NMR assignment of thein vivodaphnia magna metabolome

Abstract

<p> <italic>Daphnia </italic>(freshwater fleas) are among the most widely used organisms in regulatory aquatic toxicology/ecology, while their recent listing as an NIH model organism is stimulating research for understanding human diseases...</p>

NMR spectroscopy of a single mammalian embryo

“…The simplest way to achieve this would be via multidimensional homonuclear spectroscopy. Basic approaches such as 1 H-1 H COSY and TOCSY offer some potential in vivo, although spectral overlap and fast relaxation limit their resolving capabilities to only a handful of the most abundant compounds in vivo 9 . Conversely, dedicated approaches to reducing spectral inhomogeneities via intermolecular quantum interactions 57 hold great potential.…”

“…While 1 H NMR provides a theoretical peak capacity of around ~3000 peaks, a 2D 1 H-13 C correlation increases this to ~2 million, while 3D NMR approaches 100 million 59 . With such great improvements in spectral dispersion, even a moderate field of 500 MHz can be used to provide comprehensive assignment of a living metabolome 9 . However, such in vivo analysis generally requires isotopic enrichment ( 13 C, or 13 C and 15 N) to increase signal due to the relative low biomass content of small biological entities.…”

“…Such enrichment opens up many novel approaches including: amino-acid only NMR 60 (essential as amino acids are involved in most stress and growth processes); moving window non-uniform sampling (close to real-time 2D NMR) 61 ; and even isotope sub-editing which renders the organism "invisible" allowing only the 13 C probe molecule (drug, toxin, nutrient) to be seen and monitored inside the organism 62 . However, while it is relatively easy to fully 13 C enrich small aquatic species such Hyalella azteca (freshwater shrimp) 63 and Daphnia magna (water fleas) 9 , it becomes near impossible to fully enrich cows or humans thus limiting this approach. This stated, Stable Isotope Resolved Metabolomics, where only specific pathways are targeted, holds great potential, and is making profound in-roads into understanding the complex processes behind complex diseases such as cancer 64 .…”

“…It is the only molecular-level tool that can provide non-invasive and comprehensive information as to the structure and interactions inside living organisms. For example, the first metabolome of a living organism (Daphnia Magna, essential keystone species used in toxicity testing) was recently assigned in 2020 9 . Further, NMR holds unique capabilities to study non-covalent binding 10 , including mechanisms and receptors, essential life processes and pharmaceutical development, toxicity and fate.…”

Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small 13C enriched biological samples using 1H‐13C 2D heteronuclear correlation NMR spectroscopy

“…Indeed, this additional spectral dispersion helps overcome overlap, and 2D and 3D NMR were recently applied to provide the first metabolome assignment of a living organism. [20] Enriching small aquatic organisms via a 13 C enriched diet is relatively simple [10,11] and can increase signal by as much as 10,000 times for 13 C- 13 C correlation. Microcoil technology to date has been used in both 1 H one-dimensional NMR [14,16,17,[21][22][23] and heteronuclear two-dimensional NMR.…”