Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of γ-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Δ6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Δ5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

Essential fatty acids for dry eye:a review

Purpose Dry eye is a common complaint often encountered in optometric practice. However, it is a difficult condition to treat as clinical signs do not always correlate with patient symptoms. Essential fatty acids (EFA), particularly omega-3 EFA, may be effective in dealing with the underlying causes. Methods A literature review was carried out on the PubMed, ScienceDirect and Ovid databases. Searches included keywords such as ‘dry eye’, ‘essential fatty acids’ and ‘nutrition’ to find articles relating to the treatment of dry eye syndrome (DES) with omega-3 EFAs. Results Omega-3 and -6 EFAs need to be consumed together within a reasonable ratio to be effective. Currently, typical diets in developed countries lack omega-3 EFA and this results in an overexposure to omega-6. Omega-3 supplementation has an anti-inflammatory effect, inhibiting creation of omega-6 prostaglandin precursors. Omega-3 EFAs also demonstrate anti-inflammatory action in the lacrimal gland preventing apoptosis of the secretory epithelial cells. Supplementation clears meibomitis, allowing a thinner, more elastic lipid layer to protect the tear film and cornea. Conclusion Dietary supplementation of omega-3 EFA has already proven to be effective in coronary heart disease and arthritis. Safety is not a concern as it works synergistically with omega-6 in the body. Evidence suggests that supplementation with omega-3 EFA may be beneficial in the treatment and prevention of DES.

Quality of polyunsaturated fatty acids in Nile tilapias (Oreochromis niloticus) fed with vitamin E supplementation

Freshwater fish are an important source of protein, but they also contain other highly nutritive components such as fats. Polyunsaturated fatty acids (PUFAs) are essential for normal growth, development and reproduction of vertebrates. The antioxidant role of vitamin E in cell membranes prevents fatty acid and cholesterol oxidation, thereby promoting PUFA and subcellular particle stabilization. The effects of vitamin E supplementation on the quality of Nile tilapia (Oreochromis niloticus) carcass were investigated. The experiments were carried out in an experimental laboratory over 106 d. After sex reversal, 400 early juvenile O. niloticus were tested in a completely randomized experiment with 5 treatments (4 repetitions each), consisting of vitamin E monophosphate supplementation at 0, 50, 100, 150 or 200 mg/kg of a base diet. Treatment diets contained equal amounts of protein and energy. Tilapias supplemented with vitamin E contained arachidonic acid (20:4 omega-6; AA) which participates in inflammatory response. Nile tilapia carcasses that received vitamin E at 100 and 150 mg/kg diet had improved carcass quality by increasing the PUFA:SFA ratio and had the highest levels of polyunsaturated fatty acids from the omega-3 (linolenic acid; 18:3 omega-3) and omega-6 (linoleic acid; 18:2 omega-6) series. (C) 2012 Elsevier Ltd. All rights reserved.

Mass spectrometric study of the dissociation of Group XI metal complexes with fatty acids and glycerolipids : Ag2H+ and Cu2H+ ion formation in the presence of a double bond

Results of mass spectrometric studies are reported for the collisional dissociation of Group XI (Cu, Ag, Au) metal ion complexes with fatty acids (palmitic, oleic, linoleic and a-linolenic) and glycerolipids. Remarkably, the formation of M2H+ ions (M = Cu, Ag) is observed as a dissociation product of the ion complexes containing more than one metal cation and only if the lipid in the complex contains a double bond. Ag2H+ is formed as the main dissociation channel for all three of the fatty acids containing double bonds that were investigated while Cu2H+ is formed with one of the fatty acids and, although abundant, is not the dominant dissociation channel. Also. Cu(I) and Ag(I) ion complexes were observed with glycerolipids (including triacylglycerols and glycerophospholipids) containing either saturated or unsaturated fatty acid substituents. Interestingly. Ag2H+ ion is formed in a major fragmentation channel with the lipids that are able to form the complex with two metal cations (triacylglycerols and glycerophosphoglycerols), while lipids containing a fixed positive charge (glycerophospocholines) complex only with a single metal cation. The formation of Ag2H+ ion is a significant dissociation channel from the complex ion Ag-2(L-H)(+) where L = Glycerophospholipid (GP) (18:1/18:1). Cu(I) also forms complexes of two metal cations with glycerophospholipids but these do not produce Cu2H+ upon dissociation. Rather organic fragments, not containing Cu(I), are formed, perhaps due to different interactions of these metal cations with lipids resulting from the much smaller ionic radius of Cu(I) compared to Ag(I) (C).

