Metabolomics The Basics David Wishart Depts Comp Sci

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

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

Metabolomics Perturbation Fil Secondary Molecules tra tio n Primary Molecules on pti sor Re on i t lu i D Co nce ntr Chemical Fingerprint ati o n

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

Metabolomics Is Growing

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, amino acids, lipids, steroids, alkaloids and drugs (xenobiotics) • Includes human & microbial products • Concentration > 1 m. M

Why 1 m. M? • Equals ~200 ng/m. L • 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 m. M • Need to draw the line somewhere

Why Are Metabolites Relevant? Metabolites are the Canaries of the Genome

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

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

Traditional Metabolite Analysis HPLC, GC, CE, MS

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)

What’s the Difference Between Metabolomics and Traditional Clinical Chemistry? Throughput (more metabolites, greater accuracy, higher speed)

New Metabolomics Approaches

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

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 2 D 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

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

Quantitative vs. Chemometric • Identifies compounds • • Quantifies compds • • Concentration range of • 1 m. M to 1 M • Handles wide range of • samples/conditions • Allows identification of • diagnostic patterns • Limited by DB size • No compound ID No compound concentration range Requires strict sample uniformity Allows identification of diagnostic patterns Limited by training set

Principles of Quantitative Metabolomics Mixture Compound A Compound B Compound C

Quantitative Metabolomics with Eclipse

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-N-Oxide Urea

Metabolic Profiling: The Possibilities • Toxicology Testing • Genetic Disease Tests • Clinical Trial Testing • Nutritional Analysis • Fermentation Monitoring • Clinical Blood Analysis • Food & Beverage Tests • Clinical Urinalysis • Nutraceutical Analysis • Cholesterol Testing • Drug Phenotyping • Drug Compliance • Water Quality Testing • Dialysis Monitoring • Organ Transplantation • MRS and f. MRI

Metabolomics and Drug Toxicology 25 PAP PC 2 20 15 10 ANIT 5 0 -5 Control -10 Control -15 PAP -20 PC 1 -25 -30 -20 -10 Principal Component Analysis 0 10

Disease Diagnosis via NMR (140+ Detectable Conditions) · · · · · · Adenine Phosphoribosyltransferase Deficency Adenylosuccinase Deficiency Alcaptonuria a-Aminoadipic Aciduria b-Aminoisobutyric Aciduria a-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 hydroxylase. Deficiency 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-Hyperammonemia. Homocitrullinuria Syndrome (HHH) Hyperoxaluria Types I & 2 · · · Hyperphenylalaninemia Hyperprolinemia Hyperthreoninemia · · · ·

Applications in Clinical Analysis • • • • 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 Co. A lyase deficiency 2 carbamoyl PO 4 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).

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

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 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

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)

Applications In Transplantation Organ Condition 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) 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 Cs. A 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 Citrate, Glucose, Urea Allantoin (urine & serum) Nitrates, Nitric oxide metabolites (urine)

Applications In Transplantation Organ Condition Metabolite(s) Increased Metabolite(s) Decreased 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) Liver (Human) Post-transplant Phosphatidylcholine (bile) Heart (Human) Rejection Nitrate (urine) Heart (Human) Rejection General lipids, Lipoproteins, VLDL, Phosphatidylcholine (serum) Heart (Mouse) Acute Rejection Phosphocreatine, PO 4 (in vivo) Heart (Human) Ischemia Phosphocreatine, PO 4 (in vivo) Heart (Human) Congestive Heart Failure N-acetylaspartate, Creatine, Choline Myo-inositol (in vivo) Urea (urine) 2 2

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, acetone – Oxidative stress, apoptosis, anoxia, ischemia • Histamine, chlorotyrosine, thromoxane, NO 3 – Immune response, inflammation

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

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

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

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

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

Thanks to. . .