High-resolution Magic Angle Spinning Spectra Research Articles

Molecular Characterization of the Organic Fraction of Municipal Solid Waste and Compositional Evolution during Oxidative Processes Assessed by HR-MAS 13C NMR Spectroscopy

The use of high-resolution magic angle spinning (HR-MAS) 13C NMR spectroscopy is proposed here as an innovative and non-destructive approach to investigate the chemical composition of the organic fraction of municipal solid waste (OFMSW) and for monitoring the evolution of their composition during the oxidative iron-based Fenton treatment to which the initial matrix is subjected. The high quality and the good resolution of the 13C HR-MAS NMR spectra allowed an accurate assignment and quantification of the various types of carbon present in the analyzed organic matrix. Moreover, the HR-MAS has also shown its effectiveness in monitoring the different oxidative processes to which the same initially organic matrix has been subjected. The results obtained from the HR-MAS spectra on the collected samples during the different oxidative experiments, indicate that Fenton treatment is able to modify the percentage of the different types of carbons as a function of the concentrations of both Fenton reactants, H2O2 and Fe2+ salt, and of the oxidative process time.

Abstract 3721: Improved fitting of HRMAS NMR spectra for ex vivo metabolomic analysis of glioma tissue

Abstract Gliomas are aggressive brain tumors with high rates of treatment resistance and low survival rates. High-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy can quantify metabolite concentrations in glioma tissue, but most analysis techniques require manual peak selection and integration that prevents accurate and reliable quantitation of overlapping peaks and large macromolecule baselines. Our goal was to compare the effectiveness of operator-independent LCModel analysis of glioma spectra acquired from free induction decay (FID) and Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences. HRMAS NMR spectra were acquired using FID and CPMG sequences from 14 histologically-confirmed glioma tissue samples (WHO grade II (n=5), III (n=5), and IV (n=4)) collected during surgical resection from human brain tumor patients. Metabolite concentration ratios and Cramer-Rao lower bounds (CRLBs) were estimated using LCModel. Metabolite CRLBs were compared using a paired two sample t-test. Differences in concentrations as a function of WHO grade were determined with ANOVA and Tukey-Kramer post-hoc tests. Significance was determined by p<.05. Metabolite CRLBs for lactate and myo-inositol were significantly lower for CPMG compared to FID spectra (p<.05). Most metabolites could be quantified with LCModel from spectra acquired with CPMG where many metabolites acquired with the FID sequence were not detected. For example, lactate was quantifiable from 3 of the spectra acquired with FID compared to 12 spectra with CPMG. The use of the CPMG sequence reduces quantification errors by eliminating confounding baseline signals. LCModel facilitates improved separation of overlapping resonances compared to manual peak integration, supporting the use of operator-independent methods for metabolic spectral analysis. Comparison of metabolite concentrations as a function of WHO grade revealed significant differences in lactate and glutamine plus glutamate normalized to creatine (p<.05). 2-hydroxyglutarate (2-HG) was also detected in 9 out of 13 isocitrate dehydrogenase (IDH)-mutated samples using both sequences. IDH-mutated tissue should produce 2-HG; however, these results are promising as 2-HG can be difficult to quantify in 1D NMR due to spectral overlap. In conclusion, LCModel can reliably quantify HRMAS spectra acquired with the CPMG sequence but is less reliable with the FID sequence. Increases in lactate and glutamine plus glutamate concentrations as a function of tumor grade were consistent with previous results using HRMAS for glioma metabolic analysis, and 2-HG was detected in 1D HRMAS spectra acquired with both sequences. We expect that improved spectral fitting will contribute to future NMR-based metabolomics studies in glioma. Note: This abstract was not presented at the meeting. Citation Format: Selin Ekici, Ren Geryak, Stewart G. Neill, Hui-Kuo Shu, Candace C. Fleischer. Improved fitting of HRMAS NMR spectra for ex vivo metabolomic analysis of glioma tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3721.

