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Summary The rate of 32 P incorporation into the phospholipids of rat plasma and aorta was studied in vivo from 4 to 14 hours. While the rate of incorporation is similar during the first 4 hours, after that time it becomes greater in plasma phospholipids than in those of the aorta. The rate of incorporation of 32 P into sphingomyelin and phosphatidylethanolamine is higher in plasma than in aorta. For phosphatidylinositol the maximum of incorporation is reached much later in plasma than in aortic tissue. While phosphatidylglycerol is not detected in plasma, this phospholipid shows a high specific activity in aorta and is rapidly labelled. The specific activities of phosphatidylcholine and lysophosphatidylcholine are higher in rat plasma than in aorta and are very similar, whereas in aortic tissue phosphatidylcholine is more highly labelled than lysophosphatidylcholine and in our experiments only reached its maximum specific activity after about 14 hours. Comparisons between phospholipid turnover in rat plasma and aorta gave evidence of the metabolic activity of aortic tissue.

1. A detailed composition of phospholipid has been examined in the rat submaxillary gland. It was recognized that there is phosphatidylcholine, phosphatidyl-ethanolamine (possibly containing phosphatidylserine) and sphingomyeline as a major constituent of phospholipids, that there is trace of lysophosphatidylcholine and unknown phospholipid. 2. The phospholipase A activity of unpurified tissue extract (fraction A) or purified tissue extract (fraction D), as the source of enzyme has been examined. The fraction A or fraction D hydrolysed phosphatidylcholine as a substrate, and liberate free fatty acid and formed lysophosphatidylcholine. From above results, it was established that phospholipase A activity is present in the rat submaxillary gland.

After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.

Membrane metabolism was studied during the initiation of compensatory growth after acute reduction in renal mass. The rate of [14C]choline incorporation into phospholipid in renal cortical slices was increased by 37% at 5 min of compensatory growth in mice. The rate increased to the maximal value of 68% by 20 min and remained there for 3 h. The rate then remained increased at 28-34% above normal for 2 days and returned to normal by the 6th day. The increase in rate of choline incorporation into renal phospholipid was independent of choline uptake. [14C]Choline was found to be a specific precursor of the three renal phospholipids, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, which comprise over half the amount of the phospholipids. The relative distribution of the label in each of the three phospholipid classes did not change with compensatory growth. An increased rate of choline incorporation was also observed in kidneys of rats during compensatory growth and in the compensating kidneys of mice treated with indomethacin before uninephrectomy. The rate was increased 24% at 3 h after uninephrectomy in vivo. The increase appeared to be specific for the kidney, since it did not occur in the livers of these mice. The results indicate that the onset of renal compensatory growth is associated with a specific enhancement of the synthesis of renal choline-containing phospholipids. Since the phospholipids largely occur in the cell membrane, early alterations in cell membrane metabolism may thus play a role in the initiation of cell growth.

The role of several parameters in the regulation of human diploid fibroblasts in dense culture is assessed. The controls appear to be the result of several interacting influences. Depletion of essential amino acids and glucose from the medium contributes to the inhibition of replication, and this nutrient limitation is modified by the density of cell packing. Serum proteins alter the response of the cells to the available nutrients. The effect of serum proteins is proportional to their concentration, and they leave an imprint on the cell which influences the response of the cells to a subsequent stimulus to replication. This effect, denoted serum imprinting, alters the rapidity of the cell response (i.e. changes the pre-DNA synthetic gap) rather than the overall magnitude of the cell response. There appear to be positive and negative factors in serum which influence the serum imprint. One function of the serum proteins is to act as a sink for the products of cell metabolism. We examined the desorption of phospholipids from cells during exposure to various media and found a preferential desorption of lysophosphatidylcholine from the cell surface. The mechanisms by which these factors might redirect the cell activity are discussed.

Phospholipid Metabolism in Alveolar Macrophages of Lungs [32P]Orthophosphate Incorporation in Pulmonary Surfactant Phospholipids in Rat in vivo and in vitro Pulmonary surfactant phospholipids play an important role in the surface tension activity of the lung. Incorporation of [32P]orthophosphate into rat-lung pulmonary-surfactant phospholipids was studied in vitro and in vivo after separation on silicic-acid-column chromatography and by one and two-dimensional thin-layer chromatography. While they represent a small part of the phospholipids of the whole lung, pulmonary-surfactant phospholipids show a rapid turnover, particularly in vitro. After in vitro incubation, phosphatidylcholine and lysophosphatidylcholine show very important specific activities; by contrast, phosphatidylethanolamine and sphingomyelin have a very low turnover. Thus phosphatidyldimethylmethanolamine-methylation pathway, demonstrated in the lung, is not very important. Phosphatidylglycerol is labeled more markedly in vivo than in vitro indicating, for this lipid, a possible transfer from lung tissue to pulmonary-surfactant cells. In diphosphatidylglycerol specific activities are always very low. The incorporation of radioactive phosphate into phosphatidylinositol, more important in vitro than in vivo, is probably related to the phagocytosis activity of these cells. Our work demonstrates the existence of a phospholipid biosynthesis into pulmonary surfactant but cannot explain the metabolic relations between lung tissue and macrophagic alveolar cells investigated as a possibility in our results.

