Isolation and assay of noradrenaline, dopamine, 5-hydroxy-tryptamine, and several metabolites from brain tissue using disposable bio-rad columns packed with sephadex G-10

Abstract

Using disposable Bio-Rad columns packed with Sephadex G-10, the methods for isolation and determination of NA, DA, 5HT, 5HIAA, and GABA from the same brain tissue are described. Preparation and maintenance of the columns requires a minimum amount of time, and once prepared, the columns can be easily regenerated by washing the resin with 3 ml of 0.01 N NH 3 and 6 ml of 0.01 N HCl. Brain tissues are prepared by homogenizing in 1 ml of 0.4 N HCLO 4. After centrifugation, the supernatant is poured onto the resin. The columns can be placed directly on top of test tubes (15 × 150 mm) to allow collection of the eluate. GABA (96–98%) is removed in the first 2.2 ml of acid washed through the columns. NA and DA (100%) are contained in 2.8 ml of acid eluate. 5HT (94–98%) is washed from the column with 4 ml of an acid-ethanol solution. Finally, 5HIAA (100%) is removed with 2.0 ml of Na 2CO 3 solution. All determinations were made fluorimetrically. Also, a manual method, using Bio-Rad columns packed with G-10 resin, for isolating NA, DA, HVA, DOPAC and GABA from the same brain tissue is given as a modification of the semiautomated methods of Westernik and Korf (1976). The procedures for column preparation, maintenance, and regeneration are identical for both methods described in this paper. However, after homogenizing the brain tissue in HCLO 4, a KOH/HCOOH buffer is added to precipitate excess perchlorate ions. This procedure appears to destroy about 8–10% of the catecholamines and metabolites present in the homogenate. These methods provide recovery values, reproducibility, and sensitivity compatible with cation exchange and some solvent extraction methods.