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Theillet FX, Luchinat E. In-cell NMR: Why and how? PROGRESS IN NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 2022; 132-133:1-112. [PMID: 36496255 DOI: 10.1016/j.pnmrs.2022.04.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 04/19/2022] [Accepted: 04/27/2022] [Indexed: 06/17/2023]
Abstract
NMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies. NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR… All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success. We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the "why", oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the "how", where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology.
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Affiliation(s)
- Francois-Xavier Theillet
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France.
| | - Enrico Luchinat
- Dipartimento di Scienze e Tecnologie Agro-Alimentari, Alma Mater Studiorum - Università di Bologna, Piazza Goidanich 60, 47521 Cesena, Italy; CERM - Magnetic Resonance Center, and Neurofarba Department, Università degli Studi di Firenze, 50019 Sesto Fiorentino, Italy
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Srour H, Moussallieh FM, Elbayed K, Giménez-Arnau E, Lepoittevin JP. In Situ Alkylation of Reconstructed Human Epidermis by Methyl Methanesulfonate: A Quantitative HRMAS NMR Chemical Reactivity Mapping. Chem Res Toxicol 2020; 33:3023-3030. [PMID: 33190492 DOI: 10.1021/acs.chemrestox.0c00362] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Allergic contact dermatitis (ACD) is a reaction of the immune system resulting from skin sensitization to an exogenous hazardous chemical and leading to the activation of antigen-specific T-lymphocytes. The adverse outcome pathway (AOP) for skin sensitization identified four key events (KEs) associated with the mechanisms of this pathology, the first one being the ability of skin chemical sensitizers to modify epidermal proteins to form antigenic structures that will further trigger the immune system. So far, these interactions have been studied in solution using model nucleophiles such as amino acids or peptides. As a part of our efforts to better understand chemistry taking place during the sensitization process, we have developed a method based on the use of high-resolution magic angle spinning (HRMAS) NMR to monitor in situ the reactions of 13C substituted chemical sensitizers with nucleophilic amino acids of epidermal proteins in reconstructed human epidermis. A quantitative approach, developed so far for liquid NMR applications, has not been developed to our knowledge in a context of a semisolid nonanisotropic environment like the epidermis. We now report a quantitative chemical reactivity mapping of methyl methanesulfonate (MMS), a sensitizing methylating agent, in reconstructed human epidermis by quantitative HRMAS (qHRMAS) NMR. First, the haptenation process appeared to be much faster in RHE than in solution with a maximum concentration of adducts reached between 4 and 8 h. Second, it was observed that the concentration of cysteine adducts did not significantly increase with the dose (2.07 nmol/mg at 0.4 M and 2.14 nmol/mg at 1 M) nor with the incubation time (maximum of 2.27 nmol/mg at 4 h) compared to other nucleophiles, indicating a fast reaction and a potential saturation of targets. Third, when increasing the exposure dose, we observed an increase of adducts up to 12.5 nmol/mg of RHE, excluding cysteine adducts, for 3112 μg/cm2 (1 M solution) of (13C)MMS. This methodology applied to other skin sensitizers could allow for better understanding of the potential links between the amount of chemical modifications formed in the epidermis in relation to exposure and the sensitization potency.
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Affiliation(s)
- Hassan Srour
- CNRS, Institute of Chemistry UMR 7177, University of Strasbourg, F-67000 Strasbourg, France
| | | | - Karim Elbayed
- CNRS, ICube UMR 7357, University of Strasbourg, F-67000 Strasbourg, France
| | - Elena Giménez-Arnau
- CNRS, Institute of Chemistry UMR 7177, University of Strasbourg, F-67000 Strasbourg, France
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Windler C, Gey C, Seeger K. Skin melanocytes and fibroblasts show different changes in choline metabolism during cellular senescence. Mech Ageing Dev 2017; 164:82-90. [DOI: 10.1016/j.mad.2017.05.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Revised: 04/25/2017] [Accepted: 05/02/2017] [Indexed: 11/25/2022]
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Kaebisch E, Fuss TL, Vandergrift L, Toews K, Habbel P, Cheng LL. Applications of high-resolution magic angle spinning MRS in biomedical studies I-cell line and animal models. NMR IN BIOMEDICINE 2017; 30:10.1002/nbm.3700. [PMID: 28301071 PMCID: PMC5501085 DOI: 10.1002/nbm.3700] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/06/2016] [Revised: 10/04/2016] [Accepted: 12/31/2016] [Indexed: 05/09/2023]
Abstract
High-resolution magic angle spinning (HRMAS) MRS allows for direct measurements of non-liquid tissue and cell specimens to present valuable insights into the cellular metabolisms of physiological and pathological processes. HRMAS produces high-resolution spectra comparable to those obtained from solutions of specimen extracts but without complex metabolite extraction processes, and preserves the tissue cellular structure in a form suitable for pathological examinations following spectroscopic analysis. The technique has been applied in a wide variety of biomedical and biochemical studies and become one of the major platforms of metabolomic studies. By quantifying single metabolites, metabolite ratios, or metabolic profiles in their entirety, HRMAS presents promising possibilities for diagnosis and prediction of clinical outcomes for various diseases, as well as deciphering of metabolic changes resulting from drug therapies or xenobiotic interactions. In this review, we evaluate HRMAS MRS results on animal models and cell lines reported in the literature, and present the diverse applications of the method for the understanding of pathological processes and the effectiveness of therapies, development of disease animal models, and new progress in HRMAS methodology.
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Affiliation(s)
- Eva Kaebisch
- Departments of Radiology and Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114 USA
- Department of Hematology and Oncology, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
| | - Taylor L. Fuss
- Departments of Radiology and Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114 USA
| | - Lindsey Vandergrift
- Departments of Radiology and Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114 USA
| | - Karin Toews
- Departments of Radiology and Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114 USA
- Department of Hematology and Oncology, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
| | - Piet Habbel
- Department of Hematology and Oncology, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
| | - Leo L. Cheng
- Departments of Radiology and Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114 USA
- Corresponding Author: Leo L. Cheng, PhD, 149 13 Street, CNY-6, Charlestown, MA 02129, Ph.617-724-6593, Fax.617-726-5684,
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Bandyopadhaya A, Constantinou C, Psychogios N, Ueki R, Yasuhara S, Martyn JAJ, Wilhelmy J, Mindrinos M, Rahme LG, Tzika AA. Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle. Int J Mol Med 2016; 37:867-78. [PMID: 26935176 PMCID: PMC4790710 DOI: 10.3892/ijmm.2016.2487] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 11/04/2015] [Indexed: 12/18/2022] Open
Abstract
Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also compromising muscle function and dampens the pathogen-induced innate immune response, promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.
