Abstract
Sphingolipid metabolites function as signaling molecules in mammalian cells, influencing cell proliferation, migration, and death. Recently, sphingolipid signaling has been implicated in the regulation of developmental processes in Drosophila melanogaster. However, biochemical analysis of endogenous Drosophila sphingoid bases has not been reported. In this study, a rapid HPLC-based method was developed for the analysis of free sphingoid bases endogenous to Drosophila. Four molecular species of endogenous free sphingoid bases were observed in adult flies and identified as C14 and C16 sphingosine (Sph) and C14 and C16 dihydrosphingosine (DHS). The C14 molecular species were the most prevalent, accounting for approximately 94% of the total free sphingoid bases in adult wild-type flies. An Sph kinase (SK) mutant demonstrated significant accumulation of all four sphingoid bases, whereas a serine palmitoyltransferase mutant demonstrated low but detectable levels. When endogenous sphingoid bases were evaluated at different stages of development, the observed ratio of Sph to DHS increased significantly from early embryo to adulthood. Throughout development, this ratio was significantly lower in the SK mutant as compared with the wild-type. This is the first report describing analysis of free C14 and C16 sphingoid bases from Drosophila. The biochemical characterization of these lipids from mutant models of sphingolipid metabolism should greatly facilitate the analysis of the biological significance of these signaling molecules.
Highlights
Sphingolipid metabolites function as signaling molecules in mammalian cells, influencing cell proliferation, migration, and death
When methanol was employed as the eluting solvent, recovery of all the free long-chain sphingoid base (LCB) standards was less than 2%
In agreement with the results obtained with methanol, the addition of ammonium acetate vastly improved the recovery of all the LCB standards from the column
Summary
Sphingolipid metabolites function as signaling molecules in mammalian cells, influencing cell proliferation, migration, and death. The inadequate recovery of the LCB standards from the Strata C18-E column was vastly improved by addition of 10% by volume of a 20 mM ammonium acetate solution to the methanol elution solvent. Elution of adult fly lipids from the Strata C18-E column with chloroform-methanol-20 mM ammonium acetate (4.5:4.5:1; v/v/v), resulted in an HPLC spectrum with significant unwanted background fluorescence (data not shown).
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