Abstract

Biotinidase (EC 3.5.1.12) is required for the recycling of biotin and for the utilization of protein bound biotin from the diet. Biotinidase deficiency (MIM 253260) is inherited as an autosomal recessively trait. Patients become progressively biotin deficient which results in reduced activity of the 4 biotin-dependent carboxylases existing in man, and severe life-threatening illness. Oral biotin substitution effectively protects against disease or reverses symptoms. Delayed treatment may result in irreversible neurological damage. Time of onset and severity of illness depend on the level of residual enzyme activity necessitating early (preferably neonatal) assessment of biotinidase activity. Patients are classified as having profound (0-10% residual activity) or partial (residual activity >10-30%) deficiency, or a Km defect due to reduced affinity of biotinidase for its substrate biocytin. Heterozygous individuals show activities intermediate between the deficient and normal activity. The natural substrate of biotinidase is biocytin but it can also act on artificial biotinyl-derivatives. Biotinidase activity in plasma is usually assayed using biotinyl-p-aminobenzoic acid (biotinyl-PABA) as substrate. Liberated PABA is converted to a purple azo dye and quantitated spectrophotometrically. This simple, reproducible and easy to perform colorimetric assay for the diagnosis of patients with different forms of biotinidase deficiency, including those with a Km defect, is described.