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.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Studies on liver phosphatidyl cholines: I. Effects of fatty liver induction on phosphatidyl cholines from liver mitochondria and microsomes

Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl₄ (0.3 ml of CCl₄ suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl₄. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.     

Studies on tamarind kernel oil II1L : analysis of phospholipids

Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phospha-tidic acid, and lyso derivatives of phosphotidyl choline and phosphatidyl ethanolamine were identi-fied from phospholipids of Tamarind kernel oil. Phosphatidyl choline and phosphatidyl ethanolamine were isolated and their fatty acid composition was determined by GLC. Reference 2 is considered as paper I of the series. Presented at Annual Convention of Chemists, Dec. 1977, at Jaipur, India.     

Analysis of subcellular phosphatidyl choline in developing rabbit lung

Phosphatidyl choline is a major lung surfactant. Insufficient development of the surfactant in neonates is often associated with the Respiratory Distress Syndrome. The concentration and fatty acid composition of phosphatidyl choline have not been studied in the subcellular organelles of the developing lung. This study has investigated the development of the concentration and fatty acid composition of phosphatidyl choline in subcellular fractions of 28-day and 30-day fetal and maternal New Zealand rabbit lungs. The concentration of total phospholipids in lamellar bodies increased four to five fold from 28-day fetus to 30-day fetus which, in turn, was similar to the maternal level. Total phospholipid content increased only about 50% in mitochondria and microsomes. The percentage of phosphatidyl choline among total phospholipids in lamellar bodies increased successively from 60% at 28 days gestation to 84% at 30 days gestation and leveled at 84% in maternal lamellar bodies. Microsomal PC increased steadily from 52% in the 28-day fetus to 65% in the adult. Analysis of the fatty acid composition of phosphatidyl choline in lamellar bodies confirmed 16∶0 as the major fatty acid, and its content remained constant from 28 days gestation to adult. In contrast, the content of 16∶0 of the microsomal phosphatidyl choline decreased with increasing gestation. Changes of several unsaturated fatty acid components were observed in both lamellar bodies and microsomes in the developing lungs. Maturational development of phosphatidyl choline is reflected in an increase in the concentration of this surfactant, particularly in lamellar bodies, and possibly in remodeling of fatty acid composition in both lamellar bodies and microsomes.     

On the phospholipids ofCulex pipiens fatigans

The lipids of different developmental stages ofCulex pipiens fatigans, vector of bancroftian filariasis, have been investigated. The phospholipid composition of the developmental stages and of the subcellular fractions of fourth instar larvae of the insects were analyzed. The composition of fatty acids and their positional distribution have also been examined in the major phospholipids of the larvae. The insect eggs contained higher amounts of lipids than larvae suggesting that they were utilized during embryogenesis. Phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) comprised over 75% of the insect phospholipids. Of these, PE was present in the greatest amounts during all stages of growth and in the subcellular fractions of larvae. An ethanolamine containing sphingolipid was found as a component of the phospholipids of the insects. About 50% of the lipids of the larvae were localized in the cell debris and nuclei fraction which also contained most of the lysolipids of the insects. As in other Diptera 16∶0, 16∶1 and 18∶1 were the major fatty acids present in the insect lipids of which the fatty acid found in greatest amounts was 16∶1. Similar to the phospholipids of animal species, saturated fatty acids were predominantly linked to the 1 position of the major phospholipids of the insects while the unsaturated fatty acids were in higher amounts at the 2 position.     

Studies on the glycolipids and phospholipids of immature soybeans

The lipid of immature soybeans was extracted with chloroform-methanol and fractions containing the glycolipids and phospholipids were separated by column chromatography. Phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidic acid (PA) phosphatidyl glycerol (PG), n-acyl phosphatidyl ethanolamine (APE) and sulfolipid (SL) were identified by thin layer chromatography (TLC). Sterol glucoside (SG), esterified sterol glucoside (ESG), digalactosyl diglyceride (DGDG) and cerebrosides (CE) were isolated by TLC and identified by color reactions, chemical degradation and spectral analysis.     

