Biochemical Pharmacology, Vol. 27, pp. 1519-1520. 0006-2952/78/0601-1~19/$02.00/0 © Pergamon Press Ltd. 1978. Printed in Great Britain. MODIFICATION OF THE RADIOENZYMAT1C ASSAY FOR THE CATECHOLAMINES IAN L. MARTIN and GLEN B. BAKER MRC Neuropharmacology Unit and SUSAN M. FLEETWOOD-WALKER Department of Physiology, Medical School, Birmingham B15 2TJ, England (Received 26 October 1977; accepted 20 December 1977) Abstract--There have been several reports describing the use of catechoI-O-methyl transferase (COMT; EC 2.1. I. 1) together with radioactively labelled S-adenosyl methionine (SAM) for the quantification of the catecholamines in biological material[I-4]. The procedure involves the enzymatic transfer of the radioactively labelled methyl group of SAM to the phenolic function at the 3-position of the catecholamine, the subsequent purification of the radioactively labelled product and its quantification by liquid scintillation counting. While Nikodijevik et al. [1] used a highly purified COMT preparation for the determination of nora- drenaline (NA) Cuello et al. [2] found this to be unnecessary in the quantification of dopamine (DA). The main disadvantage with the procedure of Cuello et al. [2] is the final stage in which purification of the radioactively labelled O-methylated products of the catecholamines is carried out by paper chromatography; we have found this to be both time consuming and also to result in loss of the amine derivatives. Fry and co-workers [3], subse- quently improved the method by converting the O-methylated amines to their acetyl derivatives prior to their separation by paper chromatography. In order to facilitate the more rapid analysis of these catecholamines by the radio-enzymatic pro- cedure we have modified the methods of Cuello [2] and Fry [3] to allow the separation and purification of the catecholamines using thin-layer chroma- tography. MATERIALS AND METHODS Water was redistilled in an all glass apparatus. All materials were Analar reagents (B.D.H. Chemicals Ltd., Poole, Dorset) except where stated and they were used without further purification. Acetic anhy- dride was redistiiled from potassium hydroxide, the fraction boiling between 139° and 140° was collected and stored in an all glass container at 4 ° until required. The partially purified COMT preparation was obtained from rat liver, basically according to the method of Axelrod and Tomchick[4]. The activity of the enzyme preparation was between 300 and 500 nmoles product/rag protein/hr as deter- mined by the method of McCaman [5]. S-Adenosyl [3H]Me L-methionine (specific radio- activity about 8 Ci/mmole) was obtained from New England Nuclear, West Germany. LQD silica plates were obtained from Scientific Industries Ltd., 1519 Loughborough, Leicestershire. The solvent system was prepared by taking 30 ml of the upper organic phase from a mixture of toluene-methanol-water- ethyl acetate in the ratios 10:5:5:4, and adding to it 4 ml ethyl acetate, 2 ml methanol and 6.5 ml butan-2-one; the tank was also saturated with the aqueous phase from the first system by placing a beaker of this aqueous phase in the tank. Tissue samples were homogenised in 0.1 M perchloric acid containing 10 mg per cent disodium EDTA using an all glass homogeniser to give 10 per cent homo- genates and the samples were centrifuged in a Beckman microfuge (type B) at room temperature for 90 sec. The incubation mixture for the catecholamine assay was made up just before use and consisted of 50/zl 20 mM EGTA (adjusted to pH 7.2), 250/~1 rat liver COMT preparation in 0.5 mM sodium phos- phate buffer pH 7.0 (20 mg protein/ml), 150 #1 ['~H]Me SAM (250/zCi/ml), 50/zl of a solution con- taining 16 mg/ml of pargyline in water containing 10% (v/v) 2-mercaptoethanol, 325/zl 1 M Tris base containing 3 mM magnesium chloride adjusted to pH 10.4. To 25/zl of this incubation mixture in glass tubes (50 × 10 ram) on ice was added either 10/zl 0. I M perchloric acid containing 10 mg per cent disodium EDTA as blanks, various concentrations of the cate- cholamines in 10 #1 0.1 M perchloric acid to provide the standard curve (normally 1 to 5 ng) or 10/zl of the tissue sample supernatant. In addition 1 ng of catecholamine standards in 2 ~1 of water were added to certain tissue samples to provide internal stan- dards. All determinations were performed in dupli- cate. The contents of the tubes were mixed care- fully and incubated at 37° for 40 min. The tubes were then removed and cooled to 0 ° and to each was added 30 #1 of a mixture of 5 vols of 0.5 M borate buffer pH 10.0 and 1 volume of carrier methoxyamine mix containing 3-methoxytyramine,