The Synthesis And Purification Of Deuterium Labeled X-Gal-Neu5Gc~D And The Application Of Neu5Gc Detection In Red Meat

Sialic acids are a family of nine-carbon acidic monosaccharides which exist in the form of sugar-encrusted substance in the organism.The sialic acid family includes 50 derivatives of the nine-carbon sugar neuraminic acid.N-acetylneuraminic acid(Neu5Ac)and N-glycolylneuraminic acid(Neu5Gc)are the two major kinds of sialic acids.Neu5Ac is endogenous sialic acids for human which can be found on the surface of the brain and nerve cells frequently.It has beneficial effects by accelerating brain intelligence development.Neu5Gc is harmful for human,because Neu5Gc is an exogenous sialic acid that our bodies cannot make,so intake of combined Neu5Gc would induce inflammation before coming to a disease.The combined Neu5Gc is mainly existing in red meat of our daily diet,so it is a vitally foundation for the proportion of diet to investigate the content of Neu5Gc in various kinds of meat.For the quantification of Neu5Gc in red meat,our lab used X-Gal-Neu5Prop as internal standard and detected by UPLC.This method has a limitation because of the molecular structure of Neu5Gc and Neu5Prop is different,then they will have different response values from the fluorescence detector.In order to quantify the Neu5Gc in red meat accurately,we synthesized a new internal standard named X-Gal-Neu5GcD.Then used X-Gal-Neu5GcD as a internal standard to qutifited Neu5Gc in red meat.1.The method of synthesis,purification and detection for deuterium labeled X-Gal-Neu5GcDX-Gal-Neu5Gc can be synthetized by the enzyme of isomerase(Epimerase),sialic acid aldehyde reductase(Sialic acid Aldolase),CMP-Sia synthase(CMP-Sialic acid Synthetase)and sialic acid-glucose radical transfer enzyme(Sialyltransferase)from GlcNGc and X-Gal.If the whole H2O in the reaction is replaced by D2O,there are three hydrogen atoms on the Neu5Gc will be replaced by deuterium atoms,which can eventually synthesize deuterium labeled X-Gal-Neu5GcD.The normal X-Gal-Neu5Gc and deuterium labeled X-Gal-Neu5GcD was purified by C18 SPE column and silica column,and detected by UPLC and LC-MS.The pure X-Gal-Neu5Gc and deuterium labeled X-Gal-Neu5GcD can be acquired.2.The quantification of deuterium labeled X-Gal-Neu5GcDDeuterium labeled X-Gal-Neu5GcD can be quantified by commercial Neu5Gc.The comparison of SIM and SCAN mode of LC-MS showed that the SIM mode is more sensitive than the SCAN mode.When the quantification was measured by commercial Neu5Gc,Neu5GcD was hydrolyzed from X-Gal-Neu5GcD,and then used the SIM mode of LC-MS to detect the molecular weight of commercial Neu5Gc and deuterium labeled Neu5GcD.The concentration of three deuterium atom labeled X-Gal-Neu5GcD is 3.82 mmol/L.3.Quantitative analysis of Neu5Gc in red meatQuantify the contents of Neu5Gc in red meat by using X-Gal-Neu5Prop which synthesis before and deuterium labeled X-Gal-Neu5GcD as the internal standard,the result indicated that the use of deuterium labeled X-Gal-Neu5GcD as internal standard is feasible.The different responses of different substrate can be clear when used UPLC to detect.The proportion of free Neu5Gc and combined Neu5Gc in different species of red meat are different,and the effect to human is also individual.The free and combined sialic acid in red meat was quantified by deuterium labeled X-Gal-Neu5GcD,and the content of free and combined Neu5Gc in red meat was accurately determined.