phospholipids.     

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.