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

Abstract

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 13C-1H experiments on 13C-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 ~6–7 fold over the last two years, making 13C-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 1H-1H 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.

13C‐depleted algae as food: Permitting background free in‐vivo nuclear magnetic resonance of Daphnia magna at natural abundance

“…A culture of NA C. reinhardtii was grown in-lab according to published protocols. 9 Algae was lyophilized for long term storage.…”

“…Living organisms are chemically complex and inhomogeneous, resulting in broad lineshapes and spectral overlap in 1D NMR that makes extracting metabolic information challenging. [9][10][11] As a result, over the last decade the majority of in-vivo NMR studies have used multidimensional experiments, most commonly the 1 H 13 C Heteronuclear Single Quantum Coherence (HSQC). 3 However, such experiments are less sensitive than 1D NMR and require the organisms to be 13 Cenriched (by feeding them a diet of 13 C-labelled algae) to permit faster and more in-depth analysis.…”

“…3 However, such experiments are less sensitive than 1D NMR and require the organisms to be 13 Cenriched (by feeding them a diet of 13 C-labelled algae) to permit faster and more in-depth analysis. 8,9,12 When 13 C-labelled organisms are used, they are generally fed natural abundance (NA) algae during analysis to avoid background metabolite signals from their gut contents. 8 As the signal from the 13 C-enriched organisms is 100-fold larger than the NA algae in their stomachs, it tends not to be a problem when using 1 H- 13 C HSQC detection.…”

“…7 While 13 C-enrichment permits a broader scope of pulse sequences and greater sensitivity for in-vivo studies, 1,8 isotopic labelling is very costly, and restricts studies only to organisms that have been raised in a lab (vs. directly from the environment), which has limited the proliferation of environmental in-vivo NMR. 9 William W. Wolff and Jacob Pellizzari contributed equally to this work.…”

Development of a Simple Cost Effective Oxygenation System for In Vivo Solution State NMR in 10 mm NMR Tubes

“…In contrast, the complexity and inhomogeneity of living organisms (e.g., different body organs, physical structures, etc.) yield varying magnetic susceptibilities across the sample. , As a result, chemical shifts of the same molecule may differ depending on its localization within the organism (e.g., in the exoskeleton, eye, or heart). When summed across the entire organism this manifests as spectral broadening and loss of splitting information .…”