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Phospholipids of goat and sheep origin: Structural and functional studies
Small Ruminant Research 167 (2018) 39–47
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Phospholipids of goat and sheep origin: Structural and functional studies a
b
c
Stylianos Poutzalis , Ronan Lordan , Constantina Nasopoulou , Ioannis Zabetakis a b c
b,⁎
T
Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771, Athens, Greece Department of Biological Sciences, University of Limerick, V94 T9PX, Limerick, Ireland Department of Food Science and Nutrition, School of the Environment, University of the Aegean, Myrina, Lemnos, 81400, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherogenesis Anti-atherogenic activity Polar lipids Phosphatidylcholine Meat
The lipidomic profiles of goat and sheep meat were studied. Polar lipid fractions of raw and baked meat samples were tested for their in vitro anti-atherogenic properties. The total lipid (TL) content was extracted using the Bligh-Dyer method and was subsequently separated into total polar lipids (TPL) and total neutral lipids (TNL). The fatty acid profiles of the TPL and TNL of all three samples were determined by GC-FID. The TPL of all samples were further separated by preparative TLC into their constituent phospholipid and sphingolipid fractions. In all samples, polar lipid fraction 3 had a similar Rf value to phosphatidylcholine. These phosphatidylcholine fractions were tested for their in vitro capacity to inhibit platelet-activating factor (PAF) induced platelet aggregation (anti-inflammatory activity) using human platelets. The phospholipid content of each fraction 3 was determined using LC–MS. These results provide a novel insight into the structure of phosphatidylcholine derivatives in goat and sheep meat and highlight the nutritional value of these meats in terms of their antithrombotic and cardioprotective properties before and after the baking process.
1. Introduction Foods and fats of animal origin receive undue criticism from society and scientific communities due to their perceived negative effects on health upon consumption. Recent research trends indicate that these negative perceptions may be unwarranted as numerous studies suggest that meat consumption may be associated with a positive effect on health when eaten in moderation despite their SFA and cholesterol content (Lordan et al., 2017). Red meat is a highly nutritious and valuable source of proteins, iron, phosphorus, zinc, selenium and B group vitamins. Meat from ruminant animals, such as sheep and goat, contain trans fatty acids that are, unlike industrial fatty acids, beneficial for the human health (Bendsen et al., 2011; Mulvihill, 2001). The most common source of trans fatty acids in ruminant meat is conjugated linoleic acid (CLA), which has been linked to a protective role against cancer, cardiovascular diseases (CVD) and has been associated with weight loss (Bendsen et al., 2011; Blankson et al., 2000; Schmid et al., 2006). According to the lipid hypothesis, which stemmed from the Seven Countries Study, saturated fatty acids (SFA), cholesterol and LDL levels are responsible for atherogenesis and consequently their presence in excessive levels can increase the risk of CVD development (Steinberg, 2006). Several studies have implicated red meat in the development of
⁎
CVD due to their effect on blood lipid parameters but also the effect of additives and preservatives (Pan et al., 2012; Wolk, 2016). However, there are meta-analyses and reviews that refute these claims and they have shown that red meat consumption does not increase the risk for CVD development (Connor et al., 2016; Mcafee et al., 2010). Recent studies that separated red meat into processed and unprocessed forms, have brought to light the fact that unprocessed red meat has a minor association or no association with increased mortality and CVD (Larsson and Orsini, 2014; Micha et al., 2012, 2010). It has also been suggested that potential bias in many of the studies investigating the consumption of meat and its effect on atherosclerosis may have influenced the conclusions (Larsson and Orsini, 2014; Micha et al., 2012). Other studies have shown that a diet containing meat and cheese can lead to increased levels of HDL cholesterol and thus, it appears to be less atherogenic than a low-fat, high-carbohydrate diet (National Obesity Forum and Public Health Collaboration, 2016). There is considerable concern that red meat consumption elevates levels of choline and L-carnitine. Phosphatidylcholine (PC) is broken down to choline, which is transformed by the intestinal microbiota to trimethylamine (TMA), which along with L-carnitine is metabolised to trimethylamine N-oxide (TMAO) (Wang et al., 2011). It is proposed that excess dietary PC increases the levels of TMAO resulting in a pro-inflammatory and
Corresponding author at: Department of Biological Sciences, University of Limerick, Limerick, Ireland. E-mail address: [email protected] (I. Zabetakis).
https://doi.org/10.1016/j.smallrumres.2018.07.015 Received 23 November 2017; Received in revised form 29 June 2018; Accepted 20 July 2018 Available online 30 July 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
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2.2. Meat samples
prothrombotic state that may lead to eventual insulin resistance, type II diabetes mellitus, and CVD (Lordan et al., 2017; Zhu et al., 2016). However, some results indicate that dietary choline may not be to blame, and that the presence of specific gut bacteria promotes the conversion of choline into TMAO (Clouatre and Bell, 2013; Koeth et al., 2013). Other researchers suggested that dietary choline from PC derivatives in dairy and marine sources possess antithrombotic properties, contrary to the effects of TMAO (Lordan et al., 2017; Lordan and Zabetakis, 2017a). With regards to atherogenesis, platelet-activating factor (PAF) is a crucial potent inflammatory phospholipid mediator (Demopoulos et al., 1979), which is produced by many cells and is involved in the activation of leukocytes and their binding to endothelial cells (Demopoulos et al., 2003; Lordan and Zabetakis, 2017b). CVD such as coronary heart disease and stroke are clinical events as a consequence of atherosclerosis, which is a chronic inflammatory condition mediated by PAF and other molecules (Nasopoulou et al., 2013c) Our previous work has shown that polar lipids are strong inhibitors of the inflammatory and atherogenic actions of PAF (Megson et al., 2016; Zabetakis, 2013). Recently, polar lipids from sheep and goat dairy products have been evaluated for their inhibitory activities against PAF (Lordan and Zabetakis, 2017b; Megalemou et al., 2017; Poutzalis et al., 2016; Tsorotioti et al., 2014); the most potent inhibitory properties were exhibited by TLC polar lipid fractions with Rf similar to that of PC (Megalemou et al., 2017; Poutzalis et al., 2016). However, little is known about the bioactivity of phospholipids present in sheep or goat meat. Muscles from mammals are excellent sources of phospholipids (Christie, 1978) and especially of PC which often constitutes to almost 50% of the total phospholipids (Perez-Palacios et al., 2007; Sinanoglou et al., 2013). In lamb, the phospholipid content can constitute 42% of the total lipids, with PC and phosphatidylethanolamine being the predominant lipid species (38–55% and 25–31% respectively) (Lordan et al., 2017). Lipid microconstituents that exert in vitro anti-PAF activities could retard and/or regress atherosclerosis and consequently CVD, as several in vivo studies have demonstrated (Karantonis et al., 2006; Nasopoulou et al., 2010; Tsantila et al., 2010, 2007). The aim of our present work was to explore the lipidomic profiles of raw and baked goat and sheep meat and to evaluate the anti-atherogenic properties of PC derivatives obtained from these food sources. Our data suggests that goat and sheep meat possess potential functional cardioprotective lipid microconstituents that warrant further research, which assist in re-assessing the nutritional value of these meats.
Meat samples were purchased from a local market in Athens. Both the sheep and the goat were male and were 6–10 weeks of age. The sheep originated from Amphilochia region in central Greece, whereas the goat originated from Lakonia region in southern Greece. The meat samples used were untrimmed rib steaks, as typically consumed in Greece. Initially, the bone was removed. The baked meat samples were prepared as follows: the meat was cut into 2 × 2×1 cm3 pieces, wrapped in aluminium foil, and cooked in a preheated oven at 180 °C for 20 min. The raw and baked meat samples used for analysis were further chopped into as small as possible pieces using a knife. The remaining meat juices left after the baking process were discarded. Four groups of meat samples have been generated, namely goat raw (GR), goat baked (GB), sheep raw (SR) and sheep baked (SB) and these meat sample codes are used throughout the manuscript. 2.3. Isolation of lipids Extraction of the total lipids (TL) from each 100 g sample (kid and lamb, raw or baked) was carried out using the Bligh and Dyer method (Bligh and Dyer, 1959). The TL were weighed and one fourth of the sample was stored in sealed vials under a nitrogen atmosphere at -20 °C, while the rest of the TL was further separated by counter-current distribution (Galanos and Kapoulas, 1962) into total polar lipids (TPL) and total neutral lipids (TNL). In brief, petroleum ether and 87% aqueous ethanol were pre-equilibrated in a separatory funnel. The lower phase containing equilibrated 87% ethanol and the upper phase containing equilibrated petroleum ether were collected separately. Approximately 2 g of TL were dissolved in 9 mL pre-equilibrated petroleum ether and afterwards 3 mL pre-equilibrated 87% ethanol were added and stirred. The lower phase (ethanolic phase) was collected and transferred to a second test tube containing 9 mL of pre-equilibrated petroleum ether and was stirred again. The ethanolic phase in the second test tube was transferred to a round-bottom flask. The procedure was repeated eight times in total. Finally, the ethanolic phase (8 × 3 mL), containing the TPL, and the petroleum ether phase (2 × 9 mL), containing TNL, were evaporated to dryness, weighed and stored under nitrogen in sealed vials at −20 °C. Each extraction was carried out in triplicate. 2.4. Fractionation of TPL by preparative TLC The TPL from all samples were further separated by preparative TLC. The TLC glass plates (20 × 20 cm, thickness 1.0 mm) were coated with silica gel G-60 and activated by heating at 120 °C for 60 min. Approximately 40 mg of TPL was applied to the TLC plates. The developing system consisted of chloroform/methanol/water 65:35:6 (v/ v/v). After development, the plates were stained under iodine vapors. Eight bands appeared after the separation of TPL of the samples. Subsequently, the bands were scraped off, and the lipids from the desirable bands were extracted according to the Bligh-Dyer method (Bligh and Dyer, 1959). The chloroform phase was evaporated to dryness under nitrogen, and lipids were weighed and stored at −20 °C.
