Ceramide Aminoethylphosphonate Research Articles

Spingophosphonolipids in microsomes of Diplodon delodontus. Distribution and effect on the membrane microviscosity

1. 1. The distribution of ceramide aminoethylphosphonate (CAEP) in microsomal membranes obtained from different tissues of the bivalve mollusc Diplodon delodontus was determined. 2. 2. The concentration of CAEP reached from 9 to 19% of the total microsomal polar lipids, depending on the kind of tissue. 3. 3. Palmitic acid was the main fatty acid in the ceramide moiety, followed by stearic and eicosamonoenoic acids. 4. 4. Artificial membranes were prepared with microsomal phospholipids or phospholipids plus sterols, with and without the addition of CAEP. 5. 5. It was shown that the phosphonate confers minor mobility to the membranes. This effect is more effective when the membrane contains the natural sterols and the phospholipids are unsaturated.

Lipid Composition and Structural Studies on Lipids from the Land Snail Eobania vermiculata

The total lipids of the commercial land snail Eobania vermiculata (Gastropoda, Pulm onata, Stylom m atophora) are found to constitute a small percentage (0.8% ) of the wet tissue, which is comparable to that reported for other gastropods. Polar lipid components comprise 61.4% of the total lipids. The individual lipid classes obtained by column chromatographic fractionation were purified by preparative TLC or by column chromatography and their structure was confirmed by a combination of chromatographic and analytical determinations before and after mild alkaline hydrolysis and/or (dry) acid methanolysis and by IR analysis. Neutral lipids represent 36.4% of total lipids, containing cholesterol, cholesterol esters and triglycerides as their major components (26.2% , 29.1% and 25.5% respectively). They contain also a significant amount (14%) of free glyceryl ethers, which are found in a mollusc for the first time. The overall composition of the polar lipids (mol/100 mol lipid-P) was found as follows: Cardiolipin, 2.9; phosphatidylethanolamine, 24.9 (of which 19.8% plasmalogen analog); phosphatidylcholine, 49.2 (of which 45.6% glycerylether analog); ceramide aminoethylphosphonate, 7.5 plus 0.01 (another three minor species); diglyceride-am noethylphosphonate, 6.3; Sphingoethanolamine 1.65 (for the first time found and structurally studied in a land gastropod); and phosphatidic acid 1.1. Unsaturated fatty acyl groups represent about 72.6 and 44.1 respectively in phosphatidylethanolamine and phosphatidylcholine. A significant amount (70.5% ) of unsaturated fatty acids is concentrated in neutral lipids. The C16:0 alk-1-enyl chain was found to predominate (55.6% ) in the side chains of ethanolamine plasmalogen. Batyl alcohol was found as the main glycerylether bound to choline phosphate (97.5% ). Saturated fatty acyl groups with 16 carbon atoms were main components (54%) of the major ceramide aminoethylphosphonate species.

Phospho- and phosphonolipids of the aegean pelagic scyphomedusa Pelagia noctiluca

The total lipid content found in Pelagia noctiluca (0.19% of the whole-body weight) is comparable to that reported for other stinging jellyfish species. Its polar lipid components were found to represent 26.2% of the total lipids and to contain 75.7% glycerophospholipids and 24.3% sphingophosphonolipids. The individual lipid classes obtained by column Chromatographic fractionation were purified by preparative TLC, and their structure was confirmed by a combination of Chromatographic and analytical determinations before and after mild alkaline hydrolysis or dry acid methanolysis, and by IR analysis. The overall composition of the polar lipids was: cardiolipin, 9.1%; phosphatidylethanolamine, 24.8% (of which 55% was glyceryl ether analog); phosphatidylcholine, 36.3% (of which 62% was glyceryl ether analog); lysophosphatidylethanolamine and lysophosphatidylcholine, 5.0%; ceramide aminoethylphosphonate, 21.0% plus 3.3% (another two minor species). Unsaturated fatty acyl groups represented about 52 and 33%, respectively, in phosphatidylethanolamine and phosphatidylcholine, while their glyceryl ether analogs were found to contain about 41 and 91% unsaturated fatty alkyl chains, respectively (total unsaturation 49 and 51%, respectively). In contrast, saturated fatty acyl groups with 14–16 carbon atoms were almost exclusive components (about 96%) of the major ceramide aminoethylphosphonate species.