The formation of glycerylphosphorylcholine (GPC) by enzymatic hydrolysis of phosphatidylcholine (PC) was studied in carp Cyprinus carpio ordinary muscle by using PC-Me-14C and GPC-Me-14C as the substrates.Nearly all the decomposition products of PC were GPC, and the rate of choline liberated from GPC was only about five percent of that from PC. Moreover, there was scarcely any accumulation of lysophosphatidylcholine in the decomposition pathway of PC to GPC. Thus it was concluded that the formation of GPC by enzymatic hydrolysis of PC was nearly quantitative in carp ordinary muscle.During storage of carp and yellow tail Serbia quinqueradiata ordinary muscle, the formation of GPC increased progressively for 12 days at 0°C. The amount of GPC in yellow tail reached a plateau of approximately 68 μmoles/100g muscle after 30 days at -10°C.

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.

Incorporation of [32P]orthophosphate into chicken and chick-erythrocyte and plasma phospholipids in vivo was studied by means of one and two-dimensional thin-layer chromatography between 3 and 144 h. Radiophosphate was actively incorporated into plasma phospholipids, more rapidly into chick than chicken (maximum specific activity after 6 and 12 h, respectively) and more slowly into erythrocyte phospholipids. After 12 h, the chicken-plasma phospholipids were labelled in the order: phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, phosphatidylinositol, sphingomyelin and phosphatidylserine; while the chick-plasma phospholipids, after 6 h, were labelled in the order: phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. In chick- and chicken-erythrocyte phospholipids, higher specific activities, very similar for all the phospholipids studied, were observed at 144 h. These results strongly differ from those observed in in vitro incorporation: phosphatidic acid and phosphatidylglycerol were lowly labeled while phosphatidylserine showed high specific activities in the two experiments. Exchange reactions between plasma and erythrocyte phospholipids are not the same as in mammals; the high specific activities of the different erythrocyte phospholipids were too late for an exchange with plasma phospholipids. De novo biosynthesis was probably effective in the erythrocyte. The turnover of lysophosphatidyllcholine was low and the acylation of plasma phospholipids appeared less active than in mammal erythrocyte.

In order to study the mechanism of the accumulation of phosphatidyl choline (PC) in erythrocytes with abnormal erythrocyte phospholipids from patients with a hereditary hemolytic anemia, the phospholipids of the erythrocytes were labeled radioactively. Labeling of phosphatides was achieved by both passive equilibration with preformed phosphatides, and active "acylase"-mediated incorporation of fatty acid (FA) in the presence of glucose, ATP and coenzyme A. The labeled cells were then reincubated in fresh compatible sera and the catabolism of the labeled erythrocyte phospholipids was followed. In addition, total acylase capacity of erythrocyte stroma was determined under optimal conditions in a system with excess lysophosphatide, FA, ATP, CoA, and Mg++. No differences in passive uptake or release of phosphatides were found between the patients’ erythrocytes and comparable reticulocyte-rich controls. On the other hand, overall active incorporation of FA into PC was abnormally increased in the patients’ erythrocytes, whereas incorporation of FA into phosphatidyl-ethanolamine (PE) was decreased. However, acylase capacity for both lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) was normal in the patients’ cells. This apparent paradox could be explained by the subsequent turnover of actively incorporated PC-FA which was found to be reduced. A brief labeling experiment designed to approximate pulse-chase conditions and to label primarily PC showed a considerable inhibition of the subsequent transfer of PC-FA to PE upon reincubation in fresh serum. This transfer has previously been shown to be responsible for a significant portion of PC-FA catabolism. Reincubation in hyperlipemic sera obtained from patients with liver disease or artificially enriched with PC did not influence the abnormal outflow of phosphatide-FA in actively labeled cells. The findings were consistent with the concept that PC accumulated in these cells because of a defect in the catabolism of actively incorporated PC-FA. This defect appeared to be in the transfer of PC-FA to PE prior to final release from the cell. Passive exchange pathways and the active anabolic acylase pathway were not abnormal in these patients’ erythrocytes.

