Publications
116 results found
Sigurdsson HP, Hunter H, Alcock L, et al., 2023, Safety and tolerability of adjunct non-invasive vagus nerve stimulation in people with parkinson's: a study protocol, BMC Neurology, Vol: 23, Pages: 1-11, ISSN: 1471-2377
BACKGROUND: Parkinson's disease (PD) is the fastest growing neurological condition worldwide. Recent theories suggest that symptoms of PD may arise due to spread of Lewy-body pathology where the process begins in the gut and propagate transynaptically via the vagus nerve to the central nervous system. In PD, gait impairments are common motor manifestations that are progressive and can appear early in the disease course. As therapies to mitigate gait impairments are limited, novel interventions targeting these and their consequences, i.e., reducing the risk of falls, are urgently needed. Non-invasive vagus nerve stimulation (nVNS) is a neuromodulation technique targeting the vagus nerve. We recently showed in a small pilot trial that a single dose of nVNS improved (decreased) discrete gait variability characteristics in those receiving active stimulation relative to those receiving sham stimulation. Further multi-dose, multi-session studies are needed to assess the safety and tolerability of the stimulation and if improvement in gait is sustained over time. DESIGN: This will be an investigator-initiated, single-site, proof-of-concept, double-blind sham-controlled randomised pilot trial in 40 people with PD. Participants will be randomly assigned on a 1:1 ratio to receive either active or sham transcutaneous cervical VNS. All participants will undergo comprehensive cognitive, autonomic and gait assessments during three sessions over 24 weeks, in addition to remote monitoring of ambulatory activity and falls, and exploratory analyses of cholinergic peripheral plasma markers. The primary outcome measure is the safety and tolerability of multi-dose nVNS in PD. Secondary outcomes include improvements in gait, cognition and autonomic function that will be summarised using descriptive statistics. DISCUSSION: This study will report on the proportion of eligible and enrolled patients, rates of eligibility and reasons for ineligibility. Adverse events will be recorde
Nagy I, Friston D, Cuddihy J, et al., 2023, Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury, Pain, Vol: 164, Pages: e103-e115, ISSN: 0304-3959
Tissue injuries, including burns, are major causes of death and morbidity worldwide. These injuries result in the release of intracellular molecules and subsequent inflammatory reactions, changing the tissues’ chemical milieu and leading to the development of persistent pain through activating pain-sensing primary sensory neurons. However, the majority of pain-inducing agents in injured tissues are unknown. Here, we report that, amongst other important metabolite changes, lysophosphatidylcholines (LPCs) including 18:0 LPC exhibit significant and consistent local burn injury-induced changes in concentration. 18:0 LPC induces immediate pain and the development of hypersensitivities to mechanical and heat stimuli through molecules including the transient receptor potential ion channel, vanilloid sub-family, member 1 and member 2 at least partly via increasing lateral pressure in the membrane. As levels of LPCs including 18:0 LPC increase in other tissue injuries, our data reveal a novel role for these lipids in injury-associated pain. These findings have high potential to improve patient care.
Pires ML, Ferreira P, Shala F, et al., 2022, Using Renal-Specific Cyclo-Oxygenase-2-Deficient Mice to Understand the Pro-Thrombotic Effects of Non-Steroidal Anti-Inflammatory Drugs, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Symington J, Perryman R, Morse S, et al., 2022, ADI-PEG20 RESTORES IMMUNITY IN THE TUMOR MICROENVIRONMENT AND ERADICATES GBM TUMORS IN MICE WHEN COMBINED WITH RADIATION, 27th Annual Scientific Meeting and Education Day of the Society-for-Neuro-Oncology (SNO), Publisher: OXFORD UNIV PRESS INC, Pages: 278-278, ISSN: 1522-8517
Sagi-Kiss V, Li Y, Carey MR, et al., 2022, Ion-pairing chromatography and amine derivatization provide complementary approaches for the targeted LC-MS analysis of the polar metabolome., Journal of Proteome Research, Vol: 21, Pages: 1428-1437, ISSN: 1535-3893
Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.
