Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster)

Chemistry and Physics of Lipids 140 (2006) 55–65

Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster) Theodosia F. Garofalaki a , Sofia Miniadis-Meimaroglou a,∗ , Vassilia J. Sinanoglou b a b

Food Chemistry Laboratory, Department of Chemistry, University of Athens, Greece Food Chemistry Laboratory of Technological Educational Institution of Athens, Greece

Received 7 September 2005; received in revised form 22 December 2005; accepted 13 January 2006 Available online 7 February 2006

Abstract The total lipids of muscle and cephalothorax of Mediterranean lobster Palinurus vulgaris were found to be 1.0% and 2.4% of the wet tissue of which the phospholipids represented 66.5% and 47.5%, respectively. The main PhL saturated, monounsaturated and polyunsaturated fatty acids in muscle and cephalothorax were C16:0, C18:0, C18:1␻-9, C18:1␻-7, C20:4␻-6, C20:5␻-3 and C22:6␻-3. 2-OH C14:0 and cyclo-17:0 fatty acids were also identified though in low percentages. The main individual PhL in muscle were found to be phosphatidylcholine (53.5%), 72.0% of which corresponded to the structure of 1,2-diacyl-glycerocholine while the rest 28.0% to 1-O-alkyl-2-acyl-glycerocholine or 1-O-(1-alkenyl)-2-acyl-glycerocholine and phosphatidylethanolamine (19.3%), 75.0% of which corresponded to the structure of 1,2-diacyl-glyceroethanolamine and 25% to 1-O-alkyl-2-acyl-glyceroethanolamine or 1-O-(1-alkenyl)-2-acyl-glyceroethanolamine. Cephalothorax main PhL were found to be PC and PE (66.4% and 18.8%, respectively). In muscle and cephalothorax PC ␻-3 fatty acids amounted 7.78% and 8.60%, while in PE amounted 30.77% and 23.65% respectively. Furthermore, in both tissues PhL, cardiolipine phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatidylcholine, were also found. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Crustacean; Fatty acids; Lipids; Lobster; Palinurus vulgaris; Phospholipids

Abbreviations: CL, cardiolipine; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EI, electron ionization; ES, electrospray; GC, gas chromatography; GL, glucolipids HPTLC high performance thin layer chromatography; LPC, lyso-phosphatidylcholine; LPE, lysophosphatidylethanolamine; MS, mass spectrometry; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PhL, phospholipids; PL, polar lipids; PS, phosphatidylserine; PI, phosphatidylinositol; PUFA, polyunsaturated fatty acids; Sph, sphingomyelin ∗ Corresponding author at: Food Chemistry Laboratory, Department of Chemistry, University of Athens, Panepistimioupolis Zographou, 15701 Athens, Greece. Tel.: +30 210 7274486; fax: +30 210 7228815/483415/3624870. E-mail addresses: [email protected] (S. Miniadis-Meimaroglou), v [email protected] (V.J. Sinanoglou).

1. Introduction Phospholipids, located mainly in biological membranes, have an essential role in regulating biophysical properties, protein sorting and cell signalling pathways. The phospholipids of a majority of marine species including fish, crustaceans and molluscs are rich in PUFA, especially in the ␻-3 acids family, the latter being concentrated on the sn-2 position of the glyceryl backbone of the molecule (Soudant et al., 1995). These PUFA with 20 and 22 carbon atoms and more than three double bonds (EPA and DHA) have a vital role in human nutrition, disease prevention and health promotion.

0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2006.01.006

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The study of the crustaceans presents an increasing nutritional, commercial and economical importance due to the fact that they represent some of the widely consumed species of the Mediterranean. These species spread from Iceland and Norway to the Atlantic Ocean and the Eastern Mediterranean Sea. The ideal environment for their survival is a soft, muddy bottom, between 10 and 300 m of depth. In general, lobster of the Palinuridae family are thought to be omnivorous showing preference to sloth organisms, scavenge easily, thus meaning wounded or recently killed fish, gastropods, mollusks, several crustaceans, echinoderms etc (Kanciruk, 1980; Papoutsoglou, 1989). Nutritional habits, age and sex of the crustaceans, season of the year, temperature and salinity of the water where they survive, as well as the way of their fishing, influence the distribution and variation in total lipids, lipid classes and fatty acids composition (Chapelle, 1986; Clarke and Holmes, 1986; Chen and Jenn, 1991; Kanazawa and Koshio, 1994; Montano and Navarro, 1996; Sargent et al., 2002). The present study has been orientated towards the determination of the PhL composition of the edible crustacean Palinurus vulgaris (spiny lobster), as well as the identification of the main individual PhL classes and their fatty acid profile. 2. Experimental procedures 2.1. Reagents and standards The lipid standards used (purity > 98%) were galactosyldiglycerides, cerebrosides, cardiolipine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine and sphingomyelin and they were purchased from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). Fatty acid methyl esters used as GC and GC/MS standard were: lauric acid M-E, L7272, cis5,8,11,14,17-eicosapentaenoic acid M-E, E2012 and cis-4,7,10,13,16,19-docosahexaenoic acid M-E, D2659 (purity ≥ 98%) purchased also from Sigma Chemical Co.; Matreya Bacterial Acid Methyl Esters CPTM Mix, Catalog No. 1114; SupelcoTM 37 Component FAME Mix, Catalog No. 47885-U. All solvents used for sample preparation were of analytical grade and the solvents used for MS analyses were of HPLC grade from Merck (Darmstadt, Germany). Double distilled water was used throughout this work, while all reagents used were of analytical grade purchased from Mallinckrodt Chemical Works (St. Louis,

