Spatial Distribution Of Metabolites Research Articles

Light-Dependent Changes in the Spatial Localization of Metabolites in Solenostemon scutellarioides (Coleus Henna) Visualized by Matrix-Free Atmospheric Pressure Electrospray Laser Desorption Ionization Mass Spectrometry Imaging.

The visualization of foliage color in plants provides immediate insight into some of the compounds that exist in the leaf. However, many non-colored compounds are also present; their cellular distributions are not readily identifiable optically. In this study we evaluate the applicability of mass spectrometry imaging (MSI) via electrospray laser desorption ionization (ELDI) to reveal the spatial distribution of metabolites. ELDI-MSI is a matrix free, atmospheric pressure ionization method that utilizes a UV laser coupled with supplemental ionization by electrospray. We specifically applied ELDI-MSI to determine the spatial distribution of metabolites in Coleus Henna half leaves that were grown with half-sections either fully illuminated or shaded. We monitored dynamic changes in the spatial distribution of metabolites in response to the change of illumination every 7 days for a 28 day period. A novel source-sink relationship was observed between the 2 halves of the experimental leaf. Furthermore, Coleus Henna leaves present visually recognizable sectors associated with the differential accumulation of flavonoids. Thus, we correlated the effect of differential illumination and presence or absence of flavonoids with metabolic changes revealed by the accumulation of carbohydrates, amino acids, and organic acids. The results show the potential of ELDI-MSI to provide spatial information for a variety of plant metabolites with little sample preparation.

Imaging mass spectrometry-guided fast identification of antifungal secondary metabolites from Penicillium polonicum.

The discovery of antibiotics from microorganisms using classic bioactivity screens suffers from heavy labor and high re-discovery rate. Recently, largely uncovered biosynthetic potentials were unveiled by new approaches, such as genetic manipulation of "silent" biosynthetic gene clusters, innovative data acquisition, and processing methods. In this work, a fast and efficient antibiotic identification pipeline based on the MALDI-TOF imaging mass spectrometry was applied to study the antifungal metabolites during the confrontation of two fungal species, Penicillium polonicum and wilt-inducing fungus Fusarium oxysporum. By visualizing the spatial distribution of metabolites directly on the microbial colony and surrounding media, we predicted the antifungal candidates before isolating pure compounds and individually testing their bioactivity, which subsequently guided the identification of target molecules using classic chromatographic methods. Via this procedure, we successfully identified two antifungal metabolites, fructigenine A and B, which belong to indole alkaloid class and were not reported for antifungal activity. Our work assigned new bioactivity to previously reported compounds and more importantly showed the efficiency of this approach towards quick discovery of bioactive compounds, which can help study the vast unexploited synthetic potential of microbial secondary metabolites.

High-mass-resolution MALDI mass spectrometry imaging reveals detailed spatial distribution of metabolites and lipids in roots of barley seedlings in response to salinity stress

IntroductionMass spectrometry imaging (MSI) is a technology that enables the visualization of the spatial distribution of hundreds to thousands of metabolites in the same tissue section simultaneously. Roots are below-ground plant organs that anchor plants to the soil, take up water and nutrients, and sense and respond to external stresses. Physiological responses to salinity are multifaceted and have predominantly been studied using whole plant tissues that cannot resolve plant salinity responses spatially.ObjectivesThis study aimed to use a comprehensive approach to study the spatial distribution and profiles of metabolites, and to quantify the changes in the elemental content in young developing barley seminal roots before and after salinity stress.MethodsHere, we used a combination of liquid chromatography–mass spectrometry (LC–MS), inductively coupled plasma mass spectrometry (ICP–MS), and matrix-assisted laser desorption/ionization (MALDI–MSI) platforms to profile and analyze the spatial distribution of ions, metabolites and lipids across three anatomically different barley root zones before and after a short-term salinity stress (150 mM NaCl).ResultsWe localized, visualized and discriminated compounds in fine detail along longitudinal root sections and compared ion, metabolite, and lipid composition before and after salt stress. Large changes in the phosphatidylcholine (PC) profiles were observed as a response to salt stress with PC 34:n showing an overall reduction in salt treated roots. ICP–MS analysis quantified changes in the elemental content of roots with increases of Na+ and decreases of K+ content.ConclusionOur results established the suitability of combining three mass spectrometry platforms to analyze and map ionic and metabolic responses to salinity stress in plant roots and to elucidate tolerance mechanisms in response to abiotic stress, such as salinity stress.

