Semisynthetic preparation of choline and ethanolamine plasmalogens

Choline and ethanolamine plasmalogens containing defined acyl chains are prepared by deacylation and reacylation of beef heart plasmalogens. During the reactions, the amino group of ethanolamine plasmalogens is protected by the trityl group. Deacylation is achieved by mild alkaline hydrolysis, and the lysoplasmalogens are reacylated with oleoylimidazolide in the presence of the methylsulfinylmethide anion. The protective group is removed from N-trityl ethanolamine plasmalogen by treatment with silicic acid in hexane. The choline and ethanolamine plasmalogens prepared by the procedures described are free of geometric, positional and steric isomers.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemipheres in culture. II. Incorporation of [U-14C]ethanolamine into 1-alkenyl,2-acyl-and 1,2 diacyl-ethanolamine phosphoglycerides.

Cultured dissociated cells from rat embryo cerebral hemisphere incorporate [3H]-and [U-14C]ethanolamine into cellular lipids. Nearly all radioactivity in the lipid fractions is incorporated into 1,2-diacylethanolamine phosphoglycerides and 1-alkenyl,2-acylethanolamine phosphoglycerides (plasmalogen). Kinetic data suggest that the rate of labeling of both ethanolamine phospholipids from the phosphorylethanolamine is similar. A relative increase of the plasmalogen labeling is observed when free ethanolamine is continually present in the medium. The rate of incorporation of label from ethanolamine and phosphorylethanolamine into lipids was measured using a double label technique. Based upon these studies, an independent labeling pattern of the ethanolamine moiety of plasmalogens is suggested. A relative delay for the incorporation of label in plasmalogens could be explained by the presence of a variety of cell types which may differ in their capacity for phospholipid biosynthesis. The rate of incorporation of phosphorylethanolamine into the phosphatidylethanolamine was not affected by the presence of high concentrations of either choline or serine.     

Composition and fatty acid content of rat ventral prostate phospholipids

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphorus. The prostate also contained small amounts of serine glycerophospholipids and sphingomyelin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and stearic aldehydes. Ethanolamine plasmalogens also contained the vinyl ether of oleic aldehyde.     

High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization

Canine myocardial sarcolemma was purified, and its phospholipid constituents were determined by gas chromatography-mass spectrometry, fast atom bombardment mass spectrometry, and conventional techniques. Canine myocardial sarcolemma contained 2.7 mumol of lipid Pi/mg of protein which was comprised predominantly of choline glycerophospholipids (47%), ethanolamine glycerophospholipids (28%), and sphingomyelin (11%). Sarcolemmal phospholipids contained 40% plasmalogen which was quantitatively accounted for by choline (57% of choline glycerophospholipid) and ethanolamine (64% of ethanolamine glycerophospholipid) plasmalogens. Choline plasmalogens contained predominantly the vinyl ether of palmitic aldehyde though ethanolamine plasmalogens were composed predominantly of the vinyl ethers of stearic and oleic aldehydes. The majority of sarcolemmal ethanolamine glycerophospholipids (75%) contained arachidonic acid esterified to the sn-2 carbon. Sphingomyelin was composed predominantly of long-chain saturated fatty acids (stearic and arachidic) as well as substantial amounts (8%) of odd chain length saturated fatty acids. The possible functional role of these unusual phospholipid constituents is discussed.     

Polyunsaturated fatty acid metabolism in neuroblastoma cells in culture.

Neuroblastoma cell cultures took up linoleic and linolenic acids at approximately equal rates, and incorporated them into a variety of lipid fractions, principally cellular phospholipids. Linoleic acid was preferentially incorporated into choline phosphoglycerides, while most of the radioactivity derived from linolenic acid entered ethanolamine phosphoglycerides. There was no evidence for direct transfer of fatty acids between these two phosphoglyceride fractions. When, after the addition of cytosine arabinoside, cell division was arrested, the entry of labelled fatty acids into ethanolamine and serine phosphoglycerides was reduced, suggesting that these lipids are involved in the formation of new cell membranes. In the ethanolamine phosphoglyceride fraction, phosphatidal ethanolamine (plasmalogen) was the principal acceptor for the higher polyunsaturated fatty acids of the φ 3 series. The ratio of labelled fatty acids entering ethanolamine plasmalogens to that entering ethanolamine phosphoglycerides increased following the addition of cytosine arabinoside, suggesting plasmalogens to be involved in formation of cell processes. The first step in the metabolism of both linoleic and linolenic acid was the addition of a two-carbon unit. Conversion of linoleic acid to higher polyunsaturated fatty acids was slower than the conversion of linolenic acid to its higher analogues. This contrasted with the behaviour of dissociated cultures of normal brain cells which were able to form higher analogues of linoleic and linolenic acids at nearly equal rates.     

