Abstract: A method for synthesizing a 2-alkanoylamino-4-nitrophenyl phosphorylcholine-hydroxide useful as a sphingomyelinase-specific chromogenic artificial substrate in the diagnostic testing for Niemann-Pick disease. The method comprises reacting 2-amino-4-nitrophenol with an alkanoyl halide to form the alkananilide; phosphorylating the alkali phenolate salt of the alkananilide with .beta.-bromoethylphosphoryl dichloride to form a mixture of mono- and di-alkananilide phosphoric acid esters; recovering from the mixture the mono-alkananilide phosphoric acid ester and quaternizing it with trimethylamine; and treating the resulting quaternary salt with a mixture of weak acidic and weak basic ion exchange resins to convert it into the 2-alkanoylamino-4-nitrophenyl phosphoryl-choline-hydroxide.

Abstract: An artificial substrate consisting of a 2-alkanoylamino-4-nitrophenyl phosphorylcholine hydroxide is used for determining the sphingomyelinase activity in an extract of human cells or tissues by incubating the substrate together with the extract, whereby sphingomyelinase-catalyzed hydrolysis of the substrate into the corresponding 2-alkanoylamino-4-nitrophenol and phosphorylcholine takes place in an amount proportional to the sphingomyelinase activity in the extract. Alkalinization of the 2-alkanoylamino-4-nitrophenol thereby produced to convert it into the alkali salt results in the development of a bright yellow color whose intensity is proportional to the amount of the substrate that has been hydrolyzed. Measurement of the intensity of the yellow color, for example, by means of a simple colorimeter or photometer, provides an accurate indication of the sphingomyelinase activity in the extract.