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O C T O B E R feature stor y Immune Cell Function and Fate Regulated by Metabolism Upon exposure to foreign antigens, relatively quiescent cells of the immune system are stimulated to undergo a highly coordinated and scalable response critical to combating infection. A lthough initial activation events are orchestrated by established physiological processes such as transcription and post-translational modifications, growing evidence demonstrates both an instructive and responsive role for metabolic changes in directing the proliferative potential, differentiation, and function of immune cells1, 2. The growing evidence for a precisely engineered metabolic-immune connection is also illuminating that, in addition to traditional cell and molecular biological approaches to understanding the immune system, metabolism research also is an important avenue to pursue to complement and add to the understanding of immunology with the ultimate goal of facilitating treatments/modalities for immune system modulation. keep reading this story REFERENCES 2 0 1 3 I S S U E News & EVENTS Press Release: DHMRI and Metabolon Announce Strategic Partnership to Provide Expanded Metabolomics Capabilities August 27th, 2013 | Read Press Release Health Ingredients Congress September 26 – 27th à Poitiers, France 10th International ISSX Meeting September 29th – October 3rd Toronto, Canada Cell Culture World Congress September 30th – October 1st Boston, MA Skin Forum Skin Metabolism October 10 – 11th Valbonne, Nice France HAPPI Anti-Aging Conference October 29 – 30th Brunswick, NJ Recent Advances in Fermentation Technology Conference November 3 – 5th Marco Isle, Fl publications in the news An Integrated Clinico-Metabolomic Model Improves Prediction of Death in Sepsis Langley et. al. | Science Translational Medicine, 2013 MORE INFO The Mycobacterium Tuberculosis Regulatory Network and Hypoxia Galagan, J. et. al. | Nature, 2013 MORE INFO Leptin Engages a Hypothalamic Neurocircuitry to Permit Survival in the Absence of Insulin Fujikawa, T. et. al. | Cell Metabolism, 2013 MORE INFO Metabolomics Uncovers Dietary Omega-3 Fatty Acid-Derived Metabolites Implicated in Anti-Nonciceptive Responses after Experimental Spinal Cord Injury Figueroa, J. et. al. | Neuroscience, 2013 MORE INFO october the path forward A Technical Commentary by Michael Milburn, CSO As we continue strive to mine the genome for clues that can assist in understanding susceptibility to disease, selection of better targets for combating disease, and the biomarkers delineating response, metabolism provides a key source of data that can either lead or strengthen this pursuit. That’s right – metabolism. As metabolism research continues to expand into areas traditionally dominated by cell and molecular biology approaches (e.g. immunology research, see feature article), it is becoming increasingly clear that there are few areas in biology that metabolism does not offer a say in. Thus, despite the intense interest in molecular biology the last 30 years, we are re-awakening to the notion that, if one is interested in understanding a biological phenomena, it necessitates investigating the metabolism of the system. keep reading this story Serum Metabolomics Indicate Altered Cellular Energy Metabolism in Children with Cystic Fibrosis Joseloff, E. et. al. | Pediatric Pulmonology, 2013 VIEW SUMMARY Listeria Monocytogenes Infection Causes Metabolic Shifts in Drosophila Melanogastor Chambers, M. et. al. | PloS One, 2012 VIEW SUMMARY HuR is a Post-Transcriptional Regulator of Core Metabolic Enzymes in Pancreatic Cancer Burkhart, R. et. al. | RNA Biology, 2013 VIEW SUMMARY Infectious Disease Serum Metabolomics Indicate Altered Cellular Energy Metabolism in Children with Cystic Fibrosis Joseloff, E. et. al. Pediatric Pulmonology, 2013 VIEW SUMMARY Metabolic Disturbances Associated with Systemic Lupus Erythematosus Wu, T. et. al. | PloS One, 2012 VIEW SUMMARY Metabolomics Reveals Elevated Macromolecular Degradation in Periodontal Disease Barnes, V., et. al. | J Dent Res, 2012 VIEW SUMMARY Exposure of Clinical MRSA Heterogeneous Strains to β-Lactams Redirects Metabolism to Optimize Energy Production through the TCA Cycle Keaton, M. et. al. | PLoS One, 2013 VIEW SUMMARY Metabolism/Cardiovascular Disease Metabolite Profiling Reveals New Insights into the Regulation of Serum Urate in Humans Albrecht, E. et. al. | Metabolomics, 2013 The Transcription Factor Myc Controls Metabolic Reprogramming upon T Lymphocyte Activation Wang, R. et. al. | Immunity, 2011 VIEW SUMMARY Neuroscience/Neurology VIEW SUMMARY Detrimental Effects of Adenosine Signaling in Sickle Cell Disease Zhang, Y., et. al. | Nat Med, 2011 VIEW SUMMARY Profile of Circulatory Metabolites in an Animal Model of Multiple Sclerosis Using Global Metabolomics Mangalam, A. et. al. Journal of Clinical and Cellular Immunology, 2013 VIEW SUMMARY Reducing Endoplasmic Reticulum Stress Through a Macrophage Lipid Chaperone Alleviates Atherosclerosis Erbay, E. et. al. | Nat Med, 2009 Lipidomics Reveals Early Metabolic Changes in Subjects with Schizophrenia: Effects of Atypical Antipsychotics McEvoy, J. et. al. | PLoS One, 2013 VIEW SUMMARY VIEW SUMMARY Nutrition & Consumer Goods 3-Hydroxykynurenine and Other Parkinson’s Disease Biomarkers Discovered by Metabolomic Analysis LeWitt, P. et. al. | Movement Disorders, 2013 Metabolic Profiling in Nutrition and Metabolic Disorders LeMieux, M. et. al. Advances in Nutrition, 2013 VIEW SUMMARY VIEW SUMMARY A Diet Rich in High-Glucoraphanin Broccoli Interacts with Genotype to Reduce Discordance in Plasma Metabolite Profiles by Modulating Mitochondrial Function Armah, C. et. al. | The American Journal of Clinical Nutrition, 2013 VIEW SUMMARY Cancer/Oncology For a complete list of publications, please visit our website at www.metabolon.com/news/Publications.aspx. I S S U E publications Inflammation/Immunity Unappreciated Power of Metabolites for New Insight into Signaling Networks 2 0 1 3 Metabolomics Identifies Pyrimidine Starvation as the Mechanism of 5-aminoimidazole4-carboxamide-1-beta-riboside (AlCAr) Induced Apoptosis in Multiple Myeloma Cells Bardeleben, C. et. al. Molecular Cancer Therapeutics, 2013 Respiratory Serum Metabolomics Indicate Altered Cellular Energy Metabolism in Children with Cystic Fibrosis Joseloff, E. et. al. | Pediatric Pulmonology, 2013 VIEW SUMMARY Technology Metabolomics in Epidemiology: Sources of Variability in Metabolite Measurements and Implications Sampson. J et. al. | Cancer Epidemiology, Biomarkers & Prevention, 2013 VIEW SUMMARY VIEW SUMMARY A bimonthly publication of: © Copyright Metabolon, Inc. 2013 +1.919.572.1711 | www.metabolon.com | [email protected] F eature stor y — continued Immune Cell Function and Fate Regulated by Metabolism Highlighting this, the following brief synopsis discusses recent findings that show the impact of intracellular metabolism and metabolite availability on immune cell fate and the effect these cells can have on systemic metabolism. consumption due to enhanced