Improved 2D 1H–13C NMR permits in vivo analysis of Daphnia magna metabolism without isotopic enrichment

Daphnia magna are small crustaceans, used commonly in aquatic toxicity due to their sensitivity to pollutants. In vivo NMR provides unique insights into real-time metabolic responses, information needed to understand "why" at the biochemical level, contaminants are toxic. Due to overlap caused by the magnetic susceptibility distortions in 1D ¹H NMR, 2D is required for metabolic fingerprinting in vivo. In particular, 2D ¹H-¹³C NMR offers excellent spectral dispersion but is time-consuming and has intrinsic low sensitivity. As such, to date nearly all studies have used ¹³C isotopically enriched D. magna, but this limits studies to lab-raised organisms. Here, in order to assess the feasibility of studying Daphnia at natural abundance, symmetric ASAP HSQC in combination with time-resolved non-uniform sampling (TR-NUS) is explored. The combined approach (TR-NUS ASAP HSQC) improved Signal-to-Noise Ratios (SNRs) up to 3 times versus standard HSQC, while reconstruction of TR-NUS data provided information on a 4 min time scale. In turn, this allowed anoxia (and recovery from anoxia) to be studied for the first time on unlabelled Daphnia using HSQC. TR-NUS ASAP ¹H-¹³C HSQC is a key step towards to investigate environmental adaptability and exposure in living organisms in close to real-time. As the approach does not require isotopic enrichment it affords future possibilities to sample organisms directly from the environment.