Qualitative high field 1H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential

Abstract

This study was designed to provide a database of the endogenous metabolites in earthworm extracts of the species, Eisenia veneta and Lumbricus terrestris by high resolution 1 H-NMR spectroscopy in view of identifying biomarkers of toxicity or stress in environmental metabolomics studies. 1D and 2D NMR spectroscopic techniques enabled the identification and confirmation of the organic components in the tissue extracts of whole and segmented earthworms, dissected organs, and coelomic fluid. The extracts gave rise to characteristic 1 H-NMR spectral fingerprints of the low MW metabolites contained, specific to the species of earthworm, and to the specific regions or dissected organs of the earthworms under investigation. Distinct changes in the normal biochemistry were observed upon starvation and cooling, such as markedly decreased glucose and maltose, but increased lactate, acetate, succinate, formate and acetone. Additionally, slightly decreased threonine, arginine, lysine, leucine, citrate, asparagine and glycine were observed. Furthermore, lactate could be identified as a biomarker of acute toxic stress in expressed coelomic fluid following exposure to a model ecotoxin (3-trifluoromethylaniline). This work supports the application of 1 H-NMR spectroscopy for the study of changes in the normal invertebrate biochemistry in order to allow for the reliable assessment of biomarker responses following toxicity testing.

Metabolomics: Methodologies and applications in the environmental sciences

“…Combining NMR-based metabolomics and pattern recognition, several environmental contaminants were examined in earthworm species (Lumbricus rubellus, L. terrestris, and Eisenia andrei) exposed to metal contaminated soil, 9) in L. rubellus and E. andrei exposed to copper, 49) and in E. veneta exposed to 3-trifluoromethyl-aniline, 47) 3-fluoro-4-nitrophenol, 50) 4-fluoroaniline, 3,5-difluoroaniline, and 2-fluoro-4-methylaniline, 51) and 3-trifluoromethylaniline. 22) These studies suggest potential biomarkers for ecotoxicological testing, such as increased levels of maltose in L. rubellus after exposure to metal contaminants and decreased levels of acetate and malonate after exposure to 3-fluoro-4-nitrophenol. They also found that these metabolic effects are chemical dependent; 3,5-difluoroaniline and 2-fluoro-4-methylaniline produce similar metabolic effects, but 4-fluoroaniline does not.…”

“…Moreover, relatively lower increases of succinate and lactate in D. octaedra suggest anoxic metabolism to be lowest in the freeze-tolerant species. Lenz et al 22) also found distinct metabolic changes (i.e. decreased glucose and increased succinate and lactate) in normal earthworms under cool temperature stress than in tolerant ones.…”

“…An increasing number of published methods apply different analytical techniques to metabolomic analyses, such as high-performance liquid chromatography (HPLC) with UV detection, 17) Fourier transformed infrared (FT-IR) spectroscopy, 18,19) mass spectrometry (MS) 20) and nuclear magnetic resonance (NMR). 10,21,22) MS and NMR are the techniques utilized most commonly since both are able to detect a wide range of metabolites with relatively high specificity and reproducibility. Current MS and NMR techniques in metabolomics have been discussed in some depth [23][24][25] and so our discussion will be limited to only pertinent details in this paper.…”

“…In order to better understand the basal metabolic status and biochemical composition of this potential indicator of soil contamination, metabolic profiles of different species, specific regions or dissected organs of earthworms were studied. 22,52,53) Metabolomics allowed differentiation between two morphologically similar earthworm species and highlighted characteristics of specific organs or regions. For example, the metabolic profiles are variable in the coelomic fluids of three Eisenia earthworm species.…”

1H NMR metabolomics of earthworm responses to polychlorinated biphenyl (PCB) exposure in soil

“…Spectra were apodized through multiplication with an exponential decay corresponding to 0.3 Hz line broadening in the transformed spectrum and a zero filling factor of 2, then manually phased and calibrated to the DSS methyl singlet, set to a chemical shift (d) of 0.00 ppm. Metabolite peaks were identified in each spectrum by comparison with previously published assignments (Brown et al 2008;Cui et al 2008;Lenz et al 2005). …”