C18 PUFA Research Articles

The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata)

The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers.

Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.

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 C18–20 PUFA substrates and not C22 PUFA. In contrast, Elovl2 was active with C20–22 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.

GC-EI-MS analysis of fatty acid composition in brain and serum of twitcher mouse.

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase gene. The objective of the study was to present information about the fatty acid (FA) composition of the brain and serum of twitcher mice, a mouse model of Krabbe disease, compared to wild type, in order to identify biomarker of disease progression. We defined the FA profiles by identifying the main components present in serum and brain using GC-EI-MS analysis. The FA percentage composition was measured and data were analyzed considering the disease and the mouse age as experimental factors. Significant correlations were established, both in brain and in serum, in the fatty acid percentage composition of twitcher compared to wild type mice. The most abundant saturated fatty acid in brain was the palmitic acid (C16:0) with mean values significantly increased in twitcher mouse (p=0.0142); moreover, three monounsaturated, three polyunsaturated (PUFA) and a plasmalogen were significantly correlated to disease. In the serum highly significant differences were observed between the two groups for three polyunsaturated fatty acids. In fact, the docosahexaenoic acid (C22:6n3c) content was significantly increased (p=0.0116), while the C20 PUFA (C20:3n6c and C20:5n3c) were significantly decreased in twitcher serum samples. Our study shows a specific FA profile that may help to define a possible pattern that could distinguish between twitcher and wild type; these data are likely to provide insight in the identification of new biomarkers to monitor the disease progression and thereby permit the critical analysis of therapeutic approaches.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

Investigating long-chain polyunsaturated fatty acid biosynthesis in teleost fish: Functional characterization of fatty acyl desaturase (Fads2) and Elovl5 elongase in the catadromous species, Japanese eel Anguilla japonica

The capacity for endogenous production of LC-PUFA from PUFA in euryhaline or diadromous fish is largely unknown other than for Atlantic salmon (Salmo salar), an anadromous species, which displays a freshwater pattern. The aim of the present study was to characterize the enzymes of the LC-PUFA pathway in Japanese eel (Anguilla japonica), the most important catadromous species currently being farmed. cDNAs of two key genes were cloned and functional assays showed that they encoded a desaturase (Fads2) with Δ6 and Δ8 activity and an elongase (Elovl5) with activity towards C18 and C20 PUFA, with activities similar to marine fish and an Δ6/Δ8 activity ratio similar to Atlantic salmon. Furthermore, tissue distribution of the mRNA showed a clear marine pattern with the highest expression in the brain and the eyes. Phylogenetic analysis placed the eel cDNAs in line with classical taxonomy. The data suggest that diadromous species display a pattern of LC-PUFA biosynthesis capacity that likely reflects the environmental and nutritional influence of their early life stages rather than those of adult fish. Future studies aim to establish the full range of PUFA desaturases and elongases in Japanese eel and to provide further insight into the importance and relevance of LC-PUFA biosynthesis in fish species and the influence of diadromy.

Changes in fatty acid profiles of three tissue types in channel catfishIctalurus punctatus(Rafinesque, 1818) transferred from river to pond environments

Summary Little information is available regarding the time scale over which tissue fatty acid (FA) profiles change when fish transition between prey resources with different FA profiles, specifically when fish move from a main river channel to a floodplain habitat. The objective of this study was to evaluate changes in the FA composition of muscle, liver, and adipose fin tissues of channel catfish Ictalurus punctatus transferred from the Kaskaskia River, Illinois to earthen ponds at the Center for Fisheries, Aquaculture, and Aquatic Sciences Pond Facility, Makanda, Illinois, mimicking migration from river to floodplain habitat. Over time, n-3 and C18 polyunsaturated FA (C18 PUFA) decreased in all tissues, whereas monounsaturated fatty acids (MUFA) increased with pond residence time. Liver profiles changed more rapidly than the muscle or adipose fin profiles (significant change in <2 weeks vs ~10 weeks). The results provide some insight regarding the timing of tissue FA profile change in channel catfish moving between distinct habitats with different prey resources. Results also suggest that adipose fin tissue samples may be a less invasive alternative to muscle tissue for analysis of FA profiles in channel catfish; however, issues with reclassification/identification accuracy using adipose fin tissue must be addressed in order for this approach to be fully justified.

