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

Højfeldt, G, Helge, BW ORCID: 0009-0001-3359-981X, van Hall, G, Schjerling, P, Kjær, M, Mackey, AL, Burniston, JG ORCID: 0000-0001-7303-9318 and Agergaard, J ORCID: 0000-0003-3401-3144 (2026) 2H2O labeling methods for bulk and single muscle protein synthesis measures, and measures of integrated muscle protein synthesis and breakdown rates: A pilot study. Physiological Reports, 14 (13). ISSN 2051-817X

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	D2O; dynamic proteome profiling; muscle protein breakdown; muscle protein synthesis; Muscle, Skeletal; Humans; Deuterium Oxide; Muscle Proteins; Pilot Projects; Isotope Labeling; Proteomics; Protein Biosynthesis; Adult; Male; Young Adult; D2O; dynamic proteome profiling; muscle protein breakdown; muscle protein synthesis; Humans; Male; Pilot Projects; Muscle Proteins; Deuterium Oxide; Muscle, Skeletal; Adult; Protein Biosynthesis; Isotope Labeling; Proteomics; Young Adult; 3208 Medical Physiology; 32 Biomedical and Clinical Sciences; Clinical Research; Bioengineering; Humans; Male; Pilot Projects; Muscle Proteins; Deuterium Oxide; Muscle, Skeletal; Adult; Protein Biosynthesis; Isotope Labeling; Proteomics; Young Adult; 0606 Physiology; 1103 Clinical Sciences; 1116 Medical Physiology; 3208 Medical physiology
Subjects:	R Medicine > RC Internal medicine
Divisions:	Sport and Exercise Sciences
Publisher:	Wiley
Date of acceptance:	30 June 2026
Date of first compliant Open Access:	26 August 2026
Date Deposited:	26 Aug 2026 09:23
Last Modified:	26 Aug 2026 09:23
DOI or ID number:	10.14814/phy2.71013
URI:	https://researchonline.ljmu.ac.uk/id/eprint/29197

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