采用高效液相色谱-荧光检测法测定大鼠血清和肾上腺髓质中的多巴胺β-羟化酶。

Assay for dopamine beta-hydroxylase in rat serum and adrenal medulla by high-performance liquid chromatography with fluorescence detection.

作者信息

Nohta H, Ohtsubo K, Zaitsu K, Ohkura Y

出版信息

J Chromatogr. 1982 Feb 12;227(2):415-22. doi: 10.1016/s0378-4347(00)80394-1.

Abstract

A highly sensitive method for the assay of dopamine beta-hydroxylase in rat serum and in sample solution prepared from rat adrenal medulla is described which employs high-performance liquid chromatography with fluorescence detection. Octopamine, formed enzymatically from the substrate tyramine, is separated by Dowex 50/-X4 column chromatography and oxidized with periodate to p-hydroxybenzaldehyde, which is then converted into a fluorescent compound with 2,2'-dithiobis(1-aminonaphthalene). The derivative, after extraction with n-hexane--chloroform, is separated by normal-phase chromatography on Alox T. The limit of detection for octopamine formed enzymatically is 10 pmol. This method requires as little as 5 microliter of rat serum.

摘要

描述了一种用于测定大鼠血清和大鼠肾上腺髓质制备的样品溶液中多巴胺β-羟化酶的高灵敏度方法，该方法采用带荧光检测的高效液相色谱法。由底物酪胺酶促形成的去甲伪麻黄碱通过Dowex 50/-X4柱色谱分离，并用高碘酸盐氧化为对羟基苯甲醛，然后用2,2'-二硫代双（1-氨基萘）转化为荧光化合物。用正己烷 - 氯仿萃取后，衍生物在Alox T上通过正相色谱分离。酶促形成的去甲伪麻黄碱的检测限为10皮摩尔。该方法仅需5微升大鼠血清。