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major in nervous tissues. Phosphatidylethanolamine contained about 3% of its amount of plasmalogen forin. Phosphatidylserine and -inositol, sphingomyelin and eeramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2–3% of totle lipids) and steliate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamaine, dylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acid, namely 20:4, 20:5 and 22:6 in the n−3 family. On the other hand, phosphatidylcholine, eardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C 16-C 18 fatty acids. The aldehyde moieties of cthanolamine plasmalogen were also C 16-C 18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.

Thin Layer Chromatographic and Infra Red Spectral Evidence for the Presence of Phosphonolipids in Cicada oni

Abstract The phosphonolipids from whole Cicada oni have been isolated by preparative thin-layer chromatography (TLC) in methanol-water (2:1, v/v) and have been identified by TLC, infra-red spectroscopy, phosphono phosphorus determinations and elemental microanalyses. The presence has been confirmed of the phosphono analogues of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin and of ceramide aminoethyl phosphonate.

Characterization of the 31 P NMR Spectrum of the Schistosome Vector Biomphalaria glabrata and of the Changes Following Infection by Schistosoma mansoni

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.

Characterization and biological activity of Phytophthora infestans phospholipids in the hypersensitive response of potato tuber

Phospholipids from the mycelium of Phytophthora infestans were extracted, characterized by liquid and thin-layer chromatography, and evaluated for their fatty acid composition and phytoalexin elicitor activity in potato tuber tissue. Neutral lipids were also characterized and evaluated for elicitor activity. Triglycerides were the predominant lipid class present, comprising 77·8% of the total lipids by weight, and phosphatidyl ethanolamine and ceramide aminoethylphosphonate were the most abundant phospholipids by weight, accounting for 2·8 and 3·1% of the total lipid, respectively. The predominant fatty acids present in the mycelial lipids were palmitic, linoleic, arachidonic, and eicosapentaenoic acids. All principal acyl lipids extracted from the mycelium contained arachidonate and eicosapentaenoate and elicited the production of rishitin and lubimin in potato tuber discs. However, free arachidonic acid elicited the production of more phytoalexins than any lipid fraction isolated from the fungus. Lipolytic acyl hydrolase purified from potato tuber tissue hydrolysed phosphatidyl ethanolamine from P. infestans but was inactive on triglycerides and ceramide aminoethylphosphonate.

Occurrence of ceramide aminoethylphosphonate in edible shellfish, AGEMAKI, Sinonovacula constricta.

Ceramide aminoethylphosphonate was isolated from the shellfish, AGEMAKI, Sinonovacula constricta. The compound was purified through a combination of mild alkaline hydrolysis of the total lipids, QAE-Sephadex-A25 column chromatography and two dimensional thin layer chromatography. The infrared spectrum showed an absorption band at 1180cm-1 due to a C-P bond, and was essentially identical with that of the standard ceramide aminoethylphosphonate. Upon hydrolysis of the substance with a strong acid, neither a change in its chromatographic behavior nor liberation of inorganic phosphate was observed. The stability of the compound as to acid hydrolysis suggested the presence of a C-P bond. On comparison with the synthetic compound, the aqueous hydrolysis product was found to behave like 2-aminoethylphosphonic acid on thin layer chromatography. The predominant fatty acids were palmitic acid and stearic acid. The results obtained suggest that a person may ingest and absorb 2-aminoethylphosphonic acid, through the food chain, on the consumption of edible shellfish.

Analysis of phospho- and phosphonosphingolipids by high-performance liquid chromatography.

A simple and efficient method for the separation of phosphosphingolipids including phosphonosphingolipids by high-performance liquid chromatography is described. A mixture of authentic lipids consisting of sphingomyelin, ceramide phosphorylethanolamine, ceramide 2-aminoethylphosphonate, and ceramide N-methylaminoethylphosphonate was completely separated using a silica gel (Zorbax SIL) column with acetonitrile-methanol-water 72:40:10 (v/v) as eluting solvent. The elution of these sphingolipids was monitored directly with an ultraviolet spectromonitor at 207 nm. The practical limit of detection of each sphingolipid was about 0.2 microgram or 0.3 nmol. Using this method, we found that from one to four different phosphono- and/or phosphosphingolipids in fresh-water shellfish can be routinely identified and reproducibly quantified.

