A method for obtaining ¹³C isotopomer populations in ¹³C-enriched glucose

¹³C NMR analysis of ¹³C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting ¹³C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each ¹³C isotopomer by ¹³C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing ¹³C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-¹³C₃]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing ¹³C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.