2. Materials and methods 2.1. Reagents and instruments All reagents and solvents were of analytical grade purchased from Merck (Darmstadt, Germany). Fatty acid methyl ester standards were of GC-quality and supplied by Sigma-Aldrich (St. Louis, MO, USA), as well as bovine serum albumin (BSA) and PAF. The Chromatographic material used for thin-layer chromatography (TLC) was silica gel G-60 supplied by Merck (Darmstadt, Germany) and the polar lipid standards used for TLC were supplied by Sigma-Aldrich as a standard mix of hen egg yolk (St. Louis, MO, USA). Gas chromatographic analysis was carried out on a Shimadzu CLASS-VP (GC-17 A) (Kyoto, Japan) gas chromatograph equipped with a split/splitless injector and flame ionization detector. Liquid chromatography-mass spectrometry (LC–MS) was carried out using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempstead, UK), equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 UHPLC system. Platelet aggregation was measured on a Chrono-Log (Havertown, PA, USA) aggregometer (model 400-VS) coupled to a Chrono-Log recorder (Havertown, PA, USA).
2.5. Gas chromatographic analysis Fatty acid methyl esters (FAME) were prepared from 35 mg of TPL and 35 mg of TNL of all four samples (GR, GB, SR and SB) using a solution of 0.5 N KOH in CH3OH (KOH-CH3OH method with reaction time 5 min) and extracted with n-hexane. Analysis was carried out on a Shimadzu CLASS-VP (GC-17 A) (Kyoto, Japan) gas chromatograph equipped with a split/splitless injector and flame ionization detector. Separation of FAMEs was achieved using an Agilent J&W DB-23 fused silica capillary column (60 m × 0.251 mm i.d., 0.25 μm; Agilent, Santa Clara, CA, USA). The oven temperature was initially set at 120 °C for 5 min, raised to 180 °C at 10 °C min−1, then to 220 °C at 20 °C min−1, 40
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and finally kept at 220 °C for 30 min. The temperatures of injector and detector were maintained at 220 and 225 °C, respectively. The carrier gas was high purity helium with a linear flow rate of 1 mL.min−1 and split ratio of 1:50, as described before (Poutzalis et al., 2016). Fatty acid methyl esters were identified using Supelco 37-Component FAME Mix and additionally docosapentanoic acid (22:5) methyl ester standard. FAME 21:0 was used as internal standard for quantifying FAMEs.
Table 1 Content of total lipids (TL), expressed in grams per 100 g sample (mean ± SD, n = 3), total polar lipids (TPL), and total neutral lipids (TNL), expressed as percentages of TL in raw and baked kid and lamb. Samples Goat meat (kid) raw, GR Sheep meat (lamb) raw, SR Goat meat (kid) baked, GB Sheep meat (lamb) baked, SB
2.6. Analysis of Lipids by Liquid Chromatography-Mass Spectrometry (LC–MS) For each sample, 10 mg of dried TLC polar lipid fraction 3, from all samples, were dissolved by addition of 0.5 mL methanol and 0.5 mL chloroform. A dilution of 1 in 100 was made from the mixture in methanol containing 5 mM ammonium formate. All samples were centrifuged at 650× g for 5 min at 4 °C to sediment any particulate matter. The lipids were analysed by liquid chromatography-mass spectrometry (LC–MS) using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempstead, UK), equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 UHPLC system. All samples were analysed in both positive and negative ion mode over the mass to charge (m/z) range 250–2000 with a resolution of 25,000. The samples were injected (1 μL and 2 μL, in positive and negative modes respectively) on to a Thermo Hypersil Gold C18 column (2.1 mm x 100 mm, 1.9 μm). Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% (v/v) formic acid. Mobile phase B consisted of 90:10 isopropanol/acetonitrile containing 10 mM ammonium formate and 0.1% (v/v) formic acid. The initial conditions for analysis were 65% A / 35% B. The percentage of mobile phase B was increased to 65% over 4 min and then 100% over 15 min. These conditions were held for 2 min before re-equilibration with the starting conditions for 6 min. The flow rate was 400 μL/min with a column temperature of 40 °C. All solvents were LC–MS grade. LC–MS data was processed with Progenesis QI v2.0 software (Nonlinear Dynamics, Newcastle, UK) and searched against HMDB (www. hmdb.ca) and LIPID MAPS (www.lipidmaps.org) for identification.
TL (g/100 g sample) 10.51 ± 0.312
b,c
11.44 ± 0.254
d,e
16.54 ± 0.286
b,d
17.76 ± 0.318c,e
TPL (%TL)
TNL (%TL)
9.78 ± 0.179
a,b,c
87.11 ± 1.826
7.28 ± 0.136
a,d,e
90.85 ± 2.201
b,d,f
10.83 ± 0.319
85.71 ± 1.905
8.53 ± 0.242c,e,f
87.63 ± 1.711
Superscripts indicate statistical significant differences between: a) GR-SR, b) GR-GB, c) GR-SB, d) SR-GB, e) SR-SB, f) GB-SB, P < 0.05.
2.8. Statistical analysis All experimental analyses were carried out in triplicate, and all results were expressed as mean value ± SEM. One-way analysis of variance (ANOVA) was used in order to find the statistically significant differences. Differences were considered to be statistically significant when p was lower than 0.05. The data was analyzed using a statistical software package (PASW 18 for Windows, SPSS Inc., Chicago, IL, USA). 3. Results 3.1. TL, TPL, and TNL contents of goat and sheep meat samples The amounts of TL, TPL, and TNL of all samples (raw and baked) are shown in Table 1 [samples are coded as follows: goat raw (GR), goat baked (GB), sheep raw (SR) and sheep baked (SB)]. Sheep meat had a higher content of TL than goat meat, in both raw (SR) and baked (SB) samples, but there was no statistical significance (p > 0.05). This is attributed to the fact that the studied samples were both from young animals. The TPL content of the goat meat was significantly higher than the sheep meat, in both raw (GR as opposed to SR) and baked (GB as opposed to SB) samples (p < 0.05). Baking also caused a significant increase of TPL expressed as percentage of TL (p < 0.05). TNL content expressed as percentage of TL was similar for all samples (p < 0.05).
2.7. Biological assay in vitro on human platelet rich plasma (PRP) Healthy human volunteers (n = 6) within the university donated blood. Fasting blood samples were obtained via venipuncture using a 20 G needle (Sarstedt, Nümbrecht, Germany) attached to S-monovettes containing 0.106 mol/L trisodium citrate solution in a 1:10 ratio of citrate to blood (Sarstedt, Nümbrecht, Germany). Blood was collected from the median cubital vein or cephalic vein. The blood was centrifuged at 180 x g for 15 min at 24 °C to obtain the upper phase containing the platelet rich plasma (PRP). The lower phase was centrifuged at 1465× g for 20 min at 24 °C in order to obtain the platelet poor plasma (PPP). PRP was prepared for final analysis by standardising to a platelet count of 5.0 × 106 cells mL−1, by diluting with PPP. All analyses were carried out within 2.5 h of the initial blood draw and PRP was stored at 24 °C before use. The biological activity of all polar lipid fractions as obtained from TLC, were tested for their ability to inhibit PAF-induced platelet aggregation. Briefly PAF and all examined samples were dissolved in 2.5 mg mL−1 saline (0.90% w/v NaCl). The PAFinduced platelet aggregation was measured in PRP before (considered as 0% inhibition) and after the addition of various amounts of the sample being examined. Consequently, the plot of percent inhibition (ranging from 20 to 80%) versus different concentrations of the sample is linear. From this curve, the concentration of the sample, which inhibited 50% the PAF-induced aggregation, was calculated. The concentration for 50% inhibition is termed the IC50 value. All samples were tested in triplicate and this study received ethical approval from the National and Kapodistrian University of Athens.
