Docosahexaenoic acid: one molecule diverse functions

Docosahexaenoic acid (DHA, C22:6, ω-3) is a highly polyunsaturated omega-3 fatty acid. It is concentrated in neuronal brain membranes, for which reason it is also referred to as a “brain food”. DHA is essential for brain development and function. It plays an important role in improving antioxidant and cognitive activities of the brain. DHA deficiency occurs during aging and dementia, impairs memory and learning, and promotes age-related neurodegenerative diseases, including Alzheimer’s disease (AD). For about two decades, we have reported that oral administration of DHA increases spatial memory acquisition, stimulates neurogenesis, and protects against and reverses memory impairment in amyloid β peptide-infused AD rat models by decreasing amyloidogenesis and protects against age-related cognitive decline in the elderly. These results demonstrate a robust link between DHA and cognitive health. Rodents that were fed a diet low in ω-3 polyunsaturated fatty acids, particularly those that were DHA-deficient, frequently suffered from anxiety, depression and memory impairment. Although the exact mechanisms of action of DHA in brain functions are still elusive, a host of mechanisms have been proposed. For example, DHA, which inherently has a characteristic three-dimensional structure, increases membrane fluidity, strengthens antioxidant activity and enhances the expression of several proteins that act as substrates for improving memory functions. It reduces the brain amyloid burden and inhibits in vitro fibrillation and amyloid-induced neurotoxicity in cell-culture model. In this review, we discuss how DHA acts as a molecule with diverse functions.     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.     

DNA damage and mutations induced by arachidonic acid peroxidation

Endogenous cellular oxidation of omega6-polyunsaturated fatty acids (PUFAs) has long been recognized as a contributing factor in the development of various cancers. The accrual of DNA damage as a result of reaction with free radical and electrophilic aldehyde products of lipid peroxidation is believed to be involved; however, the genotoxic and mutation-inducing potential of specific membrane PUFAs remains poorly defined. In the present study we have examined the ability of peroxidizing arachidonic acid (AA, 20:4omega6) to induce DNA strand breaks, base modifications, and mutations. The time-dependent induction of single-strand breaks and oxidative base modifications by AA in genomic DNA was quantified using denaturing glyoxal gel electrophoresis. Mutation spectra were determined in XP-G fibroblasts and a repair-proficient line corrected for this defect by c-DNA complementation (XP-G(+)). Mutation frequencies were elevated from approximately 5- to 30-fold over the background following reaction of DNA with AA for various times. The XPG gene product was found to be involved in the suppression of mutations after extended reaction of DNA with AA. Arachidonic acid-induced base substitutions were consistent with the presence of both oxidized and aldehyde base adducts in DNA. The frequency of multiple-base substitutions induced by AA was significantly reduced upon correction for the XPG defect (14% vs 2%, P = 0.0015). Evidence is also presented which suggests that the induced frequency of multiple mutations is lesion dependent. These results are compared to published data for mutations stimulated by alpha,beta-unsaturated aldehydes identified as products of lipid peroxidation.     

Flavonoids and related compounds as inhibitors of arachidonic acid peroxidation

Until now only few data have been reported on biochemically explicable pharmacological effects of flavonoid structures. When tested against arachidonic acid metabolism many flavonoids were found to be effective against the lipoxygenase and cyclo-oxygenase pathways. Some flavonoids were predominant inhibitors of either cyclo-oxygenase or lipoxygenase, others were equally effective against both enzymes. Therefore, these compounds proved to be useful tools to elucidate fatty acid peroxidation problems.     

Molecular evolution of enzymes involved in the arachidonic acid cascade

The metabolic pathway called the arachidonic acid cascade produces a wide range of eicosanoids, such as prostaglandins, thromboxanes and leukotrienes with potent biological activities. Recombinant DNA techniques have made it possible to determine the nucleotide sequences of cDNAs and/or genomic structures for the enzymes involved in the pathway. Sequence comparison analyses of the accumulated sequence data have brought great insights into the structure, function and molecular evolution of the enzymes. This paper reviews the sequence comparison analyses of the enzymes involved in the arachidonic acid cascade.     