METABOLOMIC PROFILE AT THE DIFFERENT STAGES OF PRUNELLA VULGARIS (LAMIACEAE) ONTOGENESIS AT GROWING IN THE CLIMATE CHAMBER

By gaz chromatography-mass spectrometry (GC-MS) technique based metabolite profiling of the methanol extract derived from leaves of Prunella vulgaris L. (Lamiaceae) was carried out. P. vulgaris was grown in climatic chamber. For every metabolomics profile, we estimated 102 target substances, and 41 of them were identified. Metabolite spectrum on vegetation stage is different than other stage by absent of groups: amino acids, terpenes and lipids. Comparison of the results of statistical analysis based on the data array, including all detected metabolites (metabolite profiling) show, that metabolomes of blossom and fructification stage of plants match often and only a small number of metabolites P. vulgaris at the this stage make it specific. It was studied the spatial heterogeneity in the structure of the metabolic network. The results of it are showed that metabolomes variety of the leaves from one plants much more than local variety in the range of parts of one leaf. Spatial distributions of metabolites of heterogeneity in a leaf have been illustrated on the example of threonic acid.

In Vivo 1H Magnetic Resonance Spectroscopy.

In vivo Magnetic Resonance Spectroscopy (MRS) allows the non-invasive detection and quantification of a number of metabolites from localized volumes within a living organism. MRS localization techniques can be divided into two main groups, single voxel and multi-voxel. Single voxel techniques provide the metabolic profile from a specific small volume, whereas multi-voxel techniques are used to obtain the spatial distribution of metabolites throughout a large volume subdivided into small contiguous voxels. This chapter describes standard protocols for the acquisition and processing of in vivo single voxel1H MRS data from the rodent brain.

The Spatial Distribution of Alkaloids in Psychotria prunifolia (Kunth) Steyerm and Palicourea coriacea (Cham.) K. Schum Leaves Analysed by Desorption Electrospray Ionisation Mass Spectrometry Imaging.

Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques. To apply DESI-MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC-MS and histochemical analyses. Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI-MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC-MS/MS analysis performed using multiple-reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer. Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10-hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10-hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface. Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC-MS analysis and histochemical results. The DESI-MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd.

Spatially resolved metabolic distribution for unraveling the physiological change and responses in tomato fruit using matrix-assisted laser desorption/ionization\u2013mass spectrometry imaging (MALDI\u2013MSI)

Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI–MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI–MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI–MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations.Graphical abstractᅟ

Spatial mapping of lichen specialized metabolites using LDI-MSI: chemical ecology issues for Ophioparma ventosa.

Imaging mass spectrometry techniques have become a powerful strategy to assess the spatial distribution of metabolites in biological systems. Based on auto-ionisability of lichen metabolites using LDI-MS, we herein image the distribution of major secondary metabolites (specialized metabolites) from the lichen Ophioparma ventosa by LDI-MSI (Mass Spectrometry Imaging). Such technologies offer tremendous opportunities to discuss the role of natural products through spatial mapping, their distribution patterns being consistent with previous chemical ecology reports. A special attention was dedicated to miriquidic acid, an unexpected molecule we first reported in Ophioparma ventosa. The analytical strategy presented herein offers new perspectives to access the sharp distribution of lichen metabolites from regular razor blade-sectioned slices.

Spatial distribution of metabolites in the human lens

Spatial distribution of 34 metabolites along the optical and equatorial axes of the human lens has been determined. For the majority of metabolites, the homogeneous distribution has been observed. That suggests that the rate of the metabolite transformation in the lens is low due to the general metabolic passivity of the lens fiber cells. However, the redox processes are active in the lens; as a result, some metabolites, including antioxidants, demonstrate the “nucleus-depleted” type of distribution, whereas secondary UV filters show the “nucleus-enriched” type. The metabolite concentrations at the lens poles and equator are similar for all metabolites under study. The concentric pattern of the “nucleus-depleted” and “nucleus-enriched” distributions testifies that the metabolite distribution inside the lens is mostly governed by a passive diffusion, relatively free along the fiber cells and retarded in the radial direction across the cells. No significant difference in the metabolite distribution between the normal and cataractous human lenses was found.

