Food Chemistry 138 (2013) 1135–1144

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Food Chemistry

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Analytical Methods

Multidisciplinary analytical investigation of phospholipids and triglycerides inoffshore farmed gilthead sea bream (Sparus aurata) fed commercial diets

Roberto Anedda ⇑, Carlo Piga, Viviana Santercole, Simona Spada, Elia Bonaglini, Roberto Cappuccinelli,Gilberto Mulas, Tonina Roggio, Sergio UzzauPorto Conte Ricerche S.r.l. SP 55 Porto Conte-Capo Caccia, 07041 Tramariglio, Alghero (SS), Italy

a r t i c l e i n f o

Article history:Received 2 April 2012Received in revised form 21 November 2012Accepted 22 November 2012Available online 6 December 2012

Keywords:Nuclear magnetic resonancePhospholipidsTriglyceridesFish nutrition

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.11.098

⇑ Corresponding author. Tel.: +39 079998578; fax:E-mail address: anedda@portocontericerche.it (R.

a b s t r a c t

In this work, a quantitative characterisation of lipid (both triglycerides and phospholipids) rearrange-ments in the muscle of offshore-raised gilthead sea bream was carried out as a function of fish growthbetween April and September. Relative percentages of lipid classes and fatty acids/acyls compositionof the commercial feeds and fish dorsal muscles were assessed by means of an interdisciplinary analyticalapproach. A combination of preparative chemistry and experimental results from NMR spectroscopy, GC,3D-TLC as well as proximate analysis permitted the observed growth parameters in key metabolic eventsto be linked with fish fattening and lipid turnover. While defined effects of feed composition on fatty acidprofiles of fillets were ascertained, the relative increase of fatty acyls in triglycerides and phospholipidswere also estimated enabling detailed evaluation of TAG:PL ratio in adult offshore-farmed gilthead seabream. NMR was also used to quantify PUFA regiospecific distribution in TAG and PL.

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1. Introduction

Aquaculture, the fastest growing food-producing sector, nowaccounts for more than half the world’s fish/fish-based foods, andmay be able to meet the growing demand for aquatic food in thefuture. Given projected population growth over the next twodecades, it is estimated an additional 40 million metric tons ofaquatic food will be required by 2030 just to maintain the currentper capita consumption. In 2010, the worldwide aquacultureproduction was close to 60 million metric tons, and worth aboutUS$ 119 billion (FAO, 2012).

Fish quality is perceived through organoleptic traits such asodour, appearance, texture and flavour. Other parameters includ-ing essential polyunsatured fatty acids (PUFA), proteins, carote-noids and vitamins are not perceived directly but they arerecognised and appreciated by consumers for their putative healthbenefits. The nutritional value of fish and its quality at the time ofcapture are strictly related to feed composition and feedingstrategy (Bondad-Reantaso & Subasinghe, 2008) while processing,storage and transport variably influence the final commercialproduct. For small and large-scale production alike, feed and feed-ing account for over 50% of total costs (Commission of EuropeanCommunities, 2009). However, costs vary due to market fluctua-tions in raw materials prices such as fishmeal, oil and vegetables,which in variable amounts typically make up fish feed. In fact,

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+39 079998567.Anedda).

sustainability issues have already forced replacement of somefishmeal and oils with plant-derived alternatives (Turchini, Wingkeong, & Tocher, 2011).

In this multifaceted panorama, scientific research assumes arole in defining quality parameters on the basis of unbiased andobjective observations. Among other factors, muscle proximatecomposition of cultured gilthead sea bream depends on the rearingsystem, feed composition and intake, season and fish size. Lipids, inparticular, and more than proteins, influence the sensory charac-teristics of cooked fish. Furthermore, they have a significant effecton product marketability (Grigorakis, 2007). Lipids are storedmainly as triacylglicerols (TAG) in fat deposits, but they are alsoessential components of cell membranes where they are usuallyfound in the form of phospholipid (PL) bilayers.

Growth and quality of gilthead sea bream are significantly af-fected by physiology, environmental factors (above all, as in otherpoikilotherms, water temperature), and nutrition (Gurr, Harwood,& Frayn, 2002). But, mechanisms of lipid absorption, transport,storage and mobilisation in adult fish are less well studied thanthose of mammals (Tocher, 2003). This is particularly true of therole of nutrition in PL of adult marine fish (Tocher, Bendiksen,Campbell, & Gordon Bell, 2008). Only a few scientific reports haveexplored whether fatty acid composition of structural PL fractionreflect feed constituents, particularly the evolution of different PL(e.g. phosphatidylcholine = PC, phosphatidylethanolamine = PE)during fish growth (Benedito-Palos, Navarro, Kaushik, &Pérez-Sánchez, 2010). Rather than simultaneously monitoring theevolution of TAG and PL in fish fillets, these studies describe fatty

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acid composition after chromatographic separation. Such an ap-proach, although well established and highly informative, cannotprovide lipid profiles at particular stages of growth and is, there-fore, not wholly suitable for studying the rapid and complexchanges involved in fish metabolism.

Among other analytical techniques, gas chromatography (GC)analysis of fatty acid methyl esters (FAME) is applied routinely forfatty acid composition of tissues and foods with or without solidphase extraction (SPE) separation of oils. Alternative techniquessuch as nuclear magnetic resonance (NMR) have been reserved formore specialised research but, more recently, due to improvedaccessibility, this technique has become more popular and increas-ingly functional in food science (Guðjónsdóttir, Belton, & Webb,2008). NMR spectroscopy, by means of non-destructive analyses, al-lows complementary information about lipid fraction compositionto be obtained (Aursand, Jørgensen, & Grasdalen, 1995; Manninaet al., 2008). NMR can be used to identify lipid classes and esterifiedfatty acyl chains in both TAG and PL, and specify positional distribu-tion (Lien, Byl, Yuhas, Tomarelli, & Quinlan, 1997) since it allows dis-crimination of signals from fatty acyl chains esterified at sn-1,3 orsn-2 positions as well as hydrolyzed free fatty acids (FFA), (Aursandet al., 1995; Falch, Størseth, & Aursand, 2007; Standal & Axelson,2009; Standal, Axelson, & Aursand, 2010).

