NMR Spectroscopy Based Metabolomics and Its Multidisciplinary Research Applications G. A. NAGANA GOWDA ANALYTICAL CHEMISTRY DIVISION West Lafayette, IN--47907 Genes Metabolic Profiling mRNA enzymes small molecules (Metabolites) Causes of metabolic disturbance In this era of omic scices, •Genetic defects •mRNA defects •Enzyme defects •Drug effects Modest change in enzyme activity > substantial changes in metabolites What are Metabolites? • • • • Any organic molecule detectable in a biological system with a MW < 1000 Da Includes peptides, oligonucleotides, sugars, nucleosides, organic acids, ketones, amines, amino acids, lipids, steroids, alkaloids and drugs Includes both human & microbial products Concentration > 1μM Metebolomics/metabonomics/ metabolic profiling Metabolomics also commonly known as metabonomics or metabolic profiling deals with detection and quantitative analyses of hundreds or thousands of metabolites from biological samples in a single step. Biological samples may be from Human Animal Plant Food Potential Metabolomics Applications • Early disease diagnosis and prognosis • To determine whether a treatment is working or not • Monitor healthy people to detect early signs of disease: Predicting the risk of disease • Clinical Chemistry of 21st Century! • • Drug development: To speed the discovery and development of drugs Predict potentially harmful effects of drugs (toxicity) • • • Identification of perturbed metabolic pathways in disease Discovering new functions of gene When combined with genomics, transcriptomics and proteomics analysis, metabolomics offers potential to understand the whole biological system. • Food and Nutrition • investigations of plants • Animal studies Personalized Medicine Individual’s health or disease profile from body fluid sample One of the goals of metabolomics is to personalize the medicine Finger print Urine NMR Urine samples Metabolic finger print Designing effective Treatment protocol based on individuals metabolic finger print What are the advantages? Measures multiple (10’s to 100’s) metabolites simultaneously No sample preparation or separation!! Allows metabolic profiles or “fingerprints” to be generated Highly quantitative (NMR) Can be automated High throughput < 60 s Metabolomics requires powerful information rich analytical technique to detect a high number of metabolites in one step Non-selective Sensitivity Resolution Reproducibility Metabolite Identity Quantitative MS NMR Commonly used Analytical Techniques Mass spectrometry (MS) Nuclear magnetic resonance (NMR) spectroscopy NMR Spectroscopy • Strengths of NMR • Detection of almost all the metabolites • Reproducibile • Quantitative • Non-invasive: Enables Repeated measurements • Only technique available for the analysis of intact samples such as tissue, food, plants • Challenges • Resolution • Sensitivity • Metabolites identification in the complex spectrum NMR Facets of NMR Spectroscopy- Lets you visualize molecular species and their 3D structures inside the objects Imaging- Lets you visualize organs and inside of the human body and brain function 1D-Spectroscopy 2D Spectroscopy 3D Spectroscopy ppm -8.98 -9.00 -9.02 -9.04 -9.06 -9.08 -9.10 -9.12 -9.14 5.0 4.5 4.0 3.5 3.0 DOSY 2.5 2.0 1.5 ppm Imaging Functional MRI NMR Spectroscopy Metabolites are identified through atomic nuclei •Metabolites are detected through Hydrogen (H), Carbon (C), Nitrogen (N) or Phosphorus (P) •1H- almost all compounds in biological samples contain hydrogen atoms High Natural abundance (99.98%) High sensitivity Commonly used •13C-almost all compounds in biological samples contain carbon atoms Low natural abundance (1.1%) Low sensitivity •15N-many compounds in biological samples contain nitrogen atoms Very low natural abundance (0.37%) Very low sensitivity •31P-many compounds in biological samples contain nitrogen atoms 100% Natural abundance High sensitivity 1H Water molecule Absence of magnetic field NMR spectrum Tiny magnets Inside the magnet water Magnetic field Random orientation Observable part Magnetic field z ALIGNED along or Opposite to the applied magnetic Field x z z Difference Observable part Mz Lower magnetic field y x z x Mz z Observable part y x x Higher magnetic field NMR Signal Magnetic field z z Mz My y x RF pulse (6 μs) y x z water y NMR Signal x FT FID f Frequency > identity of the metabolite r Intensity > concentration of the metabolite e How do you get thousands of NMR signals for bodyfluids? z z Mz My y x RF