Simultaneous Preparation of Purified Plasmalogens and Sphingomyelin in Human Erythrocytes with Phospholipase A1 from Aspergillus orizae

A method for the simultaneous purification of plasmalogens and sphingomyelin (SM) in human erythrocytes is described. Treatment of total lipids with n-hexane/acetone (1:1 v/v) resulted in selective precipitation of SM. Both the supernatant and the precipitate fractions were incubated with a phospholipase A, (PLA1) from Aspergillus orizae for 3.5h. The PLA1-treated lipids were extracted with n-hexane/isopropanol, the hexane layer was obtained using a Na2SO4 solution, and the hexane layer was further washed with water. At this step, the relative concentration of the plasmalogens was 92% of the total phospholipids in the supernatant fraction, and that of SM was 97.7% in the precipitate fraction. Each fraction was applied to high performance liquid chromatography (HPLC) for further purification. The plasmalogen and SM obtained were almost free of the other lipids. The purity of the plasmalogens and SM was monitored by HPLC, which can separate intact plasmalogens from their diacyl analogs.