High-resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Research Articles

The Relationship between Histological Composition and Metabolic Profile in Breast Tumors and Peritumoral Tissue Determined with 1H HR-MAS NMR Spectroscopy.

Breast tumors constitute the complex entities composed of cancer cells and stromal components. The compositional heterogeneity should be taken into account in bulk tissue metabolomics studies. The aim of this work was to find the relation between the histological content and 1H HR-MAS (high-resolution magic angle spinning nuclear magnetic resonance) metabolic profiles of the tissue samples excised from the breast tumors and the peritumoral areas in 39 patients diagnosed with invasive breast carcinoma. The total number of the histologically verified specimens was 140. The classification accuracy of the OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model differentiating the cancerous from non-involved samples was 87% (sensitivity of 72.2%, specificity of 92.3%). The metabolic contents of the epithelial and stromal compartments were determined from a linear regression analysis of the levels of the evaluated compounds against the cancer cell fraction in 39 samples composed mainly of cancer cells and intratumoral fibrosis. The correlation coefficients between the levels of several metabolites and a tumor purity were found to be dependent on the tumor grade (I vs II/III). The comparison of the levels of the metabolites in the intratumoral fibrosis (obtained from the extrapolation of the regression lines to 0% cancer content) to those levels in the fibrous connective tissue beyond the tumors revealed a profound metabolic reprogramming in the former tissue. The joint analysis of the metabolic profiles of the stromal and epithelial compartments in the breast tumors contributes to the increased understanding of breast cancer biology.

Leaf tissue metabolomics fingerprinting of Citronella gongonha Mart. by 1H HR-MAS NMR

This research characterizes key metabolites in the leaf from Citronella gongonha Martius (Mart.) Howard (Cardiopteridaceae). All metabolites were assessed in intact leaf tissue by proton (1H) high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy integrated with the principal component analysis (PCA) to depict molecular association with the seasonal change. The major ‘known unknown’ metabolites detected in 1H HR-MAS NMR were derivatives of flavonoid, polyphenolic and monoterpenoid compounds such as kaempferol-3-O-dihexoside, caffeoyl glucoside (2), 3-O-caffeoylquinic acid (3), 5-O-caffeoylquinic acid (4), kingiside (5), 8-epi-kingisidic acid (6), (7α)-7-O-methylmorroniside (7), (7β)-7-O-methylmorroniside (8) and alpigenoside (9) together with the universally occurring sucrose (10), α-glucoses (11, 12), alanine (13), and fatty (linolenic) acid (14). Several of the major metabolites (1, 2–9) were additionally confirmed by liquid chromatography tandem mass spectrometry (LC–MS/MS). In regard with the PCA results, metabolites 1, 2–9 and 14 were influenced by seasonal variation and/or from further (a) biotic environmental conditions. The findings in this work indicate that C. gongonha Mart. is an effective medicinal plant by preserving particularly compounds 2, 3–9 in abundant amounts. Because of close susceptibility with seasonal shift and ecological trends, further longitudinal studies are needed to realize the physiology and mechanism involved in the production of these and new metabolites in this plant under controlled conditions. Also, future studies are recommended to classify different epimers, especially of the phenolics and monoterpenoids in the given plant.

Metabolomic analysis of endometrial cancer by high-resolution magic angle spinning NMR spectroscopy.

To analyze endometrial metabolite profiles between patients with endometrial cancer and controls. Seventeen (17) women with endometrium cancer and 18 controls were enrolled in this study. 1H HR-MAS (High Resolution-Magic Angle Spinning) NMR (Nuclear Magnetic Resonance) spectroscopy data obtained from endometrial tissue samples of patients with endometrial cancer and control group were analyzed with bioinformatics methods. Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA) score plots obtained with the multivariate statistical analysis of pre-processed spectral data shows a separation between the samples from patients with endometrial cancer and controls. Analysis results suggest that the levels of lactate, glucose, o-phosphoethanolamine, choline, glycerophosphocholine, phosphocholine, leucine, isoleucine, valine, glutamate, glutamine, n-acetyltyrosine, methionine, taurine, alanine, aspartate and phenylalanine are increased in patients with endometrial cancer compared to the controls. The metabolomics signature of patients with endometrial cancer is different from that of benign endometrial tissue.

