Micromethod for the fluorimetric determination of plasma N-acetyl-alpha-D-galactosaminidase and study of some of its characteristics.

We have studied some characteristics of N-acetyl-alpha-D-galactosaminidase in human plasma using a sensitive and very simple fluorimetric (single tube incubation/fixation) micromethod. The enzyme has a pH optimum at 4.5, is linear on incubation during at least 6 h, is protected from inactivation at room temperature by acidification, and is stable on freezing (about 85% residual activity after 1 year at -20 degrees C). Enzyme kinetics indicate that the affinity for the substrate is low (K(m) value 7 mmol/l). By studying about 10 possible effectors, no activation was found by detergents. As expected, the colorigenic p-nitrophenyl derivative substrate, N-acetylgalactosamine and galactose are low-affinity inhibitors. A histogram of 108 control samples showed a unimodal distribution pattern with a slight bias to the right. No pseudodeficients were found on analysis of 220 control plasma samples. Patients with alpha-galactosaminidosis had residual activity between 0.7 and 2.1%. In patients with 17 different lysosomal storage diseases, no increase was found except in mucolipidosis II and III. The main advantages of the method are its simplicity sensitivity, short incubation time requirement and economy in substrate consumption. The method can be used either for screening or diagnostic purposes of genetic N-acetyl-alpha-D-galactosaminidase deficiency.