Cerebral deficiency of vitamin B5 (d-pantothenic acid; pantothenate) as a potentially-reversible cause of neurodegeneration and dementia in sporadic Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of age-related neurodegeneration and dementia, and there are no available treatments with proven disease-modifying actions. It is therefore appropriate to study hitherto-unknown aspects of brain structure/function in AD to seek alternative disease-related mechanisms that might be targeted by new therapeutic interventions with disease-modifying actions. During hypothesis-generating metabolomic studies of brain, we identified apparent differences in levels of vitamin B5 between AD cases and controls. We therefore developed a method based on gas chromatography-mass spectrometry by which we quantitated vitamin B5 concentrations in seven brain regions from nine AD cases and nine controls. We found that widespread, severe cerebral deficiency of vitamin B5 occurs in AD. This deficiency was worse in those regions known to undergo severe damage, including the hippocampus, entorhinal cortex, and middle temporal gyrus. Vitamin B5 is the obligate precursor of CoA/acetyl-CoA (acetyl-coenzyme A), which plays myriad key roles in the metabolism of all organs, including the brain. In brain, acetyl-CoA is the obligate precursor of the neurotransmitter acetylcholine, and the complex fatty-acyl groups that mediate the essential insulator role of myelin, both processes being defective in AD; moreover, the large cerebral vitamin B5 concentrations co-localize almost entirely to white matter. Vitamin B5 is well tolerated when administered orally to humans and other mammals. We conclude that cerebral vitamin B5 deficiency may well cause neurodegeneration and dementia in AD, which might be preventable or even reversible in its early stages, by treatment with suitable oral doses of vitamin B5.

Plasma metals as potential biomarkers in dementia: a case-control study in patients with sporadic Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent form of age-related dementia but its pathogenesis remains obscure. Altered regulation of metals, particularly pan-cerebral copper deficiency, and more regionally-localized perturbation of other metals, are prominent in AD brain although data on how these CNS perturbations are reflected in the peripheral bloodstream are inconsistent to date. To assess the potential use of metal dysregulation to generate biomarkers in AD, we performed a case-control study of seven essential metals and selenium, measured by inductively coupled plasma mass-spectrometry, in samples from AD and matched control cases. Metals were sodium, potassium, calcium, magnesium, iron, zinc, and copper. In the whole study-group and in female participants, plasma metal levels did not differ between cases and controls. In males by contrast, there was moderate evidence that zinc levels trended towards increase in AD [10.8 (10.2-11.5)] µmol/L, mean (± 95% CI; P = 0.021) compared with controls [10.2 (9.6-10.4)]. Thus alterations in plasma zinc levels differed between genders in AD. In correlational analysis, there was evidence for an increased number of 'strong' metal co-regulations in AD cases and differential co-modulations of metal pairs: copper-sodium (Rcontrol = - 0.03, RAD = 0.65; P = 0.009), and copper-calcium (Rcontrol = - 0.01, RAD = 0.65; P = 0.01) were significant in AD males, potentially consistent with reported evidence for dysregulation of copper in severely damaged brain regions in AD. In conclusion, our data suggest that the measurement of metals co-regulation in plasma may provide a useful representation of those metal perturbations taking place in the AD brain and therefore might be useful as plasma-based biomarkers.

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain.

Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.

Graded perturbations of metabolism in multiple regions of human brain in Alzheimer's disease: Snapshot of a pervasive metabolic disorder.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that displays pathological characteristics including senile plaques and neurofibrillary tangles. Metabolic defects are also present in AD-brain: for example, signs of deficient cerebral glucose uptake may occur decades before onset of cognitive dysfunction and tissue damage. There have been few systematic studies of the metabolite content of AD human brain, possibly due to scarcity of high-quality brain tissue and/or lack of reliable experimental methodologies. Here we sought to: 1) elucidate the molecular basis of metabolic defects in human AD-brain; and 2) identify endogenous metabolites that might guide new approaches for therapeutic intervention, diagnosis or monitoring of AD. Brains were obtained from nine cases with confirmed clinical/neuropathological AD and nine controls matched for age, sex and post-mortem delay. Metabolite levels were measured in post-mortem tissue from seven regions: three that undergo severe neuronal damage (hippocampus, entorhinal cortex and middle-temporal gyrus); three less severely affected (cingulate gyrus, sensory cortex and motor cortex); and one (cerebellum) that is relatively spared. We report a total of 55 metabolites that were altered in at least one AD-brain region, with different regions showing alterations in between 16 and 33 metabolites. Overall, we detected prominent global alterations in metabolites from several pathways involved in glucose clearance/utilization, the urea cycle, and amino-acid metabolism. The finding that potentially toxigenic molecular perturbations are widespread throughout all brain regions including the cerebellum is consistent with a global brain disease process rather than a localized effect of AD on regional brain metabolism.