Integrating High-Resolution and Solid-State Magic Angle Spinning NMR Spectroscopy and a Transcriptomic Analysis of Soybean Tissues in Response to Water Deficiency

Solid-state NMR (SSNMR) spectroscopy methods provide chemical environment and ultrastructural details that are not easily accessible by other non-destructive, high-resolution spectral techniques. High-resolution magic angle spinning (HR-MAS) has been widely used to obtain the metabolic profile of a heterogeneous sample, combining the resolution enhancement provided by MAS in SSNMR with the shimming and locking procedures in liquid-state NMR. In this work, we explored the feasibility of using the HR-MAS and SSNMR techniques to identify metabolic changes in soybean leaves subjected to water-deficient conditions. Control and water-deficient soybean leaves were analysed using one-dimensional (1D) HR-MAS and SSNMR. Total RNA was extracted from the leaves for the transcriptomic analysis. The 1 H HR-MAS and CP-MAS 13 C{1 H} spectra of soybean leaves grown with and without water deficiency stress revealed striking differences in metabolites. A total of 30 metabolites were identified, and the impact of water deficiency on the metabolite profile of soybean leaves was to induce amino acid synthesis. High expression levels of genes required for amino acid biosynthesis were highly correlated with the compounds identified by 1 H HR-MAS. The integration of the 1 H HR-MAS and SSNMR spectra with the transcriptomic data provided a complete picture of the major changes in the metabolic profile of soybeans in response to water deficiency. Copyright © 2017 John Wiley & Sons, Ltd.

Gelified Biofluids for High-Resolution Magic Angle Spinning 1H NMR Analysis: The Case of Urine.

In this letter, we propose an alternative, effective protocol for metabolomic characterization of biofluids based on their gelification and subsequent application of high-resolution magic angle spinning (HRMAS) 1H nuclear magnetic resonance (NMR). The sample handling is very rapid and reproducible, and much less than 40 μL of neat urine are needed to obtain a sample. Our results indicate that the HRMAS spectra of gelified urine encompass all metabolites in the NMR fingerprint, as observed by solution NMR. The proposed approach can be efficiently integrated into the NMR based metabolomics analyses routines: multivariate statistical analysis of both solution and HRMAS data produced very similar statistical models, with high classification accuracy. One of the key advantages offered by the gelification approach is the improved short-term (up to 24 h) preservation of nonfrozen HRMAS NMR gel urine samples compared to the solution samples, which could lead to an alternative way for transportation or domestic collection of biofluids, without the need of cold-storage and reducing the risks of leakage.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

Complete protocol for slow-spinning high-resolution magic-angle spinning NMR analysis of fragile tissues.

High-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) is an essential tool to characterize a variety of semisolid systems, including biological tissues, with virtually no sample preparation. The "non-destructive" nature of NMR is typically compromised, however, by the extreme centrifugal forces experienced under conventional HR-MAS frequencies of several kilohertz. These features limit the usefulness of current HR-MAS approaches for fragile samples. Here, we introduce a full protocol for acquiring high-quality HR-MAS NMR spectra of biological tissues at low spinning rates (down to a few hundred hertz). The protocol first consists of a carefully designed sample preparation, which yields spectra without significant spinning sidebands at low spinning frequency for several types of sample holders, including the standard disposable inserts classically used in HR-MAS NMR-based metabolomics. Suppression of broad spectral features is then achieved using a modified version of the recently introduced PROJECT experiment with added water suppression and rotor synchronization, which deposits limited power in the sample and which can be suitably rotor-synchronized at low spinning rates. The performance of the slow HR-MAS NMR procedure is demonstrated on conventional (liver tissue) and very delicate (fish eggs) samples, for which the slow-spinning conditions are shown to preserve the structural integrity and to minimize intercompartmental leaks of metabolites. Taken together, these results expand the applicability and reliability of HR-MAS NMR spectroscopy. These results have been obtained at 400 and 600 MHz and suggest that high-quality slow HR-MAS spectra can be expected at higher magnetic fields using the described protocol.

Study of metabonomic profiles of human esophageal carcinoma by use of high-resolution magic-angle spinning 1H NMR spectroscopy and multivariate data analysis

Esophageal carcinoma (EC) is one of the most common malignant tumors. EC survival has remained disappointingly low because of the high malignancy of esophageal cancer and the lack of obvious clinical symptoms at an early stage. Early diagnosis is often difficult because the small tumor nodules are frequently missed. Metabonomics based on high-resolution magic-angle spinning (HRMAS) NMR has been popular for tumor detection because it is highly sensitive, provides rich biochemical information and requires no sample pretreatment. (1)H HRMAS spectra of non-involved adjacent esophageal tissues and of well differentiated and moderately differentiated esophageal carcinoma tumors were recorded and analyzed by use of multivariate and statistical analysis techniques. Moderately differentiated EC tumors were found to have increased total choline, alanine, and glutamate and reduced creatine, myo-inositol, and taurine compared with non-involved adjacent tissues. Moreover, clear differences between the metabonomic profiles of EC tissues enabled tumor differentiation. Furthermore, the integral Gly/MI ratio for samples of different tissue types were statistically significantly different; this was sufficient both for distinguishing non-involved tissues from esophageal carcinoma and for classification of well differentiated and moderately differentiated EC tumors.

