A simple, anesthesia-free infusion technique for in vivo metabolic tracing

Accurate metabolic flux analysis requires tracer delivery that preserves physiological metabolism. Current methods may distort metabolism through isoflurane anesthesia, surgical stress, or complex procedures. We demonstrate that isoflurane anesthesia profoundly alters serum and tissue metabolism across multiple pathways. In serum, acylcarnitines and fatty acids were broadly decreased, whereas amino acid metabolites and select nucleotide species were increased. Across multiple organs, isoflurane induced coordinated metabolic remodeling and distinct tissue-specific responses, including glycolytic remodeling in the brain and amino acid accumulation in the pancreas. To address these metabolic disturbances, we established a nonsurgical tail vein catheterization method completed in minutes under brief isoflurane anesthesia that enables multihour tracer infusion in awake, freely moving mice. Using U-¹³C₆-cystine infusion, this method achieved robust cysteine labeling and downstream labeling comparable to jugular infusion while maintaining circulating cystine pools closer to physiological levels. This platform provides a practical approach for in vivo stable isotope tracing under more physiological conditions.