Docosahexaenoic acid: membrane properties of a unique fatty acid

Docosahexaenoic acid (DHA) with 22-carbons and 6 double bonds is the extreme example of an omega-3 polyunsaturated fatty acid (PUFA). DHA has strong medical implications since its dietary presence has been positively linked to the prevention of numerous human afflictions including cancer and heart disease. The PUFA, moreover, is essential to neurological function. It is remarkable that one simple molecule has been reported to affect so many seemingly unrelated biological processes. Although details of a molecular mode of action remain elusive, DHA must be acting at a fundamental level common to many tissues that is related to the high degree of conformational flexibility that the multiple double bonds have been identified to confer. One likely target for DHA action is at the cell membrane where the fatty acid is known to readily incorporate into membrane phospholipids. Once esterified into phospholipids DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity. It is concluded that DHA's interaction with other membrane lipids, particularly cholesterol, may play a prominent role in modulating the local structure and function of cell membranes.     

The isoprostanes: Novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid. This review summarizes our current knowledge regarding these compounds. Novel aspects of the biochemistry and bioactivity of IsoPs are detailed and methods by which these compounds are analyzed are discussed. A considerable portion of this review deals with the utility of measuring IsoPs as markers of oxidant injury in human diseases particularly in association with risk factors that predispose to atherosclerosis, a condition in which excessive oxidative stress has been causally implicated.     

Docosahexaenoic acid and lactation

Docosahexaenoic acid (DHA) is an important component of membrane phospholipids in the retina and brain and accumulates rapidly in these tissues during early infancy. DHA is present in human milk, but the amount varies considerably and is largely dependent on maternal diet. This article reviews data addressing the impact of different DHA intakes by lactating women on infant and maternal outcomes to determine if available data are sufficient to estimate optimal breast milk DHA content and estimate dietary reference intakes (DRIs) for DHA by breast-feeding mothers. Results of published observational studies and interventional trials assessing the impact of maternal DHA intake (or breast milk DHA content) on infant visual function, neurodevelopment, and immunologic status were reviewed. Studies related to the potential impact of DHA intake on depression or cognitive function of lactating women also were reviewed. Although only a limited number of studies are available in the current medical literature, and study results have not been consistent, better infant neurodevelopment and/or visual function have been reported with higher vs. lower levels of breast milk DHA. The effect of DHA intake on the incidence or severity of depression in lactating women is not clear. Increasing breast milk DHA content above that typically found in the US, by increasing maternal DHA intake, may confer neurodevelopmental benefits to the recipient breast-fed infant. However, current data are insufficient to permit determination of specific DRIs during this period.     

Metabolism of arachidonic acid to eicosanoids within the nucleus

The eicosanoids are a diverse family of molecules that have powerful effects on cell function. They are best known as intercellular messengers, having autocrine and paracrine effects following their secretion from the cells that synthesize them. Many of the eicosanoids are produced from one polyunsaturated fatty acid, arachidonic acid. The diversity of possible products that can be synthesized from arachidonic acid is due, in part to the variety of enzymes that can act on it. Over the past 15 years, studies have placed many, but not all, of these enzymes at or inside the nucleus. In some cases, the nuclear import or export of arachidonic acid-processing enzymes is highly regulated. Furthermore, nuclear receptors that are activated by specific eicosanoids are known to exist. Taken together, these findings indicate that the enzymatic conversion of arachidonic acid to specific signaling molecules can occur in the nucleus, that it is regulated, and that the synthesized products may act within the nucleus. The objectives of this commentary are to review what is known about the metabolism of arachidonic acid to eicosanoids within the nucleus and to point to important areas for future discovery.     

Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination

Phospholipases A₂ (PLA₂) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS).Here, we aimed to determine whether brain expression PLA₂ enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase.Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination.We found that after 4-6 weeks of cuprizone, sPLA₂(V) and cPLA₂, but not iPLA₂(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA₂(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE₂, PGD₂, PGI₂ and TXB₂ were also increased during demyelination. During remyelination, none of the PLA₂ isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE₂, PGI₂ and PGD₂ levels returned to normal, whereas TXB₂ was still upregulated after 3 weeks of cuprizone withdrawal.Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA₂(V) is the major isoform contributing to AA release.     