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C18 PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C18 PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.

Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua)

For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod.

Fatty acid desaturase 3 null mouse biochemical phenotype (246.5)

Introduction. We report here generation of the first Fads3 knockout (KO) mouse and initial observations about its biochemical phenotype. Fatty acid desaturase 3 (FADS3) is a member of the FADS gene cluster. FADS1 and FASDS2 code for the rate‐limiting enzymes in LCPUFA biosynthesis. FADS3 has no known function.Methods. All mice (wild type (WT) and KO) were fed standard rodent chow devoid of LCPUFA. Brain and livers were harvested on day 1, 3, 7, 13, 21, and 30 of age for fatty acid and gene expression analysis. Gene expression levels were measured by quantitative and semi‐quantitative real time PCR.Preliminary Results. No differences in overt phenotypes (survival, fertility, growth rate) were observed between WT and KO. Docosahexaenoic acid (DHA, 22:6n‐3) levels in the brain of postnatal day 1 (P1) KO mice were significantly (P&lt;0.05) lower than the WT. No significant Fads1 and Fads2 mRNA expression changes were detected. Elovl2 is known to catalyze the conversion of C20 PUFA. Hepatic Elovl2 expression was significantly upregulated in P1 KO mice compared to WT, and the 22:6n‐3‐precursor 22:5n‐3 was about double in KO vs WT mice.Conclusion. Fads3 null mice have lower brain 22:6n‐3, greater liver 22:5n‐3, and greater liver Elovl2 expression on Day1, though all levels normalize within days. Fads3 appears to influence liver‐mediated 22:6n‐3 synthesis and brain 22:6n‐3 accretion in perinatal life in the Fads3 KO mouse.Grant Funding Source: NIH R01 AT007003

Cloning, Functional Characterization and Nutritional Regulation of Δ6 Fatty Acyl Desaturase in the Herbivorous Euryhaline Teleost Scatophagus Argus

Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating Δ6 desaturation activity. Quantitative real-time PCR showed that highest Δ6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of Δ6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts.

Metabolic regulation in meagre, Argyrosomus regius (Asso, 1801): Study of gene-diet interactions on lipid metabolism

Metabolic regulation in meagre, Argyrosomus regius (Asso, 1801): Study of gene-diet interactions on lipid metabolism

Effect of varying dietary levels of LC-PUFA and vegetable oil sources on performance and fatty acids of Senegalese sole post larvae: Puzzling results suggest complete biosynthesis pathway from C18 PUFA to DHA

Lipid nutrition of marine fish larvae has focused on supplying essential fatty acids (EFA) at high levels to meet requirements for survival, growth and development. However, some deleterious effects have been reported suggesting that excessive supply of EFA might result in insufficient supply of energy substrates, particularly in species with lower EFA requirements such as Senegalese sole (Solea senegalensis). This study addressed how the balance between EFA and non-EFA (better energy sources) affects larval performance, body composition and metabolism and retention of DHA, by formulating enrichment emulsions containing two different vegetable oil sources (olive oil or soybean oil) and three DHA levels. DHA positively affected growth and survival, independent of oil source, confirming that for sole post-larvae it is advantageous to base enrichments on vegetable oils supplying higher levels of energy, and supplement these with a DHA-rich oil. In addition, body DHA levels were generally comparable considering the large differences in their dietary supply, suggesting that the previously reported ∆4 fatty acyl desaturase (fad) operates in vivo and that DHA was synthesized at physiologically significant rates through a mechanism involving transcriptional up-regulation of ∆4fad, which was significantly up-regulated in the low DHA treatments. Furthermore, data suggested that DHA biosynthesis may be regulated by an interaction between dietary n-3 and n-6 PUFA, as well as by levels of LC-PUFA, and this may, under certain nutritional conditions, lead to DHA production from C18 precursors. The molecular basis of putative fatty acyl ∆5 and ∆6 desaturation activities remains to be fully determined as thorough searches have found only a single (∆4) Fads2-type transcript. Therefore, further studies are required but this might represent a unique activity described within vertebrate fads.

