Erucic acid and phospholipids of newborn rat heart cells in culture

Erucic acid (Δ¹³-docosenoic acid), labeled with¹⁴C in the 1-or 14-position, was incorporated into fetal calf serum and fed to beating, neonatal rat myocardial cells in culture. Uptake of the docosenoic acid during the first 6 hr of incubation was 41 nM/hr/mg protein in 7-day old cells and 29 nM/hr/mg protein in 14-day old cells. Fifty-seven percent of the¹⁴C-activity was taken up from the medium in 24 hr, of which 77% was in the cells and 23% was unaccounted for. Of the¹⁴C-activity taken up, 26% was in extractable lipid, with two-thirds in neutral lipid and one-third in phospholipid. Within the neutral lipid fraction, 88% of the¹⁴C-activity was present in triglycerides; while in phospholipids, 66% of the¹⁴C-activity was in phosphatidylcholine (PC); 14% in phosphatidylethanolamine (PE); 6% in sphingomyelin (SPH) and 1% or less in cardiolipin (DPG). PC had the highest specific activity, followed by SPH and PE. The specific activity of PE was one-half that of SPH when the¹⁴C-erucic acid substrate was labeled at the carboxyl position, but increased to equal that of SPH when the substrate was labeled at the double bond. The fatty acids of PC, PE, and SPH were influenced by erucic acid in the growth medium, but the amounts of each phospholipid were not affected. It is proposed that the altered fatty acid composition associated with incorporation of erucic acid or its metabolites into PC, PE, and SPH may affect integrity and function of heart cell membranes.     

Influence of dietary fat on metabolism of (14-14C)erucic acid in the perfused rat liver. Distribution of metabolites in lipid classes

Two groups of rats were fed diets containing 20% by weight of either partially hydrogenated marine oil supplemented with sunflower seed oil (PHMO) or palm oil (PO) for 8 wk. Using a liver perfusion system, the effect of dietary long chain monoenoic fatty acids on the uptake and metabolism of [14-¹⁴C]erucic acid was studied. The perfusion times were 15 and 60 min, respectively. The two groups showed equal ability for erucic acid uptake in the liver but differed in the channeling of the fatty acids into various metabolic pathways. A higher metabolic turnover of 22∶1 in the PHMO livers relative to the PO livers was demonstrated by an increased recovery of total [¹⁴C]labeling in the triglyceride (TG) and phospholipid (PL) fractions, already evident after 15 min of perfusion. The chainshortening capacity was highest in the PHMO group, reflected by a higher [¹⁴C]18∶1 incorporation in both TG and PL, and increasing from 15 to 60 min of perfusion. The amount of [¹⁴C]18∶1 found in PL and TG after 60 min of perfusion of livers from rats fed PO corresponded to that shown for the PHMO group after 15 min. The PL demonstrated a discrimination against 22∶1 compared to TG, and, when available, 18∶1 was highly preferred for PL-synthesis. The total fatty acid distribution in the TG, as determined by gas liquid chromatography (GLC), reflected the composition of the dietary fats. In the total liver PL, 22∶1 and 20∶1 were present in negligible amounts, although the PHMO diet contained 12–13% of both 22∶1 and 20∶1. In the free fatty acid fraction (FFA), the major part of the radioactivity (≈80%) was [14-¹⁴C]erucic acid, and only small amounts of [¹⁴C]18∶1(<2%) were presents, even after 60 min of perfusion. The shortened-chain 18∶1 was readily removed from the FFA pool and preferentially used for lipid esterification.     

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.     

Competitive deposition oftrans-12- andcis-9-octadecenoates into egg yolk lipids

The deposition oftrans-12-octadecenoate-12(13)-³H (12t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids.trans-12-Octadecenoate was preferentially incorporated into cholesteryl esters (CE), phosphatidylcholines (PC), and phosphatidylethanolamines (PE) but was discriminated against in triglycerides (TG). Isotopic ratios indicate that 5.9 and 5.6 times more 12t-18∶1-³H than 9c-18∶1-¹⁴C was esterified at the 1-acyl position of PE and PC, respectively. The combined 1- and 3-acyl positions of TG and the 2-acyl position of TG, PE and PC were each preferentially esterified with 9c-18∶1-¹⁴C.     

