HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC RADIOENZYMATIC ASSAY FOR PLASMA CATECHOLAMINES

Abstract

A new assay method for plasma [human] CA [catecholamine] requiring only 50 .mu.l was developed, which uses HPLC [high-performance liquid chromatography]. The NE [norepinephrine], D [dopamine] and E [epinephrine] compounds found in plasma are radioactively o-methylated with 3H-SAM [S-methyl-adenosyl-L-methionine] by the reaction of COMT [catechol-O-methyl transferase]. The reaction is terminated and a standard mixture of nonradioactive o-methylated analogs of NE, D and E is added to act as a carrier. Following separation by HPLC, the NMN [D,L-normetanephrine], 3-MOT [3-methoxytyramine] and MN [metanephrine] radioactive peaks are collected which represent NE, D and E, respectively. Then MNM and MN are oxidized to vanillin, and 3-MOT is acetylated. The products are subsequently separated by solvent extraction. This is necessary in order to avoid high radioactive blanks and to allow quantitation of the radioactivity by liquid scintillation spectrometry. The mean supine levels of NE, D and E in normal subjects were, respectively, 182, 33 and 87 pg/ml of plasma. Similar assays on patients with pheochromocytoma revealed 797, 80 and 470 pg/ml.