Identification of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice

A new class of alkyl glycerolipids, 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, was identified in lipid extracts prepared from harderian gland tumors of mice. After saponification, this lipid class yielded 1-alkyl-3-(1'-glycerol)glycerols. Identification was based on mass spectrometry, proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, and chromatography of various derivatives and appropriate standards that were synthesized. The alkyl moieties of this unique lipid class consisted of saturated aliphatic chains with chain lengths of 14 to 20 carbon atoms. The acyl moieties were mostly saturated and monounsaturated aliphatic chains ranging from 14 to 24 carbon atoms. The alkyl and acyl moieties of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols were similar to those of alkyldiacylglycerols present in the same tissue, except for the presence of monounsaturated alkyl moieties in the latter. 1-Alkyl-2-acyl-3-(2', 3'-diacylglycerol)glycerols were only found in trace amounts in the normal harderian glands of mice. The total quantity of the alkyl and acyl moieties with a chain length greater than 20 carbon atoms in the alkyldiacylglycerols from tumors were considerably lower than those found in normal harderian glands of mice. This is the first report of the presence of bisglyceryl ether lipids in mammalian tissue; its unique chemical structure is consistent with the type of ether-linked lipid products that could be synthesized in the reaction catalyzed by alkyldihydroxyacetone-P synthase.     

Gas-liquid chromatography of dialkyl, alkyl acyl, and diacyl ddrivatives of glycerol

Dialkyl, alkyl acyl, and diacyl glycerols were resolved as trimethylsilyl ethers and as acetates by gas-liquid chromatography on a nonpolar stationary phase (OV-1). The two types of derivatives proved suitable for quantitative gas chromatographic analysis.     

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.     

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.     

Occurrence of lysophosphatidic acid and its alkyl ether-linked analog in rat brain and comparison of their biological activities toward cultured neural cells.

Rat brain was found to contain substantial amounts of potent bioactive lipids lysophosphatidic acid (acyl LPA) (3.73 nmol/g tissue) and lysoplasmanic acid (alkyl LPA) (0.44 nmol/g tissue). The presence of alkyl LPA was confirmed by mild alkaline hydrolysis analysis and by gas chromatography/mass spectrometry analysis of the trimethylsilyl derivative. This is the first clear evidence of the occurrence of an alkyl LPA in nature. The predominant molecular species of acyl LPA are 18:1-, 18:0- and 16:0-containing species (46. 9, 22.5 and 18.8%, respectively). A significant amount of a 20:4-containing species (7.2%) was also detected in the acyl LPA fraction. We also confirmed that rat brain alkyl LPA consists of 16:0-, 18:0- and 18:1-containing species. Noticeably, either acyl or alkyl LPA is capable of stimulating neuroblastomaxglioma hybrid NG108-15 cells to elicit a Ca(2+) transient, the potencies being almost the same. Both acyl and alkyl LPAs also induce cell rounding upon addition to the cells. These results suggest that acyl and alkyl LPAs play important physiological roles as intercellular signaling molecules as well as the roles as metabolic intermediates in the nervous system.     

Fatty acid composition of diacyl, alkylacyl, and alkenylacyl phospholipids of control and arachidonate-depleted rat polymorphonuclear leukocytes

Phospholipid fatty acid composition and phospholipid subclass distribution of control and arachidonate-depleted rat polymorphonuclear leukocytes (PMN) were compared. The 20:4-depleted PMN contained significantly higher amounts of 16:1, 18:1 and 20:3 (delta 5,8,11) and lower amounts of 18:2 and 20:4 than the phospholipids from control cells. Choline-containing glycerophospholipids (CGP) were the major phospholipids of both control and 20:4-depleted cells representing 34% and 37% of the total phospholipids, respectively. Significant amounts of ethanolamine-containing glycerophospholipids (EGP) (29% and 30%) and sphingolipids (20% and 18%) were also present in both cell types. Serine-containing glycerophospholipids (SGP) together with inositol-containing glycerophospholipids (IGP) constituted 16% and 13% of the phospholipids in control and 20:4-depleted cells, respectively. CGP from control cells had significantly higher amounts of 16:0 and 18:2 and lower amounts of 18:0 and 20:4 than EGP, whereas CGP from 20:4-depleted cells has higher amounts of 16:0 and 16:1 and lower amounts of 20:3 than EGP. Analysis of the subclass composition of CGP and EGP revealed that both control and 20:4-depleted cells contained significantly large amounts of alkylacyl-GPC and alkenylacyl-GPE. Small amounts of alkylacyl-GPE and alkenylacyl-GPC were also observed. The predominant fatty acyl residues found in the 1,2-diacyl-GPC, alkylacyl-GPC of control cells were 16:0, 18:0, 18:1, 18:2, and 20:4, while those of 20:4-depleted cells were 16:0, 16:1, 18:0, 18:1, and 20:3. More than 60% of CGP-bound 20:4 of control cells and about 70% of the CGP-bound 20:3 of 20:4-depleted cells were found in their alkylacyl species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regio- and stereoselective enzymatic esterification of glycerol and its derivatives.