Deficient copper concentrations in dried-defatted hepatic tissue from ob/ob mice: A potential model for study of defective copper regulation in metabolic liver disease.

Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.

Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

Development of a robust and repeatable UPLC-MS method for the long-term metabolomic study of human serum.

A method for performing untargeted metabolomic analysis of human serum has been developed based on protein precipitation followed by Ultra Performance Liquid Chromatography and Time-of-Flight mass spectrometry (UPLC-TOF-MS). This method was specifically designed to fulfill the requirements of a long-term metabolomic study, spanning more than 3 years, and it was subsequently thoroughly evaluated for robustness and repeatability. We describe here the observed drift in instrumental performance over time and its improvement with adjustment of the length of analytical block. The optimal setup for our purpose was further validated against a set of serum samples from 30 healthy individuals. We also assessed the reproducibility of chromatographic columns with the same chemistry of stationary phase from the same manufacturer but from different production batches. The results have allowed the authors to prepare SOPs for "fit for purpose" long-term UPLC-MS metabolomic studies, such as are being employed in the HUSERMET project. This method allows the acquisition of data and subsequent comparison of data collected across many months or years.

Development and performance of a gas chromatography-time-of-flight mass spectrometry analysis for large-scale nontargeted metabolomic studies of human serum.

A method for the preparation and GC-TOF-MS analysis of human serum samples has been developed and evaluated for application in long-term metabolomic studies. Serum samples were deproteinized using 3:1 methanol/serum, dried in a vacuum concentrator, and chemically derivatized in a two-stage process. Samples were analyzed by GC-TOF-MS with a 25 min analysis time. In addition, quality control (QC) samples were used to quantify process variability. Optimization of chemical derivatization was performed. Products were found to be stable for 30 h after derivatization. An assessment of within-day repeatability and within-week reproducibility demonstrates that excellent performance is observed with our developed method. Analyses were consistent over a 5 month period. Additional method testing, using spiked serum samples, showed the ability to define metabolite differences between samples from a population and samples spiked with metabolites standards. This methodology allows the continuous acquisition and application of data acquired over many months in long-term metabolomic studies, including the HUSERMET project (http://www.husermet.org/).

Cerebral Vitamin B5 (D-Pantothenic Acid) Deficiency as a Potential Cause of Metabolic Perturbation and Neurodegeneration in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the HTT gene. HD usually manifests in mid-life with loss of GABAergic projection neurons from the striatum accompanied by progressive atrophy of the putamen followed by other brain regions, but linkages between the genetics and neurodegeneration are not understood. We measured metabolic perturbations in HD-human brain in a case-control study, identifying pervasive lowering of vitamin B5, the obligatory precursor of coenzyme A (CoA) that is essential for normal intermediary metabolism. Cerebral pantothenate deficiency is a newly-identified metabolic defect in human HD that could potentially: (i) impair neuronal CoA biosynthesis; (ii) stimulate polyol-pathway activity; (iii) impair glycolysis and tricarboxylic acid cycle activity; and (iv) modify brain-urea metabolism. Pantothenate deficiency could lead to neurodegeneration/dementia in HD that might be preventable by treatment with vitamin B5.

Cognitive dysfunction in diabetic rats is prevented by pyridoxamine treatment. A multidisciplinary investigation.

OBJECTIVE: The impact of diabetes mellitus on the central nervous system is less widely studied than in the peripheral nervous system, but there is increasing evidence that it elevates the risk of developing cognitive deficits. The aim of this study was to characterize the impact of experimental diabetes on the proteome and metabolome of the hippocampus. We tested the hypothesis that the vitamin B6 isoform pyridoxamine is protective against functional and molecular changes in diabetes. METHODS: We tested recognition memory using the novel object recognition (NOR) test in streptozotocin (STZ)-induced diabetic, age-matched control, and pyridoxamine- or insulin-treated diabetic male Wistar rats. Comprehensive untargeted metabolomic and proteomic analyses, using gas chromatography-mass spectrometry and iTRAQ-enabled protein quantitation respectively, were utilized to characterize the molecular changes in the hippocampus in diabetes. RESULTS: We demonstrated diabetes-specific, long-term (but not short-term) recognition memory impairment and that this deficit was prevented by insulin or pyridoxamine treatment. Metabolomic analysis showed diabetes-associated changes in 13/82 identified metabolites including polyol pathway intermediates glucose (9.2-fold), fructose (4.9-fold) and sorbitol (5.2-fold). We identified and quantified 4807 hippocampal proteins; 806 were significantly altered in diabetes. Pathway analysis revealed significant alterations in cytoskeletal components associated with synaptic plasticity, glutamatergic signaling, oxidative stress, DNA damage and FXR/RXR activation pathways in the diabetic rat hippocampus. CONCLUSIONS: Our data indicate a protective effect of pyridoxamine against diabetes-induced cognitive deficits, and our comprehensive 'omics datasets provide insight into the pathogenesis of cognitive dysfunction enabling development of further mechanistic and therapeutic studies.