nuclear receptor PGC1 and PPARδ expression that facilitates fatty acid oxidation and mitochondrial respiration10, 11. Thus, metabolic pathway choice supports the function of these distinct pro- and anti-inflammatory macrophage subsets. Although it has long been appreciated that metabolites are key elements of immune cell signaling and functional execution (e.g., histamine, eicosanoids, leukotrienes and prostaglandins), recent results have also illuminated that fundamental metabolic pathways (e.g. glycolysis) are central to primary functions across both innate and adaptive immunity. Similar to macrophages, the choice between glycolytic and oxidative metabolism has a critical impact on the adaptive immune response. Naïve quiescent T cells can be stimulated by macrophages and dendritic cells in response to cognate antigen recognition. This initial event triggers a cascade of signaling networks that coordinate the expansion and differentiation of T cells to combat pathogens, tumors, and confer immunological memory. Considering the specificity of these processes, T cell subsets predominantly consist of effector, helper, memory, and regulatory types that promote inflammation and cytotoxicity, assist humoral immunity, prevent reinfection, and regulate the immune response respectively. Similar to M1 and M2 macrophages, inflammatory T effector cells upregulate glycolytic metabolism to support rapid cell division and acquire cytolytic capacity, while immune suppressive T regulatory cells and memory cells are characterized by an enhanced rate of fatty acid oxidation. Furthermore, these metabolic phenotypes are a requirement for fate considering genetic and pharmacological enhancement of fatty acid metabolism can increase T regulatory and memory cell development, while limiting the development and function of effector T cells12, 13. Thus, metabolic manipulation may be critical in the design of future vaccine and autoimmune therapeutic strategies. The innate and adaptive branches of the immune system function in concert to generate a productive response. Consisting of granulocytes, macrophages, and dendritic cells; the innate immune response is dependent upon genetically encoded (“innate”) pathogen pattern recognition receptors and serves as the first line of defense against infection. Adaptive immune T and B lymphocytes provide pathogen and tumor specificity and are stimulated by innate cells following antigen presentation. As opposed to the broad non-targeted response of innate cells, these lymphocytes “adapt” to initial infection to confer immunological memory that protects the host from reinfection by the same pathogen. Given their distinct roles in the immune system, specific metabolic features are tailored to the precise function of each of these cell types as described below. Granulocytes such as neutrophils are relatively short-lived cells that often produce an oxidative burst (release of free radical species) to promote pathogen clearance and release metabolic regulators of inflammation such as histamine and eicosanoids. Consequently, these cells are often characterized by a highly glycolytic phenotype to rapidly support changes in growth and function3. In contrast to the relative simplicity of neutrophils, macrophages exhibit a more complex role in the regulation of both pro- and anti-inflammatory processes compared to granulocytes. Specifically, classical M1 macrophages produce the cytokine IL-12 to facilitate effector T cell responses, TNFα to induce inflammation and tissue damage, and anti-microbial nitric oxide (NO). Alternatively activated M2 macrophages can suppress inflammation, promote tissue repair, and regulate adaptive immunity4. These differing roles in innate immunity are predicated by metabolic pathway choices. For example, M1 macrophages upregulate inducible nitric oxide synthase (iNOS) and consequently utilize arginine for the production of inflammatory NO. In contrast, M2 cells increase arginase 1 expression to inhibit NO synthesis and support polyamine and hydroxyproline generation for wound healing5. M1 cells also exhibit a high rate of arachidonic acid processing for the generation of inflammatory eicosanoids such as leukotrienes and prostaglandins and utilize a glycolytic Warburg metabolism proposed to be partially mediated by elevated NO capable of nitrosylating and inactivating iron-sulfur containing electron transport proteins6-8. Indeed, a well-established connection between increased NO and reduced respiratory rate is reported in the literature9. In opposition, M2 macrophages have a high basal rate of mitochondrial oxygen BACK TO NEWSLETTER Aside from pathogen clearance, immune cells can also have a profound impact on host systemic metabolism. For example, lean adipose and liver tissue possess M2 macrophages and T regulatory cells that promote insulin sensitivity by inhibiting M1 macrophage from releasing inflammatory cytokines and chemokines that can stimulate aberrant JNK and IKK signaling typically upregulated in insulinresistance skeletal muscle and other tissues14-16. Indeed, an abundance of M1 macrophages are often found in the tissues of obese individuals. Similarly, dendritic cells can regulate the gut microbiome via the production of the vitamin A metabolite retinoic acid (RA)17. RA can promote T regulatory cell development to control immune tolerance to symbiotic bacteria and facilitate the production of IgA antibodies to maintain intestinal barrier integrity18. Despite the insight that these discoveries have provided regarding how metabolic pathway choices affect immune cell fate and contribute to systemic metabolism, understanding the extent of these regulatory networks and their implications in various diseases remains to be determined. Recent advancements in hypothesis-generating, unbiased metabolomics approaches have laid the foundation for answering these questions and coupled with the power of computational tools for not only analyzing these datasets, but marrying them with microarray, genomic, and/or proteomic datasets, holds the potential to facilitate the development of medical and therapeutic breakthroughs. REFERENCES F eature stor y — R E F E R E N C E S References 1. Pearce, E.L. & Pearce, E.J. Metabolic pathways in immune cell activation and quiescence. Immunity 38, 633-43 (2013). 10. Vats, D. et al. Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation. Cell Metab 4, 13-24 (2006). 2. MacIver, N.J., Michalek, R.D. & Rathmell, J.C. Metabolic regulation of T lymphocytes. Annu Rev Immunol 31, 259-83 (2013). 11. Thomas, G.D. et al. The biology of nematode- and IL4Ralpha-dependent murine macrophage polarization in vivo as defined by RNA-Seq and targeted lipidomics. Blood 120, e93-e104 (2012). 3. Dale, D.C., Boxer, L. & Liles, W.C. The phagocytes: neutrophils and monocytes. Blood 112, 935-45 (2008). 4. Murray, P.J. & Wynn, T.A. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11, 723-37 (2011). 5. Stout, R.D. Editorial: macrophage functional phenotypes: no alternatives in dermal wound healing? J Leukoc Biol 87, 19-21 (2010). 