Effect of the environment and fasting on the lipid and fatty acid composition ofDiplodom patagonicus

Some effects of food, habitat and temperature on the lipid composition of a freshwater mollusk,Diplodom patagonicus, were studied. Animals kept and fasted up to 60 days in an aquarium at 9 C and 20 C showed a decrease of the total lipid content that corresponded to a decrease of triacylglycerols and diacylglycerol ethers. This decrease evoked an increase of polar-to-nonpolar lipid ratio. However, no significant change in the total fatty acid composition was shown. Moreover, a decrease of temperature from 20 C to 9 C decreased the incorporation of labeled linoleic and α-linolenic acid into the lipids, but did not modify the unsaturated: saturated acid ratio of the mollusk lipids during this period. A change of habitat from lake to estuary changed very significantly the fatty acid composition of the animal. The ω6 acids, linoleic and arachidonic, typical ofD. patagonicus living in the lake, were partially replaced by ω3 acids. That this change was due to a change of food composition was indicated by the fatty acids of corresponding sediments. Therefore, the fatty acid composition ofD. patagonicus is highly sensitive to food composition and varies little with temperature and seasonal changes. Member of Carrera del Investigador Científico de la Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Member of Carrera del Investigador Científico del Consejo Nacional de Investigaciones Ciéntíficas y Técnicas, Argentina.     

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.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat adrenal gland

Delta-6 and Δ5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active Δ6 desaturase for linoleic and α-linoleic acids and a Δ5 desaturase for eicosa-8,11,14-trienoic acid. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-¹⁴C]linoleic acid to γ-linolenic acid, [1-¹⁴C]α-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-¹⁴C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in Δ5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.     

Composition and biosynthesis of fatty acids inPyramimonas grossii

The green algaPyramimonas grossii orginating in the coastal waters of the Atlantic Ocean Argentina was subcultured until a monoalgal culture was obtained. The fatty acid composition of the alga grown in a mineral medium at 12 C was determined by gas liquid chromatography (GLC) on 2 columns. The major fatty acids were oleic, linoleic, palmitic and α-linolenic acids, but the 20-carbon polyunsaturated acids, 20∶4ω6 and 20∶5ω3, respectively, belonging to the linoleic and α-linolenic series, were also found. Incubation with [¹⁴C] oleate, [¹⁴C] acetate, [¹⁴C] linoleate and [¹⁴C] α-linolenate suggests that linoleate is not directly converted to α-linolenate. [¹⁴C] Acetate was easily converted to palmitic, palmitoleic and oleic acids. However, after 48 hr of incubation, only traces of radioactivity were detected in linoleic acid and no label was found in α-linolenic acid.     

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.     

Age-related changes in Δ6 and Δ5 desaturase activities in rat liver microsomes

Age-related changes in Δ6 desaturation of [1-¹⁴C]α-linolenic acid and [1-¹⁴C]linoleic acid and in Δ5 desaturation of [2-¹⁴C]dihomo-γ-linolenic acid were studied in liver microsomes from Wistar male rats at various ages ranging from 1.5 to 24 mon. Desaturase activities were expressed both as specific activity of liver microsomes and as the capacity of whole liver to desaturate by taking into account the total amount of liver microsomal protein. Δ6 Desaturation of α-linolenic acid increased from 1.5 to 3 mon and then decreased linearly up to 24 mon to reach the same desaturation capacity of liver measured at 1.5 mon. The capacity of liver to desaturate linoleic acid increased up to 6 mon and then remained constant, whereas microsomal specific activity was equal at 1.5 and 24 mon of age. The capacity of liver to convert dihomo-γ-linolenic acid to arachidonic acid by Δ5 desaturation decreased markedly from 1.5 to 3 mon. It then increased to reach, at 24 mon, the same level as that observed at 1.5 mon. Age-related changes in the fatty acid composition of liver microsomal phospholipids at the seven time points studied and of erythrocyte lipids at 1.5 and 24 mon were consistent with the variations in desaturation capacity of liver. In particular, arachidonic acid content in old rats was slightly higher than in young rats whereas contents in linoleic and docosahexaenoic acids varied little throughout the life span. The results suggest that, in liver, the activity of desaturases may be regulated in the course of aging to maintain a constant level of polyunsaturated fatty acids in cellular membranes.     