Nijhuis A, Sikka A, Yogev O, et al., 2022, Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma, Nature Communications, Vol: 13, ISSN: 2041-1723
Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.
Graca G, Cai Y, Lau C-H, et al., 2022, Automated annotation of untargeted all-ion fragmentation LC-MS metabolomics data with MetaboAnnotatoR, Analytical Chemistry, Vol: 94, Pages: 3446-3455, ISSN: 0003-2700
Untargeted metabolomics and lipidomics LC-MS experiments produce complex datasets, usually containing tens of thousands of features from thousands of metabolites whose annotation requires additional MS/MS experiments and expert knowledge. All-ion fragmentation (AIF) LC-MS/MS acquisition provides fragmentation data at no additional experimental time cost. However, analysis of such datasets requires reconstruction of parent fragment relationships and annotation of the resulting pseudo-MS/MS spectra. Here we propose a novel approach for automated annotation of isotopologues, adducts and in-source fragments from AIF LC-MS datasets by combining correlation-based parent-fragment linking with molecular fragment matching. Our workflow focuses on a subset of features rather than trying to annotate the full dataset, saving time and simplifying the process. We demonstrate the workflow in three human serum datasets containing 599 features manually annotated by experts. Precision and recall values of 82- 92% and 82-85% respectively, were obtained for features found in the highest-rank scores (1-5). These results equal or outperform those obtained using MS-DIAL software, the current state-of-the-art for AIF data annotation. Further validation for other biological matrices and different instrument types showed variable precision (60-89%) and recall (10-88%) particularly for datasets dominated by non-lipid metabolites. The workflow is freely available as an open-source R package, MetaboAnnotatoR, together with the fragment libraries from Github (https://github.com/gggraca/MetaboAnnotatoR).
Jukes Z, Freier A, Glymenaki M, et al., 2021, Lipid profiling of mouse intestinal organoids for studying APC mutations, Bioscience Reports: molecular and cellular biology of the cell surface, Vol: 41, Pages: 1-11, ISSN: 0144-8463
Inactivating mutations including both germline and somatic mutations in the adenomatous polyposis coli (APC) gene drives most familial and sporadic colorectal cancers. Understanding the metabolic implications of this mutation will aid to establish its wider impact on cellular behaviour and potentially inform clinical decisions. However, to date, alterations in lipid metabolism induced by APC mutations remain unclear. Intestinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their 3D structure more accurately mimics an in vivo environment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liquid chromatography mass spectrometry (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from either wild-type (WT) or mice with APC mutations (Lgr5–EGFP-IRES-CreERT2Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Levels of phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and hexosylceramides (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), SM(d18:1/16:0)) were lower compared with WT. These observations indicate that cellular metabolism of sphingomyelin was up-regulated, resulting in the cellular accumulation of ceramides and production of HexCer due to the absence of Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of organoids and provided an enhanced insight into the effects of the APC mutations on lipid metabolism, making for a valuable addition to screening options of the organoid lipidome.
