Process development of eicosapentaenoic acid production

Eicosapentaenoic acid (EPA), a well-known member of omega-3 fatty acids, is considered to have a significant health promoting role in the human body. It is an essential fatty acid as the human body lacks the ability to produce it in vivo and must be supplemented through diet. Microbial EPA represents a potential commercial source. GC/MS analyses confirmed that bacterial isolate 717, similar to Shewanella pacifica on the basis of 16S rRNA sequencing, is a potential high EPA producer. Two types of bioreactors, a Stirred Tank Reactor (STR) and an Oscillatory Baffled Reactor (OBR), were investigated in order to choose the optimum system for EPA production. The EPA production media was optimised through the selection of media components in a Plackett–Burman (PB) design of experiment followed by a Central Composite Design (CCD) to optimise the concentration of medium components identified as significant in the Plackett–Burman experiment. The growth conditions for the bioreactor, using artificial sea water (ASW) medium, were optimised by applying Response Surface Methodology (RSM). This optimisation strategy resulted in an increase in EPA from 33 mg/l (10 mg/g biomass), representing 8% of the total fatty acids at shake flask level, to 350 mg/l (46 mg/g biomass) representing 25% of the total fatty acids at bioreactor level. During this study the main effects and the interactions between the bioreactor growth conditions were revealed and a polynomial model of EPA production was generated. Chemostat experiments were performed to test the effect of growth rate and temperature on EPA production.     

Vascular reactivity and high dietary eicosapentaenoic acid

Epidemiologic studies suggest that high dietary intake of eicosapentaenoic acid (EPA), a precursor of the trienoic prostaglandins, is associated with a low incidence and reduced extent of myocardial infarction. Vascular reactivity of isolated aortic strips from rats maintained for 3 weeks on a control diet or on a diet supplemented with menhaden fish oil (17% EPA) was examined with norepinephrine, sodium arachidonate, KC1, PGF2 alpha and nitroprusside. Aortic strips from rats fed the fish oil diet were significantly less responsive to the contractile effects of norepinephrine and arachidonate compared to those from control diet rats. Treatment of aortic strips with indomethacin decreased responsiveness to norepinephrine. The magnitude of the decrease was greater in control rats resulting in a similar vascular response between the 2 groups after blockade. Contractions to arachidonate were abolished by indomethacin. There were no differences in vascular responses to KC1, PGF2 alpha and nitroprusside in aortic strips from control diet rats and those from the fish oil diet rats. Aortic strips from the fish oil diet rats contained more EPA than those from the control diet rats. Thus, the contractile effect of norepinephrine in isolated rat aortic strips is normally augmented by intrinsic prostaglandins, and this augmentation is diminished by dietary intake of EPA.     

Comparative levels of arachidonic acid and eicosapentaenoic acid in Malaysian fish

Total lipids were extracted from 22 species of Malaysian fish and the constituent fatty acids were analysed by gas chromatography. Malaysian fish generally contained high levels of saturated fatty acids (range 36-55% total fatty acids) and contained variable amounts of monounsaturates, chiefly palmitic and stearic acids, but only trace levels of 20:1 and 22:1. Unlike fish caught in colder northern hemisphere waters, Malaysian fish were found to contain arachidonic acid (20:4 omega 6, range 2-12%) in addition to the expected eicosapentaenoic acid (20:5 omega 3, range 1-13%) and docosahexaenoic acid (22:6 omega 3, range 6.6-40.4%).     

Heterotrophic production of eicosapentaenoic acid by microalgae

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that plays an important role in the regulation of biological functions and prevention and treatment of a number of human diseases such as heart and inflammatory diseases. As fish oil fails to meet the increasing demand for purified EPA, alternative sources are being sought. Microalgae contain large quantities of high-quality EPA and they are considered a potential source of this important fatty acid. Some microalgae can be grown heterotrophically on cheap organic substrate without light. This mode of cultivation can be well controlled and provides the possibility to maximize EPA production on a large scale. Numerous strategies have been investigated for commercial production of EPA by microalgae. These include screening of high EPA-yielding microalgal strains, improvement of strains by genetic manipulation, optimization of culture conditions, and development of efficient cultivation systems. This paper reviews recent advances in heterotrophic production of EPA by microalgae with an emphasis on the use of diatoms as producing organisms.     