Imaging Mass Spectrometric Analysis of Neurotransmitters: A Review.

Imaging mass spectrometry (IMS) is a toolbox of versatile techniques that enable us to investigate analytes in samples at molecular level. In recent years, IMS, and especially matrix-assisted laser desorption/ionisation (MALDI), has been used to visualise a wide range of metabolites in biological samples. Simultaneous visualisation of the spatial distribution of metabolites in a single sample with little tissue disruption can be considered as one important advantage of MALDI over other techniques. However, several technical hurdles including low concentrations and rapid degradation rates of small molecule metabolites, matrix interference of signals and poor ionisation, need to be addressed before MALDI can be considered as a reliable tool for the analysis of metabolites such as neurotransmitters in brain tissues from different sources including humans. In the present review we will briefly describe current MALDI IMS techniques used to study neurotransmitters and discuss their current status, challenges, as well as future prospects.

‘Plug and Play’ assembly of a low-temperature plasma ionization mass spectrometry imaging (LTP-MSI) system

Mass spectrometry imaging (MSI) is of high and growing interest in life science research, but the investment for necessary equipment is often prohibitive for small research groups. Therefore, we developed a basic MSI system from low cost 'Plug and Play' components, which are connected to the Universal Serial Bus (USB) of a standard computer. Our open source software OpenMZxy (http://www.bioprocess.org/openmzxy) enables automatic and manual sampling, as well as the recording of position data. For ionization we used a low-temperature plasma probe (LTP), coupled to a quadrupole mass analyzer. The current set-up has a practical resolution of 1mm, and a sampling area of 100×100mm, resulting in up to 10,000 sampling points. Our prototype is easy and economical to adopt for different types of mass analyzers. We prove the usability of the LTP-MSI system for macroscopic samples by imaging the distribution of metabolites in the longitudinal cross-cut of a chili (Capsicum annuum, 'Jalapeño pepper') fruit. The localization of capsaicin in the placenta could be confirmed. But additionally, yet unknown low molecular weight compounds were detected in defined areas, which underline the potential of LTP-MSI for the imaging of volatile and semi-volatile metabolites and for the discovery of new natural products. Biological significance Knowledge about the spatial distribution of metabolites, proteins, or lipids in a given tissue often leads to novel findings in medicine and biology. Therefore, mass spectrometry based imaging (MSI) is becoming increasingly popular in life science research. However, the investment for necessary equipment is often prohibitive for small research groups. We built a prototype with an ambient ionization source, which is easy and economical to adopt for different types of mass analyzers. Therefore, we hope that our system contributes to a broader use of mass spectrometry imaging for answering biological questions.

Abstract 719: Accumulation of amino acids in metastatic foci of human colon cancer xenografts revealed by newly developed method for imaging mass spectrometry of amino acids.

Abstract MALDI imaging mass spectrometry (IMS) provides information about the spatial distribution of metabolites within thin slices of tissue, and has been used to elucidate complex phenotypes under various physiological conditions. Previously, our group reported distinct spatial distribution of a variety of metabolites in experimental model of hepatic micrometastasis of the solid tumor. However the metabolic properties in tumor and host tissues are not fully characterized, because of difficulty in visualizing most amino acids, due to their lower ionization efficiency. Here, by using of p-N,N,N-trimethylammonioanily N’-hydroxysuccinimidyl carbamate iodide (TAHS) as a derivatizing reagent, we have successfully developed the method for the detection of various amino acids simultaneously by MALDI-MS on tissue section. We prepared liver section from hepatic metastasis model of human colon cancer, and then performed MALDI-IMS. To compare the signal intensities of amino acids among the different sections, we normalized the MALDI-IMS data with the quantified value of each amino acid obtained from capillary electrophoresis-mass spectrometry. The result indicated the amount of glycine, phenylalanine, leucine, glutamate, and glutamine were significantly elevated in metastatic foci compared to liver parenchyma. Other metabolites such as ATP and glutathione also accumulated in metastatic foci, and these results suggested that increased amino acid pool were required to maintain ATP and GSH levels in tumor. In conclusion, we have demonstrated that novel imaging technique by using of IMS combined with on-tissue TAHS derivatization enables the visualization of amino acid distribution in tissue. The current method could be powerful tool to elucidate the mechanism for metabolic changes of cancer disease in vivo. Citation Format: Sakino Toue, Yuki Sugiura, Akiko Kubo, Mitsuyo Ohmura, Sachise Karakawa, Toshimi Mizukoshi, Junya Yoneda, Hiroshi Miyano, Yasushi Noguchi, Tsuyoshi Kobayashi, Yasuaki Kabe, Makoto Suematsu. Accumulation of amino acids in metastatic foci of human colon cancer xenografts revealed by newly developed method for imaging mass spectrometry of amino acids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 719. doi:10.1158/1538-7445.AM2013-719

Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy

Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm−1 phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles.