MO) and from Sigma Chemical Co (Sigma–Aldrich Company, St. Louis, MO). 2.2. Experimental animals—total lipid extraction and separation An average of 20 adult lobsters (P. vulgaris, two batches, each including ten adult lobsters) were collected from Argolicos Bay (one of the most important fishing places) during 2000. They were brought to the laboratory alive, where they were weighed and immediately processed. More explicitly the organisms were dissected and the cephalothorax and muscle were weighed and separated. Muscle and cephalothorax parts were homogenized separately and extraction of lipids was performed according to the Bligh and Dyer method (1959). After phase equilibration, the lower chloroform layer (total lipids) was removed and dried in a rotary evaporator. The extracted lipids were weighed to determine the total lipid (TL), then redissolved in chloroform/methanol (9:1, v/v.) and finally stored at a temperature of 0 ◦ C. Total lipids were afterwards separated into neutral and polar lipids on silicic acid columns (Merck and Co., Kieselgel 60), by solid phase extraction (SPE), using the modified method of Mastronicolis et al. (1996). Polar lipid fractions were quantitated by weight after being eluted from solid-phase extraction columns. In order to determine the polar lipids composition percentage, they were separated on silicic acid-coated quartz rods (Chromarods, Type SII) and they quantitated by passing the rods through a hydrogen flame ionization detector (F.I.D.) (Sinanoglou and Miniadis-Meimaroglou, 2000). Chromarods were developed with a solvent system consisting of chloroform/methanol/acetic acid/water (50:25:6:2, v/v/v/v) to a height of 17 cm to detect the polar lipids. The rods were then scanned in an Iatroscan TH-10 Analyser; Mark II (Iatron Laboratories, Inc., Tokyo, Japan.) and connected to an integrator. The flame ionization detector was operated with a hydrogen flow-rate of 160 mL/min and an air flow-rate of 2 L/min. 2.3. High performance thin layer chromatography (HPTLC) of polar lipids and individual phospholipids Polar lipid composition was determined by onedimensional HPTLC (1D-HPTLC), using the following development solvent systems: solvent A, consisting of chloroform/methanol/water (65:25:4, v/v/v) and solvent B, consisting of chloroform/methanol/glacial acetic acid/water (50:25:6:2, v/v/v/v). Individual phos-

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pholipids were further separated by two-dimensional HPTLC analysis. Quantities of 10–20 ␮g of sample were applied as spots at the lower right-hand corner of the plate −1.0 cm from each edge. The first dimension was developed with solvent B and the second dimension with solvent A. Silica gel 60-G HPTLC plates (E. Merck, Darmstadt, Germany) with a size of 10 cm × 10 cm and a layer thickness of 0.25 mm were used. Substances were visualised by exposure to iodine vapours, glycolipids, aminogroups, choline and phospholipids were detected by spraying with: a-naphtholsulfuric acid reagent (Jacin and Mishkin, 1965) ninhydrin reagent [solution 0.2% (w/v) of ninhydrin in ethanol] Dragendorff reagent (Kates, 1986) and phosphomolybdenum blue reagent (Dittmer and Lester, 1964), respectively. Phosphonolipids were detected after spraying with phosphomolybdenum blue reagent combined with the Stillway and Harmon (1980) heating test. 2.4. Fractionation of polar lipids by preparative TLC The polar lipid extract was applied at the head of a silica gel plate as a narrow band. The chromatogram was developed using solvent A. Bands, usually containing more than one lipid, were visualised with iodine vapour, scraped off and then extracted from the silica gel using the solvent system of the Bligh-Dyer procedure. After phase separation, chloroform extracts were evaporated and the residual phospholipids were subjected to a twodimensional HPTLC fractionation. 2.5. Quantitative analysis of polar lipids and individual phospholipids Total phosphorus and phosphonate phosphorus were determined according to Kapoulas et al. (1984) method, esters were determined by the Snyder and Stephens (1959) method, glyceryl ethers by the Hanahan and Watts (1961) method and plasmalogens by the Gottfried and Rapport (1963) method. Mild alkaline hydrolysis was performed with 0.1 mL NaOH (1.2 M) in a methanol solution (50%, v/v) and 45 ◦ C for 20 min (Wells and Dittmer, 1966). Chloroform-soluble products of phospholipid components were analysed by one-dimensional TLC analysis carried out on precoated 20 × 20 silica gel 60 G plates (E. Merck, Darmstadt, Germany) using solvents A and B. Visualization of spots was effected by exposure to iodine vapours, by spraying with ninhydrin reagent and with phosphomolybdenum blue reagent (Dittmer and Lester reagent). Water-soluble products of phospholipid components