Production of eicosapentaenoic acid by marine bacteria

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.     

Screening of diatoms for heterotrophic eicosapentaenoic acid production

Nine strains of diatoms (representing four species) were screened for their ability to produce eicosapentaenoic acid (EPA) when cultured heterotrophically on glucose. Four strains were able to produce EPA heterotrophically using glucose as its carbon and energy source. Of the four,Nitzschia laevis was the best EPA producer, yielding 0.017 g g^–1 dry cell weight.N. laevis was the only species tested which synthesised more EPA heterotrophically than photosynthetically. This study shows thatN. laevis is a potential source of EPA production using heterotrophic culture conditions with glucose as the carbon and energy substrate.Author for correspondence     

Nomenclature of lipoxins and related compounds derived from arachidonic acid and eicosapentaenoic acid

Oxygenated derivates of arachidonic acid and eicosapentaenoic acid which contain conjugated tetraene structures and are non-cyclized C20 carboxylic acids were first isolated and characterized from human and porcine leukocytes (Serhan, C.N. et al, 1984, Biochem. Biophys. Res. Commun. 118, 943-949; Wong, P.Y.-K., et al, 1985, Biochem. Biophys. Res. Commun. 126, 765-775). The trivial names lipoxins and lipoxenes have been introduced for compounds belonging to each of these series. Here, we propose that tetraene-containing compounds derived from arachidonic acid be denoted as lipoxins (LX) of the four series (i.e. lipoxin A4 or LXA4 and lipoxin B4 or LXB4) and those derived from eicosapentaenoic be termed lipoxins of the five series (i.e. lipoxin A5 or LXA5 and lipoxin B5 or LXB5).     

Enhanced eicosapentaenoic acid production by Navicula saprophila

Changes in the eicosapentaenoic acid (EPA) content of Navicula saprophila grown photoautotrophically and mixotrophically with the addition of acetate were examined in terms of their growth progress. Among four conditions, mixotrophic conditions in CO₂-enriched (about 2%) atmosphere gave a maximum EPA content. As sodium acetate was added to the growth medium, the EPA content increased and reached a maximum value of 34.6 mg EPA g^-1 biomass in the early stationary growth phase. In contrast, under photoautotrophic conditions in CO₂ enriched atmosphere, the EPA content decreased during this phase. EPA was localized as the fatty acid esters of monogalactosyl diacylglycerol, phosphatidylethanolamine and phosphatidylcholine, and the addition of acetate strongly enhanced production of the PC ester of EPA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chain elongation of eicosapentaenoic acid in the macrophage

In order to elucidate the metabolic fate of eicosapentaenoic acid (20:5 (n-3], a major n-3 fatty acid constituent of fish oil, resident and casein-elicited mouse peritoneal macrophages were incubated with [3H]20:5 (n-3). Comparative experiments with arachidonic acid (20:4 (n-6] were also conducted. After 4, 8 and 18 h incubation, [3H]20:5 (n-3) was extensively elongated into [3H]22:5(n-3) while [3H]20:4(n-6) was only moderately elongated into [3H]22:4(n-6) in both resident and elicited macrophages. No measurable conversion of [3H]22:5(n-3) into [3H]22:6(n-3) (delta 4 desaturation) could be demonstrated. These data demonstrate that the highly active chain elongation of 20:5(n-3) by macrophage elongase, as well as the lack of detectable delta 4 desaturase activity, are responsible for the accumulation of 22:5(n-3) in this cell.     

