Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Abstract

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.