Ethanolamine Phosphatides Research Articles

Studies on the Lipids of Japanese Littleneck, Tapes japonica Deshayes. II.

The composition of the acetone-insoluble lipids obtained from Japanese Littleneck, Tapes japonica (Deshayes) was studied. The acetone-insoluble lipids were mostly composed of phospholipids. Procedures used for the analysis of the constituents of phospholipids were as follows; silicic acid column chromatography of intact lipids, and paper chromatography of the water-soluble hydrolysis products of phospholipids according to Dawson, were found to be unsatisfactory. Thin-layer chromatography was not able to separate plasmalogens from analogous diacylphospholipids. Two-dimentional thin-layer chromatography according to Owens, which based on the specific hydrolysis of plasmalogens to lysophospholipids in the presence of mercuric chloride spray reagent was effective for the analysis of plasmalogens. By means of this method, phospholipids were separated into individual phospholipid, and lipid phosphorus was determined. The results of the analysis were as follows; phospholipids were rich in ethanolamine phospho-lipids, and these were 23.1% phosphatidyl ethanolamine, 9.2% phosphatidal ethanolamine (ethanolamine plasmalogen), and 17.8% sphingo-ethanolamine. Other phospholipids determined were 19.8% phosphatidyl choline, 10.8% phosphatidyl serine, and 6.0% phosphatidyl inositol.

Analysis of Individual Molecular Species of Phospholipids. V. Separation of Dinitrophenylated and Methylated Ethanolamine Phosphatides of Hens' Eggs.

Analysis of Individual Molecular Species of Phospholipids. V. Separation of Dinitrophenylated and Methylated Ethanolamine Phosphatides of Hens' Eggs.

Methylation of Ethanolamine Phosphatides by Microsomes from Normal and Mutant Strains of Neurospora crassa

Abstract The phospholipid N-methyltransferase system in microsomes of normal Neurospora crassa was compared with those in two choline-deficient mutant strains. The normal transferase system catalyzed the three transmethylations leading to the synthesis of lecithin, and the three phosphatide bases involved in this sequence of reactions were isolated as isotope-labeled products. The microsomes of a mutant, strain 34486, proved to have a subnormal phosphatidylethanolamine N-methyltransferase activity. The microsomes of a second mutant, strain 47904, could not catalyze the twostep conversion of phosphatidylmonomethylethanolamine to lecithin at a measurable rate and therefore were deficient in the phosphatidylmonomethylethanolamine N-methyltransferase found in the normal strain. Evidence was presented to corroborate the view based on previous studies that a single enzyme in N. crassa converts phosphatidylmonomethylethanolamine to lecithin.

Subacute Sclerosing Leukoencephalitis*†‡

Chemical studies were done on cerebral white matter, gray matter, and myelin of a case of subacute sclerosing leukoencephalitis. Myelin was obtained in one-third the normal yield and was found to have an abnormal lipid composition but a normal lipid-protein ratio, normal solubility properties in CHCl3-CH3OH and normal morphology. The myelin cholesterol was increased by 50 per cent with a concomitant decrease of cerebrosides, sulfatides and ethanolamine phosphatides. The white matter showed a pattern typical of severe demyelination, whereas the gray matter was relatively normal. Total solids of white matter were 50 per cent of normal; the cholesterol ester was 27 per cent of total lipid with large decreases in proteolipid protein, cerebrosides and ethanolamine phosphatides and smaller decreases in other lipids. During the myelin isolation a light, abnormal fraction was found which was 57 per cent cholesterol ester and presumably represents the sudanophilic material. A ganglioside abnormality was found only in the white matter. The total amount of gangliosides were twice normal on a dry weight basis and were found to have greatly increased amounts of the usually minor gangliosides, G2A, G3A, G5 and G6. These 4 gangliosides accounted for 57 per cent of the total lipid neuraminic acid.

An ethanolamine plasmalogen artifact formed by acetone extraction of freeze‐dried tissue

Extraction of freeze-dried tissues by acetone results in the in vitro production of an acetone derivative (imine) of the ethanolamine phosphatides. Some of the properties of the acetone imine of ethanolamine plasmalogen are discussed.