Sampling Method Affects HR-MAS NMR Spectra of Healthy Caprine Brain Biopsies.

The metabolic profiling of tissue biopsies using high-resolution–magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy may be influenced by experimental factors such as the sampling method. Therefore, we compared the effects of two different sampling methods on the metabolome of brain tissue obtained from the brainstem and thalamus of healthy goats by 1H HR-MAS NMR spectroscopy—in vivo-harvested biopsy by a minimally invasive stereotactic approach compared with postmortem-harvested sample by dissection with a scalpel. Lactate and creatine were elevated, and choline-containing compounds were altered in the postmortem compared to the in vivo-harvested samples, demonstrating rapid changes most likely due to sample ischemia. In addition, in the brainstem samples acetate and inositols, and in the thalamus samples ƴ-aminobutyric acid, were relatively increased postmortem, demonstrating regional differences in tissue degradation. In conclusion, in vivo-harvested brain biopsies show different metabolic alterations compared to postmortem-harvested samples, reflecting less tissue degradation. Sampling method and brain region should be taken into account in the analysis of metabolic profiles. To be as close as possible to the actual situation in the living individual, it is desirable to use brain samples obtained by stereotactic biopsy whenever possible.

Spatially-resolved metabolic profiling of living Drosophila in neurodegenerative conditions using 1H magic angle spinning NMR

Drosophila flies are versatile animal models for the study of gene mutations in neuronal pathologies. Their small size allows performing in vivo Magic Angle Spinning (MAS) experiments to obtain high-resolution 1H nuclear magnetic resonance (NMR) spectra. Here, we use spatially-resolved 1H high-resolution MAS NMR to investigate in vivo metabolite contents in different segments of the fly body. A comparative study of metabolic changes was performed for three neurodegenerative disorders: two cell-specific neuronal and glial models of Huntington disease (HD) and a model of glutamate excitotoxicity. It is shown that these pathologies are characterized by specific and sometimes anatomically localized variations in metabolite concentrations. In two cases, the modifications of 1H MAS NMR spectra localized in fly heads were significant enough to allow the creation of a predictive model.

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities.

In this study, the 1H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that 1H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.

Metabolite Profile of Alzheimer's Disease in the Frontal Cortex as Analyzed by HRMAS 1H NMR.

Background: Investigation on neurochemical changes in the frontal cortex in individuals with Alzheimer’s disease (AD) and different Apolipoprotein E (APOE) genotypes, using ex vivo solid-state high-resolution NMR analysis, may lead to a better understanding of the neurochemistry associated with AD as well as new AD-specific metabolite biomarkers that might potentially improve the clinical diagnosis of AD.Methods: Intact tissue samples of the frontal cortex were obtained from 11 patients and 11 age-matched non-demented controls. Metabolite profiles in all samples were analyzed ex vivo, using solid-state high-resolution magic angle spinning (HRMAS) 600 MHz 1H nuclear magnetic resonance (NMR). A logistic regression analysis was used to rank metabolites based on their level of contribution in differentiating the AD patient tissues and the controls, and different AD-associated APOE genotypes (APOE ε4 vs. APOE ε3).Results: Tissue samples from the AD patients showed significantly lower NAA/Cr (p = 0.011), Ace/Cr (p = 0.027), GABA/Cr (p = 0.005), Asp/Cr (p < 0.0001), mI/Cr (p < 0.0001), and Tau/Cr (p = 0.021), and higher PCho/Cr (p < 0.0001), GPCho/Cr (p < 0.0001), and α&β-Glc/Cr (p < 0.0001) than the controls did. Specifically, a newly observed resonance at 3.71 ppm, referred to as α&β-Glc, was observed in 90.9% of the AD samples (10/11). Samples with APOE ε4 also exhibited higher PCho/Cr (p = 0.0002), GPCho/Cr (p = 0.0001), α&β-Glc/Cr (p < 0.0001), and lower Asp/Cr (p = 0.004) and GABA/Cr (p = 0.04) than the samples with APOE ε3 did. In the logistic regression analysis, PCho, GPCho, ASP, and α&β-Glc were found to be the most relevant metabolites for differentiating the AD patient tissues and the controls, and different APOE genotypes.Conclusion: HRMAS 1H NMR with high spectral resolution and sensitivity offers a powerful tool to gain quantitative information on AD associated neurochemical changes. There are important neurochemical differences in the frontal cortex between the AD patient tissues and the controls, and between those with different APOE genotypes. The resonance (α&β-Glc) found at 3.71 ppm in the AD patient tissues may be further investigated for its potential in the diagnosis and monitoring of AD.

