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Ultrahigh resolution mass spectrometry-based metabolic characterization reveals cerebellum as a disturbed region in two animal models
... Besides that, due to brain heterogeneity during sample collection, care should be taken to collect sample from the same region for each sampling to avoid factors such as biological variability. In AD, hippocampus and cerebellum are two brain parts commonly studied because they are the first to suffer damage [4,18,56,[69][70][71] although other authors have analysed the whole brain [72][73][74][75]. In PD the region is not specified or the whole brain is used [76][77][78] and in SCZ, dorsolateral prefrontal cortex Broadmann Area 9 is frequently studied [50,[79][80][81]. ...
... The selection of solvent for homogenization depends on the category of metabolites to be analysed and separation techniques to be applied. If the aqueous phase is removed and after evaporation it is reconstituted with an aqueous phase, then polar compounds as amino acids (AAs), short chain fatty acids (SCFA), amines, cholines (Cho), myo-inositol, etc. will be analysed [67,71,72,78,81,[87][88][89] ...
... For instance, compounds with basic groups in their structure form a soluble salt in the presence of a weak acid, becoming more water soluble. The extraction of these compounds can be significantly improved by adding a diluted acid, such as formic acid to the aqueous phase (pH = 2), which is miscible with water and with several polar organic solvents [56,70,71,108,113]. ...
Brain is still an organ with a composition to be discovered but beyond that, mental disorders and especially all diseases that curse with dementia are devastating for the patient, the family and the society. Metabolomics can offer an alternative tool for unveiling new insights in the discovery of new treatments and biomarkers of mental disorders. Until now, most of metabolomic studies have been based on biofluids: serum/plasma or urine, because brain tissue accessibility is limited to animal models or . post mortem studies, but even so it is crucial for understanding the pathological processes. Metabolomics studies of brain tissue imply several challenges due to sample extraction, along with brain heterogeneity, sample storage, and sample treatment for a wide coverage of metabolites with a wide range of concentrations of many lipophilic and some polar compounds. In this review, the current analytical practices for target and non-targeted metabolomics are described and discussed with emphasis on critical aspects: sample treatment (quenching, homogenization, filtration, centrifugation and extraction), analytical methods, as well as findings considering the used strategies. Besides that, the altered analytes in the different brain regions have been associated with their corresponding pathways to obtain a global overview of their dysregulation, trying to establish the link between altered biological pathways and pathophysiological conditions.
... Metabolomic analysis can be performed in a variety of easily accessible biofluids such as cerebrospinal fluid, blood or urine, thus highlighting the clinical utility of this approach. However, although numerous studies have been performed to assess the pathophysiological status of transgenic animals by postmortem analysis of brain tissue samples [4][5][6][7][8][9], the use of peripheral samples has been only scarcely considered. In this sense, Graham et al. analyzed comparatively brain and plasma samples from APP/PS1 and wild type mice using 1 H-NMR with the aim of discovering new AD biomarkers [10]. ...
... Direct infusion electrospray mass spectrometry (DI-ESI-MS) has proved to be a suitable tool for fast and comprehensive "first pass" screening of metabolic abnormalities, which has been successfully applied to AD research in previous investigations using human serum [17][18][19] and brain tissue from transgenic mice [5,6,9]. On the other hand, González-Domínguez et al. described in a recent work that flow injection atmospheric pressure photoionization mass spectrometry (FI-APPI-MS) may complement ESI-MS for the analysis of little polar or non-polar metabolites, and it is able to differentiate serum samples from AD patients and healthy control subjects [20]. ...
... Metabolic alterations detected in this study (Table 1) offer the opportunity to elucidate possible biochemical pathways affected in the APP/PS1 mice, thus allowing a better understanding about disease pathology. Furthermore, these findings demonstrate the potential of using peripheral samples such as blood serum in order to characterize AD-mechanisms, providing complementary results to those observed in brain tissue, the matrix commonly employed in previous metabolomics investigations with AD models [4][5][6][7][8][9]. It is noteworthy that serum from APP/PS1 mice showed reduced levels of different amino acids (serine, valine, threonine, pyroglutamate, histidine, tyrosine, tryptophan), which may suggest that perturbations of amino acid metabolism play a critical role in the pathogenesis of AD in this mouse model. ...
Metabolomic analysis of brain tissue from transgenic mouse models of Alzheimer's disease has demonstrated a great potential for the study of pathological mechanisms and the development of new therapies and biomarkers for diagnosis. However, in order to translate these investigations to the clinical practice it is necessary to corroborate these findings in peripheral samples. To this end, this work considers the application of a novel metabolomic platform based on the combination of a two-steps extraction procedure with complementary analysis by direct infusion electrospray mass spectrometry and flow infusion atmospheric pressure photoionization mass spectrometry for a holistic investigation of metabolic abnormalities in serum samples from APP/PS1 mice. A number of metabolites were found to be perturbed in this mouse model, including increased levels of di- and tri-acylglycerols, eicosanoids, inosine, choline and glycerophosphoethanolamine; reduced content of cholesteryl esters, free fatty acids, lysophosphocholines, amino acids, energy-related metabolites, phosphoethanolamine and urea, as well as abnormal distribution of phosphocholines depending on the fatty acid linked to the molecular moiety. This allowed the elucidation of possible pathways disturbed underlying to disease (abnormal homeostasis of phospholipids leading to membrane breakdown, energy-related failures, hyperammonemia and hyperlipidemia, among others), thus demonstrating the utility of peripheral samples to investigate pathology in the APP/PS1 model.
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... Fatty acid metabolism (eicosanoids), amino acid metabolism, nucleotide metabolism (purines) [29] AD (N = 9)/healthy controls (N = 9) GC-MS ...
... Complementarily, other peripheral organs, including the liver, kidneys, spleen, and thymus, were also studied to evaluate the systemic manifestations of the molecular mechanisms behind the AD pathology [26]. In this line, Lin et al. applied DMS metabolomics to characterize the metabolic perturbations in hippocampus [28] and cerebellum [29] of the CRND8 transgenic mouse. Interestingly, the most important findings were related to an altered metabolism of arachidonic acid and eicosanoids, amino acids, nucleotides, and other metabolite classes, which were in great agreement with the studies performed on the APP × PS1 model. ...
Hydrophilic metabolites are closely involved in multiple primary metabolic pathways and, consequently, play an essential role in the onset and progression of multifactorial human disorders, such as Alzheimer’s disease. This review article provides a comprehensive revision of the literature published on the use of mass spectrometry-based metabolomics platforms for approaching the central metabolome in Alzheimer’s disease research, including direct mass spectrometry, gas chromatography-mass spectrometry, hydrophilic interaction liquid chromatography-mass spectrometry, and capillary electrophoresis-mass spectrometry. Overall, mounting evidence points to profound disturbances that affect a multitude of central metabolic pathways, such as the energy-related metabolism, the urea cycle, the homeostasis of amino acids, fatty acids and nucleotides, neurotransmission, and others.
... serum up-regulated production of eicosanoids, altered metabolism of amino acids and urea cycle [26] CRND8 (N = 6) WT (N = 6) hippocampus altered metabolism of arachidonic acid, carbohydrates and nucleotides [27] CRND8 (N = 6) WT (N = 6) cerebellum up-regulated production of eicosanoids; altered metabolism of amino acids and nucleotides [28] APP MCI (N = 28) HC (N = 73) plasma discovery of a panel of 24 metabolites mainly phospholipids and acyl-carnitines) [40] AD (N = 143) MCI (N = 145) HC (N = 153) plasma impairments in phospholipid homeostasis [41] AD (N = 53) MCI (N = 33) HC (N = 35) plasma impairments in phospholipid homeostasis [42] AD, MCI, HC brain, serum impairments in the homeostasis of phospholipids and sphingolipids [43] APP × PS1 (N = 9) WT (N = 9) brain, plasma impairments in the homeostasis of phospholipids, acyl-carnitines, amino acids and polyamines [44] Numerous non-targeted DMS-based metabolomic studies have been conducted in serum samples, which is a very useful biofluid in clinical practice for the identification of diagnostic biomarkers in a non-invasive manner. González-Domínguez et al. employed a DIMS platform based on a two-step treatment of serum samples from AD patients to obtain a holistic snapshot of metabolite alterations associated with the early development of this neurodegenerative disorder [20,21]. ...
... Other published studies on DMS-based metabolomics have focused on the characterization of metabolic impairments observed in brain from various transgenic mice models, a tissue of great interest in AD research, since it enables the in situ investigation of neuropathological processes related to this neurodegenerative disorder. Lin et al. applied an optimized DIMS platform to look for characteristic metabolic impairments in the hippocampus [27] and cerebellum [28] of the CRND8 mouse model. Major findings were observed with regard to an abnormal metabolism of amino acids and nucleotides, as well as the over-production of eicosanoids. ...
Direct mass spectrometry-based metabolomics has been widely employed in recent years to characterize the metabolic alterations underlying Alzheimer’s disease development and progression. This high-throughput approach presents great potential for fast and simultaneous fingerprinting of a vast number of metabolites, which can be applied to multiple biological matrices including serum/plasma, urine, cerebrospinal fluid and tissues. In this review article, we present the main advantages and drawbacks of metabolomics based on direct mass spectrometry compared with conventional analytical techniques, and provide a comprehensive revision of the literature on the use of these tools in the investigation of Alzheimer’s disease.
... Direct MS analysis on APP transgenic mouse cortex and cerebellum showed lower sulfatide levels (Cheng et al., 2010), while perturbations in homeostasis of lipids, energy management, and metabolism of amino acids and nucleotides were visible on APPxPS1 hippocampus and cortex (Gonzalez-Dominguez et al., 2015h). Employing ultra high resolution MS instruments, Lin et al., (2013) were able to observe over-production of eicosanoids indicating neuroinflammation on hippocampal tissues (Lin et al., 2013), and enhanced biosynthesis of amino acid and amino acid derivatives in cerebellum (Lin et al., 2014). Blood metabolomics using MS has identified numerous disturbed pathways in serum samples from APPxPS1 mouse, including perturbed phospholipid homeostasis, energy-related failures, hyperammonemia, inflammation (increased eicosanoids), among others (Gonzalez-Dominguez et al., 2015c). ...