Evidence for widespread, severe brain copper deficiency in Alzheimer's dementia.

Datasets comprising simultaneous measurements of many essential metals in Alzheimer's disease (AD) brain are sparse, and available studies are not entirely in agreement. To further elucidate this matter, we employed inductively-coupled-plasma mass spectrometry to measure post-mortem levels of 8 essential metals and selenium, in 7 brain regions from 9 cases with AD (neuropathological severity Braak IV-VI), and 13 controls who had normal ante-mortem mental function and no evidence of brain disease. Of the regions studied, three undergo severe neuronal damage in AD (hippocampus, entorhinal cortex and middle-temporal gyrus); three are less-severely affected (sensory cortex, motor cortex and cingulate gyrus); and one (cerebellum) is relatively spared. Metal concentrations in the controls differed among brain regions, and AD-associated perturbations in most metals occurred in only a few: regions more severely affected by neurodegeneration generally showed alterations in more metals, and cerebellum displayed a distinctive pattern. By contrast, copper levels were substantively decreased in all AD-brain regions, to 52.8-70.2% of corresponding control values, consistent with pan-cerebral copper deficiency. This copper deficiency could be pathogenic in AD, since levels are lowered to values approximating those in Menkes' disease, an X-linked recessive disorder where brain-copper deficiency is the accepted cause of severe brain damage. Our study reinforces others reporting deficient brain copper in AD, and indicates that interventions aimed at safely and effectively elevating brain copper could provide a new experimental-therapeutic approach.

The metabolomics of necrotising enterocolitis in preterm babies: an exploratory study.

OBJECTIVE: No single diagnostic investigation is currently available for necrotising enterocolitis (NEC). We implemented a novel, untargeted, exploratory study to determine whether metabolomics can reveal early biomarker(s) of NEC. The effect of gestational age on the metabolome was also investigated. METHODS: Two serum samples were obtained from 12 preterm babies (born <30 weeks gestation) and eight term controls: sample "A" at ≤1 week of age and sample "B" once fully fed. Samples were subjected to gas chromatography-mass spectrometry. Metabolomic data was analysed by principal component analysis (PCA), univariate and network analysis. RESULTS: Sixteen metabolite features significantly differed when B samples were compared between preterm babies who subsequently developed NEC and preterm/term controls (p value <0.05). Of these seven metabolites were linked to up-regulation of IL-1ß. Significant differences in 54 metabolite features (p value <0.05) were observed between preterm and term metabolomes. Of these, 12 metabolite features were linked to one network involved in carbohydrate/lipid metabolism (p = 1 × 10(-30)). CONCLUSIONS: Metabolomic differences were observed in preterm babies at risk of NEC. However, sample sizes were insufficient to confidently identify a biomarker. Network modelling of preterm and term metabolomes suggest possible nutritional deficiency and altered pro-insulin action in preterm babies.

Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental Diabetic Neuropathy.

High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However, our understanding of the molecular mechanisms that cause the marked distal pathology is incomplete. We performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN. We integrated proteomics and metabolomics from the sciatic nerve (SN), the lumbar 4/5 dorsal root ganglia (DRG), and the trigeminal ganglia (TG) of streptozotocin-diabetic and healthy control rats. Even though all tissues showed a dramatic increase in glucose and polyol pathway intermediates in diabetes, a striking upregulation of mitochondrial oxidative phosphorylation and perturbation of lipid metabolism was found in the distal SN that was not present in the corresponding cell bodies of the DRG or the cranial TG. This finding suggests that the most severe molecular consequences of diabetes in the nervous system present in the SN, the region most affected by neuropathy. Such spatial metabolic dysfunction suggests a failure of energy homeostasis and/or oxidative stress, specifically in the distal axon/Schwann cell-rich SN. These data provide a detailed molecular description of the distinct compartmental effects of diabetes on the PNS that could underlie the distal-proximal distribution of pathology.

Elevation of brain glucose and polyol-pathway intermediates with accompanying brain-copper deficiency in patients with Alzheimer's disease: metabolic basis for dementia.

Impairment of brain-glucose uptake and brain-copper regulation occurs in Alzheimer's disease (AD). Here we sought to further elucidate the processes that cause neurodegeneration in AD by measuring levels of metabolites and metals in brain regions that undergo different degrees of damage. We employed mass spectrometry (MS) to measure metabolites and metals in seven post-mortem brain regions of nine AD patients and nine controls, and plasma-glucose and plasma-copper levels in an ante-mortem case-control study. Glucose, sorbitol and fructose were markedly elevated in all AD brain regions, whereas copper was correspondingly deficient throughout (all P < 0.0001). In the ante-mortem case-control study, by contrast, plasma-glucose and plasma-copper levels did not differ between patients and controls. There were pervasive defects in regulation of glucose and copper in AD brain but no evidence for corresponding systemic abnormalities in plasma. Elevation of brain glucose and deficient brain copper potentially contribute to the pathogenesis of neurodegeneration in AD.

