Metabolism of sphingolipids by normal and atherosclerotic aorta of squirrel monkeys

We studied the synthesis and hydrolysis of sphingomyelin by homogenates of aortic intima plus inner media from normal squirrel monkeys and from monkeys with nutritionally-induced atherosclerosis (6-10 mo on a semi-purified diet containing butter and cholesterol). The concentrations of sphingomyelin in the aortas and plasmas of the atherosclerotic monkeys were higher than those for the normal monkeys. Palmitoyl-1-(14)C coenzyme A was actively utilized for the synthesis of ceramide (N-palmitoyl sphingosine). The addition of sphingosylphosphorylcholine increased the utilization of palmitoyl CoA in sphingomyelin synthesis, and the addition of psychosine (sphingosyl galactoside) increased the incorporation of palmitate into cerebrosides. Rates of sphingomyelin and ceramide synthesis were significantly higher in the atherosclerotic than in the control aortas. Hydrolysis of labeled sphingomyelin to ceramide was also increased in homogenates of the atherosclerotic aortas. Labeled sphingomyelin was taken up from plasma by everted carotid arteries, and this process was also enhanced by atherosclerosis. Increased rates of synthesis and of uptake from plasma of sphingomyelin may account for the increased concentrations of sphingomyelin in the atherosclerotic arteries, even though the ability to degrade sphingomyelin is also enhanced in the atherosclerotic aorta.     

Lipids of the adrenal medulla: Lysolecithin, a characteristic constituent of chromaffin granules

1. The lipid composition of microsomes, mitochondria and chromaffin granules, obtained from homogenates of bovine adrenal medulla, has been investigated. 2. The three types of particle showed characteristic differences of phospholipid and cholesterol content. The lipid composition of microsomes and mitochondria resembled that of corresponding particles from other tissues. The chromaffin granules contained 19% of the cholesterol and 14% of the phospholipids of the low-speed supernatant. 3. Thin-layer chromatography indicated the presence of these phospholipids in extracts from each particle: lecithin, lysolecithin, phosphatidylethanolamine (partly plasmalogen), phosphatidylserine, phosphatidylinositol and sphingomyelin. 4. On quantitative analysis of the phospholipids, chromaffin granules were found to contain a high concentration of lysolecithin (17% of the lipid phosphorus). Mitochondria and microsomes, on the other hand, contained very little lysolecithin (less than 2% of the lipid phosphorus).     

Phospholipid binding and self-association of the major apoprotein of human and baboon high-density lipoproteins.

The purpose of this study was to establish a relationship between self-association and phospholipid binding of the human and the baboon apoA-I protein. The enthalpy changes on binding dimyristoyl lecithin and lysolecithin to either the human or the baboon native apoA-I protein were measured in a microcalorimeter. An endothermal process, most pronounced for the human apoprotein, was observed at low phospholipid levels. At higher phospholipid to protein ratios the binding was exothermal. Gel filtration experiments on Sephadex G-200 showed that the native apoprotein of both species consists of dimers and tetramers. The baboon native apoA-I protein contained a higher amount of dimers. After preincubation of the apoA-I protein with lysolecithin, the enthalpy changes measured on subsequent binding of dimyristoyl lecithin were shifted towards more exothermal values compared to the curve for the native apoprotein. The amplitude of this shift corresponds to that of the endothermal process observed on binding dimyristoyl lecithin to the native apoprotein. This process was attributed to a phospholipid-induced disaggregation of the apoA-I protein. Gel filtration data showed a decreased extent of aggregation in the apoA-I protein preincubated with lysolecithin. This sample consisted exclusively of dimers. Ultracentrifugal flotation of the complexes formed between the apoA-I protein, and respectively dimyristoyl lecithin and sphingomyelin indicated that preincubation with lysolecithin increased the extent of complex formation. These results suggest that the dimeric form of the apoA-I protein possesses the highest affinity for phospholipids. Any dissociation of higher polymers enhances the phospholipid-binding capacity of the human and the baboon apoA-I protein.     

