Exploring Proton-Only NMR Experiments and Filters for Daphnia In Vivo: Potential and Limitations

Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism's metabolome. Typically, these studies use 2D C-13-H-1 experiments on C-13-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased similar to 6-7 fold over the last two years, making C-13-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, Can any metabolic information be obtained from Daphnia using proton-only experiments?. Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D H-1-H-1 experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Automated summary

Environmental metabolomics sheds light on the impact of human activities on organism health at the molecular level. This paper focuses on the use of in vivo NMR as a powerful tool for monitoring real-time changes in the metabolome of organisms. It also investigates the applicability of proton-only experiments on non-enriched samples, specifically using Daphnia as a model organism.