Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

The ether lipid-deficient mouse: tracking down plasmalogen functions

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed.     

Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: disparate utilization of sodium halides in the production of alpha-halo fatty aldehydes

Plasmalogens are a phospholipid molecular subclass that are enriched in the plasma membrane of many mammalian cells. The present study demonstrates that reactive brominating species produced by myeloperoxidase, as well as activated neutrophils, attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens, resulting in the production of a neutral lipid and lysophosphatidylcholine. Gas chromatography-mass spectrometry and proton NMR analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). In comparison to myeloperoxidase-generated reactive chlorinating species, reactive brominating species attacked the plasmalogen vinyl ether bond at neutral pH. In the presence of a 20-fold molar excess of NaCl compared with NaBr, myeloperoxidase-derived reactive halogenating species favored the production of 2-BrHDA over that of 2-chlorohexadecanal. Additionally, 2-BrHDA was preferentially produced from plasmalogen treated with hypochlorous acid in the presence of NaBr. The potential physiological significance of this pathway was suggested by the demonstration that both 2-BrHDA and 2-bromooctadecanal were produced by PMA-stimulated neutrophils. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by myeloperoxidase and by activated neutrophils, resulting in the production of novel brominated fatty aldehydes.     

Biosynthesis of phospholipids in Clostridium butyricum: kinetics of synthesis of plasmalogens and the glycerol acetal of ethanolamine plasmalogen

The biosynthesis of the plasmalogen forms of phosphatidylethanolamine (plasmenylethanolamine) and phosphatidylglycerol (plasmenylglycerol) and of the glycerol acetal of plasmenylethanolamine has been studied in cultures of Clostridium butyricum IFO 3852. When growing cells were pulsed with [32P]orthophosphate, there was a lag of 5 to 7 min between the rapid incorporation of label into the acylphosphatides and the rapid incorporation of label into the corresponding plasmalogens. The labeling of the glycerol acetal of plasmenylethanolamine was even slower. In pulse-chase experiments with 32Pi, the kinetics of labeling indicated precursor-product relationships between phosphatidylethanolamine and plasmenylethanolamine and between the latter and its glycerol acetal. A precursor-product relationship was also seen between phosphatidylglycerol and cardiolipin, but the kinetics of labeling of the alkenyl-containing forms of these lipids were not consistent with direct precursor-product relationships with the acyl lipids. In the presence of hydroxylamine and 32Pi, both phosphatidylserine and plasmenylserine accumulated 32P in a ratio of ca. 15:1. Upon release of the inhibition of phosphatidylserine decarboxylase, label appeared in the following sequence: phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine. Acyl phosphatidylglycerol was identified as a major phospholipid (17% of lipid phosphorus) in C. butyricum grown in low-phosphate (1.13 mM) medium with 50 mM Tris buffer. Of the acyl phosphatidylglycerol, 13% was acid labile. There appear to be two plasmalogen forms of acyl phosphatidylglycerol. One of these has a single alkenyl ether group, and the other has alkenyl ether groups on both glycerols.     

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant,alpha-bromo fatty aldehyde

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.     

Myeloperoxidase-derived reactive chlorinating species from human monocytes target plasmalogens in low density lipoprotein

A role for myeloperoxidase (MPO) in atherosclerosis has received considerable attention recently. To identify potential chlorinated lipid products in human low density lipoprotein (LDL), studies were designed to demonstrate that MPO-derived reactive chlorinating species (RCS) target the plasmalogen pool of LDL isolated from peripheral human blood in vitro. The vinyl ether bond of LDL plasmalogens was targeted by MPO-derived RCS, resulting in the release of the 16- and 18-carbon-containing alpha-chloro fatty aldehydes, 2-chlorohexadecanal and 2-chlorooctadecanal, respectively, from the plasmalogen glycerol backbone. Targeting of the LDL plasmalogen vinyl ether bond was dependent on the presence of MPO-derived RCS. Electrospray ionization mass spectrometric analysis of MPO-treated LDL demonstrated that a novel population of unsaturated lysophosphatidylcholine molecular species was produced by a phospholipase A2-independent mechanism. Unsaturated lysophosphatidylcholine molecular species elicited cyclic AMP response element binding protein phosphorylation in RAW 264.7 cells. Additionally, MPO-mediated targeting of both monocyte and LDL plasmalogen pools was demonstrated in phorbol myristate acetate-stimulated human monocytes, resulting in the production of both 2-chlorohexadecanal and 2-chlorooctadecanal. In contrast, alpha-chloro fatty aldehydes were not produced in phorbol myristate acetate-stimulated mouse monocytes. Collectively, the present studies demonstrate a novel MPO-specific mechanism that mediates the production of a novel group of unsaturated lysophosphatidylcholine molecular species and chlorinated aldehydes from both LDL and monocyte plasmalogen pools that may have important effects during inflammatory reactions mediated by monocytes, most notably atherosclerosis.     

