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.     

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.     

Metabolism of long-chain polyunsaturated alcohols in myelinating brain

cis-9-[1-(14)C]Octadecenol, cis,cis-9,12-[1-(14)C]octadecadienol, and cis,cis,cis-9,12,15-[1-(14)C]octadecatrienol were administered intracerebrally to 18-day-old rats. Incorporation of radioactivity into the constituent alkyl, alk-1-enyl, and acyl moieties of the ethanolamine phosphatides of brain was determined after 3, 6, 24, and 48 hr. Incorporation of radioactivity from each precursor proceeded at approximately the same rate leading to mono-, di-, and triunsaturated alkyl and alk-1-enyl glycerols. In addition, the labeled alcohols were found to be oxidized to the corresponding fatty acids which were incorporated into acyl groups; radioactivity derived from di- and triunsaturated alcohols was found mainly in acyl moieties produced through chain elongation and desaturation reactions of di- and triunsaturated fatty acids.     

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.     

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.     

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.     

Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.

Branched-chain fatty acids of the iso and anteiso series occur in many bacteria as the major acyl constituents of membrane lipids. In addition, omega-cyclohexyl and omega-cycloheptyl fatty acids are present in several bacterial species. These two types of fatty acids are synthesized by the repeated condensation of malonyl coenzyme A with one of the branched-chain and cyclic primers by the same enzyme system. The pathway of de novo branched-chain fatty acid synthesis differs only in initial steps of synthesis from that of the common straight-chain fatty acid (palmitic acid) present in most organisms. The cell membranes composed largely of iso-, anteiso-, and omega-alicyclic acids support growth of bacteria, which inhabit normal as well as extreme environments. The occurrence of these types of fatty acids as major cellular fatty acids is an important criterion used to aid identification and classification of bacteria.     

The alkyl and alk-1-enyl glycerols in the liver of rats fed long-chain alcohols or alkyl glycerols

Dietary long-chain alcohols and alkyl glycerols, including polyunsaturated compounds, are incorporated into the alkyl and alk-1-enyl moieties of the ionic alkoxylipids of rat liver, whereas polyunsaturated fatty acids are not.     

Composition of alkoxylipids of human heart and aorta

The major alkoxylipids of human heart and aorta are alkyl and alk-1-enyl diacyl glycerols, alkyl acyl and alk-1-enyl acyl glycerophosphoryl cholines, and the corresponding glycerophosphoryl ethanolamines. There are no pronounced differences in the composition of corresponding classes of alkoxylipids from heart, aorta, and other human tissues previously reported.     

Positional Distribution of Acyl and Alk-1-enyl Groups in Grey and White Matter Ethanolamine and Choline Phosphoglycerides of a Marsupial, the Koala (Phascolarctos cinereus)

The major phosphoglycerides in grey and white matter from the brain of the koala have been separated and examined. The major polyunsaturated fatty acids present in both the diacyl- and alk-1-enyl acylglycerophosphorylethanolamines from grey matter were 22:6 omega 3, 20:4 omega 6, and 22:4 omega 6. In both grey and white matter, 22:6 omega 3 and 20:4 omega 6 were concentrated in the 2-position of diacylglycerophosphorylethanolamines and 22:4 omega 6 in the 2-position of alk-1-enylacylglycerophosphorylethanolamines; polyunsaturated fatty acid levels were higher in diacylglycerophosphorylethanolamines. Ethanolamine phosphoglyceride fractions from grey matter were enriched in polyunsaturated fatty acids compared with those from white matter. The acyl groups 18:0, 18:1, and 16:0 and their alk-1-enyl analogues were prominent in grey and white matter ethanolamine phosphoglycerides; 18:1 was dominant in white matter alk-1-enylacylglycerophosphorylethanolamines. The plasmalogen composition of ethanolamine phosphoglycerides was 55% in grey matter and 76% in white matter. Choline phosphoglycerides contained negligible plasmalogen and low polyunsaturated fatty acid levels. Diacylglycerophosphorylcholine was characterized by high levels of 16:0 and 18:1. Similar acyl group distributions were estimated in the 1-position in both grey and white matter, 16:0 being present at greater than 50%. The presence of the molecular species 18:0/22:6 omega 3 was indicated in grey matter diacylglycerophosphorylethanolamine, 18:1/18:1 in white matter alk-1-enylcylglycerophosphorylethanolamine, and 16:0/18:1 in white matter diacylglycerophosphorylcholine.     

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.     

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.     

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.     

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.     

Lipid diversity in clostridia

Studies of the lipidomes of twenty-one species of clostridia have revealed considerable diversity. Even among those species now defined as Clostridium sensu stricto, which are related to Clostridium butyricum, the type species, lipid analysis has shown that a number of distinct clades have characteristic polar lipids. All species of Clostridium sensu stricto have phosphatidylethanolamine, phosphatidylglycerol and cardiolipin which are present as all acyl or alk-1′-enyl acyl (plasmalogen) species. In addition, almost every clade has specialized polar lipids. For example, the group closely related to Clostridium beijerinckii and several other solventogenic species has glycerol acetals of plasmenylethanolamine, which protects the membrane bilayer arrangement when the lipids are highly unsaturated or in the presence of solvents. The group related to Clostridium novyi has aminoacyl-phosphatidylglycerol, which protects these pathogens from cationic antimicrobial peptides (CAMPs) of innate immunity. Clostridium botulinum species, which fall into several groups, align with these clades, and have the same specific lipids. This review will present the current state of knowledge on clostridial lipids.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes.

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.