Structural characterization and profiling of lyso‐phospholipids in fresh and in thermally stressed mussels by hydrophilic interaction liquid chromatography—electrospray ionization—Fourier transform mass spectrometry

Abstract

The separation efficiency of hydrophilic interaction liquid chromatography and the high resolution/accuracy of electrospray ionization-Fourier transform MS were successfully applied to the detailed characterization of lyso-phosphatidylcholines (LPCs) and lyso-phosphatidylethanolamines (LPEs) contained in the lipid extracts of Mytilus galloprovincialis (Mediterranean mussel). As a result, 57 LPCs, including regio- and positional isomers, and 45 LPEs, including acyl and plasma(e)nyl species, were identified. Four lyso-phosphonocholines were also identified among mussel Lyso-Phospholipids. To the best of our knowledge this represents the first characterization, at a molecular level, ever reported for LPEs in mussels. No significant variation was observed in the composition of both LPCs and LPEs when mussels were refrigerated at +4°C for up to 48 h, i.e. under conditions usually employed for seafood transportation and storage. Treatments mimicking more severe thermal stresses, namely eight day-refrigeration at + 4°C, two week-freezing at -15°C and 6 h-storage at 25°C, resulted in a significant increase in the molar abundance of LPCs and LPEs (expressed with respect to that of their precursors, PCs and PEs, respectively) and was accompanied by the death of all or part of the molluscs. These results were interpreted invoking the generation of lyso-phospholipids, mediated by endogenous phospholipases, as an intermediate process toward the partial replacement of side chains in phospholipids, perhaps functional to a better adaptation of mussels to adverse temperature conditions. Interestingly, the relative abundances of specific compounds belonging to the LPC and LPE classes were found to follow the seasonal variations of sea temperature.

Seasonal variations in the profile of main phospholipids in Mytilus galloprovincialis mussels: A study by hydrophilic interaction liquid chromatography–electrospray ionization Fourier transform mass spectrometry

“…Such a process would imply the preliminary generation (catalyzed by endogenous phospholipases) of lyso ‐PL (ie PL missing one of their acyl chains), that would be subsequently converted, through esterification mediated by further enzymes, into new PL with appropriate side chains composition. A study recently performed in our laboratory on M galloprovincialis showed that refrigeration at 4°C of alive mussels for several days led to a progressive increase of the incidence of lyso forms of their major PL classes, namely, phosphatidylcholines (PC) and PE, with respect to their diacylic precursors . Actually, a not negligible presence of lyso ‐PL was also found in the case of gilthead sea bream ( Sparus aurata ) fillets subjected to low temperatures .…”

Ceramide lipids in alive and thermally stressed mussels: an investigation by hydrophilic interaction liquid chromatography‐electrospray ionization Fourier transform mass spectrometry

“…These findings are consistent with those inferred previously from the LPC/PC and LPE/PE concentration ratios (see Fig. 6 in ref …”

“…, the presence of compounds bearing an additional OH group was inferred also in the case of CPE. Moreover, some accurate m/z values detected in the MS spectra of CPE and CAEP classes could be assigned to sodium adducts ([M + Na] + ), as already observed for lysophosholipids detected in the lipid extracts of mussels and gilthead sea breams . The perfect alignment found for peaks detected in the extracted ion current (XIC) chromatograms related to m/z ratios of proton and sodium adducts of the same CPE or CAEP species suggested that sodium adducts were formed during the ESI process.…”

“…As a result, the lyso forms of phosphatidylcholines (LPCs) and phosphatidylethanolamines (LPEs), arising from the hydrolytic cleavage of one of the acyl chains of such phospholipids, have been found to be significantly increased after long‐term refrigeration or freezing. A similar effect has been observed upon storage at room temperature for prolonged times …”

“…In particular, a XIC chromatogram was obtained, using a 0.001 m/z unit wide extraction window centered on each of the m/z values reported in Table , from the HILIC‐ESI‐FTMS trace related to each mussel lipid extract. The same approach was adopted for LPC and LPE (see ref . for the lists of m/z ratios found for these species in the lipid extracts of M. galloprovincialis ).…”