Separation And Purification Of Phosphatidylcholine In Swine Liver And The Research On Its Biology Function

Phosphatidylcholine (PC) is also known as lecithin and 1, 2-diacyl-sn-glycero-3-phosphocholine. Many results have been proved that phosphatidylcholine is a major constituent of cell membranes and also regarded as one of the methyl donators in metabolism. Moreover phosphatidylcholine was involved in the process of maintaining the cell normal structure and function, also, it was indeed related with the cell growth, proliferation, differentiation, and carcinogenesis. Its hydrolyzed products participate in the cell signal transduction and metabolic mediation with many physiological functions like preventing senescence, reducing blood fat, keeping brain healthy, and protecting against liver injury, and being anticancer. Plasmanylcholine are a unique subset of phosphatidylcholine in which the sn-1 carbon of the glycerol back bone contains a vinyl ether–linked (a cis double bond adjacent to an ether bond) long chain hydrocarbon instead of the typical ester-linked fatty acid. Plasmanylcholine are widely distributed in mammalian tissues and some monad. Different tissues and even different cell types of the same tissue may have variable amount of plasmanylcholine. The physiological role of plasmanylcholine is not fully understood so far. It has been postulated that plasmanylcholine can serve as endogenous antioxidants, storages of polyunsaturated fatty acids (PUFAs), lipid mediators and in the procession of functions on membrane dynamics. In addition, for the synthetic plasmanylcholine, there is selective toxin on cancer cells which attracts more and more attentions.Our experiments have been proved that phosphatidylethanolamine N-methyltransferase which catalyzes the transfer of a methyl group from S-adenosylmethionine to the polar head group of phosphatidylethanolamine to form phosphatidylcholine in the liver,Moreover, the apoptosis have been found in hepatoma cells during PEMT over-expressing. These results showed that phosphatidylcholine from swine liver could inhibit cell proliferation. In order to open out the function of phosphatidylcholine, the flow of the separation and purification has been studied on the point. In addition, we also detect the nature structure and special function of plasmanylcholine which belonged to liver phosphatidylcholineFirst, Phosphatidylcholine in crude phospholipids from swine liver was separated and purified by using Al₂O₃ column chromatography with 95% alcohol as eluent. The purity was determined by thin layer chromatography on GF254 silica plate and with chloroform-methanol-water (65:25:4V/V) as developing agent. The results showed that the purity and yield rate of the PC, separated from phosphatidylethanolamine (PE) completely, reached a degree of more than 90% and 80% respectively eluted with 225ml of 95% alcohol, and 87.6% and 87.3% with a volume of 425ml.The second, The effect of the PC with different concentration(12.5,25,50μmol/L) on the proliferation of rat hepatoma cell line (CBRH-7919) was determined by micro-culture tetrazolium (MTT) assays in vitro and was compared with that of human leukemia cell line K562. Results shows that the PC derived from the liver inhibited the growth of CBRH-7919 cell significantly after 12,24 and 36 hours treatment of the PC. It suggested that PC derived from animal liver might function as specific inhibitor for hepatoma cells in concentration dependent manner. The third, an effective method for the separation of phosphatidylcholine molecules from swine liver was developed using the reversed-phase high performance liquid chromatography (RP-HPLC).The influences of organic modifiers, the composition of mobile phase and the flow rate on PC molecules separation were studied. It was found that the separation could be benefited with adding hexane and ammonium acetate in mobile phase which contained a majority of methanol. It was not appropriate for the separation of PC molecules using the methanol-acetonitrile-water gradient elution. The results showed that the RP-HPLC method for the separation of PC molecules on prepared column (4.6mm×200mm,i.d.,5μm) was established with simplicity and good reproducibility, by using methanol-hexane-0.05mol/L ammonium acetate-glycerol(84:6:8:0.6,V/V)as mobile phase at a flow rate of 1.0ml/min and 35℃.This also can lay a foundation for the analysis of PC molecules structure. The last, Quattro Micro Tandem mass spectrometer (Micromass, Waters, USA), fitted with an electrospray ion-source operated at atmospheric pressure, was used for the identification of components eluting from the column. Nitrogen was used as nebulizer gas (0.83L/min) and as curtain gas (6.7L/min). The capillary voltage was set to 3.0 kV and the cone voltage to 25V. The mass spectrum was from 100 to 1000. In conclusion, the HPLC method described here separates a wide variety of molecular species of PC in an intact form using solvents that enable UV detection. Furthermore, unequivocal species identification, including the assignment of acyl chain position, can be achieved by online electrospray MS. The method can be applied for the isolation of individual PC species in order to detect their biological activities. According to the method, the exact molecular weight of plasmanylcholine (plas-PC) was 794Da, and its structure contained sn1-18:0 (acyl chain), sn2-20:4 (ether chain).The conclusions:1. The purity of the PC, separated from phosphatidylethanolamine (PE) completely, reached a degree of more than 90% by using Al2O3 column chromatography.2. PC derived from animal liver might function as specific inhibitor for hepatoma cells in concentration dependent manner.3. The exact molecular weight of plasmanylcholine (plas-PC) was 794Da, and its structure contained sn1-18:0 (acyl chain), sn2-20:4 (ether chain).