RESUMO
p-Phenylenediamine (PPD) has been classified as a strong skin allergen, but when it comes to toxicological concerns, benzoquinone diamine (BQDI), the primary oxidation derivative of PPD, is frequently considered and was shown to covalently bind nucleophilic residues on model peptides. However, tests in solution are far from providing a reliable model, as the cutaneous metabolism of PPD is not covered. We now report the synthesis of two 13C substituted isotopomers of PPD, 1,4-(13C)p-phenylenediamine 1 and 2,5-(13C)p-phenylenediamine 2, and the investigation of their reactivity in reconstructed human epidermis (RHE) using the high resolution magic angle spinning (HRMAS) NMR technique. RHE samples were first treated with 1 or 2 and incubated for 1 to 48 h. Compared to the control, spectra clearly showed only the signals of 1 or 2 gradually decreasing with time to disappear after 48 h of incubation. However, the culture media of RHE incubated with 1 for 1 and 24 h, respectively, showed the presence of both monoacetylated- and diacetylated-PPD as major products. Therefore, the acetylation reaction catalyzed by N-acetyltransferase (NAT) enzymes appeared to be the main process taking place in RHE. With the aim of increasing the reactivity by oxidation, 1 and 2 were treated with 0.5 equiv of H2O2 prior to their application to RHE and incubated for different times. Under these conditions, new peaks having close chemical shifts to those of PPD-cysteine adducts previously observed in solution were detected. Under such oxidative conditions, we were thus able to detect and quantify cysteine adducts in RHE (maximum of 0.2 nmol/mg of RHE at 8 h of incubation) while no reaction with other nucleophilic amino acid residues could be observed.
Assuntos
Cisteína , Peróxido de Hidrogênio , Acetiltransferases/metabolismo , Alérgenos , Aminoácidos/metabolismo , Benzoquinonas/metabolismo , Meios de Cultura , Cisteína/química , Epiderme/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Fenilenodiaminas/metabolismoRESUMO
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.
Assuntos
Epiderme/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Alquilação , Células Cultivadas , Dermatite Alérgica de Contato/metabolismo , Epiderme/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Metanossulfonato de Metila/síntese química , Metanossulfonato de Metila/química , Estrutura MolecularRESUMO
BACKGROUND: High-resolution magic angle spinning (HRMAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. OBJECTIVES: To assess the impact of chemical skin sensitizers vs non-sensitizers on the metabolome of three-dimensional reconstructed human epidermis (RHE) by HRMAS NMR. METHODS: Based on the SENS-IS assay, 12 skin sensitizers and five non-sensitizing chemicals were investigated and applied on EpiSkin RHE at the published maximal non-irritating concentrations under the conditions of the test. The metabolome of RHE samples was then analyzed by HRMAS NMR. RESULTS: A total of 32 different metabolites were identified; 20 of these were quantified for all samples. Statistical univariate analysis showed that the tissue content of most measured metabolites (with the exception of acetate and glucose) was different in the untreated, treated with non-sensitizers, and treated with sensitizers samples. In RHE samples in contact with sensitizing chemicals, concentrations of 18 metabolites were significantly decreased. Alanine and tyrosine could not discriminate between sensitizer- and non-sensitizer-treated groups. A multivariate partial least-squares-discriminant analysis was performed on the two treated groups, discriminating sensitizing and non-sensitizing chemicals with a very good R2Y value of 0.87 and a good Q2Y value of 0.70. CONCLUSIONS: Data suggest that HRMAS NMR could be used to monitor the impact of chemicals, skin allergens vs non-sensitizers, on the metabolome of three-dimensional RHE.
Assuntos
Alérgenos/toxicidade , Dermatite Alérgica de Contato/metabolismo , Epiderme/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Biomarcadores/metabolismo , Dermatite Alérgica de Contato/etiologia , Análise Discriminante , Humanos , Análise Multivariada , Projetos Piloto , Testes de Irritação da PeleRESUMO
Chemical modification of epidermal proteins by skin sensitizers is the molecular event which initiates the induction of contact allergy. However, not all chemical skin allergens react directly as haptens with epidermal proteins but need either a chemical (prehaptens) or metabolic (prohaptens) activation step to become reactive. Cinnamyl alcohol has been considered a model prohapten, as this skin sensitizer has no intrinsic reactivity. Therefore, the prevailing theory is that cinnamyl alcohol is enzymatically oxidized into the protein-reactive cinnamaldehyde, which is the sensitizing agent. Knowing that reconstructed human epidermis (RHE) models have been demonstrated to be quite similar to the normal human epidermis in terms of metabolic enzymes, use of RHE may be useful to investigate the in situ metabolism/activation of cinnamyl alcohol, particularly when coupled with high-resolution magic angle spinning nuclear magnetic resonance. Incubation of carbon-13 substituted cinnamyl derivatives with RHE did not result in the formation of cinnamaldehyde. The metabolites formed suggest the formation of an epoxy-alcohol and an allylic sulfate as potential electrophiles. These data suggest that cinnamyl alcohol is inducing skin sensitization through a route independent of the one involving cinnamaldehyde and should therefore be considered as a skin sensitizer on its own.
