Session PJ. There are 37 abstracts in this session.

Session: Metabolomics, poster number: 269
Diagnostic Utility of Propionate to Identify Myocardial Dysfunction as Probed Using Hyperpolarized [1–13C] Pyruvate
Mukundan Ragavan; Matthew Merritt
University of Florida, Gainesville, FL
Modulation of pyruvate dehydrogenase (PDH) flux by propionate was studied in healthy and hypertrophied mouse hearts using hyperpolarized [1-13C] pyruvate. Perfusions were performed in Langendorff mode, with hearts suspended inside a 14.1 T NMR system equipped with a 10 mm 13C cryoprobe. Propionate increases PDH flux by 2 – 3 times in healthy hearts in perfusate containing fatty acids whereas in hypertrophied hearts, propionate has no effect. The presence of propionate did not perturb O2 consumption or mechanical function of the heart. Differential modulation of PDH flux using propionate paired with hyperpolarized metabolic imaging is a powerful tool for diagnosis of myocardial dysfunction leading to heart failure.

Session: Metabolomics, poster number: 270
Hyperpolarized [2–13C] Dihydroxyacetone as a Probe to Observe Metabolism in Perfused Livers
Mukundan Ragavan; Tatsiana Tsarova; Matthew Merritt
University of Florida, Gainesville, FL
Imaging of hepatic metabolism using MRI is advantageous for both diagnosis and monitoring treatment progression. Hyperpolarized (HP) 13C based MRI provides the ability to monitor metabolic changes in tissues in real time. Here, we report use of HP [2–13C] dihydroxyacetone to observer metabolism in perfused liver. We can detect low concentration metabolites like phosphoenolpyruvate with ease as well as determine hepatic gluconeogenesis from the 13C NMR spectra.

Session: Metabolomics, poster number: 271
Pyruvate is a Superior Substrate for Oxidation in Murine Muscle: A Carbon-13 Isotopomer Analysis
Ram Khattri; Jason Puglise; Elisabeth Barton; Matthew Merritt
University of Florida, Gainesville, 0
Muscle tissue can utilize either glucose or fatty acids as a fuel source, depending on workload and substrate availability. C57BL/10SnJ mouse hind limb muscles were studied using isotopically labeled tracers. Isotopomer analysis of 13C labeled glutamate was performed on extracts from isolated soleus and EDL muscles, using uniformly labeled 13C6 glucose and 13C3 pyruvate, with or without insulin. Glucose was able to label glutamate, but with high anaplerotic flux. Pyruvate oxidation was elevated compared to glucose, and anaplerotic flux was lower. Pyruvate also labeled alanine much more efficiently than the glucose. Deposition of label into alanine increases with insulin treatment. Pyruvate is more avidly consumed in the muscle than glucose, even under insulin treatment, with resultant increases in glutamate labeling.

Session: Metabolomics, poster number: 272
Suppression of Lipid Signals for Solution-state and HR-MAS 1H NMR-based Metabolomics in natural and in-vivo samples
Andre J Simpson1; Qusai Hassan1; Rudraksha Dutta Majumdar2; Bing Wu1; Daniel Lane1; Maryam Tabatabaei-Anraki1; Ronald Soong1; Myrna Simpson1
1University of Toronto, Toronto, Canada; 2Bruker Ltd, Milton, Canada
1H NMR spectra of whole natural samples often show strong contributions from lipids which overlap signals of interest from small metabolites. Here a technique is presented which exploits the relative difference in diffusivity and relaxation of lipids vs small metabolites to suppress the lipid signals, both in standard solution-state and HR-MAS NMR spectroscopy. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in-vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high lipid content and adds to the suite of experiments for in vivo environmental metabolomics.

Session: Metabolomics, poster number: 273
Non-polar Phytometabolites as Chemosensory Markers by NMR Metabolomics
Aruna Singh; Dushyant Kumar; Rama Jayasundar
Department of NMR, AIIMS, New Delhi, New Delhi, India
With the emerging concept of fat as the sixth taste, research on the chemosensory aspect of non-polar compounds like fatty acids and lipids is gaining importance. The present study has analyzed using NMR metabolomics, non-polar phytometabolites (extracted by dual phase chloroform/methanol-water separation) from forty eight medicinal plants (from four chemosensory groups) in context of their chemosensory property. While fatty acids and lipids were observed in all groups to varying levels, specific metabolites in each chemosensory group were also observed. Partial Least Square Discriminant Analysis of NMR spectral data showed distinct differentiation between the chemosensory groups indicating that NMR can play an important role in understanding the chemosensory nature of non-polar compounds.

