Very Long Chain Polyunsaturated Fatty Acids Research Articles

Distinctive lipid composition of the copepod Limnocalanus macrurus with a high abundance of polyunsaturated fatty acids.

We studied the copepod Limnocalanus macrurus for seasonal variation in the composition of fatty acids, wax esters and sterols in large boreal lakes, where it occurs as a glacial-relict. Vast wax ester reserves of Limnocalanus were accumulated in a period of only two months, and comprised mono- and polyunsaturated fatty acids (PUFA) and saturated fatty alcohols. In winter, the mobilization of wax esters was selective, and the proportion of long-chain polyunsaturated wax esters declined first. PUFA accounted for >50% of all fatty acids throughout the year reaching up to ca. 65% during late summer and fall. Long-chain PUFA 20:5n-3 and 22:6n-3 together comprised 17-40% of all fatty acids. The rarely reported C24 and C26 very-long-chain PUFA (VLC-PUFA) comprised 6.2 ± 3.4 % of all fatty acids in August and 2.1 ± 1.7% in September. The VLC-PUFA are presumably synthesized by Limnocalanus from shorter chain-length precursors because they were not found in the potential food sources. We hypothesize that these VLC-PUFA help Limnocalanus to maximize lipid reserves when food is abundant. Sterol content of Limnocalanus, consisting ca. 90% of cholesterol, did not show great seasonal variation. As a lipid-rich copepod with high abundance of PUFA, Limnocalanus is excellent quality food for fish. The VLC-PUFA were also detected in planktivorous fish, suggesting that these compounds can be used as a trophic marker indicating feeding on Limnocalanus.

Coexpression of multiple genes reconstitutes two pathways of very long-chain polyunsaturated fatty acid biosynthesis in Pichia pastoris.

The introduction of novel traits to cells often requires the stable coexpression of multiple genes within the same cell. Herein, we report that C22 very long-chain polyunsaturated fatty acids (VLC-PUFAs) were synthesized from C18 precursors by reactions catalyzed by delta 6-desaturase, an ELOVL5 involved in VLC-PUFA elongation, and delta 5-desaturase. The coexpression of McD6DES, AsELOVL5, and PtD5DES encoding the corresponding enzymes, produced docosatetraenoic acid (C22:4 n-6) and docosapentaenoic acid (C22:5 n-3), as well as arachidonic acid (C20:4 n-6) and eicosapentaenoic acid (C20:5 n-3) in the methylotrophic yeast Pichia pastoris. The expression of each gene increased within 24h, with high transcript levels after induction with 0.5 or 1% methanol. High levels of the newly expressed VLC-PUFAs occurred after 144h. This expression system exemplifies the recent progress and future possibilities of the metabolic engineering of VLC-PUFAs in oilseed crops.

Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO).

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω-3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ∆(6)-, ∆(8)-, ∆(5)- and ∆(4)-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.

Atypical surface behavior of ceramides with nonhydroxy and 2-hydroxy very long-chain (C28–C32) PUFAs

Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28–32 carbon atoms, 4–5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.

Expression of a type 2 diacylglycerol acyltransferase from Thalassiosira pseudonana in yeast leads to incorporation of docosahexaenoic acid β‐oxidation intermediates into triacylglycerol

Glycerolipids of the marine diatom Thalassiosira pseudonana are enriched particularly with eicosapentaenoic acid (EPA), and also with an appreciable level of docosahexaenoic acid (DHA). The present study describes the functional characterization of a type 2 diacylglycerol acyltransferase (DGAT2, EC 2.3.1.20) from T. pseudonana, designated TpDGAT2, which catalyzes the final step of triacylglycerol (TAG) synthesis. Heterologous expression of this gene restored TAG formation in a yeast mutant devoid of TAG biosynthesis. TpDGAT2 was also shown to exert a large impact on the fatty acid profile of TAG. Its expression caused a 10-12% increase of 18:1 and a concomitant decrease of 16:0 relative to that of AtDGAT1(Arabidopsis thaliana). Furthermore, in the presence of the very-long-chain polyunsaturated fatty acids (VLCPUFA) EPA and DHA, TAG formed by TpDGAT2 displayed three- to six-fold increases in the percentage of VLCPUFA relative to that of AtDGAT1 even though TpDGAT2 conferred much lower TAG-synthetic activities than Arabidopsis DGAT1. Strikingly, when fed DHA, the yeast mutant expressing TpDGAT2 incorporated high levels of EPA and DHA isomers derived from DHA β-oxidation. In contrast, no such phenomenon occurred in the cells expressing AtDGAT1. These results suggested that, in addition to the role in breaking down storage lipids, yeast peroxisomes also contribute to lipid synthesis by recycling acyl-CoAs when a fatty acyl assembly system is available to capture and utilize the fatty acyl pools generated via β-oxidation. Our study hence illustrated a case where the efficiency and substrate preference of an acyltransferase can elicit far reaching metabolic adjustments that affect TAG composition.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

