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Biotech2012 spring 3_metabolomics
Metabolomics is the quantitative measurement of metabolites in a biological system. It can be used to generate metabolic profiles or "fingerprints" that characterize phenotypes. There are over 1,400 endogenous water-soluble metabolites that can be detected using NMR or MS-based metabolomics. Quantitative metabolomics allows identifying and measuring hundreds of metabolites simultaneously without separation. It provides a holistic view of metabolic changes and can be used in various medical and clinical applications such as disease diagnosis, drug toxicity assessment, and transplant monitoring.
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Biotech2012 spring 3_metabolomics
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- 2. The Pyramid ofLife 25,000 Genes25,000 Genes 2500 Enzymes2500 Enzymes 1400 Chemicals Metabolomics Proteomics Genomics
- 3. Perturbation Primary Molecules SecondaryMolecules Resorption Dilution Filtration Concentration Metabolomics Chemical Fingerprint
- 4. Metabonomics & Metabolomics •Metabonomics:The quantitative measurement of the time-related “total” metabolic response of vertebrates to pathophysiological (nutritional, xenobiotic, surgical or toxic stimuli) • Metabolomics:The quantitative measurement of the metabolic profiles of model organisms to characterize their phenotype or phenotypic response to genetic or nutritional perturbations
- 5. Metabolomics Is GrowingGrowthin Metabolomics 0 20 40 60 80 100 120 140 160 180 200 1998 1999 2000 2001 2002 2003 2004 2005 Year #References
- 6. What is aMetabolite? • Any organic molecule detectable in the body with a MW < 1000 Da • Includes peptides, oligonucleotides, sugars, nucelosides, organic acids, ketones, aldehydes, amines, amino acids, lipids, steroids, alkaloids and drugs (xenobiotics) • Includes human & microbial products • Concentration > 1µM
- 7. Why 1 µM? •Equals ~200 ng/mL • Limit of detection by NMR • Limit of facile isolation/separation by many analytical methods • Excludes environmental pollutants • Most IEM indicators and other disease indicators have concentrations >1 µM • Need to draw the line somewhere
- 8. Why Are Metabolites Relevant? Metabolitesare the Canaries of the Genome
- 9. Why is Metabolomics Relevant? •Generate metabolic “signatures” • Monitor/measure metabolite flux • Monitor enzyme/pathway kinetics • Assess/identify phenotypes • Monitor gene/environment interactions • Track effects from toxins/drugs/surgery • Monitor consequences from gene KOs • Identify functions of unknown genes
- 10. Medical Metabolomics • Generatemetabolic “signatures” for disease states or host responses • Obtain a more “holistic” view of metabolism (and treatment) • Accelerate assessment & diagnosis • More rapidly and accurately (and cheaply) assess/identify disease phenotypes • Monitor gene/environment interactions • Rapidly track effects from drugs/surgery
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- 12. Problems with Traditional Methods •Requires separation followed by identification (coupled methodology) • Requires optimization of separation conditions each time • Often requires multiple separations • Slow (up to 72 hours per sample) • Manually intensive (constant supervision, high skill, tedious)
- 13. What’s the Difference BetweenMetabolomics and Traditional Clinical Chemistry? Throughput (more metabolites, greater accuracy, higher speed)
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- 15. Advantages • Measure multiple(10’s to 100’s) of metabolites at once – no separation!! • Allows metabolic profiles or “fingerprints” to be generated • Mostly automated, relatively little sample preparation or derivitization • Can be quantitative (esp. NMR) • Analysis & results in < 60 s
- 16. NMR versus MS •Quantitative, fast • Requires no work up or separation • Allows ID of 300+ cmpds at once • Good for CHO’s • Not sensitive • Needs MS or 2D NMR for positive ID • Very fast • Very sensitive • Allows analysis or ID of 3000+ cmpds at once • Not quantitative • Not good for CHOs • Requires work-up • Needs NMR for ID
- 17. 2 Routes toMetabolomics 1234567ppm hippurate urea allantoin creatinine hippurate 2-oxoglutarate citrate TMAO succinatefumarate water creatinine taurine 1234567ppm -25 -20 -15 -10 -5 0 5 10 15 20 25 -30 -20 -10 0 10 PC1 PC2 PAP ANIT Control Quantitative Methods Chemometric (Pattern) Methods
- 18. Quantitative vs. Chemometric •Identifies compounds • Quantifies compds • Concentration range of 1 µM to 1 M • Handles wide range of samples/conditions • Allows identification of diagnostic patterns • Limited by DB size • No compound ID • No compound conc. • No compound concentration range • Requires strict sample uniformity • Allows identification of diagnostic patterns • Limited by training set
