Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.     

Metabolism of n−3 and n−6 fatty acids in Atlantic salmon liver: Stimulation by essential fatty acid deficiency

Oxidation, esterification, desaturation, and elongation of [1-¹⁴C]18∶2n−6 and [1-¹⁴C]18∶3n−3 were studied using hepatocytes from Atlantic salmon (Salmo salar I.) maintained on diets deficient in n−3 and n−6 polyunsaturated fatty acids (PUFA) or supplemented with n−3 PUFA. For both dietary groups, radioactivity from 18∶3n−3 was incoporated into lipid fractions two to three times faster than from 18∶2n−6, and essential fatty acids (FFA) deficiency doubled the incorporation. Oxidation to CO₂ was very low and was independent of substrate or diet, whereas oxidation to acid-soluble products was stimulated by EFA deficiency. Products from 18∶2n−6 were mainly 18∶3n−6, 20∶3n−6, and 20∶4n−6, with minor amounts of 20∶2n−6 and 22∶5n−6. Products from 18∶3n−3 were mainly 18∶4n−3, 20∶5n−3, and 22∶6n−3, with small amounts of 20∶3n−3. The percentage of 22∶6n−3 in the polar lipid fraction of EFA-deficient hepatocytes was fourfold higher than in n−3 PUFA-supplemented cells. This correlated well with our other results obtained after abdominal injection of [1-¹⁴C]18∶3n−3 and [1-¹⁴C]18∶2n−6. In hepatocytes incubated with [4,5-³H]-22∶6n−3, 20∶5n−3 was the main product. This retrocon-version was increased by EFA deficiency, as was peroxisomal β-oxidation activity. This study shows that 18∶2n−6 and 18∶3n−3 can be elongated and desaturated in Atlantic salmon liver, and that this conversion and the activity of retroconversion of very long chain PUFA is markedly enhanced by FFA deficiency.     

Effect of n−3 fatty acid deficiency on fatty acid composition and metabolism of aminophospholipids in rat brain synaptosomes

Docosahexaenoic acid (DHA, 22∶6n−3) is one of the major polyunsaturated fatty acids esterified predominantly in aminophospholipids such as ethanolamine glycerophospholipid (EtnGpl) and serine glycerophospholipid (SerGpl) in the brain. Synaptosomes prepared from rats fed an n−3 fatty acid-deficient safflower oil (Saf) diet had significantly decreased 22∶6n−3 content with a compensatory increased 22∶5n−6 content when compared with rats fed an n−3 fatty acid-sufficient perilla oil (Per) diet. When the Saf group was shifted to a diet supplemented with safflower oil plus 22∶6n−3 (Saf+DHA) after weaning, 22∶6n−3 content was found to be restored to the level of the Per group. The uptake of [³H]ethanolamine and its conversion to [³H]EtnGpl did not differ significantly among the three dietary groups, whereas the formation of [³H]lysoEtnGpl from [³H]ethanolamine was significantly lower in the Saf group than in the other groups. The uptake of [³H]serine, its incorporation into [³H]SerGpl, and the conversion into [³H]EtnGpl by decarboxylation of [³H]SerGpl did not differ among the three dietary groups. The observed decrease in lysoEtnGpl formation associated with a reduction of 22∶6n−3 content in rat brain synaptosomes by n−3 fatty acid deprivation may provide a clue to reveal biochemical bases for the dietary fatty acids-behavior link.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

Metabolism of very long chain polyunsaturated fatty acids in isolated rat germ cells