Mass Spectrometry Based Imaging Techniques for Spatially Resolved Analysis of Molecules

Higher plants are composed of a multitude of tissues with specific functions, reflected by distinct profiles for transcripts, proteins, and metabolites. Comprehensive analysis of metabolites and proteins has advanced tremendously within recent years, and this progress has been driven by the rapid development of sophisticated mass spectrometric techniques. In most of the current “omics”-studies, analysis is performed on whole organ or whole plant extracts, rendering to the loss of spatial information. Mass spectrometry imaging (MSI) techniques have opened a new avenue to obtain information on the spatial distribution of metabolites and of proteins. Pioneered in the field of medicine, the approaches are now applied to study the spatial profiles of molecules in plant systems. A range of different plant organs and tissues have been successfully analyzed by MSI, and patterns of various classes of metabolites from primary and secondary metabolism could be obtained. It can be envisaged that MSI approaches will substantially contribute to build spatially resolved biochemical networks.

In vivo magnetic resonance spectroscopy of cancer

Magnetic resonance (MR) methods provide anatomical, physiological and metabolic assessment of the cancer non- invasively. In vivo MR spectroscopy (MRS) provides information on endogenous metabolites present in tissues which is useful not only for diagnosis but also for monitoring the tumor response to therapy. Technical developments in field of in vivo MRS have resulted in mapping spatial distribution of metabolites (spectroscopic imaging), improvement in spectral sensitivity and resolution, and determination of absolute concentration of metabolites. The localized proton MRS first reported two decades ago has now emerged as a non-invasive methodology, to be used in clinical settings for the evaluation of brain and other tumors. The method has shown tremendous potential in the assessment of cancer of different organs, notably, in brain, breast and prostate cancers. Recent developments in MRS technology has made possible to expand its application to other malignancies such as musculoskeletal system, head and neck, and liver. In this review we briefly summarize the potential applications of MRS in diagnosis, staging, monitoring treatment, guiding biopsies and prediction of malignancy in brain, breast and prostate cancers. We also present briefly the current status and limitations of the applications of MRS in musculoskeletal system and liver, as two examples.

Plant metabolomics--meeting the analytical challenges of comprehensive metabolite analysis

Plant metabolomics has benefited from a rich array of pre-existing methodological approaches and bioanalytical knowledge for the characterization of the many chemically diverse classes of metabolites. While the field has pushed the implementation of unbiased and generally applicable strategies for metabolite extraction, fractionation and detection, significant challenges in fundamental activities such as compound identification and quantification still exist. This review provides an introduction to metabolomics terminology, methods and resources and discusses analytical limitations. Progress in the application of stable isotope dilution and stable isotopic metabolic labeling to metabolite identification and quantification are discussed. New strategies to address spatial distribution of metabolites via mass spectrometry-based imaging are also reviewed.

Co-localisation of hypoxia and perfusion markers with parameters of glucose metabolism in human squamous cell carcinoma (hSCC) xenografts

Purpose: To examine relationships between tumour hypoxia, perfusion and metabolic microenvironment at the microregional level in three different human squamous cell carcinomas (hSCC).Materials and methods: Nude mice bearing FaDu, UT-SCC-15, and UT-SCC-5 hSCC were injected with pimonidazole hypoxia and Hoechst perfusion markers. Bioluminescence imaging was used to determine spatial distribution of glucose and lactate content in serial tumour sections. Metabolite levels were grouped in 10 concentration ranges. Images were co-registered and at each concentration range the proportion of area stained for pimonidazole and Hoechst was determined in 11–13 tumours per tumour line.Results: The spatial distribution of metabolites in pimonidazole hypoxic and Hoechst perfused areas is characterised by pronounced heterogeneity. In all three tumour lines glucose concentration decreased with increasing pimonidazole hypoxic fraction and increased with increasing perfused area at the microregional level. A weak albeit significant positive correlation between lactate concentration and pimonidazole hypoxic fraction was found only in UT-SCC-5. Lactate concentration consistently decreased with increasing perfused area in all three tumour lines.Conclusions: Both glucose consumption and supply may contribute to the microregional glucose levels. Microregional lactate accumulation in tumours may be governed by clearance potential. The extent of microregional hypoxia cannot be predicted from the lactate concentration indicating that both parameters need to be measured independently.

