Observation and identification of metabolites emerging during postmortem decomposition of brain tissue by means of in situ 1H-magnetic resonance spectroscopy

Postmortem decomposition of brain tissue was investigated by (1)H-magnetic resonance spectroscopy (MRS) in a sheep head model and selected human cases. Aiming at the eventual estimation of postmortem intervals in forensic medicine, this study focuses on the characterization and identification of newly observed metabolites. In situ single-voxel (1)H-MRS at 1.5 T was complemented by multidimensional homo- and heteronuclear high-resolution NMR spectroscopy of an extract of sheep brain tissue. The inclusion of spectra of model solutions in the program LC Model confirmed the assignments in situ. The first postmortem phase was characterized mainly by changes in the concentrations of metabolites usually observed in vivo and by the appearance of previously reported decay products. About 3 days postmortem, new metabolites, including free trimethylammonium, propionate, butyrate, and iso-butyrate, started to appear in situ. Since the observed metabolites and the time course is comparable in sheep and human brain tissue, the model system seems to be appropriate.

Issues of spectral quality in clinical 1H-magnetic resonance spectroscopy and a gallery of artifacts

In spite of the facts that magnetic resonance spectroscopy (MRS) is applied as clinical tool in non-specialized institutions and that semi-automatic acquisition and processing tools can be used to produce quantitative information from MRS exams without expert information, issues of spectral quality and quality assessment are neglected in the literature of MR spectroscopy. Even worse, there is no consensus among experts on concepts or detailed criteria of quality assessment for MR spectra. Furthermore, artifacts are not at all conspicuous in MRS and can easily be taken for true, interpretable features. This article aims to increase interest in issues of spectral quality and quality assessment, to start a larger debate on generally accepted criteria that spectra must fulfil to be clinically and scientifically acceptable, and to provide a sample gallery of artifacts, which can be used to raise awareness for potential pitfalls in MRS.

Quantitative 1H-magnetic resonance spectroscopy of human brain: Influence of composition and parameterization of the basis set in linear combination model-fitting

Localized short-echo-time (1)H-MR spectra of human brain contain contributions of many low-molecular-weight metabolites and baseline contributions of macromolecules. Two approaches to model such spectra are compared and the data acquisition sequence, optimized for reproducibility, is presented. Modeling relies on prior knowledge constraints and linear combination of metabolite spectra. Investigated was what can be gained by basis parameterization, i.e., description of basis spectra as sums of parametric lineshapes. Effects of basis composition and addition of experimentally measured macromolecular baselines were investigated also. Both fitting methods yielded quantitatively similar values, model deviations, error estimates, and reproducibility in the evaluation of 64 spectra of human gray and white matter from 40 subjects. Major advantages of parameterized basis functions are the possibilities to evaluate fitting parameters separately, to treat subgroup spectra as independent moieties, and to incorporate deviations from straightforward metabolite models. It was found that most of the 22 basis metabolites used may provide meaningful data when comparing patient cohorts. In individual spectra, sums of closely related metabolites are often more meaningful. Inclusion of a macromolecular basis component leads to relatively small, but significantly different tissue content for most metabolites. It provides a means to quantitate baseline contributions that may contain crucial clinical information.

Brain metabolite composition during early human brain development as measured by quantitative in vivo 1H magnetic resonance spectroscopy

Biochemical maturation of the brain can be studied noninvasively by (1)H magnetic resonance spectroscopy (MRS) in human infants. Detailed time courses of cerebral tissue contents are known for the most abundant metabolites only, and whether or not premature birth affects biochemical maturation of the brain is disputed. Hence, the last trimester of gestation was observed in infants born prematurely, and their cerebral metabolite contents at birth and at expected term were compared with those of fullterm infants. Successful quantitative short-TE (1)H MRS was performed in three cerebral locations in 21 infants in 28 sessions (gestational age 32-43 weeks). The spectra were analyzed with linear combination model fitting, considerably extending the range of observable metabolites to include acetate, alanine, aspartate, cholines, creatines, gamma-aminobutyrate, glucose, glutamine, glutamate, glutathione, glycine, lactate, myo-inositol, macromolecular contributions, N-acetylaspartate, N-acetylaspartylglutamate, o-phosphoethanolamine, scyllo-inositol, taurine, and threonine. Significant effects of age and location were found for many metabolites, including the previously observed neuronal maturation reflected by an increase in N-acetylaspartate. Absolute brain metabolite content in premature infants at term was not considerably different from that in fullterm infants, indicating that prematurity did not affect biochemical brain maturation substantially in the studied population, which did not include infants of extremely low birthweight.