On the specificity of a phospholipase A2 purified from the 106,000×g pellet of bovine brain

Assessment has been made of the specificity of a purified phospholipase A₂ from the 106,000×g pellet (microsomal fraction) of bovine grey matter which shows strong activity toward phosphatidylinositol (PI). In the first series of experiments involving the utilization as substrates of PI with different¹⁴C- or³H-labeled fatty acids in the 2-position, the purified phospholipase A₂ most readily removed 16∶0 palmitic acid, followed by 18∶0 stearic acid, 18∶1 oleic acid and 20∶4 arachidonic acid. In the second series of experiments, the purified phospholipase A₂ showed preferential action toward PI (100%) compared to phosphatidylcholine (PC, 62.5%), phosphatidic acid (PA, 32.6%), phosphatidylethanolamine (PE, 25.1%) and phosphatidylserine (PS, 21.5%), where each phosphoglyceride was labeled in the 2-position with [1-¹⁴C] oleic acid. In the third series of experiments, fatty acids were shown to cause inhibition of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C] oleoyl PI in the order 20∶4>18∶1>18∶0>16∶0 which is the reverse order to that just noted. In the final series of experiments, the addition of the phosphoglycerides PC, PE, PS and PA in amounts of 5 or 10 μM caused either no inhibition (PE, 2%), slight inhibition (PC, 15%) or reasonably significant inhibition (PA, 20% and PS, 40%) of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C]-oleoyl PI. The pattern of specificity observed for the purified phospholipase A₂ combined with its microsomal location are the expected properties of a phospholipase A₂ that might function in a deacylation-reacylation cycle for modifying the fatty acid distribution in PI.     

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.     

Selective deposition oftrans-8- andcis-9-octadecenoates in egg and tissue lipids of the laying hen

The deposition oftrans-8-octadecenoate-8(9)-³H (8t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids and in organ lipids from the laying hen.trans-8-Octadecenoate was preferentially incorporated into only the phosphatidylethanolamines (PE), whereas discrimination against 8t-18∶1-³H occurred in the phosphatidylcholines (PC), triglycerides (TG) and cholesteryl esters (CE). The 1-acyl position of both PE and PC contained three times more 8t-18∶1-³H than 9c-18∶1-¹⁴C. Almost total exclusion of the 8t-18∶1-³H from the 2-acyl position of these phospholipids was found. Preferential incorporation of 9c-18∶1-¹⁴C occurred at the combined 1- and 3-acyl positions and at the 2-acyl position of yolk TG. Tissue lipid analyses indicated that there was preferential deposition of 9c-18∶1-¹⁴C into all organs. Individual liver lipid classes displayed the same relative order of discrimination against 8t-18∶1-³H as did egg yolk lipids (CE>TG>PC>PE).     

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.     

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.     

Chain length specificity in the utilization of long chain alcohols for ether lipid biosynthesis in rat brain

A mixture ofcis-9-[1-¹⁴C] octadecenol and [1-¹⁴C] docosanol was injected into the brains of 19-day-old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more radily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18∶1 alkyl and alk-1-enyl moieties of the ethanolamine phosphoglycerides and into 18∶1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18∶1 alcohol from the substrate mixture (12 hr), the 22∶0 alcohol was not used to any measurable extent for alkyl and alk-1-enyl glycerol formation.     

Effects of phosphatidylcholines on de novo synthesis and excretion of sterol by L-929 fibroblasts