This paper reports results from a multi-technique study of lipidextracts from fish feed and offshore farm-raised gilthead sea breamdorsal muscle, which provide a complete description of lipid frac-tions during growth and fattening. This approach compared datafrom routine and more specialised analytical techniques. Musclefat composition was assessed as a function of growth and took intoaccount feed composition. Quantitative NMR was also used to exam-ine PUFA regiospecific distribution in TAG and PL during growth. Asa result, useful insights arising from optimisation of multidisciplin-ary analytical approaches provide in-depth information about theprocesses implicit in feeding and growth, and muscle fat content.

2. Materials and methods

2.1. Sampling plan

Cultured gilthead sea bream (Sparus aurata) were collected froma local farm in Alghero (SS), Italy between April and September2010. The fish were fed (with a ration corresponding to 1–1.5% ofthe biomass, adjusted for water temperature and fish size) once aday during the growth trial using the commercial diets describedin Section 3.1. Additional data on climatic parameters are given inSupplementary materials (Table S3). Gilthead sea bream reared inthe same floating offshore sea-cage were selected for analysis. Atthe beginning of the trial (22nd April 2010), the average weightwas 74.01 ± 16.9 g. Fish growth was followed by analyzing samplesfrom four fish harvests (10 fish/harvest) until the commercial sizewas reached (234.82 ± 35.9 g/fish). Sampling dates were chosen tomatch feed-lot/feed-composition changes, which depended onfarming practices, feed (feed-lot) availability, and resource manage-ment. On each sampling date, both fish and feed were collected. Fishwere killed by immersion in an ice-salt bath, according to fish farmprocedures, and ensured analytical samples closely resembled com-mercial products. Sample processing and extraction protocols wereoptimised, and care was taken to avoid any chemical or enzymaticoxidation during transport, storage, extraction and measurement(Christie & Han, 2010; Kramer & Hulan, 1978; Mulas et al., 2011).

2.2. Sample preparation and storage

Gilthead sea bream were caught and delivered to the laboratory(within 30 min), in an ice-salt bath, where biometric parameters

(total weight, total length, liver weight, edible part weight) weremeasured. Approximately 5 g of dorsal muscles (left side) were ex-cised from each fish and the fillets deep-frozen in liquid nitrogen ina Petri dish. Frozen samples were transferred, one at a time, to apre-cooled stainless-steel mortar, filled with liquid N2, as describedby Kramer and Hulan (1978), and crushed to obtain a fine frozenpowder. Pulverised fillets were freeze-dried for 20 h (VirTis, War-minster, PA, USA, mod. Genesis 12ES). Preliminary experiments(not shown) demonstrated some individual variability and hetero-geneity in both fish size and composition. Therefore, in order to ob-tain a representative analytical sample, 10 sea bream were caughton each sampling date and the 150 g of lyophilised dorsal musclesfrom each pooled to obtain a single analytical sample. On eachsampling date, to ensure statistical significance, three dorsal mus-cle pools and two (pooled) feed samples were prepared and ex-tracted. To avoid sample deterioration, samples were kept at�80 �C until extraction, which was carried out following a detailedprotocol. Samples were analyzed within four months of extraction.

2.3. Extraction protocol

Lipid extraction was performed following a modified Bligh andDyer method. Chloroform (CHCl3 >= 99.8%) and methanol (CH3OHCHROMASOLV�) were purchased from Sigma Aldrich, ammoniafrom Carlo Erba (Ammonia solution 30%, Carlo Erba, Milan, Italy).Each (pooled) dorsal muscle sample was dissolved in 40 ml of achloroform:methanol (CHCl3:CH3OH, 1:1) and, under a nitrogen(N2) gas stream, stirred for 30 min in the dark. The extract was fil-tered through a Whatman (class II, porosity 8 lm) filter paperusing a custom-made Buchner flask and funnel (16–40 lm poros-ity) and the filtrate collected in a round bottom flask. Extractionwas repeated with further 40 ml of chloroform:methanol (CHCl3:-CH3OH, 1:1). The solvent was subsequently evaporated using aRotavapor (Buchi Rotavapor R-210 equipped with Vacuum pumpV-700, Buchi Italia S.r.l., Milano, Italy). Operating conditions forevaporating solvents were chosen so as to limit lipid oxidation.For this reason, the water bath temperature was kept at 45 �C,the vacuum electronically controlled (Buchi Vacuum controllerV-850 Buchi Italia S.r.l., Milano, Italy), N2 gas was used to breakthe vacuum, and oil samples were always kept in a N2 gas atmo-sphere. Extracted oils were quantitatively transferred to a custommade glass centrifuge tube with a Teflon screw cap, and dilutedin chloroform:methanol (CHCl3:CH3OH, 1:2); the round bottomflask was washed three times with 5 ml aliquots. Sodium chloride(0.9%, pH = 3) was added to the filtrate, stirred vigorously and keptin the dark for 30 min. Chloroform (5 ml) and water (5 ml) wereadded to the filtrate to induce phase separation, and the samplescentrifuged (Centrifuge ALC PK121R) at 2500 rpm for seven min-utes at 4 �C. The organic phase was quantitatively transferred toa 50 ml round bottom flask while the supernatant aqueous phasewas repeatedly washed with 3–5 ml of chloroform, which wastransferred to the 50 ml flask. Finally, the chloroform wasevaporated as previously described and the oil suspended in3 � 1 ml aliquots of chloroform before being stored at �80 �C untilanalysis (4 ml clear screw-top glass vials, black caps and PTFE/sil-icone septa, Agilent Technologies Italia S.p.A., Milano, Italy).

Each sample was identified by a code where the first four num-bers refer to the sampling date (e.g. 2204 = April 22nd), followedby a letter for the tissue/specimen (M = muscle, F1,2 = feed, wheresubscript 1 and 2 refer to the two diets used). Proximate analyseson dorsal muscle and feed samples were performed according toAOAC guidelines (AOAC, 2000). On each sampling date (April22nd, June 24th, July 27th, September 1st), three (pooled) dorsalmuscle and two (pooled) feed samples were analyzed by GC,three-dimensional thin layer chromatography (3D TLC) and NMR.

