A novel assay for determination of sulfated bile acids in urine by use of flow-injection chemiluminescence principle with immobilized enzymes

Abstract

Multistep enzymatic reactions take place when sulfated bile acids (SBAs) pass through an immobilized enzyme reactor. First, SBAs take place in the reaction of desulfation under catalytical action of a bile salt sulfatase immobilized in the reactor and formed 3β-hydroxyl bile acids. Formed 3β-hydroxyl bile acids react with nicotinamide adenine dinucleotide (β-NAD+) under catalysis of another enzyme of 3β-hydroxysteroid dehydrogenase coimmobilized in the column and are converted to 3-ketosteroid. At the same time, β-NAD+ is changed into reduced nicotinamide adenine dinucleotide (NADH). Looking as if chain reaction, 1-MPMS taken as an electron mediator reacts immediately with NADH coexisting in the carrier solution and is turned into 1-MPMSH2. Formed 1-MPMSH2 again reacts with dissolved oxygen existing in the carrier solution and produces hydrogen peroxide. Last, the hydrogen peroxide reacts with the luminol reagent and gives out light in the presence of POD. Consequently, SBA can be determined by the luminous intensity. Based on the above-mentioned principle, applying the technology of flow-injection analysis, we established an accurate, simple, less time-consuming and sensitive approach for the determination of SBAs. According to the FIA manifold and experimental results optimized in the work, a new type of analytical instrument for the clinical assay of SBAs in urine or blood can be developed. We also think this method is very useful for routine analysis in clinical laboratory and long-period monitoring to patients with acute hepatitis, liver cirrhosis, and intra- and extrahepatic biliary obstruction of the urinary tract. Sampling frequency of the method is 30 samples/h, and its relative standard deviation is smaller than 2.2%. It can be used to determine SBAs in the ranges of 0.1–2.5 or 2–25 μM. © 1997 John Wiley & Sons, Inc. Lab Robotics and Automation 9: 69–79, 1997.