Δ5-Olefinic acids in the seed lipids from four Ephedra species and their distribution between the α and β positions of triacylglycerols. Characteristics common to coniferophytes and cycadophytes

The fatty acid compositions of the seed lipids from four Ephedra species, E. nevadensis, E. viridis, E. przewalskii, and E. gerardiana (four gymnosperm species belonging to the Cycadophytes), have been established with an emphasis on Δ5-unsaturated polymethylene-interrupted fatty acids (Δ5-UPIFA). Mass spectrometry of the picolinyl ester derivatives allowed characterization of 5,9- and 5,11–18∶2; 5,9,12–18∶3; 5,9,12,15–18∶4; 5,11–20∶2; 5,11,14–20∶3; and 5,11,14,17–20∶4 acids. Δ5-UPIFA with a Δ11-ethylenic bond (mostly C₂₀ acids) were in higher proportions than δ5-UPIFA with a δ9 double bond (exclusively C₁₈ acids) in all species. The total δ5-UPIFA content was 17–31% of the total fatty acids, with 5, 11, 14–20∶3 and 5, 11, 14, 17–20∶4 acids being the principal δ5-UPFIA isomers. The relatively high level of cis-vaccenic (11–18∶1) acid found in Ephedra spp. seeds, the presence of its δ5-desaturation product, 5, 11–18∶2 acid (proposed trivial name: ephedrenic acid), and of its elongation product, 13–20∶1 acid, were previously shown to occur in a single other species, Ginkgo biloba, among the approximately 170 gymnosperm species analyzed so far. Consequently, Ephedraceae and Coniferophytes (including Ginkgoatae), which have evolved separately since the Devonian period (≈300 million yr ago), have kept in common the ability to synthesize C₁₈ and C₂₀ δ5-UPIFA. We postulate the existence of two δ5-desaturases in gymnosperm seeds, one possibly specific for unsaturated acids with a δ9-ethylenic bond, and the other possibly specific for unsaturated acids with a δ11-ethylenic bond. Alternatively, the δ5-desaturases might be specific for the chain length with C₁₈ unsaturated acids on the one hand and C₂₀ unsaturated acids on the other hand. The resulting hypothetical pathways for the biosynthesis of δ5-UPIFA in gymnosperm seeds are only distinguished by the position of 11–18∶1 acid. Moreover, ¹³C nuclear magnetic resonance spectroscopy of the seed oil from two Ephedra species has shown that δ5-UPIFA are essentially excluded from the internal position of triacylglycerols, a characteristic common to all of the Coniferophytes analyzed so far (more than 30 species), with the possibility of an exclusive esterification at the sn-3 position. This structural feature would also date back to the Devonian period, but might have been lost in those rare angiosperm species containing δ5-UPIFA.     

The effect of isomerictrans-18∶1 acids on the desaturation of palmitic,linoleic and eicosa-8,11,14-trienoic acids by rat liver microsomes

The inhibitory effects of the positional isomers oftrans-18∶1 acids on the desaturation of palmitic acid to palmitoleic (Δ9-desaturase), linoleic to γ-linolenic (Δ6-desaturase) and eicosa-8,11,14-trienoic to arachidonic acid (Δ5-desaturase) were investigated. Thesetrans-18∶1 acids were found to be inhibitory for the microsomal Δ6-, Δ9- and Δ5-desaturases of rat liver. The position of the double bond in thetrans-18∶1 acids seems to be important in determining the degree of inhibition. At inhibitor/substrate ratio of 3∶1, the Δ6-desaturase was most strongly inhibited bytrans-Δ3,-Δ4,-Δ7 and-Δ15-18∶1 isomers, whereas the Δ9-desaturase was most strongly inhibited bytrans-Δ3,-Δ5,-Δ7,-Δ10,-Δ12,-Δ13 and-Δ16 isomers. At inhibitor/substrate ratio of 6∶1, the Δ5-desaturase was most strongly inhibited by Δ3-, Δ9-, Δ13- and Δ15-isomers. When 18∶0 was added to the incubations of 16∶0, 18∶2 and 20∶3 at the same I/S ratios used for thetrans-18∶1 acids, weak inhibition for Δ9-desaturase and no inhibition for Δ5-and Δ6-desaturases was observed.     