Combination of NMR Methods To Reveal the Interfacial Structure of a Pharmaceutical Nanocrystal and Nanococrystal in the Suspended State.

The detailed structure of a pharmaceutical nanosuspension was investigated using three nuclear magnetic resonance (NMR) methods: solid-state, solution-state, and high resolution-magic angle spinning (HR-MAS) NMR. Carbamazepine (CBZ) and CBZ-saccharin (SAC) cocrystal nanosuspensions were prepared by wet-milling with hydroxypropyl methylcellulose (HPMC) and sodium dodecyl sulfate (SDS) as stabilizing agents. Solid-state 13C NMR indicated the presence of not only the crystalline drug substance but also solid-state HPMC, even though HPMC was used as an aqueous solution to prepare the nanosuspensions. Solution-state 1H NMR of the nanosuspensions with and without ultracentrifugation pretreatment indicated that a fraction of the CBZ, SAC, and SDS formed a solid or semisolid phase on the surface of the nanoparticles and was in equilibrium between the dissolved and undissolved states. 1H HR-MAS NMR was highly effective in detecting and quantifying the semisolid phase on the surface of the nanoparticles. From these comprehensive NMR studies, it was concluded that the nanosuspension was composed of crystalline drug core particles surrounded by a semisolid phase consisting of the drug and stabilizing agents. The semisolid phase on the nanoparticle surface was in equilibrium with the solution phase and contributed to the stabilization of the nanoparticle by steric hindrance and electrostatic repulsion.

Effects of a small, volatile bacterial molecule on Pseudomonas aeruginosa bacteria using whole cell high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and genomics.

In the present study, high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was applied to live Pseudomonas aeruginosa (PA) bacterial cells to determine the metabolome of this opportunistic Gram-negative human pathogen, and in particular, its response to the volatile aromatic low molecular weight signaling molecule, 2-aminoacetophenone (2-AA). Multi-dimensional HRMAS NMR is a promising method which may be used to determine the in vivo metabolome of live intact bacterial cells; 2-AA is produced by PA and triggers the emergence of phenotypes that promote chronic infection phenotypes in in vitro and in vivo (animal) models. In the present study, we applied one-dimensional and two-dimensional proton (1H) HRMAS NMR to PA cells which were grown with or without 2-AA in order to examine the associations between metabolites and cellular processes in response to 2-AA. We also compared whole-genome transcriptome profiles of PA cells grown with or without 2-AA and found that 2-AA promoted profound metabolic changes in the PA cells. By comparing the whole-genome transcriptome profiles and metabolomic analysis, we demonstrated that 2-AA profoundly reprogramed the gene expression and metabolic profiles of the cells. Our in vivo 1H HRMAS NMR spectroscopy may prove to be a helpful tool in the validation of gene functions, the study of pathogenic mechanisms, the classification of microbial strains into functional/clinical groups and the testing of anti-bacterial agents.