The lipid composition of the concentrated hemagglutininating active fraction (HF) of allantoic fluid from infected eggs, but free from Chlamydia psittaci 6BC, was compared to concentrated normal allantoic fluid (NAF). Phosphatidyl-choline (PC) and phosphatidylethanolamine were the major lipid classes of the total phospholipid fraction. Some quantitative differences were found in the amount of PC and phosphatidylserine present in HF and NAF. Lysophosphatidylcholine was present in HF but absent in NAF. Triglycerides and sterols were the major lipid classes found in neutral lipids of HF and NAF. Quantitative data showed distinct differences in the amount of different neutral lipid classes present between HF and NAF. The fatty acids of various classes of lipids were examined, and differences were noted in a number of phospholipids, sterol esters, and the free fatty acids. Branched-chain saturated fatty acids were found in many lipid classes of the HF, particularly in the phosphatidylethanolamine fraction, but were absent in the NAF.

The concentration of total and individual phospholipids was determined in erythrocytes and plasma of adults, neonates, and newborn infants with Rh erythroblastosis. After extraction the phospholipids were separated by one-dimensional, thin layer chromatography into six fractions: phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and nonidentified phospholipids. In normal neonates the total amount of red cell and plasma phospholipids was significantly decreased (1.32±0.20 µmoles/ml plasma, p

Summary Mechanisms involved in the incorporation of fatty acids into the phospholipids by cell-free preparations of the intima plus inner media of aorta were studied in the rat and squirrel monkey. Stein et al.8 first demonstrated this activity in cell-free preparations of aorta. The cell-free system used in the present study had an absolute requirement for ATP and CoA and was markedly stimulated (in the incorporation of [1-14C]linoleic acid into phosphatidylcholine) by lysophosphatidylcholine but not by glycerol-3-phosphate. Apparent Michaelis constants for linoleic acid and for lysophosphatidylcholine were determined. The incorporation of fatty acids into different phospholipids and different positions was studied. Linoleic acid was shown to be more efficiently incorporated into phosphatidylcholine than oleic or palmitic acids. There appeared to be competition of the different fatty acids for some common aspect of the enzyme system. The greatest fatty acid-incorporating activity was in the microsomes. Phospholipid, RNA, and protein concentrations of the aortic and liver microsome fractions were compared. Aortic cell-free preparations from squirrel monkeys (Sqimiri sciureus) with atherosclerosis had greater [l-14C]linoleic acid incorporation into aortic phosphatidylcholine than controls when lysophosphatidylcholine was not added. With levels of added lysophosphatidylcholine which resulted in maximum rates of fatty acid incorporation there was no difference in activity between diet groups.

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4·4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18–50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.

The incorporation of [ 14 C]linoleic acid, stearic acid and palmitic acid in the lipids of slices from biopsy samples of human muscle in four cases of progressive muscular dystrophy was studied by incubation of the tissue over 40 min and compared with normal muscle and adipose tissue. 1. 1. The influx of fatty acids is different in the three tissues. Considering the different specific activities of fatty acids there are comparable conditions for the lipid synthesis m all three tissues. 2. 2. The highest incorporation of fatty acids of the tissues studied was found in muscle. In adipose tissue the turnover averaged thirty times lower. The incorporation in muscular dystrophy hardly comes up to the values of normal muscle. 3. 3. The incorporation of linoleic acid and palmitic acid in sphingomyelin is decreased in dystrophic muscle, also the incorporation of linoleic acid in triglycerides is diminished. Palmitic acid incorporation was enlarged in muscular dystrophy. 4. 4. The incorporation of linoleic acid in phosphatidylcholine of dystrophic muscle is decreased. Therefore the relation of lysophosphatidylcholine/phosphatidylcholine is nearly one. The results were interpreted as signs of defective incorporation of linoleic acid in progressive muscular dystrophy.

The arterial circulation of human placentas was perfused to remove fetal erythrocytes. This perfused tissue as well as amnion and chorion laeve were homogenized and incubated with C14 oleic acid and lysolecithin to demonstrate the activity of acyl GPC acyltransferase. This enzymatic activity was present in all three tissues. These results allow the speculation that placental enzymatic activity may influence maternal and fetal lipid patterns.

A method has been developed to measure the phospholipase A activity of the duodenal juice; its technical variation coefficient is 5%. This method is based on hydrolysis of [ 32 P]phosphatidylcholine with thin-layer chromatography and determination of the radioactivity of [ 32 P]lysophosphatidylcholine formed without previous lipid extraction. Chromatographic plates radioactivity can be recorded. The peaks are integrated, their surface being proportional to radioactivity.

1. 1. Four dipolar steroids, testosterone, estradiol and their ethinyl derivatives have been solubilised by ultrasonic dispersion with lysophosphatidylcholine in water. The effect of temperature on the extent of this solubilisation has been examined and quantitative estimations made. The solubility of the steroids in water has been determined. 2. 2. All compounds showed a very low solubility in water, testosterone being the most soluble (84.6 μmoles/l at 20°). 17α substitution of an ethinyl group in testosterone decreased the solubilisation in lysophosphatidylcholine 5-fold; in contrast 17α-ethinyl estradiol was solubilised to a 5-fold-greater extent than estradiol. 3. 3. Estimates of the polarities of the A and D rings of the compounds using the CNDO/2 molecular orbital method to assess the dipole moments associated with these fragments, provide an explanation for the solubilisation results in terms of the relative polarities of the terminal rings of the compound.