Phosphatidyltransferase of Clostridium butyricum: specificity for diacylphosphoglycerides

Phosphatidyltransferase from Clostridium butyricum, which catalyzes transfer of the phosphatidyl moiety of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) or phosphatidylserine to primary alcohols such as glycerol, serine and ethanolamine, was tested for its ability to catalyze transfer of the plasmenyl moiety from plasmalogen analogs of PE or PG to glycerol or ethanolamine, respectively. The cell membrane of C. butyricum contains high proportions of these plasmalogens. When diacyl and plasmalogen forms of PE or PG were supplied as donors in equimolar amounts to membrane particles, the diacyl forms were the preferred donors by approx. 7 and 10 to 1, respectively. When the molar ratio of PE its plasmalogen was 1:3.3, the ratio of PG formed to its plasmalogen was 1:0.66. These results show that the enzyme(s) can catalyze transfer of both the diacyl and alkenyl acyl forms of glycerophospholipids, but the diacyl forms are used preferentially.     

Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: identification of 2-chlorohexadecanal

Plasmalogens contain a vinyl ether bond linking the sn-1 aliphatic chain to the glycerol backbone of this predominant phospholipid molecular subclass, which is found in many mammalian tissues. The present study demonstrates that the vinyl ether bond of plasmalogens is a molecular target of the reactive chlorinating species produced by myeloperoxidase. Analysis by thin layer chromatography revealed that reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of the plasmalogen, lysoplasmenylcholine (1-O-hexadec-1'-enyl-sn-glycero-3-phosphorylcholine), resulting in the production of a neutral lipid. Capillary gas chromatographic analyses demonstrated that the neutral lipid generated from lysoplasmenylcholine was neither hexadecanal nor did it contain masked hexadecanal (i.e. the vinyl ether) because the dimethyl acetal of hexadecanal produced by acid methanolysis derivatization was no longer present. Electrospray ionization mass spectrometry of the myeloperoxidase-generated neutral lipid product was consistent with the production of a 16-carbon fatty aldehyde containing one chlorine atom. Furthermore, proton NMR analysis indicated that this neutral lipid product was a 2-chloro-fatty aldehyde. Additional structural analysis of this neutral lipid by gas chromatography-mass spectrometry of the underivatized product as well as its pentafluorobenzyl oxime-derivative product was consistent with the neutral lipid being 2-chlorohexadecanal. The reactive chlorinating species, hypochlorous acid and chlorine gas, both attacked the vinyl ether bond of lysoplasmenylcholine resulting in the production of 2-chlorohexadecanal. The production of 2-chlorohexadecanal was dependent on the presence of the plasmalogen masked aldehyde (i.e. the vinyl ether) in the substrate because the free fatty aldehyde, hexadecanal, was not converted to 2-chlorohexadecanal by the reactive chlorinating species generated by myeloperoxidase. Taken together, the present studies demonstrate for the first time the targeting of the vinyl ether bond of plasmalogens by the reactive chlorinating species produced by myeloperoxidase resulting in the production of novel chlorinated fatty aldehydes.     

Lipid composition of human bone marrow

1. A modified method for the analysis of phospholipid mixtures by selective hydrolysis is described. 2. The phospholipid compositions of normal human bone marrow and of the bone marrows of patients who died with anaemia or various forms of leukaemia were investigated. 3. Phospholipids from normal bone marrow comprised about 44% of lecithin, 4% of choline plasmalogen, 7% of glyceryl ether phospholipid (choline base), 10% of sphingomyelin, 22% of phosphatidylethanolamine plus phosphatidylserine, 8% of ethanolamine plasmalogen and 5% of glyceryl ether phospholipid (ethanolamine base). 4. The proportion of kephalin (i.e. phosphatidylethanolamine plus phosphatidylserine) in the pathological bone marrows tended to be lower than normal. No other consistent differences were observed between the normal and pathological samples. 4. A ceramide dihexoside was isolated from normal bone marrow.     

Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation

Plasmenyl phospholipids (1-alk-1′-enyl-2-acyl-3-glycerophospholipids, plasmalogens) are a structurally unique class of lipids that contain an α-unsaturated ether substituent at the sn-1 position of the glycerol backbone. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanisms responsible for the antioxidant properties of plasmenyl phospholipids are not fully understood, the oxidation of plasmalogens in natural mixtures of phospholipids was studied using electrospray tandem mass spectrometry. Glycerophosphoethanolamine (GPE) lipids from bovine brain were found to contain six major molecular species (16:0p/18:1-, 18:1p/18:1-, 18:0p/20:4-, 16:0p/20:4, 18:0a/20:4-, and 18:0a/22:6-GPE). Oxidation of GPE yielded lyso phospholipid products derived from plasmalogen species containing only monounsaturated sn-2 substituents and diacyl-GPE with oxidized polyunsaturated fatty acyl substituents at sn-2. The only plasmalogen species remaining intact following oxidation contained monounsaturated fatty acyl groups esterified at sn-2. The mechanism responsible for the rapid and specific destruction of plasmalogen GPE may likely involve unique reactivity imparted by a polyunsaturated fatty acyl group esterified at sn-2. This structural feature may play a central role determining the antioxidant properties ascribed to this class of phospholipids.     

The modulation of choline phosphoglyceride metabolism in human colon cancer

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

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.     

The cerebro-hepato-renal (Zellweger) syndrome. Impaired de novo biosynthesis of plasmalogens in cultured skin fibroblasts

In tissues of patients with the cerebro-hepato-renal (Zellweger) syndrome the plasmalogen content is very low. In order to study the biosynthesis of plasmalogens, skin fibroblasts of Zellweger patients, controls and heterozygotes, and amniotic fluid cells of controls were cultured in a medium supplemented with [1-14 C]hexadecanol or 1-O-[9,10-3H2]octadecylglycerol. The incorporation of 14C-label into the alkenyl moiety of plasmalogens was strongly reduced in Zellweger patients as compared to controls. The low concentration of 14C-labeled plasmalogens was not compensated for by an elevated levels of 14C-labeled alkyl phospholipids. Hexadecanol was partly oxidized to fatty acid in all cell lines and the incorporation of 14C-labeled fatty acid into phospholipids was comparable for patients and controls. [3H]Alkylglycerol was incorporated into plasmalogens with the same efficiency in Zellweger patients as in controls. These results indicate that only the reaction(s) involved in the introduction of the ether bond in the process of plasmalogen synthesis are deficient in Zellweger patients. The results also suggest that the hexadecanol incorporation patterns can be used for the (prenatal) diagnosis of the Zellweger syndrome.     

A phosphoethanolamine-modified glycosyl diradylglycerol in the polar lipids of Clostridium tetani

The polar lipids of the anaerobic bacterium Clostridium tetani, the causative agent of tetanus, have been examined by two-dimensional thin layer chromatography, ESI mass spectrometry, and NMR spectroscopy. Plasmalogen and di- and tetra-acylated species of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and N-acetylglucosaminyl diradylglycerol were the major lipids present in most strains examined except for strain ATCC 10779, the parent of strain E88, the first C. tetani strain to have its genome sequenced. This strain contained the same di- and tetra-acylated species but did not contain plasmalogens. All strains contained a novel derivative of N-acetylglucosaminyl diradylglycerol in which a phosphoethanolamine unit is attached to the 6’-position of the sugar, as judged by selective ³¹P-decoupled, ¹H-detected NMR difference spectroscopy. The N-acetylglucosamine (GlcNAc) residue is presumably linked to the 3-positon of the diradylglycerol moiety, and it has the β-anomeric configuration. Very little plasmalogen component was detected by mass spectrometry in the precursors phosphatidic acid and phosphatidylserine, consistent with the idea that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid assembly in anaerobic clostridia.     

Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals

The absence of peroxisomes in patients with the cerebrohepatorenal syndrome of Zellweger leads to several biochemical abnormalities, including deficient synthesis of plasmalogens as well as accumulation of very long-chain fatty acids and intermediates in bile acid biosynthesis. Accumulation of very long-chain fatty acids in serum and fibroblasts has hitherto been used most extensively for diagnosis. Due to the relatively small amounts of the very long-chain fatty acids also in the Zellweger patients, and the risk for interfering impurities, such analyses are difficult. Direct assay of plasmalogens is also relatively difficult and time-consuming. In this report, we describe a relatively simple method for diagnosis, based on gas-liquid chromatography of a lipid extract of erythrocytes after methyl transesterification. The alpha, beta-unsaturated ether in the plasmalogen is converted to the dimethylacetal of the corresponding aldehyde, and the relative amount of plasmalogen is thus reflected by the ratio between 18:0 dimethylacetal and methyl stearate as well as by the ratio between 16:0 dimethylacetal and methyl palmitate. The ratio 18:0 dimethylacetal/methyl stearate was found to be 0.28 +/- 0.03 (mean +/- SD) after analyses of erythrocytes from healthy or non-Zellweger infants, but less than 0.02 in erythrocytes from three infants with the Zellweger syndrome. Preliminary work with amniotic fluid suggests that this analysis may be suitable also for prenatal diagnosis of the Zellweger syndrome.     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.     