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were analysed by paper chromatographic analysis. The latter was carried out on Whatman paper No. 1 with an ascending (6–7 h) and descending (7–8 h) technique using solvent systems that consisted of phenol solution/water/ethanol/acetic acid (80:20:12:10, v/v/v/v) and of phenol solution/ethanol/acetic acid (100:12:10, v/v/v), respectively. Spots were once sprayed with ninhydrin reagent, then with a Hanes–Isherwood reagent for phospholipid detection (Hanes and Isherwood, 1949) and finally with a Dragendorff reagent. 2.6. Electrospray ionization mass spectrometry of phospholipids Phospholipids were identified by electrospray ionization mass spectrometry. ESI mass spectra were acquired on a Fisons VG Quatro SQ, SN 5147 triple quadrupole mass spectrometer (Fisons Instruments, Altrincham, WA) (Conditions: Q, SN 5147), equipped with an electrospray interface and data-handling system (Fisons Instruments). Aliquots (10 ␮L) of the sample solution containing 10−3 –10−4 mol L−1 , were injected directly into the electrospray source via a Rheodyne loop 7125 (Fisons Instruments) into a stream of solvent (methanol/water 80:20, v/v. containing 1% ammonium acetate) at a flow-rate of 2 ␮L/min. Parameters for anions (ES−): capillary 3.00 kV, HV lens 0.46 kV, focus 40 kV, skimmer 46 kV, source temperature 80 ◦ C, ion energy 1.9 V, mass range 100–1000. Parameters for cations (ES+): capillary 3.30 kV, HV lens 0.38 kV, focus 40 kV, skimmer 45 kV, source temperature 70 ◦ C, ion energy 2.4 V, mass range 100–1000. The mass spectrometer was scanned over the m/z range of interest and the mass scale was calibrated by injected solutions of standards in methanol/water (80:20, v/v) containing 1% ammonium acetate. 2.7. Gas chromatography/mass spectrometry analysis of fatty acid methyl esters Methyl esters of the fatty acids contained in phospholipid fractions were prepared as follows: a sample containing 20–50 mg of lipids was dried before transesterification and the residue was redissolved in 0.75 mL n-hexane; then 0.1 mL of 2 N potassium hydroxide in methanol was added and the solution was mixed for 2 min in a vortex mixer (Velp scientifica, Italy), dried over anhydrous sodium sulphate and left for 25 min. After phase separation the upper layer of n-hexane containing the fatty acid methyl esters was removed and immediately injected into the gas chromatograph (Sinanoglou and Miniadis-Meimaroglou, 1998).

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Quantitative and qualitative analysis were performed on a Hewlett-Packard 6890 gas chromatograph, equipped with a flame ionization detector. The following capillary columns were used: column A, capillary SGE BPX-70, Model Number 054606, (70% cyanopropyl liquid phase; 25 m × 0.32 mm id × 0.25 ␮m film thickness} and column B, capillary HP-23 cis/trans FAME Column, Model Number HP 19091 H-133, (30 m × 0.25 mm i.d. × 0.25 ␮m film thickness}. Helium was used as carrier and make up gas at a pressure of 15 psi. The split less injector temperatures for column A and B were 220 and 250 ◦ C and the detector temperatures were 275 and 300 ◦ C, respectively. For column A the temperature was programmed at 100 ◦ C for 0 min, raised from 100 to 200 ◦ C at a rate of 5 ◦ C min−1 and held constant at 200 ◦ C for 2 min, then raised from 200 to 230 ◦ C at 10 ◦ C min−1 and held at 230 ◦ C for 5 min. The duration of the analysis was 30 min whilst the He flow-rate was 1.0 mL/min. For column B the temperature was programmed at 100 ◦ C for 0 min, raised from 100 to 200 ◦ C at a rate of 4 ◦ C min−1 and held constant at 200 ◦ C for 3 min. The duration of the analysis was 28 min and the He flow-rate was 6.7 mL/min. The make-up gas was atmospheric air at a flowrate of 400 and 450 mL/min, respectively. Meanwhile, hydrogen was supplied to the FID at a flow-rate of 40 mL/min. The qualitative analysis was performed on a HRGC Mega 2 Series 8560 MFC 800 (Fisons Instruments) gas chromatograph, equipped with a mass spectrometer VG Trio-2000 Mass Spectrometer (Fisons Instruments) replacing the previously used FID; a fused silica capillary column of high polarity was used (Supelco SP 2340; 60 m × 0.32 mm i.d. and 0.2 ␮m film thickness; Supelco Inc., Bellefonte, PA, USA). This polymeric stationary phase was a non-bonded poly(biscyanopropylsiloxane). Hydrogen used as carrier gas at a pressure of 15 psi. The make-up gas was atmospheric air at a pressure of 15 psi. The injector’s and detector’s temperature was 350 and 250 ◦ C, respectively. The temperature was programmed at 150 ◦ C for 15 min, raised from 150 to 170 ◦ C at a rate of 2 ◦ C min−1 , held constant at 170 ◦ C for 10 min, raised from 170 to 215 ◦ C at 4 ◦ C min−1 and held at 215 ◦ C for 15 min. The duration of the analysis was in total 62 min (Sinanoglou and Miniadis-Meimaroglou, 1998). Electron ionization (E.I.) was produced by accelerating electrons from a hot filament through a potential difference at the standard value of 70 eV. In both GC and GC/MS methods, the fatty acid methyl esters peaks were identified by comparison of their retention times to those of the standard mixtures.

