A direct method for the simultaneous measurement of ceramide and phospholipase D activity

Both ceramide and phospholipase D (PLD) have important roles in a variety of signal transduction pathways. Recent evidence suggests that ceramide is a novel second messenger with specific biological effects. Publications in this field have increased rapidly in the last few years. However, a method to directly and rapidly measure cermide production has been lacking. Herein, we report on a novel, inexpensive, direct and rapid assay for the measurement of ceramide and the simultaneous measurement of PLD activity. This method uses labeling of cells with [(14)C]myristic acid and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. This method avoids the loss of radioactivity and variability due to changes in DAG kinase activity that are associated with the commonly-used DAG kinase assay.