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The individual phosphatides of human plasma were analyzed qualitatively and quantitatively by paper chromatography. The phosphatides which were always present were lecithin, sphingomyelin, lysolecithin phosphatidylethanolamine and a component having the properties of inositol phosphatide. Phosphatidylserine was not detected by this procedure and hence is at most a trace constituent in plasma. Very small amounts of other unidentified phosphatides also occur in plasma. One of these may be lysophosphatidylethanolamine. A study was made of the plasma phosphatides of persons having recent cardiac infarctions. There is suggestive evidence that plasma lysolecithin is significantly diminished in some of these persons. The advantages and limitations of the paper chromatographic method for the analysis of plasma phosphatides are discussed.

Phospholtptds in human liver and bile were analyzed using a neutron activation paper chromatographic technique. Phosphatidylserine, phosphoinositide, and choline plasmalogen as well as lecithin, phosphatidylethanolamine, and sphingomyelin were found to be present. Further evidence for the presence of lysolecithin in normal human bile is also presented. Similar results were obtained with rat bile using radioactive phosphorus. Lecithin comprises more than 80% of the phospholipids in human bile in contrast to liver, where it comprises 45%, indicating a selective secretion of phospholipid classes from liver into bile. The difference found in the fatty acid composition of the total phospholipids in human liver and bile by gas chromatography, coincides with the findings on the differences in phospholipid composition.

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

The composition of the lipids extracted from fresh normal human brain and from brains preserved up to 24 years in unbuffered formaldehyde solutions was analysed quantitatively using column and paper chromatography, followed by various determinations. The amounts of cholesterol, cerebrosides, sulphatides, phosphoinositides and sphingomyelin remain unaffected. Lecithin, phosphatidylethanolamine and phosphatidylserine are broken down to the corresponding lyso compounds, fatty acids, phosphatidic acid and probably lysophosphatidic acid. The lyso compounds are further broken down by the liberation of the second fatty acid. By this process the fatty acid content is markedly increased. The total lipid content decreases slightly by the formation of water soluble phosphoryl compounds. Consequently the phosphorus content of the total lipid extract suffers marked diminution. Histochemical reactions on lipids in formaldehyde stored tissues should preferably be performed in combination with an analysis of the lipid extract.

1. 1. Nuclei have been isolated from rat liver by the citric acid method of Mirsky and Pollister and their purity investigated by electron microscopy. 2. 2. Nuclear phospholipid, cholesterol, DNA and protein have been determined; the composition and appearance of the nuclei have been compared with those of nuclei prepared in sucrose by the method of Chauveau . 3. 3. The phospholipid composition of the nuclei has been investigated by hydrolysis and two-dimensional paper chromatography and compared with the phospholipid composition of whole liver. 4. 4. The amount of lipid in the nucleus is small, but the pattern of lipids is similar to that in the whole cell with the exception that cardiolipin is absent from the nucleus. 5. 5. Evidence is presented that citric acid removes the outer membrane from the nucleus whilst in sucrose the double-membrane structure remains intact. By means of Langmuir trough experiments, the amount of lipid found in the nuclear fraction has been related to the amount required to cover the surface of a single nucleus. This has been found sufficient to constitute two lipid monolayers in the case of citric acid-isolated nuclei.

1. 1. The in vitro metabolism of [3H]serine into phospholipids of proliferating and calcifying epiphyseal cartilage, and of liver, of chickens has been investigated for periods up to 6 h using pulse-labeling techniques. 2. 2. Serine was very rapidly incorporated into both epiphyseal cartilage and liver phospholipids, at roughly equivalent rates, but in markedly different ways. In liver the label was passed rapidly from phosphatidylserine to phosphatidylethanolamine, and to a minor extent into phosphatidylcholine and sphingomyelin. In contrast, in cartilage more of the label remained in phosphatidylserine with much slower transfer to phosphatidylethanolamine. However, a much larger proportion of label became incorporated into sphingomyelin. 3. 3. Comparison of the two epiphyseal cartilage layers revealed that there was greater synthesis and more rapid turnover of phosphatidylserine in the calcifying zone. In contrast, the incorporation of serine into sphingomyelin by proliferating cartilage was double that of calcifying cartilage. 4. 4. We interpret the unusually active synthesis of sphingomyelin by proliferating cartilage to be related to the expansion of plasma membranes which would be associated with cell proliferation and hypertrophy. On the other hand, the rapid synthesis of phosphatidylserine by calcifying cartilage appears to be associated with the exfoliation from chondrocytes of matrix vesicles whose lipids have been shown to be markedly enriched in phosphatidylserine. 5. 5. Data are presented which suggest that the incorporation of serine into the phosphatidylserine of calcifying cartilage is probably by a base-exchange mechanism. In contrast to liver, enzymes in epiphyseal cartilage capable of decarboxylating phosphatidylserine to phosphatidylethanolamine are relatively inactive.