Which cell type is responsible for the high levels of very long chain polyunsaturated fatty acids in testis and whether this fatty acid pattern is a result of a local synthesis are not presently known. In this study, fatty acid conversion from 20∶4n−6 to 22∶5n−6 and from 20∶5n−3 to 22∶6n−3 was investigated in isolated rat germ cells incubated with [1-¹⁴C]-labeled fatty acids. The germ cells elongated the fatty acids from 20- to 22-carbon atoms and from 22- to 24-carbon atoms but had a low Δ6 desaturation activity. Thus, little [¹⁴C]22∶5n−6 and [¹⁴C]22∶6n−3 were synthesized. When Sertoli cells were incubated with [1-¹⁴C]20∶5n−3 for 24 h, an active fatty acid elongation and desaturation were observed. In vivo germ cells normally have a higher content of 22∶5n−6 or 22∶6n−3 than Sertoli cells. An eventual transport of essential fatty acids from Sertoli cells to germ cells was thus studied. Different co-culture systems were used in which germ cells were on one side of a filter and Sertoli cells on the opposite side. When isolated pachytene spermatocytes or round spermatids were added to the opposite side of a semipermeable filter, approximately 1 nmol [¹⁴C]-22∶6n−3 crossed the filter. Little of this was esterified in the germ cells. Similarly, in using [1-¹⁴C]20∶4n−6 in identical experiments, very little [¹⁴C]22∶5n−6 was esterified in germ cells on the opposite side of the filter. Although the very active synthesis of 22∶5n−6 and 22∶6n−3 observed in Sertoli cells suggests a transport of these compounds to germ cells, this was not experimentally determined.     

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.     

Interaction of (n−3) and (n−6) fatty acids in desaturation and chain elongation of essential fatty acids in cultured glioma cells

Recent research in various biological systems has revived interest in interactions between the (n−6) and (n−3) essential fatty acids. We have utilized cultured glioma cells to show that linolenic acid, 18∶3(n−3), is rapidly desaturated and chain elongated; 20∶5(n−3) is the major product and accumulates almost exclusively in phospholipids. We examined effects of various (n−6), (n−3), (n−9) and (n−7) fatty acids at 40 μM concentration on desaturation and chain elongation processes using [1-¹⁴C]18∶3(n−3) as substrate. In general, monoenoic fatty acids were without effect. The (n−6) fatty acids (18∶2, 18∶3, 20∶3, 20∶4 and 22∶4) had little effect on total product formed. There was a shift of labeled product to triacylglycerol, and in phospholipids, slightly enhanced conversion of 20∶5 to 22∶5 was evident. In contrast, 22∶6(n−3) was inhibitory, whereas 20∶3(n−3) and 20∶5(n−3) had much less effect. At concentrations <75 μM, all acids were inhibitory. Most products were esterified to phosphatidylcholine, but phosphatidylethanolamine also contained a major portion of 20∶5 and 22∶5. We provide a condensed overview of how the (n−6) and (n−3) fatty acids interact to modify relative rates of desaturation and chain elongation, depending on the essential fatty acid precursor. Thus, the balance between these dietary acids can markedly influence enzymes providing crucial membrane components and substrates for biologically active oxygenated derivatives.     

Effects of dietary supplementation of rapeseed oil on metabolism of [1-<Superscript>14</Superscript>C]18∶1n−9, [1-<Superscript>14</Superscript>C]20∶3n−6, and [1-<Superscript>14</Superscript>C]20∶4n−3 in atlantic salmon heaptocytes