Whole-brain patterns of 1H-magnetic resonance spectroscopy imaging in Alzheimer's disease and dementia with Lewy bodies

Acknowledgments We thank Craig Lambert for his help in processing the MRS data. The study was funded by the Sir Jules Thorn Charitable Trust (grant ref: 05/JTA) and was supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre and the Biomedical Research Unit in Lewy Body Dementia based at Newcastle upon Tyne Hospitals National Health Service (NHS) Foundation Trust and Newcastle University and the NIHR Biomedical Research Centre and Biomedical Research Unit in Dementia based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

In vivo brain macromolecule signals in healthy and glioblastoma mouse models: (1) H magnetic resonance spectroscopy, post-processing and metabolite quantification at 14.1 T.

In (1) H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion-recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post-process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.

Quantitative (1)H magnetic resonance spectroscopy of myoglobin de- and reoxygenation in skeletal muscle: reproducibility and effects of location and disease

1H-magnetic resonance spectroscopy ((1)H-MRS) of deoxymyoglobin (DMb) provides a means to noninvasively monitor the oxygenation state of human skeletal muscle in work and disease. As shown in this work, it also offers the opportunity to measure the absolute tissue content of DMb, the basic oxygen consumption of resting muscle, and the reperfusion characteristics after release of a pressure cuff. The methodology to determine these tissue properties simultaneously at two positions along the calf is presented. The obtained values are in agreement with invasive determinations. The reproducibility of the (1)H-MRS measurements is established for healthy controls and patients with peripheral arterial disease (PAD). A location dependence in axial direction, as well as differences between controls and patients are demonstrated for all parameters. The reoxygenation time in particular is expected to provide a means to quantitatively monitor therapies aimed at improving muscular perfusion in these patients.

Identification of early metabolic markers of various degrees of ischemia in the mouse brain by localized H-1 magnetic resonance spectroscopy

Objectives: Magnetic resonance (MR) imaging and spectroscopy (MRS) allow the establishment of the anatomical evolution and neurochemical profiles of ischemic lesions. The aim of the present study was to identify markers of reversible and irreversible damage by comparing the effects of 10-mins middle cerebral artery occlusion (MCAO), mimicking a transient ischemic attack, with the effects of 30-mins MCAO, inducing a striatal lesion. Methods: ICR-CD1 mice were subjected to 10-mins (n = 11) or 30-mins (n = 9) endoluminal MCAO by filament technique at 0 h. The regional cerebral blood flow (CBF) was monitored in all animals by laser- Doppler flowmetry with a flexible probe fixed on the skull with < 20% of baseline CBF during ischemia and > 70% during reperfusion. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were acquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6 to 8 microliters). Six animals (sham group) underwent nearly identical procedures without MCAO. Results: The 10-mins MCAO induced no MR- or histologically detectable lesion in most of the mice and a small lesion in some of them. We thus had two groups with the same duration of ischemia but a different outcome, which could be compared to sham-operated mice and more severe ischemic mice (30-mins MCAO). Lactate increase, a hallmark of ischemic insult, was only detected significantly after 30-mins MCAO, whereas at 3 h post ischemia, glutamine was increased in all ischemic mice independently of duration and outcome. In contrast, glutamate, and even more so, N-acetyl-aspartate, decreased only in those mice exhibiting visible lesions on T2-weighted images at 24 h. Conclusions: These results suggest that an increased glutamine/glutamate ratio is a sensitive marker indicating the presence of an excitotoxic insult. Glutamate and NAA, on the other hand, appear to predict permanent neuronal damage. In conclusion, as early as 3 h post ischemia, it is possible to identify early metabolic markers manifesting the presence of a mild ischemic insult as well as the lesion outcome at 24 h.

A double-quadrature radiofrequency coil design for proton-decoupled carbon-13 magnetic resonance spectroscopy in humans at 7T

Purpose Carbon-13 magnetic resonance spectroscopy (13C-MRS) is challenging because of the inherent low sensitivity of 13C detection and the need for radiofrequency transmission at the 1H frequency while receiving the 13C signal, the latter requiring electrical decoupling of the 13C and 1H radiofrequency channels. In this study, we added traps to the 13C coil to construct a quadrature-13C/quadrature-1H surface coil, with sufficient isolation between channels to allow simultaneous operation at both frequencies without compromise in coil performance. Methods Isolation between channels was evaluated on the bench by measuring all coupling parameters. The quadrature mode of the quadrature-13C coil was assessed using in vitro 23Na gradient echo images. The signal-to-noise ratio (SNR) was measured on the glycogen and glucose resonances by 13C-MRS in vitro, compared with that obtained with a linear-13C/quadrature-1H coil, and validated by 13C-MRS in vivo in the human calf at 7T. Results Isolation between channels was better than â^'30 dB. The 23Na gradient echo images indicate a region where the field is strongly circularly polarized. The quadrature coil provided an SNR enhancement over a linear coil of 1.4, in vitro and in vivo. Conclusion It is feasible to construct a double-quadrature 13C-1H surface coil for proton decoupled sensitivity enhanced 13C-NMR spectroscopy in humans at 7T. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