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy for the Metabolic Assessment of Acute Rejection After Cardiac Transplantation in Rats

PurposeTo evaluate the potential of high-resolution magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy for metabolite characterization and the differentiation of acute rejection after heart transplantation in rat models. MethodsWe transplanted syngeneic heart grafts from Lewis rats (n = 4) and allogeneic heart grafts from F344 rats (n = 4) heterotopically into Lewis recipients. On day 7 postoperatively, the transplanted hearts were harvested for ex vivo 1H NMR spectroscopy and HR-MAS 1H NMR spectroscopy. 1H NMR spectroscopy and HR-MAS 1H NMR spectroscopy were performed at 4.7 T and 11.7 T, respectively. Metabolomic profiles contributing to the differentiation of allogeneic and syngeneic graft groups were statistically assessed by orthogonal partial least squares discriminant analysis (OPLS/O2PLS-DA). Metabolite concentrations were normalized by total spectral intensities and were compared using Mann-Whitney U tests. ResultsOne allogeneic graft that showed extensive necrotic change suggesting graft failure was excluded from the statistical analysis of the NMR spectroscopy. In the 4.7-T 1H NMR spectroscopy, the creatine peak was decreased in the allogeneic group. The PLS-DA and OPLS/O2PLS-DA score plot demonstrated good discrimination of the allogeneic graft group from syngeneic graft group. The concentrations of creatine, myo-inositol, glucose, niacinamide, hypoxanthine, inosine, and glutamine were significantly decreased in the allogeneic graft group, whereas the concentrations of glycine, phosphoethanolamine, xanthine, sn-glycero-3-phosphocholine, leucine, valine, and tyrosine were significantly increased (P < .05). ConclusionsHR-MAS 1H NMR spectroscopy can metabolically characterize the acute rejection of heart transplantation.

Quest for hydrogen environments in hydrogen-bonded systems

We performed proton high-resolution nuclear magnetic resonance (NMR) measurements on KH2PO4 ferroelectrics. The 1H high-resolution NMR measurements were performed using the magic-angle spinning technique, which removes the proton-proton dipolar coupling and the chemical shift anisotropy in solid-state materials. High-resolution magic-angle spinning 1H NMR spectroscopy provides information on the chemical structure in the KH2PO4 system. From the measured chemical shift data, we determined the oxygen separation distance of the O-H···O hydrogen bond. The localized hydrogen position of the environments near heavy atoms can be depicted using Morse potentials, which provide the wave function of the eigenstate and the probability distribution of hydrogen in the local structure based on the solutions of the Schr¨odinger equation.

The Role of High-Resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy for Predicting the Invasive Component in Patients with Ductal Carcinoma In Situ Diagnosed on Preoperative Biopsy.

The purpose of this study was to evaluate the role of high-resolution magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy in patients with ductal carcinoma in situ (DCIS) diagnosed on preoperative biopsy. We investigated whether the metabolic profiling of tissue samples using HR-MAS 1H NMR spectroscopy could be used to distinguish between DCIS lesions with or without an invasive component. Our institutional review board approved this combined retrospective and prospective study. Tissue samples were collected from 30 patients with pure DCIS and from 30 with DCIS accompanying invasive carcinoma. All patients were diagnosed with DCIS by preoperative core-needle biopsy and underwent surgical resection. The metabolic profiling of tissue samples was performed by HR-MAS 1H NMR spectroscopy. All observable metabolite signals were identified and quantified in all tissue samples. Metabolite intensity normalized by total spectral intensities was compared according to the tumor type using the Mann-Whitney test. Multivariate analysis was performed with orthogonal projections to latent structure-discriminant analysis (OPLS-DA). By univariate analysis, the metabolite concentrations of choline-containing compounds obtained with HR-MAS 1H NMR spectroscopy did not differ significantly between the pure DCIS and DCIS accompanying invasive carcinoma groups. However, the GPC/PC ratio was higher in the pure DCIS group than in the DCIS accompanying invasive carcinoma group (p = 0.004, Bonferroni-corrected p = 0.064), as well as the concentration of myo-inositol and succinate. By multivariate analysis, the OPLS-DA models built with HR-MAS MR metabolic profiles could clearly discriminate between pure DCIS and DCIS accompanying invasive carcinoma. Our preliminary results suggest that HR-MAS MR metabolomics on breast tissue may be able to distinguish between DCIS lesions with or without an invasive component.

