Radioactive Fatty Acids Research Articles

Synthesis of lipids in isolated nuclei from rat thymus and liver cells.

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-14C]acetate and [1-14C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-14C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by 14C label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20: 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2−14C] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG., 14C-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG., 14C-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG., the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of 14C-labeled fatty acids between sn-1 and sn-2 positions might be related to 20: 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDG.

Acyl specificity of phospholipases A 2 and C in thrombin-stimulated human platelets

Platelets were labelled separately with six different, radioactive unsaturated fatty acids. The cells were isolated from the radioactive precursors and treated with and without 2 U/ml of thrombin. The formation of radioactive free fatty acid+oxygenated fatty acids and of radioactive radioactive phosphatidic acid+diacylglycerol was taken as a measure of the PLA 2 and PLC reactions, respectively. We found that that in intact platelets PLA 2 prefers phospholipid molecular species containing unsaturated acyls, most likely in the sn -2 position, in the priority order: 20:4>20:5>18:2 = 18:3 = 22:6>>18:1, while PLC prefers its substrates in the priority order 20:5>20:4>18:2>18:3 = 22:6>18:1.

Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells

Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out. The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, α -linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 μ M, 24 h). The best incorporation capacity was observed in the linoleic acid followed by α -linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active Δ 9 desaturase activity. With respect to polyunsaturated acid metabolism, the conversion of α -linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells. This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the Δ 5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death.

Microsomal fatty aldehyde dehydrogenase catalyzes the oxidation of aliphatic aldehyde derived from ether glycerolipid catabolism: implications for Sjögren–Larsson syndrome

The enzyme that catalyzes the oxidation of fatty aldehyde derived from ether glycerolipid catabolism has not been identified. To determine whether microsomal fatty aldehyde dehydrogenase (FALDH) is responsible, we investigated the metabolism of 1- O-[9,10- 3H-octadecyl]-glycerol ([ 3H]OG) in FALDH-deficient cultured cells from patients with Sjögren–Larsson syndrome (SLS) and in mutant Chinese hamster ovary (CHO) cells. Intact fibroblasts from SLS patients incubated with [ 3H]OG showed a selective deficiency (38±7% of normal) in the incorporation of radioactivity into fatty acid, but no decrease in incorporation of radioactivity into fatty alcohol, total lipids and phosphatidylethanolamine (PE). Consistent with fatty aldehyde accumulation, incorporation of radioactivity into N-alkyl-phosphatidylethanolamine, which is derived from Schiff base formation of free aldehyde with PE, was 4-fold higher in SLS fibroblasts compared to normal controls. Similar results were seen with SLS keratinocytes, whereas FALDH-deficient CHO cells showed a more profound reduction in radioactive fatty acid to 12±2% of normal. These results implicate FALDH in the oxidation of ether-derived fatty aldehyde in human and rodent cells. Metabolism of ether glycerolipids is a previously unrecognized source of fatty aldehyde that may contribute to the pathogenesis of SLS.

Triacylglycerols of the red microalga Porphyridium cruentum can contribute to the biosynthesis of eukaryotic galactolipids.

A mutant of the red microalga Porphyridium cruentum was selected on the basis of impaired growth at suboptimal temperatures (15 vs. 25 degrees C). Fatty acid and lipid analyses revealed diminished proportions of eicosapentaenoic acid (from 41 to 30%) and of the eukaryotic molecular species (from 38 to 28% of monogalactosyldiacylglycerol (MGDG) and elevated proportion (10 vs. 2%) of triacylglycerols (TAG) in the mutant, as compared with the wild type. Pulse labeling of the wild type cells with radioactive fatty acid precursors indicated an initial incorporation of the fatty acids into phosphatidylcholine (PC) and TAG. Following the pulse, the label of PC and TAG decreased with time (from 25 to 5% of the total dpm in TAG) while that of chloroplastic polar lipids, mainly MGDG, continued to increase. In the mutant, however, the labeling of TAG after the pulse was higher (30% of the total dpm) than that of the wild type and decreased only slightly to 20%. This may indicate that in P. cruentum, TAG can contribute to the biosynthesis of eukaryotic species of MGDG.

Acylation of monolysocardiolipin in rat heart

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.

