Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils

Heterotrophic growth of thraustochytrids has potential in co-producing a feedstock for biodiesel and long-chain (LC, ≥C₂₀) omega-3 oils. Biodiscovery of thraustochytrids from Tasmania (temperate) and Queensland (tropical), Australia, covered a biogeographic range of habitats including fresh, brackish, and marine waters. A total of 36 thraustochytrid strains were isolated and separated into eight chemotaxonomic groups (A–H) based on fatty acid (FA) and sterol composition which clustered closely with four different genera obtained by 18S rDNA molecular identification. Differences in the relative proportions (%FA) of long-chain C₂₀, C₂₂, omega-3, and omega-6 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA), docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid (EPA), and saturated FA, as well as the presence of odd-chain PUFA (OC-PUFA) were the major factors influencing the separation of these groups. OC-PUFA were detected in temperate strains of groups A, B, and C (Schizochytrium and Thraustochytrium). Group D (Ulkenia) had high omega-3 LC-PUFA (53% total fatty acids (TFA)) and EPA up to 11.2% TFA. Strains from groups E and F (Aurantiochytrium) contained DHA levels of 50–61% TFA after 7 days of growth in basal medium at 20 °C. Groups G and H (Aurantiochytrium) strains had high levels of 15:0 (20–30% TFA) and the sum of saturated FA was in the range of 32–51%. β,β-Carotene, canthaxanthin, and astaxanthin were identified in selected strains. Phylogenetic and chemotaxonomic groupings demonstrated similar patterns for the majority of strains. Our results demonstrate the potential of these new Australian thraustochytrids for the production of biodiesel in addition to omega-3 LC-PUFA-rich oils.     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.     

Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum

The proximate composition, vitamin C, α-tocopherol, dietary fibers, minerals, fatty acid and amino acid profiles of three tropical edible seaweeds, Eucheuma cottonii (Rhodophyta), Caulerpa lentillifera (Chlorophyta) and Sargassum polycystum (Phaeophyta) were studied. The seaweeds were high in ash (37.15–46.19%) and dietary fibers (25.05–39.67%) and low in lipid content (0.29–1.11%) on dry weight (DW) basis. These seaweeds contained 12.01–15.53% macro-minerals (Na, K, Ca and Mg) and 7.53–71.53 mg.100 g⁻¹ trace minerals (Fe, Zn, Cu, Se and I). The crude protein content of E. cottonii (9.76% DW) and C. lentillifera (10.41% DW) were higher than that of S. polycystum (5.4% DW), and protein chemical scores are between 20 and 67%. The PUFA content of E. cottonii was 51.55%, C. lentillifera 16.76% and S. polycystum 20.34%. Eicosapentaenoic acid (EPA), accounted for 24.98% of all fatty acids in E. cottonii. These seaweeds have significant vitamin C (∼35 mg.100 g⁻¹) and α-tocopherol (5.85–11.29 mg.100 g⁻¹) contents.     

Comparative Analysis of Content of Omega-3 Polyunsaturated Fatty Acids in Food and Muscle Tissue of Fish from Aquaculture and Natural Habitats

Two fish species reared in aquaculture (pink salmon Oncorhynchus gorbuscha and whitefish Coregonus lavaretus) and ten fish species from natural habitats (whitefish C. lavaretus, tugun Coregonus tugun, broad whitefish Coregonus nasus, least cisco Coregonus sardinella, vendace Coregonus albula, boganid charr Salvelinus boganidae, charr Salvelinus alpinus complex, northern pike Esox lucius, sharp-snouted lenok Brachymystax lenok, and taimen Hucho taimen) have been studied. The content of two long-chain polyunsaturated omega-3 fatty acids (PUFAs), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), in the muscle tissue of the fish and in their food (intestine contents) are compared. In the aquacultures of whitefish and pink salmon, the total content of EPA and DHA is significantly higher in feed than in the muscle tissue of the fish, which indicates losses of PUFA in the two-link food chain of the aquaculture during their transfer to the upper trophic level. EPA and DHA losses in aquaculture, which are confirmed by numerous literature data, mean an inefficient usage of the available sources of PUFAs and the aggravation of the global deficit of these biochemicals in the human diet. A study of natural fish populations reveals the accumulation of EPA and DHA in their biomass compared to food in many cases, although opposite phenomena are also observed. An assumption on the presence of an optimal, physiologically adequate species-specific level of PUFA in the fish muscle tissue has been made based on our data and literature data. If the level of PUFAs in the muscles is lower than optimal, their accumulation (bioaccumulation) from food and/or de novo synthesis are observed. When the optimal level is exceeded, the content of EPA and DHA in biomass approaches maximum species-specific values; however, part of these PUFAs entering from food is not digested or is catabolized. According to the obtained data, the species of the order Salmoniformes have an optimal level of 2 to 6 mg/g of wet weight. It has been found that in aquaculture approaching to maximum values of EPA + DHA content was accompanied by their losses (scattering) in the food chains, while in natural ecosystems the maximum values of PUFA content in the fish biomass are achieved by their accumulation from the lower trophic level. Boganid charr S. boganidae had the highest content of EPA + DHA in the muscle tissue among all known fish species (32.78 mg/g of wet weight).     

