Simultaneous Treatment of Long-chain Monounsaturated Fatty Acid and n-3 Polyunsaturated Fatty Acid Decreases Lipid and Cholesterol Levels in HepG2 Cell.

The n-3 type polyunsaturated fatty acids (n-3PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from fish oil exhibit health benefits such as triacylglycerol- and cholesterol-lowering effects. Some pelagic fishes contain long-chain monounsaturated fatty acids (LC-MUFAs) such as eicosenoic acid (C20:1), which exert health-promoting effects. However, no study has evaluated beneficial effects of n-3PUFA and LC-MUFA combination. Here, we investigated effects of simultaneous treatment with n-3PUFA (EPA and DHA) and LC-MUFA (cis-5-C20:1 and cis-7-C20:1) and found that n-3PUFA and LC-MUFA combination significantly decreased lipid accumulation and reduced total cholesterol in HepG2 cells. Cholesterol level was significantly lower in DHA + cis-7-C20:1 group than in DHA + EPA group. These results suggest the importance of LC-MUFA as a functional molecule in fish oil.

Co-production of DHA and squalene by thraustochytrid from forest biomass.

Omega-3 fatty acids, and specifically docosahexaenoic acid (DHA), are important and essential nutrients for human health. Thraustochytrids are recognised as commercial strains for nutraceuticals production, they are group of marine oleaginous microorganisms capable of co-synthesis of DHA and other valuable carotenoids in their cellular compartment. The present study sought to optimize DHA and squalene production by the thraustochytrid Schizochytrium limacinum SR21. The highest biomass yield (0.46 g/gsubstrate) and lipid productivity (0.239 g/gsubstrate) were observed with 60 g/L of glucose, following cultivation in a bioreactor, with the DHA content to be 67.76% w/wtotal lipids. To reduce costs, cheaper feedstocks and simultaneous production of various value-added products for pharmaceutical or energy use should be attempted. To this end, we replaced pure glucose with organosolv-pretreated spruce hydrolysate and assessed the simultaneous production of DHA and squalene from S. limacinum SR21. After the 72 h of cultivation period in bioreactor, the maximum DHA content was observed to 66.72% w/wtotal lipids that was corresponded to 10.15 g/L of DHA concentration. While the highest DHA productivity was 3.38 ± 0.27 g/L/d and squalene reached a total of 933.72 ± 6.53 mg/L (16.34 ± 1.81 mg/gCDW). In summary, we show that the co-production of DHA and squalene makes S. limacinum SR21 appropriate strain for commercial-scale production of nutraceuticals.

N-docosahexaenoylethanolamine detected in human breast milk.

PURPOSE: Measure concentrations of the neurogenic, pro-neurogenic, pro-synaptogenic and anti-inflammatory mediator N-docosahexaenoylethanolamine (synaptamide) in relation to its precursor docosahexaenoic acid (DHA) in breast milk. DESIGN AND METHODS: Postpartum women were recruited prior to discharge. We supplemented half the subjects with omega-3 fatty acids. Breast milk samples were collected at 1, 4 and 8 weeks. Synaptamide and DHA concentrations were determined by liquidchromatography/tandem mass spectrometry (LC-MS/MS) and gas chromatography, respectively. RESULTS: Synaptamide was detected in all breast milk samples. The concentration ranged from 44 to 257 fmol/mL. Omega-3 fatty acid supplementation did not affect DHA or synaptamide concentration in breast milk due to a high-DHA-containing diet self-selected by control mothers. Nevertheless, synaptamide levels significantly correlated with DHA concentration in breast milk (r = 0.624, P < 0.001). CONCLUSION: This is the first demonstration of detectable concentrations of synaptamide in human breast milk. Although the attempt to raise the milk DHA content by omega-3 fatty acid supplementation was not successful in the current study, the positive correlation observed between synaptamide and DHA concentration suggests that synaptamide levels in human milk can be raised by proper omega-3 fatty acid supplementation that is known to increase DHA.

The Association of Fatty Acid Levels and Gleason Grade among Men Undergoing Radical Prostatectomy.