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Affiliation(s)
- Arunava Bandyopadhaya
- Department of Surgery, Microbiology and Immunobiology, Harvard Medical School and Molecular Surgery Laboratory, Center for Surgery, Innovation and Bioengineering, Department of Surgery, Massachusetts General and Shriners Burns Hospitals, Harvard Medical School, Boston, MA 02114, USA
| | - Caterina Constantinou
- Department of Surgery, Microbiology and Immunobiology, Harvard Medical School and Molecular Surgery Laboratory, Center for Surgery, Innovation and Bioengineering, Department of Surgery, Massachusetts General and Shriners Burns Hospitals, Harvard Medical School, Boston, MA 02114, USA
| | - Nikolaos Psychogios
- NMR Surgical Laboratory, Center for Surgery, Innovation and Bioengineering, Department of Surgery, Massachusetts General and Shriners Burns Hospitals, Harvard Medical School, Boston, MA 02114, USA
| | - Ryusuke Ueki
- Department of Anesthesiology and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
| | - Shingo Yasuhara
- Department of Anesthesiology and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
| | - J A Jeevendra Martyn
- Department of Anesthesiology and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
| | - Julie Wilhelmy
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Michael Mindrinos
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Laurence G Rahme
- Department of Surgery, Microbiology and Immunobiology, Harvard Medical School and Molecular Surgery Laboratory, Center for Surgery, Innovation and Bioengineering, Department of Surgery, Massachusetts General and Shriners Burns Hospitals, Harvard Medical School, Boston, MA 02114, USA
| | - A Aria Tzika
- NMR Surgical Laboratory, Center for Surgery, Innovation and Bioengineering, Department of Surgery, Massachusetts General and Shriners Burns Hospitals, Harvard Medical School, Boston, MA 02114, USA
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Pelizzo G, Mimmi MC, Ballico M, Marotta M, Goruppi I, Peiro JL, Zambaiti E, Costanzo F, Andreatta E, Tonin E, Calcaterra V. Congenital pulmonary malformations: metabolomic profile of lung phenotype in infants. J Matern Fetal Neonatal Med 2014; 29:143-7. [PMID: 25471172 DOI: 10.3109/14767058.2014.991708] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
BACKGROUND The main hydrosoluble metabolites in three different human congenital pulmonary malformations are described by nuclear magnetic resonance (NMR) spectroscopy. METHODS Bronchogenic cyst (BC), congenital lobar emphysema (CLE) and intrapulmonary sequestration (IPS), were analyzed with respect to a control sample. The extracted metabolites were submitted to high-resolution (1)H NMR-spectroscopy. RESULTS Congenital lung malformations showed free choline, phosphocoline and myoinositol high levels. IPS and CLE were found increased in lactic acid/glucose ratio. Lactic acid and glucose values resulted to be more elevated in control sample. CONCLUSIONS Congenital lung lesions showed different metabolomic profiles useful for early diagnosis.
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Affiliation(s)
- Gloria Pelizzo
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Maria Chiara Mimmi
- b Department of Medical and Biological Sciences , University of Udine , Udine , Italy
| | - Maurizio Ballico
- b Department of Medical and Biological Sciences , University of Udine , Udine , Italy
| | - Mario Marotta
- c Cincinnati Fetal Center, Pediatric Surgery Division, CCHMC , Cincinnati , OH , USA .,d Fetal Surgery Program, Congenital Malformations Research Group, Research Institute of Hospital Universitari Vall d'Hebron , Edifici Infantil , Barcelona , Spain
| | - Ilaria Goruppi
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Jose Louis Peiro
- c Cincinnati Fetal Center, Pediatric Surgery Division, CCHMC , Cincinnati , OH , USA .,d Fetal Surgery Program, Congenital Malformations Research Group, Research Institute of Hospital Universitari Vall d'Hebron , Edifici Infantil , Barcelona , Spain
| | - Elisa Zambaiti
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Federico Costanzo
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Erika Andreatta
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Elena Tonin
- a Department of the Mother and Child Health, Pediatric Surgery Unit , IRCCS Policlinico San Matteo Foundation Pavia and University of Pavia , Pavia , Italy
| | - Valeria Calcaterra
- e Department of the Mother and Child Health, Pediatric Unit , IRCCS Policlinico San Matteo Foundation Pavia , Pavia , Italy , and.,f Department of Internal Medicine University of Pavia , Pavia , Italy
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Abstract
Melanoma is a malignant tumor of melanocytes. Although extensive investigations have been done to study metabolic changes in primary melanoma in vivo and in vitro, little effort has been devoted to metabolic profiling of metastatic tumors in organs other than lymph nodes. In this work, NMR-based metabolomics combined with multivariate data analysis is used to study metastatic B16-F10 melanoma in C57BL/6J mouse spleen. Principal Component Analysis (PCA), an unsupervised multivariate data analysis method, is used to detect possible outliers, while Orthogonal Projection to Latent Structure (OPLS), a supervised multivariate data analysis method, is employed to find important metabolites responsible for discriminating the control and the melanoma groups. Two different strategies, i.e. spectral binning and spectral deconvolution, are used to reduce the original spectral data before statistical analysis. Spectral deconvolution is found to be superior for identifying a set of discriminatory metabolites between the control and the melanoma groups, especially when the sample size is small. OPLS results show that the melanoma group can be well separated from its control group. It is found that taurine, glutamate, aspartate, O-Phosphoethanolamine, niacinamide,ATP, lipids and glycerol derivatives are decreased statistically and significantly while alanine, malate, xanthine, histamine, dCTP, GTP, thymidine, 2'-Deoxyguanosine are statistically and significantly elevated. These significantly changed metabolites are associated with multiple biological pathways and may be potential biomarkers for metastatic melanoma in spleen.
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Affiliation(s)
- Xuan Wang
- Pacific Northwest National Laboratory, Richland, WA 99352, USA
- Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan, 430071, PR China
| | - Mary Hu
- Pacific Northwest National Laboratory, Richland, WA 99352, USA
| | - Ju Feng
- Pacific Northwest National Laboratory, Richland, WA 99352, USA
| | - Maili Liu
- Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan, 430071, PR China
| | - Jian Zhi Hu
- Pacific Northwest National Laboratory, Richland, WA 99352, USA
- To whom correspondence should be addressed: Jian Zhi Hu; ; Phone: (509) 371-6544; Fax: (509) 371-6546
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Mesti T, Savarin P, Triba MN, Le Moyec L, Ocvirk J, Banissi C, Carpentier AF. Metabolic impact of anti-angiogenic agents on U87 glioma cells. PLoS One 2014; 9:e99198. [PMID: 24922514 PMCID: PMC4055646 DOI: 10.1371/journal.pone.0099198] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Accepted: 05/12/2014] [Indexed: 01/02/2023] Open
Abstract
BACKGROUND Glioma cells not only secrete high levels of vascular endothelial growth factor (VEGF) but also express VEGF receptors (VEGFR), supporting the existence of an autocrine loop. The direct impact on glioma cells metabolism of drugs targeting the VEGF pathway, such as Bevacizumab (Bev) or VEGFR Tyrosine Kinase Inhibitor (TKI), is poorly known. MATERIAL AND METHODS U87 cells were treated with Bev or SU1498, a selective VEGFR2 TKI. VEGFR expression was checked with FACS flow cytometry and Quantitative Real-Time PCR. VEGF secretion into the medium was assessed with an ELISA kit. Metabolomic studies on cells were performed using High Resolution Magic Angle Spinning Spectroscopy (HR-MAS). RESULTS U87 cells secreted VEGF and expressed low level of VEGFR2, but no detectable VEGFR1. Exposure to SU1498, but not Bev, significantly impacted cell proliferation and apoptosis. Metabolomic studies with HR MAS showed that Bev had no significant effect on cell metabolism, while SU1498 induced a marked increase in lipids and a decrease in glycerophosphocholine. Accordingly, accumulation of lipid droplets was seen in the cytoplasm of SU1498-treated U87 cells. CONCLUSION Although both drugs target the VEGF pathway, only SU1498 showed a clear impact on cell proliferation, cell morphology and metabolism. Bevacizumab is thus less likely to modify glioma cells phenotype due to a direct therapeutic pressure on the VEGF autocrine loop. In patients treated with VEGFR TKI, monitoring lipids with magnetic resonance spectroscopic (MRS) might be a valuable marker to assess drug cytotoxicity.
