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Metabolomics: The Basics
David Wishart
Depts. Comp. Sci and Bio. Sci.
University of Alberta
[email protected]
July 16, 2005, 8th Banff Symposium
The Pyramid of Life
Metabolomics
1400
Chemicals
Proteomics
2500 Enzymes
Genomics
25,000 Genes
Metabolomics
Primary Molecules
Secondary Molecules
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
Growth in Metabolomics
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Year
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# References
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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 > 1mM
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 >1 mM
• Need to draw the line somewhere
Why Are Metabolites
Relevant?
Metabolites are the Canaries of the Genome
Why is Metabolomics
Relevant?
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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
ppm
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Quantitative
Methods
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2
Chemometric (Pattern)
Methods
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TMAO
hippurate
allantoin creatinine taurine
1
PC2
20
creatinine
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10
citrate
ANIT
5
hippurate
urea
2-oxoglutarate
water
succinate
fumarate
0
-5
-10
ppm
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6
5
4
3
2
1
Control
-15
PAP
-20
-25
-30
PC1
-20
-10
0
10
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
Principles of Quantitative
Metabolomics
Mixture
Compound A
Compound B
Compound C
Quantitative Metabolomics with
Eclipse
Sample Compound List
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(+)-(-)-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
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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
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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
• 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
• Organ Transplantation
• MRS and fMRI
Metabolomics and Drug
Toxicology
25
PAP
PC2
20
15
10
ANIT
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ANIT
0
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Control
-10
Control
-15
PAP
-20
PC1
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-30
-20
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Principal Component Analysis
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Disease Diagnosis via NMR
(140+ Detectable Conditions)
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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
Canavan’s Disease
Carcinoid Syndrome
Carnosinemia
Cerebrotendinous
Xanthomatosis/sterol 27hydroxylaseDeficiency
Citrullinemia
Cystathioninemia
Cystinosis
Cystinuria (Hypercystinuria)
Diabetes
Dibasic Aminoaciduria
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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
GuanidinoacetateMethyltransferase Deficiency
Hartnup Disorder
Hawkinsinuria
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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
HyperornithinemiaHyperammonemia-Homocitrullinuria
Syndrome (HHH)
Hyperoxaluria Types I & 2
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Hyperphenylalaninemia
Hyperprolinemia
Hyperthreoninemia
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Applications in Clinical Analysis
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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
Lactate
N-acetyl-aspartate
Glutamate
Citrate
Alanine
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
Acetic Acid
Betaine
Carnitine
Citric Acid
Creatinine
Dimethylglycine
Dimethylamine
Hippulric Acid
Lactic Acid
Succinic Acid
Trimethylamine
Trimn-N-Oxide
Urea
Lactose
Suberic Acid
Sebacic Acid
Homovanillic Acid
Threonine
Alanine
Glycine
Glucose
Metabolic Microarrays
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 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)
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
Citrate, Glucose, Urea
Allantoin (urine & serum)
Nitrates, Nitrites, Nitric oxide
metabolites (urine)
Applications In Transplantation
Organ
Condition
Metabolite(s) Increased
Metabolite(s) Decreased
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)
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)
Urea (urine)
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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
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 Sykes
Biochemistry
U of Alberta
NMR spect.
Fiona Bamforth
Clin. Chemistry
U of Alberta
Sample Acq.
Russ Greiner
Comp. Sci.
U of Alberta
Bioinformatics
David Wishart
Comp. Sci.
U of Alberta
Proj. Leader
Hans Vogel
Biochemistry
U of Calgary
NMR spect.
Derrick Clive
Chemistry
U of Alberta
Synthesis
Liang Li
Chemistry.
U of Alberta
MS/Separation
Mike Ellison
Biochemistry
U of Alberta
MS/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...