Code: PP	Time Slot/Poster Number: 300	Session: Screening & "omics", Poster
Quantitative Analysis of Blood Plasma Metabolites Using Isotope Enhanced NMR Methods
Nagana Gowda1; Fariba Tayyari1; Tao Ye1; Yuliana Suryani1; Siwei Wei1; Narasimhamurthy Shanaiah2; Daniel Raftery1
1Purdue University, West Lafayette, IN; 2Matrix Bio, Inc, West Lafayette, IN 47906
Abstract
We have recently shown that 15N and 13C isotope tagging and heteronuclear 2D experiments provide a convenient approach for obtaining accurate metabolites concentrations in plasma. Carboxylic acids and amides represent a majority of the metabolites in body fluids and their analysis utilizing isotope labeling significantly enhances the metabolic pool for biomarker discovery applications. Here, we demonstrate application of such approaches to accurately analyze human plasma metabolites. The methods were first tested on a mixture of synthetic compounds and extended to analyze metabolites commonly found in human blood plasma. Combination of improved sensitivity and resolution, and significantly less time required compared to conventional NMR methods at natural abundance is attractive for the quantitative metabolic profiling of complex biological samples, routinely.

Code: PP	Time Slot/Poster Number: 301	Session: Screening & "omics", Poster
Optimization of Mammalian Cell Growth and Protein Expression by NMR Quantification of Metabolites Using Quantum Mechanic Total Line Shape Analysis
Nelly Aranibar1; Michael Borys1; Bernhard Schilling1; Matthias Niemitz2; Nancy Mackin1; Zheng Jian Li1; Michael Reily1; Adrienne Tymiak1; Reb Russell1
1Bristol-Myers Squibb, Princeton, NJ; 2PERCH Solutions, Kuopio, Finland
Abstract
NMR-based metabolomics analysis was performed on a recombinant mammalian cell line expressing a therapeutic protein. The cells were grown under different conditions, at different scales, and sampled at multiple time points. Cell count, viability, titer of the target protein, and other parameters were also measured throughout the culture period. A quantum mechanical total line shape analysis (QMTLS) algorithm was used to quantify 30 metabolites such as amino acids, Krebs cycle metabolites, activated sugars, cofactors, and others. The trajectories of the metabolites were utilized to investigate changes in metabolic pathways of nutrient utilization and differences on the apoptotic response. The results shed light into culture parameters that can be manipulated to optimize cell growth and protein production.

Code: PP	Time Slot/Poster Number: 302	Session: Screening & "omics", Poster
Statistical Recoupling of Variables for the Identification of Candidate Biomarkers and Perturbed Metabolic Networks: Application to Cancer.
Benjamin Blaise1; Cécile Vercherat2; Vincent Navratil1; Annie Lacheretz-Bernigaud2; Laetitia Shintu1; Zeinab Hamze2; Céline Domange1; Marc-Emmanuel Dumas1; Martial Piotto3; Jean-Yves Scoazec2; Martine Cordier-Bussat2; Bénédicte Elena1; Pierre Toulhoat1; Lyndon Emsley1
1Université de Lyon - CRMN, Villeurbanne, France; 2Université de Lyon - INSERM U865, Lyon, France; 3Bruker, Wissembourg, France
Abstract
We have developed an automated variable-sized bucketing procedure (Statistical Recoupling of Variables) based on statistical relationships between neighboring variables, to recouple into clusters spectral points belonging to metabolic NMR signals. We show how SRV extracts individual biomarkers from metabolic phenotypes. We then show that, associated with Statistical Total Correlation Spectroscopy, it sums up metabolic data in a clear 2D pseudo-spectrum displaying spin and metabolic correlations, identified as metabolic connectivities and consequently perturbed metabolic networks. Its use in a study discriminating cell pellets over-expressing wild type or mutant forms of menin, responsible for the MEN1 cancer syndrome, opens up perspectives for a better understanding of the tumorogenesis, the identification of pathogenic mutants and discovery of potential therapeutic targets.

Code: PP	Time Slot/Poster Number: 303	Session: Screening & "omics", Poster
Taking a Semi-targeted Approach to Plant Metabolomics: NMR-guided Fractionation of Complex Mixtures by Solid-phase Extraction and Reversed-phase Liquid Chromatography
Kayla A Kaiser1; John F. K. Limtiaco1; Szabolcs Bení1, 2; Cynthia K. Larive1; Julia Bailey-Serres1
1UC Riverside, Riverside, CA; 2Semmelweis University, Budapest, Hungary
Abstract
NMR was employed to optimize the conditions for solid-phase extraction (SPE) as pre-treatment both to remove interfering lipids and enrich for secondary metabolites relative to the abundant primary metabolites which are more easily observed in crude extracts of various plant tissues. Persistent spectral overlap, even in SPE pre-treated extracts, was addressed by employing both on- and off-line reversed-phase liquid chromatographic separations prior to NMR detection. 2D NMR and tandem MS experiments were carried out to characterize individual and structurally-related classes of coeluting compounds enriched by SPE pre-treatment. The non-selective nature of NMR served to facilitate the development of this semi-targeted sample preparation method. Secondary metabolites important in the hypoxic stress response of the model plant Arabidopsis thaliana were thus identified.

