A metabolomics perspective of human brain tumours

During the past decade or so, a wealth of information about metabolites in various human brain tumour preparations (cultured cells, tissue specimens, tumours in vivo) has been accumulated by global profiling tools. Such holistic approaches to cellular biochemistry have been termed metabolomics. Inherent and specific metabolic profiles of major brain tumour cell types, as determined by proton nuclear magnetic resonance spectroscopy ((1)H MRS), have also been used to define metabolite phenotypes in tumours in vivo. This minireview examines the recent advances in the field of human brain tumour metabolomics research, including advances in MRS and mass spectrometry technologies, and data analysis.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

In vitro expression of N-acetyl aspartate by oligodendrocytes: implications for proton magnetic resonance spectroscopy signal in vivo

Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N-Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA-specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte-type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.     

New magnetic resonance spectroscopy biomarker for monitoring neurodegenerative diseases: animal models

Creatine kinase (CK) plays a central role in energy transfer in cells with high-energy demands, and the enzyme is rather susceptible to oxidative inactivation. The aim of the present study was to investigate whether the rate constant of forward CK reaction (k(for)) is a suitable indicator of alterations in cerebral energy metabolism. We monitored k(for) in the rat brain non-invasively by in vivo phosphorus ((31)P) magnetic resonance spectroscopy (MRS). To alter energy metabolism, we applied following experimental models: Huntington's disease, diabetes mellitus, chronic alcohol intoxication and chronic cerebral hypoperfusion (vascular dementia model). Results of our (31)P MRS experiment confirm importance of creatine kinase/phosphocreatinine (CK/PCr) system in the regulation of brain energy metabolism in vivo because a kinetic parameter k(for) was significantly changed in all above animal models that simulate neurodegenerative diseases or commonly during oxidative stress. Using this method we distinguished vascular dementia (VD) and Huntington disease (HD), because in VD model a kinetic parameter k(for) decreased and in the case HD increased. Considering the importance of CK for the maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which various neurodegenerative diseases might be monitored just by means saturation transfer method (31)P MRS.     

Biological 1H NMR spectroscopy

Proton nuclear magnetic resonance spectroscopy (1H NMR) is a powerful analytical method used to identify and quantitate chemical compounds. In recent years, it has been used to study rates of metabolism in microbes, isolated perfused tissues, intact animals, and human beings. This review highlights some of the more recent biological applications of 1H NMR in the study of metabolic pathophysiology in animals and man. 1H NMR can rapidly analyze complex mixtures of metabolites found in body fluid and biopsy specimens. In vivo 1H NMR methods can measure intracellular pH, a wide variety of metabolites, tissue perfusion, and rates of metabolism of endogenous and exogenous compounds. Using 13C labeled compounds or magnetization transfer techniques metabolic fluxes may be measured in vivo during virtually all normal and abnormal physiological conditions.     

Magnetic resonance spectroscopy: an in vivo tool for monitoring cerebral injury in SIV-infected macaques

The purpose of our study was to demonstrate the feasibility of using in vivo proton Magnetic Resonance Spectroscopy (MRS) to monitor the brain manifestations of SIV infection in the macaque model of AIDS. Previous spectroscopy work on macaque brain tissue and in vivo work in humans is reviewed to provide the motivation and context for this study. We collected 34 MRS data sets on 14 uninfected rhesus macaques. From this data, we demonstrate that we are capable of detecting changes similar to those observed in human MRS studies for most metabolites using less than 10 animals. The juvenile macaques utilized in this study demonstrate age-related changes in the levels of N-acetyl aspartate (NAA), a neuronal marker. The quantity and distribution of neurochemicals in the macaque are found to be slightly, but significantly, different than in the human.     