Pilla R, Law TH, Pan Y, et al., 2020, The effects of a ketogenic medium-chain triglyceride diet on the feces in dogs with idiopathic epilepsy, Frontiers in Veterinary Science, Vol: 7, Pages: 1-12, ISSN: 2297-1769
Consumption of diets containing medium chain triglycerides have been shown to confer neuroprotective and behavior modulating effects. We aimed to identify metabolic and microbiome perturbations in feces that are associated with consumption of a medium chain triglyceride ketogenic diet (MCT-KD) in dogs with idiopathic epilepsy. We used 16S rRNA gene sequencing to generate microbiome profiles and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to generate lipidomic profiles of canine feces. We made comparisons between the MCT-KD, standardized placebo diet and baseline pre-trial diet phases. Consumption of the MCT-KD resulted in a significant increase in the species richness (α-diversity) of bacterial communities found in the feces when compared to the baseline diet. However, phylogenetical diversity between samples (beta-diversity) was not affected by diet. An unnamed Bacteroidaceae species within genus 5-7N15 was identified by LEfSe as a potential biomarker associated with consumption of the MCT-KD, showing an increased abundance (p = 0.005, q = 0.230) during consumption of MCT-KD. In addition, unclassified members of families Erysipelotrichaceae (p = 0.013, q = 0.335) and Fusobacteriaceae (p = 0.022, q = 0.358) were significantly increased during MCT-KD consumption compared to baseline. Blautia sp. and Megamonas sp. instead were decreased during consumption of either placebo or MCT-KD (p = 0.045, q = 0.449, and p = 0.039, q = 0.449, respectively). Bacteroidaceae, including genus 5-7N15, have previously been associated with non-aggressive behavior in dogs. In addition, 5-7N15 is correlated in humans with Akkermansia, a genus known to be involved in the neuroprotective effect of ketogenic diets in mice models of seizures. Five metabolite features, tentatively identified as long chain triglycerides, were significantly higher after consumption of the placebo diet, but no unique features were identified after consumption of the MCT-KD. The data
Friston D, Junttila S, Borges Paes Lemes J, et al., 2020, Leptin and fractalkine: novel subcutaneous cytokines in burn injury, Disease Models and Mechanisms, Vol: 13, ISSN: 1754-8403
Burn injury is a pathology underpinned by progressive and aberrant inflammation. It is a major clinical challenge to survival and quality of life. While burn injury’s complex local and disseminating pathological processes ultimately stem from local tissue damage, to date relatively few studies have attempted to characterise the local inflammatory mediator profile. Here, cytokine content and associated transcriptional changes were measured in rat skin for three hours immediately following induction of a scald-type (60oC, 2 minutes) burn injury model. Leptin (p = 0.0002) and fractalkine (p = 0.0478) concentrations were significantly elevated post-burn above pre-burn and control site values, coinciding with the development of burn site oedema and differential expression of leptin mRNA (p = 0.0004). Further, gene sequencing enrichment analysis indicated cytokine-cytokine receptor interaction (p = 1.45x10-6). Subsequent behavioural studies demonstrated that, following subcutaneous injection into the dorsum of the paw, both leptin and fractalkine induced mechanical allodynia, heat hyperalgesia and the recruitment of macrophages. This is the first report of leptin’s elevation specifically at the burn site and the first report of fractalkine’s elevation in any tissue post-burn which, together with the functional findings, calls for exploration of the influence of these cytokines on pain, inflammation and burn wound progression. Additionally targeting these signalling molecules represents a therapeutic potential as early formative mediators of these pathological processes.
Friston D, Junttila S, Lemes JBP, et al., 2020, Leptin and fractalkine: Novel subcutaneous cytokines in burn injury., Dis Model Mech
Burn injury is a pathology underpinned by progressive and aberrant inflammation. It is a major clinical challenge to survival and quality of life. While burn injury's complex local and disseminating pathological processes ultimately stem from local tissue damage, to date relatively few studies have attempted to characterise the local inflammatory mediator profile. Here, cytokine content and associated transcriptional changes were measured in rat skin for three hours immediately following induction of a scald-type (60°C, 2 minutes) burn injury model. Leptin (p=0.0002) and fractalkine (p=0.0478) concentrations were significantly elevated post-burn above pre-burn and control site values, coinciding with the development of burn site oedema and differential expression of leptin mRNA (p=0.0004). Further, gene sequencing enrichment analysis indicated cytokine-cytokine receptor interaction (p=1.45x10-6). Subsequent behavioural studies demonstrated that, following subcutaneous injection into the dorsum of the paw, both leptin and fractalkine induced mechanical allodynia, heat hyperalgesia and the recruitment of macrophages. This is the first report of leptin's elevation specifically at the burn site and the first report of fractalkine's elevation in any tissue post-burn which, together with the functional findings, calls for exploration of the influence of these cytokines on pain, inflammation and burn wound progression. Additionally targeting these signalling molecules represents a therapeutic potential as early formative mediators of these pathological processes.
