Hypothalamic catecholamine biosynthesis in vitro as measured by liquid chromatography and electrochemical detection

A procedure is described for monitoring the biosynthesis of hypothalamic catecholamines in vitro, using the incorporation of the labeled precursor, 3H-tyrosine, into norepinephrine (NE), L-DOPAS and dopamine (DA). The technique involves separation of amines by high performance liquid chromatography, quantiation by electrochemical detection (LCEC), and the collection and measurement of labeled eluant. Rat hypothalami incubated in vitro were found to rapidly synthesize labeled DA while L-DOPA and NE synthesis were less apparent. The monoamine oxidase inhibitor, iproniazid, reduced incorporation. The tyrosine hydroxylase inhibitor, alpha methyl para-tyrosine (alpha mpt), virtually abolished DA synthesis while not appreciably affecting tissue content. The advantages of this technique over others is that it allows for the concurrent quantitation of tissue catecholamine levels, as well as their biosynthetic rate.