Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.     

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.     

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.     

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.     

Behaviour of plasmalogens during high-performance liquid chromatography on a silica column with a mobile phase containing phosphoric acid

Silica high-performance liquid chromatographic separation of phospho- and sphingolipids of biological origin using a mobile phase containing phosphoric acid leads to gradual hydrolysis of plasmalogens during their passage through the column. The resulting 2-acyl lyso analogues give rise to peaks that tail in the direction of the parent intact plasmalogen. Tailing can be prevented by previous complete acid hydrolysis of plasmalogens. Direct high-performance liquid chromatographic profiling of phospholipids, their plasmalogens (as 2-acyl lyso analogues) and sphingolipids is probably the method of choice for the diagnosis of patients with deficient plasmalogen biosynthesis caused by peroxisomal abnormalities.     

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.     

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.     

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.     

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.     

Effect of peroxisome proliferation on ether phospholipid biosynthesizing enzymes in rat liver

1. The effect of the peroxisome proliferators clofibrate and plasticizer on the activities of the first two enzymes involved in either phospholipid biosynthesis, i.e. dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone-phosphate synthase, were studied in rat liver homogenates and purified peroxisomes. 2. DHAP-AT in homogenates increased by 2 to 3-fold both in total and specific activity. However, the specific activity in purified peroxisomes showed no significant increase demonstrating for the first time that there is no specific induction of this enzyme that exceeds the induction of total peroxisomal protein. 3. Alkyldihydroxyacetone-phosphate synthase showed no significant increase in total and specific activity in homogenates and a slight decrease of its specific activity in purified peroxisomes was observed. 4. The total amount of plasmalogens did not increase upon proliferation and a slight decrease in the percentage plasmalogens in total phospholipids was observed. 5. Proliferation did not influence the phospholipid composition of the peroxisomal membrane.     

Total plasmalogens and O-(acylalkylglycerophosphoryl) ethanolamine from labelled hexadecanol and palmitate during hypoxia and anoxia in rat heart.

By the use of the Langendorff technique, surviving isolated rat hearts were perfused with [1-14 C] palmitate, [1-14C] hexadecanol or [1-14C,1-3H] hexadecanol under normal or anoxic conditions. After perfusion for 30min with either precursor, when oxygenated or in an hypoxic condition, or when 1mM-KCN was included in the system, the heart tissues showed no significant chemical changes in their content of total lipids, total phospholipids or total ethanolamine-containing phospholipids. Changes were observed in the ratio of alkyl-to alk-1-enyl-glycerophosphorylethanolamine in the tissue perfused with N2+CO1 plus CN-. A slight increase from 4.0+/-0.3 to 4.9+/-0.2% in alkyl derivatives and a decrease from 11.2+/-0.4 to 9.4+/-0.3% in alk-1-enyl derivatives was observed. The incorporation of the [14C] palmitate and the [14C] hexadecanol into the recovered phospholipids and plasmalogens was severely decreased in the tissues perfused with CN-: in the hypoxic state only a mild inhibition was observed compared with the oxygenated systems. Considerable 3H from [1-14C, 1-3H] hexadecanol was retained (25-35%) in the alk-1-enylether chains of plasmalogens under both the oxygenated conditions and with CN-, suggesting that the same mechanism of incorporation is operational at high or low O2 concentrations. The results are consistent with an O2-dependent, CN-sensitive step in the biosynthesis of plasmalogens in the rat heart.     

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.     

Clostridium difficile contains plasmalogen species of phospholipids and glycolipids

Analysis of the polar lipids of many pathogenic and non-pathogenic clostridia has revealed the presence of plasmalogens, alk-1′-enyl ether-containing phospholipids and glycolipids. An exception to this finding so far has been Clostridium difficile, an important human pathogen which is the cause of antibiotic-associated diarrhea and other more serious complications. We have examined the polar lipids of three strains of C. difficile by thin-layer chromatography and have found acid-labile polar lipids indicative of the presence of plasmalogens. The lipids from one of these strains were subjected to further analysis by liquid chromatography coupled to electrospray ionization-mass spectrometry (LC/ESI-MS), which revealed the presence of phosphatidylglycerol, cardiolipin, monohexosyldiradylglycerol, dihexosyldiradylglycerol, and two unusual glycolipids identified as an aminohexosyl-hexosyldiradylglycerol, and a trihexosyldiradylglycerol. High resolution tandem mass spectrometry determined that monohexosyldiradylglycerol, cardiolipin and phosphatidylglycerol contained significant amounts of plasmalogens. C. difficile thus joins the growing list of clostridia that have plasmalogens. Since plasmalogens in clostridia are formed by an anaerobic pathway distinct from those in animal cells, their formation represents a potential novel target for antibiotic action.     

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa

Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32.     

Enzymes in brain phospholipid docosahexaenoic acid accretion: a PL-ethora of potential PL-ayers

Neural tissue is highly enriched in docosahexaenoic acid (DHA) that is primarily found in the sn-2 position of ethanolamine-containing phospholipids and plasmalogens. Current knowledge on the activity of enzymes in brain phospholipid synthesis does not fully explain this composition and stereospecificity. It is likely that a host of enzyme-mediated processes play roles in brain DHA accumulation to develop this unique enrichment and phospholipid profile. This review examines current knowledge on the spectrum of enzymes that may be involved in brain DHA uptake and utilization in the synthesis and remodeling of phospholipids. It also highlights gaps in that knowledge, including missing information on the activity of known brain enzymes towards DHA as a substrate, and missing identities of brain enzymes that catalyze orphan reactions utilizing DHA for phospholipid formation.     