Synthesis and content of ether-linked glycerophospholipids in the harderian gland of rabbits.

Although harderian glands are rich in neutral glycerolipids with ether bonds, less than 20% of the choline glycerophospholipids have ether bonds in the white and pink portions of the adult rabbit harderian gland. Only 6% of these are plasmalogens while 94% are alkylacyl glycerophosphocholines. The ethanolamine glycerophospholipids include 37% with ether bonds in both white and pink portions. In the white portion 96% are plasmalogens but only 19% are plasmalogens in the pink portion. The microsomal ethanolaminephosphotransferase (EC 2.7.8.1) is more active with diacylglycerols than with alkylacylglycerols. The microsomal cholinephosphotransferase (EC 2.7.8.2) is equally active with both diradylglycerols. Particularly with microsomes from the pink portion, the apparent Km values for CDPethanolamine and CDPcholine are ower in the presence of alkylacylglycerols than in the presence of diacylglycerols. The incorporation of radioactivity from CDP[14C]ethanolamine and CDP[14C]choline into ethanolamine and choline plasmalogens was increased several-fold by addition of alkylacylglycerols but was not increased substantially by addition of diacylglycerols.     

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.     

Kinetic properties of plasmalogenase from bovine brain.

Abstract— Plasmalogenase was assayed by measuring the disappearance of the plasmalogen by two-dimensional thin-layer chromatography. The enzyme was present in a glycerol-bicarbonate extract of an acetone-dried powder from bovine brain. With ethanolamine plasmalogens as the substrate, the K_m was 180 μM. Diacyl glycerophosphorylcholines, diacyl glycerophosphorylethanolamines and choline plasmalogens were competitive inhibitors. With choline plasmalogens as the substrate, the K_m was 208 μM and competitive inhibition was observed with diacyl glycerophosphorylcholines and ethanolamine plasmalogens. The same enzyme may be responsible for the hydrolysis of the alk-1-enyl moiety from both plasmalogens. Plasmalogenase activity was 5.1 μmol/h/g of dog brain, 3.9 μmol/h/g of rat brain and 3.4 μmol/h/g of gerbil brain. A lysophospholipase was also found in the glycerol-bicarbonate extract from the acetone-dried powder. The lysophospholipase was more active in hydrolysing acyl groups from 2-acyl-sn-glycero-3-phosphorylethanolamines than the plasmalogenase was active in hydrolyzing alk-1-enyl groups from 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.     

Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion (125I3-)

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.     

Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport

Plasmalogens are a major sub-class of ethanolamine and choline phospholipids in which the sn-1 position has a long chain fatty alcohol attached through a vinyl ether bond. These phospholipids are proposed to play a role in membrane fusion-mediated events. In this study, we investigated the role of the ethanolamine plasmalogen plasmenylethanolamine (PlsEtn) in intracellular cholesterol transport in Chinese hamster ovary cell mutants NRel-4 and NZel-1, which have single gene defects in PlsEtn biosynthesis. We found that PlsEtn was essential for specific cholesterol transport pathways, those from the cell surface or endocytic compartments to acyl-CoA/cholesterol acyltransferase in the endoplasmic reticulum. The movement of cholesterol from the endoplasmic reticulum or endocytic compartments to the cell surface was normal in PlsEtn-deficient cells. Also, vesicle trafficking was normal in PlsEtn-deficient cells, as measured by fluid phase endocytosis and exocytosis, as was the movement of newly-synthesized proteins to the cell surface. The mutant cholesterol transport phenotype was due to the lack of PlsEtn, since it was corrected when NRel-4 cells were transfected with a cDNA encoding the missing enzyme or supplied with a metabolic intermediate that enters the PlsEtn biosynthetic pathway downstream of the defect. Future work must determine the precise role that plasmalogens have on cholesterol transport to the endoplasmic reticulum.     