Session: Metabolomics, poster number: 274
Isotopomer analysis for PNEC cell lines in normoxic and hypoxic conditions
Ram Khattri1; Zhepeng Wang2; Joseph Ippolito2; Matthew Merritt1
1University of Florida, Gainesville, 0; 2Washington University, St. Louis, MO
Intermediary metabolism was studied in PNEC cells (a model of small cell neuroendocrine prostate cancer) in hypoxic and anoxic conditions using a [U-13C3] alanine tracer. Isotopomer analysis of 13C labeled metabolites was executed on extracts of 5-10 million cells using an extremely sensitive, high temperature superconducting probe. Higher 13C incorporation into TCA cycle intermediates was observed in normoxic samples. Results suggest that unlike other aggressive cancer cell lines, PNEC cells under these conditions do not exhibit the typical Warburg phenotype.

Session: Metabolomics, poster number: 275
NMR spectroscopy and electron microscopy identification of metabolic and ultrastructural changes to the kidney following ischemia-reperfusion injury
Tafadzwa Chihanga1; Qing Ma2; Jenna Nicholson1; Hannah Ruby1; Richard Edelmann1; Prasad Devarajan2; Michael A. Kennedy1
1Miami University, Oxford,, Ohio; 2Cincinnati Children's Hospital Medical Center, Cincinnati, OH
A multidisciplinary study was used to identify nephron changes in a mouse model of ischemia reperfusion injury-induced acute kidney injury (IRI-AKI). Histology indicated distended Bowman’s glomerular spaces and proximal and distal tubules caused by increased filtrate volume due to reduced water reabsorption by damaged proximal tubule brush borders and blocked filtrate flow into collecting tubules by mucoprotein casts in distal tubules. Immunohistochemistry revealed protein AKI biomarkers in proximal tubules and glomeruli but not in distal tubules. NMR revealed several metabolites that increased, decreased, or were absent, in urine following IRI due to altered kidney function or metabolism, including increased urinary glucose due to reduced reabsorption by damaged proximal tubule brush borders. Scanning and transmission electron microscopy revealed ultrastructural changes to nephrons.

Session: Metabolomics, poster number: 276
Evaluation of 13C isotopic suppression to follow in vivo processes involving unlabelled molecules
Daniel Lane 1; Ronald Soong1; Wolfgang Bermel2; Maryam Tabatabaei Anaraki1; Bing Wu1; Hermann Heumann3; Sebastian Schmidt3; André J. Simpson1
1University of Toronto, Toronto, Canada; 2Bruker BioSpin GmbH, Rheinstetten, Germany; 3Silantes GmbH, München, Germany
Nuclear magnetic resonance (NMR) is a key tool to understand biological processes and response of organisms to their surroundings. In vivo NMR is especially powerful as it provides real-time metabolic information important for deconvoluting complex response pathways. Due to its low biomass, small organisms such as D.magna (water flea, ~ 40ug per organism) are raised on 13C enriched diets to increase NMR signal. Herein, a reverse isotopic filtering strategy was applied. Unlike conventional in vivo studies, which focus on tracking the fate of the 13C labelled organism solely, this newly adopted strategy suppresses 13C signal from the fully labelled 13C organism, thereby facilitating the ability to monitor changes in a 12C probe molecule (unlabelled contaminant or food) in real time.

Session: Metabolomics, poster number: 277
Evaluation of 1H-13C Double Tuned Planar Microcoils for 1H-13C 2D NMR Analysis of Tiny Biological Samples
Vincent Moxley-Paquette1; Bing Wu1; Daniel Lane1; Monica Bastawrous1; Paris Ning1; Ronald Soong1; Peter de Castro2; Ivan Kovacevic2; Thomas Frei2; Juerg Stuessi2; Franck Vincent2; Daniel Schmidig2; Armin Beck2; Werner Maas3; Michael Fey3; Andre J Simpson1
1University of Toronto, Toronto, Canada; 2Bruker Biospin AG (Switzerland), Fällanden, Switzerland; 3Bruker BioSpin, Billerica, MA
Most tiny aquatic organisms commonly used for toxicity testing have eggs below 1mm in diameter. One method to improve mass sensitivity is through decreasing the coil diameter. Single sided planar coils are of particular interest as they are not restricted to specific sample sizes. One notable drawback for single sided planar microcoils is that the B1 field is inhomogeneous, yet because of their versatility it is important to investigate if 2D 1H-13C NMR is feasible. Overall, the results of this study show that 1H-13C 2D NMR experiments can be performed on double tuned planar microcoils. The main presentation will compare the performance of the planar microcoil technology described here with more conventional coil designs for in vivo 2D NMR studies.

