Phospholipids in Mediterranean Cephalopods Vassilia J. Sinanoglou and Sofia Miniadis-Meimaroglou* Food Chemistry Laboratory, Department of Chemistry, University of Athens, Panepistimiopolis Zographou, 15771 Athens, Greece. Fax: (301) 7228815; (301) 7483415. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 55c, 245-255 (2000); received September 29/November 2, 1999 Cephalopods, Octopus Lipids, Phospholipids Polar lipids of the cephalopods Eledone moschata, Sepia officinalis and Todarodes sagittatus mantle, represent 50.5%, 66.1% and 74.2% of wet tissue respectively. On the other hand the polar lipids of these three species of cephalopods constitute of 80.8%, 94.8% and 93.7% of phospholipids, respectively. The main phospholipids identified were phosphatidylcholine (52.2, 51.3 and 58.4% of total phospholipids respectively in the above mentioned species), phosphatidylethanolamine (18.1, 19.7 and 23.9%), sphingomyelin (10.7, 15.2 and 6.7%), lyso- phosphatidylcholine (3.1, 3.8 and 1.8%) and the unusual lipid ceramide aminoethylphospho- nic acid (15.9, 10 and 9.2%). The 56.8% of phosphatidylcholine in Eledone moschata, the 46% in Sepia officinalis and the 74.1% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glycerocholine and the remaining percentage refer to the structure of l-oalkyl-2-acyl-glycerocholine or l-o-alkyl-l- enyl-2-acyl-glycerocholine. The 87.2% of phosphatidylethanolamine in Eledone moschata , the 81% in Sepia officinalis and the 90.7% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glyceroethanolamine and the remaining percentage refer to the structure of l-o-alkyl-2-acyl-glyceroethanolamine or l-oalkyl-l-enyl-2-acyl-glyceroethanolamine. The major saturated fatty acids in phosphatidylcholine and phosphatidylethanolamine were C16:0 (30.3-67.5% and 23.2-54.5%) and C18:0 (3.6-17% and 15.4-28%), respectively, while the major unsaturated fatty acids in these lipids were C18:ln-9, n-7 (1.0-7.3% and 5.3-10.5%), C20:5n-3 (1.5-9.8% and 4,5-15.8%) and C22:6n-3 (12.5-42.0% and 7.0- 11.3%), respectively. Introduction a limited amount of information on the complete Cephalopod molluscs (octopuses, squids, etc) composition and structure of the molecular species comprise one of the main fishery food resources of mollusc flesh phospholipids (De Koning, 1993). commercially available in Greece, representing a Membrane phospholipids are a complex mixture high proportion of fishery products consumed. of molecular species containing a variety of fatty Phospholipids are the major structural lipids in acyl acid head group compositions. In general, fish classes and one of the major constituents in phospholipids contain higher proportions of the the cell membrane of plant animal and fungal or­ longer chain polyunsaturated fatty acids. It is ganisms. Although the lipid composition of many widely accepted that chemical and physical prop­ marine molluscs is well documented, there is only erties of cell membranes depend on the phospho­ lipid composition, which can affect membrane flu­ idity which in turn alter the activity of many Abbreviations: El electron ionization, ES electrospray, membrane-bound proteins. Therefore, altering the FA fatty acids, GC gas chromatography, HPLC high per­ phospholipid profile may bring about significant formance liquid chromatography, HPTLC high perfor­ biological consequences (Stubbs and Smith, 1984; mance thin layer chromatography, 1-PC lyso-phosphati- dylcholine, MS mass spectrometry, NL neutral lipids, PC Muderhwa and Brockman, 1992; Zakim et al., phosphatidylcholine, PE phosphatidylethanolamine, 1992). PhL phospholipids, PL polar lipids, PnL phosphonoli- Objective of the present work is the quantitative pids, PUFA polyunsaturated fatty acids, Shm sphingo­ and qualitative determination of mantle phospho­ myelin, SPE solid phase extraction, TFA total fatty acids, TLC thin layer chromatography, TL total lipids, lipid composition of three edible species of cepha­ TPhL total phospholipids. lopods (Eledone moschata, Sepia officinalis and 0939-5075/2000/0300-0245 $ 06.00 © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D 246 V. J. Sinanoglou and S. Miniadis-Meimaroglou • Phospholipids in Mediterranean Cephalopods Todarodes sagittatus ) from Saronicos Bay 10 silica gel Chromarods (Type SII). The polar lip­ (Greece), focusing on the isolation and identifica­ ids were developed in chloroform/methanol/water tion of the structure of the molecular species of (70:35:3.5, by vol.) to a height of 17 cm, and the the main phospholipids [phosphatidylethanolam­ rods were scanned in an Iatroscan TH-10 Ana­ ine (PE) and phosphatidylcholine (PC)]. lyzer, Mark II (Iatron Laboratories, Inc., Tokyo, Japan.), equipped with a hydrogen flame ioniza­ Materials and