Methodological Developments for Metabolic NMR Spectroscopy from Cultured Cells to Tissue Extracts: Achievements, Progress and Pitfalls.

This is a broad overview and critical review of a particular group of closely related ex vivo and in vitro metabolic NMR spectroscopic methods. The scope of interest comprises studies of cultured cells and excised tissue, either intact or after physicochemical extraction of metabolites. Our detailed discussion includes pitfalls that have led to erroneous statements in the published literature, some of which may cause serious problems in metabolic and biological interpretation of results. To cover a wide range of work from relevant research areas, we consider not only the most recent achievements in the field, but also techniques that proved to be valid and successful in the past, although they may not have generated a very significant number of papers more recently. Thus, this comparative review also aims at providing background information useful for judiciously choosing between the metabolic ex vivo/in vitro NMR methods presented. Finally, the methods of interest are discussed in the context of, and in relation to, other metabolic analysis protocols such as HR-MAS and cell perfusion NMR, as well as the mass spectrometry approach.

Magnetic resonance spectroscopy in the rodent brain: Experts' consensus recommendations.

In vivo MRS is a non-invasive measurement technique used not only in humans, but also in animal models using high-field magnets. MRS enables the measurement of metabolite concentrations as well as metabolic rates and their modifications in healthy animals and disease models. Such data open the way to a deeper understanding of the underlying biochemistry, related disturbances and mechanisms taking place during or prior to symptoms and tissue changes. In this work, we focus on the main preclinical 1H, 31P and 13C MRS approaches to study brain metabolism in rodent models, with the aim of providing general experts' consensus recommendations (animal models, anesthesia, data acquisition protocols). An overview of the main practical differences in preclinical compared with clinical MRS studies is presented, as well as the additional biochemical information that can be obtained in animal models in terms of metabolite concentrations and metabolic flux measurements. The properties of high-field preclinical MRS and the technical limitations are also described.

A method for multiparametric statistical quantification of the heterogeneity of free Na+ concentration by 19 F MR spectroscopy: Proof of principle in silico and in vitro.

Sodium(I) (Na+ ) is one of the most important cations in mammalian tissues. Since Na+ plays a key role in basic cell function, noninvasive methods for measuring intracellular concentrations of free sodium ions in biological tissue have been developed on the basis of 19 F NMR spectroscopy. However, intracellular Na+ levels are often not uniform throughout a tissue volume (or voxel) being measured. In such cases, [Na+ ] heterogeneity is not reflected in results obtained by the classical technique, and may even result in biased average values. For this reason, we have designed an approach for quantifying [Na+ ] heterogeneity. First, the 19 F MRS resonance from FCrown-1 serving as a "Na+ probe" is transformed into a [Na+ ] curve. Then the digital points of the resulting [Na+ ] profile are used to construct a histogram with specially developed algorithms. From each [Na+ ] histogram, at least eight quantitative parameters describing the underlying statistical [Na+ ] distribution were computed: weighted median, weighted mean, standard deviation, range, mode(s), kurtosis, skewness, and entropy. In addition to our new paradigm, we present a first validation based on (i) computer simulations and (ii) experimentally obtained 19 F MR spectra of model solutions. This basic proof of principle warrants future in vivo experiments, in particular because of its ability to provide quantitative information complementary to that made available by commonly used 23 Na MRI: (i) multiparametric statistical characterization of [Na+ ] distributions; (ii) total [Na+ ] heterogeneity analysis not intrinsically limited by the size of any MRI voxels; and (iii) analysis of unequivocally intracellular [Na+ ], as opposed to measurement of a combination of intra- and extracellular [Na+ ].

Multiparametric statistical quantification of pH heterogeneity by 1 H MRS and MRSI of extracellular pH markers: Proof of principle.

