TY - JOUR
T1 - 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
AU - Hamase, Kenji
AU - Nagayasu, Reiko
AU - Morikawa, Akiko
AU - Konno, Ryuichi
AU - Zaitsu, Kiyoshi
N1 - Funding Information:
The authors gratefully acknowledge Professor Wolfgang Lindner (University of Vienna, Austria) for his valuable help in the preparation of d -FTP, and Professor Kihachiro Horiike (Shiga University of Medical Science, Japan) for his advice concerning the DAO reaction. This work was supported in part by the grant in aid from Ministry of Education, Culture, Sports, Science and Technology of Japan, and TAKEDA SCIENCE FOUNDATION.
PY - 2006/2/17
Y1 - 2006/2/17
N2 - 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.
AB - 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.
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U2 - 10.1016/j.chroma.2005.08.043
DO - 10.1016/j.chroma.2005.08.043
M3 - Article
C2 - 16443460
AN - SCOPUS:31344450586
SN - 0021-9673
VL - 1106
SP - 159
EP - 164
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1-2
ER -