Ethanolamine plasmalogens protect cholesterol-rich liposomal membranes from oxidation caused by free radicals

The aim of the present study is to investigate the effect of ethanolamine plasmalogens on the oxidative stability of cholesterol-rich membranes by comparing it with that of diacyl glycerophosphoethanolamine, using bovine brain ethanolamine plasmalogen (BBEP) or egg yolk phosphatidylethanolamine (EYPE)-containing large unilamellar vesicles (LUVs) and the water-soluble radical initiator AAPH. Electron microscopic observation and particle size measurement visually demonstrated that ethanolamine plasmalogens protect cholesterol-rich phospholipid bilayers from oxidative collapse. Lipid analyses suggested that the effect of ethanolamine plasmalogens in stabilizing membranes against oxidation is partly due to the antioxidative action of plasmalogens involved in scavenging radicals at vinyl ether linkage.     

Choline plasmalogen synthesis by the methylation pathway in chick neurons in culture

Choline plasmalogens represent a minor component of lipid membranes in most tissues. In spite of this, their rapid turnover indicates a possible functional role in the cell. The present study demonstrates that these compounds can be synthesized in neuronal cell cultures from chick embryo hemispheres by methylation of ethanolamine plasmalogens since choline plasmalogens were labeled after incubation of cells with tritiated ethanolamine or methionine. This finding could be of a particular interest since it has been suggested that choline plasmalogens, synthesized by methylation, might be involved in receptor activation.     

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.     

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.     

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).     

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.     

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.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes

Abstract— Three groups of six mice each were killed 1, 4 and 7 days after an intracerebral injection of [1,2-¹⁴C]ethanolamine. The specific radioactivities of the acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and of the acid-stable ethanolamine phosphoglycerides (diacyl and alkyl acyl glycerophosphoryletholamines) from myelin and microsomal fractions were determined. All of these brain ethanolamine phosphoglycerides turn over rapidly with an apparent half-life of less than 3 days. The biosynthesis of alkenyl acyl glycerophosphorylethanolamines from diacyl glycerophosphorylethanolamines in mouse brain myelin or microsomes is unlikely.     

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.     

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.     

Essential fatty acids and serine as plasmalogen precursors in relation to competing metabolic pathways.

Interest in altered ether-lipid metabolism, associated with peroxisomal disorders including adrenoleukodystrophy and Zellweger's syndrome, has highlighted present limitations in our understanding of the biosynthesis and turnover of plasmalogens. These 1-alkenyl ethanolamine phosphoglycerides are major phospholipids in brain, vascular tissue, neutrophils, and most tumors, and they constitute 15-20% of total phospholipids in cultured glioma cell. In glioma, turnover of polyunsaturated acyl chains in the sn-2 position of plasmalogens was examined in relation to selectivity for the (n - 3) and (n - 6) families. Remodeling of acyl chains was more dependent on chain length than on selectivity between families, consistent with plasmalogens enriched in polyunsaturated, but not specifically (n - 3), fatty acids. Extracellular serine was a precursor of serine and ethanolamine phosphoglycerides and was associated with plasmalogens due to decarboxylation and headgroup exchange. Incorporation of extracellular serine ceased within 8 h, even though more than 50% of the label remain in the medium. Analyses of medium and cellular water-soluble components indicated rapid conversion of serine to glycine and other metabolites not used in phospholipid biosynthesis. Thus, nutrient molecules as precursors of plasmalogens are involved in complex competitive interactions. As functions of plasmalogens are clarified, regulation of plasmalogen turnover becomes an increasingly important issue and elucidation of these processes is essential.     

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.     

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.     

Lipid composition and sphingolipid fatty acids of myelin in fruit bat brain

Myelin was isolated from the brain of adult fruit bats (Rousettus aegyptiacus) in a discontinuous sucrose gradient. Cholesterol comprised 189.0 mol/100 mol lipid phosphorus, galactolipids 60.3 mol/100 mol phosphorus and plasmalogens 32.5 mol/100 mol phosphorus. Choline and ethanolamine glycerophosphatide were present in nearly equal amounts followed by serine glycerophosphatide, sphingomyelin and inositol glycerophosphatides.The fatty acid composition of sphingomyelin and non-hydroxy cerebroside was determined by gas-liquid chromatography. Fatty acids were mainly saturated or mono-unsaturated with a small percentage of polyunsaturated fatty acids present.The lipid composition and sphingolipid fatty acid distribution in bat myelin was fairly similar to that of other species.     

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.     

Separation of intact plasmalogens and all other phospholipids by a single run of high-performance liquid chromatography

Plasmalogens are a unique subclass of glycerophospholipids characterized by the presence of a vinyl ether bond at the sn-1 position of the glycerol backbone, and they are found in high concentration in cellular membranes of many mammalian tissues. However, separation of plasmalogens as intact phospholipids has not been reported. This article describes a high-performance liquid chromatographic method that can separate intact ethanolamine plasmalogens (pl-PEs) and choline plasmalogens (pl-PCs) as well as all other phospholipid classes usually found in mammalian tissues by a single chromatographic run. The separation was obtained using an HPLC diol column and a gradient of a hexane/isopropanol/water system containing 1% acetic acid and 0.08% triethylamine. The HPLC method allowed a clear separation of plasmalogens from their diacyl analogues. The HPLC method, as applied to the study of peroxidation in human erythrocytes by a hydroperoxide, demonstrated that pl-PEs were targeted twice as much as their diacyl analogues.     

SYNTHESIS OF ETHANOLAMINE PHOSPHOGLYCERIDES BY MICROSOMES FROM THE BRAINS OF JIMPY AND QUAKING MICE

Abstract— —Brains of jimpy and quaking mice are known to be deficient in myelin and alkenylacyl-glycero-phosphorylethanolamines (alkenylacyl-GPE, ethanolamine plasmalogens). Ethanolamine plasmalogen synthetic activity appeared to be normal and ethanolamine phosphotransferase (EC 2.7.8.1) activities are higher in the brain microsomes from jimpy and quaking mice than in their littermate controls when the activities are assayed with alkylacylglycerols and CDP[¹⁴C]ethanolamine. When endogenous diradylglycerols were the substrate, the rate of synthesis of diacyl-GPE was normal but the rate of synthesis of the ether lipids, alkenylacyl-GPE and alkylacyl-GPE, was 33% and 8% below control levels for jimpy brain microsomes and quaking brain microsomes respectively. This difference is probably due to a normal content of diacylglycerols and a deficient content of alkylacylglycerols in the mutant brain microsomes. The apparent alkylacylglycerol deficiencies in the microsomes correspond with the ethanolamine plasmalogen deficiencies in the brains of these mutant mice.     

Temporal and Quantitative Expression of the Myelin-Associated Lipids,Ethanolamine Plasmalogen,Galactocerebroside, and Sulfatide,in the Differentiating CG-4 Glial Cell Line

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [³H]ethanolamine to label ethanolamine plasmalogens or with [³H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.