Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based ¹³C Isotopic Suppression Selects Only the Molecule of Interest within ¹³C-Enriched Organisms

Abstract

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as Daphnia magna (water fleas) and Hyalella azteca (freshwater shrimps) are commonly 13C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized. Conventionally, these studies would normally involve using a 13C-enriched probe molecule and feeding this to an organism at natural abundance, in turn allowing the fate of the probe molecule to be selectively analyzed. The drawback of such an approach is that there is a limited range of 13C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1H detection in combination with a 13C filter on living organisms. The purpose is to suppress all 1H signals from the organism (i.e., 1H attached to 13C), leaving only the probe molecule (1H attached to 12C). Because the probe molecule can be selectively observed using this approach, it then makes it possible to follow and discern processes such as bioconversion, bioaccumulation, and excretion in vivo. As the approach uses 1H detection, it provides excellent detection limits in the nanogram range. In this article, the approach is introduced, optimized on standards, and then applied to follow nicotine biotransformation and lipid assimilation in vivo to demonstrate the concept.

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics

“…Organisms used for this experiment were fed 13 C Chlamydomonas rheinhardtii (purchased from Silantes GmbH) from birth until ∼2 weeks old. Cultures were at least two generations old (born from 13 C enriched parents), thus considered 99% isotopically enriched . Prior to experimentation, organisms were switched to natural abundance algae, grown in house, to clear the gut and ensure that NMR signals are exclusively from the organism.…”

“…Additionally, due to their small size, D. magna must be isotopically 13 C enriched for 2D 1 H- 13 C, which can become cost prohibitive for large colonies.…”

“…14 This is because magnetic susceptibility distortions from the intact organisms lead to broad lineshape in standard 1D 1 H NMR preventing metabolite monitoring. 24 Additionally, due to their small size, D. magna must be isotopically 13 C enriched for 2D 1 H- 13 C, 25 which can become cost prohibitive for large colonies.…”

“…Cultures were at least two generations old (born from 13 C enriched parents), thus considered 99% isotopically enriched. 25 Prior to experimentation, organisms were switched to natural abundance algae, grown in house, to clear the gut and ensure that NMR signals are exclusively from the organism. Once the gut is cleared, organisms are ready for in vivo NMR experiments, with their media/food placed as droplets on the DMF device (and in the sample well) to provide oxygen to the organisms during experimentation, and ensure that stress responses were not from starvation.…”

Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small 13C enriched biological samples using 1H‐13C 2D heteronuclear correlation NMR spectroscopy

“…D. magna were cultured as described by Lane et al [11] To create uniformly labeled D. magna organisms (97.4% 13 C), [37] 13 C enriched Chlamydomonas reinhardtii was used as the only carbon source. The 13 C enriched C. reinhardtii was grown by Silantes GmbH as described by Akhter et al [38] To create a water-soluble extract of D. magna, freeze-dried organisms were crushed in D 2 O (6.67 μl per organism, leading to roughly 11 μg of biomass within the 25% RF field lines, i.e., approximate volume seen by HMQC; see Section 3) and vortexed for 30 s. The concentration for the D. magna extract is roughly twice that observed for a single organism.…”