Structural elucidation of olive pomace fed sea bass (Dicentrarchus labrax) polar lipids with cardioprotective activities

Food Chemistry 145 (2014) 1097–1105

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Structural elucidation of olive pomace fed sea bass (Dicentrarchus labrax) polar lipids with cardioprotective activities Constantina Nasopoulou a, Terry Smith b, Maria Detopoulou a, Constantina Tsikrika a, Leonidas Papaharisis c, Dimitris Barkas c, Ioannis Zabetakis a,⇑ a b c

Laboratory of Food Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Athens 15771, Greece Biomedical Sciences Research Complex, School of Biology, The North Haugh, The University, St. Andrews, Fife, KY16 9ST Scotland, UK Research and Development Department, Nireus Aquaculture SA, Koropi 19400, Greece

a r t i c l e

i n f o

Article history: Received 27 March 2013 Received in revised form 25 June 2013 Accepted 22 August 2013 Available online 31 August 2013 Keywords: Sea bass Olive pomace Cardioprotection Fatty acid analysis Polar lipids

a b s t r a c t The purpose of this study was to structurally characterise the polar lipids of sea bass (Dicentrarchus labrax), fed with an experimental diet containing olive pomace (OP), that exhibit cardioprotective activities. OP has been added to conventional fish oil (FO) feed at 4% and this was the OP diet, having been supplemented as finishing diet to fish. Sea bass was aquacultured using either FO or OP diet. At the end of the dietary experiment, lipids in both samples of fish muscle were quantified and HPLC fractionated. The in vitro cardioprotective properties of the polar lipid fractions, using washed rabbit’s platelets, have been assessed and the two most biologically active fractions were further analysed by mass spectrometry. The gas-chromatrograpy–mass spectrometric data shows that these two fractions contain low levels of myristic (14:0), oleic (18:1 cis x-9) and linoleic acids (18:2 x-6), but high levels of palmitic (16:0) and stearic acids (18:0) as well as eicosadienoic acid (20:2 x-6). The first fraction (MS1) also contained significant levels of arachidonic acid (20:4 x-6) and the omega-3 fatty acids: eicosapentaenoic acid (22:5) and docosahexaenoic acid (22:6). Electrospray-mass spectrometry elucidated that the lipid composition of the two fractions contained various diacyl-glycerophospholipids species, where the majority of them have either 18:0 or 18:1 fatty acids in the sn-1 position and either 22:6 or 20:2 fatty acids in the sn-2 position for MS1 and MS2, respectively. Our research focuses on the structure/function relationship of fish muscle polar lipids and cardiovascular diseases and structural data are given for polar lipid HPLC fractions with strong cardioprotective properties. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The growing aquaculture sector has increasing needs for fish oil; recent data suggest that about one million tonnes of fish oil are produced every year (Pike & Jackson, 2010) while about half of global production of fish meal and fish oil are utilised in aquaculture (Nasopoulou & Zabetakis, 2012). According to FAO (2010), about 50% of world marine fish stocks have recently been estimated as fully exploited, 32% as overexploited and only 15% as under-exploited; data that suggests new management procedures are

Abbreviations: PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol; PL, phospholipid; FAME, fatty acid methyl ester; PAF, platelet activating factor; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IC50, inhibitory concentration at fifty percent. ⇑ Corresponding author. Address: Laboratory of Food Chemistry, Faculty of Chemistry, School of Sciences, National and Kapodistrian University of Athens, Athens 15771, Greece. Tel.: +30 210 7274 663; fax: +30 210 7274 476. E-mail address: [email protected] (I. Zabetakis). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.091

urgently needed. This data also underlines the diminishing levels of available wild fish worldwide and this trend, combined to the fact that aquacultured carnivorous species require large amounts of wild fish in their feed (Naylor et al., 2000), indicates an emerging necessity to find alternative and sustainable lipid sources to achieve secure production of fish feeds and, subsequently, aquacultured fish. Regarding fish growth; marine fish, such as sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), need three long chain polyunsaturated fatty acids (PUFA) for their normal growth rate: docosahexaenoic acid (DHA) 22:6 x-3, eicosapentaenoic acid (EPA) 20:5 x-3 and arachidonic acid (AA) 20:4 x-6 (Sargent, Bell, McEvoy, Tocher, & Estevez, 1999). However, recent studies have shown that fish oil (FO) replacement of up to 60% by vegetable oil (VO) does not indicate a negative impact on the growth, survival or health status of European sea bass and gilthead sea bream (Mourente, Dick, Bell, & Tocher, 2005a; Richard, Mourente, Kaushik, & Corraze, 2006; Fountoulaki et al., 2009; Nasopoulou & Zabetakis, 2012). On the other hand, when the fish feed

