Lipid composition and erucic acid in rat liver cells in culture

Erucic acid (Δ¹³-docosenoic acid) was added to fetal calf serum, then fed to rat liver epithelial cells in culture, and uptake measured at intervals over 24 hr. During the first 6 hr. of incubation, uptake of the docosenoic acid was 21 nmoles/hr/mg protein in 7-day cells, and 15 mmoles/hr/mg protein in 14-day cells. Of¹⁴C-labeled erucic acid taken up by the cells in 24 hr, radioactivity measurements showed 60% of the total lipid¹⁴C activity derived from [1-¹⁴C] 22∶1 in neutral lipid (NL) and 40% in phospholipid (PL); whereas 55% of lipid¹⁴C activity was in NL and 45% in PL when the substrate was [14-¹⁴C] 22∶1. Within the NL fraction, 75% of¹⁴C activity derived from [1-¹⁴C] 22∶1 was in triglyceride (TG) and 11% in cholesterol (CHL), while 79% was in TG and 6.5% in CHL when the substrate was [14-¹⁴C] 22∶1. Triglycerides and cholesteryl esters accumulated in the cells during incubation with erucic acid. Among phospholipids separated by thin layer chromatography, 75% of¹⁴C activity was in lecithin (PC), 10% in phosphatidylethanolamine (PE), 5% in sphingomyelin (SPH), and 1% or less in cardiolipin (DPG). The highest specific activity (SA) was in PC, followed by SPH and PE. Incubation with erucic acid altered fatty acid composition of PC, PE, and SPH, although amounts of phospholipids were unaffected. Gas liquid chromatography analyses detected 18% erucic acid in PC, 2% in PE, and 4–5% in SPH.     

Metabolism of erucic acid in perfused rat liver: Increased chain shortening after feeding partially hydrogenated marine oil and rapeseed oil

The metabolism of [14-¹⁴C] erucic acid was studied in perfused livers from rats fed on diets containing partially hydrogenated marine oil or rapeseed oil for three days or three weeks. Control rats were given groundnut oil. Chain-shortening of erucic acid, mainly to 18∶1, was found in all dietary groups. In the marine oil and rapeseed oil groups, the percentage of chain-shortened fatty acids in very low density lipoproteins-triacylglycerols (VLDL-TG) exported from the liver increased after prolonged feeding. A similar increase was found in liver TG only with partially hydrogenated marine oil. This oil, rich intrans fatty acids, thus seemed to be more effective in promoting chain-shortening. The fatty acid composition of the secreted and stored TG differed both with respect to total fatty acids and radioactively labeled fatty acids, indicating that at least 2 different pools of TG exist in the liver. The lack of lipidosis in livers from rats fed dietary oils rich in 22∶1 fatty acids is discussed in relation to these findings. In conclusion, a discussion is presented expressing the view that the reversal of the acute lipidosis in the hearts of rats fed rapeseed oil or partially hydrogenated marine oils is, to a large extent, derived from the increased chain-shortening capacity of erucic acid in liver.     

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats

The differential uptake and targeting of intravenously infused [1-¹⁴C]palmitic ([1-¹⁴C] 16∶0) and [1-¹⁴C]arachidonic ([1-¹⁴C]20∶4n−6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 μCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-¹⁴C]20∶4n−6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-¹⁴C]16∶0. The unilateral incorporation coefficient, k ^*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-¹⁴C]20∶4n−6 than for [1-¹⁴C]16∶0. In contrast, [1-¹⁴C]16∶0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-¹⁴C]20∶4n−6 was primarily targeted for esterification into phospholipids, while [1-¹⁴C]16∶0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.     

Plasma clerance and hepatic utilization of stearic,myristic and linoleic acids introducedvia chylomicrons in rats