3.2. Fatty acid profiles of TPL and TNL of goat and sheep meat samples The fatty acid profiles of TPL of all four samples are given in Table 2a. The major fatty acids of all samples in TPL were palmitic (16:0), stearic (18:0), oleic (18:1 cis), linoleic (18:2 cis) and arachidonic (20:4). Notably, eicosadienoic (20:2), eicosapentaenoic (20:5 or EPA), docosapentaenoic (22:5 or DPA) and docosahexaenoic (22:6 or DHA) fatty acids were also present. Interestingly, the TPL samples had a rather high content of PUFA and consequently a high PUFA/SFA ratio. The highest ratio was found for goat meat, where baked kid (GB) had a higher value than raw kid (GR) (p < 0.05). In contrast, raw lamb (SR) had a higher ratio than baked lamb (SB) (p < 0.05). The TNL fatty acid profiles of all four samples are presented in Table 2b. The major fatty acids in goat meat (raw or baked) were myristic (14:0), palmitic (16:0), stearic (18:0) and oleic (18:1 cis) fatty acids, while linoleic acid (18:2 cis) was also present in notable amounts. In sheep meat (raw or baked) the major fatty acids were palmitic (16:0), stearic (18:0) and oleic (18:1 cis) fatty acids, while in noteworthy amounts myristic (14:0), vaccenic (18:1 trans) and linoleic (18:2 cis) acids were found. In sheep meat samples, we noticed an absence of fatty acids with a chain length of 20 or 22 carbons, and in goat meat samples absence of fatty acids with 22 carbons. The content of PUFA in all TNL samples is very low, and thus there is a low PUFA/SFA ratio, which is 41
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Table 2a Fatty acid composition (%) (mean ± SD, n = 3) of total polar lipids (TPL) of all samples (GR: goat meat, raw; GB: goat meat, baked; SR: sheep meat, raw and SB: sheep meat, baked). Fatty acid
GR
GB
SR
SB
10:0 12:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 18:1 18:2 18:2 18:3 18:3 20:0 20:1 20:2 20:3 20:4 20:5 22:1 22:2 22:5 22:6
0.03 ± 0.01a,c 0.14 ± 0.01a,b,c 1.13 ± 0.06a,b,c 0.02 ± 0.01 0.27 ± 0.01a,c 0.21 ± 0.01a,b,c 15.29 ± 0.36b,c 0.49 ± 0.04c 0.85 ± 0.04b,c 0.35 ± 0.02a,b,c 15.48 ± 0.22b 0.15 ± 0.02b,c 25.77 ± 0.81a 0.10 ± 0.01a,b 15.03 ± 0.20b,c 0.13 ± 0.01b,c 1.30 ± 0.04c 0.21 ± 0.01a,c 0.32 ± 0.01a,b,c 1.75 ± 0.02a,b,c 0.96 ± 0.01a,b 13.14 ± 0.16a,b,c 1.58 ± 0.02a,b,c 0.10 ± 0.01b 0.10 ± 0.01a 3.81 ± 0.05a,b,c 1.32 ± 0.02a,b,c
0.01 ± 0.01a,d,e 0.09 ± 0.01a,d,e 0.81 ± 0.01a,d,e 0.02 ± 0.01e 0.22 ± 0.02a,d,e 0.36 ± 0.01a,d,e 15.50 ± 0.16d,e 0.42 ± 0.02d,e 0.88 ± 0.01d,e 0.34 ± 0.02a,d,e 16.03 ± 0.03d,e 0.19 ± 0.04e 21.43 ± 0.03a,d,e 0.07 ± 0.01a 15.13 ± 0.05d,e 0.16 ± 0.01 1.37 ± 0.03d,e 0.17 ± 0.01a,e 0.21 ± 0.02a,d 2.07 ± 0.03a,d,e 1.07 ± 0.01a,d,e 14.95 ± 0.04a,d,e 2.25 ± 0.03a,d,e 0.07 ± 0.01d 0.10 ± 0.01a 4.46 ± 0.01a 1.62 ± 0.01a,d
0.03 ± 0.01d,f 0.04 ± 0.01b,d 0.44 ± 0.02b,d,f 0.01 ± 0.01 0.29 ± 0.01d,f 0.47 ± 0.02b,d,f 17.41 ± 0.03b,d,f 0.52 ± 0.03d,f 1.55 ± 0.02b,d,f 1.03 ± 0.01b,d,f 14.70 ± 0.03b,d 0.26 ± 0.03b,f 24.52 ± 0.07d,f 0.06 ± 0.01b 17.97 ± 0.02b,d,f 0.14 ± 0.01b 1.40 ± 0.02d,f 0.18 ± 0.01e,f 0.49 ± 0.01b,d,f 1.82 ± 0.02b,d,f 1.02 ± 0.01b,d,f 8.34 ± 0.01b,d,f 1.99 ± 0.02b,d 0.27 ± 0.01b,d,f ND 3.22 ± 0.02b 1.82 ± 0.02b,d,f
0.03 ± 0.01c,e,f 0.04 ± 0.01c,e 0.57 ± 0.01c,e,f 0.03 ± 0.01e 0.37 ± 0.01c,e,f 0.08 ± 0.01c,e,f 17.93 ± 0.25c,e,f 0.69 ± 0.01c,e,f 1.90 ± 0.02c,e,f 1.22 ± 0.01c,e,f 14.88 ± 0.06e 0.58 ± 0.08c,e,f 25.65 ± 0.06e,f 0.08 ± 0.01 17.03 ± 0.01c,e,f 0.14 ± 0.01c 1.65 ± 0.02c,e,f 0.09 ± 0.01c,f 0.19 ± 0.01c,f 1.54 ± 0.02c,e,f 0.95 ± 0.01e,f 7.93 ± 0.09c,e,f 2.00 ± 0.02c,e 0.09 ± 0.01f ND 2.76 ± 0.06c 1.58 ± 0.05c,f
33.40 ± 0.69c 27.39 ± 0.74a 39.21 ± 0.05a,b,c 1.17 ± 0.02a,b,c
33.72 ± 0.13d,e 23.04 ± 0.01a,d,e 43.25 ± 0.12a,d,e 1.28 ± 0.01a,d,e
34.64 ± 0.02d,f 27.57 ± 0.01d,f 37.78 ± 0.02b,d,f 1.09 ± 0.01b,d,f
35.79 ± 0.12c,e,f 28.53 ± 0.14e,f 35.66 ± 0.26c,e,f 1.00 ± 0.01c,e,f
trans cis trans cis ω6 ω3
SFA MUFA PUFA PUFA/SFA
ND Non-detectable. Superscripts indicate statistical significant differences between a) GR-GB, b) GR-SR, c) GR-SB, d) GB-SR, e) GB-SB, f) SR-SB, P < 0.05.
three samples (GB, SR, SB) appeared to have similar IC50 values (GB = 217.53 ± 6.83; SR = 206.79 ± 8.47; SB = 198.68 ± 6.22: p > 0.05).
approximately 10 times lower than that of the TPL PUFA/SFA ratio. 3.3. Isolation of polar lipid fraction with Rf similar to that of phosphatidylcholine from TPL with preparative TLC
3.5. Phospholipid analysis The TPL were further separated into polar lipid fractions with preparative TLC. All four samples showed a similar profile, which consisted of 8 lipid fractions. The Rf of these fractions were compared with the Rf of a standard containing lyso-phosphatidylcholine (L-PC), sphingomyelin (SM), phosphatidylcholine (PC), lyso-phosphatidylethanolamine (L-PE), and phosphatidylethanolamine (PE) (Fig. 1). In previous work (data not shown), the most potent biological activities against platelet aggregation of all fractions have been assessed and the most active fraction was fraction 3. We have, thus, focused our subsequent work to the most biologically active fraction, i.e. fraction 3, which had a Rf similar to that of PC. This fraction was notably more extensive than the others, as was noted in other studies (Perez-Palacios et al., 2007; Sinanoglou et al., 2013).
The phospholipid composition of the TLC polar lipid fraction 3 of all samples (GR, GB, SR and SB) was further screened by LC–MS analysis. In the positive ion survey scans (Fig. 3a), the overwhelming majority of species were diacyl PC species. These species contained primarily stearic (18:0) fatty acids at the sn-1 position. At the sn-2 position, monounsaturated fatty acids (16:1 or 18:1) or poly-unsaturated fatty acid (18:2 cis or 18:3) were present. The fatty acid (18:3) was less abundant in sheep meat samples in comparison to the goat meat samples. Baking did not show to have a considerable effect on the relative abundancies of fatty acid species. In the negative ion survey scans (Fig. 3b), diacyl PC species were predominant, but a range of species from other classes of phospholipids, mainly PI were also detected. At the sn-1 position, primarily palmitic (16:0) fatty acids were present, while at the sn-2 position, several fatty acids such as vaccenic acid (18:1 trans), CLA or odd-chain fatty acids were found. These fatty acids were detected in insignificant amounts. Several other phospholipids classes of minor biological value were detected, which tends to suggest that fatty acid species detected in negative ion scans were of lower analytical importance than those detected in positive ion survey scans for these PC fractions.
3.4. Biological activity of TLC polar lipid fraction 3 from all meat samples The TLC polar lipid fraction 3 from all samples was tested for their ability to inhibit PAF-induced platelet aggregation. The biological activities of each lipid fraction are expressed as an IC50 in micrograms, which are shown in Fig. 2. When comparing IC50 values, lower IC50 values correspond to stronger activities since a smaller amount of lipids is required for the same inhibitory activity. According to Fig. 2 lipid fractions from all samples exhibited inhibitory activity against PAFinduced platelet aggregation. Lipids from the raw goat meat sample (GR) had a significantly lower IC50 value (130.44 ± 4.65: p < 0.05) compared to lipids of the other three samples. Lipids from the other
4. Discussion Both animals had similar amount of TL (p > 0.05). Older sheep would be expected to have a statistically significant higher content of 42
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Table 2b Fatty acid composition (%) (mean ± SD, n = 3) of total neutral lipids (TNL) of all samples (GR: goat meat, raw; GB: goat meat, baked; SR: sheep meat, raw and SB: sheep meat, baked). Fatty acid
GR
GB
SR
GB
10:0 12:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 18:1 18:2 18:2 18:3 18:3 20:0 20:1 20:2 20:3 20:4 20:5 22:1 22:2 22:5 22:6
0.27 ± 0.01a,c 1.62 ± 0.03b,c 10.85 ± 0.11b,c 0.34 ± 0.01a,b,c 0.83 ± 0.01a,b,c 0.16 ± 0.01a,b,c 28.60 ± 0.12a,b,c 2.00 ± 0.01a,b,c 1.26 ± 0.01a,b,c 0.58 ± 0.01a,b,c 11.67 ± 0.03a,b,c 0.99 ± 0.05b,c 34.72 ± 0.26a,c 0.30 ± 0.01b,c 4.30 ± 0.03a,b,c ND 0.71 ± 0.01a,b,c 0.08 ± 0.03 0.11 ± 0.02 0.09 ± 0.01 0.05 ± 0.01 0.48 ± 0.01a ND ND ND ND ND
0.22 ± 0.01a,d 1.51 ± 0.03d,e 10.84 ± 0.12d,e 0.29 ± 0.01a,d,e 0.78 ± 0.01a,d,e 0.17 ± 0.01a,d,e 27.50 ± 0.08a,d,e 1.59 ± 0.04a,d,e 1.00 ± 0.02a,d,e 0.47 ± 0.02a,d,e 12.53 ± 0.07a,d,e 0.73 ± 0.42d,e 36.19 ± 0.21a,d 0.30 ± 0.01d,e 3.88 ± 0.03a,e ND 0.64 ± 0.01a,d,e 0.12 ± 0.01 0.68 ± 0.55 0.12 ± 0.04 0.06 ± 0.01 0.36 ± 0.01a ND ND ND ND ND
0.28 ± 0.02d,f 0.49 ± 0.01b,d,f 4.67 ± 0.02b,d,f 0.21 ± 0.01b,d,f 1.01 ± 0.01b,d,f 0.18 ± 0.01b,d,f 23.76 ± 0.30b,d 1.36 ± 0.01b,d,f 2.60 ± 0.03b,d,f 1.35 ± 0.10b,d,f 18.93 ± 0.11b,d,f 4.66 ± 0.02b,d,f 35.38 ± 0.08d,f 0.24 ± 0.02b,d 3.91 ± 0.01b,f ND 0.96 ± 0.01b,d ND ND ND ND ND ND ND ND ND ND
0.21 ± 0.01c,f 0.36 ± 0.02c,e,f 4.44 ± 0.02c,e,f 0.20 ± 0.01c,e,f 1.13 ± 0.01c,e,f 0.20 ± 0.01c,e,f 24.16 ± 0.12c,e,f 1.78 ± 0.01c,e,f 3.00 ± 0.01c,e,f 1.77 ± 0.03c,e,f 16.45 ± 0.03c,e,f 3.98 ± 0.04c,e,f 36.76 ± 0.12c,f 0.24 ± 0.01c,e 4.37 ± 0.04c,e,f ND 0.97 ± 0.02c,e ND ND ND ND ND ND ND ND ND ND
55.16 ± 0.24b,c 38.90 ± 0.22a,b,c 5.93 ± 0.02a,b,c 0.11 ± 0.01a,b,c
54.51 ± 0.16d,e 40.13 ± 0.10a,d,e 5.36 ± 0.06a,d,e 0.10 ± 0.01a,e
51.74 ± 0.24b,d,f 43.14 ± 0.22b,d,f 5.12 ± 0.02b,d,f 0.10 ± 0.01b,f
49.53 ± 0.16c,e,f 44.68 ± 0.16c,e,f 5.58 ± 0.03c,e,f 0.11 ± 0.01c,e,f
trans cis trans cis ω-6 ω-3
SFA MUFA PUFA PUFA/SFA
ND Non-detectable. Superscripts indicate statistical significant differences between a) GR-GB, b) GR-SR, c) GR-SB, d) GB-SR, e) GB-SB, f) SR-SB, P < 0.05.