Assuntos
Propanóis/metabolismo , Pele/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Propanóis/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Pele/efeitos dos fármacosRESUMO
BACKGROUND: Methylisothiazolinone (MI) [with methylchloroisothiazolinone (MCI) in a ratio of 1:3, a well-recognized allergenic preservative] was released as an individual preservative in the 2000s for industrial products and in 2005 for cosmetics. The high level of exposure to MI since then has provoked an epidemic of contact allergy to MI, and an increase in MI/MCI allergy. There are questions concerning the MI/MCI cross-reaction pattern. OBJECTIVES: To bring a new perspective on the MI/MCI cross-reactivity issue by studying their in situ chemical behaviour in 3D reconstructed human epidermis (RHE). METHODS: MI and MCI were synthesized with (13) C substitution at positions C-4/C-5 and C-5, respectively. Their in situ chemical behaviours in an RHE model were followed by use of the high-resolution magic angle spinning nuclear magnetic resonance technique. RESULTS: MI was found to react exclusively with cysteine thiol residues, whereas MCI reacted with histidines and lysines. The reaction mechanisms were found to be different for MI and MCI, and the adducts formed had different molecular structures. CONCLUSION: In RHE, different MI/MCI reactions towards different nucleophilic amino acids were observed, making it difficult to explain cross-reactivity between MI and MCI.
Assuntos
Epiderme/química , Tiazóis/química , Reações Cruzadas , Cisteína/química , Histidina/análogos & derivados , Histidina/química , Humanos , Lisina/análogos & derivados , Lisina/química , Espectroscopia de Ressonância Magnética , Compostos de Sulfidrila/química , Tiazóis/imunologiaRESUMO
Adoption of new legislations and social pressure are pushing toward the development of alternative methods to the use of animals for the assessment of most toxicological end-points including skin sensitization. To that respect, much efforts have been put in the first step of the adverse outcome pathway focusing on chemical interactions taking place between sensitizing chemicals or haptens and epidermal proteins. However, these in chemico approaches have been so far only based on the use of model nucleophiles, amino acids, peptides, or proteins in water/buffer solution and focused mainly on thiol reactivity. These studies even if bringing a valuable set of information are very far from reflecting chemical interactions that may happen between a xenobiotic and nucleophiles present in a complex heterogeneous tissue such as the epidermis. Recently, we have shown that using a high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) technique it was possible to characterize chemical interactions taking place between a skin sensitizer and nucleophilic amino acids present in a 3-D reconstructed human epidermis (RHE). We have now compared the chemical reactivity and chemoselectivity of a sensitizing α-methylene-γ-butyrolactone toward human serum albumin used as a model protein and RHE. Using this technique, we showed that amino acid modifications by this hapten was different according to the model used and that in RHE histidine residues seem to have an important role in the formation of adducts. Obviously, the role of histidine in the induction of skin sensitization has been so far neglected and should probably be taken into account for the refinement of in chemico approaches for the detection and potency classification of skin sensitizers.
Assuntos
4-Butirolactona/análogos & derivados , Alérgenos/toxicidade , Aminoácidos/metabolismo , Epiderme/metabolismo , Haptenos/toxicidade , Albumina Sérica/metabolismo , 4-Butirolactona/toxicidade , Alternativas aos Testes com Animais , Humanos , Espectroscopia de Ressonância Magnética/métodosRESUMO
PURPOSE: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies. METHODS: The experiments were conducted on intact lung biopsy samples of pig, rat, mouse, and human using a Bruker Advance III 500 spectrometer. Thirty-five to 39 metabolites were identified and 23 metabolites were quantified. Principal component analysis, partial least-squares discriminant analysis, and analysis of variance tests were performed in order to compare the metabolic profiles of each animal lung biopsies to those of the human lung. RESULTS: The metabolic composition between human and pig lung was similar. However, human lung was distinguishable from mouse and rat regarding: Trimethylamine N-oxide and betaïne which were present in rodents but not in human lung, carnitine, and glycerophosphocholine which were present in mouse but not in human lung. Conversely, succinic acid was undetected in rat lung. Furthermore, fatty acids concentration was significantly higher in rodent lungs compared to human lung. CONCLUSION: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy on lung biopsy, samples allowed to highlight that pig lung seems to be close to human lung as regarding its metabolite composition with more similarities than dissimilarities.