14C from 14CCl4 irreversibly binds to lipids from the smooth (SER) and rough (RER) endoplasmic reticulum. Most of the label is associated with the phospholipid fraction (> 95%). Prior cystamine administration decreased the extent of that binding but does not change its pattern of distribution. About the half of the label in phospholipids is in the phosphatidylcholine fraction; the other half is distributed similarly among lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine, while only a very minor fraction is associated with diphosphatidyl glycerol. No differences were found in the pattern of labeling of phospholipids in SER and RER.

The phospholipids in cows milk are known to be high in unsaturated fatty acids content. Taking of this point, the authors considered that if these unsaturated fatty acids were lilerated by hydrolysis, they developed the off-flavor of milk by autoxidation. However, no study was reported concerning the presence of phospholipase in milk. The present study deals with the occurrence and the origin of the phospholipase.(1) The presence of phospholipase was suggested by the increase of lysophosphatidylethanolamine (LPE) during the incubation of milk.(2) Addition of toluene to milk as a preservative resulted in repressing the production of LPE. But free fatty acids and LPE were increased by the incubation of milk-bacteria with milk-phospholipids. These results indicated that the phospholipase was not present originally in milk but was produced by multiplied bacteria.(3) During the incubation of milk, the increase of lysophosphatidylcholine was not observed. Therefore, this enzyme seemed to have specific activity against phosphatidylethanolamine.

死後の肝臓における脂質および脂肪酸構成の変化を検索する目的から,原発性肝癌の一例について肝葉切除のさい得られた正常肝組織部分と剖検時に採取した肝組織の脂質の変化を比較分析した。剖検肝ではトリグリセリドの増量による総脂質の増量のほか,リン脂質中でのリゾホスファチジルコリンの増率,総脂質および各脂質成分でのoctadecenoic acidの増率が示された。, In order to investigate the postmortem changes in the profiles of hepatic lipids and fatty acid, comparisons were made between the lipids of surgically resected liver specimen and of the corresponding specimen obtained at autopsy in a case of primary liver cancer. Total lipids were higher in the liver obtained at autopsy reflecting the significant increase of triglycerides. The higher percentage of lysophosphatidylcholine fraction was obtained in the postmortem specimen. The octadecenoic acid concentrations in the total lipids and in all the lipid classes were higher in the postmortem liver than in the surgically resected specimen.

When bile was diverted from the intestine of four sheep for 12–48 hr, they secreted 0·31 ± 0·019 ml. bile/min. This contained 5·0 ± 0·55 µEq total fatty acids, principally in phosphatidylcholine (48 ± 1·9%) and lysophosphatidylcholine (31 ± 5·0%). The output of total fatty acids increased when taurocholate was infused into the portal vein or duodenum, and this was due to an increased secretion of phosphatidylcholine. Stimulation of total fatty acid secretion by bile salts was most marked at low rates of bile salt transfer, and reached a maximum when taurocholate was infused into the portal vein at 37 µM/min or more. The mean maximum output of total fatty acids in the bile of 12 sheep was 15·2 ± 1·41 µM/min. When 14C-palmitic acid was infused into the portal vein with the taurocholate, it appeared in biliary phospholipids, and by the sixth hour of the infusion the level of radioactivity in bile was 8·3% of the rate of infusion. When taurocholate was infused into the portal vein at 45 µM/min or less it was transferred quantitatively into bile, but the efficiency of transfer decreased at higher infusion rates. Secretion of bile salts into bile was accompanied by increased secretion of water, but the volume of water accompanying each micromole of bile salt decreased with increases in the rate of secretion. The bile flow did not increase further when more than 127 µM/min taurocholate was infused into the portal vein, and in four sheep the maximum rate was 1·06 ± 0·052 ml./min. It is suggested that availability of bile salt may limit secretion of phospholipid into bile when the rate of secretion of bile salt is low, but that when large amounts of bile salt are secreted into bile some other factor, possibly the rate of phospholipid synthesis, limits the secretion of phospholipids into bile.