Methods tion detector and connected to an integrator. The flame ionization detector was operated with a hy­ Animals used drogen flow-rate of 160 ml min ' 1 and an air flow- Thirty speciments (six composite samples, five rate of 2 1 min- 1 The scanning speed was 240 mm cephalopods per composite) from three classes of min-1. A two-pen linear recorder (Fischer Recor- cephalopod molluscs Eledone moschata (Mollusca, ball, Model 5000) was used at 10 mV full-scale de­ Cephalopoda, Octopodidae), Sepia officinalis flection and a chart speed of 0.47 cm s_1. The stan­ (Mollusca, Cephalopoda, Sepiodea) and Todar­ dards used were ceramides, cardiolipine, odes sagittatus (Mollusca, Cephalopoda, Teuthoi- phosphatidylcholine, phosphatidylethanolamine, dae) were collected from Saronicos Bay (Greece) lyso-phosphatidylcholine, lyso-phosphatidyletha- in November 1996. They were brought to the labo­ nolamine, phosphatidylserine, sphingomyelin stan­ ratory alive in sea water and immediately analysed dards of the Sigma Chemical Co (Sigma-Aldrich as described below. Company, Dorset, U. K. and St. Louis, MO). Extraction and separation of polar from Isolation and purification of individual neutral lipids phospholipids The mantle from each of the above inverte­ Individual phospholipid components were reco­ brates, after measuring the total weight of wet tis­ vered from chromatoplates after polar lipids sepa­ sue, were homogenized in an Omni-Mixer (Sorvall ration by preparative TLC using a solvent system Inc. Newtown, Connecticut, USA; 0 °C, 5 min at consisting of chloroform/methanol/glacial acetic medium speed). Extraction of lipids was essen­ acid/water (50:25:6:2, by vol.). The individual tially effected according to Bligh and Dyer (1959). spots, visualized by exposure to iodine vapors, After phase equilibration, the lower chloroform were scraped into a vial and extracted from the layer (total lipids) was removed, concentrated in a silica gel using the solvent system of the Bligh- rotary evaporator to dryness, redissolved in 2 0 0 ml Dyer procedure, i.e. chloroform/ methanol/ water of chloroform/methanol (9:1, by vol.) and stored (2:2:1, by vol.). After phase separation, chloroform at 0 °C. extracts were evaporated and the residual lipids Total lipids were fractionated by solid phase ex­ were used for further chromatographic analysis. traction (SPE) using the method of Berger et al. The extracts of individual lipids were rechromato­ (1992). Phase separation of neutral and polar lip­ graphed on HPLC for confirmation of purity. ids was achieved by the same method as modified by Mastronicolis et al. (1996). HPLC of phospholipids (Andrikopoulos et al., 1986) Iatroscan Analysis of polar lipids Individual phospholipid components containing Lipid mixtures were separated on silicic acid- 1 0 mg of lipids were dried and the residue was re­ coated quartz rods, Chromarods (Type SII), and dissolved in chloroform to a solution 10%. Quanti­ then quantitated by passing the rods through a hy­ tative and qualitative analysis, was performed with drogen flame ionization detector (F. I. D.) a Jasco 880-PU HPLC, (Intelligent HPLC Pump (Kramer, 1980; Tanaka et al ., 1980). Mixtures of Jasco 880-31 Solvent Mixing Module) chromatog- polar lipids were evaporated to dryness and redis­ rapher, equipped with a Jasco 875 UV spectromet- solved in chloroform/methanol (9:1, by vol.) at a ric detector at 214 nm. Phospholipid components concentration of 0.03 g ml-1. From each solution were extracted on a Partisil SCX 10 |im column of polar lipids, 0 .5 - 1.0 /d was spotted on each of (250x4.6, 25 mm i.d.) (Jones Chromatography Ltd, V. J. Sinanoglou and S. Miniadis-Meimaroglou • Phospholipids in Mediterranean Cephalopods 247 Mid Glamorgan, UK). The isocratic system aceto- Merck, Darmstadt, Germany). Solvent systems nitrile/methanol/water (350:150:35 v/v/v) was used consisted of chloroform/methanol/acetic acid / as the mobile phase at a flow-rate of 1.5 ml min- 1 water (50:25:6:2, by vol.) and of chloroform/meth­ in a press of 34-40 kg cm '2. A Hewlett Packard anol/water (65:25:4, by vol.). Visualization of spots HP3396A integrator (Hewlett Packard, Palo, Alto, was effected by exposure to iodine vapors, by CA.) was used. spraying with ninhydrin reagent (solution 0 .2 % The solvents used were Lichrosolv solvents gra­ (w/v) of ninhydrin in ethanol) for detection of dient grade (E. Merck, Darmstadt, Germany). The amino-groups and by spraying with phospho­ standards used were phosphatidylcholine, phos- molybdenum blue reagent (Dittmer and Lester phatidylethanolamine, lyso-phosphatidylcholine, reagent) for detection of phospholipids. The stan­ lyso-phosphatidylethanolamine, phosphatidyls- dards used were phosphatidylcholine, phosphati-