Determination of Sphingomyelinase Activity in Cells and Body Fluids Using a New Fluorescent Derivative Of Sphingomyelin: Application to Diagnosis of Niemann-Pick Disease

Abstract

A new fluorescent derivative of sphingomyelin (PSA12-SPM) containing a pyrene-sulfonylamide residue was synthesized by covalently linking 12-((l-pyrenesulfonyl)amido)-dodecanoic acid (PSA12) to sphingosylphosphorylcholine. PSA12-SPM was a substrate for human and murine sphingomyelinases and sensitive assays were developed for these enzymatic activities. The product, PSA12-ceramide, could be detected in picomole quantities due to a fluorescence intensity which was 10-35 fold greater than that of other fluorescent ceramides (such as pyrene or nitrobenzoxadiazole derivatives). PSA12-SPM could be used in pure form or admixed with natural sphingomyelin; in the latter case, the enzyme hydrolyzed the fluorescent and non-fluorescent species at equal rates. Reliable assays of PSA12-SPM hydrolysis permitted the determination of sphingomyelinase activity in cells (e.g., blood lymphocytes, lymphoid cell lines or cultured skin fibroblasts) as well as hair follicles and body fluids (e.g., urine and tears). The diagnostic use of this assay was demonstrated in the prenatal diagnosis of Niemann-Pick disease using chorionic villi samples and in detecting carriers of the mutant gene using skin fibroblasts or lymphocytes.***for insert abbr