Synthesis of pyrenesulfonylamido-sphingomyelin and its use as substrate for determining sphingomyelinase activity and diagnosing Niemann-Pick disease.

A new fluorescent derivative of sphingomyelin (PSA12-sphingomyelin) containing a pyrene-sulfonylamide residue was synthesized by covalently linking 12-((1-pyrenesulfonyl)amido)-dodecanoic acid (PSA12) to sphingosylphosphorylcholine. It was used as substrate for acidic and neutral human and murine sphingomyelinases permitting development of sensitive assays for these enzymatic activities. The product of the sphingomyelinase assay, 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-sphingomyelin 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. Use of PSA12-sphingomyelin permitted determination of sphingomyelinase activity in cell extracts (eg human blood lymphocytes, lymphoid cell lines or cultured skin fibroblasts) as well as in hair follicles and urine. This new fluorescent derivative of sphingomyelin also permitted the detection of acid sphingomyelinase deficiency in cells derived from patients with Niemann-Pick disease.