R. Anedda et al. / Food Chemistry 138 (2013) 1135–1144 1137

2.4. NMR analysis

All NMR spectra were acquired using a Bruker Avance instru-ment, 14.09 T UltraShield magnet and 600 MHz proton frequency(Bruker BioSpin GmbH, Germany). Two SB probes, a Bruker 5 mmBBO and a Bruker 5 mm QXI, were used to perform 1D 13C, and1D or 2D 1H–1H and 2D 1H–13C NMR analysis, respectively. AllNMR measurements were performed at T = 298 K (BrukerBVT3000 and BCU05 temperature control units, N2 gas flow).NMR assignments were made according to the literature and byperforming 1D 1H NMR and 2D 1H–13C HSQC and 1H–1H TOCSY(Mannina et al., 2008) spectra. 2D 1H–13C heteronuclear correlationspectra (HSQC) were acquired using JCH = 145 Hz.

One hundred milligram/sample of extracted oil were weighedaccurately and dissolved in 0.8 ml of deuterated chloroform(CDCl3), containing 0.05% v/v Tetramethylsilane (TMS) (CambridgeIsotope Laboratories Inc., Andover, MA, USA) as a concentrationstandard and chemical shift reference (fixed at 0 ppm). Quantita-tive 1D 13C NMR spectra were acquired with 6.2 ls carbon pulse(70� Ernst angle) and inverse-gated proton decoupling using anacquisition time of 4 s, a relaxation delay of 30 s and 37878 Hzspectral width. NMR Free Induction Decays were Fourier trans-formed with 0.5 Hz line broadening, phased, and baseline-corrected using Bruker Topspin 2.1 (Bruker BioSpin GmbH,Germany). Processed spectra were exported to MNova 7.1.0(Mestrelab research S.L., Santiago de Compostela, Spain), andsignals in the carbonyl region (from 172.00 to 173.7 ppm) fitted(deconvoluted) by simulating several Lorentzian lineshapes, thesum of which matched the experimental spectrum in an auto-mated iterative fitting procedure implemented in MNova (Fig. S3in Supplementary materials). Chemical shifts, height, width andarea of each deconvoluted signal are reported by the fitting proce-dure, allowing quantitative examination of the TAG-PL mixture.The chemical shifts of signals with very low intensity [e.g. thoseassigned to unsaturated fatty acyls in PL (173.48 ppm), n-3 docosa-hexaenoic acid (n-3 DHA) in sn-2 of PE (172.4 ppm) or n-3 eicosa-tetraenoic acid (n-3 ETA) in sn-2 of TAG] were applied as fittingconstraints. Line widths of intense signals were allowed to varyin the iterative procedure while chemical shifts varied aroundassigned maxima. Final values were found to be in agreement withthe literature data (Alemany, 2002). Integrals relative to the 13CNMR carbonyl region were derived from the aforementionedfitting while those corresponding to n-3, n-6 FA and all fatty acylsin the aliphatic region (13.5–14.5 ppm spectral window, Figure 5)of 13C NMR spectra were integrated manually using MNova.

2.5. 3D-TLC and GC analysis

3D TLC was performed according to Kramer, Fouchard, andFarnworth (1983). Briefly, approximately 3–4 mg of total lipid ex-tract was applied to a Silica Gel H plate (Analtech Inc., Newark, DE,USA) and developed in three different chambers each in a differentdirection; the first contained chloroform/methanol/ammoniumhydroxide (65:25:5, v/v/v), the second chloroform/acetone/metha-nol/acetic acid/water (50:20:10:15:5, v/v/v/v/v) and the thirdhexane/diethyl ether/acetic acid (85:15:1, v/v/v). Each (pooled)dorsal muscle lipid sample was run three times to demonstratethe repeatability of spot intensity and distribution. Lipid classwas assigned on the basis of previous studies (Kramer et al., 1983).

GC analysis was performed on 4 mg of total lipid extract follow-ing basic methylation with NaOCH3 at 50 �C for 30 min using themethod of Cruz-Hernandez et al. (2006). Samples were analyzedusing an Agilent 7890A gas chromatograph (Agilent Technologies,Wilmington, DE) equipped with FID detector, split/splitless injec-tion port, an autosampler and a Supelco SP-2560 GC column(100 m � 0.25 mm internal diameter � 0.20 lm film thickness).

The system was controlled by the Agilent ChemStation (VersionB.04.02) chromatography manager. The carrier gas was H2. Theinjection volume was set to 1 lL and the split ratio was set to100:1 for GC standards and to 20:1 for fatty acid methyl esters(FAME). Two different temperature programmes labelled 175 �Cand 150 �C (based on their plateau temperature) were applied toseparate co-eluting FAMEs (Santercole, Delmonte, & Kramer,2012).

Pure FAME reference materials and reference mixtures werepurchased from Nu-Chek Prep (Elysian, MN, STD #463 and#674). Menhaden fish oil analytical standard (#47116) and 0.5 Nmethanolic base (#33080) were purchased from Supelco Inc.(Bellefonte, PA).

2.6. Statistical analysis

Statistical analyses of experimental data were performed usingGraphPad Prism 5.03 (GraphPad Software Inc., La Jolla, CA, USA)and Microsoft Excel. Experimental values are reported as meanand standard deviation. Kruskal–Wallis test was used to ascertainthe significance of observed 13C NMR integral variations. One-wayANOVA was applied for the analysis of regiospecific fatty acid (FA)position in TAG. Tukey test was used to determine the significanceof positional isomeric variations for selected FA in TAG from NMRdata and similarly applied to all GC data.

3. Results and discussion

3.1. Analysis of feeds

The feeds were grouped into two classes: those used during thefirst growth period from February to the end of June (F1) and theother, with a different composition (F2), which was used fromthe end of July to September 2010. The three lots were used duringthe four-months trial (i.e. two lots of F1 and one lot of F2). Each lotand both feeds were characterised. According to the label, F1

contained 40:20 protein:lipid relative percentage while F2, whichwas produced with the addition of purified haemoglobin,contained a relative protein and lipid content of 43 and 21, respec-tively. Additional details about feed composition are reported inSupplementary materials (Table S1). F1 appeared khaki-browncolour while F2 was dark brown.