Evidence for two distinct lysophospholipase activities that degrade lysophosphatidylcholine and lysophosphatidic acid in neuronal nuclei of cerebral cortex

Neuronal nuclei were isolated from immature rabbit cerebral cortex and nuclear lysophospholipase activities studied using two different 1-acyl lysophospholipids: lysophosphatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA). Our interest in these two lysolipids arose from the observation that lysoPA could promote the acetylation of lysoPC by substantially inhibiting a very active nuclear lysoPC lysophospholipase activity, in a competitive manner (R.R. Baker, H.-y. Chang, Mol. Cell. Biochem. (1999) in press). As there was also evidence for nuclear lysoPA deacylation, it was of interest to see whether one activity could possibly utilize both lysolipid substrates. We now have evidence for two separate lysophospholipase activities in neuronal nuclei. The lysoPC lysophospholipase activity was the more active, more highly enriched in the neuronal nuclei, and showed optimal activity at pH 8.4–9, while the lysoPA lysophospholipase activity was maintained over a much broader pH range. The lysoPC activity was substantially inhibited by free fatty acid, and showed considerable stimulation by serum albumin, while the activity utilizing lysoPA was much less affected by these agents. When lysoPC was added to incubations containing radioactive lysoPA, there was no significant inhibition found in rates of release of radioactive fatty acid, indicating that the lysoPA lysophospholipase activity did not utilize the lysoPC substrate. In incubations with lysoPC, MgATP and CoA brought about a sizable formation of phosphatidylcholine whose radioactivity was equally distributed between the sn-1 and sn-2 positions suggesting labelling both directly from the lysoPC substrate and from fatty acid produced by the lysophospholipase activity. By comparison, with the radioactive lysoPA substrate, MgATP and CoA promoted relatively lower levels of phosphatidic acid formation whose principal labelling came directly from the radioactive lysoPA. Largely because of the high activity of the nuclear lysoPC lysophospholipase, there is considerable potential in the neuronal nucleus to limit the use of lysoPC in other reactions, such as the formation of acylPAF (1-acyl analogue of platelet activating factor). It is of interest that conditions associated with brain ischaemia such as increased free fatty acid levels, falling pH and declines in MgATP may allow a preservation of neuronal nuclear lysoPC levels for acetylation. The existence of a separate lysophospholipase activity for lysoPA allows an independent control of lysoPA which can serve as an important regulator of the nuclear lysoPC lysophospholipase.

Mycoplasma penetrans infection of Molt-3 lymphocytes induces changes in the lipid composition of host cells.

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG). The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 degrees C). Incubation of [14C]oleic-acid-labelled Molt-3 cells with viable M. penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M. penetrans membrane preparations, or M. penetrans growth medium, had no effect. Total lipid analysis of Molt-3 lymphocytes infected by M. penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylglycerol. Analysis of the neutral lipid fraction in the infected Molt-3 cells revealed a fivefold increase in the relative amount of diacylglycerol and a marked increase in the free fatty acid (FFA) fraction. The profile of the FFAs released was dominated by a relatively high concentration of the polyunsaturated fatty acid docosahexaenoic acid. The release of lipid intermediates suggests that the degradation of Molt-3 cell phospholipids induced by M. penetrans may initiate a signal transmission cascade in the host cell.

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood–brain barrier endothelial cells in vitro

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n–6 and 18:3n–3 into 20:4n–6 and 22:6n–3, respectively, a coculture of the two cell types mimicking the in vivo blood–brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n–6 and 18:3n–3 (10 μm each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n–6 and 22:6n–3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum.▪ These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.—Bernoud, N., L. Fenart, C. Bénistant, J. F. Pageaux, M. P. Dehouck, P. Molière, M. Lagarde, R. Cecchelli, and J. Lecerf. Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood–brain barrier endothelial cells in vitro.

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-14C]Stearic (18:0), [1-14C]lin 18:2n-6) and [1-14C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial Δ9, Δ6 and Δ5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-14C]18:3n-3, the cells were unusually active towards [1-14C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit Δ9 and Δ6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.

