Reductive and oxidative biosynthesis of plasmalogens in myelinating brain

Palmitic acid-1-(14)C and hexadecanol-1-(14)C were administered intracerebrally to 18-day-old rats. Incorporation of radioactivity into the constituent alkyl, alk-1-enyl, and 1-acyl moieties, as well as into the 2-acyl moieties, of the ethanolamine phosphatides of brain was determined after 1, 2, 3, 6, and 22 hr. Incorporation of radioactivity from hexadecanol into both alkyl ethers and alk-1-enyl ethers proceeded at a rate more than 10 times higher than from palmitic acid. Hexadecanol was rapidly oxidized to fatty acids which were incorporated into the acyl moieties of the ethanolamine phosphatides. When palmitic acid was used as a precursor, labeled long-chain alcohols could be isolated from the lipid extract. As labeled long-chain aldehydes could not be detected in any of the lipid extracts, alcohols appear to be key intermediates for the biosynthesis of both alkyl and alk-1-enyl glycerophosphatides.     

Ehrlich ascites tumor cell surface membranes: an abnormality in ether lipid content

Most mammalian neoplasms have a defect in ether lipid content manifested by the presence of abnormally large quantities of 0-alkyl glyceryl ethers, in contrast to normal tissues in which the alk-1-enyl structure predominates. These lipids are for the most part structural. The manner in which tumor cell plasma membranes differ from normal may be important, and it has been hitherto unclear whether or not the 0-alkyl lipid abnormality of neoplasms includes the plasma membrane. The present investigation reveals that 0-alkyl lipids are present in the membranes of Ehrlich ascites tumor cells isolated by several different methods. The amount of 0-alkyl lipid, on a weight basis, represents 1-3 percent of the total phospholipids and 1-4 percent of the total aliphatic lipid. These quantities are the same as or greater than the amount of 0-alkyl lipid found in microsomes, mitochondria, and whole cell homogenate. As is generally the case for intact neoplastic tissues, the quantity of 0-alkyl lipids of Ehrlich ascites tumor plasma membrane is greater than the amount of alk-1-enyl lipids.     

Natural occurrence of free fatty aldehydes in bovine cardiac muscle

Free fatty acids, aldehydes, alcohols, and 1-O-alkyl and alk-1-enyl glycerols were identified and quantified in lipid extracts from bovine cardiac muscle. Although a number of components present in the free fatty aldehydes were also noted in the fatty chains in the 1-O-alk-1-enyl glycerols, a direct qualitative similarity did not exist as would be expected if the free fatty aldehydes were artifactual in origin. Also, a qualitative similarity did not exist between the fatty chains of the 1-O-alkyl and alk-1-enyl glycerols. This latter observation would suggest a mechanism other than biodehydrogenation of the alkyl ethers for the origin of the alk-1-enyl glycerols. Free fatty aldehydes were distributed evenly between the 105,000 g supernatant and particulate fractions of cardiac muscle, while the 1-O-alk-1-enyl glycerols were associated primarily with the particulate fraction. Free fatty alcohols were noted only in the supernatant fraction, while the 1-O-alkyl glycerols were present in both fractions.     

Detection and isolation of minor lipid constituents

The use of thick layers of adsorbent for the concentration and subsequent isolation of neutral lipid constituents is described. Lipids not perceptible by conventional methods are demonstrated in concentrates of bovine heart extracts and identified as fatty aldehydes, O-alk-1-enyl diglycerides (neutral plasmalogens), and O-alkyl diglycerides (alkoxydiglycerides).     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

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.     

Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids.

The effect of exogenous unsaturated fatty acids on the acyl and alk-1-enyl group composition of the phospholipids of Clostridium butyricum has been examined. Unsaturated fatty acids support the growth of this organism in the absence of biotin. When cells were grown at 37 degrees in media containing oleate or linoleate and a Casamino acid mixture containing traces of biotin, the exogenous fatty acids were found mainly in the alk-1-enyl chains of the plasmalogens with less pronounced incorporation into the acyl chains. However, at 25 degrees in this medium, both the acyl and alk-1-enyl chains contained substantial amounts of the 18:1 supplement plus the C19-cyclopropane chains derived from it. Ak-1-enyl chains in all the major phosphatide classes showed a uniformly high substitution by the oleate supplement in cells grown at 37 degrees. The oleate and C19-cyclopropane content of the acyl chains was more variable among the phosphatide classes. At 37 degrees, trans-9-octadecenoic acid (elaidic acid) also supported growth and was incorporated into both acyl and alk-1-enyl chains at a high level. When cells were grown on oleate at 37 degrees in media containing biotin-free Casamino acids, both the acyl and alk-1-enyl chains had a high level of 18:1 plus C19-cyclopropane chains. In the cells grown at 37 degrees with oleate substantial changes were seen in the phospholipid class composition. There was a large decrease in the ethanolamine plus N-methylethanolamine plasmalogens with a corresponding increase in the glycerol acetals of these plasmalogens. The glycerol phosphoglycerides were also significantly lower with the appearance of an unknown, relatively nonpolar phospholipid fraction.     

Synthesis of alkoxylipids

Improved and new methods for the chemical synthesis of alkoxylipids are described. In addition, some applications of synthetic compounds in biological and physicochemical studies as well as recent developments in the biosynthesis of alkyl and alk-1-enyl glycerolipids are discussed briefly.     

