BiotinidaseK _m-variants: detection and detailed biochemical investigations

Summary

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.