A methodology for regio- and stereoselective preparation of acyl glycerol derivatives is presented. It offers easy access to specific 1,2-, 1,3-diglycerides and triglycerides as well as alkyl glycerol esters, phospholipids and glycolipids. These compounds are prepared by esterification of the corresponding glycerol derivatives such as 2-monoglycerides, alkyl glycerols, glyceryl glycosides, glyceryl phosphate esters, or unsubstituted glycerol. The regio- and stereoselectivity in the esterification is achieved by using fatty acid anhydrides and an enzymatic catalyst, 1,3-specific lipase. NMR methods for determining the regio- and stereoselectivity of esterification are discussed.     

The alkyl moieties in wax esters and alkyl diacyl glycerols of sharks

The alkyl moieties in wax esters and alkyl diacyl glycerols from the liver of the dogfish, soupfin shark, and silky shark are almost exclusively saturated and monounsaturated, the main alkyl moieties being the C(16) and C(18) chains in both lipid classes. However, the alkyl moieties in wax esters occur in a wider range of chain lengths. The unsaturated alkyl moieties in the two classes of lipids are mixtures of isomers. The distribution of isomeric octadecenyl moieties in wax esters and alkyl diacyl glycerols is almost the same.     

Positional distribution of fatty acids in glycerophosphatides of bovine gray matter

Glycerophosphatides were isolated from ox brain gray matter by column chromatography. The fatty acid compositions of ethanolamine glycerophosphatides (EGP), serine glycerophosphatides (SGP), and choline glycerophosphatides (CGP) were determined by gas-liquid chromatography. The positional distribution of fatty acids in these glycerophosphatides were determined by phospholipase A hydrolysis (Habu habu venom). C(20) and C(22) polyunsaturated acids were confined almost exclusively to the 2-position of these lipids, where they comprised the majority of 2-substituents in EGP and SGP (oleic acid predominated in this position in CGP). In the 1-position, palmitoyl was the major substituent in CGP, stearoyl in SGP, and stearoyl or the corresponding alk-1-enyl group in EGP.     

A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity

The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1-6) by gas chromatography-mass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4-6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1-4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18-26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and -6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1-4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and -6), respectively. GIPL-2 and -3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.     

Occurrence of glyceryl ethers in the phosphatidylcholine fraction of surfactant from dog lungs

The molecular species of ether-linked lipids in the phosphatidylcholine (PC) fraction of the pulmonary surfactant obtained from the lavage fluid of dog were characterized. A combination of base-catalyzed methanolysis, phospholipase C treatment, gas-liquid chromatography, and mass spectrometry procedures were applied. The phospholipid composition of the surfactant, obtained by phosphorus assay of lipids separated by silica gel G thin-layer chromatography (TLC), was: PC (75%), phosphatidylglycerol (10%), phosphatidylethanolamine (7%), plus small amounts of sphingomyelin, phosphatidylinositol, and phosphatidylserine. The major components of the PC were 1,2-diacylPC (95%), and 1-O-alkyl-2-acylPC (5%). No detectable amounts of 1-O-alkyl-1'-enyl-2-acylPC or di-alkyl-1-enylPC were observed. The acyl groups present in the diacylPC were 14:0 (5%), 16:0 (68%), 16:1 (12%), 18:0 (6%), 18:1 (7%) and 18:2 (2%). The predominant alkyl ether chains located at the carbon 1 position of the 1-O-alkyl-2-acylPC were 16:0 (84%), 18:0 (5%) and 18:1 (14%). At the carbon 2 position only a 16:0 fatty acyl residue was detected. In three out of seven animals platelet-activating factor-like activity, as determined by a platelet aggregation assay, was isolated by TLC. This aggregating activity was lost upon base-catalyzed methanolysis, but was restored by functional levels after acetylation.     