Assuntos
Pulmão/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metaboloma/fisiologia , Camundongos/metabolismo , Ratos/metabolismo , Suínos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Feminino , Humanos , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
High-resolution magic angle spinning (HR-MAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. In this article, we explore a new domain of application of HR-MAS, namely, reconstructed human epidermis (RHE) and the in situ observation of chemical interactions between skin sensitizers and nucleophilic amino acids. First, the preparation, storage, and analysis of RHE were optimized, and this work demonstrated that HR-MAS NMR was well adapted for investigating RHE with spectra of good quality allowing qualitative as well as quantitative studies of metabolites. Second, in order to study the response of RHE to chemical sensitizers, the ((13)C)methyldodecanesulfonate was chosen as an NMR probe, and we compared adducts formed on human serum albumin (HSA) in solution and adducts formed in RHE. Thus, while the modification of proteins or peptides in solution takes several days to lead to a significant amount of modification, in RHE the modifications of nucleophilic amino acids were observable already at 24 h. The chemioselectivity also appeared to be different with major modifications taking place on histidine, methionine, and cysteine residues in RHE, while on HSA, significant modifications were observed on lysine residues with the formation of methylated and dimethylated amino groups. We thus demonstrated that RHE could be used to investigate in situ chemical interactions taking place between skin sensitizers and nucleophilic amino acids. This opens perspectives for the molecular understanding of the skin immune system activation by sensitizing chemicals.
Assuntos
Alérgenos/química , Aminoácidos/química , Alcanossulfonatos/química , Alcanossulfonatos/toxicidade , Alérgenos/metabolismo , Aminoácidos/metabolismo , Isótopos de Carbono/química , Técnicas de Cultura de Células , Linhagem Celular , Cisteína/química , Cisteína/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Histidina/química , Histidina/metabolismo , Humanos , Metionina/química , Metionina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Albumina Sérica/química , Albumina Sérica/metabolismoRESUMO
Standards are needed to control the quality of the lungs from nonheart-beating donors as potential grafts. This was here assessed using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy. Selective perfusion of the porcine bilung block was set up 30 min after cardiac arrest with cold Perfadex®. Lung alterations were analyzed at 3, 6, and 8 h of cold ischemia as compared to baseline and to nonperfused lung. Metabolomics analysis of lung biopsies allowed identification of 35 metabolites. Levels of the majority of the metabolites increased over time at 4°C without perfusion, indicating cellular degradation, whereas levels of glutathione decreased. When lung was perfused at 4°C, levels of the majority of the metabolites remained stable, including levels of glutathione. Levels of uracil by contrast showed a reverse profile, as its signal increased over time in the absence of perfusion while being totally absent in perfused samples. Our results showed glutathione and uracil as potential biomarkers for the quality of the lung. The metabolomics 1H high-resolution magic angle spinning NMR spectroscopy can be efficiently applied for the assessment of the quality of the lung as an original technique characterized by a rapid assessment of intact biopsy samples without extraction and can be implemented in hospital environment.
