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.     

The Ca2+-dependent incorporation of nitrogenous bases into brain microsomal phospholipid subspecies in vitro.

The specificity of the Ca2+-stimulated choline and ethanolamine incorporation into the molecular subspecies of the correspondent choline and ethanolamine phosphoglycerides has been investigated in vitro in rat brain microsomes. In the presence of 5.0 mM Ca2+-ions and at pH 8.1, choline was incorporated 6 times faster into the tetraenoic diacyl-glycero-3-phosphorylcholines (diacyl-GPCs or lecithins) than into the saturated subspecies. The specific activities of the other species were intermediary, and decreased with increasing saturation. Hexaenoic species of lecithins were however weakly labelled. The rate of labelling of diacyl-GPC molecular subspecies was affected noticeably by changing the pH and the Ca2+-ion concentration of the incubation medium. Ethanolamine was incorporated in the presence of 2.5 mM Ca2+-ions and at pH 8.1 preferentially into the monoenoic species of total ethanolamine phosphoglycerides of rat brain microsomes. The rate of incorporation into the monoenoic species was twice that into the trienoic, tetraenoic and hexaenoic and 4 times faster that into the dienoic species. When the pattern of labelling was determined specifically for the molecular subspecies of diacyl-glycero-3-phosphorylethanolamines (diacyl-GPEs or phosphatidylethanolamines), the rate of incorporation of ethanolamine into the hexaenoic species resulted three times faster that into the saturated and monoenoic species and about twice that into the trienoic and tetraenoic species, in accordance with data for liver microsomes. The pattern of labelling of the molecular subspecies of ethanolamine phosphoglycerides and of diacyl-GPEs was not influenced by changing the pH and the Ca2+-ion concentration of the incubation medium.     

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.     

Phospholipids and acyl groups in subcellular fractions from human cerebral cortex

Subcellular fractionation of human brain cortex obtained at autopsy yielded microsomal and synaptosome-rich fractions from the gray matter and microsomal and purified myelin fractions from the white matter. The phospholipids of myelin were high in plasmalogens, and the molar ratio of alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 4. The acyl groups of the myelin phosphoglycerides were enriched in monoenes (mainly 18:1 and 20:1) and a tetraene, 22:4(n - 6). The phospholipids in the synaptosome-rich fraction were high in diacyl sn-glycero-3-phosphorylcholine, and the molar ratio of the alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 0.88. The acyl groups of synaptosomal ethanolamine phosphoglycerides were rich in 22:6(n - 3) but contained a very low amount of 20:1. The lipid composition of microsomes from the gray matter was different from that of microsomes from the white matter but was nearly identical with that of the synaptosome-rich fraction. Except for a slightly lower proportion of alkenyl acyl sn-glycero-3-phosphorylethanolamine and sphingomyelin, the lipid composition of microsomes from the white matter was also similar to that of the myelin. There were also species-related differences between the brain lipid composition of human and subhuman primates and that of the rodents. Furthermore, the brain lipid composition in normal human subjects is rather constant and does not seem to be affected much by individual variations.     

COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Myelin was isolated from the brains of mice at ages of 14, 24, 41, 44, 47, and 182 days and the contents of lipid phosphorus, cholesterol, lipid galactose, alkenyl groups, ethanolamine phosphoglycerides, choline phosphoglycerides, sphingomyelin, and serine and inositol phosphoglycerides were determined. Significant differences in the composition relative to total lipid phosphorus were found in the myelin. At 14 days of age, the myelin had lower relative amounts of cholesterol, galactolipids, alkenyl groups, and ethanolamine phosphoglycerides and a higher relative amount of choline phosphoglycerides.     

Membrane lipids of human peripheral nerve and spinal cord.

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.     

METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1-¹⁴C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half-lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half-life for turnover of synaptosomal lipids was 9 days.
The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl-GPC and diacyl-GPI, since radioactivity of other phosphoglycerides (diacyl-GPS, diacyl-GPE and alkenyl-acyl-GPE) continued to increase for 12-24 h. In this fraction, half-lives of 10-14 h were obtained for the fast turnover pools of diacyl-GPC and diacyl-GPI, and slow turnover pools with half-lives of 7 days for diacyl-GPI and 10-14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl-GPI declined in a biphasic mode, thus exhibiting half-lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl-GPE and diacyl-GPS in the synaptosomal fractions was observed for a period of 24 h. The half-lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl-GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.     

