Assay for tryptophan hydroxylase activity in rat brain by high-performance liquid chromatography with fluorescence detection.
A highly sensitive and selective liquid chromatographic (HPLC) method with post-column fluorescence detection has been developed for the determination of tryptophan 5-monooxygenase activity in rat brain tissue homogenate. 5-Hydroxytryptophan, formed enzymatically from tryptophan (incubation time, 20 min), is extracted with perchloric acid and determined by HPLC. Detection is performed fluorometrically after post-column derivatization by a reaction with benzylamine in the presence of potassium hexacyanoferrate (III). The lower limit of detection for 5-hydroxytryptophan formed enzymatically is 100 fmol at a signal-to-noise ratio of three.
Iizuka
R
R
Chemical Biotesting Center, Chemical Inspection and Testing Institute, Oita, Japan.
Ishida
J
J
Yoshitake
T
T
Nakamura
M
M
Yamaguchi
M
M
D006916:5-Hydroxytryptophan / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D014365:Tryptophan Hydroxylase / Q000378:metabolism*
D006916:5-Hydroxytryptophan; D014365:Tryptophan Hydroxylase