The primary objective of the present study was to compare the rates of plasma clearance and hepatic utilization of stearic (18∶0), myristic (14∶0) and linoleic (18∶2) acids, as introducedvia chylomicrons. Lymph chylomicrons were specifically labeledin vivo with [¹⁴C]stearic and (SA), [¹⁴C]myristic acid (MA), or [¹⁴C]linoleic acid (LA) by infusing donor rats intraduodenally with the labeled fatty acids in a lipid emulsion. Following intravenous injection of recipient rats with the labeled chylomicrons, the rates of plasma clearance and incorporation of the label in triglycerides (TG), phospholipids (PL) and other lipids in the liver were compared at 5, 15 and 30 min. [¹⁴C]SA was cleared at a slightly faster rate (t_1/2=7.0 min) than [¹⁴C]MA (t_1/2=8.1 min) and [¹⁴C]LA (t_1/2=8.0 min) (P<0.05). [¹⁴C]SA was accumulated in the liver at a significantly faster rate than [¹⁴C]MA and [¹⁴C]LA. At the peak (15 min) of hepatic uptake, 30.3% of [¹⁴C]SA, 26.2% of [¹⁴C]LA and 21.9% of [¹⁴C]MA were recovered in the liver. At 30 min, 33.5% of [¹⁴C]SA was taken up by the liver, whereas 27.8% of [¹⁴C]LA and only 15.2% of [¹⁴C]MA were removed. In the liver, the percentage of [¹⁴C]SA incorporated into PL steadily increased with time, whereas the percent-age incorporated into TG decreased. [¹⁴C]SA was preferentially incorporated into PL at all time intervals, as compared with [¹⁴C]MA and [¹⁴C]LA. At 30 min, 38.6% of [¹⁴C]SA was found in PL, and only 5.2% of [¹⁴C]MA and 12.0% of [¹⁴C]LA were present in PL. A large proportion of hepatic [¹⁴C]MA remained unesterified (free fatty acid) throughout the 30-min period, with a small proportion incorporated into PL and TG. Of the total liver¹⁴C radioactivity recovered at 30 min, 63.8% of [¹⁴C]MA, 48.8% of [¹⁴C]LA and 25.5% of [¹⁴C]SA were found unesterified. During 30 min, a significantly greater amount of [¹⁴C]MA (76.9%) was oxidized in both the liver and the peripheral tissue combined, compared with [¹⁴C]LA (64.7%) and [¹⁴C]SA (61.2%). A higher proportion of [¹⁴C]LA was incorporated into TG than into PL at all time intervals. No differences were noted in the relative distribution of¹⁴C in cholesterol and other lipids among the three fatty acids. Using labeled fatty acids incorporatedin vivo into chylomicrons, the present study demonstrated that SA, MA and LA are distinctly different in their metabolic behavior. During the initial 30 min after their entry into the blood, 92–95% of the fatty acids were cleared. During this early phase of metabolism, [¹⁴C]SA was preferentially utilized for liver PL synthesis, whereas [¹⁴C]LA was better incorporated into TG. [¹⁴C]MA was poorly incorporated into hepatic lipids, but was preferentially oxidized in the liver or utilized by the peripheral tissue.     

Plasma free fatty acid incorporation into the outer and inner myocardium of unanesthetized dogs

¹⁴C-Palmitate (16∶0) and³H-oleate (18∶1) were infused into unanesthetized dogs for 90 min. Lipid and isotopic analyses were then performed on the left ventricular outer and inner walls. Average values in μmoles per gram wet weight tissue for both outer wall trigly ceride (TG) (7.0±2.6 (S.E.)) and phospholipid (PL) (17.3±2.4) were higher then the inner wall TG (1.4±0.3) and PL (12.0±2.4). TG fatty acid distribution was similar in the outer and inner myocardium. Thus those factors regulating incorporation of various fatty acids may operate at comparable rates in both heart segments. The same observation and relationship was observed for PL, but the two classes had markedly different fatty acid spectra. The transmural gradients for these classes may be related to relatively hypoxic conditions in the inner wall. Uniform¹⁴C DPM concentrations were found in both TG and PL of the outer and inner myocardium. A similar distribution pattern was found for³H. This may indicate that an individual species of plasma free fatty acid (FFA) undergoes uniform initial incorporation into TG and PL despite the existence of transmural gradients and fatty acid distribution differences. The mean outer and inner wall TG and PL³H to¹⁴C DPM concentration ratio (range of 3.9 to 4.8) was similar to a calculated plasma³H-18∶1 to¹⁴C-16∶0 specific activity ratio of 4.23. This indicates that net incorporation of fatty acids into TG and PL over 90 min was proportional to their plasma FFA concentrations, rather than to endogenous tissue lipid concentrations. The lipid gradients and fatty acid spectrum differences observed may thus be caused by recycling and catabolic pathways rather than to direct control of plasma FFA entry into TG and PL. The research presented here was performed in partial fulfillment of the requirements for the M.Sc. degree in Physiology at Hahnemann Medical College by J.S. Steinberg. Two abstracts related to this study were published previously (1,2)     

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.     

