Structural Elucidation of Irish Organic Farmed Salmon (Salmo salar) Polar Lipids with Antithrombotic Activities

Alexandros Tsoupras, Ronan Lordan, Martina Demuru, Katie Shiels, Sushanta Kumar Saha, Constantina Nasopoulou, Ioannis Zabetakis, Alexandros Tsoupras, Ronan Lordan, Martina Demuru, Katie Shiels, Sushanta Kumar Saha, Constantina Nasopoulou, Ioannis Zabetakis

Abstract

While several marine polar lipids (PL) have exhibited cardioprotective properties through their effects on the platelet-activating factor (PAF) pathways, salmon PL have not been tested so far. In this study, the antithrombotic activities of salmon PL were assessed in human platelets and the structural characterisation of bioactive salmon PL was performed by GC-MS and LC-MS analyses. PL from fillets of Irish organic farmed salmon (Salmo salar) were extracted and separated into several lipid subclasses by thin-layer chromatography (TLC), while their fatty acid profile was fully characterised by GC-MS. Salmon total lipids (TL), total neutral lipids (TNL), total polar lipids (TPL), and each PL subclass obtained by TLC were further assessed for their in vitro effects towards PAF-induced and thrombin-induced platelet aggregation in human platelets. Salmon PL exhibited antithrombotic effects on human platelet aggregation, mostly through their strong inhibitory effects against the PAF pathway with IC50 values comparable to other marine PL, but with lower effects towards the thrombin pathway. PL fractions corresponding to phosphatidylcholine and phosphatidylethanolamine derivatives exhibited the most potent anti-PAF effects, while LC-MS analysis putatively elucidated their structure/function relationship. Several diacyl-PC/PE and alkyl-acyl-PC/PE species containing mostly docosahexaenoic acid at their sn-2 glycerol-backbone may be responsible for the bioactivity. The data presented suggests that salmon contains PL with strong antithrombotic bioactivities.

