Compound-specific δ2H analysis highlights the relationship between direct assimilation and de novo synthesis of amino acids from food and water in a terrestrial mammalian omnivore

Hydrogen isotope (delta H-2) analysis has been routinely used as an ecological tracer for animal movement and migration, yet a biochemical understanding of how animals incorporate this element in the synthesis of tissues is poorly resolved. Here, we apply a new analytical tool, amino acid (AA) delta H-2 analysis, in a controlled setting to trace the influence of drinking water and dietary macromolecules on the hydrogen in muscle tissue. We varied the delta H-2 of drinking water and the proportions of dietary protein and carbohydrates with distinct hydrogen and carbon isotope compositions fed to house mice among nine treatments. Our results show that hydrogen in the non-essential (AA(NESS)) and essential (AA(ESS)) AAs of mouse muscle is not readily exchanged with body water, but rather patterns among these compounds can be described through consideration of the major biochemical pathway(s) used by organisms to synthesize or route them from available sources. Dietary carbohydrates contributed more hydrogen than drinking water to the synthesis of AA(NESS)in muscle. While neither drinking water nor dietary carbohydrates directly contributed to muscle AA(ESS), we did find that a minor but measurable proportion (10-30%) of the AA(ESS)in muscle was synthesized by the gut microbiome using hydrogen and carbon from dietary carbohydrates. delta H-2 patterns among individual AAs in mice muscle are similar to those we previously reported for bacteria, which provides additional support that this approach may allow for the simultaneous analysis of different AAs that are more influenced by drinking water (AA(NESS)) versus dietary (AA(ESS)) sources of hydrogen.