13C tracer analysis identifies extensive recycling of endogenous CO2 in vivo

13C tracing analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. We have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by liquid chromatography coupled with high-resolution mass spectrometer (LC-HRMS). As expected, citrate M+2 or M+4 is the dominant mass isotopologue in vitro. However, citrate M+1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion. One plausible explanation is that 13CO2 produced from the oxidation of 13C tracers in vitro is negligible due to the dilution of HCO3 - supplemented to cell culture when sodium bicarbonante is used and diffusible volume of CO2 in the culture incubator, while endogenous 13CO2 in vivo is substantial and is fixed into the TCA cycle, purine, and serine, resulting in M+1 isotopologues. A time course study shows the generation of high abundance citrate M+1 early in plasma, which may serve as a potent non-invasive biomarker of tissue pyruvate carboxylase activity. Altogether, our results show that recycling of endogenous CO2 is substantial in vivo and provides important insights into the experimental design and data interpretation of 13C tracing assays.