On the formation of alpha-hydroxy fatty acids. Evidence for a direct hydroxylation of nonhydroxy fatty acid-containing sphingolipids.

When Tetrahymena pyriformis, strain NT-1, was warmed from a growth temperature of 15 degrees C to 39 degrees C, there was a rapid conversion of its sphingolipids from the nonhydroxy fatty acid-containing ceramide aminoethylphosphonate (NCAEP) predominant at low temperature to the alpha-hydroxy fatty acid-containing analog (HCAEP). The fatty acid composition of the freshly formed HCAEP strongly resembled that of the original NCAEP pool. An extensive utilization of NCAEP long chain bases for HCAEP formation was shown by prelabeling the NCAEP bases with [14C]serine. The specific radioactivities of both fatty acids and long chain bases of the two sphingolipid classes of cells prelabeled with [3H]palmitic acid at 15 degrees C and then warmed to 39 degrees C were compatible with a direct hydroxylation of the intact ceramide aminoethylphosphonate or free ceramide formed from it. Exogenously added alpha-hydroxypalmitic acid was not incorporated into the sphingolipids intact, but there was an active alpha-oxidation of alpha-hydroxy fatty acids, yielding nonhydroxy fatty acids one carbon atom shorter in length. In vitro experiments failed to demonstrate a direct hydroxylation of NCAEP, and nonhydroxy fatty acid-containing free ceramides were hydroxylated only sparingly. This inefficient hydroxylation is attributed to the rapid enzymatic hydrolysis of these substrates in vitro and to a limited availability of the added ceramides to the hydroxylating enzymes. The weight of the evidence from this study strongly favors the alpha-hydroxylation of fatty acids only when they are bound as elements of sphingolipids.

Phosphono-sphingomyelins. I: Synthesis of ceramide (trimethyl) aminoethyl phosphonate

The synthesis of the phosphono-analogue of sphingomyelin is described. The N-acyl-D-erythro-sphingosyl-1-( N, N, N-trimethyl-2-aminoethyl) phosphonate was obtained by phosphonylation of N-acyl-3- O-benzoyl-D-erythro-sphingosine with (2-bromoethyl)phosphonic acid chloride and triethylamine, subsequent quaternisation with anhydrous trimethylamine and benzene at 55–60°C for four days, and finally, consecutive removal of the protective group by mild alkaline hydrolysis. Comparison of the CD spectra of both, natural sphingomyelin and its phosphono-analogue, confirmed that their structures and configurations were identical.

Seasonal changes in lipid and fatty acid composition of the freshwater mollusk,Diplodom patagonicus

AbstractDiplodom patagonicus is a freshwater bivalve mollusk living in lakes of the patagonian Andes mountains in Argentina. Lipid composition and seasonal changes in the mollusk were studied in the natural habitat. In addition to common nonpolar and polar lipids, small amounts of alk‐1‐enyldiacylglycerol ethers and significant quantities of ceramide aminoethyl phosphonate were present. Total lipid content changed during the year, primarily because of decreased triacylglycerols in winter. The fatty acid composition of the lipids, remarkably different from that of marine bivalves, and even from other fresh water animals, was especially rich in the ω6 fatty acids, linoleic and arachidonic (ca. 25%), and poor in the ω3 acids, 20∶5 and 22∶6. The ω6/ω3 acid ratio was ca. 2, which is very high compared to marine bivalves. The fatty acid composition and the ω6/ω3 acid ratio were constant during the whole year, suggesting a very stable diet, rich in vegetal detritus and poor in diatoms. The influence of environmental temperature fluctuation with season on fatty acid composition also was negible. Modest proportions of 22∶2 nonmethylene‐interrupted (NMI) acids were detected and confirmed by mass spectrometry. It was shown that 20∶2 NMI acids were absent.

Phospholipid composition and turnover of pathogenic amebas

1. 1. Evidence is presented for identifying and estimating the sphingophospholipids that predominate in Pathogenic amebas (ceramide ethanolaminephosphate, ceramide aminoethylphosphonate and sphingomyelin). 2. 2. The absence of these compounds in free living ameba suggest their use as a taxonomic criterium for identifying Entamoeba. 3. 3. All the phospholipids can be synthesized by the amebas and there is an important turnover of ceramide ethanolaminephosphate and phosphatidylethanolamine which ceases during encystment. This is the first report on the turnover of ceramides. 4. 4. Phospholipase C hydrolyzed only ceramide ethanolaminephosphate in Ameba trophozoites which are resistant to phospholipase A, suggesting ceramides are located at the outer plasma membrane.