INCORPORATION OF OMEGA-3 FATTY ACID AND SYNTHESIS OF THEIR METABOLITES IN MUSCLE TISSUE IN MICE FED WITH FLAXSEED AND PERILLA-ENRICHED DIET

Polyunsaturated fatty acids omega 3 and omega 6 are believed to have beneficial physiological effects on the human body. The present study aimed to evaluate the incorporation and synthesis of omega-3 fatty a cid and their metabolites in muscle tissue in mice fed with a flaxseed and perilla-enriched diet. The anim als were fed a diet supplemented with flaxseed and peri lla meals for fifty-six days and control animals re ceived a commercial diet. Mice were sacrificed on the 7th, 28th and 56th days and the muscle tissue was colle cted. The FA concentration was analyzed by gas chromatography. The levels of alpha-Linolenic Acid (LNA) and their metabolites, Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) increased as a function of feeding length in the groups given the supplemen ted diet. LNA was incorporated into muscle tissue a nd conversion into Very Long Chain Polyunsaturated Fatty Acids (VLC-PUFA) was observed. The muscle tissue of animals fed with enriched diets presented a high sum of n-3 FA and high concentrations of LNA, EPA and DHA, as compared to mice fed commercial diet. The intake of flaxseed and perilla-meal enriched diets by mice may result in the deposition of LNA a nd its metabolites EPA and DHA into muscle tissue.

Role of ELOVL4 and very long-chain polyunsaturated fatty acids in mouse models of Stargardt type 3 retinal degeneration

Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the ELOVL4 (Elongation of very long chain fatty acids 4) gene. Its protein product, ELOVL4, is an elongase required for the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs). It is unclear whether photoreceptor degeneration in STGD3 is caused by loss of VLC-PUFAs or by mutated ELOVL4 protein trafficking/aggregation. We therefore generated conditional knockout (cKO) mice with Elovl4 ablated in rods or cones and compared their phenotypes to transgenic (TG) animals that express the human STGD3-causing ELOVL4(STGD3) allele. Gas chromatography-mass spectrometry was used to assess C30-C34 VLC-PUFA and N-retinylidene-N-retinylethanolamine content; electroretinography was used to measure phototransduction and outer retinal function; electron microscopy was used for retinal ultrastructure; and the optomotor tracking response was used to test scotopic and photopic visual performance. Elovl4 transcription and biosynthesis of C30-C34 VLC-PUFAs in rod cKO and TG retinas were reduced up to 98%, whereas the content of docosahexaenoic acid was diminished in TG, but not rod cKO, retinas. Despite the near-total loss of the retinal VLC-PUFA content, rod and cone cKO animals exhibited no electrophysiological or behavioral deficits, whereas the typical rod-cone dystrophic pattern was observed in TG animals. Our data suggest that photoreceptor-specific VLC-PUFA depletion is not sufficient to induce the STGD3 phenotype, because depletion alone had little effect on photoreceptor survival, phototransduction, synaptic transmission, and visual behavior.

Phytophthora infestans cholinephosphotransferase with substrate specificity for very-long-chain polyunsaturated fatty acids.