BACKGROUND: Epidemiological data suggest that omega-6 (ω-6) fatty acids (FAs) may be associated with cancer incidence and/or cancer mortality, whereas ω-3 FAs are potentially protective. We examined the association of the ratio of ω-6 to ω-3 FA (ω-6:ω-3) and individual FA components with pathological results among men with prostate cancer (PCa) undergoing radical prostatectomy. METHODS: Sixty-nine men were included in the study. Components of ω-6 (linoleic acid (LA), arachidonic acid (AA), and dihomo-γ-linolenic acid (DGLA)) and ω-3 (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) were analyzed by liquid chromatography/mass selective detector separation. Logistic regression analysis was performed to determine association of FA with pathological high grade (Gleason ≥4+3) disease. RESULTS: The were 35 men with low grade disease (Gleason ≤3+4) and 34 men with high grade disease. Men with low grade disease were significantly younger (58y vs 61y, p = 0.012) and had lower D'Amico clinical classification (p = 0.001) compared to men with high grade disease. There was no significant association of ω-6:ω-3 with high grade disease (OR 0.93, p = 0.78), however overall ω-6, ω-3, and individual components of ω-6 and ω-3 FAs except EPA were significantly associated with high grade disease (ω-6: OR 3.37, 95% CI: 1.27,8.98; LA: OR 3.33, 95% CI:1.24,8.94; AA: OR 2.93, 95% CI:1.24,6.94; DGLA: OR 3.21, 95% CI:1.28,8.04; ω-3: OR 3.47, 95% CI:1.22,9.83; DHA: OR 3.13, 95% CI:1.26,7.74). ω-6 and ω-3 FA components were highly correlated (Spearman ρ = 0.77). CONCLUSION: Higher levels of individual components of ω-6 and ω-3FAs may be associated with higher-grade PCa. IMPACT: Studies into the causative factors/pathways regarding FAs and prostate carcinogenesis may prove a potential association with PCa aggressiveness.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Heterotrophic Production of Omega-3 Long-Chain Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom Phaeodactylum tricornutum.

We have created via metabolic engineering a heterotrophic strain of Phaeodactylum tricornutum that accumulates enhanced levels of the high value omega-3 long chain polyunsaturated fatty acid (LC-PUFAs) docosahexaenoic acid (DHA). This was achieved by generation of transgenic strains in which the Δ5-elongase from Ostreococcus tauri was co-expressed with a glucose transporter from the moss Physcomitrella patens. This double transformant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulate substantial levels of omega-3 LC-PUFAs. The effects of glucose concentrations on growth and LC-PUFA production of wild type and transformed strains cultivated in the light and dark were studied. The highest omega-3 LC-PUFAs accumulation was observed in cultures grown under mixotrophic conditions in the presence of 1% glucose (up to 32.2% of total fatty acids, TFA). Both DHA and EPA are detected at high levels in the neutral lipids of transgenic cells grown under phototrophic conditions, averaging 36.5% and 23.6% of TFA, respectively. This study demonstrates the potential for P. tricornutum to be developed as a viable commercial strain for both EPA and DHA production under mixo- and heterotrophic conditions.

Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 µg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 µmol photons m-2s-1) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.

Development of botanical and fish oil standard reference materials for fatty acids.

As part of a collaboration with the National Institutes of Health's Office of Dietary Supplements and the Food and Drug Administration's Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60% long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.

A meta-analysis shows that docosahexaenoic acid from algal oil reduces serum triglycerides and increases HDL-cholesterol and LDL-cholesterol in persons without coronary heart disease.

