Eobania Vermiculata

Lipid Composition and Structural Studies on Lipids from the Land Snail Eobania vermiculata Helen J. Stavrakakis*, Sofia K. Mastronicolis, and Vassilios M. Kapoulas Laboratory of Food Chemistry, School of Natural Science, National University of Athens, 13a Navarinou str., Athens 10680, Greece Z. Naturforsch. 44c, 597—608 (1989); received January 3/ April 27, 1989 Aminoethylphosphonate, Sphingophosphonolipids, Glycerylethers, Phospholipids, Mollusca The total lipids of the commercial land snail Eobania vermiculata (Gastropoda, Pulmonata, Stylommatophora) 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-aminoethylphosphonate, 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 phos phatidylcholine. A significant amount (70.5%) of unsaturated fatty acids is concentrated in neu tral lipids. The C,6:() alk-l-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. Introduction phospholipids which are abundant in this phylum. Mollusca phospholipids have been studied rather Thus, the phosphonolipid ceramide aminoethyl phosphonate, originally discovered by Rouser et al. intensively with respect to some interesting compo [1] in the sea anemona Anthopleura elegantissima, nents such as phosphonolipids and glycerylether appears to be widely distributed within the phylum Mollusca [2—11], usually accompanied by a variety Abbreviations: STE, sterol esters; TG, triglycerides; FA, of other sphingophosphonolipids [12—17]. More fatty acids; FA1, fatty alcohol; Choi, cholesterol; GE, gly over, mollusca are very rich in ether-containing ceryl ethers; PE, phosphatidylethanolamine; PC, phos phatidylcholine; SPM, sphingomyeline; Car, cardiolipin; phospholipids of both, the alkyl and alk-enyl types AEPn, 2-aminoethylphosphonic acid; TMSi, trimethylsilyl- [18-22], derivatives; Pnl, phosphonolipids; SPnL, sphingophos Although the lipid composition of marine mollusca phonolipids; TLC, thin layer chromatography; IR, in is well documented, there is only a limited amount of frared; NL, neutral lipids; TL, total lipids; PL, phos pholipids; P, phosphorus; GLC, gas liquid chromatography; information on the lipid composition of the terres MAH, mild alkaline hydrolysis; MAS, mild alkaline stable; trial mollusca. Cepaea nemoralis is the only land snail GPE, glycerylphosphorylethanolamine; GPC, glyceryl- which has been extensively studied concerning its phosphorylcholine; DMA, dimethyl acetals; St, standards; Cerb, cerebrosides; GPnl, glycerylphosphonolipids; L-PE, lipid composition and metabolism [23—28]. Some lyso-PE; L-PC, lyso-PC. other representatives of this order (Stylommatopho * Present address: General State Chemical Laboratory, ra) such as Euhadra herklotsi [29], Arianta arbus- Corfu, Greece. torum [30], Succinea putris [31], and Helix pomatia Reprint requests to Dr. S. Mastronicolis. [32—34] have been studied with regard to their fatty Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen acid composition. Althaus et al. [35] have investi 0341 - 0382/89/0700 - 0597 $01.30/0 gated the lipid composition of the single nerve cells 598 H. J. Stavrakakis et al. • Lipids from the Land Snail Eobania vermiculata of Helix pomatia and Thompson and Hanahan [18] homogenized with, 1800 ml chloroform —methanol— have studied the glyceryl ether phospholipids of two water 1:2:0.8 (v/v/v), centrifuged again, and the two species of terrestrial slugs, Arion ater and Ariolimax extracts were combined. To the pooled extracts columbianus. (1530+ 1770 ml), 1100 ml chloroform and 1100 ml In a long-term research program on the distribu water were added. They were vigorously mixed and tion of phosphonolipids in “Jelly fishes” [72] and in left for phase equilibration. The lower chloroform edible animals, we were tempted to investigate the layer was evaporated to dryness in a rotary evap lipids of the edible land snail, E. vermiculatac phy orator and redissolved in 100 ml of chloroform — lum: Mollusca, class: Gastropoda, order: Stylomma- methanol 9:1 (v/v). tophora) which is widespread in Greece and other A portion of