Serum analysis by 1H nuclear magnetic resonance spectroscopy: a new tool for distinguishing neuromyelitis optica from multiple sclerosis.

BACKGROUND: Neuromyelitis optica (NMO) and multiple sclerosis (MS), two inflammatory demyelinating diseases, are characterized by different therapeutic strategies. Currently, the only biological diagnostic tool available to distinguish NMO from MS is the specific serum autoantibody that targets aquaporin 4, but its sensitivity is low. OBJECTIVE: To assess the diagnostic accuracy of metabolomic biomarker profiles in these two neurological conditions, compared to control patients. METHODS: We acquired serum spectra (47 MS, 44 NMO and 42 controls) using proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used multivariate pattern recognition analysis to identify disease-specific metabolic profiles. RESULTS: The (1)H-NMR spectroscopic analysis evidenced two metabolites, originating probably from astrocytes, scyllo-inositol and acetate, as promising serum biomarkers of MS and NMO, respectively. In 87.8% of MS patients, scyllo-inositol increased 0.15 to 3-fold, compared to controls and in 74.3% of NMO patients, acetate increased 0.4 to 7-fold, compared to controls. Using these two metabolites simultaneously, we can discriminate MS versus NMO patients (sensitivity, 94.3%; specificity, 90.2%). CONCLUSION: This study demonstrates the potential of (1)H-NMR spectroscopy of serum as a novel, promising analytical tool to discriminate populations of patients affected by NMO or MS.

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells.

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20 %) as the number of retinal ganglion cells (19 %). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.

Ultrafast in vivo microwave irradiation for enhanced metabolic stability of brain biopsy samples during HRMAS NMR analysis.

High resolution magic-angle spinning (HRMAS) NMR spectroscopy is a well established technique for ex vivo metabolite investigations but experimental factors such as ischemic delay or mechanical stress due to continuous spinning deserve further investigations. Cortical brain samples from rats that underwent ultrafast in vivo microwave irradiation (MWp group) were compared to similar samples that underwent standard nitrogen freezing with and without exposure to domestic microwaves (FN and FN+MWd groups). One dimensional (1)H HRMAS NMR spectra were acquired and 16 metabolites of interest were quantified. Within each group 3 samples underwent long lasting acquisition (up to 15 h). Statistically significant differences in metabolite concentrations were observed between groups for metabolites associated to post mortem biochemical changes and/or anaerobic glycolysis including several neurotransmitters. Spectral assessment over time showed a drastic reduction of biochemical variations in both MW groups. Only 2/16 metabolites exhibited significant signal variations after 15 h of continuous spinning and acquisition in the MWp group. This number increased to 10 in the FN group. We confirmed limited anaerobic metabolism and post mortem degradation after ultra fast in vivo MW irradiation. Furthermore, spectra obtained after MWp and MWd irradiation exhibited an extremely stable spectral pattern over extended periods of continuous acquisition.

Metabolomic Characterization of Ovarian Epithelial Carcinomas by HRMAS-NMR Spectroscopy.

Objectives. The objectives of the present study are to determine if a metabolomic study by HRMAS-NMR can (i) discriminate between different histological types of epithelial ovarian carcinomas and healthy ovarian tissue, (ii) generate statistical models capable of classifying borderline tumors and (iii) establish a potential relationship with patient's survival or response to chemotherapy. Methods. 36 human epithelial ovarian tumor biopsies and 3 healthy ovarian tissues were studied using (1)H HRMAS NMR spectroscopy and multivariate statistical analysis. Results. The results presented in this study demonstrate that the three histological types of epithelial ovarian carcinomas present an effective metabolic pattern difference. Furthermore, a metabolic signature specific of serous (N-acetyl-aspartate) and mucinous (N-acetyl-lysine) carcinomas was found. The statistical models generated in this study are able to predict borderline tumors characterized by an intermediate metabolic pattern similar to the normal ovarian tissue. Finally and importantly, the statistical model of serous carcinomas provided good predictions of both patient's survival rates and the patient's response to chemotherapy. Conclusions. Despite the small number of samples used in this study, the results indicate that metabolomic analysis of intact tissues by HRMAS-NMR is a promising technique which might be applicable to the therapeutic management of patients.

Toward improved grading of malignancy in oligodendrogliomas using metabolomics.

In spite of having been the object of considerable attention, the histopathological grading of oligodendrogliomas is still controversial. The determination of reliable biomarkers capable of improving the malignancy grading remains an essential step in working toward better therapeutic management of patients. Therefore the metabolome of 34 human brain biopsies, histopathologically classified as low-grade (LGO, N = 10) and high-grade (HGO, N = 24) oligodendrogliomas, was studied using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) and multivariate statistical analysis. The classification model obtained afforded a clear distinction between LGOs and HGOs and provided some useful insights into the different metabolic pathways that underlie malignancy grading. The analysis of the most discriminant metabolites in the model revealed the presence of tumoral hypoxia in HGOs. The statistical model was then used to study biopsy samples that were classified as intermediate oligodendrogliomas (N = 6) and glioblastomas (GBMs) (N = 30) by histopathology. The results revealed a gradient of tumoral hypoxia increasing in the following direction: LGOs, intermediate oligodendrogliomas, HGOs, and GBMs. Moreover upon analysis of the clinical evolution of the patients, the metabolic classification seems to provide a closer correlation with the actual patient evolution than the histopathological analysis.

