Sphingolipid biosynthesis by particulate fractions of normal and “Quaking” mouse brain

No differences were detected in the ability of brain particulate fractions isolated from “Quaking” and normal mice for the incorporation of:¹⁴C-L-serine into 3-keto-dihydrosphingosine; 1-¹⁴C-stearoyl acid and 1-¹⁴C-stearoyl CoA into ceramide and¹⁴C-choline from CDP¹⁴C-choline into sphingomyelin.     

Preferential labeling of phosphatidylcholine during phospholipid synthesis by bovine mammary tissue

Bovine mammary cells and tissue synthesize de novo the classes of phospholipids, found in mammary tissue and milk, from various precursor molecules. Several short term experiments were carried out in vitro, using labeled precursors, i.e., 1-¹⁴C-fatty acids; 2-¹⁴C-acetate; U-¹⁴C-glycerol; 1,2-¹⁴C-choline; 1,2-¹⁴C-ethanolamine; 2-¹⁴C-serine; and Me-¹⁴C-methionine. All the phospholipid classes were labeled. The specific activity of tissue phosphatidylcholine was consistently three to six-fold greater than that of phosphatidylethanolamine. The results indicated that stepwise methylation of phosphatidylethanolamine with labeled methyl group of methionine was occurring to a minor extent, as was a negligible amount of choline exchange. Serine was incorporated into phosphatidylserine and sphingomyelin. Significant quantities of labeled phosphatidylserine were decarboxylated to phosphatidylethanolamine. Apparently phosphatidylcholine was synthesized de novo from choline via phosphorylcholine and CDP-choline. Based on the present observations and other data, it is suggested that there may be two pools of phosphatidylcholine in lactating mammary cells.     

Phospholipid metabolism in cells in culture

The proportions of the different lecithin fractions have been determined in HeLa and KB tissue culture cells and Ehrlich Ascites tumor. 82.8% of the total phosphatidyl choline phosphorus is found in fraction 3 of HeLa cells. The major phosphatidyl cholines found in KB cells and Ehrlich Ascites tumor are in fractions 3 and 4 and representing 66.6% and 88.7% of the total phosphatidyl choline P, respectively. The incorporation of 1,2-¹⁴C-choline and 1,2-¹⁴C-ethanolamine into the various phosphatidyl choline fractions has been assayed to determine their biosynthesis in Ehrlich Ascites tumor. The incorporation of 1,2-¹⁴C-choline into fractions 3 and 4 is 100 times the 1,2-¹⁴C-ethanolamine. This evidence indicates that the methylation pathway of phosphatidyl choline synthesis is very low in HeLa, KB and Ehrlich Ascites cells. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment of the Degree of Master of Science.     

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.     

Uptake of [14C] choline and incorporation into lung phospholipid by the isolated perfused rat lung

We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 mM. The rate of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extracellular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Km.     

Differential trypsin effect upon (1-14C) acetate incorporation into choline and ethanolamine glycerophosphatides of rat brain and liver

Preincubation of rat brain and liver slices in a medium (5% glucose, 5% fructose, 1% albumin, 1% trypsin, 10 mM phosphate buffer pH 6.0) used to pretreat brain tissue for the separation of cell types was found to uncouple the incorporation of (1-¹⁴C) acetate into ethanolamine phosphoglycerides from that of the choline phosphoglycerides. Incorporation into ethanolamine phosphoglycerides was stimulated in both brain (330%) and liver (780%) slices, while the incorporation of (1-¹⁴C) acetate into choline phosphoglycerides was reduced for both brain (71%) and liver (63%) slices, compared to control values from nonpreincubated material. With (1-¹⁴C) linolenic acid as a precursor, no significant differences were found in incorporation into ethanolamine phosphoglycerides and choline phosphoglycerides.     

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).     

Effect of chlorphentermine on incorporation of [14C]choline in the rat lung phospholipids

The effect of chlorphentermine (CP) treatment (50 mg/kg/ day, per os [po]) on the incorporation of [¹⁴C]choline into rat lung phospholipid was studied.Total phospholipid content was increased 2.0-fold and 1.7-fold after seven and 14 days, respectively, compared with the pair-fed rats. The incorporation of [¹⁴C]choline into phosphatidylcholine (PC) was significantly inhibited by either seven or 14 days of CP treatment. Nevertheless, the PC content was significantly increased by day 7 and stayed elevated at day 14 of CP treatment. Choline and phosphorylcholine contents were significantly decreased by the CP treatment. These results suggest that the higher accumulation of PC is due to inhibition of enzymes involved in the hydrolysis of phospholipids rather than to a stimulation of the phospholipid synthesis. Presented in part at the SOT Meeting, Atlanta, GA, March 1984 (abstracted inThe Toxicologist 4[1], 64).     