cis-5-olefinic unusual fatty acids in seed lipids of gymnospermae and their distribution in triacylglycerols

Open-tubular gas chromatographic analysis of fatty acids in the lipids from the seeds of 20 species of Gymnospermae showed that they all contained nonmethylene-interrupted polyenoic (NMIP) acids as minor components and palmitic, oleic, linoleic and α-linolenic acids as major components. The NMIP acids have an additional 5,6-ethylenic bond in ordinary plant unsaturated fatty acids and the following C₂ elongation acids:cis-5,cis-9-octadecadienoic acid (5,9–18∶2) (I); 5,9,12–18∶3 (II); 5,9,12,15–18∶4, 5,11–20∶2, 5,11,14–20∶3 (III); and 5,11,14,17–20∶4 (IV). The main NMIP acids found in neutral lipids are I in two species ofTaxus, II in seven species of Pinaceae, III in two species of Podocarpaceae,Torreya nucifera, Cycas revoluta, andGinkgo biloba, and III and IV in each of three species of Taxodiaceae, and Cupressaceae. The polar lipids constitute the minor fraction of seed lipids in general. The content and composition of NMIP acids in these lipids differe considerably from those in neutral lipids. Analysis of the partial cleavage products of triacylglycerols showed that the NMIP acids distribute mainly in the 1,3-position.     

General characteristics of Pinus spp. Seed fatty acid compositions, and importance of Δ5-olefinic acids in the taxonomy and phylogeny of the genus

The Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) contents and profiles of gymnosperm seeds are useful chemometric data for the taxonomy and phylogeny of that division, and these acids may also have some biomedical or nutritional applications. We recapitulate here all data available on pine (Pinus; the largest genus in the family Pinaceae) seed fatty acid (SFA) compositions, including 28 unpublished compositions. This overview encompasses 76 species, subspecies, and varieties, which is approximately one-half of all extant pines officially recognized at these taxon levels. Qualitatively, the SFA from all pine species analyzed so far are identical. The genus Pinus is coherently united—but this qualitative feature can be extended to the whole family Pinaceae—by the presence of Δ5-UPIFA with C₁₈ [taxoleic (5,9–18∶2) and pinolenic (5,9,12–18∶3) acids] and C₂₀ chains [5,11–20∶2, and sciadonic (5,11,14–20∶3) acids]. Not a single pine species was found so far with any of these acids missing. Linoleic acid is almost always, except in a few cases, the prominent SFA, in the range 40–60% of total fatty acids. The second habitual SFA is oleic acid, from 12 to 30%. Exceptions, however, occur, particularly in the Cembroides subsection, where oleic acid reaches ca. 45%, a value higher than that of linoleic acid. α-Linolenic acid, on the other hand, is a minor constituent of pine SFA, almost always less than 1%, but that would reach 2.7% in one species (P. merkusii). The sum of saturated acids [16∶0 (major) and 18∶0 (minor) acids principally] is most often less than 10% of total SFA, and anteiso-17∶0 acid is present in all species in amounts up to 0.3%. Regarding C₁₈ Δ5-UPIFA, taxoleic acid reaches a maximum of 4.5% of total SFA, whereas pinolenic acid varies from 0.1 to 25.3%. The very minor coniferonic (5,9,12,15–18∶4) acid is less than 0.2% in all species. The C₂₀ elongation product of pinolenic acid, bishomo-pinolenic (7,11,14–20∶3) acid, is a frequent though minor SFA constituent (maximum, 0.7%). When considering C₂₀ Δ5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11–20∶2 and sciadonic acids are ≤0.3 and ≤1.9%, respectively, whereas in the latter subgenus, they are most often ≥0.3 and ≥2.0%, respectively. The highest values for 5,11–20∶2 and sciadonic acids are 0.5% (many species) and 7.0% (P. pinaster). The 5,11,14,17–20∶4 (juniperonic) acid is present occasionally in trace amounts. The highest level of total Δ5-UPIFA is 30–31% (P. sylvestris), and the lowest level is 0.6% (P. monophylla). Uniting as well as discriminating features that may complement the knowledge about the taxonomy and phylogeny of pines are emphasized.     

Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream (Sparus aurata) cells

Marine fish have an absolute dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C_18–20 elongase activity. Recent research in turbot cells found low C_18–20 elongase but high Δ5 desaturase activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabeled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-¹⁴C]18∶2n−6 and [1-¹⁴C]18∶3n−3), C_18–20 elongase ([U-¹⁴C]18∶4n−3), Δ5 desaturase ([1-¹⁴C]20∶3n−6 and [1-¹⁴C]20∶5n−3), and C_20–22 elongase ([1-¹⁴C]20∶4n−6 and [1-¹⁴C]20∶5n−3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and that C_18–20 elongase and C_20–22 elongase activities were substantial. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase, which was very low, particularly with 20∶4n−3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C_18–20 elongase, and C_20–22 elongase were approximately 94-, 27-, and 16-fold greater than that for Δ5 desaturase toward their respective n−3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids in the marine fish the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity.     

Occurrence of γ-linolenic acid in compositae: A study of Youngia tenuicaulis seed oil

Seeds of Youngia tenuicaulis and other species from the plant family Compositae (Asteraceae) were studied for their oil content and fatty acid composition. The seed oil of Y. tenuicaulis growing in Mongolia was found to contain 5.6% γ-linolenic acid (18∶3Δ6cis,9cis,12cis) in addition to common fatty acids. The oil was analyzed using chromatographic [capillary gas-liquid chromatography (GLC), thin-layer chromatography] and spectroscopic (infrared, gas chromatography-mass spectrometry) techniques. Seed oil fatty acids of Saussurea amara (containing γ-linolenic acid) and of Arctium minus (containing 18∶3Δ3trans,9cis,12cis), as well as Δ5cis- and Δ5trans-18∶3 were used as GLC reference substances. The evolution in this plant family of a large number of different 18∶3 acids as well as the corresponding evolution of unusual desaturases should be investigated. On the other hand, the Δ6cis-desaturase required for the biosynthesis of γ-linolenic acid may have evolved independently several times in unrelated families of the plant kingdom.     

Diabetes-induced and age-related changes in fatty acid proportions of plasma lipids in rats

Diabetes-induced and age-related proportional changes in plasma fatty acids of triglycerides (TG), phospholipids (PL), and cholesteryl esters (CE) were investigated using streptozotocin-induced diabetic and control rats. Among n-6 fatty acids from diabetic rat plasma, increased proportions of 18∶2n-6 and 20∶3n-6 in all three lipid classes and of 18∶3n-6 in PL at 1–3 months old and in TG at 3–5 months old were observed. The proportions of 20∶4n-6 decreased in both PL and CE, but were unchanged in diabetic TG. Among the n-3 fatty acids, in the early stage, diabetes caused increases in the proportions of 18∶3n-3 in PL and CE and of 20∶5n-3 and 22∶6n-3 in TG, while 22∶5n-3 was decreased later in the disease course. These results suggest reduced Δ5-desaturase activities on 20∶3n-6 but not on 20∶4n-3, while Δ6-desaturase activity on 18∶2n-6 was essentially unaffected. Furthermore, the reduction in Δ9-desaturase activity in diabetic rats may well explain the decreases in the proportions of 16∶1n-7 and 18∶1n-7. However, the proportion of 18∶1n-9, another product of Δ9-desaturase, was significantly increased in CE and PL as compared to the controls. Thus, there was a discrepancy between our results and those of earlier studies with respect to the n-9, n-6, and n-3 fatty acid proportions of plasma lipids in diabetic rats. We also investigated age-related changes in the proportions of plasma fatty acids. Although rather small, age-related changes were evident in both diabetic and control rats.     

