13 C tracer analysis suggests extensive recycling of endogenous CO 2 in vivo

Background 13 C tracer 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. The distinct 13 C labeling patterns (e.g., M + 1 species in vivo but not in vitro) of metabolites from [U- 13 C]-glucose or [U- 13 C]-glutamine tracing in vivo and in vitro have been previously reported by multiple groups. However, the reason for the difference in the M + 1 species between in vivo and in vitro experiments remains poorly understood. Methods We have performed [U- 13 C]-glucose and [U- 13 C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13 C enrichment of metabolites in cultured cells and tissues was determined by LC coupled with high-resolution mass spectrometry (LC-HRMS). All p -values are obtained from the Student’s t -test two-tailed using GraphPad Prism 8 unless otherwise noted. Results We observed distinct enrichment patterns of tricarboxylic acid cycle intermediates in vivo and in vitro. As expected, citrate M + 2 or M + 4 was the dominant mass isotopologue in vitro. However, citrate M + 1 was unexpectedly the dominant isotopologue in mice receiving [U- 13 C]-glucose or [U- 13 C]-glutamine infusion, but not in cultured cells. Our results are consistent with a model where the difference in M + 1 species is due to the different sources of CO 2 in vivo and in vitro, which was largely overlooked in the past. In addition, a time course study shows the generation of high abundance citrate M + 1 in plasma of mice as early as few minutes after [U- 13 C]-glucose infusion. Conclusions Altogether, our results show that recycling of endogenous CO 2 is substantial in vivo. The production and recycling of 13 CO 2 from the decarboxylation of [U- 13 C]-glucose or [U- 13 C]-glutamine is negligible in vitro partially due to dilution by the exogenous HCO 3 − /CO 2 source, but in vivo incorporation of endogenous 13 CO 2 into M + 1 metabolites is substantial and should be considered. These findings provide a new paradigm to understand carbon atom transformations in vivo and should be taken into account when developing mathematical models to better reflect carbon flux.