Biotinidase Km-variants: detection and detailed biochemical investigations.

We describe a simple method for the detection of biotinidaseK m-variants and detailed biochemical investigations in 5 such patient. They were detected among 103 patients with plasma biotinidase activity which ranged from undetectable to 30% of the mean normal value. Two different types of biotinidaseK m-variants were found. (1) In 3 infants biotinidase had a single 105–430-fold elevatedK m for biocytin. Biotinidase showed very low activities (0.2–4% of the mean normal value) in the routine colorimetric assay and was not functionalin 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 normalK m and reducedV max (1.7% and 12%), and another with 330- and 59-fold elevatedK m, respectively. In these two patients, biotinidase proved to be at least partially functionalin vivo. However, the first patient developed severe symptoms and biotin deficiency late, at the age of 10–15 years, and the second had marginal biotin deficiency at the age of 2 years but no clinical symptoms. Comparative studies revealed that both patients had more severe biotin deficiency than age-matched patients with similar levels of residual biotinidase activity and a single normalK m. Therefore, all patients with residual biotinidase activity should be evaluated for the presence of aK m-mutation, since such patients should be treated with biotin. These can easily be detected by including a second substrate concentration (1.5 mmol/L) in the routine colorimetric biotinidase assay which is performed with 0.15 mmol/L biotin. Increased activity with the higher substrate concentration indicates the presence of aK m-mutation. Detailed kinetic studies are needed to evaluate the distinct forms ofK m-variants.