Session: Metabolomics, poster number: 278
Comprehensive Multiphase NMR on Larger Samples using a 7mm CMP Probehead
Paris Ning1; Daniel Lane1; Ronald Soong1; Daniel Schmidig2; Thomas Frei2; Peter De Castro2; Stephan Graf2; Werner Maas3; Jochem Struppe3; Michael Fey3; Henry J. Stronks4; Martine Monette4; Yalda Liaghati Mobarhan1; Amy Jenne1; Monica Bastawrous1; Myrna J. Simpson1; Andre J Simpson1
1University of Toronto, Toronto, Canada; 2Bruker BioSpin AG, Fällanden, Switzerland; 3Bruker BioSpin Corp., Billerica, MA; 4Bruker Ltd., Milton, Canada
Comprehensive multiphase (CMP) NMR is a useful experimental technique to study liquids/gels/solids in intact natural samples, however, studies thus far have been restricted to 4mm probeheads. A 7mm CMP probe was developed, featuring 3 times more sample volume compared to 4mm probes (5.7mm rotor inner diameter vs 3.2mm). Spectra with 13C-labelled algae showed a 2.3 times increase of signal-to-noise representing a time-saving factor of ~5. We demonstrate that 13C NMR of non-enriched chicken heart and 13C-1H HETCOR of whole freshwater shrimps become feasible in a reasonable amount of time with a 7mm probe but are not possible using 4mm probes. Further studies will demonstrate natural, non-labelled intact samples such as seedlings and living organisms that wouldn’t fit in 4mm rotors.

Session: Metabolomics, poster number: 279
Toward Invivo Metabolic Pathway Monitoring through Dual Receiver Acquisition Statistical Correlation (DRASTIC) Spectroscopy: Revealing Real-Time Temporal Correlation in Complex Mixtures
Ronald Soong1; Daniel Lane-Coplen1; Wolfgang Bermel2; Werner Maas3; Andre J Simpson1
1University of Toronto, Toronto, Canada; 2Bruker BioSpin, Rheinstetten, N/A; 3Bruker BioSpin, Billerica, MA
The ability to use multiple receivers to simultaneously acquire signals from two different nuclei, 1H and 13C, can open new avenues in heteronuclear correlation experiments. For instance, resonances that vary at the same rate in both 13C and 1H spectra are likely belong to the same molecule, allowing for the potential of mixture deconvolution through statistical correlation analysis such as STOCSY (Statistical Total Correlation Spectroscopy). In this present work, a combined DRA and STOCSY approach, namely DRASTIC (Dual Receiver Acquisition StatIstical Correlation) will be investigated and tested on various dynamic processes. Through this approach, correlations between the components in the mixtures can be clearly illustrated, potentially providing essential information on reactions dynamics and metabolic flux of interconnected biological pathways.

Session: Metabolomics, poster number: 280
Reducing impacts of individual organism variability in metabolomics via time trajectory in vivo NMR 
Maryam Tabatabaei Anaraki1; Myrna Simpson1, 2; Sebastian Schmidt3; Hermann Heumann3; Andre J. Simpson1, 2
1University of Toronto, Scarborough, Canada; 2University of Toronto, Toronto, Canada; 3Silantes GmbH, Gollierstr, München , Germany
Metabolomics is the quantitative and qualitative analysis of biological small molecules. Individual organism variability is a complication in metabolomics studies, especially when different populations of control vs exposed organisms are compared. This in-vivo study demonstrates that controls consisting of the same organisms prior to exposure overcome complications from individual organism variability and permit metabolic impacts of stress to be more easily differentiated.1 Here fully 13C labelled Daphnia magna are monitored using a continuous flow system built in-house.2 Trajectory analysis shows that when control and anoxic sets are the same organisms before and after exposure (rather than separate populations) the individual organisms variability is greatly reduced allowing the metabolic impacts from the stress process itself to be more clearly delineated.

Session: Metabolomics, poster number: 281
The potential of 2H-13C enrichment combined with in vivo low speed MAS NMR to emphasize metabolites in dynamic flux
Yalda Liaghati Mobarhan1; Ronald Soong1; Wolfgang Bermel2; Myrna J. Simpson1; Hermann Heumann3; Sebastian Schmidt3; Andre J Simpson1
1University of Toronto, Toronto, Canada; 2Bruker BioSpin, Rheinstetten, N/A; 3Silantes GmbH, München, N/A
The limitation of HRMAS for in vivo applications is the stress induced by spinning. With 1H detection, slow spinning introduces side bands that obscure the isotropic information, while fast spinning leads to stress and eventual death of the organisms. Here, a new approach using 2H detection is introduced on 2H and 13C enriched organisms. While 2H possess a significant quadrupole coupling, for metabolites under isotopic condition, their quadrupole couplings are often averaged out to zero. Larger structural components exhibit wide 2H profiles, not detected via 2D 2H-13C experiments. As such, we find 2D 2H-13C NMR provides a selective window emphasizing the dynamic fully dissolved metabolites (i.e those most likely in flux) while suppressing the structural framework of the living organism.