1098

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

contains more than 60% of VO, a reduction in growth was observed, which is probably related to their limited capacity to convert C18 fatty acids into PUFA’s (Izquierdo, 2005; Mourente et al., 2005a; Richard et al., 2006). The different concentrations of EPA, AA and a-linolenic acid (18:3 x-3) between VO and FO did not cause any differences in lipogenesis nor tissue lipid uptake in sea bass, but resulted in a decrease in hepatic lipogenesis in gilthead sea bream (Richard et al., 2006). The differential effects of VO on the two species may be due to the fact that sea bass use their liver as lipid storage, contrary to salmonoids which store their lipids in the perivisceral adipose tissue (Dias et al., 2005). Moreover, sea bass, which is a carnivorous marine fish, seems not to have quantitative nutritional effects by the substitution of FO by VO in the diet as far as the desaturation/elongation and b-oxidation activities of 14C-LNA and 14C-EPA in isolated hepatocytes and pyloric caeca enterocytes is concerned (Mourente, Dı´az-Salvago, Bell, & Tocher, 2002; Mourente et al., 2005a). Enterocytes and hepatocytes are very important in lipid metabolism including uptake oxidation, conversion of fatty acids and supply of PUFA to the tissues. Valuable information on the biosynthesis of PUFAs has been obtained by incubating isolated fish cells with radiolabeled fatty acids (Mourente et al., 2005a). Mechanisms involved in lipid deposition (lipogenesis and tissue lipid uptake) in sea bass were not affected by dietary oil source (Richard et al., 2006). Our scope is to valorize an alternative and sustainable vegetable lipid source, i.e. the olive industry by-product, named as ‘‘olive pomace’’ (OP). OP and olive mill waste water (OMWW) are the two major by-products of the olive oil extraction when using the three-phase centrifugal technology. On the other hand the modern two-phase centrifugal extraction technology merges OP with OMWW to produce a single by-product named olive mill waste (OMW) (Alburquerque, Gonzalvez, Garcia, & Cegarra, 2004). OMW, being acidic and with a high content in phenols and lipids, is phytotoxic, rendering its management problematic for all Mediterranean countries, given that significant amounts of OMW are produced every year (e.g. 5 million tonnes of OMW were produced in Spain for the olive campaign 2010–2011). Research efforts have focused on valorizing OMW in various ways, such as an energy source (Gogebakan & Selçuk, 2009) or as a fertiliser in composting (Tortosa, Alburquerque, Ait-Baddi, & Cegarra, 2012). Additionally, OP has important cardioprotective properties in terms of atheroprotective activity against Platelet Activating Factor (PAF); namely PAF-inhibitors (Karantonis et al., 2008; Nasopoulou & Zabetakis, 2013). Food components that act as PAF-inhibitors or PAF-antagonists can delay the process of atherogenesis and thus prevent the subsequent development of cardiovascular diseases (CVDs). Therefore, the presence of PAF-inhibitors or PAF-antagonists in OP is very important in terms of cardio-protection. It was found that OP contains polar lipids that inhibit or antagonise PAF in vitro (Karantonis et al., 2008) and also inhibit both specific PAF binding and PAF activity (in vitro and in vivo), consequently inhibiting early atherosclerosis development (Tsantila et al., 2007). OP polar lipids inhibit favourably the atherogenic progress (dyslipidemia and lesions): it has been found that the polar lipids of OP exert cardioprotective effects comparable to those of simvastatin, a very popular drug used to treat hypercholesterolemia (Tsantila et al., 2010). These data, coupled to the cost-effectiveness of OP polar lipids extraction and the environmental pollution problem that is caused by OP, has prompted us to seek further valorisation ways of OP in the production of food with higher nutritional value. Moreover the effect of olive OP on the growth performance and cardio protective properties of gilthead sea bream (S. aurata) and sea bass (D. labrax) has been studied (Nasopoulou, Stamatakis, Demopoulos, & Zabetakis, 2011) and it was found that OP

inclusion in fish feed at 8% resulted in poor growth rate and mortality for sea bass which has prompted us to work further on sea bass. Therefore the objective of the present study was firstly to optimise the levels of OP in fish feed, secondly to study sea bass growth performance and thirdly to elucidate the structures of sea bass muscle polar lipids that exerted cardioprotective activity, aiming to utilise this way a by-product of olive industry in the sustainable production of a novel functional food, possessing additional nutritional value in terms of cardio-protective properties, reaping the dual benefits of reduced environmental pollution and sustainable productivity. 2. Materials and methods 2.1. Reagents All chemicals and reagents were of analytical grade purchased from Merck (Darmstadt, Germany), while bovine serum albumin (BSA) and PAF were obtained from Sigma (St Louis, MO). 2.2. Fish diets Two different fish feeds were used for the dietary experimental trial of sea bass. The reference fish feed – fish oil diet (FO diet) – containing 100% fish oil (anchovy oil) and the experimental fish feed enriched with OP (OP diet), compounded by adding 4% OP prior to the extrusion, following the principles of fish nutrition (Gatlin, 2010). The chemical compositions of FO and OP diets are shown in Table 1. Protein digestibility determination took place according to van Leeuwen, van Kleef, van Kempen, Huisman, and Verstegen (1991) and energy determination took place according to the following equation (Gatlin, 2010): Energy ðMJ=KgÞ ¼ fðCPg  23:6KJÞ þ ðCFg  39:5KJÞ þ ð½CFig þ NFEg   17:4KJÞg=1000, where CP: crude protein; CF: crude fat; CFi: crude fibre; NFE = 1000  (CP + CF + Ash + Moist). OP originated from a local olive oil production. Both diets were formulated at the facilities of the marine farm where the dietary experiment took place using a twin-screw extruder creating pellets, followed by the addition of oil mixture. The pellets were dried, sealed and kept in air-tight bags until use. 2.3. Dietary experimental trial Farmed fish species sea bass (D. labrax), of initial mean body weight 150 g, was obtained from a commercial marine farm. The fish of each species were randomly distributed into two tanks (15 m3 each) in groups of 600 fish per tank at the facilities of the Table 1 Chemical composition of FO diet and OP diet (% wet weight). Ingredient (% wet weight)

FO diet

OP diet

Crude protein Fat Moisture Dietary fibre Ash Energy (MJ/kg) Protein digestibility (%) Vitamin A (IU/kg) Vitamin D (IU/kg) Vitamin E (mg/kg) Vitamin K3 (mg/kg) Vitamin C (mg/kg) Cu (mg/kg)

45.5 ± 2.1 20.2 ± 1.1 10 ± 1.3 2.2 ± 0.2 7.0 ± 0.7 21.5 ± 2.3 89 ± 4.0 7000 ± 233 3150 ± 108 180 ± 14 10 ± 0.9 200 ± 17 7.5 ± 1.4

45.5 ± 2.1 20.2 ± 1.1 7.1 ± 0.7 3.5 ± 0.2 6.2 ± 0.7 21.5 ± 2.3 89 ± 4.0 7000 ± 233 3150 ± 108 180 ± 14 10 ± 0.9 200 ± 17 7.5 ± 1.4

Values are means of three individual measurements, results are expressed as mean ± SD.