Biochemistry in healthy and neoplastic human tissues: metabolic alteration revealed by HR-MAS nuclear magnetic resonance spectroscopy

This thesis is focused on the metabolomic study of human cancer tissues by ex vivo High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. This new technique allows for the acquisition of spectra directly on intact tissues (biopsy or surgery), and it has become very important for integrated metabonomics studies. The objective is to identify metabolites that can be used as markers for the discrimination of the different types of cancer, for the grading, and for the assessment of the evolution of the tumour. Furthermore, an attempt to recognize metabolites, that although involved in the metabolism of tumoral tissues in low concentration, can be important modulators of neoplastic proliferation, was performed. In addition, NMR data was integrated with statistical techniques in order to obtain semi-quantitative information about the metabolite markers. In the case of gliomas, the NMR study was correlated with gene expression of neoplastic tissues. Chapter 1 begins with a general description of a new “omics” study, the metabolomics. The study of metabolism can contribute significantly to biomedical research and, ultimately, to clinical medical practice. This rapidly developing discipline involves the study of the metabolome: the total repertoire of small molecules present in cells, tissues, organs, and biological fluids. Metabolomic approaches are becoming increasingly popular in disease diagnosis and will play an important role on improving our understanding of cancer mechanism. Chapter 2 addresses in more detail the basis of NMR Spectroscopy, presenting the new HR-MAS NMR tool, that is gaining importance in the examination of tumour tissues, and in the assessment of tumour grade. Some advanced chemometric methods were used in an attempt to enhance the interpretation and quantitative information of the HR-MAS NMR data are and presented in chapter 3. Chemometric methods seem to have a high potential in the study of human diseases, as it permits the extraction of new and relevant information from spectroscopic data, allowing a better interpretation of the results. Chapter 4 reports results obtained from HR-MAS NMR analyses performed on different brain tumours: medulloblastoma, meningioms and gliomas. The medulloblastoma study is a case report of primitive neuroectodermal tumor (PNET) localised in the cerebellar region by Magnetic Resonance Imaging (MRI) in a 3-year-old child. In vivo single voxel 1H MRS shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; which consist of very high amounts of the choline-containing compounds and of very low levels of creatine derivatives and N-acetylaspartate. Ex vivo HR-MAS NMR, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS NMR spectra show higher detail than that obtained in vivo. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. The present study shows that ex vivo HR-MAS 1H NMR is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes. Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were analysed both by in vivo and ex vivo MRS experiments. The ex vivo HR-MAS investigations are very helpful for the assignment of the in vivo resonances of human meningiomas and for the validation of the quantification procedure of in vivo MR spectra. By using one- and two dimensional experiments, several metabolites in different histological subtypes of meningiomas, were identified. The spectroscopic data confirmed the presence of the typical metabolites of these benign neoplasms and, at the same time, that meningomas with different morphological characteristics have different metabolic profiles, particularly regarding macromolecules and lipids. The profile of total choline metabolites (tCho) and the expression of the Kennedy pathway genes in biopsies of human gliomas were also investigated using HR-MAS NMR, and microfluidic genomic cards. 1H HR-MAS spectra, allowed the resolution and relative quantification by LCModel of the resonances from choline (Cho), phosphorylcholine (PC) and glycerolphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H MRS spectroscopy. All glioma biopsies depicted an increase in tCho as calculated from the addition of Cho, PC and GPC HR-MAS resonances. However, the increase was constantly derived from augmented GPC in low grade NMR gliomas or increased PC content in the high grade gliomas, respectively. This circumstance allowed the unambiguous discrimination of high and low grade gliomas by 1H HR-MAS, which could not be achieved by calculating the tCho/Cr ratio commonly used by in vivo 1H MR spectroscopy. The expression of the genes involved in choline metabolism was investigated in the same biopsies. The present findings offer a convenient procedure to classify accurately glioma grade using 1H HR-MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low gliomas grade. Chapter 5 reports the study on human gastrointestinal tract (stomach and colon) neoplasms. The human healthy gastric mucosa, and the characteristics of the biochemical profile of human gastric adenocarcinoma in comparison with that of healthy gastric mucosa were analyzed using ex vivo HR-MAS NMR. Healthy human mucosa is mainly characterized by the presence of small metabolites (more than 50 identified) and macromolecules. The adenocarcinoma spectra were dominated by the presence of signals due to triglycerides, that are usually very low in healthy gastric mucosa. The use of spin-echo experiments enable us to detect some metabolites in the unhealthy tissues and to determine their variation with respect to the healthy ones. Then, the ex vivo HR-MAS NMR analysis was applied to human gastric tissue, to obtain information on the molecular steps involved in the gastric carcinogenesis. A microscopic investigation was also carried out in order to identify and locate the lipids in the cellular and extra-cellular environments. Correlation of the morphological changes detected by transmission (TEM) and scanning (SEM) electron microscopy, with the metabolic profile of gastric mucosa in healthy, gastric atrophy autoimmune diseases (AAG), Helicobacter pylori-related gastritis and adenocarcinoma subjects, were obtained. These ultrastructural studies of AAG and gastric adenocarcinoma revealed lipid intra- and extra-cellularly accumulation associated with a severe prenecrotic hypoxia and mitochondrial degeneration. A deep insight into the metabolic profile of human healthy and neoplastic colon tissues was gained using ex vivo HR-MAS NMR spectroscopy in combination with multivariate methods: Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The NMR spectra of healthy tissues highlight different metabolic profiles with respect to those of neoplastic and microscopically normal colon specimens (these last obtained at least 15 cm far from the adenocarcinoma). Furthermore, metabolic variations are detected not only for neoplastic tissues with different histological diagnosis, but also for those classified identical by histological analysis. These findings suggest that the same subclass of colon carcinoma is characterized, at a certain degree, by metabolic heterogeneity. The statistical multivariate approach applied to the NMR data is crucial in order to find metabolic markers of the neoplastic state of colon tissues, and to correctly classify the samples. Significant different levels of choline containing compounds, taurine and myoinositol, were observed. Chapter 6 deals with the metabolic profile of normal and tumoral renal human tissues obtained by ex vivo HR-MAS NMR. The spectra of human normal cortex and medulla show the presence of differently distributed osmolytes as markers of physiological renal condition. The marked decrease or disappearance of these metabolites and the high lipid content (triglycerides and cholesteryl esters) is typical of clear cell renal carcinoma (RCC), while papillary RCC is characterized by the absence of lipids and very high amounts of taurine. This research is a contribution to the biochemical classification of renal neoplastic pathologies, especially for RCCs, which can be evaluated by in vivo MRS for clinical purposes. Moreover, these data help to gain a better knowledge of the molecular processes envolved in the onset of renal carcinogenesis.