6. Greene, E.R., Huang, S., Serhan, C.N. & Panigrahy, D. Regulation of inflammation in cancer by eicosanoids. Prostaglandins Other Lipid Mediat 96, 27-36 (2011). 7. Beltran, B., Mathur, A., Duchen, M.R., Erusalimsky, J.D. & Moncada, S. The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. Proc Natl Acad Sci U S A 97, 14602-7 (2000). 8. Cleeter, M.W., Cooper, J.M., Darley-Usmar, V.M., Moncada, S. & Schapira, A.H. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345, 50-4 (1994). 9. Rees, D.D., Monkhouse, J.E., Cambridge, D. & Moncada, S. Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse. Br J Pharmacol 124, 540-6 (1998). BACK TO NEWSLETTER 12. Michalek, R.D. et al. Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets. J Immunol 186, 3299-303 (2011). 13. Pearce, E.L. et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature 460, 103-7 (2009). 14. Feuerer, M. et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med 15, 930-9 (2009). 15. Olefsky, J.M. & Glass, C.K. Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 72, 219-46 (2010). 16. Patel, P.S., Buras, E.D. & Balasubramanyam, A. The role of the immune system in obesity and insulin resistance. J Obes 2013, 616193 (2013). 17. Coombes, J.L. et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 204, 1757-64 (2007). 18. Mora, J.R. et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314, 1157-60 (2006). P R E S S R E L E A S E Press Release: DHMRI and Metabolon Announce Strategic Partnership to Provide Expanded Metabolomics Capabilities KANNAPOLIS & RESEARCH TRIANGLE PARK, N.C. (August 27, About the David H. Murdock Research Institute 2013)–The David H. Murdock Research Institute (DHMRI) at the The David H. Murdock Research Institute (DHMRI) is a not-for-profit, North Carolina Research Campus and Metabolon, Inc., announced research institute committed to the improvement of human life. today that they have entered into a strategic agreement to align DHMRI provides collaborators groundbreaking research and metabolomics research services. Metabolon is a pioneering leader development solutions at the intersection of human health, nutrition in discovery metabolomics and specializes in rapidly assessing and agriculture through investigations in genomics, proteomics, metabolism in biological samples to quickly identify prospective metabolomics, NMR, light microscopy and cell biology. DHMRI biomarkers and to understand metabolic effects of treatments, combines collaboration and customized solutions with a interventions, nutritionals, etc. The agreement complements DHMRI’s multidisciplinary, systems biology approach to find answers to mass spectrometry and NMR-based metabolomics services through advance research and product development. To learn more, access to Metabolon’s industry-leading high-throughput biomarker please visit www.dhmri.org or contact [email protected]. discovery and profiling platform which provides an extensive, untargeted, broad metabolite survey of more than 4000 About Metabolon biochemicals. Metabolon, Inc. is a world leader in the field of metabolomics by pioneering and patenting the industry’s leading biochemical “This agreement leverages the strengths of our organizations to best biomarker discovery and profiling platform. It has developed the serve DHMRI collaborators to understand metabolism and ultimately technology to quickly identify and measure the biochemicals in a answer key research questions and deliver healthy food products biological sample through its proprietary global processing method. for consumers. We have worked extensively in food science and Metabolon has a broad pipeline of diagnostic products in the nutrition and recognize that our combined resources are indeed fields of obesity-related conditions and cancer. Quantose™ is the complementary. DHMRI is a center of excellence in nutrition first diagnostic test developed by Metabolon using its technology. research and we are delighted to do our part to meet their growing Metabolon’s expertise is embraced by a wide range of needs,” says Chris Bernard, Senior Vice President of Sales & pharmaceutical, biotechnology, food and agricultural companies. Marketing for Metabolon. Metabolytics, its biomarker discovery and analysis business, has completed over 3,000 client studies with more than 550 customers. DHMRI is an established not-for-profit research institute that provides For more information about Metabolon, please visit www. flexible, client-focused research services to academia, government metabolon.com or contact Matt Zaske at 919-595-2200 or and industry collaborators. This agreement, consistent with the [email protected]. DHMRI’s collaborative approach to scientific inquiry, provides partners with additional resources for metabolomics. Metabolon’s global metabolomic approach can pinpoint active pathways which Media Contact: Mackenzie Mills, SpecOps Communications can be further interrogated by targeted and or customized 212.518.7721 Ph. | 913.558.2492 Cell approaches offered by the DHMRI Metabolomics team. www.SpecOpsComm.com | @SpecOpsComm “We are excited to broaden the breadth of services offered by the Benjamin D. Machon, David H Murdock Research Institute DHMRI to our collaborators. The benefits stemming from the 704.250.2600 Ph. | www.dhmri.org collective knowledge of our organizations will be readily realized by researchers, companies and consumers,” said Steve Lommel Ph.D., Interim President, David H. Murdock Research Institute. BACK TO NEWSLETTER An Integrated Clinico-Metabolomic Model Improves Prediction of Death in Sepsis Langley et. al. | Science Translational Medicine, 2013 Metabolon results led to: • Biomarkers for identifying sepsis patients who merit intensive treatment due to risk of death Key metabolomic observations: • Fatty acid transport and b-oxidation, gluconeogenesis, and the citric acid cycle alterations Synopsis Differentiating mild infections from life-threatening ones is a complex decision that is made millions of times a year in U.S. emergency rooms. Should a patient be sent home with antibiotics and chicken soup? Or should the patient be hospitalized for intensive treatment? Sepsis—a serious infection that is associated with a generalized inflammatory response—is one of the leading causes of death. In two prospective clinical studies reported by Langley et. al., patients arriving at four urban emergency departments with symptoms of sepsis were evaluated clinically and by analysis of their plasma proteome and metabolome. Survivors and nonsurvivors at 28 days were compared, and a molecular signature was detected that appeared to differentiate these outcomes– even as early as the time of hospital arrival. The signature was part of a large set of differences between these groups, showing that better energy-producing fatty acid catabolism was associated with survival of the fittest in sepsis. A test developed from the signature was able to predict sepsis survival and nonsurvival reproducibly and better than current methods. This test could aid in improving the accuracy of emergency room