y Urine z 6 μs y Urine 1H NMR spectrum x FID FT Biosamples (Serum, Urine, Tissue, etc.) Metabolomics: Flow diagram Sample preparation (Extraction, derivatization, buffering for NMR, GC-MS, etc.) 1D/2D NMR (1D 1H NOESY, 2D TOCSY, HRMAS, etc.) 10 8 6 4 ppm 2 1D/2D MS (LC-MS, GC-MS, DESI-MS, etc.) 100 0 200 300 400 m/z Data preprocessing (Baseline correction, binning, scaling, normalization, etc.) Unsupervised/supervised statistical analysis (PCA, PLS-DA, O-PLS, etc.) Scores PC2 Loadings Disease Control 5 4 3 2 PC1 Putative Biomarkers (Identity, quantity) Validation and Biological Understanding Clinical Trails 1 0 Samples ¾ Body Fluids Body fluids ¾ ¾ Cell Cultures ¾ Serum/plasma ¾ Tissue ¾ Urine ¾ Perfused organs ¾ Saliva ¾ small animals ¾ Bile ¾ Humans Food fruit, meat, fish, cheese, pasta ¾ Plants Samples from animals or humans Urine samples • Collect in plain tubes on ice or in a refrigerator • Use 0.05 to 0.1 % wt/vol NaN3 • Refrigerate frozen at -40oC until analyzed by NMR Blood • Plasma: Collect in lithium heparin to get plasma (not in EDTA or citrate) • Serum: Collect in plain tube (no additive) leave to coagulate for about 30 min. Centrifuge at 1600 g for 15 min at 4o C to separate blood cells • Store the supernatant at -40oC until analyzed by NMR Samples from animals or humans Tissue Freeze the tissue samples rapidly in liquid nitrogen (~100mg) to arrest biochemical reactions Store the samples at -80oC Phosphate Buffer (pH = 7.4) Na2HPO4 28.85 g NaH2PO4 5.25 g NaN3 3mM Fill in 1 liter flask, make up with water and dissolve the contents Sample preparation for NMR experiments Urine • 300 μl urine and 300 μl of phosphate buffer (pH=7.4) • • Centrifuge and remove particulate matter. 550 μl in 5 mm NMR tube; Insert a coaxial tube filled with D2O+TSP Serum/plasma 500 μl and 50 μl phosphate buffer Tissue Intact Tissue: High Resolution NMR with Magic angle sample spinning or Extract polar and lipid metabolites 5mm NMR tube Nature Protocols (2007) 2(11), 2692-2703 Is the sample amount fixed? • NO • If the available sample quantity is less, you can use less • Need to use appropriate NMR accessories • As little as 5 μl can be used with micro-coil NMR probes Untreated Serum/ urine Superconducting magnet Higher sensitivity by reducing the temperature of the NMR coil and preamplifier DRX500 with Cryo-probe at Purdue Higher sensitivity and resolution by increasing the magnetic field strength Avance III 800 at Purdue Temperature should be constant for the samples: 298K Achieve good solvent (water) suppression Normal 1D 1H NMR experiment- 1D NOESY presaturated NMR experiments: Urine •One dimensional NMR experiment •There are several pulse techniques for 1D NMR Residual water NOESY-presat NMR experiments: serum/plasma 1d-NOESY-presat Relaxation Edited CPMG Diffusion edited NMR High Resolution Magic Angle Spinning (HR-MAS NMR) Samples such as tissue, food or plants NMR is the method of choice • Additional interactions through space between nuclear magnets (spins) which are orientation dependent • Sample inhomogeneity leads differences in magnetic susceptibility • Solution: magic angle sample spinning • Magic angle: 54.7o • • • • • • B0 54.70 4 mm o.d. zirconium oxide rotor Cut to a suitable size to fit into the rotor Sample size ~ 10-15 mg, Insert into the rotor with additional D2O spun at the magic angle to Bo spin rate ~ 4 kHz Rotors (sample tubes) High Resolution Magic Angle Spinning (HR-MAS NMR) B0 54.70 •Biochemical changes can occur during NMR experiments •Keep the sample cooled during NMR experiment High Resolution Magic Angle Spinning NMR of tissue 400 MHz 1H CPMG MAS NMR spectra of cells and tissues (spin rate 4.2 kHz) 1D MAS NMR experiments require ~ 5 min HRMAS NMR Spectra 15 mg tissue samples Liver (cancer adjacent tissue) Liver Cancer J. Proteome Res. 6,2605, 2007 NMR of Extracts: Metabolic profiling of food Fruit, meat, fish, cheese, pasta •Soft cheese degradation in different packaging conditions by 1H-NMR • Deals with the influence of packaging on the degradation of soft cheese • NMR spectra of aqueous cheese extracts were acquired as a function of time during storage of cheese inside and outside the original package. • 15 metabolites are quantified and used in a kinetic degradation model. Magn. Reson. Chem. 2008, 46, 828–831 Chemometric analysis Multivariate Statistical Analysis 10000 G-1 G-2 PC2 Disease PC1 -8000 0 Control -10000 NMR PCA 8000 Chemometric analysis Baseline correction Data alignment Chemometric