Microtubular integrity differentially modifies the saturated and unsaturated fatty acid metabolism in cultured hep G2 human hepatoma cells

The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained unaltered. These microtubule-disrupting drugs also diminished Δ9-, Δ5-, and Δ6-desaturase capacities. The effects produced by COL were dose (0–50 μM) and time (0–300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO). Dihydrocytochalasin B (20μM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation of [¹⁴C]FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation of unsaturated FA biosynthesis. The authors are members of the Carrera del Investigador Científico del Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

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.     

Lipid metabolism of the yellow clam,Mesodesma mactroides: 2-Polyunsaturated fatty acid metabolism

The fate of labeled linoleic, α-linolenic, and higher homologs of α-linolenic acid administered to the yellow clam,Mesodesma mactroides, was investigated. It was found that the clam incorporated the acids dissolved in sea water and converted 18∶2 (n−6) into 20∶2 (n−6) and 18∶3 (n−3) into 18∶4 (n−3) and 20∶3 (n−3). The addition of casein hydrolysate to the sea water increased the desaturation capacity of the clam and allowed the conversion of 18∶2 (n−6) into 18∶3 (n−6) to be demonstrated. An enhanced desaturation of 18∶3 (n−3) into 18∶4 (n−3) was also demonstrated. After 12 hr administration of the acid, no radioactivity was found in arachidonic, 20∶5 (n−3), or 22∶6 (n−3). Feeding the clams a culture ofPhaeodactylum tricornutum previously incubated with 1-¹⁴C-α-linolenic acid demonstrated that all the homologs of the α-linolenic series were found in the clam without any important changes. Six hour administration of labeled linolenic acid resulted in the incorporation of the acid into diglycerides and phospholipids. Member of the carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientificas y Tecnicas     

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.     

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.     

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.     

Metabolic transformation of intracraneally injected [1-14C]linoleic and [1-14C]α-linolenic acids in malnourished developing rats

The effect of a low protein diet during pregnancy and lactation on the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from brains of ten-day-old rats was studied. The results indicated that partial deprivation of protein during early development was associated with an increase in the fatty acids of the n−9 family in PC. The fatty acids of the linoleic acid series decreased in PE but were not modified in PC. These minor changes did not affect the double bond index values either in PC or in PE. The effect of protein depletion on thein vivo metabolic transformation of intracraneally injected [1-¹⁴C]linoleic and [1-¹⁴C]α-linolenic acids was also studied. The percentage distribution of the labeled precursors and their derivatives among PC and PE differed from that of mass distribution. These results indicate that the direct uptake of polyunsaturated fatty acids from the blood and/or the low turnover rate of these acids incorporated into PC and PE might be involved in maintaining the fatty acid pattern of these brain lipids.     

A pathway for biosynthesis of divinyl ether fatty acids in green leaves

[1-¹⁴C]α-Linolenic acid was incubated with a particulate fraction of homogenate of leaves of the meadow buttercup (Ranunculus acris L.). The main product was a divinyl ether fatty acid, which was identified as 12-[1′(Z),3′(Z)-hexadienyloxy]-9(Z), 11(E)-dodecadienoic acid. Addition of glutathione peroxidase and reduced glutathione to incubations of α-linolenic acid almost completely suppressed formation of the divinyl ether acid and resulted in the appearance of 13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid as the main product. This result, together with the finding that 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid served as an efficient precursor of the divinyl ether fatty acid, indicated that divinyl ether biosynthesis in leaves of R. acris occurred by a two-step pathway involving an ω6-lipoxygenase and a divinyl ether synthase. Incubations of isomeric hydroperoxides derived from α-linolenic and linoleic acids with the enzyme preparation from R. acris showed that 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid was transformed into the divinyl ether 12-[1′(Z)-hexenyloxy]-9(Z), 11(E)-dodecadienoic acid. In contrast, neither the 9(S)-hydroperoxides of linoleic or α-linolenic acids nor the 13(R)-hydroperoxide of α-linolenic acid served as precursors of divinyl ethers.     

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.     

Reciprocal interactions in the desaturation of linoleic acid into γ-linolenic and eicosa-8,11,14-trienoic into arachidonic

Variable concentrations of [I¹⁴C] linoleic acid and [I¹⁴C] eicosa-8,11,14-trienoic acid were incubated with liver microsomes in a medium containing the necessary cofactors for fatty acid desaturation. The conversion of linoleic into γ-linolenic acid and eicosatrienoic into arachidonic acid were mutually inhibited and the inhibition depended on the concentration of the fatty acids incubated.     