Human high denisty apolipoprotein A-I-lysolecithin-lecithin and sphingomyelin complexes. A method for high yield recombinations to lipoprotein complexes of reproducible stoichiometry.

High denisty apolipoprotein A-1 (apoLp A-I) has been prepared in a chromatographically and immunochemically homogeneous form. This apoprotein forms trimeric and tetrameric aggregates in aqueous solutions at higher concentrations. ApoLp A-I has been recombined in almost quantitative yield in the presence of lysolecithin with phosphatidylcholine and sphingomyelin to particles of reproducible stoichiometry. Lysolecithin is not required for the interactions of lecithin and sphingomyelin with the apoprotein A-I or for the stability of these complexes. Dialysis removes most of the lysolecithin without the loss of lecithin and sphingomyelin. ApoLp A-I-lecithin particles have a molecular weight of 200 000 and contain 50 molecules lecithin and 25 of lysolecithin. ApoLp A-I-sphingomyelin complexes contain 50 sphingomyelin and 13 lysolecithin molecules. The former particles show up as discs of 100 A diameter, and the latter particles are 250 A in diameter. Their thickness was estimated as 25 A in the apoLp A-I lecithin and 60 A in the apoLp A-I-sphingomyelin particles. ApoLp A-I and lysolecithin form complexes whose densities depend on the lysolecithin concentration. Lysolecithin enhances the binding of phosphatidylcholine to apoLP A-I, yielding lipoprotein complexes with decreasing density. The yield of apoLp A-I-sphingomyelin-lysolecithin complexes is proportional to the lysolecithin concentration. The ratio of apoLp A-I to sphingomyelin in all these complexes remains constant.     

Composition of phospholipids and of phospholipid fatty acids of human plasma

The composition of the phospholipids and of the total phospholipid fatty acids was determined in the plasma of 10 normal subjects. In addition the fatty acid composition of the plasma phosphatidyl ethanolamine, phosphatidyl serine, lecithin, sphingomyelin, and lysolecithin of 6 of the subjects was measured. A wide array of fatty acids was found in the plasma total phospholipid similar to that found previously in red cell total phospholipid. The fatty acid composition in the plasma phospholipids of a given subject reflected that in his red cell phospholipids. Each individual phospholipid displayed a distinctive fatty acid pattern, which was generally similar to that of the corresponding phospholipid of red cells, although some marked differences in individual fatty acid levels between the corresponding phospholipids of plasma and red cells were evident. The high percentage of unsaturated fatty acids found in plasma lysolecithin suggests that this phospholipid did not arise entirely through the enzymatic cleavage of the -fatty acid of lecithin.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.     

Biosynthesis of retinal phospholipids: incorporation of radioactivity from labeled phosphorylcholine and cytidine diphosphate choline

Phosphorylcholine-1,2-(14)C and choline-1,2-(14)C-labeled cytidine diphosphate choline are incorporated into lecithin by whole homogenates and particulate fractions of rat retina with optimal incorporation of label by the microsomal fraction. The soluble fraction contains a factor(s) which stimulates incorporation of label with release of inorganic phosphate. Mg(++) is required for optimal incorporation of intermediates into lecithin in the presence of added diglycerides; without added diglycerides, incorporation of phosphorylcholine or cytidine diphosphate choline was moderately stimulated by preincubating the system in the absence of Mg(++) with added phosphatidic acid and by adding this mixture to fresh enzyme and the complete incubation mixture (including Mg(++)). The results show that the retina is capable of de novo synthesis of phosphatides and suggest that the rod outer segments depend on the pigment epithelium and(or) the inner rod segments for a source of phospholipids. Coenzyme A and ATP added to whole homogenate of retina did not significantly increase the incorporation of CDP-choline-1,2-(14)C into lecithin but slightly increased the radioactivity found in lysolecithin and sphingomyelin. Rats with hereditary retinitis pigmentosa have an abnormally high lipid phosphorus content of the retina, but they do not incorporate labeled CDP-choline into lecithin of retina at a higher rate than do normal animals.     