Code: PP	Time Slot/Poster Number: 304	Session: Screening & "omics", Poster
NMR Discrimination of Ginseng Landraces
Joshua Hicks1; Kristina McIntyre2; John Arnason2; Kim Colson1
1Bruker-BioSpin, Billerica, MA; 2University of Ottawa, Ottawa, Canada
Abstract
Raw material from plants vary widely due to agricultural, harvesting, or processing methods. Nuclear Magnetic Resonance (NMR) provides a powerful non-destructive technique for analyzing both pure compounds and mixtures. Efforts are underway to establish a non-targeted quality control screen for plant extracts used as dietary supplements. Such a screen will improve the safety and efficacy to the consumer. Here screening of ginseng extracts is used as an example of discriminating geographically separate and naturally divergent landraces.

Code: PP	Time Slot/Poster Number: 305	Session: Screening & "omics", Poster
NMR-based metabonomics analysis of mouse urine and fecal extracts following oral treatment with the broad-spectrum antibiotic enrofloxacin (Baytril)
Lindsey Romick-Rosendale; Aaron Goodpaster; Michael Kennedy
Miami University, Oxford, OH
Abstract
The human gastrointestinal tract is home to hundreds of bacterial species and the balance between beneficial and pathogenic bacteria is critical to human health and disease. Here, we conducted an 850 MHz NMR study to monitor changes in urine and fecal metabolic profiles of 15 mice following gut sterilization. Ten metabolites changed in urine including decreased acetate due to loss of microbial catabolism of sugars and polysaccharides, decreased trimethylamine-N-oxide due to loss of microbial catabolism of choline, and increased creatine and creatinine due to loss of microbial enzyme degradation. Eight metabolites changed in fecal extracts including depletion of amino acids produced by microbial proteases, reduction in metabolites generated by lactate-utilizing bacteria, and increased urea caused by loss of microbial ureases.

Code: PP	Time Slot/Poster Number: 306	Session: Screening & "omics", Poster
Statistical Significance Analysis of NMR-Based Metabonomics Data
Aaron M. Goodpaster; Lindsey E. Romick-Rosendale; Michael A. Kennedy
Miami University, Oxford, OH
Abstract
Use of NMR-based metabonomics to search for human disease biomarkers is becoming increasingly common. For many researchers, the ultimate goal is translation from biomarker discovery to clinical application. Clinicians routinely use the language of statistical significance testing, whereas academicians typically use analysis techniques that do not perform statistical significance evaluation. Here, we demonstrate a novel approach to integrate statistical significance testing with conventional principal components analysis data representation. A decision tree algorithm is introduced to select and apply appropriate statistical tests to loadings plot data, which are then heat-map color-coded according to p-score enabling direct visual assessment of statistical significance. The integrated metabonomics data assessment methodology should facilitate translation of NMR-based metabonomics discovery of human disease biomarkers to clinical use.

Code: PP	Time Slot/Poster Number: 307	Session: Screening & "omics", Poster
Strategies to Optimize Food Quality Control by NMR
Manfred Spraul; Birk Schütz; Hartmut Schaefer; fang fang; Eberhard Humpfer
Bruker BioSpin GmbH, Rheinstetten, Germany
Abstract
Food Quality Control by NMR has been established for fruitjuices already. Other areas of major interest are wine, edible oils and honey. In such samples, receiver gain is limited by ethanol, lipids and sugars respectivly, when measuring proton NMR. By suppressing the large signals, it is possible to work with optimized receiver gain and increased sensitivity. Shaped pulse suppression with very low power only disturbs a small area around the signals suppressed and leaves important areas like the aromatic region with polyphenol- and flavanoid signals unchanged. This region is especially important for product origin, variety and purity. Examples are shown about reproducibility and statistical results under such conditions for wine and edible oils.

Code: PP	Time Slot/Poster Number: 308	Session: Screening & "omics", Poster
Quantitative metabolic profiling by natural abundance 1H – 13C HSQC spectra of body fluids: Application to Human Urine Sample
Neeraj Sinha; Ratan Kumar Rai; Pratima Tripathi
Centre of Biomedical Magnetic Resonance, Lucknow, India
Abstract
We present a general scheme for the metabolite quantification and profiling from two – dimensional (2D) 1H–13C HSQC Nuclear Magnetic Resonance (NMR) experiment of body fluids observed in natural abundance. The measured cross peak volume from 2D HSQC NMR spectra is correlated with concentration, relaxation and experimental parameters. The relation is derived from Bloch equation and product operator formalism. The accuracy of scheme is tested on known mixture of amino acids solution and with spike-in experiments for human urine samples. The scheme is general in nature and can be applied to any other body fluid samples for metabonomic studies. We also test the applicability of scheme for multivariate analysis, making scheme complete for metabolic profiling.