Dissolution and Crystallization of Polyamides in Superheated Water and Concentrated Ionic Solutions

The dissolution and recrystallization of Polyamide 46 (PA46) from “superheated” (i) water and (ii) concentrated ionic solutions of strong solubilizing mono- and divalent Hofmeister ions are studied, utilizing in situ high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) supported by wide-angle X-ray diffraction (WAXD), attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR), and gel permeation chromatography (GPC). The samples are sealed in glass capillaries, and employing variable-temperature 1H HR-MAS NMR spectroscopy, the dissolution process of PA46 as a function of temperature and pressure can be followed in situ. The purpose of such a study is to obtain molecular insight into the dissolution process of these hydrogen-bonded synthetic polymers in the different aqueous solutions. In pure water, at temperatures close to the dissolution of PA46, two distinct 1H resonances from water are observed. These resonances are associated with water molecules in the vicinity of PA46 and water in the bulk state. On further heating, the signal from water molecules in the vicinity of PA46 dominates. This sudden change in the environment suggests that water molecules, which have escaped the dense hydrogen-bonded network of bulk water, can diffuse into the structure of PA46, triggering dissolution of the polymer. This happens at a temperature that is more than 100 °C below the melting temperature of the polymer, notably without hydrolysis as verified by GPC performed prior to and after the dissolution experiments. On cooling, recrystallization of PA46 from aqueous solution is observed where water molecules are incorporated in the crystal structure. In the presence of salts, such as LiI and Cal2, weakening of the hydrogen-bonding network of the water molecules occurs. However, above room temperature, independent of the choice of salt, depopulation of the hydrogen bonding between the water molecules occurs, observed as a decrease in the 1H chemical shift value. The reduced hydrogen bonding in the presence of ions facilitates the dissolution of PA46 at much lower temperatures compared to pure water and ultimately results in the complete suppression of crystallization from solution even at room temperature. Depending on the valency of the cation a more mobile or frozen amorphous state of PA46 is obtained at low temperatures as verified by 13C HR-MAS NMR, ATR-FTIR, and WAXD.

1H HRMAS-NMR metabolomic to assess quality and traceability of mozzarella cheese from Campania buffalo milk

The production of Mozzarella di Bufala Campana (MBC) is relevant for the agro-food economy of the Campania Region of Italy and the mark of Protected Designation of Origin (PDO) has been assigned to MBC in relation to its geographical origin. Advanced analytical methods must be then employed to assess authenticity, traceability, and quality of MBC. 1H HRMAS-NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) spectroscopy was applied here to directly identify specific metabolites in MBC intact samples without time-consuming sample pre-treatments. Overcrowded conventional 1H HRMAS-NMR spectra were selectively simplified with two NMR pulse sequences: eCPMG and eDiff, by modulating spin–spin relaxation times and diffusion of MBC molecular components, respectively. Signal elaboration of edited spectra was combined with multivariate analyses to enable significant metabolic differentiation between MBC samples from two different production sites in Campania. Principal Components Analysis (PCA) for eCPMG spectra explained 97.54% of total variance between the two MBC groups for four metabolites (β-galactose, β-lactose, acetic acid, and glycerol). Less efficient was groups distinction by PCA for eDiff spectra, although differences in polyunsaturated acids, such as linoleic and linolenic acids, were highlighted. Similarly, Discriminant Analysis (DA) provided MBC group classification with 100% success in validation tests for eCPMG spectra, while DA prediction ability was reduced to 94.12% for eDiff spectra. Hierarchical Cluster Analysis (HCA) gave a totally correct classification between the two MBC groups only for eCPMG spectra. eCPMG spectra were also used to identify metabolites during MBC aging. As compared to fresh samples, 2 days old MBC samples showed increasing signals for isobutylic alcohol, lactic acid, and acetic acid. This work shows that 1H HRMAS-NMR spectroscopy can rapidly characterise the metabolic profile of intact MBC samples and statistically distinguish the geographical origin of buffalo milk mozzarella and its freshness.