decisions regarding treatment of patients at risk of sepsis. For more information, visit here. BACK TO NEWSLETTER The Mycobacterium Tuberculosis Regulatory Network and Hypoxia Galagan, J. et. al. | Nature, 2013 Abstract We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub. For more information, visit here. BACK TO NEWSLETTER Leptin Engages a Hypothalamic Neurocircuitry to Permit Survival in the Absence of Insulin Fujikawa, T. et. al. | Cell Metabolism, 2013 Abstract The dogma that life without insulin is incompatible has recently been challenged by results showing the viability of insulin-deficient rodents undergoing leptin monotherapy. Yet, the mechanisms underlying these actions of leptin are unknown. Here, the metabolic outcomes of intracerebroventricular (i.c.v.) administration of leptin in mice devoid of insulin and lacking or re-expressing leptin receptors (LEPRs) only in selected neuronal groups were assessed. Our results demonstrate that concomitant re-expression of LEPRs only in hypothalamic g-aminobutyric acid (GABA) and pro-opiomelanocortin (POMC) neurons is sufficient to fully mediate the lifesaving and antidiabetic actions of leptin in insulin deficiency. Our analyses indicate that enhanced glucose uptake by brown adipose tissue and soleus muscle, as well as improved hepatic metabolism, underlies these effects of leptin. Collectively, our data elucidate a hypothalamicdependent pathway enabling life without insulin and hence pave the way for developing better treatments for diseases of insulin deficiency. For more information, visit here. BACK TO NEWSLETTER Metabolomics Uncovers Dietary Omega-3 Fatty Acid-Derived Metabolites Implicated in AntiNonciceptive Responses after Experimental Spinal Cord Injury Figueroa, J. et. al. | Nueroscience, 2013 Abstract Chronic neuropathic pain is a frequent comorbidity following spinal cord injury (SCI) and often fails to respond to conventional pain management strategies. Preventive administration of docosahexaenoic acid (DHA) or consumption of a diet rich in omega-3 polyunsaturated fatty acids (O3PUFAs) confers potent prophylaxis against SCI and improves functional recovery. The present study examines whether this novel dietary strategy provides significant antinociceptive benefits in rats experiencing SCI-induced pain. Rats were fed control chow or chow enriched with O3PUFAs for 8 weeks before being subjected to sham or cord contusion surgeries, continuing the same diets after surgery for another 8 more weeks. The paw sensitivity to noxious heat was quantified for at least 8 weeks post-SCI using the Hargreaves test. We found that SCI rats consuming the preventive O3PUFA-enriched diet exhibited a significant reduction in thermal hyperalgesia compared to those consuming the normal diet. Functional neurometabolomic profiling revealed a distinctive deregulation in the metabolism of endocannabinoids (eCB) and related N-acyl ethanolamines (NAEs) at 8 weeks post-SCI. We found that O3PUFAs consumption led to a robust accumulation of novel NAE precursors, including the glycerophosphocontaining docosahexaenoyl ethanolamine (DHEA), docosapentaenoyl ethanolamine (DPEA), and eicosapentaenoyl ethanolamine (EPEA). The tissue levels of these metabolites were significantly correlated with the antihyperalgesic phenotype. In addition, rats consuming the O3PUFA-rich diet showed reduced sprouting of nociceptive fibers containing CGRP and dorsal horn neuron p38 MAPK expression, well-established biomarkers of pain. The spinal cord levels of inositols were positively correlated with thermal hyperalgesia, supporting their role as biomarkers of chronic neuropathic pain. Notably, the O3PUFA-rich dietary intervention reduced the levels of these metabolites. Collectively, these results demonstrate the prophylactic value of dietary O3PUFA against SCImediated chronic pain. For more information, visit here. BACK TO NEWSLETTER the path forward — continued Unappreciated Power of Metabolites for New Insight into Signaling Networks A Technical Commentary by Michael Milburn, CSO Obviously, to many, this is not a new concept as this understanding formed the basis of almost a century of metabolic investigation from the likes of Krebs, Warburg, Cori, and Pasteur. Until recently, technologies that could fully exploit the information embedded within metabolites did not exist. But, now, metabolomics offers a more effective means for surveying metabolism and has confronted us with reminders of many of the lessons learned in the past. Perhaps the best current example of this is in the area of the regulation of metabolism where metabolite concentrations have been shown to be pivotal inputs for directing signaling and metabolic enzymes (e.g. histone acetylases/deacetylases, sirtuins, AMPK, mTOR, DNA methyltransferases)1. Although regulation of metabolic enzymes by either covalent modification or allostery has a deep history, many new findings are extending this deep history and starting to connect these principles to more advanced aspects of biological regulation. This includes the field highlighted in our feature article this month–immunology. Recent work by Chang et al.2 offers an explanation how metabolism influences immune cell fate by suggesting that metabolic substrate availability can govern non-metabolic functions for enzymes. Specifically, the authors demonstrate that T cells can utilize glycerate as an alternative fuel source to glucose for proliferation, but not inflammatory cytokine production. Instead, glycerate utilization limits glycolysis and results in a decline in the GAPDH substrate glyceraldehydes 3-phosphate. Consequently, GAPDH acquires a non-metabolic function by interacting with the 3’UTR of IFNγ mRNA and inhibiting translation. Thus, effector T cell mediated inflammation is compromised. Non-metabolic functions have also been previously described for the hexokinase 2 and PKM2 as these enzymes can regulate cytochrome c release and HDAC removal respectively. Notably, these findings have broad implications highlighting the importance of biochemical substrate availability in dictating traditional and nonmetabolic signaling networks. Future studies may reveal similar mechanisms in other cell types that potentially regulate complex diseases such as cancer, obesity, and heart disease. A hot off the press example is recent work by Moellering and Cravatt showing how cells use an intrinsically reactive glycolytic intermediate to modify lysine residues as a means to regulate glycolysis and redirect carbon to alternate pathways.3-4 Thus, these results continue to remind us that the basic unit that all life evolved around–metabolism–is central to even the most complex biological phenomena and the use of metabolomics as a vehicle for gaining a comprehensive fingerprint of the system under study will serve to integrate and advance this understanding. References 1. Wellen, K.E. & Thompson, C.B. A two-way street: reciprocal regulation of metabolism and signalling. Nat Rev Mol Cell Biol 13, 270-276 (2012). 