Functional characterisation of a Fads2 fatty acyl desaturase with Δ6/Δ8 activity and an Elovl5 with C16, C18 and C20 elongase activity in the anadromous teleost meagre (Argyrosomus regius)

The meagre, Argyrosomus regius, is a carnivorous fish with great potential to diversify finfish aquaculture in the Mediterranean. However, currently nothing is known about their essential fatty acid requirements. Meagres are marine fish but also display anadromous behaviour migrating to river estuaries to spawn, and thus may provide an insight to the influence of diadromy on biosynthetic ability for long-chain polyunsaturated fatty acid (LC-PUFA). Our primary aim was to characterise key cDNAs (fatty acyl desaturases and elongases) of the biosynthetic pathway as a key step to establish the capacity of meagre for LC-PUFA biosynthesis from shorter chain PUFA. The cDNA sequences of a fatty acyl desaturase (Fads) and elongase of very long-chain fatty acids (Elovl) were obtained using PCR-based methodologies, and function of the proteins was investigated by expression of the coding sequences of the putative desaturase and elongase in the yeast Saccharomyces cerevisiae. The tissue distribution of both cDNAs was studied by reverse transcription PCR. Our results demonstrated that meagre possesses at least one fatty acyl desaturase and one elongase involved in the endogenous production of LC-PUFA. The meagre desaturase and elongase were identified as orthologues of Fads2 and Elovl5, respectively. Functionally, the desaturase had dual Δ6/Δ8 activity, whereas the elongase exhibited high elongation efficiency for C18 and C20 PUFA with low activity towards C22 PUFA. However, we also showed that the meagre Elovl5 elongated 16:3n−3 to 18:3n−3, the first time that C16 elongation activity had been demonstrated for a fish elongase. Similar to other marine teleosts, expression of fads2 and elovl5 transcripts was highest in brain. The functions and expression of the meagre Fads2 and Elovl5 proteins suggest that the meagre has a ‘marine type’ LC-PUFA biosynthetic pathway, and that its anadromous behaviour has no major influence.

Sparing Fish Oil with Soybean Oil in Feeds for White Seabass: Effects of Inclusion Rate and Soybean Oil Composition

AbstractFish oil sparing has proven difficult for some fish species, especially marine carnivores like White Seabass Atractoscion nobilis that require one or more long‐chain polyunsaturated fatty acids (LC‐PUFAs). Recent studies have suggested that the use of saturated fatty acid (SFA)–rich lipids instead of C18 polyunsaturated fatty acid–rich (C18 PUFA) lipids may be advantageous in maintaining tissue levels of LC‐PUFAs; SFA‐rich lipids may also offer a strategic advantage in terms of meeting the LC‐PUFA requirements of marine carnivores while minimizing dietary fish oil inclusion. Accordingly, we assessed the performance and tissue fatty acid composition of White Seabass (3.8 ± 0.2 g [mean ± SE]) fed diets containing fish oil or graded levels of C18 PUFA–rich standard soy oil or SFA‐rich hydrogenated soy oil (replacing 25, 50, 75, or 100% of dietary fish oil) for 8 weeks. Feed conversion ratio, weight gain, and specific growth rate were not impaired by partial or complete replacement of dietary fish oil with hydrogenated soy oil; however, fish oil sparing with standard soy oil was associated with declining performance. The tissue fatty acid profiles of fish fed the hydrogenated soy oil–based diets were very similar to those of fish fed the fish oil–based feed, but the standard soy oil–based feeds resulted in concomitant loss of n‐3 fatty acids and LC‐PUFAs. In all cases, the magnitude of the dietary effect was greater among liver and fillet tissues than among brain and eye tissues. These data suggest a limitation, potentially related to LC‐PUFA deficiency, associated with replacing fish oil with standard soybean oil, but not with hydrogenated soybean oil. Our data suggest that the LC‐PUFA requirements of White Seabass can be effectively reduced by feeding SFA‐rich alternative lipids, allowing for a greater level of fish oil sparing without growth impairment or tissue profile modification than is possible with C18 PUFA–rich lipids.

Jumping on the Omega-3 Bandwagon: Distinguishing the Role of Long-Chain and Short-Chain Omega-3 Fatty Acids

Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are almost unanimously recognized for their health benefits, while only limited evidence of any health benefit is currently available specifically for the main precursor of these fatty acids, namely α-linolenic acid (ALA, 18:3n-3). However, both the n-3 LC-PUFA and the short-chain C18 PUFA (i.e., ALA) are commonly referred to as “omega-3” fatty acids, and it is difficult for consumers to recognize this difference. A current gap of many food labelling legislations worldwide allow products containing only ALA and without n-3 LC-PUFA to be marketed as “omega-3 source” and this misleading information can negatively impact the ability of consumers to choose more healthy diets. Within the context of the documented nutritional and health promoting roles of omega-3 fatty acids, we briefly review the different metabolic fates of dietary ALA and n-3 LC-PUFA. We also review food sources rich in n-3 LC-PUFA, some characteristics of LC-PUFA and current industry and regulatory trends. A further objective is to present a case for regulatory bodies to clearly distinguish food products containing only ALA from foods containing n-3 LC-PUFA. Such information, when available, would then avoid misleading information and empower consumers to make a more informed choice in their food purchasing behavior.

Echium Oil Provides No Benefit over Linseed Oil for (n-3) Long-Chain PUFA Biosynthesis in Rainbow Trout1–3

AbstractThe implementation of alternative lipid sources for use in aquaculture is of considerable interest globally. However, the possible benefit of using stearidonic acid (SDA)–rich fish oil (FO) alternatives has led to scientific confusion. Two hundred and forty rainbow trout (Oncorhynchus mykiss) were fed 1 of 4 diets (3 replicate tanks/treatment) containing either FO,linseed oil (LO), echium oil, or mixed vegetable oil (72% LO, 23% sunflower oil, and 6% canola oil) as the dietary lipid source (16.5%) for 73 d to investigate the competition and long-chain PUFA (LC-PUFA) biosynthesis between the fatty acid substrates a-linolenic acid (ALA) and SDA. SDA was more efficiently bioconverted to LC-PUFA compared with ALA.However, when the dietary lipid sources were directly compared, the increased provision of C18 PUFA within the LO diet resulted in no significant differences in (n-3) LC-PUFA content compared with fish fed the other diets. This study therefore shows that, rather than the previously speculated substrate competition, the limiting process in the apparent in vivo (n-3)LC-PUFA biosynthesis appears to be substrate availability. Rainbow trout fed the SDA- and ALA-rich dietary lipid sourcessubsequently had similar significant reductions in (n-3) LC-PUFA compared with fish fed the FO diet, therefore providingno additional dietary benefit on (n-3) LC-PUFA concentrations.