Acyl and alk-1-enyl group compositions of the alk-1'-enyl acyl and the diacyl glycerophosphoryl ethanolamines of mouse and ox brain

The ethanolamine phosphoglycerides were prepared from lipid extracts of ox and mouse brains by preparative thin-layer chromatography. The cyclic acetal derivatives of the alk-1-enyl groups were made by treating the ethanolamine phosphoglycerides with 1,3-propanediol. The resulting monoacyl glycerophosphoryl ethanolamines were separated from the unchanged ethanolamine phosphoglycerides by preparative thin-layer chromatography. Methyl ester derivatives of the acyl groups from both of these fractions were prepared by alkaline methanolysis. The cyclic acetal and methyl ester derivatives were analyzed by gas-liquid chromatography. Substantial differences were found in the composition of the side chains when the combined alk-1-enyl and acyl side chains of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were compared with the side chains of the diacyl glycerophosphoryl ethanolamines. The side chains from the 1-position of these two ethanolamine phosphoglycerides are different in chain length and unsaturation as well as in chemical bonding. The acyl groups from the 2-position of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were predominantly unsaturated. Therefore, acyl group compositions of the total ethanolamine phosphoglyceride from brain are of limited value and individual types should be analyzed.     

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.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the n-3 and n-6 families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.     

Evidence for the existence of cholesteryl alk-1-enyl ethers in bovine and porcine cardiac muscle

Evidence is presented for the existence of cholesteryl alk-1-enyl ethers in bovine and porcine cardiac muscle. Several different fatty chains are present in the cholesteryl ethers, the major species having 16 and 18 carbon atoms. The cholesteryl alk-1-enyl ether concentration was found to be 0.08 and 0.01 micro moles/100 mg of neutral lipid in bovine and porcine cardiac muscle, respectively.     

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.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

Composition of myelin from peripheral and central nervous systems of the squirrel monkey

Myelin was prepared from the brachial plexus and cervical spinal cord of adult squirrel monkeys (Saimiri sciureus). Brachial plexus myelin contained a larger amount of sphingomyelin and smaller amounts of cholesterol, lipid galactose, ethanolamine phosphoglyceride, choline phosphoglyceride, and alk-1-enyl ether than spinal cord myelin when compared as ratios to total lipid phosphorus. The peripheral nervous system myelin had a higher proportion of protein. All of these differences were statistically significant. Thus peripheral nervous system myelin and central nervous system myelin differ in protein content and lipid composition in this subhuman primate.     

Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants

Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols. The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain-length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids. The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one-half of the corresponding physiological membrane lipids, i.e. diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells. (2) The composition of acyl moieties in total lipids of rape cells was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of either glycerolipids. (3) In 1-O-alkyl-2-acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2-diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids. (4) Enzymes involved in synthesizing molecular species of 1-O-alkyl-2-acylglycerophosphocholines or 2-O-alkyl-1-acylglycerophosphocholines as well as 1,2-diacylglycerophosphocholines showed similar specificities with regard to chain-length and degree of unsaturation of both alkyl and corresponding acyl moieties. Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.     

Formation of 1-O-2'-hydroxyalkyl glycerophosphatides from 1,2-heptadecanediol in myelinating brain

1,2-Heptadecanediol-2-(14)C was administered intracerebrally to 18-day-old rats, and its incorporation, after 8 hr, into the individual aliphatic moieties of the ethanolamine glycerophosphatides was determined. Much of the radioactivity was found in a lipid fraction identified as 1-O-2'-hydroxyheptadecyl glycerol. Evidence is presented that a major portion of the precursor was incorporated into 1-O-2'-hydroxyheptadecyl-2-acyl ethanolamine phosphatides. Some of the diol administered was degraded to palmitic acid. The palmitic acid-1-(14)C derived from 1,2-heptadecanediol-2-(14)C apparently served as precursor for stearic and oleic acids, which were found as acyl groups, and for the biosynthesis of the corresponding O-alkyl and O-alk-1-enyl glycerols. The data presented prove that biological dehydration of 1-O-2'-hydroxyalkyl glycerophosphatides to the corresponding plasmalogens does not occur in myelinating brain.     

ACYL AND ALK-1'-ENYL GROUP COMPOSITION OF ETHANOLAMINE PHOSPHOGLYCERIDES OF HUMAN BRAIN

Ethanolamine phosphogylcerides (EPG) of human brain gray and white matter were analyzed for their alk-1′-enyl group and fatty acid compositions in sn-glycerol positions 1 and 2. Gray matter contained more 18:0 (54%) and less 18:1 (24.5%) alk-1′-enyl residues than white matter (16% and 57%. Sixty per cent of alk-1′-enyl 18.1 in gray matter was the (n-7), against 71%, in white matter. Both gray and white matter contained small amounts of 18:1 (n-5) and (n-3) isomers. The fatty acids in position I of the phosphatidylethanolamines were more saturated than the corresponding alk-1′-enyl groups of the plasmalogens. The ratios of monoenoic fatty acid isomers in position 1 were markedly different from those of the corresponding alk-1′-enyl groups in gray matter. The fatty acid patterns in position 2 of plasmalogen and phosphatidylethanolamines of white matter were similar except for 22:4(n-6) which was concentrated in the plasmalogen (16% against 10%, in the phosphatidyl ethanolamine). In gray matter, the same trend was noted. The data suggest that alk-1′-enyl residues and the fatty acids in position 1 as well as the fatty acids in position 2 of alk-1′-enyl acyl and diacyl type EPG in both gray and white matter are, at least in part, of different provenance.