Mapping the mammalian dark metabolome by in vivo isotope tracing

Despite decades of biochemical study, a comprehensive map of the mammalian metabolome remains elusive. Mass spectrometry-based metabolomics detects thousands of small molecule-associated signals in mammalian tissues, but it is currently unclear how many of these reflect products of endogenous metabolism. Here, we leverage systematic in vivo isotope tracing to infer the biosynthetic origins of unidentified metabolites. We administered 26 different isotopically labelled nutrients to mice, measured circulating and tissue metabolite labelling by mass spectrometry, and developed a statistical framework to infer the number of carbon atoms incorporated from each of these precursors into more than 4,000 putative metabolites. We show this information can be harnessed for biosynthesis-aware structure elucidation using a multimodal AI model that co-embeds isotopic labelling patterns with chemical structures. This approach revealed several previously unrecognized families of mammalian metabolites, including cysteine-derived alkylthiazolidines, dithioacetal mercapturic acid derivatives, short-chain N-acyltaurines, acylglycyltaurines, and N-oxidized taurines. It further uncovered a family of mevalonate-derived isoprenoid metabolites that includes 2,3-dihydrofarnesoic acid, which is markedly depleted in both mouse and human aging. Age-related depletion of these isoprenoids is driven by impaired coenzyme A synthesis. Our work establishes the biosynthetic precursors for thousands of unidentified metabolites and reveals multiple previously unrecognized branches of mammalian metabolism.