2.8. Statistical analysis All measurements were performed at least in triplicate (n = 3) and values were averaged and reported along with the standard deviation (S.D.). 3. Results and discussion 3.1. Lipid content and polar lipid composition of P. vulgaris The total lipid content of the whole organism, the muscle as well as the cephalothorax of the studied crustacean, represented 1.6%, 1.0% and 2.4% of the wet tissue, respectively. Since cephalothorax contains the hepatopancreas of the lobster, its concentration in total lipids was found significantly higher than the one found in the muscle tissue. Similar data concerning the lipid content (whole body) of 11 lobster and prawn species (0.7–1.5% of the wet tissue) as well as the lipid content of other decapoda crustacean from the Gulf of Mexico (0.5–8.9% of the wet tissue) have also been reported by Pearson (1977) and Donnelly et al. (1993), respectively. The lipid content of the muscle tissue was comparable to that reported for the New Zeeland lobster, Jasus lalandii (Bannatyne and Thomas, 1969) as well as five other lobsters from Sydney Retail (Krzynowek and Murphy, 1987), (1–1.2% of the wet tissue), for Homarus vulgaris (0.83% of the wet tissue) (Chapelle, 1977) while a slightly higher lipid content was found for Homarus americanus (1.84% of the wet tissue) (Papoutsoglou, 1989). It should be noted that data on cephalothorax total lipid content are not available; there is only a limited amount of information on the total lipid content of the hepatopancreas, which were for J. lalandii (de Koning and McMullan, 1966) and H. vulgaris (Chapelle, 1977) 12.5% and 10.5% of the wet tissue, respectively. The polar lipid proportion in muscle total lipids was rather different to that found in the cephalothorax (68.6 ± 0.7% and 54.1 ± 0.4% of TL). Polar lipids in both tissues consist mainly of phospholipids (97.0 ± 0.5% and 87.8 ± 0.7% of PL, respectively). The results of the quantitative analysis of phospholipid and glycolipid classes based on the Iatroscan analysis are given in Table 1. Phosphatidylethanolamine and phosphatidylcholine were the major phospholipid components. The PE content in both, muscle and cephalothorax PL was quite similar (18.7 ± 1.5% and16.5 ± 0.2% of PL, respectively) whereas the PC content was different (51.9 ± 0.6% and 58.3 ± 0.5% of PL, respectively).

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Table 1 Polar lipids profile of the crustaceans studied as determined by Iatroscan Sample

GL % of PL

CL % of PL

PE % of PL

PS, PI % of PL

PC % of PL

Sph % of PL

l-PC % of PL

Muscle Cephalothorax

3.0 ± 0.5 12.2 ± 1.5

14.5 ± 1.2 3.4 ± 0.1

18.7 ± 1.5 16.5 ± 0.2

3.2 ± 0.4 4.7 ± 0.1

51.9 ± 0.6 58.3 ± 0.5

8.2 ± 0.2 3.4 ± 0.2

0.5 ± 0.1 1.5 ± 0.1

FID area percentages were calculated according to [total weight (area lipid/area total lipid) × 100]. Data are expressed as wt. % of polar lipids and represent means ± standard deviation of four replicate determinations (confidence interval: 99%).