Docosahexaenoic acid-enriched egg phospholipids supplementation induces accretion of arachidonic acid in rat blood lipids.

Animal and humans studies have shown that supplementation with triacylglycerides containing omega3 fatty acids, mainly docosahexaenoic acid (DHA) and eicosapentaenoic acid, can induce a decrease in arachidonic acid (AA) in blood lipids. Interestingly, we observed in a previous work that a supplementation with DHA enriched eggs in a healthy elderly population induced an accretion of AA in their blood lipids. The present study investigates whether purified DHA enriched egg phospholipids could be responsible for this effect. Four groups of rats were supplemented daily, for eight weeks, with DHA phospholipids (10, 30 or 60 mg/kg) or with soybean phospholipids. Red blood cell membranes and plasma fatty acid levels were compared with that of rats without supplementation. Soybean phospholipids supplementation increased the level of AA in blood lipids but decreased that of DHA. The doses of DHA phospholipids, 30 and 60 mg/kg, induced greater amounts of AA without affecting significantly DHA levels. In contrast, DHA phospholipids supplementation, 10 mg/kg, in which there was the greatest amount of AA, induced only a slight increase in AA levels. Moreover, DHA levels were decreased by this supplementation. These results demonstrate that specific increases in AA levels are preferentially associated with DHA phospholipids levels in supplementation.     

Retrograde modulation at developing neuromuscular synapses: involvement of G protein and arachidonic acid cascade.

Intracellular loading of nonhydrolyzable GTP analogs into innervated muscle cells in Xenopus cultures led to a marked increase in the frequency of spontaneous synaptic currents (SSCs), while extracellular application of the drugs at the same concentration was without effect. The increase in SSC frequency appeared to be unrelated to changes in the muscle membrane sensitivity toward acetylcholine (ACh), but resulted from an elevated spontaneous ACh secretion from the presynaptic nerve terminal. Postsynaptic loading of arachidonic acid (AA) produced a similar effect as the GTP analogs, and the potentiation effect of both GTP analogs and AA was reversed by an inhibitor of AA metabolism, AA861. Further studies indicate that a lipoxygenase metabolite, 5-HPETE, appears to be a likely candidate for the retrograde factor involved in modulating ACh secretion. These results suggest that G protein activation of the AA cascade in the postsynaptic cell could produce a retrograde signal to modulate transmitter secretion from the presynaptic nerve terminal at developing synapses.     

Age-related changes in arachidonic acid peroxidation and glutathione-peroxidase activity in human platelets.

Lipid peroxidation, vitamin E level and glutathione-peroxidase activity were determined in platelets from elderly (greater than 68 years) and young (21-43 years) people. To further assess the platelet lipid peroxidation, the metabolism of endogenous arachidonic acid in unstimulated platelets as well as that of the exogenous one were measured in the two groups. The oxygenated metabolites of arachidonic acid were enhanced in the elder population under both conditions tested. In addition, the platelet malondialdehyde content, a marker of the overall lipid peroxidation, was also found significantly increased in platelets from aged subjects. On the other hand, the platelet vitamin E level and the glutathione-peroxidase activity were significantly depressed in the elder group compared to the young one. These results suggest that the increased platelet activation observed with age could be linked to the accumulation of lipoxygenase-dependent peroxides associated with the decreased antioxidative defence of the cells, especially glutathione-peroxidase activity.     