of lipids section. The data from phospholipid analysis strongly correlates with previous work from our group on the lipodomic studies of fish with antiinflammatory activities, where PC species containing primarily 18:1 and 18:2 were identified (Nasopoulou et al., 2014). Therefore, it could be suggested that PC-species have strong anti-inflammatory activities and the presence of these lipid species in both types of meat can be linked to potential cardioprotective activities that may aid in the prevention of CVD in humans. Dietary guidelines of the partial replacement of saturated fat with other energy sources (carbohydrates, monounsaturated or polyunsaturated fatty acids) have been suggested in the past by many organisations and are still advised today by the American Heart Association (Lordan et al., 2018; Sacks et al., 2017). These directives do not necessarily yield beneficial results against CVD, except for the case of replacement of saturated fatty acids with polyunsaturated fatty acids (Micha and Mozaffarian, 2010; Siri-Tarino et al., 2010). Foods with a high ratio of PUFA/SFA are generally regarded as nutritionally valuable. However there is a discrepancy between findings from omega-3 PUFAs, fish or fish oil consumption, regarding protection against the development of CVD. Fish and fish oils contain a complex mixture of lipids, only a small component of which is omega-3 PUFAs. Consequently, there is potential for other lipids in the mixture to be beneficial, either in an additive or even a synergistic way (Megson et al., 2016; Zabetakis, 2013). in vivo studies of polar lipids isolated from fish, olive oil, and olive pomace in hyperlipidemic rabbits have shown retard (Karantonis et al., 2006; Nasopoulou et al., 2010; Tsantila et al., 2007) or regress atheromatic plaque (Tsantila et al., 2010). It is suggested that the inflammatory action of PAF can be inhibited by dietary lipid microconstituents, as several in vitro studies have shown that polar lipids
TL (Brzostowski et al., 1997). Baking induced a statistically significant increase of TL due to moisture loss (p < 0.05) (Badiani et al., 1998). Baking caused a significant increase of TPL expressed as percentage of TL (p < 0.05), because TPL is mainly present in the connective tissue of the muscle, since the phospholipid fraction is probably fairly representative of that of the membranous structures of the muscle itself (Christie, 1978), thus their loss during baking is low, whereas the TNL is predominantly present in the adipose tissue (Christie, 1978), and consequently follows TL’s content changes due to drip-fat loss and moisture loss. So, we notice no significant change of TNL, when expressed as percentage of TL (p < 0.05) after baking. This study has some limitations, including the fact that the fatty acid profile of ruminant meat is influenced by several factors such as breed, age, genotype, nutrition, and the environment (Banskalieva et al., 2000; Santos et al., 2007). Thus more than one animal and animals of different breeds from different areas may have enhanced the study. Furthermore, the meat samples used in the study were chosen because they are the most consumed cut in both animals in Greece, thus are the most representative sample of the animal. Further research should focus on other cuts of meat in order to obtain a comprehensive view of the potential anti-inflammatory and antithrombotic effects of consuming these animals. The fatty acid composition analysis of the meats revealed the contrast between the TPL and TNL of all samples. The TPL, which corresponds mainly to the intramuscular fat, has an extremely high content of PUFA and accordingly a high PUFA/SFA ratio. Whereas, the TNL, which correspond mainly to adipose tissue, have a PUFA/SFA ratio that is approximately 10 times lower than that of the TPL ratio. This result may be explained on the basis that PUFA present in meat have a polar head, therefore, they have been extracted by the ethanolic TPL fraction during counter-current distribution, which is described in the isolation 43
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Fig. 2. in vitro biological activity of TLC polar lipid fractions, with Rf similar to that of phosphatidylcholine, of goat raw meat (GR), goat baked meat (GB), sheep raw meat (SR), and sheep baked meat (SB) samples toward platelet aggregation in human PRP, expressed as IC50 in micrograms (mean ± SEM, n = 3). Goat raw (GR) meat sample had a significantly lower (p < 0.05) IC50 value compared to IC50 values of the other three meat lipid samples.
atherogenic and thrombotic action of PAF (Megalemou et al., 2017; Poutzalis et al., 2016). The same TLC polar lipid fraction, which is the most prominent polar lipid fraction of muscle tissue (Perez-Palacios et al., 2007; Sinanoglou et al., 2013), was investigated in raw and baked goat and sheep meat, and it is clear that these PC derivatives possess similar inhibitory action against PAF-induced platelet aggregation to those found in the literature. The structures of these PC molecules differ slightly from other PC molecules of marine origin, including olive pomace fed seabass (Nasopoulou et al., 2014), olive pomace-enriched fish feed and olive pomace fed gilthead seabream (Sioriki et al., 2016). This fact emphasises the need to correlate the structural variances of different lipid species with their biological activity in order to understand the molecular mechanisms involved in inhibiting the actions of PAF on the PAF-receptor. 5. Conclusion Our previous research has shown that yogurts and cheese derived from caprine and ovine milk have strong antithrombotic properties (Lordan and Zabetakis, 2017b; Megalemou et al., 2017; Poutzalis et al., 2016; Tsorotioti et al., 2014). In our quest to determine the origin of these important bioactivities, we expanded our research to investigate the lipid composition of goat and sheep meat. The cardioprotective activities found in goat and ewe yogurts had been linked to lipids that are PC (Megalemou et al., 2017; Poutzalis et al., 2016). The data reported in this novel research suggests that goat and sheep meat also contain PC derivatives that have strong antithrombotic activities. Further studies are required to elucidate the structural role of bioactive lipids found in our diet and their effects upon consumption, in order to set realistic nutritional guidelines in order to prevent the onset of atherosclerosis. Our results underline the nutritional value of goat and sheep meat as potent sources of polar lipids with strong inhibitory properties against PAF-induced platelet aggregation, and consequently atherosclerosis. It could be concluded that sheep and goats have the enzymatic capacity to biosynthesize antithrombotic polar lipids, present in both meat and milk. This study has also for the first time demonstrated that these activities are not lost under baking conditions, highlighting that baked goat and sheep meat have potential functional and potent cardioprotective properties.
Fig. 1. Typical profile of total polar lipids (TPL) separation of our meat samples on preparative TLC. The eight bands obtained along with the corresponding (in terms of Rf) standard compounds: L-PC lyso-phosphatidylcholine, SM sphingomyelin, PC phosphatidylcholine, L-PE lyso-phosphatidylethanolamine and PE phosphatidylethanolamine, are shown on the left.
isolated from fish and olive pomace are capable of inhibiting PAF-induced platelet aggregation (Nasopoulou et al., 2014, 2013b; Nomikos et al., 2006; Sioriki et al., 2016). Polar lipids with inhibitory action against PAF have been found in various foods, even of animal origin (Lordan et al., 2017; Megalemou et al., 2017; Nasopoulou et al., 2013a; Poutzalis et al., 2016; Tsorotioti et al., 2014). Polar lipids from different foodstuffs can vary in structure, abundance, and inhibitory strength. Also it should be highlighted that different foods vary in composition for a number of molecules, that contribute to CVD and general health in a positive or a negative way. These results demonstrate that PC derivatives exert antithrombotic and anti-inflammatory properties, which are not lost under baking conditions. Previous research has shown that the TLC polar lipid fraction 3, with a Rf value similar to that of PC, from goat and sheep dairy sources possess potent anti-inflammatory properties against the
Author contributions C. Nasopoulou and I. Zabetakis designed the study and interpreted the results. R. Lordan interpreted the data and drafted the manuscript. S. Poutzalis collected data and drafted the manuscript. 44
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Fig. 3. a) Lipidomic analysis of TLC polar lipid fraction 3, with Rf similar to that of phosphatidylcholine by liquid chromatography-mass spectrometry (positive ion survey). Inserts show percentage of the most abundant phospholipids in each sample. b) Lipidomic analysis of TLC polar lipid fraction 3, with Rf similar to that of phosphatidylcholine by liquid chromatography-mass spectrometry (negative ion survey). Inserts show percentage of the most abundant phospholipids in each sample.