Lipid composition and some aspects of aminophospholipid organization in erythrocyte membrane of the marine bivalve mollusc Scapharca broughtoni (Schrenck)

1. 1. Lipid composition of plasma membranes isolated from erythrocytes of the marine bivalve mollusc Scapharca broughtoni (Schrenck) was studied. 2. 2. The main components of lipid fraction of the mollusc membrane were phospholipids and cholesterol. The major part of phospholipids consisted of aminophospholipids: phosphatidylethanolamine (PE), phosphatidylserine (PS), ceramide aminoethylphosphonate (CAEP). Nearly 50% of the total phospholipids were presented as plasmalogenes. Phospholipids contained a large amount of unsaturated (80%) fatty acids, mainly polyene acids of the ω3 family. 3. 3. Aminophospholipids are asymmetrically distributed betwee the two surfaces of lipid bilayer of the erythrocyte membrane. PE and PS are localized primarily on the inner surface of the membrane. 4. 4. The results obtained were compared with recent data on the mammalian erythrocytes. Molluscs erythrocytes are proposed as a new model in membrane biology.

Plasma membrane of Entamoeba histolytica.

Axenically propagated Entamoeba histolytica (HK9:NIH strain) were employed as starting material for the isoation of plasma membrane by a novel procedure. In the absence of known enzymatic markers, the externally disposed polypeptides of intact amoebae were iodinated and the incorporated label used to monitor membrane separation and recovery. 12 major plasma membrane polypeptides (12 x 10(3)-200 x 10(3) mol wt) were labeled and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Each of these was a glycoprotein. Preincubation of amoebae with concanavalin A stabilized the plasma membranes as large sheets, facilitating its separation by low-speed centrifugation. Dissociation of the lectin with alpha-methyl mannoside, followed by additional homogenization led to vesiculation and further purification. The isolated plasma membrane was recovered in high yield (28%) and enriched 30-fold in terms of incorporated iodide. All iodinated surface glycoproteins of the intact organism were present in the plasma membrane fraction. A Ca++-dependent ATPase was enriched in the plasma membrane to a similar extent, but over one-half of the total activity was associated with internal, unlabeled membranes, suggesting a dual localization of this activity. The isolated plasma membrane was enriched in cholesterol and had a cholesterol:molar ratio of 0.87. It also contained larger amounts of an unusual phospholipid--ceramide aminoethyl phosphonate--a phospholipase-resistant species.

Characteristics of phospholipids in microvillar membranes of octopus photoreceptor cells.

Characteristics of lipids in the microvillar membranes of octopus photoreceptor cells were studied in order to obtain some information on the membrane environment with rhodopsin in the invertebrate. (1) The membranes contain lipid and protein in almost equal proportion. The majority of lipids are phospholipids. Neutral lipids make up 16% of the total lipids, the major constituent of which is cholesterol. (2) Phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. Phosphatidylserine, ceramide 2-aminoethylphosphonate and sphingomyelin occur as minor components. An unidentified alkaline and acid stable phospholipid was found. (3) The predominant fatty acids of phosphatidylethanolamine and phosphatidylcholine are highly unsaturated such as 22 : 6, 20 : 5 and 20 : 4. The 22 : 6 and 20 : 5 are exclusively linked at the 2-position, but the 20 : 4 is linked significantly at the 1-position of the phospholipids. (4) Major molecular species are 16 : 0/22 : 6 (48.4%) and 16 : 0/20 : 4 (19.6%) in phosphatidylcholine, and 20 : 4/22 : 6 (50.7%) and 16 : 0/22 : 6 (25.6%) in phosphatidylethanolamine.

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.

Characterizations of phospholipids from Paramecium tetraurelia cells and cilia.