EFFECTS OF DIFFERENT DIETARY LEVELS OF ESSENTIAL FATTY ACIDS ON THE FATTY ACID COMPOSITION OF ETHANOLAMINE PHOSPHOGLYCERIDES IN MYELIN AND SYNAPTOSOMAL PLASMA MEMBRANES

Abstract— Three dietary levels of essential fatty acids (EFA), 3 0, 0 75 and 0 07 calorie-% were fed to rats for two generations or more. Myelin was isolated at the ages of 18, 30, 45 and 120 days and synaptosomal plasma membranes at 18, 30 and 45 days. No difference was found in the lipid composition between the dietary groups in either subcellular fraction. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) were analysed. In myelin the proportions of 18:1 and 20:1 increased with age, while those of 20:4 (n-6) and 22:6 (n-3) decreased, in synaptosomal plasma membranes the proportions of 20:4 (n-6) decreased with age, but 22:6 (n-3) increased and the sum of the polyunsaturated fatty acids was constant. At no age were significant differences found between the proportions of saturated and monounsaturated fatty acids, in either myelin or the synaptosomal plasma membrane fraction, when the different dietary groups were compared. In myelin from rats fed 007 calorie-% EFA the proportions of 20:4 (n-6) were slightly lower than in the two other groups, while those of 22 6 (n-3) were considerably lower. The synaptosomal plasma membranes fraction of rats fed O-07 calorie-% EFA had equal or slightly larger amounts of 20:4 (n-6) than in the two other groups, while 22:6 (n-3) was considerably smaller. In both subcellular fractions the decreased proportion of fatty acids of linoleic and linolenic acid series was compensated for by an increase in 20:3 (n-9) and 22:3 (n-9). The sum of these two fatty acids was equal in the EPG of myelin and synaptosomal plasma membranes at 18 days of age. At 30 and 45 days of age a lower value was found in the synaptosomal plasma membranes, while in the myelin fraction a slight decrease was found only at 120 days of age.     

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     

Fatty acid composition of phospholipids of myelin and synaptosomal proteolipid complexes from vertebrate brain.

1. Fatty acid composition of five main phospholipids of vertebrate brain myelin and synaptosomal proteolipids and membranes was studied. 2. Higher content of monoenoic and lower content of saturated and polyenoic fatty acids was found to be characteristic of phospholipids from myelin and myelin proteolipids as compared to phospholipids from synaptosomal proteolipids and membranes of vertebrates (from fishes to mammalians). Fatty acid composition of phospholipids of proteolipid complexes and of the membranes, from which they were isolated, were found to be similar in various species studied. 3. Microviscosity was found to be higher in myelin as compared to synaptosomal membranes of frog Rana temporaria and in rabbit Lepus cuniculus. It appears to be due to the difference in proteolipid content and in lipid composition of myelin and synaptosomal membranes.     

SYNAPTOSOMAL PLASMA MEMBRANES. ACYL GROUP COMPOSITION OF PHOSPHOGLYCERIDES and (Na++ K+)-ATPase ACTIVITY DURING FATTY ACID DEFICIENCY

Abstract— Essential fatty acid deficiency was induced in mice after feeding a fatty acid deficient diet for 6 months. Activity of the (Na⁺+ K⁺)-ATPase in the total brain homogenates and in isolated synaptosomal plasma membranes was significantly higher ( P & lt; 0 05) in the deficient mice than the controls. Analysis of the acyl group composition of phosphoglycerides in brain as well as in the synaptosomal plasma membranes showed that mice fed the deficient diet had increased levels of 20:3(n-9) and 22:3(n-9) and decreased levels of 20:4(n-6) and 22:4(n-6). However, acyl group changes varied among individual phosphoglycerides and were most obvious in the two species of ethanolamine phosphoglycerides. A decrease in 22:6(n-3) level was also observed in some phosphoglycerides of the synaptosomal plasma membranes especially the diacyl- sn -glycerophosphorylserine. In this experiment, a new solvent system for chromatographic separation of the diacyl- sn -glycerophosphorylserine and diacyl- sn -glycerophosphorylinositol was reported. The separation technique was suitable for analysis of acyl group composition of individual phosphoglycerides by gas-liquid chromatography. The results were consislent with a positive correlation of the non-polar acyl groups of brain membranes with the active ion transport activity. The increase in enzymic activity during deficient state may be the result of a biological adaptation due to structural alteration of the brain membranes.     