Compartmentalized regulation of lipid signaling in oxidative stress and inflammation: Plasmalogens,oxidized lipids and ferroptosis as new paradigms of bioactive lipid research

Perturbations in lipid homeostasis combined with conditions favoring oxidative stress constitute a hallmark of the inflammatory response. In this review we focus on the most recent results concerning lipid signaling in various oxidative stress-mediated responses and inflammation. These include phagocytosis and ferroptosis. The best characterized event, common to these responses, is the synthesis of oxygenated metabolites of arachidonic acid and other polyunsaturated fatty acids. Major developments in this area have highlighted the importance of compartmentalization of the enzymes and lipid substrates in shaping the appropriate response. In parallel, other relevant lipid metabolic pathways are also activated and, until recently, there has been a general lack of knowledge on the enzyme regulation and molecular mechanisms operating in these pathways. Specifically, data accumulated in recent years on the regulation and biological significance of plasmalogens and oxidized phospholipids have expanded our knowledge on the involvement of lipid metabolism in the progression of disease and the return to homeostasis. These recent major developments have helped to establish the concept of membrane phospholipids as cellular repositories for the compartmentalized production of bioactive lipids involved in cellular regulation. Importantly, an enzyme classically described as being involved in regulating the homeostatic turnover of phospholipids, namely the group VIA Ca²⁺-independent phospholipase A₂ (iPLA₂β), has taken center stage in oxidative stress and inflammation research owing to its key involvement in regulating metabolic and ferroptotic signals arising from membrane phospholipids. Understanding the role of iPLA₂β in ferroptosis and metabolism not only broadens our knowledge of disease but also opens possible new horizons for this enzyme as a target for therapeutic intervention.     

Regulation of intracellular lipid metabolism by the preparation of omega-3 phospholipids from marine organisms in deficiency of essential fatty acids in rats

It has been established that a deficit of essential fatty acids (EFA) in the animal organism induces specific modifications of composition of fatty acid (FA) of general phospholipids and plasmalogenic P1 in microsomal tissue membranes with various functions and affects the activity of phospholipase A2. It has been shown that arachidonic (AA), docosapentaenoic (DPA) and docosahexaenoic (DHA) acids in the composition of general phospholipids - phosphatidylcholine (PC), phosphatidylethanolamine (PEA) and plasmalogens PC and PEA react to EFA deficit in the organism. Quantitative redistribution of AA, DPA, DHA of FA in general phospholipids and plasmalogenic microsomal membranes depending on their functions was found under EFA deficit in the organism. Deficit of DHA and plasmalogenic phospholipids evidences that the status of cell plasmalogens affects the level of PUFA at EFA deficit in the organism. AA and DHA can be a selective target for plasmalogens. The drug of omega-3 phospholipids, considerable amount of DHA and eicosapentaenoic (EPA)FA being present in their structure, increases the amount of plasmalogens and decreases the amount of AA in the brain, heart and reproductive organs. It was also found that EFA deficit in the organism favours the increase of lisoPEA, lisoPC, free FA (FFA) connected with the increase of activity of endogenic phospholippase A2 and plasmalogen-selective phospholipase A2. The omega-3 phospholipase from marine organisms at EFA defecit decreases the amount of FFA, lisophospholipids and activity of phospholipase A2 in the microsomas of the studied tissues. The drug of omega-3 phospholipids normalizes the state and functions of the brain, liver, and heart tissues, reproductive organs against a background of EFA defecit and regulates the synthesis of biologocically active metabolites of AA in the organism.     

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.     

The protective role of plasmalogens in iron-induced lipid peroxidation

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.     

Comparative study of serine-plasmalogens in human retina and optic nerve: identification of atypical species with odd carbon chains

The objective of this work was to detect and identify phosphatidylserine plasmalogen species in human ocular neurons represented by the retina and the optic nerve. Plasmalogens (vinyl-ether bearing phospholipids) are commonly found in the forms of phosphatidylcholine and phosphatidylethanolamine in numerous mammalian cell types, including the retina. Although their biological functions are unclear, the alteration of cellular plasmalogen content has been associated with several human disorders such as rhizomelic chondrodysplasia punctata Type 2 and primary open-angle glaucoma. By using liquid chromatography coupled to high-resolution and tandem mass spectrometry, we have identified for the first time several species of phosphatidylserine plasmalogens, including atypical forms having moieties with odd numbers of carbons and unsaturation in sn-2 position. Structural elucidation of the potential phosphatidylserine ether linked species was pursued by performing MS(3) experiments, and three fragments are proposed as marker ions to deduce which fatty acid is linked as ether or ester on the glycerol backbone. Interpretation of the fragmentation patterns based on this scheme enabled the assignment of structures to the m/z values, thereby identifying the phosphatidylserine plasmalogens.