Session: Metabolomics, poster number: 282
Tracking Galactose Metabolism in HepG2 Cells using Sensitive 1H-NMR with 13C6-Galactose
Qiuwei Xu
Merck Research Laboratories, West Point, PA

Galactose metabolism by the Leloir pathway explains the conversion of galactose to glucose for normal cellular metabolisms. Rapidly dividing cancer cell HepG2 is often used in the presence of either glucose or galactose to assess mitochondrial toxicity in vitro for pharmaceutical drug candidates. When mitochondria in HepG2 cells are inhibited, ATP IC50 shifts significantly from media containing glucose to galactose (so called glc-gal shift). We utilized the sensitive 1H-NMR with 13C-labeled galactose to understand its mechanism in HepG2 cells. In this poster, we will present our experimental results of 13C6-labeled galactose metabolism in HepG2 cells; and discuss whether and how galactose plays any role in sensitizing HepG2 cells for detecting mitochondrial toxicity.

Session: Metabolomics, poster number: 283
New NMR tools for Isotopomics at 13C Natural Abundance
Tangi Jezequel1; Virginie SILVESTRE1; Katy DINIS1; Patrick GIRAUDEAU1, 2; Serge AKOKA1
1CEISAM, UMR 6230, Université de nantes, Nantes, France; 2Institut Universitaire de France, Paris, FRANCE
Position-Specific Isotope Analysis is a powerful tool to track the origin of biological or synthetic molecules. Up to now, the conventional method requires the combination of isotopic ratio mass spectrometry with quantitative 13C NMR to accurately measure position-specific isotopic variations around their natural abundance value. However this analytical method suffers from several drawbacks and is limited to pure and concentrated molecules. To improve the generality of 13C isotopic NMR, we developed spatially-encoded experiments for highly accurate quantitative 13C-NMR using an internal reference method. The analysis of complex mixtures should now become possible regardless of potential peak overlap.

Session: Metabolomics, poster number: 284
NMR Metabolomics Analysis of Stem Cell Culture Media:  Human Umbilical Cord Stem Cell Fate, Differentiation and Metabolic Pathways.
S. Raghothama1; Veena Hegde1; H. Purushothama2; Jyothsna Rao2; Gururaj Rao2
1Indian Institute of Science, Bengaluru, India; 2Sri Raghavendra Biotechnologies Stem Cell Research, Bengaluru, India
Mesenchymal stem cells derived from human umbilical cord (HUCS) were grown in culture media and differentiation done at different stages of passages. NMR spectra were recorded at regular passage intervals on the spent media to monitor the metabolomic changes that occurs during various stages of cell regulation, multiplication and differentiation. It revealed novel metabolic profile and distinctive biomarkers. About 14 metabolites were monitored and quantified at different stages of the cell growth and differentiation. They divulged specific stem cell growth metabolic pathway and cell potency. Metabolic profile of HUCS cell differentiation to Adipocytes, Chondrocytes and Osteocytes and comparing with Hela cells conferred distinct metabolic fingerprint of all the cell type.

Session: Metabolomics, poster number: 285
Targeting the Formation of 13C-12C Bonds In-Vivo: A Unique Approach to Probe Complex Processes
Amy Jenne1; Ronald Soong1; Wolfgang Bermel2; Andre J Simpson1
1University of Toronto, Scarborough, Canada; 2Bruker BioSpin, Rheinstetten, N/A
NMR is unique in its ability to provide a wide range of metabolic information in-vivo, especially when combined with isotopic labelling. However, 2D 1H-13C in-vivo datasets are complex, making it challenging to monitor specific processes of interest. Here, a new experiment is introduced that targets the formation of 12C-13C bonds in-vivo. As 12C cannot be directly detected in NMR it must be observed via its attached protons. This approach can easily be extended to follow the fate of drugs, contaminants, or target nutrients in-vivo. The main presentation will demonstrate the approach on a range of standards and simple transformations before moving onto in-vivo processes.

Session: Metabolomics, poster number: 286
Improvement of Pre-clinical Coil Sensitivity at 7T using an Inductively Coupled Secondary Resonator
Bu Sik Park1; Joe Murphy-Boesch2; Ge Ma1; Brent McCright1
1FDA, Silver Spring, MD; 2NIH, Bethesda, MD
We present numerical simulations and experimental validation data testing the feasibility to improve 19F image sensitivity of perfluorocarbon labeled cells using the secondary resonator tuned at 287 MHz to make an enhancing induced RF magnetic field (B1) at 7.0T 19F/1H MRI. The numerical simulation results of |B1+| and corresponding experimental 19F images without and with the secondary resonator tuned at 287 MHz show the improvement of |B1+| and 19F image uniformity. To model a potential clinical application, we used inductive coupling MR to image 19F perfluorocarbon labeled cells encapsulated in polyethylene glycol (PEG) after their transplantation into mice.