Brain metabolism in Alzheimer disease and vascular dementia assessed by in vivo proton magnetic resonance spectroscopy

Proton magnetic resonance spectroscopy (MRS) allows the assessment of various cerebral metabolites non-invasively in vivo. Among 1H MRS-detectable metabolites, N-acetyl-aspartate and N-acetyl-aspartyl-glutamate (tNAA), trimethylamines (TMA), creatine and creatine phosphate (tCr), inositol (Ins) and glutamate (Gla) are of particular interest, since these moieties can be assigned to specific neuronal and glial metabolic pathways, membrane constituents, and energy metabolism. In this study on 94 subjects from a memory clinic population, 1H MRS results (single voxel STEAM: TE 20 ms, TR 1500 ms) on the above metabolites were assessed for five different brain regions in probable vascular dementia (VD), probable Alzheimer's disease (AD), and age-matched healthy controls. In both VD and AD, ratios of tNAA/tCr were decreased, which may be attributed to neuronal atrophy and loss, and Ins/tCr-ratios were increased indicating either enhanced gliosis or alteration of the cerebral inositol metabolism. However, the topographical distribution of the metabolic alterations in both diseases differed, revealing a temporoparietal pattern for AD and a global, subcortically pronounced pattern for VD. Furthermore, patients suffering from vascular dementia (VD) had remarkably enhanced TMA/tCr ratios, potentially due to ongoing degradation of myelin. Thus, the metabolic alterations obtained by 1H MRS in vivo allow insights into the pathophysiology of the different dementias and may be useful for diagnostic classification.

Neuronal and axonal degeneration in experimental spinal cord injury: in vivo proton magnetic resonance spectroscopy and histology.