CHANGES IN LIPID CONCENTRATIONS AND FATTY ACID COMPOSITIONS IN RAT CEREBRUM DURING MATURATION

Abstract— Rat cerebrum was analysed at 20 different ages from birth to 45 days of age, for its concentration of protein, cholesterol, cerebrosides, phospholipids and gangliosides, and for the concentration of fatty acids of the linoleic and linolenic acid series. The fatty acid patterns of choline phosphoglycerides and ethanolamine phosphoglycerides were determined at the same ages. Phases of rapid accretion were found for protein, phospholipids, gangliosides and cholesterol. The accretion of the fatty acids of the linoleic acid series ceased at 20 days of age, while that of the fatty acids of the linolenic acid series continued. The fatty acid composition of the phosphoglycerides changed during the maturation of rat cerebrum and these changes consisted of chain elongation, increased unsaturation and variation in the pattern of the polyenoic acids. These changes varied irregularly with age and each developmental stage had characteristic fatty acid patterns of choline and ethanolamine phosphoglycerides.     

CHANGES IN ACYL GROUP COMPOSITION OF DIACYL-GLYCEROPHOSPHORYLETHANOLAMINE, ALKENYLACYL-GLYCEROPHOSPHORYLETHANOLAMINE AND DIACYL-GLYCEROPHOSPHORYLCHOLINE IN MYELIN AND MICROSOMAL FRACTIONS OF MOUSE BRAIN DURING DEVELOPMENT

—Age-related changes in acyl group composition of diacyl-glycerophosphorylethanolamine (GPE), alkenylacyl-GPE and diacyl-glycerophosphorylcholine (GPC) were examined in myelin and microsomal fractions of mouse brain during development. In general, the phosphoglycerides in the myelin fraction showed an increase in the proportions of 18:1 and 20:1 and a decrease in the proportions of 16:0, 20:4(n-6) and 22:6(n-3) with increasing age. These changes were especially obvious with the acyl groups of alkenylacyl-GPE. The acyl group profiles of phosphoglycerides in the microsomal fraction were different from those in the myelin fraction. During development, there was an increase in 22:6 and a decrease in 20:4 in the phosphoglycerides of microsomes. These changes were especially obvious with the diacyl-GPE. Starting from 2 weeks of age, there was also an increase in the proportions of 18:1 and 20:1 in alkenylacyl-GPE in the microsomal fraction but this change was not as dramatic as that in the myelin fraction. In general, the acyl groups of diacyl-GPC in both myelin and microsomal fractions showed only little age-related changes as compared to the ethanolamine phosphoglycerides. Results suggest an induction in the synthesis of monoenoic fatty acids in brain during development. The monoenoic fatty acids synthesized during this period are rapidly and preferentially incorporated into the ethanolamine plasmalogen for further utilization in synthesis of the myelin membranes.     

Adult rat brain synaptic vesicles II. Lipid composition

The lipid composition of synaptic vesicles isolated from adult rat brain was determined. Vesicles contained cholesterol and phospholipid but very little ganglioside, galactolipid, free fatty acid and triglyceride was detected. Ethanolamine and choline phosphoglycerides were the dominant phospholipids. Lysophosphatidyl choline was present in very low amounts. The fatty acid composition of the phosphoglycerides was characterized by high levels of docosahexaenoic acid in the ethanolamine and serine phosphoglycerides, and the absence of long chain fatty acids from the sphingomyelins. All the characteristic features of the lipid composition of the synaptosomal plasma membrane (with the exception of the ganglioside content) were seen in the synaptic vesicle lipids. The results are discussed in terms of the exocytosis mechanism of transmitter release.     

The turnover of phosphoglycerides in the subcellular fractions of mouse brain: a study using (1-14C)oleic acid as precursor

Abstract— The turnover of phosphoglycerides in subcellular fractions of adult mouse brain was examined after intracerebral injection of [1-¹⁴C]oleic acid. Radioactivity of the total brain homogenate decreased rapidly thereafter, with only 4 per cent of the radioactivity remaining at the end of 3 months. The rate of decrease of radioactivity in the subcellular fractions was in the order: cytosol, microsomes, synaptosomes and myelin. Increasing amounts of radioactivity were detected in the alkenyl groups and cerebrosides, but metabolic conversions were not as extensive as found previously with the palmitoyl group. The specific radioactivities for diacyl sn-glycero-3-phosphorylcholine and diacyl sn-glycero-3-phosphorylethanolamine were highest in the microsomal fraction and decreased with time. The apparent half-lives for the diacyl sn-glycero-3-phosphorylcholine and the diacyl sn-glycero-3-phosphorylethanolamine in the microsome and synaptosome-rich fractions were 1-3.5 days when estimated between 1 and 7 days after injection. The rate of decay for the brain membrane phosphoglycerides was not linear with time, probably because of the extensive amount of recycling occurring within the system. Radioactivity was incorporated into the phosphoglycerides of the myelin but equilibration of radioactivity between microsomes and myelin required 7–14 days.     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