Frictional response of fatty acids on steel

Self-assembled monolayers of fatty acids were formed on stainless steel by room-temperature solution deposition. The acids are covalently bound to the Surface as carboxylate in a bidentate manner. To explore the effect Of Saturation in the carbon backbone on friction in sliding tribology, we Study the response of saturated stearic acid (SA) and unsaturated linoleic acid (LA) as self-assembled monolayers using lateral force microscopy and nanotribometry and when the molecules are dispersed in hexadecane, using pin-on-disc tribometry. Over a very wide range (10 MPa-2.5 GPa) of contact pressures it is consistently demonstrated that the unsaturated linoleic acid molecules yield friction which is significantly lower than that of the saturated stearic acid. it is argued, using density functional theory predictions and XPS of slid track, that when the molecular backbone of unsaturated fatty acids are tilted and pressed strongly by a probe, in tribological contact, the high charge density of the double bond region of the backbone allows coupling with the steel Substrate. The interaction yields a low friction carboxylate soap film on the substrate. The saturated fatty acid does not show this effect.

Solubilities of palmitic and stearic fatty acids in supercritical carbon dioxide

The solubilities of two fatty acids, namely hexadecanoic acid (palmitic acid) and octadecanoic acid (stearic acid) in supercritical carbon dioxide (SCCO2), were determined at T = (328 and 338) K from 12.8 MPa to 22.6 MPa. Three models, namely a thermodynamic model based on the Peng-Robinson equation of state with Kwak and Mansoori mixing rules, a model based on dilute solution theory proposed by Mendez-Santiago and Teja and a new reformulated Chrastil equation model, were used to correlate the solubilities. In all the models, the correlation constants are temperature independent. All the models successfully correlated the experimental results for the solubilities of hexadecanoic acid within 3%.

The triglyceride fatty acids from heart lipids of Puntius sarana and their variation with different sizes

The triglyceride fatty acid components from the heart lipid of Puntius sarana of different sizes have been characterized by thin-layer and gas liquid chromatography. Csub(10) to Csub(24) acids including both odd-numbered and branched chain acids were detected. The major constituents were ante-iso Csub(10), Csub(10), Csub(12:2), Csub(14), Csub(16), Csub(16:1),Csub(17), Csub(18) , Csub(18:1), Csub(18:2), Csub(18:3) and Csub(20:4) while twenty other acids were detected in lower proportion. The composition of these acids and their variation with size of fish have been investigated and discussed.

Influence of cold storage time on the protein changes and fatty acids deterioration of (Rutilus frisi kutum) during frozen storage

The main aim of this research was to identify fatty acids composition of Caspian sea of White fish Rutilus frisi kutum tissue and their changes during one year cold storage (-18Ċ).The secondary aim was to determine the changes of moisture, ash, protein, fat, and to investigate the effects of storage time on peroxide, TBAi, FFA, and extractability of myofibrillar proteins of the fish tissue during one year cold storage (-18 Ċ). 10 samples of (Rutilus frisi kutum) were randomly collected from Anzali landings. The samples were frozen at -30 Ċ and kept in cold storage at -18Ċ for one year. According to time table, the samples were examined. The results showed that 27 fatty acids were identified. The unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were 74/09 and 21/63 %, respectively, in fresh tissue. So that DHA (C22:6) oleic acid (C18:1c) had high amounts (15/07 ,20/57 ) among the UFA and palmitic acid (C16:0) was the most (13/09 %) among the SFA. The effects of freezing and cold storage on fish tissue showed that UFA and SFA contents have reached to 58/79 and 22/17 %, respectively, at the end of cold storage. It indicated that these compound change to each other during frozen storage. Also ω-3 and ω-6 series of fatty acids was 24/22 and 15/56% in fresh tissue, but their contents decreased to 8/68 and 5/11% at the end of period. Among the fatty acids C22:6, C18:1c and C16:0 had the most changes. The changes of fatty acids were significantly at 95% level expected for C18:0. Results showed that moisture, ash, protein, and fat contents were 75/9±0/03, 1/28±0/012, 21/8±0/2, and 4/1±0/01 % respectively, in fresh tissue. The moisture, ash, protein, and fat contents were 72/3±0/04, 1/83±0/05, 1/91±0/01 and 19/9±0/01 % respectively, at the end of storage period. Lipid damage was measured on the basis of free fatty acids (FFA), peroxide value (PV), and Thiobarbituric acid index (TBA-i). PV, TBARS and FFA concentration of frozen Caspian Sea white fish stored at -18 Ċ the temporal variation of these three variables were statistically significant (p<0.001). Results of White fish myofibrillar proteins showed aggregation of bound reduced for stored at 12 months. SDS-PAGE analysis revealed that, the intensity of the myosin heavy chain and actin bound was reduced with increasing storage time. SDS-PAGE patterns showed that myosin heavy chain was much more susceptible to hydrolysis than actin. Key words: Rutilus frisi kutum, frozen storage, ω-3, ω-6, protein myofibrillar