フローインジェクション/固定化酵素分光光度法による尿中硫酸抱合胆汁酸の測定法の開発  [in Japanese] 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  [in Japanese]

Author(s)

    • 高 秀峰 GAO Xiu-Feng
    • 北華大学医学院医学検験部 Department of Medical Test, College of Medicine, University of Beihua
    • 李 永生 LI Yong-Sheng
    • 東北電力学院(大学)応用化学部 Department of Applied Chemistry, Northeast China Institute of Electrical Power Engineering
    • 軽部 征夫 KARUBE Isao
    • 東京大学先端科学技術研究センター Research Center for Advanced Science and Technology, University of Tokyo

Abstract

本論文は,新規酵素胆汁酸硫酸スルフェターゼ(BSS)とWST-5を用いて,迅速,簡便,高感度な硫酸抱合胆汁酸(SBA)のFIA/分光光度分析法を開発した.測定原理は次のとおりである.SBAはBSSにより脱硫酸化し,3β-ヒドロキシステロイドが生成する.この3β-ヒドロキシステロイドが3β-ヒドロキシステロイドデヒドロゲナーゼ(3β-Hydroxysteroid dehydrogenase: 3β-HSD)の触媒作用下でNAD<sup>+</sup>と反応し,NADHを生成する.次にジアホラーゼによりWST-5がNADHと反応して水溶性のホルマザンブルーを生成する.この生成物は,550 nmに吸収極大があるので,その吸光度によってSBAを定量した.本法は分析速度が15検体/h,相対標準偏差は1~5.4% であり,測定の範囲は1~75 μMであった.<br>

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 3β-hydroxyl bile acids; the produced 3β-hydroxyl bile acid reacts with nicotinamide adenine dinucleotide (NAD<sup>+</sup>) under the catalysis of co-immobilized 3β-hydroxylsteroid dehydrogenase (3β-HSD), and is converted to the 3-ketosteroid. Meanwhile, β-NAD<sup>+</sup> 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∼75 μM 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∼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.<br>

Journal

  • Bunseki kagaku

    Bunseki kagaku 52(3), 195-200, 2003-03-05

    The Japan Society for Analytical Chemistry

References:  8

Codes

  • NII Article ID (NAID)
    110002905204
  • NII NACSIS-CAT ID (NCID)
    AN00222633
  • Text Lang
    JPN
  • Article Type
    ART
  • ISSN
    05251931
  • NDL Article ID
    6491110
  • NDL Source Classification
    ZP4(科学技術--化学・化学工業--分析化学)
  • NDL Call No.
    Z17-9
  • Data Source
    CJP  NDL  NII-ELS  J-STAGE  NDL-Digital 
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