The surfactant and solubilizing properties of lysophosphatidylcholine (LPC) could have industrial uses if it became available in technical quantity and purity. The two most promising routes for obtaining it are isolation from cereal starches and mono-deacylation of egg phosphatidylcholine (PC). Baker's yeast autolysed by Letters's method yielded up to 0·3 % of LPC. The phospholipase A activity of pancreatin powder gave 80 % of the theoretical yield of LPC under optimum conditions; modifications designed to facilitate scale-up reduced the yield. Base-catalysed glycerolysis at 75° gave 23 % of the theoretical yield of LPC, and a reaction between PC and ethanolamine up to 45%. The crude product from ethanolamine after acetone precipitation gave an emulsifier containing LPC, PC and long-chain N-2-hydroxyethylamide.

Evidence has been presented to show that the DPNH-cytochrome c reductase, but not the TPNH-cytochrome c reductase, activity of microsomes requires phospholipids. Two different phospholipids, phosphatidylcholine and lysophosphatidylcholine, when solubilized together, completely restore the DPNH-cytochrome c reductase activity to acetone-extracted microsomes. Micelles containing 40 to 70% (by weight) of phosphatidylcholine, when added to acetone-extracted microsomes, stimulate the DPNH-cytochrome c reductase activity to levels 4 times higher than that of the original microsomes. Micelles formed from the synthetic phospholipid, γ-oleyl-β-butyrylphosphatidylcholine, are also able to restore the DPNH-cytochrome c reductase activity to acetone-extracted microsomes, presumably because this phospholipid forms micelles having physicochemical characteristics the same as, or closely related to, those produced by mixtures of phosphatidylcholine and lysophosphatidylcholine. When ferricyanide was used as the electron acceptor, no such lipid requirement could be demonstrated, indicating that lipids may be required for the transfer of electrons in the region beyond the flavoprotein of the DPNH-cytochrome c reductase system of intact microsomes.

1. Total lipids were extracted from digesta obtained from the rumen, abomasum and upper small intestine (jejunum) of each of four slaughtered sheep. The lipids were fractionated into unesterified fatty acids, neutral lipids and phospholipids and the proportional contribution of each fraction to the total fatty acids was determined.2. The contribution made by phospholipids to the total fatty acids in the digesta showed a marked increase in the samples from the small intestine compared with those from the rumen and abomasum. This increase was apparently due to the presence of biliary phospholipids.3. Total lipids and conjugated bile acids were extracted from sheep bile, the lipids were fractionated and their fatty-acid composition was determined. Phospholipids predominated and these consisted mainly of phosphatidylcholine, together with some lysophosphatidylcholine.4. Both phospholipids contained significant amounts of unsaturated C18components which could account, at least in part, for the previously reported increament to the proportion of these acids in the digesta when it enters the upper jejunum.5. The overall fatty acid compositions of the two biliary phospholipids were very similar and, in common with other naturally occurring phosphatidylcholines, the fatty acids present in position 2 of the phosphatidylcholine of bile were found to consist almost entirely of unsaturated components.6. Total lipids and conjugated bile acids were extracted from samples of digesta obtained from three sheep with cannulas in different positions in the jejunum. Analysis of the lipids indicated that biliary phospholipids, in particular phosphatidylcholine, underwent progressive hydrolysis in the intestinal lumen.7. The distribution of conjugated bile acids, unesterified fatty acids and phospholipids between the solid (particulate) and liquid (micellar) phases of the intestinal digesta was determined. These chyme constituents were, for the most part, associated with the particulate matter and thus, at any given time, it appears that only a small fraction of the total fatty acids is available for absorption in micellar form. It is suggested that the micellar solubilization of fatty acids may be facilitated by the presence of lysophosphatidylcholine.

The changing phospholipid pattern of the last instar larva and during metamorphosis has been studied by a new method. After extraction by the usual method of Folch et al. (1957), the total lipids were separated into polar and neutral fractions by a fixation technique on silicic acid. The phospholipids were subfractionated into classes and quantities estimated by thin-layer chromatography. Phosphatidylcholine and phosphatidylethanolamine were the major constitutents. Sphingomyelin was found in relatively high concentration but other constituents identified were lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, and cardiolipin. A relationship was found between this last group of compounds and oxygen consumption. The major changes were noted at the time of pupation and adult emergence. At the beginning of pupal period, a considerable amount of phosphatidylcholine is transformed into lysophosphatidylcholine. Reacylation of lysophosphatidylcholine to form phosphatidylcholine is suggested for the first time in an insect. There is a probability of phosphatide transfer between serine and phosphatidylethanolamine on adult emergence. The significance of these variations is discussed.

Diced turkey leg and breast muscle stored for 22 days at 0° and —3°, and for up to one year at —10°, —20° and —60° was examined at intervals for lipid composition. Free fatty acids increased at all temperatures except —60°, with a Q10 of 3-4 between 0° and —20° 90% of the fatty acids liberated were unsaturated, matching in composition the unsaturated acids of the muscle glycerophospholipids. Demonstration of linear relationships between increase in free fatty acids and decrease in phosphatidylethanolamine or increase in lysophosphatidylcholine confirmed phospholipase A2 as the enzyme mainly responsible. The composition of the fatty acids liberated during storage at 0° and —3° showed that both lipase and phospholipase were active. A slight decrease in extractability, resulting in an apparent loss of phospholipid-P, was observed after storage at low temperatures.

