High-Performance Liquid Chromatographic Assay for Biotinidase Activity in the Human Urine

Abstract

To date, no detectable biotinidase activity has been reported in the human urine. In the present study, a simple and convenient HPLC-fluorimetric assay method was applied to biotinidase activity measurement of urine samples. Biotinyl-6-aminoquinoline was utilized as the enzyme substrate, and the liberated product (6-aminoquinoline) was monitored with a fluorimetric detector. Biotinidase activity was assessed in urine specimens from: twenty-five patients with various renal disorders associated with proteinuria, and forty age- and sex-matched healthy control subjects. The examined samples from the patients with renal diseases exhibited biotinidase activity with the exception of six of the patients (enzyme activity, median: 49.9 pmol · min⁻¹ · ml⁻¹ of urine; range: 0–2498 pmol · min⁻¹ · ml⁻¹); specific activity, median: 19.4 pmol · min⁻¹ · mg⁻¹ of protein; range: 0–176 pmol · min⁻¹ · mg⁻¹) while none of the urine specimens from the control subjects showed detectable enzyme activity. Thus, this method was shown to be a successfully applicable for the quantitative analysis of biotinidase activity in the human urine.