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Affiliation(s)
- Tanja Mesti
- Laboratoire de Recherches Biochirurgicales, Université Paris Descartes, Hôpital Européen Georges Pompidou, Paris, France
| | - Philippe Savarin
- Chemistry, Structure and Properties of Biomaterials and Therapeutic Agents, Unité Mixte de Recherche 7244, Centre National de la Recherche Scientifique, Université Paris 13 Sorbonne Paris Cité, Bobigny, France
| | - Mohamed N. Triba
- Chemistry, Structure and Properties of Biomaterials and Therapeutic Agents, Unité Mixte de Recherche 7244, Centre National de la Recherche Scientifique, Université Paris 13 Sorbonne Paris Cité, Bobigny, France
| | - Laurence Le Moyec
- Unité de Biologie Intégrative des Adaptations à l'Exercice, Unité 902, Institut National de la Santé et de la Recherche Médicale, Université d'Evry, Evry, France
| | - Janja Ocvirk
- Division of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
| | - Claire Banissi
- Laboratoire de Recherches Biochirurgicales, Université Paris Descartes, Hôpital Européen Georges Pompidou, Paris, France
| | - Antoine F. Carpentier
- Unité de Formation et de Recherche de Santé, Médecine et Biologie Humaine, Université Paris 13, Bobigny, France
- Hôpital Avicenne, Assistance Publique-Hôpitaux de Paris, Bobigny, France
- * E-mail:
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Studies of Secondary Melanoma on C57BL/6J Mouse Liver Using 1H NMR Metabolomics. Metabolites 2013; 3:1011-35. [PMID: 24958263 PMCID: PMC3937829 DOI: 10.3390/metabo3041011] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2013] [Revised: 09/24/2013] [Accepted: 10/10/2013] [Indexed: 12/20/2022] Open
Abstract
NMR metabolomics, consisting of solid state high resolution magic angle spinning (HR-MAS) 1H-NMR, liquid state high resolution 1H-NMR, and principal components analysis (PCA) has been used to study secondary metastatic B16-F10 melanoma in C57BL/6J mouse liver. The melanoma group can be differentiated from its control group by PCA analysis of the estimates of absolute concentrations from liquid state 1H-NMR spectra on liver tissue extracts or by the estimates of absolute peak intensities of metabolites from 1H HR-MAS-NMR data on intact liver tissues. In particular, we found that the estimates of absolute concentrations of glutamate, creatine, fumarate and cholesterol are elevated in the melanoma group as compared to controls, while the estimates of absolute concentrations of succinate, glycine, glucose, and the family of linear lipids including long chain fatty acids, total choline and acyl glycerol are decreased. The ratio of glycerophosphocholine (GPC) to phosphocholine (PCho) is increased by about 1.5 fold in the melanoma group, while the estimate of absolute concentration of total choline is actually lower in melanoma mice. These results suggest the following picture in secondary melanoma metastasis: Linear lipid levels are decreased by beta oxidation in the melanoma group, which contributes to an increase in the synthesis of cholesterol, and also provides an energy source input for TCA cycle. These findings suggest a link between lipid oxidation, the TCA cycle and the hypoxia-inducible factors (HIF) signal pathway in tumor metastases. Thus, this study indicates that the metabolic profile derived from NMR analysis can provide a valuable bio-signature of malignancy and cell hypoxia in metastatic melanoma.
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Tzika AA, Constantinou C, Bandyopadhaya A, Psychogios N, Lee S, Mindrinos M, Martyn JAJ, Tompkins RG, Rahme LG. A small volatile bacterial molecule triggers mitochondrial dysfunction in murine skeletal muscle. PLoS One 2013; 8:e74528. [PMID: 24098655 PMCID: PMC3787027 DOI: 10.1371/journal.pone.0074528] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Accepted: 08/03/2013] [Indexed: 01/06/2023] Open
Abstract
Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) metabolomics, in vivo31P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by 1H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo31P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections.
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Affiliation(s)
- A. Aria Tzika
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Athinoula A. Martinos Center of Biomedical Imaging, Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
- * E-mail: (AAT); (LGR)
| | - Caterina Constantinou
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
| | - Arunava Bandyopadhaya
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
| | - Nikolaos Psychogios
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Athinoula A. Martinos Center of Biomedical Imaging, Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
| | - Sangseok Lee
- Department of Anesthesiology and Critical Care, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
| | - Michael Mindrinos
- Department of Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America
| | - J. A. Jeevendra Martyn
- Department of Anesthesiology and Critical Care, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
| | - Ronald G. Tompkins
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
| | - Laurence G. Rahme
- Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, United States of America
- Shriners Hospitals for Children Boston, Boston, Massachusetts, United States of America
- * E-mail: (AAT); (LGR)
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He XH, Li WT, Gu YJ, Yang BF, Deng HW, Yu YH, Peng WJ. Metabonomic studies of pancreatic cancer response to radiotherapy in a mouse xenograft model using magnetic resonance spectroscopy and principal components analysis. World J Gastroenterol 2013; 19:4200-4208. [PMID: 23864784 PMCID: PMC3710423 DOI: 10.3748/wjg.v19.i26.4200] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/14/2012] [Accepted: 04/28/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the metabolic profiles of xenograft pancreatic cancer before and after radiotherapy by high-resolution magic angle spinning proton magnetic resonance spectroscopy (HRMAS 1H NMR) combined with principal components analysis (PCA) and evaluate the radiotherapeutic effect.
METHODS: The nude mouse xenograft model of human pancreatic cancer was established by injecting human pancreatic cancer cell SW1990 subcutaneously into the nude mice. When the tumors volume reached 800 mm3, the mice received various radiation doses. Two weeks later, tumor tissue sections were prepared for running the NMR measurements. 1H NMR and PCA were used to determine the changes in the metabolic profiles of tumor tissues after radiotherapy. Metabolic profiles of normal pancreas, pancreatic tumor tissues, and radiation- treated pancreatic tumor tissues were compared.
RESULTS: Compared with 1H NMR spectra of the normal nude mouse pancreas, the levels of choline, taurine, alanine, isoleucine, leucine, valine, lactate, and glutamic acid of the pancreatic cancer group were increased, whereas an opposite trend for phosphocholine, glycerophosphocholine, and betaine was observed. The ratio of phosphocholine to creatine, and glycerophosphocholine to creatine showed noticeable decrease in the pancreatic cancer group. After further evaluation of the tissue metabolic profile after treatment with three different radiation doses, no significant change in metabolites was observed in the 1H NMR spectra, while the inhibition of tumor growth was in proportion to the radiation doses. However, PCA results showed that the levels of choline and betaine were decreased with the increased radiation dose, and conversely, the level of acetic acid was dramatically increased.