Reinvestigation of the polymethylene-interrupted 18:2 and 20:2 acids of Ginkgo biloba seed lipids

The fatty acid composition of Ginkgo biloba seed lipids was reinvestigated with particular emphasis on the polymethylene-interrupted octadecadienoic and eicosadienoic acids. Analysis of the picolinyl esters and 4,4-dimethyloxazoline derivatives by capillary gas-liquid chromatography on a highly polar cyanopropyl polysiloxane stationary phase coupled with mass spectrometry revealed the presence of three such acids, with the structures 5,9–18:2, 5,11–18:2, and 5,11–20:2. This indicated that in G. biloba seeds, cis-vaccenic (11–18:1) acid may be a substrate for the Δ5-desaturase characteristic of gymnosperms. The 5,11-18:2 acid was not limited to G. biloba, as it may occur in a few other species. The 5,11-20:2 acid is a common component of the seed lipids from almost all gymnosperm species analyzed so far.     

Conifer seeds: Oil content and fatty acid composition

The seed oils from twenty-five Conifer species (from four families—Pinaceae, Cupressaceae, Taxodiaceae, and Taxaceae) have been analyzed, and their fatty acid compositions were established by capillary gas-liquid chromatography on two columns with different polarities. The oil content of the seeds varied from less than 1% up to 50%. Conifer seed oils were characterized by the presence of several Δ5-unsaturated polymethylene-interrupted polyunsaturated fatty acids (Δ5-acids) with either 18 (cis-5,cis-9, 18∶2,cis-5,cis-9,cis-12 18∶3, andcis-5,cis-9,cis-12,cis-15 18∶4 acids) or 20 carbon atoms (cis-5,cis-11 20∶2,cis-5,cis-11,cis-14, 20∶3, andcis-5,cis-11,cis-14,cis-17 20∶4 acids). Pinaceae seed oils contained 17–31% of Δ5-acids, mainly with 18 carbon atoms. The 20-carbon acids present were structurally derived from 20∶1n-9 and 20∶2n-6 acids. Pinaceae seed oils were practically devoid of 18∶3n-3 acid and did not contain either Δ5-18∶4 or Δ5-20∶4 acids. Several Pinaceae seeds had a Δ5-acid content higher than 50 mg/g of seed. The only Taxaceae seed oil studied (Taxus baccata) had a fatty acid composition related to those of Pinaceae seed oils. Cupressaceae seed oils differed from Pinaceae seed oils by the absence of Δ5-acids with 18 carbon atoms and high concentrations in 18∶3n-3 acid and in Δ5-acids with 20 carbon atoms (Δ5-20∶3 and Δ5-20∶4 acids). Δ5-18∶4 Acid was present in minute amounts. The highest level of Δ5-20∶4 acid was found inJuniperus communis seed oil, but the best source of Δ5-acids among Cupressaceae wasThuja occidentalis. Taxodiaceae seed oils had more heterogeneous fatty acid compositions, but the distribution of Δ5-acids resembled that found in Cupressaceae seed oils. Except forSciadopytis verticillata, other Taxodiaceae species are not interesting sources of Δ5-acids. The distribution profile of Δ5-acids among different Conifer families appeared to be linked to the occurrence of 18∶3n-3 acid in the seed oils.     

Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): Radiolabeling studies

The fresh-water green alga Parietochloris incisa is the richest plant source of the PUFA arachidonic acid (20∶4n−6, AA). To elucidate the biosynthesis of AA in this alga we labeled cultures of P. incisa with radioactive precursors. Pulse chase labeling with acetate resulted in its incorporation via the de novo biosynthesis pathway of FA. However, labeled acetate was also utilized for the elongation of C₁₆ and C₁₈ PUFA. Labeling with [1-¹⁴C]oleic acid has shown that the first steps of the lipid-linked FA desaturations utilize cytoplasmic lipids. PC and diacylglyceryltrimethylhomoserine are the major lipids involved as acyl carriers for the Δ12 and Δ6 desaturations of oleic acid, leading sequentially to linoleic and γ-linolenic acids. The latter is released from its lipid carrier and elongated to 20∶3n−6, which is reincorporated primarily into PF and PC and finally desaturated to AA. Galactolipids, mostly monogalctosyldiacylglycerol (MGDG), serve as substrates for the chloroplastic Δ12 desaturase and, apparently, the ω3 desaturation, common to higher plants and many green algae. The predominant sequence desaturates the 18∶1/16∶0 molecular species of MGDG stepwise to the 18∶3n−3/16∶3n−3 molecular species similar to the prokaryotic pathway of higher plants and green algae.     