Differentiating Diffuse World Health Organization Grade II and IV Astrocytomas With Ex Vivo Magnetic Resonance Spectroscopy

The prognosis and treatment of astrocytomas, which are primary brain tumors, vary depending on the grade of the tumor, necessitating a precise preoperative classification. Magnetic resonance spectroscopy (MRS) provides information about metabolites in tissues and is an emerging noninvasive tool to improve diagnostic accuracy in patients with intracranial neoplasia. To investigate whether ex vivo MRS could differentiate World Health Organization grade II (A-II) and IV astrocytomas (glioblastomas; GBM) and to correlate MR spectral profiles with clinical parameters. Patients with A-II and GBM (n = 58) scheduled for surgical resection were enrolled. Tumor specimens were collected during surgery and stored in liquid nitrogen before being analyzed with high-resolution magic angle spinning MRS. The tumors were histopathologically classified according to World Health Organization criteria as GBM (n = 48) and A-II (n = 10). Multivariate analysis of ex vivo proton high-resolution magic angle spinning spectra MRS showed differences in the metabolic profiles of different grades of astrocytomas. A-II had higher levels of glycerophosphocholine and myo-inositol than GBM. The latter had more phosphocholine, glycine, and lipids. We observed a significant metabolic difference between recurrent and nonrecurrent GBM (P < .001). Primary GBM had more phosphocholine than recurrent GBM. A significant correlation (P < .001) between lipid and lactate signals and histologically estimated percentage of necrosis was observed in GBM. Spectral profiles were not correlated with age, survival, or magnetic resonance imaging-defined tumor volume. Ex vivo MRS can differentiate astrocytomas based on their metabolic profiles.

MRS study of meningeal hemangiopericytoma and edema: A comparison with meningothelial meningioma

Intracranial hemangiopericytomas (HPCs) are rare tumors and their radiological appearance resembles that of meningiomas, especially meningothelial meningiomas. To increase the knowledge on the biochemical composition of this type of tumor for better diagnosis and prognosis, we performed a molecular study using exvivo high resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) perfomed on HPC and peritumoral edematous tissues. Moreover, to help in the discrimination between HPC and meningothelial meningioma we compared the exvivo HR-MAS spectra of samples from one patient with HPC and 5 patients affected by meningothelial meningioma. Magnetic resonance imaging (MRI), invivo localized single voxel 1H-MRS was also performed on the same patients prior to surgery and the invivo and exvivo MRS spectra were compared. We observed the presence of OH-butyrate, together with glucose in HPC and a low amount of N-acetylaspartate in the edema, that may reflect neuronal alteration responsible for associated epilepsy. Many differences between HPC and meningothelial meningioma were identified. The relative ratios of myo-inositol, glucose and gluthatione with respect to glutamate are higher in HPC compared to meningioma; whereas the relative ratios of creatine, glutamine, alanine, glycine and choline-containing compounds with respect to glutamate are lower in HPC compared to meningioma. These data will be useful to improve the interpretation of invivo MRS spectra resulting in a more accurate diagnosis of these rare tumors.

Quantifying brain tumor tissue abundance in HR-MAS spectra using non-negative blind source separation techniques

Given high-resolution magic angle spinning (HR-MAS) spectra from several glial tumor subjects, our goal is to differentiate between tumor tissue types by separating the different sources that contribute to the profile of each spectrum. Blind source separation techniques are applied for obtaining characteristic profiles for necrosis, highly cellular tumor and border tumor tissue and providing the contribution (abundance) of each of these tumor tissue types to the profile of each spectrum. The problem is formulated as a non-negative source separation problem. Non-negative matrix factorization, convex analysis of non-negative sources and non-negative independent component analysis methods are considered. The results are in agreement with the pathology obtained by the histopathological examination that succeeded the HR-MAS measurements. Furthermore, an analysis to verify to which extent the dimension of the input space, the input features and the number of sources to be extracted from the HR-MAS data could influence the performance of the source separation is presented. Copyright © 2012 John Wiley & Sons, Ltd.

