Altered phospholipid metabolism in sodium butyrate-induced differentiation of C6 glioma cells

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP₃) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP₃ production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP₃ formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP₃ formation and its subsequent action, i.e., Ca²⁺ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.     

Mifepristone Treatment Results in Differential Regulation of Glycerolipid Biosynthesis in Baby Hamster Kidney Cells Expressing a Mifepristone-Inducible ABCA1

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [³H]serine or [³H]inositol or [³H]ethanolamine or [methyl-³H]choline or [³H]glycerol or [¹⁴C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-³H]glycerol or [¹⁴C]oleate or [³H]ethanolamine or [methyl-³H]choline uptake in BHK cells. In contrast, [³H]glycerol and [¹⁴C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [³H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [³H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [³H]glycerol and [¹⁴C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.     

The labeling of brain ethanolamine phosphoglycerides from cytidine diphosphate ethanolamine in vitro

Chicken brain microsomes convert¹⁴C-ethanolamine-labeled cytidine diphosphate ethanolamine (CDPE) to ethanolamine lipids (EPG) at a measurable rate. Mitochondria and particle-free supernatant fractions are almost inactive. On adding saturating amounts of diacyl glycerol to the microsomes, formation of EPG increases 10-fold, and is almost totally confined to the diacylsn-glycero-3-phosphorylethanolamine (GPE), whose synthesis increases 20-fold (from 13.1 to 249 mμmoles/mg protein/hr). The addition of the 1-alkenyl 2-acyl glycerol also produces a noticeable increase in the rate of EPG labeling, which is almost exclusively confined to the alkenyl acyl GPE, whose synthesis is stimulated 30-fold (from 3.1 to 90 mμmoles/mg protein/hr). Synthesis of alkyl acyl GPE from CDPE was not detected in the brain microsomes. Synthesis of EPG from CDPE by chicken brain homogenates has also been examined and results similar to those reported for the brain microsomes were obtained. In addition, small amounts of labeled alkyl acyl GPE are produced from CDPE in the homogenates, but in no case does this synthesis increase on adding various lipid acceptors to the incubation system. Various properties of the phosphorylethanolaminediacyl glycerol phosphotransferase (E.C. 2.7.8.1) of brain microsomes were examined. It is concluded that the enzymic activities which convert CDPE to diacyl GPE and alkenyl acyl GPE, respectively, are similar.     

The effect of lidocaine onde novo phospholipid biosynthesis in the isolated hamster heart

Lidocaine is used clinically as an antiarrhythmic agent, but its effect on cardiac phospholipid metabolism has not been defined. In this study, hamster hearts were perfused with [1,3-³H]glycerol in the presence of 0.5 mg/mL lidocaine. The incorporation of radioactivities into lysophosphatidic acid, phosphatidic acid, phosphatidylethanolamine, cytidine diphosphate diacylglycerol, phosphatidylinositol, phosphatidylserine, diacylglycerol and triacylglycerol were enhanced by lidocaine treatment, whereas the labelling of phosphatidylcholine was reduced. Analyses of enzyme activities in the heart after perfusion with lidocaine revealed that the activities of phosphatidate phosphatase and acyl-coenzyme A (CoA):1,2-diacylglycerol acyltransferase were enhanced. The presence of lidocaine in the assay did not directly stimulate these enzymes. However, the activity of acyl-CoA:glycerol-3-phosphate acyltransferase was stimulated by lidocaine whereas the activity of cytidine diphosphocholine:1,2-diacylglycerol cholinephosphotransferase was inhibited by lidocaine. We conclude that lidocaine affects the regulation of phospholipid biosynthesis in the heart by both direct and indirect modulation of phospholipid biosynthetic enzymes.     

Effects of sodium butyrate on the transfer of arachidonic acid to phosphatidylcholine in a clonal oligodendrocyte cell line (CB-II)

