Occurrence and characterization of oils rich in gamma-linolenic acid (III): the taxonomical value of the fatty acids in Echium (Boraginaceae)

Fourteen species of the genus Echium (Fam. Boraginaceae) collected in the Macaronesia were surveyed in a search for high levels of gamma-linolenic acid (GLA, 18:3omega6) in the seed oil. High amounts of this fatty acid were found in all of them, ranging from 18.85% (E. pitardii var. pitardii) to 27.42% (E. gentianoides) on total seed fatty acids. The GLA content related to total seed weight was also significant, ranging from 1.26% (E. handiense) to 8.22% (E. gentianoides). In addition, considerable amounts of stearidonic acid (SA, 18:4omega3) were detected, ranging from 3.78% (E. bonnetii var. bonnetii) to 8.81% (E. pininana) on total fatty acids. Besides all the perennial species, the four herbaceous Echium taxa endemic to the Macaronesia also showed high GLA percentages. This is in contrast to the low GLA level found in continental Echium species, all of them bearing an herbaceous habit. These results are in good agreement with the available genetic data and show the ability of GLA to discriminate between Macaronesian and continental Echium species. The analysis of five other Macaronesian species belonging to plant families rich in GLA are also reported.     

Occurrence and characterization of oils rich in gamma-linolenic acid part II: fatty acids and squalene from Macaronesian Echium leaves

Leaves from 25 Macaronesian Echium (Boraginaceae) species have been surveyed for hydrocarbon compounds. These plants were previously reported as the major source of gamma-linolenic acid so far found in nature. In addition, six European Echium species and the common Borago officinalis have been analysed for comparative purposes. High squalene amounts were found in all Echium plants from the Macaronesia, ranging from 3.73%, in E. simplex to 20.1% in E. fastousum. Squalene was almost absent from all European Echium species, and the same is true for B. officinalis. The relatively high oil content (2.27%) in leaves of E. fastuosum raises the total squalene amount to about 0.46% within this tissue. The main fatty acid component in the leaf was alpha-linolenic acid (ALA, 18:3omega3), ranging in the Macaronesian Echium from 9.32% in E. acanthocarpum to 54.45% in E. simplex. Possible utilisation of these plants as a commercial source of squalene and hypotheses about its physiological role in the plant are discussed.     

Substrate specificity of acyl-Delta(6)-desaturases from Continental versus Macaronesian Echium species

Echium (Boraginaceae) species from the Macaronesian islands exhibit an unusually high level of gamma-linolenic acid (18:3n-6; GLA) and relatively low content of octadecatetraenoic acid (18:4n-3; OTA) in the seed, while the amounts of both fatty acids in their Continental (European) relatives are rather similar. We have tested the hypothesis of whether a different specificity of the acyl-Delta(6)-desaturases (D6DES) towards their respective usual substrates, linoleic acid (18:2n-6; LA) for GLA and alpha-linolenic acid (18:3n-3; ALA) for OTA, was partly responsible for this composition pattern. To this aim we have expressed in yeast the coding sequences of the D6DES genes for the Continental species Echium sabulicola, and the Macaronesian Echium gentianoides. When the yeast cultures are supplemented with the two fatty acid substrates (LA and ALA), a similar utilization of both compounds was found for the D6DES of E. sabulicola, while a preference for LA over ALA was observed for the enzyme of E. gentianoides. This substrate preference must contribute to the increased accumulation of GLA in the seeds of the Macaronesian Echium species. Comparison among the amino acid sequences of these desaturases and other related enzymes, allowed us the discussion about the possible involvement of some specific positions in the determination of substrate specificity.     

Replacement of dietary fish oil for Atlantic salmon parr (Salmo salar L.) with a stearidonic acid containing oil has no effect on omega-3 long-chain polyunsaturated fatty acid concentrations

The worldwide increase in aquaculture production and the concurrent decrease of wild fish stocks has made the replacement of fish oil in aquafeeds an industry priority. Oil from a plant source Echium plantagineum L., Boraginaceae, has high levels of stearidonic acid (SDA, 18:4omega3, 14%) a biosynthetic precursor of omega-3 long-chain (> or =C(20)) polyunsaturated fatty acids (omega3 LC-PUFA). Atlantic salmon (Salmo salar L.) parr were fed a control fish oil diet (FO) or one of 3 experimental diets with 100% canola oil (CO) 100% SDA oil (SO), and a 1:1 mix of CO and SDA oil (MX) for 42 days. There were no differences in the growth or feed efficiency between the four diets. However, there were significant differences in the fatty acid (FA) profiles of the red and white muscle tissues. Significantly higher amounts of SDA, eicosapentaenoic acid (20:5omega3, EPA), docosahexaenoic acid (22:6omega3, DHA) and total omega3 FA occurred in both red and white muscle tissues of fish fed SO and FO compared with those fed CO. Feeding SO diet resulted in omega3 LC-PUFA amounts in the white and red muscle being comparable to the FO diet. This study shows that absolute concentration (mug/g) of EPA, DHA and total omega3 have been maintained over 6 weeks for Atlantic salmon fed 14% SDA oil. The balance between increased biosynthesis and retention of omega3 LC-PUFA to maintain the concentrations observed in the SO fed fish remains to be conclusively determined, and further studies are needed to ascertain this.     