The biosynthesis of docosahexaenoic acid [22:6(n-3)] from linolenic acid in primary hepatocytes isolated from wild northern pike

Primary hepatocytes from wild northern pike Esox lucius were incubated with radiolabelled linolenic acid ([l-14C]-18:3(n-3)) to assess their ability to synthesize docosahexaenoic acid [22:6(n-3)]. The distribution of radioactivity in lipid classes and hepatocyte polyunsaturated fatty acids (PUFA) was measured over the time-course of 24h. The majority of radioactivity from [l-14C]-18:3(n-3) was recovered in hepatocyte triacylglycerols (TAG) and phosphatidylcholine (PC). The levels of radioactivity in TAG and in most of phospholipids, including PC, increased significantly over the incubation period. Radioactivity from [1-14C]-18:3(n-3) was recovered in several hepatocyte PUFA, including 22:6(n-3), and the Δ6 and Δ5-desaturation products 18:4(n-3) and 20:5(n-3). The presence of radioactivity in C24 (n-3) PUFA may be evidence that the biosynthesis of 22:6(n-3) in pike proceeds via a pathway independent of Δ4-desaturation. Analysis by radio gas chromatography revealed that radiolabelled 24:6(n-3) was present among the desaturation and elongation products of [l-14C]-18:3(n-3). The results establish that, under the in vitro conditions employed, pike hepatocytes are able to convert linolenic acid to 20:5(n-3) and 22:6(n-3).

The biosynthesis of docosahexaenoic acid [22:6(n-3)] from linolenic acid in primary hepatocytes isolated from wild northern pike.

Primary hepatocytes from wild northern pike Esox lucius were incubated with radiolabelled linolenic acid ([l-14 C]-18:3(n-3)) to assess their ability to synthesize docosahexaenoic acid [22:6(n-3)]. The distribution of radioactivity in lipid classes and hepatocyte polyunsaturated fatty acids (PUFA) was measured over the time-course of 24h. The majority of radioactivity from [l-14 C]-18:3(n-3) was recovered in hepatocyte triacylglycerols (TAG) and phosphatidylcholine (PC). The levels of radioactivity in TAG and in most of phospholipids, including PC, increased significantly over the incubation period. Radioactivity from [1-14 C]-18:3(n-3) was recovered in several hepatocyte PUFA, including 22:6(n-3), and the Δ6 and Δ5-desaturation products 18:4(n-3) and 20:5(n-3). The presence of radioactivity in C24 (n-3) PUFA may be evidence that the biosynthesis of 22:6(n-3) in pike proceeds via a pathway independent of Δ4-desaturation. Analysis by radio gas chromatography revealed that radiolabelled 24:6(n-3) was present among the desaturation and elongation products of [l-14 C]-18:3(n-3). The results establish that, under the in vitro conditions employed, pike hepatocytes are able to convert linolenic acid to 20:5(n-3) and 22:6(n-3).

Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza

Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [ 3H]ethanolamine, [ 3H]myristic acid and [ 3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [ 3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.

141 HUMAN MILK FAT SYNTHESIS BY MAMMARY GLAND EPITHELIAL CELLS IN VITRO

In contrast to the exstensive research done on milk synthesis in animal models, there are only few studies on human milk synthesis. Using human mammary epithelial cells (HMEC) from mammoplasty specimens, we first studied different aspects of growth and differentiation with the aim of creating a cell culture model for studies on human milk synthesis. The cells, which are commercially available (Mammary Pack®, Clonetics Corp.), were cultured in serum-free media on different substrates. By culturing the cells on top of a reconstituted basal membrane matrix (Matrigel®, Beckton Dickinson Labware) a morphological differentiation was achieved with development of alveolar and duct-like structures. We have also studied lipid synthesis using a radioactive fatty acid precursor (3H-acetate) while stimulating the cells with different lactogenic hormones. Acetate was incorporated into lipids at a rate of 0,8-1,3 nmoles/million cells/hour in media without epidermal growth factor (EGF), insulin (I), hydrocortison (HC) and prolactin-rich bovine pituitary extract (BPE). This incorporation rate is similar to the rate reported in a previous study on HMEC from human breast milk. The incorporation rate was highest during the first 2 hours of incubation. Addition of EGF, I, HC and BPE caused an increase in the acetate incorporation rate to 2,4-3,4 nmoles/million cells/hour. Most of this increase was caused by EGF and I, while HC and BPE seemed to have minor effects, In some cases even inhibitory. The composition of the synthesised lipids was studied by thin layer chromatography and subsequent radiomaging, showing that there was a mixture dominated by triglycerides and phospholipids (preliminary results). Gas chromatography of lipid extracts from the cells showed that the chain lenghts of the main fatty acids were 18 and 16 carbon atoms. We conclude that this system could be a useful cell culture model for studying human milk fat synthesis.