1. 1. Evidence is given for reactions leading to the formation of all the major glycerol containing phospholipids of Saccharomyces cerevisiae. A cell free particulate fraction prepared from S. cerevisiae is capable of incorporating sn-[14C]glycero-3-phosphoric acid into phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerophosphate, phosphatidylglycerol, cardiolipin, CDP-diglyceride and diglyceride. These products were separated by a two-dimensional chromatography system. 2. 2. The incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidic acid requires CoA and ATP and is greatly stimulated by the addition of divalent cations. The incorporation of this label into CDP-diglyceride, phosphatidylserine, phosphatidylinositol, phosphatidylglycerophosphate, phosphatidylglycerol and cardiolipin has an additional requirement for CTP. Inositol and serine enhance the incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidylinositol and phosphatidylserine, respectively. Evidence is given for two pathways for phosphatidylethanolamine synthesis, from the reaction of CDP-ethanolamine and diglyceride and by the decarboxylation of phosphatidylserine. There are also two pathways for the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine and the reaction of CDP-choline and diglyceride. CDP-diglyceride may therefore be a precursor, directly or indirectly, of all the phospholipids under study. The direct conversion of labeled CDP-diglyceride to some of the phospholipids is demonstrated. 3. 3. Formation of CDP-diglyceride from endogenous precursors appears to be a major reaction.

1. 1. A study has been made of the contaminating effects of phospholipids in the titrimetric procedure of DOLE1. 2. 2. Phosphatides were found to be extracted to varying degrees in the heptane phase of the method of Dole. None of the lysophosphatides was extracted. 3. 3. Unlike lecithin, phosphatidylethanolamine and phosphatidylserine were found to be titratable under the conditions used in the above method. 4. 4. Exclusion of cephalins by adsorption on silicic acid removed the interference and allowed for accurate free fatty acid estimation. 5. 5. The significance of these findings is discussed in relation to the extensive use of the Dole procedure without modification.

The phospholipids ofCorynebacterium diphtheriae, Corynebacterium xerosis, Corynebacterium equi andCorynebacterium ovis were examined, largely by chromatographic procedures. In all of these, lipids of the phosphoinositide and mannophosphoinositide type were prominent. In contrast to the mycobacteria, the mannophosphoinositides of the corynebacteria were all dimannophosphoinositides; however, as in mycobacteria, these dimannophosphoinositides apparently occurred in the diacylated and triacylated forms—the tetraacylated component prominent in mycobacteria was absent. Phosphatidylethanolamine and phosphatidylserine were also absent. InCorynebacterium diphtheriae the major single phospholipid corresponded to phosphatidylglycerol: cardiolipin also appeared to be a major lipid. The fatty acids of the corynebacterial phospholipids were distinguished by the presence of branched chain isomers of medium chain length. The importance of phospholipids in the taxonomy of the actinomycetes and related eubacteria is discussed.

1. 1. Phosphatidylserine and phosphatidylethanolamine were found to be the principal phospholipids in the strictly anaerobic bacterium Megasphaera elsdenii . 2. 2. The identity of phosphatidylserine, unique as a major bacterial phospholipid, was based on the following observations: (a) it cochromatographed with reference phosphatidylserine on one- and two-dimensional thin-layer chromatograms; (b) its deacylation product after mild alkaline hydrolysis cochromatographed with reference sn -glycero-3-phosphorylserine on paper chromatograms in two solvent systems; (c) it was the only phospholipid labelled during growth in the presence of [1- 14 C]serine; (d) it was labelled prior to phosphatidylethanolamine during growth in the presence of [3- 14 C]serine; (e) it could be converted into phosphatidylethanolamine by treatment with phosphatidylserine decarboxylase from Escherichia coli . 3. 3. Both phosphatidylserine and phosphatidylethanolamine of M. elsdenii comprise high levels of plasmalogen analogues (72 and 87%, respectively), implying that this organism is very suitable for studies on the pathways involved in the biosynthesis of plasmalogens in microorganisms.

