Extended Data Figure 1: Probing the fractional contribution of the oxidative pentose phosphate pathway to NADPH production with [2H]glucose.
From: Quantitative flux analysis reveals folate-dependent NADPH production
a, Example of LC–MS chromatogram of M+0 and M+1 forms of NADPH and NADP+. Plotted values are 5 p.p.m. mass window around each compound. b, Extent of NADPH labelling must be corrected for extent of glucose-6-phosphate labelling. Incomplete labelling can occur due to influx from glycogen or hydrogen-deuterium exchange. c, Labelling 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, Labelling fraction of fructose-1,6-phosphate and 6-phosphogluconate after feeding [1-2H]glucose. Labelling fraction of fructose-1,6-phosphate reflects the labelling 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 quantification of its labelling directly. The difference in the labelling fraction between glucose-6-phosphate and 6-phosphogluconate reflects the fraction of deuterium labelling 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 oxidative pentose phosphate pathway, we measured the relative concentration of oxidative pentose phosphate pathway intermediates with or without feeding of [1-2H]glucose. No significant changes were observed. f, Effect of different mechanisms of correcting for the deuterium kinetic isotope effect on fractional contribution of oxidative pentose phosphate pathway to NADPH production. g, Effect of different mechanisms of correcting for the deuterium kinetic isotope effect on calculated total NADPH production rate. The correction mechanisms are: (1) no kinetic isotope effect (CKIE = 1), (2) no effect on total pathway flux but preferential utilization of 1H over 2H-labelled substrate (equation (4) of main text) (the smallest reasonable correction, and the one applied in the main text), or (3) full kinetic isotope effect observed for the isolate enzyme with associated decrease in total pathway flux (Eqn. 6 of Methods) (the largest reasonable correction). All results are mean ± s.d., n ≥ 2 biological replicates from a single experiment and results were confirmed in multiple experiments.
