Analysis of sphingolipid synthesis and transport by metabolic labeling of cultured cells with [³H]serine

Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM biosynthesis in cultured cells based on [³H]serine incorporation at the first step in the pathway. The method can be used to assay the effect of pharmacological and genetic manipulations on ceramide synthesis and transport to the Golgi apparatus.