Assuntos
Transplante de Pulmão , Pulmão/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Metaboloma/fisiologia , Proteoma/análise , Sobrevivência de Tecidos/fisiologia , Animais , Prótons , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Marcadores de Spin , SuínosRESUMO
BACKGROUND: The aim of this preliminary study is to characterize by ¹H high-resolution magic angle spinning NMR spectroscopy (HRMAS) the metabolic content of intact biopsy samples obtained from 12 patients suffering from neuroblastoma (NB). PROCEDURE: The biochemical NB profile was first compared to normal adrenal medulla. In a second step, the relationship between the tumor metabolic profile and the patients' clinical data was investigated. RESULTS: A higher level of creatine, glutamine/glutamate, acetate and glycine characterized NB biopsies while healthy adrenal medulla tissue contained adrenaline and a larger amount of ascorbic acid. Adrenaline, which was undetectable in NB spectra, represented the metabolic signature of normal adrenal medulla. NB from patients younger than 12 months contained a higher level of acetate and lysine. Conversely, higher amounts of glutathione, glutamate, myo-inositol, glycine, serine and ascorbic acid were detected in NB samples belonging to younger children. Glutamine/glutamate, aspartate, creatine, glycine were characteristic of stage I-II NB. Acetate and creatine were characteristic of stage IV NB. Finally, a relatively higher amount of aspartate, succinate, and glutathione was detected in patients alive without active disease after a mean follow-up of 7 years whereas a higher concentration of acetate and taurine was characteristic of patients with worse prognosis. CONCLUSIONS: Our preliminary results suggest the existence of a complex metabolic reality in NB, probably representative of tumor behavior. However, the real impact of these promising results should be assessed by long-term prospective studies on a larger cohort of patients.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodos , Neuroblastoma/metabolismo , Medula Suprarrenal/metabolismo , Biópsia , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estadiamento de Neoplasias , Neuroblastoma/patologiaRESUMO
A novel coil, called Z coil, is presented. Its function is to reduce the strong thermal effects produced by rf heating at high frequencies. The results obtained at 500MHz in a 50 microl sample prove that the Z coil can cope with salt concentrations that are one order of magnitude higher than in traditional solenoidal coils. The evaluation of the rf field is performed by numerical analysis based on first principles and by carrying out rf field measurements. Reduction of rf heating is probed with a DMPC/DHPC membrane prepared in buffers of increasing salt concentrations. The intricate correlation that exists between the magnetic and electric field is presented. It is demonstrated that, in a multiply tuned traditional MAS coil, the rf electric field E(1) cannot be reduced without altering the rf magnetic field. Since the detailed distribution differs when changing the coil geometry, a comparison involving the following three distinct designs is discussed: (1) a regular coil of 5.5 turns, (2) a variable pitch coil with the same number of turns, (3) the new Z coil structure. For each of these coils loaded with samples of different salt concentrations, the nutation fields obtained at a certain power level provide a basis to discuss the impact of the dielectric and conductive losses on the rf efficiency.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Simulação por Computador , Dimiristoilfosfatidilcolina/química , Campos Eletromagnéticos , Membranas Artificiais , Modelos Químicos , Éteres Fosfolipídicos/química , TemperaturaRESUMO
BACKGROUND: Primary hyperparathyroidism (PHPT) may be related to a single gland disease or multiglandular disease, which requires specific treatments. At present, an operation is the only curative treatment for PHPT. Currently, there are no biomarkers available to identify these 2 entities (single vs. multiple gland disease). The aims of the present study were to compare (1) the tissue metabolomics profiles between PHPT and renal hyperparathyroidism (secondary and tertiary) and (2) single gland disease with multiglandular disease in PHPT using metabolomics analysis. METHODS: The method used was (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Forty-three samples from 32 patients suffering from hyperparathyroidism were included in this study. RESULTS: Significant differences in the metabolomics profile were assessed according to PHPT and renal hyperparathyroidism. A bicomponent orthogonal partial least square-discriminant analysis showed a clear distinction between PHPT and renal hyperparathyroidism (R(2)Y = 0.85, Q(2) = 0.63). Interestingly, the model also distinguished single gland disease from multiglandular disease (R(2)Y = 0.96, Q(2) = 0.55). A network analysis was also performed using the Algorithm to Determine Expected Metabolite Level Alterations Using Mutual Information (ADEMA). Single gland disease was accurately predicted by ADEMA and was associated with higher levels of phosphorylcholine, choline, glycerophosphocholine, fumarate, succinate, lactate, glucose, glutamine, and ascorbate compared with multiglandular disease. CONCLUSION: This study shows for the first time that (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy is a reliable and fast technique to distinguish single gland disease from multiglandular disease in patients with PHPT. The potential use of this method as an intraoperative tool requires specific further studies.