Atlantic salmon were fed fish meal-based diets supplemented with either 100% fish oil (FO) or 100% rapeseed oil (RO) from an initial weight of 85 g to a final average weight of 280 g. The effects of these diets on the capacity of Atlantic salmon hepatocytes to elogate, desaturate, and esterify [1-¹⁴C]18∶1n−9 and the immediate substrates for the Δ⁵ desaturase, [1-¹⁴C]20∶3 n−6 and [1-¹⁴C]20∶4n−3, were investigated. Radiolabeled 18∶1n−9 was mainly esterified into cellular TAG, whereas the more polyunsaturated FA, [1-¹⁴C]20∶3n−6 and [1-¹⁴C]20∶4n−3, were primarily esterified into cellular PL. More of the elongation product, [1-¹⁴C]20∶1n−9, was produced from 18∶1n−9 and more of the desaturation and elongation products, 22∶5n−6 and 22∶6n−3, were produced from [1-¹⁴C]20∶3n−6 and [1-¹⁴C]20∶4n−3, respectively, in RO hepatocytes than in FO hepatocytes. Further, we studied whether increased addition of [1-¹⁴C]18∶1n−9 to the hepatocyte culture media would affect the capacity of hepatocytes to oxidize 18∶1n−9 to acid-soluble products and CO₂. An increase in exogenous concentration of 18∶1n−9 from 7 to 100 μM resulted in a nearly twofold increase in the amount of 18∶1n−9 that was oxidized. The conversion of 20∶4n−3 and 20∶3n−6 to the longer-chain 22∶6n−3 and 22∶5n−6 was enhanced by RO feeding in Atlantic salmon hepatocytes. The increased capacity of RO hepatocytes to produce 22∶6n−3 was, however, not enought to achieve the levels found in FO hepatocytes. Our data further showed that there were no differences in the hepatocyte FA oxidation capacity and the lipid deposition of carcass and liver between the two groups.     

The metabolism of 20- and 22-carbon unsaturated acids in rat heart and myocytes as mediated by feeding fish oil

When rats were fed 5% corn oil, the heart phospholipids contained large amounts of 22-carbon (n−6) acids. When half of the corn oil was replaced with fish oil, the reduced level of arachidonate and 22-carbon (n−6) acids in phospholipids was accompanied by increases in the levels of 22-carbon (n−3) acids while only small amounts of 20∶5(n−3) were acylated. Heart myocytes readily took up and acylated [1-¹⁴C]-labeled 20∶4(n−6), 20∶5(n−3) and 22∶6(n−3) into phospholipids. The uptake and acylation of 20∶4(n−6) was greater than for 20∶5(n−3) but the intracellular labeling profiles were similar. Uptake and acylation of 22∶6(n−3) was somewhat lower. In addition the intracellular labeling profile differed in that more 22∶6(n−3) was incorporated into the ethanolamine-containing phospholipids than when 20∶4(n−6) or 20∶5(n−3) were the substrates. Neither 20∶4(n−6) nor 20∶5(n−3) was chain elongated. When [3-¹⁴C]-labeled 22∶4(n−6) and 22∶5(n−3) were the substrates, it was not possible to detect radioactive 22∶5(n−6) or 22∶6(n−3). Both [3-¹⁴]-labeled substrates were acylated into phospholipids and retroconverted with the subsequent esterification of radioactive 20∶4(n−6) and 20∶5(n−3) into triglycerides and phospholipids. These studies show that cardiomyocytes lack the ability to make 22-carbon acids from 20-carbon precursors but they retroconvert 22-carbon acids to 20-carbon acids. The high levels of 22-carbon polyunsaturated acids in total heart lipids thus cannot be attributed to the synthetic capacities of cardiomyocytes.     

Essential fatty acid uptake and metabolism in the developing rodent brain

Studies were carried out to determine whether the brain takes up and metabolizes essential fatty acids during early postnatal development in rodents. Rats and mice were dosed with deuterium-labeled linoleic and linolenic acids either by intraperitoneal injection or by gavage. Animals were killed at different times thereafter, and organs were removed. Brains, livers, and blood were analyzed by gas chromatography— negative-ion-mass spectrometry for labeled fatty acids. To determine whether fatty acids were present in the brain apart from cerebral blood, a subset of animals was exsanguinated by perfusion with buffered saline, and the brain was then fractionated into subcellular components. Results demonstrated that the brain took up both labeled essential fatty acids within 8 h from the time of dosing. There was on average a greater uptake of linolenic acid into the cerebellum than into the cerebral cortex during the first 8 d of life in rats. The amount of linoleic acid taken into either region was similar, however. Docosahexaenoic acid intermediates, 20∶5n−3 and 22∶5n−3, were also found labeled in the brain. Time-course labeling experiments indicated that these intermediates may be converted to 22∶6n−3 within the brain. A rise of labeled 22∶6n−3 in the brain at 24 h appeared to be due to uptake of this fatty acid from the blood. The Amount of labeled 22∶6n−3 in the brain continued to increase beyond 24 h, and this did not appear to be correlated with its blood concentration. These results suggest that, during development in the rodent, different regions within the brain may vary in their capacity to synthesize 22∶6n−3, and this may be correlated with regional growth rates.     

