A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingo sine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease, This study reports a new as say method to detect acid ceramidase activity in vitro using Bodipy or liss amine rhodamine-conjuggate ceramide (C12 ceramide; dodecanoylsphingosine), Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[C -14]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substra te provided data very similar to those of the radioactive substrate, but un der the experimental conditions tested, it was significantly more sensitive , Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy do decanoic acid, could be detected, providing an accurate measure of acid cer amidase activity as low as 0.1 pmol/mg protein/h, Acid ceramidase activitie s in shin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, co nfirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatogr aphic technique is a reliable, rapid, and highly sensitive method to determ ine acid ceramidase activity, and that it could be useful wherever the in v itro detection of acid ceramidase activity is of importance. (C) 1999 Acade mic Press.