... The findings demonstrated significant impairments in energy metabolism, lipid homeostasis, oxidative stress and amino acids metabolism. These findings were largely consistent with those found from Tg mouse brain tissue samples (Lin et al., 2014;Lin et al., 2013;Gonzalez-Dominguez et al., 2015h). These findings highlighted the systemic nature of AD as metabolite dysregulation was seen in multi-organs, not just the brain. ...
Alzheimer’s disease (AD) is a progressive neurodegenerative disease which affects a growing number of people as the population ages worldwide. Alzheimer’s Disease International estimated that more than 100 million people will be living with dementia by 2050. At present there are no disease-modifying therapies and research has expanded to the −omic sciences with scientists aiming to get a holistic view of the disease using systems medicine. Metabolomics and Lipidomics give a snap-shot of the metabolism. As analyzing the brain in vivo is difficult, the metabolic information of the periphery has potential to unravel mechanisms that have not been considered, such as those that link the brain to the liver and the gut or other organs. With that in mind we have produced a mini-review, to record a number of studies in the field and the molecular pathways that have been flagged in animal and human models of AD. Human studies deal with cohorts in the order of the hundreds due to the difficulty of organizing AD studies, however it is possible that these first pilots point towards important mechanisms. The trend in these small studies is the involvement of many organs and pathways. Some findings, that have been reproduced, are ceramides being increased, phospholipids and neurotransmitters depleted and sterols being found depleted too. Initial findings point to an important role to lipid homeostasis in AD, this is not surprising as the brain’s main constituents are water and lipids.
... Eicosanoids, amino acids, histamine and derivatives [55] DI-ESI-FTICR-MS Hippocampus (CRND8) Eicosanoids, amino acids and derivatives [56] DI-ESI-FTICR-MS Cerebellum (CRND8) Eicosanoids, purines, pyrimidines, amino acids and derivatives [57] DI-ESI-QQQ-MS (MDMS-SL) ...
... Authors found important changes related to the homeostasis of histamine and several amino acids, the urea cycle and the production of eicosanoids, thus demonstrating that depletion of IL-4 might potentiate AD pathology in the APP/PS1 model. Alternatively, Lin et al. also employed DIMS to address metabolic changes in hippocampus [56] and cerebellum [57] of the CRND8 transgenic mice. Similarly to findings from the APP/PS1 mouse model, these studies also revealed significant neuroinflammation, with increased production of eicosanoids, as well as abnormal metabolism of amino acids and purines. ...
Metabolomics based on direct mass spectrometry (MS) analysis, either by direct infusion or flow injection of crude sample extracts, shows a great potential for metabolic fingerprinting because of its high-throughput screening capability, wide metabolite coverage and reduced time of analysis. Considering that numerous metabolic pathways are significantly perturbed during the initiation and progression of diseases, these metabolomic tools can be used to get a deeper understanding about disease pathogenesis and discover potential biomarkers for early diagnosis. In this work, we describe the most common metabolomic platforms used in biomedical research, with special focus on strategies based on direct MS analysis. Then, a comprehensive review on the application of direct MS fingerprinting in clinical issues is provided.
... It is, therefore, likely that such a change in metabolome is directly linked to the toxicity. To address this issue, many researchers have conducted com-prehensive metabolomic analyses, the results of which have suggested that dioxins alter the levels of a number of compounds and components in the liver, [10][11][12] skeletal muscle, 12) hippocampus, 13) cerebellum, 14) blood, 15,16) and urine 17) in mice and rats, and in HepG2 cells. 18) However, in spite of these research findings, the component(s) contributing to dioxin toxicity appears to remain largely unidentified. ...
... The operating conditions of LC were as follows: column: HSS T3 column (2.1×100 mm, 1.8 mm i.d., Waters); column temperature: 40°C; sample room temperature: 4°C (urine) and 10°C (feces); mobile phase: A: 0.1% formic acid (positive ion mode) or 10 mM 4-methylmorphorin (negative ion mode), and B: methanol (urine) or acetonitrile (feces); elution program [% of B in A (min)]: 0% (0-5), 0 to 40% (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), 40 to 90% (20-29), 90% (29)(30), 90 to 0% (30-31) and 0% (31)(32)(33)(34)(35); flow rate, 0.2 mL/min. ...
This study investigated dioxin-induced changes in metabolomes in pubertal rat excrement. The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or restricting dietary intake (pair-fed group) markedly altered the metabolomic profile including lipids, hormones, and vitamins in the urine and feces. TCDD caused an increase in the fecal chenodeoxycholic acid and taurocholic acid content and in urinary adrenaline and 17β-estradiol, while the urinary melatonin level was reduced by TCDD. These changes were not observed in the pair-fed group. In accordance with the elevated level of fecal bile acids, TCDD reduced the intestinal expression of the apical sodium-dependent bile salt transporter, which plays a role in resorbing bile acids from the bile duct. In addition, CYP7A1, a rate-limiting enzyme for bile acid biosynthesis, was attenuated by TCDD treatment, although TCDD induced hepatic CYP8B1, an enzyme essential for cholic acid synthesis. Supplying cholic acid or chenodeoxycholic acid to TCDD-exposed rats tended to restore the TCDD-produced reduction in serum triglycerides, whereas no similar trend was observed in wasting syndrome and lipid accumulation in the liver. These results suggest that: 1) TCDD alters the circulating levels of bile acids and hormones via a mechanism distinct from an attenuation in dietary intake, although the majority of TCDDinduced changes in nutrient contents in the excrement is due to a reduction in food intake; and 2) TCDD facilitates the excretion of bile acids and disrupts their biosynthesis, resulting in the disturbance of lipid homeostasis.
... Numerous efforts have been made in the last years to identify metabolic failures associated with pathological mechanisms underlying to Alzheimer's disease. To this end, different authors have previously addressed the metabolomic investigation of several mouse models of AD using both brain tissue and blood samples, such as the APP/PS1 E9 (Graham et al., 2013), TASTPM (Hu et al., 2012;Forster et al., 2012), CRND8 (Salek et al., 2010;Lin et al., 2013;Lin et al., 2014), APP/PS1 M146L (Woo et al., 2010;Trushina et al., 2012), APP Tg2576 (Dedeoglu et al., 2004;Lalande et al., 2014) or SAMP8 (Jiang et al., 2008;Wang et al., 2014), among others. Thereby, multiple associations have been described between Alzheimer's disease and metabolic perturbations such as oxidative stress, mitochondrial dysfunction, abnormal lipid metabolism or inflammatory processes. ...
... Finally, a considerable accumulation of several eicosanoids was also found in serum from these transgenic models, including prostaglandins (PG), hydroxy-eicosapentaenoic acid (HEPE) and leukotriene B4 (LTB4), with the highest fold changes with regard to wild type controls (Table 1). The increase of these pro-inflammatory mediators in brain and biofluids has been repeatedly described in both human AD (Bazan et al., 2002;Trushina et al., 2013) and transgenic models (Piro et al., 2012;Lin et al., 2013;Lin et al., 2014). This inflammatory process has been traditionally associated with the overactivation of phospholipases, principally PLA 2 , leading to the release of polyunsaturated fatty acids from the hydrolysis of phospholipids (Farooqui et al., 2004), and subsequent oxidation by cyclooxygenases and 5-lipoxygenase (Manev et al., 2011), generating a complex meshwork of lipid mediators closely associated with neuronal pathways involved in AD neurobiology (Frisardi et al., 2011). ...
Inflammation plays a prominent role in the pathogenesis of Alzheimer’s disease, affecting both brain and the peripheral system. Thus, modulation of inflammation in animal models of this neurodegenerative disorder may be of great interest to elucidate the pathological mechanisms underlying this inflammatory component. To this end, a metabolomic investigation on a triple transgenic mouse model obtained by crossing the APP/PS1 mice with interleukin-4 knockout mice (a model of impaired immune function) was performed for the first time. Serum samples from transgenic mice and wild type animals were analyzed by direct infusion mass spectrometry followed by multivariate statistics in order to identify altered metabolites. Subsequently, metabolic pathway analysis allowed the elucidation of potential pathological mechanisms associated with the development of Alzheimer-type disorders in response to interleukin-4 deficiency, such as impaired homeostasis of histamine, altered metabolism of amino acids (threonine, aspartate and tyrosine), deregulated urea cycle and increased production of eicosanoids. Therefore, this work demonstrates the potential of this triple transgenic model with modulated immunity for the study of pathological mechanisms associated with inflammation in Alzheimer’s disease.
... Furthermore, the role of a dysregulated endocannabinoid-eicosanoid network in the pathogenesis of AD has been recently demonstrated in the APP/PS1 mice with inactivated monoacylglycerol lipase [52]. On the other hand, other studies focused on individual brain areas including the hippocampus [42,65,72], cortex [11] and cerebellum [1,43], because metabolic perturbations induced by AD-type disorders could be region-specific in the brain. In this sense, the characterization of regional metabolomic perturbations may be of greater interest in order to investigate the impact of disease on different brain regions and determine the most affected ones in AD mice. ...
... Concentrations of nucleosides (adenosine, guanosine, inosine) tended to be higher in the brains from APP/PS1 mice, which might suggest a disturbed neuroprotective function leading to neural damage because purine nucleosides exert important neuromodulator roles in the central nervous system [64]. In addition, this abnormal recycling of brain nucleosides is finally reflected in altered levels of other purine metabolites such as adenine, hypoxanthine and xanthine (Table 2), in agreement with previous studies [33,34,43]. Therefore, the metabolism of purines highlights as a candidate pathway for the search of potential markers of pathological processes occurring in the APP/PS1 transgenic mice, as schematized in Fig. 2 (simplified scheme of biochemical pathways related to metabolism of purines according to the Kyoto Encyclopedia of Genes and Genomes). ...
Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide, but its etiology is still not completely understood. The identification of underlying pathological mechanisms is becoming increasingly important for the discovery of biomarkers and therapies, for which metabolomics presents a great potential. In this work, we studied metabolic alterations in different brain regions of the APP/PS1 mice by using a high-throughput metabolomic approach based on the combination of gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry. Multivariate statistics showed that metabolomic perturbations are widespread, affecting mainly to hippocampus and cortex, but also present in regions not primarily associated with AD such as striatum, cerebellum and olfactory bulbs. Multiple metabolic pathways could be linked to the development of AD-type disorders in this mouse model, including abnormal purine metabolism, bioenergetic failures, dyshomeostasis of amino acids and disturbances in membrane lipids, among others. Interestingly, region-specific alterations were observed for some of the potential markers identified, associated with abnormal fatty acid composition of phospholipids and sphingomyelins, or differential regulation of neurotransmitter amino acids (e.g. glutamate, glycine, serine, N-acetyl-aspartate), not previously described to our knowledge. Therefore, these findings could provide a new insight into brain pathology in Alzheimer's disease.
... liver, kidney, spleen, thymus), 100 with results in agreement with earlier studies performed in human AD cohorts [91][92][93][94][95] and complementary works carried out in the same animal model by using orthogonal GC-MS and RPLC-MS. [64][65][66][67] Similarly, Lin et al. also employed direct infusion MS with the aim of characterizing metabolic perturbations in the hippocampus 101 and cerebellum 102 of the CRND8 transgenic model, which were mainly related to altered metabolism of arachidonic acid and eicosanoids, amino acids, nucleotides and other metabolite classes. ...
The efficient analysis of polar and charged metabolites in biological samples remains a huge challenge in the field of metabolomics. Over the past years, novel mass spectrometry-based analytical tools have been developed to enable the sensitive and efficient profiling of polar ionogenic metabolites in various biological samples. This book gives the reader a comprehensive overview of these recent technological developments.
Topics covered include the use of chemical labelling strategies for allowing the analysis of polar metabolites using reversed-phase liquid chromatography–mass spectrometry (RPLC-MS) and the latest methodological developments in RPLC-MS, hydrophilic interaction liquid chromatography (HILIC)-MS and ion-pair LC-MS approaches. Attention is also paid to developments in nano-LC-MS and capillary electrophoresis–mass spectrometry methods specifically for profiling polar metabolites in small volume biological samples. The utility of ion-mobility MS and NMR spectroscopy will also be outlined. Sample preparation is the key part in the analytical workflow employed for metabolomics. Therefore, ample emphasis will be given on recent solid-phase extraction and solid-phase micro-extraction methods. Finally, analytical techniques for chiral metabolic profiling will also be considered.
Discussing the state-of-the-art of the proposed topics in one single book for probing the polar metabolome, using relevant examples, is unique and needed in the metabolomics field. This book has relevance and appeal to an international audience of analytical and biomedical researchers in industry and academia.
... It should be noted that the deposition of senile plaques containing Aβ peptides and the formation of neurofibrillary tangles are mainly localized in medial temporal lobe structures, specifically the cortex and the hippocampus [51]. While numerous studies in transgenic models of AD have been performed to study the whole brain, other studies have focused on individual brain areas [10,[52][53][54][55][56], as metabolic perturbations induced by AD-type disorders can be region-specific in the brain [57,58]. Most of the findings from these studies suggest that the cortex and the hippocampus are the most sensitive regions during early-stage AD [53,59]. ...
Alzheimer's disease (AD) is characterized, amongst other features, by the pathologic accumulation of abnormally phosphorylated tau filaments in neurons that lead to neurofibrillary tangles. However, the molecular mechanisms by which the abnormal processing of tau leads to neurodegeneration and cognitive impairment remain unknown. Metabolomic techniques can comprehensively assess disturbances in metabolic pathways that reflect changes downstream from genomic, transcriptomic and proteomic systems. In the present study, we undertook a targeted metabolomic approach to determine a total of 187 prenominated metabolites in brain cortex tissue from wild type and rTg4510 animals (a mice model of tauopathy), in order to establish the association of metabolic pathways with cognitive impairment. This targeted metabolomic approach revealed significant differences in metabolite concentrations of transgenic mice. Brain glutamine, serotonin and sphingomyelin C18:0 were found to be predictors of memory impairment. These findings provide informative data for future research on AD, since some of them agree with pathological alterations observed in diseased humans.
... Interestingly, these findings were then validated by applying orthogonal hyphenated approaches, such as UHPLC-MS [34][35][36][37][38], GC-MS [35][36][37][38][39], and CE-MS [40], thus demonstrating the reliability of direct MS for metabolomic fingerprinting. In this line, Lin et al. reported the use of DIMS techniques to address metabolic alterations in the hippocampus [41] and cerebellum [42] of CRND8 transgenic mice, revealing significant perturbations in the regulation of neuroinflammatory processes. Finally, it is also noteworthy the recent development of a novel metabolomic approach based on flow injection analysis using an atmospheric pressure photoionization mass spectrometer (FI-APPI-MS) with the aim to complement the ionization capabilities of the electrospray source (ESI), usually employed in DIMS-based metabolomics [43]. ...
Metabolomics based on direct mass spectrometry analysis shows a great potential in biomedical research because of its high-throughput screening capability and wide metabolome coverage. This chapter contains detailed protocols to perform comprehensive metabolomic fingerprinting of multiple biological samples (serum, plasma, urine, brain, liver, spleen, thymus) by using complementary analytical platforms. The most important issues to be considered are discussed, including sample treatment, metabolomic analysis, raw data preprocessing, and data analysis.
... Decreased pyroglutamate and uric acid in Tg-ctrl mice may reflect glutathione insufficiency [70] and a failure in scavenging of peroxynitrite in neurons [71]. Moreover, increased prostaglandin which is a product of the arachidonic acid cascade, possibly reflecting damaging pro-oxidant cascades [72,73]. Thus, oxidative stress is strongly implicated in AD, as has been reported previously [74][75][76], in contrast, treatment with the antioxidant TRF reduces the stress. ...
We have recently shown that the tocotrienol-rich fraction (TRF) of palm oil, a mixture of vitamin E analogs, improves amyloid pathology in vitro and in vivo. However, precise mechanisms remain unknown. In this study, we examined the effects of long-term (10 months) TRF treatment on behavioral impairments and brain metabolites in (15 months old) AβPP/PS1 double transgenic (Tg) Alzheimer's disease (AD) mice. The open field test, Morris water maze, and novel object recognition tasks revealed improved exploratory activity, spatial learning, and recognition memory, respectively, in TRF-treated Tg mice. Brain metabolite profiling of wild-type and Tg mice treated with and without TRF was performed using ultrahigh performance liquid chromatography (UHPLC) coupled to high-resolution accurate mass (HRAM)-orbitrap tandem mass spectrometry (MS/MS). Metabolic pathway analysis found perturbed metabolic pathways that linked to AD. TRF treatment partly ameliorated metabolic perturbations in Tg mouse hippocampus. The mechanism of this pre-emptive activity may occur via modulation of metabolic pathways dependent on Aβ interaction or independent of Aβ interaction.
... Lin et al. also described the application of DIMS-based metabolomics to investigate the characteristic neurochemical profile of the CRND8 transgenic mice. They found increased hippocampal production of neuroinflammatory eicosanoids [44], as well as profound metabolic perturbations related to abnormal metabolism of purines and fatty acids in the cerebellum [45]. Furthermore, it is also worth noting the utility of combining complementary ionization sources in order to maximize metabolomic coverage, as recently demonstrated by applying FI-APPI-MS fingerprinting on serum samples from AD patients [46] and APP 9 PS1 mice [34]. ...
Correction to: J. Anal. Test. (2017) 1:17 DOI 10.1007/s41664-017-0018-4.
... Altered purine metabolism associated with neurodegenerative processes has been described in AD [46,47]. Decreased levels of energy-rich diphosphates and triphosphates (ATP, ADP, inosine 5′-diphosphate, guanosine 5′-diphosphate) and increased levels of purine bases (hypoxanthine, inosine, xanthine, xanthosine) were found in the brains of Tg animals compared with controls (Fig. 4). ...
Background
Tauopathies represent heterogeneous groups of neurodegenerative diseases that are characterised by abnormal deposition of the microtubule-associated protein tau. Alzheimer’s disease is the most prevalent tauopathy, affecting more than 35 million people worldwide. In this study we investigated changes in metabolic pathways associated with tau-induced neurodegeneration.
Methods
Cerebrospinal fluid (CSF), plasma and brain tissue were collected from a transgenic rat model for tauopathies and from age-matched control animals. The samples were analysed by targeted and untargeted metabolomic methods using high-performance liquid chromatography coupled to mass spectrometry. Unsupervised and supervised statistical analysis revealed biochemical changes associated with the tauopathy process.
Results
Energy deprivation and potentially neural apoptosis were reflected in increased purine nucleotide catabolism and decreased levels of citric acid cycle intermediates and glucose. However, in CSF, increased levels of citrate and aconitate that can be attributed to glial activation were observed. Other significant changes were found in arginine and phosphatidylcholine metabolism.
Conclusions
Despite an enormous effort invested in development of biomarkers for tauopathies during the last 20 years, there is no clinically used biomarker or assay on the market. One of the most promising strategies is to create a panel of markers (e.g., small molecules, proteins) that will be continuously monitored and correlated with patients’ clinical outcome. In this study, we identified several metabolic changes that are affected during the tauopathy process and may be considered as potential markers of tauopathies in humans.
Electronic supplementary material
The online version of this article (doi:10.1186/s13195-017-0303-5) contains supplementary material, which is available to authorized users.
... Lin et al. also described the application of DIMS-based metabolomics to investigate the characteristic neurochemical profile of the CRND8 transgenic mice. They found increased hippocampal production of neuroinflammatory eicosanoids [44], as well as profound metabolic perturbations related to abnormal metabolism of purines and fatty acids in the cerebellum [45]. Furthermore, it is also worth noting the utility of combining complementary ionization sources in order to maximize metabolomic coverage, as recently demonstrated by applying FI-APPI-MS fingerprinting on serum samples from AD patients [46] and APP 9 PS1 mice [34]. ...