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Conflict of Interest
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Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Phospholipids of goat and sheep origin: Structural and functional studies a
b
c
Stylianos Poutzalis , Ronan Lordan , Constantina Nasopoulou , Ioannis Zabetakis a b c
b,⁎
T
Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771, Athens, Greece Department of Biological Sciences, University of Limerick, V94 T9PX, Limerick, Ireland Department of Food Science and Nutrition, School of the Environment, University of the Aegean, Myrina, Lemnos, 81400, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherogenesis Anti-atherogenic activity Polar lipids Phosphatidylcholine Meat
The lipidomic profiles of goat and sheep meat were studied. Polar lipid fractions of raw and baked meat samples were tested for their in vitro anti-atherogenic properties. The total lipid (TL) content was extracted using the Bligh-Dyer method and was subsequently separated into total polar lipids (TPL) and total neutral lipids (TNL). The fatty acid profiles of the TPL and TNL of all three samples were determined by GC-FID. The TPL of all samples were further separated by preparative TLC into their constituent phospholipid and sphingolipid fractions. In all samples, polar lipid fraction 3 had a similar Rf value to phosphatidylcholine. These phosphatidylcholine fractions were tested for their in vitro capacity to inhibit platelet-activating factor (PAF) induced platelet aggregation (anti-inflammatory activity) using human platelets. The phospholipid content of each fraction 3 was determined using LC–MS. These results provide a novel insight into the structure of phosphatidylcholine derivatives in goat and sheep meat and highlight the nutritional value of these meats in terms of their antithrombotic and cardioprotective properties before and after the baking process.
1. Introduction Foods and fats of animal origin receive undue criticism from society and scientific communities due to their perceived negative effects on health upon consumption. Recent research trends indicate that these negative perceptions may be unwarranted as numerous studies suggest that meat consumption may be associated with a positive effect on health when eaten in moderation despite their SFA and cholesterol content (Lordan et al., 2017). Red meat is a highly nutritious and valuable source of proteins, iron, phosphorus, zinc, selenium and B group vitamins. Meat from ruminant animals, such as sheep and goat, contain trans fatty acids that are, unlike industrial fatty acids, beneficial for the human health (Bendsen et al., 2011; Mulvihill, 2001). The most common source of trans fatty acids in ruminant meat is conjugated linoleic acid (CLA), which has been linked to a protective role against cancer, cardiovascular diseases (CVD) and has been associated with weight loss (Bendsen et al., 2011; Blankson et al., 2000; Schmid et al., 2006). According to the lipid hypothesis, which stemmed from the Seven Countries Study, saturated fatty acids (SFA), cholesterol and LDL levels are responsible for atherogenesis and consequently their presence in excessive levels can increase the risk of CVD development (Steinberg, 2006). Several studies have implicated red meat in the development of
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CVD due to their effect on blood lipid parameters but also the effect of additives and preservatives (Pan et al., 2012; Wolk, 2016). However, there are meta-analyses and reviews that refute these claims and they have shown that red meat consumption does not increase the risk for CVD development (Connor et al., 2016; Mcafee et al., 2010). Recent studies that separated red meat into processed and unprocessed forms, have brought to light the fact that unprocessed red meat has a minor association or no association with increased mortality and CVD (Larsson and Orsini, 2014; Micha et al., 2012, 2010). It has also been suggested that potential bias in many of the studies investigating the consumption of meat and its effect on atherosclerosis may have influenced the conclusions (Larsson and Orsini, 2014; Micha et al., 2012). Other studies have shown that a diet containing meat and cheese can lead to increased levels of HDL cholesterol and thus, it appears to be less atherogenic than a low-fat, high-carbohydrate diet (National Obesity Forum and Public Health Collaboration, 2016). There is considerable concern that red meat consumption elevates levels of choline and L-carnitine. Phosphatidylcholine (PC) is broken down to choline, which is transformed by the intestinal microbiota to trimethylamine (TMA), which along with L-carnitine is metabolised to trimethylamine N-oxide (TMAO) (Wang et al., 2011). It is proposed that excess dietary PC increases the levels of TMAO resulting in a pro-inflammatory and
Corresponding author at: Department of Biological Sciences, University of Limerick, Limerick, Ireland. E-mail address: [email protected] (I. Zabetakis).
https://doi.org/10.1016/j.smallrumres.2018.07.015 Received 23 November 2017; Received in revised form 29 June 2018; Accepted 20 July 2018 Available online 30 July 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
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2.2. Meat samples
prothrombotic state that may lead to eventual insulin resistance, type II diabetes mellitus, and CVD (Lordan et al., 2017; Zhu et al., 2016). However, some results indicate that dietary choline may not be to blame, and that the presence of specific gut bacteria promotes the conversion of choline into TMAO (Clouatre and Bell, 2013; Koeth et al., 2013). Other researchers suggested that dietary choline from PC derivatives in dairy and marine sources possess antithrombotic properties, contrary to the effects of TMAO (Lordan et al., 2017; Lordan and Zabetakis, 2017a). With regards to atherogenesis, platelet-activating factor (PAF) is a crucial potent inflammatory phospholipid mediator (Demopoulos et al., 1979), which is produced by many cells and is involved in the activation of leukocytes and their binding to endothelial cells (Demopoulos et al., 2003; Lordan and Zabetakis, 2017b). CVD such as coronary heart disease and stroke are clinical events as a consequence of atherosclerosis, which is a chronic inflammatory condition mediated by PAF and other molecules (Nasopoulou et al., 2013c) Our previous work has shown that polar lipids are strong inhibitors of the inflammatory and atherogenic actions of PAF (Megson et al., 2016; Zabetakis, 2013). Recently, polar lipids from sheep and goat dairy products have been evaluated for their inhibitory activities against PAF (Lordan and Zabetakis, 2017b; Megalemou et al., 2017; Poutzalis et al., 2016; Tsorotioti et al., 2014); the most potent inhibitory properties were exhibited by TLC polar lipid fractions with Rf similar to that of PC (Megalemou et al., 2017; Poutzalis et al., 2016). However, little is known about the bioactivity of phospholipids present in sheep or goat meat. Muscles from mammals are excellent sources of phospholipids (Christie, 1978) and especially of PC which often constitutes to almost 50% of the total phospholipids (Perez-Palacios et al., 2007; Sinanoglou et al., 2013). In lamb, the phospholipid content can constitute 42% of the total lipids, with PC and phosphatidylethanolamine being the predominant lipid species (38–55% and 25–31% respectively) (Lordan et al., 2017). Lipid microconstituents that exert in vitro anti-PAF activities could retard and/or regress atherosclerosis and consequently CVD, as several in vivo studies have demonstrated (Karantonis et al., 2006; Nasopoulou et al., 2010; Tsantila et al., 2010, 2007). The aim of our present work was to explore the lipidomic profiles of raw and baked goat and sheep meat and to evaluate the anti-atherogenic properties of PC derivatives obtained from these food sources. Our data suggests that goat and sheep meat possess potential functional cardioprotective lipid microconstituents that warrant further research, which assist in re-assessing the nutritional value of these meats.
Meat samples were purchased from a local market in Athens. Both the sheep and the goat were male and were 6–10 weeks of age. The sheep originated from Amphilochia region in central Greece, whereas the goat originated from Lakonia region in southern Greece. The meat samples used were untrimmed rib steaks, as typically consumed in Greece. Initially, the bone was removed. The baked meat samples were prepared as follows: the meat was cut into 2 × 2×1 cm3 pieces, wrapped in aluminium foil, and cooked in a preheated oven at 180 °C for 20 min. The raw and baked meat samples used for analysis were further chopped into as small as possible pieces using a knife. The remaining meat juices left after the baking process were discarded. Four groups of meat samples have been generated, namely goat raw (GR), goat baked (GB), sheep raw (SR) and sheep baked (SB) and these meat sample codes are used throughout the manuscript. 2.3. Isolation of lipids Extraction of the total lipids (TL) from each 100 g sample (kid and lamb, raw or baked) was carried out using the Bligh and Dyer method (Bligh and Dyer, 1959). The TL were weighed and one fourth of the sample was stored in sealed vials under a nitrogen atmosphere at -20 °C, while the rest of the TL was further separated by counter-current distribution (Galanos and Kapoulas, 1962) into total polar lipids (TPL) and total neutral lipids (TNL). In brief, petroleum ether and 87% aqueous ethanol were pre-equilibrated in a separatory funnel. The lower phase containing equilibrated 87% ethanol and the upper phase containing equilibrated petroleum ether were collected separately. Approximately 2 g of TL were dissolved in 9 mL pre-equilibrated petroleum ether and afterwards 3 mL pre-equilibrated 87% ethanol were added and stirred. The lower phase (ethanolic phase) was collected and transferred to a second test tube containing 9 mL of pre-equilibrated petroleum ether and was stirred again. The ethanolic phase in the second test tube was transferred to a round-bottom flask. The procedure was repeated eight times in total. Finally, the ethanolic phase (8 × 3 mL), containing the TPL, and the petroleum ether phase (2 × 9 mL), containing TNL, were evaporated to dryness, weighed and stored under nitrogen in sealed vials at −20 °C. Each extraction was carried out in triplicate. 2.4. Fractionation of TPL by preparative TLC The TPL from all samples were further separated by preparative TLC. The TLC glass plates (20 × 20 cm, thickness 1.0 mm) were coated with silica gel G-60 and activated by heating at 120 °C for 60 min. Approximately 40 mg of TPL was applied to the TLC plates. The developing system consisted of chloroform/methanol/water 65:35:6 (v/ v/v). After development, the plates were stained under iodine vapors. Eight bands appeared after the separation of TPL of the samples. Subsequently, the bands were scraped off, and the lipids from the desirable bands were extracted according to the Bligh-Dyer method (Bligh and Dyer, 1959). The chloroform phase was evaporated to dryness under nitrogen, and lipids were weighed and stored at −20 °C.
