Metabolomics: The Basics

1. Metabolomics: The Basics David Wishart Depts. Comp. Sci and Bio. Sci. University of Alberta david.wishart@ualberta.ca July 16, 2005, 8th Banff Symposium

2. The Pyramid of Life Metabolomics Proteomics Genomics 1400 Chemicals 2500 Enzymes 25,000 Genes

3. Metabolomics Perturbation Primary Molecules Filtration Secondary Molecules Dilution Concentration Resorption 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 Growing

6. What is a Metabolite? • 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 > 1mM

7. Why 1 mM? • 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 mM • Need to draw the line somewhere

8. Why Are Metabolites Relevant? Metabolites are 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 • Generate metabolic “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

11. Traditional Metabolite Analysis HPLC, GC, CE, MS

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 Between Metabolomics and Traditional Clinical Chemistry? Throughput (more metabolites, greater accuracy, higher speed)

14. New Metabolomics Approaches

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. 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 NMR versus MS

17. 25 PC2 20 15 ANIT 10 5 0 -5 Control -10 -15 PAP -20 PC1 -25 -30 -20 -10 0 10 ppm 7 6 5 4 3 2 1 TMAO creatinine hippurate allantoin creatinine taurine citrate urea hippurate 2-oxoglutarate water succinate fumarate ppm 7 6 5 4 3 2 1 2 Routes to Metabolomics Quantitative Methods Chemometric (Pattern) Methods

18. Identifies compounds Quantifies compds Concentration range of 1 mM 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 Quantitative vs. Chemometric

19. Mixture Compound A Compound B Compound C Principles of Quantitative Metabolomics

20. Quantitative Metabolomics with Eclipse

21. (+)-(-)-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 Sample Compound List • 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 • 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

22. 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 Metabolic Profiling: The Possibilities

23. 25 PC2 20 15 ANIT 10 5 0 -5 Control -10 -15 PAP -20 PC1 -25 -30 -20 -10 0 10 PAP ANIT Control Metabolomics and Drug Toxicology Principal Component Analysis

24. Adenine Phosphoribosyltransferase Deficency Adenylosuccinase Deficiency Alcaptonuria a-Aminoadipic Aciduria b-Aminoisobutyric Aciduria a-AminoketoadipicAciduria 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 Disease Diagnosis via NMR (140+ Detectable Conditions) • 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. 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 Applications in Clinical Analysis Clinical Chemistry 47, 1918-1921 (2001).

26. Applications in Metabolite Imaging Lactate N-acetyl-aspartate Glutamate Citrate Alanine

27. Acetic Acid Betaine Carnitine Citric Acid Creatinine Dimethylglycine Dimethylamine Hippulric Acid Lactic Acid Succinic Acid Trimethylamine Trimn-N-Oxide Urea Lactose Suberic Acid Sebacic Acid Homovanillic Acid Threonine Alanine Glycine Glucose Metabolic Microarrays Normal Below Normal Above Norrmal Absent Patient 1 Patient 2 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

28. 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)

29. Applications In Transplantation

30. Applications In Transplantation

31. 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

32. Why NOT Metabolomics For 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

33. Human Metabolome Project • $7.5 million Genome Canada Project launched in Jan. 2005 • Mandate to quantify (normal and abnormal ranges) and identify all metabolites in urine, CSF, plasma and WBC’s • Make all data freely and electronically accessible (HMDB) • Make all cmpds publicly available (HML) www.hmdb.ca

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. David Wishart Comp. Sci. U of Alberta Proj. Leader Brian Sykes Biochemistry U of Alberta NMR spect. Russ Greiner Comp. Sci. U of Alberta Bioinformatics Hans Vogel Biochemistry U of Calgary NMR spect. Fiona Bamforth Clin. Chemistry U of Alberta Sample Acq. Derrick Clive Chemistry U of Alberta Synthesis Liang Li Chemistry. U of Alberta MS/Separation Mike Ellison Biochemistry U of Alberta MS/Separation.

36. Concluding Comments • Metabolomics is 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

37. Thanks to...