The effect of sodium butyrate on membrane phospholipid metabolism in a neonate rat cerebellum derived clonal oligodendrocyte cell line (CB-II) was investigated. Sodium butyrate is an agent known to induce cell differentiation and morphological transformations. A comparison of thein vivo phospholipid labeling patterns obtained by incubating CB-II cells with [³H]choline, [¹⁴C]myristic acid or [³H]arachidonic acid indicated that butyrate altered the route of acylation-deacylation in phosphatidylcholine (PC) biosynthesis. Using anin vitro incubation system containing homogenates of CB-II cells, the largest proportion of radioactivity was found in PC, and addition of sodium butyrate resulted in a further increase in the transfer of arachidonic acid to PC, but not to phosphatidylinositol. Similar results were obtained when thisin vitro acylation activity was tested using homogenates from sodium butyrate pretreated cells. The butyrate effect was observed regardless of whether or not exogenous lysophosphatidylcholine (LPC) was added to the incubation system. Addition of butyrate did not result in a change in the activity of LPC:acyl-CoA (coenzyme A) acyltransferase (EC 2.3.1.23) in CB-II cells upon incubating cell homogenates with [1-¹⁴C]arachidonoyl-CoA and LPC. However, when cell homogenates were incubated with [³H]arachidonic acid in the presence of 2.5–10 mM sodium butyrate, arachidonoyl-CoA synthesis was stimulated. A time course study demonstrated that significant stimulation occurred after three minutes. Taken together, the results suggest that in CB-II cells, sodium butyrate stimulates the transfer of arachidonic acid into PC and that this effect is at least partially due to a stimulation of arachidonoyl-CoA ligase (EC 6.2.1.3).     

Source of lung surfactant phospholipids: Comparison of palmitate and acetate as precursors

The phospholipids and the fatty acid compositions of major phospholipids in rat lung parenchyma, microsomes, lamellar bodies and alveolar wash were quantified. Adult rats were injected simultaneously with [³H] palmitate and [¹⁴C] acetate into the femoral vein. The appearance of labeled phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) in each lung fraction was measured during short periods of time (5 min to 2 hr) after isotope administration. Relatively more PC, DSPC and PG labeled with acetate radioactivity in lung microsomes entered lamellar body and alveolar wash fractions than those labeled with palmitate radioactivity. However, there was no difference between palmitate and acetate labeled phospholipids in the transport from microsomes to lamellar bodies by phospholipid exchange proteins. On the other hand, prior injection of colchicine resulted in decrease in the transport of PC from microsomes to alveolar space to a relatively greater extent in the acetate radioactivity than in the palmitate radioactivity.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.     

Phosphatidylcholine as the choline donor in sphingomyelin synthesis

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [¹⁴C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [Methyl-¹⁴C]choline resulted in rapid accumulation of essentially all the label (≈95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 μM choline), loss of lable from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 μM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 μM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9–12 hr until it reached the specific radioactivity of PC. In the presence of 300 μM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion, we conclude that sphingomyelin synthesis in Novikoff cells circumvents the water-soluble choline pool and that phosphatidylcholine serves as the immediate choline source. All our data support the phosphatidylcholine:ceramide cholinephosphotransferase pathway of sphingomyelin synthesis. Presented in part at the annual meeting of the Federation of American Societies for Experimental Biology, Anaheim, April 1985 (1).     

Prolonged retention of doubly labeled phosphatidylcholine in human plasma and erythrocytes after oral administration

The plasma kinetics of a preparation of dilinoleoyl phosphatidylcholine (DLPC) specifically labeled with³H in the choline moiety and with¹⁴C in the 2-fatty acid (FA) were evaluated in six healthy volunteers after oral administration. Retention of both isotopes in plasma exceeded expectations, with a half-life in the elimination phase of 172.2 h for³H and 69.7 h for¹⁴C. Up to 60 d after administration, there were still significant levels of radioactivity present in plasma. The relative stability of the [¹⁴C]FA label was demonstrated by the retention for more than 12 h of an isotope ratio close to that of the compound administered. The¹⁴C label of DLPC remained in position-2, as assessed by cleavage of plasma phospholipids with phospholipase A₂. The [³H]choline label showed an early incorporation into high density lipoproteins and subsequently into low density lipoproteins (LDL); conversely, the¹⁴C radioactivity was rapidly incorporated into triacylglycerols that were mainly associated with very low density lipoproteins. Radioactivity measurements revealed that both isotopes remained the longest time in LDL. In red blood cell (RBC) lipids, [³H]choline radioactivity accumulated over time, with a plateau after 48 h, whereas FA radioactivity accumulated more rapidly and was followed by a progressive decay. Analysis of the isotope ratio in these cells suggested an early incorporation of lyso products followed by rapid transfer of FA from plasma. The RBC maintained considerable radioactivity for a prolonged time, thus acting as a possible reservoir for the DLPC administered. Our study showed that dilinoleoyl PC remained in plasma longer than predicted based on earlier studies, and that after absorption the FA label was found in position 2.     

