Internal standard based absolute quantitation of glycolytic intermediates was performed to characterize the thermodynamic states of Saccharomyces cerevisiae metabolism. A mixture of C-13-labeled glycolytic intermediates was prepared via extraction from S. cerevisiae cells cultivated using a synthetic medium containing [U-C-13] glucose as the sole carbon source. The C-13-labeled metabolite mixture was used as an internal standard for the analysis of S. cerevisiae cultivated in a medium containing natural glucose. The methodology was employed for the absolute quantitation of glycolytic intermediates of BY4742, pfk1 Delta, and zwf1 Delta strains of S. cerevisiae. Fructose-1,6-bisphosphate was he most abundant intermediate in the BY4742 strains in the log phase of growth. Estimation of the Gibbs free energy change (Delta G) from the absolute concentration revealed that several reactions, such as those catalyzed by ribose-5-phosphate ketoisomerase and phosphoglucose isomerase, were commonly at near-equilibrium in all three strains. A significant shift in thermodynamic state was also observed for the transketolase-transaldolase reaction, for which Delta G was -6.6 +/- 0.5 kJ mol(-1) in the BY4742 strain and 5.4 +/- 0.3 kJ mol(-1) in the zwf1 Delta strain. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.