Synthesis of methylated ethanolamine moieties: regulation by choline in lemna

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[(3)H(3)C]methionine, l-[(14)CH(3)]methionine, or [1,2-(14)C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

The conversion of ethanolamine and of its metabolites to choline in human neuroblastoma clones: Effect of differentiation induced by retinoic acid

The sequential methylation of ethanolamine (Etn) or phosphorylethanolamine to the corresponding choline (Cho) derivatives was studied in both undifferentiated and retinoic acid (RA) differentiated human neuroblastoma clones LA-N-1 and LA-N-2. Conversion of Etn derivatives to the respective Cho metabolites was low in both cell types. However, after treatment of the cultures with ethanol or RA, the methylation of phosphoryl-Etn was stimulated while that of phosphatidyl-Etn was severely reduced in both cholinergic LA-N-2 and catecholaminergic LA-N-1 cells.Abbreviations Etn ethanolamine - MeEtn monomethylethanolamine - Me₂Etn dimethylethanolamine - Cho choline - PEtn phosphorylethanolamine - PtdEtn phosphatidylethanolamine - LPtdEtn lysophosphatidylethanolamine - RA retinoic acid     

In vivo studies on pathways for the biosynthesis of lecithin in the rat

The in vivo biosynthesis of lecithin in rats has been studied with the precursors choline-1,2-(14)C, ethanolamine-1,2-(14)C and methionine-CH(3)-(14)C or -CH(3)-(3)H. Lecithin synthesis from choline is rapid in all organs. No sex difference was observed in this pathway. The biosynthesis of lecithin by methylation of phosphatidyl ethanolamine is of quantitative significance in the liver, but not in extrahepatic tissues. More lecithin is synthesized by this pathway in female rats. In liver the lecithin synthesized via both pathways enters a common pool which is in rapid equilibrium with lecithin of blood plasma. A sex difference in the utilization of radioactive ethanolamine for the formation of phosphatidyl ethanolamine was observed (greater utilization in the female). Incorporation of ethanolamine into phospholipids of extrahepatic tissues was slow in both sexes. With labeled methionine as precursor the liver cytidine diphosphate (CDP) choline had a specific activity identical with that of liver lecithin after 20 min, while the specific activity of phosphoryl choline remained low. With labeled choline as precursor the phosphoryl choline reached a specific activity 50 times that of lecithin after 20 min, while the specific activity of CDP choline was only four times that of lecithin. These findings indicate that the reaction: CDP choline + diglyceride right harpoon over left harpoon phosphatidyl choline + CMP is freely reversible in vivo.     

Reactivity of plasmalogens: kinetics of acid-catalyzed hydrolysis

The acid-catalyzed hydrolysis of the alpha,-unsaturated ether group of two plasmalogens, lysophosphatidal choline and lysophosphatidal ethanolamine, and several model compounds (isobutyl vinyl ether, 1-butenyl ethyl ether, and dihydropyran) was studied by determining the true second-order rate constants. The results indicate that the chemical reactivity of the substituted vinyl ether group in plasmalogens is not appreciably affected by the presence of a bulky substituent on the -carbon. Activation energies, enthalpies, and entropies were also determined (from measurements of the rate constants at different temperatures).     

Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium

The role of nuclear membrane phospholipids as targets of phospholipases resulting in the generation of nuclear signaling messengers has received attention. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from perfused hearts. Rat heart nuclei contained choline glycerophospholipids composed of palmitoyl and stearoyl residues at the sn-1 position with oleoyl, linoleoyl, and arachidonoyl residues at the sn-2 position. Diacyl molecular species were the predominant molecular subclass in the choline glycerophospholipids, with the balance of the molecular species being plasmalogens. In the ethanolamine glycerophospholipid pool from rat heart nuclei approximately 50% of the molecular species were plasmalogens, which were enriched with arachidonic acid at the sn-2 position. A 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids was observed in hearts rendered globally ischemic for 15 min followed by 90 min of reperfusion in comparisons with the content of these phospholipids in control perfused hearts. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium was partially reversed by the calcium-independent phospholipase A(2) (iPLA(2)) inhibitor bromoenol lactone (BEL), suggesting that the loss of nuclear phospholipids during ischemia/reperfusion is mediated, in part, by iPLA(2). Western blot analyses of isolated nuclei from ischemic hearts demonstrated that iPLA(2) is translocated to the nucleus after myocardial ischemia. Taken toghether, these studies have demonstrated that nuclear phospholipid mass decreases after myocardial ischemia by a mechanism that involves, at least in part, phospholipolysis mediated by iPLA2.     

Conversion of ethanolamine, monomethylethanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain.