The formation of lysophosphatidylinositol phosphate in human platelet microsomes

The formation of lysophosphatidylinositol phosphate (lysoPIP) from lysophosphatidylinositol (lysoPI) via kinase activity was studied in microsomal preparations from human platelets. For this purpose, [³H]lysoPI or [³H]phosphatidylinositol ([³H]PI) was prepared and incubated in the presence or absence of ATP, MgCl₂ and Triton X-100, and the appearances of radioactivity in [³H]lysoPIP and [³H]phosphatidylinositol phosphate ([³H]PIP), respectively, were monitored using thin layer chromatography. Both lysoPI and PI phosphorylations were completely dependent upon the presence of ATP and MgCl₂ in the incubation medium; Triton X-100 addition stimulated both reactions, with the stimulation of PI conversion being considerably greater than that for lysoPI conversion. The present results demonstrate that lysoPI can be converted to lysoPIP by phosphorylation in human platelet microsomes. The potential significance of this enzymatic reaction in stimulated cells is discussed in relation to the generation of inositol-1,4,5-trisphosphate, an important intracellular second messenger.     

Formation of diacyl and alkylacyl glycerophosphocholine in rabbit alveolar macrophages

The incorporation of various labeled precursors into alkenylacyl, alkylacyl and diacyl phospholipids in rabbit alveolar macrophages was studied. The incorporation rates of the individual precursors were shown to be quite different among the three subclasses of phospholipids. [³H] Glycerol, [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P]-orthophosphate were preferentially incorporated into choline glycerophospholipids (CGP), especially into diacyl glycerophosphocholine (GPC), indicating that the de novo synthesis of diacyl GPC is extremely high. Considerable portions of the radioactiveties of [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P] orthophosphate were also found in alkylacyl GPC, the incorporation being higher than or comparable to that in the case of diacyl glycerophosphoethanolamine (GPE). We then examined the activities of cholinephosphotransferase and ethanolaminephosphotransferase, and found that the activity of cholinephosphotransferase was remarkably high in macrophage microsomes compared with that in microsomes from several other tissues. This suggests that diradylglycerols were preferentially utilized by cholinephosphotransferase, which is consistent with the results obtained for intact cells. We confirmed that a considerably higher amount of diacyl GPC as well as alkylacyl GPC was formed through this enzyme reaction with macrophage microsomes than with brain microsomes. The high formation of alkylacyl GPC could be responsible, at least in part, for the accumulation of this unique ether phospholipid, a stored precursor form of plateletactivating factor in macrophages. Fatty chains are designated in terms of number of carbon atoms: number of double bonds, e.g., 18:1 for oleic acid.     

The effect of bleomycin on the uptake and incorporation of [14C] choline into phospholipids in hamster lung tissue slices

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [¹⁴C] choline into phospholipids of lung slicesin vitro. The stimulatory effect is opposite to the results obtained previously using [¹⁴C] acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.     

Phospholipid synthesis in human embryo fibroblasts infected with herpes simplex virus type 2

The effect of herpes simplex virus type 2 infection on the synthesis of phospholipids in human embryo fibroblasts was determined at temperatures permissive (35 C) or nonpermissive (42 C) for virus replication. Incorporation of [³²P]i was decreased by herpes simplex virus type 2 in fection after 6 hr, which corresponds to the time of initiation of progeny virus production. No differences were observed in the relative incorporation of [³²P]i into phospholipid classes. In another series of experiments, cells were labeled with [³H] ethanolamine before infection and with [¹⁴C] ethanolamine after infection. The incorporation of [¹⁴C] ethanolamine was also decreased after 6 hr of infection. When choline was substituted for ethanolamine, a similar, although less pronounced, decrease in incorporation was seen in infected cells compared to mock-infected cells. During abortive infection at 42 C, incorporation of [³H] thymidine into cellular DNA was stimulated, but the incorporation of phospholipid precursors was decreased. Total phospholipid composition and phospholipid acyl group composition were not changed appreciably during abortive or productive infection, regardless of whether the cells were labeled before or after infection. In conclusion, these data indicated that, during herpes simplex virus type 2 infection, the incorporation of lipid prescursors into phospholipid was decreased. The stimulation of cellular DNA synthesis previously observed during abortive infection at 42 C was not paralleled by a detectable stimulation of total phospholipid synthesis. Neither productive nor abortive infection resulted in significant phospholipid compositional changes in the host cell; however, both resulted in a marked inhibition of phospholipid synthesis.     

