Extended Data Figure 3: The extent of carbon scrambling via non-oxidative pentose phosphate pathway is insufficient to substantially affect oxidative pentose phosphate pathway flux determination using [1-¹⁴C]glucose and [6-¹⁴C]glucose, with most carbon entering oxidative pentose phosphate pathway directed towards nucleotide synthesis.

a, Schematic of glycolysis and pentose phosphate pathway showing fate of glucose C6. Note that glucose C6 occupies the phosphorylated position (that is, the last carbon) in every intermediate. Thus, upon catabolism to pyruvate, glucose C6 always becomes pyruvate C3, irrespective of any potential scrambling reactions. b, Schematic of glycolysis and pentose phosphate pathway showing fate of glucose C1. Glucose C1 can be scrambled via the non-oxidative pentose phosphate pathway, moving to C3 (red boxes) or C6 as shown here. The forms shown in the green boxes were not experimentally observed. As glucose C3 becomes pyruvate C1 (the carboxylic acid carbon of pyruvate), which is selectively released as CO₂ by pyruvate dehydrogenase, scrambling of C1 to C3 can potentially increase CO₂ release from glucose C1 relative to C6. This is ruled out in panels d and e. c, Feeding [1-¹³C]glucose or [6-¹³C]glucose results in 50% labelling of 3-phosphoglycerate without any double labelling (that is, M+2), as expected in the absence of scrambling. d, MS/MS method to analyse positional labelling of 1-labelled pyruvate. Collision induced dissociation breaks pyruvate to release the carboxylic acid group as CO₂. If the daughter peak of 1-labelled pyruvate does not contain labelled carbon (m/z = 43), the labelling is at the C1 position; otherwise, it is at C2 or C3. e, After feeding [1-¹³C]glucose or [6-¹³C]glucose, pyruvate is not labelled at the C1 position (< 0.5%), ruling out extensive scrambling. f, Oxidative pentose phosphate pathway flux is similar to or smaller than ribose demand for nucleotide synthesis. Mean ± s.d., n = 3.