Processing of high resolution magic angle spinning spectra of breast cancer cells by the filter diagonalization method

Proton nuclear magnetic resonance ((1)H NMR) spectroscopy for detection of biochemical changes in biological samples is a successful technique. However, the achieved NMR resolution is not sufficiently high when the analysis is performed with intact cells. To improve spectral resolution, high resolution magic angle spinning (HR-MAS) is used and the broad signals are separated by a T(2) filter based on the CPMG pulse sequence. Additionally, HR-MAS experiments with a T(2) filter are preceded by a water suppression procedure. The goal of this work is to demonstrate that the experimental procedures of water suppression and T(2) or diffusing filters are unnecessary steps when the filter diagonalization method (FDM) is used to process the time domain HR-MAS signals. Manipulation of the FDM results, represented as a tabular list of peak positions, widths, amplitudes and phases, allows the removal of water signals without the disturbing overlapping or nearby signals. Additionally, the FDM can also be used for phase correction and noise suppression, and to discriminate between sharp and broad lines. Results demonstrate the applicability of the FDM post-acquisition processing to obtain high quality HR-MAS spectra of heterogeneous biological materials.

Metabolic profile of chronic liver disease by NMR spectroscopy of human biopsies

Among the different processes occurring during the evolution of liver disease, fibrosis has a predominant role. Liver fibrosis mechanisms are fairly constant irrespective of the underlying etiology. Cirrhosis is the end-stage of this reaction. Metabolic profiles, which are affected by many physiological and pathological processes, may provide further insight into the metabolic consequences of this severe liver disease. The aim of this study was to demonstrate the applicability of 1H high resolution magic angle spinning (HR-MAS) NMR spectroscopy in the biochemical profile determination of human liver needle biopsy samples for the characterization of metabolic alterations related to the severity of liver disease. We recorded and analyzed HR-MAS spectra of 68 liver tissue samples obtained by needle biopsy from patients with chronic liver disease. Multivariate analysis was applied to these data to obtain discrimination patterns and to reveal relevant metabolites. The metabolic characterization of liver tissue from needle biopsies by HR-MAS NMR spectroscopy provided differential patterns for cirrhotic and non-cirrhotic chronic liver disease tissue. Metabolites closely related to the liver metabolism such as some fatty acids, glucose and amino acids show differences between the two groups. Phospholipid precursors, which have been previously correlated with hepatic lesions also show differences. Furthermore, the correlation between histologically assessed liver disease stages and the levels of the most discriminative metabolites show that liver dysfunction is present at the initial stages of chronic hepatic lesions. Overall, this work suggests that the additional information obtained by NMR metabolomics applied to needle biopsies of human liver may be useful for assessing metabolic alterations and liver dysfunction in chronic liver disease.

NMR Structural Characterization of Quercus alba (White Oak) Degraded by the Brown Rot Fungus, Laetiporus sulphureus

High resolution magic angle spinning (HRMAS) is utilized here to characterize the structure of solid lignin from Quercus alba (white oak) wood that has been biodegraded by the brown rot (BR) fungus Laetiporus sulphureus. The ground wood sample is swelled in deuterated solvent, allowing an enhanced molecular motion that is required for obtaining 2D HRMAS spectra. This technique provides for direct, non-invasive analysis of solid biodegraded lignin. Emphasis is placed on the characterization of the lignin side chains in an effort to elucidate the types of linkages connecting the various monomers. The HRMAS spectra obtained provide evidence for the presence of structures in this biodegraded lignin that have been commonly found in chemically extracted lignins and model compounds. This information supports the existence of several of the six major types of lignin linkages (β-O-4, β-5, β-1, 5-5, 4-O-5, and β-β) as well as three other structures: dibenzodioxocin, isochroman, and spirodienone.

