Demonstration of glucose-6-phosphate hydrogen 5 enrichment from deuterated water by transaldolase-mediated exchange alone

Abstract

Purpose Enrichment of glucose position 5 (H5) from deuterated water (2H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from 2H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent. Methods Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-13C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% 2H2O. G6P 2H-enrichment and 13C-isotopomer distributions were analyzed by 2H and 13C NMR following derivatization to monoacetone glucose. Results 2H NMR analysis revealed high 2H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. 13C NMR isotopomer analysis revealed that [U-13C]G6P was converted to [1,2,3-13C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-13C2]G6P and [3-13C]G6P, predicted products of combined transaldolase and transketolase exchanges. Conclusion Hydrogen 5 of G6P was enriched from 2H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by 2H2O independently of gluconeogenesis.