BIOTINIDASE K-M-VARIANTS - DETECTION AND DETAILED BIOCHEMICAL INVESTIGATIONS

We describe a simple method for the detection of biotinidase K-m-varia nts and detailed biochemical investigations in 5 such patient. They we re detected among 103 patients with plasma biotinidase activity which ranged from undetectable to 30% of the mean normal value. Two differen t types of biotinidase K-m-variants were found. (1) In 3 infants bioti nidase had a single 105-430-fold elevated K-m for biocytin. Biotinidas e showed very low activities (0.2-4% of the mean normal value) in the routine colorimetric assay and was not functional in vivo. Accordingly , these patients presented with classical clinical illness. (2) In two patients biotinidase showed biphasic kinetics indicating the presence of one component with a normal K-m and reduced V-max (1.7% and 12%), and another with 330- and 59-fold elevated K-m, respectively. In these two patients, biotinidase proved to be at least partially functional in vivo. However, the first patient developed severe symptoms and biot in deficiency late, at the age of 10-15 years, and the second had marg inal biotin deficiency at the age of 2 years but no clinical symptoms. Comparative studies revealed that both patients had more severe bioti n deficiency than age-matched patients with similar levels of residual biotinidase activity and a single normal K-m. Therefore, all patients with residual biotinidase activity should be evaluated for the presen ce of a K-m-mutation, since such patients should be treated with bioti n. These can easily be detected by including a second substrate concen tration (1.5mmol/L) in the routine colorimetric biotinidase assay whic h is performed with 0.15mmol/L biotin. Increased activity with the hig her substrate concentration indicates the presence of a K-m-mutation. Detailed kinetic studies are needed to evaluate the distinct forms of K-m-variants.