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

Abstract

In vivo NMR of small 13 C-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. 1 H– 13 C 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.

Ex vivo Comprehensive Multiphase NMR of whole organisms: A complementary tool to in vivo NMR

“…In summary, if costs are ignored, F 1 generation organisms are ideal for most in vivo NMR as the contributions from key metabolites, such as amino acids and sugars, are on a relative basis more pronounced, and it is often these categories of metabolites that flux during important processes or stress responses [ 36 , 65 , 66 ]. The reproducibility of CMP-NMR across the F 1 -F 3 generations is discussed in Fig.…”

“…However, the only drawback of using the F 0 generation is the over emphasized lipid signal. While this is not ideal, as lipids often mask other interesting signals [ 67 ], all components are labelled during 3 weeks of growth [ 14 ] and F 0 generations are commonly used successfully in vivo response studies [ 36 , 65 , 66 ]. As such, the use of F 0 vs. F 1 will likely come down to the cost of raising multiple generations of organisms, which will be driven by the reproduction rate, and food consumption rate of the organisms.…”

Unsaturated fatty acids profiling in liveC. elegansusing real-time NMR spectroscopy

“…To avoid the preparation of multiple lipid extracts at individual time-points, we resorted to NMR routines on live worms. Over the past years, great progress has been made in the metabolic analysis of 13 C-isotope enriched, intact multicellular organisms, with breakthrough studies in the water flea Daphnia magna 25,26 , the mold Neurospora crassa 39 and more recently in C. elegans 30 .…”

“…Notably, these methods have been successfully applied to perform high-resolution metabolic studies in vivo. By introducing 13 C-isotope enrichment in live multicellular organisms, such as the water flea Daphnia magna, metabolic compositions were analyzed using 2D 1 H- 13 C correlation NMR spectra, thus expanding the number of compounds that can be identified and analyzed compared with standard 1D 1 H NMR metabolomics studies 25,26 .…”

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

“…However, its utility is limited by much poorer resolution (regional level) and lower sensitivity. The sensitivity of stable isotope-resolved MRI can be substantially improved by the use of hyperpolarized substrates [209][210][211], post-acquisition processing [11], and proton detection [201,[212][213][214] but still at relatively low spatial resolution and metabolic coverage (typically < 10 metabolites).…”