3. Green, Douglas R. & Rathmell, Jeffrey. Sweet Nothings: Sensing of Sugar Metabolites Controls T Cell Function. Cell Metabolism 18, 7-8 (2013). 2. Chih-Hao Chang, et. al. Posttranscriptional Control of T Cell Effector Function by Aerobic Glycolysis. Cell 153, 12391251 (2013). 4. Moellering, R. E. and Cravett, B. F. Functional Lysine Modification by an Intrinsically Reactive Primary Glycolytic Metabolite. Science 2, 549-553 (2013). BACK TO NEWSLETTER Serum Metabolomics Indicate Altered Cellular Energy Metabolism in Children with Cystic Fibrosis Joseloff, E. et. al | Pediatric Pulmonology, 2013 Abstract Background: Cystic fibrosis (CF) is a multi-system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis. Aim: We hypothesized that metabolomics using serum from children would differentiate CF from non-CF lung disease subjects and would provide insight into metabolism in CF. Methods: Serum collected from children with CF (n ¼ 31) and 31 age and gender matched children with other lung diseases was used for metabolomic profiling by gas-and liquid-chromatography. Relative concentration of metabolites was compared between the groups using partial least square discriminant analyses (PLS-DA) and linear modeling. Results: A clear separation of the two groups was seen in PLS-DA. Linear model found that among the 459 detected metabolites 92 differed between CF and non-CF. These included known biochemicals in lipid metabolism, oxidants, and markers consistent with abnormalities in bile acid processing. Bacterial metabolites were identified and differed between the groups indicating intestinal dysbiosis in CF. As a novel finding several pathways were markedly different in CF, which jointly point towards decreased activity in the b-oxidation of fatty acids. These pathways include low ketone bodies, low medium chain carnitines, elevated di-carboxylic acids and decreased 2-hydroxybutyrate from amino acid metabolism in CF compared to non-CF. Conclusion: Serum metabolomics discriminated CF from non-CF and show altered cellular energy metabolism in CF potentially reflecting mitochondrial dysfunction. Future studies are indicated to examine their relation to the underlying CF defect and their use as biomarkers for disease severity or for cystic fibrosis transmembrane regulator (CFTR) function in an era of CFTR modifying drugs. BACK TO NEWSLETTER Listeria Monocytogenes Infection Causes Metabolic Shifts in Drosophila Melanogaster Chambers et. al. | PLoS One, 2012 Metabolon results led to: • Important insights into how metabolism and innate immunity are coupled • Novel pathways that offer possible avenues for manipulating the innate immune response for combating infection Key metabolomic observations: • Wide-scale changes in energy metabolism with Listeria monocytogenes infection • Novel pathway changes with infection (uric acid metabolism) Synopsis Although immunity and metabolism are tightly linked, this connection is not well understood and often unappreciated. Deeper understanding may allow for more effective manipulation (through diet, environment or drugs) to improve from infection. Thus, investigators used a model of innate immunity (lethal L. monocytogenes infection of Drosophila melanogaster) to investigate metabolic changes coupled to the event. Metabolomics revealed profound changes in pathways of energy metabolism (beta-oxidation and glycolysis). In addition to energy pathway changes, significant reductions in the anti-oxidant uric acid were noted and uricase mutants were shown to be more resistant to infection by some strains of bacteria and had improved survival upon wounding. This study lays a foundation for further investigation of the metabolic-immune connection with the ultimate goal of facilitating treatments/modalities for combating infection. BACK TO NEWSLETTER Metabolic Disturbances Associated with Systemic Lupus Erythematosus Wu T et. al. | PLoS One, 2012 Metabolon results led to: • New therapeutic targets • New biomarkers • A potential treatment via dietary correction Key metabolomic observations: • Major changes related to inflammation and oxidative stress • Metabolic changes suggesting dysregulated energy metabolism Synopsis Systemic lupus erythematosus (SLE) is a highly diverse autoimmune disease. Reliable and specific biomarkers hinder clinical management and the development of new therapies. Metabolomic analysis of plasma from SLE subjects discovered biomarkers with remarkably high sensitivity and specificity compared to normal subjects or subjects with Rheumatoid arthritis (another pro-inflammatory, chronic systemic autoimmune disease). These results were then validated in an independent assay and cohort. Oxidized lipids were among the top differentiating metabolites and they also correlated well with disease grade. Many of the markers intuitively connect to the clinical features of the disease. For example, it is likely that the lipid peroxidation markers are a combination of the impaired beta-oxidation of fatty acids (and the reported lower energy status of SLE subjects) and the oxidative stress associated with the disease. BACK TO NEWSLETTER Metabolomics Reveals Elevated Macromolecular Degradation in Periodontal Disease Barnes, V., et. al | J Dent Res, 2012 Abstract Periodontitis is a chronic inflammatory disease characterized by tissue destruction. In the diseased oral environment, saliva has primarily been considered to act as a protectant by lubricating the tissue, mineralizing the bones, neutralizing the pH, and combating microbes. To understand the metabolic role that saliva plays in the diseased state, we performed untargeted metabolomic profiling of saliva from healthy and periodontitic individuals. Several classes of biochemicals, including dipeptide, amino acid, carbohydrate, lipids, and nucleotide metabolites, were altered, consistent with increased macromolecular degradation of proteins, triacylglycerol, glycerolphospholipids, polysaccharides, and polynucleotides in the individuals with periodontal disease. These changes partially reflected the enhanced host-bacterial interactions in the diseased state as supported by increased levels of bacterially modified amino acids and creatine metabolite. More importantly, the increased lipase, protease, and glycosidase activities associated with periodontitis generated a more favorable energy environment for oral bacteria, potentially exacerbating the disease state. BACK TO NEWSLETTER The Transcription Factor Myc Controls Metabolic Reprogramming upon T Lymphocyte Activation Wang, R. et. al. | Immunity, 2011 Abstract To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid β-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions. BACK TO NEWSLETTER Detrimental Effects of Adenosine Signaling in Sickle Cell Disease Zhang et al. | Nature Medicine, 2011 Metabolon results led to: • A novel therapeutic target • A novel biomarkers that also translate to humans • Mechanistic understanding of signaling events that induce sickle cell disease Key metabolomic observations: • Adenosine was markedly increased in the SCD mouse and human subjects suggesting disease