Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

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.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Docosapentaenoic acid (22:5n-3): A review of its biological effects

This article summarizes the current knowledge available on metabolism and the biological effects of n-3 docosapentaenoic acid (DPA). n-3 DPA has not been extensively studied because of the limited availability of the pure compound. n-3 DPA is an elongated metabolite of EPA and is an intermediary product between EPA and DHA. The literature on n-3 DPA is limited, however the available data suggests it has beneficial health effects. In vitro n-3 DPA is retro-converted back to EPA, however it does not appear to be readily metabolised to DHA. In vivo studies have shown limited conversion of n-3 DPA to DHA, mainly in liver, but in addition retro-conversion to EPA is evident in a number of tissues. n-3 DPA can be metabolised by lipoxygenase, in platelets, to form ll-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-DPA. It has also been reported that n-3 DPA is effective (more so than EPA and DHA) in inhibition of aggregation in platelets obtained from rabbit blood. In addition, there is evidence that n-3 DPA possesses 10-fold greater endothelial cell migration ability than EPA, which is important in wound-healing processes. An in vivo study has reported that n-3 DPA reduces the fatty acid synthase and malic enzyme activity levels in n-3 DPA-supplemented mice and these effects were stronger than the EPA-supplemented mice. Another recent in vivo study has reported that n-3 DPA may have a role in attenuating age-related decrease in spatial learning and long-term potentiation. However, more research remains to be done to further investigate the biological effects of this n-3 VLCPUFA.     

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n −3 PUFAs

We examined the effect of n ?3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML‐I. On day 3 of the culture, n ?3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n ?6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML‐I cells at concentrations between 250 and 500 μM by Annexin V‐FITC staining. In Western blotting analysis, the loss of NF‐κB, Bcl‐2 and p‐Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA‐treated cells. Exposure of AML‐I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle‐treated cells in a time‐dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR‐γ (peroxisome proliferator‐activated receptor‐γ) were increased in EPA‐treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML‐I.     

Spray-dried milk supplemented with alpha-linolenic acid or eicosapentaenoic acid and docosahexaenoic acid decreases HMG Co A reductase activity and increases biliary secretion of lipids in rats

In our earlier study, we have shown that rats fed spray-dried milk containing alpha-linolenic acid (LNA 18:3 n-3) or eicosapentaenoic acid (EPA 20:5 n-3) and docosahexaenoic acid (DHA 22:6 n-3) had significantly lower amounts of serum and liver cholesterol. To evaluate the mechanism for hypocholesterolemic effect of n-3 fatty acids containing milk formulation, we fed male Wistar rats with spray-dried milk containing linseed oil (LSO) (source of LNA) or fish oil (FO) (source of EPA+DHA) for 8 weeks. Feeding n-3 fatty acid containing milk formulation lowered the hepatic 3-hydroxy-methylglutaryl coenzyme A (HMG Co A) activity by 17-22% compared to rats given control diet devoid of n-3 fatty acids. The cholesterol level in liver microsomes was found to be decreased by 16% and 20%, respectively, in LSO and FO containing formulation fed rats. The bile flow was enhanced to an extent of 19-23% in experimental groups compared to control animals. The biliary cholesterol and phospholipid secretion was increased to an extent of 49-55% and 140-146%, respectively, in rats fed n-3 fatty acid containing formulation. The increase in the total bile acids secretion in bile was mainly reflected on an increase in the levels of taurine conjugated bile acids. These results indicated that n-3 fatty acid containing spray-dried milk formulation would bring about the hypocholesterolemic effect by lowering HMG Co A reductase activity in liver and by increasing the secretion of bile constituents.     

Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis

The high commercial values of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have driven a strain-improvement program, aimed at increasing the content of those fatty acids in the microalga Pavlova lutheri (SMBA 60) as parent strain. After a round of mutation using UV-light as mutagenic agent, an isolate strain (tentatively called II#2) was obtained, the EPA and DHA contents of which (in % dry biomass) were 32.8% and 32.9% higher than those of the control, native strain. The final EPA yields, when the cultures were maintained under appropriate conditions, were 17.4 and 23.1 mg. g(-1) dry biomass, for the wild-type and the II#2 strain, respectively, whereas the final DHA yields were 8.0 and 10.6 mg. g(-1) dry biomass, respectively. These results suggest that random mutagenesis can successfully be applied to increase the yield of n-3 fatty acids by microalgae.     