The turnover of the lipid components of myelin

AbstractThe rate of loss of radioactivity in the lipid components of rat myelin labeled with acetate‐1‐C14 was determined over a period of one year. Rats were injected with acetate‐1‐C14 at 15舑16 days of age and purified myelin was prepared by differential ultracentrifugation from brain and spinal cord of this group at 1 day, 2 weeks, 1 month, 2 months, 3 months, 6 months and 1 year after injection. Total lipid was extracted from the myelin preparations and the lipids were separated into their components by thin‐layer chromatography. Cholesterol, galactolipid, ethanolamine phosphatide, choline phosphatide, inositol phosphatide, serine phosphatide and sphingomyelin specific activities at each age were measured. Three of the myelin lipid components, serine phosphatide, inositol phosphatide, and choline phosphatide decreased in specific activity faster than cerebroside, cholesterol, sphingomyelin, and ethanolamine phosphatide. Acetate‐1‐C14 injected into adult animals, though incorporated into myelin to a very small extent, is taken up primarly in the choline phosphatide. These experiments suggests that myelin does not behave as a fixed entity but that certain constituents may be more actively metabolized than others.

The phospholipid pattern of Tenebrio molitor larvae

The phospholipids of Tenebrio molitor larvae have been examined by chromatography on columns of DEAE-cellulose and silicic acid-Hyflo Super Cel. The major constituents consist of choline and ethanolamine phospholipids. Inositol phosphatides represented a relatively small portion of the total phospholipids. Phosphatidyl serine and lysolecithin compounds were not detected. A choline sphingolipid was demonstrated to be present, and only ethanolamine phosphatides were detected in the plasmalogen form. The overall phospholipid pattern was qualitatively similar to other insects.

Lipid composition of the normal human brain: gray matter, white matter, and myelin

Gray matter, white matter, and myelin were isolated from the frontal lobes of humans aged 10 months, 6 yr, 9 yr, and 55 yr and the lipid compositions of each were determined. Myelin had a much higher lipid content (78–81% of the dry weight) than white matter (49–66%) or gray matter (36–40%). Myelin contained much higher molar percentages of cerebroside and cerebroside sulfate, slightly higher molar percentages of cholesterol, and lower molar percentages of ethanolamine glycerophosphatides and choline glycerophosphatides than gray matter. The molar percentages of serine glycerophosphatides and sphingomyelin were about the same in each tissue. The aldehyde content of glycerophosphatides, expressed as molar percentage of the total lipoidal residues in each lipid, were as follows: ethanolamine glycerophosphatides from myelin 40–50%; ethanolamine glycerophosphatides from gray matter 21–27%; serine glycerophosphatides from myelin 21–36%; serine glycerophosphatides from gray matter 0.3–3.8%. Choline glycerophosphatides from either tissue contained only traces of aldehydes. The extra-myelin portion of white matter had a lipid composition that was very similar to that of myelin, but quite different from that of gray matter. Assuming a molecular weight of 28,000 for myelin protein(s), it was calculated that for each protein molecule in human myelin there are 186 lipid molecules, 111 of which are polar lipids and 75 of which consist of cholesterol. The over-all molar ratios of the polar lipids are phosphatidal ethanolamine:serine glycerophosphatides:choline glycerophosphatides:sphingomyelin:cerebroside:cerebroside sulfate:ceramide:uncharacterized lipids 25:9:20:9:29:7:3:9. It was calculated that the molar ratio of protein amino acids to polar lipids in human myelin is 2.38 to 1.

Biochemical changes in denervated skeletal muscle: III. The effect of denervation atrophy on the concentration and 32P labelling of the individual phospholipids of the rat gastrocnemius