The incubation of neurons from chick embryos in primary culture with [3H]ethanolamine revealed the conversion of this base into monomethyl, dimethyl and choline derivatives, including the corresponding free bases. Labelling with [methyl-3H]monomethylethanolamine and [methyl-3H]dimethylethanolamine supported the conclusion that in chick neuron cultures, phosphoethanolamine appears to be the preferential substrate for methylation, rather than ethanolamine or phosphatidylethanolamine. The methylation of the latter two compounds, in particular that of phosphatidylethanolamine, was seemingly stopped at the level of their monomethyl derivatives. Fetal rat neurons in primary culture incubated with [3H]ethanolamine showed similar results to those observed with chick neurones. However, phosphoethanolamine and phosphatidylethanolamine and, to a lesser extent, free ethanolamine, appeared to be possible substrates for methylation reactions. The methylation of water-soluble ethanolamine compounds de novo was further confirmed by experiments performed in vivo by intraventricular injection of [3H]ethanolamine. Phosphocholine and the monomethyl and dimethyl derivatives of ethanolamine were detected in the brain 15 min after injection.     

Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-L-alanine amidase of Pneumococcus.

Pneumococci growing on choline-containing medium are known to incorporate this amino alcohol into the wall teichoic acid and produce autolysin-sensitive cell walls. In contrast, bacteria grown on the choline analogue, ethanolamine, incorporate ethanolamine into the teichoic acid and synthesize cell walls that are resistant to the homologous autolysin. In this communication, we report experiments aimed at understanding the biochemical mechanism of this phenomenon. Ethanolamine-containing (autolysin-resistant) cell walls were methylated in vitro with methyl iodide. Under appropriate conditions, virtually all of the ethanolamine residues could be converted to choline. After methylation, the formerly autolysin-resistant walls could be quantitatively hydrolyzed by the pneumococcal autolysin. Methylated walls also recovered another property typical of cell walls isolated from choline-grown bacteria: they could induce the in vitro "conversion" of an inactive form of autolysin to the catalytically active form (Tomasz, A., and Westphal, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 2627-2630). The results suggest that the autolysin-catalyzed hydrolysis of amide bonds in the peptidoglycan requires an additional interaction between the enzyme protein and choline residues in the teichoric acid portion of the cell wall.     

The effect of isoprenaline on the phospholipid content of the compact and spongious musculature of the carp ventricular myocardium

1. After a single injection of 40 mg kg-1 of isoprenaline to the carp, lysophospholipids appear in the tissue of the heart ventricle, ethanolamine plasmalogens increase and choline plasmalogens decrease; phosphatidylinositol is lowered in the spongious layer only. 2. Daily administration of 5 mg kg-1 of the drug leads, after 5 doses, to a dramatic decrease of the diphosphatidylglycerol content; during the subsequent 5 and 10 doses a return to normal values occurs. Shifts in plasmalogens are similar to those found after a single high dose. Some other phospholipids change significantly. 3. All changes reveal that the spongious musculature is more sensitive to the drug than the compact one.     

Biosynthesis of major phospholipids inCandida albicans

Biosynthesis of major phospholipids was examined by identifying enzymes and in vitro uptake of specific labeled precursors through various pathways inCandida albicans. The presence of PS synthetase, choline kinase, and ethanolamine kinase was demonstrated in this organism. Phosphatidylcholine was found to be synthesized mainly through cytidine diphosphate-choline (CDP)-choline and methylation pathways. The presence of a methylation pathway was further confirmed by blocking methyltransferases with 2-hydroxyethyl hydrazine. Phosphatidylethanolamine was synthesized by all three, CDP-ethanolamine, Phosphatidylserine (PS)-decarboxylase, and base exchange pathways, while PS was formed by PS-synthetase and base exchange mechanisms.     

The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography

Myelin phospholipids have been examined by a separation-reaction-separation procedure for two-dimensional thin-layer chromatography on silica gel. After separation in one dimension, alk-1-enyl groups are cleaved by exposure of the plates to HCl fumes. Development in the second dimension quantitatively separates acid-labile and acid-stable phosphoglycerides as well as the aldehydes released from the acid-labile phosphoglycerides. Myelin phospholipids from the central nervous systems of the rhesus monkey, squirrel monkey, ox, and mouse contain 32-36% acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and 8-14% acid-stable ethanolamine phosphoglycerides. Acid-labile choline and serine phosphoglycerides account for less than 1% of the myelin phospholipids.