Phospholipids of the pulmonate land snailCepaea nemoralis (L.)

The phospholipids of the snailCepaea nemoralis, comprising the major lipid fraction (65%) in this terrestrial pulmonate, were investigated by thin-layer and column chromatography. Detailed gas chromatographic analyses of liberated fatty acid fractions and amino acid analyses of the water soluble moieties of isolated phospholipid classes were carried out. Phosphatidyl choline (47%) and phosphatidyl ethanolamine (21%) were found to be the predominant phospholipid classes, while phosphatidyl serine (8%), phosphatidyl inositol (6%), diphosphatidyl glycerol (3%), ceramide amino-ethylphosphonate (7%), lysophosphatidyl choline (1%), and phosphatidic acid (1%) were present in lesser amounts. In the phosphatidyl ethanolamine and phosphatidyl serine fractions, minor quantities of plasmalogen analogues were detected. Fatty acid profiles of the various phospholipid classes appeared to be strikingly diverse, e.g. a characteristic component, such as linoleic (18∶2ω6) acid, ranging from 3–54%. In vivo radioisotope studies using 1-¹⁴C-acetate demonstrated the high biosynthetic rate of all phospholipid classes and their respective fatty acid fractions. Results are discussed in relation to data on the phospholipids from other invertebrate species.     

Lipids of subcellular particles

Methods for isolation and characterization of subcellular particles as well as procedures for analysis of lipid class composition are discussed. The literature on distribution of lipids in subcellular particles is then reviewed. Pertinent new data from our laboratories are presented as well. The isolated particles are related to the organelles to which they correspond in the cell and are discussed with regard to heterogeneity and morphological integrity. Confusion can arise with regard to subcellular particles unless it is appreciated that: 1) preparation of particles of high purity generally requires more than the classical differential centrifugation scheme (both differential and gradient centrifugation may be required); 2) it is hazardous to apply exactly the same procedure for all tissues; 3) all subcellular fractions must be thoroughly characterized. The more recently devised DEAE cellulose column and thin-layer chromatographic procedures for analysis of lipid class composition are more reliable than the older hydrolytic or silicie acid column or paper chromatographic techniques. The chief lipid components of mitochondria from all organs and species are lecithin, phosphatidyl ethanolamine, and cardiolipin (diphosphatidyl glycerol). Despite the fact that reports in the literature are in agreement that phosphatidyl inositol is a major component of mitochondria, it is concluded on the basis of new data obtained from highly purified mitochondria and improved analytical methods that phosphatidyl inositol is not a major component of mitochondria. The presence of a relatively large amount of phosphatidyl inositol in mitochondrial preparations is probably related in part to contamination with other particles. Some analytical procedures are demonstrated to give erroneous values for this lipid class. It is also concluded that phosphatidyl serine, phosphatidic acid, sphingomyelin, cerebrosides, and lysophosphatides, reported to occur in mitochondria, are not characteristic mitochondrial components and furthermore that the large amount of uncharacterized mitochondrial phospholipid reported is actually an analytical artifact. Microsomes appear to be similar to mitochondria except that cardiolipin is either low in or absent from microsomes. Available data indicate nuclei to be rather similar to mitochondria and microsomes, at least in some organs. Studies of the fatty acids of subcellular particles indicate that different particles from one organ have very similar fatty acid compositions. It is clear that there are marked variations in fatty acid composition of particles from different organs and from different species. Differences in dietary fat may be associated with marked changes in fatty acid composition, although brain mitochondrial lipids are largely unchanged. Each lipid class from mitochondria of most organs appears to have a fairly characteristic fatty acid composition. Cardiolipin from some organs contains primarily linoleic acid, phosphatidyl ethanolamine contains large amounts of linoleic and higher polyunsaturates, and lecithin is similar to phosphatidyl ethanolamine except that it does not contain as much arachidonic acid and/or other highly unsaturated fatty acids. New data, the first to be reported, are presented for heart mitochondrial cardiolipin, phosphatidyl ethanolamine, and lecithin. It is concluded that there are two basically different types of membranous structures. Myelin is the chief representative of the metabolically stable type of membrane structure while mitochondria represent the more labile type. The two types of membranes have very different in vivo properties and very different lipid compositions. Myelin is characterized by a high content of cholesterol and sphingolipids with more long chain saturated or monoenoic fatty acids while mitochondria are characterized by a low cholesterol content, little or no sphingolipid, and highly unsaturated fatty acids. It is clear that formulations of the myelin type membrane structure such as that of Vandenheuvel cannot apply to mitochondria. It is postulated that membrane structures intermediate between the extremes represented by myelin and mitochondria exist.     