Biological Response in Various Compartments of the Rat Lens after In Vivo Exposure to UVR-B Analyzed by HR-MAS1H NMR Spectroscopy

The purpose of the present study was to investigate metabolic changes in different compartments of the rat lens (anterior, nuclear, posterior, and equatorial) after exposure to an acute double threshold dose of ultraviolet-B radiation (UVR-B) by using high-resolution magic angle spinning (HR-MAS) (1)H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition (PR) METHODS: methods. One eye in each of 28 6-week-old female albino Sprague-Dawley rats was exposed to in vivo 7.5 kJ/m2 UVR-B for 15 minutes. The contralateral eye was left unexposed. One week after irradiation, all rats were killed, and both lenses were isolated. Each lens was cored by a trephine, and the cylinder was sliced into three portions (anterior, nuclear, and posterior). The lens material that remained after the coring process was analyzed as the equatorial region. Analysis of lens metabolism was performed by HR-MAS 1H NMR spectroscopy (14.1 T; Avance DRX600; Bruker BioSpin GmbH, Rheinstetten, Germany), and the metabolic profiles were statistically analyzed by the PR method of principal component analysis (PCA). Metabolic differences were detected among the compartments in the lens, both in samples from the contralateral nonexposed lenses and in samples from lenses exposed to in vivo UVR-B. In the rat lens, exposure to UVR-B caused changes in GSH, phosphocholine, myo-inositol, succinate, formate, and adenosine triphosphate (ATP)/adenosine diphosphate (ADP) and in levels of the amino acids phenylalanine, taurine, hypo-taurine, tyrosine, alanine, valine, isoleucine, and glutamate, that varied among lens compartments. HR-MAS 1H NMR spectroscopy, combined with PR methods (PCA), is effective for analysis of separate parts of the intact rat lens. To understand the biochemistry of the lens, it is important to divide the lens into sections, representing functionally and anatomically distinct compartments.

In vivo 31P and 1H HR-MAS NMR spectroscopy analysis of the unstarved Aporrectodea caliginosa (Lumbricidae)

This study demonstrates that 31P and 1H high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR), a recently developed NMR technique, can be applied to the in vivo analysis of metabolites from an unstarved earthworm. The endogeic earthworm Aporrectodea caliginosa was cut into lengths and then the different body parts (anterior, middle, posterior) with the gut content were analyzed. With the HR-MAS NMR, metabolites show well-resolved signals, whereas, with conventional NMR, spectra are highly dependent on the gut content. 31P HR-MAS NMR has been used to evaluate the effect of an acute exposure of the earthworm to glyphosate. Our observations support a low toxicity of the herbicide and suggest that glyphosate could be trapped in the cutaneous mucus. Earthworms could therefore play a role in horizontal dispersion and stabilization of glyphosate in the drilosphere. Phosphorylated metabolites, such as phospholombricine and lombricine, were clearly identified and their amount measured during experiments. The 1H HR-MAS NMR method offers the opportunity to measure, on the same sample and simultaneously, both the hydrosoluble metabolites and lipids. The data on lipid location and relative succinate concentration shed light on the physiological and metabolic functions of the different body parts of the earthworm.