Conversion of 18∶3 Δ9cis, 12cis, 15trans in rat liver microsomes

Several years ago, it was established that the Δ15 trans isomer of α-linolenic acid is converted in vivo into fatty acids containing 20 and 22 carbons (geometrical isomers of eicosapentaenoic and docosahexaenoic acids). The present study focused on the in vitro Δ6 desaturation, the first step of the biosynthesis of the n-3 long-chain polyunsaturated fatty acids from 18:3n-3. For that purpose, rat liver microsomes were prepared and incubated with radiolabeled 18∶3 Δ9cis, 12cis, 15cis (18∶3 c,c,c) or 18∶3 Δ9cis, 12cis, 15trans (18∶3c,c,t) under desaturation conditions. The data show that 18∶3c,c,t is converted at a lower rate compared with α-linolenic acid. The product of conversion of 18∶3 c,c,t may be 18∶4 Δ6cis, 9cis, 12cis, 15trans resulting from a Δ6 desaturation of the trans substrate. Moreover, the conversion of radiolabeled 18∶3c,c,t was strongly decreased by the presence of 18∶3c,c,c (up to 48%) while the 18∶3c,c,t only slightly decreased the conversion of radiolabeled 18∶3c,c,c. Thus, the desaturation enzyme presented a higher affinity for the native all-cis n-3 substrate.     

Norflurazon—An inhibitor of essential fatty acid desaturation in isolated liver cells

Norflurazon is a herbicide known to inhibit carotene biosynthesis and linolenic acid biosynthesis in plants. In the present work, the effect of norflurazon on the metabolism of essential fatty acids was studied in isolated rat liver cells and in rat liver microsomes, incubated with [1-¹⁴C] labeled linolenic acid (18∶3, n−3), dihomogammalinolenic acid (20∶3, n−6) and eicosapentaenoic acid (20∶5, n−3). Norflurazon (0.1 mM, 1.0 mM) was found to inhibit essential fatty acid desaturation. The Δ6 desaturation is inhibited more efficiently than the Δ5 and Δ4 desaturation. The chain elongation of essential C₁₈ fatty acids to their C₂₀ and C₂₂ homoglogs was not inhibited by norflurazon.     

Trans,n-3, and n-6 fatty acids in canadian human milk

The presence oftrans fatty acids in human milk may be a concern because of their possible adverse nutritional and physiological effects on the recipient infant. The mother's diet is the source of human milktrans fatty acids, and since these fatty acids are prevalent in many common foods of the Canadian diet, thetrans fatty acid content and the fatty acid composition of Canadian human milk were measured by gas-liquid chromatography coupled with silver nitrate-thin layer chromatography. In samples obtained from 198 lactating mothers across Canada, the average percentage of totaltrans (sum oft18∶1,t18∶2, andt18∶3) was 7.2% of breast milk fatty acids with a range of 0.1–17.2%. Analysis oft18∶1 isomer distribution indicated that partially hydrogenated vegetable oils are the major source of thesetrans fatty acids in human milk, whereas contribution from dairy products appeared to be relatively minor. Linoleci and α-linolenic acid levels were inversely related to the totaltrans fatty acids, indicating that the elevation oftrans fatty acids in Canadian human milk is at the expense of n-3 and n-6 essential fatty acids. Levels of arachidonic and docosahexaenoic acids did not correlate with their parent fatty acids, indicating that it might be difficult to elevate the levels of n-6 and n-3 C_20–22 polyunsaturated fatty acids in breast milk by increasing levels of linoleic and α-linolenic acids in the mother's diet.     