Changes in the content of gamma-linolenic C18:3 (n-6) and stearidonic C18:4 (n-3) acids in developing seeds of viper's bugloss Echium vulgare L

Changes in the composition of fatty acids (FA) were determined in lipid extracts isolated from developing ovaries containing ovules and developing seeds of Echium vulgare L. The samples were collected successively over 20 days beginning with the first day after flowering. The contents of the n-6 FA family members, i.e., gamma-linolenic (GLA) (C(18:3)) and linoleic (LA) (C(18:2)) acids changed in a parallel manner and reached the maximum of 13.9% and 24%, respectively, on the 12th day, after which they fell systematically down to 8.6% and 18.2%, respectively, on the 20th day after flowering. Starting with day 13, the content of alpha-linolenic acid (ALA) (C(18:3) n-3) begins to grow intensively, from 24.2% to 39.3% on the 20th day after flowering. The increase in the content of stearidonic acid (SDA) (C(18:4) n-3), up to 10.5% on the 20th day after flowering, occurred steadily as the seeds developed, and was independent of the changes in the content of GLA and LA. The pattern of changes in the content of SDA, GLA, LA and ALA during the development of seeds, and the occurrence of SDA in the seed oil of other plants, demonstrate that the biosynthesis of SDA in the seeds is critically dependent on the presence of ALA. The above condition indicates that SDA biosynthesis in the seeds of Echium vulgare follows the scheme LA --> simultaneous, competitive, action of Delta(6) and Delta(15) desaturases, leading to the formation of GLA and ALA, respectively, and then ALA (Delta(6) des) --> SDA. The biosynthesis according to the scheme: GLA (Delta(15) des) --> SDA is highly unlikely.     

Dose-dependent effects of dietary gamma-linolenic acid on rat spleen lymphocyte functions.

Feeding rodents a diet rich in evening primrose oil (EPO), which contains 5-10 g gamma-linolenic acid (GLA)/100 g total fatty acids, has been shown to decrease lymphocyte proliferation and natural killer cell activity. However, EPO contains a very high level of linoleic acid which itself can affect lymphocyte functions and it is not clear to what extent the effects of EPO can be attributed to GLA. The current study investigated the effect of two levels of GLA in the rat diet upon immune cell functions; the level of linoleic acid was maintained below 30 g/100 g total fatty acids. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g or 10 g GLA/100 g total fatty acids in place of a proportion of linoleic acid. The total polyunsaturated fatty acid content and the n-6 to n-3 polyunsaturated fatty acid ratio of the diet were maintained at 35 g/100 g total fatty acids and 7, respectively. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet, with an increase in the proportions of GLA and dihomo-gamma-linolenic acid when the diets containing GLA were fed; these diets also increased the proportion of arachidonic acid in spleen leukocytes. Spleen lymphocyte proliferation in response to concanavalin A was significantly reduced (by 60%) by feeding the diet containing the higher level of GLA, but not by the diet containing the lower level of GLA. Spleen natural killer cell activity and prostaglandin E (PGE) production by spleen leukocytes were not significantly affected by inclusion of GLA in the diet, although there was a tendency towards decreased natural killer cell activity by cells from rats fed the high GLA diet. Thus, this study shows that dietary GLA is capable of altering the fatty acid composition of cells of the immune system and of exerting some immunomodulatory effects, but that the level of GLA in the diet must exceed 4.4 g/100 g total fatty acids for these effects to become apparent.     

gamma-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals

Oleaginous fungi of the genus Mucorales were screened for gamma-linolenic acid (GLA) production on solid substrates containing moistened cereals. Cunninghamella elegans CCF 1318 produced the highest yields of GLA when cultivated on barley. Substrate moisture and cultivation temperature proved critical for effective GLA production. Vegetable oil supplied to the cultures improved GLA production. Rotating bottles and plastic bags were used as cultivation vessels to reproduce the conditions found in rotating drums and tray bioreactors, respectively. After 11 days of cultivation at 21 degrees C, C. elegans produced 14.2 mg of GLA per gram of dry substrate, composed of a mixture of barley, spent malt grains (SMG) and peanut oil. GLA represented 15.6% of the total fatty acids in the lipid extract.     