1099

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

farm. Both tanks were supplied with flow-through natural sea water system and were provided with continuous aeration and filters. The water temperature was 14–25.5 °C and oxygen content was kept close to saturation. The fish were forced to fast for three days before being transported to the tanks and then were acclimatised to the new environment for three days. The fish were fed with a 1% diet of body weight per day. Fish were acclimatised for 7 days to the experimental diets, prior to feeding trial initiation. Fish were weighed under moderate anaesthesia twice throughout the experimental period (at the beginning and at the end), which lasted for 180 days (April–September). The feed-conversion ratio and specific growth rate were calculated, while feed intake and mortality were recorded daily. At the beginning of the on-growing period, fish of both tanks were fed only with FO diet (reference diet) and 50 fish samples were collected and weighed. Ten of these fish samples provided the muscle samples to extract the fish muscle total polar lipids (TPL) that have been fractionated by HPLC; the biological activity of the obtained polar lipid fractions was then measured. At the end of the on-growing period (180 days), 200 fish samples were collected from each dietary treatment, 50 of them weighed and 10 of them provided the muscle samples to extract the fish muscle total polar lipids (TPL) that have been fractionated by HPLC; the biological activity of the obtained polar lipid fractions was then measured.

Table 2 Inhibitory and aggregatory activities against PAF, expressed as IC50 or EC50 values (lg TPL), of TPL of sea bass muscle fed with FO and OP diet and of DHA, EPA and their methyl esters (mean ± SD, 95% confidence levels, n = 3). All data are given in lg.

Inhibition

3.6 ± 0.6

–

Inhibition

3.8 ± 0.7

–

2.0 ± 0.3  50.0 ± 2.0  

– 120 ± 3.0

70.0 ± 5.0   260 ± 10.0 

– –

indicate statistically significantly different inhibitory activity within the same column (p < 0.05), according to Wilcoxon sign test.

Table 3 HPLC polar lipid fractions of fish fed with FO and OP diet, according to retention time (Rt) and their biological activity against washed platelets’ aggregation, or the PAFcaused washed platelets’ aggregation (mean ± SD, 95% confidence levels, n = 3). HPLC polar lipid fraction

Rt (min)

1 2 3 4 5 6

0–4 4–5 5–12 12–18 18–28.5 28.5– 39.5 39.5–47 47–58 58–62 62–68.3 68.3– 70.9 70.9– 71.3 71.3– 75.2 75.2– 77.1 77.1– 78.3 78.3–84 84–88 88–105

7 8 9 10 11 12 13 14

2.5. Instrumentation

The separation of fish TPL was performed on a reverse-phase HPLC with Nucleosil-300 C-18 column with a gradient elution system. The solvents and the elution solvent system were: solvent A: methanol: acetic acid (90:1, v/v), solvent B: acetonitrile (100%) and solvent C: water: acetic acid (100:1, v/v); elution profile: 0–30 min from 9:1:90 (A:B:C) to 27:3:70 (A:B:C) (gradient linear) and held from 30 to 35 min, followed by a linear gradient to 36:4:60 (A:B:C) over 35–45 min, then a linear gradient to 45:5:50 (A:B:C) from 45 to 60 min followed by a linear gradient

TPL of sea bass fed with FO diet TPL of sea bass fed with OP diet DHA EPA

Inhibition Inhibition and aggregation Inhibition Inhibition

EC50 (lg)

 ,   

TL of fish fillets of farmed sea bass fed with FO diet and OP diet were extracted by the Bligh and Dyer method (Bligh & Dyer, 1959). The separation of TNL and TPL was achieved with the countercurrent distribution (CCD) extraction procedure (Galanos & Kapoulas, 1962). The upper phase of petroleum ether contained the TNL while the lower phase of ethanol contained the TPL which were selected in a glass-stoppered flask, evaporated at 30 °C on a rotary evaporator, dissolved in chloroform/methanol (1:1) and stored at 20 °C until further analysis.

2.6. HPLC separation of TPL

Action towards PAF

Methyl-ester of DHA Methyl-ester of EPA

2.4. Separation of total lipids (TL) to total polar lipids (TPL) and total neutral lipids (TNL)

HPLC separation was conducted on TPL obtained from the samples at room temperature, on an HP HPLC Series 1100 liquid chromatographer (Hewlett–Packard, Waidronnn, German) equipped with a 100 lL Loop Rheodyne (7725 i) loop valve injector, a degasser G1322A, a gradient pump G1311A, a HP UV spectrophotometer G1322A, as a detection system, and a normal phase column (YMCPack Amino, 250  20 mm, S-5 lm, 12 nm (internal diameter)). The analysis of the chromatograph was performed via the Agilent Chemstation software. PAF-induced aggregation was measured in a Chrono-Log (Havertown, PA) aggregometer coupled to a Chrono-Log recorder (Havertown, PA).

IC50 (lg)

Analyte

15 (MS1) 16 17 (MS2) 18

Fish fed with FO

Fish fed with OP

Aggregation (peq PAF g1)

% Inhibition

Aggregation (peq PAF g1)

% Inhibition

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

100 ± 8.0 91 ± 7.8 40 ± 3.6  56 ± 4.1  56 ± 4.6  68 ± 7.3 

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

100 ± 9.1 80 ± 6.7 27 ± 2.2  73 ± 5.7  72 ± 5.9  24 ± 3.5 

0.0 ± 0.0   0.0 ± 0.0 0.0 ± 0.0   0.0 ± 0.0 0.0 ± 0.0

71 ± 7.1  35 ± 3.1 32 ± 2.8  56 ± 4.8  32 ± 2.2 

1.3 ± 0.3   0.0 ± 0.0 6.5 ± 2.1   0.0 ± 0.0 0.0 ± 0.0

0.0 ± 0.0  36 ± 2.9 0.0 ± 0.0  5.0 ± 1.2  70 ± 5.3 

0.0 ± 0.0

0.0 ± 0.0 

0.0 ± 0.0

47 ± 3.9 

0.0 ± 0.0

17 ± 2.2 

0.0 ± 0.0

100 ± 8.9 

66 ± 6.9

0.0 ± 0.0

0.0 ± 0.0 0.0 ± 0.0

  