Uptake of fluorescent plasmalogen analogs by cultured human skin fibroblasts deficient in plasmalogen

One of the consequences of hereditary peroxisomal dysfunction in the cerebro-hepato-renal (Zellweger) syndrome (CHRS) is a dramatic decrease in the biosynthesis and cellular content of ether lipids. In the present study effects of reduced cellular plasmalogen levels on membrane-membrane interactions were investigated. Cultured CHRS fibroblasts were incubated with unilamellar phospholipid vesicles consisting of 1-O-alkenyl-2-acyl- or 1,2-diacyl-sn-glycerophosphocholines and ethanolamines, carrying either the trans-parinaroyl or the 1,6-diphenyl-1,3,5-hexatriene propionyl group in position 2. Transfer of the fluorogenic phospholipids from vesicles to cells was followed by measuring the concomitant increase in fluorescence intensity. Transfer of phospholipids from cells to vesicles was monitored by incubating cells, prelabeled with [3H]oleic acid, in the presence of phospholipid vesicles. Fibroblasts from healthy donors or CHRS fibroblasts supplemented with the plasmalogen precursor 1-O-hexadecylglycerol served as controls. Plasmalogen-deficient cells exhibited a significantly increased tendency to take up exogenous choline or ethanolamine plasmalogens. Cellular plasmalogens were transferred from control cells to vesicles at a higher rate if the acceptor vesicles consisted of plasmalogens as compared to diacylglycerophosphocholine. Thus, it appears as if mechanisms existed which preserve cellular plasmalogen levels during interaction with exogenous phospholipid pools. Preliminary experimental evidence suggests that the observed exchange of phospholipids between cultured fibroblasts and vesicles occurs by a protein-catalyzed process.     

Increasing plasmalogen levels protects human endothelial cells during hypoxia

Supplementation of cultured human pulmonary arterial endothelial cells (PAEC) with sn-1-O-hexadecylglycerol (HG) resulted in an approximately twofold increase in cellular levels of plasmalogens, a subclass of phospholipids known to have antioxidant properties; this was due, primarily, to a fourfold increase in the choline plasmalogens. Exposure of unsupplemented human PAEC to hypoxia (PO(2) = 20-25 mmHg) caused an increase in cellular reactive oxygen species (ROS) over a period of 5 days with a coincident decrease in viability. In contrast, HG-supplemented cells survived for at least 2 wk under these conditions with no evidence of increased ROS. Hypoxia resulted in a selective increase in the turnover of the plasmalogen plasmenylethanolamine. Human PAEC with elevated plasmalogen levels were also more resistant to H(2)O(2), hyperoxia, and the superoxide generator plumbagin. This protection was seemingly specific to cellular stresses in which significant ROS were generated because the sensitivity to lethal heat shock or glucose deprivation was not altered in HG-treated human PAEC. HG, by itself, was not sufficient for protection; HG supplementation of bovine PAEC had no effect upon plasmalogen levels and did not rescue these cells from the cytotoxic effects of hypoxia. This is the initial demonstration that plasmalogen content can be substantially enhanced in a normal cell. These data also demonstrate that HG can protect cells during hypoxia and other ROS-mediated stress, likely due to the resulting increase in these antioxidant phospholipids.     

Quantification of long-chain aldehydes by gas chromatography coupled to mass spectrometry as a tool for simultaneous measurement of plasmalogens and their aldehydic breakdown products

The cleavage of the specific vinyl ether linkage at the sn-1 position of plasmalogens leads to the formation of two products: the 1-lyso-2-acyl glycerophospholipid and a long-chain fatty aldehyde. Plasmalogens are measured by quantifying one of these two products. In this paper, we describe a rapid and sensitive procedure for measuring plasmalogens via quantification of long-chain fatty aldehydes. After lipid extraction, the sn-1 vinyl ether bond of plasmalogens is cleaved by acidic hydrolysis. The produced aldehydes are then derivatized with (pentafluorobenzyl)hydroxylamine hydrochloride and analyzed by gas chromatography/mass spectrometry in selected-ion mode. Plasmalogens are then indirectly quantified by subtracting the free aldehydes obtained without prior HCl treatment from the total aldehydes obtained after acidic hydrolysis. This method is applied to three rat brain areas selected for this study. Two of these are affected in neurodegenerative diseases (cerebral cortex and hippocampus) and one is rich in white matter (cerebellum). In comparison to other procedures, the advantages of this method are not only its usefulness in plasmalogen quantification but also the identification of aldehydic breakdown products.