Estimating pentose phosphate pathway activity from the analysis of hepatic glycogen (13) C-isotopomers derived from [U-(13) C]fructose and [U-(13) C]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.