The Human Metabolome Project: Its Application to Disease Diagnosis

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The Human Metabolome Project Its Application to
Disease Diagnosis Monitoring

• David Wishart
• University of Alberta
• david.wishart_at_ualberta.ca

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The Pyramid of Life
Metabolomics Proteomics Genomics
1400 Chemicals
2500 Enzymes
25,000 Genes
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Why Are Metabolites Relevant?
Metabolites are the Canaries of the Genome
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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)

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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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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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HGP vs. HMP

• Focus on genome
• US/UK Effort
• 3,000,000,000 bases
• Public Repository (GenBank)
• Lots of pre-existing electronic data
• 1 billion
• Start 1990, end 2003

• Focus on metabolome
• Canadian Effort
• 1400 compounds
• Public Repository (HMDB)
• Almost no pre-existing electronic records
• 7.5 million
• Start 2005, end 2008

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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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Newer Metabolomics Technologies
iStat 22 chemistries
Cyra-Nose 320
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Coupled ES-IMS Systems
IMS Column
Electro Spray Mechanism
Chris Backhouse U of A
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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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The HMDB the HML

• HML is the Human Metabolome Library
• Repository of chemical samples for public
  redistribution
• Includes purchased, isolated synthesized cmpds
  (many unique or rare cmpds)

• HMDB is the public face of the HMP
• Freely web-accessible database providing detailed
  information on metabolites, chemistry, enzymes,
  diseases, pathways
• Links metabolome to genome

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Human Metabolome Database
www.hmdb.ca
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The HMDB Allows One To

• Learn more about the markers used in standard
  diagnoses
• Understand metabolism at many levels
• Link chemistry to genetics
• Link cmpd concentrations with disease
• Query and compare newly IDd compounds with
  existing compounds
• Simulate the consequences of knock-outs or
  deletions on metabolic flux

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The Human Metabolome Library
10 mg 10 g of 1400 human metabolites
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The HML Allows One To

• Access rare or unusual metabolites as references
  or standards for MS, HPLC, GC-MS or NMR analyses
• Compare newly isolated compounds with known cmpds
  (saves on reinventing the wheel)
• Use these compounds as precursors to synthesize
  new metabolites
• Screen for potential transcr. modulators

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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
• Petrochemical Analysis

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NMR Metabolic Profiling and Drug Toxicology
Principal Component Analysis
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Genetic Disease Testing via NMR
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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 Cancer
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
Metabolic Microarray - 35 min.
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Concluding Comments

• Metabolomics is here to stay
• Canada is actually leading the way in this field
  with several metabolomics companies and the Human
  Metabolome Project based entirely in Canada
• Think of the HMDB and the HML as new tools and
  reagents to do genomic research and to assist in
  diagnoses
• Characterized by rapidly evolving technologies
  (MS, NMR, MEMs, mFS)

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Thanks to...