Variation in fatty acid composition of <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (Spirulina) strains

A study of the fatty acid composition was made for 35 Arthrospira strains, concentrating on the most abundant fatty acids, the two polyunsaturated C₁₈ acids, linoleic and γ-linolenic acid, and palmitic acid. When grown at 30 ^∘C and low irradiance (10 μmol photon m⁻² s⁻¹), these three acids together formed 88–92% of total fatty acids. There were considerable differences in the composition of the two polyunsaturated acids. Depending on the strain, linoleic acid formed 13.1–31.5% and γ-linolenic acid formed 12.9–29.4% total fatty acids. In contrast, the range for palmitic acid was narrow: 42.3–47.6% of total fatty acids. Repeat experiments on several strains under defined conditions led to closely similar results for any particular environment, suggesting that fatty acid composition can be used as an aid in differentiating between strains. Five additional strains, which had apparently originated from the same original stock cultures as 3 of the 35 in the main study, but from different culture collections, were also assayed. With four strains the results were similar, irrespective of culture source, but with one strain marked differences occurred, especially in the polyunsaturated C₁₈ fatty acid fraction. These differences were independent of the age of the culture. In addition, straight morphotypes derived during repeat subcultures of four strains; each showed a similar fatty acid composition to that of the helical morphotypes of the same strains. A decrease in temperature from 30 to 20 ^∘C, an increase in irradiance (at 30 ^∘C) from 10 to 70 μmol photon m⁻² s⁻¹ and transfer to dark heterotrophy all favoured an increase in polyunsaturated C₁₈ fatty acids. The highest γ-linolenic acid content of any conditions was found for three strains grown heterotrophically on glucose in the dark at 30 ^∘C. A comparative study of six strains of Spirulina confirmed a previous study showing the absence of γ-linolenic acid in all Spirulina strains, thus permitting the separation of these two genera.     

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage delta6-desaturase

The improvement of nutritional quality is one potential application for the genetic modification of plants. One possible target for such manipulation is the modification of fatty acid metabolism. In this work, expression of a borage Δ⁶-desaturase cDNA in tomato (Lycopersicon esculentum L.) has been shown to produce γ-linolenic acid (GLA; 18:3 Δ^6,9,12) and octadecatetraenoic acid (OTA; 18:4 Δ^6,9,12,15) in transgenic leaf and fruit tissue. This genetic modification has also, unexpectedly, resulted in a reduction in the percentage of linoleic acid (LA 18:2 Δ^9,12) and a concomitant increase in the percentage of α-linolenic acid (ALA; 18:3 Δ^9,12,15) in fruit tissue. These changes in fatty acid composition are thought to be beneficial for human health.     

Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Effects of CO(2) Concentration during Growth on Fatty Acid Composition in Microalgae

The degree of unsaturation of fatty acids was higher in Chlorella vulgaris 11h cells grown with air (low-CO₂ cells) than in the cells grown with air enriched with 2% CO₂ (high-CO₂ cells). The change in the ratio of linoleic acid to α-linolenic acid was particularly significant. This change of the ratio was observed in four major lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine, and phosphatidylethanolamine). The relative contents of lipid classes were essentially the same both in high-CO₂ and low-CO₂ cells. After high-CO₂ cells were transferred to low CO₂ condition, total amount of fatty acids remained constant but the relative content of α-linolenic acid increased during a 6-hour lag phase in growth with concomitant decreases in linoleic and oleic acids. When low-CO₂ cells were transferred to high CO₂ condition, total amount of fatty acids and relative content of oleic acid increased significantly. The amount of α-linolenic acid remained almost constant, while the amounts of palmitic, oleic, and linoleic acids increased. Similar, but smaller, changes in fatty acid compositions were observed in two species of green algae Chlamydomonas reinhardtii and Dunaliella tertiolecta. However, no difference was found in Euglena gracilis, Porphyridium cruentum, Anabaena variabilis, and Anacystis nidulans.     

Isolation and functional characterization of a delta 6-desaturase gene from the pike eel (Muraenesox cinereus)

Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR methods. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.     

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

Plant desaturases comprise two independently evolved classes, a structurally well characterized soluble class responsible for the production of monoenes in the plastids of higher plants and the poorly structurally characterized integral membrane class that has members in the plastid and endoplasmic reticulum that are responsible for producing mono- and polyunsaturated fatty acids. Both require iron and oxygen for activity and are inhibited by azide and cyanide underscoring their common chemical imperatives. We previously showed that the Δ⁹ acyl-CoA integral membrane desaturase Ole1p from Saccharomyces cerevisiae exhibits dimeric organization, like the soluble plastidial acyl-ACP desaturases. Here we use two independent bimolecular complementation assays, i.e. yeast two-hybrid analysis and Arabidopsis leaf protoplast split luciferase assay, to demonstrate that members of the plant integral membrane fatty acid desaturase (FAD) family, FAD2, FAD3, FAD6, FAD7, and FAD8, self-associate. Further, the endoplasmic reticulum-localized desaturase FAD2 can associate with FAD3, as can the plastid-localized FAD6 desaturase with either FAD7 or FAD8. These pairings appear to be specific because pairs such as FAD3 and FAD7 (or FAD8) and FAD2 and FAD6 do not interact despite their high amino acid similarity. These results are consistent also with their known endoplasmic reticulum and plastid subcellular localizations. Chemical cross-linking experiments confirm that FAD2 and FAD3 can form dimers like the yeast Ole1p and, when coexpressed, can form FAD2-FAD3 heterodimers. Metabolic flux analysis of yeast coexpressing FAD2 and FAD3 indicates that heterodimers can form a metabolic channel in which 18:1-PC is converted to 18:3-PC without releasing a free 18:2-PC intermediate.     

