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- Title
Metabolic incorporation of H<sub>2</sub><sup>18</sup>O into specific glucose‐6‐phosphate oxygens by red‐blood‐cell lysates as observed by <sup>13</sup>C isotope‐shifted NMR signals.
- Authors
Coelho, Margarida; Barosa, Cristina; Tavares, Ludgero; Jones, John G.
- Abstract
Water enriched with oxygen‐18 (H218O) 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 2H, 18O does not experience significant isotope effects for any of these processes. Therefore, H218O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18O enrichments of glucose can be measured. As a proof of concept, H218O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13C NMR was applied to quantify positional 18O 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) H218O. Enrichment of glucose‐6‐phosphate was analyzed by 13C NMR analysis of 18O‐shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13C NMR MAG spectra from hemolysate revealed resolved 18O‐shifted signals in Positions 1‐5. Mean 18O 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 18O‐shifted glucose‐6‐phosphate signals were detected in preparations containing sugar phosphate precursors only. H218O 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.
- Subjects
SUGAR phosphates; GLUCOSE synthesis; PHOSPHATE metabolism; ISOTOPE shift
- Publication
NMR in Biomedicine, 2020, Vol 33, Issue 11, p1
- ISSN
0952-3480
- Publication type
Article
- DOI
10.1002/nbm.4395