CONCLUSION: The combined methods were demonstrated to have the potential for allowing early diagnosis and assessment of pancreatic cancer response to radiotherapy.
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12
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Fedele TA, Galdos-Riveros AC, Jose de Farias e Melo H, Magalhães A, Maria DA. Prognostic relationship of metabolic profile obtained of melanoma B16F10. Biomed Pharmacother 2013; 67:146-56. [DOI: 10.1016/j.biopha.2012.10.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Accepted: 10/23/2012] [Indexed: 12/20/2022] Open
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13
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Davila M, Candiota AP, Pumarola M, Arus C. Minimization of spectral pattern changes during HRMAS experiments at 37 degrees celsius by prior focused microwave irradiation. MAGNETIC RESONANCE MATERIALS IN PHYSICS BIOLOGY AND MEDICINE 2013; 25:401-10. [PMID: 22286777 DOI: 10.1007/s10334-012-0303-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/20/2011] [Revised: 12/21/2011] [Accepted: 01/07/2012] [Indexed: 10/14/2022]
Abstract
OBJECT High-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy provides detailed metabolomic information from intact tissue. However, long acquisition times and high rotation speed may lead to timedependent spectral pattern changes, which may affect proper interpretation of results. We report a strategy to minimize those changes, even at physiological recording temperature. MATERIALS AND METHODS Glioblastoma(Gbm) tumours were induced in 12 mice by stereotactic injection of GL261 cells. Animals were sacrificed and tumours were removed and stored in liquid N2. Half of the samples were exposed to focused microwave (FMW) irradiation prior to HRMAS while the other half was not. Time-course experiments (374 min at 37°C, 9.4T, 3,000 Hz spinning rate) were carried out to monitor spectral pattern changes. Differences were assessed with Unianova test while post-HRMAS histopathology analysis was performed to assess tissue integrity. RESULTS Significant changes (up to 1.7 fold) were observed in samples without FMW irradiation in several spectral regions e.g. mobile lipids/lactate (0.90-1.30 ppm), acetate (1.90 ppm), N-acetyl aspartate (2.00 ppm), and Choline-containing compounds (3.19-3.25 ppm). No significant changes in the spectral pattern of FMW-irradiated samples were recorded. CONCLUSION We describe here a successful strategy to minimize spectral pattern changes in mouse Gbm samples using a FMW irradiation system.
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Affiliation(s)
- Myriam Davila
- Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Edifici Cs, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Valle`s, Spain
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McIntyre DJO, Madhu B, Lee SH, Griffiths JR. Magnetic resonance spectroscopy of cancer metabolism and response to therapy. Radiat Res 2012; 177:398-435. [PMID: 22401303 DOI: 10.1667/rr2903.1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Magnetic resonance spectroscopy allows noninvasive in vivo measurements of biochemical information from living systems, ranging from cultured cells through experimental animals to humans. Studies of biopsies or extracts offer deeper insights by detecting more metabolites and resolving metabolites that cannot be distinguished in vivo. The pharmacokinetics of certain drugs, especially fluorinated drugs, can be directly measured in vivo. This review briefly describes these methods and their applications to cancer metabolism, including glycolysis, hypoxia, bioenergetics, tumor pH, and tumor responses to radiotherapy and chemotherapy.
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Affiliation(s)
- Dominick J O McIntyre
- Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
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15
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The metabolic features of normal pancreas and pancreatic adenocarcinoma: preliminary result of in vivo proton magnetic resonance spectroscopy at 3.0 T. J Comput Assist Tomogr 2011; 35:539-43. [PMID: 21926845 DOI: 10.1097/rct.0b013e318227a545] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVE The objective of the study was to analyze the metabolic features and distribution of normal pancreas and pancreatic adenocarcinoma while determining the biomarker of pancreatic cancerous process. METHODS Twenty-seven control and 29 pancreatic adenocarcinoma patients underwent breath-hold 3-T proton magnetic resonance spectroscopy. The ratios of lipid (lipid/InW), choline-containing compounds (CCCs/InW), and fatty acids (FAs/InW) to nonsaturated internal water (InW) of the normal pancreas head and body-tail region, with cancerous and noncancerous regions in pancreatic adenocarcinoma, were calculated. RESULTS In normal pancreas, there were no statistical difference in the ratios of FAs to InW and lipid to InW of different regions, but CCCs/InW of body-tail area was greater than that of head (7.28 × 10⁻⁴ vs 3.23 × 10⁻⁴). In pancreatic cancer, FAs/InW and lipid/InW between cancerous and noncancerous region were different (3.44 × 10⁻⁴ vs 16.3 × 10⁻⁴ and 7.78 × 10⁻⁴ vs 36.3 × 10⁻⁴, respectively). Choline-containing compounds/InW in cancerous region was smaller than that in noncancerous region of pancreatic head cancer (1.62 × 10⁻⁴ vs 5.69 × 10⁻⁴) but similar to such region in body-tail cancer. Lipid/InW dropped in noncancerous regions (from 0.67 to 0.36), whereas there were no differences in FAs/InW and CCCs/InW between normal pancreas regions and noncancerous regions in pancreatic cancer. CONCLUSIONS In normal pancreas, CCCs of body-tail region was greater than that of head. Whereas in pancreatic adenocarcinoma, CCCs, FAs, and lipid were all decreased in cancerous region, lipid in the noncancerous region was also decreased compared with normal pancreas. Lipid may be the potential sensitive biomarker for pancreatic cancer.
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16
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Keshari KR, Tsachres H, Iman R, Delos Santos L, Tabatabai ZL, Shinohara K, Vigneron DB, Kurhanewicz J. Correlation of phospholipid metabolites with prostate cancer pathologic grade, proliferative status and surgical stage - impact of tissue environment. NMR IN BIOMEDICINE 2011; 24:691-9. [PMID: 21793074 PMCID: PMC3653775 DOI: 10.1002/nbm.1738] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
This study investigates the relationship between phospholipid metabolite concentrations, Gleason score, rate of cellular proliferation and surgical stage in malignant prostatectomy samples by performing one- and two-dimensional, high-resolution magic angle spinning, total correlation spectroscopy, pathology and Ki-67 staining on the same surgical samples. At radical prostatectomy, surgical samples were obtained from 49 patients [41 with localized TNM stage T1 and T2, and eight with local cancer spread (TNM stage T3)]. Thirteen of the tissue samples were high-grade prostate cancer [Gleason score: 4 + 3 (n = 7); 4 + 4 (n = 6)], 22 low-grade prostate cancer [Gleason score: 3 + 3 (n = 17); 3 + 4 (n = 5)] and 14 benign prostate tissues. This study demonstrates that high-grade prostate cancer shows significantly higher Ki-67 staining and concentrations of phosphocholine (PC) and glycerophosphocholine (GPC) than does low-grade prostate cancer (2.4 ± 2.8% versus 7.6 ± 3.5%, p < 0.005, and 0.671 ± 0.461 versus 1.87 ± 2.15 mmolal, p < 0.005, respectively). In patients with local cancer spread, increases in [PC + GPC + PE + GPE] (PE, phosphoethanolamine; GPE, glycerophosphoethanolamine] and Ki-67 index approached significance (4.2 ± 2.5 versus 2.7 ± 2.4 mmolal, p = 0.07, and 5.3 ± 3.8% versus 2.9 ± 3.8%, p = 0.07, respectively). PC and Ki-67 were significantly lower and GPC higher in prostate tissues when compared with cell cultures, presumably because of a lack of important stromal-epithelial interactions in cell cultures. The findings of this study will need to be validated in a larger cohort of surgical patients with clinical outcome data, but support the role of in vivo (1)H MRSI in discriminating between low- and high-grade prostate cancer based on the magnitude of elevation of the in vivo total choline resonance.
