Improved methods for the simultaneous separation of free diacylglycerol species from ceramides containing phytosphingosine and sphingosine bases with non hydroxy and alpha-hydroxy fatty acids

Second messenger ceramides differ in their composition according to the nature of their sphingoid bases and their amide linked fatty acids. These different species may be involved in many different biological responses, as may be the diacylglycerol species. For this reason, it is interesting to have a simple method which allows for the separation of these compounds into classes of species for further analysis. We describe here a simple procedure which resolves ceramides containing phytosphingosine and normal fatty acids from ceramides with sphingosine and alpha-hydroxy fatty acids, and ceramides with sphingosine and normal fatty acids. Moreover, by means of a three-dimensional thin-layer chromatography, an enhanced simultaneous resolution between diacylglycerol species was obtained. This improved method was used to obtain the separation of free ceramides extracted from fish gills where the presence of ceramides with phytosphingosine was shown. Chemical and gas liquid chromatography analyses of the separated species showed the efficiency of the described TLC procedure.