2. Materials and methods 2.1. Reagents and instruments All reagents and solvents were of analytical grade purchased from Merck (Darmstadt, Germany). Fatty acid methyl ester standards were of GC-quality and supplied by Sigma-Aldrich (St. Louis, MO, USA), as well as bovine serum albumin (BSA) and PAF. The Chromatographic material used for thin-layer chromatography (TLC) was silica gel G-60 supplied by Merck (Darmstadt, Germany) and the polar lipid standards used for TLC were supplied by Sigma-Aldrich as a standard mix of hen egg yolk (St. Louis, MO, USA). Gas chromatographic analysis was carried out on a Shimadzu CLASS-VP (GC-17 A) (Kyoto, Japan) gas chromatograph equipped with a split/splitless injector and flame ionization detector. Liquid chromatography-mass spectrometry (LC–MS) was carried out using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempstead, UK), equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 UHPLC system. Platelet aggregation was measured on a Chrono-Log (Havertown, PA, USA) aggregometer (model 400-VS) coupled to a Chrono-Log recorder (Havertown, PA, USA).
2.5. Gas chromatographic analysis Fatty acid methyl esters (FAME) were prepared from 35 mg of TPL and 35 mg of TNL of all four samples (GR, GB, SR and SB) using a solution of 0.5 N KOH in CH3OH (KOH-CH3OH method with reaction time 5 min) and extracted with n-hexane. Analysis was carried out on a Shimadzu CLASS-VP (GC-17 A) (Kyoto, Japan) gas chromatograph equipped with a split/splitless injector and flame ionization detector. Separation of FAMEs was achieved using an Agilent J&W DB-23 fused silica capillary column (60 m × 0.251 mm i.d., 0.25 μm; Agilent, Santa Clara, CA, USA). The oven temperature was initially set at 120 °C for 5 min, raised to 180 °C at 10 °C min−1, then to 220 °C at 20 °C min−1, 40
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and finally kept at 220 °C for 30 min. The temperatures of injector and detector were maintained at 220 and 225 °C, respectively. The carrier gas was high purity helium with a linear flow rate of 1 mL.min−1 and split ratio of 1:50, as described before (Poutzalis et al., 2016). Fatty acid methyl esters were identified using Supelco 37-Component FAME Mix and additionally docosapentanoic acid (22:5) methyl ester standard. FAME 21:0 was used as internal standard for quantifying FAMEs.
Table 1 Content of total lipids (TL), expressed in grams per 100 g sample (mean ± SD, n = 3), total polar lipids (TPL), and total neutral lipids (TNL), expressed as percentages of TL in raw and baked kid and lamb. Samples Goat meat (kid) raw, GR Sheep meat (lamb) raw, SR Goat meat (kid) baked, GB Sheep meat (lamb) baked, SB
2.6. Analysis of Lipids by Liquid Chromatography-Mass Spectrometry (LC–MS) For each sample, 10 mg of dried TLC polar lipid fraction 3, from all samples, were dissolved by addition of 0.5 mL methanol and 0.5 mL chloroform. A dilution of 1 in 100 was made from the mixture in methanol containing 5 mM ammonium formate. All samples were centrifuged at 650× g for 5 min at 4 °C to sediment any particulate matter. The lipids were analysed by liquid chromatography-mass spectrometry (LC–MS) using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempstead, UK), equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 UHPLC system. All samples were analysed in both positive and negative ion mode over the mass to charge (m/z) range 250–2000 with a resolution of 25,000. The samples were injected (1 μL and 2 μL, in positive and negative modes respectively) on to a Thermo Hypersil Gold C18 column (2.1 mm x 100 mm, 1.9 μm). Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% (v/v) formic acid. Mobile phase B consisted of 90:10 isopropanol/acetonitrile containing 10 mM ammonium formate and 0.1% (v/v) formic acid. The initial conditions for analysis were 65% A / 35% B. The percentage of mobile phase B was increased to 65% over 4 min and then 100% over 15 min. These conditions were held for 2 min before re-equilibration with the starting conditions for 6 min. The flow rate was 400 μL/min with a column temperature of 40 °C. All solvents were LC–MS grade. LC–MS data was processed with Progenesis QI v2.0 software (Nonlinear Dynamics, Newcastle, UK) and searched against HMDB (www. hmdb.ca) and LIPID MAPS (www.lipidmaps.org) for identification.
TL (g/100 g sample) 10.51 ± 0.312
b,c
11.44 ± 0.254
d,e
16.54 ± 0.286
b,d
17.76 ± 0.318c,e
TPL (%TL)
TNL (%TL)
9.78 ± 0.179
a,b,c
87.11 ± 1.826
7.28 ± 0.136
a,d,e
90.85 ± 2.201
b,d,f
10.83 ± 0.319
85.71 ± 1.905
8.53 ± 0.242c,e,f
87.63 ± 1.711
Superscripts indicate statistical significant differences between: a) GR-SR, b) GR-GB, c) GR-SB, d) SR-GB, e) SR-SB, f) GB-SB, P < 0.05.
2.8. Statistical analysis All experimental analyses were carried out in triplicate, and all results were expressed as mean value ± SEM. One-way analysis of variance (ANOVA) was used in order to find the statistically significant differences. Differences were considered to be statistically significant when p was lower than 0.05. The data was analyzed using a statistical software package (PASW 18 for Windows, SPSS Inc., Chicago, IL, USA). 3. Results 3.1. TL, TPL, and TNL contents of goat and sheep meat samples The amounts of TL, TPL, and TNL of all samples (raw and baked) are shown in Table 1 [samples are coded as follows: goat raw (GR), goat baked (GB), sheep raw (SR) and sheep baked (SB)]. Sheep meat had a higher content of TL than goat meat, in both raw (SR) and baked (SB) samples, but there was no statistical significance (p > 0.05). This is attributed to the fact that the studied samples were both from young animals. The TPL content of the goat meat was significantly higher than the sheep meat, in both raw (GR as opposed to SR) and baked (GB as opposed to SB) samples (p < 0.05). Baking also caused a significant increase of TPL expressed as percentage of TL (p < 0.05). TNL content expressed as percentage of TL was similar for all samples (p < 0.05).
2.7. Biological assay in vitro on human platelet rich plasma (PRP) Healthy human volunteers (n = 6) within the university donated blood. Fasting blood samples were obtained via venipuncture using a 20 G needle (Sarstedt, Nümbrecht, Germany) attached to S-monovettes containing 0.106 mol/L trisodium citrate solution in a 1:10 ratio of citrate to blood (Sarstedt, Nümbrecht, Germany). Blood was collected from the median cubital vein or cephalic vein. The blood was centrifuged at 180 x g for 15 min at 24 °C to obtain the upper phase containing the platelet rich plasma (PRP). The lower phase was centrifuged at 1465× g for 20 min at 24 °C in order to obtain the platelet poor plasma (PPP). PRP was prepared for final analysis by standardising to a platelet count of 5.0 × 106 cells mL−1, by diluting with PPP. All analyses were carried out within 2.5 h of the initial blood draw and PRP was stored at 24 °C before use. The biological activity of all polar lipid fractions as obtained from TLC, were tested for their ability to inhibit PAF-induced platelet aggregation. Briefly PAF and all examined samples were dissolved in 2.5 mg mL−1 saline (0.90% w/v NaCl). The PAFinduced platelet aggregation was measured in PRP before (considered as 0% inhibition) and after the addition of various amounts of the sample being examined. Consequently, the plot of percent inhibition (ranging from 20 to 80%) versus different concentrations of the sample is linear. From this curve, the concentration of the sample, which inhibited 50% the PAF-induced aggregation, was calculated. The concentration for 50% inhibition is termed the IC50 value. All samples were tested in triplicate and this study received ethical approval from the National and Kapodistrian University of Athens.