Detection of cancer in cervical tissue biopsies using mobile lipid resonances measured with diffusion-weighted 1 H magnetic resonance spectroscopy

The purpose of this study was to implement a diffusion-weighted sequence for visualisation of mobile lipid resonances (MLR) using high resolution magic angle spinning (HR-MAS) (1)H MRS and to evaluate its use in establishing differences between tissues from patients with cervical carcinoma that contain cancer from those that do not. A stimulated echo sequence with bipolar gradients was modified to allow T(1) and T(2) measurements and optimised by recording signal loss in HR-MAS spectra as a function of gradient strength in model lipids and tissues. Diffusion coefficients, T(1) and apparent T(2) relaxation times were measured in model lipid systems. MLR profiles were characterised in relation to T(1) and apparent T(2) relaxation in human cervical cancer tissue samples. Diffusion-weighted (DW) spectra of cervical biopsies were quantified and peak areas analysed using linear discriminant analysis (LDA). The optimised sequence reduced spectral overlap by suppressing signals originating from low molecular weight metabolites and non-lipid contributions. Significantly improved MLR visualisation allowed visualisation of peaks at 0.9, 1.3, 1.6, 2.0, 2.3, 2.8, 4.3 and 5.3 ppm. MLR analysis of DW spectra showed at least six peaks arising from saturated and unsaturated lipids and those arising from triglycerides. Significant differences in samples containing histologically confirmed cancer were seen for peaks at 0.9 (p < 0.006), 1.3 (p < 0.04), 2.0 (p < 0.03), 2.8 (p < 0.003) and 4.3 ppm (p < 0.0002). LDA analysis of MLR peaks from DW spectra almost completely separated two clusters of cervical biopsies (cancer, 'no-cancer'), reflecting underlying differences in MLR composition. Generated Receiver Operating Characteristic (ROC) curves and calculated area under the curve (0.962) validated high sensitivity and specificity of the technique. Diffusion-weighting of HR-MAS spectroscopic sequences is a useful method for characterising MLR in cancer tissues and displays an accumulation of lipids arising during tumourigenesis and an increase in the unsaturated lipid and triglyceride peaks with respect to saturated MLR.

Cerebral activation by fasting induces lactate accumulation in the hypothalamus

Carbon-13 ((13)C) high-resolution magic angle spinning (HR-MAS) spectroscopy was used to investigate the neuroglial coupling mechanisms underlying appetite regulation in the brain of C57BL/6J mice metabolizing [1-(13)C]glucose. Control fed or overnight fasted mice received [1-(13)C]glucose (20 micromol/g intraperitoneally [i.p.]), 15 min prior to brain fixation by focused microwaves. The hypothalamic region was dissected from the rest of the brain and (13)C HR-MAS spectra were obtained from both biopsies. Fasting resulted in a significant increase in hypothalamic [3-(13)C]lactate and [2-(13)C]gamma-aminobutyric acid (GABA) relative to the remaining brain. Administration of the orexigenic peptide ghrelin (0.3 nmol/g i.p.) did not increase hypothalamic [3-(13)C]lactate or [2-(13)C]GABA, suggesting that ghrelin signaling is not sufficient to elicit all the metabolic consequences of hypothalamic activation by fasting. Our results indicate that the hypothalamic regulation of appetite involves, in addition to the well-known neuropeptide signaling, increased neuroglial lactate shuttling and augmented GABA concentrations.

Toward accurate quantification of metabolites, lipids, and macromolecules in HRMAS spectra of human brain tumor biopsies using LCModel

High-resolution magic angle spinning (HRMAS) (1)H MR spectroscopy of biopsy samples provides detailed biochemical profiles that can be related to the lower-resolution spectra obtained in vivo. Nevertheless, there is still significant overlap of many resonance peaks and contributions from broad lipid and macromolecule resonances that impede accurate quantification. We determined a minimum set of in vitro metabolite and simulated lipid and macromolecule resonances needed for LCModel analysis and quantification of brain tumor biopsy HRMAS spectra. We also demonstrate the quality of the LCModel fit for the four main brain tumor types (astrocytoma grade II, glioblastoma, metastasis, and meningioma). Our data suggest that when fitting resonances of coupled spins systems in high-resolution spectra, interactions between metabolites and the macromolecular environment of the biopsy may cause small peak shifts not found in the solution spectra. However, LCModel is shown to provide a user-independent method of analyzing HRMAS brain tumor spectra.