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Affiliation(s)
- K R Keshari
- Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, USA
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17
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Abstract
The detailed knowledge of mammalian cell metabolism and its adjustments to different cell properties and perturbations, such as disease and drug exposure, is of enormous value in the deeper understanding of pathological processes and drug mechanisms, as well as in the development of new and improved methods for diagnosis, follow-up of disease progression and treatment response. This review covers recent developments in the use of NMR-based metabonomics to characterize cellular metabolomes and interpret them in terms of metabolic changes taking place in a wide range of situations. The analytical methodology available is briefly presented and the applications developed so far are reviewed. These include differences in cell properties (e.g., drug resistance, cell cycle stage, specific growth conditions and genetic characteristics) and changes induced in response to different perturbations (e.g., disease, drug exposure and irradiation).
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18
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Triba MN, Starzec A, Bouchemal N, Guenin E, Perret GY, Le Moyec L. Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells. NMR IN BIOMEDICINE 2010; 23:1009-1016. [PMID: 20963798 DOI: 10.1002/nbm.1516] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca(2+) chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies.
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Affiliation(s)
- M N Triba
- Universités Paris 13 and Paris 6, Bobigny, France
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19
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Duarte IF, Lamego I, Marques J, Marques MPM, Blaise BJ, Gil AM. Nuclear Magnetic Resonance (NMR) Study of the Effect of Cisplatin on the Metabolic Profile of MG-63 Osteosarcoma Cells. J Proteome Res 2010; 9:5877-86. [DOI: 10.1021/pr100635n] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Iola F. Duarte
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
| | - Inês Lamego
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
| | - Joana Marques
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
| | - M. Paula M. Marques
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
| | - Benjamin J. Blaise
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
| | - Ana M. Gil
- CICECO−Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, R&D Unit “Molecular Physical-Chemistry”, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal, and Université de Lyon, Centre de RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France
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20
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Bayet-Robert M, Loiseau D, Rio P, Demidem A, Barthomeuf C, Stepien G, Morvan D. Quantitative two-dimensional HRMAS 1H-NMR spectroscopy-based metabolite profiling of human cancer cell lines and response to chemotherapy. Magn Reson Med 2010; 63:1172-83. [PMID: 20432288 DOI: 10.1002/mrm.22303] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping.
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21
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Čuperlović-Culf M, Barnett DA, Culf AS, Chute I. Cell culture metabolomics: applications and future directions. Drug Discov Today 2010; 15:610-21. [DOI: 10.1016/j.drudis.2010.06.012] [Citation(s) in RCA: 138] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2009] [Revised: 05/18/2010] [Accepted: 06/23/2010] [Indexed: 01/20/2023]
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22
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RIGHI VALERIA, APIDIANAKIS YIORGOS, MINTZOPOULOS DIONYSSIOS, ASTRAKAS LOUKAS, RAHME LAURENCEG, TZIKA AARIA. In vivo high-resolution magic angle spinning magnetic resonance spectroscopy of Drosophila melanogaster at 14.1 T shows trauma in aging and in innate immune-deficiency is linked to reduced insulin signaling. Int J Mol Med 2010; 26:175-84. [PMID: 20596596 PMCID: PMC3722717 DOI: 10.3892/ijmm_00000450] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2010] [Accepted: 03/12/2010] [Indexed: 01/07/2023] Open
Abstract
In vivo magnetic resonance spectroscopy (MRS), a non-destructive biochemical tool for investigating live organisms, has yet to be used in the fruit fly Drosophila melanogaster, a useful model organism for investigating genetics and physiology. We developed and implemented a high-resolution magic-angle-spinning (HRMAS) MRS method to investigate live Drosophila at 14.1 T. We demonstrated, for the first time, the feasibility of using HRMAS MRS for molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. We showed that the metabolic HRMAS MRS profiles of injured, aged wild-type (wt) flies and of immune deficient (imd) flies were more similar to chico flies mutated at the chico gene in the insulin signaling pathway, which is analogous to insulin receptor substrate1-4 (IRS1-4) in mammals and less to those of adipokinetic hormone receptor (akhr) mutant flies, which have an obese phenotype. We thus provide evidence for the hypothesis that trauma in aging and in innate immune-deficiency is linked to insulin signaling. This link may explain the mitochondrial dysfunction that accompanies insulin resistance and muscle wasting that occurs in trauma, aging and immune system deficiencies, leading to higher susceptibility to infection. Our approach advances the development of novel in vivo non-destructive research approaches in Drosophila, suggests biomarkers for investigation of biomedical paradigms, and thus may contribute to novel therapeutic development.
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Affiliation(s)
- VALERIA RIGHI
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School
- Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital
| | - YIORGOS APIDIANAKIS
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School
- Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Boston, MA 02114,
USA
| | - DIONYSSIOS MINTZOPOULOS
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School
- Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital
| | - LOUKAS ASTRAKAS
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School
- Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital
| | - LAURENCE G. RAHME
- Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Boston, MA 02114,
USA
| | - A. ARIA TZIKA
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School
- Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital
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Lewis IA, Karsten RH, Norton ME, Tonelli M, Westler WM, Markley JL. NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions. Anal Chem 2010; 82:4558-63. [PMID: 20459129 PMCID: PMC2878175 DOI: 10.1021/ac100565b] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2010] [Accepted: 04/27/2010] [Indexed: 12/27/2022]
Abstract
Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure (13)C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional (1)H-(1)H NMR spectra from (12)C- and (13)C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli . We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 muM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling.
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Affiliation(s)
- Ian A. Lewis
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
| | - Ryan H. Karsten
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
| | - Mark E. Norton
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
| | - Marco Tonelli
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
| | - William M. Westler
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
| | - John L. Markley
- National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706
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Righi V, Andronesi O, Mintzopoulos D, Tzika AA. Molecular characterization and quantification using state of the art solid-state adiabatic TOBSY NMR in burn trauma. Int J Mol Med 2010; 24:749-57. [PMID: 19885614 PMCID: PMC3722686 DOI: 10.3892/ijmm_00000288] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
We describe a novel solid-state nuclear magnetic resonance (NMR) method that maximizes the advantages of high-resolution magic-angle-spinning (HRMAS), relative conventional liquid-state NMR approaches, when applied to intact biopsies of skeletal muscle specimens collected from burn trauma patients. This novel method, termed optimized adiabatic TOtal through Bond correlation SpectroscopY (TOBSY) solid-state NMR pulse sequence for two-dimensional (2D)1H-1H homonuclear scalar-coupling longitudinal isotropic mixing, was demonstrated to provide a 40–60% improvement in signal-to-noise ratio (SNR) relative to its liquid-state analogue TOCSY (TOtal Correlation SpectroscopY). Using 1-and 2-dimensional HRMAS NMR experiments, we identified several metabolites in burned tissues. Quantification of metabolites in burned tissues showed increased levels of lipid compounds, intracellular metabolites (e.g., taurine and phosphocreatine) and substantially decreased water-soluble metabolites (e.g., glutathione, carnosine, glucose, glutamine/glutamate and alanine). These findings demonstrate that HRMAS NMR Spectroscopy using TOBSY is a feasible technique that reveals new insights into the pathophysiology of burn trauma. Moreover, this method has applications that facilitate the development of novel therapeutic strategies.