Evidence for,and taxonomic value of,an arachidonic acid cascade in the lipomycetaceae

By using specific inhibitors of the lipoxygenase and cyclo-oxygenase pathways, arachidonic acid metabolites with similar sensitivities towards these inhibitors as in humans, were detected inDipodascopsis uninucleata. The taxonomic value of aspirin sensitive arachidonic acid metabolites in the Lipomycetaceae was next assessed. No metabolites of which the production is inhibited by aspirin were detected in strains representing the following species:Lipomyces starkeyi, Lipomyces kononenkoae, Lipomyces tetrasporus, Myxozyma melibiosi, Myxozyma mucilagina, Myxozyma kluyveri, Waltomyces lipofer, Zygozyma oligophaga andZygozyma arxii. The detection of such aspirin sensitive arachidonic acid metabolites in representative strains ofLipomyces anomalus and the genusDipodascopsis, emphasises the isolated position of these taxa in the genusLipomyces and the family Lipomycetaceae, respectively. Finally using long chain fatty acid analyses, electrophoretic karyotyping and other phenotypic characters, a phylogenetic scheme is proposed for some genera in the Lipomycetaceae.     

A2B adenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade

Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2B receptors, 5'-(N-ethylcarboxamido)adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A2A receptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A3 receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3 receptor antagonist was ineffective to antagonize adenosine-induced contraction, A2 or A1 receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50-70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A2B receptors mediate the adenosine-induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.     

Lipid peroxidation of a human hepatoma cell line (HepG2) after incorporation of linoleic acid, arachidonic acid, and docosahexaenoic acid

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography-mass spectrometry (GC-MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H(2)O(2) (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H(2)O(2). However, the enhancement of lipid peroxidation by H(2)O(2) was not observed in DHA-supplemented cells. The same result was obtained in the GC-MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA-MHP and a small amount of DHA-MHP were observed in AA- and DHA-supplemented cells respectively. GC-MS analysis also indicated the specific positional distribution of DHA-MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.     

Influence of the arachidonic acid cascade on the in vitro hepatic response to hypoxia

Studies were undertaken to determine the effect of arachidonic acid, the precursor of bisenoic prostanoic acid derivatives, on the response of the isolated, perfused rabbit liver to hypoxia. Two and one half hours of severe hypoxia resulted in significant increases in hepatic vascular perfusion pressure, tissue wet weight, and the rates of cellular loss of lactic dehydrogenase, malic dehydrogenase, and acid phosphatase into the perfusing medium. Hypoxia also increased the rate of hepatic PGF_2α production by 25% after 2 hours (p<0.05, hypoxia vs sham). The addition of arachidonic acid (0.1 μg/g/min for 150 minutes) to the perfusion medium of hypoxic livers significantly attenuated the changes in perfusion pressure, tissue wet weight, and loss of cellular enzymes. Arachidonic acid administration increased the rate of PGF_2α production by 100% (p<0.05, sham vs hypoxia + arachidonic acid) within 30 min after hypoxia and maintained this rate for the duration of the study. These results demonstrate that hypoxia mediated prostaglandin F_2α synthesis in the rabbit liver can occur in the absence of neural and blood borne components and that significant activation of the arachidonic acid cascade via the administration of exogenous arachidonic acid has a salutary effect on hepatic hemodynamics and cellular integrity during hypoxia.     

Docosahexaenoic acid: Measurements in food and dietary exposure

The Dietary Reference Intakes (DRIs) were established to be an indicator of adequacy of dietary nutrients as well as providing levels for adequacy in reducing risk of chronic diseases such as neurodegenerative diseases, cardiovascular diseases, cancers, diabetes mellitus, etc. One particular nutrient that is increasingly discussed as a potential candidate for the generation of a DRI is the omega-3 (n-3) fatty acid docosahexaenoic acid (DHA) due to its potential benefits in reducing risk for cardiovascular disease, role in resolution of inflammation, its importance in cognitive function in infants and inhibiting the progression of neurodegenerative diseases in the elderly. Each reference value refers to and is predicated on estimates of daily nutrient intake and the goal of this paper is to review these intakes. The confidence of these values is critical in establishing dose–response relationships. This paper reports intake values for DHA and examines how these data were generated and the relative confidence in these values. The adult US population is estimated to consume 80–100 mg/d of DHA based on a nationally representative sample of >8400 individuals as part of the National Health and Nutrition Examination Survey (NHANES). This value and those presented for women and men at various ages appear reasonable and should be used as the basis for establishing an Adequate Intake (AI) for DHA.