Investigation of metabolite changes in the transition from pre‐invasive to invasive cervical cancer measured using 1H and 31P magic angle spinning MRS of intact tissue

The aim of this study was to determine the metabolic changes in the transition from pre-invasive to invasive cervical cancer using high-resolution magic angle spinning (HR-MAS) MRS. Biopsy specimens were obtained from women with histologically normal cervix (n = 5), cervical intraepithelial neoplasia (CIN; mild, n = 5; moderate/severe, n = 40), and invasive cancer (n = 23). (1)H HR-MAS MRS data were acquired using a Bruker Avance 11.74 T spectrometer (Carr-Purcell-Meiboom-Gill sequence; TR = 4.8 s; TE = 135 ms; 512 scans; 41 min acquisition). (31)P HR-MAS spectra were obtained from the normal subjects and cancer patients only (as acetic acid applied before tissue sampling in patients with CIN impaired spectral quality) using a (1)H-decoupled pulse-acquire sequence (TR = 2.82 s; 2048 scans; 96 min acquisition). Peak assignments were based on values reported in the literature. Peak areas were measured using the AMARES algorithm. Estimated metabolite concentrations were compared between patient diagnostic categories and tissue histology using independent samples t tests. Comparisons based on patient category at diagnosis showed significantly higher estimated concentrations of choline (P = 0.0001) and phosphocholine (P = 0.002) in tissue from patients with cancer than from patients with high-grade dyskaryosis, but no differences between non-cancer groups. Division by histology of the sample also showed increases in choline (P = 0.002) and phosphocholine (P = 0.002) in cancer compared with high-grade CIN tissue. Phosphoethanolamine was increased in cancer compared with normal tissue (P = 0.0001). Estimated concentrations of alanine (P = 0.01) and creatine (P = 0.008) were significantly reduced in normal tissue from cancer patients compared with normal tissue from non-cancer patients. The estimated concentration of choline was significantly increased in CIN tissue from cancer patients compared with CIN tissue from non-cancer patients (P = 0.0001). Estimated concentrations of choline-containing metabolites increased from pre-invasive to invasive cervical cancer. Concurrent metabolite depletion occurs in normal tissue adjacent to cancer tissue.

Characterization of brain metastases using high‐resolution magic angle spinning MRS

The objectives of this study were to (a) explore the spectral characteristics of brain metastases, focusing on the origin of the primary cancer, and (b) evaluate the correlation with clinical outcome using multivariate analysis. High-resolution magic angle spinning (HR-MAS) MR spectra (n = 26) were obtained from 16 patients with brain metastases using a Bruker Avance DRX600 instrument. Standard pulse-acquired and spin-echo (TE 32 and 285 ms) (1)H spectra were obtained. These were examined using principal component analysis (PCA) and partial least squares regression analysis (PLS) relating spectral data to clinical outcome. The PCA score plot of pulse-acquired HR-MAS spectra showed a trend of clustering due to the origin of the metastases, mainly based on differences in the lipid signals at 1.3 and 0.9 ppm. With PLS, spectra of patients who died less than 5 months after surgery appeared to cluster in the lower left quadrant of the score plot. These preliminary results on brain metastasis classification and prediction of survival must be validated in a larger patient cohort. However, the possibility of differentiating metastases according to origin and predicting survival on the basis of HR-MAS spectra suggests that this method may be useful for diagnosing and planning treatment for brain metastases and also for guiding decisions about terminating further treatment.

A Functional Analysis of Mouse Models of Cardiac Disease through Metabolic Profiling

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.

MR spectroscopy of muscle and brain in guanidinoacetate methyltransferase (GAMT)-deficient mice: validation of an animal model to study creatine deficiency.

As a model for guanidinoacetate methyltransferase (GAMT) deficiency in humans, a gene knockout mouse model was generated. Here we report on several metabolic abnormalities in these mice, observed by in vivo and in vitro MR spectroscopy. In (1)H MR spectra of brain and hindleg muscle a clearly reduced signal of creatine (Cr) was observed in GAMT-deficient (GAMT-/-) animals. Analysis of the (1)H MR spectra of GAMT-/- brain indicated little or no increase of a signal for guanidinoacetate (Gua). In proton MR spectra of muscle, a broad signal of low intensity was observed for Gua. However, substantial Gua accumulation in intact muscle tissue was unequivocally confirmed in high-resolution magic angle spinning spectra, in which the Gua signal was resolved as one clear sharp singlet. In (31)P MR analysis of brain and hindleg muscle a strongly reduced phosphocreatine (PCr) content was shown. In addition, a signal of phosphorylated Gua at 0.5 ppm upfield of PCr was observed, with much higher intensity in muscle than in brain. This signal decreased when ischemia was applied to the muscle and recovered after ischemia was released. Overall, the in vivo (31)P and (1)H MR spectroscopy of GAMT-/- mice is similar to that of human GAMT deficiency. This opens up new avenues for the fundamental study of tissue-type dependence of creatine synthesis and transport and for diagnostic and therapeutic aspects of creatine deficiencies in humans.