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.     

Tissue culture of cocoa bean (Theobroma cacao L.): Incorporation of fatty acids into lipids of cultured cells

Suspension cell cultures of cocoa bean rapidly incorporated palmitic, stearic, oleic and linoleic acids into cellular lipids. Thus, 75 and 20% of [1-¹⁴C] palmitic acid was incorporated into polar lipids and triglycerides, respectively, after 48 hr. When [1-¹⁴C] oleic and [1-¹⁴C] linoleic acid were added separately, polar lipids consistently contained most of the radioactive fatty acids. Ca. 60% of the stearic acid accumulated as unesterified fatty acid in the cells. Palmitic and stearic acid were not desaturated, but oleic acid and linoleic acid were further desaturated. The kinetics of conversion of oleic acid and linoleic acid suggested a sequential desaturation pathway of 18∶1→18∶2→18∶3 in cocoa bean cell suspensions.     

Lipid and fatty acid characterization and metabolism in the sea anemonePhymactis clematis (Dana)

Neutral lipid, phospholipids and fatty acids of the sea anemonePhymactis clematis from the south-west Atlantic were characterized and quantified in spring and autumn. Neutral lipids predominated over phospholipids in both seasons. Triacylglycerol and diacylglycerol ethers were the major lipids. In spring, an increase of esterified sterols was noted. The major fatty acids found were 22∶5ω3, 20∶5ω3 and 16∶0. The sea anemones were also incubated in vivo with either [1-¹⁴C]linoleate or [1-¹⁴C] α-linolenate for 2 hr. Isotope incorporation into lipids and their transformations into higher fatty acids were examined. Both precursors were incorporated into the lipids, mainly in triacylglycerols and mono-acylglycerols, while α-linolenate was also incorporated into phospholipids. The radioactive linoleate was elongated to 20∶2, 22∶2 and 24∶2 fatty acids, but not desaturated to 18∶3ω6. α-Linolenate was desaturated by Δ6 desaturase to 18∶4ω3. The specificity of Δ6-desaturase is discussed.     

Metabolism of linoleic acid in the cat

Cats fed a diet containing linoleate as the only polyunsaturated fatty acid showed extremely low levels of arachidonate in the plasma lipids, as well as an increase in linoleate, eicosadienoate and an unknown fatty acid. Administration of [1-¹⁴C] linoleic acid and [2-¹⁴C] eicosa-8,11,14-trienoic acid to cats showed that in the liver there was no conversion of the [1-¹⁴C] 18∶2 to arachidonate, whereas there was significant metabolism of [2-¹⁴C] 20∶3 to arachidonate. It was found when methyl-γ-linolenate was fed to cats that the level of 20∶3ω6 and 20∶4ω6 in the erythrocytes increased significantly. These results show that there is no significant Δ6 desaturase activity in the cat, whereas chain elongation and Δ5 desaturase enzymes are operative. The unknown fatty acid was isolated from the liver lipids and shown to be a 20-carbon fatty acid with 3 double bonds and which by gas liquid chromatography could be separated from 20∶3ω9 and 20∶3ω6. The presence of the Δ5-desaturase activity and the results of the ozonolysis studies indicated that this unknown fatty acid was eicosa-5,11,14-trienoic acid.     

Dietary linoleic, α-linolenic and oleic acids are oxidized at similar rates in rats fed a diet containing these acids in equal proportions

The objective of this study was to examine whether whole body oxidation rates of dietary linoleic, α-linolenic and oleic acids differ when the acids are provided in identical quantities. Male rats were fed for 10 wk a 15% fat (w/w) diet containing equal amounts of linoleic, α-linolenic and oleic acids (22.7, 23.0 and 23.2% of total fatty acids, respectively). At week 10, after overnight fasting, rats were intragastrically administered 20 μCi of either [1-¹⁴C]-labelled linoleic, α-linolenic or oleic acid in a 200-μL bolus of oil containing equal quantities of each fatty acid. The appearance of¹⁴CO₂ in expired air was then monitored hourly for 12h for each animal. A preliminary study had shown that growth and food consumption patterns in animals consuming the oil containing equal quantities of each of the fatty acids paralleled the patterns of animals that were self-selecting among separate diets, each of which contained one of the component oils. The appearance of¹⁴C, expressed as percent dose administered, peaked at 2–3 h post-dose for¹⁴C-labelled linoleic (5.28±0.37%/h), α-linolenic (6.92±0.51%/h) and oleic (5.98±0.44%/h) acids. Statistically these values were not significantly different. Cumulative¹⁴CO₂ excretion rates over 12 h were also similar for linoleic (27.2±0.9%), α-linolenic (26.8±1.2%) and oleic (25.9±1.2%) acids. The results suggest that the rat's capacity to oxidize 18-carbon unsaturated fatty acids is not affected by fatty acid unsaturation when these fatty acids are provided at equal dietary levels.     