0.0 ± 0.0 0.0 ± 0.0   0.0 ± 0.0

 

80 ± 7.3

93 ± 7.9

  

2.9 ± 0.7

0.0 ± 0.0 

97 ± 8.2 100 ± 9.3 100 ± 8.7 

0.0 ± 0.0 10.9 ± 1.8   0.0 ± 0.0

100 ± 9.5 100 ± 9.9 0.0 ± 0.0 

 

Indicates statistically significantly different inhibitory activity within the same row between HPLC polar lipid fractions of fish fed with FO and OP diet (p < 0.05), according to Wilcoxon sign test.    Indicates statistically significantly different aggregatory activity within the same row between HPLC polar lipid fractions of fish fed with FO and OP diet (p < 0.05), according to Wilcoxon sign test.

90:10 (A:B) from 60 to 70 min and held at this ratio from 70 to 80 min, then a linear gradient back down to 9:1:90 (A:B:C) from 80 to 90 min and maintained from 90 to 95 min. (Stamatakis et al., 2009). Injections of 100 ll of TPL samples were applied each time. The flow rate was 1 ml min-1 and the eluted substances were detected spectrophotometrically by UV detection at 208 nm at room temperature. The separation was achieved on a stepped gradient elution system. 2.7. Biological assay The purified polar lipid fractions of fish fillets of farmed fish fed with FO and OP diet, obtained by the above HPLC separations, were tested for their biological activity according to the washed platelet

1100

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

aggregation assay (Demopoulos, Pinckard, & Hanahan, 1979). PAF, as well as the examined samples, were dissolved in 2.5 mg of bovine serum albumin (BSA) ml1 of saline. The aggregatory activity of each HPLC lipid fraction to induce platelet aggregation was expressed in peq PAF g1 in reference to an eight points’ regression curve of standard PAF (Demopoulos et al., 1979), while the inhibitory activity of TPL was expressed as IC50 value (in mg) (Nasopoulou, Nomikos, Demopoulos, & Zabetakis, 2007) and the inhibitory activity of HPLC lipid fractions was expressed as a percentage of inhibition of the PAF-induced platelet aggregation.

out by comparison of the retention time and fragmentation pattern with a Bacterial FAME standard (Supelco). 2.10. Statistical analysis Chemical analyses were carried out in triplicates and all results were expressed as mean value ± SD. The Wilcoxon sign test was used to determine significant differences in the same group. Differences were considered significant for p < 0.05. The data were analysed using a statistical software package (IBM SPSS Statistics 19.0, SPSS Inc., Chicago, IL, USA).

2.8. Lipidomic analysis Total lipid extracts were dissolved in 15 ll of chloroform: methanol (1:2) and 15 ll of acetonitrile: isopropanol: water (6:7:2) and analysed with an Absceix 4000 QTrap, a triple quadrupole mass spectrometer equipped with a nanoelectrospray source. Samples were delivered using a Nanomate interface in direct infusion mode (125 nl min1). The lipid extracts were analysed in both positive and negative ion modes using a capillary voltage of 1.25 kV. MS/MS scanning (daughter, precursor and neutral loss scans) were performed using nitrogen as the collision gas with collision energies between 35 and 90 V. Each spectrum encompasses at least 50 repetitive scans. Tandem mass spectra (MS/MS) were obtained with collision energies as follows: 35–45 V, PC in positive ion mode, parent-ion scanning of m/z 184; 35–55 V, PI in negative ion mode, parent-ion scanning of m/z 241; 35–65 V, PE in negative ion mode, parent-ion scanning of m/z 196; 20–35 V, PS in negative ion mode, neutral loss scanning of m/z 87; and 40–90 V, for all glycerophospholipids (including PA and PG) detected by precursor scanning for m/z 153 in negative ion mode. MS/MS daughter ion scanning was performed with collision energies between 35 and 90 V. Assignment of phospholipid species is based upon a combination of survey, daughter, precursor and neutral loss scans. The identity of phospholipid species were verified using the LIPID MAPS: Nature Lipidomics Gateway (www.lipidmaps.org). 2.9. Quantification of fatty acid content Full characterisation and quantification of the fatty acids was conducted by conversion to the corresponding fatty acid methyl esters (FAME) followed by GC–MS analysis. Briefly, the samples were spiked with an internal standard fatty acid 17:0 (20 ll of 1 mM) and dried under nitrogen. The fatty acids from the lipids (neutral and phospholipid) were released by base hydrolysis to release fatty acids were released by base hydrolysis using 500 ll of concentrated ammonia and 50% propan-1-ol (1:1), followed by incubation for 5 h at 50 °C. After cooling the samples are evaporated to dryness with nitrogen and dried twice more from 200 ll of methanol: water (1:1) to remove all traces of ammonia. The protonated fatty acids were then extracted by partitioning between 500 ll of 20 mM HCl and 500 ll of ether, the aqueous phase was re-extracted with fresh ether (500 ll) and the combined ether phases were dried under nitrogen in a glass tube. The fatty acids were then converted to methyl esters by adding an ethereal solution of diazomethane (3  20 ll aliquots) to the dried residue, while on ice. After 30 min the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood. The FAME products were dissolved in 10–20 ll dichloromethane and 1–2 ll analysed by GC–MS on a Agilent Technologies (GC-6890 N, MS detector-5973) with a ZB-5 column (30 M  25 mm  25 mm, Phenomenex), with a temperature program of at 70 °C for 10 min followed by a gradient to 220 °C at 5 °C/min and held at 220 °C for a further 15 min. Mass spectra were acquired from 50 to 500 amu. The identity of FAMEs was carried

