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

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 13 C-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 13 C-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 13 C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13 C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1 H detection in combination with a 13 C filter on living organisms. The purpose is to suppress all 1 H signals from the organism (i.e., 1 H attached to 13 C), leaving only the probe molecule ( 1 H attached to 12 C). 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 1 H 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.