In fact, according to our analysis, different lots of the same feed(2204F1 vs. 2406F1) did not differ significantly from each other.However, the lipid composition of different feeds (F1 vs. F2) wassignificantly different using GC data (P < 0�0001) or comparisonof 13C NMR spectra profiles (Table S2 and Fig. S1, Supplementarymaterials). As shown in Fig. 1a, F2 was characterised by a more in-tense 13C NMR signals due to linoleic acid (n-6 LA) (in sn-1,3 of TAGat 173.22 ppm, and in sn-2 of TAG at 172.80 ppm), lower n-3 DHAat sn-2 (172.11 ppm) and sn-1,3 (172.51 ppm) in TAG, and n-3 eico-sapentaenoic acid (n-3 EPA) at sn-2 (172.58 ppm) and sn-1,3(172.98 ppm) compared with F1. Signals from fatty acyls (172.37,172.44 ppm) in PL (Fig. 1b) were very low intensity in both F1

and F2 compared with corresponding signals in 13C NMR spectraof pooled dorsal muscle samples.

NMR spectra also gave useful hints about FFA composition infeeds. Fig. 1b shows that F1 contained higher amounts of FFA,and suggests more saturated fatty acids and a lower percentageof n-3 DHA were hydrolyzed in F1 than in F2. However, FFA signalswere low in both F1 and F2 suggesting TAG hydrolysis did not sig-nificantly affect the quality of feed.

3D TLC of feed lipid extracts produced spots with very lowintensity, predominantly due to the presence of PC in the feeds(Fig. S2, Supplementary materials).

Fig. 1. (a) Carbonyl region of the superimposed 13C NMR spectra of the lipid extract of the feed used in April and June (2204F1 and 2406F1, black line) and in July (2607F2, greyline). (b) Relevant regions between 172.9 ppm and 177.9 ppm, showing the signals from fatty acids in sn-1,3 position of PL, reported in the onset. Signals from FFA, resonate athigher frequency in the same spectra.

Table 1Lipid contents of dorsal muscles in farmed gilthead sea bream(% w/w – extracted lipids per gram of lyophilised muscle).

LIPIDS (% of DM)

Sample Mean SD

2204M 8.91 0.402406M 9.55 0.112607M 14.74 1.210109M 18.39 1.46

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Some differences in proximate analysis were found comparedwith feed label values, especially lipid content of F2 (Table S1, Sup-plementary materials). This can, hypothetically, be ascribed to oilleaking during pellet storage rather than any reduced quality. GCanalysis (Table S2, Supplementary materials) on the lipid extractsof the three (pooled) feed samples confirmed NMR observations.

A reduction in fish meal-derived PUFA, especially n-3 EPA andn-3 DHA, and increased plant-derived n-6 LA and a-linolenic(n-3 ALA) acids were in F2. Thus, whilst the lipid profile character-ised for F1 appears balanced, the composition of F2 reflects highlevels of substitution for fish oil/meal with vegetable oils.

Although GC attained a complete description of fatty acids, itcould not measure lipid classes or FFA. Thus, our results underlinehow an interdisciplinary approach is essential for obtaining com-plete characterisation of feed composition, quality and evolutionduring storage and use.

3.2. Muscle/feed lipid composition and diet-growth correlations

Growth and fattening of fish are two related processes, whichresult in overall weight gain. A quantitative indication of musclefattening is provided in Table 1. The increase in total muscle lipidwas expected (Company, Calduch-Giner, Kaushik, & Perez-Sanchez,1999), but only a more detailed analysis of structural (PL) and stor-age (TAG) lipids allows relevant information about lean and fattymuscle mass to be gathered.

Fig. 2a shows the expanded carbonyl region of a typical 13CNMR spectrum for (pooled) dorsal muscle lipids (from 172 ppmto 174 ppm) with full assignments. The NMR profile in this spectralregion is the sum of NMR signals from all carbonyl fatty acylsesterified to glycerol. This region of 13C spectra is highly informa-tive as it reflects overall composition of the oil and provides

information about the lipid classes (TAG and PL), chemical compo-sition and positional distribution (sn-1,3, sn-2) in TAG and PL. Pre-vious NMR work on animal and vegetable oils have succeeded inassigning carbon spectra of PL and TAG in complex mixtures(Alemany, 2002; Aursand, Standal, & Axelson, 2007; Aursandet al., 1995; Casu, Anderson, Gregory, & Gibbons, 1991; Falchet al., 2007; Gunstone, 1991; Mannina et al., 2008; Scano et al.,2011; Standal & Axelson, 2009; Standal et al., 2010).

No FFAs were detected in gilthead sea bream muscle by 13CNMR (Fig. 2b) or 3D TLC (Fig. 2c). This corroborates the validityof sample extraction and storage procedures adopted in this study,the freshness and quality of farmed fish at capture, and the rela-tionship between these fish and the samples used for analysis. Fur-thermore, the carbonyl spectral region of 13C NMR spectra providesa snapshot of the evolution of lipid composition in sea bream mus-cle as a function of fish growth (Fig. 2b). The gradual increase ofTAG and a corresponding decrease of PL in the muscle during thefeeding trial is evident using 13C NMR (Fig. 2b). Most likely aconsequence of muscle fattening, this change may be induced byreduced activity in farm cages (Davison, 1997) and use of high-energy diets (Company et al., 1999; Grigorakis & Alexis, 2005).Increased water temperature in the summer (Table S3,

Fig. 2. (a) The carbonyl region of a typical 13C NMR spectrum is reported to schematically summarize the assignments of the signals observed. (b) Carbonyl region of the 13CNMR spectra of lipid from dorsal muscle as a function of growth, from April to September. A net relative increase of triglycerides with a correspondent decrease ofphospholipids is observed. (c) 3D-TLC of dorsal muscle lipids at April (2204M) and September (0109M). The increase in TAG and a corresponding decrease in PL are clearlyobservable, in agreement with NMR data. TAG = triglycerides; DPG = diphosphoglycerides; Cho = cholesterol; CE = cholesteryl esters; PI = phosphatidylinositol; PS = phos-phatidylserine; SP = sphingomyieline; PC = phosphatidylcholine; PE = phosphatidylethanolamine. AA = arachidonic acid; EPA = eicosapentaenoic acid; DPA = docosapenta-enoic acid; ETA = eicosatetraenoic acid; SDA = stearidonic acid; DHA = docosahexaenoic acid; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids;UFA = unsaturated fatty acids.

