Metabolomics: The Basics

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Metabolomics The Basics

• David Wishart
• Depts. Comp. Sci and Bio. Sci.
• University of Alberta
• david.wishart_at_ualberta.ca

July 16, 2005, 8th Banff Symposium
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The Pyramid of Life
Metabolomics Proteomics Genomics
1400 Chemicals
2500 Enzymes
25,000 Genes
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Metabolomics
Perturbation
Primary Molecules
Filtration
Secondary Molecules
Dilution
Concentration
Resorption
Chemical Fingerprint
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Metabonomics Metabolomics

• MetabonomicsThe quantitative measurement of the
  time-related total metabolic response of
  vertebrates to pathophysiological (nutritional,
  xenobiotic, surgical or toxic stimuli)
• MetabolomicsThe quantitative measurement of the
  metabolic profiles of model organisms to
  characterize their phenotype or phenotypic
  response to genetic or nutritional perturbations

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Metabolomics Is Growing
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What is a Metabolite?

• Any organic molecule detectable in the body with
  a MW lt 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 gt 1mM

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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 gt1 mM
• Need to draw the line somewhere

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

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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

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Traditional Metabolite Analysis
HPLC, GC, CE, MS
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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)

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Whats the Difference Between Metabolomics and
Traditional Clinical Chemistry?

• Throughput
• (more metabolites, greater accuracy, higher speed)

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New Metabolomics Approaches
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Advantages

• Measure multiple (10s to 100s) 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 lt 60 s

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NMR versus MS

• Quantitative, fast
• Requires no work up or separation
• Allows ID of 300 cmpds at once
• Good for CHOs
• 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

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2 Routes to Metabolomics
Quantitative Methods
Chemometric (Pattern) Methods
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Quantitative vs. Chemometric

• 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

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Principles of Quantitative Metabolomics
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Quantitative Metabolomics with Eclipse
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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

• ()-(-)-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

• 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

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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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Metabolomics and Drug Toxicology
Principal Component Analysis
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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
• Canavans Disease
• Carcinoid Syndrome
• Carnosinemia
• Cerebrotendinous Xanthomatosis/sterol
  27-hydroxylaseDeficiency
• Citrullinemia
• Cystathioninemia

• Dicarboxylic Aminoaciduria
• Dichloromethane Ingestion
• Dihydrolipoyl Dehydrogenase Deficiency
• Dihydropyrimidine Dehydrogenase Deficiency
• Dimethylglycine Dehydrogenase Deficiency
• Essential Fructosuria
• Ethanolaminosis
• Ethylmalonic Aciduria
• Familial Iminoglycinuria
• Fanconis Syndrome
• Folate Disorder
• Fructose Intolerance
• Fulminant Hepatitis
• Fumarase Deficiency
• Galactosemia
• Glucoglycinuria
• Glutaric Aciduria Types 1 2
• Glutathionuria
• Glyceroluria (GKD)

• 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)

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Applications in Clinical Analysis

• 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

• 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

Clinical Chemistry 47, 1918-1921 (2001).
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Applications in Metabolite Imaging
Lactate
N-acetyl-aspartate
Glutamate
Citrate
Alanine
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Metabolic Microarrays
Acetic Acid Betaine Carnitine Citric
Acid Creatinine Dimethylglycine Dimethylamine Hipp
ulric Acid Lactic Acid Succinic
Acid Trimethylamine Trimn-N-Oxide Urea Lactose Sub
eric Acid Sebacic Acid Homovanillic
Acid Threonine Alanine Glycine Glucose
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
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Why Metabolomics For Transplants?

• Relatively non-invasive (no need for biopsy, just
  collect urine, blood or bile)
• Can be quite organ specific
• Very fast (lt60 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 (prepost op)

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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) 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)
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Applications In Transplantation
Organ Condition Metabolite(s) Increased Metabolite(s) Decreased 22
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)
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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

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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 dont have a complete list of human
  metabolites

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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 WBCs
• Make all data freely and electronically
  accessible (HMDB)
• Make all cmpds publicly available (HML)

www.hmdb.ca
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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

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David Wishart Comp. Sci. U of Alberta Proj. Leader
Brian Sykes Biochemistry U of Alberta NMR spect.
Russ Greiner Comp. Sci. U of Alberta Bioinformatic
s
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/Separati
on.
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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

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Thanks to...