A rapid method for quantifying free sphingoid bases and complex sphingolipids in microgram amounts of cells following...

Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase), key enzymes in sphingolipid metabolism. The purpose of this study was to develop and validate rapid methods for the determination of free sphingoid bases and total sphingolipids in small quantities of cells exposed to pure fumonisin B(1). The developed rapid methods were a modification of an earlier 'original' method and used a single CHCl(3) extraction subsequent to base or acid hydrolysis of cell suspensions. The average recovery of the C(20)-sphinganine internal standard using the rapid extraction method for free sphingoid bases was 48% and 84% for control and fumonisin B(1)-treated LLC-PK(1) cells (approx. 100 mug protein), respectively, while the recovery using the original extraction method was 1% or less. The average total sphingolipid concentrations (free sphingoid bases plus complex sphingolipids) determined by the rapid and original method were similar. The rapid extraction method provided simpler and shorter extractions with improved recovery, and was more economical by allowing experimental designs using smaller quantities of cells (10-100 mug protein) and fumonisins. In conclusion, the rapid method is useful for the study of fumonisin-induced disruption of sphingolipid metabolism in cultured cells where free sphingoid base concentration increases and complex sphingolipids decrease markedly.