Virtual decoupling to break the simplification versus resolution trade-off in nuclear magnetic resonance of complex metabolic mixtures.

The heteronuclear single quantum correlation (HSQC) experiment developed by Bodenhausen and Ruben (1980) in the early days of modern nuclear magnetic resonance (NMR) is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment, however, always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier and demonstrate its application towards metabolite identification in a complex mixture. Based on the measurement of clean in-phase and clean anti-phase (CLIP/CLAP) HSQC spectra (Enthart et al., 2008), we construct a virtually decoupled HSQC (vd-HSQC) spectrum that maintains the highest possible resolution in the proton dimension. Combining this vd-HSQC spectrum with a J-resolved spectrum (Pell and Keeler, 2007) provides useful information for the one-dimensional proton spectrum assignment and for the identification of metabolites in Dreissena polymorpha (Prud'homme et al., 2020).

1H-NMR metabolomics profiling of zebra mussel (Dreissena polymorpha): A field-scale monitoring tool in ecotoxicological studies.

Biomonitoring of aquatic environments requires new tools to characterize the effects of pollutants on living organisms. Zebra mussels (Dreissena polymorpha) from the same site in north-eastern France were caged for two months, upstream and downstream of three wastewater treatment plants (WWTPs) in the international watershed of the Meuse (Charleville-Mézières "CM" in France, Namur "Nam" and Charleroi "Cr" in Belgium). The aim was to test 1H-NMR metabolomics for the assessment of water bodies' quality. The metabolomic approach was combined with a more "classical" one, i.e., the measurement of a range of energy biomarkers: lactate dehydrogenase (LDH), lipase, acid phosphatase (ACP) and amylase activities, condition index (CI), total reserves, electron transport system (ETS) activity and cellular energy allocation (CEA). Five of the eight energy biomarkers were significantly impacted (LDH, ACP, lipase, total reserves and ETS), without a clear pattern between sites (Up and Down) and stations (CM, Nam and Cr). The metabolomic approach revealed variations among the three stations, and also between the upstream and downstream of Nam and CM WWTPs. A total of 28 known metabolites was detected, among which four (lactate, glycine, maltose and glutamate) explained the observed metabolome variations between sites and stations, in accordance with chemical exposure levels. Metabolome changes suggest that zebra mussel exposure to field contamination could alter their osmoregulation and anaerobic metabolism capacities. This study reveals that lactate is a potential biomarker of interest, and 1H-NMR metabolomics can be an efficient approach to assess the health status of zebra mussels in the biomonitoring of aquatic environments.

The Zebra Mussel (Dreissena polymorpha) as a Model Organism for Ecotoxicological Studies: A Prior 1H NMR Spectrum Interpretation of a Whole Body Extract for Metabolism Monitoring.

The zebra mussel (Dreissena polymorpha) represents a useful reference organism for the ecotoxicological study of inland waters, especially for the characterization of the disturbances induced by human activities. A nuclear magnetic resonance (NMR)-based metabolomic approach was developed on this species. The investigation of its informative potential required the prior interpretation of a reference 1H NMR spectrum of a lipid-free zebra mussel extract. After the extraction of polar metabolites from a pool of whole-body D. polymorpha powder, the resulting highly complex 1D 1H NMR spectrum was interpreted and annotated through the analysis of the corresponding 2D homonuclear and heteronuclear NMR spectra. The spectrum interpretation was completed and validated by means of sample spiking with 24 commercial compounds. Among the 238 detected 1H signals, 53% were assigned, resulting in the identification of 37 metabolites with certainty or high confidence, while 5 metabolites were only putatively identified. The description of such a reference spectrum and its annotation are expected to speed up future analyses and interpretations of NMR-based metabolomic studies on D. polymorpha and to facilitate further explorations of the impact of environmental changes on its physiological state, more particularly in the context of large-scale ecological and ecotoxicological studies.