Estimating sphingolipid metabolism and trafficking in cultured cells using radiolabeled compounds

The use of radioactive metabolites, particularly radiolabeled complex sphingolipids, represents an experimental approach to investigating different aspects of sphingolipid metabolism. The rationale of this approach is based on the fact that when complex sphingolipids—sphingomyelin and gangliosides—are added to cultured cells, they are partly taken up and inserted into the outer plasma membrane leaflet (where most cell sphingolipids are located); they are then processed metabolically through the intracellular traffic and metabolizing pathways of the corresponding endogenous components. Assessing the metabolic products formed by cells from an exogenously administered sphingolipid can be instrumental in (1) recognizing the metabolic processing and intracellular trafficking of a specific sphingolipid, (2) ascertaining cell specificity in metabolic processes, and (3) establishing the subcellular aspects of metabolic events involved in sphingolipid processing. It can also be useful in investigating the role of sphingolipids in signaling processes and evaluating the effects exerted by different molecules on sphingolipid metabolism. All this information can be gained using appropriately radiolabeled complex sphingolipids, adequate pulse-chase conditions, and carefully separating, identifying, and quantifying the radiolabeled metabolites. This chapter describes the experimental procedures useful for a global investigation of radiolabeled sphingolipid metabolism in cultured cells and then the specific protocols suitable for studying specific pathways involved in sphingolipid metabolism and intracellular sphingolipid trafficking.