Fatty acid metabolism of the calanoid copepodParacalanus parvus: 2. Palmitate,stearate, oleate and acetate

The de novo biosynthesis of fatty acids in the wild, calanoid copepodParacalanus parvus was studied. The incubation of labeled acetate proved the de novo biosynthesis of saturated and monounsaturated even fatty acids from 14 to 20 carbons and the 22∶1 acid. Saturated and monounsaturated uneven fatty acids from 15 to 21 carbons were also synthesized. The copepod could not synthesize linoleic and α-linolenic acids. By administration of [1-¹⁴C]palmitate, [1-¹⁴C] stearate and [1-¹⁴C]oleate, it was possible to elucidate the general pattern of the de novo biosynthesis of fatty acids in the wildP. parvus.     

Linoleic acid uptake by isolated enterocytes: Influence of α-linolenic acid on absorption

In a previous study we showed that intestinal uptake of α-linolenic acid (18∶3n−3) was carrier-mediated and we suggested that a plasma membrane fatty acid protein was involved in the transport of long-chain fatty acids. To further test this hypothesis, the mechanism of linoleic acid (18∶2n−6) uptake by isolated intestinal cells was examined using a rapid filtration method and 20 mM sodium taurocholate as solubilizing agent. Under these experimental conditions transport of [1-¹⁴C]linoleic acid monomers in the concentration range of 2 to 2220 nM was saturable with a V_m of 5.1±0.6 nmol/mg protein/min and a K_m of 183±7 nM. Experiments carried out in the presence of metabolic inhibitors, such as 2,4-dinitrophenol and antimycin A, suggested that an active, carriermediated mechanism was involved in the intestinal uptake of this essential fatty acid. The addition of excess unlabeled linoleic acid to the incubation medium led to a 89% decrease in the uptake of [1-¹⁴C]linoleic acid, whiled-glucose did not compete for transport into the cell. Other long-chain polyunsaturated fatty acids added to the incubation mixture inhibited linoleic acid uptake by more than 80%. The presence of α-linolenic acid (18∶3n−3) in the incubation medium caused the competitive inhibition (K_i=353 nM) of linoleic acid uptake. The data are compatible with the hypothesis that intestinal uptake of both linoleic, and α-linolenic acid is mediated by a membrane carrier common to long-chain fatty acids.     

Effect of different acids with Δ9,12-dienoic structures on Δ9 desaturation activity in rat liver microsomes

The effect of oral administration, for 24 or 48 hr, of different octadeca fatty acids containing a 9,12-dienoic structure on the fatty acid composition and Δ9 desaturation activity of liver microsomes of rat fed a fat-free diet was studied. The ethyl esters of linoelaidic and γ-linolenic acids, the methyl ester of linoleic acid and free columbinic acid were administered to rats maintained on a fat-free diet. The supplementation of the fat-free diet with linoelaidate produced no relevant changes in the fatty acid composition pattern of liver microsomes and did not modify the percentage of conversion of palmitic to palmitoleic acid. The addition of linoleate or γ-linolenate to the fat-free diet returned liver microsome Δ9 desaturation activity toward the control and partially restored the liver microsome fatty acid spectrum found in the fat-free diet. Columbinic acid (5-trans-9-cis,12-cis-18∶3), which cannot be transformed into arachidonic acid, also decreased the Δ9 desaturation activity enhanced by the fat-free diet and evoked changes in the microsomal fatty acid composition similar to those produced by the ω6 fatty acids. These results suggest that the modulation of Δ9 desaturase activity evoked by dietary administration of unsaturated acids of ω6 series would depend on thecis double bond configuration of these acids.     