Ivanisevic J, Want EJ, 2019, From samples to insights into metabolism: uncovering biologically relevant information in LC-HRMS metabolomics data, Metabolites, Vol: 9, Pages: 1-30, ISSN: 2218-1989
Untargeted metabolomics (including lipidomics) is a holistic approach to biomarker discovery and mechanistic insights into disease onset and progression, and response to intervention. Each step of the analytical and statistical pipeline is crucial for the generation of high-quality, robust data. Metabolite identification remains the bottleneck in these studies; therefore, confidence in the data produced is paramount in order to maximize the biological output. Here, we outline the key steps of the metabolomics workflow and provide details on important parameters and considerations. Studies should be designed carefully to ensure appropriate statistical power and adequate controls. Subsequent sample handling and preparation should avoid the introduction of bias, which can significantly affect downstream data interpretation. It is not possible to cover the entire metabolome with a single platform; therefore, the analytical platform should reflect the biological sample under investigation and the question(s) under consideration. The large, complex datasets produced need to be pre-processed in order to extract meaningful information. Finally, the most time-consuming steps are metabolite identification, as well as metabolic pathway and network analysis. Here we discuss some widely used tools and the pitfalls of each step of the workflow, with the ultimate aim of guiding the reader towards the most efficient pipeline for their metabolomics studies.
Friston D, Laycock H, Nagy I, et al., 2019, Microdialysis workflow for metabotyping superficial pathologies: application to burn injury, Analytical Chemistry, Vol: 91, Pages: 6541-6548, ISSN: 0003-2700
Burn injury can be a devastating traumatic injury, with long-term personal and social implications for the patient. The many complex local and disseminating pathological processes underlying burn injury's clinical challenges are orchestrated from the site of injury and develop over time, yet few studies of the molecular basis of these mechanisms specifically explore the local signaling environment. Those that do are typically destructive in nature and preclude the collection of longitudinal temporal data. Burn injury therefore exemplifies a superficial temporally dynamic pathology for which experimental sampling typically prioritizes either specificity to the local burn site or continuous collection from circulation. Here, we present an exploratory approach to the targeted elucidation of complex, local, acutely temporally dynamic interstitia through its application to burn injury. Subcutaneous microdialysis is coupled with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis, permitting the application of high-throughput metabolomic profiling to samples collected both continuously and specifically from the burn site. We demonstrate this workflow's high yield of burn-altered metabolites including the complete structural elucidation of niacinamide and uric acid, two compounds potentially involved in the pathology of burn injury. Further understanding the metabolic changes induced by burn injury will help to guide therapeutic intervention in the future. This approach is equally applicable to the analysis of other tissues and pathological conditions, so it may further improve our understanding of the metabolic changes underlying a wide variety of pathological processes.
Przystal JM, Hajji N, Khozoie C, et al., 2018, Efficacy of arginine depletion by ADI-PEG20 in an intracranial model of GBM, Cell Death and Disease, Vol: 9, ISSN: 2041-4889
Glioblastoma multiforme (GBM) remains a cancer with a poor prognosis and few effective therapeutic options. Successful medical management of GBM is limited by the restricted access of drugs to the central nervous system (CNS) caused by the blood brain barrier (BBB). We previously showed that a subset of GBM are arginine auxotrophic because of transcriptional silencing of ASS1 and/or ASL and are sensitive to pegylated arginine deiminase (ADI-PEG20). However, it is unknown whether depletion of arginine in peripheral blood in vivo has therapeutic activity against intracranial disease. In the present work, we describe the efficacy of ADI-PEG20 in an intracranial model of human GBM in which tumour growth and regression are assessed in real time by measurement of luciferase activity. Animals bearing intracranial human GBM tumours of varying ASS status were treated with ADI-PEG20 alone or in combination with temozolomide and monitored for tumour growth and regression. Monotherapy ADI-PEG20 significantly reduces the intracranial growth of ASS1 negative GBM and extends survival of mice carrying ASS1 negative GBM without obvious toxicity. The combination of ADI-PEG20 with temozolomide (TMZ) demonstrates enhanced effects in both ASS1 negative and ASS1 positive backgrounds.Our data provide proof of principle for a therapeutic strategy for GBM using peripheral blood arginine depletion that does not require BBB passage of drug and is well tolerated. The ability of ADI-PEG20 to cytoreduce GBM and enhance the effects of temozolomide argues strongly for its early clinical evaluation in the treatment of GBM.
