Purpose: Enrichment of glucose position 5 (H5) from deuterated water ((2)H2O) 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 (2)H2O 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-(13)C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% (2)H2O. G6P (2)H-enrichment and (13)C-isotopomer distributions were analyzed by (2)H and (13)C NMR following derivatization to monoacetone glucose.
Results: (2)H NMR analysis revealed high (2)H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. (13)C NMR isotopomer analysis revealed that [U-(13)C]G6P was converted to [1,2,3-(13)C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-(13)C2]G6P and [3-(13)C]G6P, predicted products of combined transaldolase and transketolase exchanges.
Conclusion: Hydrogen 5 of G6P was enriched from (2)H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by (2)H2O independently of gluconeogenesis.
Keywords: 13C-NMR isotopomer analysis; deuterated water; gluconeogenesis; pentose phosphate pathway; transaldolase exchange.
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