Bilateral adrenalectomy altered phospholipid distribution in various tissues of the rat. Total phospholipids were drastically reduced in liver, heart and kidney; the change in brain was much less significant. Phospholipid fractionation was performed by paper chromatography on extracts of these tissues. Phosphatidylcholine content decreased after adrenalectomy whereas phosphatidylserine and phosphatidylethanolamine content increased relatively. Phosphatidylinositol disappeared from the chromatograms of liver and heart extracts 12 days after adrenalectomy. The significance of these observations is discussed. We observed that brain phospholipid metabolism was much less affected by adrenalectomy than the other tissues we studied. (Endocrinology 80: 765, 1967)

The phospholipids of Desulfovibrio desulfuricans , Norway strain, D. vulgaris , and D. gigas were examined in relationship to their qualitative and quantitative composition. D. desulfuricans and D. vulgaris exhibited an essentially identical phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and lysophosphatidylserine. Phosphatidylserine (10.9%) was present in D. desulfuricans but was not detected in D. vulgaris. D. gigas was found to contain only two phospholipids, phosphatidylethanolamine (30%) and phosphatidylglycerol (70%). An ornithine-containing lipid was detected in D. gigas which was not present in the other two Desulfovibrio species.

1. 1. Normal and glucose-6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) deficient red cells have been compared in regard to their lipid content and its stability during incubation with hemolytic compounds. 2. 2. In fresh samples no significant differences in cholesterol or total lipid phosphorus were detected between the two kinds of red cells. Slight differences were detected in the composition of phosphatides. The mean sphingomyelin content was lower, and phosphatidylserine content higher, in the enzyme-deficient than in the normal cells. The differences were statistically significant at the 5% level in a material consisting of about 20 normal and 20 enzyme-deficient blood samples. However, most of the single values fell within overlapping regions, not permitting individual differentiation between the two kinds of cells. 3. 3. Incubation with nitrofurantoin or acetylphenylhydrazine did not lead to any alterations in the cholesterol or total lipid phosphorus. On the other hand such incubation resulted in a significant decrease of phosphatidylserine and increase of phosphatidylethanolamine in the majority of the enzyme-deficient red cell samples. The possible mechanisms and significance of these changes are discussed.

The time course of incorporation of [14C]formate into the lipids of the nuclear and cytoplasmic fractions of Ehrlich ascites tumor cells was investigated. By paper-chormatographic analysis and hydrolysis studies it was shown that phosphatidylserine, and to a lesser extent, phosphatidylethanolamine were the most active phosphatides. However, a ninhydrin-negative, phosphorus-free lipid conatined most of the radioactivity after 3 h incubation. This lipid releases fatty acids and ninhydrin-positive, water-soluble labeled compounds only after prolonged hydrolysis. These lipids moreover have paper-chromatographic properties which differentiate them from all the known common neutral lipids and phosphatides. They are tentatively identified as fatty acid amines of amino acids or peptides. The data in these experiments indicate that the decarboxylation of phosphatidylserine to phosphatidylethanolamine and the direct methylation of phosphatidylethanolamine to lecithin are not major metabolic pathways in Ehrlich ascites tumor cells.

Dikstein and Sulman (1) reported that the treatment of the rectus abdominis muscle of Bufo viridis toad with 3.4 M (25%) acetone for 2 minutes eliminated the response of the muscle to acetylcholine (ACh), but 5 to 20 μg/ml of phospholipids such as phosphatidylserine could restore the response of the treated muscle to ACh. They also observed similar effects of phospholipids on the rabbit uterus treated with acetone and restoration of the response of the ileum to norepinephrine after the treatment with phospholipids, and postulated that the phospholipids of receptors were extracted by acetone and could be replaced by those from an exogenous source. In a recent report, however, Ehrenpreis, Hazra and Bigo-Gullino (2) presented evidence that smooth muscles of various organs treated with 3.4 M (25%) acetone were not restored in their responses to agonists by application of phospholipids, and the effect of acetone was concerned with the denaturation of the receptor protein by acetone, but not with lipid extraction from the receptor. We have found in this paper that the mode of action of acetone is very similar to that of papaverine. The aims of this paper are to study the mechanism of antiacetylcholine action of acetone and papaverine, and to test the restoration of the response of the papaverine or acetone-treated ileum to ACh by phospholipids. Also in our experiments, as described by Ehrenpreis, Hazra and Bigo-Gullino (2), 3.4 M (25%) acetone applied for 2 minutes at 32°C abolished the response of ileum to ACh, and even large doses of phospholipids could not restore the responsiveness of the ileum after the acetone treatment. In an ileum maintaining a moderate response to ACh after the acetone treatment under milder conditions, however, various phospholipids could restore distinctly the responsiveness of the ileum.