Assuntos
Hiperparatireoidismo Primário/metabolismo , Hiperparatireoidismo Secundário/metabolismo , Espectroscopia de Ressonância Magnética , Metabolômica , Adulto , Idoso , Feminino , Humanos , Hiperparatireoidismo Primário/diagnóstico , Hiperparatireoidismo Secundário/diagnóstico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Fígado/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Alanina/metabolismo , Animais , Ácido Aspártico/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Granulócitos/metabolismo , Imuno-Histoquímica , Macrófagos/metabolismo , Masculino , RatosRESUMO
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 and on pulse sequences originally developed for liquid state NMR. In most cases the outcome of the experiment is conform to what is expected from high-resolution liquid state NMR spectroscopy. However in some instances, experiments run under MAS can produce some very puzzling results. After reviewing the basic hardware which is at the heart of HRMAS spectroscopy, we show that the origin of this behavior lies in the natural time-dependence of some physical quantities imparted by the rotation. We focus in particular on the effects of B1 inhomogeneities on the nutation, the (90 degrees)+x-t-(90 degrees )-x and the MLEV16 experiments. Different models of radiofrequency distribution of B1 fields in a solenoidal coil are derived from simple geometrical considerations. These models are shown by NMR spin dynamics calculations to reproduce the experimental NMR results. They are also consistent with electromagnetic simulations of the B1 field distribution inside a solenoidal coil.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Fenômenos Eletromagnéticos , Espectroscopia de Ressonância Magnética/instrumentação , Matemática , Processamento de Sinais Assistido por ComputadorRESUMO
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.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Politetrafluoretileno/química , Processamento de Sinais Assistido por Computador , Temperatura , Água/química , Zircônio/químicaRESUMO
Succinate dehydrogenase gene (SDHx) mutations increase susceptibility to develop pheochromocytomas/paragangliomas (PHEOs/PGLs). In the present study, we evaluate the performance and clinical applications of (1)H high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy-based global metabolomic profiling in a large series of PHEOs/PGLs of different genetic backgrounds. Eighty-seven PHEOs/PGLs (48 sporadic/23 SDHx/7 von Hippel-Lindau/5 REarranged during Transfection/3 neurofibromatosis type 1/1 hypoxia-inducible factor 2α), one SDHD variant of unknown significance, and two Carney triad (CTr)-related tumors were analyzed by HRMAS-NMR spectroscopy. Compared to sporadic, SDHx-related PHEOs/PGLs exhibit a specific metabolic signature characterized by increased levels of succinate (P < .0001), methionine (P = .002), glutamine (P = .002), and myoinositol (P < .0007) and decreased levels of glutamate (P < .0007), regardless of their location and catecholamine levels. Uniquely, ATP/ascorbate/glutathione was found to be associated with the secretory phenotype of PHEOs/PGLs, regardless of their genotype (P < .0007). The use of succinate as a single screening test retained excellent accuracy in distinguishing SDHx versus non-SDHx-related tumors (sensitivity/specificity: 100/100%). Moreover, the quantification of succinate could be considered a diagnostic alternative for assessing SDHx-related mutations of unknown pathogenicity. We were also able, for the first time, to uncover an SDH-like pattern in the two CTr-related PGLs. The present study demonstrates that HRMAS-NMR provides important information for SDHx-related PHEO/PGL characterization. Besides the high succinate-low glutamate hallmark, SDHx tumors also exhibit high values of methionine, a finding consistent with the hypermethylation pattern of these tumors. We also found important levels of glutamine, suggesting that glutamine metabolism might be involved in the pathogenesis of SDHx-related PHEOs/PGLs.
Assuntos
Metaboloma , Metabolômica , Paraganglioma/genética , Paraganglioma/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Redes e Vias Metabólicas , Metabolômica/métodos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , Tomografia por Emissão de Pósitrons , Estrutura Secundária de Proteína , Curva ROC , Sensibilidade e Especificidade , Succinato Desidrogenase/química , Succinato Desidrogenase/deficiência , Ácido Succínico/química , Ácido Succínico/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Rotational resonance and radiofrequency-driven dipolar recoupling (RFDR) experiments have been used to recover the weak proton dipolar interaction present in peptides bound to swollen resins spun at the magic angle. The intensity of the correlation peaks obtained using these sequences is shown to be significantly stronger than the one obtained using the classical NOESY experiment. In addition, it is found that during the relatively long mixing times required to transfer magnetization in such soft materials, the RFDR sequence also achieves magnetization transfer via the scalar J-coupling.
