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LIMS for the Masses The Human Metabolome Project Its Application to

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LIMS for the Masses The Human Metabolome Project Its Application to Powered By Docstoc
					The Human Metabolome
Project: Its Application to
  Disease Diagnosis &
       Monitoring
           David Wishart
       University of Alberta
     david.wishart@ualberta.ca
The Pyramid of Life

                Metabolomics
    1400
  Chemicals

                Proteomics
 2500 Enzymes

                Genomics
25,000 Genes
    Why Are Metabolites
        Relevant?




Metabolites are the Canaries of the Genome
 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)
Brian Sykes       Russ Greiner     David Wishart   Hans Vogel
Biochemistry      Comp. Sci.       Comp. Sci.      Biochemistry
U of Alberta      U of Alberta     U of Alberta    U of Calgary
NMR spect.        Bioinformatics   Proj. Leader    NMR spect.




Fiona Bamforth    Derrick Clive    Liang Li        Mike Ellison
Clin. Chemistry   Chemistry        Chemistry.      Biochemistry
U of Alberta      U of Alberta     U of Alberta    U of Alberta
Sample Acq.       Synthesis        MS/Separation   MS/Separation.
 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
             HGP vs. HMP
• Focus on genome        • Focus on metabolome
• US/UK Effort           • Canadian Effort
• 3,000,000,000 bases    • 1400 compounds
• Public Repository      • Public Repository
  (GenBank)                (HMDB)
• Lots of pre-existing   • Almost no pre-existing
  electronic data          electronic records
• $1 billion             • $7.5 million
• Start 1990, end 2003   • Start 2005, end 2008
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
Newer Metabolomics
   Technologies




iStat – 22 chemistries   Cyra-Nose 320
Coupled ES-IMS Systems




 IMS Column            Electro Spray Mechanism
          Chris Backhouse – U of A
            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
      The HMDB & the HML
• HMDB is the public    • HML is the Human
  face of the HMP         Metabolome Library
• Freely web-           • Repository of
  accessible database     chemical samples
  providing detailed      for public
  information on          redistribution
  metabolites,          • Includes purchased,
  chemistry, enzymes,     isolated &
  diseases, pathways      synthesized cmpds
• Links metabolome        (many unique or
  to genome               rare cmpds)
Human Metabolome Database




         www.hmdb.ca
 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 ID’d
  compounds with existing compounds
• Simulate the consequences of knock-
  outs or deletions on metabolic flux
The Human Metabolome
       Library




10 mg – 10 g of ~1400 human metabolites
   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
      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
• Petrochemical Analysis   • MRS and fMRI
NMR Metabolic Profiling and
    Drug Toxicology
                           25
                                 PC2
                  PAP      20

                           15

                           10          ANIT
                            5
                 ANIT       0

                           -5

                           -10
                                                      Control
                           -15
                 Control                      PAP
                           -20
                                                           PC1
                           -25
                             -30        -20     -10   0         10




  Principal Component Analysis
Genetic Disease
Testing via NMR
    140+ Detectable Conditions
   Adenine                           Dicarboxylic Aminoaciduria         Histidinemia
    Phosphoribosyltransferase         Dichloromethane Ingestion          Histidinuria
    Deficency                         Dihydrolipoyl Dehydrogenase        Homocystinsufonuria
   Adenylosuccinase Deficiency        Deficiency                         Homocystinuria
   Alcaptonuria                      Dihydropyrimidine                  4-Hydroxybutyric Aciduria
   a-Aminoadipic Aciduria             Dehydrogenase Deficiency           2-Hydroxyglutaric Aciduria
   b-Aminoisobutyric Aciduria        Dimethylglycine                    Hydroxykynureninuria
   a-Aminoketoadipic Aciduria         Dehydrogenase Deficiency
                                                                          Hydroxylysinemia
   Anorexia Nervosa                  Essential Fructosuria
                                                                          Hydroxylysinuria
   Argininemia                       Ethanolaminosis
                                                                          3-Hydroxy-3-methylglutaric Aciduria
   Argininosuccinic Aciduria         Ethylmalonic Aciduria
                                                                          3-Hydroxy-3-methylglutaryl-Co A
   Aspartylglycosaminuria            Familial Iminoglycinuria            Lyase Deficiency
   Asphyxia                          Fanconi’s Syndrome                 Hydroxyprolinemia
   Biopterin Disorders               Folate Disorder                    Hyperalaninemia
   Biotin-responsive Multiple        Fructose Intolerance               Hyperargininemia (Argininemia)
    Carboxylase Deficiency            Fulminant Hepatitis                Hyperglycinuria
   Canavan’s Disease                 Fumarase Deficiency                Hyperleucine-Isoleucinemia
   Carcinoid Syndrome                Galactosemia                       Hyperlysinemia
   Carnosinemia                      Glucoglycinuria                    Hyperornithinemia
   Cerebrotendinous                  Glutaric Aciduria Types 1 & 2      Hyperornithinemia-
    Xanthomatosis/sterol 27-          Glutathionuria                      Hyperammonemia-Homocitrullinuria
    hydroxylaseDeficiency             Glyceroluria (GKD)                  Syndrome (HHH)
   Citrullinemia                     D-Glyceric Aciduria                Hyperoxaluria Types I & 2
   Cystathioninemia                  Guanidinoacetate-
   Cystinosis                         Methyltransferase Deficiency       Hyperphenylalaninemia
   Cystinuria (Hypercystinuria)      Hartnup Disorder                   Hyperprolinemia
   Diabetes                          Hawkinsinuria                      Hyperthreoninemia
   Dibasic Aminoaciduria
Applications in Clinical Analysis
•   14 propionic acidemia                   • 96% sensitivity and 100%
•   11 methylmalonic aciduria
                                              specificity in ID of
•   11 cystinuria
•   6 alkaptonuria                            abnormal from normal by
•   4 glutaric aciduria I
                                              metabolite concentrations
•   3 pyruvate decarboxylase deficiency
•   3 ketosis                               • 95.5% sensitivity and
•   3 Hartnup disorder
•   3 cystinosis                              92.4% specificity in ID of
•   3 neuroblastoma                           disease or condition by
•   3 phenylketonuria
•   3 ethanol toxicity                        characteristic metabolite
•   3 glycerol kinase deficiency              concentrations
•   3 HMG CoA lyase deficiency
•   2 carbamoyl PO4 synthetase deficiency   • 120 sec per sample

            Clinical Chemistry 47, 1918-1921 (2001).
         Applications in Cancer




                       Homovanillic Acid
                       Dimethylglycine




                       Trimethylamine
                       Trimn-N-Oxide
                       Dimethylamine
Normal




                       Hippulric Acid

                       Succinic Acid




                       Sebacic Acid
                       Suberic Acid
                       Acetic Acid
Below Normal




                       Lactic Acid
                       Citric Acid
                       Creatinine




                       Threonine
                       Carnitine




                       Glucose
Above Norrmal




                       Alanine
                       Glycine
                       Lactose
                       Betaine




                       Urea
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.
    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)
Thanks to...

				
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posted:12/3/2011
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