Glyceryl ethers in insects: Biosynthesis of ethanolamine phosphoglycerides containing alkyl and alk-1-enyl glyceryl ether linkages

When¹⁴C-labeled acetate, fatty acids or fatty alcohols were injected into or fed to the tobacco budworm, acyl, alkyl and alk-1-enyl moieties of the phospholipids incorporated radioactivity. Fatty acids were the principal precursor in acyl bond formation and fatty alcohols in the synthesis of alkyl and alk-1-enyl glyceryl ethers. Detailed analysis of the etherlinked phosphoglycerides revealed that most of the radioactivity was in the ethanolamine phosphoglycerides, and very little¹⁴C was found in the choline phosphoglycerides. In experiments of a short duration, the alkyl glyceryl ethers incorporated more radioactivity than the alk-1-enyl glyceryl ethers. The reverse was found with long term experiments, when the alk-1-enyl ethers had higher radioactivity. In addition to demonstrating the synthesis of ether-linked ethanolamine phosphoglycerides, the data suggested that fatty alcohols and acids were interconverted by insects and that the alk-1-enyl ethers were derived from the alkyl ethers. Presented at the AOCS Meeting, Houston, May 1971. The following abbreviations and terminology will be used: PE, PC, PI and PS for the generic terms ethanolamine, choline, inositol and serine phosphoglycerides, respectfully. Alkyl glyceryl ether for 1-alkyl-2-acyl-sn-glycerol-3-phosphoryl-, and alk-1-enyl glyceryl ether for 1-alk-1′-enyl-2-acyl-sn-glycerol-3-phosphoryl-(commonly called plasmalogen). These are adapted from the tentative rules published inJ. Lipid Res. 8:522–528 (1967).     

Glyceryl ether,wax ester and triglyceride composition of the mouse preputial gland

Major lipid classes of the preputial gland of the mouse have been identified as wax ester, neutral plasmalogen, glyceryl ether diester and triglyceride. The chain lengths and degree of unsaturation in the aliphatic moieties of the alk-1-enyl and alkyl glyceryl ethers are similar to those of the fatty alcohols of the wax ester fraction. This lends support to the theory that long chain fatty alcohols can be direct precursors of the aliphatic chains of glyceryl ethers. The striking qualitative, as well as quantitative, similarities between the alkyl and alk-1-enyl moieties of the glyceryl ethers in the neutral lipid fraction suggest that they share a common pathway of biosynthesis or are interconvertible. Neutral plasmalogens and glyceryl ether diesters contain significant amounts of odd-numbered and branched fatty acids, unlike the fatty acids of the triglycerides; therefore, the biosynthesis of neutral plasmalogens and glyceryl ether diesters may not be related to the biosynthesis of triglycerides. Presented at the AOCS Meeting, Chicago, October, 1967. Taken from a dissertation submitted to Tulane University in partial fulfillment of the requirement for the degree of Doctor of Philosophy by Gail Sansone.     

On the levels of alkyl and alk-1-enyl glycerolipids in normal and neoplastic tissues: A method of quantification

A method is described for the quantification of the constituentO-alkyl andO-alk-1-enyl glycerols of neutral lipids or phospholipids. The method involves chemical degradation, the preparation of derivatives, and quantification by gas chromatography using internal standards. Alk-1-enyl moieties are converted to alkyl substituted dioxanes in the presence of 1,1-dimethoxyheptadecane as standard; alkyl glycerols are analyzed as isopropylidene derivatives using 1-O-heptadecyl glycerol as internal standard. The method is applied to the quantification of alkyl and alk-1-enyl glycerols derived from total lipids of rat heart, liver, testes, and brain and of various transplantable tumors, i.e. amelanotic melanoma, melanoma B16, sarcoma T241, and Novikoff hepatoma. The levels of alkyl glycerols range from 0.11–1.07% total lipids and those of alk-1-enyl glycerols from 0.49–5.03%. The data are compared to those obtained by other methods.     