3. Results and discussion 3.1. Total and total polar lipid content of fish muscle The Total Lipid (TL) content percentage of sea bass muscle fed with FO and OP diet was 4.3 ± 0.9 and 4.8 ± 1.1, respectively, while the Total Polar Lipid (TPL) content percentage was 0.9 ± 0.3 and 1.6 ± 0.3, respectively. According to these data, both fish samples contain similar TL content, however sea bass fed with OP diet exhibited statistical increased TPL content, (p < 0.05) according to the Wilcoxon test, compared to the one of sea bass fed with FO diet. This statistical difference can be attributed to the OP diet, which has increased phospholipids’ levels. 3.2. Growth performance The enrichment of fish feed with OP (8%) has been successful in the case of gilthead sea bream (Nasopoulou et al., 2011) but was deemed unsatisfactory for sea bass. Therefore, in this study, the percentage of OP enrichment in the fish feed was halved (4% as opposed to 8%) while doubling the duration of the provision of the finishing diet (to 180 days as opposed to 90 days in our previous work, Nasopoulou et al., 2011). With these modifications, satisfactory results have been obtained both in terms of growth rate and also on the biologically activity of fish lipids against atheromatosis and thus the prevention of CVDs. The growth performance factors of sea bass fed with FO diet in comparison with sea bass fed with OP diet exhibited no statistical differences, p > 0.05 according to the Wilcoxon test, indicating similar feed conversion ratio and specific growth rate. These data suggest that sea bass fed with the experimental OP diet exhibited satisfactory growth performance, similar to that of sea bass fed with FO diet. More specifically, the feed conversion ratio for sea bass fed with OP diet was 3.42 ± 1.18, while for sea bass fed with FO diet was 1.75 ± 0.62 (values are means of three individual measurements and results are expressed as mean ± SD). The feed conversion ratio of sea bass fed with OP diet (3.42 ± 1.18) was satisfactory under the specified experimental conditions, considering the fact that other researchers – conducting dietary treatment in sea bass with diet manufactured with partial substitution of dietary fish oil with blends of vegetable oils – reported mean feed conversion ratio values ranging from 0.9 to 1.5 (Mourente & Bell, 2006), from 0.9 to 2.0 (Mourente, Good, Thompson, & Bell, 2007) and from 1.7 to 2.1 (Eroldog˘an et al., 2012a). Specific growth rate of sea bass fed with FO and OP diet was 0.60 ± 0.08 %/day and 0.49 ± 0.05 %/day, respectively (values are means of three individual measurements and results are expressed as mean ± SD). These results show that sea bass accepted the experimental OP diet, exhibiting satisfactory specific growth rate (0.49 ± 0.05 %/day), indicating that the replacement of 4% of FO

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

1101

Fig. 1. Total fatty acid analysis by gas chromatography–mass spectrometry. (A) Chromatogram of FAMEs obtained from fraction MS1, (B) chromatogram of FAMEs obtained from fraction MS2, (inserts show percentage of each fatty acid), see Experimental for details.

by OP does not moderate growth performance. On the contrary, it enhanced the development of the specific fish species. Similar specific growth rate values of sea bass fed with experimental diets containing plant origin oils has previously reported (Eroldog˘an et al., 2012b; Montero, Robaina, Caballero, Ginés, & Izquierdo, 2005; Mourente, Good, & Bell, 2005b). However, previous research (Nasopoulou et al., 2011) with farmed sea bass fed with OP diet showed a statistical decrease in specific growth rate and a statistical increase in mortality in comparison with sea bass fed with FO diet, although the feed intake was acceptable. The lack of growth performance in sea bass at the aforementioned study could be attributed to different percentage of enrichment of the experimental diet (8% vs 4% at the present study) and to different duration of the finishing diet (90 days vs 180 days at the present study). The growth performance factors measured in sea bass fed with FO and OP diets have shown some not statistically significant differences on feed conversion rate (FCR) and special growth rate (SGR). More specifically, FCR for sea bass fed OP diet were

3.42 ± 1.18, while for sea bass fed with FO diet was 1.75 ± 0.62. Sea bass accepted the experimental OP diet exhibiting lower SGR (0.49 ± 0.05% day) compared to the one fed the FO diet (0.60 + 0.08% day), indicating that the introduction of 4% OP does moderate growth performance. Possibly, the lowest growth performance in OP diet results from the significantly higher percentage of fibres in OP diet than that in FO diet. Olive pomace features a very high percentage of indigestible fibres which maybe affected negatively the growth effectiveness of OP diet. This disadvantage is possibly balanced by the enrichment of sea bass’ flesh with polar lipids.

3.3. Biological activity of TPL and HPLC fish muscle polar lipids The inhibitory activity of TPL of sea bass muscle fed with FO and OP diet, IC50 value expressed in lg of TPL, against PAF-induced platelet aggregation is shown in Table 2.

1102

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

Fig. 2. Lipidomic analysis of fraction MS1. (A) Negative ion ES-MS survey scans (600–1000 m/z), (B) positive ion ES-MS survey scans (600–1000 m/z). Annotation of the major lipid species are based upon daughter ion fragmentation analysis by ES-MS–MS, see Experimental for details.

According to Table 2, the inhibitory activity of TPL of both fish samples (i.e. FO diet as opposed to OP diet) did not show any statistical difference, however – as is discussed below – the HPLC lipid fraction of both fish species exhibited significant differences regarding their biological activity against platelets. More specifically, TPL from both fish samples were fractionated by reverse-phase HPLC in three sequential separations of 100 lL (the HPLC chromatograms are given in the Supplementary material). The fractions with the same retention times (Rt) from each

injection were unified and examined for their biological activity to cause PAF-induced washed rabbit platelet aggregation. The aggregatory activities, expressed in peq PAF g1 and inhibitory activities, expressed as % inhibition of the PAF-induced platelet aggregation, of obtained polar lipid fractions after HPLC separation of fish fed with FO and OP diet are shown in Table 3. Regarding the inhibitory activity against the PAF-induced platelet aggregation the HPLC polar lipid fractions 1, 2, 16 and 17 of both fish fed with the FO and OP diets exhibited similar, potent,