involvement • Adenosine leads to the release of a specific red blood cell metabolite that lower oxygen affinity for hemoglobin, promoting sicklingand tissue damage. Synopsis Despite precise genetic understanding of sickle cell disease (SCD) and decades of study, therapies are limited. In a mouse model of SCD, metabolomics identified adenosine as a disease potentiator and led to the identification of the adenosine 2B receptor (A2BR) as a novel therapeutic target. Specifically, antagonism of the A2BR attenuated SCD. Metabolomic analysis revealed signaling events downstream of the A2BR that promote SCD (PKA, 2,3-DPG). These results are translatable since adenosine and 2,3-DPG were discovered to be increased in humans with SCD. Finally, the results have impact in current drugs targeting the inflammatory arm of SCD through agonismof a different adenosine receptor (the A2AR). Thus, given the dual nature of adenosine receptor activation in SCD, agonists to the A2AR may need to be exquisitely selective across the receptor sub-classes. BACK TO NEWSLETTER Reducing Endoplasmic Reticulum Stress Through a Macrophage Lipid Chaperone Alleviates Atherosclerosis Erbayet al. | Nature Medicine, 2009 Metabolon results led to: • Characterization of the mechanism of action • Validation of potential target Key metabolomic observations: • Characterization of the mechanisms underlying lipid chaperone activation to macrophage ER stress • Reveal the lipid chaperone aP2 and steroylCoAdesaturase-1 (SCD-1) as potential targets to combat macrophage ER stress and minimize atherosclerosis Synopsis Activation of the ER stress is characteristic of lipid-laden macrophages in atherosclerotic lesions and is proposed to have a role in plaque vulnerability and acute cardiac death. It has been proposed that lipid chaperones may be a link between toxic lipids and organelle stress in macrophages. Lipidomic analysis implicated regulation of de novo lipogenesis and desaturation, a rate-limiting step catalyzed by SCD-1, as a potential mechanism underlying chaperone driven changes in lipid composition in macrophage thus affecting lipid toxic potential. Further chaperone mediated regulation of SCD-1 activity was causally linked to lipid-induced ER stress responses in macrophage. Overall, this study shows that de novo fatty acid synthesis and desaturation can be highly beneficial if not essential for defending ER function when macrophages are exposed to toxic lipids and it highlights how examining lipids as a class was able to identify novel targets. BACK TO NEWSLETTER Metabolic Profiling in Nutrition and Metabolic Disorders LeMieux, M. et. al. | Advances in Nutrition, 2013 Abstract Nutrients exert potent effects on metabolism through a variety of regulatory mechanisms, resulting in local and systemic changes in metabolite levels. Numerous studies have focused on mechanisms by which nutrients and disease states regulate metabolism at the gene or protein levels using genomic and proteomic approaches, respectively. However, few studies have investigated nutritional regulation of the whole metabolome. Thus, metabolomic approaches have recently emerged to complement the genomics and proteomics research and to help identify biologically meaningful metabolites and metabolic networks that control cellular responses to genetic and environmental factors, including diet, and to identify metabolic diseases that are influenced by genetic and dietary factors. These large-scale studies expedite our ability to develop targeted treatments. The goal of this symposium was to provide a forum to introduce the metabolomics field to nutrition researchers. An overview of the state-of-the-art metabolomic technologies used was provided. The impact of some specific nutrients, disease states, or genetic variations and their interaction with the metabolome was discussed by the speakers. Our objectives were as follows: 1) to educate the audience about the use of metabolomics as an innovative tool for linking changes in cell metabolites and genetic variations to nutrient metabolism, energy balance, and the overlying effects on health and disease; 2) to understand the concept of metabolomics and describe the analytical tools and resources available in this area; 3) to introduce the potential application of metabolomics in the field of nutrition research; and 4) to provide specific nutrition-relevant metabolomics study examples in investigating regulation of the metabolic network or metabolic changes resulting from disease states by dietary factors. Adv. Nutr. 4: 548–550, 2013. BACK TO NEWSLETTER A Diet Rich in High-Glucoraphanin Broccoli Interacts with Genotype to Reduce Discordance in Plasma Metabolite Profiles by Modulating Mitochondrial Function Armah, C. et. al. | The American Journal of Clinical Nutrition, 2013 Abstract Background: Observational and experimental studies suggest that diets rich in cruciferous vegetables and glucosinolates may reduce the risk of cancer and cardiovascular disease (CVD). Objective: We tested the hypothesis that a 12-wk dietary intervention with high-glucoraphanin (HG) broccoli would modify biomarkers of CVD risk and plasma metabolite profiles to a greater extent than interventions with standard broccoli or peas. Design: Subjects were randomly assigned to consume 400 g standard broccoli, 400 g HG broccoli, or 400 g peas each week for 12 wk, with no other dietary restrictions. Biomarkers of CVD risk and 347 plasma metabolites were quantified before and after the intervention. Results: No significant differences in the effects of the diets on biomarkers of CVD risk were found. Multivariate analyses of plasma metabolites identified 2 discrete phenotypic responses to diet in individuals within the HG broccoli arm, differentiated by single nucleotide polymorphisms associated with the PAPOLG gene. Univariate analysis showed effects of sex (P , 0.001), PAPOLG genotype (P , 0.001), and PAPOLG genotype 3 diet (P , 0.001) on the plasma metabolic profile. In the HG broccoli arm, the consequence of the intervention was to reduce variation in lipid and amino acid metabolites, tricarboxylic acid (TCA) cycle intermediates, and acylcarnitines between the 2 PAPOLG genotypes. Conclusions: The metabolic changes observed with the HG broccoli diet are consistent with a rebalancing of anaplerotic and cataplerotic reactions and enhanced integration of fatty acid b-oxidation with TCA cycle activity. These modifications may contribute to the reduction in cancer risk associated with diets that are rich in cruciferous vegetables. This trial was registered at clinicaltrials.gov as NCT01114399. BACK TO NEWSLETTER Metabolomics Identifies PyrimidineStarvation as the Mechanism of 5-Aminoimidazole-4Carboxamide-1-β-Riboside-Induced Apoptosis in Multiple Myeloma Cells Bardelebenet al. | Molecular Cancer Therapeutics, 2013 Metabolon results led to: • Mechanism of action of a compound that induces apoptosis in multiple myeloma cells • A potential new target in multiple myeloma and a patient stratification strategy Key metabolomic observations: • Profound