Metabolomics: The Basics

David Wishart

Depts. Comp. Sci and Bio. Sci.

University of Alberta

david.wishart@ualberta.ca

July 16, 2005, 8th Banff Symposium

The Pyramid of Life

25,000 Genes25,000 Genes

2500 Enzymes2500 Enzymes

1400 Chemicals

Metabolomics

Proteomics

Genomics

PerturbationPrimary Molecules

Secondary Molecules

Resorption

DilutionFiltr

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Concentration

Metabolomics

Chemical Fingerprint

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

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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 > 1M

Why 1 M?

• 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 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 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

2 Routes to Metabolomics

1234567ppm

hippurate urea

allantoin creatininehippurate

2-oxoglutarate

citrate

TMAO

succinatefumarate

water

creatinine

taurine

1234567ppm

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QuantitativeMethods

Chemometric (Pattern)Methods

Quantitative vs. Chemometric

• Identifies compounds• Quantifies compds• Concentration range of

1 M 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

Mixture

Compound A

Compound B

Compound C

Principles of Quantitative Metabolomics

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

• Trimethylamine-N-Oxide

• Urea

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

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Principal Component Analysis

Metabolomics and Drug Toxicology

Disease Diagnosis via NMR (140+ Detectable Conditions)

Adenine Phosphoribosyltransferase Deficency

Adenylosuccinase Deficiency Alcaptonuria -Aminoadipic Aciduria -Aminoisobutyric Aciduria -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

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

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 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).

Applications in Metabolite Imaging

N-acetyl-aspartateLactate

Glutamate

Citrate

Alanine

Metabolic Microarrays

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Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient 10Patient 11Patient 12Patient 13Patient 14Patient 15

NormalBelow NormalAbove NorrmalAbsent

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 TransplantationOrgan 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)

Citrate, Glucose, UreaAllantoin (urine & serum)

Kidney (Human) Graft Dysfunction TMAO, DimetheylamineLactate, Acetate, Succinate, Glycine, Alanine, (urine)

Kidney (Rat) Graft DysfunctionReperfusion Injury

TMAO, Citrate, Lactate, Dimetheylamine, Acetate (urine)

Kidney (Rat) Reperfusion Injury(ischemia)

TMAO, Allantoin (serum)

Kidney (Human) Graft DysfunctionCsA 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)

Applications In TransplantationOrgan Condition 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 MethylarginineDimethylarginine(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)

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

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 SykesBiochemistryU of AlbertaNMR spect.

Russ GreinerComp. Sci.U of AlbertaBioinformatics

David WishartComp. Sci.U of AlbertaProj. Leader

Hans VogelBiochemistryU of CalgaryNMR spect.

Fiona BamforthClin. ChemistryU of AlbertaSample Acq.

Derrick CliveChemistryU of AlbertaSynthesis

Liang LiChemistry.U of AlbertaMS/Separation

Mike EllisonBiochemistryU of AlbertaMS/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...