Enzymatic quantification of sphingosine in the picomole range in cultured cells

An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-[32P]sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.