Dietary low linolenic acid compared with docosahexaenoic acid alter synaptic plasma membrane phospholipid fatty acid composition and sodium-potassium ATPase kinetics in developing rats

The objective of this study was to investigate if maternal dietary 20:4n-6 arachidonic acid (AA) and 22:6n-3 compared with adequate or low levels of 18:3n-3 linolenic acid (LNA) increases synaptic plasma membrane (SPM) cholesterol and phospholipid content, phospholipid 20:4n-6 and 22:6n-3 content, and Na,K-ATPase kinetics in rat pups at two and five weeks of age. At parturition, Sprague-Dawley rats were fed semi-purified diets containing either AA + docosahexaenoic acid (DHA), adequate LNA (control; 18:2n-6 : 18:3n-3 ratio of 7.1 : 1) or low LNA (18:2n-6 : 18:3n-39 ratio of 835 : 1). During the first two weeks of life, the rat pups received only their dams' milk. After weaning, pups received the same diet as their respective dams to five weeks of age. No significant difference was observed among rat pups fed the diet treatments for SPM cholesterol or total and individual phospholipid content at two and five weeks of age. Fatty acid analysis revealed that maternal dietary AA + DHA, compared with feeding the dams the control diet or the low LNA diet, increased 20:4n-6 in phosphatidylserine and 22:6n-3 content of SPM phospholipids. Rats fed dietary AA + DHA or the control diet exhibited a significantly increased Vmax for SPM Na,K-ATPase. Diet treatment did not alter the Km (affinity) of SPM Na,K-ATPase in rat pups at two and five weeks of age. It is concluded that dietary AA + DHA does not alter SPM cholesterol and phospholipid content but increases the 22:6n-3 content of SPM phospholipids modulating activity of Na,K-ATPase.     

真菌发酵生产EPA及DHA影响因素的研究进展

对真菌发酵生产EPA及DHA的影响因素进行综述 ,介绍了菌种、碳源、氮源、C/N比、pH值、温度、发酵时间、通气量、代谢途径的调控、种龄和接种量等因素对EPA及DHA产量的影响。     

产二十二碳六烯酸等多不饱和脂肪酸真菌的筛选*

从土壤中筛选出一株产二十二碳六烯酸（DHA）的丝状真菌,菌丝含油21.23％,DHA占总脂肪酸2.51％;同时含二十碳五烯酸（EPA）,占总脂肪酸的0．41％;不饱和脂肪酸占总脂肪酸的80％。经鉴定为头孢霉属（Caphalosporiumsp．）真菌。同时发现两株菌含EPA,经鉴定为小克银汉霉（Cunninghamellasp.）和毛霉（Mucorsp．）。在这几个属中发现DHA和EPA尚属首次。头孢霉菌DHA产量及百分含量和斜面菌种在不同温度下储藏有关。菌种在20℃储藏10天,在液体PDA培养基上发酵,DHA可占总脂肪酸11．27％,产量达63．35mg／L。     

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.     

ELOVL2 gene polymorphisms are associated with increases in plasma eicosapentaenoic and docosahexaenoic acid proportions after fish oil supplement

Fish oil supplementation provides an inconsistent degree of protection from cardiovascular disease (CVD), which may be attributed to genetic variation. Single nucleotide polymorphisms (SNPs) in the elongation-of-very-long-chain-fatty-acids-2 (ELOVL2) gene have been strongly associated with plasma proportions of n-3 long-chain polyunsaturated fatty acids (LC-PUFA). We investigated the effect of genotype interaction with fish oil dosage on plasma n-3 LC-PUFA proportions in a parallel double-blind controlled trial, involving 367 subjects randomised to treatment with 0.45, 0.9 and 1.8 g/day eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1.51:1) or olive oil placebo for 6 months. We genotyped 310 subjects for ELOVL2 gene SNPs rs3734398, rs2236212 and rs953413. At baseline, carriers of all minor alleles had lower proportions of plasma DHA than non-carriers (P = 0.021–0.030). Interaction between genotype and treatment was a significant determinant of plasma EPA (P < 0.0001) and DHA (P = 0.004–0.032). After the 1.8 g/day dose, carriers of ELOVL2 SNP minor alleles had approximately 30 % higher proportions of EPA (P = 0.002–0.004) and 9 % higher DHA (P = 0.013–0.017) than non-carriers. Minor allele carriers could therefore particularly benefit from a high intake of EPA and DHA in maintaining high levels of plasma n-3 PUFA conducive to protection from CVD.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0362-6) contains supplementary material, which is available to authorized users.     

