In Vivo Real-Time Metabolic Isotopic Tracing via Breath Analysis upon Deuterated Water Ingestion: A Proof-of-Concept Study.

Stable isotope tracing with deuterium oxide (D2O) offers a powerful approach for probing metabolic dynamics, yet noninvasive and real-time tools for monitoring in vivo deuterium (D) incorporation remain scarce. This study introduces secondary electrospray ionization-high-resolution mass spectrometry (SESI-HRMS) as a noninvasive, real-time approach for tracking D incorporation in human exhaled breath metabolites. A total of 11 healthy volunteers were enrolled across two independent study centers. Each participant provided up to 20 breath samples across fasting, post-D2O intake, and postprandial phases, yielding 213 samples in total. SESI-HRMS enabled the detection of 2,691 deuterium/hydrogen (D/H) pairs and revealed distinct metabolic trajectories associated with D labeling. Short-chain fatty acids (SCFAs) exhibited biphasic kinetics consistent with rapid orogastric exchange followed by microbial fermentation. Transient increases in pyruvaldehyde were associated with glycemic fluctuations, while dynamic alterations in medium-chain acylcarnitines suggested shifts in mitochondrial energy metabolism. Notably, SESI-HRMS unambiguously resolved D-labeled peaks from natural 13C isotopologues, demonstrating high analytical specificity for stable isotope tracing. Collectively, these findings demonstrate the feasibility of SESI-HRMS real-time breath analysis for noninvasive investigation of D-labeled metabolic dynamics in humans.