—During an extensive decomposition of phospholipids, at the end of the second week of Wallerian degeneration, the decomposition of glycerophosphatides were studied in detail. In a degenerative process lasting for 2 weeks about one-third of the choline phosphatides, two-thirds of the ethanolamine phosphatides, one-third of the serine phosphatides and one-quarter of the inositol phosphatides, were destroyed. The amount of lysophosphatidylcholine decreased proportionally to the destruction of choline phosphatide. On the other hand, the amount of lysophosphatides formed from‘kephalin'-containing fatty aldehyde, during the marked destruction of these phospholipids, remained constant or increased to a small extent and its percentage distribution increased 2 or 3 times compared with other phospholipids. Ethanolamine phosphatides having a high fatty aldehyde content can be regarded as mainly responsible for the relative accumulation of lysophosphatides.

1. 1. Enzymes similar to mammalian phospholipases A and B are present in several subcellular fractions prepared from the house fly, Musca domestica , and the blow fly, Phormia regina . 2. 2. In the house fly phospholipase A activity depends on calcium ion concentration and both phosphatidylethanolamine and phosphatidylcholine are hydrolyzed. 3. 3. In both insects the phospholipase A activity in the supernatant and microsomes in higher than that in the mitochondria. 4. 4. House fly supernatant phospholipase A activity shows a pH optimum of 6·4 with phosphatidylcholine as the substrate and 7·3 with phosphatidylethanolamine. The K m is 0·225 × 10 −3 molar phosphatidylethanolamine. 5. 5. House fly phospholipase B activity, highest in mitochondria, can also be detected in the microsomal fraction. The deacylation of lysophosphatidylcholine is unaffected by calcium ions while the deacylation of lysophosphatidylethanolamine is inhibited.

1. 1. The activation by phospholipids of particulate mitochondrial ATPase from rat liver was studied on a preparation extracted from submitochondrial particles with cholate. 2. 2. Lysophosphatidylcholine and the highly acidic phospholipids diphosphatidylglycerol, phosphatidylinositol and phosphatidylserine stimulated the ATPase at low concentration (50–60 μg/mg protein); phosphatidylethanolamine was less effective whereas phosphatidylcholine exhibited the lowest activity. 3. 3. The saturation curves of phospholipids acting at low concentration showed in most cases a deviation from normal Michaelis-Menten kinetics. This devation was absent or less manifest with phosphatidylethanolamine and phosphatidylcholine. Competition between phospholipids and oligomycin was seen with acidic phospholipids or mixtures of neutral and acidic phospholipids. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide, tributyltin and chlorpromazine competitively inhibited the activation produced by phosphatidylserine, phosphatidylinositol and diphosphatidylglycerol. 5. 5. It is concluded that both the hydrophobic-hydrophilic balance and the negative charge of phospholipids are important in the interaction between phospholipids and protein at the level of particulate mitochondrial ATPase. The competition between phospholipids and inhibitors of mitochondrial ATPase suggests that the activation induced by phospholipids is involved in the inhibitory effect.

Summary The incorporation of C 14 -chlorcholinchlorid into the lipids of kernels of wheat plants, grown in field under normal conditions, was studied. The results indicate that CCC is incorporated partially into the phosphatidylcholine and lysophosphatidylcholine. Accordingly, two different pathways for the synthesis of the phosphatidylcholine and its lysocompound are proposed. It is noteworthy that the same ratio of the active lysolecithin to active lecithin is the same as the ratio of these two lipid constituents, naturally synthesized in the wheat kernel in the corresponding stage of maturity.

I. Several elution techniques for the quantitative recovery of phospholipids from thin-layer chromatograms were compared. Chloroform-methanol (I:1) containing 10% water was a most effective elution agent. 2. A methanol, Cab-O-Sil in toluene—fluor radioassay technique and a water-Aquasol radioassay technique for the quantitative recovery of 14 C-phospholipids from thin-layer chromatograms is described. 3. Dual-labelled ( 3H : 14C ) phospholipids were radioassayed, after thin-layer chromatography, by the water-Aquasol technique. A quantitative recovery was obtained for several phospholipid classes examined, including phosphatidylcholine and lysophosphatidylcholine. The position of the labels had little effect, and the ratios of 3H : 14C had not effect on the recovery rate.

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.