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.     

Effects of aging on the composition and metabolism of docosahexaenoate-containing lipids of retina

The amount of docosahexaenoate (22∶6n−3)-containing phospholipid species decreases with aging in the rat retina. Most lipids, but especially choline and serine glycerophospholipids, show a significant fall in 22∶6n−3, which is not compensated by increases in other polyenoic fatty acids. The decrease not only affects 22∶6 but also various very long chain n−3 hexaenoic fatty acids which, in phosphatidylcholine, have up to 36 carbon atoms, and which are probably synthesized by successive elongations of 22∶6n−3. The in vitro incorporation of [2-³H] glycerol into retinal lipids indicates that the de novo biosynthetic pathways are not impaired by aging. The incorporation of [1-¹⁴C]docosahexaenoate is significantly stimulated into all lipids of aged retinas, but to the largest extent in those showing the largest decreases in 22∶6, especially in choline glycerophospholipids. The results indicate that the decreased levels of 22∶6 with aging are due not to an impaired activity of the enzymes involved in the synthesis and turnover of phospholipids but to a decreased availability of this polyene in the retina. It is suggested that this may stem from a defect in some of the enzymatic steps that lead to the synthesis of 22∶6n−3, probably that catalyzed by Δ₄ desaturase, the effect on longer hexaenes being secondary to the decreased synthesis of 22∶6.     

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).     

Elongation predominates over desaturation in the metabolism of 18∶3n−3 and 20∶5n−3 in turbot (Scophthalmus maximus) brain astroglial cells in primary culture

The origin of docosahexaenoic acid (DHA, 22∶6n−3) that accumulates in turbot brain during development was investigated by studying the incorporation and metabolismvia the desaturase/elongase pathways of [1-¹⁴C]-labelled polyunsaturated fatty acids (PUFA) in primary cultures of brain astrocytic glial cells. There was little specificity evident in the total incorporation of PUFAs into the turbot astrocytes. However, specificity was apparent in the distribution of the various PUFAs among the individual lipid classes. In particular, there was very specific incorporation of [¹⁴C]arachidonic acid (AA, 20∶4n−6) into phosphatidylinositol balanced by a lower incorporation of this acid into total diradyl glycerophosphocholines. [¹⁴C]-Linolenic acid (LNA, 18∶3n−3) and [¹⁴C]eicosapentaenoic acid (EPA, 20∶5n−3) were metabolizedvia the desaturase/elongase pathways to a significantly greater extent than [¹⁴C]linoleic acid (18∶2n−6) and [¹⁴C]AA. The turbot astrocytes expressed very little Δ5 desaturase activity and only low levels of Δ4 desaturation activity. Although the percentages were small, approximately 4–5 times as much labelled DHA was produced from [¹⁴C]EPA compared with [¹⁴C]LNA. However, it was concluded that very little DHA in the turbot brain could result from the metabolism of LNA and EPA in astrocytic glial cells.     

Effects of dietary n−3 polyunsaturated fatty acids on phospholipid composition and calcium transport in mouse cardiac sarcoplasmic reticulum

