A method for the estimation of the catecholamines and their metabolites in brain tissue

Abstract

1 A method is described whereby the catecholamines, their precursors and metabolites may be estimated in the same brain sample. The method, outlined below, was rigorously tested in pure solution before being applied to the estimation of the catechol and methoxyamines in extracts of rat brain. (i) The brain tissue was homogenized in perchloric acid and the perchloric acid removed as its insoluble potassium salt. (ii) The acid metabolites of the amines were extracted from the acidified extract into ethyl acetate. (iii) After extraction with ethyl acetate, the aqueous extract was treated with acetic anhydride and sodium hydrogen carbonate and the acetylated derivatives of the amines extracted into dichloromethane. (iv) The aqueous solution remaining after extraction with dichloromethane contains the acetylated amino-acids. Acetyl-dopa could be extracted into dichloromethane only after acidification of this aqueous solution. The acetyl-dopa in this dichloromethane extract could be estimated fluorimetrically. (v) The dichloromethane extract containing the acetylated amines was chromatographed on paper in the organic phase of a mixture of toluene: ethyl acetate: methanol: water, 10: 1: 5: 5. (vi) The acetylated amines were eluted and estimated fluorimetrically. A new method was developed for the estimation of 3-methoxytyramine and its acetylated derivative. 2 The reproducibility of the method was demonstrated by the consistent estimates obtained for the concentrations of noradrenaline, dopamine and 3-methoxytyramine in whole rat brain. Adrenaline and metanephrine were not detected. The concentration of normetanephrine in rat brain was at the limit of detection of the method. 3 Although the estimation for the levels of endogenous amines in whole rat brain were consistent, the recoveries of exogenous amines taken through the procedure were low and very variable. Control experiments demonstrated that the exogenous amines were lost at the perchloric acid extraction stage of the procedure.