Development and Application of a Low-Volume Flow System for Solution-State <i>in Vivo</i> NMR

<i>In vivo</i> nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for <i>in vivo</i> NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors. The use of a double pump design with a sump slurry pump return allows algal food suspensions to be continually supplied without the need for filters, eliminating the possibility of clogging and leaks. Using the flow system, the living organism can be kept alive without stress indefinitely. To evaluate the feasibility and applicability of the flow system, changes in the metabolite profile of ¹³C enriched <i>Daphnia magna</i> over a 24-h period are compared when feeding laboratory food vs exposing them to a natural algal bloom sample. Clear metabolic changes are observed over a range of metabolites including carbohydrates, lipids, amino acids, and a nucleotide demonstrating <i>in vivo</i> NMR as a powerful tool to monitor environmental stress. The particular bloom used here was low in microcystins, and the metabolic stress impacts are consistent with the bloom being a poor food source forcing the <i>Daphnia</i> to utilize their own energy reserves