Lau CH, Siskos AP, Maitre L, et al., 2018, Determinants of the urinary and serum metabolome in children from six European populations, BMC Medicine, Vol: 16, ISSN: 1741-7015
BackgroundEnvironment and diet in early life can affect development and health throughout the life course. Metabolic phenotyping of urine and serum represents a complementary systems-wide approach to elucidate environment–health interactions. However, large-scale metabolome studies in children combining analyses of these biological fluids are lacking. Here, we sought to characterise the major determinants of the child metabolome and to define metabolite associations with age, sex, BMI and dietary habits in European children, by exploiting a unique biobank established as part of the Human Early-Life Exposome project (http://www.projecthelix.eu).MethodsMetabolic phenotypes of matched urine and serum samples from 1192 children (aged 6–11) recruited from birth cohorts in six European countries were measured using high-throughput 1H nuclear magnetic resonance (NMR) spectroscopy and a targeted LC-MS/MS metabolomic assay (Biocrates AbsoluteIDQ p180 kit).ResultsWe identified both urinary and serum creatinine to be positively associated with age. Metabolic associations to BMI z-score included a novel association with urinary 4-deoxyerythronic acid in addition to valine, serum carnitine, short-chain acylcarnitines (C3, C5), glutamate, BCAAs, lysophosphatidylcholines (lysoPC a C14:0, lysoPC a C16:1, lysoPC a C18:1, lysoPC a C18:2) and sphingolipids (SM C16:0, SM C16:1, SM C18:1). Dietary-metabolite associations included urinary creatine and serum phosphatidylcholines (4) with meat intake, serum phosphatidylcholines (12) with fish, urinary hippurate with vegetables, and urinary proline betaine and hippurate with fruit intake. Population-specific variance (age, sex, BMI, ethnicity, dietary and country of origin) was better captured in the serum than in the urine profile; these factors explained a median of 9.0% variance amongst serum metabolites versus a median of 5.1% amongst urinary metabolites. Metabolic pathway correlations were identified, and concentrations of
Ahmetaj-Shala B, Olanipekun M, Tesfai A, et al., 2018, Development of a novel UPLC-MS/MS-based platform to quantify amines, amino acids and methylarginines for applications in human disease phenotyping, Scientific Reports, Vol: 8, ISSN: 2045-2322
Amine quantification is an important strategy in patient stratification and personalised medicine. This is because amines, including amino acids and methylarginines impact on many homeostatic processes. One important pathway regulated by amine levels is nitric oxide synthase (NOS). NOS is regulated by levels of (i) the substrate, arginine, (ii) amino acids which cycle with arginine and (iii) methylarginine inhibitors of NOS. However, biomarker research in this area is hindered by the lack of a unified analytical platform. Thus, the development of a common metabolomics platform, where a wide range of amino acids and methylarginines can be measured constitutes an important unmet need. Here we report a novel high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) platform where ≈40 amine analytes, including arginine and methylarginines can be detected and quantified on a molar basis, in a single sample of human plasma. To validate the platform and to generate biomarkers, human plasma from a well-defined cohort of patients before and after coronary artery bypass surgery, who developed systemic inflammatory response syndrome (SIRS), were analysed. Bypass surgery with SIRS significantly altered 26 amine analytes, including arginine and ADMA. Consequently, pathway analysis revealed significant changes in a range of pathways including those associated with NOS.