1. 1. The phosphatidyl compounds containing inositol, serine, ethanolamine and choline, the phosphatidal (plasmalogen) compounds containing serine, ethanolamine and choline, a lipid containing “polyglycerophosphate”, the protein-bound phosphoinositides and an alkali-acid stable fraction have been isolated and separated in both normal and denervated rat-gastrocnemius muscles and their labelling patterns from 32P i determined. 2. 2. The denervated muscle incurred losses on a whole muscle basis in all the phospholipids studied, with the exception of phosphatidal choline. The losses, however, ranged from very marked for protein-bound phosphoinositides and “polyglycerophosphate” to slight for phosphatidal ethanolamine and phosphatidal serine. 3. 3. The normal rat gastrocnemius actively incorporated 32P i into each of the phospholipids. For all fractions except phosphatidyl inositol and the protein-bound phosphoinositides, there is a progressive rise in incorporation with time. Phosphatidyl inositol showed rapid initial labelling to a plateau corresponding to approx. 20% of the labelling of muscle-inorganic phosphate. The protein-bound phosphoinositides also showed rapid initial labelling from muscle-inorganic phosphate which eventually reached a plateau at 12 h. The patterns of labelling of both of these fractions is consistent with the presence of more than one pool with at least one pool being labelled rapidly from inorganic phosphate. 4. 4. Fifteen days after denervation, a number of changes are observed in the incorporation of 32P into the individual phospholipids with time. By 96 h there was an increase in the specific activity of each of the phospholipids relative to the specific activity of muscle-inorganic phosphate. At 12 h these increases were not evident. 5. 5. After denervation the incorporation of label expressed as a corrected relative uptake ( i.e. a measure of 32P i incorporated into whole denervated muscle relative to 32P i incorporated into whole normal muscle corrected for differences in the specific activities of inorganic phosphate in normal and denervated muscle) was decreased in all phospholipids with the exception of the phosphatidal compounds. 6. 6. Comparison of the ratio of specific activities of each phospholipid fraction with that for muscle-inorganic phosphate showed that incorporation of 32P i was increased into phosphatidal ethanolamine and the “polyglycerophosphate” containing lipid and was decreased into phosphatidal choline and protein-bound phosphoinositide. When restricted to a unit weight of muscle these results have been interpreted in terms of changes either in the enzyme activity concerned with incorporation of 32P i or in the size of the product pool.

Effect of Simple and Complex Carbohydrates upon Total Lipids, Nonphospholipids, and Different Fractions of Phospholipids of Serum in Young Men and Women

Eight young, healthy persons, 4 men and 4 women, were fed experimental diets for 4 dietary periods of 4 weeks each. Except for the type of carbohydrate and total protein in the second period, diets were constant in composition. Total fats, proteins, and carbohydrates constituted 40, 16 and 44% of total calories of the basic diet, respectively. The ratio of complex to simple carbohydrates was 1:4 in periods 1 and 3 and this was reversed in periods 2 and 4. Serum total lipids, nonphospholipids, and the different fractions of phospholipids (ethanolamine phosphatides, inositides, lecithins, lysolecithins, and sphingomyelins) were determined using silicic acid column chromatography. Serum total lipids, phospholipids, and nonphospholipids were found to be significantly reduced with the high cereal diet and increased with the high sugar diet when the total calories and fats were held constant for both men and women. The percentage of the ethanolamine fraction to the total phospholipids increased with the high sugar diet and decreased with the high cereal diet. The proportion of lecithins to total phospholipids showed the opposite trend. The other phospholipids showed minimal changes with dietary change. The possible importance of the changes in the phosphatides in relation to the coagulation of the blood was pointed out. An hypothesis was suggested for the mechanism for the lipid-lowering effect of complex carbohydrates in contrast with the lipemic effect of high sucrose diet.

INDIVIDUAL MOLECULAR SPECIES OF DIFFERENT PHOSPHOLIPID CLASSES. II. A METHOD OF ANALYSIS.

AbstractNew analytical possibilities arise when glycerosphophatides are converted into diglyceride acetates or analogous compounds: In this less polar form the phospholipids can be subjected to the usual methods of triglyceride fractionation, including chromatography on silica gel mixed with silver nitrate. This opens a route to subfractionation of various glycerophosphatide classes, and makes analysis of the individual molecular species potentially possible in many cases. The same approach can also be applied to the analysis of sphingomyelins.Two methods are suitable for the conversion of glycerophosphatides into 舠diglyceride acetates舡: 1) Acetolysis in a mixture of acetic anhydride and acetic acid, and 2) treatment with phospholipase C (E.C. 3.1.4.3.) followed by acetylation. Acetolysis was used successfully with phosphatidyl choline, phosphatidyl ethanolamine and corresponding alkoxy phosphatide (native cephalin B), phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid and cardiolipin. Phospholipase C fromClostridium welchii was used for native choline and ethanolamine plasmalogens as well as for sphingomyelins. Although this enzyme does not attack pure ethanolamine phosphatides it did so in the presence of added (dipalmitoyl) phosphatidyl choline or serum sphingomyelin. The mixed substrates containing sphingomyelin proved particularly valuable, since the plasmalogenic diglyceride acetates were easily separated from the usual diglyceride acetates and ceramide diacetates on silicic acid.The diglyceride acetates obtained from lecithins of hens舗 eggs, ox‐brain and human serum were subjected to preparative TLC on Kieselgel G mixed with silver nitrate. The resulting subfractions were characterized by GLC analysis of the fatty acid methyl esters. When these data were combined with enzymic analysis of the acids occupying the alpha and beta positions of the molecules a rather detailed description of the lecithins became possible.The use of diglyceride acetates provides a route to analysis of fatty acids in many phosphatidyl compounds which have been obtained previously only as mixtures with the alkoxy analogs.

