Selective Amino Acid-Only in Vivo NMR: A Powerful Tool To Follow Stress Processes

In vivo NMR of small ¹³C-enriched aquatic organisms is developing as a powerful tool to detect and explain toxic stress at the biochemical level. Amino acids are a very important category of metabolites for stress detection as they are involved in the vast majority of stress response pathways. As such, they are a useful proxy for stress detection in general, which could then be a trigger for more in-depth analysis of the metabolome. ¹H-¹³C heteronuclear single quantum coherence (HSQC) is commonly used to provide additional spectral dispersion in vivo and permit metabolite assignment. While some amino acids can be assigned from HSQC, spectral overlap makes monitoring them in vivo challenging. Here, an experiment typically used to study protein structures is adapted for the selective detection of amino acids inside living Daphnia magna (water fleas). All 20 common amino acids can be selectively detected in both extracts and in vivo. By monitoring bisphenol-A exposure, the in vivo amino acid-only approach identified larger fluxes in a greater number of amino acids when compared to published works using extracts from whole organism homogenates. This suggests that amino acid-only NMR of living organisms may be a very sensitive tool in the detection of stress in vivo and is highly complementary to more traditional metabolomics-based methods. The ability of selective NMR experiments to help researchers to "look inside" living organisms and only detect specific molecules of interest is quite profound and paves the way for the future development of additional targeted experiments for in vivo research and monitoring.