Identification of mobile cholesterol compounds in experimental gliomas by (1)H MRS in vivo: effects of ganciclovir-induced apoptosis on lipids

This letter presents a novel identification and analysis of mobile cholesterol compounds in an experimental glioma model by (1)H MRS in vivo. The cholesterol compounds turned out to comprise as much as 17 mol% of MRS visible total lipids. The results also imply partly associated accumulation of (1)H MRS detectable cholesterol compounds and unsaturated lipids during gene therapy-induced apoptosis, and indicate that the contribution of cholesterol compounds cannot be bypassed in spectral lipid analysis. The introduced (1)H MRS approach facilitates a non-invasive follow-up of mobile cholesterol compounds, paving way for studies of tumour cholesterol metabolism in vivo.     

Proton magnetic resonance spectroscopy and its diagnostically important metabolites in the brain

This review provides a brief summary of the physical basis of magnetic resonance spectroscopy ((1)H MRS) and its application in the human brain. We discuss the chemical structure, signal properties, biological function, normal spatial distribution and diagnostic potential of the more significant metabolites detectable in brain tissue: N-acetylaspartate, N-acetylaspartylglutamate, choline-containing substances, creatine, phosphocreatine, myo-inositol, glutamine and glutamate. We also present a few notes on the importance of proper spectral quantification and contemporary trends in 1H MRS. [corrected].     

Metabolic characterization of neurological diseases by proton localized NMR spectroscopy of the human brain.

Proton localized Magnetic Resonance Spectroscopy (MRS) of the brain allows the non invasive detection of intracellular cerebral metabolites. Localized MRS has been performed using short stimulated-echo times in various neurological diseases including stroke, multiple sclerosis, and AIDS-related leukoencephalopathies. Principal component analysis (PCA) was used to determine the critical parameters defining the metabolic profile of normal and diseased brain. PCA clearly differentiates the demyelinating processes from ischaemic lesions and leukoencephalopathies. Localized MRS of the brain appears growingly as a tool of choice to discriminate, quantitate and assess cerebral metabolic damage in patients with neurological disorders.     

^H磁共振波谱在脑胶质瘤的应用进展

磁共振波谱（magnetic resonance spectroscopy,MRS）技术的出现使活体检测组织的代谢和生化信息成为可能,随着其技术的不断成熟,其在临床的应用范围日益扩大。脑胶质瘤具有与正常脑组织不同的代谢特征,借助MRS技术一方面可以反映其代谢特征,另外可将其与正常脑组织区分,因此MRS技术特别是^1H-MRS在脑胶质瘤的诊断、鉴别诊断、分级及预后评估中应用日益广泛。本文就相关进展进行综述。     

Metabolic heterogeneity of the normal human brain: multivariate analysis of <Superscript>1</Superscript>H MRS in vivo spectra acquired at 3T

Introduction

In recent years multivariate projection techniques of data analysis (PCA, PLS-DA) have been increasingly used for detection of complex ¹H MRS derived metabolic signatures in pathologic conditions. However, these techniques have not been applied in the studies of metabolic heterogeneity of the normal human brain.

Objective

In this work we extended current knowledge about regional distribution of metabolites by multivariate analysis of metabolite levels obtained from various cortical and subcortical regions.

Methods

The studied group consisted of 71 volunteers with no neurological disorders. The metabolite levels obtained from short echo time ¹H MRS in vivo spectra were subjected to univariate and multivariate analysis.

Results

The major variance direction in the dataset was dominated by glutamine?+?glutamate, creatine, myo-inositol and was successful in differentiation of the cortical grey matter and cerebellar vermis from the cortical white matter, pons, basal ganglia, hippocampus and thalamus. The projection plane formed by the second and third variance directions was dominated by N-acetylaspartate?+?N-acetylaspartylglutamate, choline and glutamine?+?glutamate variation not explained by the first direction. This plane revealed a huge metabolic contrast between the pons and basal ganglia, differentiation between the cortical grey matter regions and cerebellar vermis as well as biochemical heterogeneity between the regions such as: thalamus, basal ganglia and hippocampus.

Conclusion

Multivariate approach to ¹H MRS data analysis provides an insight into the normal brain biochemistry and is helpful in understanding the regional heterogeneity of the normal brain. Such knowledge is crucial for a proper interpretation of altered metabolic pathways in diseases.