Studies on the methylation of ethanolamine phosphatides by liver preparations

The phosphatidylmethyltransferase enzyme system of rat liver microsomes has been dispersed by treatment with deoxycholate and sonic oscillation. With the dispersed preparation, complete removal of endogenous phospholipid substrates could be achieved. The latter showed an absolute dependency on added N-monomethyl aminoethanol or N,N-dimethylaminoethanol phosphatides for methylation to lecithin. Kinetic study of the methylation reaction indicated that a single enzyme is involved which catalyzes the stepwise methylation of N-methylaminoethanol phosphatide to lecithin. Tests were made of the suggestion that phosphatidyl serine might be methylated prior to decarboxylation and, in this manner, might yield the first observed substrate, the monomethylethanolamine phosphatide. It was demonstrated that this reaction does not occur. The only decarboxylation product detected was ethanolamine phosphatide.

The reaction of diazomethane with synthetic phosphatides and with insect lipid extracts

1. 1. The reaction of diazomethane with synthetic phosphatidylethanolamine, N-methylphosphatidylethanolamine, phosphatidylcholine and insect-lipid extracts was studied in order to find out if the reaction was specific to one type of phosphatide. 2. 2. The three synthetic phosphatides reacted in essentially the same manner, although with different rates of reaction, forming dimethyl esters of phosphatidic acids and a nitrogenous compound similar to neurine. The results with insect extracts gave evidence of the reaction of diazomethane with many natural phosphatides. 3. 3. N-methylation of the intact ethanolamine phosphatides occurred to a limited extent and was increased by the presnece of alcohols in the reaction medium. 4. 4. Techniques used included column chromatography on silicic acid, paper chromatography and infrared spectroscopy.

Improved determination of glycerol and fatty acids in glycerides and ethanolamine phosphatides by gas–liquid chromatography

The fatty acid and glycerol content of glycerides were estimated by a simplified hydrogenolysis–acetylation procedure and GLC. When fatty acids interfered in the analysis, glycerol alone was estimated by saponification–acetylation and GLC. Preliminary acetolysis was necessary for the estimation of glycerol in ethanolamine phosphatides by these methods.

INFLUENCE OF TEMPERATURE ON FATTY ACID COMPOSITION OF PSYCHROPHILIC AND MESOPHILIC SERRATIA SPECIES.

Growth of Serratia marcescens at 10° resulted in a decrease in the amounts of cyclopropane and palmitic acids, relative to those at 30°, and corresponding increases in monoenoic (hexadecenoic and octadecenoic) acids. A Serratia-like psychrophile grown at 5° or 10° had large proportions of monoenoic acids (chiefly hexadecenoic acid but not octadecenoic acid) but no cyclopropane acids. When grown in media containing L-methionine or S-adenosyl methionine (known cofactors for biosynthesis of the cyclopropane ring), the psychrophile still did not produce any cyclopropane acids. The phosphatide composition of the psychrophile was similar to that of S. marcescens with respect to the presence of phosphatidyl ethanolamine, phosphatidyl glycerol, and other acidic phosphatides, but the lipo-amino acids found in S. marcescens were not detected in the psychrophile.The present data support the classification of the psychrophile as a species of Serratia, but suggest that it has lost the ability to synthesize cyclopropane acids, perhaps as a result of adaptation to growth at low temperatures.

Post-heparin phospholipase

Egg phosphatidyl ethanolamine is degraded to lysophosphatidyl ethanolamine and fatty acids by a phospholipase in post-heparin plasma at rates of 10 to 30 μmoles/hr per ml plasma. The optimal system for measuring this enzyme contained albumin and (NH4)2SO4, and the optimal pH was 8.8–9.1. Only slight degradation of egg or beef lecithin occurred. Post-heparin and pancreatic phospholipase differ in heat stability; in the inhibiting effects of EDTA, HgCl2, and diethyl-p-nitrophenyl phosphate; in the requirement for deoxycholate or albumin; and in an apparent specificity of the post-heparin enzyme for the ethanolamine phosphatide.