Abnormal lipid composition of fat tissue in human mesenteric panniculitis

Mesenteric fat tissue obtained at autopsy from 6 patients with mesenteric panniculitis (MP) were found to contain significant amounts of cholesteryl esters (CE). In addition, samples from 3 of these cases were found to contain 0.5–1.3% free cholesterol, 0.9–1.9% free fatty acids (FFA), 0.6–2.5% 1-alkyl glyceryl ether diesters and small amounts of squalene. Two of these tissues also contained alk-1-enyl glyceryl ether diesters. The fatty acid compositions of the CE, FFA, triacylglycerides and glyceryl ether diesters (GEDE) were determined and oleic acid (18∶1) was found to be the major fatty acid. The alkyl group composition of the GEDE consisted essentially of 16∶0 and 18∶0 and 18∶1 carbon atoms in both types of ethers.     

Chain length specificity in the utilization of long chain alcohols for ether lipid biosynthesis in rat brain

A mixture ofcis-9-[1-¹⁴C] octadecenol and [1-¹⁴C] docosanol was injected into the brains of 19-day-old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more radily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18∶1 alkyl and alk-1-enyl moieties of the ethanolamine phosphoglycerides and into 18∶1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18∶1 alcohol from the substrate mixture (12 hr), the 22∶0 alcohol was not used to any measurable extent for alkyl and alk-1-enyl glycerol formation.     

Glyceryl ethers in insects: identification of alkyl and alk-1-enyl glyceryl ether phospholipids

Alkyl and alk-1-enyl glyceryl ethers have been identified in the phospholipids of three insect species, the American cockroach (Periplaneta americana), the tobacco budworm (Heliothis virescens), and the boll weevil (Anthonomus grandis). Glyceryl ethers were not detected in the neutral lipids. The ethers were found in the phospholipid fraction of whole insects and in isolated fat body tissue. The ether content varied among the three insect species, and fluctuated during various developmental stages. Gas liquid chromatographic analysis of the alkyl glyceryl ethers and aldehydes derived from the alk-1-enyl glyceryl ethers of the cockroach and budworm whowed striking differences in chain length. However, the hydrocarbon side-chain of the two ether fractions were similar in length for each species. Preliminary evidence indicates that 1-¹⁴C-acetate can be incorporated into alkyl ethers but not into alk-1-enyl ethers ofHeliothis pupae. Presented at the AOCS-AACC joint meeting, Washington, D.C., 1968. Under contract with the U.S. Atomic Energy Commission.     

Lipid composition of yoshida ascites hepatoma and of livers and blood plasma from host and normal rats

The lipid composition of Yoshida ascites hepatoma cells was analyzed together with that of ascitic plasma and of livers and blood plasma from host and normal rats. In comparison to normal livers, host livers showed no significant differences in the content of the various lipid classes, but contained a higher percentage of palmitic acid and a lower proportion of arachidonic acid in the major phospholipid classes. In addition, tumor growth induced a marked hypertriglyceridemia in host animals; changes in the concentration of other plasma lipid classes were not statistically significant. The ascitic plasma contained small amounts of lipids mainly constituted by cholesteryl esters and phospholipids. Yoshida hepatoma cells contained less phospholipids in comparison to both host and normal liver, while the increased level of triglycerides and the decrease of free fatty acids were not statistically significant. Hepatoma cells showed appreciable amounts of ether-linked lipids associated in part to neutral lipids (as glyceryl ether diesters) and, in part, to ethanolamine and choline phosphoglycerides. The alkyl groups in GEDE as well as in ethanolamine and choline phosphoglycerides were mainly constituted by C_16∶0 and C_18∶0 followed by C_18∶1. The alk-1-enyl groups in ethanolamine and choline phosphoglycerides were C_16∶0 and C_18∶0 with only a minor proportion of C_18∶1. In comparison to both host and normal liver, Yoshida hepatoma cells showed significant changes in the fatty acid composition of neutral lipids and phospholipids. Some of the major changes consisted of an increase of monoenoic acids associated with a decrease of arachidonic and docosahexaenoic acids in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol.     

