Metabolic incorporation of H₂¹⁸O into specific glucose‐6‐phosphate oxygens by red‐blood‐cell lysates as observed by ¹³C isotope‐shifted NMR signals

Abstract

Water enriched with hydrogen isotopes (2 H, 3 H) has been used extensively as a metabolic tracer for carbohydrate biosynthesis in animals and humans. 1-4 While these tracers can be easily delivered and are rapidly and homogenously distributed throughout tissues, incorporation of 2 H or Abbreviations: 2 H2O, water enriched with deuterium; H2 18 O, water enriched with oxygen-18; MAG, monoacetone glucose; KIE, kinetic isotope effect.

Enrichment of hepatic glycogen and plasma glucose from H₂18O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice

“…In contrast to hydrogen isotopes, the much smaller relative mass difference between 18 O and 16 O means the absence of significant isotope discrimination because of KIE. We recently demonstrated 18 O‐incorporation from H 2 18 O into the oxygens of glucose‐6‐phosphate in hemolysate preparations that was in accordance with known exchange mechanisms acting at the triose and hexose phosphate levels 16 . These measurements were performed by 13 C NMR analysis of the monoacetone glucose derivative and quantification of the isotope‐shifted 18 O‐signals.…”

“…3.1 | Analysis of glycogen 18 O-enrichment by 13 C NMR Both groups of mice had similar liver glycogen content (146 ± 27 μmol/g wet weight NC and 117 ± 34 μmol/g wet weight HS) and the 18 Oenrichment levels of plasma water were also similar (4.75% ± 0.32% NC and 4.95% ± 0.51% HS). The TAMAG derivatives of liver glycogen presented intense and narrow signals for the 13 C-enriched carboxyls bound to oxygens 3, 5, and 6 of the TAMAG hexose moiety with well-resolved 18 O-shifted signals (chemical shift distance between 16 O and 18 O signals of 2.65, 2.47, and 2.47 Hz, respectively) (Figure 2A). The positional 18 O-enrichments estimated from these signals are shown in Figure 2B.…”

“…7,[13][14][15] The oxygens of water also undergo exchanges and/or additions with precursors of glucose and glycogen synthesis, hence in the presence of O), the hexose oxygens become enriched with 18 O (Figure 1). In contrast to hydrogen isotopes, the much smaller relative mass difference between 18 O and 16 O means the absence of significant isotope discrimination because of KIE. We recently demonstrated O into the oxygens of glucose-6-phosphate in hemolysate preparations that was in accordance with known exchange mechanisms acting at the triose and hexose phosphate levels.…”

“…We recently demonstrated O into the oxygens of glucose-6-phosphate in hemolysate preparations that was in accordance with known exchange mechanisms acting at the triose and hexose phosphate levels. 16 These measurements were performed by 13 C NMR analysis of the monoacetone glucose derivative and quantification of the isotope-shifted 18 O-signals. The objective of the present study was to translate this approach into animal models and test the applicability of H 2 18 O as a tracer for hepatic carbohydrate biosynthesis.…”