Delta-6 desaturase makes a different fatty acid profile of seed oil in Iranian borage (Echium amoenum Fisch. and Mey.)

Iranian borage (Echium amoenum) from Boraginaceae is a valuable medicinal plant native to Iran and Syria. We determined fatty acid profile and individual fatty acid contents in E. amoenum seed oil using gas chromatography. Nevertheless, the cumulative amount of ω-6 and ω-3 fatty acids in E. amoenum (78.5 %) was in great accordance with those of other species of Echium, Stearidonic acid (SDA, C18:4ω3) and gamma-linolenic acid (GLA, C18:3ω6) constituted only 9.7 % of seed oil in this plant. This observation elucidated the weak activity of delta-6 desaturase (D6DES) in E. amoenum compared with D6DESs of other species. As D6DES enzyme is responsible for converting linoleic acid (LA, C18:2ω6) and alpha-linolenic acid (ALA, C18:3ω3) to GLA and SDA, we isolated coding sequence of D6DES gene and characterized primary structure of the translated protein to probably find some evidences explaining the weak activity of D6DES enzyme in E. amoenum. Gene sequence from E. amoenum showed a high identity of 94–96 % with the other Echium species and the amino acid homology increased by 98 %. All the expected signatures including cytochrome b5 domain and three conserved histidine-rich motifs were found in the translated amino acid sequence. Protein alignment revealed that all the conserved motifs in D6DES sequence from E. amoenum are coincident with its counterparts from other Echium species. However, secondary structure of the enzyme deduced from its primary structure using computational simulation represented obvious differences with D6DES proteins of the other species.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

High-level production of gamma-linolenic acid in Brassica juncea using a delta6 desaturase from Pythium irregulare

gamma-Linolenic acid (GLA), a nutritionally important fatty acid in mammals, is synthesized by a delta6 desaturase. Here, we report identification of PiD6, a new cDNA from the oleaginous fungus, Pythium irregulare, encoding a 459-amino acid protein that shares sequence similarity to carboxyl-directed desaturases from various species. Expression of PiD6 in yeast (Saccharomyces cerevisiae) revealed that it converts exogenously supplied linoleic acid into GLA, indicating that it encodes a delta6 fatty acid desaturase. Expression of the desaturase in Brassica juncea under the control of the Brassica napus napin promoter resulted in production of three delta6 unsaturated fatty acids (18:2-6, 9; 18:3-6, 9, 12; and 18:4-6, 9, 12, 15) in seeds. Among them, GLA (18:3-6, 9, 12) is the most abundant and accounts for up to 40% of the total seed fatty acids. Lipid class and positional analysis indicated that GLA is almost exclusively incorporated into triacylglycerol (98.5%) with only trace amounts found in the other lipids. Within triacylglycerols, GLA is more abundant at the sn-2 position.     

Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid

The delta 6-desaturase gene was cloned from Rhizopus stolonifer, which could accumulate up to 49% of gamma-linolenic acid (GLA, C18:3 Δ^6,9,12) to the total fatty acids. The cloned DNA contains a 1,380 bp open reading frame encoding a protein of 460 amino acids, which showed high similarity to those of fungal delta 6-desaturases with three conserved histidine-rich motifs and HPGG motif. Notably, this deduced sequence had a shorter C-terminus. Results demonstrated that the cDNA sequence exhibited delta 6-desaturase activity by accumulation of about 22.4 % of GLA to the total fatty acids in the recombinant Pichia pastoris strain GS115.     

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova?400.     

Association of calmodulin inhibition, erythrocyte membrane stabilization and pharmacological effects of drugs

Isolated liver cells from rats fed a diet deficient in essential fatty acids were used to study the oxidation, esterification and, especially, the desaturation and chain elongation of [1-14C]linoleic acid. 14C-labelled arachidonic acid (20:4) and smaller amounts of eicosatrienoic acid (20:3) were recovered mainly in the phospholipids, while gamma-linolenic acid (18:3) was found in both the phospholipids and the triacylglycerol fraction. Lactate strongly increased the formation of arachidonic acid, which was found mainly in the phosphatidylcholine and the phosphatidylinositol fractions. Lactate reduced the amounts of gamma-linolenic acid. Glucagon and (+)-decanoylcarnitine reduced the formation of arachidonic acid, and (+)-decanoylcarnitine increased the incorporation of gamma-linolenic acid especially, in the triacylglycerol fraction. Increasing concentrations of the [1-14C]linoleic acid substrate increased the formation of arachidonic acid and of the other chain-elongated or desaturated fatty acids. Lactate also stimulated the formation of arachidonic acid in liver cells from animals fed adequate amounts of essential fatty acids. It is suggested that dietary and hormonal factors which can change the intracellular levels of malonyl-CoA may influence both the ratio of arachidonic acid/gamma-linolenic acid formed and the total amounts of desaturated and chain-elongated fatty acids formed from linoleic acid.     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.     

