Isolation of tryptophan, 5-HT, and 5HIAA from single brain areas using disposable columns packed with sephadex G-10...

Using disposable Bio-Rad Columns packed with Sephadex G-10 resin (7 x 40 mm resin column), tryptophan, 5HT, and 5HIAA can be isolated from single discrete brain areas. The preparation of Sephadex G-10 columns requires a minimum amount of time, and once prepared, the columns require very little maintenance. Since the procedures involve gel filtration and not affinity chromatography, equilibration of the resin to its appropriate ionic strength or pH is not required. Brain tissue weighing 25-200 mg is homogenized in 1 ml of 0.4 M HClO4. After centrifugation at 4800 g, the supernatant is decanted onto the columns. Washing and eluting involve twelve 1 ml applications of either a phosphate buffer or a carbonate-NH4OH solution. Tryptophan and 5HIAA are eluted with the third, fourth, and fifth washings while 5HT is eluted with the last five washings. Tryptophan, 5HT and 5HIAA are all assayed by fluorometric techniques. These methods provide recovery values, reproducibility, and sensitivity comparable with cation exchange and some solvent extract methods.