Female rats of the Wistar strain received six separate intraperitoneal injections of D-galactosamine (250 mg/kg body wt per single injection). The rats were sacrificed 48 hr after the initial injection and the livers analyzed for their lipid compositions. A marked increase in the levels of total lipids and triglyceride with a pronounced decrease in phospholipid content in the liver was observed. However the percentage of individual phospholipid fractions obtained by thin layer chromatography showed no significant changes after D-galactosamine administration, except for a slight increase in lysophosphatidylcholine. Fatty acid analyses of total lipids and phospholipids demonstrated an increase in the percentage of oleic and linoleic acids, with a decrease in the percentage of arachidonic acid after D-galactosamine administration. In the triglyceride fraction, a slight increase in the percentage of myristic, palmitoleic and linolenic acids was observed.

Ruminant mammary tissues utilized cytidinediphosphocholine-l,2- 14 C for biosynthesis of the choline-containing lipids, i.e., phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, which were secreted into milk. Experiments performed both in vivo and in vitro revealed that phosphatidylcholine always attained the highest specific activity. The radioactivity in the phosphatidylcholine fraction decreased rapidly compared to that of the lysophosphatidylcholine and sphingomyelin fractions. The rate of decrease of the specific activity of phosphatidylcholine was consistent with the suggestion that two discrete pools of this compound are present in lactating mammary tissue.

SUMMARY Variation was noted in lipid and lipid fractions obtained from muscles of maturing turkeys. The lipid content of thigh muscles increased with advancing maturity, whereas breast muscle lipid remained constant throughout the test period. Total phospholipid levels showed no significant variation with age, but comprised a decreasing percentage of total lipid as bird age advanced. Of the individual phospholipids studied, phosphatidylethanolamine had an inverse relationship with age in both tissues studied. Phosphatidylcholine and phosphatidylethanolamine accounted for approximately 50 and 22%, respectively, of the total phospholipid. Lysophosphatidylcholine (5%), sphingomyelin (10%), phosphatidylinositol plus phosphatidylserine (10%) and other unidentified phospholipids comprised the remainder.

The major phospholipids of hepatitis B antigen, subtype adw , were characterized as phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. In addition, two carbohydrate-containing lipids were observed, one of which was characterized as a non-sialic acid-containing, water-soluble glycosphingolipid. Upon hydrolysis the glycosphingolipid yielded both hydroxy and non-hydroxy fatty acid as well as a long-chain base which was putatively identified as dihydrosphingosine.

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettree cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettree cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.

Activation of rat liver microsomal UDPglucuronyltransferase (EC 2.4.1.17) by phospholipase A (EC 3.1.1.4) was reversed by micellar dispersions of several phospholipids and by serum albumin. When microsomal membranes were treated with the phospholipase in presence of albumin, which binds and removes surface-active phospholipid degradation products (lysophosphatides and unsaturated fatty acids), activation was delayed by albumin at a concentration of 1 mg/mg of microsomal protein and completely prevented by higher concentrations of albumin. The UDPglucuronyltransferase of intact microsomal membranes was activated by lysophosphatidylcholine and unsaturated fatty acids at concentrations which were produced by phospholipase treatment, and the activation by lysophosphatidylcholine most closely resembled that by phospholipase A. It is concluded that the activation of UDPglucuronyltransferase by phospholipase A is due to the detergent activity of phosphilopid hydrolysis products and that the enzyme in intact microsomal membranes probably is not constrained by interaction with a specific phospholipid.

Rat liver microsomes contain an enzyme which catalyzes the acylation by acyl Coenzyme A thioesters of di(1(1'')acyl-sn-glycero-3-phosphoryl)1',3'-sn-glycerol(di(lysophosphatidyl)glycerol) to a compound identified as either 1'(1-acyl-sn-glycero-3-phosphoryl)-3'(1'',2" diacyl-sn-glycero-3-phosphoryl)-sn-glycerol or the corresponding 1'(1,2 diacyl isomer) (phosphatidyllysophosphatidylglycerol). A small amount of diphosphatidylglycerol is also formed. Evidence was obtained for the presence of radioactivity in both 1(1'') acyl and 2(2'') acyl positions of the reaction products. The enzyme has a pH optimum of pH 8.5 and displays considerable activity at an even more alkaline pH. In this regard, the acyltransferase resembles the enzyme which catalyzes the acylation of the 1-acyl isomer of lysophosphatidylglycerol, but differs from that which carries out the acylation of the 1-acyl isomer of lysophosphatidylcholine. Under the conditions used, linoleate was incorporated less readily than either oleate or stearate. Rat liver mitochondria displayed slight acyltransferase activity at pH 7.4 and virtually none at pH 9.0.