Evidence of secondary structure by high-resolution magic angle spinning NMR spectroscopy of a bioactive peptide bound to different solid supports.

The structure of the 19-amino acid peptide epitope, corresponding to the 141-159 sequence of capsid viral protein VP1 of foot-and-mouth disease virus (FMDV), bound to three different resins, namely, polystyrene-MBHA, PEGA, and POEPOP, has been determined by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. A combination of homonuclear and heteronuclear bidimensional experiments was used for the complete peptide resonance assignment and the qualitative characterization of the peptide folding. The influence of the chemicophysical nature of the different polymers on the secondary structure of the covalently attached FMDV peptide was studied in detail. In the case of polystyrene-MBHA and polyacrylamide-PEGA resins, the analysis of the 2D spectra was hampered by missing signals and extensive overlaps, and only a propensity toward a peptide secondary structure could be derived from the assigned NOE correlations. When the FMDV peptide was linked to the polyoxyethylene-based POEPOP resin, it was found to adopt in dimethylformamide a helical conformation encompassing the C-terminal domain from residues 152 to 159. This conformation is very close to that of the free peptide previously analyzed in 2,2,2-trifluoroethanol. Our study clearly demonstrates that a regular helical structure can be adopted by a resin-bound bioactive peptide. Moreover, a change in the folding was observed when the same peptide-POEPOP conjugate was swollen in aqueous solution, displaying the same conformational features as the free peptide in water. The possibility of studying solid-supported ordered secondary structures by the HRMAS NMR technique in a wide range of solvents can be extended either to other biologically relevant peptides and proteins or to new synthetic oligomers.

Multistep synthesis of 2,5-diketopiperazines on different solid supports monitored by high resolution magic angle spinning NMR spectroscopy.

The solid-phase synthesis of 2,5-diketopiperazines containing the trans-4-hydroxy-L-proline amino acid residue (Hyp) was performed on Ellman polystyrene, polyoxyethylene-polyoxypropylene (POEPOP), polystyrene-polyoxyethylene NovaSyn, and Wang resins, respectively. The reaction pathway allowed the introduction of different functional groups around the bicyclic scaffold in a combinatorial approach, and it generated mixtures of isomers. A detailed characterization of the single reaction steps by high resolution magic angle spinning (HRMAS) NMR spectroscopy was performed. The NMR spectral resolution of the resin-bound intermediates and final products was greatly influenced by the polymer matrix. The POEPOP resin permitted to obtain HRMAS NMR spectra with a resolution comparable with that of the spectra of the molecules in solution. Moreover, configurational and conformational isomers formed during the solid-phase reaction steps could be detected and easily assigned. Therefore, the combination of the HRMAS NMR technique with the use of nonaromatic resins may become an extremely powerful tool in solid-phase organic synthesis. This approach will allow the monitoring of multistep reactions and the conception of on-bead structural studies either on small molecules or on natural and/or synthetic oligomers.

Theoretical study of two-dimensional COSY experiments for S = 7/2 spins: application to (59)Co in the tetrahedral cluster HFeCo(3)(CO)(11)PPh(2)H.

The first investigation and analysis of (59)Co 2D NMR homonuclear chemical shift correlation spectra are reported for the tetrahedral mixed-metal cluster HFeCo(3)(CO)(11)PPh(2)H. For this cluster in solution, the (59)Co 2D COSY and DQF COSY NMR spectra prove the existence of a scalar coupling between (59)Co nuclei. In order to obtain a value of this coupling, the 2D COSY and DQF COSY NMR spectra for a three-spin 7/2 AX(2) system have been simulated by numerical density matrix calculations. The comparison between experimental and theoretical 2D NMR spectra gives a spin-coupling constant |(1)J((59)Co - (59)Co)| = (115 +/- 20) Hz for this cluster. Experimental measurements of T(1) and of the line widths for cobalt 59 as well as theoretical (59)Co 1D NMR spectra are reported and support our findings.

Origin of the residual NMR linewidth of a peptide bound to a resin under magic angle spinning.

The tetrapeptide Ala-lle-Gly-Met bound to a Wang resin via the methionine residue was studied by NMR under MAS conditions and compared to the same peptide in solution. The bound peptide exhibits average linewidths superior to those observed for the peptide in solution. The origin of the residual NMR linewidth observed for the bound form was investigated. The dynamics of the peptide is shown to be only marginally responsible for the increased linewidth; the major cause of the line broadening appears to be nonaveraged magnetic susceptibility differences.

Differential Line Broadening in the Presence of Quadrupolar-CSA Interference

The formalism for calculating the lineshape of a spin 1/2 J-coupled to a high-spin nucleus undergoing quadrupolar and chemical shift anisotropy (CSA) relaxations is derived in the case where the tensors of both interactions are noncoincident and nonaxial. The expressions show that the CSA-quadrupolar interference term which is responsible for the asymmetry of lines involves a term depending on tensorial parameters. The effect of this term on the lineshapes is discussed with respect to three cases, namely coincident-axially symmetric, noncoincident-axially symmetric, and general noncoincident quadrupolar and CSA tensors. These cases are considered in the analysis of the lineshape of the 1H-decoupled spectra of the 31P nucleus J-coupled to the 59Co nucleus encountered in the tetrahedral cluster HFeCo3(CO)11PPh2H. Copyright 1998 Academic Press.