DOC PREVIEW
UNC-Chapel Hill ENVR 442 - Metabolomics

This preview shows page 1-2-3 out of 9 pages.

Save
View full document
View full document
Premium Document
Do you want full access? Go Premium and unlock all 9 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 9 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 9 pages.
Access to all documents
Download any document
Ad free experience
Premium Document
Do you want full access? Go Premium and unlock all 9 pages.
Access to all documents
Download any document
Ad free experience

Unformatted text preview:

1Nature Reviews: Drug Discovery Nicholson et al. (2002)EfficacyToxicityPrimary MoleculesSecondary MoleculesResorptionDilutionFiltrationhippurateureaallantoincreatininehippurate2-oxoglutaratecitrateTMAOsuccinatefumaratewatercreatininetaurineConcentrationAdapted from D. Robertson, Pfizer Global Research and DevelopmentMetabolomicsComprehensive Definition:The quantitative measurement of the time-related multiparametric metabolic response of living systems to pathophysiological exogenous or endogenous stimuli or genetic modificationOperational Definition:The systematic exploration of biofluid composition using NMR/pattern recognition technology in order to associate target organ toxicity with NMR spectral patterns and identify novel surrogate markers of toxicity.Adapted from D. Robertson, Pfizer Global Research and DevelopmentMetabolomics:The study of the total metabolite pool (metabolome), metabolic regulation and fluxes in individual cells or cell types. Can be achieved through a wide spectrum of technologic methods including LC-MS, GC-MS, and nuclear magnetic resonance (NMR)Metabonomics: The study of the systemic biochemical profiles and regulation of function in whole organisms by analyzing a metabolite pool (metabolome) in biofluids and tissues. Usually implies that the study is done specifically through nuclear magnetic resonance profiling2Metabolome: The quantitative complement of all the low molecular weight molecules present in cells in a particular physiological or developmental state Biofluid: A fluid sample obtained from a living system. The donor might typically be a human or an animal. Fluids can be excreted (such as urine, sweat), expressed or secreted (such as milk, bile), obtained by intervention (such as blood plasma, serum or cerebrospinal fluid), develop as a result of a pathological process (such as blister or cyst fluid), or be applied and collected (such as dialysis fluid)From Metabometrix, Ltd.Advantages of Metabolomics• Identification of target organ, severity, onset, duration and reversal of the effects (time-course)• Classify sample as “normal” vs. “abnormal”• Determine mechanisms of action within the organ• Potential for identifying novel biomarkers of toxic effect• Non-invasive•No a priori decisions about samples need be made• No sample processing necessary other than cold collection• Complete time course data can readily be obtained• Minimization of compound requirements• Relatively fast analysis (200-300 samples/day)• Useful tool for modeling physiological variation and exposure conditions in animals and humansAdapted from D. Robertson, Pfizer Global Research and DevelopmentNMR spectroscopySpectroscopy deals with the interactions between electromagnetic radiation and matter. Spectroscopy is used to derive the properties of matter at the molecular level. Nuclear magnetic resonance (NMR) exploits the magnetic properties of atomic nuclei. The method functions as follows: A substance is placed in a magnetic field. Some atomic nuclei (e.g. protons, nuclei of hydrogen atoms) then behave like microscopic compass needles, called nuclear spins. Each nuclear spin orientation corresponds to a different energy level. The spins may jump between the levels when the sample is exposed to radio waves whose frequency exactly matches the energy spacing. This is called resonance. One way of measuring the energy is to change the irradiation frequency. At resonance, the spins flip causing an electric signal. The strength of the signal is plotted as a function of frequency in a diagram, the NMR spectrum. From: www.nobel.seIn metabolomics, it is the patterns that occur when many different biochemical entities are detected simultaneously in a mixture using 1H NMR that are interpreted.3NMR in Metabolomics:Pro:• Non-destructive• Applicable to intact biomaterials• More information rich in complex-mixture analyses• No extraction/derivatization is necessaryCon:• Less sensitive than MSHistory:• NMR has been used to study metabolites in biofluids for over a decade• Metabolomics technology as it is known today (600 MHz 1H NMR) was pioneered by Jeremy Nicholson, Elaine Holmes and John Lindon of Imperial College in London• Only recently have advances in flow-through NMR hardware and pattern recognition software made the possibility of “high”-throughput in vivotoxicity assessment a practical possibility NMR Acquisition andGilson 215 Control SystemNMR flow probeN2gasVarian Inova 600Shielded magnet120 ul flow probeGilson 215 autosamplerBiomek® RobotRefrigeratedMetabolismCage (0oC)Frozen Storage+ NaN3Deuterated Buffer TSPData ProcessingAdapted from D. Robertson, Pfizer Global Research and DevelopmentAdapted from D. Robertson, Pfizer Global Research and Development Adapted from D. Robertson, Pfizer Global Research and Development4Day 1Day 2Day 3Day 4Day 5Normal Metabolic ProfilesAdapted from D. Robertson, Pfizer Global Research and DevelopmentFunctional NMR Spectrum of Rat UrineNature Reviews: Drug Discovery Nicholson et al. (2002)“Biomarker Windows”Toxicogenomics, Hamadeh & Afshari (eds.) Wiley-Liss, 2004Mass Spectrometry-based Metabolomic Analysis5Mass Spectrometry-based Metabolomic Analysis Mass Spectrometry-based Metabolomic AnalysisMass Spectrometry-based Metabolomic AnalysisHigh Throughput Metabolomics Using UltraHigh Throughput Metabolomics Using Ultra--high Field Fourier high Field Fourier Transform Ion Cyclotron Resonance Mass SpectrometryTransform Ion Cyclotron Resonance Mass SpectrometryHuman plasma (100-500 Da range)Small molecule fingerprinting based on accurate mass aloneBorchers et al. In review6Data Processing Steps in Metabolomic AnalysisPathway MappingNature Reviews: Drug Discovery Nicholson et al. (2002)Data Analysis in MetabolomicsNMR SpectraPrimary Data ProcessingUnsupervised mapping of data in 3D spaceSupervised classification and calculation of confidence intervalsPattern Recognition (PR) Methods:PR and related multivariate statistical approaches can be used to discern significant patterns in complex data sets and are particularly appropriate in situations where there are more variables than samples in the data set. The general aim of PR is to classify objects (in this case 1H NMR spectra) or to predict the origin of objects based on identification of inherent patterns in a set of indirect measurements. PR methods can reduce the dimensionality of complex data sets via 2 or 3D mapping procedures, thereby


View Full Document

UNC-Chapel Hill ENVR 442 - Metabolomics

Documents in this Course
Load more
Download Metabolomics
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Metabolomics and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Metabolomics 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?