Lipid metabolism of the yellow clam,Mesodesma mactroides: 3-Saturated fatty acids and acetate metabolism

The fate of labeled palmitate, stearate, and acetate administered to the yellow clam,Mesodesma mactroides, was investigated. 1-¹⁴C palmitic and 1-¹⁴C stearic acids were oxidized to CO₂ to a limited extent. They were mainly incorporated in diacylglycerols and triacylglycerols and were converted to higher homologs. After administration, palmitic acid was converted to stearic and oleic acids, whereas administered stearic acid was converted to 18∶1, 18∶2, 20∶1, and 20∶2 acids. Labeled acetate was readily included by the clam in 12∶0, 14∶0, 14∶1, 15∶0, 16∶1, 16∶1, 16∶2, 18∶2, 18∶1, 18∶2, 20∶1, 20∶2, and 20∶3 acids.     

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

Comparative effect of glucagon,dibutyryl cyclic AMP,and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes

The effect of glucagon, dibutyryl cyclic adenosine 3′,5′-monophosphate, and epinephrine on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied. The incubations were performed with rat liver microsomes and labeled linoleic acid under desaturating and elongating conditions. Under desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas under elongating conditions it was converted to 20∶2ω6. Glucagon, dibutyryl cyclic AMP, and epinephrine decreased the oxidative desaturation of linoleic acid to γ-linolenic acid while the elongating reaction was not modified in the experimental conditions tested. Consequently, the results support the hypothesis that the oxidative desaturation of linoleic acid to γ-linolenic acid is the main controllable step in the biosynthesis of polyunsaturated fatty acids of the linoleic acid family in the microsomes.     

The effects oftrans fatty acids on fatty acyl Δ5 desaturation by human skin fibroblasts

The effectiveness of different fatty acids as inhibitors of fatty acyl Δ5 desaturation activity in human skin fibroblasts has been investigated. When incubated with 2.25 μM [¹⁴C] eicosatrienoate (20∶3ω6) in otherwise lipid-free medium, these cells rapidly incorporate the radiolabeled fatty acid into cellular glycerolipids and desaturate it to produce both [¹⁴C] arachidonate and [¹⁴C] docosatetraenoate. The Δ5 desaturation activity can be enhanced by prior growth of the cells without serum lipids. Elaidate (9t–18∶1) is a potent inhibitor of Δ5 desaturation whiletrans-vaccenate (11t–18∶1) is virtually without effect. Oleate and linoleate are only mildly inhibitory. Linoelaidate (9t, 12t–18∶2) is more inhibitory than linoleate but significantly less effective than elaidate. The effects of elaidate can be readily overcome by increasing the concentration of exogenous eicosatrienoate. Studies with a variety oftrans monounsaturates of differing chain lengths indicate that the ω9trans fatty acids are potent inhibitors of Δ5 desaturation, while ω7trans fatty acids are relatively ineffective. Intact human fibroblasts could thus be important in characterizing novel fatty acids as selective inhibitors of arachidonate synthesis in vivo.     

Biosynthesis of [14C]arachidonic acid from [14C]linoleate in primary cultures of rat Sertoli cells

The conversion of [¹⁴C]linoleate to [¹⁴C]arachidonate by rat Sertoli cells was established by use of primary cultures. Most of the¹⁴C from [1-¹⁴C]linoleate was located in C-3 of the synthesized arachidonate, indicating that the labeled tetraene had originated largely by elongation and desaturation of the intact labeled substrate rather than by mere addition of¹⁴C-acetate generated by bio-oxidation of the radioactive substrate to an already existing 18-carbon precursor. Although a relatively small amount of¹⁴C was present in 18∶3ω6 and a relatively large amount of¹⁴C was present in 20∶2, it was not possible from these data to establish the relative importance of 20∶2 in the biosynthesis of arachidonic acid in rat Sertoli cells.     