TH-D-304A-05: Novel Hyperpolarized C-13 Metabolic Imaging Technique to Monitor Radiation-Induced Tissue Damage in Vivo at Moderate Radiation Doses

Purpose: Recent introduction of nuclear hyperpolarization technique to liquid state enabled in vivo detection of C labeled endogenous metabolites with MR spectroscopy imaging (MRSI). We investigate feasibility of employing this novel technique to assess radiation-induced tissue damage, which could result in radiotherapy, at moderate doses. Method and Materials: In this study we used Wistar male rats weighing 200–450 g to make the model by unilaterally irradiating one kidney with a collimated beam of X-rays (200 kVp, HVL 1.0 mm Cu). Kidney was chosen because it is a radiosensitive organ and provides internal control. Initially, rats received dose of 15 Gy and were subjected to magnetic resonance spectroscopy imaging (MRSI) at 21 days of postirradiation time. In MRSI, rats were intravenously injected with a hyperpolarized solution of 80 mM []pyruvate. Hyperpolarization was performed by using DNP technique in HyperSense™ polarizer (Oxford instruments). Fast chemical shift imaging (CSI) was performed at 3 T to acquire axial slices through both kidneys in every 6th second to measure spatial distribution of metabolites, pyruvate, lactate, and alanine. Animals were sacrificed after imaging to harvest kidney tissues for histopathology. Results: MR spectra in kidneys clearly show metabolism of pyruvate and its endogenous downstream products, lactate and alanine. Perfusion was visible as initial high concentration of pyruvate in vena cava vain subsequently distributed in kidneys producing lactate and alanine. We observe low lactate production is in the irradiated kidney in comparison to non-irradiated kidney. Further analysis is being performed, including histopathology, to justify imaging results. Conclusion: In over preliminary data, observed reduction of lactate production in the irradiated kidney in comparison to non-irradiated kidney suggests potential use of hyperpolarized metabolic imaging technique to distinguish and assess radiation-induced tissue damage in kidneys. Further analysis is necessary to assess radiation effects to confirm observations.

Imaging of Metabolites by MALDI Mass Spectrometry

Visualization of endogenous molecules in small experimental animals is very important in order to investigate the localization of specific molecules. Conventional imaging techniques are not suitable for simultaneous visualization of endogenous molecules. We recently developed a new imaging method-imaging mass spectrometry (IMS) using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF). In this study, we applied IMS using MALDI-QIT-TOF to visualize the metabolites in medaka (Oryzias latipes) sections and determined the spatial distribution of metabolites including lipids.

Introduction to proton (H) magnetic resonance spectroscopy of the breast

Proton magnetic resonance spectroscopy (1H MRS) provides biochemical information about the tissue under investigation. The diagnostic value of 1H MRS in cancer is typically based on the detection of elevated levels of choline compounds, which is a marker of active tumor [1]. Breast 1H MRS is performed on a clinical magnet of 1.5 T or higher in field strength. A four or more channel breast coil is also needed, just as for imaging. The most widely used technique is the single voxel technique, which is limited to scan one lesion at a time. Magnetic resonance spectroscopic imaging provides information about the spatial distribution of metabolites and is useful for studying multiple lesions [2,3]. The two main potential clinical applications of 1H MRS include its use as an adjunct to breast MRI to improve the specificity in differentiating benign from malignant lesions and in monitoring or even predicting response to treatment in patients undergoing neoadjuvant chemotherapy. Studies have suggested that 1H MRS may decrease the number of benign biopsies recommended by MRI [4,5]. Also, in patients undergoing neoadjuvant chemotherapy, 1H MRS may be able to predict response as early as 24 hours after the first dose [6]. Currently, several limitations exist that make the technique premature for clinical use [7]. Preliminary data are promising, warranting further evaluation with larger, preferably multicenter, trials.