Trocewicz J, Oka K, Nagatsu T
J Chromatogr. 1982 Feb 12;227(2):407-13. doi: 10.1016/s0378-4347(00)80393-x.
This paper describes a new and highly sensitive assay for phenylethanolamine N-methyltransferase (PNMT) activity with noradrenaline as substrate in various rat brain regions by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% of contaminating adrenaline, which was removed to reduce the blank value. Enzymatically formed adrenaline was adsorbed on an aluminum oxide column, eluted with 0.5 M hydrochloric acid, separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. 3,4-Dihydroxybenzylamine was added to the incubation mixture as an internal standard after the reaction. This assay was very sensitive and 0.5 pmol of adrenaline formed enzymatically could be detected. This assay method was applied to measure PNMT activity in various rat brain regions. The highest activity was observed in the hypothalamus, pons plus medulla oblongata, septum, lower brain stem, and cerebral cortex; the lowest activity was in the striatum, hippocampus, cerebellum, and limbic brain.
本文描述了一种通过高效液相色谱电化学检测法，以去甲肾上腺素为底物，测定大鼠不同脑区苯乙醇胺N-甲基转移酶（PNMT）活性的新型高灵敏度检测方法。市售的去甲肾上腺素含有约0.27%的肾上腺素污染物，将其去除以降低空白值。酶促生成的肾上腺素吸附在氧化铝柱上，用0.5 M盐酸洗脱，通过高效反相离子对色谱法分离，并用电化学检测法测定。反应后向孵育混合物中加入3,4-二羟基苄胺作为内标。该检测方法非常灵敏，能够检测到酶促生成的0.5 pmol肾上腺素。该检测方法被应用于测量大鼠不同脑区的PNMT活性。在下丘脑、脑桥加延髓、隔区、低位脑干和大脑皮层观察到最高活性；最低活性在纹状体、海马体、小脑和边缘脑区。
