A simple, anesthesia-free infusion technique for <i>in vivo</i> metabolic tracing

Accurate metabolic flux analysis requires tracer delivery that preserves physiological metabolism. Current methods may distort metabolism through anesthesia, surgical stress, or complex procedures. We demonstrate that isoflurane anesthesia profoundly alters serum and tissue metabolism across multiple pathways. Glycolytic and TCA cycle intermediates, sulfur and aromatic amino acid metabolites, acylcarnitines, and nucleotide pools decreased, while branched-chain amino acids, their ketoacids, ketone bodies, and fatty acids increased. These coordinated changes were suggestive of mitochondrial complex I inhibition and reduced oxidative catabolism, leading to shifts in metabolite pool sizes that compromise isotopologue-based flux interpretation. We established a tail vein catheterization method completed in minutes under brief anesthesia that enables multi-hour tracer infusion in awake, freely moving mice. This method achieved steady-state labeling of cystine and downstream products comparable to jugular infusion without supraphysiologic cystine accumulation. This platform provides a practical, physiologically accurate method for <i>in vivo</i> steady-state isotope tracing.