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Determination of Sulfate-Conjugated 3β-Hydroxy Bile Acid in Urine by HPLC with Postcolumn Fluorescence Detection Using an Immobilized β-Hydroxysteroid Dehydrogenase Column

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Abstract

A highly sensitive and specific method was established for quantification of 3β-hydroxy-5-cholenoic acid by high-performance liquid chromatography using an immobilized β-hydroxysteroid dehydrogenase column (β-HSD column) and a postcolumn fluorescent detection. The β-HSD column shows maximum enzyme activity and stability at 20° C, and has a very short life at 25°C. The detection limit was 7 pmol/injection. We also developed an efficient method for extracting the sulfate conjugates at position 3 from urine and conducting their enzymatic hydrolyses. This method was used to analyze urine from patients with congenital biliary atresia. A high concentration of 3β,12α-dihydroxy-5-cholenoic acid was also present in the urine from these patients.

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Authors and Affiliations

  1. Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima, Osaka, 553, Japan

    Kazuo Kuruma

  2. Diagnostic Science Department, Shionogi & Co., Ltd., Settsu, Osaka, 566, Japan

    Yasuhide Morioka, Kazuyuki Sasakura, Hirotune Igimi & Kiyohisa Uchida

Authors
  1. Kazuo Kuruma
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  2. Yasuhide Morioka
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  3. Kazuyuki Sasakura
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  4. Hirotune Igimi
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  5. Kiyohisa Uchida
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Kuruma, K., Morioka, Y., Sasakura, K. et al. Determination of Sulfate-Conjugated 3β-Hydroxy Bile Acid in Urine by HPLC with Postcolumn Fluorescence Detection Using an Immobilized β-Hydroxysteroid Dehydrogenase Column. ANAL. SCI. 12, 847–851 (1996). https://doi.org/10.2116/analsci.12.847

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  • DOI: https://doi.org/10.2116/analsci.12.847

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