Lipase specificity towards eicosapentaenoic acid and docosahexaenoic acid depends on substrate structure

The fatty acid specificity of five lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated in the hydrolysis of fish oil, squid oil and a model system. The model system contained methyl esters of EPA, DHA and palmitic acid. All the investigated lipases discriminated against both EPA and DHA more in the model system than in the natural oils. Thus both EPA and DHA were more easily hydrolysed from a glyceride than from a methyl ester. In the model system, the lipase from Candida rugosa showed the highest discrimination against DHA, while the lipases from Pseudomonas fluorescens and Pseudomonas cepacia discriminated against EPA the most. In a glyceride, the fatty acid specificity of lipases towards EPA and DHA was affected by the positional distribution of the fatty acids and the glyceride structure due to the regiospecificity and triglyceride specificity of the lipase. In the oils, the Pseudomonas lipases also discriminated against EPA the most, while DHA was initially discriminated the most by the lipase from Thermomyces lanuginosus. However, after longer reaction times the enrichment of DHA in the glyceride fraction of the oils was greatest for the lipase from C. rugosa.     

Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.     

Dietary docosahexaenoic and eicosapentaenoic acid: Emerging mediators of inflammation

The inflammatory response is designed to help fight and clear infection, remove harmful chemicals, and repair damaged tissue and organ systems. Although this process, in general, is protective, the failure to resolve the inflammation and return the target tissue to homeostasis can result in disease, including the promotion of cancer. A plethora of published literature supports the contention that dietary n-3 polyunsaturated fatty acids (PUFA), and eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in particular, are important modulators of a host's inflammatory/immune responses. The following review describes a mechanistic model that may explain, in part, the pleiotropic anti-inflammatory and immunosuppressive properties of EPA and DHA. In this review, we focus on salient studies that address three overarching mechanisms of n-3 PUFA action: (i) modulation of nuclear receptor activation, i.e., nuclear factor-κB (NF-κB) suppression; (ii) suppression of arachidonic acid–cyclooxygenase-derived eicosanoids, primarily prostaglandin E₂-dependent signaling; and (iii) alteration of the plasma membrane micro-organization (lipid rafts), particularly as it relates to the function of Toll-like receptors (TLRs), and T-lymphocyte signaling molecule recruitment to the immunological synapse (IS). We propose that lipid rafts may be targets for the development of n-3 PUFA-containing dietary bioactive agents to down-modulate inflammatory and immune responses and for the treatment of autoimmune and chronic inflammatory diseases.     

Hydrolysis of anandamide and eicosapentaenoic acid ethanolamide in mouse splenocytes

The hydrolysis of anandamide has been studied in mouse splenocytes using tritiated anandamide analogs labeled in the acyl- or ethanolamide parts of the molecule. [3H]Anandamide undergoes rapid (t(1/2) = 2.5 min) uptake and hydrolysis, yielding ethanolamine and arachidonic acid. The anandamide hydrolysis in splenocytes is sensitive to inhibition by phenylmethylsulfonyl fluoride, and it is assumed that the observed activity is due to fatty acid amide hydrolase, which inactivates anandamide in central and peripheral tissues. Eicosapentaenoic acid ethanolamide and the 15-hydroxy-derivative of anandamide are shown to be amidohydrolase substrates as well. The fatty acids derived from hydrolytic cleavage of acylethanolamines undergo rapid oxidation by splenocyte lipoxygenase, yielding the corresponding 12-hydroxy-derivatives. Oxygenated ethanolamide derivatives were not found. The data suggest that polyenoic fatty acid ethanolamides are metabolic precursors of eicosanoids in splenocytes and that amide bond hydrolysis is the key point in switching of biological activity spectra between endocannabinoids and oxylipins.     