Sphingomyelin metabolism is linked to salt transport in the gills of euryhaline fish

Byin vivo andin vitro studies ofl-(3-³H)serine and [9,10(n)-³H]palmitic acid incorporation into phospholipids, we show a change in the renewal of the ceramide moiety of sphingomyelin in the gills of euryhaline fish (sea bass and eels) when the animals were subjected to abrupt alterations in environmental salinity.In vivo, decrease of the salinity from sea water (salinity 3.7%) to diluted sea water (salinity 1%) induced an increase of label incorporation into gill sphingomyelin. The same was true when gills from sea water-adapted sea bass or sea water-adapted eels were incubated in diluted sea water. A decrease in free ceramides synthesis was also observed in the gills of sea water-adapted sea bass when the salinity of the incubation medium was reduced. Direct inhibition of Na⁺/K⁺-ATPase activity with ouabain decreased the sphingomyelin synthesis in the gills of sea bass duringin vitro incubation in diluted sea water, whereas treatment with furosemide stimulated sphingomyelin synthesis in the same gills incubated in sea water. These findings indicate that changes in Na⁺ fluxes modify the sphingomyelin turnover and control the production of free ceramides and sphingosine in gill cells of euryhaline fish. In view of the well-known effects of these sphingomyelin degradation products on isolated tumor cell differentiation, we suggest that they play a very important role in modulating chloride cell distribution and metabolism of fish gills during abrupt changes in environmental salinity.     

In vitro incorporation of acetate-1-14C into the phospholipids of rabbit and human endometria

Endometria from nonpregnant and 6-day pregnant rabbits and from humans in the proliferative and secretory phases were incubated with 1-¹⁴C-acetate.¹⁴CO₂ was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of¹⁴C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of¹⁴C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria. Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolamine of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease. This investigation was supported by a grant from the Ford Foundation.     

Studies on phospholipid biosynthesis in hepatocytes from alcoholic rats by using radiolabeled exogenous precursors

We have studied the synthesis of phospholipids in hepatocytes isolated from chronically ethanol-treated rats by using isotopically labelled serine, ethanolamine, and choline as exogenous precursors. Our results demonstrate that ethanol induces specific effects on the biosynthesis of phosphatidyl-ethanolamine and phosphatidylcholinevia CDP-derivatives and also on the synthesis of phosphatidylserinevia the Ca⁺⁺-dependent base-exchange reaction. Thus, the synthesis of phosphatidylethanolamine from [³-H]ethanolamine and the incorporation of [³H]serine into phosphatidylserine were clearly higher in hepatocytes from ethanol-treated rats compared to controls. The synthesis of phosphatidylcholine from [methyl-¹⁴C] choline, on the other hand, decreased markedly, suggesting a specific inhibition of cholinephosphotransferase activity. We have also demonstrated that the phosphatidylcholine levels are markedly decreased in hepatocytes isolated from chronically ethanol-treated rats as a consequence of the lower phosphatidylcholine biosynthesis. The decrease in the incorporation of radioactivity from choline to betaine, which we also found, is interpreted as being the result of a higher use of betaine as methyl donor instead of methionine to maintain the hepaticS-adenosylmethionine levels in chronic alcoholism.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Inhibition of sterologenesis in brain and liver of fetal and suckling rats from dams fed di-2-ethylhexyl phthalate plasticizer