Acid production and transport in numerous biological tissues and medical conditions are active areas of research. Heterogeneity of pH within a given homogeneous-appearing tissue volume has been reported, but none of the conventional methods currently available for measuring tissue pH provides quantitative parameters describing the frequency of occurrence of pH values within such a volume. We have previously presented a multiparametric noninvasive in vivo approach, providing at least 10 different statistical descriptors of pH heterogeneity based on a novel type of line shape analysis developed for pH-sensitive 31 P MRS resonances. However, this method suffers from lack of sensitivity, thus making rapid and spatially resolved measurements difficult. We present here the proof of principle of a new, more sensitive approach to statistical characterization of extracellular pH heterogeneity based on 1 H MRS, with the potential of being combined with spatial resolution. We experimentally study a range of test solutions of a reporter molecule that has previously been shown to possess a 1 H MRS resonance whose chemical shift varies with pH, including when injected intravenously into experimental animals (imidazole ethoxycarbonylpropionic acid, [IEPA]). Statistical pH heterogeneity descriptors are determined for phantoms mimicking tissue pH heterogeneity. To this end, the pH-sensitive 1 H MRS resonance is transformed into a pH curve. Subsequently, the digital points of this pH profile are used to build a histogram using dedicated algorithms. The following descriptors are computed from this histogram: weighted mean pH and median pH, pH standard deviation, pH range, pH mode(s), pH kurtosis, pH skewness and pH entropy. Our new method is also validated by analyzing previously published in vivo MRSI spectra. The proof of principle provided in this work should form the basis of further in vivo studies in physiology and medicine, eg in cancer research, but also in other fields such as kidney and muscle research.

In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH.

We present an investigation of tumor pH regulation, designed to support a new anticancer therapy concept that we had previously proposed. Our study uses a tumor model of ras-transformed hamster fibroblasts, CCL39, xenografted in the thighs of nude mice. We demonstrate, for the first time, that genetic modifications of specific mechanisms of proton production and/or proton transport result in distinct, reproducible changes in intracellular and extracellular tumor pH that can be detected and quantified noninvasively in vivo, simultaneously with determinations of tumor energetic status and necrosis in the same experiment. The CCL39 variants used were deficient in the sodium/proton exchanger, NHE-1, and/or in the monocarboxylate transporter, MCT4; further, variants were deficient in glycolysis or respiration. MCT4 expression markedly increased the gradient between intracellular and extracellular pH from 0.14 to 0.43 when compared to CCL39 wild-type tumors not expressing MCT4. The other genetic modifications studied produced smaller but significant increases in intracellular and decreases in extracellular pH. In general, increased pH gradients were paralleled by increased tumor growth performance and diminished necrotic regions, and 50% of the CCL39 variant expressing neither MCT4 nor NHE-1, but possessing full genetic capacity for glycolysis and oxidative phosphorylation, underwent regression before reaching a 1-cm diameter. Except for CCL39 wild-type tumors, no significant HIF-1α expression was detected. Our in vivo results support a multipronged approach to tumor treatment based on minimizing intracellular pH by targeting several proton production and proton transport processes, among which the very efficient MCT4 proton/lactate co-transport deserves particular attention.

Multiparametric optimization of (31)P NMR spectroscopic analysis of phospholipids in crude tissue extracts. 1. Chemical shift and signal separation.

(31)P NMR spectroscopy is known to be a fast and accurate method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantitated separately in (31)P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. For solvent systems resulting in the formation of two phases, the effects of varying water and methanol content on chemical shift and line width of phospholipid signals have been previously determined. However, little attention has been paid to the influence that other extract components may exert on signal separation. We present, for the first time, a systematic and comprehensive study of (31)P NMR chemical shift as a function of four experimental parameters: (i) extract concentration, (ii) concentration of chelating agent, (iii) pH value of the aqueous component of the solvent system, and (iv) temperature of the NMR measurement. This multiparametric study provides methodological guidelines for predictable and reproducible manipulation of (31)P NMR spectra of brain phospholipids. It also provides a database for rational and efficient optimization of phospholipid spectra from other body tissues, cultured cells, and phospholipid-containing biofluids.

Multiparametric optimization of (31)P NMR spectroscopic analysis of phospholipids in crude tissue extracts. 2. Line width and spectral resolution.

The quality of NMR spectra in general and of spectra to be used for analysis of compound mixtures in particular is essentially defined by two basic parameters: signal-to-noise ratio and spectral resolution. The latter is determined by signal dispersion (chemical shift differences) and line widths. The present study focuses on multiparametric optimization of spectral resolution in (31)P NMR spectra of phospholipids from brain tissue extracts. This report presents, for the first time, a systematic and comprehensive study of phospholipid (31)P NMR line widths as a function of four experimental parameters: (i) extract concentration, (ii) concentration of a chelating agent, (iii) pH of the aqueous component of the solvent system, and (iv) temperature of the NMR measurement. Theoretical underpinnings of observed line width variations (transversal relaxation effects) are briefly discussed. In conjunction with an analogous, concurrently published report on chemical shift effects in the same tissue extract system, this multiparametric line width study provides a complete set of methodological guidelines for (i) generating well-defined tissue extracts, and (ii) choosing matched and optimized measurement conditions for highly reproducible and well-resolved (31)P NMR spectra of brain phospholipids. This study also offers a comprehensive database and a strategy for rational and efficient optimization of phospholipid spectra from other tissue extracts.