Fatty acid and prostaglandin metabolism in children with diabetes mellitus. II. The effect of evening primrose oil supplementation on serum fatty acid and plasma prostaglandin levels

Our previous study demonstrated that levels of dihomo-gamma-linolenic acid (DGLA) and arachidonic acid in serum total lipids decreased in association with increased plasma levels of prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) in patients with insulin-dependent diabetes mellitus. In this study, 11 children with insulin-dependent diabetes mellitus completed a double-blind, placebo-controlled study to assess the effect of dietary supplementation with gamma-linolenic acid (GLA) on serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. GLA was given as the seed oil from the evening primrose (EPO) and all patients received either EPO capsules (containing 45 mg of GLA and 360 mg of linoleic acid) or indistinguishable placebo capsules for 8 months. Initially patients took 2 capsules daily for 4 months then 4 capsules daily for a further 4 months. All patients were assessed at the start of the study, after 4 months and at the end of the study, by measuring serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. After administration of 4 capsules daily the DGLA levels increased and PGE2 levels decreased significantly (p less than 0.01) in the EPO compared with the placebo group. Neither fatty acid nor PGE2 and PGF2 alpha levels were altered by administration of 2 EPO capsules daily. This suggests that the altered essential fatty acid and PG metabolism in diabetes may be reversed by direct GLA supplementation.     

Lipids and fatty acids in muscle, liver and skin of three edible fish from the Senegalese coast: Sardinella maderensis, Sardinella aurita and Cephalopholis taeniops

Lipid content and fatty acid composition were determined in three species of edible fish caught in Senegalese waters during the upwelling season (January, 1993). Sardinella maderensis and Sardinella aurita are fat fish containing more than 5% (fresh wt.) of lipids, whereas Cephalopholis taeniops is a lean fish with approximately 1% of lipids. Skin, liver and muscle were studied for each fish species. About 40 fatty acids were identified by GC and GC/MS as methyl esters and N-acyl pyrrolidides. Palmitic acid was the main acid in the muscle and skin of all samples studied (20-33% of total fatty acids). Oleic acid was the main fatty acid in the liver of S. maderensis (27.2%+/-0.1) and S. aurita (44.7%+/-0.1). Arachidonic acid was a minor component in all samples. The flesh (muscle) of the three fish species contained high concentrations of omega3 polyunsaturated fatty acids (PUFA), ranging from 16.0 to 29.1% and including 20:5 omega3 (eicosapentaenoic acid, EPA) and 22:6 omega3 (docosahexaenoic acid, DHA) acids as major components. These two acids together accounted for 24.7%+/-0.1 and 12.9%+/-0.1 of total acids in the skin of S. maderensis and S. aurita, respectively. The percentages of PUFA found in the fish studied were very similar to those in fish used commercially as sources of PUFA. Muscle sterols, which accounted for 9-11% of total lipids, consisted mainly of cholesterol (up to 97% of total sterols).     

Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins

During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased. During fatty acid synthesis Rhizopus sp. produced only gamma-linolenic acid (GLA) instead of ALA. The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined. There was a distinct increase in the amount of free amino acids during tempe fermentation. Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids. In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans. In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid. The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe. In the presence of the Rhizopus mold, the vitamin B(12) formation by C. freundii was three times higher than that of a fermentation without the mold.     

Comparative levels of arachidonic acid and eicosapentaenoic acid in Malaysian fish

Total lipids were extracted from 22 species of Malaysian fish and the constituent fatty acids were analysed by gas chromatography. Malaysian fish generally contained high levels of saturated fatty acids (range 36-55% total fatty acids) and contained variable amounts of monounsaturates, chiefly palmitic and stearic acids, but only trace levels of 20:1 and 22:1. Unlike fish caught in colder northern hemisphere waters, Malaysian fish were found to contain arachidonic acid (20:4 omega 6, range 2-12%) in addition to the expected eicosapentaenoic acid (20:5 omega 3, range 1-13%) and docosahexaenoic acid (22:6 omega 3, range 6.6-40.4%).