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

1103

Fig. 3. Lipidomic analysis of fraction MS2. (A) Positive ion ES-MS survey scans (600–1000 m/z), (B) negative ion ES-MS survey scans (600–1000 m/z). Annotation of the major lipid species are based upon daughter ion fragmentation analysis by ES-MS–MS, see Experimental for details.

inhibitory activity (Table 3). Moreover polar lipid fractions 3, 6, 7, 9, 10, 15 and 18 of fish fed with OP diet showed significantly less potent inhibitory activity against PAF-induced platelet aggregation compared with the respective polar lipid fractions of fish fed with FO diet. The rest of the polar lipid fractions of fish fed with OP diet had significantly increased inhibitory properties compared with the respective polar lipid fractions of fish fed with FO diet (Table 3),

indicating that the components of OP with proved anti-atherogenic and cardio-protective qualities were transferred to the OP diet and thus to fish maintaining their strong biological activity. It is worth mentioning that the lipid fraction of fish fed with OP diet induced a noticeable platelet aggregation while the respective lipid fraction of fish fed with OF diet did not exhibited any aggregatory activity (Table 3). More specifically the HPLC lipid fractions

1104

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

of fish fed with OP diet that exhibited aggregatory properties were fractions 7, 9, 15 and 17, while the most potent platelet aggregatory activity was attributed to fraction 17. Such diversity in biological activity (potent or less potent inhibitory or aggregatory activity) could be attributed to the fact that fish polar lipids are a mixture of lipid molecules that can potentially have aggregatory or inhibitory properties. The final activity observed depends: (a) on the relative ability of each molecule to aggregate platelets or inhibit the PAF-induced platelet aggregation and (b) the relative amount of each molecule in the mixture.

3.4. Fatty acid analysis The total fatty acid content of the lipids (neutral and phospholipid) present in fraction MS1 and MS2 were determined by GC–MS (Fig. 1). Both fractions contain low levels of myristic (14:0), oleic (18:1) and linoleic acids (18:2) and high levels of palmitic (16:0) and stearic acids (18:0) as well as eicosadienoic acid (20:2 x-6). However fraction MS1 also contained significant levels of AA (20:4 x-6) and the omega-3 fatty acids EPA (22:5) and DHA (22:6). The phospholipid composition of the two fractions MS1 and MS2 were investigated in detail using electrospray mass spectrometry. In negative ion mode, survey scans between 600 and 1000 m/z of MS1 (Fig. 2A) showed a wide range of molecular species from all of the major classes of phospholipid (PI, PS, PE, PA and PG). ES-MS– MS daughter ion fragmentation analysis allowed full characterisation of all of the major lipid species. Many of these were diacylglycerol species containing either 18:0 or 18:1 fatty acids at the sn-1 position and 22:6 fatty acids in the sn-2 position, this includes PA (748 m/z), PG (819 m/z), PE (789 m/z), PS (833 m/z) and PI (910 m/z). The only exception to this were PI (18:0, 20:2) at 889 m/z and two acyl–alkyl PE species, at 775 m/z and 803 m/z corresponding to a-18:0, 22:6 and a-20:0, 22:6, respectively, both of which still containing a DHA (22:6) at the sn-2 position. In positive ion survey scans (Fig. 2B) the lipid species were predominantly diacyl PC species containing primarily either 18:1, 18:2, 20:4, 22:5 or 22:6 fatty acids, with the only exception being a minor acyl-alkyl-PC species at 848 m/z (a-20, 22:6). In contrast to this, lipidomic analysis of MS2 showed that the vast majority of the lipid species were diacyl PC species contained either 18:2, and/or C20:2 (Fig. 3A). The negative ion mode survey scan (Fig. 3B), which in contrast showed very few other low abundance lipid species, the only significant one being PG (18:2, 20:5) at 819 m/z. Further lipidomic analysis of both MS1 and MS2 fractions at lower mass range showed no evidence of lyso-PC species. In Table 2, the IC50 values of DHA and EPA and their corresponding methyl esters are given in lg. In brief, the free acids (especially DHA) exhibited stronger PAF inhibition activities than the corresponding methyl esters. The IC50 value of DHA has been found to be 2 lg, similar to the one for total lipids of olive pomace fed sea bream (5 lg, as reported in our previous work, Nasopoulou et al., 2011). Methyl esters are most likely acting as pro-drugs, allowing passive uptake of these molecules by the human body; they are converted in situ to the corresponding free fatty acids by esterases allowing them to exhibit their cardioprotective activities. The PC species identified in this work (Figs. 2 and 3) could serve as natural precursors, whereby they are taken up/endocytosed by normal mechanisms, where upon phospholipase A2 s activities allow liberation of free DHA and EPA fatty acid. Due to the very low level of acyl-alkyl-PC species which could potentially form PAF species by the concerted actions of a phospholipase A2 and lysoPAF acetyltransferase, forming a PAF species (Fragopoulou et al., 2006; Prescott, Zimmerman, Stafforini, & McIntyre, 2000; Yost, Weyrich, & Zimmerman, 2010). This means that, unlike many

