Development of a rapid analytical method for the determination of sulfated bile acids in urine based on flow-injection spectrophotometry by using immobilized enzyme technology

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