- 19. Mixture Compound A Compound B CompoundC Principles of Quantitative Metabolomics
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- 21. Sample Compound List •(+)-(-)-Methylsuccinic Acid • 2,5-Dihydroxyphenylacetic Acid • 2-hydroxy-3-methylbutyric acid • 2-Oxoglutaric acid • 3-Hydroxy-3-methylglutaric acid • 3-Indoxyl Sulfate • 5-Hydroxyindole-3-acetic Acid • Acetamide • Acetic Acid • Acetoacetic Acid • Acetone • Acetyl-L-carnitine • Alpha-Glucose • Alpha-ketoisocaproic acid • Benzoic Acid • Betaine • Beta-Lactose • Citric Acid • Creatine • Creatinine • D(-)Fructose • D-(+)-Glyceric Acid • D(+)-Xylose • Dimethylamine • DL-B-Aminoisobutyric Acid • DL-Carnitine • DL-Citrulline • DL-Malic Acid • Ethanol • Formic Acid • Fumaric Acid • Gamma-Amino-N-Butyric Acid • Gamma-Hydroxybutyric Acid • Gentisic Acid • Glutaric acid • Glycerol • Glycine • Glycolic Acid • Hippuric acid • Homovanillic acid • Hypoxanthine • Imidazole • Inositol • isovaleric acid • L(-) Fucose • L-alanine • L-asparagine • L-aspartic acid • L-Histidine • L-homocitrulline • L-Isoleucine • L-Lactic Acid • L-Lysine • L-Methionine • L-phenylalanine • L-Serine • L-Threonine • L-Valine • Malonic Acid • Methylamine • Mono-methylmalonate • N,N-dimethylglycine • N-Butyric Acid • Pimelic Acid • Propionic Acid • Pyruvic Acid • Salicylic acid • Sarcosine • Succinic Acid • Sucrose • Taurine • trans-4-hydroxy-L-Proline • Trimethylamine • Trimethylamine-N-Oxide • Urea
- 22. Metabolic Profiling: The Possibilities •Genetic Disease Tests • Nutritional Analysis • Clinical Blood Analysis • Clinical Urinalysis • Cholesterol Testing • Drug Compliance • Dialysis Monitoring • MRS and fMRI • Toxicology Testing • Clinical Trial Testing • Fermentation Monitoring • Food & Beverage Tests • Nutraceutical Analysis • Drug Phenotyping • Water Quality Testing • Organ Transplantation
- 23. -25 -20 -15 -10 -5 0 5 10 15 20 25 -30 -20 -100 10 PC1 PC2 PAP ANIT Control ANIT PAP Control Principal Component Analysis Metabolomics and Drug Toxicology
- 24. Disease Diagnosis viaNMR (140+ Detectable Conditions) • Adenine Phosphoribosyltransferase Deficency • Adenylosuccinase Deficiency • Alcaptonuria • α-Aminoadipic Aciduria • β-Aminoisobutyric Aciduria • α-Aminoketoadipic Aciduria • Anorexia Nervosa • Argininemia • Argininosuccinic Aciduria • Aspartylglycosaminuria • Asphyxia • Biopterin Disorders • Biotin-responsive Multiple Carboxylase Deficiency • Canavan’s Disease • Carcinoid Syndrome • Carnosinemia • Cerebrotendinous Xanthomatosis/sterol 27- hydroxylaseDeficiency • Citrullinemia • Cystathioninemia • Cystinosis • Cystinuria (Hypercystinuria) • Diabetes • Dibasic Aminoaciduria • Dicarboxylic Aminoaciduria • Dichloromethane Ingestion • Dihydrolipoyl Dehydrogenase Deficiency • Dihydropyrimidine Dehydrogenase Deficiency • Dimethylglycine Dehydrogenase Deficiency • Essential Fructosuria • Ethanolaminosis • Ethylmalonic Aciduria • Familial Iminoglycinuria • Fanconi’s Syndrome • Folate Disorder • Fructose Intolerance • Fulminant Hepatitis • Fumarase Deficiency • Galactosemia • Glucoglycinuria • Glutaric Aciduria Types 1 & 2 • Glutathionuria • Glyceroluria (GKD) • D-Glyceric Aciduria • Guanidinoacetate- Methyltransferase Deficiency • Hartnup Disorder • Hawkinsinuria • Histidinemia • Histidinuria • Homocystinsufonuria • Homocystinuria • 4-Hydroxybutyric Aciduria • 2-Hydroxyglutaric Aciduria • Hydroxykynureninuria • Hydroxylysinemia • Hydroxylysinuria • 3-Hydroxy-3-methylglutaric Aciduria • 3-Hydroxy-3-methylglutaryl-Co A Lyase Deficiency • Hydroxyprolinemia • Hyperalaninemia • Hyperargininemia (Argininemia) • Hyperglycinuria • Hyperleucine-Isoleucinemia • Hyperlysinemia • Hyperornithinemia • Hyperornithinemia- Hyperammonemia-Homocitrullinuria Syndrome (HHH) • Hyperoxaluria Types I & 2 • Hyperphenylalaninemia • Hyperprolinemia • Hyperthreoninemia
- 25. Applications in ClinicalAnalysis • 14 propionic acidemia • 11 methylmalonic aciduria • 11 cystinuria • 6 alkaptonuria • 4 glutaric aciduria I • 3 pyruvate decarboxylase deficiency • 3 ketosis • 3 Hartnup disorder • 3 cystinosis • 3 neuroblastoma • 3 phenylketonuria • 3 ethanol toxicity • 3 glycerol kinase deficiency • 3 HMG CoA lyase deficiency • 2 carbamoyl PO4 synthetase deficiency • 96% sensitivity and 100% specificity in ID of abnormal from normal by metabolite concentrations • 95.5% sensitivity and 92.4% specificity in ID of disease or condition by characteristic metabolite concentrations • 120 sec per sample Clinical Chemistry 47, 1918-1921 (2001).