EFFECTS OF HORMONES ON NA AND H2O TRANSPORT AND ON PHOSPHOLIPID METABOLISM IN TOAD BLADDER.

Incubation of tied paired lobes of bladder from Bufo marinus with 0.025–0.25 U/ml of oxytocin led to appreciable stimulations of both sodium transport and of water transport from the mucosal (luminal) to serosal side. Incubation with aldosterone led to a very slight stimulation of sodium transport, which was statistically significant, in one out of three series. P32 from orthophosphate was incorporated into phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidyl inositol. There were no statistically significant stimulations in the incorporation of P32 into phosphatidic acid, phosphatidyl choline, or phosphatidyl inositol at any concentration of oxytocin. However, there was a statistically significant stimulation of P32 incorporation into phosphatidyl ethanolamine with 0.025 U/ml oxytocin. There was a statistically significant inhibition in incorporation in phosphatidyl choline and phosphatidyl inositol with 0.25 U/ml oxytocin. Cholinergic agents did not produce statistically significant stimulations of either water or sodium transport in toad or turtle bladders, but they led to significant stimulations of incorporation of P32 into some of the phosphatides (phosphatidic acid, phosphatidyl choline, and phosphatidyl inositol in the toad bladder, and phosphatidic acid and phosphatidyl inositol in turtle bladder). On the basis of the fine structure of the epithelium of the toad bladder it is suggested that the phospholipid effect in response to cholinergic agents may be related to stimulation of the secretion of an organic material by the epithelial and/or goblet cells.

Effect of Fasting on the Incorporation in Vitro of Palmitate-C14 into Glycerolipids of Mitochondria

The effect of a 24-hour fast and a re-feeding of glucose, corn oil, or egg albumin by a stomach tube following the fast upon the incorporation of palmitate-1-C14 into various lipids of rat liver mitochondria was determined. Both normal animals and animals with regenerating livers were used. Fasting depressed the incorporation of palmitate-1-C14 into both triglycerides and total phospholipids. The latter was reflected in an almost complete cessation of the incorporation of C14 into ethanolamine phosphatide, and little change in that of the inositol and choline phosphatide. When the fasted rats were fed glucose, the incorporation of C14 into triglycerides and ethanolamine and choline phosphatides increased. Re-feeding corn oil depressed the incorporation of palmitate-1-C14 into the lipids and re-feeding egg albumin increased C14 uptake by the phospholidips but not by the triglycerides. With mitochondria from regenerating livers, the incorporation of palmitate-1-C14 into the choline phosphatides was markedly increased, and that in the other lipids decreased when compared with the values found employing mitochondria from normal livers. Fasting the partially hepatectomized animals resulted in lower incorporation of C14 into choline phosphatides and triglycerides. Glucose, corn oil, and egg albumin re-feedings generally had the same effects on regenerating liver mitochondria as on normal ones.

The incorporation in vitro of [ 14C]palmitate into glycerolipids by various cell components

1. 1. The incorporation in vitro of [ 14C]palmitate into triglycerides, ethanolamine phosphatides, and choline phosphatides has been studied. Whole homogenates, mitochondria, and microsomes plus cytoplasmic supernatant from normal and 3-day “regenerating” rat livers were employed as enzyme. 2. 2. The relative uptakes of 14C in the various lipids differed, depending on the part of the cell used as enzyme, and whether or not regenerating liver or normal liver was employed. 3. 3. The data suggest a multiplicity of pathways of incorporation of fatty acid into the glycerolipids, and indicate changes in lipid metabolism in response to altered tissue physiology.

The incorporation in vitro of [ 14C]palmitate into glycerolipids by various cell components

1. 1. The incorporation in vitro of [ 14C]palmitate into triglycerides, ethanolamine phosphatides, and choline phosphatides has been studied. Whole homogenates, mitochondria, and microsomes plus cytoplasmic supernatant from normal and 3-day “regenerating” rat livers were employed as enzyme. 2. 2. The relative uptakes of 14C in the various lipids differed, depending on the part of the cell used as enzyme, and whether or not regenerating liver or normal liver was employed. 3. 3. The data suggest a multiplicity of pathways of incorporation of fatty acid into the glycerolipids, and indicate changes in lipid metabolism in response to altered tissue physiology.