Lipogenesis in the developing brain from intracranially administered [1-14C] acetate and [U-14C] glucose

Fifteen-day-old rats divided into two groups were given [1-¹⁴C]acetate or [U-¹⁴C] glucose by intracranial injection and were sacrificed after 1 hr. Analysis of lipids from the two groups showed differences in the incorporation of radioactivity in the polar lipids and cholesterol. Analysis of brain fatty acid showed that whereas radioactivity from acetate was incorporated into saturated, monoand polyunsaturated fatty acids, the radioactivity from [U-¹⁴C] glucose was found only in 16∶0, 18∶0, and 18∶1. No radioactivity was found in polyunsaturated fatty acids even after concentration of this fraction by AgNO₃:SiO₂ thin layer chromatographic method. This difference is discussed in hypothetical terms of nonhomogeneous acetyl CoA pool, formation of acetyl CoA from glucose exclusively inside the mitochondria, and activation of injected acetate to acetyl CoA.     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

Placental transport oftrans fatty acids in the rat

Placental transport of 9-trans [1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans [1-¹⁴C] octadecadienoic (linoelaidic) acids was demonstrated in rats. On the 18th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparison, 9-cis [1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis [1-¹⁴C] octadecadienoic (linoleic) acid also was injected into the maternal circulation of rats. All animals were sacrificed 1 hr following injection. Lipid composition and distribution of label were determined in maternal plasma, placental and fetal tissues. Differences in specific activities of plasma, placental and fetal total lipids indicated a decreasing concentration gradient for bothcis andtrans isomers of octadecenoic and octadecadienoic acids. Distribution of radioactivity in various lipid components was determined by thin layer chromatography. Irrespective of the label, the highest percentage of total radioactivity was carried by triglycerides (TG) in maternal plasma (∼60–80%), and was incorporated mainly in phospholipids (PL) of fetal tissues (∼50–60%). A nearly equal distribution of the label was found between PL and TG of placental lipids (∼40%). Radioactivity of fatty acid methyl esters (FAME) determined by radiogas liquid chromatography indicated that after injection of linoelaidate, radioactivity of maternal plasma, placental and fetal tissue FAME was associated only witht,t-18∶2. Following injection of elaidate, all the radioactivity in placental FAME was associated witht-18∶1; however, in fetal tissues, the label was distributed between 16∶0 andt-18∶1. These findings suggest that, in contrast to linoelaidic acid, rat fetal tissues can metabolize elaidic acid via β oxidation to form acetyl CoA and palmitic acid.     

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.     

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.     

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.     

Incorporation and metabolic conversion of erucic acid in various tissues of the rat in short term experiments

Rats were intravenously injected with a mixture of free (14-¹⁴C) erucic acid (22∶1) and (9–10³H) oleic acid (18∶1). After 2, 4, 8, 16, and 30 min, radioactivity was examined in blood, liver, heart, kidneys, and spleen. Free (¹⁴C) 22∶1 disappeared from the blood more rapidly than free (¹⁴C) 18∶1 between 0 and 8 min. Incorporation of label into triglycerides only appeared after 16 min and at 30 min they represented 4% of the injected radioactivity. In this fraction, 63% of¹⁴C radioactivity was present as 18∶1 and not as the original 22∶1, while almost all³H radioactivity was recovered as unchanged 18∶1. At all times studied, the majority of radioactivity was found in the liver, primarily as triglycerides (60% of radioactivity in total lipids) and as phospholipids (20–30%).¹⁴C was present in nearly the same proportion as³H (13% of injected radioactivity after only 2 min, 11% at 30 min).¹⁴C radioactivity was contained in 18∶1 in higher proportion than 22∶1 (45% in triglycerides, 65% in phospholipids). Since labeled triglycerides of blood, rich in (¹⁴C) 18∶1, mainly originate from the liver triglycerides, it appears that 18∶1 is the major form of utilization of 22∶1 in the tissues after its conversion in liver. In the other organs tested, radioactivity was found 10–15 times lower than in liver. In the heart,¹⁴C was 3 to 4 times higher than³H. More than 80% was recovered as 22∶1 in triglycerides. In spleen and kidneys, the¹⁴C:³H ratio was particularly high in free fatty acids and monoglycerides. In kidneys, 60% of¹⁴C was present as nervonic acid (24∶1) in monoglycerides and 40% in phospholipids, suggesting that the mononervonin formed was used for phospholipid biosynthesis. A preliminary report of this work was presented at the 10th International Congress of Nutrition, Kyoto, Japan, August 1975.     