The effective flux between phospholipids and neutral lipids is critical for a high level of biosynthesis and accumulation of very-long-chain polyunsaturated fatty acids (VLCPUFAs), such as arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3). Here we describe a cDNA (PiCPT1) from Phytophthora infestans, a VLCPUFA-producing oomycete, that may have a role in acyl trafficking between diacylglycerol (DAG) and phosphatidylcholine (PC) during the biosynthesis of VLCPUFAs. The cDNA encodes a polypeptide of 393 amino acids with a conserved CDP-alcohol phosphotransferase motif and approximately 27% amino acid identity to the Saccharomyces cerevisiae cholinephosphotransferase (ScCPT1). In vitro assays indicate that PiCPT1 has high cholinephosphotransferase (CPT) activity but no ethanolaminephosphotransferase (EPT) activity. Substrate specificity assays show that it prefers VLCPUFA-containing DAGs, such as ARA DAG and DHA DAG, as substrates. Real-time PCR analysis reveals that expression of PiCPT1 was upregulated in P. infestans organisms fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFA-specific CPT which may play an important role in shuffling VLCPUFAs from DAG to PC in the biosynthesis of VLCPUFAs in P. infestans.

Cicrulating markers of retinal and optic nerve lipids

AbstractPurpose Blood lipids are frequently used as a surrogate of lipid composition of peripheral tissues. Even if it is well accepted such a relationship has never been clearly demonstrated for the eye. The aim of this study was to determine in human samples whether a lipidomic approach based on red blood cells could reveal associations between circulating and ocular lipid profiles.Methods Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on these tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source‐mass spectrometer (LC‐ESI‐MS).Results Gas chromatographic approach did not shpw any relevant association between circulating and ocular lipids except for arachidonic acid whose circulating amounts were positively associated with its retinal and optic nerve levels. However, significant associations emerged from LC‐ESI‐MS analyses. Indeed, phospholipid species in red blood cells were positively or negatively associated with representative pools of retinal DHA (docosahexaenoic acid), retinal VLC‐PUFA (very‐long chain polyunsaturated fatty acids) or optic nerve plasmalogens.Conclusion First, our results show that LC‐ESI‐MS methodology is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. Second, this study has identified several individual lipid species as good candidates to represent circulating biomarkers of ocular lipids.

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

BackgroundThe assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics.Methodology and Principal FindingsRed blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens.Conclusions and SignificanceLC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.

Essential Role of ELOVL4 Protein in Very Long Chain Fatty Acid Synthesis and Retinal Function

Very long chain polyunsaturated fatty acid (VLC-PUFA)-containing glycerophospholipids are highly enriched in the retina; however, details regarding the specific synthesis and function of these highly unusual retinal glycerophospholipids are lacking. Elongation of very long chain fatty acids-4 (ELOVL4) has been identified as a fatty acid elongase protein involved in the synthesis of VLC-PUFAs. Mutations in ELOVL4 have also been implicated in an autosomal dominant form of Stargardt disease (STGD3), a type of juvenile macular degeneration. We have generated photoreceptor-specific conditional knock-out mice and used high performance liquid chromatography-mass spectrometry (HPLC-MS) to examine and analyze the fatty acid composition of retinal membrane glycerophosphatidylcholine and glycerophosphatidylethanolamine species. We also used immunofluorescent staining and histology coupled with electrophysiological data to assess retinal morphology and visual response. The conditional knock-out mice showed a significant decrease in retinal glycerophospholipids containing VLC-PUFAs, specifically contained in the sn-1 position of glycerophosphatidylcholine, implicating the role of Elovl4 in their synthesis. Conditional knock-out mice were also found to have abnormal accumulation of lipid droplets and lipofuscin-like granules while demonstrating photoreceptor-specific abnormalities in visual response, indicating the critical role of Elovl4 for proper rod or cone photoreceptor function. Altogether, this study demonstrates the essential role of ELOVL4 in VLC-PUFA synthesis and retinal function.

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Biosynthesis of Triacylglycerols (TAGs) in Plants and algae

Triacylglycerols (TAGs), which consist of three fatty acids bound to a glycerol backbone, are major storage lipids that accumulate in developing seeds, flower petals, pollen grains, and fruits of innumerous plant species. These storage lipids are of great nutritional and nutraceutical value and, thus, are a common source of edible oils for human consumption and industrial purposes. Two metabolic pathways for the production of TAGs have been clarified: an acyl CoA-dependent pathway and an acyl-CoA-independent pathway. Lipid metabolism, specially the pathways to fatty acids and TAG biosynthesis, is relatively well understood in plants, but poorly known in algae. It is generally accepted that the basic pathways of fatty acid and TAG biosynthesis in algae are analogous to those of higher plants. However, unlike higher plants where individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, the complete pathway, from carbon dioxide fixation to TAG synthesis and sequestration, takes place within a single algal cell. Another distinguishing feature of some algae is the large amounts of very long-chain polyunsaturated fatty acids (VLC-PUFAs) as major fatty acid components. Nowadays, the focus of attention in biotechnology is the isolation of novel fatty acid metabolizing genes, especially elongases and desaturases that are responsible for PUFAs synthesis, from different species of algae, and its transfer to plants. The aim is to boost the seed oil content and to generate desirable fatty acids in oilseed crops through genetic engineering approaches. This paper presents the current knowledge of the neutral storage lipids in plants and algae from fatty acid biosynthesis to TAG accumulation.

