Relative estimates of TCA cycle pool size from 14CO2 production profiles

In metabolic and isotopic steady state, the rate of 14CO2 production by TCA cycle intermediates labeled at different positions is linear. However, before the system reaches isotopic steady state, the rate of 14CO2 production is non-linear. The x-intercept extrapolated from the linear phase indicates the turnover rate of all metabolic pools the tracer must pass through. By exposing identical systems to 14C succinate labeled in different positions, the contribution of TCA cycle pools to the non-linear phase may be considered. Specifically, the extrapolated x-intercept for [2,314C] succinate will be greater than the x-intercept for [1,414C] succinate if the TCA cycle pools are a contributing factor to the non-linear phase. The authors have used this method to analyze pyruvate oxidation in AS 30D hepatoma cells. They found that the extrapolated x-intercepts for the two tracers were identical. This indicates that the non-linear phase resulted from equilibration of the tracer with pools prior to entering the TCA cycle, i.e. lactate. Using this technique, it may be possible to estimate the variations in TCA cycle pool sizes in vivo