Estimating pentose phosphate pathway activity from the analysis of hepatic glycogen 13C‐isotopomers derived from [U‐13C]fructose and [U‐13C]glucose

Purpose The pentose phosphate pathway (PPP) is an important component of hepatic intermediary metabolism. Jin et al developed an elegant 13 C‐NMR method for measuring hepatic PPP flux by quantifying the distribution of glucose 13 C‐isotopomers formed from [U‐ 13 C]glycerol. We demonstrate that this approach can be extended to exogenous [U‐ 13 C]fructose and [U‐ 13 C]glucose precursors by 13 C‐NMR analysis of glycogen. Methods Twelve male C57BL/6 mice fed standard chow were provided a 55/45 mixture of fructose and glucose at 30% w/v in the drinking water for 18 wk. On the evening before sacrifice, the fructose component was enriched with 20% [U‐ 13 C]fructose for 6 mice, while the glucose component was enriched with 20% [U‐ 13 C]glucose for the remaining 6 mice. Mice were allowed to feed and drink naturally overnight, and then, euthanized. Livers were freeze‐clamped and glycogen was extracted and derivatized for 13 C NMR spectroscopy. Flux of each sugar into the PPP relative to its incorporation into glycogen was quantified from selected 13 C glycogen isotopomer ratios. Results Both [U‐ 13 C]fructose and [U‐ 13 C]glucose precursors yielded glycogen 13 C‐isotopomer distributions that were characteristic of PPP activity. The fraction of [U‐ 13 C]glucose utilized by the PPP relative to its conversion to glycogen via the direct pathway was 14 ± 1%, while that from [U‐ 13 C]fructose relative to its conversion to glycogen via the indirect pathway was significantly lower (10 ± 1%, P = .00032). Conclusions Hepatic PPP fluxes from both [U‐ 13 C]glucose and [U‐ 13 C]fructose precursors were assessed by 13 C NMR analysis of glycogen 13 C‐isotopomers. Glucose‐6‐phosphate generated via glucokinase and the direct pathway is preferentially utilized by the PPP.