     

Traumatic Brain Injury in the Rat: Alterations in Brain Lactate and pH as Characterized by 1H and 31P Nuclear Magnetic Resonance

Application of both phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) to the study of brain metabolism permits the noninvasive measurement of intracellular pH and brain lactate level. We have used water-suppression 1H MRS with novel lactate-editing techniques, together with 31P MRS, to characterize sequential changes in brain lactate level and pH in vivo over an 8-h period following fluid-percussion brain injury of graded severity in the rat. A transient fall in intracellular pH (from 7.09 +/- 0.07 at baseline to 6.88 +/- 0.09 at 40 min postinjury) occurred in animals subjected to moderate- (1.5-2.2 atm) and high- (2.5-3.3 atm) but not low-level (0.1-1.2 atm) injury; intracellular pH returned to baseline by 90 min postinjury. Transient elevations in brain lactate level were observed that temporally paralleled and were significantly correlated with the pH changes for all injury levels (r = 0.93, p less than 0.001). Postinjury alterations in intracellular brain pH and lactate level were identical in magnitude in animals subjected to either moderate or high-level injury. However, animals subjected to moderate injury had a moderate chronic neurological deficit that persisted up to 4 weeks postinjury, whereas animals subjected to a high level of injury showed greater histopathological damage and a more severe chronic neurological deficit. These data suggest that the extent of posttraumatic intracellular cerebral acidosis in our model of experimental head injury is not directly related to the severity of functional neurological deficit.     

Biochemical characterization of human brain and kidney tissues by magnetic resonance spectroscopy

In vivo and in vitro Magnetic Resonance Spectroscopy is useful for monitoring changes in intracellular metabolites of human cerebral and renal tissues. Healthy and tumoral tissues of different histologic types have been characterized from a biochemical point of view. In vitro molecular characterization is performed on both the aqueous and lipid extracts of surgically removed tissue biopsies, after in vivo MRS, yielding a full picture of tissue biochemistry. Biochemical markers of healthy brain and kidney and of their relative neoplastic lesions have been disclosed. Moreover, some biochemical features can differentiate neoplasm within the same histological type. Ex vivo MRS also gives molecular information related to necrotic phenomena in glial tumors. MRS finding paralleled histologic data and new knowledge about the molecular base of proliferative neoplastic phenomena can be obtained.     

Radionuclide labeling and evaluation of candidate radioligands for PET imaging of histone deacetylase in the brain

Histone deacetylases (HDACs) regulate gene expression by inducing conformational changes in chromatin. Ever since the discovery of a naturally occurring HDAC inhibitor, trichostatin A (TSA) stimulated the recent development of suberoylanilide (SAHA, Zolinza®), HDAC has become an important molecular target for drug development. This has created the need to develop specific in vivo radioligands to study epigenetic regulation and HDAC engagement for drug development for diseases including cancer and psychiatric disorders. 6-([¹⁸F]Fluoroacetamido)-1-hexanoicanilide ([¹⁸F]FAHA) was recently developed as a HDAC substrate and shows moderate blood–brain barrier (BBB) permeability and specific signal (by metabolic trapping/or deacetylation) but rapid metabolism. Here, we report the radiosynthesis of two carbon-11 labeled candidate radiotracers (substrate- and inhibitor-based radioligand) for HDAC and their evaluation in non-human primate brain. PET studies showed very low brain uptake and rapid metabolism of both labeled compounds but revealed a surprising enhancement of brain penetration by F for H substitution when comparing one of these to [¹⁸F]FAHA. Further structural refinement is needed for the development of brain-penetrant, metabolically stable HDAC radiotracers and to understand the role of fluorine substitution on brain penetration.     

Magnetic resonance spectroscopy: a powerful tool for drug metabolism studies.

