Flow injection spectrophotometric determination of sulfated bile acids in urine with immobilized enzyme reactors using water soluble tetrazolium blue-5

A simple assay of sulfated bile acid (SBA) in urine using flow injection (FI)-spectrophotometry with immobilized enzyme reactors is proposed. The system consists of an injection valve, a switch valve, two immobilized enzyme reactors and a UV-VIS detector with a flow cell. The multi-step enzymatic reactions will occur when an injected sample containing SBA passes through the immobilized enzyme reactors. First, SBA will desulfate under catalysis of immobilized bile acid sulphate sulfatase (BSS), to form 3 beta -hydroxyl bile acids; the produced 3 beta -hydroxyl bile acid reacts with nicotinamide adenine dinucleotide (NAD(+)) under catalysis of co-immobilized 3 beta -hydroxylsteroid dehydrogenase (3 beta -HSD), and is converted to the 3-ketosteroid. Meanwhile, beta -NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). Then by catalysis of immobilized diaphorase, NADH reacts with a novel reagent called "water soluble tetrazolium blue-5" (WST-5) to generate a blue diformazan dye, which is detected at 550 nm. By using FI-spectrophotometry manifold and optimized conditions, we have obtained a linear response for 1-75 muM glycolithocholate sulfate (GLCA-S) with a correlation coefficient of 0.999 and an analytical rate of 15 samples per hour. The R.S.D. was less than 1%. The recoveries (91-108%) of GLCA-S added into urine were satisfactory and the assay correlated well with the manual UBASTEC method. Therefore, it will be applicable for urine tests on patients suffering from hepatobiliary disease. (C) 2001 Elsevier Science B.V. All rights reserved.