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Affiliation(s)
- Valeria Righi
- NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital and Shriners Burn Institute, Harvard Medical School, Boston, MA 02114, USA
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Guénin S, Morvan D, Thivat E, Stepien G, Demidem A. Combined methionine deprivation and chloroethylnitrosourea have time-dependent therapeutic synergy on melanoma tumors that NMR spectroscopy-based metabolomics explains by methionine and phospholipid metabolism reprogramming. Nutr Cancer 2009; 61:518-29. [PMID: 19838924 DOI: 10.1080/01635580902803727] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met-sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.
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26
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Loiseau D, Morvan D, Chevrollier A, Demidem A, Douay O, Reynier P, Stepien G. Mitochondrial bioenergetic background confers a survival advantage to HepG2 cells in response to chemotherapy. Mol Carcinog 2009; 48:733-41. [PMID: 19347860 DOI: 10.1002/mc.20539] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (DeltaPsim). It thus may be predicted that DeltaPsim regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O(2) consumption/ATP production coupling, DeltaPsim maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered DeltaPsim, leading to an increased energy request to regenerate DeltaPsim in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.
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27
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Bayet-Robert M, Morvan D, Chollet P, Barthomeuf C. Pharmacometabolomics of docetaxel-treated human MCF7 breast cancer cells provides evidence of varying cellular responses at high and low doses. Breast Cancer Res Treat 2009; 120:613-26. [DOI: 10.1007/s10549-009-0430-1] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2009] [Accepted: 05/23/2009] [Indexed: 12/13/2022]
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28
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Duarte IF, Marques J, Ladeirinha AF, Rocha C, Lamego I, Calheiros R, Silva TM, Marques MPM, Melo JB, Carreira IM, Gil AM. Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy. Anal Chem 2009; 81:5023-32. [DOI: 10.1021/ac900545q] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Iola F. Duarte
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Joana Marques
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Ana F. Ladeirinha
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Cláudia Rocha
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Inês Lamego
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Rita Calheiros
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Tânia M. Silva
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - M. Paula M. Marques
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Joana B. Melo
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Isabel M. Carreira
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
| | - Ana M. Gil
- CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, and Research Unit “Molecular Physical-Chemistry”, University of Coimbra, Portugal
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29
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Giraudeau P, Remaud GS, Akoka S. Evaluation of Ultrafast 2D NMR for Quantitative Analysis. Anal Chem 2008; 81:479-84. [DOI: 10.1021/ac8021168] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Patrick Giraudeau
- Université de Nantes, CNRS, CEISAM UMR 6230, B. P. 92208, 2 Rue de la Houssinière, F-44322 Nantes Cedex 03, France
| | - Gérald S. Remaud
- Université de Nantes, CNRS, CEISAM UMR 6230, B. P. 92208, 2 Rue de la Houssinière, F-44322 Nantes Cedex 03, France
| | - Serge Akoka
- Université de Nantes, CNRS, CEISAM UMR 6230, B. P. 92208, 2 Rue de la Houssinière, F-44322 Nantes Cedex 03, France
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30
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Swanson MG, Keshari KR, Tabatabai ZL, Simko JP, Shinohara K, Carroll PR, Zektzer AS, Kurhanewicz J. Quantification of choline- and ethanolamine-containing metabolites in human prostate tissues using 1H HR-MAS total correlation spectroscopy. Magn Reson Med 2008; 60:33-40. [PMID: 18581409 DOI: 10.1002/mrm.21647] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
A fast and quantitative 2D high-resolution magic angle spinning (HR-MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline- and ethanolamine-containing metabolites in human prostate tissues in approximately 1 hr prior to pathologic analysis. At a 40-ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. PC was significantly higher (0.39 +/- 0.40 mmol/kg vs. 0.02 +/- 0.07 mmol/kg, z = 3.5), while ethanolamine (Eth) was significantly lower in cancer versus benign prostate tissues (1.0 +/- 0.8 mmol/kg vs. 2.3 +/- 1.9 mmol/kg, z = 3.3). Glycerophosphocholine (GPC) (0.57 +/- 0.87 mmol/kg vs. 0.29 +/- 0.26 mmol/kg, z = 1.2), phosphoethanolamine (PE) (4.4 +/- 2.2 mmol/kg vs. 3.4 +/- 2.6 mmol/kg, z = 1.4), and glycerophosphoethanolamine (GPE) (0.54 +/- 0.82 mmol/kg vs. 0.15 +/- 0.15 mmol/kg, z = 1.8) were higher in cancer versus benign prostate tissues. The ratios of PC/GPC (3.5 +/- 4.5 vs. 0.32 +/- 1.4, z = 2.6), PC/PE (0.08 +/- 0.08 vs. 0.01 +/- 0.03, z = 3.5), PE/Eth (16 +/- 22 vs. 2.2 +/- 2.0, z = 2.4), and GPE/Eth (0.41 +/- 0.51 vs. 0.06 +/- 0.06, z = 2.6) were also significantly higher in cancer versus benign tissues. All samples were pathologically interpretable following HR-MAS analysis; however, degradation experiments showed that PC, GPC, PE, and GPE decreased 7.7 +/- 2.2%, while Cho+mI and Eth increased 18% in 1 hr at 1 degrees C and a 2250 Hz spin rate.
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Affiliation(s)
- Mark G Swanson
- Department of Radiology, University of California-San Francisco, 1700 4th Street, San Francisco, CA 94158, USA.
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31
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Morvan D, Demidem A. Metabolomics by proton nuclear magnetic resonance spectroscopy of the response to chloroethylnitrosourea reveals drug efficacy and tumor adaptive metabolic pathways. Cancer Res 2007; 67:2150-9. [PMID: 17332345 DOI: 10.1158/0008-5472.can-06-2346] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Metabolomics of tumors may allow discovery of tumor biomarkers and metabolic therapeutic targets. Metabolomics by two-dimensional proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy was applied to investigate metabolite disorders following treatment by chloroethylnitrosourea of murine B16 melanoma (n = 33) and 3LL pulmonary carcinoma (n = 31) in vivo. Treated tumors of both types resumed growth after a delay. Nitrosoureas provoke DNA damage but the metabolic consequences of genotoxic stress are little known yet. Although some differences were observed in the metabolite profile of untreated tumor types, the prominent metabolic features of the response to nitrosourea were common to both. During the growth inhibition phase, there was an accumulation of glucose (more than x10; P < 0.05), glutamine (x3 to 4; P < 0.01), and aspartate (x2 to 5; P < 0.01). This response testified to nucleoside de novo synthesis down-regulation and drug efficacy. However, this phase also involved the increase in alanine (P < 0.001 in B16 melanoma), the decrease in succinate (P < 0.001), and the accumulation of serine-derived metabolites (glycine, phosphoethanolamine, and formate; P < 0.01). This response witnessed the activation of pathways implicated in energy production and resumption of nucleotide de novo synthesis, thus metabolic pathways of DNA repair and adaptation to treatment. During the growth recovery phase, it remained polyunsaturated fatty acid accumulation (x1.5 to 2; P < 0.05) and reduced utilization of glucose compared with glutamine (P < 0.05), a metabolic fingerprint of adaptation. Thus, this study provides the proof of principle that metabolomics of tumor response to an anticancer agent may help discover metabolic pathways of drug efficacy and adaptation to treatment.