1 The (Na++ K+)-stimulated ATPase of human erythrocyte ghosts is completely inactivated by treatment with pure phospholipase A2 or phospholipase C. 2 These phospholipases also cause considerable or even complete loss of Mg2+-dependent ATPase activity. 3 Enzymatic hydrolysis of sphingomyelin does not significantly influence either of the ATPase activities. 4 Conversion of the glycero-phospholipids into phosphatidic acid by phospholipase D causes a drastic decrease in the Mg2+-dependent ATPase activity, whereas the (Na++ K+)-stimulated activity is increased. 5 Enzymatic decarboxylation of the phosphatidylserine fraction of the ghosts does not influence the (Na++ K+)-stimulated ATPase, unless the last 13% of this fraction is converted into phosphatidyethanolamine, when there is a complete loss of this particular ATPase activity. On the other hand, the Mg2+-dependent activity tends to be increased by this treatment. 6 The (Na++ K+)-stimulated ATPase activity of erythrocyte ghosts treated with phospholipase C and subsequently extracted with dry ether, can be completely reconstituted by the addition of phosphatidylserine. Phosphatidylcholine, phosphatidylethanolamine and phospholipid mixtures are found to be ineffective. Phosphatidic acid produced a partial restoration of activity. 7 It is concluded that the (Na++ K+)-stimulated ATPase activity strongly depends upon the presence of phosphatidylserine, of which only a minor fraction in the erythrocyte membrane is directly involved. It cannot be concluded from the experiments described in this paper that the Mg2+-dependent ATPase also requires a specific phospholipid. However, a direct involvement of either cholesterol or sphingomyelin in this activity can be precluded.

1. 1. Mixed lipides of the skin of rabbits have been used for the separation of their phospholipide components on a silicic acid column. Through use of a gradient elution with a number of chloroform-methanol mixtures and pure methanol, satisfactory separations have been obtained in an 8-hr. period. The elution patterns have been constant and the phosphorus recoveries have been high (91%). Combined chemical, infrared, and paper chromatographic techniques have been used for analysis. 2. 2. Lecithin, phosphatidylethanolamine, and phosphatidylserine comprise the major phospholipide components. Polyglycerolphosphatides, lysolecithin, and sphingomyelin occur in sufficient quantity to be of interest. Small amounts of plasmalogens and traces of two unidentified lipides, one of which appears to be a “lysocephalin,” make up the rest of the skin phosphatides.

The inositol phosphatide fraction of rat brain has been separated by chromatography on formaldehyde-treated paper into monophosphoinositide and two other inositol-containing phosphatides named phosphoinositide 1 and 2. Quantitative determination of the ether-soluble phosphatides gave the following values for rat brains of 450 to 500 mg dry weight: 14 to 21 mg phosphatidylcholine and phosphatidylethanolamine, 3.5 to 4.6 mg phosphatidylserine, 0.9 to 1.3 mg monophosphoinositide, and 0.1 to 0.2 mg each of phosphoinositide 1 and 2. All three inositol phosphatides incorporated P32 at a significantly higher rate than the other brain phosphatides. Of the three, monophosphoinositide always had the lowest specific activity; after 16 and 32 hours, phosphoinositide 2, which is probably identical with the triphosphoinositide of Ballou, had the highest specific activity.

Yeast lipids have been isolated from whole cells by solvent extraction procedures. The components of the lipid extract have been fractionated by gradient elution chromatography on silicic acid columns, using chloroform-methanol mixtures as eluents. Combined chemical, ultraviolet absorption, paper chromatographic and thin-layer chromatographic techniques have been used to characterize the lipid samples. The results indicate that the neutral lipid fractions contained coenzyme Q, in addition to sterol esters, glycerides, free fatty acids and sterol. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and carbohydrate-containing lipids were present in the phospholipid fractions.