Demonstration of enzymatic conversion of lysolecithin to lecithin in normal human plasma

An enzyme in normal human plasma that converts [1-acyl ¹⁴C] lysolecithin to lecithin is demonstrated. This enzyme is inhibited by heparin and is not derived from platelets or other blood elements. The synthesis of lecithin from labeled lysolecithin was not stimulated by ATP and CoA or by oleyl CoA and there was nearly an equal distribution of labeled fatty acid between the two positions of lecithin indicating that the enzyme may be a lysolecithin: lysolecithin acyl transferase (LLAT). The enzyme is associated with the lipoproteins of the plasma, and may have a physiological role in the formation of saturated cholesterol esters in plasma.     

Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis

The concentration of lysophosphatidylcholine (monoacyl sn-glycerol 3-phosphorylcholine) in intima plus inner media of atherosclerotic aorta from squirrel monkeys was nearly eight times that in comparable control tissue. Plasma levels of the same compound were somewhat elevated in the atherosclerotic group. The metabolism of fatty acyl CoA's and lysophosphatides was studied in cell-free preparations of intima plus inner media from squirrel monkey aorta. Linoleic acid was incorporated predominantly into phosphatidylcholine (as opposed to other phospholipids) when linoleoyl-1-(14)C CoA was the substrate. The extent of this reaction was dependent on the concentration of lysophosphatidylcholine. Lysophosphatidylethanolamine (monoacyl sn-glycerol 3-phosphorylethanolamine) stimulated the incorporation of linoleate into phosphatidylethanolamine. 1-Palmitoyl-1'-(14)C sn-glycerol 3-phosphorylcholine ((14)C-lysophosphatidylcholine) was incorporated into phosphatidylcholine only in the presence of acyl CoA's or ATP plus CoA. Incorporation of (14)C with (14)C-lysophosphatidylcholine plus linoleoyl CoA equaled that with linoleoyl-1-(14)C CoA and lysophosphatidylcholine. Various other lines of evidence are presented to support the importance of the fatty acyl CoA:lysophosphatide fatty acyl transferase mechanism in aortic phospholipid metabolism. Cell-free preparations of aortic intima plus inner media from squirrel monkeys with early, nutritionally-induced atherosclerosis utilized linoleoyl-1-(14)C CoA more than preparations from control monkeys when incubations were carried out without added lysophosphatidylcholine and for long periods (30 min). With optimum levels of labeled linoleoyl CoA and unlabeled lysophosphatidylcholine, or unlabeled linoleoyl CoA and labeled lysophosphatidylcholine, there were no differences in substrate utilization between control and atherosclerotic tissues. We conclude that the concentrations of lysophosphatidylcholine, which are higher in atherosclerotic than in control aortic tissues, could be a factor controlling rates of fatty acid incorporation into phosphatidylcholine.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

ON THE PHOSPHOLIPASE A2 ACTIVITY OF HUMAN CEREBRAL CORTEX

Abstract— Preparations of phospholipase Az have been obtained from human cerebral cortex. The enzyme was extracted from acetone-dried tissue and purified by heat-treatment and gel filtration on Sephadex.
Although heating at 65°C or 70°C destroys most of the phospholipase A₁ activity that is present in crude extracts, a small proportion remains associated with the A₂ activity during these procedures. The heat-treated extracts hydrolyse lecithin in preference to phosphatidyl-ethanolamine but have no action on lysolecithin or neutral lipids. The results suggest that A₂ activity and the heat-stable component of A₁ may both be due to a single phospholipase A that can hydrolyse diacylglycerophosphatides at either the 2-or the 1-position, to form a mixture of isomeric lysoderivatives.
A molecular weight of 55,000 was calculated for the enzyme.     