Law TH, Volk HA, Pan Y, et al., 2018, Metabolic perturbations associated with the consumption of a ketogenic medium-chain TAG diet in dogs with idiopathic epilepsy, British Journal of Nutrition, Vol: 120, Pages: 484-490, ISSN: 1475-2662
Consumption of diets containing medium-chain TAG (MCT) has been shown to confer neuroprotective effects. We aim to identify the global metabolic perturbations associated with consumption of a ketogenic diet (medium-chain TAG diet (MCTD)) in dogs with idiopathic epilepsy. We used ultra-performance liquid chromatography-MS (UPLC-MS) to generate metabolic and lipidomic profiles of fasted canine serum and made comparisons between the MCTD and standardised placebo diet phases. We identified metabolites that differed significantly between diet phases using metabolite fragmentation profiles generated by tandem MS (UPLC–MS/MS). Consumption of the MCTD resulted in significant differences in serum metabolic profiles when compared with the placebo diet, where sixteen altered lipid metabolites were identified. Consumption of the MCTD resulted in reduced abundances of palmitoylcarnitine, octadecenoylcarnitine, stearoylcarnitine and significant changes, both reduced and increased abundances, of phosphatidylcholine (PC) metabolites. There was a significant increase in abundance of the saturated C17 : 0 fatty acyl moieties during the MCTD phase. Lysophosphatidylcholine (17 : 0) (P=0·01) and PC (17:0/20:4) (P=0·03) were both significantly higher in abundance during the MCTD. The data presented in this study highlight global changes in lipid metabolism, and, of particular interest, in the C17 : 0 moieties, as a result of MCT consumption. Elucidating the global metabolic response of MCT consumption will not only improve the administration of current ketogenic diets for neurological disease models but also provides new avenues for research to develop better diet therapies with improved neuroprotective efficacies. Future studies should clarify the involvement and importance of C17 : 0 moieties in endogenous MCT metabolic pathways.
Martin G, Kolida S, Marchesi J, et al., 2018, In vitro modeling of bile acid processing by the human fecal microbiota, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X
Bile acids, the products of concerted host and gut bacterial metabolism, have important signaling functions within the mammalian metabolic system and a key role in digestion. Given the complexity of the mega-variate bacterial community residing in the gastrointestinal tract, studying associations between individual bacterial genera and bile acid processing remains a challenge. Here, we present a novel in vitro approach to determine the bacterial genera associated with the metabolism of different primary bile acids and their potential to contribute to inter-individual variation in this processing. Anaerobic, pH-controlled batch cultures were inoculated with human fecal microbiota and treated with individual conjugated primary bile acids (500 μg/ml) to serve as the sole substrate for 24 h. Samples were collected throughout the experiment (0, 5, 10, and 24 h) and the bacterial composition was determined by 16S rRNA gene sequencing and the bile acid signatures were characterized using a targeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) approach. Data fusion techniques were used to identify statistical bacterial-metabolic linkages. An increase in gut bacteria associated bile acids was observed over 24 h with variation in the rate of bile acid metabolism across the volunteers (n = 7). Correlation analysis identified a significant association between the Gemmiger genus and the deconjugation of glycine conjugated bile acids while the deconjugation of taurocholic acid was associated with bacteria from the Eubacterium and Ruminococcus genera. A positive correlation between Dorea and deoxycholic acid production suggest a potential role for this genus in cholic acid dehydroxylation. A slower deconjugation of taurocholic acid was observed in individuals with a greater abundance of Parasutterella and Akkermansia. This work demonstrates the utility of integrating compositional (metataxonomics) and functional (metabonomics) systems biology approaches
Want EJ, 2018, LC-MS Untargeted Analysis., Methods Mol Biol, Vol: 1738, Pages: 99-116
LC-MS untargeted analysis is a valuable tool in the field of metabolic profiling (metabonomics/metabolomics), and the applications of this technology have grown rapidly over the past decade. LC-MS offers advantages over other analytical platforms such as speed, sensitivity, relative ease of sample preparation, and large dynamic range. As with any analytical approach, there are still drawbacks and challenges to overcome, but advances are constantly being made regarding both column chemistries and instrumentation. There are numerous untargeted LC-MS approaches which can be used in this ever-growing research field; these can be optimized depending on sample type and the nature of the study or biological question. Some of the main LC-MS approaches for the untargeted analysis of biological samples will be described in detail in the following protocol.