Plasma transport forms of ingested fatty alcohols in the rat

Previous studies have shown that ingested fatty alcohols are absorbed as fatty acids and fatty acid esters, particularly triglycerides. The present study was carried out to determine whether fatty alcohols are also transported as 0-alkyl glyceryl ethers, alk-1-enyl glyceryl ethers, and as wax esters. Oxidation of fatty alcohols to other lipids was assessed by using a mixture of [1-³H] hexadecanol and [1-¹⁴C] hexadecanol of predetermined ratio. The results indicate that the absorption of fatty alcohol, and of its transport forms, parallels the absorption of labeled fatty acids. Six to 25% of plasma radioactivity was present as 1-0-alkyl diacylglyceryl ethers with a smaller proportion of ether lipids in the phospholipid fraction. In addition, 4–13% of the ingested hexadecanol appeared in the plasma as a material having the chromatographic properties of wax ester. Fatty alcohols were not detected in the plasma as alk-1-enyl lipids.     

Tumor lipids: Comparison of glyceryl ethers of mouse and insect tumors

Examination of the lipids of two tumor-bearing mutant strains of fruit fly reveal that they do not have the elevated content of glyceryl ethers typical of many neoplasms of higher animals. Glyceryl ether diesters were absent, and the amounts of alkyl and alk-1-enyl glyceryl ethers of tumorous and normal tissues were similar.     

Incorporation of [1-14C]octadecanol into the lipids ofLeishmania donovani

After incubation of stationary phaseLeishmania donovani with [1-¹⁴C] octadecanol, about 70% of the precursor was taken up within 3 hr. Wax esters and acyl moieties of glycerolipids contained most of the¹⁴C-activity from 3 to 6 hr, because octadecanol was partly oxidized to stearate. Ether moieties were only weakly labeled. After 40 hr, 1-0-aklyl and 1-0-alk-1′-enyl diacylglycerols as well as 1-0-alkyl and 1-0-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines contained nearly all of the radioactivity. Most of the label in the neutral ether lipids was located in the alkyl ether side chain, whereas, in the phosphatidylethanolamine fraction, most of the label was found in the alkenyl ether side chain. Administration of 1-0-[1-¹⁴C] hexadecyl glycerol resulted in rapid labeling of the vinyl ether side chain of phosphatidylethanolamine plasmalogen (1 hr) increasing further at 2.5 hr. Most of the radioactivity in the alkoxy diacylglycerols was found in the 1-0-alkyl moiety.     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Occurrence of ethanolamine- and choline-containing plasmalogens in adipose tissue

Analysis of phospholipids in porcine, bovine and rat adipose tissue revealed a relatively high level of plasmalogens (O-alk-1-enyl lipids). About 50% of the ethanolamine phospholipids in the pig and beef samples consisted of alk-1-enylacylglycerophosphorylethanolamine, and the corresponding value for the rat sample was near 35%. In the ethanolamine and choline phospholipid fractions, theO-alk-1-enyl moieties were almost exclusively 16∶0, 18∶0 and 18∶1, whereas the acyl moieties had chain lengths ranging from 16 to 22 carbon atoms with a high degree of unsaturation.     

Effect of pentadecan-2-one on lipid metabolism in HeLa cells

HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1-¹⁴C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased¹⁴C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.     

Liberation of aldehydes from alk-1-enyl glyceryl ethers by acid hydrolysis

Labeled alk-1-enyl glyceryl ethers were used in conjunction with thin layer chromatography to study the liberation of aldehydes from alk-1-enyl glyceryl ethers by two acid hydrolysis procedures. Both methods gave similar results, but neither liberated the aldehydes quantitatively. Only 75–85% of the alk-1-enyl glyceryl ether radioactivity was liberated as free aldehydes. Several nonaldehyde products were detected and one appeared to be a cyclic acetal. Under contract with the U.S. Atomic Energy Commission.     