Code: PP	Time Slot/Poster Number: 309	Session: Screening & "omics", Poster
Multinuclear NMR Studies of Metabolites Associated with Prostate Cancer for Non-Invasive Screening
Neil Mackinnon; Vivekanandan Subramanian; Amjad Khan; Thekkelnaycke Rajendiran; Arul Chinnaiyan; Ayyalusamy Ramamoorthy
University of Michigan, Ann Arbor, MI
Abstract
The high prevalence of prostate cancer in aging men necessitates accurate, robust and non-invasive diagnostic techniques to ensure potentially fatal forms are identified early. Ultra-high field, multi-nuclear NMR experiments are utilized to identify metabolites differentiating normal versus cancerous (localized and metastatic) states associated with the human prostate from a series of model cell lines. A comparison of these metabolites with those identified from biological fluids, which may be collected with minimal inconvenience to the patient, is done with the goal of improving the ability to diagnose this disease. Ultimately, identification of the metabolic pathways important in prostate cancer is anticipated to be critical in monitoring the development and progression of prostate cancer, in addition to aiding the search for treatments.

Code: PP	Time Slot/Poster Number: 310	Session: Screening & "omics", Poster
Hadamard NMR in Automation
Ron Crouch; Bert Heise; Eriks Kupce
Varian, Inc., Oxford, United Kingdom
Abstract
A set of basic NMR experiments that are typically used for structure determination in small organic molecules - COSY, DQ COSY, TOCSY, NOESY, ROESY, multiplicity edited HSQC, HSQC-TOCSY, HMBC and HETCOR - have been modified to incorporate Hadamard encoding. A suite of setup and processing macros has been developed that allow fully automatic setup, processing and plotting of a user-selected series of the Hadamard experiments. The construct is then placed into the Study Queue - a standard protocol for automation on Varian NMR systems. This allows investigating multiple samples unattended and in full automation. Provided sensitivity is adequate, all the essential information that is required for structure determination of small organic molecules can be recorded in about three minutes.

Code: PP	Time Slot/Poster Number: 311	Session: Screening & "omics", Poster
Perturbational Metabolomics of Human Breast Cancer Cells
Quincy Teng1; Wenlin Huang1; Timothy Collette1; Drew Ekman1; Chalet Tan2
1US EPA, Athens, GA; 2Mercer University, Atlanta, GA
Abstract
Breast cancer is the second leading cause of cancer death in women. Estrogen receptor (ER) is an important predictive and prognostic marker in human breast cancer, expressed in over 60% cases. Binding of estrogens to ER leads to transcriptional activation of ER target genes that are important in cell proliferation, angiogenesis and survival of breast cancer cells. We have developed a cellular metabolomics approach and applied it to characterize metabolic changes caused by a synthetic estrogen 17alpha-ethynylestradiol in ER+ MCF-7 and ER- MDA-MB-231 breast cancer cells. The method is effective, accurate, and exhibits greater metabolite retention 50x more than the conventional method. Using this approach, we have observed metabolic changes of MCF-7 cells caused by EE2, but not MDA-MB-231 cells.

Code: PP	Time Slot/Poster Number: 312	Session: Screening & "omics", Poster
Exploring the Metabolome of the Temperature-Dependent Coral Pathogen Vibrio coralliilyticus with NMR-Based Microbial Metabolomics
Arezue Boroujerdi1; Maria Vizcaino2; Tracey Schock1; Pamela Morris3; Daniel Bearden1
1National Institute of Standards and Techonology, Charleston, SC; 2Medical University of South Carolina, Charleston, Charleston, SC; 3College of Charleston, Charleston, SC
Abstract
Metabolomics provides a ‘snapshot’ of an organism’s metabolic state after a physiological response to stress. The bacterium Vibrio coralliilyticus exhibits a temperature-dependent pathogenicity similar to many other Vibrio species. NMR spectra were obtained of methanol-water extracts of intracellular metabolites of the bacteria at both 27 °C (virulent form) and 24 °C (avirulent form). Principal components analysis of the spectra consistently showed significant temperature-based separations largely due to the metabolites betaine, succinate, and glutamate. Optimization of stressor response analysis involved reducing the amount of time required to obtain suitable samples and addressed data quality issues. Controlling the consistency between batches is challenging. We report on Vibrio coralliilyticus’ metabolomic changes during growth to further understand the biology of this organism.