The effect of puromycin on phosphatidylcholine and triacylglycerol synthesis was studied in isolated cells of rat intestinal mucosa using radioactive palmitate, glycerol, 2-hexadecylglycerol, and lysophosphatidylcholine as markers. Puromycin caused a 60–65% inhibition of phosphatidylcholine biosynthesis but did not affect the formation of triacylglycerols. Under comparable conditions protein synthesis was inhibited 90–95% and glycoprotein synthesis 60–70%. The utilization of the various lipid precursors indicated that puromycin inhibited the biosynthesis of phosphatidylcholine via both the CDP-choline and the lysophosphatidylcholine pathways, without interfering with triacylglycerol synthesis from either phosphatidic acid or monoacylglycerol precursors. Since both phosphatidylcholines and proteins are involved in the assembly of chylomicrons, it is suggested that the effect of puromycin on chylomicron formation could be due to an inhibition of the biosynthesis of any one or all three of the membrane components: proteins, glycoproteins, and phosphatidylcholines.

1. When unlabelled mitochondria from guinea-pig brain were incubated with a 32P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from 32P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. 32P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [32P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a 32P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.

1. Immunoglobulin G was isolated from sera of non-immunized rabbits or rabbits immunized with whole HeLa cell homogenate. The anti-HeLa immunoglobulin G and its Fab fragment precipitated the particulate 400000g-min. fraction of HeLa cell homogenate. 2. Immunoglobulin G from immunized or non-immunized rabbits and fresh or inactivated complement were added to HeLa cell cultures. Changes in the cell count and cellular contents of DNA, RNA, protein, total and individual phospholipids, cholesterol (and esters) and ganglioside were followed. 3. Addition of immunoglobulin G from non-immunized rabbits and guinea-pig serum (complement) caused a transient increase in DNA followed by a permanent increase in RNA, protein, dry weight and number of cells per culture. 4. Addition of anti-HeLa immunoglobulin G and active complement caused an increase in the cellular content of cholesterol, total phospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine greater than the increase of the controls and a decrease in the molar percentages of phosphatidylcholine and phosphatidylethanolamine as compared with the controls. 5. The cholesterol/phospholipid ratio remained constant. 6. The appearance of lysophosphoglycerides was transient, reaching a maximum 3hr. after addition of anti-HeLa immunoglobulin G. 7. The content of lysophosphoglycerides in HeLa cultures exposed to immunoglobulin G from non-immunized rabbits ranged from 50% to 30% of the values obtained from cultures exposed to the anti-HeLa immunoglobulin G and complement. 8. The changes in the lipid pattern of the HeLa cells were associated with the appearance of juxta-nuclear vacuoles in cells, but were apparently not specifically related to the presence of active complement.

1. 1. A new enzyme belonging to the group of phospholipases D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was found in Corynebacterium pseudotuberculosis (ovis). It splits sphingomyelins to N- acylsphingosylphosphates and choline, and lysophosphatidylcholine to lysophosphatidic acids and choline. 2. 2. Methods of purification of the enzyme by (NH4)2SO4 and methanol precipitation and elution from CM Sephadex columns are described. 3. 3. The behaviour of the enzyme upon Sephadex columns, ion-exchangers and electrophoresis was investigated. 4. 4. Optimal conditions of the enzymic reaction are studied and the K m determined using sphingomyelins as a substrate (K m = 6.2 mM ) and lysophosphatidylcholines ( K m = 8.3 mM ) . 5. 5. Using a new modification of paper and thin-layer chromatography the product of enzymic hydrolysis of sphingomyelins, N- acylsphingosylphosphates , were separated from other related compounds. 6. 6. A new colour reaction for the characterization of N- acylsphingosylphosphates is described. 7. 7. The spectrum of sphingosine bases in sphingomyelins and N- acylsphingosylphosphates is characterized by 4-sphingenine (as the main component) and sphinganine (present only in a trace amount). 8. 8. The main fatty acids are in both sphingolipids C 24:1 and C 16:0 acids. The minor components are composed from C 24:0 , C 22:0 , C 18:0 , C 18:1 , C 22:1 , C 23:0 and C 20:0 acids.

1. 1. Purified lysophospholipase from beef pancreas was found to deacylate 1-acyllysophosphatidate, 1-acyl-lysophosphatidylethanolamine, 1-acyl-lysophosphatidylserine, 1-acyl-lysophosphatidylglycerol and 1-acyl-lysophosphatidylcholine at comparable rates. 2. 2. The enzyme was found to be active on lysophosphatidylcholine generated in microsomal membranes by the action of exogenous phospholipase A 2 . Thus, the combined action of phospholipase A 2 and lysophospholipase can be used as an enzymatic tool for delipidating biological membranes. 3. 3. The lysophospholipase from beef pancreas also hydrolyzed triacetin, tributyrin, p -nitrophenylacetate and phosphatidylcholines containing short-chain fatty acids. Evidence is presented indicating that the hydrolysis of these substrates is brought about by the same enzyme. 4. 4. The properties of the lysophospholipase and these of carboxylesterases are discussed.

Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh-Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N-acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids.