A RAPID METHOD FOR QUANTIFYING FREE SPHINGOID BASES AND COMPLEX SPHINGOLIPIDS IN MICROGRAM AMOUNTS OF CELLS FOLLOWING EXPOSURE TO FUMONISINB-1

Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyl transferase (ceramide synthase), key enzymes in sphingolipid metabolis m. 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 devel oped rapid methods were a modification of an earlier 'original' method and used a single CHCl3 extraction subsequent to base or acid hydroly sis 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-PK1 c ells (approx. 100 mu g protein), respectively, while the recovery usin g the original extraction method was 1% or less. The average total sph ingolipid concentrations (free sphingoid bases plus complex sphingolip ids) determined by the rapid and original method were similar. The rap id extraction method provided simpler and shorter extractions with imp roved recovery, and was more economical by allowing experimental desig ns using smaller quantities of cells (10-100 mu g protein) and fumonis ins. In conclusion, the rapid method is useful for the study of fumoni sin-induced disruption of sphingolipid metabolism in cultured cells wh ere free sphingoid base concentration increases and complex sphingolip ids decrease markedly.