Glyceryl ether containing lipids of whole brains from germ-free and conventional rats

The percentage alkyl and alk-1’-enyl glyceryl ethers of the polar lipids isolated from germ-free and conventional rat brain tissue has been determined. No difference could be detected in the quantity of the ether-containing lipids in these animals. Similarly, the composition of the aldehydic chain of the alk-1’-enyl glyceryl ethers from germ-free and conventional rat brain tissue was shown to be the same. The microbial flora, therefore, do not influence either the quantity of the ether-containing lipids or the composition of the aldehyde chain of the alk-1’-enyl glyceryl ethers in the rat brain.     

The acyl and Alk-1-enyl groups of the major phosphoglycerides from ox brain myelin and mouse brain microsomal,mitochondrial and myelin fractions

The major phosphoglycerides from ox brain myelin and mouse brain microsomal, mitochondrial and myelin fractions were separated by preparative thin layer chromatography. Alk-1-enyl groups from the alk-1-enyl acyl glycerophosphorylethanolamines were reacted with 1,3-propanediol to form the 1,3-dioxolane derivatives. Acyl groups were converted to the methyl ester derivatives and the acyl groups from alk-1-enyl acyl glycerophosphorylethanolamines and diacyl glycerophosphorylethanolamines were also determined separately. The acyl and alk-1-enyl group compositions of the phosphoglycerides from microsomal and mitochondrial fractions were quite similar. The ethanolamine and serine phosphoglycerides contained large amounts of 18∶0, 18∶1, 20∶4 and 22∶6 acyl groups. The choline phosphoglycerides had small amounts of polyunsaturated acyl groups and large amounts of 16∶0, 18∶1 and 18∶0 acyl groups. The mitochondrial cardiolipins contained unusual amounts of several acyl groups including 18∶1, 52%; 18∶2, 6%; and 16.1, 4%. A large portion of the mouse brain 18∶2 is in that fraction. The myelin phosphoglycerides were deficient in saturated and 22∶6 groups and markedly enriched in 18∶1 and 20∶1 groups when compared with the corresponding microsomal or mitochondrial phosphoglycerides. Presented in part at the AOCS Meetings, New York, October 1968 and San Francisco, April 1969.     

Ether-containing lipids of the slime mold,Physarum polycephalum: II. Rates of biosynthesis

The in vivo incorporation of 1-¹⁴C-palmitic acid and 1-0-[8,9-³H] hexadecyl glycerol (chimyl alcohol) by plasmodia of the slime mold,Physarum polycephalum has been studied.¹⁴C-palmitate rapidly enters ester and alkyl ether side chains of phospholipids, but alk-1-enyl side chains are labeled more slowly.³H-chimyl alcohol is incorporated into the alkyl ether phospholipids, which appear to undergo enzymatic desaturation, producing plasmalogens. The feasibility ofPhysarum as the source of a cell-free enzyme system for plasmalogen synthesis is discussed.     

Total Lipids of Sarda Sheep Meat that Include the Fatty Acid and Alkenyl Composition and the CLA and <Emphasis Type="Italic">Trans</Emphasis>-18:1 Isomers