Fatty acids composition of macroalgae from Bulgarian Black Sea coast

Abstract Lipids and fatty acids (FA) composition of three Black Sea macroalgae Cladophora vagabunda,Ceramium rubrum and Cystoseira barbata were studied. Fatty acids composition was analyzed by GC/MS. Total lipids content varied widely among the species and ranged between 0.66 and 0.98 g per 100 g fresh weight. Generally, saturated fatty acids were major components (62-71%), with 16:0 as the most abundant saturate (41- 57%). Total polyunsaturated FAs and monounsaturated FAs ranged from 28% to 38%. The green alga Cladophora vagabunda showed higher C18 PUFAs contents than did C20 PUFAs while for red alga Ceramiumrubrum the trend was opposite. Cystoseira barbata belonging to the group of brown algae showed similar amounts of C18 and C20 PUFAs contents. Cladophora vagabunda was rich in linoleic acid and Ceramiumrubrum in arachidonic acid (AA) while Cystoseira barbata was rich in both linoleic acid and eicosopentaenoic acid. All of the studied species had a nutritionally beneficial n6/n3 ratio (1.24-2.84:1).

Orally administered [14C]DPA and [14C]DHA are metabolised differently to [14C]EPA in rats

Previous studies have revealed that C20 PUFA are significantly less oxidised to CO₂ in whole-body studies compared with SFA, MUFA and C18 PUFA. The present study determined the extent to which three long-chain PUFA, namely 20:5n-3 EPA, 22:5n-3 docosapentaenoic acid (DPA) and 22:6n-3 DHA, were catabolised to CO₂ or, conversely, incorporated into tissue lipids. Rats were administered a single oral dose of 2·5 μCi [1-¹⁴C]DPA, [1-¹⁴C]EPA, [1-¹⁴C]DHA or [1-¹⁴C]oleic acid (18:1n-9; OA), and were placed in a metabolism chamber for 6 h where exhaled ¹⁴CO₂ was trapped and counted for radioactivity. Rats were euthanised after 24 h and tissues were removed for analysis of radioactivity in tissue lipids. The results showed that DPA and DHA were catabolised to CO₂ significantly less compared with EPA and OA (P<0·05). The phospholipid (PL) fraction was the most labelled for all three n-3 PUFA compared with OA in all tissues, and there was no difference between C20 and C22 n-3 PUFA in the proportion of label in the PL fraction. The DHA and DPA groups showed significantly more label than the EPA group in both skeletal muscle and heart. In the brain and heart tissue, there was significantly less label in the cholesterol fraction from the C22 n-3 PUFA group compared with the C20 n-3 PUFA group. The higher incorporation of DHA and DPA into the heart and skeletal muscle, compared with EPA, suggests that these C22 n-3 PUFA might play an important role in these tissues.

Elongation of long-chain fatty acids in rabbitfish Siganus canaliculatus: Cloning, functional characterisation and tissue distribution of Elovl5- and Elovl4-like elongases

Elongases of very long-chain fatty acids (Elovl) catalyse the rate-limiting step of the elongation pathway that results in net 2C elongation of pre-existing fatty acyl chains. As the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) is particularly relevant in fish, Elovl involved in the pathway have been investigated in various studies. Here we report the molecular cloning, functional characterisation and tissue distribution of two distinct elovl-like cDNAs isolated from the herbivorous marine teleost Siganus canaliculatus. Unlike the carnivorous marine fish previously investigated, we hypothesise that the rabbitfish has an enhanced LC-PUFA biosynthetic capability as previously anticipated in a former study on fatty acyl desaturases (Fad). The results of the present study showed that rabbitfish expresses at least two elovl cDNAs, which have high homology in sequence and function to Elovl5 and Elovl4 elongases that have been investigated previously in other fish species. Furthermore, the results confirm that the activities of the Elovl5 and Elovl4 enzymes enable rabbitfish to perform all the elongation reactions required for the biosynthesis of the physiologically essential C20–22 LC-PUFA including eicosapentaenoic (20:5n-3), arachidonic (20:4n-6) and docosahexaenoic (22:6n-3, DHA) acids, as well as the less common very long-chain fatty acids (>C24). Rabbitfish is thus the first marine teleost in which genes encoding Fad and Elovl enzymes, with all the activities required for the production of DHA from C18 PUFA, have been characterised.