R. Anedda et al. / Food Chemistry 138 (2013) 1135–1144 1139

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Supplementary materials) is also likely to influence fat storage(Ibarz, Blasco, Gallardo, & Fernández-Borràs, 2010).

3D TLC of muscle lipids (Fig. 2c) confirmed PL (PC, PE, PI and PS)was at higher concentrations in fish muscle, relative to TAG, inApril (2204M) than in September (0109M). It is worth noting thatquantification of PL and TAG, together with their FA composition,assumes primary importance in determining the evolution andquality of fish muscle during fattening. Moreover, NMR character-isation also offers evidence of possible TAG and PL hydrolysis.

TAG:PL ratio has been studied previously in other fish speciessuch as Atlantic salmon although the investigation produced con-troversial results in vitro and in vivo (Torstensen & Tocher, 2011).It was suggested the differential increase in TAG relative to PLmight be closely related to an increase in numbers of TAG-rich adi-pocytes in muscle for a variety of fish species (Fontagné, Geurden,Escaffre, & Bergot, 1998; Nanton et al., 2007) including gilthead seabream (Benedito-Palos et al., 2008; Caballero, Izquierdo, Kjørsvik,Fernández, & Rosenlund, 2004).

Observations in this study confirm that gilthead sea bream storea significant percentage of fat in lean muscle and not only in spec-ialised storage tissues or organs (i.e. liver, mesenteric fat, ventral orperivisceral adipose tissue). Further quantitative evidence of TAGand PL evolution during fish growth can be gained indirectly byassessing 13C NMR signal areas for specific fatty acyls, which arepresumed to be present in both TAG and PL.

There are some suggestions in the literature about the role ofn-3 highly unsaturated fatty acids (n-3 HUFA), which make up PLmembranes, in modulating membrane fluidity in vivo (Hashimoto,Hossain, & Shido, 2006). These studies indicated fatty acid compo-sition of commercial diets in aquaculture may have a significantimpact on non-specific immune factors (i.e. affecting levels of pros-taglandins, leukotrienes, lipoxins, i.e. modulating inflammatoryactivity) (Özyurt, Polat, & Özkütük, 2005), stress response (Mon-tero et al., 2003) and consequently fish welfare.

Table 2 presents the quantification of sn-2 n-3 DHA from 13CNMR. Variations in the relative quantity of sn-2 n-3 DHA can be ob-served in both TAG and PL (PC and PE). The gradual increase in sn-2n-3 DHA in TAG and corresponding decrease in sn-2 n-3 DHA inboth PC and PE could be ascribed to the overall increase in theTAG:PL ratio. Changes in the relative amounts of PL and TAG arerelevant to the overall composition of fillets, and should be consid-ered using a multi-technique approach. This can be easily done bycomparing 13C NMR carbonyl spectral region and GC analysis ofFAME to get information on lipid classes and total lipids, respec-tively. It is worth noting, however, that NMR data reported in Ta-ble 2, also shows a 1.2-fold decrease in the PC:PE ratio,confirming – as previous studies suggest – rearrangement of fattyacyls in both TAG and PL also occurs as a function of growth(Benedito-Palos et al., 2008).

The alteration of the PC:PE ratio has previously been associatedwith homeoviscous adaptation of biological membranes and accli-matisation to cold. However, while previous studies in rainbowtrout (Hazel, 1990) demonstrated PC:PE ratio synthesis is posi-tively correlated with water temperature (Tocher et al., 2008), no

Table 2TAG/PL distribution of sn-2 n-3 DHA (mean values of three muscle pools, % of total lipids)

Sample sn-2n-3 DHAin TAG (%)

sn-2n-3 DHA in PC (%)

sn-2n-3 DHA in P

2204M 2.80 4.59 2.202406M 3.41 3.25 1.702607M 3.85 1.39 0.700109M 3.87 1.23 0.77

detailed or systematic work on the phospholipid biosynthesisand turnover in adult gilthead sea bream has been carried out todate. Interest in the role of PL classes in adult fish nutrition isincreasing since PL dietary supplementation is believed to have apositive impact on growth rate and health (Benedito-Palos et al.,2008; Olsen, Myklebust, Kaino, & Ringø, 1999), resistance to stress(Liu et al., 2002) and feed conversion (Benedito-Palos et al., 2008).

In the literature only a few reports analyze the effect of diet andrearing system on structural phospholipids in adult gilthead seabream and other marine fish while several published studies baseconclusions on the necessity of dietary phospholipids in larvae andjuveniles (Benedito-Palos et al., 2008; Liu et al., 2002; Olsen et al.,1999; Tocher et al., 2008). Structural and chemical properties ofmembrane phospholipids are known to have a role in determiningaverage membrane lipid order, more commonly referred to as‘‘membrane fluidity’’, by regulating fish response to temperaturechanges through physiological adaptation (Arts & Kohler, 2008,chap. 10; Dey, Buda, Wiik, Halver, & Farkas, 1993). Our data sug-gest major changes principally affect the quantity of TAG in farmedgilthead sea bream muscle during fattening. However, structuralphospholipids can be influenced as well through diet and the envi-ronment. Indeed, it is evident from our 3D TLC and 13C NMR datathat TAG:PL ratio is principally affected by muscle fattening. But,13C NMR analyses also showed PC:PE ratio changed during growthalbeit to a lesser extent. If this remodelling and turnover of PL takesplace, a similar process involving FA redistribution should be ex-pected. In previous studies on the lipid fraction of fish muscle,PUFA, and especially n-3 HUFA, generally, showed sn-2 specificityin TAG (Gunstone & Seth, 1994).