Contactless Thermometry by MRI and MRS: Advanced Methods for Thermotherapy and Biomaterials.

Control of temperature variation is of primordial importance in particular areas of biomedicine. In this context, medical treatments such as hyperthermia and cryotherapy, and also the development and use of hydrogel-based biomaterials, are of particular concern. To enable accurate temperature measurement without perturbing or even destroying the biological tissue or material to be monitored, contactless thermometry methods are preferred. Among these, the most suitable are based on magnetic resonance imaging and spectroscopy (MRI, MRS). Here, we address the latest developments in this field as well as their current and anticipated practical applications. We highlight recent progress aimed at rendering MR thermometry faster and more reproducible, versatile, and sophisticated and provide our perspective on how these new techniques broaden the range of applications in medical treatments and biomaterial development by enabling insight into finer details of thermal behavior. Thus, these methods facilitate optimization of clinical and industrial heating and cooling protocols.

Multiparametric quantification of heterogeneity of metal ion concentrations, as demonstrated for [Mg2+] by way of 31P MRS.

Magnesium(II) is the second most abundant intracellular cation in mammals. Non-invasive 31P MRS is currently used to measure intracellular free Mg2+ levels in studies of magnesium deficiency disorders. However, this technique only provides one [Mg2+] value for a given tissue volume (or voxel), based on the chemical shift of the ATP-ß (or NTP-ß) resonance. We present here an approach for quantifying tissue heterogeneity in regard to [Mg2+], by way of multiple 31P MRS-derived descriptors characterizing the statistical intra-volume distribution of free [Mg2+] values. Our novel paradigm exploits the fact that the lineshape of the ATP-ß 31P MRS resonance reflects the statistical distribution of [Mg2+] values within the observed volume (or voxel). Appropriate lineshape analysis reveals multiple quantitative statistical parameters (descriptors) characterizing the [Mg2+] distribution. First, the ATP-ß 31P MRS resonance is transformed into a [Mg2+] curve that is used to construct a histogram with our specially developed algorithms. From this histogram, at least eight [Mg2+] descriptors are computed: weighted mean concentration and median concentration, standard deviation of concentration, range of concentration, concentration mode(s), concentration kurtosis, concentration skewness, and concentration entropy. Comprehensive evaluation based on in silico and experimental models demonstrates the validity of this new method. This basic feasibility study should open new avenues for future in vivo studies in physiology and medicine.

Thermal heterogeneity within aqueous materials quantified by 1H NMR spectroscopy: Multiparametric validation in silico and in vitro.

We recently suggested a new paradigm for statistical analysis of thermal heterogeneity in (semi-)aqueous materials by 1H NMR spectroscopy, using water as a temperature probe. Here, we present a comprehensive in silico and in vitro validation that demonstrates the ability of this new technique to provide accurate quantitative parameters characterizing the statistical distribution of temperature values in a volume of (semi-)aqueous matter. First, line shape parameters of numerically simulated water 1H NMR spectra are systematically varied to study a range of mathematically well-defined temperature distributions. Then, corresponding models based on measured 1H NMR spectra of agarose gel are analyzed. In addition, dedicated samples based on hydrogels or biological tissue are designed to produce temperature gradients changing over time, and dynamic NMR spectroscopy is employed to analyze the resulting temperature profiles at sub-second temporal resolution. Accuracy and consistency of the previously introduced statistical descriptors of temperature heterogeneity are determined: weighted median and mean temperature, standard deviation, temperature range, temperature mode(s), kurtosis, skewness, entropy, and relative areas under temperature curves. Potential and limitations of this method for quantitative analysis of thermal heterogeneity in (semi-)aqueous materials are discussed in view of prospective applications in materials science as well as biology and medicine.

Multiparametric quantification of the heterogeneity of free Ca2+ concentration by 19F MR spectroscopy.

