A procedure for the isolation of noradrenaline (together with adrenaline) , dopamine, 5-hydroxytryptamine and histamine from the same tissue sample using a single column of strongly acidic cation exchange resin

Atack, C.; Magnusson, T.

Acta Pharmacologica et Toxicologica 42(1): 35-57


ISSN/ISBN: 0001-6683
PMID: 579711
Accession: 004611721

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Using a single column of a strongly acidic cation exchange resin (Dowex 50W, X-4, 200-400 mesh, resin bed 4.0 mm diameter .times. 75 mm long in sodium form), noradrenaline (NA) ), dopamine (DA), 5-hydroxytryptamine (5-HT), and histamine (Hm), were recovered in separate eluates after a single extraction of the same tissue sample into perchloric acid. Recoveries of the amines from homogenates and filtrates were approximately the same: for NA (together with A), about 90% in a 7 ml eluate; for DA, about 95% in 3.5 ml; for 5-HT, about 75% in 7 ml; and for Hm, about 85% in 4.5 ml. The establishment and routine use of the procedure were presented in detail. Several metabolites of the amines, including 5-hydroxyindole-3-acetic acid (5-HIAA), the precursor amino acids and 3-methoxytyramine (3-MT), could be recovered from the same Dowex 50 column by incorporating the procedures developed by Carlsson, Kehr and Lindqvist in this laboratory. All the amines and many of their metabolites can be purified successively into their eluates in a normal working day. For catecholamines these compounds could be monitored: tyrosine .fwdarw. dopa .fwdarw. DA .fwdarw. NA .fwdarw. A and also DA .fwdarw. 3-MT; and for 5-HT, the metabolic pathway tryptophan .fwdarw. 5-hydroxytryptophan .fwdarw. 5-HT .fwdarw. 5-HIAA. The column purification contributed considerable specificity to the overall procedure. Most compounds which are acidic; e.g., 5-HIAA, neutral or amphoteric, e.g., all the precursor amino acids, were eluted before the amines. Considerable resolution of basic compounds into separate fractions was also achieved: Hm was eluted before spermidine, and DA was separated from NA plus A. Many compounds were prevented from interfering in fluorimetric assays especially adapted for measuring these amines in the Dowex 50 eluates. Salt equivalent to at least 10 g of tissue placed on the column did not affect the recoveries of the amines. For quantitative determinations of endogenous amines, approximately 10 ng of each amine on the Dowex 50 column were sufficient, and smaller amounts could be detected. The procedure was developed particularly for measuring amines in nervous tissue, but it has been applied successfully to several peripheral tissues and body fluids.