The effects of dietary n−3 and n−6 polyunsaturated fatty acids on the fatty acid composition of phospholipid, Ca⁺⁺· Mg⁺⁺ ATPase and Ca⁺⁺ transport activities of mouse sarcoplasmic reticulum were investigated. Mice were fed a 2 weight percent fat diet containing either 0.5 weight percent ethyl esters of 18∶3n−3, 20∶5n−3 or 22∶6n−3 as a source of n−3 polyusaturated fatty acid or 0.5 weight percent safflower oil as a cource of n−6 polyunsaturated fatty acid for 10 days. Olive oil (2 weight percent) was used as a control diet. Although feeding n−6 polyunsaturated fatty acid induced very little modifications of the phospholipid sarcoplasmic reticulum fatty acid composition, feeding n−3 polyunsaturated fatty acid altered it markedly. Inclusion of 18∶−3, 20∶5n−3 or 22∶6n−3 in the diet caused an accumulation of 22∶6n−3, which replaced 20∶4n−6 and 18∶2n−6 in phospholipid sarcoplasmic reticulum. The saturated fatty acids were significantly increased with a concurrent reduction of 18∶1n−9. These changes in the fatty acid composition resulted in a decrease in the values of the n−6/n−3 polyunsaturated fatty acid ratio and a decrease in the ratio of 20 carbon to 22 carbon fatty acids esterified in the phospholipid sarcoplasmic reticulum. This was associated with a decrease in Ca⁺⁺ uptake by n−3 polyunsaturated fatty acid enriched sarcoplasmic reticulum vesicles as compared with n−6 fatty acid and control diet sarcoplasmic reticulum vesicles. However, neither the affinity for Ca⁺⁺ nor the maximal velocity of ATP hydrolysis activity of Ca⁺⁺·MG⁺⁺ ATPase were altered by the different diets. The data suggest that the incorporation of 22∶6n−3 and/or the decrease of 20∶4n−6 plus 18∶2n−6 in the phospholipid sarcoplasmic reticulum may affect the membrane lipid bilayer structure and make it more permeable to Ca⁺⁺.     

Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects

The effect of dietary docosahexaenoic acid (22∶6n−3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n=6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18∶1n−9[d6], 18∶2n−6[d2], and 18∶3n−3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20∶5n−3[d4] and 22∶6n−3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20∶3n−6[d2] and 20∶4n−6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18∶1n−9[d6], 18∶2n−6[d2], and 18∶3n−3[d4]. The results indicate that accumulation of n−3 LCFA metabolites synthesized from 18∶3n−3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n−6 LCFA metabolites synthesized from 18∶2n−6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n−6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n−6 LCFA metabolites and an increase in n−3 LCFA levels in tissue lipids.     

Effect of the Δ<Superscript>6</Superscript>-desaturase inhibitor SC-26196 on PUFA metabolism in human cellsinhibitor SC-26196 on PUFA metabolism in human cells

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-{[(1E)-pyridin-3-yl-methylidene]-amino}piperazin-1-yl)pentanenitrile (SC-26196), a Δ⁶-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 μM [1-¹⁴C] 18∶2n−6 by 87–95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-¹⁴C]20∶3n−6 to 20∶4 in the fibroblasts, demonstrating that it is selective for Δ⁶-desaturase. The IC₅₀ values for inhibition of the desaturation of 2 μM [1-¹⁴C] 18∶3n−3 and [3-¹⁴C]24∶5n−3 in the fibroblasts, 0.2–0.4 μM, were similar to those for the inhibition of [1-¹⁴C] 18∶2n−6 desaturation, and the rates of recovery of [1-¹⁴C] 18∶2n−6 and [3-¹⁴C] 24∶5n−3 desaturation after removal of SC-26196 from the culture medium also were similar. SC-26196 reduced the conversion of [3-¹⁴C] 22∶5n−3 and [3-¹⁴C] 24∶5n−3 to DHA by 75 and 84%, respectively, but it had no effect on the retroconversion of [3-¹⁴C] 24∶6n−3 to DHA. These results demonstrate that SC-26196 effectively inhibits the desaturation of 18- and 24-carbon PUFA and, therefore, decreases the synthesis of arachidonic acid, EPA, and DHA in human cells. Furthermore, they provide additional evidence that the conversion of 22∶5n−3 to DHA involves Δ⁶-desaturation.     