The muscle PL were found to contain a quite high percentage of CL and Sph (14.5 ± 1.2% and 8.2 ± 0.2% of PL, respectively) and a rather low of GL (3.0 ± 0.5% of PL) while the cephalothorax PL, contain a high percentage of Gl (12.2 ± 1.5% of PL and a low one of Cl and Sph (3.4 ± 0.2% and 3.4 ± 0.1% of PL, respectively). The high glycolipid content of the cephalothorax is probably due to the lobsters diet, part of which is composed of echinoderms, which themselves are rich in glycolipids (Joseph, 1989). It should be noted that phosphonolipids were not found. The lipid phosphorous, ester, glyceryl-ether and plasmalogen content of muscle polar lipids were found to be 1.2 ± 0.02, 2.14 ± 0.07, 0.249 ± 0.006 and 0.181 ± 0.005 ␮mol/mg PL, respectively, which can only mean that phospholipid molecules are mainly glyceryl-ester analogues (79% of total PhL). The glyceryl-ether analogues represented the remaining 21% of total PhL, 71.4% of which were vinyl-ether analogues. 3.2. Fatty acid composition in muscle and cephalothorax polar lipids Data on fatty acid composition of polar lipids, as well as the proportion of the saturated, monounsaturated and polyunsaturated fatty acids of the polar lipid fractions are given in Table 2. Seven of the fatty acids identified in the muscle PL, were found in percentages that reached or exceeded 4.37% of the TFA: C16:0, C16:1 ␻-7, C18:0, C18:1 ␻-9, C20:4␻-6, C20:5 ␻-3 and C22:6 ␻-3. In the cephalothorax PL eight fatty acids were determined at levels higher than 3.15% of the TFA: C16:0, C16:1 ␻-7, C17:0, C18:0, C18:1 ␻-9, C18:1 ␻-7, C20:4 ␻-6 and C22:6 ␻-3. The main differences observed between muscle and cephalothorax PL fatty acids were: (a) the proportion of saturated, monounsaturated and polyunsaturated fatty acids traced in muscle (28.35%, 19.46% and 52.19%, respectively) and cephalothorax (45.45%, 38.55% and 16.00%, respectively). (b) The fact that C16:0 found in cephalothorax was almost twice as much as that found

in muscle PL. (c) The significantly higher levels of oleic acid (C18:1 ␻-9) (19.92 ± 0.45) and cis-vaccenic acid (C18:1 ␻-7) (6.23 ± 0.04) in cephalothorax PL, where the C18:1 ␻-9/C18:1 ␻-7 isomer ratio had a normal value (3.19), while the same ratio in muscle PL had an unusual Table 2 Fatty acid composition (w/w %) of polar lipid of the crustacean muscle and cephalothorax Fatty Acids

Palinurus vulgaris Muscle

Cephalothorax

C14:0 C14:1 ␻-5 C15:0 C16:0 C16:1 ␻-9 C16:1 ␻-7 Iso-C17:0 C17:0 C17:0 cyclo 2-OH C14:0 C17:1 ␻-7 C18:0 C18:1 ␻-9 C18:1 ␻-7 C18:2 ␻-6 cis C18:2 ␻-6 trans C18:3 ␻-6 C18:3 ␻-3 C20:0 C20:1 ␻-9 C20:2 ␻-6 C20:4 ␻-6 C20:3 ␻-3 C22:1 ␻-9 C20:5 ␻-3 C22:5 ␻-3 C24:1 C22:6 ␻-3

1.00 ± 0.14 0.10 ± 0.01 1.14 ± 0.11 12.16 ± 1.06 – 4.37 ± 0.41 0.33 ± 0.02 1.71 ± 0.10 1.14 ± 0.04 0.57 ± 0.03 – 9.27 ± 1.20 12.05 ± 1.32 1.31 ± 0.25 2.05 ± 0.04 – 0.97 ± 0.07 0.50 ± 0.06 – 1.05 ± 1.08 1.84 ± 0.13 23.02 ± 0.46 – 0.53 ± 0.03 12.79 ± 0.34 0.47 ± 0.03 0.74 ± 0.03 10.56 ± 0.87

2.66 ± 0.02 – 1.93 ± 0.03 23.84 ± 0.07 0.99 ± 0.01 6.92 ± 0.06 0.91 ± 0.02 3.15 ± 0.03 – – 1.43 ± 0.02 13.11 ± 0.23 19.92 ± 0.45 6.23 ± 0.04 – 1.17 ± 0.01 0.98 ± 0.01 0.73 ± 0.03 0.76 ± 0.02 – – 3.84 ± 0.05 1.34 ± 0.01 – 2.15 ± 0.25 – 0.99 ± 0.01 6.85 ± 0.67


␻:0
␻:1
␻:n
␻:3
␻:6

28.35 19.46 52.19 24.32 27.87

45.45 37.39 17.16 9.73 5.99

FID area percententages were calculated according to [total weight (area lipid/area total lipid) × 100]. Data are expressed as wt. % of total fatty acids and represent means ± standard deviation of three replicate determinations (confidence interval: 99%).