3.2. Fatty acid profiles of TPL and TNL of goat and sheep meat samples The fatty acid profiles of TPL of all four samples are given in Table 2a. The major fatty acids of all samples in TPL were palmitic (16:0), stearic (18:0), oleic (18:1 cis), linoleic (18:2 cis) and arachidonic (20:4). Notably, eicosadienoic (20:2), eicosapentaenoic (20:5 or EPA), docosapentaenoic (22:5 or DPA) and docosahexaenoic (22:6 or DHA) fatty acids were also present. Interestingly, the TPL samples had a rather high content of PUFA and consequently a high PUFA/SFA ratio. The highest ratio was found for goat meat, where baked kid (GB) had a higher value than raw kid (GR) (p < 0.05). In contrast, raw lamb (SR) had a higher ratio than baked lamb (SB) (p < 0.05). The TNL fatty acid profiles of all four samples are presented in Table 2b. The major fatty acids in goat meat (raw or baked) were myristic (14:0), palmitic (16:0), stearic (18:0) and oleic (18:1 cis) fatty acids, while linoleic acid (18:2 cis) was also present in notable amounts. In sheep meat (raw or baked) the major fatty acids were palmitic (16:0), stearic (18:0) and oleic (18:1 cis) fatty acids, while in noteworthy amounts myristic (14:0), vaccenic (18:1 trans) and linoleic (18:2 cis) acids were found. In sheep meat samples, we noticed an absence of fatty acids with a chain length of 20 or 22 carbons, and in goat meat samples absence of fatty acids with 22 carbons. The content of PUFA in all TNL samples is very low, and thus there is a low PUFA/SFA ratio, which is 41
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Table 2a Fatty acid composition (%) (mean ± SD, n = 3) of total polar lipids (TPL) of all samples (GR: goat meat, raw; GB: goat meat, baked; SR: sheep meat, raw and SB: sheep meat, baked). Fatty acid
GR
GB
SR
SB
10:0 12:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 18:1 18:2 18:2 18:3 18:3 20:0 20:1 20:2 20:3 20:4 20:5 22:1 22:2 22:5 22:6
0.03 ± 0.01a,c 0.14 ± 0.01a,b,c 1.13 ± 0.06a,b,c 0.02 ± 0.01 0.27 ± 0.01a,c 0.21 ± 0.01a,b,c 15.29 ± 0.36b,c 0.49 ± 0.04c 0.85 ± 0.04b,c 0.35 ± 0.02a,b,c 15.48 ± 0.22b 0.15 ± 0.02b,c 25.77 ± 0.81a 0.10 ± 0.01a,b 15.03 ± 0.20b,c 0.13 ± 0.01b,c 1.30 ± 0.04c 0.21 ± 0.01a,c 0.32 ± 0.01a,b,c 1.75 ± 0.02a,b,c 0.96 ± 0.01a,b 13.14 ± 0.16a,b,c 1.58 ± 0.02a,b,c 0.10 ± 0.01b 0.10 ± 0.01a 3.81 ± 0.05a,b,c 1.32 ± 0.02a,b,c
0.01 ± 0.01a,d,e 0.09 ± 0.01a,d,e 0.81 ± 0.01a,d,e 0.02 ± 0.01e 0.22 ± 0.02a,d,e 0.36 ± 0.01a,d,e 15.50 ± 0.16d,e 0.42 ± 0.02d,e 0.88 ± 0.01d,e 0.34 ± 0.02a,d,e 16.03 ± 0.03d,e 0.19 ± 0.04e 21.43 ± 0.03a,d,e 0.07 ± 0.01a 15.13 ± 0.05d,e 0.16 ± 0.01 1.37 ± 0.03d,e 0.17 ± 0.01a,e 0.21 ± 0.02a,d 2.07 ± 0.03a,d,e 1.07 ± 0.01a,d,e 14.95 ± 0.04a,d,e 2.25 ± 0.03a,d,e 0.07 ± 0.01d 0.10 ± 0.01a 4.46 ± 0.01a 1.62 ± 0.01a,d
0.03 ± 0.01d,f 0.04 ± 0.01b,d 0.44 ± 0.02b,d,f 0.01 ± 0.01 0.29 ± 0.01d,f 0.47 ± 0.02b,d,f 17.41 ± 0.03b,d,f 0.52 ± 0.03d,f 1.55 ± 0.02b,d,f 1.03 ± 0.01b,d,f 14.70 ± 0.03b,d 0.26 ± 0.03b,f 24.52 ± 0.07d,f 0.06 ± 0.01b 17.97 ± 0.02b,d,f 0.14 ± 0.01b 1.40 ± 0.02d,f 0.18 ± 0.01e,f 0.49 ± 0.01b,d,f 1.82 ± 0.02b,d,f 1.02 ± 0.01b,d,f 8.34 ± 0.01b,d,f 1.99 ± 0.02b,d 0.27 ± 0.01b,d,f ND 3.22 ± 0.02b 1.82 ± 0.02b,d,f
0.03 ± 0.01c,e,f 0.04 ± 0.01c,e 0.57 ± 0.01c,e,f 0.03 ± 0.01e 0.37 ± 0.01c,e,f 0.08 ± 0.01c,e,f 17.93 ± 0.25c,e,f 0.69 ± 0.01c,e,f 1.90 ± 0.02c,e,f 1.22 ± 0.01c,e,f 14.88 ± 0.06e 0.58 ± 0.08c,e,f 25.65 ± 0.06e,f 0.08 ± 0.01 17.03 ± 0.01c,e,f 0.14 ± 0.01c 1.65 ± 0.02c,e,f 0.09 ± 0.01c,f 0.19 ± 0.01c,f 1.54 ± 0.02c,e,f 0.95 ± 0.01e,f 7.93 ± 0.09c,e,f 2.00 ± 0.02c,e 0.09 ± 0.01f ND 2.76 ± 0.06c 1.58 ± 0.05c,f
33.40 ± 0.69c 27.39 ± 0.74a 39.21 ± 0.05a,b,c 1.17 ± 0.02a,b,c
33.72 ± 0.13d,e 23.04 ± 0.01a,d,e 43.25 ± 0.12a,d,e 1.28 ± 0.01a,d,e
34.64 ± 0.02d,f 27.57 ± 0.01d,f 37.78 ± 0.02b,d,f 1.09 ± 0.01b,d,f
35.79 ± 0.12c,e,f 28.53 ± 0.14e,f 35.66 ± 0.26c,e,f 1.00 ± 0.01c,e,f
trans cis trans cis ω6 ω3
SFA MUFA PUFA PUFA/SFA
ND Non-detectable. Superscripts indicate statistical significant differences between a) GR-GB, b) GR-SR, c) GR-SB, d) GB-SR, e) GB-SB, f) SR-SB, P < 0.05.
three samples (GB, SR, SB) appeared to have similar IC50 values (GB = 217.53 ± 6.83; SR = 206.79 ± 8.47; SB = 198.68 ± 6.22: p > 0.05).
approximately 10 times lower than that of the TPL PUFA/SFA ratio. 3.3. Isolation of polar lipid fraction with Rf similar to that of phosphatidylcholine from TPL with preparative TLC
3.5. Phospholipid analysis The TPL were further separated into polar lipid fractions with preparative TLC. All four samples showed a similar profile, which consisted of 8 lipid fractions. The Rf of these fractions were compared with the Rf of a standard containing lyso-phosphatidylcholine (L-PC), sphingomyelin (SM), phosphatidylcholine (PC), lyso-phosphatidylethanolamine (L-PE), and phosphatidylethanolamine (PE) (Fig. 1). In previous work (data not shown), the most potent biological activities against platelet aggregation of all fractions have been assessed and the most active fraction was fraction 3. We have, thus, focused our subsequent work to the most biologically active fraction, i.e. fraction 3, which had a Rf similar to that of PC. This fraction was notably more extensive than the others, as was noted in other studies (Perez-Palacios et al., 2007; Sinanoglou et al., 2013).
The phospholipid composition of the TLC polar lipid fraction 3 of all samples (GR, GB, SR and SB) was further screened by LC–MS analysis. In the positive ion survey scans (Fig. 3a), the overwhelming majority of species were diacyl PC species. These species contained primarily stearic (18:0) fatty acids at the sn-1 position. At the sn-2 position, monounsaturated fatty acids (16:1 or 18:1) or poly-unsaturated fatty acid (18:2 cis or 18:3) were present. The fatty acid (18:3) was less abundant in sheep meat samples in comparison to the goat meat samples. Baking did not show to have a considerable effect on the relative abundancies of fatty acid species. In the negative ion survey scans (Fig. 3b), diacyl PC species were predominant, but a range of species from other classes of phospholipids, mainly PI were also detected. At the sn-1 position, primarily palmitic (16:0) fatty acids were present, while at the sn-2 position, several fatty acids such as vaccenic acid (18:1 trans), CLA or odd-chain fatty acids were found. These fatty acids were detected in insignificant amounts. Several other phospholipids classes of minor biological value were detected, which tends to suggest that fatty acid species detected in negative ion scans were of lower analytical importance than those detected in positive ion survey scans for these PC fractions.
3.4. Biological activity of TLC polar lipid fraction 3 from all meat samples The TLC polar lipid fraction 3 from all samples was tested for their ability to inhibit PAF-induced platelet aggregation. The biological activities of each lipid fraction are expressed as an IC50 in micrograms, which are shown in Fig. 2. When comparing IC50 values, lower IC50 values correspond to stronger activities since a smaller amount of lipids is required for the same inhibitory activity. According to Fig. 2 lipid fractions from all samples exhibited inhibitory activity against PAFinduced platelet aggregation. Lipids from the raw goat meat sample (GR) had a significantly lower IC50 value (130.44 ± 4.65: p < 0.05) compared to lipids of the other three samples. Lipids from the other
4. Discussion Both animals had similar amount of TL (p > 0.05). Older sheep would be expected to have a statistically significant higher content of 42
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Table 2b Fatty acid composition (%) (mean ± SD, n = 3) of total neutral lipids (TNL) of all samples (GR: goat meat, raw; GB: goat meat, baked; SR: sheep meat, raw and SB: sheep meat, baked). Fatty acid
GR
GB
SR
GB
10:0 12:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 18:1 18:2 18:2 18:3 18:3 20:0 20:1 20:2 20:3 20:4 20:5 22:1 22:2 22:5 22:6
0.27 ± 0.01a,c 1.62 ± 0.03b,c 10.85 ± 0.11b,c 0.34 ± 0.01a,b,c 0.83 ± 0.01a,b,c 0.16 ± 0.01a,b,c 28.60 ± 0.12a,b,c 2.00 ± 0.01a,b,c 1.26 ± 0.01a,b,c 0.58 ± 0.01a,b,c 11.67 ± 0.03a,b,c 0.99 ± 0.05b,c 34.72 ± 0.26a,c 0.30 ± 0.01b,c 4.30 ± 0.03a,b,c ND 0.71 ± 0.01a,b,c 0.08 ± 0.03 0.11 ± 0.02 0.09 ± 0.01 0.05 ± 0.01 0.48 ± 0.01a ND ND ND ND ND
0.22 ± 0.01a,d 1.51 ± 0.03d,e 10.84 ± 0.12d,e 0.29 ± 0.01a,d,e 0.78 ± 0.01a,d,e 0.17 ± 0.01a,d,e 27.50 ± 0.08a,d,e 1.59 ± 0.04a,d,e 1.00 ± 0.02a,d,e 0.47 ± 0.02a,d,e 12.53 ± 0.07a,d,e 0.73 ± 0.42d,e 36.19 ± 0.21a,d 0.30 ± 0.01d,e 3.88 ± 0.03a,e ND 0.64 ± 0.01a,d,e 0.12 ± 0.01 0.68 ± 0.55 0.12 ± 0.04 0.06 ± 0.01 0.36 ± 0.01a ND ND ND ND ND
0.28 ± 0.02d,f 0.49 ± 0.01b,d,f 4.67 ± 0.02b,d,f 0.21 ± 0.01b,d,f 1.01 ± 0.01b,d,f 0.18 ± 0.01b,d,f 23.76 ± 0.30b,d 1.36 ± 0.01b,d,f 2.60 ± 0.03b,d,f 1.35 ± 0.10b,d,f 18.93 ± 0.11b,d,f 4.66 ± 0.02b,d,f 35.38 ± 0.08d,f 0.24 ± 0.02b,d 3.91 ± 0.01b,f ND 0.96 ± 0.01b,d ND ND ND ND ND ND ND ND ND ND
0.21 ± 0.01c,f 0.36 ± 0.02c,e,f 4.44 ± 0.02c,e,f 0.20 ± 0.01c,e,f 1.13 ± 0.01c,e,f 0.20 ± 0.01c,e,f 24.16 ± 0.12c,e,f 1.78 ± 0.01c,e,f 3.00 ± 0.01c,e,f 1.77 ± 0.03c,e,f 16.45 ± 0.03c,e,f 3.98 ± 0.04c,e,f 36.76 ± 0.12c,f 0.24 ± 0.01c,e 4.37 ± 0.04c,e,f ND 0.97 ± 0.02c,e ND ND ND ND ND ND ND ND ND ND
55.16 ± 0.24b,c 38.90 ± 0.22a,b,c 5.93 ± 0.02a,b,c 0.11 ± 0.01a,b,c
54.51 ± 0.16d,e 40.13 ± 0.10a,d,e 5.36 ± 0.06a,d,e 0.10 ± 0.01a,e
51.74 ± 0.24b,d,f 43.14 ± 0.22b,d,f 5.12 ± 0.02b,d,f 0.10 ± 0.01b,f
49.53 ± 0.16c,e,f 44.68 ± 0.16c,e,f 5.58 ± 0.03c,e,f 0.11 ± 0.01c,e,f
trans cis trans cis ω-6 ω-3
SFA MUFA PUFA PUFA/SFA
ND Non-detectable. Superscripts indicate statistical significant differences between a) GR-GB, b) GR-SR, c) GR-SB, d) GB-SR, e) GB-SB, f) SR-SB, P < 0.05.