Assuntos
Vírus da Febre Aftosa/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Resinas Vegetais/química , Sequência de AminoácidosRESUMO
In this study, we i) assessed the metabolic profile of the normal adrenal cortex and medulla of adult human subjects by means of (1)H-high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy; ii) compared the biochemical profile of adenoma (Ad), adrenal cortical carcinoma (ACC), and pheochromocytoma (PCC) samples with that of healthy adrenal tissue samples; and iii) investigated the metabolic differences between ACCs and Ads as well as between ACCs and PCCs. Sixty-six tissue samples (13 adrenal cortical tissue, eight medullary tissue, 13 Ad, 12 ACC, and 20 PCC samples) were analyzed. Adrenaline and noradrenaline were undetectable in cortical samples representing the metabolic signature of the tissue derived from neural crest. Similarity between the metabolic profile of Ads and that of the normal adrenal cortex was shown. Inversely, ACC samples clearly made up a detached group exhibiting the typical stigmata of neoplastic tissue such as choline-containing compounds, biochemical markers of anaerobic processes, and increased glycolysis. Significantly higher levels of lactate, acetate, and total choline-containing compounds played a major role in the differentiation of ACCs from Ads. Moreover, the high fatty acid content of ACCs contributed to the cluster identification of ACCs. Of the 14 sporadic PCC samples, 12 exhibited predominant or exclusive noradrenaline secretion. The noradrenaline:adrenaline ratio was inverted in the normal medullary tissue samples. Multiple endocrine neoplasia type 2- and NF1-related PCC samples exhibited both adrenaline and noradrenaline secretion. In the von Hippel-Lindau disease-related PCC samples, only noradrenaline secretion was detected by HRMAS NMR spectroscopy. This study is one of the first applications of metabolomics to adrenal pathophysiology and it is the largest study to report HRMAS NMR data related to the adrenal cortex and adrenal cortical tumors.
Assuntos
Adenoma/metabolismo , Córtex Suprarrenal/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Medula Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Feocromocitoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Metabolômica , Pessoa de Meia-IdadeRESUMO
Paragangliomas (PGLs) are frequently associated with germline mutations in genes involved in energy metabolism. The purpose of the present study was to assess whether the tumor metabolomic profile of patients with hereditary and apparently sporadic PGLs enables the distinction of different subtypes of tumors. Twenty-eight unrelated patients with a histological diagnosis of PGLs were included in the present study. Twelve had germline mutations in SDHx genes (5 SDHB, 7 SDHD), 6 VHL, and 10 were apparently sporadic. Intact tumor samples from these patients (one per patient) were evaluated with (1)H high-resolution magic angle spinning (HRMAS) NMR spectroscopy. SDHx-related tumors were characterized by an increase in succinate levels in comparison to other tumor subtypes (pâ=â0.0001 vs VHL and pâ=â0.000003 vs apparently sporadic). Furthermore, we found significantly lower values of glutamate in SDHx-related tumors compared to other subtypes (pâ=â0.0007 vs VHL and pâ=â0.003 vs apparently sporadic). Moreover, SDHx-tumors also exhibited lower values of ATP/ADP/AMP (pâ=â0.01) compared to VHL. VHL tumors were found to have the highest values of glutathione (GSH) compared to other tumors. Based on 4 metabolites (succinate, glutamate, GSH, and ATP/ADP/AMP), tumors were accurately distinguished from the other ones on both 3- and 2-class PLS-DA models. The present study shows that HRMAS NMR spectroscopy is a very promising method for investigating the metabolomic profile of various PGLs. The present data suggest the existence of a specific succinate-glutamate hallmark of SDHx PGLs. The relevance of such a metabolomic hallmark is expected to be very useful in designing novel treatment options as well as improving the diagnosis and follow-up of these tumors, including metastatic ones.
Assuntos
Ácido Glutâmico/metabolismo , Metabolômica , Paraganglioma/metabolismo , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Humanos , Metabolômica/métodos , Modelos Biológicos , Paraganglioma/genética , Succinato Desidrogenase/genéticaRESUMO
INTRODUCTION: Providing information on cancerous tissue samples during a surgical operation can help surgeons delineate the limits of a tumoral invasion more reliably. Here, we describe the use of metabolic profiling of a colon biopsy specimen by high resolution magic angle spinning nuclear magnetic resonance spectroscopy to evaluate tumoral invasion during a simulated surgical operation. CASE PRESENTATION: Biopsy specimens (n = 9) originating from the excised right colon of a 66-year-old Caucasian women with an adenocarcinoma were automatically analyzed using a previously built statistical model. CONCLUSIONS: Metabolic profiling results were in full agreement with those of a histopathological analysis. The time-response of the technique is sufficiently fast for it to be used effectively during a real operation (17 min/sample). Metabolic profiling has the potential to become a method to rapidly characterize cancerous biopsies in the operation theater.