Estimating Pentose Phosphate Pathway Activity From The Analysis Of Hepatic Glycogen C-13-Isotopomers Derived From [U-C-13]Fructose And [U-C-13]Glucose

Purpose The pentose phosphate pathway (PPP) is an important component of hepatic intermediary metabolism. Jin et al developed an elegant C-13-NMR method for measuring hepatic PPP flux by quantifying the distribution of glucose C-13-isotopomers formed from [U-C-13]glycerol. We demonstrate that this approach can be extended to exogenous [U-C-13]fructose and [U-C-13]glucose precursors by C-13-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-C-13]fructose for 6 mice, while the glucose component was enriched with 20% [U-C-13]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 C-13 NMR spectroscopy. Flux of each sugar into the PPP relative to its incorporation into glycogen was quantified from selected C-13 glycogen isotopomer ratios.Results Both [U-C-13]fructose and [U-C-13]glucose precursors yielded glycogen C-13-isotopomer distributions that were characteristic of PPP activity. The fraction of [U-C-13]glucose utilized by the PPP relative to its conversion to glycogen via the direct pathway was 14 +/- 1%, while that from [U-C-13]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-C-13]glucose and [U-C-13]fructose precursors were assessed by C-13 NMR analysis of glycogen C-13-isotopomers. Glucose-6-phosphate generated via glucokinase and the direct pathway is preferentially utilized by the PPP.