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
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Applications In Transplantation
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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...