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

Second messenger ceramides differ in their composition according to the nat ure of their sphingoid bases and their amide linked fatty acids. These diff erent species may be involved in many different biological responses, as ma y be the diacylglycerol species. For this reason, it is interesting to have a simple method which allows for the separation of these compounds into cl asses of species for further analysis. We describe here a simple procedure which resolves ceramides containing phy tosphingosine and normal fatty acids from ceramides with sphingosine and al pha-hydroxy fatty acids, and ceramides with sphingosine and normal fatty ac ids. Moreover, by means of a three-dimensional thin-layer chromatography, a n enhanced simultaneous resolution between diacylglycerol species was obtai ned. This improved method was used to obtain the separation of free ceramides ex tracted from fish gills where the presence of ceramides with phytosphingosi ne was shown. Chemical and gas liquid chromatography analyses of the separa ted species showed the efficiency of the described TLC procedure.