Very-long-chain polyunsaturated fatty acids in the retina: analysis and clinical relevance in physiological and pathological conditions

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal glycerophospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called Elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STGD3), a dominantly inherited juvenile macular degeneration leading to vision loss. In this context, PC molecular species containing VLC-PUFA in retina must be precisely characterized to improve our understanding of the pathogenesis of STGD3. For 25 years, several current approaches using gas chromatography or gas chromatography-mass spectrometry have been used for the characterisation and quantification of VLC-PUFA. These methods allowed characterizing with precision the structures (carbon chain length and double bond positions) of the different VLC-PUFA in bovine retina. Thus, retinal C28-C36 fatty acids are polyunsaturated and belong to the n-3 and n-6 families. The n-6 family of C28-C36 fatty acids has four or five double bonds, while the n-3 family of VLC-PUFA contains five or six double bonds. Moreover, some studies allowed determining the position of VLC-PUFA in the VLC-PC species. But most of these conventional approaches are time-consuming, requiring successive extraction, chromatographic steps and often a derivatization step before. Recently, a normal LC-ESI-MS/MS method was proposed for the structural characterization and the quantification of VLC-PC species in retinas from bovines and human donors. It directly analyses phospholipids as intact molecules and preserves the information based on the relative position of acyl radicals on the glycerol backbone. Since the results from molecular and biochemical studies led to the conclusion that VLC-PUFA are involved in the pathogenesis of STGD3, this specific and accurate method may be useful to more precisely investigate the molecular mechanisms leading to photoreceptor dysfunction and death in STD3.

Occurrence of all-cis-5,8,11,14,17,20,23-hexacosaheptaenoic acid (26:7n-3) in roughscale sole Clidoderma asperrimum flesh lipids

Fatty acid analysis of roughscale sole Clidoderma asperrimum flesh lipids was carried out by gas chromatography. An unidentified peak appeared in the chromatogram in the elution region of ≥C24 fatty acids. After enrichment by solvent partitioning, reversed-phase thin-layer chromatography (TLC), and argentation TLC, the peak component was subjected to structural analyses. The partially hydrogenated products after reaction with hydrazine hydrate gave seven isomers of cis-hexacosenoic acid (26:1). Gas chromatography-mass spectrometry (GC–MS) analyses of their dimethyl disulfide adducts identified the monounsaturates as 5-, 8-, 11-, 14-, 17-, 20-, and 23-26:1. The peak component was assigned to all-cis-5,8,11,14,17,20,23-hexacosaheptaenoic acid (26:7n-3). GC–MS analyses of the 4,4-dimethyloxazoline derivative and methyl ester confirmed this structure. This fatty acid is a rare, very long-chain polyunsaturated fatty acid (VLCPUFA). The concentrations of the acid found in roughscale sole were 0.69 ± 0.34% (N = 5) of total fatty acids in flesh lipids. Roughscale sole appears to be characterized by the occurrence of 26:7n-3, which is lacking in popular sources of methylene-interrupted VLCPUFA, such as vertebrate retina, spermatozoa, and herring.

Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants

The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

Molecular analysis of ∆6 desaturase and ∆6 elongase from Conidiobolus obscurus in the biosynthesis of eicosatetraenoic acid, a ω3 fatty acid with nutraceutical potentials

Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a ∆6 desaturase cDNA (CoD6) and a ∆6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Δ6 double bond into α-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Δ6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20 °C to 10 °C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20 °C to 30 °C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Δ12 desaturase) and CpDesX (a ω3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.

Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24–36 carbon atoms (C24–C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC–ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/ sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24–C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/ z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC–ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.