Session: Metabolomics, poster number: 287
Nitrogen-Based Metabolomics for functional analysis of urease in Staphylococcus aureus
Fatema Bhinderwala1, 2; Samantha Lonergan1; Chunyi Zhou4; Jade Woods1; Paul Fey4; Robert Powers3
1University of Nebraska Lincoln, Lincoln, Nebraska; 2Nebraska Center for Integrated Biomolecular Commun, Lincoln, Nebraska; 3University of Nebraska-Lincoln, Lincoln, NE; 4Department of Pathology and Microbiology, Univers, Omaha, NE
We present a protocol for 15N-labeled metabolite tracer experiments that may also be combined with routine 13C-tracer experiments to simultaneously detect both 15N- and 13C-labeled metabolites in metabolic samples. A database consisting of 2D 1H-15N HSQC natural abundance spectra of 54 nitrogen-containing metabolites are also presented to facilitate the assignment of 15N-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes with 15N1-ammonium chloride and 15N4-arginine. Using this methodology, we evaluate the role of urease mutants in Staphylococcus aureus and its role in acid-induced cell death via ammonia generation and pH homeostasis.

Session: Metabolomics, poster number: 288
Extractive Ratio Analysis NMR Spectroscopy for Improved Unknown Metabolite Identification in Crowded Spectra
G. A. Nagana Gowda1; Liladhar Paudel1; Daniel Raftery1, 2
1University of Washington, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Identification of unknowns based on peaks in crowded spectra represents a significant bottleneck in metabolomics. NMR spectra of many biological systems are highly complex due to signal overlap from different classes of compounds. Here we show that the combination of selective extraction of a compound class followed by ratio analysis by NMR spectroscopy (RANSY) offers a promising approach for improved compound identification. In RANSY, spectra of individual metabolites are obtained by dividing peak ratios by their standard deviations over a set of NMR spectra. Here, we manipulate the extraction conditions to limit the compounds extracted from a single urine sample, which give rise to NMR spectra with exploitable varying peak intensities that are then suitable for RANSY analysis.

Session: Metabolomics, poster number: 289
Application of F1-PSYCHE-TOCSY for identification of metabolites in Coriander sativum L. Seeds Extract  
Ajay Verma1; Rashmi Parihar1; Subrato Bhattacharya2; Bikash Baishya1
1CBMR, Lucknow, India; 2Banaras Hindu University, Banaras, U.P.
Application of one of the most sensitive pure shift method, F1-PSYCHE-TOCSY1¬¬, for the easy identification of metabolites in crowded regions of 1H NMR spectrum from Coriander2 (Coriander sativum L.) seed extract is presented here. For aqueous extraction standard protocol3 is followed.
In Figure 1A, regions marked with rectangle b shows severe crowding of metabolites signal due to multiplet pattern which hampers the m¬¬¬etabolites identification. In Figure 1B, these signals are well resolved due to the ultrahigh resolution along F1 and it can be seen that two signals, one from β-fructofuranose and another from α-fructopyranose are present. Other regions are also compared and investigated where 2D F1-PSYCHE-TOCSY allows better identification of metabolites in crowded region.

Session: Metabolomics, poster number: 290

NMR spectroscopy of living human (tumor) stem cells under hypoxic and normoxic conditions.

Hans Robert Kalbitzer; Christian Dobner; Werner Kremer; Peter Hau; Ulrich Bogdahn
University of Regensburg, Regensburg, Germany

A meaningful study of living mammalian cells by NMR spectroscopy requires to provide nutrients and growth factors and to remove metabolic waste. Most important, O2, CO2, and N2 partial pressures have to set to physiological values. We have devised a perfusion system where all these external parameters can be controlled during the NMR experiments. Oxygen consumption and metabolic answers can be quantified precisely. Under optimal condition the survival time of the cells in the NMR spectrometer is significantly extended (up to 72 h). We have studied the metabolic answer including intracellular lipids and proteins in glioblastoma cancer stem cells and neural stem cells under different external conditions as well as after application of drug compounds.


Session: Metabolomics, poster number: 291
Amplification in 13C Hyperpolarization by the Addition of Spin=21/2 Gd-based Metallofullerene to Trityl OX063 at 7 Tesla
Alisa Leavesley1; Xiaoling Wang2; Tinghui Li3; Bimala Lama4; Zhehong Gan2; Joanna R. Long4; Harry Dorn3; Song-I Han1
1University of California Santa Barbara, Santa Barbara, CA; 2NHMFL, Tallahassee, FL; 3Virginia Tech, Blacksburg, VA; 4University of Florida, Gainesville, FL
We report here the application of a high spin S = 21/2 compound – a gadolinium-based endohedral metallofullerene (EMF) Gd3N@C80 for dissolution DNP at 7 Tesla. It enhanced the 13C nuclear hyperpolarization from that achieved under d-DNP conditions significantly, by adding its water soluble derivative Gd3N@C80[DiPEG2000(OH)45] to trityl OX063. The phenomenal gain in 13C DNP enhancement is attributed to the efficient cross-effect (CE) DNP mechanism that results from the strong coupling between trityl and Gd3N@C80[DiPEG2000(OH)45] electron spins. The 13C DNP frequency profile, its power dependence, EPR spectra, pump-probe ELDOR spectra, and electron and nuclear relaxation rates at various concentrations of Gd3N@C80[DiPEG2000(OH)45] will be presented to dissect the mechanism for the notable increase.