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bands, respectively (Table 3). The lipid phosphorous determination in each one of the extracted lipid bands confirmed the individual phospholipid proportion as shown in Table 1. The data on fatty acids as well as the proportion of the saturated, monounsaturated and polyunsaturated fatty acids of the total fatty acids contained in each of the PE and PC phospholipid fractions of both tissues are given in Table 4. The major saturated fatty acids in muscle and cephalothorax PE were C16:0 and C16:0, C18:0, respectively, while the major unsaturated fatty acids in muscle and cephalothorax PE were C18:1 ␻-9, C20:4␻-6, C20:5 ␻-3 and C22:6-␻ 3. In muscle PE the less common fatty acid C24:1 was identified and quantified in a percentage of 6.70 ± 0.36%. C16:1 ␻-7, C18:1 ␻-7 and C20:1 ␻-9 represented a lower percentage. The major saturated fatty acids in muscle and cephalothorax PC were C16:0 and C18:0, while the major unsaturated fatty acids were C16:1 ␻-7, C18:1 ␻-9, C18:1 ␻-7, C20:4 ␻-6, C20:5 ␻-3 and C22:6 ␻-3. PE having a greater amount of polyunsaturated fatty acids is considerable more unsaturated than PC, which is in agreement with reported values (Clarke, 1979). The muscle and cephalothorax PE contained markedly more C20:4␻-6, C20:5␻-3 and C22:6 ␻-3 while the PC contained significantly more C16:0, C16:1 ␻-7 and C18:1␻9 than the corresponding PE.

high value (9.19) (Clarke and Wickins, 1980). (d) The higher quantities of ␻-3 fatty acids (EPA and DHA) in the muscle PL. (e) In muscle PL, EPA was found in more significant amounts than DHA. The latter was found in considerably higher amounts in the cephalothorax PL. (f) Finally, the high levels of C20:4 ␻-6 in the muscle PL (␻3/␻-6 ratio <1). Similar high levels of arachidonic acid have been reported in muscle tissue for the New Zeeland lobster J. lalanddi (Bannatyne and Thomas, 1969). High percentage of C20:4 ␻-6 in PhL fraction were also identified in amphipod crustacean as reported by Dembitsky et al. (1994b). On the contrary, in the cephalothorax of the studied lobster as well as in the muscle tissue of the H. vulgaris as reported by (Chapelle, 1977), the ␻-3/␻-6 ratio >1. Some less common fatty acids have also been detected, such as those having an odd number of carbon atoms [15:0, 17:0, 17:1 (␻-7)], hydroxy (2-OHC14:0), branched (iso C17:0) and cyclo fatty acids (17:0 cyclo); the last two also detected in amphipod crustaceans (Dembitsky et al., 1994a,b,c). 3.3. Determination of individual phospholipids and their bound fatty acid composition in muscle and cephalothorax of P. vulgaris The preparative TLC of P. vulgaris muscle and cephalothorax PL revealed the presence of 12 and 11

Table 3 Identification and relative composition of muscle and cephalothorax polar lipid mixture of P. vulgaris by one-dimensional preparative TLC Spot number on TLC plate by decreasing Rf

Reagent

Identification of spots

N

P

D

S

1-D TLC of muscle PL 12 10, 11 8, 9 7 6 5 4 3 1, 2

− − + + − − + − −

− + + + + + + + +

− − − − − + − − +

+ − − − + − − − −

GDG, CERB CL, PA PE PS PI, l-PE PC Unknown Shm l-PC

1-D TLC of cephalothorax PL 11 9, 10 8 6, 7 4, 5 3 2 1

− + − − − − − +

− + + + + + + +

− − − − + + + −

+ − − + − − − −

GDG, CERB CL, PE N-MMPE PI, l-PE PC Shm, l-PC l-PC Unknown

Reagents: N: ninhydrin, P: molybdenum blue (reagent for phosphorus), D: Dragendorff (reagent for choline); S: a-naphthol (reagent for sugars). +: positive reaction; −: negative reaction.

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Table 4 Fatty acid composition (w/w, %) of phosphatidylethanolamine and phosphatidylcholine of P. vulgaris FA % (w/w) of total phospholipid fatty acids Muscle

Cephalothorax

PE

PC

PE

PC

C14:0 C14:1 ␻-5 C15:0 C16:0 C16:1 ␻-7 Iso-C17:0 C17:0 Cyclo-C17:0 2-OH C14:0 C17:1 ␻-7 C18:0 C18:1 ␻-9 C18:1 ␻-7 C18:2 ␻-6 C18:3 ␻-6 C18:3 ␻-3 C20:0 C20:1 ␻-9 C20:2␻-6 C20:4 ␻-6 C20:3 ␻-3 C22:1 ␻-9 C20:5 ␻-3 C22:5 ␻-3 C24:1 C22:6 ␻-3

1.36 ± 0.51 2.51 ± 0.73 1.00 ± 0.12 13.85 ± 2.91 2.46 ± 0.41 0.46 ± 0.11 3.20 ± 0.38 0.86 ± 0.37 1.46 ± 0.12 – 0.86 ± 0.13 6.04 ± 0.25 0.53 ± 0.14 0.70 ± 0.02 0.46 ± 0.02 4.11 ± 0.02 1.07 ± 0.13 1.35 ± 0.42 1.64 ± 0.26 25.19 ± 0.07 – 0.80 ± 0.18 14.33 ± 2.21 0.76 ± 0.33 6.70 ± 0.36 7.84 ± 1.89