Metabolomics is largely employed in numerous biomedical research fields, such as the study of the underlying pathology of diseases, discovery of diagnostic biomarkers, or drug development. Nowadays, the main challenge is to obtain comprehensive and unbiased metabolomic profiles due to the huge complexity, heterogeneity, and dynamism of the metabolome. To this end, mass spectrometry represents a very interesting analytical platform, since the complexity of metabolome may be overcome through the use of different orthogonal separation techniques, including liquid chromatography, gas chromatography, and capillary electrophoresis. Alternatively, direct mass spectrometry analysis has been postulated as a complementary choice to hyphenated approaches. This technique exhibits several advantages such as the ability for high-throughput screening, fast analysis, and wide metabolomic coverage, since there is not exclusion of compounds due to the separation device. The present work explores the utility of metabolomics based on direct infusion mass spectrometry for analyzing blood samples. The most important analytical concerns to be considered are discussed, including sample handling, comprehensive fingerprinting, as well as subsequent identification of metabolites, and global characterization of metabolomic profiles. To conclude, a brief review on the application of these metabolomic platforms in Alzheimer’s disease research is also provided.
... MS-fingerprinting of serum samples from these mice revealed significant changes in circulating levels of different eicosanoids, amino acids and related compounds, thus evidencing that depletion of interleukin 4 might potentiate AD pathology in the APP × PS1 model [64]. To conclude, Lin et al. employed ultra-high resolution mass spectrometry to identify metabolomic perturbations in hippocampus [65] and cerebellum [66] from CRND8 transgenic mice; and similarly to previous works with the APP × PS1 mouse model, metabolomic profiles exhibited characteristic features associated with abnormal homeostasis of arachidonic acid leading to the over-production of eicosanoids, as well as abnormal metabolism of amino acids, nucleotides and other metabolites. ...
Alzheimer’s disease is a complex neurodegenerative disorder characterized by a multi-factorial etiology, not completely understood to date. In this context, the application of metabolomics is emerging in the last years because of its potential to monitor molecular alterations associated with disease pathogenesis and progression, as well as to discover candidate diagnostic biomarkers. However, the huge heterogeneity and dynamism of the human metabolome makes impossible the simultaneous determination of the entire set of metabolites from complex biological samples. Thus, the most common strategy to get a comprehensive overview of the organism’s phenotypic expression is the combined use of complementary metabolomic platforms. In this review, we summarize the advantages and limitations of the most important analytical techniques usually employed in metabolomics, including nuclear magnetic resonance, direct infusion mass spectrometry and hyphenated approaches based on the coupling of orthogonal separation mechanisms (i.e. liquid chromatography, gas chromatography, capillary electrophoresis) with mass spectrometry. Moreover, the suitability of metabolomics to unravel the complex pathology underlying to Alzheimer’s disease is also presented.
... Each mass typically yielded a unique molecular formula within a mass tolerance of 1.0 ppm with a 13 C-isotope because of high mass accuracy. Nevertheless, the molecular formulas must be validated using the rules described by Lin et al. (2014). The sum formulas of the true discriminant masses were calculated and validated through isotope pattern matching using Bruker Daltonics Data Analysis version 3.4. ...
Biosurfactant producers are crucial for incremental oil production in microbial enhanced oil recovery (MEOR) processes. The isolation of biosurfactant-producing bacteria from oil reservoirs is important because they are considered suitable for the extreme conditions of the reservoir. In this work, a novel biosurfactant-producing strain Acinetobacter junii BD was isolated from a reservoir to reduce surface tension and emulsify crude oil. The biosurfactants produced by the strain were purified and then identified via electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR-MS). The biosurfactants generated by the strain were concluded to be rhamnolipids, the dominant rhamnolipids were C26H48O9, C28H52O9, and C32H58O13. The optimal carbon source and nitrogen source for biomass and biosurfactant production were NaNO3 and soybean oil. The results showed that the content of acid components increased with the progress of crude oil biodegradation. A glass micromodel test demonstrated that the strain significantly increased oil recovery through interfacial tension reduction, wettability alteration and the mobility of microorganisms. In summary, the findings of this study indicate that the newly developed BD strain and its metabolites have great potential in MEOR.
... Statistical analysis and metabolic annotation. Metabolomic data analysis followed our previous publications 33,34 . Briefly, the DI-ICR-FT-MS mass spectra were exported to peak lists for alignment. ...
Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.
... The Tg CRND8 mouse is a widely used transgenic mouse model, which exhibit early-onset Aβ deposition, cognitive deficits as well as behavioral abnormalities that parallel AD in humans. Previous tissue metabolomics studies from our laboratories have shown that widespread metabolic perturbations can occur as early as 2-3 months, prior to any histologically detectable symptoms appear [13,14]. More recently, several reports found that a panel of urinary metabolites is sufficient to allow metabolic discrimination of AD mouse models as well as delineating the oxidative stress response in a non-invasive manner [15,16]. ...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with amyloid plaques accumulation as the key feature involved in its pathology. To date, however, the biochemical changes in AD have not been clearly characterized. Here, we present that urinary metabolomics based on high resolution mass spectrometry was employed for delineation of metabolic alterations in transgenic CRND8 mice. In this noninvasive approach, urinary metabolome reveals the biochemical changes in early onset of this AD mouse model. In virtue of comprehensive metabolite profiling and multivariate statistical analysis, a total of 73 differential metabolites of urine sample sets was identified in 12-week and 18-week transgenic mice compared to wild-type littermates, covering perturbations of aromatic amino acids metabolism, the Krebs cycle and one-carbon metabolism. Of particular interest is that divergent tryptophan metabolism, such as upregulation of serotonin pathway while downregulation of kynurenine pathway, was observed. Meanwhile, the accumulation of both N-acetylvanilalanine and 3-methoxytyrosine indicated aromatic L-amino acid decarboxylase deficiency. And the microbial metabolites derived from aromatic amino acid metabolism and drug-like phase II metabolic response via the glycine conjugation reactions were also highlighted, indicating that genetic modification in mouse brain not only alters genotype but also perturbs the gut microbiome. Together, our study demonstrated that the integrative approach employing mass spectrometry-based metabolomics and a transgenic mouse model for AD may provide new evidence for distinct metabolic signatures. The perturbations of metabolic pathways may have far-reaching implications for early diagnosis and intervention in AD.
... The decrease observed in adenosine monophosphate levels (Table 1) and the complementary increase of uric acid (Table 2) suggests an impaired purine metabolism. In this sense, Lin et al. found a perturbed catabolism of different purine derivatives toward the accumulation of uric acid in the cerebellum of transgenic CRND8 mice [45]. Moreover, the degradation of AMP may have important consequences in cellular energy homeostasis, since it plays a central role in glucose and lipid metabolism through the AMP-activated protein kinase, which is known to be decreased in AD brain [46]. ...
The transgenic mouse APP/PS1 is widely employed by neuroscientists because reproduces well some of the neuropathological and cognitive deficits observed in human Alzheimer's disease. In this study, serum samples from APP/PS1 mice (n = 30) and wild-type controls (n = 30) were analyzed using a metabolomic multiplatform based on the combination of gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry, in order to obtain wide information about serum metabolome. Metabolic profiles showed significant differences between the groups of study, and numerous metabolites were identified as potential players in the development of Alzheimer-type disorders in this transgenic model. Pathway analysis revealed the involvement of multiple metabolic networks in the underlying pathology, such as deficiencies in energy metabolism, altered amino acid homeostasis, abnormal membrane lipid metabolism, and other impairments related to the integrity of the central nervous system. It is noteworthy that some of these metabolomic markers are in accordance with pathological alterations observed in human Alzheimer's disease, while others have not been previously described. Therefore, these results demonstrate the potential of metabolomics and the use of transgenic animal models to understand the pathogenesis of Alzheimer's disease.
Copyright © 2015 Elsevier B.V. and Sociétéfrançaise de biochimie et biologie Moléculaire (SFBBM). All rights reserved.
... Alternatively, metabolomics based on direct infusion mass spectrometry (DIMS) has proved to be a useful screening tool due to its wide metabolome coverage and fast analysis, despite having several drawbacks such as the lack of resolution for isobars differentiation and difficulty of quantification without stable-isotope internal standards [19]. However, this high-throughput approach is not as widespread as hyphenated techniques in routine metabolomic studies, and it was only previously applied twice to address metabolic changes in hippocampus [20] and cerebellum [21] of the CRND8 transgenic mice. ...
The identification of pathological mechanisms underlying to Alzheimer's disease is of great importance for the discovery of potential markers for diagnosis and disease monitoring. In this study, we investigated regional metabolic alterations in brain from the APP/PS1 mice, a transgenic model that reproduces well some of the neuropathological and cognitive deficits observed in human Alzheimer's disease. For this purpose, hippocampus, cortex, cerebellum and olfactory bulbs were analyzed using a high-throughput metabolomic approach based on direct infusion mass spectrometry. Metabolic fingerprints showed significant differences between transgenic and wild-type mice in all brain tissues, being hippocampus and cortex the most affected regions. Alterations in numerous metabolites were detected including phospholipids, fatty acids, purine and pyrimidine metabolites, acylcarnitines, sterols and amino acids, among others. Furthermore, metabolic pathway analysis revealed important alterations in homeostasis of lipids, energy management, and metabolism of amino acids and nucleotides. Therefore, these findings demonstrate the potential of metabolomic screening and the use of transgenic models for understanding pathogenesis of Alzheimer's disease.
Copyright © 2014 Elsevier B.V. All rights reserved.
... AMPactivated protein kinase, responsible for regulation of glucose and lipid metabolism, is implicated in pathogenesis of AD [53]. Thereby, previous authors found numerous alterations in purine and pyrimidine metabolites in AD brain and cerebrospinal fluid in response to enhanced nucleotide degra-dation [21,[54][55][56]. Alternatively, the reduction of urea levels in both spleen and thymus denotes perturbed ammonia recycling through the urea cycle. ...
