Inverse or direct detect experiments and probes: Which are "best" for in-vivo NMR research of 13C enriched organisms?

In-vivo Nuclear Magnetic Resonance (NMR) spectroscopy is a unique and powerful approach for understanding sublethal toxicity, recovery, and elucidating a contaminant's toxic mode of action. However, magnetic susceptibility distortions caused by the organisms, along with sample complexity, lead to broad and overlapping 1D NMR spectra. As such, 2D NMR in combination with C-13 enrichment (to increase signal) is a requirement for metabolite assignment and monitoring using high field in-vivo flow based NMR. Despite this, it is not clear which NMR experiment and probe combinations are the most appropriate for such studies. In terms of experiments, H-1-C-13 Heteronuclear Single Quantum Coherence (HSQC) and C-13-H-1 Heteronuclear Correlation Spectroscopy (HETCOR) experiments are logical choices for molecular fingerprinting. HSQC uses H-1 for detection and thus will be the most sensitive, while HETCOR uses C-13 for detection, which benefits from improved spectral dispersion (i.e. a larger chemical shift range) and avoids detection of the huge in-vivo water signal which can be problematic in HSQC. NMR probes are available in two variations, inverse (inner coil H-1) which is best suited to H-1 detection and observe (inner coil C-13) which is ideal for C-13 detection. To further complicate matters, the low biomass in many aquatic organisms makes cryoprobes desirable, however, changing cryoprobes is time prohibitive, requiring at least a day to warmup and cool down, meaning only a single probe can be used to monitor "real-time" in-vivo responses. The key questions become: Is it best to use HSQC on an inverse cryoprobe and accept a compromised HETCOR? Or is it best to use HETCOR on an observe cryoprobe and accept a compromised HSQC? Here these questions are explored using living C-13 enriched Daphnia as the test case. The number of metabolites identified across the different probe/experiment combinations are compared over a range of experiment times. Finally, the probes/experiments are compared to monitor an anoxic stress response. Both probes and experiments prove to be quite robust, albeit HSQC identified slightly more metabolites in most cases. HETCOR did nearly as-well and because of the lack of water complications would be the most accessible approach for researchers to apply in-vivo NMR to C-13 enriched organisms, both in terms of experimental setup and flow system design. This said, when using an optimized flow system, HSQC did identify the most metabolites and an inverse probe design offers the most potential for H-1-only approaches which are continuously being developed and have the potential to eventually overcome the current limitation that requires C-13 enriched organisms. (C) 2020 Elsevier B.V. All rights reserved.