Acyl and alkenyl group composition of brain subcellular fractions of goldfish (Carassius auratus L.) acclimated to different environmental temperatures

Goldfish were acclimated to 5, 15, and 30°C, and the acyl group composition of choline phosphoglycerides (CPG) and ethanolamine phosphoglycerides (EPG) from whole brain and brain subcellular particles was examined. With the exception of synaptosomal CPG, the acyl group composition of CPG from whole brain and subcellular particles, including myelin, from cold-acclimated fish showed little response to the change in environmental temperature. Those changes that did occur were consistent with the expected trend toward a higher degree of unsaturation of the CPG acyl groups in fish acclimated to 5°C. The acyl group composition of CPG from synaptosomes of the cold-acclimated fish did, however, differ markedly in having a reduced unsaturation index (U.I.) and unsaturated: saturated fatty acid ratio (UFA:SFA) which was caused mainly by the decrease in 226n-3 content. In contrast, changes in the acyl group composition of EPG on cold acclimation were greater than those observed in any CPG fraction. The generally expected trend toward greater unsaturation was observed only in mitochondrial and myelin EPG. Moreover, in all fractions the amount of 226n-3 in EPG was lower at decreased environmental temperatures. In the synaptosomal and microsomal EPG, the reduction in 226n-3 was such that a markedly reduced U.I. was obtained. It is suggested that two compensatory mechanisms maintain the necessary degree of membrane permeability and fluidity in order to prevent transition to a crystalline state at lower temperatures.     

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.     

Turnover Rates of the Molecular Species of Ethanolamine Plasmalogen of Rat Brain

1,2-Diradyl-3-acetylglycerols prepared from 1-O-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (alkenylacyl-GPE, ethanolamine plasmalogen) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3-impregnated TLC. The percent compositions of substractions were compared with that of 1,2-diacylglycero-3-phosphoethanolamine (diacyl-GPE). The incorporation rate of [1-3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyunsaturated fatty chains, and the sum of tetraene-, pentaene-, and hexaene-containing species was greater than 65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time-dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl-GPE is synthesized from the corresponding species of alkylacyl-GPE. The observed turnover time of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene greater than or equal to hexaene greater than pentaene greater than or equal to monoene greater than or equal to diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl-GPE from alkylacyl-GPE, were of the order: hexaene greater than tetraene greater than pentaene greater than diene greater than or equal to monoene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. A novel pathway for the metabolism of ether-linked phosphoglycerides.

Madin Darby canine kidney (MDCK) cells convert 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine [( 3H]alkylacylGPC) to a product tentatively identified as an ethanolamine-containing phosphoglyceride (PE) (Daniel, L. W., Waite, B. M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132). In the present study, analysis of the radiolabeled phosphoglycerides as diradylglycerobenzoate derivatives indicated that [3H] alkylacylGPC was initially converted to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkylacylGPE) which was subsequently desaturated to 1-O-[3H]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkenylacylGPE). The conversion of [3H]/[32P]alkyl-lysoGPC to [3H]alkenylacylGPE indicated that base exchange enzymes were not involved in this pathway. A phosphono analog of alkyl-lysoGPC, resistant to phospholipase D hydrolysis and radiolabeled in the 1-O-alkyl chain was readily incorporated, acylated, and subsequently metabolized to [3H]alkylacylGPC and [3H]alkenylacylGPE. Therefore, the involvement of phospholipase D in the conversion pathway was ruled out. The conversion of [3H]alkylacylGPC or its phosphono analog to [3H]alkenylacylGPE was significantly enhanced by the addition of 100 microM ethanolamine to the culture media, suggesting that [3H]alkylacylglycerol is an intermediate in the cytidine-dependent pathway of PE synthesis. MDCK cell cytosol and microsomes contained no detectable phospholipase C activity. However, incubation of microsomes with CMP resulted in the degradation of [3H]alkylacylGPC and accumulation of [3H]alkylacylglycerol. Furthermore, the addition of CDP-ethanolamine to microsomes following preincubation with CMP, resulted in a decrease in [3H]alkylacylglycerol with a concomitant increase in [3H]alkenylacylGPE. Overall, these results suggest that the reverse reaction of choline phosphotransferase may be responsible for the conversion of alkylacylGPC to alkylacylGPE.