1.35 ± 0.14 0.34 ± 0.02 1.80 ± 0.17 18.94 ± 2.22 8.98 ± 0.54 0.87 ± 0.15 2.11 ± 0.29 2.06 ± 0.18 0.73 ± 0.02 – 5.44 ± 0.23 24.59 ± 0.59 6.47 ± 0.77 2.20 ± 0.11 0.58 ± 0.11 0.30 ± 0.01 0.43 ± 0.92 0.45 ± 0.04 1.07 ± 0.78 11.64 ± 0.20 – 0.54 ± 0.05 4.16 ± 0.37 0.38 ± 0.03 0.96 ± 0.40 3.62 ± 0.47

1.19 ± 0.04 – 1.98 ± 0.01 12.91 ± 0.13 4.78 ± 0.08 – – – – 1.74 ± 0.05 9.63 ± 0.46 15.59 ± 0.25 3.61 ± 0.10 1.04 ± 0.03 0.66 ± 0.02 – – 2.57 ± 0.07 0.84 ± 0.01 17.68 ± 0.15 1.33 ± 0.07 – 18.78 ± 0.48 – – 4.87 ± 0.09

2.10 ± 0.02 – – 21.70 ± 0.20 10.24 ± 0.10 – 2.63 ± 0.03 – – 2.63 ± 0.07 9.53 ± 0.32 25.58 ± 0.56 3.18 ± 0.02 1.16 ± 0.01 0.77 ± 0.04 0.70 ± 0.03 – 2.09 ± 0.13 0.85 ± 0.01 5.97 ± 0.20 1.01 ± 0.01 – 5.50 ± 0.14 – – 3.10 ± 0.03


Saturated
Monounsaturated
Polyunsaturated

24.12 20.39 55.49

33.73 42.33 23.94

25.71 28.29 45.97

35.96 44.18 20.14

FID area percentages were calculated according to [total weight (area lipid/area total lipid) × 100]. Data are expressed as wt. % of total fatty acids and represent means ± standard deviation of three replicate determinations (confidence interval: 99%).

Two-dimensional HPTLC of isolated PE and PC revealed during first dimension only one component, which after second dimension resolved into four components: PE1 (26.3% of total PE), PE2 (0.5% of total PE), PE3 (31.3% of total PE) and PE4 (41.9% of total PE) according to increasing Rf. This could be attributed to different fatty acids. All the components (PE1 , PE3 and PE4 ) responded positively to the ninhydrin and to the phosphomolybdenum blue reagent. PC in the first dimension revealed one component witch in the second dimension resolved into four components, PC1 (8.0% of total PC), PC2 (14.2% of total PC), PC3 (8.5% of total PC) and PC4 (69.3% of total PC) according to increasing Rf, resulting from their constituent fatty acids nature. All the PC components responded positively to the phosphomolybdenum blue reagent (phosphocompounds).

The alkaline stable fraction of muscle PE and PC (after MAH) in HPTLC, gave one spot positive to ninhydrin and to phosphomolybdenum blue, which was co-chromatographed with standard of LPE and one spot (positive to phosphomolybdenum blue), which was cochromatographed with the standard of LPC, respectively. The water-soluble fraction of PE in paper chromatography gave one spot (positive to ninhydrin and to Hanes–Isherwood reagent), which was co-chromatographed with l-␣-glycerylphosphorylethanolamine standard while the water-soluble fraction of PC gave one spot (positive to Hanes–Isherwood reagent and to Dragendorff reagent), which was co-chromatographed with l-␣-glycerylphosphorylcholine standard. The results of lipid phosphorus, esters, glycerylethers and plasmalogens analytical determination of muscle PE and PC fractions before and after MAH,

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Fig. 1. Negative ion spectra of phosphatidylethanolamine molecular species.

revealed that 75.0% of PE correspond to the structure of 1,2-diacyl-glyceroethanolamine and the remaining percentage correspond to the structure of 1-O-alkyl2-acyl-glyceroethanolamine or 1-O-(1-alkenyl)-2-acylglyceroethanolamine, while 72.0% of PC correspond to the structure of 1,2-diacyl-glycerocholine and the remaining percentage correspond to the structure of 1-Oalkyl-2-acyl-glycerocholine or 1-O-(1-alkenyl)-2-acylglycerocholine. 3.4. PE and PC species of muscle PhL characterisation by ESI-MS analysis The negative ion spectra of PE fractions yielded a peak at m/z 140 corresponding to the ethanolamine