Longitudinal in vivo proton magnetic resonance spectroscopy (1H-MRS) and immunohistochemistry were performed to investigate the tissue degeneration in traumatically injured rat spinal cord rostral and caudal to the lesion epicenter. On 1H-MRS significant decreases in N-acetyl aspartate (NAA) and total creatine (Cr) levels in the rostral, epicenter, and caudal segments were observed by 14 days, and levels remained depressed up to 56 days post-injury (PI). In contrast, the total choline (Cho) levels increased significantly in all three segments by 14 days PI, but recovered in the epicenter and caudal, but not the rostral region, at 56 days PI. Immunohistochemistry demonstrated neuronal cell death in the gray matter, and reactive astrocytes and axonal degeneration in the dorsal, lateral, and ventral white-matter columns. These results suggest delayed tissue degeneration in regions both rostrally and caudally from the epicenter in the injured spinal cord tissue. A rostral-caudal asymmetry in tissue recovery was seen both on MRI-observed hyperintense lesion volume and the Cho, but not NAA and Cr, levels at 56 days PI. These studies suggest that dynamic metabolic changes take place in regions away from the epicenter in injured spinal cord.

Nuclear magnetic resonance spectroscopy and ultrasound

The work described in this thesis is directed to the examination of the hypothesis that ultrasound may be used to perturb molecular motion in the liquid phase. These changes can then be detected by nuclear magnetic resonance (NMR) in spin-lattice and spin-spin relaxation times. The objective being to develop a method capable of reducing the pulsed NMR acquisition times of slowly relaxing nuclei. The thesis describes the theoretical principles underlying both NMR spectroscopy and ultrasonics with particular attention being paid to factors that impinge on testing the above hypothesis. Apparatus has been constructed to enable ultrasound at frequencies between 1 and 10 mega-hertz with a variable power up to 100W/cm-2 to be introduced in the NMR sample. A broadband high frequency generator is used to drive PZT piezo-electric transducer via various transducer to liquid coupling arrangements. A commercial instrument of 20 kilo-hertz has also been employed to test the above hypothesis and also to demonstrate the usefulness of ultrasound in sonochemistry. The latter objective being, detection of radical formation in monomer and polymer ultrasonic degradation. The principle features of the results obtained are: Ultrasonic perturbation of T1 is far smaller for pure liquids than is for mixtures. The effects appear to be greater on protons (1H) than on carbon-13 nuclei (13C) relaxation times. The observed effect of ultrasonics is not due to temperature changes in the sample. As the power applied to the transducer is progressively increased T1 decreases to a minimum and then increases. The T1's of the same nuclei in different functional groups are influenced to different extents by ultrasound. Studies of the 14N resonances from an equimolar mixture of N, N-dimethylformamide and deuterated chloroform with ultrasonic frequencies at 1.115, 6, 6.42 and 10 MHz show that as the frequency is increased the NMR signal to noise ratio decreases to zero at the Larmor frequency of 6.42 MHz and then again rises. This reveals the surprising indication that an effect corresponding to nuclear acoustic saturation in the liquid may be observable. Ultrasonic irradiation of acidified ammonium chloride solution at and around 6.42 MHz appears to cause distinctive changes in the proton-nitrogen J coupling resonance at 89.56 MHz. Ultrasonic irradiation of N, N-dimethylacetamide at 2 KHz using the lowest stable power revealed the onset of coalescence in the proton spectrum. The corresponding effect achieved by direct heating required a temperature rise of approximately 30oC. The effects of low frequency (20 KHz) on relaxation times appear to be nil. Detection of radical formation proved difficult but is still regarded as the principle route for monomer and polymer degradation. The initial hypothesis is considered proven with the results showing significant changes in the mega-hertz region and none at 20 KHz.

Quantitative Determination of Dimethylaminoethanol in Cosmetic Formulations by Nuclear Magnetic Resonance Spectroscopy

A nuclear magnetic resonance (NMR) spectroscopic method was validated for the quantitative determination of dimethylaminoethanol (DMAE) in cosmetic formulations. The linearity in the range from 0.5000 to 1.5000 g (DMAE salt/mass maleic acid) presents a correlation coefficient > 0.99 for all DMAE salts. The repeatability (intraday), expressed as relative standard deviation, ranged from 1.08 to 1.44% for samples and 1.31 to 1.88% for raw materials. The detection limit and quantitation limit were 0.0017 and 0.0051 g for DMAE, 0.0018 and 0.0054 g for DMAE bitartrate, and 0.0023 and 0.0071 g for DMAE acetamidobenzoate, respectively. The proposed method is simple, precise, and accurate and can be used in the quality control of raw materials and cosmetic gels containing these compounds as active substances.