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MESH Headings
- Animals
- Antineoplastic Agents/pharmacokinetics
- Carcinoma, Lewis Lung/drug therapy
- Carcinoma, Lewis Lung/metabolism
- Carcinoma, Lewis Lung/pathology
- Drug Resistance, Neoplasm
- Inactivation, Metabolic
- Magnetic Resonance Spectroscopy/methods
- Male
- Melanoma, Experimental/drug therapy
- Melanoma, Experimental/metabolism
- Melanoma, Experimental/pathology
- Metabolic Networks and Pathways
- Mice
- Mice, Inbred C57BL
- Models, Biological
- Neoplasm Transplantation
- Nitrosourea Compounds/pharmacokinetics
- Nitrosourea Compounds/pharmacology
- Proteomics/methods
- Treatment Outcome
- Tumor Cells, Cultured
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Affiliation(s)
- Daniel Morvan
- Institut National de la Santé et de la Recherche Médicale and Centre Jean Perrin, 484 rue Montalembert, F-63005 Clermont-Ferrand, France.
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32
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Swanson MG, Zektzer AS, Tabatabai ZL, Simko J, Jarso S, Keshari KR, Schmitt L, Carroll PR, Shinohara K, Vigneron DB, Kurhanewicz J. Quantitative analysis of prostate metabolites using 1H HR-MAS spectroscopy. Magn Reson Med 2006; 55:1257-64. [PMID: 16685733 DOI: 10.1002/mrm.20909] [Citation(s) in RCA: 182] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
A method was developed to quantify prostate metabolite concentrations using (1)H high-resolution magic angle spinning (HR-MAS) spectroscopy. T(1) and T(2) relaxation times (in milliseconds) were determined for the major prostate metabolites and an internal TSP standard, and used to optimize the acquisition and repetition times (TRs) at 11.7 T. At 1 degrees C, polyamines (PAs; T(1mean) = 100 +/- 13, T(2mean) = 30.8 +/- 7.4) and citrate (Cit; T(1mean) = 237 +/- 39, T(2mean) = 68.1 +/- 8.2) demonstrated the shortest relaxation times, while taurine (Tau; T(1mean) = 636 +/- 78, T(2mean) = 331 +/- 71) and choline (Cho; T(1mean) = 608 +/- 60, T(2mean) = 393 +/- 81) demonstrated the longest relaxation times. Millimolal metabolite concentrations were calculated for 60 postsurgical tissues using metabolite and TSP peak areas, and the mass of tissue and TSP. Phosphocholine plus glycerophosphocholine (PC+GPC), total choline (tCho), lactate (Lac), and alanine (Ala) concentrations were higher in prostate cancer ([PC+GPC](mean) = 9.34 +/- 6.43, [tCho](mean) = 13.8 +/- 7.4, [Lac](mean) = 69.8 +/- 27.1, [Ala](mean) = 12.6 +/- 6.8) than in healthy glandular ([PC+GPC](mean) = 3.55 +/- 1.53, P < 0.01; [tCho](mean) = 7.06 +/- 2.36, P < 0.01; [Lac](mean) = 46.5 +/- 17.4, P < 0.01; [Ala](mean) = 8.63 +/- 4.91, P = 0.051) and healthy stromal tissues ([PC+GPC](mean) = 4.34 +/- 2.46, P < 0.01; [tCho](mean) = 7.04 +/- 3.10, P < 0.01; [Lac](mean) = 45.1 +/- 18.6, P < 0.01; [Ala](mean) = 6.80 +/- 2.95, P < 0.01), while Cit and PA concentrations were significantly higher in healthy glandular tissues ([Cit](mean) = 43.1 +/- 21.2, [PAs](mean) = 18.5 +/- 15.6) than in healthy stromal ([Cit](mean) = 16.1 +/- 5.6, P < 0.01; [PAs](mean) = 3.15 +/- 1.81, P < 0.01) and prostate cancer tissues ([Cit](mean) = 19.6 +/- 12.7, P < 0.01; [PAs](mean) = 5.28 +/- 5.44, P < 0.01). Serial spectra acquired over 12 hr indicated that the degradation of Cho-containing metabolites was minimized by acquiring HR-MAS data at 1 degree C compared to 20 degrees C.
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Affiliation(s)
- Mark G Swanson
- Department of Radiology, University of California-San Francisco, 94107, USA.
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33
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Demidem A, Morvan D, Madelmont JC. Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells: a relay for chemotherapy? Int J Cancer 2006; 119:992-1004. [PMID: 16557598 DOI: 10.1002/ijc.21761] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
In a previous study, it was reported that secondary untreated melanoma tumors implanted several weeks after and at distance from primary chloroethylnitrosourea (CENU)-treated tumors underwent differentiation and growth inhibition. To see whether the primary treated tumor released soluble factors that mediated the secondary tumor response, serum transfer experiments were performed in vivo. Administration of serum from CENU-treated tumor-bearing donors arrested tumor proliferation, decreased vessel formation and induced tumor metabolite alterations encompassing glutathione decrease and polyunsaturated fatty acid and phosphoethanolamine increase. These changes mimicked secondary tumor phenotype. To reproduce the model in vitro, cell culture supernatant transfer experiments were performed. CENU-treated cell cultures showed polyploidy and reactive oxygen species (ROS) production. Cell cultures challenged by a conditioned medium of CENU-treated cells underwent growth inhibition, cytoskeleton disorders, cytokinesis retardation, metabolite alterations, glutathione decrease and phosphoethanolamine increase, without ROS elicitation. Proteomics of CENU-treated cell conditioned media revealed altered protein secretion activity by CENU-treated cells. Among de novo secreted proteins, the most expressed were phosphatidylethanolamine-binding protein (PEBP), cardiovascular heat shock protein (cHsp), Rho-associated coiled-coil forming kinase 2 (ROCK) and actin fragments. These proteins testified of cytoskeleton disorders, growth inhibition and metabolite alterations. This article demonstrates the release by CENU-treated tumors of growth inhibitory differentiation-inducing soluble factors. These factors mediate remote bystander effects and attest persistent biological activity of residual tumors after chemotherapy.