Session: Metabolomics, poster number: 292
Sensitive, High-Resolution 1D and 2D NMR for 13C Metabolomics Studies of Small-Abundance Mouse Tissue
Jinny Sun1; Renuka Sriram2; Robert Bok2; Romelyn Delos Santos2; Justin Delos Santos2; Mark Van Criekinge2; Daniel Vigneron2; John Kurhanewicz2
1UC Berkeley–UCSF Bioengineering Graduate Program, University of California, San Francisco, CA; 2Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA

This study demonstrates significant increases in flux through aerobic glycolysis and oxidative phosphorylation with development of therapeutic resistance to androgen deprivation therapy using a transgenic murine model. Fractional enrichment and relative 13C isotopomers areas of small-abundance tissue extracts were quantified using high-resolution 1D and 2D NMR. Based on these metabolic differences between androgen-sensitive and insensitive prostate cancer, a combination of hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate can be used to noninvasively predict therapeutic resistance in future patient studies using HP 13C MRI.

Session: Metabolomics, poster number: 293

NMR Quantification of Glucose Consumption and Lactate Efflux in Living Human Prostate Biopsies

Jeremy Bancroft Brown1; Subramaniam Sukumar1; Mark VanCriekinge1; Romelyn Delos Santos1; Rosalie Nolley2; Jinny Sun1; Justin Delos Santos1; Laura Tabatabai1; Katsuto Shinohara1; Hao Nguyen1; Donna Peehl1; Renuka Sriram1; John Kurhanewicz1
1University of California, San Francisco, San Francisco, CA; 2Stanford University, Palo Alto, CA

To our knowledge, glucose consumption and lactate efflux have not been previously quantified in living human prostate biopsies. In this abstract, we present proof-of-concept of this NMR-based technique, which in the future will enable metabolic measurements in biopsy samples of treatment-resistant prostate cancer to better understand the pathobiology of the disease and develop novel hyperpolarized 13C-labeled MRI agents. In n=2 benign and n=2 malignant biopsy samples, we observed a trend towards higher lactate efflux and fractional enrichment coming from 1,6-13C2 glucose in the malignant samples. Future work will focus on combining these measurements with oxygen consumption measurements, quantification of intracellular metabolites such as glutamate, and use of the technique in biopsies of treatment-resistant prostate cancer such as CRPC and NEPC.

Session: Metabolomics, poster number: 294
The Development of a uL Volume Flow System for the Analysis of Daphnia Eggs via In Vivo NMR Spectroscopy
Monica Bastawrous1; Daniel Lane1; Ronald Soong1; Maryam Tabatabaei Anaraki1; Wolfgang Bermel2, 3; Werner E. Maas3; Michael Fey3; Daniel Schmidig4; Hermann Heumann5; Sebastian Schmidt5; Andre J. Simpson1
1University of Toronto, Scarborough, Canada; 2Bruker BioSpin GmbH, Rheinstetten, Germany; 3Bruker BioSpin Corp., Billerica, MA; 4Bruker BioSpin AG., Fällanden, Switzerland; 5Silantes GmbH, München , Germany
Recent work argues dormant egg banks are the most critical for long-term population survival and community dynamics in aquatic environments. The size of these eggs (often ~200 um in diameter) makes analysis using traditional 5mm NMR probes challenging. Here a novel uL volume flow system is created to fit inside a 1mm NMR micro-coil probe. With this flow system, the eggs can be studied with minimal oxidative stress as oxygenated water flows continuously throughout the system. High quality metabolic fingerprints can be extracted from 2D 1H-13C and pseudo 3D NMR of 13C enriched eggs. The main presentation will show applications of the micro-flow system on tiny living organisms and eggs and demonstrate the potential to detect and explain environmental stress.

Session: Metabolomics, poster number: 295
Prebiotic effects of white button mushroom feeding on succinate and intestinal gluconeogenesis
Yuan Tian; Robert Nichols; Pratiti Roy; Wei Gui; Jingtao Zhang; Veronika Weaver; Jingwei Cai; Andrew Patterson; Margherita Cantorna
Dr, State College, PA
White button mushrooms have been reported to have health benefits, through poorly described mechanisms. WB feeding (1%) resulted in changes to the composition of microbiota in conventional mice to expand a population of Bacteroidetes subtype Prevotella that produce propionate and succinate. Microbial propionate and succinate production induced expression of genes important for intestinal gluconeogenesis (IGN) via the gut-brain neural circuit. Reduced hepatic glucose production was a metabolic benefit of IGN that was found in WB fed CV mice. In absence of microbiota or in mice with disruptions in the ability to sense microbiota there was no WB mediated effect. Daily consumption of WB mushrooms is a simple way to improve the microbiota, metabolic fitness and prevent the development of diabetes.

