Rapid assay for one-run determination of purine and pyrimidine nucleotide contents in neocortical slices and cell cultures

The present study describes the measurement of endogenous nucleoside di- and triphosphate contents (ATP, GTP, UTP, CTP, ADP, GDP and UDP) in rat neocortical brain slices and mixed neuronal/astrocytic corticoencephalic cultures. Determination was by means of anion-exchange HPLC using a binary gradient of 0.3 M ammonium carbonate and water. In addition, a new method is described for the identification of nucleoside triphosphates, using digestion of the nucleotides by phosphoglycerate kinase and partial splitting of nucleoside diphosphates to shift the equilibrium of the phosphoglycerate kinase reaction in direction of breakdown of nucleoside triphosphates. Finally, the determination of the sum of creatine and creatine phosphate is suggested as an alternative reference value instead of protein under conditions when cells are cultured in protein-containing medium. (C) 1999 Elsevier Science B.V. All rights reserved.