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34
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Morvan D, Demidem A, Guenin S, Madelmont JC. Methionine-dependence phenotype of tumors: Metabolite profiling in a melanoma model usingL-[methyl-13C]methionine and high-resolution magic angle spinning1H–13C nuclear magnetic resonance spectroscopy. Magn Reson Med 2006; 55:984-96. [PMID: 16598721 DOI: 10.1002/mrm.20869] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Tumors frequently have abnormal L-methionine (Met) metabolism, the so-called Met-dependence phenotype that refers to the inability to proliferate in the absence of Met. However, the origin of this phenotype is still unknown and may arise from one of several pathways of Met metabolism. To help characterize the metabolic features of Met-dependent/independent phenotypes, the fate of the methyl carbon of L-[methyl-13C]Met was chased in a murine model of malignant melanoma (B16-F1) in vitro and in vivo. Growth curves under Met restriction showed that melanoma cells in vitro were Met-independent, whereas implanted melanoma tumors in vivo were Met-dependent. Label-assisted high-resolution magic angle spinning 1H-13C NMR spectroscopy metabolite profiling showed that, in vitro, creatine and phosphatidylcholine 13C-enrichments were poor, but S-adenosyl-Met and posttranslationally N-methylated protein signals were strong. In contrast, in vivo, creatine and phosphatidylcholine enrichments were strong but S-adenosyl-Met and N-methylated protein signals were poor. In addition, in vivo, transsulfuration was very efficient, consumed one-carbon units originating from the methyl carbon of Met, and yielded taurine labeling. From these data, the Met-dependent/independent phenotypes appear closely related to the source of one-carbon units. Thus, L-[methyl-13C]Met-assisted NMR spectroscopy metabolite profiling allowed the discrimination between Met-dependence and Met-independence and provided novel mechanistic information on their origin.
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35
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Loening NM, Chamberlin AM, Zepeda AG, Gonzalez RG, Cheng LL. Quantification of phosphocholine and glycerophosphocholine with 31P edited 1H NMR spectroscopy. NMR IN BIOMEDICINE 2005; 18:413-20. [PMID: 16075415 DOI: 10.1002/nbm.973] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
Choline and the related compounds phosphocholine (PC) and glycerophosphocholine (GPC) are considered to be important metabolites in oncology. Past studies have demonstrated correlations linking the relative ratios and concentrations of these metabolites with the development and progression of cancer. Currently, in vivo and tissue ex vivo magnetic resonance spectroscopy methods have mostly centered on measuring the total concentration of these metabolites and have difficulty in differentiating between them. Here, a new scheme that uses (31)P edited (1)H spectroscopy to quantify the concentrations of choline, PC and GPC in biological samples is reported and its applicability is demonstrated using samples of human brain tumor extracts. This method is particularly well-suited for analytical situations where the PC and GPC resonances are not sufficiently resolved and/or are obscured by other metabolites. Consequently, this scheme has the potential to be used for the analysis of choline compounds in ex vivo tissue samples.
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Affiliation(s)
- Nikolaus M Loening
- Department of Chemistry, Lewis & Clark College, Portland, OR 97219, USA.
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36
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Piotto M, Elbayed K, Wieruszeski JM, Lippens G. Practical aspects of shimming a high resolution magic angle spinning probe. JOURNAL OF MAGNETIC RESONANCE (SAN DIEGO, CALIF. : 1997) 2005; 173:84-89. [PMID: 15705516 DOI: 10.1016/j.jmr.2004.11.022] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/07/2004] [Revised: 11/16/2004] [Indexed: 05/24/2023]
Abstract
High resolution magic angle spinning (HRMAS) has become an extremely versatile tool to study heterogeneous systems. HRMAS relies on magic angle spinning of the sample to average out to zero magnetic susceptibility differences in the sample and to obtain resonance linewidths approaching those of liquid state NMR. Shimming such samples therefore becomes an important issue. By analyzing the different sources of magnetic field perturbations present in a sample under MAS conditions, we propose a simple protocol to obtain optimum shim settings in HRMAS. In the case of aqueous samples, we show that the lock level cannot be used as a reliable indicator of the quality of the shims at high spinning speeds. This effect is explained by the presence of temperature gradients imparted by the sample rotation.
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Affiliation(s)
- Martial Piotto
- Institut de Chimie, FRE 2446, 4 rue Blaise Pascal, Université Louis Pasteur, 67084 Strasbourg, France.
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37
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Zektzer AS, Swanson MG, Jarso S, Nelson SJ, Vigneron DB, Kurhanewicz J. Improved signal to noise in high-resolution magic angle spinning total correlation spectroscopy studies of prostate tissues using rotor-synchronized adiabatic pulses. Magn Reson Med 2004; 53:41-8. [PMID: 15690501 DOI: 10.1002/mrm.20335] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
A rotor-synchronized WURST-8 adiabatic pulse scheme was compared to the conventional MLEV-17 hard pulse scheme for isotropic mixing in total correlation spectroscopy (TOCSY) studies of intact human prostate tissues under high-resolution magic angle spinning (HR-MAS) conditions. Both mixing schemes were extremely sensitive to the rotational resonance condition and dramatic reductions in signal to noise were observed when pulse durations deviated from 1/(spin rate). A significant increase in cross-peak intensities was observed using rotor-synchronized WURST-8 adiabatic pulses versus those observed using the rotor-synchronized MLEV-17 hard pulse scheme in both solution and tissue. In tissue, absolute signal intensities ranged from 1.5x to 10.5x greater (average: 4.75x) when WURST-8 was used in place of MLEV-17. Moreover, the difference was so dramatic that several metabolite cross peaks observed using WURST-8 pulses were not observed using MLEV-17 pulses, including cross peaks corresponding to many of the choline- and ethanolamine-containing metabolites. Due to the complex modulation of TOCSY cross peaks for multiply coupled spins and the shorter T(2) relaxation times of tissue metabolites, maximum cross-peak intensities occurred at shorter mixing times than predicted by theory. In summary, a WURST-8 adiabatic mixing scheme produced significantly greater absolute cross-peak signal intensities than MLEV-17 hard pulse mixing, and maximum cross-peak intensity versus mixing time must be established for specific spin systems and T(2) relaxation times.
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Affiliation(s)
- Andrew S Zektzer
- Center for Molecular and Functional Imaging, Department of Radiology, University of California, 185 Berry Street, San Francisco, CA 94017, USA
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38
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Soubias O, Piotto M, Saurel O, Assemat O, Réat V, Milon A. Detection of natural abundance 1H-13C correlations of cholesterol in its membrane environment using a gradient enhanced HSQC experiment under high resolution magic angle spinning. JOURNAL OF MAGNETIC RESONANCE (SAN DIEGO, CALIF. : 1997) 2003; 165:303-308. [PMID: 14643713 DOI: 10.1016/j.jmr.2003.08.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The quality and signal to noise ratio of a J-based HETCOR performed on a standard MAS probe have been compared with a gradient enhanced HSQC performed on a HR-MAS probe at 500 MHz. The sample selected was cholesterol, inserted at 30 mol% in acyl chain deuterated phospholipids (DMPC-d54), at a temperature where the bilayer is in a liquid crystalline phase (310 K). It is representative of any rigid molecule undergoing fast axial diffusion in a bilayer as the main movement. After optimization of the spinning rate and carbon decoupling conditions, it is shown that the ge-HSQC/MAS approach is far superior to the more conventional J-HETCOR/MAS in terms of signal to noise ratio, and that it allows the detection of all the natural abundance cross peaks of cholesterol in a membrane environment. Clear differences between the 1H and 13C chemical shifts of cholesterol in a membrane and in chloroform solution were thus revealed.
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Affiliation(s)
- O Soubias
- Institut de Pharmacologie et de Biologie Structurale, CNRS and UPS, UMR 5089 205 rte de Narbonne, 31077 Toulouse, Cedex 4, France
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