| Sphingolipids are a highly diverse class of lipids that serve not only as components of cellular membranes but also as signaling molecules capable of eliciting cell differentiation,proliferation,immunization,senescence and apoptosis.Because of their biological activity,sphingolipids are gradually applied as food nutritional and medicinal composition to improve human health.Nowadays the researches of sphingolipids mostly focus on the high animals and plants,but few involves in marine organisms.Marine microalgaes have the advantages of short life cycle,large species and biomass,and many species have the capacity to produce many kinds of active substances,so they have been the main source of new natural products.Emiliania huxleyi is a marine eukaryotic microalgae,and can produce abundant secondary metabolism active substances.Recently,an unusual class of glycosphingolipids from virus infected E.huxleyi were isolated and analyzed,implicating a novel structure with highly bioactivity,which will have important scientific significance and broad application prospects in natural food nutrition and medicine research.In this study,E.huxleyi BOF92 and its specific virus EhV99B1 were taken as research materials.Fragmentation studies of sphingolipids revealed the principles of characteristic fragments with mass spectrometry by using standards of sphingolipids.Then a lipid extraction method and an ultra high performance liquid chromatography-quadrupole orbitrap fourier transform-exactive mass spectrometry(UPLC-Q-Exactive-MS)method were developed.Based on these methods,the composition and content of sphingolipids were analized in normal and viral infected E.huxleyi cells.The main results were as follows:Firstly,the total lipids of E.huxleyi were detected by fluorescence microscopy and thin layer chromatography(TLC).Neutral lipids accumulated significantly after virus infection along with the nuclear fragmentation at late stage(45hpi).The composition of total lipids also changed to some extent.Antioxidant test in vitro indicated a variable degree of antioxidant activities in E.huxleyi lipid extracts and that of virus infected cells possessed more significant reducing power.In addition,the total lipids extracts had inhibitory or stimulatory effects on normal liver cells LO2 and hepatoma cells HepG2 at concentration-dependent manner.Secontly,the mass spectrometry fragmentation principles of sphingolipids were studied.Standards of sphingolipids were analysed directly by mass spectrometry in the positive andnegative mode.The results showed that it was easier for sphingolipids to be ionized in positive mode,and ion fragmentations or neutral loss can provide information about sphingolipid's structure.Therefore,the type and structure of sphingolipids could be determined by analysing mass chromatogram.The characteristic ion fragments of sphingolipids were yielded by the removal of two waters from long chain bases(LCB)of sphingolipids.For example,if the LCBs are d18:1 and t18:0,the m/z of characteristic ion fragmentations in the positive mode are 264.3,282.3.Lastly,UPLC-Q-Exactive-MS method was established to study the changes of the composition and content of sphingolipids in E.huxleyi BOF92.13 sphingolipids were identified in E.huxleyi BOF92.There were a small amount of glycosphingolipids with one hexose group and content of ceramides were the highest,reaching 99% of the total sphingolipids.Most of ceramides have C18 long-chain base with two hydroxyl groups and C20-C23 fatty acid chain with no hydroxyl group.Virus infection did not change the composition of sphingolipids in host cells,but the levels of sphingolipids was reduced in different degrees.It suggested that some releated enzymes coded by viral genome involved in the metabolic pathway of host sphingolipids. |
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