the total lipid extract was evaporated European countries. to dryness, redissolved in a minimal amount of In a previous paper [36] we reported the presence chloroform—methanol 98:2 (v/v) and applied on a of complex glyco- and phosphonolipids among the column (4.5 x 13.3 cm) packed with 10 g of silicic water-methanol soluble lipids (“acidic lipids”) of acid activated overnight at 120 °C and mixed with 5 g Eobania. In this paper we describe the qualitative of Hyflo Super Cel (filter aid). Elution was per and quantitative composition of the lipids of E. ver formed with solvent mixtures of increasing polarity miculata, which were found to be rich in phos as indicated in Table I. Monitoring was effected by phonolipids and glyceryl ether phospholipids. TLC (see Fig. 1 and 2). Experimental Preparation of derivatives Materials Fatty acid methyl esters and aldehyde dimethyl- acetals for GLC analysis were prepared by dry-acid Silica gel G, type 60 was purchased from Merck: methanolysis [38] of the lipid fractions followed by silicic acid for column chromatography, 100 mesh, extraction with petr.ether (b.p. 40—60 °C), three from Mallinckrodt; cardiolipin, sphingomyelin, times, and purification by preparative TLC with phosphatidylethanolamine, lyso-phosphatidyletha- petr. ether—diethyl ether—acetic acid 70:30:1 (v/v/v) nolamine, lyso-phosphatidylcholine, phosphatidic as developing solvent. acid, phosphatidylserine, phosphatidyl inositol were For the preparation of isopropylidene derivatives from SERVA; L-a-glycerophosphorylethanolamine, of glycerylethers, the phosphatidylcholine fraction phosphatidylcholine, L-a-glycerophosphorylcholine, was submitted to prolonged acid hydrolysis [39, 40], 2-aminoethyl-phosphonic acid, d , L-chimyl alcohol, followed by extraction of the fatty acid methylesters d , L-batyl alcohol, ceramide, and sphingosine were for GLC analysis. The remaining methanol layer was from SIGMA. made strongly alkaline and the glycerylethers were Adult organisms of E. vermiculata were collected extracted with ethyl acetate (three times). They were in coastal areas of East Crete (Greece), in August. purified by preparative TLC with petr. ether—diethyl ether—acetic acid 30:70:1 (v/v/v) as developing sol Extraction and fractionation of lipids vent mixture and they were converted to their iso The animals were fed with flour for two days and propylidene derivatives according to Hanahan et al. kept in a perforated box at room temperature for [41]- approximately one month before lipid extraction. For preparation of trimethylsilyl derivatives of The total weight (with shells) of 390 specimen was long chain bases, a portion of purified sphingolipid 1276 g (wet tissue, 850 g). was submitted to acid methanolysis [42] followed by Their lipids were extracted by a modification of extraction of fatty acid methylesters (three times) the Bligh-Dyer method [37] as follows: To the intact with petr.ether (b.p. 40—60 °C). The remaining snails (with shells) 1800 ml chloroform—methanol aqueous-methanolic layer was evaporated to dryness 1:2 (v/v) were added and immediately homogenized in a stream of nitrogen. The residue was dissolved in in an Omni-Mixer (0 °C, 5 min at medium speed). 2 ml 2 n NaOH and the long chain bases were ex The liquid phase was separated by centrifugation tracted with ethyl acetate (three times). After purifi ( —4 °C, 10 min, 3000 rpm) and the residue was re cation by preparative TLC, they were converted to H. J. Stavrakakis et al. • Lipids from the Land Snail Eobania vermiculata 599 their TMS derivatives according to Sweeley et al. IR analysis was performed in a Perkin-Elmer IR [43]. spectrometer, model 157 with sodium chloride Mild alkaline hydrolysis (deacylation) [44] was prisms. used for the preparation of mild alkali stable compo Paper chromatographic analysis of water soluble nents and of the respective water soluble products products was carried out on paper whatman No. 1 for further analysis or identification. with the ascending

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