THE METABOLISM OF PALMITIC ACID IN THE PHOSPHOLIPIDS, NEUTRAL GLYCERIDES AND GALACTOLIPIDS OF MOUSE BRAIN

Abstract— Following intracerebral injection, [¹⁴C]palmitic acid was rapidly incorporated into a variety of brain lipids. After 12 hr, 78 per cent of the lipid radioactivity was in phospholipids, 15 per cent was in triacylglycerols, 1 per cent each was in free fatty acids and galactolipids, and the remainder was in other neutral glycerides. Over 65 per cent of the phospholipid radioactivity was found in the choline phosphoglycerides but this proportion decreased substantially with time. At later times, increasing portions of the radioactivity were present in the monounsaturated acyl groups and the alkenyl groups but no radioactivity was detected in cholesterol or polyunsaturated acyl groups. These results indicate that most of the extensive recycling of radioactivity took place without oxidative degradation of the palmitoyl groups. The relative rates of incorporation of radioactivity were compared at 12 hr after injection. The specific radioactivities of the serine, ethanolamine, and choline phosphoglycerides had ratios of 6:3:2 based on the palmitoyl group content and 1:2:4 based on their phosphorus content. The specific radioactivities of galactolipids with O -acyl groups were higher than the specific radioactivitiesof cerebrosides or cerebroside sulphates. A new solvent mixture for thin-layer chromatography of brain galactolipids was described (chloroform-acetone-methanol-water, 60:20:20:1, by vol.).     

Lipid composition of membranes of aminestorage organelles

1. The lipid composition of the membranes from isolated 5-hydroxytryptamine-storage organelles of blood platelets of rabbits and of those from chromaffin granules of bovine adrenal medulla was compared. 2. In contrast with the membranes of the chromaffin granules, those of the 5-hydroxytryptamine organelles did not contain lysophosphatidylcholine (lysolecithin). 3. Both the cholesterol/phospholipid ratio and the relative proportions of phosphatidylethanolamine (kephalin), phosphatidylinositol and phosphatidylserine were about the same in both membranes, whereas phosphatidylcholine (lecithin) and sphingomyelin showed somewhat higher values in the membranes of the 5-hydroxytryptamine organelles. 4. In conclusion, the release of 5-hydroxytryptamine from blood platelets is probably not correlated with the presence of lysophosphatidylcholine in the membranes of the storage organelles and may thus differ from the mechanism of catecholamine release in adrenal medulla.     

ACYLATION OF LYSOPHOSPHATIDES BY PLASMA MEMBRANE FRACTIONS OF RAT LIVER

The plasma membrane fraction of rat liver was isolated and incubated with labeled lysophosphatides in the presence of cofactors; the acylation of lysolecithin to lecithin by the fraction was compared to that of the rough and smooth microsomes. The purity of the isolated fractions was ascertained by enzyme markers and electron microscopy, and the maximal contamination of the plasma membrane fraction by microsomes did not exceed 20%. Under conditions at which the reaction was proportional to the amount of enzyme used, the plasma membrane had a specific activity similar to that of the smooth and rough microsomes. With doubly labeled lysolecithin (containing palmitic acid-¹⁴C and choline-³H) it was shown that the lecithin formed retained the same ratio of the two labels, which indicated that lysolecithin was converted to lecithin through an acylation reaction. The newly formed lecithin was shown to be bound to the plasma membrane fraction; this suggested that it is incorporated into the structure of the membrane itself.     

PHOSPHOLIPIDS IN THE NERVOUS SYSTEM OF MOLLUSCS

Abstract The phospholipid composition of nervous ganglia of the bivalve Unio crassa , the gastropod Helix pomatia and the cephalopods Octopus sp. and Ommastrephes sloanei pacificus have been investigated.
The ganglia of cephalopods contain considerably more phospholipids than do gastropod and bivalve ganglia. Especially rich in phospholipids are the optic ganglion of the squid Ommastrephes and the cerebral ganglion of Octopus , where their content is of the same order as in the brain of teleosts and amphibia.
In the ganglia of the lower molluscs, the bivalve and the gastropod, no sphingomyelin nor X-phospholipid could be detected.
No sphingomyelin nor X-phospholipid were found in the optic ganglion of Octopus , whereas in its cerebral ganglion sphingomyelin but no X-phospholipid was present.
In both the optic and the cerebral ganglia of the squid Ommastrephes both sphingomyelin and X-phospholipid were found.     

Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

Exchange of phospholipids between low and high density lipoproteins of squirrel monkeys

Ultracentrifugal analysis of the plasma of squirrel monkeys at various times after the injection of [Me-(14)C]choline revealed the specific activities of lecithin in both high (HDL) and low (LDL) density lipoproteins to be similar. This was also true for sphingomyelin. The exchange of phospholipids in vitro was studied by incubating unlabeled plasma with labeled LDL and HDL isolated 40 hr after the injection of [Me-(14)C]choline. Recentrifugation of plasma immediately after the addition of either (14)C-labeled LDL or HDL demonstrated that significant exchanges of both lecithin and sphingomyelin had occurred. In further studies, (14)C-labeled LDL or HDL were incubated with plasma and the low density lipoproteins were rapidly isolated by precipitation with heparin-Mn(2+). Complete equilibration of lecithin and sphingomyelin between LDL and HDL was attained after 4 and 5 hr, respectively. The fractional exchange rates for lecithin and sphingomyelin of LDL to HDL were 0.60 hr(-1) and 0.45 hr(-1). Corresponding values for HDL to LDL were 0.51 hr(-1) and 0.53 hr(-1). Inhibition of plasma lecithin:cholesterol acyltransferase reduced the exchange of sphingomyelin but had no effect on lecithin exchange. The rates of exchange of four lecithin subfractions of different unsaturation between LDL and HDL were the same.     

The incorporation of long-chain fatty acids into phospholipids of respiring slices of rat cerebrum

1. Respiring slices of adult rat cerebrum have been shown to incorporate long-chain (14)C-labelled fatty acids into phospholipid. 2. Labelling was almost entirely confined to lecithin and ethanolamine phospholipid, only traces being present in serine phospholipid. 3. Palmitic acid, oleic acid and linoleic acid were incorporated more actively into lecithin than into ethanolamine phospholipid, but the converse was found with stearic acid. 4. All four acids labelled the 1- and 2-positions of both lipids; palmitic acid, oleic acid and linoleic acid were approximately evenly distributed, but stearic acid was incorporated predominantly at the 1-position. 5. It is considered that incorporation is most likely brought about through acylation of endogenously derived lysophosphatides. 6. The possible implications of this pathway of lipid metabolism in nervous tissue are discussed.     

Phospholipid removal during degradation of rat plasma very low density lipoprotein in vitro.

The hydrolysis of glycerophospholipids in very low density lipoprotein by enzyme(s) released into circulation after the injection of heparin to rats was studied. [32P]Lysolecithin was formed rapidly from [32P]lecithin when very low density lipoprotein, labeled biosynthetically with 32P, was incubated with postheparin plasma. The [32P]lysolecithin was associated with the plasma protein fraction of density greater than 1.21 g/ml, whereas [32P]lecithin exchanged between very low and high density lipoproteins. Inhibition of the plasma lecithin: cholesterol acyl transferase activity did not change the excess [32P]lysolecithin formation in postheparin plasma, and only a negligible amount of radioactivity was associated with blood cells when the incubation was repeated in whole blood. Analysis of the results has demonstrated that phospholipids are removed from VLDL by two pathways: hydrolysis of glycerophospholipids by the heparin-releasable phospholipase activity (greater than50%) and transfer to high density lipoproteins (less than50%). The tissue origin of the postheparin phospholipase was studied in plasma obtained from intact rats and supradiaphragmatic rats using specific inhibitors of the extrahepatic lipase system (protamine sulfate and 0.5 M NaCl). The phospholipase activity could be ascribed to both the hepatic and extrahepatic lipase systems. It is concluded that hydrolysis of glycerophospholipids is the major mechanism responsible for the removal of phospholipids from very low density lipoprotein during the degradation of the lipoprotein. It is suggested that phospholipid hydrolysis occurs concomitantly with triglyceride hydrolysis, predominantly in extrahepatic tissues.