Synthesis of (9Z, 12E)-, (9E, 12Z)-[1-14C]-linoleic acid, (9Z, 12Z, 15E)-, (9E, 12Z, 15Z)-[1-14C]-linolenic acid and (5Z, 8Z, 11Z, 14E)-[1-14C]-arachidonic acid

Trans polyunsaturated fatty acids are produced in vegetable oils during heat treatment (240-250 °C).ln order to study the metabolic pathway of 9c, 12t and 9t, 12c linoleic acid and 9c, 12c, 15t and 9t, 12c, 15c linolenic acid, these products were prepared labelled with carbon 14 in the carboxylic position. 5c, 8c, 11c, 14t-Arachidonic acid was also labelled on the carboxylic position with carbon 14 in order to study its physiological effects. To introduce the labelling (E)-bromo precursors with a 17 carbons chain or a 19 carbon chain were needed. The different syntheses were done by elongation steps and creation of cis double bonds via highly stereospecific Wittig reactions. The radioactive carbon atom was introduced from [ 14 C]-potassium cyanide. The final radioactive fatty acids had a specific activity greater than 50 mCi/mmol and a radioactive purity better than 99 % for linoleic and linolenic and better than 98.6 % for arachidonic acid.

High performance liquid chromatography of long-chain acylcarnitine and phospholipids in fatty acid turnover studies

In this paper we describe a rapid, isocratic high performance liquid chromatography (HPLC) method for the study of radioactive fatty acid incorporation into complex lipids of human erythrocytes, which allows the simultaneous separation of the major phospholipid classes and long-chain acylcarnitines. The lipid extract of erythrocytes pulsed with radioactive fatty acids was injected into an HPLC system equipped with a silica column. The individual components eluted were monitored by ultraviolet absorption and radioactive emission. With respect to the UV profile, the radioactive profile showed an additional peak between phosphatidyl-choline and phosphatidylethanolamine, which was identified as long-chain acylcarnitine by different experimental approaches. The radioactivity recovered in the long-chain acylcarnitines contains essential information enabling definition of acyl trafficking in red cells.

The Incorporation of Arachidonic Acid into Triacylglycerol in P388D1 Macrophage‐Like Cells

When 388D1 cells are incubated in media containing 10 microM [3H]arachidonic acid (delta4Ach), the label is rapidly incorporated into phospholipids and triacylglycerol. However, incorporation of [3H]delta4Ach into phospholipids clearly precedes incorporation into triacylglycerol, indicating that the phospholipid pool constitutes the primary metabolic fate of the delta4Ach via a remodelling pathway. In contrast, [3H]delta4Ach is incorporated into triacylglycerol almost exclusively via de novo synthesis, as evidenced by studies using propranolol, a phosphatidate phosphohydrolase inhibitor. This compound induced a time-dependent and concentration-dependent increase in the levels of [3H]delta4Ach-containing phosphatidate that is directly correlated with a decrease in the levels of [3H]delta4Ach-containing triacylglycerol. In addition, no change in the levels of [3H]delta4Ach-containing diacylglycerol and monoacylglycerol were apparent along the time course of fatty acid incorporation into triacylglycerol. However, a sharp and transient accumulation of cell-associated free [3H]delta4Ach was detected shortly after exposure of the cells to the radioactive fatty acid. Collectively, the results reported herein suggest that free delta4Ach availability determines the patterns of incorporation and distribution of this fatty acid among the various lipid classes of P338D1 cells.

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes.

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition, LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.

Glutamate stimulates the formation of N-acylphosphatidylethanolamine and N-acylphosphatidylethanolamine in cortical neurons in culture

The formation of anandamide N-armarachidonoylethanolamine, N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [ 14C]ethanolamine, [U- 14C]arachidonic acid, [ 3H]arachidonic acid, [ 32P]phosphate, [ 14C]stearic acid, or [ 3H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [ 14C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N-acylethanolamine (0.06-0.55% of total radioactivity) and N-acylphosphatidylethanolamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [ 32P]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D ( Streptomyces chromofuscus) resulted in two compounds, one comigrating as phosphatidic acid and the other as N-acylethanolamine. Compound I could be labelled with [ 14C]stearic acid and [ 3H]myristic acid, but not with [ 3H]- or [ I4C]arachidonic acid. Exogenous [ 3H]anandamide was metabolised with a t 1 2 of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced the older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED 50 = 40 μM) the formation of both compounds. It is suggested that N-acylethanol amine and N-acylphosphatidylethanolamine are formed in relation to the cytotoxicity induced by glutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachidonic acid.