Inflammatory processes and other failures related to the immune system are common features associated with Alzheimer's disease, in both brain and the peripheral system. Thus, the study of the main organs of the immune system may have a great potential for the elucidation of pathological mechanisms underlying these abnormalities. This is the first metabolomic investigation performed in spleen and thymus from transgenic mice of Alzheimer's disease. Tissues were fingerprinted using a metabolomic platform comprising gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry. Multivariate statistics demonstrated significant differences in numerous metabolites between the APP/PS1 mice and wild type controls, and it was proven that multiple biochemical pathways are disturbed in these organs including abnormal metabolism of phospholipids, energy deficiencies, altered homeostasis of amino acids, oxidative stress, and others. Therefore, these findings highlight the importance of the proper metabolic functioning of peripheral immune system in the development of neurodegenerative disorders such as Alzheimer's disease. This article is protected by copyright. All rights reserved.
... In these studies, cerebrospinal fluid (CSF) is usually the biofluid of choice as its composition is directly related to metabolite production in the brain, but the use of noninvasive samples such as blood serum or plasma is gaining importance in order to get easier and cheaper methods for diagnosis. However, the use of shotgun metabolomics has not been yet reported for AD diagnosis, except in recent studies with brain of transgenic mice [19,20], which could contribute to deepen into our understanding of the pathological mechanisms occurring in this disorder. ...
Currently, there is no cure for Alzheimer’s disease and early diagnosis is very difficult, since no biomarkers have been established with the necessary reliability and specificity. For the discovery of new biomarkers, the application of omics is emerging, especially metabolomics based on the use of mass spectrometry. In this work, an analytical approach based on direct infusion electrospray mass spectrometry was applied for the first time to blood serum samples in order to elucidate discriminant metabolites. Complementary methodologies of extraction and mass spectrometry analysis were employed for comprehensive metabolic fingerprinting. Finally, the application of multivariate statistical tools allowed us to discriminate Alzheimer patients and healthy controls, and identify some compounds as potential markers of disease. This approach provided a global vision of disease, given that some important metabolic pathways could be studied, such as membrane destabilization processes, oxidative stress, hypometabolism, or neurotransmission alterations. Most remarkable results are the high levels of phospholipids containing saturated fatty acids, respectively, polyunsaturated ones and the high concentration of whole free fatty acids in Alzheimer’s serum samples. Thus, these results represent an interesting approximation to understand the pathogenesis of disease and the identification of potential biomarkers.
Graphical Abstract
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Amino acids’ neuroactivity, and roles in excitotoxity and oxidative stress are linked to dementia. We aimed to investigate whether circulating amino acid concentrations were associated with cognitive decline in patients with mild Alzheimer’s disease (AD) and Lewy body dementia (LBD). Baseline serum amino acid concentrations were measured in 89 patients with AD and 65 with LBD (13 with Parkinson’s disease dementia and 52 with dementia with Lewy bodies). The Mini-Mental State Examination (MMSE) was administered at baseline and annually for five years. Associations between baseline amino acid concentrations and longitudinal MMSE score were assessed using a linear-mixed effects model stratified by diagnosis with adjustment for multiple comparisons. The results of the study indicated that serum tyrosine was positively associated with MMSE performance during the five-year follow-up period in patients with LBD (q-value=0.012), but not AD. In conclusion, higher baseline serum concentrations of tyrosine, the precursor amino acid in dopamine and norepinephrine synthesis, was associated with better cognitive performance in patients with LBD, but not AD, throughout the 5-year follow-up period.
In order to investigate the protective effects of Rhodiola crenulata extract on Alzheimer's disease, a brain metabolomics study in rats was conducted by HPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry. Rat model was constructed by bilateral hippocampal injection of amyloid‐β peptide and immunohistochemistry was performed to evaluate the pharmacological effect of Rhodiola crenulata extract. Multivariate statistical analysis was used to discover potential biomarkers in rat brain and related metabolic pathways analysis was conducted to elucidate the action mechanism of Rhodiola crenulata extract. As a result, a total of 19 metabolites contributing to Alzheimer's disease progress were identified and 9 of them were restored to the normal levels after drug administration. Pathway analysis revealed that the protective effects of Rhodiola crenulata extract are related to the regulation of glutathione metabolism and arachidonic acid metabolism in rat brain. In conclusion, this work demonstrates that the developed metabolomics method is useful to investigate the protective effects of Rhodiola crenulata extract against Alzheimer's disease. These outcomes may further provide reliable evidence to illuminate the intervention mechanism of other traditional Chinese medicines on Alzheimer's disease.
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A metabolomic strategy based on accurate mass and isotopic fine structures (IFSs) by dual mode combined-Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was established to explore the effects of Rhodiola crenulata extract (RCE) on Alzheimer disease (AD) in rats. Experimental AD model was induced in rats by bilateral hippocampal injection of Aβ1-42, and Morris water maze task (MWM) was used to evaluate the effects of RCE on AD. Subsequently, the metabolomic study was performed using HPLC-FT-ICR-MS, fraction collector and direct infusion (DI)-FT-ICR-MS to screen and identify the potential biomarkers. A total of 20 metabolites contributing to AD progress were identified, and 17 metabolites of them were restored to the control-like levels after RCE treatment (daily dose: 2.24 g/kg). The metabolic pathway analysis revealed that the disturbed pathways including tryptophan metabolism, sphingolipid metabolism and glycerophospholipid metabolism in AD model rats were regulated after high dose RCE application. It is the first time that the dual mode combined-FT-ICR-MS based metabolomic strategy was applied to biochemically profile the serum metabolic pathways of AD rats affected by RCE. These outcomes provide reliable evidence to illuminate the biochemical mechanisms of AD and facilitate investigation of the therapeutic benefits of RCE in AD treatment. Notably, it indicated that the developed method based on accurate mass and IFSs has sufficient performance for identification of biomarkers in metabolomic studies.
Resolving power is a critical factor determining the quality of ultrahigh-resolving power mass spectra of crude oil. In this study, 7T Fourier-transform ion cyclotron mass spectrometry (FT-ICR MS), equipped with quadrupole detection, was applied and evaluated for crude oil analysis for the first time. Four spectra were obtained from two oil samples using two ionization methods. Resolving power of 1,500,000 was observed at m/z 400 with 4 seconds transient signal. Comparison with literature reports revealed that the achieved resolving power was comparable with or superior to those obtained from instruments using higher magnetic fields but without quadrupole detection. 6,000–10,000 peaks with an S/N ratio of 3 or higher were observed from the obtained
spectra and over 97% of the peaks could be assigned to appropriate chemical formulas with an error within 1 ppm. Double bond equivalents vs. carbon number plots generated from the obtained data agreed well with those previously reported without quadrupole detection. Mass accuracy values of the assigned elemental formulas were examined and the average root mean square error was calculated to be only 160 ppb. Low unassignment rate of the observed peaks and strong agreement with previously reported results suggests that unwanted harmonics of reduced frequency are not significant for the data obtained with quadrupole detection. Overall, the data presented in this study show that FT-ICR MS equipped with quadrupole detection can be powerful a tool to examine complex mixtures like crude oil. To the best of our knowledge, this is the first paper reporting application of FT-ICR MSequipped with quadrupole detection for the oil analysis.
an in-depth treatise identifying hundreds of causes of “AD” identified by profiling an exhaustive compilation of experimental results from the literature
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin elicits many harmful effects in tissues. Metabolomic changes and the associated pathway alterations caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the cerebellum, an area thought to be less affected by environmental alterations, remain unknown. Here, metabolomics was performed to identify endogenous metabolites that were associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin in the cerebellum of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated mice using Fourier transform ion cyclotron resonance mass spectrometry. Distinct peaks were located in two mass ranges, 210 m/z–420 m/z and 450 m/z–570 m/z. In principal component space, the high-dose group was clearly separated from the control group. Six metabolites associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin dose were identified. The metabolite 1-palmitoyl lysophosphatidic acid increased with increasing doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, indicating activation of the rat sarcoma pathway. Biosynthesis of the unsaturated fatty acid 18-hydroxyoleate was inhibited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. The decrease in N-arachidonoyl taurine, implies that taurine increased, suggesting inhibition of neuronal signal transmission. A decrease in N-acetyl-aspartyl-glutamate has been associated with injury of the cerebellum through activation of N-methyl-D-aspartic acid receptors. An increase in glycerophosphoinositol suggests damage to blood–brain barrier function, and changes in purine metabolism were observed because inosine increased following 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. These results suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin activates the rat sarcoma pathway, alters fatty acid biosynthesis and purine metabolism, inhibits neurotransmitter systems, and is harmful to blood–brain barrier function in the cerebellum.
Metabolomics has demonstrated a great potential for the study of pathological mechanisms occurring in brain from Alzheimer's disease patients and transgenic models. However, its application to peripheral samples is not so common, although it can provide interesting information about systemic abnormalities underlying to disease. This work represents the first metabolomic investigation of multiple peripheral organs (liver, kidney, spleen and thymus) from the APP/PS1 mice by using a high-throughput approach based on direct infusion mass spectrometry. Our findings demonstrated that these organs suffer significant metabolic impairments related to energy metabolism (e.g. glycolysis, Krebs cycle, β-oxidation), lipid homeostasis (e.g. cellular membrane breakdown, fatty acid metabolism), degradation of nucleotides, oxidative stress, hyperammonemia and metabolism of amino acids. It is noteworthy that many of these alterations have been previously described in brain, confirming the systemic character of this neurodegenerative disorder and the utility of peripheral samples to understand its pathogenesis.This article is protected by copyright. All rights reserved
In the wake of genomics, metabolomics characterizes the small molecular metabolites revealing the phenotypes induced by gene mutants. To address the metabolic signatures in the hippocampus of the amyloid-beta (Aβ) peptides produced in transgenic (Tg) CRND8 mice, high-field ion cyclotron resonance-Fourier transform mass spectrometry supported by LC-LTQ-Orbitrap was introduced to profile the extracted metabolites. More than 10,000 ions were detected in the mass profile for each sample. Subsequently, peak alignment and the 80 % rule followed by feature selection based on T score computation were performed. The putative identification was also conducted using the highly accurate masses with isotopic distribution by interfacing the MassTRIX database as well as MS/MS fragmentation generated in the LTQ-Orbitrap after chromatographic separation. Consequently, 58 differentiating masses were tentatively identified while up to 44 differentiating elemental compositions could not be biologically annotated in the databases. Nonetheless, of the putatively annotated masses, eicosanoids in arachidonic acid metabolism, fatty acid beta-oxidation disorders as well as disturbed glucose metabolism were highlighted as metabolic traits of Aβ toxicity in Tg CRND8 mice. Furthermore, a web-based bioinformatic tool was used for simulation of the metabolic pathways. As a result of the obtained metabolic signatures, the arachidonic acid metabolism dominates the metabolic perturbation in hippocampal tissues of Tg CRND8 mice compared to non-Tg littermates, indicating that Aβ toxicity functions neuroinflammation in hippocampal tissue and new theranostic opportunities might be offered by characterization of altered arachidonic acid metabolism for Alzheimer's disease.