Dietary Enrichment with Medium Chain Triglycerides (AC-1203) Elevates Polyunsaturated Fatty Acids in the Parietal Cortex of Aged Dogs: Implications for Treating Age-Related Cognitive Decline

Dogs demonstrate an age-related cognitive decline, which may be related to a decrease in the concentration of omega-3 polyunsaturated fatty acids (n-3 PUFA) in the brain. Medium chain triglycerides (MCT) increase fatty acid oxidation, and it has been suggested that this may raise brain n-3 PUFA levels by increasing mobilization of n-3 PUFA from adipose tissue to the brain. The goal of the present study was to determine whether dietary MCT would raise n-3 PUFA concentrations in the brains of aged dogs. Eight Beagle dogs were randomized to a control diet (n = 4) or an MCT (AC-1203) enriched diet (n = 4) for 2 months. The animals were then euthanized and the parietal cortex was removed for phospholipid, cholesterol and fatty acid determinations by gas-chromatography. Dietary enrichment with MCT (AC-1203) resulted in a significant increase in brain phospholipid and total lipid concentrations (P < 0.05). In particular, n-3 PUFA within the phospholipid, unesterified fatty acid, and total lipid fractions were elevated in AC-1203 treated subjects as compared to controls (P < 0.05). Brain cholesterol concentrations did not differ significantly between the groups (P > 0.05). These results indicate that dietary enrichment with MCT, raises n-3 PUFA concentrations in the parietal cortex of aged dogs.     

Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12–18:2) substantially. The most common product was vaccenic acid (trans-11–18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15–18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n − 3)) and docosahexaenoic acid (DHA; 22:6(n − 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml⁻¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11–18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.     

Associations of maternal prenatal dietary intake of n-3 and n-6 fatty acids with maternal and umbilical cord blood levels

Maternal n-3 and n-6 polyunsaturated fatty acid (PUFA) status may influence birth outcomes and child health. We assessed second trimester maternal diet with food frequency questionnaires (FFQs) (n=1666), mid-pregnancy maternal erythrocyte PUFA concentrations (n=1550), and umbilical cord plasma PUFA concentrations (n=449). Mean (SD) maternal intake of total n-3 PUFA was 1.17 g/d (0.43), docosahexaenoic and eicosapentaenoic acids (DHA+EPA) 0.16 g/d (0.17), and total n-6 PUFA 12.25 g/d (3.25). Mean maternal erythrocyte and cord plasma PUFA concentrations were 7.0% and 5.2% (total n-3), 5.0% and 4.6% (DHA+EPA), and 27.9% and 31.4% (total n-6). Mid-pregnancy diet–blood and blood–blood correlations were strongest for DHA+EPA (r=0.38 for diet with maternal blood, r=0.34 for diet with cord blood, r=0.36 for maternal blood with cord blood), and less strong for n-6 PUFA. The FFQ is a reliable measure of elongated PUFA intake, although inter-individual variation is present     