As shown in Table 3, n-3 DHA always showed positionalspecificity for sn-2 in TAG. However, 2204M samples significantlydiffered from 2607M and 0109M in positional distribution ofn-3 DHA in TAG (P = 0.0088) with significantly higher specificitytoward sn-2 observed only from July (i.e. in 2607M and 0109M).n-3 EPA, in comparison, always showed distinct specificity for sn-1,3 positions in TAG (P = 0.578). 2204M, 2406M, 2607M differedsignificantly from 0109M for n-3 stearidonic acid (n-3 18:4, n-3SDA) positional isomerism (P = 0.002), which showed specificitytowards sn-2 on TAG only in September. Similar behaviour forn-3 SDA was observed for n-3 docosapentaenoic acid (n-3 DPA)(P = 0.0129). In contrast, neither n-3 DHA nor n-3 EPA showed clearspecificity towards sn-2 on TAG and, in particular, n-3 EPA was al-ways selectively esterified to the sn-1,3 positions. The regiospecificbehaviour of n-3 DHA progressively changed during muscle fatten-ing until September when n-3 DHA showed specificity to sn-2.

The positional distribution of n-6 LA, SFA and MUFA did notchange during growth. In particular, the positional distribution ofn-6 LA, one of the main components of F2, showed marked specific-ity toward the sn-1,3 positions at the beginning of the trial andthrough all the phases of muscle growth and fattening(P = 0.162). It has been reported that positional isomerism, to-gether with chain length and unsaturation of fatty acids, influencesignificantly fatty acid mobilisation and storage in rats to supplyperipheral tissues and organs (Raclot & Groscolas, 1993).

in gilthead sea bream dorsal muscle during growth, as evidenced by 13C NMR.

E (%)TAG/PL TAG/PC TAG/PE PC/PE

0.41 0.61 1.29 2.120.69 1.05 2.02 1.921.88 2.82 5.61 1.992.04 3.32 5.50 1.69

Table 3Positional isomery (%) of several fatty acyls in TAG of dorsal muscle as a function of fish growth, calculated from 13C NMR signal areas. Mean values and standard deviations arecalculated on three pools of 10 fish each. Different letters in the same row indicate statistically significant differences (P < 0�05). EPA = eicosapentaenoic acid;DHA = docosahexaenoic acid; DPA = docosapentaenoic acid; SDA = stearidonic acid; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; LA = linoleic acid.

FA 2204M 2406M 2607M 0109M

sn-2 sn-1.3 SD sn-2 sn-1.3 SD sn-2 sn-1.3 SD sn-2 sn-1.3 SD

n-3 DHA 54.46a 45.54 4.32 59.73ab 40.27 0.79 62.94b 37.06 1.21 61.61b 38.39 0.44n-3 EPA 23.32a 76.68 6.82 19.57a 80.43 0.95 19.97a 80.03 1.71 19.98a 80.02 1.52n-3 18:4 (SDA) 41.75a 58.25 2.39 40.91a 59.09 2.06 47.71ab 52.29 3.17 51.47b 48.53 1.86n-3 DPA 29.62a 70.38 5.89 30.07a 69.93 3.73 36.24ab 63.76 2.63 43.06b 56.94 3.66n-6 18:2 (LA) 20.37a 79.63 2.52 22.50a 77.50 1.26 19.87a 80.13 0.39 19.93a 80.07 0.48SFA 57.54 42.46 1.88 56.56 43.44 0.90 56.38 43.62 0.22 58.17 41.83 0.94MUFA 30.99 69.01 6.17 26.28 73.72 1.22 30.11 69.89 0.34 31.95 68.05 0.83

R. Anedda et al. / Food Chemistry 138 (2013) 1135–1144 1141

3.3. Meeting EFA requirements and proper PL supply by feedingcommercial diets

Several recent studies have sought to predict the influence of die-tary lipid composition on lipid fractions from commercial giltheadsea bream fillets (Benedito-Palos et al., 2009; Benedito-Palos et al.,2011). Our results are in agreement with previous reports, whichsuggest fatty acids components of gilthead sea bream fillets arepalmitic (16:0), myristic (14:0), stearic (18:0), palmitoleic (16:1)and oleic (n-9 OA) acids, n-6 LA, n-3 EPA, and n-3 DHA (Grigorakis,Alexis, Anthony, & Hole, 2002; Özyurt et al., 2005). Tables 4 and 5 de-scribe relative percentages of FA in total lipids and the distributionbetween TAG and PL from gilthead sea bream muscle as obtainedby GC analysis of FAME and 13C NMR, respectively.

More specifically, Table 4 shows high percentages of n-6 LA andn-9 OA were accumulated in the muscle during growth. This couldreasonably be ascribed to the high dietary intake of these FA, whichin turn may imply feeds made from vegetable sources (e.g. fromsoybean) were used (Benedito-Palos et al., 2008, 2010; Grigorakiset al., 2002; Menoyo et al., 2004). Similar observations have beenmade for several fish species including Arctic char (Olsen et al.,1999) and Caspian brown trout (Kenari, Mozanzadeh, & Pourgho-lam, 2011). Several reports demonstrate the necessity of essentialfatty acids (EFA) such as n-3 DHA and n-3 EPA in gilthead seabream nutrition (Benedito-Palos et al., 2010; Tocher & Ghioni,1999). These FA must be included in aquafeed because marine fishhave limited capacity to convert C18 PUFA to C20 PUFA. Indeed, ithas been reported that several markers of deficiency can be ob-served in gilthead sea bream fed with a restricted supply of essen-tial FA (EPA + DHA: <0.7–0.9% of DM) such as lipid liver disease,

Table 4Changes in the relative quantity of most relevant fatty acids in sea bream dorsal muscle dsignificant differences (P < 0�05). AA = arachidonic acid; EPA = eicosapentaenoic acid; DPAMUFA = monounsaturated fatty acids; ALA = a-linolenic acid; LA = linoleic acid; OA = oleic

Fatty acid 2204M 2406M

Mean SD Mean

14:0 3.02 0.12 3.2315:0 0.25 0.02 0.2616:0 14.35 1.21 13.9118:0 4.40 0.19 4.26n-7 9c-16:1 4.25 0.36 4.68n-9 9c-18:1 (OA) 14.54 1.20 15.83n-6 18:2 (LA) 16.09a 1.35 17.02ab

n-3 18:3 (ALA) 1.47 0.10 0.99n-6 20:4 (AA) 0.91a 0.07 0.89a

n-3 20:5 (EPA) 7.44a 0.50 8.94b

n-3 22:5 (DPA) 3.53 0.25 3.65n-3 22:6 (DHA) 14.99a 0.94 14.00a

SFA 22.88 22.77MUFA 23.54 25.02n-3 HUFA 29.27 30.07n-6 PUFA 18.29 19.66n-3/n-6 ratio 1.60 1.53

reduced growth rates and oxidative stress (Sitjà-Bobadilla et al.,2005).