Lipid and fatty acid composition of testes of quaking mice

Testes of quaking mice (sterile mutants) and of controls were analyzed for major lipid classes and fatty acid composition. Of the main lipid classes, only cholesterol esters differed significantly in concentration between the two groups (1.01 for quakers vs 0.69 mg/g wet wt of tissue for controls). The concentration of triglycerides was 4.5–5.0, that of total phosphatides 18–19, and that of free cholesterol 1.9–2.0 mg/g for mutants and controls. The concentrations of phosphatidyl ethanolamine and of sphingomyelin were both lower in quaking than in normal mice, but only the change in the former was statistically significant. Phosphatidyl choline was the major phosphatide (43–45% of total phosphatides) followed by phosphatidyl ethanolamine (24–26%) and sphingomyelin, phosphatidyl serine, and phosphatidyl inositol (all ca. 7% of total phosphatides). Minor differences between the mutants and controls were observed in concentrations of fatty acids of major lipid classes. The mutants, sterile because of faulty spermatid differentiation, had normal quantities of 22∶6 w3 and 22∶5 w6. These data are consistent with the hypothesis that the 22-carbon polyenes are associated with the formation of spermatids, rather than with their final differentiation into spermatozoa.     

Viral stimulation of choline phosphotransferase in spleen microsomes

Choline phosphotransferase and phosphatidyl ethanolamine methyltransferase enzymatic activities (nmoles phosphatidyl choline/min/mg protein) have been determined in spleen microsomes of Rauscher virus infected balb/c male mice at 5, 10, 14, and 21 days following inoculation of the virus. There is a significant stimulation of the choline phosphotransferase activity in the virus infected spleens with the peak of activity at about 10 days of viral infection. The specific activity of choline phosphotransferase is 10 times that of the phosphatidyl ethanolamine methyltransferase at 10 days of viral infection. There is a 51-fold increase over controls for the total microsomal choline phosphotransferase at 14 days of viral infection and only an 18-fold increase over controls for the phosphatidyl ethanolamine methyltransferase activity. There is a significant (P less than 0.001) increase over controls in the concentration of total phospholipid-P, phosphatidyl choline-P, and phosphatidyl choline-P fractions as separated by argentation chromatography of microsomes from spleens of mice infected with Friend virus of Rauscher virus for 14 days. The choline phosphotransferase and phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of 14 day Friend and/or Rauscher virus are unaltered during viral infection.     

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.     

Studies on liver phosphatidyl cholines: II. Effect of sex,age and species differences on phosphatidyl cholines from liver mitochondria and microsomes