Divergent incorporation of dietary trans fatty acids in different serum lipid fractions

Trans fatty acids may be involved in atherosclerotic vascular diseases. We investigated the incorporation of dietary trans fatty acids and oleic acid into the serum triglycerides (TG), cholesterol esters (CE), and phospholipids (PL). Fourteen healthy female volunteers, aged 23.2±3.1 yr (mean±SD), body mass index 20.8±2.1 kg/m² participated in this study. All subjects consumed both a trans fatty acid-enriched diet (TRANS diet) and an oleic acid-enriched diet (OLEIC diet) for 4 wk according to a randomized crossover design. Both experimental diet periods were preceded by consumption of a baseline diet for 2 wk which supplied 37% of total energy (E%) as fat: 18 E% from saturated fatty acids (SFA), 12 E% from monounsaturated fatty acids, and 6 E% from polyunsaturated fatty acids. Five E% of the SFA in the baseline diet was replaced by trans fatty acids (18∶1 t and 18∶2 c,t+18∶2t,t, where c is cis and t is trans) in the TRANS diet and by oleic acid (18∶1n-9) in the OLEIC diet. After the TRANS diet, the proportions of 18∶1t and 18∶2t increased (P <0.001) in all serum lipid fractions analyzed. The increase of 18∶1 t in TG and PL (1.80±0.28 vs. 5.26±1.40; 1.07±0.34 vs. 3.39±0.76 mol% of total fatty acids, respectively) was markedly higher than that in CE (0.44±0.07 vs. 0.92±0.26), whereas that of 18∶2t was nearly the same in all three fractions. The proportions of palmitic, stearic, arachidonic, and eicosapentaenoic acids in TG, CE, and PL and that of oleic acid in TG and CE were decreased when compared with the baseline value. In contrast, the proportion of palmitoleic acid in TG and PL and that of linoleic acid in PL increased on the TRANS diet. After consumption of the OLEIC diet, the proportion of oleic acid increased in all three lipid fractions analyzed, and the percentage increase was nearly the same in all fractions. In contrast, the proportions of 18∶1 t in TG and PL and 18∶2 t in TG and CE decreased when compared with the baseline value. In conclusion, a moderate increase in dietary trans fatty acids resulted in a marked incorporation into serum lipids and decreased the conversion of linoleic acid to its more unsaturated long-chain metabolites. Analysis of 18∶1 t from serum TG and PL seems to reflect reliably the dietary intake of this fatty acid.     

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.     

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.     

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).     

Uptake of fatty acids by the developing rat brain

Polyunsaturated fatty acids are avidly taken up by the developing rat brain. To explore the specificity of this process, [1-¹⁴C]labeled 16∶0, 18∶2n−6, 18∶3n−3, and 22∶6n−3 each were co-injected with [³H]18∶1n−9 into the jugular vein of two-wk-old functionally hepatectomized and shamoperated control rats. The radioactivities present in the brain, liver and serum were assessed 30 min after injection. Uptake of labeled fatty acids into brain lipids steadily increased with increasing degree of unsaturation, with more than twice as much uptake of 22∶6n−3 compared to 16∶0. Phosphatidylcholine was the principal radioactive species in the brain except for animals injected with [1-¹⁴C]22∶6n−3, in which more of the label was incorporated into phosphatidylethanolamine. Determination of watersoluble oxidation products in the brain and serum revealed that the greater uptake of the more unsatrated fatty acids did not result from differences in rates of degradation.     

Mass fragmentographic determination of docosenoic acid in rapeseed oils

A highly sensitive and accurate reference method for determination of docosenoic acid (mainly erucic acid, 22∶1n−9) in different rapeseed oils is described. A fixed amount of [1-¹⁴C]erucic acid methyl ester (about 1 μg) is added to a fixed amount of oil. After treatment with sodium methoxide/methanol reagent and extraction with hexane, the amount of unlabeled erucic acid is determined from the ratio between the recordings at m/e 320 and m/e 322 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with an MID (multiple ion detector). The two ions used correspond to the M-32 peak in the mass spectrum of unlabeled and [1-¹⁴C]labeled erucic acid methyl ester. The relative standard deviation of the method is about 1.8%. The method was compared with a gas chromatographic method for determination of erucic acid.     

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.