In vitro desaturation and elongation of rumenic acid by rat liver microsomes

Various nutritional studies on CLA, a mixture of isomers of linoleic acid, have reported the occurrence of conjugated long-chain PUFA after feeding experimental animals with rumenic acid, 9c,11t–18∶2, the major CLA isomer, probably as a result of successive desaturation and chain elongation. In the present work, in vitro studies were carried out to obtain information on the conversion of rumenic acid. Experiments were first focused on the in vitro Δ6-desaturation of rumenic acid, the regulatory step in the biosynthesis of long-chain n−6 PUFA. The conversion of rumenic acid was compared to that of linoleic acid (9c,12c–18∶2). Isolated rat liver microsomes were incubated with radiolabeled 9c,12c–18∶2 and 9c,11t–18∶2 under desaturation conditions. The data indicated that [1-¹⁴C]9c,11t–18∶2 was a poorer substrate for Δ6-desaturase than [1-¹⁴C]-9c,12c–18∶2. Next, in vitro elongation of 6c,9c,11t–18∶3 and 6c,9c,12c–18∶3 (γ-linolenic acid) was investigated in rat liver microsomes. Under elongation conditions, [1-¹⁴C]6c,9c,11t–18∶3 was 1.5-fold better converted into [3-¹⁴C]8c,11c,13t–20∶3 than [1-¹⁴C]6c,9c,12c–18∶3 into [3-¹⁴C]8c,11c,14c–20∶3. Finally, in vitro Δ5-desaturation of 8c,11c,13t–20∶3 compared to 8c,11c,14c–20∶3 was investigated. The conversion level of [1-¹⁴C]8c,11c,13t–20∶3 into [1-¹⁴C]5c,8c,11c,13t–20∶4 was 10 times lower than that of [1-¹⁴C]8c,11c,14c–20∶3 into [1-¹⁴C]5c,8c,11c,14c–20∶4 at low substrate concentrations and 4 times lower at the saturating substrate level, suggesting that conjugated 20∶3 is a poor substrate for the Δ5-desaturase.     

Investigation of γ-Linolenic Acid and Stearidonic Acid Biosynthesis During a Life Cycle of <Emphasis Type="Italic">Borago officinalis</Emphasis> L.

This study investigated the levels of γ-linolenic (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3) in various parts of the borage plant (Borago officinalis L.) to elucidate Δ6-desaturase activity. Lipid class and fatty acid (FA) composition during germination of the seeds and FA composition of various borage parts were determined as well as FA compositions of neutral lipids (NL), polar lipids (PL), glycolipids (GL) and monogalactosyl diacylglycerols (MGDG) of borage leaves. When seeds were germinated for 12 days in the dark then exposed to light for 8 h, an overall decrease in oil content was seen with a significant increase in PL from 0.2 to 39.1%. An increase in SDA (from 0.2 to 0.6 g/100 g oil) indicated minor FA synthesis during germination and therefore, Δ6-desaturase activity with the most marked increase after light exposure. The FA compositions of developing and mature seeds were similar, suggesting oil/FA synthesis takes place at the initial stage of seed development. Among all the borage parts, the leaves had the highest amounts of α-linolenic acid (ALA, 18:3n-3) (36.2%), indicating Δ15-desaturase activity, and SDA (25.2%), indicating Δ6-desaturase activity. In leaves, the GL and especially, MGDG fractions had the highest amounts of SDA (31.8 and 39.8%, respectively), indicating that Δ6-desaturase is most active in chloroplasts. Leaves and developing seeds appear to be the major sources of Δ6-desaturase in borage, associated with different organelles in the different tissues.     