Effect of eicosapentaenoic acid on central systolic blood pressure

Central systolic blood pressure (C-SBP) has been shown to be a better predictor of cardiovascular risk than brachial SBP. In this study, the effects of eicosapentaenoic acid (EPA) on C-SBP were compared with pravastatin. Twenty-four patients with hyperlipidemia were assigned 13 to receive 1800 mg/day EPA (EPA group) and 11 to receive 10 mg/day pravastatin (pravastatin group) for 3 months. In the EPA group, there were no changes in the LDL–cholesterol level. However, the radial augmentation index (AI) and C-SBP decreased after treatment by 5.7% (p<0.01) and 8.7% (p<0.001), respectively. Moreover, systolic and diastolic brachial BPs decreased by 7.1% and 8.0%, respectively (p<0.01 for both). In the pravastatin group, the LDL–cholesterol level decreased by 29.5% (p<0.001). However, there were no significant changes in brachial BP, AI and C-SBP between. These results suggested that EPA but not pravastatin may reduce cardiac afterload by reducing vascular reflected waves and lowering C-SBP.     

Effect of docosahexaenoic acid and eicosapentaenoic acid supplementation on oxidative stress levels during pregnancy

Docosahexaenoic acid (DHA) is an indispensable component of cell membranes with high requirements during pregnancy. DHA supplementation is thought to enhance oxidative stress because of increased likelihood of lipid peroxidation. We estimated the oxidative stress levels in two groups of pregnant women who received daily supply of required vitamins with (n = 23) or without (n = 23) 500 mg of DHA and 150 mg of eicosapentaenoic acid (EPA) from 20 weeks of gestation to the time of delivery. Urinary excretions of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage and of malondialdehyde (MDA), a marker of lipid peroxidation, were measured at 20, 30 weeks and at the time of delivery. Urinary MDA excretion remained unchanged throughout the study period in both groups. Urinary 8-OHdG excretion at delivery was significantly higher than at 20 and 30 weeks (p < 0.05), but there were no group differences at the three time points. There were no differences between the two groups in plasma a-tocopherol levels. We conclude that under the conditions studied, a daily supplementation of 500 mg DHA and 150 mg EPA with vitamins to pregnant women did not enhance lipid peroxidation or oxidative DNA damage.     

Effects of eicosapentaenoic acid and docosahexaenoic acid on anxiety-like behavior in socially isolated rats

Abstract

The effects of fish oil for improving mental health have been reported. The present study was undertaken to compare the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on anxiety-like behavior using a rat model. Experimental diets enriched in EPA or DHA as glycerides were prepared. Rats were exposed to social isolation stress and fed the experimental diet for 14 days. The results of behavioral tests revealed that rats fed the EPA-enriched diet exhibited less anxiety-like behavior than rats fed the control or DHA-enriched diets. Furthermore, EPA suppressed anxiety-like behavior only in socially isolated rats. The increase in EPA contents in the brain phospholipid fraction by feeding EPA-enriched diet was more significant than that of DHA by feeding DHA-enriched diet. These results suggest that dietary EPA is more anxiolytic than DHA in rats exposed to social isolation stress and is effective in increasing EPA content in brain membranes.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A₂ during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosaspentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPa are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of ¹⁴C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a ‘Max EPA’ fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F_1α but no difference was seen in leukotriene B₄ formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA grou than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as ¹⁴C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Production of eicosapentaenoic acid by the filamentous fungus Pythium irregulare

Because of the diversity of their lipids and fatty acid biosynthetic pathways, lower fungi may find utilization as sources of omega-3 or other polyunsaturated fatty acids (PUFA). Production of eicosapentaenoic acid (EPA) by the filamentous fungus, Pythium irregulare, has been demonstrated in 14-1 fermentors. Sweet whey permeate (lactose) was preferred over glucose as a substrate for production of a high-EPA-content lipid. Characterization of the lipid indicated that approximately 90% of the EPA was contained in the neutral lipid fraction. A specific productivity of 24.9 mg EPA/g dry biomass was achieved at 14°C, at which temperature the lipid contained 25.5% EPA and 54.2% PUFA. This is the highest mycelial EPA content for a fungal lipid that has been reported in the literature. Correspondence to: D. J. O'Brien