Studies on the metabolism and disposition of drugs using nuclear magnetic resonance spectroscopy (MRS) as the analytical technique are reviewed. An overview of the main studies classed in terms of the observed magnetic nucleus (1H, 2H, 7Li, 13C, 19F, 31P, 77Se) is followed by some typical examples of the way in which 19F and 31P MRS can be profitably employed to gain more understanding about the metabolism and disposition of the anticancer fluoropyrimidines (5-fluorouracil (FU) and its prodrugs) and ifosfamide (IF). The results of three recent studies carried out in our laboratory are developed. They concern the direct quantitative monitoring of the hepatic metabolism of FU in the isolated perfused mouse liver, the elucidation of the origin of the cardiotoxicity of FU and the metabolism of IF from an analysis of biofluids of patients. Finally, the advantages and limitations of MRS for investigations on drug metabolism are discussed.     

Phosphorus magnetic resonance spectroscopy: its utility in examining the membrane hypothesis of schizophrenia

A novel approach to understanding the pathophysiology of schizophrenia has been the investigation of membrane composition and functional perturbations, referred to as the "Membrane Hypothesis of Schizophrenia." The evidence in support of this hypothesis has been accumulating in findings in patients with schizophrenia of reductions in phospholipids and essential fatty acids various peripheral tissues. Postmortem studies indicate similar reductions in essential fatty acids in the brain. However, the use of magnetic resonance spectroscopy (MRS) has provided an opportunity to examine aspects of membrane biochemistry in vivo in the living brain. MRS is a powerful, albeit complex, noninvasive quantitative imaging tool that offers several advantages over other methods of in vivo biochemical investigations. It has been used extensively in investigating brain biochemistry in schizophrenia. Phosphorus MRS (31P MRS) can provide important information about neuronal membranes, such as levels of phosphomonoesters that reflect the building blocks of neuronal membranes and phosphodiesters that reflect breakdown products. 31P MRS can also provide information about bioenergetics. Studies in patients with chronic schizophrenia as well as at first episode prior to treatment show a variety of alterations in neuronal membrane biochemistry, supportive of the membrane hypothesis of schizophrenia. Below, we will briefly review the principles underlying 31P MRS and findings to date. Magnetic resonance spectroscopy (MRS) is a powerful, albeit complex, imaging tool that permits investigation of brain biochemistry in vivo. It utilizes the magnetic resonance imaging hardware. It offers several advantages over other methods of in vivo biochemical investigations. MRS is noninvasive, there is no radiation exposure, does not require the use of tracer ligands or contrast media. Because of it is relatively benign, repeated measures are possible. It has been used extensively in investigating brain biochemistry in schizophrenia.     

Chemical oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its in vivo metabolism in rat brain and liver

We investigated in vivo the metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the brain and liver of rats 45 min after the systemic administration of 50 mg/kg of the neurotoxin. The metabolites present in brain and liver extracts were identified through multiple analytical methods by comparison to authentic compounds obtained from a number of chemical oxidations of MPTP. Our results indicate the presence of approximately 15% unreacted MPTP and relatively large amounts of both 1-methyl-4-phenylpyridinium (MPP+) and a mixture of three nonpolar lactams: 1-methyl-4-phenyl-5,6-dihydro-2(1H)-pyridinone, 1-methyl-4-phenyl-2(1H)-pyridinone, and a previously unreported metabolite 1-methyl-4-phenyl-2-piperidinone. Whereas MPP+ was more prevalent in the brain than in the liver, the lactam metabolites were more predominant in the liver. The amounts of the N-oxide and N-demethylated metabolites of MPTP were minimal.     

Evaluation of cardiac energetics by non-invasive 31P magnetic resonance spectroscopy

Alterations in myocardial energy metabolism have been implicated in the pathophysiology of cardiac diseases such as heart failure and diabetic cardiomyopathy. ³¹P magnetic resonance spectroscopy (MRS) is a powerful tool to investigate cardiac energetics non-invasively in vivo, by detecting phosphorus (³¹P)-containing metabolites involved in energy supply and buffering. In this article, we review the historical development of cardiac ³¹P MRS, the readouts used to assess cardiac energetics from ³¹P MRS, and how ³¹P MRS studies have contributed to the understanding of cardiac energy metabolism in heart failure and diabetes.This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.