analysis: Data pretreatment • Data binning: 32K to 4K: -pH effects • Data Normalization: to compensate -Dilution effects of urine • Data scaling -equal weight for small and big peaks variance scaling: (division by the standard deviation of the peak intensities across the set of spectra) and Parato scaling (division by the square root of the standard deviations). Approaches of Data analysis ppm 7 6 5 4 3 2 Chemometric (Pattern recognition) Quantitative Methods 25 TMAO hippurate allantoin creatinine taurine PC2 20 creatinine 15 10 citrate 5 urea hippurate water 1 2-oxoglutarate 0 succinate -5 fumarate Diet1 -10 ppm 7 6 5 4 3 2 1 Diet3 -15 Diet2 -20 -25 -30 PC1 -20 -10 0 10 Quantitative Chemometric • Metabolite Identification No • Metabolite quantification No • Handles wide range of samples/conditions Requires strict sample uniformity • Allows identification of diagnostic patterns Allows identification of diagnostic patterns • Use database for identification • More NMR for identifying new metabolites Comparison of patients with different severity of coronary atherosclerosis Predict the occurrence and severity of coronary artery disease using blood plasma. Brindle et al., Nature medicine 8, 2002. Example: personalized medicine Administration of galactosamine hydrochloride (induce liver disease) to a group of ten rats. Effects observed from NMR were variable enough that the rats could be classified as either ‘responders’ or ‘non-responders’. PCA analysis showed some discrimination between responder and non-responder groups in terms of their pre-dose metabolite profiles. PCA (predose) Andrew et al., Nature letters 440, April 2006. Metabolic Disorder: Aminoacylase I deficiency Handbook of 1H-NMR spectroscopy in inborn errors of metabolism: body fluid NMR spectroscopy and in vivo MR spectroscopy / von U. Engelke, S. Moolenaar, S. Hoenderop, E. Morava, M. van der Graaf, A. Heerschap, R. Wevers Heilbronn: SPS Verlagsgesellschaft, 2007 ISBN 3-936145-49-0 Some 2D NMR experiments for metabonomics Experiment Detected nucleus Indirect axis NMR interaction responsible COSY δH δH DQF-COSY δH δH TOCSY δH δH 2J NOESY δH δH H-H Dipole rHH, conformation ROESY δH δH H-H Dipole rHH, conformation HMQC δH δC 1J CH C-H HSQC δH δC 1J CH C-H HMBC δH δC 2J CH JRES (homo) δH JHH Measurement of JHH JRES (hetero) δC JCH Measurement of JCH 2J HH 2J & 3JHH connectivity HH HH & 3JHH connectivity & 3JHH all in a spin system & 3JCH C-C-H & C-C-C-H Metabolites identification using 2D NMR Double Quantum Filtered-COSY experiment DQF-COSY Jejunal aspirate (fluid from small intestine) More NMR experiments for identifying new metabolites: Total Correlation Spectroscopy (TOCSY -Rat kidney 17 mg From Bruker Identification of unknown metabolites using 2D NMR methods HMBC HSQC DQF-COSY SEFT NMR metabolite Databases ¾ Several freely available electronic database ¾ Information on individual metabolite ¾ Human Metabolome Database (HMDB) (http://www.hmdb.ca/) ¾ HMDB currently contains nearly 2500 metabolite entries Cryoprobe 13C NMR spectra 500 MHz 13C/1H dual probe Rat urine diluted 2:1 with buffer, 512 scans, 30 minute acquisition Control Dosed 90 mg/kg hydrazine, 48 h Lindon JC Combining NMR and MS Methods: Nuclear Magnetic Resonance (NMR) Spectroscopy Sample High resolution NMR Mass Spectrometry (MS) New MS technologies High-Throughput Screening by NMR Metabolomics: Future Clinical chemistry tool? Analytical Laboratory at LipoSciences Assessment of Risk of coronary heart disease blood plasma Turnover with BEST-NMR ~ 150 sec including NMR-measurement In vivo NMR 0.5, 1.0, 3.0,…….now it is 7 Tesla Metabolites detection in vivo Future Directions: Integration of these approaches Improvements for NMR are needed for better sensitivity and resolution z Isotope tags z Cryoprobes z Micro-coil NMR z High magnetic fields z HRMAS z LC-NMR z LC-MS z GC-MS Conclusions • Metabolomics is rapidly growing with its applications spread in many areas of science: Biomedicine, Drug discovery, Food and Nutrition, etc. • Metabolic analysis is a promising approach for the noninvasive monitoring of human health, by detecting early stages of illness and applying the right treatment. • Metabolomics complements genomics, proteomics and histology • Metabolomics allows probing of rapid physiological changes or events that are not easily detected by microarrays or histological methods • Metabolomics based personalized medicine will eventually improve the health of the whole population.