Sensitive high-performance liquid chromatographic assay for D-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro-D-tryptophan

Kenji Hamase, Reiko Nagayasu, Akiko Morikawa, Ryuichi Konno, Kiyoshi Zaitsu

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

A sensitive assay for d-amino-acid oxidase (DAO) activity in mammalian tissues has been established. d-Tryptophan (d-Trp) analogs were tested as substrates for DAO, and 5-fluoro-d-tryptophan (d-FTP) was found to be the best substrate. By the enzymatic reaction, d-FTP was converted to 5-fluoroindole-3-acetic acid (FIAA), a highly fluorescent product, and the product was determined by an RP-HPLC system with a fluorescence detector. The detection limit for purified DAO (from hog kidney) was 0.25 μU, and the within-day and day-to-day precisions of the assays were 4.6% (RSD, n = 5), and 13.8% (RSD, 5 days), respectively. By the present method, the detailed distribution of DAO activity in the mouse brain was determined using individual animals for the first time, and significant activities were observed in the cerebellum, medulla oblongata and midbrain. Because sensitive DAO assay is frequently required in small tissues or in limited-tissue regions, the present method is useful for various research studies concerning DAO and the related d-amino acids.

Original languageEnglish
Pages (from-to)159-164
Number of pages6
JournalJournal of Chromatography A
Volume1106
Issue number1-2
DOIs
Publication statusPublished - Feb 17 2006

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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