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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Nature. 2014 May 4;510(7504):298–302. doi: 10.1038/nature13236

Extended Figure 1. Probing the fractional contribution of the oxPPP to NADPH production with 2H-glucose.

Extended Figure 1

(a) Example of LC-MS chromatogram of M+0 and M+1 forms of NADPH and NADP+. Plotted values are 5 ppm mass window around each compound. (b) Extent of NADPH labeling must be corrected for extent of glucose-6-phosphate labeling. Incomplete labeling can occur due to influx from glycogen or H/D exchange. (c) Labeling fraction of glucose-6-phosphate and fructose-1,6-phosphate in iBMK cells with and without activated Akt (20 min after switching into 1-2H-glucose). (d) Labeling fraction of fructose-1,6-phosphate and 6-phosphogluconate after feeding 1-2H-glucose. Labeling fraction of fructose-1,6-phosphate reflects the labeling of glucose-6-phosphate, whose peak after addition of the 2H-glucose was not sufficiently resolved from other LC-MS peaks in HEK293T and MDA-MB-468 cells to allow precise quantitation of its labeling directly. The difference in the labeling fraction between glucose-6-phosphate and 6-phosphogluconate reflects the fraction of deuterium labeling specifically at position 1 of glucose-6-phosphate. (e) Due to the kinetic isotope effect, feeding of deuterium tracer can potentially alter pathway fluxes. To assess whether the feeding of 1-2H-glucose creates a bottleneck in the oxPPP, we measured the relative concentration of oxPPP intermediates with or without feeding of 1-2H-glucose. No significant changes were observed. (f) Impact of different mechanisms of correcting for the deuterium kinetic isotope effect on fractional contribution of oxPPP to NADPH production. (g) Impact of different mechanisms of correcting for the deuterium kinetic isotope effect on calculated total NADPH production rate. The correction mechanisms are (i) no kinetic isotope effect (CKIE = 1), (ii) no impact on total pathway flux but preferential utilization of 1H over 2H-labeled substrate (Eqn. 4 of main text) (the smallest reasonable correction, and the one applied in the main text), or (iii) full kinetic isotope effect observed for the isolate enzyme with associated decrease in total pathway flux (Eqn. M5 of Methods) (the largest reasonable correction). All results are mean ± SD, N ≥ 2 biological replicates from a single experiment and were confirmed in multiple experiments.