Heavy water labeling reveals metabolic flexibility of amino acid and polyamine pathways in mammalian cells

Amino acid and polyamine metabolism underpins many cellular processes, such as cell growth, stress adaptation, and signaling. However, the usage of specific metabolic pathways is highly context-dependent, and there are many compensatory mechanisms in place for the biosynthesis of Amino acids. Here, we establish low-dose heavy water (D₂O) labeling as a tracer to monitor amino acid and polyamine metabolism in mammalian systems. Using targeted HPLC-MS of primary amines, we quantified deuterium incorporation in mouse embryonic fibroblasts, pancreatic β-cell-derived MIN6 cells, and mouse tissues, which we then benchmarked with orthogonal tracers (¹³C-glucose and ¹⁵NH₄⁺). We demonstrated D₂O labels nonessential Amino acids and polyamines. We validated specificity, as inhibition of key metabolic steps altered deuterium incorporation into Ala/Ser/Gly and polyamines and revealed differential engagement of branched-chain amino acid metabolism. We found that glutamine starvation induces integrated stress response-linked remodeling, increasing deuterium incorporation into Glu and glycolytic Amino acids while identifying changes in Amino acids efflux. Finally, in vivo short-term D₂O exposure distinguishes tissue-specific biosynthetic capacities. Collectively, these data challenge the assumption of uniform alanine labeling by D₂O and demonstrate that D₂O provides a sensitive readout of metabolic flexibility, transport crosstalk, and pathway regulation across cell types and tissues.