SINGLE-RUN SEPARATION AND DETECTION OF MULTIPLE METABOLIC INTERMEDIATES BY ANION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND APPLICATION TO CELL POOL EXTRACTS PREPARED FROM ESCHERICHIA-COLI

A method is described for analysis of the intracellular concentrations of metabolic intermediates of the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, the pentose phosphate pathway, and the tric arboxylic acid cycle in cell pool extracts of Escherichia coil. A sing le anion-exchange HPLC run of 40 min allowed resolution of 27 anionic metabolite standards. Detection limits of 0.1 nmol per injection were achieved by use of a conductivity detector equipped with an anion self -regenerating suppressor and a uv detector. A boiling water extraction procedure was used to prepare cell pool extracts. Cochromatography of cell pool extracts and metabolite standards was used to confirm the i dentities of metabolites in the cell pool, As many as 16 metabolites c ould be detected and quantified in the cell pool extracts by using the described HPLC method. An analysis of metabolite concentrations in E. coil showed the dynamics of glucose metabolism during a 8-min transit ion from starvation to steady-state metabolism following addition of g lucose. The ease and power of this method suggests general utility for in vivo metabolite analysis in a variety of experimental systems. (C) 1995 Academic Press, Inc.