Antiphospholipid Antibodies Alter Cell-Death-Regulating Lipid Metabolites in First and Third Trimester Human Placentae.

PROBLEM: Antiphospholipid antibodies (aPL) are maternal autoantibodies that increase the risk of a woman developing preeclampsia 10-fold. aPL are internalized into the syncytiotrophoblast and increase extrusion of necrotic trophoblast debris into the maternal blood. This necrotic trophoblast debris may trigger endothelial cell dysfunction contributing to the pathogenesis of preeclampsia. We hypothesize that aPL directly affect placental metabolism, leading to increased syncytiotrophoblast death. METHODS OF STUDY: First and third trimester human placental explants were cultured with aPL, a control antibody, or media only, and placental conditioned culture media was examined by mass spectroscopy. Molecular targets of interest were investigated using qRTPCR and immunohistochemistry. RESULTS: The levels of 79 and 132 metabolites, respectively, were altered due to the treatment of first and third trimester placental explants with aPL. These included ceramides and diacylglycerols, which play important roles in cell death regulatory pathways. Antiphospholipid antibodies also decreased the expression of protein kinase C-epsilon (PRKCE) in placental explants, possibly due to the disrupted balance between ceramides and diacylglycerols caused by aPL. CONCLUSION: One mechanism by which aPL cause aberrant cell death in the syncytiotrophoblast in the first and third trimester is by disruption of placental lipid signaling and decreased expression of PRKCE.

Molecular phenotyping of a UK population: defining the human serum metabolome.

Phenotyping of 1,200 'healthy' adults from the UK has been performed through the investigation of diverse classes of hydrophilic and lipophilic metabolites present in serum by applying a series of chromatography-mass spectrometry platforms. These data were made robust to instrumental drift by numerical correction; this was prerequisite to allow detection of subtle metabolic differences. The variation in observed metabolite relative concentrations between the 1,200 subjects ranged from less than 5 % to more than 200 %. Variations in metabolites could be related to differences in gender, age, BMI, blood pressure, and smoking. Investigations suggest that a sample size of 600 subjects is both necessary and sufficient for robust analysis of these data. Overall, this is a large scale and non-targeted chromatographic MS-based metabolomics study, using samples from over 1,000 individuals, to provide a comprehensive measurement of their serum metabolomes. This work provides an important baseline or reference dataset for understanding the 'normal' relative concentrations and variation in the human serum metabolome. These may be related to our increasing knowledge of the human metabolic network map. Information on the Husermet study is available at http://www.husermet.org/. Importantly, all of the data are made freely available at MetaboLights (http://www.ebi.ac.uk/metabolights/).

Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.

Atmospheric trends and radiative forcings of CF4 and C2F6 inferred from firn air.

The atmospheric histories of two potent greenhouse gases, tetrafluoromethane (CF4) and hexafluoroethane (C2F6), have been reconstructed for the 20th century based on firn air measurements from both hemispheres. The reconstructed atmospheric trends show that the mixing ratios of both CF4 and C2F6 have increased during the 20th century by factors of approximately 2 and approximately 10, respectively. Initially, the increasing mixing ratios coincided with the rise in primary aluminum production. However, a slower atmospheric growth rate for CF4 appears to be evident during the 1990s, which supports recent aluminum industry reports of reduced CF4 emissions. This work illustrates the changing relationship between CF4 and C2F6 that is likely to be largely the result of both reduced emissions from the aluminum industry and faster growing emissions of C2F6 from the semiconductor industry. Measurements of C2F6 in the older firn air indicate a natural background mixing ratio of <0.3 parts per trillion (ppt), demonstrating that natural sources of this gas are negligible. However, CF4 was deduced to have a preindustrial mixing ratio of 34 -1 ppt (-50% of contemporary levels). This is in good agreement with the previous work of Harnisch et al. (18) and provides independent confirmation of their results. As a result of the large global warming potentials of CF4 and C2F6, these results have important implications for radiative forcing calculations. The radiative forcings of CF4 and C2F6 are shown to have increased over the past 50 years to values in 2001 of 4.1 x 10(-3) Wm(-2) and 7.5 x 10(-4) Wm(-2), respectively, relative to preindustrial concentrations. These forcings are small compared to present day forcings due to the major greenhouse gases but, if the current trends continue, they will continue to increase since both gases have essentially infinite lifetimes. There is, therefore, a large incentive to reduce perfluorocarbon emissions such that through the implementation of the Kyoto Protocol, the atmospheric growth rates may decline in the future.