Incorporation of n−3 fatty acids of fish oil into tissue and serum lipids of ruminants

This study examines the biohydrogenation and utilization of the C₂₀ and C₂₂ polyenoic fatty acids in ruminants. Eicosapentaenoic (20∶5n−3) and docosahexaenoic (22∶6n−3) acids were not biohydrogenated to any significant extent by rumen microorganisms, whereas C₁₈ polyenoic fatty acids were extensively hydrogenated. The feeding of protected fish oil increased the proportion of 20∶5 from 1% to 13–18% and 22∶6 from 2% to 7–9% in serum lipids and there were reductions in the proportion of stearic (18∶0) and linoleic (18∶2) acids. The proportion of 20∶5 in muscle phospholipids (PL) increased from 1.5% to 14.7% and 22∶6 from 1.0% to 4.2%; these acids were not incorporated into muscle or adipose tissue triacylglycerols (TAG). In the total PL of muscle, the incorporated 20∶5 and 22∶6 substituted primarily for oleic (18∶1) and/or linoleic (18∶2) acid, and there was no consistent change in the porportion of arachidonic (20∶4) acid.     

Chain elongation of polyunsaturated fatty acids by vascular endothelial cells: Studies with arachidonate analogues

This study has utilized radiolabeled analogues of arachidonic acid to study the substrate specificity of elongation of long-chain polyunsaturated fatty acids. Human umbilical vein endothelial cells were incubated for 2–72 hr in medium supplemented with 0.9–2.6 μM [¹⁴C]fatty acid, and cellular glycerolipids were analyzed by gas-liquid chromatography with radioactivity detection. Elongation of naturally occurring C₂₀ polyunsaturated fatty acids occurred with eicosapentaenoate (20∶5(n−3))>Mead acid (20∶3(n−9))>arachidonate (20∶4(n−6)). Chain length markedly influenced the extent of elongation of 5,8,11,14-tetraenoates (18∶4>19∶4>20∶4>21∶4); effects of initial double bond position were also observed (6,9,12,15–20∶4>4,7,10,13–20∶4. Neither 5,8,14- nor 5,11,14–20∶3 was elongated to the extent of 5,8,11–20∶3. Differences between polyunsaturated fatty acids were observed both in the initial rates and in the maximal percentages of elongation, suggesting that the content of cellular C₂₀ and C₂₂ fatty acids may represent a balance between chain elongation and retroconversion. Umbilical vein endothelial cells do not exhibit significant desaturation of either 22∶4(n−6) or 22∶5(n−3). By contrast, incubation with 5,8,11,14-[¹⁴C]18∶4(n−4) resulted in formation of both [¹⁴C]20∶5(n−4) and [¹⁴C]22∶5(n−4). The respective time courses for the appearances of [¹⁴C]22∶5(n−4) and [¹⁴C]20∶5(n−5) suggests Δ6 desaturation of [¹⁴C]22∶4(n−4) rather than Δ4 desaturation of [¹⁴C]20∶4(n−4).     

Biosynthesis of unsaturated fatty acids in the diatomPhaeodactylum tricornutum

The biosynthesis of fatty acids in the diatomPhaeodactylum tricornutum was studied. The diatom was incubated with sodium [1¹⁴C] acetate and the acids [1-¹⁴C] palmitic, [1-¹⁴C] stearic, [1-¹⁴C] linoleic and [1-¹⁴C] α-linolenic. The distribution of radioactivity in the products was determined by gas liquid radiochromatography. The diatom synthesized “de novo” not only saturated and monounsaturated fatty acids, but also linoleic, α-linolenic and other fatty acids including the highly polyunsaturated 20∶5ω3 and 22∶6ω3. When labeled acetate, stearic, α-linolenic or even linoleic acid were incubated with the diatom, the polyunsaturated C₂₀ fatty acids synthesized belonged predominantly to the ω 3 family. The existence of Δ9, Δ6, Δ5, Δ4, ω6 and possibly ω3 desaturases inP. tricornutum is suggested. Member of the Carrera del Investigador Científico of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Member of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientificas y Técnicas.     