phosphate head group-H+ : (O-PO3 HCH2 CH2 NH2 )− and a peak at m/z 154 corresponding to the Nmonomethylethanolamine phosphate head group (OPO3 HCH2 CH2 NHCH3 )− (Fig. 1). The positive ion spectra of PC classes yielded a characteristic peak at m/z 184 corresponding to the choline phosphate head group + H+ : (HO-PO3 HCH2 CH2 N(CH3 )3 )+ (Fig. 2). All the ion spectra of phosphatidylethanolamine moieties yielded major peaks at m/z 227, 253, 255, 269, 279, 281, 283 and 311 corresponding to (RCOO− ) for C14:0, C16:1, C16:0, C17:0, C18:2, C18:1, C18:0 and C20:0, respectively and of phosphatidylcholine moieties at m/z 256, 270, 280, 282, 284, 312 and 340 corresponding to (RCOO− + H+ ) for C16:0, C17:0, C18:2, C18:1, C18:0, C20:0 and C22:0, respectively. The molecular weights

Fig. 2. Positive ion spectra of phosphatidylcholine molecular species.

T.F. Garofalaki et al. / Chemistry and Physics of Lipids 140 (2006) 55–65

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Table 5 Molecular species of phosphatidylethanolamine of the crustacean studied MW

m/z (M − H+ )

689

688

763

762

789 791 793 795 799 803 805 807 809 813 815 817 819

788 790 792 794 898 802 804 806 808 812 814 816 818

Molecular species Diacyl PE

Alkenyl-acyl PE

Alkyl-acyl PE

C15:0/C17:0cyclo; C14:1/C18:0; C16:1/C16:0; C18:1/C14:0 C16:0/C22:6; C16:1/C22:5; C18:1/C20:5; C18:2/C20:4 C18:1/C22:6; C18:2/C22:5 C18:0/C22:6; C18:1/C22:5 C18:0/C22:5; C20:0/C20:5 C20:0/C20:4 C16:1/C24:1; C20:0/C20:2 C17:0cyclo/C24:1; C20:3/C22:6 C17:0/C24:1; C20:2/C22:6 C20:1/C22:6 C20:1/C22:5 C18:3/C24:1 C18:2/C24:1 C18:1/C24:1 C18:0/C24:1

C15:0/C18:0; C16:0/C17:0 C17:0/C22:5

C16:0/C17:0cyclo; C15:0/C18:1; C16:1/C17:0 C17:0/C22:6; C17:0cyclo/C22:5

– – – – C17:0/C24:1 – – – – – – – –

– – – – C24:1/C17:0cyclo – – – – – – – –

Table 6 Molecular species of phosphatidylcholine of the crustacean studied MW

m/z (M + H+ )

Molecular species Diacyl PC

Alkenyl-acyl PC

Alkyl-acyl PC

C14:0/C17:0cyclo; C14:1/C17:0; C15:0/C16:1 C16:1/C17:0cyclo; C15:0/C18:2 C14:0/C20:0; C16:0/C18:0

C14:1/C17:0cyclo

701

702

C14:1/C16:1

727

728

C14:1/C18:2; C14:0/C18:3

745

746

767 771

768 772

783

784

789

790

C16:1/C17:0; C15:0/C18:1; C16:0/C17:0cyclo; 2-OH-C14:0/C18:2 C15:0/C20:4 C18:1/C17:0cyclo; C15:0/C20:2; 2-OH-C14:0/C20:5 C18:1/C18:2; C16:0/C20:3; C18:0/C18:3 –

811

810

833

834

C18:0/C20:3; C18:1/C20:2; C18:2/C20:1 C20:2/C20:4; C18:1/C22:5; C20:1/C20:5; C18:0/C22:6

of phosphatidylethanolamine moieties ranged from 689 to 819 (Fig. 1) and of phosphatidylcholine moieties from 701 to 833 (Fig. 2). By examining the molecular weights for combinations of fatty acid moieties, head groups and acyl, alkyl or alkenyl linkages, we evaluated the possible combinations that will generate the parent ions of PE moieties (Kerwin et al., 1994) (Table 5) and PC moieties (Table 6). Over 40 molecular species of PE and PC were

C16:0/C20:3; C18:1/C18:2 C16:1/C20:0; C18:1/C18:0; C16:0/C20:1

C15:0/C18:3 C17:0/C17:0cyclo; C14:1/C20:0; C16:1/C18:0; C16:0/C18:1; C14:0/C20:1 C16:0/C20:4 C16:1/C20:1; C16:0/C20:2; C18:0/C18:2

C17:0 cyclo/C20:1; C17:0/C20:2 C18:2/C20:4; C16:1/C22:5; C18:1/C20:5; C16:0/C22:6 –

C17:0/C20:3 C17:0/C20:0; C18:2/C20:5; C18:3/C20:4; C16:1/C22:6 –

–

–

found in P. vulgaris muscle and cephalothorax, including diacyl, alkyl-acyl and alkenyl-acyl moieties. 3.5. Overall discussion The analytical procedure previously developed, provides a powerful tool for the study of intact phospholipids in muscle and cephalothorax of edible invertebrates as

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