Liver mitochondrial and microsomal phosphatidyl cholines differing in the degree of unsaturation of their fatty acids have been separated into four fractions by silver ion silica gel TLC. The levels of the four phosphatidyl choline fractions were determined for male and female rats and mice, fetal and young rabbits, and female hamsters and guinea pigs. The sum of phosphatidyl choline fractions 1, 2, and 3 of mitochondria and microsomes was greater in the female rat than in the male rat with the difference being a reflection of a higher level of fraction 3 which contains arachidonic acid. The female rat has greater concentration of phosphatidyl choline fractions 1 and 3 of mitochondria. Similar results were seen in mouse liver microsomes but not in mitochondria. The levels of the individual four fractions varied from species to species. No change occurred in the levels of the phosphatidyl choline fractions of fetal (−9 and −3 days) rabbits, but an increase was seen in the level of fraction 4 between day 3 and day 35 in both the mitochondria and microsomal fractions of liver. The concentration of mitochondrial and microsomal protein, total phospholipid and total lecithin phosphorus were determined in rat, mouse, hamster and guinea pig. The total phospholipid phosphorus/protein (μg/mg) of microsomes was greater in all species than that observed in mitochondria. Liver microsomes contain 45–50% of total phospholipid phosphorus as lecithin whereas mitochondria contains 32–37%. The fatty acid patterns of mitochondria and microsomal phosphatidyl cholines were determined and the ratio of palmitate to stearate was greater than two for mice and hamsters and approximately 0.5 for rat and guinea pigs.     

Phospholipids of human serum

Phospholipids extracted from normal human serum were fractionated into lecithin, lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol. Identification of each was established by thin-layer chromatography and infrared spectrophotometry. The content of plasmalogen was determined in both lecithin and phosphatidyl ethanolamine fractions. The composition of fatty acids and fatty aldehydes in isolated phospholipids is presented. The degree of unsaturation as reflected in the average content of double bonds per molecule of the fatty acids in phospholipids was: lecithin 1.2, choline plasmalogen 2.1, lysolecithin 0.6, sphingomyelin 0.2, phosphatidyl ethanolamine 2.8, lysophosphatidyl ethanolamine 1.0, phosphatidyl serine 1.0, and phosphatidyl inositol 1.8. Both chlline and ethanolamine plasmalogen aldehydes were predominantly saturated. Molecular weight of each phospholipid was calculated from determined fatty acid and fatty aldehyde compositions; the phosphorus factor for each phospholipid was computed. On a weight percent basis, lecithin, sphingomyelin, and lysolecithin accounted for 95% of the total phospholipids. The ethanolamine-containing phospholipids accounted for 2.5%, and the remainder was divided among phosphatidyl inositol, choline plasmalogen and phosphatidyl serine. Presented in part at the AOCS Meeting, Houston, April, 1965. Dept. of Health, Education and Welfare, USPHS.     

Lipids ofKlebsiella pneumoniae: The presence of phosphatidyl choline in succinate-grown cells

The carbon and energy source for aerobically grown cultures ofKlebsiella penumoniae profoundly influenced the total lipid content and phosphatide composition. Glucose-grown cells contained 13% lipid, 56% of which was phospholipids. Succinate-grow cells contained 8% lipid, 66% of which was phospholipids. The predominant phosphatides of glucose-grown cells were phosphatidyl ethanolamine, 82%; phosphatidyl glycerol, 4.5%; phosphatidic acid, 5%; cardiolipin, 6.5%; phosphatidyl serine; and trace amounts of unidentified phosphatides. Phosphatides of succinate-grown cells were phosphatidyl ethanolamine, 38%; diphosphatidyl glycerol, 14%; phosphatidyl glycerol, 13%; phosphatidyl choline, 14.5%; phosphatidyl serine, 6%; phosphatidic acid, 4%; and 10% unknown lipids. No trace of phosphatidyl choline was found in glucose-grown cells. Paper 75-11-170 of the Kentucky Agricultural Experiment Station.     