sources of lipids, this is an excellent source of molecules with inhibitory and agonistic effects against PAF. Polar lipids in food could show either a weak agonistic (aggregatory) or an inhibitory effect on the PAF-induced biological activities. Some lipids, at low levels, could have a rather weak agonistic activity but, at higher levels, their inhibitory activities prevail. However, some other lipids might not show any agonistic activities but only inhibitory ones. In the case of lipid fractions with aggregatory activities (i.e. this is the case of the MS1 and MS2 fractions studied here), they act as weak agonists to PAF-receptors, being thousand times less active than PAF. However, in this case, these components are practically acting as PAF-inhibitors. Weak PAF agonists (with aggregatory activity) have been found to exhibit in vivo higher antiatherogenic activity than PAF – inhibitors (Tsantila et. al., 2007; Nasopoulou et al., 2010). The detection of polar lipids in MS1 and MS2 fractions that exhibit aggregatory actions is a potent indication that these lipid fractions contain biologically active compounds against PAF and consequently against atherogenesis. The association of omega-3 PUFAs and CVDs has been revised recently by evaluating all randomised trials on the supplementation of omega-3 PUFAs to adults (Rizos, Ntzani, Bika, Kostapanos, & Elisaf, 2012). In this review, the results of 20 studies on 68.680 patients were evaluated and omega-3 PUFAs were not found to be statistically significantly associated with CVDs in various patient populations. Therefore, the latest evidence on the in vivo activities of omega-3 prescribed drugs (where the fatty acids DHA and EPA have been esterified hence they are neutral molecules) suggests that they do not offer strong protection against CVDs. The implications of the data reported here can be viewed as follows. Firstly, the prescribed drugs (e.g. OmacorÒ in UK, containing the methyl esters of DHA and EPA) are less potent against PAF (than the free fatty acids), but they serve as precursors (pro-drugs) of free fatty acids with strong cardioprotective activites. Secondly, the cardioprotective properties of the lipids within MS1 and MS2 fractions contain considerable amounts of EPA and DHA which can be liberated by the activity of a phospholipase A2, but do so without liberation of potential harmful lyso-alkyl-PC, a PAF precursor (Fragopoulou et al., 2006; Prescott et al., 2000; Yost et al., 2010).

4. Conclusions Our present study aims to structurally characterise the fractions of polar lipids present in OP fed sea bass, namely fractions MS1 and MS2 that have shown strong cardioprotective properties, and understand the relationship between their structure and (biological) function of these lipids as agonists and inhibitors of PAF activities, hence reducing CVDs. The aforementioned HPLC fish polar lipid fractions (MS1 and MS2) contained considerable amounts of EPA and DHA that could be liberated by the activity of the enzyme phospholipase A2. Possible limitations of OP inclusion in aquaculture fish feed could be potential variation of OP’s chemical composition, due to the agronomic and technological conditions of production and due to the olive cultivar, which mainly affects the phenolic content of olive oil and by-products. However, in previous work of our group (unpublished data), such variation does not affect the cardioprotective properties of OP. Another possible limitation could be the high indigestible fibres’ content of OP, which maybe moderate the growth effectiveness of OP diet. Our current and future work is towards the identification of fish polar lipids that inhibit atherosclerosis, applying a novel pro-drug/ precursor approach (Qandil, 2012) by exploiting phospholipids containing PUFA in order to reduce PAF activity.

C. Nasopoulou et al. / Food Chemistry 145 (2014) 1097–1105

Acknowledgements This work is supported in part by the aquaculture company NIREUS SA and TKS’s Wellcome Trust project grants 086658 and 093228. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 08.091. References Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Demopoulos, C. A., Pinckard, R. N., & Hanahan, D. J. (1979). Platelet activating factor. Evidence for 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine as the active component (a new class of lipid chemical mediators). Journal of Biological Chemistry, 254, 9355–9358. Dias, J., Alvarez, M. J., Arzel, J., Corraze, G., Diez, A., Bautista, J. M., et al. (2005). Dietary protein source affects lipid metabolism in the European seabass (Dicentrarchus labrax). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 142, 19–31. Eroldog˘an, T., Turchini, G. M., Yılmaz, A. H., Tasßbozan, O., Engin, K., Ölçülü, A., et al. (2012b). Potential of cottonseed oil as fish oil replacer in European sea bass feed formulation. Turkish Journal of Fisheries and Aquatic Sciences, 12, 787–797. Eroldog˘an, T., Yılmaz, A. H., Turchini, G. M., Arslan, M., Sirkeciog˘lu, N. A., Engin, K., et al. (2012a). Fatty acid metabolism in European sea bass (Dicentrarchus labrax): Effects of n  6 PUFA and MUFA in fish oil replaced diets. Fish Physiology and Biochemistry. http://dx.doi.org/10.1007/s10695-012-9753-7. FAO (Food and Agriculture Organization of the United Nations) (2010). The State of World Fisheries and Aquaculture. Rome: FAO, Italy. Fountoulaki, E., Vasilaki, A., Hurtado, R., Grigorakis, K., Karacostas, I., Nengas, I., et al. (2009). Fish oil substitution by vegetable oils in commercial diets for gilthead sea bream (Sparus aurata L.); effects on growth performance, flesh quality and fillet fatty acid profile: Recovery of fatty acid profiles by a fish oil finishing diet under fluctuating water temperatures. Aquaculture, 289, 317–326. Fragopoulou, E., Iatrou, C., Antonopoulou, S., Ruan, X. Z., Fernando, R. L., Powis, S. H., et al. (2006). Platelet-activating factor (PAF) increase intracellular lipid accumulation by increasing LDL and scavenger receptors in human mesangial cells. Journal of Laboratory and Clinical Medicine, 147, 281–289. Galanos, D. S., & Kapoulas, V. M. (1962). Isolation of polar lipids from triglyceride mixtures. Journal of Lipid Research, 3, 134–137. Gatlin, D. M. (2010). Principles of fish nutrition. USDA, Southern Regional Aquaculture Center. Publication No. 5003, 1-8. Gogebakan, Z., & Selçuk, N. (2009). Trace elements partitioning during co-firing biomass with lignite in a pilot-scale fluidized bed combustor. Journal of Hazardous Materials, 162, 1129–1134. Izquierdo, M. (2005). Essential fatty acid requirements in Mediterranean fish species. Cahiers Options Méditerranéennes, 63, 91–102. Montero, D., Robaina, L., Caballero, M. J., Ginés, R., & Izquierdo, M. S. (2005). Growth, feed utilization and flesh quality of European sea bass (Dicentrarchus labrax) fed diets containing vegetable oils: A time-course study on the effect of a re-feeding period with a 100% fish oil diet. Aquaculture, 248, 121–134. Mourente, G. J., & Bell, J. G. (2006). Partial replacement of dietary fish oil with blends of vegetable oils (rapeseed, linseed and palm oils) in diets for European sea bass (Dicentrarchus labrax L.) over a long term growth study: Effects on muscle and liver fatty acid composition and effectiveness of a fish oil finishing diet. Comparative Biochemistry and Physiology, Part B, 145, 389–399. Mourente, G., Dı´az-Salvago, E., Bell, J., & Tocher, D. R. (2002). Increased activities of hepatic antioxidant defence enzymes in juvenile gilthead sea bream (Sparus aurata L.) fed dietary oxidised oil: Attenuation by dietary vitamin E. Aquaculture, 214, 343–361. Mourente, G., Dick, J. R., Bell, J., & Tocher, D. R. (2005a). Effect of partial substitution of dietary fish oil by vegetable oils on desaturation and b-oxidation of [1-