Mapping Quantitative Trait Loci for Omega-3 Fatty Acids in Asian Seabass

Omega-3 fatty acids are essential fatty acids for human health. Therefore, increasing both percentage of omega-3 and a better fatty acid profile in fish fillets is one of the breeding goals in aquaculture. However, it is difficult to increase the omega-3 content in fish fillets, as the phenotypic selection of these traits is not easily feasible. To facilitate the genetic improvement of the Asian seabass for optimal fatty acid profiles, a genome-wide scan for quantitative trait loci (QTL) affecting fatty acid level in the flesh of the Asian seabass was performed on an F₂ family containing 314 offspring. All family members were genotyped using 123 informative microsatellites and 22 SNPs. High percentages of n-3 polyunsaturated fatty acids (PUFA), especially C22:6 (DHA 16.48?±?3.09 %) and C20:5 (EPA 7.19?±?0.86 %) were detected in the flesh. One significant and 54 suggestive QTL for different fatty acids and a water content trait were detected on the whole genome. QTL for C18:0b was located on linkage groups (LG) 5. QTL for total n-3 PUFA content in flesh were mapped onto LG6 and LG23 with the phenotypic variance explained ranging from 3.8 to 6.3 %. Four QTL for C22:6 were detected on LG6, LG23, and LG24, explaining 3.9 to 4.9 % of the phenotypic variance, respectively. Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.     

Rainbow trout (Oncorhynchus mykiss) Elovl5 and Elovl2 differ in selectivity for elongation of omega-3 docosapentaenoic acid

The synthesis of the omega-3 long-chain polyunsaturated fatty acids (LCPUFA)  eicosapentaenoic acid (EPA; 20:5n− 3) and docosahexaenoic acid (DHA; 22:6n − 3) from dietary α-linolenic acid (ALA; 18:3n − 3) requires three desaturation and three elongation steps in vertebrates. The elongation of EPA to docosapentaenoic acid (DPA; 22:5n − 3) can be catalysed by the elongase enzymes Elovl5 or Elovl2, but further elongation of DPA to 24:5n − 3, the penultimate precursor of DHA, is limited to Elovl2, at least in mammals. Elovl5 enzymes have been characterised from seventeen fish species but Elovl2 enzymes have only been characterised in two of these fish. The essentiality of Elovl2 for DHA synthesis is unknown in fish. This study is the first to identify an Elovl2 in rainbow trout (Oncorhynchus mykiss) and functionally characterise the Elovl5 and Elovl2 using a yeast expression system. Elovl5 was active with C_18–20 PUFA substrates and not C₂₂ PUFA. In contrast, Elovl2 was active with C_20–22 PUFA substrates and not C₁₈ PUFA. Thus, rainbow trout is dependent on Elovl2 for DPA to 24:5n − 3 synthesis and ultimately DHA synthesis. The expression of elovl5 was significantly higher than elovl2 in liver. Elucidating this dependence on Elovl2 to elongate DPA and the low elovl2 gene expression compared with elovl5 are critical findings in understanding the potential for rainbow trout to synthesize DHA.     

Feeding spectra of <Emphasis Type="Italic">Arctodiaptomus salinus</Emphasis> (Calanoida,Copepoda) using fatty acid trophic markers in seston food in two salt lakes in South Siberia (Khakasia,Russia)

During two vegetation seasons (2004–2005), we compared feeding spectra of Arctodiaptomus salinus (Calanoida, Copepoda) populations inhabiting two neighboring salt lakes, Shira and Shunet, Khakasia, Russia, using fatty acid (FA) trophic markers. Sestonic FA composition in two lakes moderately differed, whereas levels of diatom FA markers were higher in Lake Shunet and of Cyanobacteria and green algae markers in Lake Shira. In general, markers in storage lipids—triacylglycerols (TAG) of A. salinus—reflected the differences in sestonic composition of the two lakes. Nevertheless, TAG fraction was also enriched by FA trophic markers of the minor components of seston, which were selectively ingested by the animals. In Lake Shira, A. salinus had significantly higher concentrations of bacterial FA markers in TAG. In Lake Shunet, TAG of A. salinus contained significantly higher relative amounts of 18:4ω3, 18:5ω3 and C22 polyunsaturated fatty acids (PUFA), which indicated marked contribution of cryptophytes or (and) flagellates into the diet. Laboratory experiments showed feeding on Cryptomonas and sulfur purple bacteria in Lake Shunet and ciliates and colonial picoplankton in both lakes, and generally confirmed the differences in FA trophic markers in A. salinus between the lakes. The two populations of A. salinus markedly differed in levels of essential long-chain PUFA, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, although the levels of these FA in seston were quite similar between the two lakes. The higher levels of the essential ω3 PUFA in A. salinus in Lake Shunet may be an adaptive response of the animals to a vertical stratification of physico-chemical conditions and significantly higher salinity levels at the boundary of adjacent bottom layer in this lake.     