Phosphatides isolated from seeds of commercial and experimental safflower varieties

Studies on phosphatides from several safflower varieties show the following five major results. The total phosphatide contents of the various safflower seeds are quite similar (0.48% for a commercial and 0.58% for a brown-striped variety). The same three major and one minor phosphatides were present in all varieties: phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS). The amounts of these lipids present in the crude phosphatide mixture were quite similar in all varieties tested (}36% for PC, }15% for PE, }23% for PI, and less than 2% for PS). The fatty acid composition of the phosphatides of UC-1 high oleic safflower is very different from that of the other varieties, but it reflects the composition of the corresponding oil triglycerides as far as the major acid is concerned. All other safflower seed phosphatides investigated have linoleic acid as the major fatty acid constituent. A simple and very sensitive color test has been found which can differentiate phosphatides of the high linoleic from the high oleic type. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes

The effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities, the two enzymes involved in phosphatidyl choline biosynthesis in liver microsomes, has been investigated. Female rats were fed a 5% ethanol-liquid diet containing amino acids, minerals, vitamins, with and without choline, for 2, 6, and 10 weeks. Control animals were pair-fed the same isocaloric diet with 5% sucrose with and without choline. Ethanol administration with or without dietary choline stimulated significantly (P<0.001) the specific activities of phosphatidyl ethanolamine methyltransferase in liver microsomes in the animals fed 5% ethanol for 2, 6, and 10 weeks, when compared to those control animals pairfed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P<0.02) the specific activities of choline phosphotransferase. The specific activities of phosphatidyl ethanolamine methyltransferase continued to increase in the liver microsomes from the animals in which dietary choline was omitted for 2, 6, and 10 weeks in the sucrose controls and alcohol-fed animals. Ethanol administration stimulates significantly (P<0.001) the phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of animals fed the liquid diet with dietary omission of choline and methionine for 2 weeks.     

Studies on the lipids of sheep red blood cells. I. Lipid composition in low and high potassium red cells

The lipid composition of whole red blood cells was investigated in five sheep with red cells containing a low concentration of potassium (LK) and in five sheep with red cells containing a high concentration of potassium (HK). No apparent differences within the limit of error of the experiment were detected in the lipid class composition between the HK and LK red cells. Cholesterol, the only nonpolar lipid detected in the tissue, was present in oneto-one molar ratio to the total phospholipids. Phosphatidyl ethanolamine and sphingomyelin accounted for 85% of the total phospholipids; phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, and lysolecithin were present in lesser amounts. No lecithin was detected in any of the animals in this investigation. Plasmalogen compounds were found only in the ethanolamine lipids. The molar ratio of choline to noncholine phospholipids was also approximately one to one. It was concluded that the major lipid class distribution in the two types of red cells cannot be directly responsible for the differences observed in the cation concentrations in these cells in the two species of sheep.     

Study of free and bound lipids ofBrassica campestris,var yellow sarson

Mature seeds ofBrassica campestris var. yellow sarson were extracted with hexane to yield free lipid. The residue then was extracted with chloroform-methanol to release bound lipid. Free and bound lipids were separated into polar and nonpolar fractions chromatographically. The nonpolar fraction of both free and bound lipid consisted mainly of triglycerides with small amounts of steryl esters, free sterols, mono- and di-glycerides, and free fatty acids. The principal components of polar bound lipid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and steryl glycoside. In the free polar lipid, there was more phosphatidyl inositol and less phosphatidyl choline and phosphatidyl ethanolamine. Erucic acid content was much greater in the nonpolar fractions and in the polar free lipid than in the polar bound lipid.     

Molecular species of phosphatidyl ethanolamine from egg yolk

Phosphatidyl ethanolamine was isolated from total egg yolk lipids by preparative thin layer chromatography (TLC). The purified phosphatide contained 3% of the alkoxy derivative. It was degraded to diglycerides in the presence of purified sphingomyelin by phospholipase C fromClostridium welchii. The diglycerides were acetylated and resolved on the basis of unsaturation by argentation TLC. The fatty acid composition of the original phosphatidyl ethanolamine and the derived acetates was determined by gas chromatography, as was the molecular weight distribution of the diglyceride acetates. The placement of the fatty acids in the parent phosphatide was deduced by hydrolysis with phospholipase A fromCrotalus atrox, and in the acetates with pancreatic lipase. Some 33 major species of phosphatidyl ethanolamine were identified and compared to those for egg yolk lecithins. Presented in part at the Canadian Federation of Biological Societies Meeting, Kingston, June 1968.