According to our NMR analysis, n-3 DHA and n-3 EPA reserveswere relatively high in dorsal muscle TAG during fattening. In fact,the intensity of both sn-1,3 and sn-2 n-3 DHA and n-3 EPA in TAGincreased during growth (Fig. 2b and Table 5). Nevertheless, the to-tal concentration of n-3 DHA content in muscle decreased overall,as determined by 13C NMR and GC analyses (Tables 4 and 5). IfNMR and GC results are compared, the balance of essential FAn-3 DHA and n-3 EPA, which are stored in and/or mobilised frommuscle, can be followed during growth. While both techniques re-port an overall stability of n-3 EPA content, GC results show anoverall decrease in n-3 DHA (Table 4). However, 13C NMR offers amore complete picture; as the fish muscle fattened n-3 DHA-richTAG were stored in muscle adipocytes and PL concentrations de-creased leading to an overall decrease in n-3 DHA in the muscle(Table 5). Thus, it is reasonable to assume that fattening in farmedgilthead sea bream is likely to lead to an overall reduction in n-3DHA due reduced PL and increased TAG:PL ratio even if feedsand, consequently, the new TAG are composed of a relatively a highpercentage of this FA.

It is well known that PL in fish contain more n-3 HUFA than TAG(Ackman, 2000), and the observed decrease in PL cannot be consid-ered as positive in terms of fish quality. These observations implyn-3 FA-poor feeds, together with unsuitable farming procedures,have a negative effect on quality during fattening, specifically n-3FA content and nutritional content.

It is, however, largely unknown whether marine fish are able tosynthesize PL de novo. The mechanisms implied in absorption andtransport of PL, and the products of their digestion, are not fully

uring growth, detected by GC. Different letters in the same row indicate statistically= docosapentaenoic acid; DHA = docosahexaenoic acid; SFA = saturated fatty acids;

acid; HUFA = highly unsaturated fatty acid; PUFA = poly unsaturated fatty acid.

2607M 0109M

SD Mean SD Mean SD

0.22 3.44 0.09 3.95 0.180.01 0.28 0.00 0.29 0.010.09 14.57 0.15 13.91 0.260.09 4.22 0.06 3.68 0.120.06 4.95 0.07 5.28 0.130.18 18.77 0.57 17.65 0.280.08 17.29abc 0.12 19.03c 0.200.03 0.87 0.02 1.45 0.050.00 0.76bc 0.02 0.69c 0.020.09 8.33b 0.21 8.36b 0.210.04 3.64 0.03 3.56 0.080.08 10.26bc 0.35 9.53c 0.17

23.93 22.9428.38 27.5525.56 25.6119.76 21.441.29 1.19

Table 5NMR quantitative analysis of gilthead seabream dorsal muscle lipid extracts. Different letters in the same row indicate statistically significant differences (P < 0.05).AA = arachidonic acid; EPA = eicosapentaenoic acid; DPA = docosapentaenoic acid; DHA = docosahexaenoic acid; SFA = saturated fatty acids; MUFA = monounsaturated fattyacids; UFA = unsaturated fatty acids; LA = linoleic acid; ETA = eicosatetraenoic acid; SDA = stearidonic acid.

Lipid class FA 2204M 2406M 2607M 0109M

mol % SD mol % SD mol % SD mol % SD

TAG n-3 DHA 5.13 0.19 5.70 0.18 6.21 0.14 6.33 0.11n-3 EPA 5.80 0.62 6.43 0.13 7.07 0.17 7.35 0.10n-3 SDA 5.35 0.12 4.4 0.26 4.14 0.23 4.49 0.12n-3 DPA 5.89 1.24 5.89 0.44 4.95 0.48 4.55 0.54n-3 ETA + AA 2.24 0.21 2.11 0.72 2.33 0.09 2.32 0.62n-6 LA 22.74 2.49 24.06 1.25 29.25 0.62 31.69 0.83SFA 13.45 0.76 15.32 0.72 17.59 0.33 17.09 0.39MUFA 5.62 0.27 6.23 0.21 7.58 0.45 7.29 0.03D9 18:1 + 16:1 12.43 2.08 13.95 1.21 14.44 0.54 13.19 0.89

PL PE DHA 2.20 0.41 1.70 0.16 0.70 0.11 0.77 0.28PC DHA 4.59 0.24 3.25 0.04 1.40 0.25 1.24 0.34PC EPA + AA 2.66 0.34 1.83 0.11 1�11 0.12 0.94 0.14PL SFA 7.04 0.27 5.51 0.26 2.62 0.15 1.84 0.35PL UFA 4.75 0.03 3.51 0.03 1.52 0.51 1.41 0.13

n-3/n-6 1.54a 0.08 1.44ab 0.06 1.31bc 0.02 1.22c 0.02

Fig. 3. Aliphatic carbon region of 13C NMR spectra showing terminal methylene of lipid extracts from dorsal muscles as a function of fish growth. Distinctive signals fromcholesterol, n-6 FA and n-3 FA are indicated.

1142 R. Anedda et al. / Food Chemistry 138 (2013) 1135–1144

elucidated (Tocher et al., 2008). Moreover, PL requirements inadult marine fish are, as yet, undefined even though several bene-ficial effects of dietary PL in adult fish have been reported. Further-more, since recent studies suggest consumption of essential fattyacids (n-3 EPA + n-3 DHA) in the form of PL leads to the highestincorporation of these FA in human plasma PL compared withTAG and ethyl-esters forms (Schuchardt et al., 2011), there is inter-est in characterising both TAG:PL ratio and the FA composition ofTAG and PL in farmed fish. This would also offer promising criteriato identify high-grade quality farmed fish for human consumption.

3.4. 13C NMR assignments of PL aliphatic carbons

Additional information describing the lipid profile of giltheadsea bream muscle can be obtained by analyzing aliphatic carbonsignals from 13C NMR spectra. Fig. 3 shows the aliphatic region ofthe 13C NMR spectra from muscle lipid extracts, as a function offish growth, with peak assignments. The high-frequency-shiftedshoulders at 14.08 ppm and 14.28 ppm may be reasonably as-signed to FA esterified PL: FA in PL resonate at a higher NMR fre-quencies than those of TAG. In fact, a decrease in PL corresponds

R. Anedda et al. / Food Chemistry 138 (2013) 1135–1144 1143

to an increase in TAG in this region, as observed in the 13C NMR sig-nals for the carbonyl region. Fig. 3 provides snapshot of TAG:PL bal-ance in sea bream muscle during growth and could be a candidatefor quantification. However, the carbonyl region may be more suit-able for this as these signals show a faster NMR relaxation timethan aliphatic carbons, and consequently more rapid NMR acquisi-tion and quantification (Standal & Axelson, 2009). Due to the com-plexity of the spectra, caused by signal overlap, fitting anddeconvolution of these NMR signals may be biased. Nevertheless,NMR signal profile in this region provides useful hints about lipidcomposition. As previously discussed, and summarised in Tables4 and 5, Fig. 3 also suggests a progressive decrease in n-3:n-6 ratioin farmed gilthead sea bream muscle as a function of fattening.Cholesterol concentration (signal at 11.8 ppm, assigned to C18 ofa methyl group of cholesterol in Fig. 3) decreased during growth,which was to be expected given the low n-6 cholesterol contentof F2 compared with F1 (Fig. S1, Supplementary materials).

4. Conclusions

Our experimental data suggest a multi-technique analytical ap-proach is informative, if not mandatory, for understanding thecomplex process of lipid storage and turnover in adult giltheadsea bream. We have demonstrated that NMR, GC, 3D TLC and prox-imate analysis of fish fillets are able to delineate precisely the com-position of fat stored in fish dorsal muscle provided thepreparation protocol is optimised appropriately. Such an approachdescribes, quantitatively and simultaneously, TAG:PL ratio, TAGand PL FA composition, and positional distribution. We alsoshowed the distribution of n-3 DHA suggests the TAG:PL ratio in-creased during fattening from April to September while the PC:PEratio decreased, indicating lipid turnover and redistributioninfluenced TAG – specifically – but also PL fractions. Esterificationof TAG was preferentially at sn-2 for n-3 DHA but not n-3 EPA,which was always selectively esterified at the sn-1,3 positions.Conversely, SFA showed regiospecificity for sn-2 position, whichwas unexpected. An overall decrease in n-3 HUFA in the total lipidfraction can be ascribed to the dilution of PL and corresponding in-crease in TAG although newly stored TAG were rich in n-3 HUFA.

From an analytical point of view, the results showed GCaccurately quantified FA composition of total lipid extracts afterhydrolysis of TAG and PL, but lacked data describing lipid classesand the regiospecific distribution of FAs (Table 4). On the otherhand, NMR provided valuable insights into the FA composition ofTAG and the most relevant PL classes (i.e. PC and PE) (Table 5).3D TLC completed the analysis with qualitative or semi-quantitative information on lipid class composition and cholesterolcontent. The results from these different experimental approachescharacterising lipid profiles of farmed gilthead sea bream muscleoffer complementary information facilitating a more completeoverview than more commonly used methods. Moreover, such acombined approach substantiated and completed the molecularpicture offered thus far by the conventional techniques used toexamine marine fish growth and nutrition, particular farmed gilt-head sea bream.

Although NMR was prone to modest errors due to signal overlapand low sensitivity, it was useful in defining lipid classes and FAprofiles associated with growth and lipid storage patterns in Sparusaurata, which may be suitable for defining differences throughmultivariate statistical analysis (Standal & Axelson, 2009). Poten-tial applications include the study of gilthead sea bream growthand development, in particular the balance of TAG and PL in fillets,and acyl distribution and partitioning.

In our opinion, the observations discussed in this paper mayhelp clarify metabolic processes as yet unstudied such as

enzymatic regulation of phospholipids synthesis and turnover inadult gilthead sea bream, which involve acylglycerol biosynthesisand metabolism, membrane formation, storage and structural lipidmetabolism, nutrient partitioning and fuel economy. Moreover,this approach can add relevant detail on the influence of fatty acylcomposition in TAG and diacylglycerols (DAG) on the biosyntheticactivity of the relevant enzymes (Oxley, Torstensen, Rustan, &Olsen, 2005). New 13C NMR assignments suggested, assigned toterminal –CH3 groups from n-6 and n-3 FA in PL, offer additionalinformation about the TAG:PL ratio in fish muscle (Fig. 3). Thedescription of TAG and PL composition in farmed fish is paramountsince these lipid classes, influenced by fattening, determine nutri-tional and organoleptic traits of commercial products. Our studyenables determination of the TAG:PL ratio as a function of growth,and defines quantitatively to what extent the fat stored in adultgilthead muscle as TAG and PL is influenced by diet. This has sev-eral implications for nutritional quality and organoleptic traits, andhence value for consumers.

Our data, in agreement with previous studies (Benedito-Paloset al., 2010), showed TAG composition resembled the dietary fattyacid profile. As TAG-rich tissues contribute to the overall nutri-tional quality of the fish, TAG:PL ratio is closely related to the com-position of feed and nutritional quality. Moreover, since musclelipid composition is known to influence significantly sensorialcharacteristics of fish flesh (Grigorakis et al., 2002), the higherthe TAG:PL ratio the more the sensory description of cooked fleshwill be influenced by the vegetable oils fed to farmed fish. Thus,our results allow delineation of quality standards in farmed gilt-head sea bream, and give valuable information directing farmingpractices towards production of high-quality gilthead sea bream.

Acknowledgments

This work was supported by Sardinia Region Government bymeans of Sardegna Ricerche Technology Park and FondazioneBanco di Sardegna. We also acknowledge the ‘‘AssociazioneAcquacoltori Sardi’’ for providing fish samples and for makingavailable the farm for research activities. Special thanks are dueto prof Kramer JKG, NRC Guelph, Canada for helpful discussions.The authors declare that there are no conflicts of interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2012.11.098.

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