Flow‐based in vivo NMR spectroscopy of small aquatic organisms

Abstract

NMR applied to living organisms is arguably the ultimate tool for understanding environmental stress responses and can provide desperately needed information on toxic mechanisms, synergistic effects, sublethal impacts, recovery, and biotransformation of xenobiotics. To perform in vivo NMR spectroscopy, a flow cell system is required to deliver oxygen and food to the organisms while maintaining optimal line shape for NMR spectroscopy. In this tutorial, two such flow cell systems and their constructions are discussed: (a) a single pump high‐volume flow cell design is simple to build and ideal for organisms that do not require feeding (i.e., eggs) and (b) a more advanced low‐volume double pump flow cell design that permits feeding, maintains optimal water height for water suppression, improves locking and shimming, and uses only a small recirculating volume, thus reducing the amount of xenobiotic required for testing. In addition, key experimental aspects including isotopic enrichment, water suppression, and 2D experiments for both 13C enriched and natural abundance organisms are discussed.

Ex vivo Comprehensive Multiphase NMR of whole organisms: A complementary tool to in vivo NMR

“…Before moving onto the biological differences detected by CMP-NMR, it is worth discussing the differences between ex vivo over in vivo CMP-NMR. While in vivo NMR is the obvious choice for studying real-time responses and the most biologically active state, when such information is not critical to the study, ex vivo NMR can offer some distinct advantages that can be summarized as: Increased Resolution and Signal Intensity: The resolution from ex vivo under MAS is superior to that obtained in vivo (both under MAS [ 10 , 11 , 18 ] and static in vivo systems [ 5 , 6 , [12] , [13] , [14] , [36] , [37] , [38] ]) in large part due to the ability to spin faster. Further, ex vivo experiments can be run for much longer (i.e.…”

“…S4a and S4b in the supporting information compares 13 C and 1 H spectrum collected ex vivo under MAS to that of collected in a flow system in vivo. Note flow systems are commonly used for toxicity based in-vivo studies [ 12 , [39] , [40] , [41] ]. The increase in resolution, and thus, the information content of the ex vivo spectrum, is impressive and clearly shows how ex vivo NMR is complementary to in vivo.…”

“…Increased Resolution and Signal Intensity: The resolution from ex vivo under MAS is superior to that obtained in vivo (both under MAS [ 10 , 11 , 18 ] and static in vivo systems [ 5 , 6 , [12] , [13] , [14] , [36] , [37] , [38] ]) in large part due to the ability to spin faster. Further, ex vivo experiments can be run for much longer (i.e.…”

“…From an environmental toxicology perspective, in vivo research is the ultimate biological goal to fully comprehend the impacts of pollutants and toxins on life. In a sense, living organisms become the ultimate biosensors responding, in real-time, to environmental stressors, with their metabolic profile providing vital information that describes how the contaminants impact the organisms at the molecular level [ [12] , [13] , [14] ]. However, living organisms are heterogeneous multi-phase mixtures of solutions (blood, fluids), gels (lipids, tissue) and solids (bones, shells), which require an NMR approach that can study all phases in a sample holistically.…”

“…As they are primary consumers, they play a major role in freshwater ecosystems and are responsible for the transfer of nutrients and toxins across trophic levels [ 20 , 21 ]. Daphnia are highly susceptible to stressors and thus can provide key insight into the toxic-mode-of-action of various environmental toxins [ 12 , 14 ]. As they can reproduce asexually in ideal conditions [ 22 ] (i.e., lab setting), the progenies are all genetically identical to their parents.…”