Cytochrome b5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2^Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b₅ protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b₅ domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b₅s were coexpressed with the desaturase. Coexpression of a cytochrome b₅ from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b₅ proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.     

Oleic,Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47^phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47^phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.     

Functional characterization of a delta 6-desaturase gene from the black seabream (<Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>)

Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

Metabolic engineering to produce γ-linolenic acid in <Emphasis Type="Italic">Brassica napus</Emphasis> using a Δ6-desaturase from pike eel

We generated γ-linolenic acid (GLA, C18:3^Δ6,9,12)-producing transgenic Brassica napus transformed with McD6DES, the Δ6-desaturase gene identified from pike eel (Muraenesox cinereus) under the control of the seed-specific vicilin promoter. Seed-specific expression of McD6DES in B. napus produced up to 8.4% of GLA by creating a double bond at the sixth position from the carboxyl end of linoleic acid (LA, C18:2^Δ9,12) in seeds. These results demonstrate that McD6DES expression enables to reconstitute in polyunsaturated fatty acid biosynthetic pathways, highlighting the potential of GLA biosynthesis as a target for metabolic engineering of oilseed crops.     

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways,and Antifungal Activity in Bread

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C_18:1 fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter⁻¹. Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter⁻¹. L. hammesii accumulated the monohydroxy C_18:1 fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter⁻¹ (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.     

Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans

Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l^?1 linoleic acid, 23 g l^?1 cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l^?1 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l^?1 h^?1. This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.     

Excess Linoleic Acid Increases Collagen I/III Ratio and “Stiffens” the Heart Muscle Following High Fat Diets

Controversy exists on the benefits versus harms of n-6 polyunsaturated fatty acids (n-6 PUFA). Although n-6 PUFA demonstrates anti-atherosclerotic properties, survival following cardiac remodeling may be compromised. We hypothesized that n-6 PUFA like linoleic acid (LA) or other downstream PUFAs like γ-linolenic acid or arachidonic acid alter the transforming growth factor-β (TGFβ)-collagen axis in the heart. Excess dietary LA increased the collagen I/III ratio in the mouse myocardium, leading to cardiac “stiffening” characterized by impaired transmitral flow indicative of early diastolic dysfunction within 5 weeks. In vitro, LA under TGFβ1 stimulation increased collagen I and lysyl oxidase (LOX), the enzyme that cross-links soluble collagen resulting in deposited collagen. Overexpression of fatty acid desaturase 2 (fads2), which metabolizes LA to downstream PUFAs, reduced collagen deposits, LOX maturation, and activity with LA, whereas overexpressing fads1, unrelated to LA desaturation, did not. Furthermore, fads2 knockdown by RNAi elevated LOX activity and collagen deposits in fibroblasts with LA but not oleic acid, implying a buildup of LA for aggravating such pro-fibrotic effects. As direct incubation with γ-linolenic acid or arachidonic acid also attenuated collagen deposits and LOX activity, we concluded that LA itself, independent of other downstream PUFAs, promotes the pro-fibrotic effects of n-6 PUFA. Overall, these results attempt to reconcile opposing views of n-6 PUFA on the cardiovascular system and present evidence supporting a cardiac muscle-specific effect of n-6 PUFAs. Therefore, aggravation of the collagen I/III ratio and cardiac stiffening by excess n-6 PUFA represent a novel pathway of cardiac lipotoxicity caused by high n-6 PUFA diets.     

Evaluation of fatty acid composition among selected amaranth grains grown in two consecutive years

The oil of amaranth grain (Amaranthus spp.) is a rich source of poly-unsaturated fatty acids. In this study, we tested 10 amaranth samples representing two species (Amaranthus cruentus and Amaranthus hypochondriacus) in two consecutive years (2010, 2011). Grain oils were analysed by gas chromatography for their fatty acids profile. In 2010, oil content ranged from 6.4–8.2% for A. cruentus and 6.3–7.9% for A. hypochondriacus. In 2011, the level was 7.1–8.2% and 6.6–8.7% for A. cruentus and A. hypochondriacus, respectively. Linoleic, palmitic, and oleic acids were dominant fatty acids in all of the oil samples. The essential linoleic acid level was 33.3–38.7% (A. cruentus) and 31.7–47.5% (A. hypochondriacus) in 2010 and 34.6–39.9% (A. cruentus) and 34–44.5% (A. hypochondriacus) in 2011. The minority fatty acids, i.e. stearic, α-linolenic, and arachidic acids were also observed. Eicosenoic and behenic acids were present in the grain in trace amounts. Statistical evaluation showed a significant effect of year and species of amaranth on the levels of certain fatty acids. There was a strong positive correlation between oil content and oleic acid, and a negative correlation between oleic acid and either of the other two fatty acids, linoleic and α-linolenic ones.