Direct SpectrophotometricDeterminationof a-Amylase Activityin Saliva, with p-Nitrophenyla-Maltoside as Substrate

We describe a simple, direct kinetic method for determination of salivary a-amylase (1,4 a-o-glucan 4-glucanohydrolase, EC 3.2.1.1). The assay makes use of a well-defined substrate, p-nitrophenyl a-maltoside, which is hydrolyzed by a-amylase to a chromogenic product, p-nitrophenol. Activity is determined by directly monitoring the increase in absorbance of the reaction mixture. Amylase activity can be defined in international (IUB) units of micromoles of product/mm per liter of saliva. For 22 healthy subjects, the mean ± SD of amylase activity in mixed saliva was 2.77 ± 1.12 U/liter. Activity and instrumental response were linearly related over the entire range tested (0.224 to 11.90 U/liter). The within-run precision (CV) over this range was better than 3% for all but the lowest activities. Values obtained with this assay correlate well with those obtained with a modified Nelson-Somogyi saccharogenic method (r = 0.979). The precision and simplicity of this assay suggest that it is the method of choice for determining amylase activity in human saliva.