Certain algae contain the (n-3) fatty acid DHA, yet the relation between algal oil supplementation and cardiovascular disease risk factors has not been systematically examined. Our objective was to examine the relation between algal oil supplementation and cardiovascular disease risk factors. We conducted a systematic review of randomized controlled trials published between 1996 and 2011 examining the relation between algal oil supplementation and cardiovascular disease risk factors and performed a meta-analysis of the association between algal oil DHA supplementation and changes in the concentrations of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C). We identified 11 randomized controlled trials with 485 healthy participants that evaluated the relation between algal oil DHA supplementation and TG, LDL-C, and HDL-C. The median dose of algal DHA was 1.68 g/d. The pooled estimate for the change in TG concentration was -0.20 mmol/L (95% CI: -0.27 to -0.14), 0.23 mmol/L (95% CI: 0.16-0.30) for LDL-C, and 0.07 mmol/L (95% CI: 0.05-0.10) for HDL-C. DHA supplementation from algal oil, a marine source of (n-3) fatty acids not extracted from fish, may reduce serum TG and increase HDL-C and LDL-C in persons without coronary heart disease.

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value.

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.

Enrichment of eicosapentaenoic acid and docosahexaenoic acid from sardine by-products by supercritical fluid fractionation.

Based on the principle of supercritical chromatography, a fractionation process for fatty acid methyl esters (FAMEs) under supercritical conditions was studied with the aim of obtaining in extracts highly enriched eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish cannery waste. Yields of the fractionation process were enhanced by using adjuvant material and modifying CO(2) volumetric density and temperature. The combination of adjuvant properties with optimized pressure and temperature conditions permitted efficient fractionations of FAMEs, according to chain length and degree of unsaturation, in high purity (up to 95% of FAME) and good yields (45% of EPA and DHA starting potential). This work shows how green technologies, such as supercritical processes, can constitute a good alternative to the use of organic solvents in classical methods for valorisation of complex food industry waste.

In vitro percutaneous absorption studies and in vivo evaluation of anti-inflammatory activity of essential fatty acids (EFA) from fish oil extracts.

The aim of the present study was to evaluate the in vitro percutaneous absorption and the in vivo anti-inflammatory activity of EPA and DHA fatty acids from three oily extracts, obtained by acetonic extractions from the entrails of different varieties of Mediterranean fishes such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus). In the first part of our research, we focused our attention on the characterization of the oily extracts to determine their omega-3 polyunsaturated fatty acid content, then, we evaluated the in vitro percutaneous absorption through excised human skin (stratum corneum/epidermis membranes; SCE) of EPA and DHA contained in the extracts. In the second part, the fish oil which guaranteed the best in vitro permeation profile of these omega-3 fatty acids was studied in order to evaluate its inhibiting ability towards the in vivo UVB-induced skin erythema. From the results obtained, all the fish oils tested in this study presented significant amounts of omega-3 fatty acids EPA and DHA, and particularly sardine oil extract showed higher concentrations of these substances compared to the other two fish oils. The in vitro experiments revealed interesting fluxes of these compounds from sardine extract through the stratum corneum/epidermis membranes and an appreciable anti-inflammatory activity against UVB-induced erythema in human volunteers was also observed.

Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data.

An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition. This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product. The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat. No known mycotoxins were produced by the production strains under the conditions tested. Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight. In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA). This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects. Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1. No obvious signs of toxicity were observed. Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups. The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day. This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk.

Evaluation of single cell sources of docosahexaenoic acid and arachidonic acid: a 4-week oral safety study in rats.

Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are secreted in human milk and consumed by the nursing neonate but are not present in infant formulas currently available in the US. Supplementation of formulas with DHA and ARA may be particularly important for premature infants, who have less accretion of these fatty acids in utero than term infants. Some experts suggest that DHA and ARA should be added to infant formulas. Common sources of these fatty acids (e.g. fish oils, egg yolk lipids) are not optimal for infants in that they contain disproportionate amounts of other fatty acids. This 4-wk study examined the safety of a high-DHA algal oil and a high-ARA fungal oil, blended so that the DHA:ARA ratio approximates that in human milk. Rats were fed the blend at levels representing three, 11 and 22 times the anticipated infant exposure. Control animals were fed either a high-fat diet (13.1%, w/w; equivalent to the fat content of the treated groups) or a low-fat diet (5%, w/w). There were no treatment-related differences in body weight, food intake, organ weights, haematology or clinical chemistry. Thus, this study indicates that a blend of algal and fungal oils is a safe source of DHA and ARA as it produced no adverse effects in rats when administered for 4 wk at levels up to 22 times the expected infant exposure.