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes

The biosynthesis of [¹⁴C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [¹⁴C] phosphatidic acid by preincubation of unlabeled microsomes withsn-[¹⁴C]glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgC1₂. The newly formed [¹⁴C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [¹⁴C] phosphatidate and [¹⁴C]CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP:phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation ofsn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol. Presented in part at the Annual Meeting of the Canadian Federation of Biological Societies, Winnipeg, June 1975.     

Phospholipid exchange between subcellular organelles of rabbit lung

A soluble protein fraction (PLEP) prepared from rabbit lung can catalyze the exchange of phospholipids between subcellular organelles of the lung and between these subcellular organelles and synthetic liposomes. Phospholipid exchange between microsomes and synthetic liposomes and between mitochondria and synthetic liposomes was stimulated 8-fold and 2.5-fold, respectively, in the presence of the protein fraction. Lung exchange protein could also catalyze phospholipid exchange between subcellular organelles of the liver and synthetic liposomes. Phospholipid transfer between microsomes and lamellar bodies of the lung was stimulated 2-fold by the exchange protein. Both radiolabeled phosphatidylcholine (PC) and phosphatidylinositol (PI) were transferred from³²P-labeled microsomes to lamellar bodies, but the exchange protein exhibited no transfer activity for phosphatidylglycerol (PG) and that for phosphatidylethanolamine (PE) was insignificant compared to the transfer activity for phosphatidylcholine and phosphatidylinositol. While the physiological role of the phospholipid exchange proteins in the lung is unknown, it is possible that they participate in the distribution of the newly synthesized phospholipids from the site of synthesis to lamellar bodies and other membrane compartments of cells.     

The synthesis in vivo of choline and ethanolamine phosphoglycerides in different brain areas during aging

The biosynthesis of choline and ethanolamine phosphoglycerides was tested in vivo in different brain areas of the rat during aging. Mixtures of [2−³H] glycerol and [Me-¹⁴C] choline or [2-³H] glycerol and [2-¹⁴H] ethanolamine were injected into lateral ventricle of the brain as lipid precursors and their incorporation into corresponding phospholipid was examined. A significant decrease of synthesis of both phosphoglycerides takes place in cerebral cortex and in the striatum, and is already apparent at 9 months of age with no further decrease or change therafter. No significant change takes place in the cerebellum. The unchanged absorption of injected water-soluble precursors, together with the lack of any significant change of phospholipid/protein ratio in all examined brain areas, suggests that the incorporation of both glycerol and nitrogen bases are affected by aging.     

Lipid products formed during desaturation of [1-14C] stearyl CoA by hen liver microsomes

A number of lipid products are formed during the desaturation of stearyl CoA by hen liver microsomes. This article presents an analysis of the products formed when [1-¹⁴C] stearyl CoA is incubated with hen liver microsomes for various time periods. [1-¹⁴C] Oleyl CoA was the first radioactive unsaturated product formed. Synthesis of phospholipids containing [1-¹⁴C] oleic acid occurs only after the desaturase activity has begun to decline. The specific radioactivity of [1-¹⁴C] oleyl CoA was similar to the specific radioactivity of [1-¹⁴C] stearyl CoA at all time periods tested. The specific radioactivities of [1-¹⁴C] oleic acid and phospholipids containing [1-¹⁴C] oleic acid were much lower than that of the [1-¹⁴C] stearyl CoA.     

Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-¹⁴C]glycerol and [1-¹⁴C]arachidonic acid incorporation. By contrast, a higher amount of [1-¹⁴C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.     

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.     

The turnover of the lipid components of myelin

The rate of loss of radioactivity in the lipid components of rat myelin labeled with acetate-1-C¹⁴ was determined over a period of one year. Rats were injected with acetate-1-C¹⁴ at 15–16 days of age and purified myelin was prepared by differential ultracentrifugation from brain and spinal cord of this group at 1 day, 2 weeks, 1 month, 2 months, 3 months, 6 months and 1 year after injection. Total lipid was extracted from the myelin preparations and the lipids were separated into their components by thin-layer chromatography. Cholesterol, galactolipid, ethanolamine phosphatide, choline phosphatide, inositol phosphatide, serine phosphatide and sphingomyelin specific activities at each age were measured. Three of the myelin lipid components, serine phosphatide, inositol phosphatide, and choline phosphatide decreased in specific activity faster than cerebroside, cholesterol, sphingomyelin, and ethanolamine phosphatide. Acetate-1-C¹⁴ injected into adult animals, though incorporated into myelin to a very small extent, is taken up primarly in the choline phosphatide. These experiments suggests that myelin does not behave as a fixed entity but that certain constituents may be more actively metabolized than others.