Polyunsaturated fatty acids in stream food webs – high dissimilarity among producers and consumers

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Size-related dietary changes observed in young-of-the-year pumpkinseed (<Emphasis Type="Italic">Lepomis gibbosus</Emphasis>): stomach contents and fatty acid analyses

Changes in dietary composition and its effects on the fatty acid (FA) composition of young-of-the-year (YOY) pumpkinseed (Lepomis gibbosus) from an artificial reservoir (Mirgenbach—northeastern France) were related to body size as shown by stomach content and FA analyses. Comparisons were made between three size classes of fishes: 25–35 mm total length (TL), 35–45 mm TL and 45–55 mm TL. Diets of the youngest L. gibbosus (TL ≤ 35 mm) consisted mainly of zooplanktonic microcrustaceans and Chironomidae. Ontogenetic development influenced the FA composition of L. gibbosus. Older YOY L. gibbosus showed an increase in proportions of monounsaturated FA proportions and a decrease in polyunsaturated FA and mainly essential FA (particularly docosahexaenoic acid). The low ω3/ω6 ratio and low PUFA content (mainly on DHA) suggested that L. gibbosus would not transfer the benefits of consuming ω3 PUFA up the food web.     

Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding

Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions of enzymes involved in PUFA biosynthesis.     

Screening for marine nanoplanktic microalgae from Greek coastal lagoons (Ionian Sea) for use in mariculture

Mediterranean mariculture uses imported strains of marine phytoplankton, raising questions of ecological risk and ability to adapt to local conditions for mass culture outdoors. In this context, we report here on the mass-culture potential and chemical composition of six strains of Prasinophyceae (five strains of Tetraselmis sp. and one Pyramimonas sp.) isolated from a Greek coastal lagoon. Proximate composition had a pattern of 10–20% ash, 35–65% protein, 6–10% lipids, and 25–45% other organics including carbohydrates. The amino acid profiles were typical for the marine representatives of the class. All strains had a high PUFA content with dominant the ω3 fraction in four of them. The fatty acid profiles indicated a Tetraselmis strain with high EPA (14%) and a Pyramimonas strain with high DHA (6%). These strains might be a good alternative for the common commercial strains used in Mediterranean aquaculture.     

Enhancement of Polyunsaturated Fatty Acid Production by Tn5 Transposon in Shewanella baltica

Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3–5 times more eicosapentaenoic acid (EPA 20:5 n−3) compared to the wild type at 10°C. One of the mutants produced 0.3 mg EPA g⁻¹ when grown at high temperature (30°C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n−3) g⁻¹ was produced by S. baltica mutants at 4°C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid

Spray-dried milk enriched with n-3 fatty acids from linseed oil or fish oil were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of α linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25–36% and glutathione transferase activity increased to an extent of 34–39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2–4.5 fold compared to control. The serum thromboxane B₂ level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas, 6-keto- prostaglandin F1α level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase. (Mol Cell Biochem xxx: 9–16, 2005)     

Inhibition of platelet aggregation by omega-3 polyunsaturated fatty acids is gender specific—Redefining platelet response to fish oils

Existence of gender differences in cardiovascular disease (CVD) following long-chain omega-3 polyunsaturated fatty acid (LCn-3 PUFA) supplementation have suggested that sex hormones play a role in cardio-protection. The objective of this study was to determine gender specific responses in the efficacy of LCn-3 PUFA to inhibit platelet aggregation in vitro. Blood was analyzed for collagen-induced platelet aggregation following pre-incubation with LCn-3 PUFA in healthy adults (n=42). Eicosapentaenoic acid (EPA) was significantly more effective in reducing platelet aggregation compared with docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). When grouped by gender, this differential pattern was followed in males only. In females, DHA, DPA and EPA were all equally effective. Between group analyses (LCn-3 PUFA vs. gender) showed that both DHA and DPA were significantly less effective in males compared with females. EPA was equally effective in reducing platelet aggregation in both groups. These findings show that significant gender differences exist in platelet aggregation in response to various LCn-3 PUFA treatments.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.