For biological tissue that is heterogeneous with respect to free intracellular Ca2+ concentration ([Ca2+]i), the lineshape of the 19F MRS resonance of injected [Ca2+]-sensitive 4-FBAPTA or BAPTA-FF reflects the statistical distribution of [Ca2+]i values. While conventional 19F MRS of these fluorinated Ca2+ reporter molecules only provides one [Ca2+]i value per spectrum, our specially designed lineshape analysis reveals at least eight quantitative statistical parameters (descriptors) characterizing the [Ca2+]i distribution within the observed tissue volume. To this end, the [Ca2+]-sensitive 19F MRS resonance is transformed into a [Ca2+]i curve. Subsequently, the digital points of this [Ca2+]i profile are used to build a histogram using dedicated algorithms. The following statistical descriptors are computed from this histogram: weighted mean and median, standard deviation, range, mode(s), kurtosis, skewness, and entropy. Our new method is thoroughly validated through in silico and experimental models. The potential of combining statistical [Ca2+] information with spatial resolution is demonstrated by simulated statistical CSI maps. This proof of principle should form the basis of future in vivo studies in physiology and medicine, notably in heart and muscle research.

Multiparametric quantification of thermal heterogeneity within aqueous materials by water 1H NMR spectroscopy: Paradigms and algorithms.

Processes involving heat generation and dissipation play an important role in the performance of numerous materials. The behavior of (semi-)aqueous materials such as hydrogels during production and application, but also properties of biological tissue in disease and therapy (e.g., hyperthermia) critically depend on heat regulation. However, currently available thermometry methods do not provide quantitative parameters characterizing the overall temperature distribution within a volume of soft matter. To this end, we present here a new paradigm enabling accurate, contactless quantification of thermal heterogeneity based on the line shape of a water proton nuclear magnetic resonance (1H NMR) spectrum. First, the 1H NMR resonance from water serving as a "temperature probe" is transformed into a temperature curve. Then, the digital points of this temperature profile are used to construct a histogram by way of specifically developed algorithms. We demonstrate that from this histogram, at least eight quantitative parameters describing the underlying statistical temperature distribution can be computed: weighted median, weighted mean, standard deviation, range, mode(s), kurtosis, skewness, and entropy. All mathematical transformations and calculations are performed using specifically programmed EXCEL spreadsheets. Our new paradigm is helpful in detailed investigations of thermal heterogeneity, including dynamic characteristics of heat exchange at sub-second temporal resolution.

Expression of Cell-Surface Marker ABCB5 Causes Characteristic Modifications of Glucose, Amino Acid and Phospholipid Metabolism in the G3361 Melanoma-Initiating Cell Line.

We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids. Parametric and non-parametric statistical analysis of absolute and relative metabolite levels yielded significant differences for compounds involved in glucose, amino acid and phospholipid (PL) metabolism. By contrast, energy metabolism was virtually unaffected by ABCB5 expression. The sum of water-soluble metabolites per total protein was 17% higher in ABCB5-WT vs. ABCB5-KD G3361 variants, but no difference was found for the sum of PLs. Enhanced abundance was particularly pronounced for lactate (+ 23%) and alanine (+ 26%), suggesting an increase in glycolysis and potentially glutaminolysis. Increases in PL degradation products, glycerophosphocholine and glycerophosphoethanolamine (+ 85 and 123%, respectively), and redistributions within the PL pool suggested enhanced membrane PL turnover as a consequence of ABCB5 expression. The possibility of glycolysis modulation by an ABCB5-dependent IL1ß-mediated mechanism was supported by functional studies employing monoclonal antibody (mAb)-dependent ABCB5 protein inhibition in wildtype G3361 melanoma cells. Our metabolomic results suggest that the underlying biochemical pathways may offer targets for melanoma therapy, potentially in combination with other treatment forms.

Overexpression of catalase or Bcl-2 delays or prevents alterations in phospholipid metabolism during glucocorticoid-induced apoptosis in WEHI7.2 cells.

Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.

Overexpression of catalase or Bcl-2 alters glucose and energy metabolism concomitant with dexamethasone resistance.

