High-performance liquid chromatographic radioenzymatic assay for plasma catecholamines

A new assay method for plasma CA's requiring only 50 microliter has been developed, which uses HPLC. The NE, D, and E compounds found in plasma are radioactively o-methylated with 3H-SAM by the reaction of COMT. The reaction is terminated and a standard mixture of nonradioactive o-methylated analogues of NE, D, and E is addded to act as a carrier. Following separation by HPLC, the NMN, 3-MOT, and MN 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.