The effect of di-2-ethylhexyl phthalate (DEHP) on lipid metabolism was studied in liver and brain from fetal rats taken 3 days before parturition from dams receiving dietary DEHP during gestation. In fetuses from rats receiving 0.5% or 1.0% DEHP in a stock diet, the incorporation of¹⁴C-acetate and labeled mevalonate (³H or¹⁴C) into the C₂₇ sterols, C₃₀ sterols, and squalene fractions of brain tissue incubated in vitro was significantly reduced between the confidence limits P<0.05 to P<0.001. When liver from fetuses was incubated with labeled mevalonate, incorporation of label into the C₂₇ sterol and C₃₀ sterol fractions was significantly reduced as well (P<0.02 to P<0.001), whereas incorporation of labeled mevalonate into the squalene fraction was not significantly altered. The incorporation of¹⁴C-acetate into total hepatic lipids of the fetal rats was also studied, and statistically significant reductions in incorporation were observed in the lanosterol fraction (P<0.001), the combined fraction of sterol esters + squalene (P<0.02), and the combined fraction of cholesterol + diglycerides (P<0.01). No significant changes were observed in the incorporation of¹⁴C-acetate into phospholipids, free fatty acids, or triglycerides. In 8-day old suckling rats delivered from dams fed 0.5% DEHP for the last 16 days of gestation and maintained on the same diet during the nursing period, the incorporation of¹⁴C-mevalonate into hepatic C₂₇ sterols, in vitro, was significantly depressed (P<0.05) whereas in corporation into C₃₀ sterols and squalene was similar to control values. In these same suckling rats, body weights were significantly lower in the control group (21.7 vs. 18.8 g, P<0.01), whereas liver weight as a % of body weight was significantly higher (P<0.01) in rats nursing from the DEHP-fed dams. The results indicate that the inhibitory effect of dietary DEHP on lipid metabolism in the mature rat is transmitted across the placental barrier to the developing fetus and that the abnormal pattern of lipid metabolism in rats delivered from DEHP-fed females is only partially restored to normal during the suckling periods.     

Effect of dietary di-2-ethylhexyl phthalate on lipid biosynthesis in selected tissues from the rat,in vitro

Di-2-ethylhexyl phthalate (DEHP), a plasticizer commonly used in the production of polyvinyl chloride plastics, has become an environmental pollutant. At the present time, the biological significance of phthalates in the environment is unknown. In the present studies, we observed that addition of DEHP to a stock diet of rats resulted in marked effects on incorporation of¹⁴C-acetate into lipid by liver and kidney slices; other organs, such as heart, testes, and aorta were unaffected. Incorporation of¹⁴C-acetate into total lipid of liver (dpm/mg wet wt) from rats, fed 0.5% or 1.0% DEHP for 10 or 18 days, respectively, was decreased to ca. 50% of control values. The decreased incorporation into liver lipid is not attributable to any one lipid fraction, inasmuch as incorporation into the phospholipid, sterol+diglyceride, free fatty acid, triglyceride, and sterol ester+hydrocarbon fractions was decreased 30–70% with respect to controls. In addition, the percent distribution of¹⁴C-acetate among the individual phospholipids was ca. 25% lower in phosphatidyl choline of the DEHP-fed rats. In rats fed 0.5% DEHP, incorporation of¹⁴C-acetate into total lipid of kidney was similar to control values, but incorporation into the triglyceride and sterol ester+hydrocarbon fraction was decreased 30–40%, whereas incorporation into the sterol+diglyceride fraction was increased 38%. Livers from DEHP-fed rats were ca. 20% larger than livers from control rats and, at the 0.%% level of DEHP feeding, testes wts were elevated; no significant changes were noted in wts of spleen, heart, aorta, kidney, or body wt gains in rats fed DEHP. These studies emphasize a subtle toxicity of phthalate esters not previously reported and emphasize the need for further biochemical studies to evaluate the effect of phthalates on biological systems.     

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO₂/13%O₂/82% N₂ for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-¹⁴C] choline chloride into phosphocholine, [methyl-¹⁴C]phosphocholine into CDPcholine, and [¹⁴C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.     

Metabolic fate of sphingomyelin of high-density lipoprotein in rat plasma

The metabolic fate of high density lipoprotein (HDL) sphingomyelin in plasma was studied in rats over a 24-hr period after injection of HDL containing sphingomyelin which was¹⁴C-labeled in the stearic (18∶0) or lignoceric acid (24∶0) moiety and³H-labeled in the choline methyl groups. Decay of label in plasma followed three phases. The first two phases were similar for both isotopes and both types of sphingomyelin (t_1/2≃10 and 110 min). However, during the third phase (from 10 hr after injection),³H label disappeared more slowly than¹⁴C label from 18∶0 sphingomyelin, whereas the³H/¹⁴C ratio remained relatively constant when 24∶0 sphingomyelin was used. Intact, doubly-labeled 18∶0 sphingomyelin disappeared from HDL rapidly (t_1/2=38 min) by tissue uptake and by transfer to very low density lipoprotein (VLDL). VLDL contained up to 12% of the sphingomyelin 1 hr after injection. This is the first demonstration of a transferin vivo of sphingomyelin from HDL to VLDL. A similarly rapid transfer was also observedin vitro. Some nontritated, [¹⁴C]18∶0 or [¹⁴C]24∶0 sphingomyelin was redistributed more slowly into HDL. Doubly-labeled phosphatidylcholine appeared in VLDL and HDL within 1 hr after injection and reached 1.8 and 2.1% of the injected¹⁴C and³H in VLDL at 1 hr, and 4.8 and 6.9% in HDL at 3 hr, respectively.     

Pyruvate metabolism by rat lung in vitro

Rat lung slices were used to examine the net uptake of pyruvate and the incorporation of specifically labeled pyruvate-¹⁴C into CO₂ and lipid fractions. Pyruvate uptake and pyruvate-2-¹⁴C incorporation into these fractions was increased by raising the pyruvate concentration in the incubation medium from 1 to 25 mM. The addition of 5 mM glucose to the incubation medium decreased the apparent oxidation of pyruvate-2-¹⁴C to CO₂ but increased its incorporation into lung lipids. Glucose did not increase net pyruvate uptake by lung slices. The incorporation of pyruvate-1-¹⁴C and pyruvate-3-¹⁴C into CO₂ and lung lipids was also examined. The results indicated that a major portion of the pyruvate utilized was oxidized to CO₂ with substantial levels found in the fatty acid moiety of lung phospholipids. The low level of pyruvate conversion to phospholipid glycerol, possibly via the dicarboxylic acid shuttle, is of uncertain physiological significance. Journal Series Paper No. 4258 of the Agricultural Experiment Station, Pennsylvania State University.     

Biosynthesis of phospholipids in subcellular particles from cultured cells of human tissue

A time study of the incorporation of³²P_i into the phospholipids of HeLa, KB, human heart, and liver tissue-culture cell lines has been carried out. The incorporation of³²P_i at various time-intervals into the phospholipids of nuclei, mitochondria, and microsomes of HeLa and KB cells was investigated. The labeling of the isotope into the phospholipids was divided into three groups. The first had two components: phosphatidyl inositol and polyglycerol phosphatides, which showed the greatest incorporation of the isotope as demonstrated in the specific activity values and the percentage of total radioactivity after 15 to 30 minutes of incubation. A second group was composed of the major phospholipids of all tissue-culture cell lines studied, phosphatidyl choline, and phosphatidyl ethanolamine. At first, there was a delayed labeling of these phospholipids; however, after one hour of incubation, a rapid increase was shown in the incorporation of³²P_i. A third group of lipids containing sphingomyelin and phosphatidyl serine demonstrated low specific activity values. The phospholipids of the subcellular fractions, nuclei, mitochondria, and microsomes, had a high degree of incorporation of the isotope into the individual phospholipids and probably represented an active process in the membranes of these cellular units or a renewal of the biological membrane structures. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

Sterol synthesis in the liver intestine,and lung of the guinea pig

The relative rates of sterol synthesis in the liver, ileum, and lung of the guinea pig have been studied by measuring the incorporation by tissue slices of¹⁴C-labeled acetate into digitonin-precipitable sterols. The liver showed maximum incorporation of acetate at pH 6.5, the ileum at pH 7.5, and the lung at pH 6.0. The incorporation of acetate approached the maximum rate at a concentration of 10 mM with the liver and lung and 5 mM with the ileum. Using these conditions of assay, sterol synthesis was measured in the liver, ileum, and lung of four groups of guinea pigs killed at 6-hourly intervals. Depending on the time of day, the rate of sterol synthesis in the ileum was from 6 to 14 times that in the liver, while in the lung the rate was up to 3 times that shown by the liver. Additional studies showed that all regions of the small intestine synthesized sterol at a higher rate than the liver, with the highest rate of synthesis occurring in the ileum. The rates observed in the adrenal, testis, muscle, adipose tissue, and skin indicated that these tissues are not quantitatively important sites of sterol synthesis in the guinea pig.