The effects on [¹⁴C] sterol synthesis and excretion by exposure of L-929 cells to several phosphatidylcholines (PC) has been examined. No significant effects were noted on either parameter during a 6-hr period if exposure of cells to the phospholipid preceded the addition of [1-¹⁴C] acetate by just 30 min. However, if cultures were grown in media containing delipidized serum and 2×10⁻⁵ M PC through 2 or more subculturings prior to adding [1-¹⁴C] acetate, the amount of [¹⁴C] sterol increased in both cells and medium by 70–200% when saturated or monounsaturated PC were used. Dilinoleylphosphatidylcholine (18∶2 PC) at the same concentration did not stimulate synthesis or excretion of newly synthesized sterol. Total cellular sterol was determined by gas chromatography, and was only marginally affected by long-term exposure to dipalmitylphosphatidylcholine (16∶0 PC), whereas the total sterol of the medium increased by 4-fold over a 19-hr period. Cultures which had been exposed to 16∶0 PC through 3 subculturings continued to display enhanced de novo sterol synthesis, but not excretion, for up to 5 hr after replacement with fresh medium lacking 16∶0 PC. The disparity in response to 2×10⁻⁵ M levels of 16∶0 PC and 18∶2 PC may relate to differences in metabolism of cellular fatty acids, whereas relatively small changes in the cellular fatty acid composition were noted with 16∶0 PC-treated cells. The results indicate that extracellular PC can promote sterol synthesis and excretion by L-929 cells, and that the magnitude of this response is influenced by the time of exposure to the phospholipid and by its fatty acid composition.     

Phospholipid composition of the granular amebocyte from the horseshoe crab, Limulus polyphemus

The phospholipid composition was determined for the amebocyte of the primitive arthropod Limulus polyphemus. The total fatty acid composition of the cells' lipids was analyzed by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters (FAME). The FAME analysis revealed high levels of 20-carbon polyunsaturated fatty acids (PUFA), especially arachidonic (20∶4n-6) and eicosapentaenoic (20∶5n-3) acids. Almost 20% of the total lipid profile was comprised of dimethyl acetals of 16- to 20-carbon chain lengths, indicative of plasmalogens in the phospholipid pool. Phospholipids, analyzed by high-pressure liquid chromatography, included phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SPH), and cardiolipin (CL). PE and PC levels predominated at 42.2 and 36.3%, respectively. Smaller amounts of PS (9.0%) and PI (6.2%) were present, as well as low levels of SPH (4.6%), CL (1.6%), and trace amounts of lysophosphatidylcholine. The major phospholipid species, PE, PC, PS and PI, were collected and their molecular species were examined by electrospray-ionization mass spectrometry. The molecular species within the phospholipid classes reflected the high levels of PUFA seen in the total lipid profile. PI was mainly composed of 18∶0a/20∶4. Over half of the PS consisted of 18∶0a/18∶1 and 18∶0a/20∶4. The major PE species were 20∶1p/20∶5, 20∶1p/20∶4, 18∶0p/20∶5, and 18∶0p/20∶4. PC had the largest distribution of molecular species, and its most abundant species were 16∶0e/20∶5, 16∶0e/20∶4, and 16∶0p/20∶4. The presence of 16∶0e/20∶4 is the first documentation of a specific precursor to platelet-activating factor in an invertebrate hemocyte. Note: at the sn-1 position: [a=1=O-acyl, e=1-O-alkylether, and p=1-O-alk-1′-enyl (plasmalogen)].     

Fatty acid synthesis in rat testes injected intratesticularly or incubated with 1-14C acetate

Fatty acid synthesis was studied in testes of young and adult rats either injected intratesticularly or incubated with 1-¹⁴C acetate. The pattern of¹⁴C incorporation into lipids and individual fatty acids in the two age groups was similar but results obtained with intratesticular injection differed considerably from those obtained in the in vitro studies. In the former more than 70% of the¹⁴C incorporated in total lipids was in phosphatides, with about 15% in triglycerides and only minor amounts in cholesteryl esters and free fatty acids. Most of the¹⁴C incorporated into total fatty acids was in saturated acids (predominantly 16∶0). A small amount of¹⁴C was in the higher polyenes and there was a progressive increase with time after acetate injection in the¹⁴C content of 22∶5 W6. In testes incubated with 1-¹⁴C acetate, the phosphatide, triglyceride, and free fatty acid fractions had similar amounts of¹⁴C. In the total fatty acid fraction about 40% of the incorporated¹⁴C was in saturated acids (predominantly 14∶0 and 16∶0) and about 50% in the higher polyenes. Twenty carbon polyenes and 22∶5 W6 had significant¹⁴C incorporation, but most of the¹⁴C was in 22∶4 W6. About 80% of the¹⁴C in the latter compound was in the carboxyl carbon, indicating its origin from endogenous 20∶4 W6 elongated by the added 1-¹⁴C acetate used as substrate. The¹⁴C 22∶4 was present predominantly in the triglyceride and phosphatide fractions with minor amounts in other lipids.¹⁴C-labeled compounds of retention time greater than 22∶5 were also present in all lipid fractions. Presented at the ISF-AOCS World Congress Chicago, September 1970.     