二十二碳六烯酸对心房颤动大鼠心房肌生理改变及双孔钾通道TASK-1蛋白表达的影响

目的：探讨二十二碳六烯酸（DHA）对大鼠心房颤动（AF）模型心房肌生理特性的影响及相关机制研究。方法：80只乙酰胆碱-氯化钙混合液敏感的SD大鼠分为对照组（CTL组）、DHA处理组（DHA组）、房颤组（AF组）和房颤+DHA处理组（DHA+AF组）,观察房颤持续时间;采用全细胞膜片钳技术记录大鼠心房肌细胞动作电位时程（APD）和双孔钾通道TASK-1电流,Western blot测定大鼠心房组织TASK-1蛋白表达。结果：大鼠尾静脉注射乙酰胆碱-氯化钙混合液后,房颤持续时间随实验天数增加而逐渐延长,DHA干预缩短房颤持续时间。与CTL组相比,AF组大鼠心房肌细胞复极50%时的动作电位时程（APD₅₀）和复极90%时的动作电位时程（APD₉₀）明显缩短,心房肌细胞TASK-1电流密度升高,蛋白表达升高（P<0.05）。与AF组相比,DHA+AF组大鼠心房肌细胞APD₅₀和APD₉₀明显延长,TASK-1电流密度和蛋白表达降低（P<0.05）。结论：DHA具有延长房颤大鼠心房肌细胞APD的作用,可能与其下调心房肌TASK-1蛋白的表达从而降低心房肌细胞TASK-1电流密度有关。     

Screening and characterization of eicosapentaenoic acid-producing marine bacteria

To obtain eicosapentaenoic acid-producing bacteria, some 3,500 strains of bacteria were isolated from the intestine of marine animals from sea around the Korean peninsula. Forty two eicosapentaenoic acid-producing bacteria were obtained through screening with TLC and gas chromatography, and the strain with highest content of eicosapentaenoic acid (up to 33% of total fatty acid when grown at 5°C ) as the sole polyunsaturated fatty acid was identified as an Aeromonas sp. on the basis of physiological properties.     

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.     

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

Differential expression of parvalbumin in neonatal phencyclidine‐treated rats and socially isolated rats

Decreased parvalbumin expression is a hallmark of the pathophysiology of schizophrenia and has been associated with abnormal cognitive processing and decreased network specificity. It is not known whether this decrease is due to reduced expression of the parvalbumin protein or degeneration of parvalbumin‐positive interneurons (PV⁺ interneurons). In this study, we examined PV⁺ expression in two rat models of cognitive dysfunction in schizophrenia: the environmental social isolation (SI) and pharmacological neonatal phencyclidine (neoPCP) models. Using a stereological method, the optical fractionator, we counted neurons, PV⁺ interneurons, and glial cells in the medial prefrontal cortex (mPFC) and hippocampus (HPC). In addition, we quantified the mRNA level of parvalbumin in the mPFC. There was a statistically significant reduction in the number of PV⁺ interneurons (p = 0.021) and glial cells (p = 0.024) in the mPFC of neonatal phencyclidine rats, but not in SI rats. We observed no alterations in the total number of neurons, hippocampal PV⁺ interneurons, parvalbumin mRNA expression or volume of the mPFC or HPC in the two models. Thus, as the total number of neurons remains unchanged following phencyclidine (PCP) treatment, we suggest that the decreased number of counted PV⁺ interneurons represents a reduced parvalbumin protein expression below immunohistochemical detection limit rather than a true cell loss. Furthermore, these results indicate that the effect of neonatal PCP treatment is not limited to neuronal populations.