A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue.
A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue.
复制标题
血浆和组织中多巴胺、去甲肾上腺素和肾上腺素的灵敏放射性酶测定。
作者：
N. Ben;J. C. Porter
描述了用于测量一个样品中的多巴胺、去甲肾上腺素和肾上腺素的双同位素放射酶测定法。测定程序包括孵育、溶剂提取和薄层色谱法。将多巴胺、去甲肾上腺素和肾上腺素与儿茶酚-O-甲基转移酶（COMT）和[3 H] S-腺苷甲硫氨酸（[3 H]SAM）孵育，并分别转化为O-甲基化的氚化衍生物：[3 H]甲氧基酪胺、[3 H]去甲甲间胺和[3 H]间胺。经过几个提取步骤后，O-甲基化产物通过二维薄层色谱法使用硅胶进行纯化。薄层色谱系统导致三种O-甲基化化合物的完全分离，重叠仅为1- 2%。该测定是线性的，从0到5 ng的每一个儿茶酚胺，并有一个灵敏度为10-30 pg。大量的血浆中的加入降低了COMT的活性，但增加镁浓度的孵育混合物和EGTA的血浆样品中加入提高了回收率。通过使用孵育结束时加入的[14 C]甲氧基酪胺、[14 C]去甲间烷胺和[14 C]间烷胺，校正各样品在提取和色谱分析期间发生的损失。几种儿茶酚化合物被称为O-甲基化的COMT进行了检查的交叉反应性。在测试的物质中，只有二羟基苯丙氨酸（多巴）表现出交叉反应性。然而，DOPA与多巴胺的明显30%交叉反应性是由于COMT制剂中存在脱羧酶活性。断头大鼠的躯干血浆只要50穆尔就足以测定三种儿茶酚胺。
A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepinephrine, and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]S-acenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytyramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps the O-methylated products were purified by means of two-dimensional, thin-layer chromatography using silica gel. The thin-layer chromatographic system resulted complete separation of the three O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14Ci1metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 mul of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines.