A nitrogen metabolism profiling platform simultaneously tracing 30 15N-labeled metabolites reveals that primitive cells constitutively engage de novo pyrimidine synthesis through CAD Ser1900 phosphorylation, while differentiated cells repress this pathway in a uridine-dependent manner and rely nearly exclusively on pyrimidine salvage despite expressing de novo pathway enzymes, establishing cell differentiation state as a fundamental determinant of pyrimidine synthesis pathway choice across cultured cells, primary tissues, and human tumors.

Conventional stable isotope tracing assays track one or several metabolites. However, cells use an array of nutrients to sustain nitrogen metabolic pathways. This incongruency hampers a system level understanding of cellular nitrogen metabolism. Therefore, we created a platform to simultaneously trace 30 nitrogen isotope-labeled metabolites. This platform revealed that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine despite expressing de novo pathway enzymes. This link between cell state and pyrimidine synthesis routes persisted in physiological contexts, including primary murine and human tissues and tumor xenografts. Mechanistically, we found that Ser1900 phosphorylation of CAD, the first enzyme of the de novo pathway, was enriched in primitive cells and that mimicking this modification in differentiated cells abrogated their preference for pyrimidine salvage. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.