²H₂O labeling methods for bulk and single muscle protein synthesis measures, and measures of integrated muscle protein synthesis and breakdown rates: A pilot study

This methodological pilot study explored the feasibility of an integrated tracer approach for simultaneous measurement of human skeletal muscle protein synthesis and breakdown during prolonged deuterium oxide (²H₂O) labeling. Four healthy men completed a five-week protocol combining daily oral ²H₂O intake with selected stable isotope amino acid tracers to assess bulk fractional synthesis rates, fractional breakdown rates, and single-protein turnover. The study specifically addressed the methodological question of whether bulk or individual protein synthesis measurements remain consistent over a prolonged ²H₂O labeling period. We suggest that measurements of mixed-protein fractions in muscle might be vulnerable to error due to the rise-to-plateau kinetics associated with longer-duration labeling. Mean mixed-protein synthesis rates could be underestimated when labeling exceeds approximately 4 weeks. Increasing precursor enrichment mitigated this effect but highlights the need for controlled enrichment strategies in long-term studies. In contrast, dynamic proteome profiling (DPP) provided more robust single-protein turnover estimates across the full period because the different isotopic plateaus of individual peptides could be modeled. The study also tested the feasibility of combining ²H₂O labeling with ¹⁵N-alanine-based breakdown measurements and evaluated potential interference from multiple tracers on DPP outputs. Together, these findings outline practical considerations, limitations, and feasibility of an integrated multi-tracer framework for studying human muscle protein turnover over extended periods.