Development of a determination method for sulfated bile acids in urine

Abstract

This dissertation gives a brief description of two new approachs for the assay of sulfated bile acid(SBA)in urine based on flow-injection chemiluminescence and spectrophotometry with immobilized enzyme reactors.The analytical procedure of FIA-chemiluminescence is as follows.SBAs take place in the reaction of desulphation under a catalytical action of a bile salt sulfatase immobilized in the column and formed 3β-hydroxyl bile acids;the formed 3β-hydroxyl bile acids react with nicotinamide adenine dinucleotide(β-NAD+)under the catalysis of 3β-hydroxysteroid dehydrogenase coimmobilized in the same column, and are converted to 3-ketosteroid.At the same time, β-NAD+ is changed into reduced nicotinamide adenine dinucleotide(NADH).Looking like a chain reaction, 1-MPMS is taken as an electron mediator and reacts immediately with NADH coexisting in the carrier solution, and is turned into 1-MPMSH2;the 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.SBA can be determined by the luminous intensity.Based on this principle, an approach was established for the determination of SBAs.Its sampling frequency is 30 sample/h, and the relative standard deviation(RSD)is smaller than 2.2%.It can be used to determine SBAs in the range of 0.1∼12μM.Flow-injection spectrophotometry with immobilized enzyme reactors is based on a part of the principle mentioned above, namely, after β-NAD+ is converted to NADH, by catalysis of diaphorase immobilized, NADH reacts with a novel colouring reagent, called Water Soluble Tetrazolium Blue(WST-5), to generate a blue diformazan dye.Last, SBA is determined from the absorbance of the formed formazan(λmax=550nm).By using the manifold and optimized conditions, the author has obtained the linear response in the range of 1∼75μM GLCA-S with a correlation coefficient of 0.999 and an analytical rate of 15 sample/h.RSD was less than 1%.The recoveries(91∼108%)of GLCA-S added into urine were satisfactory and the assay had a good correlativity with manual the UBASTEC method.As a consequence, based on the method, a new-type of analytical instrument for the assay of SBAs in urine has been developed.The author also thinks that this method is very useful for routine analyses in a clinic laboratory and long-period monitoring to patients with acute hepatitis, liver cirrhosis, and intra-and extra-hepatic biliary obstruction urinary.