1-14C-Linoleic acid distribution in various tissue lipids of guinea pigs following an oral dose

A recent study on the metabolism of 1-¹⁴C-α-linolenic acid in the guinea pig revealed that the fur had the highest specific activity of all tissues examined, 48 h after dosing. The present study investigated the pattern of tissue lipid labeling following an oral dose of 1-¹⁴C-linoleic acid after the animals had been dosed for the same time as above. Guinea pigs were fed one of two diets with a constant linoleic acid content (18% total fatty acids) and a different content of α-linolenic acid (0.3 or 17.3%) from weaning for 3 wk and 1-¹⁴C-linoleic acid was given orally to each animal for 48 h prior to sacrifice. The most highly labeled tissues (dpm/mg of linoleic acid) were liver, followed by brain, lung and spleen, heart, kidney and adrenal and intestines, in both diet groups. The liver had almost a three-fold higher specific activity than skin and fur which was more extensively labeled than the adipose and carcass. Approximately two-thirds of the label in skin plus fur was found in the fur which, because of a low lipid mass, would indicate that the fur was highly labeled. All tissues derived from animals on the diet with the low α-linolenic acid level were significantly more labeled than the tissues from the animals on the high α-linolenic acid diet, by a factor of 1.5 to 3. The phospholipid fraction was the most highly labeled fraction in the liver, free fatty acids were the most labeled fraction in skin & fur, while triacyglycerols were the most labeled in the carcass and adipose tissue. In these tissues, more than 90% of the radioactivity was found in fatty acids with 2-double bonds in the tissue lipids. These data indicate that the majority of label found in guinea pig tissues 48 h after dosing was still associated with a fatty acid fraction with 2-double bonds, which suggests there was little metabolism of linoleic acid to more highly unsaturated fatty acids in this time frame. In this study, the labeling of guinea pig tissues with linoleic acid, 48 h after dosing, was quite different from the labeling with α-linolenic acid reported previously. The retention of the administered radioactivity from ¹⁴C-linoleic acid in the whole body lipids was 1.6 times higher in the group fed the low α-linolenic acid diet (diet contained a total of 1.8 g PUFA/100 g diet)compared with the group fed the high α-linolenic acid diet (diet contained 3.6 g PUFA/100 g diet). The lack of retention of ¹⁴C-labeled lipids in the whole body would be consistent with an increased rate of β-oxidation of the labeled fatty acid on the diet rich in PUFA, a result supported by other studies using direct measurement of labeled carbon dioxide.     

Zinc deficiency increases the rate of Δ6 desaturation of linoleic acid in rat mammary tissue

The effect of zinc deficiency on the Δ⁶-desaturation of [1-¹⁴C] linoleic acid was studied in mammary tissue microsomes from lactating rats. The rats were maintained on zinc-adequate (20 ppm zinc) or zinc-deficient (10 ppm zinc changing to 0.5 ppm zinc during last trimester) diets throughout gestation and for the first 3 days of lactation. Mammary tissue microsomes were incubated with [1-¹⁴C] linoleic acid and other samples of mammary tissue, mammary milk and the milk in the stomachs of the pups were analyzed for total fatty acid composition. In mammary microsomes from zinc-deficient rats, Δ⁶-desaturation of linoleic acid was 3.4 times greater than in microsomes from zinc-adequate rats. This change in metabolism of linoleic acid was reflected by comparable changes in the relative tissue and milk composition of linoleic and arachidonic acids and in the ratios of palmitic to palmitoleic acid, stearic to oleic acid and linoleic and arachidonic acid.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

Modulation of fatty acid incorporation and desaturation by trifluoperazine in fungi

The effects of trifluoperazine (TFP) on [1-¹⁴C]fatty acid incorporation into the lipids ofMortierella ramanniana var.angulispora were studied. TFP decreased [1-¹⁴C]-fatty acid incorporation into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol, but greatly increased¹⁴C-labeling in phosphatidic acid. These changes in [1-¹⁴C]fatty acid incorporation induced by TFP were accompanied by a decrease in desaturation of some [1-¹⁴C]fatty acids taken up by the fungal cells. When [1-¹⁴C]lioleic acid (LA) was incubated with the fungal cells, total γ-linolenic acid (GLA) formation from incorporated [1-¹⁴C]LA decreased, but the¹⁴C-labeled GLA conent in individual lipid classes was essentially unchanged. This suggests that the site of the TFP effect on GLA formation from [1-¹⁴C]LA taken up from the medium is not the desaturase acting on LA linked to complex lipids. On the other hand, GLA formation from [1-¹⁴C]oleic acid was much less susceptile to TFP, which suggests that in this fungus Δ6 desaturation to GLA has at least two different pathways with different degrees of susceptibility to TFP.