Synthesis of linoleate and α-linolenate by chain elongation in the rat

The objective was to determine whether rats could synthesize longer chain polyunsaturates from hexadecadienoate (16∶2n−6) and hexadecatrienoate (16∶3n−3). Rats were gavaged with uniformly¹³C-labelled hexadecadienoate or hexadecatrienoate, euthanized 24 h later, and total lipids were extracted from liver and carcass. Gas chromatogrpahy/combustion/isotope ratio mass spectrometry was used to measure¹³C levels in individual liver, carcass, and whole body fatty acids.¹³C Enrichment was present in desaturated and chain-elongated polyunsaturates, including linoleate, arachidonate, α-linolenate, and docosahexaenoate at 12–13% of the dose of tracer given.¹³C Enrichment from hexadecatrienoate was highest in carcass and liver α-linolenate, representing 3.5 and 17.9% of the total α-linolenate pool, respectively. For linoleate, arachidonate, or docosahexaenoate, the contribution of¹³C did not exceed 0.2% of the total body pool. Green leafy vegetables common in the human diet were shown to contain up to 1.2% of total fatty acids as hexadecadienoate and 11.6% as hexadecatrienoate. Hence, humans consuming green vegetables probably synthesize a small proportion of their total body content of linoleate and α-linolenate.     

Application of new methods and analytical approaches to research on polyunsaturated fatty acid homeostasis

New methods and analytical approaches are important to challenge and/or validate established beliefs in any field including the metabolism of polyunsaturated fatty acids (PUFA; polyunsaturates). Four methods that have recently been applied toward obtaining a better understanding of the homeostasis of PUFA include the following: whole-body fatty acid balance analysis, magnetic resonance imaging (MRI), ¹³C nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Whole-boby balance studies permit the measurement of both the percentage of oxidation of linoleate and α-linolenate and their conversion to long-chain PUFA. This method has shown that β-oxidation to CO₂ is normally the predominant metabolic fate of linoleate and α-linolenate. Furthermore, models of experimental undernutrition in both humans and animals show that β-oxidation of linoleate and α-linolenate markedly exceeds their intake, despite theoretically sufficient intake of linoleate or α-linolenate. Preliminary results suggest that by using MRI to measure body fat content, indirect whole-body linoleate balance can be done in living humans, ¹³C NMR spectroscopy provided unexpected evidence that linoleate and α-linolenate were metabolized into lipids synthesized de novo, an observation later quantified by tracer mass balance done using GC-C-IRMS. This latter method showed that within 48 h of dosing with ¹³C-α-linolenate, >80% underwent β-oxidation to CO₂ by suckling rats, whereas 8–9% was converted to newly synthesized lipids and <1% to docosahexaenoate. Further application of these recently developed methods in different models should clarify the emerging importance of β-oxidation and carbon recycling in PUFA homeostasis in mammals including humans.     

Fatty acid metabolism in the calanoid copepodParacalanus parvus: 1. Polyunsaturated fatty acids

The metabolic fate of radioactive linoleate and α-linolenate administered to the South Atlantic copepodParacalanus parvus was studied. The wild copepod was able to incorporate the labeled acids dissolved in seawater. The radioactive linoleate was elongated to 20∶2ω6 and 22∶2ω6 and desaturated by a Δ6 desaturase to 18∶3ω6. α-Linolenate was also desaturated by a Δ6 desaturase to 18∶4ω3 and elongated to 20∶3ω3. The copepod was able to convert α-18∶3 to 20∶5ω3 and 22∶6ω3. 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 Científico of the Consejo Nacional de Investigaciones Cientificas y Tècnicas.     

Changes in linoleic acid metabolism and membrane fatty acids of LLC-PK cells in culture induced by 5α-cholestane-3β, 5, 6β-triol

The aim of this study was to investigate the effect of the oxysterol 5α-cholestane-3β, 5, 6β-triol (triol) on the metabolism of linoleic acid (18∶2n−6) to arachidonic acid (20∶4n−6) and on the cell membrane fatty acid composition. Porcine kidney cells were incubated in medium with or without 10 μg/mL of triol for 24 h, then incubated for 1, 6, or 12 h in a medium which contained 50 μM of either [¹⁴C]linoleic acid or unlabeled linoleic acid. The cellular uptake of [¹⁴C] linoleic acid was significantly higher in the triol-treated cells than in control cells. After 1- and 6-h incubations despite the increase of [¹⁴C]linoleic acid pool size in the triol-treated cells, neither total n−6 polyunsaturated fatty acids (PUFA) metabolites nor arachidonic acid were increased in the triol-treated cells as compared to the control cells, but trienoic acids accumulated to a greater extent in the triol-treated cells. Therefore, the ratios of n−6 PUFA metabolites vs. pool size of linoleic acid and of tetraenoic acids vs. dienoic acids were significantly decreased in triol-treated cells as compared to the control cells. The cellular fatty acid composition also showed that linoleic acid percentage was significantly increased while arachidonic acid percentage was significantly decreased in the triol-treated cells, and that the accumulation of trienoic acids (18∶3n−6+20∶3n−6) observed from the [¹⁴C]linoleic acid experiment was due solely to increased 20∶3n−6 content. This latter finding indicates that a decrease of elongase activity by triol is unlikely. Our results also showed that the triol-treated cells had a lower level of free cholesterol but higher levels of phospholipid and triol in their membranes, suggesting that triol displaced free cholesterol from the cell membrane.     

