NAD^+と固定化3α-およびβ-ヒドロキシステロイド脱水素酵素を用いる高速液体クロマトグラフィーによる硫酸抱合型胆汁酸分画の新測定法

概要

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We present a new analytical method for sulfated individual bile acids in serum by high-performance liquid chromatography, immobilized 3α- and β-hydroxysteroid dehydrogenase in a column form and NAD^+ as a coenzyme, using enzymatic hydrolysis by sulfatase The principle of the enzyme reaction is as follows: 3α(β)-sulfated bile acid+[numberical formula]-hydroxy bile acid +SO_4^<2->+H^+ 3α(β)-hydroxy bile acid+[numberical formula]-keto bile acid+NADH NADH produced was determined fluorometrically. The optimum conditions for the enzymatic hydrolysis was an incubation for 48 hours at 37℃ with 0.5ml of 0.2M acetate buffer (pH5.0), 0.4ml of 25×10^<-7>M D-saccharic acid-1,4-lactone solution, and 0.1ml of enzyme solution (32units/ml sulfatase in 0.2% NaCl). It is considered that the new analytical method for sulfated individual bile acids, which is much easier than solvolysis, is capable of giving reliable and reproducible results, and can be applied clinically to patients with hepatobiliary diseases for the study of the metabolism of sulfated bile acids.
日本ビタミン学会の論文
1986-04-25