¹H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite—Malylglutamate

Abstract

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

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

“…Among all the detected signals, 38% (89 peaks) remained unassigned to any known or unknown metabolites, due to the lack of concordant information obtained for them. The D. polymorpha metabolome during the resting period is dominated by an intense unknown singlet (M5, 78), putrescine (33 and 77), trimethylamine (71), succinate (55) and betaine (91) (Figure 1, File S1). Smaller signals are due to the low-concentration polar metabolites belonging to several functional classes: 15 proteinogenic amino acids, 7 compounds with amine and amide functional groups, 3 carboxylic acids, 7 compounds in the nucleotide or nucleoside family, 2 coenzymes and 5 carbohydrates (Table 1).…”

“…By exploiting the vast analytical potential of NMR spectrometry and associating several homonuclear and heteronuclear acquisition sequences (JRES, COSY, TOCSY, HSQC), we were able to provide a thoroughly argued description and annotation of a representative spectrum of D. polymorpha (Supplementary Files 1-3). Despite the interest of combining those approaches for the metabolomic spectra interpretation [30,31], the use of these 2D acquisition sequences to strengthen spectral interpretation remains scarce in NMR-based ecotoxicological metabolomic studies [55,56], especially on bivalve species [57,58]. However, even in those studies producing a higher confidence spectral interpretation, a validation of the signal assignment through standard metabolites spiking into the sample has rarely be performed.…”

In-Vivo NMR Spectroscopy: A Powerful and Complimentary Tool for Understanding Environmental Toxicity

“…However, since 2001 there has been an interest in environmental studies as a method to detect and explain toxicity [ 23 , 30 ]. Environmental metabolomics studies changes in the metabolite profile with changing environmental conditions [ 30 ], and have been used on a host of different species including: worms [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ], Drosophila melanogaster [ 39 ], Daphnia magna [ 20 , 40 , 41 , 42 , 43 , 44 , 45 ], Hyalella azteca [ 46 , 47 , 48 ], Caenorhabditis elegans [ 49 , 50 , 51 ], rodents [ 52 , 53 , 54 , 55 ], and plants [ 56 , 57 , 58 , 59 , 60 ]. Species are often chosen either due to their susceptibility to external stressors or their abundance geographically.…”

Effects of the Chiral Fungicides Metalaxyl and Metalaxyl-M on the Earthworm Eisenia fetida as Determined by 1H-NMR-Based Untargeted Metabolomics

“…Metabolomics is potentially a powerful tool to accomplish this purpose; it has been used to elucidate the toxic effects of such substances as metals [10], nanoparticles [11], and pharmaceuticals [12]. In particular, metabolomic studies using 1 H nuclear magnetic resonance (NMR) have proven effective in evaluating the response of organisms to pesticide exposure [13,14,15,16,17,18,19,20], and have become a widely used tool for the study of the effects of soil contaminants on earthworms [21,22,23,24,25]. This method is fast and can determine quantitative changes in the levels of metabolites, such as amino acids and sugars, provoked by an external stressor [26,27,28,29,30].…”