Desaturation of fatty acids inTrypanosoma cruzi

Uptake and metabolism of saturated (16∶0, 18∶0) and unsaturated [18∶1(n−9), 18∶2(n−6), 18∶3(n−3)] fatty acids by cultured epimastigotes ofTrypanosoma cruzi were studied. Between 17.5 and 33.5% of the total radioactivity of [1-¹⁴C]labeled fatty acids initially added to the culture medium was incorporated into the lipids ofT. cruzi and mostly choline and ethanolamine phospholipids. As demonstrated by argentation thin layer chromatography, gas liquid chromatography and ozonolysis of the fatty acids synthesized, exogenous palmitic acid was elongated to stearic acid, and the latter was desaturated to oleic acid and 18∶2 fatty acid. The 18∶2 fatty acid was tentatively identified as linoleic acid with the first bond in the Δ9 position and the second bond toward the terminal methyl end. Exogenous stearic acid was also desaturated to oleic and 18∶2 fatty acid, while oleic acid was only converted into 18∶2. All of the saturated and unsaturated fatty acids investigated were also converted to a small extent (2–4%) into polyunsaturated fatty acids. No radioactive aldehyde methyl ester fragments of less than nine carbon atoms were detected after ozonolysis of any of the fatty acids studied. These results demonstrate the existence of Δ9 and either Δ12 or Δ15 desaturases, or both, inT. cruzi and suggest that Δ6 desaturase or other desaturases of the animal type are likely absent in cultured forms of this organism.     

Dietary saturated fat level alters the competition between α-linolenic and linoleic acid

Male weanling rats were fed semi-synthetic diets high in saturated fat (beef tallow) vs high in linoleic acid (safflower oil) with or without high levels of α-linolenic acid (linseed oil) for a period of 28 days. The effect of feeding these diets on cholesterol content and fatty acid composition of serum and liver lipids was examined. Feeding linseed oil with beef tallow or safflower oil had no significant effect on serum levels of cholesterol. Serum cholesterol concentration was higher in animals fed the safflower oil diet than in animals fed the beef tallow diet without linseed oil. Feeding linseed oil lowered the cholesterol content in liver tissue for all dietary treatments tested. Consumption of linseed oil reduced the arachidonic acid content with concomitant increase in linoleic acid in serum and liver lipid fractions only when fed in combination with beef tallow, but not when fed with safflower oil. Similarly, ω3 fatty acids (18∶3ω3, 20∶5ω3, 22∶5ω3, 22∶6ω3) replaced ω6 fatty acids (20∶4ω6, 22∶4ω6) in serum and liver lipid fractions to a greater extent when linseed oil was fed with beef tallow than with safflower oil. The results suggest that the dietary ratio of linoleic acid to saturated fatty acids or of 18∶3ω3 to 18∶2ω6 may be important to determine the cholesterol and arachidonic acid lowering effect of dietary α-linolenic acid.     

Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant ofMortierella alpina 1S-4

A Δ12 desaturase-defective mutant of an arachidonic acid (AA)-producing fungus,Mortierella alpina 1S-4, converted α-linolenic acid (18:3ω3) to 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (EPA). On submerged cultivation at 20°C for 10 d in a 5-L fermentor containing medium comprising 1% glucose, 1% yeast extract and 3% (vol/vol) linseed oil, EPA production amounted toca. 1 g/L culture broth (64 mg/g dry mycelium), which accounted forca. 20% of the total mycelial fatty acids. AA content was 26 mg/g dry mycelium (0.4 g/L), accounting for 7.8% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (4.5%), oleic acid (20.4%), linoleic acid (10.0%), 18:3ω3 (20.3%) and lignoceric acid (4.3%). Most of the EPA produced (ca. 90 mol%) was in triglyceride form.