Glucocorticoids induce apoptosis in lymphocytes by causing the release of cytochrome c into the cytosol; however, the events in the signaling phase between translocation of the steroid-receptor complex to the nucleus and the release of cytochrome c have not been elucidated. Previously, we found that, in response to steroid treatment, WEHI7.2 mouse thymic lymphoma cells overexpressing catalase (CAT38) show delayed apoptosis (delayed cytochrome c release) compared to the parental cells, while Bcl-2 overexpressing cells (Hb12) are protected from steroid-induced apoptosis. In lymphocytes, glucocorticoid treatment decreases glucose uptake. Both glucose deprivation and the attendant ATP drop are known inducers of apoptosis. Therefore, we used (31)P and (1)H NMR spectroscopy to compare metabolic profiles of WEHI7.2, CAT38 and Hb12 cells in the presence and absence of dexamethasone to determine: (1) whether glucocorticoid effects on glucose metabolism contribute to the mechanism of steroid-induced apoptosis; and (2) whether catalase or Bcl-2 overexpression altered metabolism thereby providing a mechanism of steroid resistance. Loss of mitochondrial hexokinase activity was correlated to the induction of apoptosis in WEHI7.2 and CAT38 cells. CAT38 and Hb12 cells have an altered basal metabolism which includes increases in hexokinase activity, lactate production when subcultured into new medium, use of mitochondria for ATP production and potentially increased glutaminolysis. These data suggest that: (1) glucocorticoid effects on glucose metabolism may contribute to the mechanism of steroid-induced lymphocyte apoptosis; and (2) the altered metabolism seen in catalase and Bcl-2 overexpressing cells may contribute to both the steroid resistance and increased tumorigenicity of these variants.

Increasing the antioxidant defense in WEHI7.2 cells results in a more tumor-like metabolic profile.

Chronic inflammation is often associated with increased cancer frequency. Continuous exposure to reactive oxygen species, as at the site of chronic inflammation, can result in cells with increased antioxidant defense enzymes. In WEHI7.2 cells, overexpression of catalase or thioredoxin by transfection or selection of a cell population resistant to hydrogen peroxide has resulted in WEHI7.2 variants with altered glucose and energy metabolism. This metabolic change would favor survival in a tumor environment. We conclude that metabolic alterations, due to increased antioxidant enzyme expression, may underlie the increased tumorigenicity seen previously in the variants and contribute to the increased tumor risk associated with chronic inflammation.

Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN-gamma/TNF-alpha in HT-29 cells.

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-alpha for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-gamma for 7 h. Perchloric acid extracts were analyzed by (1)H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5'-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-gamma/TNF-alpha treatment, but not in cells which were TNF-alpha-treated without IFN-gamma preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors.

Metabolomic analysis of rat brain by high resolution nuclear magnetic resonance spectroscopy of tissue extracts.

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy.

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell-cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. 31P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in 31P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid 31P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible 31P NMR spectra of phospholipids.

Cerebral biochemical pathways in experimental autoimmune encephalomyelitis and adjuvant arthritis: a comparative metabolomic study.

Many diseases, including brain disorders, are associated with perturbations of tissue metabolism. However, an often overlooked issue is the impact that inflammations outside the brain may have on brain metabolism. Our main goal was to study similarities and differences between brain metabolite profiles of animals suffering from experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rat models. Our principal objective was the determination of molecular protagonists involved in the metabolism underlying these diseases. EAE was induced by intraplantar injection of complete Freund's adjuvant (CFA) and spinal-cord homogenate (SC-H), whereas AA was induced by CFA only. Naive rats served as controls (n = 9 for each group). Two weeks after inoculation, animals were sacrificed, and brains were removed and processed for metabolomic analysis by NMR spectroscopy or for immunohistochemistry. Interestingly, both inflammatory diseases caused similar, though not identical, changes in metabolites involved in regulation of brain cell size and membrane production: among the osmolytes, taurine and the neuronal marker, N-acetylaspartate, were decreased, and the astrocyte marker, myo-inositol, slightly increased in both inoculated groups compared with controls. Also ethanolamine-containing phospholipids, sources of inflammatory agents, and several glycolytic metabolites were increased in both inoculated groups. By contrast, the amino acids, aspartate and isoleucine, were less concentrated in CFA/SC-H and control vs. CFA rats. Our results suggest that inflammatory brain metabolite profiles may indicate the existence of either cerebral (EAE) or extra-cerebral (AA) inflammation. These inflammatory processes may act through distinct pathways that converge toward similar brain metabolic profiles. Our findings open new avenues for future studies aimed at demonstrating whether brain metabolic effects provoked by AA are pain/stress-mediated and/or due to the presence of systemic proinflammatory molecules. Regardless of the nature of these mechanisms, our findings may be of interest for future clinical studies, e.g. by in-vivo magnetic resonance spectroscopy.