1105

14C]18:3n–3 (LNA) and [1-14C]20:5n–3 (EPA) in hepatocytes and enterocytes of European sea bass (Dicentrarchus labrax L.). Aquaculture, 248, 173–186. Mourente, G., Good, J. E., & Bell, J. G. (2005b). Partial substitution of fish oil with rapeseed, linseed and olive oils in diets for European sea bass (Dicentrarchus labrax L.): Effects on flesh fatty acid composition, plasma prostaglandins E2 and E2a immune function and effectiveness of a fish oil finishing diet. Aquaculture Nutrition, 11, 25–40. Mourente, G., Good, J. E., Thompson, K. D., & Bell, J. G. (2007). Effects of partial substitution of dietary fish oil with blends of vegetable oils, on blood leukocyte fatty acid compositions, immune function and histology in European sea bass, (Dicentrarchus labrax L.). British Journal of Nutrition, 98, 770–779. Nasopoulou, C., Karantonis, H. C., Perrea, D. N., Theocharis, S. E., Iliopoulos, D. G., Demopoulos, C. A., et al. (2010). In vivo anti-atherogenic properties of cultured gilthead sea bream (Sparus aurata) polar lipid extracts in hypercholesterolaemic rabbits. Food Chemistry, 120, 831–836. Nasopoulou, C., Nomikos, T., Demopoulos, C. A., & Zabetakis, I. (2007). Comparison of antiatherogenic properties of lipids obtained from wild and cultured sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata). Food Chemistry, 100, 560–567. Nasopoulou, C., Stamatakis, G., Demopoulos, C. A., & Zabetakis, I. (2011). Effects of olive pomace and olive pomace oil on growth performance, fatty acid composition and cardio protective properties of gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). Food Chemistry, 129, 1108–1113. Nasopoulou, C., & Zabetakis, I. (2012). Benefits of fish oil replacement by plant originated oils in compounded fish feeds. A review. LWT – Food Science and Technology, 47, 217–224. Nasopoulou, C., & Zabetakis, I. (2013). Agricultural and aquacultural potential of olive pomace. A review. Journal of Agricultural Science, 5, 116–127. Naylor, R. L., Goldburg, R. J., Primavera, J. H., Kautsky, N., Beveridge, M. C. M., Clay, J., et al. (2000). Effect of aquaculture on world fish supplies. Nature, 405, 1017–1024. Pike, I. H., & Jackson, A. (2010). Fish oil: Production and use now and in the future. Lipid Technology, 22, 59–61. Prescott, S. M., Zimmerman, G. A., Stafforini, D. M., & McIntyre, T. M. (2000). Platelet-activating factor and related lipid mediators. Annual Review of Biochemistry, 69, 419–445. Qandil, A. M. (2012). Prodrugs of nonsteroidal anti-inflammatory drugs (NSAIDs), more than meets the eye: A critical review. International Journal of Molecular Sciences, 13, 17244–17274. Richard, N., Mourente, G., Kaushik, S., & Corraze, G. (2006). Replacement of a large portion of fish oil by vegetable oils does not affect lipogenesis, lipid transport and tissue lipid uptake in European seabass (Dicentrarchus labrax L.). Aquaculture, 261, 1077–1087. Rizos, E. C., Ntzani, E. E., Bika, E., Kostapanos, M. S., & Elisaf, M. S. (2012). Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events. A systematic review and meta-analysis. JAMA, 308, 1024–1033. Sargent, J., Bell, G., McEvoy, L., Tocher, D., & Estevez, A. (1999). Recent developments in the essential fatty acid nutrition of fish. Aquaculture, 177, 191–199. Stamatakis, G., Tsantila, N., Samiotaki, M., Panayotou, G. N., Demopoulos, A. C., Halvadakis, C. P., et al. (2009). Detection and isolation of antioxidant substances present in olive mill wastes by a novel filtration system. Journal of Agricultural and Food Chemistry, 57, 10554–10564. Tortosa, G., Alburquerque, J. A., Ait-Baddi, G., & Cegarra, J. (2012). The production of commercial organic amendments and fertilisers by composting of two-phase olive mill waste (‘‘alperujo’’). Journal of Cleaner Production, 26, 48–55. Tsantila, N., Karantonis, H. C., Perrea, D. N., Theocharis, S. E., Iliopoulos, D. G., Antonopoulou, S., et al. (2007). Antithrombotic and antiatherosclerotic properties of olive oil and pomace polar extracts in rabbits. Mediators of Inflammation, 2007, 1–11. Tsantila, N., Karantonis, H. C., Perrea, D. N., Theocharis, S. E., Iliopoulos, D. G., Iatrou, C., et al. (2010). Atherosclerosis regression study in rabbits upon olive pomace polar lipid extract administration. Nutrition, Metabolism & Cardiovascular Diseases, 20, 740–747. van Leeuwen, P., van Kleef, D. J., van Kempen, G. J. M., Huisman, J. & Verstegen, M. W. A. (1991). The post valve T-caecum cannulation technique in pigs applicated to determine the digestibility of amino acids in maize, groundnut and sunflower meal. Journal of Animal Physiology and Animal Nutrition, 65, 183–193.Yost, C. C., Weyrich, A. S., & Zimmerman, G. A. (2010). The platelet activating factor (PAF) signaling cascade in systemic inflammatory responses. Biochimie, 92, 692–697.