Session: Metabolomics, poster number: 296
Minimum-Rank (MR) Criterion for NMR Solvent Signal Removal
Yu Yang1; Hongwei Yao2; Jiyang Dong1; Xiaobo Qu1; Zhong Chen1
1Department of Electronic Science, Xiamen Universit, Xiamen, China; 2Department of Chemistry, Xiamen University, Xiamen, China
For many NMR applications, solvent signal is required to be removed from the spectrum, while the removal should cause at least as possible influence on other peaks in the spectrum. With a sample of solvent signal obtained from a separated experiment, the removal objective is equal to the determination of the intensity and phase shift of the solvent signal within the solution signal. To estimate the removal effect, we proposed a minimum rank criterion, i.e. the removal of the signal should make the Hankel matrix of the residual data to have the minimized rank. Through an optimization of the minimum rank objective, the clean signal removal could be attained. The results showed the efficacy of the proposed minimum rank method.

Session: Metabolomics, poster number: 297
CMOS transceivers for compact architectures and nL to sub-nL high performance NMR micro-sensors.
Marco Grisi; Giovanni Boero
EPFL, Lausanne, Switzerland
In this poster we focus on the advantages of CMOS ultra-compact architectures with respect to alternatives by comparing performance and geometrical features, indicating the path towards high-sensitivity CMOS-based micro-sensors that will enable NMR on samples that are currently out of reach, such as large unicellular microorganisms, micro-tissues derived from stem-cells, and even mammalian embryos (humans included).

Session: Metabolomics, poster number: 298

NMR analysis of 13C carboxylate isotopomers of metabolites using 15N-Cholamine chemoselection

Penghui Lin1; Sara Vicente-Munoz2; Teresa Fan1; Andrew Lane1
1University of Kentucky, Lexington, KY; 2Cincinnati Children's Hospital Medical Center, Cincinnati, OH

Isotopomer analysis of carboxyl-containing metabolites by NMR is hampered by the low sensitivity of the 13C nucleus and the relatively low chemical shift dispersion of carboxylates. 15N-Cholamine has been shown to be a useful chemoselective derivatizing agent for carboxylates, producing a peptide bond that can be detected via the HN with high sensitivity in 1H{15N}HSQC experiments. Here we report an improved method of 15N-Cholamine derivatization and show how the 13C-15N coupling patterns in the high resolution 1H{15N} HSQC spectrum can be used to identify and quantify the 13C isotopomer distributions of the carboxylate and adjacent carbon in metabolite mixtures.  

Session: Metabolomics, poster number: 299
A non-invasive longitudinal study of phosphorous metabolites in young mdx mice
Christopher Lopez; Kimberly Guice; Harneet Arora; Abhinandan Batra; Glenn Walter; Sean Forbes
university of florida, Gainesville, UNITED STATES
Introduction - Duchenne muscular dystrophy (DMD) is a fatal X linked recessive disease. The purpose of this study was to determine whether 31P-MRS could detect differences in dystrophic and wild-type mice.
Methods - Dystrophic mice were examined with 31P-MRS for relative concentrations of phosphorous containing metabolites in the lower mouse hind limb.
Results - PCr/ATP was lower (p<0.05) at 4 weeks of age in dystrophic mice compared to wild-type and there was relatively greater magnitude of difference between dystrophic and wild-type mice at this age.
Conclusion - Significant differences in 31P metabolites were observed between dystrophic and wild-type mice at a young age, consistent with early inflammatory development dystrophic mice.

Session: Metabolomics, poster number: 300
High Fidelity Sampling Schedules for NMR Spectra of High Dynamic Range.

Sven G. Hyberts; Gerhard Wagner
Harvard Medical School, Boston, MA
Some sampling schedules yield better results than others. Finding a useful schedule is easy for a low dynamic range spectrum and a conservative sparsity. Not so when the dynamic range is large and/or when extreme sparsity is desired. In order to evaluate the goodness of a sampling schedule, we create a Signal-to-Artifact Ratio metric, (SAR), based on the resulting hmsIST reconstructed spectrum of a simulated Lorentzian signal located either on or off the Nyquist grid. We find that this ratio (SAR) may range between 10:1 to 10,000,000:1 depending on the sampling strategy, seed value selection, length of the resulting recreated FID and the sparsity. Finally, we verify that the SAR measure is predictable of the quality of the resulting reconstruction.