SYNOPSISPhospholipids from Paramecium tetraurelia strains 51s and d,95 cultures and isolated cilia were characterized. The following classes of phospholipids were identified in whole cell lipids: the 1‐alkyl‐2–acylglyceryl and the 1,2–diacylglyceryl forms of phosphonylethanolamine, phosphorylethanolamine. and phosphorylcholine; cardiolipin: ceramide aminoethylphosphonate and 5 other sphingolipids: phosphatidylserine; phosphatidylinositol; and lyso derivatives of the major glycerophospholipids. Cilia lipids were rich in ether lipids, phosphonolipids. and sphingolipids. Net lipid biosynthesis did not occur, as determined by the weight of lipids extracted from culture medium compared with the weight of lipids extracted from culture medium and ciliates after 7 days of growth. Total lipids/cell decreased with culture age. changes in the neutral lipid fraction accounting for the major decrease. Phospholipid class distributions changed with culture age—the glyceryl phosphorylethanolamine and choline content of cells decreased, while the glyceryl ohosphonylethanolamine content remained relatively constant: hence, the ratio of phosphonolipids to total phospholipids increased. All fatty acids observed in total lipids from cells and cilia were also present in the glycerophospholipids. Total lipids from cilia contained a greater percent of polyunsaturated fatty acid than those of whole cells. Whole cells and cilia glyceryl phosphonolipids contained up to 93% eicosatetraenoic plus eicosapentaenoic fatty acids. The enrichment of phosphonolipids in cilia accounted for most of the polyunsaturated fatty acid enrichment observed in cilia total lipids. The fatty acid composition of all major whole cell glycerophospholipid classes changed dramatically with culture age, while only small changes occurred in cilia glycerophospholipid fatty acids.

Biochemical studies of the excitable membrane of Paramecium tetraurelia. II. Phospholipids of ciliary and other membranes

The phospholipids of cilia and deciliated bodies of Paramecium tetraurelia were isolated and characterized. 1-alkyl-2-acyl- sn-glycero-3-(2′-aminoethyl) phosphonate (GAEPL), phosphatidylethanolamine, and 1-alkyl-2-acyl- sn-glycero-3-phosphorylcholine (GPC) were the major lipids of Paramecium, and the minor lipids included phosphatidylinositol, cardiolipin, ceramide-(2-aminoethyl) phosphonate (CAEP), ceramide phosphorylethanolamine (COPE) and four sphingolipids whose identity was not established. The deciliated bodies contained 4% cardiolipin, 15% GAEPL, 41% phosphatidylethanolamine, 30% GPC and 3% each of CAEP and phosphatidylinositol; the cilia contained no cardiolipin, 24% GAEPL, 37% phosphatidylethanolamine, 15% GPC, 15% CAEP, 3% phosphatidylinositol, 2% COPE and small amounts (approx. 1%) of the four uncharacterized sphingolipids. No alteration in phospholipid composition was found among cells harvested in the various stages of growth. The phospholipids of six Paramecium mutants of three distinct phenotypes (pawn, paranoiac and fast) were also examined. Only one significant difference was found on comparison of the whole cell, deciliated body and cilia fraction of the mutants with the analogous fractions from wild type cells: the fast mutant, fA 97, had two extra, minor phospholipids (approx. 2%) in the deciliated body fraction that were tentatively identified as 1,2-diacyl- sn-glycero-3-(2′-aminoethyl) phosphonate (AEPL) and 1-alkyl-2-acyl- sn-glycero-3-phosphorylethanolamine (GPE).

Studies on Sphingolipids of Barnea (Umitakea) dilatata japonica. I.

1. The complex lipids were extracted from the muscle of a marine bivalve, Barnea (Umitakea) dilatata japonica with chloroform-methanol (2 : 1, vol/vol).2. The lipids were treated mildly with diluted alkali and the alkaline stable lipids were fractionated on silicic acid column chromatography.3. Sphingolipids were isolated and characterized as ceramide monohexoside (CMH) and ceramide 2-aminoethylphosphonate (CAEP). Sphingomyelin and ceramide 2-N-methylaminoethylphosphonate were not found in the alkaline stable lipids fraction.4. CMH was composed of galactosyl ceramide which contained octadecasphingadienine as the main long chain bases and hexadecanoic acid as the main fatty acids.5. Ceramide moiety of CAEP was mainly composed of hexadecanoic acid and octadecasphingadienine, and only 2-aminoathylphosphonic acid was detected in the water-soluble components of CAEP.