4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined.
In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding.
The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.
Text mining methods have added considerably to our capacity to extract biological knowledge from the literature. Recently the field of systems biology has begun to model and simulate metabolic networks, requiring knowledge of the set of molecules involved. While genomics and proteomics technologies are able to supply the macromolecular parts list, the metabolites are less easily assembled. Most metabolites are known and reported through the scientific literature, rather than through large-scale experimental surveys. Thus it is important to recover them from the literature. Here we present a novel tool to automatically identify metabolite names in the literature, and associate structures where possible, to define the reported yeast metabolome. With ten-fold cross validation on a manually annotated corpus, our recognition tool generates an f-score of 78.49 (precision of 83.02) and demonstrates greater suitability in identifying metabolite names than other existing recognition tools for general chemical molecules. The metabolite recognition tool has been applied to the literature covering an important model organism, the yeast Saccharomyces cerevisiae, to define its reported metabolome. By coupling to ChemSpider, a major chemical database, we have identified structures for much of the reported metabolome and, where structure identification fails, been able to suggest extensions to ChemSpider. Our manually annotated gold-standard data on 296 abstracts are available as supplementary materials. Metabolite names and, where appropriate, structures are also available as supplementary materials.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-010-0251-6) contains supplementary material, which is available to authorized users.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to have the adverse effects on human health. In this study, we applied a metabolomic approach in conjunction with unsupervised and supervised machine learning methods to investigate the toxic effects of TCDD. By using liquid chromatography/quadrupole time-of-flight mass spectrometry, non-targeted metabolomic analysis revealed the metabolic signatures of the toxicity in aryl hydrocarbon receptor (AhR)-high affinity C57BL/6J (C6) mice as well as low affinity strain-DBA/2J (D2) mice. Lysophospholipids and long chain fatty acids were strikingly elevated in the C6 mice exposed to TCDD in both liver and skeletal muscle tissues. Meanwhile, the level of palmitoylcarnitine, which is one of the important indicators in fatty acid β-oxidation, increased significantly. Moreover, several nucleosides and amino acids decreased markedly. On the other hand, much less differentiating metabolites were highlighted in another strain-D2 mouse model. Taking liver and skeletal muscle tissues together, the levels of inosine, valine and glutamine decreased significantly. One lysophospholipid and two fatty acids were found to be enhanced. The principal components analysis and support vector machine clustering results also exhibited discriminations in the liver and skeletal muscle tissues of the mice. The obtained results indicated that TCDD could disrupt several metabolic pathways, including fatty acid biosynthesis and amino acid metabolism in both C6 and D2 mice. The increased rate of fatty acid beta-oxidation, however, was only observed in the liver and skeletal muscle tissues of C6 mice. The perturbation of the tricarboxylic acid (TCA) cycle was testified in two strains but the change was much slighter in D2 mice. It was of particular interest to note that the succinate level was enhanced in the liver tissues of both strains, and particularly, the change was up to 11.49-fold in the liver of C6 mice treated with TCDD. Collectively, the discrimination of D2 mice was not as distinct as that of C6 mice when exposed to the same dosage. Furthermore, D2 was confirmed to be less-sensitive rather than resistant to a high dose of TCDD.
Background:
A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISI(Matsuda)) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns.
Methodology and principal findings:
By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISI(Matsuda) of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISI(Matsuda) value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX.
Conclusions:
Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISI(Matsuda) are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase.
The causes and etiology of Crohn's disease (CD) are currently unknown although both host genetics and environmental factors play a role. Here we used non-targeted metabolic profiling to determine the contribution of metabolites produced by the gut microbiota towards disease status of the host. Ion Cyclotron Resonance Fourier Transform Mass Spectrometry (ICR-FT/MS) was used to discern the masses of thousands of metabolites in fecal samples collected from 17 identical twin pairs, including healthy individuals and those with CD. Pathways with differentiating metabolites included those involved in the metabolism and or synthesis of amino acids, fatty acids, bile acids and arachidonic acid. Several metabolites were positively or negatively correlated to the disease phenotype and to specific microbes previously characterized in the same samples. Our data reveal novel differentiating metabolites for CD that may provide diagnostic biomarkers and/or monitoring tools as well as insight into potential targets for disease therapy and prevention.
Wine chemical compositions, which result from a complex interplay between environmental factors, genetic factors, and viticultural practices, have mostly been studied using targeted analyses of selected families of metabolites. Detailed studies have particularly concerned volatile and polyphenolic compounds because of their acknowledged roles in the organoleptic and therapeutic properties. However, we show that an unprecedented chemical diversity of wine composition can be unraveled through a nontargeted approach by ultrahigh-resolution mass spectrometry, which provides an instantaneous image of complex interacting processes, not easily or possibly resolvable into their unambiguous individual contributions. In particular, the statistical analysis of a series of barrel-aged wines revealed that 10-year-old wines still express a metabologeographic signature of the forest location where oaks of the barrel in which they were aged have grown.
Metabolites of neural cells, is known to have a significant effect on the normal physiology and function of neurons in brain. However, whether they play a role in pathogenesis of neurodegenerative diseases is unknown. Here, we show that metabolites of neurons play essential role in the pathogenesis of Alzheimer's disease (AD). Firstly, in vivo and in vitro metabolites of cerebellar neurons both significantly induced the expression of Abeta-degrading enzymes in the hippocampus and cerebral cortex and promoted Abeta clearance. Moreover, metabolites of cerebellar neurons significantly reduced brain Abeta levels and reversed cognitive impairments and other AD-like phenotypes of APP/PS1 transgenic mice, in both early and late stages of AD pathology. On the other hand, metabolites of hippocampal neurons reduced the expression of Abeta-degrading enzymes in the cerebellum and caused cerebellar neurodegeneration in APP/PS1 transgenic mice. Thus, we report, for the first time, that metabolites of neurons not only are required for maintaining the normal physiology of neurons but also play essential role in the pathogenesis of AD and may be responsible for the regional-specificity of Abeta deposition and AD pathology.
We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.
The prevailing amyloid hypothesis for Alzheimer's disease (AD) holds that amyloid beta-protein (Abeta) causes neuronal degeneration by forming neurotoxic fibrillar structures. Yet, many aspects of AD pathology and symptoms are not well explained by this hypothesis. Here, we present evidence that neurotoxicity of soluble oligomeric Abeta closely corresponds to the selective neurodegeneration so distinctly manifest in AD. Selectivity was first observed in vitro, where only the human central nervous system neuronal cells were susceptible to soluble oligomeric Abeta. Furthermore, in mouse cerebral slice treated with soluble oligomeric Abeta, selective regiospecific toxicity was evident in the hippocampal CA1, a division important for memory, but not in the CA3 subfield. The fibrillar Abeta, however, killed neurons in all regions of the cerebral slice cultures and also in cerebellar slices. Remarkably, even at the highest soluble oligomeric Abeta concentrations, cerebellar neurons were completely spared, consistent with one of the hallmark features of AD pathology. Our observation of the selective neurodegeneration of soluble oligomeric Abeta to neurons involved in cognitive function may provide a new opportunity for the development of an effective AD therapy as well as elucidating the pathological mechanism of AD.
Structure elucidation of unknown small molecules by mass spectrometry is a challenge despite advances in instrumentation. The first crucial step is to obtain correct elemental compositions. In order to automatically constrain the thousands of possible candidate structures, rules need to be developed to select the most likely and chemically correct molecular formulas.
An algorithm for filtering molecular formulas is derived from seven heuristic rules: (1) restrictions for the number of elements, (2) LEWIS and SENIOR chemical rules, (3) isotopic patterns, (4) hydrogen/carbon ratios, (5) element ratio of nitrogen, oxygen, phosphor, and sulphur versus carbon, (6) element ratio probabilities and (7) presence of trimethylsilylated compounds. Formulas are ranked according to their isotopic patterns and subsequently constrained by presence in public chemical databases. The seven rules were developed on 68,237 existing molecular formulas and were validated in four experiments. First, 432,968 formulas covering five million PubChem database entries were checked for consistency. Only 0.6% of these compounds did not pass all rules. Next, the rules were shown to effectively reducing the complement all eight billion theoretically possible C, H, N, S, O, P-formulas up to 2000 Da to only 623 million most probable elemental compositions. Thirdly 6,000 pharmaceutical, toxic and natural compounds were selected from DrugBank, TSCA and DNP databases. The correct formulas were retrieved as top hit at 80-99% probability when assuming data acquisition with complete resolution of unique compounds and 5% absolute isotope ratio deviation and 3 ppm mass accuracy. Last, some exemplary compounds were analyzed by Fourier transform ion cyclotron resonance mass spectrometry and by gas chromatography-time of flight mass spectrometry. In each case, the correct formula was ranked as top hit when combining the seven rules with database queries.
The seven rules enable an automatic exclusion of molecular formulas which are either wrong or which contain unlikely high or low number of elements. The correct molecular formula is assigned with a probability of 98% if the formula exists in a compound database. For truly novel compounds that are not present in databases, the correct formula is found in the first three hits with a probability of 65-81%. Corresponding software and supplemental data are available for downloads from the authors' website.
Recent technical advances in mass spectrometry (MS) have brought the field of metabolomics to a point where large numbers
of metabolites from numerous prokaryotic and eukaryotic organisms can now be easily and precisely detected. The challenge
today lies in the correct annotation of these metabolites on the basis of their accurate measured masses. Assignment of bulk
chemical formula is generally possible, but without consideration of the biological and genomic context, concrete metabolite
annotations remain difficult and uncertain. MassTRIX responds to this challenge by providing a hypothesis-driven approach
to high precision MS data annotation. It presents the identified chemical compounds in their genomic context as differentially
colored objects on KEGG pathway maps. Information on gene transcription or differences in the gene complement (e.g. samples
from different bacterial strains) can be easily added. The user can thus interpret the metabolic state of the organism in
the context of its potential and, in the case of submitted transcriptomics data, real enzymatic capacities. The MassTRIX web
server is freely accessible at http://masstrix.org
Although numerous studies have been performed for the toxicological mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the metabolic changes of TCDD toxicity is less well understood. In this study, liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOFMS) was used for non-targeted metabolomics for understanding the different metabolic patterns associated with TCDD exposure in aryl hydrocarbon receptor (AhR) sensitive C57BL/6J (C6) and less sensitive DBA/2J (D2) mouse strains. The serum samples were analyzed and treated with metabolomic analysis in conjunction with multivariate data analysis. Metabolite identification was performed with interpreting high resolution MS data and MS/MS fragmentation, searching against databases and comparing with authentic compounds. Twelve differentiating metabolites (defined as a ≥1.5-fold change with a P≤0.001) were highlighted in C6 mice versus control group, revealing lipid accumulation, fatty acid beta-oxidation, inflammation and alteration of amino acids as well as phase II drug-like metabolism. In contrast, only 2 differentiating metabolites were detected in D2 mouse model.
Alteration of hormones has long been known to affect development. TCDD and related PHAHs modulate the levels of many hormonal systems. Dioxins cause a spectrum of morphological and functional developmental deficits. Fetotoxicity, thymic atrophy, and structural malformations are often noted. Delayed genitourinary tract effects have been observed, and recent studies reported behavioral effects. Highly exposed human offspring have exhibited developmental problems as well. Recently, hormonal and neurological abnormalities have been reported in infants from the general population. The complex alteration of multiple endocrine systems is likely associated with the spectrum of adverse developmental effects caused by dioxin and related compounds.
An upper elemental compositional boundary for fossil hydrocarbons has previously been established as double-bond equivalents (i.e., DBE = rings plus double bonds) not exceeding 90% of the number of carbons. For heteroatom-containing fossil compounds, the 90% rule still applies if each N atom is counted as a C atom. The 90% rule eliminates more than 10% of the possible elemental compositions at a given mass for fossil database molecules. However, some synthetic compounds can fall outside the upper boundary defined for naturally occurring compounds. Their inclusion defines an "absolute" upper boundary as DBE (rings plus double bonds to carbon) equal to carbon number plus one, and applies to all organic compounds including fullerenes and other molecules containing no hydrogen. Finally, the DBE definition can fail for molecules with particular atomic valences. Therefore, we also present a generalized DBE definition that includes atomic valence to enable calculation of the correct total number of rings, double bonds, and triple bonds for heteroatom-containing compounds.
Metabolomics, the exponentially developing technique, could provide a systemic mapping in toxicology by directly measuring small molecular metabolites. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to be neurotoxic in mammalian animals. In this study, we employed liquid chromatography/quadrupole time-of-flight mass spectrometry for non-targeted analysis of metabolic profiling in hippocampal sample sets of the rats exposed to TCDD. Hippocampal metabolome from different ages of the healthy rats (4-week, 12-week and 20-week) was also deciphered. The relationship between the two tested cases was unlocked to delineate TCDD toxicity associated with ageing. Tandem mass spectrometry fragmentation in conjunction with metabolic database searching and compared to authentic standards was utilized for metabolite identification. As a consequence, the reduced levels of phenylalanine and leucine/isoleucine as well as the up-regulation of inosine and hypoxanthine were highlighted for understanding of TCDD toxicity related to age in rats and the trajectory was depicted by principal components analysis.
In this work, metabonomic methods utilizing (1)H NMR spectroscopy and multivariate statistical technique have been applied to investigate the metabolic profiles of SAM. The serum metabolome of senescence-prone 8 (SAMP8), a murine model of age-related learning and memory deficits and Alzheimer's disease (AD), was compared with that of control, senescence-resistant 1 (SAMR1), which shows normal aging process. Serum samples were collected for study from both male and female 12-month-old SAMP8 and age matched SAMR1 ( n = 5). (1)H NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The results showed that the serum metabolic patterns of SAMP8 and SAMR1 were significantly different due to strains and genders. Subtle differences in the endogenous metabolite profiles in serum between SAMP8 and SAMR1 were observed. The most important metabolite responsible for the strain separation was lack of inosine, which meant the protective function of anti-inflammation, immunomodulation and neuroprotection might be attenuated in SAMP8. Other differential metabolites observed between strains included decreased glucose, PUFA, choline, phosphocholine, HDL, LDL, D-3-hydoxybutyrate, citrate and pyruvate and increased lactate, SFA, alanine, methionine, glutamine and VLDL in serum of SAMP8 compared with those of SAMR1, suggesting perturbed glucose and lipid metabolisms in SAMP8. Besides the differences observed between the strains, an impact of gender on metabolism was also found. The females exhibited larger metabolic deviations than males and these gender differences in SAMP8 were much larger than in SAMR1. Higher levels of VLDL, lactate and amino acids and lower levels of HDL, LDL and unsaturated lipids were detected in female than in male SAMP8. These facts indicated that the metabolism disequilibrium in female and male SAMP8 was different and this may partly explain that females were more prone to AD than males. The results of this work may provide valuable clues to the understanding of the mechanisms of the senile impairment and the pathological changes of AD, as well as show the potential power of the combination of the NMR technique and the pattern recognition method for the analysis of the biochemical changes of certain pathophysiologic conditions.
We studied the significance of alterations in the metabolomics of sulfur-containing substances in rapidly regenerating rat livers. Male rats were subjected to two-thirds partial hepatectomy (PHx), and the changes in hepatic levels of major sulfur-containing amino acids and related substances were monitored for 2 weeks. Liver weight began to increase from 24 h after the surgery, and appeared to recover fully in 2 weeks. Serum alanine aminotransferase and aspartate aminotransferase activities were elevated immediately after the surgery and returned slowly to normal levels in 2 weeks. Methionine, S-adenosylmethionine (SAM), cystathionine and cysteine were increased rapidly and remained elevated for longer than 1 week. Hepatic glutathione concentration was increased gradually for 24 h, and then decreased thereafter, whereas hypotaurine was elevated drastically right after the surgery. Hepatic concentrations of polyamines were altered significantly by PHx. In the hepatectomized livers putrescine concentration was elevated rapidly, reaching a level 40- to 50-fold greater than normal in 6-12 h. Ornithine, the metabolic substrate for putrescine synthesis, was also elevated markedly. Spermidine was increased significantly, whereas spermine was depressed below normal, which appeared to be due to the increased consumption of decarboxylated SAM for spermidine biosynthesis. The results show that the metabolomics of sulfur-containing amino acids and related substances is altered profoundly in regenerating rat livers until the original weight is recovered. Hepatic concentrations of polyamines after PHx are closely associated with the alteration in the metabolomics of sulfur-containing substances. The implication of these changes in the progression of liver regeneration is discussed.
A strong linear relationship was observed between the average double bond equivalence (DBE) and the ratio of carbon to oxygen atoms in oxygenated compounds of dissolved organic matter (DOM). Data were acquired by a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), equipped with a negative-mode electrospray ionization source. The slope and y-intercepts extracted from the linear relationship can be used to compare DOM samples originating from different locations. Significant differences in these parameters were observed between inland riverine and offshore coastal DOM samples. Offshore coastal DOM molecules underwent a change of one DBE for each removal or addition of two oxygen atoms. This suggested the existence of multiple carboxyl groups, each of which contains a double bond and two oxygen atoms. Inland riverine samples exhibited a change of ~1.5 DBE following the addition or removal of two oxygen atoms. This extra change in DBE was attributed to cyclic structures or unsaturated chemical bonds. The DBE value with maximum relative abundance and the minimum DBE value for each class of oxygenated compounds showed that approximately two oxygen atoms contributed to a unity change in DBE. The qualitative analyses given here are in a good agreement with results obtained from analyses using orthogonal analytical techniques. This study demonstrates that DBE and the carbon number distribution, observed by high resolution mass spectrometry, can be valuable in elucidating and comparing structural features of oxygenated molecules of DOM.
Metabolomics, as an essential part of genomics studies, intends holistic understanding of metabolic networks through simultaneous analysis of a myriad of both known and unknown metabolites occurring in living organisms. The initial stage of metabolomics was designed for the reproducible analyses of known metabolites based on their comparison to available authentic compounds. Such metabolomics platforms were mostly based on mass spectrometry (MS) technologies enabled by a combination of different ionization methods together with a variety of separation steps including LC, GC, and CE. Among these, Fourier-transform ion cyclotron resonance MS (FT-ICR/MS) is distinguished from other MS technologies by its ultrahigh resolution power in mass to charge ratio (m/z). The potential of FT-ICR/MS as a distinctive metabolomics tool has been demonstrated in nontargeted metabolic profiling and functional characterization of novel genes. Here, we discuss both the advantages and difficulties encountered in the FT-ICR/MS metabolomics studies.
