Studies on ether phospholipids: I. A new method of determination using phospholipase A1 from guinea pig pancreas: Application to krebs ii ascites cells

Abstract

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A 1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42° C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A 1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.