1. 1. The preparation of the phospholipide fractions found to be active as in vivo anticoagulants (1) is described. 2. 2. The active material is probably an unsaturated phosphatidylserine. The most important evidence of this was obtained by a paper Chromatographic study of the composition of the active fractions and by deamination with pyridoxal producing inactivation. Of the contaminating substances, inositol phosphatide and acetal phosphatide were shown to be inactive. Only phosphatidylserine and lysophosphatidylserine remain. 3. 3. The activity of the anticoagulant is destroyed or decreased by deamination, acylation, hydrolysis, bromination, diazomethanolysis, reduction with lithium aluminum hydride, reduction of unsaturation by catalytic hydrogenation, and epoxide formation.

PS concentrations (5,6j has been reported. It was ta~lor,'~~~p,"~a ~~~~~~~;entially similar results suggested that PS molecules containing unsatu- this rated fatty acid chains have antithromboplastic he p~ preparations were stored at - 14 *e: in tightly activity (7, 8). closed cslatair&s under nitrogen, unless stated othe~kise. This paper reports our study of synthetic and SaturatedPS's beef brain PS preparationse We confirmed the These were prepared as reported in an earlier publics- correlation between the degree of unsaturation of (''). the PS-fatty acid residues and the antithrombo- ~~~,":f.~-~~~~yl -, ,tdioleoyl-, and ,tdilinolenoyl- plastic We found that beef brain PS PS were synthesized essentially according to the scheme oxidizes very readily on storage and that there of de Haas et as. (13) following the route B. may be a relationship between the degree of oxi- dation and the antithromboplastic activity of PS. Wydrogena~ion of the PS Preparations On the basis of these results, a chemical treatment of the beef brain PS was developed to yield prep- arations with high and constant antihrombo- plastic activity.

Utilizing paper, thin layer and gas chromatography, the phospholipids of dormant and germinating spores have been isolated and identified. The identifications were based upon agreements of retardation factor values between the unknowns and reference compounds and their derivatives. Quantitative analysis of components, color reactions and specific labeling experiments were also used to support certain identifications. At least three, and usually more, criteria were used for each phospholipid that was definitively identified.The major phospholipids of Uromyces phaseoli are phosphatidylcholine and phosphatidylethanolamine. Diphosphatidylglycerol, or cardiolipin, phosphatidylinositol, and another phosphoinositide were present as minor components. In germinating spores, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylserine, and phosphatidic acid were detected in trace amounts. The presence of these common intermediates in the biosynthesis of phospholipids indicates that phospholipid synthesis is active during the germination process. Except for the absence of phosphatidylglycerol, the types of phospholipids present are similar to the host plant. Germ tube wall preparations were found to contain phosphatidylcholine and phosphatidylethanolamine in about the same proportion as that observed in resting spores, while the proportion of diphosphatidylglycerol was about three times higher. An unidentified phosphorous-containing lipid was also a significant component of the total phospholipids extracted from germ tube walls.

We have re-examined the question of phospholipid activation of latent (Na+ + K+)-ATPase activity in delipidized preparations. The influence of both polar head-groups and fatty acyl chain fluidity have been examined. The results indicate the following: 1. 1. The enzyme can be reactivated not only by phosphatidylserine, but also equally well by phosphatidylglycerol. A large variety of other purified phospholipids were found to have little or no effect. 2. 2. This specificity for (Na+ + K+)-ATPase reactivation correlates with the observation that only phosphatidylserine and phosphatidylglycerol vesicles show substantial discrimination for K+ over Na+ in terms of permeability. 3. 3. Maximal reactivation is obtained only when the fatty acyl chains are fluid. Activation by dipalmitoyl phosphatidylglycerol is inhibited below its transition temperature, or in the presence of cholesterol. Arrhenius plots for the activation by dipalmitoyl phosphatidylglycerol show a break for (Na+ + K+-ATPase activity corresponding to the presumed transition temperature for the gel to liquid-crystalline transition for this phospholipid. We suggest the term “viscotropic” to describe such effects of membrane fluidity on enzyme activity. 4. 4. The ATPase preparations used in this paper were obtained by deoxycholate treatment of microsomal fractions of rabbit kidney and beef brain. This treatment results in the removal of more than 90% of the phospholipid and inhibition of (Na+ + K+-ATPase activity. It was noted that exclusive specificity for reactivation by phosphatidylserine and phosphatidylglycerol was obtained only with those preparations where the inhibition of (Na+ + K+)-ATPase activity was essentially complete. 5. 5. These results are discussed in relation to previous work on the phospholipid requirement for (Na+ + K+)-ATPase activity, the ability of phospholipids to exhibit K+Na+ discrimination and current concepts of protein-lipid interactions in biological membranes.