increase in the nucleotide precursor orotate Synopsis AICAr (5-aminoimidazole-4-carboxamide-1-b-riboside) has been studied as an antitumor agent with the suspected mechanism of action (MOA) through AMPK activation and mTOR inhibition. However, cytotoxic affects do not always correlate with this putative MOA. Thus, investigators used a metabolomics screen to elucidate the MOA and this screen revealed a >26 fold increase in orotate, a precursor of de novo synthesis of pyrimidines. An array of follow-up experimentation showed that inhibiting or rescuing steps governing pyrimidine synthesis were clearly at the epicenter of the MOA of AICAr. This finding can be coupled to the gene expression profiles of multiple myeloma (MM) patients that reveal clones with deficiencies in this pathway suggesting that MM may be particularly sensitive to pyrimidine starvation and thus, inhibitors to this pathway. Hence, a metabolomics screen revealed a new target area (novel from the putative target) that may be particularly promising against MM. BACK TO NEWSLETTER HuR is a Post-Transcriptional Regulator of Core Metabolic Enzymes in Pancreatic Cancer Burkhart, R. et. al. | RNA Biology, 2013 Abstract Cancer cell metabolism differs from normal cells, yet the regulatory mechanisms responsible for these differences are incompletely understood, particularly in response to acute changes in the tumor microenvironment. HuR, an RNA binding protein, acts under acute stress to regulate core signaling pathways in cancer through post-transcriptional regulation of mRNA targets. We demonstrate that HuR regulates the metabolic phenotype in pancreatic cancer cells and is critical for survival under acute glucose deprivation. Using three pancreatic cancer cell line models, HuR-proficient cells demonstrated superior survival under glucose deprivation when compared with isogenic cells with siRNA-silencing of HuR expression (HuR-deficient cells). We found that HuR-proficient cells utilized less glucose, but produced greater lactate, as compared with HuR-deficient cells. Acute glucose deprivation was found to act as a potent stimulus for HuR translocation from the nucleus to the cytoplasm, where HuR stabilizes its mRNA targets. We performed a gene expression array on ribonucleoproteinimmuno precipitated mRNAs bound to HuR and identified 11 novel HuR target transcripts that encode enzymes central to glucose metabolism. Three (GPI, PRPS2 and IDH1) were selected for validation studies, and confirmed as bona fide HuR targets. These findings establish HuR as a critical regulator of pancreatic cancer cell metabolism and survival under acute glucose deprivation. Further explorations into HuR’s role in cancer cell metabolism should uncover novel therapeutic targets that are critical for cancer cell survival in a metabolically compromised tumor microenvironment. BACK TO NEWSLETTER Exposure of Clinical MRSA Heterogeneous Strains to β-Lactams Redirects Metabolism to Optimize Energy Production through the TCA Cycle Keaton et. al. | PLoS One, 2013 Metabolon results led to: • Identification of key metabolic adaptation to resistance development • Potential targets to prevent the acquisition of resistance or resistant strains Key metabolomic observations: • An increase in TCA cycle intermediates • Changes in carbon sources that feed the TCA cycle (fatty acids, amino acids) Synopsis Methicillin-resistant Staphylococcus aureus (MRSA) is a severe threat in both hospital and community-acquired infections so there is a continuing need to expand our understanding of the cellular physiology that allow strains to adapt and survive in the presence of beta-lactam antibiotics. To this end, metabolomics and gene expression profiling were preformed on strains during resistance development. Collectively, the data indicated that an upregulation of TCA cycle activity was a key feature of adaptation (presumably for cell wall synthesis/ metabolism). The importance of this upregulation was confirmed when the fate of strain adaptation was shown to follow the presence or absence of the TCA cycle enzyme aconitase. These novel insights into the precise metabolic adaptations of MRSA provide potential new targets for the design of new drugs against MRSA infections. BACK TO NEWSLETTER Metabolite Profiling Reveals New Insights into the Regulation of Serum Urate in Humans Albrecht, E. et. al. | Metabolomics, 2013 Abstract Serum urate, the final breakdown product of purine metabolism, is causally involved in the pathogenesis of gout, and implicated in cardiovascular disease and type 2 diabetes. Serum urate levels highly differ between men and women; however the underlying biological processes in its regulation are still not completely understood and are assumed to result from a complex interplay between genetic, environmental and lifestyle factors. In order to describe the metabolic vicinity of serum urate, we analyzed 355 metabolites in 1,764 individuals of the population based KORA F4 study and constructed a metabolite network around serum urate using Gaussian Graphical Modeling in a hypothesis-free approach. We subsequently investigated the effect of sex and urate lowering medication on all 38 metabolites assigned to the network. Within the resulting network three main clusters could be detected around urate, including the well-known pathway of purine metabolism, as well as several dipeptides, a group of essential amino acids, and a group of steroids. Of the 38 assigned metabolites, 25 showed strong differences between sexes. Association with uricostatic medication intake was not only confined to purine metabolism but seen for seven metabolites within the network. Our findings highlight pathways that are important in the regulation of serum urate and suggest that dipeptides, amino acids, and steroid hormones are playing a role in its regulation. The findings might have an impact on the development of specific targets in the treatment and prevention of hyperuricemia. BACK TO NEWSLETTER Profile of Circulatory Metabolites in an Animal Model of Multiple Sclerosis Using Global Metabolomics Mangalam, A. et. al. | Journal of Clinical and Cellular Immunology, 2013 Abstract Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Recently, metabolic aberrations have been defined in various disease processes either as contributing to the disease, as potential biomarkers, or as therapeutic targets. Thus in an attempt to define the metabolic alterations that may be associated with MS disease progression, we profiled the plasma metabolites at the chronic phase of disease utilizing relapsing remitting experimental autoimmune encephalomyelitis (RR-EAE) model in SJL mice. At the chronic phase of the disease (day 45), untargeted global metabolomic profiling of plasma collected from EAE diseased SJL and healthy mice was performed, using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry. A total of 282 metabolites were identified, with significant changes observed in 44 metabolites (32 up-regulated and 12 down-regulated), that mapped to lipid, amino acid, nucleotide and