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-¹⁴C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-¹⁴C]LA. [1-¹⁴C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-¹⁴C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-¹⁴C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-¹⁴C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-¹⁴C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.     

The effects of partially hydrogenated marine oils on the mitochondrial function and membrane phospholipid fatty acids in rat heart

The influence of dietary partially hydrogenated marine oils containing docosenoic acid on rat heart mitochondrial membrane phospholipid fatty acid composition was studied with particular reference to cardiolipin and oxidative phosphorylation. Five groups of male weanling rats were fed diets containing 20% (w/w) peanut oil (PO), partially hydrogenated peanut oil (HPO), partially hydrogenated Norwegian capelin oil (HCO), partially hydrogenated herring oil (HHO), and rapeseed oil (RSO) for 10 weeks. All the cardiac phospholipids investigated were influenced by the experimental diets. An increased amount of arachidonic acid observed in phosphatidylethanolamine (PE) after feeding partially hydrogenated oils suggests a changed regulation of the arachidonic acid metabolism in comparison with PO treatment. 22∶1 originating from the dietary oils was incorporated only to a small extent into phosphatidylcholine (PC) and PE. A selective incorporation of 18∶1 isomers into the 1- and 2-positions of PC and PE with respect to geometry and position of the double bond was observed. Large amounts of 18∶1trans were incorporated into the 1-position of PC and PE, irrespective of the amount of 18∶2 supplemented to the diets, replacing a considerable proportion of stearic acid in this position. After feeding HHO and RSO, the content of 22∶1 in mitochondrial cardiolipin of rat heart was found to be 3% (mainly cetoleic acid) and 10% (mainly erucic acid), respectively, indicating a high affinity forcis isomers of 22∶1, but also a considerable resistance against incorporation oftrans isomers was observed. The ability of rat cardiac mitochondria to oxidize palmitoylcarnitine and to synthesize ATP was depressed after feeding HHO and RSO. Dietarycis isomers of 22∶1 seem to have a specific ability to interfere with cardiac ATP synthesis and also to alter the fatty acid composition of cardiolipin of rat heart.     

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.     

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids

Freshly isolated rat hepatocytes were incubated for 20 min with [U-¹⁴C]glycerol in the presence or absence of unlabeled linoleic (18∶2n-6), arachidonic (20∶4n-6), or docosahexaenoic (22∶6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (≈95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PF), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-¹⁴C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.     

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.     

Molecular species composition of the major diacyl glycerophospholipids from muscle,liver, retina and brain of cod (Gadus morhua)

The molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from white muscle, liver, retina and brain of cod (Gadus morhua) were determined by high-performance liquid chromatography of the respective 1,2-diacylglycerol 3,5-dinitrobenzoyl derivatives. A minimum of 69 diacyl species was identified. In muscle and liver saturated fatty acid/polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid/PUFA molecular species were predominant, particularly 16∶0/20∶5 and 16∶0/22∶6 in PC, 16∶0/22∶6 and 18∶1/22∶6 in PE and 18∶0/22∶6 and 18∶1/22∶6 in PS. Didocosahexaenoyl species were major components of PC, PE and PS from retina, comprising 29.3, 71.8 and 59.7% of the respective totals. Didocosahexaenoyl species were also abundant in PE and PS from brain, accounting for 13.8 and 24.0% of the totals, respectively. DiPUFA species were important in muscle, totalling 21.2% in PC and 38.3% in PE. PC from all tissues had the largest amounts of species containing only saturated or monounsaturated fatty acids, accounting for 59.8% of PC from brain, including 12.8% of 18∶1/24∶1 plus 24∶1/18∶1.     

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.