Synthesis and desulfurization-decarboxylation of cyclic thionocarbonates of glycerol 1-ethers

A method is described for the synthesis of cyclic thionocarbonates of 1-O-alkyl glycerols in quantitative yield. These derivatives of glycerol ethers can be quantitated by UV absorbance, analyzed by gas-liquid chromatography, or quantitatively converted to allyl alkyl ethers for gas-chromatographic analysis.     

Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei which contains C14 acyl and alkyl chains shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.     

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.     

Core aldehydes of alkyl glycerophosphocholines in atheroma induce platelet aggregation and inhibit endothelium-dependent arterial relaxation.

Plaque disruption with superimposed thrombosis is considered to be responsible for precipitating acute coronary syndrome. We identified sn-1-alkyl- and sn-1-acyl-type glycerophosphocholine (GroPCho) core aldehydes from human atheromas and demonstrated their activities on platelets and arteries. The naturally occurring core aldehydes were identified and quantified in relation to synthetic standards by high performance liquid chromatography with on-line electrospray mass spectrometry. 1-O-Hexadecyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) alkyl GroPCho core aldehyde), occurring in atheroma at less than 0.1% of total phosphatide, induced aggregation of washed rabbit platelets (50% effective dose was approximately 50 nM). Aggregations induced by C(5) alkyl GroPCho core aldehydes were completely inhibited by two different platelet-activating factor receptor antagonists. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) acyl GroPCho core aldehyde) induced platelet shape change, but not aggregation. By contrast, 10 microM C(5) alkyl and C(5) acyl GroPCho core aldehydes both inhibited endothelium-dependent relaxation of rabbit artery by 50% (endothelium-independent relaxation was not affected). The present demonstration of platelet aggregation by physiologically relevant concentrations of alkyl GroPCho core aldehydes suggests that alkyl GroPCho core aldehyde generated in atheroma could be involved in precipitating acute coronary events, in which thrombus formation following lipid-rich plaque disruption plays an important role.     

Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by ³¹P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ⁵-unsaturation (30∶3^5,21,24) and 30∶2^21,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.     

Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana.

The Arabidopsis thaliana delta-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter. S. cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose. Gas-liquid chromatography analysis of total lipids indicated that the transformed S. cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues. Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC. A marked effect on the unsaturation of 16:1 and 18:1 was observed when S. cerevisiae harboring the FAD2 gene was cultured at 8 degrees C. To assess the ethanol tolerance of S. cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined. The results indicated that S. cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells.     

Use of t-butyldimethylchlorosilane/imidazole reagent for identification of molecular species of phospholipids by gas-liquid chromatography mass spectrometry

The t-butyldimethylsilyl derivatives of 1,2-diakyl, 1-alk-1'-enyl-2-acyl, 1-alkyl-2-acyl and 1,2-diacyl glycerols were analysed with a gas chromatograph mass spectrometer system. The characteristic fragment ions were as follows. The molecular weight determining ion was [M-57]+, which was formed by cleavage of the t-butyl radical from the molecular ion. The nature of the alk-1'-enyl residue could be determined by the presence of ions at [RCH-CH 56]+ and [RCH = CH + 130]+ (RCH = CH = alk-1'-enyl), and the alkyl residue by the ion at [R + 130]+(R = alkyl group). Ions giving information about the acyl group, [RCO]+, [RCO + 74]+ and [M-RCH = CHO, -RO or -RCOO]+ were also observed. The mass spectra of pairs of trimethylsilyl and t-butyldimethylsilyl derivatives showed differences in several respects. The t-butyldimethylsilyl derivatives gave more effective information for elucidating the structure of phosphoglycerides.     

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.     

Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.