xenobiotic metabolism and distinguished EAE from healthy group [p<0.05, false discovery rate (FDR)<0.23]. Mapping the differential metabolite signature to their respective biochemical pathways using the Kyoto Encyclopedia of Genes and Genomics (KEGG) database, we found six major pathways that were significantly altered (containing concerted alterations) or impacted (containing alteration in key junctions). These included bile acid biosynthesis, taurine metabolism, tryptophan and histidine metabolism, linoleic acid and D-arginine metabolism pathways. Overall, this study identified a 44 metabolite signature drawn from various metabolic pathways which correlated well with severity of the EAE disease, suggesting that these metabolic changes could be exploited as (1) biomarkers for EAE/MS progression and (2) to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment of MS. BACK TO NEWSLETTER Lipidomics Reveals Early Metabolic Changes in Subjects with Schizophrenia: Effects of Atypical Antipsychotics McEvoy, J. et. al. | PLoS One, 2013 Abstract There is a critical need for mapping early metabolic changes in schizophrenia to capture failures in regulation of biochemical pathways and networks. This information could provide valuable insights about disease mechanisms, trajectory of disease progression, and diagnostic biomarkers. We used a lipidomics platform to measure individual lipid species in 20 drug-naïve patients with a first episode of schizophrenia (FE group), 20 patients with chronic schizophrenia that had not adhered to prescribed medications (RE group), and 29 race-matched control subjects without schizophrenia. Lipid metabolic profiles were evaluated and compared between study groups and within groups before and after treatment with atypical antipsychotics, risperidone and aripiprazole. Finally, we mapped lipid profiles to n3 and n6 fatty acid synthesis pathways to elucidate which enzymes might be affected by disease and treatment. Compared to controls, the FE group showed significant down-regulation of several n3 polyunsaturated fatty acids (PUFAs), including 20:5n3, 22:5n3, and 22:6n3 within the phosphatidylcholine and phosphatidylethanolamine lipid classes. Differences between FE and controls were only observed in the n3 class PUFAs; no differences where noted in n6 class PUFAs. The RE group was not significantly different from controls, although some compositional differences within PUFAs were noted. Drug treatment was able to correct the aberrant PUFA levels noted in FE patients, but changes in re patients were not corrective. Treatment caused increases in both n3 and n6 class lipids. These results supported the hypothesis that phospholipid n3 fatty acid deficits are present early in the course of schizophrenia and tend not to persist throughout its course. These changes in lipid metabolism could indicate a metabolic vulnerability in patients with schizophrenia that occurs early in development of the disease. BACK TO NEWSLETTER 3-Hydroxykynurenine and Other Parkinson’s Disease Biomarkers Discovered by Metabolomic Analysis LeWitt, P. et. al. | Movement Disorders, 2013 Abstract Parkinson’s disease (PD) biomarkers are needed to enhance therapeutics research and to understand PD pathogenesis. Methods that simultaneously measure hundreds of small molecular-weight compounds–metabolomic analysis–“fingerprint” disease specific alterations in individual compounds or metabolic pathways. Beyond a nontargeted search for PD biomarkers, we hypothesized that PD cerebrospinal fluid would show increased formation of the excitotoxin 3-hydroxykynurenine and diminished concentration of the antioxidant glutathione. Cerebrospinal fluid was collected at <4 hours postmortem from 48 pathologically verified PD subjects and 57 comparably-aged controls. Assays involved ultra-high-performance liquid and gas chromatography linked to mass spectrometry. We used univariate techniques to determine fold-changes in concentrations of biochemicals; false-discovery rates were calculated to exclude spurious findings. Data was modeled using a Support Vector Machine for analyzing compounds selected by Welch’s t test. Classification accuracy was determined by cross-validation. Of 243 structurally-identified biochemicals,19 compounds differentiated PD from controls at a 20% false-discovery level. In PD, mean 3-hydroxykynurenine concentration was increased by one-third, and mean oxidized glutathione was decreased by 40% (for each, P<.01). Four of the 19 compounds differentiating PD from controls were Nacetylated amino acids, suggesting a generalized alteration in N-acetylation activity. The Support Vector Machine classification model distinguished between groups at 83% sensitivity and 91% specificity for the learning data, and at 65% and 79% from cross-validation. In this study, the first for metabolomic profiling of PD cerebrospinal fluid, we found several novel biomarkers and offer new directions for recognizing disease-specific biochemical indicators. The findings support involvement of excitotoxicity and oxidative stress in the pathogenesis of PD. BACK TO NEWSLETTER Metabolomics in Epidemiology: Sources of Variability in Metabolite Measurements and Implications Sampson. J et. al. | Cancer Epidemiology, Biomarkers & Prevention, 2013 Abstract Background: Metabolite levels within an individual vary over time. This withinindividual variability, coupled with technical variability, reduces the power for epidemiologic studies to detect associations with disease. Here, the authors assess the variability of a large subset of metabolites and evaluate the implications for epidemiologic studies. Methods: Using liquid chromatography/mass spectrometry (LC/MS) and gas chromatography-mass spectroscopy (GC/MS) platforms, 385 metabolites were measured in 60 women at baseline and year-one of the Shanghai Physical Activity Study, and observed patterns were confirmed in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening study. Results: Although the authors found high technical reliability (median intraclass correlation ¼ 0.8), reliability over time within an individual was low. Taken together, variability in the assay and variability within the individual accounted for the majority of variability for 64% of metabolites. Given this, a metabolite would need, on average, a relative risk of 3 (comparing upper and lower quartiles of “usual” levels) or 2 (comparing quartiles of observed levels) to be detected in 38%, 74%, and 97% of studies including 500, 1,000, and 5,000 individuals. Age, gender, and fasting status factors, which are often of less interest in epidemiologic studies, were associated with 30%, 67%, and 34% of metabolites, respectively, but the associations were weak and explained only a small proportion of the total metabolite variability. Conclusion: Metabolomics will require large, but feasible, sample sizes to detect the moderate effect sizes typical for epidemiologic studies. Impact: We offer guidelines for determining the sample sizes needed to conduct metabolomic studies in epidemiology. BACK TO NEWSLETTER