Table 1 (columns ‘measured’) gives the mass fractions of 10 metabolic intermediates that were measured in the extracts of the biomass that was sampled 0, 40 and 60 min after the switch from naturally-labeled medium to 13C-labeled medium.
3.1.1Mass isotopomer fractions of naturally-labeled biomass
The mass fractions of the sample taken at t= 0 represent the 13C-labeling distribution of metabolites formed from naturally-labeled glucose and ethanol. The mass fractions greater than M+ 0 are caused by naturally-occurring isotopes of carbon, oxygen and hydrogen. In the cumomer model used to simulate the mass isotopomer fractions, the natural occurrence of the 13C-isotope is taken into account when defining the cumomer distribution of the carbon substrates. The occurrence of natural isotopes of the atom species other than carbon is accounted for by applying the correction procedure proposed in .
The cumomer model can be used to generate the mass isotopomers of all metabolic intermediates formed from naturally-labeled carbon substrates by setting the 13C-labeled fractions of the substrates at zero. These calculated mass isotopomer fractions can be compared to the corresponding measured mass isotopomer fractions of the sample taken at t= 0. Since these mass isotopomer fractions are fully independent of both the topology of the metabolic reaction network and of the fluxes therein, this comparison allows verification of the accuracy of the mass isotopomer measurements. Table 1 (column ‘calculated, time = 0 min’) shows that the calculated fractions agree well with the measured ones. Seeing the small standard deviations of the mass fractions, the observed correspondence between the measured and theoretically expected values at t= 0 demonstrates the accuracy of the MS measurements.
3.1.2Mass isotopomer fractions of biomass grown on 1-13C1-glucose
The biomass was sampled both at t= 40 and at t= 60 min in order to check whether the 13C-labeling distributions of the intracellular metabolites were in isotopic steady-state after more than 30 minutes, as was estimated on the basis of literature values of intracellular pool sizes and estimated fluxes. It can be verified in Table 1 (columns ‘measured, time = 40 and 60’) that the mass fractions do not change significantly after 40 min, indicating that these metabolic intermediates may indeed be assumed in isotopic steady-state.
The similarity of the mass isotopomer fractions in the two independently sampled, washed, extracted and analyzed biomass samples (t= 40, respectively, t= 60) further indicates that the procedure is reproducible. If the metabolism were not instantaneously quenched and turnover of metabolites would still occur in an uncontrolled way in the sampled biomass, the outcomes of the two samples would very unlikely show the same consistency.
Inspecting the mass isotopomer distributions further shows that prior to any calculation, the distributions already hold information on the metabolic fluxes. For example: the near-identity of the mass fractions of glucose 6-phosphate and fructose 6-phosphate at t= 40 and t= 60 demonstrates that either fructose 6-phosphate is uniquely synthesized from glucose 6-phosphate or, in case the fructose 6-phosphate pool has additional influxes, that the phosphoglucose isomerase catalyzes an exchange flux that is considerably larger than its net flux. Under the current experimental conditions, it is to be expected that part of the glucose will be converted via the pentose phosphate pathway. By consequence, fructose 6-phosphate will have influxes catalyzed by transaldolase and transketolase (v9 through v11, v13 and v15 in Fig. 1), suggesting that the phosphoglucose isomerase reaction is highly reversible.
No such large exchange flux is found for phosphoglucomutase that interconverts glucose 6-phosphate and glucose 1-phosphate. The latter metabolite has a larger M+ 0 fraction and lower M+ 1 and M+ 2 fractions than glucose 6-phosphate, which indicates that the reaction between the two components is not at equilibrium. A tentative explanation for the fact that glucose 1-phosphate is 13C-labeled to a lower extent than glucose 6-phosphate is that the glucose 1-phosphate that stems from glucose 6-phosphate is diluted by glucose 1-phosphate molecules that originate from turnover of the large intracellular glycogen pool, which is not yet in isotopic steady-state after 40 and 60 min of 13C-labeled medium supply. This explanation is supported by the finding of Mashego et al.  who switched a chemostat culture of the same strain from naturally-labeled to 100% uniformly 13C-labeled feed and observed that the fraction of remaining unlabeled glucose 1-phosphate rapidly decreased, but leveled off and stayed constant at around 20% during several hours after the onset of 13C-labeled feeding.
The occurrence of simultaneous glycogen synthesis and degradation is in fact a futile cycle, wasting ATP. The occurrence of this phenomenon in S. cerevisiae was discussed by François and Parrou  who suggested that it could act as a ‘glycolytic safety valve’ preventing death of the cells by ATP imbalance under stress conditions. It remains an open question why glycogen cycling would then occur under the undisturbed conditions of the present study.
Because of the suspected influx of unlabeled glucose 1-phosphate, the apparent isotopic steady-state within 40 min is in fact not truly steady: it will take considerably more time before the glycogen storage pool will be in true isotopic steady-state. In the model, the continuing turnover of partially unlabeled glycogen that dilutes the 13C-labeling of the glucose 1-phosphate pool is accounted for by adding an influx with unlabeled compound (v21 f) and an equally large efflux (v21 b) of the same pool.
From the data in Table 1 it can furthermore be inferred that the reactions interconverting fructose 1,6-bisphosphate and two molecules of 2/3-phosphoglycerate are not in equilibrium. The mass isotopomer distribution of fructose 1,6-bisphosphate that can be calculated by multiplying two mass isotopomer distributions of 2/3-phosphoglycerate at t= 60 is: M+ 0= 0.394, M+ 1= 0.431 and M+ 2= 0.145. This does not correspond very well to the actually measured values, as should be the case at equilibrium.
The mass isotopomer fractions of the combined 2/3-phosphoglycerate pool are very similar to those of phosphoenolpyruvate at both t= 0 and t= 60. This demonstrates that the phosphoglycerate mutase and enolase also catalyze exchange fluxes that are significantly higher than the net glycolytic flux.
Finally, the good agreement of the glucose 6-phosphate and 6-phosphogluconate metabolites that is observed in Table 1 serves as a quality control, as in S. cerevisiae the latter compound uniquely originates from the former, and thus must have an identical 13C-label distribution.