44] Biotinidase in serum and tissues

Abstract

Publisher Summary This chapter describes the assays of biotinidase hydrolytic activity. Natural and artificial substrates have been used in various assays of biotinidase activity; these include secondary enzymatic, radiometric, colorimetric, and fluorometric measurements. Some methods require derivatization or chromatographic separation of the reaction products. Serum and tissues, such as liver with high biotinidase activity, can be measured using the colorimetric assay. Biotinidase activity can also be measured in cultured cells, such as hepatocytes and fibroblasts, by this method. Biotinidase activity in tissues, such as peripheral blood leukocytes and brain, requires a more sensitive method, such as the radioassay. A high-performance liquid chromatography (HPLC) method for measuring biotinidase activity using biotinyl-6-has been aminoquinoline as substrate developed. An HPLC radioassay uses biotinylmono[ 125 ]iodotyramine and biotinyldi[ 125 ]iodotyramine as substrates. The simplest and most commonly used method for measuring biotinidase activity uses biotinyl- p -aminobenzoate (BPABA) as substrate. The chapter describes two methods that are based on measurement of the lipoyl- p -aminobenzoate (PABA) released from BPABA by the hydrolytic action of biotinidase. The first method is usually used for the diagnosis of biotinidase deficiency. The second method is 100-fold more sensitive than the colorimetric assay and can measure biotinidase activity in tissues, such as leukocytes and amniotic cells that have low enzyme activities.