Microsomal Δ5 desaturation of eicosa-8,11,14-trienoic acid is activated by a cytosolic fraction

Δ5 Desaturation of eicosa-8,11,14-trienoic acid to arachidonic acid was studied in rat liver microsomes. It was shown that Δ5 desaturation of fatty acids in vitro requires the participation of a peripheral component of cytosolic origin. Desaturation of 20∶3n−6 to 20∶4n−6 decreases in washed microsomes as they lose an adsorbed cytosolic fraction (CF), but the enzymatic activity can be recovered as a function of CF concentration in the incubation medium. Albumin does not substitute for CF. Δ5 Desaturation of 20∶3n−6 is inhibited by arachidonic acid by a product inhibition effect, but CF prevents retroinhibition of Δ5-desaturase by 20∶4n−6. This ability of CF is eliminated by preincubation of CF with 20∶4n−6, but not with γ−18∶3n−6, the product of Δ6 desaturation of 18∶2n−6, thus indicating that CF impairs the retroinhibitory effect of arachidonic acid on Δ5-desaturase in a specific manner. Δ6 Desaturation of linoleic acid to γ−18∶3n−6 is also activated by CF and retroinhibited by γ−18∶3n−6. CF activity on Δ6 desaturation is retained after preincubation with 20∶4n−6, but it is lost after preincubation with γ−18∶3n−6. Activation of Δ6-desaturase by CF is associated with the removal of the reaction product in a specific manner. Chromatography of CF by Sephacryl S-200 separates two major subfractions which show different efficiency in reactivating Δ5- and Δ6-desaturase activities in washed microsomes. Therefore, CF may contain subfractions that can prevent Δ5- and δ6-desaturase retroinhibition by apparently binding their respective reaction products specifically.     

Regiospecific analysis of conifer seed triacylglycerols by gas-liquid chromatography with particular emphasis on Δ5-olefinic acids

Dibutyroyl derivatives of monoacylglycerols (DBMAG) from conifer seed oil triacylglycerols (TAG) were prepared by partial deacylation of TAG with ethylmagnesium bromide followed by diesterification with n-butyryl chloride. The resulting mixtures were analyzed by gas-liquid chromatography (GCL) with 65% phenylmethyl silicon open tubular fused-silica capillary column operated under optimal conditions and separated according to both their fatty acid structures and their regiospecific distribution. Seed oils of 18 species from 5 conifer families (Pinaceae, Taxaceae, Cupressaceae, Cephalotaxaceae, and Podocarpaceae) were analyzed. The chromatograms showed a satisfactory resolution of DBMAG containing palmitic (16∶0) stearic (18∶0), taxoleic (cis-5, cis-9 18∶2), oleic (cis-9 18∶1), cis-vaccenic (cis-11 18∶1), pinolenic (cis-5, cis-9, cis-12 18∶3), linoleic (cis-9, cis-12 18∶2), α-linolenic (cis-9 cis-12, cis-15 18∶3), and an almost baseline resolution of DBMAG containing gondoic (cis-11 20∶1), cis-5, cis-11 20∶2, sciadonic (cis-5, cis-11, cis-14 20∶3), dihomolinoleic (cis-11 cis-14 20∶2), juniperonic (cis-5, cis-11, cis-14, cis-17 20∶4), and dihomo-α-linolenic (cis-11, cis-14, cis-17 20∶3) acids. We have observed that results for Pinus pinaster and P. koraiensis seed oils obtained with this new simple method compared favorably with literature data established with other usual regiospecific analytical techniques. Δ5-Olefinic acids are esterified mainly at the external positions of the glycerol backbone in all cases, in agreement with data obtained by other methodologies allowing validation of the GLC regiospecific method. To date, 45 gymnosperm species (mostly Coniferophytes) from 21 genera belonging to 9 families have been analyzed, all of them showing a definite enrichment of Δ5-olefinic acids in the external positions of TAG. These fatty acids (FA), with one exception only, represent between-2 and 8% of FA esterified to the internal positions. For some species, i.e., P. koraiensis and P. pinaster, this asymmetrical distribution was established by at least three analytical procedures and confirmed by stereospecific analysis of their seed TAG.