Tesfai A, MacCallum N, Kirkby NS, et al., 2017, Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways, PLoS ONE, Vol: 12, ISSN: 1932-6203
RationaleNitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known.ObjectiveOur objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity.Methods and measurements34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay.Main resultsOf all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis.ConclusionsIn early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.
Maitre L, Lau C-HE, Vizcaino E, et al., 2017, Assessment of metabolic phenotypic variability in children's urine using H-1 NMR spectroscopy, Scientific Reports, Vol: 7, ISSN: 2045-2322
The application of metabolic phenotyping in clinical and epidemiological studies is limited by a poor understanding of inter-individual, intra-individual and temporal variability in metabolic phenotypes. Using 1H NMR spectroscopy we characterised short-term variability in urinary metabolites measured from 20 children aged 8–9 years old. Daily spot morning, night-time and pooled (50:50 morning and night-time) urine samples across six days (18 samples per child) were analysed, and 44 metabolites quantified. Intraclass correlation coefficients (ICC) and mixed effect models were applied to assess the reproducibility and biological variance of metabolic phenotypes. Excellent analytical reproducibility and precision was demonstrated for the 1H NMR spectroscopic platform (median CV 7.2%). Pooled samples captured the best inter-individual variability with an ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid, 3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate, tyrosine, valine and 3-hydroxyisovalerate exhibited the highest stability with over 50% of variance specific to the child. The pooled sample was shown to capture the most inter-individual variance in the metabolic phenotype, which is of importance for molecular epidemiology study design. A substantial proportion of the variation in the urinary metabolome of children is specific to the individual, underlining the potential of such data to inform clinical and exposome studies conducted early in life.
Want EJ, Metz TO, 2017, MS Based Metabonomics, ENCYCLOPEDIA OF SPECTROSCOPY AND SPECTROMETRY, 3RD EDITION, VOL 2: G-M, Editors: Lindon, Tranter, Koppenaal, Publisher: ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD, Pages: 926-935
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Tesfai A, Shala BA, Shala F, et al., 2017, Plasma Taurine Levels At Early Diagnosis Predict Survival, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Robinson O, Toledano MB, Sands C, et al., 2016, Global metabolic changes induced by plant-derived pyrrolizidine alkaloids following a human poisoning outbreak and in a mouse model, Toxicology Research, Vol: 5, Pages: 1594-1603, ISSN: 2045-4538
Several hundred cases of Hirmi Valley Liver Disease (HVLD), an often fatal liver injury, occurred from 2001 to 2011 in a cluster of rural villages in Tigray, Ethiopia. HVLD is principally caused by contamination of the food supply with plant derived pyrrolizidine alkaloids (PAs), with high exposure to the pesticide DDT among villagers increasing their susceptibility. In an untargeted global approach we aimed to identify metabolic changes induced by PA exposure through 1H NMR spectroscopic based metabolic profiling. We analysed spectra acquired from urine collected from HVLD cases and controls and a murine model of PA exposure and PA/DDT co-exposure, using multivariate partial least squares discriminant analysis. In the human models we identified changes in urinary concentrations of tyrosine, pyruvate, bile acids, N-acetylglycoproteins, N-methylnicotinamide and formate, hippurate, p-cresol sulphate, p-hydroxybenzoate and 3-(3-hydroxyphenyl) propionic acid. Tyrosine and p-cresol sulphate were associated with both exposure and disease. Similar changes to tyrosine, one-carbon intermediates and microbial associated metabolites were observed in the mouse model, with tyrosine correlated with the extent of liver damage. These results provide mechanistic insight and implicate the gut microflora in the human response to challenge with toxins. Pathways identified here may be useful in translational research and as “exposome” signals.