Session: Metabolomics, poster number: 301
Development of New NIST Quality Control Reference Materials for Metabolomics Harmonization
Tracey Schock1; Christina Jones2; Clay Davis1; Yamil Simon-Manso2; John Bowden1; David Sheen2; Blaza Toman2; Benjamin Neely1; Lisa Kilpatrick2; Deb Ellisor1; Katrice Lippa2
1National Institute of Standards and Technology, Charleston, SC; 2National Institute of Standards and Technology, Gaithersburg , MD

The inherent qualities of nuclear magnetic resonance (NMR) lend to the success of the analytical instrumentation in metabolomics research, both in discovery and targeted workflows. As metabolomics continues to progress, it has become vital to engage the metabolomics community in measurement harmonization efforts, encouraging repeatable, reproducible, and comparable measurements to impart confidence in distinguishing phenotypic markers. NIST is developing both human biofluid (plasma) and tissue-based (liver) quality control materials that suit multiple omics measurements and platforms, including metabolomic (NMR and MS), lipidomic and proteomic measurements. These materials will include highly confident molecular identifications and will serve as a tool for intra- and inter-laboratory quality control, characterization of new technology and protocols, as well as, a training tool for analyst proficiency.

Session: Metabolomics, poster number: 302
Hyperpolarization of Nanodiamond with X-Band DNP Polarizer
Jeong Hyun Shim; Sangwon Oh; Kiwoong Kim
KRISS, Daejeon, South Korea
Hyperpolarized nanodiamonds (ND) are promising imaging agent, having surface functionality, decent hyperpolarization efficiency, and relatively long T1 relaxation time. In this work, hyperpolarization study of ND was conducted at 0.3 T and 3.3 K with X-band DNP polarizer. Polarization time was measured to be surprisingly short, around 20 s. DNP spectrum exhibited two peaks separated by around 0.4 mT, corresponding to 11 MHz, indicating that solid effect mechanism is still dominant at 0.3 T region. The dependence of microwave power was also measured for optimizing DNP condition. Finally, hyperpolarized ND sample was transferred to 6 T superconducting magnet remotely located. 13C NMR of the hyperpolarized ND revealed 40-fold enhancement compared to thermal polarization at 6 T and 300 K.

Session: Hyperpolarization, poster number: 303
In vitro and In cell kinetic studies by Dissolution Dynamic Nuclear Polarization
David Guarin1; Sadet Aude1; Emanuelle Webber1; Emeric Miclet3; Delphine Ravault3; Sagan Sandrine3; Geoffrey Bodenhausen1; Dennis Kurzbach2; Daniel Abergel1
1ENS, Departement de Chimie, Paris, France; 2ENS Paris, Paris, N/A; 3UPMC, Labooratoir des Biomolecules, Paris, 75
The study of enzyme kinetics, and of cell metabolism, is central for the understanding of cellular function and metabolic diseases. In this project we will use dissolution-DNP techniques to perform a detailed time-domain study of the oxidative stage of the Pentose Phosphate Pathway. We will investigate in vitro and in cell suspension the metabolism of different cells, such as the Trypanosoma Brucei.

Session: Metabolomics, poster number: 304
Use of LC/MS-microcoil NMR in characterizing limited quantities of unknown synthetic cannabinoids metabolites
Chandrashekhar Honrao1; Xiaoyu Ma1; Shashank Kulkarni1; JodiAnne Wood1; Alexander Zvonok2; MIchael Malamas1; Jason Guo3; Alexandros Makriyannis1
1Center for Drug Discovery, Northeastern University, Boston, MA; 2MAK Scientific LLC, Boston, MA; 3Department of Chemistry, Northeastern University, Boston, MA
LC/MS combined with NMR is often applied for unambiguous structural characterization of unknown drug metabolites, which can contribute to efficacy and toxicity of lead candidates in drug discovery. However, limited quantities of drug metabolites are available at drug discovery stage & often not sufficient for NMR studies. We have optimized a workflow involving LC/MS and microcoil NMR for characterizing relatively low quantities of drug metabolites (nanograms to micrograms), where purified metabolites were dissolved in 3-5 uL of deuterated solvents and delivered to the microcoil NMR flow cell using segmented flow technique. We have applied this workflow for characterizing metabolites of two novel synthetic cannabinoids, MAK1616 & AM1710, which have been indicated for the treatment of pain and inflammation.

Session: Metabolomics, poster number: 305
Advanced Methods for Enhancing NMR-based Metabolomics
István Timári; Cheng Wang; Dawei Li; Alexandar L Hansen; Lei Bruschweiler-Li; Rafael Bruschweiler
Ohio State University, Columbus, OH
Over the last few years, important developments have emerged both instrumentally and methodologically for improving sensitivity, resolution and spectral simplicity in NMR spectroscopy. However, the comprehensive analysis of complex mixtures without prior chromatographic separation, as is typically the case in metabolomics, is not without challenges. Therefore, there is a clear demand for advanced approaches that can provide maximal information in the shortest possible time frame from sample preparation through data collection and analysis. To further boost the power of NMR-based metabolomics, we present recent NMR and computational tools, including pure shift experiments, non-uniform absolute minimal sampling (AMS) and the maximal clique approach for the analysis of complex metabolite mixtures.