Acylation of docosahexaenoic acid into phospholipids by intact human neutrophils

Docosahexaenoic acid was not only acylated into phospholipids but also into triacylglycerols by intact human neutrophils. The distribution of radiolabeled docosahexaenoic acid among individual phospholipids was dependent on the incubation time. [1-¹⁴C]Docosahexaenoic acid at all concentrations (1 to 8 μM) was acylated mainly into phosphatidic acid after 1–2 min incubation, and the radioactivity of phosphatidic acid started to decline after a longer period of incubation, suggesting the participation of docosahexaenoyl-phosphatidic acid in the synthesis of other glycerolipids. It was acylated primarily into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) after a 2-hr incubation. The labeled phosphatidic acid may be rapidly deacylated and the 22∶6(n−3) moiety is then reacylated into other lysophospholipids. The low levels of [¹⁴C]22∶6(n−3) in 1,2-diacylglycerol suggest that the deacylation-reacylation cycle may be a major pathway in the formation of [¹⁴C]22∶6(n−3)-PC and-PE in intact neutrophils. This n−3 fatty acid was a relatively poor substrate for acylation into phosphatidyl-inositol as compared to arachidonic acid and eicosapentaenoic acid. However, the patterns of distribution of all three polyunsaturated fatty acids among the diacyl-and ether-linked class compositions of PC and PE were similar. These data suggest the potential of increasing the content of docosahexaenoic acid of membrane lipids in neutrophils by dietary supplement of this fatty acid.     

Metabolism of arachidonate in rat testis: Characterization of 26–30 carbon polyenoic acids

Fatty acid methyl esters of long-chain polyenoic fatty acids (LCPA) from rat testis injected with [1-¹⁴C] arachidonate were analyzed and separated by reversed-phase high performance liquid chromatography (RP-HPLC). Earlier, all previously identified LCPA were prepared in high purity along with 4 previously unidentified fatty acids, which were further characterized by capillary gas chromatography (GC), catalytic hydrogenation and alkaline isomerization. Unidentified fatty acids proved to be 26∶4, 26∶5, 28∶5 and 30∶5. All of these LCPA incorporated¹⁴C from arachidonate (20∶4) to specific activities that were comparable to that of 20∶4 and previously identified metabolites of 20∶4 and much greater than specific activities of 18∶1n−9 or 22∶6n−3. LCPA were analyzed on a capillary GC system capable of resolving knowncis-trans and positional isomers of the n−3, n−6, n−7 and n−9 families of unsaturated fatty acids. Log plots of isothermal retention times and normal plots of temperature programmed retention times were linear (r=0.999) in carbon number when values for known and previously unidentified LCPA of 4 or 5 double bonds, respectively, were coplotted. Thus, the newly identified fatty acids belong to the n−6 family of fatty acids synthesized from arachidonic acid.     

β-Oxidation of 18∶3n−3 in atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) hepatocytes treated with different fatty acids

To study whether Atlantic salmon β-oxidation was affected by dietary FA composition, an in vitro study with primary hepatocytes was undertaken. Isolated hepatocyte cultures were stimulated with either 16∶0, 18∶1n−9, 18∶2n−6, 18∶3n−3, 20∶5n−3, or 22∶6n−3 in triplicate for 24 h. In addition, a control was included where no FA stimulation was performed, also in triplicate. After stimulation, radiolabeled [1-¹⁴C]18∶3n−3 was added and the cells were incubated for 2 h at 20°C. The cells were then harvested, and radioactivity was determined in the acid-soluble part of the cells and medium, i.e., the end products of the β-oxidation pathway. Specific β-oxidation activity was significantly higher in hepatocytes stimulated with 18∶3n−3. Further, when taking into account the amount of radiolabeled [1-¹⁴C]18∶3n−3 taken up by the cells—the relative amount of β-oxidized [1-¹⁴C]18∶3n−3 of the total FA taken up by the hepatocytes—no significant differences were found. Thus, the regulation of β-oxidation activity in the primary Atlantic salmon hepatocytes seems to be at the level of FA uptake and transport into the cell. This in vitro study shows that the catabolism processes in salmon hepatocytes are affected by the FA available and probably already regulated at the level of FA uptake.