A Rapid and Simple Determination of Histamine and Polyamines

Abstract

The authors have already reported a method to separate catecholamines, serotonin, histamine and polyamines by a single P-cellulose column chromatography (1, 2). As the adsorption of histamine and polyamines to the column is significantly strong, the weak acidic cation exchanger, CM-cellulose, was utilized to separate these amines and the method was successfully applied to rat tissues.CM-Cellulose (capacity: 0.70 meq/g) was obtained from Brown Co., U.S.A. Phosphate buffer (pH 6.2 and 7.5) was prepared from 0.1 M NaH2PO4 and 0.1 M Na2HPO4. Borate buffer (pH 9.0) was prepared from 0.2 M H3B03 (containing 0.2 M KCI) and 0.2 M Na2CO3. and each tissue was homogenized in 5 volumes of ice-cold 0.4 N HCIO4. The homogenate was centrifuged at 3000 rpm for 5 min. The supernatant was neutralized to pH 5-6 with 2 N KOH through a microtube with stirring in an ice bath, and the precipitate was removed by centrifugation (3000 rpm, 5 min). The neutralized tissue extract (1-4 nil) was applied to a CM-cellulose column (0.6 x 10 cm) equilibrated with buffer-l. The elution was per formed at room temperature (20-25'C) by increasing stepwise both concentration and pH of the buffer (buffer-l to 6). Histamine and polyamines were determined by OPT-reaction and TNBS-reaction, respectively, as described previously (1).The standard substances showed the following separation in the CM-cellulose column chromatography.Buffer-1 Fraction (15 ml): tryptophan, tyrosine, and dopa, Buffer-2 Fraction (15 ml): adrenaline, noradrenaline, dopamine, tyramine, metanephrine, nor metanephrine, tryptamine, serotonin, lysine, histidine and arginine, Buffer-3 Fraction The substances interfering with the fluorometric determination of histamine such as arginine, agmatine and spermidine (3) were removed from the histamine fraction. In the determi nation of polyamine only, tissue extract was applied to a column equilibrated with 0.02 M