Keywords: GC-MS; LC-MS; antithrombotic; docosahexaenoic acid (DHA); phosphatidylcholine; phosphatidylethanolamine; platelet aggregation; platelet-activating factor (PAF); polar lipids; polyunsaturated fatty acids (PUFA); salmon; thrombin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TLC analysis of the TPL of Irish organic farmed salmon fillets and the biological activity of each PL subclass towards PAF-induced platelet aggregation in hPRP. (A): Typical profile of the PL separation of salmon fillet on preparative TLC (Silica G). The elution system used for the separation of the TPL was chloroform:methanol:water 65:35:6 (v/v/v). PL: Polar lipids; E: mixture of standard PL from egg; Lyso-PC: lyso-phosphatidylcholine; SM: sphingomyelin; PC: phosphatidylcholine; Lyso-PE: lyso-phosphatidylethanolamine; PE: phosphatidylethanolamine; CL: cardiolipin; TLC; thin-layer chromatography. (B): Biological activities of the PL subclasses of the TLC bands towards PAF-induced platelet aggregation in hPRP. The results reflect the inhibitory strength of each lipid sample and are expressed as mean values of IC50 (μg of lipids in the aggregometer cuvette that causes 50% inhibition of PAF-induced aggregation of hPRP); The low IC50 value indicates strong inhibition of PAF-induced aggregation of hPRP; Lipid fractions of TLC bands 1 and 4 (corresponding to Lyso-PC and Lyso-PE) did not exhibit these bioactivities. hPRP: human platelet-rich plasma; PAF: platelet-activating factor; PL: polar lipids; TPL: total polar lipids.
Figure 2
Figure 2
A representative chart of the 50% inhibitory effect of Irish organic farmed salmon fillet PL towards PAF-induced platelet aggregation in hPRP. Trace 1 (blue) in both A and B demonstrate the maximum-reversible aggregation of hPRP induced by 2.6 × 10−8 M of PAF; Trace 2 (black) demonstrates the effect of preincubation of hPRP with the appropriate concentration (IC50 value) of salmon TPL (A) and the corresponding TLC Band of PC (B), which causes 50% inhibition of PAF-induced hPRP aggregation. PAF: platelet-activating factor; hPRP: human platelet-rich plasma; TPL: total polar lipids; PC: phosphatidylcholine.
Figure 3
Figure 3
Representative HPLC chromatograms of salmon PL LC-MS analysis. (A) Depicts a representative chromatogram of the TLC fraction corresponding to salmon phosphatidylethanolamine (PE) derivatives, whereas (B) depicts a representative chromatogram of the TLC fraction corresponding to salmon phosphatidylcholine (PC) derivatives. Analysis was performed using a HPLC (Agilent 1260 series) equipped with Q-TOF mass spectrometer (Agilent 6520). The column used for the separations was an Agilent C18 Poroshell 120 column (2.7 µm, 3.0 × 150 mm). The composition of mobile phase (A) was 2 mM ammonium acetate in water and 2 mM ammonium acetate in 95% acetonitrile for mobile phase (B). Chromatographic separation was performed by gradient elution starting with 60% B for 1 min, then increasing to 90% B over 2.5 min. Subsequently, 90% B was held for 1.5 min and increased afterward to 100% over 5 min. Then, 100% B was held for 4 min, reducing afterward to 60% B over 0.5 min and hold for 1 min until the next run. The mobile phase flow rate was 0.3 mL/min until 5 min elapsed, increasing up to 0.6 mL/min until 10 min had elapsed and held at this flow until the end of the run. The injection volume was 10 μL.
Figure 4
Figure 4
Representative mass spectra of PC species present in the relative TLC fraction of bioactive salmon PC. The LC-MS analysis demonstrated that the TLC fraction of the bioactive salmon PC contains a mixture of several PC species. (A–C) Mass spectra depicting the most abundant PC molecules eluted at peaks 1, 2, and 3 of the LC-MS analysis, respectively. The most probable and proposed identities of the PC species for each acquired m/z values were verified using the LIPID MAPS: Nature Lipidomics Gateway (www.lipidmaps.org), by using the lowest delta values during identification, in combination with their content of FA that was acquired by both GC-MS and LC-MS analyses. In all 3 peaks, several PC molecules are proposed baring either EPA or DHA in the sn-2 position of the glycerol backbone, whereas lower but considerable quantities of alkyl-acyl moieties also seem to be present in all spectra (A–C) that are outlined in squares. Interestingly, at peak 3c (elution time 3.2 min), a 1-O-alkyl (18:0)-2-sn-akyl (22:6, DHA)-3-glycerophosphocholine seems to be present in the TLC fraction of the bioactive salmon PC. FA: fatty acids; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; PC: phosphatidylcholine.
Figure 4
Figure 4
Representative mass spectra of PC species present in the relative TLC fraction of bioactive salmon PC. The LC-MS analysis demonstrated that the TLC fraction of the bioactive salmon PC contains a mixture of several PC species. (A–C) Mass spectra depicting the most abundant PC molecules eluted at peaks 1, 2, and 3 of the LC-MS analysis, respectively. The most probable and proposed identities of the PC species for each acquired m/z values were verified using the LIPID MAPS: Nature Lipidomics Gateway (www.lipidmaps.org), by using the lowest delta values during identification, in combination with their content of FA that was acquired by both GC-MS and LC-MS analyses. In all 3 peaks, several PC molecules are proposed baring either EPA or DHA in the sn-2 position of the glycerol backbone, whereas lower but considerable quantities of alkyl-acyl moieties also seem to be present in all spectra (A–C) that are outlined in squares. Interestingly, at peak 3c (elution time 3.2 min), a 1-O-alkyl (18:0)-2-sn-akyl (22:6, DHA)-3-glycerophosphocholine seems to be present in the TLC fraction of the bioactive salmon PC. FA: fatty acids; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; PC: phosphatidylcholine.
Figure 5
Figure 5
Representative mass spectra of PE species present in the relative TLC fraction of bioactive salmon PE. The LC-MS analysis showed that the TLC fraction of bioactive salmon PE contains a mixture of several PE species. (A–C) Mass spectra depicting the most abundant PE molecules eluted at peaks 1, 3a, and 3b of the LC-MS analysis, respectively. The most probable and proposed identities of PE species for each acquired m/z values were verified using the LIPID MAPS: Nature Lipidomics Gateway (www.lipidmaps.org), by using the lowest delta values during identification, in combination with their content in FA that was acquired by both GC-MS and LC-MS analyses. In all 3 peaks, several PE molecules are proposed with DHA in the sn-2 position of the glycerol backbone, whereas lower but considerable quantities of alkyl-acyl moieties seem to be present only at B and C spectra of the PE molecules eluted at peak 3, which are outlined in squares. FA: fatty acids; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; PE: phosphatidylethanolamine.
Figure 5
Figure 5
Representative mass spectra of PE species present in the relative TLC fraction of bioactive salmon PE. The LC-MS analysis showed that the TLC fraction of bioactive salmon PE contains a mixture of several PE species. (A–C) Mass spectra depicting the most abundant PE molecules eluted at peaks 1, 3a, and 3b of the LC-MS analysis, respectively. The most probable and proposed identities of PE species for each acquired m/z values were verified using the LIPID MAPS: Nature Lipidomics Gateway (www.lipidmaps.org), by using the lowest delta values during identification, in combination with their content in FA that was acquired by both GC-MS and LC-MS analyses. In all 3 peaks, several PE molecules are proposed with DHA in the sn-2 position of the glycerol backbone, whereas lower but considerable quantities of alkyl-acyl moieties seem to be present only at B and C spectra of the PE molecules eluted at peak 3, which are outlined in squares. FA: fatty acids; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; PE: phosphatidylethanolamine.

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