Metabolomics: The Basics
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Transcript of Metabolomics: The Basics
Metabolomics: The Basics
David Wishart
Depts. Comp. Sci and Bio. Sci.
University of Alberta
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
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taurine
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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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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
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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...