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- 27. Metabolic Microarrays AceticAcid Betaine Carnitine CitricAcid Creatinine Dimethylglycine Dimethylamine HippulricAcid LacticAcid SuccinicAcid Trimethylamine Trimn-N-Oxide Urea Lactose SubericAcid SebacicAcid HomovanillicAcid Threonine Alanine Glycine Glucose Patient 1 Patient2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Normal Below Normal Above Norrmal Absent
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- 29. Why Metabolomics For Transplants? •Relatively non-invasive (no need for biopsy, just collect urine, blood or bile) • Can be quite organ specific • Very fast (<60 s for an answer) & cheap • Metabolic changes happen in seconds, gene, protein and tissue changes happen in minutes, hours or days • Allows easy longitudinal monitoring of patient (or organ) function (pre&post op)
- 30. Applications In Transplantation OrganCondition Metabolite(s) Increased Metabolite(s) Decreased Kidney (Human) Chronic Renal Failure TMAO, Dimethylamine, Urea, Creatinine (serum) Kidney (Rat) Renal Damage (chemical) Acetone, Lactate, Ethanol, Succinate, TMAO, Dimethylamine, Taurine (urine & serum) Citrate, Glucose, Urea Allantoin (urine & serum) Kidney (Human) Graft Dysfunction TMAO, Dimetheylamine Lactate, Acetate, Succinate, Glycine, Alanine, (urine) Kidney (Rat) Graft Dysfunction Reperfusion Injury TMAO, Citrate, Lactate, Dimetheylamine, Acetate (urine) Kidney (Rat) Reperfusion Injury (ischemia) TMAO, Allantoin (serum) Kidney (Human) Graft Dysfunction CsA toxicity TMAO, Alanine, Lactate, Citrate (urine & serum) Kidney (Mouse) Nephrectomy Methionine, Citrulline, Arginine, Alanine (urine & serum) Serine (serum) Kidney (Mouse) Nephrectomy Guanidinosuccinate, Guanidine, Creatinine, Guanidinovalearate, (urine & serum) Guanidinoacetate (urine) Kidney (Human) Acute Rejection Nitrates, Nitrites, Nitric oxide metabolites (urine)
- 31. Applications In Transplantation OrganCondition Metabolite(s) Increased Metabolite(s) Decreased 2 2 Liver (Rat) Reperfusion Injury Citrate, Succinate, Ketone bodies (good function) Citrate, Succinate, Ketone bodies (poor function) Liver (Human) Ischemia Methylarginine Dimethylarginine (liver catheter) Liver (Human) Graft Dysfunction Glutamine (serum & urine) Urea (urine) Liver (Human) Post-transplant Phosphatidylcholine (bile) Heart (Human) Rejection Nitrate (urine) Heart (Human) Rejection General lipids, Lipoproteins, VLDL, LDL, Phosphatidylcholine (serum) Heart (Mouse) Acute Rejection Phosphocreatine, PO4 (in vivo) Heart (Human) Ischemia Phosphocreatine, PO4 (in vivo) Heart (Human) Congestive Heart Failure N-acetylaspartate, Creatine, Choline Myo-inositol (in vivo)
- 32. Metabolites & Function •Serum Creatinine – Late stage organ stress and tissue breakdown • TMAO – Early stage buffering response • Creatine, methyl-histidine, taurine, glycine – Tissue damage, muscle breakdown, remodelling • Citrate, lactate, acetate, acetone – Oxidative stress, apoptosis, anoxia, ischemia • Histamine, chlorotyrosine, thromoxane, NO3 – Immune response, inflammation
- 33. Why NOT MetabolomicsFor Transplants? • Still an early stage technology – not “ready for prime time” • Metabolites are not always organ specific and not always as informative as protein or gene measures • Still defining signature metabolites and their meaning • Still don’t have a complete list of human metabolites
- 34. Human Metabolome Project •Purpose is to facilitate Metabolomics • Objective is to improve – Disease identification – Disease prognosis & prediction – Disease monitoring – Drug metabolism and toxicology – Linkage between metabolome & genome – Development of software for metabolomics
- 35. Concluding Comments • Metabolomicsis rapidly becoming the “new clinical chemistry” • Metabolomics complements genomics, proteomics and histology • Metabolomics allows probing of rapid physiological changes or events that are not as easily detected by microarrays or histological methods • Canada is actually leading the way (at least for now) in this field