Anwar MA, Vorkas PA, Li J, et al., 2016, Prolonged Mechanical Circumferential Stretch Induces Metabolic Changes in Rat Inferior Vena Cava, EUROPEAN JOURNAL OF VASCULAR AND ENDOVASCULAR SURGERY, Vol: 52, Pages: 544-552, ISSN: 1078-5884
Vorkas PA, Shalhoub J, Lewis MR, et al., 2016, Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk – An Exploratory Study, European Journal of Vascular and Endovascular Surgery, Vol: 52, Pages: 5-10, ISSN: 1532-2165
Objectives: Stroke is a major cause of death and disability. The fact that three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, we assess the utility of comprehensive metabolic phenotyping to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples.Design: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n=5), and asymptomatic patients (n=5). Two adjacent biological replicates were obtained from each tissue.Materials and Methods: Organic and aqueous metabolite extracts were separately obtained and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were utilised for statistical analysis.Results: The two studied groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p=10-4-10-5). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine and palmitoylcarnitine), intermediates of the β-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected.Conclusions: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and β-oxidation) were implicated and will be further investigated. These results indicate that metabolic
Wesseling H, Xu B, Want EJ, et al., 2016, System-based proteomic and metabonomic analysis of the Df(16)A(+/-) mouse identifies potential miR-185 targets and molecular pathway alterations, Molecular Psychiatry, Vol: 22, Pages: 384-395, ISSN: 1476-5578
Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography-mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A(+/-) mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were
Want E, Pape J, Nye L, et al., 2016, Placental Metabolomics: Methods and Applications., 63rd Annual Scientific Meeting of the Society-for-Reproductive-Investigation, Publisher: SAGE PUBLICATIONS INC, Pages: 151A-152A, ISSN: 1933-7191
Law TH, Davies ES, Pan Y, et al., 2016, A randomised trial of a medium-chain TAG diet as treatment for dogs with idiopathic epilepsy - CORRIGENDUM., British Journal of Nutrition, Vol: 115, Pages: 1696-1696, ISSN: 1475-2662
McPhail MJW, Shawcross D, Lewis MR, et al., 2016, Mutlivariate metabotyping of plasma accurately predicts survival in decompensated cirrhosis, Journal of Hepatology, Vol: 64, Pages: 1058-1067, ISSN: 1600-0641
Background & AimsPredicting survival in decompensated cirrhosis (DC) is important in decision making for liver transplantation and resource allocation. We investigated whether high-resolution metabolic profiling can determine a metabolic phenotype associated with 90-day survival.MethodsTwo hundred and forty-eight subjects underwent plasma metabotyping by 1H nuclear magnetic resonance (NMR) spectroscopy and reversed-phase ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS; DC: 80-derivation set, 101-validation; stable cirrhosis (CLD) 20 and 47 healthy controls (HC)).Results1H NMR metabotyping accurately discriminated between surviving and non-surviving patients with DC. The NMR plasma profiles of non-survivors were attributed to reduced phosphatidylcholines and lipid resonances, with increased lactate, tyrosine, methionine and phenylalanine signal intensities. This was confirmed on external validation (area under the receiver operating curve [AUROC] = 0.96 (95% CI 0.90–1.00, sensitivity 98%, specificity 89%). UPLC-TOF-MS confirmed that lysophosphatidylcholines and phosphatidylcholines [LPC/PC] were downregulated in non-survivors (UPLC-TOF-MS profiles AUROC of 0.94 (95% CI 0.89–0.98, sensitivity 100%, specificity 85% [positive ion detection])). LPC concentrations negatively correlated with circulating markers of cell death (M30 and M65) levels in DC. Histological examination of liver tissue from DC patients confirmed increased hepatocyte cell death compared to controls. Cross liver sampling at time of liver transplantation demonstrated that hepatic endothelial beds are a source of increased circulating total cytokeratin-18 in DC.ConclusionPlasma metabotyping accurately predicts mortality in DC. LPC and amino acid dysregulation is associated with increased mortality and severity of disease reflecting hepatocyte cell death.
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