of lipids section. The data from phospholipid analysis strongly correlates with previous work from our group on the lipodomic studies of fish with antiinflammatory activities, where PC species containing primarily 18:1 and 18:2 were identified (Nasopoulou et al., 2014). Therefore, it could be suggested that PC-species have strong anti-inflammatory activities and the presence of these lipid species in both types of meat can be linked to potential cardioprotective activities that may aid in the prevention of CVD in humans. Dietary guidelines of the partial replacement of saturated fat with other energy sources (carbohydrates, monounsaturated or polyunsaturated fatty acids) have been suggested in the past by many organisations and are still advised today by the American Heart Association (Lordan et al., 2018; Sacks et al., 2017). These directives do not necessarily yield beneficial results against CVD, except for the case of replacement of saturated fatty acids with polyunsaturated fatty acids (Micha and Mozaffarian, 2010; Siri-Tarino et al., 2010). Foods with a high ratio of PUFA/SFA are generally regarded as nutritionally valuable. However there is a discrepancy between findings from omega-3 PUFAs, fish or fish oil consumption, regarding protection against the development of CVD. Fish and fish oils contain a complex mixture of lipids, only a small component of which is omega-3 PUFAs. Consequently, there is potential for other lipids in the mixture to be beneficial, either in an additive or even a synergistic way (Megson et al., 2016; Zabetakis, 2013). in vivo studies of polar lipids isolated from fish, olive oil, and olive pomace in hyperlipidemic rabbits have shown retard (Karantonis et al., 2006; Nasopoulou et al., 2010; Tsantila et al., 2007) or regress atheromatic plaque (Tsantila et al., 2010). It is suggested that the inflammatory action of PAF can be inhibited by dietary lipid microconstituents, as several in vitro studies have shown that polar lipids
TL (Brzostowski et al., 1997). Baking induced a statistically significant increase of TL due to moisture loss (p < 0.05) (Badiani et al., 1998). Baking caused a significant increase of TPL expressed as percentage of TL (p < 0.05), because TPL is mainly present in the connective tissue of the muscle, since the phospholipid fraction is probably fairly representative of that of the membranous structures of the muscle itself (Christie, 1978), thus their loss during baking is low, whereas the TNL is predominantly present in the adipose tissue (Christie, 1978), and consequently follows TL’s content changes due to drip-fat loss and moisture loss. So, we notice no significant change of TNL, when expressed as percentage of TL (p < 0.05) after baking. This study has some limitations, including the fact that the fatty acid profile of ruminant meat is influenced by several factors such as breed, age, genotype, nutrition, and the environment (Banskalieva et al., 2000; Santos et al., 2007). Thus more than one animal and animals of different breeds from different areas may have enhanced the study. Furthermore, the meat samples used in the study were chosen because they are the most consumed cut in both animals in Greece, thus are the most representative sample of the animal. Further research should focus on other cuts of meat in order to obtain a comprehensive view of the potential anti-inflammatory and antithrombotic effects of consuming these animals. The fatty acid composition analysis of the meats revealed the contrast between the TPL and TNL of all samples. The TPL, which corresponds mainly to the intramuscular fat, has an extremely high content of PUFA and accordingly a high PUFA/SFA ratio. Whereas, the TNL, which correspond mainly to adipose tissue, have a PUFA/SFA ratio that is approximately 10 times lower than that of the TPL ratio. This result may be explained on the basis that PUFA present in meat have a polar head, therefore, they have been extracted by the ethanolic TPL fraction during counter-current distribution, which is described in the isolation 43
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Fig. 2. in vitro biological activity of TLC polar lipid fractions, with Rf similar to that of phosphatidylcholine, of goat raw meat (GR), goat baked meat (GB), sheep raw meat (SR), and sheep baked meat (SB) samples toward platelet aggregation in human PRP, expressed as IC50 in micrograms (mean ± SEM, n = 3). Goat raw (GR) meat sample had a significantly lower (p < 0.05) IC50 value compared to IC50 values of the other three meat lipid samples.
atherogenic and thrombotic action of PAF (Megalemou et al., 2017; Poutzalis et al., 2016). The same TLC polar lipid fraction, which is the most prominent polar lipid fraction of muscle tissue (Perez-Palacios et al., 2007; Sinanoglou et al., 2013), was investigated in raw and baked goat and sheep meat, and it is clear that these PC derivatives possess similar inhibitory action against PAF-induced platelet aggregation to those found in the literature. The structures of these PC molecules differ slightly from other PC molecules of marine origin, including olive pomace fed seabass (Nasopoulou et al., 2014), olive pomace-enriched fish feed and olive pomace fed gilthead seabream (Sioriki et al., 2016). This fact emphasises the need to correlate the structural variances of different lipid species with their biological activity in order to understand the molecular mechanisms involved in inhibiting the actions of PAF on the PAF-receptor. 5. Conclusion Our previous research has shown that yogurts and cheese derived from caprine and ovine milk have strong antithrombotic properties (Lordan and Zabetakis, 2017b; Megalemou et al., 2017; Poutzalis et al., 2016; Tsorotioti et al., 2014). In our quest to determine the origin of these important bioactivities, we expanded our research to investigate the lipid composition of goat and sheep meat. The cardioprotective activities found in goat and ewe yogurts had been linked to lipids that are PC (Megalemou et al., 2017; Poutzalis et al., 2016). The data reported in this novel research suggests that goat and sheep meat also contain PC derivatives that have strong antithrombotic activities. Further studies are required to elucidate the structural role of bioactive lipids found in our diet and their effects upon consumption, in order to set realistic nutritional guidelines in order to prevent the onset of atherosclerosis. Our results underline the nutritional value of goat and sheep meat as potent sources of polar lipids with strong inhibitory properties against PAF-induced platelet aggregation, and consequently atherosclerosis. It could be concluded that sheep and goats have the enzymatic capacity to biosynthesize antithrombotic polar lipids, present in both meat and milk. This study has also for the first time demonstrated that these activities are not lost under baking conditions, highlighting that baked goat and sheep meat have potential functional and potent cardioprotective properties.
Fig. 1. Typical profile of total polar lipids (TPL) separation of our meat samples on preparative TLC. The eight bands obtained along with the corresponding (in terms of Rf) standard compounds: L-PC lyso-phosphatidylcholine, SM sphingomyelin, PC phosphatidylcholine, L-PE lyso-phosphatidylethanolamine and PE phosphatidylethanolamine, are shown on the left.
isolated from fish and olive pomace are capable of inhibiting PAF-induced platelet aggregation (Nasopoulou et al., 2014, 2013b; Nomikos et al., 2006; Sioriki et al., 2016). Polar lipids with inhibitory action against PAF have been found in various foods, even of animal origin (Lordan et al., 2017; Megalemou et al., 2017; Nasopoulou et al., 2013a; Poutzalis et al., 2016; Tsorotioti et al., 2014). Polar lipids from different foodstuffs can vary in structure, abundance, and inhibitory strength. Also it should be highlighted that different foods vary in composition for a number of molecules, that contribute to CVD and general health in a positive or a negative way. These results demonstrate that PC derivatives exert antithrombotic and anti-inflammatory properties, which are not lost under baking conditions. Previous research has shown that the TLC polar lipid fraction 3, with a Rf value similar to that of PC, from goat and sheep dairy sources possess potent anti-inflammatory properties against the
Author contributions C. Nasopoulou and I. Zabetakis designed the study and interpreted the results. R. Lordan interpreted the data and drafted the manuscript. S. Poutzalis collected data and drafted the manuscript. 44
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Fig. 3. a) Lipidomic analysis of TLC polar lipid fraction 3, with Rf similar to that of phosphatidylcholine by liquid chromatography-mass spectrometry (positive ion survey). Inserts show percentage of the most abundant phospholipids in each sample. b) Lipidomic analysis of TLC polar lipid fraction 3, with Rf similar to that of phosphatidylcholine by liquid chromatography-mass spectrometry (negative ion survey). Inserts show percentage of the most abundant phospholipids in each sample.
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Conflict of Interest
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