In this work, the potential of combining capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS) and Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) for metabolomics of genetically modified organisms (GMOs) is demonstrated. Thus, six different varieties of maize, three of them transgenic (PR33P66 Bt, Tietar Bt and Aristis Bt) and their corresponding isogenic lines (PR33P66, Tietar and Aristis) grown under the same field conditions, were analyzed. Based on the ultrahigh resolution and remarkable mass accuracy provided by the 12-T FT-ICR-MS it was possible to directly analyze a good number of metabolites whose identity could be proposed based on their specific isotopic pattern. For identification of metabolite isomers, CE-TOF-MS was also used combining the information on nominal mass with electrophoretic mobility corroborating in that way the identity of several new biomarkers. Furthermore, PLE extractions were evaluated in order to establish selective extraction as an additional criterion to obtain useful information in maize metabolomics. Differences in the metabolite levels were found between the three transgenic maize varieties compared with their wild isogenic lines in some specific metabolic pathways. To our knowledge, this is the first time that an approach as the one presented in this work (pressurized liquid extraction+FT-ICR-MS+CE-TOF-MS) is shown for a metabolomic study.
Alzheimer's disease (AD) can be diagnosed with a considerable degree of accuracy. In some centers, clinical diagnosis predicts the autopsy diagnosis with 90% certainty in series reported from academic centers. The characteristic histopathologic changes at autopsy include neurofibrillary tangles, neuritic plaques, neuronal loss, and amyloid angiopathy. Mutations on chromosomes 21, 14, and 1 cause familial AD. Risk factors for AD include advanced age, lower intelligence, small head size, and history of head trauma; female gender may confer additional risks. Susceptibility genes do not cause the disease by themselves but, in combination with other genes or epigenetic factors, modulate the age of onset and increase the probability of developing AD. Among several putative susceptibility genes (on chromosomes 19, 12, and 6), the role of apolipoprotein E (ApoE) on chromosome 19 has been repeatedly confirmed. Protective factors include ApoE-2 genotype, history of estrogen replacement therapy in postmenopausal women, higher educational level, and history of use of nonsteroidal anti-inflammatory agents. The most proximal brain events associated with the clinical expression of dementia are progressive neuronal dysfunction and loss of neurons in specific regions of the brain. Although the cascade of antecedent events leading to the final common path of neurodegeneration must be determined in greater detail, the accumulation of stable amyloid is increasingly widely accepted as a central pathogenetic event. All mutations known to cause AD increase the production of beta-amyloid peptide. This protein is derived from amyloid precursor protein and, when aggregated in a beta-pleated sheet configuration, is neurotoxic and forms the core of neuritic plaques. Nerve cell loss in selected nuclei leads to neurochemical deficiencies, and the combination of neuronal loss and neurotransmitter deficits leads to the appearance of the dementia syndrome. The destructive aspects include neurochemical deficits that disrupt cell-to-cell communications, abnormal synthesis and accumulation of cytoskeletal proteins (e.g., tau), loss of synapses, pruning of dendrites, damage through oxidative metabolism, and cell death. The concepts of cognitive reserve and symptom thresholds may explain the effects of education, intelligence, and brain size on the occurrence and timing of AD symptoms. Advances in understanding the pathogenetic cascade of events that characterize AD provide a framework for early detection and therapeutic interventions, including transmitter replacement therapies, antioxidants, anti-inflammatory agents, estrogens, nerve growth factor, and drugs that prevent amyloid formation in the brain.
Previous studies have shown that the pathophysiology of Alzheimer's disease (AD) is linked to oxidative stress. Oxidative damage to different biomolecular components of the brain is a characteristic feature of AD. Recent evidence suggests that methionine may act as an antioxidant defense molecule in proteins by its ability to scavenge oxidants and, in the process, undergo oxidation to form methionine sulfoxide. The enzyme peptide, methionine sulfoxide reductase (MsrA), reverses methionine sulfoxide back to methionine, which once again is able to scavenge oxidants. The purpose of this study was to measure the activity of MsrA in the brain of AD patients compared with control subjects. Our results showed that there was a decline in MsrA activity in all brain regions studied in AD and this decline reached statistical significance in the superior and middle temporal gyri (p < 0.001), inferior parietal lobule (p < 0.05), and the hippocampus (p < 0.05) in AD. An elevation of protein carbonyl content was found in all brain regions except the cerebellum in AD and reached statistical significance in the superior and middle temporal gyri and hippocampus. Messenger RNA analysis suggests that the loss in enzyme activity may be the result of a posttranslational modification of MsrA or a defect of translation resulting in inferior processing of the MsrA mRNA. Our results suggest that a decline in MsrA activity could reduce the antioxidant defenses and increase the oxidation of critical proteins in neurons in the brain in AD.
We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.
Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.
Alzheimer's disease (AD) is characterized by a progressive cognitive decline leading to dementia and involves the deposition of amyloid-beta (Abeta) peptides into senile plaques. Other neuropathological features that accompany progression of the disease include a decrease in synaptic density, neurofibrillary tangles, dystrophic neurites, inflammation, and neuronal cell loss. In this study, we report the early kinetics of brain amyloid deposition and its associated inflammation in an early onset transgenic mouse model of AD (TgCRND8) harboring the human amyloid precursor protein gene with the Indiana and Swedish mutations. Both diffuse and compact plaques were detected as early as 9-10 weeks of age. Abeta-immunoreactive (Abeta-IR) plaques (4G8-positive) appeared first in the neocortex and amygdala, then in the hippocampal formation, and lastly in the thalamus. Compact plaques (ThioS-positive) with an amyloid core were observed as early as diffuse plaques were detected, but in lower numbers. Amyloid deposition increased progressively with age. The formation of plaques was concurrent with the appearance of activated microglial cells and shortly followed by the clustering of activated astrocytes around plaques at 13-14 weeks of age. This TgCRND8 mouse model allows for a rapid, time-dependent study of the relationship between the fibrillogenic process and the inflammatory response during the brain amyloidogenic process.
Extracellular guanine-based purines, namely the nucleotides GTP, GDP, GMP and the nucleoside guanosine, exert important neuroprotective and neuromodulator roles in the central nervous system, which may be related to inhibition of the glutamatergic neurotransmission activity. In this study, we investigated GMP effects on mice inhibitory avoidance performance and the dependence on its conversion to guanosine for such effect, by using the ecto-5'-nucleotidase specific inhibitor AOPCP. We also investigated if this conversion occurs in the central nervous system or peripherally, and if guanosine and GMP affect nociception by the tail-flick test. I.p. GMP or guanosine (7.5 mg/kg) or i.c.v. GMP (480 nmol) pretraining administration was amnesic for the inhibitory avoidance task. I.c.v. AOPCP (1 nmol) administration completely reversed the amnesic effect of i.c.v. GMP, but not of i.p. GMP, indicating that peripheral conversion of GMP to guanosine is probably relevant to this effect. AOPCP alone did not interfere with the performance. Furthermore, tail-flick measurement was unaffected by i.p. GMP and guanosine, suggesting that the amnesic effect of both purines was not due to some antinociceptive effect against the footshock used in the task. All these data together, in accordance to those previously observed in studies involving glutamate uptake and seizures reinforce the idea that guanosine is the specific extracellular guanine-based purines effector and indicate that its conversion occurs not only in the central nervous system but also peripherally.
As the scope of the problem of Alzheimer's disease (AD) grows due to an aging population, research into the devastating condition has taken on added urgency. Rare inherited forms of AD provide insight into the molecular pathways leading to degeneration and have made possible the development of transgenic animal models. Several of these models are based on the overexpression of amyloid precursor protein (APP), presenilins, or tau to cause production and accumulation of amyloid-beta into plaques or hyperphosphorylated tau into neurofibrillary tangles. Producing these characteristic neuropathological lesions in animals causes progressive neurodegeneration and in some cases similar behavioral disruptions to those seen in AD patients. Knockout models of proteins involved in AD have also been generated to explore the native functions of these genes and examine whether pathogenesis is due to loss of function or toxic gain of function in these systems. Although none of the transgenic lines models the human condition exactly, the ability to study similar pathological processes in living animals have provided numerous insights into disease mechanisms and opportunities to test therapeutic agents. This chapter reviews animal models of AD and their contributions to developing therapeutic approaches for AD.
A large metabolomics study was performed on 600 plasma samples taken at four time points before and after a single intake of a high fat test meal by obese and lean subjects. All samples were analyzed by a liquid chromatography-mass spectrometry (LC-MS) lipidomic method for metabolic profiling. A pragmatic approach combining several well-established statistical methods was developed for processing this large data set in order to detect small differences in metabolic profiles in combination with a large biological variation. Such metabolomics studies require a careful analytical and statistical protocol. The strategy included data preprocessing, data analysis, and validation of statistical models. After several data preprocessing steps, partial least-squares discriminant analysis (PLS-DA) was used for finding biomarkers. To validate the found biomarkers statistically, the PLS-DA models were validated by means of a permutation test, biomarker models, and noninformative models. Univariate plots of potential biomarkers were used to obtain insight in up- or downregulation. The strategy proposed proved to be applicable for dealing with large-scale human metabolomics studies.
Genes responsible for the generation of 3-dehydroquinate (DHQ), an early metabolite in the established shikimic pathway of aromatic amino acid biosynthesis, are absent in most euryarchaeotes. Alternative gene products, Mj0400 and Mj1249, have been identified in Methanocaldococcus jannaschii as the enzymes involved in the synthesis of DHQ. 2-Amino-3,7-dideoxy-d-threo-hept-6-ulosonic acid (ADH) synthase, the product of the Mj0400 gene, catalyzes a transaldol reaction between 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde to yield ADH. Dehydroquinate synthase II, the product of the Mj1249 gene, then catalyzes deamination and cyclization of ADH, resulting in DHQ, which is fed into the canonical pathway. Three crystal structures of ADH synthase were determined in this work: a complex with a substrate analogue, fructose 1,6-bisphosphate, a complex with dihydroxyacetone phosphate (DHAP), thought to be a product of fructose 1-phosphate cleavage, and a native structure containing copurified ligands, modeled as DHAP and glycerol. On the basis of the structural analysis and comparison of the enzyme with related aldolases, ADH synthase is classified as a new member of the class I aldolase superfamily. The description of the active site allows for the identification and characterization of possible catalytic residues, Lys184, which is responsible for formation of the Schiff base intermediate, and Asp33 and Tyr153, which are candidates for the general acid/base catalysis.
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