The total lipids of the longissimus dorsi muscle were analyzed from commercial adult Sarda sheep in Sardina taken from local abattoirs, and in the subsequent year from three local farms in the Sassari region that provided some information on the amount and type of supplements fed to the pasture-fed sheep. The complete lipid analysis of sheep meat included the fatty acids from O-acyl and N-acyl lipids, including the trans- and conjugated linoleic acid (CLA) isomers and the alk-1-enyl ethers from the plasmalogenic lipids. This analysis required the use of a combination of acid- and base-catalyzed methylation procedures, the former to quantitate the O-acyl, N-acyl and alkenyl ethers, and the latter to determine the content of CLA isomers and their metabolites. A combination of gas chromatographic and silver-ion separation techniques was necessary to quantitate all of the meat lipid constituents, which included a prior separation of the trans-octadecenoic acids (18:1) and a separation of fatty acid methyl esters and the dimethylacetals (DMAs) from the acyl and alk-1-enyl ethers, respectively. The alk-1-enyl moieties of the DMAs were analyzed as their stable cyclic acetals. In general, about half of the meat lipids were triacylglycerols, even though excess fat was trimmed from the meat. The higher fat content in the meat appears to be related to the older age of these animals. The variation in the trans-18:1 and CLA isomer profiles of the Sarda sheep obtained from the abattoirs was much greater than in the profiles from the sheep from the three selected farms. Higher levels of 10t-18:1, 7t9c-18:2, 9t11c-18:2 and 10t12c-18:2 were observed in the commercial sheep meat, which reflected the poorer quality diets of these sheep compared to those from the three farms, which consistently showed higher levels of 11t-18:1, 9c11t-18:2 and 11t13c-18:2. In the second study, sheep were provided with supplements during the spring and summer grazing season, which contributed to higher levels of 11t-18:1 and 9c11t-18:2. The farm that provided a small amount of supplements during the spring had the better lipid profile at both time periods. The polyunsaturated fatty acid (PUFA) content was higher in the meat from Sarda sheep from the three farms than in the meat from those sheep obtained from commercial slaughter operations. The plasmalogenic lipid content ranged from 2 to 3% of total lipids, the alk-1-enyl ethers consisted mainly of saturated and monounsaturated moieties, and the trans-18:1 profile was similar to that of the FA. The n-6 (6–8%) and n-3 PUFA (2–3%) contents, the n-6/n-3 ratio (3:1), as well as the saturated fatty acid (SFA) content (42–45%) and the SFA to PUFA ratio (4:1 to 5:1) of the Sarda sheep from the three farms were comparable to sheep meat lipids found in similar commercial operations in Europe. Inclusion of small amounts of supplements for the grazing Sarda sheep resulted in improved quality of sheep meat lipids.     

Lipid and fatty acyl composition of rat brain capillary endothelia isolated by a new technique

A method is described for the isolation of pure capillary endothelia from rat brain and the phospholipid composition of these cells is reported. This method is rapid and requires only a small amount of starting material. It involves: (a) tissue disruption by high speed homogenization, (b) separation of the capillary endothelia from other brain structures using sucrose gradients, and (c) a final purification using a glass bead column. Choline and ethanolamine phosphoglycerides were found to be the predominant lipid classes of these cells amounting to 31.9% and 24.4%, respectively, of total phospholipids. The choline phosphoglycerides consisted almost exclusively of 1,2-diacyl glycerophosphorylcholine, whereas the ethanolamine phosphoglycerides consisted of approximately equal amounts of 1,2-diacyl and 1-alk-l’-enyl-2-acyl glycerophosphorylethanolamine. The composition of the constituent fatty acids of both choline and ethanolamine phosphoglycerides and the alk-1-enyl composition of ethanolamine phosphoglycerides is reported. Saturated fatty acids accounted for 45% of the total fatty acids in choline phosphoglycerides and for 53% in ethanolamine phosphoglycerides. Arachidonic acid accounted for approximately 48% of the total fatty acids in alk-1-enyl ethanolamine phosphoglyceride.     

Mass spectrometric analysis of long chain alk-1-enyl ether esters and alkyl ether esters of diols

The mass spectra of homologous series of long chain alk-1-enyl ether esters and alkyl ether esters of short chain diols were determined, and general patterns of fragmentation were established. Both types of diol lipids yielded ions characteristic of the alkoxy or alk-1-enyloxy moiety and the acyl moiety, as well as ions indicative of the constituent short chain diol. Prominent ions were formed from both types of ether esters due to the loss of the alkoxy or alk-1-enyloxy moieties giving rise to ions for which cyclic structures are proposed. High resolution mass spectrometry and deuterium labeling techniques were used to verify the composition of ions and to substantiate fragmentation mechanisms. This is part VII in the series “Naturally Occurring Diol Lipids” (part VI is Reference 1) and part VIII in the series “Mass Spectrometry of Lipids” (part VII is Reference 23).